Hepatitis c virus- seroprevalence, genotypic identification, and comparative assessment of diagnostic techniques in a tertiary care hospital in Kanchipuram.

Rahman A et al | DOI: 10.65188/nurexus.1005

Nurexus | Journal of MedVerse Research and Practice | Volume 2 | Issue – 1 | June 2024

Page 13

Journal of MedVerse Research & Practice

nurexus.com

Hepatitis C virus- seroprevalence, genotypic identification, and

comparative assessment of diagnostic techniques in a tertiary care

hospital in Kanchipuram.

Dr. Abdul Rahman

Department of Microbiology,

Mahatma Gandhi Medical College and Research Institute, Pondicherry, India

Email ID: abdulrahman@gmail.com

Submission Date: 19.05.2024

Accepted Date: 16.06.2024

Published Date: 30.06.2024

DOI: 10.65188/nurexus.1005

article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits

unrestricted use, distribution, and reproduction in any medium, provided the original author(s) and source are credited

Abstract

Introduction: Hepatitis C virus (HCV) infection constitutes a significant global public health concern. Given that

numerous infected individuals remain asymptomatic, primary diagnostic modalities encompass Rapid Diagnostic

Tests, Enzyme-Linked Immunosorbent Assay, Chemiluminescent Immunoassay, and real-time Polymerase Chain

Reaction for HCV RNA detection. This investigation sought to evaluate HCV seroprevalence utilizing diverse

techniques, including real-time PCR, CLIA, third-generation ELISA, and RDTs, in conjunction with identifying

prevalent genotypes.

Materials and methods: The Virology Section of the Department of Microbiology at the Mahatma Gandhi

Medical College and Research Institute, Pondicherry, India, conducted an observational cross-sectional study

from September 2022 to November 2023, involving 700 samples from individuals suspected of HCV. After

processing, samples were tested via RDTs, ELISA, CLIA, and real-time PCR according to established protocols,

with PCR employed to identify genetic material in positive cases.

Results: Among the 700 samples, anti-HCV antibodies were detected in 9 (1.28%) via RDTs, 9 (1.28%) via

ELISA, and 11 (1.5%) via CLIA, and HCV RNA was identified in 11 (1.5%) by RT-PCR, with genotype 3b

constituting 55% of positive results, followed by 1a type at 36%.

Conclusion: Some samples initially deemed negative by other assays tested positive by RT-PCR, reinforcing its

status as the gold standard diagnostic method. The significance of genotyping in diagnosis, treatment planning,

and monitoring is underscored by timely interventions capable of preventing complications.

Keywords: Hepatitis C, seroprevalence, genotype, diagnosis.

Introduction

The Hepatitis C virus (HCV) belongs to the Flaviviridae family and is characterized by its single-

stranded RNA structure [1]. The HCV virus was first identified in the United States in 1989 and causes

hepatitis in over three to four million people annually. According to estimates from the World Health

Organization (WHO), approximately 3% of the global population, or around 210 million people, are

chronically infected with HCV [2]. HCV transmission primarily occurs among intravenous drug users,

hemodialysis patients, and individuals receiving contaminated blood products. In addition, nosocomial

transmission plays a significant role in the spread of the virus. Each year, more than 350,000 deaths are

Rahman A et al | DOI: 10.65188/nurexus.1005

Nurexus | Journal of MedVerse Research and Practice | Volume 2 | Issue – 1 | June 2024

Page 14

attributed to HCV infection, with 25% resulting from hepatocellular carcinoma and 27% from cirrhosis

[3].

HCV remains a major global health concern due to its high chronicity rate and the absence of an

effective vaccine. In India, WHO estimates suggest that 10–24 million individuals are actively infected

with HCV, with a seroprevalence ranging from 0.09% to 2.02% among healthy populations [4]. The

virus exhibits remarkable genetic heterogeneity, comprising multiple subtypes and at least six major

genotypes, designated 1 through 6. Genotype 1a is distributed worldwide, while genotype 3

predominates in India [5].

With advancements in molecular diagnostics, the use of techniques such as real-time polymerase chain

reaction (PCR), chemiluminescent immunoassay (CLIA), third-generation enzyme-linked

immunosorbent assay (ELISA), and rapid diagnostic tests (RDTs) has become essential for detecting,

quantifying, and characterizing HCV infections. The present study aims to determine the most

prevalent HCV genotype and to estimate the seroprevalence of HCV within the target population using

these advanced diagnostic methods.

