BACKGROUND:Deep sequencing could improve understanding of HIV treatment failure and viral population dynamics. However, this tool is often inaccessible in low- and middle-income countries.OBJECTIVES:To determine the genetic patterns of resistance emerging in West African HIV-1 subtypes during first-line virological failure, and the implications for future antiretroviral options.PATIENTS AND METHODS:Participants were selected from a Nigerian cohort of people living with HIV who had failed first-line ART and subsequently switched to second-line therapy. Whole HIV-1 genome sequences were generated from first-line virological failure samples with Illumina MiSeq. Mutations detected at ≥2% frequency were analysed and compared by subtype.RESULTS:HIV-1 sequences were obtained from 101 participants (65% female, median age 30 years, median 32.9 months of nevirapine- or efavirenz-based ART). Thymidine analogue mutations (TAMs) were detected in 61%, other core NRTI mutations in 92% and NNRTI mutations in 99%. Minority variants (<20% frequency) comprised 18% of all mutations. K65R was more prevalent in CRF02_AG than G subtypes (33% versus 7%; P = 0.002), and ≥3 TAMs were more common in G than CRF02_AG (52% versus 24%; P = 0.004). Subtype G viruses also contained more RT cleavage site mutations. Cross-resistance to at least one of the newer NNRTIs, doravirine, etravirine or rilpivirine, was predicted in 81% of participants.CONCLUSIONS:Extensive drug resistance had accumulated in people with West African HIV-1 subtypes, prior to second-line ART. Deep sequencing significantly increased the detection of resistance-associated mutations. Caution should be used if considering newer-generation NNRTI agents in this setting.

Background: Recently emerging SARS-CoV-2 variants have been associated with an increased rate of transmission within the community. Little is known about the impact their increased infectivity has on transmission within hospitals.Methods: We collected viral sequences and epidemiological data of patients with community and healthcare associated SARS-CoV-2 infections, sampled from 16th November 2020 to 10th January 2021, from nine hospitals participating in the COG-UK HOCI study. Outbreaks were identified using ward information, lineage and pairwise genetic differences between viral sequences.Findings: Mixed effects logistic regression analysis of 4184 sequences showed healthcare-acquired infections were no more likely to be identified as the Alpha variant than community acquired infections. Nosocomial outbreaks were investigated based on overlapping ward stay and SARS-CoV-2 genome sequence similarity. There was no significant difference in the number of patients involved in outbreaks caused by the Alpha variant compared to outbreaks caused by other lineages.Interpretation: Notwithstanding evidence from community studies that the Alpha variant is more transmissible, we find no evidence to support it causing more nosocomial transmission than previous lineages. This suggests that the stringent infection prevention measures already in place in UK hospitals contained the spread of the Alpha variant as effectively as other less transmissible lineages, providing reassurance of their efficacy against emerging variants of concern.Funding Information: COG-UK HOCI funded by COG-UK consortium. The COG-UK consortium is supported by funding from the Medical Research Council (MRC) part of UK Research & Innovation (UKRI), the National Institute of Health Research (NIHR) and Genome Research Limited, operating as the Wellcome Sanger Institute.Declaration of Interests: None to declare. Ethics Approval Statement: Ethical approval for the HOCI study was provided by REC 20/EE/0118.

