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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~ Conclusion
 ~  References
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  Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 36  |  Issue : 4  |  Page : 532-536
 

A study on the circulating genotypes of hepatitis C virus in a tertiary care hospital in Central Kerala


1 Department of Microbiology, PRS Hospital, Thiruvananthapuram, Kerala, India
2 Department of Microbiology, Pushpagiri Institute of Medical Sciences, Thiruvalla, Kerala, India
3 Department of Microbiology, Amrita Institute of Medical Sciences, Kochi, Kerala, India

Date of Web Publication18-Mar-2019

Correspondence Address:
Dr. Seema Oommen
Department of Microbiology, Pushpagiri Institute of Medical Sciences and Research Centre, Thiruvalla - 689 101, Kerala
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_18_239

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 ~ Abstract 

Background: Hepatitis C is an emerging infection in India, which is known to progresses to liver cirrhosis and hepatocellular carcinoma. The persistence of chronic HCV infection is due to the existence of various genotypes and its various subtypes. There are seven different genotypes of HCV. These genotypes vary in their severity to cause infections as well as their response to treatment. Aim: This study aims at identifying the predominant genotypes of HCV in a population of patients presenting in a tertiary care center in Central Kerala. Settings and Design: This study was conducted at a tertiary care hospital and medical college, located in Central Kerala in the Department of Microbiology from January 2014 to June 2015.The sample size was 600 and a high risk group of patients attending the gastroenterology department, deaddiction centre and health care workers were screened. Materials and Methods: Serum samples were subjected to EIA, either rapid card or ELISA. Serum samples that were positive for HCV antibodies were confirmed by PCR. Twenty seven samples were positive for HCV antibodies by ELISA/rapid card, out of which 16 were confirmed by PCR. These 16 samples were subjected to gene sequencing to identify the genotype. Results: The prevalent genotypes isolated in this study was genotype 1, 3 and 4. Genotype 1 and 3 was predominantly seen transmitted by blood transfusions and multiple hemodialysis. The variability in laboratory parameters like SGOT and SGPT and its ratio with each genotype was also evaluated. Conclusion: To conclude, the occurrence of genotype 4 at a similar level to genotype 1 shows diffusion of new genotype in Kerala.


Keywords: Genotypes, hepatitis C virus, high-risk group


How to cite this article:
John M, Oommen S, Jagan OA, George S, Pillai S. A study on the circulating genotypes of hepatitis C virus in a tertiary care hospital in Central Kerala. Indian J Med Microbiol 2018;36:532-6

How to cite this URL:
John M, Oommen S, Jagan OA, George S, Pillai S. A study on the circulating genotypes of hepatitis C virus in a tertiary care hospital in Central Kerala. Indian J Med Microbiol [serial online] 2018 [cited 2019 May 21];36:532-6. Available from: http://www.ijmm.org/text.asp?2018/36/4/532/254387



 ~ Introduction Top


Hepatitis C is a positive-stranded RNA virus, which belongs to the family Flaviviridae. The global prevalence of hepatitis C is 177 million. It is estimated that around 2.5% of the world is infected with HCV.[1] In India, it is estimated that around 12.5 million people are suffering from chronic hepatitis.[2] Hepatitis C is an emerging infection in India. The risk factors are poor screening of blood donors, aseptic therapeutic practices, reuse of needles and professional blood donations. Prospective studies have shown that 80% of cases of acute hepatitis C progress to chronic infection. The persistence of chronic hepatitis C virus (HCV) infection is due to the existence of various genotypes and its various subtypes. About 10%–20% of these acute infections will develop complications of chronic liver disease, such as liver cirrhosis within two to three decades of onset, and 1%–5% will develop hepatocellular carcinoma.[3]

A peculiar characteristic of HCV genome is its genetic heterogeneity due to the accumulation of mutations during viral replications. This high mutation rate is due to RNA-dependent RNA polymerase which lacks proofreading activity.[3] Like other RNA viruses, HCV circulates in an infected person as a population of 'closely related swarm of genetically distinct species,' called as quasispecies.[3] The viral population is composed of a master sequence i.e., dominant sequence and few other viral sequences which vary from the master sequence. Variations predominantly occur in the envelope protein E2, at the amino acid terminal. This region is called as the hyper-variable region; this has been sequenced to characterize the different genotypes in infected individuals. The high mutation rates in the viral population result in escape mutants, alteration in cell tropism and change in the virulence pattern and the development of resistance to viral drugs.[3] Changes in the quasispecies result in emergence of new strains and also fluctuations in the existing viral populations. The chronicity of HCV infection is due to viral persistence, which is, in turn, due to the existence of quasispecies of HCV. The reinfection in HCV patients is also explained by the existence of quasispecies, which allows evasion of host immune response.

