European Journal of Cardiovascular Medicine

Hepatitis is the term which describes the inflammation of liver that results from various reasons which include both infectious and non-infectious. Hepatitis viruses are the most common cause of hepatitis all over the world. The major etiological agents are the hepatotropic viruses which are of 5 types termed as A to E. (2). Hepatitis A and E are typically caused by consumption of contaminated food and water. Hepatitis B, C and D can cause both acute and chronic hepatitis and get primarily transmitted through blood and body fluids. Together Hepatitis B virus and Hepatitis C virus cause approximately 90% of fatalities (3). Hepatitis C virus is a single stranded RNA virus belonging to the Flaviviridae family. Hepatitis C virus (HCV) is a bloodborne virus (6) and is the primary agent of posttransfusion hepatitis. The estimated prevalence of HCV infection in India is about 1 to 1.9%. Government of India has launched a National Viral Hepatitis Control Program, and this program aims at a nationwide elimination of Hepatitis C by 2030. The major component of NVHCP is to provide early diagnosis and management of viral hepatitis at all levels of healthcare. Early diagnosis can prevent health problems that may result from infection and prevents transmission of the virus. (6)

Aims and Objectives

 To qualitatively and quantitatively estimate the HCVRNA in seropositive Hepatitis C patients attending tertiary center in Central Kerala and to describe their clinico- epidemiologic profile.

Study Design-Hospital-based descriptive cross-sectional study

Study Setting- Department of Microbiology and NVHCP unit, GMC Ernakulum

Study Population-All patients whose samples received and tested positive for anti HCV antibody at Department of Microbiology

Study Duration-From December 2021 to April 2023

Inclusion Criteria-Blood samples for anti HCV antibody test received at Department of Microbiology

Exclusion Criteria- Haemolysed samples, Insufficient samples

Sample Size- All samples received and tested positive for anti HCV antibody at the Department of Microbiology during the study period were included in the study. Consecutive sampling method was used.

Data Collection

The primary patient details and clinical details were collected from the patients who were approached by the principal investigator and the proforma were filled. Informed consent was obtained from all these patients before data collection. The patient’s information were entered into NVHCP official site. All the patients were provided with a unique NVHCP identification number. Informed consent was taken from all the patients before data collection.

Study Procedure

Blood samples received in clot activator tubes at serology lab in the Microbiology Department for anti HCV antibody testing were centrifuged, serum separated and used for anti HCV antibody (IgG) test.

• Antibody Detection

The 3^rd generation ELISA (HCV Microlisa kit) was used. The HCV proteins are present in serum at levels well below the limits of detection.  The 3^rd generation HCV ELSA utilizes a combination of antigen with the sequence of both HCV structural and non-structural antigen i.e. CORE, E1, E2, NS3, NS4, and NS5.

• RNA detection

Sample collection

From patients who were positive for HCV IgG ELISA, blood was collected in 2 EDTA bottles from 2 each from one patient. Plasma was separated from the blood by centrifugation which were stored in a vial at -80ºc. HCV RNA detection (qualitative) and viral load estimation (quantitative) by Polymerase chain reaction were done once a week.

HCV RNA DETECTION (Alto star kit (ASO211513, LOT-037700., EXP-2023/7/31)

 HCV RNA detection was done as a 2-step procedure

1. RNA extraction -( QIAamp Viral RNA Mini kit, A48383, LOT-2592604, EX25/08/2024)

Viral RNA can be purified from plasma (treated with anticoagulants other than heparin), serum and other cell-free body fluid. The sample (140-280µl is first lysed under highly denaturing conditions to inactivate RNases and to ensure isolation of intact viral RNA. Buffering conditions are then adjusted and the sample is loaded onto the QIAamp Mini spin column. High quality RNA is eluted in a special RNase-free buffer, ready for direct use or safe storage.

b)REAL TIME RT PCR(AltoStar HCV RT_PCR Kit)-

The AltoStar HCV RT_PCR kit is an invitro diagnostic kit , for the detection and quantification of HCV specific RNA in human EDTA plasma within the Altostar workflow .It is based on real time PCR technology, using reverse transcriptase reaction to convert RNA to complimentary DNA, polymerase chain reaction for the amplification of HCV specific target sequences and fluorescently labelled target specific probes for the for the detection of amplified DNA . Fluorescent detectors are FAM and VIC. Probes specific for HCV RNA are labelled with fluorophore FAM. The probe specific for internal control is labelled with a fluorophore detectable in the VIC.