BackgroundEmergence of variants with specific mutations in key epitopes in the spike protein of SARS-CoV-2 raises concerns pertinent to mass vaccination campaigns and use of monoclonal antibodies. We aimed to describe the emergence of the B.1.1.7 variant of concern (VOC), including virological characteristics and clinical severity in contemporaneous patients with and without the variant.MethodsIn this cohort study, samples positive for SARS-CoV-2 on PCR that were collected from Nov 9, 2020, for patients acutely admitted to one of two hospitals on or before Dec 20, 2020, in London, UK, were sequenced and analysed for the presence of VOC-defining mutations. We fitted Poisson regression models to investigate the association between B.1.1.7 infection and severe disease (defined as point 6 or higher on the WHO ordinal scale within 14 days of symptoms or positive test) and death within 28 days of a positive test and did supplementary genomic analyses in a cohort of chronically shedding patients and in a cohort of remdesivir-treated patients. Viral load was compared by proxy, using PCR cycle threshold values and sequencing read depths.FindingsOf 496 patients with samples positive for SARS-CoV-2 on PCR and who met inclusion criteria, 341 had samples that could be sequenced. 198 (58%) of 341 had B.1.1.7 infection and 143 (42%) had non-B.1.1.7 infection. We found no evidence of an association between severe disease and death and lineage (B.1.1.7 vs non-B.1.1.7) in unadjusted analyses (prevalence ratio [PR] 0·97 [95% CI 0·72–1·31]), or in analyses adjusted for hospital, sex, age, comorbidities, and ethnicity (adjusted PR 1·02 [0·76–1·38]). We detected no B.1.1.7 VOC-defining mutations in 123 chronically shedding immunocompromised patients or in 32 remdesivir-treated patients. Viral load by proxy was higher in B.1.1.7 samples than in non-B.1.1.7 samples, as measured by cycle threshold value (mean 28·8 [SD 4·7] vs 32·0 [4·8]; p=0·0085) and genomic read depth (1280 [1004] vs 831 [682]; p=0·0011).InterpretationEmerging evidence exists of increased transmissibility of B.1.1.7, and we found increased virus load by proxy for B.1.1.7 in our data. We did not identify an association of the variant with severe disease in this hospitalised cohort.FundingUniversity College London Hospitals NHS Trust, University College London/University College London Hospitals NIHR Biomedical Research Centre, Engineering and Physical Sciences Research Council.

Objectives: Recently emerging SARS-CoV-2 variants have been associated with an increased rate of transmission within the community. We sought to determine whether this also resulted in increased transmission within hospitals. Methods: We collected viral sequences and epidemiological data of patients with community and healthcare associated SARS-CoV-2 infections, sampled from 16th November 2020 to 10th January 2021, from nine hospitals participating in the COG-UK HOCI study. Outbreaks were identified using ward information, lineage and pairwise genetic differences between viral sequences. Results: Mixed effects logistic regression analysis of 4184 sequences showed healthcare-acquired infections were no more likely to be identified as the Alpha variant than community acquired infections. Nosocomial outbreaks were investigated based on overlapping ward stay and SARS-CoV-2 genome sequence similarity. There was no significant difference in the number of patients involved in outbreaks caused by the Alpha variant compared to outbreaks caused by other lineages. Conclusions: We find no evidence to support it causing more nosocomial transmission than previous lineages. This suggests that the stringent infection prevention measures already in place in UK hospitals contained the spread of the Alpha variant as effectively as other less transmissible lineages, providing reassurance of their efficacy against emerging variants of concern. (c) 2021 The Authors. Published by Elsevier Ltd on behalf of The British Infection Association. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )

Current methods for diagnosing drug-resistant tuberculosis are time consuming, resulting in delays in patients receiving treatment and in transmission onwards. They also require a high level of laboratory infrastructure, which is often only available at centralized facilities, resulting in further delays to diagnosis and additional barriers to deployment in resource-limited settings.

