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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

Founding Editor

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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

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S.K. Lam, Kuala Lumpur, Malaysia

Bruce R. Levin, Atlanta, Georgia, USA

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Rosanna W. Peeling, Geneva, Switzerland

David H. Persing, Seattle, Washington, USA

Gianfranco Pezzino, Topeka, Kansas, USA

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Mario Raviglione, Geneva, Switzerland

Leslie Real, Atlanta, Georgia, USA

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Nancy Rosenstein, Atlanta, Georgia, USA

Connie Schmaljohn, Frederick, Maryland, USA

Tom Schwan, Hamilton, Montana, USA

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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

Antibiotics for FRIf
Medical care§

$30.00

$64.72

$80.00

Ambulatory clinic visit

$40.00

$60.03

$80.00

Hospitalization for FRI

$5,000

$1 1 ,645

$15,000

Hospitalization for influenza

$7,500

$17,465

$25,000

Hospitalization for PUI

$15,000

$19,441

$25,000

Hospitalization for SARS
Diagnostic tests

$20,000

$28,502

$40,000

14,15

Rapid influenza test

$15.00

$26.86

$40.00

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

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

Specificity of influenza test

0.80

0.95

0.99

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

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

Miscellaneous probabilities

Probability of an FRI

0.10

0.33

0.50

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

Annual probability of poor match between
vaccine and circulating influenza strains

0.05

0.20

0.50

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

Miscellaneous values

Patient time for outpatient medical visit (min)

Estimate

Influenza length of illness (d)

Other FRIjf length of illness (d)

Estimate

Average duration of hospitalization, influenza

(d)

Average duration of hospitalization, FRI^j (d)

10.2

7.7

Average duration of hospitalization, SARS
(d)

HRQL scores

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#

9,10

Contact investigations (per SARS case)

100

*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

Influenza testing

2.14

5,286

$1,702

Flome isolation

2.13

Influenza testing

2.14

5,286

Dominated

Multiplex RT-PCR testing!

2.05

8,474

Savings

Flome isolation

2.13

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

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

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.

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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

To receive tables of contents of new issues send an email to iistserve@dc.gov with subscribe eid-toc in the body of your message.

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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.

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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.

577

Median age (y) (range)

12(2-58)

33(1-67)

12(1-92)

Sex (% male)

Poultry contact (%)

Adequate* specimen (%)

100

Death (%)

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

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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%)

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

(xl 0 3 )
Treatment

Oseltamivir

Corticosteroids

Outcome

ARDS

Inotropic support

Peak AST (U)

129

Peak ALT (U)

Peak BUN (mg/dL)

10.7

Peak creatinine

0.8

1.07

0.7

(mg/dL)

6, M

7, M

13, M

39, F

58, F

67 (M)

100

1,200

2,200

4,900

4,100

2,000

3,300

5,680

624

638

1,763

1,435

580

660

454

140

111

304

150

380

185

790

175

280

120

394

150

106

132

1.7

1.1

0.7

8.1

3.6

2.3

-(20)

-(18)

-(8)

-(29)

-(16)

-(13)

-(8)

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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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

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.

Uttradit/F/27

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

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.

Lopburi/F/46

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

Khonkaen/M/5

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

Kanchanaburi/M/6

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

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.

Kanchanaburi/M/6

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

Supanburi/M/7

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

Chaiyapoum/M/13

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

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.

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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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.

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al. Clinical features and rapid viral diagnosis of human disease asso-
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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
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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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209

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)

3 (3.7)

31 (37.8)

32 (39.0)

4 (4.9)

5 (all)

8 (9.8)

Changed bedding

Yes

46 (59.0)

32 (41 .0)

Touched patients

Yes

75 (96.2)

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.

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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.

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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

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

2/1

11/3

HACEK§

0/4

0/0

1/1

5/1

Enterococcus spp.

1/1

0/0

1/0

2/1

Brucella melitensis

0/1

0/0

2/0

Coxiella burnetii

0/0

2/0

0/0

1/NP

2/0

Corynebacterium spp.

0/2

0/1

1/0

2/0

Mycoplasma hominis

0/0

1/0

Enterobacteria spp.

1/1

0/0

1/1

Alcaligenes faecalis

0/1

0/0

1/0

Pseudomonas aeruginosa

0/1

0/0

1/0

Bacillus cereus

0/0

1/0

Candida spp.

