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Background   Streptococcus pneumoniae  and  Haemophilus influenzae  cause pneumonia and as  Neisseria meningitidis  they are important agents of meningitis. Although several PCR methods have been described for these bacteria the specificity is an underestimated problem. Here we present a quantitative multiplex real-time PCR (qmPCR) for detection of  S. pneumoniae  (9802 gene fragment),  H. influenzae  ( omp P6  gene) and  N. meningitidis (ctrA  gene). The method was evaluated on bronchoalveolar lavage (BAL) samples from 156 adults with lower respiratory tract infection (LRTI) and 31 controls, and on 87 cerebrospinal fluid (CSF) samples from meningitis patients.    Results  The analytical sensitivity was not affected by using a combined mixture of reagents and a combined DNA standard ( S. pneumoniae/H. influenzae/N. meningitidis ) in single tubes. By blood- and BAL-culture and  S. pneumoniae  urinary antigen test,  S. pneumoniae  and  H. influenzae  were aetiological agents in 21 and 31 of the LTRI patients, respectively. These pathogens were identified by qmPCR in 52 and 72 of the cases, respectively, yielding sensitivities and specificities of 95% and 75% for  S. pneumoniae , and 90% and 65% for  H. influenzae , respectively. When using a cut-off of 10 5  genome copies/mL for clinical positivity the sensitivities and specificities were 90% and 80% for  S. pneumoniae , and 81% and 85% for  H. influenzae , respectively. Of 44 culture negative but qmPCR positive for  H. influenzae , 41 were confirmed by  fucK  PCR as  H. influenzae . Of the 103 patients who had taken antibiotics prior to sampling,  S. pneumoniae  and  H. influenzae  were identified by culture in 6% and 20% of the cases, respectively, and by the qmPCR in 36% and 53% of the cases, respectively.  In 87 CSF samples  S. pneumoniae  and  N. meningitidis  were identified by culture and/or  16 S rRNA  in 14 and 10 samples and by qmPCR in 14 and 10 samples, respectively, giving a sensitivity of 100% and a specificity of 100% for both bacteria.    Conclusions  The PCR provides increased sensitivity and the multiplex format facilitates diagnosis of  S. pneumoniae ,  H. influenzae  and  N. meningitidis  and the assay enable detection after antibiotic treatment has been installed. Quantification increases the specificity of the etiology for pneumonia.

bmc microbiology

Multiplex quantitative PCR for detection of lower respiratory tract infection and meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis