Mrigendra P. Shrestha

Rates of Hepatitis E Virus Infection and Disease among Adolescents and Adults in Kathmandu, Nepal

To determine hepatitis E virus (HEV) infection and disease rates in the Kathmandu Valley of Nepal, serum was collected from 757 healthy Nepalese (ages 12-48 years) during March and September 1992 and September 1993. At each visit, reports of interval illness were obtained. Sera were examined for IgG to HEV, using a commercially available kit. Seroconversion was used as a marker for HEV infection, and an episode of hepatitis E was defined as a history of jaundice with seroconversion. Seroprevalence ranged from 16% to 31% and increased with age, whereas both infection and disease rates decreased with age. Infection and disease rates were as high as 99/1000 and 45/1000 person-years, respectively. These results highlight the importance of sporadic hepatitis E as a public health problem among adolescents and young adults in this region.

Clinical and Epidemiological Relevance of Quantitating Hepatitis E Virus-Specific Immunoglobulin M

ABSTRACT Diagnosis of acute hepatitis E by detection of hepatitis E virus (HEV)-specific immunoglobulin M (IgM) is an established procedure. We investigated whether quantitation of HEV IgM and its ratio to HEV total Ig furnished more information than conventional IgM tests that are interpreted as positive or negative. A previously described indirect immunoassay for total Ig against a baculovirus-expressed HEV capsid protein was modified to quantitate HEV-specific IgM in Walter Reed (WR) antibody units by using a reference antiserum and the four-parameter logistic model. A receiver-operating characteristics curve derived from 197 true-positive specimens and 449 true-negative specimens identified 30 WR units/ml as an optimum cut point. The median HEV IgM level in 36 patients with acute hepatitis E fell from 3,000 to 100 WR units/ml over 6 months, suggesting that 100 WR units/ml would be a more appropriate cut point for distinguishing recent from remote IgM responses. Among three hepatitis E case series, determination of the HEV IgM-to-total-Ig ratio in acute-phase serum revealed that most patients had high ratios consistent with primary infections whereas a few had low ratios, suggesting that they had sustained reinfections that elicited anamnestic antibody responses. The diagnostic utility of the new IgM test was similar to that of a commercially available test that uses different HEV antigens. In conclusion, we found that HEV IgM can be detected specifically in >95% of acute hepatitis E cases defined by detection of the virus genome in serum and that quantitation of HEV IgM and its ratio to total Ig provides insight into infection timing and prior immunity.

Association of hepatitis E virus with an outbreak of hepatitis at a military training camp in Nepal

From 29 January 1995 to 15 March 1995, an outbreak of hepatitis occurred among 692 soldiers at an isolated training camp 25 km east of Kathmandu. Thirty‐two cases occurred approximately 8 weeks after arrival of soldiers at the camp. To determine the etiology of the outbreak, patient sera were examined for evidence of infection with hepatitis A, B, C, and E viruses using commercially available enzyme‐linked immunosorbent assay (ELISA) kits. The polymerase chain reaction (PCR) was used to detect hepatitis E virus (HEV) RNA. Evidence of recent infection (IgM to HEV and/or HEV RNA) was found in all but two patients, whereas none had evidence of recent infection with hepatitis A, B, or C viruses. Therefore, the outbreak was attributed to HEV. Fecally contaminated drinking water was suspected as the source of the outbreak. To determine the extent of HEV infections among those without clinical hepatitis, sera from the remaining soldiers were examined for markers of HEV infection. Evidence of past infection (IgG to HEV in the absence of IgM or HEV RNA) was found among 204 soldiers (prevalence = 30%), leaving 488 individuals susceptible to infection at the onset of the outbreak. Evidence of recent infection was found among another 83 individuals. We conclude that most exposed, susceptible soldiers sustained HEV infection without experiencing overt hepatitis. If the levels of virus inoculum and prior immunity in this population were typical, inapparent infection may be the usual adult response to virus exposure in an endemic area. J. Med. Virol. 54:178–182, 1998.

