Characterization of HBV Genotypes and Response Rate of Antiviral Therapy in KP, Pakistan

Abstract

Hepatitis B virus (HBV) infection is a criticalglobal health problem includingPakistan where an estimated prevalence rate of 3% has been reported in the general population. HBV belongs to family Hepadnaviridae and can be categorized into 9 genotypes (A-I) till now, by sequence divergence of more than 8% depending on the complete genome. Infection with different genotypes possesses different clinical manifestations, characteristics and responses to antiviral treatment as reported from different geographical regions of the world. Although Pakistan has a tremendous burden of HBV infection according to recent reports, yet molecular epidemiological aspects and response of various antiviral therapies in our ethnic population have drawn very little attention. The present study was therefore designed to investigate the current pattern of HBV genotypes distribution in Khyber Pakhtunkhwa (KP) province of Pakistan; which is the third most populous province of the country. HBV infected patients having a history of clinical disease in the form of positive serology, molecular identification and liver function tests(LFTs) were enrolled in the study at different healthcare units of KP. Confirmation of the infection in each subject was carried out by serological and molecular techniques at AWKUM and other collaborating institutes. Furthermore, due to lack of published literature on the response rates of different antivirals being used for the treatment of HBV infection in KP province, second part of this study focused on characterization of treatment response among the chronically infected patients. A total of 3000 HBV positive samples belonging to chronic HBV patients with a clinical history of the disease and who were enrolled for antiviral treatment at different healthcare units were collected from seven different districts of the KP province including Peshawar, Charsadda, Mardan, Swat, Swabi, Hazara and Tribal region. All samples were screened by ICT and ELISA followed by confirmation through qualitative polymerase chain reaction (PCR). Type-specific PCR or Restriction Fragment Length Polymorphism (RFLP) and random sequencing of the partial S gene were employed for characterization of the HBV genotypes. A total of 100S gene sequences were obtained out of which 28 sequences representing the entire diversity of the sequenced types were used for phylogenetic analysis. For assessment of response rates of different antivirals, 2875 HBV infected patients receiving different antiviral therapies (Entecavir 1.0 mg once/day and Tenofovir Disoproxil Fumarate 300 mg once/day), both as mono or combination therapy for six and twelve months at different health-care centers in KP were recorded. xx Out of 3000 samples, 1680 (90% male; 10% female) were found positive for both HBsAg and HBeAg while 1170 were detected positive for HBsAg and negative for HBeAg (65.9% male; 34% female). Qualitative PCR confirmed active infection of HBV in all patients (56.3% male; 43% female). Three genotypes were confirmed in our study, genotype D was detected as the most predominant genotype in 2065 patients (75.66% male; 60% female) followed by genotype A in 679 patients (18.11% male; 28.5% female) and C in 256 patients (6.21% male; 11.51% female). The ratio of HBV genotype D was recorded high in male patients as compared to female patients in age group (71-85 years). District wise distribution of genotypes showed that infection with genotype D was found more in districts Peshawar (90%), Mardan (85.71%), Charsadda (81.81%), Swat (79.2%) and Hazara (77.77%). While lower in distrcitSwabi (64.28%) and Tribal region (61%). Genotype A was high in Swabi (35.71%) followed by Tribal region (28.10%) whereas genotype C was high in tribal region and Peshawar (10.89%, 10%) and followed by Charsadda (8%). Phylogenetic study revealed that some of our HBV sequences showed a close homology with previously reported Pakistani isolates NCVI-JN132120.1, NCVI-JN1321132 and EF584653.1 while others clustered with some foreign isolates of HBV fromCanada, Hungary, Argentina and Syria. Also in our analysis one sample didn’t show any similarity with any of HBV isolates available in literature that indicated to a great divergence and a distinct origin of the genotype. In case of Entecavir monotherapy for six months, 30.55% (32% male; 28.75% female) of patients among 900 (55.55% male; 44.44% female) achieved end of treatment response (ETR) while 35.3% (39% male; 30% female) patients achieved ETR after 12 months of treatment. 0.77% (1% male; 0.5% female) virologic relapse (VR) was observed in patients treated with ETV monotherapy. The overall ETR achieved in case of monotherapy was 35%. ETR achieved remained high in male patients (61.90%) as compared to female patients (38%). High ETR was observed 71.45% in male patients of age group 71-85 years and low ETR 42.51% was observed in male patients of age group 26-40 years while in female patients high ETR 42.85% was achieved by patients of age group 71-85 years and low ETR 25% was shown by age group 56-70 years. As a whole high ETR (40.31%) was recorded in patients of age group 26-40 years and lowest 2.22% was observed in patients of age group 71-85 years. In ETV-TDF combination therapy comparatively higher number 51.89% (57% male; 44.40% female) of patients among 1975 achieved ETR after six months of therapy while 53.42% (58.7% xxi male; 47.2% female) showed ETR after 12 months of therapy and the virologic relapse recorded was 0.10% (0.09% male; 0.11% female). The overall ETR achieved in combination therapy remained high 53.41% (59.71% male; 40.28% female) as compared to ETV monotherapy 35% (61.90% male; 38% female). High ETR was recorded in male patients (70.38%) of age group 26- 40 years while low ETR was achieved by male patients of age group 10-25 years. In case of female patients, high ETR 54.54% was recorded in patients of age group 1 (10-25 years) and lower ETR 27.18% was recorded in patients of age group 2 (26-40 years). Our study revealed HBsAg as the better serological marker in the detection of active HBV infection as compared to HBeAg. HBV genotype D was found as the most prevalent genotype in male patients mostly of age group 71-85 yearsand remained high in district Peshawar. Phylogenetic analysis also showed high sequence similarity of our sequences with those already reported from Pakistan as well as from other parts of the world. While one isolate didn’t cluster with any sequence showing a great divergence and distinct origin of the strain.Our study confirmed that response of ETV-TDF combination therapy was good as compared to ETV monotherapyso it may be a good choice of treatment for the patients.