Materials & Methods

The Microbiology Department at Mahatma Gandhi Medical College and Research Institute, located in

Pondicherry, India, conducted an observational cross-sectional study from September 2022 to

November 2023. Blood specimens were obtained from 700 patients who presented to the outpatient

department for hepatitis evaluation within the research protocol.

Informed consent was procured from all participants following ethical committee approval. Individuals

of all ages and sexes, including those diagnosed with chronic hepatitis, patients undergoing

hemodialysis, and those with previous blood transfusions, needle stick injuries, or referrals from various

departments, were deemed eligible for sample collection. Those who declined to participate in the study

were excluded. Participants were thoroughly informed about the study procedures, and their

demographic and medical information was documented in detail.

A comprehensive survey gathered data on various symptoms, including jaundice, decreased appetite,

elevated body temperature, weight loss, and coexisting health conditions (such as diabetes, cancer, HIV,

HBV, and HAV). The study also evaluated potential risk factors among the participants, including

previous blood transfusions, kidney dialysis, accidental needle injuries, organ transplantation, surgical

procedures, tattoos, alcohol consumption, intravenous drug use, and multiple sexual partners.

After obtaining informed consent and following sterile techniques, 5 mL of blood was collected and

processed according to established protocols. The blood samples underwent analysis using CLIA (Matrix

Lab Pvt, Ltd.), ELISA (QUALPRO Pvt Ltd.), and RDTs (HCV Tridot, Diagnostic Enterprises, Pvt

Ltd.). Samples that tested positive were further verified using RT-PCR (Helini Biomolecules Pvt. Ltd).

The study obtained approval from the Institutional Ethics Committee of Mahatma Gandhi Medical

College and Research Institute, Pondicherry, India (Ref No: MGMCRI/IEC/2022/91837). A detailed

Participant Information Sheet was provided to all participants, and written informed consent was

obtained prior to their participation in the study.

Rahman A et al | DOI: 10.65188/nurexus.1005

Nurexus | Journal of MedVerse Research and Practice | Volume 2 | Issue – 1 | June 2024

Page 15

Results

Table 1: Sero-positivity shown in different diagnostic methods and the sample used

Study

subjects

No of the

samples

tested

RDT(%)

generation

ELISA (%)

CLIA(%)

PCR

Dialysis

469

5(55.5%)

6(54.5%)

Medicine

Surgery

101

2(22.3%)

2(18.2%)

Orthopedics

1(9.1%)

Obstetrics &

gynecology

1(11.1)

1(11.1%)

1(9.1%)

Blood bank

1(11,1%)

1(11.1%)

1(9.1%)

All 700 samples included in this study underwent ELISA, CLIA, and RDT testing. Eleven (1.5%)

samples tested positive for CLIA, and nine (1.2%) tested positive for RDT and ELISA. These 11

samples were used to detect the genes using HCV RNA RT-PCR. Six (54.5%) of the 11 seropositive

cases came from the dialysis department, followed by the surgery, orthopedics, and obstetrics and

gynecology departments.

Table 2: Age distribution of HCV-positive subjects (N=11)

Table 2 : Shows that the maximum age group was between 30 and 40 years (36.4%). This

was followed by 40-50 years (27.2%), 50-60 years (27.2%), and > 60 years of age (9.2%).

None of the patients aged < 30 years showed HCV seropositivity.

Age group

Frequency (%)

30-40

4(36.4%)

40-50

3(27.2%)

50-60

3(27.2%)

>60

1(9.2%)

Rahman A et al | DOI: 10.65188/nurexus.1005

Nurexus | Journal of MedVerse Research and Practice | Volume 2 | Issue – 1 | June 2024

Page 16

36%

56%

GENOTYPE 1

GENOTYPE 2

GENOTYPE 3

22.3%

72.7%

Male Female

Figure 1: Gender distribution

The above pie chart shows that HCV was positive in more males, with a proportion of 72.7%,

which is more than two-thirds of the cases that have shown seropositivity for HCV.

Table 3:Risk factors among HCV-positive study subjects (N=11)

Risk factors

Frequency (%)

Tattoo

4(36.4%)

Surgery

2(18.1%)

Blood transfusion

2(18.1%)

Dialysis

3(27.2%)

Injecting drug users

2(18.1%)

Sexual exposure

2(18.1%)

As indicated in Table 4, tattooing (36.4%) was the primary risk factor among HCV-positive

subjects. This was followed by dialysis (27.2%).

