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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.

SUBMITTER: Tavares DA

PROVIDER: S-EPMC6397248 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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

Tavares Débora A DA Handem Sara S Carvalho Ricardo J RJ Paulo A Cristina AC de Lencastre Hermínia H Hinds Jason J Sá-Leão Raquel R

Scientific reports 20190301 1

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 ...[more]

PMID: 30824850

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Project description:We have developed a duplex real-time PCR for the rapid diagnosis of Streptococcus pneumoniae infection from culture-negative clinical samples with the simultaneous determination of penicillin susceptibility. The assay amplifies a lytA gene target and a penicillin binding protein 2b (pbp2b) gene target in penicillin-susceptible organisms. The assay was shown to be sensitive (detects 0.5 CFU per PCR) and specific for the detection of S. pneumoniae DNA. The assay was validated by comparing pbp2b PCR results with MIC data for 27 S. pneumoniae isolates. All 5 isolates with penicillin MICs of > 1.0 mg/liter were pbp2b real-time PCR negative, as were 9 of the 10 isolates with penicillin MICs of 0.12 to 1.0 mg/liter. One isolate with a penicillin MIC of 0.12 to 1.0 mg/liter gave an equivocal pbp2b real-time PCR result. Twelve isolates were penicillin susceptible (MICs of < or = 0.06 mg/liter) and pbp2b real-time PCR positive. These data were used to establish an algorithm for the interpretation of penicillin susceptibility from the duplex PCR result. pbp2b real-time PCR results were also compared to an established PCR-restriction fragment length polymorphism (RFLP) method previously applied to these 27 isolates and 46 culture-negative clinical samples (containing S. pneumoniae DNA by broad-range 16S rRNA gene PCR). Discordant results were seen for four isolates and six culture-negative clinical samples, as PCR-RFLP could not reliably detect penicillin MICs of 0.12 to 1.0 mg/liter. We report prospective application of the duplex PCR assay to the diagnosis of S. pneumoniae infection from 200 culture-negative clinical specimens sent to the laboratory for diagnostic broad-range 16S rRNA gene PCR. One hundred six were negative in the duplex PCR. Ninety-four were lytA PCR positive, and 70 of these were also pbp2b PCR positive and interpreted as penicillin susceptible. Fourteen were pbp2b PCR negative and interpreted as having reduced susceptibility to penicillin. For the remaining 10 samples, susceptibility to penicillin was not determined.

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

Publications

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

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