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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
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
Pharmacometric analyses of time series viral load data may detect drug effects with greater power than approaches using single time points. Because SARS-CoV-2 viral load rapidly rises and then falls, viral dynamic models have been used. We compared different modelling approaches when analysing Phase II-type viral dynamic data. Using two SARS-CoV-2 datasets of viral load starting within 7 days of symptoms, we fitted the slope-intercept exponential decay (SI), reduced target cell limited (rTCL), target cell limited (TCL) and TCL with eclipse phase (TCLE) models using nlmixr. Model performance was assessed via Bayesian information criterion (BIC), visual predictive checks (VPCs), goodness-of-fit plots, and parameter precision. The most complex (TCLE) model had the highest BIC for both datasets. The estimated viral decline rate was similar for all models except the TCL model for dataset A with a higher rate (median [range] day-1 : dataset A; 0.63 [0.56-1.84]; dataset B: 0.81 [0.74-0.85]). Our findings suggest simple models should be considered during pharmacodynamic model development.
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Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Humanos , Teorema de Bayes , Carga ViralRESUMO
A combination of favipiravir and zanamivir successfully cleared influenza B infection in a child who had undergone bone marrow transplant for X-linked severe combined immunodeficiency, with no recovery of T lymphocytes. Deep sequencing of viral samples illuminated the within-host dynamics of infection, demonstrating the effectiveness of favipiravir in this case.
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Influenza Humana , Zanamivir , Amidas , Antivirais/uso terapêutico , Criança , Humanos , Influenza Humana/tratamento farmacológico , Pirazinas/uso terapêutico , Zanamivir/uso terapêuticoRESUMO
Background: Norovirus is a leading cause of worldwide and nosocomial gastroenteritis. The study aim was to assess the utility of molecular epidemiology using full genome sequences compared to routine infection prevention and control (IPC) investigations. Methods: Norovirus genomes were generated from new episodes of norovirus at a pediatric tertiary referral hospital over a 19-month period (n = 182). Phylogeny identified clusters of related sequences that were verified using epidemiological and clinical data. Results: Twenty-four clusters of related norovirus sequences ("sequence clusters") were observed, including 8 previously identified by IPC investigations ("IPC outbreaks"). Seventeen sequence clusters (involving 77/182 patients) were corroborated by epidemiological data ("epidemiologically supported clusters"), suggesting transmission between patients. Linked infections were identified among 44 patients who were missed by IPC investigations. Thirty-three percent of norovirus sequences were linked, suggesting nosocomial transmission; 24% of patients had nosocomial infections from an unknown source; and 43% were norovirus positive on admission. Conclusions: We show there are frequent introductions of multiple norovirus strains with extensive onward nosocomial transmission of norovirus in a pediatric hospital with a high proportion of immunosuppressed patients nursed in isolation. Phylogenetic analysis using full genome sequences is more sensitive than classic IPC investigations for identifying linked cases and should be considered when investigating norovirus nosocomial transmission. Sampling of staff, visitors, and the environment may be required for complete understanding of infection sources and transmission routes in patients with nosocomial infections not linked to other patients and among patients with phylogenetically linked cases but no evidence of direct contact.
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Infecções por Caliciviridae/transmissão , Infecções por Caliciviridae/virologia , Infecção Hospitalar/transmissão , Infecção Hospitalar/virologia , Genoma Viral , Norovirus/genética , Criança , Surtos de Doenças , Gastroenterite/virologia , Genótipo , Hospitais Pediátricos , Humanos , FilogeniaRESUMO
The original version of this article unfortunately did not display the appropriate captions in the figure. The correct version is displayed below.
