Intra-familial transmission of Hepatitis B virus in a peri-urban community from the Democratic Republic of the Congo

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Despite global elimination targets set for 2030, Hepatitis B virus (HBV) infection remains a major public health challenge in low-income countries, including the Democratic Republic of Congo (DRC). Limited evidence on the regional transmission pathways preclude progress towards HBV elimination. This study aimed to assess the prevalence, molecular characteristics, and transmission dynamics of HBV in the Lukelenge health district, a peri-urban area in central DRC. Methods. We employed a two-tiered recruitment strategy: community member volunteers were enrolled during the first phase, and upon notification of HBV positivity in an index case, family contacts were subsequently recruited in the second phase. Participants were screened for hepatitis B surface antigen (HBsAg), followed by PCR amplification of HBV DNA and sequencing. Genotyping and phylogenetic analysis of preS/S sequences were performed to explore regional HBV diversity and transmission patterns. Results. A total of 751 participants from 677 households were included. The overall HBsAg prevalence was 3.8% [95% CI: 2.6–5.5], with the highest rate (10.1%) found in children aged 5 years and younger. All 42 HBV isolates belonged to genotype E, with 97.6% sharing the ayw4 serotype. Mutations with relevancy to immune escape were detected in 9.5% of strains, while those possibly linked to antiviral resistance were found in 4.7%. Maximum likelihood phylogenetic analysis showed intra-familial clustering of preS/S sequences, suggesting that parent-to-child transmission was the most frequent mode of HBV spread in the study population. Conclusions. HBV in Lukelenge shows intermediate endemicity, especially affecting young children. Intra-familial transmission revealed to be predominant, likely involving both vertical and horizontal pathways. Family-targeted interventions, including maternal screening and universal birth-dose vaccination, should be prioritized to eliminate HBV in this region. Intra-familial Transmission Hepatitis B virus Peri-urban Community Democratic Republic of Congo Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Hepatitis B virus (HBV) infection remains a major worldwide health challenge, with an estimated 254 million chronically infected people, causing substantial mortality from liver cirrhosis and hepatocellular carcinoma (HCC) [ 1 ]. In response, the World Health Organization (WHO) set a goal to eliminate HBV infection by 2030, with a 90% reduction in new infections and a 65% reduction in deaths through expanded vaccination, early diagnosis, enhanced antiviral access, and the development of emerging curative therapies[ 2 , 3 ]. In spite of these worldwide efforts, HBV infection persists, particularly in sub-Saharan Africa, which accounts for 63% of global new infections annually [ 4 ]. Africa faces substantial gaps in vaccination, diagnostics, and treatment, exacerbating the global burden of HBV, with significant disparities observed both across and within countries in the region[ 4 , 5 ]. In the Democratic Republic of the Congo (DRC), HBV prevalence is in line with broader African trends. The national prevalence is reported to be around 3.3%, with approximately 300.000 children living with chronic hepatitis B, far exceeding the WHO's 2030 elimination target of ≤ 0.1% prevalence in children under five[ 6 – 8 ]. Rural and peri-urban areas face the most notable gaps in HBV control strategies, necessitating urgent region-specific interventions[ 4 , 9 , 10 ]. Since HBV can be transmitted through blood, semen, or vaginal fluids via either horizontal or vertical routes, and the relative importance of each pathway varies geographically[ 11 ], it is essential to understand regional transmission dynamics in order to implement context-specific interventions. Horizontal transmission has been widely recognized in Africa, especially through close contact within households and communities[ 12 ]. However, with increased coverage of the three-dose hepatitis B vaccination series (HepB3) in infants, the significance of horizontal transmission would be diminishing[ 13 ]. In contrast, the relative importance of vertical transmission remains debated especially in the DRC, where a study in pregnant women and their offspring have suggested a crucial role of mother-to-child transmission in the persistence and spread of HBV within households[ 14 ]. Population-based studies providing insight into this hypothesis remain limited in DRC. Genetic analysis of HBV gives valuable insights into viral strain typing and transmission dynamics. The HBV genome is a circular, partially double-stranded DNA (~ 3.2 kb) comprising four overlapping open reading frames (ORFs): preS/S, preCore/Core, Pol, and X[ 15 ]. The preS/S ORF, which encodes for the surface antigen of hepatitis B (HBsAg), is highly variable and has been thoroughly studied for phylogenetic purposes[ 15 , 16 ]. This region's variability is partly driven by the overlapping Pol ORF, encoding a polymerase with low replication fidelity, yielding a high mutation rate. As a result, ten HBV genotypes (A-J), nine serotypes, and over forty sub-genotypes have been identified [ 15 , 17 ]. Mutations in the preS/S region, particularly within the "a" determinant of the Small S protein, are of critical clinical importance as they can give rise to immune escaping strains capable of infecting vaccinated individuals and causing occult HBV infections[ 15 ]. These mutations can also be accountable for antiviral drug resistance and disease progression to cirrhosis or HCC[ 18 ]. Overall, genetic analysis of the preS/S region is useful for understanding HBV diversity, transmission dynamics, and its implications on disease progression and treatment resistance[ 15 , 16 ]. Comprehensive genetic analyses of HBV strains circulating among the African population is crucial for guiding implementation of targeted public health interventions. However, such studies detailing HBV genotypes, mutations, and transmission pathways remain scarce in the DRC[ 19 , 20 ]. To address this critical knowledge gap, the present study aimed to assess HBV prevalence, genetic diversity, and potentials transmission patterns within a peri-urban community in central DRC. Methods Design and setting of the study This community-based cross-sectional study was conducted between April and November 2023 in the Lukelenge health district (HD), located in Mbujimayi, Kasaï-Oriental province, DRC, an area endemic for HBV (Fig. 1 ). Lukelenge, a peri-urban area on the outskirts of Mbujimayi, spans 35 km² and has an estimated population of 341,591, resulting in a population density of approximately 9,760 inhabitants per km². The local economy is primarily based on artisanal diamond mining; however, declining profitability has considerably impacted household income, making Lukelenge one of the most impoverished HDs in the region[ 21 ]. Sampling strategy and study population A simple random sampling method was used to select households in the study area, based on the household registry provided by the Central Office of the Lukelenge HD. Participant recruitment followed a two-tiered approach, with visits spaced six months apart. During the first visit, all selected households were visited, and within each household, one eligible individual, aged one year or older and residing in the study area for at least three months, was randomly selected using a lottery method, to be screened for HBsAg. Those who tested positive for HBsAg were designated as "index cases", and their households were subsequently invited to participate in the second-phase, family-based screening. During the second visit, up to five household members, including the index case's parents and siblings, were selected as "contact cases" for HBsAg screening. Eligibility required written consent from adults, or permission from a parent or legal guardian for minors (under 18 years). Additionally, verbal assent was obtained from minors aged 15 to 18 years. Using Cochran’s formula[ 22 ], the required sample size for the community survey was calculated assuming a 3.3% estimated prevalence of HBsAg[ 6 ], a 95% confidence level (Z = 1.96), and an absolute margin of error of 1.4%. This yielded a minimum required sample size of 626 participants. To account for possible non-response, a final sample size of 700 was targeted. Field investigation and sample collection Each visit involved a brief interview to assess eligibility, gather demographic data, and document the family pedigree. Eligible participants underwent a rapid HBsAg test (One Step HBsAg Rapid Test Strip, RapidLab, China) following the manufacturer's instructions. Briefly, 50 µL of blood was collected via fingerprick, applied to the test strip, and allowed to develop for 15 minutes. For HBV DNA extraction, an additional 50 µL of blood was collected, spotted onto a Whatman 903 filter card (Cytiva, USA), and allowed to dry. The dried blood spot sample (DBSs) was stored in a zip-lock bag with desiccants at ambient temperature. The samples were later transferred to the Biobank of the Institut National de Recherche Biomédicale (INRB) in Kinshasa, DRC, and stored at -20°C. These DBSs were then used for molecular analyses. Field data were recorded using a semi-structured electronic questionnaire, designed with the KoboToolBox application ( https://kf.kobotoolbox.org/ ). Participants who tested positive for HBsAg received counseling and were referred to the Lukelenge HD’s reference hospital for appropriate care and follow-up. HBV DNA extraction, PCR amplification, and sequencing Total DNA was extracted from dried blood spot samples using the DNeasy® Blood and Tissue Kit (Qiagen, Hilden, Germany). The DNA was eluted in 30 µL of final volume. PCR reactions included 2 µL of DNA solution as template in a 25 µL total volume, and PrimeSTAR Max DNA Polymerase (TaKaRa, Japan), and was run on a C1000 Touch thermal cycler (Bio-Rad, USA). Previously established sets of primers were used ( Supplementary Table S1 ), with PCR conditions that we optimized for HBV genomic DNA amplification, as indicated below. PreS/S region amplification (~ 1200 bp): A semi-nested PCR was performed using primers PS1 and P3 for the first round, followed by PS1 and S2 for the second round [ 22 ], with a final primer concentration of 0.2 µM. Thermal cycling conditions consisted of 30 cycles at 98°C for 10 seconds, 55°C for 15 seconds, and 72°C for 5 seconds. Whole genome amplification (~ 3200 bp): Universal primers WA-L and WA-R [ 26 ], with a final concentration of 0.5 µM, were used. Thermal cycling conditions included 40 cycles at 98°C for 10 seconds, 60°C for 5 seconds, and 72°C for 30 seconds. This was performed only on the index cases specimens. Successfully amplified PCR products were purified using the NucleoFast® 96 PCR Clean-up kit (Macherey-Nagel, France) and subsequently sequenced using the 3730XL DNA Analyzer (Applied Biosystems) with specific sequencing primers detailed in Supplementary Table S1 . HBV molecular analysis Nucleotide sequences were inspected and trimmed using Chromas Software v2.6.6[ 23 ]. They were assembled on the CLC Genomics Workbench platform v.23 (CLC bio, Denmark), and then aligned using MAFFT v7[ 24 ]. Phylogenetic analysis was performed using maximum likelihood trees reconstructed with IQ-TREE v2.3.6 [ 25 ] and visualized as midpoint-rooted trees on the Interactive Tree of Life (iTOL) platform v7 [ 26 ]. Node support was evaluated using 1,000 bootstrap replicates to assess topology robustness. The most appropriate nucleotide substitution model was selected based on the Bayesian Information Criterion (BIC). Reference sequences of HBV genotypes A to J were retrieved from the Genbank database ( https://www.ncbi.nlm.nih.gov/genbank/ ) and incorporated into the phylogenetic trees. Genotyping was cross validated using the Hepatitis B Virus Phylogenetic Typing Tool v2.59[ 27 ]. Genetic distances within and between families were calculated using the Kimura-2-Parameter (K2P) substitution model in MEGA v11[ 28 ]. HBV serotypes were inferred by analyzing key amino acid residues within the small S protein sequence (i.e., positions 122, 127, 140, 159, and 160), following established classification algorithms[ 29 ]. Mutations linked to immune escape or antiviral resistance were further predicted using the HIV-Grade: HBV-Tool[ 30 ], assessing clinically relevant viral polymorphisms. Statistical analysis Statistical analyses were performed using R software version 4.4.3 (The R Development Core Team, Austria). Categorical variables were summarized as absolute