Recurrence of hepatitis C virus after treatment with pegylated interferon and direct acting antivirals in Punjab Pakistan

Although increased response rates concomitant in hepatitis C virus but relapse after treatment is threatened. Therefore, it is terrible requirement to evaluate the response of Pegylated interferon and direct acting antivirals in Punjab Pakistan. The study was conducted to find the rate of recurrence of HCV infection after treatment with Pegylated Interferon and Direct Acting Antivirals in Punjab Pakistan. This study was conducted at Department of Pathology, Nawaz Sharif Medical College Gujrat, while treatment effects monitored in different Government and Private Hospitals of Punjab, Pakistan. Total 973 patients who administered the recommended dose and divided in two groups (i) Interferon based therapy (ii) direct acting antivirals (DAAs).Other parameters like ALT and viral load studied. The rate of recurrence was higher in female infected with genotype 2b and in male with mixed genotype 3a/2b after six month of antiviral therapy. Genotype 3a showed significant response to therapy after three month. 32 among 374 (8.5%) were positive after 24 weeks of treatment with interferon, 29 (7.7%) patients have same genotype while 3 patients were re-infected with different HCV strains. With DAAs, only 27 (4.8%) patients were positive among 558 after 2 weeks and one patient re-infected with different genotype. Early and sustained virological response noted in DAAs. ALT and viral load decreased faster with DAAs that not achieved after 4 weeks with pegylated interferon. Sustained virological response appears in DAAs and recurrence rate is high in interferon therapy compared to DAAs. Therefore, reinfection has implications for correct treatment efficiency and to select strategies for retreatment cases.

Prevalence, Genotypic Distribution and the associated Risk Factors of Hepatitis C Infection in Pakistan Pediatric Patients

Hepatitis C virus (HCV) is an important contributor to chronic morbidity and mortality in developing countries. The study’s objective was to determine the genotype distribution and risk factors associated with the transmission of HCV infections in pediatric patients. Rapid screening and confirmation by the enzyme-linked immunosorbent assay (ELISA) were used to analyze 585 pediatric blood specimens hospitalized and visited the outpatient department of the largest tertiary care hospital in Pakistan. Detection and genotyping of HCV RNA were performed using a real-time polymerase chain reaction (RT-PCR). Demographic data and a history of risk factors were gathered through a survey questionnaire. HCV RNA was detected in 323 (72.4%) cases which showed viral load ranging from Log10 IU/mL < 3 to > 6 IU/mL. HCV genotype 3a was detected in 256 (79.3%) cases while type 3b and 1a was observed in 36 (11.1%) and 31 (9.6%) patients, respectively. HCV positivity was significantly associated with the cases from rural areas [p = 0.005; odds ratio (OR) 1.65; 95% CI 1.16-2.23] and also significantly associated with low-income group [p < 0.001; OR 5.75; 95% CI 3.90-8.40]. The primary risk factors associated with HCV transmission in children were family history (p = 0.002), blood transfusion (p = 0.03), surgical procedures (p = 0.02), and history of injections (p = 0.05). HCV genotype 3a is the most common genotype in children. The main risk factors for HCV transmission in children are blood transfusion, surgical procedures, and injection practices by informal health care providers.

Development of full-length cell-culture infectious clone and subgenomic replicon for a genotype 3a isolate of hepatitis C virus

