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Persistent SARS-CoV-2 infections may act as viral reservoirs that could seed future outbreaks1-5, give rise to highly divergent lineages6-8 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 people11-14. This work has profound implications for understanding and characterizing SARS-CoV-2 infection, epidemiology and evolution.
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COVID-19 , Inquéritos Epidemiológicos , Infecção Persistente , SARS-CoV-2 , Humanos , Substituição de Aminoácidos , Anticorpos Monoclonais/imunologia , COVID-19/epidemiologia , COVID-19/virologia , Evolução Molecular , Hospedeiro Imunocomprometido/imunologia , Mutação , Infecção Persistente/epidemiologia , Infecção Persistente/virologia , Síndrome de COVID-19 Pós-Aguda/epidemiologia , Síndrome de COVID-19 Pós-Aguda/virologia , Prevalência , RNA Viral/análise , RNA Viral/genética , SARS-CoV-2/química , SARS-CoV-2/classificação , SARS-CoV-2/genética , SARS-CoV-2/imunologia , SARS-CoV-2/isolamento & purificação , Seleção Genética , Autorrelato , Fatores de Tempo , Carga Viral , Replicação ViralRESUMO
[This corrects the article DOI: 10.1371/journal.ppat.1011461.].
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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.
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COVID-19 , SARS-CoV-2 , Humanos , Viés de Seleção , SARS-CoV-2/genética , Carga Viral , COVID-19/epidemiologia , COVID-19/prevenção & controle , VacinaçãoRESUMO
Factor VIII and IX clotting factor concentrates manufactured from pooled plasma have been identified as potent sources of virus infection in persons with hemophilia (PWHs) in the 1970s and 1980s. To investigate the range and diversity of viruses over this period, we analysed 24 clotting factor concentrates for several blood-borne viruses. Nucleic acid was extracted from 14 commercially produced clotting factors and 10 from nonremunerated donors, preserved in lyophilized form (expiry dates: 1974-1992). Clotting factors were tested by commercial and in-house quantitative PCRs for blood-borne viruses hepatitis A, B, C and E viruses (HAV, HBV, HCV, HEV), HIV- types 1/2, parvoviruses B19V and PARV4, and human pegiviruses types 1 and 2 (HPgV-1,-2). HCV and HPgV-1 were the most frequently detected viruses (both 14/24 tested) primarily in commercial clotting factors, with frequently extremely high viral loads in the late 1970s-1985 and a diverse range of HCV genotypes. Detection frequencies sharply declined following introduction of virus inactivation. HIV-1, HBV, and HAV were less frequently detected (3/24, 1/24, and 1/24 respectively); none were positive for HEV. Contrastingly, B19V and PARV4 were detected throughout the study period, even after introduction of dry heat treatment, consistent with ongoing documented transmission to PWHs into the early 1990s. While hemophilia treatment is now largely based on recombinant factor VIII/IX in the UK and elsewhere, the comprehensive screen of historical plasma-derived clotting factors reveals extensive exposure of PWHs to blood-borne viruses throughout 1970s-early 1990s, and the epidemiological and manufacturing parameters that influenced clotting factor contamination.
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Fatores de Coagulação Sanguínea , Patógenos Transmitidos pelo Sangue , Humanos , Patógenos Transmitidos pelo Sangue/isolamento & purificação , Infecções Transmitidas por Sangue/epidemiologia , Infecções Transmitidas por Sangue/virologia , Contaminação de Medicamentos , História do Século XX , Hemofilia A , Vírus/classificação , Vírus/isolamento & purificação , Vírus/genética , Reação em Cadeia da Polimerase , Fator VIII , Fatores de TempoRESUMO
BACKGROUND AND OBJECTIVES: Exclusion of blood donors with hepatitis B virus (HBV) core antibodies (anti-HBc) prevents transfusion-transmitted HBV infection but can lead to significant donor loss. As isolated anti-HBc positivity does not always indicate true past HBV infection, we have investigated the effectiveness of confirmatory anti-HBc testing and the representation of rare blood groups in anti-HBc-positive donors. MATERIALS AND METHODS: Three hundred ninety-seven HBV surface antigen-negative and anti-HBc initially reactive blood donor samples were tested by five different anti-HBc assays. RESULTS: Eighty percentage of samples reactive in Architect anti-HBc assay were positive by the Murex assay and anti-HBc neutralization. Eleven out of 397 samples showed discordant results in supplementary testing from the Murex confirmatory test result, and five remained undetermined following extensive serological testing. Thirty-eight percentage of anti-HBc-positive donors identified as minority ethnic groups compared with 11% representation in anti-HBc-negative donors (p < 0.0001); the frequency of the Ro blood group in anti-HBc-positive donors was 18 times higher in non-white ethnic groups. CONCLUSION: Using two anti-HBc assays effectively enabled the identification of HBV-exposed and potentially infectious donors, their deferral and potential clinical follow-up. However, the exclusion of confirmed anti-HBc-positive donors will still impact the supply of rare blood such as Ro.
