Investigation of a hepatitis A outbreak from Shimla Himachal Pradesh.

BACKGROUND & OBJECTIVE: Hepatitis A is an enterically transmitted viral disease, highly prevalent in India and mainly presents as a paediatric sporadic disease. This study investigated an outbreak of viral hepatitis at Shimla, Himachal Pradesh, India, during January-March 2007. METHODS: Eighty seven blood samples, 3 water samples and 2 sewage samples were collected. Serum samples were tested for IgM and IgG anti HAV and IgM and IgG anti HEV antibodies. Serum, sewage and water samples were tested for HAV-RNA by nested RT- PCR. Nearly complete full genome (excluding extreme 5' end) was amplified from one serum sample. RESULTS: The hepatitis cases were mainly seen among children and young adults and 63.2 per cent (55/88) were positive for anti-HAV IgM. These cases were reported from the areas getting water supply from Ashwani Khud water supply system. This water purification system received water from a natural stream in which treated sewage water was let into 4 km upstream the collection point since one year. HAV-RNA present in serum, sewage and water samples showed 100 per cent sequence homology. Phylogenetic analysis based on 5' non coding (5' NC) and nearly complete genome showed the evidence of HAV genotype IIIA in all the samples. INTERPRETATION & CONCLUSION: The aetiological agent of the present outbreak was hepatitis A virus which is emerging in an outbreak form in India, emphasizing a definite need for formulating vaccination/control strategies.

Evaluation of human (genotype 1) and swine (genotype 4)-ORF2-based ELISAs for anti-HEV IgM and IgG detection in an endemic country and search for type 4 human HEV infections.

Open reading frame 2 proteins (ORF2) from swine (genotype 4, S-ORF2) and human (genotype 1, H-ORF2) hepatitis E virus (HEV) having 91.4% identity at amino acid level were expressed using baculovirus expression system. Comparison of ELISAs based on the two proteins yielded identical results when sequential serum samples from monkeys and pigs experimentally infected with genotypes 1 and 4 HEV, respectively, were tested. Samples from patients (n = 258) suffering from non-A, non-B hepatitis during outbreaks of the disease and 180 sera from apparently healthy children were screened by H-ORF2-, S-ORF2-based ELISAs and Genelabs ELISA, a widely used commercial test for HEV diagnosis. Specificity of all three tests in detecting IgM and IgG antibodies in healthy children was comparable. Excellent correlation was noted in detecting both IgM (98.7% concordance) and IgG (97.7% concordance) anti-HEV antibodies when H-ORF2 and S-ORF2 ELISAs were compared. When compared with Genelabs ELISA, both H-ORF2 and S-ORF2 ELISAs identified 34 and 18 additional positives, respectively, in IgM and IgG anti-HEV tests showing comparatively less sensitivity of the commercial assay. The concordance of Genelabs ELISA in IgM detection was 86.4% and 85.6%, respectively, with H-ORF2 and S-ORF2 ELISAs. The concordance between Genelabs ELISA and H-ORF2 decreased further to 73.6% when 129 human samples from recent HEV epidemics (2002-2004) were tested for IgM. Similar results were obtained when sequential samples from 11 hepatitis E patients were examined. Screening of serum samples from 137 sporadic non-A, non-B hepatitis cases further confirmed the superiority of the H-ORF2 and S-ORF2 ELISAs. All 36/137 HEV-RNA-positive samples from sporadic cases belonged to genotype 1 confirming absence/rarity of type 4 human infections. H-ORF2 and S-ORF2 antigens were swappable in ELISAs for detecting both genotypes 1 and 4 HEV infections.

Type-IV Indian swine HEV infects rhesus monkeys.

In contrast to countries reporting zoonotic spread of hepatitis E virus (HEV), distinct genotypes circulate in humans (genotype 1) and pigs (genotype 4) from India indicating rarity of such spread. Pigs were refractory to human HEV. As rhesus is an excellent animal model for human HEV, an attempt was made to infect rhesus monkeys with swine HEV. Experimental infection of both the rhesus monkeys with swine-HEV as evidenced by seroconversion to anti-HEV antibodies and presence of viraemia suggests possibility of human infections or differential susceptibility. Comparison of Open Reading Frame-2 and hypervariable regions of HEV genomes showed identity of swine and monkey-derived HEV.

Swine HEV infection in south India and phylogenetic analysis (1985-1999).

