Hepatitis B virus genetic heterogeneity and drug resistance among jaundiced patients at Coast General Teaching and Referral Hospital, Mombasa County, Kenya

Introduction

In spite of the existing control and preventive measures, hepatitis B virus (HBV) infections have remained a public health problem worldwide. In the recent past, great advances have been made toward management of these infections. HBV treatment at the onset involved the use of interferon alpha allowing change in natural progression of HBV.^[1] The Kenyan Ministry of Health treatment guidelines for HBV (2014) recommends the use of entecavir and tenofovir as preferred nucleoside analogs treatment with one immune-modulating drug; pegylated interferon alpha 2a. However, long time use of nucleoside analogs can result into emergence of drug-resistant strains.^[2] Therefore, the use of drug combination of entecavir and tenofovir is preferred.^[2-4]

To date, HBV is differentiated into 10 genotypes (A-J) based on the divergence of HBV genomic sequence by either more than 8.0% or greater than 4.0% in the S gene.^[5] The HBV genotypes show specific pathogenesis which result to diverse clinical results among HBV chronic patients world over.^[6] HBV genotype A and B patients respond better to therapeutic interferon based with genotype A having greater tendency of chronicity.^[7] Further, patients infected with genotypes C and D tend to have reduced rate of seroconversion with genotype D having a higher rate of chronicity and mutations.^[6] However, genotypes C and D have higher chances of mutations on core promoter and pre-S genes and disease progression to hepatocellular carcinoma.^[7] HBV genotypes are further segregated into subgenotypes based on the divergence of the genomic sequence between 4.0% and 8.0%.^[5] In Africa, mainly genotype A and to a varying extend genotypes C, D, and E have been detected.^[5,6] East and Central African countries are invaded predominantly by HBV genotype A of subtype A1 with North Africa countries having D1 and D7 subgenotypes.^[6] In addition, HBV subgenotype A2 has also been detected in South Africa.^[8] In Kenya, HBV genotypes A-E have been detected in some parts of the country.^[9-12] Despite the information on HBV genetic diversity and drug resistance being critical in patients’ management, this information is still elusive. Mombasa is populated with high-risk populations to HBV which could drive this infections transmission networks. The information on distribution of HBV genotypes and drug resistance among jaundiced patients in Mombasa County is not clear yet albeit Mombasa being known to be populated with high-risk population to these infections. Therefore, this study was aimed at determining HBV genetic diversity and drug resistance among patients presenting with jaundice and other related symptoms at Coast General Hospital, Mombasa County, Kenya.

Patients and Methods

A hospital-based cross-sectional study was done and a total of 222 samples collected among jaundice patients at the casualty, pediatric clinic, hepatic clinic of outpatient departments ,and inpatients seeking medical services at Coast General Teaching and Referral Hospital, Mombasa County, Kenya. The study participants of which were both in and outpatients were recruited during the period between February 2018 and August 2018. Ethical approval was obtained from Kenyatta University Ethical Review Committee; Ref. KU/ERC/APPROVAL/VOL.I (104) and National Ethical Review Committee (NACOSTI/P/18/46294/25080) before execution. Structured questionnaire was administered to patients who consented to obtain demographic data, clinical history of jaundiced patients due to HBV infection, and clinical symptoms. Venous blood samples were obtained and screened for HBV infections.

HBV amplifications

Approximately 5 ml blood samples were used and viral DNA extracted using QIAamp DNA Blood Mini Viral Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions,^[4,10] HBV pol gene (HBV-pol) was amplified by nested polymerase chain reaction (PCR) using primers; F₁ 5’-CCTGCTGGTGGCTCCAGTTC-3’ and R₁ 5’-CGTCCCGCG (AC) AGGATCCAGTT-3’ in the first PCR and the second PCR primers were; F₂ 5’-CYTGGCCWAAATTCGCAGTCCC -3’and R₂ 5’-GCAAANCCCAAAAGACCACAAT-3’.^[9] This was performed in 50 μL reaction volume constituting; 20 μL genomic DNA, 10 μL 10× PCR buffer (Qiagen), 4 μL dNTPs (Thermo Fisher Scientific), 2.5 μL of each primer sequence, 0.5 μL Hot Start Taq DNA Polymerase, (Qiagen GmbH, Hilden, Germany), and 10.5 μL of 2 mM MgCl₂. The amplifications conditions were similar Hot Start; initial activation at 95°C for 15 min in 35 cycles with each cycle 94°C for 45 s denaturation, annealing at 60°C for 45 s, extension at 72°C for 60 s, and final extension at 72°C for 10 min were used in both the first and second round PCRs.^[10,12] The PCR amplification was confirmed by visualization with ethidium bromide staining of the gel. The confirmed products from second round PCR were then purified using QIAquick kit (Qiagen Inc., Valencia, CA).^[10,12,13] followed by bidirectional population sequencing using an automated sequencer ABI 377 (Applied Biosystems, Foster City, CA) [Figure 1].^[10] The generated sequences were phylogenetically analyzed using MEGA X version 10.0.4.^[10,12,13] [Figure 2] Genotypic drug resistance in the HBV pol region was defined as the presence of one or more resistance mutations as specified by the consensus mutations in the HBV http://hbv.geno2pheno.org/index.php.^[4,10] The HBV isolate sequences were submitted to a web-based software prediction to genotypic resistance for mutations which are genotype-specific and polymerase gene (RT mutation) (Max-Planck-Institute for Informatik, Germany) at https://hivdb.stanford.edu/HBV/HBVseq/development/hbvseq.^[10]

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Figure 1:

Flowchart of protocol used in the study

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Figure 2:

