Background: Surveillance of invasive pneumococcal disease (IPD) is essential to monitor the efficacy of conjugate vaccines. Because antibiotic treatment for suspected IPD is frequently initiated before specimen collection, diagnosis and serotype identification, increasingly, rely on molecular methods.
Aim: We review the performance of molecular Streptococcus pneumoniae serotyping methods targeting serotype-specific sequences in the cps gene cluster (cpsA-cpsB, wzy and wzx), using multiplex PCR-based reverse line blot (mPCR/RLB) and PCR/sequencing for use on culture-negative/lytA positive specimens. We compared our results with those of published studies since 2000 of other culture-independent methods of direct pneumococcal serotype identification.
Methods: The methods consisted of a mPCR/RLB (58 primer pairs, targeting >90 pneumococcal serotypes in 2 separate assays) and cpsA cps B PCR/sequencing. Clinical specimens tested included blood, cerebrospinal fluid (CSF), joint and lung aspirates, pleural fluid (PF), nasopharyngeal (NP) swabs and pus.
Results: mPCR/RLB assay alone identified serotypes, individually or to within a small group, in 25% and 50% of batched culture-negative PF and NP samples. Addition of cpsA-cpsB for NP swabs increased serotype identification to 46% of specimens. In individual culture-negative diagnostic specimens, cpsA-cpsB PCR/sequencing, alone, identified serotypes in 88% of CSF and 71% of PF specimens.
Conclusion: mPCR/RLB was designed to identify serotypes in cultures, but it is also useful for screening large numbers of culture-negative/PCR positive specimens, inexpensively. cpsA-cpsB PCR/sequencing, is significantly more sensitive, because of the use of nested primers and a single target. Literature review indicated that multiple serotype-specific multiplex real-time-PCR (with 3-5 targets each) is generally more sensitive than cpsA-cpsB PCR/sequencing for identification of a limited number of serotypes. However, the multiple reactions required to identify even the 13 serotypes in the most recent conjugate vaccine limits its efficiency. Microarray is promising with 81% and 70% serotype detection rate in culture negative CSF and NP swabs respectively (i.e. similar to ours for CSF but more sensitive for NP swabs), but this must be balanced against greater expense .