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Hazards of travel 


EMERGING 
INFECTIOUS DISEASES 


A Peer-Reviewed Journal Tracking and Analyzing Disease Trends 


Vol.'ll, No.2, February 2005 




A Peer-Reviewed Journal Tracking and Analyzing Disease Trends 


pages 191-360 


EMERGING 
INFECTIOUS DISEASES 

EDITOR-IN-CHIEF 
D. Peter Drotman 


EDITORIAL STAFF 

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EDITORIAL BOARD 

Dennis Alexander, Addlestone Surrey, United Kingdom 

Ban Alios, Nashville, Tennessee, USA 

Michael Apicella, Iowa City, Iowa, USA 

Barry J. Beaty, Ft. Collins, Colorado, USA 

Martin J. Blaser, New York, New York, USA 

David Brandling-Bennet, Washington, D.C., USA 

Donald S. Burke, Baltimore, Maryland, USA 

Jay C. Butler, Anchorage, Alaska 

Charles H. Calisher, Ft. Collins, Colorado, USA 

Arturo Casadevall, New York, New York, USA 

Kenneth C. Castro, Atlanta, Georgia, USA 

Thomas Cleary, Houston, Texas, USA 

Anne DeGroot, Providence, Rhode Island, USA 

Vincent Deubel, Shanghai, China 

Ed Eitzen, Washington, D.C., USA 

Duane J. Gubler, Honolulu, Hawaii, USA 

Scott Halstead, Arlington, Virginia, USA 

David L. Heymann, Geneva, Switzerland 

Sakae Inouye, Tokyo, Japan 

Charles King, Cleveland, Ohio, USA 

Keith Klugman, Atlanta, Georgia, USA 

S.K. Lam, Kuala Lumpur, Malaysia 

Bruce R. Levin, Atlanta, Georgia, USA 

Myron Levine, Baltimore, Maryland, USA 

Stuart Levy, Boston, Massachusetts, USA 

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Tom Marrie, Edmonton, Alberta, Canada 

John E. McGowan, Jr., Atlanta, Georgia, USA 

Stephen S. Morse, New York, New York, USA 

Philip P. Mortimer, London, United Kingdom 

Fred A. Murphy, Davis, California, USA 

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P. Keith Murray, Ames, Iowa, USA 

Stephen Ostroff, Atlanta, Georgia, USA 

Rosanna W. Peeling, Geneva, Switzerland 

David H. Persing, Seattle, Washington, USA 

Gianfranco Pezzino, Topeka, Kansas, USA 

Richard Platt, Boston, Massachusetts, USA 

Mario Raviglione, Geneva, Switzerland 

Leslie Real, Atlanta, Georgia, USA 

David Reiman, Palo Alto, California, USA 

Nancy Rosenstein, Atlanta, Georgia, USA 

Connie Schmaljohn, Frederick, Maryland, USA 

Tom Schwan, Hamilton, Montana, USA 

Ira Schwartz, Valhalla, New York, USA 

Tom Shinnick, Atlanta, Georgia, USA 

Patricia Simone, Atlanta, Georgia, USA 

Bonnie Smoak, Bethesda, Maryland, USA 

Rosemary Soave, New York, New York, USA 

P. Frederick Sparling, Chapel Hill, North Carolina, USA 

Jan Svoboda, Prague, Czech Republic 

Bala Swaminathan, Atlanta, Georgia, USA 

Robert Swanepoel, Johannesburg, South Africa 

Phillip Tarr, Seattle, Washington, USA 

Timothy Tucker, Cape Town, South Africa 

Elaine Tuomanen, Memphis, Tennessee, USA 

Mary E. Wilson, Cambridge, Massachusetts, USA 

John Ward, Atlanta, Georgia, USA 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 




EMERGING 
INFECTIOUS DISEASES 

A Peer-Reviewed Journal Tracking and Analyzing Disease Trends Vol. 1 1, No. 2, February 2005 



On the Cover 

Romare Bearden (1911-1988). 

The Sea Nymph (1977) 

Collage on various papers with paint and 
graphite on fiberboard 
(111.8 cm x 81.3 cm). 

Permanent collection: Glen and Lynn Tobias. 

Cover art copyright 

Romare Bearden Foundation / 

Licensed by VAGA, New York, New York 

About the Cover p. 358 


Perspective 

Distinguishing Febrile Respiratory 
Illnesses from SARS 191 

K. Khan et al. 

Optimal management of febrile respiratory illnesses 
during a hypothetical SARS outbreak varies, but 
increasing influenza vaccination rates would save 
money and lives. 

Research 

Human Disease from Influenza A 
(H5N1), Thailand, 2004 201 

T. Chotpitayasunondh et al. 

Direct contact with sick poultry, young age, 
pneumonia and lymphopenia, and acute respiratory 
distress syndrome should prompt specific laboratory 
testing for H5 influenza. 



No H5N1 Transmission to Hospital 
Employees, Hanoi 210 

N.T. Liem et al. 

A seroprevalence study found no transmission of 
avian influenza H5N1 viruses from patients to 
hospital employees in Vietnam, 2004. 

Bacterial Zoonoses and Infective 
Endocarditis, Algeria 216 

A. Benslimani et al. 

Serologic and molecular tools are important for the 
diagnosis of blood culture-negative endocarditis. 



Novel Flavivirus or New 

Lineage of West Nile Virus 225 

T. Bakonyi et al. 

Rabensburg virus, isolated from Culex pipiens 
mosquitoes in central Europe, represents a new 
lineage of West Nile virus or a novel flavivirus of the 
Japanese encephalitis virus group. 

Survey for Bat Lyssaviruses, 

Thailand 232 

B. Lumlertdacha et al. 

Existence of lyssavirus infection in Thai bats has 
been confirmed by demonstration of serum 
neutralizing antibodies. 

Spotted Fever and Typhus 

Group Rickettsioses 237 

Y.-J. Choi et al. 

Multiplex nested PCR and sequencing analysis 
indicated rickettsialike agents in serum specimens 
from febrile patients. 

Pneumocystis jirovecii in 

General Population 245 

F.J. Medrano et al. 

P. jirovecii colonization can be frequently detected 
in immunocompetent adults, which suggests that 
the general population could be a source of this 
infection. 

Cryptosporidiosis Decline after 
Membrane Filtration 251 

S. Goh et al. 

Sporadic cryptosporidiosis and associated hospital 
admissions of children declined after membrane 
filtration of public drinking water supplies was 
introduced. 

Carbapenemase in 

Enterobacteriaceae, U.S. Rivers . . .260 

C. Aubron et al. 

Identification of imipenem-resistant Enterobacter 
asburiae isolates from distant rivers indicates an 
environmental reservoir for carbapenemase genes. 

Investigation of Rickettsial 
Infection in Brazil 265 

L.A. Sangioni et al. 

Surveys of horse serum are a useful method of 
surveillance for Brazilian spotted fever in areas 
where humans are exposed to Amblyomma 
cajennense ticks. 






Waddlia malaysiensis: New 
Chlamydialike Bacterium 271 

P.K.B. Chua et al. 

A novel obligate intracellular bacterium was isolated 
from urine samples from fruit bats ( Eonycteris 
spelaea) in peninsular Malaysia. 

Quarantine for SARS, Taiwan 278 

Y.-H. Hsieh et al. 

Quarantine for SARS during the 2003 Taiwan 
outbreak expedited case detection, thereby 
indirectly preventing infections. 

Wild Animal Mortality Monitoring 
and Human Ebola 283 

P. Rouquet et al. 

An animal mortality monitoring network in Gabon 
and the Republic of Congo has demonstrated 
potential to predict and possibly prevent human 
Ebola outbreaks. 


EMERGING 

INFECTIOUS DISEASES 

A Peer-Reviewed Journal Tracking and Analyzing Disease Trends Vol. 1 1, No. 2, February 2005 



330 Pneumocystis Transmission in 
Pediatric Transplant Unit 

B. Hocker et al. 

Another Dimension 

333 First Self 

G. Callahan 

Letters 

339 Schistosoma mansoni in Family 
5 Years after Safari 


Historical Review 


p. 288 341 Methicillin-resistant 

Staphylococcus aureus, Singapore 


Smallpox Surveillance and 
Control Measures 291 

E. Kerrod et al. 

Targeted surveillance and containment 
interventions have been successful for outbreak 
control and should be explored as alternatives to 
mass vaccination. 


343 Mumps Virus-associated 

Hemophagocytic Syndrome 

343 Imported Cutaneous Diphtheria, 
Germany 

344 Antimicrobial Drug Consumption 
in Companion Animals 


Dispatches 

298 In Vitro Host-Cell Susceptibility 
to Usutu Virus 

T. Bakonyi et al. 

302 Bat Incidents at Children’s 
Camps, New York 

A. Robbins et al. 

306 West Nile Virus in Morocco 

I. S chuff enecker et al. 

310 Differential Detection of 
Multiple Respiratory 
Pathogens 

T. Briese et al. 

314 Comparing Aberration 
Detection Methods 

L. Hutwagner et al. 

317 Malaria Epidemic and Drug 
Resistance, Djibouti 

C. Rogier et al. 

322 Late Recognition of SARS in 
Nosocomial Outbreak 

T. Wong et al. 



346 

Vibrio cholerae SXT Element, Laos 


347 

Impact of Influenza Pandemic on 



Pacific Islands 


349 

Mycotic Brain Abscess 


351 

Tuberculosis in Undocumented 

p. 299 


Migrants, Geneva 

A 9 & 

352 

Mycobacterium chelonae in 



Kidney-Pancreas Recipient 

m m % 

354 

Psychological Effects of 



Quarantine (Replies) 


Book Reviews 


356 

Community-based Health 



Research 


356 

The Pneumococcus 


357 

DNA Amplification 


News & Notes 



About the Cover 


358 

Hazards of Travel 


326 Typhoid Fever in Children in 
Urban Slum, Bangladesh 

W.A. Brooks et al. 


PERSPECTIVE 


Managing Febrile Respiratory 
Illnesses during a Hypothetical 
SARS Outbreak 

Kamran Khan,* Peter Muennig,t Michael Gardam4 and Joshua Graff Zivinj- 


Since the World Health Organization declared the 
global outbreak of severe acute respiratory syndrome 
(SARS) contained in July 2003, new cases have periodical- 
ly reemerged in Asia. This situation has placed hospitals 
and health officials worldwide on heightened alert. In a 
future outbreak, rapidly and accurately distinguishing 
SARS from other common febrile respiratory illnesses 
(FRIs) could be difficult. We constructed a decision-analy- 
sis model to identify the most efficient strategies for manag- 
ing undifferentiated FRIs within a hypothetical SARS 
outbreak in New York City during the season of respiratory 
infections. If establishing reliable epidemiologic links were 
not possible, societal costs would exceed $2.0 billion per 
month. SARS testing with existing polymerase chain reac- 
tion assays would have harmful public health and econom- 
ic consequences if SARS made up <0.1% of circulating 
FRIs. Increasing influenza vaccination rates among the 
general population before the onset of respiratory season 
would save both money and lives. 

O n July 5, 2003, the World Health Organization 
(WHO) declared that human chains of transmission of 
severe acute respiratory syndrome (SARS) had ended. 
Since then, new cases of SARS have resurfaced in Asia, 
including several in the absence of laboratory exposures. 
This reemergence of the SARS -associated coronavirus 
(SARS-CoV) has sparked international concern and has 
prompted heightened surveillance by hospitals and health 
officials worldwide. Such concerns have been amplified 
by fears that a future SARS outbreak could coincide with 
respiratory infection season, when influenza infections and 
other febrile respiratory illnesses (FRIs) develop in large 
segments of the population. 

Current SARS case-definition and case-exclusion crite- 
ria encompass clinical, epidemiologic, and laboratory fea- 


*St. Michael’s Hospital, Toronto, Ontario, Canada; fColumbia 
University, New York, New York, USA; and ^University Health 
Network, Toronto, Ontario, Canada 


tures (1). Should the timely establishment of epidemiolog- 
ic links between SARS cases be lost in a future outbreak, 
frontline healthcare providers would be forced to rely on 
clinical signs and symptoms or diagnostic testing to con- 
firm or exclude infections with SARS-CoV (2). 
Unfortunately, the signs and symptoms of SARS are non- 
specific and cannot be used reliably to differentiate SARS 
from other FRIs. Moreover, existing serologic tests for 
SARS-CoV cannot definitively exclude infection until at 
least 4 weeks has elapsed from the onset of symptoms and 
thus have no role in early clinical decision making (1). 
Although reverse transcriptase-polymerase chain reaction 
(RT-PCR) assays used to detect SARS-CoV can provide 
test results within a matter of hours, their suboptimal sen- 
sitivity makes them inadequate for ruling out SARS (3). 
Furthermore, since SARS infections would likely make up 
a minute fraction of FRIs circulating among the general 
population, the pretest probability, and thus the positive 
predictive value of RT-PCR tests, would be extremely low, 
even if future generation assays had better test sensitivity 
and specificity. 

In 2003 and 2004, the emergence of SARS-CoV in 
China coincided with respiratory illness season, which 
suggests that the virus may resurface during winter 
months, like many other respiratory pathogens. Should this 
seasonal pattern recur, rapidly and accurately differentiat- 
ing SARS infections from other FRIs would become a crit- 
ical component of any future outbreak containment efforts 
(2,3). This distinction will also continue to be an important 
issue among travelers in whom FRIs develop after their 
return from SARS -affected areas. However, existing diag- 
nostic limitations place frontline healthcare practitioners in 
a precarious position, since clinical decisions with poten- 
tially dangerous consequences must be made in the face of 
uncertainty. Recognizing such limitations, WHO recently 
called for the development of evidence-based clinical 
algorithms to help address these diagnostic dilemmas (4). 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


191 


PERSPECTIVE 


Methods 

Overview and Definitions 

A hypothetical cohort comprising all residents of New 
York City was entered into a decision-analysis model. The 
model is premised on a SARS outbreak during respirato- 
ry season where person-to-person transmission of SARS 
is documented and epidemiologic links between cases are 
poorly defined. The outbreak was designed to be consis- 
tent in size and duration with the Toronto outbreak (5). 
The analytic horizon of the analysis was defined as the 
expected lifetime of persons living in New York City dur- 
ing the 2004-2005 respiratory illness season. FRIs are 
defined herein as nonspecific infections caused by 
pathogens other than SARS-CoV for which the microbio- 
logic origin cannot be determined on the basis of clinical 
grounds alone. The model was designed to identify the 
most effective and cost-effective uses of societal 
resources in managing FRIs of undetermined origin dur- 
ing a SARS outbreak. 

The analysis was conducted in adherence with the ref- 
erence case scenario as defined by the Panel on Cost- 
Effectiveness in Health and Medicine (6). All relevant 
costs and benefits were considered from the societal per- 
spective of New York City, including those related to sec- 
ondary transmission of SARS. Since costs and changes in 
health-related quality of life in the analysis were limited to 
a single respiratory season, no discounting was performed 
on these 2 parameters. However, all future years of life lost 
due to premature death from infections were discounted at 
an annual rate of 3%. 

Decision-Analysis Model 

A decision-analysis model was constructed by using 
DATA 4.0 (TreeAge Software, Williamstown, MA, USA) 
that examined 2 competing strategies in the context of a 
SARS outbreak coinciding with respiratory season: 1) 
home isolation for persons with FRIs of undetermined ori- 
gin, pending fever and symptom resolution for at least 24 
hours and 2) outpatient diagnostic testing of FRIs to ascer- 
tain a microbiologic diagnosis with subsequent test-driven 
management. A third complementary strategy entailing 
mass influenza vaccination among the general population 
before the onset of respiratory season was considered in 
conjunction with the above competing strategies. 

Primary assumptions of the model were as follows: 1) 
epidemiologic linkages between SARS cases are not well 
defined; 2) SARS cannot reliably be distinguished from 
other FRIs on clinical grounds alone; 3) current SARS 
tests cannot definitively rule out infection early in the 
course of illness (1,7); 4) public nonadherence to home 
isolation guidelines during a SARS outbreak would be 
negligible (5,8); 5) positive SARS (RT-PCR) test requires 


isolation precautions pending confirmation of the diagno- 
sis (2); 6) patients with confirmed SARS cases will be 
managed as inpatients pending resolution of the clinical ill- 
ness; 7) patients with confirmed SARS cases require isola- 
tion precautions for 10 days after resolution of illness (2); 
8) persons with FRIs of undetermined origin must be 
afebrile and symptom-free for 24 hours before returning to 
work; 9) negative SARS (RT-PCR) test alone will have no 
influence on SARS isolation precautions (2); 10) negative 
SARS (RT-PCR) test result combined with a positive test 
for another respiratory pathogen will result in the discon- 
tinuation of SARS isolation precautions (2); 11) in the 
absence of appropriate isolation precautions, persons with 
SARS will transmit infection to 3 additional persons 
(9,10); 12) SARS, influenza, respiratory syncytial virus, 
and community-acquired pneumonia are the primary caus- 
es of death from FRIs; 13) a future SARS outbreak would 
be managed by using existing healthcare infrastructure; 
and 14) no proven effective treatment for SARS currently 
exists. 

A plausible range of high and low values for each vari- 
able was used to conduct sensitivity analyses, which exam- 
ined the influence of parameter error on the results of the 
analysis. Selected variables in the model are listed in 
Tables 1 and 2. 

Composition of FRIs 

We used nationally representative data (25,27) in con- 
junction with studies published in the medical literature 
(11,28-30) to derive our base estimates for an “average” 
respiratory season. In our model, the microbiologic origin 
of an FRI was categorized into 1 of 4 mutually exclusive 
groups: 1) SARS-CoV and coronaviruses OC43 and 229E; 
2) influenza viruses A and B; 3) a panel of common respi- 
ratory pathogens, including respiratory syncytial viruses A 
and B, parainfluenza viruses 1-3, human metapneu- 
movirus, Bordetella pertussis , Chlamydia pneumoniae , 
Mycoplasma pneumoniae , Legionella pneumophila , and L. 
micdadev, and 4) all other causes. 

In our base-case analysis, we assigned the proportion of 
FRIs due to SARS to be 0.01%, which was estimated 
assuming a SARS outbreak of similar size and duration to 
the Toronto outbreak. The proportion of FRIs due to 
influenza was derived from 2 large observational studies 
conducted over multiple respiratory seasons (11,28) and 
was corroborated by dividing the expected proportion of 
the U.S. population who get influenza each season (25) by 
the proportion of the U.S. population having influenzalike 
infections (27). The proportion of FRIs due to the common 
respiratory pathogen panel listed above was estimated 
from the medical literature (29,30). In our base-case sce- 
nario, we estimated that approximately one third of FRIs 
would be due to influenza, one third would be due to the 


192 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


Distinguishing Febrile Respiratory Illnesses from SARS 


panel of common respiratory pathogens, and the remaining 
one third would be due to other miscellaneous pathogens 
not indicated above. 

Diagnostic Tests 

We evaluated 3 categories of rapid diagnostic tests with 
optimal turnaround times of <24 hours. The first category 
constitutes RT-PCR assays capable of detecting SARS- 
CoV as well as coronaviruses OC43 and 229E (23,24). A 
second category includes 2 multiplex PCR assays, which, 
when used in combination, can detect 13 different respira- 
tory pathogens, including influenza viruses A and B, respi- 
ratory syncytial viruses A and B, parainfluenza viruses 
1-3, human metapneumo virus, C. pneumoniae , M. pneu- 
moniae , L. pneumophila , L. micdadei , and B. pertussis 
(20-22). The third category comprises a widely available 
enzyme immunoassay capable of rapidly detecting infec- 
tions with influenza A and B (19). 

The sensitivity and specificity of these tests were 
obtained from the medical literature (19-24), while the 
positive predictive value of each diagnostic test was calcu- 
lated by incorporating the estimated prevalence of specific 
pathogens into Bayes’ equation. 

Influenza Vaccination 

The effectiveness of the influenza vaccine was derived 
from the medical literature (31). To account for seasonal 
variation between circulating strains of influenza and the 
composition of the trivalent vaccine, we varied the effec- 
tiveness of the vaccine over a wide range of plausible val- 
ues in our sensitivity analysis. The average seasonal 


effectiveness of the influenza vaccine was adjusted by 
assuming that the vaccine would be poorly matched to cir- 
culating influenza strains approximately twice every 10 
years (31). 

We used data from the U.S. Behavioral Risk Factor 
Surveillance System to estimate seasonal influenza vacci- 
nation rates among the population of New York City (33). 
In our sensitivity analyses, we evaluated the incremental 
costs and benefits of raising vaccination rates above this 
seasonal average. 

Management Algorithms 

In our model, the home isolation strategy required per- 
sons with FRIs of undetermined origin to remain at home 
for at least 24 hours after resolution of illness. We assumed 
that adherence to public health guidelines in the setting of 
a widespread SARS outbreak would be near universal 
(5,8). Under this strategy, we assumed that persons would 
attempt to manage their illness at home by using self-care, 
visit a healthcare provider if the illness were serious or per- 
sistent, or proceed to a hospital if their illness became pro- 
gressively severe. 

The diagnostic evaluation strategy involved outpatient 
testing of persons with FRIs to ascertain a microbiologic 
origin. In this strategy, persons with FRIs of undetermined 
cause would observe home isolation precautions until the 
results of diagnostic tests were available. We assumed that 
a positive SARS RT-PCR test would require isolation pre- 
cautions for the patient, public health intervention, and 
additional testing to confirm the diagnosis (2). We also 
assumed that a negative SARS RT-PCR test in conjunction 


Table 1 . Selected costs in the decision-analysis model* 

Costsf 

Low 

Base 

High 

Source 

Vaccines and medications 





Influenza vaccine 

$10.00 

$27.78 

$40.00 

11 

Antibiotics for FRIf 
Medical care§ 

$30.00 

$64.72 

$80.00 

12 

Ambulatory clinic visit 

$40.00 

$60.03 

$80.00 

13 

Hospitalization for FRI 

$5,000 

$1 1 ,645 

$15,000 

14 

Hospitalization for influenza 

$7,500 

$17,465 

$25,000 

14 

Hospitalization for PUI 

$15,000 

$19,441 

$25,000 

14 

Hospitalization for SARS 
Diagnostic tests 

$20,000 

$28,502 

$40,000 

14,15 

Rapid influenza test 

$15.00 

$26.86 

$40.00 

16 

Multiplex^} RT-PCR 

$50.00 

$154.02 

$200.00 

Prodesse Inc., pers. comm. 

SARS# RT-PCR 

$20.00 

$54.80 

$100.00 

Prodesse Inc., pers. comm. 

Miscellaneous 





Patient time (per hour) 

$15.00 

$24.55 

$30.00 

17 

Contact investigation (per SARS contact) 

$100.00 

$222.94 

$300.00 

5,18 

*FRI, febrile respiratory illness; PUI, person under investigation (for SARS); SARS, severe acute respiratory syndrome; RT-PCR, reverse transcription- 
polymerase chain reaction. 

fMedical and nonmedical costs were adjusted to 2004 U.S. dollars by using the Consumer Price Index. 


^Antimicrobial drug costs are based on a 7-day course of oral levofloxacin. 

§lncludes laboratory tests, transportation costs, and patient time. 

TfDetects influenza viruses A and B, respiratory syncytial viruses A and B, parainfluenza viruses 1-3, human metapneumovirus, Legionella pneumophila, 
L. micdadei, Mycoplasma pneumoniae, Chlamydia pneumoniae, and Bordetella pertussis. 

#Detects SARS-associated coronavirus and coronaviruses OC43 and 229E. 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


193 


PERSPECTIVE 


Table 2. Selected probabilities in the decision-analysis model* 

Selected probabilities 

Low 

Base 

High 

Source 

Diagnostic tests 





Sensitivity of influenza test 

0.50 

0.70 

0.90 

19 

Specificity of influenza test 

0.80 

0.95 

0.99 

19 

Sensitivity of multiplex! RT-PCR 

0.70 

0.85 

0.95 

20-22 

Specificity of multiplex! RT-PCR 

0.80 

0.987 

0.99 

20-22 

Sensitivity of SARSf RT-PCR 

0.25 

0.70 

0.95 

4,23 

Specificity of SARSf RT-PCR 
Morbidity and mortality 

0.95 

0.994 

1.00 

23,24 

Hospitalization due to influenza 

0.002 

0.004 

0.01 

25 

Death due to influenza 

0.0 

0.0012 

0.01 

25,26 

Hospitalization due to FRI 

0.010 

0.015 

0.02 

Calculated 

Death due to FRI 

0.0 

0.0009 

0.01 

Calculated 

Death due to SARS 

0.10 

0.15 

0.20 

24 

Miscellaneous probabilities 





Probability of an FRI 

0.10 

0.33 

0.50 

27 

Due to influenza 

0.20 

0.33 

0.50 

11,25,27,28 

Due to multiplex! organisms other than 
influenza 

0.20 

0.33 

0.50 

29,30 

Due to other causes§ 

0.20 

0.33 

0.50 

Calculated 

Due to SARS 

0.0 

0.0001 

0.01 

Assigned 

Influenza vaccine effectiveness 

0.35 

0.67 

0.85 

31 

Annual probability of poor match between 
vaccine and circulating influenza strains 

0.05 

0.20 

0.50 

31 

Probability of successful self-care 
management of an FRI at home 

0.33 

0.67 

1.00 

Assumption 

Probability of receiving outpatient 
antimicrobial drugs for an FRI 

0.33 

0.67 

1.00 

32 

Miscellaneous values 





Patient time for outpatient medical visit (min) 

30 

50 

90 

Estimate 

Influenza length of illness (d) 

3 

7 

10 

31 

Other FRIjf length of illness (d) 

1 

3 

5 

Estimate 

Average duration of hospitalization, influenza 

(d) 

Average duration of hospitalization, FRI^j (d) 

5 

10.2 

15 

14 

3 

7.7 

10 

14 

Average duration of hospitalization, SARS 
(d) 

HRQL scores 

10 

16 

30 

15 

SARS, hospitalized 

0.05 

0.160 

0.50 

HUI 

SARS, outpatient 

0.25 

0.670 

0.75 

HUI 

SARS, contact 

0.50 

0.785 

0.95 

HUI 

FRI, hospitalized 

0.25 

0.511 

0.75 

HUI 

FRI, outpatient 

0.50 

0.804 

0.95 

HUI 

Reproductive number for SARS# 

2 

3 

4 

9,10 

Contact investigations (per SARS case) 

25 

50 

100 

5 


*RT-PCR, reverse transcriptase-polymerase chain reaction; SARS, severe acute respiratory syndrome; FRI, febrile respiratory illness; HRQL, health- 
related quality of life; HUI, Health Utilities Index. 

fRefers to influenza viruses A and B, respiratory syncytial viruses A and B, parainfluenza viruses 1-3, human metapneumovirus, Legionella pneumophila, 
L. micdadei, Mycoplasma pneumoniae, Chlamydia pneumoniae, and Bordetella pertussis. 

^Detects SARS-associated coronavirus and coronaviruses OC43 and 229E. 

§Febrile respiratory illnesses not due to SARS, influenza viruses A and B, respiratory syncytial viruses A and B, parainfluenza viruses 1-3, human 
metapneumovirus, L. pneumophila, L. micdadei, M. pneumoniae, C. pneumoniae, and B. pertussis. 

IJFebrile respiratory illnesses not due to SARS or influenza viruses A and B. 

#ln the absence of public health interventions. 


with a positive test for an alternate respiratory pathogen 
would lead to the elimination of isolation precautions (2). 
If all test results were negative, we assumed that isolation 
precautions would remain in effect, since current SARS 
RT-PCR assays are not sufficiently sensitive to rule out 
SARS (2). We also assumed that persons with FRIs, for 


which the microbiologic origin was confirmed to be due to 
a pathogen other than SARS-CoV, would return to work 
only after resolution of their illness. 

Under each strategy, we considered the possibility that 
persons with FRIs seeking medical care might receive 
antimicrobial drugs during their evaluation. We estimated 


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Distinguishing Febrile Respiratory Illnesses from SARS 


this probability by using data from the National 
Ambulatory Medical Care Survey (32). 

Illness and Death 

Changes in health-related quality of life (HRQL), 
including the impact of isolation, due to SARS and other 
FRIs were derived by using the Health Utilities Index 
Mark 3 (HUI) (34). We used the HUI to minimize double 
counting of productivity losses, since HRQL scores gener- 
ated from this instrument do not include productivity loss- 
es (William Furlong, pers. comm.). Parameters for the HUI 
were derived from a panel of 4 specialist physicians with 
clinical experience managing SARS patients in Toronto. 
These physicians did not directly value health states, but 
rather functioned as expert “describers,” who facilitated 
the mapping of heath states to community-based prefer- 
ence scores from the HUI. 

SARS, influenza, respiratory syncytial virus, and com- 
munity-acquired pneumonia due to typical and atypical 
bacteria were assumed to be the primary contributors of 
death from FRIs on a population level. Mortality data for 
community-acquired pneumonia were obtained from the 
National Center for Health Statistics (35); data for SARS, 
influenza, and respiratory syncytial virus were obtained 
from the medical literature (24,26,36). We estimated that 
patients with SARS would each transmit infection to 3 
other persons if appropriate isolation precautions were not 
observed (e.g., false-negative SARS RT-PCR test com- 
bined with a false-positive test for an alternate diagnosis) 
(9,10). 

Costs and Charges 

Costs attributable to transportation, ambulatory care 
(13), laboratory tests (16), influenza vaccination (11), 
antimicrobial agents (12), hospitalization (14,15), public 
health investigation (5,18), and patient time (17) were 
included in the analysis. Transportation costs to see a med- 
ical provider were derived by using U.S. national data and 
were adjusted to account for the estimated proportion of 
the population driving, using public transportation, or trav- 
eling by other means such as biking or walking. The base 
cost of an ambulatory care visit was estimated by using the 
national average 2000 Medicare reimbursement rates for a 
focused medical evaluation (CPT-code 99213); the cost of 
the rapid influenza test was derived from the Centers for 
Medicare and Medicaid Services (16). The costs of the 
SARS RT-PCR assay and the multiplex PCR assays used 
to detect the common respiratory pathogen panel were 
obtained from a test manufacturer and included 15 minutes 
of technician time (Prodesse Inc., pers. comm.)(18). 

Influenza vaccination and antimicrobial drug costs 
were obtained by using average wholesale prices of phar- 


maceuticals (11,12). The costs and frequency of adverse 
reactions to influenza vaccination were estimated from the 
medical literature and incorporated into the net costs and 
benefits of the vaccine (37). 

Hospital charges and the average length of stay for 
patients with influenza and other respiratory infections 
requiring hospitalization were estimated from the 
Healthcare Cost and Utilization Project (14). The 
Medicare Provider Analysis and Review system was used 
to derive cost-to-charge ratios and subsequently convert 
hospital charges into societal costs (38). Per diem hospital- 
ization costs for SARS were approximated by using ICD- 
9 code 769, “respiratory distress syndrome,” which was 
subsequently multiplied by the average length of stay for 
hospitalized patients with SARS (15). Public health costs, 
including contact investigation, were estimated from the 
Toronto SARS experience (5). 

Patient time costs were estimated from data on the 
median salary of persons living in New York City and 
included time spent in travel and receiving medical care 
(17). When applicable, medical and nonmedical costs were 
adjusted to 2004 U.S. dollars by using the Consumer Price 
Index. The potential economic effects of a SARS outbreak 
on tourism or other commercial industries were not consid- 
ered in the analysis. 

Results 

If SARS were to resurface during the 2004-2005 respi- 
ratory season and the timely establishment of epidemio- 
logic links between SARS cases was not possible, our 
analysis estimates that the societal costs for New York City 
would exceed $2.0 billion for each month in which the 
SARS outbreak and respiratory season coincided. 

In our base-case analysis, we found the use of multiplex 
PCR assays to detect infections with a broad panel of com- 
mon respiratory pathogens to be the dominant strategy, 
saving $79 million and resulting in the gain of 8,474 qual- 
ity-adjusted life-years (QALYs) relative to a strategy of 
home isolation. If SARS RT-PCR testing were used in con- 
junction with multiplex PCR assays in our base-case sce- 
nario, however, we estimate that costs would increase by 
about $87 million and have lower effectiveness than mul- 
tiplex PCR testing alone. These findings are directly 
related to the very low positive predictive value of the 
SARS RT-PCR test under low prevalence conditions and 
the harm resulting from false-positive test results. 

If SARS testing were unavailable, confirming an alter- 
nate diagnosis for an FRI would be the most effective and 
least expensive strategy, dominating a strategy of influen- 
za testing alone or home isolation. However, if multiplex 
PCR testing were also unavailable, home isolation would 
be the least expensive strategy, albeit less effective than 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


195 


PERSPECTIVE 


testing for influenza alone. Rapid influenza testing would 
be accomplished at an incremental cost of $9.0 million but 
would result in gains of 5,286 QALYs (incremental cost- 
effectiveness ratio of $1,702 per QALY gained). If the 
described outbreak were to unfold, a campaign to increase 
influenza vaccination rates among the general population 
before the onset of respiratory season would save an esti- 
mated $5.0 million and lead to the gain of 128 QALYs for 
each percentage of New York City’s population vaccinated 
above the seasonal baseline. 

The total costs, the number of QALYs gained, and the 
incremental cost-effectiveness of each strategy in the 
model is shown in Table 3. The results of sensitivity analy- 
ses are shown in Table 4 and Figure 1 . Algorithms outlin- 
ing optimal treatment strategies under different testing 
capabilities are shown in Figure 2. 

Discussion 

Our analysis indicates that current diagnostic limita- 
tions in discriminating SARS from other common FRIs 
could have enormous public health and economic conse- 
quences, particularly if epidemiologic links between 
SARS cases were to become tenuous. Under such condi- 
tions, we found that most costs would not be related to 
SARS infections themselves, but rather to procedural 
changes in the management of other FRIs due to the 
known or perceived presence of SARS. 

We report 3 key findings with direct policy relevance. 
First, in our base analysis, the most efficient mechanism 
for discriminating SARS infections from other FRIs 
involves excluding SARS by confirming an alternate diag- 
nosis. This approach is the most cost-effective strategy 
under low prevalence conditions since the positive predic- 
tive value of SARS RT-PCR tests would be extremely low, 
and false-positive SARS tests would have deleterious soci- 
etal repercussions. While the Centers for Disease Control 
and Prevention supports an approach of excluding SARS 


by confirming an alternate diagnosis (2), caution is advised 
since SARS coinfection with other respiratory pathogens, 
including the human metapneumo virus, has been docu- 
mented (39). 

Second, we demonstrate that SARS testing under low 
prevalence conditions would be detrimental from both a 
public health and an economic perspective. In our analysis, 
the low positive predictive value of the SARS RT-PCR test 
translates into unnecessary costs from diagnostic testing, 
public health interventions, and lost opportunity costs for 
persons with false-positive test results. Moreover, negative 
consequences on quality of life would occur when persons 
are incorrectly diagnosed as having an infection with 
SARS. Our sensitivity analyses indicate that SARS diag- 
nostic testing should not be performed unless the preva- 
lence or pretest probability of SARS among persons 
presenting with FRIs exceeds 0.1%. 

Third, the use of influenza vaccination as a means to 
distinguish SARS from influenza has been debated (40). In 
our analysis, we find that if SARS reemerged during respi- 
ratory season, higher rates of influenza vaccination among 
the general population would lead to both health benefits 
and economic savings. These savings would occur by 
reductions in influenza illness and death, reductions in 
costs related to the investigation and isolation of persons 
with FRIs, and increases in the pretest probability of SARS 
and, therefore, the positive predictive value of SARS diag- 
nostic testing. The policy implications of these findings, 
however, must be carefully considered in the context of 
available influenza vaccine supplies and must ensure their 
prioritization for groups at high risk (40). 

Our analysis has several limitations. Foremost was our 
inability to derive specific estimates of the proportion of 
FRIs due to specific pathogens. Since the seasonal compo- 
sition of respiratory viruses and bacteria varies across 
regions and seasons, we attempted to derive estimates that 
best reflected seasonal averages. Although national 


Table 3. Cost-effectiveness of strategies for managing FRIs of undetermined etiology* 


Available public health strategies 

Monthly total 

Costs ($ billion)! QALY gained Incremental cost-effectiveness (cost per QALY gained) 

Flome isolation 

2.13 

0 

- 

Influenza testing 

2.14 

5,286 

$1,702 

Flome isolation 

2.13 

0 

- 

Influenza testing 

2.14 

5,286 

Dominated 

Multiplex RT-PCR testing! 

2.05 

8,474 

Savings 

Flome isolation 

2.13 

0 

- 

SARS + influenza testing 

2.19 

5,280 

Dominated 

Influenza testing 

2.14 

5,286 

Dominated 

SARS + multiplex RT-PCR testing! 

2.14 

8,429 

Dominated 

Multiplex RT-PCR testing! 

2.05 

8,474 

Savings 


*FRI, febrile respiratory illness; QALY, quality-adjusted life-year; RT-PCR, reverse transcription-polymerase chain reaction; -, reference category. 
fShown in 2004 U.S. dollars rounded to the nearest 10 million. 

^Multiplex RT-PCR testing to detect influenza viruses A and B, respiratory syncytial viruses A and B, parainfluenza viruses 1-3, human 
metapneumovirus, Bordetella pertussis, Chlamydia pneumoniae, Mycoplasma pneumoniae, Legionella pneumophila, and L. micdadei. 


196 


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Table 4. Threshold values from one-way sensitivity analyses* 

Distinguishing Febrile Respiratory Illnesses from SARS 

SARS prevalence (%)f 

Appropriate strategy 


Broad testing capabilities^ 

<0.1% Multiplex§ RT-PCR testing alone is the most effective and least expensive (i.e., dominant) 

strategy. 

0.1 %-0.9% Combination of SARS and multiplex§ RT-PCR testing is the most effective strategy, while 

multiplex PCR testing alone is the least expensive strategy. 

>0.9% Combination of SARS and multiplex}: RT-PCR§ testing is the most effective strategy, while home 

isolation is the least expensive strategy. 


Intermediate testing capabilities^ 

<0.9% Multiplex§ RT-PCR testing alone is the most effective and least expensive (i.e., dominant) 

strategy. 

>0.9% Multiplex§ RT-PCR testing alone is the most effective strategy, while home isolation is the least 

expensive strategy. 

Minimal testing capabilities# 

<1 .9% Rapid influenza testing is more effective than home isolation. 


Any Home isolation is less expensive than rapid influenza testing. 

Influenza is >36% of FRIs Rapid influenza testing is the dominant strategy. 

*SARS, severe acute respiratory syndrome; FRI, febrile respiratory illness; RT-PCR, reverse transcription-polymerase chain reaction. 

■{■Prevalence or pretest probability of SARS among circulating FRIs. 

^Capable of performing rapid influenza antigen detection tests, multiplex polymerase chain reaction assays to detect influenza viruses A and B, 
respiratory syncytial viruses A and B, parainfluenza viruses 1-3, human metapneumovirus, Bordetella pertussis, Chlamydia pneumoniae, Mycoplasma 
pneumoniae, Legionella pneumophila, and L, micdadei, and coronavirus assays to detect SARS-associated coronavirus and coronaviruses OC43 and 
229E (with test turnaround times <24 hours). 

§Refers to influenza viruses A and B, respiratory syncytial viruses A and B, parainfluenza viruses 1-3, human metapneumovirus, B, pertussis, C. 
pneumoniae, M. pneumoniae, L. pneumophila, and L. micdadei. 

IJCapable of performing rapid influenza antigen detection tests, multiplex PCR assays to detect influenza viruses A and B, respiratory syncytial viruses A 
and B, parainfluenza viruses 1-3, human metapneumovirus, B. pertussis, C. pneumoniae, M. pneumoniae, L. pneumophila, and L. micdadei (with test 
turnaround times of <24 hours). 

#Capable of performing rapid influenza antigen detection tests (with test turnaround times of <24 hours). 


surveillance data on influenza are available, information 
on other common respiratory pathogens are more limited, 
since most of these pathogens are self-limited, nonre- 
portable diseases, for which treatment is infrequently 
sought. 

We estimated the sensitivity of current SARS RT-PCR 
assays to be -70% (4); however, we recognize that the type 
of specimen tested and the timing of collection can influ- 
ence the test’s sensitivity (4,36). In our base-case scenario, 
in which SARS represented 0.01% of all circulating FRIs, 
changes in SARS RT-PCR test sensitivity had a negligible 
impact on overall societal costs and population health. If 
the pretest probability of SARS were to increase substan- 
tially above our baseline, however, SARS RT-PCR test 
sensitivity would have an increasingly important influence 
on the effectiveness of strategies involving SARS testing. 

Our reported test sensitivity for the multiplex PCR 
assays, which detect common respiratory viruses and bac- 
teria, is lower than values reported in the medical literature 
(20-22). Since estimates in the literature reflect experi- 
mental conditions and are essentially measures of test effi- 
cacy, we wished to estimate real-world effectiveness of 
these tests by taking into account factors such as ineffec- 
tive specimen collection methods, delays in laboratory 
testing, or other related factors. 

Our analysis demonstrates that influenza vaccination 
would lead to cost-savings, which has been reported in 
other studies of healthy adults in the pre-SARS era (31,37). 


However, the specific benefits quantified in our analysis 
would only be realized if the conditions of the model were 
to occur, i.e., the reemergence of SARS during a respirato- 
ry season, when epidemiologic links between cases are 
poorly defined. 

Finally, our analysis does not adequately address the 
complexities of microbiologic coinfection in the develop- 
ment of FRIs. While our model allows for multiple posi- 
tive test results, we assume that only 1 organism is 
responsible for causing an FRI. This issue is particularly 
relevant when considering SARS coinfection with other 
respiratory organisms (39). Nonetheless, in our analysis 
the effect of SARS coinfection on a population level is 
minimal given that SARS -Co V infections make up only 
0.01% of all FRIs. 

Speculation about the reemergence of SARS has 
prompted heightened surveillance by health officials 
worldwide. Given that SARS has resurfaced in each of the 
past 2 respiratory seasons in the absence of accidental lab- 
oratory exposures, SARS -Co V may reappear annually at 
times when FRIs are widely prevalent among the general 
population. Even if the world does not experience another 
large-scale, multinational outbreak, healthcare providers 
around the globe will continue to see patients with nonspe- 
cific FRIs who are incidentally returning from SARS- 
affected areas. This fact underscores the importance of 
having evidence-based guidelines to facilitate the timely 
and accurate distinction of SARS infections from other 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


197 


PERSPECTIVE 




prgtwbitft y <at SARS iimnog undiff .w.mtLrtlnrl FRk 


Figure 1. Two-way sensitivity analysis on the prevalence (i.e., 
pretest probability) of severe acute respiratory syndrome and 
influenza among undifferentiated febrile respiratory illnesses. A) 
Preferred strategies to minimize societal costs. B) Preferred strate- 
gies to maximize societal health. 


FRIs of lesser public health importance. Our analysis pro- 
vides guidance on the most effective and efficient use of 
resources when managing persons with FRIs of undeter- 
mined etiology when the epidemiologic history for SARS 
is either unavailable or unreliable. Our findings will help 
policy makers and healthcare practitioners make decisions 
based on available evidence and avoid decisions that are 
driven by fear and misinformation. 

Acknowledgments 

We are indebted to James Brunton, Kevin Gough, Mona 
Loutfy, and Sharon Walmsely for sharing their SARS knowledge 
and experience; Arthur Slutsky for his insightful comments on the 
manuscript; Marisa Creatore and Peter Gozdyra for their assis- 
tance in developing the decision analysis model; and Mohammad 
Keshoofy for his assistance in preparing this manuscript. 

This research was jointly conducted by the Inner City Health 
Research Unit, St. Michael’s Hospital, Toronto, and the Mailman 
School of Public Health, Columbia University, New York City. 

Funding for this study was provided by the Canadian 
Institutes of Health Research and Columbia University. Joshua 



Figure 2. Optimal management of undifferentiated febrile respira- 
tory illnesses under different testing capabilities. pSARS, preva- 
lence (i.e., pretest probability) of severe acute respiratory 
syndrome among febrile respiratory illnesses. Values are rounded 
to the nearest fraction. 


Graff Zivin is the recipient of an unrestricted research grant from 
the Merck Company Foundation to study medical innovation and 
health policy. None of the authors bears financial conflicts of 
interest pertaining to influenza vaccination, diagnostic tests, or 
any other elements of the study. 

Dr. Khan is an infectious diseases specialist with advanced 
training in preventive medicine and public health. He is an assis- 
tant professor of medicine at St. Michael’s Hospital, University 
of Toronto, where he conducts research on infectious diseases in 
new immigrant and refugee populations. His additional research 
interests include population mobility, the global movement of 
infectious diseases, health economics, and decision and cost- 
effectiveness analyses. 

References 

1. Centers for Disease Control and Prevention. Revised U.S. surveil- 
lance case definition for severe acute respiratory syndrome (SARS) 
and update on SARS cases — United States and worldwide, December 
2003. MMWR Morb Mortal Wkly Rep. 2003;52:1202-6. 

2. Centers for Disease Control and Prevention. Clinical guidance on the 
identification and evaluation of possible SARS-CoV disease among 
persons presenting with community-acquired illness [monograph on 
the Internet]. 2004 Jan 8 [cited 2004 Jan 28]. Available from 
http ://www. cdc . go v/ncidod/ sars/pdf/clinicalguidance .pdf 


198 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 



PERSPECTIVE 


3. Centers for Disease Control and Prevention. Public health guidance 
for community-level preparedness and response to severe acute res- 
piratory syndrome (SARS) version 2/3 [monograph on the Internet]. 
2004 Jul 20 [cited 2004 Nov 23]. Available from http://www.cdc. 
go v/ncidod/ sars/ guidance 

4. World Health Organization. Summary of the discussion and recom- 
mendations of the SARS laboratory workshop [monograph on the 
Internet]. 2003 Oct 22 [cited 2004 Jan 28]. Available from 
http ://w ww. who .int/csr/ sars/ guidelines/ en/S ARSLabmeeting .pdf 

5. Basrur SV. Toronto Public Health’s response to the severe acute res- 
piratory syndrome (SARS) outbreak 2003. Report to board of health 
[monograph on the Internet]. 2003 Sep 9 [cited 2004 Jan 28]. 
Available from http://www.city.toronto.on.ca/health/pdf/boh_2003 
septl5_tph_response_to_sars.pdf 

6. Gold MR, Siegel JE, Russel LB, Weinstein MC, editors. Cost-effec- 
tiveness in health and medicine. New York: Oxford University Press; 
1996. 

7. Centers for Disease Control and Prevention. Interpreting SARS-CoV 
test results from CDC and other public health laboratories [mono- 
graph on the Internet]. 2004 Jan 8 [cited 2004 Jan 28]. Available from 
http://www.cdc.gOv/ncidod/sars/guidance/F/pdf/app7.pdf 

8. Blendon RJ, Benson JM, DesRoches CM, Raleigh E, Taylor-Clark K. 
The public’s response to severe acute respiratory syndrome in 
Toronto and the United States. Clin Infect Dis. 2004;38:925-31. 

9. Lipsitch M, Cohen T, Cooper B, Robins J, Ma S, James L, et al. 
Transmission dynamics and control of severe acute respiratory syn- 
drome. Science. 2003;300:1966-70. 

10. Riley S, Fraser C, Donnelly CA, Ghani AC, Abu-Raddad LJ, Hedley 
AJ, et al. Transmission dynamics of the etiological agent of SARS in 
Hong Kong: impact of public health interventions. Science. 
2003;300:1961-6. 

11. Bridges C, Thompson W, Meltzer M, Reeve G, Talamonti W, Cox N, 
et al. Effectiveness and cost-benefit of influenza vaccination of 
healthy working adults. JAMA. 2000;284:1655-63. 

12. Drug topics red book: pharmacy’s fundamental resource. Montvale 
(NJ): Medical Economics; 2003. 

13. Centers for Medicare and Medicaid Services. Medicare payment sys- 

tems and coding files - physician fee schedule [monograph on the 
Internet]. [cited 2004 Jan 28]. Available from 

http://www.cms.gov/physicians/pfs/default.asp 

14. Agency for Healthcare Research and Quality. Healthcare cost and uti- 
lization project [database on the Internet]. 2001 [cited 2004 Jan 28]. 
Available from http://hcup.ahrq.gov/HCUPnet.asp 

15. Ontario Ministry of Health and Long Term Care, Public Health 
Division, Epi-Centre. SARS database. 2004. Toronto, Canada. 

16. Centers for Medicare and Medicaid Services. Medicare payment sys- 
tems and coding files - clinical laboratory fee schedule [monograph 
on the Internet], [cited 2004 Jan 28]. Available from 
http ://w w w. cms .gov/ pro vider s/puf do wnload/def ault . asp#labf ee 

17. Bureau of Labor Statistics. Mean hourly earnings and weekly hours 
by selected characteristics, private industry and state and local gov- 
ernment [monograph on the Internet]. 2002 Apr [cited 2004 Jan 28]. 
Available from http://www.bls.gov/ncs/ocs/sp/ncbl0497.txd 

18. Bureau of Labor Statistics. National occupational employment and 
wage estimates; healthcare practitioner and technical occupations 
[monograph on the Internet]. 2003 Nov 26 [cited 2004 Jan 28]. 
Available from http://www.bls.gov/oes/2002/oes_29He.htm 

19. Centers for Disease Control and Prevention. Laboratory diagnostic 
procedures for influenza [monograph on the Internet], [cited 2004 Jan 
28]. Available from: http://www.cdc.gov/flu/professionals/labdiagno- 
sis.htm 

20. Hindiyeh M. Hillyard DR. Carroll KC. Evaluation of the Prodesse 
Hexaplex multiplex PCR assay for direct detection of seven respira- 
tory viruses in clinical specimens. Am J Clin Pathol. 
2001;116:218-24. 


21. Fan J. Henrickson KJ. Savatski LL. Rapid simultaneous diagnosis of 
infections with respiratory syncytial viruses A and B, influenza virus- 
es A and B, and human parainfluenza virus types 1, 2, and 3 by mul- 
tiplex quantitative reverse transcription-polymerase chain 
reaction- enzyme hybridization assay (Hexaplex). Clin Infect Dis. 
1998;26:1397-402. 

22. Welti M, Jaton K, Altwegg M, Sahli R, Wenger A, Bille J. 
Development of a multiplex real-time quantitative PCR assay to 
detect Chlamydia pneumoniae, Legionella pneumophila and 
Mycoplasma pneumoniae in respiratory tract secretions. Diagn 
Microbiol Infect Dis. 2003;45:85-95. 

23. Chan HK, Poon LLLM, Cheng VCC, Guan Y, Hung IFN, Kong J, et 
al. Detection of SARS coronavirus in patients with suspected SARS. 
Emerg Infect Dis [serial on the Internet]. 2004 Feb [cited 2004 Jan 
28]. Available from http://www.cdc.gov/ncidod/EID/voll0no2/03- 
0610.htm 

24. World Health Organization. Consensus document on the epidemiolo- 
gy of severe acute respiratory syndrome [monograph on the Internet]. 
2003 [cited 2004 Jan 28]. Available from http://www.who.int/csr/ 
sars/en/WHOconsensus.pdf 

25. Centers for Disease Control and Prevention. Influenza: the disease 
[monograph on the Internet]. 2004 Nov 15 [cited 2004 Nov 23]. 
Available from http://www.cdc.gov/flu/about/disease.htm 

26. Thompson WW, Shay DK, Weintraub E, Brammer L, Cox N, 
Anderson LJ, et al. Mortality associated with influenza and respirato- 
ry syncytial virus in the United States. JAMA. 2003;289:179-86. 

27. Adams PF, Hendershot GE, Marano MA. Current estimates from the 
National Health Interview Survey, 1996. National Center for Health 
Statistics. Vital Health Stat. 1099; 10. 

28. Zambon M, Stockton J, Clewley J, Fleming D. Contribution of 

influenza and respiratory syncytial virus to community cases of 
influenza-like illness: an observational study. Lancet. 

2001;358:1410-6. 

29. Lieberman D, Shvartzman P, Ben-Yaakov M, Lazarovich Z, Hoffman 
S, Mosckovitz R, et al. Etiology of respiratory tract infection in adults 
in a general practice setting. Eur J Clin Microbiol Infect Dis. 
1998;17:685-9. 

30. Lieberman D, Lieberman D, Korsonsky I, Ben-Yaakov M, 
Lazarovich Z, Friedman MG, et al. A comparative study of the etiol- 
ogy of adult upper and lower respiratory tract infections in the com- 
munity. Diagn Microbiol Infect Dis. 2002;42:21-8. 

31. Nichol K. Cost-benefit analysis of a strategy to vaccinate healthy 
working adults against influenza. Arch Intern Med. 
2001;161:749-59. 

32. Steinman M, Landefeld C, Gonzales R. Predictors of broad-spectrum 
antibiotic prescribing for acute respiratory tract infections in adult 
primary care. JAMA. 2003;289:719-25. 

33. Centers for Disease Control and Prevention. Behavioral Risk Factor 
Surveillance System [database on the Internet], [cited 2004 Jan 28]. 
Available from http://www.cdc.gov/brfss/ 

34. Feeny DH, Furlong W, Boyle M, Torrance GW. Multiattribute health 

status classification systems: Health Utilities Index. 

Pharmacoeconomics. 1 995 ;7 :490-502. 

35. Multiple cause of death public use file for 2000 data [software]. 
Hyattsville (MD): National Center for Health Statistics; 2000. 

36. Peiris JSM, Yuen KY, Osterhaus ADME, Stohr K. The severe acute 
respiratory syndrome. N Engl J Med. 2003;349:2431-41. 

37. Muennig P, Khan K. Cost-effectiveness of vaccination versus treat- 
ment of influenza in healthy adolescents and adults. Clin Infect Dis. 
2001;33:1879-85. 

38. Centers for Medicare and Medicaid Services. Medicare provider and 
analysis review (MEDPAR) of short stay hospitals, 2001 [monograph 
on the Internet]. 2004 Sep 17 [cited 2004 Nov 23]. Available from 
http://cms.hhs.gov/statistics/medpar/default.asp 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


199 


PERSPECTIVE 


39. Chan PK, Tam JS, Lam CW, Chan E, Wu A, Li CK, et al. Human 
metapneumovirus detection in patients with severe acute respiratory 
syndrome. Emerg Infect Dis. 2003;9:1058-63. 


The opinions expressed by authors contributing to this journal do 
not necessarily reflect the opinions of the Centers for Disease 
Control and Prevention or the institutions with which the authors 
are affiliated. 


40. Centers for Disease Control and Prevention. SARS, influenza, and 
use of influenza vaccine. MMWR Morb Mortal Wkly Rep. 
2003;52:941-2. 


Address for correspondence: Kamran Khan, Inner City Health Research 
Unit, St. Michael’s Hospital, 30 Bond St, Toronto, Ontario, Canada M5B 
1W8; fax: 416-864-5485; email: km.khan@utoronto.ca 




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Human Disease from Influenza A 
(H5N 1), Thailand, 2004 

Tawee Chotpitayasunondh,* Kumnuan Ungchusak,t Wanna Hanshaoworakul,f 
Supamit Chunsuthiwat,t Pathom Sawanpanyalert,t Rungruen Kijphati,t Sorasak Lochindarat,* 
Panida Srisan,* Pongsan Suwan,t Yutthasak Osotthanakorn,f Tanakorn Anantasetagoon,t 
Supornchai Kanjanawasri,t Sureeporn Tanupattarachai,t Jiranun Weerakul,t 
Ruangsri Chaiwirattana,t Monthira Maneerattanaporn,t Rapol Poolsavatkitikool,t 
Kulkunya Chokephaibulkit,± Anucha Apisarnthanarak,§ and Scott F. Dowell^ 


Influenza A (H5N1) is endemic in poultry across much 
of Southeast Asia, but limited information exists on the dis- 
tinctive features of the few human cases. In Thailand, we 
instituted nationwide surveillance and tested respiratory 
specimens by polymerase chain reaction and viral isola- 
tion. From January 1 to March 31, 2004, we reviewed 610 
reports and identified 12 confirmed and 21 suspected 
cases. All 12 confirmed case-patients resided in villages 
that experienced abnormal chicken deaths, 9 lived in 
households whose backyard chickens died, and 8 reported 
direct contact with dead chickens. Seven were children <14 
years of age. Fever preceded dyspnea by a median of 5 
days, and lymphopenia significantly predicted acute respi- 
ratory distress syndrome development and death. Among 
hundreds of thousands of potential human cases of influen- 
za A (H5N1 ) in Asia, a history of direct contact with sick 
poultry, young age, pneumonia and lymphopenia, and pro- 
gression to acute respiratory distress syndrome should 
prompt specific laboratory testing for H5 influenza. 

T he 1997 outbreak of avian influenza in Hong Kong 
challenged the prevailing hypothesis that avian 
influenza viruses could infect humans only after passing 
through pigs or other intermediate hosts. In that outbreak, 
18 persons were infected with influenza A (H5N1) virus, 6 
died (1), and the epidemiologic and virologic evidence 
strongly suggested that direct contact with infected poultry 
was the route of transmission (1-3). All known influenza 
A virus subtypes that express hemagglutinins HI to HI 5 
and neuraminidases N1 to N9 are found in wild waterfowl 
(4,5), but only HI, H2, or H3 hemagglutinin subtypes had 


*Queen Sirikit National Institute of Child Health, Bangkok, 
Thailand; fMinistry of Public Health, Nonthaburi, Thailand; 4Siriraj 
Hospital, Bangkok, Thailand; §Thammasat University Hospital, 
Bangkok, Thailand; and ^International Emerging Infections 
Program, Nonthaburi, Thailand 


previously been known to cause human illness. Since 
1997, avian outbreaks with some subtypes of influenza A 
viruses have been reported to cause mostly mild or inap- 
parent infection in humans. For example, 2 mild clinical 
cases of H9N2 infection occurred in Hong Kong (6), and a 
large outbreak of conjunctivitis caused by H7N7 occurred 
in the Netherlands (7). 

In late 2003 and early 2004, outbreaks of highly patho- 
genic avian influenza A (H5N1) virus infection were 
reported to cause lethal illness among poultry in at least 8 
Asian countries (Cambodia, Indonesia, Japan, Laos, South 
Korea, China, Vietnam, and Thailand) (8). The first human 
cases were confirmed in Vietnam and Thailand in January 
2004, and some clinical features of the first 5 Thai cases 
and 10 Vietnamese cases have been reported (9,10). 
Despite the fact that new outbreaks among poultry contin- 
ued to be reported through the time of this writing (August 
2004), human cases have not been recognized outside of 
Thailand and Vietnam. This finding may be in part because 
pneumonia is very common, and the distinguishing fea- 
tures of pneumonia caused by influenza A (H5N1) are not 
widely appreciated. We report the clinical details of 12 
confirmed cases in Thailand and compare these with 21 
suspected but unconfirmed cases and 577 reported cases 
that were later excluded. In addition, predictors of severe 
disease, pathologic features, and epidemiologic exposures 
are analyzed and discussed. 

Methods 

Epidemiologic Investigations 

Nationwide surveillance to detect influenza A (H5N1) 
was initiated by the Thai Ministry of Public Health in 
December 2003, after outbreaks of sudden death in poultry 
were reported in some provinces in the central region. 


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Under this newly established surveillance system, all 
patients visiting the health services with pneumonia or 
influenzalike illness were asked if they had been exposed 
to ill poultry during the preceding 7 days or had resided in 
an area where abnormal poultry deaths occurred during the 
preceding 14 days. Influenzalike illness was defined 
according to the World Health Organization (WHO) rec- 
ommendations, which require acute fever (temperature 
>38.0°C) and either cough or sore throat in the absence of 
other diagnoses. Patients admitted with pneumonia or 
influenza and either of these poultry exposures were 
reported through the provincial public health office to the 
regional disease prevention and control centers and also to 
Bureau of Epidemiology at the Ministry of Public Health. 
Throat or nasopharyngeal swabs and serum samples were 
collected for viral study at the Thai National Institute of 
Health, Department of Medical Sciences. Staff members 
from the provincial health office visited family members to 
confirm history of exposure and assess the household envi- 
ronment. 

Patients with confirmed cases of H5N 1 were defined as 
patients reported to the system who had laboratory evi- 
dence of influenza A (H5N1) infection. Suspected case- 
patients were defined as patients with reported exposure to 
ill poultry and severe pneumonia, or patients with expo- 
sure and laboratory evidence of influenza A infection not 
confirmed as H5N1. Excluded case-patients were all 
remaining patients reported through the system who did 
not meet the exposure criteria or who lacked laboratory 
evidence of influenza A (H5N1) infection, including those 
with infections caused by influenza A H3 or HI, as well as 
other laboratory-confirmed pneumonia pathogens. 

We performed comparisons of dichotomous variables 
by using chi-square or Fisher exact tests, as appropriate, 
and t tests for continuous variables that were normally 
distributed, or Wilcoxon rank-sum tests for other continu- 
ous variables. We considered p values of <0.05 to be 
significant. 

Laboratory Investigations 

Respiratory specimens (including nasopharyngeal aspi- 
rates, nasopharyngeal swabs, nasal swabs, or throat swabs) 
were collected and stored in viral transport medium. Blood 
cultures were obtained from all patients on admission, and 
serum samples for mycoplasma titer and cold agglutinin 
testing were obtained when available. Paired serum sam- 
ples taken at least 14 days apart, if available, were collect- 
ed for serologic confirmation of H5N1 infection. An 
adequate sample was defined as any of the above respira- 
tory specimens collected from day 2 to day 14 after onset 
of fever. 

All specimens were submitted for testing at the National 
Institute of Health of Thailand, except 1, which was tested 


at Virology Laboratory at Siriraj Hospital, Mahidol 
University. Methods used for H5 identification were in 
accordance with those recommended by the WHO refer- 
ence laboratories for influenza (11). Specifically, specimens 
in transport medium were tested by reverse transcrip- 
tion-polymerase chain reaction (RT-PCR) to detect nucleic 
acids of influenza A and B and injected onto a Madin- 
Darby canine kidney (MDCK) cell monolayer for viral iso- 
lation. Nasopharyngeal aspirates were agitated and 
centrifuged to separate the epithelial cells. Sediments of 
epithelial cells were tested for influenza A and B by 
immunofluorescence assay (IFA) with specific monoclonal 
antibodies. Specimens positive for influenza A were further 
tested for subtypes HI, H3, and H5 with specific mono- 
clonal antibodies. The supernatant was tested by RT-PCR 
and viral isolation for the other types of specimens (12). 

Specimens positive for influenza A by RT-PCR were 
further tested for subtypes HI, H3, and H5 by using spe- 
cific primer sets. The H5- specific primer set was as fol- 
lows: H5-1 GCC ATT CCA CAA CAT ACA CCC, and 
H5-2 TAA ATT CTC TAT CCT CCT TTC CAA, with an 
expected product size of 358 bp (12,13). If results were 
negative for all subtypes or positive for H5, they were con- 
firmed by real-time RT-PCR using primer/probe H5 as fol- 
lows: InfA_TH5_A, InfA_TH5_F, InfA_TH_Ic, and 
InfA_TH5_fl (14). For viral isolation, if a cytopathic 
effect was observed, IFA was performed to identify the 
virus in infected cell cultures by using specific monoclon- 
al antibodies to HI, H3, and H5. If a cytopathic effect was 
not observed in the first passage, the culture medium pas- 
saged in MDCK for a second time. If no cytopathic effect 
occurred, the negative cell culture was confirmed by IFA 
with pooled viral monoclonal antibodies. 

Specimens were considered positive for avian influen- 
za virus if the viral culture was positive and was confirmed 
by IFA with H5- specific monoclonal antibody provided by 
the WHO, if epithelial cells in clinical specimens were IFA 
positive for H5, or if the RT-PCR was positive with H5 
specific primers (RT-PCR or real-time RT-PCR). A speci- 
men was negative for avian influenza virus if IFA, RT-PCR 
or real-time RT-PCR, and viral isolation (second passage) 
were negative. 

Clinical Investigations 

All potential case-patients reported through the surveil- 
lance system needed basic demographic, exposure, and 
clinical information recorded, as well as specimens sub- 
mitted, for the purpose of case classification. Patients with 
suspected cases were reviewed in more detail by telephone 
or written correspondence with the attending physician. 
Laboratory-confirmed case-patients had a thorough review 
with standardized forms of all medical records, chest radi- 
ographs, and laboratory data by the attending physicians. 


202 


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Respiratory failure was defined as requiring ventilatory 
support and cardiac failure as requiring inotropic drug sup- 
port. Liver dysfunction was diagnosed when serum aspar- 
tate aminotransferase (AST) or alamin aminotransferase 
(ALT) was >8 times the upper limit of normal. Renal dys- 
function was diagnosed when serum creatinine was >1.5 
mg/dL. Bone marrow dysfunction was diagnosed when all 
3 of the cell lines in the peripheral blood (erythrocytes, 
leukocytes, and platelets) were below the lower limit of 
normal. Leukopenia was defined as a total leukocyte count 
below the following age-specific cutoffs; 1-3 years 
<6,000, 4-7 years <5,500, and >8 years <4,500 cells/mm 3 . 
Lymphopenia was defined as an absolute lymphocyte 
count <1,500 cells/mm 3 , and thrombocytopenia was 
defined as a platelet count <150, 000/mm 3 (15). 

The attending radiologist classified chest radiograph 
findings as normal, interstitial infiltrates, lobar infiltrates, 
or combinations of these by using standard criteria. Acute 
respiratory distress syndrome (ARDS) was defined when 
clinical deterioration was associated with chest radi- 
ographs showing diffuse bilateral infiltrates accompanied 
by severe arterial hypoxemia. 

Results 

From January 1 to March 31, 2004, a total of 610 cases 
were reported from 67 of 76 provinces in Thailand. After 
thorough review of the clinical, epidemiologic, and labora- 
tory findings, we identified 12 confirmed and 21 suspect- 
ed cases. The onset of illness of the first confirmed case 
was on January 3, and the last was on March 2 (Figure 1). 
A total of 577 cases were excluded, including 38 who had 
positive RT-PCR tests for influenza A (H3) infection, 48 
seropositive for Mycoplasma pneumoniae , and 10 for 
Chlamydophila pneumoniae. 

Table 1 compares characteristics of patients with con- 
firmed, suspected, and excluded cases. Confirmed case- 
patients tended to be younger than suspected case-patients 
and more often had fatal disease than excluded patients 
(p < 0.0001). Reported poultry exposure was similar in all 
groups, but all confirmed patients had an adequate labora- 
tory specimen, whereas 10% of suspected patients and 
19% of excluded patients did not. All patients with an ade- 
quate laboratory specimen had testing completed. 


Human Disease from Influenza A (H5N1), Thailand, 2004 



Figure 1. Epidemic curve showing the dates of onset for 12 con- 
firmed and 21 suspected human cases of avian influenza A 
(H5N1) infection, Thailand, 2004. 


Of the 12 confirmed cases, 7 were in children <14 years 
of age, and 5 were in adults (Table 2). Fever was often the 
first symptom, and dyspnea often occurred a median of 5 
days after illness onset (range 1-16). During the initial 
evaluation at hospital, all patients were found to have 
fever, cough, and dyspnea, and almost half had myalgia 
and diarrhea. The hospital course was characterized by 
intermittent high fevers and persistent cough productive of 
thick sputum. One patient had a small amount of hemopt- 
ysis. Later in the course of the disease, organ failure or 
dysfunction was commonly observed, including respirato- 
ry failure in 9 (75%) patients, cardiac failure in 5 (42%), 
and renal dysfunction in 4 (33%). 

Routine laboratory tests on admission showed leukope- 
nia in 7 (58%) patients, lymphopenia in 7 (58%), and 
thrombocytopenia in 4 (33%) (Table 2). During the course 
of illness, elevated serum transaminase values were docu- 
mented in 67% of patients, although they were >8 times 
normal in only 17%. Serum creatinine rose to >1.5 mg/dL 
in 4 (33%) patients. Blood cultures were negative in all 
patients. One adult patient was found to be HIV seroposi- 
tive, and 1 pediatric patient had a mycoplasma titer of 
1:160. 

Admission leukocyte and platelet counts tended to be 
more depressed in the 8 patients who died than in the 4 
patients who survived (Figure 2). ARDS was associated 
with a fatal outcome (p = 0.02), and depressed admission 
leukocyte and platelet counts were also associated with 


Table 1. Characteristics of 12 confirmed, 

21 suspected, and 577 excluded human cases of avian influenza A (H5N1) in Thailand, 2004 

Characteristic 

Confirmed 

Suspected 

Excluded 

No. 

12 

21 

577 

Median age (y) (range) 

12(2-58) 

33(1-67) 

12(1-92) 

Sex (% male) 

67 

71 

59 

Poultry contact (%) 

58 

52 

48 

Adequate* specimen (%) 

100 

90 

81 

Death (%) 

67 

38 

4 


*Adequate was defined as a respiratory specimen obtained 2-14 days after onset of fever. 


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203 


RESEARCH 


Table 2. Characteristics and clinical findings of confirmed avian influenza A (H5N1) cases in Thailand, 2004* 

Patient no. 

Characteristics 1 2 3 4 5 6 7 8 9 10 V\ 12 % 

Age (y), sex 2JJ\ 27, F 31, M 46, F 5JM 


Symptoms 


Fever 

+ 

+ 

+ 

+ 

+ 

Rhinorrhea 

- 

+ 

- 

- 

+ 

Cough 

+ 

+ 

+ 

+ 

+ 

Sore throat 

+ 

+ 

- 

+ 

+ 

Myalgia 

- 

+ 

+ 

+ 

- 

Dyspnea 

+ 

+ 

+ 

+ 

+ 

Diarrhea 

+ 

- 

+ 

- 

+ 

Abdominal pain 

- 

- 

- 

- 

+ 

Conjunctivitis 

- 

- 

- 

- 

- 

Vomiting 

- 

- 

- 

- 

- 

Laboratory values 

Flematocrit (vol%) 

30 

39 

38 

46 

39 

Total leukocyte 

4,200 

13,600 

4,660 

7,360 

5,600 

count 

Total lymphocyte 

2,646 

3,400 

513 

2,429 

2,296 

count 

Platelet count 

214 

306 

171 

272 

94 

(xl 0 3 ) 
Treatment 

Oseltamivir 

+ 

- 

+ 

- 

+ 

Corticosteroids 

+ 

- 

+ 

- 

- 

Outcome 

ARDS 

- 

- 

+ 

- 

+ 

Inotropic support 

- 

- 

- 

- 

+ 

Peak AST (U) 

129 

18 

74 

NA 

70 

Peak ALT (U) 

57 

23 

41 

NA 

47 

Peak BUN (mg/dL) 

NA 

8 

10.7 

NA 

12 

Peak creatinine 

NA 

0.8 

1.07 

NA 

0.7 


(mg/dL) 


6, M 

6, M 

6, M 

7, M 

13, M 

39, F 

58, F 

67 (M) 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

100 

+ 

+ 

- 

- 

- 

- 

- 

33 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

100 

- 

+ 

+ 

+ 

+ 

- 

+ 

75 

- 

- 

+ 

- 

- 

+ 

- 

42 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

100 

- 

- 

+ 

- 

- 

+ 

- 

42 

- 

+ 

- 

- 

- 

- 

- 

17 

- 

- 

+ 

- 

+ 

+ 

- 

u 

25 

32 

39 

40 

41 

37 

33 

38 


1,200 

2,200 

4,900 

4,100 

2,000 

3,300 

5,680 


624 

638 

1,763 

1,435 

580 

660 

454 


89 

140 

111 

304 

150 

380 

185 



+ 

- 

+ 

+ 

+ 

- 

- 

58 

+ 

+ 

+ 

+ 

+ 

+ 

- 

67 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

75 

- 

- 

+ 

+ 

+ 

- 

+ 

42 

790 

175 

280 

120 

34 

394 

NA 


150 

43 

50 

52 

47 

106 

NA 


NA 

14 

22 

10 

132 

37 

39 


NA 

1.7 

1.1 

0.7 

8.1 

3.6 

2.3 


-(20) 

-(18) 

-(8) 

-(29) 

-(16) 

-(13) 

-(8) 

33 


Survival + + + + - (13) 

(day of death) 

*M, male; f, female; +, yes, -, no, NA, not applicable; ARDS, acute respiratory distress syndrome; AST, aspartate aminotransferase; ALT, alanine 
aminotransferase; BUN, blood urea nitrogen. 


ARDS development. The most pronounced difference was 
in the absolute lymphocyte count, with a mean of 995 in 
those with ARDS vs. 2,825 in those without (p = 0.002). A 
low absolute lymphocyte count on admission was also 
associated with death (mean of 1,056/mm 3 in those who 
died compared to 2,247/mm 3 in those who survived, p = 
0.05). In addition, the median total leukocyte count was 
3,700/mm 3 for those who died compared with 6,010/mm 3 
for those who survived (p = 0.09), and the median platelet 
count was 145,000/mm 3 in those who died and 
243,000/mm 3 in those who survived (p = 0.17). 

All 12 patients had abnormal chest radiographs a medi- 
an of 7 days after onset of fever (range 3-17 days). Two 
patients had interstitial infiltration, and 10 had patchy 
lobar infiltrates in a variety of patterns (single lobe, multi- 
ple lobes, unilateral or bilateral distributions). The radi- 
ographic pattern progressed to diffuse bilateral 
ground-glass appearance, with clinical features compatible 
with ARDS, in all 8 patients who died and in 1 patient who 


survived (Figure 3). A pneumothorax developed in 1 
patient during mechanical ventilation. The median time 
from onset to ARDS development was 6 days (range 
4-13). 

Treatment for all patients included broad- spectrum 
antimicrobial drugs aiming to cover most of the usual and 
unusual respiratory pathogens. Eight patients were treated 
with corticosteroid drugs, including 2 patients who sur- 
vived and 6 patients who died. Seven patients were treated 
with the neuraminidase inhibitor oseltamivir at various 
stages of illness. Treatment tended to have been started 
earlier in those who survived (a median of 4.5 days from 
onset compared with 9 days for those who died), and both 
survivors who were treated received the complete 5 -day 
course of drug, whereas 2 of 5 patients who died received 
the complete 5 -day course (Figure 4). 

Pathologic tissues from the lungs and spleen of 3 
patients were available for analysis in the current report. A 
fourth patient (number 6) was autopsied but is the subject 


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Figure 2. Distribution of the absolute lymphocyte count (ALC), total 
leukocyte count, and platelet count on admission for 4 patients 
who survived and 8 who died of human influenza A (H5N1) infec- 
tion, Thailand, 2004. ARDS, acute respiratory distress syndrome. 


of a separate report. The lungs showed diffuse alveolar 
damage, with hyaline membrane formation, reactive 
fibroblasts, and areas of hemorrhage. The spleen had 
numerous atypical lymphocytes but no viral inclusions 
(Figure 5). 

All 12 confirmed patients resided in a village with 
abnormal chicken deaths (Table 3). Nine lived in a house 
whose backyard chickens died unexpectedly. Direct con- 
tact with dead chickens was reported in 8 patients, with a 
median of 4 days between the last exposure and the onset 
of symptoms (range 2-8 days). The details of exposures in 
these case-patients and in groups of matched controls are 
the subject of a separate investigation. 

Discussion 

The detection of a few human infections with influenza 
A (H5N1) in the context of an avian epizootic involving at 
least 8 countries has proven to be a considerable challenge. 


Human Disease from Influenza A (H5N1), Thailand, 2004 

The history of direct contact with sick and dying poultry, 
young age of many patients, pneumonia and lymphopenia, 
and progression to ARDS in spite of broad- spectrum 
antimicrobial treatment indicate that specific laboratory 
testing for H5 influenza should be sought. Ideally, such 
information should be routinely collected and used to min- 
imize opportunities for recombination of this virulent new 
pathogen with existing human influenza viruses. 

The optimal treatment for case-patients with suspected 
H5 infection is not known, but in vitro susceptibility test- 
ing suggests that resistance to adamantanes is a common 
feature of H5 isolates from 2004 (11), whereas these iso- 
lates remain susceptible to the neuraminidase inhibitors. 
Although no controlled data are available on which to base 
treatment recommendations, our observations were that 
the 4 patients who survived tended to have been treated 
with oseltamivir earlier in the course of their disease. We 
advocate using this agent in the early treatment of case- 
patients with suspected H5N 1 influenza, in agreement with 
the recommendations of WHO (16). Controlled trials of 
oseltamivir and corticosteroid treatment would be helpful 
in confirming or refuting any specific benefit. 

Approximately 1,820,387,000 persons live in the 8 
countries in Asia that reported poultry epidemics with 
avian influenza A (H5N1) in 2004 (-30% of the world’s 
population). One community survey in Thailand found that 
12%-61% of rural residents had regular contact with back- 
yard birds (17). Thus, the 12 cases we report likely repre- 
sent the end result of hundreds of thousands of potential 
exposures and an unknown number of human cases. 
Perhaps in part because few distinctive features of human 
disease caused by avian influenza have been reported, and 
specific diagnostic tests for H5 disease are not widely 
available, human cases have been few and have been 
reported only from Vietnam and Thailand. 

Among >600 possible case-patients reported to the Thai 
Ministry of Public Health, most reported clear exposure to 



Figure 3. Chest radiographs from patients 8 and 9. Panel A demonstrates patchy alveolar infiltration of the right lower lung on day 5 of 
illness for patient 9; panel B demonstrates the progression to acute respiratory disease syndrome (ARDS) on day 8. panel C shows inter- 
stitial infiltration of both lungs of patient 8 on day 4 of illness; panel D shows the rapid progression to ARDS by day 6. 


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205 


RESEARCH 



Figure 4. Timing of the clinical course and oseltamivir treatment for 
4 patients who survived and 8 patients who died of human influen- 
za A (H5N1 ) infection, Thailand, 2004. 


sick poultry, and the demographic characteristics were 
similar among confirmed, suspected, and excluded groups. 
All confirmed patients had an adequate specimen submit- 
ted and processed, whereas 10% of the suspected patients 
and 19% of those excluded had inadequate specimens. The 
availability of properly collected specimens and use of 
specific laboratory tests for influenza A (H5N1) will be 
essential for monitoring the ongoing risk from this 
pathogen in East Asia. 

Human infections with highly pathogenic avian 
influenza may be easy to miss in the context of the regular 
incidence of pneumonia in much of rural Asia, where the 


capacity to make specific etiologic diagnoses remains lim- 
ited. We found certain features to be helpful, as have inves- 
tigators in Vietnam (9). Eight of the 12 patients had direct 
exposures to ill poultry 2-8 days before onset. Seven of the 
12 were young children, and routine laboratory testing at 
the time of admission to hospital identified marked lym- 
phopenia in 8. Although the initial chest radiographs 
would not immediately identify these cases as unusual, 
deaths in children and younger adults from hospitalized, 
radiographically confirmed pneumonia typically range 
from 1% to 10% and from l%-5% among patients with 
radiographically confirmed pneumonia in rural Thailand 
(18-20). Thus, the progression in 9 of the 12 patients to 
ARDS, followed by the death of 8 patients, separates these 
cases as a form of unusually severe pneumonia. 

The disease may in fact be more severe than that seen in 
Hong Kong in 1997. Of the 34 cases officially reported to 
the WHO in 2004, 23 (68%) patients died compared to 6 
(33%) of those in Hong Kong (p = 0.02). Several lines of 
evidence indicate that the H5N1 viruses have evolved to 
more virulent forms since 1997, with different antigenic 
structure (21), internal gene constellations (22), and an 
expanded host range (23,24). This virologic evolution may 
be a factor in the persistence of H5N 1 viruses in the avian 
populations. Since the 1997 outbreak, Hong Kong has 
experienced a series of reintroductions of H5 viruses, 
despite instituting unusually stringent control measures, 
including the culling of all poultry in the territory, strict reg- 
ulations of live poultry markets, and monthly “off days,” in 
which all markets are emptied and cleaned (22,25). H5 out- 
breaks in poultry have also recurred repeatedly in Thailand, 



Figure 5. Pathologic findings from a patient 
(number 6) with confirmed influenza A (H5N1) 
infection. All slides are stained with hematoxylin 
and eosin, shown at 40x objective. Panel A 
shows hyaline membrane formation lining the 
alveolar spaces of the lung and vascular con- 
gestion with a few infiltrating lymphocytes in the 
interstitial areas. Reactive fibroblasts are also 
present. Panel B is an area of lung with prolifer- 
ating reactive fibroblasts within the interstitial 
areas. Few lymphocytes are seen, and no viral 
intranuclear inclusions are visible. Panel C 
shows fibrinous exudates filling the alveolar 
spaces, with organizing formation and few hya- 
line membranes. The surrounding alveolar 
spaces contain hemorrhage. Panel D is from a 
section of spleen, showing numerous atypical 
lymphoid cells scattered around the white pulp. 
No viral intranuclear inclusions are seen. 


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Table 3: Brief history of exposure of the 1 2 confirmed case-patients 

Human Disease from Influenza A (H5N1), Thailand, 2004 

Patient no. Province/sex/age (y) 

Exposure history 


1 

Supanburi/M/2 

Raised chickens in backyard. Chickens died unexpectedly 5 days before illness onset. 
Frequently played with chickens and had direct contact with carcasses. 

2 

Uttradit/F/27 

Raised chickens in backyard, but chickens did not die. Two months before onset, ducks in a 
nearby area died unexpectedly. 

3 

Nakornratchasima/M/31 

Raised chickens in backyard. Three days before onset, chickens started to die. The last 
patient died on the date he became sick. He buried all carcasses. 

4 

Lopburi/F/46 

Raised 60 chickens in back yard. All chickens died unexpectedly 1 month before onset. She 
burned and buried carcasses without protection. 

5 

Khonkaen/M/5 

Raised fighting cocks that died 4 days before onset. Reported direct contact with carcasses. 
Ate chicken with suspected H5N1 influenza. 

6 

Kanchanaburi/M/6 

No poultry in family. Helped slaughter one ill chicken 2 days before onset. 

7 

Sukhothai/M/6 

Mother slaughtered 2 ill chickens in house 4 days before onset. No direct contact with 
chickens. Mother got sick on same day and died without laboratory confirmation. 

8 

Kanchanaburi/M/6 

Chickens in backyard died unexpectedly. Grandfather slaughtered ill chickens. No direct 
contact with chickens but played near slaughtering area. 

9 

Supanburi/M/7 

No poultry in family. Frequently played on ground near a chicken farm that reported 
unexpected poultry deaths. 

10 

Chaiyapoum/M/13 

Helped raise chickens in backyard. Eight days before onset, chickens died unexpectedly 
and patient assisted with slaughtering. 

11 

Patumthani/F/39 

Factory worker living in province A during weekdays but in province B on weekends. 

Fighting cocks lived at a neighboring house. Province B reported outbreaks 2 months before 
onset. No contact with live or dead chickens. 

12 

Supanburi/F/58 

Raised 40-50 chickens in backyard. Chickens started to die 5 days before onset. Buried and 
slaughtered ill chickens every day until onset date. 


Vietnam, and elsewhere despite intensive control measures 
(26), and recurrences should be anticipated for the foresee- 
able future. 

If H5 viruses do persist, they will likely continue to 
evolve, potentially to forms more easily transmitted from 
person to person. We identified no suspected or confirmed 
cases among Thai health personnel, supporting the experi- 
ence from Vietnam and Hong Kong that efficient human- 
to-human transmission has not occurred (9,27). Serologic 
studies of healthcare workers and household contacts of 
patients in the 1997 Hong Kong outbreak provided evi- 
dence of occasional seroconversions associated with close 
exposures. These findings indicate that inefficient trans- 
mission is possible and reinforce the importance of infec- 
tion control precautions (28,29). Studies of healthcare 
workers and poultry cullers in Thailand are under way to 
determine whether similar seroconversions may have 
occurred after exposure to patients with the 2004 viruses. 

In addition to gradual mutational changes, H5 viruses 
have the potential to reassort with existing human influen- 
za viruses to produce a strain with high virulence and effi- 
cient transmissibility. In this context, the known pattern of 
human influenza isolations in Thailand raises particular 
concerns about control of avian influenza during the 
months from June to August, when human influenza can 
be expected to peak (Figure 6). 

After the official announcement of the first human case 
on January 23, a national public education campaign was 
carried out through the mass media and thousands of vil- 
lage health volunteers. Villagers, especially children, were 


informed to avoid exposure to ill poultry. According to the 
Department of Livestock, ~40 million chickens in 160 
affected villages of 41 provinces were slaughtered from 
January to May 2004. Within 2 months of implementing 
widespread poultry culling, quarantine measures, and the 
public education campaign, the number of potential cases 
reported to the surveillance system decreased dramatically 
and confirmed human cases ceased, despite interim 
improvement in the quality of surveillance and laboratory 
testing. The course of this outbreak reconfirms observa- 
tions from the smaller 1997 outbreak in Hong Kong that 
early detection of human cases and aggressive public 
health and agricultural interventions can save lives (30). 

We believe this outbreak of H5N 1 is unlikely to be the 
last because of the formidable challenges in eradicating the 
virus, and the potential reservoir in waterfowl (31). We 
must be well prepared for a future surge of either small or 



Figure 6. Seasonal variation in viral isolations of human influenza 
A (H3N2), A (H INI), and B, in Thailand. 


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207 


RESEARCH 


large outbreaks, early detection must be ensured, informa- 
tion shared, and control measures for both animals and 
humans promptly implemented. 

Acknowledgments 

We thank Jaran Tinwutthipongse and Kith Kittiampon for 
strong policy support; the provincial chief medical officers and 
epidemiology staff of Suphanburi, Kanchanaburi, Sukhothai, 
Chaiyapoum, Uttradit, Khonkaen, Lopburi, Nakhonratchasima, 
Pathumthani, and Ayuthaya for being directly involved in the 
investigations at the village level; the laboratory staff at the 
Department of Medical Sciences, including Pranee Thawatsupha, 
Wattana Auwanit, Malinee Chittaganpitch, Sunthareeya 
Waicharoen, Siriphan Saeng-Aroon, Wattanapong Wootta, and 
Wimol Petkanchanapong, for working long hours processing 
hundreds of specimens during the outbreak; Rungnapa 
Prasanthong, Ubonrat Naruponjirakul, Chuleeporn Jirapongsa, 
Potjaman Siriarayaporn, Yongjur Laosirithaworn, trainees of the 
Field Epidemiology Training Program, and the directors of the 
disease control offices in regions 1-12, who supported the field 
investigation teams from the central level; and Cathy E. Roth and 
Teresa Tam, Khanchit Limpakarnjanarat, Sonja Olsen, and Mark 
Simmerman for technical assistance. 

The Ministry of Public Health, Thailand, supported the out- 
break response as a part of its routine public health function. 

Dr. Chotpitayasunondh is a pediatric infectious disease spe- 
cialist at Queen Sirikit National Institute of Child Health, 
Bangkok, Thailand. He serves as a senior medical consultant for 
the Thai Ministry of Public Health on emerging and reemerging 
infectious diseases, including SARS and avian influenza. 

References 

1. Chan PK. Outbreak of avian influenza A (H5N1) vims infection in 
Hong Kong in 1997. Clin Infect Dis. 2002;34:S58-64. 

2. Subbarao K, Klimov A, Katz J, Regnery H, Lim W, Hall H, et al. 
Characterization of an avian influenza A (H5N1) vims isolated from 
a child with a fatal respiratory illness. Science. 1998;279:393-6. 

3. Bridges CB, Lim W, Hu-Primmer J, Sims L, Fukuda K, Mak KH, et 
al. Risk of influenza A (H5N1) infection among poultry workers, 
Hong Kong, 1997-1998. J Infect Dis. 2002;185:1005-10. 

4. Shortridge KF, Gao P, Guan Y, Ito T, Kawaoka Y, Markwell D, et al. 
Interspecies transmission of influenza viruses: H5N1 vims and a 
Hong Kong SAR perspective. Vet Microbiol. 2000;74:141-7. 

5. Nicholson KG, Wood JM, Zambon M. Influenza. Lancet. 
2003;362:1733-45. 

6. Peiris M, Yuen KY, Leung CW, Chan KH, Ip PL, Lai RW, et al. 
Human infection with influenza H9N2. Lancet. 1999;354:916-7. 

7. Fouchier RA, Schneeberger PM, Rozendaal FW, Broekman JM, 
Kemink SA, Munster V, et al. Avian influenza A vims (H7N7) asso- 
ciated with human conjunctivitis and a fatal case of acute respiratory 
distress syndrome. Proc Natl Acad Sci USA. 2004;101:1356-61. 

8. World Health Organization. Avian influenza A (H5N1) — situation 
(poultry) in Asia as at 2 March 2004: need for a long-term response, 
comparison with previous outbreaks. Wkly Epidemiol Rec. 
2004;79:96-9. 


9. Tran TH, Nguyen TL, Nguyen TD, Luong TS, Pham PM, Nguyen 
VC, et al. Avian influenza A (H5N1) in 10 patients in Vietnam. N 
Engl J Med. 2004;350:1179-88. 

10. Centers for Disease Control and Prevention. Cases of influenza A 
(H5N1)— Thailand, 2004. MMWR Morb Mortal Wkly Rep. 
2004;53:100-3. 

11. Centers for Disease Control and Prevention. Outbreaks of avian 
influenza A (H5N1) in Asia and interim recommendations for evalu- 
ation and reporting of suspected cases — United States, 2004. MMWR 
Morb Mortal Wkly Rep. 2004;53:97-100. 

12. World Health Organization. Animal influenza training manual. 
Harbin, China: The Organization; 2001. 

13. Ministry of Public Health T. Influenza A (H5N1) laboratory training 
manual. Bangkok, Thailand: Ministry of Public Health; 2004. 

14. Spackman E, Senne DA, Myers TJ, Bulaga LL, Garber LP, Perdue 
ML, et al. Development of a real-time reverse transcriptase PCR 
assay for type A influenza virus and the avian H5 and H7 hemagglu- 
tinin subtypes. J Clin Microbiol. 2002;40:3256-60. 

15. Berhman R, Kliegman R, Jenson H. Nelson textbook of pediatrics. 
Philadelphia: Saunders; 2000. p. 2186-7. 

16. World Health Organization. WHO interim guidelines on clinical man- 
agement of humans infected by influenza A (H5N1). Vol. 2004. 
Geneva: The Organization; 2004. 

17. Olsen S, Wannachaiwong Y, Chotpitayasunondh T, Chittaganpitch M, 
Limpakarnjanarat K, Dowell S. Human and avian influenza in 
Thailand: reducing opportunities for reassortment. Boston: Infectious 
Diseases Society of America; 2004. 

18. Nascimento-Carvalho CM, Rocha H, Santos-Jesus R, Benguigui Y. 
Childhood pneumonia: clinical aspects associated with hospitaliza- 
tion or death. Braz J Infect Dis. 2002;6:22-8. 

19. Marrie TJ, Carriere KC, Jin Y, Johnson DH. Factors associated with 
death among adults <55 years of age hospitalized for community- 
acquired pneumonia. Clin Infect Dis. 2003;36:413-21. 

20. Kanlayanaphotporn J, Brady M, Chantate P, Chantra S, 
Siasiriwattana S, Dowell S, et al. Pneumonia surveillance in 
Thailand: current practice and future needs. Southeast Asian J Trop 
Med Public Health. 2004;35:711-6. 

21. Horimoto T, Fukuda N, Iwatsuki-Horimoto K, Guan Y, Lim W, Peiris 
M, et al. Antigenic differences between H5N 1 human influenza virus- 
es isolated in 1997 and 2003. J Vet Med Sci. 2004;66:303-5. 

22. Sims LD, Ellis TM, Liu KK, Dyrting K, Wong H, Peiris M, et al. 
Avian influenza in Hong Kong 1997-2002. Avian Dis. 
2003;47:832-8. 

23. Sturm-Ramirez KM, Ellis T, Bousfield B, Bissett L, Dyrting K, Rehg 
JE, et al. Reemerging H5N 1 influenza viruses in Hong Kong in 2002 
are highly pathogenic to ducks. J Virol. 2004;78:4892-901. 

24. Perkins LE, Swayne DE. Pathogenicity of a Hong Kong-origin H5N 1 
highly pathogenic avian influenza virus for emus, geese, ducks, and 
pigeons. Avian Dis. 2002;46:53-63. 

25. Guan Y, Poon LL, Cheung CY, Ellis TM, Lim W, Lipatov AS, et al. 
H5N 1 influenza: a protean pandemic threat. Proc Natl Acad Sci U S 
A. 2004; 101:8 156-61. 26. 

26. Normile D, Enserink M. Infectious diseases. Avian influenza makes a 
comeback, reviving pandemic worries. Science. 2004;305:321. 

27. Yuen KY, Chan PK, Peiris M, Tsang DN, Que TL, Shortridge KF, et 
al. Clinical features and rapid viral diagnosis of human disease asso- 
ciated with avian influenza A H5N1 virus. Lancet. 1998;351:467-71. 

28. Buxton Bridges C, Katz JM, Seto WH, Chan PK, Tsang D, Ho W, et 
al. Risk of influenza A (H5N1) infection among health care workers 
exposed to patients with influenza A (H5N1), Hong Kong. J Infect 
Dis. 2000;181:344-8. 

29. Katz JM, Lim W, Bridges CB, Rowe T, Hu-Primmer J, Lu X, et al. 
Antibody response in individuals infected with avian influenza A 
(H5N1) viruses and detection of anti-H5 antibody among household 
and social contacts. J Infect Dis. 1999;180:1763-70. 


208 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


Human Disease from Influenza A (H5N1), Thailand, 2004 


30. Tam JS. Influenza A (H5N1) in Hong Kong: an overview. Vaccine. 
2002;20:S77-81. 


Use of trade names is for identification only and does not imply 
endorsement by the Public Health Service or by the U.S. 
Department of Health and Human Services. 


31. Chen H, Deng G, Li Z, Tian G, Li Y, Jiao P, et al. The evolution of 
H5N 1 influenza viruses in ducks in southern China. Proc Natl Acad 
Sci USA. 2004; 101:10452-7. 


Address for correspondence: Scott F. Dowell, Department of Disease 
Control Building 7, Ministry of Public Health, Tivanon Road, Nonthaburi 
11000, Thailand; fax +66-2-580-0911; email sdowell@cdc.gov 



EMERGING 
INFECTIOUS DISEASES 

A Peer-Reviewed Journal Tracking and Analyzing Disease Trends Vol.8, No.l, January 2002 


sMol- 


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RESEARCH 


Lack of H5N1 Avian Influenza 
Transmission to Hospital 
Employees, Hanoi, 2004 

Nguyen Thanh Liem,* World Health Organization International Avian Influenza 
Investigation Team, Vietnam , 1 and Wilina Limj- 


To establish whether human-to-human transmission of 
influenza A H5N1 occurred in the healthcare setting in 
Vietnam, we conducted a cross-sectional seroprevalence 
survey among hospital employees exposed to 4 confirmed 
and 1 probable H5N1 case-patients or their clinical speci- 
mens. Eighty-three (95.4%) of 87 eligible employees com- 
pleted a questionnaire and provided a serum sample, 
which was tested for antibodies to influenza A H5N1. 
Ninety-five percent reported exposure to >1 H5N1 case- 
patients; 59 (72.0%) reported symptoms, and 2 (2.4%) ful- 
filled the definition for a possible H5N1 secondary 
case-patient. No study participants had detectable antibod- 
ies to influenza A H5N1. The data suggest that the H5N1 
viruses responsible for human cases in Vietnam in January 
2004 are not readily transmitted from person to person. 
However, influenza viruses are genetically variable, and 
transmissibility is difficult to predict. Therefore, persons pro- 
viding care for H5N1 patients should continue to take 
measures to protect themselves. 

D irect transmission of H5N1 viruses of purely avian 
origin from birds to humans was first described dur- 
ing an outbreak among poultry in Hong Kong in 1997. In 
that outbreak, 6 of 18 confirmed human H5N1 case- 
patients died (1), and serologic evidence was found for 
asymptomatic infection in humans after exposure to infect- 
ed poultry (2).Avian-to-human transmission of influenza 
viruses is believed to be infrequent because of host barri- 
ers to infection, such as cell receptor specificities, and 
because the acquisition by avian viruses of the ability for 
human-to-human transmission requires either genetic reas- 
sortment with a human influenza strain or genetic mutation 
(3). However, a study of household and social contacts of 


*National Pediatric Hospital, Hanoi, Vietnam; and fDepartment of 
Health, Hong Kong, Special Administrative Region, China 


Hong Kong H5N 1 case-patients found evidence, although 
limited, for human-to-human transmission (4). Further evi- 
dence was provided by a study of healthcare workers 
(HCWs), which found that significantly more HCWs 
exposed to patients with H5N 1 infection were positive for 
H5 antibody than nonexposed HCWs (3.7% vs. 0.7%); 2 
HCWs seroconverted after exposure to H5N1 -infected 
patients, in the absence of known poultry exposure (5). 
These 2 studies provided the first evidence, although lim- 
ited, of human-to-human transmission of H5N1 viruses of 
purely avian origin. 

On December 12, 2003, influenza A H5N1 viruses were 
detected among poultry at a farm near Seoul, the Republic 
of Korea (6), and outbreaks of H5N1 in poultry were sub- 
sequently reported in 8 other Asian countries (Japan, 


World Health Organization (WHO) International Avian Influenza 
Investigation Team, Vietnam: Bach Huy Anh (Hanoi Medical 
University), Philippe Barboza (Institut de Veille Sanitaire, France), 
Niranjan Bhat (Centers for Disease Control and Prevention, USA 
[CDC]), Arnold Bosman (EPIET, National Institute for Public Health 
and the Environment, Netherlands), Sofia Boqvist 
(Smittskyddsinstitutet, Sweden), Rick Brown (Asian Development 
Bank), Pascale Brudon (WHO), Philippe Calain (WHO), Maria 
Cheng (WHO), Aaron Curns (CDC), Valerie Delpech (Health 
Protection Agency, UK), Robert Dietz (WHO), Nguyen Cong Doan 
(CDC), United States), Rodger Doran (WHO), Mirna Du Ry van 
Beest Holle (European Programme for Intervention Epidemiology 
Training, EPIET), Joel Francart, Keiji Fukuda (CDC), Amy Wolkin 
(CDC), Patrice Gautier (Veterinaires sans frontieres, Vietnam), 
Futoshi Hasebe (Asian Development Bank), Peter Horby (WHO), 
Shigeyuki Itamura (National Institute for Infectious Diseases, 
Japan), Veronique Jestin (OIE), Donna Mak (Centre for 
International Health, Australia), Noel Miranda (SERVAC, 
Philippines), Hitoshi Oshitani (WHO), Takehiko Saito (National 
Institute for Infectious Diseases, Japan), Taronna Maines (CDC), 
Reiko Saito (Nigata University, Japan), James Mark Simmerman 
(CDC), Terry Tumpey (CDC), Timothy Uyeki (CDC). 


210 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


No H5N1 Influenza Hospital Transmission, Hanoi, 2004 


Indonesia, Vietnam, Thailand, Laos, Cambodia, China, 
and Malaysia); a situation that the Office International des 
Epizooties has called “a crisis of global importance” (7). 
Human case-patients infected with H5N1 related to these 
poultry outbreaks were identified in Vietnam and Thailand 
in January 2004, and on September 28, 2004, possible 
human-to-human transmission was reported in a family 
cluster in Thailand (8). 

Concern is widespread that the current situation in 
Asia favors the emergence of a highly pathogenic 
influenza virus with the ability for efficient transmission 
from person to person, which would lead to an influenza 
pandemic. While experiences from Hong Kong in 1997 
indicate that human-to-human transmission of purely 
avian H5N1 viruses is possible but not sustainable, genet- 
ic alterations over time may lead to subsequent H5N1 
infections behaving quite differently. An understanding 
of the current and absolute risk for human-to-human 
transmission of circulating avian H5N1 viruses is vital to 
guide appropriate public health and infection control 
responses and to inform pandemic preparedness. 
Unfortunately, little data are available to quantify the 
transmissibility of the H5N1 strains currently circulating 
in poultry in Asia. To investigate the risk for human-to- 
human transmission of avian H5N1 viruses to hospital 
employees, we undertook a cross-sectional seropreva- 
lence study among employees of 1 hospital in Vietnam, 
who were exposed to confirmed and probable H5N1 
case-patients or their clinical samples. 

Methods 

From December 27, 2003, to January 19, 2004, 4 chil- 
dren, 4-12 years of age, with confirmed H5N1 infection 
and 1 with probable H5N1 infection were admitted and 
treated at the National Pediatric Hospital (NPH), Hanoi, 
Vietnam. Detailed information regarding the 4 confirmed 
H5N1 patients has been published elsewhere (9). Eligible 
study participants were hospital employees who had possi- 
ble exposure to the patients with confirmed or probable 
H5N1 infections, such as by working in wards or entering 
rooms where H5N 1 patients were admitted, or having han- 
dled clinical specimens from these patients. To allow suf- 
ficient time for seroconversion in any infected HCWs, the 
study took place 29 days after discharge of the last con- 
firmed H5N1 patient. All eligible participants were pro- 
vided with written and verbal information about the study 
and gave written consent for participation. 

Definitions 

We used the following definitions in our study: study 
period, from date of admission of first confirmed case- 
patient (December 27, 2003) to 29 days after discharge of 
the last confirmed case-patient (February 17, 2004); con- 


firmed H5N1 primary case patient, a patient admitted to 
NPH, Hanoi, from December 27, 2003, to January 19, 
2004, inclusive with a respiratory illness and influenza A 
H5N1 virus detected in clinical specimens by either viral 
culture or reverse transcriptase-polymerase chain reaction; 
probable H5N 1 primary case patient, a patient admitted to 
NPH, Hanoi, from December 27, 2003, to January 19, 
2004, inclusive with a respiratory illness and high titer of 
antibodies to influenza A/H5 detected in a single serum 
sample; possible H5N1 secondary case, a hospital employ- 
ee who had fever (if measured >38°C), and at least 1 of 3 
symptoms (cough, shortness of breath, sore throat), and 
contact with a confirmed or probable influenza A H5N1 
case-patient, in the absence of exposure to poultry. 

Questionnaires 

Information was collected by using a self-administered 
questionnaire in Vietnamese. Participants were asked their 
age, sex, residence address, occupation, department where 
they worked, whether they smoked, their medical history, 
whether they had symptoms during the study period, 
whether they had taken hygienic measures while caring for 
H5N1 case-patients, their influenza vaccination status, use 
of oseltamivir prophylaxis, and potential risk factors for 
H5N1. These risk factors included duration and type of 
exposure to H5N1 case-patients, contact with ill poultry or 
poultry that died of an illness, and whether they shopped at 
live-poultry markets or had freshly butchered or live poul- 
try in their home in the previous month. 