Background: Emergence of variants with specific mutations in key epitopes in the spike protein of SARS-CoV-2 raise concerns pertinent to the current mass vaccination campaigns and use of monoclonal antibodies. We describe emergence of the variant of concern (VOC) B.1.1.7 in two North Central London (NCL) hospitals including virological characteristics and clinical severity in contemporaneous patients with and without the new variant. The strength of our study is the depth and granularity of clinical data.Methods: SARS-CoV-2 PCR positive samples collected from 9th November 2020 for patients acutely admitted to one of 2 NCL hospitals on or before 20th December 2020 were sequenced and analysed for the presence of VOC-defining mutations. We compared illness severity in those with variant of concern (VOC) and non-VOC infections and conducted supplementary genomic analyses in a cohort of chronically shedding immunocompromised/treated patients. Viral loads were compared by Ct value and sequencing read-depth.Findings: Of 341 successfully sequenced patients, 198 (58%) had VOC infections. There was no evidence of an association with severe disease by lineage (VOC vs non-VOC) in unadjusted analyses (PR 0.97, 95% CI 0.72-1.31), or analyses adjusted for sex, age, comorbidities and ethnicity (aPR 1.07, 95% CI 0.79-1.43). We detected no VOC-defining mutations in immunocompromised/treated patients; viral loads were higher in VOC samples.Interpretation: Despite emerging evidence of increased transmissibility of the VOC, we did not identify an association with severe disease, suggesting the surge in hospitalisations was more likely related to a significant increase in cases overall rather than virus characteristics.Funding Statement: UCLH APDU receives funding from the University College London Hospitals NHS Trust. DF was funded by the i-sense EPSRC IRC in Agile Early Warning Sensing Systems for Infectious Diseases and Antimicrobial Resistance (EP/R00529X/1).Declaration of Interests: We declare no competing interests.Ethics Approval Statement: The clinical information and SARS-CoV-2 PCR samples were collected as part of routine clinical care. Data were extracted and analysed using permission granted by the NHS REC (IRAS:284088, REC reference:20/HRA/2505).

Recombination is an important feature of HIV evolution, occurring both within and between the major branches of diversity (subtypes). The Ugandan epidemic is primarily composed of two subtypes, A1 and D, that have been co-circulating for 50 years, frequently recombining in dually infected patients. Here, we investigate the frequency of recombinants in this population and the location of breakpoints along the genome. As part of the PANGEA-HIV consortium, 1,472 consensus genome sequences over 5 kb have been obtained from 1,857 samples collected by the MRC/UVRI & LSHTM Research unit in Uganda, 465 (31.6 per cent) of which were near full-length sequences (>8 kb). Using the subtyping tool SCUEAL, we find that of the near full-length dataset, 233 (50.1 per cent) genomes contained only one subtype, 30.8 per cent A1 (n = 143), 17.6 per cent D (n = 82), and 1.7 per cent C (n = 8), while 49.9 per cent (n = 232) contained more than one subtype (including A1/D (n = 164), A1/C (n = 13), C/D (n = 9); A1/C/D (n = 13), and 33 complex types). K-means clustering of the recombinant A1/D genomes revealed a section of envelope (C2gp120-TMgp41) is often inherited intact, whilst a generalized linear model was used to demonstrate significantly fewer breakpoints in the gag-pol and envelope C2-TM regions compared with accessory gene regions. Despite similar recombination patterns in many recombinants, no clearly supported circulating recombinant form (CRF) was found, there was limited evidence of the transmission of breakpoints, and the vast majority (153/164; 93 per cent) of the A1/D recombinants appear to be unique recombinant forms. Thus, recombination is pervasive with clear biases in breakpoint location, but CRFs are not a significant feature, characteristic of a complex, and diverse epidemic.

OBJECTIVES:HIV-1 integrase inhibitors are recommended as first-line therapy by WHO, though efficacy and resistance data for non-B subtypes are limited. Two recent trials have identified the integrase L74I mutation to be associated with integrase inhibitor treatment failure in HIV-1 non-B subtypes. We sought to define the prevalence of integrase resistance mutations, including L74I, in West Africa. METHODS:We studied a Nigerian cohort of recipients prior to and during receipt of second-line PI-based therapy, who were integrase inhibitor-naive. Illumina next-generation sequencing with target enrichment was used on stored plasma samples. Drug resistance was interpreted using the Stanford Resistance Database and the IAS-USA 2019 mutation lists. RESULTS:Of 115 individuals, 59.1% harboured CRF02_AG HIV-1 and 40.9% harboured subtype G HIV-1. Four participants had major IAS-USA integrase resistance-associated mutations detected at low levels (2%-5% frequency). Two had Q148K minority variants and two had R263K (one of whom also had L74I). L74I was detected in plasma samples at over 2% frequency in 40% (46/115). Twelve (26.1%) had low-level minority variants of between 2% and 20% of the viral population sampled. The remaining 34 (73.9%) had L74I present at >20% frequency. L74I was more common among those with subtype G infection (55.3%, 26/47) than those with CRF02_AG infection (29.4%, 20/68) (P = 0.005). CONCLUSIONS:HIV-1 subtypes circulating in West Africa appear to have very low prevalence of major integrase mutations, but significant prevalence of L74I. A combination of in vitro and clinical studies is warranted to understand the potential implications.

