Evidence of hepatitis A virus infection in the patients with acute encephalitis syndrome in Gorakhpur region, North India.

The etiological agent remained unidentified in a large number of patients hospitalized for acute encephalitis syndrome (AES) in 2008-2009 in Uttar Pradesh and Bihar, north India. All patients were found to present with fever and altered sensorium, while 28%, 19% and 13% showed hepatomegaly, splenomegaly and meningeal signs, respectively. Involvement mostly of children with abnormal hepatic features prompted us to undertake an exploratory study on viral hepatitis A to determine its association, if any, with hepatic derangements. AES patients (n = 2515) and healthy children (n = 167) were investigated for the presence of serum anti-hepatitis A virus (anti-HAV) IgM and anti-Japanese encephalitis (anti-JE) virus IgM by ELISA. Cerebrospinal fluids (CSFs, n = 595) and rectal swabs (n = 182) were examined for anti-HAV IgM and/or HAV RNA. Anti-HAV IgM was detected in the sera of 14.6% patients as against 6.6% of healthy children (p = 0.0042). Anti-JE virus IgM positivity was Keywords: acute encephalitis syndrome; cerebrospinal fluid; hepatitis A virus; anti-HAV IgM; non-Japanese encephalitis.

Group B rotavirus infection in patients with acute gastroenteritis from India: 1994-1995 and 2004-2010.

Faecal specimens collected from 2101 patients with acute gastroenteritis from three cities (Pune, Alappuzha, Belgaum) in India during 1994-1995 and 2004-2010 were tested for group B rotavirus (RVB) by amplification of the NSP2 gene using RT-PCR. Seventy-five (3.6%) specimens were shown to contain RVB RNA. The positivity rate in Pune, Alappuzha and Belgaum was 4.1%, 7.3% and 4.1%, respectively, in the 2000s which was not significantly different from the detection rate in the 1990s in Pune (2.5%, P>0.05). RVB infections prevailed in adolescents and adults (62/1082, 5.7%) compared to children (13/1019, 1.3%, P<0.001) and were detected throughout the year. Phylogenetically, all strains clustered in an NSP2 lineage together with Indian-Bangladeshi RVB strains belonging to VP7 genotype G2. The study confirmed the occurrence of RVB infections in western India and reported for the first time circulation of RVB strains in southern India, suggesting that an increased awareness and monitoring for RVB infections is necessary in India.

Circulation of Aichi virus genotype B strains in children with acute gastroenteritis in India.

Acute gastroenteritis (AG) is considered as one of the major health problems affecting humans of all ages. A number of viruses have been recognized as important causes of this disease. Recently, Aichi virus has been shown to play an aetiological role in sporadic infections and outbreaks of AG. A study on surveillance of enteric viruses was conducted during 2004-2008 in three cities in Maharashtra state, western India. A total of 1240 stool specimens from children aged ≤8 years hospitalized for AG were screened for the presence of Aichi virus by RT-PCR of the 3C-3D junction region followed by sequencing for the identification of genotype. Aichi virus was detected at a prevalence of 1·1% in the <5 years age group and characterized as genotype B. This is the first report on the circulation of Aichi virus genotype B in India.

A cross-sectional study on aetiology of diarrhoeal disease, India.

BACKGROUND: Global, regional and national estimates clearly place diarrhoeal diseases as a major, albeit to an extant neglected public health problem. Deaths of children aged <5 years owing to diarrhoea was estimated to be 1.87 million at the global level (uncertainty range from 1.56 to 2.19 million), which is approximately 19% of total child deaths. OBJECTIVES: The present report is a cross-sectional study undertaken to estimate the role of various aetiological agents causing diarrhoea in North Karnataka and adjoining areas of Maharashtra and Goa. METHODS: Three hundred stool samples were collected from patients seeking health care at KLES Dr. Prabhakar Kore Hospital and Medical Research Centre, Belgaum; and processed for detection of various bacterial, viral and parasitic agents. RESULTS: Bacterial pathogens attributed to 65.7% of diarrhoea cases, followed by viral infection (22%), parasitic infection (16.3%) and infection by Candida spp. (5.6%). The study identified Escherichia coli in general and Enteropathogenic E. coli in particular, and Group A Rotavirus to be the most frequently isolated pathogens among diarrhoea patients. CONCLUSION: The data generated from the current study will help the health officials for better interventional and treatment strategies for diarrhoeal diseases.

Fecal shedding of hepatitis A virus in Indian patients with hepatitis A and in experimentally infected Rhesus monkey.

