A Cross-Sectional Study to Detect the Prevalence of Hepatitis C Virus Infection among Dialysis Patients in a Tertiary Care Hospital in Bangalore

INTRODUCTION

Hepatitis C Virus (HCV) is a single-stranded, enveloped RNA virus measuring about 55-65 nanometers in diameter and belongs to the family Flaviviridae. According to the WHO, there are 58 million chronic HCV carriers worldwide with 1.5 million new HCV infections every year and 2,90,000 deaths resulting from HCV infection [1]. About 75-85% of patients infected with the HCV virus become chronic carriers [2]. Chronic Hepatitis C carriers have a 15-20% risk of developing cirrhosis within 20 years [3]. 

Chronic HCV carriers run 15-20 times higher risk of developing hepatocellular carcinoma (HCC) [4] and account for 30% of total cases of HCC worldwide [5]. A systematic review conducted by SuY et al. found high incidence rates of HCV infection among dialysis patients [6]. The prevalence of HCV in hemodialysis patients is found to be higher than in the general population, with male predominance in studies conducted in tertiary care hospitals in various states across India [7,8].

Hepatitis C Virus (HCV) is a major blood-borne viral infection in patients on dialysis with significant morbidity and mortality. The prevalence of HCV infection is higher in dialysis patients than general population due to underlying impaired immunity, prolonged vascular access, exposure to contaminated fluids, handling by medical staff, frequent blood transfusions, and frequent hospitalization, which increases the risk of exposure to nosocomial HCV infection [9]. The prevalence of HCV infection among dialysis patients ranges from 6% to 60% worldwide, but in India, it ranges between 4.3% and 48% [10]. The presence of HCV infection in dialysis patients is linked to higher mortality rates owing to hepatic and extra-hepatic complications, which include renal manifestations such as cryoglobulinemia, membrane nephropathy, and glomerulonephritis [11]. The high mortality rate among HCV-positive dialysis patients is also linked to the acceleration of atherosclerosis, immune deficiencies, and sepsis [12].

Detection of HCV infection among dialysis patients plays an important role in reducing morbidity and mortality by preventing the development of various hepatic and extra-hepatic complications and in cutting down transmission rates of HCV infection among dialysis patients. The methods that are currently available for detecting HCV infection include serological methods, such as CLIA, and molecular methods, such as RT PCR. CLIA has a sensitivity of 96.1% and a specificity of 91.9% in detecting HCV antibodies [13]. However, there are chances of false negative results by CLIA even with high sensitivity due to a decline in humoral and cellular immunity in dialysis and the absence of antibodies during the initial 2-6 weeks of the window period [14,15]. These disadvantages of CLIA can be overcome by RT PCR, which detects even low levels of HCV RNA during the initial stages of HCV infection [16].

This study aims to detect the incidence and prevalence of HCV infection and associated risk factors among dialysis patients by CLIA and RT PCR methods.

PATIENTS AND METHODS

Study design and source of data

This prospective study was conducted in the Department of Microbiology of M.S. Ramaiah Medical College, Bangalore for a period of 1 year from January 2022 to December 2022.

Sample size estimation

 The prevalence of Hepatitis C among renal dialysis patients was reported to be 22% [17]. Assuming similar proportions in our study with an absolute precision of 7.5% and the desired confidence interval of 95% (Alpha error -5%), the required sample size is 117 patients.

Method of Collection of Samples      

The requisite minimum sample size for this investigation was established at 117 participants. However, the current study encompassed a total of 120 individuals undergoing dialysis. About 4 mL of blood was collected under aseptic precautions, each in the serum separator (Yellow top) and K2 EDTA (Purple top) vacutainer from the patients who were receiving dialysis therapy in M S Ramaiah Memorial Hospital. These patients were selected based on the following inclusion and exclusion criteria. Informed consent was obtained from all patients after a detailed explanation of the purpose and procedure.

Inclusion Criteria

•           CKD patients who were above 18 years of age on dialysis therapy.

Exclusion Criteria

•           CKD patients with a history of HCV infection before starting dialysis therapy.

•           CKD patients who are co-infected with Hepatitis B Virus (HBV) or Human Immunodeficiency Virus (HIV) as they can act as independent risk factors.

The blood collected in vacutainers was subjected to centrifugation at a speed of 4000 rotations per minute (rpm), and serum and plasma were separately obtained in the yellow-top and the purple-top vacutainers, respectively. The serum and plasma samples were stored at -20℃.

