Distribution of different hepatitis C virus genotypes in patients with hepatitis C virus infection

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http://www.wjgnet.com/1007-9327/16/2005.asp

ISSN 1007-9327 CN 14-1219/R World J Gastroenterol 2010 April 28; 16(16):

2005-2009

BRIEF ARTICLE

Distribution of different hepatitis C virus genotypes in patients with hepatitis

C virus infection

Farah Bokharaei Salim, Hossein Keyvani, Afsaneh Amiri, Fatemeh Jahanbakhsh

Sefidi, Ramin Shakeri, Farhad Zamani

Farah Bokharaei Salim, Hossein Keyvani, Fatemeh Jahan­bakhsh Sefidi, Virology

Department, Iran University of Medical Sciences, Tehran 14114, Iran

Afsaneh Amiri, Farhad Zamani, GI and Liver Disease Research Centre, Iran

University of Medical Sciences, Tehran 14114, Iran

Ramin Shakeri, Digestive Disease Research Centre, Tehran University of Medical

Sciences, Tehran 14114, Iran

Author contributions: Bokharaei Salim F and Keyvani H desi­gned the research;

Zamani F, Bokharaei Salim F and Jahanbakhsh Sefidi F performed the research;

Amiri A, Bokharaei Salim F and Zamani F wrote the paper.

Supported by Local Fund from Iran University of Medical Sciences

Correspondence to: Farhad Zamani, Assistant Professor, GI and Liver Disease

Research Centre, Iran University of Medical Sciences, Firouzgar hospital, Valadi

street, Valiasr square, Tehran, Iran. zamani.farhad@...

Telephone: +98-21-88940489 Fax: +98-21-88942622

Received: January 4, 2010  Revised: February 3, 2010

Accepted: February 10, 2010

Published online: April 28, 2010

Abstract

AIM: To investigate the presence of mixed infection and discrepancy between

hepatitis C virus (HCV) genotypes in plasma, peripheral blood mononuclear cells

(PBMCs), and liver biopsy specimens.

METHODS: From September 2008 up to April 2009, 133 patients with chronic

hepatitis C referred to Firouzgar hospital for initiation of an antiviral

therapy were recruited in the study. Five milliliters of peripheral blood was

collected from each patient and liver biopsy was performed in those who gave

consent or had indications. HCV genotyping was done using INNO-LiPAâ„¢ HCV â…¡

in serum, PBMCs, and liver biopsy specimens and then confirmed by sequencing of

5’-UTR fragments.

RESULTS: The mean age of patients was 30.3 ± 17.1 years. Multiple transfusion

was seen in 124 (93.2%) of patients. Multiple HCV genotypes were found in 3

(2.3%) of 133 plasma samples, 9 (6.8%) of 133 PBMC samples, and 8 (18.2%) of 44

liver biopsy specimens. It is notable that the different genotypes found in

PBMCs were not the same as those found in plasma and liver biopsy specimens.

CONCLUSION: Our study shows that a significant proportion of patients with

chronic hepatitis C are affected by multiple HCV genotypes which may not be

detectable only in serum of patients.

© 2010 Baishideng. All rights reserved.

Key words: Chronic hepatitis C virus infection; Mixed hepa­titis C virus

infection; Peripheral blood mononuclear cells; hepatocyte

Peer reviewer: Sabine Mihm, Professor, Department of Gastro­enterology,

Georg-August-University, -Koch-Str.40, Göttingen D-37099, Germany

Bokharaei Salim F, Keyvani H, Amiri A, Jahanbakhsh Sefidi F, Shakeri R, Zamani

F. Distribution of different hepatitis C virus genotypes in patients with

hepatitis C virus infection. World J Gastroenterol 2010; 16(16): 2005-2009

Available from: URL: http://www.wjgnet.com/1007-9327/full/v16/i16/2005.htm DOI:

http://dx.doi.org/10.3748/wjg.v16.i16.2005

INTRODUCTION

Hepatitis C virus (HCV) is an enveloped positive-stranded RNA virus of the

family Flaviviridae and genus Hepacivirus that causes both acute and chronic

hepatitis[1-3]. HCV is a major health problem affecting 170 million people

worldwide[4]. It is estimated that chronic HCV infection is responsible for

approximately 250 000 to 350 000 deaths per year, mainly related to

decompensation of cirrhosis, end-stage liver disease, and hepatocellular

carcinoma[5].

Phylogenetic analysis of HCV sequences resulted in a nomenclature that

recognizes distinct virus types and subtypes[6]. Six large groups of viral

genotypes[7-12], in addition to over 70 different subtypes (termed a, b, c,

etc.) are distributed worldwide[6,13]. The HCV genotypes should be

systematically determined before treatment, as it determines the indication, the

duration of treatment, the dose of ribavirin and the virological monitoring

procedure[14].

The HCV is considered as essentially hepatotropic[15], but virus sequences have

also been found in other impor­tant extrahepatic sites, including peripheral

blood mono­nuclear cells (PBMCs), the central nervous system, and bone marrow

from chronically-infected patients[16-19].

