Abstract
Real-time PCR targeting lytA (the major autolysin gene) and piaB (permease gene of the pia ABC transporter) are currently used as the gold-standard culture-independent assays for Streptococcus pneumoniae identification. We evaluated the performance of a new real-time PCR assay – targeting SP2020 (putative transcriptional regulator gene) – and compared its performance with the assays previously described. A collection of 150 pneumococci, 433 non-pneumococci and 240 polymicrobial samples (obtained from nasopharynx, oropharynx, and saliva; 80 from each site) was tested. SP2020 and lytA-CDC assays had the best performance (sensitivity of 100% for each compared to 95.3% for piaB). The specificity for lytA and piaB was 99.5% and for SP2020 was 99.8%. Misidentifications occurred for the three genes: lytA, piaB and SP2020 were found in non-pneumococcal strains; piaB was absent in some pneumococci including a serotype 6B strain. Combining lytA and SP2020 assays resulted in no misidentifications. Most polymicrobial samples (88.8%) yielded concordant results for the three molecular targets. The remaining samples seemed to contain non-typeable pneumococci (0.8%), and non-pneumococci positive for lytA (1.7%) or SP2020 (8.7%). We propose that combined detection of both lytA-CDC and SP2020 is a powerful strategy for the identification of pneumococcus either in pure cultures or in polymicrobial samples.
Highlights
Real-time PCR targeting lytA and piaB are currently used as the gold-standard culture-independent assays for Streptococcus pneumoniae identification
The performance of this real-time PCR assay was initially tested with a collection of 67S. pneumoniae and 104 non-pneumococcal isolates
Strains yielded cycle threshold (CT) values for piaB far higher than those obtained for lytA and SP2020
Summary
Real-time PCR targeting lytA (the major autolysin gene) and piaB (permease gene of the pia ABC transporter) are currently used as the gold-standard culture-independent assays for Streptococcus pneumoniae identification. We propose that combined detection of both lytA-CDC and SP2020 is a powerful strategy for the identification of pneumococcus either in pure cultures or in polymicrobial samples. For culture-independent assays the current method of choice is a real-time PCR assay targeting the gene lytA (lytA-CDC)[7,8]. LytA is the major autolysin of pneumococcus and has been described as ubiquitous and specific of this species[9] The performance of this real-time PCR assay was initially tested with a collection of 67S. The latter group included 13 viridans group streptococci not identified to the species level. This method has been extensively used by different laboratories in both disease and carriage studies[10,11]
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