RESUMO
Hepatitis B virus (HBV) genotypes are important in both the clinical manifestation of disease and treatment response. Although Kenya belongs to the African Region (AFR-E) characterized by high mortality and hyperendemicity of HBV, there is a paucity of HBV genotyping data. The aim of this study was to molecularly characterize the basic core promoter/precore (BCP/PC) and complete surface (S) regions of HBV isolated from 61 HBsAg-positive liver disease patients attending Kenyatta National Hospital in Nairobi. HBsAg, HBeAg and viral loads were determined. HBV DNA was amplified and sequenced from 58/61 patients. In addition to the complete genome of two isolates, the BCP/PC and the complete S regions of 43 and 38 isolates, respectively were sequenced. Following phylogenetic analysis of the S region, 38 isolates clustered with subgenotype A1, whereas two isolates clustered with genotype D, one with subgenotype D1 and another as an outlier of the clade containing subgenotype D6 and the D/E recombinant. When the complete genome of the latter isolate was sequenced it clustered with D6. The majority of isolates belonged to serological subtype adw2 and only four to ayw2. Three distinct groups of subgenotype A1, distinguished by different amino acid motifs, circulate in Kenya: two in the African cluster and a monophyletic clade in the "Asian" cluster. HBeAg-negativity was a result of G1896A in genotype D isolates, whereas in subgenotype A1, the HBeAg-negativity was a result of mutations in the Kozak region (1809-1812) or precore start codon (1814-1816). Mutations at positions 1762 and 1764 occurred more frequently in HCC patients (p<0.05). In conclusion, subgenotypes A1, D1 and D6 circulate in liver disease patients in Kenya, with A1 predominating.
Assuntos
Carcinoma Hepatocelular/virologia , Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Cirrose Hepática/virologia , Neoplasias Hepáticas/virologia , Adulto , Sequência de Bases , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/genética , Estudos Transversais , DNA Viral/sangue , DNA Viral/genética , Feminino , Genótipo , Técnicas de Genotipagem , Antígenos de Superfície da Hepatite B/genética , Antígenos E da Hepatite B/genética , Vírus da Hepatite B/classificação , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/diagnóstico , Hepatite B Crônica/genética , Humanos , Quênia , Cirrose Hepática/diagnóstico , Cirrose Hepática/genética , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/genética , Masculino , Filogenia , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Carga ViralRESUMO
AIM: To molecularly characterize hepatitis B virus (HBV) isolates from Kerala and to relate them to the clinical manifestation of infection. METHODS: Sera and clinical data were collected from 91 patients diagnosed with chronic HBV infection and HBV-related hepatocellular carcinoma (HCC). HBV from 44 HCC, 22 cirrhotic and 25 chronic hepatitis patients were genotyped by sequencing of the complete S region or by restriction fragment length polymorphism assays. The basic core promoter/precore region was sequenced. The complete surface DNA sequences were assembled and aligned manually, and then compared with the sequences of HBV of genotypes (A-J) from GenBank. The evolutionary history was inferred using the Neighbor-Joining method and the evolutionary distances computed using the Kimura 2-parameter method. Bootstrapping was performed using 1000 replicates. The TaqMan BS-1 probe was used to quantify HBV DNA at a lower detection limit of approximately 20 IU/mL. Continuous variables were compared using an independent Student's t test. The χ² test or Fisher's exact test was used to compare categorical variables. The differences were considered statistically significant at P < 0.05. RESULTS: Irrespective of disease status, the predominant genotype was A (72%); 95% belonging to subgenotype A1, followed by genotypes D (27%) and C (1%). HCC patients infected with subgenotype A1 were significantly younger than those infected with D. Mutation A1762T/G1764A was significantly associated with HCC in both genotypes A and D. Mutation G1862T was more frequent in subgenotype A1 (P < 0.0001), and in combination with A1762T/G1764A, it was significantly associated with HBV from HCC patients. Mutation C1766T/T1768A was significantly associated with genotype A (P = 0.05) and HCC (P = 0.03). The preS2 start codon M1T/I mutation was unique to genotype A strains (15.6%) from all disease groups and occurred at a higher frequency in isolates from HCC patients (P = 0.076). A higher frequency of preS deletion mutants (33.3%) was observed in genotype A from HCC compared with non-HCC patients, but did not reach statistical significance. The preS2:F22L mutation was found in genotypes A and D. CONCLUSION: Kerala is the first Indian state in which subgenotype A1 has been found to predominate in liver disease patients who developed HCC at a relatively young age.
