Molecular characterization of hepatitis B virus in blood donors in Botswana.

Hepatitis B virus (HBV) poses a significant threat to blood transfusion safety in sub-Saharan Africa (SSA) where allogeneic blood donations are screened serologically, and more sensitive nucleic acid tests (NATs) are utilized infrequently. HBV strains circulating among blood donors in Botswana are not yet characterized. We designed a cross-sectional study to determine the HBV sub-genotypes and prevalence of hepatitis B surface antigen (HBsAg) among blood donors between November 2014 and October 2015. A total of 12,575 blood donations were screened for HBsAg and 50 consecutive plasma samples were selected for genotyping from confirmed HBsAg+ donations. Overlapping Pol and complete S (Pol/S) open reading frames (ORFs) were sequenced from extracted HBV DNA. To identify any signature amino acids, mutations were compared to sequences from a cohort of chronic HBV patients co-infected with HIV and were treatment naïve. The prevalence of HBsAg+ blood donors was 1.02% (95% CI 0.9-1.2%), and the circulating sub-genotypes were A1 serotype adw2 (36.1%), D2 serotype ayw2 (2.9%), and D3 serotypes ayw 1/2 (58.3%). Prevalence of escape mutations was 14% from HBV isolates of blood donors and 15% from isolates of HBV/HIV co-infected patients (p = 0.6926). The escape mutations sP120L, sG130R, sY134H, and sD144A were identified predominantly among HBV isolates from blood donors. These escape mutations have been associated with accelerated HBV sequelae [e.g., liver cirrhosis (LC) and hepatocellular carcinoma (HCC)], failure to detect HBsAg, inability to respond to immunoglobulin (Ig) therapy, and HBV vaccine escape. Characterizing the HBV burden, circulating sub-genotypes, and clinically relevant mutations among blood donors in Botswana is important to elucidate the efficacy of currently available vaccines, predicting HBV-transmission patterns, understanding the cohort's risk to HBV-related complications, and to developing prevention strategies and effective genotype-based antiretroviral therapies.

Molecular characterization of hepatitis C virus in liver disease patients in Botswana: a retrospective cross-sectional study.

BACKGROUND: Hepatitis C virus (HCV) infection is a major cause of chronic liver disease globally. Direct acting antivirals (DAAs) have proven effective in curing HCV. However, the current standard of care (SOC) in Botswana remains PEGylated interferon-α (IFN-α) with ribavirin. Several mutations have been reported to confer resistance to interferon-based treatments. Therefore, there is a need to determine HCV genotypes in Botswana, as these data will guide new treatment guidelines and understanding of HCV epidemiology in Botswana. METHODS: This was a retrospective cross-sectional pilot study utilizing plasma obtained from 55 participants from Princess Marina Hospital in Gaborone, Botswana. The partial core region of HCV was amplified, and genotypes were determined using phylogenetic analysis. RESULTS: Four genotype 5a and two genotype 4v sequences were identified. Two significant mutations - K10Q and R70Q - were observed in genotype 5a sequences and have been associated with increased risk of hepatocellular carcinoma (HCC), while R70Q confers resistance to interferon-based treatments. CONCLUSION: Genotypes 5a and 4v are circulating in Botswana. The presence of mutations in genotype 5 suggests that some patients may not respond to IFN-based regimens. The information obtained in this study, in addition to the World health organization (WHO) recommendations, can be utilized by policy makers to implement DAAs as the new SOC for HCV treatment in Botswana.

OPTIMIZATION OF DRIED BLOOD SPOT ASSAYS FOR HEPATITIS B VIRUS SURFACE ANTIBODY QUANTIFICATION.

