In this study, the performance characteristics of the new FPMC analysis method were evaluated by assessing agreement with results of the sequencing method, a generally accepted standard method. The FPMC analysis method could provide the accessible options for clinical laboratories focusing on the rapid diagnosis of respiratory tract infections using multiplexed molecular method on NPS and sputum samples. Our results demonstrate that the FPMC analysis method provides a large panel of both viral and other respiratory pathogens in a simple design pattern with a relatively less time to obtain results.
Viruses always contribute to the etiology of RTIs and are regarded as the leading cause of several severe RTIs [9]. Our study found that human rhinovirus was the most common in RTI, and that human rhinovirus was the only identified virus in a substantial part of patients. Human rhinovirus are traditionally regarded as be linked to upper respiratory tract infection [10], otitis media [11], and sinusitis [12]. Recently human rhinovirus has been increasingly recognized as one of the lower respiratory tract pathogens, especially in patients with asthma [13], infants [14], elderly patients [15], and immunocompromised adults [16] with the emerging application of PCR assays for detection of respiratory viruses in clinical laboratories. It was reported that human rhinovirus is the etiology of one-half to two-thirds of common colds, which can also bring about considerable economic burden in respect of medical visit [17, 18]. Given the frequency and consequence of human rhinovirus infections, effective control of the virus by prevention and treatment would make significant impacts on public health. Moreover, atypical respiratory pathogens such as mycoplasma pneumoniae and chlamydia pneumoniae have become a public health problem in many countries of the world [19]. There are some reports depicting that symptoms of atypical respiratory infections is identical to viral respiratory infections and that co-infection of atypical respiratory pathogens with other viruses could be also detected [20].
Recently, co-infection with multiple pathogens is growingly acknowledged as be both common and important for disease manifestation. Our study showed that 37 patients had two pathogens, with human rhinovirus plus parainfluenza virus or coronavirus being the most types. Additionally, three patients were found to have three organisms including human rhinovirus, coronavirus and parainfluenza virus/adenovirus. The treatment may be more difficult for patients with co-infection of several organisms relative to those with infection of only one organism. Previous studies have shown that co-infections of viruses with other pathogens were detected in patients with RTIs [21] and this phenomenon was also found in 3 specimens with mycoplasma pneumoniae plus viruses like adenovirus, parainfluenza virus or influenza B virus.
At present, the main methods for detecting viruses include virus isolation and culture, electron microscopy, direct immunofluorescence (IF), indirect IF, alkaline phosphatase and anti-alkaline phosphatase bridge linked enzyme labeling (AP-AAP), biotin streptavidin peroxidase, enzyme-linked immunosorbent assay, and molecular biology methods like multiple reverse transcription polymerase chain reaction (mRT-PCR), nested PCR and nucleic acid hybridization, multiple real-time PCR, gene chip technology, suspension array technology etc. Virus isolation and culture is a most classic method, by which the existence and type of viruses can be objectively exhibited. However, the long culture cycle and low sensitivity of the method greatly limit its application in clinical diagnosis. Although the virus particles could be detected by electron microscopy, it is not suitable for rapid diagnosis in clinical practice owing to several factors such as being time-intensive and relatively low in the positivity rate. For AP-AAP method, although it could be used to detect viral antigens, non-specific results are usual due to many operating procedures and it is difficult to evaluate the accuracy of the positive results. The detection technology of nucleic acid qualitative PCR is mainly based on fluorescence qualitative polymerase chain reaction. Its key steps involve the effective extraction, isolation and purification of nucleic acid in viruses from a sample, and designing the corresponding specific primer sequence. As a new developed technique, multiple real-time qualitative fluorescent PCR analysis could realize rapid screening of nucleic acid detection and typing of multiple pathogens with strong sensitivity and high specificity by using the technology of hybridization or polymerase chain reaction. Moreover, on the basis of ensuring the sensitivity and specificity of analysis, it could perform detection by micro-sample handling and make operation procedures more easily.
Our results demonstrate that the overall performance of the FPMC analysis method has an overall percent agreement (true-positive and true-negative results) of > 99% for all available targets tested compared with the sequencing method. Discrepancy between the FPMC analysis method and the sequencing method may be due to three main factors. Firstly, the sensitivity of the sequencing method may be low, which will lead to the negative results for those weakly positive samples with CT value being near the cut off of the FPMC analysis method. Secondly, primers of the sequencing method may not be able to cover all sub-types of organisms, and thus some organisms in a sample could not be detected using the sequencing method. Finally, FPMC analysis method is a new assay based on PCR reaction. So there are occasionally false-positive results due to the PCR contamination during the process of experiments. In this study, the performance characteristics of the new FPMC analysis method were evaluated by assessing agreement with the results of the sequencing method, a generally accepted standard method.
However, there are still some limitations in our study. Firstly, this study is lack of another molecular-based method for discordant sample adjudications. Comparisons of this FPMC analysis method with another multiplex panel would provide useful information about discordant results with the sequencing method. But this is beyond the designs of our current study. Secondly, the pathogen spectrum of the FPMC analysis method does not include all pathogens. Therefore, combination of the FPMC analysis method and other molecular methods detecting bacteria could help to improve ability in diagnostic testing of respiratory pathogens. Finally, the lack of detection of influenza A virus and Covid-2019 in this study limits the data on the performance for these targets. In our subsequent research, relevant samples will be collected to elucidate the diagnostic efficacy of the new assay kit for influenza A virus and Covid-19. Overall, the FPMC analysis method is a rapid, accurate, and easy-to-use assay for detection of organisms in clinical specimens from the respiratory tract in clinical laboratories.