Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 28, Num. 4, 2010, pp. 358-362

Indian Journal of Medical Microbiology, Vol. 28, No. 4, October-December, 2010, pp. 358-362

Brief Communication

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 Mishra¹, A Bhargava¹, S Khan², N Pathak², RP Punde², S Varshney²

¹ Bhopal Memorial Hospital and Research Centre, Bhopal - 462 038; Division of Translational Research, Tata Memorial Centre, ACTREC, Navi Mumbai - 410 210, India
² Bhopal Memorial Hospital and Research Centre, Bhopal - 462 038, India
Correspondence Address:
P K Mishra, Bhopal Memorial Hospital and Research Centre, Bhopal - 462 038; Division of Translational Research, Tata Memorial Centre, ACTREC, Navi Mumbai - 410 210, India, pkm_8bh@yahoo.co. uk

Date of Submission: 28-Jun-2010
Date of Acceptance: 02-Sep-2010

Code Number: mb10105

PMID: 20966569
DOI: 10.4103/0255-0857.71813

Abstract

Keywords: 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 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 (IL-2), interferon-γ (IFN-γ), tumor necrosis factor (TNF-α), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10)] 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 (second-generation 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 blood sample conjugated with ethylenediaminetetraacetic acid was collected from each patient by routine veni-puncture 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.

Detection of HCV RNA

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 RT-PCR Kit (Shanghai ZJ Bio-Tech, Shanghai, China) through Light Cycler 2.0 following all the necessary instructions from the supplier. ^[5]

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 + H ₂ O ₂ ) 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 levels of cytokines, IL-2, TNF-α, IFN-γ, IL-4, IL-6 and IL-10. 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]

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 <0.05 was considered to be significant.

Results

Patient characteristics

A total of 68 patients were enrolled for the present study. [Table - 1] and [Table - 2] shows the biochemical features and details of the chronic HCV patients included in the study.

HCV genotyping

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].

Efficacy of treatment

Out of 68 patients enrolled in the present study, 60 responded significantly to the therapy. RVR, EVR and LVR were observed in 73.68, 15.79 and 10.53%, respectively, of the genotype-3 infected patients. While in patients infected with genotype-1, RVR and LVR were observed in 36.36% and 63.64% patients, respectively. SVR was observed was 94.74% and 45.45% in genotype-3 and -1 patients, respectively [Table - 3].

T helper cytokine response

Multiplex CBA assay for human Th1/Th2 cytokines showed that virological response in HCV infections is strongly associated with Th2 to Th1 cytokine shift. A significant increase in the levels of Th1 cytokines (IL-2, TNF-α, IFN-γ) following therapy was observed during RVR in comparison to their baseline levels (before initiation of therapy). These levels were also higher during SVR. In spite of a significant Th1 response among genotype-1 and -3 patients who achieved SVR, a weak Th1 response was observed in the patients with no SVR, further indicating a strong correlation of Th1 cytokines with the SVR [Figure - 1] and [Figure - 2].

Discussion

HCV infection is a worldwide public health problem and major cause of hepatic fibrosis, cirrhosis, end-stage liver disease and hepatocellular carcinoma (HCC). ^[8] The death-defying liver diseases associated with chronic HCV infection have led investigators to look for correlates between viral properties and disease progression, severity of disease and the response to antiviral therapy. The study is first of its kind to assess HCV genotypes in a cohort from central India. HCV genotype has been identified as an important determinant of virological response to interferon-based antiviral therapies. Knowledge of genotypes helps in predicting therapeutic response and the choice of treatment duration. ^[9] Our study observed a high prevalence of genotype-3 infection in the central part of the country. From the total 68 patients examined, 57 were found to be suffering from genotype-3 infections and 11 were found to be with from genotype-1 infection, with no case of co-infection (two or more HCV genotypes).

The issue of best treatment for HCV infection is in constant flux throughout the world. Currently, the combination of interferon plus ribivirin is considered as the first line treatment. Previous studies have reported that with the recommended "standard dose and duration treatment regimens", SVR is achieved in Asia for around 70% of HCV genotype-1 infected cases, approximately 90% of HCV genotype 2/3, 65% of HCV genotype 4, and 80% of HCV genotype 6 patients. ^[10] Genotype-1 patients with a RVR may have higher SVR when treated for 24 weeks with pegylated interferon plus ribavirin therapy. ^[11] On the other hand, a shorter 12-16 week course of therapy with peg interferon alpha-2b and ribavirin was reported to be effective in patients with genotype-3. ^[12],[13] In the present study, the patients with genotype-3 responded more efficiently to the combination therapy in comparison to the genotype-1 infected patients. A RVR was observed in 73.68% patients with genotype-3. Moreover, EVR and LVR were observed in 15.79 and 10.53%, respectively, and SVR was achieved by 94.74% genotype-3 infected patients. While in patients with genotype-1, the RVR observed was 36.36% and LVR was observed in the remaining 63.64% patients. SVR was achieved in 45.45% of genotype-1 infected patients [Table - 3].

The appropriate mechanism responsible for the synergistic effect of two drugs used in combination therapy still remains to be elucidated. In addition to direct antiviral mechanisms, the immunemodulatory effects of both the drugs seem to be important. ^[14] The characterisation of host immune system to different HCV strains has important prognostic, therapeutic, and epidemiologic implications. ^[15] Thus, identifying reliable predictors of treatment response is essential. As chronic HCV infections are associated with higher Th2 cytokine levels and a shift from Th2 to Th1 cytokine is necessary for successful recovery, ^[16] we evaluated the levels of circulating cytokines in the patients infected from both the HCV genotypes prior to therapy and after SVR. Following therapy during RVR, a significant increase in the levels of Th1 cytokines was observed in genotype-3 infected patients, indicating a shift from Th2 to Th1 cytokine profile. The Th1 cytokine profile remains to be dominant during SVR but was poor in no sustained virological response (NSVR) patients with genotype-1 and -3, further indicating a strong correlation of Th1 cytokines with the SVR [Figure - 1] and [Figure - 2].

Given the lack of an effective vaccine, optimal treatment of chronic HCV infection is now perceived as a "must" in terms of public health strategies, as well as of the clinical setting for individual patients. Knowledge of the local or regional prevalence and the spectrum of HCV genotype manifestations is crucial. A prior recognition of the infecting genotype will assist in scheduling the optimal treatment duration in a cost-effective manner. The present study revealed that genotype-3 is more prevalent in the central Indian population, along with traces of genotype-1, and clinicians must also keep in mind of their possible occurrence. Our study also indicated that immune therapeutic response is weaker in genotype-1 in comparison to genotype-3 and these immune responses are directly related to the recovery (SVR). Due to small sample size of genotype-1 (n = 11), the conclusions obtained from the findings on this group are limited and further studies with a larger number of samples are clearly needed. Besides providing the knowledge of local prevalence of HCV genotypes and their therapeutic response, the current study might also assist in understanding the host immune mechanisms involved to achieve SVR during combination therapy.

Acknowledgements

The authors are thankful to the Bhopal Memorial Hospital Trust for providing the necessary financial support and Mr. Naveen Kumar Khare for providing technical assistance.

References

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