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Introduction and Objectives: Chronic hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC). In African populations, HCC frequently presents at an advanced stage with poor outcomes. We applied whole genome sequencing (WGS) to compare HBV genomes in individuals with and without HCC., Materials and Methods: We identified adults with HBV infection, with and without complicating HCC, in Cape Town, South Africa. We generated HBV WGS using pan-genotypic probe-based enrichment followed by Illumina sequencing., Results: Compared to the non-HCC group, HCC patients were more likely to be male (p < 0.0001), older (p = 0.01), HIV-negative (p = 0.006), and have higher HBV viral loads (p < 0.0001). Among 19 HCC and 12 non-HCC patients for whom WGS was obtained, genotype A dominated (74 %), of which 96 % were subgenotype A1. PreS2 deletions (Δ38-55) were enriched in HBV sequences from HCC patients (n = 7). The sequence motif most strongly associated with HCC comprised either a deletion or polymorphism at site T53 in PreS2 - collectively coined 'non-T53' - together with a basal core promoter (BCP) mutation G1764A (AUROC = 0.79)., Conclusions: In this setting, HBV sequence polymorphisms and deletions are associated with HCC, and 'non-T53 + G1764A' represents a putative signature motif for HCC. Additional investigations are needed to disaggregate the impact of other demographic, clinical, and environmental influences, to ascertain the extent to which viral polymorphisms contribute to oncogenesis, and to determine whether HBV sequence is a useful biomarker for HCC risk stratification., Competing Interests: Declaration of competing interest PCM has participated in projects supported by GSK, outside the direct scope of the work presented here. MIA has participated in projects supported by Prenetics, J&J, and Pfizer outside the direct scope of this work. AK is an Associate Editor for the journal Annals of Hepatology. All other authors declare no conflicts of interest., (Copyright © 2025 Fundación Clínica Médica Sur, A.C. Published by Elsevier España, S.L.U. All rights reserved.)

Background: Clinical studies have reported rising pre-treatment HIV drug resistance during antiretroviral treatment (ART) scale-up in Africa, but representative data are limited. We estimated population-level drug resistance trends during ART expansion in Uganda., Methods: We analyzed data from the population-based open Rakai Community Cohort Study conducted at agrarian, trading, and fishing communities in southern Uganda between 2012 and 2019. Consenting participants aged 15-49 were HIV tested and completed questionnaires. Persons living with HIV (PLHIV) provided samples for viral load quantification and virus deep-sequencing. Sequence data were used to predict resistance. Population prevalence of class-specific resistance and resistance-conferring substitutions were estimated using robust log-Poisson regression., Findings: Data from 93,622 participant-visits, including 4,702 deep-sequencing measurements, showed that the prevalence of NNRTI resistance among pre-treatment viremic PLHIV doubled between 2012 and 2017 (PR:1.98, 95%CI:1.34-2.91), rising to 9.61% (7.27-12.7%). The overall population prevalence of pre-treatment viremic NNRTI and NRTI resistance among all participants decreased during the same period, reaching 0.25% (0.18% - 0.33%) and 0.05% (0.02% - 0.10%), respectively ( p- values for trend = 0.00015, 0.002), coincident with increasing treatment coverage and viral suppression. By the final survey, population prevalence of resistance contributed by treatment-experienced PLHIV exceeded that from pre-treatment PLHIV, with NNRTI resistance at 0.54% (0.44%-0.66%) and NRTI resistance at 0.42% (0.33%-0.53%). Overall, NNRTI and NRTI resistance was predominantly attributable to rtK103N and rtM184V. While 10.52% (7.97%-13.87%) and 9.95% (6.41%-15.43%) of viremic pre-treatment and treatment-experienced PLHIV harbored the inT97A mutation, no major dolutegravir resistance mutations were observed., Interpretation: Despite rising NNRTI resistance among pre-treatment PLHIV, overall population prevalence of pre-treatment resistance decreased due to treatment uptake. Most NNRTI and NRTI resistance is now contributed by treatment-experienced PLHIV. The high prevalence of mutations conferring resistance to components of current first-line ART regimens among PLHIV with viremia is potentially concerning., Competing Interests: Declaration of interests We declare no competing interests.

