Simultaneous Detection of Neisseria meningitidis, Haemophilus influenzae,and Streptococcus pneumoniaein Suspected Cases of Meningitis and Septicemia Using Real-Time PCR

ABSTRACTA single-tube 5' nuclease multiplex PCR assay was developed on the ABI 7700 Sequence Detection System (TaqMan) for the detection of Neisseria meningitidis, Haemophilus influenzae,and Streptococcus pneumoniaefrom clinical samples of cerebrospinal fluid (CSF), plasma, serum, and whole blood. Capsular transport (ctrA),capsulation (bexA), and pneumolysin (ply) gene targets specific for N. meningitidis, H. influenzae,and S. pneumoniae,respectively, were selected. Using sequence-specific fluorescent-dye-labeled probes and continuous real-time monitoring, accumulation of amplified product was measured. Sensitivity was assessed using clinical samples (CSF, serum, plasma, and whole blood) from culture-confirmed cases for the three organisms. The respective sensitivities (as percentages) for N. meningitidis, H. influenzae,and S. pneumoniaewere 88.4, 100, and 91.8. The primer sets were 100% specific for the selected culture isolates. The ctrAprimers amplified meningococcal serogroups A, B, C, 29E, W135, X, Y, and Z; the plyprimers amplified pneumococcal serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10A, 11A, 12, 14, 15B, 17F, 18C, 19, 20, 22, 23, 24, 31, and 33; and thebexAprimers amplified H. influenzaetypes b and c. Coamplification of two target genes without a loss of sensitivity was demonstrated. The multiplex assay was then used to test a large number (n= 4,113) of culture-negative samples for the three pathogens. Cases of meningococcal, H. influenzae,and pneumococcal disease that had not previously been confirmed by culture were identified with this assay. The ctrAprimer set used in the multiplex PCR was found to be more sensitive (P< 0.0001) than the ctrAprimers that had been used for meningococcal PCR testing at that time.