and relative frequencies, while continuous variables were described using medians and ranges (minimum to maximum). The prevalence of HBsAg was estimated at both community and intra-familial levels using Jeffreys’ approximate Bayesian confidence intervals. Age-specific prevalences were calculated based on data from index case screening, without considering intra-familial screening data. The chi-square test was used to compare HBsAg positivity rates between groups, and the odds ratio indicating the trend in the likelihood of positivity. Genetic distances of HBV within and between families were compared using the Wilcoxon rank-sum test. Receiver Operating Characteristic (ROC) curve analysis was applied to predict monophyletic clusters based on sequence similarity, in order to determine optimal cutoff value for viral genetic divergence as the threshold for defining similarity between HBV sequences. A p-value of less than 0.05 was considered statistically significant. Results Community survey (among index cases) In the community survey, a total of 700 households were randomly selected, and 700 eligible participants were recruited, with 677 providing complete survey responses, yielding a response rate of 97% (Fig. 2 ). The study participants had a sex ratio of 0.7 (male: n = 293, female: n = 384), and a median age of 16 years (range: 1 to 81 years). Among the 677 participants, 26 individuals were tested positive for HBsAg, leading to an estimated HBsAg prevalence of 3.8% (95% CI: 2.6–5.5). Children aged 5 years and younger showed the highest prevalence at 10.1% (n = 8/79 [OR = 3.8; 95% CI: 1.4–10.6]; p = 0.019) compared to adults aged 18 years and older (2.8%, n = 8/282). There was no significant difference in HBsAg positivity between males (3.8% [95% CI: 2.0-6.4]; n = 11/293) and females (3.9% [95% CI: 2.3–6.2]; n = 15/384) (Fig. 3 ). Intra-familial survey (among familial contacts of index cases) Of the 26 index households, five had relocated and six declined to participate, leaving 15 households in which HBsAg screening was ultimately conducted (Fig. 2 ). From each of these 15 households, five case contacts were included, except for family #24, where only four individuals participated. In total, 74 individuals (11 fathers, 12 mothers, and 51 children) were included in the intra-familial screening, of whom 17 tested positives for HBsAg, resulting in a prevalence of 23.0% (95% CI: 14.5–33.5). Compared to the prevalence observed in the community survey, the prevalence among familial contacts of index cases was significantly higher (23.0% vs 3.8%, OR = 5.8 [95% CI: 2.9–11.1]; p < 0.001) (Fig. 3 ). None of the participants had previously tested positive for HBV or received antiviral treatment, and only 10% of participants had heard of hepatitis B, typically referring to it using the local terms mutshima muhula (hepatomegaly) or difu diuhula (abdominal bloating). Molecular profile of HBV strains circulating in the study population A total of 42 HBV preS/S sequences were successfully obtained, including 25 from the 26 index cases and all 17 HBsAg-positive family contacts. All isolates were assigned to HBV genotype E (HBV/E) (Fig. 4 ). Notably, 41 of 42 HBV/E strains (97.6%) were classified as serotype ayw4 , demonstrating high serotype concordance. One strain exhibited a discordant, yet-unclassified serotype, with an L127Q substitution alongside wild-type R122 and K160 positions. A detailed mutational analysis is provided in Supplementary Table S2 . Furthermore, mutational analysis identified six mutations (i.e., P120S, C121Y, G130R, G145R, C147S, and T126N) within the "a" determinant of the major hydrophilic region of the HBsAg in 4 of 42 viral strains (9.5%), which have been reported as potentially associated with immune escape. Additionally, two mutations, rtI169T and rtM204K, were each detected in a different sequence (4.7%), within the overlapping RT gene, with previous reports suggesting possible links to antiviral drug resistance [ 31 , 32 ]. Whole-genome amplification was successful in 96.1% (25/26) of index cases samples, and phylogenetic reconstruction of whole-genomic sequences revealed close ancestral relationships, consistent with that of the preS/S region ( Supplementary Figure S1 ). HBV transmission dynamics within studied families and community To explore transmission patterns, phylogenetic analysis, genetic distance calculations, and family pedigree reconstructions were employed using preS/S sequences. Among the 15 HBV-positive families studied, 73.3% (11/15) of families had an infected parent; maternal and paternal infections were present in 60% (9/15) and 53.8% (7/13) of the families, respectively, and 38.4% (5/13) had both parents infected (Fig. 5 ). Within families, genetic distances were significantly smaller in preS/S sequences than those between unrelated families (0.1 [range: 0-0.7] vs. 0.8 [range: 0.1–2.9]; p < 0.001), as shown in Fig. 6 . Multiple infections were detected in 80% (12/15) of the households. In 66.7% (10/15) of these families, sequences clustered monophyletically (≥ established best cutoff value of 99.65% similarity) (Fig. 7 ). These findings suggest a predominant mode of HBV intra-familial transmission, including mother-child (26.7% [n = 4/15]; families #1, #3, #8 and #20), father-child (15.4% [n = 2/13]; families #15 and #22), sibling-to-sibling (20% [n = 3/15]; families #2, #7, and #14), and spouse-to-spouse (7.6% [n = 1/13]; family #25) patterns. In contrast, 26.7% (4/15) of families harbored multiple infected individuals carrying genetically divergent HBV strains with sequence similarity < 99.65%, suggesting independent viral introductions rather than intra-familial transmission. In families #3 and #8, the parents were infected with distinct strains, but the mother-child pairs shared closely related strains, indicating possible mother-to-child transmission. In family #22, the parents carried divergent strains, but the father and child shared closely related strains, suggesting a possible father-to-child transmission. In family #13, the mother and child were infected with genetically distant strains, suggesting acquisition from separate sources (Fig. 7 ). Discussion This study assessed the epidemiological landscape and transmission dynamics of HBV in Lukelenge, a peri-urban community of central DRC. Two key findings emerged: first, the region exhibited intermediate HBV endemicity, characterized by a markedly high prevalence among children aged 5 years and younger. Second, phylogenetic analysis combined with family pedigree reconstructions strongly suggested that intra-familial spread is the predominant mode of HBV transmission, particularly involving parent-to-child and sibling-to-sibling routes. These findings offer cornerstone evidence to guide local public health strategies. Overall, the estimated HBsAg prevalence was 3.8% (95% CI: 2.6–5.5; n = 26/677) in the study population, slightly exceeding the national estimate of 3.3% for the DRC[ 6 ]. This positions the region within the WHO’s intermediate endemicity category for HBV, suggesting a persistent infection burden[ 33 ]. Contributing factors to this situation likely involve limited access to healthcare services, insufficient screening programs, and delays in diagnosis[ 2 , 4 , 5 ]. One in ten children aged five years and younger was infected with HBV. This finding is of particular concern and provides key insight into local transmission dynamics. It suggests substantial HBV transmission during early life likely due to major gaps in prevention, including low HepB3 vaccine coverage (estimated at just 65%) and the absence of a universal timely birth dose HBV vaccine program throughout the DRC[ 34 ]. Given that early-life HBV infections carry a higher risk of progressing towards chronic infection and subsequent severe liver diseases like cirrhosis and HCC[ 35 ], this high prevalence in young children represents a major public health threat. Similar trends of elevated childhood HBV prevalence have been reported in other sub-Saharan African countries, including Cameroon, South Africa, and Nigeria[ 16 , 36 , 37 ]. To tackle this issue, essential interventions should include the enforcement of the birth-dose HBV vaccination, enhancing maternal HBV screening, and improving access to antiviral prophylaxis for infected mothers. Regarding HBV genetic characterization, this study exclusively identified the HBV genotype E and the ayw4 serotype in the study population. This genotype has been previously described in southeastern region of the DRC[ 19 ] and is known to be prevalent across Central and West Africa, including countries such as Cameroon and The Gambia[ 16 , 38 , 39 ]. The observed restricted genetic diversity suggests localized transmission patterns. Interestingly, the unique L127Q substitution found in one isolate suggests a potential emergence of yet-unclassified serotype. Located in the S gene, this mutation could affect antigenicity, immune recognition, and diagnostic accuracy, and warrants further investigation. Immune and vaccine escape mutations were identified in approximately one out of ten viral strains, including G145R, P120S, and T126N, which have been reported to be associated with reduced vaccine efficacy[ 40 , 41 ]. The G145R mutation, in particular, is notable for its stability and ability to be transmitted both vertically and horizontally[ 15 , 41 ]. These mutations enable HBV to evade immune responses from vaccination, immunoglobulin treatments, and natural immunity, leading to breakthrough infections and diminished vaccine effectiveness[ 15 ]. The detection of vaccine escape variants in this endemic area raises concerns about the risk of breakthrough infections, as seen previously in the DRC among vaccinated children aged 6 to 59 months[ 42 ]. Additionally, mutations such as rtI169T and rtM204K, possibly linked to antiviral resistance, were detected in nearly 5% of viral strains. An isolated rtI169T mutation may contribute to entecavir resistance when coexisting with additional mutations[ 32 ]. The rtM204K mutation confers resistance to adefovir and tenofovir in vitro , although this has not been confirmed in clinical outcomes[ 31 ]. This resistance could limit treatment options for chronic HBV infection, particularly in resource-limited settings where these drugs are commonly used[ 43 ]. The presence of drug-resistant strains in an antiviral treatment-naïve population presents a significant challenge for HBV control[ 44 ]. Regardless of the presence of these mutations, there is an urgent need for comprehensive control strategies, including enhanced viral surveillance, expanded access to antiviral treatments, and post-exposure prophylaxis. This study provided also compelling evidence pointing towards the household as the primary setting for HBV transmission in the study area. Approximately 80% of affected families had multiple infected members, and genetically similar strains were detected within 66.7% of households, supporting the predominance of intra-familial transmission of HBV in the study community. Parent-to-child transmission emerged as the most common route, with maternal transmission likely playing a more significant role than paternal transmission. This aligns with findings from other HBV-endemic regions where perinatal and early childhood exposure are recognized as major contributors to the persistence of HBV infection[ 45 ]. Sibling-to-sibling transmission was also evident, likely facilitated by close contact and shared personal items, which are well-documented transmission routes in endemic settings[ 46 ]. Although spousal transmission was identified in one family, most infections appeared to be acquired during childhood, suggesting a limited local role for sexual transmission in the study community. Remarkably, horizontal acquisition of HBV through extra-familial route was limited, suggesting that exposure within the broader community plays a less significant role in its spread. Collectively, these findings provide important public health implications, indicating that targeted interventions within households - such as routine HBV screening for family members of infected individuals and the identification of HBsAg-positive individuals who may be eligible for antiviral therapy – could effectively reduce HBV disease burden[ 47 ]. Particularly given the growing momentum to integrate antenatal HBsAg screening into the triple elimination initiative for HIV, syphilis, and hepatitis B, this approach presents a valuable opportunity. Antenatal maternal screening could serve as an effective entry point for identifying infected households, enabling timely familial screening and follow-up interventions, as has been suggested in similar