Feasibility of a quantitative polymerase chain reaction assay for diagnosing pneumococcal pneumonia using oropharyngeal swabs
Streptococcus pneumoniae is the most important pathogen causing community-acquired pneumonia (CAP). The current diagnostic microbial standard detects S. pneumoniae in less than 30% of CAP cases. A quantitative polymerase chain reaction (PCR) targeting autolysin (lytA) is able to increase the rate of detection. The aim of this study is validation of this quantitative PCR in vitro using different available strains and in vivo using clinical samples (oropharyngeal swabs). The PCR autolysin (lytA) was validated by testing the intra- and inter-run variability. Also, the in vitro specificity and sensitivity, including the lower limit of detection was determined. In addition, a pilot-study was performed using samples from patients (n = 28) with pneumococcal pneumonia and patients (n = 28) with a pneumonia without detection of S. pneumoniae with the current diagnostic microbial standard, but with detection of either a viral and or another bacterial pathogen to validate this test further. The intra- and inter-run variability were relatively low (SD’s ranging from 0.08 to 0.96 cycle thresholds). The lower limit of detection turned out to be 1–10 DNA copies/reaction. In-vitro sensitivity and specificity of the tested specimens (8 strains carrying lytA and 6 strains negative for lytA) were both 100%. In patients with pneumococcal and non-pneumococcal pneumonia a cut-off value of 6.000 copies/mL would lead to a sensitivity of 57.1% and a specificity of 85.7%. We were able to develop a quantitative PCR targeting lytA with good in-vitro test characteristics.
KeywordsStreptococcus pneumoniae Quantitative PCR Pneumonia LytA Community-acquired pneumonia
RD, WvdR, WR, MvS and WB were involved in the conception and design of the study. WvdR, RJ and MvS were involved in method development and molecular testing for the study. MvS, RD, WvdR, NP and WB participated in the drafting of the manuscript. All authors interpreted the data and revised the manuscript critically for important intellectual content. All authors approved the final manuscript.
No sponsors were involved in the study design, sample collection, analysis and interpretation of data, writing or decision to submit the paper for publication. Glaxo Smith Kline and Chiesi both provided an unrestricted grant.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflicts of interest.
For the REDUCE study ethical approval was obtained through the METC Noord-Holland (Postbus 501, 1800 AM, Alkmaar, The Netherlands) which is now part of the METC of the Amsterdam University Medical Centres.
For the REDUCE study informed consent was obtained for taking oropharyngeal swabs.
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