Background Molecular assays are important tools for pathogen detection but need to be periodically re-evaluated with the discovery of additional genetic diversity that may cause assays to exclude target taxa or include non-target taxa. A single well-developed assay can find broad application across research, clinical, and industrial settings. Pathogen prevalence within a population is estimated using such assays and accurate results are critical for formulating effective public health policies and guiding future research. A variety of assays for the detection of Staphylococcus aureus are currently available. The utility of commercial assays for research is limited, given proprietary signatures and lack of transparent validation. Results In silico testing of existing peer-reviewed assays show that most suffer from a lack of sensitivity and specificity. We found no assays that were specifically designed and validated for quantitative use. Here we present a qPCR assay, SaQuant, for the detection and quantification of S. aureus as might be collected on sampling swabs. Sensitivity and specificity of the assay was 95.6 and 99.9 %, respectively, with a limit of detection of between 3 and 5 genome equivalents and a limit of quantification of 8.27 genome equivalents. The presence of DNA from non-target species likely to be found in a swab sample, did not impact qualitative or quantitative abilities of the assay. Conclusions This assay has the potential to serve as a valuable tool for the accurate detection and quantification of S. aureus collected from human body sites in order to better understand the dynamics of prevalence and transmission in community settings.

Disease control relies on pathogen identification and understanding reservoirs. Staphylococcus aureus infection prevention is based upon decades of research on colonization and infection, but diminishing returns from mitigation efforts suggest significant knowledge gaps. Existing knowledge and mitigation protocols are founded upon culture-based detection, with almost no information about pathogen quantities. We employed a qPCR assay on samples from three body sites to characterize colonization more comprehensively than previous studies by describing both prevalence and pathogen quantity. We show a much higher overall prevalence (65.9%) than previously documented, with higher quantities and prevalence associated with the nares, non-Hispanic males (86.9%), and correlating with colonization in other body sites. These results suggest that research and clinical practices likely misclassify over half of colonized persons, limiting mitigation measures and their impact. This work begins the process of rebuilding foundational knowledge of S. aureus carriage with more accurate and wholistic approaches.

Aims To understand the impact of storage temperature on recovery of Staphylococcus aureus on sampling swabs. S. aureus is a common cause of skin and soft tissue infections, but also causes a variety of life-threatening diseases. With a large pool of asymptomatic carriers and transmission that can occur even through indirect contact, mitigation efforts have had limited success. Swab sampling, followed by culturing, is a cornerstone of epidemiological studies, however S. aureus viability on swabs stored at different temperatures has not been characterized. Methods and Results We determined survival rates on swabs stored at five different temperatures. Samples stored at-70°C had no decay over time while samples stored at higher temperatures showed an exponential decay in viability. Mortality rates were greatest for swabs stored at 37°C. Survival at intermediate temperatures (-20°C-20.5°C) did not differ significantly, however we observed more variation at higher temperatures. Conclusions To maximize recovery of S. aureus cells, samples should be stored at-70°C or processed for culturing without delay. Significance and Impact of Study Epidemiological studies of bacterial diseases are typically limited to determination of pathogen presence/absence, yet quantitative assessments of pathogen load and genetic diversity can provide insights into disease progression and severity, likelihood of transmission, and adaptive evolutionary potential. For studies of S. aureus where time or access to a microbiology laboratory may delay culturing, deep freezing or timely culturing will maximize the degree to which sampling results reflect source status.

Staphylococcus aureus exists as a pathogen and commensal. Individuals with asymptomatic carriage serve as a reservoir for transmission and are at increased risk of infecting themselves. In order to characterize the genomic diversity of S. aureus circulating in the community, we sequenced 166 genomes collected from individuals in Yuma, AZ.

Monitoring the emergence of new SARS-CoV-2 variants is important to detect potential risks of increased transmission or disease severity. We investigated the identification of SARS-CoV-2 variants from real-time reverse transcriptase polymerase chain reaction (RT-PCR) routine diagnostics data. Cycle threshold (Ct) values of positive samples were collected from April 2021 to January 2022 in the Northern Metropolitan Area of Barcelona (n = 15,254). Viral lineage identification from whole genome sequencing (WGS) was available for 4618 (30.3%) of these samples. Pairwise differences in the Ct values between gene targets (ΔCt) were analyzed for variants of concern or interest circulating in our area. A specific delay in the Ct of the N-gene compared to the RdRp-gene (ΔCtNR) was observed for Alpha, Delta, Eta and Omicron. Temporal differences in ΔCtNR correlated with the dynamics of viral replacement of Alpha by Delta and of Delta by Omicron according to WGS results. Using ΔCtNR, prediction of new variants of concern at early stages of circulation was achieved with high sensitivity and specificity (91.1% and 97.8% for Delta; 98.5% and 90.8% for Omicron). Thus, tracking population-wide trends in ΔCt values obtained from routine diagnostics testing in combination with WGS could be useful for real-time management and response to local epidemics.