Serologic Testing 

All participants were asked to provide a single blood 
specimen. Serum samples were collected on February 17, 
2004, immediately processed, stored at -25 °C, and 
shipped frozen on dry ice to the Government Virus Unit, 
Department of Health, Hong Kong, China. Serum samples 
were tested for antibodies to influenza A H5N 1 virus by 
microneutralization test as described by Rowe et al. (10) 
with H5N1 viruses A/Vietnam/ 11 94/2004 and A/Vietnam/ 
3212/2004. Serum was considered to be positive in the 
microneutralization test if an anti-H5 titer of >40 was 
obtained in 2 independent assays. Microneutralization 
antibody-positive serum was adsorbed with influenza A 
H1N1 virus to eliminate the possibility of detecting anti- 
body that was cross-reactive among influenza virus of dif- 
ferent subtypes, and the microneutralization test was 
repeated. No change in antibody titer after adsorption indi- 
cated the presence of anti-H5 antibody, while a >4-fold 
reduction in microneutralization after adsortion was inter- 
preted as evidence for significant cross-reaction. 
Microneutralization antibody-positive serum was subject- 
ed to Western blot analysis by using recombinant protein 
from A/HK/ 15 6/97 virus. 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


211 


RESEARCH 


Table 1 . Demographic and exposure characteristics of study 

participants 

Characteristic n (%)* 


Age group (y) 


<30 

20 (24.1) 

30-39 

26 (31.3) 

40-49 

26 (31.3) 

>49 

11 (13.3) 

Male sex 

30 (36.1) 

Residence in Hanoi City 

81 (97.6) 

Department 

ICUf 

37 (45.1) 

Infectious diseases 

30 (36.6) 

Laboratory 

8 (9.8) 

Radiology 

6 (7.3) 

Hematology 

1 (1-2) 

Years smoked 

None 

64 (78.1) 

<10 

6 (7.3) 

10-30 

12(14.6) 

Chronic medical condition 

22 (26.5) 

Influenza vaccination in 2004 

56 (68.3) 

Oseltamivir since Dec 27, 2003 

1 (1-2) 

No. of H5N1 patients visited 

0 (none) 

4 (4.9) 

1 

3 (3.7) 

2 

31 (37.8) 

3 

32 (39.0) 

4 

4 (4.9) 

5 (all) 

8 (9.8) 

Changed bedding 

Yes 

46 (59.0) 

No 

32 (41 .0) 

Touched patients 

Yes 

75 (96.2) 

No 

3 (3.9) 

Exposure to H5N1 patient(s) who 

57 (73.1) 


did not wear masks 

*The no. of study participants (n) for each characteristic ranged from 78 to 
83; percentage provided is based on the actual number of participants. 
flCU, intensive care unit. 


Results 

Study Participants 

Of 87 eligible staff members who had possible expo- 
sure to H5N1 patients, 83 (95.4%) completed a question- 
naire and provided a serum sample (Table 1). The median 
age of employees was 37.4 years (range 22-55 years), and 
53 (64%) were female. Most employees (97.6%) were res- 
idents of Hanoi City, Vietnam. Of the 83 employees, 51 
(61%) were nurses or nurse’s aides, 19 doctors (23%), 7 
(8%) laboratory employees, and 6 (7%) other. Thirty-seven 
(45.1%) worked in the intensive care unit (ICU), 30 
(36.6%) in the infectious diseases department, 8 (9.8%) in 
the laboratory, 6 (7.3%) in radiology, and 1 in the hema- 
tology department. More than two thirds (68.3%) of the 


employees reported receiving influenza vaccine in 2004, 
and 1 person reported taking oseltamivir for treatment of 
influenzalike illness since December 27, 2003. No respon- 
dents took oseltamivir as prophylaxis against influenza 
infection. In total, 76.8% of participants reported contact 
with 2 or 3 influenza A H5N1 patients. Four hospital 
employees (4.9%) reported no contact with H5N1 patients; 
they were all laboratory personnel who had handled clini- 
cal material from H5N1 patients. Median duration of expo- 
sure to the hospitalized H5N1 primary case-patients 
reported was 82 hours, ranging from 1 to 299 hours (N = 
78). Most participants reported always wearing protective 
masks (94.8%), gloves (61.5%), and eye-protection 
(31.6%) while caring for H5N1 patients (Table 2). 

Clinical Symptoms 

The figure summarizes the symptoms reported by hos- 
pital employees during the study period. Overall, 59 
(72.0%) employees reported symptoms during the study 
period; 66.0% of these had onset of symptoms within 1 to 
7 days after exposure to a H5N 1 patient. Median duration 
of reported illness was 5 days (range CM10 days). Three 
persons (5.4%) were too ill to work; none were admitted to 
the hospital. Two persons (2.4%) who worked in ICU met 
the possible secondary H5N1 case-patient definition. They 
reported contact with patients but not with sick poultry or 
pigs, and neither worked in the laboratory. Both reported 
receiving the 2003-2004 influenza vaccine and denied tak- 
ing oseltamivir. Table 3 summarizes reported contact with 
poultry and pigs by participants. Approximately 1 quarter 
of participants (25.6%) reported the presence of poultry 
outside their homes, and 2 HCWs (9.5%) reported that 
poultry had died in the past month. The 2 possible H5N1 
secondary case-patients did not report have poultry dying 
outside their homes within the previous month. 

H5N1 Antibody Prevalence 

Samples were obtained from all 83 participants, includ- 
ing the 2 with possible secondary cases, and none were 
positive for antibodies to influenza A H5N 1 . One sample 
initially had an antibody titer of 160 and 640 against 
A/Vietnam/1 194/2004 and A/Vietnam/3212/2004, respec- 
tively. However, microneutralization tests using influenza 
A H1N1 viruses showed a high titer of 10,240, and 
microneutralization repeated after adsorption with influen- 
za A H1N1 virus showed an 8-fold reduction in the anti- 
body titer, which was interpreted as indicating a 
cross-reacting anti-Nl antibody. 

Discussion 

No evidence was found of nosocomial transmission of 
H5N1 viruses among 83 hospital employees with exposure 


212 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


No H5N1 Influenza Hospital Transmission, Hanoi, 2004 


Table 2. Protective equipment used by hospital employees while 

examining or caring for H5N1 patients 

Equipment n (%) 


Mask (N = 77) 


Always 

73 (94.8) 

Not always 

2 (2.6) 

Never 

2 (2.6) 

Types of masks (N = 75)* 

N95 

65 (86.7) 

Surgical 

55 (73.3) 

N92 

2(2.7 

Other 

8(10.7) 

Eye protection (N = 76) 

Always 

24 (31.6) 

Not always 

15(19.7) 

Never 

37 (55.2) 

Type of eye protection (N = 39) 

Glasses 

36 (92.3) 

Face shield 

3 (7.7) 

Gloves (N = 78) 

Always 

48 (61.5) 

Not always 

21 (26.9 

Never 

9(11.5) 


*Use of multiple respirators or masks at different times possible. 


to 4 confirmed and 1 probable H5N 1 case-patients or their 
clinical samples. A number of possible factors may explain 
these findings: a lack of infectivity of the patients at the 
time of admission; the effective use of personal protective 
equipment (PPE) and infection control; low sensitivity of 
the antibody detection method; lack of susceptibility of 
HCWs, or a lack of transmissibility of this particular H5N1 
strain. 

No data are available on the duration of H5N1 virus 
shedding in children. However, for human influenza 
viruses, viral shedding at high titers is generally more pro- 
longed in children, and virus can be recovered up to 6 
days before and 21 days after the onset of symptoms. The 
H5N1 patients in this study were admitted with severe ill- 
ness 3-7 days after onset of symptoms and PCR-positive 
specimens were obtained from the 4 confirmed case- 
patients on the day 1(1 patient), day 2(1 patient), and day 
3 (2 patients) after admission. In addition, live virus was 
cultured from samples taken from 2 of the patients on 
days 1 and 3 after admission, respectively. None of the 
patients were treated with oseltamivir because this was 
not available at the time (9). Two of the patients were 
treated orally with the nucleoside analogue ribavirin dur- 
ing their admission, 1 on day 4 after admission, and the 
other on day 1 (9). However, the 2 other confirmed case- 
patients and the probable case-patient did not receive 
antiviral treatment and, if human infection with H5N 1 is 
associated with viral shedding, these patients would be 
expected to be contagious during their admission. 

Most hospital employees (94.8%) reported that they 
always wore masks while caring for H5N1 patients, and 


often the reported type of mask was an N95 respirator. 
However, N95 respirators were first available in NPH on 
January 7, and some employees reported wearing N95s 
before this date. Therefore, reported PPE use in this study 
may be biased by inaccurate recall or a tendency to report 
behavior that HCWs know is recommended. Enhanced 
infection control practices and PPE were instituted on 
January 7, and the diagnosis of avian influenza was first 
confirmed on January 9. Therefore some HCWs in this 
study were likely exposed to H5N 1 patients without opti- 
mal PPE or infection control. 

Oseltamivir prophylaxis was not used by any of the 
staff in this study and therefore did not play a role in pro- 
tecting HCWs. Whether the HCWs in the study were pro- 
tected by cross-reactive immunity to other influenza A 
subtypes is hard to assess. One possible explanation for the 
observation that most confirmed H5N1 case-patients are 
reported in children or young adults is that older adults are 
protected by cross-reactive immunity from previous expo- 
sure to other influenza A viruses. This hypothesis requires 
further investigation. 

Serum samples were taken from HCWs at least 29 days 
after last possible exposure and at a time when the anti- 
body response to exposure would be expected to be 
detectable (4). Based on a small number of samples, the 
sensitivity of microneutralization test in detecting antibod- 
ies to H5N1 in children and adults is 88% and 80%, 
respectively, while the specificity is 100% and 93%, 
respectively (10). Also, the microneutralization assay uti- 
lized H5N1 strains isolated from human patients in North 
Vietnam, so the negative results are unlikely to be false 
negatives due to a poor match between antigen and anti- 
body. False-positive results are perhaps more likely, and 1 
sample was initially positive but appeared to be due to 
cross-reacting anti-Nl antibody. 

Epidemiologic evidence from Vietnam and Thailand 
clearly indicates that sustained human-to-human trans- 
mission of H5N1 has not yet occurred. Most reports of 


Table 3. Possible non-healthcare-related H5N1 
among study participants 

exposures 

Exposure 

n (%)* 

Poultry outside the home in last 4 weeks 

21 f (25.6) 

Do not know 

6 (7.3) 

Pigs outside the home in the last 4 weeks 

10t (12.2) 

Do not know 

7 (8.5) 

Visited market with sick poultry in last 4 weeks 

3 (3.7) 

Do not know 

18(22.2) 

<1 m from sick or dead poultry since July 2003 

8(10.) 

Do not know 

11 (13.6) 

Anyone sick in the household in the last week 

11 (13.4) 

Do not know 

2 (2.4) 


*The no. (N) of study participants for each characteristic was 80 to 82 with 
the percentage provided based on the actual number of respondents. 
fOf which 2 persons had dying poultry outside their home. 

JOf which none had dying pigs outside their home. 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


213 


RESEARCH 



% of respondents 

Figure. Reported symptoms and percentage of hospital employ- 
ees with symptoms (N = 82). 


H5N1 -infected patients have been sporadic, and despite 
the evidence from Hong Kong of human-to-human trans- 
mission and the occurrence of family clusters of H5N1 in 
Vietnam and Thailand, no evidence indicates that 
influenza A H5N1 has ever caused >1 generation of 
human-to-human transmission. Although this study has 
not distinguished the inherent transmissibility of the virus 
from the influence of infection control or host resistance, 
the data provides further reassurance that the risk for 
human-to-human transmission of currently circulating 
avian H5N1 viruses is low. Studies among household 
members of confirmed H5N1 case-patients will provide 
additional information on the risk for human-to-human 
transmission in the absence of infection control meas- 
ures. 

While the absolute risk for human-to-human transmis- 
sion of avian H5N1 viruses may be low at this time, the 
high case-fatality proportion among recent human H5N1 
patients demonstrates that the individual consequences of 
infection are very serious, and intensive measures to pro- 
tect healthcare workers and laboratory staff against infec- 
tion remain warranted. The risk of person-to-person 
transmission of H5N1 viruses could increase in the future. 
Consequently, every H5N 1 case should be managed by cli- 
nicians and public health professionals with the assump- 
tion that human-to-human transmission can occur and that 
the risk for such transmission is unpredictable. 

Acknowledgments 

We acknowledge the invaluable support and cooperation of 
the staff of NHP, Hanoi, and of the Ministry of Health of the 
Socialist Republic of Vietnam. We also thank Jackie Katz for pro- 
viding advice on testing and the recombinant protein for 


A/HK/ 156/97 for Western blot analysis, and Alain Moren for 
reviewing the manuscript. 

This document has been produced with the financial assis- 
tance of the European Union and of the Italian Government. The 
views expressed herein can in no way be taken to reflect the offi- 
cial opinion of the European Union or the Italian Government. 

The authors of this article participated as follows: Nguyen 
Thanh Liem contributed to the conception, planning, and imple- 
mentation of the study and to the preparation and review of the 
manuscript. Wilina Lim tested the biological samples, interpreted 
the results, and contributed to the preparation of the manuscript. 
Mirna Du Ry van Beest Holle and Arnold B osman contributed to 
the study design, planning and implementation, and drafting of 
the manuscript. Bach Huy Anh contributed to the study design 
and implementation. Timothy Uyeki and Peter Horby contributed 
to study conception, design, and manuscript drafting. Tom Grein, 
Keiji Fukuda, Aaron Curns, and Valerie Delpech contributed to 
the study design. 

Dr. Liem is director of the National Pediatric Hospital, 
Hanoi, Vietnam. 

References 

1. Yuen KY, Chan PK, Peiris M, Tsang DNC, Que TL, Shortridge KF, 
et al. Clinical features and rapid viral diagnosis of human disease 
associated with avian influenza A H5N1 virus. Lancet. 
1998;351:467-71. 

2. Buxton Bridges C, Lim W, Hu-Primmer J, Sims L, Fukuda K, Mak 
KH, et al. Risk of influenza A (H5N1) infection among poultry work- 
ers, Hong Kong, 1997-1998. J Infect Dis. 2002;185:1005-10. Epub 
Mar 19, 2002. 

3. Webster RG. Influenza virus: transmission between species and rele- 
vance to emergence of the next human pandemic. Arch Virol. 
1997;120 (Suppl 120):105-13. 

4. Katz JM, Lim W, Bridges CB, Rowe T, Hu-Primmer J, Lu X, et al. 
Antibody response in individuals infected with avian influenza A 
(H5N1) viruses and detection of anti-H5 antibody among household 
and social contacts. J Infect Dis. 1999;180:1763-70. 

5. Buxton Bridges C, Katz JM, Seto WH, Chan PK, Tsang D, Ho W, et 
al. Risk of influenza A (H5N1) infection among health care workers 
exposed to patients with influenza A (H5N1), Hong Kong. J Infect 
Dis. 2000;181:344-8. 

6. Office International des Epizooties. Disease information bulletin. 12 
December 2003. Vol. 16 - No. 50. [accessed October 11.2004]. 
Available from http://www.oie.int/eng/info/hebdo/AIS_67.HTM#Sec2 

7. Office International des Epizooties. Press release. Update on highly 
pathogenic avian influenza control methods in Asia including use of 
vaccination [accessed October 11, 2004], Available from 
http://www.oie.int/eng/press/en_040927.htm 

8. Thailand Ministry of Public Health. Press Release: Avian influenza 
infectious of patients in Kamphaeng-Phet (Sept 28, 2004) [accessed 
October 11, 2004]. Available from http://thaigcd.ddc.moph.go.th/ 
download/AI_press_280904_en.pdf 

9. Hien TT, Liem NT, Dung NT, San LT, Mai PP, Chau NvV, et al. Avian 
influenza A (H5N1) in 10 patients in Vietnam. N Engl J Med. 
2004;350:1179-88. 


214 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


No H5N1 Influenza Hospital Transmission, Hanoi, 2004 


10. Rowe T, Abernathy RA, Hu-Primmer J, Thompson WW, Lu X, Lim 
W, et al. Detection of human serum antibody to avian influenza A 
(H5N1) virus using a combination of serologic assays. J Clin 
Microbiol. 1999;37:937-43. 


Mirna Du Ry van Beest Holle, Center for Infectious Disease 
Epidemiology, National Institute for Public Health and the Environment, 
PO Box 1, 3720 BA, Bilthoven, the Netherlands; fax: 31-30-2744409; 
email: mirna.du.ry@rivm.nl 


The opinions expressed by authors contributing to this journal do 
not necessarily reflect the opinions of the Centers for Disease 
Control and Prevention or the institutions with which the authors 
are affiliated. 



Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


215 




RESEARCH 


Bacterial Zoonoses and Infective 
Endocarditis, Algeria 

Akila Benslimani,* 1 Florence Fenollar^ 1 Hubert Lepidi,t and Didier Raoultf 


Blood culture-negative endocarditis is common in 
Algeria. We describe the etiology of infective endocarditis 
in this country. Samples from 110 cases in 108 patients 
were collected in Algiers. Blood cultures were performed in 
Algeria. Serologic and molecular analysis of valves was 
performed in France. Infective endocarditis was classified 
as definite in 77 cases and possible in 33. Causative 
agents were detected by blood cultures in 48 cases. All 62 
blood culture-negative endocarditis cases were tested by 
serologic or molecular methods or both. Of these, 34 test- 
ed negative and 28 had an etiologic agent identified. A total 
of 18 infective endocarditis cases were caused by zoonot- 
ic and arthropodborne bacteria, including Bartonella quin- 
tana (14 cases), Brucella melitensis (2 cases), and Coxiella 
burnetii (2 cases). Our data underline the high prevalence 
of infective endocarditis caused by Bartonella quintana in 
northern Africa and the role of serologic and molecular 
tools for the diagnosis of blood culture-negative endo- 
carditis. 


I n Algeria, infective endocarditis is common. Vegetations 
graft primarily on lesions of rheumatic heart disease 
(1,2). The rate of blood culture-negative endocarditis in 
Algeria is as high as 76% (2), which leads to difficulty in 
antimicrobial treatment. Most cases of blood culture-neg- 
ative endocarditis have been thought to be caused by pre- 
vious antimicrobial therapy. Infective endocarditis 
prognosis is often obscured by delayed diagnosis and a 
lack of specific treatment. In Algeria, poor socioeconomic 
level and lack of medical follow-up of patients are among 
the factors associated with endocarditis. The concentration 
of medical infrastructures in the northern part of the coun- 
try leads to the referral of patients with serious illnesses, 
such as endocarditis, to northern hospitals, especially with- 
in Algiers (Figure 1). Algiers, the capital and largest city 
with ~5 million inhabitants, has 7 hospitals, including 6 
cardiology and 5 cardiac surgery wards. These wards 
receive patients with endocarditis, either for diagnosis and 


*Service de Biologie Clinique, Alger, Algerie; and fUniversite de la 
Mediterranee, Marseille, France 


treatment or for corrective surgery of postendocarditis 
lesions. A retrospective analysis of Algerian infective 
endocarditis cases showed streptococci and staphylococci 
were the leading causes, followed by less frequent causes, 
such as enterobacteria and Haemophilus spp. (2). A high 
percentage of blood culture-negative endocarditis was 
noted. However, no study has evaluated the agents respon- 
sible for blood culture-negative endocarditis. New sero- 
logic and molecular tools, which have improved the 
etiologic diagnosis of infective endocarditis, have not been 
used to clarify the unknown role of fastidious bacteria 
(3-11). In our study, samples were collected from 110 
patients with suspected cases of endocarditis. All samples 
were analyzed prospectively by using conventional micro- 
biologic methods in Algiers. When available, cardiac 
valves and serum samples were stored to perform retro- 
spective analysis at the Unite des Rickettsies (Marseille, 
France). 

Material and Methods 
Patients 

Clinicians usually diagnose infective endocarditis by 
using the modified Duke criteria, which includes 3 major 
criteria (blood cultures typical of infective endocarditis, 
vegetations on echocardiography, and Coxiella burnetii 
serologic testing with immunoglobulin [Ig] G phase I titer 
>1:800) and 7 minor criteria (positive blood cultures, 
fever, previous heart disease, arterial embolism, positive 
results on serologic examination for endocarditis bacterial 
pathogens, immunologic disorders, and atypical but com- 
patible findings on echocardiography) (12). Definite infec- 
tive endocarditis is diagnosed if any of the following 
conditions is met: 2 major criteria exist; 1 major criterion 
and 3 minor criteria; or 5 minor criteria. Possible infective 
endocarditis is considered if 1 major criterion and 1 minor 
criterion or 3 minor criteria exist. On the basis of these 
criteria, we could locate 110 cases in 108 patients with 


Hhese 2 authors have contributed equally to the manuscript. 


216 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


Bacterial Zoonoses and Infective Endocarditis, Algeria 



Figure 1. Map of Algeria. Courtesy of Wikipedia Encyclopedia 
(http://en.wikipedia.org/wiki). 


definite or possible infective endocarditis in 5 cardiology 
wards and 2 cardiac surgery wards in Algiers during a 42- 
month period (June 2000-December 2003). For each 
patient, an information sheet with epidemiologic, clinical, 
echocardiographic, and biologic data was filled out. A min- 
imum of 3 blood cultures were sampled per patient. Thirty- 
eight cardiac valve specimens from 38 (35.4%) patients 
were sampled and stored at -80°C. Thirty-seven cardiac 
valve specimens from another 30 (27.3%) patients were 
formalin-fixed for pathologic testing. Sixty-one serum 
samples from 61 (55.5%) patients were available. 

Blood Cultures 

Either Castaneda Aer/Anaer (Bio-Rad, Marnes-La- 
Coquette, France) or broth for blood culture (Institut 
Pasteur d’Algerie, Algiers, Algeria) were used as blood- 
culture medium and were incubated at 37°C. If signs of 
culture appeared, a blood sample was taken from the cul- 
ture bottle and Gram staining on Columbia blood agar 
(BioMerieux, Marcy L’Etoile, France) and chocolate agar 
(BioMerieux) was performed. Agar plates were incubated 
in 5% C0 2 at 37 °C. In the event of culture, the microor- 
ganism was identified by API identification tests 
(BioMerieux). At day 15 of incubation, if cultures 
remained negative, an enrichment of each bottle was 
processed on Todd-Hewitt broth (Institut Pasteur 
d’Algerie) supplemented with 0.01% L-cysteine (Sigma- 
Aldrich, Lausanne, Switzerland) and 0.001% hypochloride 


pyridoxal (Sigma- Aldrich). In cases of broth turbidity, 
microscopic examinations were performed as described 
above. If culture was positive, the strain was identified. 

Valve Analysis 

Axenic Culture 

Thirty-eight excised cardiac valves were examined. If 
macroscopic lesions of infective endocarditis were detect- 
ed, we attempted to divide the valve into 3 parts to be used 
for bacteriologic analysis, storage at -80°C, and histologic 
analysis. Portions of valve tissue were ground with a mor- 
tar and pestle and cultured on Columbia blood agar and 
chocolate agar supplemented with Polyvitaminic 
Supplement (Bio-Rad) at 35°C for 15 days in 5% C0 2 . We 
performed direct Gram staining and identified colonies as 
described above. 

Cell Culture 

Cell cultures were performed in France. Specimens 
from 12 cardiac valves positive on polymerase chain reac- 
tion (PCR) for Bartonella quintana or Brucella melitensis 
were spread onto cells grown within a shell vial as previ- 
ously described (13,14). After 3 weeks of incubation at 
37°C, the bacteria were detected by using Gimenez stain- 
ing, a direct immunofluorescence test incorporating poly- 
clonal antibodies directed against Bartonella , and by PCR 
targeting the 16S rRNA sequence. 

Molecular Biology 

For the 38 cardiac samples stored at -80°C, molecular 
analysis was performed in France. After 18 hours of pro- 
teinase K digestion at 55°C, DNA was extracted from tis- 
sue by using the MagNA Pure LC instrument (Roche 
Molecular Biochemicals, Manheim, Germany) and 
MagNA Pure LC DNA Isolation Kit III (Roche Molecular 
Biochemicals), as described by the manufacturer. A PCR- 
positive valve sample taken from a patient with 
Staphylococcus aureus endocarditis was used as a positive 
control. A mixture of all reagents used for DNA extraction 
and DNA extracted from normal heart tissue were 
processed as negative controls. One negative control was 
included for every 5 samples tested. PCR amplification 
and sequencing were performed, as previously described 
(15), by using primers in Table 1. PCR targeting the 16S 
rRNA sequence was systematically performed. When a 
negative result occurred, additional PCR was performed 
targeting the 18S and 28S rRNA internal transcribed spac- 
er to search for fungal infections. All positive PCR prod- 
ucts were sequenced. The sequences were compared to 
those available in GenBank. Positive PCR results were 
considered as certain, when congruence existed between 
the results obtained with PCR and those obtained with 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


217 



RESEARCH 


Table 1. Primers used for broad-range 16S rRNA polymerase chain reaction (PCR) and, according to species identified by 
sequencing, primers targeting a second gene for confirmation of positive 1 6S rRNA PCR results and primers used for fungal PCR 


Microorganisms Gene 


Eubacteria 

16S rRNA 

Staphylococcus spp. 

RpoB 

Streptococcus spp. 

RpoB 

Enterococcus spp. 

RpoB 

Streptococcus spp. 

SOD 

Enterobacteriaceae 

RpoB 

Mycoplasma homlnls 

FtsY 

Coxlella burnetii 

IS111 

Bartonella spp. 

ITS 

Bacillus spp. 

RpoB 

Cory ne bacterium spp. 

RpoB 

Fungi 

18S-28S ITS 


Forward primer 

536f 5' CAGCAGCCGCGGTAATAC 
StphF 5' AAACCIATACGCAATTGGTT 
StrpF 5' AARYTIGGMCCTGAAGAAAT 
StrpF 5' AARYTIGGMCCTGAAGAAAT 
dl 5' CCITAYICITAYGAYGCIYTIGARCC 
CM7 5' AACCAGTTCCGCGTTGGCCTGG 
MH1 F 5' GT GTT GT ATCGACAACAG 
Trans3 5' CAACT GT GTGGAATT GAT GA 
ITSF1 5' GCGACTGGGGTGAAGTGG 
Bc55F 5' T CTCGTATGGAACGT GTT GT 
C2700F 5' GWATGAACATYGGBCAGGT 
FCU 5' TCCGTAGGTGAACCTGCGG 


Reverse primer 

RP2 5' ACGGCTACCTTGTTACGACTT 
StphR 5' GTTTCATGACTTGGGACGG 
StrpR 5' TGIARTTTRT CAT CAACCAT GT G 
StrpR 5' TGIARTTTRT CAT CAACCAT GT G 
d2 5' ARRTARTAIGCRTGYTCCCAIACRTC 
CM31 b 5' CCTGAACAACACGCTCGGA 
MH1 R 5' GT GTT GT ATCGACAA 
TransS 5' TTTACATGACGCAATAGCGC 
ITSR1 5' AGGCTTGGGAT CAT CAT C 
Bc260R 5' T GAACGT CACGYACTT CAAA 
C3130R 5' TCCATYTCRCCRAARCGCT 
RCU 5' GCTGCGTTCTTCATCGATGC 


other analyses. With a positive result interpreted as a pos- 
sible case, we performed additional PCR, targeting a sec- 
ond gene with genus-specific primers (Table 1). When the 
PCR was positive and the sequence gave the same result, 
the case was reclassified as certain. When the second PCR 
was negative, we performed a PCR targeting a third gene. 
When both PCRs targeting the second and the third gene 
were negative, the result was classified as negative. 

Histologic and Immunohistologic Analysis 

Thirty- seven valve samples underwent fixation by for- 
malin and were paraffin-embedded. Valve specimens were 
cut to 3-qm thickness serial sections. Hematoxylin-eosin- 
saffron, periodic acid-Schiff, Giemsa, Brown- 
Hopps/Brown-Brenn Gram, Grocott-Gomori methena- 
mine silver, and Warthin- Starry stains were used (16). On 
the basis of the histologic findings, valve specimens were 
divided into 3 groups: A, B, and C. Group A samples 
showed histologic features of infective endocarditis con- 
sisting of vegetations or polymorphonuclear leukocyte- 
rich valvular inflammation. Group B specimens showed 
valvular inflammation composed of mainly inflammatory 
mononuclear cells, macrophages, and lymphocytes with- 
out vegetations and microorganisms. Group C samples 
were devoid of inflammation, vegetations, or microorgan- 
isms. When Bartonella endocarditis was suspected, 
immunohistochemical analysis was performed on valve 
sections with an anti- Bartonella rabbit polyclonal antibody 
as previously described (17). 

Serum Sample Analysis 

Serologic Testing 

Brucella serologic analysis was performed by Rose- 
Bengale agglutination (Bio-Rad, Marnes-La-Coquette, 
France) for 61 serum samples from 61 patients in Algiers, 
and the samples were stored at -20°C for further study. 
The confirmation was observed by Wright Serology (Bio- 
Rad). In the case of endocarditis, specific antibody titers 


exceeded 1:800. Bartonella and C. burnetii serologic test- 
ing was performed in France on all 61 samples. For 
Bartonella serologic testing, B. quintana and B. henselae 
were used as antigens in a microimmunofluorescence 
(MIF) assay performed as previously described (18). A 
patient was considered to have Bartonella endocarditis 
when IgG titers >1:800 were observed (18). The species 
identification was performed with Western blot performed 
before and after serum cross-adsorption as previously 
described (19). For C. burnetii serologic testing, 
immunoglobulin (Ig) G, IgM, and IgA antibody titers were 
estimated by using an MIF test as previously described 
(20). A diagnosis of chronic endocarditis was made when 
a patient had an IgG phase I titer >1:800 (20). A Light 
Cycler nested PCR was performed on positive serum sam- 
ples for Bartonella and C. burnetii as previously described 
( 21 , 22 ). 

Results 

Patient Characteristics 

Our prospective study led to identification of 110 cases 
from 108 patients. The 110 episodes were classified as 77 
(70%) definite infective endocarditis and 33 (30%) possi- 
ble infective endocarditis (12). A second episode of infec- 
tive endocarditis developed in 2 patients during our survey. 
The patients included 64 men and 40 women with a mean 
age of 35.3 years (range 17-72 years). The series included 
4 children, 2 boys (6 and 8 years of age) and 2 girls (9 and 
14 years of age). Among the patients, 34 came from rural 
areas, 61 lived in urban areas, 1 was in prison, and no 
information could be obtained for 12. Among 96 patients 
whose living conditions were known, 59 (61.5%) lived in 
poor and crowded families of at least 10 persons. Among 
the 110 cases, 87 (79%) episodes were diagnosed on native 
valve and 23 (21%) on prosthetic valve. The mitral valve 
was affected in 31 (28.2%) cases, the aortic in 29 (26.3%), 
and both in 41 (37.2%). The tricuspid valve was affected 
in 3 (2.7%) patients, and 4 (3.6%) had aortic, mitral, and 


218 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


tricuspid involvement. We reported 1 case with mitral and 
pulmonary valves affected, with the persistence of an arte- 
rial canal, and 1 patient on a pacemaker. 

Blood Cultures 

Blood cultures identified 48 microorganisms (Table 2). 
Of the 22 Streptococcus spp. cultures, 5 Streptococcus 
mitis , 6 Streptococcus sp., 3 S. agalactiae , 3 

Granulicatella adiacens , 2 a- Streptococcus, 1 S. oralis , 1 
S. intermedius, and 1 Gemella morbillorum were identi- 
fied. Seven Staphylococcus aureus and 5 coagulase-nega- 
tive Staphylococcus were observed. One Haemophilus 
influenzae , 1 H. aphrophilus, 1 Haemophilus sp., 1 
Kingella kingae , and 1 Actinobacillus actinomycetemcomi- 
tans were identified among the HACEK group 
( Haemophilus , Actinobacillus , Cardiobacterium , 
Eikenella , Kingella.). One Brucella melitensis , a zoonotic 
agent, was isolated. 

Serum Analysis 

Using serologic testing, infective endocarditis could be 
diagnosed in 11 (18%) of 61 serum samples. A positive 
Brucella serologic result with titers of 1:3,200 was 
observed for 2 patients (1 sample was also culture posi- 
tive). Two other patients had a typical profile of Q fever 
endocarditis (Phase I: IgG 1:3,200; IgM 1:25; IgA 
1: 1,600/Phase II: IgG 1:6,400; IgM 1:25; IgA 1:1,600 for 
1 patient and Phase I: IgG 1:6,400; IgM 1:800; IgA 
1: 50/Phase II: IgG 1:12,800; IgM 1:800; IgA 1:100 for the 
other patient). Among these 2 patients, C. burnetii Light 
Cycler nested-PCR performed on serum samples was 


Bacterial Zoonoses and Infective Endocarditis, Algeria 

positive for the sample from 1 patient. A positive 
Bartonella serologic result, with IgG >1:800, was 
observed for 7 patients (Table 3). The Western-blot analy- 
sis of the 7 serum samples allowed the specific diagnosis 
of B. quintana (Figure 2). Of these 7 patients, B. quintana 
Light Cycler nested-PCR performed on serum samples 
was positive for 5 patients (Table 3). 

Cardiac Valve Analysis 

Axenic culture of cardiac valves was positive for 9 
samples. The growth of 2 coagulase-negative 
Staphylococcus , 2 Streptococcus sp., 1 Staphylococcus 
aureus , 1 Streptococcus mitis , 1 S. intermedius , 1 
Corynebacterium sp., and 1 Candida kruzei was observed. 
Another sample was polymicrobial. Cell culture allowed 
the growth of B. quintana , an arthropodborne disease 
agent, from 3 valve samples (Tables 2 and 3). The num- 
bers of valve specimens classified into groups A, B, and C 
were 21, 5, and 11, respectively. With the exception of 
Bartonella endocarditis, the samples with histologic fea- 
tures of infective endocarditis had vegetations in most 
cases, moderate fibrosis, calcifications in some cases, and 
numerous inflammatory infiltrates composed predomi- 
nantly of polymorphonuclear leukocytes and abundant 
neovascularization. By using special stains, microorgan- 
isms were visualized in 16 samples from group A, gram- 
positive cocci and gram-negative bacilli in 8 cases each. 
In samples from group B, the inflammatory infiltrates 
were rare and focal and consisted mainly of macrophages 
and lymphocytes with discrete neovascularization. The 
specimens from group C showed noninflammatory 


Table 2. Distribution of 1 10 infective endocarditis cases* diagnosed in Algeria using blood culture, cardiac valve culture, serologic 
testing, cardiac valve polymerase chain reaction (PCR), and PCR on serum samples 


Positive samples/tested samples 


Identified microorganisms 

Blood culture 
(N = 110) 

Cardiac valve 
culture (N = 38) 

Serologic 
testing (N = 61) 

Cardiac valve 
PCR (N = 38) 

PCR on serum 
sample (N = 9) 

Total 

Streptococcus spp. and related 
genera 

0/22 

0/4 

NP 

7/0 

NP| 

24/0 

Bartonella quintana 

0/1 J 

0/3 

5/2 

10/0 

3/2 

12/2 

Staphylococcus spp. 

2/10 

0/3 

NP 

2/1 

NP 

11/3 

HACEK§ 

0/4 

0/0 

NP 

1/1 

NP 

5/1 

Enterococcus spp. 

1/1 

0/0 

NP 

1/0 

NP 

2/1 

Brucella melitensis 

0/1 

0/0 

2/0 

2/0 

NP 

2/0 

Coxiella burnetii 

0/0 

0/0 

2/0 

0/0 

1/NP 

2/0 

Corynebacterium spp. 

0/2 

0/1 

NP 

1/0 

NP 

2/0 

Mycoplasma hominis 

0/0 

0/0 

NP 

1/0 

NP 

1/0 

Enterobacteria spp. 

1/1 

0/0 

NP 

0/0 

NP 

1/1 

Alcaligenes faecalis 

0/1 

0/0 

NP 

0/0 

NP 

1/0 

Pseudomonas aeruginosa 

0/1 

0/0 

NP 

0/0 

NP 

1/0 

Bacillus cereus 

0/0 

0/0 

NP 

1/0 

NP 

1/0 

Candida spp. 

0/0 

0/1 

NP 

1/0 

NP 

1/0 


Negative samples for definite infective endocarditis/negative samples 
for possible infective endocarditis 

No etiology 10/25 211 8/20 211 NP/NP 

*77 definite and 33 possible. 
fNP, not performed. 

jlf we consider that Bartonella quintana was misidentified as Haemophilus influenzae. 

§ HACEK, Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, Kingella. 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


219 


RESEARCH 


Table 3. Living conditions, involved cardiac valves, and diagnostic tools for Bartonella qulntana endocarditis cases in 14 patients* 

Bartonella Cardiac 


Patient 

Living conditions 

Involved cardiac 
valves 

Blood 

culture 

serologic 

testing 

PCR on serum 
sample 

valve 

culture 

Cardiac 
valve PCR 

Histologic 

analysis 

1 

Poor rural area 

Aortic 

- 

1:800 

B. quintana 

B. quintana 

B. quintana 

NP 

2 

Poor rural area 

Mitral 

- 

1:1,600 

- 

- 

B. quintana 

WS+/IC+ 

3 

Poor urban area 

Mitral 

- 

1:800 

B. quintana 

NP 

NP 

WS+/IC+ 

4 

Poor rural area 

Aortic 

- 

NP 

NP 

- 

B. quintana 

NP 

5 

Poor urban area 

Tricuspid 

+ t 

1:1,600 

B. quintana 

NP 

NP 

NP 

6 

Poor rural area 

Aortic + mitral 

- 

NP 

NP 

- 

B. quintana 

WS+/IC+ 

7 

Unknown 

Aortic + mitral 

- 

NP 

NP 

- 

B. quintana 

WS+/IC+ 

8 

Poor urban area 

Aortic 

- 

1:800 

B. quintana 

- 

B. quintana 

WS+/IC+ 

9 

Good urban area 

Aortic 

- 

NP 

NP 

B. quintana 

B. quintana 

WS+/IC+ 

10 

Good rural area 

Aortic + mitral 

- 

NP 

NP 

B. quintana 

B. quintana 

WS+/IC+ 

11 

Poor urban area 

Mitral 

- 

1:3,200 

- 

NP 

NP 

NP 

12 

Poor rural area 

Aortic 

- 

1:3,200 

B. quintana 

NP 

NP 

NP 

13 

Poor rural area 

Aortic 

- 

NP 

NP 

- 

B. quintana 

NP 

14 

Poor rural area 

Aortic 

- 

NP 

NP 

- 

B. quintana 

NP 


*NP, not performed; WS+, Warth in-Starry positive; IC+, immunochemistry positive; PCR, polymerase chain reaction, 
flf we consider that B, quintana was misidentified as Haemophilus influenzae. 


degenerative damage with extensive fibrosis and often 
calcifications. The histologic features of Bartonella endo- 
carditis were different from the other infective endocardi- 
tis. Samples from 7 cases with Bartonella endocarditis 
were examined. The valve tissues showed degenerative 
damage with extensive fibrosis. The valve tissues were 
poorly inflamed with rare mononuclear inflammatory cell 
infiltrates composed of lymphocytes and macrophages 
and discrete neovascularization. Vegetations, present in 
all samples, were small in size. In all cases, the Warthin- 
Starry stain detected Bartonella , mainly in vegetations as 
small bacillary organisms (Figure 3). 

The 16S rRNA PCR was positive for 29 cardiac valves 
(Tables 2 and 4). B. quintana was detected on 10 speci- 
mens (Table 3). Among the Streptococcus spp. and related 
genera, 3 Streptococcus sp., 1 S. mitis , 1 S. mutans , 1 S. 
gordonii , 1 S. pneumoniae , and 1 Granulicatella adiacens 
were detected. Two Staphylococcus aureus and 1 coagu- 
lase-negative Staphylococcus were identified. Among the 
2 bacteria from the HACEK group, 1 H. paraphrophilus 
and 1 Cardiobacterium hominis were identified. PCR per- 
formed with a second gene confirmed the previous PCR 
results with 1 exception. One Streptococcus sp. was not 
retrieved by PCR targeting a second or third gene and was 
considered as contamination. The PCR targeting the 
18S-28S rRNA ITS allowed the detection of 1 Candida 
parapsilosis. Finally, Bartonella spp. were also specifical- 
ly visualized in vegetations by immunohistochemistry in 
all the cases of B. quintana endocarditis (Figure 3). 

Causative Microorganisms and Discordant Results 

The overall distribution of causative microorganisms 
and their identification, depending on the diagnostic tools 
used, are displayed in Table 2. An etiologic agent could not 
be determined for 10 (13%) of definite cases and 28 (76%) 


of possible cases. For the 2 patients with recurring infective 
endocarditis, the cause for the first episode was different 
than that of the second episode. One patient had endocardi- 
tis caused by Streptococcus oralis , and 1 year later, endo- 
carditis caused by K. kingae developed. For the other 
patient, no etiologic diagnosis was established for the first 
episode, during which a valve removal was necessary. Four 
months after cardiac surgery, the patient had endocarditis 


12345 12345 12345 

207 ► 

129 * 

85 ► 

39 ► 

32 ► 



17.5 ► 
7.1 ► 




A B 


L 


) 

c 


Figure 2. Western blot performed with a serum sample from a 
patient with an endocarditis caused by Bartonella quintana. 
Molecular masses (in kilodaltons) are given to the left of the pan- 
els. A) Untreated serum sample analyzed with B. quintana (lane 
1), B. henselae (lane 2), B. elizabethae (lane 3), B. vinsonii subsp. 
arupensis (lane 4), and B. vinsonii subsp. berkhoffii (lane 5) anti- 
gens. B) B. quintana - adsorbed serum sample analyzed with B. 
quintana (lane 1), B. henselae (lane 2), B. elizabethae (lane 3), B. 
vinsonii subsp. arupensis (lane 4), and B. vinsonii subsp. berkhoffii 
(lane 5) antigens. C) B. fre/ise/ae-adsorbed serum analyzed with 
B. quintana (lane 1), B. henselae (lane 2), B. elizabethae (lane 3), 
B. vinsonii subsp. arupensis (lane 4), and B. vinsonii subsp. berk- 
hoffii (lane 5) antigens. 


220 


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Bacterial Zoonoses and Infective Endocarditis, Algeria 



Figure 3. A) Section of an aortic valve from a patient with 
Bartonella endocarditis. Note the extensive fibrosis of the connec- 
tive valve tissue (arrowhead), the vegetation (*), and the low 
inflammatory infiltrate of the valve tissue (hematoxylin-phloxine- 
saffron, original magnification lOOx). B) Resected valve with 
Bartonella quintana infection showing darkly stained bacilli consis- 
tent with Bartonella. Note the numerous clusters of argyrophilic 
bacteria present in the valvular vegetation (Warth in-Starry silver, 
original magnification 1,000x). C) Immunohistochemical detection 
of B. quintana in a resected valve from a patient with Bartonella 
endocarditis. Note the extracellular distribution of the bacterial 
colonies (*) in the valvular vegetation (polyclonal antibody and 
hematoxylin counterstain, original magnification 250x). 


caused by Staphylococcus epidermidis. Nine discrepant 
results were also observed and are summarized in Table 4. 

Discussion 

Endocarditis cases with fastidious agents escape micro- 
biologic diagnosis classically applied in Algerian laborato- 
ries. For the first time, we established a profile of the 
microbiologic etiology of infective endocarditis in Algeria. 
Our conclusions concerning PCR results were submitted to 
a rigorous strategy of validation. All of the controls must 
be correct for validating each assay. The result was con- 
sidered true if confirmation was obtained by successfully 
amplifying bacterial DNA when targeting another gene, 
the PCR result was congruent with the results of other 
diagnostic tools, or both. 

Of the 77 cases of definite infective endocarditis, the 
cause was found for 67 (87%) cases. The diagnosis was 
performed on the basis of positive blood cultures for 44 
cases. For 20 (26%) cases, no etiologic diagnosis was 
obtained in Algeria but was performed in France on the 
basis of cardiac valve PCR, and Bartonella and Coxiella 
burnetii serologic testing. These data show improvement 
in the etiologic diagnosis of endocarditis when molecular 
or serologic tools are used. The rate of remaining infective 
endocarditis without cause is comparable to the prevalence 
in western countries (16). As in other countries, the etio- 
logic distribution is dominated by the bacteria responsible 
for infective endocarditis, such as Streptococcus spp. and 
related genera, Staphylococcus spp., and bacteria from the 
HACEK group. The difference in comparison to other 
countries is that blood culture-negative endocarditis is 
mainly linked to zoonotic and arthropodborne agents. 

For the 33 cases of possible infective endocarditis, the 
number of etiologic diagnoses was fewer than those for 
definitive infective endocarditis However, in this group, 
some cases are infective endocarditis and others are not. If 
we consider a Bartonella serologic result >1:800 as a 
major criterion (5), the 2 possible cases of B. quintana 
infective endocarditis will be classified as definite. 
Therefore, Bartonella serologic results should be taken 
into account in future revisions of the Duke criteria. Of the 
48 case-patients with positive blood cultures, 19 had addi- 
tional samples tested through a second analysis (serologic 
or molecular methods). Of the 19, 11 had negative results, 
5 were concordant, and 3 were discordant. Of these 48 cul- 
tures, 1 corresponds to brucellosis. 

Of the 62 blood culture-negative endocarditis cases, 
samples from all were tested by serologic or molecular 
methods. Of these, 34 were negative, and 28 had an etio- 
logic agent identified. Seventeen of those were due to 
zoonoses or arthropodborne bacterial diseases. 

Discrepancies were observed between the results ob- 
tained by using the various techniques. Some discrepancies 


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221 



RESEARCH 


Table 4. Discrepant results between blood culture, cardiac valve culture, cardiac valve PCR, and serologic testing for 9 patients* 


Patient 

Blood culture 

Cardiac valve 
culture 

16S rRNA PCR 

PCR targeting 
another gene 

Histologic 

analysis 

Serologic 

testing 

Conclusions 

n 

Negative 

Candida krusei 

Streptococcus 

spp. 

RpoB: negative 
SOD: negative 
18-28S ITS: C. 
parapsilosis 

NP 

Negative 

C. parapsilosis 

2t 

Negative 

Polymicrobial 

Streptococcus 

mitis 

S. mitis 

A 

Negative 

S. mitis 

3t 

Negative 

CNS 

Haemophilus 

paraphrophilus 

NP 

A /BGN 

Negative 

H. paraphrophilus 

4t 

Negative 

CNS 

Bartonella 

quintana 

B. quintana 

NP 

NP 

B. quintana 

5t 

S. mitis 

Staphylococcus 

aureus 

Streptococcus 

gordonii 

S. gordonii 

A /CGP 

Negative 

S. gordonii 

6t 

H. influenzae 

NP 

NP 

NP 

NP 

1:1,600 
Positive 
PCR on 
serum 
samples 

B. quintana 

7t 

Streptococcus 

intermedius 

Streptococcus 

intermedius 

S. mutans 

S. mutans 

A 

NP 

S. mutans 

at 

Negative 

Cory ne bacterium 
spp. 

Bacillus cereus 

B. cereus 

A 

Negative 

B. cereus 

at 

Negative 

S. mitis 

Enterococcus 
ga Hina rum 

E. gallinarum 

A /CGP 

Negative 

E. gallinarum 

*PCR, polymerase chain reaction; NP, not performed; CNS, coagulase-negative Staphylococcus', BGN, bacillus gram negative; CGP, cocci gram positive. 
■fThe microorganisms detected in valve culture were contaminants. 

JThe microorganisms were misidentified. 


resulted from culture contamination with the cutaneous 
flora. A significant rate of contamination has been already 
reported, and the low specificity of valve culture that we 
observed confirms these results (23-25). One discrepancy 
was caused by identification problems at the species level 
for Streptococcus. This fact has been previously reported 
(7). Another discrepancy was linked to a Candida species 
misidentification by phenotypic analysis, which was cor- 
rected by using molecular tools. The last discordant case 
corresponded to a patient for whom blood cultures were 
positive for H. influenzae. When serum samples were ana- 
lyzed, a diagnosis of B. quintana endocarditis has been 
established in the presence of positive Bartonella MIF, 
Western blot, and PCR. We do not know if B. quintana was 
misidentified as H. influenzae , which is possible as both 
are slow-growing, hemin-dependent, small, gram-negative 
bacteria (26). We believe that as fastidious, small, gram- 
negative bacteria growing in blood agar, the 2 organisms 
may be confused. 

In Algeria, cases of infective endocarditis caused by 
zoonotic and arthropodborne disease agents, such as 
Coxiella burnetii , Brucella melitensis , and Bartonella 
quintana are frequently observed and correspond to one 
quarter of the performed diagnoses. B. quintana would be 
one of the most common agents of infective endocarditis in 
our Algiers series (15.6% of definite infective endocardi- 
tis). The prevalence of endocarditis caused by Bartonella 
varies depends on the country. In Canada, Bartonella caus- 


es 3% of endocarditis cases (27). In Sweden, no Bartonella 
endocarditis was identified in an analysis of 334 infective 
endocarditis cases (28). In the United Kingdom, 
Bartonella endocarditis accounts for 1.1% of infective 
endocarditis cases (29). In Germany and in France, 
Bartonella endocarditis accounts for 3% of all infective 
endocarditis (A. Sander et al. unpub. data) (27). The fre- 
quency of Bartonella endocarditis is <1% for Sweden and 
higher in France, Germany, the United Kingdom (3%), and 
North Africa (15%). Such differences may be linked to dif- 
ferences in living conditions. 

Homeless people are at risk for B. quintana endocardi- 
tis (30,31). Indeed, B. quintana, like Rickettsia 
prowazekii , the agent of epidemic typhus, is transmitted 
by body lice. Those who live in extreme poverty are often 
the persons who are infested. The recent description of 
typhus in Algeria confirms that poor socioeconomic con- 
ditions still exist in this country (32-34). In our studies, B. 
quintana endocarditis cases occurred in patients living in 
poor conditions. Although the only known reservoir for B. 
quintana is humans, the bacterium has recently been asso- 
ciated with fleas (35). Moreover, some cases of B. quin- 
tana infections have been linked to contact with cats and 
cat fleas in patients who were not homeless and did not 
have body lice (36). 

Brucella melitensis , well known in northern Africa, 
where bucellosis is endemic in certain areas, accounts for 
2.6% of all infective endocarditis cases for which an etio- 


222 


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Bacterial Zoonoses and Infective Endocarditis, Algeria 


logic diagnosis has been performed (37). Be cause C. bur- 
netii detection requires specialized tests not normally 
found in most laboratories, it is not often diagnosed in 
Algeria (38). Two cases were retrospectively detected. 

Importance of infective endocarditis caused by zoonot- 
ic and arthropodborne agents in Algeria leads to 2 consid- 
erations. First, specific serologic tests need to be used for 
diagnosis. Indeed, 25% of our etiologic diagnoses corre- 
spond to microorganisms for which the diagnosis is usual- 
ly based on serologic testing. Secondly, the therapeutic 
impact of Brucella and Coxiella diagnosis is important 
because the antimicrobial treatment of endocarditis caused 
by these agents must include doxy cy cline. The 2 patients 
with Q fever endocarditis died during their hospitalization 
because of inadequate antimicrobial therapy. Finally, the 
high rate of blood culture-negative endocarditis was not 
linked to prior antimicrobial therapy but rather to fastidi- 
ous microorganisms for which serologic testing (as for 
zoonotic and arthropodborne disease agents) or molecular 
analysis (as for Mycoplasma hominis [39] and 
Corynebacterium spp.) are diagnostic tools. 

Our study underlines the need to perform serologic 
analysis to determine for the etiology of infective endo- 
carditis. Bartonella serologic testing is an important tool 
for diagnosis of blood culture-negative endocarditis and 
should be taken into account in future revisions of the 
Duke criteria. This study made it possible to show that 
zoonotic and arthropodborne disease agents cause one 
quarter of infective endocarditis in Algeria; B. quintana 
caused 13% of our cases. 

Acknowledgments 

We thank Kelly Johnston for reviewing the manuscript. 

Dr. Benslimani is a physician working at the E.H.S. Dr 
Maouche, Algiers, Algeria. Her research interests include the 
clinical features and diagnosis of endocarditis. 

References 

1. Moreillon P, Que YA. Infective endocarditis. Lancet. 
2004;363:139-49. 

2. Kezzal K. Importance of blood culture in septicemia, particularly 
bacterial endocarditis. Arch Inst Pasteur Alger. 1986;55:41-60. 

3. Bentley S, Maiwald M, Murphy L, Pallen M, Yeats C, Dover L, et al. 
Sequencing and analysis of the genome of the Whipple’s disease bac- 
terium Tropheryma whipplei. Lancet. 2003;361:637-44. 

4. Gauduchon V, Chalabreysse L, Etienne J, Celard M, Benito Y, Lepidi 
H, et al. Molecular diagnosis of infective endocarditis by PCR ampli- 
fication and direct sequencing of DNA from valves tissue. J Clin 
Microbiol. 2003;41:763-6. 

5. Rolain JM, Lecam C, Raoult D. Simplified serological diagnosis of 
endocarditis due to Coxiella burnetii and Bartonella. Clin Diagn Lab 
Immunol. 2003;10:1147-8. 

6. Watkin RA, Lang S, Lambert PA, Littler WA, Elliott TSJ. The micro- 
bial diagnosis of infective endocarditis. J Infect. 2003;47:1-11. 


7. Podglajen I, Bellery F, Poyart C, Coudol P, Buu-Hoi A, Bruneval P, 
et al. Comparative molecular and microbiologic diagnosis of bacteri- 
al endocarditis. Emerg Infect Dis. 2003;9:1543-7. 

8. Goldenberger D, Kunzli A, Vogt P, Zbinden R, Altwegg M. Molecular 
diagnosis of bacterial endocarditis by broad-range PCR amplification 
and direct sequencing. J Clin Microbiol. 1997;35:2733-9. 

9. Mylonakis E, Calderwood S. Infective endocarditis in adults. N Engl 
J Med. 2001;345:1318-30. 

10. Millar BC, Moore JE. Emerging issues in infective endocarditis. 
Emerg Infect Dis. 2004;10:1110-6. 

11. Millar BC, Moore JE. Current trends in the molecular diagnosis of 
infective endocarditis. Eur J Clin Microbiol Infect Dis. 
2004;23:353-65. 

12. Li JS, Sexton DJ, Mick N, Nettles R, Fowler VG Jr, Ryan T, et al. 
Proposed modifications to the Duke criteria for the diagnosis of infec- 
tive endocarditis. Clin Infect Dis. 2000;30:633-8. 

13. Raoult D, Vestris G, Enea M. Isolation of 16 strains of Coxiella bur- 
netii from patients by using a sensitive centrifugation cell culture sys- 
tem and establishment of the strains in HEL cells. J Clin Microbiol. 
1990;28:2482-4. 

14. La Scola B, Raoult D. Culture of Bartonella quintana and Bartonella 
henselae from human samples: a 5-year experience (1993 to 1998). J 
Clin Microbiol. 1999;37:1899-905. 

15. La Scola B, Michel G, Raoult D. Use of amplification and sequenc- 
ing of the 16S rRNA gene to diagnose Mycoplasma pneumoniae 
osteomyelitis in a patient with hypogammaglobulinemia. Clin Infect 
Dis. 1997;24:1161-3. 

16. Lepidi H, Durack DT, Raoult D. Diagnostic methods current best 
practices and guidelines for histologic evaluation in infective endo- 
carditis. Infect Dis Clin North Am. 2002;16:339-61. 

17. Lepidi H, Fournier P, Raoult D. Quantitative analysis of valvular 
lesions during Bartonella endocarditis. A case control study. Am J 
Clin Pathol. 2000;114:880-9. 

18. Fournier P, Mainardi J, Raoult D. Value of microimmunofluorescence 
for the diagnosis and follow-up of Bartonella endocarditis. Clin Diag 
Lab Immunol. 2002;9:795-801. 

19. Houpikian P, Raoult D. Western Immunoblotting for Bartonella 
endocarditis. Clin Diag Lab Immunol. 2003;10:95-102. 

20. Tissot-Dupont H, Thirion X, Raoult D. Q fever serology: cutoff deter- 
mination for microimmunofluorescence. Clin Diag Lab Immunol. 
1994;1:189-96. 

21. Zeaiter Z, Fournier P, Greub G, Raoult D. Diagnosis of Bartonella 
endocarditis by a real-time nested-PCR assay using serum. J Clin 
Microbiol. 2003;41:919-25. 

22. Fournier P, Raoult D. Comparison of PCR and serology for the diag- 
nosis of acute Q fever. J Clin Microbiol. 2003;41:5094-8. 

23. Giladi M, Szold O, Elami A, Bruckner D, Johnson BL Jr. 
Microbiological cultures of heart valves and valve tags are not valu- 
able for patients without infective endocarditis who are undergoing 
valve replacement. Clin Infect Dis. 1997;24:884-8. 

24. Chuard C, Antley CM, Reller LB. Clinical utility of cardiac valve 
Gram stain and culture in patients undergoing native valve replace- 
ment. Arch Pathol Lab Med. 1998;122:412-5. 

25. Campbell WN, Tsai W, Mispireta LA. Evaluation of the practice of 
routine culturing of native valves during valve replacement surgery. 
Ann Thorac Surg. 2000;69:548-50. 

26. Colson P, Lebrun L, Drancourt M, Boue F, Raoult D, Nordmann P. 
Multiple recurrent bacillary angiomatosis due to Bartonella quintana 
in an HIV-infected patient. Eur J Clin Microbiol Infect Dis. 
1996;15:178-80. 

27. Raoult D, Fournier P, Drancourt M, Marrie T, Etienne J, Cosserat P, 
et al. Diagnosis of 22 new cases of Bartonella endocarditis. Ann 
Intern Med. 1996;125:646-53. 

28. Werner M, Fournier PE, Andersson R, Hogevik H, Raoult D. 
Bartonella and Coxiella antibodies in 334 prospectively studied 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


223 


RESEARCH 


episodes of infective endocarditis in Sweden. Scand J Infect Dis. 
2003;35:724-7. 

29. Lamas CC, Eykyn SJ. Blood culture negative endocarditis: analysis 
of 63 cases presenting over 25 years. Heart. 2003;89:258-62. 

30. Klein JL, Nair SK, Harrison TG, Hunt I, Fry NK, Friedland JS. 
Prosthetic valve endocarditis caused by Bartonella quintana. Emerg 
Infect Dis. 2002;8:202-3. 

31. Posfay Barbe K, Jaeggi E, Ninet B, Liassine N, Donatiello C, Gervaix 
A, et al. Bartonella quintana endocarditis in a child. N Engl J Med. 
2000;342:1841-2. 

32. Niang M, Brouqui P, Raoult D. Epidemic typhus imported from 
Algeria. Emerg Infect Dis. 1999;5:716-8. 

33. Birg ML, La Scola B, Roux V, Brouqui P, Raoult D. Isolation of 
Rickettsia prowazekii from blood by shell vial cell culture. J Clin 
Microbiol. 1999;37:3722-4. 

34. Mokrani K, Fournier PE, Dalichaouche M, Tebbal S, Aouati A, 
Raoult D. Epidemic typhus is a reemerging threat in Algeria. J Clin 
Microbiol. 2004;42:3898-900. 


35. Rolain JM, Franc M, Davoust B, Raoult D. Molecular detection of 
Bartonella quintana , B. koehlerae, B. henselae , B. clarridgeiae, 
Rickettsia felis, and Wolbachia pipientis in cat fleas, France. Emerg 
Infect Dis. 2003;9:338-42. 

36. Fournier P, Lelievre H, Eykyn S, Mainardi J, Marrie T, Bruneel F, et 
al. Epidemiologic and clinical characteristics of Bartonella quintana 
and Bartonella henselae endocarditis: a study of 48 patients. 
Medicine. 2001;80:245-51. 

37. Memish ZA, Balkhy HH. Brucellosis and international travel. J 
Travel Med. 2004;11:49-55. 

38. Maurin M, Raoult D. Q fever. Clin Microbiol Rev. 1999;12:518-53. 

39. Fenollar F, Gauduchon V, Casalta JP, Lepidi H, Vandenesch F, Raoult 
D. Mycoplasma endocarditis: two case reports and a review. Clin 
Infect Dis. 2004;38:e21-4. 


Address for correspondence: Didier Raoult, CNRS UMR 6020, Unite des 
Rickettsies, IFR, 48 Universite de la Mediterranee, Faculte de medecine, 
27 Boulevard Jean Moulin, 13385 Marseille cedex 5, France; fax: 33- 
491-83-03 90; email: Didier.Raoult@medecine.univ-mrs.fr 



224 


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Novel Flavivirus or New Lineage of 
West Nile Virus, Central Europe 

Tamas Bakonyi,*t Zdenek Hubalek,± Ivo Rudolf^ and Norbert Nowotny *§ 1 


A flavivirus (strain 97-103) was isolated from Culex 
pipens mosquitoes in 1997 following floods in South 
Moravia, Czech Republic. The strain exhibited close anti- 
genic relationship to West Nile virus (WNV) prototype strain 
Eg-101 in a cross-neutralization test. In this study, mouse 
pathogenicity characteristics and the complete nucleotide 
and putative amino acid sequences of isolate 97-103, 
named Rabensburg virus (RabV) after a nearby Austrian 
city, were determined. RabV shares only 75%-77% 
nucleotide identity and 89%-90% amino acid identity with 
representative strains of WNV lineages 1 and 2. Another 
RabV strain (99-222) was isolated in the same location 2 
years later; it showed >99% nucleotide identity to strain 97- 
103. Phylogenetic analyses of RabV, WNV strains, and 
other members of the Japanese encephalitis virus (JEV) 
complex clearly demonstrated that RabV is either a new 
(third) lineage of WNV or a novel flavivirus of the JEV 
group. 

W est Nile vims (WNV), a member of the Japanese 
encephalitis vims (JEV) group within the genus 
Flavivirus , family Flaviviridae , is the most widespread 
flavivirus, occurring in Africa, Eurasia, Australia, and 
North America. Other members of the JEV group fla- 
vivimses are Cacipacore virus (CPCV), Koutango virus 
(KOUV), JEV, Murray Valley encephalitis virus (MVEV), 
Alfuy virus (ALFV), St. Louis encephalitis virus (SLEV), 
Usutu virus (USUV), and Yaounde vims (YAOV) (1). 
Although initially WNV was considered to have minor 
human health impact, the human and equine outbreaks in 
Europe (Romania, Russia, France, Italy), Africa (Algeria, 
Tunisia, Morocco), and Asia (Israel) within the last 10 
years, and especially the virus’s emergence and spread in 
North America since 1999, put it into the focus of scientif- 
ic interest. The distribution and ecology of WNV, as well 
as clinical features, pathogenesis, and epidemiology of 
West Nile disease have been reviewed (2-6). Phylogenetic 


*University of Veterinary Medicine, Vienna, Austria; fSzent Istvan 
University, Budapest, Hungary; ^Institute of Vertebrate Biology 
ASCR, Brno, Czech Republic; and §United Arab Emirates 
University, Al Ain, United Arab Emirates 


analyses showed 2 distinct lineages of WNV strains 
(which themselves subdivide into several subclades or 
clusters), isolated in different geographic regions (7-10). 

The presence of WNV in central Europe has been 
known for some time. Serologic surveys have detected 
specific antibodies to WNV in several vertebrate hosts in 
Austria, Czech Republic, Hungary, and Slovakia during 
the past 40 years, and several virus strains were isolated 
from mosquitoes, rodents, and migrating birds (3). Human 
cases of West Nile fever were reported in the Czech 
Republic in 1997 (11) and in Hungary in 2003 (12). 
Although these countries are important transit areas or 
final destinations for migratory birds from the African con- 
tinent, and hence may play an important role in the circu- 
lation and conservation of different WNV strains, genetic 
information about the strains isolated in central Europe has 
not been available. We report the complete genome 
sequence and phylogenetic analyses, as well as antigenic 
and mouse virulence characteristics, of a unique flavivirus 
strain (97-103), closely related to WNV, which was isolat- 
ed by intracranial injection of suckling mice with 
homogenates of female Culex pipiens mosquitoes collect- 
ed 10 km from Lanzhot, Czech Republic, after a flood in 
1997 (11,13,14). The collection site was very close to the 
Czech-Austrian border, ~2 km from the small Austrian 
town of Rabensburg. Consequently, the isolate 97-103 was 
later tentatively called Rabensburg virus (RabV). Another 
antigenically identical or very closely related strain (99- 
222) was isolated from Cx. pipiens mosquitoes in the same 
location 2 years later (14). 

Methods 

Isolates 97-103 (passage 5 in suckling mouse brain 
[SMB]) and 99-222 (passage 4 in SMB) were freeze-dried 
in October 2000 (14). Viral RNA was extracted from 
140 |lL of virus resuspended in diethylpyrocarbonate 


Hhis study will be presented at the International Conference on 
Emerging Infectious Diseases, February 26-March 1 , 2005, Al Ain, 
United Arab Emirates. 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


225 


RESEARCH 


(DEPC)-treated water, using the QIAamp viral RNA Mini 
Kit (Qiagen, Hilden, Germany), according to the manufac- 
turer’s instructions. For amplification of the complete 
genome, oligonucleotide primers were designed with the 
help of the Primer Designer 4 for Windows 95 program 
(Scientific and Educational Software, version 4.10) and 
were synthesized by GibcoBRL Life Technologies, Ltd. 
(Paisley, Scotland, UK). A complete list of the 35 primers 
used in reverse transcription-polymerase chain reaction 
(RT-PCR) and sequencing reactions is available upon 
request. Reverse transcription and amplification were per- 
formed with a continuous RT-PCR method with the Qiagen 
OneStep RT-PCR Kit (Qiagen) following the manufactur- 
er’s instructions. Reverse transcription (at 50°C for 30 min) 
was followed by a denaturation step at 95 °C for 15 min, and 
40 cycles of amplification (94°C for 40 s, 57°C for 50 s, 
72°C for 1 min). Reactions were completed by a final 
extension for 7 min at 72°C, and the amplicons were kept 
at 4°C until electrophoresis was carried out. The reactions 
were performed in a Perkin-Elmer GeneAmp PCR System 
2400 thermocycler (Perkin-Elmer Corp., Wellesley, MA, 
USA). After RT-PCR, the amplicons were electrophoresed 
in agarose gel, stained with ethidium bromide, and bands 
were visualized under UV light. Gels were photographed 
with a Kodak DS Electrophoresis Documentation and 
Analysis System (Eastman Kodak Company, New Haven, 
CT, USA). Product sizes were determined with reference to 
a 100 - bp DNA Ladder (Promega, Madison, WI, USA). 
Fluorescence-based direct sequencings were performed in 
both directions on the PCR products with the ABI Prism 
Big Dye Terminator cycle sequencing ready reaction kit 
(Perkin-Elmer) and an ABI Prism 310 genetic analyzer 
(Perkin-Elmer) automated sequencing system (15). 

The nucleotide sequences were identified by BLAST 
search against GenBank databases and were compiled and 
aligned with the help of the Align Plus 4 for Windows 95 
(Scientific and Educational Software, version 4.00) and 
ClustalX Multiple Sequence Alignment (version 1.81) pro- 
grams. Phylogenetic analysis was performed with the 
Phylogeny Inference Program Package (PHYLIP) version 
3.57c. Distance matrices were generated by the Fitch pro- 
gram, with a translation/transversion ratio of 2.0. 
Phylogenetic trees were delineated by using the Tree View 
(Win32) program version 1.6.6. 


Results 

Both virus strains were identified as WNV by comple- 
ment fixation and neutralization tests (11,13). Strain 97- 
103 was compared antigenically in detail with the 
Egyptian Eg- 101 topotype strain of WNV (16), a represen- 
tative of WNV lineage 1 (clade la). In plaque-reduction 
cross-neutralization tests (PRNT) with homologous and 
heterologous antisera (produced by injection of ICR mice 
with 3 intraperitoneal doses at weekly intervals), the serum 
raised against Eg- 101 neutralized both the homologous 
virus and 97-103 at a titer of 512, while the strain 97-103 
specific serum was effective against strain Eg- 101 only at 
a titer of 64, although it neutralized the homologous virus 
at 512. The average 4-fold difference in cross-PRNT titers 
indicates certain antigenic heterogeneity of the 2 strains, 
and the 97-103 isolate was therefore regarded as a subtype 
of WNV (14). 

Virulence of RabV strains 97-103 and 99-222 was deter- 
mined by intracranial and intraperitoneal injection of spe- 
cific-pathogen-free (SPF) outbred ICR mice. Central 
nervous system symptoms (e.g., pareses of hind limbs) 
developed in suckling mice, which died 7-15 days after 
intracranial injection (Table 1). Adult mice did not show 
any clinical symptoms and survived the experimental infec- 
tion. On the other hand, the WNV topotype strain Eg- 101 
caused fatal illness in intracranially injected mice, killing 
them within 4 to 6 days after infection, regardless of their 
age (11,13). After intraperitoneal injection, strain Eg- 101 
killed all suckling mice but a <10% of adult mice; RabV 
strains 97-103 and 99-222 killed approximately one third of 
suckling mice, and the average survival time was 11 days 
(range 10-14 days). Thus, both Rabensburg virus strains 
exhibit clearly lower virulence for mice than the Egyptian 
WNV topotype strain. In addition, average survival time of 
suckling ICR mice injected intracranially with RabV was 
significantly longer than with strain Eg- 101. 

The genome of strain 97-103 Rabensburg virus (RabV) 
was investigated by RT-PCR and subsequent direct 
sequencing of the amplicons. Initially, oligonucleotide 
primers designed on the consensus sequences of linage 1 
and 2 WNV strains were applied to the viral nucleic acid 
of RabV. On the basis of the sequence information 
obtained from these PCR products, specific primer pairs 
were designed to produce overlapping amplicons covering 


Table 1 . Survival time (days) of suckling mice injected intracranially with Rabensburg virus isolates 97-103 and 99-222 

Suckling mouse brain 

Strain 97-103 


Strain 99-222 


(SMB) passage no. 

Average survival time 

Range 

Average survival time 

Range 

smb 0 * 

12.1 

12-13 

12.2 

9-15 

SMB 1 

8.5 

7-10 

11.8 

11-13 

smb 2 

8.5 

7-11 

10.0 

9-11 

smb 3 

8.1 

7-9 

8.7 

7-10 


*Represents the original mosquito suspension during virus isolation attempts. 


226 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


the entire genome. The RT-PCR products were sequenced, 
and the sequences were compiled, resulting in a 10,972 - 
nucleotide (nt-) sequence that represented the complete 
genome of the virus. The sequence was identified by 
BLAST search against GenBank databases. The highest 
identity rates of RabV to other flavi viruses (78%-90%) 
were found with certain regions of WNV strains of lineage 
1 and 2. 

From the second isolate (99-222), 5 genomic regions 
have been amplified and sequenced so far, showing a total 
of 3656 nt. They represent partial coding sections from the 
core (C), anchored C, premembrane (PreM), and mem- 
brane (M) protein regions (between nucleotide positions 
117 and 752); NS 3 protein region (between nucleotide 
positions 5294 and 5536, and between nucleotide positions 
5565 and 6343); NS4b and NS5 regions (between 
nucleotide positions 7321 and 8112); and NS5 protein 
region (between nucleotide positions 9095 and 10305). 
Partial sequence analysis of isolate 99-222 showed >99% 
identity to 97-103. Aligned to strain 97-103, only a few 
nucleotide substitutions were observed, in the following 
positions: C 609 to T; C 720 to A; G 5727 to A (resulting in an 
amino acid change Met to lie); T 5910 to C (resulting in an 


Novel Flavivirus or New Lineage of West Nile Virus 

amino acid change lie to Thr); T 5961 to C; C 9630 to A; and 
^9843 t° T. 

Similar to other flavi viruses (17), the nucleotide 
sequence of RabV contains 1 open reading frame (ORF) 
encoding the viral proteins as a large polyprotein precur- 
sor. The ORF starts at nucleotide position 97, and codes for 
a 3,433-amino acid (aa) polypeptide. The putative amino 
acid sequence of the polyprotein precursor gene of RabV 
97-103 has been translated; based on the amino acid align- 
ment with WNV, the putative mature proteins, conserved 
structural elements, and putative enzyme motifs were 
localized. The anchored C protein is located between nt 97 
and 465; within this region, the C protein is located 
between nt 97 and 411. The PreM protein is encoded from 
nt 466 to nt 966, with the M protein between nt 742 and 
966. The envelope (E) protein is encoded between 
nucleotide positions 967 and 2469, followed by the non- 
structural proteins NS1 (nt 2470-3525), NS2a (nt 
3526-4218), NS2b (nt 421SM611), NS3 (nt 4612-6468), 
NS4a (nt 6469-6846), 2K (nt 6847-6915), NS4b (nt 
6916-7680), and NS5 (nt 7681-10395), respectively. 
Amino acid identities with WNV were found at the known 
conserved positions (i.e., Cys residues involved in 


Table 2. Sequences of West Nile virus (WNV) strains and other members of the Japanese encephalitis virus group used for 
phylogenetic analyses 

Virus name 

Code 

Accession 

no.* 

Year 

Isolation 

Host 

Geographic origin 

WNV lineage, 
clade 

WNV HNY1999 

NY99a 

AF202541 

1999 

Human 

New York 

la 

WNV NY99flamingo38299 

NY99b 

AF1 96835 

1999 

Flamingo 

New York 

la 

WNV IS98STD 

Is98 

AF481864 

1998 

Stork 

Israel 

la 

WNV Italyl 998Equine 

It98 

AF404757 

1998 

Horse 

Italy 

la 

WNV RO9750 

Ro96 

AF260969 

1996 

Culex pipiens 

Romania 

la 

WNV VLG4 

Rus99a 

AF317203 

1999 

Human 

Volgograd 

la 

WNV LEIV-Vlg99-27889 

Rus99b 

AY277252 

1999 

Human 

Volgograd 

la 

WNV PaHOOl 

Tu97 

AY268133 

1997 

Human 

Tunisia 

la 

WNV PaAnOOl 

FrOO 

AY268132 

2000 

Horse 

France 

la 

WNV EglOl 

Eg51 

AF260968 

1951 

Human 

Egypt 

la 

WNV Chin-01 

ChinOI 

AY490240 

Unknown 

Unknown! 

China 

la 

WNV Kunjin MRM61C 
WNV Sarafend 

Kunjin 

Sarafend 

D00246 

AY688948 

1960 

Cx. armulirostris 
Laboratory strain 

Australia 

1b 

2 

WNV B956 (WNFCG) 

Ug37 

Ml 2294 

1937 

Human 

Uganda 

2 

WNV LEIV-Krnd88-1 90 

Rus98 

AY277251 

1998 

Dermacentor 

marginatus 

Caucasus 

4t 

Rabensburg virus (97-103) 

RabV 

AY765264 

1997 

Cx. pipiens 

Czech Republic 

3t 

Japanese encephalitis virus 

JEV 

NC_001 437 

- 

- 

- 

- 

Murray Valley 
encephalitis virus 

MVEV 

NC_000943 

— 

— 

— 

— 

Usutu virus 

USUV 

AY45341 1 

- 

- 

- 

- 

Saint Louis 
encephalitis virus 

SLEV 

AF013416 

- 

- 

- 

- 

Alfuy virus 

ALFV 

AF0 13360 

- 

- 

- 

- 

Cacipacore virus 

CPCV 

AF013367 

- 

- 

- 

- 

Koutango virus 

KOUV 

AF0 13384 

- 

- 

- 

- 

Yaounde virus 

YAOV 

AF013413 

- 

- 

- 

- 


*Partial nucleotide sequences (NS5 protein region) are indicated in italics. 
fUnknown, tentative speciation. 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


227 


RESEARCH 


intramolecular bonds in the E and NS1 protein, putative 
integrin binding motif of the E protein, catalytic triad and 
substrate binding pocket of the trypsin-like serine protease, 
RNAhelicase motif of the NS3 protein, and RNA-depend- 
ent RNA polymerase motif of the NS 5 protein; 15). 

To investigate the phylogenetic relationship of RabV to 
other WNV isolates, multiple nucleotide and putative 
amino acid sequence alignments were made involving 16 
WNV strains (listed in Table 2). Although several 
complete WNV nucleotide sequences from previously 
published studies (10,18) have been deposited in the 
GenBank databases, only selected representatives of line- 
ages and clades have been included in our alignments, in 
order to obtain more precise and demonstrative trees. 

RabV exhibited 73%-77% nucleotide identity rates to 
the different WNV strains (Table 3). The relationships 
between the strains are demonstrated in Figure 1. The 2 
lineages of WNV are obviously separated in the tree. Clade 
la viruses form a tight cluster with close genetic relation- 
ship among the members. Kunjin virus, the representative 
of clade lb, appears as a separate branch of lineage 1. 
Unfortunately, no complete genome sequence information 
is available on clade lc (Indian strains); thus, they are not 
represented in the tree. The prototype Uganda strain B956 
(WNFCG) of lineage 2 is grouped together with the 


Sarafend strain, a laboratory strain with uncertain origin 
and passage history. Two viruses proved to be clearly dis- 
tinct with significant genetic distances to all other WNV 
strains and also from each other: RabV and strain LEIV- 
Kmd88-190 (in the phylogenetic trees designated Rus98). 
The latter virus was isolated from Dermacentor margina- 
tus ticks in the northwest Caucasus Mountain valley in 
1998 and was regarded as a new variant of WNV (19-21). 
Because these 2 viruses differ considerably from all WNV 
strains, the issue is raised about whether classifying these 
2 viruses as separate members of the JEV group might be 
more appropriate. 

To elucidate this question, a comprehensive phyloge- 
netic analysis was performed on all representatives of the 
JEV group. Because only partial common sequence infor- 
mation of the NS 5 protein gene region is currently avail- 
able from SLEV, ALV, CPCV, KOUV, and YAOUV (22), 
the phylogenetic analysis had to be restricted to this 
region (Figure 2). Within the investigated genome stretch, 
RabV showed 77%-78% identity to lineage 1 and 2 WNV 
strains, 77% identity to strain LEIV-Krnd88-190, and 
71%-76% identity to other representatives of the JEV 
group. In the phylogenetic tree (Figure 2), the separation 
of the 2 unique strains (RabV and LEIV-Krnd88-190 = 
Rus98) from WNV is clearly visible. Although RabV 


Table 3. 

Nucleotide and amino acid identity rates between RabV* 

and other flaviviruses 



Code 



Identity to RabV (%) 



Nucleotide 

Amino acid 

WNV lineage and clade 

Complete 

Partial! 

Complete 

Partial! 

NY99a 

la 

77 

78 

90 

95 

NY99b 

la 

77 

78 

90 

95 

Is98 

la 

77 

78 

90 

95 

It98 

la 

77 

78 

90 

95 

Ro96 

la 

77 

78 

90 

95 

Rus99a 

la 

77 

78 

90 

95 

Rus99b 

la 

77 

78 

90 

95 

Tu97 

la 

76 

78 

90 

95 

FrOO 

la 

77 

78 

90 

95 

Eg51 

la 

77 

78 

90 

95 

ChinOI 

la 

77 

78 

90 

95 

Kunjin 

1b 

75 

77 

89 

94 

Sarafend 2 

77 

78 

90 

96 

Ug37 

2 

77 

78 

90 

96 

Rus98 

4 (speculation) 

73 

77 

87 

95 

JEV 

- 

68 

74 

75 

86 

MVEV 

- 

69 

74 

76 

86 

USUV 

- 

68 

72 

75 

83 

SLEV 

- 

- 

71 

- 

78 

ALFV 

- 

- 

74 

- 

88 

CPCV 

- 

- 

71 

- 

79 

KOUV 

- 

- 

76 

- 

90 

YAOV 

- 

- 

75 

- 

87 


*RabV, Rabensburg virus; JEV, Japanese encephalitis virus; MVEV, Murray Valley encephalitis virus; USUV, Usutu virus; SLEV, St. Louis encephalitis 


virus; ALFV, Alfuy virus; CPCV, Cacipacore virus; KOUV, Koutango virus; YAOV, Yaounde virus. 
tPartial alignment between nucleotide positions 9067 and 10101. 

^Partial alignment between amino acid positions 2991 and 3335. 


228 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


Novel Flavivirus or New Lineage of West Nile Virus 




Figure 1. Phylogenetic tree illustrating the genetic relationship 
between selected West Nile virus strains based on their complete 
genome sequences. Bar on the left demonstrates the genetic dis- 
tance. (Abbreviations and accession numbers are listed in Table 2.) 


exhibits the closest relationship to the WNV representa- 
tives, similar identity rates (76%) exist between MVEV 
and USUV, as well as between JEV and ALFV, and these 
viruses have been taxonomically classified as separate 
viruses. The Rus98 virus clusters together with KOUV, a 
virus isolated originally from a Kemp’s gerbil ( Tatera 
kempi) in Senegal 1968 and subsequently recovered from 
other rodent species and several genera of ticks 
( Rhipicephalus , Hyalomma , Alectorobius ) in central 
Africa (23). The Rus98 strain was also isolated from 
ticks. 

The putative amino acid sequence of RabV was also 
compared with the corresponding sequences of representa- 
tives of WNV lineages and clades, as well as with other 
JEV group viruses on the available polypeptide sequence 


regions. RabV shared 89%-90% identity on the complete 
polypeptide precursor region with the WNV strains, 87% 
identity with the Rus89 strain, and 75%-76% identity with 
JEV, USUV, and MVEV. The alignments of the partial 
amino acid sequences of the NS5 region (between aa 2991 
and 3335) showed 94%-96% identity rates with the WNV 
strains, 95% with strain Rus98, and 78%-90% with the 
other members of the JEV group (Table 3). Phylogenetic 
trees, based on the amino acid alignments, displayed near- 
ly identical topology to nucleotide sequence-based trees 
(data not shown). The complete genome sequence of RabV 
(flavivirus strain 97-103) has been deposited in GenBank 
under accession no. AY765264. 

Discussion 

WNV strains of different lineages exhibit considerable 
genomic diversity (76%-77% nucleotide identity only). At 
the same time, WNV is not sharply delimited genomically 
from the other members of the JEV group. The available 
partial sequences of the NS 5 gene region from other virus- 
es of the group show 71%-76% nucleotide and 78%-90% 
amino acid identities to WNV strains. The closest relatives 
of WNV are KOUV and YAOV (10,22-24). 

Lineage 1 of WNV comprises strains from several con- 
tinents and is subdivided into at least 3 clades. In clade la, 
several subclades or clusters are formed by closely related 
strains, such as strains isolated 40-50 years ago in Europe 
and Africa; strains isolated 20-30 years ago in Africa; 
strains isolated within the last 10 years in Europe and 
Africa; and strains isolated within the last 5 years in the 
United States and Israel. Clade lb consists of the 
Australian isolates (Kunjin), while clade lc contains 
strains from India. Lineage 2 is composed of WNV strains 
that have been isolated, so far exclusively, in the sub- 
Saharan region of Africa and in Madagascar (18). The 
genetic distance between the 2 lineages is relatively great 
in contrast to that within some representatives of lineage 1 
that were isolated in distant geographic locations and with- 
in considerable time intervals. While the viruses in clade 
la share 95.2%-99.9% nucleotide and 99.3%-100% 
amino acid identity to each other, and also 86.6%-87.8% 
nucleotide and 97.4%-97.7% amino acid identity to the 
clade lb viruses, the overall identity rates between lineage 
1 and 2 are only 75.7%-76.8% on nucleotide level and 
93.2%-94.0% on amino acid level (18), identity rates that 
resemble those between RabV and either lineage 1 or line- 
age 2 WNV strains. Besides genomic differences, anti- 
genic variability can be observed in cross-neutralization 
analyses and monoclonal antibody binding assays (8,18). 

The results of the phylogenetic analyses indicate that 
viruses closely related to WNV are present in central 
Europe and southern Russia. Although these viruses have 
initially been identified as WNV, they can be regarded, on 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


229 


RESEARCH 




Figure 2. Phylogenetic tree illustrating the genetic relationship 
between representatives of the Japanese encephalitis virus com- 
plex and selected West Nile virus strains based on partial genome 
sequences of the NS5 protein gene. Bar on the left demonstrates 
the genetic distance. (Abbreviations and accession numbers are 
listed in Table 2.) 


the basis of their genetic distances, either as separate line- 
ages of WNV (RabV: lineage 3; LEIV-Krnd88-190 = 
Rus98: lineage 4) or as new viruses within the JEV group. 
The antigenic and biologic differences between RabV and 
the WNV reference strain Eg- 101 also support this opin- 
ion. Isolation of RabV in 1997 was obviously not an iso- 
lated event; rather, flaviviruses of the RabV type seem to 
be present or persist in this area, as demonstrated by the 
isolation of an almost identical virus strain (99-222) 2 
years later (14). The ecology of RabV needs further inves- 
tigation. Other unanswered questions concern the patho- 
genicity and host spectrum of the virus, especially 
regarding possible human infections. 

To summarize, a novel flavivirus strain of unknown 
human pathogenicity, repeatedly isolated from Cx. pipiens 
mosquitoes in central Europe, has been molecularly char- 
acterized, including determination of its complete 


nucleotide and deduced amino acid sequences. Based on 
the analysis of the virus and comparison with related virus- 
es including phylogenetic relationships, we suggest that 
RabV be classified either as a new (third) lineage of WNV 
or as a novel flavivirus within the JEV group. 


This study was funded by a grant of the Austrian Federal 
Ministry for Health and Women’s Issues, and it was also sup- 
ported by the Czech Science Foundation (206/03/0726). 

Dr. Bakonyi is a lecturer in virology at the Faculty of 
Veterinary Science, Budapest, and also works as a guest 
researcher at the University of Veterinary Medicine, Vienna. He 
is interested in the molecular diagnosis and epidemiology of ani- 
mal and human viruses. 

References 

1. Heinz FX, Collett MS, Purcell RH, Gould EA, Howard CR, 
Houghton M, et al. Family Flaviviridae. In: van Regenmortel MHV, 
Faquet CM, Bishop DHL, editors. Virus taxonomy, Seventh 
International Committee for the Taxonomy of Viruses. San Diego: 
Academic Press; 2000. p. 859-78. 

2. Hayes CG. West Nile fever. In: Monath TP, editor. The arboviruses: 
epidemiology and ecology. Vol. V. Boca Raton (FL): CRC Press; 
1989. p. 59-88. 

3. Hubalek Z, Halouzka J. West Nile fever — a reemerging mosquito- 
borne viral disease in Europe. Emerg Infect Dis. 1999;5:643-50. 

4. Hubalek Z. European experience with the West Nile virus ecology 
and epidemiology: could it be relevant for the New World? Viral 
Immunol. 2000;13:415-26. 

5. Murgue B, Zeller H, Deubel V. The ecology and epidemiology of 
West Nile virus in Africa, Europe. In: Mackenzie JS, Barrett ADT, 
Deubel V, editors. Japanese encephalitis and West Nile viruses. 
Current topics in Microbiology. Vol. 267: West Nile. Berlin: Springer; 
2002. p. 195-221. 

6. Zeller HG, Schuffnecker I. West Nile virus: an overview of its spread 
in Europe and the Mediterranean basin in contrast to its spread in the 
Americas. Eur J Clin Microbiol Infect Dis. 2004;23:147-56. 

7. Berthet F, Zeller HG, Drouet M, Rauzier J, Digoutte J, Deubel V. 
Extensive nucleotide changes and deletions within the envelope gene 
of Euro-African West Nile viruses. J Gen Virol. 1997;78:2293-7. 

8. Savage HM, Ceianu C, Nicolescu G, Karabatsos N, Lanciotti RS, 
Vladimirescu A, et al. Entomologic and avian investigations of an 
epidemic of West Nile fever in Romania, 1996, with serologic and 
molecular characterization of a virus from mosquitoes. Am J Trop 
MedHyg. 1999;61:600-11. 

9. Lanciotti RS, Roehrig JT, Deubel V, Smith J, Parker M, Steele K, et 
al. Origin of the West Nile virus responsible for an outbreak of 
encephalitis in the northeastern U.S. Science. 1999;286:2333-7. 

10. Charrel RN, Brault AC, Gallian P, Lemasson JJ, Murgue B, Murri S, 
et al. Evolutionary relationship between Old World West Nile virus 
strains evidence for viral gene flow between Africa, the Middle East, 
and Europe. Virology. 2003;315:381-8. 

11. Hubalek Z, Halouzka J, Juricova Z. West Nile fever in Czechland. 
Emerg Infect Dis. 1999;5:594-5. 

12. Ferenzi E, Bakonyi T, Toth-Mittler E, Czegledi A, Ban E. Emergence 
of an old-new virus: domestic West Nile virus infections with central 
nervous system symptoms, 2003-2004. In: Abstracts of the 2004 year 
Congress of the Hungarian Society for Microbiology; 2004. Oct. 7-9, 
Keszthely, Hungary. Abstract p. 36-7 [in Hungarian]. Budapest: 
Hungarian Soceity for Microbiology; 2004. 


230 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


Novel Flavivirus or New Lineage of West Nile Virus 


13. Hubalek Z, Halouzka J, Juricova Z, Sebesta O. First isolation of mos- 
quito-borne West Nile vims in the Czech Republic. Acta Virol. 
1998;42:119-20. 

14. Hubalek Z, Savage HM, Halouzka J, Juricova Z, Sanogo YO, Lusk S. 
West Nile virus investigations in South Moravia, Czechland. Viral 
Immunol. 2000;13:427-33. 

15. Bakonyi T, Gould EA, Kolodziejek J, Weissenbock H, Nowotny N. 
Complete genome analysis and molecular characterization of Usutu 
virus that emerged in Austria in 2001; comparing with the South 
African strain SAAR- 1776 and other flaviviruses. Virology. 
2004;328:301-10. 

16. Melnick JL, Paul JR, Riordan JT, Barnett VH, Goldblum N, Zabin E. 
Isolation from human sera in Egypt of a virus apparently identical to 
West Nile vims. Proc Soc Exp Biol Med. 1951;77:661-5. 

17. Rice CM, Lenches EM, Eddy SR, Shin SJ, Sheets RL, Strauss JH. 
Nucleotide sequence of yellow fever virus: Implications for flavivirus 
gene expression and evolution. Science. 1985;229:726-33. 

18. Lanciotti RS, Ebel GD, Deubel V, Kerst AJ, Murri S, Meyer R, et al. 
Complete genome sequences and phylogenetic analysis of West Nile 
virus strains isolated from the United States, Europe, and the Middle 
East. Virology. 2002;298:96-105. 

19. Prilipov AG, Kinney RM, Samokhvalov El, Savage HM, Al’khovskii 
SV, Tsuchiya KR, et al. Analysis of new variants of West Nile fever 
virus [in Russian], Vopr Virusol. 2002;47:36-41. 


20. Lvov DK, Kovtunov Al, Iashkulov KB, Gromashevskii VL, 
Dzharkenov AF, Shchelkanov MI, et al. Circulation of West Nile 
virus (. Flaviviridae , Flavivirus ) and some other arbovimses in the 
ecosystems of Volga delta, Volga- Akhtuba flood-lands and adjoining 
arid regions (2000-2002) [in Russian]. Vopr Virusol. 2004;49:45-51. 

21. Lvov DK, Butenko AM, Gromashevsky VL, Kovtunov Al, Prilipov 
AG, Kinney R, et al. West Nile virus and other zoonotic viruses in 
Russia: examples of emerging-reemerging situations. Arch Virol 
Suppl. 2004;18:85-96. 

22. Kuno G, Chang GJ, Tsuchiya KR, Karabatsos N, Cropp CB. 
Phylogeny of the genus Flavivirus. J Virol. 1998;72:73-83. 

23. Burke DS, Monath TP. Flavivimses. In: Knipe DM, Howley PM, 
Griffin DE, Lamb RA, Martin MA, Roizman B, et al. editors. Fields 
virology, vol. 1. Philadelphia: Lippincott Williams & Wilkins; 2001. 
p. 1055-109. 

24. Gaunt MW, Sail AA, de Lamballerie X, Falconar AK, Dzhivanian TI, 
Gould EA. Phylogenetic relationships of flavivimses correlate with 
their epidemiology, disease association and biogeography. J Gen 
Virol. 2001;82:1867-76. 


Address for correspondence: Norbert Nowotny, Zoonoses and Emerging 
Infections Group, Clinical Virology, Clinical Department of Diagnostic 
Imaging, Infectious Diseases and Clinical Pathology, University of 
Veterinary Medicine, Vienna, A- 12 10 Vienna, Austria; fax: 43 1 
250772790; email: Norbert.Nowotny@vu-wien.ac.at 


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RESEARCH 


Survey for Bat Lyssaviruses, 

Thailand 

Boonlert Lumlertdacha,* Kalyanee Boongird,t Sawai Wanghongsa,t Supaporn Wacharapluesadee4 
Lawan Chanhome,* Pkamatz Khawplod,* Thiravat Hemachudha4 Ivan Kuzmin, § 

and Charles E. Rupprecht§ 


Surveillance for lyssaviruses was conducted among 
bat populations in 8 provinces in Thailand. In 2002 and 
2003, a total of 932 bats of 11 species were captured and 
released after serum collection. Lyssavirus infection was 
determined by conducting virus neutralization assays on 
bat serum samples. Of collected samples, 538 were either 
hemolysed or insufficient in volume, which left 394 suitable 
for analysis. These samples included the following: 
Pteropus lylei (n = 335), Eonycteris spelaea (n = 45), 
Hipposideros armiger (n = 13), and Rousettus leschen- 
naulti (n = 1). No serum samples had evidence of neutral- 
izing antibodies when tested against rabies virus. However, 
16 samples had detectable neutralizing antibodies against 
Aravan virus, Khujand virus, Irkut virus, or Australian bat 
lyssavirus; all were specifically associated with fruit bats P. 
lylei (n = 15) and E. spelaea (n = 1). These results are con- 
sistent with the presence of naturally occurring viruses 
related to new putative lyssavirus genotypes. 

R abies is an acute encephalitis caused by a lyssavirus. 

On a global basis, bats have been associated with sev- 
eral different genotypes of lyssavirus (1-5). Two human 
infections with Australian bat lyssavirus (ABLV) have 
been reported, the clinical signs of which were consistent 
with classical rabies infection, namely a diffuse, nonsup- 
purative encephalitis (3). A serosurvey for agents similar to 
ABLV among bats in the Philippines detected a prevalence 
of 9.5% (22/231) (6). Six of 14 species (fruit- and insect- 
eating bats) were seropositive for reactivity against ABLV. 
These included Taphozous melanopogan (4/30), 
Mineopterus schreibersi (4/11), Philetor brachypterus 
(1/13), Scotophilus kuhlii (4/63), Pteropus hypomelanus 
(3/14), and Rousettus amplexicaudatus (6/50) (6). 


*Thai Red Cross Society, Bangkok, Thailand; fMinistry of 
Agriculture, Bangkok, Thailand; 4Chulalongkorn University, 
Bangkok, Thailand; and §Centers for Disease Control and 
Prevention, Atlanta, Georgia, USA 


However, Asian bat lyssaviruses (1,2,4) were unavailable 
at that time to check for cross-reactivity. 

Canine rabies is enzootic in Thailand. No bat- associated 
rabies or lyssavirus deaths in have been reported in humans 
or other animals (7).This lack of data for other agents, how- 
ever, does not exclude their existence (1). Rabies statistics 
in humans and animals are underreported (8). Moreover, 
without a history of dog bite, rabies may be dismissed, or 
clinical manifestations of bat-related cases may be variable 
(8). In the context of bat lyssavirus as an emerging global 
infectious disease, baseline data are necessary to allow for 
future public health assessment of its impact. This active 
surveillance sought to determine whether bats in Thailand 
had evidence of lyssavirus infections. 

Methods 

Collection of Specimens 

From March 2002 through August 2003, bats were col- 
lected from 8 provinces throughout central, eastern, and 
southern Thailand (Figure 1). Sites were chosen on the 
basis of local reports of known bat colonies or after inves- 
tigation by the Royal Department of Forestry, Ministry of 
Agriculture. Insectivorous bats in caves were captured dur- 
ing the day by using fine-mesh, long-handled butterfly 
nets. Larger fruit bats were captured with nets near sunset, 
as the bats flew for foraging activities, or before dawn 
when returning to their roosts (Figure 2). Thick leather 
gloves were worn when bats were handled and transferred 
into individual cotton pouches for transportation and pro- 
cessing. 

Of the 932 bats collected, all were identified to 11 dif- 
ferent species of both insectivorous and frugivorous bats 
(Table 1). Forty percent were female. All bats appeared 
healthy. At least 110 bat species (>20 million) are believed 
to be present in Thailand, according to estimates from a 


232 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


Survey for Bat Lyssaviruses, Thailand 



Figure 1. Map of Thailand showing bat collection sites from 8 
provinces and locations of bats found seropositive. 1 = Chonburi, 
2 = Singburi, 3 = Ayuttaya, 4 = Chachongsao, 5 = Rayong, 6 = 
Prachinburi, 7 = Ratchaburi, 8 = Suratthani. 


Royal Department of Forestry survey in 2003. Eighty five 
percent are insectivorous; the rest are frugivorous. 

Bats were anesthetized by administering a 0.2- to 0.5- 
mg intramuscular injection of ketamine hydrochloride. 
Animals were identified to sex and by species, based on 
gross morphology, as described (9). Animals were marked 
by hair or claw clipping. Blood, obtained from wing veins 
or by direct cardiac puncture, was transferred from the col- 
lecting syringe into 1.5-mL microtubes (Axygen 
Scientific, Union City, CA, USA) and stored in an icebox 
until centrifugation. Serum was frozen at -20°C during 
transportation and stored in a freezer at -70°C. After 
recovery from sedation, bats were allowed to fly to their 
roosts. Sixteen of 932 died during the capture process. 
Inspection of the capture sites 1-2 months later included 
an assessment of whether the local ecology was disturbed. 
No additional bats died after the procedure, according to 
residents living near roosts. 


Serologic Testing for Neutralizing Antibodies 

Serum specimens were obtained from blood samples 
after clotting. In general, 394 samples from 4 different 
species were of sufficient volume and quality (Table 2). 
The samples originated from Chonburi (n = 167), Ayuttaya 
(n = 105), Chachoengsao (n = 36), Singburi (n = 81), and 
Surattani (n = 5). For adequate volume during testing, they 
were diluted 1:5 in Eagle’s minimum essential medium 
(Invitrogen, Carlsbad, CA, USA) supplemented with 2% 
fetal bovine serum (Invitrogen). Serum samples were heat- 
inactivated for 30 min at 56°C before testing. 

Initially, all 394 samples were screened in a modified 
rapid fluorescent focus inhibition test (6) against rabies 
virus (RABV, strain CVS- 11) and ABLV (pteropid sub- 
type; 40 50% tissue culture infective dose), with World 
Health Organization standard serum as a source for posi- 
tive control antibody with 50% endpoint dilution of 1 IU = 
1:20. Approximately 50 qL of diluted serum at 1:5, 1:10, 
and 1:20 dilutions was incubated with 50 |lL of ABLV in 
96- well microtiter plates for 90 min at 37 °C in a C0 2 incu- 
bator. Murine neuroblastoma cells (50 qL) were added to 
each serum-virus mixture, which was incubated for 20 h. 
Culture medium was removed after incubation, and the 
plates were fixed with 90% acetone, air-dried, and then 
stained with fluorescein isothiocyanate-conjugated anti- 
rabies monoclonal antibodies (Fujirebio Diagnostic, Inc, 
Malvern, PA, USA). Samples were considered positive if 
the number of fluorescent foci was reduced by 50% at the 
1:5 dilution. 

Those samples that demonstrated positive or suspicious 
activity were additionally tested against a broader panel of 
other lyssaviruses, including Aravan, Khujand, and Irkut 
virus isolates. Twofold serum dilutions, from 1:25 to 1:100, 
were tested, and virus doses varied from 32 to 100 infec- 
tious units. These reactions were performed by using drops 
of cell culture medium on 4-well (6-mm) Teflon-coated 
glass slides (Cell-line/Erie Scientific Co., Portsmouth, NH, 
USA), incubated in a moist chamber for 48 h. 

Direct Fluorescent Antibody (DFA) 

and Mouse Inoculation (Ml) Testing of Brains 

Brains from 16 dead bats (2 P. lylei and 14 P. hypome- 
lanus) were collected in iceboxes at the capture sites for 
transportation and then were stored at -70°C until testing. 
Each brain was tested for lyssavirus antigen by DFA. 
Multiple impressions were prepared, and slides were fixed 
in acetone, allowed to dry at room temperature, and stained 
with commercial fluorescein isothiocyanate-conjugated 
anti-rabies monoclonal antibodies (Fujirebio Diagnostic, 
Inc). These brain impressions were examined with a fluo- 
rescent microscope. 

For MI testing, pooled 20% brain suspensions from all 
16 bats were prepared by mixing ~0.5 g of each bat brain 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


233 


RESEARCH 



Figure 2. Thai Flying foxes (Pteropus lylei) at their roost. 


in 32 mL of normal saline solution. No antimicrobial 
preparations were added. The mixture was left to sediment 
at room temperature for 30 min, and the supernatant was 
used to inject into the brains of 1 -month-old Swiss albino 
strain mice. Approximately 0.03 mL of each suspension 
was injected into each of 30 mouse brains. They were kept 
in 6 glass jars (5 in each) with a diameter of 15 cm and 
were observed for 60 days. 

Results 

Serologic Testing 

All 394 serum samples were negative against RABV, 
but 16 (4%) were positive or suggestive of ABLV (Table 2). 
Further tests of these samples demonstrated neutralizing 
activity against Aravan, Khujand, or Irkut viruses or ABLV 


(Table 3). These 16 samples originated from 2 species, P. 
lylei (n = 15) and Eonycteris spelaea (n = 1), collected at 
Chonburi (n = 9), Singburi (n = 4), Ayuttaya (n = 2), and 
Chachoengsao (n =1) Provinces (Table 2). 

Chonburi is adjacent to Chachoengsao Province in the 
east, whereas Singburi and Ayuttaya are both located in the 
central part of the country (Figure 1). Approximately 5% 
of positive bat serum specimens were found in 2 eastern 
provinces (Chonburi, 9/158 and Chachoengsao, 1/36) ver- 
sus 3% in 2 central provinces (Singburi, 4/81, and 
Ayuttaya, 2/105). Antibody-positive bats were dispersed 
throughout the collection period (March 2002 through 
August 2003). Most (15 of 16) positive samples came from 
P. lylei. One of 45 E. spelaea (versus 15 of 335 P. lylei) 
tested positive. 

DFA and Ml Testing 

Sixteen bat brains tested by DFA had no detectable 
lyssavirus antigen. After intracerebral injection, 4 of 30 
mice died, on days 11, 12, 14, and 21, respectively. None 
of these 4 brains tested positive with DFA for evidence of 
lyssavirus antigens. 

Discussion 

This study presents evidence of neutralization of 
lyssaviruses other than RABV and ABLV by sera from 
Thai bats. These findings are consistent with the presence 
of naturally induced antibodies against >1 lyssavirus geno- 
type in the Thai bat populations studied. 

