Indian Journal of Pathology and Microbiology
Home About us Instructions Submission Subscribe Advertise Contact e-Alerts Ahead Of Print Login 
Users Online: 385
Print this page  Email this page Bookmark this page Small font sizeDefault font sizeIncrease font size


 
  Table of Contents    
ORIGINAL ARTICLE  
Year : 2013  |  Volume : 56  |  Issue : 4  |  Page : 384-387
Hepatitis C virus genotypes: A plausible association with viral loads


1 Department of Pathology, Islamabad Medical and Dental College, Islamabad, Pakistan
2 Department of Medicine, Islamabad Medical and Dental College, Islamabad, Pakistan
3 Department of Pathology, Army Medical College, National University of Science and Technology, Islamabad, Pakistan
4 Department of Pathology, Shaukat Khanum Memorial Cancer Hospital and Research Centre, Lahore, Pakistan

Click here for correspondence address and email

Date of Web Publication18-Jan-2014
 

   Abstract 

Background and Aim: The basic aim of this study was to find out the association of genotypes with host age, gender and viral load. Material and Methods: The present study was conducted at Social Security Hospital, Pakistan. This study included 320 patients with chronic hepatitis C virus (HCV) infection who were referred to the hospital between November 2011 and July 2012. HCV viral detection and genotyping was performed and the association was seen between genotypes and host age, gender and viral load. Results : The analysis revealed the presence of genotypes 1 and 3 with further subtypes 1a, 1b, 3a, 3b and mixed genotypes 1b + 3a, 1b + 3b and 3a + 3b. Viral load quantification was carried out in all 151 HCV ribonucleic acid (RNA) positive patients. The genotype 3a was observed in 124 (82.12%) patients, 3b was found in 21 (13.91%), 1a was seen in 2 (1.32%), 1b in 1 (0.66%), mixed infection with 1b + 3a in 1 (0.66%), 1b + 3b in 1 (0.66%) and 3a + 3b was also found in 1 (0.66%) patient. Viral load quantification was carried out in all 151 HCV RNA positive patients and was compared between the various genotypes. The mean viral load in patients infected with genotype 1a was 2.75 × 10 6 , 1b 3.9 × 10 6 , 3a 2.65 × 10 6 , 3b 2.51 × 10 6 , 1b + 3a 3.4 × 106, 1b + 3b 2.7 × 106 and 3a + 3b 3.5 × 10 6 . An association between different types of genotypes and viral load was observed. Conclusion : Further studies should be carried out to determine the association of viral load with different genotypes so that sufficient data is available and can be used to determine the type and duration of therapy needed and predict disease outcome.

Keywords: Genotype, hepatitis C virus, viral load

How to cite this article:
Nabi SG, Zaffar G, Sheikh NI, Hassan K, Hassan U. Hepatitis C virus genotypes: A plausible association with viral loads. Indian J Pathol Microbiol 2013;56:384-7

How to cite this URL:
Nabi SG, Zaffar G, Sheikh NI, Hassan K, Hassan U. Hepatitis C virus genotypes: A plausible association with viral loads. Indian J Pathol Microbiol [serial online] 2013 [cited 2018 Nov 17];56:384-7. Available from: http://www.ijpmonline.org/text.asp?2013/56/4/384/125319



   Introduction Top


Hepatitis C virus (HCV) infection is found world-wide. [1] It is estimated that there are more than 170 million people chronically infected with HCV and 3-4 million persons are newly infected each year. Approximately 280,000 deaths/year are related to HCV infection. [2],[3] HCV is the main cause of liver fibrosis, cirrhosis and hepatocellular carcinoma in a substantial number of patients .[4],[5] Countries with high rates of chronic infection are Egypt (22%), Pakistan (4.8%) and China (3.2%). The main mode of transmission in these countries is attributed to unsafe injections using contaminated equipment. [1] The prevalence of HCV infection is high (8% of the population) in Pakistan. [6] The rate of disease progression is variable and several factors have been identified as important in predicting the outcome of progression. These include age at infection, gender, genotype/subtype, viral load and mode of infection. [7] Due to considerable sequence diversity HCV is classified into a series of genotypes. These genotypes have shown distinct geographical and frequency distribution across the whole world. [6],[8],[9],[10] In patients infected with HCV, clinical findings, genotypes and viral load are strong predictors for the outcome of antiviral therapy [6],[11],[12] Therefore, genotype specific correlations are of high clinical relevance. HCV is a ribonucleic acid (RNA) virus that has a high mutation rate. This high rate results in extensive genetic heterogeneity and HCV isolates are found as either quasi species or genotypes. [13] Humans can be co-infected with >1 genotype (mixed genotype infection) of this virus. [14] The rate of HCV mixed genotype infections is extremely variable for different regions and for the same group of patients tested by using different assays. [15] Thus, it is difficult to determine the prevalence of mixed genotype infections by currently available assays, including direct deoxyribonucleic acid (DNA) sequencing, because they are designed to identify only the HCV genotype dominant in that particular population. Consequently, genotypes present at lower frequencies could be missed or mistyped. [16]

