The global incidence of respiratory tract infections is high, leading to millions of deaths annually due to respiratory tract infections. Respiratory tract infections are becoming one of the major infectious diseases threatening human health and are particularly harmful to the frail elderly population and immunocompromised children. In one hospital, 36.7% of deaths in patients <12 years old were due to pneumonia caused by respiratory tract infections [24]. The wide variety of pathogens and similar clinical symptoms of respiratory tract infections pose great difficulties to clinicians for diagnosis and treatment. Although isolation and culture are the gold standards for pathogen diagnosis, the positive detection rate is low because of the cumbersome methods involved and the long turnaround time. Furthermore, some microorganisms (such as mycoplasmas, chlamydiae, and viruses) have a low success rate of in vitro culture. Although there is a corresponding window period for serum immunological examination, the effect of the body’s ability to produce antibodies production ability is of little significance for the early diagnosis of pathogens. Multiplex PCR technology is simple, rapid, and highly sensitive and has been gradually applied to respiratory pathogen detection in clinical practice [25]. Due to the limitation of fluorescence types in each reaction, multiplex real-time PCR can only detect 3–4 pathogens in a single well. If there are many types of pathogens, multi-well detection is bound to be required, making the operation is more complex, requiring more nucleic acid samples, and reducing the throughput. MassARRAY combined with multiplex PCR and mass spectrometry can provide high throughput detection using 96- or 384-well plates with one reaction capable of achieving 40 gene amplifications.
Of the 27 pathogens in our study, only influenza B virus and respiratory syncytial virus had a detection sensitivity of 1000 copies/µl, and the remainder was detected at a sensitivity of 100 copies/μl. Thus, the RT-PCR MassARRAY method established in this study had high detection sensitivity. In the analysis of 207 clinical samples for the detection of respiratory tract pathogens, the positive detection rate was 27.54% (57/207). S. pneumoniae, H. influenzae, M. pneumoniae, and M. catarrhalis are the main pathogens of community-acquired pneumonia and acute bacterial infections [26], and the detection rates of these four pathogens were found to be higher than those of other bacteria, such as Bordetella pertussis and Legionella pneumoniae, in this study. It was also found that among the 20 cases of S. pneumoniae infection, there were only 9 (45.0%) cases of single pathogen infection, and among 11 cases of dual and multiple infections, there were five cases of M. catarrhalis co-infection and four of H. influenzae co-infection, which indicated that co-infections between bacteria were relatively common. Furthermore, the antibiotic resistance of these three bacteria is different [27]. Comprehensive and detailed pathogen diagnosis is of great significance in guiding the rational use of antibiotics to avoid the inappropriate prescription of antibiotics. There were 21/207 (10.1%) samples that tested positive for viruses (note: there were two samples co-infected by three viruses). In China, viral infections are highly prevalent in autumn and winter and relatively low in spring and summer [28]. The samples in this study were collected from clinical patients from April to July (during spring and summer), which may be one of the reasons for the low positive rate of viral respiratory tract infections. PIV was detected in ten cases, including two cases of PIV1, three of PIV2, two of PIV3, and three of PIV4. There was no significant difference in the number of subtypes, which was different from the positive detection rates of PIV types 1, 2, 3, and 4 reported by Wang et al. (2019) in Shanghai, of 2.74%, 0.62%, 8.59%, and 3.40%, respectively [29]. This may be because that Wang et al.’s study population was mainly children, and PIV3 is one of the leading causes of lower respiratory tract infections in infants as well as immunocompromised people [30]. In this study, seven cases (3.38%) of HMPV were detected, which was close to the positive detection rate of HMPV of 3.53% reported in 2018 by Zhong et al. (2019) [31]. We also detected six (2.90%) cases of influenza A virus, all of which were H1N1. Additionally, respiratory syncytial virus was detected in two cases and adenovirus and human coronavirus in one case each. Due to the limited number of samples and sample types, some subtypes of human bocavirus; human enterovirus; influenza B; and influenzae A H3, H3N2 seasonal, H5, and H7 were not detected in this study.
The MRT-MassARRAY respiratory pathogen detection system established in this study detected 27 respiratory pathogens and showed sensitivity and specificity similar to the results obtained using consensus PCR/RT-PCR. The advantage over real-time PCR is mainly reflected in the multiplicity of nucleic acid mass spectrometry amplification. In this study, a 27-plex one tube amplification mode was used, and the test cost was as low as 15 dollars per sample. MassARRAY detection uses 96/384 chips, with a high throughput of up to 384 samples. Based on this feature, our detection system is highly suited to early clinical pathogen screening and regional respiratory pathogen epidemiological investigations.