key: cord-0903671-flwsxr7d
authors: Chiu, Yu-ting; Tien, Ni; Lin, Hsiao-Chuan; Wei, Hsiu-Mei; Lai, Huan-Cheng; Chen, Jiun-An; Low, Yan-Yi; Lin, Hsiu-Hsien; Hsu, Yu-Lung; Hwang, Kao-Pin
title: Detection of Respiratory Pathogens by Application of Multiplex PCR Panel During Early Period of COVID-19 Pandemic in a Tertiary Hospital in Central Taiwan
date: 2021-10-06
journal: J Microbiol Immunol Infect
DOI: 10.1016/j.jmii.2021.09.011
sha: 0e3207cd1c696cb465653ef1616c9ec9cbb93dcf
doc_id: 903671
cord_uid: flwsxr7d

Background Respiratory tract infections (RTIs) represent a major cause of clinical visits worldwide. Viral epidemiology of RTIs in adults has been less studied compared to children. FilmArray respiratory panel (FA-RP), a multiplex, real time polymerase chain reaction method can simultaneously detect the nucleic acids of multiple pathogens. The purpose of this study is to analyze the epidemiology and clinical presentations of an RTI cohort. Methods This retrospective cohort study was conducted at China Medical University Hospital (CMUH) and China Medical University Children’s Hospital (CMUCH), from January 2020 to June 2020. The FA-RP results were collected and analyzed according to upper versus lower RTIs. Results Among 253 respiratory samples tested, 135 (53.4%) were from adults and 118 (46.6%) from children. A total positive rate of 33.9% (86/253) was found, with 21.48% (29/135) in adults and 48.31% (57/118) in children. Human rhinovirus/Enterovirus (HRV/EV) was detected in most of the age groups and was more common in URIs. HRV/EV was found as a frequent co-detection virus. Among children, HRV/EV was the most detected pathogen of URIs, while the most predominant pathogen in LRIs was M. pneumoniae. Conclusions FA-RP has the potential to improve the detection rate of respiratory pathogens. The positive rate of FA-RP was higher in children compared to adults, which likely corresponds to the higher incidence of viral RTIs in children. Different pathogens may lead to different types of respiratory infections.

Respiratory tract infections (RTIs), which range from a common cold to severe pneumonia, represent a major cause of clinical visits worldwide. Although bacterial etiology remains the common cause of pneumonia, viral respiratory infections are responsible for a number of hospital admissions. 1, 2, 3 Viruses and atypical pathogens are major causes of pediatric RTIs. Adenovirus (ADV), human rhinovirus (HRV), influenza virus (FLU), and respiratory syncytial virus (RSV) are the most common pathogens. In addition to viruses, Mycoplasma pneumoniae (M. pneumoniae) is the most common atypical pathogen affecting children. 4 In comparison, the viral epidemiology of RTIs in adults has been less studied compared to the children population. 5 Conventional diagnostic tests for the identification of pathogens of RTIs, including culturebased and immunological methods, are widely used. However, these methods are time-consuming and cumbersome. New diagnostic methods, such as multiplex nucleic acid amplification tests (NAAT) and molecular point-of-care testing (POCT) are faster and highly sensitive and have the potential to improve the detection rate of respiratory pathogens. 6, 7, 8 One study reported a 30% to 50% increase in the diagnosis of respiratory viruses with the use of a multiplexed molecular test compared to culture-based and immunological methods. 9 The accurate and rapid detection of causative pathogens plays a significant role in selecting appropriate therapy, minimizing therapy costs, and controlling the disease.

In December 2019, infections with the new pandemic pathogen Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), emerged in Wuhan, China, 10 resulting in a global J o u r n a l P r e -p r o o f outbreak by the following year. Some pandemic-prevention policies, including border controls and mandatory quarantines, had been implemented in Taiwan. People have also been required to wear face masks when taking public transportation and to reduce participation in cluster activities. Thus, the viral epidemiology of RTIs in Taiwan seemed to undergo huge changes in 2020. 11 In this study, we investigate the epidemiology of respiratory viruses identified in the respiratory specimens of patients with respiratory tract infections by using the BioFire FilmArray ® Respiratory Panel (FA-RP). Viral distributions in different age groups and the locations of RTIs are discussed separately.

This observational retrospective study was conducted at China Medical University Hospital (CMUH) and the adjacent China Medical University Children's Hospital (CMUCH), a tertiary hospital in Taichung, Taiwan. From January 1, 2020 to June 30, 2020, the early period of the COVID-19 pandemic in Taiwan, we investigated the testing results of nasopharyngeal specimens from patients who visited our hospitals. Those who had fever or symptoms of RTIs were included in the study; otherwise, patients were excluded. The tested samples were collected from the outpatient department (OPD), emergency department (ED), general wards, and intensive care units 

This study was approved by the Institutional Review Board of CMUH (CMUH19-REC3-108), with a waiver regarding informed consent. All methods of this study were performed in accordance with the relevant guidelines and regulations.

