Investigation of hepatitis B virus mutations associated with immune escape and drug resistance in human immunodeficiency virus-infected patients

Lorato Modise; Nomathamsanqa Sithebe; Hazel Mufhandu

doi:10.12688/f1000research.132498.1

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Research Article

Investigation of hepatitis B virus mutations associated with immune escape and drug resistance in human immunodeficiency virus-infected patients

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

Lorato Modise ¹, Nomathamsanqa Sithebe¹, Hazel Mufhandu ¹

PUBLISHED 27 Sep 2023

Author details Author details

¹ Biological Sciences, North West University, Mahikeng, North West, 2735, South Africa

Lorato Modise
Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Validation, Writing – Original Draft Preparation, Writing – Review & Editing

Nomathamsanqa Sithebe
Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing

Hazel Mufhandu
Roles: Data Curation, Formal Analysis, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

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Abstract

Background: HBV/HIV co-infection impact on high HBV replication, progression to liver cancer and high mortality. Co-infection may lead to cross-resistance of HBV and HIV drugs due to immune therapy pressure or hepatotoxicity. These challenges necessitate continuous monitoring of HBV variants to aid better diagnosis and treatment strategies. We conducted this study to characterise HBV among HIV infected individuals.
Methods: Serum was screened for HBsAg using ELISA, followed by DNA extraction, PCR amplification, Sanger sequencing and phylogenetic analysis.
Results: Of the 50 samples in this study 100% (N=50/50) were HBsAg positive; 78% (N=41/50) HBV/HIV co-infection and 92% (N=38/41) of the amplicons were successfully sequenced. Samples nucleotide sequences were identified as genotype A. Mutations prevalence in the HBsAg region was 47% (N=18/38); including mutations associated with diagnostic failure (K122R and T143S) and 7 vaccines escape mutations (P127T, G145R, S207N, Y200T, E164D, Y206H and L209V). Mutations showed resistance to lamivudine 71% (n=5/7), telbivudine 57% (n=4/7), 14% (n=1/7) for entecavir and 43% (n=3/7) for adefovir. Mutations causing resistance to lamivudine and telbivudine were M204V, L180M, V163I, and S202K; with S202K also causing resistance to entecavir and adefovir resistance mutation were I253Y, I223V and M250I. Multiple drug resistance mutations within a single sample contained L180M, M204V, S202K and M250I mutations. There was no statistical significance between the RT mutations associated with drug resistance at P>0.005. The correlation test exhibited a weak statistical association between SHB and RT mutations (0.877**).
Conclusions: This study shows the predominance of HBV genotype A in HIV-infected patients. We discovered HBV mutations linked to immune evasion and drug resistance. Although there is no statistical significance amongst the mutations associated with drug resistance and vaccine escape. These mutations could have clinical implications that could have therapeutic repercussions by influencing the correct clinical diagnosis and treatment in HBV/ HIV co-infected individuals.

Keywords

Hepatitis B virus, HIV, Drug resistance, Vaccine escape, Mutation, Co-infection,

Corresponding authors: Lorato Modise, Hazel Mufhandu

Competing interests: No competing interests were disclosed.

Grant information: 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.

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

How to cite: Modise L, Sithebe N and Mufhandu H. Investigation of hepatitis B virus mutations associated with immune escape and drug resistance in human immunodeficiency virus-infected patients [version 1; peer review: 1 approved with reservations, 1 not approved]. F1000Research 2023, 12:1232 (https://doi.org/10.12688/f1000research.132498.1) First published: 27 Sep 2023, 12:1232 (https://doi.org/10.12688/f1000research.132498.1) Latest published: 21 Aug 2025, 12:1232 (https://doi.org/10.12688/f1000research.132498.4)

