F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.132498.4 Research Article Articles Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients

[version 4; peer review: 1 approved, 2 approved with reservations, 1 not approved]

 Modise Lorato Conceptualization Data Curation Formal Analysis Funding Acquisition Investigation Methodology Project Administration Resources Validation Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0001-7008-157X a 1 Sithebe Nomathamsanqa Conceptualization Data Curation Formal Analysis Funding Acquisition Project Administration Writing – Original Draft Preparation Writing – Review & Editing 1 Mufhandu Hazel Data Curation Formal Analysis Supervision Validation Visualization Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-1551-3314 b 1 Biological Sciences, North West University, Mahikeng, North West, 2735, South Africa lorato.modise@smu.ac.za hazel.mufhandu@nwu.ac.za

No competing interests were disclosed.

 21 8 2025 2023 12 1232 15 8 2025 Copyright: © 2025 Modise L et al. 2025 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background

Co-infection of hepatitis B virus (HBV) and human immunodeficiency virus (HIV) has an impact on high HBV replication and progression to liver cancer. These may lead to cross-resistance of drugs due to natural mutations or therapeutic pressure. These require continuous monitoring of HBV variants for better diagnosis and treatment strategies.

 Methods

Convenience sampling was used to collect fifty archival sera from Inkosi Albert Luthuli Central Hospital. Sera were subjected to HBsAg screening using ELISA, DNA extraction, PCR amplification, Sanger sequencing, genotype prediction and mutation analysis.

 Results

Of the 50 samples, 86% (43/50) were HBsAg positive; 82% (41/50) PCR positive with 92% (38/41) sequenced and only 26 sequences were subjected to molecular characterization. The HBV sequences showed similarity to genotype A (73% [19/26]), genotypes G (5% [3/26]) and genotype C (15% [4/26]). Prevalence of the mutations in the surface region was (47% [18/38]); including diagnostic failure (K122R and T143S) and immune escape mutations (P127T, G145R, S207N, Y200T, E164D, Y206H and L209V). The mutations in the RT were at (36% [14/38]) with drug resistance mutations (DRM) at (50% [7/14]). Mutations showed resistance to lamivudine (LMV) at (35% [5/14]), telbivudine (LdT) at (29% [4/14]), (14% [2/14]) for entecavir (ETV) and (21% [3/14]) for adefovir (ADV). One sample had a combination of L180M, M204V, S202K, and M250I mutations.

 Conclusions

Our findings highlight the prevalence of HBV genotype A in HIV-infected patients in South Africa. The study provides evidence of mutations linked to immune evasion and drug resistance; this infers that these mutations may have clinical implications for the diagnosis and treatment of HBV in HBV/HIV co-infected individuals. Further in vitro studies must be conducted to explore the impact of the identified mutation on the surface protein expression during diagnosis; phenotype impact of the mutant virus towards the antiviral drugs.

 Hepatitis B virus HIV Drug resistance Vaccine escape Mutation Co-infection South African Centre for Epidemiological Modelling North-West University National Research foundation Organisation for Women in Science for the Developing Worlds This work was supported by funds from the North-West University, National Research foundation Innovation bursary (Grant No: SFH150727131319), Health and Welfare Sector Education and Training Authority, South African Centre for Epidemiological Modelling and Analysis and Organisation for Women in Science for the Developing Worlds (Grant No: 3240287283). These funding agencies were not involved and responsible in the study design and writing of the thesis and manuscript. The Authors in this study are solely responsible for the content and not the funding agencies involved. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Amendments from Version 3

The title of the study has been revised to make it more descriptive and specific clearly stating that the investigation of the study is on the mutations in the overlapping partial sequences of the Surface (S) and Polymerase (pol) genes of hepatitis B virus which are associated with immune escape and drug resistance in HIV-Infected patients. Under the introduction, the HBV genome features are briefly characterized; context is provided on the surface and polymerase genes, the proteins they encode for  including surface antigen (containing the major hydrophilic region) and polymerase. Description is briefly given on the roles of the encoded proteins and on the mutations in the genes and their association with causing viral immune escape and drug resistance. An update is done under table 1, since the N=50 mentioned and removed as see at (Table 1). I have changed N (study population) to n (sub-sample size of each demographic variable and not for total sample population (50). The same on the abstract.

 Introduction

The Orthohepadnavirus genus is a significant group of human viruses, and the HBV is a prototype of the Hepadnaviridae family ( Summers et al., 1975). More than 300 million people worldwide are chronically infected with hepatitis B, which can lead to severe liver disease and hepatocellular carcinoma (HCC), which accounts for more than 1 million annual deaths ( Musyoki et al., 2015). HBV/HIV co-infections in the region of South Africa (SA) are estimated to range from 5% to 23% ( Anastasiou et al., 2017; Kaswa & de Villiers, 2023; King & Hagemeister, 2016). However, only limited evidence is available for co-infection in KwaZulu-Natal (KZN) province with a hepatitis B surface antigen (HBsAg) seroprevalence of 8.5% reported in HIV-infected people ( Msomi et al., 2020). The management of Hepatitis B include prevention by vaccination and treatment using the antiviral drugs. The introduction of the hepatitis B vaccine into the South African Expanded Program of Immunization (EPI) in 1995 led to a reduction in liver diseases ( Spearman et al., 2013). Treatment for co-infection with HBV/HIV uses drugs that are effective against both HIV and HBV including, a combination of tenofovir/(TDF) and lamivudine (LMV)/emtricitabine (FTC) /efavirenz (EFV) as outlined by Maponga et al. (2020). However, HBV vaccination and treatment may be threatened by the appearance of mutations on the HBV genome that could cause unfavourable clinical effects, such as vaccine escape and drug resistance. The HBV genome features are characterized by overlapping genes, notably the surface (S) gene within the polymerase (pol) gene. The S gene encodes the surface antigen (HBsAg), contains the major hydrophilic region (MHR) along with ‘α’ determinant (aa124-aa147) region, which is crucial for immune recognition and diagnostic purposes ( Liu et al., 2017). This overlap causes mutations in one gene to affect the other, impacting HBV evolution, replication, antiviral effectiveness, and immune escape. Mutations in the ‘α’ determinant hinder anti-HBs binding and can lead to the emergence of drug resistance mutations (DRM) in the YMDD motif of pol region ( Adesina et al., 2021; Cooreman et al., 2001). Moreover, changes near the YMDD motif contribute to drug resistance in treatments such as lamivudine, adefovir, and entecavir. Several studies have revealed M204V/I in the YMDD motif (amino acid 203±206) among HIV-infected drug-naive and drug-experienced people who received long term LMV therapy ( Mokaya et al., 2018; Selabe et al., 2007; Selabe et al., 2009). Also, previous studies showed drug resistance resulting from a single or combination of the following mutations: V173L, L180M, M204V/I, and A181V/T to lamivudine, rtA181V/T and rtN236T to adefovir, or rtI169T, rtI184G, rtS202G/I and rtM250V to entecavir, in treatment experienced and treatment naïve people ( Colagrossi et al., 2018; Lai et al., 2003; Torresi, 2002; Zöllner et al., 2001). Mutations in the pol gene also impact the surface gene with the previously identified DRM in pol leading to the emergence of immune escape mutations in the MHR ( Torresi, 2002). The prevalence of HBV has been documented in several studies among HIV positive people in KZN ( Millar et al., 2023; Samsunder et al., 2019). However, investigations on the HBV genotype, mutations associated with immune escape and drug resistance are still scarce in this region, and most studies have focused on reporting seroprevalence. The aim of the study was to describe the prevalence and molecular characterization of HBV mutations associated with immune escape and drug resistance in HIV-infected individuals in Durban, KZN, South Africa.

 Methods Study design and population

We applied a convenience sampling method to collect fifty archival sera in this descriptive exploratory investigation. The sample size was not determined, and we used samples that were already available. Sera were from people who underwent HIV testing at the Inkosi Albert Luthuli Central Hospital (NHLS-IALCH-NHLS) in Durban, KwaZulu-Natal Province, South Africa. Based on the sample size of 50 sera, we used the confidence level of 95% and the margin of error for our study was 14%. The 50 participants included men and women who previously tested positive for HBsAg and HIV. Participants received a written informed consent form, the information on the consent form was given in a language the patient understands, which is english and their native language (Zulu). Demographic data (age, sex, and ethnicity) were also collected. The specimen’s codes were de-linked to keep patients anonymous; with only additional data on the age and gender of the study participants provided.

