Suspected spread of hepatitis A virus from a restaurant among adults in rural area of the Kerala state, India.

India is experiencing a substantial decrease in early childhood exposure to hepatitis A virus (HAV). Kerala has experienced several hepatitis A outbreaks in young adults/adults in the recent past. The current hepatitis outbreak occurred in Nellikuzhi, Kerala state, India in December 2016. Investigation was carried by preparing a line list of suspected hepatitis cases. The blood and stool samples collected from patients were tested for anti-HAV/anti-Hepatitis E virus (HEV) immunoglobulin (IgM) antibodies and RNA respectively. A total of 562 suspected hepatitis cases were reported during the outbreak. Along with the first case (35 years, male), 86.1% (484/562) of the cases gave history of consuming food/water/cold drinks from one restaurant. Anti-HAV IgM positivity was 74.5% (73/98) in tested samples and amongst the positives, 81% were adults/young adults and adolescents. None of the samples tested positive for anti-HEV IgM. There were three HAV associated deaths without any co-morbidity. Sequence analysis of HAV RNA positive stool samples showed the presence of genotype IIIA HAV. The suspected source of the infection was a private well situated in the premise of a restaurant. Considering increasing HAV naive population in Kerala, there is a need to introduce hepatitis A vaccine in high-risk age groups.

An outbreak of hepatitis B with high mortality in India: association with precore, basal core promoter mutants and improperly sterilized syringes.

In 2009, an outbreak of hepatitis B with high mortality was observed in Sabarkantha district, Gujarat state, India with 456 cases and 89 deaths. Hospitalized patients with self-limiting disease (152, AVH)) and fulminant hepatic failure (39, FHF including 27 fatal and 12 survivals) were investigated. These were screened for diagnostic markers for hepatitis viruses, hepatitis B virus (HBV) genotyping and mutant analysis. Complete HBV genomes from 22 FHF and 17 AVH cases were sequenced. Serosurveys were carried out in the most and least affected blocks for the prevalence of HBV and identification of mutants. History of injection from a physician was associated with FHF and AVH cases. Co-infection with other hepatitis viruses or higher HBV DNA load was not responsible for mortality. Four blocks contributed to 85.7% (391/456) of the cases and 95.5% (85/89) mortality while two adjacent blocks had negligible mortality. Sequence analysis showed the presence of pre-core and basal core promoter mutants and 4 amino acid substitutions exclusively among FHF cases. None of the self-limiting patients exhibited these dual mutations. Genotype D was predominant, D1 being present in all FHF cases while D2 was most prevalent in AVH cases. Probably due to violation of accepted infection control procedures by the qualified medical practitioners, HBV prevalence was higher in the affected blocks before the outbreak. Gross and continued use of HBV contaminated (mutant and wild viruses) injection devices led to an explosive outbreak with high mortality with a striking association with pre-C/BCP mutants and D1 genotype.

Outbreaks of hepatitis A among children in western India.

Hepatitis A in most developing countries is a sporadic childhood disease, but lately focal outbreaks have been observed among children in India. During 2004, we investigated a large-scale outbreak of hepatitis among children living in a residential colony in Daund Taluka of District Pune in the western region of India. In total, 123 overt and 56 sub-clinical cases were detected. All the patients were reactive for IgM antibodies against hepatitis A virus (IgM anti-HAV) and were negative for IgM anti-hepatitis E virus, confirming HAV to be the etiological agent of the outbreak. Serum samples, feces and sewage samples were tested for HAV RNA and molecular characterization of the positives showed the presence of genotype IIIA. Further, IgM anti-HAV-positive sera from eight focal outbreaks were analyzed. The causative HAV in all these small-scale outbreaks also belonged to genotype IIIA, indicating the predominance of genotype IIIA in this region. This report of a large-scale, explosive outbreak of hepatitis A in Indian children once again emphasizes the need to evolve proper public health strategies, especially for vaccination, in countries in the transitional phase from hyperendemicity to intermediate endemicity.

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.

Molecular characterization of hepatitis A virus from a large outbreak from Kerala, India.