Motivation: Target enrichment strategies generate genomic data from multiple pathogens in a single process, greatly improving sensitivity over metagenomic sequencing and enabling cost-effective, high-throughput surveillance and clinical applications. However, uptake by research and clinical laboratories is constrained by an absence of computational tools that are specifically designed for the analysis of multi-pathogen enrichment sequence data. Here we present an analysis pipeline, Castanet, for use with multi-pathogen enrichment sequencing data. Castanet is designed to work with short-read data produced by existing targeted enrichment strategies, but can be readily deployed on any BAM file generated by another methodology. Also included are an optional graphical interface and installer script., Results: In addition to genome reconstruction, Castanet reports method-specific metrics that enable quantification of capture efficiency, estimation of pathogen load, differentiation of low-level positives from contamination, and assessment of sequencing quality. Castanet can be used as a traditional end-to-end pipeline for consensus generation, but its strength lies in the ability to process a flexible, pre-defined set of pathogens of interest directly from multi-pathogen enrichment experiments. In our tests, Castanet consensus sequences were accurate reconstructions of reference sequences, including in instances where multiple strains of the same pathogen were present. Castanet performs effectively on standard computers and can process the entire output of a 96-sample enrichment sequencing run (50M reads) using a single batch process command, in $<$2 h., Availability and Implementation: Source code freely available under GPL-3 license at https://github.com/MultipathogenGenomics/castanet, implemented in Python 3.10 and supported in Ubuntu Linux 22.04. The data underlying this article are available in Europe Nucleotide Archives, at https://www.ebi.ac.uk/ena/browser/view/PRJEB77004., (© The Author(s) 2024. Published by Oxford University Press.)

There is limited data on human immunodeficiency virus (HIV) evolutionary trends in African populations. We evaluated changes in HIV viral diversity and genetic divergence in southern Uganda over a 24-year period spanning the introduction and scale-up of HIV prevention and treatment programs using HIV sequence and survey data from the Rakai Community Cohort Study, an open longitudinal population-based HIV surveillance cohort. Gag (p24) and env (gp41) HIV data were generated from people living with HIV (PLHIV) in 31 inland semi-urban trading and agrarian communities (1994-2018) and four hyperendemic Lake Victoria fishing communities (2011-2018) under continuous surveillance. HIV subtype was assigned using the Recombination Identification Program with phylogenetic confirmation. Inter-subtype diversity was evaluated using the Shannon diversity index, and intra-subtype diversity with the nucleotide diversity and pairwise TN93 genetic distance. Genetic divergence was measured using root-to-tip distance and pairwise TN93 genetic distance analyses. Demographic history of HIV was inferred using a coalescent-based Bayesian Skygrid model. Evolutionary dynamics were assessed among demographic and behavioral population subgroups, including by migration status. 9931 HIV sequences were available from 4999 PLHIV, including 3060 and 1939 persons residing in inland and fishing communities, respectively. In inland communities, subtype A1 viruses proportionately increased from 14.3% in 1995 to 25.9% in 2017 ( P  < .001), while those of subtype D declined from 73.2% in 1995 to 28.2% in 2017 ( P  < .001). The proportion of viruses classified as recombinants significantly increased by nearly four-fold from 12.2% in 1995 to 44.8% in 2017. Inter-subtype HIV diversity has generally increased. While intra-subtype p24 genetic diversity and divergence leveled off after 2014, intra-subtype gp41 diversity, effective population size, and divergence increased through 2017. Intra- and inter-subtype viral diversity increased across all demographic and behavioral population subgroups, including among individuals with no recent migration history or extra-community sexual partners. This study provides insights into population-level HIV evolutionary dynamics following the scale-up of HIV prevention and treatment programs. Continued molecular surveillance may provide a better understanding of the dynamics driving population HIV evolution and yield important insights for epidemic control and vaccine development., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press.)