Varicella zoster virus (VZV) is a skin-tropic virus that infects epidermal keratinocytes and causes chickenpox. Although common, VZV infection can be life-threatening, particularly in the immunocompromized. Therefore, understanding VZV-keratinocyte interactions is important to find new treatments beyond vaccination and antiviral drugs. In VZV-infected skin, kallikrein 6 and the ubiquitin ligase MDM2 are upregulated concomitant with keratin 10 (KRT10) downregulation. MDM2 binds to KRT10, targeting it for degradation via the ubiquitin-proteasome pathway. Preventing KRT10 degradation reduced VZV propagation in culture and prevented epidermal disruption in skin explants. KRT10 knockdown induced expression of NR4A1 and enhanced viral propagation in culture. NR4A1 knockdown prevented viral propagation in culture, reduced LC3 levels, and increased LAMP2 expression. We therefore describe a drug-able pathway whereby MDM2 ubiquitinates and degrades KRT10, increasing NR4A1 expression and allowing VZV replication and propagation.

Background The extent of transmission of influenza in hospital settings is poorly understood. Next generation sequencing may improve this by providing information on the genetic relatedness of viral strains. Objectives We aimed to apply next generation sequencing to describe transmission in hospital and compare with methods based on routinely-collected data. Methods All influenza samples taken through routine care from patients at University College London Hospitals NHS Foundation Trust (September 2012 to March 2014) were included. We conducted Illumina sequencing and identified genetic clusters. We compared nosocomial transmission estimates defined using classical methods (based on time from admission to sample) and genetic clustering. We identified pairs of cases with space-time links and assessed genetic relatedness. Results We sequenced influenza sampled from 214 patients. There were 180 unique genetic strains, 16 (8.8%) of which seeded a new transmission chain. Nosocomial transmission was indicated for 32 (15.0%) cases using the classical definition and 34 (15.8%) based on genetic clustering. Of the 50 patients in a genetic cluster, 11 (22.0%) had known space-time links with other cases in the same cluster. Genetic distances between pairs of cases with space-time links were lower than for pairs without spatial links (P < .001). Conclusions Genetic data confirmed that nosocomial transmission contributes significantly to the hospital burden of influenza and elucidated transmission chains. Prospective next generation sequencing could support outbreak investigations and monitor the impact of infection and control measures.

Background & methods The ICONIC project has developed an automated high-throughput pipeline to generate HIV nearly full-length genomes (NFLG, i.e. from gag to nef) from next-generation sequencing (NGS) data. The pipeline was applied to 420 HIV samples collected at University College London Hospitals NHS Trust and Barts Health NHS Trust (London) and sequenced using an Illumina MiSeq at the Wellcome Trust Sanger Institute (Cambridge). Consensus genomes were generated and subtyped using COMET, and unique recombinants were studied with jpHMM and SimPlot. Maximum-likelihood phylogenetic trees were constructed using RAxML to identify transmission networks using the Cluster Picker. Results The pipeline generated sequences of at least 1Kb of length (median = 7.46Kb, IQR = 4.01Kb) for 375 out of the 420 samples (89%), with 174 (46.4%) being NFLG. A total of 365 sequences (169 of them NFLG) corresponded to unique subjects and were included in the down-stream analyses. The most frequent HIV subtypes were B (n = 149, 40.8%) and C (n = 77, 21.1%) and the circulating recombinant form CRF02_AG (n = 32, 8.8%). We found 14 different CRFs (n = 66, 18.1%) and multiple URFs (n = 32, 8.8%) that involved recombination between 12 different subtypes/CRFs. The most frequent URFs were B/CRF01_AE (4 cases) and A1/D, B/C, and B/CRF02_AG (3 cases each). Most URFs (19/26, 73%) lacked breakpoints in the PR+RT pol region, rendering them undetectable if only that was sequenced. Twelve (37.5%) of the URFs could have emerged within the UK, whereas the rest were probably imported from sub-Saharan Africa, South East Asia and South America. For 2 URFs we found highly similar pol sequences circulating in the UK. We detected 31 phylogenetic clusters using the full dataset: 25 pairs (mostly subtypes B and C), 4 triplets and 2 quadruplets. Some of these were not consistent across different genes due to interand intra-subtype recombination. Clusters involved 70 sequences, 19.2% of the dataset. Conclusions The initial analysis of genome sequences detected substantial hidden variability in the London HIV epidemic. Analysing full genome sequences, as opposed to only PR+RT, identified previously undetected recombinants. It provided a more reliable description of CRFs (that would be otherwise misclassified) and transmission clusters.