settings such as Burkina Faso[ 48 ]. This study has the following limitations. First, being a community-based survey conducted in a single location, the findings may not be generalizable to other regions. Second, the limited number of tested family contacts may have led to over-/under-estimations of intra-familial HBV prevalence. Third, the individual vaccination status of children was not assessed due to issues with parental recall, missing records, and limited knowledge about HBV. However, these children were presumed to be vaccinated in accordance with the national immunization program in the DRC. HIV status was not determined. Fourth, the cross-sectional design of the study precludes the determination of infection chronology within families, making it difficult to identify HBV transmission directions and primary sources. Fifth, genetic analyses were limited to the preS/S region of the HBsAg, this could limit insights into broader HBV genetic diversity and evolutionary patterns. Lastly, reliance on self-reported data may have introduced recall bias. Conclusions This study provides valuable insights into HBV epidemiology in the central DRC with significant public health implications. The findings confirm an intermediate endemicity profile, with transmission occurring predominantly within families through vertical (parent-to-child) and horizontal (sibling-to-sibling) routes. The exclusive detection of HBV genotype E, ayw4 serotype and limited genetic diversity suggest stable, localized transmission patterns. The identification of immune escape and possible drug-resistant variants highlights the need for improved HBV surveillance, enhanced vaccination strategies, and expanded access to antiviral treatment. Moving forward, targeted interventions, including universal birth-dose vaccination, maternal screening, and household-based prevention measures, will be essential in reducing the HBV burden in the study population. Abbreviations BIC: Bayesian Information Criterion CI: Confidence Intervals DRC: Democratic Republic of Congo HBsAg: Hepatitis B surface antigen HBV/E: HBV genotype E HBV: Hepatitis B virus HCC: Hepatocellular Carcinoma HD: Health District HepB3: Three-dose hepatitis B vaccination series INRB : Institut National de Recherche Biomédicale iTOL: Interactive Tree of Life K2P: Kimura-2-Parameter OR: Odd Ratio ORFs: Open Reading Frames PCR: Polymerase Chain Reaction ROC: Receiver Operating Characteristic WHO: World Health Organization Declarations Ethics approval and consent to participate Informed consent was obtained from adult participants and legal guardian for minors (under 18 years). Additionally, verbal assent was obtained from minors aged 15 to 18 years. The study followed the Declaration of Helsinki principles and was approved by the ethics committee of the University of Mbujimayi, the DRC ( n°002/CEI/UM/2023 ). Consent for publication All co‑authors have read and approved this manuscript for publication. Data availability and materials All data generated during this study are included in the published article and its additional file. The genetic data we generated and analyzed during this study are available in the DNA Data Bank of Japan (DDBJ) and GenBank database under accession numbers LC858535 to LC858576. Competing interests The authors do not have a commercial or other association that might pose a conflict of interest. Funding This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI under the grant numbers 22KK0279 (to Yu Nakagama). Additional funding was provided by KAKENHI under Grant Number JP18KK0454 and 23H02951, JSPS Core-to-Core Program (Grant Number: JPJSCCB20240010), and the Japan Agency for Medical Research and Development (AMED) under Grant numbers JP25wm0125003 and JP19fm0208020 (all to Yasutoshi Kido). Authors' contributions FCK, ETK, NKK, YN, and YK conceived the study. FCK and ETK wrote the study protocol. FCK, ETK, SN and YN did the data analyses and made the figures. FCK, YN, ETK, SN, NKK, YS, and YK wrote the initial manuscript. All co-authors made a major contribution in revising the manuscript. They read and approved the final manuscript. Acknowledgements This study was conducted as part of a collaborative research initiative between Japan and the Democratic Republic of Congo through the International Research Center for Infectious Diseases Collaboration Program (J-GRID+) program. We also acknowledge the contributions of the Network Core Center at the National Center for Global Health and Medicine (NCGM), which provided critical institutional support under this initiative (https://jgrid-plus.ncgm.go.jp/). We gratefully appreciate the field surveyors for their technical work. We are especially grateful to Prof. Masaru Enomoto for his valuable discussions and insights that greatly contributed to the development of this research. Presentation in previous meetings . This research was previously presented at the 25th International Conference on Emerging Infectious Diseases (EID) under the U.S.-Japan Cooperative Medical Sciences Program (USJCMSP), held in Tokyo, Japan, from March 11 to 15, 2025. References Easterbrook PJ, Luhmann N, Bajis S, Min MS, Newman M, Lesi O, et al. WHO 2024 hepatitis B guidelines: an opportunity to transform care. The Lancet Gastroenterology and Hepatology [Internet]. 2024 [cited 2025 May 9];9:493–5. Available from: https://www.thelancet.com/action/showFullText?pii=S246812532400089X Global hepatitis report 2024: action for access in low- and middle-income countries [Internet]. [cited 2025 May 9]. Available from: https://www.who.int/publications/i/item/9789240091672 Global health sector strategy on viral hepatitis 2016-2021. Towards ending viral hepatitis [Internet]. [cited 2025 May 9]. Available from: https://iris.who.int/handle/10665/246177 Sonderup MW, Spearman CW. HBV elimination in Africa—Current status and challenges. Clinical Liver Disease [Internet]. 2024 [cited 2025 May 9];23:e0166. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC11068139/ Sonderup MW, Spearman CW. Global Disparities in Hepatitis B Elimination—A Focus on Africa. Viruses [Internet]. 2022 [cited 2025 May 9];14. Available from: https://pubmed.ncbi.nlm.nih.gov/35062286/ Thompson P, Parr JB, Holzmayer V, Carrel M, Tshefu A, Mwandagalirwa K, et al. Seroepidemiology of Hepatitis B in the Democratic Republic of the Congo. The American Journal of Tropical Medicine and Hygiene [Internet]. 2019 [cited 2025 May 9];101:226. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC6609197/ Thompson P, Morgan CE, Ngimbi P, Mwandagalirwa K, Ravelomanana NLR, Tabala M, et al. Arresting vertical transmission of hepatitis B virus (AVERT-HBV) in pregnant women and their neonates in the Democratic Republic of the Congo: a feasibility study. The Lancet Global Health [Internet]. 2021 [cited 2025 May 9];9:e1600–9. Available from: https://pubmed.ncbi.nlm.nih.gov/34416175/ Kabore HJ, Li X, Alleman MM, Manzengo CM, Mumba M, Biey J, et al. Progress Toward Hepatitis B Control and Elimination of Mother-to-Child Transmission of Hepatitis B Virus — World Health Organization African Region, 2016–2021. MMWR Morbidity and Mortality Weekly Report [Internet]. 2023 [cited 2025 May 9];72:782–7. Available from: https://www.cdc.gov/mmwr/volumes/72/wr/mm7229a2.htm Anderson M, Mangogola T, Phinius BB, Mpebe G, Aimakhu CO, Choga WT, et al. Hepatitis B Virus Prevalence among HIV-Uninfected People Living in Rural and Peri-Urban Areas in Botswana. Microorganisms [Internet]. 2024 [cited 2025 May 9];12:1207. Available from: https://www.mdpi.com/2076-2607/12/6/1207/htm Nlombi CM, Natuhoyila AN, Tudiakuilayi MM. Evaluation of Seroprevalence and Factors Associated With the Portage of HBsAg in Pregnant Women in Rural Vanga and Semi-Urban Maluku International Journal of Health Sciences and Research Evaluation of Seroprevalence and Factors Associated With the Portage. 2019; Moutchia J, Njouom R, Rumpler E, Besombes C, Texier G, Tejiokem M, et al. Maternal age at first childbirth and geographical variation in HBV prevalence in Cameroon: Important role of mother-to-child transmission. Clinical Infectious Diseases [Internet]. 2022 [cited 2025 May 9];74:836–45. Available from: https://hal.science/hal-03283690 Whittle H, Inskip H, Bradley AK, McLaughlan K, Shenton F, Lamb W, et al. The Pattern of Childhood Hepatitis B Infection in Two Gambian Villages. The Journal of Infectious Diseases [Internet]. 1990 [cited 2025 May 9];161:1112–5. Available from: https://dx.doi.org/10.1093/infdis/161.6.1112 Ansari A, Vincent JP, Moorhouse L, Shimakawa Y, Nayagam S. Risk of early horizontal transmission of hepatitis B virus in children of uninfected mothers in sub-Saharan Africa: a systematic review and meta-analysis. The Lancet Global Health [Internet]. 2023 [cited 2025 May 9];11:e715–28. Available from: https://www.sciencedirect.com/science/article/pii/S2214109X23001316?via%3Dihub Morgan CE, Ngimbi P, Boisson-Walsh AJN, Ntambua S, Matondo J, Tabala M, et al. Hepatitis B Virus Prevalence and Transmission in the Households of Pregnant Women in Kinshasa, Democratic Republic of Congo. Open Forum Infectious Diseases [Internet]. 2024;11:1–10. Available from: https://doi.org/10.1093/ofid/ofae150 Pollicino T, Cacciola I, Saffioti F, Raimondo G. Hepatitis B virus PreS/S gene variants: Pathobiology and clinical implications. Journal of Hepatology [Internet]. 2014 [cited 2025 May 9];61:408–17. Available from: https://pubmed.ncbi.nlm.nih.gov/24801416/ Pinho-Nascimento CA, Bratschi MW, Höfer R, Soares CC, Warryn L, Pečerska J, et al. Transmission of Hepatitis B and D Viruses in an African Rural Community. mSystems [Internet]. 2018 [cited 2025 May 9];3:e00120-18. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC6143728/ Chen J, Li L, Yin Q, Shen T. A review of epidemiology and clinical relevance of Hepatitis B virus genotypes and subgenotypes. Clinics and Research in Hepatology and Gastroenterology [Internet]. 2023 [cited 2025 May 9];47. Available from: https://pubmed.ncbi.nlm.nih.gov/37479136/ Chen BF. Hepatitis B virus pre-S/S variants in liver diseases. World Journal of Gastroenterology [Internet]. 2018 [cited 2025 May 9];24:1507–20. Available from: https://pubmed.ncbi.nlm.nih.gov/29662289/ Kabamba AT, Kalunga BT, Mwamba CM, Nyembo CM, Dufrasne F, Dessilly G, et al. Epidemiological aspects and molecular characterization of the hepatitis B virus among blood donors in Lubumbashi, Democratic Republic of Congo. Transfusion Clinique et Biologique [Internet]. 2021 [cited 2025 May 9];28:30–7. Available from: https://pubmed.ncbi.nlm.nih.gov/33232802/ Shindano TA, Horsmans Y, Kabamba BM. Genetic and phylogenic characterization of hepatitis B virus in the eastern part of the Democratic Republic of Congo. Journal of Medical Virology [Internet]. 2018 [cited 2025 May 9];90:250–4. Available from: https://pubmed.ncbi.nlm.nih.gov/28460156/ Démocratique R, Congo DU. Enquête nutritionnelle anthropométrique et de mortalité selon la méthodologie smart. 2021; Nanjundeswaraswamy TS, Divakar S. Determination of Sample Size and Sampling Methods in Applied Research. Proceedings on Engineering Sciences. 2021;3:25–32. Chromas | Technelysium Pty Ltd [Internet]. [cited 2025 May 9]. Available from: https://technelysium.com.au/wp/chromas/ Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution [Internet]. 2013 [cited 2025 May 9];30:772–80. Available from: https://pubmed.ncbi.nlm.nih.gov/23329690/ Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, Von Haeseler A, et al. IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Molecular Biology and Evolution [Internet]. 2020 [cited 2025 May 9];37:1530. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC7182206/ Letunic I, Bork P. Interactive Tree of Life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool. Nucleic Acids Research [Internet]. 2024 [cited 2025 May 9];52:W78–82. Available from: https://dx.doi.org/10.1093/nar/gkae268 Hepatitis B Virus Phylogenetic Typing Tool [Internet]. [cited 2025 May 9]. Available from: https://www.genomedetective.com/app/typingtool/hbv/how-to-use Tamura K, Stecher G, Kumar S. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Molecular Biology and Evolution [Internet]. 2021 [cited 2025 May 9];38:3022–7. Available from: https://dx.doi.org/10.1093/molbev/msab120 Bell TG, Kramvis A. Bioinformatics Tools for Small Genomes, Such as Hepatitis B Virus. Viruses [Internet]. 2015 [cited 2025 May 9];7:781. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC4353916/ HIV-GRADE - HIV-GRADE viral Genotypic resistance algorithms [Internet]. [cited 2025 May 9]. Available from: https://www.hiv-grade.de/cms/grade/ Zhang K, Bach C, Fraune MN, Xia Y, Beggel B, Kaiser R, et al. Novel rtM204 Mutations in HBV Polymerase Confer Reduced Susceptibility to Adefovir and Tenofovir. Journal of Antivirals & Antiretrovirals. 