 Viral load (sample) [IU/ml] =

The viral load of an unknown sample is determined by comparing its fluorescence signal (or cycle threshold) to the standard curve, (Standard plasmids containing known concentrations of HCV RNA are used to create the standard curve), allowing for the calculation of the HCV RNA concentration in IU/mL (International Units per millilitre) or copies/mL

Reports entered in proforma and NVHCP portal. Patients who are positive for HCV RNA are guided to the NVHCP Nodal officer for treatment and follow up.

Data Management And Statistical Analysis

The data in the proforma were numerically coded and entered in Microsoft Excel spreadsheet. The data was analysed using SPSS software 20.0. The level of statistical significance was taken as P value. P value <0.05 is taken as the significant value. Quantitative variables were tested using independent t test.

A total of 55 seropositive patients were included in the study. Highest number of seropositive Hepatitis C patients belonged to the age group of 31-45 years in our study. Males were predominant in our study population compared to females.

Figure 1: Pie chart showing distribution of seropositive Hepatitis C patients based on HCV RNA

In this study, among the seropositive Hepatitis C patients 90.9% s were positive for HCV RNA

Table 1: Distribution of seropositive Hepatitis C patients based on viral load

In viral load estimation 5 samples of the study gave a viral load of ≤200IU/ml which are considered negative. Among the remaining positive samples (50 numbers),52.7% had >1,00,000IU/ml viral load  who required aggressive therapy

Figure 2: Pie chart showing distribution of seropositive Hepatitis C patients based    on History of alcohol consumption. 

Among study population 52.7% persons gave history of alcoholism. Hence there was statistically significant association between Hepatitis C seropositivity and alcohol intake in this study and the P value 0.013

Figure 3: Pie chart showing distribution of seropositive Hepatitis C patients based on    High-risk behaviour

Among the high-risk group persons (N=33) 48% were MSM people,39.3% with multiple sexual partners,12.1% had sexual contact with seropositive HCV persons. Statistically significant association between high-risk behaviour and Hepatitis C seropositivity was present in our study (P value=0.041)

Table 2-Correlation between HCV seropositivity and various other risk factors

Figure 4: Doughnut diagram showing distribution of seropositive Hepatitis C patients based on Elevated ALT

Among HCV RNA positive persons 34.5% showed elevated ALT levels. The association between Elevated ALT levels and seropositive Hepatitis C patients was not found to be statistically significant in this study.

The study was conducted at the Department of Microbiology, Government Medical college Ernakulam with 55 seropositive samples for Hepatitis C from December 2021 to April 2023.The aim of the study was to detect HCV RNA qualitatively and quantitatively in the study population and also to describe their clinico epidemiological profile. Among the 55 seropositive samples 50 were positive for HCV RNA. Out of 50 positive samples, more than 1 lakh IU/ml viral load was obtained in 29 samples.

In our study 45.5% of Hepatitis C seropositive patients were in the age group between 31-45 years. A similar finding was observed in a study conducted in Odisha (37.5%) Punjab (32.61%) and Chennai (35%). Another study conducted in Punjab in the year 2014 observed that the largest population were belonged to 5-18 years which was 20.2%.

Our study found that 74.5% of seropositive Hepatitis C patients were male, consistent with a study conducted in Tamil Nadu (2014–2017), where 73% of cases were male (105). In contrast, a 2014 study in Punjab reported a higher proportion of female seropositive Hepatitis C patients at 53.8% (42).

Analysis of our study population showed that 90.9% of seropositive Hepatitis C patients tested positive for HCV RNA. We observed similar finding in a study conducted in Kolkata in the year 2005 where HCV RNA positive percentage was 86.1%. (106). Studies from Punjab and Surat reported HCV RNA positivity rates of 72.3% and 66%, respectively.

