Research
Hospital-Based Diagnosis
of Hemorrhagic Fever, Encephalitis, and Hepatitis in Cambodian Children
Y. Meng Chhour,* Gaye Ruble,† Rathavuth Hong,* Kyi Minn,‡ Yuvatha
Kdan,* Touch Sok,* Ananda Nisalak,† Khin Saw Aye Myint,† David W.
Vaughn,§ and Timothy P. Endy†
*National Pediatric Hospital, Phnom Penh, Cambodia; †Armed Forces
Research Institute of Medical Sciences, Bangkok, Thailand; ‡World
Vision International, Phnom Penh, Cambodia; and §Walter Reed Army
Institute of Research, Silver Spring, Maryland, USA
Surveillance
was conducted for three clinical syndromes (hemorrhagic fever,
encephalitis, and hepatitis) in Cambodian children admitted to
the National Pediatric Hospital in Phnom Penh from July 1996 through
September 1998. Acute- and convalescent-phase sera, and cerebrospinal
fluid, when applicable, underwent diagnostic evaluation for infections
with Dengue virus (DENV), Japanese encephalitis virus
(JEV), and Hepatitis A, B, C, and E viruses. Of 621 children
admitted with hemorrhagic fever, 499 (80%) were confirmed to have
either primary or secondary DENV infection. DENV rates were as
high as 10.6/100 hospital admissions in September 1998. Of 50
children with clinical encephalitis, 9 (18%) had serologic evidence
of JEV infection. Forty-four children had clinical hepatitis,
most (55%) due to Hepatitis A virus (HAV). One patient
had Hepatitis B virus, and no patients had hepatitis C
or E. This study identified a large number of children with vaccine-preventable
diseases (JEV and HAV).
Infectious diseases continue to pose a major threat to populations
in developing countries in tropical regions. Dengue is the most
important arbovirus infection in Southeast Asia (1).
It is spread by the bite of the vector mosquito, Aedes aegypti,
and causes asymptomatic infection, mild to severe influenzalike
symptoms (dengue fever), and plasma leakage and hemorrhage, which
are sometimes fatal (dengue hemorrhagic fever). In 1995, Rathavuth
et al. conducted 2 days of surveillance for hemorrhagic fever in
children admitted to the National Pediatric Hospital (NPH) in Phnom
Penh (2). Their findings of a high frequency of
secondary Dengue virus (DENV) infection, a low mean age of
admission, and the presence of all four dengue serotypes suggested
that dengue was highly endemic in Cambodia.
Bacterial, viral, fungal, and parasitic agents are all causes of
encephalitis or encephalopathy in children in Southeast Asia. Tsai
reported that the main causes of encephalitis in rural Asia included
tuberculosis, typhoid fever, cerebral malaria, and viruses such
as DENV, herpes simplex, measles, Enterovirus, and HIV (3).
Few reports have been published about the incidence or possible
causes of encephalitis in Cambodian children. In a report by Sunara
et al., surveillance at two pediatric hospitals in Phnom Penh from
1990 through 1994 showed >300 cases of acute encephalitis in
children (4). While the cause for many of these
cases was suspected to be Japanese encephalitis virus (JEV),
laboratory confirmation was lacking.
Only one report has discussed the prevalence of markers for Hepatitis
viruses A, B, and C (HAV, HBV, HCV), in Cambodia. In 1991, Thuring
et al. conducted a serologic study for markers of viral hepatitides
and HIV in Takeo, a southern province (5). In that
study adults and children, both healthy and those ill with liver
or kidney disease, were examined. HAV was the most frequent cause
of acute hepatitis in these children, occurring in 11 (32%) of 34
pediatric patients. Ongoing infection with HBV accounted for 18%,
and one child tested positive for HCV-specific antibody. That study
did not screen for Hepatitis E virus (HEV) infection.
Our study was undertaken to characterize the extent of disease
in Cambodian children, specifically for the following three syndromes:
hemorrhagic fever (HF), encephalitis, and hepatitis.