Hepatitis A is highly endemic in India. The surveillance reports for the disease from this region are primarily based on the demonstration of hepatitis A virus (HAV) specific serum IgM and IgG antibodies. The present study was conducted to assess the presence and duration of fecal shedding of HAV in patients with hepatitis A and in an experimentally infected rhesus monkey. Nested reverse transcription polymerase chain reaction (RT-PCR) was applied to fecal specimens from 67 sporadic cases of hepatitis A. Recent infection with HAV in these cases was evidenced by the presence of serum anti-HAV IgM. Fecal HAV RNA positivity was observed in nearly 40% patients. The proportion of HAV RNA positivity in fecal specimens obtained within the first week (36.58%) was not different from those collected in 2-12 weeks post onset (42.42%) (P>0.05). A significant number of HAV RNA positive stool specimens showed presence of full virus particles by immune electron microscopy (IEM). Extended fecal shedding of HAV could be a major contributory factor for high circulation of virus thereby maintaining hyperendemicity of the disease. One of the IEM positive samples was inoculated into an anti-HAV negative rhesus monkey. Serum alanine amino transferase levels of the monkey remained within the normal limits. However, HAV RNA positivity in the feces was noted from 3 to 50 days post inoculation. The monkey seroconverted to anti-HAV IgM on day 31. This study records prolonged excretion of HAV in humans as well as in experimentally infected rhesus monkey.

Detection of IgM against hepatitis A virus using tissue culture derived antigen.

Sera from healthy donors and four groups of subjects with acute viral hepatitis were tested for anti-HAV IgM by ELISA with hepatitis A virus antigen grown in tissue culture. The results were compared with those obtained by formalin inactivated HAV from 'HAVAB-M' test kit (Abbott Laboratories, USA). With both preparations of antigens, sera from healthy donors and patients suffering from acute hepatitis B or non-A, non-B did not show anti-HAV IgM antibodies whereas acute phase sera from hepatitis A patients showed strong reactions indicating the presence of anti-HAV IgM antibodies. The results indicate similarity in the specificity of both preparations of HAV against anti-HAV antibodies and encourage the use of tissue culture derived HAV for serological diagnosis of hepatitis A.

Cultivation of buffalo green monkey kidney cells persistently infected with hepatitis A virus.

Studies were carried out to determine the effect of prolongation of incubation periods, cocultivation with normal buffalo green monkey kidney (BGMK) cells and different concentrations of foetal calf serum (FCS) on the production of hepatitis A virus (HAV) by BGMK cell line persistently infected with HAV strain HM175. HAV could be detected from week 1 onwards. However, maintenance of cultures beyond this period was found to yield substantially higher quantities of virus. Cocultivation of persistently infected cells with normal BGMK cells also improved the antigen yields. Different concentrations of FCS did not show any effect on the amount of virus produced. The cell line was maintained up to 46 passages during which there was continuous production of HAV in the cells and release of small amounts of virus in the culture supernatants. Cell associated and cell free viral particles were found to be infectious. Supernatant derived virus was a highly suitable inoculum for infecting other susceptible cell lines. Persistently infected BGMK cell line appears to be a reliable and economical source to derive HAV in adequate amounts for diagnostic and research purposes.

Indigenous anti-hepatitis A virus IgM capture ELISA for the diagnosis of hepatitis A.

Anti-hepatitis A virus IgM capture ELISA was developed by using the reagents produced in the NIV laboratory. The major reagents of the assay were anti-human IgM antibody, hepatitis A virus (HAV) and anti-HAV IgG-horse radish peroxidase (HRP) conjugate. Of these, anti-human IgM antibodies were generated in rabbit against IgM secreted by human hybridoma clone(G3). HAV was derived from buffalo green money kidney cell line infected with HM-175 strain. Virus purified from the cell lysates was used for immunization of rabbits and guinea-pigs. There was very low anti-HAV response. A seropositive rhesus monkey was inoculated with monkey adapted strain of HAV to boost the anti-HAV antibody titre. Anti-HAV IgGs derived from hyperimmune sera of monkey and hepatitis A patient were conjugated with HRP. The preparations of conjugate--particularly human antibody--HRP conjugate yielded highly satisfactory results in anti-HAV capture ELISA. The assay appears to be specific, sensitive and quick and is useful in differentiating acute HAV infection from other acute infections caused by B, E and non-A non-B hepatitis viruses.

Exposure of Indian children to hepatitis A virus & vaccination age.

It is known that 90 per cent of children in India are exposed to hepatitis A virus (HAV) by the age of six years. The aim of the study was to determine when in early childhood maximum HAV infections take place and to deduce an appropriate age for vaccination against HAV. Blood samples of 499 children between the ages of three days and six years were collected and tested for the presence of antibodies against hepatitis A. A statistically significant negative correlation between IgG anti-HAV and age was observed (P < 0.01) up to 11.67 months when IgG anti-HAV positivity was found to be minimum (9.25%). Subsequently a significant positive correlation was noted (P < 0.01). Exposure to HAV was 28.9 per cent soon after the waning of maternal antibodies in the 13-15 month age group which increased to 52.5 per cent by two years of age and 90.9 per cent by 6 yr. It is concluded that in addition to other preventive measures, if children in India are to be vaccinated against hepatitis A they should be immunised against HAV by 9-10 months of age when the maternal antibodies disappear.