Dried blood spot (DBS) cards can be used as an alternative sample collection method to plasma, however, there is no optimized elution protocol for DBS cards specifically for hepatitis B surface antibody (anti-HBs) testing. The study aimed to develop a DBS elution protocol for anti-HBs quantification. Our study sought to determine the ideal phosphate-buffered saline (PBS) buffer volume to use by comparing three PBS volumes (300uL, 450uL, and 500uL), and the optimal time to agitate DBS discs on a plate shaker (1hr, 2hrs, 3hrs, and 4hrs) to yield DBS anti-HBs concentrations that are comparable to corresponding plasma anti-HBs concentrations. The optimal DBS storage temperature (25°C, -20°C, and -80°C) was investigated to determine the ideal long-term storage temperature of the cards. Residual samples were used for optimization (2019-2021). A total of 50 DBS-plasma pairs was used throughout the study, with plasma anti-HBs concentrations being used as the golden standard to compare. The analysis of results was carried out by determining the p-values of the Wilcoxon sign rank. A two-way analysis of variance (ANOVA) was also performed to determine the impact of PBS elution volumes, elution time, and storage temperature on the anti-HBs concentration of DBS samples on STATA Version 15.0. No statistically significant difference between the DBS-plasma anti-HBs pairs was observed when using 450 or 500uL of PBS buffer and when samples were agitated for 3 hours (p=0.594, p=0.499 respectively). The optimal storage temperature for DBS cards was 25°C because the results showed no statistically significant difference between DBS-plasma anti-HBs titers (p=0.594). The two-way ANOVA analysis showed that elution volumes and time had no statistically significant impact on the DBS anti-HBs concentrations, p=0.948 and p=0.381 respectively. Storage temperature had a statistically significant impact on the DBS anti-HBs concentrations, p=0.002. The optimized DBS elution protocol for anti-HBs quantification will help monitor vaccine efficacy in infants due to the low sample volumes required compared to plasma and also can be used for anti-HBs testing in resource-limited areas around the country.

Hepatitis B surface antigen loss in individuals with chronic hepatitis B virus and HIV-1 infections in Botswana.

OBJECTIVES: We sought to determine hepatitis B surface antigen (HBsAg) loss and its predictors among people with chronic hepatitis B (CHB) infections and HIV (PWH) in Botswana. METHODS: Archived plasma samples from a cohort of PWH in Botswana (2013-2018) with 3 yearly time-points were used. Samples were screened for HBsAg, immunoglobulin M HBV core antibodies (anti-HBc IgM) and HBV e-antigen (HBeAg) at all time points. HBV deoxyribonucleic acid (DNA) quantification was done at baseline. The Wilcoxon rank-sum was used to compare continuous variables while the chi-squared test and Fishers exact test were used for categorical data wherever appropriate. Logistic regression was used to assess predictors of seroclearance. RESULTS: Of 141 participants with HBsAg-positive serology (HBsAg+) at baseline, 92.2% (131/141) [95% confidence interval (CI) 87.4-96.1] were persistently HBsAg+ at year 1. We report a HBsAg loss of 7.1% (10/141) (95% CI 3.9-12.6) among participants with negative HBeAg and negative IgM serologies. HBsAg loss was 6.3% (7/111) among antiretroviral therapy (ART)-experienced participants and 10.7% (3/28) (95% CI 0.4-5.0) in ART-naive participants. Most participants who had positive anti-HBc IgM serology and did not lose HBsAg were on either lamivudine (3TC)-based therapy or non-tenofovir disoproxil fumarate (TDF)-based therapy, except for one participant. The participants also had varying HBeAg status. HBsAg loss was independent of HIV viral load, CD4 + cell count, age, and sex. CONCLUSION: We report a HBsAg loss of 6.3% over a 3-year period among ART-experienced CHB participants. Future studies that focus on HBsAg loss in mono-infected patients and the possible correlation between HBeAg status and HBsAg loss are warranted.

Impact of Hepatitis Delta Virus Infection on the Selection of Hepatitis B Surface Antigen Mutations.