Quantitation of Immunoglobulin to Hepatitis E Virus by Enzyme Immunoassay

ABSTRACT We developed a quantitative enzyme immunoassay (EIA) for antibody to hepatitis E virus (HEV) by using truncated HEV capsid protein expressed in the baculovirus system to improve seroepidemiology, to contribute to hepatitis E diagnosis, and to enable vaccine evaluations. Five antigen lots were characterized; we used a reference antiserum to standardize antigen potency. We defined Walter Reed antibody units (WR U) with a reference antiserum by using the four-parameter logistic model, established other reference pools as assay standards, and determined the conversion factor: 1 WR U/ml = 0.125 World Health Organization unit (WHO U) per ml. The EIA performed consistently; median intra- and intertest coefficients of variation were 9 and 12%, respectively. The accurate minimum detection limit with serum diluted 1:1,000 was 5.6 WR U/ml; the test could detect reliably a fourfold antibody change. In six people followed from health to onset of hepatitis E, the geometric mean antibody level rose from 7.1 WR U/ml to 1,924.6 WR U/ml. We used the presence of 56- and 180-kDa bands by Western blotting as a confirmatory test and to define true-negative and -positive serum specimens. A receiver-operating characteristics plot identified 30 WR U/ml as an optimum cut-point (sensitivity, 86%; specificity, 89%). The EIA detected antibody more sensitively than a commercially available test. The EIA was transferred to another laboratory, where four operators matched reference laboratory results for a panel of unknowns. Quantitation of antibody to HEV and confirmation of its specificity by Western blotting make HEV serology more meaningful.

Prevalence and Genetic Diversity of Bartonella Species Detected in Different Tissues of Small Mammals in Nepal

ABSTRACT Bartonellae were detected in a total of 152 (23.7%) of 642 tissues from 108 (48.4%) of 223 small mammals trapped in several urban areas of Nepal. Based on rpoB and gltA sequence analyses, genotypes belonging to seven known Bartonella species and five genotypes not belonging to previously known species were identified in these animals.

Epidemiology of Hepatitis E Virus (HEV): A Cohort Study in Kathmandu, Nepal

Hepatitis E is endemic to Kathmandu. To determine age- and sex-specific prevalence and risk factors for antibody to HEV (anti-HEV), blood and historic information was obtained from 4486 volunteers in March 1992 and again in September. Sera were assayed for anti-HEV IgG by enzyme immunoassay. The anti-HEV prevalence (10.2%) was greater in males than in females, and was shown to increase with age in males but not females. Anti-HEV prevalence was greater in subjects with a recent history of jaundice, but most anti-HEV positive subjects had no such history. We concluded that anti-HEV prevalence in female and male children was similar, prevalence in males increased during the young adult years, prevalence was greater in subjects with a recent history of jaundice, and most HEV infection was subclinical.

Genetic Diversity of Thottapalayam Virus, a Hantavirus Harbored by the Asian House Shrew (Suncus murinus) in Nepal

Despite the recent discovery of genetically divergent hantaviruses in shrews of multiple species in widely separated geographic regions, data are unavailable about the genetic diversity and phylogeography of Thottapalayam virus (TPMV), a hantavirus originally isolated from an Asian house shrew (Suncus murinus) captured in southern India more than four decades ago. To bridge this knowledge gap, the S, M, and L segments of hantavirus RNA were amplified by reverse transcription polymerase chain reaction from archival lung tissues of Asian house shrews captured in Nepal from January to September 1996. Pair-wise alignment and comparison revealed approximately 80% nucleotide and > 94% amino acid sequence similarity to prototype TPMV. Phylogenetic analyses, generated by maximum likelihood and Bayesian methods, showed geographic-specific clustering of TPMV, similar to that observed for rodent- and soricid-borne hantaviruses. These findings confirm that the Asian house shrew is the natural reservoir of TPMV and suggest a long-standing virus-host relationship.