Molecular Characterization Of Acute And Chronic Hepatitis B Virus Among Patients Attending Selected Hospitals In Kenya

Abstract

Background: Hepatitis B Virus accounts for majority of liver related deaths in the world with 2 billion persons infected worldwide. It’s primarily transmitted through contact with body fluids of the infected person. The virus is categorized into ten (A-J) geographically distributed genotypes. The infection starts as acute infection and may lead to chronic infection and liver cancer. The virus has four phases; the immune-tolerant phase, immune-clearance phase, Immune control Phase and Immune escape Phase. What exactly influences HBV natural disease development from acute state to chronic state and to liver cancer is not so clear, however, the progression may depends on changes on the viral genome and the host’s immunity clonal selection mechanism. The changes occur through genome mutations for the host and virus. These genome changes can be detected early before the onset of chronic infection. Objectives: The aim of the study was to characterize and compare hepatitis B Virus and Human TP53 tumor suppressor gene responsible for acute and chronic viral hepatitis among patients attending selected clinics in Nairobi, Kisumu and Eldoret. The study also established a consensus nucleotide sequences from the sequences generated, to be used in development of a kit that may detect and/or predict Hepatitis B Viruses likely to develop chronic infection. Methodology: Blood samples were taken for serological and molecular analysis from patients suspected as having viral hepatitis and Liver cancer in the selected clinics in Nairobi, Kisumu and Eldoret. The samples were transported to KEMRI where they were screened for Hepatitis B surface antigen (HBsAg), antibody to the Hepatitis B core protein (HBcAb), anti-HIV and anti-HCV using commercially available kits. Viral DNA of BCP/PC, polymerase and Surface regions was extracted, amplified and sequenced from all HBsAg positive. Similarly human TP53 exon 4 and 7 was amplified and sequenced. Antibodies to HBV core protein (HBcAb) IgM positive and HBsAg were considered as acute infection whereas HBV core protein (HBcAb) IgG positive and HBsAg positive were Chronic infection. Statistical data analysis of factors will be performed using SPSS, Univariate descriptive analyses using proportions for categorical variables and measures of central tendency for continuous variables was done. The sequences were aligned and mutation detected using Bioedit software. The Phylogenetic trees drawn using Mega 7 software. Results and Discussion A total of 389 samples from patients with jaundice were collected from the three selected sites; of which Nairobi (KN) and its environs had 285 , Eldoret (ELD) and Kisumu had 28 and 76 samples respectively. The mean age (±SD) of patients was 39.8 years (±14.1) for Nairobi, 35.2 years xii (±11.9) for Kisumu, 32.1 years (±10.4) for Eldoret. The prevalence of Hepatitis B virus was found in 50.6 % using HBsAg and only 2.3% had probable acute infection with IgM antibody to the core protein positive.. 132 samples were successfully sequenced. Among HBV DNA positive specimens HBV genotype A was the most prevalent (90.0 %) then genotype D (9.7 %) and E (0.7%). Analysis of HBV/D full genome isolate had close similarity to subgenotype D6, subgenotype D4 and HBV D/E recombinant. Two isolated recombinant sequences had greater than 4 % nucleotide divergence from other previously known HBV D/E recombinants. Among 61 HBsAg negative randomly selected samples, Occult hepatitis B (OBI) was determined to be 32.2% and was significantly associated with a HBc positive status and a lower mean a HBs titre in jaundiced patients. A1762T/G1764A and G1896A pre core and core mutations were common among Hepatitis B chronic patients. Two known drug resistance mutations (A194T and V191I) were detected in sequence from two chronic patients; one genotype D and the other genotype A. Among TP53 positive samples it was observed that at Codon 72, Homozygous Proline (P) amino acid was the most common (54.5%), as compared to homozygous Arg/Arg (R) (12.1%,) which was the least common. Other patients had the heterozygous Arg/Pro (33.3%) indicating that the patients had both the Proline and arginine in either the forward or reverse strand. The homozygous state was observed in the extreme ends with homozygous Arg/Arg being common among those patients without HCC and homozygous Pro/Pro being common among with HCC At Codon 249, patients who had serine mutation (Arginine changing to Serine) were 24.2% of which 75.0% of them had HCC and the remaining 25.0% had only hepatitis without HCC. All pre-core and core mutations A1762T/G1764A (33.3%) and G1896A (83.3%) were among HCC with TP53 Codon 249 mutation. Generally, the study did not observe any association between patients with HCC and either codon 7 or 249 polymorphisms (Fisher test=3.5 and p=0.12). Conclusions: From the results, Hepatitis B virus is prevalent among jaundice patients, seeking health care at Kenyan hospitals. The prevalence observed in the study was higher than the national Hepatitis B prevalence. There are new recombinant strains of Hepatitis B virus among the selected Kenyan population that very little information is known about them. Similarly, OBI was determined to be highly prevalent among jaundiced patients in Kenya presenting for medical care (32.2%) with mutations T116N and T118A observed within the HBsAg major hydrophilic region. Kenyan population have variant Codon 72 and Codon 249 polymorphisms, however there was no association between HCC and the codons polymorphisms. xiii Recommendation: A number of mutations and viral molecular diversity have been observed in this study, we recommend that HBV Diversity in the country should be considered when designing interventions for HBV.