The canine transmissible venereal tumor (CTVT) is a clonally transmissible cancer that regresses spontaneously or after treatment with vincristine, but we know little about the regression mechanisms. We performed global transcriptional, methylation, and functional pathway analyses on serial biopsies of vincristine-treated CTVTs and found that regression occurs in sequential steps; activation of the innate immune system and host epithelial tissue remodeling followed by immune infiltration of the tumor, arrest in the cell cycle, and repair of tissue damage. We identified CCL5 as a possible driver of CTVT regression. Changes in gene expression are associated with methylation changes at specific intragenic sites. Our results underscore the critical role of host innate immunity in triggering cancer regression.

Traditional epidemiological investigation of nosocomial transmission of influenza involves the identification of patients who have the same influenza virus type and who have overlapped in time and place. This method may misidentify transmission where it has not occurred or miss transmission when it has. We used influenza virus whole-genome sequencing (WGS) to investigate an outbreak of influenza A virus infection in a hematology/oncology ward and identified 2 separate introductions, one of which resulted in 5 additional infections and 79 bed-days lost. Results from WGS are becoming rapidly available and may supplement traditional infection control procedures in the investigation and management of nosocomial outbreaks.

Dolutegravir (DTG), a second-generation integrase strand-transfer inhibitor (INSTI), is equivalent or superior to current non-nucleotide reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), and first-generation INSTI-based antiretroviral regimens (ARVs). It has the potential to make big improvements in HIV control globally and within patients. This is perhaps the most "precious" HIV drug available. The integrase mutation R263K has been observed in tissue culture experiments and in patients treated with dolutegravir monotherapy in clinical trials. Globally, adherence and monitoring may be less than optimal and therefore DTG resistance more common. This is particularly important in low-middle-income countries, where patients may remain on failing regimens for longer periods of time and accumulate drug resistance. Data on this mutation in non-subtype B infections do not exist. We describe the first report of the R263K integrase mutation in a dolutegravir-exposed subtype D-infected individual with vertically acquired HIV. We have used deep sequencing of longitudinal samples to highlight the change in resistance over time while on a failing regimen. The case highlights that poorly adherent patients should not be offered dolutegravir even as part of a combination regimen and that protease inhibitors should be used preferentially.

We report the molecular investigations of a large influenza A(H3N2) outbreak, in a season characterised by sharp increase in influenza admissions since December 2016. Analysis of haemagglutinin (HA) sequences demonstrated co-circulation of multiple clades (3C.3a, 3C.2a and 3C.2a1). Most variants fell into a novel subclade (proposed as 3C.2a2); they possessed four unique amino acid substitutions in the HA protein and loss of a potential glycosylation site. These changes potentially modify the H3N2 strain antigenicity.