Hepatitis C virus (HCV) genotype 3 is widely distributed, and genotype 3-infected patients achieve a lower cure rate in direct-acting antiviral (DAA) therapy and are associated with a higher risk of hepatic steatosis than patients with other genotypes. Thus, the study of the virology and pathogenesis of genotype 3 HCV is increasingly relevant. Here, we developed a full-length infectious clone and a subgenomic replicon for the genotype 3a isolate, CH3a. From an infected serum, we constructed a full-length CH3a clone, however, it was nonviable in Huh7.5.1 cells. Next, we systematically adapted several intergenotypic recombinants containing Core-NS2 and 5′UTR-NS5A from CH3a, and other sequences from a replication-competent genotype 2 a clone JFH1. Adaptive mutations were identified, of which several combinations facilitated the replication of CH3a-JFH1 recombinants; however, they failed to adapt to the full-length CH3a and the recombinants containing CH3a NS5B. Thus, we attempted to separately adapt CH3a NS5B-3′UTR by constructing an intragenotypic recombinant using 5′UTR-NS5A from an infectious genotype 3a clone, DBN3acc, from which L3004P/M in NS5B and a deletion of 11 nucleotides (Δ11nt) downstream of the polyU/UC tract of the 3′UTR were identified and demonstrated to efficiently improve virus production. Finally, we combined functional 5′UTR-NS5A and NS5B-3′UTR sequences that carried the selected mutations to generate full-length CH3a with 26 or 27 substitutions (CH3acc), and both revealed efficient replication and virus spread in transfected and infected cells, releasing HCV of 104.2 f.f.u. ml−1. CH3acc was inhibited by DAAs targeting NS3/4A, NS5A and NS5B in a dose-dependent manner. The selected mutations permitted the development of subgenomic replicon CH3a-SGRep, by which L3004P, L3004M and Δ11nt were proven, together with a single-cycle virus production assay, to facilitate virus assembly, release, and RNA replication. CH3acc clones and CH3a-SGRep replicon provide new tools for the study of HCV genotype 3.

Prevalence of Genotype 3a in Different Regions of Pakistan: A systematic Review and Meta-Analysis

The present study aims to explore the prevalence of genotype 3a under hepatitis C virus (HCV) infections among all the provinces of Pakistan. It is alarming to note that Pakistan stands in the second position for having a large number of cases of HCV every year. Six major genotypes characterize HCV. To study the overall prevalence of HCV and its associated genotype 3a in all the provinces of Pakistan, a systematic review and meta-analysis were performed using STATA version 14.2. The published studies conducted in all the regions of Pakistan reported the incidence of HCV genotype 3a were shortlisted. The pooled summary estimates were calculated along with their confidence interval by using the "Metaprop" command. The literature review showed that the prevalence of HCV genotype 3a is most common in all the provinces of Pakistan. It is revealed that the prevalence of HCV genotype 3a was 86.46% in Punjab, which is the highest among all the regions.

Frequency Distribution of Hepatitis C Virus Genotypes with Reference to Age and Sex in Various Districts of Khyber Pakhtunkhwa, Pakistan

Background: Pakistan has the second highest prevalence of hepatitis C in the world after Egypt. Viral hepatitis is a leading cause of morbidity and mortality in Pakistan and, worryingly, reinfection rates are also on the rise. This cross-sectional study was aimed at finding the most common genotypes of hepatitis C in terms of age and sex in a Pakistani cohort. Materials and methods: The authors collected blood samples from 1,260 patients with diagnosed hepatitis C visiting a primary teaching hospital affiliated with Peshawar Medical College, Pakistan, from different districts of Khyber Pakhtunkhwa, Pakistan, between January 2017 and April 2019. Hepatitis C virus RNA was quantified by real-time polymerase chain reaction and genotyping was then performed. Results: The authors found that genotype 3a was the most prevalent type followed by 1a, mixed, and 3b, respectively. Genotypes 2a and 1b were the least prevalent in Khyber Pakhtunkhwa. The most common genotype was 3a, observed in 75.87% of cases. The most common mixed genotype was 3a+1a, observed in 39 cases (3.10%); it had a prevalence of 3.49% in females compared with 2.70% in males. Overall, the most common age group affected by hepatitis C virus was 41–50 years (31.35%), followed by the 51–60 years group (24.45%). Infection rate was comparatively low in other age groups. A significant difference was observed in the prevalence of genotype 3a and 2a among different districts. Conclusion: The authors concluded that genotype 3a was the most prevalent genotype and it was observed more frequently in the female population, with a median age of 45 years.

Assessment of Hepatitis C virus genotypes in regular dialysis patients of Khyber Pakhtunkhwa, Pakistan

Globally viral hepatitis is a major health problem. HCV is a causative agent of hepatitis and is responsible for acute and chronic hepatitis leads to cirrhosis and hepatocellular carcinoma. This study was carried out to know the HCV genotypes in Dialysis patients in NWFP (Pakistan). The age ranged from 15-65 years. During this study a total of 63 samples were collected and were analyzed for HCV genotypes. RNA was extracted from whole blood; reverse transcribed into cDNA and was subjected to multiplex PCR. Of these 63 samples, 14 were genotyped as genotype 3a was found in 9(64.28%) patients, followed by genotype 3b (21.42%) in 3 and 2a in 2(14.28%) patients. Three positive samples remained untyped. In age group 31 to 40 years, the number of positive patients were comparatively greater.

