A semi automated real-time duplex PCR assay for rapid detection of Streptococcus pneumoniae directly from clinical samples

Abstract number: P1849

Sidhu M., Zhao P., Deatly A., Huijts S., Pride M., Jansen K.

Background: Accurate diagnosis of pneumococcal infections is critical in assessing the effectiveness of pneumococcal-conjugate vaccination on colonization in light of both serotype switching and replacement. Sensitive, rapid, and reliable detection is essential for surveillance of nasopharyngeal carriage. We developed and characterized a semi-automated, real-time duplex PCR assay to detect S. pneumoniae (SPn) directly from clinical samples.

Methods: Targeting the lytA and ply genes simultaneously, we tested 91 SPn serotypes in addition to 26 non-related bacterial strains. Assay sensitivity was established by spiking different concentrations of SPn into naïve human blood. The method was then applied to DNAs extracted by automation from 100 mL of 150 blood (chest X-ray confirmed CAP), 22 pleural fluid (PF), and 147 cerebrospinal fluid (CSF) specimens. In positive DNA, the Prevnar 13 vaccine serotypes were identified by capsule specific real-time PCR.

Results: Detection limits for lytA and ply genes were reproducibly established at 160 CFU/mL of blood. Only SPn strains were amplified showing an assay specificity of 100%. Of the 150 blood samples, 13% were detected by blood culture while 14% by PCR. Detection rates in PF and CSF were 91% and 12%, respectively.

Conclusion: Our highly sensitive (1.6 CFU per reaction) and specific PCR assay offers rapid detection of SPn in clinical specimens. Substantially increased detection rates observed in PF swabs indicate the usefulness of this assay on samples obtained from sites with higher bacterial burden. Details of the method and a comparison between PCR and blood culture method will be presented at the meeting.

Session Details

Date: 10/04/2010
Time: 00:00-00:00
Session name: Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases
Location: Vienna, Austria, 10 - 13 April 2010
Presentation type:
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