Fig -2: Distribution of different HCV genotypes among study subjects

RT-PCR was used to identify the genotypes of all the 11 positive cases. Fig -2 displays the

Rahman A et al | DOI: 10.65188/nurexus.1005

Nurexus | Journal of MedVerse Research and Practice | Volume 2 | Issue – 1 | June 2024

Page 17

genotypes that were found to be most common in our study: 3b (56%) and 1a (33%).In this

study, the prevalent genotype was 3b (55%), followed by 1a (36%), and 2 (9%).

Discussion

Studies conducted across India have demonstrated considerable regional variation in Hepatitis C

Virus (HCV) seroprevalence, with reported rates of 0.22% in South India, 0.3% in Western India,

1.8% in Central India, and 1.9% in Northern India. In developing countries, unsafe therapeutic

injections and blood transfusions are considered the major routes of HCV transmission, as reported

by Rajkumar et al., Ramya et al., and Badge et al. [4–6]. In the present study, the observed HCV

seroprevalence was 1.5%, which lies between the higher prevalence reported by Sivagamasundari et

al. and the lower prevalence documented by Mathur et al. [7,8]. A higher prevalence was noted

among males, which is consistent with findings reported by Verma et al., Rajasekaran et al., and

Mahajan et al. [9,10,3]. However, this contrasts with observations by Roy et al., who reported a

higher prevalence among females. The most affected age group in the present study was 30–40 years,

whereas Roy et al. reported the highest prevalence in the 50–60-year age group [11].

During the study period, the majority of samples were obtained from patients attending the dialysis

unit. Tattooing, which is a common cultural practice, emerged as a significant risk factor among the

study population. In contrast, Rajkumar et al. identified injecting drug users as the predominant high-

risk group [4]. Polymerase chain reaction (PCR) detected HCV RNA in eleven samples that were

negative by rapid diagnostic tests (RDTs), supporting previous reports that PCR can detect HCV

RNA during the early acute phase of infection, even before hepatic enzyme elevation occurs, as noted

by Ramya et al. [5]. Additionally, PCR identified two positive cases among seronegative samples,

which may be explained by the limited sensitivity of ELISA during the first three weeks of infection,

potentially missing early acute HCV cases.

The widespread availability and ease of use of anti-HCV antibody tests in resource-limited settings

such as India have led to the acceptance of RDTs as practical screening tools for HCV infection.

However, molecular diagnostic techniques remain essential for confirmatory diagnosis and for

monitoring treatment outcomes. These techniques were instrumental in the initial identification of

HCV, as the virus could not be cultivated easily in laboratory settings, as described by Ramya et al.

and Sivagamasundari et al. [6,7]. Nucleic acid testing (NAT), including CLIA-based assays, is

considered the gold standard for detecting active HCV replication. NAT is particularly valuable in

diagnosing acute HCV infection, as it can detect viral RNA as early as one week following exposure

and several weeks before seroconversion, as reported by Mathur et al. [8].

Genotypic analysis in the present study revealed genotype 3b (56%) and genotype 1a (33%) as the

predominant circulating genotypes. Similar genotype distribution patterns have been reported by

Thanjavur et al. [1]. In contrast, Appalaraju et al. reported genotype 4 as the most prevalent genotype

in South India, followed by genotype 3 [12]. Overall, genotype 3 remains the most common genotype

in India, accounting for approximately 63.85% of cases, while genotype 1 accounts for 25.72% [12].

Given that the incidence of HCV infection is nearly four times higher than that of HIV infection,

there is an urgent need for enhanced public awareness and preventive strategies. Although HCV

genotype influences therapeutic decision-making, recent advances have led to the development of

direct-acting antiviral agents targeting the NS5B region, which are effective against multiple

genotypes, as reported by Appalaraju et al. and Preethi et al. [12,13]. While genotyping is not

currently mandated in treatment guidelines, it remains crucial for epidemiological surveillance and

Rahman A et al | DOI: 10.65188/nurexus.1005

Nurexus | Journal of MedVerse Research and Practice | Volume 2 | Issue – 1 | June 2024

Page 18

vaccine development, as emphasized by Gowri et al. and Anoop Kumar et al. [14,15].

Conclusion

Tests for detecting HCV infection using serology are simple, reliable, widely available, and cost-

effective. However, these methods are not effective in identifying early-stage HCV infections. The

most accurate technique for diagnosing HCV infection remains HCV RNA RT-PCR. Additionally,

genotyping plays a vital role in managing HCV treatment and monitoring disease progression. Some

research has identified genetic variations that may be attributed to population movement or travel. As

a result, it is essential to implement rigorous infection control measures in both critical and non-

critical settings.