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Doadores de Sangue , Anticorpos Anti-Hepatite B , Antígenos do Núcleo do Vírus da Hepatite B , Vírus da Hepatite B , Hepatite B , Humanos , Anticorpos Anti-Hepatite B/sangue , Hepatite B/sangue , Hepatite B/prevenção & controle , Feminino , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/sangue , Masculino , Vírus da Hepatite B/imunologia , Seleção do Doador/métodos , Antígenos de Grupos Sanguíneos/imunologia , Doação de SangueRESUMO
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.
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COVID-19 , Epidemias , Humanos , COVID-19/epidemiologia , SARS-CoV-2 , Reino Unido/epidemiologia , Inquéritos e QuestionáriosRESUMO
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.
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Fármacos Anti-HIV , Infecções por HIV , HIV-1 , Humanos , HIV-1/genética , Austrália , Infecções por HIV/tratamento farmacológico , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/uso terapêutico , Mutação , Sequenciamento Completo do Genoma , Farmacorresistência Viral/genética , GenótipoRESUMO
Occult hepatitis B (HBV) infection (OBI), characterized by low viral loads, accounts for much of the risk of HBV transfusion-transmitted infection. With anticore antibodies (anti-HBc) screening introduced in England, the imperative to identify OBI donors has increased. We aimed to develop an ultra-sensitive PCR system and investigate risk factors for HBV DNA presence in blood donations. Seven extraction methods and three PCR assays were compared. The optimal system was sought to determine HBV DNA presence in anti-HBc-positive donations. Predictors of DNA positivity were subsequently investigated. Extraction from 5 mL of plasma increased sample representation and resulted in HBV DNA detection in low viral load samples (~0.5 IU/mL). Screening of 487 763 donations in 2022 identified two OBI donors and 2042 anti-HBc-positive donors, 412 of the latter with anti-HBs < 100 mIU/mL. Testing of 134 anti-HBc-positive donations utilizing the 5 mL extraction method identified two further HBV DNA-positive donations. Higher anti-HBc titer and anti-HBs negativity were significant predictors of DNA detectability in anti-HBc-positive donations. An ultrasensitive PCR assay identified potentially infectious donations increasing HBV DNA detection in anti-HBc-positive donors from 0.5% to 1.9%. Anti-HBc titers may further complement the risk stratification for DNA positivity in anti-HBc screening and minimize unnecessary donor deferral.