Introduction

The Orthohepadnavirus genus is a significant group of human viruses, and the Hepatitis B virus (HBV) is a prototype of the Hepadnaviridae family (Summers et al., 1975). Globally, it is thought that more than 300 million people are infected with HBV, which can lead to chronic liver disease and hepatocellular carcinoma (HCC), which accounts for over 1 million annual fatalities (Musyoki et al., 2015). The Baltimore virus taxonomy places HBV in the class VII of viruses (Gust et al., 1986). It is an enclosed virus with 3200 nucleotide base pairs of partially antagonistic relaxed and circular double stranded DNA (RC-dsDNA), and it replicates through an intermediary ssRNA (Liu et al., 2021). Hepatocytes are the cells that the hepatitis B virus infects to cause liver infection, which can then clinically show as symptomatic or asymptomatic acute hepatitis marked by liver inflammation to fulminant hepatitis. Acute HBV hepatitis that is not treated may progress to chronic HBV hepatitis, which can cause cirrhosis and HCC (Liang, 2009). Many tests, including clinical, biochemical, serological, and molecular approaches, are used to detect HBV infection, illness associated with HBV, and to differentiate between acute and chronic infections. The hepatitis B surface antigen (HBsAg), the hepatitis envelope antigen (HBeAg), the antibody to the hepatitis surface antigen (anti-HBs), the antibody to the hepatitis B core antigen (anti-HBc), and the immuglubullin antibody sub-class of the anti-HBc are the HBV antibodies and antigens that are determined by the serological testing (IgM anti-HBc). The key clinical marker for acute or chronic infection, prevalence, and endemicity of HBV infection is the presence of HBsAg, which can be identified two weeks after exposure. By identifying the qualitative and quantitative HBV-DNA, molecular methods can diagnose HBV. Based on the entire genome genetic variability of roughly 8%, HBV genomes have been divided into ten primary genotypes designating A to J (Tatematsu et al., 2009). The lack of a viral polymerase proofreading mechanism during viral replication causes the genetic heterogeneity (Rahman et al., 2016). Moreover, immunization and antiviral medication have an external and internal impact on HBV sequence heterogeneity. Geographical differences in the distribution of the various HBV genotypes, however, influence the pathogenicity of HBV and the administration of treatment. Due to their shared method of blood-born transmission, HBV, and the human immunodeficiency virus (HIV) frequently co-infect individuals (Audsley et al., 2010). Depending on where HBV is endemic, different geographic areas have different common pathways of transmission. Lamivudine (3TC) is used in the treatment of HBV and HIV (Benson et al., 2009). However, due to the development of drug resistance after 4 years of single-drug treatment, HIV-infected persons should not receive 3TC or emtricitabine (FTC) monotherapy for HBV infection (Di Martino et al., 1999). The emergence of the mutation rtm204 is a hallmark of 3TC resistance (YMDD mutation). The 3TC should only be administered to individuals on totally suppressive ART since it accelerates unchecked HBV DNA replication and raises the risk of death from HBV infection or illness. HBV prevalence has been documented in several studies among HIV-positive people from Durban, KwaZulu-Natal. However, investigations on the HBV genotype, HBV vaccine escape, and treatment resistance are still scarce in this field and most studies have concentrated on the seroprevalence. To ascertain the prevalence and genotypic characteristics of HBV among HIV-infected people in Durban, we undertook this study.

Methods

Study design, population, and ethical clearance

We employed the convenience sampling method and used samples that were available for use in this descriptive exploratory investigation; the sample size was not determined. Fifty preserved frozen sera samples from people who underwent HIV testing at the National Health Laboratory Services’ Inkosi Albert Luthuli Central Hospital (NHLS-IALCH-NHLS) in Durban, KwaZulu-Natal Province, South Africa, were utilized in this investigation. The 50 participants included both men and women who had HIV-positive test results. Participants were given a written informed consent form, the information on the consent form was given in a language that the patient understands which is English and Setswana. After receiving the written informed consent of all participants, samples were taken, tested for HIV positivity, and the residual sera were archived at the NHLS-IALCH-NHLS. At that moment, the subjects were not taking ART. The North-West University Research Ethics Regulation Committee issued an ethical certificate (Ref no: NWU-00068-15-A9) on 2015-05-22. Samples receival and data collection on the samples began in October 2015 to March 2016. 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

Samples collection and processing

A total of 50 stored frozen serum specimens from HIV-infected individuals in Durban were donated from NHLS-IALCH-NHLS and travelled on ice to the virology laboratory at the North-West University. Upon arrival the specimens were aliquoted into numerous 1.5 mL Eppendorf tubes and stored at -80 °C until further use.

