Evaluation of two commercial multiplex PCR tests for the diagnosis of acute respiratory infections in hospitalized children

Acute respiratory tract infections (ARTI), including the common cold, pharyngitis, sinusitis, otitis media, tonsillitis, bronchiolitis and pneumonia are the most common diagnoses among pediatric patients and account for the majority of antibiotic prescriptions. A clear and rapid diagnosis is the key to preventing antibiotic abuse. Recently, based on different detection principles, many multi-target molecular analyses that can simultaneously detect dozens of pathogens have been developed, thereby greatly improving sensitivity and shortening turnaround time. In this work, we conducted a head-to-head comparative study between melting curve analysis (MCA) and capillary electrophoresis assay (CE) in the detection of nine respiratory pathogens in sputum samples collected from hospitalized ARTI childre. The XYres-MCA kit (Geneworks was used for amplication and MCA analysis. Probes are labeled with FAM, ROX and Cy5. MCA was performed after PCR amplication; thus, each hybrid will show unique characteristics through the combination of Tm and its corresponding uorescent probes. The RT-PCR was completed on a thermocycler (Veriti Thermal Cycler, Applied Biosystems China, Beijing, China). Step 1: RT-PCR amplication. Mix 5µL RNA/DNA extraction with 4.3µL Res-reaction mixture1 and 0.7µL Res-enzyme1. The thermal prole was 10 min at 25 °C; 30 min at 50 °C; 15 min at 95 °C; 40 cycles of 30 s at 94 °C, 30 s at 55 °C, 30 s at 72 °C; 10 min at 72 °C. Step 2: PCR Amplication and MCA. 10µL of the RT-PCR product was mixed with 40µL PCR-MCA mixture, containing 39.8µL Res-reaction mixture2 and 0.2µL Res-enzyme2. The thermal prole was 2 min at 95 °C; 10 cycles of 15 s at 94 °C, 15 s at 60 °C, 15 s at 72 °C; 30 cycles of 15 s at 94 °C, 15 s at 55 °C, 15 s at 72 °C; and then, the MCA began with denaturation for 2 min at 95 °C, hybridization for 90 s at 40 °C, and a stepwise temperature increase (1 °C per 20 s) from 40 °C to 90 °C. The levels of FAM, ROX, and Cy5 uorescence were collected and recorded during the MCA procedure.

sensitivity was observed in the melting curve assay. By using this sensitive and rapid test, it may improv patient prognosis and antimicrobial management.

Background
Acute respiratory tract infections (ARTI) is acknowledged as one of the greatest threats to pediatric health [1,2]. Nearly 1.9 million children died from ARTI, most of them from developing country [3]. Accurate early etiologic diagnosis is crucial to the outcome of ARTI [4]. Multiplexed PCR methods enable highthroughput pathogen detection in a short time using limited amount of samples, and this detection strategy may have a signi cant impact on the management of infectious diseases [5][6][7]. Our previous research report pointed out that multiplex-PCR assays applied on an automated capillary electrophoresis system can simultaneously detect human rhinovirus (HRV), in uenza A (FluA), FluA-H1N1, FluA-H3N2, in uenza B (FluB), adenovirus (ADV), human Bocavirus (HBoV), human metapneumovirus (HMPV), parain uenza virus (HPIV), human coronavirus (HCoV), respiratory syncytial virus (RSV), M. pneumoniae and Chlamydia, all in one reaction [8,9]. However, many clinical laboratories, especially lab in the primary care, are not equipped with such highly specialized laboratory equipment, which limits their widespread use in developing countries like China.
In this regard, melting curve analysis (MCA) was developed based on multicolor probe-based uorescence. Tm is a highly reproducible physical parameter, combining it with uorophore signature, MCA assay is feasible identifying certain pathogenic bacteria or viruses [10]. This simple measurement is performed in a closed tube, reducing the possibility of contamination. It takes about 50 minutes to complete, ensuring fast turn-around time. Recently, a new kit based on MCA analysis has been commercialized and can detect FluA, FluB, ADV, HBoV, HMPV, HPIV-1, HPIV-2, HPIV-3, HCoV-229E, HCoV-OC43, RSV and M. pneumoniae. To our knowledge, no studies have been conducted to evaluate the clinical performance of this new method [11][12][13].
This study describes the rst evaluation of the clinical performance of the XYres-MCA kit, compared against the ResP-CE method in a head-to-head manner using 237 sputum specimens from children with ARTI.

Ethics Statement
All sputum samples were collected for their original testing purposes and provided to the study without any patient identity. All aspects of the study were conducted according to the Institutional Review Boards of Children's Hospital of Hebei Province.

Study population and specimen collection
Between April to September 2019, patients who had developed ARTI signs and symptoms were enrolled in this study. Patients were asked to cough, and the expectorated sputum was collected. If the child is too young to cough, a sterile negative pressure suction catheter can be used to obtain the oropharyngeal suction into a transport tube containing VTM (Hopebio Technologies, Qingdao, China). On the same day, the samples were stored at 4 °C for nucleic acid extraction. Before extraction, a pipette tip was used to gently mix the sputum and VTM to liquefy it.
Nucleic acid extraction DNA/RNA extraction is performed on an automated workstation (Smart LabAssist-16/32, Health Gene Technologies, China) [14]. A total of 200µL of lique ed sputum was used for nucleic acid extraction and eluted in 30µL, which was divided into two parts for ResP-CE and XYres-MCA analysis, respectively. ResP-CE analysis has been described in previous work, and we performed the experiments according to previously reported methods [14].

