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An emm-type specific qPCR to track bacterial load during experimental human Streptococcus pyogenes pharyngitis

BACKGROUND: Streptococcus pyogenes causes a profound global burden of morbidity and mortality across its diverse clinical spectrum. To support a new controlled human infection (challenge) model seeking to accelerate S. pyogenes vaccine development, we aimed to develop an accurate and reliable molecu...

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Autores principales: Fabri, Loraine V., Azzopardi, Kristy I., Osowicki, Joshua, Frost, Hannah R., Smeesters, Pierre R., Steer, Andrew C.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8138111/
https://www.ncbi.nlm.nih.gov/pubmed/34020607
http://dx.doi.org/10.1186/s12879-021-06173-w
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author Fabri, Loraine V.
Azzopardi, Kristy I.
Osowicki, Joshua
Frost, Hannah R.
Smeesters, Pierre R.
Steer, Andrew C.
author_facet Fabri, Loraine V.
Azzopardi, Kristy I.
Osowicki, Joshua
Frost, Hannah R.
Smeesters, Pierre R.
Steer, Andrew C.
author_sort Fabri, Loraine V.
collection PubMed
description BACKGROUND: Streptococcus pyogenes causes a profound global burden of morbidity and mortality across its diverse clinical spectrum. To support a new controlled human infection (challenge) model seeking to accelerate S. pyogenes vaccine development, we aimed to develop an accurate and reliable molecular method for quantifying bacterial load from pharyngeal swabs collected during experimental human pharyngitis. METHODS: Combined sequential RNA+DNA extraction from throat swabs was compared to traditional separate RNA-only and DNA-only extractions. An emm-type specific qPCR was developed to detect the emm75 challenge strain. Results from the qPCR were compared to culture, using throat swab samples collected in a human challenge study. RESULTS: The qPCR was 100% specific for the emm75 challenge strain when tested against a panel of S. pyogenes emm-types and other respiratory pathogens. Combined RNA+ DNA extraction had similar yield to traditional separate extractions. The combined extraction method and emm75 qPCR had 98.8% sensitivity compared to culture for throat swabs collected from challenge study participants. CONCLUSIONS: We have developed a reliable molecular method for measuring S. pyogenes bacterial load from throat swabs collected in a controlled human infection model of S. pyogenes pharyngitis. TRIAL REGISTRATION: NCT03361163 on 4th December 2017. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12879-021-06173-w.
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spelling pubmed-81381112021-05-21 An emm-type specific qPCR to track bacterial load during experimental human Streptococcus pyogenes pharyngitis Fabri, Loraine V. Azzopardi, Kristy I. Osowicki, Joshua Frost, Hannah R. Smeesters, Pierre R. Steer, Andrew C. BMC Infect Dis Technical Advance BACKGROUND: Streptococcus pyogenes causes a profound global burden of morbidity and mortality across its diverse clinical spectrum. To support a new controlled human infection (challenge) model seeking to accelerate S. pyogenes vaccine development, we aimed to develop an accurate and reliable molecular method for quantifying bacterial load from pharyngeal swabs collected during experimental human pharyngitis. METHODS: Combined sequential RNA+DNA extraction from throat swabs was compared to traditional separate RNA-only and DNA-only extractions. An emm-type specific qPCR was developed to detect the emm75 challenge strain. Results from the qPCR were compared to culture, using throat swab samples collected in a human challenge study. RESULTS: The qPCR was 100% specific for the emm75 challenge strain when tested against a panel of S. pyogenes emm-types and other respiratory pathogens. Combined RNA+ DNA extraction had similar yield to traditional separate extractions. The combined extraction method and emm75 qPCR had 98.8% sensitivity compared to culture for throat swabs collected from challenge study participants. CONCLUSIONS: We have developed a reliable molecular method for measuring S. pyogenes bacterial load from throat swabs collected in a controlled human infection model of S. pyogenes pharyngitis. TRIAL REGISTRATION: NCT03361163 on 4th December 2017. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12879-021-06173-w. BioMed Central 2021-05-21 /pmc/articles/PMC8138111/ /pubmed/34020607 http://dx.doi.org/10.1186/s12879-021-06173-w Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Technical Advance
Fabri, Loraine V.
Azzopardi, Kristy I.
Osowicki, Joshua
Frost, Hannah R.
Smeesters, Pierre R.
Steer, Andrew C.
An emm-type specific qPCR to track bacterial load during experimental human Streptococcus pyogenes pharyngitis
title An emm-type specific qPCR to track bacterial load during experimental human Streptococcus pyogenes pharyngitis
title_full An emm-type specific qPCR to track bacterial load during experimental human Streptococcus pyogenes pharyngitis
title_fullStr An emm-type specific qPCR to track bacterial load during experimental human Streptococcus pyogenes pharyngitis
title_full_unstemmed An emm-type specific qPCR to track bacterial load during experimental human Streptococcus pyogenes pharyngitis
title_short An emm-type specific qPCR to track bacterial load during experimental human Streptococcus pyogenes pharyngitis
title_sort emm-type specific qpcr to track bacterial load during experimental human streptococcus pyogenes pharyngitis
topic Technical Advance
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8138111/
https://www.ncbi.nlm.nih.gov/pubmed/34020607
http://dx.doi.org/10.1186/s12879-021-06173-w
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