Hepatitis B surface antigen (HBsAg) assay

The Monolisa HBsAg ultra confirmatory kit was used in accordance with the manufacturer’s instructions to conduct an enzyme-linked immunosorbent test (ELISA) to identify the presence of HBV HBsAg (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 L of undiluted sera specimens. At 450 nm, the optical density (OD) index of the specimens was determined and compared to the 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

Serum obtained from patients was 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 sample was added into 1.5 μL Eppendorf tubes, to which 20 μL of proteinase K and 200 μL Buffer AL (binding buffer mixed with poly [A] carrier RNA) was added. An aliquot of 200 μL of the serum sample was added into 1.5 μL Eppendorf tubes, to which 20 μL of proteinase K and 200 μL of buffer AL (binding buffer mixed with poly (A) carrier RNA) were 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 antisense strand reverse primer, S2Na (5′-CCA CAA TTC KTTGAC ATA CTT TCC A-3′) were used. The master mix were prepared using AmpliTaq 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 MgCl₂, 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 22.5 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 (25.5 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 and loaded into the sequencing machine, the ABI 3130XL genetic analyser (Applied Biosystems, Foster City, CA). An aliquot of 10 μL of the Hi-Di formamide was added into the sample for 5 minutes and loaded into a 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 complementary genotype sequences from the GenBank using the Basic local alignment search tool (BLAST). Representative sequences belonging to distinct genotypes were redeemed from GenBank to make comparisons with study sequences. Multiple sequence alignment of the sequenced nucleotide region was performed with ClustalW within the MEGA software package version 7.0 (TomHall, North Carolina State University). The aligned nucleotide base sequences were subjected to phylogeny inference on MEGA 7.0 (Kumar et al., 2004). The neighbour-joining method was used to generate dendograms, and the evolutionary relationship was performed using pairwise genetic distance with 1000 bootstrap replicates (Saitou & Nei, 1987). Frequency estimates of evolutionary divergence between nucleotide sequences were then estimated using the Kimura 2-parameter model (Kimura, 1980).

Mutations analysis

The 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 drug resistance mutations.

Statistical analyses

Microsoft Excel and the data science statistical program STATA were used for data analyses (version 15). Excel was used to calculate the frequency of age as numeric values and the frequency of HBsAg and mutations as categorical and numeric variables. STATA was used to import the Excel (.csv) file to conduct additional research on the distribution and associations of the mutations. The likelihood of a link between the two categorical variables (mutations and age) was evaluated using the Fisher’s exact test. The Pearson correlation coefficient was used to examine the relationship between the surface region and the HBV virus’s reverse transcriptase mutations. In this study, a significant p value was defined as one with a 5% level of significance.

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 from patients of black African ethnicity most of the study participants were female at 66% (N = 33/50) and 34% (N = 17/50) were males as shown in (Table 1). The median age and standard deviation of the study population were 33 years (range of 18-55).

Table 1. Demographics data of the patients by age, ethic group and ELISA screening of HBV and HIV antigens.

Age group by years	Ethnic group		HIV p24 antigen		HBsAg antigen
Age group by years	Female	Male	HIV + female	HIV + male	HBsAg + female	HBsAg + male
18-35	Black African	Black African	20	06	16	3
36-55	Black African	Black African	13	11	13	08
Total	33	17	33	17	29	11

Laboratory testing

HBsAg assay

The HBsAg was positive in all HIV positive samples resulting in a 100% (N=50/50) HBV seroprevalence. Of the 50 HBsAg positive patients (Table 1). Primarily, the study female’s participant had the highest HBsAg at (N=29/50) when compared to the males at (N=11/50) as shown in (Table 1).

PCR amplification

The PCR amplification of HBV DNA amplicons was successful in 78% (N=41/50) of the HBsAg and HIV positive samples. HBV overlapping surface/polymerase region amplicons are shown as 547 bp bands below (Figure 1). PCR amplification could not be obtained for the other 22% (N=11/50) samples.

Figure 1. PCR amplification products of overlapping surface/polymerase HBV gene of this study.

PCR amplicons are shown as 547 bp bands.

Sequence analyses of overlapping surface/polymerase gene region

Only 92% (N=38/41) of the overlapping surface/polymerase nucleotide sequence products could be obtained by Sanger sequencing. The sample sequence showed a 98% to 99% homology similarity index with previously deposited HBV genotype A sequences in the GenBank. Phylogenetic tree analysis identified nucleotide sequences from this study as genotype A as depicted in (Figure 2). The sub-genotypes of sequences were confirmed by depositing all the surface gene nucleotides sequenced into the Genotype2pheno database. The 38 individuals’ sequence were identified as sub-genotype A1 based on the results retrieved from the Geno2Pheno database with the percentage of similarity to sub-genotype profile of 96.85% -99.0%.