Phylogenetic tree of hepatitis B virus (HBV) pol gene sequences from Coast General Teaching and Referral Hospital, Mombasa, Kenya. Neighbor-joining method at 1000 bootstrap replicates was used. Chimpanzee HBV (Orangutan Samad – AF193863) was the outgroup used. Bootstrap values >70% are shown and the HBV isolates from participants of the study are specified in

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Results

Discussion

The HBV overall infection level was higher than those previously detected in Kenya; 2.36%,^[14] 3%,^[10] 3.8%,^[14] 6.0%,^[15] 7.25%,^[10] 13.3%,^[16] and 14.6%,^[17,18] Uganda 14.9%,^[19] and in other African countries such as Zambia 9.9%, Malawi 6.7%, Uganda 4.9%, Ethiopia 4.7%, and Rwanda 2.4%.^[15] This high HBV infection rate in this study is attributed to high likelihood of study participants being of HBV high-risk groups, especially intravenous drug users, therefore, treatment and management among this group are of necessity.^[20] In comparison, the finding was lower than some studies conducted in Ghana 54.2%^[21] and Kenya 50.6%.^[11] The variations depicted in the prevalence rates were related to the sample size and the study populations.

Kenyan Ministry of Health directs the use of entecavir and tenofovir for the treatment of HBV through the clinical protocol for HBV therapeutic guidelines for hepatitis B (2014). On the other hand, long duration use of these drugs could be a factor to emergence of HBV-resistant strains. HBV drug resistance mutations have been highlighted in the treatment and management of chronic HBV patients,^[3] therefore, tracking of primary and secondary resistance mutations among patients is of great value in monitoring, optimizing, and choosing the suitable treatment in achieving sustainable virological response, thus reducing disease progression to hepatocellular carcinoma.

This study found high overall drug resistance prevalence of 12 (26.7%) [Table 2] which could be linked to very low genetic barrier to lamivudine. This prevalence was higher compared to the previous studies.^[10,22,23] This disparity could be due to different therapeutic management periods of patients and low genetic barrier to LAM.^[10] Mutation rtM204V occurred predominantly followed by rtL180M; rtM204V [Table 2] mutation known to be primary mutation since it is susceptible to HBV and lamivudine while rtL180M mutation is taken to be secondary mutation due to its ability to enhance viral replication.^[3] This finding concurs with observations in the previous studies in Kenya and other countries.^[3,10,13,24] This is in contrary to a study in Ethiopia on HIV/HBV patients on second-line therapy.^[25] In addition, there was no rtM204I mutation detected in the study; absence of this mutation with detection of rtM204V mutation confirms the predominance of genotype A in drug resistance mutations.^[22] This finding is similar to the previous studies.^[10,22]

Cross-resistance to 3CT, ETV, and LdT was detected in 11 samples in this study [Table 2], however, data on prior exposure to therapies were not captured. Therefore, there could be long-term 3CT therapy failure and exposure to viral strains harboring resistance mutational patterns associated with cross-resistance.^[2] In addition, resistance mutation rtV173L, rtL180M, and rtM204V can cause alterations in the S gene resulting to immune escape variants hence greater number of viral strains with cross-resistance.^[3,26] In addition, the presence of secondary mutation in amino acid position 180 (rtL180M) in pol gene can reinstate hepatitis B capacity to replicate.^[2] Further, hepatitis B has the ability to stay for long as covalently closed circular DNA even under seroclearance or treatment making the covalently closed circular HBV DNA to act as a store for drug resistance strains.^[2] This finding is similar to observations in the previous studies.^[2,10]

There was no detected resistance mutation to adefovir and tenofovir in this study. This concur with finding in the previous studies.^[10,13,25] This is linked to their high anti-HBV potency with high genetic barrier to drug resistance.^[26] Therefore, this supports the reference by the World Health Organization that tenofovir be used in treatment patients with HIV/HBV coinfection.^[13] However, this was in contrary to an observation from the previous study.^[22] This discrepancy in observations could be associated to diverse treatment periods.

The detected genotypes concur with the previous studies that have also shown the same distribution.^[10-12] The phylogenetic analysis revealed that genotype A1 was the most predominantly circulating among the studied subjects [Figure 2]. These findings agree with the previous studies that have been conducted in the country and other surrounding countries.^[6,9-12] HBV genotype A1 sequences were clustered with reference sequences from Kenya and Uganda.^[6] This trend indicates its possible East African origin with an implication of these strains circulating locally among Kenyan residents. By virtue of population, migration across East African countries could explain this alignment. Subgenotype A2 sequences were aligned with those from Martinique and Belgium as an indication of its possible origins [Figure 2].^[1,6] In addition, sequences of subgenotypes A3, B, and genotype C2 clustered with references sequences from Nigeria, China, and Japan.^[6,8] The detected viral genotypes confirm the persistent and stability of this viral strains circulating within the country. In contrary to the previous studies conducted in the country, this study confirmed possible existing of other HBV genotypes circulating among Kenyan population. HBV genotypes A3, B, and C2 were detected even though at low proportion. Despite low proportion of the sample used, there are newly detected genotypes in this study. As a result, there is a likelihood of existence of possible other genotypes in this region.^[9,10,12] The genotypes D and E in the previous studies were detected among patients who had a history of intravenous drug use.^[9,10,12]

Conclusion

The high HBV drug resistance was detected among hepatitis B patients, high resistance to LAM and cross-resistance to entecavir. As a result, we recommend a combination of tenofovir and lamivudine for the treatment and management of HBV patients. HBV genotypes A1 remains the most predominant genotype 35 (74.5%) with new genotypes A3, B and C2 1(0.02%) being detected in this region. Detection of new HBV genotypes in Kenya calls for continuous surveillance of HBV infections and circulating trends of HBV genotypes.