Hepatitis E is endemic in India. It was recently noted that although all the Indian human hepatitis E virus (HEV) isolates (1976-2001) were placed in genotype I, the swine HEV recovered from western India (2000) belonged to genotype IV. This was in contrast to reports from the United States and Taiwan wherein both human and swine HEV belonged to the same genotype, i.e., genotypes III and IV, respectively. In order to validate these findings further, we retrospectively examined serum samples collected from pigs from southern India. Sequential serum samples from 45 (1985-1987) and 12 (1999) pigs from Karnataka state, south India, were screened for the presence of HEV RNA (nested PCR) and IgG-anti-HEV (ELISA). PCR products (Open Reading Frame-2 region) were sequenced and subjected to phylogenetic analysis. In this study, 42/45 (1985-1987) and 12/12 (1999) pigs showed seroconversion to IgG anti-HEV antibodies, with a mean age at seroconversion of 4.8 +/- 1.6 months. Four samples collected in 1999 and two samples collected during 1985 were HEV RNA positive. All swine HEV sequences clustered with genotype IV, demonstrating that swine HEV was prevalent among south Indian pigs for at least for 16 years and, similar to western India, belonged to genotype IV. Thus, genotype I and IV HEV continue to circulate in humans and pigs, respectively, from India. Whether swine HEV infects humans remains to be determined.

Changing epidemiology of hepatitis A and hepatitis E in urban and rural India (1982-98).

The epidemiology of hepatitis A virus (HAV) and hepatitis E virus (HEV) was assessed among age-stratified urban high socioeconomic, lower middle socioeconomic status and rural populations from western India in 1998. When compared with previous surveys, a clear shift from high to intermediate endemicity of HAV was evident only for higher socioeconomic population (1982-98), raising the possibility of outbreaks of hepatitis A in this category. A decrease in anti-HAV positivity was noted in rural children aged 6-10 years. Lower circulation of HEV was noted among < 25-year-old urban higher socioeconomic and rural individuals. For both viruses, the lower middle socioeconomic populations were comparable in 1982 and 1998. Socioeconomic status and family size (odds ratio = 23 and 1.6, respectively) were independently associated with anti-HAV positivity. Age, lower middle socioeconomic status and well water were significant independent variables for HEV infection (odds ratio = 5.7, 2.4 and 1.9, respectively). Hence, vaccination policy for hepatitis A needs to be reviewed.

Prevalence of anti-hepatitis E virus antibodies in different Indian animal species.

Prevalence of IgG antibodies to hepatitis E virus (IgG-anti-HEV) was determined among different animal species from India. Seropositivity varied from 4.4% to 6.9% in cattle, 54.6-74.4% in pigs and 2.1-21.5% in rodents. Of the 44 dogs screened, 10 were positive (22.7%). None of the 250 goat sera tested were found to be anti-HEV positive. Among rodents, over 50% serum samples collected in 1985 from Bandicota bengalensis were positive for anti-HEV antibodies. No evidence of HEV infection was obtained following experimental inoculation of an Indian strain (AKL-90) of HEV into anti-HEV negative pigs and goats. The results document varied prevalence of anti-HEV antibodies in different animal species from India and of inability of Indian pigs and goats to support replication of at least one human strain of HEV.

Hepatitis G virus infection in India: prevalence and phylogenetic analysis based on 5' non-coding region.

OBJECTIVES: To determine the prevalence of hepatitis G virus (HGV) infection in western India and to carry out phylogenetic analysis of HGV isolates. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) assay was used to detect HGV RNA in serum samples obtained from paid plasma donors, patients with hemophilia and voluntary blood donors. Nine Indian and one Kenyan HGV RNA-positive samples were sequenced in the 5' non-coding region (5'-NCR). Phylogenetic analysis based on the comparison of a 101 nucleotide fragment from a large number of HGV isolates from 22 countries (including Indian and Kenyan sequences obtained during the present study) was carried out. RESULTS: HGV RNA positivity rates among paid plasma donors from a commercial plasmapheresis unit (7/43, 16.3%) and patients with hemophilia (5/44, 11.4%) were significantly higher than that in voluntary blood donors (0/51; p=0.003 and 0.019, respectively). Among patients with acute non-A to E hepatitis and fulminant hepatic failure, 1 of 50 and 1 of 28 were HGV RNA-positive, whereas 6 of 49 (12%) patients with chronic liver disease had circulating HGV RNA. All Indian isolates belonged to genotype 2, whereas the Kenyan isolate formed a distinct branch within genotype 1 consisting of African isolates. CONCLUSION: Our results suggest existence of parenteral transmission of HGV in the Indian population. HGV was not an important cause of acute non-A to E hepatitis or fulminant hepatic failure among the patients investigated. Genotype 2 seems to be the most prevalent genotype in western India.

Retrospective analysis of blood transfusion recipients: evidence for post-transfusion hepatitis E.