Lyssaviruses are classified into groups on the basis of 
their genetic, antigenic, and relative pathogenic attributes. 
At least 7 putative genotypes and 2 major phylogroups are 
recognized on the basis of their overall phylogenetic 


Table 1 . Bat species captured in Thailand 



Province 





Species 

Chonburi Rayong Ayuttaya 

Chachoengsao Singburi 

Prachinburi 

Ratchaburi 

Suratthani 

Total 

Hipposideros 

lavatus 

46 




40 

86 

H. armiger 




103 


103 

Eonycteris 

spelaea 

28 

36 




64 

Rousettus 

1 

5 



5 

11 

leschennault 







Pteropus lylei 

150 242 

110 58 

28 



588 

P. hypomelanus 

16 3 





19 

P. vampyrus 





23 

23 

Emballonura 





14 

14 

monticola 







Scotophillus 
heath I 

3 





3 

Mega derm a 




13 


13 

spasma 







Cynopterus 

sphinx 




8 


8 

Total 

241 3 245 

110 99 

28 

124 

82 

932 


234 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


Survey for Bat Lyssaviruses, Thailand 


Table 2. Bat sera screened and positive for neutralizing antibodies (positive/screened) 


Species 



Site 



Total 

Chonburi 

Singburi 

Ayuttaya 

Chachoengsao 

Suratthani 

Hipposideros armiger 

1/8 




0/5 

1/13 

Eonycteris spelaea 

0/22 

0/23 




0/45 

Rousettus leschermault 

0/1 





0/1 

Pteropus lylei 

8/1 36 

4/58 

2/1 05 

1/36 


15/335 

Total 

9/1 67 

4/81 

2/1 05 

1/36 

0/5 

16/394 


relatedness (1). Phylogroup I includes RABV (genotype 
1), Duvenhage vims (DUVV) (genotype 4), European bat 
lyssavirus (EBLV) 1 (genotype 5), EBLV-2 (genotype 6), 
and ABLV (genotype 7). Phylogroup II includes Lagos bat 
vims (LBV) (genotype 2) and Mokola virus (MOKV) 
(genotype 3) (10). In this study, neutralization titers to new 
putative genotypes, namely, Irkut, Khujand, and Aravan 
vimses, and of much lesser degree to ABLV but not to 
RABV, were evident. Khujand virus is related to genotype 
6, while Aravan virus is related to Khujand virus, with 
moderate similarity to genotypes 4, 5, and 6 (2,4). ABLV 
is more closely related to RABV (3). When a comparative 
phylogenetic analysis was performed, Irkut virus was rec- 
ognized as a member of a cluster joining lyssavirus geno- 
types 4 and 5 (76% bootstrap support) (1). 

This preliminary study demonstrates that which vims is 
used for a serologic test is critical. All Thai samples were 
negative to RABV and most to ABLV, findings which help 
explain why lyssavirus infection has not previously been 
reported in Thai bats. A relatively low prevalence of 
lyssavirus infection in Thai bats in the current study (4% 
as compared to 9.5% in the Philippines survey [6]) may be 
explained by the fact that as many as 43 samples had a 1:5 
(some of them, both 1:5 and 1:10) dilution considered 
unreadable because of the effect of hemolysis. Moreover, 


another 13 samples with equivocal result were seropositive 
for ABLV after subsequent testing. Lurther testing of these 
additional 13 samples against Irkut, Khujand, and Aravan 
viruses was not possible because of insufficient volume. 
Therefore, the actual positive number might be 29 (7.3%) 
of 396. Nevertheless, without a Thai lyssavirus isolate, 
concluding to which virus these bats have been exposed is 
difficult. These data also suggest that several lyssaviruses 
are in circulation throughout Thailand as well as other 
Asian countries, such as in the Philippines, Central Asia, 
and portions of Russia (1,2, 4, 6). 

Lurther studies throughout the year should be expanded 
to other species of bats, as well as a focus upon bats such 
as P. lylei and in locations with the highest prevalence of 
neutralizing antibodies. Whether P. lylei is the single most 
important species is not known. Surveillance among sick 
and dying bats and collection of their brains would assist 
in identifying infecting viruses. 

Public health authorities need to be aware of the poten- 
tial for bats to transmit lyssaviruses, and to increase sur- 
veillance and public education. Attention should focus on 
the protective efficacy of commercially available vaccines 
and immune globulins against these novel nonrabies 
lyssaviruses after exposure, before fatal human infection 
occurs. 


Table 3. Neutralization of lyssaviruses by Thai bat sera*| 


Antibody titers against different viruses 


Serum ID 

Aravan 

Khujand 

Irkut 

ABLV 

CVS-1 1 

78B 

<10 

<10 

>200 

<10 

<10 

733 

<10 

<10 

>200 

<10 

<10 

688 

1:56 

1:25 

<10 

<10 

<10 

615 

1:13 

1:25 

<10 

<10 

<10 

120 

<10 

<10 

1:65 

<10 

<10 

0/69 

<10 

<10 

1:170 

<10 

<10 

96 

<10 

1:21 

1:33 

<10 

<10 

125 

1:20 

1:56 

1:13 

<10 

<10 

741 

1:12 

<10 

>200 

1:13 

<10 

731 

<10 

<10 

>200 

<10 

<10 

724 

1:35 

1:56 

1:29 

<10 

<10 

303 

1:35 

1:50 

1:50 

<10 

<10 

740 

1:35 

<10 

>200 

<10 

<10 

729 

1:40 

>200 

>200 

1:20 

<10 

461 

1:56 

<10 

1:16 

<10 

<10 

519 

1:35 

<10 

1:50 

<10 

<10 


*ABLV, Australian bat lyssavirus; CVS, challenge virus standard. 
fBoldface indicates statistical significance. 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


235 


RESEARCH 


Acknowledgments 

We thank our colleagues at the Thai Red Cross Society, 
Ministry of Agriculture, and Chulalongkorn University for their 
input and expertise, Denny Constantine and Richard Luce, and 
staff members in the Viral and Rickettsial Zoonoses Branch of the 
Centers for Disease Control and Prevention. Special thanks to the 
family of Joachim-Sutthiporn Bulian for the field information. 

This research was supported in part by a grant from 
Thailand Research Fund. 

Dr. Lumlertdacha is a staff member in the Rabies Diagnostic 
and Quarantine Unit, Queen Saovabha Memorial Institute, Thai 
Red Cross Society. His areas of interest are rabies epidemiology, 
zoonoses, including surveys of Nipah virus and lyssaviruses, and 
the diagnosis of rabies in animals. 

References 

1. Botvinkin AD, Poleschuk EM, Kuzmin IV, Borisova TI, Gazaryan 
SV, Yager P, et al. Novel lyssaviruses isolated from bats in Russia. 
Emerg Infect Dis. 2003;9:1623-5. 

2. Arai YT, Kuzmin IV, Kameoka Y, Botvinkin AD. New lyssavirus 
genotype from the Lesser Mouse-eared Bat ( Myotis blythi ), 
Kyrghyzstan. Emerg Infect Dis. 2003;9:333-7. 


3. Gould AR, Hyatt AD, Lunt R, Kattenbelt JA, Hengstberger S, 
Blacksell SD. Characterization of a novel lyssavirus isolated from 
pteropid bats in Australia. Virus Res. 1998;54:165-87. 

4. Kuzmin IV, Orciari LA, Arai YT, Smith JS, Hanlon CA, Kameoka Y, 
et al. Bat lyssaviruses (Aravan and Khujand) from Central Asia: phy- 
logenetic relationships according to N, P and G gene sequences. Virus 
Res. 203;97:65-79. 

5. Iwasaki T, Inoue S, Tanaka K, Sato Y, Morikawa S, Hayasaka D, et 
al. Characterization of Oita virus 296/1972 of Rhabdoviridae isolat- 
ed from a horseshoe bat bearing characteristics of both lyssavirus and 
vesiculovirus. Arch Virol. 2004;149:1139-54. 

6. Arguin PM, Murray-Lillibridge K, Miranda ME, Smith JS, Calaor 
AB, Rupprecht CE. Serologic evidence of lyssavirus infections 
among bats, the Philippines. Emerg Infect Dis. 2002;8:258-62. 

7. Smith P, Lawhaswasdi K, Vick W. Isolation of rabies virus from fruit 
bats in Thailand. Nature. 1967;216:384. 

8. Hemachudha T, Laothamatas J, Rupprecht CE. Human rabies: a dis- 
ease of complex neuropathogenetic mechanisms and diagnostic chal- 
lenges. Lancet Neurol. 2002;1:101-9. 

9. Lekagul B, McNeely J. Mammals of Thailand. Bangkok: Darnsutha 
Press; 1988. 

10. Badrane H, Bahloul C, Perrin P, Tordo N. Evidence of two lyssavirus 
phylogroups with distinct pathogenicity and immunogenicity. J Virol. 
2001;75:3268-76. 


Address for correspondence: Boonlert Lumlertdacha, Queen Saovabha 
Memorial Institute, Thai Red Cross Society, Rama 4 Rd, Bangkok 10330, 
Thailand; fax: 662-2540212; email: Qsmibld@yahoo.com 




EMERGING 
INFECTIOUS DISEASES 

A Peer-Reviewed Journal Tracking and Analyzing Disease Trends Vol.8, No.3, March 2002 


236 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


Spotted Fever Group and Typhus 
Group Rickettsioses in Humans, 

South Korea 

Yeon-Joo Choi,* 1 Won-Jong Jang, 1 * Jong-Hyun Kim,* Ji-Sun Ryu,* Seung-Hyun Lee,* Kyung-Hee 
Park,* Hyung-Suk Paik,t Young-Sang Koh4 Myung-Sik Choi,§ and Ik-Sang Kim§ 


The presence of the nucleic acid of the spotted fever 
group (SPG) and typhus group (TG) rickettsiae was inves- 
tigated in 200 serum specimens seropositive for SFG rick- 
ettsiae by multiplex-nested polymerase chain reaction with 
primers derived from the rickettsial outer membrane protein 
B gene. The DNAof SFG, TG, or both rickettsiae was ampli- 
fied in the 24 serum specimens, and sequence analysis 
showed Rickettsia conorii, R. japonica, and R. felis in the 
specimens. R. conorii and R. typhi were found in 7 serum 
specimens, which indicated the possibility of dual infection 
in these patients. These findings suggest that several kinds 
of rickettsial diseases, including boutonneuse fever, rick- 
ettsialpox, R. felis infection, and Japanese spotted fever, as 
well as scrub typhus and murine typhus, are occurring in 
Korea. 


H uman rickettsioses, known to occur in Korea, include 
mainly scrub typhus, murine typhus, and epidemic 
typhus. Scrub typhus, caused by Orientia tsutsugamushi , a 
major rickettsial disease in Korea, is transmitted through 
the bites of mite larvae. An earlier study by Choi and col- 
leagues reported that 34.3% of febrile hospital patients in 
autumn were seropositive for the disease (1). Rickettsia 
typhi , transmitted by the fleas of various rodents, causes 
murine typhus, which is a milder form of typhus than 
human typhus (2). The first patient with murine typhus in 
Korea was reported in 1959. Two cases of murine typhus 
confirmed by culture were reported since 1988 (3,4), and 
now >200 cases of murine typhus are presumed to occur 
annually in South Korea. Epidemic typhus is caused by R. 


*Konkuk University, Choongbuk, Republic of Korea; fPusan 
National University, Pusan, Republic of Korea; 4Cheju National 
University College of Medicine, Jeju, Republic of Korea; and 
§Seoul National University College of Medicine and Institute of 
Endemic Disease, Seoul, Republic of Korea 


prowazekii and is transmitted by the body louse (5). The 
disease is fatal in 10% to 30% of patients, depending on 
underlying diseases and the nutritional state of the host (2). 
The disease appeared after the end of the Korean War. 
Since 1951, however, no other cases have been reported in 
Korea (6). 

Spotted fever group (SFG) rickettsioses are associated 
with arthropods, such as ticks, mites, and fleas (2). SFG 
comprises several divergent lineages: the R. rickettsii 
group, R. japonica , R. montana , the R. massiliae group, R. 
Helvetica , R. felis, and the R. akari group (2). Recently, the 
nucleic acids of R. japonica and R. rickettsii were found in 
Haemaphysalis longicornis in Korea (7). A previous sero- 
epidemiologic study demonstrated that SFG rickettsioses 
were highly likely in Korea (8). No clinical human case of 
SFG rickettsioses, however, has been reported in Korea 
until now. 

In this study, to check whether SFG rickettsioses were 
present in humans, serum specimens from patients with 
acute febrile disease were studied by using molecular 
sequence-based identification techniques. We report the 
presence of the rompB gene of SFG rickettsiae, similar to 
R. akari , R. conorii , R. japonica , and R. felis , in serum 
specimens from Korean patients with acute febrile disease. 
The nucleic acids of both R. conorii and R. typhi were 
found to coexist in 7 serum specimens. This study presents 
the first molecular evidence of SFG rickettsioses in 
humans. 

Materials and Methods 
Rickettsial Strains 

The following strains were obtained from the American 


W.-J. Choi and W.-J. Jang contributed equally to this work. 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


237 


RESEARCH 


Type Culture Collection (ATCC; Manassas, VA, USA): R. 
typhi Wilmington (VR-144), R. prowazekii Breinl (VR- 
142), R. akari MK (VR-148), R. japonica YH (VR-1363), 
R. conorii Indian Tick Typhus (VR-597), and R. sibirica 
246 (VR-151). These rickettsial agents were propagated in 
Vero (CRL-1586) or L929 (CCL-1) cell monolayers. 

Serum Samples and Serologic Testing 

The serum specimens analyzed in this study were 
obtained from South Korean patients with acute febrile ill- 
ness from 1993 to 1999. The specimens were submitted to 
the Institute of Endemic Disease at Seoul National 
University’s Medical Research Center for laboratory diag- 
nosis for scrub typhus, leptospirosis, and hemorrhagic 
fever with renal syndrome caused by hantavirus. Some of 
the serum specimens were used for the nucleic acid detec- 
tion study of SFG rickettsial agents. The rationale for 
selecting the samples for polymerase chain reaction (PCR) 
analysis included the presence of immunoglobulin (Ig) M 
antibodies with titers from 1:40 to 1:160 against any of the 
tested antigens in the samples. Serologic testing was per- 
formed by indirect immunofluorescence assay (IFA) with 
a panel of 4 SFG rickettsial antigens, R. japonica, R. akari , 
R. conorii , and R. sibirica , as previously described (8). 

Oligonucleotide Primers 

The oligonucleotide primers used for priming the PCRs 
are shown in Table 1 . The primers were developed on the 
basis of the rompB gene sequences of R. conorii strain 
Seven (GenBank accession no. AF123721), and the citrate 
synthase (git A) gene sequence of R. prowazekii (GenBank 
accession no. M17149) was synthesized. The selection of 
the primers was based on the “primer 3” program 
(http://www-genome.wi.mit.edu/cgi-bin/primer/ 
primer3_www.cgi/), to obtain the optimal melting temper- 
ature and GC content and to avoid hairpin loop structures. 
The selected sequences were analyzed through the BEAST 
program (http : //w w w. ncbi . nlm. nih. go v/B FAST) . 


Detection of rompB Gene in Human Sera 

DNA for PCR analysis was extracted from 200 p L of 
serum samples by using QIAamp Blood Mini Kit (Qiagen 
GmbH, Hilden, Germany) according to the manufacturer’s 
instructions. SFG and typhus group (TG) rickettsia rompB 
gene in human sera were detected with multiplex nested 
PCR. The primary amplification of the specimen was per- 
formed in a final reaction volume of 50 pD. The reaction 
mixture contained 5 pD of prepared DNA sample, 20 pmol 
of rompB outer forward primer (OF) and outer reverse 
primer (OR), 200 pM of deoxynucleoside triphosphate 
mixture (dNTP, Takara, Otsu, Japan), 1 x PCR buffer, 1.25 
U Taq polymerase (Takara EX Taq, Takara), and distilled 
water. First, PCR reactions were incubated at 95 °C for 5 
min, subjected to 35 cycles of 95°C for 15 s, 54°C for 15 
s, and 72°C for 30 s, and final extension at 72°C for 3 min 
in a GeneAmp PCR system 9600 (Perkin-Elmer Applied 
Biosystems, Foster City, CA, USA). After this, 2 pD of the 
amplified product was again amplified in a nested fashion 
with inner primer sets (rompB SFG IF, rompB SFG/TG IR, 
and rompB TG IF). The nested PCR reaction mixture con- 
tained 10 pmol of each primer in a PCR premixture tube 
(AccuPower PCR PreMix, Bioneer Corp., Daejon, Korea) 
that contained 1 U of Taq DNA polymerase, 250 qmol/F 
each of dNTP, 50 mmol/F of Tris-HCl (pH 8.3), 40 
mmol/F of KC1, 1.5 mmol/F of MgCl 2 , and gel loading 
dye. The volume was then adjusted to 20 pD with distilled 
water. Nested PCR reactions were incubated at 95 °C for 5 
min, subjected to 35 cycles of 95°C for 15 s, 56°C for 15 
s, and 72°C for 30 s, and final extension at 72°C for 3 min. 
PCR amplification of the gltA gene of SFG and TG rick- 
ettsiae was performed by using the oligonucleotide pairs 
RpCS.877p and RpCS.l,258n for the primary PCR ampli- 
fication and RpCS.896p and RpCS.l,233n for the second- 
ary amplification. The primary PCR cycling condition 
consisted of incubation at 95 °C for 5 min, then 35 cycles 
each of 15 s at 95°C, 15 s at 54°C, and 30 s at 72°C, fol- 
lowed by a final extension cycle of 3 min at 72°C. The 
nested PCR cycling condition consisted of incubation at 


Table 1 . Oligonucleotide primers for amplification of partial rickettsial genes* 

Primer 

Target rickettsia group 

Gene 

Position 

Nucleotide sequence (5-3') 

rompB OF 

SFG and TG 

rompB} 

3,620-3,643 

GTAACCGGAAGTAATCGTTTCGTAA 

rompB OR 

SFG and TG 

rompB 

4,131-4,109 

GCTTTATAACCAGCTAAACCACC 

rompB SFG IF 

SFG 

rompB 

3,652-3,674 

GTTTAATACGTGCTGCTAACCAA 

rompB SFG/TG IR 

SFG and TG 

rompB 

4,077-4,057 

GGTTTGGCCCATATACCATAAG 

rompB TG IF 

TG 

rompB 

3,828-3,850 

AAGATCCTT CT GAT GTTGCAACA 

RpCS.877p§ 

SFG and TG 

gltAt 

877-895 

GGGGGCCTGCTCACGGCGG 

RpCS.1 ,258n§ 

SFG and TG 

gltA 

1 ,258-1 ,237 

ATTGCAAAAAGTACAGTGAACA 

RpCS.896p 

SFG and TG 

gltA 

896-91 5 

GGCTAATGAAGCAGTGATAA 

RpCS.1 ,233n 

SFG and TG 

gltA 

1,233-1,215 

GCGACGGTATACCCATAGC 


*SFG, spotted fever group; TG, typhus group; OR, outer reverse primer; OF, outer forward primer. 
fOligonucleotide primer sequences derived from Rickettsia conorii genes (accession no. AF123721). 
^Oligonucleotide primer sequences derived from R. prowazekii genes (accession no. Ml 7149). 
§Primer sequences derived from (9). 


238 


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Spotted Fever and Typhus Group Rickettsioses 


95°C for 5 min, then 35 cycles each of 15 s at 95°C, 15 s 
at 54°C, and 30 s at 72°C, followed by a final extension 
cycle of 3 min at 72°C. To avoid cross-contamination, 3 
separate rooms with entirely separate equipment and solu- 
tions were used. Thus, the handling and treatment of sam- 
ples and the addition of a template, the handling of 
DNA-free PCR reagents, and the post-PCR work were 
strictly separated. Aerosol-resistant tips (Axigen 
Scientific, Inc., Union City, CA, USA) were used for the 
handling of all reagents in the PCR study. The amplifica- 
tion products were visualized by electrophoresis on a 1.5% 
agarose gel stained with ethidium bromide (0.5 |lg/mL) 
and using 1 x TAE migration buffer (pH 8.0; 40 mmol/L 
Tris-acetate, 1 mmol/L EDTA). 

Restriction Fragment Length 
Polymorphism (RFLP) Analysis 

The PCR products were purified by using an AccuPrep 
PCR purification kit (Bioneer Corp.), according to the 
manufacturer’s instructions. Restriction endonuclease 
digestions were performed with 10 (iL of amplified prod- 
ucts by using Alul (New England Biolabs, Beverly, MA, 
USA). The digested DNA was resolved by electrophoresis 
through a 10% polyacrylamide gel at 100 V for 4 h in a 1 
x TBE buffer (pH 8.0; 90 mmol/L Tris-borate, 2 mmol/L 
EDTA), and was visualized after staining with ethidium 
bromide. 

Cloning, Sequencing, and Analysis of Nucleotide 

All positive PCR products were cloned by using 
pGEM-T Easy Vector System I (Promega). Verifying 
whether the clones contained inserts was accomplished by 
digestion of plasmid DNA with EcoRI (New England 
Biolabs) and separation in 1.5% agarose gels. Plasmids 
containing DNA inserts were sequenced for both strands 
by using Big Dye Terminator Sequence Kit and ABI Prism 
377 Automated DNA Sequencer (Perkin-Elmer Applied 
Biosystems), according to the manufacturer’s protocol. 
The obtained sequences, except for the primer regions, 
were aligned with the corresponding sequences of other 
rickettsiae deposited in the GenBank database to identify 
known sequences with a high degree of similarity using 
multisequence alignment programs, the Phydit software 
(10), and the Meg Align software package (Windows ver- 
sion 3.12e; DNASTAR, DYNASTAR Inc., Madison, WI, 
USA). Phylogenetic trees were generated by using the 
neighbor-joining algorithms and the Jukes and Cantor 
matrix. Bootstrap analysis was performed to investigate 
the stability of the trees obtained through the neighbor- 
joining method. The percentages of similarity were deter- 
mined using the FASTA network service (European 
Bioinformatics Institute Fasta Service; available from 
http : //www. ebi . ac.uk/fasta) . 


Nucleotide Sequence Accession Numbers Used 

GenBank accession numbers of the rompB gene 
sequences used for sequence comparisons are AB003681 
for R. japonica , AF123705 for R. aeschlimannii , 
AF123706 for R. africae , AF123707 for R. akari , 
AF123708 for Astrakhan rickettsia strain A-167, 
AF 123709 for R. australis , AF123711 for R. honei strain 
RB, AF123712 for Israeli tick typhus rickettsia, AF123714 
for R. massiliae , AF123715 R. mongolotimonae , 
AF 1237 16 for R. montanensis , AF 1237 17 for R. parkeri , 
AF 1237 19 for R. rhipicephali , AF 123721 for R. conorii 
strain Seven, AF123722 for R. sibirica , AF123723 for R. 
slovaca, AF123725 forR. Helvetica, AF 182279 for R.felis, 
AF211820 for R. prowazekii strain Florida, AF211821 for 
R. prowazekii strain Virginia, AF123718 for R. prowazekii, 
AF161079 for R. prowazekii, AF479763 for R. ambly- 
ommii strain WB-8-2 rompB pseudogene, AY260451 for 
R. heilongjiangensis, AY260452 for R. hulinensis, L04661 
for R. typhi crystalline surface layer protein (slpT) gene, 
and X16353 for R. rickettsii. The GenBank accession num- 
ber of the git A gene sequence used for developing primers 
is M17149 for R. prowazekii. 

Results 

Multiplex Nested PCR Amplification of rompB Gene 

Nested PCR assay, with primer pairs rompB OF and 
rompB OR in primary reactions and rompB SFG IF, rompB 
SFG/TG IR, and rompB TG IF in multiplex-nested reac- 
tions, was performed to identify the unknown rickettsial 
agents in the seropositive serum specimens and to differen- 
tiate between SFG and TG rickettsiae in terms of size. When 
the primers previously mentioned were used, the nested 
PCR assay generated -420 bp for SFG rickettsiae and about 
230 bp for TG rickettsiae. The negative controls consistent- 
ly failed to yield detectable PCR products, whereas the pos- 
itive controls always gave the expected PCR products. 
Overall, 200 serum specimens from febrile patients from all 
areas of South Korea were tested. After the nested PCR was 
performed, the expected rompB gene products were 
obtained from 24 seropositive serum samples. Figure 1 
shows the result of electrophoresis of 24 PCR-amplified 
samples. Of the 24 amplified products, 16 showed the elec- 
trophoretic pattern of 1 DNA band of -420 bp, which cor- 
responded to SFG. The amplified size of only 1 sample was 
-230 bp for TG. The 7 other amplified products showed an 
electrophoretic pattern of 2 bands of -420 bp for SFG and 
230 bp for TG. Therefore, the 23 amplified products corre- 
sponding to SFG rickettsial agents were named HI product, 
while the 8 products corresponding to TG were named H2 
product. The HI products included HI to H24 (except HI 9), 
while the H2 products were H3-2, H7-2, H8-2, H13-2, H14- 
2, H15-2, H18-2, and H19 (Figure 1). 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


239 


RESEARCH 


Ml 2 3 4 5 6 7 8 910 11 12 



M 13 14 15 16 17 18 19 20 21 22 23 24 



Figure 1. Agarose gel electrophoresis analysis on 1.5% agarose 
gel of DNA sequences amplified by multiplex-nested polymerase 
chain reaction (PCR) assay by using outer and inner primer sets 
targeted rompB gene and template DNAs from serum samples. 
Lanes: M, size marker DNA (100-bp DNA ladder); 1-24, each 
number of amplified H products. The number on the left indicates 
the molecular size (in base pairs) of the amplified PCR products. 


RFLP Analysis and Sequencing Analysis 

RFLP analysis of the 23 HI products corresponding to 
SFG rickettsial agents using Alul demonstrated that the 
restriction patterns of 17 HI products were identical with 
that of R. conorii , 2 with that of R. akari , 1 with that of R. 
japonica , and 3 with that of R. felis (Figure 2). RFLP 


analysis of the 8 H2 products corresponding to TG rick- 
ettsial agents by using Alul showed that the restriction pat- 
terns of all the H2 products were identical with that of R. 
typhi (Figure 3). 

Sequencing Analysis 

To identify the SFG and TG rickettsiae detected in 
human serum specimens, nucleotide sequences of the 
PCR-amplified products were determined and compared 
with partial rompB gene sequences of various rickettsial 
agents obtained from the GenBank database. Table 2 
shows the similarity between the partial rompB gene 
sequences of various rickettsial agents and 6 of the 
sequenced HI products (clones HI, H3, H5, H10, H20, 
and H22). Clones HI, H3, and H20 showed 100%, 
99.72%, and 98.87% degrees of similarity to R. conorii , 
respectively. Clone H10 showed 100% similarity to R. 
japonica , and clone H5 showed 100% similarity to R. 
akari. In particular, clone H22 showed 99.44% similarity 
to R. felis. All the compared HI products showed low lev- 
els of similarity (70.90%-74.01%) to the TG species. The 
clones that clustered partially with the rompB gene of R. 
conorii were differentiated in 3 groups by their levels of 
similarity: group 1 (12 HI products with 100% similarity), 
group 2 (4 HI products with 99.72% similarity), and group 
3 (1 HI product with 98.87% similarity). Clones H22, 
H23, and H24 clustered as the R. felis group. Table 3 
shows the similarity between the partial rompB gene 
sequences of various rickettsial species and H2 product 
sequences. All H2 products showed low levels of similari- 
ty (67.05%-69.94%) to SFG rickettsial species, such as R. 
sibirica , R. akari , R. conorii , R. felis , and R. japonica. 
They also showed high levels of similarity 
(93.64%-100%) to TG rickettsial species, such as R. 
prowazekii and R. typhi. The H2 products’ levels of simi- 
larity to R. typhi ranged from 99.42% to 100%. A neigh- 
bor-joining analysis based on partial rompB gene 
sequences demonstrated that 17 HI products formed a 
cluster with R. conorii , 2 with R. akari , 1 with R. japoni- 
ca , and 3 with R. felis (data not shown). The analysis of the 



Figure 2. Restriction fragment length polymor- 
phism analysis of HI products amplified with 
multiplex-nested primer set from seropositive 
sera. Ethidium bromide-stained polyacrylamide 
gels of Alul restriction endonuclease digestion 
of =420 bp rickettsial DNA amplified by using 
the nested primer H set WJ77/80 in the primary 
reactions and WJ79/83/78 in the nested reac- 
tions. Lanes: M, size marker DNA (25-bp DNA 
ladder); 1-18: HI -HI 8; 19-23: H20-24; C, 
Rickettsia conorii ; A, R. akari ; J, R. japonica ; F, 
R. felis. J-S; predicted fragments after digestion. 
The number on the left indicates the molecular 
size (in base pairs) of restriction fragments. 


240 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 







Spotted Fever and Typhus Group Rickettsioses 


M1 2345 678PT 



Figure 3. Restriction fragment length polymorphism analysis of 
H2-products amplified with multiplex-nested primer set from 
seropositive sera. Ethidium bromide-stained polyacrylamide gels 
of Alul restriction endonuclease digestion of «230-bp rickettsial 
DNA amplified by using the nested primer H set WJ77/80 in the 
primary reactions and WJ79/83/78 in the nested reactions. Lanes: 
M, size marker DNA (25-bp DNA ladder); 1, H3-2; 2, H7-2; 3, H8- 
2; 4, HI 3-2; 5, HI 4-2; 6, HI 5-2; 7, HI 8-2; 8, HI 9; P, Rickettsia 
prowazekii ; T, R. typhi. P and T; predicted fragments after diges- 
tion. The number on the left indicates the molecular size (in base 
pairs) of restriction fragments. 


8 H2 product sequences showed that the sequences of all 
H2 products formed a cluster with R. typhi and were sepa- 
rated from the SFG rickettsial strains (data not shown). 

Nested PCR Amplification of gltA Gene 

The results of the multiplex nested PCR of the rompB 
gene were confirmed by a second PCR assay with specific 
primer pairs RpCS.877p and RpCS. 1,258 in primary reac- 
tions and RpCS.896p and RpCS. 1,233 in nested reactions. 


The primer sets generated ~338 bp for SFG and TG rick- 
ettsiae. The expected size of the gltA gene fragment was 
generated in 22 of 24 samples that were positive for the 
PCR detection of the rompB gene (Figure 4). All positive 
PCR products were cloned, and their sequences were 
determined. Since the PCR assay using primer sets for the 
amplification of the gltA gene could not discriminate 
between the SFG rickettsia and TG rickettsia by size dif- 
ference, the sequences of 3 clones for each PCR product 
were determined. The results of the sequencing analysis 
for gltA- PCR amplifications were identical to those of the 
analysis of the rompB -PCR product (data not shown). 
Seven samples that were positive for both the rompB genes 
of R. conorii and R. typhi were also positive for both of 
their gltA genes (data not shown). 

Discussion 

SFG and TG rickettsial infections occur worldwide and 
may cause serious diseases in humans. These pathogenic 
bacteria are transmitted to people by arthropod vectors, 
such as ticks, fleas, and lice. In this study, multiplex-nest- 
ed PCR was conducted to detect and identify SFG and TG 
rickettsial antigens in patient sera with positive results 
from the serosurvey. The rompB gene domain II region, 
which is a highly conserved region of rompB , was targeted 
for PCR amplification for the specific detection of SFG 
and TG rickettsiae. Amplified DNA sequences were ana- 
lyzed by using nucleotide-sequencing methods, and RFLP 
analysis was used to confirm the PCR results. The results 
indicated the presence of several SFG rickettsiae, R. 
conorii , R. akari , R. japonica , and R. felis, in the serum 
specimens. The results were also confirmed by a second 
PCR with specific primer pairs for the gltA gene and by 
sequence analysis of its DNA amplicons. 


Table 2. Similarity matrix between partial rompB gene sequence of various rickettsial strains and nested polymerase chain reaction (HI 
products) 


1* 

2 

3 

4 

5 

6 

7 

H1| 

H3 

H5 H10 H20 H22 

1* 

2 

93.79 










3 

96.05 

94.92 

- 








4 

91.81 

96.89 

92.94 

- 







5 

92.94 

98.59 

94.07 

96.61 

- 






6 

74.01 

73.16 

74.29 

72.32 

73.45 

- 





7 

72.03 

70.9 

72.32 

70.34 

71.19 

93.22 

- 




H1| 

93.79f 

100 

94.92 

96.89 

98.59 

73.16 

70.9 

- 



H3 

93.5 

99.72 

94.63 

96.61 

98.31 

72.88 

70.62 

99.72 

- 


H5 

100 

93.79 

96.05 

91.81 

92.94 

74.01 

72.03 

93.79 

93.5 

- 

H10 

91.81 

96.89 

92.94 

100 

96.61 

72.32 

70.34 

96.89 

96.61 

91.81 

H20 

92.66 

98.87 

93.79 

96.33 

97.46 

72.6 

70.34 

98.87 

98.59 

92.66 96.33 

H22 

95.76 

94.63 

99.44 

92.66 

93.79 

74.01 

72.03 

94.63 

94.35 

95.76 92.66 93.5 


*1 , partial rompB of Rickettsia akari (AF123707), 2; R. conorii (AF123721); 3, R. felis (AF182279); 4, R. japonica (AB003681); 5, R. sibirica (AF12322); 6, 
R. prowazekii (AF21 1820); 7, R. typhi (L04661). 
fH, HI products amplified from patient sera. 

JThe similarity values (%) on the lower left are the levels of similarity between partial rompB gene sequences. 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


241 


RESEARCH 


Table 3. Similarity matrix between partial rompB gene sequence of various rickettsial strains and nested polymerase chain reaction (H2 
products) [Table is separated into 2 parts because of print limitations; see 1-piece version available at http://www.cdc.gov/ncidod/eid/vol1 1no02/04-0603.htm#table3] 



1* 

2 

3 

4 

5 

6 

7 

8 

9 

1* 

2 

92.49 









3 

98.84 

93.64 

- 







4 

94.8 

95.38 

95.95 

- 






5 

95.38 

90.17 

96.53 

92.49* 

- 





6 

67.63 

70.52 

67.63 

68.79 

67.05 

- 




7 

67.63 

70.52 

67.63 

68.79 

67.05 

100 

- 



8 

67.63 

70.52 

67.63 

68.79 

67.05 

100 

100 

- 


9 

67.63 

70.52 

67.63 

68.79 

67.05 

100 

100 

100 

- 

10 

67.63 

69.94 

67.63 

69.36 

67.05 

94.22 

94.22 

94.22 

94.22 

H8-2| 

67.63 

69.94 

67.63 

69.36 

67.05 

94.22 

94.22 

94.22 

94.22 

HI 3-2 

67.05 

69.36 

67.05 

68.79 

66.47 

93.64 

93.64 

93.64 

93.64 

HI 4-2 

67.63 

69.94 

67.63 

69.36 

67.05 

94.22 

94.22 

94.22 

94.22 

HI 5-2 

67.63 

69.94 

67.63 

69.36 

67.05 

94.22 

94.22 

94.22 

94.22 

HI 8-2 

68.21 

70.52 

68.21 

69.94 

67.63 

93.64 

93.64 

93.64 

93.64 

H19 

67.63 

69.94 

67.63 

69.36 

67.05 

94.22 

94.22 

94.22 

94.22 

H3-2 

67.63 

69.94 

67.63 

69.36 

67.05 

94.22 

94.22 

94.22 

94.22 

H7-2 

67.63 

69.94 

67.63 

69.36 

67.05 

94.22 

94.22 

94.22 

94.22 


10* 

H8-2| 

HI 3-2 

HI 4-2 

HI 5-2 

HI 8-2 

H19 

H3-2 

H7-2 


1 * 

2 

3 

4 

5 

6 

7 

8 
9 


10 

- 







H8-2| 

100 

- 






HI 3-2 

99.42 

99.42 

- 





HI 4-2 

100 

100 

99.42 

- 




HI 5-2 

100 

100 

99.42 

100 

- 



HI 8-2 

99.42 

99.42 

98.84 

99.42 

99.42 

- 


H19 

100 

100 

99.42 

100 

100 

99.42 

- 

H3-2 

100 

100 

99.42 

100 

100 

99.42 

100 

H7-2 

100 

100 

99.42 

100 

100 

99.42 

100 100 

*1 , partial rompB genes of Rickettsia sibirica (AF 12322); 2, R. akari (AF 123707), 3; R. conorii (AF 123721); 4, R. felis (AF 182279); 5, R. japonica 
(AB003681); 6, R. prowazekii (AF21 1820); 7, R. prowazekii (AF123718); 8, R. prowazekii (AF161079); 9, R. prowazekii (AF21 1821); 10, R. typhi (L04661). 
fH, H2 products amplified from patient sera. 

*The similarity values (%) on the lower left are the levels of similarity between partial rompB gene sequences and the values (bp) on the upper right are the 
number of different bases out of compared total bases in partial rompB gene sequences. 


For the first time, SFG rickettsiae in human serum 
specimens in South Korea have been reported. R. akari is 
a member of the spotted fever group rickettsiae and is a 
causative agent of rickettsial pox, a disease transmitted by 
the bite of Allodermanyssus sanguineus , a mite ectopara- 
site of the domestic mouse ( Mus muscularis) (2). The dis- 
ease was first described in New York City in 1946. R. akari 
was isolated from the Korean vole in 1957. The previous 
seroepidemiologic study conducted by the authors on 
3,401 patients with febrile disease indicated that the 
seropositive rate was 16.24% for the rickettsial antigen 


through IFA. R. conorii is an etiologic agent of the 
Mediterranean spotted fever or boutonneuse fever (2). Our 
previous study indicated that the seropositive rate was 
14.34% for the antigen. R. japonica, the causative agent of 
Oriental spotted fever, was first isolated from a patient 
with febrile, exanthematous illness in Japan in 1985 (2). 
The disease is now endemic in the southwestern part of 
Japan, where >100 cases have been described (2). Previous 
studies showed the presence of nucleic acids of R. japoni- 
ca and R. rickettsii in H. longicornis by PCR. Our seroepi- 
demiologic study demonstrated that the seropositive rate 


242 


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Spotted Fever and Typhus Group Rickettsioses 


Ml 2 3 4 5 6 7 8 9101112 



M 13 14 15 16 17 18 19 20 21 22 23 24 



Figure 4. Agarose gel electrophoresis analysis on 1.5% agarose 
gel of DNA sequences amplified by nested polymerase chain reac- 
tion (PCR) assay using primer sets targeted partial gltA gene and 
template DNA sequences from 24 serum samples. Lanes: M, size 
marker DNA (100-bp DNA ladder); 1-24, each number of amplified 
gltA products. The number on the left indicates the molecular size 
(in base pairs) of the amplified PCR products. 


was 19.9%. Although no clinical human case of SFG rick- 
ettsioses has been reported in Korea until now, this study’s 
findings strongly suggest the prevalence of SFG rick- 
ettsiosis in Korea. 

R. felis is an emerging pathogen responsible for 
fleaborne spotted fever and had been considered a member 
of the TG rickettsiae based on its reactivity with anti -R. 
typhi antibodies. A genetic analysis of the 16S rRNA, cit- 
rate synthase, rompA , and rompB genes, however, placed 
R. felis as a member of SFG. R. felis has been reported in 
various countries, including the United States, Mexico, 
Brazil, Germany, and France (11,12). In Asia, the first case 
of R. felis infection was reported in 2003 (13). R. typhi was 
also among those detected in the SFG rickettsiae in the 
febrile disease patients’ sera. Fleas are also found to be 
vectors for R. typhi (2). Of major importance to the epi- 
demiology of the rickettsioses caused by R. typhi and R. 
felis is the maintenance of both rickettsial agents in their 


hosts by transovarial transmission, and the fact that neither 
organism is lethal for fleas (14). 

Finally, we report the presence of both R. conorii and R. 
typhi in serum from Korean patients. Sera from patients 
with SFG rickettsiosis have been reported to react with TG 
rickettsiae by using serologic analysis methods (15). The 
serum specimens from patients with TG rickettsiosis were 
also demonstrated to contain cross -reactive antibodies 
against SFG rickettsiae (15,16). In a previous study, 
approximately one third of specimens seropositive for 
antibodies against SFG rickettsiae had antibodies against 
TG rickettsiae (unpub. data). Therefore, the multiplex- 
nested PCR was designed to detect and differentiate SFG 
rickettsial agents from TG rickettsial agents in the patient 
serum specimens with positive results from the serosurvey 
with SFG rickettsial antigens. SFG rickettsiae and TG 
rickettsiae were differentiated in terms of the size of ampli- 
fied products. PCR results also confirmed the RFLP and 
sequencing analysis. In sera taken from 7 patients, both 
SFG and TG rickettsial antigens were detected, which 
indicated dual infection. Previously, a case of dual infec- 
tion with Ehrlichia chaffeensis and an SPG rickettsia was 
reported in a human patient. Cases of dual infection with 
Bartonella clarridgeiae and B. henselae in cats have also 
been reported, as well as infection with the 2 different 
genotypes of B. henselae (17,18). A recent report suggest- 
ed that coinfection of R. felis with either B. clarridgeiae or 
B. quintana in fleas may cause dual infection in a human 
that comes in contact with flea feces (14). These reports 
support this study’s findings regarding the dual infection of 
SFG and TG rickettsiae in 7 patients. The differences 
between R. conorii and R. typhi vectors, however, still can- 
not be explained, and further studies are needed. 

In conclusion, this study confirmed, by using PCR- 
based amplification methods, that several SFG rickettsiae, 
R. conorii , R. akari , R. japonica, and R. felis, existed in the 
sera of Korean patients with febrile episodes. Our findings 
indicate that SFG rickettsiae, including R. felis , should be 
used in serologic tests on Korean patients suspected of 
having rickettsiosis. TG rickettsiae existed in 8 patients, 
and 7 of them were also infected with R. conorii. The evi- 
dence of double infection is expected to help describe the 
cross-reactivity between the patient sera of SFG rick- 
ettsioses and TG rickettsioses. 

This study was supported by a grant from the Korea Health 
21 Research and Development Project, Ministry of Health and 
Welfare, Republic of Korea (01-PJ10-PG6-01GM0 1-0004). This 
study was conducted in the Department of Microbiology, College 
of Medicine, Konkuk University. 

Dr. Choi is a postdoctoral fellow in the Department of 
Microbiology, College of Medicine, Konkuk University, 


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243 




RESEARCH 


Choong-cheongbuk-do, Korea. This work is part of her doctoral 
thesis. Her research focuses on the serologic and molecular epi- 
demiology of various rickettsial diseases and the development of 
diagnostic tools. 

References 

1. Choi MS, Park SK, Chang WJ, Huh MS, Kim HR, Han TH, et al. A 
seroepidemiological survey on the scrub typhus in Korea, 1994. J 
Korean Soc Microbiol. 1994;30:593-602. 

2. Raoult D, Roux V. Rickettsioses as paradigms of new or emerging 
infectious diseases. Clin Microbiol Rev. 1997;10:694-719. 

3. Kim YW, Cho MK, Min CH, Yoon CS. Isolation and characterization 
of Rickettsia typhi from patients in Korea. J Korean Soc Microbiol. 
1988;23:265-75. 

4. Song JW, Baek LJ, Lee YJ, Song KJ, Han SH. Seroepidemiologic 
analysis of acute rebrile illness from Korea in 1996. J Korean Soc 
Virol. 1998;28:377-82. 

5. Gross L. How Charles Nicolle of the Pasteur Institute discovered that 
epidemic typhus is transmitted by lice: reminiscence from my years 
at the Pasteur Institute in Paris. Proc Natl Acad Sci USA. 
1996;93:10539-40. 

6. Chung HY. Suspected human infectious diseases in Korea; 
Rickettsial infections. Korean J Infect Dis. 1985;18:93-7. 

7. Lee JH, Park HS, Jung KD, Jang WJ, Koh SE, Kang SS, et al. 
Identification of spotted fever group rickettsiae detected from 
Haemaphysalis longicornis in Korea. Microbiol Immunol. 
2003;47:301-4. 

8. Jang WJ, Kim JH, Choi YJ, Jung KD, Kim YG, Lee SH, et al. First 
serologic evidence of human spotted fever group rickettsiosis in 
Korea. J Clin Microbiol. 2004;42:2310-3. 

9. Roux V, Rydkina E, Eremeeva M, Raoult D. Citrate synthase gene 
comparison, a new tool for phylogenetic analysis, and its application 
for the rickettsiae. Int J Syst Bacteriol. 1997;47:252-61. 


10. Chun J. Computer-assisted classification and identification of actino- 
mycetes. [Ph.D. thesis]. Newcastle Tyne, United Kingdom: 
University of Newcastle upon Tyne; 1995. 

11. Zavala- Velazquez JE, Ruiz-Sosa JA, Sanchez-Elias RA, Becerra- 
Carmona G, Walker DH. Rickettsia felis rickettsiosis in Yucatan. 
Lancet. 2000;356:1079-80. 

12. Richter J, Fournier PE, Petridou J, Haussinger D, Raoult D. Rickettsia 
felis infection acquired in Europe and documented by polymerase 
chain reaction. Emerg Infect Dis. 2002;8:207-8. 

13. Parola, P, Miller RS, McDaneiel P, Telford SR 3rd, Rolain JM, 
Wongsrichanalai C, et al. Emerging rickettsioses of the Thai- 
Myanmar border. Emerg Infect Dis. 2003;9:592-5. 

14. Rolain JM, Franc M, Davousr B, Raoult D. Molecular detection of 
pathogenic Bartonella and Rickettsia in cat fleas from France. Emerg 
Infect Dis. 2003;9:338-42. 

15. Hechemy KE, Raoult D, Fox J, Han Y, Elliotte LB, Rawlings J. 
Cross-reaction of immune sera from patients with rickettsial disease. 
J Med Microbiol. 1989;29:199-202. 

16. Uchiyama T, Zhao L, Yan Y, Uchida T. Cross-reacttivity of Rickettsia 
japonica and Rickettsia typhi demonstrated by immunofluorescence 
and Western immunoblotting. Microbiol Immunol. 1995;39:951-7. 

17. Gurfield AN, Boulouis HJ, Chomel BB, Kasten RW, Heller R, 
Bouillin C, et al. Epidemiology of Bartonella infection in domestic 
cats in France. Vet Microbiol. 2001;80:185-98. 

18. Abbott RC, Chomel BB, Kasten RW, Floyd-Hawkins KA, Kikuchi Y, 
Koehler JE, et al. Experimental and natural infection with Bartonella 
henselae in domestic cats. Comp Immunol Microbiol Infect Dis. 
1997;20:41-51. 


Address for correspondence: Ik-Sang Kim, Department of Microbiology 
and Immunology, Seoul National University College of Medicine and 
Institute of Endemic Disease, Seoul, 110-799, Republic of Korea; fax: 82- 
43-851-9329; email: molecule @ plaza. snu.ac.kr 


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Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


Pneumocystis jirovecii in 
General Population 

Francisco J. Medrano,* Marco Montes-Cano,* Manuel Conde,* Carmen de la Horra,* 
Nieves Respaldiza,* Antonia Gasch,* Maria J. Perez-Lozano,* Jose M. Varela,* 

and Enrique J. Calderon* 


The possible presence of Pneumocystis among 
healthy adults was examined by detecting Pneumocystis 
jirovecii- specific DNA in prospectively obtained oropharyn- 
geal wash samples from 50 persons without underlying 
lung disease or immunosuppression. Pneumocystis car- 
riage, defined by detecting Pneumocystis DNA by nested 
polymerase chain reaction in 2 independent analyses plus 
successful mitochondrial large subunit ribosomal RNA typ- 
ing by direct sequencing, was found in 20% of cases. All 
carriers were asymptomatic, anti-HIV negative, and had 
normal total lymphocyte and CD4+ cell counts. A second 
sample obtained in the 6-month follow-up was positive in 2 
of 9 available carriers. Genotype analysis showed different 
polymorphisms; 85A/248C (40%) and 85C/248C (30%) 
were most frequently observed. This study provides the 
first evidence that P jirovecii DNA can be frequently detect- 
ed in the respiratory tract of immunocompetent adults, 
which agrees with the hypothesis that the general popula- 
tion could be a reservoir and source of this infection. 

neumocystis jirovecii (formerly known as 
Pneumocystis carinii f. sp. hominis) (1) is the 
causative agent of Pneumocystis pneumonia (PCP), one of 
the most frequent and severe opportunistic infections in 
immunocompromised patients (2). Pneumocystis organ- 
isms represent a large group of species of atypical fungi 
with universal distribution and pulmonary tropism, and 
each species has a strong specificity for a given mam- 
malian host species (3,4). 

Despite the advances made in understanding human 
Pneumocystis infection, many aspects about its epidemiol- 
ogy and natural history remain unclear. Serologic studies 
have shown that specific antibodies to the pathogen can be 
detected in most children early in life (5-7), which indi- 
cates frequent exposure to this organism. Based on this 


*Virgen del Rocio University Hospital, Seville, Spain 


finding, disease in immunocompromised persons has long 
been thought to result from reactivation of latent infection 
acquired in childhood. However, animal and human stud- 
ies have shown that elimination of Pneumocystis often 
occurs after infection (8-9), which implies that the persist- 
ence of latent organisms is limited. 

Alternatively, the possibility that Pneumocystis can be 
transmitted from person to person has been raised after the 
reports of cluster outbreaks of PCP among solid-organ 
transplant and oncology patients (10,11). Evidence sup- 
porting the active or de novo airborne acquisition of the 
organism from human sources has accumulated in the last 
few years, including evidence for different Pneumocystis 
genotypes in different episodes of PCP in the same patient 
(12,13). Also, Pneumocystis DNA was detected in the 
upper respiratory tract of healthy participants after close 
contact with patients with PCP (14-16) and in air samples 
from the rooms of PCP patients (17,18). Pneumocystis has 
also been found in immunosuppressed patients without 
PCP (19); these situations have been described as 
Pneumocystis colonization or carriage. 

PCP patients, immunodeficient carriers, or transiently 
parasitized immunocompetent persons have been hypothe- 
sized to play a role as sources of Pneumocystis infection 
(4). Although some earlier studies failed to detect the 
organism in postmortem lung samples or bronchoalveolar 
samples from immunocompetent adults (20,21), a recent 
report indicates that Pneumocystis DNA can be frequently 
detected in healthy infants (22). 

The ability to detect Pneumocystis in normal, healthy 
persons is due to the development of more sensitive meth- 
ods. Pneumocystis can now be detected in respiratory sam- 
ples obtained by noninvasive methods using immuno- 
fluorescence staining and polymerase chain reaction 
(PCR) (23,24). By using these methods, Pneumocystis 
carriage was found in 10% to 40% of immunocompetent 



Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


245 


RESEARCH 


patients with different chronic lung diseases (25,26). From 
an epidemiologic point of view, this high prevalence is dif- 
ficult to determine if PCP or colonized immunosuppressed 
patients and their close contacts are the only sources of 
infection, since the persistence of latent organism in lung 
appears to be time-limited (8,9). We tested the hypothesis 
that in a normal community environment healthy adults 
can be transiently colonized by Pneumocystis , and these 
persons play a role in the persistence of the organism in the 
human ecosystem. Identifying Pneumocystis sources is 
essential to developing proper measures to prevent a dis- 
ease that still causes substantial illness and death among 
immunosuppressed patients. This study attempts to deter- 
minate whether P. jirovecii can be detected in the general 
normal, healthy population. 

Methods 

Study Population 

This prospective study included persons who 1) had not 
been exposed to patients in a hospital environment within 
the year before the study or 2) had not been diagnosed with 
or were not suspected to have chronic lung disease, neo- 
plasm, or immunosuppression of any cause. The first 50 
persons evaluated in the Occupational Health Service of 
the Virgen del Rocio University Hospital from February to 
July 2003 who had not been excluded by the above crite- 
ria were enrolled in this study. 

The mean age of persons in this study group was 33.9 
+ 9.45 years. Nineteen (31.6%) were male. Distribution 
according to professional standing was 28 (56%) newly 
employed physician residents, 13 (26%) university or 
common services staff members, and 9 (18%) administra- 
tive staff. 

Each participant underwent a clinical-epidemiologic 
examination, and oropharyngeal samples were collected 
for analysis in the Occupational Health Service, a building 
located outside the hospital environment. Demographic 
variables, underlying medical conditions, habits, and 
antimicrobial therapy were recorded by using a standard- 
ized form. Informed consent was obtained from partici- 
pants. The study protocol was designed and performed 
according to the Helsinki Declaration and was approved by 
the ethics committee, Virgen del Rocio University 
Hospital, Seville, Spain. 

For all Pneumocystis carriers, a complete clinical and 
biologic evaluation was performed, including physical 
examination, chest x-ray, conventional blood work, anti- 
HIV serologic examination, and peripheral blood lympho- 
cyte subsets analyses. Volunteers who were designated P. 
jirovecii carriers were reexamined after 6 months, when 
oropharyngeal samples were again obtained. 


Case Definition 

A Pneumocystis carrier was defined as a person who 
met all of the following conditions: 1) no clinical history 
of PCP, 2) respiratory specimen with detectable P. jirovecii 
DNA by nested PCR in 2 independent analyses, and 3) 
successful mitochondrial large subunit ribosomal RNA 
(mtLSU-rRNA) typing of the respiratory specimen by 
direct sequencing at least once. Persons who did not meet 
these criteria were considered Pneumocystis- negative. 

Sampling and Detecting P. jirovecii 

Oropharyngeal wash samples were obtained by gar- 
gling with 10 mL of sterile physiologic serum (0.9% NaCl) 
for a period of 1 min. Samples were centrifuged at 2,900 x 
g for 5 min and kept frozen at -20°C until DNA was 
extracted. 

After digestion with proteinase K at 56°C for 2 h, DNA 
from 2 aliquots of each oropharyngeal wash sample was 
extracted on 2 days by using a commercial kit from Qiagen 
(Hilden, Germany). Sham extractions, carried out in paral- 
lel with the processing of samples, were also included to 
control for contamination in the DNA extraction step. The 
purified DNA was used as a template to amplify the region 
containing mtLSU-rRNA by nested PCR, as described 
elsewhere (27,28). The sensitivity of this nested PCR assay 
is 1 organism/qL. Briefly, in the first amplification round, 
the external primers pAZ102-E (5'-GAT GGC TGT TTC 
CAA GCC CA-3') and pAZ102-H (5'-GTG TAC GTT 
GCA AAG TAC TC-3') were used. This amplification 
yields a 346-base pair (bp) fragment. The second round of 
amplification used the primers pAZ102-X (5'-GTG AAA 
TAC AAATCG GAC TAG G-3') and pAZ102-Y (5'-TCA 
CTT AAT ATT AAT TGG GGA GC-3') and yielded a 260- 
bp product. Forty cycles of amplification were carried out 
for both rounds. 

The amplification products were analyzed by elec- 
trophoresis on a 1.5% agarose gel containing ethidium bro- 
mide, and the bands were visualized by UV light. To 
prevent false-positives from contamination, pipettes with 
filters were used in all manipulations. DNA extraction, 
preparation of the reaction mixture, PCR amplification, 
and detection were performed in different areas under a 
laminar flow hood. To detect any cross-contamination, all 
PCRs were performed with negative controls and sterile 
water. 

The products from nested PCR amplification were puri- 
fied by using Sephacryl S-400 columns (Amersham 
Pharmacia Biotech AB, Uppsala, Sweden) and reamplified 
by using ABI Prism dRhodamine Terminator Cycle 
Sequencing Ready Reaction Kit (PE Applied Biosystems, 
Foster City, CA, USA). Then, for each reaction, 5 qL of 
PCR product, 4 pH terminator-ready reaction mix, and 3 
pmol of primer were added. The extension products were 


246 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


Pneumocystis jirovecii in General Population 


purified by ethanol precipitation to remove excess dye ter- 
minators. Each sample pellet was resuspended in 12.5 |iL 
of template suppression reagent and heated at 95°C for 3 
min for denaturation. Electrophoresis was carried out on 
the ABI prism 310 sequencer (PE Applied Biosystems) 
according to manufacturer’s recommendations. The 
sequenced DNA fragments were analyzed by using 
Sequence Navigator v. 1.0.1 (PE Applied Biosystems). 

In the specimens of carriers, the P. jirovecii dihy- 
dropteroate synthase (DHPS) locus was analyzed by PCR 
restriction fragment length polymorphism (RFLP), as pre- 
viously described (28). In brief, the single-copy gene of 
DHPS was amplified by the primers DHPS-3 (5'-GCG 
CCT ACA CAT ATT ATG GCC ATT TTA AAT C-3') and 
DHPS -4 (5'-GGA ACT TTC AAC TTG GCA ACC AC-3') 
by using a touchdown-PCR protocol, yielding a 370-bp 
fragment. The PCR product was divided into 3 aliquots. 
One was used to confirm the presence of a 370-bp frag- 
ment from the DHPS gene. The second and third aliquots 
were used to identify wild-type sequences versus muta- 
tions in codons 55 and 57 by RFLP with AccI and Haelll 
(Roche Diagnostics, Mannheim, Germany), respectively. 
When the mutation is present, a 370-bp band appears. 
After RFLP in wild-type samples, bands appear at 229 bp 
and 141 bp with AccI and at 221 bp and 149 bp with 
Haelll. 

Laboratory Studies 

Peripheral blood lymphocyte subsets were determined 
by using a flow cytometer (Cytoron Absolute, Ortho, 
Raritan, NJ, USA) after incubation with monoclonal anti- 
bodies OKT3, OKT4, and OKT8 (Ortho). Serum anti-HIV 
antibodies were determined by a commercial enzyme- 
linked immunosorbent assay (ELISA) (Multispot HIV- 
l/HIV-2 rapid test, BioRad, Hercules, CA, USA). The 
results were interpreted according to the manufacturer’s 
recommendations . 

Statistical Analysis 

The chi-square test was used for assessing differences 
between proportions. Results were considered significant 
at p < 0.05. Statistical analyses were performed by using 
the Statistical Package for Serial Studies for personal com- 
puters (SPSS version 12, SPSS Inc., Chicago, IL, USA). 

Results 

P. jirovecii DNA in 12 of 50 samples was successfully 
amplified twice by nested PCR. The mtLSU-rRNA frag- 
ment locus was successfully typed in 10 of the 12 samples 
in which mtLSU-rRNA had been amplified. Thus, P. 
jirovecii carriage was detected in 20% of the participants. 
To assess the reproducibility and consistency of results, 
serial samples were obtained with a 2-day interval in 5 par- 


ticipants (1 carrier and 4 noncarriers). In all of them, con- 
sistent positive and negative patterns of results were 
obtained (positive PCR test after a positive result and neg- 
ative PCR test after a negative result). 

The DHPS primer sets amplified a 370-bp band in 7 
(70%) of 10 carriers. In all positive specimens with the 
DHPS-based PCR assay, the RFLP technique identified a 
wild DHPS genotype (Table). 

Results of physical examination were normal for 
Pneumocystis carriers. All were anti-HIV negative and had 
normal total lymphocyte and CD4+ cell counts. Chest radi- 
ographs were normal in 8 participants, and 1 participant 
had an apical cystic bullae (Table). Only 1 person had 
taken steroids for a brief period in the 6 months before the 
study. No differences were detected due to age, sex, pro- 
fession, alcohol intake, and smoking habit between P. 
jirovecii carriers and noncarriers. 

Five known mtLSU-rRNA types are described for this 
Pneumocystis gene locus (15); 4 genotypes were isolated 
in the current study. Genotypes at this locus were identi- 
fied on the basis of polymorphisms at nucleotide positions 
85 and 248. Genotype 2 (85:A/248:C) was observed in 4 
cases, genotype 1 (85:C/248:C) in 3 cases, genotype 3 
(85:T/248:C) in 2 cases, and genotype 4 (85:C/248:T) in 
the remaining case (Table). 

During the follow-up, all carriers were asymptomatic 
for pulmonary disease. Pneumocystis DNA was detected 
by PCR in a second oropharyngeal wash obtained 6 
months after the first in 2 (22.2%) of the 9 available carri- 
ers. Neither sample was typed because of insufficient 
quantity of PCR product. 

Discussion 

This study on immunocompetent healthy adults docu- 
ments that P. jirovecii DNA can be detected by sensitive 
DNA amplification techniques by using noninvasive sam- 
pling of the respiratory tract. DNA detection does not 
establish the existence of infectious intact organisms. 
However, in animal models, detecting Pneumocystis DNA 
in nasal and oral samples is a good indicator that the organ- 
ism is in the lung (29). Also, experiments show that 
Pneumocystis organisms can replicate in the lung alveolus 
of immunocompetent hosts and remain infectious (30). 
Thus, our results agree with the hypothesis that the gener- 
al human population could play an important role as a 
reservoir and source of P. jirovecii infection and support 
the saprophytic nature of this pathogen in humans. 

An important finding of this study is that Pneumocystis 
DNA was not detected in >75% of the immunocompetent 
colonized adults within 6 months, which suggests the pos- 
sible transience of the carrier state in healthy persons. 
This observation agrees with previous reports that show 
that most immunocompetent healthcare workers who 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


247 


RESEARCH 


Table. Epidemiologic, biologic, and microbiologic features of healthy Pneumocystis carriers* 


Participant 
(age [y], 
sex)f 

Profession 

Alcohol 

intake 

>40 

g/day 

Smoking 

habit 

Total 

lymphocyte 

count 

(cells/pL) 

CD4+ cell 
count 
(cells/pL) 

mtLSU-rRNA 
genotype 
(nucleotide 
position: identity) 

DHPS genotype 
(codon position: 
identity) by PCR- 
RFLP assay 

PCR result 
at month 
6 of 

follow-up 

1 (28, F) 

Administrative 

No 

No 

2,327 

653 

3 (85:T/248:C) 

1 (55:Thr/57:Pro) 

+ 

2 (42, F) 

Administrative 

No 

No 

2,455 

1,168 

1 (85:C/248:C) 

1 (55:Thr/57:Pro) 

- 

3 (43, F) 

Administrative 

No 

No 

NA 

NA 

4 (85:C/248:T) 

Not amplified 

- 

4 (40, M) 

Administrative 

Yes 

No 

1,528 

520 

3 (85:T/248:C) 

Not amplified 

- 

5 (41 , F) 

New resident 

No 

No 

NA 

NA 

2 (85:A/248:C) 

1 (55:Thr/57:Pro) 

NA 

6 (27, F) 

New resident 

No 

No 

1,354 

550 

2 (85:A/248:C) 

1 (55:Thr/57:Pro) 

- 

7 (32, F) 

New resident 

No 

No 

2,300 

604 

1 (85:C/248:C) 

1 (55:Thr/57:Pro) 

- 

8 (40, M) 

University 

staff 

Yes 

Yes 

1,730 

924 

2 (85:A/248:C) 

1 (55:Thr/57:Pro) 

+ 

9 (26, M) 

University 

staff 

Yes 

Yes 

3,602 

924 

2 (85:A/248:C) 

1 (55:Thr/57:Pro) 

- 

10(42, M) 

University 

staff 

No 

No 

1,518 

432 

1 (85:C/248:C) 

Not amplified 

- 


*mtLSU-rRNA, mitochondrial large subunit ribosomal RNA; DHPS, dihydropteroate synthase; PCR, polymerase chain reaction; RFLP, restriction fragment 
length polymorphism; F, female; M, male; +, positive result; -, negative result; NA, not available. 

fChest radiograph results were normal for all participants, except participant 5, for whom results were not available, and participant 9, who showed an 
apical cystic bullae. 


were colonized with the pathogen cleared the infection 
(15,16). 

The number of persons examined in this study suffi- 
ciently demonstrated that P. jirovecii is an organism fre- 
quently found in healthy adults in the normal community. 
Since participants were all affiliated in some way with the 
Virgen del Rocio University Hospital in Seville, Spain, a 
broader group of healthy adults would need to be exam- 
ined to estimate the prevalence of the carriers in the gener- 
al population. Carrying out the study in a hospital could 
have somewhat biased the results, although we excluded 
persons with prior exposure to patients within the hospital 
and collected samples in a building outside the hospital. 

The accepted current diagnostic standard for 
Pneumocystis infection is the direct demonstration of the 
stained microorganism in respiratory samples. Techniques 
based on PCR amplification of specific genome regions 
that provide high sensitivity are now widely used to diag- 
nose several infectious diseases. These technical advances 
have allowed us to detect infections with samples obtained 
by noninvasive methods and in samples with low pathogen 
load. Different studies involving both sputum and bron- 
choalveolar lavage specimens have demonstrated the high- 
er sensitivity of these techniques compared to 
conventional staining and monoclonal antibody immuno- 
histochemical techniques (31,32). The main drawback to 
PCR is the possibility of false-positives (usually because 
of contamination) and the absence of rapid culture meth- 
ods to confirm the PCR amplification results obtained. We 
avoided potential false-positives by adopting stringent pre- 
cautionary measures and by examining the PCR signal 
from 2 different genes of P. jirovecii. 

The mtLSU-rRNA gene was selected for genotyping 
because it has a high degree of genetic conservation and is 


useful for detecting intraspecific differences between pop- 
ulations (33). The allelic frequency distribution patterns at 
this gene seen in the present study are similar to those 
reported in AIDS-associated PCP cases in a large study 
conducted in 5 cities in the United States (33). In that 
study, P. jirovecii genotypes were correlated with the place 
of diagnosis, rather than the person’s place of birth. Our 
results support the concept of a community source of the 
infectious agents. Furthermore, an epidemiologic study 
was recently performed in patients in Spain with various 
pulmonary diseases. By also analyzing mtLSU-rRNA 
types, we found a high prevalence of genotype 1 (45%) 
and genotype 3 (40%) and lower of genotype 2 (10%) (28). 
In contrast, in the present study, genotype 2 was the most 
prevalent, whereas it was low in the pulmonary patient 
study, and genotype 3 was more frequently found in pul- 
monary patients than in normal healthy adults. Patients 
with pulmonary disorders may have a greater susceptibili- 
ty to genotype 3. 

In our study, the rate of carriers identified as having the 
DHPS gene is 70%. The low amplification rate obtained is 
perhaps related to a low pathogen load in the samples. 
However, this rate is similar to that reported for AIDS 
patients with PCP in previously published studies (33). 

In the last decade, PCR technologies have shown that 
immunocompromised patients without PCP can be sub- 
clinically infected with Pneumocystis (19). In addition, a 
high prevalence of Pneumocystis is seen among immuno- 
competent patients with chronic pulmonary disorders 
(25,26,34), patients with small-cell lung carcinoma (35), 
or pregnant women (36). The pathogen has been detected 
in immunocompetent contacts of patients with PCP and in 
immunocompetent healthcare workers, whether or not they 
had contact with immunocompromised patients (15,16). 


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Pneumocystis jirovecii in General Population 


Also, immunocompetent animal model hosts are common- 
ly transiently colonized; Pneumocystis can replicate 
actively in their lungs and can be transmitted to another 
host (30,37). Moreover, postmortem lung screening using 
conventional staining for Pneumocystis showed small 
numbers of the organism in the lungs of immunocompetent 
individuals (38). 

Thus, substantial evidence exists of Pneumocystis colo- 
nization of healthy persons, and our findings are consistent 
with these observations. Most previous studies that failed 
to find the organism on autopsy (21) or in respiratory sam- 
ples (14,29,31,36,39) from immunocompetent persons 
were performed on only a few persons. In some cases, fail- 
ure was probably related to the use of either single PCR 
(31) or a less sensitive PCR (39). In others, failure could 
be related to the use of sputum or nasal secretions 
(14,29,36) that may have had lower numbers of organisms 
than in the oropharyngeal wash we analyzed or because of 
different procedures used for analyses (29). Protocols for 
acquiring and processing respiratory samples and analyti- 
cal probes and methods should be standardized to enable 
better comparisons between studies performed in different 
laboratories. 

In summary, immunocompetent healthy adults might 
harbor short-lived infections that could be transmitted to 
other immunocompetent host in whom a transient infec- 
tion can develop. Similarly, infants can become infected 
and a primary infection can develop, and immunosup- 
pressed, susceptible people can become infected and clini- 
cal PCP can develop. Today, we know that human 
pneumocystosis is anthroponotic. Our findings may sug- 
gest that healthy adults represent a new dynamic reservoir 
and source of infection for human Pneumocystis species. 
Immunocompetent carriers in community ecosystems 
might present a public health issue that merits further 
research. 

Acknowledgment 

We thank Edna S. Kaneshiro for assistance during the prepa- 
ration of the manuscript. 

This study was partially supported by the fifth Framework 
Program of the European Commission contract No. QLK2-2000- 
01369, the Research Project 32/02 of the Ministry of Health, 
Junta de Andalucia, and the Research Project FIS 03/1743 of the 
Spanish Ministry of Health. 

Dr. Medrano is an instructor and fellow in the Department of 
Internal Medicine, Virgen del Rocio University Hospital, Seville, 
Spain. His main areas of interest include physiopathologic and 
epidemiologic research on human pathogens, such as Leishmania 
and Pneumocystis spp. 


References 

1. Stringer JR, Beard CB, Miller RF, Wakefield AE. A new name 
( Pneumocystis jirovecii ) for Pneumocystis from humans. Emerg 
Infect Dis. 2002;8:891-6. 

2. Calderon- Sandubete EJ, Varela- Aguilar JM, Medrano-Ortega FJ, 
Nieto-Guerrero V, Respaldiza-Salas N, de la Horra-Padilla C, et al. 
Historical perspective on Pneumocystis carinii infection. Protist. 
2002;153:303-10. 

3. Gigliotti F, Harmsen AG, Haidaris CG, Haidaris PJ. Pneumocystis 
carinii is not universally transmissible between mammalian species. 
Infect Immun. 1993;61:2886-90. 

4. Dei-Cas E. Pneumocystis infections: the iceberg? Med Mycol. 
2000;38(Suppl l):23-32. 

5. Meuwissen JHE Th, Tauber I, Leeuwenberg ADEM, Beckers PJA, 
Sieben M. Parasitologic and serologic observations of infection with 
Pneumocystis in humans. J Infect Dis. 1977;136:43-9. 

6. Pifer LL, Hughes WT, Stagno S, Woods D. Pneumocystis carinii 
infection: evidence for high prevalence in normal and immunosup- 
pressed children. Pediatrics. 1978;61:35-41. 

7. Medrano FJ, Respaldiza N, Medrano A, Varela JM, Montes-Cano M, 
de La Horra C, et al. Seroprevalence of Pneumocystis human infec- 
tion in southern Spain. J Eukaryot Microbiol. 2003;50(Suppl): 
649-50. 

8. Chen F, Gigliotti F, Harmsen AG. Latency is not an inevitable out- 
come of infection with Pneumocystis carinii. Infect Immun. 
1993;61:5406-9. 

9. O’Donnell WJ, Pieciak W, Chertow GM, Sanabria J, Lahive KC. 
Clearance of Pneumocystis carinii cystic in acute P. carinii pneumo- 
nia: assessment serial sputum induction. Chest. 1998;114:1264-8. 

10. Chaves JP, David S, Wauters JP, Van Melle G, Francioli P. 
Transmission of Pneumocystis carinii from AIDS patients to other 
immunosuppressed patients: a cluster of Pneumocystis carinii pneu- 
monia in a renal transplant recipient. AIDS. 1991;5:927-32. 

11. Vrathalitis I, Aoun M, Daneau D, Meunier F. Pneumocystis carinii 
pneumonia in patients with cancer. An increasing incidence. Cancer. 
1993;71:481-5. 

12. Keely SP, Stringer JR, Baughman RP, Linke MJ, Walzer PD, Smulian 
AG. Genetic variation among Pneumocystis carinii hominis isolates 
in recurrent pneumocystosis. J Infect Dis. 1995;172:595-8. 

13. Keely SP, Stringer JR. Sequences of Pneumocystis carinii s.f. homin- 
is strains associated with recurrent pneumonia vary at multiple loci. J 
Clin Microbiol. 1997;35:2745-7. 

14. Vargas SL, Ponce CA, Giglotti F, Ulloa AV, Prieto S, Munoz MP, et 
al. Transmission of Pneumocystis carinii DNA from a patient with P. 
carinii pneumonia to immunocompetent contact health care workers. 
J Clin Microbiol. 2000;38:1536-8. 

15. Miller RF, Ambrose HE, Wakefield AE. Pneumocystis carinii f. sp 
hominis DNA in immunocompetent health care workers in contact 
with patients with P. carinii pneumonia. J Clin Microbiol. 
2001;39:3877-82. 

16. Durand- Joly I, Soula F, Chabe M, Dalle JH, Lafitte JJ, Senechal M, 
et al. Long-term colonization with Pneumocystis jirovecii in hospital 
staffs: a challenge to prevent nosocomial pneumocystosis. J Eukaryot 
Microbiol. 2003;50(Suppl):614-5. 

17. Bartlett MS, Vermund SH, Jacobs R, Durant PJ, Shaw MM, Smith 
JW, et al. Detection of Pneumocystis carinii DNA in air samples: 
likely environmental risk to susceptible persons. J Clin Microbiol. 
1997;35:2511-3. 

18. Olsson M, Lidman C, Latouche S, Bjorkman A, Roux P, Linder E, et 
al. Identification of Pneumocystis carinii f. sp. hominis gene 
sequences in filtered air in hospital environments. J Clin Microbiol. 
1998;36:1737-40. 

19. Nevez G, Raccurt C, Jounieaux V, Dei-Cas E, Mazars E. 
Pneumocystosis versus pulmonary Pneumocystis carinii colonization 
in HIV-negative and HIV-positive patients. AIDS. 1999;13:535-56. 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


249 


RESEARCH 


20. Peters SE, Wakefield AE, Sinclair K, Millard PR, Hopkin JM. A 
search for Pneumocystis carinii in postmortem lungs by DNA ampli- 
fication. J Pathol. 1992;166:195-8. 

21. Wakefield AE, Pixley FJ, Banerji S, Sinclair K, Millard PR, Hopkin 
JM. Detection of Pneumocystis carinii with DNA amplification. 
Lancet. 1990;336:451-3. 

22. Vargas SL, Hughes WT, Santolaya ME, Ulloa AV, Ponce CA, Cabrera 
CE, et al. Search for primary infection by Pneumocystis carinii in a 
cohort of normal health children. Clin Infect Dis. 2001;32:855-61. 

23. Eisen D, Ross BC, Fairbairn J, Warren RJ, Baird RW, Dwyer B. 
Comparison of Pneumocystis carinii detection by toluidine blue O 
staining, direct immunofluorescence and DNA amplification in spu- 
tum specimens from HIV patients. Pathology. 1994;26:198-200. 

24. Helweg-Larsen J, Jensen JS, Benfield T, Svendsen UG, Lundgren JD, 
Lundgren B. Diagnostic use of PCR for detection of Pneumocystis 
carinii in oral wash samples. J Clin Microbiol. 1998;36:2068-72. 

25. Calderon EJ, Regordan C, Medrano FJ, Ollero M, Varela JM. 
Pneumocystis carinii infection in patients with chronic bronchial dis- 
ease. Lancet. 1996;347:977. 

26. Sing A, Roggenkamp A, Autenrieth IB, Heesemann J. Pneumocystis 
carinii carriage in immunocompetent patients with primary pul- 
monary disorders as detected by single or nested PCR. J Clin 
Microbiol. 1999;37:3409-10. 

27. Wakefield AE, Pixley FJ, Banerji S, Sinclair K, Miller RF, Moxon 
ER, et al. Amplification of mitochondrial ribosomal RNA sequences 
from Pneumocystis carinii DNA of rat and human origin. Mol 
Biochem Parasitol. 1990;43:69-76. 

28. Montes-Cano MA, de la Horra C, Martin- Juan J, Varela JM, 
Torronteras R, Respaldiza N, et al. Pneumocystis jiroveci genotypes 
in Spanish population. Clin Infect Dis. 2004;39:123-8. 

29. Oz HS, Hughes WT. DNA amplification of nasopharyngeal aspirates 
in rats: a procedure to detect Pneumocystis carinii. Microb Pathog. 
1999;27:119-21. 

30. Chabe M, Dei-Cas E, Creusy C, Fleurisse L, Respaldiza N, Camus D, 
et al. Immunocompetent host as a reservoir of Pneumocystis organ- 
ism: histological and RT-PCR data demonstrate active replication. 
Eur J Clin Microbiol Infect Dis. 2004;23:89-97. 

31. Wakefield AE, Guiver L, Miller RF, Hopkin JM. DNA amplification 
on induced sputum samples for diagnosis of Pneumocystis carinii 
pneumonia. Lancet. 1991;337:1378-9. 


32. Leibovitz E, Pollack H, Moore T, Papellas J, Gallo L, Krasinski K, et 
al. Comparison of PCR and standard cytological staining for detec- 
tion of Pneumocystis carinii from respiratory specimens from 
patients with or at high risk for infection by human immunodeficien- 
cy virus. J Clin Microbiol. 1995;33:3004-7. 

33. Beard CB, Carter JL, Keely SP, Huang L, Pieniazek NJ, Moura INS, 
et al. Genetic variation in Pneumocystis carinii isolates from differ- 
ent geographic regions: implications for transmission. Emerg Infect 
Dis. 2000;6:265-72. 

34. Vargas SL, Ponce CA, Sanchez CA, Ulloa AV, Bustamante R, Juarez 
G. Pregnancy and asymptomatic carriage of Pneumocystis jiroveci. 
Emerg Infect Dis. 2003;9:605-6. 

35. Calderon EC, de la Horra C, Medrano FJ, Lopez-Suarez A, Montes- 
Cano M, Respaldiza N, et al. Pneumocystis jirovecii isolates with 
dihidropteroate synthase (DHPS) mutations in patients with chronic 
bronchial diseases. Eur J Clin Microbiol Infect Dis. 2004;23:545-9. 

36. de la Horra C, Varela JM, Fernandez-Alonso J, Medrano FJ, 
Respaliza N, Montes-Cano M, et al. Association between human- 
Pneumocytis infection and small-cell lung carcinoma. Eur J Clin 
Invest. 2004;34:229-35. 

37. Dumolin A, Mazars E, Segury N, Gargallo- Viola V, Vargas S, Cailliez 
JC, et al. Transmission of Pneumocystis carinii disease from 
immunocompetent contacts of infected host to susceptible. Eur J Clin 
Microbiol Infect Dis. 2000;19:671-8. 

38. Sheldon WH. Subclinical Pneumocystis pneumonitis. Am J Dis 
Child. 1959;97:287-97. 

39. Atzori C, Agostine F, Angeli E, Mainini A, Orlando G, Cargnel A. 
Combined use of blood and oropharyngeal samples for noninvasive 
diagnosis of Pneumocystis pneumonia using the polymerase chain 
reaction. Eur J Clin Microbiol Infect Dis. 1998;17:241-6. 

Address for correspondence: Francisco J. Medrano, Department of 

Internal Medicine, Virgen del Rocfo University Hospital, Avda Manuel 

Siurot s/n, 41013 Seville, Spain; fax: 34-95-501-4278; email: medra- 

no@cica.es 

All material published in Emerging Infectious Diseases is in the 
public domain and may be used and reprinted without special per- 
mission; proper citation, however, is appreciated. 



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Sporadic Cryptosporidiosis 
Decline after Membrane Filtration 
of Public Water Supplies, 
England, 1996-2002 

Stella Goh,* Mark Reacher,f David P. Casemore^ Neville Q. Verlander,§ Andre Charlett,§ 
Rachel M. Chalmers, H Margaret Knowles,# Anthony Pennington,* Joy Williams,* Keith Osborn,** 

and Sarah Richardstt 


The incidence of sporadic cryptosporidiosis among 
106,000 residents of 2 local government districts in north- 
west England before and after installation of membrane fil- 
tration of public water supplies was compared to that of 
59,700 residents whose public water supplies remained 
unchanged. A national outbreak of foot and mouth disease 
in livestock during 2001 was associated with a decline in 
sporadic human cryptosporidiosis in all regions of the 
United Kingdom. In a Poisson regression model, mem- 
brane filtration was associated with an estimated 79% 
reduction (incidence ratio 0.207, 95% confidence intervals 
0.099-0.431 , p < 0.0001 ) after adjustment for the interval of 
the foot and mouth disease epidemic and the water source. 
Despite the confounding effect of that epidemic, membrane 
filtration of the public water supply was effective in reduc- 
ing the risk for sporadic human Cryptosporidium infection in 
this population. 

C ryptosporidium is a genus of enteric parasites that 
cause diarrhea in humans and many animal species 
worldwide; it is the third most common cause of non viral 
infectious diarrhea reported in England and Wales (1,2). 
Oocysts are shed in large numbers in feces of infected 
humans and animals and contain highly infectious sporo- 
zoites when ingested (1,3). Disease may be prolonged and 
fatal in immunocompromised persons (1). Crypto- 
sporidium hominis (previously designated C. parvum 


*Carlisle and District Primary Care Trust, Carlisle, United 
Kingdom; fHealth Protection Agency, Cambridge, United 
Kingdom; ^University of Wales, Aberystwyth, Ceredigion, Wales, 
United Kingdom; §Health Protection Agency Centre for Infections, 

Colindale, London, United Kingdom; ^Singleton Hospital, 
Swansea, Wales, United Kingdom; #Cumberland Infirmary, 
Carlisle, United Kingdom; **United Utilities, Great Sankey, 
Warrington, United Kingdom; and tfWest Cumberland Hospital, 
Whitehaven, United Kingdom 


genotype 1) is found in humans but occurs naturally in 
livestock animals very rarely; C. parvum (previously des- 
ignated C. parvum genotype 2) infects humans and live- 
stock (4-6). 

Cryptosporidium oocysts are a threat to the safety of 
drinking water supplies because they remain viable in 
water and damp soils for prolonged periods and are resist- 
ant to concentrations of disinfectants, including chlorine, 
used in conventional water treatment. Removal of these 
oocysts depends on sedimentation, coagulation, and filtra- 
tion (1,7,8). We have previously reported a prospective 
case-control study of risk factors for sporadic cryp- 
tosporidiosis in residents of Allerdale and Copeland local 
government districts in North Cumbria, rural northwest 
England, from March 1, 1996, to February 29, 2000. That 
study showed a strong association with the usual daily vol- 
ume of cold unboiled tap water drunk and with short visits 
to farms (9). We present extended observation of the pop- 
ulation to August 31, 2002, during which time membrane 
filtration of public drinking water supplies was introduced 
for two thirds of the study population and a national out- 
break of foot and mouth disease (FMD) in livestock 
occurred. 

Materials and Methods 

The study area comprises part of the Lake District 
National Park. The lakes act as natural reservoirs for local 
public water supplies and have livestock farms and open 
grazing land abutting them. Approximately one third of the 
study population receive public water supplies from 
Ennerdale Lake, one third from Crummock Lake, and one 
third from a number of smaller sources. From March 1, 
1996, to February 29, 2000, water from Ennerdale and 
Crummock Lakes was disinfected with chlorine, but unfil- 
tered, because the low level of particulate matter in these 


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251 


RESEARCH 


sources precluded chemically assisted flocculation. 
Membrane filtration began on March 1, 2000, at works 
treating water from Ennerdale and Crummock Lakes and 
remained active until the end of the study. The remaining 
third of the population received water from a number of 
smaller sources undergoing a variety of conventional treat- 
ments, including coagulation, filtration and chlorination, 
and chlorination alone. No changes in the treatment of 
water from these other sources occurred at any time. 

FMD Epidemic in Livestock 

The first FMD case in livestock was confirmed on 
February 21, 2001, in southeast England and the last case 
on September 30, 2001, in northwest England (10). 
Epidemic controls were enforced throughout the United 
Kingdom; they included culling livestock, excluding live- 
stock from traditional pastures, limiting livestock move- 
ments, and excluding the public from the countryside. The 
FMD epidemic was associated with marked attenuation of 
the usual spring peak in human cryptosporidiosis reporting 
from all regions of England and Wales and with a decline 
predominantly of C. parvum (livestock and human 
species). FMD epidemic controls were applied uniformly 
across the Allerdale and Copeland Districts and ended on 
January 21, 2002. 

Analysis of risk factors and laboratory testing for 
Cryptosporidium were undertaken as previously 
described; cases with date of onset from March 1 , 2000, to 
August 31, 2002, and associated controls were added (5,9). 
To determine if introduction of membrane filtration and 
the FMD epidemic in livestock were associated with a 
change in risk factors or incidence of human cryp- 
tosporidiosis, observations were divided into 5 intervals: 
before commissioning of membrane filtration (March 1, 
1996, to February 29, 2000); membrane filtration commis- 
sioning (March 1, 2000, to July 31, 2000); established 
membrane filtration before the FMD epidemic (August 1 , 
2000, to February 20, 2001); the FMD livestock epidemic 
to ending of local FMD epidemic controls (February 21, 
2001 to January 20, 2002); and post-FMD epidemic 
(January 21, 2002, to August 31, 2002). 

Case Definition 

Case-patients were residents of Allerdale or Copeland 
who had diarrhea (>3 loose stools in a 24-hour period) 
with onset from March 1, 1996, to August 31, 2002; were 
fecal smear positive for Cryptosporidium oocysts, but 
feces negative for other enteric pathogens; and had spent at 
least 1 night within the study area in the 14 days before 
onset. Patients were excluded if, within 14 days of onset, 
they had contact with another household patient with cryp- 
tosporidiosis or any diarrhea illness, traveled outside the 
United Kingdom, or traveled within the United Kingdom 


and stayed outside the study area during the entire 14-day 
period before onset of illness; or if they, or a household 
member, had already been enrolled as a case-patient or 
control at any time during the study. 

Control Definition 

Controls were residents of Allerdale or Copeland who 
had no history of diarrhea (defined as >3 loose stools in a 
24-hour period) and had spent at least 1 night within the 
study area in the 14 days before interview. Potential con- 
trols were excluded if they had traveled outside the United 
Kingdom in the 14 days before the date of interview or if 
they had traveled within the United Kingdom and stayed 
outside the study area during the entire 14 day period 
before the date of interview. Potential controls were also 
excluded if they or a household member had already been 
enrolled as a case-patient or control at any time during the 
study. 

The local water company provided details of the water 
sources, water supply zones, number of houses and resi- 
dents served by each water source and water supply zone, 
and treatment of water to each zone. The study team and 
water company maintained vigilance for changes in the 
water supplies to the population at all stages of the study. 
In a minority of participants, the water source changed; 
these case-patients and controls were categorized as 
receiving mixed supplies from >1 source. 

Risk Factor Analysis 

Five sets of contingency tables, 1 for each interval of 
observation defined by introduction of membrane filtration 
and the FMD epidemic, were constructed for each expo- 
sure variable, and odds ratios were calculated. Main effects 
variables were defined as those significant at p < 0.2 in any 
of the 5 sets of contingency tables. These variables — inter- 
val, age, sex, and water supply zone — were put into a mul- 
tivariable model. Stepwise sequential removal of variables 
with p > 0.05 was undertaken retaining time interval, age, 
sex, and water supply zone. The significance of interaction 
terms between time interval and the remaining main 
effects variables was tested in separate models with step- 
wise sequential removal of terms with p > 0.05. 

Incidence Rates and Incident Rate Modeling 

Incidence rates were determined for residents by water 
supply zone and modeled by Poisson regression using the 
number of cases as the predictor variable and the number 
of person-years of observation as the offset (11,12). The 
models had 3 predictor variables: membrane filtration 
(before and after), FMD epidemic (before, during, and 
after local FMD controls), and water source (Ennerdale, 
Crummock, and “other” water supplies). The interaction 
between these predictors was explored. The models 


252 


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Cryptosporidiosis Decline after Membrane Filtration 


provided estimates of the incidence rate ratio (IRR). The 
goodness-of-fit of the models was assessed. The species of 
Cryptosporidium isolates before and after membrane 
filtration were compared. 

Results 

Population and Water Supplies 

Public water supplies derived from Crummock Lake 
served 58,295 residents; from Ennerdale Lake, 47,780 res- 
idents; and from a variety of other smaller water sources, 
including a few private water supplies, the remaining 
59,699 residents of Allerdale and Copeland (Appendix 
Table 1; available from http://www.cdc.gov/ncidod/EID/ 
volllno2/04-0274_app.htm). Public drinking water sup- 
plies derived from Crummock and Ennerdale Lakes before 
March 1, 2000, were chlorinated but not filtered 
(Appendix Table 1). Separate membrane filtration plants at 
water treatment works at Crummock and Ennerdale Lakes 
were commissioned from March 1, 2000, to July 31, 2000; 
full operation was achieved by August 1, 2000. These 
plants remained fully operational until the end of the study, 
August 31, 2002. The treatment of water derived from 
other sources remained unchanged for the study period. 
These multiple smaller sources received a variety of con- 
ventional treatments, including coagulation, filtration and 
chlorination, and chlorination alone. In addition, a small 
number of houses had private water supplies, which were 
untreated (Appendix Table 1). 

Recruitment and Exclusion of Patients 

A total of 249 patients identified as having sporadic 
cryptosporidiosis were ascertained during the study peri- 
od; 74 (30%) were excluded, and 175 (70%) were enrolled 
(Table 1). Of the 175 primary cases of cryptosporidiosis 
enrolled, 153 (87%) had onset dates from March 1, 1996, 
to February 29, 2000, before the commissioning of mem- 
brane filtration at Crummock and Ennerdale Lakes; 22 
(13%) patients had onset from March 1, 2000, to August 
31, 2002, after the membrane filtration plants were intro- 
duced (Table 1). 

Recruitment and Exclusion of Controls 

A total of 929 potential controls were approached dur- 
ing the study; 392 (42%) were excluded, and 537 (58%) 
were enrolled. Two hundred and twenty one (24%) persons 
either refused to participate or were repeatedly unavailable 
for interview (Table 1). The address was not found for 3 
(<1%). One hundred twelve (12%) were excluded because 
they did not meet the study control definition for a variety 
of reasons (Table 1). Fifty- six (6%) were not enrolled for 
administrative reasons. The study team cancelled inter- 
views for 44 (5%) because 3 control interviews had been 


completed for the associated case and 9 (1%) interviews 
because the potential controls were found to be in the 
wrong age band; the reason for exclusion was not record- 
ed for 3 potential controls (<1%). Of the 537 controls 
enrolled, 468 (87%) had interview dates from March 1, 
1996, to February 29, 2000; and 69 (12.9%) had interviews 
from March 1, 2000, to August 31, 2002 (Table 1). 

Study Population 

Patients 

Of the 175 case-patients, 150 (86%) were <16 years of 
age, and 96 (55%) were <6 years of age. Ninety (51%) 
were male (Table 2). The proportion of cases <16 years of 
age and the proportion who were male were lower after 
membrane filtration was introduced into Crummock and 
Ennerdale Lake water. The proportion of case-patients 
served by water from other sources that never received 
membrane filtration was higher after introduction of mem- 
brane filtration (Table 2). 

In addition to diarrhea, a substantial proportion of 
patients had abdominal pain, vomiting, fever, loss of 
appetite, and weight loss (Appendix Table 2). Forty-two 
(24%) patients remained symptomatic at interview. Of the 
133 (76%) whose symptoms had abated at interview, the 
median duration of illness was 9 days (range 2-21 days) 
(Appendix Table 2). In children <6 years of age, 14 (25%) 
of 55 of boys and 6 (15%) of 41 girls were admitted to hos- 
pital because of diarrhea (Appendix Table 3). The admis- 
sion rates in children 6-15 years of age were 3 (11%) of 28 
boys and 3 (12%) of 26 girls. Most of the patients and all 
male patients <6 years of age had onset dates before the 
membrane filtration was introduced (Appendix Table 3). 
Twenty-six (17%) of the 150 patients who were <16 years 
of age were admitted to hospital, but none over this age. 

Species identification was undertaken for 68 fecal spec- 
imens from patients with onset from January 1, 1998, to 
February 29, 2000 (Appendix Table 4). Fifty-seven (84%) 
were C. parvum. Thirteen (81%) of the 16 smears derived 
from patients with onset dates from March 1, 2000, to 
August 31, 2002, were also C. parvum. Overall, 70 
(83.3%) of the 84 specimens for which the species was 
identified were C. parvum. 

Controls 

The 537 controls had similar age, sex, and drinking 
water supplies as the 175 patients (Table 2). The time 
between notification of a case by a microbiology laborato- 
ry to the study-coordinating center to enrollment of the 
patient and his or her associated controls was a median of 
2 weeks (range 1-8 weeks) and was similar before and 
after the introduction of membrane filtration. 


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253 


RESEARCH 




Table 1. Exclusions and recruitment of case-patients and controls 





n(%) 





Before and after 


Before membrane 

After membrane filtration, 

membrane filtration, 


filtration, March 1 , 1996- 

March 1 , 2000-August 31 , 

March 1, 1996- 

Exclusion criteria 

February 29, 2000 

2002 

August 31, 2002 

Case-patients 

Refused to participate 

1 (0.5) 

0(0) 

1 (0.4) 

Could not complete adequate interview 

1 (0.5) 

1 (2.4) 

2 (0.8) 

Did not respond to letters or phone calls 
Did not meet study case definition 

2(1.0) 

0(0) 

2 (0.8) 

No history of diarrhea 

1 (0.5) 

1 (2.4) 

2 (0.8) 

Mixed enteric infection 

1 (0.5) 

0 

1 (0.4) 

Secondary case 

36(17.4) 

10(23.8) 

46(18.5) 

Travel outside UK in 14 days before onset 

8 (3.9) 

8(19.0) 

16(6.4) 

Visitor to study area 

1 (0.5) 

0(0) 

1 (0.4) 

Residence outside study area 

1 (0.5) 

0(0) 

1 (0.4) 

Case-patient or household member previously 
interviewed as case or control 

2(1.0) 

0(0) 

2 (0.8) 

Potential case-patients approached 

207(100) 

42(100) 

249(100) 

Potential cases excluded 

54 (26.1) 

20 (47.6) 

74 (29.7) 

Total case-patients enrolled 

Controls 

Refused or unavailable for interview 

153 (73.9) 

22 (52.4) 

175 (70.3) 

Refused to participate 

23 (3.0) 

12(7.9) 

35 (3.8) 

Unavailable at requested interview times 

125(17.1) 

23(15.2) 

148(15.9) 

Said interview times were not convenient 

35 (4.5) 

3 (2.0) 

38 (4.1) 

Address not found 

Did not meet study control definition 

3 (0.4) 

0(0) 

3 (0.3) 

History of diarrhea 

46 (5.9) 

6 (4.0) 

52 (5.6) 

Travel outside UK in 14 days before interview 

8 (1 .0) 

4(2.6) 

12(1.3) 

Not resident in study area in 14 days before 
interview 

3 (0.4) 

1 (0.7) 

4 (0.4) 

Moved from study area 

27 (3.5) 

7(4.6) 

34 (3.7) 

Residence outside study area 

2 (0.3) 

0(0) 

2 (0.2) 

Control or household member already interviewed 
as a case or control 

7 (0.9) 

1 (0.7) 

8 (0.9) 

Not enrolled for administrative reasons or reason 
not recorded 




Interview cancelled; 3 controls already enrolled 
for associated case 

19(2.4) 

25(16.6) 

44 (4.7) 

Interview cancelled; potential control found to be 
in wrong age group 

9(1.2) 

0(0) 

9 (1 .0) 

Reason for exclusion not recorded 

3 (0.4) 

0(0) 

3 (0.3) 

Potential controls approached 

778(100) 

151 (100) 

929(100) 

Potential controls excluded 

310(39.8) 

82 (54.3) 

392 (42.2) 

Total controls enrolled 

468 (60.2) 

69 (45.7) 

537 (57.8) 


Risk Factor Analysis 

None of the interaction terms between the main effects 
variables and time intervals of observation, defined by 
introduction of membrane filtration and the FMD epidem- 
ic in livestock, were significant, including the term for the 
usual volume of cold unboiled tap water drunk per day (p 
= 0.12). These interaction terms were therefore excluded 
from the final multivariable risk factor model (Table 3). 
The risk for sporadic cryptosporidiosis was independently 
associated with the usual volume of cold unboiled tap 
water drunk each day, with contact with cattle farms and 


noncattle farms, and with feeding pets leftovers. Water 
supply zones, the time interval of observation, age, and 
gender were not independently associated with having a 
case (Table 3). 

Incidence and Seasonality 

The incidence within the populations served by public 
water supplies derived from Ennerdale Lake, Crummock 
Lake, and other water sources was similar before March 
2000 at ~22 cases per 100,000 person years but declined to 
<10 per 100,000 person years after March 1, 2000 


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Cryptosporidiosis Decline after Membrane Filtration 


(Table 4) (Figure 1). The decline was more marked in the 
populations served by water derived from treatment works 
at Ennerdale and Crummock Lakes, where membrane fil- 
tration plants had been installed, than in the population 


served by other water sources, where membrane filtration 
was not installed. A well-defined spring peak in cases was 
apparent from 1996 to 1999, but not from 2000 to 2002 
(Figures 2 and 3). 


Table 2. Baseline characteristics of case-patients and controls 

Characteristics 


n (%) 


Before membrane 
filtration, March 1, 1996- 
February 29, 2000 

After membrane 
filtration, March 1, 2000- 
August 31 , 2002 

Before and after membrane 
filtration, March 1, 1996- 
August 31 , 2002 

Case-patients, total 

153(100) 

22(100) 

175(100) 

Sex 




Female 

70 (45.8) 

15(68.2) 

85 (48.6) 

Male 

83 (54.2) 

7(31.8) 

90 (51.4) 

Age 




<1-5 

87 (56.9) 

9 (40.9) 

96 (54.9) 

6-15 

47 (30.7) 

7(31.8) 

54 (30.9) 

16+ 

19(12.4) 

6 (27.3) 

25(14.3) 

Water sources and water supply zones 




Crummock Lake 




Crummock North 

37 (24.2) 

2(9.1) 

39 (22.3) 

Crummock South 

19(12.4) 

4(18.2) 

23(13.1) 

Ennerdale Lake 




Ennerdale North 

30(19.6) 

2(9.1) 

32(18.3) 

Ennerdale South 

13(8.5) 

1 (4.5) 

14(8.0) 

Other sources 




Millom 

19(12.4) 

3(13.6) 

22(12.6) 

Quarry Hill 

16(10.5) 

6 (27.3) 

22(12.6) 

Hausegill 

3 (2.0) 

0 

3(1.7) 

Hayknott 

2(1.3) 

0 

2(1.1) 

Underscar 

1 (0.7) 

2(9.1) 

3(1.7) 

Fellside 

0 

0 

0 

Mixed >1 source 

11 (7.2) 

1 (4.5) 

12(6.9) 

Different private water supplies 

2(1.3) 

1 (4.5) 

3(1.7) 

Controls, total 

468(100) 

69(100) 

537(100) 

Sex 




Female 

234 (50) 

31 (44.9) 

265 (49.3) 

Male 

234 (50) 

38 (55.1) 

272 (50.7) 

Age 




<1-5 

273 (58.3) 

27 (39.1) 

300 (55.9) 

6-15 

137 (29.3) 

20 (29.0) 

157 (29.2) 

16+ 

58(12.4) 

12(17.4) 

70(13.0) 

Water sources and water supply zones 




Crummock Lake 




Crummock North 

104 (22.2) 

7(10.1) 

111 (20.7) 

Crummock South 

49(10.5) 

13(18.8) 

62(11.5) 

Ennerdale Lake 




Ennerdale North 

100 (21.4) 

7(10.1) 

107(19.9) 

Ennerdale South 

43 (9.2) 

3 (4.3) 

46 (8.6) 

Other sources 




Millom 

54(11.5) 

10(14.5) 

64(11.9) 

Quarry Hill 

42 (9.0) 

16(23.2) 

58(10.8) 

Hausegill 

5(1.1) 

0 

5 (0.9) 

Hayknott 

6(1.3) 

1 (1 .4) 

7(1.3) 

Underscar 

3 (0.6) 

6 (8.7) 

8(1.5) 

Fellside 

0 

2 (2.9) 

2 (0.4) 

Mixed >1 source 

1 (0.2) 

0 

1 (0.2) 

Different private water supplies 

51 (10.9) 

4(5.8) 

55(10.2) 

Crummock Lake 

10(2.1) 

0 

10(1.9) 


*Mixed: mixed supply derived from Ennerdale and Crummock, or from Ennerdale and another source, or from Crummock and another source. 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


255 


RESEARCH 


After membrane filtration was introduced for 
Crummock and Ennerdale supplies, an estimated reduction 
in incidence of -79% occurred (IRR 0.207, 95% confi- 
dence intervals [Cl] 0.099-0.431), p < 0.0001, after adjust- 
ment for the FMD epidemic interval and water source in a 
Poisson regression model (Table 5). The decrease attrib- 
uted to the FMD interval was -60% (IRR 0.394, 95% Cl 


0.167-0.925), with some evidence of a residual effect after 
the end of local FMD epidemic controls (IRR 0.686, 95% 
Cl 0.292-1.61). No additional effect was contributed by 
water source (p = 0.6). The data for this model are pre- 
sented graphically in Figure 1 and detailed in Table 4. 
Fittle difference was made by modeling the intervals for 
commissioning and postcommissioning of membrane 


Table 3. Final multivariable model of risk factors for sporadic cryptosporidiosis, Allerdale and Copeland residents, March 1 , 1 996, to 
August 31 , 2002 


Risk factors 

Case-patients 

Controls 

Adjusted odds 
ratio* 

Lower 95% Clf 

Upper 
95% Cl 

p value 

Sex 







Female 

85 

265 

1 

0.45 

1.184 

0.202 

Male 

90 

272 

0.73 




Age 







<1-5 

96 

300 

1 ,002/y 

0.938 

1.021 

0.872 

6-15 

54 

157 





16+ 

25 

70 





Water sources and water supply zones 






0.556 

Crummock Lake 







Crummock North 

39 

111 

1 




Crummock South 

23 

62 

1.262 

0.547 

2.913 


Ennerdale Lake 







Ennerdale North 

32 

107 

1.25 

0.594 

2.63 


Ennerdale South 

14 

46 

0.556 

0.164 

1.881 


Other sources 







Millom 

22 

64 

1.502 

0.686 

3.288 


Quarry Hill 

22 

58 

0.703 

0.273 

1.81 


Hausegill 

3 

5 

1.356 

0.22 

8.34 


Hayknott 

2 

7 

1.016 

0.1 

10.29 


Underscar 

3 

9 

0.983 

0.1 

9.18 


Fellside 

0 

1 

0.005 

0 

OO 


Bridgend 

0 

2 

0.004 

0 

OO 


Mixed public supplies 

12 

55 

0.893 

0.32 

2.494 


Private water supplies 

3 

10 

0.1 

0.007 

1 


Usual daily volume of cold unboiled tap 



1 .543 per pint 

1.212 

1.965 

< 0.001 

water drunk at home 







<1/4 pint 

25 

122 





1/4-1 pint 

78 

260 





>1-2 pints 

46 

106 





>2 pints 

22 

42 





Contact with a cattle farm 







Yes 

19 

29 

4.532 

1.757 

11.69 

0.002 

No 

144 

475 





Contact with a noncattle farm 







Yes 

17 

24 

3.809 

1.677 

8.651 

0.002 

No 

146 

471 





Feed pet leftovers 







Yes 

14 

19 

3.746 

1.214 

11.56 

0.021 

No 

161 

515 





Interval of study 






0.585 

March 1 , 1996-February 29, 2000 

153 

468 

1 




March 1, 2000-July 31,2000 

4 

16 

0.965 

0.235 

3.958 


August 1 , 2000-February 20, 2001 

6 

16 

1.115 

0.319 

3.895 


February 21 , 2001 -January 21 , 2002 

6 

18 

0.367 

0.078 

1.72 


January 1 1 , 2002-August 31 , 2002 

6 

19 

0.485 

0.138 

1.701 



*Adjusted for accidentally touching animal feces, feeding pets biscuits, feeding pets raw vegetables, contact with anyone outside the household with a 
history of diarrhea, type of sewage system to the house, consumption of mixed salad, and local authority of residence. 
fCI, confidence interval. 