Phylogenetic evaluation of HCV sequence recovered from various geographical areas suggests that there are seven different genotypes of HCV. These genotypes have only 50% identity with each other in their nucleotide sequence. Each genotype is further divided into various subtypes. Each subtype shares 75%–85% nucleotide sequence identity in their core E1 and NS5B regions of the genome.[3] Genotypes 1 and 2 are prevalent all over the world, but their relative prevalence varies from one geographical region to another. The common genotypes found in western countries are 1a, 1b, 2a, 2b, 3a, 4a and 6a.[4] In India, genotype 3 predominates in north, west and east, while genotype 1 predominates in the south of India.[5] This study aims at identifying the predominant genotypes of HCV in a population of patients presenting in a tertiary care centre in Central Kerala.


 ~ Materials and Methods Top


Patient selection and sample collection

This study was conducted at a tertiary care hospital and medical college, located in Central Kerala in the Department of Microbiology from January 2014 to June 2015. Human ethical clearance for this study was obtained from the Institution Ethics Committee before the collection of samples and a detailed pro forma was collected from patients included in the study. The sample size was calculated to be 600, using the confidence level of 99% and tolerance error of 1% after considering the prevalence of HCV from the previous studies. Patients visiting the hospital, whose sample was sent to the Microbiology Department, were included as high-risk group. High-risk group included current or former injecting drug users; people on long-term haemodialysis; health care workers; patients who have received multiple blood transfusions; people living with human immunodeficiency virus and/or hepatitis B virus; people with abnormal liver tests; alcoholics and patients attending sexually transmitted diseases clinics. Antenatal cases and children <16 years have been excluded from the study.

Serological assessment

Serum samples were subjected to EIA, either rapid card or ELISA. Patients attending the deaddiction centre were screened by rapid card test HCV TRI-DOT (by J. Mitra and Co., Pvt., Ltd.). Other samples were subjected to HCV Microlisa third-generation ELISA (by J. Mitra and Co., Pvt., Ltd.) for the detection of HCV antibodies. Serum samples that were positive for HCV antibodies were stored at −70° for further confirmation by polymerase chain reaction (PCR).

Molecular assessment, sequencing and phylogenetic analysis

Samples in which HCV antibodies were detected were subjected to HCV RNA extraction. QIAamp Viral RNA Mini Kit (Qiagen Inc., Germany) was used for the detection of HCV RNA (Panigrahi et al., 1994). PCR products were run on 2% agarose gel, stained with SYBR Safe DNA gel stain (Invitrogen by Life Technologies, USA) and analysed.

Amplified products of HCV were sequenced and analysed. HCV genotypes were subjected to phylogenetic analysis by using MEGA version 7.0.26 (which is licensed as proprietary freeware. The orginal authors are Masatoshi Nei, Sudhir Kumar, Koichiro Tamura from Pennsylvania State University). Phylogenetic analysis was performed using maximum likelihood tree method with a 1000 bootstrap replications, as seen in [Figure 1]. Nucleotide sequences analysed in this study have been deposited in the GeneBank accession numbers: KT833131 to KT833145.
Figure 1: Phylogenetic dendrogram of hepatitis C virus 5' untranslated region sequences isolated from humans constructed using the maximum likelihood method based on the Tamura-Nei model with 1000 bootstrap resampling. The strains of the present study are highlighted

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 ~ Results Top


Of the 600 samples screened, 27 samples were positive for HCV antibodies by ELISA/rapid card. These 27 samples were subjected to PCR, of which 16 were positive for HCV RNA [Table 1]. These 16 cases had active HCV infection, with the presence of HCV RNA in the peripheral blood. Genotypes of 16 samples were identified and phylogenetic tree was constructed with 15 sequences to assess HCV genotype distribution. In this study, phylogenetic analysis of HCV sequences showed three distinct clusters corresponding to HCV genotype 1 (n = 6, 37.50%), genotype 3 (n = 5, 31.25%) and genotype 4 (n = 5, 31.25%). The risk factors associated with each genotype were analysed [Table 1]. The risk factors commonly associated with each genotype were different. Genotypes 1 and 3 were predominantly seen transmitted by blood transfusions and multiple haemodialysis. The variability in laboratory parameters such as serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) and its ratio with each genotype was also evaluated to assess the prognosis of the patient [Table 1].
Table 1: Genotype distribution of hepatitis C virus detected with risk factors and laboratory parameters

