title: 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.
authors.affiliation: Division of Bacterial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA. xwang2@cdc.gov
authors.forename: Xin
authors.initials: X
authors.lastname: Wang
authors.forename: M Jordan
authors.initials: MJ
authors.lastname: Theodore
authors.forename: Raydel
authors.initials: R
authors.lastname: Mair
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Trujillo-Lopez
authors.forename: Mignon
authors.initials: M
authors.lastname: du Plessis
authors.forename: Nicole
authors.initials: N
authors.lastname: Wolter
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Baughman
authors.forename: Cynthia
authors.initials: C
authors.lastname: Hatcher
authors.forename: Jeni
authors.initials: J
authors.lastname: Vuong
authors.forename: Lisa
authors.initials: L
authors.lastname: Lott
authors.forename: Anne
authors.initials: A
authors.lastname: von Gottberg
authors.forename: Claudio
authors.initials: C
authors.lastname: Sacchi
authors.forename: J Matthew
authors.initials: JM
authors.lastname: McDonald
authors.forename: Nancy E
authors.initials: NE
authors.lastname: Messonnier
authors.forename: Leonard W
authors.initials: LW
authors.lastname: Mayer
mesh_terms: D006193:Haemophilus influenzae / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016920:Meningitis, Bacterial / Q000175:diagnosis* / Q000382:microbiology*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009345:Neisseria meningitidis / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013296:Streptococcus pneumoniae / Q000235:genetics / Q000302:isolation & purification*