Materials
and Methods
Surveillance was conducted at NPH in Phnom Penh. This hospital,
one of two pediatric referral hospitals in Cambodia, serves a population
of approximately 2.7 million children ages <14. Enrolled
in this study were children admitted with any of the following clinical
signs: HF (fever, headache, or rash, and on physical examination,
a positive tourniquet test, ascites, pleural effusion bleeding,
or shock); encephalitis (headache, fever, or neck stiffness, and
alteration of consciousness or focal neurologic signs); and hepatitis
(lethargy, anorexia, nausea or vomiting, abdominal pain, hepatomegaly,
scleral icterus, or jaundice). Case definitions were kept broad
to capture as many cases as possible.
On the basis of published criteria from the World Health Organization,
cases of DHF were classified into one of four grades of severity.
Grade 1 includes fever with nonspecific symptoms; the only hemorrhagic
manifestation is a positive tourniquet test, easy bruising, or both.
Grade 2 includes Grade 1 manifestations plus spontaneous bleeding
(usually skin hemorrhages). Grade 3 includes circulatory failure
(rapid, weak pulse, hypotension) and cold, clammy skin. Grade 4
is manifested by profound shock with undetectable blood pressure
or pulse. The last two grades are considered to be dengue shock
syndrome (DSS).
Sera were collected on the day of admission, at the time of discharge,
and, in some cases, on follow-up exam. However, due to the nature
of the population, a follow-up visit was not always possible, and
therefore diagnosis relied on only an admission and discharge sample.
Sera were stored at -70°C until transported to the Armed Forces
Research Institute of Medical Sciences in Bangkok, Thailand, on
dry ice. Clinical criteria for admission diagnosis directed the
subsequent diagnostic workup. All DHF and encephalitis cases were
tested for both JEV and DENV. Sera were tested for immunoglobulin
(Ig) G and IgM antibody against DENV and JEV by use of an antibody-capture
enzyme-linked immunoassay (EIA) and previously published criteria
of acute and primary or secondary dengue (6). Virus
isolation was attempted with acute-phase serum specimens, as previously
described (7).
Sera were screened for IgM antibody to HAV, IgM antibody to hepatitis
B core antigen (HbcAg), hepatitis B surface antigen (HbsAg), and
total Ig to HCV by using commercially available kits (HAVAB-EIA,
Corzyme-M, AUZYME Monoclonal, and HCV EIA Third Generation; Abbott
Laboratories, Abbott Park, IL). Assays were performed as recommended
by the manufacturer.
All samples were tested for total Ig and IgM to HEV by an indirect
second-generation EIA developed at the Department of Virus Diseases,
Walter Reed Army Institute of Research. The assay quantifies total
Ig and IgM reactive with recombinant HEV capsid protein expressed
using a baculovirus system expressed in U/mL (8).
To control interassay variation, all specimens were tested in duplicate
wells, with all specimens from a single patient tested together
on the same plate. A patient was considered to have HEV infection
if there was virologic (HEV RNA positive) or serologic (IgM >100
U/mL, total Ig >500 U/mL) evidence of acute infection.
Statistics
All statistical procedures were performed by using SPSS for Windows,
Version 10.0 (SPSS Inc., Chicago, IL). Fisher’s exact test (two-tailed)
was used to determine significant difference in the number of boys
with encephalitis and diagnosed as having JEV compared with girls
with the same syndrome and final diagnosis.
Results
Figure 1 shows the rates of HF, encephalitis,
and hepatitis per 100 hospital admissions, by month and year. From
July 1996 through September 1998, 621 children were admitted
with a diagnosis of HF (Table 1). Of those,
495 were confirmed to have a secondary DENV infection by serologic
tests; 14 had primary dengue. Figure 2 illustrates
the number of confirmed DENV patients compared with the total number
of patients with HF.
The severity of DHF can be classified into four grades based on
two pathophysiologic findings: hemorrhage and shock. In this study,
41 of the 509 total confirmed dengue patients had DHF Grade 1; 145
patients had DHF Grade 2; 180 patients had DHF Grade 3; and 29 patients
had DHF Grade 4.
