Clinical validation of multiplex real-time PCR assays for detection of bacterial meningitis pathogens

Abstract

Neisseria meningitidis, Haemophilus influenzae, and Streptococcus 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 detect N. meningitidis ctrA, H. influenzae hpd, and S. pneumoniae lytA and serogroup-specific genes in the cap locus for N. 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 B synD, W135 synG, and Y synF showed 100% specificity for detecting N. meningitidis species, with high sensitivity (serogroup B synD, 99% [75/76]; W135 synG, 97% [38/39]; and Y synF, 100% [66/66]). The lower limits of detection (LLD) were 9, 43, and 10 copies/reaction for serogroup B synD, W135 synG, and Y synF 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, and S. 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.

Fig 1

Pairwise comparison analyses for species-specific PCR assays for N. meningitidis (Nm) ctrA, H. influenzae (Hi) hpd, and S. pneumoniae (Sp) lytA carried out using ABCs isolates. Each assay was compared between multiplex and singleplex (A, B, and C) and between singleplex duplicates (D, E, and F). Shown is the Bland-Altman plot that depicts the cycle threshold difference (ΔC_T) between the two assays (ΔC_{Tmultiplex-singleplex} [ΔC_Tm-s] or ΔC_{Tsingleplex1-singleplex2} [ΔC_Ts1-s2]) as a function of the average C_T value of the two assays [(C_{Tmultiplex or singleplex} + C_Tsingleplex)/2]. The reference lines on the plot indicate the ideal zero difference (solid line) and the observed mean C_T difference (mean ΔC_Tm-s or mean ΔC_Ts1-s2) (middle dashed line), as well as the upper and lower limits of agreement defined by the mean ΔC_T ± 1.96 standard deviations (top and bottom dashed lines) (–3).

Fig 2

Pairwise comparison analyses for N. meningitidis serogroup-specific PCR assays carried out using ABCs isolates. Each of the six PCR assays was compared between multiplex and singleplex platforms. The Bland-Altman plot for each assay is shown.