Real-time PCR is a powerful tool for detection of human pathogens because of the high sensitivity and throughput capability. Introduction of fluorescent probes improves the assay specificity and enables development of a multiplex platform for amplification and detection of multiple target genes. Multiplex real-time PCR allows simultaneous detection of multiple organisms in a single reaction, which conserves limited quantities of clinical specimens and significantly reduces costs, such as reagents and person time, which is particularly important in resource-limited settings. However, concerns that the sensitivity of a single assay may be compromised when it is run with other PCR assays in a single reaction remain. In this study, we found that the sensitivity of three multiplex real-time PCR assays was indistinguishable from that of the singleplex real-time PCR assays for detection of bacterial meningitis pathogens as evaluated using ABCs clinical isolates as well as clinical specimens collected from different geographic locations. The sensitivities and specificities of each assay varied among the different laboratories due to the use of different reference standards. Culture is still considered the “gold standard.” Since none of the reference standards used in this study is considered the “gold standard,” the sensitivity and specificity in this analysis may not be accurately assessed.
A multiplex real-time PCR assay using N. meningitidis ctrA
, H. influenzae bexA
, and S. pneumoniae ply
as target genes has previously been developed (9
). However, the bexA
gene is present only in encapsulated H. influenzae
, and the bexA
-based real-time PCR assay is not able to detect nontypeable H. influenzae
. Additionally, the lytA
PCR assay has been shown to be more specific than the ply
assay for detection of S. pneumoniae
). The multiplex assay described in this study improves upon these previously developed assays by using the protein D gene as a target for detection of both typeable and nontypeable H. influenzae
and the lytA
gene as a target for detection of S. pneumoniae
). Both multiplex real-time PCR assays use the ctrA
gene as a target for detection of N. meningitidis
. While the ctrA
-based real-time PCR assay is a suitable tool for capturing most of the encapsulated N. meningitidis
strains, it fails to detect strains that lack ctrA
or that have significant variation in this gene (5
). A recently developed real-time PCR assay targeting the N. meningitidis sodC
gene can capture groupable and nongroupable N. meningitidis
and is very useful for analyzing isolates or specimens collected from nasopharyngeal carriage surveys (12
). The performance of the sodC
assay in the multiplex platform remains to be investigated.
A CSF specimen from a meningitis patient typically has bacterial counts in excess of 103
CFU per milliliter (17
). The LLDs of the multiplex real-time PCR assays described in this study range from 1 to 210 genome equivalents per real-time PCR (250 to 52,500 CFU equivalents per milliliter), which is within the range of bacterial counts in a meningitis CSF specimen. The real-time PCR assays described in this study should be able to detect the target pathogen from meningitis CSF specimens with typical bacterial load. This study used a CT
cutoff value of 35 for all real-time PCR assays. We recommend that specimens with high CT
values (>35 and ≤40) should be analyzed individually. We have observed that nonspecific amplification results in high CT
values and that specimens with high CT
values often have poor replicate reproducibility, which leads to higher rates of false positives (data not shown). High CT
values could also result from the presence of PCR inhibitors or target DNA degradation or low DNA quantity due to antibiotic treatment prior to CSF specimen collection or suboptimal specimen transport and storage conditions. Some of these possibilities can be evaluated by diluting specimens which have high CT
values or by performing antibiotic detection. In any case, interpretation of specimens with high CT
value should include consideration of clinical data and other laboratory results.
Several factors may influence the multiplex real-time PCR performance compared to the singleplex performance, including PCR reagents, DNA quality, and fluorescence reporter dyes. We observed lower sensitivity of some assays in multiplex than in singleplex when different commercial PCR reagents were used. In addition, the serogroup A sacB assay failed to detect N. meningitidis A when the probe was labeled with Cy5 in multiplex (data not shown) but performed well when the probe was labeled with Hex. This underscores the importance of assay optimization when developing multiplex real-time PCR assays from the singleplex real-time PCR assays. The optimal conditions for singleplex real-time PCR may not be optimal for the multiplex PCR. The performance of the multiplex real-time PCR assay does not seem to be affected by different PCR instruments or methods used for DNA extraction.
The three multiplex real-time PCR assays described here have demonstrated sensitivities similar to that of the singleplex real-time PCR for detecting bacterial meningitis pathogens in clinical isolates, CSF specimens, blood, nasal washes, throat swabs, and inoculated transport medium specimens. Multiplex real-time PCR also conserves valuable resources, which makes it particularly useful in resource-limited settings. These advantages make the multiplex real-time PCR assays developed in this study a widely useful tool for disease surveillance and research into the causative pathogens of bacterial meningitis.