Detection and clinical characteristics analysis of respiratory viruses in hospitalized children with acute respiratory tract infections by a GeXP‐based multiplex‐PCR assay

Abstract Background The information regarding viral epidemiology and clinical characteristics in hospitalized children with acute respiratory tract infection (ARTI) in central Fujian is limited. In this study, we aimed at analyzing the viral epidemiology and clinical characteristics of ARTI in hospitalized children admitted to The First Affiliated Hospital of Fujian Medical University. Methods Cohort of 386 hospitalized children (31 days to 15 years) diagnosed with ARTI admitted to the Department of Pediatrics from January 1, 2018, to December 31, 2018, was enrolled in this study. Nasopharyngeal swab or sputum samples on the day of hospitalization were tested for 11 viruses via a GeXP‐based multiplex‐PCR assay. The viral profiles and clinical characteristics were analyzed. Results The overall positive rate of the samples was 43.26% (167/386). Among the 167 positive samples, 134 (80.24%, 134/167) had a single virus and 33 (19.76%, 33/167) had multiple viruses. There was a significant difference in the frequency of single vs mixed infections among positive samples (80.24% vs 19.76%; χ 2 = 122.168, P = .000) as well as among the total examined samples (34.72% vs 8.55%; χ 2 = 77.945, P = .000). Human rhinovirus was the most prevalent virus (17.36%, 67/386), followed by influenza A (5.96%, 23/386) and human adenovirus (5.70%, 22/386). There was no significant difference in the etiological distribution of viral pathogens between males and females (χ 2 = 0.480, P = .489). Viral infections were more likely to occur in the winter‐spring months than in the summer‐autumn months (52.51% vs 33.53%, χ 2 = 13.830, P = .000). Conclusions The GeXP‐based multiplex PCR is an accurate and high‐throughput assay allows us to quickly detect multiple respiratory viruses simultaneously in pediatric patients. Our study provides information on the viral profiles and clinical characteristics in hospitalized children with ARTI, which would help better effective prevention strategies.


| INTRODUC TI ON
Acute respiratory tract infection (ARTI) is a leading cause of hospitalization, morbidity, and mortality in children in pediatrics throughout the world. 1 [HADV]) has been identified as a major cause in ARTI in children. 3 Identifying the pathogens of viral infection timely is especially important for early diagnosis and clinical decision-making for the pediatricians.
Presently, several diagnostic methods for the detection of respiratory virus, including virus culture, viral-antigen detection, and viral-antibody detection have been described. 4,5 Virus culture is a gold standard method; however, this method is labor-intensive and time-consuming, making it impractical to be used in the clinical laboratory. 5 Antigen and antibody detection methods are easy-to-perform; however, they exhibit poor sensitivity and may have a false negative or positive reaction. 6 In contrast, molecular techniques, such as polymerase chain reaction (PCR) and real-time fluorescent PCR assays, are sensitive and specific for virus detection 7 ; however, only one virus can be detected by conventional PCR at a time. It should be noted that multiplex-PCR technology can simultaneously detect multiple pathogens at the same time, which is also easy-to-operate and need less workforce. 7 It is well known that ARTI prevalence in children may vary in different geographic regions and different seasons. 4,8 However, information regarding viral ARTI in pediatric hospitalized children in Fuzhou city (central Fujian) is still limited. Thus, to better understand the information about the epidemiology of the pathogens in pediatric hospitalized patients with ARTI and provide effective prevention strategies, we aimed in this study to investigate the epidemiology of respiratory viruses via a GeXP-based multiplex-PCR assay in children under 15 years of age in pediatrics.

| Study design and study population
This study was conducted from January 1, 2018, to December 31, 2018, in The First Affiliated Hospital of Fujian Medical University.
The inclusion criteria were as follows: patients under 15 years old, acute fever, and symptoms of ARTI. The definition of ARTI was according to diagnostic criteria of "Zhu Futang Practical of Pediatrics". 9 Briefly, patients with ARTI appear at least one of the following symptoms: sore throat, cough, shortness of breath, or coryza as an acute onset of symptoms within two days. The exclusion criteria for all participants were as follows: antiviral, antibiotic, or hormonal drug treatment prior to admission; and patients receiving radiotherapy, chemotherapy, or immunosuppressive therapy.

| Sample collection
Nasopharyngeal swab (NPS) or sputum samples were obtained from patients with symptoms of ARTI on the day of hospitalization.

| Total nucleic acid extraction
Total nucleic acid was extracted using a nucleic acid extraction kit following the manufacturer's instruction (Ningbo ZD Biotechnology Co., Ltd).

| Reverse transcription PCR
The 20-μL PCR amplification reaction mixtures contained 14 μL of Premix, 1 μL of RT-PCR reverse transcriptase, and 5 μL of nucleic acid. RT-PCR conditions were as follows: 25°C for 5 minutes and then 50°C for 15 minutes; the reaction was terminated by incubation at 95°C for 2 minutes.