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 ~ Discussion Top


Molecular studies have unveiled the existence of seven genotypes. These genotypes have found to show geographic differences. These genotypes also differ in their pathogenicity and their potential to cause hepatocellular cancers. These seven genotypes show 50% differences in their nucleotide sequences.[3]

Genotype 1 is the most common genotype worldwide; 2% of the world is infected with this genotype.[5] It is also the most predominant genotype in Central Asia.[6] Genotype 1 is further subtyped into 1a and 1b; in America, 60%–70% of hepatitis C belongs to either genotype 1a or 1b. In Japan, 1b is responsible for 73% of HCV infections. Genotype 1a is predominantly seen in intravenous drug users (IVDUs).[7] There were no IVDUs in this study; genotype 1b is more prevalent amongst the older population throughout Europe and Asia and is associated with blood transfusion.[8] In this study, two of three cases of genotype 1b had a history of blood transfusion. Medical treatment with unsterilised needles, large-scale vaccination programme and blood transfusions has resulted in spread of genotype 1b infections between 1940 and 1950. IVDU has resulted in the dissemination of genotype 1b from 1960 onward.[5] Genotype 1b is predominantly associated with hepatocellular carcinoma.[3] Genotype 1 requires a prolonged treatment with pegylated ribavirin and interferon for 48 weeks to attain sustained viral response while compared to genotypes 2 and 3 which requires only 24 weeks of treatment.[9] Newer drugs such as sofosbuvir and ledipasvir have reduced the duration of treatment to 12 weeks without ribavirin.[10] Three of our cases were of genotype 1a and 1b each (6/16), suggesting that genotype 1 and its subtypes are most common in this part of Kerala accounting for 37.5% of cases in this study population. The risk factors most commonly associated with genotype 1 in our study were a history of blood transfusions and multiple haemodialysis. The five of six cases (83.3%) with genotype 1 underwent both of these interventions. History of surgical history was seen in four cases (66.7%) and a history of renal transplant seen in three cases (50%). This falls in line with other studies which suggest that medical treatment with unsterilised needles, large-scale vaccination programme and blood transfusions has resulted in spread of genotype 1.[5]

Genotype 2 accounts for 9.1% of the cases worldwide.[6] This genotype is most predominant in West Africa.[3] Subtypes 2a and 2b are commonly found in North America, Europe and Japan. Subtype 2c is commonly found in Northern Italy.[11] Genotype 2a and 2b are also prevalent amongst the older age group in Europe and Asia. In 1940–1950, genotype 2 was spread due to unhygienic therapeutic practices, large-scale vaccinations and blood transfusion in the past. After 1960, the spread of genotype 2 is associated with IVDU.[12] Genotype 2 is one of the easiest genotypes to treat and is not aggressive as genotype 1; it requires treatment with pegylated interferon and ribavirin for 24 weeks.[9] We did not encounter this genotype in our study.

Genotype 3 is the second most common genotype, which accounts for 30% of the worlds' infection.[6] Genotype type 3 commonly occurs in Indian subcontinent and Southeast Asia.[13] In Northern Europe and the USA, genotype 3a has been found common with IVDU.[14] Genotype 3 is less virulent and requires treatment with pegylated interferon and ribavirin for 24 weeks.[9] It was found that 31% (5/16) of cases were tagged with genotype. When the subtypes of genotype 3 were analysed, it was found that there were two cases of 3a and 3b each. One genotype was not subtyped. The risk factor commonly associated with genotype 3 in our study was multiple haemodialysis seen in two cases (40%), blood transfusion seen in one case (20%) and history of multiple sexual partners seen in one case (20%). In Northern Europe and the USA, genotype 3a has been found common with IVDU.[14]

Genotype 4 is prevalent in North Africa and Middle Eastern countries.[15] More than 90% of the cases in Egypt belong to this genotype.[6] This genotype is associated with intermediate drug resistance to pegylated interferon and ribavirin and requires a treatment duration of 48 weeks.[10] Genotype 4a was found in four cases and genotype 4 in one case. Overall, the percentage of genotype 4 was 31% (5/16). The risk factors associated with genotype 4 in our study were a history of blood transfusion and a history of multiple haemodialysis. In a study from Egypt, where more than 90% of the infected belongs to genotype 4, the common risk factor isolated was improper therapeutic practice.[15] As genotype 4 was previously seen predominantly in the Middle Eastern countries, a rise in genotype 4 in our locality is alarming. It could be explained due to the increased migration of the local population to the oil rich Middle East countries for employment.[16] Amongst the five cases of genotype 4, none had a history of working in Middle Eastern countries. In an era globalised world, as the migration continues, an increasing number of individuals get exposed with these new HCV genotypes. The entry of HCV genotype 4 to the Indian population may be result of infection from these returning emigrants. Genotype 4 has not been reported as yet from Kerala, and the presence of this genotype is significant in terms of disease management with patients requiring 48 weeks therapy.

