Impact of Temporary Storage Conditions on the Viability of Streptococcus pneumoniae in Saliva

Allicock, Orchid M; York, Anna; Waghela, Pari; Yolda-Carr, Devyn; Weinberger, Daniel M; Wyllie, Anne L.

doi:10.1128/msphere.00331-22

Cited by 7 publications

(2 citation statements)

References 26 publications

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“…Saliva samples were collected from healthy volunteers and screened for the absence of pneumococcus using qPCR targeting piaB , as previously described. 16 Samples negative for both targets were considered negative for pneumococcus and were pooled together. A serotype 19A pneumococcus strain was serially diluted into the pooled saliva, from 5X10 7 CFU/ml to 5X10 1 CFU/mL, in duplicate (Supplementary Figure 1).…”

Section: Methodsmentioning

confidence: 99%

“…Overall, this leads to lower sample collection burden to study participants (minimizing potential testing aversion), and/or clinical personnel, which is critical particularly in longitudinal studies. Additionally, we have previously demonstrated that encapsulated pneumococci remain viable in raw, unsupplemented saliva with a stable bacterial load seen for 24 hours in the absence of cold-chain transport (i.e., ~19-30°C), 16 further alleviating collection and transport burden for studies conducted in remote or resource-limited settings.…”

Section: Introductionmentioning

confidence: 99%

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A low-cost culture- and DNA extraction-free method for the molecular detection of pneumococcal carriage in saliva

Peno,

Lin,

Hislop

et al. 2023

Preprint

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BackgroundMolecular methods have improved the sensitivity of detection of pneumococcal carriage in saliva. However, they typically require sample culture-enrichment and nucleic acid extraction, prior to performing the detection assay. These factors may limit scalability for extensive surveillance of pneumococcus, particularly in low-resource settings. In this study, we evaluated the performance of a DNA-extraction-free method for the detection of pneumococcus in saliva.MethodsWe developed a streamlined qPCR-based protocol for the detection of pneumococcus, omitting culture-enrichment and DNA extraction. Using saliva samples collected from children attending childcare centers (New Haven, CT, USA), we evaluated detection of pneumococcus using saliva lysates as compared to purified DNA extracted from culture-enriched aliquots of the paired samples using qPCR targeting the pneumococcalpiaBgene.ResultsOf 759 saliva samples tested from 92 children (median age 3.65 years; IQR (2.46-4.78), pneumococcus was detected in 358 (47.2%) saliva lysates prepared using the extraction-free protocol and in 369 (48.6%) DNA extracted from the culture-enriched samples. We observed a near-perfect agreement between the two protocols (Cohen’s kappa: 0.92; 95%CI: 0.90-0.95). While we also observed a high correlation between the qPCR CTvalues generated by the two methods (r=0.93,p<0.0001), the CTvalues generated from the extraction-free, saliva lysates were higher (lower concentration) than those obtained from DNA extracted from culture-enriched samples (ΔCT= 6.68,p<0.00001).ConclusionsFor pneumococcal carriage surveillance in children, our findings suggest that a DNA extraction-free approach may offer a cost-effective alternative to the resource-intensive culture-enrichment method. While, as expected, we observed higher qPCR CTvalues (lower bacterial load) in the absence of culture-enrichment, the overall rate of detection remained unaffected.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%