The present study was undertaken to investigate the distribution pattern of HCV genotypes in patients with chronic hepatitis and their association with viral load.


   Materials and Methods Top


The present study was conducted at social security hospital, Rawalpindi. This study included 320 patients with chronic HCV infection who were referred to the hospital between November 2011 and July 2012. HCV viral detection and genotyping was performed at Lab One Garden Town Lahore. Methodology used was Standard Gel Method for polymerase chain reaction (PCR) and RNA Extraction Nested PCR Examination on Gel for HCV genotyping. The basic aim of this study was to find out the association of genotypes with host age, gender and viral load.

Viral RNA extraction

Sera of all patients were subjected to viral RNA extraction from 140 ml serum samples using Qiagen (Germany) RNA kit according to manufacturer's protocol.

Qualitative analysis (PCR based detection)

All the 320 ELISA samples were tested for HCV presence or absence by PCR, as ELISA is not as reliable as PCR-based analysis. Viral RNA was taken to reverse transcribe the 5' NCR of HCV using Moloney murine leukemia virus reverse transcriptase (M-MLV RTase, Frementas) in a total reaction volume of 20 μL. The reaction mixture contained 4 μL M-MLV (×5) buffer, 1 μL of M-MLV RTase enzyme, 1 μL dNTPs (10 mM), 0.5 μL RNase inhibiter (Fermentas), 1.5 μL RNase free water, 1 μL specific anti-sense primer and 10 μL template (viral RNA). Complementary DNA (cDNA) was synthesized using ABI Veriti 96 well thermocycler. Cycling condition for cDNA were as follows: 42°C for 55 min followed by 70°C for 10 min. The cDNA produced was stored at 4°C for short term storage or –20°C for prolonged storage. cDNA product was used for qualitative analysis of HCV infection. The first round PCR was performed using sense and anti-sense primers followed by second round PCR (nested PCR), using the first round product with an inner sense and antisense primers. PCRs were carried out using Taq polymerase (Fermentas) for 35 cycles. Finally, 5 μL of the second round PCR product was mixed with 2 μL of ×6 loading dye and electrophoresed on 1.5% agarose gel with commercially available 100 bp DNA marker (Fermentas). Specific bands of HCV detections were visualized under ultraviolet light of Wealtec gel doc system.

HCV genotyping

A total of 151 PCR positive samples were then processed for HCV genotyping by the method described by Ohno et al. [17] Confirmation was based on specific PCR bands.


   Results Top


Out of 320 HCV antibody positive patients tested for the presence of HCV RNA, 151 patients were found to be HCV RNA positive. All HCV RNA positive samples were subjected to genotype determination using restriction fragment length polymorphism and type specific PCR followed by direct sequencing. The analysis revealed the presence of genotypes 1 and 3 with further subtypes 1a, 1b, 3a, 3b and mixed genotypes 1b + 3a, 1b + 3b and 3a + 3b ([Figure 1] and [Figure 2]. Viral load quantification was carried out in all 151 HCV RNA positive patients and was compared between the groups of genotypes revealed in the study.
Figure 1: The electrophoresis patt ern of genotype. Lane 1: 100 bp deoxyribonucleic acid ladder, Lane 2-4: Polymerase chain reacti on (PCR) product of genotype 1b (234 bp), Lane 5: negati ve control. Lane 6-10: PCR product HCV genotype 3.Lan 7 shows PCR product of genotype 3b (176 bp) lane 8 and lane 10 show PCR product of genotype 3a (232 bp)

Click here to view
Figure 2: The electrophoresis patt ern of genotype. Lane 1: 100 bp deoxyribonucleic acid ladder, Lane 2: Polymerase chain reacti on product of genotype 1a (209 bp)

Click here to view


Genotypes 3a and 3b were observed in 124 (82.12%) and 21 (13.91%) patients respectively whereas types 1a and 1b were seen in 2 (1.32%) and 1 (0.66%) patient respectively. Mixed infection with 1b + 3a was found in 1 (0.66%), with 1b + 3b in 1 (0.66%) and with 3a + 3b was found in 1 (0.66%) patient.