Nearly full genome sequencing (NFGS) of HIV is increasingly available in Western countries, but its application in sub-Saharan Africa is scarce. The PANGEA_HIV project is producing HIV NFGS in African sites, including Uganda. The Ugandan epidemic features a long-term co-circulation of subtypes A1 and D, with the consequent emergence of many recombinants. We collected 685 contemporary (2009-2014) samples from Ugandan populations (a rural cohort in Masaka, several Lake Victoria fisherfolk communities, and female sex workers from Kampala); as well as 53 historical samples taken in 1986 from AIDS patients. They were analysed using Illumina MiSeq next-generation sequencing; 609 (565 contemporary, 44 historical) yielded sequences ≥1Kb, with half of them (320, 52%) being NFGS. To identify recombinants, we adapted the SCUEAL subtyping tool for full-genome analysis. Maximum-likelihood phylogenies were built with RAxML to detect clusters using Cluster Picker. Drug-resistance mutations (DRM) and co-receptor usage were analysed using Stanford HIVdb and Geno2pheno tools, respectively. Unique recombinant forms (URF) between subtypes A1 and D accounted for 169 (29.9%) of the contemporary sequences, followed by subtypes A1 (147, 26.0%) and D (113, 20.0%). The rest belonged to other subtypes (10 C, 1 G), other URF (70, 12.4%), and recombinants showing unclassified segments (55, 9.7%), most of them due to the presence of gaps. A1/D recombinants also predominated among the historical sequences (21, 47.7%), followed by subtype A1 (10, 22.7%) and other URF (10, 22.7%). We detected 54 clusters (44 pairs, 10 triplets) among contemporary sequences, showing a clustering rate of 21.1%. Most clusters (77.8%) involved subjects from the same population only; 63.0% involved subjects from the same sampling location only. Contemporary sequences showed low DRM rates at PR+RT (3.3%) and integrase (0.5%); historical sequences showed no DRM. X4 co-receptor usage, which confers resistance to entry inhibitors, was rare (0.5%) among contemporary samples but frequent (47.4%) among historical ones, probably due to an advanced infection stage. HIV recombinants dominate the Ugandan epidemics. Their prevalence analysing NFGS was higher than in earlier studies of partial sequences. We found low levels of resistance, as expected in low-income settings, and a low rate of phylogenetic clustering, with most clusters being intra-population. However, almost 40% of the clusters involved geographical mobility.

SignificanceIn this report we provide evidence of a source of macrophage (Mφ) populations that are derived from unique biphenotypic early pro-B cells with non-rearranged B-cell receptors. These early precursors give rise to either tissue resident- or monocyte-derived Mφs during homeostasis and inflammatory responses, thereby demonstrating functional plasticity depending on the environmental cues in adult mice. We suggest that these findings significantly advance and expand our understanding of Mφ biology and hematopoiesis, the plasticity of hematopoietic precursors, and the heterogeneity of Mφ subsets.

BackgroundWhole-genome sequencing, and other molecular methods, can be used to identify potential chains of transmission of infections in hospitals. However, integrating this information with epidemiological data to guide control measures is challenging. We aimed to produce an interactive application for visualisation of hospital infections for use by infection control teams and researchers.MethodsWe developed this application using Shiny, the web application framework for R. The minimum data set required to run the application comprises admission and sample dates and the ward on which the sample was taken. Optional additional data are dates of patient ward transfers, descriptive patient characteristics including genetically defined clusters, and genetic distances between infections. We demonstrate this application with a case-study of 242 influenza samples from a UK hospital (September, 2012, to March, 2014), which were sequenced as part of the ICONIC project.FindingsThe application presents three data visualisations: one displays epidemic curves of the numbers of cases of the infection over time, both overall and separately for each ward. Interactive options allow the user to change graph scales and display subsets of the data. The second visualisation is a schematic representation of hospital wards that shows patient locations on a given date. It then highlights epidemiological and genetic links between patients. The third visualisation is an interactive timeline displaying the wards that patients were staying on and on which dates they were likely to be exposed to, or transmitting, an infection.InterpretationThis novel application produces visual displays to aid interpretation of complex epidemiological and genomic data in hospital settings. It can be used to highlight areas in a hospital in which infections may have been transmitted, and to trace possible chains of transmission by highlighting patients who share epidemiological or genetic links. Advantages include its user-friendly interface and flexibility for use in any setting with similar data on any pathogen. A challenge is integration with existing hospital systems to facilitate data import, since some expertise is needed to extract appropriately formatted data from hospital systems.FundingSupported by the Health Innovation Challenge Fund (T5-355) (ICONIC), a parallel funding partnership between the Department of Health and the Wellcome Trust.