Use of filter paper disks for hepatitis A surveillance.

BACKGROUND: Venous blood collection is a cumbersome and uncomfortable procedure during hepatitis A surveillance. Collection of capillary blood by finger prick is an alternative method. AIM: To evaluate the reactivity of capillary blood/anti-hepatitis A virus (HAV) IgG stored on filter paper disks for detection of anti-HAV antibody. METHODS: Venous blood specimens were collected from healthy individuals. Simultaneous capillary blood specimens obtained by finger prick were stored on filter paper disks. A reference standard of anti-HAV IgG in known concentrations was spotted on filter paper disks. The reactivities of anti-HAV IgG and capillary blood specimens eluted from filter paper disks were tested by blocking ELISA for detection of anti-HAV antibody. The results were evaluated by comparing optical density (OD) and neutralization values with those obtained for WHO anti-HAV IgG stored in liquid phase and homologous venous blood specimens, respectively. RESULTS: Among both venous and capillary-blood specimens stored for 10 days, percent neutralization shown by the same 46 specimens was > 50 and that of the same 3 specimens was < 50, indicating anti-HAV positivity and negativity, respectively. There was significant correlation between the OD values displayed by anti-HAV IgG from liquid phase and that eluted from filter paper disk (p < 0.01). Sixteen serum specimens stored for a period of 2 months showed results similar to those of the corresponding filter paper disk elutes. CONCLUSION: Use of filter paper disks could be a suitable choice for pre- and post-immunization collection of blood specimens during hepatitis A surveillance.

Prevalence of hepatitis a antibodies in western Indian population: changing pattern.

This report pertains to a retrospective study conducted between 1983 and 1995 at three time points to evaluate the prevalence of hepatitis A virus (HAV) infection in the population of Bhor Taluk, situated in western India. Serum samples from children and adults were tested for anti-HAV antibodies using blocking ELISA test. There was a significant decrease in anti-HAV prevalence among children aged 5-10 years in 1995 (87.36%) as compared to that of 1983 (97.58%) and 1987 (96.48%). All individuals >11 years of age were seropositive for anti-HAV antibodies. Anti-HAV prevalence was similar in the users of well water, but was significantly reduced in individuals supplied with piped water in 1995 (88.61%) compared with that in 1983 (98.77%). A significant decrease in anti-HAV positivity was noted in children from Bhor Taluk as compared to children from Pune bled in 1992. These results underline the need for periodic surveillance of seroepidemiology of hepatitis A to determine the measures for prevention and control of the disease.

Vertical transmission of hepatitis A.

Two hospital delivered full term newborn babies were detected to have cholestatic jaundice in the first week of life. They had raised liver enzyme levels, which gradually declined over a period of one month. Both babies were anti HAV IgM positive on 6th day of life in Case 1 and on 7th day of life in Case 2 respectively. Both the mothers had jaundice 20 and 26 days before delivery and had anti HAV IgM positivity two and three weeks prior to delivery in Case 1 and 2 respectively. Hepatitis A virus is not transmitted vertically from mother to baby. However, there are 3 such case reports in literature stating vertical transmission of HAV infection. We are reporting it in two neonates for the first time in India.

Hepatitis A in day care centre.

A focal outbreak of hepatitis was detected in a day-care centre for children centrally located in Pune. The source of infection was suspected to be an 11-year-old child who probably got the infection from his school. Seven out of 15 children from day-care centre developed clinical hepatitis. Two cases of secondary infection were identified among the family contacts of infected children. Sera from all the nine sick children were positive for anti-hepatitis A virus-IgM antibodies. A stool sample from a case of secondary infection showed presence of HAV-RNA by RT-nested PCR. These findings proved that the outbreak was caused by hepatitis A virus.

Fulminant hepatic failure: etiology, viral markers and outcome.

