It Takes Two to Tango: Combining Conventional Culture With Molecular Diagnostics Enhances Accuracy of Streptococcus pneumoniae Detection and Pneumococcal Serogroup/Serotype Determination in Carriage
Miellet, Willem R.; van Veldhuizen, Janieke; Litt, David; Mariman, Rob; Wijmenga-Monsuur, Alienke J.; Badoux, Paul; Nieuwenhuijsen, Tessa; Thombre, Rebecca; Mayet, Sanaa; Eletu, Seyi; Sheppard, Carmen; van Houten, Marianne Alice; Rots, Nynke Y.; Miller, Elizabeth; Fry, Norman K.; Sanders, Elisabeth A.M.; Trzciński, Krzysztof
(2022) Frontiers in Microbiology, volume 13
(Article)
Abstract
BACKGROUND: The specificity of molecular methods for the detection of Streptococcus pneumoniae carriage is under debate. We propose a procedure for carriage surveillance and vaccine impact studies that increases the accuracy of molecular detection of live pneumococci in polymicrobial respiratory samples. METHODS: Culture and qPCR methods were applied to detect
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pneumococcus and pneumococcal serotypes in 1,549 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 signal specific for pneumococcus in qPCRs were re-examined with a second, qPCR-guided culture. Optimal C q 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. RESULTS: Detection of pneumococcus 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 displayed increased sensitivity of detection for multiple serotype carriage, and implementation of qPCR-guided culturing significantly increased the proportion of nasopharyngeal and oropharyngeal samples from which live pneumococcus was recovered ( p < 0.0001). For paired nasopharyngeal and oropharyngeal samples from adults none of the methods applied to a single sample type exhibited good agreement with results for primary and qPCR-guided nasopharyngeal and oropharyngeal cultures combined (Cohens kappa; 0.13-0.55). However, molecular detection of pneumococcus displayed increased sensitivity with culture-enriched oropharyngeal samples when compared with either nasopharyngeal or oropharyngeal primary cultures ( p < 0.05). CONCLUSION: The accuracy of pneumococcal carriage surveillance can be greatly improved by complementing conventional culture with qPCR and vice versa, by using results of conventional and qPCR-guided cultures to interpret qPCR data. 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 for future carriage surveillance and vaccine impact studies improves detection of pneumococcal carriage in adults in particular and enhances the specificity of serotype carriage detection.
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Keywords: carriage, conventional culture, diagnostic accuracy, qPCR (quantitative PCR), Streptococcus pneumoniae (pneumococcus), Microbiology, Microbiology (medical)
ISSN: 1664-302X
Publisher: Frontiers Media S. A.
Note: Funding Information: ES and KT had an idea and initiated the study. NR, EM, NF, ES, and KT secured financial support. NF and KT led the project. AW-M, PB, CS, MH, NR, EM, and NF conducted carriage studies, collected the data, and provided study materials. WM, JV, and KT developed, validated laboratory methods, and wrote the laboratory protocol. WM, JV, DL, PB, TN, SM, RT, SE, and CS analyzed samples and collected the data. WM, RM, and KT contributed analytical tools. WM, JV, and DL curated the data. WM, DL, NF, and KT managed the study. WM and KT performed formal analysis of study data, visualized presentation of the results, and drafted the manuscript. All authors amended, critically reviewed, and commented on the final manuscript. Publisher Copyright: Copyright © 2022 Miellet, van Veldhuizen, Litt, Mariman, Wijmenga-Monsuur, Badoux, Nieuwenhuijsen, Thombre, Mayet, Eletu, Sheppard, van Houten, Rots, Miller, Fry, Sanders and Trzciński.
(Peer reviewed)