HCV replicates through a negative-strand interme­diary. Although hepatocytes

are the primary sites for HCV replication, there is evidence of negative-strand

HCV RNA in PBMCs. The HCV genomic sequences present in PBMCs have been found to

differ from those found in serum and the liver biopsy specimens[20-24].

Detection of HCV RNA in extrahepatic reservoirs has important implications for

transmission, disease progression, and effective treat­ment[25]. Furthermore,

the PBMC compartment may be a privileged site for HCV, which is able to

reinitiate viral replication after termination of HCV treatment when conditions

again become more favorable. Re-infection of HCV after orthotropic liver

transplantation has postulated that extrahepatic sites are suitable for HCV

replication[26]. Thus, even if clearance of HCV from hepatocytes is achieved by

treatment, re-infection from extrahepatic sites, such as the PBMC compartment,

may occur[27].

The purpose of the present study is to determine the presence and frequency of

different HCV genotypes in plasma, PBMC, and liver biopsy specimens of

chronically infected patients.

MATERIALS AND METHODS

Patients

In this cross-sectional study, 133 consecutive patients with established chronic

hepatitis C referred to Firouzgar Hospital from September 2008 to July 2009 were

enrolled. Informed consents were obtained from the patients, and the study was

approved by the local ethics committee of GI and Liver Disease Research centre

(GILDRC) of Iran University of Medical Sciences.

Inclusion criteria were positive anti-HCV antibodies along with positive plasma

HCV RNA. Also none of the included patients had been treated for HCV previously.

Collection and preparation of samples

About 5 mL of peripheral blood were collected from each patient into

EDTA-containing vacutainer tubes. Plasma was stored at -70℃ until analysis.

PBMCs were isolated from EDTA-treated blood by centrifugation over density

gradient (Lymphoprep, Oslo, Norway). PBMCs were then washed three times with

phosphate-buffered saline (pH = 7.4), counted and stored at -70℃ for later

detection. Some of the patients who gave consent underwent a liver biopsy for

diagnostic purpose. Liver biopsy specimens were divided into 2 portions: one

used for histological diagnosis, and the second was submerged into RNALater

(Ambion Inc., Austin, TX) and stored at -70℃ for HCV genotyping. Samples of

serum and PBMCs were collected from all patients on the same day the liver

biopsy was performed.

Isolation of RNA from serum, PBMCs, and liver

RNA was extracted from 140 mL of plasma by using the QIAamp Viral RNA Extraction

kit (Qiagen GmbH, Hilden, Germany), from a pellet of approximately 3-5 × 106

PBMCs using the RNA Virus Mini Extraction Kit (Invitek GmbH, Germany), and from

approximately 2 mm3 of liver biopsy specimens using the DNA/RNA Virus Mini

Extraction Kit (Invitek GmbH, Germany), according to the manufacturer’s

instructions.

HCV genotyping

RNA was isolated from plasma, PBMC, and liver biopsy specimens as described

above. 5’-untranslated region (5’-

UTR) genotyping was done using a standard methodology with the INNO-LiPAâ„¢ HCV

Ⅱ kit (Innogenetics, Ghent, Belgium) according to the manufacturer’s

instructions. Briefly, the 5’-UTR is amplified with biotinylated primers.

Biotin-labeled PCR products are reverse hybridized to specific probes attached

to nitrocellulose strips and then incubated with a chromogen. Developing results

in a purple precipitate that forms a line on the strip. The HCV type is deduced

on the basis of the patterns of hybridizing bands, using the line probe assay

interpretation chart[28].

INNO-LiPAâ„¢ HCV â…¡ genotyping has also been confirmed by sequencing of

5’-UTR fragments. The HCV 5’-UTR was amplified from total RNA isolated from

plasma, PBMC, and liver biopsy specimens of 10 randomly selected patients by

reverse transcriptase-PCR as described previously[29]. The 211-bp 5’-UTR PCR

products were sequenced by dye termination method using the ABI 3130 XL

sequencer. The sequencing results were compared with other sequences posited in

gene bank by Blast software.

Statistical analysis

Data analysis was done by SPSS software version 16.0, using descriptive

statistical indexes such as mean and standard deviation (SD), 95% confidence

interval (CI) and t test. P value less than 0.05 was considered statistically

significant.

RESULTS

One hundred and thirty three patients with established chronic hepatitis C were

recruited in this study. Multiple blood transfusion was the most important

related risk factor found in 124 (93.2%) of patients, 87 (65.4%) with

thalassemia and 37 (27.8%) with hemophilia. The source of infection was unknown

in 9 (6.8%) patients. The mean age of patients was 30.3 ± 17.1 years. Out of

133 patients, 100 (75.2%) were male. The mean viral load of all of the patients

was 6.3 × 105 ± 7.8 × 105 (range from 45 000 to 5.59 × 106). Out of 133

patients, 44 patients gave consent to undergo liver biopsy. Genotype 1a was the

most frequent genotype in serum (63.9%), PBMCs (61.0%), and in liver biopsy

specimens (54.5%). HCV genotypes in serum, PBMCs, and liver biopsy specimens of

all patients were similar except for 20 patients (15%).