Figure 2. Phylogenetic tree comparing the overlapping surface/polymerase gene sequences of this study with representative sequences obtained from the GenBank (designated by accession numbers).

Study sequences are represented by letters Q, P and followed by numeric values.

Mutations within the surface gene

The prevalence of mutations in the surface gene was 47% (N=18/38) and mutations were found in the “α” epitope, “β”-cell epitope, “T” helper cell epitope and outside the “α” epitope as shown in (Table 2). The most common mutations on the surface region of HBV were S207N at 71% (27/38), followed by L216V and A194V at 23%, P70H at 21% L209V at 18%, P217L at 8%, F134L, E164D and T189I at 5% and S204R, S117N, T143S, G145R, Y206H, P127T, Y200T, F129T and K122R all at 3% (Table 2).

Table 2. Mutations within the surface gene region.

Epitope	Mutation	Frequency (%)	Function
“α” epitope	K122R	3	Sub-serotype change (d/y)
	F134L	5	Unclear
	S117N	3	Unclear
	T143S	3	Vaccine escape selection
“β”-cell epitope	S207N	71	Unknown in genotype A
	Y200T	3	Unknown in genotype A
	G145A	3	Vaccine escape selection
T-helper epitope	P127T	3	Lower reactivity in HBsAg assay
Outside “α”	E164D	5	Vaccine-escape mutation
	L209V	18
	Y206H	3
	L216V	23
	A194V	23
	P70H	21	Overt HBV infections
	P127L	8
	T189I	5
	S204R	3
	F129T	3

Mutations within the polymerase gene region

Mutations prevalence were reported at 36% (N=14/38) in different positions within the reverse transcriptase (RT) region. The M129L mutation was the most common in this study, accounting for 84% (32/38), followed by V163I at 78%; I253Y at 50% and S105T at 40%. Other mutations identified from sequences included L217R, A233S, Q125E, T128A, V214A, V204I, M204V, L180M, V173L and S202K at 21%, 16%, 13%, 8% and 3% (Table 3). The prevalence of mutations associated with drug resistance was 57% (8/14) within the RT region (Table 3). Drug resistance mutations included lamivudine (LMV) resistance at 71% (5/7), telbivudine (LdT) at 57% (4/7), 14% for entecavir (ETV) and 43% for adefovir (ADV) resistance. Mutations causing resistance to LMV and LdT were M204V, L180M, V163I, and S202K. S202K mutation also causes resistance to ETV. ADV resistance mutations were I253Y, I223V and M250I (Table 3). Multiple drug resistance mutations within a single sample were identified from one sample containing L180M, M204V, S202K and M250I mutations.

Table 3. Distribution of amino acids substitution in reverse transcriptase region of polymerase of HBV positive patients co-infected with HIV.

Amino acids substitutions	Frequency
Drug resistance mutation
M129L	84%
M204V	3%
L180M	3%
V163I	78%
V173L	3%
A223S	16%
Q125E	13%
S202K	3%
L217R	21%
V214A	3%
V204I	3%
I253Y	50%
T128A	8%
S105T	40%

Frequency of mutations

The prevalence of the mutations on the sequences of surface protein region was (N=39) and RT mutations were (N=41). The mean and standard deviation of the SHB mutations was 7.66±4.79; for RT mutations the mean and standard deviation was 11.21±5.44 as depicted in (Table 4).

Table 4. Frequencies of the resistance of mutations in the polymerase of HBV samples.

Drug	Resistance		Susceptible		Resistance	Susceptible	Probability
Drug	Age 18 – 35	Age 36-55	Age 18-35	Age 36-55	Total	Total	Fishers extract test
Lamivudine	3 12.00	3 17.65	22 88.00	14 82.35	6	36	0.6720
Adefovir	1 4.00	0 0.00	24 96.00	17 100.00	1	42	1.0000
Entecavir	3 12.00	3 17.65	22 88.00	14 82.35	6	36	0.6720
Tenofovir	0	0	25 100.00	17 100.00	0	42	0.6355

Association of mutations on the surface and polymerase

A correlation test was done between the SHB and RT mutations was done using and the Pearson Correlation. There was no statistical significance between the RT mutations at P>0.005 (Table 4). The correlation test exhibited a weak statistical association between SHB and RT mutations (0.877**) as shown in Figure 3.

