It Takes Two to Tango: Combining Conventional Culture with Molecular Diagnostics Enhances Accuracy of Streptococcus pneumoniae Detection and Pneumococcal Serogroup/Serotype determination in Carriage

Abstract

ABSTRACTBackgroundThe specificity of molecular methods for the detection of Streptococcus pneumoniae carriage is under debate. We propose a procedure that increases the accuracy of molecular detection of live pneumococci in polymicrobial respiratory samples.MethodsCulture and qPCR methods were applied to detect S. pneumoniae and pneumococcal serotypes in 1549 nasopharyngeal samples collected in the Netherlands (n=972) and England (n=577) from 946 toddlers and 603 adults, and in paired oropharyngeal samples collected exclusively from 319 Dutch adults. Samples with no live pneumococci isolated at primary diagnostic culture yet generating pneumococcus-specific signal in qPCRs were re-examined with a second, qPCR-guided culture. Optimal Cq cut-offs for positivity in qPCRs were determined via receiver operating characteristic (ROC) curve analysis using isolation of live pneumococci from the primary and qPCR-guided cultures as reference.ResultsDetection of S. pneumoniae and pneumococcal serotypes with qPCRs in cultured (culture-enriched) nasopharyngeal samples exhibited near-perfect agreement with conventional culture (Cohen’s kappa: 0.95). Molecular methods also displayed increased sensitivity of detection for multiple serotype carriage. Among paired samples from adults, the sensitivity of S. pneumoniae detection in primary nasopharyngeal plus oropharyngeal cultures was significantly lower compared with molecular detection in both culture-enriched samples together (p<0.0001) and also in culture-enriched oropharyngeal samples alone (p<0.05).ConclusionsThe sensitivity of S. pneumoniae carriage surveillance can be greatly improved by complementing conventional culture with qPCR and vice versa. The specificity of molecular methods for the detection of live pneumococci can be enhanced by incorporating statistical procedures based on ROC curve analysis. The procedure we propose improves detection of S. pneumoniae carriage in adults in particular and enhances specificity of serotype carriage detection.