Identification of Streptococcus pneumoniae by a real-time PCR assay targeting SP2020.
Débora A Tavares, Sara Handem, Ricardo J Carvalho, A Cristina Paulo, Hermínia de Lencastre, Jason Hinds, Raquel Sá-Leão
Author Information
Débora A Tavares: Laboratory of Molecular Microbiology of Human Pathogens, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB-NOVA), Oeiras, Portugal.
Sara Handem: Laboratory of Molecular Microbiology of Human Pathogens, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB-NOVA), Oeiras, Portugal.
Ricardo J Carvalho: Laboratory of Molecular Microbiology of Human Pathogens, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB-NOVA), Oeiras, Portugal.
A Cristina Paulo: Laboratory of Molecular Microbiology of Human Pathogens, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB-NOVA), Oeiras, Portugal.
Hermínia de Lencastre: Laboratory of Molecular Genetics, ITQB-NOVA, Oeiras, Portugal.
Jason Hinds: Institute for Infection and Immunity, St George's University of London, London, UK.
Raquel Sá-Leão: Laboratory of Molecular Microbiology of Human Pathogens, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB-NOVA), Oeiras, Portugal. rsaleao@itqb.unl.pt.
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.