256 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


Cryptosporidiosis Decline after Membrane Filtration 

Table 4. Incidence of sporadic cryptosporidiosis by water source, March 1 , 1 996-August 31 , 2002 

Rate per 100,000 


Water source and time intervals* 

Membrane filtration (MF) 

Cases (n) 

Person-years 

person-years 

95% Clf 

Crummock Lake 

Before MF interval 

No 

56 

233,623 

23.97 

(18.11, 31.13) 

After MF interval 

Commissioning MF 

Yes 

1 

25,726 

3.89 

(0.10, 21.66) 

Established MF pre-FMDE 

Yes 

1 

33,970 

2.94 

(0.07, 16.40) 

Established MF and FMDE 

Yes 

1 

54,902 

1.82 

(0.05, 10.15) 

Established MF and post-FMDE 

Yes 

3 

31,490 

9.53 

(1.96, 27.84) 

Total after MF 

Yes 

6 

146,088 

4.11 

(1.51, 8.91) 

Ennerdale Lake 

Before MF interval 

No 

43 

191,053 

22.51 

(16.29, 30.32) 

After MF interval 

Commissioning MF 

Yes 

0 

20,258 

0 

(0, 18.21) 

Established MF pre-FMDE 

Yes 

1 

27,223 

3.67 

(0.09, 20.47) 

Established MF and FMDE 

Yes 

2 

46,387 

4.31 

(0.52, 15.57) 

Established MF and post-FMDE 

Yes 

0 

26,606 

0 

(0, 13.86) 

Total after MF 

Yes 

3 

120,474 

2.49 

(0.51, 7.28) 

Other sources 

Before MF interval 

No 

54 

238,265 

22.66 

(17.03, 29.57) 

After MF interval 

Commissioning MF 

No 

3 

24,449 

12.27 

(2.53, 35.86) 

Established MF pre-FMDE 

No 

4 

32,257 

12.4 

(3.38,31.75) 

Established MF and FMDE 

No 

3 

51,997 

5.77 

(1.19, 16.86) 

Established MF and post-FMDE 

No 

3 

29,824 

10.06 

(2.07, 29.40) 

Total after MF 

No 

13 

138,527 

9.38 

(5.00,16.05) 


*Time intervals. pre-MF (membrane filtration) March 1 , 1996-February 29, 2000; post-MF March 1 , 2000-August 31 , 2002. Post-MF comprises the 
following: commissioning MF, March 1 , 2000-July 31 , 2000; established MF before foot and mouth disease epidemic (FMDE), August 1 , 2000-February 
20, 2001 ; established MF and FMDE, February 21 , 2001-January 20, 2002; established MF and post-FMDE, January 21 , 2002-August31 , 2002. 
fCI, confidence intervals. 


filtration separately, and no significant difference was seen 
between these rate estimates when this modeling was done 
(p = 0.35) 

Discussion 

Consumption of cold unboiled tap water from public 
drinking water supplies was shown to be a leading inde- 
pendent risk factor for sporadic cryptosporidiosis with a 
highly significant increase in risk with the usual volume 
drunk each day (Table 3). Risk was also increased by con- 
tact with cattle farms and noncattle farms and with feeding 
pets leftovers. Fifty five percent of patients were <6 years 
of age, and 31% were 6-15 years. Infection was predomi- 
nantly with C. parvum (livestock and human species). The 
results of the risk factor analysis for the entire study peri- 
od were similar to those obtained for the interval before 
installation of membrane filtration, when most cases arose 
(9). Illness was prolonged and almost one fifth of children 
<6 years of age required hospital admission. The excess in 
hospitalization in boys <6 years of age may suggest that 
young boys are more vulnerable to Cryptosporidium than 
young girls, a bias in favor of admitting young boys, or a 
combination of these factors. 

The 2001 FMD epidemic in livestock, which occurred 
after membrane filtration was introduced in Allerdale and 


Crummock Lake water, affected all regions of the United 
Kingdom (10). This livestock epidemic was associated 
with a highly significant decline in laboratory reports of 
human cryptosporidiosis from all regions of England and 
Wales and was more marked in Northwest England. The 
decline in reports was most marked for C. parvum (the 
species infectious in humans and livestock species) than 
for C. hominis (infectious only in humans) (10). The FMD 
epidemic control measures of excluding the public from 
the countryside, extensively culling farm animals, and lim- 
iting animal movements probably decreased direct and 
indirect exposure of the human population to livestock and 
livestock feces. 

The annual agricultural and horticultural census con- 
ducted by the U.K. Department for Environment, Food and 
Rural Affairs and its predecessor, the Ministry of 
Agriculture, Food and Fisheries showed >600,000 sheep, 
300,000 lambs, 100,000 total cattle and calves, and 40,000 
calves <1 year of age in each of the years 1996-2000 with- 
in the 135,000 hectares of agricultural land in Allerdale 
and Copeland local government districts (13,14). A sub- 
stantial decline occurred in 2001 and 2002 associated with 
the FMD epidemic, but no evidence suggested that the 
decline in animal densities or change in human contact 
with livestock and with the countryside differed within 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


257 


RESEARCH 


30 



Mar 1 , 1 996 - Feb 29. 2000 Mar 1 . 2000 - Aug 31 . 2002 


Figure 1. Cases of primary cryptosporidiosis per 100,000 person- 
years before and after membrane filtration introduced into public 
water supplies, derived from Crummock Lake, Ennerdale Lake, 
and other water sources. 


Allerdale and Copeland, according to the sources or distri- 
bution of the public drinking water supplies (13,14). The 
decline in incidence attributable to the FMD epidemic 
effect was therefore expected for our entire study popula- 
tion, regardless of its household water supply. We therefore 
believe that the experience of the population served by 
other supplies provided a valid measure of the impact of 
the FMD epidemic in livestock, whereas the population 
served by water from Ennerdale and Crummock Lakes 
experienced the effect of both membrane filtration and the 
FMD epidemic. The results of the Poisson regression 
model indicated a marked reduction of incidence in spo- 
radic cryptosporidiosis following introduction of mem- 
brane filtration after adjustment for the FMD epidemic 
interval and water source (Table 5). Despite the confound- 
ing effect of the FMD epidemic, our study provides con- 
vincing evidence that membrane filtration was highly 
effective in reducing the risk for sporadic cryptosporidio- 
sis in this population; this measure was also associated 
with a decline in hospital admissions for cryptosporidiosis 
in children, especially of boys <6 years of age. 

The incidence rates associated with other supplies from 
a number of different sources and treatment works, some 
using conventional flocculation and filtration, were similar 
from March 1996 to February 2001 to the rates in the pop- 



Figure 2. Average number of cases by month of onset before 
membrane filtration, March 1, 1996-February 29, 2000, Allerdale 
and Copeland local government districts. 



Figure 3. Average number of cases by month of onset after mem- 
brane filtration introduced March 1, 2000, to August 31, 2002, 
Allerdale and Copeland local government districts. 


ulation served by Crummock and Ennerdale Lakes, whose 
water was unfiltered at this time. This finding supports the 
notion that conventional sand filtration and flocculation 
may be insufficient to prevent intermittent low-level 
Cryptosporidium oocyst contamination of treated water. 
The local water company has since closed higher risk 
sources and substituted them with water from lower risk 
catchments. 

Our observations strongly support recent revision of the 
UK drinking water regulations requiring water companies 
to undertake risk assessments of water sources, and where 
judged to be a risk, to implement continuous monitoring of 
Cryptosporidium oocyst concentrations in treated water 
(15). A minimum standard of an average of <1 oocyst per 
10 L of water in any 24-hour period is required. 


Table 5. Poisson regression model of the incidence of sporadic cryptosporidiosis* 

Predictor 

Category 

IRR 

95% Cl 

P 

Membrane filtration 

No 

Reference 




Yes 

0.207 

0.099-0.431 

<0.0001 

Foot and mouth disease epidemic 

Pre 

Reference 




During 

0.394 

0.167-0.925 



Post 

0.686 

0.292-1.612 

0.05 

Water supply 

Crummock 

Reference 




Ennerdale 

0.907 

0.620-1 .329 



Other 

0.820 

0.573-1.174 

0.6 


*IRR, incidence rate ratio; Cl, confidence interval. Goodness-of-fit test (chi square 10.84, 9 df, p = 0.3). 


258 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


Cryptosporidiosis Decline after Membrane Filtration 


The substantial negative impact of waterborne cryp- 
tosporidiosis leading to potentially life-threatening diar- 
rhea and stunting in childhood is well-recognized in 
developing countries (16,17). Our findings show that 
Cryptosporidium remains an obstacle in water and sanita- 
tion infrastructure and a threat to child health in industrial- 
ized countries as well. The scale of this effect will continue 
to be underestimated if adequate surveillance of cryp- 
tosporidiosis by testing diarrheal feces specimens for 
Cryptosporidium and collation of positive test results, 
especially in children, is omitted by health services (18). 
Although the study population was located in an area of 
livestock farming with high historic rates of cryp- 
tosporidiosis in England, the demonstration that public 
drinking water supplies were a leading independent risk 
factor for sporadic cryptosporidiosis and that introduction 
of membrane filtration at water treatment works was effec- 
tive in substantially lowering this risk, may have relevance 
to water companies, regulators, policymakers, and con- 
sumers in other countries. 

Acknowledgments 

We thank colleagues in the steering group and in the many 
local and national agencies that contributed to this research. In 
particular, we thank Brian White, Vic Emmerson, Mary 
Cosgrove, Peter Daley, John Cain, Rachel Horton, Susan 
Partridge, Judith Hilton, Patrick Wall, Ros Stanwell-Smith, 
James Stuart, Claire Gilham, Alan Godfree, Matthew Wilkinson, 
Charmian Kerr, David Counter, Andrew Holliman, John Gray, 
Tony Lloyd, Mark Smith, David Holt, Joy Graham, Jennifer 
Clay, Paul Blaylock, Richard Lamb, Dennis Massey, Emma 
Wigginton, Kristin El win, and Anne Thomas. 

This study was funded by the Department of Environment, 
Food and Rural Affairs, Department Health, United Kingdom 
Water Industry Research Limited and was supervised by the 
Drinking Water Inspectorate. The Membrane Filtration plant 
installed in this study was provided by Memcor Ltd. None of the 
authors has any financial links with Memcor Ltd. No funding was 
obtained from Memcor Ltd. for this study. 

Dr. Goh is an honorary consultant to the Carlisle and District 
Primary Care Trust and was formerly the consultant in 
Communicable Disease Control to North Cumbria Health 
Authority, in northwest England. She specialized in public health 
medicine and developed an interest in childhood immunization, 
enteric infection, Cryptosporidium , and water supplies. 


References 

1. Guerrant RL. Cryptosporidiosis: an emerging highly infectious 
threat. Emerg Infect Dis. 1997;3:51-7. 

2. Health Protection Agency. Trends in selected gastrointestinal infec- 
tions-2001. Comm Dis Rep CDR Weekly. 2003 [accessed 14 Dec 
2002]. Available from http://www.hpa.org.uk/cdr/PDfiles/2002/ 
cdr0702.pdf 

3. Fayer R, Morgan.U, Upton SJ. Epidemiology of Cryptosporidium : 
transmission, detection, and identification. Int J Parasitol. 
2000;30:1305-22. 

4. Peng MM, Xiao L, Freeman AR, Arrowood MJ, Escalante AA, 
Weltman AC, et al. Genetic polymorphism among Cryptosporidium 
parvum isolates: evidence of two distinct human transmission cycles. 
Emerg Infect Dis. 1997;3:567-73. 

5. Spano F, Putigagni L, McLauchlin J, Casemore DP, Crisanti A. PCR- 
RFLP analysis of the Cryptosporidium oocyst wall protein (COWP) 
gene discriminates between C. wrairi and C. parvum , and between C. 
parvum isolates of human and animal origin. FEMS Microbiol Lett. 
1997;150:209-17. 

6. Xiao L, Fayer R, Upton SJ. Cryptosporidium taxonomy: recent 
advances and implications for public health. Clin Microbiol Rev. 
2004;17:72-97. 

7. Korich DG, Mead JR, Madore MS, Sinclair NA, Sterling CR. Effects 
of ozone, chlorine dioxide, chlorine, and monochloramine on 
Cryptosporidium parvum oocyst viability. Appl Environ Microbiol. 
1990;56:1423-8. 

8. Meinhardt PL, Casemore DP, Miller KB. Epidemiologic aspects of 
human cryptosporidiosis and the role of waterborne transmission. 
Epidemiol Rev. 1996;18:118-36. 

9. Goh S, Reacher M, Casemore DP, Verlander NQ, Chalmers R, 
Knowles M, et al. Sporadic cryptosporidiosis, North Cumbria, 
England, 1996-2000. Emerg Infect Dis. 2004;10:1007-15. 

10. Smerdon WJ, Nichols T, Chalmers RM, Heine H, Reacher MH. Foot 
and mouth disease in livestock and reduced cryptosporidiosis in 
humans, England and Wales. Emerg Infect Dis. 2003;9:22-8. 

11. Clayton D, Hills M. Chapter 23: Poisson and logistic regression. 
Statistical models in epidemiology. Oxford: Oxford University Press; 
1993. p. 227-36. 

12. Stata Corp. Poisson regression. College Station (TX): Stata 
Corporation; 2003. 

13. Goh S. Animal cryptosporidiosis. North Cumbria sporadic cryp- 
tosporidiosis study. Carlisle, U.K.: Carlisle and District Primary Care 
Trust; 2004. p. 209-24. 

14. Agricultural and Horticultural Annual Census Parish group data 
(excluding minor holdings). York: Department for Environment, 
Food and Rural Affairs; 2004. 

15. The Stationery Office. The water supply (water quality) 
(Amendment) regulations 2000 statutory instrument No. 3184. 
London: The Stationery Office; 1999. [accessed 12 Dec 2004]. 
Available from http://www.dwi.gov.uk/regs/si3184. 

16. Checkley W, Gilman RH, Black RE, Epstein L, Carbrera L, Sterling 
CR. Effect of water and sanitation on childhood health in a poor 
Peruvian peri-urban community. Lancet. 2004;363:112-8. 

17. Dillingham R, Guerrant RL. Childhood stunting: measuring and 
stemming the staggering costs of inadequate water and sanitation. 
Lancet. 2004;363:94-5. 

18. Crook P, Mayon-White R, Reacher M. Enhancing surveillance of 
cryptosporidiosis: test all faecal specimens from children. Commun 
Dis Public Health. 2002;5:112-3. 


The opinions expressed by authors contributing to this journal do 
not necessarily reflect the opinions of the Centers for Disease 
Control and Prevention or the institutions with which the authors 
are affiliated. 


Address for correspondence: Mark Reacher, Health Protection Agency, 
East of England, Institute of Public Health, University Forvie Site, 
Robinson Way, Cambridge CB2 2SR, United Kingdom; fax: 44 0 1223 
331865; email: mark.reacher@hpa.org.uk 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


259 


RESEARCH 


Carbapenemase-producing 
Enterobacteriaceae, U.S. Rivers 

Cecile Aubron,* Laurent Poirel,* Ronald J. Ash,f and Patrice Nordmann* 


Our study was initiated by previous isolation of 30 
imipenem-resistant, gram-negative rods from 7 of 16 U.S. 
rivers sampled from 1999 to 2001. Imipenem hydrolysis 
was detected in 22 of those isolates identified as 
Enterobacter asburiae. Random amplified polymorphism 
DNA analysis showed that these E. asburiae isolates were 
genetically indistinguishable. An identical clavulanic 
acid-inhibited p-lactamase IMI-2 was identified from each 
isolate that shared 99% and 97% amino acid identity with 
the chromosome-encoded p-lactamases IMI-1 and NmcA, 
respectively, from E. cloacae clinical isolates. The b/alMI-2 
gene was located on a self-transferable 66-kb plasmid. 
Sequence analysis of a cloned 5.5-kb DNA fragment 
obtained from 1 of the imipenem-resistant E. asburiae iso- 
lates identified an upstream LysR-type regulator gene that 
explained inducibility of IMI-2 expression. p-Lactamase IMI- 
2 is the first inducible and plasmid-encoded carbapene- 
mase. Identification of clonally related E. asburiae isolates 
from distant rivers indicates an environmental and enter- 
obacterial reservoir for carbapenemase genes. 

C arbapenems, such as imipenem and meropenem, are 
the most potent p-lactam antimicrobial drugs for 
avoiding resistance in gram-negative rods. Resistance to 
carbapenems is rare in Enterobacteriaceae and may be 
mediated by 3 mechanisms: hyperproduction of an AmpC- 
type cephalosporinase combined with decreased drug per- 
meability through the outer membrane, decreased affinity 
of penicillin-binding proteins that constitute target proteins 
for carbapenems, and carbapenem-hydrolyzing p-lacta- 
mases (1-3). These rare carbapenemases may be either 
plasmid-mediated metallo- p-lactamases (IMP- and VIM- 
type) or chromosomally encoded and clavulanate-inhibited 
enzymes (NmcA, IMI-1, Sme-l/Sme-2) (2,4-9). The latter 
group of enzymes shares consistent percentage of identity 
and belongs to the Ambler class A of p-lactamases (2,10). 
Very recently, plasmid-mediated and clavulanate-inhibited 
carbapenemases have been reported as a source of nosoco- 
mial infections in U.S. hospitals (11-15). 


*University Paris XI, Paris, France; and fWashburn University, 
Topeka, Kansas, USA 


While the role of animals in the emergence of clinical- 
ly important, antimicrobial-resistant strains has been 
extensively shown (e.g., in Salmonella spp.), the role of 
aquatic environment as a reservoir of antimicrobial-resist- 
ance genes is less established (16-21). A recent study 
described high levels of antimicrobial-resistant strains 
from U.S. rivers (22). We identified the imipenem-resist- 
ant, gram-negative strains recovered from that study and 
analyzed the molecular mechanism involved in carbapen- 
em resistance of the imipenem-resistant enterobacterial 
strains. Clonally related Enterobacter asburiae strains 
were identified in midwestern U.S. rivers. E. asburiae nat- 
urally produces a cephalosporinase but no carbapenemase 
and may be responsible for nosocomial infections (23). 
Here, the strains expressed a novel plasmid-encoded and 
clavulanate-inhibited carbapenemase. 

Materials and Methods 

Bacterial Isolates 

A previous study identified 30 imipenem-resistant, 
gram-negative strains out of 1,861 ampicillin-resistant, 
gram-negative isolates from 7 out of 16 U.S. rivers that 
were sampled from 1999 to 2001 (22). Identification of 
these imipenem-resistant isolates was performed by con- 
ventional biochemical techniques (API-20E and API-NE 
systems [bioMerieux, Marcy-l’Etoile, France]), and con- 
firmed by 16S rDNA sequencing (24). 

E. asburiae CIP 103358 and E. asburiae CIP 105006 
were used as reference strains (Institut Pasteur strain col- 
lection, Paris, France). E. cloacae NOR-1 and E. cloacae 
1413B were used as strains that produce the chromosome- 
encoded, clavulanate-inhibited carbapenemases NmcA 
and IMI-1, respectively (5,8). One of the E. asburiae iso- 
lates recovered from a river (strain MS7) was used for 
cloning experiments. Streptomycin-resistant Escherichia 
coli DH10B strain was used in cloning and conjugation 
experiments (Life Technologies, Eragny, France). 

Antimicrobial Agents and Resistance Study 

The antimicrobial agents and their sources were as 


260 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


Carbapenemase in Enterobacteriaceae, U.S. Rivers 


follows: amoxicillin, ceftazidime, clavulanic acid, and 
ticarcillin (GlaxoSmithKline, Nanterre, France); aztreon- 
am (Bristol-Myers Squibb, Paris La Defense, France); 
cephalothin (Eli Lilly, Saint-Cloud, France); piperacillin 
and tazobactam (Lederle, Les Oullins, France); cefotaxime 
(Aventis, Romainville, France); imipenem (without cilas- 
tatin) (Merck Sharp and Dohme, Paris, France); meropen- 
em (AstraZeneca, Paris, France); ampicillin and 
streptomycin (Sigma, Paris, France). 

MICs were determined by an agar dilution technique on 
Mueller-Hinton (MH) agar (Sanofi Diagnostics Pasteur, 
Marnes-La-Coquette, France) with an inoculum of 10 4 
CFU per spot (25). Carbapenemase activity was deter- 
mined by UV spectrophotometry with culture extracts of 
each of the imipenem-resistant, gram-negative rods and 
imipenem (100 pmol) as substrate, as reported previously 

(26) . One unit of enzyme activity corresponded to the 
hydrolysis of 1 pmol of substrate per min. Inducibility of 
the (3-lactamase expression was determined with imipen- 
em and cefoxitin as (3-lactamase inducers, as described 

(27) . Briefly overnight culture of each imipenem-resistant 
E. asburiae isolate was diluted (1: 10) in a prewarmed tryp- 
ticase soy broth, allowed to culture in an antimicrobial-free 
medium for 2 h, and further cultured for 6 h with cefoxitin 
(2-50 mg/L) or imipenem (10-50 mg/L). (3-Lactamase 
culture extracts were obtained after centrifugation and son- 
ication, as detailed (26). 

Nucleic Acid Techniques and Conjugation 

Genotype comparison of the imipenem-resistant E. 
asburiae strains was performed by using the random 
amplified polymorphism detection (RAPD) technique as 
described with primer 6MW (CCGACTCGAG 
NNNNNNATGTGG) and primers UBC 245 and UBC 282 
(26,28,29). Transfer of the imipenem resistance marker 
from each imipenem-resistant E. asburiae isolate to E. coli 
DH10B was attempted by using the immobilization filter 
mating out technique, as described (26). Briefly, equal vol- 
ume (0.1 mL) of overnight cultures of each E. asburiae 
isolate and E. coli DH10B were put onto a paper filter that 
was placed on an MH agar plate. Twenty-four hours later, 
the filter was removed, washed with water (0.2 mL), and 
the bacterial suspension was spread onto MH agar plates 
containing ampicillin (100 mg/L) and streptomycin (50 
mg/L) for selecting transconjugants after 24 h (26). 

Plasmid extraction was performed for each E. asburiae 
strain and their transconjugants and compared to reference 
plasmid sizes of E. coli NCTC 50192 by using the Kieser 
technique designed to extract large size plasmids (30,31). 
Whole-cell DNA of Enterobacter spp. reference strains 
and of an E. asburiae strain MS7 was extracted as 
described (26). 


Southern hybridization of plasmid DNA (26) of the 
transconjugants was performed as described by the manu- 
facturer with the ECL nonradioactive kit (Amersham, Les 
Ulis, France). An 818-bp internal probe for bla lM M was 
obtained by using primers IMI- A (5'-ATAGCCATC- 
CTTGTTTAGCTC-3') and IMI-B (5'-TCTGCGAT- 
TACTTTATCCTC-3') and standard polymerase chain 
reaction (PCR) amplification procedures (5,26). 

Primers designed to hybridize to the ends of the 
bla NmcA , bla miA , and bla Sme _ l/Sme _ 2 genes were used for 
standard PCR amplification experiments (5,7,8) with plas- 
mid DNA of each imipenem-resistant E. asburiae isolate 
and of their transconjugants as templates. Cloning experi- 
ments were then performed with BamRl restricted whole- 
cell DNA of E. asburiae MS7 followed by ligation of 
DNA fragments into the ZtaraHI-site of cloning vector 
pGB2 (32). Recombinant plasmids were transformed by 
electroporation into E. coli DH10B electrocompetent cells 
(26). E. coli DH10B harboring recombinant plasmids was 
selected on MH agar plates containing ampicillin (100 
mg/L) and streptomycin (100 mg/L). 

DNA sequencing of both strands of PCR fragments 
amplified with the primers for bla lMlA and plasmid DNA of 
E. asburiae isolates as templates and of the cloned frag- 
ment of a recombinant plasmid was determined with an 
Applied Biosystems sequencer (ABI377). The nucleotide 
sequences and the deduced protein sequences were ana- 
lyzed with software available on the Internet from the 
National Center for Biotechnology Information Web site 
(http://www.ncbi.nlm.nih.gov/BLAST). 

Results 

Bacterial Identification 

Twenty-nine of the 30 imipenem-resistant isolates sub- 
stantially hydrolyzed imipenem, i.e., 10.5 + 1.6 U/mg of 
protein of culture extracts. These isolates were a single 
Aeromonas hydrophila isolate, 6 Stenotrophomonas mal- 
tophilia isolates known to naturally produce carbapene- 
mases, and 22 Enterobacter spp. isolates identified as E. 
asburiae that were further analyzed. 

As reported in Table 1 , E. asburiae strains were isolat- 
ed at different times from several rivers in the midwest. 
Other tested rivers had ampicillin-resistant isolates that 
were not imipenem-resistant (Figure). These rivers were 
Arkansas (Little Rock), Canadian (Oklahoma City), 
Hudson (New York), Chicago (Chicago), Colorado 
(Glenwood Springs), Missouri (Parkville), Cuyahoga 
(Cleveland), Mississippi (New Orleans, St. Louis), Ohio 
(Cincinnati, Louisville, Pittsburgh, Wheeling), Platte 
(Grand Island), Scioto (Columbus), Wabash (Terre Haute), 
and White (Indianapolis). RAPD analysis was then 


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261 


RESEARCH 


Table 1 . Origin and date of isolation of imipenem-resistant Enterobacter asburiae environmental isolates 


River (city) 

Isolate 

Date 

Arkansas River (Wichita, KS) 

E. asburiae AK1 

September 1999 

Kansas River (Topeka, KS) 

E. asburiae K1-K5 

September 2000 

Des Moines River (Des Moines, IA) 

E. asburiae DM1 -DM8 

August 2001 

Mississippi River (Minneapolis, MN) 

E. asburiae MS1-MS8 

August 2001 


performed to compare all imipenem-resistant E. asburiae 
isolates. Using a series of different primers, this genotyp- 
ing technique identified clonally indistinguishable E. 
asburiae isolates, although they were from various geo- 
graphic origins (data not shown). 

p-Lactam Resistance Marker 

The imipenem resistance marker was transferred from 
each imipenem-resistant E. asburiae isolate to E. coli 
DH10B by conjugation. Plasmid analysis identified a 66- 
kb plasmid (pNat) from cultures of each imipenem-resist- 
ant E. asburiae isolate, whereas this plasmid was not 
isolated from E. cloacae and E. asburiae reference strains 
(data not shown). PCR experiments with primers for the 
bid imi_i gene were positive with plasmid DNA of each E. 
asburiae isolate and transconjugants as templates, where- 
as primers designed to amplify bla NmcA and bla Sme _ 1/Sme _ 2 
failed to give PCR product. The Southern blot analysis 
confirmed that the Z?/< 2 IMI -like gene was located on the nat- 
ural plasmid pNat (data not shown). 

Sequencing PCR products with primers hybridizing at 
the ends of the bla lMlA gene and plasmid DNA of each 
imipenem-resistant E. asburiae isolate identified the same 
p-lactamase IMI-2 in all cases. This novel enzyme had 2 
amino acid substitutions (tyrosine to histidine at position 
Ambler 105 and asparagine to aspartic acid at position 
Ambler 35) compared to the chromosomally encoded car- 
bapenemase IMI-1 (5). p-Lactamase IMI-1 had been iso- 
lated from an E. cloacae isolate from Minnesota close to 
locations where IMI-2-producing isolates have been found 
(5). However, the bla lMl _ 2 gene was not just a point-mutant 
derivative of the bla lMlA gene, since these genes differ by 
11 nucleotide substitutions. p-Lactamase IMI-2 was also 
related to NmcA (97% amino acid identity) (8). 



Figure. Sites of isolation of IMI-2-producing Enterobacter asburiae 
isolates (black circles) and ampicillin-resistant, gram-negative rods 
(white circles). 


Cloning ZtomHI-restricted DNA of whole-cell DNA of 
E. asburiae MS7 gave recombinant plasmid pIMI-2 that 
had a 5. 5 -kb insert that allowed identification of the sur- 
rounding sequence of the bla lMl _ 2 gene. A gene encoding a 
LysR-type regulator named IMIR-2 was found just 
upstream of bla lMl _ 2 . It shared 95% amino acid identity 
with IMIR-1, which is located upstream of the bla lMlA 
gene (5). The surrounding sequences of bla lMl _ 2 shared sig- 
nificant nucleotide identity with transposable elements. 
Part of an open reading frame that shared 97% nucleotide 
identity with that of the transposase gene tnpA of the trans- 
poson Tn2501 (TnJ family) was identified downstream of 
bl a imi -2 ( 3 ^)- Upstream of imiR-2 , a 142-bp sequence 
shared 76% nucleotide identity with part of the insertion 
sequence IS2. 

Susceptibility Testing and Expression of Resistance 

MICs of several P-lactams, including carbapenems for 
the IMI-2-positive E. asburiae MS7 and for E. coli 
DH10B expressing the bla lMl _ 2 gene were high (Table 2). 
The MICs of p-lactams for all imipenem-resistant clinical 
isolates were identical (data not shown). Much higher level 
of resistance to aztreonam than to expanded- spectrum 
cephalosporins was found for the IMI-2-positive strains, 
as reported for the other producers of class A carbapene- 
mases (2). The activity of p-lactamase IMI-2 was partially 
inhibited by clavulanate and tazobactam. Induction studies 
showed increase of p-lactamase expression from 17- to 30- 
fold (170 to 300 U/mg of protein) (for each E. asburiae 
isolate when imipenem (50 mg/L) and cefoxitin (50 mg/L) 
were used as inducers, respectively. These induction 
results were consistent with location and functionality of a 
LysR-type regulator gene upstream of the bla lMl _ 2 gene in 
the imipenem-resistant E. asburiae isolates. No other 
antimicrobial resistance marker was carried by natural 
plasmid pNat. 

Discussion 

This report indicates that several U.S. rivers may be a 
reservoir for broad- spectrum carbapenemases. Here, we 
report a novel clavulanic-acid inhibited Ambler class A p- 
lactamase IMI-2 that has an usual spectrum of hydrolysis 
for this type of p-lactamase, including penicillins, car- 
bapenems, and aztreonam (2). p-Lactamase IMI-2 is close- 
ly related to several Ambler class A carbapenemases whose 
genes are chromosomally located, including bla lMlA and 
bla N mcA , and found in several clinical isolates (5,8). While 


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Carbapenemase in Enterobacteriaceae, U.S. Rivers 


Table 2. MICs (mg/L) of p-lactams for several carbapenemase producers and reference strain Escherichia coli DH1 OB 


p-Lactam(s)* 

Enterohacter 
asburiae MS7| 

E. cloacae 
1413BT 

Escherichia coli 
DH10B (pNat)t 

E. coli DH10B 
(plMI-2)t 

E. coil DH10B 

Amoxicillin 

>512 

>512 

>512 

>512 

4 

Amoxicillin + CLA 

>512 

>512 

>512 

>512 

4 

Ticarcillin 

128 

>256 

128 

256 

4 

Ticarcillin + CLA 

16 

>256 

16 

32 

4 

Piperacillin 

16 

>256 

8 

128 

2 

Piperacillin + TZB 

4 

>256 

2 

16 

2 

Cephalothin 

512 

>256 

64 

512 

4 

Cefotaxime 

0.06 

1 

0.06 

1 

0.06 

Ceftazidime 

0.12 

2 

0.06 

0.5 

0.25 

Aztreonam 

4 

8 

4 

64 

0.12 

Imipenem 

>64 

>64 

16 

>64 

0.06 

Meropenem 

32 

4 

2 

32 

0.06 


*CLA, clavulanic acid at a fixed concentration of 2 mg/L; TZB, tazobactam at a fixed concentration of 4 mg/L. 

^Enterohacter asburiae MS7 produces acquired p-lactamase IMI-2, whereas E, cloacae 1413B produces acquired p-lactamases TEM-1 and IMI-1 (5). 
^Natural plasmid pNat harbors the Wai M i -2 gene, whereas plMI-2 is a recombinant plasmid that has the same p-lactamase gene. 


this work was in progress, a clinical case of an NmcA- 
producing E. cloacae isolate was reported from Seattle 
(34). An extended epidemiologic survey identified Sme-1 
type-producing Serratia marcescens isolates from the 
West Coast to the East Coast, which indicates that these 
isolates may also represent a reservoir for carbapenemases 
in Enterobacteriaceae (9). Thus, identification of car- 
bapenemase genes in enterobacterial strains from rivers 
may have clinical importance. 

In the present study, the (3-lactamase gene was plasmid- 
encoded and was adjacent to mobile sequences that may 
play an additional role in gene transfer. The E. asburiae 
isolates were clonally related and may correspond to a sin- 
gle clone, although they were obtained from distantly 
related midwestern rivers. The reason for the presence of 
these antimicrobial-resistant strains in this region is 
unknown. Taking into account the small number of speci- 
mens withdrawn from the rivers and the selection tech- 
nique for imipenem-resistant isolates (ampicillin- and not 
imipenem-containing plates), the prevalence of carbapene- 
mase-producing enterobacterial strains may be high in the 
environment, at least in the Midwest. 

Cloning experiments led to identification of a regulato- 
ry gene from an E. asburiae strain (found in the other E. 
asburiae strains as well [data not shown]) that explained 
inducibility of carbapenemase expression. Whatever the 
level of imipenem resistance is, failure of an imipenem- 
containing regimen may occur when treating infections 
caused by similar carbapenemase-producing strains, as 
deduced from results obtained with an animal model of 
pneumonia (35). Finally, this study raises the question of 
the importance of this reservoir in Enterobacteriaceae as 
well as the origin of this plasmid-located carbapenemase 
gene that may be transferred among other enterobacterial 
pathogens. 


Acknowledgement 

We thank K. Bush for providing E. cloacae 1413B that pro- 
duced the chromosome-encoded, clavulanate-inhibited carbapen- 
emase IMI- 1 . 

This work was funded by a grant from the Ministere de 
T Education Nationale et de la Recherche, (UPRES EA 3539) 
Universite Paris XI, Paris, France, and by the European 
Community (6th PCRD, LSHM-CT-2003-503-335). 

Dr. Aubron is studying antimicrobial resistance mechanisms 
at the Hospital Bicetre, South-Paris Medical School, University 
Paris XI, France. She is a resident specializing in infectious dis- 
eases. 

References 

1. Lee EH, Nicolas MH, Kitzis MD, Pialoux G, Collatz E, Gutmann L. 
Association of two resistance mechanisms in a clinical isolate of 
Enterohacter cloacae with high level resistance to imipenem. 
Antimicrob Agents Chemother. 1991;35:1093-8. 

2. Nordmann P, Poirel L. Emerging carbapenemases in gram-negative 
aerobes. Clin Microbiol Infect. 2002;8:321-31. 

3. De Champs C, Henquell C, Guelon D, Sirot D, Gazuy N, Sirot J. 
Clinical and bacteriological study of nosocomial infections due to 
Enterohacter aerogenes resistant to imipenem. J Clin Microbiol. 
1993;31:123-7. 

4. Nordmann P, Mariotte S, Naas T, Labia R, Nicolas MH. Biochemical 
properties of a carbapenem-hydrolyzing (3-lactamase from 
Enterohacter cloacae and cloning of the gene into Escherichia coli. 
Antimicrob Agents Chemother. 1993;37:939-46. 

5. Rasmussen BA, Bush K, Keeney D, Yang Y, Hare R, O’Gara C, et al. 
Characterization of IMI-1 (3-lactamase, a class A carbapenem- 
hydrolyzing enzyme from Enterohacter cloacae. Antimicrob Agents 
Chemother. 1996;40:2080-6. 

6. Naas T, Livermore DM, Nordmann P. Characterization of an LysR 
family protein, SmeR from Serratia marcescens S6, its effect on 
expression of the carbapenem-hydrolyzing (3-lactamase Sme-1, and 
comparison of this regulator with other (3-lactamase regulators. 
Antimicrob Agents Chemother. 1995;39:629-37. 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


263 


RESEARCH 


7. Naas T, Vandel L, Sougakoff W, Livermore DM, Nordmann P. 
Cloning and sequence analysis of the gene for a carbapenem- 
hydrolyzing class A (3-lactamase, Sme-1, from Serratia marcescens 
S6. Antimicrob Agents Chemother. 1994;38:1262-70. 

8. Naas T, Nordmann P. Analysis of a carbapenem- hydrolyzing class A 
(3-lactamase from Enterobacter cloacae and of its LysR-type regula- 
tory protein. Proc Natl Acad Sci USA. 1994;91:7693-7. 

9. Queenan AM, Torres- Vierra C, Gold HS, Carmeli Y, Eliopoulos GM, 
Moellering Jr RC, et al. SME-type carbapenem-hydrolyzing class A 
(3-lactamases from geographically diverse Serratia marcescens 
strains. Antimicrob Agents Chemother. 2000;44:3035-9. 

10. Ambler RP, Coulson AF, Frere JM, Ghuyssen JM, Joris B, Forsman 
M, et al. A standard numbering scheme for the class A beta-lactamase. 
Biochem J. 1991;276:269-72. 

11. Miriagou V, Tzouvelekis LS, Rossiter S, Tzelepi E, Angulo FJ, 
Whichard JM. Imipenem resistance in Salmonella clinical strain due 
to plasmid-mediated class A carbapenemase KPC-2. Antimicrob 
Agents Chemother. 2003;47:1297-300. 

12. Yigit H, Queenan AM, Anderson GJ, Domenech-Sanchez A, Biddle 
JW, Steward CD, et al. Novel carbapenem-hydrolyzing (3-lactamase, 
KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae. 
Antimicrob Agents Chemother. 200 1 ;45: 1151—61. 

13. Smith-Moland E, Black JA, Ourada J, Reisbig MD, Hanson ND, 
Thomson KS. Occurrence of newer (3-lactamases in Klebsiella pneu- 
moniae isolates from 24 U.S. hospitals. Antimicrob Agents 
Chemother. 2002;46:3837-42. 

14. Smith-Moland E, Hanson ND, Herrera VL, Black AJ, Lockhart T, 
Hossain A, et al. Plasmid-mediated, carbapenem-hydrolysing (3-lacta- 
mase, KPC-2, in Klebsiella pneumoniae isolates. J Antimicrob 
Chemother. 2003;51:711-4. 

15. Poirel L, Weldhagen GF, Naas T, De Champs C, Dove MG, 
Nordmann P. GES-2, class A (3-lactamase from Pseudomonas aerug- 
inosa with increased hydrolysis of imipenem. Antimicrob Agents 
Chemother. 2001;45:2598-603. 

16. Sherwood L. Antimicrobial use in animal feed — time to stop. N Engl 
J Med. 2001;345:1202-3. 

17. White DG, Shaohua Z, Sudler R, Sherry A, Friedman S, Chen S, et al. 
The isolation of antibiotic-resistant salmonella from retail ground 
meats. N Engl J Med. 2001;345:1147-54. 

18. Mac Arthur JV, Tuckfield RC. Spatial patterns in antibiotic resistance 
among stream bacteria; effect of industrial pollution. Appl Environ 
Microbiol. 2000;66:3722-6. 

19. Munesia M, Garcia A, Miro E, Prats G, Jofre J, Navarro F. 
Bacteriophages and diffusion of (3-lactamase genes. Emerg Infect 
Dis. 2004;10:1134-7. 

20. Goni-Urriza M, Capdepuy M, Arpin C, Raymond N, Caumette P, 
Quentin C. Impact of an urban effluent on antibiotic resistance of 
riverine Enterobacteriaceae and Aeromonas spp. Appl Environ 
Microbiol. 2000;66:125-32. 

21. Baya AM, Brayton PR, Brown VL, Grimes DJ, Russek-Cohen E, 
Colwell RR. Coincident plasmids and antimicrobial resistance in 
marine bacteria isolated from polluted and unpolluted Atlantic Ocean 
samples. Appl Environ Microbiol. 1986;51:1285-92. 

22. Ash RJ, Mauck B, Morgan M. Antibiotic resistance of gram-negative 
bacteria in rivers, United States. Emerg Infect Dis. 2002;8:713-6. 


23. Brenner DJ, McWorther AC, Kai A, Steigerwalt AG, Farmer JJ. 
Enterobacter asburiae sp. nov. a new species found in clinical speci- 
mens and reassignement of Erwinia dissolvens and Erwinia nimi- 
pressuralis to the genus Enterobacter as Enterobacter dissolvens 
comb. nov. and Enterobacter nimipressuralis comb. nov. J Clin 
Microbiol. 1986;23:1114-20. 

24. Avidor B, Kletter Y, Abulafia S, Golan Y, Ephros M, Giladi M. 
Molecular diagnosis of cat scratch disease: a two-step approach. J 
Clin Microbiol. 1997;35:1924-30. 

25. National Committee for Clinical Laboratory Standards. Methods for 
dilution antimicrobial susceptibility tests for bacteria that grow aero- 
bically. 6th ed. Approved standard M7-A5. Wayne (PA): The 
Committee; 2003. 

26. Poirel L, Guibert M, Bellais S, Naas T, Nordmann P. Integron- and 
carbenicillinase-mediated reduced susceptibility to amoxicillin- 
clavulanic acid in isolates of multidrug-resistant Salmonella enterica 
serotype Typhimurium DTI 04 from French patients. Antimicrob 
Agents Chemother. 1999;43:1098-104. 

27. Poirel L, Naas T, Guibert M, Chaibi EB, Labia R, Nordmann P. 
Molecular and biochemical characterization of VEB-1, a novel class 
A extended-spectrum (3-lactamase encoded by an Escherichia coli 
integron gene. Antimicrob Agents Chemother. 1999;43:573-81. 

28. Nazarowec-White M, Farber JM. Phenotypic and genotypic typing 
food and clinical isolates of Enterobacter sakazakii. J Med Microbiol. 
1999;48:559-67. 

29. Telenius H, Carter NP, Bebb CE, Nordenskjold M, Ponder BA, 
Tunnacliffe A. Degenerate oligonucleotide-primer PCR: general 
amplification of target DNA by single degenerate primer. Genomics. 
1992;13:718-25. 

30. Kieser T. Factors affecting the isolation of CCC DNA from 
Streptomyces lividans and Escherichia coli. Plasmid. 1984;12:19-36. 

31. Danel F, Hall LM, Gur D, Livermore DM. OXA-14, another extend- 
ed-spectrum variant of OXA-10 (PSE-2) (3-lactamase from 
Pseudomonas aeruginosa. Antimicrob Agents Chemother. 
1995;39:1881-4. 

32. Churchward G, Belin D, Nagamine Y. A pSC 101 -derived plasmid 
which shows no sequence homology to other commonly used cloning 
vectors. Gene. 1984;31:165-71. 

33. Michiels T, Cornells G, Ellis K, Grinsted J. Tn2501, a component of 
the lactose transposon Tn951, is an example of a new category of 
class II transposable elements. J Bacteriol. 1987;169:624-31. 

34. Pottumarthy S, Smith-Moland E, Juretschko S, Swanzy SR, Thomson 
KS, Fritsche TR. NmcA carbapenem-hydrolyzing enzyme in 
Enterobacter cloacae in North America. Emerg Infect Dis. 
2003;9:999-1002 

35. Mimoz O, Leotard S, Jacolot A, Padoin C, Louchahi K, Petitjean O, 
et al. Efficacies of imipenem, meropenem, cefepime, and ceftazidime 
in rats with experimental pneumonia due to a carbapenem-hydrolyz- 
ing (3-lactamase-producing strain of Enterobacter cloacae. 
Antimicrob Agents Chemother. 2000;44:885-90. 

Address for correspondence: Patrice Nordmann, Service de 

Bacteriologie-Virologie, Hopital de Bicetre, 78 Rue du General Leclerc, 

94275 Le Kremlin-Bicetre, France; fax: 33-1-45-21-63-40; email: nord- 
mann. patrice@bct.ap-hop-paris.fr 


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Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


Rickettsial Infection in Animals and 
Brazilian Spotted Fever Endemicity 

Luis A. Sangioni,*t Mauricio C. Horta,* Manoella C.B. Vianna,* Solange M. Gennari,* 
Rodrigo M. Soares,* Marcio A.M. Galvao4 Teresinha T.S. Schumaker,* Fernando Ferreira,* 

Odilon Vidotto,§ and Marcelo B. Labruna* 


We compared the rickettsial infection status of 
Amblyomma cajennense ticks, humans, dogs, and horses 
in both Brazilian spotted fever (BSF)-endemic and -nonen- 
demic areas in the state of Sao Paulo, Brazil. Most of the 
horses and few dogs from BSF-endemic areas had sero- 
logic titers against Rickettsia rickettsii antigens. In contrast, 
no dogs or horses from BSF-nonendemic areas had sero- 
logic titers against R. rickettsii antigens, although they were 
continually exposed to A. cajennense ticks. All human 
serum samples and ticks from both areas were negative by 
serologic assay and polymerase chain reaction, respec- 
tively. Our results indicate that surveys of horse serum are 
a useful method of BSF surveillance in areas where 
humans are exposed to A. cajennense ticks. In addition, we 
successfully performed experimental infection of A. cajen- 
nense ticks with R. parkeri. 

B razilian spotted fever (BSF) is an acute, febrile, tick- 
borne disease caused by the bacterium Rickettsia rick- 
ettsii. The disease is transmitted by Amblyomma ticks and 
has been considered endemic in some areas of the states of 
Sao Paulo, Minas Gerais, Rio de Janeiro, and Espirito 
Santo (1-7). Although the tick species Amblyomma aureo- 
latum is the main vector of BSF in few areas of the state of 
Sao Paulo (8, A. Pinter, unpub data), A. cajennense is the 
most common tick vector associated with the disease in 
Brazil (9-11). 

A. cajennense is a common tick in rural areas of the 
state of Sao Paulo, where it is also the main tick species 
infesting humans (12,13). In contrast, BSF cases have been 
reported at only a few locations within the geographic 
range of this tick species (14). Although unreported cases 


*University of Sao Paulo, Sao Paulo, Sao Paulo, Brazil; fCentro 
Integrado de Ensino Superior - Campus Universitario, Campo 
Mourao, Parana, Brazil; ^Federal University of Ouro Preto, Ouro 
Preto, Minas Gerais, Brazil; and §Londrina State University, 
Londrina, Parana, Brazil 


may have occurred in other areas where BSF is not known 
to be endemic, this possibility is unlikely for such a high- 
ly lethal disease. Ecologic differences might be the main 
factor regulating the occurrence of R. rickettsii among 
ticks and, consequently, the occurrence of the disease. 

The infection rate by R. rickettsii within a tick popula- 
tion can be diminished or even suppressed when a second 
Rickettsia species infects most of the members of that tick 
population (15,16). Thus, we hypothesize that the absence 
of human cases of BSF in some areas of the state of Sao 
Paulo (where human parasitism by A. cajennense is 
intense) is related to the presence of other, less pathogenic 
Rickettsia species infecting A. cajennense tick populations. 
In this regard, our study evaluated the rickettsial infection 
status of A. cajennense populations from both BSF-endem- 
ic and -nonendemic areas in the state of Sao Paulo. We also 
serologically evaluated humans and domestic animals 
from these BSF-nonendemic areas to compare it to a recent 
evaluation that we performed in BSF-endemic areas (17). 

Materials and Methods 

Study Area 

The study was conducted on 6 farms in the state of Sao 
Paulo. Three of these farms (farms 1, 2, and 3) were con- 
sidered endemic for BSF because of the recent occurrence 
of several laboratory-confirmed human cases of the dis- 
ease among residents (4,14). These farms were the same 
ones evaluated in a study of Horta et al. (17). The remain- 
ing 3 farms (4, 5, and 6) were considered nonendemic for 
BSF because they had never had human cases of this dis- 
ease. However, A. cajennense ticks were abundant there, 
and human infestation by this tick was a normal finding 
year-round among farm residents. Farms 1 (22°44'19"S, 
46°55'27"W), 2 (22°47'03"S, 46°54'10"W) and 3 
(22°41'14"S, 46°53'17"W) were located in the Pedreira 
Municipality whereas farms 4 (23°23'15"S, 47°26'14"W), 


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RESEARCH 


5 (23°36'43"S, 46°57'29"W), and 6 (21°57'07"S, 
47°27'05"W) were located in Porto Feliz, Cotia, and 
Pirassununga Municipalities, respectively. 

In all 6 farms, human occupations were basically divid- 
ed between livestock-raising activities for men and house- 
hold activities for women and children. Nevertheless, 
children spent substantial time in outdoor activities. All 6 
farms had horses grazing on mixed overgrowth pastures, 
interspersed with remote forest areas. However, the major 
ecologic difference was large populations of free-living 
capybaras that inhabited livestock pastures on farms 1, 2, 
and 3 and the absence of this animal from horse pastures 
on farms 4, 5, and 6. All farms, except farm 4, had free- 
roaming dogs with free access to pasture and forest areas. 
Recent studies on ticks collected on the pastures and on 
horses and dogs from these 6 farms allowed the tick 
species A. cajennense and Dermacentor nitens to be iden- 
tified on the 6 farms. In addition, the capybara tick, A. 
cooperi , was present on farms 1, 2, and 3 but absent in the 
pastures of farms 4, 5, and 6 (13,17-19). Human infesta- 
tion by Amblyomma ticks was frequent on all the farms. 

Ticks 

From December 2000 to March 2001, free-living A. 
cajennense adult ticks were collected from horse pastures 
of the 6 farms by dragging and by using C0 2 traps. Totals 
of ticks collected from the farms are as follows: farm 1 
(244), farm 2 (353), farm 3 (213), farm 4 (222), farm 5 
(206), and farm 6 (230). All ticks were brought alive to the 
laboratory, where their surfaces were disinfected by 
immersion in 70% alcohol for 10 min followed by wash- 
ing in sterile water; they were then individually tested by 
the hemolymph test (20). Briefly, a drop of hemolymph of 
each tick was dried on a glass slide and stained by the 
Gimenez method (21). Thereafter, ticks were frozen at 
-80° C until processed for DNA extraction. 

DNA Extraction 

All ticks were processed individually for DNA extrac- 
tion. Each tick was cut into 2 symmetric halves through its 
median axis. One half was returned to the -80°C freezer 
for further studies, and the other half was used for DNA 
extraction according to a modification of a previously 
described protocol (22). For this purpose, each tick half 
was placed in a 1.5-mL microtube containing 150 qL of TE 
buffer (Tris HC1 10 mmol/L, EDTA 1 mmol/L, pH 7.4) and 
homogenized by using a sterile micropestle. Microtubes 
containing the homogenized, triturated ticks were then 
vortexed vigorously. Next, 450 qL of guanidine thio- 
cyanate (5 mol/L) were added to the tube, which was vor- 
texed again and incubated for 10 min at room temperature 
with short vortexing every 2 min. Thereafter, 100 |lL of 
chloroform was added to the tube, which was inverted sev- 


eral times and left resting for 2 min. The tube was cen- 
trifuged at 12,000 x g for 5 min to separate the aqueous 
phase, which was transferred to a clean 1.5-mL microtube. 
Next, 600 |iL of isopropanol was added to the aqueous 
phase (400 |lL), which was homogenized by inverting the 
tube several times and then incubated at -20°C for 2 to 1 8 
h. Thereafter, the tube was centrifuged at 12,000 x g for 15 
min; the supernatant was discarded, and the pellet was 
dried at room temperature and then resuspended with 30 
pL of buffer TE. Finally, the microtubes were incubated at 
56°C for 15 min to facilitate DNA homogenization and 
then stored at -20°C until tested by polymerase chain reac- 
tion (PCR). 

PCR 

Five microliters of the extracted DNA from tick speci- 
men was used as template for amplification of fragments 
of the rickettsial git A (citrate synthase gene) and 17-kDa 
protein gene. A 381-bp portion of the Rickettsia git A gene 
was targeted from each extracted tick DNA by using 
primers RpCS.877 and RpCS.1258n (23), and a 434-bp 
portion of the Rickettsia genus-specific 17-kDa protein 
gene was targeted as previously described (24). Ten micro- 
liters of the PCR product underwent electrophoresis in 
1.5% agarose gel, stained with ethidium bromide, and 
examined with UV transillumination. For the 10 individual 
ticks that were tested by PCR, a negative control (5 qL of 
water) and positive control (5 qL of DNA extracted from 
an A. cajennense tick experimentally infected with R. 
parkeri) were included. Procedures to obtain R. parkeri 
experimentally infected ticks are described below. PCR 
results were statistically analyzed by the program @Risk 
Software - Risk Analysis Add-in for Microsoft Excel 
(Palisade Corporation, Newfield, NY, USA), which adopt- 
ed Monte Carlo techniques to determine the confidence 
level of the prevalence of ticks infected by Rickettsia in 
each tick population (farm), considering a = 0.05. 

R. parkeri Experimentally Infected Ticks 

Purified R. parkeri organisms (Maculatum strain) were 
obtained by the renografin purification method from 
infected Vero cells (25). The resultant purified rickettsiae 
were resuspended in sucrose-phosphate-glutamic acid 
buffer and stored frozen at -80°C until tick infection. 
Seventy adult specimens of A. cajennense were obtained 
from the third generation of our laboratory colony at the 
University of Sao Paulo. This colony was established 15 
months earlier from engorged females collected on horses 
on farm 6 of the present study. Adult ticks had their dor- 
sum attached to double-face adhesive tape, which was 
taped onto petri dishes. Purified stock of R. parkeri was 
thawed at room temperature, and each tick was injected by 
using a 28-gauge microfine insulin needle. Under a stereo- 


266 


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Investigation of Rickettsial Infection in Brazil 


scopic microscope, a small drop (~2 |iL) of R. parkeri sus- 
pension was injected into the coelom of the tick, through 
the articulation of coxa IV with trochanter IV, in each of 50 
adult ticks. A control group of 20 ticks were injected by the 
same procedure with phosphate-buffered saline (PBS). 
Ticks were removed from the adhesive tape and held in an 
incubator at 35°C and relative humidity >95% for 5 days. 
Ticks were tested by the hemolymph test as described 
above, at days 3 and 5 after infection. Thereafter, ticks 
were frozen at -80°C. DNA of hemolymph-positive ticks 
was extracted, as described above, to be used as positive 
control for our PCR assays. A sample of 10 PBS-injected 
ticks were also tested by the PCR method described above. 

Domestic Animals and Humans 

During our visit to farms 1 to 6, blood samples were 
collected from 100% of the dogs and horses on each farm 
and -90% of the resident humans. Blood samples were 
transported at room temperature to the laboratory, where 
samples were centrifuged (1,500 x g , 10 min), and the sera 
were aliquoted into labeled microtubes and stored at 
-20°C until tested by the indirect immunofluorescence 
assay (IFA) with R. rickettsii antigen, as described (17). 
The serologic results of farms 1 to 3 have been reported by 
Horta et al. (17) and will be compared with our results for 
farms 4 to 6. Collection of animal and human samples was 
previously approved by ethical principles in animal and 
human research of the University of Sao Paulo. 

Results 

Field Ticks 

A total of 1,468 A. cajennense adult ticks (810 from dis- 
ease-endemic and 658 from disease-nonendemic areas) 
were tested by the hemolymph test. They were all nega- 
tive. These same ticks were also negative by the PCR pro- 
tocols targeting the rickettsial genes git A and 17-kDa 
protein. In all PCR assays, DNA of A. cajennense ticks 
experimentally infected with R. parkeri (positive controls) 
yielded the expected bands whereas no bands were 
obtained for the negative controls. 

Our results, after being analyzed by the Monte Carlo 
techniques, are that on farm 5, where 206 ticks (our small- 
est sample) were tested, the prevalence of A. cajennense 
ticks infected by Rickettsia was at most 1.43% (upper limit 
of 95% confidence interval). If the prevalence was higher 
than this value, infection in at least 1 tick would have been 
detected by our PCR. Similarly, in farm 2, where 353 ticks 
were tested (our largest sample), the prevalence of ticks 
infected by Rickettsia was at most 0.8% (upper limit of 
95% confidence interval). Overall, these analyses indicat- 
ed that the prevalence of rickettsial infection on the 6 
farms was no more than 0.8%-1.43%. As we used 


Rickettsia genus specific primers in the PCR, this infection 
could be due to any Rickettsia species. 

Ticks Experimentally Infected with R. parkeri 

Of 50 ticks infected with R. parkeri , 10 (20%) showed 
typical Rickettsia- like organisms within the hemocytes 3 
days after injection. On day 5, the number of ticks show- 
ing typical Rickettsia- like organisms in their hemocytes 
increased to 28 (56%). None of the 20 ticks injected with 
PBS showed Rickettsia- like organisms in their hemolymph 
3 or 5 days after injection. All 28 hemolymph-positive 
ticks yielded expected bands in both PCR protocols (gltA 
and 17-kDa protein) whereas no PBS-injected ticks yield- 
ed amplified DNA bands. 

Serologic Assays 

Serum samples were collected from horses, dogs, and 
humans from the 6 farms, as shown in the Table. From the 
BSF-nonendemic areas (farms 4-6), no sample from a dog, 
horse, or human reacted positively with R. rickettsii anti- 
gens. The serologic results for the BSF-endemic areas 
(farms 1-3) were reported by Horta et al. (17). The pro- 
portion of horses that reacted positively with R. rickettsii 
antigens (titer >64) varied from 57.1% to 80%; for dogs, 
these proportions varied from 0% to 66.7%. Like farms 
4-6, no human serum sample from farms 1 to 3 reacted 
positively with R. rickettsii antigens. 

Discussion 

Our study evaluated A. cajennense ticks in BSF-endem- 
ic and -nonendemic areas in the state of Sao Paulo. In addi- 
tion, we serologically evaluated domestic animals and 
humans from BSF-nonendemic areas and compared the 
results with a previous serologic evaluation in BSF-endem- 
ic areas (17). Our results for the nonendemic areas showed 
no evidence of a pathogenic Rickettsia species circulating 
in A. cajennense ticks in farms 4 to 6, since all animals, 
humans, and ticks were negative. In contrast, Horta et al. 
(17) showed serologic evidence of R. rickettsii infection by 
cross-absorption and IFA analyses in most of the horses and 
some dogs in the 3 BSF-endemic areas (farms 1-3), a find- 
ing that is supported by the recent occurrence of human 
BSF cases in those farms. The serologic reactivity of hors- 
es, dogs, and humans to R. rickettsii antigen in BSF-endem- 
ic areas where A. cajennense is the main vector is 
characterized by a high frequency of serologically positive 
horses, followed by a lower frequency in dogs, and an even 
lower frequency or absence of serologically positive 
humans (17). This pattern has been observed in several 
BSF-endemic areas in which A. cajennense has been 
incriminated as the vector (3,17,26,27). The absence of 
serologic reactivity among the human residents whom we 
tested is supported by their lack of history of the disease; 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


267 


RESEARCH 


Table. Results of indirect immunofluorescence assay for antibodies to Rickettsia rickettsii in humans and domestic animals from 3 
BSF-endemic areas (farms 1-3)* and 3 BSF-nonendemic areas (farms 4-6), Sao Paulo, Brazil! 





Reactive seraf/total sera tested (% reactive) 



Source 

Farm 1 

Farm 2 

Farm 3 

Farm 4 

Farm 5 

Farm 6 

Humans 

0/20 (0) 

0/21 (0) 

0/9 (0) 

0/4 (0) 

0/2 (0) 

0/1 0 (0) 

Horses 

9/1 0 (90) 

4/7 (57.1) 

4/5 (80) 

0/1 6 (0) 

0/1 0 (0) 

0/21 (0) 

Dogs 

1/4 (25) 

4/6 (66.7) 

0/6 (0) 

No dogs 

0/4 (0) 

0/1 (0) 

*Data from Horta et al . (17). 
fBSF, Brazilian spotted fever. 

JSera showing titers >64 for R. rickettsii antigen. 


previous cases reported in this area were lethal or if not, the 
survivors do not live in the BSF-endemic area anymore. 

Horses are one of the most important hosts for A. cajen- 
nense in the state of Sao Paulo; both immature and adult 
ticks will successfully feed on this animal (18). This fact 
makes the horse an excellent sentinel for BSF surveillance. 
Once the A. cajennense population increases in an area, 
parasitic stages will have a greater chance to successfully 
feed on other host species, including dogs and humans. As 
dogs are naturally infested with ticks more frequently than 
humans, they are also a good sentinel for BSF surveillance. 
Results of our study support this statement because our 
serologic survey of horses and dogs from 3 areas, where no 
BSF case has been reported, indicated that neither R. rick- 
ettsii nor a closely related species circulated in the local A. 
cajennense ticks. Thus, we recommend surveys of horse 
sera as a useful method for BSF surveillance in areas 
where humans are exposed to A. cajennense ticks. This 
procedure would allow potentially BSF-endemic areas to 
be identified before human cases occur. 

We failed to detect any rickettsial DNA in the field-col- 
lected A. cajennense ticks. Although this result is support- 
ed by the serologic results in the BSF-nonendemic areas, it 
was not expected for the BSF-endemic areas, where infec- 
tion by R. rickettsii in horses and dogs has been indirectly 
proven by serologic cross-absorption methods (17). 
Finding R. rickettsii- infected ticks in spotted fever-endem- 
ic areas can be difficult. In North Carolina, a U.S. state 
with a high incidence of Rocky Mountain spotted fever 
(caused by R. rickettsii ), only 1 of 2,123 Dermacentor 
variabilis ticks studied was infected by R. rickettsii (15). 
Thus, further studies in Sao Paulo should encompass a 
much larger number of A. cajennense ticks. 

The major ecologic differences between the BSF- 
endemic and -nonendemic areas of our study were the 
presence of capybaras and their main tick species (A. 
cooperi ), found solely in the BSF-endemic areas. In a 
recent survey of rickettsiae in A. cooperi ticks collected on 
farms 1, 2, and 3 (19), 2 rickettsiae were isolated from 
these ticks: R. bellii and a Rickettsia species (strain 
COOPERI) closely related to R. parkeri and R. africae. 
Similar to the present study, no R. rickettsii was found 
infecting A. cooperi ticks. 


Burgdorfer et al. (16) found that high infection rates (up 
to 80%) by a less pathogenic rickettsia were the limiting 
factor for establishing R. rickettsii in the D. andersoni tick 
population of the east side of the Bitterroot Valley in 
Montana, USA. On the west side of this valley, where 
8%-16% of the ticks were infected by the less pathogenic 
rickettsia, disease caused by R. rickettsii was endemic. 
Based on these observations, the results of our study sug- 
gest that unknown factors other than the presence of dif- 
ferent Rickettsia species are responsible for the absence of 
a pathogenic spotted fever group rickettsia’ s infection of 
populations of A. cajennense populations in farms 4, 5, and 
to 6 (BSF-nonendemic areas). 

In a recent study performed in our laboratory (A. Pinter 
and M.B. Labruna, unpub. data) R. rickettsii was detected 
in 6 (0.89%) of 669 A. aureolatum adult ticks by using the 
same PCR protocols as the present study. These ticks were 
collected in a different BSF-endemic area, in which A. 
aureolatum is the main vector of the disease. As our results 
showed that the highest predictable infection rate of R. rick- 
ettsii in the A. cajennense population of farm 3 (where 353 
ticks were tested) was 0.8%, we might have found a R. rick- 
ettsii- infected A. cajennense tick if we had tested a larger 
sample of ticks from that farm. Even though recent studies 
have failed to detect or isolate R. rickettsii from A. cajen- 
nense ticks in Brazil, earlier studies detected it efficiently in 
the states of Sao Paulo (28) and Minas Gerais (9,10), as 
well as in Colombia (29), Mexico (30), and Panama (31). 

Our study showed that R. parkeri could experimentally 
infect A. cajennense ticks. A previous, more extensive, 
study showed that A. americanum ticks experimentally 
infected with R. parkeri were able to maintain this infec- 
tion for 2 generations and were able to transmit it to guinea 
pigs through tick feeding (32). Natural infection of ticks by 
this agent has been reported in A. maculatum (33) and A. 
triste (34). The Rickettsia species (strain COOPERI), 
found to be infecting A. cooperi ticks in Sao Paulo state 
(19), seems to be another strain of R. parkeri or a closely 
related species. These results show that R. parkeri can 
infect different Amblyomma species under experimental or 
natural conditions. The potential role of A. cajennense to 
transmit R. parkeri in nature requires further investigation, 
especially since R. parkeri was recently shown to be path- 
ogenic for humans (35). 


268 


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Investigation of Rickettsial Infection in Brazil 


Acknowledgments 

We are grateful to the owners of farms 1-6 for making our 
study possible and to David H. Walker for providing IFA slides 
and the R. parkeri - purified stock. 

This work was supported by the Fundagao de Amparo a 
Pesquisa do Estado de Sao Paulo (grant 00/02711-1 to S.M.G.) 
and performed at the Faculty of Veterinary Medicine, University 
of Sao Paulo, Sao Paulo, Sao Paulo, under the coordination of 
M.B. Labruna. 

Dr. Sangioni is a professor of veterinary parasitology at the 
Veterinary School of the Centro Integrado de Ensino Superior at 
Campo Mourao, Brazil. His research interests have focused on 
the ecology of tickborne diseases. 

References 

1. Gongalves AJR, Lopes PFA, Melo JCP, Pereira AA, Pinto AMM, 
Lazera MS, et al. Rickettsioses. A proposito de quatro casos diagnos- 
ticados no Rio de Janeiro de febre amculosa barsileira. Folha Medica. 
1981;82:127-34. 

2. Sexton DJ, Muniz M, Corey GR, Breitschwerdt EB, Hegarty BC, 
Dumler S, et al. Brazilian spotted fever in Espirito Santo, Brazil: 
description of a focus of infection in a new endemic region. Am J 
Trop Med Hyg. 1993;49:222-6. 

3. Lemos ERS, Machado RD, Coura JR. Rocky Mountain spotted fever 
in an endemic area in Minas Gerais, Brazil. Mem Inst Oswaldo Cruz. 
1994;89:497-501. 

4. Lemos ER, Alvarenga FB, Cintra ML, Ramos MC, Paddock CD, 
Ferebee TL, et al. Spotted fever in Brazil: a seroepidemiological 
study and description of clinical cases in an endemic area in the state 
of Sao Paulo. Am J Trop Med Hyg. 2001;65:329-34. 

5. Lemos ER, Rozental T, Villela CL. Brazilian spotted fever: descrip- 
tion of a fatal clinical case in the State of Rio de Janeiro. Rev Soc 
Bras Med Trop. 2002;35:523-5. 

6. Galvao MA, Lamounier JA, Bonomo E, Tropia MS, Rezende EG, 
Calic SB, et al. Rickettsioses emergentes e reemergentes numa regiao 
endemica do estado de Minas Gerais, Brazil. Cad Saude Publica. 
2002;18:1593-7. 

7. Galvao MA, Calic SB, Chamone CB, Mafra SCL, Cesarino Filho G, 
Olano JP, et al. Spotted fever rickettsiosis in Coronel Fabriciano, 
Minas Gerais State. Rev Soc Bras Med Trop. 2003;36:479-81. 

8. Gomes LS. Thypho exanthematico de Sao Paulo. Brasil Medico. 
1933; 17(52):9 19-21. 

9. Moreira JA, Magalhaes O. Thypho exanthematico em Minas Gerais. 
Brasil Medico. 1935;44:465-70. 

10. Dias E, Martins A, Ribeiro DJ. Thypho exanthematico no Oeste de 
Minas Gerais. Brasil Medico. 1937;51:651-5. 

11. Lima VLC, Figueiredo AC, Pignatti MG, Modolo M. 1995. Febre 
maculosa no municfpio de Pedreira, Estado de Sao Paulo, Brasil: 
Relagao entre ocorrencia de casos e parasitismo humano por 
ixodfdeos. Rev Soc Bras Med Trop. 1995;28:135-7. 

12. Guimaraes JH, Tucci EC, Barros-Battesti DM. Ectoparasitos de 
importancia veterinaria. Sao Paulo: Editora Pleiade; 2001. 

13. Labruna MB, Kerber CE, Ferreira F, Faccini JLH, De Waal DT, 
Gennari SM. Risk factors to tick infestations and their occurrence on 
horses in the State of Sao Paulo, Brazil. Vet Parasitol. 2001;97:1-14. 

14. Lima VL, Souza SS, Souza CE, Vilela MF, Papaiordanou PM, Del 
Guercio VM, et al. Situagao da febre maculosa na regiao administra- 
tiva de Campinas, Sao Paulo, Brasil. Cad Saude Publica. 
2003;19:331-4. 


15. Burgdorfer W. Ecological and epidemiological considerations of 
Rocky Mountain spotted fever and scrub typhus. In: Walker DH, edi- 
tor. Biology of rickettsial diseases. Vol. 1. Boca Raton (FL): CRC 
Inc.; 1988. p. 33-50. 

16. Burgdorfer W, Hayes SF, Mavros AJ. Nonpathogenic rickettsiae in 
Dermacentor andersoni : a limiting factor for the distribution of 
Rickettsia rickettsii. In: Burgdorfer W, Anacker RL, editors. 
Rickettsiae and rickettsial diseases. New York: Academic Press; 
1981. p.585-94. 

17. Horta MC, Labruna MB, Sangioni LA, Vianna MCB, Gennari MS, 
Galvao MAM, et al. Prevalence of antibodies to spotted fever group 
rickettsiae in humans and domestic animals in a Brazilian Spotted 
fever endemic area in the state of Sao Paulo, Brazil: serological evi- 
dence for infection by Rickettsia rickettsii and another spotted fever 
group rickettsia. Am J Trop Med Hyg. 2004;71:93-7. 

18. Labruna MB, Kasai N, Ferreira F, Faccini JLH, Gennari SM. 
Seasonal dynamics of ticks (Acari: Ixodidae) on horses in the state of 
Sao Paulo, Brazil. Vet Parasitol. 2002;105:65-72. 

19. Labruna MB, Whitworth T, Horta MC, Bouyer DH, McBride JW, 
Pinter A, et al. Rickettsia species infecting Amblyomma cooperi ticks 
from an endemic area for Brazilian spotted fever in the state of Sao 
Paulo, Brazil. J Clin Microbiol. 2004;42:90-8. 

20. Burgdorfer W. The hemolymph test. Am J Trop Med Hyg. 
1970;19:1010-4. 

21. Gimemez DF. Staining rickettsiae in yolk-sac cultures. Stain 
Technology. 1964;39:135-40. 

22. Chomkzynski P. A reagent for the single-step simultaneous isolation 
of RNA, DNA and proteins from cell and tissue samples. 
Biotechniques. 1993;15:532-7. 

23. Regnery RL, Spruill CL, Plikaytis BD. Genotypic identification of 
rickettsiae and estimation of intraspecies sequence divergence for 
portions of two rickettsial genes. J Bacteriol. 1991;173:1576-89. 

24. Webb L, Carl M, Malloy DC, Dasch GA, Azad AF. Detection of 
murine typhus infection in fleas by using the polymerase chain reac- 
tion. J Clin Microbiol. 1990;28:530-4. 

25. Hanson BA, Wisseman CL Jr, Waddell A, Silverman DJ. Some char- 
acteristics of heavy and light bands of Rickettsia prowazekii on 
Renografin gradients. Infect Immun. 1981;34:596-604. 

26. Lemos ERS, Machado RD, Coura JR, Guimaraes MAA, Chagas N. 
Epidemiological aspects of the Brazilian spotted fever: serological 
survey of dogs and horses in an endemic area in the state of Sao 
Paulo, Brazil. Rev Inst Med Trop de Sao Paulo. 1996;38:427-30. 

27. Galvao MAM. Febre maculosa em Minas Gerais: um estudo sobre a 
distribuigao da doenga no Estado e seu comportamento em area de 
foco peri-urbano [Doctoral thesis]. Belo Horizonte: Faculdade de 
Medicina da UFMG; 1996. p. 114. 

28. Vallejo-Freire A. Spotted fever in Mexico. Memorias dolnstituto 
Butantan. 1946; 19: 159-80. 

29. Patino-Camargo L. Nuevas observaciones sobre un tercer foco de 
fiebre petequial (maculosa) en el hemisferio americano. Bol Oficina 
SanitPanam. 1941;20:1112-4. 

30. Bustamante ME, Varela G. Estudios de fiebre manchada en Mexico. 
Hallazgo del Amblyomma cajennense naturalmente infectado, en 
Veracruz. Rev Inst Salubr Enferm Trop. 1946;7:75-8. 

31. Rodaniche EC. Natural infection of the tick Amblyomma cajennense 
with Rickettsia rickettsii in Panama. Am J Trop Med Hyg. 
1953;2:696-9. 

32. Goddard J. Experimental infection of lone star ticks, Amblyomma 
americanum (L.), with Rickettsia parkeri and exposure of guinea pigs 
to the agent. J Med Entomol. 2003;40:686-9. 

33. Lackman D, Parker R, Geloff R. Serological characteristics of a path- 
ogenic rickettsia occuring in Amblyomma maculatum. Public Health 
Rep. 1949;64:1342-9. 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


269 


RESEARCH 


34. Venzal JM, Portillo A, Estrada-Pena A, Castro O, Cabrera PA, Oteo 
JA. Rickettsia parkeri in Amblyomma triste from Uruguay. Emerg 
Infect Dis. 2004;10:1493-5. 


All material published in Emerging Infectious Diseases is in the 
public domain and may be used and reprinted without special per- 
mission; proper citation, however, is appreciated. 


35. Paddock CD, Sumner JW, Comer JA, Zaki SR, Goldsmith CS, 
Goddard J, et al. Rickettsia parkeri : a newly recognized cause of spot- 
ted fever rickettsiosis in the United States. Clin Infect Dis 
2004;38:15-21. 


Address for correspondence: Marcelo B. Labruna, Departamento de 
Medicina Veterinaria Preventiva e Saude Animal, Faculdade de Medicina 
Veterinaria e Zootecnia, Universidade de Sao Paulo, Sao Paulo, SP, Brazil 
05508-000; fax: 55-11-3091 7928; email: labruna@usp.br 


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Isolation of Waddlia malaysiensis, A 
Novel Intracellular Bacterium, from 
Fruit Bat (Eonycteris spelaea) 

Paul K.B. Chua,*t John E. Corkill,* Poh Sim Hooi4 Soo Choon Cheng4 Craig Winstanley,* 

and C. Anthony Hart* 


An obligate intracellular bacterium was isolated from 
urine samples from 7 (3.5%) of 202 fruit bats ( Eonycteris 
spelaea) in peninsular Malaysia. The bacterium produced 
large membrane-bound inclusions in human, simian, and 
rodent cell lines, including epithelial, fibroblastlike, and lym- 
phoid cells. Thin-section electron microscopy showed retic- 
ulate bodies dividing by binary fission and elementary 
bodies in the inclusions; mitochondria surrounded the inclu- 
sions. The inclusions were positive for periodic acid-Schiff 
stain but could not be stained by fluorescein-labeled 
an\\-Chlamydia trachomatis major outer membrane protein 
monoclonal antibody. The bacterium was resistant to peni- 
cillin and streptomycin (MICs >256 mg/L) but susceptible to 
tetracycline (MIC = 0.25 mg/L) and chloramphenicol (MIC = 
0.5 mg/L). Sequence analysis of the 16SrRNAgene indicat- 
ed that it was most closely related to 2 isolates of Waddlia 
chondrophila (94% and 96% identity). The 16S and 23S 
rRNAgene signatures were only 91% identical. We propose 
this novel bacterium be called W. malaysiensis. 

A n estimated 1,415 microbes are infectious for humans 
(1). Of these, 868 (61%), are considered to be zoonot- 
ic; overall, zoonotic pathogens are twice as likely to be 
associated with emerging diseases (1). Wildlife have been 
increasingly recognized as important reservoirs of poten- 
tially zoonotic microorganisms (2,3). In particular, bats 
have been shown to be both important reservoirs and vec- 
tors of pathogens. These pathogens include viruses such as 
rabies (4), European lyssavirus (5), Hendra (6) and 
Menangle (7) viruses in Australia, Nipah and Tioman 
viruses in Malaysia (8,9), hantaviruses in Korea (10), a 
number of different bunyaviruses, flaviviruses, and 


*University of Liverpool, Liverpool, United Kingdom; fNational 
Public Health Laboratory, Kuala Lumpur, Malaysia; and 
^University of Malaya, Kuala Lumpur, Malaysia 


alphaviruses. Moreover, solitary microchiropteran bats are 
prime contenders as reservoirs of Marburg and Ebola 
viruses. In addition, bats have been identified as reservoirs 
of fungi such as Histoplasma capsulatum and 
Coccidioides immitis. However, apart from leptospirosis 
(11) and some studies on enteric flora and pathogens 
(12-14), little is known of the bacteria that infect and are 
excreted by bats. 

As part of an investigation into the reservoir of Nipah 
virus in Malaysia (8,9,15), a novel chlamydialike bacteri- 
um was isolated from the urine of Eonycteris spelaea ; the 
Lesser Dawn Bat (16). This bat is a generalist nectivore 
that travels tens of kilometers from its cave-roosting sites 
to feed (16). It is found throughout Burma, Indonchina, the 
Philippines, Malaysia, Indonesia, Nepal, and northern 
India. Little is known of the potential pathogens harbored 
by E. spelaea , but 1 survey of lyssavirus infection of bats 
in the Philippines did not detect virus in brain sections or 
neutralizing antibody to rabies or Australian bat lyssavirus 
in serum from E. spelaea (17). Neither Nipah nor Tioman 
viruses have been isolated from E. spelaea , and detecting 
this chlamydialike bacterium was a chance finding (15). 
We describe the isolation and characterization of this novel 
bacterium and propose that it be given the name Waddlia 
malaysiensis since it was first isolated in Malaysia. 

Material and Methods 

Collection of Samples and Isolation of the Bacterium 

As part of an investigation into the reservoir of Nipah 
virus (8,9), we made 3 field trips from May to July 1999 to 
a colony of fruit bats (. E . spelaea) roosting in a cave (Gua 
Tempurong) situated 25 km from the initial Nipah out- 
break in Perak, northern peninsular Malaysia. The first 
visit was to observe the fruit bats’ roosting behavior, in 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


271 


RESEARCH 


particular, timing of return to roost, leaving for feeding, 
and urination and defecation habits. In the second and third 
visits, clean plastic sheets (1.5 x 3 m) were suspended over 
areas where the bats had been observed previously to uri- 
nate and defecate (15). The sheets were suspended ~0.5 m 
above the ground and held taut with 4 metal rods. The 
sheets and rods were put in place 30 min before the bats 
were expected to return to roost. Sterile cotton swabs were 
used to collect the urine as soon as it fell onto the plastic 
sheets. The swabs were then placed into virus transport 
medium (2 mL: ICN Biomedicals Inc, Irvine, CA, USA), 
containing 1% bovine albumin hydrolysate, amphotericin 
B (20 |lg/mL), penicillin G (100 U/mL), and streptomycin 
(50 qg/mL). The samples were transported at 4°C to the 
laboratory on the day of collection. Each swab, in transport 
medium, was gently vortexed, and 200 pH of the medium 
was transferred into individual wells of a 24- well tissue 
culture plate (Sterilin, Stone, U.K.) preseeded with 1 x 10 5 
Vero cells in Eagle’s minimal essential medium (Sigma, 
Basingtoke, U.K.). The plates were sealed and incubated at 
37°C. The culture was examined daily for cytopathic effect 
(CPE) with phase-contrast microscopy. Isolates were 
stored at -70°C, and 1 strain was chosen at random for fur- 
ther characterization and transported to Liverpool at 
-20°C. 

Microbiologic Characteristics 

To determine the range of cells susceptible to infection, 
different cells were cultured in 25 -m 2 plastic flasks 
(Becton Dickinson, Basingstoke, U.K.) in 199 medium 
(Sigma) with 2% (vol/vol) fetal calf serum but no added 
antimicrobial agents. Because the bacterium replicated so 
rapidly, including chlorhexidine, normally used in culture 
of Chlamydia trachomatis to prevent overgrowth of Vero 
cells, was not necessary. Approximately 10 7 bacterial cells 
(as determined by electron microscopic count) were added 
to each flask of cells and incubated at 37°C in air with 5% 
C0 2 and examined daily for CPE. For each cell line, 
growth was determined by both phase-contrast microscopy 
and demonstration of inclusions by thin- section electron 
microscopy. A variety of human (Hep-2, HEK, MRC-5, 
A549 and an Epstein Barr virus (EBV)-transformed 
human B-lymphoblastoid line), simian (Vero, LLC-MK2), 
and rodent (3T3, BHK) cell lines were used. Attempts 
were also made to grow the bacteria on 7% horse blood 
Columbia agar plates in air with 5% C0 2 and anaerobical- 
ly at 37°C for 72 h. 

To determine antimicrobial susceptibility, coverslip 
cultures of Vero cells were prepared as described previ- 
ously except that chlorhexidine was omitted from the 
growth medium (18,19). After 48 h of incubation, the 
growth medium was removed and -10 5 bacteria (in 0.5 mL 
medium) were added to each vial containing the coverslip 


monolayer of Vero cells. After absorption (without cen- 
trifugation) for 30 min, fresh 199 medium with 2% fetal 
calf serum, which incorporated doubling dilutions of 
antimicrobial agents from 1 mg/L down to 0.06 mg/L and 
doubling increases in concentration from 1 mg/L to 256 
mg/L, was added. The antimicrobial agents used were 
chloramphenicol, tetracycline, penicillin G, and strepto- 
mycin. The coverslip cultures were incubated at 37 °C for 
72 h; they were then methanol-fixed and Giems a- stained 
as described previously (19). The MIC of an antimicrobial 
agent was defined as the lowest concentration required to 
inhibit the formation of inclusions. 

To determine staining characteristics, coverslip cultures 
of Vero cells were infected with -10 5 bacteria. After 48 h of 
culture, the cells were methanol-fixed and stained by 
Giemsa, periodic acid-Schiff (PAS), or immunofluores- 
cence staining by using a monoclonal antibody directed 
against the major outer membrane protein of C. trachoma- 
tis (Microtrak, Trinity Biotech, Bray, Ireland) as described 
previously (18,19). For thin-section electron microscopy, 
infected cells were fixed in cacodylate-buffered glutaralde- 
hyde (2%), scraped from the flask, postfixed through 
increasing concentrations of ethanol (to 100% vol/vol), and 
then araldite embedded. Thin- sections were stained in 
uranyl acetate and Reynold’s lead citrate and examined 
with a Philips 301 electron microscope. For negative- stain 
electron microscopy, suspensions were placed on a 
Formvar-coated grid and stained in phosphotungstic acid. 

Genomic Characteristics 

Total DNA was extracted from a 72-h culture of the 
bacterium in Vero cells. The infected cells were scraped 
from a 25 -cm 2 tissue culture flask (Becton Dickinson, 
Basingstoke, U.K.) into 2 mL 199 medium without fetal 
calf serum. One milliliter of this mixture was centrifuged 
at 13,000 X g for 30 min, and the pellet was suspended in 
250 (iL of 5% wt/vol Chelex-100 resin slurry (BioRad, 
Hemel Hempstead, U.K.). This suspension was boiled for 
15 min, followed by centrifugation at 13,000 X g for 10 
min; the supernatant was then removed and stored at 
-20°C until used. 

For analysis of the 16S rRNA gene, a 1,526-bp ampli- 
con was produced by using primers 16S-FOR and 168- 
REV (Table 1) as described by Rurangirwa et al. (20). The 
amplicon was excised from the agarose gel and purified by 
using a gel purification kit (Qiagen, West Sussex, U.K.). 
The amplicon was cloned into a cloning vector, pGEM-T 
(Promega, Southampton, U.K.) and transformed into 
Escherichia coli. Full-length sequencing of the 1,526-bp 
amplicon within the cloning vector was achieved by using 
overlapping internal primers (F1-F4 forward and R1-R4 
reverse, Table 1). 16S rRNA signature sequence, 16S-23S 
rRNA intergenic space, and 23 S rRNA domain I signature 


272 


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Table 1 . Oligonucleotide primers for PCR and sequencing* 

Waddlia malaysiensis, New Chlamydialike Bacterium 

Gene target 

Primer sequence 

PCR 

1 6S rRNA (1 ,526 bp from ref. 20) 

16S-FOR 

16S-REV 

Tm = 55°C 

5' AGA GTT TGA TCC TGG 3' 
5' TAC CTT GTT ACG ACT T 3' 

16S rRNA signature sequence (298 bp from ref. 21) 
16S1GF 
16S1GR 
Tm = 51° C 

5' CGG CGT GGA TGA GGC AT 3' 
5' TCA GTC CCA GTG TTG GC 3' 

16S - 23S rRNA signature sequence (1 kbp from ref. 21) 
16SF2 
23S1GR 
Tm = 61° C 

5' CCG CCC GTC ACA TCA TGG 3' 

5' TGG CTC ATC ATG CAA AAG GCA 3' 

23S rRNA signature sequence (627 bp: domain 1 from ref. 21) 
23S1GR 
Tm = 61° C 

5' TGG CTC ATC ATG CAA AAG GCA 3' 

MurA signature sequence (690 bp from ref. 22) 
murA - for 
murA - rev 
Tm = 55° C 

5' GTN GGN GCN ACN GAR AA 3' 

5' GCC ATN ACR TAN GCR AAN CCN GC 3' 

sctN (331 bp) 
sctN FOR 
sctN REV 
Tm = 58° C 
Sequencing 

16S rRNA (1,526 bp) 
Forward: 

5' AGA RGG AAT GAA ACG TTC 3' 
5' GGC TCR TTC ATA TCA TC 3' 

FI (Ml 3) 
F2 
F3 
F4 

Reverse: 

5 GTT TTC CCA GTC ACG ACG TTG TA 3' 

5' GCT CAC CAA GGC TAA GAC GTC 3' (277-298) 

5' CTA GCT TTG ACC TGA CGC TGA T 3' (752-774) 
5' GAA TCT GCA ACT CGG CTC CAT G 3' (1 323-1 345) 

R1 (Ml 3) 

R2 

R3 

R4 

*PCR, polymerase chain reaction; Tm, melting temperature. 

5' TTG TGA GCG GAT AAC AAT TTC 3' 

5' CAT CCT AAA TGC TGG CAA C 3' (392-373) 

5' CAC CGC TAC ATG TGG AAT TCC 3' (843-822) 

5' GAT CCT CTC TAG CAC CAT ATC C 3' (1358-1336) 

sequence polymerase chain reaction (PCR) were carried 
out by using the method of Everett et al. (21) with the 
primers shown in Table 1 . In each case, PCR amplification 
was performed in 50-|lL volumes. All primers were added 
at 20 pmol per assay; PCR buffer (plus 1.5 mmol/L 
MgCl 2 ), Q solution, and Taq polymerase were obtained 
from Qiagen Ltd (Crawley, U.K.). The presence of the 
murA protein signature was sought by PCR by using 
primer murA- for and murA- rev (Table 1), which amplifies 
a 690-bp fragment of the UDP-N-acetylglucosamine 1- 
carboxyvinyltransferase gene of Waddlia chondrophila 
(22). In this case, PCR was attempted by using a range of 
Mg 2+ concentrations from 1.5 to 4.0 mmol/L . Primers to 
amplify a 331 -bp segment of the sctN gene were designed 
by alignment of the sctN genes of C. trachomatis 
(AE001337), C. pneumoniae (AE002167), and C. muri- 
darum (AE002271). The sctN gene encodes a type III 

secretion system ATPase, which is highly conserved 
among these bacteria (23). Sequence determination was 
performed by using an automated DNA sequencer (ABI 
PRISM 377; Perkin-Elmer, Warrington, U.K.) and was 
analyzed by using commercial software (Lasergene: 
DNAStar Inc., Madison, WI, USA). 

Lor phylogenetic analyses, sequence data on complete 
16S rRNA genes for each of the Chlamy diales genera 
were retrieved from GenBank and aligned with ClustalW 
(24). The phylogenetic tree was generated from the align- 
ment by using the genetic distance-based neighbor-joining 
algorithms of the Data Analysis in Molecular Biology 
software (DAMBE; http://web.hku.hk/~xxia/software/ 
software.htm). Sequence input order was randomized, and 
100 datasets were examined by bootstrapping resampling 
statistics. 


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273 


RESEARCH 


Results 

During the second and third field visits to Gua 
Tempurong, 206 urine samples were obtained (93 in the 
second and 113 in the third field visit) from individual 
bats. A total of 7 urine samples (all from the third visit) 
produced a characteristic CPE on Vero cells after 5 to 7 
days of culture. The same CPE was identified for each of 
the 7 isolates. One (G817) was therefore selected at ran- 
dom for further characterization. 

On negative- stain electron microscopy of the super- 
natant from G817 cultured on Vero cells, small bacterial 
cells (0.4-0.6 pm) resembling chlamydial elementary bod- 
ies were seen (Figure 1A). Inclusions visible by phase- 
contrast microscopy could be detected within 48 to 72 h 
postinfection of Vero cells. Similar inclusions could be 
seen after infection of human lung (MRC-5, A549), kidney 
(human embryo kidney [HEK]), laryngeal (HEp-2), and B- 
lymphoblastoid cells lines; and of simian kidney (LLC- 
MK2) and rodent epithelial (3T3, BHK) cell lines. Figures 
IB and IB show large inclusions in HEK- and the EBV- 
transformed human B-lymphoblastoid cell lines, respec- 
tively. In Figure IB, mixtures of reticulate and elementary 
bodies are visible. A thin- section electron micrograph of an 
earlier stage of infection of HEK cells (48 h postinfection, 
Figure ID) shows a collection of reticulate bodies with 
evidence of replication by binary fission. Mitochondria 
can be seen in close proximity. The bacterium could not be 
cultured on blood or chocolate agar, aerobically or anaero- 
bically, when incubated for up to 7 days, nor did it have 
catalase or oxidase activities. 


Inclusions could be stained by both Giemsa and PAS 
but not by the Mikrotrak immunofluorescence system, 
which recognizes the C. trachomatis major outer mem- 
brane protein. MICs of tetracycline and chloramphenicol 
were 0.25 mg/L and 0.5 mg/L, respectively, but strepto- 
mycin (256 mg/L) and penicillin G (256 mg/L) did not 
inhibit the formation of inclusions at therapeutically 
achievable levels. 

All of the 16S rRNA gene, the 16S-23S rRNA inter- 
genic spacer region, and the 627-bp domain I of the 23 S 
rRNA gene were sequenced in both directions. This 
sequence of 2379 bp has been lodged in GenBank with the 
accession number AY1 84804. A BLAST search indicated 
that a 1,552-bp sequence of the bacterium’s 16S rRNA 
gene had 96% and 94% identity with two 16S rRNA 
sequences from W. chondrophila (AF 346001 and AF 
042496). The 16S rRNA (298-bp) and 23S rRNA (627-bp) 
gene signatures had 91% identity with the 2 W. chon- 
drophila sequences deposited in GenBank. The 16S-23S 
rRNA intergenic space of the bat isolate was 223 bp com- 
pared to 213 bp (AF042496) and 217 bp (AF346001) for 
W. chondrophila. Figure 2 shows a neighbor-joining den- 
dogram demonstrating the relationships of the novel bat 
bacterial isolate to other members the Chlamy diales. This 
indicates that the novel bacterium is most closely related 
to, but distinct from, W. chondrophila. No PCR amplicons 
were detected on amplification of either murA or sctN. 
When DNA from C. trachomatis (lymphogranuloma 
venereum strain LI) was used as positive control, ampli- 
cons of the correct size were detected. 




Figure 1 . A, Negative stain electronmicro- 
graph of Waddlia malaysiensis elementary 
bodies. B-D, Thin-section electronmicro- 
graphs of cells infected with W. malaysien- 
sis. B, large inclusion with elementary(e) 
and reticulate(r) bodies in HEK cells 72 h 
postinfection. C, a large inclusion in 
Epstein Barr virus-transformed human 
B-lymphocytes. D, dividing reticulate bod- 
ies in HEK cells 48 h postinfection in an 
inclusion with numerous surrounding mito- 
chondria (arrow). 


274 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


Waddlia malaysiensis, New Chlamydialike Bacterium 


Chlamydia trachomatis (U68443) 
Chlamydia psittad (U 6844 7) 


I af: 

W n 


- Parachlamydia UWE25 
Parachlamydia acanthamoebae (Y07556) 
AF042496 \ , 

AF346001 J 

W. malaysiensis 


W. chondrophiia 


0.02 


- Simkania negevensis (U 684 60) 

R. porceliionis (AY223862) 

Escherichia coli 
(J01S59) 


Figure 2. Phylogenetic relationships of Waddlia malaysiensis to 
other Chlamydiales. 


Discussion 

Members of the order Chlamydiales are obligate intra- 
cellular bacteria. Recently, a suggestion to revise and 
update their classification has been made (21). This revi- 
sion was based on comparisons of 16S rRNA and 23 S 
rRNA genes, and it split the Chalmy diales into 4 families, 
Chlamydiaceae , Simkaniaceae , Parachlamydiaceae , and a 
family now named Waddliaceae (20), which has W. chon- 
drophiia as the prime member (Table 2). This scheme of 
nomenclature has largely been accepted, although splitting 
the family Chlamydiaceae into 2 genera, Chlamydia and 
Chlamydophila , raised some concerns (25). The 
Chlamydiales are an expanding group of bacteria with new 
genera and species increasingly being described and 
detected in a wide array of hosts (26,27). Recent examples 
include Rhab do chlamydia porceliionis , isolated from ter- 


restrial isopods, which is related to but not entirely within 
the family Simkaniaceae (28), and 2 insect-associated 
chlamydia, Fritschea bemesia and F. eriococci in the fam- 
ily Simkaniaceae (29). In addition, a number of 
Chlamydiales endosymbionts have been recovered from 
human clinical and environmental isolates of 
Acanthamoeba spp. that are related to the 
Parachlamydiaceae (30). Indeed for one of these, UWE25, 
the full genome has been sequenced (31). Analyses of 16S 
rRNA, 23S rRNA genes, and the 16S-23S intergenic space 
indicate that the bacterium we have isolated from fruit bats 
is most closely related to the Waddliaceae (Figure 2). 
There are, however, some similarities and differences 
between our isolate and W. chondrophiia. W. chrondrophi- 
la has been isolated twice from cattle, and the bacteria 
were obtained from a first-trimester bovine abortion in the 
United States (20) and a septic stillborn calf in Germany 
(32). The bacterium from the United States was isolated 
initially by culture on bovine turbinate cells (20), but the 
German isolate was able to grow in human diploid fibrob- 
lasts, simian (Buffalo Green Monkey, and murine 
[McCoy]) cells lines (32). Our bat isolate was able to grow 
in a wide range of cell types from different anatomic sites 
and animal species. Some evidence suggests that W. chon- 
drophiia also has a wide host cell range, but not all possi- 
bilities have been tested. There is also recent evidence, 
based on 16S rDNA amplification, of W. chondrophiia in 
an Australian mammal, Gilbert’s Potoroo (33). Like W. 
chondrophiia , our isolate was resistant to penicillin G and 
streptomycin (MICs >256 mg/L) and could not be stained 
by immunofluorescence using monoclonal anti-C. tra- 


Table 2. Current status of the Chlamydiales 

Family 

Genus and species 

Biovars 

Host/animal disease* 

1. Chlamydiacae 

Chlamydia trachomatis 

Serovars A-K 

Humans: trachoma, STI 



Serovars Li-L 3 

Humans: STI 


C. muridarum 

- 

Mice: proliferative ileitis 


C. suis 

- 

Swine: conjunctivitis: pneumonia 


Chlamydophila psittaci 

Serovars A-H 

Birds, cattle: pneumonia! 


C. pneumoniae 

3 biovars 

Humans, koala, equines: 
pneumonia, conjunctivitis 


C. pecorum 


Wide host range and disease manifestation 


C. felis 

? 2 biovars 

Cats: rhinitis! 


C. caviae 


Guinea pigs: conjunctivitis 


C. abortus 


Sheep, cattle, goats: abortion! 

II. Parachlamydiacieae 

Parachlamydia acanthamoebae 


Amoebae: RTI 


Neochlamydia hartmannellae 
Numerous others including UWE25 


Amoebae 

III. Waddliaceae 

Waddlia chondrophiia 


Cattle, potoroos: abortion 

IV. Simkaniae 

Simkania negevensis 


Amoebae, humans: RTI 


“Candidates Fritschea bemesiae” 


Whitefly 


“Candidates F. eriococci” 


Whitefly 

V. Clamydia-like organisms 

“Candidates Rhabdochlamydia 




porceliionis” 




*STI, sexually transmitted infection; RTI, respiratory tract infection; -perhaps (i.e., disputed); ?, may be 2 biovars but not confirmed, 
indicates zoonotic potential. 


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275 


RESEARCH 


chomatis antibodies. However, in contrast to 1 report (20), 
the bat bacterial inclusions stained intensely with PAS 
stain. This stain reacts with the glycogen matrix elaborat- 
ed by Chlamydiaceae when growing intracellularly. The 
bat isolate is sensitive to tetracycline (MIC 0.25 mg/L) and 
chloramphenicol (MIC 0.5 mg/L) at concentrations that 
are clinically achievable and similar to those needed to 
cure infections by C. trachomatis. No evidence for the 
presence of one of the key genes (, sctT) of the pathogenic- 
ity island-associated type III secretion system of C. tra- 
chomatis was found in W. malaysiensis ; however, this does 
not mean that no such island is present. Three genes ( sctT, 
sctN , and sctV) from a type III secretion system have been 
described in the Parachlamydia- like endosymbiont 
UWE25, and sufficient differences exist in the nucleotide 
and putative amino acid sequences of these, when com- 
pared to those of C. trachomatis (31), that our primers 
would not amplify it. 

Negative-stain electron microscopic examination of the 
bat bacterium released from Vero cells showed small cocci 
indistinguishable from the elementary bodies of C. tra- 
chomatis. On thin-section electron microscopy of infected 
cells, large numbers of intracellular bacteria could be seen 
within membrane-bound inclusions. In mature inclusions 
in all cell types tested, mixtures of elementary and reticu- 
late bodies were found. In less mature inclusions, dividing 
reticulate bodies were present, and mitochondria could be 
seen around the inclusion (Figure ID). The species name 
of W. chondrophila was derived from the collections of 
mitochondria around the intracellular inclusions. The bat 
isolate was closest to the 2 W. chondrophila isolates made 
from cattle on the basis of 16S rRNA gene comparisons 
(96% and 94% identity). The 16S rDNA and 23S rDNA 
gene signature sequences also placed the bat bacterium 
close, to but not identical to, W. chondrophila (91%); in 
addition, the 16S - 23 S rRNA intergenic space was slight- 
ly longer than for W. chondrophila. Thus, the bat isolate is 
part of the genus Waddlia , and we propose the name 
Waddlia malaysiensis for it since it was first detected in 
Malaysia. The organism appears sufficiently distinct from 
W. chondrophila to justify a different species assignment. 
It is PAS positive, does not have the murA signature of W. 
chondrophila , and has differences in the 16S - 23 S rRNA 
genomic regions. The collection of mitochondria in prox- 
imity to inclusions that gave W. chondrophila its species 
name was also exhibited by W. malaysiensis and might 
therefore be a characteristic of the genus Waddlia. 

The Chlamydiales infect a wide range of animals 
including humans (27,34). Some pathogens such as C. tra- 
chomatis appear to solely affect humans; others affect only 
animals; and a sizeable number are zoonotic pathogens 
(Table 2). W. chondrophila has been isolated from aborted 
cattle fetuses in the United States and German (20,32) but 


has also been detected in an apparently healthy Potoroo in 
Australia (33). Recent serologic evidence has suggested a 
strong statistical association between high titers of W. 
chondrophila antibodies and bovine abortion (35). 
Members of the genera Parachlamydia and Simkania 
infect protozoa such as amoebae and can cause disease in 
humans (30,36,37). In this respect, evidence exists for 
replication of W. chondrophila in amoebae (38), which 
suggests that it might fall into the group of environmental- 
ly preadapted pathogens, as has been suggested for S. 
negevensis (39) and C. pneumoniae (40). Whether W. 
malaysiensis can grow in amoebae and has zoonotic poten- 
tial remains to be determined. 

Dr. Chua is a pediatrician and medical microbiologist. He 
was the first to isolate Nipah vims in Malaysia. 

References 

1 . Taylor LH, Latham SM, Woolhouse ME. Risk factors for human dis- 
ease emergence. Philos Trans R Soc Lond B Bio Sci. 
2001;356:983-9. 

2. Frohlich K, Thiede S, Kozikowski T, Jakob W. A review of mutual 
transmission of important infectious diseases between livestock and 
wildlife in Europe. Ann N Y Acad Sci. 2002;969:4-13. 

3. Simpson VR. Wild animals as reservoirs of infectious diseases in the 
UK. Vet J. 2002;163:128-46. 

4. Paez A, Nunez C, Garcia C, Boshell J. Molecular epidemiology of 
rabies enzootics in Colombia: evidence for human and dog rabies 
associated with bats. J Gen Virol. 2003;84:795-802. 

5. Fooks AR, Finnegan C, Johnson N, Mansfield K, McElhinney L, 
Manser P. Human case of EL type 2 following exposure to bats in 
Angus, Scotland. Vet Rec. 2002; 15 1:679. 

6. Halpin K, Young PL, Field HE, Mackenzie JS. Isolation of Hendra 
virus from pteropid bats: a natural reservoir of Hendra virus. J Gen 
Virol. 2000;81:1927-32. 

7. Bowden TR, Westenberg M, Wang L-F, Eaton BT, Boyle DB. 
Molecular characterization of Menangle virus, a novel paramyx- 
ovirus which infects pigs, fruit bats and humans. Virology. 
2001;283:358-73. 

8. Chua KB, Koh CL, Hooi PS, Wee KF, Khong JH, Chu BH, et al. 
Isolation of Nipah virus from Malaysian Island flying-foxes. 
Microbes Infect. 2002;4:145-51. 

9. Chua KB, Wang LF, Lam SK, Eaton BT. Full length genome 
sequence of Tioman virus, a novel paramyxovirus in the genus 
Rubulavirus isolated from fruit bats in Malaysia. Arch Virol. 
2002;147:1323-48. 

10. Kim GR, Lee YT, Park CH. A new natural reservoir of hantavirus: 
isolation of hantaviruses from lung tissue of bats. Arch Virol. 
1994;134:85-95. 

11. Bunnell JE, Hice CL, Watts DM, Montrueil V, Tesh RB, Vinetz JM. 
Detection of pathogenic Leptospira spp infections among mammals 
captured in the Peruvian Amazon basin region. Am J Trop Med Hyg. 
2000;63:255-8. 

12. Arata AA, Vaughn JB, Newell KW, Barth RA, Gracian M. 
Salmonella and Shigella infections in bats in selected areas of 
Colombia. Am J Trop Med Hyg. 1968;17:92-5. 

