Genotypes of hepatitis C virus in the Indian sub-continent: A decade-long experience from a tertiary care hospital in South India.

Background: Hepatitis C virus (HCV) is a leading cause of chronic liver disease (CLD) that can progress to cirrhosis and hepatocellular carcinoma. Genotypes of HCV can vary in pathogenicity and can impact on treatment outcome. Objectives: To study the different genotypes among patients with HCV related CLD attending a tertiary care hospital in south India during 2002-2012. Study Design: Study subjects were those referred to clinical virology from the liver clinic. Genotyping was performed using the genotype specifi c core primers in nested polymerase chain reaction (PCR), 5′ non-coding regions based PCR- restriction fragment length polymorphism and NS5B sequencing methods. With the latter method, obtained sequences were compared with published GenBank sequences to determine the genotype. Results: Of the 451 samples tested, HCV genotype 3 was found to be the most predominant (63.85%). Other genotypes detected were genotype 1 (25.72%), genotype 2 (0.002%), genotype 4 (7.5%) and genotype 6 (2.7%). Genotype 3 was the common genotype in patients from Eastern India while genotype 1 and 4 were mainly seen in South Indian patients. Genotype 6 was seen exclusively in patients from North-Eastern India. Two other patients were infected with recombinants of genotype 1 and 2. Conclusions: In this study spanning a decade, HCV genotype 3 and genotype 1 were found to be the predominant genotypes in the Indian sub-continent. Genotype 4 and genotype 6 appeared to show some geographic restriction. A continued monitoring of HCV genotypes is essential for the optimum management of these chronically infected patients. In addition, knowledge of circulating genotypes could impact on future vaccine formulations.

Infection with hepatitis C virus genotype 3 - Experience of a tertiary health care centre in south India.

To analyse the response rate and the predictive values of virological, biochemical and histological factors on HCV antiviral therapy in HCV genotype 3 infected patients, we retrospectively studied 21 HCV genotype 3 infected patients, who underwent HCV antiviral therapy. Low (57%) sustained viral response (SVR) rate and significant association of SVR with normalization of alanine transaminase (ALT) levels were observed in our study. Absence of early viral response (EVR) showed high (80%) predictive value on SVR. Absence of EVR and normalisation of the ALT levels can predict the outcome of HCV antiviral therapy.

Detection of opportunistic DNA viral infections by multiplex PCR among HIV infected individuals receiving care at a tertiary care hospital in South India.

Purpose: Opportunistic viral infections cause increased morbidity and mortality among human immunodeficiency virus (HIV) infected individuals, especially those who are not on antiretroviral treatment. Early diagnosis of these opportunistic viruses will be able to reduce the risk of disease progression with appropriate intervention. Materials and Methods: Multiplex PCR was attempted to detect the opportunistic herpes viruses (HSV-1, HSV-2, VZV, EBV, and CMV), adenovirus and polyoma viruses (JC and BK) in three cocktails of PCR reactions. Subsequently, all the viruses detected were quantitated by testing using monoplex real time PCR. Whole blood samples collected between 2006 and 2007 from 68 treatment naοve HIV-1 infected and 30 normal healthy individuals were tested for these eight viruses. Among the 68 HIV -1 infected individuals 35 had CD4+ T cell count less than or equal to 200 while the other 33 had greater than 200 CD4+ T cells. Results: Among the 68 HIV-1 infected individuals, 49 (72%) were positive for EBV, 5 (7%) samples were positive for CMV. All the five CMV positive individuals had CD4+ T cell count of less than or equal to 200 cells/µL. The mean EBV load among the individuals with a CD4+ T cells of less than or equal to 200 cells/µL was 3.88 log 10 while among those with greater than 200 CD4+ T cells it was 3.75 log 10 . The mean CMV load was 6.98 log 10. Three samples were positive for both CMV & EBV. None of the samples was positive for HSV-1, HSV-2, VZV, Adenovirus, JC and BK viruses. Conclusions: In our study, multiplex PCR based detection system was found useful in detecting opportunistic viruses in HIV infected individuals. Though EBV is the most prevalent opportunistic viral infection among HIV infected individuals, there was no significant association between EBV load, CD4+ T cell counts and HIV-1 virus load. CMV was seen in HIV infected individuals with low CD4+ T cell counts (less than 200 cells/μL).

