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

BackgroundThe 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.

Longitudinal dynamics ofStreptococcus pneumoniaecarriage and SARS-CoV-2 infection in households with children

“…Samples were considered positive by qPCR for piaB, lytA and Egene when Cqs were below 40. For qPCR-based serotyping results concordance with piaB/lytA was required [26]. Intraclass correlations (ICCs (3,1)) were calculated in a two-way mixed-effects model with consistency relationship (piaB Cq = lytA Cq + systematic error) and of single measurement type [27], and Bland-Altman plots [28] were used to assess agreement between qPCR measurements using the "ïrr" and "blandr" R packages, respectively.…”

“…Moreover, the overall study size limited our ability to adjust for potential confounding factors in Cox regression analysis. Pneumococcal carriage episodes may have been missed due to the omission of a culture-enrichment procedure for pneumococcal detection [39].…”

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“…Cq thresholds for positivity were determined with receiver operating characteristic (ROC) curve analysis. The specificity of metA and ctrA qPCR assays were evaluated in Bland-Altman analysis and by calculating intraclass correlation coefficients, as previously described [6].…”

“…Samples were regarded as positive for a genogroup by qPCR when the Cq was lower than the cut-off value set for ctrA. The specificity of genogroup-specific qPCR assays was determined in Bland-Altman analyses with upper limit of agreement between metA and ctrA as a priori acceptable limit for genogroup-specific Cqs [6].…”