 Laboratory testing

Hepatitis B surface antigen (HBsAg) assay

The Monolisa HBsAg ultra-confirmatory kit was used in according to the manufacturer’s instructions to perform an enzyme-linked immunosorbent assay (ELISA) on samples previously positive for HBsAg to confirm the presence of HBsAg marker (BioRad, Raymond Poincare, Marnes-la-Coquette, France). To identify and measure the presence of HBsAg, excess antibodies (anti-HBs; anti-HBs diluent: neutralization reagent) were used to neutralize 200 μL of undiluted sera specimens. At 450 nm, the optical density (OD) index of the sample was determined and compared to the cut-off value (COV) mean of a negative control. Reactive specimens for HBsAg were defined as those with an index greater than or equal to the COV.

DNA extraction of HBV

The sera obtained from patients were used to extract HBV deoxyribonucleic acid (DNA) using the QIAamp DNA Mini kit (catalog number: 51304) from (Qiagen, Hilden, Germany) following the manufacturer’s instructions. This technique allows the isolation and purification of total DNA from contaminants, inhibitors, and nucleases in the serum. An aliquot of 200 μL of the serum was added into 1.5 μL Eppendorf tube, to which 20 μL of proteinase K and 200 μL buffer AL (binding buffer mixed with poly [A] carrier RNA) was added. The mixture was pulse-vortex for 15 seconds to allow lysis of the mixture and destroy RNA, followed by a 10-minute incubation at 56 °C. The mixture was then transferred to a QIAamp spin column to allow binding of the DNA and centrifuged for 1 minute at 8 000 rpm. The column was placed into a clean collection tube, then 500 μL of buffer AW1 was added, and it was centrifuged for 1 minute at 8 000 rpm. The solution was aspirated, 500 μL of buffer AW2 was added to purify the DNA, and it was followed by centrifugation for 3 minutes at 14 000 rpm. The QIAamp spin column was placed in a sterile 1.5 μL Eppendorf tube, and 50 μL of elution buffer (provided by the kit as buffer AE) was added directly into the column and incubated at room temperature for 5 minutes to precipitate the DNA. The DNA was eluted by centrifugation at 8 000 rpm for 1 minute and stored at -20 °C until further analyses were performed. The negative control, consisting of nuclease-free water, was included in the extraction procedure to identify contamination.

 Polymerase chain reaction

First round and nested-PCR

A nested polymerase chain reaction (PCR) amplification of the overlapping surface/polymerase gene covering nucleotides 256 to 796 from EcoRI site was done as described previously ( Mphahlele, 2008) with slight modification. Outer sense strand (forward primer) S1 (5′-CCT GCT GGT GGC TCC AGT TC-3′), and anti-sense strand (reverse primer) S2Na (5′-CCA CAA TTC KTTGAC ATA CTT TCC A-3′) were used. The master mix were prepared using Ampli Taq Gold DNA polymerase (ThermoFisher Scientific, Waltham, Massachusetts, United States). For each sample the following reagent volumes and concentration of the master mix were prepared as follows: 18.5 μL nuclease-free water, 2.5 μL of 1x PCR buffer with MgCl 2, 0.5 μL (0.2 mM dNTP mix), 0.5 μL (10 μM) forward primer S1; 0.5 μL (10 μM) reverse S2Na anti-sense primer, 0.125 Taq DNA polymerase. A total of 22.1 μL of master mix was aliquoted into a 0.5 mL thin-walled PCR tube and 3 μL of DNA template was added. The PCR reaction mixtures (25.1 μL) was subjected to amplification of HBV DNA, carried out in an automated touch down thermal cycler CFX96 (Bio-Rad, Raymond Poincare, Marnes-la-Coquette, France). The HBV DNA amplification conditions were initial denaturation at 95 °C for 4 minutes, followed by 40× cycles involving denaturation at 95 °C for 4 minutes, annealing at 58 °C for 30 seconds, elongation at 72 °C for 1 minute, and final extension at 72 °C for 10 minutes.

Nested PCR

First round PCR product was used as a template for nested PCR. An aliquot of 3 μL of the first round PCR reaction was subjected to a nested PCR, the master mix volume and concentration were prepared as same for the first round PCR. The nested PCR conditions used were the same as first round PCR protocol except the annealing temperature at 55 °C for 45 seconds. Forward primers S6E (5′-GAGAAT TCCGAGGACTGG GGA CCC TG-3′) and reverse primer S7B (5′-CGG GAT CCT TAG GGT TTA AAT GTATAC C-3′) were used during nested PCR. The negative control consisting of nuclease-free water and a positive control were included in the PCR amplification assays.

PCR products verification

PCR amplification products were verified using 1% agarose gel (ThermoFischer, Waltham, Massachusetts) stained with 0.15 U/μL ethidium bromide (Biorad, California, USA). Aliquot of 10 μL PCR amplicon product was mixed with 2 μL 10x loading buffer (ThermoFischer, Waltham, Massachusetts). The mixtures were run on 1% gel along with a 1 Kb Invitrogen molecular weight maker (ThermoFischer, Waltham, Massachusetts) as a band size reference. The agarose gel was run at 100 V for 45 minutes. The gel was placed inside the ultraviolet (UV) transilluminator (Bio-rad, Hercules, California, United States) to visualise and image capturing.

Sequencing reaction

The PCR products and the nested PCR primers S6E and S7B were sent for bi-directional Sanger sequencing at the Inqaba (Inqaba Biotechnological Industry, Pretoria, South Africa). The amplicons were prepared for direct sequencing using the BigDye terminator v3.0 cycle sequencing ready reaction kit (catalog number: 4458687) from (ThermoFischer, Waltham, Massachusetts). Briefly, an aliquot of 50 μL of the 1:1 ratio of sodium acetate: ethanol (NaAc:EtOH) was added to the amplicons solution and centrifuged at 2000 g for 30 minutes. The well plates were inverted and centrifuged at 150 g for 1 minute. Pre-chilled 70% ethanol was added into the wells and then centrifuged at 2000 g for 5 minutes. The pellets were dried at 65 °C for 5 minutes followed by an addition of 10 μL Hi-Di formamide for 5 minutes and loaded into the sequencing machine ABI 3130XL genetic analyser (Applied Biosystems, Foster City, CA).

 Sequences analysis

Nucleotide sequences of HBV chromatograms were viewed and edited by removing unwanted and mixed nucleotides character from the sequences by ChromasPro, version.1. The contiguous sequences were formed by joining overlapping DNA sequences of a gene using BioEdit. The consensus sequences were compared with the GenBank complementary genotype sequences using the basic local alignment search tool (BLAST). Representative sequences belonging to different genotypes were redeemed from GenBank to make comparisons with the study sequences. Multiple sequences alignment was performed with ClustalW within the MEGA software package version 7.0 (TomHall, North Carolina State University). Genotyping and identification of the sequence was done on Geno2pheno ( Geno2pheno, 2023). Sequences were uploaded into the website where they were aligned at the sequence position 1 to 3182 (~3.18 kbp) relative to the Hepatitis B virus (taxon:10407) and a boostscan analysis of ≥ 80.0 was considered positive.

 Mutations analysis

The aligned sequences were uploaded into the BioEdit and analysed for nucleotide base and amino acids changes. The sequences were further uploaded into the Geno2pheno to identify DRMs in the reverse transcriptase (RT) of polymerase and mutations in the overlapping S region.

 Statistical analyses

Microsoft Excel and the data science statistical program STATA were used for data analyses (version 15). Excel (.csv.) file was imported into STATA and was used to calculate the frequency of age as numeric values and the frequency of HBsAg and mutations as categorical and numeric variables. The proportions of data reported as medians with interquartile ranges (IQR) for continuous variables (age) as frequencies and percentages for categorical variables (sex, ethnicity and mutations).

 Ethical approval

The study ethics certificate was provided by the North-West University research ethics regulatory committee (ref. NWU-00068-15-A9).

 Results Baseline demographic data

The baseline demographics of the study showed that all 50 HIV positive samples included were female at 64% (n=32/50) and 36% (n=18/50) male as shown in Table 1. The median age was 33 years [IQR: 18-55] and there was no statistical difference on age between the female and male black Africans at p=0.8.

