Optimization and validation of a high-throughput nanofluidic real-time PCR assay to evaluate nasopharyngeal carriage of 15 bacterial species and 92 Streptococcus pneumoniae serotypes

Abstract

Abstract Background: Sensitive tools for detecting concurrent colonizing pneumococcal serotypes are needed for detailed evaluation of the direct and indirect impact of routine pneumococcal conjugate vaccine (PCV) immunization. Method: A high-throughput quantitative nanofluidic real-time PCR (Fluidigm) reaction-set was developed to detect and quantify 92 pneumococcal serotypes in archived clinical samples. Nasopharyngeal swabs collected in 2009-2011 from South African children ≤5years-old, previously serotyped with standard culture-based methods were used for comparison. Results: The reaction-set within the Fluidigm® effectively amplified all targets with high efficiency (90-110%), reproducibility (R2≥0.98), and at low limit-of-detection (<102 CFU/ml). A blind analysis of 1973 nasopharyngeal swab samples showed diagnostic sensitivity >80% and specificity >95 compared with the referent standard, culture-based Quellung method. The Fluidigm method was able to serotype pneumococcal types with good discrimination compared with Quellung (ROC-AUC: >0.73). Conclusion: The high-throughput nanofluidic real-time PCR method simultaneously detects 57 individual serotypes, and 35 serotypes within 16 serogroups in 96 samples (including controls), within a single qPCR run. This method can be used to evaluate the impact of current PCV formulations on vaccine-serotype and non-vaccine-serotype colonization, including detection of multiple concurrently colonizing serotypes. Interpretation: The Fluidigm method can allow for monitoring of serotype-specific bacterial load, as well as emergence or ongoing transmission of minor or co-colonizing serotypes that may have invasive disease potential.