Abstract
Pneumonia is a severe infectious disease. In addition to common viruses and bacterial pathogens (e.g. Streptococcus pneumoniae), fastidious respiratory pathogens like Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella spp. can cause severe atypical pneumonia. They do not respond to penicillin derivatives, which may cause failure of antibiotic empirical therapy. The same applies for infections with B. pertussis and B. parapertussis, the cause of pertussis disease, that may present atypically and need to be treated with macrolides. Moreover, these fastidious bacteria are difficult to identify by culture or serology, and therefore often remain undetected. Thus, rapid and accurate identification of bacterial pathogens causing atypical pneumonia is crucial. We performed a retrospective method evaluation study to evaluate the diagnostic performance of the new, commercially available Lightmix® multiplex RT-PCR assay that detects these fastidious bacterial pathogens causing atypical pneumonia. In this retrospective study, 368 clinical respiratory specimens, obtained from patients suffering from atypical pneumonia that have been tested negative for the presence of common agents of pneumonia by culture and viral PCR, were investigated. These clinical specimens have been previously characterized by singleplex RT-PCR assays in our diagnostic laboratory and were used to evaluate the diagnostic performance of the respiratory multiplex Lightmix® RT-PCR. The multiplex RT-PCR displayed a limit of detection between 5 and 10 DNA copies for different in-panel organisms and showed identical performance characteristics with respect to specificity and sensitivity as in-house singleplex RT-PCRs for pathogen detection. The Lightmix® multiplex RT-PCR assay represents a low-cost, time-saving and accurate diagnostic tool with high throughput potential. The time-to-result using an automated DNA extraction device for respiratory specimens followed by multiplex RT-PCR detection was below 4 h, which is expected to significantly improve diagnostics for atypical pneumonia-associated bacterial pathogens.
Highlights
Community-acquired pneumonia (CAP) is a severe infectious disease
We present the first evaluation of the multiplex Lightmix® RT-PCR for simultaneous detection of the most important bacterial pathogens causing atypical pneumonia, namely Bordetella
We investigated the performance of the multiplex RT-PCR with clinical respiratory specimens that have been previously characterized by singleplex in-house RT-PCR assays (N = 368) (Fig. 1 and Table 1)
Summary
The most common causative agents include viruses (like influenza) and bacterial pathogens (like Streptococcus pneumoniae and Haemophilus influenzae) (WHO, 2016). Besides these “common” causes of CAP, fastidious bacterial pathogens (like Chlamydia pneumoniae, Mycoplasma pneumoniae, Legionella pneumophila and Coxiella burnetii) can cause so-called atypical pneumonia that accounts for up to 15% of all CAP cases (Cunha, 2006). The detection of atypical bacterial agents of CAP by culture or serology remains a challenge. Rapid and accurate molecular methods are required for fast identification of bacterial pathogens causing atypical pneumonia and to subsequently adapt the antibiotic regimen of the patients
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