An unusual seroconversion profile in a pregnant woman infected with the human immunodeficiency virus-1: need for using later generations HIV screening assays.

The first HIV-1 marker that appears in blood following infection is HIV-1 RNA and usually the load is in millions of copies/ ml preceding seroconversion. A 24-year-old pregnant woman, gravida 2, parity 1 was tested for HIV as part of antenatal screening. Three samples were collected and tested from this individual over a period 70 days. The HIV-1 RNA level during seroconversion phase was very low, contrary to the well understood natural history of HIV infection. The reactivity rate in the ELISA and the Western Blot profile showed a gradual increase over the 70 days with a weak reactivity in a second generation assay (detects IgG only) for the third sample. This case illustrates the uncertainties regarding the serological window period in HIV infection and the need to use at least a third generation assay in testing centres for early detection of HIV infection.

An evaluation of saliva as an alternative to plasma for the detection of hepatitis C virus antibodies.

PURPOSE: Seroepidemiological studies on the prevalence of Hepatitis C virus (HCV) in India have been hampered by reluctance of subjects to provide blood samples for testing. We evaluated the use of saliva as an alternate specimen to blood for the detection of antibodies to HCV. METHODS: Chronic liver disease (CLD) patients attending the liver clinic were recruited for this study. A saliva and plasma sample (sample pair) was collected from each patient included in the study. Saliva samples were collected using a commercially available collection device--OmniSal. Sample pairs were tested with an in-use ELISA for the detection of antibodies to HCV (HCV-Ab), with a minor modification in the manufacturer's protocol while testing saliva. The cut-off absorbance value for declaring a sample as positive was determined by receiver operating curve (ROC) analysis. HCV-Ab positivity in saliva was compared with that in plasma as well as with viral load in plasma and infecting genotype of the virus. Sensitivity, specificity, positive and negative predictive values, and correlation coefficients were calculated using Medcalc statistical software. RESULTS: The optimal accuracy indices were: sensitivity-81.6%; specificity-92.5%; PPV-85.1% and NPV-90.5%. No correlation was found between salivary positivity and HCV viral load in plasma or infecting genotype. CONCLUSIONS: The accuracy indices indicate that the assay must be optimized further before it can be recommended for routine use in epidemiological surveys for HCV-Ab.

Significance of the hepatitis C virus (HCV) core antigen as an alternative plasma marker of active HCV infection.

PURPOSE: To evaluate the role of core antigen (Ortho trak-C assay) as a marker of active HCV infection in comparison to HCV RNA as detected by reverse transcription polymerase chain reaction (RT-PCR). METHODS: This evaluation was carried out during January 2000 to December 2003 in HCV infected individuals who were treatment naomicronve or were on anti-viral therapy. Additionally, sequential plasma samples from patients on clinical follow-up were included in this study. A total of 167 samples from 61 patients were tested by trak-C and RT-PCR. HCV RNA detection was achieved by a RT-PCR. Trak-C assay results were also compared in a limited proportion of these samples with known HCV viral load and genotype. RESULTS: Of 167 samples tested, 56.9% were RNA positive and 43.1% were RNA negative while 50.3% were trak-C positive and 49.7% were trak-C negative, yielding a sensitivity of 85.3% and a specificity of 95.8% for the trak-C assay (Kappa co-efficient = 0.8). The concentration of HCVcAg and HCV RNA showed significant correlation (n=38, r=0.334, P =0.04). The trak-C assay detected the most prevalent HCV genotypes in India without significant difference (P =0.335). The difference between mean absorbance values of HCV RNA positive samples compared to HCV RNA negative samples in the trak-C assay was highly significant (P < 0.000). Qualitative results of trak-C assay and RT-PCR were comparable in 93% of follow-up samples. CONCLUSIONS: Trak-C assay can be recommended for confirmation of HCV infection and follow-up in laboratories with resource-poor facilities.