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Agamaglobulinemia/imunologia , Agamaglobulinemia/virologia , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Doenças Genéticas Ligadas ao Cromossomo X/virologia , Kobuvirus/imunologia , Infecções por Picornaviridae/imunologia , Infecções por Picornaviridae/virologia , Viroses/imunologia , Adolescente , Doença Crônica , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/virologia , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Masculino , Viroses/virologiaRESUMO
Norovirus full-genome sequencing is challenging due to sequence heterogeneity among genomes. Previous methods have relied on PCR amplification, which is problematic due to primer design, and transcriptome sequencing (RNA-Seq), which nonspecifically sequences all RNA, including host and bacterial RNA, in stool specimens. Target enrichment uses a panel of custom-designed 120-mer RNA baits that are complementary to all publicly available norovirus sequences, with multiple baits targeting each position of the genome, which overcomes the challenge of primer design. Norovirus genomes are enriched from stool RNA extracts to minimize the sequencing of nontarget RNA. SureSelect target enrichment and Illumina sequencing were used to sequence full genomes from 507 norovirus-positive stool samples with reverse transcription-real-time PCR cycle threshold (CT) values of 10 to 43. Sequencing on an Illumina MiSeq system in batches of 48 generated, on average, 81% on-target reads per sample and 100% genome coverage with >12,000-fold read depth. Samples included genotypes GI.1, GI.2, GI.3, GI.6, GI.7, GII.1, GII.2, GII.3, GII.4, GII.5, GII.6, GII.7, GII.13, GII.14, and GII.17. When outliers were accounted for, we generated >80% genome coverage for all positive samples, regardless of CT values. A total of 164 samples were tested in parallel with conventional PCR genotyping of the capsid shell domain; 164/164 samples were successfully sequenced, compared to 158/164 samples that were amplified by PCR. Four of the samples that failed capsid PCR analysis had low titers, which suggests that target enrichment is more sensitive than gel-based PCR. Two samples failed PCR due to primer mismatches; target enrichment uses multiple baits targeting each position, thus accommodating sequence heterogeneity among norovirus genomes.
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Fezes/virologia , Genoma Viral , Norovirus/isolamento & purificação , Hibridização de Ácido Nucleico/métodos , RNA Viral/genética , Análise de Sequência de DNA/métodos , Manejo de Espécimes/métodos , Infecções por Caliciviridae/virologia , Humanos , Masculino , Norovirus/genéticaRESUMO
VZV is a ubiquitous member of the Herpesviridae family that causes varicella (chicken pox) and herpes zoster (shingles). Both manifestations can cause great morbidity and mortality and are therefore of significant economic burden. The introduction of varicella vaccination as part of childhood immunization programs has resulted in a remarkable decline in varicella incidence, and associated hospitalizations and deaths, particularly in the USA. The vaccine preparation, vOka, is a live attenuated virus produced by serial passage of a wild-type clinical isolate termed pOka in human and guinea pig cell lines. Although vOka is clinically attenuated, it can cause mild varicella, establish latency, and reactivate to cause herpes zoster. Sequence analysis has shown that vOka differs from pOka by at least 42 loci; however, not all genomes possess the novel vOka change at all positions, creating a heterogeneous population of genetically distinct haplotypes. This, together with the extreme cell-associated nature of VZV replication in cell culture and the lack of an animal model, in which the complete VZV life cycle can be replicated, has limited studies into the molecular basis for vOka attenuation. Comparative studies of vOka with pOka replication in T cells, dorsal root ganglia, and skin indicate that attenuation likely involves multiple mutations within ORF 62 and several other genes. This article presents an overview of the clinical aspects of the vaccine and current progress on understanding the molecular mechanisms that account for the clinical phenotype of reduced virulence.