Demographics data of the patients by age, sex, HBV and HIV antigens markers.
Age in years (median, IQR) Sex HIV p24 antigen HBsAg antigen
[%; n/50] 33[9.25,2736.25] Female Male HIV (+) Female HIV (+) Male HBsAg (+) Female HBsAg (+) Male
18-35 59% (n=19) 66% (n=12) 40% (n=20) 14% (n=7) 34% (n=17) 12% (n=6)
36-55 41% (n=13) 33% (n=6) 12% (n=12) 22% (n=11) 24% (n=12) 16% (n=8)
Total 64% (n=32) 36% (n=18) 64% (n=32) 36% (n=18) 58% (n=29) 28% (n=14)
p-value 0.8 0.1 0.6
Laboratory testing

HBsAg assay

The confirmatory screening of HBsAg reported a prevalence of 86% (n=43/50) with 12% (n=6/50) being negative and 2% (n=1/50) missing data ( Table 1). Majority of the female’s participant were HBsAg positive at 58% (n=29/50) when compared to the males at 28% (n=14/50) as shown in Table 1.

PCR amplification and genotyping

The PCR amplification of HBV DNA amplicons was successful in 82% (n=41/50) of the samples and consisting of overlapping surface/polymerase region. PCR amplification could not be obtained for 18% (n=09/50) samples. Only 92% (n=38/41) sequence products could be obtained by Sanger sequencing with only 26 sequences used to perform genotyping and 12 sequences were excluded due to the poor quality of the sequence reads. The genotypes of the sequences were confirmed by depositing nucleotides sequences into the Genotype2pheno database which showed that most of the nucleotide sequences had homology similarity to genotype A at 73% (n=19/26) and 12% (n=3/26) as genotype G and 15% (n=4/26) being genotype C ( Table 2). The study sequences with the highest similarity identity to genotype A were: (Q4P7 and Q7P7 to KX520697.1|South Africa; Q9P7 to U87728.1|South Africa; Q27P7 to AY233274.1|South Africa. Reference sequence of AY233277.1|South Africa had between 97%-99% similarity to fifteen sequences (Q5P7, Q8P7, Q10P7, Q13P7, Q18P7, Q19P7, Q20P7, Q21P7, Q22P7, Q23P7, Q24P7, Q26P7, Q27P7, Q29P7 and Q39P7). The sequences Q6P7, Q17P7, and Q42P7 had similarity to genotype G (EU694179.1|South Africa and sequences Q11P7, Q43P7, Q44P7, Q45P7 showed similarity to genotype C AB562444.1|Vietnam ( Table 2). Sub-genotypes of the sequences were confirmed by depositing nucleotides sequences into the Genotype2pheno database. The results retrieved from the Geno2Pheno database had a 96.85%-99.0% percentage of similarity to sub-genotype A1 for the genotype A sequences only.

Genotype identity of the study sequences by comparing with standard representative sequences obtained from GenBank.
Sample code Reference accession no 1 Genotype identity Homology similarity (%)
Q4P7 KX520697.1|South Africa A 99.46
Q5P7 AY233277.1|South Africa A 98.96
Q6P7 EU694179.1|South Africa G 99.83
Q7P7 KX520697.1|South Africa A 98.76
Q8P7 AY233277.1|South Africa A 98.42
Q9P7 U87728.1|South Africa A 98.98
Q10P7 AY233277.1|South Africa A 97.62
Q11P7 AB562444.1|Vietnam C 99.39
Q13P7 AY233277.1|South Africa A 97.95
Q17P7 EU694179.1|South Africa G 99.32
Q18P7 AY233277.1|South Africa A 98.56
Q19P7 AY233277.1|South Africa A 99.18
Q20P7 AY233277.1|South Africa A 99.78
Q21P7 AY233277.1|South Africa A 99.96
Q22P7 AY233277.1|South Africa A 98.49
Q23P7 AY233277.1|South Africa A 99.28
Q24P7 AY233277.1|South Africa A 99.94
Q26P7 AY233277.1|South Africa A 97.96
Q27P7 AY233274.1|South Africa A 99.08
Q29P7 AY233277.1|South Africa A 99.20
Q39P7 AY233277.1|South Africa A 97.50
Q42P7 EU694179.1|South Africa G 98.82
Q43P7 AB562444.1|Vietnam C 99.28
Q44P7 AB562444.1|Vietnam C 97.18
Q45P7 AB562444.1|Vietnam C 99.42

Reference sequences obtained from GenBank (designated by accession numbers). The study sequences are represented by the letters Q, P and are followed by numeric values.

 Mutations within the surface region

The prevalence of mutations in the surface gene was 47% (n=18/38) and mutations were found in the “α”, “β”, “T” and outside the “α” epitope as shown in Table 3. The most common mutations on the surface region and their frequencies were S207N at 71% (27/38), followed by L216V and A194V at 23%, and the least prevalent being S204R, S117N, T143S, G145R, Y206H, P127T, Y200T, F129T and K122R all at 3% as shown in Table 3.

Amino acids substitution in surface, polymerase and their associated functions.
Surface region Reverse transcriptase
Epitope Mutation Frequency Function Mutation Drugs of resistance
“α” epitope K122R 3 Diagnostic failure M129L Compensatory/epistasis to LMV
F134L 5 Diagnostic failure M204V LMV, LdT
S117N 3 Diagnostic failure L180M LMV, LdT
T143S 3 Diagnostic failure V163I LMV, LdT
“β” epitope S207N 71 Vaccine escape V173L LMV
Y200T 3 Vaccine escape A236T ADV
G145R 3 Vaccine escape Diagnostic failure Q125E Compensatory/epistasis to LMV
T-helper epitope P127T 3 Vaccine escape Diagnostic failure S202K LMV, LdT, ETV
Outside “α” E164D 5 Vaccine escape L217R Compensatory/epistasis to LMV
L209V 18 Vaccine escape/ cause RT mutations (M204V/M204I/V173L) V214A Compensatory/epistasis to LMV
Y206H 3 Vaccine escape V204I, S169T LMV, ETV
L216V 23 Vaccine escape I253Y/M205I ADV
A194V 23 Vaccine escape L181T/L80V LMV,
P70H 21 Unknown L80V LMV
P127L 8 Vaccine escape L180M+M204V+S202K+ M250I LMV+LdT+ADV and EFV
T189I 5 Vaccine escape
S204R 3 Vaccine escape
F129T 3 Unknown

Mutations within the polymerase region

The prevalence of mutations in the RT of polymerase was reported at 36% (n=14/38). Mutations showed resistance to lamivudine (LMV) at (35% [5/14]), telbivudine (LdT) at (29% [4/14]), (14% [2/14]) for entecavir (ETV) and (21% [3/14]) for adefovir (ADV). Mutations causing resistance to LMV and LdT were M204V, L180M, V163I, and S202K. The S202K mutation causes resistance to ETV in addition to V204I and S169T. The ADV resistance mutations were I253Y, A236T and M250I in addition to complementary resistant mutation (L80I) not shown in the table. Multiple DRMs within a single sample were identified in one sample containing L180M, M204V, S202K and M250I mutations ( Table 3).