The data in this study were analyzed by the chi-square test, Fisher's exact tests and Kruskal-Wallis H test. All statistical analyses were performed using IBM SPSS Statistics version 25.0 (IBM Co., Armonk, NY, USA).

A total of 253 respiratory samples were collected, among which 104 pathogens were detected.

Among 253 respiratory samples tested, 118/253 (46.6%) were from children and 135/253 (53.4%) were from adults. The numbers of each age group are evenly distributed, apart from the age group between 18-44 years. The percentage of patients with LRIs (56.9%) is greater than that of patients with URIs (36.0%). The overall positive rate is 33.9% (86/253), which is higher in the children's group (57/118, 48.31%) compared to that in the adult group (29/135, 21.48%). The positive rate among 2-4 age group is the highest (22/28, 78.57%), followed by the age groups of ≦2 years ( 20/29, 68.97%) and 18-44 years (19/67, 28.36%). The negative samples were mostly attributed to patients in the older age group ( Table 1) .

The number of positive samples by specific viral agent and month of diagnosis are shown in Table 1 ). The results vary between children and adults, as shown in Table 2 . In the children's group, HRV/EV were the most common isolated pathogens, followed by ADV and M. pneumoniae. In the adult group, HRV/EV were the most common, followed by FLU, ADV, and M. pneumoniae.

Detection of more than one respiratory pathogen was found in 17/86 (19.77%) of the positive samples, with a higher co-detection rate in the children's group (14/57, 24.56%) than in the adult group (3/29, 10.34%). However, this was not found to be statistically significant with a p value of 0.156. Fifteen samples detected two respiratory pathogens, one sample detected three respiratory pathogens, and one sample detected four respiratory pathogens. HRV/EV is found in most of the multiple isolations, making it the most frequent isolated pathogen. The most common combinations of isolated pathogens are HRV/EV and ADV (total 6/17, 35.29%), as shown in Supplementary   Table 2 . 

Respiratory infections are a common cause of morbidity worldwide. In particular, viruses and atypical pathogens are major causes of pediatric RTIs. 12 Multiplex nucleic acid-based molecular detection techniques have the potential to improve the detection rate of respiratory pathogens, and reduce antibiotic use. 8, 13 With the increasing availability of multiplex PCR panels, some viruses, such as HMPV, human coronavirus, and HRV, which have previously been difficult to cultivate, are now being detected frequently using the NAAT methods.

In our study, 253 nasopharyngeal specimens were collected and analyzed with FA-PR over a In our study, results showed that HRV/EV was the most commonly identified virus in almost all age groups, especially infected young children, in accordance with previously published data. 14, 19-21 HRV is difficult to culture, while multiplex PCR panel allows the rapid detection of HRV. 22 Human Rhinoviruses (HRVs) are small (15-30nm), non-enveloped viruses containing a single- Therefore, recurrent or overlapping infections caused by different genotypes of HRVs are frequent.

The above reasons may explain the predominant detected respiratory pathogens. 25 Influenza virus, previously thought to be the most common pathogen, 3 pandemic, wearing face masks became a daily routine for people living in Taiwan. We thus observed a decline in the proportion of viral infections afterward. Although a study demonstrated that surgical masks can efficaciously reduce the spread of FLU particles within respiratory droplets into the environment, 24 a Cochrane study demonstrated no clear reduction in respiratory viral infections from using surgical masks during seasonal influenza. 27 Adenovirus, a non-enveloped virus, represents the second most common pathogen in children's group. In our study, the age group younger than 5 years seem to be at a higher risk of getting infected.

Taiwan, with a high prevalence amongst school-aged children and adolescents. When infected with M. pneumoniae, children younger than 3 years tend to develop upper airway infections, whereas children between 5 -20 years tend to develop acute bronchitis and pneumonia. 28 Co-infection has been found in 31-51.8% of positive respiratory samples in previous studies. 19, 21, 23, [29] [30] [31] However, in our study, a lower rate of 19.77% co-infection was detected, with a higher rate of co-infection noted in children (24.56%). The largest proportion of co-detected pathogens was J o u r n a l P r e -p r o o f HRV/EV. Combinations of HRV/ADV and HRV/RSV have been previously found as the most frequent co-infection viruses. 14, 32 However, one study suggested that the presence of RSV reduces the probability of HRV infection. 25 In our study, the most common combination of co-infections occurred with HRV/EV and adenovirus (35.7% in the children's group), and the combination of HRV/EV and RSV was found in one child presenting with lower RTI. HRV causes clinical symptoms in patients with co-infections, rather than being just an incidental finding. 25 Although the high sensitivity of PCR allows the detection of minute amounts of viral nucleic acids, questions remain concerning the clinical relevance of positive test results. 33 Detection of respiratory viruses could be the result of post-infectious shedding or asymptomatic colonization, and may not represent acute infection. 33 Viral cultures must be performed to document long durations of virus shedding.