BACKGROUND AND OBJECTIVES: Of 200 volunteer blood donors we had screened earlier for hepatitis E virus (HEV) RNA, using reverse-transcription polymerase chain reaction, 3 were positive, raising the possibility of transfusion-associated hepatitis E in areas endemic for this virus. This retrospective study was to reassess the extent of post-transfusion hepatitis E among transfusion recipients, investigated in 1982. MATERIALS AND METHODS: We re-evaluated 56 recipients followed biweekly for 3 months after transfusion. The controls were 51 normal, healthy persons who gave blood at a 2-month interval, as well as 412 blood donors from whom blood was taken once in 1982. RESULTS: Of the 56 transfusion recipients, 19 were positive for IgG antibodies against HEV (anti-HEV) in the pretransfusion sample. Two of the 37 IgG anti-HEV-negative recipients seroconverted to IgM and IgG anti-HEV 5 and 4 weeks after transfusion, 1 with raised serum alanine aminotransferase levels. None showed symptoms of hepatitis. Attempts to detect HEV RNA in transfused blood, from aliquot units stored at -20 degrees C for over 17 years, were not successful. Of the controls, 17 out of 51 were IgG anti-HEV positive in the initial sample itself. None of the 34 IgG anti-HEV-negative controls seroconverted during the 2-month follow-up. Of the blood donors, 154 out of 412 were IgG anti-HEV positive. None of the 412 donors had circulating IgM anti-HEV antibodies. A significantly higher (p<0.03) proportion of susceptible transfusion recipients were IgM anti-HEV positive as compared with susceptible blood donors. CONCLUSION: The results suggest that, in countries where HEV is endemic, the transmission of hepatitis E may be associated with blood transfusion.

Hepatitis E virus: can it be transmitted parenterally?

Of 200 voluntary blood donors screened for hepatitis E virus (HEV) RNA, employing the reverse transcription-polymerase chain reaction (RT-PCR), three were found to be positive (1.5%). None of the HEV RNA-positive blood donors had any symptoms at the time of blood donation or during subsequent follow-up. One donor was positive for immunoglobulin M (IgM) antibodies to HEV, with a raised serum alanine aminotransferase (ALT) level, whereas the other two donors were negative for both immunoglobulin G (IgG) and IgM antibodies to HEV. Follow-up blood samples collected 2-5 months later from HEV RNA-positive blood donors demonstrated the presence of IgG anti-HEV antibodies. Overall seroprevalence of IgG anti-HEV was 18.6%. Retrospective studies on samples collected from commercial blood donors and haemophiliacs revealed IgG anti-HEV positivity to be 24. 6% (46/191) and 24.4% (22/90) and statistically not different (P>0. 1) from the prevalence among voluntary blood donors and an age-matched normal population, respectively. However, a highly significant proportion of the paid plasma donors, with a high prevalence of IgG antibodies to human immunodeficiency virus and hepatitis C virus, were positive for IgG antibodies to HEV (54/71, 76%, P<0.001), indicating a possible role of blood-derived HEV in the transmission of the virus among plasma donors. These results demonstrate the possible risk of transfusion-associated hepatitis E in hyperendemic areas.

Long-term serological follow up and cross-challenge studies in rhesus monkeys experimentally infected with hepatitis E virus.

BACKGROUND/AIMS: The aims of this study were to examine the decline of IgG anti-HEV antibodies over a period of 7 years in rhesus monkeys experimentally infected with hepatitis E virus, and to assess the protectivity of these antibodies by challenging the monkeys with a heterologous isolate of hepatitis E virus, 5 years after the primary inoculation. METHODS: Nine rhesus monkeys (six non-pregnant and three pregnant at the time of hepatitis E virus inoculation) were followed serologically and biochemically for 7 years post-inoculation. Based on regression analysis, estimated time for IgG anti-HEV titers to reach 1:100 or 1:50 was calculated. Three of the monkeys inoculated initially with AKL-90 isolate and challenged 2 years later with PUN-85 isolate of hepatitis E virus were rechallenged with KOL-91 isolate of the virus, 5 years post-primary inoculation. Evidence of viral replication was assessed by measuring serum alanine aminotransferase levels, excretion of the virus in feces or bile (reverse-transcription polymerase chain reaction) and rise in IgG anti-HEV titers (ELISA). RESULTS: None of the challenged monkeys showed evidence of disease. In contrast to extensive replication of the virus in anti-HEV-negative control monkeys, limited replication was noted in one of the challenged monkeys. The estimated time for the titers to reach 1:100 or 1:50 varied from 3.15 to 44.9 years (19.4+/-11.6 years) and 6.9 to 84.3 years (35.4+/-21.3 years), respectively. Decline in titers was independent of the pregnancy status at the time of infection or reexposure of the monkeys to HEV CONCLUSION: The results show persistence of IgG anti-HEV antibodies for a long time and protectivity of low titered antibodies against reinfection, leading to disease even after intravenous exposure to a heterologous isolate of hepatitis E virus.