Out of 151 PCR positive patients, 81 (53.64%) were females and 70 (46.36%) were males. Distribution pattern of HCV genotypes according to the gender is given in [Table 1]. Distribution pattern of HCV genotypes according to the age is given in [Table 2].
Table 1: Distributi on patt ern of hepati ti s C virus genotypes according to gender N = 151

Click here to view
Table 2: Distributi on patt ern of hepati ti s C virus genotypes according to age N = 151

Click here to view


Viral load quantification was carried out in all 151 HCV RNA positive patients and was compared between various genotypes. The mean viral load of the patients infected with genotype 1a was 2.75 × 10 6 , 1b 3.9 × 10 6 , 3a 2.65 × 10 6 , 3b 2.51 × 10 6 , 1b+3a 3.4 × 106, 1b+3b 2.7 × 106 and 3a+3b 3.5 × 10 6 .


   Disscussion Top


HCV has been classified into 1-6 genotypes on the basis of the identification of their different genomic sequences. [18],[19] The distribution of HCV genotypes vary according to the geographical region. Genotypes 1-3 are widely distributed throughout the world. [20],[21] Subtype 1a is prevalent in North and South America, Europe and Australia. [21],[22] Subtype 1b is common in North America and Europe, [21],[23] and is also found in parts of Asia. [21] Genotype 2 is present in most developed countries, [21],[24] but is less common than genotype 1. [21]

HCV genotype 3a was most common in our study followed by genotype 1. The results of our study were similar to a collaborated study conducted in Lahore and Faisalabad by Ijaz et al. in which prevalence of genotypes was as follows: genotype 3 (n = 4287, 70.9%), followed by genotype 1 (n = 802, 13.3%) and genotype 4 (n = 446, 7.4%). [4] A similar study showed type 3 (63.38%) to be the most common followed by type 1 (30.98%) and type 2 (5.63%). [25] A study conducted at Agha Khan University, Karachi, Pakistan found HCV type 3 genotype to be the most common (68%), followed by type 1 in 14% cases; the rest of the cases were undefined. [26]

In a French study conducted in Department of Microbiology, Compiegne, France the distribution frequency of HCV genotypes was as follows: subtype 1a, 16.4%; subtype 1b, 46.3%; subtype 2a, 7.5%; subtype 3a, 20.9%; type 4, 4.5%; type 5, 0.7%; and co-infections, 3.7% [26]

According to our study in the group of patients of age less than 30 years 65.21% of patients were found to be infected with HCV genotype 3a while 28.26% of patients were infected with genotype 3b. No significant percentage was shown by other genotypes, i.e., 1a, 3a + 3b and 1a + 1b. In the age group 31-40 years: 86.04% of patients had 3a genotype infection, while 11.62% showed 3b genotype infection of HCV, whereas patients of age group 41-50 years showed the percentage to be 88% of 3a and 10% of 3b. Lower percentages of genotype 3b infections were noted with increasing age groups. Genotype 1b was not found in patients less than 50 years while its prevalence was 8.33% in patients of age more than 50 years. Prevalence of genotype 1a was lower (2.17%) in the age group less than 30 and also in the age group 41-50 (1%). No patient of genotype 1b was found in the age group above 50. A study conducted in China in 2009 described the recent distribution and evolution of HCV genotypes in southwest china. It was shown that the relative prevalence of genotypes 3 and 6 has increased significantly and that of genotypes 1b and 2a has declined significantly among young patients. Previous finnding in European countries showed a correlation between genotype 3a with a younger age group and genotype 1b with an older age group [23] This shift in distribution was associated with changes in the modes of HCV acquisition in China. [19],[25]

Our patient's data showed no significant differences in genotype distribution in relation to gender. Various genotypes were equally distributed in relation to gender.