The interaction of multiple viruses in one host is thought to enhance the development of mutations. However, the impact of hepatitis D virus (HDV) positivity on the development of unique hepatitis B virus (HBV) mutations among people living with human immunodeficiency virus (HIV) (PLWH) remains poorly understood in African countries, including Botswana. We used HBV sequences generated from the Botswana Combination Prevention Project (BCPP), which is the largest pair-matched cluster-randomized HIV trial in Botswana. Only participants with available HBV sequences (n = 55) were included in our study ([HIV/HBV-positive (n = 50) and HIV/HBV/HDV-positive (n = 5)]. Geno2pheno was used to determine HBV genotypes, and HBV surface region sequences (all subgenotype A1) were aligned in AliView for mutational analysis, while the impact of mutations was assessed using Phyre2. Our results identified 182 common mutations between the two groups. In the HIV/HBV/HDV cohort, only three mutations (L95W, W156Q, C221Y) were classified as deleterious, with only L95W being the most frequent. In the HIV/HBV cohort, four mutations (W199R, C221A, C221S, W223G) were also classified as deleterious. Our results demonstrate the presence of unique HBV mutations among the HIV/HBV/HDV-positive cohort. Functional characterization of these mutations is recommended to determine their effect on HDV.

High Prevalence of Hepatitis B Virus Drug Resistance Mutations to Lamivudine among People with HIV/HBV Coinfection in Rural and Peri-Urban Communities in Botswana.

(1) Background: We aimed to determine the prevalence of hepatitis B virus (HBV) resistance-associated mutations (RAMs) in people with HBV and human immunodeficiency virus (HBV/HIV) in Botswana. (2) Methods: We sequenced HBV deoxyribonucleic acid (DNA) from participants with HBV/HIV from the Botswana Combination Prevention Project study (2013-2018) using the Oxford Nanopore GridION platform. Consensus sequences were analyzed for genotypic and mutational profiles. (3) Results: Overall, 98 HBV sequences had evaluable reverse transcriptase region coverage. The median participant age was 43 years (IQR: 37, 49) and 66/98 (67.4%) were female. Most participants, i.e., 86/98 (87.8%) had suppressed HIV viral load (VL). HBV RAMs were identified in 61/98 (62.2%) participants. Most RAMs were in positions 204 (60.3%), 180 (50.5%), and 173 (33.3%), mostly associated with lamivudine resistance. The triple mutations rtM204V/L180M/V173L were the most predominant (17/61 [27.9%]). Most participants (96.7%) with RAMs were on antiretroviral therapy for a median duration of 7.5 years (IQR: 4.8, 10.5). Approximately 27.9% (17/61) of participants with RAMs had undetectable HBV VL, 50.8% (31/61) had VL < 2000 IU/mL, and 13/61 (21.3%) had VL ≥ 2000 IU/mL. (4) Conclusions: The high prevalence of lamivudine RAMs discourages the use of ART regimens with 3TC as the only HBV-active drug in people with HIV/HBV.

Molecular Characterization of Hepatitis B Virus in People Living with HIV in Rural and Peri-Urban Communities in Botswana.

(1) Background: Hepatitis B virus (HBV) sequencing data are important for monitoring HBV evolution. We aimed to molecularly characterize HBV sequences from participants with HBV surface antigen-positive (HBsAg+) serology and occult hepatitis B infection (OBI+). (2) Methods: We utilized archived plasma samples from people living with human immunodeficiency virus (PLWH) in Botswana. HBV DNA was sequenced, genotyped and analyzed for mutations. We compared mutations from study sequences to those from previously generated HBV sequences in Botswana. The impact of OBI-associated mutations on protein function was assessed using the Protein Variation Effect Analyzer. (3) Results: Sequencing success was higher in HBsAg+ than in OBI+ samples [86/128 (67.2%) vs. 21/71 (29.2%)]. Overall, 93.5% (100/107) of sequences were genotype A1, 2.8% (3/107) were D3 and 3.7% (4/107) were E. We identified 13 escape mutations in 18/90 (20%) sequences with HBsAg coverage, with K122R having the highest frequency. The mutational profile of current sequences differed from previous Botswana HBV sequences, suggesting possible mutational changes over time. Mutations deemed to have an impact on protein function were tpQ6H, surfaceV194A and preCW28L. (4) Conclusions: We characterized HBV sequences from PLWH in Botswana. Escape mutations were prevalent and were not associated with OBI. Longitudinal HBV studies are needed to investigate HBV natural evolution.