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Hepatite B Crônica , Hepatite B , Humanos , Vírus da Hepatite B/genética , DNA Viral , Doadores de Sangue , Antígenos do Núcleo do Vírus da Hepatite B , Anticorpos Anti-Hepatite B , Antígenos de Superfície da Hepatite B , Reação em Cadeia da Polimerase/métodos , Medição de RiscoRESUMO
Over 1000 cases of unexplained severe acute hepatitis in children have been reported to date worldwide. An association with adeno-associated virus type 2 (AAV2) infection, a human parvovirus, prompted us to investigate the epidemiology of AAV in the United Kingdom. Three hundred pediatric respiratory samples collected before (April 03, 2009-April 03, 2013) and during (April 03, 2022) the COVID-19 pandemic were obtained. Wastewater samples were collected from 50 locations in London (August 2021-March 2022). Samples were tested for AAV using real-time polymerase chain reaction followed by sequencing. Selected adenovirus (AdV)-positive samples were also sequenced. The detection frequency of AAV2 was a sevenfold higher in 2022 samples compared with 2009-2013 samples (10% vs. 1.4%) and highest in AdV-positive samples compared with negatives (10/37, 27% vs. 5/94, 5.3%, respectively). AAV2-positive samples displayed high genetic diversity. AAV2 sequences were either very low or absent in wastewater collected in 2021 but increased in January 2022 and peaked in March 2022. AAV2 was detected in children in association with AdV of species C, with a highest frequency in 2022. Our findings are consistent with the expansion of the population of children unexposed to AAV2, leading to greater spread of the virus once distancing restrictions were lifted.
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Infecções por Adenoviridae , COVID-19 , Hepatite , Humanos , Criança , Dependovirus/genética , Pandemias , Águas Residuárias , Adenoviridae/genéticaRESUMO
BACKGROUND: A large, worldwide outbreak of mpox (formerly referred to as monkeypox) involving mainly men who have sex with men commenced in May 2022. We evaluated the frequency of positivity for the causative agent, monkeypox virus (MPXV), in blood donations collected in August 2022, during the outbreak period in Southern England. METHODS/MATERIALS: The sensitivity and specificity of an MPXV-specific PCR and a generic non-variola orthopoxvirus (NVO) PCR were evaluated using samples from mpox cases and synthetic DNA standards. Residual minipools from nucleic acid testing were obtained from 10,896 blood donors in Southern England, with 21% from London. RESULTS: MPXV and NVO PCRs were both capable of detection of single copies of target sequence with calculated limits of detection (LOD)90 s of 2.3 and 2.1 DNA copies and analytical sample sensitivities of 46 and 42 MPXV DNA copies/ml, respectively. 454 minipools produced from 10,896 unique donors were assayed for MPXV DNA by both methods. No positive minipools were detected by either PCR. CONCLUSIONS: Although blood donors are unrepresentative of the UK population in terms of MPXV infection risk, the uniformly negative MPXV DNA testing results provide reassurance that MPXV viraemia and potential transmission risk were rare or absent in donors during the outbreak period. Minipools from blood donors allow rapid implementation of large-scale population-based screening for emerging pathogens and represent an important resource for pandemic preparedness.
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Mpox , Minorias Sexuais e de Gênero , Masculino , Humanos , Feminino , Monkeypox virus/genética , Mpox/epidemiologia , Mpox/diagnóstico , Doação de Sangue , Homossexualidade Masculina , Surtos de DoençasRESUMO
BACKGROUND: Human immunodeficiency virus (HIV)-1 genetic diversity increases during infection and can help infer the time elapsed since infection. However, the effect of antiretroviral treatment (ART) on the inference remains unknown. METHODS: Participants with estimated duration of HIV-1 infection based on repeated testing were sourced from cohorts in Botswana (n = 1944). Full-length HIV genome sequencing was performed from proviral deoxyribonucleic acid. We optimized a machine learning model to classify infections as < or >1 year based on viral genetic diversity, demographic, and clinical data. RESULTS: The best predictive model included variables for genetic diversity of HIV-1 gag, pol, and env, viral load, age, sex, and ART status. Most participants were on ART. Balanced accuracy was 90.6% (95% confidence interval, 86.7%-94.1%). We tested the algorithm among newly diagnosed participants with or without documented negative HIV tests. Among those without records, those who self-reported a negative HIV test within <1 year were more frequently classified as recent than those who reported a test >1 year previously. There was no difference in classification between those self-reporting a negative HIV test <1 year, whether or not they had a record. CONCLUSIONS: These results indicate that recency of HIV-1 infection can be inferred from viral sequence diversity even among patients on suppressive ART.