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Vacina contra Varicela/efeitos adversos , Vacina contra Varicela/genética , Animais , Linhagem Celular , Varicela/virologia , Vacina contra Varicela/imunologia , DNA Viral/química , DNA Viral/genética , Cobaias , Herpes Zoster/virologia , Humanos , Mutação Puntual , Análise de Sequência de DNA , Inoculações Seriadas , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Ativação Viral , Cultura de Vírus , Latência ViralAssuntos
Agamaglobulinemia/complicações , Doenças Genéticas Ligadas ao Cromossomo X/complicações , Infecções por Picornaviridae/diagnóstico , Infecções por Picornaviridae/etiologia , Tirosina Quinase da Agamaglobulinemia/genética , Agamaglobulinemia/diagnóstico , Agamaglobulinemia/genética , Agamaglobulinemia/imunologia , Biomarcadores , Pré-Escolar , Doença Crônica , Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Humanos , Imunofenotipagem , Kobuvirus/imunologia , Masculino , Mutação , Infecções por Picornaviridae/tratamento farmacológico , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Introduction: Studying antibody dynamics following re-exposure to infection and/or vaccination is crucial for a better understanding of fundamental immunological processes, vaccine development, and health policy research. Methods: We adopted a nonlinear mixed modeling approach based on ordinary differential equations (ODE) to characterize varicella-zoster virus specific antibody dynamics during and after clinical herpes zoster. Our ODEs models convert underlying immunological processes into mathematical formulations, allowing for testable data analysis. In order to cope with inter- and intra-individual variability, mixed models include population-averaged parameters (fixed effects) and individual-specific parameters (random effects). We explored the use of various ODE-based nonlinear mixed models to describe longitudinally collected markers of immunological response in 61 herpes zoster patients. Results: Starting from a general formulation of such models, we study different plausible processes underlying observed antibody titer concentrations over time, including various individual-specific parameters. Among the converged models, the best fitting and most parsimonious model implies that once Varicella-zoster virus (VZV) reactivation is clinically apparent (i.e., Herpes-zoster (HZ) can be diagnosed), short-living and long-living antibody secreting cells (SASC and LASC, respectively) will not expand anymore. Additionally, we investigated the relationship between age and viral load on SASC using a covariate model to gain a deeper understanding of the population's characteristics. Conclusion: The results of this study provide crucial and unique insights that can aid in improving our understanding of VZV antibody dynamics and in making more accurate projections regarding the potential impact of vaccines.
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Herpes Zoster , Herpesvirus Humano 3 , Humanos , Viremia , Anticorpos Antivirais , Células Produtoras de AnticorposRESUMO
Metagenomic next-generation sequencing (mNGS) is an untargeted technique capable of detecting all microbial nucleic acid within a sample. This protocol outlines our wet laboratory method for mNGS of cerebrospinal fluid (CSF) specimens and tissues from sterile sites. We use this method routinely in our clinical service, processing 178 specimens over the past 2.5 years in a laboratory that adheres to ISO:15189 standards. We have successfully used this protocol to diagnose multiple cases of encephalitis and hepatitis.
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BACKGROUND: Varicella zoster virus infection (VZV) is widespread and clinically important as the cause of varicella pneumonitis and meningoencephalitis (a complication of primary infection/zoster) and post-herpetic neuralgia (a complication of zoster/secondary infection). The use of live-attenuated varicella vaccine to reduce the burden of these diseases has been established in many countries for a number of years. SOURCES OF DATA: Original papers and review articles including guidelines and recommendations by the American Academy of Paediatrics Committee on Infectious Diseases, the Advisory Committee on Immunization Practices and EuroSurveillance. AREAS OF AGREEMENT: Immunoassay of VZV IgG by enzyme immunosorbent assay is used as a surrogate marker for previous primary infection or successful immunization. Patients who have had natural primary infection do not require vaccination against varicella. Live VZV vaccines are safe and effective at protecting against disease caused by VZV. To ensure long-term protection, a two-dose immunization regime is strongly recommended, due to significant waning of protection following a single dose. Universal two-dose immunization has been shown to be cost-effective in Western temperate countries. In many countries, routine vaccination of children is recommended but, due to cost, often not provided by universal programmes. Cost-effectiveness of a universal programme will be determined by the baseline rate of severe varicella disease. AREAS OF CONTROVERSY: No international consensus exists: measurement of VZV immunity or cost-effectiveness of introducing VZV vaccination to a country. Decisive factors will include the pre-vaccination burden of VZV-associated disease.