 Discussion

HBV continues to be endemic in South Africa ( Maepa et al., 2022). However, there are still areas in our country with limited published data on circulating genotypes. Therefore, this study considered to determine the HBV genotype and mutations circulating in HIV-infected people from KZN. The partially overlapping surface/polymerase gene region was successfully amplified in 78% (41/50), confirming the active replication of HBV in HIV co-infected people. Geno2pheno characterized majority of the sequences as HBV genotype A, followed by fewer sequences showing similar homology to genotype C and G. The predominance of HBV genotype A over other genotypes is consistent with previous studies in South Africa ( Bowyer et al., 1997; Kimbi et al., 2004; Kramvis, 2014; Kramvis et al., 2005; Kramvis & Kew, 2007; Selabe et al., 2009). Genotype A was found to be the most prevalent among the antiretroviral therapy (ART) naive HIV-infected people in South Africa ( Audsley et al., 2010). This suggest that the mutant HBV is common in both naive and experience HIV positive people. The genotype A mutant virus may have a high transmission rate and low response to ART by an individual. The uncommon genotype G showed similarity to a reference sequence from South Africa with HIV co-infection ( Lukhwareni et al., 2020). The occurrence of genotype C may be due to migration into South Africa. The predominance of HBV genotype A in our study indicates a less occurrence of genetic diversity and suggests that these strains are circulating in this geographic location due to the substandard immigration of new strains into the study region ( Kramvis et al., 2008; Kramvis & Kew, 2007). A total of 47% variants were observed from all sequences at different locations within the surface region (upstream and downstream of MHR). We identified K122R and T143S which are characterized to be associated with diagnostic failure. Also, we found G145R, P127T, S207N, Y200T, E164D, Y206H and L209V which are immune escape mutations, and the findings correlate to previous studies ( El-Mokhtar et al., 2020; Ireland et al., 2000; Mokaya et al., 2018). Contrary some of the mutations have been previously reported to have dual function as diagnostic failure and immune escape such as the G145R and P127T ( Archampong et al., 2019; Colagrossi et al., 2018; Kuzin et al., 2012; Mokaya et al., 2018; Yan et al., 2017; Yousif et al., 2014). We identified some mutations on the upstream position (< aa147) of the S region such as F129T and P70H but their functions have not been elucidated or reported in vitro. Other mutations were identified downstream position >aa147 (to aa216) including: T189I, A194V, S207N, Y200T, Y206H, L209V, L216V and S204R. These mutations were previously linked with vaccination escape diagnostic failure ( Colagrossi et al., 2018). The G145R and E164D mutations either occurring alone or in combination have shown to result from a substitution change in the overlapping polymerase region caused by the mutation M204V, M204I, and V173L which cause lamivudine resistance ( Colagrossi et al., 2018; Torresi, 2002). The frequency of DRMs in the RT region was 50% (n=7/14). M204V, L180M, V163I, and S202K mutations are linked to LMV and LdT resistance; S202K in addition confer resistance to EFV together with V204I. ADV resistance mutations included the I253Y, A236T, and complementary M250I. A single sample sequence contained a combination of L180M, M204V, S202K, and M250I mutations that showed to be associated with high resistance to LMV, LdT, EFV and ADV drugs. This is not opposing to previous studies on combination of DRMs ( Anastasiou et al., 2017; Mokaya et al., 2018). Contrary, the presence of multiple mutations combination may also induce cross-resistance to other drugs. Both L180M and M204V increased cross-resistance to other drugs and decreased sensitivity to ETV ( Sheldon et al., 2005). On the contrary, these combined L180M + M204V + S202K + M250I and L180M + M204V + V173L + S202K + M250I mutations are reported to increase susceptibility to ADV due to the presence of the secondary mutation V173L which is missing from our combination of DRMs. However, the in vitro functional studies on the combined mutations L180M + M204V + S202K + M250I have not been established but we suggest they could have implication on the treatment of HBV in HIV-coinfected people. The following compensatory mutations S202K, Q125E, L217R, V124A, V204I, and T128A were identified, and other identified DRMs were T189I, Y206H, L216V, and S209N. These mutations have effects on treatment resistance; further research is required to fully comprehend how they affect antiviral drugs. The identification of DRMs in HBV treatment-naive patient increases the possibility that the treatment-naive patient may be infected with a mutant HBV virus that was either naturally occurring resistance mutations or derived from a patient who developed resistance after antiviral treatment ( Belyhun et al., 2017; Tan et al., 2012; Vutien et al., 2014; Zhao et al., 2016). We conclude that additional research is required to identify the mechanism producing the DRMs in HIV-positive patients who have never received HBV therapy. Although they can’t extrapolate on past treatment histories, the DRMs identified in this study provide information on the present status of treatment resistance. Second, the DRMs identified in this study suggest that those with mutations resistance to ARTs may have fewer options for therapy. The study does not support whether dual HBV/HIV therapy was effective on HBV or the effect of HIV treatment on ART-naïve HBV. This does not change how the DRMs sequences results are interpreted, which highlights the need of HBV testing by genotyping in addition to HBV and HCV testing prior to starting ART in HIV patients, as recommended by WHO et al. (2017).

 Limitations

The findings of the study should be considered, bearing in mind limitations, including the design of a cross-sectional study with a small sample size. Genotyping of the HBV was not based on full genome sequences which could have identified clearly the mixed base clustering of the sequences to the genotypes. The results encourage a larger sample size to provide a true representative of dominant mutations in the study site and other areas, which improves the generalization of the results. There is no availability of the presumptuous treatment history, and it effect on the understanding the impact of HIV treatment on the HBV mutations profile and treatment.

 Conclusion

This study reports on the prevalence of HBV genotype A among HIV-infected patients. Furthermore, it provides evidence on the presence of HBV mutations related to immune escape and drug resistance in people co-infected with HIV in KwaZulu-Natal, South Africa. A thorough-synchronizes diagnosis and antiviral therapy for HBV patients with HIV co-infection should include proper HBV diagnosis by resistance testing to minimize the emergence and spread of antiviral drug resistance. We recommend that further in vitro studies be conducted to explore the impact and functions of the S and pol mutation on the protein expression and DRMs towards the antiviral resistance.

 Data availability Underlying data

Figshare: data set for patients’ demographics, HIV and HBV test 2017-2019.xlsx, https://doi.org/10.6084/m9.figshare.23946621.v1 ( Modise, 2023a).

Figshare: PCR amplicon gel electrophoresis of HBV overlapping surface region, https://doi.org/10.6084/m9.figshare.23815278.v1 ( Modise, 2023b).

Figshare: PCR amplicon gel image of HBV overlapping surface region.pdf, https://doi.org/10.6084/m9.figshare.23946639.v1 ( Modise, 2023c).

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

 Acknowledgements

The authors would like to thank the NHLS-IALCH-NHLS in KZN for donating samples for this study. We thank the State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences for providing training on HBV genotyping and training on cell culture techniques.

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Dis. 2017;61:13–19. 10.1016/j.ijid.2017.05.015 Lukhwareni A Gededzha MP Amponsah-Dacosta E : Impact of Lamivudine-Based Antiretroviral Treatment on Hepatitis B Viremia in HIV-Coinfected South Africans. Viruses. 2020 Jun 11;12(6):634. 32545313 10.3390/v12060634 PMC7354521 Maepa MB Ely A Kramvis A : Hepatitis B Virus Research in South Africa. Viruses. 2022;14(9). 36146747 10.3390/v14091939 PMC9503375 Maponga TG McNaughton AL Schalkwyk M van : Treatment advantage in HBV/HIV coinfection compared to HBV monoinfection in a South African cohort. J. Infect. 2020;81(1):121–130. 32360882 https://doi.org/10.1016/j.jinf.2020.04.037 PMC7308798 Millar J Cromhout GZL Mchunu N : Hepatitis B Virus Prevalence and Mother-to-Child Transmission Risk in an HIV Early Intervention Cohort in KwaZulu-Natal, South Africa. Open Forum Infect. Dis. 2023;10(8). 37547854 https://doi.org/10.1093/ofid/ofad366 PMC10400149 Modise L : data set for patients’ demographics, HIV and HBV test 2017-2019.xlsx.Dataset. figshare. 2023a. 10.6084/m9.figshare.23946621.v1 Modise L : PCR amplicon gel electrophoresis of HBV overlapping surface region. figshare. Figure. 2023b. 10.6084/m9.figshare.23815278.v1 Modise L : PCR amplicon gel image of HBV overlapping surface region.pdf. figshare. Figure. 2023c. 10.6084/m9.figshare.23946639.v1 Mokaya J McNaughton AL Hadley MJ : A systematic review of hepatitis B virus (HBV) drug and vaccine escape mutations in Africa: A call for urgent action. PLoS Negl. Trop. Dis. 2018;12(8): e0006629. 30080852 https://doi.org/10.1371/journal.pntd.0006629 PMC6095632 Mphahlele MJ : Impact of HIV co-infection on hepatitis B prevention and control: a view from sub-Saharan Africa. South Afr. J. Epidemiol. 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A. 1975;72(11):4597–4601. 1060140 10.1073/pnas.72.11.4597 PMC388770 Tan Y Ding K Su J : The naturally occurring YMDD mutation among patients chronically infected HBV and untreated with lamivudine: a systematic review and meta-analysis. PLoS One. 2012;7(3): e32789. https://doi.org/10.1371/journal.pone.0032789 Torresi J : The virological and clinical significance of mutations in the overlapping envelope and polymerase genes of hepatitis B virus. J. Clin. Virol. 2002;25(2):97–106. 10.1016/s1386-6532(02)00049-5 Vutien P Trinh HN Garcia RT : Mutations in HBV DNA polymerase associated with nucleos(t) ide resistance are rare in treatment-naive patients. Clin. Gastroenterol. Hepatol. 2014;12(8):1363–1370. 10.1016/j.cgh.2013.11.036 World Health Organization : Guidelines on Hepatiti B and C Testing.2017. Reference Source Yan B Lv J Feng Y : Temporal trend of hepatitis B surface mutations in the post-immunization period: 9 years of surveillance (2005-2013) in eastern China. Sci. 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Lancet. 2001;357(9260):934–935. 11289355 10.1016/s0140-6736(00)04219-7 10.5256/f1000research.186606.r407676 Reviewer response for version 4 Gelanew Tesfaye 1 Referee https://orcid.org/0000-0002-0696-3786 Armauer Hansen Research Institute, Addis Ababa, Addis Ababa, Ethiopia

Competing interests: No competing interests were disclosed.