Figure 3. Scatter plot showing the correlation between the SHB and RT mutations.

Plot of SHB_protein *RT_mutations, symbol used is ‘+’. 11 obs had missing values. 7 obs hidden.

Discussion

In the current study, the HBsAg seroprevalence in HIV-infected patients was 100%. The HBsAg prevalence in HIV-infected people is higher in this study as compared to previous studies in South Africa, with an estimation between 3% and 23% reported from the Western Cape, KwaZulu-Natal, and Gauteng provinces (Andersson et al., 2013; King & Hagemeister, 2016; Lukhwareni et al., 2009; Maponga, 2012; Mphahlele, 2008; Msomi et al., 2020; Samsunder et al., 2019). The HBsAg seroprevalence from this study is not coherent with published data from South Africa; comparing with previous studies, they used larger sample cohort in their studies, whereas our study used a small sample size, which influenced the high prevalence rate. However, the study report supports previous studies that endemic HBV is still present in South Africa and that HBV seroprevalence among HIV-infected people is presently high among adults (Amponsah-Dacosta, 2021; Maepa et al., 2022). All the HBsAg-positive samples were subjected to PCR amplification of the HBV DNA. The partially overlapping surface/polymerase gene region was successfully amplified in 78% (41/50), confirming the active replication of HBV and HBV/HIV co-infection on the input study samples. The presence of HBV/HIV co-infection is attributed to the shared route of viral transmission since both viruses share a similar route of transmission. Furthermore, it was also reported that females were the majority infected with HBV infection at 66% (N=33/50) compared to their male counterparts at 34% (N=17/50) in this population. This does not correlate with the findings of (Kew et al., 2008), who reported a prevalence of 4% in women compared with 9% in men. Similarly (Moonsamy et al., 2022), reported higher prevalence rates in males than females aged 25 to 49 years. The phylogenetic analysis shows the predominance of genotype A from our study sequences. The identification of HBV genotype A is coherent with previous studies in South Africa (Kimbi et al., 2004; Kramvis, 2014). The patient’s sequences were further identified as sub-genotype A1 by Geno2Pheno, indicating that HBV showed significant diversity, suggesting an African origin. The HBsAg serves is part of the S region which is employed in the development of recombinant vaccines; this region consists of α, β and T–cell epitopes. The “α” epitope is the primary area that neutralizing antibodies attacks and it contains the β and T–cell epitopes (Caligiuri et al., 2016). The “a” determinant, also known as the primary hydrophilic region of HBsAg, is the collection of B cell epitopes and is made up of the amino acids 124–147 (Liu et al., 2017). The “α” epitope domain of HBsAg may undergo mutations due to nucleotide and amino acid substitutions, which may result in structural and functional alterations in the S protein (Zuckerman & Zuckerman, 2003). This could alter the antigenicity of the vaccination and result in mutations that escape the immunization, allowing mutant HBV to replicate in the body of the vaccinated population (Yan et al., 2017). We determined the mutations on the partial surface gene of HBV/HIV co-infected individuals from our study. A total of 18 nucleotide substitutions were observed within the HBsAg region in all 38 partial surface sequences. The study reports mutations at sequence locations at the at α, β and T–cell epitopes that have clinical ramifications. The primary hydrophilic region (aa79 to aa147) of the surface antigen had the “α” epitope and contained four mutations (K122R, F134L, T143S, and S117N). G145R, S207N, and Y200T variations were found in the "T" epitope, whereas P127T was found in the “β” epitope of the “α” determinant region. The following mutations were found to be absent from the “α” epitope: E164D, L209V, Y206H, A194V, P70H, P127L, T189I, S204R, and F129T. We found escape mutations from vaccines including P127T, G145R, S207N, Y200T, Y206H, and L209V. Among 27 samples, the S207N mutation was the most prevalent one. P127T has been linked to vaccination escape selection in the past (Colagrossi et al., 2018); which contradicts the findings of Kuzin et al., (2012), who reported this mutation to causing diagnostic failure. The clinical implications of this mutations must further be explored in vitro. One patient sequence contained the mutation G145R, which has already been identified by (Colson et al., 2007; Yan et al., 2017). In the “β”-cell epitope of HBsAg, the G145R mutation is a significant vaccine escape mutation that is linked to a point mutation that results in the substitution of arginine for glycine at position 145 (G145R). The HBsAg antigenicity is decreased because of the interference with antibody binding (Kao & Chen, 2002; Su et al., 2008). Given that it was the first vaccine-escape mutation discovered in children who received vaccinations in