28 research outputs found
Internal validation of STRmix⢠â A multi laboratory response to PCAST
We report a large compilation of the internal validations of the probabilistic genotyping software STRmixâ˘. Thirty one laboratories contributed data resulting in 2825 mixtures comprising three to six donors and a wide range of multiplex, equipment, mixture proportions and templates. Previously reported trends in the LR were confirmed including less discriminatory LRs occurring both for donors and non-donors at low template (for the donor in question) and at high contributor number. We were unable to isolate an effect of allelic sharing. Any apparent effect appears to be largely confounded with increased contributor number
Development of an amplicon-based sequencing approach in response to the global emergence of mpox
The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.This publication was made possible by
CTSA Grant Number UL1 TR001863 from the
National Center for Advancing Translational
Science (NCATS), a component of the National
Institutes of Health (NIH) awarded to CBFV. INSA
was partially funded by the HERA project (Grant/
2021/PHF/23776) supported by the European
Commission through the European Centre for
Disease Control (to VB).info:eu-repo/semantics/publishedVersio
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Evaluation of C-Reactive Protein Point of Care Testing, and Associated Research Challenges, to Improve the Quality of Antibiotic Prescribing in the Community in Northern Thailand
Background
Antimicrobial resistance (AMR) is a global health challenge, disproportionally affecting low- and middle-income countries (LMICs). Antibiotic use is a key driver of AMR, yet data on their use in LMICs, and in the community where most antibiotics are consumed, are comparatively scarce. C-reactive protein (CRP) is used in some high-income countries to guide antibiotic prescription for community respiratory tract infections (RTIs). Little evaluation has taken place in LMICs.
Methods
A two year retrospective review of antibiotic use in primary care units (PCUs) across a northern Thai district was conducted. A RCT was carried out in ten Thai and Myanmar primary care clinics evaluating CRP testing to optimise antibiotic use in patients with a history of fever. CRP testing was reviewed in a subgroup with sore throats to determine its ability to identify Group A Streptococcus (GAS) infection.
Results
Few participants took antibiotics before attending PCUs. RTIs were the commonest infection presentation. Antibiotics appear to be overused in some self-limiting infections. Particularly high proportions were prescribed for sore throats, where the correlations between CRP levels and GAS were poor.
In the trial context, CRP testing significantly reduced the proportion of antibiotics prescribed in Thailand and Myanmar, although a non-significant reduction was seen when Thai participants were considered separately. CRP testing improved antibiotic targeting with respect to high CRP levels. Clinical outcomes and health-seeking behaviour during the study period were unaffected. Most healthcare workers and participants supported CRP testing.
Conclusions
While not uncommon, antibiotic overuse in routine primary care in Thailand was of lower magnitude than anticipated. CRP testing is unlikely to contribute to further large scale reduction in antibiotic prescribing but could better target their use. Identifying who needs antibiotics for sore throats remains challenging. Antimicrobial stewardship interventions in PCUs could have a large impact on prescribing but need to be multi-faceted in nature
Development of an amplicon-based sequencing approach in response to the global emergence of mpox.
The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response
Pregnancy and neonatal outcomes of COVID -19: coreporting of common outcomes from PAN-COVID and AAP-SONPM registries
Objective
Few large cohort studies have reported data on maternal, fetal, perinatal and neonatal outcomes associated with severe acute respiratory syndrome coronavirus 2 (SARSâCoVâ2) infection in pregnancy. We report the outcome of infected pregnancies from a collaboration formed early during the pandemic between the investigators of two registries, the UK and Global Pregnancy and Neonatal outcomes in COVIDâ19 (PANâCOVID) study and the American Academy of Pediatrics (AAP) Section on NeonatalâPerinatal Medicine (SONPM) National Perinatal COVIDâ19 Registry.