HCV plasma viral load, also called HCV viremia, is expressed in copies per milliliter or viral particle equivalents per milliliter. The methods used to measure HCV viremia are based on two principles: the quantitation of viral proteins (measure of antigenemia using monoclonal antibodies) and the quantitation of viral nucleic acid using PCR after reverse transcription (RT-PCR), or signal amplification (branched DNA). [23] In our study we used PCR to quantify HCV viremia. In our study, mean HCV RNA were lower in patients infected with HCV type 3 than in patients infected with HCV type 1 (2.51 × 10 6 for genotype 3b, 2.65 × 10 6 for genotype 3a vs. 2.75 × 10 6 for genotype 1a, 3.9 × 10 6 for genotype 1b). The result is totally in agreement with another study conducted in Agha Khan university by Moatter et al. in 2002 where they documented mean HCV RNA levels were lower in patients infected with HCV type 3 than in patients infected with HCV type 1 (8.63 MEq/mL vs. 37 MEq/mL; P < 0.001). [26] A study conducted in India showed viral loads of genotypes 1a and genotype 3 to be 1.9 × 10 5 and 1.6 × 10 5 respectively. Both of these figures were less than the values of our study. [21]


   Conclusion Top


There is an association between different types of genotypes and viral load. This is just an assumption. The current study does not prove it or justify it. Further studies should be carried out to determine the association of viral load with different genotypes so that sufficient data is available and can be used to determine the type and duration of therapy needed and predict disease outcome.

 
   References Top

1.Fact sheet N°164. Available from: http://www.who.int/topics/hepatitis/factsheets/en/index.html. [2011 Jun 28/05/2013].  Back to cited text no. 1
    
2.National Institutes of Health. National Institutes of Health Consensus Development Conference Statement: Management of hepatitis C:2002 - June 10-12, 2002. Hepatology 2002;36:S3-20.  Back to cited text no. 2
    
3.Yu ML, Chuang WL. Treatment of chronic hepatitis C in Asia: When East meets West. J Gastroenterol Hepatol 2009;24:336-45.  Back to cited text no. 3
    
4.Ijaz B, Ahmad W, Javed FT, Gull S, Sarwar MT, Kausar H, et al. Association of laboratory parameters with viral factors in patients with hepatitis C. Virol J 2011;8:361.  Back to cited text no. 4
    
5.Giannini C, Bréchot C. Hepatitis C virus biology. Cell Death Differ 2003;10 Suppl 1:S27-38.  Back to cited text no. 5
    
6.Idrees M, Ur Rehman I, Manzoor S, Akbar H, Butt S, Afzal S, et al. Evaluation of three different hepatitis C virus typing methods for detection of mixed-genotype infections. J Dig Dis 2011;12: 199-203.  Back to cited text no. 6
    
7.Berry V, Arora R, Paul P. Hepatitis C - Clinical outcome and diagnosis. J K Sci 2005;7:129-32.  Back to cited text no. 7
    
8.Abid K, Quadri R, Veuthey AL, Hadengue A, Negro F. A novel hepatitis C virus (HCV) subtype from Somalia and its classification into HCV clade 3. J Gen Virol 2000;81:1485-93.  Back to cited text no. 8
    
9.Ndjomou J, Pybus OG, Matz B. Phylogenetic analysis of hepatitis C virus isolates indicates a unique pattern of endemic infection in Cameroon. J Gen Virol 2003;84:2333-41.  Back to cited text no. 9
    
10.Simmonds P. Genetic diversity and evolution of hepatitis C virus - 15 years on. J Gen Virol 2004;85:3173-88.  Back to cited text no. 10
    
11.Kumar D, Farrell GC, Fung C, George J. Hepatitis C virus genotype 3 is cytopathic to hepatocytes: Reversal of hepatic steatosis after sustained therapeutic response. Hepatology 2002;36:1266-72.  Back to cited text no. 11
    
12.Manns MP, McHutchison JG, Gordon SC, Rustgi VK, Shiffman M, Reindollar R, et al. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: A randomised trial. Lancet 2001;358:958-65.  Back to cited text no. 12
    
13.Martell M, Esteban JI, Quer J, Genescà J, Weiner A, Esteban R, et al. Hepatitis C virus (HCV) circulates as a population of different but closely related genomes: Quasispecies nature of HCV genome distribution. J Virol 1992;66:3225-9.  Back to cited text no. 13
    
14.Jarvis LM, Ludlam CA, Simmonds P. Hepatitis C virus genotypes in multi-transfused individuals. Haemophilia 1995;1:3-7.  Back to cited text no. 14
    