HCV antibody detection        

The VITROS Anti-HCV test was performed using the VITROS Anti-HCV Reagent Pack and VITROS Immunodiagnostic products, Anti-HCV calibrator on the VITROS ECIQ Immunodiagnostic system. An immunometric technique was used. In the first stage, HCV antibodies present in the sample bind with HCV recombinant antigens coated on the wells. The Unbound sample is removed by washing. In the second stage, horse radish peroxidase (HRP) labeled antibody conjugate binds to any human IgG captured on the well in the first stage. The unbound conjugate was removed by washing. The bound HRP conjugate was measured by a luminescent reaction. The amount of HRP conjugate bound was indicative of the level of HCV antibodies present in the sample. The HCV reactivity of tested serum samples was decided based on VITROS ECIQ CLIA kit literature as – values below 0.9 as non-reactive, values between 0.9 to 0.99 as intermediate reactive and retested using fresh serum collected from the patient and reclassified accordingly and values above or equal to 1 as reactive. In this study, we didn't get any intermediate reactive values. The serum samples were then stored at -20℃ for further reference.

HCV RNA detection by PCR using the Thermo Fisher Quant Studio 3 Real-Time PCR Machine

Steps include-

1)                  Nucleic acid extraction HCV RNA extraction from plasma samples was done immediately using a BIOBEE viral DNA extraction kit. 250 microliters of plasma were mixed with 250 microliters of lysis buffer, 5 microliters of carrier RNA, and 10 microliters of Proteinase in the sterile microcentrifuge tube and incubated at room temperature for 5 minutes. The tube is then centrifuged at 8000 rotations per minute (rpm) for 5 minutes to get lysate. 400 microliters of this lysate are mixed with 400 microliters of binding buffer in a new 1.5 mL. Microcentrifuge tube and then centrifuged at 8000 rpm for 30 seconds. After discarding the flow-through, 450 microliters of wash buffer 1, is added and centrifuged at 8000 rpm for 30 seconds. The process is repeated with wash buffer 2 also. After discarding the flow through, the spin column is air-dried at 8000 rpm for 3 minutes. Then the spin column is shifted to a new 1.5 mL. A Microcentrifuge tube and 50 microliters of elution buffer are added and centrifuged at 8000 rpm for 1 minute. The lysate collected in the microcentrifuge tube will have viral RNA if present. The extracted samples (templates) were stored at -80℃. HCV RNA detection from those templates is done using the NEODX HCV RNA qualitative RT PCR detection kit.

2)                  Master Mix preparation (120 reactions) (Table 1 and 2) – Thaw the contents of the kit at room temperature till equilibrated and the reaction mix is prepared for 100 and 20 reactions as below. According to the NeoDx HCV PCR Detection Kit used in the current study, the cDNA is available inbuilt in the kit and does not require additional preparation.

3)                  Template addition- 15 microliters of reaction mix are prepared for each of 120 PCR reaction tubes with 1 tube each for negative and positive controls to which 10 microliters each of sample, negative and positive control is added to respective tubes and centrifuged and transferred to PCR room where tubes are placed in the sample holder and programmed as below (Table 3) and the result is generated and data is interpreted for each run (Table 4 and 5).

Follow-up – All the participants were tested for HCV antibodies by CLIA and HCV RNA by PCR at the beginning and at the end of a 1-year study and results were noted down and positive patients were treated with direct-acting antivirals (DAAs) and were put on separate dialysis machines dedicated for HCV positive patients.

Statistical Method

The Sample size was estimated using the n-master version 2.0 software developed by CMC Vellore. Descriptive statistics of HCV positives were analyzed and summarized in terms of percentage. Statistical data were generated using IBM SPSS Statistical Software. Statistical significance between categorical variables was tested using the chi-square test of independence, and continuous variables using the Paired T-test. A p-value

RESULTS

Demographic characteristics of the patients (Table 6)

The incidence and prevalence rate of HCV in dialysis patients in this study, by CLIA and RT PCR methods, is calculated as follows

Incidence rate = number of new cases over time/total population* 100 patient-years

Incidence rate = 5/120*100=4.1% per 100 patient-years

Prevalence rate = number of cases (existing and new) during specified time/total number of cases * 100

Prevalence rate = 7(2+5)/120=0.58*100=5.8%. The HCV antibody and HCV RNA of the dialysis patients were measured at the beginning and at the end of the study to compare the prevalence rate of HCV infection in that year which is depicted in Table 7.  

 Master Mix preparation (total 120 reactions)

Table 1 – for 100 reactions (1^st batch)

Table 2 – For 20 reactions

Table 3 – Steps of programming PCR instrument

Table 4 -Result interpretation for HCV

Ct – cycle threshold

Invalid – (Repeat extraction and detection)

Table 5 – Data interpretation for HCV

HEX/VIC – HCV genes tested, IC – Internal control.

Table 6 – Patient demographic characteristics

Table 7 – Prevalence of HCV infection among dialysis patients by CLIA and RT PCR.

 DISCUSSION