However, concomitant viral culture with PCR is not performed in co-detection patients to document this idea. In contrast to RSV, hMPV, and ADV, viruses such as HRV and hCoV have been frequently found in asymptomatic children. This suggests that a causal inference based on the detection of these viruses in symptomatic patients should be made with caution. 33 In this study, we demonstrated that HRV/EV was the most common detected pathogen in URIs, while M. pneumoniae is predominant in LRIs, which is found to be statistically significant in the children's group (p=0.035). It is well known that HRV is a frequently detected respiratory virus in children with mild respiratory infections and can commonly cause URIs. However, HRV may also lead to more severe respiratory tract symptoms, such as pneumonia, bronchiolitis, and asthma. 34 In our study, seven children infected with HRV suffered from dyspnea.

This study has some limitations that should be addressed in future works. First, our sample size is based on specimens collected from a single location in the period of six months. This may limit the general applicability of the findings and viral epidemiology may also be affected by seasonal was not available in our hospital during the study period.

In conclusion, FA-RP has the potential to improve the detection rate of respiratory pathogens. Data are presented as case number (percentages).

J o u r n a l P r e -p r o o f Data are presented as case number (percentages).

Detection of more than one respiratory pathogen was found in 17/86 (19.77%) of the positive samples, with a higher co-detection rate in the children's group (14/57, 24.56%) than in the adult group (3/29, 10.34%). However, this was not found to be statistically significant with a p value = 0.156.

J o u r n a l P r e -p r o o f 

Epidemiology and etiology of childhood pneumonia

Respiratory viral infections among hospitalized adults: Experience of a single tertiary healthcare hospital. Influenza Other Respir Viruses

Viruses of Respiratory Tract: An Observational Retrospective Study on Hospitalized Patients in Rome

Community-acquired pneumonia requiring hospitalization among U.S. children

Viral respiratory tract infections in adult patients attending outpatient and emergency departments

Recent advances in the detection of respiratory virus infection in humans

Multiplex PCR system for the rapid diagnosis of respiratory virus infection: systematic review and meta-analysis

Molecular point-of-care testing for respiratory viruses versus routine clinical care in adults with acute respiratory illness presenting to secondary care: a pragmatic randomised controlled trial protocol (ResPOC)

The FilmArray® respiratory panel: an automated, broadly multiplexed molecular test for the rapid and accurate detection of respiratory pathogens

Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study

Systematic review and meta-analysis of respiratory viral coinfections in children

Multicenter Evaluation of BioFire FilmArray Respiratory Panel 2 for Detection of Viruses and Bacteria in Nasopharyngeal Swab Samples

Age-related prevalence of common upper respiratory pathogens, based on the application of the FilmArray Respiratory panel in a tertiary hospital in Greece

Rapid detection of respiratory organisms with the FilmArray respiratory panel in a large children's hospital in China

Contribution of the FilmArray Respiratory Panel in the management of adult and pediatric patients attending the emergency room during 2015-2016 influenza epidemics: An interventional study

Pathogens Causing Respiratory Tract Infections in Children Less Than 5 Years of

Seasonal variations of 15 respiratory agents illustrated by the application of a multiplex polymerase chain reaction assay

Bacterial and viral pathogen spectra of acute respiratory infections in under-5 children in hospital settings in Dhaka city

Rhinovirus associated with severe lower respiratory tract infections in children

Seasonal variations of respiratory viruses and etiology of human rhinovirus infection in children

Rhinovirus-from bench to bedside

Survival of rhinoviruses on human fingers

Respiratory virus shedding in exhaled breath and efficacy of face masks

Interference between respiratory syncytial virus and rhinovirus in respiratory tract infections in children

Prevalence of respiratory virus in symptomatic children in private physician office settings in five communities of the state of Veracruz

Physical interventions to interrupt or reduce the spread of respiratory viruses (Review)

Clinical and epidemiological characteristics in children with community-acquired mycoplasma pneumonia in Taiwan: A nationwide surveillance

codetection and seasonal distribution of upper airway viruses and bacteria in children with acute respiratory illnesses with cough as a symptom

Etiology, seasonality, and clinical characterization of viral respiratory infections among hospitalized children in Beirut, Lebanon

Multiple viral infection detected from influenza-like illness cases in Indonesia

Viral co-infections in pediatric patients hospitalized with lower tract acute respiratory infections

Frequent detection of respiratory viruses without symptoms: toward defining clinically relevant cutoff values

Genotypic Diversity and Epidemiology of Human Rhinovirus Among Children with Severe Acute Respiratory Tract Infection in Shanghai