| Statistical analysis
The statistical analysis was performed using statistical analysis software SPSS package version 23.0 for Mac OS X (SPSS Inc). Continuous variables were expressed as means ± standard deviations. Positive percentages in male and female patients and positive percentages among different age groups or different season groups were analyzed with the chi-square test. A P-value <.05 was considered to be statistically significant.

| Confirmation of the GeXP-based assay
RNA-free water and the plasmid carrying the corresponding viral nucleic acid were used as a no-template control (NTC) and positive control (PC), respectively. As indicated in Figure 1, no corresponding peak except the internal reference was observed in the NTC. Whereas 11 virus peaks appeared in the PC, and the peaks were well separated. No crossover reaction was found in PC. These indicated that the results of GeXP-based assay were accurate and reliable.

| Results of respiratory viruses detected overview
The overall positive rate of the samples was 43.26% (167/386).

| Age and gender distributions of the respiratory viruses
The positive rates of viral infection in male and female patients were 44.49% (105/236) and 40.67% (61/150), respectively. There was no significant difference in the etiological distribution of viral pathogens between males and females (χ 2 = 0.480, P = .489). In this study, all the patients were grouped by age as follows: infants (age: <1 year), toddlers (age: 1-3 years), preschoolers (age: 3-5 years), and school-aged children (age: 5-15 years). The distributions of the viral etiologies of the four age groups were shown in Table 2; however, no significant difference among the different age groups was observed.

| Seasonal distribution of the respiratory viruses
The hospitalized children were divided into four groups according to  Table 3. Overall, the positive detection rate was 50.33%, 36.11%, 28.81%, and 57.14% in spring, summer, autumn, and winter, respectively. Viral infections were more likely to occur in winter-spring months than in the summer-autumn months (52.51% vs 33.53%, χ 2 = 13.830, P = .000). It should be noted that HADV occurred more frequently in summer. HRSV was detected almost throughout the year. Other viruses occurred almost sporadically throughout the year without obvious seasonal trends. Currently, nucleic acid amplification techniques (NAATs) (such as PCR and multiplex PCR) have been increasingly used for identification of pathogens in infectious respiratory diseases 7 ; and one of the multiplex-PCR technology, GenomeLab gene expression profiler genetic analysis system (GeXP)-based assay (developed by Beckman Coulter), has been adopted in the infectious pathogens diagnostics [10][11][12] ; this detection technique has been applied in clinical for over 5 years; however, more clinical application studies are still necessary, especially in southeast China. GenomeLab gene expression profiler genetic analysis system is a high-sensitive and specific analytical platform for high-throughput nucleic acid detection based on capillary electrophoresis separation technology, multiple gene analysis technology, and high-sensitivity laser-induced fluorescence technology. 10 The GeXP-based multiplex-PCR assay can easily detect and analyze up to 30 genes simultaneously in a single analysis by converting amplification with multiple primers to amplification with a pair of universal primers. 10 In this study, we used GeXP-based multiplex-PCR assay to detect 11 kinds of virus, as well as mycoplasma pneumonia and chlamydia pneumoniae in 386 samples from hospitalized children with acute respiratory tract infection over a period of 1 year. It was a rapid, accurate, and high-throughput analytical method which can finish the detection process within 4 hours and may aid the pediatricians for early medical decision-making.

| D ISCUSS I ON
Overall, the positive rate for the detection of viruses in the present study was 43.26%; HRV, IFVA, HADV, and H1N1 ranked top four in all the infected viruses. With the application of this multiplex-PCR assay, the detection of multiple co-infecting viruses had become common which reflected the high sensitivity of this multiplex-PCR assay. Kouni S described that the prevalence of viral co-infections ranged between 15% and 44% in children seen in the hospital or emergency department. 13 In the present study, the detection rate of  In our study, gender had no influence on the detection rate of respiratory viruses, which was consistent with other reports. 8,14,15 Many studies documented that respiratory viral infection was characterized by seasonal distributions, with their peaks lasting from winter to early spring. 15,16 In the present study, the detection rate of respiratory viruses was also higher in winter and spring. One possible reason was that climate change may be an important factor contributing to the variability in seasonal trends and viral etiologies of ARTI; the cold weather during this period may increase the risk of respiratory infections. When the analysis was carried out according to the ages, we However, there are several limitations to our study. First, all data for this study were obtained from a single center and may not be representative of the entire pediatric population. Second, the sample size was relatively small, and more samples are needed to confirm these observations in future.
In summary, the GeXP-based multiplex-PCR assay allows us to quickly detect multiple respiratory infections caused by viruses.
The array can detect 11 respiratory viruses simultaneously in only 4 hours. Our study provides information on the viral profiles and clinical characteristics in hospitalized children with ARTI, which would help better effective prevention strategies.

ACK N OWLED G M ENTS
The study was supported by the grant from National Natural Science Foundation of China (81601834). The authors thank all the participants in this study for their cooperation in samples preparation. Also, Huanhuan Huang wants to thank, in particular, the care and love in daily work and life from Professor Miaohui Huang over the past years.