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Infecções por HIV , HIV-1 , Antirretrovirais/farmacologia , Antirretrovirais/uso terapêutico , Botsuana/epidemiologia , Variação Genética , Infecções por HIV/tratamento farmacológico , HIV-1/genética , Humanos , Carga ViralRESUMO
Respiratory syncytial virus (RSV) causes substantial morbidity and mortality in infants and young children worldwide. Here we evaluated host demographic and viral factors associated with RSV disease severity in 325 RSV-infected infants under 1 year of age from 3 European countries during 2017-2020. Younger infants had a higher clinical severity (ReSViNET) score and were more likely to require hospitalization, intensive care, respiratory support, and/or mechanical ventilation than older infants (<3 months vs 3 to <6 months and 3 to <6 months vs ≥6 months). Older age (≥6 months vs <3 months), higher viral load, and RSV-A were associated with a greater probability of fever. RSV-A and RSV-B caused similar disease severity and had similar viral dynamics. Infants with a more severe RSV infection, demonstrated by having a higher ReSViNET score, fever, and requiring hospitalization and intensive care, were more likely to have developed subsequent wheezing at 1 year of age. CLINICAL TRIALS REGISTRATION: NCT03756766.
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Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Hospitalização , Humanos , Lactente , Sons Respiratórios/etiologia , Infecções por Vírus Respiratório Sincicial/complicações , Infecções por Vírus Respiratório Sincicial/epidemiologia , Índice de Gravidade de DoençaRESUMO
BACKGROUND: A new variant of SARS-CoV-2, B.1.1.7, emerged as the dominant cause of COVID-19 disease in the UK from November, 2020. We report a post-hoc analysis of the efficacy of the adenoviral vector vaccine, ChAdOx1 nCoV-19 (AZD1222), against this variant. METHODS: Volunteers (aged ≥18 years) who were enrolled in phase 2/3 vaccine efficacy studies in the UK, and who were randomly assigned (1:1) to receive ChAdOx1 nCoV-19 or a meningococcal conjugate control (MenACWY) vaccine, provided upper airway swabs on a weekly basis and also if they developed symptoms of COVID-19 disease (a cough, a fever of 37·8°C or higher, shortness of breath, anosmia, or ageusia). Swabs were tested by nucleic acid amplification test (NAAT) for SARS-CoV-2 and positive samples were sequenced through the COVID-19 Genomics UK consortium. Neutralising antibody responses were measured using a live-virus microneutralisation assay against the B.1.1.7 lineage and a canonical non-B.1.1.7 lineage (Victoria). The efficacy analysis included symptomatic COVID-19 in seronegative participants with a NAAT positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to vaccine received. Vaccine efficacy was calculated as 1 - relative risk (ChAdOx1 nCoV-19 vs MenACWY groups) derived from a robust Poisson regression model. This study is continuing and is registered with ClinicalTrials.gov, NCT04400838, and ISRCTN, 15281137. FINDINGS: Participants in efficacy cohorts were recruited between May 31 and Nov 13, 2020, and received booster doses between Aug 3 and Dec 30, 2020. Of 8534 participants in the primary efficacy cohort, 6636 (78%) were aged 18-55 years and 5065 (59%) were female. Between Oct 1, 2020, and Jan 14, 2021, 520 participants developed SARS-CoV-2 infection. 1466 NAAT positive nose and throat swabs were collected from these participants during the trial. Of these, 401 swabs from 311 participants were successfully sequenced. Laboratory virus neutralisation activity by vaccine-induced antibodies was lower against the B.1.1.7 variant than against the Victoria lineage (geometric mean ratio 8·9, 95% CI 7·2-11·0). Clinical vaccine efficacy against symptomatic NAAT positive infection was 70·4% (95% CI 43·6-84·5) for B.1.1.7 and 81·5% (67·9-89·4) for non-B.1.1.7 lineages. INTERPRETATION: ChAdOx1 nCoV-19 showed reduced neutralisation activity against the B.1.1.7 variant compared with a non-B.1.1.7 variant in vitro, but the vaccine showed efficacy against the B.1.1.7 variant of SARS-CoV-2. FUNDING: UK Research and Innovation, National Institute for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midlands NIHR Clinical Research Network, and AstraZeneca.