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Vacina contra Varicela/administração & dosagem , Varicela/prevenção & controle , Varicela/imunologia , Vacina contra Varicela/efeitos adversos , Vacina contra Varicela/imunologia , Criança , Serviços de Saúde da Criança/métodos , Pré-Escolar , Saúde Global , Humanos , Lactente , Guias de Prática Clínica como Assunto , Resultado do Tratamento , Vacinação/métodos , Vacinas Atenuadas/imunologiaRESUMO
Purpose: Descemet's membrane endothelial keratoplasty (DMEK) is becoming the gold standard to treat corneal endothelial dysfunctions worldwide. Compared with conventional penetrating keratoplasty, infectious complications after DMEK are ill defined. We describe the clinical picture of 2 DMEK recipients, operated on the same day and in the same clinic, who developed atypical herpes simplex virus type 1 (HSV-1) infection in the transplant recipient eye within days post-DMEK. Because recipients received cornea tissue from 2 different donors prepared by the same eye bank, the likelihood of a common HSV-1 source was determined. Design: Case series. Participants: Two DMEK recipients who developed atypical intraocular HSV-1 disease shortly after surgery and surplus cornea specimens of 6 donors. Methods: Surplus cornea donor (pre-DMEK cornea remnants and conditioned cornea storage and transport media) and recipient samples (post-DMEK aqueous humor) were assayed for HSV-1 DNA and infectious virus by real-time polymerase chain reaction (RT-PCR) and cell culture, respectively. Target-enriched whole viral genome sequencing was performed on HSV-1 DNA-positive ocular specimens. Main Outcome Measures: Clinical picture of atypical intraocular HSV-1 infection post-DMEK and presence and homology of HSV-1 genomes between ocular specimens of DMEK donors and recipients. Results: Herpes simplex virus type 1 DNA was detected in aqueous humor and donor cornea specimens of both DMEK cases, but not in the cornea remnants of 6 randomly selected donors processed by the same eye bank. Infectious HSV-1 was isolated from the cornea remnant and corresponding culture medium of 1 cornea donor. Notably, whole-genome sequencing of virus DNA-positive specimens demonstrated exceptionally high genetic similarity between HSV-1 strains in recipient and donor specimens of both DMEK cases. Conclusions: Data indicate cross-contamination of cornea grafts during DMEK preparation with subsequent graft-to-host HSV-1 transmission that caused atypical sight-threatening herpetic eye disease shortly after DMEK. Ophthalmologists should be aware that HSV-1 transmission by DMEK is possible and can lead to atypical ocular disease, a condition that can easily be prevented by taking appropriate technical and clinical measures at both eye bank and surgical levels.
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OBJECTIVES: The objective of this trial is to assess whether early antiviral therapy in outpatients with COVID-19 with either favipiravir plus lopinavir/ritonavir, lopinavir/ritonavir alone, or favipiravir alone, is associated with a decrease in viral load of SARS-CoV-2 compared with placebo. TRIAL DESIGN: FLARE is a phase IIA randomised, double-blind, 2x2 factorial placebo-controlled, interventional trial. PARTICIPANTS: This trial is being conducted in the United Kingdom, with Royal Free Hospital, London as the lead site. Participants are non-hospitalised adults with highly suspected COVID-19 within the first 5 days of symptom onset, or who have tested positive with SARS-CoV-2 causing COVID-19 within the first 7 days of symptom onset, or who are asymptomatic but tested positive for SARS-CoV-2 for the first time within the last 48 hours. Inclusion criteria are as follows: 1. Any adult with the following: Symptoms compatible with COVID-19 disease (Fever >37.8°C on at least one occasion AND either cough and/ or anosmia) within the first 5 days of symptom onset (date/time of enrolment must be within the first 5 days of symptom onset) OR ANY symptoms compatible with COVID-19 disease (may include, but are not limited to fever, cough, shortness of breath, malaise, myalgia, headache, coryza) and tested positive for SARS-CoV-2 within the first 7 days of symptom onset) (date/time of enrolment must be within the first 7 days of symptom onset) OR no symptoms but tested positive for SARS-CoV-2 within the last 48 hours (date/time of test must be within 48 hours of enrolment) 2. Male or female aged 18 years to 70 years old inclusive at screening 3. Willing and able to take daily saliva samples 4. Able to provide full informed consent and willing to comply with trial-related procedures Exclusion criteria are as follows: 1. Known hypersensitivity to any of the active ingredients or excipients in favipiravir and matched placebo, and in lopinavir/ritonavir and matched placebo (See Appendix 2) 2. Chronic liver disease at screening (known cirrhosis of any aetiology, chronic hepatitis (e.g. autoimmune, viral, steatohepatitis), cholangitis