Demographic characteristics of the study population
This study comprised 254 HCV sero-reactive individuals with 133 males (52.36%) and 121 females (47.63%). The age of the study population ranged from 20 to 83 years with a mean age of 51.91(± 11.82) years. The mean viral load of the RNA-positive individuals was 6.06(± 6.18) log IU/ml, and no significant difference in viral load was observed between genotype 3 and genotype 1.
Occurrence of HCV viremia
The frequency of active HCV infection in this study population was evaluated. Overall, HCV RNA positivity was 58.26%. HCV viremia was observed among 54.14% and 62.81% of males and females, respectively. The study found that HCV RNA positivity was highest (66.67%) in people aged 52 to 59. Blood transfusion, medical procedures, and improper use of syringes/needles were the common risk factors associated with active infection in the study, with blood transfusion accounting for the majority of patients (68.83%) (p < 0.05). Of the total 254 patients, 110 were cirrhotic and 144 non-cirrhotic. Among the cirrhotic patients, 69 (62.7%) were having decompensated cirrhosis. Decompensated cirrhosis of liver was associated with HCV viremia in 72.46% of cases (p < 0.05) (Table 1).
Genotype distribution among the study population
Two HCV genotypes and seven subtypes were predominating in this study population (Fig- 1b). Of the 148 individuals that tested positive for HCV RNA, 120 (81%) had infection with genotype-3 (3a- 50%, 3b- 46.67%, 3 g- 2.5%, and 3i- 0.83%), while 28 (19%) had infection with genotype-1 (1a- 21.43%, 1b- 67.86%, and 1c- 10.71%). When the prevalence of HCV genotypes was compared across the seven age categories, those between the ages of 52 and 59 were most likely to have genotype 3 infection (82.22%, n = 37) (p < 0.05). Genotype distribution across gender, mode of transmission and status of cirrhosis is listed in Table 2.
Phylogenetic analysis of the study
A phylogenetic tree was constructed with the representative sequences obtained from this study population (Supplementary Fig – 2) with reference sequences from the NCBI database. Corresponding representative sequences were found to cluster with the respective reference sequences.
The phylogenetic tree was constructed by using MEGA X software package with 34 reference sequences downloaded from NCBI and 65 representative sequences from in-house sequencing. Prefix “NICED-VL/CLD” denoted in-house sequences.
Assessment of DAA therapy
This study monitored DAA therapy in 148 HCV RNA-positive individuals for 48 weeks. Of these 148 individuals, 120 individuals infected with genotype 3 were treated with Sofosbuvir (SOF) + Daclatasvir (DCV), and 28 individuals infected with genotype 1 were offered Sofosbuvir (SOF) + Ledipasvir (LDV) [19, 20]. After 4 weeks of treatment, individuals were followed up for assessing the attainment of RVR, 8 patients (5.40%) (Male = 3, Female = 5) had lost follow-up within the first month of DAA treatment and the rest (n = 140) showed 100% RVR. During the 12-week follow-up, 15 patients (10.71%) (Male = 7, Female = 8) had lost follow-up. Out of the remaining 125 patients, 4 patients (3.2%) tested positive again, and remaining 121 patients showed EVR (96.8%). In the next follow-up visit, 112 patients (92.56%) showed SVR24, while 9 patients (7.44%) tested RNA positive again. Hence, the SVR24 achievement rate was 92.56% (Fig 2).
Schematic diagram of DAA treatment follow-up
Out of 112 patients who achieved SVR24, 58 (51.78%) were male and 54(48.21%) were female. Gender-wise HCV genotype distribution of these 112 patients was gen-3a (42, 37.5%) (Female- 59.5% and male- 40.5%), gen-3b (50, 44.64%) (Female- 50% and male- 50%), gen-1a (2, 1.79%) (Male- 100%), gen-1b (15, 13.39%) (Female- 20% and male- 80%), and gen-1c (3, 2.68%) (Female- 33.33% and male- 66.67%) (Fig 3a) and among 13 relapse patients, 9 (69.24%) were male and 4 (30.76%) were female. Genotypically, these 13 relapse patients were distributed as follows- gen-3a (3, 23%) (Female- 33.3% and male- 66.7%), gen-3b (8, 62%) (Female- 37.5% and male- 62.5%), gen-1a (1, 7.69%) (Male- 100%), and gen 1b (1, 7.69%) (Male-100%) (Fig 3b).
Comparative genotype distribution of HCV among patients based on treatment outcome; (a) Pie-donut chart representing the genotype distribution of the SVR24 achieved patients, (b) Pie-donut chart representing the genotype distribution of the patients who could not achieve SVR24
Patients (n = 13) who failed to achieve SVR24 (relapsed) were re-interviewed and counseled again. A modified treatment regime with SOF + velpatasvir (VEL) + ribavirin (Riba) was prescribed to everyone re-interviewed for an additional 6 months. Of the 13 relapse patients taking SOF + VEL + Riba for another 24 weeks, 12 patients (92.3%) did not show active infection (considered as SVR’24 achieved), and in contrast, 1 patient (7.69%) continued to be nonresponsive to DAA. Overall, 99.2% of patients achieved either SVR24 or SVR’24. Patients who have achieved SVR24 were requested to revisit after 48 weeks for SVR48 evaluation. At the SVR48 assessment, only 65 patients (58.04%) attended, and upon evaluating the status of viremia, undetectable HCV RNA was found in all patients. Therefore, it can be inferred that among the attended patients the SVR48 achievement rate is 100%. Fig-2 represents the treatment follow-up for 48 weeks.
Furthermore, Supplementary Fig-3 provides an overview of DAA efficacy by bifurcating the study population on the basis of the status of cirrhosis. Of the 77 SVR achieved patients, 57.14% were cirrhotic and 42.86% were non-cirrhotic, while out of 70 lost follow-up cases, 34.29% were cirrhotic and 65.71% were non-cirrhotic.
Clinical assessment before and after DAA treatment
Liver parameters were assessed for any significant changes between the baseline and post-treatment condition. We found that ALP, SGPT and Total Bilirubin returned to normal post-treatment. Significant differences (p < 0.001) were observed between two conditions in ALP, SGOT, SGPT, albumin, globulin and total bilirubin levels (Table 3).
Liver related adverse events
Among the 65 patients who completed 48 weeks of follow up, 37 were cirrhotic and 28 were non cirrhotic. Liver related adverse events were observed in 2 non-cirrhotic and 17 cirrhotic patients. Hepatomegaly and portal hypertension were the most common adverse effects. Cirrhotic patients had higher incidence of portal hypertension (13.5%) and hepatomegaly (13.5%). Occurrence of ascites (8.1%), hepatic encephalopathy (2.7%) and hepatocellular carcinoma (8.1%) were majorly observed in cirrhotic patients Table 4.