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Anticorpos Neutralizantes/sangue , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , COVID-19/virologia , SARS-CoV-2/imunologia , Adolescente , Adulto , COVID-19/epidemiologia , Teste de Ácido Nucleico para COVID-19 , Vacinas contra COVID-19/efeitos adversos , ChAdOx1 nCoV-19 , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Amplificação de Ácido Nucleico , Pandemias/prevenção & controle , Método Simples-Cego , Reino Unido/epidemiologia , Carga Viral , Adulto JovemRESUMO
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 (CT) 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.
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COVID-19 , SARS-CoV-2 , Alelos , COVID-19/diagnóstico , Humanos , Reação em Cadeia da Polimerase , SARS-CoV-2/genéticaRESUMO
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.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , RNA Viral/genética , Genoma Viral , Mutação , GenômicaRESUMO
BACKGROUND: Convalescent plasma containing neutralizing antibody to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is under investigation for coronavirus disease 2019 (COVID-19) treatment. We report diverse virological characteristics of UK intensive care patients enrolled in the Immunoglobulin Domain of the REMAP-CAP randomized controlled trial that potentially influence treatment outcomes. METHODS: SARS-CoV-2 RNA in nasopharyngeal swabs collected pretreatment was quantified by PCR. Antibody status was determined by spike-protein ELISA. B.1.1.7 was differentiated from other SARS-CoV-2 strains using allele-specific probes or restriction site polymorphism (SfcI) targeting D1118H. RESULTS: Of 1274 subjects, 90% were PCR positive with viral loads 118-1.7â ×â 1011IU/mL. Median viral loads were 40-fold higher in those IgG seronegative (nâ =â 354; 28%) compared to seropositives (nâ =â 939; 72%). Frequencies of B.1.1.7 increased from <1% in November 2020 to 82% of subjects in January 2021. Seronegative individuals with wild-type SARS-CoV-2 had significantly higher viral loads than seropositives (medians 5.8â ×â 106 and 2.0â ×â 105 IU/mL, respectively; Pâ =â 2â ×â 10-15). CONCLUSIONS: High viral loads in seropositive B.1.1.7-infected subjects and resistance to seroconversion indicate less effective clearance by innate and adaptive immune responses. SARS-CoV-2 strain, viral loads, and antibody status define subgroups for analysis of treatment efficacy.
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Anticorpos Antivirais/imunologia , COVID-19/imunologia , COVID-19/terapia , SARS-CoV-2/imunologia , Carga Viral/imunologia , Idoso , Anticorpos Neutralizantes/imunologia , COVID-19/virologia , Estado Terminal , Feminino , Humanos , Imunização Passiva , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , RNA Viral/imunologia , Testes Sorológicos/métodos , Glicoproteína da Espícula de Coronavírus/imunologia , Reino Unido , Soroterapia para COVID-19RESUMO
BACKGROUND: The World Health Organization recommends drug-susceptibility testing of Mycobacterium tuberculosis complex for all patients with tuberculosis to guide treatment decisions and improve outcomes. Whether DNA sequencing can be used to accurately predict profiles of susceptibility to first-line antituberculosis drugs has not been clear. METHODS: We obtained whole-genome sequences and associated phenotypes of resistance or susceptibility to the first-line antituberculosis drugs isoniazid, rifampin, ethambutol, and pyrazinamide for isolates from 16 countries across six continents. For each isolate, mutations associated with drug resistance and drug susceptibility were identified across nine genes, and individual phenotypes were predicted unless mutations of unknown association were also present. To identify how whole-genome sequencing might direct first-line drug therapy, complete susceptibility profiles were predicted. These profiles were predicted to be susceptible to all four drugs (i.e., pansusceptible) if they were predicted to be susceptible to isoniazid and to the other drugs or if they contained mutations of unknown association in genes that affect susceptibility to the other drugs. We simulated the way in which the negative predictive value changed with the prevalence of drug resistance. RESULTS: A total of 10,209 isolates were analyzed. The largest proportion of phenotypes was predicted for rifampin (9660 [95.4%] of 10,130) and the smallest was predicted for ethambutol (8794 [89.8%] of 9794). Resistance to isoniazid, rifampin, ethambutol, and pyrazinamide was correctly predicted with 97.1%, 97.5%, 94.6%, and 91.3% sensitivity, respectively, and susceptibility to these drugs was correctly predicted with 99.0%, 98.8%, 93.6%, and 96.8% specificity. Of the 7516 isolates with complete phenotypic drug-susceptibility profiles, 5865 (78.0%) had complete genotypic predictions, among which 5250 profiles (89.5%) were correctly predicted. Among the 4037 phenotypic profiles that were predicted to be pansusceptible, 3952 (97.9%) were correctly predicted. CONCLUSIONS: Genotypic predictions of the susceptibility of M. tuberculosis to first-line drugs were found to be correlated with phenotypic susceptibility to these drugs. (Funded by the Bill and Melinda Gates Foundation and others.).
