In China, the misuse of antibiotics is acknowledged as one of the greatest threats to people health, as well as a major contributor to rising healthcare costs [16]. Due to the clinical similarity between bacterial and viral symptoms, the rapid and comprehensive pathogen diagnostic testing should be used to prevent empiric or unnecessary antimicrobial treatment [17]. This study demonstrates for the first time the clinical performance of a new XYres-MCA analysis on the detection of 12 types/subtypes respiratory pathogens. Compared with a CFDA approval multiplex-PCR assay, XYres-MCA can detect a higher percentage of positive results. The agreement between the two tests was strong for all pathogens (kappa test > 0.60), except for coronavirus and influenza B virus.
An increasing number of recently published studies have focused on the development of molecular methods to solve multiple detection in one reaction [5, 13, 14, 18, 19]. The multiplex-PCR methods had been previously evaluated versus conventional techniques [1, 6, 8, 20] or with mono-plex PCR [21]. Because of their advantage of automated, highly reproducible, cost-effective and excellent sensitivity, the use of multiplex PCR testing is recommended as first-line tests for detection of respiratory pathogens [7]. The comparative study is worthy of performed as the multiplex-PCR kits vary widely from a manufactory to another, on the scope of tested pathogens, detection principles and the equipment used [6, 22–24]. Only one multiplex PCR kit has been approved by CFDA, and this kit needs to be used on a highly specialized equipment, worth millions of RMB, namely automated capillary electrophoresis system, which exceeds the limit for equipment purchasing in most primary hospitals. MCA technology requires only one PCR instrument with four fluorescent channels. It is hoped that the comparative research data and the low price of equipment can help promote the application of multiple PCR technologies in primary hospitals.
An important aspect of this study was the observation that a higher clinical sensitivity of the MCA in the detection of virus infections in clinical samples in contrast to the CE. The reason for the inconsistent results of influenza B virus is unclear but may potentially be explained by different targeted gene regions of the virus. Furthermore, the following two aspects of observation may explain why the XYres-MCA showed an advanced detection of parainfluenza virus and coronavirus, whereas a drop in sensitivity for the detection of Influenza A virus. First, the sensitivity may vary when the test is applicable to the entire family or is specific to a single type [25]. In the present study, subtypes of parainfluenza and coronavirus can be distinguished by XYres-MCA, and subtypes of influenza A can be distinguished by ResP-CE. Second, 12 types of pathogens and one internal control (IC) are set in the same reaction in XYres-MCA, whereas CE detects 13 pathogens, one IC, one human DNA and one human RNA sequences. Fewer targets in one reaction results in reduced competition for primers, nucleotides or enzymes, which may lead to the increased sensitivity [25, 26]. In addition, By using the fluorophore and Tm value as dual labels, XYres-MCA has the combined advantages of improved flexibility in probe design and expanded cross-platform compatibility, and if required, the assay can be changed to accommodate more probes to detect new viruses or their subtypes.
In terms of co-detection, the core question is how to interpret it in a clinical sense. By molecular methods, a positive result may indicate the true pathogen causing the disease or a harmless colonizer. It Is known that most common respiratory pathogens can be existed in asymptomatic carriers [27] and a high co-detection rate was observed in our previous study and other studies using PCR methods [18, 28]. In routine clinical settings, children with mixed infections of two or more respiratory viruses are common due to undeveloped immune system but are not easily detected by conventional methods. Whether viral co-infection causes the illness to be more severe is still under debate [29, 30]. Therefore, results of additional detected viruses must be interpreted with caution, and their clinical relevance needs to be correlated in further studies.
Limitations
There were several limitations in our study. First, due to the budget restrictions the study lasted 6 months. This may weaken the ability to capture epidemic pathogens during seasons not covered by this study. Second, as several pathogens, eg, Rhinovirus, Chlamydia, were not included into the target of XYres-MCA, the performance of XYres-MCA assay for detection of these pathogens is uncertain. Third, compared with ResP-CE, the MCA panel does not contain a human DNA or RNA to monitor the quality of respiratory specimens.