Hepatitis Delta Virus Clade 8 Is the Predominant Clade Circulating in Botswana amongst People Living with HIV.

Hepatitis delta virus (HDV) co-infections more often result in severe hepatitis compared to hepatitis B virus (HBV) infections alone. Despite a high HDV prevalence (7.1%), information regarding circulating HDV clades is very limited in Botswana. We extracted total nucleic acid from confirmed HDV-positive samples and quantified their viral load. We then sequenced the large hepatitis delta antigen (L-HDAg) using Oxford Nanopore Technology (ONT). Genotyping was performed using the HDV Database, and HDV mutation profiling was performed on AliView. All participants with HBV genotypic information belonged to sub-genotype A1, and 80% (4/5) of them had a higher HDV viral load and a lower HBV viral load. We sequenced 75% (9/12) of the HDV-positive samples, which belonged to HDV clade 8. A total of 54 mutations were discovered, with the most prevalent being Q148R (16%), D149P (16%) and G151D (16%). Known mutations such as S117A, K131R, R139K and G151D were detected, while the other mutations were novel. Our results reveal that HDV clade 8 is the predominant clade in Botswana. The significance of all mutations remains unclear. Future studies with a larger sample size to detect other HDV clades that might be circulating in Botswana and functionally characterize the detected mutations are warranted.

Persistence and risk factors of occult hepatitis B virus infections among antiretroviral therapy-naïve people living with HIV in Botswana.

Aim: This study aimed to determine the kinetics of occult hepatitis B virus infections (OBI) among people with HIV (PWH). Methods: The study used archived plasma samples from longitudinal HIV natural history studies. We identified new OBI cases and assessed risk factors for OBI using Cox proportional hazards regression analysis. Results: At baseline, 8 of 382 [(2.1%) (95% CI: 1.06-4.1)] samples tested positive for hepatitis B surface antigen (HBsAg+). Of the 374 HBsAg-negative samples, 76 had sufficient sample volume for HBV DNA screening. OBI positivity (OBI+) at baseline was reported in 11 of 76 [14.7 95% CI (8.3-24.1)] HBsAg-negative (HBsAg-) participants. Baseline HBsAg-negative samples with sufficient follow-up samples (n = 90) were used for analysis of newly identified OBI cases. Participants contributed 129.74 person-years to the study and were followed for a median of 1.02 years (IQR: 1.00-2.00). Cumulatively, there were 34 newly identified OBI cases from the 90 participants, at the rate of 26.2/100 person-years (95% CI: 18.7-36.7). Newly identified OBI cases were more common among men than women (61.1% vs. 31.9%) and among participants with CD4+ T-cell counts ≤450 cells/mL (p-value = 0.02). Most of the newly identified OBI cases [55.9% (19/34)] were possible reactivations as they were previously HBV core antibody positive. Conclusion: There was a high rate of newly identified OBI among young PWH in Botswana, especially in men and in participants with lower CD4+ T-cell counts. OBI screening in PWH should be considered because of the risk of transmission, possible reactivation, and risk factors for the development of chronic liver disease, including hepatocellular carcinoma.

Hepatitis B Virus Prevalence among HIV-Uninfected People Living in Rural and Peri-Urban Areas in Botswana.

(1) Background: we determined the prevalence of the hepatitis B virus (HBV) amongst people without human immunodeficiency virus (HIV) in rural and peri-urban areas in Botswana. (2) Methods: We screened for the hepatitis B surface antigen (HBsAg) from archived plasma samples of people without HIV (n = 2135) randomly selected from the Botswana Combination Prevention Program (BCPP) (2013-2018). We sequenced 415 bp of the surface region using BigDye sequencing chemistry. (3) Results: The median age of participants was 31 (IQR: 24-46) and 64% (1360/2135) were female. HBV prevalence was 4.0% (86/2135) [95% CI: 3.3-4.9]) and ranged between 0-9.2%. Older participants (>35 years) had increased odds of HBV positivity (OR: 1.94; 95% CI: [1.32-2.86]; p = 0.001). Thirteen samples were sequenced and seven (53.8%) were genotype A, three (23.1%) were genotype D and genotype E each. Clinically significant mutations were identified in the surface region, but no classic drug resistance mutations were identified. (4) Conclusions: We report an HBV prevalence of 4.0% (95% CI 3.3-4.9) among people without HIV in rural and peri-urban communities in Botswana with varying rates in different communities. A comprehensive national HBV program is required in Botswana to guide HBV prevention, testing and management.