Italy, the G145R mutation is a typical mutation (Zanetti et al., 1988). Its occurrence and stability have increased over the years with an increase of HBV endemicity and use of universal immunization (Caligiuri et al., 2016). This mutation is crucial in causing failure on the detection of HBV by serological routine assays (Cooreman et al., 2001). The long-term success of mass vaccination, however, has been previously reported to be threatened by G145R, which has grown more dominant with vaccine escape selection (Yan et al., 2017). Thus, it is advised that it be incorporated into the design of future vaccines (Yan et al., 2017). It is said that this mutation has a possible horizontal transmission pattern (Colagrossi et al., 2018). Globally, the G145R mutations have previously been linked to HBV breakthrough infections in the population who had received vaccinations (Cooreman et al., 2001; Theamboonlers et al., 2001). On the other hand, mutations G145R and F143S have also been discovered in people with occult hepatitis B infection (OBI), and they have been associated to the clinical implications. On the other hand, mutations G145R and F143S have also been found in individuals with occult hepatitis B infection (OBI), and they have been linked to the clinical implications of diagnostic failure due to suboptimal HBsAg detection when using commercial ELISA kits (Coppola et al., 2016). It was previously reported as a nucleos (tide) analog (NA-induced) immune escape mutation (Colagrossi et al., 2018; Lai et al., 2003), demonstrated that the E164D result from the substitution change in the overlapping polymerase region caused by the mutation rtM204V, rtM204I, and rtV173L) (Lai et al., 2003; Zöllner et al., 2001). E164D was another significant mutation discovered from this study. These mutations produce a decrease in anti-HBs and are linked to lamivudine resistance therapy (Colagrossi et al., 2018). Additional mutations found in this study included K122R, T143S, Y206H, L209V, S207N, Y200T, and P70H. Nevertheless, there is little information available about these mutations’ effects on surface protein structure and activities, and more research is needed to determine how they may affect patients’ health. K122R and T143S mutations, on the other hand, have reportedly been linked to diagnostic adequacy (Cooreman et al., 2001). With certain ELISA diagnostic methods failing to identify HBsAg from samples bearing the K122R in prior research (Malik et al., 2012). All the HBsAg mutations mentioned above have clinical implications that are connected to immunological escape and have an impact on the antibodies’ ability to recognize HBsAg. whilst others are connected to diagnostic failure. Antiviral medications that prevent reverse transcription specifically target the HBV polymerase to prevent HBV DNA replication. The nucleoside/nucleotide analogues LMV, ADV, TDF, LdT, and ETV are antiviral medications that are used to treat HBV polymerase. Despite the introduction of TDF, lamivudine treatment is still favoured among patients with HBV/HIV co-infection in South Africa. Antiviral medications may potentially contribute to the development of drug resistance due to selection pressure during long-term use of the antiviral treatment, in addition to the high mutation rate caused by the HBV error-prone (Fares & Holmes, 2002). The frequency of drug resistance-related mutations in the RT region was 50% (N=7/14). M204V, L180M, V163I, and S202K were mutations linked to LdT resistance; S202K also conferred ETF resistance. Combining L180M and M204V results in increased cross-resistance to other nucleosides and decreased sensitivity to ETV but not ADV. It may also result in vaccine escape mutations in the overlapping S-region, which would stop HBsAg from secreting (Sheldon et al., 2005). ADV resistance mutations included the I253Y, I223V, and M250I mutations. A single sample from one patient that contained the mutations L180M, M204V, S202K, and M250I was shown to have several drug resistance mutations. All the drug resistance mutations found in the RT of the pol region were found in genotype A sequences. Moreover, the following compensatory mutations were found: S202K, Q125E, L217R, V124A, V204I, I253Y, T128A, and S105T. We still need to investigate how these changes affect RT functions. As this study found no mutations linked to tenofovir resistance, we advise using a tenofovir-based regimen to treat HBV in people who also have HIV. At P>0.05, there was no statistical difference between the mutations linked to medication resistance. The Pearson Correlation was used to conduct a correlation test between the SHB and RT mutations. The connection between SHB and RT mutations was not strong. Further in vitro studies must be carried out to investigate the effects of the detected alterations on the antibody neutralization and nucleoside/tide drug analogues. This does not rule out the possibility that these mutations have major clinical implications towards the diagnosis and therapy.

