Duplex Real-Time PCR Assay for Detection of Streptococcus pneumoniae in Clinical Samples and Determination of Penicillin Susceptibility
- 1 August 2008
- journal article
- research article
- Published by American Society for Microbiology in Journal of Clinical Microbiology
- Vol. 46 (8), 2751-2758
- https://doi.org/10.1128/jcm.02462-07
Abstract
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 ≤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.Keywords
This publication has 14 references indexed in Scilit:
- Genotypic identification of presumptive Streptococcus pneumoniae by PCR using four genes highly specific for S. pneumoniaeJournal of Medical Microbiology, 2006
- Empyema: the use of broad range 16S rDNA PCR for pathogen detectionArchives of Disease in Childhood, 2005
- Comparison of four polymerase chain reaction assays for specificity in the identification of Streptococcus pneumoniaeDiagnostic Microbiology and Infectious Disease, 2004
- Clinical features, aetiology and outcome of empyema in children in the north east of EnglandThorax, 2004
- Development of broad-range 16S rDNA PCR for use in the routine diagnostic clinical microbiology serviceJournal of Medical Microbiology, 2003
- Rapid Real-Time PCR for Determination of Penicillin Susceptibility in Pneumococcal Meningitis, Including Culture-Negative CasesJournal of Clinical Microbiology, 2002
- Sensitive and Specific Method for Rapid Identification of Streptococcus pneumoniae Using Real-Time Fluorescence PCRJournal of Clinical Microbiology, 2001
- Genetic Relationships between Clinical Isolates of Streptococcus pneumoniae , Streptococcus oralis , and Streptococcus mitis : Characterization of “Atypical” Pneumococci and Organisms Allied to S. mitis Harboring S. pneumoniae Virulence Factor-Encoding GenesInfection and Immunity, 2000
- Diagnosis of Bacteremic Pneumococcal Pneumonia by Amplification of Pneumolysin Gene Fragment in SerumThe Journal of Infectious Diseases, 1995
- The estimation of the bactericidal power of the bloodEpidemiology and Infection, 1938