 11 9 2025 Copyright: © 2025 Gelanew T 2025 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

I have no further comment. I endorsed the manuscript for indexing.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

NA

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

 10.5256/f1000research.179250.r380330 Reviewer response for version 3 Prabdial-Sing Nishi 1 Referee Centre for Vaccines and Immunology, Division of the National Health Laboratory Service, National Institute for Communicable Diseases, Johannesburg, South Africa

Competing interests: No competing interests were disclosed.

 5 8 2025 Copyright: © 2025 Prabdial-Sing N 2025 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

Thank you to the authors for addressing some of the reviewer’s comments.

There are still grammatical errors in the abstract.

Abstract: Background

‘”cross-resistance of drugs”” what does this mean exactly, that sites on the HIV and HBV genome have shown cross-resistance of HIV antiviral drugs and to HBV antiviral drugs? I have not seen this and should be better written

Begin the next sentence “Continuous monitoring of HBV variants is required for better…”

Results:

Also as you found all genotype A to be subgenotype A1, then indicate A1 in your findings and conclusions

Genotype c, should be C

Introduction:

The abbreviation “DRM”, needs to be explained when used for the first time

Method

English should be English

used in according should be used according

Results:

Mutations showed resistance to lamivudine (LMV) at (35% [5/14]), telbivudine (LdT) at (29% [4/14]), (14% [2/14]) for entecavir (ETV) and (21% [3/14]) for adefovir (ADV). Mutations causing resistance to LMV and LdT were M204V, L180M, V163I, and S202K”

“the statement implies that the patients were treated for HBV and these DRMs were found. However, it was mentioned that these patients were Treatment naïve? Please clarify whether the patients were treatment naïve for HIV or HBV or both?

Discussion:

This suggest that the mutant HBV is common in both naive and

experience HIV positive people””. This sentence is not referenced. There is only one reference for naïve, but not HIV treated persons.

Conclusion

thorough-synchronizes diagnosis” what does this mean?

What do the authors mean by substandard immigration of new strains?

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

NA

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

 10.5256/f1000research.179250.r393935 Reviewer response for version 3 Gelanew Tesfaye 1 Referee https://orcid.org/0000-0002-0696-3786 Armauer Hansen Research Institute, Addis Ababa, Addis Ababa, Ethiopia

Competing interests: No competing interests were disclosed.

 28 7 2025 Copyright: © 2025 Gelanew T 2025 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

The manuscript by Modise et al entitled "Investigation of Hepatitis B Virus Mutations Associated with Immune Escape and Drug Resistance in HIV-Infected Patients" is highly informative, and I concur with the authors’ observation that most studies conducted in Africa focus primarily on prevalence, while only a limited number address genotyping and drug resistance profiling. The paper is well written, with sound methodological approaches. The conclusions are appropriately toned down, clearly derived from the data, and the limitations are well articulated. I recommend acceptance in its current form. However, I have the following few minor comments.

1. - The title lacks specificity. It should be revised to:

Investigation of Mutations in the Partial Sequences of the S and Polymerase Genes of Hepatitis B Virus Associated with Immune Escape and Drug Resistance in HIV-Infected Patients

2. - The introduction would benefit from expanding the brief overview of the specific genes targeted in this study—namely those encoding the surface antigen and polymerase—and their roles in immune escape and antiviral drug resistance.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

My expertise is clinical microbiology and Immunology/molecular biology. My ongoing research areas are dengue virus, HBV, RSV, and pathogens causing STIs.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

 10.5256/f1000research.179250.r386531 Reviewer response for version 3 Ike Anthony C. 1 Referee https://orcid.org/0000-0003-2706-6141 University of Nigeria, Nsukka, Enugu, Nigeria

Competing interests: No competing interests were disclosed.

 14 6 2025 Copyright: © 2025 Ike AC 2025 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

The article is well designed and carried out and was also well written or corrected to bring it to its present level. I have made few observations as listed below:

In Page 4 under DNA extraction of HBV in Lines 4 and 10, the Eppendorf tube is 1.5 mL and not 1.5 μL.

In the same page under Polymerase chain reaction First round and nested-PCR, in Line 8 of that section, the authors need to state whether the 0.125 of Taq DNA polymerase is the concentration (units) or volume (μL) of the enzyme.

Under Nested PCR, the last line of the section, Line 6, the authors should state what was used as positive control and state the source if possible.  

In page 6 under laboratory testing HBsAg assay Line 2 of the section, it should be females and not female's. Also the HBsAg positive for the females should be 91% (N=29/32) and not 58% (N=29/50), since there were only 32 females and not 50. similarly that of the males should be 78% (N=14/18) and not 28% (N=14/50), males ere 18 and not 50. This should be corrected in Table 1 and be extended to the results of HIV (+) Female and HIV (+) Male.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Medical Microbiology with Virology bias. I have researched and published works on Noroviruses, Rotaviruses, influenza virus, hepatitis B and C viruses, Dengue virus among others.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

 10.5256/f1000research.163315.r284916 Reviewer response for version 2 McNaughton Anna 1 Referee https://orcid.org/0000-0002-7436-8727 Nuffield Department of Medicine, University of Oxford Medawar Building, Oxford, England, UK

Competing interests: No competing interests were disclosed.

 6 6 2024 Copyright: © 2024 McNaughton A 2024 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation reject

The authors have clearly revised the manuscript, taking into account some of the feedback provided previously and their metadata now looks to be more accurate. However, there are still several issues with the revised document, and particularly the analysis and presentation of the results which need correcting before the manuscript can be approved.

HBsAg assay – COV needs defining at first use.

Baseline demographic data – the median age is presented and the range is but I can’t see the standard deviation, despite the authors stating it in the paragraph. Table 1 should be revised to be more informative (i.e. present proportions) – if all individuals were black africans and HIV positive, this is not particularly useful information to present? I would encourage the authors to revise the data in table 1 to make it more informative, such as is there any data on their HIV treatment?

Phylogenetic analysis – the authors state that sequences were obtained for 38/41 samples that were PCR positive, but only analysed n=26 sequences. Why were 12 sequences excluded from the analysis?

The sequences analysed are relatively short, and as the authors state, this can affect the clustering but the phylogenetic analysis presented remains very problematic for genotyping. The reference sequences do not cluster by genotype, making it difficult to use the tree to confidently genotype the sequences presented. Whilst the authors checked a number of gtA the sequences with geno2pheno, I am puzzled by the interpretation of the other sequences (assumed not to be gtA). A number of sequences do not clearly cluster with any of the reference sequences, and the authors describe these as “24% (N=7/26) as genotypes B, C, D, E, F and G”, which is difficult to interpret. It is also a little unclear why sequences at the bottom of the tree ‘Q6P7, Q17P7, and Q42P7’ were decided to be closer to genotype H, D, B, and E than to genotype A without presenting bootstrapping data? This analysis needs revising, and perhaps the authors should consider a maximum-likelihood analysis as it may be more discriminatory for genotyping than the approach currently used. Alternatively, they may consider using geno2pheno to genotype their sequences, and present this data in a table rather than a tree?

Association of mutations on the surface and polymerase regions – authors state the mean and standard deviation of the SHB mutations, but only provide a single number? Are the numbers presented also the number of mutations per sequence, as this is not clear?