Of the 75 samples tested, 22 were polymerase chain reaction (PCR)-positive
for virus. DENV-2 was isolated from 14 samples, DENV-3 from seven
samples, and DENV-4 from one sample. All three serotypes were found
in children living in Phnom Penh. DENV-2 and -3 were found in Kampong
Speu and Kampong Cham. The other provinces appeared to have only
one circulating dengue serotype, based on the small number of positive
samples.
During this same period, 50 children were admitted to NPH with
a diagnosis of encephalitis; 9 (18%) were due to JEV (Table
1) and 2 to acute secondary dengue infection. There was no evidence
of concurrent infections. Figure 3 illustrates
the number of cases of JEV, by month and year, compared with the
total encephalitis cases. While over twice as many girls as boys
had this syndrome, significantly more boys were diagnosed with JEV
infection (p=0.015). The final outcome for children seen at NPH
with encephalitis was poor: death or disability occurred in 29 (58%)
of the children.
Forty-four children were admitted with a diagnosis of hepatitis.
Twenty-four cases were confirmed to be due to acute HAV infection
on the basis of elevated HAV IgM antibodies (Table
1). One patient had serologic evidence of acute hepatitis B,
and no serologic evidence for HCV or HEV infections. Figure
4 illustrates the number of HAV patients compared with the total
number of patients admitted to NPH with a clinical diagnosis of
hepatitis. Of the 24 children hospitalized with hepatitis A, 17
(71%) had onset in July, August, and September. Most children admitted
to NPH came from Phnom Penh (339 patients) or its adjacent province,
Kandal (146 patients). Table 2 shows the distribution
of inpatients by province and syndrome on admission.
Discussion
This surveillance was undertaken to characterize the extent of
disease in Cambodian children with respect to three specific syndromes:
hemorrhagic fever, encephalitis, and hepatitis. To our knowledge,
this is the first such study conducted in Cambodia. As in other
Southeast Asian countries, DHF accounted for a large percentage
of hospitalizations and deaths of Cambodian children. Dengue was
confirmed in 82% of children admitted to NPH with symptoms that
suggested dengue fever. Serologic results for the remaining 112
HF cases suggested DENV infection in most instances, but lack of
a convalescent-phase sample prevented definitive diagnosis. DHF
has been reported as a leading cause of hospitalization and death
of children throughout Asia in 1998 (9). Our surveillance
shows that in August and September 1998, the hospitalization rate
for children with HF exceeded 10 per 100 hospital admissions.
Similarly, Japanese encephalitis has been reported to occur in
nearly every country in Asia (10). While the disease
is presumed to be endemic in Cambodia, no laboratory-confirmed data
on disease frequency have been published until now. During this
2-year study, 18% of children admitted to NPH with encephalitis
had JEV infection. Similar to reports elsewhere (11),
more cases of JEV infection occurred in boys than in girls (six
versus three, respectively). Transmission of disease is usually
seasonal, from late summer to early fall. In our study, 67% of cases
were reported from May to October; the remainder occurred in January
or February.
HAV infection is highly endemic in developing countries that lack
adequate clean water and have poor sanitary conditions (12).
According to the 1998 Cambodian General Population Census, only
29% of the population has access to safe water (range 23.7%-60.3%)
(13). In poorer countries, most children develop
antibodies to HAV by age 10. A seroprevalence study conducted on
200 healthy Cambodian children from 1990 through 1991 showed that
97% were positive for anti-HAV IgG by 15 years of age (5).
While hepatitis A is largely considered a subclinical infection
in children in Thuring’s study, it accounted for 32% of acute hepatitis
in hospitalized children in our study; hepatitis A accounted for
55% of the pediatric patients hospitalized with suspected hepatitis.
One (2%) of 44 children showed evidence of ongoing HBV infection.
This contrasts with Thuring’s earlier study, in which 18% of children
with acute hepatitis were actively infected with HBV (HbsAg positive).
However, similar to Thuring’s study, we saw no evidence of HCV in
this population.