13. Heard DJ, Young JL, Goodyear B, Ellis GA. Comparative rectal bac- 
terial flora of four species of flying fox (Pteropus sp). J Zoo Wildl 
Med. 1997;28:471-5. 


276 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


Waddlia malaysiensis, New Chlamydialike Bacterium 


14. Souza V, Rocha M, Valera A, Eguiarte LE. Genetic structure of natu- 
ral populations of Escherichia coli in wild hosts on different conti- 
nents. Appl Environ Microbiol. 1999;65:3373-85. 

15. Chua KB. A novel approach for collecting samples from fruit bats for 
isolation of infectious agents. Microbes Infect. 2003;5:487-90. 

16. Heideman PD, Utzurrum RCB. Seasonality and synchrony of repro- 
duction in three species of nectarivorous Philippines bats. 
Biomedcentral Ecology. 2003. Available from http://www.biomed- 
central.com/1472-6785/3/1 1 

17. Arguin PM, Murray-Lillibridge K, Mirand MEG, Smith JS, Calaor 
AB, Rupprecht CE. Serologic evidence of lyssavirus infection among 
bats, the Philippines. Emerg Infect Dis. 2002;8:258-62. 

18. How SJ, Hobson D, Hart CA. Studies in vitro of the nature and syn- 
thesis of the cell wall of Chlamydia trachomatis. Current 
Microbiology. 1984;10:269-74. 

19. How SJ, Hobson D, Hart CA, Quayle E. A comparison of the in vitro 
activity of antimicrobials against Chlamydia trachomatis examined 
by Giemsa and a fluorescent antibody stain. J Antimicrob Chemother. 
1985;15:399-404. 

20. Rurangirwa FR, Dilbeck PM, Crawford TB, McGuire TC, McElwain 
TF. Analysis of the 16S rRNA gene of microorganism WSU8-1044 
from an aborted bovine foetus reveals that it is a member of the order 
Chlamy diales proposal of Waddliaceae fam. nov., Waddlia chon- 
drophila gen. nov., sp. nov. Int J Syst Bacteriol. 1999;49:577-81. 

21. Everett KDF, Bush RM, Anderson AA. Emended description of the 
order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and 
Simkaniaceae fam. nov. each containing one monotypic genus, 
revised taxonomy of the family Chlamy diaceae, including a new 
genus and five new species, and standards for the identification of 
organisms. Int J Syst Bacteriol. 1999;49:415-40. 

22. Griffiths E, Gupta RS. Protein signatures distinctive of chlamydial 
species: horizontal transfers of cell wall biosynthesis genes glmU 
from archaea to chlamydiae and murA between chlamydiae and 
Streptomyces. Microbiology. 2002;148:2541-9. 

23. Subtil A, Dautry-Varsat A. Type III secretion system in Chlamydia 
species: identified members and candidates. Microbes Infect. 
2000;2:367-9. 

24. Thomson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the 
sensitivity of progressive multiple sequence alignment through 
sequence weighting position-specific gap penalties and weight matrix 
choice. Nucleic Acids Res. 1994;22:4673-80. 

25. Schachter J, Stephens RS, Timms P, Kuo C, Bavoil PM, Birkelund S, 
et al. Radical changes to chlamydial taxonomy are not necessary just 
yet. Int J Syst Evol Microbiol. 2001 ;5 1:249. 

26. Corsaro D, Vallassina M, Venditti D. Increasing diversity within 
Chlamydiae. Crit Rev Microbiol. 2003;29:37-78. 

27. Corsaro D, Venditti D. Emerging chlamydial infections. Crit Rev 
Microbiol. 2004;30:75-106. 


28. Kostanjsek R, Stras J, Drobne D, Avgustin G. “ Candidatus 
Rhabdochlamydia porcellionis,” an intracellular bacterium from the 
hepatopancreas of the terrestrial isopod Porcellio scaber (Crustacea: 
Isopoda). Int J Syst Evol Microbiol. 2004;54:543-9. 

29. Thao ML, Baumann L, Hess JM, Falk BW, Ng JCK, Gullan PJ, et al. 
Phylogenetic evidence for two insect-associated chlamydia of the 
family Simkaniaceae. Curr Microbiol. 2003;47:46-50. 

30. Fritsche TR, Horn M, Wagner M, Herwig RP, Schleifer K-H, Gautom 
RK. Phylogenetic diversity among geographically dispersed 
endosymbionts recovered from clinical and environmental isolates of 
Acanthamoeba spp. Appl Environ Microbiol. 2000;66:2613-9. 

31. Horn M, Collingro A, Schmitz-Esser S, Beier CL, Puckhold U, 
Fartmann B, et al. Illuminating the evolutionary history of chlamydi- 
ae. Science.2004;304:728-30. 

32. Henning K, Schares G, Granzow H, Polster U, Hartmann M, Hotzel 
H, et al. Neospora caninum and Waddlia chondrophila strain 2032/99 
in a septic stillborn calf. Vet Microbiol. 2002;85:285-92. 

33. Bodett TJ, Viggers K, Warren K, Swan R, Conaghty S, Sims C, et al. 
Wide range of Chlamydiale types detected in native Australian mam- 
mals. Vet Microbiol. 2003;96:177-87. 

34. Longbottom D, Coulter LJ. Animal chlamydioses and zoonotic impli- 
cations. J Comp Pathol. 2003;128:217-44. 

35. Dilbeck-Robertson P, McAllister MM, Bradway D, Evermann JF. 
Results of a new serologic test suggest an association of Waddlia 
chondrophila with bovine abortion. J Vet Diagn Invest. 
2003;15:568-9. 

36. Friedman MG, Dvoskin B, Kahane S. Infections with chlamydia-like 
microorganism Simkania negevensis, a possible emerging pathogen. 
Microbes Infect. 2003;5:1013-9. 

37. Birtles RJ, Rowbotham TJ, Storey C, Marrie TJ, Raoult D. 
Chlamydia-like obligate parasite of free living amoebae. Lancet. 
1997;349:925-6. 

38. Michel R, Steinert M, Zoller L, Hauroder B, Henning K. Free-living 
amoebae may serve as hosts for the Chlamydia-like bacterium 
Waddlia chondrophila isolated from an aborted bovine foetus. Acta 
Protozool. 2004;43:37-42. 

39. Kahane S, Dvoskin B, Mathias M, Friedmann MG. Infection of 
Acanthamoeba polyphaga with Simkania negevensis and S. negeven- 
sis survival within amoebal cysts. Appl Environ Microbiol. 
2001;67:4789-95. 

40. Essig A, Heinemann M, Simnacher U, Marre R. Infection of 
Acanthamoeba castellani by Chlamydia pneumoniae. Appl Environ 
Microbiol. 1997;63:1396-9. 

Address for correspondence: C. A. Hart, Department of Medical 

Microbiology, University of Liverpool, Duncan Building, Daulby St, 

Liverpool, L69 3GA, United Kingdom; fax: 0151 706 5805; email: 

cahmm@liv.ac.uk 


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Quarantine for SARS, Taiwan 

Ying-Hen Hsieh,* Chwan-Chuan King,t Cathy W. S. Chen4 Mei-Shang Ho,§ Jen-Yu Lee,* 
Feng-Chi Liu,* Yi-Chun Wu,K and Jiunn-Shyan JulianWufl 


During the 2003 outbreak of severe acute respiratory 
syndrome (SARS) in Taiwan, >150,000 persons were quar- 
antined, 24 of whom were later found to have laboratory- 
confirmed SARS-coronavirus (SARS-CoV) infection. Since 
no evidence exists that SARS-CoV is infective before the 
onset of symptoms and the quarantined persons were 
exposed but not symptomatic, we thought the quarantine’s 
effectiveness should be investigated. Using the Taiwan 
quarantine data, we found that the onset-to-diagnosis time 
of previously quarantined confirmed case-patients was sig- 
nificantly shortened compared to that for those who had not 
been quarantined. Thus, quarantine for SARS in Taiwan 
screened potentially infective persons for swift diagnosis 
and hospitalization after onset, thereby indirectly reducing 
infections. Full-scale quarantine measures implemented on 
April 28 led to a significant improvement in onset-to-diagno- 
sis time of all SARS patients, regardless of previous quar- 
antine status. We discuss the temporal effects of quarantine 
measures and other interventions on detection and isolation 
as well as the potential usefulness of quarantine in faster 
identification of persons with SARS and in improving isola- 
tion measures. 


T he severe acute respiratory syndrome (SARS) epidem- 
ic from November 2002 to June 2003 came with much 
public attention and left swiftly, resulting in >8,000 proba- 
ble cases worldwide and 774 deaths (1). Prominent among 
retrospective analyses is the belief that the simple ancient 
system of placing persons suspected of being infected 
under quarantine was instrumental in the quick contain- 
ment of the outbreak (2-5). However, questions persist 
regarding how quarantine worked to control this disease, 
given the time-tested axiom that quarantine is most useful 
only when patients are infectious before becoming symp- 
tomatic, thus directly preventing secondary infections (6). 
Moreover, due to early confusion resulting from imprecise 
clinical diagnosis and case definition (7), correct clinical 
diagnosis and prompt isolation were often impossible, 


*National Chung Hsing University, Taichung, Taiwan; fNational 
Taiwan University, Taipei, Taiwan; fFeng Cha University, Taichung, 
Taiwan; §Academia Sinica, Taipei, Taiwan; and ^Center for 
Disease Control, Taipei, Taiwan 


which resulted in insufficient isolation and gaps in the con- 
tainment strategy for hospital infection control (8). Since 
all available evidence indicates that SARS patients were 
only infectious after symptom onset (9), one may argue 
that quarantine provides a window of several days during 
which illnesses can be diagnosed swiftly and persons iso- 
lated accordingly. In this study, we used data from the 
Taiwan SARS outbreak to explore whether quarantine was 
effective in expediting the time from onset to clinical diag- 
nosis and hospitalization, and the time from clinical diag- 
nosis to classification as a probable case-patient, thus 
contributing indirectly to prevention of possible infections. 

Methods 

Data 

During the outbreak of 2003, 346 SARS cases were 
officially confirmed in Taiwan, among which were 37 
direct SARS deaths (cause of death was recorded as 
SARS) and 36 SARS-related deaths (cause of death was 
not directly attributed to SARS) as reported by the World 
Health Organization (WHO) (1). To guard against the 
potential threat of a large-scale epidemic, the government 
attempted to place >150,000 people under home quaran- 
tine. Two distinct levels of quarantine were implemented 
in Taiwan. Level A quarantine, aimed at people having 
close contact with a suspected SARS case-patient, was 
implemented on March 18, 2003. Level B quarantine, 
aimed at travelers from affected areas, was implemented 
on April 28, in the aftermath of the first SARS death on 
April 26 (10,11). Most of the quarantined persons were 
confined to their homes for 10-14 days. Public health 
nurses would bring the quarantined persons 3 meals every 
day and sometimes helped them with odd jobs such as 
washing clothes or taking care of pets. Center for Disease 
Control-Taiwan officially confirmed 346 SARS- 
CoV-positive cases, of which 17 case-patients had been 
previously quarantined; 134 additional laboratory-con- 
firmed antibody-positive SARS cases occurred, of which 7 
case-patients had previously been quarantined. The total 
number of confirmed SARS case-patients in Taiwan by the 


278 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


Quarantine for SARS, Taiwan 


Table 1 . Cumulative numbers of persons under quarantine during the SARS outbreak, Taiwan, 2003, and the quarantined SARS 
patients classified by their status* 


Level and reason for 
quarantine 

No. quarantined 
persons 

No. quarantined officially confirmed 
SARS-CoV case-patients 

No. quarantined laboratory confirmed, 
antibody positive SARS case-patients with 

Level A 

Family members 

7,921 

8 

2 

Classmates and teachers 

16,564 

1 

0 

Healthcare workers 

2,409 

0 

3 

Others! 

19,224 

6f 

1 

All others§ 

9,514 

2 

1 

Subtotal 

55,632 

17 

7 

Level B 

95,828 

0 

0 

Total 

151,460 

17 

7 


*Updated December 2004. 

tPassengers and drivers of domestic public transportation traveling for >1 hour in the same bus or train cabin with a SARS case-patient, persons who 
had contacts with someone under quarantine or receiving care in a medical facility where cluster infection had occurred, and homeless persons. 
JCo-workers and friends of SARS case-patients, airplane passengers who sat within 3 rows of or stayed in the same room as SARS patients, and 
persons with missing information. 

§One case-patient had onset of symptoms 2 days after the end of quarantine. 


end of December 2004 was 480, of which 24 had been 
quarantined previously. Details regarding the persons 
quarantined during the SARS outbreak are itemized in 
Table 1. 

The 134 patients with laboratory-confirmed SARS 
either had milder symptoms, and SARS was therefore clin- 
ically diagnosed as suspected, ruled out at the time of the 
outbreak, or considered probable in patients whose speci- 
mens had previously tested negative by polymerase chain 
reaction (PCR) or anti-SARS-CoV antibody, perhaps due 
to wrong timing, but later were reconfirmed by >2 differ- 
ent laboratory tests in a follow-up epidemiologic study. 
Seven people in this group had been previously quaran- 
tined. Our criterion for a quarantined person was someone 
who had been placed under official quarantine for >1 day 
before the onset of symptoms. Thus, persons in whom 
symptoms developed on the same date or before the noti- 
fication of quarantine were considered not quarantined and 
were therefore excluded. Persons who were known to have 
had a record of close contacts with others during the sup- 
posed quarantine period were also excluded. One of the 24 
case-patients actually had an imported case but was quar- 
antined before implementation of level B quarantine on 
April 28 for reasons other than simply being a traveler 
from an affected area. 

Statistical Analyses 

We compared the mean time from onset of symptoms to 
clinical diagnosis (and admission) for the 24 patients with 
laboratory-confirmed SARS -Co V who had been quaran- 
tined before symptom onset to that of the 451 SARS- 
CoV-positive case-patients who had not. Note that 5 cases 
were deleted from the data of 480 total cases for our com- 
parison test because of missing information on their rele- 
vant dates. (We will use the term “diagnosis” to mean 
clinical diagnosis hereafter.) For the mean time from diag- 
nosis to classification as probable case, we only used the 


officially confirmed cases for comparison, since the labo- 
ratory confirmed cases were either ruled out or classified 
as suspected cases only and thus had no classification of 
probable time. Again, 2 of these cases were deleted from 
the data for our comparison test because of missing infor- 
mation on their relevant dates; therefore, 344 case-patients 
(17 quarantined and 327 nonquarantined) were used. Due 
to the skewed data, we used the nonparametric Mann- 
Whitney test. 

To investigate the effect of large-scale quarantine on the 
changes in the efficiency of the public health system to 
identify SARS patients for isolation, we considered the 
temporal effect of important events for intervention and 
control of SARS in Taiwan. On April 28, level B quaran- 
tine was implemented, which marked the start of large- 
scale home quarantine (12). 

A second important date in SARS prevention and con- 
trol was May 10, when changes in the review and classifi- 
cation procedures were implemented by the cabinet-level 
SARS Prevention and Extrication Committee in Taiwan to 
expedite the review and classification of SARS cases (13). 
Before May 9, the relevant medical records (including any 
available laboratory test results) of all reported SARS 
patients were reviewed by a central SARS Advisory 
Committee of the Center for Disease Control-Taiwan in 
Taipei. Due to the rapid increase in the number of reported 
cases caused by the hospital cluster outbreaks in Taipei in 
late April, the SARS Advisory Committee in CDC-Taiwan 
could not handle the rapidly increasing caseload. 
Consequently, after May 10, 3 regional offices of the 
Bureau of National Health Insurance in northern, central, 
and southern Taiwan took over the responsibility of case 
review. Local SARS expert committees were established 
in the 3 regions with each committee consisting of special- 
ists similar to the central committee in Taipei. The experi- 
ences and the standard operation procedures of case review 
and case classification used by the central committee were 


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279 


RESEARCH 


transferred to the 3 regional SARS expert committees in 
the Bureau of National Health Insurance through several 
consensus meetings (14). 

We used the Mann- Whitney test to compare the time 
intervals from onset to clinical diagnosis of SARS symp- 
toms of the patients with confirmed SARS -Co V with onset 
occurring during the 3 periods of February 25-April 27 
(period 1), April 28-May 9 (period 2), and May 10-June 
15 (period 3). Five patients were deleted from the data for 
our comparison test due to missing information on their 
relevant dates, and 2 patients were deleted because their 
onsets of SARS did not occur during the 3 time periods. 

We also compared, using the Mann- Whitney test, the 
intervals from diagnosis to classification as probable 
SARS of the 343 officially confirmed SARS-CoV case- 
patients (by dividing the cases into 3 groups, according to 
the time period in which the date of diagnosis occurred). 
Again, the laboratory-confirmed cases had never been 
classified as probable cases. Moreover, 2 cases were 
deleted from the data for our comparison test due to miss- 
ing information on their relevant dates, and 1 case was 
deleted because classification as a probable case-patient 
did not occur during the 3 periods from February 25 to 
June 15. 

Results 

The mean time from onset to diagnosis for the previ- 
ously quarantined persons (1.20 days) was significantly 
shorter than that of those who were not quarantined (2.89 
days) (Table 2). However, the respective mean times from 
diagnosis to classification (6.21 days and 7.34 days) (Table 
2), though slightly reduced for the quarantined persons, 
were not significantly different. For the mean onset-to- 
diagnosis time, period 1 was significantly longer (3.60 
days to 2.49 days) than period 2 (p < 0.0001), while the 
mean difference before and after May 10 was not signifi- 
cant (p = 0.0722) (Table 3). The mean diagnosis-to-classi- 
fication time (Table 4) was not significantly different from 
period 1 to period 2. However, the time was significantly 
shortened after May 10 (from period 2 to period 3). 

Discussion and Conclusions 

The experience in the affected areas has shown that the 
transmission of SARS can be prevented by adherence to 
basic public health measures, including rapid case detec- 


tion, isolation of patients with suspected and probable 
cases, contact tracing, and good infection control (9). The 
effect of possible delays in effective isolation of probable 
case-patients has been studied in some modeling work on 
SARS (15-17). In Taiwan, all patients were supposed to be 
placed in the isolation room and negative pressure room, if 
available, as soon as they were reported as having proba- 
ble or suspected SARS. For most of May, the number of 
suspected case-patients alone remained well above 1,000, 
partly because of confusion in diagnosis and the tendency 
to overdiagnosis because of heightened alertness on the 
part of physicians and legal punishment for underreport- 
ing. At times, however, due to the lack of available isola- 
tion rooms or the number of suspected cases pending 
review, patients with suspected but unconfirmed SARS 
were kept for days in an observation room or emergency 
department under crude isolation, where nosocomial infec- 
tions readily occurred. At other times, patients scheduled 
to transfer to another hospital with negative pressure isola- 
tion rooms were temporarily kept in the observation room 
in the emergency department where nosocomial infections 
might occur because of insufficient isolation and protec- 
tion procedures (18). When full isolation facilities were 
not available to all patients, those classified as probable 
SARS case-patients likely received higher priority and 
were observed more closely during their isolation by 
healthcare workers than were the suspected case-patients. 

For some case-patients, delays occurred because of the 
patient’s uncertain status or urgent need for intubation 
without comprehensive information on the patient’s con- 
tact and clinical history; these delays led to insufficient 
protection and isolation. One well-known case-patient was 
the index patient at Hoping Hospital in Taipei, where the 
largest cluster infection in Taiwan occurred. Her condition 
was diagnosed and reported as suspected SARS on April 9. 
However, because the patient had no apparent contact with 
another known SARS case-patient, her case was reviewed 
but not reclassified as probable until April 25, by which 
time the clustered cases, which included medical staff 
members and an x-ray technician who had contact with 
her, had already forced the hospital to shut down on the 
previous day. More strict infection control would have 
been in place had the index patient been confirmed as a 
probable SARS patient. Several other similar cases 
occurred in Taiwan, some more than 1 month later. 


Table 2. Comparison of mean time intervals by using the Mann-Whitney test for the onset-to-diagnosis and diagnosis-to-classification 
times for quarantined and nonquarantined SARS patients, Taiwan, 2003 


Onset-to-diagnosis interval (d) 

Diagnosis-to-classification interval (d) 

Quarantined persons 

1 .203 (n = 24) 

7.7647 (n = 17) 

Nonquarantined persons 

2.891 4 (n = 451)* 

7.5443 (n = 327)f 

Mean difference 

1 .6831 1 (0.0061) 

0.2204 (0.7864) 


*5 cases deleted because of missing information on the relevant dates. 
f2 cases deleted because of missing information on the relevant dates. 

^Denotes significance at the 1% level; p values of Mann-Whitney test are in parentheses. 


280 


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Quarantine for SARS, Taiwan 


Table 3. Results of Mann-Whitney test for temporal changes in onset-to-diagnosis time of 473* confirmed SARS case-patients with 
onset of illness during period 1 ,| period 2, and period 3, 2003 


Interval (d) 

Period 1 (N = 161) 

Period 2 (N = 146) 

Period 3 (N = 166) 

Mean difference (p value) 

Onset to diagnosis 

3.6398 

2.0959 

- 

1.5439* (< 0.0001) 

Onset to diagnosis 

- 

2.0959 

2.6024 

0.5065 (0.0722) 


*5 cases deleted because of missing information on the relevant dates and 2 case-patients deleted because their onsets of SARS did not occur during the 
3 periods of 2/25-6/15. 

fPeriod 1 (2/25-4/27), time from onset of first case to the day before implementation of intervention measures including level B quarantine; period 2 
(4/28-5/9), time from implementation of intervention measures to the implementation of expedited classification procedure; period 3 (5/10-6/15), time of 
the expedited classification procedure to the date of onset of the last SARS case. 

^Denotes significance at 1% level. 


Therefore, the importance of rapid classification as proba- 
ble case-patients cannot be ignored. 

Our results show that quarantine reduced the time from 
onset to diagnosis but did not significantly reduce the time 
from diagnosis to classification. Thus, a previously quar- 
antined person could expect his or her condition to be diag- 
nosed and to be hospitalized more quickly once clinical 
symptoms appeared. However, the same person would not 
receive higher priority in the classification process to 
determine candidates for effective isolation. Nevertheless, 
in many hospitals with available isolation rooms, patients 
with suspected cases were effectively isolated as soon as 
chest radiographic evidence of infiltrates consistent with 
pneumonia or acute respiratory distress syndrome became 
available. Moreover, in the latter stages of the epidemic 
when a reliable laboratory test for SARS -Co V became 
more available, many patients were isolated in negative 
pressure chambers immediately if results of reverse tran- 
scription-PCR for SARS-CoV from 2 different laborato- 
ries were positive. Therefore, the effect of classification as 
a probable case-patient might not be as pronounced as it 
would have been otherwise. 

For all laboratory confirmed case-patients, regardless 
of whether they were quarantined previously, the imple- 
mentation of full-scale intervention measures, including 
level B quarantine on April 28, significantly decreased the 
time from onset to diagnosis, but it only slightly improved 
the time from diagnosis to classification. However, the 
small sample size of 24 previously quarantined SARS 
case-patients did not permit a meaningful test of whether a 
significant difference existed for the previously quaran- 
tined persons during each of the 3 periods. 


By comparison, the change in the review and classifi- 
cation procedure initiated on May 10 helped shorten the 
diagnosis-to-classification time for all SARS patients, 
indicating that the action by the SARS Prevention and 
Extrication Committee to expedite the review process had 
indeed worked. However, by separating the analyses of 
data into discrete epochs marked by significant events, we 
have included those cases whose illnesses straddle epochs. 

In the future, when facing newly emerging infectious 
diseases like SARS, in which the patient’s infectivity in the 
incubation period is unknown, precise clinical diagnosis 
cannot be made, and modes of transmission are uncertain, 
quarantine should be used not only to directly prevent pos- 
sible asymptomatic infections but also to screen out poten- 
tially infective persons and thus prevent secondary or even 
tertiary infections. 

The quarantine in Taiwan was indeed useful in helping 
to identify persons who are likely to develop symptoms 
and isolate them more quickly if and when they did, 
although its effect on isolation and infection control could 
perhaps be improved by quicker classification or confir- 
mation of previously quarantined patients. No conclusion 
was drawn regarding whether better outbreak control 
would be achieved by placing fewer persons in quarantine 
or by concentrating on improving the efficiency of detec- 
tion and isolation procedures. In fact, each area may be 
improved in efficiency without jeopardizing the other’s 
improvement. 

Acknowledgments 

We are grateful to Roy Anderson, John Glasser, and Fred 
Brauer for constructive discussions that helped formulate some 


Table 4. Mann-Whitney test results for temporal changes in the diagnosis-to-classification time of 343* officially confirmed SARS-CoV 
cases with classification during period 1 ,f period 2, and period 3, Taiwan, 2003 

Interval (d) 

Period 1 (N = 103) 

Period 2 (N = 114) 

Period 3 (N = 126) Mean difference (p value) 

Diagnosis to classification 

9.1845 

8.2368 

0.9477 (0.7729) 

Diagnosis to classification 

- 

8.2368 

5.6508 2.5860t (<0.0001 ) 


*2 cases were deleted from the data for our comparison test because of missing information on the relevant dates, and 1 case was deleted because 
classification as probable case did not occur during the 3 time periods of 2/25-6/15. 

fPeriod 1 , time from onset of first case to the day before implementation of intervention measures including Level B quarantine; period 2, time from 
implementation of intervention measures to the implementation of expedited classification procedure; period 3, time of the expedited classification 
procedure to the date of onset of the last SARS case. 

^Denotes significance at the 1% level. 


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of the ideas for this work and to the anonymous referees for their 
many valuable comments. Sincere thanks are also extended to 
central and local public health personnel and medical staff who 
devoted all of their efforts to the quarantine and prevention of 
SARS in Taiwan. Y.H.H. would like to thank Mathematics of 
Information Technology and Complex Systems (MITACS) for 
their generous financial support for Y.H.H. to attend MITACS 
SARS meetings at Banff, Alberta, Canada, where several of the 
above-mentioned discussions took place. 

Y.H.H. (NSC 92-275 1-B005-001-Y), C.C.K. (NSC 92- 
2751-B002-020-Y), and M.S.H. were supported by SARS 
research grants from the National Science Council of Taiwan. 

Dr. Hsieh is a professor of applied mathematics at National 
Chung Hsing University. His primary research interests are 
focused on mathematical and statistical modeling of infectious 
diseases epidemiology. 

References 

1 . World Health Organization. Summary of probable SARS cases with 
onset of illness from 1 November 2002 to 31 July 2003. 2003 Sep 26 
[cited 2005 Jan6]. Available at http://www.who.int/csr/sars/ coun- 
try/table2003_ 09_23/en/. 

2. Enserink M. SARS: a pandemic prevented. Science. 2003;302:2045. 

3. Ou J, Li Q, Zeng G. Efficiency of quarantine during an epidemic of 
severe acute respiratory syndrome — Beijing, China, 2003. MMWR 
Morb Mortal Wkly Rep. 2003;52:1037-40. 

4. Pang X, Zhu Z, Xu F, Guo J, Gong X, Liu D, et al. Evaluation of con- 
trol measures implemented in the severe acute respiratory syndrome 
outbreak in Beijing, 2003. JAMA. 2003;290:3215-21. 

5. Chau PH, Yip PSF. Monitoring the severe acute respiratory syndrome 
epidemic and assessing effectiveness of interventions in Hong Kong 
Special Administrative Region. J Epidemiol Community Health. 
2003;57:766-9. 

6. Diamond B. SARS spread new outlook on quarantine models. Nature 
Med. 2003 ;9: 1441. 

7. Hon KL, Li AM, Cheng FW, Leung TF, Ng PC. Personal view of 

SARS: confusing definition, confusing diagnoses. Lancet. 

2003;361:1984-5. 


8. Chow KY, Lee CE, Ling ML, Heng DMK, Yap SG. Outbreak of 
severe acute respiratory syndrome in a tertiary hospital in Singapore, 
linked to an index patient with atypical presentation: epidemiological 
study. BMJ. 2004;328:195-8. 

9. World Health Organization. Consensus document on the epidemiolo- 
gy of severe acute respiratory syndrome (SARS). 2003 Oct 17 [cited 
2005 Jan 6]. Available at http://www.who.int/csr/sars/en/WHO 
consensus.pdf. 

10. Twu SJ, Chen TJ, Chen CJ, Olsen SJ, Lee LT, Fisk T, et al. Control 
measures for severe acute respiratory syndrome (SARS) in Taiwan. 
Emerg Infect Dis. 2003;9:718-20. 

11. Centers for Disease Control and Prevention. Use of quarantine to pre- 
vent transmission of severe acute respiratory syndrome — Taiwan 
2003. MMWR Morb Mortal Wkly Rep. 2003;52:680-3. 

12. Hsieh YH, Lee JY, Chang HL. On SARS epidemiology, cumulative 
case curve, and logistic-type model: ascertaining effectiveness of 
intervention and predicting case number, Emerg Infect Dis. 
2004;10:1165-7. 

13. Center for Disease Control-Taiwan. SARS major timeline. In: 
Memoir of severe acute respiratory syndrome control in Taiwan, 
2003. Taipei: CDC-Taiwan; 2003. p. 67-81. 

14. Hsieh YH, Chen CWS, Hsu SB. SARS outbreak in Taiwan [reply to 
Hsueh and Yang]. Emerg Infect Dis. 2004;10:1515-6. 

15. Lloyd-Smith JO, Galvani AP, Getz WM. Curtailing transmission of 
severe acute respiratory syndrome within a community and its hospi- 
tal. Proc R Soc Lond B Biol Sci. 2003;270:1979-89. 

16. Hsieh YH, Chen CWS, Hsu SB. SARS outbreak, Taiwan 2003. 
Emerg Infect Dis. 2004;10:201-6. 

17. Fraser C, Riley S, Anderson RM, Ferguson NM. Factors that make an 
infectious disease outbreak controllable. Proc Natl Acad Sci USA. 
2004;101:6146-51. 

18. Wu JS, Ho MS, Huang TM, Chen KT, Hsu KH, Su IJ, et al. 
Epidemiological investigation of the SARS outbreak in the Taipei 
Municipal Hoping Hospital. Memoir of severe acute respiratory syn- 
drome control in Taiwan, 2003. CDC-Taiwan: Taipei;2003. p. 45-8. 

Address for correspondence: Prof. Ying-Hen Hsieh, Department of 

Applied Mathematics, National Chung Hsing University, 250 Kuo-Kuang 

Rd., Taichung, Taiwan 402; fax: 886-4-22853949; email: hsieh@ 

amath.nchu.edu.tw 


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282 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


Wild Animal Mortality Monitoring 
and Human Ebola Outbreaks, Gabon 
and Republic of Congo, 2001-2003 

Pierre Rouquet,* Jean-Marc Fromentjt 1 Magdalena Bermejo ,^ 1 Annelisa Kilbourn,§ 

William Karesh,§ Patricia Reed,§ Brice Kumulungui,* Philippe Yaba,* Andre Delicat,* 

Pierre E. Rolling and Eric M. Leroy*# 


All human Ebola virus outbreaks during 2001-2003 in 
the forest zone between Gabon and Republic of Congo 
resulted from handling infected wild animal carcasses. After 
the first outbreak, we created an Animal Mortality 
Monitoring Network in collaboration with the Gabonese and 
Congolese Ministries of Forestry and Environment and 
wildlife organizations (Wildlife Conservation Society and 
Programme de Conservation et Utilisation Rationnelle des 
Ecosystemes Forestiers en Afrique Centrale) to predict and 
possibly prevent human Ebola outbreaks. Since August 
2001, 98 wild animal carcasses have been recovered by 
the network, including 65 great apes. Analysis of 21 car- 
casses found that 10 gorillas, 3 chimpanzees, and 1 duiker 
tested positive for Ebola virus. Wild animal outbreaks 
began before each of the 5 human Ebola outbreaks. Twice 
we alerted the health authorities to an imminent risk for 
human outbreaks, weeks before they occurred. 

E bola vims, a member of the Filoviridae family, causes 
severe hemorrhagic fever in humans and nonhuman 
primates. The human case-fatality rate ranged from 50% to 
89%, according to the viral subtype, from the first out- 
breaks in Zaire and Sudan in 1976 to the 2003 outbreaks in 
the Republic of Congo (1-4). No effective therapy or pro- 
phylaxis exists, and Ebola is a major public health concern. 
The first recorded human Ebola outbreaks (Yambuku Zaire 
1976; Nzara, Sudan, 1976 and 1979; Tandala, Zaire, 1977) 
occurred abruptly, from an unidentified source, with sub- 


centre International de Recherches Medicales de Franceville, 
Franceville, Gabon; fEuropean Union Project Cybertracker 
Monitoring Programme, Libreville, Gabon; tUniversidad de 
Barcelona, Barcelona, Spain; §Wildlife Conservation Society, 
Bronx, New York, USA; ^Centers for Disease Control and 
Prevention, Atlanta, Georgia, USA; and #lnstitut de Recherche 
pour le Developpement, Franceville, Gabon 


sequent person-to-person spread (1,2, 5, 6). No trace of the 
virus was initially found in wild animals close to the out- 
breaks (7-9). In 1989, for the first time, a nonhuman pri- 
mate outbreak due to a new subtype of Ebola virus, Ebola 
subtype Reston, occurred in a colony of Macaca fascicu- 
laris in a quarantine facility in Reston, Virginia, USA, after 
the introduction of monkeys from the Philippines (10). 
Ebola Reston caused severe hemorrhagic fever in mon- 
keys, but no clinical cases of human infection were identi- 
fied, even though anti-filovirus antibodies were found in 
quarantine facility personnel (11). Later, in 1994, Ebola- 
specific immunohistochemical staining was positive on 
necropsy specimens from 1 of 12 chimpanzees that died in 
the Tai forest of Cote d’Ivoire (12). During this outbreak, 
an ethnologist was infected while performing an autopsy 
on a chimpanzee carcass; this was the first documented 
case of human infection transmitted by a nonhuman pri- 
mate (13). During the 1996 outbreak in Mayibout (Gabon), 
an epidemiologic survey showed that the index case- 
patients had been infected by contact with a chimpanzee 
carcass. Concurrently, many nonhuman primate carcasses 
were reported in the area close to the outbreak, but none 
was recovered (14,15). Recently, we showed that all the 
human Ebola virus outbreaks that occurred in the past 3 
years in Gabon and the Republic of Congo resulted from 
multiple introductions of the virus from different infected 
animal carcasses (16). We describe the development, test- 
ing, and evaluation of an Animal Mortality Monitoring 
Network (AMMN) in northeastern Gabon and northwest- 
ern Republic of Congo designed to alert human and animal 
health authorities on emerging epidemics. 


^hese authors contributed equally to this work. 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


283 


RESEARCH 


Materials and Methods 

Epidemiologic Surveillance Network 

An alert network was set up by the Ministries of Health 
in hospitals and clinics in the different regions of Gabon 
and Republic of Congo, designed to report all human cases 
of viral hemorrhagic syndromes. Particular attention was 
paid to the northeastern region of Gabon, which had 
already been affected by outbreaks, and to its border region 
with Republic of Congo. Wildlife organizations such as the 
Wildlife Conservation Society (WCS), Programme de 
Conservation et Utilisation Rationnelle des Ecosystemes 
Forestiers en Afrique Centrale (ECOFAC), and the World 
Wildlife Fund (WWF) were chosen to form the backbone 
of AMMN, in close collaboration with the Ministries of 
Forestry and Environment of the 2 countries. WWF was 
present in the Minkebe Reserve in Gabon, while ECOFAC 
was in charge of the Odzala National Park and the Fossi 
gorilla sanctuary in Republic of Congo (Figure 1). 

All information on human cases of viral hemorrhagic 
syndrome or on the presence of dead animals in affected 
areas was centralized by a Viral Hemorrhagic Fever 
Committee (VHFC), composed of representatives of the 
Ministries of Health, Forestry, and Environment, the World 
Health Organization (WHO), wildlife agencies, and the 
Centre International de Recherches Medicales de 
Franceville (CIRMF). VHFC was also charged with send- 
ing specialized CIRMF teams to sample animal carcasses 
for diagnostic purposes. CIRMF is the regional reference 
laboratory for viral hemorrhagic fevers, and communicates 
its results to the ministries of health, forestry, and environ- 
ment and to WHO. 

Ebola Outbreak Investigation: Human Case Data 

The Gabonese and Congolese Ministries of Health, in 
close collaboration with WHO and its partners in the 
Global Outbreak Alert and Response Network (GOARN), 
were in charge of human epidemiologic investigations. A 
case of Ebola hemorrhagic fever was defined as any prob- 
able or laboratory-confirmed case, based on international- 
ly recognized criteria (definition available from 
http ://www. who.int/emc/diseases/ebola/ebola7 .html) . 

Ebola Outbreak Investigation: Animal Data 

Collection Sites 

From August 2001 to June 2003, carcasses were found 
on both sides of the Gabon-Republic of Congo border in 
the Ogooue Ivindo (Gabon) and West Basin (Congo) 
provinces (Figure 1). This entire area is covered by a 
Marantaceae and Zingiberaceae forest, with both open 
and closed canopies. The climate is equatorial, with 2 dry 
seasons (December-February and June-August) and 2 wet 



Figure 1. Map of the forest zone straddling the border between 
Gabon and Republic of Congo, showing (red points) the location 
of Ebola virus-positive carcasses, confirmed by testing in the 
Centre International de Recherches Medicales de Franceville 
biosafety level 4 unit during the 2001-2003 outbreaks in Gabon 
and Republic of Congo. 


seasons (March-May and September-November). Mean 
rainfall is 1,500 mm per year and mean temperature is 
24°C. Relative humidity always exceeds 80% (village of 
Mboko, Republic of Congo, 1995) (17). 

Fauna 

The large-animal fauna includes Loxodonta africana 
(Elephant), Syncerus caffer (Buffalo), Tragelaphus sp. 
(Sitatunga), Cephalophus sp. (Duiker), Hylochoerus mein- 
ertzhagim (Giant Forest Hog), Potamochoerus porcus 
(Red River Hog), Gorilla gorilla , Pan troglodytes 
(Chimpanzee), Cercopithecus sp. (Guenon), Cercocebus 
sp. (Mangabey), Colobus sp., Panthera pardus (Feopard), 
Nandinia (Two-spotted Palm Civet), Civettidis civetta 
(African Civet), Genetta servalina (Genet), mongoose sp., 
Orycteropus afer (Antbear), Manis sp. (Pangolin), 
Atherurus africanus , Thryonomys swinderianus , and 
Python sebae (17,18). 

Carcass Detection 

Focal hunters (primarily adult and adolescent men of 
the Bakota, Bakola, Mboko, Mongom, and Pygmy tribes) 
were the main sources of information regarding the loca- 
tion of carcasses. Their reported sightings were confirmed 
by ECOFAC monitoring teams who recorded both the 
global positioning system (GPS) position on a Cyber 
Traker field computer (available from http ://www. cyber- 
tracker, co.za/) and carcass status before alerting VHFC. 

Sampling Team and Methods 

When wild animal carcasses were found, VHFC asked 
CIRMF to send a team to the site for diagnostic purposes. 
Sampling permits were granted by the Gabonese and 
Congolese Ministries of Forestry and Environment and 
Health. Owing to the isolated nature of the outbreak zone 


284 


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Wild Animal Mortality Monitoring and Human Ebola 


and its distance from CIRMF, a base camp was established 
nearby. GPS location of the carcasses, and the information 
provided on their state of decomposition, allowed the 
autopsy team to sample only the freshest carcasses. 

Wild animal Carcass Sampling 

Ideally, the carcass sampling teams comprised a mini- 
mum of 5 persons (3 porters and 2 persons to perform the 
autopsy). One of the porters was charged with disinfection 
procedures. Digital photographs were taken. Necropsy was 
performed with high-level precautions, including water- 
tight clothes (Pro-Tech “C,” Tyvek, Contem, Luxembourg) 
equipped with air filtration equipment and Proflow 
Automask Litehood face shields (Delta Protection, Lyon, 
France) (Figure 2), and disposable lancets and forceps. A 
2% chlorine spray was used to disinfect reusable equip- 
ment (masks and filtration apparatus), as well as the autop- 
sy site and carcass remnants. Hermetic 60-L containers 
equipped with safety tops were used to transport reusable 
equipment and waste. Waste was returned to the main 
camp for incineration. 

The nature of the samples taken depended on the state 
of the carcasses. When the carcasses were in good condi- 
tion, 0.5 -cm 3 specimens of liver, spleen, muscle, and skin 
were taken. Half of the samples were placed in Nunc 
CryoTube vials (Nalge International, Rochester, New 
York, USA), which were placed in a small liquid nitrogen 
dry-shipper container (5.4 L) for cryopreservation 
(-196°C). The other samples were placed in Nunc 
CryoTube vials containing 10% formalin, for immunohis- 
tochemical testing. Bones were placed in hermetic con- 
tainers. At the main camp, the dry- shipper contents were 
transferred into larger dry-shipper containers (20.3 L), 
which were then forwarded to the CIRMF laboratory at the 
end of the mission. 

Laboratory Studies 

Sample Preparation 

Potentially infected specimens were collected and 
manipulated according to WHO guidelines on viral hemor- 
rhagic fever agents in Africa (19). Muscle and skin tissue 
were fragmented and homogenized in phosphate- buffered 
saline, and the final supernatant was filtered for antigen 
detection and RNA amplification. Bones were cut, and 
internal tissue was scraped. Bone marrow or internal bone 
tissue was prepared in the same way as muscle and skin. 

Testing 

Muscle and skin tissue samples were tested by poly- 
merase chain reaction (PCR), antigen detection, and, in 
some cases, immunohistochemical staining. Bone marrow 
and internal bone tissue were tested by PCR only. 



Figure 2. Field watertight clothes equipped with air filtration equip- 
ment, used for high-risk wild animal necropsy. Odzala National 
Park, Republic of Congo, June 2003. Photo: P. Rouquet. 


Antigen Detection 

Samples were used for antigen detection as previously 
described (20). Briefly, Maxisorp (Nalge International) 
plates were coated with a cocktail of 7 monoclonal anti- 
bodies against Ebola virus Zaire antigens; control plates 
were coated with normal mouse ascitic fluid produced 
from a parent myeloma cell line. Sample extracts (see 
above) were then added to the wells, followed by hyper- 
immune rabbit Ebola polyvalent antiserum and then per- 
oxidase-conjugated goat antibodies against rabbit 
immunoglobulin G (IgG). The TMB detector system 
(Dynex Technologies, Issy-les Molineaux, France) was 
used to measure optical density. 

DNA Amplification 

For the detection of viral mRNA, total RNA was isolat- 
ed from sample extracts by using the RNeasy kit (Qiagen, 
Hilden, Germany), and cDNA was synthesized from 
mRNA as previously described (21). Two pairs of degen- 
erate primers corresponding to the L-gene of Ebola virus 
were used for 2 rounds of amplification, yielding a 298-bp 
fragment (5'-TATMGRAATTTTTCYTTYTCATT -3' and 
5 '-AT GT GGT GGG YTATA AWARTC ACTR AC AT- 3 ' for 
primary PCR; 5 '-GC WA A AGCMTT Y CC WAG YA AYAT - 
GATGG-3' and 5 '-ATA AWARTC ACTR ACATGCA- 
TATAACA-3' for nested PCR). 

Immunohistochemical Staining 

Formalin-fixed specimens were sent to the Centers for 
Disease Control and Prevention (Atlanta, Georgia, USA) 
for immunohistochemical staining as previously described 
( 22 ). 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


285 



RESEARCH 


Results 

Human Outbreaks 

From October 2001 to December 2003, 5 human Ebola 
virus outbreaks of the Zaire subtype occurred in the area 
straddling the border between Gabon (northeast) and 
Republic of Congo (northwest), with 313 cases and 264 
deaths (23,24). The first outbreak occurred from October 
2001 to May 2002, with a total of 92 cases and 70 deaths 
in Gabon and Republic of Congo. Epidemiologic investi- 
gations showed that at least 2 duikers, 2 chimpanzees, and 
2 gorilla carcasses were involved or suspected of being 
involved in the infection of 6 human index patients. A sec- 
ond human outbreak began in January 2002 and ended in 
June 2002 in Entsiami Republic of Congo, with a total of 
30 cases and 25 deaths. One gorilla and 1 duiker were sus- 
pected of involvement in 2 human index cases. A third out- 
break occurred from May to June 2002 in Oloba Republic 
of Congo, with 13 cases and 12 deaths. A chimpanzee was 
shown to have infected the human index patient. The 
fourth outbreak occurred from December 2002 to April 
2003 in Mbomo and Kelle, Republic of Congo, with 143 
cases and 128 deaths. Gorillas and duikers were suspected 
of infecting 3 human index patients. The last outbreak 
occurred from November 2003 to December 2003 in 
Mbanza and Mbomo, Republic of Congo, with 35 cases 
and 29 deaths. The source of infection of the human index 
patient was not clearly identified. 

Carcasses 

From August 2001 to June 2003, a total of 98 animal 
carcasses were found in an area of about 20,000 km 2 
(Figure 3). Carcasses of 3 principal species were recov- 
ered: 65 great apes (50 gorillas and 15 chimpanzees) and 
14 duikers (Figure 3). Only 6% of carcasses sampled were 
in good condition (entire body); 57% were in poor condi- 
tion (partial carcasses with muscles or skin); and 38% were 
in bad condition (bones only). Two peaks of animal deaths 
were observed (Figure 4). The first occurred in the Ekata 
region (Gabon) from November to December 2001, with 
51 carcasses, including 30 great apes and 8 duikers. The 
second occurred from December 2002 to February 2003 in 
the Los si gorilla sanctuary (Republic of Congo), with 20 
carcasses, including 17 great apes, 2 duikers, and 1 
Cercopithecus cephus. 

Laboratory Findings 

An animal carcass was considered infected by Ebola 
virus if >1 of the 3 laboratory tests (antigen detection, 
DNA amplification, and immunohistochemical staining) 
was positive. When possible, DNA amplification was con- 
firmed by sequencing the PCR products. Twenty-one 
gorilla, chimpanzee, and duiker carcasses were sampled in 



15 



Chimpanzes 


14 



Dukers 


13 

6 sw 



Other Other 

primates* species** 


Figure 3. Species distribution of carcasses found in the forest 
straddling the border between Gabon and Republic of Congo 
(2001-2003). * = other primates: Cercopithecus sp.; f = other 
species: Atherurus africanus (1), Genetta sp (3), Loxodonta 
africana (1 ), Manis sp. (1 ), Mongoose sp. (1 ), Thryonomys swinde- 
rianus (2), Tragelaphus sp. (1), Python sebae (2), and bird of prey 

(li- 


the wild and analyzed in the CIRMF biosafety level 4 
(BSL-4) laboratory. Fourteen of these carcasses tested pos- 
itive for Ebola virus, 6 in 2 or 3 tests and 8 in only 1 test 
(Table). Eight positive samples were muscles, and 6 were 
bones or bone marrow. All the muscle and skin tissue sam- 
ples were tested by both PCR and antigen detection. In 
total, 10 gorillas, 3 chimpanzees, and 1 duiker tested posi- 
tive. All the relatively well-preserved gorilla and chim- 
panzee carcasses tested positive. In contrast, 
well-preserved samples taken from carcasses of C. cephus , 
Genetta sp., and Tragelaphus sp. were negative. 

Discussion 

We describe the successful implementation of a sur- 
veillance network of Ebola outbreaks in wild large 


40 

35 

c/> 

8! 30 
(/) 

o 25 

2 20 


15 

10 

5 

0 


4 First human 
n outbreak 
begins 



4 Second human 
outbreak begins 


4 Fourth 
human 
1 outbreak 
I begins 


4 Third human 
outbreak 

B begins 

_fifl 


□ Others 

□ Duikers 

■ Great apes 


Last 

human 

outbreak 

begins 

4 


Aug Oct Dec Feb Apr Jun Aug Oct Dec Feb Apr Jun Aug Oct Dec 

Sep Nov Jan Mar May Jul Sep Nov Jan Mar May Jul Sep Nov Jan 

2001 2002 2003 2004 


Figure 4. Temporal distribution of carcasses found in the forest 
straddling the border between Gabon and the Republic of Congo 
(2001-2003). Two peaks of mortality were observed: the first 
occurred in the Ekata region (Gabon) from November to 
December 2001 and the second from December 2002 to February 
2003 in the Lossi gorilla sanctuary (Republic of Congo). 


286 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


Wild Animal Mortality Monitoring and Human Ebola 


Table. Results of laboratory analysis of animal carcasses found in forest between Gabon and the Republic of Congo, November 2001- 
June 2003* 


Location 


Animal 

Area 


GPS 

Date 

Tissue 

Death 

PCR 

Ag 

IHC 

Ebola+ by 2 or 3 tests 









Gorilla 

Zadie 

0,7055N 

14, 2747 E 

Nov 2001 

Muscle§ 

5 d 

+ 

+ 

+ 

Gorilla 

Lossi 

0,2395N 

14, 4938 E 

Dec 2002 

Muscle§ 

8 d 

+ 

+ 

+ 

Gorilla 

Lossi 

0,2354N 

14, 4839 E 

Dec 2002 

Muscle§ 

8 d 

+ 

+ 

+ 

Gorilla 

Mbanza 

0,6987N 

14, 7029 E 

Jun 2003 

Muscle§ 

5 d 

+ 

+ 

N/A 

Chimpt 

Lossi 

0,2387N 

14, 4885 E 

Dec 2002 

Muscle§ 

3d 

- 

+ 

+ 

Chimp 

Ebola+ by 1 test 

Lossi 



Feb 2003 

Musclelj 

10 d 

+ 

+ 

N/A 

Gorilla 

Zadie 

1.1669N 

14,1650E 

Feb 2002 

Bone marrow# 

1 mo 

+ 

N/A 

N/A 

Gorillatt 

Zadie 

0,731 ON 

14,2644E 

Mar 2002 

Bone# 

3 wk 

+ 

N/A 

N/A 

Gorillatt 

Zadie 

0,731 ON 

14,2644E 

Mar 2002 

Bone# 

3 wk 

+ 

N/A 

N/A 

Gorilla 

Lossi 

0,2348N 

14, 4852 E 

Dec 2002 

Bone# 

2 wk 

+ 

N/A 

N/A 

Gorilla 

Lossi 

0,2346N 

14, 4823 E 

Dec 2002 

Bone# 

2 wk 

+ 

N/A 

N/A 

Gorilla 

Lossi 

0,2987N 

14, 5075 E 

Feb 2003 

Muscle!} 

8 d 

- 

+ 

N/A 

Duiker 

Lossi 

0,2293N 

14, 4892 E 

Dec 2002 

Bone# 

2 wk 

+ 

N/A 

N/A 

Chimpt 

Lossi 

0,2387N 

14, 4885 E 

Dec 2002 

Muscle!! 

12 h 

- 

+ 

- 

Tested and Ebola- 









GorillaJ 

Zadie 

0,651 ON 

14, 2375 E 

Mar 2002 

Skull# 

1 mo 

- 

N/A 

N/A 

Duiker 

Lossi 

0,2376N 

14, 4882 E 

Dec 2002 

Bone# 

3 wk 

- 

N/A 

N/A 

Duiker 

Lossi 



Jun 2003 

Skin§ 

2d 

- 

- 

N/A 

Cercopithecus 

cephus 

Lossi 

0,2737N 

14,51 63E 

Feb 2003 

Muscle§ 

3d 

- 

- 

N/A 

Genet 

Zadie 

0,6749N 

13,8851 E 

Nov 2001 

Muscle!! 

5 d 

- 

- 

N/A 

Genet 

Zadie 

0,6771 N 

14, 2937 E 

Feb 2002 

Muscle§ 

2d 

- 

- 

N/A 

Sitatunga 

Zadie 

0,9560N 

13,7776E 

Apr 2002 

Muscle§ 

3d 

- 

- 

N/A 


*GPS, global positioning system (CyberTracker field computer); PCR, polymerase chain reaction; Ag, antigen detection; IHC, immunohistochemical tests; 
N/A, not applicable. 
fMother and infant. 

Jl-month delay between the field and the laboratory and preserved in bad conditions. 

§Sample found in good condition. 

TfSample found in poor condition. 

#Sample found in very poor condition (bone only). 


mammals. We often identified wild animal outbreaks 
before human Ebola outbreaks. Twice this enabled us to 
alert the health authorities of Republic of Congo and 
Gabon to an imminent risk for human outbreaks, after the 
discovery of carcasses of Ebola virus-infected animals. 

Human Ebola outbreaks in this region have always 
occurred in remote areas, raising major logistic problems. 
Roads are often barely passable, and means of communica- 
tion are frequently nonexistent. The carcass detection and 
investigation network therefore had to rely on teams 
already present in these forest zones, and notably those pos- 
sessing radios or satellite telephones. Conservation organi- 
zations such as ECOFAC, WCS, and WWF were thus the 
ideal partners. ECOFAC monitoring teams played a critical 
role by exploring remote forest zones, capitalizing on the 
information provided by villagers and hunters. 

Performing an autopsy on high-risk animal carcasses 
requires heavy equipment, highly qualified personnel, and 
experienced veterinarians, as illustrated by the case of the 
Swiss anthropologist who was infected after examining a 
chimpanzee carcass without adequate protective measures 
in the Tai forest (13). Carcasses decompose very rapidly in 


the equatorial forest: an adult male gorilla carcass (-150 
kg) takes only 10 days to decompose entirely, i.e., be 
reduced to a heap of bones and hair (Figure 5). Carcasses 
observed 3-4 days after death bear few signs of scavenger 
activity but are covered with fly eggs and maggots. 
Maggots consume the entire flesh within 5 to 10 days, 
while scavengers (mainly mongoose) take pieces and dis- 
seminate them around the site. Thus, after ~3weeks, only a 
few bones bearing small-mammal gnaw marks remain. 

Although the PCR technique used by CIRMF can 
detect Ebola virus genetic material in carcasses 3^1 weeks 
old, the material is often degraded and incomplete. Often, 
only a small sequence of the L-gene (RNA polymerase) 
can be analyzed, and this cannot be used for strain identi- 
fication. Furthermore, degraded samples increase the 
false-negative rate (25). Rapid sampling is therefore cru- 
cial for successful diagnosis, and the availability of a small 
aeroplane was particularly helpful in certain cases. The 
presence of the CIRMF BSF4 laboratory relatively close to 
the outbreak area was a considerable advantage. 

Using a combination of 3 laboratory techniques (PCR, 
immunohistochemical staining, and antigen capture), we 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


287 


RESEARCH 



Figure 5. State of the wild animal carcasses found in the field, Lossi gorilla sanctuary, Republic of Congo, December 2002. Carcasses 
decompose very rapidly in the equatorial forest. Photo: P. Rouquet. A) Female chimpanzee, 3 days after death. B) Female gorilla, 7 days 
after death. C) Female gorilla, 21 days after death. 


showed for the first time that wild gorillas and chim- 
panzees can be decimated by Ebola. Bones of a 
Cephalophus dorsalis carcass also tested positive for 
Ebola vims by reverse transcription (RT)-PCR, indicating 
that a third wild species may be naturally susceptible. In 
Africa, only chimpanzees had previously been diagnosed 
as positive for Ebola virus, by immunohistochemical test- 
ing, in the Tai forest of Cote d’Ivoire, and were considered 
the cause of the human outbreak in Mayibout (Gabon) 
(12,14,15). The large number of carcasses found in this 
region, together with the results of animal population cen- 
suses conducted in the Lossi reserve before and after out- 
breaks, indicates that great apes are affected massively and 
duikers to a lesser extent (16,26). The lowland gorilla pop- 
ulation density in this region (<6 times as high as the chim- 
panzee population density) is among the highest in the 
world (<10 gorillas/km 2 ) (27), which likely explains why 
more gorilla carcasses than chimpanzee carcasses were 
found. High population density can amplify outbreaks but 
cannot alone explain their severity. Small monkeys, 
although abundant in this area, do not seem to be affected. 
Only 1 carcass of Cercopithecus cephus was found; it was 
in good condition but was negative by RT-PCR and anti- 
gen capture (Table). Some Potamochoerus porcus carcass- 
es were reported by hunters but none could be sampled. 
Carcasses of large animals are more likely to be found than 
those of small animals, because the time taken for a car- 
cass to decompose depends on its size. 

The source of gorilla infection is unknown, but sever- 
al lines of evidence point to direct infection by >1 natural 
hosts. First, the detection of different strains of Ebola 
virus in gorilla carcasses located only a few kilometers 
apart argues against a major role of gorilla-to-gorilla 
transmission. Indeed, Ebola virus remains genetically sta- 
ble during a given outbreak, from the first to the last case 
(28,29), whereas we obtained 4 different glycoprotein 
sequences (E.M. Leroy, P. Rouquet, unpub. data) from 
samples of gorillas and chimps located in the Lossi sanc- 
tuary. The large distance separating positive carcasses 


found during a short period, and the existence of physical 
barriers such as roads and rivers, also supports direct 
transmission from a natural host. Finally, the occurrence 
of simultaneous outbreaks in 2 or 3 different species that 
display little interspecies contact (30) provides further 
evidence that gorillas and chimpanzees are directly infect- 
ed by >1 natural hosts. However, cases of gorilla-to-goril- 
la transmission cannot be ruled out, especially within a 
given group. Indeed, 5 gorilla carcasses belonging to the 
same group were found in a close area in the Lossi sanc- 
tuary. Ebola outbreaks in gorilla groups may result in their 
rapid dissolution, especially if the dominant male is rap- 
idly affected, which forces possibly infected females to 
integrate into another group. However, this type of inter- 
group transmission appears to be marginal. 

Chimpanzees are probably infected by the same mech- 
anisms as gorillas. During the Tai outbreak in Cote 
d’Ivoire, carnivorous behavior (especially consumption of 
Colobus monkeys) was the suspected source of infection 
(12), but this notion is challenged by the infection of goril- 
las, which are almost exclusively herbivorous. However, 
chimpanzees are considered to be the primate species 
whose behavior (mainly fighting, social grooming, sexual 
activities, and predation) carry the highest risk for both 
intra- and interspecies pathogen transmission (30). This 
idea is supported by the detection of the infected carcasses 
of a mother and her 1 -year-old offspring. Repeated contact 
between young individuals and their mothers is known to 
be a significant risk factor for Ebola virus transmission 
( 2 , 6 ). 

Duikers represent a special case. Although they are the 
most common large-mammal species in this region, few 
carcasses were found. This circumstance may be due to the 
lack of interactions among individuals, as duikers general- 
ly live alone or in pairs. Some duikers, despite being 
herbivorous, eat the flesh of decomposing carcasses 
(K. Abernethy, unpub. data). Thus, in addition to being 
directly infected by the natural host(s), duikers might also 
become infected by licking or eating fresh carcasses of 


288 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


Wild Animal Mortality Monitoring and Human Ebola 


Ebola virus-infected animals. This scenario would play a 
marginal role, however, because carcasses are only infec- 
tive for 3 or 4 days after the animal’s death (E.M. Leroy, P. 
Rollin, unpub. data). Furthermore, we observed little scav- 
enging of carcasses during the first days after the animal’s 
death. 

Serum from a survivor of the human outbreak in 
Mekambo (Grand Etoumbi, March 2002), who had direct 
contact with a gorilla carcass, was positive for Ebola 
virus-specific IgG. Ebola virus L gene sequences were 
detected in bone marrow samples of this gorilla, conclu- 
sively linking the 2 cases. Thus, the last outbreaks in 
Mekambo (Gabon, 2001) and Lossi (Republic of Congo, 
2002-2003) confirm that wild animal mortality can reveal 
Ebola virus propagation in the forest ecosystem and indi- 
cate a role of wild animals as “vectors” in human out- 
breaks. 

No effective medical treatment or vaccine exists for 
Ebola virus infection. The only way of minimizing human 
cases is to break the chain of human-human transmission. 
Humans do not seem to be at a major risk for infection by 
the unidentified natural host(s). Large outbreaks among 
wild animals can amplify human outbreaks by increasing 
the number of index transmission events. Therefore, reduc- 
ing contacts between humans and dead wildlife can reduce 
the risks for transmission. 

Epidemiologic surveillance of animal mortality rates 
can thus help prevent the emergence of the disease in 
human populations (Figure 6). At the time of the Kelle 
(Republic of Congo) outbreak, our network detected 
infected gorilla carcasses (Lossi, December 6, 2002) 3 
weeks before the disease emerged in humans (December 
25, 2002), showing active Ebola virus propagation in this 
area. We were thus able to warn health authorities of an 
imminent human outbreak in the region. Nonetheless, a 
human outbreak occurred. In June 2003, we issued a new 
alert on a risk for human outbreaks after the discovery of 
an infected gorilla carcass near the village of Mbanza 
(Republic of Congo). An outbreak occurred in this village 
in November 2003. These failures suggest that human and 
animal health authorities need to work together more 
closely. In the future, health authorities need to educate 
local populations on the risk for infection through contact 
with carcasses at all times. During expected disease out- 
breaks, health authorities need to be able to respond imme- 
diately by sending teams to affected areas (24). The early 
successes of the network in this area warrant its extension 
to all countries with known outbreaks of hemorrhagic 
fevers. The participation of new frontline partners, such as 
foresters, would be invaluable to expend logistical existing 
capacity provided largely by field conservationists. 
Finally, as the capacity of such a system to react rapidly is 



Figure 6. Schematic representation of the Ebola cycle in the equa- 
torial forest and proposed strategy to avoid Ebola virus transmis- 
sion to humans and its subsequent human-human propagation. 
Ebola virus replication in the natural host (a). Wild animal infection 
by the natural host(s) (b), no doubt the main source of infection. 
Wild animal infection by contact with live or dead wild animals (c). 
This scenario would play a marginal role. Infection of hunters by 
manipulation of infected wild animal carcasses or sick animals (d). 
Three animal species are known to be sensitive to Ebola virus and 
to act as sources of human outbreaks, gorillas, chimpanzees, and 
duikers. Person-to-person transmission from hunters to their fam- 
ily and then to hospital workers (e). The wild animal mortality sur- 
veillance network can predict and might prevent human outbreaks. 
Medical surveillance can prevent Ebola virus propagation in the 
human population. 


crucial for its success, sampling teams should be created to 
collect material and obtain virologic testing results with a 
minimum of delay in other countries harboring hemor- 
rhagic viruses. An efficient animal mortality monitoring 
network backed up by a rapid reaction system would allow 
public health authorities to predict and possibly prevent 
human Ebola outbreaks. 


Acknowledgments 

We thank the national and international teams involved in 
the control of the Ebola outbreaks that occurred in Gabon and the 
Republic of Congo. The national teams were members of the 
Gabonese Health Ministry and the Health Service of the 
Gabonese Defense Ministry during the Gabon outbreaks, and 
members of the Congolese Health Ministry during the outbreaks 
in the Republic of Congo. The international teams were mainly 
scientific and medical experts from WHO and Medecins Sans 
Frontieres. We thank all those involved in wildlife conservation 
for sample collection and case reporting, in particular the ECO- 
FAC monitoring teams and T. Smith. We are also grateful to G. 
Moussavou and L. Allela for technical assistance; D. Young, X. 
Pourrut, and J. Wickings for help in preparing the manuscript; 
and D. Drevet, P. Blot, and C. Aveling for constant support and 
encouragement. Lastly, we thank T.G. Ksiazek for generously 
providing reagents to CIRMF. 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


289 


RESEARCH 


CIRMF is supported by the Government of Gabon, Total- 
Fina-Elf Gabon, and Ministere de la Cooperation Frangaise. This 
work was also supported by a Fonds de Solidarity Prioritaire and 
a Fonds d’Aide et de Cooperation, grants from the Ministere des 
Affaires Etrangeres de la France (FSP no. 2002005700 and FAC 
no. 1999-49). 

Dr. Rouquet is the head of the Primate Center at CIRMF. He 
is experienced in the treatment and evaluation of simian immun- 
odeficiency virus and simian/HIV transmission in different pri- 
mate models of HIV infection (pathogenic and nonpathogenic). 
Since 1995, he has been involved in hemorrhagic fever research, 
particularly Ebola. 

References 

1. World Health Organization. Ebola hemorrhagic fever in Zaire. Bull 
World Health Organ. 1978;56:271-93. 

2. World Health Organization. Ebola hemorrhagic fever in Soudan. Bull 
World Health Organ. 1978;56:247-70. 

3. Peters CJ, Sanchez A, Feldman H, Rollin PE, Nichol ST, Ksiazek TG. 
Filoviruses as emerging pathogens. Semin Virol. 1994;5:147-54. 

4. WHO Wkly Epidemiol Rec. 2003;33:2003;78:285-96 [cited 2003 
Aug 15]. Available from http://www.who.int/wer/2003/en/ 
wer7833.pdf 

5. Heymann DL, Weisfeld JS, Webb PA, Johnson KM, Cairns T, 
Berquist H. Ebola haemorrhagic fever: Tandala, Zaire, 1977-1978. J 
Infect Dis. 1980;142:372-6. 

6. Baron RC, Me Cormick JB, Zubeir OA. Ebola virus disease in south- 
ern Sudan: hospital dissemination and intrafamilial spread. Bull 
World Health Organ. 1983;61:997-1003. 

7. Arata AA, Johnson B. Approaches towards studies on potential reser- 
voirs of viral haemorrhagic fever in southern Sudan (1977). In: Pattyn 
SR, editor. Ebola virus haemorrhagic fever. Amsterdam: 
Elsevier/Netherlands Biomedical; 1978. p.191-202. 

8. Germain M. Collection of mammals and arthropods during the epi- 
demic of haemorrhagic fever in Zaire. In: Pattyn SR, editor. Ebola 
virus haemorrhagic fever. Amsterdam: Elsevier/Netherlands 
Biomedical; 1978. p. 185-9. 

9. Breman JG, Johnson KM, Van Der Groen G, Robbins CB, 
Szczeniowski MV, Ruti K, et al. A search for Ebola virus in animals 
in the Democratic Republic of the Congo and Cameroon: ecologic, 
virologic, and serologic surveys, 1979-1980. J Infect Dis. 
1999;179(Suppl l):S139-47. 

10. Jahrling P, Geisbert T, Dalgard D, Johnson ED, Ksiazek TG, Hall 
WC, et al. Preliminary report: isolation of Ebola virus from monkeys 
imported to USA. Lancet. 1990;335:502-5. 

11. Becker S, Feldmann H, Will C, Slenczka W. Evidence for occurence 
of filovirus antibodies in humans and imported monkeys: do subclin- 
ical filovirus infections occur worldwide? Med Microbiol Immunol 
(B erl) . 1 992 ; 1 8 1 :43-5 5 . 

12. Formenty P, Boesch C, Wyers M, Steiner C, Donati F, Dind F, et al. 
Ebola virus outbreak among wild chimpanzees living in a rain forest 
of Cote d’Ivoire. J Infect Dis 1999;179(Suppl 1):S 120-6. 

13. Le Guenno B, Formenty P, Wyers M, Gounon P, Walker F, Boesch C. 
Isolation and partial characterization of a new strain of Ebola virus. 
Lancet. 1995;345:1271-4. 

14. Georges AJ, Leroy EM, Renaut AA, Benissan CT, Nabias RJ, Ngoc 
MT, et al. Ebola haemorrhagic fever. J.Infect Dis. 1999;179(Suppl 
l):S65-75. 


15. Amblard J, Obiang P, Edzang S, Prehaud C, Bouloy M, Le Guenno 
BL. Identification of the Ebola virus in Gabon in 1994. Lancet. 
1997;349:181-2. 

16. Leroy EM, Rouquet P, Formenty P, Souquiere S, Kilbourne A, 
Froment J-M, et al. Multiple Ebola virus transmission events and 
rapid decline of Central African wildlife. Science. 2004;303:387-90. 

17. Maisels F. Synthesis of information concerning the Park National 
d’Odzala, Congo. Projet ECOFAC, composante Congo June 1996 
[cited 2003 Jul 14]. Available from http://www.ecofac.org/Biblio/ 
Download/Odzala Synthese.pdf 

18. Bermejo M. Inventaire et recensement des petits primates diurnes 
Parc National D’Odzala Congo. Projet ECOFAC, composante 
Congo Sept 1995 [cited 2003 Jul 14]. Available from http:// 
www.ecofac.org/Biblio/Download/PrimatesDiurnesInventaire.pdf 

19. World Health Organization. Viral haemorrhagic fevers report of a 
WHO expert committee. Geneva: The Organization; 1985. 

20. Zaki SR, Shieh WJ, Greer PW, Goldsmith CS, Ferebee T, Katshitshi 
J, et al. A novel immunohistochemical assay for the detection of 
Ebola virus in skin: implications for diagnosis, spread, and surveil- 
lance of Ebola hemorrhagic fever. Commission de Lutte contre les 
Epidemies a Kikwit. J Infect Dis. 1999;179(Suppl l):S36-47. 

21. Leroy EM, Baize S, Lu CY, McCormick JB, Georges AJ, Georges- 
Courbot MC, et al. Diagnosis of Ebola haemorrhagic fever by RT- 
PCR in an epidemic setting. J Med Virol. 2000;60:463-7. 

22. Ksiazek TG, Rollin PE, Jahrling PB, Johnson E, Dalgard DW, Peters 
CJ. Enzyme immunosorbent assay for Ebola virus antigens in tissues 
of infected primates. J Clin Microbiol. 1992;30:947-50. 

23. World Health Organization. Outbreak(s) of Ebola haemorrhagic 
fever, Congo and Gabon, October 2001-July 2002. Wkly Epidemiol 
Rec. 2003;78:217-24 [cited 2003 Jun 23]. Available from http:// 
www.who.int/wer/ 2003/en/wer7826.pdf 

24. Formenty P, Libama F, Epelboin A, Allarangar Y, Leroy E, Moudzeo 
H, et al. Outbreak of Ebola hemorrhagic fever in the Republic of the 
Congo, 2003: a new strategy? Med Trop (Mars). 2003;63:291-5. 

25. Blacksell SD, Khounsy S, Westbury HA. The effect of sample degra- 
dation and RNA stabilization on classical swine fever virus RT-PCR 
and ELISA methods. J Virol Methods. 2004;118:33-7. 

26. Walsh PD, Abernethy KA, Bermejo M, Beyers R, De Wachter P, 
Akou ME, et al. Catastrophic ape decline in western equatorial 
Africa. Nature. 2003;422:611-4. 

27. Bermejo M. Status and conservation of primates in Odzala National 
Park, Republic of Congo. Oryx. 1999;33:323-31. 

28. Rodriguez LL, De Roo A, Guimard Y, Trappier S.G, Sanchez A, 
Bressler D, et al. Persistence and genetic stability of Ebola virus dur- 
ing the outbreak in Kikwit, Democratic Republic of Congo, 1995. J 
Infect Dis. 1999; 179(Suppll):S 170-6. 

29. Leroy EM, Baize S, Mavoungou E, Apetrei C. Sequence analysis of 
the GP, NP, VP40 and VP24 genes of Ebola virus isolated from 
deceased, surviving and asymptomatically infected individuals dur- 
ing the 1996 outbreak in Gabon: comparative studies and phyloge- 
netic characterization. J Gen Virol. 2002;83(Part 1):67— 73. 

30. Tutin CEG. Ecologie et organisation sociale des primates de la foret 
tropicale africaine: aide a la comprehension de la transmission des 
retrovirus. Bull Soc Pathol Exot. 2000;93, 3, 157-61. 


Address for correspondence: Pierre Rouquet, Centre International de 
Recherches Medicales de Franceville, (CIRMF) BP 769, Franceville, 
Gabon; fax: (33) 153013602; email: p.rouquet@cirmf.org 

Use of trade names is for identification only and does not imply 
endorsement by the Public Health Service or by the U.S. 
Department of Health and Human Services. 


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Surveillance and Control Measures 
during Smallpox Outbreaks 

Emma Kerrod,* Alasdair M. Geddes,t Martyn Regan, and Steve Leach* 


We reviewed historical data from 2 smallpox out- 
breaks in Liverpool and Edinburgh during the early and 
middle years of the 20th century to assess their contribu- 
tion to developing modern strategies for response to a 
deliberate release of smallpox virus. Reports contempora- 
neous to these outbreaks provide detail on the effective- 
ness of public health interventions. In both outbreaks, 
extensive contact tracing, quarantine, and staged vaccina- 
tion campaigns were initiated, and the outbreaks were 
controlled within 15 months and 3 months, respectively. In 
Edinburgh, the number of fatalities associated with vacci- 
nation exceeded the number of deaths from the disease. 
In Liverpool, ambulatory, vaccine-modified cases and mis- 
diagnosis as chickenpox resulted in problems with out- 
break control. The relatively slow spread of smallpox, as 
exemplified by the report from Liverpool, allowed for effec- 
tive implementation of targeted intervention methods. 
Targeted surveillance and containment interventions have 
been successful in the past and should be explored as 
alternatives to mass vaccination. 


H eightened awareness of the potential threat of biolog- 
ic terrorism has generated debate over the most 
appropriate modeling strategies to assist in planning pub- 
lic health interventions and the required relevant data and 
assumptions for model parameterization (1). A fundamen- 
tal issue for modeling the potential impact of a deliberate 
release of smallpox virus is the dearth of recent data. For 
these reasons, the impact of a bioterrorist release upon a 
modern population and of the subsequent attempts to con- 
tain it are difficult to predict with precision. The dynamics 
of disease outbreaks in the 21st century, and the outcomes 
of control strategies used to contain them, have been pre- 
dicted by using models parameterized with contemporary 
outbreak data (e.g., measles immunization campaigns). 
However, to obtain a better idea of how an eradicated dis- 
ease, such as smallpox, might be controlled requires an 


*Centre for Emergency Preparedness and Response, Health 
Protection Agency, Wiltshire, United Kingdom; fUniversity of 
Birmingham, Edgbaston, Birmingham, United Kingdom; and 
^Health Protection Agency, Liverpool, United Kingdom 


analysis of historical outbreak data, much as has been done 
in a number of studies (2-5). 

Inherent problems are associated with extrapolating 
past data to the modern day, such as possible differences in 
susceptibility to infection between modern and historical 
populations (e.g., immunity) and also potential differences 
in risk for disease transmission (e.g., changes in contact 
patterns) (1). Nonetheless, when these factors can be 
addressed properly, the advantages of using historical data 
as a foundation for modern assessments far outweigh the 
disadvantages. For smallpox particularly, epidemiologic 
and outbreak data from the past have been largely relied 
upon to provide insight into, and evaluation of, the effica- 
cy and efficiency of different public health control strate- 
gies for a potential bioterrorist attack. 

For example, the levels of protection afforded today by 
smallpox vaccinations carried out many years ago are dif- 
ficult to calculate, since few relevant recent assessments 
exist. A recent study reported stable antiviral antibody and 
slowly declining antiviral T-cell responses to vaccinia 
virus in volunteers 1-75 years after vaccination (6). How 
these longer lasting responses correlate with protection 
from infection itself, from more serious disease, or from 
death, remains difficult to determine. Natural exposure to 
the organism is the only way to know whether this 
response correlates to full (i.e., no disease), or partial (i.e., 
fewer deaths) protection from smallpox. Since data on nat- 
ural exposure to smallpox virus are not available for con- 
temporary populations, analysis of historical data is likely 
to provide the most convincing evidence (3). 

Historical data on this and other aspects of disease con- 
trol were published in the early 1900s after a variola major 
virus outbreak in Liverpool (1902-1903) (7) and in the 
mid- 1940s after an outbreak in Edinburgh in 1942 (8) 
(document available from http://www.cdc.gov/ncidod/eid/ 
volllno2/04-0609.htm_app). These reports form the basis 
of this article, which discusses the use of historical data in 
predictive assessments of disease events. The Liverpool 
smallpox outbreak data are included in a large section 
specifically on smallpox in the annual Health Department 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


291 


HISTORICAL REVIEW 


Report for the city written by the Medical Officer of 
Health in 1903, at a time when smallpox was still endem- 
ic in Liverpool. The report covers all aspects related to 
health, ranging from typhus and tuberculosis to rainfall, 
temperature, and demographic statistics. Supplementary 
information on this outbreak has also been taken from 
Appendix 10 of the Annual Report of the Medical Officer 
of The Local Government Board 1904-05, in Report on 
Smallpox and Smallpox Hospitals at Liverpool, 1902-03, 
which investigated specific aspects of the outbreak for the 
local government board (9). A further report has also been 
used, written in 1913 by the assistant medical officer of 
health for Liverpool; it reports in greater detail on the 
effects of the disease in relation to the impact of vaccina- 
tion and includes a large series of cases from the 
1902-1903 outbreak (10). 

The report on the 1942 outbreak in Edinburgh also pro- 
vides data on a range of important aspects of smallpox 
control, including adverse events to vaccination (8). This 
large report was written in 1944 by the medical officer of 
health and his colleagues at a time when smallpox was no 
longer endemic in the region. The stated purpose of the 
report was to provide information for medical staff in the 
event of future outbreaks. The information detailed, there- 
fore, is more descriptive than that in the Liverpool publi- 
cation but provides more data on the clinical and control 
aspects used. Again, supplementary articles have been 
consulted, primarily those concerning the contemporane- 
ous outbreaks in Glasgow and Fife that led up to the 
Edinburgh outbreak. A close evaluation of the 2 outbreaks 
illustrates the value of using historical data when consider- 
ing public health control and containment strategies for 
potential bioterrorist events. 

Outbreaks 

Since the 1860s, Liverpool had had cases of smallpox 
(7). According to the 1904-1905 report, seaports were 
prone to occurrences of smallpox, and therefore, Liverpool 
had “abundant opportunities of perfecting its administra- 
tion in regard of this disease” (9). Although the annual 
number of cases had declined considerably in the 17 years 
or so before the outbreak began in 1902, a total of 23 cases 
were imported by sea and 16 were introduced by 
“vagrants.” However, according to 1 researcher, an epi- 
demic broke out toward the end of 1902 (10). The outbreak 
lasted from October 1902 to the end of December 1903 
and resulted in 2,032 cases and 161 deaths (case-fatality 
rate = 8%). The first smallpox case occurred in 1901 and 
resulted directly from an imported case-patient, a merchant 
seaman. This importation brought the disease into 
Liverpool at the end of 1901, a year in which, until that 
time, practically no smallpox had been reported (9). The 
administrative actions of the Public Health Department of 


Liverpool checked the spread of smallpox until November 
1902, when an unrecognized case-patient (7), an infant, 
was medically attended only when the child was dying of 
the disease. In addition, 6 infected household members 
were found, and subsequent house-to-house inquiries in 
the district discovered another 20 clinical case-patients 
during the next few days, most of whom were friends of 
the infected family (7). This number of cases is assumed to 
have resulted from chains of transmission beginning with 
the infant and spreading through the family and to wider 
contacts, rather than transmission from the child directly to 
26 others. Despite attempts to prevent further spread, the 
number of cases in the locality reached 99 by the end of 
January 1903. The disease then continued to spread to the 
east and south of the city, with the monthly number of 
cases peaking at 356 in March 1903. The timeline of the 
outbreak in relation to a number of other key events and 
control measures is shown in Figure 1 . 

Similarly, until 1905, Edinburgh had also seldom been 
free from smallpox. Later, however, smallpox outbreaks 
became infrequent, with only 4 outbreak years from 1905 
to 1920, and then none at all in the 20 years before 1942. 
The outbreak, therefore, was a relatively new experience 
for a large section of the population (11). This outbreak 
was relatively small and lasted 3 months (October 
27-December 30, 1942), which resulted in 36 cases 
including 8 deaths (case-fatality rate = 22%). Smallpox 
had previously been imported into Scotland on May 29, 
1942, by a ship arriving from Bombay into Scotland’s 
other major city, Glasgow (resulting in 36 cases and 8 
deaths) (12). In August, 3 weeks after the last case in 
Glasgow, an outbreak was reported in Fife (29 cases and 8 
deaths). As the outbreak in Fife was being brought under 
control, the first case of smallpox appeared in Edinburgh 
Royal Infirmary. The disease then spread to the hospital’s 
convalescent home and then into the general public. The 


Smallpox 
spread to 
Robssrt St. and 
diffusion in city 


J. 

i- 

E 



Unrecognized case in 
Robsart Street - 
extension of outbreak* 


58 m JI y ffi H M SZ H 36 


Increased no. cases 
Fazakertey Hospital 
reopened for smallpox 


Continued increase 
in demand for 
hospital isolation. 
Park Hill Hospital 
opened to smallpox 
patients 


** *e J6 


Fortnights 


Figure 1 . Timeline of Liverpool outbreak: key events and control 
interventions, using hospital admission data (December 6, 1901- 
November 27, 1903) (9). *House-to-house visitation of the district 
"forthwith commenced" (7). Over the next few days, 20 more 
cases were found and reported. 


292 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


Smallpox Surveillance and Control Measures 


means of the spread of disease from Glasgow to Fife and 
then to Edinburgh, and from hospital settings to the gener- 
al public, was, however, never identified. Indeed, for 8 of 
13 Edinburgh community cases, the source of infection 
was never discovered. The author of the outbreak report 
conjectured that subclinical infections, i.e., “mild attacks” 
or missed cases, might have been the reason for the lost 
epidemiologic links but adds that these theories were hard 
to reconcile with the facts (8). A timeline of the Edinburgh 
outbreak, highlighting the milestone events and control 
measures employed, is shown in Figure 2. 

Vaccination Status of Population 

To appreciate the course of the outbreaks and the sub- 
sequent effects of the various control measures used, the 
vaccination history of the populations involved should be 
put into context. If we assumed that the level of infant vac- 
cination in Liverpool was similar to that for England and 
Wales as a whole in 1902 and 1903 (-75%) when solid 
immunity in the city might have ranged from 9% to 16% 
(solid immunity, as termed by Dixon [13], is either 5 or 10 
years of total protection from attack). However, at that 
time, considerable support for the antivaccination cause 
resulted in an infant vaccination rate in the late 19th cen- 
tury that varied from 0% in some districts to nearly 100% 
in others (13), and as the background rates for Liverpool 
are not reported, being more specific about the levels of 
vaccination that existed is difficult. 

Vaccination levels also varied from region to region in 
Scotland. The percentage of vaccinated infants in Scotland 
was normally -30.7% (14); however, the Registrar 
General for Scotland reported that 55% of infants were 
being vaccinated in 1941. Whether this report is for 
Scotland as a whole or for Edinburgh alone is not clear (8). 
Dixon’s estimate of solid immunity for the whole of 
England and Wales in 1947, assuming 40% of infants were 
vaccinated, was 4%-7%. However, this percentage was 
increased by the vaccination of National Service entrants 
to -20%. For Scotland, with an infant vaccination rate of 
-30%, solid immunity would have been <20% (13). The 
vaccinial state of the public as a whole was reportedly low 
in the area around Fife (Methilhill), with only 20%-30% 
of the population having been previously vaccinated; but 
in towns nearer Edinburgh (e.g., Cowdenbeath) 40%-50% 
had been vaccinated (14). 

Public Health Response 

In both Liverpool and Edinburgh, phased public health 
responses were implemented (Figures 1 and 2). In 
Liverpool, at the earliest phase of the outbreak, with the 
discovery of the first unreported case in Robsart Street 
(Figures 1 and 3), active case finding in the local area was 
instituted. One report states that, thereafter, usually within 


Outbreak in 
Glasgow 
f36 cases! 


Outbreak in Fife 
(29 cases) 


Outbreak in 
Edinburgh 
(36 cases) 


May 29- 

Notification of 1st 

# Glasgow case 

received in Edinburgh 

June 30 1 

General precautionary 
measures in Edinburgh 

I : 


Oct 31$ 

First 2 
Edinburgh cases 
diagnosed 
Nov 2 
Reception 
House 
opened 


Nov 8-Dec 8K 
' 22 Vaccination centers opened 

0 Edirtxxgh • institubonal 
■ Edinburgh - comminty 


1 


. * Novi 

j,| I Com 

■ I ■ I ■ I 


November 18 
Community cases 


123456789 K> 11 12 13 14 15 16 17 18 19 2D 21 22 23 24 25 28 Z7 2829 30 31 32 33 34 


Figure 2. Timeline of Edinburgh outbreak. *May 29: Revaccination 
of all Edinburgh medical, nursing, domestic, artisan and ambulance 
staff. Edinburgh Smallpox Hospital reconditioned and isolation units 
set-up for observation cases (8). fJune 30: great majority of other 
essential personnel vaccinated (11). Some public vaccination by 
private practitioners (=4% [20,000]). ^November 1 : quarantine and 
daily surveillance of (present and past) patients and visitors to 
Royal Infirmary (8). ^November 8-December 8: further vaccination 
centers opened after 3 more cases occurred. Sixty sessions held 
each day. One vaccination center reopened December 9-12 and 
December 21-24 to cope with a few isolated cases (8,11). 


an hour of notification, patients were removed to hospital 
by ambulance, and the clothing, bedding, and dwellings 
were immediately disinfected (9). An inspector followed 
the ambulance and immediately made inquiries about pos- 
sible sources of infection. Information about the state of 
vaccination of possible contacts was then sent to vaccina- 
tion officers; additional medical staff members were 
employed at this time to assist with vaccination. These 
vaccination officers in Liverpool first recommended 
immediate vaccination or revaccination to all close con- 
tacts of case-patients, then to related workforces, schools, 
and the general public. Special arrangements were made 
for the prompt vaccination of all vagrants coming into the 
city, who were subsequently paid a small sum for consent- 
ing. Offers of vaccination and re vaccination to contacts 



Figure 3. Spatial-temporal distribution of incidence of smallpox 
during outbreak, by district, Liverpool, 1902-1903 (7). *lncidence 
of smallpox per district (per 100,000) calculated as number of 
cases per district -f by district population x 1 00,000. New cases per 
district were counted from the locations given on the 4 maps in the 
original report for each of the periods above. District populations 
were tabulated separately (7). 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


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HISTORICAL REVIEW 


and people living close to persons with smallpox were 
reported in the Health Department Report to have been 
promptly made and almost universally accepted; these 
vaccinations were reported to have greatly limited the 
amount of smallpox in Liverpool. As the number of cases 
increased as the outbreak developed, hospital isolation 
accommodations were expanded by committing an 
increasing number of hospitals to the intake of smallpox 
patients (Figure 1). 

In Edinburgh, after notification of the first Glasgow 
case on May 29 (Figure 2), the first campaign of vaccina- 
tion and revaccination for essential personnel (e.g., med- 
ical staff, civil defense workers, and police), was agreed to 
on June 30 and promptly instituted on July 1, 4 months 
before the disease reached Edinburgh (11). Edinburgh had 
not had a smallpox case for 20 years, but at this time, the 
smallpox hospital was reopened, and satellite isolation 
units in the hospital grounds prepared to receive patients 
for observation. All contacts of Glasgow case-patients 
arriving in Edinburgh were, as was routine practice, exam- 
ined and put under surveillance. The Public Health 
Department was responsible for the medical supervision of 
contacts, and medical officers of health were responsible 
for requesting precautionary behavior in the general pub- 
lic. The second vaccination campaign took place from 
November to December 1942, when the disease had taken 
hold in Edinburgh itself. At this time the contacts of 
patients were vaccinated, and vaccination was extended 
subsequently to the general public with the opening, on 
November 8 (Figure 2), of 22 vaccination centers through- 
out the city. 

Despite the previous vaccination of infants and other 
target groups, levels of immunity contemporaneous with 
these 2 outbreaks were insufficient on their own to prevent 
expanding outbreaks. Nevertheless, the spread of infection 
over both space and time across Liverpool was character- 
istically slow, taking 3 months to significantly extend out 
of the district into which it was introduced, to more south- 
eastern districts (Figures 1 and 3). 

The first 2 cases in Edinburgh were diagnosed on 
October 31. On November 1, active case-finding was initi- 
ated with house-to-house searches, and a first aid post was 
opened subsequently, which provided 8,000 vaccinations to 
people in the area in which these patients lived. Family con- 
tacts of patients were sent for observation to a prepared 
reception house, which was opened on November 2, the day 
after the first 2 cases had been confirmed (Figure 2). 
Persons in the reception house were quarantined for 21 
days, and all but 1 of their employers agreed to pay their 
wages during this time (8). The exception to this rule attend- 
ed work during the day and returned to the reception house 
at night and was examined both upon leaving and on return- 
ing for signs of infection. The Royal Infirmary convalescent 


home also acted as an additional observation ward. 

Contact tracing was an important part of the control 
methods instituted in both outbreaks. In Edinburgh, the 
press was used extensively as a means to trace contacts of 
case-patients and to persuade large numbers of persons to 
accept vaccination; the use of the press also allowed the 
authorities to reach possible contacts with a minimum of 
delay (11). In all, ~ 1,700 contacts of the 36 cases were 
traced and observed for 18 to 21 days, which represents an 
average of ~47 contacts per case. More than 900 persons 
were traced as contacts and revaccinated from 3 cases 
alone. Despite being infected, these ambulatory cases had 
used public transport or been in contact with large numbers 
of persons because of their occupation (8). The readiness 
of the public to cooperate with all the above recommend- 
ed, routine precautions is noted in the Annual Report, 1942 
(11). The press was also used in the Liverpool outbreak. 
Circulars that detailed the movements of case-patients who 
had used public transport and the location and availability 
of public vaccinators were widely distributed. Although 
the total number of traced contacts is unclear, we know 
that contacts were visited every day for 14 days after noti- 
fication, and then every few days for a further 2 weeks. At 
the peak of the outbreak, when 356 cases existed, as many 
as 2,000 families were being visited daily, which repre- 
sents an average of ~6 families contacted per case. On the 
basis of an average household size for England, at that 
time 5, we have a rough estimate of 30 contacts traced and 
vaccinated per case. 

In the Liverpool outbreak, the occurrence of a large 
number of vaccine-modified cases caused particular prob- 
lems for those attempting to control the outbreak, espe- 
cially with respect to late or incorrect diagnoses. 
According to Hanna (10), 72.7% of those vaccinated pre- 
viously and 16.8% of unvaccinated cases were considered 
to be “modified discrete and discrete smallpox” (modified 
here meaning an accelerated clinical course compared with 
expected course of ordinary smallpox, usually with fewer 
lesions, not necessarily modified by vaccination) (13). 
Chickenpox was a notable misdiagnosis in some instances; 
2.6% of chickenpox diagnoses were found subsequently to 
be smallpox (similarly, in the 1901-1902 London out- 
break, the figure for the same misdiagnosis was 2.5%). To 
help overcome this problem, chickenpox was made a noti- 
fiable disease, provisionally in April, and permanently in 
August 1902. Reporting of smallpox itself, however, was 
not always straightforward in the Liverpool outbreak. 
According to 1 author (9), the diagnosis of smallpox was 
sometimes revoked upon admission to hospital, or vice 
versa, a nonsmallpox case-patient was often treated as hav- 
ing smallpox in the hospital. On at least 1 occasion, infor- 
mation on patients treated in the hospital did not reach the 
medical officer of health. In Edinburgh, the first 2 cases 


294 


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Smallpox Surveillance and Control Measures 


were misdiagnosed as chickenpox and meningococcal sep- 
ticemia, respectively. Misdiagnosis as chickenpox is a con- 
cern that continues to exist today. In Glasgow, in 1942, 
severe vaccinial reactions, occurring at the end of the pos- 
sible incubation period of smallpox, also complicated the 
problem of diagnosis for medical practitioners (12). 

As with the 3 ambulatory case-patients in the 
Edinburgh outbreak discussed above, such patients were 
also a problematic source of infection in Liverpool (7,10). 
Some smallpox infections were reported to be so mild 
(usually vaccine-modified) that doctors were not consult- 
ed, and patients and their household contacts continued to 
visit public areas and shops. For example, 1 unreported 
smallpox case occurred in a person whose family contin- 
ued to go to work and socialize, which gave rise to 29 other 
cases. Twenty prosecutions were instituted against mem- 
bers of this family during the outbreak. The extent to 
which ambulatory vaccine-modified cases might occur in 
any modern day U.K. outbreak is not known. However, the 
proportion of vaccine-modified cases overall would be 
much less than in Liverpool because of the length of time 
since the population was last vaccinated. This finding has 
been discussed in greater detail elsewhere (2). 

Previous vaccination status also strongly influenced the 
relationship between age at time of attack and death (Figure 
4). In a study that examined a series of 1,163 case-patients 
during the 10 years after the Liverpool outbreak (mostly 
from the epidemic period 1902-1903), 943 (81%) had been 
vaccinated in infancy, and 220 (18.9%) had not been vacci- 
nated (10). Among those vaccinated in infancy, 28 (2.9%) 
deaths occurred from smallpox, whereas among the unvac- 
cinated, 60 (27.2%) deaths occurred. The case mortality 
among the vaccinated rose steadily with age from the 20- to 
30-year age group upwards to the >60-year group (no 
deaths occurred in those <20), but never exceeded 10%. 
However, among the unvaccinated, 58% of patients <2 
years of age died, decreasing to 30.6% for those 2 to 5 years 
of age. The ratio was lower (3.2%) for those 10 to 15 years 
of age; the case-fatality rate rose (13%) for those 15 year of 
age, and it was 50% for those >40 years of age. The effect 
of vaccination on protection against death according to age 
has also been noted by others (15,16). The level of partial 
immunity to smallpox, i.e., protection from death as 
opposed to protection from infection, in a modern popula- 
tion may be higher than previously thought (1); spread of 
infection from ambulant patients with vaccine-modified 
cases may be an important and problematic means of trans- 
mission (10,13,17), as has been pointed out in more recent 
analyses (2). In the Edinburgh outbreak, 6 of the 8 deaths 
from smallpox occurred in adults >20 years of age who had 
been vaccinated in infancy. 

No data concerning vaccine-related adverse events are 
available from the Liverpool outbreak, but we know that of 


70 


□ Vaccinated 
■ Unvaccinated 



<2 2-5 5-10 10-15 15-20 20-30 30-40 40-50 50^0 >60 


A ge (y) 

Figure 4. Percentage case-patient death rate by age in the vacci- 
nated and unvaccinated, Liverpool outbreak, 1902-1903 (10). 


the estimated 360,000 vaccinations (based on lymph issue) 
performed in Edinburgh and adjacent counties (-77% of 
the local population), 10 vaccine-related deaths occurred; 
8 of these were from encephalomyelitis. Compared to vac- 
cination campaigns in England and Wales in 1951 to 1960 
(18), the numbers of postvaccinial encephalomyelitis and 
generalized vaccinia were much higher (Table 1). Indeed, 
a similarly high incidence of postvaccinial encepha- 
lomyelitis was reported during the Fife outbreak (14). 
Approximately 78% of vaccinees in and around Edinburgh 
had had a previously successful vaccination; the remain- 
der, -22%, had either a previously unsuccessful vaccina- 
tion or no vaccination at all. In neither of the outbreaks was 
an intensified national vaccination campaign reported to 
have been initiated. 

Discussion 

In both the Liverpool and Edinburgh outbreaks, phased 
public health responses were implemented, and the out- 
breaks were brought under control within 15 and 3 
months, respectively. Because smallpox arrived first in 
Glasgow, Edinburgh health authorities had time to prepare 
and implement a 2-phased vaccination campaign along 
with active surveillance. For Liverpool, the report demon- 
strates clearly that the spread of infection across the city 
was slow, which suggests a relatively low transmission 
rate and a characteristically long generation time, allow- 
ing for targeted intervention methods to be effectively 
implemented. By comparing the incidence of cases in dif- 
ferent districts across the panels shown in Figure 3, the 
outbreak appears to have taken 3 months (November 
1902-January 1903) to spread into districts adjacent to the 
origin of the outbreak and then an additional 3 months 
(February- April 1903) to spread to more eastern and 
western districts. The slow spread of smallpox described 
here is not dissimilar to that described in studies in other 
countries, for example, Pakistan and Bangladesh in the 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


295 


HISTORICAL REVIEW 


Table. Rates of adverse events to smallpox vaccination in 
Edinburgh, 1942* 



1st vaccination 

2nd vaccination 

Adverse event 

campaign 
Jun - Julf 

campaign 
Nov - Decf 

Nonspecific rashes 

20 

12.5 

Auto-inoculation and 

7.5 

7.5 

generalized vaccinia 

Postvaccinial 

encephalomyelitis 

5.0 

4.7 

*Source: reference 8. 
fPer 100,000. 


1960s (17). In Bangladesh, smallpox tended to be more 
rapidly transmitted within family units but spread more 
slowly between them (19). 

Active surveillance, vaccination of contacts, and 
prompt hospital isolation of patients were important 
aspects of disease control measures in both outbreaks. 
Indeed, the success of the surveillance-containment strate- 
gy in Liverpool, the basis of which has been discussed 
more recently elsewhere (20), was particularly noted by 
the observers of the time (7,10). Unlike the situation in the 
United Kingdom today, both Liverpool and Edinburgh had 
designated smallpox hospitals, either already open or 
ready to reopen, at the time of these outbreaks. These ded- 
icated facilities must have contributed to infection control 
efforts. However, control and containment procedures in 
the 2 cities were hampered in both outbreaks to some 
extent by reintroduction of the disease from other areas, by 
patients with ambulant cases of mild infection (probably 
vaccine-modified), and by missed cases. 

These 2 case studies draw attention to issues of current 
concern, not only to the potential impact of vaccine-modi- 
fied cases mentioned above, but also to adverse events to 
vaccination, both of which might have an impact in a mod- 
ern-day outbreak. However, in contrast to these 2 out- 
breaks, the fact that routine smallpox vaccination ceased in 
the West during the 1970s brings complications of its own. 
Persons <30 years of age have never received the vaccine 
and are immunologically naive. This 30-year time gap since 
vaccination also has implications for the immune status of 
previous vaccinees and the potential for adverse event and 
disease complications and indeed for the spread of disease 
among this population. If historical events are to be used as 
sources of evidence, and the data from them extrapolated to 
modern populations, they must be considered within the 
ethical and social context of today, by observing societal 
differences, expedited travel, waning immunity, and 
increased recognition of contraindications to vaccination. 
In particular, the number of people who are immunocom- 
promised today continues to rise with the increase of HIV 
infection, chemotherapy, immunity disorders, and trans- 
plantations. So too has the number of people with atopic 
dermatitis; in the United Kingdom alone, 2.3% of the pop- 
ulation is estimated to have this condition (21). However, 


cardiac adverse events to vaccination, such as myocarditis 
and pericarditis, were not reported in these 2 case studies, 
as has been seen in more recent vaccination efforts (22). 

The studies also illustrate that the level of background 
solid immunity in these populations was low and could 
give rise to expanding outbreaks. The response to these 
outbreaks was not to implement a national vaccination 
campaign but rather a targeted approach, expanded when 
necessary. Although these data, based on the direct experi- 
ence of infected populations, are not truly predictive for a 
modern smallpox outbreak (1), they are very instructive. 

Analysis of the Edinburgh and Liverpool outbreaks 
suggests that outbreaks after deliberate release of smallpox 
virus may evolve over time. Therefore, sufficient opportu- 
nity exists for targeted enhanced surveillance measures to 
be put in place, for additional staff to be mobilized for an 
effective follow-up, and for a containment strategy to be 
implemented. The Liverpool outbreak took 15 months to 
control; the one in Edinburgh 3 months. This time differ- 
ence probably reflects that reintroductions of smallpox 
occurred during the 1902-1903 outbreak because the dis- 
ease was still endemic in the United Kingdom, poorer 
socioeconomic conditions existed in Liverpool at this time, 
and crowding was more prevalent, particularly in the dock- 
land areas most heavily affected. By contrast in 1942, 
smallpox was no longer endemic in the United Kingdom, 
and socioeconomic conditions in Edinburgh were better. 
One might hope for at least as swift an end to a similarly 
sized modern-day outbreak as was seen in Edinburgh. 

Modeling of data from other historical outbreaks of 
smallpox may help to further develop targeted surveillance 
and containment interventions for smallpox in the present 
era (3,23). Such interventions warrant further investigation 
because of clear, accumulating evidence of the substantial 
disease and death likely to accompany any mass popula- 
tion smallpox vaccination strategy. 

Acknowledgments 

We thank the archivist of the city of Edinburgh for permis- 
sion to republish the following: Clark G, Seiter HE, Joe A, 
Gammie JL, Tait HP, Jack RP. The Edinburgh outbreak of small- 
pox, 1942. Authority of the Public Health Committee, Edinburgh, 
Scotland; 1944. 

The work was funded by the U.K. Department of Health. 
The views expressed are those of the authors and not necessarily 
those of the Department of Health. 

Miss Kerrod is a risk assessment scientist in the Microbial 
Risk Assessment Department, Centre for Emergency 
Preparedness and Response, Health Protection Agency, Porton 
Down, Salisbury, UK. Her research interests include bioterrorism 
issues and the epidemiology of infectious diseases. 


296 


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Smallpox Surveillance and Control Measures 


References 

1. Ferguson NM, Keeling MJ, Edmunds, WJ, Gani R, Grenfell BT, 
Anderson RM, et al. Planning for smallpox outbreaks. Nature. 
2003;425:681-5. 

2. Eichner M. Analysis of historical data suggests long-lasting protec- 
tive effects of smallpox vaccination. Am J Epidemiol. 
2003;158:717-23. 

3. Eichner M, Dietz K. Transmission potential of smallpox: estimates 
based on detailed data from an outbreak. Am J Epidemiol. 
2003;158:110-7. 

4. Gani R, Leach S. Transmission potential of smallpox in contempo- 
rary populations. Nature. 2001;414:748-51. 

5. Meltzer MI, Damon I, LeDuc JW, Millar JD. Modeling potential 
responses to smallpox as a bioterrorist weapon. Emerg Infect Dis. 
2001;7:959-69. 

6. Hammarlund E, Lewis MW, Hansen SG, Strelow LI, Nelson JA, 
Sexton GJ, et al. Duration of antiviral immunity after smallpox vac- 
cination. Nat Med. 2003;9:1131-7. 

7. Hope EW. Report on the health of the City of Liverpool during 1903. 
Liverpool: C. Tinling & Co.; 1904. 

8. Clark G, Seiter HE, Joe A, Gammie JL, Tait HP, Jack RP. The 
Edinburgh outbreak of smallpox, 1942. Authority of the Public 
Health Committee; 1944. 

9. Reece RJ. Report to the local government on smallpox and smallpox 
hospitals at Liverpool, 1902-03. London: HMSO. 

10. Hanna W (1913). Studies in smallpox and vaccination. Rev Med 
Virol. 2002;12:201-9. 

11. Annual report for the health of the city during 1942. Edinburgh: 
Medical Officer of Health, City and Royal Burgh of Edinburgh Public 
Health Department; 1942. 

12. MacGregor A. The outbreak of smallpox in Glasgow 1942. BMJ. 
1942;2:627-9. 


13. Dixon CW. Smallpox. London: J & A Churchill Ltd.; 1962. 

14. Fyfe GM, Fleming JB. Encephalomyelitis following vaccination in 
Fife. BMJ. 1943;ii:671-4. 

15. Rao AR. Smallpox, Bombay. Bombay, India: The Kothari Book 
Depot; 1972. 

16. Mack TM. Smallpox in Europe, 1950-71. J Infect Dis. 
1972;125:161-9. 

17. Fenner F, Henderson DA, Arita I, Jezek Z, Ladnyi ID. Smallpox and 
its eradication. Geneva: World Health Organization; 1988. 