Methods
This was an analysis of data from the PANâCOVID registry (1 January to 25 July 2020), which includes pregnancies with suspected or confirmed maternal SARSâCoVâ2 infection at any stage in pregnancy, and the AAPâSONPM National Perinatal COVIDâ19 registry (4 April to 8 August 2020), which includes pregnancies with positive maternal testing for SARSâCoVâ2 from 14âdays before delivery to 3âdays after delivery. The registries collected data on maternal, fetal, perinatal and neonatal outcomes. The PANâCOVID results are presented overall for pregnancies with suspected or confirmed SARSâCoVâ2 infection and separately in those with confirmed infection.
Results
We report on 4005 pregnant women with suspected or confirmed SARSâCoVâ2 infection (1606 from PANâCOVID and 2399 from AAPâSONPM). For obstetric outcomes, in PANâCOVID overall and in those with confirmed infection in PANâCOVID and AAPâSONPM, respectively, maternal death occurred in 0.5%, 0.5% and 0.2% of cases, early neonatal death in 0.2%, 0.3% and 0.3% of cases and stillbirth in 0.5%, 0.6% and 0.4% of cases. Delivery was preterm (<â37âweeks' gestation) in 12.0% of all women in PANâCOVID, in 16.1% of those women with confirmed infection in PANâCOVID and in 15.7% of women in AAPâSONPM. Extreme preterm delivery (<â27âweeks' gestation) occurred in 0.5% of cases in PANâCOVID and 0.3% in AAPâSONPM. Neonatal SARSâCoVâ2 infection was reported in 0.9% of all deliveries in PANâCOVID overall, in 2.0% in those with confirmed infection in PANâCOVID and in 1.8% in AAPâSONPM; the proportions of neonates tested were 9.5%, 20.7% and 87.2%, respectively. The rates of a smallâforâgestationalâage (SGA) neonate were 8.2% in PANâCOVID overall, 9.7% in those with confirmed infection and 9.6% in AAPâSONPM. Mean gestationalâageâadjusted birthâweight Zâscores were â0.03 in PANâCOVID and â0.18 in AAPâSONPM.
Conclusions
The findings from the UK and USA registries of pregnancies with SARSâCoVâ2 infection were remarkably concordant. Preterm delivery affected a higher proportion of women than expected based on historical and contemporaneous national data. The proportions of pregnancies affected by stillbirth, a SGA infant or early neonatal death were comparable to those in historical and contemporaneous UK and USA data. Although maternal death was uncommon, the rate was higher than expected based on UK and USA population data, which is likely explained by underascertainment of women affected by milder or asymptomatic infection in pregnancy in the PANâCOVID study, although not in the AAPâSONPM study. The data presented support strong guidance for enhanced precautions to prevent SARSâCoVâ2 infection in pregnancy, particularly in the context of increased risks of preterm delivery and maternal mortality, and for priority vaccination of pregnant women and women planning pregnancy. Copyright Š 2021 ISUOG. Published by John Wiley & Sons Ltd
Source data for samples included in this study.
Listed are institute, specimen code, sample type, Ct value, sequencing platform, percent genome coverage at 10Ă, and accession numbers. (XLSX)</p
Geographical distribution of public health laboratories that implemented the human monkeypox virus primer scheme with their established amplicon-based sequencing workflows.
Public health laboratories contributing data to this study include: CDPH, CEVS, DPHL, FDH, IBL, JHMI, LACPHL, MASPHL, MDH, NHS Lothian, INSA, and RISHL. The base layer of the map has been sourced from Carto (https://docs.carto.com/development-tools/carto-for-react/guides/basemaps) under an open source CC-BY license (https://github.com/CartoDB/basemap-styles/blob/master/LICENSE.md). CDPH, Connecticut Department of Public Health; CEVS, Centro Estadual de Vigilância em Saúde; DPHL, Delaware Public Health Lab; FDH, Florida Department of Health; IBL, Idaho Bureau of Laboratories; INSA, National Institute of Health Dr. Ricardo Jorge; JHMI, Johns Hopkins Medical Institutions; LACPHL, Los Angeles County Public Health Lab; MASPHL, Massachusetts State Public Health Laboratory; MDH, Minnesota Department of Health; NHS Lothian, National Health Service Lothian; RISHL, Rhode Island State Health Laboratory.</p
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The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.</div