Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR

A single-tube 5 ' nuclease multiplex PCR assay was developed on the ABI 770 0 Sequence Detection System (TaqMan) for the detection of Neisseria meningi tidis, Haemophilus influenzae, and Streptococcus pneumoniae from clinical s amples of cerebrospinal fluid (CSF), plasma, serum, and whole blood. Capsul ar transport (ctrA), capsulation (bexA), and pneumolysin (ply) gene targets specific for N, meningitidis, H. influenzae, and S. pneumoniae; respective ly, were selected. Using sequence-specific fluorescent-dye-labeled probes a nd continuous real-time monitoring, accumulation of amplified product was m easured. Sensitivity was assessed using clinical samples (CSF, serum, plasm a, and whole blood) from culture-confirmed cases for the three organisms. T he respective sensitivities (as percentages) for N, meningitidis, H. influe nzae, and S. pneumoniae were 88.4, 100, and 91.8. The primer sets were 100% specific for the selected culture isolates, The ctrA primers amplified men ingococcal sero-groups A, B, C, 29E,, W135, X, Y, and Z; the ply primers am plified pneumococcal serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10A, 11A, 12, 1 1 , 15B, 17F, 18C, 19, 20, 22, 23, 24, 31, and 33; and the bexA primers ampli fied H. influenzae types h and c, Coamplification of two target genes witho ut a loss of sensitivity was demonstrated. The multiplex assay was then use d to test a large number (n = 4,113) of culture-negative samples for the th ree pathogens, Cases of meningococcal, IJ. influenzae, and pneumococcal dis ease that had not previously been confirmed by culture were identified with this assay. The ctrA primer set used in the multiplex PCR was found to be more sensitive (P < 0.0001) than the ctrA primers that had been used for me ningococcal PCR testing at that time.