The discussion is too long and should be more concise – the discussion of the wider literature on drug resistant mutations could be reduced. Some context may also be useful to discuss – these samples were taken from HIV-positive individuals, so were any of them on treatment for their HIV infections and were these treatments active against HBV also? The authors mention that some patients are ART treatment naïve late in the discussion – but a little more detail on this would add to the interpretation of this study.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

No

Are the conclusions drawn adequately supported by the results?

No

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Virology, genomics, epidemiology, viral hepatitis

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

Modise Lorato Mosetsanagape Virology, Sefako Makgatho health Sciences University, Pretoria, Gauteng, South Africa

Competing interests: No competing interests were disclosed.

 12 3 2025

Dear reviewer

We truly appreciate knowledge, and insightful comments on our paper. We appreciate your reviews and recommendations, which have greatly enhanced the manuscript. We have carefully considered all your comments and revised them accordingly. 

Specific reviewer comment

HBsAg assay – COV needs defining at first use.

Response: Cut off value (COV) has been defined at first use.

Reviewer comments

Baseline demographic data – the median age is presented, and the range is, but I can’t see the standard deviation, despite the authors stating it in the paragraph. Table 1 should be revised to be more informative (i.e. present proportions) – if all individuals were black Africans and HIV positive, this is not particularly useful information to present. I would encourage the authors to revise the data in table 1 to make it more informative, such as is there any data on their HIV treatment?

Response: 

Since all the samples were from the same group, the information on the ethnicity of Black African has been deleted from the description in Table 1.

The description of the statistical analysis has been revised in the text and Table 1 to be specific to the standard deviation. The median age is presented with the interquartile range (IQR) the range and did not present the standard deviation because the data in not normally distributed and there is poor statistical power. Table 1 presents the demographic data collected from the participants which includes the age, sex and test done before. The data is already presented in proportions expressed by either a count by the total number and the proportion as a percentage. The age is already presented in proportions as indicated in the text (n=19/50).

The results are on demographic data which is the primary data on the participants HIV status. We can only report on the data that was collected and available for this study, The data on the treatment history of the participants specifically HIV treatment is not available hence, it is not included. This is highlighted in the limitations section. 

 Reviewer comments

Phylogenetic analysis – the authors state that sequences were obtained for 38/41 samples that were PCR positive but only analysed n=26 sequences. Why were 12 sequences excluded from the analysis? The sequences analysed are relatively short, and as the authors state, this can affect the clustering, but the phylogenetic analysis presented remains very problematic for genotyping. The reference sequences do not cluster by genotype, making it difficult to use the tree to confidently genotype the sequences presented. Whilst the authors checked a few gtA the sequences with geno2pheno, I am puzzled by the interpretation of the other sequences (assumed not to be gtA). A few sequences do not clearly cluster with any of the reference sequences, and the authors describe these as “24% (N=7/26) as genotypes B, C, D, E, F and G”, which is difficult to interpret. It is also a little unclear why sequences at the bottom of the tree ‘Q6P7, Q17P7, and Q42P7’ were decided to be closer to genotype H, D, B, and E than to genotype A without presenting bootstrapping data? This analysis needs revising, and perhaps the authors should consider a maximum-likelihood analysis as it may be more discriminatory for genotyping than the approach currently used. Alternatively, they may consider using geno2pheno to genotype their sequences,

Response:

The 12 sequences excluded from the analysis had poor sequence reads on the chromatogram, and they could not form the proper consensus sequences thus they were not considered for the analysis.

The genotyping of the sequences is revised with the use of geno2pheno software as advised and the data is presented in a table instead of a phylogenetic tree (Table 2). The sequences were uploaded onto the geno2pheno software and compared against standard (wildtype) genome with the highest nucleotide similarity to the study sequences.

The sequences that identify as genotype A have been clarified through revision. Additionally, the 24% (N=7/26) that represent a combination of other genotypes (C and G) have been broken down to demonstrate the individual and specific genotypes C and G; 12% (N=3/26) represent genotypes G, and 15% (N=4/26) represent genotype C.

 Association of the mutations

Data on the association of mutations on the surface and polymerase regions has been removed due to the poor statistical power and statistical association. We focused on reporting the data on the mutations associated with drug resistance with a focus on their frequency as depicted in Table 3.

 Reviewer comment

The discussion is too long and should be more concise – the discussion of the wider literature on drug resistant mutations could be reduced. Some contexts may also be useful to discuss – these samples were taken from HIV-positive individuals, so were any of them on treatment for their HIV infections and were these treatments active against HBV also?

Response:

The discussion has been summarised. The data on the treatment history of the participants specifically HIV treatment is not available hence, it is not included. Secondly, we don’t know if HIV treatments is active against HBV because we do not have the information currently available to support that. However, we know the treatment history of HBV and that the participants are naïve to HBV ARTs. We have also added information on the results interpretation of the mutations in HBV patients that are HBV treatment naïve (last paragraph under the discussion section).

Results

Table 1: The median age was 33 years [IQR: 18-55] and there was no statistical difference on age between the genders with p=0.8.

92% (N=38/41) sequence products could be obtained by Sanger sequencing with only 26/38 sequences used to perform genotyping, that is, 12 sequences were excluded due to the poor quality of their sequence reads.

The genotype of the sequences was confirmed by depositing nucleotide sequences into the Genotype2pheno database, which showed that most of the nucleotide sequences had homology with genotype A at 73% (N=19/26), genotype G at 12% (N=3/26), and genotype C at 15% (N=4/26) (Table 2).

The study sequences with the highest similarity to Genotype A were: (Q4P7 and Q7P7 to KX520697.1|South Africa; Q9P7 to U87728.1|South Africa; Q27P7 to AY233274.1|South Africa.  Reference sequence of AY233277.1|South Africa had between 97%-99% similarity to fifteen sequences (Q5P7, Q8P7, Q10P7, Q13P7, Q18P7, Q19P7, Q20P7, Q21P7, Q22P7, Q23P7, Q24P7, Q26P7, Q27P7, Q29P7 and Q39P7).  The sequences Q6P7, Q17P7 and Q42P7 had similarity to genotype G (EU694179.1|South Africa; sequences Q11P7, Q43P7, Q44P7 and Q45P7 showed similarity to genotype C AB562444.1|Vietnam (Table 2).  Sub-genotypes of the sequences were confirmed by depositing nucleotides sequences into the Genotype2pheno database. The results retrieved from the Geno2Pheno database had a 96.85%-99.0% percentage of similarity to sub-genotype A1 for the genotype A sequences only.

 Discussion

Discussion on the interpretation of mutations in treatment-naïve has been revised. This includes clarity on the impact of the mutations on the treatment’s status of the participants. We also discuss how the study cannot answer on whether the treatment was active against HBV (see the last paragraph under the discussion section). However, we made recommendation that further in vitro studies must investigate the mechanism of action by DRMs on ARTs in vitro assays (last sentence under conclusion).

Additional correction and response

Reference removed from the list 

1. Kimura M: A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences.

J. Mol. Evol. 1980; 16(2): 111–120. PubMed Abstract|Publisher Full Text.

2. Kumar S, Tamura K, Nei M: MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief. Bioinform. 2004; 5(2): 150–163. PubMed Abstract|Publisher Full Text.

3. Malik A, Singhal DK, Albanyan A, et al.: Hepatitis B virus gene mutations in liver diseases: a report from New Delhi. PLoS One. 2012; 7(6): e39028. PubMed Abstract|Publisher Full Text|Free Full Text.

4. Saitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 1987; 4(4): 406–425. .

Updated references and citation

1.    Belyhun, Yeshambel, Melanie Maier, and Uwe Gerd Liebert. "HIV therapy with unknown HBV status is responsible for higher rate of HBV genome variability in Ethiopia." Antiviral therapy 22.2 (2017): 97-111.

2.    Geno2pheno. HBV genotyping 2023 [Available from: https://hbv.geno2pheno.org/.

3.    World Health Organization. Guidelines on Hepatitis B and C Testing 2017 update (available at https://apps.who.int/iris/bitstream/handle/10665/254621/9789241549981- eng.pdf.).