2017;09:10–7. Caligiuri P, Cerruti R, Icardi G, Bruzzone B. Overview of hepatitis B virus mutations and their implications in the management of infection. World Journal of Gastroenterology. 2016;22:145–54. Schweitzer A, Horn J, Mikolajczyk RT, Krause G, Ott JJ. Estimations of worldwide prevalence of chronic hepatitis B virus infection: A systematic review of data published between 1965 and 2013. The Lancet [Internet]. 2015 [cited 2025 May 9];386:1546–55. Available from: https://pubmed.ncbi.nlm.nih.gov/26231459/ WHO, UNICEF. Somalia : WHO and UNICEF estimates of immunization coverage : 2022 revision Somalia : WHO and UNICEF estimates of immunization coverage : 2022 revision. 2024;1–23. Shimakawa Y, Yan HJ, Tsuchiya N, Bottomley C, Hall AJ. Association of Early Age at Establishment of Chronic Hepatitis B Infection with Persistent Viral Replication, Liver Cirrhosis and Hepatocellular Carcinoma: A Systematic Review. PLoS ONE. 2013;8. Moonsamy S, Suchard M, Pillay P, Prabdial-Sing N. Prevalence and incidence rates of laboratory-confirmed hepatitis B infection in South Africa, 2015 to 2019. BMC Public Health [Internet]. 2022;22:1–13. Available from: https://doi.org/10.1186/s12889-021-12391-3 Ezeilo MC, Engwa GA, Iroha RI, Odimegwu DC. Seroprevalence and Associated Risk Factors of Hepatitis B Virus Infection Among Children in Enugu Metropolis. Virology: Research and Treatment. 2018;9:0–6. Kafeero HM, Ndagire D, Ocama P, Kato CD, Wampande E, Walusansa A, et al. Mapping hepatitis B virus genotypes on the African continent from 1997 to 2021: a systematic review with meta-analysis. Scientific Reports [Internet]. 2023;13:1–14. Available from: https://doi.org/10.1038/s41598-023-32865-1 Dumpis U, Holmes EC, Mendy M, Hill A, Thursz M, Hall A, et al. Transmission of hepatitis B virus infection in Gambian families revealed by phylogenetic analysis. Journal of Hepatology. 2001;35:99–104. Lazarevic I. Clinical implications of hepatitis B virus mutations: Recent advances. World Journal of Gastroenterology. 2014;20:7653–64. Wang J, Zhu B, Lu M, Yang D. Hepatitis B virus preS/S gene mutations and their clinical implications. Annals of Blood. 2017;2:17–17. Morgan CE, Powers KA, Edwards JK et al. Characterizing hepatitis B virus infection in children in the Democratic Republic of Congo to inform elimination efforts. 2024; Desalegn H, Aberra H, Berhe N, Mekasha B, Stene-Johansen K, Krarup H, et al. Treatment of chronic hepatitis B in sub-Saharan Africa: 1-year results of a pilot program in Ethiopia. BMC Medicine. 2018;16:1–10. Lumley SF, Mokaya J, Maponga TG, Kramvis A, Dusheiko G, Irving W, et al. Hepatitis B virus resistance to nucleos(t)ide analogue therapy: WHO consultation on questions, challenges, and a roadmap for the field. The Lancet Microbe. 2025;1–12. Pan CQ, Zhang JX. Natural history and clinical consequences of hepatitis B virus infection. International Journal of Medical Sciences. 2005;2:36–40. Martinson FEA, Weigle KA, Royce RA, Weber DJ, Suchindran CM, Lemon SM. Risk factors for horizontal transmission of hepatitis B virus in a rural district in Ghana. American Journal of Epidemiology. 1998;147:478–87. Zhao W. Guidelines for the Prevention, Diagnosis, Care and Treatment for People with Chronic Hepatitis B Infection (Text Extract): Executive Summary. Infectious Diseases and Immunity. 2024. Guingané AN, Kaboré R, Shimakawa Y, Somé EN, Kania D, Pisoni A, et al. Screening for Hepatitis B in partners and children of women positive for surface antigen, Burkina Faso. Bulletin of the World Health Organization. 2022;100:256–67. Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterialsHBVTransmissionDRC2025.docx Cite Share Download PDF Status: Published Journal Publication published 28 Jul, 2025 Read the published version in Tropical Medicine and Health → Version 1 posted Editorial decision: Revision requested 22 Jun, 2025 Reviews received at journal 22 Jun, 2025 Reviews received at journal 21 Jun, 2025 Reviewers agreed at journal 13 Jun, 2025 Reviewers agreed at journal 11 Jun, 2025 Reviewers invited by journal 11 Jun, 2025 Editor assigned by journal 10 Jun, 2025 Submission checks completed at journal 10 Jun, 2025 First submitted to journal 06 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6836597","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":470052812,"identity":"170d1f94-615b-472a-8b97-40c2df678454","order_by":0,"name":"Florence Cindibu Kalonji","email":"","orcid":"","institution":"Department of Virology and Parasitology, Graduate School of Medicine, Osaka Metropolitan University","correspondingAuthor":false,"prefix":"","firstName":"Florence","middleName":"Cindibu","lastName":"Kalonji","suffix":""},{"id":470052813,"identity":"19564461-4d4c-4b6d-8d13-6a039861a79b","order_by":1,"name":"Yu 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11:23:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6836597/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6836597/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s41182-025-00781-x","type":"published","date":"2025-07-28T16:29:34+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84686628,"identity":"ac878187-c2fc-4d4e-a67f-1fa23d91e105","added_by":"auto","created_at":"2025-06-16 09:01:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":383109,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeographic location of the study sites\u003c/strong\u003e. Location of the Lukelenge health district at different geographical levels: the Democratic Republic of the Congo (A), the Kasaï-Oriental province (B), and the city of Mbujimayi (C).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6836597/v1/a9c6bf6c5ae773dc4042e277.png"},{"id":84685361,"identity":"afa33c11-37bd-40d7-8498-47d0d9e2c1f6","added_by":"auto","created_at":"2025-06-16 08:53:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":172474,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eParticipant recruitment and selection process in this study. \u003c/strong\u003eRDT: Rapid Diagnostic Test; HBsAg: Hepatitis B surface antigen; (+): positive.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6836597/v1/ec9a5867dbf259e65e1f9374.png"},{"id":84685343,"identity":"eafbcf49-0064-4f50-9846-5aa240579a9f","added_by":"auto","created_at":"2025-06-16 08:53:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":411257,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHBsAg positivity rates in the study population. \u003c/strong\u003eHBsAg positivity rates across different groups: between the community and household contacts (A), across age groups (B), and between genders (C).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6836597/v1/3ad66f53c32fe765add4589b.png"},{"id":84685350,"identity":"eea6b52a-c628-4ef9-bfea-11da2d6bc27f","added_by":"auto","created_at":"2025-06-16 08:53:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1194050,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMaximum likelihood phylogenetic tree of HBV strains circulating in the study population. \u003c/strong\u003eThis rooted maximum likelihood phylogenetic tree was created using 92 complete preS/S sequences. These include 50 reference sequences from GenBank, representing all HBV genotypes (A-J), with accession numbers, country of origin, and genotype labels provided. Additionally, 42 sequences from this study are marked with red dots. The branch lengths are proportional to sequence divergence. Sequences of the same genotype are color-coded, with the red clade highlighting the predominant genotype E. The optimal substitution model, identified by IQ-TREE, is the transversion model with equal base frequencies and a discrete gamma distribution with four rate categories (TVMe + G4). Bootstrap values are shown at the tree nodes.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6836597/v1/2b50fc333927e517a4990a53.png"},{"id":84685419,"identity":"7719adf3-2b09-4f15-9cf5-988b7440c561","added_by":"auto","created_at":"2025-06-16 08:53:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":511549,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFamily pedigree reconstruction for households included in the study. \u003c/strong\u003eSquares represent males, and circles represent females. The first level of the tree shows the parents, while the second level includes their direct descendants. Red symbols indicate index cases positive for HBV, and green symbols represent positive family contacts. A diagonal line through a square indicates a deceased father. \"yrs\" refers to years.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6836597/v1/93e05f106d0d0eafc7e49811.png"},{"id":84685415,"identity":"60a5779a-b914-4450-b2e3-fac9c4987029","added_by":"auto","created_at":"2025-06-16 08:53:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":57070,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMean inter- and intra-familial genetic distances\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoxplots of genetic distances: (orange) inter-familial distances and (cyan) intra-familial distances. The Wilcoxon rank-sum test was used to compare genetic distances of HBV within and between families, with a p-value \u0026lt; 0.001. \u0026nbsp;Black individual plots indicate genetic distance specific to each family.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6836597/v1/aeb037813aef996df8f04bf8.png"},{"id":84685366,"identity":"6377e00d-ee5f-489a-a5cc-fa865ac00eed","added_by":"auto","created_at":"2025-06-16 08:53:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":422603,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMaximum likelihood phylogenetic tree of preS/S sequences from local HBV isolates. \u003c/strong\u003eThis unrooted phylogenetic tree was constructed using the maximum likelihood method, based on 32 complete preS/S sequences from this study. The best-fit model, identified by IQ-TREE, is the Jukes-Cantor substitution model with invariant sites (JC + I). Families with more than two infected individuals are represented by colored diamonds, while families with only one infected individual are shown as circles. Sample labels include \"F\" for family, followed by the family number, relationship, and age. Each diamond and circle are uniquely colored to represent a different family. Bootstrap values are shown at the tree nodes.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6836597/v1/7369cb9a9ff4b4e163ab8956.png"},{"id":88268830,"identity":"94fbb581-a3ac-4fad-b64a-832a8f5524e1","added_by":"auto","created_at":"2025-08-04 16:52:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4159222,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6836597/v1/9dc5a3cb-86f2-4fe7-a0ce-fff31c430a20.pdf"},{"id":84685335,"identity":"131271f8-8b20-4507-b533-3f5b1355da92","added_by":"auto","created_at":"2025-06-16 08:53:31","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":791605,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialsHBVTransmissionDRC2025.docx","url":"https://assets-eu.researchsquare.com/files/rs-6836597/v1/1b8528c8719328d8ec04d327.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Intra-familial transmission of Hepatitis B virus in a peri-urban community from the Democratic Republic of the Congo","fulltext":[{"header":"Background","content":"\u003cp\u003eHepatitis B virus (HBV) infection remains a major worldwide health challenge, with an estimated 254\u0026nbsp;million chronically infected people, causing substantial mortality from liver cirrhosis and hepatocellular carcinoma (HCC) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In response, the World Health Organization (WHO) set a goal to eliminate HBV infection by 2030, with a 90% reduction in new infections and a 65% reduction in deaths through expanded vaccination, early diagnosis, enhanced antiviral access, and the development of emerging curative therapies[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In spite of these worldwide efforts, HBV infection persists, particularly in sub-Saharan Africa, which accounts for 63% of global new infections annually [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Africa faces substantial gaps in vaccination, diagnostics, and treatment, exacerbating the global burden of HBV, with significant disparities observed both across and within countries in the region[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In the Democratic Republic of the Congo (DRC), HBV prevalence is in line with broader African trends. The national prevalence is reported to be around 3.3%, with approximately 300.000 children living with chronic hepatitis B, far exceeding the WHO's 2030 elimination target of \u0026le;\u0026thinsp;0.1% prevalence in children under five[\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Rural and peri-urban areas face the most notable gaps in HBV control strategies, necessitating urgent region-specific interventions[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSince HBV can be transmitted through blood, semen, or vaginal fluids via either horizontal or vertical routes, and the relative importance of each pathway varies geographically[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], it is essential to understand regional transmission dynamics in order to implement context-specific interventions. Horizontal transmission has been widely recognized in Africa, especially through close contact within households and communities[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, with increased coverage of the three-dose hepatitis B vaccination series (HepB3) in infants, the significance of horizontal transmission would be diminishing[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In contrast, the relative importance of vertical transmission remains debated especially in the DRC, where a study in pregnant women and their offspring have suggested a crucial role of mother-to-child transmission in the persistence and spread of HBV within households[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Population-based studies providing insight into this hypothesis remain limited in DRC.