High prevalence of hepatitis delta virus among people with hepatitis B virus and HIV coinfection in Botswana.

BACKGROUND: Approximately 15-20 million people worldwide are infected with hepatitis delta virus (HDV), which is approximately 5 % of people with chronic hepatitis B virus (HBV). Sub-Saharan Africa has high HDV prevalence, leading to worse clinical outcomes among people who are HIV/HBV/HDV tri-infected. There are limited data on HDV prevalence among people with HIV (PWH) who are HBV-infected and uninfected in Botswana. We, therefore, determined HDV prevalence among PWH in Botswana. METHODS: This was a retrospective cross-sectional study utilizing archived plasma samples from PWH with results for HBV markers such as hepatitis B surface antigen (HBsAg), hepatitis B core antibody (anti-HBc), immunoglobulin M antibody to hepatitis B core antigen (IgM anti-HBc) and hepatitis B e antigen (HBeAg). Samples were categorized according to their HBsAg status and screened for anti-HDV antibodies. Total nucleic acid was extracted from samples with a single positive anti-HDV result, and HDV ribonucleic acid (RNA) load was quantified using the Altona Diagnostic RealStar® HDV RT-PCR kit. Statistical analysis was performed using STATA version 14.0 where p-values < 0.05 were considered statistically significant. RESULTS: The study cohort (n = 478) included both HBsAg positive (44 %) and negative (56 %) participants, with a median age of 42 [IQR; 41-43]. Anti-HDV prevalence of (15/211) [7.1 %, 95 % CI: 4.4 - 11.4] was recorded among HBsAg positive participants, all of whom were IgM anti-HBc negative, while 5/6 participants were HBeAg negative. HDV RNA load was detected in 11/12 (92 %) anti-HDV-positive participants. No HDV prevalence was recorded among participants who were HBsAg negative, therefore, the overall HDV prevalence was (15/478) [3.1 %, 95 % CI: 1.9 - 5.1]. HIV viral load suppression was statistically insignificant, irrespective of HDV status. CONCLUSIONS: We report high HDV prevalence among HBsAg-positive PWH in Botswana. Most HDV-positive participants had active HDV infection, therefore, we recommend HDV screening in this cohort to guide their clinical care.

Atypical Hepatitis B Virus Serology Profile-Hepatitis B Surface Antigen-Positive/Hepatitis B Core Antibody-Negative-In Hepatitis B Virus/HIV Coinfected Individuals in Botswana.

(1) Background: Hepatitis B core antibodies (anti-HBc) are a marker of hepatitis B virus (HBV) exposure; hence, a normal HBV serology profile is characterized by HBV surface antigen (HBsAg) and anti-HBc positivity. However, atypical HBV serologies occur, and we aimed to determine the prevalence of an atypical profile (HBsAg+/anti-HBc-) in a cohort of people with HIV-1 (PWH) in Botswana. (2) Methods: Plasma samples from an HIV-1 cohort in Botswana (2013-2018) were used. The samples were screened for HBsAg and anti-HBc. Next-generation sequencing was performed using the GridION platform. The Wilcoxon rank-sum test and Chi-squared tests were used for the comparison of continuous and categorical variables, respectively. (3) Results: HBsAg+/anti-HBc- prevalence was 13.7% (95% CI 10.1-18.4) (36/263). HBsAg+/anti-HBc- participants were significantly younger (p < 0.001), female (p = 0.02) and ART-naïve (p = 0.04) and had a detectable HIV viral load (p = 0.02). There was no statistically significant difference in the number of mutations observed in participants with HBsAg+/anti-HBc- vs. those with HBsAg+/anti-HBc+ serology. (4) Conclusions: We report a high HBsAg+/anti-HBc- atypical serology profile prevalence among PWH in Botswana. We caution against HBV-testing algorithms that consider only anti-HBc+ samples for HBsAg testing, as they are likely to underestimate HBV prevalence. Studies to elucidate the mechanisms and implications of this profile are warranted.