Despite expanded antiretroviral therapy (ART) in South Africa, HIV-1 transmission persists. Integrase strand transfer inhibitors (INSTI) and long-acting injectables offer potential for superior viral suppression, but pre-existing drug resistance could threaten their effectiveness. In a community-based study in rural KwaZulu-Natal, prior to widespread INSTI usage, we enroled 18,025 individuals to characterise HIV-1 drug resistance and transmission networks to inform public health strategies. HIV testing and reflex viral load quantification were performed, with deep sequencing (20% variant threshold) used to detect resistance mutations. Phylogenetic and geospatial analyses characterised transmission clusters. One-third of participants were HIV-positive, with 21.7% having detectable viral loads; 62.1% of those with detectable viral loads were ART-naïve. Resistance to older reverse transcriptase (RT)-targeting drugs was found, but INSTI resistance remained low (<1%). Non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance, particularly to rilpivirine (RPV) even in ART-naïve individuals, was concerning. Twenty percent of sequenced individuals belonged to transmission clusters, with geographic analysis highlighting higher clustering in peripheral and rural areas. Our findings suggest promise for INSTI-based strategies in this setting but underscore the need for RPV resistance screening before implementing long-acting cabotegravir (CAB) + RPV. The significant clustering emphasises the importance of geographically targeted interventions to effectively curb HIV-1 transmission., (© 2024. The Author(s).)

Introduction: To prioritize and tailor interventions for ending AIDS by 2030 in Africa, it is important to characterize the population groups in which HIV viraemia is concentrating., Methods: We analysed HIV testing and viral load data collected between 2013-2019 from the open, population-based Rakai Community Cohort Study (RCCS) in Uganda, to estimate HIV seroprevalence and population viral suppression over time by gender, one-year age bands and residence in inland and fishing communities. All estimates were standardized to the underlying source population using census data. We then assessed 95-95-95 targets in their ability to identify the populations in which viraemia concentrates., Results: Following the implementation of Universal Test and Treat, the proportion of individuals with viraemia decreased from 4.9% (4.6%-5.3%) in 2013 to 1.9% (1.7%-2.2%) in 2019 in inland communities and from 19.1% (18.0%-20.4%) in 2013 to 4.7% (4.0%-5.5%) in 2019 in fishing communities. Viraemia did not concentrate in the age and gender groups furthest from achieving 95-95-95 targets. Instead, in both inland and fishing communities, women aged 25-29 and men aged 30-34 were the 5-year age groups that contributed most to population-level viraemia in 2019, despite these groups being close to or had already achieved 95-95-95 targets., Conclusions: The 95-95-95 targets provide a useful benchmark for monitoring progress towards HIV epidemic control, but do not contextualize underlying population structures and so may direct interventions towards groups that represent a marginal fraction of the population with viraemia., Competing Interests: MKG, LCW report grants from the National Institutes of Health during the conduct of this study. ABr, MM report an EPSRC PhD studentship during the conduct of this study. OR acknowledges grants from the Bill and Melinda Gates Foundation (OPP1175094 to C.Fraser., OPP1084362 to D. Pillay), the Engineering and Physical Sciences Research Council (EP/X038440/1), and the Moderna Charitable Foundation during the conduct of this study. This arrangement has been reviewed and approved by the Johns Hopkins University in accordance with its conflict-of-interest policies.

Respiratory syncytial virus (RSV) is the leading cause of hospitalisation for respiratory infection in young children. RSV disease severity is known to be age-dependent and highest in young infants, but other correlates of severity, particularly the presence of additional respiratory pathogens, are less well understood. In this study, nasopharyngeal swabs were collected from two cohorts of RSV-positive infants <12 months in Spain, the UK, and the Netherlands during 2017-20. We show, using targeted metagenomic sequencing of >100 pathogens, including all common respiratory viruses and bacteria, from samples collected from 433 infants, that burden of additional viruses is common (111/433, 26%) but only modestly correlates with RSV disease severity. In contrast, there is strong evidence in both cohorts and across age groups that presence of Haemophilus bacteria (194/433, 45%) is associated with higher severity, including much higher rates of hospitalisation (odds ratio 4.25, 95% CI 2.03-9.31). There is no evidence for association between higher severity and other detected bacteria, and no difference in severity between RSV genotypes. Our findings reveal the genomic diversity of additional pathogens during RSV infection in infants, and provide an evidence base for future causal investigations of the impact of co-infection on RSV disease severity., (© 2024. The Author(s).)