Efficacy of Sofosbuvir Plus Ribavirin with and without Pegylated Interferon-Α in Patients with HCV Genotype 3A Infection

Background: Hepatitis C virus (HCV) infection is a serious problem in low and middle income countries including Pakistan. Objective: To compare the efficacy of combination therapy, Sofosbuvir plus Ribavirin with and without Pegylated Interferon-α in HCV genotype 3a infected patients. Study Design: Randomized control trial Place and Duration: Department of Gastroenterology, Pakistan Institute of Medical Sciences (PIMS), Islamabad, during from 16th April 2017 to 15th October 2017. Methodology: One hundred and fifty four HCV infected patients with genotype 3a were included in this study. Detailed demographics were recorded after taking informed written consent. Patients were divided in two groups (A and B) randomly, Group A was offered with Sofosbuvir plus Ribavirin with Pegylated Interferon-α for 12 weeks and Group B was offered with Sofosbuvir plus Ribavirin for 24 weeks. The sustained virological response (SVR) after 12 weeks of therapy was compared between the study groups. Results: The frequency of SVR12 was comparatively higher in patients treated with Sofosbuvir plus Ribavirin with Pegylated Interferon-α (94.8%) than Sofosbuvir plus Ribavirin alone (84.4%) with similar trends in gender and age groups. Conclusion: The combination therapy Sofosbuvir plus Ribavirin with Pegylated Interferon-α had higher eradication rate against HCV genotype 3a in our local setting. Key Words: HCV, genotype 3a, Sofosbuvir, Ribavirin, Pegylated Interferon-α, SVR12

Lipid Droplets Accumulation during Hepatitis C Virus Infection in Cell-Culture Varies among Genotype 1–3 Strains and Does Not Correlate with Virus Replication

Liver steatosis is a common complication of chronic hepatitis C virus (HCV) infection, which can result in accelerated liver fibrosis development, especially in patients infected with genotype 3a. The precise mechanisms of HCV-induced liver steatosis remain unclear, but it is often posited that increased intracellular lipid accumulation is the underlying cause of steatosis. To study experimentally how HCV infection in human liver derived cells by different genotypes and subtypes might affect lipid accumulation, we performed detailed cytofluorimetric and microscopy analyses of intracellular lipid droplets (LDs) in relation to the viral Core and to cell endoplasmic reticulum proteins. Following culture infection with HCV genotype 1a, 2a, 2b, 2c, and 3a strains, we found variable levels of intracellular LDs accumulation, associated to the infecting strain rather than to the specific genotype. Although two genotype 3a strains showed high levels of lipid accumulation, as previously observed, some strains of other genotypes displayed a similar phenotype. Moreover, the analyses of LDs size, number, and shape indicated that the apparent increase in lipid accumulation is due to an increase in the overall number rather than in the size of droplets. Finally, differences in total lipid content across genotypes did not correlate to differences in Core distribution nor Core levels. In conclusion, our study provides a quantitative in-depth analysis of the effect of HCV infection on LDs accumulation in cell-culture.

Damar İçi Madde Bağımlılığı Olan ve Madde Bağımlısı Olmayan Hastalar Arasında Hepatit C Virus (HCV) Genotiplerinin Dağılımı

