An emm-type specific qPCR to track bacterial load during experimental human Streptococcus pyogenes pharyngitis
Abstract 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 relia...
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doaj-a389c0e351284e819b89b2d71cf959912021-05-23T11:08:34ZengBMCBMC Infectious Diseases1471-23342021-05-012111910.1186/s12879-021-06173-wAn emm-type specific qPCR to track bacterial load during experimental human Streptococcus pyogenes pharyngitisLoraine V. Fabri0Kristy I. Azzopardi1Joshua Osowicki2Hannah R. Frost3Pierre R. Smeesters4Andrew C. Steer5Tropical Diseases Research Group, Murdoch Children’s Research InstituteTropical Diseases Research Group, Murdoch Children’s Research InstituteTropical Diseases Research Group, Murdoch Children’s Research InstituteTropical Diseases Research Group, Murdoch Children’s Research InstituteTropical Diseases Research Group, Murdoch Children’s Research InstituteTropical Diseases Research Group, Murdoch Children’s Research InstituteAbstract 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.https://doi.org/10.1186/s12879-021-06173-wPharyngitisHuman challenge studyqPCRNucleic acid extractionStreptococcus pyogenes |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Loraine V. Fabri Kristy I. Azzopardi Joshua Osowicki Hannah R. Frost Pierre R. Smeesters Andrew C. Steer |
spellingShingle |
Loraine V. Fabri Kristy I. Azzopardi Joshua Osowicki Hannah R. Frost Pierre R. Smeesters Andrew C. Steer An emm-type specific qPCR to track bacterial load during experimental human Streptococcus pyogenes pharyngitis BMC Infectious Diseases Pharyngitis Human challenge study qPCR Nucleic acid extraction Streptococcus pyogenes |
author_facet |
Loraine V. Fabri Kristy I. Azzopardi Joshua Osowicki Hannah R. Frost Pierre R. Smeesters Andrew C. Steer |
author_sort |
Loraine V. Fabri |
title |
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_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_sort |
emm-type specific qpcr to track bacterial load during experimental human streptococcus pyogenes pharyngitis |
publisher |
BMC |
series |
BMC Infectious Diseases |
issn |
1471-2334 |
publishDate |
2021-05-01 |
description |
Abstract 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. |
topic |
Pharyngitis Human challenge study qPCR Nucleic acid extraction Streptococcus pyogenes |
url |
https://doi.org/10.1186/s12879-021-06173-w |
work_keys_str_mv |
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