Detection of respiratory bacterial pathogens causing atypical pneumonia by multiplex Lightmix ® RT-PCR

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.

Epidemiology of Legionnaires’ Disease, Hong Kong, China, 2005−2015

“…Among the 18 PCR-diagnosed cases for which a UAT was performed, only 2 showed positive UAT results, which implied that 16 of these PCR-diagnosed cases would not have been diagnosed if PCR had not been performed. With the development of commercially available multiplex PCR assays for respiratory pathogens, including Legionella species ( 12 ), it is expected that the Legionnaires’ disease incidence will continue to increase, and the percentage of Legionnaires’ disease cases diagnosed by PCR will also increase.…”

Community-acquired bacterial pneumonia in adults: An update

“…Another multiplex PCR, Fast Track Diagnostics FTD-29.19, tests nine bacterial pathogens and has demonstrated more pathogen detection in 37 per cent over 11 per cent by conventional methods in elderly patients 87 . Multiplexing several gene targets for detection of atypical pathogens has shown comparable performance with in-house individual PCRs in a study demonstrating overall 52 per cent PCR positivity in patients suspected with atypical pneumonia 88 .…”

“…ICU, intensive care unit; PCR, polymerase chain reaction. Source : Refs 29 38 54 58 59 82 85 86 88 .…”

Atypical Pathogen Distribution in Chinese Hospitalized AECOPD Patients: A Multicenter Cross-Sectional Study

“…10,20,22 We believe that this variation is related to the collection method and site of the specimens subjected to PCR testing. The diagnostic performance of Roche Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella pneumophila multiplex Lightmix RT-PCR kit has been evaluated by Wagner et al 23 and showed identical performance characteristics (specificity and sensitivity) to in-house singleplex RT-PCRs for pathogen detection. Wagner 24 and Orozco-Hoyos 25 used the kit to detect Mycoplasma pneumoniae, Chlamydia pneumoniae.…”