or any known elevation of liver aminotransferases with AST or ALT > 3 X ULN)* 3. Chronic kidney disease (stage 3 or beyond) at screening: eGFR < 60 ml/min/1.73m2 * 4. HIV infection, if untreated, detectable viral load or on protease inhibitor therapy 5. Any clinical condition which the investigator considers would make the participant unsuitable for the trial 6. Concomitant medications known to interact with favipiravir and matched placebo, and with lopinavir/ritonavir and matched placebo, and carry risk of toxicity for the participant 7. Current severe illness requiring hospitalisation 8. Pregnancy and/ or breastfeeding 9. Eligible female participants of childbearing potential and male participants with a partner of childbearing potential not willing to use highly effective contraceptive measures during the trial and within the time point specified following last trial treatment dose. 10. Participants enrolled in any other interventional drug or vaccine trial (co-enrolment in observational studies is acceptable) 11. Participants who have received the COVID-19 vaccine *Considering the importance of early treatment of COVID-19 to impact viral load, the absence of known chronic liver/ kidney disease will be confirmed verbally by the participant during pre-screening and Screening/Baseline visit. Safety blood samples will be collected at Screening/Baseline visit (Day 1) and test results will be examined as soon as they become available and within 24 hours. INTERVENTION AND COMPARATOR: Participants will be randomised 1:1:1:1 using a concealed online minimisation process into one of the following four arms: Arm 1: Favipiravir + Lopinavir/ritonavir Oral favipiravir at 1800mg twice daily on Day 1, followed by 400mg four (4) times daily from Day 2 to Day 7 PLUS lopinavir/ritonavir at 400mg/100mg twice daily on Day 1, followed by 200mg/50mg four (4) times daily from Day 2 to Day 7. Arm 2: Favipiravir + Lopinavir/ritonavir placebo Oral favipiravir at 1800mg twice daily on Day 1, followed by 400mg four (4) times daily from Day 2 to Day 7 PLUS lopinavir/ritonavir matched placebo at 400mg/100mg twice daily on Day 1, followed by 200mg/50mg four (4) times daily from Day 2 to Day 7. Arm 3: Favipiravir placebo + Lopinavir/ritonavir Oral favipiravir matched placebo at 1800mg twice daily on Day 1, followed by 400mg four (4) times daily from Day 2 to Day 7 PLUS lopinavir/ritonavir at 400mg/100mg twice daily on Day 1, followed by 200mg/50mg four (4) times daily from Day 2 to Day 7. Arm 4: Favipiravir placebo + Lopinavir/ritonavir placebo Oral favipiravir matched placebo at 1800mg twice daily on Day 1, followed by 400mg four (4) times daily from Day 2 to Day 7 PLUS lopinavir/ritonavir matched placebo at 400mg/100mg twice daily on Day 1, followed by 200mg/50mg four (4) times daily from Day 2 to Day 7. MAIN OUTCOMES: The primary outcome is upper respiratory tract viral load at Day 5. SECONDARY OUTCOMES: Percentage of participants with undetectable upper respiratory tract viral load after 5 days of therapy Proportion of participants with undetectable stool viral load after 7 days of therapy Rate of decrease in upper respiratory tract viral load during 7 days of therapy Duration of fever following commencement of trial medications Proportion of participants with hepatotoxicity after 7 days of therapy Proportion of participants with other medication-related toxicity after 7 days of therapy and 14 days post-randomisation Proportion of participants admitted to hospital with COVID-19 related illness Proportion of participants admitted to ICU with COVID-19 related illness Proportion of participants who have died with COVID-19 related illness Pharmacokinetic and pharmacodynamic analysis of favipiravir Exploratory: Proportion of participants with deleterious or resistance-conferring mutations in SARS-CoV-2 RANDOMISATION: Participants will be randomised 1:1:1:1 using a concealed online minimisation process, with the following factors: trial site, age (≤ 55 vs > 55 years old), gender, obesity (BMI <30 vs ≥30), symptomatic or asymptomatic, current smoking status (Yes = current smoker, No = ex-smoker, never smoker), ethnicity (Caucasian, other) and presence or absence of comorbidity (defined as diabetes, hypertension, ischaemic heart disease (including previous myocardial infarction), other heart disease (arrhythmia and valvular heart disease), asthma, COPD, other chronic respiratory disease). BLINDING (MASKING): Participants and investigators will both be blinded to treatment allocation (double-blind). NUMBERS TO BE RANDOMISED (SAMPLE SIZE): 240 participants, 60 in each arm. TRIAL STATUS: Protocol version 4.0 dated 7th January 2021. Date of first enrolment: October 2020. Recruitment is ongoing, with anticipated finish date of 31st March 2021. TRIAL REGISTRATION: The FLARE trial is registered with Clinicaltrials.gov, trial identifying number NCT04499677 , date of registration 4th August 2020. FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.
