A latest and promising approach for prediction of viral load in hepatitis B virus infected patients.

INTRODUCTION: Designing a rapid, reliable and sensitive assay for detection of hepatitis B virus (HBV) variants by real-time PCR is challenging at best. A recent approach for quantifying the viral load using a sensitive fluorescent principle was brushed in this study. MATERIALS AND METHODS: : A total of 250 samples were collected from the outpatient unit, CLRD. Complete Human HBVDNA sequences (n = 944) were selected from the National Centre for Biotechnology Information (NCBI), primers and probes were designed and synthesized from the core, surface, and x region. Real-time based quantification was carried out using a standard kit and in-house generated standards and RT-PCR protocols. RESULTS AND DISCUSSION: The standard calibration curve was generated by using serial dilution 10(2) to 10(8). The calibration curve was linear in a range from 10(2) to 10(8) copies/ml, with an R(2) value of 0.999. Reproducibility as measured by dual testing of triplicates of serum samples was acceptable, with coefficients of variation at 6.5%, 7.5%, and 10.5%. Our results showed that amplification performance was good in the case of the x-region-based design (98%). Out of 100 negative samples screened by enzyme linked immunosorbent assay and the standard RT-PCR kit, one sample was detected as positive with the in-house developed RT-PCR assay, the positivity of the sample was confirmed by sequencing the amplified product, NCBI accession EU684022. CONCLUSION: This assay is reproducible showing limited inter- and intra-assay variability. We demonstrate that the results of our assay correlated well with the standard kit for the HBV viral load monitor.

Association of genetic variants of mannan-binding (MBL) lectin-2 gene, MBL levels and function in ulcerative colitis and Crohn's disease.

Ulcerative colitis and Crohn's disease are the two major forms of inflammatory bowel disease (IBD). A series of reports have hypothesized interplay of genetic and environmental factors in the pathogenesis of IBD. Polymorphism in the mannan-binding lectin-2 (MBL-2) gene is known to affect the structural assembly and function thereby predisposing subjects to various diseases. The present study was designed to evaluate effect of MBL-2 gene polymorphism on MBL levels and function in IBD patients. Genomic DNA was isolated from blood samples collected from 157 ulcerative colitis, 42 Crohn's disease and 204 control subjects. Genotyping for different polymorphic sites at exon1 of MBL-2 gene was performed by refractory mutation system-PCR and amplification followed by restriction digestion (PCR-RFLP). Serum MBL concentration and C4 deposition levels were estimated using ELISA. Mannan-binding lectin-2 genotypic variants were calculated in IBD and healthy controls. The frequency of single nucleotide polymorphisms at codon 54 was significantly higher in ulcerative colitis patients than controls (P < 0.0001). Ulcerative colitis patients with 'codon 54'-variation showed low serum MBL concentrations coupled with altered MBL function compared to controls. In conclusion, single nucleotide polymorphism in the MBL-2 gene is an important risk factor significantly affecting MBL levels and function in the development of ulcerative colitis among Indians.

Prevalence, risk factors and genotype distribution of HCV and HBV infection in the tribal population: a community based study in south India.

Viral hepatitis caused by the hepatitis C virus (HCV) and hepatitis B virus (HBV) represents a major public health problem in India. These viruses share common modes of transmission, such as parenteral routes. We aimed to assess the exposure of a tribal population to these viruses in south India. The present study was carried out on serum samples from 890 individuals (526 males and 324 females) belonging to the Lambada tribe residing in the state of Andhra Pradesh, south India. Anti-HCV antibody and hepatitis B surface antigen (HBsAg) status in the sera were analyzed using commercially available enzyme immunoassays (Abbott Labs, Chicago, IL). HCV-RNA and HBV-DNA in the sera was tested by reverse transcriptase polymerase chain reaction (RT-PCR) and PCR, respectively. The infecting genotype of HCV was determined using type-specific primers corresponding to the NS5 region of the virus. Out of the 890 samples, 18 (2.02%; male 11/526; female 7/364) were positive for HCV-RNA by RT-PCR and, 17 of them were positive for anti-HCV antibody. Genotyping of HCV isolates from the 18 individuals positive for HCV-RNA revealed that 66.67% (12/18) were infected with type 1 of HCV and its variants; while in the remaining (6/18), the infecting genotype was found to be type 3 and its variants. A total of 46 samples (5.16%; males 28/526; female 18/364) were positive for HBsAg; while 11 were positive only for HBV-DNA, 9 were positive for both hepatitis B e antigen (HBeAg) and HBV-DNA. Cultural practices such as tattooing, traditional medicine (e.g. blood-letting), rituals (e.g. scarification), body-piercing etc are the potential sources of spread of infection in this tribe. None of the samples analyzed revealed co-infection with the 2 viruses.