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

Loraine V Fabri, Kristy I Azzopardi, Joshua Osowicki, Hannah R Frost, Pierre R Smeesters, Andrew C Steer, Loraine V Fabri, Kristy I Azzopardi, Joshua Osowicki, Hannah R Frost, Pierre R Smeesters, Andrew C Steer

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.

Keywords: Human challenge study; Nucleic acid extraction; Pharyngitis; Streptococcus pyogenes; qPCR.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Performance of the emm75 qPCR. qPCR targeting the emm75 or speB gene was performed on genomic DNA isolated from 10-fold dilutions of S. pyogenes M75. The speB qPCR was used to provide a benchmark for the performance of the emm75 qPCR, using FAM-conjugated probes for both reactions. Each point represents the mean and standard deviation (SD) of duplicate reactions
Fig. 2
Fig. 2
Optimisation of the method for isolation of DNA and RNA from throat swabs in eNat. a Pre-lysis versus No pre-lysis extractions using the combined RNA+DNA approach. eNats were spiked with S. pyogenes M75 at three concentrations and 200l aliquots (1.8102, 1.8104, 1.8106 total GE) underwent DNA extraction using a chemical Pre-lysis step () or No pre-lysis (). A larger 600l aliquot (3x vol) of eNat medium spiked with M75 (5.4104 total GE) was processed without pre-lysis (). Data shown in total GE to account for differences in input bacteria. Each point represents the mean and SD of two eNats tested in duplicate qPCRs. b eNats containing a range of M75 were used to test separate column extractions (RNA-only or DNA-only ) versus combined RNA+DNA isolation (RNA and DNA). The emm75 qPCR was used to assess DNA whilst RNA was converted to cDNA and tested in a SYBR Green qPCR for the housekeeping gene, gyrA. Each point represents the mean and SD of three eNats tested in duplicate qPCRs targeting the emm75 gene
Fig. 3
Fig. 3
Correlation between culture and qPCR results. The number of emm75 genome equivalents (GE), extrapolated from qPCR Ct values, was compared to plate growth scores derived by culture. The median log10 emm75 GE of each score is represented by the dotted line (---), whilst the width of the shape reflects the density of data at the respective values

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