Assuntos
Amidas/uso terapêutico , Antivirais/uso terapêutico , Tratamento Farmacológico da COVID-19 , Lopinavir/uso terapêutico , Pirazinas/uso terapêutico , Ritonavir/uso terapêutico , Carga Viral , Assistência Ambulatorial , Ensaios Clínicos Fase II como Assunto , Método Duplo-Cego , Combinação de Medicamentos , Quimioterapia Combinada , Intervenção Médica Precoce , Humanos , Ensaios Clínicos Controlados Aleatórios como Assunto , SARS-CoV-2 , Reino UnidoRESUMO
Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) viral loads change rapidly following symptom onset, so to assess antivirals it is important to understand the natural history and patient factors influencing this. We undertook an individual patient-level meta-analysis of SARS-CoV-2 viral dynamics in humans to describe viral dynamics and estimate the effects of antivirals used to date. This systematic review identified case reports, case series, and clinical trial data from publications between January 1, 2020, and May 31, 2020, following Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. A multivariable Cox proportional hazards (Cox-PH) regression model of time to viral clearance was fitted to respiratory and stool samples. A simplified four parameter nonlinear mixed-effects (NLME) model was fitted to viral load trajectories in all sampling sites and covariate modeling of respiratory viral dynamics was performed to quantify time-dependent drug effects. Patient-level data from 645 individuals (age 1 month to 100 years) with 6,316 viral loads were extracted. Model-based simulations of viral load trajectories in samples from the upper and lower respiratory tract, stool, blood, urine, ocular secretions, and breast milk were generated. Cox-PH modeling showed longer time to viral clearance in older patients, men, and those with more severe disease. Remdesivir was associated with faster viral clearance (adjusted hazard ratio (AHR) = 9.19, P < 0.001), as well as interferon, particularly when combined with ribavirin (AHR = 2.2, P = 0.015; AHR = 6.04, P = 0.006). Combination therapy should be further investigated. A viral dynamic dataset and NLME model for designing and analyzing antiviral trials has been established.
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Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , COVID-19/virologia , Carga Viral/efeitos dos fármacos , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Adulto , Alanina/análogos & derivados , Alanina/farmacologia , Ensaios Clínicos como Assunto , Quimioterapia Combinada , Feminino , Humanos , Interferons/farmacologia , Masculino , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , SARS-CoV-2/patogenicidade , Eliminação de Partículas Virais/efeitos dos fármacosRESUMO
Metagenomic high-throughput sequencing (mHTS) is a hypothesis-free, universal pathogen detection technique for determination of the DNA/RNA sequences in a variety of sample types and infectious syndromes. mHTS is still in its early stages of translating into clinical application. To support the development, implementation and standardization of mHTS procedures for virus diagnostics, the European Society for Clinical Virology (ESCV) Network on Next-Generation Sequencing (ENNGS) has been established. The aim of ENNGS is to bring together professionals involved in mHTS for viral diagnostics to share methodologies and experiences, and to develop application recommendations. This manuscript aims to provide practical recommendations for the wet lab procedures necessary for implementation of mHTS for virus diagnostics and to give recommendations for development and validation of laboratory methods, including mHTS quality assurance, control and quality assessment protocols.