\u003c/p\u003e \u003cp\u003eGenetic analysis of HBV gives valuable insights into viral strain typing and transmission dynamics. The HBV genome is a circular, partially double-stranded DNA (~\u0026thinsp;3.2 kb) comprising four overlapping open reading frames (ORFs): preS/S, preCore/Core, Pol, and X[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The preS/S ORF, which encodes for the surface antigen of hepatitis B (HBsAg), is highly variable and has been thoroughly studied for phylogenetic purposes[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This region's variability is partly driven by the overlapping Pol ORF, encoding a polymerase with low replication fidelity, yielding a high mutation rate. As a result, ten HBV genotypes (A-J), nine serotypes, and over forty sub-genotypes have been identified [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Mutations in the preS/S region, particularly within the \"a\" determinant of the Small S protein, are of critical clinical importance as they can give rise to immune escaping strains capable of infecting vaccinated individuals and causing occult HBV infections[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These mutations can also be accountable for antiviral drug resistance and disease progression to cirrhosis or HCC[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Overall, genetic analysis of the preS/S region is useful for understanding HBV diversity, transmission dynamics, and its implications on disease progression and treatment resistance[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eComprehensive genetic analyses of HBV strains circulating among the African population is crucial for guiding implementation of targeted public health interventions. However, such studies detailing HBV genotypes, mutations, and transmission pathways remain scarce in the DRC[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. To address this critical knowledge gap, the present study aimed to assess HBV prevalence, genetic diversity, and potentials transmission patterns within a peri-urban community in central DRC.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDesign and setting of the study\u003c/h2\u003e \u003cp\u003eThis community-based cross-sectional study was conducted between April and November 2023 in the Lukelenge health district (HD), located in Mbujimayi, Kasa\u0026iuml;-Oriental province, DRC, an area endemic for HBV (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Lukelenge, a peri-urban area on the outskirts of Mbujimayi, spans 35 km\u0026sup2; and has an estimated population of 341,591, resulting in a population density of approximately 9,760 inhabitants per km\u0026sup2;. The local economy is primarily based on artisanal diamond mining; however, declining profitability has considerably impacted household income, making Lukelenge one of the most impoverished HDs in the region[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSampling strategy and study population\u003c/h3\u003e\n\u003cp\u003eA simple random sampling method was used to select households in the study area, based on the household registry provided by the Central Office of the Lukelenge HD. Participant recruitment followed a two-tiered approach, with visits spaced six months apart. During the first visit, all selected households were visited, and within each household, one eligible individual, aged one year or older and residing in the study area for at least three months, was randomly selected using a lottery method, to be screened for HBsAg. Those who tested positive for HBsAg were designated as \"index cases\", and their households were subsequently invited to participate in the second-phase, family-based screening. During the second visit, up to five household members, including the index case's parents and siblings, were selected as \"contact cases\" for HBsAg screening. Eligibility required written consent from adults, or permission from a parent or legal guardian for minors (under 18 years). Additionally, verbal assent was obtained from minors aged 15 to 18 years. Using Cochran\u0026rsquo;s formula[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], the required sample size for the community survey was calculated assuming a 3.3% estimated prevalence of HBsAg[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], a 95% confidence level (Z\u0026thinsp;=\u0026thinsp;1.96), and an absolute margin of error of 1.4%. This yielded a minimum required sample size of 626 participants. To account for possible non-response, a final sample size of 700 was targeted.\u003c/p\u003e\n\u003ch3\u003eField investigation and sample collection\u003c/h3\u003e\n\u003cp\u003eEach visit involved a brief interview to assess eligibility, gather demographic data, and document the family pedigree. Eligible participants underwent a rapid HBsAg test (One Step HBsAg Rapid Test Strip, RapidLab, China) following the manufacturer's instructions. Briefly, 50 \u0026micro;L of blood was collected via fingerprick, applied to the test strip, and allowed to develop for 15 minutes. For HBV DNA extraction, an additional 50 \u0026micro;L of blood was collected, spotted onto a Whatman 903 filter card (Cytiva, USA), and allowed to dry. The dried blood spot sample (DBSs) was stored in a zip-lock bag with desiccants at ambient temperature. The samples were later transferred to the Biobank of the \u003cem\u003eInstitut National de Recherche Biom\u0026eacute;dicale\u003c/em\u003e (INRB) in Kinshasa, DRC, and stored at -20\u0026deg;C. These DBSs were then used for molecular analyses. Field data were recorded using a semi-structured electronic questionnaire, designed with the KoboToolBox application (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://kf.kobotoolbox.org/\u003c/span\u003e\u003cspan address=\"https://kf.kobotoolbox.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Participants who tested positive for HBsAg received counseling and were referred to the Lukelenge HD\u0026rsquo;s reference hospital for appropriate care and follow-up.\u003c/p\u003e\n\u003ch3\u003eHBV DNA extraction, PCR amplification, and sequencing\u003c/h3\u003e\n\u003cp\u003eTotal DNA was extracted from dried blood spot samples using the DNeasy\u0026reg; Blood and Tissue Kit (Qiagen, Hilden, Germany). The DNA was eluted in 30 \u0026micro;L of final volume. PCR reactions included 2 \u0026micro;L of DNA solution as template in a 25 \u0026micro;L total volume, and PrimeSTAR Max DNA Polymerase (TaKaRa, Japan), and was run on a C1000 Touch thermal cycler (Bio-Rad, USA). Previously established sets of primers were used (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e), with PCR conditions that we optimized for HBV genomic DNA amplification, as indicated below.\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003ePreS/S region amplification (~\u0026thinsp;1200 bp): A semi-nested PCR was performed using primers PS1 and P3 for the first round, followed by PS1 and S2 for the second round [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], with a final primer concentration of 0.2 \u0026micro;M. Thermal cycling conditions consisted of 30 cycles at 98\u0026deg;C for 10 seconds, 55\u0026deg;C for 15 seconds, and 72\u0026deg;C for 5 seconds.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eWhole genome amplification (~\u0026thinsp;3200 bp): Universal primers WA-L and WA-R [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], with a final concentration of 0.5 \u0026micro;M, were used. Thermal cycling conditions included 40 cycles at 98\u0026deg;C for 10 seconds, 60\u0026deg;C for 5 seconds, and 72\u0026deg;C for 30 seconds. This was performed only on the index cases specimens.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eSuccessfully amplified PCR products were purified using the NucleoFast\u0026reg; 96 PCR Clean-up kit (Macherey-Nagel, France) and subsequently sequenced using the 3730XL DNA Analyzer (Applied Biosystems) with specific sequencing primers detailed in \u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e.\u003c/p\u003e\n\u003ch3\u003eHBV molecular analysis\u003c/h3\u003e\n\u003cp\u003eNucleotide sequences were inspected and trimmed using Chromas Software v2.6.6[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. They were assembled on the CLC Genomics Workbench platform v.23 (CLC bio, Denmark), and then aligned using MAFFT v7[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Phylogenetic analysis was performed using maximum likelihood trees reconstructed with IQ-TREE v2.3.6 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and visualized as midpoint-rooted trees on the Interactive Tree of Life (iTOL) platform v7 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Node support was evaluated using 1,000 bootstrap replicates to assess topology robustness. The most appropriate nucleotide substitution model was selected based on the Bayesian Information Criterion (BIC). Reference sequences of HBV genotypes A to J were retrieved from the Genbank database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/genbank/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/genbank/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and incorporated into the phylogenetic trees. Genotyping was cross validated using the Hepatitis B Virus Phylogenetic Typing Tool v2.59[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Genetic distances within and between families were calculated using the Kimura-2-Parameter (K2P) substitution model in MEGA v11[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. HBV serotypes were inferred by analyzing key amino acid residues within the small S protein sequence (i.e., positions 122, 127, 140, 159, and 160), following established classification algorithms[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Mutations linked to immune escape or antiviral resistance were further predicted using the HIV-Grade: HBV-Tool[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], assessing clinically relevant viral polymorphisms.