18. Conybeare ET. Illness attributed to smallpox vaccination during 
1951-60. Mon Bull Minist Health Public Health Lab Serv. 
1964a;23: 126-33. 

19. Baxby D Surveillance-containment is key to eradication of smallpox. 
BMJ. 1995;3 10:62. 

20. Thomas DB, Mack TM, Ali A, Muzaffer Khan M. Epidemiology of 
smallpox in West Pakistan. 3. Outbreak detection and interlocality 
transmission. Am J Epidemiol. 1972;95:178-89. 

21. James Britton. Eczema facts & figures-how common is eczema and 
what are the costs? [cited September 2004]. Available from 
http://www.dermatology.co.uk/eczema/diagnosisandcauses/article/art 
icle.asp?ArticleID=899 

22. Centers for Disease Control and Prevention. Update: cardiac and 
other adverse events following civilian smallpox vaccination — 
United States, 2003. MMWR Morb Mortal Wkly Rep. 
2003;52:639-42. 

23. Eichner M. Case isolation and contact tracing can prevent the spread 
of smallpox. Am J Epidemiol. 2003;158:118-28. 


Address for correspondence: Emma Kerrod, Centre for Emergency 
Preparedness and Response, Health Protection Agency, Porton Down, 
Salisbury, Wiltshire, SP4 0JG, UK; fax: +44-0-1980-612491; email: 
emma.kerrod@hpa.org.uk 



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297 




DISPATCHES 


In Vitro Host-Cell 
Susceptibility to 
Usutu Virus 

Tamas Bakonyi,*t Helga Lussy,* 

Herbert Weissenbock,* Akos Hornyak,t+ 
and Norbert Nowotny *§ 1 

We investigated the susceptibility to Usutu virus 
( Flavivirus ) of 13 permanent cell lines, 3 primary cell cul- 
tures, and chicken embryos. Vero, PK-15, and goose 
embryo fibroblast cells developed cytopathic effects; how- 
ever, viral multiplication was detected in all mammalian cell 
types by immunohistochemical tests. Chicken embryo 
fibroblast cells and chicken embryos were resistant. 

U ntil its emergence in Austria in 2001 (1), Usutu virus 
was regarded as a flavivirus found only in sub- 
Saharan Africa. The virus was first isolated from Culex 
naevei in South Africa (2); later it was detected in other 
mosquito (Cx perfuscus , Mansonia africana, Coquille- 
tidia aurites ), bird ( Turdus libonyanus , Bycanistes fiscula- 
tor ), and rodent species ( Praomys sp.) (3-5). Also, Usutu 
virus was isolated once from a man with fever and rash (3). 
In Africa, Culex mosquitoes and birds are responsible for 
transmission and circulation of the virus in nature; howev- 
er, the infection does not cause overt disease in the local 
host species. Since its introduction to Europe, Usutu virus 
has shown substantial pathogenicity for several wild bird 
species and causes severe die-offs, especially in the 
Eurasian blackbird ( T. merula) populations. Recurring 
enzootics have been observed from mid-July to the end of 
September in the affected areas in the eastern part of 
Austria within the last 4 years (6). 

Usutu virus is a member of the Japanese encephalitis 
virus (JEV) group within the mosquitoborne flaviviruses 
(7). The most important members of the group, West Nile 
virus (WNV), Murray Valley encephalitis virus (MVEV), 
St. Louis encephalitis virus (SLEV), and JEV are able to 
infect a broad spectrum of animal species. These viruses 
are transmitted by different mosquito species and frequent- 
ly cause infections in birds (all virus species), rodents 
(WNV, SLEV), swine (JEV), and horses (WNV, MVEV, 
SLEV). WNV, SLEV, JEV, and MVEV are human 
pathogens as well; they may cause epidemics of encephali- 
tis in humans in certain geographic regions. 


*University of Veterinary Medicine, Vienna, Austria; fFaculty of 
Veterinary Science, Budapest, Hungary; ^Central Veterinary 
Institute, Budapest, Hungary; and §United Arab Emirates 
University, Al Ain, United Arab Emirates 


The classic manner of flavivirus cultivation is intra- 
cerebral inoculation of suckling mice or inoculation of 
embryonated eggs (8). A variety of primary cells and 
established cell lines support the replication of flavivirus- 
es: Green monkey (Vero), hamster (BHK-21), human 
(SW-13, HeLa), porcine (PS), and mosquito cell lines, as 
well as primary chicken and duck embryo cells have been 
used for flavivirus isolation and propagation in routine 
diagnostic applications. The appearance of cytopathic 
effects (CPEs), plaque formation, and virus yields greatly 
vary with the different viruses and host cells. 

Since Usutu virus was of minor clinical importance 
until its emergence in central Europe, its biologic features, 
host spectrum, and pathogenesis had not previously been 
studied. With the changes in the clinical appearance of 
Usutu virus infection in the new environment, and the 
impact of closely related viruses on human and veterinary 
health care, the detailed characterization of the virus is of 
high priority. 

The Study 

We investigated the in vitro susceptibility of various 
cell cultures and embryonated eggs to Usutu virus infec- 
tion. Human (HeLa), green monkey (Vero), equine (ED), 
bovine (MDBK), porcine (PK-15), rabbit (RK-13), canine 
(MDCK, DK), feline (CR), hamster (BHK-21, BF), rat 
(C6), and turtle (TH1) permanent cell lines, as well as pri- 
mary horse kidney (EqK), chicken embryo fibroblast 
(CEF), and goose embryo fibroblast (GEF) cell cultures 
were tested. Cells were propagated in Earle’s minimal 
essential medium (MEM) (Gibco Invitrogen, Paisley, UK) 
containing L-glutamine, antimicrobial drugs, and 10% 
fetal calf serum (FCS). The cells were regularly subcul- 
tured by employing standard techniques. To 1 -day-old 
confluent monolayers of the permanent cell lines and pri- 
mary cell cultures, grown on the surface of chamber slides, 
the Austrian Usutu virus strain Vienna 2001 -blackbird 
(GenBank accession no. AY453411) was added at a multi- 
plicity of infection (MOI) of 3. The virus was originally 
isolated in Vero cells in 2001 from the brain homogenate 
of a blackbird found dead in the area surrounding Vienna. 
The isolate was propagated twice in Vero cells. The second 
virus passage was used for the experiments; 50% tissue 
culture infective dose (TCID 50 ) was determined, and 
aliquots of the virus were stored frozen at -80°C until used. 
The virus was added to the cells, which were then incubat- 
ed at 37°C for 1 h. Thereafter, the inoculum was removed, 
the cell cultures were washed once with phosphate- 
buffered saline (PBS), and MEM containing 2% FCS, L- 


Hhis study will be presented at the International Conference on 
Emerging Infectious Diseases, February 26-March 1 , 2005, Al Ain, 
United Arab Emirates. 


298 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


In Vitro Host-Cell Susceptibility to Usutu Virus 


glutamine, and antimicrobial drugs were added. For all cell 
types, controls were cultivated simultaneously and treated 
in the same way as the infected cultures with the exception 
that MEM was used for inoculation. All cell cultures were 
incubated at 37°C for 3 to 5 days; then the medium was 
removed and the monolayers were fixed with chilled 
(-20°C) acetone. The cells were stained with hematoxylin- 
eosin (HE) and examined microscopically. In parallel, 
immunohistochemical (IHC) testing was carried out on the 
cell cultures by using the avidin-biotin complex technique, 
with a polyclonal antiserum raised in mice against WNV 
antigens, for which cross-reactivity with Usutu virus had 
been demonstrated previously (1). The number of antigen- 
positive cells was evaluated microscopically and scored 
(see Table). 

Embryonated chicken eggs (strain LSL White, which 
was derived from the strain White Leghorn), originating 
from a specified pathogen free (SPF) herd (VALO eggs, 
Lohmann, Cuxhaven, Germany), were injected into the 
allantoic sac with 6 x 10 5 TCID 50 of Usutu virus at the age 
of 10 days. The eggs were incubated together with mock- 
infected controls at 37.5°C for further 4 days and were 
checked daily by transillumination. On day 4 postinfec- 
tion, the eggs were opened, and the embryos were fixed in 
4% buffered formaldehyde solution. Histologic sections 
were made from paraffin-embedded organs of the 
embryos, and the slides were analyzed by light microscopy 
after HE and IHC staining, respectively, as described 
above. 

Three to 4 days after inoculation, pronounced CPEs 
were observed in Usutu virus-infected Vero and PK-15 
cell cultures as well as in GEF cells. The first foci of cell 
rounding and subsequent shrinkage of the cells were 
observed on day 2 or day 3 post infection, when groups of 

4 to 8 cells, but also single cells, showed rounding and 
degeneration; within 1 day the affected cells lost their 
adherence to the bottom of the flask and floated in the 
medium. Within a further 2 days, 90%-100% of the cells 
exhibited CPE. Typical Usutu virus CPE is shown in HE- 
stained Vero cells in Figure 1. The mock-infected Vero, 
PK-15, and GEF control cell cultures did not show any 
CPE. The other investigated cell types inoculated with 
Usutu virus did not develop visible CPE within a period of 

5 days, and they were also negative by microscopy after 
HE staining. However, by IHC with cross-reactive WNV- 
antiserum, focal virus multiplication was detected in all 
cell cultures, independent of animal species and tissue 
type, except chicken embryo fibroblast cells (Figure 2). 
The percentage of Usutu virus antigen-positive cells var- 
ied from ~1% (DK) to 50% (GEF) (Table). In the case of 
HeLa cells, different clones adapted to the propagation of 
human rhinoviruses (HeLa Rhino) and herpes simplex 
viruses (HeLa HSV), respectively, were also tested, but 


Table. Semiquantitative evaluation of the number of Usutu virus 
antigen-positive cells* 


Cell line/culture IHC result 


HeLa (human) 

++ 

Vero (simian) 

++ 

ED (equine) 

++ 

MDBK (bovine) 

+ 

PK-15 (porcine) 

++ 

RK-13 (lapin) 

++ 

MDCK (canine) 

++ 

DK (canine) 

( + ) 

CR (feline) 

+ 

BHK-21 (hamster) 

+ 

BF (hamster) 

+ 

C6 (rat) 

+ 

TH1 (turtle) 

++ 

EqK (equine) 

++ 

CEF (chicken) 

- 

GEF (goose) 

++ 


*IHC, immunohistochemical; scoring criteria: (+), 1%-5% positive cells; +, 
6%-25% positive cells; ++, 26%-50% positive cells. Primary cell cultures 
are indicated in italics. 


1A 


1 ^9 
% 



9 


' ib 

Figure 1. Cytopathic effect (CPE) of Vero cells caused by Usutu 
virus infection, 4 days postinfection (hematoxylin-eosin staining). 
A) Uninfected control, B) Usutu virus infected. Bar = 100 pm. 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


299 



DISPATCHES 


2A * 2B 2C 

4 ? ; U '' i < 


k 


2D 2E ^ ■ • V* 2F 

Figure 2. Demonstration of Usutu virus antigen 3 days postinfection. Immunohistochemical (IHC) tests were performed by using a poly- 
clonal antibody to West Nile virus, which cross-reacts with Usutu virus. A) Vero control; B) Vero infected; C) CR (feline) control; D) CR 


infected; E) goose embryo fibroblast (GEF) control; F) GEF infected 

they gave the same results as the commonly used (ATCC) 
HeLa cells by HE and IHC staining. The mock-infected 
control cell cultures were clearly negative in each case. 

The Usutu virus-infected chicken embryos did not 
show any lesions when investigated by gross and 
histopathologic examination after 4 days of incubation and 
were negative by IHC as well. To rule out the slight possi- 
bility that the Usutu virus strain used for inoculation 
underwent a change in cell tropism during the 2 passages 
in Vero cells, CEF, Vero, PK-15, MDCK, and DK cells, as 
well as embryonated chicken eggs, were reinfected with 
the original Usutu virus isolate (before passaging); the 
results were identical to the results obtained with Usutu 
virus passaged twice before use. 

Conclusions 

The appearance of CPE in flavivirus-infected cell cul- 
tures depends on the virus and host cell type, as well as on 
MOI levels and incubation time employed (8). In many 
cases, the presence and multiplication of flavi viruses do 
not inhibit significantly the host cell macromolecular syn- 
thesis, resulting in noncytopathic persistent infections 
(9,10). Pathogenesis and virulence of flaviviruses are 
influenced in vivo by several virus- and host-dependent 
factors, including the role of defective interfering particles, 
viral receptors, neuro virulence, immune-response (e.g., 
antibody-dependent enhancement), and host resistance 
genes (8). Although some of these processes are not yet 
fully understood, the basic requisite of any pathogenic 


; A,B) bar = 50 mm; C-F) bar = 100 pm. IHC staining. 

effect is the host susceptibility to the virus infection. This 
study demonstrates that Usutu virus can infect cell cultures 
of various tissue types derived from a wide variety of ani- 
mal species, including cell lines of human origin. Since 
only Vero, PK-15, and GEF cells develop CPE after Usutu 
virus infection, these cell lines and cell culture are the most 
appropriate ones for diagnostic purposes (e.g., virus isola- 
tion, plaque reduction neutralization test). As demonstrat- 
ed by IHC, considerable differences have been found in the 
susceptibility of the various cell lines and cultures to Usutu 
virus infection and in the extent of spread of the infection; 
even cell lines derived from the same animal species and 
organ varied significantly in their susceptibility to Usutu 
virus infection, e.g., MDCK cells strongly support Usutu 
virus multiplication, while DK cells are far less suscepti- 
ble. Both of these cell lines, however, have been derived 
from dog kidneys. On the other hand, the differences 
between the 2 canine kidney cell lines might also be the 
consequence of different random mutations (e.g., in genes 
of the interferon or other innate defense systems) that 
allowed the cells to immortalize. Since in Austria, Usutu 
virus infects wild birds and causes high death rates, espe- 
cially in blackbirds, one would think that birds are most 
susceptible hosts for the virus. Therefore, the finding that 
both the chicken embryo fibroblast monolayers and the 
chicken embryos are apparently resistant to Usutu virus 
infection was unexpected. Usutu virus, however, is not the 
only flavivirus with such contradiction in host spectrum. 
Ilheus virus, a South American mosquitoborne flavivirus 


300 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


In Vitro Host-Cell Susceptibility to Usutu Virus 


belonging to the Ntaya vims group (7), also naturally 
affects wild birds and produces plaques in primary rhesus 
kidney cells and various established cell lines (Vero, PS, 
BHK-21, and LLC-MK2), but not in avian cells (8). 
Preliminary results of our chicken experiments with Usutu 
vims also support that idea that the domestic chicken is 
resistant to the infection, even when young. Further inves- 
tigations involving different bird and mammal species will 
be necessary to show the most important host species, nat- 
ural reservoirs, and vectors of Usutu virus and to estimate 
its epidemiologic impact and possible threat to domesticat- 
ed animals and to the human population. 

Acknowledgments 

We thank B. Murgue for kindly providing a polyclonal anti- 
serum against WNV antigens. 

This study was funded by a grant of the Austrian Federal 
Ministry for Health and Women’s Issues. 

Dr. Bakonyi is lecturer of virology at the Faculty of 
Veterinary Science, Budapest, and also a guest researcher at the 
University of Veterinary Medicine, Vienna. He is interested in the 
molecular diagnosis and epidemiology of animal and human 
viruses. 

References 

1. Weissenbock H, Kolodziejek J, Url A, Lussy H, Rebel-Bauder B, 
Nowotny N. Emergence of Usutu virus, an African mosquito-borne 
flavivirus of the Japanese encephalitis virus group, central Europe. 
Emerg Infect Dis. 2002;8:652-6. 


2. Woodall JP. The viruses isolated from arthropods at the East African 
Virus Research Institute in the 26 years ending December 1963. Proc 
EAfr Acad. 1964;2:141-6. 

3. Adam F, Diguette JP. Virus d’Afrique (base de donnes). Centre col- 
laborateur OMS de reference et de recherche pour les arbovirus et les 
virus de fievres hemorrhagiques (CRORA); Institut Pasteur de Dakar. 
Available from http://www.pasteur.fr/recherche/banques/ CRORA/ 

4. Cornet M, Robin Y, Chateau R, Heme G, Adam C, Valade M, et al. 
Isolement d’ arbovirus au Senegal Oriental a partir de moustiques 
(1972-1977) et notes sur Pepidemiologie des virus transmis par les 
Aedes en particulier du virus amaril. Cahiers ORSTOM, Serie 
Entomologie medicale et Parasitologie. 1979;17:149-63. 

5. Hubalek Z. Pathogenic microorganisms associated with free-living 
birds (a review). Acta Sci. Nat. Brno. 1994;28:1-74. 

6. Weissenbock H, Kolodziejek J, Fragner K, Kuhn R, Pfeffer M, 
Nowotny N. Usutu virus activity in Austria, 2001-2002. Microbes 
Infect. 2003;5:1132-6. 

7. Heinz FX, Collett MS, Purcell RH, Gould EA, Howard CR, 
Houghton M, et al. Family Flaviviridae . In: van Regenmortel MHV, 
Faquet CM, Bishop DHL, editors. Virus taxonomy, seventh 
International Committee for the Taxonomy of Viruses. San Diego: 
Academic Press; 2000. p. 859-78. 

8. Burke DS, Monath TP. Flaviviruses. In: Knipe DM, Howley PM, 
Griffin DE, Lamb RA, Martin MA, Roizman B, et al., editors. Fields 
virology, vol. 1. Philadelphia: Lippincott Williams & Wilkins; 2001. 
p. 1055-109. 

9. Schmaljohn C, Blair CD. Persistent infection of cultured mammalian 
cells by Japanese encephalitis virus. J Virol. 1977;24:580-9. 

10. Brinton MA. Characterization of West Nile virus persistent infections 
in genetically resistant and susceptible mouse cells. I. Generation of 
defective nonplaquing virus particles. Virology. 1982;116:84-94. 

Address for correspondence: Norbert Nowotny, Zoonoses and Emerging 
Infections Group, Clinical Virology, Clinical Department of Diagnostic 
Imaging, Infectious Diseases and Clinical Pathology, University of 
Veterinary Medicine, Vienna, A-1210 Vienna, Austria; fax: 43-1-25077- 
2790; email: Norbert.Nowotny@vu-wien.ac.at 


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301 


DISPATCHES 


Bat Incidents at 
Children's Camps, 
New York State, 
1998-2002 

Amy Robbins,* Millicent Eidson,* Mary Keegan,* 
Douglas Sackett,* and Brian Laniewicz* 

From 1998 to 2002, a total of 299 bat incidents were 
reported at 109 children’s camps in New York; 1,429 
campers and staff were involved, and 461 persons 
received rabies treatment. In 53.8% of the incidents, the bat 
was captured and samples tested negative for rabies virus, 
which resulted in 61.3% of persons not receiving rabies 
treatment. 


R abies is a neurologic disease with close to a 100% 
case-fatality rate; once clinical signs appear, it is 
almost always untreatable (1). After a person is exposed to 
rabies, death can be prevented only if treatment, common- 
ly referred to as postexposure prophylaxis (PEP), is initiat- 
ed. PEP includes an initial dose of immune globulin and a 
series of 5 doses of rabies vaccine in a 1 -month period. 
PEPs are costly in terms of money and time because of the 
5 medical visits, particularly if the person must be trans- 
ported elsewhere for the treatment. The New York State 
Department of Health (NYSDOH) has a unique program 
that requires that rabies exposures and treatments be 
reported. County expenses associated with authorized 
treatments in accordance with state and federal guidelines 
are then partially reimbursed (2). 

Despite a large number of rabid animals in the United 
States (7,967 confirmed in 2002), rabies in humans is rare 
because of the availability of PEP; 31 cases were reported 
in the United States from 1990 to 2003 (3). Twenty-nine 
(94%) of the 31 cases were associated with bat rabies vari- 
ants, and a bat bite could be definitively documented for 
only 3 of them (3). Four children in the United States (4-8) 
and 1 child in Quebec, Canada, died of bat-related rabies 
(9). The families of the children in the United States were 
unaware of the potential for rabies transmission from bats. 

Children’s summer camps share habitats favored by 
bats and other wildlife; thus, children and camp staff may 
come into contact with bats that are either roosting in camp 
buildings or flying among camp facilities while foraging. 
A camp-related rabies death occurred in Alberta, Canada, 
in 1985 in a 25 -year-old student who had been bitten and 
scratched by a bat and received no treatment (10). 


*New York State Department of Health, Albany, New York, USA 


Of the 3,827 bats tested by the NYSDOH Wadsworth 
Center’s Rabies Laboratory in 2002, 102 (2.6%) were 
rabid (11). Although the probability of an individual bat 
being rabid is relatively low, bats that can expose humans 
to rabies must be assumed rabid, when a definitive diagno- 
sis of rabies cannot be made. In 1999, the federal Advisory 
Committee on Immunization Practices (ACIP) updated the 
national PEP recommendations to include incidents with 
bats in which there was a “reasonable probability that 
exposure has occurred” (12). These types of incidents 
include direct contact with a bat; a bite, scratch, or mucous 
membrane contact with bat saliva or nervous tissue; a 
sleeping person awakening to find a bat in the room; or an 
adult witnessing a bat in the room with a previously unat- 
tended child, or a mentally disabled or intoxicated person 
( 12 ). 

The Study 

In 1998, the NYSDOH Zoonoses Program began an 
educational program to address the importance of bats in 
camp settings. This program was conducted in collabora- 
tion with the NYSDOH Center for Environmental Health 
(CEH), Bureau of Community Environmental Health and 
Food Protection (BCEHFP). NYSDOH offered training 
for all local and state health department camp inspectors 
responsible for inspecting camps before opening each 
season. Fact sheets on bats and bat-proofing camps and 
houses, bat capture kits, guidelines for managing bats, risk 
for rabies transmission (particularly in children’s camp set- 
tings), and guidance regarding human exposure to rabies 
and treatment decisions were provided. Starting in 1999, 
these materials included rabies awareness refrigerator 
magnets instructing people to contact health departments 
and not release bats when they are found in dwellings, and 
rabies awareness stickers for children to teach them not to 
touch bats (13). In 2003, -700 children’s camps received a 
videotape about keeping bats out of occupied dwellings 
and capturing bats for testing in exposure incidents. 

Children’s camp operators are required by New York 
State Public Health Law to obtain a permit, and camps 
must undergo inspection by the local health department. 
Associated regulations require camp operators to report 
certain camper injuries and illnesses within 24 hours of 
occurrence. Beginning in 1998, bat incidents were report- 
ed to the NYSDOH’s Zoonoses Program and to BCEHFP. 
In 1999, the Children’s Camp Bat Exposure Incident 
Report form was developed to standardize the reports. 
Twenty-three different types of incidents could be report- 
ed, 13 of which were considered probable rabies exposures 
requiring consideration of PEP. The form was revised in 
2000 to include additional information about the incidents, 
and in 2001 and 2002 the types of bat incidents reported 
were limited to the 13 types that require consideration of 


302 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


PEP if the bat is not tested and confirmed negative for 
rabies. These incidents include: bite; scratch; saliva or 
nervous tissue contact; direct physical contact with live or 
dead bat; person touched bat without seeing the part of bat 
touched; bat flew into person and touched person’s bare 
skin; bat flew into person and touched person’s lightweight 
clothing, and person reports feeling an unpleasant sensa- 
tion at the point of contact; person with bare feet stepped 
on bat; person awakens to find a bat in the room; live bat 
found in room with an unattended infant, child, or person 
with sensory or mental impairment; person slept in small, 
closed-in camp cabin, in which bats were swooping past 
sleeping person; bat found on ground near an unattended 
infant, child, or person with mental impairment; unidenti- 
fied flying object hits person and time of day (dusk or 
dawn), presence of mark where hit, and place where flying 
object came from (i.e., good site for roosting bats) all sup- 
port likelihood that it was a bat. The camps reported the bat 
incidents to the local health department or NYSDOH dis- 
trict offices, which submitted the incident report forms to 
BCEHFP; that bureau then forwarded the forms to the 
Zoonoses Program. Staff from the Zoonoses Program and 
Wadsworth Center taught local and district camp inspec- 
tors how to prevent human contact with bats, bat capture 
techniques, and methods of evacuating a building during 
an incident. 

Reported incidents and additional information from 3 
other reporting sources were added to the children’s camp 
database for the final analysis. Information included: 1) 
specimen history forms for camp-associated bats that were 
tested at the Rabies Laboratory; 2) the Zoonoses Program 
rabies exposure and PEP database established by a 
statewide reporting requirement; and 3) CEH’s environ- 
mental Health Information and Permitting System (eHIPS). 

From 1998 to 2002 during the summer camp season 
(June through August), 299 bat incidents were reported at 
109 of the estimated 2,600 NYS children’s camps, involv- 
ing 1,429 campers and staff (Table). The average and 
median ages of persons in bat incidents (based on the 
reported ages of 963 persons) were 14.8 and 13 years, 
respectively. During the 5-year period, 461 (32.2%) 
exposed persons (337 campers, 123 staff, 1 unknown sta- 
tus) received PEP (Figure 1). Forty-six persons refused 
PEP, and treatment status was unknown for 117. Over the 


Bat Incidents at Children’s Camps, New York 



Figure 1. Number of persons who refused, received, or avoided 
postexposure prophylaxis (PEP) in children’s camp bat incidents, 
New York State, 1998-2002. Treatment status was unknown (not 
reported to New York State Department of Health) for 117 persons: 
9 persons in 1998, 19 persons in 1999, 22 persons in 2000, 33 per- 
sons in 2001 , and 34 persons in 2002. PEP was avoided because 
the bats were captured and tested negative for rabies virus. 


5 -year period, bats were submitted for testing, and rabies 
was ruled out in 53.8% of the incidents. These test results 
prevented 805 (61.3%) exposed persons (567 campers, 
196 staff, 42 unknown status) from having PEP treatment. 
Of the 209 bats tested from 1998 to 2002, 4 bats collected 
in 2000 were rabid, and these incidents did not require any 
treatment for exposure. 

Four types of bat exposure reported most frequently 
accounted for 1,098 (77%) of persons in bat incidents at 
children’s camps (Figure 2). Exposure types were 
unknown for 69 of the incidents from 1998 to 2002. 
Specific exposure types (more than 1 type could be 
reported per incident) and numbers of persons exposed 
were sleeping where a bat was seen (797), sleeping where 
bats were swooping (205), direct physical contact with a 
bat (62), and a bat flying into them (36). The proportion 
of treatments prevented because of bats testing negative 
for rabies was 63%, 37%, 26%, and 11%, for these 4 
types of exposure, respectively. 

Conclusions 

From 1998 to 2002, almost 300 separate bat incidents 
involving ~ 1,500 children and staff at children’s camps in 
New York State were reported. Approximately one third of 


Table. Children’s camp bat incidents and number of persons reported, New York State, 1 998-2002* 



Bat incidents 

1998 

1999 

2000 

2001 

2002 

Total 

Reported incidents (June-August) 

45 

34 

74 

74 

72 

299 

No. of incidents with bat submitted for 
testing (%) 

19(42.2) 

5(14.7) 

44 (59.4) 

50 (67.5) 

43 (59.7) 

161 (53.8) 

No. of incidents with rabid bat 

0 

0 

4 

0 

0 

4 

No. of camps reporting incidents 

16 

21 

46 

42 

40 

109 

No. of persons in reported incidents 

334 

145 

386 

331 

233 

1,429 


*From 1998 to 2000, all bat incidents at children’s camps were requested for reporting. From 2001 to 2002, only bat incidents resulting in concern about 
potential rabies exposure were requested for reporting. 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


303 


DISPATCHES 



Figure 2. Number of persons exposed to bats by most frequently 
reported incident types, New York State, 1998-2002. Shown are 
the 4 most reported exposures of 23 reportable incidents of any 
type from 1998 to 2000, and of the 13 reportable exposure types 
from 2001 to 2002. Postexposure prophylaxis was avoided 
because the bats were captured and tested negative for rabies 
virus. 


these persons received PEP because the bats were not cap- 
tured and tested to rule out rabies. PEP treatment of -800 
persons was not necessary because the bats were captured 
and tested negative for rabies. 

At an estimated cost of $1,136 per PEP (2), this repre- 
sents healthcare cost savings >$900,000. This estimate 
underestimates the true cost savings of preventing 5 med- 
ical visits during a month for each treated person, trans- 
portation costs, coordinating and administering the 
treatments, opportunity and psychological costs of missing 
camp, and lost wages. 

Most of those involved in bat incidents were campers, 
which is not unexpected, as most camps have a higher 
number of campers than staff. Gender often depended on 
which camp was affected, as many camps are single sex. 
The 4 most common types of bat exposures requiring PEP 
are ones in which there is a reasonable probability that 
rabies exposure has occurred. The 2 most common types of 
incidents in which PEP was required (sleeping where a bat 
was seen or was swooping) are preventable by properly 
bat-proofing camp cabins. PEP can also be avoided with 
proper bat capture technique and cabin evacuation. In 1 
camp, after 5 incidents in a short period, PEP treatment 
was required in 42 cases. Education on bat-proofing and 
capture did not prevent 25 subsequent incidents in the 
same season but did result in bat capture and negative 
rabies test results in 24 of them, preventing 180 campers 
and staff members from receiving PEP treatment. 

Although only a few human rabies cases are diagnosed 
each year in the United States, inapparent or unreported bat 
bites appear to account for most of them (14). Equally 
important, bat exposures strongly affect healthcare costs 


when rabies cannot be ruled out by capturing and testing 
bats. Just as it is unacceptable for other wildlife to affect 
the health and safety of children at camp, keeping bats out 
of sleeping quarters and other buildings should be part of 
routine camp safety education, inspection, and certifica- 
tion programs. Although bats are part of the external camp 
environment, occupied buildings must be bat-proof. If 
exposures around or in camp buildings do occur, campers 
and staff must know how to avoid further exposures and 
how to capture the bat for rabies testing. Systems for 
reporting camp bat exposures and their consequences will 
identify this important public health problem and aid pub- 
lic health responses to reduce its impact. 

Acknowledgments 

We thank Timothy Shay, Felix Mrozek, and staff of the 
Center for Environmental Health’s Bureau of Community 
Environmental Health and Food Protection for reporting animal 
rabies cases and human exposures at children’s camps and for 
developing systems for surveillance and rabies control; Amy 
Willsey, Amy Schrom, Yoichiro Hagiwara, and staff of the 
Zoonoses Program, NYSDOH, for rabies treatment surveillance; 
Charles Trimarchi, Robert Rudd, Richard Raczkowski, and staff 
of the Rabies Laboratory, Wadsworth Center, NYSDOH, for lab- 
oratory testing; local, district, and regional health and environ- 
ment departments for rabies surveillance and education; human 
and animal healthcare providers for rabies and exposure report- 
ing; and staff from the children’s camps for incident reporting. 

Ms. Robbins’ research was supported through the Maternal 
and Child Health Graduate Assistant Program as funded by the 
HHS Maternal and Child Health Services Block Grant. 

Ms. Robbins is an MPH graduate of the University at 
Albany School of Public Health, currently working for the New 
Mexico Department of Health. She is the recipient of an applied 
epidemiology fellowship from the Council of State and 
Territorial Epidemiologists and the Centers for Disease Control 
and Prevention. Her research interest is infectious disease epi- 
demiology. 

References 

1. Plotkin SA. Rabies. Clin Infect Dis. 2000;30:4-12. 

2. Chang HH, Eidson M, Noonan-Toly C, Trimarchi CV, Rudd R, 
Wallace B, et al. Public health impact of reemergence of rabies, New 
York. Emerg Infect Dis. 2002;8:909-13. 

3. Krebs JW, Wheeling JT, Childs JE. Rabies surveillance in the United 
States during 2002. J Am Vet Med Assoc. 2003;223:1736-48. 

4. Centers for Disease Control. Human rabies — -New York, 1993. 
MMWR Morb Mortal Wkly Rep. 1993;42:805-6. 

5. Feder HM, Nelson R, Reiher HW. Bat bite? Lancet. 1997;350:1300. 

6. Centers for Disease Control. Human rabies — Washington, 1995. 
MMWR Morb Mortal Wkly Rep. 1995;5:433-7. 

7. Centers for Disease Control. Human rabies — Connecticut, 1995. 
MMWR Morb Mortal Wkly Rep. 1996;45:207-9. 


304 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


Bat Incidents at Children’s Camps, New York 


8. Centers for Disease Control. Human Rabies — Tennessee, 2002. 
MMWR Morb Mortal Wkly Rep. 2002;51:828-9. 

9. Centers for Disease Control. Human rabies — Quebec, Canada, 2000. 
MMWR Morb Mortal Wkly Rep. 2000;49: 1 1 15. 

10. Human case of rabies — British Columbia. Can Commun Dis Rep. 
1985; 1 1:213. 

11. Rudd R. 2002 Rabies annual summary, Wadsworth Center Rabies 
Laboratory, New York State Department of Health, [cited Jul 24, 
2003] . Available at http://www.wadsworth.org/rabies/2002/index.htm 

12. Centers for Disease Control and Prevention. Human rabies preven- 
tion — United States, 1999; recommendations of the Advisory 
Committee on Immunization Practices (ACIP). MMWR Morb Mortal 
Wkly Rep. 1999;48(RR-1):1-21. 


13. Eidson M, Schmit K, Keegan M, Trimarchi CV, Tserenpuntsag B, 
Willsey A. Development and evaluation of bat rabies education mate- 
rials. Evidence-Based Preventive Medicine. 2004;1:85-91. 

14. Gibbons RV. Cryptogenic rabies, bats, and the question of aerosol 
transmission. Ann Emerg Med. 2002;39:528-36. 


Address for correspondence: Millicent Eidson, New York State 
Department of Health, Corning Tower, Room 621, Albany, NY 12237, 
USA; fax: 518-473-6590; email: mxe04@health.state.ny.us 


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305 



DISPATCHES 


West Nile Virus in 
Morocco, 2003 

Isabelle Schuffenecker,* 

Christophe N. Peyrefitte,t 
Mohammed el Harrak,± Severine Murri,* 
Agnes Leblond,§ and Herve G. Zeller* 

West Nile virus (WNV) reemerged in Morocco in 
September 2003, causing an equine outbreak. A WNV 
strain isolated from a brain biopsy was completely 
sequenced. On the basis of phylogenetic analyses, 
Moroccan WNV strains isolated during the 1996 and 2003 
outbreaks were closely related to other strains responsible 
for equine outbreaks in the western Mediterranean basin. 

I n the early 1950s, scientists first recognized that West 
Nile vims (WNV) reached outbreak levels in humans in 
Egypt and Israel (1,2). Initially considered a minor 
arbovirus, WNV has recently emerged as a major public 
health and veterinary concern in southern Europe, the 
Mediterranean basin, and the United States and Canada 
(1-3). Several outbreaks of severe human meningoen- 
cephalitis with fatalities have been reported within the last 
8 years in Europe and North Africa, specifically in 
Romania (1996), Russia (1999), Israel (2000), and Tunisia 
(1997, 2003) (1,3). Epizootics in horses have also been 
documented in Morocco (1996), Italy (1998), France 
(2000), and Israel (2000) (1,4). WNV was responsible for 
a cluster of human and equine cases in southern France in 
2003 (5,6). 

On the basis of phylogenetic analyses, WNV strains 
isolated since 1996 in southern Europe and the 
Mediterranean basin belong to the clade la of lineage 1 
(7,8). Moreover, these strains belong to 2 distinct geno- 
types (8,9). One cluster includes equine strains isolated in 
Italy and France, human strains isolated in Russia and 
Israel, and mosquito strains isolated in Romania and 
Kenya. The other cluster includes most of the strains iso- 
lated from birds and horses in Israel from 1997 to 2001 
and the North American isolates. Only 5 strains isolated 
in the Mediterranean basin have been completely 
sequenced. 

Since the first WNV outbreak in Morocco in 1996, 
which caused 94 equine cases (including 42 deaths) and 1 
human case (10), no WNV infections have been reported. 
An outbreak of WNV occurred among horses stabled in 


*lnstitut Pasteur, Lyon, France; flnstitut de Medecine Tropicale du 
Service de Sante des Armees, Marseille, France; fLaboratoire 
Bio-Pharma, Rabat, Morocco; and §Ecole Veterinaire de Lyon, 
Marcy I’Etoile, France 


the Moroccan province of Kenitra in September and 
October 2003. The complete genome sequence of a WNV 
strain isolated from a brain biopsy was characterized, as 
well as the complete genome sequence of a strain isolated 
during the Morocco 1996 WNV outbreak. We studied phy- 
logenetic relationships of the 2 Moroccan strains with 
other WNV strains isolated in the Mediterranean basin. 

The Study 

During the fall of 2003, 9 equine WNV cases were 
reported to the Moroccan Ministry of Agriculture. All 
horses had acute neurologic symptoms, fever, paresis of 
the hindquarters, paralysis, or some combination of these 
symptoms (Table); 5 horses were euthanized. Clinical 
cases occurred from September 12 to October 1, 2003. No 
abnormal bird deaths were observed, and no human cases 
were reported. 

Equine clinical cases were reported from 3 locations 
~20 to 30 km northeast of Kenitra (34°18'N, 06°30'W), 
close to the Sebou River delta and the Atlantic Ocean 
(Figure 1). Irrigation networks are developed in this farm- 
ing area. In addition, a natural bird reserve, Sidi Boughaba, 
is located 15 km southeast of Kenitra, along one of the 
migratory Europe-sub- Saharan routes, where numerous 
migrating and breeding birds are found. 

Virus isolation was performed from a brain biopsy in 
the BioPharma laboratory in Rabat, Morocco. Brain sus- 
pension was injected onto BSR cells. Cytopathic effect 
was observed 4 days after infection. WNV was identified 
by immunofluorescence assay and confirmed by reverse 
transcription-polymerase chain reaction (RT-PCR). The 
complete WNV genome was sequenced in the National 
Reference Center for Arboviruses in Lyon, France, after a 
single passage of the strain (04.05) on Vero E6 cells. 
Twenty-five overlapping amplicons were amplified and 
sequenced on both strands (AY701413). The complete 
sequence of the strain 96-111 isolated during the 1996 
Moroccan equine outbreak was also determined 
(AY701412). 

Pairwise alignments of 96-111 and 04.05 sequences 
using ClustalW1.7 software (11) showed a 98.9% 
nucleotide identity and a 99.8% amino-acid identity 
between the 2 Moroccan isolates. Six amino-acid differ- 
ences were observed between the 2 strains: 1 in the E gene 
(17 32V), 2 in the NS1 gene (V979I and R1079S), 1 in the 
NS2a gene (H1262Y), and 2 in the NS3 gene (F1551L and 
A1754T). 

Multiple alignments of Moroccan WNV sequences and 
other WNV sequences available in GenBank database 
were generated by ClustalW1.7 software. Phylogenetic 
trees were constructed by using nucleotide alignments, the 
Jukes Cantor algorithm, and the neighbor-joining method 
implemented in molecular evolutionary genetics analysis 


306 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


West Nile Virus in Morocco 


Table. Clinical and epidemiologic features of 9 horses with confirmed West Nile Virus (WNV) infection, Kenitra Province, Morocco* 


Locality 

Age (y) 

Sex 

Date of symptoms 

Clinical data 

Death 

Ouled Slama 

5 

M 

Sep 1 2, 2003 

Paralysis 

- 

Ameur Seflia 

7 

M 

Sep 1 1 , 2003 

Paralysis, fever 

+ 

? 

8 

F 

Sep 07, 2003 

Paralysis 

+ 

Mograne 

7 

F 

Sep 1 4, 2003 

Paralysis 

+ 

Ameur Seflia 

6 

M 

Sep 1 9, 2003 

Ataxia, fever 

- 

Ameur Seflia 

7 

M 

Sep 23, 2003 

Ataxia 

- 

Mograne 

6 

M 

Sep 29, 2003 

Paresis, fever 

+ 

Mograne 

10 

F 

Oct 01 , 2003 

Paresis, fever 

+t 

Ameur Seflia 

10 

F 

? 

Paresis, fever 

- 

*(y), years of age; M, male; F, female. 
fSource of the Morocco 2003 WNV strain (04.05). 






(MEGA) software (12). The robustness of branching pat- 
terns was tested by 1,000 bootstrap pseudoreplications. 

Comparison of the complete genome sequences 
showed a high degree of identity between the Moroccan 
strains and those of the European/Mediterranean/Kenyan 
cluster. Paired identity at the nucleotide level ranged from 
98.2% to 98.9% and from 98.6% to 99% for 04.05 and 96- 
111 strains, respectively. Paired nucleotide identity with 
strains of the Israeli/American cluster ranged from 96.2% 
to 96.3% and 96.5% to 96.6%, respectively. The 5 amino- 
acid residues characteristic of the European/ 
Mediterranean/Kenyan genotype were conserved in both 
Moroccan strains, i.e., T416 (E protein), S861 (NS1 pro- 
tein), 11861 (NS3 protein), V2209 (NS4a protein), and 
D2522 (NS4b protein). 

On the basis of the complete genome sequences, phylo- 
genetic data showed that both Moroccan strains belonged 
to the clade la of the lineage 1 and clustered with the 
strains of the European/Mediterranean/Kenyan cluster 
(Figure 2A). On the basis of the envelope sequences, 
equine WNV strains isolated in the Mediterranean basin 
from 1996 to 2003 belonged to 2 distinct clusters, i.e., the 
European/Mediterranean/Kenyan cluster and the 
American/Israeli cluster (Figure 2B). The Moroccan 
equine strains clustered with the Italian and French equine 
strains. They were more distantly related to the 3 equine 
strains isolated in Israel in 2000. 

Conclusions 

WNV has been circulating in the Mediterranean basin 
for a long time (1-3); in the western part of the basin, only 
a few isolates have been obtained and completely 
sequenced. We report here the isolation and complete 
genome characterization of 2 WNV strains involved in 
equine outbreaks in Morocco in 1996 and more recently in 
2003. 

During the late summer of 2003, an equine outbreak 
was reported in Morocco. By contrast with the 1996 out- 
break (10), the epidemic was restricted geographically and 
temporally. Climatic and vectorial conditions might have 
been insufficient to lead to a major transmission of the 


virus. No young horses were clinically affected, probably 
because of the structure of the equine population in Kenitra 
Province, where most horses are bought at the age of 4 or 
5 years and the mean age of the equine population is 10 
years. 

High pairwise nucleotide and amino-acid identity val- 
ues indicated that the Morocco 1996 and 2003 WNV 
strains are closely related. Determining if both outbreaks 
were related to an endemic strain or to distinct introduction 
events by migratory birds was not possible. Since 1996, 
WNV-positive serologic results have been found every 
year in horses with neurologic signs, which suggests 
endemic circulation of the virus (M. el Harrak, unpub. 
data.). 

On the basis of envelope and complete genome 
sequences, we demonstrated that both Moroccan strains 
belonged to the European/Mediterranean/Kenyan cluster 
previously defined (8). The characterization of 2 new com- 
plete WNV genome sequences allowed us to demonstrate 
the genetic stability of the WNV strains involved in the 
equine outbreaks reported since 1996 in the western part of 
the Mediterranean basin. Our data also suggested the 



Figure 1. Map showing Kenitra Province, where equine clinical 
cases occurred in 2003. (adapted from the Internet site http:www. 
morocco.com/travel/map03.html) 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


307 


DISPATCHES 



Figure 2. Phylogenetic trees of West Nile 
Virus (WNV) nucleotide sequences. 
Phylograms were constructed with the 
MEGA program, by using the Jukes Cantor 
algorithm and the neighbor-joining 
method. The percentage of successful 
bootstrap replicates is indicated at nodes. 
The length of branches is proportional to 
the number of nucleotide changes (% of 
divergence). The strains sequenced in this 
study are indicated by asterisks (*). A) 
Complete nucleotide sequence GenBank 
accession no. are Italy 1998 (AF404757), 
France 2000 (AY268132), Kenya 1998 
(AY262283), Romania 1996 (AF260969), 
Volgograd 1999 (VLG4) (AF317203), 
Volgograd 1999 (27889) (AY277252), 
Volgograd 2000 (AY278442), Tunisia 1997 
(AY2681 33), Israel 1998 (AF481864), NY 
1999 H (AF202541 ), NY 1999 EQ 
(AF260967), Egypt 1951 (AF260968), 
Kunjin 1960 (D00246), Uganda 1937 
(Ml 2294). B) Envelope nucleotide 
sequence Genbank accession no. are Italy 
1998 (AF404757), France 2000 

(AY2681 32), Israel 2000 H (AF394217), 
Kenya 1998 (AY262283), Romania 1996 
(AF260969), Volgograd 1999 (VLG4) 
(AF317203), Volgograd 1999 (27889) 
(AY277252), Volgograd 2000 (AY278442), 
Tunisia 1997 (AY268133), Israel 1998 
(AF481864), Mexico 2003 (AY426741) 




existence of 2 subclusters of WNV strains in the 
European/Mediterranean/Kenyan cluster. One subcluster 
includes strains isolated in the western Mediterranean 
basin (France, Italy, Morocco) that have probably been 
introduced from West Africa. The other subcluster includes 
strains isolated in the eastern Mediterranean basin (Israel) 
and southeastern Europe (Romania, Volgograd) that have 
probably been introduced from East Africa. The molecular 
epidemiologic features of the strains in the Mediterranean 
basin appear to be more complex. Since 1997, at least 2 
lineages cocirculate in Israel, i.e., the European/ 
Mediterranean/Kenyan lineage and the more recent 
Israeli/ American lineage in birds, equines, and humans 
(9,13). Strains of the latter genotype were imported in 
1999, probably through infected birds or mosquitos, from 
the Middle East to North America, causing high rates of 
avian deaths and high rates of illness and deaths in humans 
and equines. In Israel, the emergence of the 
Israeli/American genotype has also been associated with 
avian deaths. Whether the introduction of this genotype is 
associated with the high rates of illness and death during 
the 2000 human outbreak is unclear. Five amino- acid 
residues are known to distinguish the European/ 
Mediterranean/Kenyan and the Israeli/American geno- 


types (7,8). In the future, testing the role of those specific 
residues and comparing the biologic properties of strains 
of both genotypes will be useful, knowing that only 
Israeli/American strains are responsible for avian deaths 
(14) and probable increased neuro virulence (15). No virus- 
es of the Israeli/American genotype have been isolated 
elsewhere in the Mediterranean basin or in Europe. 

During the Morocco 2003 outbreak, WNV reemerged 
in southern France, causing 7 human and 4 equine cases, 
and in Tunisia, causing approximately 200 human cases in 
Monastir province (H. Trikki, pers. comm.). No virus iso- 
lation or genome amplification was obtained or reported. 
Knowing the possibility of transmission through blood 
donations, surveillance of WNV infections must be 
enhanced in the Mediterranean basin. For the moment, in 
contrast with the situation in North America, human and 
equine outbreaks have been restricted geographically and 
temporally (3). The mechanisms of WNV reintroduction in 
Europe and in the Mediterranean basin and the cycle of 
maintenance in infected areas remain to be elucidated. 
Further studies should focus on competence of mosquito 
vectors, identifying bird species involved in the cycle of 
transmission, and the persistence mechanisms of the virus 
in WNV-endemic areas. 


308 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


West Nile Virus in Morocco 


Acknowledgments 

We thank Philippe Marianneau for fruitful discussions. 

Dr. Schuffenecker is a biologist working for the French 
National Reference Center for Arboviruses. She is involved in the 
diagnosis and epidemiology of vector-borne diseases. 

References 

1. Murgue B, Zeller H, Deubel V. West Nile in the Mediterranean basin: 
1950-2000. Ann NY Acad Sci. 2001;951:117-26. 

2. Hayes CG. West Nile Vims: Uganda, 1937, to New York City, 1999. 
Ann NY Acad Sci. 2001;951:25-37. 

3. Zeller HG, Schuffenecker I. West Nile virus: an overview of its spread 
in Europe and the Mediterranean basin in contrast to its spread in the 
Americas. Eur J Clin Microbiol Infect Dis. 2004;23:147-56. 

4. Steinman A, Banet C, Sutton GA, Yadin H, Hadar S, Brill A. Clinical 
signs of West Nile virus encephalomyelitis in horses during the out- 
break in Israel in 2000. Vet Rec. 2002;151:47-9. 

5. Mailles A, Dellamonica P, Zeller H, Durand JP, Zientara S, Goffette 
R, et al. Human and equine infections in France, August-September 
2003. Eurosurveillance Weekly. 2003 ;7: 10/23/2003. Available from 
http:www.eurosurveillance.org/ew/2003/031023.asp 

6. Del Giudice P, Schuffenecker I, Vandenbos F, Counillon E, Zeller H. 
Human West Nile virus, France [letter]. Emerg Infect Dis. 
2004;10:1885-6. 

7. Lanciotti RS, Ebel GD, Deubel V, Kerst AJ, Murri S, Meyer R, et al. 
Complete genome sequences and phylogenetic analysis of West Nile 
virus strains isolated from the United States, Europe, and the Middle 
East. Virology. 2002;298:96-105. 


8. Charrel RN, Brault AC, Gallian P, Lemasson J-J, Murgue B, Murri S, 
et al. Evolutionary relationship between Old World West Nile virus 
strains. Evidence for viral gene flow between Africa, the Middle East, 
and Europe. Virology. 2003;315:381-8. 

9. Banet-Noach C, Malkinson M, Brill A, Samina I, Yadin H, Weisman 
Y, et al. Phylogenetic relationships of West Nile vimses isolated from 
birds and horses in Israel from 1997 to 2001. Virus Genes. 
2003;26:135-41. 

10. Tber Abdelhaq A. West Nile fever in horses in Morocco. Bull Off Int 
Epizoot. 1996;11:867-9. 

1 1 . Thompson JD, Higgins DG, Gibson TJ. Clustal W: improving the sen- 
sitivity of progressive multiple sequence alignments through 
sequence weighting, position specific gap penalties and weight 
matrix choice. Nucleic Acids Res. 1994;22:4673-80. 

12. Kumar S, Tamura K, Nei M. MEGA: molecular evolutionary genet- 
ics analysis software for microcomputers. Comput Appl Biosci. 
1994;10:189-91. 

13. Hindiyeh M, Shulman LM, Mendelson E, Weiss L, Grossman Z, Bin 
H. Isolation and characterization of West Nile vims from the blood of 
viremic patients during the 2000 outbreak in Israel. Emerg Infect Dis. 
2001;7:748-50. 

14. Komar N, Langevin S, Hinten S, Nemeth N, Edwards E, Hettler D, et 
al. Experimental infection of North American birds with the New 
York 1999 strain of West Nile virus. Emerg Infect Dis. 
2003;9:311-22. 

15. Ceccaldi P-E, Lucas M, Despres P. New insights on the neuropatho- 
genicity of West Nile virus. FEMS Microbiol Lett. 2004;233:1-6. 

Address for correspondence: Isabelle Schuffenecker, Centre de Reference 

des Arbovirus, Institut Pasteur, 21 Ave Tony Gamier, 69365 Lyon cedex 

07, France; fax: 33-4-37-28-24-51; email: schuffenecker @cervi- 

lyon.inserm.fr 



Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


309 



DISPATCHES 


Diagnostic System 
for Rapid and 
Sensitive 
Differential 
Detection of 
Pathogens 

Thomas Briese,* 1 Gustavo Palacios,* 1 Mark 
Kokoris^ 1 Omar Jabado,* Zhiqiang Liu,* Neil 
Renwick,* Vishal Kapoor,* Inmaculada Casas4 
Francisco Pozo4 Ron Limberger,§ Pilar Perez- 
Brena4 Jingyue Ju,* and W. Ian Lipkin* 

Naturally emerging and deliberately released 
pathogens demand new detection strategies to allow early 
recognition and containment. We describe a diagnostic 
system for rapid, sensitive, multiplex discrimination of 
microbial gene sequences and report its application for 
detecting 22 respiratory pathogens in clinical samples. 

E fficient laboratory diagnosis of infectious diseases is 
increasingly important to clinical management and 
public health. Methods to directly detect nucleic acids of 
microbial pathogens in clinical specimens are rapid, sen- 
sitive, and may succeed when culturing the organism fails. 
Clinical syndromes are infrequently specific for single 
pathogens; thus, assays are needed that allow multiple 
agents to be simultaneously considered. Current multiplex 
assays employ gel-based formats in which products are 
distinguished by size, fluorescent reporter dyes that vary 
in color, or secondary enzyme hybridization assays. Gel- 
based assays are reported that detect 2-8 different targets 
with sensitivities of 2-100 PFU or <1-5 PFU, depending 
on whether amplification is carried out in a single or nest- 
ed format, respectively (1-4). Fluorescence reporter sys- 
tems achieve quantitative detection with sensitivity 
similar to that of nested amplification; however, their 
capacity to simultaneously query multiple targets is limit- 
ed to the number of fluorescent emission peaks that can be 
unequivocally resolved. At present, up to 4 fluorescent 
reporter dyes can be detected simultaneously (5,6). 
Multiplex detection of up to 9 pathogens has been 
achieved in hybridization enzyme systems; however, the 


*Columbia University, New York, New York, USA; fQiagen Inc., 
Valencia, California, USA; flnstituto de Salud Carlos III, 
Majadahonda, Madrid, Spain; and §New York State Department of 
Health, Albany, New York, USA 


method requires cumbersome postamplification process- 
ing (7). 

The Study 

To address the need for sensitive multiplex assays in 
diagnostic molecular microbiology, we created a poly- 
merase chain reaction (PCR) platform in which microbial 
gene targets are coded by a library of 64 distinct Masscode 
tags (Qiagen Masscode technology, Qiagen, Hilden, 
Germany). A schematic representation of this approach is 
shown in Figure 1. Microbial nucleic acids (RNA, DNA, 
or both) are amplified by multiplex reverse transcription 
(RT)-PCR using primers labeled by a photocleavable link 
to molecular tags of different molecular weight. After 
removing unincorporated primers, tags are released by UV 
irradiation and analyzed by mass spectrometry. The identi- 
ty of the microbe in the clinical sample is determined by its 
cognate tags. 

As a first test of this technology, we focused on respi- 
ratory disease because differential diagnosis is a common 
clinical challenge, with implications for outbreak control 
and individual case management. Multiplex primer sets 
were designed to identify up to 22 respiratory pathogens in 
a single Mass Tag PCR reaction; sensitivity was estab- 
lished by using synthetic DNA and RNA standards as well 
as titered viral stocks; the utility of Mass Tag PCR was 
determined in blinded analysis of previously diagnosed 
clinical specimens. 

Oligonucleotide primers were designed in conserved 
genomic regions to detect the broadest number of members 
for a given pathogen species by efficiently amplifying a 
50- to 300-bp product. In some instances, we selected 
established primer sets; in others, we used a software pro- 
gram designed to cull sequence information from 
GenBank, perform multiple alignments, and maximize 
multiplex performance by selecting primers with uniform 
melting temperatures and minimal cross -hybridization 
potential (Appendix Table, available at http://www.cdc. 
gov/ncidod/eid/volllno02/04-0492_app.htm). Primers, 
synthesized with a 5' C6 spacer and aminohexyl modifica- 
tion, were covalently conjugated by a photocleavable link 
to Masscode tags (Qiagen Masscode technology) (8,9). 
Masscode tags have a modular structure, including a tetra- 
fluorophenyl ester for tag conjugation to primary amines; 
an o-nitrobenzyl photolabile linker for photoredox cleav- 
age of the tag from the analyte; a mass spectrometry sensi- 
tivity enhancer, which improves the efficiency of 
atmospheric pressure chemical ionization of the cleaved 
tag; and a variable mass unit for variation of the cleaved 
tag mass (8,10-12). A library of 64 different tags has been 
established. Forward and reverse primers in individual 


iThese authors contributed equally to this study. 


310 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


Differential Detection of Multiple Respiratory Pathogens 


1. PCR amplification with Mass Tag primers 4. Automated sample injection, phdocieavage 




96‘WrtU thwnoc^ii*' piste 


I 

2 . Product purilication cn filler plale 



3. Eiulion mio 96-well loading ppale 
for mares spectrometer analysis 



5 Detection and pathogen identificalion 


i 

* 1 

i 

JkLliiiii 

dJL 

JiL 


Mss* 


Figure 1 . Schematic representation of Mass Tag polymerase chain 
reaction (PCR). 


primer sets are labeled with distinct molecular weight tags. 
Thus, amplification of a microbial gene target produces a 
dual signal that allows assessment of specificity. 

Gene target standards were cloned by PCR into 
pCR2.1-TOPO (Invitrogen, Carlsbad, CA, USA) by using 
DNA template (bacterial and DNA viral targets) or cDNA 
template (RNA viral targets) obtained by reverse transcrip- 
tion of extracts from infected cultured cells or by assembly 
of overlapping synthetic polynucleotides. Assays were ini- 
tially established by using plasmid standards diluted in 
2.5-qg/mL human placenta DNA (Sigma, St. Louis, MO, 
USA) and subjected to PCR amplification with a multiplex 
PCR kit (Qiagen), primers at 0.5 |imol/L each, and the fol- 
lowing cycling protocol: an annealing step with a temper- 
ature reduction in 1°C increments from 65 °C to 51°C 
during the first 15 cycles and then continuing with a 
cycling profile of 94°C for 20 s, 50°C for 20 s, and 72°C 
for 30 s in an MJ PTC200 thermal cycler (MJ Research, 
Waltham, MA, USA). Amplification products were sepa- 
rated from unused primers by using QIAquick 96 PCR 
purification cartridges (Qiagen, with modified binding and 
wash buffers). Masscode tags were decoupled from ampli- 
fied products through UV light-induced photolysis in a 
flow cell and analyzed in a single quadrapole mass spec- 
trometer using positive-mode atmospheric pressure chem- 
ical ionization (Agilent Technologies, Palo Alto, CA, 
USA). A detection threshold of 100 DNA copies was deter- 
mined for 19 of 22 cloned targets by using a 22-plex assay 
(Table 1). 

Many respiratory pathogens have RNA genomes; thus, 
where indicated, assay sensitivity was determined by using 
synthetic RNA standards or RNA extracts of viral stocks. 
Synthetic RNA standards were generated by using T7 
polymerase and linearized plasmid DNA. After quantita- 


tion by UV spectrometry, RNA was serially diluted in 2.5- 
qg/mL yeast tRNA (Sigma), reverse transcribed with ran- 
dom hexamers by using Superscript II (Invitrogen, 
Carlsbad, CA, USA), and used as template for Mass Tag 
PCR. As anticipated, sensitivity was reduced by the use of 
RNA instead of DNA templates (Table 1). The sensitivity 
of Mass Tag PCR to detect live vims was tested by using 
RNA extracted from serial dilutions of titered stocks of 
coronaviruses (severe acute respiratory syndrome [SARS] 
and OC43) and parainfluenzaviruses (HPIV 2 and 3). A 
100-qL volume of each dilution was analyzed. RNA 
extracted from a l-TCID 50 /mL dilution, representing 0.025 
TCID 50 per PCR reaction, was consistently positive in 
Mass Tag PCR. 

RNA extracted from banked sputum, nasal swabs, and 
pulmonary washes of persons with respiratory infection 
was tested by using an assay panel comprising 30 gene tar- 
gets that represented 22 respiratory pathogens. Infection in 
each of these persons had been previously diagnosed 
through vims isolation, conventional nested RT-PCR, or 
both. Reverse transcription was performed using random 
hexamers, and Mass Tag PCR results were consistent in all 
cases with the established diagnosis. Infections with respi- 
ratory syncytial vims, human parainfluenza vims, SARS 
coronavirus, adenovirus, enterovirus, metapneumovims, 
and influenza vims were correctly identified (Table 2 and 
Figure 2). A panel comprising gene targets representing 17 


Table 1 . Sensitivity of pathogen detection by Mass Tag 
polymerase chain reaction determined by using plasmid and 
synthetic RNA standards* 

Pathogen or protein 

Detection threshold 
(DNA copies/RNA copies) 

Influenza A matrix 

100/1,000 

Influenza A N1 

100/NA 

Influenza A N2 

100/NA 

Influenza A HI 

100/NA 

Influenza A H2 

100/NA 

Influenza A H3 

100/NA 

Influenza A H5 

100/NA 

Influenza B H 

500/1 ,000 

RSV group A 

100/1,000 

RSV group B 

100/500 

Metapneumovirus 

100/1,000 

CoV-SARS 

100/500 

CoV-OC43 

100/500 

CoV-229E 

100/500 

HPIV-1 

100/1,000 

HPIV-2 

100/1,000 

HPIV-3 

100/500 

Chlamydia pneumoniae 

100/NA 

Mycoplasma pneumoniae 

100/NA 

Legionella pneumophila 

100/NA 

Enterovirus (genus) 

500/1 ,000 

Adenovirus (genus) 

5,000/NA 


*NA, not assessed; RSV, respiratory syncytial virus; CoV, coronavirus; 
SARS, severe acute respiratory syndrome; HPIV, human parainfluenza 
virus. 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


311 


DISPATCHES 


Table 2. Multiplex pathogen detection by Mass Tag polymerase 
chain reaction using Masscode-labeled primers in a 30-plex 
assay with clinical specimens with previously identified 
pathogens* 


Pathogen 

No. positive/no. testedf 

RSV A 

2/2 

RSV B 

3/3 

HPIV-1 

1/1 

HPIV-3 

2/2 

HPIV-4 

2/2 

CoV-SARS 

4/4 

Metapneumovirus 

2/3 

Influenza B 

1/3 

Influenza A 

2/6 

Adenovirus 

2/2 

Enterovirus 

2/2 

*RSV, respiratory syncytial virus; HPIV, human parainfluenza virus; CoV, 
coronavirus; SARS, severe acute respiratory syndrome. 
fNo. positive and consistent with previous diagnosis/number tested (with 
respective previous diagnosis). 


pathogens related to central nervous system infectious dis- 
ease (influenza A virus matrix gene; influenza B virus; 
human coronaviruses 229E, OC43, and SARS; 

enterovirus; adenovirus; human herpesvirus- 1 and -3; West 
Nile virus; St. Louis encephalitis virus; measles virus; 
HIV-1 and -2; and Streptococcus pneumoniae , Haemo- 
philus influenzae , and Nesseria meningitidis) was applied 


to RNA obtained from banked samples of cerebrospinal 
fluid and brain tissue that had been previously character- 
ized by conventional diagnostic RT-PCR. Two of 3 cases 
of West Nile virus encephalitis were correctly identified. 
Eleven of 12 cases of entero viral meningitis were detected 
representing serotypes CV-B2, CV-B3, CV-B5, E-6, E-ll, 
E-13, E-18, and E-30 (data not shown). 

Conclusions 

Our results indicate that Mass Tag PCR is a sensitive 
and specific tool for molecular characterization of 
microflora. The advantage of Mass Tag PCR is its capaci- 
ty for multiplex analysis. Although the use of degenerate 
primers (e.g., enteroviruses and adenoviruses, Appendix 
Table and Table 1) may reduce sensitivity, the limit of mul- 
tiplexing to detect specific targets will likely be defined by 
the maximal primer concentration that can be accommo- 
dated in a PCR mix. Analysis requires the purification of 
product from unincorporated primers and mass spec- 
troscopy. Although these steps are now performed manual- 
ly, and mass spectrometers are not yet widely distributed in 
clinical laboratories, the increasing popularity of mass 
spectrometry in biomedical sciences and the advent of 
smaller, lower-cost instruments could facilitate wider use 
and integrated instrumentation. In addition to developing 



Figure 2. Analysis of clinical specimens. RNA 
extracts from clinical specimens containing known 
pathogens were reverse transcribed into cDNA 
(Superscript RT system, Invitrogen, Carlsbad, CA; 
20- L volume). Five microliters of the reaction 
were subjected to Mass Tag PCR by using 
primers coupled to Masscode tags (Qiagen 
Masscode technology, Qiagen, Hilden, Germany). 
Detection of (A) influenza virus A (HI N1 ), (B) res- 
piratory syncytial virus (RSV) group B, (C) human 
coronavirus SARS (HCoV-SARS), (D) human 
parainfluenza virus (HPIV) types 1 and (E) 3, and 
(F) enterovirus (EV) by using a 30-plex assay, 
including 60 primers targeting influenza A virus 
matrix gene (FLUAV-M), and for typing N1, N2, 
HI, H2, H3, and H5 sequences, as well as 
influenza B virus (FLUBV), RSV groups A and B, 
HCoV-229E, -OC43, and -SARS, HPIV types 1 , 2, 
3, and 4 (groups A and B combined; 4 primers), 
human metapneumovirus (HMPV, 4 primers), 
measles virus (MEV), EV (degenerate primer pair 
targeting all serogroups), human adenoviruses 
(HAdV, degenerate primer pair targeting all 
serogroups), human herpesvirus 1 (HHV-1, her- 
pes simplex virus), human herpesvirus 3 (HHV-3; 
varicella-zoster virus), Mycoplasma pneumoniae, 
Chlamydia pneumoniae, Legionella pneumo- 
phila, Streptococcus pneumoniae, Haemophilus 
influenzae. Y-axis values indicate signal to noise 
ratio. The bar indicates an arbitrary cut-off thres- 
hold of 2.7 (4 times average background deter- 
mined with random human DNA). 


312 


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Differential Detection of Multiple Respiratory Pathogens 


additional pathogen panels, our continuing work is focused 
on optimizing multiplexing, sensitivity, and throughput. 
Potential applications include differential diagnosis of 
infectious diseases, blood product surveillance, forensic 
microbiology, and biodefense. 

Acknowledgments 

We are grateful to Cinnia Huang, Jill Taylor, and Tony 
Mazzulli for providing sample materials with previously identi- 
fied pathogens for analysis. 

This work was supported by National Institutes of Health 
awards AI51292, AI056118, AI55466, U54AI057158 (Northeast 
Biodefense Center-Lipkin) and the Ellison Medical Foundation. 
M. Kokoris was a consultant for Qiagen GmbH while the work 
reported in this manuscript was pursued. He is currently a con- 
sultant for Operon Biotechnologies, Inc., which holds the rights 
on Masscode technology. 

Dr. Briese is associate professor of epidemiology at the 
Columbia University Mailman School of Public Health and asso- 
ciate director of the Jerome L. and Dawn Greene Infectious 
Disease Laboratory. His research interests include the molecular 
epidemiology of emerging viral diseases, virus-host interactions, 
and novel techniques for pathogen detection and discovery. 

References 

1 . Fan J, Henrickson KJ, Savatski LL. Rapid simultaneous diagnosis of 
infections with respiratory syncytial viruses A and B, influenza virus- 
es A and B, and human parainfluenza virus types 1, 2, and 3 by mul- 
tiplex quantitative reverse transcription-polymerase chain 
reaction-enzyme hybridization assay (Hexaplex). Clin Infect Dis. 
1998;26:1397-402. 

2. Stockton J, Ellis JS, Saville M, Clewley JP, Zambon MC. Multiplex 
PCR for typing and subtyping influenza and respiratory syncytial 
viruses. J Clin Microbiol. 1998;36:2990-5. 


3. Ellis JS, Zambon MC. Molecular diagnosis of influenza. Rev Med 
Virol. 2002;12:375-89. 

4. Coiras MT, Aguilar JC, Garcia ML, Casas I, Perez-Brena P. 
Simultaneous detection of fourteen respiratory viruses in clinical 
specimens by two multiplex reverse transcription nested-PCR assays. 
J Med Virol. 2004;72:484-95. 

5. Vet JA, Majithia AR, Marras SA, Tyagi S, Dube S, Poiesz BJ, et al. 
Multiplex detection of four pathogenic retroviruses using molecular 
beacons. Proc Natl Acad Sci USA. 1999;96:6394-9. 

6. Verweij JJ, Blange RA, Templeton K, Schinkel J, Brienen EA, van 
Rooyen MA, et al. Simultaneous detection of Entamoeba histolytica, 
Giardia lamblia, and Cryptosporidium parvum in fecal samples by 
using multiplex real-time PCR. J Clin Microbiol. 2004;42:1220-3. 

7. Grondahl B, Puppe W, Hoppe A, Kuhne I, Weigl JA, Schmitt HJ. 
Rapid identification of nine microorganisms causing acute respirato- 
ry tract infections by single-tube multiplex reverse transcription- 
PCR: feasibility study. J Clin Microbiol. 1999;37:1-7. 

8. Kokoris M, Dix K, Moynihan K, Mathis J, Erwin B, Grass P, et al. 
High-throughput SNP genotyping with the Masscode system. Mol 
Diagn. 2000;5:329-40. 

9. Lukhtanov EA, Kutyavin IV, Gamper HB, Meyer RB Jr. 
Oligodeoxyribonucleotides with conjugated dihydropyrroloindole 
oligopeptides: preparation and hybridization properties. Bioconjug 
Chem. 1995;6:418-26. 

10. Venkatesan H, Greenberg MM. Improved utility of photolabile solid 
phase synthesis supports for the synthesis of oligonucleotides con- 
taining 3'-hydroxyl termini. J Org Chem. 1996;61:525-9. 

11. Abdel-Baky S, Allam K, Giese RW. Detection of electrophore- 
labeled DNA and albumin by gas chromatography: labile amide elec- 
trophone release tags. Anal Chem. 1993;65:498-9. 

12. Saha M, Saha J, Giese RW. 4-(Trifluoromethyl)-2,3,5,6-tetrafluo- 
robenzyl bromide as a new electrophone derivatizing reagent. J 
Chromatogr. 1993;641:400-4. 


Address for correspondence: W. Ian Lipkin, Mailman School of Public 
Health, Columbia University, 722 West 168th St, New York, NY 10032, 
USA; fax: 212-342-9044; email: wil2001@columbia.edu 


All material published in Emerging Infectious Diseases is in the 
public domain and may be used and reprinted without special per- 
mission; proper citation, however, is appreciated. 


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313 


DISPATCHES 


Comparing 
Aberration 
Detection Methods 
with Simulated Data 

Lori Hutwagner,* Timothy Browne,* 

G. Matthew Seeman,* and Aaron T. Fleischauer* 

We compared aberration detection methods requiring 
historical data to those that require little background by 
using simulated data. Methods that require less historical 
data are as sensitive and specific as those that require 3-5 
years of data. These simulations can determine which 
method produces appropriate sensitivity and specificity. 

T he Early Aberration Reporting System (EARS) was 
developed to allow analysis of public health surveil- 
lance data. Several alternative aberration detection meth- 
ods are available to state and local health departments for 
syndromic surveillance. Before 2001, most statistical aber- 
ration detection methods required at least 5 years of back- 
ground data (1-6). However, with the release of Bacillus 
anthracis in the U.S. mail shortly after the September 11, 
2001, World Trade Center attacks, substantial interest has 
emerged in public health tools that could be rapidly imple- 
mented without requiring years of background data. Newly 
developed nonhistorical aberration detection methods can 
require as little as 1 week of data to begin analysis, 
although they have not been extensively evaluated against 
traditional historical methods (7,8). 

The objective of our study was to determine the sensi- 
tivity, specificity, and time to detection of 3 methods that 
require <3 years of historical baseline data, Cl -MILD 
(Cl), C2-MEDIUM (C2), and C3-ULTRA (C3), and com- 
pare the results with those of 2 methods that require 5 
years of historical baseline, the historical limits method (2) 
and the seasonally adjusted cumulative sum (CUSUM) (5), 
based on simulated data. Simulated data were used to 
avoid some of the interpretation difficulties that can come 
from making these comparisons on the basis of empirical- 
ly observed, natural disease data. All 5 of these methods 
are components of EARS (7). 

The Study 

The methods Cl, C2, and C3 were named according to 
their degree of sensitivity, with Cl being the least sensitive 
and C3 the most sensitive. All 3 methods are based on a 


*Centers for Disease Control and Prevention, Atlanta, Georgia, 
USA 


positive 1 -sided CUSUM calculation. For Cl and C2, the 
CUSUM threshold reduces to the mean plus 3 standard 
deviations (SD). The mean and SD for the Cl calculation 
are based on information from the past 7 days. The mean 
and SD for the C2 and C3 calculations are based on infor- 
mation from 7 days, ignoring the 2 most recent days. These 
methods take into consideration daily variation because 
the mean and SD used by the methods are based on a 
week’s information. These methods also take seasonality 
into consideration because the mean and SD are calculated 
in the same season as the data value in question. 

Since 1989, results from the historical limits method 
have been used to produce Figure 1 in the Morbidity and 
Mortality Weekly Report. This method compares the num- 
ber of reported cases in the 4 most recent time periods for 
a given health outcome with historical incidence data on 
the same outcome from the preceding 5 years; the method 
is based on comparing the ratio of current reports with the 
historical mean and SD. The historical mean and SD are 
derived from 15 totals of 3 intervals (including the same 4 
periods, the preceding 4 periods, and the subsequent 4 
periods over the preceding 5 years of historical data). 

The seasonally adjusted CUSUM method is based on 
the positive 1 -sided CUSUM where the count of interest is 
compared to the 5-year mean and the 5-year SD for that 
period. The seasonally adjusted CUSUM was originally 
applied to laboratory-based Salmonella serotype data. 

To calculate sensitivity, specificity, and time to detec- 
tion, all 5 detection methods of EARS were used to inde- 
pendently analyze 56,000 sets of artificially generated 
case-count data based on 56 sets of parameters. These 56 
sets of parameters each generated 1,000 iterations of 6 
years of daily data, 1994-1999, by using a negative bino- 
mial distribution with superimposed outbreaks. Means and 
standard deviations were based on observed values from 
national and local public health systems and syndromic sur- 
veillance systems. Examples of the data included national 
and state pneumonia and influenza data and hospital 
influenzalike illness. Adjustments were made for days of 
the week, holidays, postholiday periods, seasonality, and 
trend. Any 6 years could be used, but the years 1994-1999 
were used to set day of the week and holiday patterns and 
to avoid any problems that programs might have with the 
year 2000. Fifty (89%) of these datasets then had outbreaks 
superimposed throughout the data. Three types of out- 
breaks were used, each representing various types of natu- 
rally occurring events: log normal, a rapidly increasing 
outbreak; inverted log normal, a slowly starting outbreak; 
and a single-day spike. These types of outbreaks were com- 
bined with different SDs and incubation times to create 10 
different types of outbreaks that had equal probability of 
being included in the simulated data. A year of final simu- 
lated data can be seen in the Figure, with original data and 


314 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


Comparing Aberration Detection Methods 



Figure. Example of 1 year of simulated data with simulated out- 
breaks. Simulated data are based on real means and standard 
deviations with different types of simulated outbreaks randomly 
inserted. 


outbreaks that were added. As a result of these analyses, the 
statistically marked aberrations, or flags, produced by the 5 
detection methods were evaluated for their specificity, sen- 
sitivity, and time to detection. These data can be obtained at 
http ://w w w.bt . cdc . gov/surveillance/ ears/datasets . asp . 

In our study, sensitivity was defined as the number of 
outbreaks in which >1 day was flagged, divided by the 
total number of outbreaks in the data. An outbreak was 
defined as a period of consecutive days in which varying 
numbers of aberrant cases were added to the baseline num- 
ber of cases. An outbreak had days before and after it when 
no aberrant cases were added to the baseline case counts. 
Specificity was defined as the total number of days that did 
not contain aberrant cases (and that were not flagged), 
divided by the total number of days that did not contain 
aberrant cases. Based on these definitions, actual values 
for sensitivity and specificity were calculated. 

Time to detection was defined as the number of com- 
plete days that occurred between the beginning of an out- 
break and the first day the outbreak was flagged. For 
example, if a method flags an outbreak on the first day, its 
time to detection is 0. Likewise, if it flags on the second 
day, its time to detection is 1, and so on. Time to detection 
is an average of the times to detection for each outbreak 
and dataset. Only outbreaks that were flagged on at least 1 


day were included in the average. Therefore, sensitivity is 
needed to completely interpret time to detection. We calcu- 
lated 2-sided 95% confidence values, and they were rela- 
tively small and consistent. 

Overall, the CUSUM methods (the seasonally adjusted 
CUSUM, Cl, C2, and C3) had similar times to detection, 
but their sensitivities varied (Table). Specifically, Cl, C2, 
and C3 showed increasing sensitivity from 60% to 7 1 % to 
82%, respectively. The seasonally adjusted CUSUM and 
C3 methods had similar sensitivities, 82.5% and 82.3%, 
but C3 had a higher specificity, 88.7% and 95.4%. The his- 
torical limits and Cl and C2 methods showed varying sen- 
sitivities (44%— 71%), with Cl and C2 having the highest, 
but all demonstrated similar specificities (96%-97%). 

When results were stratified by outbreak type, 1-day 
outbreaks (i.e., spikes) exhibited the lowest sensitivities. 
Analysis was broken down by dataset and outbreak type 
(online Appendix Tables 1 and 2, available at http://www. 
cdc .go v/ncidod/EID/vol 1 1 no02/04-05 87_app 1 .htm and 
http://www.cdc.gov/ncidod/EID/volllno02/04-0587_ 
app2.htm). 

For the 6 datasets that contained noise but no outbreaks, 
no sensitivity or time to detection exist to calculate. The 
overall specificity for the seasonally adjusted CUSUM, 
historical limits, Cl, C2, and C3 were 88.7%, 98.3%, 
97.2%, 97.2%, and 95.2%, respectively. The specificity for 
these 6 datasets was consistent with general results. The 
historical limits method showed superior specificity in all 
but the last dataset. 

Conclusions 

These simulations demonstrate that the methods for 
aberration detection that require little baseline data, Cl, 
C2, and C3, are as sensitive and specific as the historical 
limits and seasonally adjusted CUSUM methods. As 
expected, Cl, C2, and C3 showed increasing sensitivities 
in accordance with their intended sensitivity levels (Cl 
being the least sensitive, C3 being the most), but with 
decreasing specificities as sensitivity increases. Seasonally 


Table. By method, overall sensitivity and specificity and time to detection 


Type of method 

Name 

Sensitivity (%) 

Specificity (%) 

Time to detection (d)* 

Historical methods (at least 

Seasonally adjusted CUSUM| 

82.5 

88.7 

1.272 

5 y historical data) 

Historical limits^ 

43.9 

96.3 

2.942 

Nonhistorical methods 

C1-MILD§ 

60.1 

97.0 

1.122 

(<3 y historical data) 

C2-MEDIUMU 

71.2 

97.0 

1.319 


C3-ULTRA** 

82.3 

95.4 

1.307 


*Time to detection must be interpreted with sensitivity because time to detection does not include missed outbreaks. 

fThe seasonally adjusted CUSUM method sums the positive differences of the current value from the mean for a period similar to the current value over 5 
years. 

JThe historical limits method compares the current sum of 4 time periods to the mean of the sum of 15 totals of 4 time periods surrounding the current 
point of interest over 5 years. 

§The C1-MILD method is based on CUSUM, but the calculations reduce to the current value being greater than the mean plus 3 standard deviations 
(SD), with the mean and SD based on the past 7 days. 

j|The C2-MEDIUM method is based on CUSUM, but the calculations reduce to the current value being greater than the mean plus 3 SD, with the mean 
and SD based on the past 7 days shifted by 2 days. 

**The C3-ULTRA method is based on CUSUM, summing the positive difference of the current value from the mean for 3 days, with the mean and SD 
based on the past 7 days shifted by 2 days. 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


315 


DISPATCHES 


adjusted CUSUM and the historical limits method also 
showed sensitivities and specificities as expected, with the 
seasonally adjusted CUSUM having the lower specificity 
and higher sensitivity. These findings emphasize the effec- 
tiveness of aberration detection methods without requiring 
long-term historical data as a baseline. 

Since the 10 simulated outbreaks were randomly gener- 
ated by using consistent rates, the sensitivity, specificity, 
and time to detection could be stratified by dataset and out- 
break type. The results of these analyses were largely con- 
gruent with the expected findings, with some variations. 
The simulated datasets are designed for public health offi- 
cials to select a dataset that best reflects their data of inter- 
est or the type of outbreak they are anticipating to determine 
which method provides them with the sensitivity and speci- 
ficity they would find useful. The simulated datasets can 
also be used to make comparisons with other methods. 

The aberration detection methods Cl, C2, and C3 are 
used in several states, counties, and local public health 
departments. Public health departments are able to apply 
these methods to data sources that do not have long peri- 
ods of baseline data. Public health departments are also 
able to apply 1 set of methods they understand to various 
types of diseases, covering different frequencies and sea- 
sonalities. The Cl, C2, and C3 methods have detected out- 
breaks of public health interest, including West Nile 
disease and the start of the influenza season. 

Cl, C2, and C3 demonstrate consistency over the vari- 
ous situations represented in these simulations. Other aber- 
ration detection methods exist, as do other simulated 
datasets. The simulated datasets presented in this paper 
cover a larger variety of types of data that might be expect- 
ed in public health. These simulated datasets also include 
enough past years of data so that methods that require 5 
years of historical information can also be used in the com- 
parisons. These simulations provide a method to fairly 
compare other methods among themselves and to the 
methods included in EARS. 

The simulations were based on means and SDs to help 
determine which method performs better under which cir- 
cumstances. When deciding which method to use, the 
potential user should base the decision on the sensitivity or 
specificity or the time to detection. 

A potential limitation is that the method for calculating 
average times to detection disregards undetected out- 
breaks. Therefore, times to detection should not be consid- 
ered without also taking into account the sensitivity. 
However, this method was preferred over the alternative of 
assigning arbitrary numbers of days to detection for out- 
breaks that were not detected since the alternative method 
could lead to misinterpretation of the data. Another limita- 
tion is that the artificial datasets may not fully reproduce 
the nuances of natural disease occurrences. While approx- 


imations, the simulated data were generated based on nat- 
urally observed data and included variations for trend over 
time, days of the week, seasons, and holidays. Therefore, 
while these comparisons represent relative sensitivities, 
specificities, and times to detection, we do not know 
whether results using naturally occurring data would be 
consistent. 

The results of this study suggest that the EARS histori- 
cal methods do not have a strong advantage when com- 
pared with nonhistorical methods. In fact, the lack of 
historical data does not impair the EARS outbreak detec- 
tion methods. This study also demonstrates the effective- 
ness of artificial outbreak data in comparing and 
evaluating outbreak detection methods. As aberration 
detection methods are increasingly being used by state and 
local health departments to monitor for naturally occurring 
outbreaks and bioterror events, this study contributes to the 
quest to determine the most efficient method for analyzing 
surveillance data. 

Ms. Hutwagner works with the Bioterrorism Preparedness 
and Response Program at the Centers for Disease Control and 
Prevention on developing aberration detection methods for their 
national “drop-in surveillance” system and ongoing syndromic 
surveillance. She has been implementing these methods at vari- 
ous sites in the United States and internationally. 

References 

1. Teutsch SM, Churchill RE, editors. Principles and practice of public 
health surveillance. New York: Oxford University Press; 2000. 

2. Stroup DF, Williamson GD, Herndon JL, Karon J. Detection of aber- 
rations in the occurrence of notifiable diseases surveillance data. Stat 
Med. 1989;8:323-9. 

3. Farrington CP, Andrews NJ, Beale AD, Catchpole MA. A statistical 
algorithm for the early detection of outbreaks of infectious disease. J 
R Stat Soc Ser A Stat Soc. 1996;159:547-63. 

4. Simonsen F, Clarke JM, Stroup DF, Williamson GD, Arden NH, Cox 
NJ. A method for timely assessment of influenza-associated mortali- 
ty in the United States. Epidemiology. 1997;8:390-5. 

5. Hutwagner LC, Maloney EK, Bean NH, Slutsker L, Martin SM. 
Using laboratory-based surveillance data for prevention: an algorithm 
for detecting salmonella outbreaks. Emerg Infect Dis. 
1997;3:395-400. 

6. Stern F, Lightfoot D. Automated outbreak detection: a quantitative 
retrospective analysis. Epidemiol Infect. 1999;122:103-10. 

7. Hutwagner L, Thompson W, Seeman GM, Treadwell T. The bioter- 
rorism preparedness and response Early Aberration Reporting System 
(EARS). J Urban Health. 2003;80:i89-96. 

8. Hutwagner L, Thompson W, Groseclose S, Williamson GD. An eval- 
uation of alternative methods for detecting aberrations in public 
health surveillance data. American Statistical Association, Joint 
Statistical Meetings, Proceedings of the Biometrics Section. 
Indianapolis; 2000 Aug. p. 82-5. 


Address for correspondence: Lori Hutwagner, Centers for Disease 
Control and Prevention, 1600 Clifton Rd, Mailstop Cl 8, Atlanta, GA 
30333, USA; fax: 404-639-0382; email: lhutwagner@cdc.gov 


316 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


Malaria Epidemic 
and Drug 
Resistance, Djibouti 

Christophe Rogier,* Bruno Pradines,* 

H. Bogreau,* Jean-Louis Koeck,t+ 
Mohamed-Ali Kamil, § 
and Odile Mercereau-Puijalonf 

Analysis of Plasmodium falciparum isolates collected 
before, during, and after a 1999 malaria epidemic in Djibouti 
shows that, despite a high prevalence of resistance to 
chloroquine, the epidemic cannot be attributed to a sudden 
increase in drug resistance of local parasite populations. 

F ’rom March to June 1999, an epidemic of Plasmodium 
falciparum malaria affecting all age groups spread in 
the city of Djibouti, Horn of Africa, an area with low and 
irregular transmission. Since the 1970s, autochthonous 
cases of malaria have been reported among the local pop- 
ulation, but their incidence is usually low (1). Anopheles 
arabiensis , the main malaria vector in the city (2,3), has 
been found since the 1970s, possibly from Ethiopia (1,4). 
The focused distribution and the specificity of the breeding 
sites allowed a control strategy based on treatment of the 
larval sites with a larvivorous autochthonous fish, comple- 
mented with pinpoint use of bacterial toxins (3). 
Unfortunately, malaria control activities were progressive- 
ly decreased so that, since the mid-1990s, vector control 
activity has been reduced to irregular insecticide indoor or 
outdoor spraying. Djiboutians frequently travel, and the 
Djibouti-Ethiopian railway has been suspected to be an 
effective route for propagating malaria parasites (5). 
Although some chloroquine treatment failures were report- 
ed in Djibouti in 1990 (6), most persons with P. falciparum 
were treated by chloroquine or quinine at the beginning of 
the 2000s, including during the 1999 epidemics. To deter- 
mine whether this epidemic was associated with temporary 
changes in environmental conditions or to importation of 
new (virulent) or resistant P. falciparum strains, we inves- 
tigated P. falciparum population diversity before, during, 
and after the outbreak and analyzed in vitro susceptibility 
profiles to a panel of antimalarials during the epidemics. 

The Study 

The study was conducted at the Centre Hospitalier des 
Armees Bouffard, a French military hospital in Djibouti 


*IMTSSA-IFR48, Marseille, France; fCentre Hospitalier des 
Armees Bouffard, Djibouti; fHIA R. Piquet, Bordeaux, France; 
§Ministry of Health, Djibouti; and fllnstitut Pasteur, Paris, France 


serving military and civilian natives from the entire city, 
and at other public health facilities of Djibouti. From 1997 
to 2002 clinical malaria in the hospital shows the same 
temporal fluctuations as in dispensaries in the city 
(Figure). The incidence of patients with P. falciparum 
malaria admitted to the hospital increased > 10-fold from 
March to May 1999 compared with the same period in 
1997, 1998, and 2000-2002. In contrast, the number of 
admissions, consultations at the outpatient clinic, or blood 
counts performed for other causes than fever did not vary 
over the same period. The meteorologic station of the 
international airport of Djibouti recorded heavy rainfall the 
month before the epidemic. However, similar rainfall in 
1997 or autumn 1999 was not followed by such a dramat- 
ic increase in malaria incidence in the ensuing months 
(Figure). When annual averages were compared, no partic- 
ular variations in minimal or maximal mean air tempera- 
tures were found to occur during the months preceding the 
epidemic. 

Forty-six blood samples were collected from 
September 14 to December 31, 1998 (period 1), 61 from 
April 12 to April 30, 1999 (period 2), and 32 from March 
15 to May 15, 2002 (period 3), from patients with P. falci- 
parum clinical cases who had not travelled outside the city 
of Djibouti during the preceding month and declared not 
having taken any antimalarial drug before the blood sam- 
pling. The study was cleared by the Djibouti Ministry of 
Health. Informed oral consent was obtained from patients 
before blood collection. Venous blood was collected 
before treatment administration in Vacutainer EDTA tubes 
(Becton Dickinson, Rutherford, NJ, USA). Thin blood 
smears were stained with an RAF kit (Reactifs RAF, Paris, 
France). Parasitemia was expressed as the proportion of P. 
falciparum- infected erythrocytes. Aliquots of freshly col- 
lected blood were kept at -20°C until DNA extraction. 



Figure. Rainfall (bars) and monthly incidence of Plasmodium falci- 
parum clinical malaria cases (curve) at the French Military Hospital 
- CHA Bouffard (circle) and in the dispensaries of the city 
(Department of Epidemiology and Public Hygiene. Triangle), 
Djibouti city, January 1997-May 2002. 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


317 


DISPATCHES 


P. falciparum genetic diversity was investigated by 
using mspl and msp2 encoding highly polymorphic loci 
from merozoite surface protein genes. Mspl and msp2 
were genotyped by using nested polymerase chain reaction 
(PCR), as described (7), except that family- specific fluo- 
rescent primers were used in the nested PCR for assign- 
ment to the K1-, Mad20-, or Ro33-type mspl family and to 
the 3D7- or FC27-type msp2 family. Fragment length was 
analyzed by the Genescan technology. Approximately 50% 


of the blood samples contained multiple mspl or msp2 
genotypes. The mean multiplicity of infection, i.e., the 
number of genotypes present in the blood sample, was 
~1.5 concurrent R falciparum infections per person, with a 
decreasing tendency over the study period (Table 1). For 
each locus, multi-infection cases were excluded from 
analysis of genetic diversity. We identified 9 mspl alleles 
in 83 isolates and 17 msp2 alleles in 108 isolates. The 
genetic diversity estimated by the unbiased expected 


Table 1 . Multiplicity of infections deduced from mspl and msp2 genotyping and frequency (%) of the Pfdhfr (codons 51 , 59, and 108), 
Pfdhps (codons 436, 437, and 540) and Pfcrt (codon 76) genotypes 

Locus 

Period 1 
1998 (n = 46) 

Period 2 
1999 (n = 61) 

Period 3 
2002 (n = 32) 

Total (N = 139) 

mspl 

Mean multiplicity 

1.6 

1.5 

1.3 


SD* 

0.7 

0.7 

0.5 


No of multiple infections (%) 

23 (50) 

22 (36) 

1 1 (34) 

56 (40) 

msp2-t 

Mean multiplicity 

1.4 

1.2 

1.1 


SD 

0.7 

0.4 

0.4 


No. of multiple infections (%) 

14(31) 

12(20) 

2(6) 

28 (21) 

mspl and msp2 

Mean multiplicity 

1.8 

1.6 

1.4 


SD 

0.7 

0.7 

0.6 


No. of multiple infections (%) 

28 (61) 

29 (48) 

12(38) 

69 (50) 

Pfdhfr 
Codon 51 

(Wildtype) N 

38 (83) 

60 (98) 

15(50) 

1 1 3 (83) 

N & 1 

3(6) 

0 

0 

3(2) 

1 

5(11) 

1 (2) 

15(50) 

21 (15) 

Not genotyped 

- 

- 

2 

2 

Codon 59 

(Wildtype) C 

43(94) 

61 (100) 

29 (97) 

133 (97) 

C & R 

1 (2) 

0 

0 

1 (1) 

R 

2(4) 

0 

1 (3) 

3(2) 

Not genotyped 

- 

- 

2 

2 

Codon 108 

(Wildtype) S 

37(81) 

60 (98) 

16(50) 

113(81) 

S & N 

2(4) 

0 

0 

2(2) 

N 

7(15) 

1 (2) 

16(50) 

24(17) 

Pf dhps 
Codon 436 

(Wildtype) S 

46(100) 

52 (93) 

29 (94) 

127 (95) 

F 

0 

1 (2) 

0 

1 (1) 

A 

0 

3(5) 

2(6) 

5(4) 

Not genotyped 

- 

5 

1 

6 

Codon 437 

(Wildtype) A 

45 (98) 

54 (96) 

19(61) 

1 1 8 (89) 

G 

1 (2) 

2(4) 

12(39) 

15(11) 

Not genotyped 

- 

5 

1 

6 

Codon 540 

(Wildtype) K 

44(96) 

60 (98) 

19(59) 

123 (88) 

K & E 

1 (2) 

0 

0 

1 (1) 

E 

1 (2) 

1 (2) 

13(41) 

15(11) 

Pfcrt 
Codon 76 

(Wildtype) K 

1 (2) 

1 (2) 

1 (3) 

7(5) 

K& T 

3(7) 

2(3) 

2(6) 

3(2) 

T 

42(91) 

58 (95) 

29(91) 

129 (93) 


*SD, standard deviation; The genotypes at the Pf dhfr, Pf dhps, and Pfcrt locus refer to the one-letter symbolized amino acids coded by the codons. A, 
Alanine; C, Cysteine; F, Phenylalanine; G, Glycine; I, Isoleucine; K, Lysine; N, Asparagine; R, Arginine; S, Serine; T, Threonine. 

■ fmsp2 multiplicity estimated on 45 and 59 samples in 1998 and 1999, respectively. 


318 


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Malaria Epidemic and Drug Resistance, Djibouti 


heterozygocity (8), i.e., the probability that 2 randomly 
chosen genotypes are different in the sample, before, dur- 
ing, and after the 1999 outbreak was 0.79 (n = 23), 0.37 (n 
= 39), and 0.64 (n = 21) at the mspl locus and 0.83 (n = 
31), 0.34 (n = 47) and 0.63 (n = 30) at the msp2 locus, 
respectively. During the epidemic, Ro33-131 accounted 
for 79% of the mspl allele and FC27-408 accounted for 
81% of the msp2 alleles. Both alleles were present before 
and after the epidemic but with a much lower prevalence. 
They accounted for 26% of the mspl and 35% of the msp2 
alleles in 1998 and 14% of the mspl and 10% of the msp2 
alleles in 2002 (Table 2). 

To look for resistance-associated point mutations and 
haplotypes, the complete coding region of Pfdhfr (dihydro- 
folate reductase) and Pfdhps (dihydropteroate synthase) 
was amplified and sequenced (ABI 3100 Genetic Analyser, 
Applied Biosystems, Courtaboeuf, France) as described 
(9). We focused the analysis on point mutations of Pfdhfr 
codons 16, 51, 59, 108, and 164 and Pfdhps codons 436, 
437, 540, 581, and 613, which have been associated with 
resistance to pyrimethamine and proguanil metabolite and 
to sulfadoxine, respectively (10). The prevalences of the 
Pfdhfr and Pfdhps mutations are shown in Table 1. No 
mutant was detected for Pfdhfr codons 16 and 164 and 


Pfdhps codon 581. A single isolate collected in period 2 
harbored the Pfdhps A613S mutation. No isolate harbored 
the quintuple mutant haplotype (Pfdhfr S108N, N51I, and 
C59R and Pfdhps K540E and A437G) or the Pfdhfr C59R 
and Pfdhps K540E combination that predicts sulfadoxine- 
pyrimethamine clinical failure (9). One isolate containing 
at least 2 P. falciparum populations harbored 3 Pfdhfr 
mutations (S108N, N51I, and C59R) and the Pfdhps 
K540E mutation. 

From 1998 to 1999, the frequency of isolates with 
mutated Pfdhfr codons 51, 59, and 108 decreased (not sig- 
nificantly), and Pfdhps allelic frequency did not differ sig- 
nificantly. The prevalence of isolates harboring the Pfdhfr 
N51I, Pfdhfr S108N, Pfdhps A437G, and Pfdhps K540E 
mutations increased from 1998-1999 to 2002 (Fisher exact 
test, p < 0.001 each). Presence of the chloroquine resist- 
ance-associated K76T mutation of Pfcrt (chloroquine- 
resistance transporter) (11) was analyzed by nested 
allele-specific PCR. Over the study period, 93% of the iso- 
lates harbored the Pfcrt K76T mutation (Table 1), without 
any significant temporal variation. 

Twenty seven P. falciparum isolates collected during 
the 1999 epidemic with a 0.05%-5.0% parasitemia were 
transported at 4°C to our laboratory in Marseille, France, 


Table 2. Distribution of mspl and msp2 alleles by allelic families and fragment size (in base pair) among Djibouti isolates with only 1 
allele detected by locus* 


Locus Allelic families 

Allele (base pair) 

1998 (%) 

1999 (%) 

2002 (%) 

mspl 

K1 

129 

4.3 

2.6 

14.3 


203 

0.0 

2.6 

0.0 

Mad 20 

166 

4.3 

0.0 

0.0 


184 

34.8 

2.6 

57.1 


193 

0.0 

7.7 

0.0 


202 

21.7 

5.1 

14.3 


237 

4.3 

0.0 

0.0 


241 

4.3 

0.0 

0.0 

Ro 33 

131 

26.1 

79.5 

14.3 

msp2 

3D7 

221 

9.7 

2.1 

0.0 


226 

0.0 

2.1 

0.0 


248 

16.1 

0.0 

60.0 


253 

0.0 

2.1 

0.0 


261 

0.0 

2.1 

0.0 


275 

0.0 

10.6 

0.0 


282 

3.2 

0.0 

6.7 


284 

0.0 

0.0 

3.3 


308 

3.2 

0.0 

0.0 


346 

0.0 

0.0 

3.3 


366 

3.2 

0.0 

0.0 


371 

0.0 

0.0 

3.3 

FC27 

173 

3.2 

0.0 

0.0 


373 

6.5 

0.0 

6.7 


408 

35.5 

80.9 

10.0 


444 

3.2 

0.0 

0.0 


468 

16.1 

0.0 

6.7 

isolates collected in 1998 (mspl: n = 23; msp2: 

n = 31), 1999 (mspl: 

n = 39; msp2: n = 47), and 2002 (mspl: n = 21 ; msp2: n = 30). 



Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


319 


DISPATCHES 


Table 3. In vitro drug sensitivity of 27 Plasmodium falciparum isolates collected in Djibouti, 1999 


Drugs 

Isolates studied (n) 

Mean IC 5 o* 

95% confidence interval 

Cut-off value 

% resistant isolates 

Chloroquine 

27 

326 nmol/L 

224-474 nmol/L 

>100 nmol/L 

93 

Amodiaquine 

27 

10.0 nmol/L 

8.0-12.6 nmol/L 

>80 nmol/L 

0 

Cycloguanil 

24 

13 nmol/L 

8-21 nmol/L 

>500 nmol/L 

4 

Pyrimethamine 

25 

69 nmol/L 

41-1 17 nmol/L 

>2,000 nmol/L 

4 

*The 50% inhibitory concentration (IC 50 ) of chloroquine diphosphate, amodiaquine, pyrimethamine dihydrochloride, and cycloguanil, i.e., the drug 
concentration corresponding to 50% of the uptake of 3H-hypoxanthine by the parasites in drug-free control wells, was determined by nonlinear regression 
analysis of log-dose/response curves. Mean IC 50 and proportion of resistant isolates according to cut-off values are indicated. Data were expressed as the 
geometric mean IC 50 and 95% confidence intervals were calculated. 


and analyzed for in vitro drug sensitivity by using an iso- 
topic microtest (12). Among them, 93% were classified as 
resistant to chloroquine (Table 3). No isolate was resistant 
to amodiaquine. In vitro resistance was 4% for both 
pyrimethamine and cycloguanil. 

Conclusions 

Before and after the 1999 epidemic, P. falciparum 
genetic diversity in Djibouti was large, with -80% and 63% 
heterozygocity. This finding is somewhat surprising for an 
area where disease endemicity is low (13) and probably 
reflects importation of strains from neighboring areas such 
as Ethiopia or Somalia (1,5). P. falciparum genetic diversi- 
ty was diminished during the epidemic, reflecting the circu- 
lation of a restricted number of strains during that period. 
Most of these strains harbored an mspl and msp2 genotype 
that was detected before the epidemic. The prevalence of 
Pfcrt , Pfdhfr , and Pfdhps mutant genotypes did not vary 
significantly from 1998 to 1999. Thus, our data do not sup- 
port the hypothesis of a sudden increase in the drug resist- 
ance of the local P. falciparum population as causing the 
epidemic. Our data are also not consistent with massive 
invasion by a single strain/genotype but rather suggest 
expansion during the epidemic of a few strains that were 
already prevalent. Further genotyping is needed to establish 
how many strains were circulating and their possible origin. 
What could have caused this sudden amplification? One 
possibility is a temporary increase in vector density. 
Unfortunately, no vectors were captured at that time, and 
this hypothesis is difficult to explore retrospectively. 

The low prevalence of Pfdhfr and Pfdhps resistance 
mutations in 1998 and 1999 and of proguanil or 
pyrimethamine in vitro resistance in 1999 may explain the 
very low incidence of clinical malaria among the French 
soldiers stationed in Djibouti who were taking chloro- 
quine-proguanil chemoprophylaxis. However, the sharp 
increase of Pfdhfr and Pfdhps resistance mutations 
observed in 2002 threatens sulfadoxine-pyrimethamine 
efficacy in the near future, even more so since the limited 
acquired immunity is unlikely to contribute to sustained 
drug efficacy (14). Molecular and in vitro assays point to a 


very high prevalence of chloroquine resistance. This find- 
ing calls for an urgent in vivo assessment of the antimalar- 
ials presently used in Djibouti in order to consider a rapid 
change in first-line treatment policy. 

Acknowledgments 

We are indebted to E. Garnotel, J.J. DePina, T. Fusai, H. 
Bouchiba, E. Czarnecki, P. Bigot, J. Mosnier, M. Desbordes, and 
M. Abakari for their help and technical assistance. 

This work was supported by the program PAL+ (2002) of 
the French Ministry for Research, the Delegation Generale pour 
l’Armement and Impact Malaria (Sanofi-Synthelabo groupe). 

Dr. Rogier is the chief of the Research Unit in 
Parasitological Biology and Epidemiology of the Institute for 
Tropical Medicine of the French Army, Le Pharo, Marseille, 
France. His main areas of interest include epidemiology and pop- 
ulation genetics related to malaria. 

References 

1. Carteron B, Morvan D, Rhodain F. Le probleme de l’endemie palus- 
tre dans la Republique de Djibouti. Med Trop (Mars). 
1978;38:837-46. 

2. Shidrawi GR. Rapport sur une visite en Republique de Djibouti du 14 
janvier au 11 fevrier 1982. OMS/EM/MAL/190. Geneva: World 
Health Organization; 1982. 

3. Louis JP, Albert JP. Le paludisme en Republique de Djibouti. 
Strategic de controle par la lutte antilarvaire biologique: Poissons lar- 
vivores autochtones ( Aphanius dispar) et toxines bateriennes. Med 
Trop (Mars). 1988;48:127-31. 