High Prevalence of Hepatitis B Virus Infection Among People With HIV in Rural and Periurban Communities in Botswana.

Background: We aimed to determine the prevalence of hepatitis B virus (HBV) infection among people with human immunodeficiency virus (PWH) in rural and periurban communities in Botswana. Methods: PWH from a previous population-based study, the Botswana Prevention Combination Project, which enrolled adults in 30 communities across Botswana (2013-2018), were screened for HBV surface antigen (HBsAg) and HBV core antibody (anti-HBc). HBsAg-positive (HBsAg+) samples were further screened for HBV core immunoglobulin M antibodies (anti-HBc immunoglobulin M [IgM]) and HBV e antigen (HBeAg). We quantified HBV viral load on participants who tested positive (n = 148) and negative for HBsAg (n = 381). Results: Of 3304 participants tested, 271 (8% [95% confidence interval {CI}, 7%-9%]) were HBsAg+ while 1788 (56% [95% CI, 54%-57%]) of 3218 PWH whom we tested had positive anti-HBc. Approximately 88% of HBsAg+ participants were on antiretroviral therapy (ART), 40% and 56% of whom were receiving lamivudine- and tenofovir-containing ART, respectively. Male sex (relative risk ratio [RRR], 1.8 [95% CI, 1.2-2.7]) and the northern geographic region (RRR, 2.5 [95% CI, 1.4-4.7]) were independent predictors of HBV infection (HBsAg+). Of 381 persons with negative HBsAg who were tested for occult HBV, 126 (33% [95% CI, 29%-38%]) had positive HBV DNA. Eleven participants were highly viremic with high HBV viral load while on a lamivudine- or tenofovir-containing regimen. Ten (91%) of these participants also had positive HBeAg serology, while 4 (36%) had positive anti-HBc IgM serology. Conclusions: The prevalence of HBV was high among PWH in Botswana while on ART regimens with activity against HBV.

Decreased hepatitis B virus vaccine response among HIV-positive infants compared with HIV-negative infants in Botswana.

OBJECTIVES: We sought to determine vaccine antibody titres and the prevalence of hepatitis B surface antigen (HBsAg) in both HIV-positive and HIV-negative infants born to HIV-positive mothers in Botswana. DESIGN: This was a retrospective cross-sectional study using 449 archived dried blood spot samples from both HIV-positive and HIV-negative infants collected between 2016 and 2018. METHODS: We screened dried blood spot samples for HBsAg and determined hepatitis B surface antibody titres. We determined hepatitis B virus (HBV) genotypes by amplifying 415 base-pairs of the surface region. RESULTS: HIV-positive infants mounted a significantly lower immune response to the HBV vaccine (P  < 0.001). Furthermore, a lower proportion of HIV-positive infants had protective hepatitis B surface antibody titres (74.5%) than HIV-negative infants (89.2%) (P < 0.001). HIV-positive infants were older and 50.9% of them had completed vaccination (P = 0.018). Of the 449 infant samples tested, three (0.67%) were positive for HBsAg. Of the three HBsAg-positive infants, two had protective titres (>10 mIU/ml). Two of the three HBV-positive infants were infected with genotype D3 and had no drug-resistance or escape mutations. CONCLUSION: Vaccine response was lower among HIV-positive infants compared with HIV-negative infants. HBV infections were observed in both HIV-positive and HIV-negative infants in Botswana. Studies to investigate additional preventive strategies to reduce HBV mother-to-child transmission are recommended.

Hepatitis B virus prevalence and vaccine antibody titers in children HIV exposed but uninfected in Botswana.

