Simultaneous Detection of Key Bacterial Pathogens Related to Pneumonia and Meningitis Using Multiplex PCR Coupled With Mass Spectrometry

Zhang, Chi; Xiu, Leshan; Xiao, Yan; Xie, Zhengde; Ren, Lili; Peng, Junping

Pneumonia and meningitis can be caused by various bacterial pathogens; therefore, identifying the causative organism in a clinical case is challenging. Real-time PCR is a method frequently used for pathogen detection, but the limited optical channels restrict the number of targets that can be detected simultaneously. An optimized real-time PCR assay can be used to detect three to four targets in one reaction, which means that multiple reactions are needed to cover the spectrum of potential pathogens (Edin et al., 2015; Gadsby et al., 2015) . Some sampleto-result platforms, such as the FilmArray (Biofire, Salt Lake City, UT, USA) system, provide another solution. The FilmArray system integrates sample preparation, amplification, detection, and analysis in one device, and the results can be obtained in 1 h, with 5 min of hands-on time. However, each FilmArray analyzer can process only one specimen per run, resulting in a high cost per specimen (Poritz et al., 2011; Babady, 2013) . One advantage of the BP-MS method established in this study is the comprehensive coverage of targets. Up to 11 pathogens can be detected simultaneously in one reaction, which is beneficial in respiratory illnesses with many possible etiologies and saves a substantial amount of labor. The high sample throughput of this method is another advantage. Using a 384-well PCR plate, as many as 380 clinical specimens can be analyzed within a single experiment, reducing the cost to <$4 per sample (not including nucleic acid extraction). Therefore, when many samples need to be tested, the BP-MS method is more feasible and economical than a FilmArray system. The 11 target pathogens selected for use in the BP-MS method are the etiologic organisms most frequently detected in association with bacterial respiratory diseases. For community acquired pneumonia (CAP), S. pneumoniae is most often the cause, and other common bacterial causes include H. influenzae and M. catarrhalis (Musher and Thorner, 2014) . M. pneumoniae and L. pneumophila are atypical CAP etiologies (Arnold et al., 2007) . In contrast to bacteria associated with CAP, the dominant etiologic agents of hospital-acquired bacterial pneumonia are S. aureus, P. aeruginosa, K. pneumoniae, and A. baumannii, always showing less susceptibility to antimicrobials (Jones, 2010) . In addition, N. meningitidis and B. pertussis are common causes of meningitis and pertussis. We have also designed assays for Chlamydia pneumoniae, which is an important atypical pathogen responsible for CAP. As isolates and positive clinical samples of C. pneumoniae are not available in this study, we have not evaluated its performance, so it is not included in the current panel.