Statistical analyses
The McNemar's test is used to compare the overall positive rate of pathogens between XYres-MCA and ResP-CE. The consistency between the two methods was evaluated using Kappa statistics (κ value 0.21-0.4 fair, 0.41-0.6 moderate, 0.61-0.8 substantial and 0.81-1 almost perfect) [15]. Analysis was performed using SPSS version 19.0 (SPSS Inc., Chicago, USA). If p < .05, it is statistically signi cant.

Patient demographics and diagnosis
A total of 237 patients were included in this study, and their sputum specimens were collected. The main diagnosis was pneumonia (62.4%, Table 2). Participants were more likely to be male (143; 60.3%) with a median age of 1.8 years (interquartile range 0.5-3.0 years).  (Table 3), with a total of 203 positive results (Table 4).   Table 4 shows the number of each pathogen detected by XYres-MCA according to co-infections, diagnosis and age. The most frequently detected pathogen was human parain uenza virus, which was detected in 78 samples, of which 48 (61.5%) were from infants under 1 year old. In addition, the positive detection rates of in uenza B virus, RSV and M. pneumoniae were also related with age.

Performance comparison of XYres-MCA and ResP-CE
Overall, the positive detection rate of XYRes-MCA was signi cantly higher than ResP-CE (72.2% vs. 63.7%, Table 5, p = .002).  The performance for individual targets is presented in Table 7. For most organisms, the agreement between XYRes-MCA and ResP-CE was excellent (κ > 0.61). However, for certain organisms, including FluB and HCoV, XYRes-MCA results seem to be more sensitive than ResP-CE, as the positive detection rate of FluB was 100% vs. 88%, HCoV was 95% vs. 32%.

Discussion
In China, the abuse of antibiotics is recognized as one of the greatest threats to human health, and it is also the major contributor to rising healthcare costs [16]. Due to the clinical similarity between bacterial and viral symptoms, rapid and comprehensive pathogen diagnostic tests should be used to prevent empirical or unnecessary antimicrobial treatment [17]. This study demonstrates for the rst time the clinical performance of the new XYres-MCA analysis in 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. Except for coronavirus and in uenza B virus, the two were very consistent for all pathogens (κ value > 0.61), An increasing number of recently published studies focus on the development of molecular methods to solve multiple detection in one reaction [5,13,14,18,19]. The multiplex-PCR methods has been previously evaluated versus conventional techniques [1,6,8,20] or single-plex PCR [21]. Due to their advantages of automation, high repeatability, cost-effectiveness and high sensitivity, the use of multiplex PCR are recommended as rst-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][23][24]. The CFDA has approved only one multiplex PCR kit, which requires highly specialized equipment worth millions of RMB, namely an automated capillary electrophoresis system, which exceeds the limit of purchasing equipment in most primary hospitals. MCA technology only requires a PCR instrument with four uorescent channels. It is hoped that by comparative research data and low equipment price, it is possible to promote the application of multiple PCR technologies in primary hospitals.
An important aspect of this study is the observation that a higher clinical sensitivity of the MCA in detecting viral infections in clinical samples than CE. The reason for the inconsistent results of in uenza B virus is unclear, but it may be explained by different target gene regions of the virus. In addition, the following two observations may explain why XYres-MCA showed advanced detection of parain uenza virus and coronavirus, whereas a drop in sensitivity for the detection of In uenza A virus. First, when the test is applicable to the entire family or speci c to a single type, the sensitivity may vary [25]. In this study, the subtypes of parain uenza and coronavirus can be distinguished by XYres-MCA, while the subtypes of in uenza A can be distinguished by ResP-CE. Second, 12 pathogens and one internal control (IC) were set in the same reaction in XYres-MCA, while CE detected 13 pathogens, one IC, one human DNA and one human RNA sequences. There are fewer targets in one reaction, resulting in less competition for primers, nucleotides or enzymes, which may lead to increased sensitivity [25,26]. In addition, by using uorophore and Tm value as dual labels, XYres-MCA has the following advantages: it can increases exibility of probe design and can expand cross-platform compatibility, and if required, the assay can 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. Though molecular methods, a positive result may indicate the true pathogen causing the disease or a harmless colonizer. It Is well known that most common respiratory pathogens can be existed in asymptomatic carriers [27], and high co-detection rates have been observed in our previous work and other studies using PCR methods [18,28]. In the routine clinical settings, children with mixed infections of two or more respiratory viruses are common due to the undeveloped immune system, but conventional methods are not easy to nd.
Whether the viral co-infection will cause the illness to become more serious is still controversial [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
Our research has several limitations. First, due to budget restrictions, the study lasted 6 months. This may weaken the ability to capture epidemic pathogens during seasons. Second, as several pathogens (eg, Rhinovirus, Chlamydia) were not included in the target of XYres-MCA, so the performance of XYres-MCA assay to detect these pathogens cannot be determined. Third, compared to ResP-CE, the MCA panel does not contain a human DNA or RNA for monitoring the quality of respiratory specimens.

Conclusions
In conclusion, in this rst clinical study of the new XYres-MCA analysis of 237 samples, we observed excellent sensitivity of this assay compared to another multiplex-PCR assay based on capillary electrophoresis. XYres-MCA analysis may be a new tool for respiratory virus testing in clinical laboratories and may have a positive impact on antimicrobial stewardship and isolation facilities. The exibility of MCA technology in primer design and the characteristics that do not require highly specialized laboratory equipment are expected to play an active role in the emergence of new viruses.