Prevalence of hepatitis C virus genotypes and impact

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Indian Journal of Medical Microbiology, (2010) 28(4): 358-62

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Brief communications

Prevalence of hepatitis C virus genotypes and impact of T helper cytokines in achieving sustained virological response during combination therapy: A study from Central India *PK Mishra, A Bhargava, S Khan, N Pathak, RP Punde, S Varshney

Abstract Characterisation of host immune response to hepatitis C virus (HCV) genotypes may have an important prognostic and therapeutic implication. Genotype-3 was more prevalent in the examined cohort and demonstrated a significantly higher response to combination therapy than genotype-1. Sustained virological response (SVR) was 94.74% in genotype-3 and 45.45% in genotype-1. The patients who achieved SVR reported higher levels of circulating T helper 1 cytokines in comparison to subjects with no SVR in both the studied groups. Besides providing local prevalence, our study might also assist in understanding the host immune mechanisms involved to achieve SVR during combination therapy in chronic HCV patients. Key words: Antiviral therapy, cytokines, HCV genotypes, hepatitis C virus, molecular surveillance

Introduction Hepatitis C virus (HCV) is a leading cause of chronic liver disease and has already infected at least 170 million people worldwide. In India, there are about 12–13 million HCV carriers and modeling data predict that the burden of disease could soon increase substantially.[1] Till date, six different HCV genotypes and more than 70 subtypes have been identified based on the nucleic acid sequences.[2] Due to such high levels of genetic heterogeneity, treatment options for HCV infections are poor. At the moment, the only accepted antiviral therapy with proven effectiveness is a combination therapy of (pegylated) interferon alpha and ribavirin.[3] It is suggested that early viral load decline following combination therapy is an important predictor of the treatment responses in chronic hepatitis C patients. [4] Moreover, the immune response to HCV genotypes and the region-specific prevalence of HCV genotypes in central India is still unknown, and the impacts of viral load, genotype, and immune mediators on the clinical course are poorly defined. Thus, in order to refine the current knowledge of treatment strategies, there is an ongoing need to evaluate the influence of host and viral factors on virological response. The objective of the present study was to perform *Corresponding author (email:) Bhopal Memorial Hospital and Research Centre (PKM, AB, SK, NP, RPP, SV), Bhopal - 462 038, Division of Translational Research (PKM, AB), Tata Memorial Centre, ACTREC, Navi Mumbai - 410 210, India Received: 28-06-2010 Accepted: 02-09-2010 DOI: 10.4103/0255-0857.71813 

PMID: ****

a clinical, epidemiological and immunological characterisation of chronic hepatitis C infection in a cohort from central India. HCV genotypes were characterised and the varied therapeutic response of these genotypes was studied through quantification of HCV RNA using real time polymerase chain reaction (PCR) (Light Cycler 2.0, Roche Diagnostics, Mannheim, Germany). To observe the immune response to the different genotypes prevalent in central India, the levels of cytokines [interleukin-2 (IL2), interferon-γ (IFN-γ), tumor necrosis factor (TNF-α), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL10)] were evaluated using multiplex cytometric bead array (CBA) assay for human T helper (Th)1/Th2 cytokines (BD Biosciences, San Jose, CA, USA). Materials and Methods Subject selection A total of 68 patients (males = 43 and females = 25) with chronic liver disease attending gastroenterology clinic or admitted in wards were studied. Age range was 20–80 years. Diagnosis was done by a >6-month history of liver disease together with a positive HCV antibody test (secondgeneration enzyme immunoassay) and detectable HCV RNA by real time PCR. The alanine aminotransferase (ALT) levels of these patients were also observed to be raised on at least two determinations within previous 6 months, and liver biopsy findings were consistent with chronic HCV within 18 months before therapy. There was no serological evidence of co-infection with other hepatotropic viruses. Other possible causes of hepatocellular injury, such as alcohol, were also excluded. Informed consent was obtained from all subjects included in the study, and clinical information pertaining to them was properly recorded as per institutional review board guidelines. Ten millilitres of

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Mishra, et al.: Therapy response and cytokines

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blood sample conjugated with ethylenediaminetetraacetic acid was collected from each patient by routine venipuncture method. The blood thus obtained was centrifuged at 3000 rpm for 10 minutes for the separation of plasma which was used for further investigations. All the patients received pegylated interferon subcutaneously at a dose of 180 µg or 1.5 µg/kg once weekly plus oral ribavirin given in two divided doses per day at a dose of 1000 mg/day for patients weighing 75 kg or less and 1200 mg/day for those weighing more than 75 kg. The duration of therapy was 24 weeks in case of genotype-3 and 48 weeks in genotype-1 infected cases. The detection of HCV RNA was done weekly and the patients were classified into three groups according to their response to combination therapy: rapid virological response (RVR), defined as undetectable HCV RNA at week 4 after the initiation of combination therapy; early virological response (EVR), defined as undetectable HCV RNA at weeks 5–12 of combination therapy; and late virological response (LVR), defined as undetectable HCV RNA at weeks 13–24 of combination therapy. A sustained virological response (SVR) was defined as clearance of HCV RNA by real time PCR after 6 months upon cessation of combination therapy.

levels of cytokines, IL-2, TNF-α, IFN-γ, IL-4, IL-6 and IL10. The assay was performed by following all the necessary instructions from the manufacturer. Data acquisition and analysis were carried out on a flow cytometric platform using BD CBA software (BD Biosciences, San Jose, CA, USA).[7]

Detection of HCV RNA

HCV genotyping

Extraction of HCV RNA was done using QIAamp Viral RNA Mini extraction Kit (Qiagen, Hilden, Germany), according to the standard kit protocol. Briefly, RNA was isolated from plasma by lysis of viral particles under highly denaturing conditions, followed by binding of viral RNA to a silicagel-based membrane (QIAamp membrane) in the QIAamp Mini spin column and washing with buffer AW1 and AW2. Viral RNA was then eluted using RNase-free buffer AVE and was detected using HCV Real Time RTPCR Kit (Shanghai ZJ Bio-Tech, Shanghai, China) through Light Cycler 2.0 following all the necessary instructions from the supplier.[5]

The distribution of HCV genotypes evaluated in 68 patients by genotype screening showed a prevalence of HCV genotype-3 in 83.82% cases (57 out of 68), while 16.18% (11 out of 68) patients were found to be infected with genotype-1 [Table 3].

HCV genotyping Following the quantification, identification of HCV genotypes was done using Linear Array HCV genotyping kit (Roche Diagnostics, Mannheim, Germany), as per the supplier’s protocol. Briefly, HCV RNA was amplified using biotinylated primers and the amplicons were subjected to hybridisation with the genotype specific probes attached on genotyping strips. After hybridisation, the attached amplicons were washed and enzyme (horse radish peroxidase) was added followed by the addition of substrate (3, 3′, tetra methyl benzidine + H2O2) to develop a band which is specific to a particular genotype. The developed bands obtained were analysed for genotype using a reference strip.[6] T helper cytokine response Plasma from patients and controls was subjected for the analysis of Th1/Th2 cytokines through the determination of

Statistical analysis Statistical analysis was performed by using analysis of variance (ANOVA) or Student’s t-test. The Statistical Package for Social Sciences (SPSS) software package (SPSS Inc., Chicago, IL, USA) was used to perform statistical analysis. A P value of