Clinical Validation of Multiplex Real-Time PCR Assays for Detection of Bacterial Meningitis Pathogens

Neisseria meningitidis ,Haemophilus influenzae , andStreptococcus pneumoniae are important causes of meningitis and other infections, and rapid, sensitive, and specific laboratory assays are critical for effective public health interventions. Singleplex real-time PCR assays have been developed to detectN. meningitidis ctrA ,H. influenzae hpd , andS. pneumoniae lytA and serogroup-specific genes in thecap locus forN. meningitidis serogroups A, B, C, W135, X, and Y. However, the assay sensitivity for serogroups B, W135, and Y is low. We aimed to improve assay sensitivity and develop multiplex assays to reduce time and cost. New singleplex real-time PCR assays for serogroup BsynD , W135synG , and YsynF showed 100% specificity for detectingN. meningitidis species, with high sensitivity (serogroup BsynD , 99% [75/76]; W135synG , 97% [38/39]; and YsynF , 100% [66/66]). The lower limits of detection (LLD) were 9, 43, and 10 copies/reaction for serogroup BsynD , W135synG , and YsynF assays, respectively, a significant improvement compared to results for the previous singleplex assays. We developed three multiplex real-time PCR assays for detection of (i)N. meningitidis ctrA ,H. influenzae hpd , andS. pneumoniae lytA (NHS assay); (ii)N. meningitidis serogroups A, W135, and X (AWX assay); and (iii)N. meningitidis serogroups B, C, and Y (BCY assay). Each multiplex assay was 100% specific for detecting its target organisms or serogroups, and the LLD was similar to that for the singleplex assay. Pairwise comparison of real-time PCR between multiplex and singleplex assays showed that cycle threshold values of the multiplex assay were similar to those for the singleplex assay. There were no substantial differences in sensitivity and specificity between these multiplex and singleplex real-time PCR assays.