4. Courtois D, Mouchet J. Etude des populations de culicides en TFAI. 
Med Trop (Mars). 1970;30:837-46. 

5. Fox E, Bouloumie J, Olson JG, Tible D, Lluberas M, Shakib SO, et 
al. Plasmodium falciparum voyage en train d’Ethiopie a Djibouti. 
Med Trop (Mars). 1991;51:185-9. 

6. Rodier GR, Parra JP, Kamil M, Chakib SO, Cope SE. Recurrence and 
emergence of infectious diseases in Djibouti city. Bull World Health 
Organ. 1995;73:755-9. 

7. Zwetyenga J, Rogier C, Tall A, Fontenille D, Snounou G, Trape Jf, et 
al. No influence of age on infection complexity and allelic distribu- 
tion in Plasmodium falciparum infections in Ndiop, a Senegalese vil- 
lage with seasonal, mesoendemic malaria. Am J Trop Med Hyg. 
1998;59:726-35. 

8. Nei M. Estimation of average heterozygosity and genetic distance 
from small number of individuals. Genetics. 1978;89:583-90. 


320 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


Malaria Epidemic and Drug Resistance, Djibouti 


9. Kublin JG, Dzinjalamala FK, Kamwendo DD, Malkin EM, Cortese 
JF, Martino LM, et al. Molecular markers for failure of sulfadoxine- 
pyrimethamine and chlorproguanil-dapsone treatment of Plasmodium 
falciparum malaria. J Infect Dis. 2002;185:380-8. 

10. Reeder JC, Rieckmann KH, Genton B, Lorry K, Wines B, Cowman 
AF. Point mutations in the dihydrofolate reductase and dihy- 
dropteroate synthetase genes and in vitro susceptibility to 
pyrimethamine and cycloguanil of Plasmodium falciparum isolates 
from Papua New Guinea. Am J Trop Med Hyg. 1996;55:209-13. 

11. Djimde A, Doumbo OK, Cortese JF, Kayentao K, Doumbo S, Diourte 
Y, et al. A molecular marker for chloroquine-resistant falciparum 
malaria. N Engl J Med. 2001;344:257-63. 

12. Pradines B, Rogier C, Fusai T, Tall A, Trape JF, Doury JC. In vitro 
activity of artemether and its relationship to other standard antimalar- 
ial drugs against West African isolates. Am J Trop Med Hyg. 
1998;58:354-7. 


13. Anderson TJ, Haubold B, Williams JT, Estrada-Franco JG, 
Richardson L, Mollinedo R, et al. Microsatellite markers reveal a 
spectrum of population structures in the malaria parasite Plasmodium 
falciparum. Mol Biol Evol. 2000;17:1467-82. 

14. Plowe CV, Kublin JG, Dzinjalamala FK, Kamwendo DS, Mukadam 
RA, Chimpeni P, et al. Sustained clinical efficacy of sulfadoxine- 
pyrimethamine for uncomplicated falciparum malaria in Malawi after 
10 years as first line treatment: five year prospective study. BMJ. 
2004;328:545-8. 

Address for correspondence: Christophe Rogier, IMTSSA, BP46, Parc du 
Pharo, 13998 Marseille- Armees, France; fax: 33 4 91 15 01 64; email: 
christophe.rogier@ wanadoo.fr 







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INFECTIOUS DISEASES 

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Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


321 



DISPATCHES 


Late Recognition 
of SARS in 
Nosocomial 
Outbreak, Toronto 

Thomas Wong 5 *f Tamara Wallington,! 

L. Clifford McDonald, § Zahid Abbas,* 
Michael Christian,! Donald E. Low,f 
Denise Gravel,* Marianna Ofner,*t 
Barbara Mederski,^ Lisa Berger,! Lisa Hansen,* 
Cheryl Harrison, H Arlene King,* Barbara Yaffe,! 
and Theresa Tam* 

Late recognition of severe acute respiratory syndrome 
(SARS) was associated with no known SARS contact, hos- 
pitalization before the nosocomial outbreak was recog- 
nized, symptom onset while hospitalized, wards with SARS 
clusters, and postoperative status. SARS is difficult to rec- 
ognize in hospitalized patients with a variety of underlying 
conditions in the absence of epidemiologic links. 

S evere acute respiratory syndrome (SARS) spread glob- 
ally in 2003, infecting >8,000 people and killing near- 
ly 800. In total, 438 probable or suspected SARS cases and 
44 deaths were reported in Canada (1,2). SARS was first 
recognized retrospectively in Canada in a woman who had 
returned from Hong Kong on February 23, 2003. This 
international connection ignited the outbreak in Canada, 
which affected mainly the Toronto area (1,2). 

After enhanced infection control precautions and public 
health measures were implemented in March 2003, the 
Canadian outbreak began to subside in April. On May 14, 
the World Health Organization (WHO) took Toronto off 
the list as a SARS-affected area in the absence of newly 
reported cases for at least 2 incubation periods after the last 
SARS case-patient was isolated. In accordance with public 
health principles, the enhanced measures were selectively 
relaxed in low-risk settings in Toronto area hospitals in 
early May 2003, although full precautions were still rec- 
ommended for patients with febrile respiratory illnesses. In 
the third week of May, a cluster of febrile respiratory ill- 
ness at a Toronto area rehabilitation hospital was reported 
to the health department. Traceback of these SARS cases 
identified the index patient as a postoperative patient who 


*Public Health Agency of Canada, Ottawa, Ontario, Canada; 
fUniversity of Toronto, Toronto, Ontario, Canada; f Toronto Public 
Health, Toronto, Ontario, Canada; §Centers for Disease Control 
and Prevention, Atlanta, Georgia, USA; and ^North York General 
Hospital, Toronto, Ontario, Canada 


was transferred from hospital X to the rehabilitation hospi- 
tal. This link uncovered clusters of unrecognized SARS 
infections on a surgical ward and a psychiatry ward at hos- 
pital X. Investigation determined that the ventilation sys- 
tem did not contribute to the spread of SARS at that 
hospital. On May 23, 2003, hospital X was closed to 
nonobstetric admissions other than newly identified SARS 
cases, and SARS precautions were reintroduced. As part of 
an outbreak investigation, we explored potential factors 
contributing to the late recognition of SARS infections in 
a cohort of persons with SARS admitted to hospital X. 

The Study 

Hospital X is a Toronto-area community hospital with 
425 beds. During the 2003 outbreak in Toronto, dedicated 
SARS inpatient units were created at the hospital. All non- 
healthcare workers with probable or suspected SARS, 
according to the WHO case definition (3), exposed at and 
admitted to hospital X with symptom onset from April 17, 
2003, to June 8, 2003, were included in this retrospective 
cohort investigation. Healthcare workers were excluded. If 
SARS was not recorded as a possible diagnosis in the med- 
ical chart, despite SARS -defining manifestations for at 
least 24 hours of hospitalization, recognition of SARS was 
classified as late. Otherwise, recognition was classified as 
prompt. Laboratory diagnosis of SARS was obtained by 
reverse transcriptase-polymerase chain reaction (RT-PCR) 
or serologic testing (4,5). 

SPSS version 11.0 (SPSS, Inc., Chicago, IL, USA) was 
used for statistical analysis. Continuous variables were 
dichotomized about the median. Factors associated with 
late recognition were deemed statistically significant if the 
p value was <0.05 (2-tailed), using chi-square test with 
Yates correction or Fisher exact test, when appropriate. 
Relative risks with 95% confidence intervals were listed as 
undefined when certain cell sizes were 0. The small sam- 
ple size, small number of patients with the outcome of 
interest (late SARS recognition), and small cell size for 
some dichotomous variables precludes multivariate logis- 
tic regression analysis. 

The SARS outbreak involved 88 case-patients whose 
exposure setting was hospital X (Figure). SARS occurred 
in 50 patients, family members, or visitors. Thirty-three of 
these 50 persons were admitted to hospital X, and all 33 
were included in this analysis. 

SARS-associated coronavirus (SARS-CoV) laboratory 
results were available for 29 (88%) of the 33 SARS 
patients. No samples were available for SARS-CoV testing 
from 4 deceased patients. Twenty-four (83%) of the 29 
SARS patients tested were positive by RT-PCR, serology, 
or both. The 5 remaining patients had negative acute-phase 
SARS-CoV serologic results, and their convalescent-phase 
results were not available (data not shown). 


322 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


Late Recognition of SARS in Nosocomial Outbreak 



Symptom onset 


Figure. Reported probable and suspected severe acute respirato- 
ry syndrome cases in persons (or their family members) with 
symptom onset after April 17, 2003, whose exposure setting was 
hospital X. 


Eleven (33%) patients had late recognition of SARS. 
Their mean age was 68.8 years, and 8 (73%) were postop- 
erative patients. All were admitted before May 23, 2003, 
the day when Hospital X reintroduced enhanced SARS 
precautions. No case-patients had a recognized close con- 
tact with another SARS patient initially. None were travel 
related. (Table 1). Six (55%) patients were admitted to an 
intensive care unit (ICU), and 3 (27%) required mechani- 
cal ventilation (Table 2). All patients had infiltrates on 
chest radiographs; infiltrates of 9 (81%) were bilateral. 

Using univariate analysis, we found that patients with 
late recognition of SARS were more likely to have no 
known contact with another SARS patient (p < 0.001), to 
have been a patient on a ward where SARS cluster 


occurred (p < 0.01), to be admitted before the nosocomial 
outbreak was recognized at the hospital (p < 0.01), to have 
symptom onset while hospitalized (p < 0.001), and to be a 
postoperative patient, (p = 0.001) (Tables 1 and 2). Clinical 
findings and laboratory abnormalities during hospitaliza- 
tion were not associated with late SARS recognition. The 
small sample size, small number of patients with late 
SARS recognition, and small cell size for some dichoto- 
mous variables precluded multivariate logistic regression 
analysis. 

The hospital reintroduced enhanced SARS precautions 
on May 23, 2003, under the direction of public health 
authorities promptly after the nosocomial outbreak was 
recognized. For patients admitted before that date (N = 
20), the relative risk for late recognition of SARS for post- 
operative patients was 2.7 (95% confidence interval 
0.99-7.2, p = 0.07) and was just short of statistical signif- 
icance (data not shown). Once SARS transmission was 
recognized at hospital X and enhanced infection control 
precautions were reinstituted, clinicians were more likely 
to suspect SARS, and nosocomial transmission was ended 
abruptly. 

Conclusions 

Our results highlight the difficulty clinicians can have 
in recognizing locally acquired SARS among patients with 
other underlying medical conditions but with no apparent 
epidemiologic linkage. The patients had no known contact 
with other SARS patients, did not have a travel history to 
SARS-affected areas outside of Canada when the outbreak 
was thought to be over in Toronto, and were unaware of a 
simmering outbreak associated with hospital X. Perhaps 
because of the nonspecific nature of clinical manifesta- 
tions, SARS can be especially difficult to recognize among 


Table 1 . Proportion of patients with late SARS recognition by demographic and exposure characteristics, Toronto, hospital X, April 17- 


June 8, 2003* 


Characteristics 

Late SARS recognition (%) 

RR (95% Cl) 

p value 

Age (y) 


0.6 (0.2-1 .7) 

0.5 

<58.5 

4/16(25.0) 



>58.5 

7/1 7 (41 .2) 



Sex 


0.5 (0.1-1 .4) 

0.3 

F 

3/15(20.0) 



M 

8/1 8 (44.4) 



Aware of close contact with a SARS patient 


UD 

<0.001 

Yes 

0/1 4 (0) 



No 

11/19 (57.9) 



Exposure from being an inpatient on wards at hospital X with SARS 


4.1 (1.3-12.7) 

<0.01 

clusters 




Yes 

8/1 3 (61 .5) 



No 

3/20(15.0) 



Admitted to hospital X before May 23, 2003 


UD 

<0.01 

Yes 

11/20 (55.0) 



No 

0/1 3 (0) 



SARS symptom onset while hospitalized 


UD 

<0.001 

Yes 

11/15 (73.3) 



No 

0/1 8 (0) 




*SARS, severe acute respiratory syndrome; RR, relative risk; Cl, confidence interval; F, female; M, male; UD, undefined. 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


323 



DISPATCHES 


Table 2. Proportion of patients with late SARS recognition by clinical characteristics, Toronto, hospital X, April 1 7-June 8, 2003* 


Characteristics 

Late SARS recognition/total SARS (%) RR (95% Cl) 

p value 

Postoperative 


5.3 (1.8-16.2) 

0.001 

Yes 

8/1 1 (72.7) 



No 

3/22(13.6) 



Maximum temperature during hospitalization (°C) 


0.8 (0.3-3. 1) 

1.0 

<38.8 

5/15(33.3) 



>38.8 

6/15(40.0) 



First symptom includes 




Fever 


1.0 (0.2-5. 4) 

1.0 

Yes 

10/30 (33.3) 



No 

1/3 (33.3) 



Cough 


0.7 (0.2-2. 6) 

0.7 

Yes 

2/8 (25.0) 



No 

9/25 (36.0) 



Dyspnea 


0.4 (0.1 -2.4) 

0.4 

Yes 

1/7 (14.3) 



No 

10/26(38.5) 



Diarrhea 


0.6 (0.1 -3.5) 

0.6 

Yes 

1/5 (20.0) 



No 

10/28 (35.7) 



Nausea/vomiting 


3.2 (1 .9-5.4) 

0.3 

Yes 

1/1 (100) 



No 

10/32 (31.3) 



Admitted to ICU 


1.4 (0.5-3. 8) 

0.7 

Yes 

6/15(40.0) 



No 

5/18(27.8) 



Supplemental oxygen 


5.0 (0.7-34.2) 

0.054 

Yes 

10/22 (45.5) 



No 

1/11 (9.1) 



Mechanical ventilation 


0.8 (0.3-2. 3) 

0.7 

Yes 

3/1 1 (27.3) 



No 

8/22 (36.4) 



Death 


1.3 (0.5-3. 5) 

0.7 

Yes 

4/10(40.0) 



No 

7/23 (30.4) 



Treatment with 




Ribavirin 


UD 

1.0 

Yes 

0/2 (0) 



No 

10/30 (33.3) 



Corticosteroids 


0.3 (0.1-1. 1) 

0.06 

Yes 

2/15(13.3) 



No 

8/17(47.1) 



Antimicrobial drugs 


1.0 (0. 3-3.0) 

1.0 

Yes 

8/24 (33.3) 



No 

3/9 (33.3) 



*SARS, severe acute respiratory syndrome; RR, relative risk; Cl, confidence interval; UD, undefined; ICU, intensive care unit. 


patients already hospitalized for other reasons. These 
symptoms overlap many of the symptoms of hospitalized 
febrile postoperative patients and patients with other respi- 
ratory illnesses (6-12). In addition, at the time of this noso- 
comial outbreak, the full spectrum of the clinical signs and 
symptoms of SARS had not yet been well characterized 
( 11 ). 

To detect SARS early, health professionals need to look 
not only for epidemiologic links but also clusters of unex- 
plained respiratory infection. A cluster of respiratory infec- 


tions among families and visitors may not be evident ini- 
tially because hospitals do not normally track infections in 
inpatients’ families and visitors. In addition, infected 
patients may be asymptomatic before they are transferred 
to another healthcare facility. 

Even though our investigation generated interesting 
hypotheses, it had several limitations, which included 
reliance on retrospective chart reviews to abstract data. 
Such information may have been affected by missing data 
or recall bias. Our analysis did not include hospital work- 


324 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


Late Recognition of SARS in Nosocomial Outbreak 


ers, nor did it include persons who were exposed at hospi- 
tal X but were subsequently admitted elsewhere. Studies 
that include hospital workers and persons admitted to other 
hospitals are needed. Seroprevalence studies will be help- 
ful because some infected persons may not be sympto- 
matic. The small sample size limited the power to detect 
small differences and precluded multivariate analysis. A 
small number of patients with a diagnosis of SARS may 
not actually have been infected with SARS-CoV. This pos- 
sibility could have biased the results either way. However, 
this number is likely small because most case-patients test- 
ed positive for SARS-CoV. 

Although our results highlight the challenge of recog- 
nizing SARS among hospitalized patients, the occurrence 
of seasonal respiratory infections such as influenza may 
further compound the difficulty in identifying a SARS 
case. In places where recent SARS transmission has 
occurred, SARS should be considered during the evalua- 
tion of nosocomial as well as community-acquired pneu- 
monia. This recommendation is particularly important if 
the hospital housed SARS patients within the previous 20 
days or if unexplained febrile respiratory clusters had been 
noticed within the institution. 

This nosocomial outbreak underscores the importance 
of sharing information among clinicians, laboratories, 
infection control departments, occupational health servic- 
es, and public health departments and of collaborating 
seamlessly in the search for clusters of respiratory infec- 
tions. A sensitive infectious disease surveillance system 
operated by the infection control officer must be in place 
in healthcare facilities for early detection and implementa- 
tion of appropriate measures to interrupt transmission 
(13,14). This surveillance should include the monitoring of 
increased absenteeism among healthcare workers; unusual 
fever or pneumonia clusters among patients, family, visi- 
tors, and healthcare workers; pneumonia deaths; and labo- 
ratory testing for respiratory pathogens or SARS-CoV. 
These steps require commitment, preparedness planning, 
resources, and training. The lack of a rapid, sensitive, and 
specific diagnostic test for early SARS infection hampers 
the ability of clinicians to make a prompt diagnosis in 
cases when an epidemiologic link is missing (15). 

Acknowledgments 

We acknowledge the efforts of the many clinical and public 
health professionals whose dedication stopped the spread of 
SARS in Canada. 

Dr. Wong is an epidemiologist with the Centre for Infectious 
Disease Prevention and Control at the Public Health Agency of 


Canada. His interests include infectious disease surveillance and 

emerging infectious disease response. 

References 

1. Poutanen SM, Low DE, Henry B, Finklestein S, Rose D, Green K, et 
al. Identification of severe acute respiratory syndrome in Canada. N 
Engl J Med. 2003;348:1995-2005. 

2. Varia M, Wilson S, Sarwal S, McGeer A, Gournis E, Galansis E, et al. 
Investigation of a nosocomial outbreak of severe acute respiratory 
syndrome (SARS) in Toronto, Canada. CMAJ. 2003;169:285-92. 

3. World Health Organization. Case definitions for severe acute respira- 
tory syndrome, [cited 2004 May 18]. Available from 
http ://www. who .int / c sr/sars/ casedef inition/ en/ 

4. Peiris JS, Lai ST, Poon LL, Guan Y, Yam LYC, Lim W, et al. 
Coronavirus as a possible cause of severe acute respiratory syndrome. 
Lancet. 2003;361:1319-25. 

5. World Health Organization. Recommendations for laboratories test- 
ing by PCR for presence of SARS coronavirus-RNA. [cited 2004 
May 18]. Available from www.who.int/csr/sars/coronarecommenda- 
tions/ en/index .html 

6. Booth CM, Matukas LM, Tomlinson GA, Rachlis AR, Ros DB, 
Dwosh HA, et al. Clinical features and short-term outcomes of 144 
patients with SARS in the greater Toronto area. JAMA. 
2003;289:2801-9. 

7. Lee N, Hui D, Wu A, Chan P, Cameron P, Joynt GM, et al. A major 
outbreak of severe acute respiratory syndrome in Hong Kong. N Engl 
J Med. 2003;348:1986-94. 

8. Tsang KW, Ho PL, Ooi GC, Yee WK, Wang T, Chan- Yeung M, et al. 
A cluster of cases of severe acute respiratory syndrome in Hong 
Kong. N Engl J Med. 2003;348:1977-85. 

9. Jernigan JA, Low DE, Hefland RF. Combining clinical and epidemi- 
ologic features for early recognition of SARS. Emerg Infect Dis. 
2004;10:327-33. 

10. Klinger JR, Sanchez MP, Curtin LA, Durkin M, Matyas B. Multiple 
cases of life-threatening adenovirus pneumonia in a mental health 
care center. Am J Respir Crit Care Med. 1998;157:645-9. 

11. Rainer TH, Chan PK, Ip M, Lee N, Hui DS, Smit D, et al. The spec- 
trum of severe acute respiratory syndrome-associated coronavirus 
infection. Ann Intern Med. 2004;140:614-9. 

12. Peiris JS, Chu CM, Cheng VC, Chan KS, Hung IFN, Poon LLM, et 
al. Clinical progression and viral load in a community outbreak of 
coronavirus-associated SARS pneumonia: a prospective study. 
Lancet. 2003;361:1767-72. 

13. Loutfy MR, Wallington T, Rutledge T, Mederski B, Rose K, Kwolek 
S, et al. Hospital preparedness and SARS. Emerg Infect Dis. 
2004;10:771-6. 

14. McDonald LC, Simor AE, Su IJ, Maloney S, Ofner M, Chen KT, et 
al. SARS in healthcare facilities. Emerg Infect Dis. 2004;10:777-81. 

15. Muller MP, Tomlinson GA, Matukas LM, Detsky AS, McGeer A, 
Low DE, et al. Discriminative ability of laboratory parameters in 
severe acute respiratory syndrome (SARS) [abstract V-796b]. 43rd 
Meeting of the Interscience Conference on Antimicrobial Agents and 
Chemotherapy; 2003 Sep 14-17; Chicago. Washington: American 
Society for Microbiology; 2003. 

Address for correspondence: Thomas Wong, Division of Community 

Acquired Infections, Public Health Agency of Canada, Room 3444, 

Building # 6, AL: 0603B, Tunney’s Pasture, Ottawa, Ontario K1A 0L2, 

Canada; fax: 613-941-9813; email: tom_wong@phac-aspc.gc.ca 


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Bacteremic 
Typhoid Fever in 
Children in an 
Urban Slum, 
Bangladesh 

W. Abdullah Brooks,* Anowar Hossain,* 

Doli Goswami,* Amina Tahia Sharmeen,* 
Kamrun Nahar,* Khorshed Alam,* Noor Ahmed,* 
Aliya Naheed,* G. Balakrish Nair,* Stephen Luby,* 
and Robert F. Breiman* 

We confirmed a bacteremic typhoid fever incidence of 
3.9 episodes/1,000 person-years during fever surveillance 
in a Dhaka urban slum. The relative risk for preschool chil- 
dren compared with older persons was 8.9. Our regression 
model showed that these children were clinically ill, which 
suggests a role for preschool immunization. 

T yphoid fever is a major cause of illness; the global 
incidence in 2000 was an estimated 21,650,974 cases 
with 216,510 deaths (1). The cause of typhoid fever, 
Salmonella enterica subspecies enterica serotype Typhi ( S . 
Typhi), is both waterborne and foodborne, with an annual 
incidence approaching 1% in disease-endemic areas (2-4). 
Peak incidence is reported to occur in children 5-15 years 
of age; however, in regions where the disease is highly 
endemic, children <5 years of age may have among the 
highest infection rates (1,4-6). Population-based data are 
limited (1) and would be helpful for refining estimates of 
the impact of disease and for identifying age groups at 
highest risk, thereby making it possible to optimize vacci- 
nation strategies (7,8). 

Data on disease severity and sequelae can contribute to 
estimating the impact of disease. Most complications — 
including intestinal perforation and peritonitis, 
encephalopathy, intestinal hemorrhage, hepatospleno- 
megaly, vomiting, and diarrhea (4,9) — are late onset. 
Whether children <5 years of age (preschool children) 
have silent infection or clinical disease is controversial 
(4,5,10), which has important implications for both case 
management and prevention. We report our findings from 
prospective, population-based active surveillance. 

The Study 

Since 1998, the ICDDR,B Centre for Health and 


*ICDDR,B Centre for Health and Population Research, Dhaka, 
Bangladesh 


Population Research has operated a surveillance and inter- 
vention site in Kamalapur, an urban slum in Dhaka, 
Bangladesh. We initiated fever surveillance for dengue 
fever and dengue hemorrhagic fever in August 2000. To 
identify treatable causes of fever, we obtained blood cul- 
tures from December 6, 2000, to October 8, 2001. 

The community comprises 7 geographic strata, repre- 
senting 379 clusters. We selected the surveillance cohort 
by using stratified cluster randomization and obtained 
informed written consent from all households. 

Field research assistants screened household members 
for fever in their homes once weekly with a standardized 
questionnaire. We defined fever as >3 consecutive febrile 
days (reported) for persons >5 years of age, or any duration 
of fever for preschool children (<5 years of age). This def- 
inition facilitated detection of dengue fever. Field research 
assistants referred febrile participants to our field clinic, 
where study physicians confirmed fever and collected clin- 
ical data by using a standard form. Patients with an axillary 
temperature of >38°C were designated as febrile. After col- 
lecting blood for serologic tests of dengue and dengue 
hemorrhagic fever, we collected an additional 1 mL of 
blood from preschool children and >3 mL from older 
persons for culture. 

Blood cultures were transported within 2 hours to our 
clinical microbiology laboratory (12 km from the field 
clinic). Specimens were processed by using standard meth- 
ods with in-tube lysis centrifugation (Wampole isolator 
1.5, Carter- Wallace, Inc., Cranbury, NJ, USA), plated on 
blood, chocolate, and MacConkey agar and incubated at 
37°C for 16 to 18 hours. Colonies were evaluated with bio- 
chemical tests and confirmed by serologic identification 
with commercial antisera (Denka, Sieken, Co., Ltd., 
Tokyo, Japan). Antimicrobial susceptibility was deter- 
mined by disk diffusion using standard NCCLS methods 
( 11 ). 

We confirmed typhoid fever if we isolated S. Typhi 
from blood during a febrile episode. Febrile controls were 
culture-negative for S. Typhi, Paratyphi, or Salmonella 
group D during fever. 

If S. Typhi was isolated, then we treated the infection 
with 14 days of standard therapy, adjusting for antimicro- 
bial susceptibility. First-line drugs were amoxicillin (40 
mg/kg up to 1,500 mg orally divided 3 times daily) or cot- 
rimoxazole (10 mg/kg trimethoprim divided into 2 daily 
doses). When patients remained febrile after 72 hours or 
new danger signs (e.g., lethargy, inability to drink, 
cyanosis, convulsions), developed, treatment was consid- 
ered to have failed. We treated treatment failure in persons 
>12 years of age with ciprofloxacin (500 mg orally twice a 
day) and referred younger patients to the hospital. We 
defined recovery as >7 consecutive afebrile days after 
completing therapy. 


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Typhoid Fever in Children in Urban Slum, Bangladesh 


Statistical analysis was performed by using Stata/SE 
Release 8.2 (Stata Statistical Software: Release 8.0. 2003, 
Stata Corporation, College Station, TX, USA). Incidence 
was determined by dividing the number of cases by per- 
son-years of observation, with calculation of exact 95% 
confidence intervals (CIs). Univariate analysis was per- 
formed by using 2-by-2 tables for relative odds (RO) and 
95% CIs. We obtained p values by using the Fisher 2-tailed 
exact test. Multivariate modeling was conducted by step- 
wise forward logistic regression, using all covariates sig- 
nificantly associated with typhoid fever in univariate 
analysis. Co variates that were significant when age, sex, 
and geographic location were controlled for, were retained 
in the final model. We adjusted models for clustering of 
repeat patient visits and tested for goodness-of-fit with 
either Pearson or Hosmer-Lemeshow methods (12). 
Research Review and Ethical Review Committees of 
ICDDR,B approved this study. 

During the study period, we took blood for culture from 
888 (99.9%) of 889 eligible study participants; 54 (6.1%) 
reported prior medication exposure. All specimens had 
adequate volume. A microorganism was isolated from 65 
(7.3%) cultures. Isolation rates were highest in winter. No 
positive culture reported >1 organism (Table 1), nor did 
any culture-positive patient have laboratory-confirmed 
dengue. 

S. Typhi was isolated from 26 preschool children 
(Figure 1) and 23 older study participants (age range 10 
months-50 years, median 4.0 years [95% Cl 3.0— 8.0]). 
There were 1,393 person-years of observation for pre- 
school children and 11,014 for others. Overall, typhoid 
fever incidence was 3.9 episodes/1,000 person-years. 
Typhoid fever incidence among preschool children was 
18.7 episodes/1,000 person-years and 2.1 episodes/1,000 
person-years among older participants. The incidence rate 
difference between the 2 age groups was 16.6 cases/1,000 
person-years (95% Cl 9.4-23.8; p < 0.001). Preschool chil- 
dren’s relative risk for typhoid fever was thus 8.9 (95% Cl 
4.9-16.4). Typhoid fever among preschool children varied 
by age, with 4% in the first year of life and 85% occurring 
in those 2 to 4 years of age (Figure 2). 

We investigated surveillance bias resulting from fever 
definition differences between age groups (4). Preschool 
children’s mean fever duration (days) prior to visiting the 
clinic was 4.0 (95% Cl 3. 2^1. 8) and other patients’ mean 
duration was 4.9 (95% Cl 2.9-6.8, p = 0.37). We collected 
84.6% of preschool specimens and 78.3% of others’ after 3 
febrile days, and 96.2% and 86.7%, respectively, by day 7. 

A multivariate model showed that typhoid fever patients 
were more likely than febrile controls to be preschool age 
(RO 2.04; 95% Cl 1.09-3.82; p = 0.03), have >3 days of 
fever (RO 2.55; 95% Cl 1.16-5.63; p = 0.02), have temper- 
ature >39°C (RO 1.95; Cl 1.01-3.80; p = 0.04), and have 


Table 1. Distribution of 65 blood culture isolates 


Organism 

No. (%) 

Cumulative (%) 

Salmonella Typhi 

49 (75.4) 

75.4 

Staphylococcus epldermldls 

2(3.1) 

78.5 

Aclnetobacter spp. 

4 (6.2) 

84.6 

Salmonella group D 

2(3.1) 

87.7 

Viridans-group Streptococcus 

2(3.1) 

90.8 

Salmonella Paratyphi A 

3 (4.6) 

95.4 

Streptococcus pneumoniae 

2(3.1) 

98.5 

Enterobacter spp. 

1 (1 .5) 

100.0 


mental status changes (RO 3.94; Cl 1.98-7.81; p < 0.02). 
Another model indicated preschool typhoid fever patients 
were significantly more likely than older patients to have 
fever >39°C (RO 1.62; Cl 1.21- 2.17), mental status 
changes (RO 3.54; Cl 2.25-5.55), and crepitations (rales) 
on auscultation (RO 4.44; Cl 3.11-6.33). 

All patients with culture-confirmed typhoid fever 
recovered, except for 1 child with tuberculosis. Four adults 
required ciprofloxacin. No hospitalizations, complications, 
or deaths occurred among confirmed typhoid fever 
patients. 

In vitro antimicrobial susceptibility testing (Table 2) 
showed a high prevalence of ampicillin, cotrimoxazole, 
and chloramphenicol resistance, with 27 isolates (55.1%) 
resistant to all 3; ceftriaxone resistance was found in iso- 
lates from 1 preschool child. Routine nalidixic acid testing 
was not performed, following NCCFS 2000 guidelines. 

Conclusions 

Our data indicate a high infection ratio in this urban 
population, which is highest among preschool children. 
These ratios are comparable to recent regional reports 
(4,6,13) and indicate that typhoid fever in preschool chil- 
dren may be underappreciated. That preschool children 
have 8.9 times the risk for S. Typhi infection as older per- 
sons corroborates age-specific rates in highly disease- 
endemic areas (1). The antimicrobial susceptibility data 



Age (y) 

Figure 1 . Distribution of typhoid fever by age. 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


327 


DISPATCHES 



Age (y) 

Figure 2. Age distribution of patients <5 years of age with typhoid 
fever. 


indicate high ratios of in vitro resistance to standard 
antimicrobial agents, with a high prevalence of multidrug 
resistance. 

The degree of illness of preschool children is controver- 
sial; some report benign bacteremia (5,14) and others have 
found clinical illness (4,13). Our multivariate model shows 
that preschool children are clinically ill. Coexisting condi- 
tions, particularly pneumonia, are not only more common 
in preschool typhoid fever patients but also may result in 
misclassification and underreporting, as well as contribute 
to a worsening cycle of repeated infection and deaths. 
Future studies should explore these issues in this age 
group. 

Substantial clinical illness among preschool children 
argues the need for them to be enrolled in vaccination pro- 
grams. The age- specific infection rates suggest vaccination 
in the first year of life, integrating with existing Expanded 
Programme on Immunization (EPI) schedules. This prac- 
tice would require either a polysaccharide protein-conju- 
gate vaccine to stimulate T-cell-dependent responses (15) 
or a live attenuated oral vaccine, since T-cell-independent 
responses do not mature until the child is 1 8-24 months of 
age. 

The limitations of this study could result in an underes- 
timate of the incidence of typhoid fever. First, this study 
was not designed to measure typhoid fever incidence or 
disease impact. The surveillance program was designed to 
identify dengue. Thus, febrile episodes for young children 


Table 2. Antimicrobial resistance patterns of Salmonella enterlca 
serovar Typhi, Kamalapur, 2001 


Antimicrobial agent 

% resistance 

Ampicillin 

55.1 

Cotrimoxazole 

57.1 

Chloramphenicol 

57.1 

Ciprofloxacin 

0.0 

Ceftriaxone 

2.0 


were defined differently than for older persons. Although 
we did not find evidence of preferential selection for 
young children, future studies may adopt a common fever 
definition. Second, the blood volume examined, though 
not inadequate, may not have been optimal. Third, blood 
culture sensitivity is relatively low, estimated at 25%-50% 
(1). Fourth, the 6.1% estimate of earlier medicine exposure 
may be an underestimate, as we did not validate these 
reports. If these agents were antimicrobial, the number of 
serovar Typhi isolates recovered from peripheral blood 
would be reduced. Fifth, we had only 10 months of obser- 
vation and therefore did not attempt an estimate of disease 
impact, adjustments for blood culture sensitivity, or expo- 
sure to antimicrobial agents. Ours is thus a conservative 
estimate of incidence. Further observation should allow 
the impact of disease to be estimated. 

Acknowledgments 

We gratefully acknowledge Eric Mintz and Pavani Kalluri for 
their suggestions and assistance with the manuscript preparation. 

This study was funded by the International Centre for 
Tropical Disease Research (ICIDR) of the National Institutes of 
Health, by a cooperative agreement from the U.S. Agency for 
International Development (HRN-A-00-96-90005-00), and by 
core donors to the ICDDR,B Centre for Health and Population 
Research. The funding sources had no involvement in the study 
design, interpretation, or decision to submit this paper. 

Author participation in this article was as follows: W.A. 
Brooks was the principal investigator and provided the concep- 
tion, design, execution, and principal data analysis of this study, 
as well as preparing the manuscript. A. Hossain and K. Alam per- 
formed the blood cultures and determined sensitivities. D. 
Goswami and A. Naheed provided overall supervision of the 
project operation. A.T. Sharmeen and K. Nahar were responsible 
for the clinical staff. N. Ahmed supervised field operations. B. 
Nair, S. Luby, and R. Breiman were senior team members who 
contributed to the design, interim discussions of the project’s 
progress, data analysis, and manuscript preparation. 

Dr. Brooks is a specialist in pediatrics and preventive medi- 
cine. He is on faculty at the Bloomberg School of Public Health 
at Johns Hopkins University in Baltimore, Maryland, from where 
he was seconded to ICDDR,B. He established an urban field site 
in 1998, from which he conducts surveillance and intervention 
studies on a variety of infectious diseases, primarily but not 
exclusively in children, including acute respiratory disease, 
dengue, typhoid fever, and shigellosis. 

References 

1. Crump JA, Mintz LS, editors. The global burden of typhoid fever. 

Bull World Health Organ. 2004;82:346-53. 


328 


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Typhoid Fever in Children in Urban Slum, Bangladesh 


2. Keddy KH, Klugman K, Hansford CF, Blondeau C, Bouveret le Cam 
NN. Persistence of antibodies to the Salmonella typhi Yi capsular 
polysaccharide vaccine in South African school children ten years 
after immunization. Vaccine. 1 999; 17:11 0-3 . 

3. Simanjuntak CH, Paleologo FP, Punjabi NH, Darmowigoto R, 
Soeprawoto, Totosudirjo H, et al. Oral immunisation against typhoid 
fever in Indonesia with Ty21a vaccine. Lancet. 1991;338:1055-9. 

4. Sinha A, Sazawal S, Kumar R, Sood S, Reddaiah VP, Singh B, et al. 
Typhoid fever in children aged less than 5 years. Lancet. 
1999;354:734-7. 

5. Ferreccio C, Levine MM, Manterola A, Rodriguez G, Rivara I, 
Prenzel I, et al. Benign bacteremia caused by Salmonella typhi and 
paratyphi in children younger than 2 years. J Pediatr. 1984; 104:899- 
901. 

6. Lin FY, Vo AH, Phan VB, Nguyen TT, Dry la D, Tran CR, et al. The 
epidemiology of typhoid fever in the Dong Thap Province, Mekong 
Delta region of Vietnam. Am J Trop Med Hyg. 2000;62:644-8. 

7. Clemens J, Hoffman S, Ivanoff B, Klugman K, Levine MM, Neira M, 
et al. Typhoid fever vaccines. Vaccine. 1999;17:2476-8. 

8. Levine MM, Noriega F. A review of the current status of enteric vac- 
cines. PNG Med J. 1995;38:325-31. 

9. Agarwal KS, Singh SK, Kumar N, Srivastav R, Rajkumar. A study of 
current trends in enteric fever. J Commun Dis. 1998;30:171-4. 


10. Butler T, Islam A, Kabir I, Jones PK. Patterns of morbidity and mor- 
tality in typhoid fever dependent on age and gender: review of 552 
hospitalized patients with diarrhea. Rev Infect Dis. 1991;13:85-90. 

11. NCCLS. M2-A7-disk diffusion. Performance standards for antimi- 
crobial disk susceptibility test, in CLS document M2-A7. Wayne 
(PA): NCCLS; 2000. 

12. Selvin S. Statistical analysis of epidemiological data. 2nd ed. 
Monographs in epidemiology and biostatistics. Vol. 25. New York: 
Oxford University Press, Inc.; 1996. p. 467. 

13. Saha SK, Baqui AH, Hanif M, Darmstadt GL, Rahulamin M, 
Nagatake T, et al. Typhoid fever in Bangladesh: implications for vac- 
cination policy. Pediatr Infect Dis J. 2001;20:521-4. 

14. Morris JG Jr, Ferreccio C, Garcia J, Lobos H, Black RE, Rodriguez 
H, et al. Typhoid fever in Santiago, Chile: a study of household con- 
tacts of pediatric patients. Am J Trop Med Hyg. 1984;33:1198-202. 

15. Lin FY, Ho VA, Khiem HB, Trach DD Bay PV, Thanh TC, et al. The 
efficacy of a Salmonella Typhi Vi conjugate vaccine in two-to-five- 
year-old children. N Engl J Med. 2001;344:1263-9. 


Address for correspondence: W. Abdullah Brooks, ICDDR,B: Centre for 
Health and Population Research, GPO Box 128, Mohakhali, Dhaka 1000, 
Bangladesh; fax:503. 210.0453; email: abrooks@icddrb.org 



The Ellison Medical Foundation 

Senior Scholar Award in Global Infectious Disease 


Request for Letters of Intent - Deadline: March 9, 2005 


The Ellison Medical Foundation, established by Lawrence J. Ellison, announces the fifth year of a 
program to support biomedical research on parasitic and infectious diseases caused by viral, 
bacterial, protozoal, fungal or helminthic pathogens that are of major global public health concern 
but are relatively neglected in federally funded research within the U.S. Letters of intent for the 
Senior Scholar Award in Global Infectious Disease arc due in the foundation office by March 9, 
2005. 


The intent of the Global Infectious Disease program is to focus its support by placing emphasis on: 

• Innovative research that might not be funded by traditional sources, such as projects involving 
the application of new concepts or new technologies whose feasibility is not yet proven, 
projects seeking commonalities among pathogens that might yield new insights into 
mechanisms of disease, projects seeking to bring together diverse scientific disciplines in the 
study of infectious diseases, or support to allow established investigators to move into a new 
research area. 

• Aspects of fundamental research that may significantly impact the understanding and control 
of infectious diseases, but have not found a home within traditional funding agencies. 

Those submitting successful letters of intent will be invited to submit full applications. Evaluation 
is performed by a two phase process involving the Foundation’s Global Infectious Disease Initial 
Review Group and Scientific Advisory Board. Reviewers will pay close attention to arguments as 
to why the proposed work is unlikely to be supported by established sources. Up to ten Senior 
Scholar Awards will be made in the Fall, 2005. 

Eligibility: Established investigators employed by U.S. 501(c)(3) institutions, or U.S. colleges or 
universities, are eligible to apply. There is no limit on the number of Senior Scholar letters of intent 
submitted from any one institution. Whereas the Foundation only makes awards to U.S. nonprofit 
institutions, the Global Infectious Disease program encourages formation of research consortia 
between U.S. institutions and those in other disease-endemic countries, as through a subcontract 
mechanism, when such collaborations will benefit the proposed research. Current or past Senior 
Scholar Awardees are not eligible to apply. 

Terms of the Award: Each award will be made for up to $150,000 per year direct cost, with full 
indirect cost at the institution's NIH negotiated rate added to that, for up to four years. 

Complete Application Details: For further information, sec the foundation website at 
http://\% ww.ellisonfoundation.org . 

Address any questions to: Richard L. Sprott, Ph.D. 

Executive Director, The Ellison Medical Foundation 

4710 Bcthcsda Avenue, Suite 204 

Bethesda, MD 20814-5226 

Phone: 301/657-1830 

Fax: 301/657-1828 

Emai I : rsprott@el I ison foundation .org 


EID 


OnZinz 

www.cdc.gov/eid 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


329 


DISPATCHES 


Molecular Evidence 
of Pneumocystis 
T ransmission 
in Pediatric 
Transplant Unit 

Britta Hocker,* Constanze Wendt,t 
Aimable Nahimana4 Burkhard Tonshoff,* 
and Philippe M. Hauser^ 

We describe an outbreak of Pneumocystis jirovecii 
pneumonia in a pediatric renal transplant unit, likely attrib- 
utable to patient-to-patient transmission. Single-strand con- 
formation polymorphism molecular typing showed that 3 
affected patients had acquired the same 2 strains of 
Pneumocystis, which suggests interhuman infection. An 
infant with mitochondriopathy was the probable index 
patient. 

D espite intensive medical treatment, Pneumocystis 
jirovecii pneumonia (PCP) is still a severe disease in 
immunocompromised patients, with a high death rate of 
up to 50 % (1). The first report of human Pneumocystis 
infection appeared in 1909; nevertheless, its epidemiology 
is poorly understood to date. In the 1950s, reports on PCP 
epidemics in malnourished infants in hospitals and orphan- 
ages aroused suspicion of interhuman transmission. In 
addition, animal studies have demonstrated airborne trans- 
mission of Pneumocystis (2). A case-control study con- 
ducted for a cluster of 5 PCP cases in transplant recipients 
suggested transmission of P. jirovecii from AIDS patients 
to other immunosuppressed persons (3). However, molec- 
ular typing methods for P. jirovecii were lacking so that 
patient-to-patient transmission could not be assessed at the 
molecular level. When such techniques were developed in 
the 1990s, 3 analyses showed different P. jirovecii geno- 
types within clusters (4-6). A recent analysis at the molec- 
ular level of a cluster of 10 PCP cases strongly suggested 
that HIV-infected persons with active PCP transmitted P. 
jirovecii to renal transplant recipients (7). The role of inter- 
human transmission of P. jirovecii in the epidemiology of 
PCP is still unclear. 

The Outbreak 

Having observed no occurrence of PCP in our pediatric 


*University Children’s Hospital, Heidelberg, Germany; fHygiene 
Institute Heidelberg, Germany; and ^University Hospital of 
Lausanne, Lausanne, Switzerland 


renal transplant unit for the last 20 years and only 1 case in 
all German pediatric renal transplant units during the last 
10 years, we encountered 3 consecutive incidents of PCP 
during a 5 -month period. The first patient was a 13 -year- 
old girl, who had received her second renal graft because 
of cystic kidney disease; PCP developed 4 months after 
transplantation. The second patient, a 14-year-old boy, fell 
ill in the ninth posttransplant month; he had bilateral vesi- 
coureteral reflux as underlying renal disease. The third 
patient was a 13-year-old girl, who had a transplant 2 years 
before contracting PCP because of cystic renal dysplasia 
occurring in the context of Bardet-Biedl syndrome. 

All 3 children had been given cyclosporine A (average 
dose 6.7 mg/kg/day), mycophenolate mofetil (1,060 
mg/m 2 /day), and methylprednisolone (3.2 mg/m 2 /day), as 
maintenance immunosuppression. One patient had also 
received induction therapy with the interleukin-2-receptor- 
antibody basiliximab. All 3 children had been treated with 
methylprednisolone pulses for acute rejection episodes 2, 
3, and 15 months before PCP was diagnosed. 

Clinically, all patients showed nonspecific symptoms, 
such as mild fever, dyspnea, and dry cough in the absence 
of auscultatoric anomalies. Laboratory tests showed an 
elevation of lactic dehydrogenase activity, C-reactive pro- 
tein concentration in blood, and pronounced hypercal- 
cemia (2. 7-3. 5 mmol/L), which was interpreted as an 
extrarenal production of 1.25 -dihydroxy vitamin D 3 by 
activated alveolar macrophages. We found a significant 
reduction of S-adenosylmethionine concentration in plas- 
ma (6 nmol/L; normal range 86-128 nmol/L), which 
appears to be specific to PCP, unlike bacterial or other 
atypical pneumonias (8). We measured the blood count of 
CD4+ and CD4/DR+T lymphocytes in the third patient to 
indicate the degree of immunosuppression, since antirejec- 
tion therapy had been administered 15 months before the 
occurrence of PCP. The number of CD4+T cells was nor- 
mal at the time of PCP diagnosis (1,100 cells/qL; normal 
range 505-1,151 cells/qL), while the number of activated 
T-helper cells was slightly decreased (24 CD4/DR+ 
cells/qL; normal range 29-87/|iL). Only in the course of 
PCP did the numbers of CD4+- and CD4/DR+-T lympho- 
cytes drop significantly (308 CD4+-T cells/qL and 8 
CD4/DR+ cells/qL). Chest radiographs and thorax comput- 
ed tomographic scans of the 3 children showed typical 
signs of interstitial pneumonia, e.g., ground-glass opacity. 

Diagnosis of PCP was confirmed by the presence of 
cysts and vegetative forms in bronchoalveolar lavage fluid, 
proved by immunofluorescence staining, and through 
detection of Pneumocystis DNA by means of polymerase 
chain reaction (PCR). In spite of intensive antimicrobial 
therapy, 2 of our 3 renal transplant patients died, at 10 and 
28 days, respectively, after the onset of PCP. 

To determine if PCP could have been caused by patient- 


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Pneumocystis Transmission in Pediatric Transplant Unit 


to-patient transmission, we closely examined the course of 
PCP in the infected children (Figure). Patient 1 stayed on 
the same ward and same floor, but not in the same room 
(distance between the rooms’ doors ~10 m), as an infant 
with a yet-unclassified mitochondriopathy and pneumonia, 
which later was diagnosed as PCP Patient 2’s hospital stay 
overlapped that of patient 1 ; the patients were on the same 
ward and same floor, in rooms with doors separated by 8 
m, before the onset of PCP. Patient 3 spent her holiday 
with patient 2 in a summer camp organized by our 
Pediatric Nephrology Division. 

Proceeding on the assumption that PCP in the 4 chil- 
dren resulted from patient-to-patient transmission, we 
investigated the genotypes of P. jirovecii with the multitar- 
get single-strand conformation polymorphism (SSCP) 
method. This typing procedure is based on the amplifica- 
tion by PCR of 4 variable regions of the genome, followed 
by the detection of polymorphisms by means of SSCP. 
These 4 genomic regions are as follows: internal tran- 
scribed spacer number 1 of the nuclear rRNA genes oper- 
on (ITS1), the intron of the nuclear 26S rRNA gene (26S), 
the variable region of the mitochondrial 26S rRNA gene 
(mt26S), and the region surrounding intron number 6 of 
the (3-tubulin gene ((3-tub). Typing procedures were carried 
out as described elsewhere (9). A variable region amplified 
from a bronchoalveolar lavage fluid specimen can gener- 
ate either 2 bands (simple pattern) or >2 bands (complex 
pattern). While a simple pattern corresponds to a single 
allele of the genomic region, the presence of >2 bands 
(complex pattern) indicates the existence of several alleles 
for a given region, most probably attributable to coinfec- 
tion with multiple P. jirovecii types (10). 

Our analysis showed that all 3 renal transplant patients 
had acquired the same 2 strains of Pneumocystis , types 1 
and 2. The infant with mitochondriopathy had been infect- 
ed with >2 strains, which possibly included types 1 and 2 
(Table). In contrast, 3 unrelated cases in patients (patients 
4, 5, and 6) from the same hospital harbored other P. 
jirovecii types. The index of discrimination of the method 
was high (0.93) (10), and the probability that patients 1, 2, 
and 3 were infected with the same strains by chance is 
extremely low. We observed that the proportion of coin- 
fecting strains within the clinical specimen was more 
important than the amount of template DNA to detect or 


not detect a strain by means of the SSCP typing method. A 
coinfecting strain has to represent 1 1 % of the population to 
be detected (11). Coinfecting strains are missed when very 
low amounts of template DNA are used, sometimes result- 
ing in a negative PCR; however, this was not the case for 
the specimens analyzed in the present study. 

The index patient had more strains than the other 
patients (Table) but generated smaller amounts of PCR 
products with the 4 different PCR tests used, which sug- 
gests a lower amount of DNA template. Although exclud- 
ing a common source of P. jirovecii is difficult, the results 
strongly suggest that all 3 kidney transplant recipients had 
infected each other, and the infant could have acted as 
index patient. This transmission may have occurred direct- 
ly from 1 patient to another but also indirectly through 
immunocompetent carriers. Indeed, carriage of P. jirovecii 
DNA in the nose of immunocompetent relatives and 
healthcare workers in close contact with a PCP patient has 
been described (12). 

Conclusions 

To our knowledge, this report is the first published on 
an outbreak of PCP in a pediatric renal transplant unit, 
probably attributable to patient-to-patient transmission. 
However, we cannot exclude that the cases described were 
infected by the same environmental source. The presence 
of P. jirovecii in the air of hospital corridors has been 
described (13), making an environmental reservoir in the 
hospital possible. Other potential sources of P. jirovecii 
could be asymptomatic P. jirovecii carriers, such as 
immunosuppressed patients (14,15). Our findings at the 
molecular level suggest that P. jirovecii may be transmitted 
nosocomially and be acquired by immunosuppressed pedi- 
atric transplant recipients. The incubation periods of P. 
jirovecii infection (17, 15, and 19 weeks for patients 1, 2, 
and 3, respectively) would be longer than those (2-12 
weeks) suggested by the previously described clusters of 
PCP (3,7,16). This finding may reflect a difference 
between adults and children. 

Until the outbreak of PCP outlined in this article, pedi- 
atric renal transplant recipients in our hospital and other 
pediatric renal transplant units in Germany were not given 
PCP prophylaxis routinely because of possible side effects, 
such as a rise of serum creatinine values, myelosuppression, 


Week 2002 

10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 
Index Pat. > X D 

Pat. 1 RTx + R R > X D 

Pat. 2 RTx Ja nuary 2002 R$ > X D 

Pat. 3 RTx De cember 2000 > X 


Figure. Course of Pneumocystis 
pneumonia (PCP) in 3 pediatric renal 
transplant recipients and 1 infant suf- 
fering from a yet-unclassified mito- 
chondriopathy. R, acute rejection 
episode; RTx, renal transplantation; 
4, contact; #, joint holiday; > start of 
PCP symptoms; - , hospitalization; 
X, diagnosis of PCP; D, death. 


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331 


DISPATCHES 


Table. Pneumocystis jirovecii genotyping by PCR-SSCP of 4 genomic regions* 






SSCP pattern 



Patient 

Date 

ITS1 

26S 

mt26 

p-tub 

P. jirovecii type 

Index case-patient 

3/27/02 

A 

A, B 

A, B,C 

A, B 

>2 types (nonidentifiable) 

1 

7/26/02 

A 

A, B 

A 

A 

1, 2 

2 

10/30/02 

A 

A, B 

A 

A 

1, 2 

3 

12/3/02 

A 

A, B 

A 

A 

1, 2 

4 

2/7/03 

A, B 

A, B, C 

B, C 

B, C 

>2 types (nonidentifiable) 

5 

2/1 5/03 

A 

A, B 

C 

A 

6, 44 

6 

2/1 8/03 

A, B 

A, B 

A 

C 

45, 46 

*Bold letters signify the most abundant simple pattern with the complex one, as shown by silver staining. Bold numbers signify the most abundant 
P. jirovecii type. PCR, polymerase chain reaction; SSCP, multitarget single-strand conformation polymorphism. 


and Lyell syndrome. We had not observed any case of PCP 
in our transplant recipients for the last 20 years without 
prophylaxis. In the light of the high death rate for PCP, 
prophylactic treatment with trimethoprim- sulfamethoxa- 
zole is highly recommended for the first 6 posttransplant 
months and during the 4 months after antirejection thera- 
py, in accordance with the guidelines for adults (1). 
According to these guidelines, patient 3, in whom PCP 
developed 15 months after steroid pulse therapy, would not 
have been protected by prophylaxis. Whether prophylaxis 
should be given for a longer period of time remains 
unknown, particularly since immunosuppression did not 
appear to be intensive in this patient at the onset of PCP, as 
indicated by the normal CD4+ T-lymphocyte count in 
peripheral blood and the only slightly decreased number of 
activated T-helper cells. 

Dr. Hocker is a physician and research fellow at the 
University Children’s Hospital, Heidelberg, Germany. Her 
research activities are focused on immunosuppressive therapy 
and diagnosis and treatment of opportunistic infections in pedi- 
atric renal transplant recipients. 

References 

1 . EBPG Expert Group on Renal Transplantation. European best prac- 
tice guidelines for renal transplantation. Section IV: long-term man- 
agement of the transplant recipient. IV.7.1 Late infections. 
Pneumocystis carinii pneumonia. Nephrol Dial Transplant. 
2002;17(Suppl 4):36-8. 

2. Hughes WT. Current issues in the epidemiology, transmission, and 
reactivation of Pneumocystis carinii. Semin Respir Infect. 
1998;13:283-8. 

3. Chave JP, David S, Wauters JP, Van Melle G, Francioli P. 
Transmission of Pneumocystis carinii from AIDS patients to other 
immunosuppressed patients: a cluster of Pneumocystis carinii pneu- 
monia in renal transplant recipients. AIDS. 1991;5:927-32. 

4. Olsson M, Eriksson BM, Elvin K, Strandberg M, Wahlgren M. 
Genotypes of clustered cases of Pneumocystis carinii pneumonia. 
Scand J Infect Dis. 2001;33:285-9. 


5. Latouche S, Poirot JL, Maury E, Bertrand V, Roux P. Pneumocystis 
carinii hominis sequencing to study hypothetical person-to-person 
transmission. AIDS. 1997;11:549. 

6. Helweg-Larsen J, Tsolaki AG, Miller RF, Lundgren B, Wakefield AE. 
Clusters of Pneumocystis carinii pneumonia: analysis of person-to- 
person transmission by genotyping. Q J M. 1998;91:813-20. 

7. Rabodonirina A, Vanhems P, Couray-Targe S, Gillibert RP, Ganne C, 
Nizard N, et al. Molecular evidence of interhuman transmission of 
Pneumocystis pneumonia among renal transplant recipients hospital- 
ized with HIV-infected patients. Emerg Infect Dis. 2004;10:1766-73. 

8. Skelly M, Hoffman J, Fabbri M, Holzman RS, Clarkson AB Jr, 
Merali S. S-adenosylmethionine concentrations in diagnosis of 
Pneumocystis carinii pneumonia. Lancet. 2003;361:1267-8. 

9. Hauser PM, Francioli P, Bille J, Telenti A, Blanc DS. Typing of 
Pneumocystis carinii f. sp. hominis by single-strand conformation 
polymorphism of four genomic regions. J Clin Microbiol. 
1997;35:3086-91. 

10. Hauser PM, Blanc DS, Sudre P, Senggen Manoloff E, Nahimana A, 
Bille J, et al. Genetic diversity of Pneumocystis carinii in HlV-posi- 
tive and -negative patients as revealed by PCR-SSCP typing. AIDS. 
2001;15:461-6. 

11. Nahimana A, Blanc DS, Francioli P, Bille J, Hauser PM. Typing of 
Pneumocystis carinii f.sp. hominis by PCR-SSCP to indicate high 
frequency of co-infections. J Med Microbiol. 2000;49:753-8. 

12. Vargas SL, Ponce CA, Gigliotti F, Ulloa AV, Prieto S, Munoz MP, et 
al. Transmission of Pneumocystis carinii DNA from a patient with P. 
carinii pneumonia to immunocompetent contact health care workers. 
J Clin Microbiol. 2000;38:1536-8. 

13. Bartlett MS, Vermund SH, Jacobs R, Durant PJ, Shaw MM, Smith 
JW, et al. Detection of Pneumocystis carinii DNA in air samples: 
likely environmental risk to susceptible persons. J Clin Microbiol. 
1997;35:2511-3. 

14. Hauser PM, Blanc DS, Bille J, Nahimana A, Francioli P. Carriage of 
Pneumocystis carinii by immunosuppressed patients and molecular 
typing of the organisms. AIDS. 2000;14:461-3. 

15. Vargas SL, Ponce CA, Sanchez CA, Ulloa AV, Bustamante R, Juarez 
G. Pregnancy and asymptomatic carriage of Pneumocystis jirovecii. 
Emerg Infect Dis. 2003;9:605-6. 

16. Goesch TR, Gotz G, Stellbrinck KH, Albrecht H, Weh HJ, Hossfeld 
DK. Possible transfer of Pneumocystis carinii between immunodefi- 
cient patients. Lancet. 1990;336:627. 


Address for correspondence: Britta Hocker, University Children's 
Hospital, Im Neuenheimer Feld 150, 69120 Heidelberg, Germany; fax: 
49-6221-564203; email: Britta_Hoecker@med.uni-heidelberg.de 


332 


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First Self 

Gerald N. Callahan* 


S lowly, purposefully, my mother unbuttons her blouse. 

It is blue with small white flowers, and the tail is 
tucked firmly into the elastic waistband of her salmon-pink 
pants. Beginning at the top and moving down, she works 
carefully at each of the small plastic buttons. 

“Mother,” I plead with her, “you don’t need to do that.” 
She smiles at me and continues unfastening buttons. 
Her padded cotton and elastic bra begins to appear. Her 
breasts swell pallidly above it. 

The room is not well lit. The curtains are drawn, as they 
always are, against the sun. But I can see more of my 
mother than I wish to. My father, sitting here with me, says 
nothing. My wife, Gina, and two other women in the room 
also sit silently as my mother undresses herself. 

I smell her perfume as she works at her blouse, her per- 
fume and the lotion she lathers herself with every morning. 
I see the wrinkles beneath her arms, the flaps of skin at the 
elbows. She pulls off the blouse and stands before us with 
it in her right hand. 

Her gray hair sprays in every direction. Her back is lit- 
tered with small brown moles, her skin like ice over an old 
pond. And her dark eyes, fallen far back in the sockets of 
her skull, flutter from face to face like moths. 

This is not, of course, my mother. My mother would 
never have bared this much of herself in front of strangers 
and certainly never in front of her son. My mother was 
quiet, shy, prudent. And this is, of course, my mother, her 
face, her hands, her dried out, fungus-ruined feet. But 
things have changed. 

The nondescript, nappy, brown carpet is just as it 
always has been. The counters are still lined with the detri- 
tus of middle-class life. The cheap fan still chops at the air 
overhead, and the draining board with its plastic dish rack 
still drips dishwater into the same stainless steel sink. The 
clock with its three golden balls spools out the hours like 
kites, just as it always has. But my mother is mad. And the 
six of us have gathered today to evaluate her for custodial 
care. Custodial care! It sounds as though we might turn her 
over to the janitors at the university where I work. As 
though they might know what to do with her since we 


*Colorado State University, Fort Collins, Colorado, USA 


don’t. Appalled or not, though, we have no more time to 
twist our tales. 

The Self 

I am an immunologist. I have spent my life studying the 
intricate paths by which we protect ourselves from this 
infectious world, studying self, non- self, and why the two 
should never meet. But as a son watching his mother dis- 
integrate, I am cut adrift. 

My mother’s self, the thing that was her for all these 
years, the thing I had imagined fixed as flint beneath her 
bones has fractured, shattered like a crystal vase dropped 
on concrete. It is one thing to watch feathers grow from 
chicken-skin grafts on nude mice, quite another to watch 
your mother undress herself in front of total strangers. 

Merriam Webster says that self is “the entirety of an 
individual, the realization or embodiment of an abstrac- 
tion” (1). I don’t know what that means. Even though it 
somehow feels right, it seems woefully incomplete and 
metaphysical. As though no human using ordinary lan- 
guage could truly speak of my mother’s disappearance, no 
matter how concretely and obviously she is disappearing. 

Sir Frank Macfarlane Burnet first described the neces- 
sity of biological self after watching an ameba ingest and 
digest another microorganism: “The fact that one is digest- 
ed, and the other not, demands that in some way or other 
the living substance of the ameba can distinguish between 
the chemical structure characteristics of ‘self’ and any suf- 
ficiently different chemical structure as ‘non-self’” (2). 
Later in contemplation of an immune response, Burnet 
added, “The failure of antibody production against autolo- 
gous cells demands the postulation of an active ability of 
the reticulo-endothelial cells to recognize ‘self’ patterns 
from ‘ non- self ’ patterns in organic material taken into their 
substance” (3). “Demands” for after all, even the most 
primitive of us do not regularly eat ourselves. And even the 
most complicated of us do not regularly mistake our bod- 
ies for infectious enemies and destroy the very thing that 
sustains us. Burnet’s self becomes something substantial, 
something unique that our appetites and our immune sys- 
tems ignore while they chew away at the rest of the organ- 
ic world. 


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ANOTHER DIMENSION 


The fact that on the surface these two selves — the self 
of Merriam Webster and the self of Macfarlane Burnet — 
seem incommensurate, we probably owe to the Frenchman 
Rene Descartes. 

The Divided Self 

Descartes, a mathematician and philosopher, found 
himself one day deeply concerned with the reality of 
things. What could he truly trust? What was rationally and 
irrefutably real? We all know that we make mistakes at 
times about what is real — the monster under the bed, the 
shadow in the closet, purpose. Most of us just shrug it off, 
but Descartes was not so easily mollified. He secluded 
himself in a darkened room at the back of his chateau and 
set out to discover what was demonstrably real, trustwor- 
thy, certain (4,5). 

Descartes considered what we learn through the senses, 
the stuff we see, hear, taste, touch, and smell — the physi- 
cal world that apparently surrounds us. Is any of it truly 
real, unquestionably real? No. Almost immediately, he 
realized our senses can fool us. Dreams provide hard evi- 
dence of that. While we are in a dream, we become com- 
pletely absorbed with false reality. Dreams do not 
announce to us that they are not “real.” And many things 
in the “real” world (mirages, optical illusions, sleights of 
hand) do not announce to us that they are false. 

Descartes, the inventor of analytical geometry, turned 
to the reality of mathematics, a priori knowledge — knowl- 
edge accessible without sensory perception. Because of his 
deep investment in mathematics, Descartes thought a pri- 
ori knowledge inviolable, beyond reproach, above suspi- 
cion. But as he delved deeper, he realized that some evil 
genius might have fooled us about mathematics. 
Mathematics might be nothing more than an elaborate ruse 
with nothing whatsoever to do with reality (as many of us 
suspected in grade school). He was forced to abandon 
mathematics as well as sensory knowledge. Without math- 
ematics and the physical world the only things left to 
Descartes were his own thoughts. He realized that ration- 
ally and philosophically he could not question the reality 
of the questioner. It simply wouldn’t make sense. So his 
questions proved his own existence, even if he could not 
establish the existence of anything else. Cogito ergo sum. 

Had Descartes been a microbiologist, things might have 
ended differently. But for the mathematician, the world 
devolved to one man’s thoughts. Descartes rested then, in 
the midst of an absolutely solitary universe. Two types of 
things existed, the seemingly real but demonstrably 
untrustworthy physical world ( res extans ), and the truly 
real world of the mind ( res cogitans). These were two 
completely separate worlds. The one outside our heads 
was full of machines and ghosts, including our own bod- 
ies. The one in our thoughts was concrete, real, essential. 


Reality flourished inside human thought, specifically 
Descartes’ thought. The rest was doubtful. When he was 
finished, Descartes had scalpeled the self off the body. 

The self, he claimed, was something other than the 
physical world that surrounds us. Selves did not come 
from the same stuff as trees, and stones, and arms, and 
legs, and knuckles, and immune systems. Selves came 
from somewhere else. Self stuff and body stuff were dis- 
tinct and immiscible. 

But almost 400 years later, as I watch my mother fum- 
ble with the tails on her blouse, I am little comforted by 
Rene Descartes. 

The Biological Self 

We finally convince my mother to put her blouse back 
on and button it. It takes her two tries, but she now has 
each button in its proper hole. I am embarrassed. She 
seems unabashedly pleased with herself — what remains of 
it. She smiles again at me. I turn to one of the women seat- 
ed quietly across the room. I look for forgiveness or some 
sort of reassurance that my mother’s antics haven’t ruined 
this for all of us. 

“She will do perfectly,” Jennifer says. 

“Does she wander at night?” Melissa asks. 

Lesch-Nyhan disease was first described in 1964, by 
two physicians named Michael Lesch and William Nyhan. 
Two brothers appeared one afternoon in these doctors’ 
clinic. Both boys were manifesting bizarre but identical 
symptoms. Much about these original cases turned out to 
be characteristic of most cases of Lesch-Nyhan disease, 
which affects boys almost exclusively. And the fact that the 
disease manifested identically in the twin boys suggested 
that an altered gene was involved. 

Lesch-Nyhan disease is caused by a mutant gene on the 
X chromosome. Women have two X chromosomes, men 
only one. So problems on an X chromosome are some- 
times hidden in women by the normal allele on the alter- 
nate X chromosome. But Y chromosomes have almost 
nothing in common with X chromosomes. So X-linked 
mutations are almost always apparent in men. 

A single mutation in the gene that encodes an enzyme 
called hypoxanthine-guanine phosphoribosyl transferase, 
or HGPRT, is responsible for Lesch-Nyhan disease. In its 
mutant form, the enzyme does not function. HGPRT cat- 
alyzes a biochemical process called purine salvage. 
Purines are used to make DNA and RNA — the stuff of 
genes and genetic control and transcription. Because of the 
importance of DNA synthesis, we have more than one way 
to make purines. We can synthesize purines from scratch 
or salvage purines from DNA-breakdown products in our 
blood (6). People with Lesch-Nyhan disease cannot sal- 
vage purines. These people must rely on the purines they 


334 


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First Self 


synthesize from scratch as their only source of purines 
because their purine-salvage pathways do not work. On the 
surface, that doesn’t seem like such a bad thing. Beneath 
the surface, the effects are horrifying. 

One of the manifestations of Lesch-Nyhan disease is 
unmistakable. Somewhere between three months and four 
years of age, boys with Lesch-Nyhan disease begin to self- 
mutilate, and they become very creative at it. These boys 
chew off their lips, chew their fingers to bloody stubs. If 
not restrained, they engage in head banging, arm and leg 
banging, nose and eye gouging — sometimes blinding 
themselves. And they may, against their overt wishes, 
attack their care givers, sometimes doing considerable 
harm to the ones they love most. 

A single change in a single gene results in massive 
changes in human behavior, massive changes in self-per- 
ception, massive alterations of self. After all, what is more 
basic to self-perception and self-preservation than the abil- 
ity to distinguish self from non-self and eat only non-self? 
Descartes was wrong. Wrong! Because clearly, and dra- 
matically, and horribly res extans warps res cogitans. 

We know of several infectious agents that also alter 
human and animal behavior. Toxoplasma gondii makes rats 
fond of cat urine. Wolbachia changes insects’ sexual pref- 
erences. Borna-disease virus shows up more often in peo- 
ple with certain behavioral disorders. Dicrocelium 
dendriticum , a parasitic fluke, makes ants fond of heights 
and more likely to be eaten by cows, D. dendriticum' s pri- 
mary host. Euhaplorchis calif orniensis, another fluke, 
makes fish swim in shallow waters, where the fish are 
more likely to be eaten by birds, this fluke’s primary host. 
And Streptococcus infections seem to predispose some 
children to obsessive compulsive disorders (7). We will 
probably find many more infectious microorganisms that 
alter human behavior. 

Our selves are not something ethereal, something 
forged from a separate reality. Our selves are no different 
from our livers or our hearts. Our selves are just as suscep- 
tible to the effects of breeding and infection as any other 
part of us. So, just as there is biology of reproduction or 
respiration, there must be biology of self. Who we are is 
not simply a matter of spirit or story. It is in our genes — 
those we are born with, and those we acquire. Genes arose 
and were preserved over eons to protect us, to provide each 
of us with some specific edge in the struggle for survival 
and reproduction. Our genes come from a very long line of 
survivors and reproducers. In these genes is the template 
for self. 

“She does wander at night,” I add, wishing I didn’t have 
to. “Twice, that I know of, Dad found her outside the house 
in Kanab, Utah, making her way toward town. The first 
time she wasn’t even wearing the bottoms of her pajamas. 
When he stopped her and asked where she was going, she 


said, ‘Home. I’m going home.’ My father could not make 
her understand that she was home.” 

The Evolution of Self 

If selves are born inside genes, then like all things bio- 
logical, selves must have an evolutionary advantage and an 
evolutionary history. In the beginning there was RNA 
(probably), RNA that snapped together spontaneously 
from the molecules afloat in the primitive seas. Then there 
was DNA twisted into long chains and wrapped in bits of 
fat. Later, true cells appeared. Life — bacteria, archaea, 
prokaryotes, eukaryotes — a most remarkable gift began to 
unwrap itself. Everything was suddenly possible. And 
from the outset there were only three rules that governed 
us: eat, don’t get eaten, reproduce as quickly and as often 
as possible — three rules alone that would account for all 
who followed. 

First-self had walked onto the stage. Pronouns became 
meaningful. “Us” was no longer sufficient to describe 
everything, “me” and “you” were necessary now. While 
sense of self was perhaps a long way off, self was there, 
that day, swimming in a thin broth of “other.” 

Bacteria were a major step up from the muck. They 
were, after all, living, but they suffered from one huge 
drawback: each of them had only one cell to work with. 
That meant then, and still means now, that most bacterial 
cells had to do everything, all the time, all at once. Each 
cell had to see, hear, touch, taste, and smell. Each cell had 
to eat and excrete, reproduce and think. Each cell had to 
make everything that was needed for the survival of the 
individual. Because of this, bacteria, though remarkable 
survivors, weren’t and aren’t much good at anything 
beyond simple survival — poetry probably baffles them. 

One day, all of this began to change. A few cells got 
together, cemented themselves to one another with some 
new glue. A protoplasmic hand reached into the void and 
another hand took hold. The door of opportunity swung 
wide open. For the first time, individual cells were freed 
from necessity. No longer did anyone have to be every- 
thing for everyone. No longer did anyone face everything 
alone. 

Cellular specialization took the world by storm. Some 
cells stopped eating and became eyes (or something that 
would one day become eyes), others ears, others nerves, 
others muscles — there were no limits. Taste buds, anten- 
nae, pincers, intestines, hearts, tails, legs, arms, muscles, 
bones, livers, lungs, hair, nails, claws, blood, hide, and 
horn were all within reach. 

But almost immediately, everyone saw that cellular 
specialization, alone, led nowhere. Before multicellularity 
could be had, selfishness was needed. The first few multi- 
cellular creatures probably shared everything with every- 
one. After all, they had no means to distinguish among 


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335 


ANOTHER DIMENSION 


themselves. All that I have is yours, not because of altru- 
ism, but because I cannot tell you from me. Such largesse 
defeated the whole purpose of cellular specialization. 
What benefit is there to eyes, if what I see I share with the 
blind who surround me? Remember there are rules. I 
come first. I am not to be eaten by others. I am to eat oth- 
ers. I am to reproduce first. The things I see are for me and 
me alone. 

But “I” had no means yet for such distinction. What or 
who was me and what or who was not? I could not decide. 
Before I could reach for the stars, I had to reach within and 
find some way to know myself from others. Without a 
sense of self we are little more than bacteria — maybe less. 

If I am to keep what I have earned, if I alone am to ben- 
efit from my mutations and absorptions, my specializa- 
tions, my senses, my motility, I must know self from 
non- self. My eyes must be for my self. My thoughts must 
be my own. My heart must beat only for me. I must keep 
all that I can to my self at the expense of non-self, or I have 
gained nothing. 

Selves leave no fossils, so we cannot know for certain 
how the first colonial (multicellular) organisms came to 
sense their selves. But biologically , biochemically , basi- 
cally, they had to know, everything depended on it. And 
the biology and chemistry of that knowledge were and are 
the only things between each of us and the rest of us. The 
evolved self, the self geneticized. A protein marker, per- 
haps, carried by every cell inside every one of us. A pass- 
port to be checked and rechecked at every interaction. A 
self to be validated over and over. 

For a few millennia, that was probably good enough. 
But life was changing. Microorganisms discovered para- 
sitism. Once inside another’s membrane, food cost noth- 
ing, life was simple, and reproduction was almost 
guaranteed. Now, self discrimination was not enough. 
Once others learned to hide within self, force was needed 
to maintain boundaries. Now, we needed immunity to keep 
us whole. Once again, if our own mutations and adapta- 
tions were to serve us, the integrity of self was essential. 
Infectious diseases posed the first great challenge to the 
biological preeminence of self. Immune systems quickly 
found ways to detect and destroy non-self. 

In the beginning, biological self was probably nothing 
more than a simple system for recognition of other and 
recognition of non-self as food. Now self had teeth. Now 
self rose like a shield to stand between us and those who 
would destroy us to further their own journey towards 
reproduction. We became what we served and protected. 

Infection, Immunity, and Self 

Over time, the self grew. Like the brain, layers upon 
layers of self formed inside living things. Like the cerebral 
cortex, late in evolution, psychological self arose — self- 


conception, self-perception, self-deception. But still, like 
the amygdala in the brain, beneath the complicated and 
sophisticated self beats the heart of a beast, focused only 
on food, survival, and sex. 

Unlike the brain, the layers of self are strewn through- 
out the body. In between the layers is immunity and infec- 
tion. And that, it seems, ties it all together. When the 
psychological self is stressed, the immunologic defense of 
self falters. Perceived threats — exams, other men and 
women, public speaking, air travel — stimulate the hypo- 
thalamus to produce corticotrophin-releasing hormone 
(CRH), which stimulates the pituitary gland to secrete 
adrenocorticotropic hormone (ACTH). ACTH induces the 
adrenal glands to produce cortisol. Cortisol suppresses the 
immune system (8). The two selves synergized. 
Furthermore, the consequence of this change in perception 
of self or environment is, as you might expect, accompa- 
nied by considerable increase in susceptibility to infectious 
diseases (9-13). How we think about ourselves and our 
surroundings changes our resistance to disease. 

Infection and inflammation cross the bridge between 
selves in the opposite direction. Every organ of the 
immune system is innervated. Every spot where an 
immune response takes place is hardwired to the brain. 
Immunology and neurology are irreversibly intertwined. 
Interleukins produced by activated macrophages and T 
cells act on the adrenals, the hypothalamus, the pituitary, 
and the brain stem. In response, moods change, libidos 
drop, self-perception fogs, appetites trail off, and sleep 
becomes nearly impossible (8). 

Infection, inflammation, and immunity shape self. Self- 
perception derails immunity. Cytomegalovirus and T. 
gondii have been implicated in the etiology of schizophre- 
nia (14,15). People with bipolar disorder are more fre- 
quently infected with herpesvirus type 1 than people 
without bipolar disorder (16). Mice born to mothers infect- 
ed with influenza virus never develop a lust for exploration 
(17). And of course infection by other parasites, bacteria, 
and viruses can change animal behavior in unexpected and 
consequential ways. Infection, at least at times, changes 
our mental perceptions of ourselves and our surroundings. 

Infections change our immune perceptions as well. 
Among more than 3 million U.S. military personnel fol- 
lowed from 1988 to 2000, the strongest predictors of mul- 
tiple sclerosis were serum levels of immunoglobulin (Ig) G 
antibodies to Epstein-Barr virus viral capsid antigen or 
nuclear antigen (18). Multiple sclerosis is a remarkable 
autoimmune disease in which the immune system attacks 
the nervous system — self vs. self. And viral or bacterial 
infections have been implicated in the etiologies of 
rheumatoid arthritis, diabetes mellitus, Crohn’s disease, 
and some types of thyroiditis — all autoimmune diseases. 
These examples indicate that at least some infections cloud 


336 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


First Self 


the immunologic border between self and non-self. As our 
understanding of infection and immunologic self-percep- 
tion deepens, more examples will likely surface. Further 
research will elucidate the interdependence between 
immunologic and psychological disorders and the link 
between both and infectious diseases. 

Animals protected from birth against infections never 
develop any functional sense of immunologic self (19). 
Human autoimmune diseases, such as diabetes mellitus, 
show a correlation with schizophrenia (20) and other 
behavioral disorders. Cause and effect relationships 
remain obscure, but a link may exist between infectious 
disease and immunologic as well as psychological percep- 
tion of self. Infection, immunity, and inflammation, like 
water on old plywood, sometimes split and sometimes 
cement the layers of self. But together or apart, self is a 
brick in the bulwark of human biology. 

Last Self 

All her life my mother preferred things simple. She 
liked jam better than jelly. She loved cornbread and black- 
strap molasses, white gravy, melons, “Amazing Grace.” 
She hated driving. She grew up poor, truly poor. Maybe 
poverty burned up all the fuel she was saving for complex- 
ity before she ever found any. Regardless, her tastes never 
changed. She was always most comfortable with ordinary 
things. 

I remember her simplicity. But by the start of the sec- 
ond year of her custodial existence, no matter how hard I 
tried, I could no longer remember much of anything else 
about how she once was. I couldn’t recall when her hair 
might have been brown and combed, her pants not fat with 
diapers, her smile less vacant. 

The craters of her face were gaunt and empty now. 
Every cold and flu wracked her lungs and sent mucus cas- 
cading from her nose, across her mouth, and onto to the 
knit sweaters she wore against the cold. Places where her 
self had played across the geography of her skin were 
abandoned — empty lots overgrown with weeds. With me, 
hers was the purest and truest indifference. I loved her for 
that. I would sit by her for hours simply to bathe in the low, 
warm light of that indifference. 

The smell of urine was everywhere. We were without 
pretense. She told stories, I pretended to listen. Over and 
over she spun those stories around me as though they 
might protect me from something I would have to face 
when she was gone. Layer after layer of her peeled away 
until all that was left was bare wood. The weathered boards 
scoured of paint by the raging storm. Underneath, there 
was fear, often; a fervent sexuality, always; and hunger — 
the vestiges of flames that first flickered billions of years 
ago. Life, infection, and her own defenses had stripped her 
of everything else. Unadorned self. 


The last day she spoke to me, she was dressed in red 
sweat pants and a loose purple jersey top with long 
sleeves. Her nose ran. When I came into her room, she was 
lying in bed staring at the ceiling. She rarely spoke com- 
plete sentences. She never recognized me. I sat next to her 
and for several moments said nothing. 

The room was split in two by a red blanket hung as a 
curtain. On the other side of the curtain was another bed. 
Sometimes Mom had roommates, but for the last week or 
two, no one had occupied the other bed. Lull or empty, the 
other bed held no interest for Mother. The floor was cov- 
ered in spattered beige tiles. 

I enjoyed these moments, sitting quietly next to her — 
moments stolen from reality, shielded from certainty. I 
wasn’t an immunologist here, just an old woman’s son 
wishing for things that could never be. I reached out and 
held her hand, thin now with thickened blue veins and 
knuckles fat from inflammation. She turned to me. 

“Hello,” I said. 

“Hello,” she said with obvious pleasure. 

Then she lifted my hand to her lips and kissed my fin- 
ger tips. 

“How are you?” I asked because whether someone is 
dying, bleeding to death from a severed limb, or just fin- 
ishing a pastrami sandwich, that question inexplicably 
comes to my lips. 

“Line,” she said and turned back to the ceiling, smiling. 
“I’m fine.” 

I wiped her nose. 

Today, she wore no lipstick, and the aides had taken her 
bridge from her mouth. Lour of her lower front teeth were 
missing. Her tongue fell through the opening when she 
spoke and twisted her words. 

“What would you like to do today, Mom?” I asked not 
really expecting anything. 

She looked up at me with eyes deep-brown as 
mahogany. She pursed her lips beneath her small mous- 
tache. And for a moment her eyes moved off to one side as 
though she actually thought about what I’d asked. Linally 
she looked back into my eyes and said to me: 

“I’m hungry.” 

“Then let’s eat.” 

I lifted her into her wheel chair and rolled it down the 
tiled hall to the dining room. She ate Salisbury steak and 
mashed potatoes, green beans and corn, peach pie with ice 
cream. And likely she would have eaten even more if any- 
one had offered it. As she ate, she stared across the top of 
her fork at the brown plastic tabletop. I watched her chin 
moving with the food and her eyes as they chewed slow- 
ly on nothing. Neither of us spoke. There was really no 
need. 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


337 


ANOTHER DIMENSION 


Dr. Callahan is associate professor of immunology/public 
understanding of science and associate professor of English at 
Colorado State University. His research interests focus on the 
relationship between infectious and behavioral disease. 

References 

1. Merriam- Webster’s collegiate dictionary. 10th ed. Springfield (MA): 
Merriam- Webster, Incorporated; 1998. 

2. Burnet FM. Biological aspects of infectious diseases. Cambridge, 
U.K.: Cambridge University Press; 1940. 

3. Burnet FM. The production of antibodies. 2nd ed. London: 
Macmillan; 1949. 

4. Descartes R. Discourse on the method of rightly conducting the rea- 
son, seeking truth in the sciences. Jan Leiden; 1637. 

5. Descartes R. Meditations on first philosophy. 1641. 

6. Hyder A, Jinnah M. Lesch-Nyhan syndrome [monograph on the 
Internet]. 2002 Jan 25 [cited 2005 Jan 7], Available from 
http://www.emedicine.com/neuro/topic630.htm 

7. Callahan GN. Madness. Emerg Infect Dis. 2002;8:998-1002. 

8. Wilder RL. Neuroendocrine-immune system interactions and autoim- 
munity. Annu Rev Immunol. 1995;13:307-38. 

9. Yang EV, Glaser R. Stress-associated immunomodulation and its 
implications for responses to vaccination. Expert Rev Vaccines. 
2002;1:453-9. 

10. Yang EV, Glaser R. Stress-induced immunomodulation: implications 
for tumorigenesis. Brain Behav Immun. 2003;17(Suppl l):S37-40. 

1 1 . Yang EV, Glaser R. Stress-induced immunomodulation and the impli- 
cations for health. Int Immunopharmacol. 2002;2:315-24. 

12. Yang EV, Glaser R. Stress-induced immunomodulation: impact on 
immune defenses against infectious disease. Biomed Pharmacother. 
2000;54:245-50. 


13. Glaser R, Rabin B, Chesney M, Cohen S, Natelson B. Stress-induced 
immunomodulation: implications for infectious diseases? JAMA. 
1999;281:2268-70. 

14. Torrey EF, Yolken RH. Toxoplasma gondii and schizophrenia. Emerg 
Infect Dis. 2003;9:1375-80. 

15. Leweke FM, Gerth CW, Koethe D, Klosterkotter J, Ruslanova I, 
Krivogorsky B, et al., Antibodies to infectious agents in individuals 
with recent onset schizophrenia. Eur Arch Psychiatry Clin Neurosci. 
2004;254:4-8. 

16. Dickerson FB, Boronow JJ, Stallings C, Origoni AE, Cole S, 
Krivogorsky B, et al. Infection with herpes simplex virus type 1 is 
associated with cognitive deficits in bipolar disorder. Biol Psychiatry. 
2004;55:588-93. 

17. Shi L, Fatemi SH, Sidwell RW, Patterson PH. Maternal influenza 
infection causes marked behavioral and pharmacological changes in 
the offspring. J Neurosci. 2003;23:297-302. 

18. Levin LI, Munger KL, Rubertone MV, Peck CA, Lennette ET, 
Spiegelman D, et al. Multiple sclerosis and Epstein-Barr virus. 
JAMA. 2003;289:1533-6. 

19. Callahan GN. Eating dirt. Emerg Infect Dis. 2003;9:1016-21. 

20. Wright P, Sham PC, Gilvarry CM, Jones PB, Cannon M, Sharma T, 
et al. Autoimmune diseases in the pedigrees of schizophrenic and 
control subjects. Schizophr Res. 1996;20:261-7. 


Address for correspondence: Gerald N. Callahan, Department of 
Microbiology, Immunology, and Pathology, Colorado State University, 
1619 Campus Delivery, Fort Collins, CO 80523, USA; fax: 970-491- 
0603; email: gerald.callahan@colostate.edu 


Access 

Another 
Dimension articles 

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338 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 




LETTERS 


Schistosoma 
mansoni in Family 
5 Years after Safari 

To the Editor: Each year -350,000 
Americans travel to Africa and 
-500,000 travel to Brazil and the Far 
East, all schistosomiasis-endemic 
regions. Data from the European 
Network on Imported Infectious 
Diseases Surveillance (TropNetEurop) 
suggest that most schistosomiasis 
cases imported to Europe are acquired 
in Africa; 80% of new cases world- 
wide occur in sub-Saharan Africa 
(1,2). Travelers to Africa from the 
United States are also at high risk for 
infection. Schistosoma mansoni has 
the greatest impact on residents of dis- 
ease-endemic areas who have high- 
grade infection and progressive 
hepatosplenic disease with portal 
hypertension and its manifestations. 
Most infected, short-term travelers 
sustain a low-level of fluke infestation 
with few symptoms, although serious 
complications can occur. 

We report a 38-year-old American 
man with ectopic S. mansoni fluke 
migration that led to neural schistoso- 
miasis. His symptoms prompted us to 
test family members who had accom- 
panied him on a trip to Kenya 5 years 
earlier. The family members had been 
unaware of the risk for schistosomia- 
sis that the trip posed. Five years after 
a Kenyan safari during which the 
index patient visited northeastern 
Lake Victoria and swam 1 afternoon, 
vertigo, nausea, and nystagmus devel- 
oped. The results of liver function 
tests were normal and peripheral 
blood showed no eosinophilia. Biopsy 
of a large cerebellar lesion noted on 
magnetic resonance imaging (MRI) 
was diagnostic, yielding multiple S. 
mansoni ova within large eosinophilic 
granulomas, consistent with tumoral 
neuroschistosomiasis. We tested 24 of 
25 family members who had accom- 
panied him to Kenya for schistosomi- 
asis (Figure). All of the accompanying 


family members, except 3 women, 
had gone into the water. All members 
were well, except an 8-year-old boy, 
in whom granulomatous colitis had 
developed after the trip. 

Eighteen of 25 enzyme-linked 
immunosorbent assays (ELISA) were 
positive for S. mansoni infection, 
including that of samples from the 
index patient and the boy (Figure). 
ELISA was performed on 1 8 samples 
at the Centers for Disease Control and 
Prevention (CDC) and 7 samples at 
Focus Technologies. Both tests used 
the same CDC-produced antigen, the 
microsomal fraction of adult S. man- 
soni fluke, which has both a sensitiv- 
ity and specificity for S. mansoni of 
99%. Confirmatory immunoblots 
were performed at CDC on samples 
from 19 of the 25 ELISA-tested fam- 
ily members, with 1 discordant result, 
a positive ELISA and negative S. 
mansoni and hematohium immuno- 
blots. Three of 7 ELISA-negative 
family members were the nonswim- 
mers. Analyses of single stool speci- 
mens from 7 family members, includ- 
ing the index patient, and 1 rectal 
biopsy sample were negative for ova. 

Because of the high positivity rate, 
praziquantel was prescribed for all 26 
travelers. The index patient received 
20 mg/kg of praziquantel twice daily 


for 4 days and high-dose dexametha- 
sone with subsequent 2-month taper; 
his symptoms resolved over months. 
An MRI 8 months after treatment 
demonstrated minimal residual 
inflammation. All other family mem- 
bers received 20 mg/kg of praziquan- 
tel twice in 1 day and tolerated it with- 
out adverse events. Ten months after 
treatment, the boy is growing after 
years of an inflammatory colitis char- 
acterized by hematochezia and 
growth retardation. He continues to 
have nonbloody diarrhea and consti- 
pation. 

We postulate that the mature fluke 
pair migrated from the mesenteric 
veins through Batson’s vertebral- 
venous plexus to the cerebral veins at 
the cerebellar level. There the female 
expelled multiple ova into the cere- 
bellum. An ensuing vigorous granulo- 
matous response led to posterior fossa 
mass effect and compression of 
medullary nausea centers, which 
resulted in the patient’s nausea, verti- 
go, and nystagmus. Ectopic ovum 
migration more commonly causes 
neuroschistosomiasis; however, in 
this case, multiple ova within 1 gran- 
ulomatous mass suggest fluke-pair 
migration rather than individual ovum 
migration. Neuroschistosomiasis is 
most commonly associated with 





# Positive ELISA 
O Negative ELISA 

# Negative Stool 

■ *\ Did not swim or wade, negative ELISA 
NT Not tested 


Figure. Testing for Schistosoma mansoni infection among family members 5 years after 
trip to Kenya. ELISA, enzyme-linked immunosorbent assay.See text for further description 
of testing. 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


339 


LETTERS 


S. japonicum , which has smaller ova. 
In the literature, we found 16 other 
case-patients with intracranial 
tumoral S. mansoni. Eight of the 
patients demonstrated cerebellar 
involvement, which suggests a com- 
mon fluke migratory pathway (3-15). 
Like our patient, 6 others were not 
native to disease-endemic regions. 

This unsuspected case of neural 
schistosomiasis illustrates the need 
for detailed inquiry into every fresh- 
water exposure by persons who have 
traveled to schistosomiasis-endemic 
regions. Adult Schistosoma flukes 
generally survive in venules from 6 to 
10 years but can survive <40 years; 
therefore, remote travel history is rel- 
evant. Examination of stool samples 
for ova has been considered the stan- 
dard method of diagnosing S. man- 
soni and S. japonicum infection, and 
urine examination is used to diagnose 
S. haematobium. Multiple, fresh 
morning specimens are ideal. 
However, stool examination is not 
likely to be as sensitive as current 
immunologic assays for detecting 
low levels of infection. Moreover, in 
disease-nonendemic regions, opera- 
tor variability may influence ova 
detection. Among 13 recorded cases 
of neurotumoral S. mansoni in which 
stool specimens were examined, no 
stool ova were found in 5 cases. In 
our family cohort, among the 7 
ELISA-positive members who sub- 
mitted stool specimens, no examina- 
tions performed at CDC demonstrat- 
ed eggs (Figure). 

The ELISA uses a highly sensitive 
and specific antigen for S. mansoni. 
Because the sensitivity is less for S. 
haematobium and S. japonicum , sub- 
sequent species- specific immunoblots 
are recommended based on travel his- 
tory that suggests exposure to specific 
species. Thus, we recommend ELISA, 
immunoblot if applicable, and stool or 
urine examination for travelers with 
possible exposure in disease-endemic 
regions. ELISA does not have the 
same utility in persons native to dis- 


ease-endemic regions because posi- 
tivity is also consistent with earlier 
infection. Stool or urine examination 
is diagnostic in suspected immigrant 
case-patients. 

In all cases, knowing that stool or 
urine examination shows ova is valu- 
able because repeat examination at 4 
to 6 weeks can be used to monitor 
treatment response. Because prazi- 
quantel is well tolerated and effective, 
empiric therapy among returning trav- 
elers after possible exposure is rea- 
sonable. However, diagnosing infec- 
tion when possible and demonstrating 
cleared infection after therapy are 
more prudent approaches, particularly 
as praziquantel resistance emerges 
(16). 

In conclusion, pretravel counseling 
against freshwater exposure and post- 
travel screening for schistosomiasis of 
persons with any freshwater exposure 
in disease-endemic regions are war- 
ranted. As illustrated, the diagnosis of 
schistosomiasis in a returned traveler 
should prompt screening for infection 
in fellow travelers. 

This work was presented in part at 
the American Society of Tropical 
Medicine and Hygiene 52nd annual meet- 
ing, December 3-7, 2003, as late-breaking 
abstract 2034, Philadelphia, PA. 

Valerianna Amorosa,* 

Daniel Kremens,* 

Martin S. Wolfe,t+ 

Timothy Flanigan, § 

Kevin M. Cahill, K#** Kevin Judy,* 
Scott Kasner,* 
and Emily Blumberg* 
*University of Pennsylvania, Philadelphia, 
Pennsylvania, USA; fGeorge Washington 
University, Washington, DC, USA; 
^Georgetown University, Washington, DC, 
USA; §Brown University, Providence, 
Rhode Island, USA; IJRoyal College of 
Surgeons, Dublin, Ireland; #New York 
University, New York, New York, USA; and 
**Lenox Hill Hospital, New York, New York, 
USA. 


References 

1. Grobusch MP, Muhlberger N, Jelinek T, 
Bisoffi Z, Corachan M, Harms G, et al. 
Imported schistosomiasis in Europe: sen- 
tinel surveillance data from TropNetEurop. 
J Travel Med. 2003;10:164-9. 

2. Centers for Disease Control and 
Prevention. Annex A, Fact sheets for candi- 
date diseases for elimination or eradication. 
MMWR Morb Mortal Wkly Rep. 
1999;48S1:154. 

3. Bambirra EA, de Souza AJ, Cesarini I, 
Rodrigues PA, Drummond CA. The 
tumoral form of schistosomiasis: report of a 
case with cerebellar involvement. Am J 
Trop Med Hyg. 1984;33:76-9. 

4. Gjerde IO, Mork S, Larsen JL, Huldt G, 
Skeidsvoll H, Aarli JA. Cerebral schistoso- 
miasis presenting as a brain tumor. Eur 
Neurol. 1984;23:229-36. 

5. Goasguen J, Antoine HM, Saliou P, 
Herbelleau T, Putz DM, Jallon PM, et al. 
Cerebral bilharziasis caused by 
Schistosoma mansoni. Rev Neurol. 
1984;140:293-5. 

6. Schils J, Hermanus N, Flament-Durant J, 
Van Gansbeke D, Baleriaux D. Cerebral 
schistosomiasis. Am J Neuroradiol. 
1985;6:840-1. 

7. Andrade AN, Bastos CL. Cerebral 
schistosomiasis mansoni. Arq Neuro- 
psiquitr. 1989;47:100-4. 

8. Cabral G, Pittella JE. Tumoural form of 
cerebella schistosomiasis mansoni. Report 
of a surgically treated case. Acta Neurochir 
(Wien). 1989;99:148-51. 

9. Brito DMM, Filho A, Furtado HRC, 
Carneio GJD, Filho GS, Almeida NS, et al. 
Esquistossomose cerebella. Neurobiol 
(Recife). 1993;56:69-72. 

10. Lee YK, Choi TY, Jin SY, Lee DW. 
Imported CNS schistosomiasis. J Korean 
Med Sci. 1995;10:57-61. 

11. Pittella JEH, Gusmao NDS, Carvalho GTC, 
da Silveira RL, Campos GF. Tumoral form 
of cerebral schistosomiasis mansoni. A 
report of four cases and a review of the lit- 
erature. Clin Neurol Neurosurg. 
1996;98:15-20. 

12. Case records of the Massachusetts General 
Hospital — case 39 — 1996. N Engl J Med. 
1996;335:1906-14. 

13. Ferreira L, Lima F, dos Anjor MR, Costa J. 
Tumor form of encephalic schistosomiasis: 
presentation of a case surgically treated. 
Rev Soc Bras Med Trop. 1998;31:89-93. 

14. Fowler R, Lee C, Keystone J. The role of 
corticosteroids in the treatment of cerebral 
schistosomiasis caused by Schistosoma 
mansoni: case report and discussion. Am J 
Trop Med Hyg. 1999;61:47-50. 

15. Braga BP, da Costa LB, Lambertucci JR. 
Magnetic resonance imaging of cerebellar 
Schistosomiasis mansoni. Rev Soc Bras 
Med Trop. 2003;36: 635-6. 


340 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


LETTERS 


16. Lawn SD, Lucas SB, Chiodini PL. Case 
report: Schistosoma mansoni infection: fail- 
ure of standard treatment with praziquantel 
in a returned traveler. Trans R Soc Trop 
Med Hyg. 2003;97:100-1. 

Address for correspondence: Valerianna 

Amorosa, Division of Infectious Diseases, 
Pennsylvania Hospital, 800 Spruce St, 
Philadelphia, PA 19107, USA; fax: 215-829- 
7132; email: Valerianna.amorosa@uphs. 

upenn.edu 


Community- 
associated 
Methicillin- 
resistant 
Staphylococcus 
aureus, Singapore 

To the Editor: Community-asso- 
ciated methicillin-resistant Staphylo- 
coccus aureus (CA-MRSA) is an 
emerging phenomenon that has been 
reported from almost every continent 
in the world (1-4). Such strains are 
usually characterized by multisuscep- 
tibility to non- (3-lactam antimicrobial 
drugs, production of Panton- Valentine 
leukocidin (PVL), and presence of 
staphylococcal chromosome cassette 
mec (SC Cmec) IVa, a novel smaller 
variant of the methicillin-resistance 
locus (5). The genetic backgrounds of 


CA-MRSA strains from different 
parts of the world are distinct and spe- 
cific for each geographic region 
(1-5). 

We conducted a study at our insti- 
tution, a 1,600-bed adult acute-care, 
tertiary-level public hospital, to deter- 
mine evidence and the clinical and 
molecular profile of CA-MRSA in 
Singapore. We reviewed the microbi- 
ology laboratory records at our institu- 
tion for multidrug- susceptible MRS A 
strains isolated from January 1, 2001, 
to April 15, 2004. S. aureus was iden- 
tified by colony morphologic features, 
coagulation of citrated rabbit plasma 
with EDTA (BBL Becton Dickinson 
and Co., Cockeysville, MD, USA), 
and production of clumping factor and 
protein A (BactiStaph, Remel, Lenexa, 
KS, USA). Methicillin resistance was 
determined by susceptibility testing 
and confirmed by latex agglutination 
for penicillin binding protein-2a (6). 
Multidrug- susceptible strains were 
defined by susceptibility to cotrimoxa- 
zole and gentamicin as determined by 
the Kirby-Bauer disk diffusion method 
following NCCLS guidelines (7). 

The medical records of patients 
infected by these MRSA were 
reviewed, and strains were labeled 
community-associated if they had 
been isolated within 48 hours of hos- 
pitalization from patients who had not 
been in any hospital for >1 year. 
Community-associated strains were 
tested for PVL genes (8), and the 
SC Cmec was typed by following a 


previously described method (9). 
Molecular typing was done by pulsed- 
field gel electrophoresis (PLGE) with 
restriction endonuclease Smal and 
multilocus sequence typing (10). 
These strains were sent to the Lrench 
Reference Center for Staphylococci, 
where genetic sequences encoding 
accessory gene regulator (agr) sub- 
types, enterotoxins, exfoliative toxins, 
toxic-shock syndrome toxin- 1, 

hemolysins, and LukE-LukD leuko- 
toxin were detected by polymerase 
chain reaction (PCR)-based methods. 
Comparisons with CA-MRSA strains 
worldwide in terms of toxin profile 
and PLGE patterns were also per- 
formed in Lrance; the latter was 
achieved by using Taxotron software 
(Institut Pasteur, Paris, Prance) to dig- 
itize and analyze Smal macrorestric- 
tion patterns. 

Eight of 266 multidrug- susceptible 
strains fulfilled the criteria for com- 
munity acquisition, but only 5 of these 
strains (corresponding to patients 1,3, 
and 6-8) had been archived. The 
demographic and clinical data of the 
patients are shown in the Table. Most 
were young, healthy adults with cuta- 
neous abscesses. Patient 1 had dia- 
betes mellitus but had never been hos- 
pitalized; he was the only patient with 
severe bacteremic pneumonia. Patient 
6 had early- stage endometrial cancer 
resected in 2000 but had not attended 
her follow-up appointments for >1 
year before her hospitalization. 
Patient 8 had traveled to Taipei, 


Table. Demographic and clinical data of patients with community-associated methicillin-resistant Staphylococcus aureus (MRSA) 

Patient 

1 

2 

3 

4 

5 

6 

7 

8 

Date of MRSA 
isolation 

Mar 2001 

Nov 2002 

Jan 2003 

Feb 2003 

Mar 2003 

May 2003 

Oct 2003 

Apr 2004 

Ethnicity 

Indian 

Filipino 

Chinese 

Chinese 

Filipino 

Chinese 

Filipino 

Chinese 

Age 

52 

20 

38 

37 

31 

56 

21 

33 

Sex 

M 

F 

M 

M 

F 

F 

F 

F 

Coexisting 

conditions 

Diabetes 

mellitus 

- 

- 

- 

- 

Endometrial 

cancer 

- 

- 

Infection type 

Pneumonia, 

Hand 

Hand 

Hand 

Hand 

Hand 

Chin 

Abdominal 


bacteremia 

abscess 

abscess 

abscess 

abscess 

abscess 

abscess 

wall abscess 

Therapy* 

IV vancomycin 

l&D 

l&D 

l&D 

l&D 

l&D 

l&D 

l&D 

Appropriate 
antimicrobial drug 

Yes 

No 

No 

No 

No 

No 

No 

No 

usage 










*Therapy: IV, intravenous; not applicable; l&D, incision and drainage of abscess. 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


341 


LETTERS 


Taiwan, for a month; the abscess 
developed 3 days after her return 
home. Travel history was not docu- 
mented in the other patients’ records. 
Patients 2-8 received (3-lactam 
antimicrobial drugs in addition to sur- 
gical drainage of their abscesses and 
recovered without any complications. 

All 5 archived strains had different 
molecular and toxin profiles, and the 
only consistent feature was the pres- 
ence of PVL genes. Isolates 3 and 7 
possessed SCC mec IV. Isolates 1, 6, 
and 8 were mec A positive, but their 
SCC mec belonged to none of the 4 
major structural types. Comparisons 
with published data on CA-MRSA 
strains showed that isolate 7 was iden- 
tical to the European strain of CA- 
MRSA in terms of PFGE pattern, 
toxin profile, and sequence type (ST 
80) (2,5). Isolate 3 had an identical 
PFGE pattern and sequence type (ST 
30) compared to the Oceanian 
Southwest Pacific strain but differed 
slightly in toxin profile, as the LukD- 
LukE leukocidin genes were absent 
(3,5). Isolate 8 was similar to the 
Taiwanese strains: it was ST 59 and 
had non-typable SCC mec (4). It 
belonged to agr 1 and tested positive 
for enterotoxin sek, y2-hemolysin, and 
(3-hemolysin genes. 