The viral load was measured for all seropositive Hepatitis C patients in our study, and 5 out of 55 had a viral load of ≤200 IU/ml, classifying them as negative (6). Among seropositive Hepatitis C patients in this study 52.7% were having a viral load of >1lakh IU/ml. In studies conducted in Kolkata and Delhi observed 60%and 62% of HCV RNA positive patients yielded a viral load of a viral load of ≥ 1lakh IU/ml (103).

Our findings revealed that 52.7% of seropositive Hepatitis C patients had a history of alcohol intake. This aligns with a 2011 study conducted in Chennai, which reported an alcohol usage rate of 57.8% among HCV-positive patients (43). In contrast, a 2019 study in Punjab found a much lower prevalence, with only 3.2% of seropositive patients reporting alcohol consumption (42).

 In our study, 60% of the participants reported a history of high-risk behaviour. Among them, 29.1% (16 individuals) identified as engaging in MSM behaviour, 23.6% had multiple sexual partners, and 7.3% reported sexual contact with seropositive partners. A 2003 study in Chennai found that 20% of seropositive Hepatitis C patients reported MSM behaviour, with lower contributions from other high-risk behaviours (56). Additionally, a 2015 study among jail inmates in Chennai revealed that 70% of seropositive Hepatitis C patients reported having sex exclusively with women, while 20% reported having sex with men (110).

Among the seropositive Hepatitis C patients in our study, 12.7% received blood transfusions. Studies conducted in Uttarakhand and Punjab during the time period of 2012-2014 had shown that blood transfusion history was given by 27.5% and 6.5% seropositive Hepatitis C patients respectively.  In a study conducted in multi transfused thalassemia patients at Surat in 2018, it was observed that 51% of patients were seropositive for Hepatitis C infection. (66)

Our analysis found that 9% of seropositive Hepatitis C patients had a history of haemodialysis. Similar results were reported in 2012 studies from Tripura (10.9%) and Meghalaya (14%) (109,102). A 2019 study in Punjab found a significantly lower rate of 2.3% (44). Meanwhile, a 2013 study in Hyderabad conducted among renal failure patients on haemodialysis reported a substantially higher Hepatitis C prevalence of 46% (59).

We observed that just 7.3% (4 out of 55) of seropositive patients had a history of chemotherapy for different carcinomas. which was only 7.3%. We could not find any studies regarding the significant association between seropositivity of Hepatitis C and chemotherapy.

An analysis of our study population revealed that 32.7% (18 out of 55) of seropositive Hepatitis C patients had a history of intravenous drug use. This finding is similar to a 2014 study in Punjab, where the prevalence was 34.8% (42). Meanwhile, studies conducted specifically among injectable drug users in Chennai (2005) and Delhi (2015) reported substantially higher rates of seropositivity at 70.3% and 53.8%, respectively (43, 50), likely due to their focus on high-risk populations.

In this study, an average of 15.1% of seropositive Hepatitis C patients reported a history of percutaneous injuries. A similar prevalence was observed in a 2012 study from Uttarakhand, where 13.6% of seropositive patients had a history of such injuries (108). Additionally, a 2019 study from Punjab found that 5.2% of seropositive Hepatitis C patients reported a history of tattooing (44).

The study results showed that 13 out of 55 seropositive Hepatitis C patients had a history of surgery. In a study conducted in Punjab at 2019 observed that 24.1%of Hepatitis C seropositive patients had history of surgery in the past. (44)

 Among our study population, 34.5% of Hepatitis C seropositive patients presented with elevated ALT levels. Similar findings were reported in a 2019 study from Punjab (37.6%) (44) and a 2005 study from Kolkata (33%) (106).

The study was conducted at the Department of Microbiology, Government Medical College, Ernakulam with 55 seropositive samples for Hepatitis C from December 2021 to April 2023.

1. In our study highest number (45.5%) of the seropositive Hepatitis C patients were in the age group of 31-45 years and 74.5% were males.
2. Among the total 55 seropositive samples 50 were HCV RNA positive (90.9%) who had active Hepatitis C infection requiring treatment.
3. Among the 50 HCV RNA positive patients 29 had a viral load >1 lakh IU/ml who needed intensified antiviral therapy with interferon alpha
4. We could demonstrate statistically significant association between Hepatitis C seropositivity and alcohol intake and high-risk behaviour such as MSM character, multiple sexual partners.

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