Genotypes 5, 6 and 7 were not isolated in our study. Genotype 5 is predominantly confined to South Africa.[17] Around 0.8% of the HCV infections worldwide is caused by genotype 5a.[10] A recent study conducted in Vellore, India, suggested that genotype 5 shows poor response to treatment with pegylated interferon and ribavirin.[2] Genotype 6 accounts for 5.4% of the world's HCV infection and it remains confined to Southeast Asia.[10] Genotype 6 is prevalent in Hongkong.[18] In a recent study from Vellore, India, few cases of genotype 6 have been isolated. This study also states poorer response to treatment with pegylated interferon and ribavirin in genotype 6.[15] Very few reports are available on this genotype 7. It has been isolated from few Vietnamese patients.[10]

In chronic viral illness, the ratio of SGOT/SGPT is predictive of long-term complications such as cirrhosis and fibrosis. Usually in chronic viral hepatitis, SGOT/SGPT is less than 1. But as the disease progress, and cirrhosis and fibrosis sets in the the ratio of SGOT/SGPT starts rising above 1. Hence, in chronic hepatitis C infection, raised SGOT/SGPT is suggestive of fibrosis, rather than necroinflammatory changes.[19] A raised SGOT/SGPT over 1.09 is predictive of the progression of chronic viral hepatitis to cirrhosis.[20] It was observed that all patients with genotype 1 had the ratio of SGOT/SGPT more than 1 (normal range is 0.5–0.7).[21] A raised SGOT/SGPT over 1.09 is predictive of the progression of chronic viral hepatitis to cirrhosis.[20] An increased SGOT/SGPT of over 1.16 is predictive of poor survival. It was observed that five of six 83% of patients infected with genotype 1 had SGOT/SGPT of more than 1.16. As it has already been proven that genotype 1 is more aggressive and requires prolonged treatment, our study affirms to the same. In our study, all patients with genotype 3 had the ratio of SGOT/SGPT <1, with P value 0.038, which is statistically significant. This is suggestive of good prognosis in these patients. These patients with genotype 3 have a better response to treatment than compared to patients with genotype 1.[10] In patients infected with genotype 4, the ratio of SGOT/SGPT, which is a predictor of the prognosis and outcome in the chronically infected patients, was above 1.16 in two cases (40%), which is also suggestive of fatal outcome and delayed response to treatment.


 ~ Conclusion Top


In our study, the occurrence of genotype 4 at a similar level to genotype 1 shows diffusion of new genotype in Kerala. Introduction of new HCV genotypes into the population may result in emergence of mixed genotypes or replacement of newly emerging genotypes. This results in recombinant viruses and evolution of new drug resistant genotype. In our study, we were unable to identify the subtypes for two samples; this suggests genetic diversity in genotype or a technical typing error. Genetic diversity of HCV will influence the disease progression, clinical and epidemiological management of infection. Our study reinforces the need for continuous monitoring to analysis divergence and heterogeneity of HCV viruses.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
 ~ References Top

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Petruzziello A, Marigliano S, Loquercio G, Cozzolino A, Cacciapuoti C. Global epidemiology of hepatitis C virus infection: An up-date of the distribution and circulation of hepatitis C virus genotypes. World J Gastroenterol 2016;22:7824-40.  Back to cited text no. 1
    
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Abdulkarim AS, Zein NN, Germer JJ, Kolbert CP, Kabbani L, Krajnik KL, et al. Hepatitis C virus genotypes and hepatitis G virus in hemodialysispatients from Syria: Identification of two novel hepatitis C virus subtypes. Am J Trop Med Hyg 1998;59:571-6.  Back to cited text no. 18
    
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21.
Fortunato G, Castaldo G, Oriani G, Cerini R, Intrieri M, Molinaro E, et al. Multivariate discriminant function based on six biochemical markers in blood can predict the cirrhotic evolution of chronic hepatitis. Clin Chem 2001;47:1696-700.  Back to cited text no. 21
    


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