Conclusion

This study demonstrates the prevalence of HBV genotype A in HIV-positive patients as well as the presence of HBV mutations in HBV/HIV co-infected people. We discovered HBV mutations linked to immune escape and drug resistance that could have therapeutic repercussions by influencing the correct clinical diagnosis and HBV treatment. This study has improved our understanding of the genotypes and mutations of HBV that have clinical implications in those who have HBV and HIV co-infection in South Africa. To determine how immune escape and drug resistance-related HBV mutations affect diagnosis and drug resistance treatment in the context of HIV/HVB co-infection, we recommend testing the HIV samples for these mutations.

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).

This project contains the following underlying data:

- data set for HIV 2017-2019.xlsx
- data set for demographic and virology test 2017-2019.xlsx

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

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

Figshare: Reference sequences accession number and Genotype, https://doi.org/10.6084/m9.figshare.23946642.v1 (Modise, 2023d).

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 National Health Laboratory Services in KwaZulu-Natal for donating samples for this study. We thank the State Key Lab of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences for providing training on HBV genotyping and cell culturing.

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Version 4

VERSION 4 PUBLISHED 27 Sep 2023

Author details Author details

¹ Biological Sciences, North West University, Mahikeng, North West, 2735, South Africa

Nomathamsanqa Sithebe
Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing

Hazel Mufhandu
Roles: Data Curation, Formal Analysis, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

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.

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Modise L, Sithebe N and Mufhandu H. Investigation of hepatitis B virus mutations associated with immune escape and drug resistance in human immunodeficiency virus-infected patients [version 1; peer review: 1 approved with reservations, 1 not approved]. F1000Research 2023, 12:1232 (https://doi.org/10.12688/f1000research.132498.1)

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Reviewer Report 22 Nov 2023

Anna McNaughton, Nuffield Department of Medicine, University of Oxford Medawar Building, Oxford, England, UK

Not Approved

https://doi.org/10.5256/f1000research.145424.r224163

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

No
Are sufficient details of methods and analysis provided to allow replication by others?

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?

Partly
Are the conclusions drawn adequately supported by the results?

No

Competing Interests: No competing interests were disclosed.

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.

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Author Response 31 May 2024

Lorato Mosetsanagape Modise, Biological Sciences, North West University, Mahikeng, 2735, South Africa

31 May 2024

Author Response

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 ... Continue reading 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.
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.
Competing Interests: No competing interests. Close
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COMMENTS ON THIS REPORT

Author Response 31 May 2024

Lorato Mosetsanagape Modise, Biological Sciences, North West University, Mahikeng, 2735, South Africa

31 May 2024

Author Response

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 ... Continue reading 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.
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.
Competing Interests: No competing interests. Close
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Reviewer Report 27 Oct 2023

Nishi Prabdial-Sing, Centre for Vaccines and Immunology, Division of the National Health Laboratory Service, National Institute for Communicable Diseases, Johannesburg, South Africa

Approved with Reservations

https://doi.org/10.5256/f1000research.145424.r211197

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

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

Partly
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
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

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.

