Molecular characterization of hepatitis A virus strains in a tertiary care health set up in north western India.

BACKGROUND & OBJECTIVES: Hepatitis A virus usually causes acute viral hepatitis (AVH) in the paediatric age group with a recent shift in age distribution and disease manifestations like acute liver failure (ALF). This has been attributed to mutations in 5'non-translated region (5'NTR) which affects the viral multiplication. The present study was aimed to carry out the molecular detection and phylogenetic analysis of hepatitis A virus strains circulating in north western India. METHODS: Serum samples from in patients and those attending out patient department of Pediatric Gastroenterology in a tertiary care hospital in north India during 2007-2011 with clinically suspected AVH were tested for anti-hepatitis A virus (HAV) IgM antibodies. Acute phase serum samples were subjected to nested PCR targeting the 5'NTR region followed by sequencing of the representative strains. RESULTS: A total of 1334 samples were tested, 290 (21.7%) were positive for anti-HAV IgM antibody. Of these, 78 serum samples (< 7 days old) were subjected to PCR and 47.4% (37/78) samples showed the presence of HAV RNA. Children < 15 yr of age accounted for majority (94%) of cases with highest seropositivity during rainy season. Sequencing of 15 representative strains was carried out and the circulating genotype was found to be III A. The nucleotide sequences showed high homology among the strains with a variation ranging from 0.1-1 per cent over the years. An important substitution of G to A at 324 position was shown by both AVH and ALF strains. The cumulative substitution in AVH strains Vs ALF strains as compared to GBM, Indian and prototype strain in the 200-500 region of 5' NTR was comparable. INTERPRETATION & CONCLUSION: Our results showed hepatitis A still a disease of children with III A as a circulating genotype in this region. The mutations at 5'NTR region warrant further analysis as these affect the structure of internal ribosomal entry site which is important for viral replication.

Assessment of dried blood samples as an alternative less invasive method for detection of Hepatitis E virus marker in an outbreak setting.

Hepatitis E virus (HEV) is the causative agent of hepatitis E. It can be asymptomatic, associated with acute self-limiting hepatitis or acute liver failure. The conventional diagnosis of HEV infection relies on anti-HEV IgM serology. The collection of blood samples by venepunture for laboratory confirmation is often difficult during an outbreak. Thus, testing the specimens of dried blood spots (DBS) on filter papers can prove to be a feasible alternative. The present study aimed to evaluate the applicability of anti-HEV IgM detection from DBS samples and the stability of anti-HEV IgM detection at varied time interval, at various storage temperatures. Paired blood and DBS sample were collected from 44 jaundiced patients and eight healthy controls during HEV outbreaks. The DBS were tested for anti-HEV IgM by available ELISA kit with in-house modifications. Three cut offs were determined, that is, the CO1: kit cut-off, CO2: mean of negative controls above 3SD and CO3: area under Receiver operating Curve. The sensitivity of anti-HEV IgM detection ranged from 86-91%. The maximum sensitivity (91%) and specificity (100%) was obtained using CO3. Maximum stability of anti-HEV IgM antibodies (100%) was observed till 65 days at 4°C. Storage at 37°C significantly reduced anti-HEV IgM positivity, wherein 42.85% sample became negative by 45 days. DBS showed good sensitivity and specificity for detecting anti-HEV IgM and can be considered an alternate to serum sample. Moreover, anti-HEV IgM was stable at 4°C, which makes DBS a preferred method for storage and transportation of the sample to reference laboratory.