Assuntos
Metagenômica , Vírus , Sequenciamento de Nucleotídeos em Larga Escala , Vírus/genéticaRESUMO
Protease inhibitors (PIs) are the second- and last-line therapy for the majority of HIV-infected patients worldwide. Only around 20% of individuals who fail PI regimens develop major resistance mutations in protease. We sought to explore the role of mutations in gag-pro genotypic and phenotypic changes in viruses from six Nigerian patients who failed PI-based regimens without known drug resistance-associated protease mutations in order to identify novel determinants of PI resistance. Target enrichment and next-generation sequencing (NGS) with the Illumina MiSeq system were followed by haplotype reconstruction. Full-length Gag-protease gene regions were amplified from baseline (pre-PI) and virologic failure (VF) samples, sequenced, and used to construct gag-pro-pseudotyped viruses. Phylogenetic analysis was performed using maximum-likelihood methods. Susceptibility to lopinavir (LPV) and darunavir (DRV) was measured using a single-cycle replication assay. Western blotting was used to analyze Gag cleavage. In one of six participants (subtype CRF02_AG), we found 4-fold-lower LPV susceptibility in viral clones during failure of second-line treatment. A combination of four mutations (S126del, H127del, T122A, and G123E) in the p17 matrix of baseline virus generated a similar 4-fold decrease in susceptibility to LPV but not darunavir. These four amino acid changes were also able to confer LPV resistance to a subtype B Gag-protease backbone. Western blotting demonstrated significant Gag cleavage differences between sensitive and resistant isolates in the presence of drug. Resistant viruses had around 2-fold-lower infectivity than sensitive clones in the absence of drug. NGS combined with haplotype reconstruction revealed that resistant, less fit clones emerged from a minority population at baseline and thereafter persisted alongside sensitive fitter viruses. We used a multipronged genotypic and phenotypic approach to document emergence and temporal dynamics of a novel protease inhibitor resistance signature in HIV-1 matrix, revealing the interplay between Gag-associated resistance and fitness.
Assuntos
Farmacorresistência Viral , Antígenos HIV/metabolismo , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/metabolismo , Inibidores de Proteases/farmacologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Substituição de Aminoácidos , Relação Dose-Resposta a Droga , Genoma Viral , Genótipo , Antígenos HIV/genética , Infecções por HIV/tratamento farmacológico , HIV-1/genética , Humanos , Testes de Sensibilidade Microbiana , Mutação , Fenótipo , Filogenia , Deleção de Sequência , Carga Viral , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genéticaRESUMO
Human herpesviruses (HHV) cause a variety of clinically relevant conditions upon primary infection of typically young and immunocompetent hosts. Both primary infection and reactivation after latency can lead to more severe disease, such as encephalitis, congenital defects and cancer. Infections with HHV are also associated with cardiovascular and neurodegenerative disease. However, most of the associations are based on retrospective case-control analyses and well-powered prospective cohort studies are needed for assessing temporality and causality. To enable comprehensive investigations of HHV-related disease etiology in large prospective population-based cohort studies, we developed HHV Multiplex Serology. This methodology represents a low-cost, high-throughput technology that allows simultaneous measurement of specific antibodies against five HHV species: Herpes simplex viruses 1 and 2, Varicella zoster virus, Epstein-Barr virus, and Cytomegalovirus. The newly developed HHV species-specific ('Monoplex') assays were validated against established gold-standard reference assays. The specificity and sensitivity of the HHV species-specific Monoplex Serology assays ranged from 92.3% to 100.0% (median 97.4%) and 91.8% to 98.7% (median 96.6%), respectively. Concordance with reference assays was very high with kappa values ranging from 0.86 to 0.96 (median kappa 0.93). Multiplexing the Monoplex Serology assays resulted in no loss of performance and allows simultaneous detection of antibodies against the 5 HHV species in a high-throughput manner.