Abstract
BackgroundTreatment of community acquired pneumonia (CAP) patients with antibiotics before laboratory-confirmed diagnosis leads to loss of knowledge on the causative bacterial pathogen. Therefore, an increasing number of pneumococcal infections is identified using non-culture based techniques. However, methods for serotyping directly on the clinical specimen remain scarce. Here we present three approaches for detection and serotyping of pneumococci using samples from patients with CAP.MethodsThe first approach is quantitative PCR (qPCR) analysis on blood samples (n = 211) followed by capsular sequence typing (CST) to identify the serotype. The second approach, a urinary antigen assay (n = 223), designated as inhibition multiplex immunoassay (IMIA), is based on Luminex technology targeting 14 serotypes. The third approach is a multiplex immunoassay (MIA) (n = 171) also based on Luminex technology which detects serologic antibody responses against 14 serotypes. The three alternative assays were performed on samples obtained from 309 adult hospitalized CAP patients in 2007–2010 and the results were compared with those obtained from conventional laboratory methods to detect pneumococcal CAP, i.e. blood cultures, sputum cultures and BinaxNOW® urinary antigen tests.ResultsUsing qPCR, MIA and IMIA, we were able to detect the pneumococcus in samples of 56% more patients compared to conventional methods. Furthermore, we were able to assign a serotype to the infecting pneumococcus from samples of 25% of all CAP patients, using any of the three serotyping methods (CST, IMIA and MIA).ConclusionThis study indicates the usefulness of additional molecular methods to conventional laboratory methods for the detection of pneumococcal pneumonia. Direct detection and subsequent serotyping on clinical samples will improve the accuracy of pneumococcal surveillance to monitor vaccine effectiveness.
Highlights
Treatment of community acquired pneumonia (CAP) patients with antibiotics before laboratory-confirmed diagnosis leads to loss of knowledge on the causative bacterial pathogen
Optimization and implementation of the quantitative PCR (qPCR), capsular sequence typing (CST) and inhibition multiplex immunoassay (IMIA) methods Quantitative detection using qPCR was based on two target genes, ply and lytA
The minimal bacterial load, determined using colony forming units (CFU) dilution series, that could be detected in the qPCR was 75 CFU per milliliter blood
Summary
Treatment of community acquired pneumonia (CAP) patients with antibiotics before laboratory-confirmed diagnosis leads to loss of knowledge on the causative bacterial pathogen. Streptococcus pneumoniae is a major human pathogen causing considerable morbidity and mortality throughout the world. It is considered the main cause of community acquired pneumonia (CAP) [1,2], there is little consensus in the literature on the prevalence of pneumococci in CAP, it ranges from 10 to 48% in hospitalized. A few studies described the use of PCR to identify the serotype directly from pneumococcal DNA present in blood [17,18,19]. These PCRs contain a myriad of primers and/or probes, using single- or multiplex reactions, making the method complex
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