 10.5256/f1000research.163315.r284915 Reviewer response for version 2 Prabdial-Sing Nishi 1 Referee Centre for Vaccines and Immunology, Division of the National Health Laboratory Service, National Institute for Communicable Diseases, Johannesburg, South Africa

Competing interests: No competing interests were disclosed.

 3 6 2024 Copyright: © 2024 Prabdial-Sing N 2024 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

Thank you to the authors for addressing some of the reviewer’s comments.

There are still grammatical errors in the abstract.

Abstract:

Background:

“…cross-resistance to drugs…”, not "of drugs".

Begin the next sentence “Continuous monitoring of HBV variants is required for better…”

Methods:

“Sera” is in the middle of the sentence and should be “sera”.

Results:

“Surface” is in the middle of the sentence and should be “surface”.

Also as you found all genotype A to be subgenotype A1, then indicate A1 in your findings and conclusions.

Introduction:

The abbreviation “MHR”, needs to be explained when used for the first time.

Results:

The abbreviation “SHB”, needs to be explained when used for the first time.

Discussion:

“Therefore, this study should be considered…” should be written as: “This study determined the HBV genotype…”

What do the authors mean by substandard immigration of new strains?

in vitro should be in vitro (in italics)

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

virology, diagnostics, molecular biology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

 10.5256/f1000research.145424.r224163 Reviewer response for version 1 McNaughton Anna 1 Referee https://orcid.org/0000-0002-7436-8727 Nuffield Department of Medicine, University of Oxford Medawar Building, Oxford, England, UK

Competing interests: No competing interests were disclosed.

 22 11 2023 Copyright: © 2023 McNaughton A 2023 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation reject

In this study, the authors sequenced the HBsAg region from a number of samples from individuals co-infected with HIV and HBV. Whilst this data can be informative, there are a number of issues with the analysis, results are not consistent with their metadata and the conclusions need re-evaluating before this manuscript can be reconsidered. The manuscript is also too long in several parts, and should be more focused on relevant information.

Abstract - Sampling site should be mentioned in the abstract – important to understand how/where these patients were identified, and has a link to the genotype distribution.

Introduction - Very lengthy, with a large amount of information on HBV biology that is not relevant to this study. The authors should make this section more concise, and focused on HBV/HIV coinfection in the region these patients were sampled from, and the implications of treatment.

Sampling approach – it is unexpected that 100% of n=50 people tested for HIV would randomly also test positive for HBV via convenience sampling. Were samples screened in advance as part of the clinical testing? The available metadata online (Figshare) also seems to state that 43/50 samples were HBV HBsAg positive, with 7 samples either negative or missing so the authors need to clarify this result. This sampling approach also means that discussions about prevalence and comparing with other studies (in results/particularly in discussion) are not appropriate and should be removed from the study.

Methods are too detailed and could be more concise.

Figure 1 is not required in the paper, particularly if sequences were generated.

PCR Amplification – the authors state that 41/50 amplicons were obtained but that they were not obtained for 11/50 samples – this is 52/50?

Sequence analyses of overlapping surface/polymerase gene region - The authors state that ‘Phylogenetic tree analysis identified nucleotide sequences from this study as genotype A as depicted in (Figure 2) but the current analysis has issues and cannot be used to inform genotyping assumptions as a result (see below).

Phylogenetic analysis/Figure 2 - Authors should say what type of phylogenetic analysis this was, and how many study sequences were included in it. What length were these sequences?

List of reference sequences listed in Figshare does not tally with the sequences in the tree where there appears to be more sequences? Unclear why this is.

Methods says bootstrap analysis was done but the data is not presented on the tree anywhere.

There appears to be a few issues with the clustering of the reference sequences in the tree – the clade at the bottom shows genotypes D/B/E/C clustering, but then other genotype B/C sequences can be seen to cluster with genotypes F/H at the top of the tree? Sequences of the same genotype should cluster together and they do not appear to be in this analysis. This analysis needs revising.

For the mutation analysis, it is unclear what reference sequence the authors were comparing their sequences to? I am not keen of the approach of simply comparing sequences to a reference sequence to determine ‘mutations’ as these are likely to mostly just be reflective of expected sequence diversity. I think looking at a more focused list of sequences with known phenotypic associations would be more informative (as has been done with the resistance-associated mutations). Some rephrasing might help also – for example, saying 47% of sequences had mutations could imply that 53% of sequences were identical in the region? Be clear that these are amino acid differences.

Table 3 - add the specific drug the mutation provides resistance against.

Table 4 needs revising to say n= and % for the age groups. Typo of Fisher’s exact test also.

I think figure 3 can also be removed – this is just showing that sequences more distant from a reference sequence in surface, and also more distant in RT. This is not surprising as the two regions overlap. Are the numbers listed amino acid positions in the sequences – it would be useful to know where in the genome this is?

The conclusions need re-evaluating after addressing the concerns elsewhere in the paper.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

No

Are the conclusions drawn adequately supported by the results?

No

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Virology, genomics, epidemiology, viral hepatitis

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

Modise Lorato Mosetsanagape Virology, Sefako Makgatho health Sciences University, Pretoria, Gauteng, South Africa

Competing interests: No competing interests.

 14 4 2024

Dear Reviewer

We appreciate the efforts, comments, and concerns in making the manuscript better. We have gone through the comments and edited the manuscript accordingly in the revised manuscript. The inputs made in response to your comments are listed below.

In this study, the authors sequenced the HBsAg region from a number of samples from individuals co-infected with HIV and HBV. Whilst this data can be informative, there are several issues with the analysis, results are not consistent with their metadata and the conclusions need re-evaluating.

before this manuscript can be reconsidered. The manuscript is also too long in several

parts, and should be more focused on relevant information.

Reviewer’s comments:

Abstract - Sampling site should be mentioned in the abstract – important to understand how/where these patients were identified and has a link to the genotype distribution.

Response: Yes, We have added information in the abstract on where the samples were collected (Inkosi Albert Luthuli Central Hospital) and how they were collected ( Convenience sampling).

Introduction - Very lengthy, with a large amount of information on HBV biology that is not relevant to this study. The authors should make this section more concise and focused on HBV/HIV coinfection in the region these patients were sampled from, and the implications of treatment.

Response : The introduction has been revised and shortened by removing information on the HBV biology and history. We have included information on the prevalence of HBV/HIV coinfection in the study region and implication of treatment.

Sampling approach – it is unexpected that 100% of n=50 people tested for HIV would randomly also test positive for HBV via convenience sampling. Were samples screened in advance as part of the clinical testing? The available metadata online (Figshare) also seems to state that 43/50 samples were HBV HBsAg positive, with 7 samples either negative or missing so the authors need to clarify this result. This sampling approach also means that discussions about prevalence and comparing with other studies (in results/particularly in discussion) are not appropriate and should be removed from the study.

Response : The sampling method and sample size is now clarified in the methodology section in page 3 under the title “Study design and population”. Convenience sampling was used to collect 43/50 samples that were previously tested for HIV and HBsAg. The sample size has been revised by reporting the correct size which correspond to the metadata value. The confirmatory screening of HBsAg reported a prevalence of 86% (N=43/50) with 12% (N=6/50) being negative and2% (N=1/50) missing data (Table 1).

Reviewer comment: Methods are too detailed and could be more concise.

Response : The methods have been revised and summarized.

Figure 1 is not required in the paper, particularly if sequences were generated.

Response : Figure has been removed from the paper and included as supplementary data on the repository site.

PCR Amplification – the authors state that 41/50 amplicons were obtained but that they were not obtained for 11/50 samples – this is 52/50?

Response : The result of failed PCR amplification has been corrected to 08/50 instead of 11/50.

Sequence analyses of overlapping surface/polymerase gene region

‘Phylogenetic tree analysis identified nucleotide sequences from this study as genotype A as depicted in (Figure 2) but the current analysis has issues and cannot be used to inform genotyping assumptions as a result (see below).

Phylogenetic analysis/Figure 2 - Authors should say what type of phylogenetic analysis this was,

and how many study sequences were included in it. What length were these sequences?

List of reference sequences listed in Figshare does not tally with the sequences in the tree?

there appears to be more sequences. Unclear why this is. Methods says bootstrap analysis was done but the data is not presented on the tree anywhere. There appears to be a few issues with the clustering of the reference sequences in the tree – the clade at the bottom show's genotypes D/B/E/C clustering, but then other genotype B/C sequences can be seen to cluster with genotypes F/H at the top of the tree? Sequences of the same genotype should cluster together, and they do not appear to be in this analysis. This analysis needs revising.

