Background: Molecular 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. Here, we evaluated the performance of a DNA-extraction-free method for the detection of pneumococcus in saliva. Methods: We 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 the saliva lysates as compared to purified DNA extracted from culture-enriched aliquots of the paired samples using qPCR targeting the pneumococcal piaB gene. Results: Of 759 saliva samples tested from 92 children, 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 CT values generated by the two methods (r=0.93, p<0.0001), the CT values 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). Conclusions: For 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 CT values (lower bacterial load) in the absence of culture-enrichment, the overall rate of detection remained unaffected

DMW has received consulting fees from Pfizer, Merck, and GSK and is PI on research grants from Pfizer and Merck. ALW has received consulting and/or advisory board fees from Pfizer, Merck, Diasorin, PPS Health, Co-Diagnostics, and Global Diagnostic Systems for work unrelated to this project, and is Principal Investigator on research grants from Pfizer, Merck, NIH RADx UP and SalivaDirect, Inc. to Yale University and from NIH RADx, Balvi.io and Shield T3 to SalivaDirect, Inc.

This study supported by FAST Grant to AW and Merck. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The collection of saliva samples from children in childcare centers (New Haven, CT, USA) was approved by the Institutional Review Board of the Yale Human Research Protection Program (Protocol number: 200002839). Written informed consent was obtained from parent or guardian of every participating child. The collection of de-identified saliva samples from healthy volunteers for assay validation was approved by the Institutional Review Board of the Yale Human Research Protection Program (Protocol number: 2000029374).

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