HIV incidence has been declining in Africa with scale-up of HIV interventions. However, there is limited data on HIV evolutionary trends in African populations with waning epidemics. We evaluated changes in HIV viral diversity and genetic divergence in southern Uganda over a twenty-five-year period spanning the introduction and scale-up of HIV prevention and treatment programs using HIV sequence and survey data from the Rakai Community Cohort Study, an open longitudinal population-based HIV surveillance cohort. Gag (p24) and env (gp41) HIV data were generated from persons living with HIV (PLHIV) in 31 inland semi-urban trading and agrarian communities (1994 to 2018) and four hyperendemic Lake Victoria fishing communities (2011 to 2018) under continuous surveillance. HIV subtype was assigned using the Recombination Identification Program with phylogenetic confirmation. Inter-subtype diversity was estimated using the Shannon diversity index and intra-subtype diversity with the nucleotide diversity and pairwise TN93 genetic distance. Genetic divergence was measured using root-to-tip distance and pairwise TN93 genetic distance analyses. Evolutionary dynamics were assessed among demographic and behavioral sub-groups, including by migration status. 9,931 HIV sequences were available from 4,999 PLHIV, including 3,060 and 1,939 persons residing in inland and fishing communities, respectively. In inland communities, subtype A1 viruses proportionately increased from 14.3% in 1995 to 25.9% in 2017 (p<0.001), while those of subtype D declined from 73.2% in 1995 to 28.2% in 2017 (p<0.001). The proportion of viruses classified as recombinants significantly increased by more than four-fold. Inter-subtype HIV diversity has generally increased. While p24 intra-subtype genetic diversity and divergence leveled off after 2014, diversity and divergence of gp41 increased through 2017. Inter- and intra-subtype viral diversity increased across all population sub-groups, including among individuals with no recent migration history or extra-community sexual partners. This study provides insights into population-level HIV evolutionary dynamics in declining African HIV epidemics following the scale-up of HIV prevention and treatment programs. Continued molecular surveillance may provide a better understanding of the dynamics driving population HIV evolution and yield important insights for epidemic control and vaccine development.

Persistent SARS-CoV-2 infections may act as viral reservoirs that could seed future outbreaks 1-5 , give rise to highly divergent lineages 6-8 and contribute to cases with post-acute COVID-19 sequelae (long COVID) 9,10 . However, the population prevalence of persistent infections, their viral load kinetics and evolutionary dynamics over the course of infections remain largely unknown. Here, using viral sequence data collected as part of a national infection survey, we identified 381 individuals with SARS-CoV-2 RNA at high titre persisting for at least 30 days, of which 54 had viral RNA persisting at least 60 days. We refer to these as 'persistent infections' as available evidence suggests that they represent ongoing viral replication, although the persistence of non-replicating RNA cannot be ruled out in all. Individuals with persistent infection had more than 50% higher odds of self-reporting long COVID than individuals with non-persistent infection. We estimate that 0.1-0.5% of infections may become persistent with typically rebounding high viral loads and last for at least 60 days. In some individuals, we identified many viral amino acid substitutions, indicating periods of strong positive selection, whereas others had no consensus change in the sequences for prolonged periods, consistent with weak selection. Substitutions included mutations that are lineage defining for SARS-CoV-2 variants, at target sites for monoclonal antibodies and/or are commonly found in immunocompromised people 11-14 . This work has profound implications for understanding and characterizing SARS-CoV-2 infection, epidemiology and evolution., (© 2024. The Author(s).)

HIV incidence in eastern and southern Africa has historically been concentrated among girls and women aged 15-24 years. As new cases decline with HIV interventions, population-level infection dynamics may shift by age and gender. Here, we integrated population-based surveillance of 38,749 participants in the Rakai Community Cohort Study and longitudinal deep-sequence viral phylogenetics to assess how HIV incidence and population groups driving transmission have changed from 2003 to 2018 in Uganda. We observed 1,117 individuals in the incidence cohort and 1,978 individuals in the transmission cohort. HIV viral suppression increased more rapidly in women than men, however incidence declined more slowly in women than men. We found that age-specific transmission flows shifted: whereas HIV transmission to girls and women (aged 15-24 years) from older men declined by about one-third, transmission to women (aged 25-34 years) from men that were 0-6 years older increased by half in 2003 to 2018. Based on changes in transmission flows, we estimated that closing the gender gap in viral suppression could have reduced HIV incidence in women by half in 2018. This study suggests that HIV programmes to increase HIV suppression in men are critical to reduce incidence in women, close gender gaps in infection burden and improve men's health in Africa., (© 2023. The Author(s).)