No indication of acute hepatitis E was found in our study, nor
did any of the children admitted to NPH with hepatitis have evidence
of prior exposure to HEV. Similar to hepatitis A, hepatitis E is
common in countries that lack adequate clean water and in which
general sanitation is poor. In Southeast Asia, epidemics of HEV
have been reported in Myanmar, Vietnam, and Indonesia (14).
In most disease-endemic areas, up to 5% of the children are positive
for anti-HEV antibodies. While clinical attack rates of hepatitis
E are reported to be highest in young adults (15-40 years), a recent
report from India noted anti-HEV antibodies in >60% of children
<5 years old (15). Cambodia is surrounded by
countries (except Thailand) that are endemic for HEV; therefore,
the total lack of anti-HEV IgG in this Cambodian population was
unexpected.
Currently, Cambodian children are given BCG, polio, diphtheria-tetanus-pertussis,
and measles vaccines, although coverage varies throughout the country.
Instituting childhood HAV immunizations could benefit countries
that have shown a decrease in age-related HAV seroprevalence concomitant
with improved socioeconomic development (16,17).
While the seroprevalence for HAV in Thuring’s 1991 study approached
100% in children by age 15, no HAV seroprevalence data have been
gathered since then. Further studies are warranted to determine
if additional vaccines, such as those for HAV and JEV, should be
added to the nation’s immunization program.
Acknowledgments
We thank the laboratory technicians in the Department of Virology,
Arbovirology and Hepatitis Sections, Armed Forces Research Institute
of Medical Sciences, and the nurses and staff at the National Pediatric
Hospital for their technical support. We also thank Panpaka Supakalin
for statistical support.
Funding for this project was provided by World Vision and the United
States Army Medical Research and Materiel Command.
Dr. Y. Meng Chhour is a staff member of the National Pediatric
Hospital in Phnom Penh, Cambodia, and of the Ministry of Health,
Cambodia. His research interests include the epidemiology of infectious
diseases endemic to Cambodia and occurring in the pediatric population.
References
- Lam SK. Emerging
infectious diseases—Southeast Asia. Emerg Infect Dis 1998;4:145-7.
- Rathavuth H, Vaughn DW, Minn K, Nimmannitya S, Nisalak A, Raengsakulrach
B, et al. Hemorrhagic
fever in Cambodia is caused by dengue viruses: evidence for transmission
of all four serotypes. Southeast Asian J Trop Med Public Health
1997;28:120-5.
- Tsai TF. Japanese encephalitis. In: Feigin R, Cherry J, editors.
Textbook of pediatric infectious diseases. 4th ed. Philadelphia:
W.B. Saunders Company; 1998; p. 1993-2001.
- Sunnara Y, Touch S. Japanese encephalitis in the Kingdom of
Cambodia. In: Rojanasuphot S, Tsai T, editors. Southeast Asian
J Trop Med Public Health 1995;26(Suppl 3):22-3.
- Thuring EG, Joller-Jemelka HI, Sareth H, Sokhan U, Reth C, Grob
P. Prevalence
of markers for hepatitis viruses A, B, C and of HIV in healthy
individuals and patients of a Cambodian province. Southeast
Asian J Trop Med Public Health 1993;24:239-49.
- Innis BL, Nisalak A, Nimmannitya S, Kusalerdchariya S, Chonoswasdi
V, Suntayakorn S, et al. An enzyme-linked immunosorbent assay
to characterize dengue infections where dengue and Japanese encephalitis
co-circulate. Am J Trop Med 1989;40:418-27.
- Vaughn DW, Green S, Kalayanarooj S, Innis BL, Nimmannitya S,
Suntayakorn S, et al. Dengue
in the early febrile phase: viremia and antibody responses.
J Infect Dis 1997;176:322-30.
- Innis BL, Seriwatana J, Robinson RA, Shrestha MP, Yarbough PO,
Longer CF, et al. Quantitation of immunoglobulin to hepatitis
E virus by enzyme immunoassay. J Clin Diagn Immunol. In press.
- Gubler DJ, Meltzer M. Impact
of dengue/dengue hemorrhagic fever on the developing world.
