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.

A duplex real-time PCR for the detection of Streptococcus pneumoniae and Neisseria meningitidis in cerebrospinal fluid

“…As previously described, CSF specimens from meningitis patients typically have bacterial counts in excess of 10 3 to 10 5 colony-forming units (CFU)/mL [30]. [26].…”

“…Genome equivalents were calculated assuming one molecule of N. meningitidis and S. pneumoniae DNA. Considering a genome size of 2.1 Mb for S. pneumoniae, a corresponding 2.2 fg of DNA was calculated, and 2.3 fg was determined for N. meningitidis DNA based on a 2.2 Mb genome [26], determined according to the previously published equation [27]. Considering a genome size of 2.1 Mb as determined for S. pneumoniae, the number of genomic copies in the nucleic acid extracts from each strain was determined using the following formula [28]: genome copies = quantity of DNA in extract/2.2 fg or quantity of DNA in extract/2.3 fg.…”

“…Laboratory specificity, defined as the proportion of RT-PCR negative cases from negative cases determined by a reference standard, was obtained from 30 bacterial and fungal isolates representing other species used to validate our RT-PCR [26,14] DNA extraction was performed using a boiling and freeze-thaw treatment, where 200 µL of CSF fluid was heated at 100°C for 10 minutes and immediately frozen at 0°C for 5 minutes. A volume of 150 µL supernatant was recovered after centrifugation at 14,000 g for 10 minutes and stored at -20°C.…”

Evolution of bacterial meningitis diagnosis in Sao Paulo State-Brazil and future challenges

“…After incorporation of triplex qPCR into IAL BM diagnostic routine based on ctrA, lytA and bexA genes, a new target to detect Hi cases belonging to any of the six serotypes (a, b, c, d, e, f ), including Hi-nt, has been proposed by Wang et al (2011; 26,27 . The new target hpd gene, is responsible for the Hi protein D synthesis.…”

“…The method relies on the detection of specific genes related to the biosynthesis of capsular polysaccharide responsible for different groups and types of Nm and Hi, respectively, and used in previously positive samples for each agent 21,23,24,27 .…”