Isolate 6 had a PFGE pattern that 
may be distantly related to U.S. 
strains; the similar sequence type 
(ST 1) served to emphasize this, 
although the presence of nontypable 
SC Cmec rather than SC Cmec IV 
implied that methicillin resistance 
was acquired differently. It belonged 
to agr 3 and tested positive for 
LukD-LukE leukocidin, enterotoxins 
seb and seh, and y2-hemolysin genes. 
Isolate 1 is unique to Singapore in that 
it had a novel sequence type (ST524: 
7-6-1-5-71-5-6 and S CCmec. It 
belonged to agr 1 and tested positive 
for y-hemolysin gene as well as for 
the enterotoxin gene cluster. 

Widely diversified CA-MRSA 
strains exist in Singapore. The demo- 
graphic profile and clinical symptoms 


of local patients infected with these 
strains were consistent with published 
literature (2-4). The lack of a pediatric 
unit at our institution prevented a more 
complete epidemiologic description. 

In contrast to previous reports 
(1-5), our findings are unique in that 
most of our strains do not have a dis- 
tinctive molecular profile and may be 
related to strains from different parts 
of the world. Epidemiologic and 
molecular data strongly suggest that 
isolate 8 was imported from Taiwan. 
Some of the other strains (especially 
isolates 3 and 7) may have been 
imported from other countries too, as 
Singapore is an international travel 
hub with >6 million visitors annually. 

CA-MRSA has only been isolated 
sporadically in Singapore, and no 
dominant clone was seen among our 
isolates. Singapore may be in an early 
phase of CA-MRSA emergence, and 
healthcare workers should remain 
vigilant for future outbreaks. 

Acknowledgment 

We thank the staff of the 
Microbiology Laboratory at the Singapore 
General Hospital, especially Lan-Huay 
Ong and Grace Wang, for their assistance 
in this study. 

This study was funded by a grant 
from Singapore General Hospital’s 
Department of Clinical Research. 

Li-Yang Hsu,* Anne Tristan, t 
Tse-Hsien Koh,* Michele Bes,t 
Jerome Etienne, t Asok Kurup,* 
Thuan-Tong Tan,* 
and Ban-Hock Tan* 
*Singapore General Hospital, Singapore; 
and fFaculte de Medecine Laennec, Lyon, 
France 

References 

1. Naimi TS, LeDell KH, Boxrud DJ, Groom 
AV, Steward CD, Johnson SK, et al. 
Epidemiology and clonality of community- 
acquired methicillin-resistant Staphylo- 
coccus aureus in Minnesota, 1996-98. Clin 
Infect Dis. 2001;33:990-6. 


2. Dufour P, Gillet Y, Bes M, Lina G, 
Vandenesch F, Floret D, et al. Community- 
acquired methicillin-resistant Staphylo- 
coccus aureus infections in France: emer- 
gence of a single clone that produces 
Panton- Valentine leukocidin. Clin Infect 
Dis. 2002;35:819-24. 

3. O’Brien FG, Lim TT, Chong FN, Coombs 
GW, Enright MC, Robinson DA, et al. 
Diversity among community isolates of 
methicillin-resistant Staphylococcus aureus 
in Australia. J Clin Microbiol. 2004;42: 
3185-90. 

4. Wang CC, Lo WT, Chu ML, Siu LK. 
Epidemiological typing of community- 
acquired methicillin-resistant Staphylo- 
coccus aureus from children in Taiwan. 
Clin Infect Dis. 2004;39:481-7. 

5. Vandenesch F, Naimi T, Enright MC, Lina 
G, Nimmo GR, Heffernan H, et al. 
Community-acquired methicillin-resistant 
Staphylococcus aureus carrying Panton- 
Valentine leukocidin genes: worldwide 
emergence. Emerg Infect Dis. 
2003;9:978-84. 

6. Nakatomi Y, Sugiyama J. A rapid latex 
agglutination assay for the detection of 
penicillin-binding protein 2'. Microbiol 
Immunol. 1998;42:739-43. 

7. National Committee for Clinical 
Laboratory Standards. Performance stan- 
dards for antimicrobial susceptibility test- 
ing. Fourteenth informational supplement. 
NCCLS document M100-S14. Wayne (PA): 
The Committee; 2004. 

8. Lina G, Piemont Y, Godail-Gamot F, Bes 
M, Peter MO, Gauduchon V, et al. 
Involvement of Panton-Valentine leuko- 
cidin-producing Staphylococcus aureus in 
primary skin infections and pneumonia. 
Clin Infect Dis. 1999;29:1128-32. 

9. Oliveira DC, de Lencastre H. Multiplex 
PCR strategy for rapid identification of 
structural types and variants of the mec ele- 
ment in methicillin-resistant Staphylo- 
coccus aureus. Antimicrob Agents 
Chemother. 2002;46:2155-61. 

10. Enright MC, Day NP, Davies CE, Peacock 
SJ, Spratt BG. Multilocus sequence typing 
for characterization of methicillin-resistant 
and methicillin-sensitive clones of 
Staphylococcus aureus. J Clin Microbiol. 
2000;38:1008-15. 


Address for correspondence: Li-Yang Hsu, 
Department of Internal Medicine, Singapore 
General Hospital, Outram Rd, S 169608, 
Singapore; fax: 65-67322601; email: liyang_ 
hsu@yahoo.com 


342 


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LETTERS 


Mumps Virus- 
associated 
Hemophagocytic 
Syndrome 

To the Editor: Virus-associated 
hemophagocytic syndrome (VAHS) is 
a fulminant disorder associated with 
systemic viral infection and is charac- 
terized pathologically by the prolifer- 
ation of hemophagocytic histiocytes 
in the lymphoreticular tissues. Here 
we report a case of mumps VAHS fol- 
lowing parotitis and pancreatitis. 

A 39-year-old, previously healthy 
woman sought treatment for abdomi- 
nal pain on June 14, 2002. On physical 
examination, her bilateral parotid 
glands were swollen, and her left 
upper quadrant was tender. Laboratory 
studies showed a leukocyte count of 
4,640/mm 3 , a hemoglobin concentra- 
tion of 13.9 g/dL, and a platelet count 
of 19.1 x 10 4 /mm 3 . The level of amy- 
lase was elevated in her blood (1,613 
IU/L; normal 50-160 IU/L) and urine 
(12,940 IU/L; normal 200-1,100 
IU/L). Her level of pancreatic 
enzymes was also elevated: lipase 
level was 194 IU/L (normal 7-60 
IU/L) and phospholipase A2 level was 
1,340 ng/dL (normal 130-400 ng/dL). 
Parotitis and acute pancreatitis due to 
a mumps virus infection were diag- 
nosed. After supportive therapy, the 
laboratory abnormalities improved. 

On July 1, the patient’s tempera- 
ture suddenly rose to 39°C. At that 
time, pancytopenia was evident, with 
a leukocyte count of 2,350/mm 3 , a 
hemoglobin concentration of 10.9 
g/dL, and a platelet count of 9.1 x 
10 4 /mm 3 . Laboratory studies showed 
an elevation of lactic dehydrogenase 
(1,403 IU/L; normal 180-460 IU/L), 
ferritin (12,727.0 ng/mL; normal 
4.0-64.2 ng/mL), and soluble inter- 
leukin-2 receptors (1,660 U/mL; nor- 
mal 145-519 U/mL). Hypercyto- 
kinemia was also shown, with an 
interleukin-6 of 12.7 pg/mL (normal 


<3.1 pg/mL). Her bone marrow was 
normocellular, and an increased num- 
ber of histiocytes with hemophagocy- 
tosis was found. Extensive cultures 
and serologic studies for microbial 
and viral infections were all negative, 
whereas tests for immunoglobulin G 
and immunoglobulin M antibodies 
against the mumps virus were 
both positive. Mumps VAHS was 
diagnosed. Treatment with corticos- 
teroids led to a complete remission of 
symptoms. 

VAHS was initially reported by 
Risdall et al. in 1979 (1). Although the 
precise pathogenesis of VAHS 
remains unknown, current hypotheses 
focus on the roles played by activating 
cytokines. VAHS has been reported in 
connection with a variety of viruses: 
adenovirus, cytomegalovirus, dengue, 
Epstein-Barr, hepatitis A, hepatitis B, 
hepatitis C, herpes simplex, HIV, 
human herpesvirus 6, human her- 
pesvirus 8, influenza A (antigenic type 
H1N1), measles, parainfluenza type 
III, parvovirus B 19, rubella, and vari- 
cella-zoster (2). This report is the first 
of a VAHS case associated with a 
mumps virus infection. The clinical 
course of VAHS is highly variable, 
and in some cases, especially in 
Epstein-Barr virus infection, VAHS is 
a dramatic illness with a potentially 
fatal outcome (2). This case implies 
that mumps VAHS may have a posi- 
tive prognosis. 

Kunihiko Hiraiwa,* 
Katsuyuki Obara,t 
and Atsuhisa Satof 

* Hamamatsu Red Cross Hospital, 
Hamamatsu, Japan; and fMito Red Cross 
Hospital, Mito, Japan 

References 

1. Risdall RJ, McKenna RW, Nesbit ME, 
Krivit W, Balfour HH, Simmons RD et al. 
Virus-associated hemophagocytic syn- 
drome. Cancer. 1979;44:993-1002. 

2. Fisman DN. Hemophagocytic syndromes 
and infection. Emerg Infect Dis. 
2000;6:601-8. 


Address for correspondence: Kunihiko 

Hiraiwa, Hamamatsu Red Cross Hospital, 1-5- 
30, Takabayashi, Hamamatsu, 430-0907, Japan; 
fax: 81-53-472-3751; email: hiraiwa9215@ 
hotmail.com 


Imported Cutaneous 
Diphtheria, 
Germany, 1997-2003 

To the Editor: The March 2004 
report by de Benoist et al. on the inci- 
dence of imported cutaneous diphthe- 
ria in the United Kingdom (1) 
prompted us to describe the situation 
of cutaneous diphtheria in Germany 
and to analyze the cases reported to 
the German Consiliary Laboratory on 
Diphtheria since its establishment at 
our institute in 1997. The laboratory 
provides advisory and diagnostic 
services mainly to microbiologic lab- 
oratories throughout Germany. 

From 1997 to 2003, 6 cases of 
cutaneous infections caused by toxi- 
genic Corynebacterium diphtheriae 
were documented (Table). None of 
these was accompanied by secondary 
diphtheria infection. Toxigenicity 
was determined by both dtx poly- 
merase chain reaction and Elek test 
(2). As in the United Kingdom, all 
cases for which clinical information 
was available (N = 5) were imported. 
Three were found in tourists who had 
traveled to tropical countries: a 
20-year-old diver had injured her heel 
after stepping on coral in Thailand; a 
60-year-old tourist had a chronic 
ulcer develop in the thigh after a trip 
to Indonesia (no history of an insect 
bite); and a 39-year-old traveler to 
Kenya returned with a purulent ear 
infection with no memory of trauma 
or insect bite. The remaining import- 
ed C. diphtheriae skin infections 
were reported in 2 Angolan children, 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


343 


LETTERS 


5 and 10 years of age, who were 
brought to Germany by a humanitari- 
an organization for surgery on severe 
gun wounds to their lower extremities 
(foot and thigh with chronic 
osteomyelitis, respectively). To our 
knowledge, these reports are the first 
of cutaneous diphtheria in gunshot 
wounds in recent years. Moreover, in 
the patient with the thigh wound, C. 
diphtheriae was also isolated from a 
deep fistula, which suggests involve- 
ment of C. diphtheriae in the chronic 
osteomyelitis. 

As in the United Kingdom, all 
cases of diphtheria reported since 1997 
were caused by C. diphtheriae mitis. In 
4 of 5 cutaneous diphtheria patients 
who had an available medical history, 
mixed infections with Staphylococcus 
aureus and Streptococcus pyogenes 
were found; 3 of 5 patients were not 
sufficiently vaccinated against diph- 
theria as recommended. Systemic 
symptoms, such as malaise and gener- 
al weakness, developed in the 20-year- 
old Thailand tourist, although she had 
received a booster dose just before her 
travel. Cutaneous diphtheria must be 
expected even in vaccinated patients; 
for instance, among serum samples of 
287 healthy German adults with a 
complete record of basic immuniza- 
tion against diphtheria, only 42.2% 
showed full serologic protection 
as indicated by antitoxin levels 
>0.1 IU/mL (3). 

As de Benoist et al. outline, cuta- 
neous diphtheria might be difficult to 
diagnose because of its unspecific 
clinical appearance and the presence 
of mixed infections in chronic 
nonhealing skin lesions. Because of 
the nearly complete disappearance of 
cutaneous diphtheria in many parts of 
the western world, microbiologists 
lack experience in identifying C. diph- 
theriae grown from specimens. From 
1997 to 2003, approximately one fifth 
of the strains sent to our Consiliary 
Laboratory on Diphtheria for species 
identification and toxin testing were 
either nondiphtheria Corynebacterium 


spp. or noncoryneform bacteria of dif- 
ferent genera (including lactobacilli, 
Dermabacter hominis , and 
Propionibacterium acnes). 

Clinicians (4) and microbiologists 
(5) should be aware of the possibility 
of cutaneous diphtheria in chronically 
infected skin lesions in patients 
returning from disease-endemic 
regions. Medical personnel should 
include this in civilian as well as mil- 
itary health services, since our cases 
indicate that toxigenic C. diphtheriae 
might affect not only travel-related 
skin injuries caused by leisure or 
tourist activities but also wounds in 
patients from war regions in diphthe- 
ria-endemic areas. 

Andreas Sing* 
and Jurgen Heesemann* 

*Max von Pettenkofer-lnstitut fur Hygiene 
und Medizinische Mikrobiologie, Munich, 
Germany 

References 

1. De Benoist AC, White JM, Efstratiou A, 
Kelly C, Mann G, Nazareth B, et al. 
Imported cutaneous diphtheria, United 
Kingdom. Emerg Infect Dis. 
2004;10:511-3. 

2. Sing A, Hogardt M, Bierschenk S, 
Heesemann J. Detection of differences in 
the nucleotide and amino acid sequences of 
diphtheria toxin from Corynebacterium 
diphtheriae and Corynebacterium ulcerans 
causing extrapharyngeal infections. J Clin 
Microbiol. 2003;41:4848-51. 

3. Hasselhorn HM, Nubling M, Tiller FW, 
Hofmann F. Factors influencing immunity 
against diphtheria in adults. Vaccine. 
1998;16:70-5. 

4. Bonnet JM, Begg NT. Control of diphthe- 
ria: guidance for consultants in communi- 
cable disease control. Commun Dis Public 
Health. 1999;2:242-9. 

5. Efstratiou A, George RC. Laboratory 
guidelines for the diagnosis of infections 
caused by Corynebacterium diphtheriae 
and C. ulcerans. Commun Dis Public 
Health. 1999;2:250-7. 


Address for correspondence: Andreas Sing, 
Max von Pettenkofer-lnstitut fur Hygiene und 
Medizinische Mikrobiologie, National 
Consiliary Laboratory on Diphtheria, 
Pettenkoferstrasse 9a, 80336 Munich, 

Germany; fax: 49-89-5160-5223; email: 

sing @ m340 1 . mpk. med. uni-muenchen . de 


Antimicrobial Drug 
Consumption in 
Companion Animals 

To the Editor: During the last 
decade, use of antimicrobial drugs for 
growth promotion and therapeutic 
treatment in food animals has received 
much attention. The reservoir of resist- 
ant bacteria in food animals implies a 
potential risk for transfer of resistant 
bacteria, or resistance genes, from 
food animals to humans. Subsequent 
emergence of infections in humans, 
caused by resistant bacteria originat- 
ing from the animal reservoir, is of 
great concern. These unintended con- 
sequences of antimicrobial drug use in 
animals led to termination of antimi- 
crobial growth promoters in food ani- 
mals in countries in the European 
Union, including Denmark, where the 
consumption of antimicrobial drugs 
by production animals was reduced by 
50% from 1994 to 2003 (1). 

In Denmark, the VetStat program 
monitors all veterinary use of medi- 
cines for animals. VetStat is based on 
reporting from the pharmacies and 
from veterinary practitioners and con- 
tains detailed information, such as 
animal species, reason for prescrip- 
tion, and dosage on each prescription. 
In Denmark, antimicrobial drugs can 
be obtained only by prescription and 
only at pharmacies. 

So far, use of antimicrobial drugs 
in companion animals has received 
little attention; monitoring programs 
have focused on antimicrobial drug 
consumption in food animals. 
According to data generated by the 
VetStat program in 2003, consump- 
tion of fluoroquinolones and 
cephalosporins in companion animals 
was substantial when compared to 
consumption in food animals (1). 
Fluoroquinolones and cephalosporins 
are antimicrobial drugs ranked by the 
U.S. Food and Drug Administration 
as critically important in human med- 
icine, and for which emergence of 


344 


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LETTERS 


resistant bacteria is especially unde- 
sirable (2). Considering the shared 
environment of humans and compan- 
ion animals, transfer of resistant bac- 
teria or mobile resistance determi- 
nants from companion animals to 
humans would be possible, and emer- 
gence of resistance to fluoro- 
quinolones and cephalosporins in 
companion animals should be a mat- 
ter of concern. 

Several scientific publications 
have reported the occurrence of the 
same resistance genes in companion 
animals and in humans (3-6) and the 
possible transfer of bacteria between 
companion animals and humans 
(3-9). Companion animal owners and 
their families are likely in close con- 
tact with their animals daily, which 
provides the opportunity for transfer 
of bacteria between companion ani- 
mals and humans. A large proportion 
of the human population presumably 
has daily contact with companion ani- 
mals, not only in Denmark but also in 
other countries. In Denmark, 20% of 
families own dogs and 16% own cats 
( 10 ). 

In 2002, legal restrictions aimed to 
reduce the usage of fluoroquinolones 
in food animals were imposed in 
Denmark. The total annual consump- 
tion of fluoroquinolones in animals 
(companion and food animals) in 
Denmark was reduced from 183 kg in 
2001 to 53 kg in 2003 (1). Of these 53 
kg of fluoroquinolones, almost half 
(24 kg) was used in companion ani- 
mals (data based on reporting on use 
in veterinary practice and sales from 
pharmacies on prescription). These 
data document that fluoroquinolones 
remain widely used for infections in 
companion animals, even though the 
emergence of fluoroquinolone resist- 
ance in bacteria is especially undesir- 
able and regarded as a human health 
hazard. A similar situation exists with 
cephalosporins. The total consump- 
tion of cephalosporins in animals 
(companion and food animals) in 


Denmark in 2003 was 461 kg, of 
which more than half (254 kg) was 
consumed by companion animals (1). 

Thus, a comparatively small num- 
ber of companion animals (550,000 
dogs and 650,000 cats) (10) consume 
approximately the same amount of 
fluoroquinolones and cephalosporins 
as consumed annually in the much 
larger population of food animals in 
Denmark (23 million slaughter pigs, 
130 million broiler chickens, and 1.2 
million cattle and dairy cows) (10). 
We do not believe that antimicrobial 
drugs are more generously prescribed 
for companion animals in Denmark 
than in other industrialized countries. 
Rather, the data presented here reflect 
the apparent contrast between policies 
of antimicrobial drug use for food ani- 
mals and policies for companion ani- 
mals. The use of these antimicrobial 
drugs is avoided or restricted in food 
animals to minimize spread of resist- 
ance, while in companion animals 
prescription continues unimpeded. 
This situation may create undesirable 
antimicrobial drug resistance in bacte- 
ria, which may subsequently spread to 
humans from the previously neglected 
reservoir in companion animals. 

This work is a part of The Danish 
Integrated Antimicrobial Resistance 
Monitoring and Research Programme 
(DANMAP), and was funded by the 
Danish Ministry of Food, Agriculture and 
Fisheries and the Danish Ministry of the 
Interior and Health. 

Ole E. Heuer,* 

Vibeke Frokjaer Jensen,* 
and Anette M. Harnmerumf 

*Danish Institute for Food and Veterinary 
Research, Soborg, Denmark; and 
fNational Centre for Antimicrobials and 
Infection Control, Copenhagen, Denmark 

References 

1. DANMAP 2003: use of antimicrobial 
agents and occurrence of antimicrobial 
resistance in bacteria from food animals, 


foods and humans in Denmark. Sprborg, 
Denmark: Danish Zoonoses Center;2004. 

2. U.S. Food and Drug Administration. FDA 
guidance (152): Guidance for industry: 
evaluating the safety of antimicrobial new 
animal drugs with regard to their microbio- 
logical effects on bacteria of human health 
concern. Fed Reg. 2003 ;68:6 1221. 

3. Butaye P, Devriese LA, Haesebrouck F. 
Differences in antibiotic resistance patterns 
of Enterococcus faecalis and Enterococcus 
faecium strains isolated from farm and pet 
animals. Antimicrob Agents Chemother. 
2001;45:1374-8. 

4. Lanz R, Kuhnert P, Boerlin P. Antimicrobial 
resistance and resistance gene determinants 
in clinical Escherichia coli from different 
animal species in Switzerland. Vet 
Microbiol. 2003;91:73-84. 

5. Simjee S, White DG, McDermott PF, 
Wagner DD, Zervos MJ, Donabedian SM, 
et al. Characterization of Tnl546 in van- 
comycin-resistant Enterococcus faecium 
isolated from canine urinary tract infec- 
tions: evidence of gene exchange between 
human and animal enterococci. J Clin 
Microbiol. 2002;40:4659-65. 

6. van Belkun A, van den Braak N, 
Thomassen R, Verbrugh H, Endtz H. 
Vancomycin-resistant enterococci in cats 
and dogs. Lancet. 1996;348:1038-9. 

7. Rodrigues J, Thomazini CM, Lopes CA, 
Dantas LO. Concurrent infection in a dog 
and colonization in a child with a human 
enteropathogenic Escherichia coli clone. J 
Clin Microbiol. 2004;42:1388-9. 

8. Damborg P, Olsen KE, Moller, Nielsen E, 
Guardabassi L. Occurrence of 
Campylobacter jejuni in pets living with 
human patients infected with C. jejuni. J 
Clin Microbiol. 2004;42:1363-4. 

9. Guardabassi L, Loeber ME, Jacobson A. 
Transmission of multiple antimicrobial- 
resistant Staphylococcus intermedius 
between dogs affected by deep pyoderma 
and their owners. Vet Microbiol. 2004; 
98:23-7. 

10. Nyt fra Danmarks Statistik: No. 499. Titel, 
Denmark: Familiernes Kaeledyr (publica- 
tion in Danish); 2000. 


Address for correspondence: Ole E. Heuer, 
Department of Epidemiology and Risk 
Assessment, Danish Institute for Food and 
Veterinary Research, Mprkhpj Bygade 19, DK- 
2860 Spborg, Denmark; fax: 45-7234-7028; 
email: oeh@dfvf.dk 

All material published in Emerging 
Infectious Diseases is in the public 
domain and may be used and reprinted 
without special permission; proper cita- 
tion, however, is appreciated. 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


345 


LETTERS 


Vibrio cholerae 
SXT Element, Laos 

To the Editor: The SXT element 
is a Vibrio cholerae- derived ICE 
(integrating and conjugative element), 
which has also been referred to as a 
conjugative transposon (1) or a con- 
stin (2). ICEs excise from the chromo- 
somes of their hosts, transfer to a new 
host through conjugation, and then 
integrate into the chromosome again. 
SXT element was originally isolated 
in 1993 from a V. cholerae 0139 clin- 
ical isolate (SXT M01 °) (1). The -100- 
kbp SXT element confers resistance 
to sulfamethoxazole, trimethoprim, 
chloramphenicol, and streptomycin 

(1) . Since 1994, V. cholerae isolates 
from Bangladesh, India, and 
Mozambique have also contained the 
SXT element (2-4). In SXT M01 °, 
resistance genes are embedded near 
the 5' end, in a -17.2-kbp composite 
transposon-like element that inter- 
rupts the SXT-encoded rumAB oper- 
on. In contrast, in El Tor 01 V. choler- 
ae strains isolated in India and 
Bangladesh, the resistance genes are 
located in SXT ET , which is closely 
related but not identical to SXT M01 ° 

(2) . Comparison of 2 related ICEs, 
SXT of V. cholerae and R391 of 
Providencia rettgeri (5), showed that 
the conserved backbone apparently 
contains 3 hot spots for insertions of 
additional DNA sequences: the first 
between s043 and traL , the second 
between trA and s054, and the third 
between s073 and traF. R391 con- 
tains an intact rumAB operon and a 
transposon-associated kanamycin 
resistance gene located -3.5 kbp from 
the rumAB operon (6). Mobile genetic 
elements such as SXT have a crucial 
role in spreading antimicrobial drug 
resistance genes among microbial 
populations, and our understanding of 
these genetic elements would help to 
control the emergence of antimicro- 
bial drug resistance. 

We have been monitoring the drug 
sensitivity pattern in the Lao People’s 


Democratic Republic (Laos) since 
1993, and we have found that V. 
cholerae 01 strains isolated after 
1997 were resistant to tetracycline, 
sulfamethoxazole, trimethoprim, 
chloramphenicol, and streptomycin 
(7). Analysis of the genetic determi- 
nants encoding antimicrobial drug 
resistance showed an SXT element 
(SXT LA0S ), which is different from 
the previously reported SXTs (8). 
SXT LA0S contains 2 novel open read- 
ing frames (ORFs) in the third hot 
spot (between s073 and traF). SXT ET 
contains a class 9 integron in hot spot 
s073-traF that harbors dfrAl as a 
gene cassette (2). In SXT M01 °, the 
gene encoding trimethoprim resist- 
ance ( dfrl8 ) is encoded in the -17.2- 
kbp composite transposon-like ele- 
ment that interrupts the SXT-encoded 
rumAB operon. SXT LA0S does not 
encode dfrl8 or dfrAl , and the gene 
encoding trimethoprim resistance has 
not been identified. In this study, we 
analyzed hot spot s 04 3 -traL and hot 
spot traA-s054 to better characterize 
SXT LA0S . 

Two sets of primers were designed 
to amplify the hot spot regions. 
Primer HS1-F, which anneals to 
s043, was 5' GGC TAT TCC ACC 
GGT GGT G 3'; primer HS1-R, 
which anneals to traL , was 5' TGC 
CGA TCA CTA GCC CCA AC 3'; 
primer HS2-F, which anneals to traA , 
was 5' ATG GGT CTC TAC AAT 
ACG CC 3'; and primer HS2-R, 
which anneals to s054 , was 5' GGA 
GAC AGC GCA AGC GCC AG 3'. 
Polymerase chain reaction (PCR) 
amplifications on genomic DNA 
extracted from the V. cholerae 01 
strain isolated in Laos (strain 00LA1) 
with primers HS1-F and HS1-R 
yielded an amplicon of -1100 bp, 
which is slightly different from the 
amplicon obtained with DNA extract- 
ed from V. cholerae 0139, strain 
MOIO (-1,000 bp). PCR amplifica- 
tion using primers HS2-F and HS2-R 
gave amplicons of similar size 
(-2,200 bp) for both strains. The 


-1,000-bp and -2,200-bp PCR prod- 
ucts from strain 00LA1 were cloned 
independently into the pCR 2.1 vec- 
tor and tested to determine if recom- 
binant plasmids confer trimethoprim 
resistance after transformation to 
Escherichia coli. No trimethoprim- 
resistant colonies were observed after 
transformation. The nucleotide 
sequences of the inserted fragments 
were analyzed. The region between 
s043 and traL showed 97% identity 
to the corresponding region of P. 
rettgeri R391 (accession no. 
AY090559), which encodes 2 hypo- 
thetical proteins (ORF 37 and ORF 
38). The region between traA and 
s054 showed 97% identity to the cor- 
responding region of SXT M01 ° 
(accession no. AY055428). Since the 
gene encoding trimethoprim resist- 
ance was not located in any of the hot 
spot regions proposed by Beaber et 
al. (5), we also analyzed the region 
between s026 and s027 , which in 
R391 contains the kanamycin resist- 
ance gene. Primers s026-F (5' GAG 
CAA TGG GCG AGA GTT CC) and 
s027-R (5' TCA GCG ACA ACC 
GGA GAA TG) gave an amplicon of 
409 bp for SXT M01 °, as expected, 
while no PCR product was obtained 
for SXT LA0S . This result suggested 
that the region between s026 and 
s027 in SXT LA0S is also different 
from SXT M01 °. 

V. cholerae 0139 has not been iso- 
lated in Laos, and the SXT element 
was not likely transmitted from a V. 
cholerae 0139 strain to a V. cholerae 
01 strain. Since SXT LA0S has a hot 
spot that is identical to R391, we 
show evidence for a possible inde- 
pendent emerging of SXT LA0S . 
Further analysis is needed to under- 
stand the evolution and relationship 
between different ICEs and the emer- 
gence of new variants. 

In a previous study (8), we con- 
firmed experimentally that trimetho- 
prim resistance was also transferred 
by conjugation, and we hypothesized 
that the responsible gene is located 


346 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


LETTERS 


within SXT LA0S . However, the gene 
was not found in any of the proposed 
hot spot regions. The possibility that 
the trimethoprim resistance determi- 
nant is located on the chromosome 
outside the SXT element and cotrans- 
fers with the SXT in an Hfr-like man- 
ner cannot be ruled out (9). Therefore, 
additional hot spot regions may exist 
in SXT elements for insertion of 
DNA; otherwise the trimethoprim 
resistance gene is not encoded within 
SXT LA0S . 

The nucleotide sequence data 
reported in this study will appear in 
the DDBJ/EMBL/GenBank nucleo- 
tide sequence databases with the 
accession numbers AB 185252 for the 
hot spot s043-traL and AB 186353 for 
the hot spot traA-s054. 

Claudia Toma,* Noboru Nakasone,* 
Tianyan Song,* 
and Masaaki Iwanaga* 

*University of the Ryukyus, Okinawa, 
Japan 

References 

1. Waldor MK, Tschape H, Mekalanos JJ. A 
new type of conjugative transposon 
encodes resistance to sulfamethoxazole, 
trimethoprim, and streptomycin in Vibrio 
cholerae 0139. J Bacteriol. 1996; 178: 
4157-65. 

2. Hochhut B, Lotfi Y, Mazel D, Faruque SM, 
Woodgate R, Waldor MK. Molecular analy- 
sis of antibiotic resistance gene clusters in 
Vibrio cholerae 0139 and Ol SXT con- 
stins. Antimicrob Agents Chemother. 
2001;45:2991-3000. 

3. Amita, Chowdhury SR, Thungapathra M, 
Ramamurthy T, Nair GB, Ghosh A. Class I 
integrons and SXT elements in El Tor 
strains isolated before and after 1992 Vibrio 
cholerae 0139 outbreak, Calcutta, India. 
Emerg Infect Dis. 2003;9:500-2. 

4. Dalsgaard A, Forslund A, Sandvang D, 
Arntzen L, Keddy K. Vibrio cholerae Ol 
outbreak in Mozambique and South Africa 
in 1998 are multiple-drug resistant, contain 
the SXT element and the aadA2 gene locat- 
ed on class 1 integrons. J Antimicrob 
Chemother. 2001;48:827-38. 

5. Beaber JW, Burras V, Hochhut B, Waldor 
MK. Comparison of SXT and R391, two 
conjugative integrating elements: definition 
of a genetic backbone for the mobilization 
of resistance determinants. Cell Mol Life 
Sci. 2002;59:2065-70. 


6. Boltner D, Mac Mahon C, Pembroke JT, 
Strike P, Osborn A. R391: a conjugative 
integrating mosaic comprised of phage, 
plasmid, and transposon elements. J 
Bacteriol. 2002;184:5158-69. 

7. Phantouamath B, Sithivong N, Sisavath L, 
Munnalath K, Khampheng C, Insisiengmay 
S, et al. Transition of drug susceptibilities 
of Vibrio cholerae Ol in Lao People’s 
Democratic Republic. Southeast Asian J 
Trop Med Public Health. 2001;32:95-9. 

8. Iwanaga M, Toma C, Miyazato T, 
Insisiengmay S, Nakasone N, Ehara M. 
Antibiotic resistance conferred by a class I 
integron and SXT constin in Vibrio choler- 
ae strains isolated in Laos. Antimicrob 
Agents Chemother. 2004;48:2364-9. 

9. Hochuut B, Marrero J, Waldor MK. 
Mobilization of plasmids and chromosomal 
DNA mediated by the SXT element, a con- 
stin found in Vibrio cholerae 0139. J 
Bacteriol. 2000;182:2043-7. 


Address for correspondence: Claudia Toma, 
Division of Bacterial Pathogenesis, Department 
of Microbiology, Graduate School of Medicine, 
University of the Ryukyus, Nishihara, Okinawa 
903-0215, Japan; fax: 81-98-895-1408; email: 
k950417 @med.u-ryukyu.ac.jp 


Modeling the 
Impact of 

Pandemic Influenza 
on Pacific Islands 

To the Editor: Many Pacific 
Island countries and areas have been 
severely impacted in influenza pan- 
demics. The 1918 pandemic killed 
substantial proportions of the total 
population: Fiji -5.2%, Tonga -4.2% 
to 8.4%, Guam -4.5%, Tahiti -10%, 
and Western Samoa -19% to 22% 
(1,2). Thirty-one influenza pandemics 
have occurred since the first pandem- 
ic in 1580(3); another one is likely, if 
not inevitable (4). The potential use of 
influenza as a bioweapon is an addi- 
tional concern (5). 


The scale of an influenza pandem- 
ic may be projected on the basis of the 
available historical data that have 
been built into a computer model, e.g., 
FluAid (6). Flu Aid uses a determinis- 
tic model to estimate the impact range 
of an influenza pandemic in its first 
wave. Given the lack of accessible 
data for specific Pacific Island coun- 
tries and areas, the default values used 
in FluAid were used for the propor- 
tion of the population in the high-risk 
category for each age group, for the 
death rates, hospitalizations, and ill- 
ness requiring medical consultations. 
Country-specific population data 
were obtained from the Secretariat of 
the Pacific Community, and hospital 
bed data were obtained from the 
World Health Organization (WHO) 
(7,8). The FluAid model was supple- 
mented by a model of an 8 -week pan- 
demic wave and modeling of hospital 
bed capacity. Further methodologic 
details are provided in the online 
Appendix (available from htttp:// 
w w w. cdc .gov/ncidod/EID/vol 1 1 no02 
/04-095 l_app.htm). 

The results indicate that at inci- 
dence rates of 15% and 35%, pandem- 
ic influenza would cause 650 and 
1,530 deaths, respectively, giving 
crude death rates of 22 to 52 per 
100,000 (see the Table in the online 
Appendix). Most deaths (83%) would 
occur in the high-risk group, 60% of 
whom would be 19-64 years of age, 
and 22% would be >65 years of age. 
Additionally, 3,540 to 8,250 persons 
would be hospitalized, most of whom 
(78%) would not have high-risk con- 
ditions. Also, 241,000 to 563,000 
medical consultations would occur. 
Most (87%) consultations would be 
for patients without high-risk condi- 
tions (50% birth-18 years of age and 
46% 19-64 years of age). 

In the peak week of the pandemic 
(week 4), from 15% to 34% of all hos- 
pital beds would be required for 
patients with influenza (Table). The 
upper end of impact on hospital beds 
at >40% would occur for Guam, 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


347 


LETTERS 


Table. Predicted impact on health services from the next influenza pandemic using the 

FluAid model for the peak week (at incidence rates [IR] of 15% and 35%) 

Hospital bed 

requirement in the Consultations per 
peak week physician in the 

Input data (% of bed capacity) peak week 
Physicians 
Hospital per 10,000 


Country/area 

beds (n) 

population 

15% IR 

35% IR 

15% IR 

35% IR 

Melanesia 







Fiji Islands 

2,097 

3.4 

16 

38 

78 

182 

New Caledonia 

935 

20.1 

11 

27 

13 

31 

Solomon Islands 

881 

1.3 

17 

39 

209 

487 

Vanuatu 

Micronesia 

605 

1.2 

13 

29 

224 

524 

Federated States of 
Micronesia 

329 

5.9 

13 

29 

45 

106 

Guam 

225 

11.1 

31 

73 

24 

56 

Kiribati 

140 

3.0 

24 

56 

90 

209 

Marshall Islands 

105 

4.6 

18 

42 

59 

137 

Nauru 

50 

15.7 

7 

16 

17 

40 

Northern Mariana 
Islands 

82 

4.5 

43 

100 

58 

135 

Palau 

Polynesia 

90 

11.0 

11 

25 

24 

56 

American Samoa 

140 

7.0 

17 

39 

38 

89 

Cook Islands 

128 

7.8 

5 

12 

34 

79 

French Polynesia 

1,062 

17.5 

10 

23 

15 

35 

Niue 

0 

13.0 

* 

* 

20 

48 

Samoa 

557 

3.4 

12 

29 

79 

184 

Tokelau 

36 

13.3 

2 

4 

20 

47 

Tonga 

200 

3.5 

20 

47 

76 

178 

Tuvalu 

56 

5.9 

7 

17 

45 

105 

Wallis and Futuna 

75 

9.2 

8 

19 

29 

68 

Total 

7,793 

6.3 

15 

34 

42 

99 


*The single hospital in Niue was completely destroyed in a cyclone in 2004. 


Kiribati, Marshall Islands, Northern 
Mariana Islands, and Tonga. 
Assuming all consultations required 
doctors, 42 to 99 influenza consulta- 
tions per doctor would be required 
during the peak week (Table). The 
upper end of impact on consultations 
for individual Pacific Island countries 
and areas would vary from 31 (New 
Caledonia) to 524 (Vanuatu); Fiji, 
Kiribati, Samoa, Solomon Islands, 
Tonga, and Vanuatu would have rates 
>150 consultations per week. 

The uncertainties associated with 
pandemic influenza mean that any 
modeling of its future impact is rela- 
tively crude. For example, the new 
strain may be particularly infectious, 
virulent, or both. In contrast, the use 
of international-level public health 


interventions as recommended by 
WHO (9) may prevent pandemic 
influenza from reaching some Pacific 
Island countries and areas or particu- 
larly remote island groups. These 
issues and other limitations with the 
model are detailed in the online 
Appendix. 

Nevertheless, if the death rate is in 
the range suggested by the model, this 
outcome would make it the worst 
internal demographic event since the 
1918 influenza pandemic for many 
Pacific Island countries and areas. 
The lower death rate (albeit for a sin- 
gle wave) is similar to the U.S. rates 
for the 1957 influenza pandemic (22 
per 100,000) and the 1968 influenza 
pandemic (14 per 100,000) (10). The 
upper end is considerably lower than 


for the 1918 pandemic, which sug- 
gests that the range indicated is rea- 
sonably plausible. Although relatively 
high, the death toll from pandemic 
influenza would still be less than the 
typical annual impact for some 
Pacific Island countries and areas 
from other infectious diseases 
(including malaria and diarrheal dis- 
eases) and from such fundamental 
determinants of health status such as 
poor sanitation, poor diet, and tobacco 
use. 

The predicted range of hospitaliza- 
tions attributable to pandemic influen- 
za would likely overwhelm hospital 
capacity in many of the Pacific Island 
countries and areas. Rapid response at 
the onset of the pandemic could 
ensure efficacious use of hospital beds 
and resources, e.g., cancel elective 
procedures and early discharge to 
community care. Other contingency 
plans by hospitals could facilitate 
lower hospital admission rates (e.g., 
strengthening the primary care 
response). 

Planning and capacity building 
could be provided by WHO, the 
Secretariat of the Pacific Community, 
and donor nations and agencies with 
support for improving surveillance 
and other preventive measures for 
disease control (see the online 
Appendix for details). A combination 
of national capacity building with 
international support will maximize 
the capacity to respond to the next 
influenza pandemic as well as other 
potential communicable disease 
threats. 

Acknowledgments 

Helpful comments were provided by 
Debbie Ryan, George Thomson, and Seini 
Kupu. 

This work was funded in part by the 
New Zealand Ministry of Health. The 
views expressed are those of the authors 
and do not necessarily represent those of 
the Ministry of Health or the Secretariat of 
the Pacific Community. 


348 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


LETTERS 


Nick Wilson,* Osman Mansoor,t 
Douglas Lush,± 
and Tom Kiedrzynski§ 

*Otago University, Wellington, New 
Zealand; fPublic Health Consulting Ltd, 
Wellington, New Zealand; ^New Zealand 
Ministry of Health, Wellington, New 
Zealand; and §Secretariat of the Pacific 
Community, Noumea, New Caledonia 

References 

1. Herda PS. The 1918 influenza pandemic in 
Fiji, Tonga, and the Samoas. In: Bryder L, 
Dow DA, editors. New countries and old 
medicine: proceedings of an international 
conference on the history of medicine and 
health. Auckland (NZ): Pyramid Press; 
1995. p. 46-53. 

2. Crosby AW. America’s forgotten pandemic: 
the influenza of 1918. Cambridge, UK: 
Cambridge University Press; 2003. p. 
233-6. 

3. Lazzari S, Stohr K. Avian influenza and 
influenza pandemics. Bull WHO. 
2004;82:242. 

4. Webster RG. Predictions for future human 
influenza pandemics. J Infect Dis. 
1997;176:S14-9. 

5. Madjid M, Lillibridge S, Mirhaji P, 
Casscells W. Influenza as a bioweapon. J R 
Soc Med. 2003;96:345-6. 

6. Meltzer MI, Shoemake HA, Kownaski M, 
Crosby R. FluAid 2.0: A manual to aid state 
and local-level public health officials plan, 
prepare, and practice for the next influenza 
pandemic (Beta test version). Atlanta: 
Centers for Disease Control and 
Prevention; 2000. 

7. Secretariat of the Pacific Community 
Demography/Population Programme. 
Pacific Island populations 2004, prt 1. 
Available from http://www.spc.org.nc/ 
demo g/EnglishO 1 - 02/Recent Stats/ 
2004/Pacific%20Island%20Populations%2 
02004.xls 

8. World Health Organization. The Work of 

WHO in the Western Pacific Region. 
Report of the regional director — 1 July 
2002-30 June 2003. Manila: The 

Organization; 2003 :p. 217. [cited 2004 Jul 
20]. Available from http://www.wpro. 
who.int/pdf/rcm54/en/rdr/l 9_stat_ 
annex.pdf 

9. World Health Organization. WHO consul- 
tation on priority public health interven- 
tions before and during an influenza pan- 
demic. Geneva: The Organization; 2004. 
[cited 2004 Jul 20]. Available from 
http ://www. who . int/c sr/ dis ease/ avian_influ 
enza/en/final.pdf 

10. Glezen WP. Emerging infections: pandemic 
influenza. Epidemiol Rev. 1996;18:64-76. 


Address for correspondence: Nick Wilson, 
Department of Public Health, Wellington 
School of Medicine and Health Sciences, Otago 
University, PO Box 7343, Wellington South, 
New Zealand; fax: 64-4-4763646; email: nwil- 
son@actrix.gen.nz 


Mycotic Brain 
Abscess Caused by 
Opportunistic 
Reptile Pathogen 

To the Editor: A 38-year-old, 
HIV-seropositive Nigerian man 
sought treatment with an 8-month his- 
tory of severe parietal headache, 
impaired memory, fatigue, paresthesia 
of the left arm, and left- sided focal 
seizures. He had no history of neuro- 
logic disorders, including epilepsy. 
On physical examination, the patient 
appeared well, alert, and oriented, 
with slurred speech. Evaluation of the 
visual fields showed left homony- 
mous hemianopsia. All other neuro- 
logic assessments were unremarkable. 
The patient had a blood pressure of 
120/80, a pulse of 88 beats per 
minute, and a body temperature of 
37.3°C. Leukocyte count was 
8,600/qL, total lymphocyte count was 
l,981/|iL, CD4+ cell count was 
102/|iL, and CD4/CD8 ratio was 0.07. 
HIV RNA-load was <50 copies/mL; 
all other laboratory parameters were 
normal. The patient had received anti- 
retroviral therapy (stavudine, lamivu- 
dine, nevirapine) for 5 months before 
admission, but no prophylaxis for 
opportunistic infections. Magnetic 
resonance imaging (MRI) of the brain 
disclosed 2 masses, 3.3 and 4.8 cm in 
diameter, respectively (Figure A), and 
signs of chronic sinusitis. A computed 
tomographic chest scan showed infil- 
tration of both lower segments with 
multiple, small nodules (Figure B). 


Blood cultures were repeatedly nega- 
tive. A computer-guided needle- 
aspiration of the brain lesions yielded 
yellow-brown, creamy fluid in which 
abundant septated fungal hyphae were 
detected microscopically (Figure C). 
Cytologic investigation was consis- 
tent with a necrotic abscess. The 
cycloheximide-resistant isolate was 
strongly keratinolytic and identified 
as a Chrysosporium anamorph of 
Nannizziopsis vriesii (1,2). High-dose 
antimicrobial treatment with 
voriconazole (200 mg twice daily, 
subsequently reduced to 200 mg 
daily) was added to the antiretroviral 
(ritonavir, amprenavir, trizivir), anti- 
convulsive, and adjuvant corticos- 
teroid treatment. The isolate was 
highly susceptible to voriconazole in 
vitro (MIC, <16|lg/mL [Etest, AB- 
Biodisk Solna, Sweden]). Recovery 
was complicated by a generalized 
seizure and severe, acute psychosis 
associated with rapid refilling of the 2 
lesions with mycotic abscess fluid. 
After re-aspiration, the patient’s psy- 
chosis improved gradually, and no 
further seizures occurred. When last 
seen 4 months later, the patient was 
healthy and without neurologic 
deficits. His CD4+ cell count was 
233/qL, HIV-load was <50 
copies/mL, and a MRI scan of the 
brain showed partial regression of the 
2 brain lesions (Figure D). 

Chrysosporium spp. are common 
soil saprobes, occasionally isolated 
from human skin. Invasive infection 
is very rare in humans, and most were 
observed in immunocompromised 
patients, manifesting as osteomyelitis 
(3,4) or diffuse vascular brain inva- 
sion (5). Here, we report the first case 
of brain abscesses by the 
Chrysosporium anamorph of N. 
vriesii. This fungus has been associat- 
ed with fatal mycosis in reptiles (6,7) 
and cutaneous mycosis in chameleons 
originating from Africa (2). 

In our patient, we were unable to 
determine the portal of entry and the 
sequence of fungal dissemination; no 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


349 


LETTERS 



Figure. Chrysosporium sp. brain abscess in an HIV-seropositive patient. A) T2-weighted 
magnetic resonance imaging (MRI) scan of the brain showing 2 large masses (triangles) 
surrounded by a ring of signal intensity and extensive perifocal edema (open arrows), 
global swelling of the right hemisphere, and a midline shift of 1 .2 cm. B) Computed tomo- 
graphic scan of the chest showing infiltration of the left and right lower segment. C) Mold 
mycelium in aspirate of brain abscess with calcoflour white stain. D) T2-weighted MRI 
scan of the brain performed 4 months after beginning of therapy. 


skin lesions were present at the time 
of admission. However, the multifocal 
nature, lung infiltration, and involve- 
ment of the middle cerebral artery dis- 
tribution suggest hematogenous dis- 
semination (8,9) after replication of 
airborne conidia within the respirato- 
ry tract. 

Fungi cause >90% of brain 
abscesses in immunocompromised 
transplant patients with an associated 
mortality rate of 97% (10), despite 
aggressive surgery and antifungal 
therapy (9). Our patient was treated 
successfully with abscess drainage, 
antiretroviral therapy, and oral 
voriconazole, a novel antifungal tria- 
zole drug. Despite limited data avail- 
able on voriconazole penetration into 
brain abscess cavities (9), this drug 


was clinically and radiologically 
effective in our patient. 

Acknowledgments 

We thank the patient for cooperating 
with our investigation, Pfizer Germany for 
providing voriconazole, and Heidemarie 
Losert and Elisabeth Antweiler for their 
excellent technical assistance. 

Christoph Steininger,* 

Jan van Lunzen,* 

Kathrin Tintelnot,t 
Ingo Sobottka,* Holger Rohde,* 
Matthias Ansver Horstkotte,* 
and Hans-Jurgen Stellbrink* 
*University Clinic Eppendorf, Hamburg, 
Germany; and fRobert Koch-lnstitut, 
Mykologie, Berlin, Germany 


References 

1. Van Oorschot CAN. A revision of 
Chrysosporium and allied genera. Stud 
Mycol. 1980; 1—89. 

2. Pare JA, Sigler L, Hunter DB, Summerbell 
RC, Smith DA, Machin KL. Cutaneous 
mycoses in chameleons caused by the 
Chrysosporium anamorph of Nannizziopsis 
vriesii (Apinis) Currah. J Zoo Wildl Med. 
1997;28:443-53. 

3. Stillwell WT, Rubin BD, Axelrod JL. 
Chrysosporium, a new causative agent in 
osteomyelitis. A case report. Clin Orthop. 
1984; 190-2. 

4. Roilides E, Sigler L, Bibashi E, Katsifa H, 
Flaris N, Panteliadis C. Disseminated infec- 
tion due to Chrysosporium zonatum in a 
patient with chronic granulomatous disease 
and review of non-aspergillus fungal infec- 
tions in patients with this disease. J Clin 
Microbiol. 1999;37:18-25. 

5. Warwick A, Ferrieri P, Burke B, Blazar BR. 
Presumptive invasive Chrysosporium 
infection in a bone marrow transplant recip- 
ient. Bone Marrow Transplant. 
1991;8:319-22. 

6. Nichols DK, Weyant RS, Lamirande EW, 
Sigler L, Mason RT. Fatal mycotic dermati- 
tis in captive brown tree snakes ( Boiga 
irregularis ). J Zoo Wildl Med. 
1999;30:111-8. 

7. Thomas AD, Sigler L, Peucker S, Norton 
JH, Nielan A. Chrysosporium anamorph of 
Nannizziopsis vriesii associated with fatal 
cutaneous mycoses in the salt-water croco- 
dile ( Crocodylus porosus ). Med Mycol. 
2002;40:143-51. 

8. Calfee DP, Wispelwey B. Brain abscess. 
Semin Neurol. 2000;20:353-60. 

9. Mathisen GE, Johnson JP. Brain abscess. 
Clin Infect Dis. 1997;25:763-79. 

10. Hagensee ME, Bauwens JE, Kjos B, 
Bowden RA. Brain abscess following mar- 
row transplantation: experience at the Fred 
Hutchinson Cancer Research Center, 
1984-1992. Clin Infect Dis. 
1994;19:402-8. 

Address for correspondence: Christoph 

Steininger, University Clinic Eppendorf, 
Department of Medicine I, Infectious Diseases 
Unit, Martinistrasse 52, 20246 Hamburg, 
Germany; fax: 49-40-42803-6832; email: 
c . steininger @ uke . uni-hamburg . de 


The opinions expressed by authors con- 
tributing to this journal do not necessari- 
ly reflect the opinions of the Centers for 
Disease Control and Prevention or the 
institutions with which the authors are 
affiliated. 


350 


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LETTERS 


Tuberculosis in 
Undocumented 
Migrants, Geneva 

To the Editor: In today’s global- 
ized world, a growing number of peo- 
ple are migrating in search of a better 
life. Simultaneously, industrialized 
countries are strengthening border 
controls and administrative barriers to 
contain this influx of newcomers, 
resulting in a significant increase in 
illegal migration and human traffick- 
ing. The U.S. Department of State 
estimates the annual flow of irregular 
migrants worldwide to be 700,000-2 
million (1). Many of the migrants are 
from countries where tuberculosis is 
endemic, and they contribute to the 
increasing proportion of foreign-born 
persons with tuberculosis in North 
America and Europe. These persons 
may be highly contagious in the local 
population, as they have limited 
access to healthcare and often go 
untreated (2,3). 

Of 450,000 residents in Geneva, 
Switzerland, 10,000-20,000 are 
undocumented and come from devel- 
oping countries or Eastern Europe. All 
patients treated for tuberculosis in 
Geneva are systematically registered 
by the Antituberculosis Center, a 
facility at Geneva University 
Hospital. An outpatient clinic pro- 
vides free consultations for patients 
with tuberculosis who have no health 
insurance, and patients are not 
required to disclose their immigration 
status to physicians. Patients with suf- 
ficient funds pay for their medication. 

All cases of tuberculosis in undoc- 
umented migrants (foreign residents 
with no resident permits) reported 
from 1994 to 1998 were reviewed by 
the same investigator. Their sociode- 
mographic and clinical characteristics 
were compared with those of 7 South 
American legal residents with tuber- 
culosis (representing the whole sam- 
ple of South American tuberculosis 
patients) during the same period and 


with those of a group of 50 tuberculo- 
sis patients from the general popula- 
tion in a previous study. 

From 1994 to 1998, a total of 397 
persons in Geneva were notified that 
they were infected with tuberculosis. 
Twenty-two (6%) case-patients were 
found among undocumented 
migrants. The mean age was 31 years 
(19^18 years), and 20 (91%) were 
women; 15 (68%) came from South 
America, 5 (22%) came from Africa, 
and 2 (9%) came from Europe. 
Nineteen (95%) of 20 persons had 
symptoms for >1 month preceding 
their first medical encounter. 
Approximately 27.2% had pulmonary 
manifestations only, 36.4% had extra- 
pulmonary manifestations only, and 
the remaining 36.4% had both pul- 
monary and extrapulmonary manifes- 
tations. Mycobacterium tuberculosis 
was found in 11 of 14 with pulmonary 
involvement, and chest radiograph 
was normal in 5 (22%). When com- 
pared with patients from the general 
population, women were more numer- 
ous (91% vs. 30%), and extrapul- 
monary tuberculosis was more fre- 
quent among undocumented residents 
(72% vs. 34%). The time from first 
symptoms to first consultation was 
also longer when compared to the 
general population and the registered 
South American residents (5% vs. 
30% and 40%, respectively, consult- 
ing in the first month; p = 0.008). In 4 
(19%) patients, resistance to >1 anti- 
tuberculosis drug was identified, with 
no multidrug resistance (defined as 
rifampicin) identified, a rate of resist- 
ance similar to that seen in their coun- 
tries of origin but higher than the 
Swiss rate (6.3%) (4). All patients 
were treated with a 4-drug regimen 
(HRZE: H = isoniazid, R = rifam- 
picin, Z = pyrazinamid, E = ethambu- 
tol) for 2 months, followed by a 2- 
drug therapy (HR) for 4 months. 
Eighteen (82%) patients adhered to 
the regimen, as determined by month- 
ly medical interviews and urine isoni- 
azid checks. Only the 4 remaining 


patients who missed more than one 
third of the appointments with 50% of 
negative urine checks, or who default- 
ed, were placed under directly 
observed therapy. Fifteen (68%) 
patients regularly attended their 
appointments until completion of 
treatment. Seven (32%) patients left 
Switzerland before the end of treat- 
ment, 2 of whom were deported. 

Fourteen (64%) patients were hos- 
pitalized to initiate treatment. Four 
had health insurance; the other 
patients contracted a debt for hospital- 
ization. The lack of insurance did not 
influence adherence to treatment neg- 
atively. However, as a consequence of 
tuberculosis, 8 (66%) lost their jobs. 

Of 102 identified close contacts, 
88 (87%) were evaluated by tuber- 
culin skin testing. Chest x-ray was 
performed on 21 (24%) patients with 
a positive test (>10 mm induration), 
and isoniazid was prescribed prophy- 
lactically. No secondary case of active 
tuberculosis was identified. 

Most undocumented immigrants 
with tuberculosis in Geneva are young 
South American or African women 
engaged in domestic activities. This 
finding reflects the irregular work 
opportunities in Geneva, an area with 
little agriculture and industry. As sus- 
pected, a delay of several weeks 
occurred before seeking care (5). The 
economic and social impact of tuber- 
culosis was high for this population. 
Two thirds of these patients lost their 
jobs as a consequence of tuberculosis. 
Joblessness could be an additional fac- 
tor to further deter patients from seek- 
ing care. Adherence to treatment was 
good, which suggested confidence that 
care providers would not report to 
immigration authorities and that sup- 
portive follow-up care was available. 
Of more concern, approximately one 
third of the patients left Switzerland 
before completing the full course of 
treatment. This transfer rate of undoc- 
umented migrants corresponds to that 
observed (43%) among foreign-born 
patients with unknown legal status in 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


351 


LETTERS 


Switzerland (6). Failure to complete a 
full course of treatment may lead to 
relapse and emergence of resistant 
strains. A growing proportion of cases 
of tuberculosis observed in Europe is 
in migrants, some undocumented, 
from the developing world (3). Strong 
political measures should be enforced 
to ensure access to healthcare services 
with respect to confidentiality (as 
recently stated in the Netherlands) (7). 
Much emphasis has been put on 
screening at time of arrival. Screening 
can be conducted for immigrants and 
asylum seekers, but undocumented 
migrants are not screened (8). 
Facilitated access to medical services 
and free affordable therapy is a neces- 
sity; active tuberculosis develops in 
most foreign-born residents several 
years after their arrival (2). In an era of 
high mobility, specific innovative pro- 
grams should be established to control 
and prevent tuberculosis for this high- 
risk, foreign-bom population. Early 
detection with nonidentifying tubercu- 
losis tracking systems (9), screening at 
unspecialized clinics (10), and free 
treatment with adequate administra- 
tive measures are needed. 
Industrialized countries must take 
responsibility to reduce the spread of 
resistant tuberculosis. 

Sigiriya Aebischer Perone,* 
Patrick Bovier,* 

Christian Pichonnaz,* 

Thierry Rochat,* and Louis Loutan* 

*Geneva University Hospital, Geneva, 
Switzerland 

References 

1. International Organization for Migration. 
World migration 2003: managing migration, 
challenges and responses for people on the 
move. Geneva: The Organization; 2003. 

2. Centers for Disease Control and 
Prevention. Trends in tuberculosis — United 
States, 1998-2003. MMWR Morb Mortal 
Wkly Rep. 2004;53:209-14. 

3. EuroTB. Surveillance of tuberculosis in 

Europe — EuroTB. Report on tuberculosis 
cases notified in 2001. 2003, p. 1-60. [cited 
October 20, 2004]. Available from 

http ://www. eurotb . org/repports/200 1 / etb_2 
00 l_p l_text_tables.pdf 


4. Helbing P, Altpeter E, Raeber PA, Pfyffer 
GE, Zellweger JP. Surveillance of antitu- 
berculosis drug resistance in Switzerland 
1995-1997; the central link. Eur Respir J. 
2000;16:200-2. 

5. Asch S, Leake B, Gelberg L. Does fear of 
immigration authorities deter tuberculosis 
patients from seeking care? West J Med. 
1994; 373-6. 

6. Helbing P, Medinger C, Altpeter E, Raeber 
PA, Beeli D, Zellweger JP. Outcome of 
treatment of pulmonary tuberculosis in 
Switzerland in 1996. Swiss Med Wkly. 
2002;132:517-22 

7. Sheldon T. Dutch minister warns that ille- 
gal immigrants must receive care. BMJ. 
1999;3 18: 1234. 

8. Menzies D. Controlling tuberculosis among 
foreign born within industrialised coun- 
tries. Am J Respir Crit Care Med. 
2001;164:914-5. 

9. Kim DJ, Rizdon R, Giles B, Mireles T, 
Garrity K, Hathcock AL, et al. A no-name 
tuberculosis tracking system. Am J Public 
Health. 2003;93:1637-9 

10. El-Hamad I, Casalini C, Matteelli A, Casari 
S, Bugiani M, Caputo M, et al. Screening 
for tuberculosis and latent tuberculosis 
infection among undocumented immigrants 
at an unspecialised health service unit. Int J 
Tuberc Lung Dis. 2001;5:712-6. 


Address for correspondence: Louis Loutan, 
Travel and Migration Medicine Unit, 
Department of Community Medicine, Geneva 
University Hospital, 24, rue Micheli-du-Crest, 
1211 Geneva 14, Switzerland; fax: 0041-22- 
372-96-26; email: louis. loutan @hcuge.ch 


Mycobacterium 
chelonae Skin 
Infection in Kidney- 
Pancreas Recipient 

To the Editor: Mycobacterium 
chelonae is rapid growing and is ubiq- 
uitous in the environment, including 
soil, water, domestic and wild ani- 
mals, and milk and fruit products. It 
can be associated with infections of 
the soft tissue, lung, bone, joint, cen- 
tral nervous system, and eye. M. che- 
lonae infections in an immunocom- 


promised host are disseminated in 
>50% of those infected; chronic use 
of steroids, even in low doses, seems 
to be the most important predictive 
factor for disseminated disease (1,2). 
In immunocompetent hosts, nontuber- 
culous mycobacteria can colonize 
body surfaces and be secreted for pro- 
longed periods without causing dis- 
ease. In hematopoietic stem cell and 
solid organ transplant recipients, 
infections with nontuberculous 
mycobacteria are common and may 
be a source of illness and death (3). 
We describe a case of localized cuta- 
neous M. chelonae infection after a 
dog bite in a kidney-pancreas trans- 
plant recipient. 

A 43-year-old female patient 
underwent kidney transplantation for 
diabetic nephropathy in 1985. After 
loss of organ function due to chronic 
rejection, she underwent combined 
kidney-pancreas transplantation 5 
years later, in 1990. Because of chron- 
ic rejection, the patient lost the kidney 
graft 5 years later, in 1995, and went 
back on dialysis with a well-function- 
ing pancreas graft. In 2004, the patient 
was bitten on the right forearm by a 
dog. She was on immunosuppressive 
therapy of prednisolone (5 mg/day), 
cyclosporine- A (trough levels of 100 
ng/dL), and azathioprine (50 mg/day). 
The initial lesion healed without 
major complication. After several 
days, a single firm edematous plaque 
of 3 x 5 cm developed at the site of the 
animal bite, and the patient was 
admitted to the Department of 
Dermatology. Empiric antimicrobial 
combination therapy, including clin- 
damycin (300 mg every 8 hours) and 
ciprofloxacin (500 mg every 12 
hours), was initiated. As no clinical 
improvement was achieved, a biopsy 
was performed, which showed a gran- 
ulomatous inflammation with a high 
number of mycobacteria (Figure). 
Atypical mycobacteria were cultured 
from a second biopsy (Lowenstein- 
Jensen/Stonebrink, Heidelberg, Ger- 
many); M. chelonae was identified by 


352 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


LETTERS 



Figure. Skin biopsy of the lesion showed granulomatous infection with Mycobacterium 
chelonae (Ziehl-Neelsen stain, x40) 


polymerase chain reaction. Therefore, 
antimicrobial therapy was changed to 
clarithromycin (500 mg twice daily) 
for 6 months. Although cyclosporine- 
A dosage was reduced with initiation 
of antimicrobial therapy, trough level 
increased to 350 ng/mL; therefore, 
further dose reduction was performed. 
Within a few weeks, the lesion disap- 
peared completely, and the patient 
retained good pancreatic graft func- 
tion. To rule out dissemination to 
other organs, a computed tomography 
of head, thorax, and abdomen was 
performed at time of diagnosis. 

In the immunocompromised host, 
an uncontrolled proliferation of pri- 
marily colonizing or contaminating 
pathogens or commensals can 
progress to severe disease. Diagnosis 
is often difficult because patients with 
these infections may have atypical 
symptoms due to immunosuppressive 
therapy. If diagnosis is made early, 
dissemination can likely be avoided. 
Therefore, suspicious cutaneous 
lesions should be biopsied for 
histopathologic examination, and spe- 
cial stains and tissue cultures should 
be performed for detecting fungi, 
viruses, and bacteria, including 
mycobacteria (3,4). Nontuberculous 
mycobacteria are resistant to conven- 
tional tuberculostatic therapy and 
have variable susceptibility to other 


antimicrobial agents (1,2,5). Clari- 
thromycin seems to be the most active 
drug, and azithromycin might also 
have good activity (3-6). Clari- 
thromycin has been administered suc- 
cessfully as monotherapy, and our 
case confirms these data. However, 
several cases of resistance have been 
described, and use of at least 1 other 
drug, such as an aminoglycoside or a 
quinolone, in addition to clar- 
ithromycin has been recommended 
(1,4, 7-9). Clarithromycin is a potent 
inhibitor of cytochrome P450 (3,4). 
Therefore, cyclosporine-A and tacro- 
limus levels have to be monitored 
exactly, and dose adjustments may be 
required. Duration of therapy depends 
on the isolate, site of infection, and 
clinical response to therapy, but in 
general, it should be continued for at 
least 6 months (3,8). 

Thus far only a few cases of infec- 
tions with M. chelonae in kidney, 
heart, liver, and lung transplant recip- 
ients have been described (3). Most of 
these infections were disseminated 
and often resulted in chronic infec- 
tion. To our knowledge, this report is 
the first of localized cutaneous 
disease from M. chelonae , which 
completely healed within 3 months, in 
a kidney-pancreas transplant recipi- 
ent. Although M. chelonae might be 
part of the colonizing oral flora of 


dogs, it is more likely that the bite 
contributed to translocation of the 
transient dermal flora. Any factor that 
disrupts the skin barrier, such as 
insulin self-injection in diabetes 
patients, surgical wound, insect sting, 
or animal bite, might be associated 
with this type of infection (10,11). We 
conclude that early diagnosis prevents 
dissemination, leads to rapid clinical 
response, and allows antimicrobial 
monotherapy with a macrolide. Such 
an approach preserved the function of 
the pancreatic allograft. 

Ingrid Stelzmueller,* 

Karin M. Dunst,* Silke Wiesmayr,* 

Robert Zangerle,* Paul Hengster,* 
and Hugo Bonatti* 

*lnnsbruck Medical University, Innsbruck, 
Austria 

References 

1. Wallace RJ Jr, Brown BA, Onyi GO. Skin, 
soft tissue and bone infections due to 
Mycobacterium chelonae : importance of 
prior corticosteroid therapy, frequency of 
disseminated infections and resistance to 
oral antimicrobials other than clar- 
ithromycin. J Infect Dis. 1992;166:405-12. 

2. Nathan DL, Singh SS, Kestenbaum TM, 
Casparian JM. Cutaneous Mycobacterium 
chelonae in a liver transplant patient. J Am 
Acad Dermatol. 2000;43:333-6. 

3. Doucette K, Fishman JA. Nontuberculous 
mycobacterial infection in hematopoietic 
stem cell and solid organ transplant recipi- 
ents. Clin Infect Dis. 2004;38:1428-39. 

4. Patel R, Roberts GD, Keating MR, Paya 
CV. Infections due to nontuberculous 
mycobacteria in kidney, heart and liver 
transplant recipients. Clin Infect Dis. 
1994;19:263-73. 

5. Weisdorf DJ. Typical and atypical 

Mycobacterium infections after hemopoiet- 
ic stem cell or solid organ transplantation. 
In: Bowden RA, Ljungman P, Paya CV, edi- 
tors. Transplant infections. 2nd ed. 
Philadelphia: Lippincott Williams & 

Wilkins; 2003. p. 250-8. 

6. Wallace RJ, Tanner D, Brennan PJ, Brown 
BA. Clinical trial of clarithromycin for 
cutaneous (disseminated) infection due to 
Mycobacterium chelonae. Ann Intern Med. 
1993;119:482-6. 

7. Chastain MA, Buckley J, Russo GG. 
Mycobacterium chelonae! abscessus com- 
plex infection in a liver transplant patient. 
Int J Dermatol. 2001;40:769-4. 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


353 


LETTERS 


8. Tebas P, Faisal S, Wallace RJ, Fraser Y. 
Rapid development of resistance to clar- 
ithromycin following monotherapy for dis- 
seminated Mycobacterium chelonae infec- 
tion in a heart transplant patient. Clin Infect 
Dis. 1995;20:443-4. 

9. Vemulapalli RK, Cantey JR, Steed LL, 
Knapp TL, Thielmann NM. Emergence of 
resistance to clarithromycin during treat- 
ment of disseminated cutaneous 
Mycobacterium chelonae infection: case 
report and literature review. J Infect Dis. 
2001;43:163-8. 

10. Singh S, Rattan A, Kumar S. Severe cuta- 
neous Mycobacterium chelonae infection 
following a yellow jacket sting. Tuber Lung 
Dis. 1992;73:305-6. 

11. McKinsey DS, Dykstra M, Smith DL. The 
terrier and the tendonitis. N Engl J Med. 
1995;332:338. 


Address for correspondence: Ingrid 

Stelzmueller, Medical University Innsbruck, 
Department of General and Transplant Surgery, 
AnichstraBe 35, 6020 Innsbruck, Austria; fax: 
+43-512-504-22605; email: ingrid. stelz- 

mueller @ uklibk. ac . at 


SARS Control and 
Psychological 
Effects of 
Quarantine, 
Toronto, Canada 

To the Editor: Hawryluck et al. 
(1) have published an interesting 
study that found that some persons 
subject to quarantine for severe acute 
respiratory distress syndrome (SARS) 
displayed symptoms of posttraumatic 
stress disorder and depression. They 
conclude that the psychological 
symptoms result from quarantine. I 
believe the study has serious flaws 
and that their conclusion is premature. 

First, their study sampled 129 vol- 
unteers among the >15,000 persons 
subjected to quarantine. As acknowl- 
edged by the authors, persons with the 
most severe symptoms may be more 


likely to volunteer for the study, 
resulting in an overestimation of the 
frequency and severity of the symp- 
toms. Second, more than two thirds of 
the participants were healthcare work- 
ers. Healthcare workers in Toronto 
who cared for SARS patients but were 
not subject to quarantine were experi- 
encing extreme stress because they 
were working with a poorly under- 
stood infectious disease, wearing pro- 
tective equipment for extended peri- 
ods, and watching colleagues become 
ill and die while wondering if they 
themselves were the next victims. 
Most healthcare workers subject to 
quarantine in Toronto (including 34% 
of persons on work quarantine) likely 
cared for SARS patients and would 
have experienced stresses similar to 
those not quarantined. Third, 85% of 
the study participants wore masks at 
home, indicating that they were likely 
to have been symptomatic and subject 
to isolation rather than quarantine. 
Certainly symptomatic persons would 
be undergoing stress because of their 
concerns about SARS developing, the 
possibility of dying, and the potential 
for exposing others. Increasing levels 
of stress with increasing length of iso- 
lation found in the study may be due 
to more severe or prolonged symp- 
toms rather than to isolation or quar- 
antine per se. 

Measuring the psychological 
effects of isolation and quarantine will 
require studies comparing psycholog- 
ical symptoms of healthcare workers 
subjected to quarantine with those 
who continued working, as well as 
studies comparing randomly selected 
persons subject to isolation with the 
general population living in the city 
during the outbreak. 

In the final analysis, although iso- 
lation and quarantine are stressful, 
that is an insufficient reason to hesi- 
tate when these measures are indicat- 
ed. One might wonder how stressed 
the participants would have been if 
SARS had developed and they infect- 
ed their family members or friends. 


Regardless of whether isolation and 
quarantine induce posttraumatic stress 
disorder, public health officials must 
be cognizant of and prepared to sup- 
ply appropriate emotional and social 
support to persons subject to isolation 
or quarantine. 

Harry F. Hull* 

*Minnesota Department of Health, 
Minneapolis, Minnesota, USA 

Reference 

1. Hawryluck L, Gold WL, Robinson S, 
Pogorski S, Galea S, Styra R. SARS control 
and psychological effects of quarantine, 
Toronto, Canada. Emerg Infect Dis. 
2004;10:1206-12. 


Address for correspondence: Harry F. Hull, 
State Epidemiologist and Division Director, 
Infectious Disease Epidemiology, Prevention 
and Control Division, Minnesota Department 
of Health, 717 Delaware, SE, Minneapolis, MN 
55414, USA; fax: 612-676-5666; email: 
harry, hull @ health, state . mn .us 


In Response: Dr. Hull raises con- 
cerns regarding our study design and 
the conclusions that were drawn, 
believing the conclusions to be pre- 
mature (1). To reiterate, we concluded 
that quarantine might result in consid- 
erable psychological distress in the 
forms of posttraumatic stress disorder 
(PTSD) and depressive symptoms, 
but we clearly qualify this conclusion 
by stating that the results of the study 
are hypothesis-generating and require 
further exploration. 

Dr. Hull correctly writes that more 
than two thirds of the respondents to 
our survey were healthcare workers 
and assumes that healthcare workers 
in Toronto who cared for patients 
with severe acute respiratory syn- 
drome (SARS) were extremely 
stressed. We agree with this statement 
not on the basis of data presented in 
this study, but rather on additional 


354 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


LETTERS 


work that we have conducted on non- 
quarantined, uninfected healthcare 
workers treating patients with SARS 
in a hospital in Toronto (2). The 
implication of Dr. Hull’s statement, 
however, is that being a healthcare 
worker in Toronto at the time of 
SARS, rather than being placed into 
quarantine, was responsible for the 
psychological distress that we meas- 
ured. To dispute this, we found that 
healthcare worker status was not cor- 
related with PTSD or depression 
symptoms, which indicates that 
respondents who were nonhealthcare 
workers experienced similar levels of 
distress as healthcare workers who 
responded. Furthermore, we found 
that longer durations of quarantine 
were associated with increased symp- 
toms of PTSD, which indicates that 
the physical state of being in quaran- 
tine was at least in part responsible 
for the psychological distress. 

Finally, Dr. Hull states that 85% of 
the study participants wore masks at 
home, which indicates that they were 
likely to have been symptomatic and 
subject to isolation rather than quar- 
antine. This statement is incorrect. 
The respondents to this survey were 
asymptomatic, exposed persons who 
were placed into quarantine. 
Instructions to all quarantined per- 
sons, per public health guidelines (3), 
were to wear masks while in the pres- 
ence of other household members, 
not because they were symptomatic, 
but rather because they may have 
been incubating SARS and had the 


potential to transmit infection to 
household contacts in the 24 hours 
before symptom onset. 

Although the terms isolation and 
quarantine have often been used inter- 
changeably, they actually represent 
distinct concepts (4). The strategies 
differ in that isolation applies to per- 
sons who are known to have an ill- 
ness, and quarantine applies to those 
who have been exposed to a transmis- 
sible pathogen but who may or may 
not become ill. Quarantine directives 
for SARS included the adherence to 
home infection control measures, 
including wearing masks in the pres- 
ence of other household members, not 
sharing utensils, and sleeping in sepa- 
rate quarters (3). 

We agree with Dr. Hull’s final 
statement that the psychological dis- 
tress experienced by persons in quar- 
antine is not a sufficient reason to 
refrain from invoking these measures 
when they are needed to control an 
outbreak. We did not arrive at this 
conclusion in our article. The goal of 
the study was to develop a benchmark 
for the possible distress associated 
with quarantine. While we felt that 
documenting the possible distress that 
may result from quarantine was 
important, it was not intended to 
negate the need to impose quarantine 
should it be required, but rather to 
determine the support measures that 
may be needed by quarantined per- 
sons. Public health officials must be 
cognizant of these needs and prepared 
to supply appropriate emotional and 


social support to persons in quaran- 
tine for such measures to succeed in 
halting the spread of disease. 

Rima Styra,* Laura Hawryluck,* 
and Wayne Gold* 

*University Health Network, Toronto, 
Ontario, Canada 

References 

1. Hull HF. SARS control and psychological 
effects of quarantine Toronto, Canada [let- 
ter]. Emerg Infect Dis. 2005;11:353-4. 

2. Gold WL, Hawryluck L, Robinson S, 
McGeer A, Fones C, Kennedy S, et al. Post- 
traumatic stress disorder (PTSD) among 
healthcare workers (HCW) at a hospital 
treating patients with SARS [abstract]. In: 
Program and abstracts of the 43rd meeting 
of the Interscience Conference on 
Antimicrobial Agents and Chemotherapy 
(ICAAC); 2003 Sep 14-17; Chicago. 
Washington (DC): American Society for 
Microbiology; 2003. 

3. Svoboda, T, Henry B, Shulman L, Kennedy 
E, Rea E, Ng W, et al. Public health meas- 
ures to control the spread of the severe 
acute respiratory syndrome during the out- 
break in Toronto. N Engl J Med. 
2004;350:2352-61. 

4. Centers for Disease Control and 
Prevention. Public health guidance for 
community-level preparedness and 
response to severe acute respiratory syn- 
drome (SARS) Version 2/3. 2004 Jul 20 
[cited 2004 Sep 26]. Available from 
http://www.cdc.gov/ncidod/sars/guidance 

Address for correspondence: Rima Styra, 
Toronto General Hospital, 200 Elizabeth St, 8 
Eaton North - Rm 235, Toronto, Ontario, 
Canada M5G 2C4; fax: 416-340-4198; email: 
rima.styra@uhn.on.ca 


Correction, Vol. 9, No. 12 


In "Severe Acute Respiratory Syndrome Epidemic 
in Asia," by G. Zhou and G. Yan, an error occurred in 
the Table. Under the table heading "Parameter estima- 
tion," the third subheading should be 'T/a." The cor- 
rected table appears online at http://www.cdc.gov/nci- 
dod/EID/vol9no 12/03-03 82. htm#table 

EID 

Ontlnt 

www.cdc.gov/eid 

We regret any confusion this error may have caused. 



Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


355 




BOOK REVIEWS 


Community-based 
Health Research: 
Issues and 
Methods 

Daniel S. Blumenthal and Ralph J. 
DiClemente, editors 

Springer Publishing Company, 

New York, New York 
ISBN: 0-8261-2025-3 
Pages: 240, Price: U.S.$39.95 

So many, 11 of 14, contributors to 
this volume are from the Atlanta area 
that I expected this book to speak with 
a Southern drawl. That it does not 
attests to how much this metropolis, 
growing in kudzu-fashion, has 
changed. This element of profound 
change is also a major motif in the 10 
chapters collected by the editors to 
promote community-based health 
research as a mechanism for address- 
ing historic wrongs. 

The book is aptly titled and subti- 
tled. Former Surgeon General David 
Satcher writes in a concise but illumi- 
nating foreword, “Community-based 
research is where medicine, public 
health, and science meet.” In the open- 
ing chapter, Daniel Blumenthal and 
Eileen Yancey herald the arrival of a 
“new paradigm” in which community 
members become full partners with 
“culturally competent” researchers. To 
them, community-based research is 
population centered, prevention 
focused, multidisciplinary, collabora- 
tive, enlightening, and empowering. 
Caswell Evans follows by adding 
“evidence-based” assessments, find- 
ings, and guidance to the mix. In chap- 
ter 3, Bill Jenkins, Camara Jones, and 
Blumenthal address some of the ethi- 
cal issues related to community-based 
research by describing, analyzing, and 
drawing lessons from the Tuskegee 
syphilis study. Culturally and linguis- 
tically diverse voices from the com- 
munity are heard in chapter 4. 


Attention shifts from issues to 
methods in the last 6 chapters of the 
book. In textbook fashion, Nabih R. 
Asal and Laura A. Beebe distinguish 
observational studies from experi- 
mental designs in chapter 5 and 
remind the reader of the importance of 
person, place, and time in epidemio- 
logic investigations. The strengths 
and weaknesses of the Behavioral 
Risk Factor Surveillance System are 
illustrated by Deborah Holtzman in 
chapter 6. Qualitative research meth- 
ods are described in chapter 7 and 
applied to a case study of 45 African- 
American, female crack-cocaine users 
in chapter 8. Community intervention 
trials are introduced and a half dozen 
are reviewed in chapter 9. Then the 
book rather abruptly ends with a short 
chapter on cardiovascular risk-reduc- 
tion community intervention trials. 

Instructors looking for a graduate- 
level textbook may find this recent 
addition to the preventive medicine 
literature incomplete. It fails to link 
community-based research with theo- 
ries of social and cultural change; the 
principles and practices of community 
mobilization; and the identification, 
development, implementation, and 
evaluation of culturally competent 
interventions. The editors have pro- 
duced an adequate introduction to 
community-based research issues and 
methods, but a concluding section that 
serves to pull all the components 
together would put additional copies 
of this publication in college book- 
stores. 


William W. Darrow* 

*Florida International University, North 
Miami, Florida, USA 


Address for correspondence: William W. 
Darrow, Robert R. Stempel School of Public 
Health, Florida International University, 3000 
NE 151st St, TR-7, North Miami, Florida 
33181-3600, USA; fax: 305-919-5673; email: 
darro w w @ f iu . edu 


The Pneumococcus 

Elaine I. Tuomanen, Timothy J. 
Mitchell, Donald A. Morrison, and 
Brian G. Spratt, editors 

Washington: American Society for 
Microbiology Press; 2004 
ISBN: 1 -55581 -297-X 
Pages: 466, Price: U.S.$115.95 

Streptococcus pneumoniae , known 
as the pneumococcus, remains an 
important pathogen in spite of 
tremendous advances in medical care. 
Globally, as many as 1 million chil- 
dren die of pneumococcal infections 
each year, nearly all in developing 
countries. Pneumococcal disease is 
also common in children in industrial- 
ized countries, although in those set- 
tings nearly all such deaths occur in 
older adults or adults with chronic 
medical conditions. Given its place 
near the top of the list of killer bacte- 
ria, pneumococcus is a focus of 
numerous researchers around the 
world. A new book, The Pneumo- 
coccus, edited by Elaine Tuomanen et 
al., is the latest effort to summarize 
the state of research on the organism. 

The book begins by providing a 
well-thought-out answer to a basic 
question — what is a pneumococ- 
cus? — and moves on to chapters on 
topics ranging from attachment and 
invasion of the respiratory tract to 
vaccine-induced immunity. The edi- 
tors are leaders primarily in the areas 
of molecular biology and pathogene- 
sis, and the focus of much of the book 
is on these topics, although issues 
such as treatment, carriage, disease in 
persons with immunodeficiencies, 
antimicrobial resistance, and epidemi- 
ology are also well covered. The rela- 
tively recent deciphering of several 
pneumococcal genomes has led to a 
new outburst of research activity, 
aspects of which are summarized in 
several of the chapters. 


356 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


BOOK REVIEWS 


All of the authors are recognized 
experts in their respective areas. The 
foreword by Robert Austrian, a pio- 
neer in pneumococcal microbiology, 
disease description, and vaccine 
work, provides an interesting summa- 
ry of the history of major discoveries 
in the field. While covering many 
areas of pneumococcal research, the 
book is not exhaustive; for example, 
issues specific to pneumococcal dis- 
ease in developing countries are men- 
tioned only in passing. 

The book may be most suitable as 
a tool for new researchers in the pneu- 
mococcal field, but it may also be use- 
ful for medical students, graduate stu- 
dents, and infectious disease special- 
ists. The level of detail varies among 
the chapters, but it is adequate to pro- 
vide an introduction to each of the 
topics covered, and all chapters are 
thoroughly referenced. Overall, the 
editors and writers have done a 
remarkable job of consolidating the 
latest information. The Pneumo- 
coccus is an authoritative reference in 
a rapidly changing field. 

Cynthia G. Whitney* 

*Centers for Disease Control and 
Prevention, Atlanta, Georgia, USA 

Address for correspondence: Cynthia G. 
Whitney, Respiratory Diseases Branch, 
Division of Bacterial and Mycotic Diseases, 
National Center for Infectious Diseases, 
Centers for Disease Control and Prevention, 
1600 Clifton Rd NE, Mailstop C23, Atlanta, 
GA 30333, USA; fax: 404-639-3970; email: 
c whitney @ cdc . go v 


DNA Amplification: 
Current 

Technologies and 
Applications 

Vadim V. Demidov and Natalia E. 
Broude, authors 

Horizon Bioscience, Norfolk, UK 
IBSN: 0-9545232-9-6 
Pages: 335, Price: U.S.$180 

DNA amplification is a powerful 
technique that has had an immense 
impact on scientific research in the 
past 2 decades. While polymerase 
chain reaction (PCR) is still the most 
popular method, alternative methods 
of DNA amplification are constantly 
being developed. In addition, the 
extraordinary versatility of PCR has 
led to its use in novel ways that have 
opened new avenues of research. 
These novel methods for DNA ampli- 
fication and the versatility of PCR 
are highlighted in DNA Amp- 
lification: Current Technologies and 
Applications. 

The 17 chapters in this book are 
divided into 4 sections that focus on 
enzymes (3 chapters), thermal cycling 
methods (6 chapters), isothermal 
methods (6 chapters), and the detec- 
tion of non-DNA analytes by DNA 
amplification (2 chapters). Each chap- 
ter has a thorough description of 
methods and highly detailed protocols 
for applying the technique to at least 1 
specific application. Several excellent 
chapters describe the uses of Phi29 


DNA polymerase and of applications 
using isothermal rolling circle ampli- 
fication. A chapter on multiple-dis- 
placement amplification details the 
isothermal amplification of total 
genomic DNA and should prove 
extremely useful for amplifying DNA 
in limited amounts, such as DNA 
from clinical samples. The final 2 
chapters describe use of either real- 
time PCR or rolling circle amplifica- 
tion to detect and quantify non-DNA 
analytes, such as serum cytokines, 
with much greater sensitivity than 
conventional enzyme-linked immuno- 
sorbent assay methods. 

This book is not for the novice sci- 
entist, as it does not describe basic 
DNA amplification fundamentals; 
rather, it is directed at those with a 
solid background in molecular biolo- 
gy who desire knowledge of cutting- 
edge applications. Although many of 
the detailed protocols will not be 
applicable to certain laboratory situa- 
tions, the versatility of most of the 
methods described will allow them to 
be easily adapted to other studies. 
Therefore, this book will be a good 
addition to the library of researchers 
in molecular biology or to molecular 
diagnostics laboratories planning to 
expand their horizon beyond standard 
PCR amplification techniques. 

Robert F. Massung* 

*Centers for Disease Control and 
Prevention, Atlanta, Georgia, USA 


Address for correspondence: Robert F. Massung, 
Centers for Disease Control and Prevention, 
1600 Clifton Rd, Atlanta, GA 30333, USA; fax: 
404-639-4436; email: rfm2@cdc.gov 


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357 


ABOUT THE COVER 



Romare Bearden (1911-1988). 

The Sea Nymph (1977) 

Collage on various papers with paint and 
graphite on fiberboard 
(111 .8 cm x 81 .3 cm). 

Permanent collection: Glen and 
Lynn Tobias. 

Cover art copyright 

Romare Bearden Foundation / Licensed 
by VAGA, New York, New York 


Hazards of Travel— Who Will 
Free the Contemporary 
Traveler? 


Polyxeni Potter* 


A native of Mecklenburg County, North Carolina, Romare Bearden was the 
offspring of a middle-class family established in Charlotte, where the rail- 
road and cotton industries flourished after the Civil War. His paternal great- 
grandparents, with whom he spent considerable time, were described in the 
1915 publication Colored Charlotte as “former servants of Dr. Joseph Wilson 
the father of President Woodrow Wilson....” (1). His maternal grandparents, 
who were also influential in his development, ran a boarding house in 
Pittsburgh, Pennsylvania, serving migrant steel mill workers from the South. 

Around 1914, Bearden’s family moved north to New York and settled in 
Harlem. In their apartment at 154 West 131st Street, he grew up with the artis- 
tic, intellectual, and political influences of the cultural movement of the 1920s 
and 30s known as Harlem Renaissance. His circle included writers Langston 
Hughes and Ralph Ellison, musicians Duke Ellington and Fats Waller, activist 
W.E.B. DuBois, and artists Aaron Douglas and Jacob Lawrence (2). Although 
he spent most of his school years in New York, Bearden visited Pittsburgh 
often, enjoying life in his grandparents’ boardinghouse, where mill workers 
returning from work would sit on the steps and “tell stories about down-home 
in the South” (1). 

Bearden had many talents and broad academic interests. He graduated from 
New York University with a degree in education, but he also loved mathemat- 
ics and music and was an accomplished writer and cartoonist. His editorial 
drawings on the social, political, and economic issues of his day (depression era 
soup lines, segregation, social inequality), are reminiscent of the politically 
charged work of Diego Rivera and other Mexican muralists and of Francisco 
de Goya’s caprices, which chronicled the vices of 19th-century Spain. Bearden 
studied art throughout his life. While employed in the New York City 
Department of Social Services, he satisfied his growing wish to become an 
artist by painting during evenings and weekends. By the end of the 1930s, he 
was fully engaged in art. 

“...[T]he function of the artist is to find ways of communicating, in sensi- 
ble, sensuous terms, those experiences which do not find adequate expression 
in the daily round of living and for which, therefore, no ready made means of 
communication exists. . .” wrote Bearden in his first solo exhibition pamphlet in 
1940 (1). In a career marked by continuous growth, he experimented with new 
media, always seeking the texture, form, and color that most closely embodied 
his artistic goals, and became one of the most creative and original artists of the 
20th century. 

Bearden’s early work was mostly gouaches (opaque watercolors on brown 
paper). He became increasingly interested in the human figure but gradually 
moved away from representational painting toward abstraction (3) and “those 
universals that must be digested by the mind and cannot be merely seen by the 
eye” (4). By the early 1960s, he was constructing photomontages, which he 


*Centers for Disease Control and Prevention, Atlanta, Georgia, USA 


358 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 





ABOUT THE COVER 


continued to refine through various techniques into col- 
lages, his signature style. During the 1970s and 80s, he 
synthesized elements of his earlier work into an individu- 
alized art form using brown paper, brilliant color, and 
graphite drawings. 

The collage, which dates back to medieval Persia and 
Japan, was known in Europe well before the 18th century 
and was rediscovered and used in modern times by Pablo 
Picasso and others. Bearden turned the medium into a nar- 
rative device, synthesizing color, form, photographic 
images, and patches of social commentary into intricate, 
richly textured, intensely emotional scenes. “. . .1 use many 
disparate elements to form either a figure, or part of a 
background. I build my faces... from parts of African 
masks, animal eyes, marbles, mossy vegetation....” (5). 

A prolific artist, Bearden painted the places where he 
lived and worked: the rural South, northern cities of his 
childhood, and the Caribbean islands where he spent the 
latter part of his life. His artistic goal was “to reveal 
through pictorial complexities the riches of a life I know.” 
“I do not need to go looking for ‘happenings,’ the absurd, 
or the surreal,” he said, “because I have seen things that 
neither Dali, Beckett, Ionesco nor any of the others could 
have thought possible; and to see these things I did not 
need to do more than look out of my studio window” (6). 

In 1976, after many years, Bearden traveled to the sites 
of his early childhood, only to find that everything had 
changed. Shortly afterwards, perhaps reflecting on his own 
life’s journey, he embarked on a series of 20 collages based 
on Homer’s Odyssey. Inspired by Odysseus’ epic travails 
as he wandered the Mediterranean in search of Ithaca, 
these compositions showcase the essential geometry in 


Bearden’s work. Highly finished flat panels of vivid color 
contain minimal surface manipulation or paint. Fluid char- 
coal silhouettes beneath the waves recall the dark figures 
adorning classical Greek pottery. 

“. . .[T]he sparkle and pulsations of water give men and 
women a certain energy. . .” wrote Bearden in praise of his 
Caribbean experience (1), which also might have prompt- 
ed this excursion into mythology. For most of us, fascina- 
tion with the sea and longing for the unknown prompt trav- 
el. As the graceful nymph on this month’s cover frees 
Odysseus from one more hurdle of his 10-year journey, we 
sympathize with the weary traveler. Yet, however grue- 
some, his impediments were imaginary — angry gods, 
cyclopes, sirens, Scylla and Charybdis. As we reach con- 
temporary ports of call, the threats we meet — SARS, avian 
flu, West Nile virus, Ebola — are real. 

References 

1. National Gallery of Art Washington. The art of Romare Bearden. 
New York: Harry N. Abrams, Inc.; 2003. 

2. Romare Bearden Foundation — biography, [cited 2005 Jan]. Available 
from http : //w w w.beardenf oundation . org/ artlif e/biography/html 

3. Schwartzman M. Romare Bearden his life and art. New York: Henry 
N. Abrams, Inc.; 1990. 

4. Marshall arts presents Romare Bearden, [cited 2005 Jan]. Available 
from http ://www. courses . vcu . edu/ENG-mam/bio2 . htm 

5. Campbell MS, Patton SF. Memory and metaphor: the art of Romare 
Bearden. New York: Oxford University Press; 1991. 

6. African culture online, [cited 2005 Jan]. Available from 
http://www.africancultureonline.com/forums/showpost.php ?p=4583 
&postcount=l 


Address for correspondence: Polyxeni Potter, EID Journal, Centers for 
Disease Control and Prevention, 1600 Clifton Rd NE, Mailstop D61, 
Atlanta, GA 30333, USA; fax: 404-371-5449; email: PMPl@cdc.gov 






~3 


EMERGING 

INFECTIOUS DISEASES 






OM.C r.»— 







ONLINE 


www.cdc.gov/eid 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 


359 



NEWS & NOTES 


EMERGING 

INFECTIOUS DISEASES 

A Peer-Reviewed Journal Tracking and Analyzing Disease Trends 
Vol. 11, No. 3, March 2005 

Upcoming Issue 


Look in the March issue for the following topics: 

Fly Transmission of Campylobacter 

Disease Risk from Foods, England and Wales, 1996-2000 

Probable Psittacosis Outbreak Linked to Wild Birds 

Rapid Identification of Emerging Pathogens: Coronavirus 

Outpatient Community-acquired Pneumonia 
and Antimicrobial Drugs 

Effect of Regulation and Education on Salmonellosis 

SARS Risk Perceptions in Healthcare Workers, Japan 

Logitudinally Profiling Neutralizing Antibody Response to SARS 
Coronavirus with Pseudotypes 

Methicillin-resistant Staphylococcus aureus in Horses and 
Horse Personnel, 2000 2002 

Notifiable Disease Surveillance and Practicing Physicians 

Rumor Surveillance and Avian Influenza H5N1 

Complete list of articles in the March issue at 
http://www.cdc.gov/ncidod/eid/upcoming.htm 


Upcoming Infectious 
Disease Activities 

March 16-18, 2005 

Focus on Fungal Infections 15 

Sheraton Bal Harbour 

Miami, Florida, USA 

Contact: 770-751-7332 or 

c . chase @ imedex .com 

http://www.imedex.com/calendars/ 

infectiousdisease.htm 

April 9-12, 2005 

Society for Healthcare Epidemiology 
of America (SHEA) Annual Meeting 
Los Angeles, California, USA 
Contact: 703-684-1006 
Web site: http://www.shea-online.org 

May 1, 2005 

International Society of Travel 
Medicine (ISTM) offers certificate 
of knowledge in travel medicine exam 
(Given prior to the opening of 9th 
Conference of the ISTM) 

Contact: exam@istm.org 
http://www.ISTM.org/ 

May 1-5, 2005 

9th Conference of the International 
Society of Travel Medicine 
Lisbon, Portugal 
Contact: +49-89-2180-3830 
http://www.ISTM.org/ 

November 13-18, 2005 

Fourth MIM Pan- African Malaria 

Conference 

Yaounde, Cameroon 

http ://w w w.mim. su.se/conference2005 

2006 

June 25-29, 2006 

ISHAM 2006 (International Society 
for Human and Animal Mycology) 
Palais des Congres 
Paris, France 
Contact: 770-751-7332 or 
c . chase @ imedex .com 
http://www.imedex.com/calendars/ 
infectiousdisease.htm 


360 


Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 2, February 2005 





EMERGING 

INFECTIOUS DISEASES 

I Piir-KiHivid JeuriHl Tracking oni Analrijn^ OisiDi* Tirndi 

Editorial Policy 
and Call for Articles 

Emerging Infectious Diseases is a peer- 
reviewed journal established expressly to promote 
the recognition of new and reemerging infectious 
diseases around the world and improve the under- 
standing of factors involved in disease emergence, 
prevention, and elimination. 

The journal is intended for professionals in 
infectious diseases and related sciences. We wel- 
come contributions from infectious disease spe- 
cialists in academia, industry, clinical practice, and 
public health, as well as from specialists in eco- 
nomics, social sciences, and other disciplines. 
Manuscripts in all categories should explain the 
contents in public health terms. For information on 
manuscript categories and suitability of proposed 
articles see below and visit http://www.cdc.gov/ 
eid/ncidod/ EID/instruct.htm. 

Emerging Infectious Diseases is published in 
English. To expedite publication, we post articles 
online ahead of print. Partial translations of the 
journal are available in Japanese (print only), 
Chinese, French, and Spanish (http://www.cdc. 
go v/eid/ ncido d/EID/ trans . htm) . 


Instructions to Authors 

Manuscript Preparation. For word processing, 
use MS Word. Begin each of the following sec- 
tions on a new page and in this order: title page, 
keywords, abstract, text, acknowledgments, biog- 
raphical sketch, references, tables, figure legends, 
appendixes, and figures. Each figure should be in a 
separate file. 

Title Page. Give complete information about each 
author (i.e., full name, graduate degree(s), affilia- 
tion, and the name of the institution in which the 
work was done). Clearly identify the correspon- 
ding author and provide that author's mailing 
address (include phone number, fax number, and 
email address). Include separate word counts for 
abstract and text. 

Keywords. Include up to 10 keywords; use terms 
listed in Medical Subject Headings Index Medicus. 

Text. Double-space everything, including the title 
page, abstract, references, tables, and figure leg- 
ends. Indent paragraphs; leave no extra space 
between paragraphs. After a period, leave only one 
space before beginning the next sentence. Use 12- 
point Times New Roman font and format with 
ragged right margins (left align). Italicize (rather 
than underline) scientific names when needed. 

Biographical Sketch. Include a short biographi- 
cal sketch of the first author — both authors if only 
two. Include affiliations and the author's primary 
research interests. 

References. Follow Uniform Requirements 
(www.icmje. org/index.html). Do not use endnotes 
for references. Place reference numbers in paren- 
theses, not superscripts. Number citations in order 
of appearance (including in text, figures, and 
tables). Cite personal communications, unpub- 
lished data, and manuscripts in preparation or sub- 
mitted for publication in parentheses in text. 


Consult List of Journals Indexed in Index Medicus 
for accepted journal abbreviations; if a journal is 
not listed, spell out the journal title. List the first 
six authors followed by “et al.” Do not cite refer- 
ences in the abstract. 

Tables and Figures. Create tables within MS 
Word’s table tool. Do not format tables as columns 
or tabs. Send graphics in native, high-resolution 
(200 dpi minimum) .TIF (Tagged Image File), or 
.EPS (Encapsulated Postscript) format. Graphics 
should be in a separate electronic file from the text 
file. For graphic files, use Arial font. Convert 
Macintosh files into the suggested PC format. 
Figures, symbols, letters, and numbers should be 
large enough to remain legible when reduced. 
Place figure keys within the figure. For more infor- 
mation see EID Style Guide (http://www.cdc.gov/ 
ncidod/ EID/sty le_guide.htm). 

Manuscript Submission. Include a cover letter 
indicating the proposed category of the article 
(e.g., Research, Dispatch) and verifying that the 
final manuscript has been seen and approved by all 
authors. Complete provided Authors Checklist. To 
submit a manuscript, access Manuscript Central 
from the Emerging Infectious Diseases web page 
(w w w.cdc . gov/eid) . 


Types of Articles 

Perspectives. Articles should be under 3,500 
words and should include references, not to exceed 
40. Use of subheadings in the main body of the text 
is recommended. Photographs and illustrations are 
encouraged. Provide a short abstract (150 words), 
a one-sentence summary of the conclusions, and a 
brief biographical sketch of first author. Articles in 
this section should provide insightful analysis and 
commentary about new and reemerging infectious 
diseases and related issues. Perspectives may also 
address factors known to influence the emergence 
of diseases, including microbial adaptation and 
change, human demographics and behavior, tech- 
nology and industry, economic development and 
land use, international travel and commerce, and 
the breakdown of public health measures. If 
detailed methods are included, a separate section 
on experimental procedures should immediately 
follow the body of the text. 

Synopses. Articles should be under 3,500 words 
and should include references, not to exceed 40. 
Use of subheadings in the main body of the text is 
recommended. Photographs and illustrations are 
encouraged. Provide a short abstract (150 words), 
a one-sentence summary of the conclusions, and a 
brief biographical sketch of first author — both 
authors if only two. This section comprises concise 
reviews of infectious diseases or closely related 
topics. Preference is given to reviews of new and 
emerging diseases; however, timely updates of 
other diseases or topics are also welcome. If 
detailed methods are included, a separate section 
on experimental procedures should immediately 
follow the body of the text. 

Research Studies. Articles should be under 3,500 
words and should include references, not to exceed 
40. Use of subheadings in the main body of the text 
is recommended. Photographs and illustrations are 
encouraged. Provide a short abstract (150 words), 
a one- sentence summary, and a brief biographical 
sketch of first author — both authors if only two. 
Report laboratory and epidemiologic results with- 
in a public health perspective. Explain the value of 
the research in public health terms and place the 


findings in a larger perspective (i.e., "Here is what 
we found, and here is what the findings mean"). 

Policy and Historical Reviews. Articles should 
be under 3,500 words and should include refer- 
ences, not to exceed 40. Use of subheadings in the 
main body of the text is recommended. 
Photographs and illustrations are encouraged. 
Provide a short abstract (150 words), a one- sen- 
tence summary of the conclusions, and brief biog- 
raphical sketch. Articles in this section include 
public health policy or historical reports that are 
based on research and analysis of emerging disease 
issues. 

Dispatches. Articles should be 1,000-1,500 words 
and need not be divided into sections. If subhead- 
ings are used, they should be general, e.g., “The 
Study” and “Conclusions.” Provide a brief abstract 
(50 words); references (not to exceed 15); figures 
or illustrations (not to exceed two); and a brief 
biographical sketch of first author — both authors if 
only two. Dispatches are updates on infectious dis- 
ease trends and research. The articles include 
descriptions of new methods for detecting, charac- 
terizing, or subtyping new or reemerging 
pathogens. Developments in antimicrobial drugs, 
vaccines, or infectious disease prevention or elim- 
ination programs are appropriate. Case reports are 
also welcome. 

Commentaries. Thoughtful discussions (500- 
1,000 words) of current topics. Commentaries may 
contain references but no figures or tables. 

Another Dimension. Thoughtful essays, short 
stories, or poems on philosophical issues related to 
science, medical practice, and human health. 
Topics may include science and the human condi- 
tion, the unanticipated side of epidemic investiga- 
tions, or how people perceive and cope with infec- 
tion and illness. This section is intended to evoke 
compassion for human suffering and to expand the 
science reader's literary scope. Manuscripts are 
selected for publication as much for their content 
(the experiences they describe) as for their literary 
merit. 

Letters. Letters commenting on recent articles as 
well as letters reporting cases, outbreaks, or origi- 
nal research are welcome. Letters commenting on 
articles should contain no more than 300 words 
and 5 references; they are more likely to be pub- 
lished if submitted within 4 weeks of the original 
article's publication. Letters reporting cases, out- 
breaks, or original research should contain no 
more than 800 words and 10 references. They may 
have one Figure or Table and should not be divid- 
ed into sections. All letters should contain material 
not previously published and include a word count. 

Book Reviews. Short reviews (250-500 words) of 
recently published books on emerging disease 
issues are welcome. The name of the book, pub- 
lisher, and number of pages should be included. 

Announcements. We welcome brief announce- 
ments (50-150 words) of timely events of interest 
to our readers. (Announcements may be posted on 
the journal Web page only, depending on the event 
date.) 

Conference Summaries. Summaries of emerging 
infectious disease conference activities are pub- 
lished online only (effective January 2005). 
Summaries, which should contain 500-1,000 
words, should focus on content rather than process 
and may provide illustrations, references, and links 
to full reports of conference activities.