Adv Virus Res 1999;53:35-70.
- Cherry JD, Shields WD. Encephalitis and meningoencephalitis.
In: Feigin R, Cherry J, editors. Textbook of pediatric infectious
diseases. 4th ed. Philadelphia: W.B. Saunders Company; 1998. p.
457-68.
- Tsai TF, Nadhirat S, Rojanasuphaot S. Regional
workshop on control strategies for Japanese encephalitis. Southeast
Asian J Trop Med Public Health 1995;26 (Suppl. 3):1-59.
- Kunasol P, Cooksley G, Chan VF, Isahak I, John J, Loleka S,
et al. Hepatitis
A virus: declining seroprevalence in children and adolescents
in southeast Asia. Southeast Asian J Trop Med Public Health
1998;29:255-62.
- National Institute of Statistics, Ministry of Planning. General
population census of Cambodia 1998. Phnom Penh, Cambodia: the
Institute;1999.
- Corwin AL, Tien NTK, Bounlu K, Winarno J, Putri MP, Laras K,
et al. The
unique riverine ecology of hepatitis E virus transmission in southeast
Asia. Trans R Soc Trop Med Hyg 1999;93:255-60.
- Aggarwal R, Krawczynski K. Hepatitis
E: an overview and recent advances in clinical and laboratory
research. J Gastroenterol Hepatol 2000;15:9-20.
- Kunasol P, Cooksley G, Chan VF, Isahak, I, John, J, Loleka,
S, et al. Hepatitis
A virus: declining seroprevalence in children and adolescents
in Southeast Asia. Southeast Asian J Trop Med Public Health
1998;29:255-62.
- Poovorawan Y. Changing
epidemiology and prevention of hepatitis A virus infection.
Acta Paediatrica Sinica 1998;39:139-45.
Table
1. Characteristics of pediatric patients with clinical hemorrhagic
fever, encephalitis, or hepatitis, National Pediatric Hospital,
Phnom Penh, Cambodia, July 1996–September 1998a |
|
Syndrome
|
Total no. of cases
|
Males
|
Females
|
Mean age (range)
|
Outcome
|
|
|
621
|
288
|
332
|
7 years (5 mo-15 yrs)
|
11b
|
Secondary dengue
|
|
222
|
272
|
7 years (8 mo-15 yrs)
|
4b
|
Primary dengue
|
|
8
|
6
|
4 years (5 mo-9 yrs)
|
—
|
Total encephalitis
|
50
|
15
|
35
|
4 years (3 mo-14 yrs)
|
17b
|
12c
|
JE
|
9
|
6
|
3
|
6 years (3-10 yrs)
|
2b
|
2c
|
Total hepatitis
|
44
|
21
|
23
|
6 years (2 mo-14 yrs)
|
—
|
Hepatitis A
|
24
|
12
|
12
|
5 years (2 mo-9 yrs)
|
—
|
Hepatitis B
|
1
|
1
|
0
|
10 years
|
—
|
|
aTotal number of hospital admissions
during this period was 16,492 children.
bDeaths.
cDisabled.
JE, Japanese encephalitis; —, all recovered.
|
Table
2. Distribution of inpatients by most widely represented
provinces, National Pediatric Hospital, Cambodia |
|
Syndrome upon admission |
No. (%) of patients by province
|
|
Phnom Penh
|
Kandal
|
Kampong
Speu
|
Kampong
Cham
|
Takeo
|
Prey Veng
|
Total
|
|
Hemorrhagic fever
|
309 (50)
|
116 (19)
|
9 (3)
|
40 (6)
|
20 (3)
|
|
494 (81)
|
Encephalitis
|
15 (30)
|
15 (30)
|
8 (16)
|
|
5 (11)
|
|
43 (87)
|
Hepatitis
|
15 (34)
|
15 (34)
|
3 (7)
|
3 (7)
|
|
5 (11)
|
41 (93)
|
Total
|
339
|
146
|
20
|
43
|
25
|
5
|
|
|
|
|