15.Forns X, Maluenda MD, López-Labrador FX, Ampurdanès S, Olmedo E, Costa J, et al. Comparative study of three methods for genotyping hepatitis C virus strains in samples from Spanish patients. J Clin Microbiol 1996;34:2516-21.  Back to cited text no. 15
    
16.Lau JY, Mizokami M, Kolberg JA, Davis GL, Prescott LE, Ohno T, et al. Application of six hepatitis C virus genotyping systems to sera from chronic hepatitis C patients in the United States. J Infect Dis 1995;171:281-9.  Back to cited text no. 16
    
17.Ohno O, Mizokami M, Wu RR, Saleh MG, Ohba K, Orito E, et al. New hepatitis C virus (HCV) genotyping system that allows for identification of HCV genotypes 1a, 1b, 2a, 2b, 3a, 3b, 4, 5a, and 6a. J Clin Microbiol 1997;35:201-7.  Back to cited text no. 17
    
18.Conte D, Fraquelli M, Prati D, Colucci A, Minola E. Prevalence and clinical course of chronic hepatitis C virus (HCV) infection and rate of HCV vertical transmission in a cohort of 15,250 pregnant women. Hepatology 2000;31:751-5.  Back to cited text no. 18
    
19.Dusheiko G, Schmilovitz-Weiss H, Brown D, McOmish F, Yap PL, Sherlock S, et al. Hepatitis C virus genotypes: An investigation of type-specific differences in geographic origin and disease. Hepatology 1994;19:13-8.  Back to cited text no. 19
    
20.Chakravarti A, Dogra G, Verma V, Srivastava AP. Distribution pattern of HCV genotypes & its association with viral load. Indian J Med Res 2011;133:326-31.  Back to cited text no. 20
[PUBMED]  Medknow Journal  
21.Rivas-Estilla AM, Cordero-Pérez P, Trujillo-Murillo Kdel C, Ramos-Jiménez J, Chen-López C, Garza-Rodríguez Mde L, et al. Genotyping of hepatitis C virus (HCV) in infected patients from Northeast Mexico. Ann Hepatol 2008;7:144-7.  Back to cited text no. 21
    
22.López-Labrador FX, Ampurdanés S, Forns X, Castells A, Sáiz JC, Costa J, et al. Hepatitis C virus (HCV) genotypes in Spanish patients with HCV infection: Relationship between HCV genotype 1b, cirrhosis and hepatocellular carcinoma. J Hepatol 1997;27:959-65.  Back to cited text no. 22
    
23.Pouillot R, Lachenal G, Pybus OG, Rousset D, Njouom R. Variable epidemic histories of hepatitis C virus genotype 2 infection in West Africa and Cameroon. Infect Genet Evol 2008;8:676-81.  Back to cited text no. 23
    
24.Zhou Y, Wang X, Mao Q, Fan Y, Zhu Y, Zhang X, et al. Changes in modes of hepatitis C infection acquisition and genotypes in southwest China. J Clin Virol 2009;46:230-3.  Back to cited text no. 24
    
25.Moatter T, Hussainy AS, Hamid S, Ahmad Z, Siddiqui S. Comparative analysis of viral titers and histologic features of Pakistani patients infected with hepatitis C virus type 3. Int J Infect Dis 2002;6:272-6.  Back to cited text no. 25
    
26.Castelain S, Zawadzki P, Khorsi H, Darchis JP, Capron D, Sueur JM, et al. Comparison of hepatitis C virus serotyping and genotyping in French patients. Clin Diagn Virol 1997;7:159-65.  Back to cited text no. 26
    

Top
Correspondence Address:
Usman Hassan
House No. 356, Street 4, Phase 4, Gulraiz Housing Scheme, Rawalpindi
Pakistan
Login to access the Email id

Source of Support: Supported By Hospital., Conflict of Interest: None


DOI: 10.4103/0377-4929.125319

Rights and Permissions


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2]



 

Top
 
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Email Alert *
    Add to My List *
* Registration required (free)  


    Abstract
   Introduction
    Materials and Me...
   Results
   Disscussion
   Conclusion
    References
    Article Figures
    Article Tables

 Article Access Statistics
    Viewed1999    
    Printed49    
    Emailed1    
    PDF Downloaded117    
    Comments [Add]    

Recommend this journal