Conflict of Interest: Nil

Reference

1. Thanjavur N, Reddy H, Mekala CD, Rayi R. Hepatitis C virus genotype distribution and molecular

epidemiology in chronic patients with hemodialysis and the comparative evaluation of screening

methods. J App Biol Biotech. 2021;9(4):78-84. DOI: 10.7324/JABB.2021.9410

2. Subramanian Vennila, Epidemiology and Molecular characterization of Hepatitis C virus in

Tamilnadu (India)during the year 2013 International Journal of Advanced Research (2014), Volume

2, Issue 11, 935-943

3. Supriya Mahajan, Comparative evaluation of three rapid immunochromatographic test assays with

chemiluminescent microparticle immunoassay for the detection of hepatitis C virus

antibodyVirusDis. (July–September 2019) 30(3):373–379

4. Rajkumar Manojkumar Singh, Comparative Evaluation of Three Diagnostic Tools for the Detection

of Hepatitis C Virus among High-risk Individuals in a Tertiary Care Centre of Northeast India,

Journal of Clinical and Diagnostic Research. 2022 Jul, Vol-16(7): DC13-DC17

5. Dr.Ramya S R, Hepatitis C Virus- Epidemiology and Genotyping, IOSR Journal of Dental and

Medical Sciences (IOSR-JDMS) e-ISSN: 2279-0853, p-ISSN: 2279- 0861.Volume 14, Issue 3 Ver.

VIII (Mar. 2015), PP 29-34

6. Shalini Badge, S. R. Parate and Ganvir, G. B. 2021. Correlation and Path Analysis Studies for Yield

and Quality Traits in Tomato (Solanum lycopersicum L). Int.J.Curr.Microbiol.App.Sci. 10(06): 829-

837. Doi: https://doi.org/10.20546/ijcmas.2021.1006.088.

7. Sivagamasundari TG, HOD VM. A Prospective Study To Assess The Clinical Profile Of Patients With

Hepatitis B Visiting A Tertiary Care Centre. Journal of Pharmaceutical Negative Results. 2022 Dec

31:9834-43.

8. Mathur A, Goyal LK, Sharma MK, Gupta AK, Hooja N, Yadav RN. Seroprevalence of hepatitis C

virus among patients at a tertiary health care center in Rajasthan, India. Int J Adv Med 2020;7:683-6

9. Verma RK, Radhika, Singh DP, Sethi K, and Singh S: Molecular characterization of hepatitis C virus

in a Tertiary Care Hospital in Rural India of Western Uttar Pradesh. Int J Pharm Sci & Res 2023;

14(10): 4924-28. doi: 10.13040/IJPSR.0975- 8232.14(10).4924-28.

10. Rajasekaran C, Kalpanaraj D, Banu ST, Duraivel M. Seroprevalence of Hepatitis C Virus Infection

among Hemodialysis Patients in A Tertiary Care Hospital in South India. J Pure Appl Microbiol.

2023;17(1):371-379. doi 10.22207/JPAM.17.1.2..

11. Col Partha Roy (Retd), Prevalence and genotyping pattern of hepatitis C virus among patients on

maintenance hemodialysis at five centers in Pune, India.

12. https://doi.org/10.1016/j.mjafi.2018.08.001

13. Appalaraju B, Rizwana MM. A Retrospective Study to Determine the Genotypic Distribution of

Hepatitis-C from a Tertiary Care Hospital in South India. J Pure Appl Microbiol. 2023;17(3):1863-

Rahman A et al | DOI: 10.65188/nurexus.1005

Nurexus | Journal of MedVerse Research and Practice | Volume 2 | Issue – 1 | June 2024

Page 19

1870. doi: 10.22207/JPAM.17.3.51

14. M. Preethi, comparative study on Elisa, cilia and rapid diagnostic test in detecting HCV infection in

blood donor at a tertiary care center, Journal of Applied Pharmaceutical Research 11 (3); 2023: 48 –

15. V Gowri, The Current Seroprevalence of Hepatitis C Virus in a Tertiary Care Centre in Vellore,

Tamil Nadu, Indian Journal of Community Medicine/Vol 37/Issue 2/April 2012, DOI: 10.4103/0970-

0218.96110, www.ijcm.org.in.

16. Anoop Kumar Genotyping & diagnostic methods for hepatitis C virus: A need of low-resource

countries Indian J Med Res 147, May 2018, pp 445-455 DOI:10.4103/ijmr.IJMR_1850_16