0/0

0/1

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

Poor rural area

Aortic

1:800

B. quintana

Poor rural area

Mitral

1:1,600

B. quintana

WS+/IC+

Poor urban area

Mitral

1:800

B. quintana

WS+/IC+

Poor rural area

Aortic

B. quintana

Poor urban area

Tricuspid

+ t

1:1,600

B. quintana

Poor rural area

Aortic + mitral

B. quintana

WS+/IC+

Unknown

Aortic + mitral

B. quintana

WS+/IC+

Poor urban area

Aortic

1:800

B. quintana

WS+/IC+

Good urban area

Aortic

B. quintana

WS+/IC+

Good rural area

Aortic + mitral

B. quintana

WS+/IC+

Poor urban area

Mitral

1:3,200

Poor rural area

Aortic

1:3,200

B. quintana

Poor rural area

Aortic

B. quintana

Poor rural area

Aortic

B. quintana

*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

)

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.

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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

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

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

Negative

Candida krusei

Streptococcus

spp.

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

Negative

C. parapsilosis

Negative

Polymicrobial

Streptococcus

mitis

S. mitis

Negative

S. mitis

Negative

CNS

Haemophilus

paraphrophilus

A /BGN

Negative

H. paraphrophilus

Negative

CNS

Bartonella

quintana

B. quintana

S. mitis

Staphylococcus

aureus

Streptococcus

gordonii

S. gordonii

A /CGP

Negative

S. gordonii

H. influenzae

1:1,600
Positive
PCR on
serum
samples

B. quintana

Streptococcus

intermedius

Streptococcus

intermedius

S. mutans

Negative

Cory ne bacterium
spp.

Bacillus cereus

B. cereus

Negative

B. cereus

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

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

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.

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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

WNV NY99flamingo38299

NY99b

AF1 96835

1999

Flamingo

New York

WNV IS98STD

Is98

AF481864

1998

Stork

Israel

WNV Italyl 998Equine

It98

AF404757

1998

Horse

Italy

WNV RO9750

Ro96

AF260969

1996

Culex pipiens

Romania

WNV VLG4

Rus99a

AF317203

1999

Human

Volgograd

WNV LEIV-Vlg99-27889

Rus99b

AY277252

1999

Human

Volgograd

WNV PaHOOl

Tu97

AY268133

1997

Human

Tunisia

WNV PaAnOOl

FrOO

AY268132

2000

Horse

France

WNV EglOl

Eg51

AF260968

1951

Human

Egypt

WNV Chin-01

ChinOI

AY490240

Unknown

Unknown!

China

WNV Kunjin MRM61C
WNV Sarafend

Kunjin

Sarafend

D00246

AY688948

1960

Cx. armulirostris
Laboratory strain

Australia

WNV B956 (WNFCG)

Ug37

Ml 2294

1937

Human

Uganda

WNV LEIV-Krnd88-1 90

Rus98

AY277251

1998

Dermacentor

marginatus

Caucasus

Rabensburg virus (97-103)

RabV

AY765264

1997

Cx. pipiens

Czech Republic

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

NY99b

Is98

It98

Ro96

Rus99a

Rus99b

Tu97

FrOO

Eg51

ChinOI

Kunjin

Sarafend 2

Ug37

Rus98

4 (speculation)

JEV

MVEV

USUV

SLEV

ALFV

CPCV

KOUV

YAOV

*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

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9. Lanciotti RS, Roehrig JT, Deubel V, Smith J, Parker M, Steele K, et
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12. Ferenzi E, Bakonyi T, Toth-Mittler E, Czegledi A, Ban E. Emergence
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Congress of the Hungarian Society for Microbiology; 2004. Oct. 7-9,
Keszthely, Hungary. Abstract p. 36-7 [in Hungarian]. Budapest:
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13. Hubalek Z, Halouzka J, Juricova Z, Sebesta O. First isolation of mos-
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15. Bakonyi T, Gould EA, Kolodziejek J, Weissenbock H, Nowotny N.
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21. Lvov DK, Butenko AM, Gromashevsky VL, Kovtunov Al, Prilipov
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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
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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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231

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

H. armiger

103

Eonycteris

spelaea

Rousettus

leschennault

Pteropus lylei

150 242

110 58

588

P. hypomelanus

16 3

P. vampyrus

Emballonura

monticola

Scotophillus
heath I

Mega derm a

spasma

Cynopterus

sphinx

Total

241 3 245

110 99

124

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

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

>200

<10

733

<10

>200

<10

688

1:56

1:25

<10

615

1:13

1:25

<10

120

<10

1:65

<10

0/69

<10

1:170

<10

1:21

1:33

<10

125

1:20

1:56

1:13

<10

741

1:12

<10

>200

1:13

<10

731

<10

>200

<10

724

1:35

1:56

1:29

<10

303

1:35

1:50

<10

740

1:35

<10

>200

<10

729