Background: In the last decade, universally available antiretroviral therapy (ART) has led to greatly improved health and survival of people living with HIV in sub-Saharan Africa, but new infections continue to appear. The design of effective prevention strategies requires the demographic characterisation of individuals acting as sources of infection, which is the aim of this study., Methods: Between 2014 and 2018, the HPTN 071 PopART study was conducted to quantify the public health benefits of ART. Viral samples from 7124 study participants in Zambia were deep-sequenced as part of HPTN 071-02 PopART Phylogenetics, an ancillary study. We used these sequences to identify likely transmission pairs. After demographic weighting of the recipients in these pairs to match the overall HIV-positive population, we analysed the demographic characteristics of the sources to better understand transmission in the general population., Findings: We identified a total of 300 likely transmission pairs. 178 (59·4%) were male to female, with 130 (95% CI 110-150; 43·3%) from males aged 25-40 years. Overall, men transmitted 2·09-fold (2·06-2·29) more infections per capita than women, a ratio peaking at 5·87 (2·78-15·8) in the 35-39 years source age group. 40 (26-57; 13·2%) transmissions linked individuals from different communities in the trial. Of 288 sources with recorded information on drug resistance mutations, 52 (38-69; 18·1%) carried viruses resistant to first-line ART., Interpretation: HIV-1 transmission in the HPTN 071 study communities comes from a wide range of age and sex groups, and there is no outsized contribution to new infections from importation or drug resistance mutations. Men aged 25-39 years, underserved by current treatment and prevention services, should be prioritised for HIV testing and ART., Funding: National Institute of Allergy and Infectious Diseases, US President's Emergency Plan for AIDS Relief, International Initiative for Impact Evaluation, Bill & Melinda Gates Foundation, National Institute on Drug Abuse, and National Institute of Mental Health., Competing Interests: Declaration of interests DJD, SHE, CF, EP-M, and OR report grant funding from NIH for this work. MSC reports other NIH grant funding. SHE reports NIH funding for meetings and travel. CF and OR report grant funding from the Bill & Melinda Gates Foundation for this work. HA reports honoraria from the Global Fund. MSC reports payments from Medscape and UpToDate for written material. WP reports consulting fees from WHO. All other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The hepatitis C virus (HCV) epidemic in Western countries is primarily perpetuated by the sub-populations of men who have sex with men (MSM) and people who inject drugs (PWID). Understanding the dynamics of transmission in these communities is crucial for removing the remaining hurdles towards HCV elimination. We sequenced 269 annotated HCV plasma samples using probe enrichment and next-generation sequencing, obtaining 224 open reading frames of HCV (OR497849-OR498072). Maximum likelihood phylogenies were generated on the four most prevalent subtypes in this study (HCV1a, 1b, 3a, 4d) with a subsequent transmission cluster analysis. The highest rate of clustering was observed for HCV4d samples (13/17 (76.47%)). The second highest rate of clustering was observed in HCV1a samples (42/78 (53.85%)) with significant association with HIV-positive MSM. HCV1b and HCV3a had very low rates of clustering (2/83 (2.41%) and (0/29)). The spread of the prevalent subtype HCV1b appears to have been largely curtailed, and we demonstrate the onwards transmission of HCV1a and HCV4d in the HIV-positive MSM population across municipal borders. More systematic data collection and sequencing is needed to allow a better understanding of the HCV transmission among the community of PWID and overcome the remaining barriers for HCV elimination in Belgium.