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using R software version 4.4.3 (The R Development Core Team, Austria). Categorical variables were summarized as absolute and relative frequencies, while continuous variables were described using medians and ranges (minimum to maximum). The prevalence of HBsAg was estimated at both community and intra-familial levels using Jeffreys\u0026rsquo; approximate Bayesian confidence intervals. Age-specific prevalences were calculated based on data from index case screening, without considering intra-familial screening data. The chi-square test was used to compare HBsAg positivity rates between groups, and the odds ratio indicating the trend in the likelihood of positivity. Genetic distances of HBV within and between families were compared using the Wilcoxon rank-sum test. Receiver Operating Characteristic (ROC) curve analysis was applied to predict monophyletic clusters based on sequence similarity, in order to determine optimal cutoff value for viral genetic divergence as the threshold for defining similarity between HBV sequences. A p-value of less than 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCommunity survey (among index cases)\u003c/h2\u003e \u003cp\u003eIn the community survey, a total of 700 households were randomly selected, and 700 eligible participants were recruited, with 677 providing complete survey responses, yielding a response rate of 97% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The study participants had a sex ratio of 0.7 (male: n\u0026thinsp;=\u0026thinsp;293, female: n\u0026thinsp;=\u0026thinsp;384), and a median age of 16 years (range: 1 to 81 years). Among the 677 participants, 26 individuals were tested positive for HBsAg, leading to an estimated HBsAg prevalence of 3.8% (95% CI: 2.6\u0026ndash;5.5). Children aged 5 years and younger showed the highest prevalence at 10.1% (n\u0026thinsp;=\u0026thinsp;8/79 [OR\u0026thinsp;=\u0026thinsp;3.8; 95% CI: 1.4\u0026ndash;10.6]; p\u0026thinsp;=\u0026thinsp;0.019) compared to adults aged 18 years and older (2.8%, n\u0026thinsp;=\u0026thinsp;8/282). There was no significant difference in HBsAg positivity between males (3.8% [95% CI: 2.0-6.4]; n\u0026thinsp;=\u0026thinsp;11/293) and females (3.9% [95% CI: 2.3\u0026ndash;6.2]; n\u0026thinsp;=\u0026thinsp;15/384) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eIntra-familial survey (among familial contacts of index cases)\u003c/h2\u003e \u003cp\u003eOf the 26 index households, five had relocated and six declined to participate, leaving 15 households in which HBsAg screening was ultimately conducted (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). From each of these 15 households, five case contacts were included, except for family #24, where only four individuals participated. In total, 74 individuals (11 fathers, 12 mothers, and 51 children) were included in the intra-familial screening, of whom 17 tested positives for HBsAg, resulting in a prevalence of 23.0% (95% CI: 14.5\u0026ndash;33.5). Compared to the prevalence observed in the community survey, the prevalence among familial contacts of index cases was significantly higher (23.0% vs 3.8%, OR\u0026thinsp;=\u0026thinsp;5.8 [95% CI: 2.9\u0026ndash;11.1]; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNone of the participants had previously tested positive for HBV or received antiviral treatment, and only 10% of participants had heard of hepatitis B, typically referring to it using the local terms \u003cem\u003emutshima muhula\u003c/em\u003e (hepatomegaly) or \u003cem\u003edifu diuhula\u003c/em\u003e (abdominal bloating).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMolecular profile of HBV strains circulating in the study population\u003c/h2\u003e \u003cp\u003eA total of 42 HBV preS/S sequences were successfully obtained, including 25 from the 26 index cases and all 17 HBsAg-positive family contacts. All isolates were assigned to HBV genotype E (HBV/E) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Notably, 41 of 42 HBV/E strains (97.6%) were classified as serotype \u003cem\u003eayw4\u003c/em\u003e, demonstrating high serotype concordance. One strain exhibited a discordant, yet-unclassified serotype, with an L127Q substitution alongside wild-type R122 and K160 positions. A detailed mutational analysis is provided in \u003cb\u003eSupplementary Table S2\u003c/b\u003e. Furthermore, mutational analysis identified six mutations (i.e., P120S, C121Y, G130R, G145R, C147S, and T126N) within the \"a\" determinant of the major hydrophilic region of the HBsAg in 4 of 42 viral strains (9.5%), which have been reported as potentially associated with immune escape. Additionally, two mutations, rtI169T and rtM204K, were each detected in a different sequence (4.7%), within the overlapping RT gene, with previous reports suggesting possible links to antiviral drug resistance [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Whole-genome amplification was successful in 96.1% (25/26) of index cases samples, and phylogenetic reconstruction of whole-genomic sequences revealed close ancestral relationships, consistent with that of the preS/S region (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHBV transmission dynamics within studied families and community\u003c/h2\u003e \u003cp\u003eTo explore transmission patterns, phylogenetic analysis, genetic distance calculations, and family pedigree reconstructions were employed using preS/S sequences. Among the 15 HBV-positive families studied, 73.3% (11/15) of families had an infected parent; maternal and paternal infections were present in 60% (9/15) and 53.8% (7/13) of the families, respectively, and 38.4% (5/13) had both parents infected (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Within families, genetic distances were significantly smaller in preS/S sequences than those between unrelated families (0.1 [range: 0-0.7] vs. 0.8 [range: 0.1\u0026ndash;2.9]; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Multiple infections were detected in 80% (12/15) of the households. In 66.7% (10/15) of these families, sequences clustered monophyletically (\u0026ge;\u0026thinsp;established best cutoff value of 99.65% similarity) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These findings suggest a predominant mode of HBV intra-familial transmission, including mother-child (26.7% [n\u0026thinsp;=\u0026thinsp;4/15]; families #1, #3, #8 and #20), father-child (15.4% [n\u0026thinsp;=\u0026thinsp;2/13]; families #15 and #22), sibling-to-sibling (20% [n\u0026thinsp;=\u0026thinsp;3/15]; families #2, #7, and #14), and spouse-to-spouse (7.6% [n\u0026thinsp;=\u0026thinsp;1/13]; family #25) patterns.\u003c/p\u003e \u003cp\u003eIn contrast, 26.7% (4/15) of families harbored multiple infected individuals carrying genetically divergent HBV strains with sequence similarity\u0026thinsp;\u0026lt;\u0026thinsp;99.65%, suggesting independent viral introductions rather than intra-familial transmission. In families #3 and #8, the parents were infected with distinct strains, but the mother-child pairs shared closely related strains, indicating possible mother-to-child transmission. In family #22, the parents carried divergent strains, but the father and child shared closely related strains, suggesting a possible father-to-child transmission. In family #13, the mother and child were infected with genetically distant strains, suggesting acquisition from separate sources (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study assessed the epidemiological landscape and transmission dynamics of HBV in Lukelenge, a peri-urban community of central DRC. Two key findings emerged: first, the region exhibited intermediate HBV endemicity, characterized by a markedly high prevalence among children aged 5 years and younger. Second, phylogenetic analysis combined with family pedigree reconstructions strongly suggested that intra-familial spread is the predominant mode of HBV transmission, particularly involving parent-to-child and sibling-to-sibling routes. These findings offer cornerstone evidence to guide local public health strategies.\u003c/p\u003e \u003cp\u003eOverall, the estimated HBsAg prevalence was 3.8% (95% CI: 2.6\u0026ndash;5.5; n\u0026thinsp;=\u0026thinsp;26/677) in the study population, slightly exceeding the national estimate of 3.3% for the DRC[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This positions the region within the WHO\u0026rsquo;s intermediate endemicity category for HBV, suggesting a persistent infection burden[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Contributing factors to this situation likely involve limited access to healthcare services, insufficient screening programs, and delays in diagnosis[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. One in ten children aged five years and younger was infected with HBV. This finding is of particular concern and provides key insight into local transmission dynamics. It suggests substantial HBV transmission during early life likely due to major gaps in prevention, including low HepB3 vaccine coverage (estimated at just 65%) and the absence of a universal timely birth dose HBV vaccine program throughout the DRC[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Given that early-life HBV infections carry a higher risk of progressing towards chronic infection and subsequent severe liver diseases like cirrhosis and HCC[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], this high prevalence in young children represents a major public health threat. Similar trends of elevated childhood HBV prevalence have been reported in other sub-Saharan African countries, including Cameroon, South Africa, and Nigeria[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. To tackle this issue, essential interventions should include the enforcement of the birth-dose HBV vaccination, enhancing maternal HBV screening, and improving access to antiviral prophylaxis for infected mothers.\u003c/p\u003e \u003cp\u003eRegarding HBV genetic characterization, this study exclusively identified the HBV genotype E and the \u003cem\u003eayw4\u003c/em\u003e serotype in the study population. This genotype has been previously described in southeastern region of the DRC[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and is known to be prevalent across Central and West Africa, including countries such as Cameroon and The Gambia[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The observed restricted genetic diversity suggests localized transmission patterns. Interestingly, the unique L127Q substitution found in one isolate suggests a potential emergence of yet-unclassified serotype. Located in the S gene, this mutation could affect antigenicity, immune recognition, and diagnostic accuracy, and warrants further investigation. Immune and vaccine escape mutations were identified in approximately one out of ten viral strains, including G145R, P120S, and T126N, which have been reported to be associated with reduced vaccine efficacy[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The G145R mutation, in particular, is notable for its stability and ability to be transmitted both vertically and horizontally[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. These mutations enable HBV to evade immune responses from vaccination, immunoglobulin treatments, and natural immunity, leading to breakthrough infections and diminished vaccine effectiveness[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The detection of vaccine escape variants in this endemic area raises concerns about the risk of breakthrough infections, as seen previously in the DRC among vaccinated children aged 6 to 59 months[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Additionally, mutations such as rtI169T and rtM204K, possibly linked to antiviral resistance, were detected in nearly 5% of viral strains. An isolated rtI169T mutation may contribute to entecavir resistance when coexisting with additional mutations[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The rtM204K mutation confers resistance to adefovir and tenofovir \u003cem\u003ein vitro\u003c/em\u003e, although this has not been confirmed in clinical outcomes[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. This resistance could limit treatment options for chronic HBV infection, particularly in resource-limited settings where these drugs are commonly used[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The presence of drug-resistant strains in an antiviral treatment-na\u0026iuml;ve population presents a significant challenge for HBV control[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Regardless of the presence of these mutations, there is an urgent need for comprehensive control strategies, including enhanced viral surveillance, expanded access to antiviral treatments, and post-exposure prophylaxis.