BACKGROUND: Botswana introduced the HBV vaccine at birth for all newborns in 2000. To the best of our knowledge, since the introduction of HBV vaccination, there have been limited data for vaccine response to HBV and its impact on early childhood HBV infections among children HIV exposed but uninfected in Botswana. AIMS: To determine the prevalence of hepatitis B surface antigen (HBsAg) and HBV vaccine response in 18 months old children HIV exposed but uninfected in Botswana. METHODS: Stored plasma samples from 304 children at 18 months of age and 287 mothers from delivery were tested for HBsAg. Mothers with positive HBsAg had HBV DNA level tested, and their HBV genotypes were determined by amplifying a 415-base pair (bp) region of the surface gene. Plasma samples from children exposed to HIV were tested for hepatitis B surface antibody (anti-HBs) titers. RESULTS: No children (0 of 304) were positive for HBsAg at 18 months while 5 (1.74%) of 287 HIV-positive mothers were HBsAg positive. Four of the HBsAg positive mothers were infected with genotype A1, while 1 was infected with genotype E. The median anti-HBs titer in children was 174 mIU/mL [QR: 70, 457]. Three (1.1%) of 269 children had an inadequate vaccine response (<10 mIU/mL), while 266 (98.9%) of 269 had protective immunity. However, when using the ≥100mIU/mL threshold, only 170 (63.2%) of 269 children had complete protection. CONCLUSION: No HBsAg positivity was identified in a cohort of children HIV exposed but uninfected. The absence of HBsAg positives was associated with good HBV vaccine responses and low maternal HBsAg prevalence in Botswana.

Increased Prevalence of Liver Fibrosis and HIV Viremia among Patients with HIV, HBV, and Tuberculosis in Botswana.

People with concomitant human immunodeficiency virus (HIV) and tuberculosis (TB) have an increased risk of hepatotoxic reactions due to antiretroviral therapy (ART) and anti-TB therapy (ATT). Concomitant hepatitis B virus (HBV) in these patients may lead to poorer health outcomes. To assess liver enzyme levels and immune response in adults with HIV, HBV, and TB, data from 300 antiretroviral-naïve people living with HIV (PLWHIV) were analyzed. The prevalence of HIV/HBV (cHIV/HBV) and HIV/TB (cHIV/TB) was 28% (95% CI: 23.0-33.4) and 10% (95% CI: 6.8-14.0), respectively. HIV/HBV/TB (cHIV/HBV/TB) prevalence was 5.3% (95% CI: 3.1-8.5). There was a statistically significant difference between the groups of participants in HIV viral load (p = 0.004), hemoglobin levels (p = 0.025), and body mass index (p = 0.011). A larger proportion of cHIV/HBV/TB participants (37.5%) had an aspartate aminotransferase to platelet ratio index (APRI) score ≥0.5 (p = 0.013), a lower cutoff for significant liver fibrosis. Immunological non-responders (CD4+ T-cell count <20% gain and HIV viral load <400 copies/mL at 6 months) were observed in all groups except those with cHIV/TB. Our findings support the need to screen for infections that could cause excessive liver damage prior to ATT or ART initiation, such as HBV.

In Silico Prediction of Human Leukocytes Antigen (HLA) Class II Binding Hepatitis B Virus (HBV) Peptides in Botswana.

