AbstractBackgroundStreptococcus pyogenescauses a profound global burden of morbidity and mortality across its diverse clinical spectrum. To support a new controlled human infection (‘challenge’) model seeking to accelerateS. pyogenesvaccine development, we aimed to develop an accurate and reliable molecular method for quantifying bacterial load from pharyngeal swabs collected during experimental human pharyngitis.MethodsCombined sequential RNA + DNA extraction from throat swabs was compared to traditional separate RNA-only and DNA-only extractions. Anemm-type specific qPCR was developed to detect theemm75 challenge strain. Results from the qPCR were compared to culture, using throat swab samples collected in a human challenge study.ResultsThe qPCR was 100% specific for theemm75 challenge strain when tested against a panel ofS. pyogenes emm-types and other respiratory pathogens. Combined RNA + DNA extraction had similar yield to traditional separate extractions. The combined extraction method andemm75 qPCR had 98.8% sensitivity compared to culture for throat swabs collected from challenge study participants.ConclusionsWe have developed a reliable molecular method for measuringS. pyogenesbacterial load from throat swabs collected in a controlled human infection model ofS. pyogenespharyngitis.Trial registrationNCT03361163on 4th December 2017.