\u003c/p\u003e \u003cp\u003eThis study provided also compelling evidence pointing towards the household as the primary setting for HBV transmission in the study area. Approximately 80% of affected families had multiple infected members, and genetically similar strains were detected within 66.7% of households, supporting the predominance of intra-familial transmission of HBV in the study community. Parent-to-child transmission emerged as the most common route, with maternal transmission likely playing a more significant role than paternal transmission. This aligns with findings from other HBV-endemic regions where perinatal and early childhood exposure are recognized as major contributors to the persistence of HBV infection[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Sibling-to-sibling transmission was also evident, likely facilitated by close contact and shared personal items, which are well-documented transmission routes in endemic settings[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Although spousal transmission was identified in one family, most infections appeared to be acquired during childhood, suggesting a limited local role for sexual transmission in the study community. Remarkably, horizontal acquisition of HBV through extra-familial route was limited, suggesting that exposure within the broader community plays a less significant role in its spread. Collectively, these findings provide important public health implications, indicating that targeted interventions within households - such as routine HBV screening for family members of infected individuals and the identification of HBsAg-positive individuals who may be eligible for antiviral therapy \u0026ndash; could effectively reduce HBV disease burden[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Particularly given the growing momentum to integrate antenatal HBsAg screening into the triple elimination initiative for HIV, syphilis, and hepatitis B, this approach presents a valuable opportunity. Antenatal maternal screening could serve as an effective entry point for identifying infected households, enabling timely familial screening and follow-up interventions, as has been suggested in similar settings such as Burkina Faso[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study has the following limitations. First, being a community-based survey conducted in a single location, the findings may not be generalizable to other regions. Second, the limited number of tested family contacts may have led to over-/under-estimations of intra-familial HBV prevalence. Third, the individual vaccination status of children was not assessed due to issues with parental recall, missing records, and limited knowledge about HBV. However, these children were presumed to be vaccinated in accordance with the national immunization program in the DRC. HIV status was not determined. Fourth, the cross-sectional design of the study precludes the determination of infection chronology within families, making it difficult to identify HBV transmission directions and primary sources. Fifth, genetic analyses were limited to the preS/S region of the HBsAg, this could limit insights into broader HBV genetic diversity and evolutionary patterns. Lastly, reliance on self-reported data may have introduced recall bias.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study provides valuable insights into HBV epidemiology in the central DRC with significant public health implications. The findings confirm an intermediate endemicity profile, with transmission occurring predominantly within families through vertical (parent-to-child) and horizontal (sibling-to-sibling) routes. The exclusive detection of HBV genotype E, \u003cem\u003eayw4\u003c/em\u003e serotype and limited genetic diversity suggest stable, localized transmission patterns. The identification of immune escape and possible drug-resistant variants highlights the need for improved HBV surveillance, enhanced vaccination strategies, and expanded access to antiviral treatment. Moving forward, targeted interventions, including universal birth-dose vaccination, maternal screening, and household-based prevention measures, will be essential in reducing the HBV burden in the study population.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBIC: Bayesian Information Criterion\u003c/p\u003e\n\u003cp\u003eCI: Confidence Intervals\u003c/p\u003e\n\u003cp\u003eDRC: Democratic Republic of Congo\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHBsAg: Hepatitis B surface antigen\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHBV/E: HBV genotype E\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHBV: Hepatitis B virus\u003c/p\u003e\n\u003cp\u003eHCC: Hepatocellular Carcinoma\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHD: Health District\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHepB3: Three-dose hepatitis B vaccination series\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eINRB : Institut National de Recherche Biom\u0026eacute;dicale\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eiTOL: Interactive Tree of Life\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eK2P: Kimura-2-Parameter\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOR: Odd Ratio\u003c/p\u003e\n\u003cp\u003eORFs: Open Reading Frames\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePCR: Polymerase Chain Reaction\u003c/p\u003e\n\u003cp\u003eROC: Receiver Operating Characteristic\u003c/p\u003e\n\u003cp\u003eWHO: World Health Organization\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from adult participants and legal guardian for minors (under 18 years). Additionally, verbal assent was obtained from minors aged 15 to 18 years. \u0026nbsp;The study followed the Declaration of Helsinki principles and was approved by the ethics committee of the University of Mbujimayi, the DRC (\u003cstrong\u003en\u0026deg;002/CEI/UM/2023\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll co‑authors have read and approved this manuscript for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated during this study are included in the published article and its additional file. The genetic data we generated and analyzed during this study are available in the DNA Data Bank of Japan (DDBJ) and GenBank database under accession numbers LC858535 to LC858576. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors do not have a commercial or other association that might pose a conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI under the grant numbers 22KK0279 (to Yu Nakagama). Additional funding was provided by KAKENHI under Grant Number JP18KK0454 and 23H02951, JSPS Core-to-Core Program (Grant Number: JPJSCCB20240010), and the Japan Agency for Medical Research and Development (AMED) under Grant numbers JP25wm0125003 and JP19fm0208020 (all to Yasutoshi Kido).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFCK, ETK, NKK, YN, and YK conceived the study. FCK and ETK wrote the study protocol. FCK, ETK, SN and YN did the data analyses and made the figures. FCK, YN, ETK, SN, NKK, YS, and YK wrote the initial manuscript. All co-authors made a major contribution in revising the manuscript. They read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was conducted as part of a collaborative research initiative between Japan and the Democratic Republic of Congo through the International Research Center for Infectious Diseases Collaboration Program (J-GRID+) program. We also acknowledge the contributions of the Network Core Center at the National Center for Global Health and Medicine (NCGM), which provided critical institutional support under this initiative (https://jgrid-plus.ncgm.go.jp/). We gratefully appreciate the field surveyors for their technical work. We are especially grateful to Prof. Masaru Enomoto for his valuable discussions and insights that greatly contributed to the development of this research.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePresentation in previous meetings\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis research was previously presented at the 25th International Conference on Emerging Infectious Diseases (EID) under the U.S.-Japan Cooperative Medical Sciences Program (USJCMSP), held in Tokyo, Japan, from March 11 to 15, 2025. \u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eEasterbrook PJ, Luhmann N, Bajis S, Min MS, Newman M, Lesi O, et al. WHO 2024 hepatitis B guidelines: an opportunity to transform care. The Lancet Gastroenterology and Hepatology [Internet]. 2024 [cited 2025 May 9];9:493\u0026ndash;5. Available from: https://www.thelancet.com/action/showFullText?pii=S246812532400089X\u003c/li\u003e\n\u003cli\u003eGlobal hepatitis report 2024: action for access in low- and middle-income countries [Internet]. [cited 2025 May 9]. Available from: https://www.who.int/publications/i/item/9789240091672\u003c/li\u003e\n\u003cli\u003eGlobal health sector strategy on viral hepatitis 2016-2021. Towards ending viral hepatitis [Internet]. [cited 2025 May 9]. Available from: https://iris.who.int/handle/10665/246177\u003c/li\u003e\n\u003cli\u003eSonderup MW, Spearman CW. HBV elimination in Africa\u0026mdash;Current status and challenges. Clinical Liver Disease [Internet]. 2024 [cited 2025 May 9];23:e0166. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC11068139/\u003c/li\u003e\n\u003cli\u003eSonderup MW, Spearman CW. Global Disparities in Hepatitis B Elimination\u0026mdash;A Focus on Africa. Viruses [Internet]. 2022 [cited 2025 May 9];14. Available from: https://pubmed.ncbi.nlm.nih.gov/35062286/\u003c/li\u003e\n\u003cli\u003eThompson P, Parr JB, Holzmayer V, Carrel M, Tshefu A, Mwandagalirwa K, et al. Seroepidemiology of Hepatitis B in the Democratic Republic of the Congo. The American Journal of Tropical Medicine and Hygiene [Internet]. 2019 [cited 2025 May 9];101:226. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC6609197/\u003c/li\u003e\n\u003cli\u003eThompson P, Morgan CE, Ngimbi P, Mwandagalirwa K, Ravelomanana NLR, Tabala M, et al. Arresting vertical transmission of hepatitis B virus (AVERT-HBV) in pregnant women and their neonates in the Democratic Republic of the Congo: a feasibility study. The Lancet Global Health [Internet]. 2021 [cited 2025 May 9];9:e1600\u0026ndash;9. Available from: https://pubmed.ncbi.nlm.nih.gov/34416175/\u003c/li\u003e\n\u003cli\u003eKabore HJ, Li X, Alleman MM, Manzengo CM, Mumba M, Biey J, et al. Progress Toward Hepatitis B Control and Elimination of Mother-to-Child Transmission of Hepatitis B Virus \u0026mdash; World Health Organization African Region, 2016\u0026ndash;2021. MMWR Morbidity and Mortality Weekly Report [Internet]. 2023 [cited 2025 May 9];72:782\u0026ndash;7. Available from: https://www.cdc.gov/mmwr/volumes/72/wr/mm7229a2.htm\u003c/li\u003e\n\u003cli\u003eAnderson M, Mangogola T, Phinius BB, Mpebe G, Aimakhu CO, Choga WT, et al. Hepatitis B Virus Prevalence among HIV-Uninfected People Living in Rural and Peri-Urban Areas in Botswana. Microorganisms [Internet]. 2024 [cited 2025 May 9];12:1207. Available from: https://www.mdpi.com/2076-2607/12/6/1207/htm\u003c/li\u003e\n\u003cli\u003eNlombi CM, Natuhoyila AN, Tudiakuilayi MM. Evaluation of Seroprevalence and Factors Associated With the Portage of HBsAg in Pregnant Women in Rural Vanga and Semi-Urban Maluku International Journal of Health Sciences and Research Evaluation of Seroprevalence and Factors Associated With the Portage. 2019; \u003c/li\u003e\n\u003cli\u003eMoutchia J, Njouom R, Rumpler E, Besombes C, Texier G, Tejiokem M, et al. Maternal age at first childbirth and geographical variation in HBV prevalence in Cameroon: Important role of mother-to-child transmission. Clinical Infectious Diseases [Internet]. 2022 [cited 2025 May 9];74:836\u0026ndash;45. Available from: https://hal.science/hal-03283690\u003c/li\u003e\n\u003cli\u003eWhittle H, Inskip H, Bradley AK, McLaughlan K, Shenton F, Lamb W, et al. The Pattern of Childhood Hepatitis B Infection in Two Gambian Villages. The Journal of Infectious Diseases [Internet]. 1990 [cited 2025 May 9];161:1112\u0026ndash;5. Available from: https://dx.doi.org/10.1093/infdis/161.6.1112\u003c/li\u003e\n\u003cli\u003eAnsari A, Vincent JP, Moorhouse L, Shimakawa Y, Nayagam S. Risk of early horizontal transmission of hepatitis B virus in children of uninfected mothers in sub-Saharan Africa: a systematic review and meta-analysis. The Lancet Global Health [Internet]. 2023 [cited 2025 May 9];11:e715\u0026ndash;28. Available from: https://www.sciencedirect.com/science/article/pii/S2214109X23001316?via%3Dihub\u003c/li\u003e\n\u003cli\u003eMorgan CE, Ngimbi P, Boisson-Walsh AJN, Ntambua S, Matondo J, Tabala M, et al. Hepatitis B Virus Prevalence and Transmission in the Households of Pregnant Women in Kinshasa, Democratic Republic of Congo. Open Forum Infectious Diseases [Internet]. 2024;11:1\u0026ndash;10. Available from: https://doi.org/10.1093/ofid/ofae150\u003c/li\u003e\n\u003cli\u003ePollicino T, Cacciola I, Saffioti F, Raimondo G. Hepatitis B virus PreS/S gene variants: Pathobiology and clinical implications. Journal of Hepatology [Internet]. 2014 [cited 2025 May 9];61:408\u0026ndash;17. Available from: https://pubmed.ncbi.nlm.nih.gov/24801416/\u003c/li\u003e\n\u003cli\u003ePinho-Nascimento CA, Bratschi MW, H\u0026ouml;fer R, Soares CC, Warryn L, Pečerska J, et al. Transmission of Hepatitis B and D Viruses in an African Rural Community. mSystems [Internet]. 2018 [cited 2025 May 9];3:e00120-18. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC6143728/\u003c/li\u003e\n\u003cli\u003eChen J, Li L, Yin Q, Shen T. A review of epidemiology and clinical relevance of Hepatitis B virus genotypes and subgenotypes. Clinics and Research in Hepatology and Gastroenterology [Internet]. 2023 [cited 2025 May 9];47. Available from: https://pubmed.ncbi.nlm.nih.gov/37479136/\u003c/li\u003e\n\u003cli\u003eChen BF. Hepatitis B virus pre-S/S variants in liver diseases. World Journal of Gastroenterology [Internet]. 