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


Emerging Infectious Diseases. 2002;8(5) 

In This Article

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.


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