Quantitative polymerase chain reaction methods that are validated to determine bacterial load are important for assessing the impact of vaccines in public health programs.
A nanofluidic quantitative polymerase chain reaction (qPCR) reaction-set was found to be sensitive, specific, and reproducible for the accurate detection and quantification of 92 pneumococcal serotypes, as well as other colonizing bacteria in 96 specimens, according to a study published by Scientific Reports.
“Real-time PCR methods have been developed with an increasing number of serotypes detected, including in high-throughput systems,” the study authors wrote. “Further, (qPCR) methods that are validated to determine bacterial load are important for assessing the impact of vaccines in public health programs. Increased bacterial load in the nasopharynx may predict serotype-specific invasive disease potential.”
The study noted that there are 100 biochemically and serologically distinct S. pneumoniae serotypes and that capsular diversity is a key virulence factor in S. pneumoniae, as serotypes have varying potential to cause disease in humans.
The investigators sought to address an unmet need for sensitive tools that can identify concurrent colonizing pneumococcal serotypes for detailed analysis of the direct and indirect impact of routine pneumococcal conjugate vaccine (PCV) immunization. Investigators developed the high-throughput, quantitative, nanofluidic real-time PCR (Standard BioTools ‘Fluidigm’) reaction-set to identify 92 pneumococcal serotypes in archived clinical samples.
“Pneumococcal colonization surveys are best performed using comprehensive, sensitive, and accurate serotyping methods able to detect multiple serotype co-carriage, including VT and non-vaccine serotypes (NVT),” the study authors wrote. “Consistent surveillance of circulating serotypes is needed to inform vaccination strategies and utilization of higher or alternate valency PCVs, including tailored to different geographic regions; and for the detection of emerging or replacement serotypes.”
Investigators collected nasopharyngeal swabs between 2009 and 2011 from South African children ≤ 5 years-old, who were previously serotyped with standard culture-based methods for comparison. The reaction-set within the ‘Fluidigm’ was able to effectively amplify the targets with high efficiency (90%–110%), reproducibility (R2≥ 0.98), and with a low limit-of-detection (<102 CFU/ml).
The investigators noted that culture-based Quellung methods are the gold standard for S. pneumoniae serotyping, but are time-consuming, have low sensitivity, and are expensive. PCR-based detection, on the other hand, does not rely on organism viability, which the investigators noted can be negatively impacted by antibiotic use, sample transport, and storage conditions. PCR testing is also less time consuming, provides faster diagnosis, and has higher sensitivity compared with culture-based methods, according to the study.
A blind analysis of 1973 nasopharyngeal swab samples showed diagnostic sensitivity >80% and specificity >95% vs the Quellung method. Further, the qPCR method serotyped pneumococcal types with good discrimination compared with Quellung (ROC-AUC: > 0.73), according to the study. The high-throughput nanofluidic real-time PCR method simultaneously detected 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,” the study authors wrote.
Compared with the Quellung method, the study reaction-set was 98.8% accurate in classifying pneumococcal serotypes and the average sensitivity across the qPCR reaction-set was 89.1% compared with Quellung.
“Our qPCR 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,” the study authors wrote.
Downs, S.L., Madhi, S.A., van der Merwe, L. et al. Optimization of a high-throughput nanofluidic real-time PCR to detect and quantify of 15 bacterial species and 92 Streptococcus pneumoniae serotypes. Sci Rep 13, 4588 (2023). https://doi.org/10.1038/s41598-023-31820-4