2018 [cited 2025 May 9];24:1507\u0026ndash;20. Available from: https://pubmed.ncbi.nlm.nih.gov/29662289/\u003c/li\u003e\n\u003cli\u003eKabamba AT, Kalunga BT, Mwamba CM, Nyembo CM, Dufrasne F, Dessilly G, et al. Epidemiological aspects and molecular characterization of the hepatitis B virus among blood donors in Lubumbashi, Democratic Republic of Congo. Transfusion Clinique et Biologique [Internet]. 2021 [cited 2025 May 9];28:30\u0026ndash;7. Available from: https://pubmed.ncbi.nlm.nih.gov/33232802/\u003c/li\u003e\n\u003cli\u003eShindano TA, Horsmans Y, Kabamba BM. Genetic and phylogenic characterization of hepatitis B virus in the eastern part of the Democratic Republic of Congo. Journal of Medical Virology [Internet]. 2018 [cited 2025 May 9];90:250\u0026ndash;4. Available from: https://pubmed.ncbi.nlm.nih.gov/28460156/\u003c/li\u003e\n\u003cli\u003eD\u0026eacute;mocratique R, Congo DU. Enqu\u0026ecirc;te nutritionnelle anthropom\u0026eacute;trique et de mortalit\u0026eacute; selon la m\u0026eacute;thodologie smart. 2021; \u003c/li\u003e\n\u003cli\u003eNanjundeswaraswamy TS, Divakar S. Determination of Sample Size and Sampling Methods in Applied Research. Proceedings on Engineering Sciences. 2021;3:25\u0026ndash;32. \u003c/li\u003e\n\u003cli\u003eChromas | Technelysium Pty Ltd [Internet]. [cited 2025 May 9]. Available from: https://technelysium.com.au/wp/chromas/\u003c/li\u003e\n\u003cli\u003eKatoh K, Standley DM. MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution [Internet]. 2013 [cited 2025 May 9];30:772\u0026ndash;80. Available from: https://pubmed.ncbi.nlm.nih.gov/23329690/\u003c/li\u003e\n\u003cli\u003eMinh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, Von Haeseler A, et al. IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Molecular Biology and Evolution [Internet]. 2020 [cited 2025 May 9];37:1530. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC7182206/\u003c/li\u003e\n\u003cli\u003eLetunic I, Bork P. Interactive Tree of Life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool. Nucleic Acids Research [Internet]. 2024 [cited 2025 May 9];52:W78\u0026ndash;82. Available from: https://dx.doi.org/10.1093/nar/gkae268\u003c/li\u003e\n\u003cli\u003eHepatitis B Virus Phylogenetic Typing Tool [Internet]. [cited 2025 May 9]. Available from: https://www.genomedetective.com/app/typingtool/hbv/how-to-use\u003c/li\u003e\n\u003cli\u003eTamura K, Stecher G, Kumar S. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Molecular Biology and Evolution [Internet]. 2021 [cited 2025 May 9];38:3022\u0026ndash;7. Available from: https://dx.doi.org/10.1093/molbev/msab120\u003c/li\u003e\n\u003cli\u003eBell TG, Kramvis A. Bioinformatics Tools for Small Genomes, Such as Hepatitis B Virus. Viruses [Internet]. 2015 [cited 2025 May 9];7:781. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC4353916/\u003c/li\u003e\n\u003cli\u003eHIV-GRADE - HIV-GRADE viral Genotypic resistance algorithms [Internet]. [cited 2025 May 9]. Available from: https://www.hiv-grade.de/cms/grade/\u003c/li\u003e\n\u003cli\u003eZhang K, Bach C, Fraune MN, Xia Y, Beggel B, Kaiser R, et al. Novel rtM204 Mutations in HBV Polymerase Confer Reduced Susceptibility to Adefovir and Tenofovir. Journal of Antivirals \u0026amp; Antiretrovirals. 2017;09:10\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eCaligiuri P, Cerruti R, Icardi G, Bruzzone B. Overview of hepatitis B virus mutations and their implications in the management of infection. World Journal of Gastroenterology. 2016;22:145\u0026ndash;54. \u003c/li\u003e\n\u003cli\u003eSchweitzer A, Horn J, Mikolajczyk RT, Krause G, Ott JJ. Estimations of worldwide prevalence of chronic hepatitis B virus infection: A systematic review of data published between 1965 and 2013. The Lancet [Internet]. 2015 [cited 2025 May 9];386:1546\u0026ndash;55. Available from: https://pubmed.ncbi.nlm.nih.gov/26231459/\u003c/li\u003e\n\u003cli\u003eWHO, UNICEF. Somalia : WHO and UNICEF estimates of immunization coverage : 2022 revision Somalia : WHO and UNICEF estimates of immunization coverage : 2022 revision. 2024;1\u0026ndash;23. \u003c/li\u003e\n\u003cli\u003eShimakawa Y, Yan HJ, Tsuchiya N, Bottomley C, Hall AJ. Association of Early Age at Establishment of Chronic Hepatitis B Infection with Persistent Viral Replication, Liver Cirrhosis and Hepatocellular Carcinoma: A Systematic Review. PLoS ONE. 2013;8. \u003c/li\u003e\n\u003cli\u003eMoonsamy S, Suchard M, Pillay P, Prabdial-Sing N. Prevalence and incidence rates of laboratory-confirmed hepatitis B infection in South Africa, 2015 to 2019. BMC Public Health [Internet]. 2022;22:1\u0026ndash;13. Available from: https://doi.org/10.1186/s12889-021-12391-3\u003c/li\u003e\n\u003cli\u003eEzeilo MC, Engwa GA, Iroha RI, Odimegwu DC. Seroprevalence and Associated Risk Factors of Hepatitis B Virus Infection Among Children in Enugu Metropolis. Virology: Research and Treatment. 2018;9:0\u0026ndash;6. \u003c/li\u003e\n\u003cli\u003eKafeero HM, Ndagire D, Ocama P, Kato CD, Wampande E, Walusansa A, et al. Mapping hepatitis B virus genotypes on the African continent from 1997 to 2021: a systematic review with meta-analysis. Scientific Reports [Internet]. 2023;13:1\u0026ndash;14. Available from: https://doi.org/10.1038/s41598-023-32865-1\u003c/li\u003e\n\u003cli\u003eDumpis U, Holmes EC, Mendy M, Hill A, Thursz M, Hall A, et al. Transmission of hepatitis B virus infection in Gambian families revealed by phylogenetic analysis. Journal of Hepatology. 2001;35:99\u0026ndash;104. \u003c/li\u003e\n\u003cli\u003eLazarevic I. Clinical implications of hepatitis B virus mutations: Recent advances. World Journal of Gastroenterology. 2014;20:7653\u0026ndash;64. \u003c/li\u003e\n\u003cli\u003eWang J, Zhu B, Lu M, Yang D. Hepatitis B virus preS/S gene mutations and their clinical implications. Annals of Blood. 2017;2:17\u0026ndash;17. \u003c/li\u003e\n\u003cli\u003eMorgan CE, Powers KA, Edwards JK et al. Characterizing hepatitis B virus infection in children in the Democratic Republic of Congo to inform elimination efforts. 2024; \u003c/li\u003e\n\u003cli\u003eDesalegn H, Aberra H, Berhe N, Mekasha B, Stene-Johansen K, Krarup H, et al. Treatment of chronic hepatitis B in sub-Saharan Africa: 1-year results of a pilot program in Ethiopia. BMC Medicine. 2018;16:1\u0026ndash;10. \u003c/li\u003e\n\u003cli\u003eLumley SF, Mokaya J, Maponga TG, Kramvis A, Dusheiko G, Irving W, et al. Hepatitis B virus resistance to nucleos(t)ide analogue therapy: WHO consultation on questions, challenges, and a roadmap for the field. The Lancet Microbe. 2025;1\u0026ndash;12. \u003c/li\u003e\n\u003cli\u003ePan CQ, Zhang JX. Natural history and clinical consequences of hepatitis B virus infection. International Journal of Medical Sciences. 2005;2:36\u0026ndash;40. \u003c/li\u003e\n\u003cli\u003eMartinson FEA, Weigle KA, Royce RA, Weber DJ, Suchindran CM, Lemon SM. Risk factors for horizontal transmission of hepatitis B virus in a rural district in Ghana. American Journal of Epidemiology. 1998;147:478\u0026ndash;87. \u003c/li\u003e\n\u003cli\u003eZhao W. Guidelines for the Prevention, Diagnosis, Care and Treatment for People with Chronic Hepatitis B Infection (Text Extract): Executive Summary. Infectious Diseases and Immunity. 2024. \u003c/li\u003e\n\u003cli\u003eGuingan\u0026eacute; AN, Kabor\u0026eacute; R, Shimakawa Y, Som\u0026eacute; EN, Kania D, Pisoni A, et al. Screening for Hepatitis B in partners and children of women positive for surface antigen, Burkina Faso. Bulletin of the World Health Organization. 2022;100:256\u0026ndash;67. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"tropical-medicine-and-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tmah","sideBox":"Learn more about [Tropical Medicine and Health](https://tropmedhealth.biomedcentral.com/)","snPcode":"41182","submissionUrl":"https://submission.springernature.com/new-submission/41182/3","title":"Tropical Medicine and Health","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Intra-familial, Transmission, Hepatitis B virus, Peri-urban, Community, Democratic Republic of Congo","lastPublishedDoi":"10.21203/rs.3.rs-6836597/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6836597/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground.\u003c/h2\u003e \u003cp\u003eDespite global elimination targets set for 2030, Hepatitis B virus (HBV) infection remains a major public health challenge in low-income countries, including the Democratic Republic of Congo (DRC). Limited evidence on the regional transmission pathways preclude progress towards HBV elimination. This study aimed to assess the prevalence, molecular characteristics, and transmission dynamics of HBV in the Lukelenge health district, a peri-urban area in central DRC.\u003c/p\u003e\u003ch2\u003eMethods.\u003c/h2\u003e \u003cp\u003eWe employed a two-tiered recruitment strategy: community member volunteers were enrolled during the first phase, and upon notification of HBV positivity in an index case, family contacts were subsequently recruited in the second phase. Participants were screened for hepatitis B surface antigen (HBsAg), followed by PCR amplification of HBV DNA and sequencing. Genotyping and phylogenetic analysis of preS/S sequences were performed to explore regional HBV diversity and transmission patterns.\u003c/p\u003e\u003ch2\u003eResults.\u003c/h2\u003e \u003cp\u003eA total of 751 participants from 677 households were included. The overall HBsAg prevalence was 3.8% [95% CI: 2.6\u0026ndash;5.5], with the highest rate (10.1%) found in children aged 5 years and younger. All 42 HBV isolates belonged to genotype E, with 97.6% sharing the \u003cem\u003eayw4\u003c/em\u003e serotype. Mutations with relevancy to immune escape were detected in 9.5% of strains, while those possibly linked to antiviral resistance were found in 4.7%. Maximum likelihood phylogenetic analysis showed intra-familial clustering of preS/S sequences, suggesting that parent-to-child transmission was the most frequent mode of HBV spread in the study population.\u003c/p\u003e\u003ch2\u003eConclusions.\u003c/h2\u003e \u003cp\u003eHBV in Lukelenge shows intermediate endemicity, especially affecting young children. Intra-familial transmission revealed to be predominant, likely involving both vertical and horizontal pathways. Family-targeted interventions, including maternal screening and universal birth-dose vaccination, should be prioritized to eliminate HBV in this region.\u003c/p\u003e","manuscriptTitle":"Intra-familial transmission of Hepatitis B virus in a peri-urban community from the Democratic Republic of the Congo","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-16 08:53:13","doi":"10.21203/rs.3.rs-6836597/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-22T23:07:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-22T21:49:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-21T09:20:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"164151080820673797996444826415167106846","date":"2025-06-13T05:14:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"116493739072566066820343793541368917535","date":"2025-06-12T00:26:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-11T06:33:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-10T08:47:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-10T08:45:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Tropical Medicine and Health","date":"2025-06-06T11:19:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"tropical-medicine-and-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tmah","sideBox":"Learn more about [Tropical Medicine and Health](https://tropmedhealth.biomedcentral.com/)","snPcode":"41182","submissionUrl":"https://submission.springernature.com/new-submission/41182/3","title":"Tropical Medicine and Health","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"72cd245c-04c8-4932-b261-4a9cc3f03ee6","owner":[],"postedDate":"June 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-04T16:48:07+00:00","versionOfRecord":{"articleIdentity":"rs-6836597","link":"https://doi.org/10.1186/s41182-025-00781-x","journal":{"identity":"tropical-medicine-and-health","isVorOnly":false,"title":"Tropical Medicine and Health"},"publishedOn":"2025-07-28 16:29:34","publishedOnDateReadable":"July 28th, 2025"},"versionCreatedAt":"2025-06-16 08:53:13","video":"","vorDoi":"10.1186/s41182-025-00781-x","vorDoiUrl":"https://doi.org/10.1186/s41182-025-00781-x","workflowStages":[]},"version":"v1","identity":"rs-6836597","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6836597","identity":"rs-6836597","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}