Detection of HIV drug resistance (HIVDR) is vital to successful anti-retroviral therapy (ART). HIVDR testing to determine drug-resistance mutations is routinely performed in Australia to guide ART choice in newly diagnosed people living with HIV or in cases of treatment failure. In 2022, our clinical microbiology laboratory sought to validate a next-generation sequencing (NGS)-based HIVDR assay to replace the previous Sanger-sequencing (SS)-based ViroSeq. NGS solutions for HIVDR offer higher throughput, lower costs and higher sensitivity for variant detection. We sought to validate the previously described low-cost probe-based NGS method (veSEQ-HIV) for whole-genome recovery and HIVDR-testing in a diagnostic setting. veSEQ-HIV displayed 100% and 98% accuracy in major and minor mutation detection, respectively, and 100% accuracy of subtyping (provided > 1000 mapped reads were obtained). Pairwise comparison exhibited low inter-and intrarun variability across the whole-genome (Jaccard index [J] = 0.993; J = 0.972) and the Pol gene (J = 0.999; J = 0.999), respectively. veSEQ-HIV met all our pre-set criteria based on WHO recommendations and successfully replaced ViroSeq in our laboratory. Scaling-down veSEQ-HIV to a limited batch size and sequencing on Illumina iSeq. 100, allowed easy implementation of the assay into the workflow of a small sequencing laboratory with minimal staff and equipment and the ability to meet clinically relevant test turn-around times. As HIVDR-testing moves from SS- to NGS-based methods and new ART drugs come to market (particularly those with targets outside the Pol region), whole-genome recovery using veSEQ-HIV provides a robust, cost-effective and "future-proof" NGS method for HIVDR-testing., (© 2023 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

Despite the scale-up of antiretroviral therapy (ART) in South Africa, HIV-1 incidence remains high. The anticipated use of potent integrase strand transfer inhibitors and long-acting injectables aims to enhance viral suppression at the population level and diminish transmission. Nevertheless, pre-existing drug resistance could impede the efficacy of long-acting injectable ART combinations, such as rilpivirine (an NNRTI) and cabotegravir (an INSTI). Consequently, a thorough understanding of transmission networks and geospatial distributions is vital for tailored interventions, including pre-exposure prophylaxis with long-acting injectables. However, empirical data on background resistance and transmission networks remain limited. In a community-based study in rural KwaZulu-Natal (2018-2019), prior to the widespread use of integrase inhibitor-based first-line ART, we performed HIV testing with reflex HIV-1 RNA viral load quantification on 18,025 participants. From this cohort, 6,096 (33.9%) tested positive for HIV via ELISA, with 1,323 (21.7%) exhibiting detectable viral loads (> 40 copies/mL). Of those with detectable viral loads, 62.1% were ART-naïve, and the majority of the treated were on an efavirenz + cytosine analogue + tenofovir regimen. Deep sequencing analysis, with a variant abundance threshold of 20%, revealed NRTI resistance mutations such as M184V in 2% of ART-naïve and 32% of treated individuals. Tenofovir resistance mutations K65R and K70E were found in 12% and 5% of ART-experienced individuals, respectively, and in less than 1% of ART-naïve individuals. Integrase inhibitor resistance mutations were notably infrequent (< 1%). Prevalence of pre-treatment drug resistance to NNRTIs was 10%, predominantly consisting of the K103N mutation. Among those with viraemic ART, NNRTI resistance was 50%, with rilpivirine-associated mutations observed in 9% of treated and 6% of untreated individuals. Cluster analysis revealed that 20% (205/1,050) of those sequenced were part of a cluster. We identified 171 groups with at least two linked participants; three quarters of clusters had only two individuals, and a quarter had 3-6 individuals. Integrating phylogenetic with geospatial analyses, we revealed a complex transmission network with significant clustering in specific regions, notably peripheral and rural areas. These findings derived from population scale genomic analyses are encouraging in terms of the limited resistance to DTG, but indicate that transitioning to long-acting cabotegravir + rilpivirine for transmission reduction should be accompanied by prior screening for rilpivirine resistance. Whole HIV-1 genome sequencing allowed identification of significant proportions of clusters with multiple individuals, and geospatial analyses suggesting decentralised networks can inform targeting public health interventions to effectively curb HIV-1 transmission.

HIV incidence in eastern and southern Africa has historically been concentrated among girls and women aged 15-24 years. As new cases decline with HIV interventions, population-level infection dynamics may shift by age and gender. Here, we integrated population-based surveillance of 38,749 participants in the Rakai Community Cohort Study and longitudinal deep sequence viral phylogenetics to assess how HIV incidence and population groups driving transmission have changed from 2003 to 2018 in Uganda. We observed 1,117 individuals in the incidence cohort and 1,978 individuals in the transmission cohort. HIV viral suppression increased more rapidly in women than men, however incidence declined more slowly in women than men. We found that age-specific transmission flows shifted, while HIV transmission to girls and women (aged 15-24 years) from older men declined by about one third, transmission to women (aged 25-34 years) from men that were 0-6 years older increased by half in 2003 to 2018. Based on changes in transmission flows, we estimated that closing the gender gap in viral suppression could have reduced HIV incidence in women by half in 2018. This study suggests that HIV programs to increase HIV suppression in men are critical to reduce incidence in women, close gender gaps in infection burden and improve men's health in Africa.