The complete data of these patients have been sum­marized in Table 1. As shown

in Table 2, more than one HCV genotype has been found in some patients, as 3

(2.3%) patients had different HCV genotypes in their serum samples. In 9 (6.8%)

patients, different HCV genotypes were found in PBMCs. In 8 (18.2%) patients

(who were not exactly same as the 9 patients with different HCV genotypes in

PBMCs) multiple HCV genotypes were found in liver biopsy specimens. The

INNO-LiPA™ HCV Ⅱ genotyping was confirmed with sequencing of the 5’-UTR. A

100% correlation was demonstrated between INNO-LiPAâ„¢ HCV â…¡ genotyping and

sequencing of the 5’-UTR.

DISCUSSION

The present study was performed on 133 chronically HCV infected patients to

evaluate the prevalence of potential mixed HCV infection in their plasma, PBMCs,

and liver biopsy specimens. In 15% of these patients different HCV genotypes

were found in these compartments. Multiple HCV genotypes were detected in 3

(2.3%) of 133 plasma, 9 (6.8%) of 133 PBMC, and 8 (18.2%) of 44 liver biopsy

specimens.

Mixed infection is infection of an individual with two or more distinct HCV

genotypes. Mixed viral infection is of great clinical importance as it may

result in more severe disease, unresponsiveness to antiviral therapy or

recurrence after the completion of antiviral therapy course[30].

In our study, various HCV genotypes presented in PBMCs were different from those

found in plasma or liver biopsy specimens (Table 1). It means that PBMCs may

present some divergent types, which are not detectable in liver biopsy

specimens. It reinforces the previous proposed theories in which PBMCs have been

known as an extrahepatic replication site for HCV[17,25,26,31-34].

It is suggested that infection with one HCV type doesn’t make a barrier to

acquisition of other genotypes, therefore multiple exposures to HCV especially

in poten­tial risk groups, might lead to several episodes of re-infection and

to the establishment of mixed infection in some patients. It is also well known

that super infection with a new HCV strain leads to suppression of one virus

under the detection limit of PCR while the other one prevails as under antiviral

therapy, the displaced strain may become viremic again and may alter the outcome

of therapy[30,35-39].

In our study the prevalence of mixed HCV infection was estimated about 2.3% in

plasma, 6.8% in PBMC, and 18.2% in liver biopsy specimens of chronically

infected patients with HCV. Mixed infection with two HCV genotypes have been

detected in 1% of HCV-positive patients, using type-specific primers[40,41].

Also figures of 1.6% to 31% have been reported in multi-transfused

hemophiliacs[42,43].

Our study demonstrated that a significant proportion of HCV infected patients

have divergent HCV genotypes in their PBMCs and liver biopsy specimens which

were not detectable in their plasma.

In the present study we used INNO-LiPAâ„¢ HCV â…¡ genotyping which is currently

the most applied method and has more sensitivity than RFLP. One of the problems

of INNO-LiPAâ„¢ HCV â…¡ is that it may underestimate the actual rate of mixed

infection[9,30,44]. According to this, the true prevalence of mixed infection

may be higher than estimated in this study.

The second shortcoming is that only 44 of our patients had indication or gave

consent to undergo liver biopsy, however, 18.2% of them had multiple HCV

geno­types which is a significant proportion of the total. It indicates that

hepatocytes are the main reservoirs for HCV. On the other hand since performance

of liver biopsy is not possible for all patients, we suggest the assessment of

PBMCs as another HCV reservoir for detection of HCV mixed infection[26,34,45].

In conclusion, our study shows that patients with hemo­philia and thalassemia

are from the most high risk groups in whom mixed infection is relatively common.

On the other hand failure to treatment and relapse of infection is also frequent

in these groups. So it seems that considering the plasma genotype as the target

genotype for scheduling of an anti HCV therapy may be one of the factors that

leads to the failure of treatment. HCV genotyping in PBMCs or liver biopsy

specimens might be beneficial.

COMMENTS

Background

Relapse and failure of antiviral therapy have been frequently seen in patients

with chronic hepatitis C especially in those who are on multiple blood

transfusions.

Research frontiers

It has been suggested that infection with multiple hepatitis C virus (HCV)

genotypes in patients affected by chronic HCV infection might result in frequent

relapse after antiviral therapy.

Innovations and breakthroughs

The authors found that a significant proportion of patients with chronic HCV

infection, especially who are on chronic blood transfusion, might present more

than one HCV genotype in their serum, hepatocytes and peripheral blood

mononuclear cells (PBMCs). It is notable that detected HCV genotypes in serum

may be different from those found in PBMCS or hepatocytes.

Applications

According to the above findings the authors suggest HCV genotyping in

hepatocytes and PBMCs along with serum of patients. This may result in choosing

a more appropriate antiviral therapy in these patients.

Peer review

This is a well written manuscript and it addresses a relevant topic of multiple

HCV infections/superinfection/humoral protection and others. Moreover, the

material itself is of some value.

REFERENCES