CITE

Report a concern

Author Response 30 May 2024

Lorato Mosetsanagape Modise, Biological Sciences, North West University, Mahikeng, 2735, South Africa

30 May 2024

Author Response

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 ... Continue reading 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.
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.
Competing Interests: No competing interest. Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 30 May 2024

Lorato Mosetsanagape Modise, Biological Sciences, North West University, Mahikeng, 2735, South Africa

30 May 2024

Author Response

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 ... Continue reading 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.
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.
Competing Interests: No competing interest. Close
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Nishi Prabdial-Sing, National Institute for Communicable Diseases, Johannesburg, South Africa
Anna McNaughton, University of Oxford Medawar Building, Oxford, UK
Anthony C. Ike, University of Nigeria, Nsukka, Nigeria
Tesfaye Gelanew, Armauer Hansen Research Institute, Addis Ababa, Ethiopia

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1 Views

11 Sep 2025 | for Version 4

Tesfaye Gelanew, Armauer Hansen Research Institute, Addis Ababa, Addis Ababa, Ethiopia

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Approved

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

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05 Aug 2025 | for Version 3

Nishi Prabdial-Sing, Centre for Vaccines and Immunology, Division of the National Health Laboratory Service, National Institute for Communicable Diseases, Johannesburg, South Africa

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Approved 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?

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22 Views

28 Jul 2025 | for Version 3

Tesfaye Gelanew, Armauer Hansen Research Institute, Addis Ababa, Addis Ababa, Ethiopia

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

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

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
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

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

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5 Views

14 Jun 2025 | for Version 3

Anthony C. Ike, University of Nigeria, Nsukka, Enugu, Nigeria

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

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

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
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

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.

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50 Views

06 Jun 2024 | for Version 2

Anna McNaughton, Nuffield Department of Medicine, University of Oxford Medawar Building, Oxford, England, UK

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Not Approved

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.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Virology, genomics, epidemiology, viral hepatitis

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Author Response

23 Apr 2025

Lorato Mosetsanagape Modise, Biological Sciences, North West University, Mahikeng, 2735, South Africa

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.).

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03 Jun 2024 | for Version 2

Nishi Prabdial-Sing, Centre for Vaccines and Immunology, Division of the National Health Laboratory Service, National Institute for Communicable Diseases, Johannesburg, South Africa

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Approved 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)

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

virology, diagnostics, molecular biology

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37 Views

22 Nov 2023 | for Version 1

Anna McNaughton, Nuffield Department of Medicine, University of Oxford Medawar Building, Oxford, England, UK

37 Views Cite this report Responses(1)

Not Approved

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

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

No
Are sufficient details of methods and analysis provided to allow replication by others?

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?

Partly
Are the conclusions drawn adequately supported by the results?

No

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Virology, genomics, epidemiology, viral hepatitis

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Author Response

31 May 2024

Lorato Mosetsanagape Modise, Biological Sciences, North West University, Mahikeng, 2735, South Africa

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.

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Competing Interests

No competing interests.

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Reviewer Report

41 Views

27 Oct 2023 | for Version 1

Nishi Prabdial-Sing, Centre for Vaccines and Immunology, Division of the National Health Laboratory Service, National Institute for Communicable Diseases, Johannesburg, South Africa

41 Views Cite this report Responses(1)

Approved With Reservations

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

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

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

Partly
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
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

virology, diagnostics, molecular biology

Respond to this report

Responses (1)

Author Response

30 May 2024

Lorato Mosetsanagape Modise, Biological Sciences, North West University, Mahikeng, 2735, South Africa

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.

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Competing Interests

No competing interest.

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

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Investigation of hepatitis B virus mutations associated with immune escape and drug resistance in human immunodeficiency virus-infected patients

Abstract

Keywords

Introduction

Methods

Study design, population, and ethical clearance

Laboratory testing

Polymerase chain reaction

Sequences analysis

Mutations analysis

Statistical analyses

Ethical approval

Results

Baseline demographic data

Table 1. Demographics data of the patients by age, ethic group and ELISA screening of HBV and HIV antigens.

Laboratory testing

Figure 1. PCR amplification products of overlapping surface/polymerase HBV gene of this study.

Sequence analyses of overlapping surface/polymerase gene region

Figure 2. Phylogenetic tree comparing the overlapping surface/polymerase gene sequences of this study with representative sequences obtained from the GenBank (designated by accession numbers).

Table 2. Mutations within the surface gene region.

Table 3. Distribution of amino acids substitution in reverse transcriptase region of polymerase of HBV positive patients co-infected with HIV.

Table 4. Frequencies of the resistance of mutations in the polymerase of HBV samples.

Figure 3. Scatter plot showing the correlation between the SHB and RT mutations.

Discussion

Conclusion

Data availability

Underlying data

Acknowledgements

References

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