The Office for National Statistics Coronavirus (COVID-19) Infection Survey (ONS-CIS) is the largest surveillance study of SARS-CoV-2 positivity in the community, and collected data on the United Kingdom (UK) epidemic from April 2020 until March 2023 before being paused. Here, we report on the epidemiological and evolutionary dynamics of SARS-CoV-2 determined by analysing the sequenced samples collected by the ONS-CIS during this period. We observed a series of sweeps or partial sweeps, with each sweeping lineage having a distinct growth advantage compared to their predecessors, although this was also accompanied by a gradual fall in average viral burdens from June 2021 to March 2023. The sweeps also generated an alternating pattern in which most samples had either S-gene target failure (SGTF) or non-SGTF over time. Evolution was characterized by steadily increasing divergence and diversity within lineages, but with step increases in divergence associated with each sweeping major lineage. This led to a faster overall rate of evolution when measured at the between-lineage level compared to within lineages, and fluctuating levels of diversity. These observations highlight the value of viral sequencing integrated into community surveillance studies to monitor the viral epidemiology and evolution of SARS-CoV-2, and potentially other pathogens.

In this study, we evaluated the impact of viral variant, in addition to other variables, on within-host viral burden, by analysing cycle threshold (Ct) values derived from nose and throat swabs, collected as part of the UK COVID-19 Infection Survey. Because viral burden distributions determined from community survey data can be biased due to the impact of variant epidemiology on the time-since-infection of samples, we developed a method to explicitly adjust observed Ct value distributions to account for the expected bias. By analysing the adjusted Ct values using partial least squares regression, we found that among unvaccinated individuals with no known prior exposure, viral burden was 44% lower among Alpha variant infections, compared to those with the predecessor strain, B.1.177. Vaccination reduced viral burden by 67%, and among vaccinated individuals, viral burden was 286% higher among Delta variant, compared to Alpha variant, infections. In addition, viral burden increased by 17% for every 10-year age increment of the infected individual. In summary, within-host viral burden increases with age, is reduced by vaccination, and is influenced by the interplay of vaccination status and viral variant., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Fryer et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Due to the high prevalence of resistance to NNRTI-based ART since 2018, consolidated recommendations from the WHO have indicated dolutegravir as the preferred drug of choice for HIV treatment globally. There is a paucity of resistance outcome data from HIV-1 non-B subtypes circulating across West Africa., Aims: We characterized the mutational profiles of persons living with HIV from a cross-sectional cohort in North-East Nigeria failing a dolutegravir-based ART regimen., Methods: WGS of plasma samples collected from 61 HIV-1-infected participants following virological failure of dolutegravir-based ART were sequenced using the Illumina platform. Sequencing was successfully completed for samples from 55 participants. Following quality control, 33 full genomes were analysed from participants with a median age of 40 years and median time on ART of 9 years. HIV-1 subtyping was performed using SNAPPy., Results: Most participants had mutational profiles reflective of exposure to previous first- and second-line ART regimens comprised NRTIs and NNRTIs. More than half of participants had one or more drug resistance-associated mutations (DRMs) affecting susceptibility to NRTIs (17/33; 52%) and NNRTIs (24/33; 73%). Almost a quarter of participants (8/33; 24.4%) had one or more DRMs affecting tenofovir susceptibility. Only one participant, infected with HIV-1 subtype G, had evidence of DRMs affecting dolutegravir susceptibility-this was characterized by the T66A, G118R, E138K and R263K mutations., Conclusions: This study found a low prevalence of resistance to dolutegravir; the data are therefore supportive of the continual rollout of dolutegravir as the primary first-line regimen for ART-naive participants and the preferred switch to second-line ART across the region. However, population-level, longer-term data collection on dolutegravir outcomes are required to further guide implementation and policy action across the region., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

