Abstract
AbstractBackgroundMolecular 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.
Publisher
Cold Spring Harbor Laboratory