BACKGROUND & OBJECTIVES: Hepatitis A is highly prevalent in India and mainly presents as a sporadic disease. This study investigated an outbreak of viral hepatitis at Medical College Hospital area, Kottayam, Kerala state, India during January 2005. METHODS: Blood (133), faecal (1), sewage (4), and water samples (13) were collected. Sera were tested for IgG- and IgM-anti-HAV and IgM antibodies against hepatitis E (IgM-anti-HEV). Sewage, faeces and water samples were tested for HAV RNA in nested RT-PCR and HAV RNA positive samples were further processed for RNA quantitation using Real Time PCR. RESULTS: Of the 1180 total cases, 540 were reported from Medical college area. Two deaths were reported among doctors. Patients from the community gave a previous history of visit to medical college hospital area. The sewage treatment plant at the campus was non-functional since 1990 and the untreated sewage was constantly overflowing and getting mixed with a canal. At the time of the study, all the water sources were superchlorinated. HAV RNA was present in the faeces of hepatitis A patient (1.36 x 10(7) copies/ml), sewage tank (2.57 x 10(3) copies/ml and the canal (<100 copies/ml). None of the 13 water samples concentrated 10,000-fold and the soil sample showed presence of HAV RNA. Phylogenetic analysis based on 5'-non-coding and P2 regions showed HAV-genotype IIIA in all samples. INTERPRETATION & CONCLUSION: The aetiological agent of the present outbreak was found to be HAV. Epidemic hepatitis A (genotype-IIIA) is emerging in Indian adults, emphasizing the need for definite policy for control.

Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination.

The development of an effective human immunodeficiency virus type 1 (HIV-1) vaccine is likely to depend on knowledge of circulating variants of genes other than the commonly sequenced gag and env genes. In addition, full-genome data are particularly limited for HIV-1 subtype C, currently the most commonly transmitted subtype in India and worldwide. Likewise, little is known about sequence variation of HIV-1 in India, the country facing the largest burden of HIV worldwide. Therefore, the objective of this study was to clone and characterize the complete genome of HIV-1 from seroconverters infected with subtype C variants in India. Cocultured HIV-1 isolates were obtained from six seroincident individuals from Pune, India, and virtually full-length HIV-1 genomes were amplified, cloned, and sequenced from each. Sequence analysis revealed that five of the six genomes were of subtype C, while one was a mosaic of subtypes A and C, with multiple breakpoints in env, nef, and the 3' long terminal repeat as determined by both maximal chi2 analysis and phylogenetic bootstrapping. Sequences were compared for preservation of known cytotoxic T lymphocyte (CTL) epitopes. Compared with those of the HIV-1LAI sequence, 38% of well-defined CTL epitopes were identical. The proportion of nonconservative substitutions for Env, at 61%, was higher (P < 0.001) than those for Gag (24%), Pol (18%), and Nef (32%). Therefore, characterized CTL epitopes demonstrated substantial differences from subtype B laboratory strains, which were most pronounced in Env. Because these clones were obtained from Indian seroconverters, they are likely to facilitate vaccine-related efforts in India by providing potential antigens for vaccine candidates as well as for assays of vaccine responsiveness.

Adaptive larval thermotolerance and induced cross-tolerance to propoxur insecticide in mosquitoes Anopheles stephensi and Aedes aegypti.

Fourth-instar larvae of mosquitoes Anopheles stephensi and Aedes aegypti normally died within 90 min at 43 degrees C. Pre-exposure to high but sublethal temperatures conferred adaptive thermotolerance, dependent on the temperature and the duration of pre-exposure. Adaptive cross-tolerance to propoxur (a carbamate insecticide) was also induced in larvae by pre-exposing them to sublethal temperatures. Pre-exposure to sublethal concentrations of propoxur was found to confer cross-thermotolerance to a lower extent. These results suggest that the shock proteins (e.g. heat shock proteins) induced by unrelated stress factors play an important role in the development of adaptive cross-protection (stress response) to other stress conditions.

Characterization of MboI repeat DNA sequence of Anopheles stephensi.

MboI repeat fragment of mosquito Anopheles stephensi has been isolated by molecular cloning. The restriction map and entire nucleotide sequence of the 433bp insert has been determined. Hybridization of this repeat DNA with restriction enzyme digest of mosquito DNA does not show an interspersed pattern but suggests that this repeat may be tandemly repeated at one major site and a few minor sites in the genome of Anopheles stephensi. The hybridization pattern also indicates that this repeat family comprises of many similar but non-identical sequences. An open reading frame encoding 66 amino acids with an initiation and two tandem termination codons has been identified. This putative 66 amino acid polypeptide sequence has significant homology to a small region of RNA tumour viral envelope protein.