# Simultaneous Detection of Nine Key Bacterial Respiratory Pathogens Using Luminex xTAG(r) Technology
## 1 Introduction  
Lower respiratory tract infections (LRTIs) are caused by numerous pathogens and, if untreated, these can progress to severe infections and even death. As reported by The Global Burden of Disease Study, LRTIs were the second-leading cause of deaths in 2013 [1]. The age-standardized mortality rate of LRTIs has been reported to be 41.7 per 100,000 (95% CI 37.1-44.1 per 100,000) [2]. The elderly and people with weakened immune systems are particularly susceptible to these types of infections [3].  
A large and increasing number of pathogens, including bacteria, atypical agents and viruses, are responsible for LRTIs [4; 5; 6]. The clinical presentations and radiology of LRTIs caused by different pathogens are very similar, and this makes it difficult to differentiate these pathogens using only clinical symptoms and lung imaging. The identification of pathogens in severe LRTIs would be ofgreat value in facilitating prompt and successful medical treatment. Therefore, a powerful assay that can simultaneously detect multiple pathogens both quickly and accurately is urgently needed.  
Traditional methods used to identify respiratory pathogens include microbial culture, staining methods, urinary antigen tests, and serological tests [7]. These traditional methods can help identify respiratory pathogens to some extent. However, they usually have particular disadvantages: staining methods have poor sensitivity, culture of pathogens is time-consuming, and the specialized culture media for atypical agents and viruses are not accessible in most hospitals and clinical institutions. Serological responses may occur late, thus requiring paired sera at least a week apart, and such serological assays are commonly used in retrospective diagnosis and systematic surveys [8; 9].  
Molecular methods such as polymerase chain reaction (PCR) and real-time PCR offer high sensitivity and specificity, and have been generated for the rapid identification of respiratory pathogens [10; 11; 12]. These methods can be used to analyze clinical samples, including sputum, bronchoalveolar lavage fluid (BALF) and blood. However, the throughput of most molecular methods is relatively limited, and most molecular methods cannot detect more than five pathogens in one reaction [10; 12; 13; 14]. Therefore, designing a high-throughput, labor-saving, and rapid method to identify and differentiate respiratory pathogens would be important in enhancing rapid response for the prompt treatment of LRTIs.  
Compared with other methods, the Luminex xTAG(r) technology is a high-throughput, rapid, labor-saving multiplex assay offering both high sensitivity and specificity. This technology is reported to detect up to 100 different targets in a single reaction vessel simultaneously [15; 16; 17]. This technology is also very useful because of its flexibility in detecting a broad range of pathogens, which means it can be organized into different multiplex assays for different diagnostic targets, as required.  
In this study, we aimed to develop a high-throughput and useful multiplex molecular method for the rapid detection of respiratory bacteria of public health importance. We developed an assay combining multiplex PCR and Luminex xTAG(r) technology (MPLT) for detection of nine key respiratory bacteria, including _Streptococcus pneumoniae_, _Moraxella catarralis_, _Staphylococcus aureus_, _Streptococcus pyogenes_, _Haemophilus influenzae_, _Mycoplasma pneumoniae_, _Legionella_ spp., _Pseudomonas aeruginosa_, and _Klebsiella pneumoniae_. These pathogens account for most of the respiratory bacteria that precipitate LRTIs [3]. The sensitivity and specificity of the MPLT assay was evaluated, and 86 clinical BALF specimens from LRTI patients were assayed for simultaneous bacteriological detection using the MPLT assay. Using this new method, we compared the results with the data from the nine individual real-time PCRs.