OBJECTIVE: To investigate the etiology and outcome of fulminant hepatic failure (FHF) in children. SETTING: Hospital based descriptive. METHODS: 36 children (22 males and 14 females) presenting with FHF over a period of one year were investigated. The ages ranged from 1.5 to 9 years. FHF was defined as occurrence of encephalopathy within eight weeks of onset of jaundice with no evidence of pre-existing liver disease. Detailed history, clinical examination, routine biochemical parameters and relevant diagnostic tests were carried out. Viral markers studied were anti HAV-IgM, HBsAg, anti HBc-IgM, anti-HCV and anti HEV-IgM. RESULTS: A viral etiology could be established in 22 children (61.1%). Hepatitis A (n = 12), Hepatitis B (n = 3), Hepatitis A and B (n = 2), and Hepatitis A and E (n = 4). Two children had enteric fever (1 with associated HEV), 2 children had Wilson's disease, 1 child had Indian Childhood Cirrhosis (ICC) and 2 children had drug induced hepatitis. Etiological diagnosis was not possible in 8 children (22%). Fourteen children (39%) died. Poor outcome was associated with spontaneous bleeding, raised prothrombin time, lower transaminases and higher bilirubin on admission. CONCLUSION: Viral hepatitis is the commonest cause of FHF in children. HAV alone or in combination is responsible for upto 50% of all FHF in children. Chronic liver disease can also present as FHF. Etiological diagnosis is not possible to upto one-fourth of all cases.

Electron microscopy of buffalo green monkey kidney cells persistently infected with hepatitis A virus and immunolocalization of HAV antigens.

Studies were carried out to analyse the ultrastructural changes and the distribution of hepatitis A virus (HAV)/antigens at subcellular level in buffalo green monkey kidney (BGMK) cells persistently infected with HM-175 strain of HAV. HAV infected BGMK cells showed distinct abnormalities in the endoplasmic reticulum and cytoplasmic membrane as compared to uninfected cells. The abnormalities were characterized by wavy arrays, structures like myelin, annulate lamellae, cytoplasmic inclusion bodies and vesicles. The wavy arrays within the cytoplasm of the host cells appeared to represent degenerating membranes. A complex myelin like body was found in close association with a group of virus like particles. Annulate lamellae like structures involving single paired membrane were detected infrequently whereas the cytoplasmic vesicles were numerous in these cells. An indirect immunogold technique was utilized to localize the HAV antigenin infected cells. A high density immunogold label for HIV like particles was predominantly detected in cytoplasmic vesicles. These results suggest a strong association of membrane substructure in vesicle forms with the compartmentalized replication of HAV within persistently infected host cells.

Characterization of VP7 and VP4 genes of rotavirus strains: 1990-1994 and 2000-2002.

A total of 90 rotavirus-positive faecal specimens collected from patients hospitalized with diarrhoea in 1990-1994 (n=77) and 2000-2002 (n=13) were investigated for VP7 and VP4 genotypes. The specimens included 21 typable and 69 non-reactive or multireactive rotavirus strains as monitored by monoclonal antibody-based serotyping ELISA. Genotyping was carried out by multiplex PCR/sequencing using primers specific for both VP7 and VP4 genes. The contribution of common genotypes (G1P[8], G2P[4], G3P[8], G4P[8]) in causing rotavirus diarrhoea was 79.2% and 92.3% in the years 1990-1994 and 2000-2002, respectively, while G9P[8] infections were detected at lower levels (1.3% and 7.7%) at both time-points. There was a predominance of G1P[8] in 1990-1994 and of G2P[4] in 2000-2002. The detection of unusual rotavirus strains (G1P[6], G1P[4], G1P[19], G2P[8], G3P[4], G4P[6]) in 19.5% of the patients indicated a significant contribution of reassortants in causing diarrhoea in western India.

Seroepidemiology of hepatitis A in voluntary blood donors from Pune, western India (2002 and 2004-2005).

Recently, a changing pattern of hepatitis A epidemiology has been reported in the Indian population indicating a rise in the rate of hepatitis A infection among adults. The study's objective was to assess anti-HAV prevalence in voluntary blood donors from middle and high socioeconomic strata. Serum samples collected from voluntary blood donors from Pune city and its suburbs in the years 2002 and 2004-2005 were tested for anti-HAV IgG antibodies. Serum samples collected during 2004-2005 were examined for anti-HAV IgM antibodies. Positive samples were tested for HAV-RNA. Agewise anti-HAV positivity was significantly low in adults aged 18-25 years (90.4%) compared to those aged >25 years (97.4%) (P<0.01). A decline in anti-HAV prevalence was significant in 2004-2005 compared to that in 2002 (96.5% vs. 92.1%) (P<0.01). Overall, in both adult age groups, the proportion of anti-HAV positivity was remarkably low in the high socioeconomic group (HSG) (88.96%) compared to that of the middle socioeconomic group (MSG) (95.86%) (P<0.01). Anti-HAV IgM positivity was not significant (~1%), however, presence of HAV-RNA in one of the samples indicated the possibility of horizontal transmission of HAV. Increase in seronegativity to HAV in HSG implicates a rise in the susceptible pool and indicates the need for vaccination against hepatitis A.