Detection of HEV RNA in faeces, by RT-PCR during the epidemics of hepatitis E in India (1976-1995).

Out of the 15 hepatitis E (HEV) epidemics that occurred during the years 1976-1995 in the Gujarat and Maharashtra states of India, 45.78% (76/166) stool samples showed the presence of HEV RNA. HEV RNA was found significantly more often in samples that were transported in liquid nitrogen (50.9%) compared with samples that were transported in wet ice (37.0%) (P < 0.05). Stool samples collected within 7 days after the onset of the disease (59.2%) were more often positive for HEV RNA when compared with samples that were collected 7-20 days after the onset of the disease (28.5%) (P < 0.01). It has been observed in experimentally infected Rhesus monkeys that they excrete HEV throughout the incubation period and for a variable length of time after the elevation of serum ALT levels. A similar situation is found in humans.

Cross-challenge studies in rhesus monkeys employing different Indian isolates of hepatitis E virus.

The aim of this study was to determine if rhesus monkeys infected with one isolate of hepatitis E virus (HEV) were immune to subsequent challenge with other isolates of the virus. Three epidemic and one sporadic Indian HEV isolates were employed in the study. The interval between primary inoculation and challenge varied from 1 year and 6 months to 2 years and 9 months. Evidence of HEV infection was ascertained by rise in serum alanine transaminase (ALT) levels and/or seroconversion to antibodies to HEV (anti-HEV), and the presence of HEV-RNA in the bile or faeces of the infected monkeys. No evidence for multiplication of virus in monkeys challenged with different HEV isolates was obtained. These results show that immunity generated by one isolate of HEV protects against different isolates of hepatitis E virus.

Aetiology of acute sporadic non-A, non-B viral hepatitis in India.

Non-A, non-B (NANB) hepatitis viruses are now classified as hepatitis E (enterically transmitted) and hepatitis C (parenterally transmitted). India experiences a large number of epidemics of the enteric disease every year. In addition, about 70% of the sporadic cases among adults are also due to NANB hepatitis. With the availability of an immunoblot assay for the detection of anti-HEV-IgM and the polymerase chain reaction (PCR) for the detection of HCV-RNA, serum samples from epidemic and sporadic NANB patients were screened for these markers. We found that a large number of cases from the epidemics were HEV, though a few remained undiagnosed, while of the sporadic cases only a few could be diagnosed as HCV or HEV; a large proportion remained undiagnosed.

Hepatitis E virus infection in pregnant rhesus monkeys.

Ten non-pregnant female monkeys and four pregnant monkeys (all Macaca mulatta) in the last third of their gestation period were infected intravenously with the stool sample of a patient with hepatitis E virus infection (immuno-electronmicroscopy positive for hepatitis E virus). Four more non-pregnant monkeys were inoculated with a lower dose (less number of virus particles by IEM) of a stool sample collected on a different day from the same patient. The average incubation period as evidenced by the rise of serum alanine transferase in the non-pregnant monkeys, was 36.4 +/- 4.9 days. The dose of the virus did not affect the incubation period. Two of the pregnant monkeys had incubation periods of 9 and 13 days respectively. They delivered healthy babies on 40th and 53rd day respectively after inoculation. At the age of 11 months, both babies were negative for anti-HEV antibodies. One monkey which delivered a healthy baby on the 2nd day after inoculation had incubation period of 36 days. The baby of this monkey was anti-HEV positive at the age of 11 months. The incubation period was 41 days in the fourth monkey which delivered a macerated foetus on the 36th day after infection. No fatality was recorded in the infected monkeys. Bile samples collected from all monkeys showed strong signals in nested polymerase chain reaction (PCR). It seems that the incubation period in pregnant monkeys was determined by the state of pregnancy.

Hepatitis B infection among dental personnel in Pune & Bombay (India).

To assess the risk of hepatitis B infection among dental personnel, serum samples were collected from dentists of Pune and students, staff, auxiliary staff and class D staff of a dental college in Bombay. Dentists (32.02%), dental auxiliary staff (35.89%), clinical assistants and post-graduate students (19.56%) were found to have significantly higher prevalence of HBV infection as compared to undergraduate dental students (3.94%). The prevalence of HBV infection was high among the dentists as compared to voluntary donors. A positive linear association was observed in the positivity of HBV seromarkers with increasing age and number of years spent by the workers in the dental environment. The rate of increase in HBV seropositivity with age was higher (P less than 0.05) among dental personnel when compared to voluntary donors. Vaccination against hepatitis B is recommended for all the dental students before they start their clinical phase and for susceptible dentists and dental auxillary staff.