Hepatitis B virus (HBV) is the primary cause of liver-related malignancies worldwide, and there is no effective cure for chronic HBV infection (CHB) currently. Strong immunological responses induced by T cells are associated with HBV clearance during acute infection; however, the repertoire of epitopes (epi) presented by major histocompatibility complexes (MHCs) to elicit these responses in various African populations is not well understood. In silico approaches were used to map and investigate 15-mers HBV peptides restricted to 9 HLA class II alleles with high population coverage in Botswana. Sequences from 44 HBV genotype A and 48 genotype D surface genes (PreS/S) from Botswana were used. Of the 1819 epi bindings predicted, 20.2% were strong binders (SB), and none of the putative epi bind to all the 9 alleles suggesting that multi-epitope, genotype-based, population-based vaccines will be more effective against HBV infections as opposed to previously proposed broad potency epitope-vaccines which were assumed to work for all alleles. In total, there were 297 unique epi predicted from the 3 proteins and amongst, S regions had the highest number of epi (n = 186). Epitope-densities (Depi) between genotypes A and D were similar. A number of mutations that hindered HLA-peptide binding were observed. We also identified antigenic and genotype-specific peptides with characteristics that are well suited for the development of sensitive diagnostic kits. This study identified candidate peptides that can be used for developing multi-epitope vaccines and highly sensitive diagnostic kits against HBV infection in an African population. Our results suggest that viral variability may hinder HBV peptide-MHC binding, required to initiate a cascade of immunological responses against infection.

In Silico Analysis of Hepatitis B Virus Occult Associated Mutations in Botswana Using a Novel Algorithm.

Occult hepatitis B infections (OBI) represent a reservoir of undiagnosed and untreated hepatitis B virus (HBV), hence the need to identify mutations that lead to this phenotype. Functionally characterizing these mutations by in vitro studies is time-consuming and expensive. To bridge this gap, in silico approaches, which predict the effect of amino acid (aa) variants on HBV protein function, are necessary. We developed an algorithm for determining the relevance of OBI-associated mutations using in silico approaches. A 3 kb fragment of subgenotypes A1 and D3 from 24 chronic HBV-infected (CHB) and 24 OBI participants was analyzed. To develop and validate the algorithm, the effects of 68 previously characterized occult-associated mutations were determined using three computational tools: PolyPhen2, SNAP2, and PROVEAN. The percentage of deleterious mutations (with impact on protein function) predicted were 52 (76.5%) by PolyPhen2, 55 (80.9%) by SNAP2, and 65 (95.6%) by PROVEAN. At least two tools correctly predicted 59 (86.8%) mutations as deleterious. To identify OBI-associated mutations exclusive to Botswana, study sequences were compared to CHB sequences from GenBank. Of the 43 OBI-associated mutations identified, 26 (60.5%) were predicted by at least two tools to have an impact on protein function. To our knowledge, this is the first study to use in silico approaches to determine the impact of OBI-associated mutations, thereby identifying potential candidates for functional analysis to facilitate mechanistic studies of the OBI phenotype.

Chronic and Occult Hepatitis B Virus Infection in Pregnant Women in Botswana.

The hepatitis B virus (HBV) is a global problem; however, the burden of HBV infection in pregnant women in Botswana is unknown. We sought to determine the prevalence of chronic and occult HBV infection in human immunodeficiency virus (HIV)-infected and -uninfected pregnant women in Botswana. Samples from 752 pregnant women were tested for hepatitis B surface antigen (HBsAg), and HBsAg-positive samples were tested for hepatitis B e antigen (HBeAg) and HBV DNA load. Samples that were HBsAg negative were screened for occult HBV infection by determining the HBV DNA load. HBV genotypes were determined based on a 415-base-pair fragment of the surface gene. Among the 752 women tested during pregnancy or early postpartum, 16 (2.1%) (95% confidence interval (CI): 2.0⁻2.2) were HBsAg-positive. The prevalence of chronic HBV infection was higher (3.1%) among HIV-infected (95% CI: 3.0⁻3.2) compared with HIV-uninfected women (1.1%) (95% CI: 1.07⁻1.1, p = 0.057). Among the 622 HBsAg-negative women, the prevalence of occult HBV infection was 6.6% (95% CI: 6.5⁻6.7). Three of thirteen HBsAg-positive participants were HBeAg-positive, and all were HIV-negative. Of the 11 maternal samples successfully genotyped, five (45.5%) were genotype D3, five (45.5%) were genotype A1, and one was genotype E (9%). Low and similar proportions of HIV-infected and -uninfected pregnant women in Botswana had occult or chronic HBV infection. We identified a subset of HIV-negative pregnant women who had high HBV DNA levels and were HBeAg-positive, and thus likely to transmit HBV to their infants.