Response:

The prevalence of mutations associated with drug resistance has been revised, to focus only on reporting the mutations associated with specific drug (shown in section titled: Mutations within the polymerase region and table 3)

The phylogenetic tree results have been revised. The length these sequences is reported under the PCR amplification results as 547 bp in size and included 24 sequences and 39 reference sequences. The reference sequences listed in the metadata included some of the sequences and not all the reference sequences, but the list has been revised to correspond to the phylogenetic tree reference sequences. The neighbor joining method was used to construct a tree. The clustering of the sequences was revised, and the clustering of the sequences was mixed due to the short PCR product of 547 bp, unlike the full genome which would have given a homogenous clustering pattern. The sequence clustering results have been revised.

For the mutation analysis, it is unclear what reference sequence the authors were comparing their

sequences to? I am not keen of the approach of simply comparing sequences to a reference

sequence to determine ‘mutations’ as these are likely to mostly just be reflective of expected

sequence diversity. I think looking at a more focused list of sequences with known phenotypic

associations would be more informative (as has been done with the resistance-associated

mutations). Some rephrasing might help also – for example, saying 47% of sequences had

mutations could imply that 53% of sequences were identical in the region? Be clear that these are amino acid differences.

Response: The sequences were uploaded into geno2pheno and compared against standard genome with reported mutations using bioinformatics and statistical packages to check for sequence diversity and mutations. We did not investigate the phenotypic associations these mutants have on the virus. The focus was on identifying the mutations in the overlapping pol and S and their clinical implications.

Table 3 - add the specific drug the mutation provides resistance against.

Response : Table 3 has been revised, the initial table has been removed and replaced with the showing specific drug associated with resistance

Table 4 needs revising to say n= and % for the age groups. Typo of Fisher’s exact test also. I think figure 3 can also be removed – this is just showing that sequences more distant from a reference sequence in surface, and also more distant in RT. This is not surprising as the two regions overlap. Are the numbers listed amino acid positions in the sequences – it would be useful to know where in the genome this is?

Response: Table 4 has been removed.

The conclusions need re-evaluating after addressing the concerns elsewhere in the paper.

Response: The conclusion has been revised.

The sequences of the studies Q cluster with each other and this is what is shown with my study sequences. There is a clustering between B/C and F/H reference sequences.

The sequences were compared with the reference sequences on Geno2Pheno, which is an online server tool. Sequence alignment to the deposited HBV consensus sequence of the respective genotype. We looked at genetic mutations, not phenotypic mutations. that will acquire in vitro studies which is not the focus of the study.

Under results section the subheadings 1. mutations within the surface region and 2. mutations within the polymerase region have been combined and reported under the heading: Mutations analysis.

 10.5256/f1000research.145424.r211197 Reviewer response for version 1 Prabdial-Sing Nishi 1 Referee Centre for Vaccines and Immunology, Division of the National Health Laboratory Service, National Institute for Communicable Diseases, Johannesburg, South Africa

Competing interests: No competing interests were disclosed.

 27 10 2023 Copyright: © 2023 Prabdial-Sing N 2023 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

The authors undertook a small study to characterize HBV among HIV infected individuals. The evidence and findings that the authors have revealed in the study indicates the necessity to engage in larger studies and population size to understand the extent of HBV sequence variation and diversity seen in South African patients coinfected with HIV. The study provides remarkable technical aspects for many others to undertake studies such as these and the authors are commended on their scientific output.

There were a few grammatical errors and writing of the manuscript requires improvement. It is imperative that the writing of sequence variation indicates that it is the virus that was sequenced and not host or patient genes, because this can cause confusion to the reader. This has been commented on in the manuscript but the authors need to identify more of these to correct throughout the paper. 

The discussion is too long and too much information is provided that can be summarized and placed more appropriately in the introduction, especially on the vaccine escape mutations. The discussion should focus on the findings from the study with comparison on other similar studies in other countries or within the same country.

On the results and discussion, the scatter plot needs to be explained as the X- and Y-axes are not well labeled and the interpretation and relevance of the plot does not seem to be evident in the discussion. The statistical significance is commented on for the RT gene but not shown for the HBsAg gene, but the latter was commented on. The sample size is small to conclude from these statistical significant data. It is recommended that a statistician look at the data and recommend a direction.

Overall, the manuscript requires major improvement to improve on the aim and discussion and to draw on strengths from the authors' findings.

The edits and comments are available as a PDF attachment which can be found here.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

virology, diagnostics, molecular biology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Modise Lorato Mosetsanagape Virology, Sefako Makgatho health Sciences University, Pretoria, Gauteng, South Africa

Competing interests: No competing interest.

 1 3 2024

Dear Reviewer,

We appreciate the efforts, comments, and concerns in making the manuscript better. We have gone through the comments and edited the manuscript accordingly in the revised manuscript. The inputs made in response to your comments are listed below.

Reviewer’s general comments:

The authors undertook a small study to characterize HBV among HIV infected individuals. The evidence and findings that the authors have revealed in the study indicates the necessity to engage in larger studies and population size to understand the extent of HBV sequence variation and diversity seen in South African patients coinfected with HIV. The study provides remarkable technical aspects for many others to undertake studies such as these and the authors are commended on their scientific output.

Reviewer’s comments: There were a few grammatical errors and writing of the manuscript requires improvement. It is imperative that the writing of sequence variation indicates that it is the virus that was sequenced and not host or patient genes, because this can cause confusion to the reader. This has been commented on in the manuscript, but the authors need to identify more of these to correct throughout the paper.

Response 1 : Yes, we have made the necessary improvement on the grammar and writing of the paper.

Response 2: We have made clarification on the use and writing of virus sequence and virus nucleotide sequences and HBV sequence instead of host or patients’ genes.

Reviewer’s comments: The discussion is too long and too much information is provided that can be summarized and placed more appropriately in the introduction, especially on the vaccine escape mutations. The discussion should focus on the findings from the study with comparison on other similar studies in other countries or within the same country.

Response : Thank you for noting this. We have excluded the opening of the discussion about the seroprevalence. The discussion section has been revised, summarised, and focusing more on the sequence analysis of the viruses (genotyping and mutations analysis). The information on the vaccine escape mutations has been moved to the introduction.

Reviewer’s specific comments per section

Abstract

Reviewer’s comments:

Add a line on where and how samples were collected?

Response : The type of samples and the location where the samples were collected is now clarified in the abstract and methodology section in page 3 under the title “Study design and population”

Reviewer’s comments:

So, here you indicate statistical significance of RT mutations. Were the mutations in the HBsAg statistically significant?  as compared to RT.

Response : We have provided the statistical significance of association between the RT and HBsAg mutations as outlined in the abstract and results section on page 10 “Association of mutations on the surface and polymerase”

Reviewer’s comments:

The aim of the study be revised.

Response: We have revised the aim of the study

Methods

Reviewers’ comments:

Add a line on where and how samples were collected?

Response: We added information of how and where the samples were collected. We also describe the sampling method and how the sample size was calculated.

Results

Reviewers’ comments:

Is the prevalence of HBsAg correctly reported and how were samples collected?

Response : The seroprevalence of HBV has been revised by reporting the correct percentage values of HBsAg.

Reviewers’ comments:

It is not clear what these numbers are? is the first number, the number of mutations found in the age group and the second number is a %? Figure 3 is not well explained as how to read this plot because the X-and Y- axes are not well labelled .

Response : Table 4 and Figure 3 are removed. The probability of association between the RT and SHB which was represented on Table 4 and Figure 3 is reported as depicted on page 10 under section “Association of mutations on the surface and polymerase” to report that there was no statistical significance association between the SHB and RT mutations at P > 0.005.

Phylogenetic analysis was also revised.

Discussion

Reviewer’s comments:

This is not a valid finding as it may reflect how samples were chosen for the study (previous point above). Also, the sample size is very small and not representative. I would avoid starting the discussion with this. Rather begin at the sequence analyses as this is the strength of the study.

Response 1 : Thank you for noting this. We have excluded the statement on sample size being statistically significant and included it as a study limitation (last paragraph under discussion) on page 11.

Response 2 : We begin the discussion on the sequence analyses and focus on the genotype identification and diversity.