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Antituberculosos/farmacologia , Farmacorresistência Bacteriana/genética , Genoma Bacteriano , Mycobacterium tuberculosis/genética , Tuberculose/tratamento farmacológico , Sequenciamento Completo do Genoma , Antituberculosos/uso terapêutico , Etambutol/farmacologia , Genótipo , Humanos , Isoniazida/farmacologia , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Fenótipo , Pirazinamida/farmacologia , Rifampina/farmacologia , Tuberculose/microbiologiaRESUMO
Targeted metagenomics using strand-specific libraries with target enrichment is a sensitive, generalized approach to pathogen sequencing and transcriptome profiling. Using this method, we recovered 13 (76%) complete human respiratory syncytial virus (RSV) genomes from 17 clinical respiratory samples, reconstructed the phylogeny of the infecting viruses, and detected differential gene expression between 2 RSV subgroups, specifically, a lower expression of the P gene and a higher expression of the M2 gene in RSV-A than in RSV-B. This methodology can help to relate viral genetics to clinical phenotype and facilitate ongoing population-level RSV surveillance and vaccine development. Clinical Trials Registration. NCT03627572 and NCT03756766.
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Genoma Viral , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Lactente , Filogenia , Infecções por Vírus Respiratório Sincicial/metabolismo , Vírus Sincicial Respiratório Humano/classificação , Vírus Sincicial Respiratório Humano/metabolismo , Análise de Sequência , Análise de Sequência de RNA , Sequenciamento Completo do GenomaRESUMO
Viral genetic sequencing can be used to monitor the spread of HIV drug resistance, identify appropriate antiretroviral regimes, and characterize transmission dynamics. Despite decreasing costs, next-generation sequencing (NGS) is still prohibitively costly for routine use in generalized HIV epidemics in low- and middle-income countries. Here, we present veSEQ-HIV, a high-throughput, cost-effective NGS sequencing method and computational pipeline tailored specifically to HIV, which can be performed using leftover blood drawn for routine CD4 cell count testing. This method overcomes several major technical challenges that have prevented HIV sequencing from being used routinely in public health efforts; it is fast, robust, and cost-efficient, and generates full genomic sequences of diverse strains of HIV without bias. The complete veSEQ-HIV pipeline provides viral load estimates and quantitative summaries of drug resistance mutations; it also exploits information on within-host viral diversity to construct directed transmission networks. We evaluated the method's performance using 1,620 plasma samples collected from individuals attending 10 large urban clinics in Zambia as part of the HPTN 071-2 study (PopART Phylogenetics). Whole HIV genomes were recovered from 91% of samples with a viral load of >1,000 copies/ml. The cost of the assay (30 GBP per sample) compares favorably with existing VL and HIV genotyping tests, proving an affordable option for combining HIV clinical monitoring with molecular epidemiology and drug resistance surveillance in low-income settings.
Assuntos
Fármacos Anti-HIV , Infecções por HIV , HIV-1 , Fármacos Anti-HIV/uso terapêutico , Farmacorresistência Viral/genética , Genômica , Infecções por HIV/diagnóstico , Infecções por HIV/tratamento farmacológico , Infecções por HIV/epidemiologia , Humanos , Carga Viral , ZâmbiaRESUMO
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.