Identification of Streptococcus pneumoniae by a real-time PCR assay targeting SP2020

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.

A Pangenome Approach for Discerning Species-Unique Gene Markers for Identifications of Streptococcus pneumoniae and Streptococcus pseudopneumoniae

“…The proposition of using more than one marker is based on the findings that not all of the proposed species unique gene markers for S. pseudopneumoniae were present in all the strains tested. This is also supported by previous findings that markers reported to be unique for S. pneumoniae are also found in clinical isolates of other species of the Mitis-Group of the genus Streptococcus (Tavares et al, 2019). These facts further demonstrate the importance of using multi-locus approaches for identifications and differentiation of S. pneumoniae and S. pseudopneumoniae.…”

“…Interestingly, other, previously reported gene markers for S. pneumoniae were not necessarily detected by our pangenomic approach. For instance, Tavares et al (2019) indicated that "lytA, piaB, and SP2020 were found in non-pneumococcal strains." Also, Carvalho et al (2007) reported that a psaAbased real-time PCR-assay was positive in S. pseudopneumoniae strains.…”

Identification of Streptococcus pneumoniae and other Mitis streptococci: importance of molecular methods

“…Two genes, piaA and piaB encoding components of an ABC transport system essential for iron uptake, were described as specific for S. pneumoniae but these genes are absent in some pneumococcal isolates, which limits the sensitivity of the test [79][80][81]. Moreover, piaA was detected in some S. mitis and S. pseudopneumoniae [66] and recently S. pseudopneumoniae isolates positive for piaB have been reported [70].…”

“…Comparative genomic analyses of S. pneumoniae and S. pseudopneumoniae led to the identification of markers specific for these two species, respectively SPN0001/SP2020, encoding a GtnR-family transcriptional regulator, and SPS0002, specifying an osmosensitive potassium channel histidine kinase/response regulator KdpDE [88]. The specificity of both markers was close to 100%, with SPN0001/SP2020 found in a few non-pneumococcal strains and SPS0002 occasionally present in S. pneumoniae [70,88].…”

Re-evaluation of Streptococcus pneumoniae carriage in Portuguese elderly by qPCR increases carriage estimates and unveils an expanded pool of serotypes

“…Recently, new strategies to detect pneumococcal carriage based on real-time PCR (qPCR) have been proposed, validated, and are now being increasingly used [19][20][21][22][23] . The use of qPCR enables high sensitivity in samples where pneumococci are at low density.…”