Genotype distribution of hepatitis C virus (HCV) can vary over the years between different patient groups and regions. The prevalence of intravenous drug users (IVDU) is known to increase in our country, yet there are a limited number of studies investigating the distribution of HCV genotypes in this group. These data are essential for monitorization of the changes in HCV epidemiology. The present study aimed to evaluate the five-year results of HCV genotyping among patients infected with HCV related to IVDU and unrelated to drug use. Plasma samples of 720 patients (HCV antibody, HCV RNA positive), which were sent to our laboratory for HCV genotyping between January 2014-March 2019 were analyzed. HCV RNA extraction from plasma samples was performed in the automated-extraction system of EZ1 advanced (Qiagen, Germany) using the EZ1 virus mini kit v2.0 (Qiagen, Germany). Amplicons were obtained by amplifying the 5’NCR and core gene region in the Rotorgene 6000 real-time PCR (Qiagen, Germany) device with the HCV RNA real-time quantitative 2.0 (NLM, Italy) kit. For the genotyping, a commercial line probe assay (LIPA) based on in vitro reverse hybridization GEN-C2.0 kit (NLM, Italy) which can distinguish 1, 2, 3, 4, 6 genotypes and 1a, 1b, 2a/c, 2b, 3a, 3b, 3c, 3k, 4a, 4b, 4c/d, 4e, 4f, 4h, 5a, 6a/b, 6g, 6f/q, 6m, 7a subtypes of HCV, based on variations in the 5’-NCR and core regions was used. HCV genotype distribution of 266 IVDU (93.2%: male; median age: 25 ± 6.82) and 454 non-drug users (51.3%: male; median age: 56.5 ± 16.06) were examined. In order of frequency in the group with IVDU; genotype 1a, 3a, 1b, 4c/d, 2b, 4, 3 were observed and genotype 1, 2a/c and mixed genotype (1+3a) were detected in one patient. In the group without IVDU, in order of frequency; genotype 1b, 1a, 3a, 1, 2a/c, 4 were observed and genotype 2b, 4c/d, 5a, 6a/b, 6 and mixed genotype (3+4) were detected in one patient. Genotypes 1a and 3a were significantly higher in the IVDU group (p< 0.00001, p< 0.00001), while 1b was significantly higher in patients without IVDU (p< 0.00001). Genotypes 1a and 3a were more common in young men (p< 0.00001, p= 0.000163), while 1b was higher in middleaged women (p< 0.00001). The incidence of genotypes 1b (p= 0.021) and 3a (p= 0.012) was higher in foreign nationals than the Turkish patients. When the HCV genotype distribution was examined by years, it was observed that the percentages of genotype 1b and 1a were decreasing, while the percentage of genotype 3a was increasing. As a result, in this study, HCV genotype distribution among IVDU was observed to be different from the general population without IVDU. It was found that genotypes 1a and 3a were more common in the IVDU group. As in the other regions of our country, genotype 1b was found most frequently in the general population. Genotype 3a increases significantly compared to years. In our study, the determination of genotypes existing in different parts of the world may be due to the foreign nationals living in our city and our region is a tourism center. It is also necessary to investigate whether there is an increase in IVDU over the years.

THE HEPATITIS C UNTYPE-ABLE GENOTYPES, AN EMERGENCE OF QUASI-SPECIES IN HCV INFECTED PATIENTS

Objective: To determine the frequency of Type-able and untype-able genotypes in hepatitis C infected patients,and to observe their association with gender, age, Alanine Aminotranferease and viral load. Study Design: Cross-sectional analytical study. Place and Duration of Study: Department of Microbiology, Combined Military Hospital, and Lahore, Pakistanfrom Sep 2017 to Mar 2018. Methodology: Six hundred forty seven anti HCV antibodies positive serum samples by Enzyme Linked ImmunoSorbant Assay were received from a total of 6791 serum samples. The positive sera were subjected to qualitative PCR and quantitative real time (RT) PCR to determine pre-treatment viral load. Quantitative PCR positive sera with viral load >500IU/ml were further subjected to molecular genotyping by using Ohno et al method. Result: Out of 647 positive serum samples, type-specific PCR fragments were seen in 424 sera, while 13 (3.1%) of serum samples were of untype-able genotype. In all age groups genotype 3a had emerged as a predominantgenotype 397 (93.6%), followed by 1b 8 (1.9%), 3b 4 (0.9%), 1a 2 (0.5%), while no sample detected to have 2a, 2b,5a, 6a and mixed genotypes. The highest prevalence of untype-ables were seen in 61-70 age group. Conclusion: Need of the hour is proper sequencing of untype-able genotypes via upgrading existing methodologies. It will not only help the clinicians in achieving sustained virological response but also help in identifying new genotypes/subtypes.