It Takes Two to Tango: Combining Conventional Culture with Molecular Diagnostics 1 Enhances Accuracy of Streptococcus pneumoniae Detection 2 and Pneumococcal Serogroup/Serotype determination in Carriage 3 4

Background: The specificity of molecular methods for the detection of Streptococcus pneumoniae carriage is under debate. We propose a procedure that increases the accuracy of 41 molecular detection of live pneumococci in polymicrobial respiratory samples. 42 43 Methods: Culture and qPCR methods were applied to detect S. pneumoniae and 44 pneumococcal serotypes in 1549 nasopharyngeal samples collected in the Netherlands 45 (n=972) and England (n=577) from 946 toddlers and 603 adults, and in paired oropharyngeal 46 samples collected exclusively from 319 Dutch adults. Samples with no live pneumococci 47 isolated at primary diagnostic culture yet generating pneumococcus-specific signal in qPCRs 48 were re-examined with a second, qPCR-guided culture. Optimal C q cut-offs for positivity in 49 qPCRs were determined via receiver operating characteristic (ROC) curve analysis using 50 isolation of live pneumococci from the primary and qPCR-guided cultures as reference. 51 52 Results: Detection of S. pneumoniae and pneumococcal serotypes with qPCRs in cultured 53 (culture-enriched) nasopharyngeal samples exhibited near-perfect agreement with 54 conventional culture (Cohen’s kappa: 0.95). Molecular methods also displayed increased 55 sensitivity of detection for multiple serotype carriage. Among paired samples from adults, the 56 sensitivity of S. pneumoniae detection in primary nasopharyngeal plus oropharyngeal cultures 57 was significantly lower compared with molecular detection in both culture-enriched samples 58 together ( p <0.0001) and also in culture-enriched oropharyngeal samples alone ( p <0.05). 59 60 Conclusions: The sensitivity of S. pneumoniae carriage surveillance can be greatly improved 61 by complementing conventional culture with qPCR and vice versa . The specificity of 62 molecular methods for the detection of live pneumococci can be enhanced by incorporating 63 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. complementing conventional culture with qPCRs and vice versa . We also show that testing nasopharyngeal samples alone leads to underestimation of pneumococcal carriage in adults. in Bland-Altman plots. Carriage rates between both laboratories were compared using Cohen’s kappa. We evaluated the performance of conventional and molecular methods applied to detect S. pneumoniae and pneumococcal serotypes in 1549 nasopharyngeal samples collected from 946 children aged 1-5 years (n=653 in the Netherlands and n=293 in England) and from 603 250 adults (n=319 in the Netherlands and n=284 in England), and on oropharyngeal samples from 251 319 adults in the Netherlands. All PCR results shown were generated by testing the Dutch 252 and English samples in the Dutch laboratory, except were stated otherwise. pneumoniae testing oropharyngeal samples with molecular methods and conducting qPCR-guided culturing. We highlight several statistical procedures that can be used to evaluate the reliability of molecular results and enhance the specificity of molecular methods for the detection of live S. pneumoniae