The raw material for viral evolution is provided by intra-host mutations occurring during replication, transcription or post-transcription. Replication and transcription of Coronaviridae proceed through the synthesis of negative-sense 'antigenomes' acting as templates for positive-sense genomic and subgenomic RNA. Hence, mutations in the genomes of SARS-CoV-2 and other coronaviruses can occur during (and after) the synthesis of either negative-sense or positive-sense RNA, with potentially distinct patterns and consequences. We explored for the first time the mutational spectrum of SARS-CoV-2 (sub)genomic and anti(sub)genomic RNA. We use a high-quality deep sequencing dataset produced using a quantitative strand-aware sequencing method, controlled for artefacts and sequencing errors, and scrutinized for accurate detection of within-host diversity. The nucleotide differences between negative- and positive-sense strand consensus vary between patients and do not show dependence on age or sex. Similarities and differences in mutational patterns between within-host minor variants on the two RNA strands suggested strand-specific mutations or editing by host deaminases and oxidative damage. We observe generally neutral and slight negative selection on the negative strand, contrasting with purifying selection in ORF1a, ORF1b and S genes of the positive strand of the genome.

Background: Although most currently used regimens for Hepatitis C virus (HCV) infections can be initiated without prior knowledge of genotype and subtype, genotyping is still useful to identify patients who might benefit from a personalized treatment due to resistance to direct-acting antivirals (DAA)., Objectives: To assess the utility of full-genome next-generation sequencing (FG-NGS) for HCV genotyping., Study Design: 138 HCV plasma samples previously genotyped by VERSANT HCV Genotype Assay (LiPA) were subjected to FG-NGS and phylogenetically genotyped Genome Detective. Consensuses were analysed by HCV-GLUE for resistance-associated substitutions (RASs) and their impact on treatment response was investigated., Results: 102/138 (73.9%) samples were sequenced to a genome coverage and depth of >90% of the HCV open reading frame covered by >100 reads/site. Concordant genotype and subtype results were assigned in 97.1% and 79.4% of samples, respectively. FG-NGS resolved the subtype of 13.7% samples that had ambiguous calls by LiPA and identified one dual infection and one recombinant strain. At least one RAS was found for the HCV genes NS3, NS5A, and NS5B in 2.91%, 36.98% and 27.3% samples, respectively. Irrespective of the observed RAS, all patients responded well to DAA treatment, except for HCV1b-infected patients treated with Zepatier (33.3% failure rate (5/15))., Conclusion: While LiPA and FG-NGS showed overall good concordance, FG-NGS improved specificity for subtypes, recombinant and mixed infections. FG-NGS enabled the detection of RAS, but its predictive value for treatment outcome in DAA-naïve patients remains uncertain. With additional refinements, FG-NGS may be the way forward for HCV genotyping., Competing Interests: Declaration of Competing Interest The authors declare no commercial or financial relationship that could be construed as potential conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Tools to detect SARS-CoV-2 variants of concern and track the ongoing evolution of the virus are necessary to support public health efforts and the design and evaluation of novel COVID-19 therapeutics and vaccines. Although next-generation sequencing (NGS) has been adopted as the gold standard method for discriminating SARS-CoV-2 lineages, alternative methods may be required when processing samples with low viral loads or low RNA quality. To this aim, an allele-specific probe PCR (ASP-PCR) targeting lineage-specific single nucleotide polymorphisms (SNPs) was developed and used to screen 1,082 samples from two clinical trials in the United Kingdom and Brazil. Probit regression models were developed to compare ASP-PCR performance against 1,771 NGS results for the same cohorts. Individual SNPs were shown to readily identify specific variants of concern. ASP-PCR was shown to discriminate SARS-CoV-2 lineages with a higher likelihood than NGS over a wide range of viral loads. The comparative advantage for ASP-PCR over NGS was most pronounced in samples with cycle threshold ( C T ) values between 26 and 30 and in samples that showed evidence of degradation. Results for samples screened by ASP-PCR and NGS showed 99% concordant results. ASP-PCR is well suited to augment but not replace NGS. The method can differentiate SARS-CoV-2 lineages with high accuracy and would be best deployed to screen samples with lower viral loads or that may suffer from degradation. Future work should investigate further destabilization from primer-target base mismatch through altered oligonucleotide chemistry or chemical additives.