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Paragraph: Furthermore, the ability of PCR/electrospray ionization mass spectrometry (PCR/ESI-MS) to detect enteroviruses and rhinovirus es in respiratory samples has not been well evaluated. We sought to use PCR/ESI-MS to comprehensively investigate the viral epidemiology of adult RTIs, including testing for  es and enteroviruses. Nasopharyngeal or throat swabs from 267 adults with acute RTIs (212 upper RTIs and 55 lower RTIs) who visited a local clinic or the outpatient or emergency departments of a medical center in Taiwan between October 2012 and June 2013 were tested for respiratory viruses by both virus isolation and PCR/ESI-MS. Throat swabs from 15 patients with bacterial infections and 27 individuals without active infections were included as control samples. Respiratory viruses were found in 23.6%, 47.2%, and 47.9% of the 267 cases by virus isolation, PCR/ESI-MS, and both methods, respectively. When both methods were used, the influenza A virus (24.3%) and  es (9.4%) were the most frequently identified viruses, whereas human coronaviruses, human metapneumovirus (hMPV), enteroviruses, adenoviruses, respiratory syncytial virus, and parainfluenza viruses were identified in small proportions of cases (<5% of cases for each type of virus). Coinfection was observed in 4.1% of cases. In the control group, only 1 (2.4%) sample tested positive for a respiratory virus by PCR/ESI-MS. Patients who were undergoing steroid treatment, had an active malignancy, or suffered from chronic obstructive pulmonary disease (COPD) were at risk for  , hMPV, or parainfluenza infections, respectively. Overall, immunocompromised patients, patients with COPD, and patients receiving dialysis were at risk for noninfluenza respiratory virus infection. Rhinoviruses (12.7%), influenza A virus (10.9%), and parainfluenza viruses (7.3%) were the most common viruses involved in the 55 cases of lower RTIs. The factors of parainfluenza infection, old age, and immunosuppression were independently associated with lower RTIs. In conclusion, PCR/ESI-MS improved the diagnostic yield for viral RTIs. Non-influenza respiratory virus infections were associated with patients with comorbidities and with lower RTIs. Additional studies that delineate the clinical need for including non-influenza respiratory viruses in the diagnostic work-up in these populations are warranted. Text: V iral respiratory tract infections (RTIs) in humans occur throughout the year and represent a major cause of clinical visits worldwide. In the past, the viral causes of RTIs were largely unknown, primarily due to the insensitivity of culturebased methods for the detection of viruses or to the narrow spectrum of viral detection using singleplex nucleic acid tests (NATs). Recently, the development of multiplex respiratory NATs has allowed for the simultaneous, rapid, and sensitive detection of multiple viruses, which facilitates comprehensive studies regarding the epidemiology of viral RTIs. Currently, the viral epidemiology of RTIs has been studied more extensively among pediatric populations compared with adult populations throughout the world. 1 Similarly, most studies describing the viral etiology of respiratory illness in Taiwan, a subtropical country in Eastern Asia, were limited to pediatric populations. [2] [3] [4] Thus, studies among adult patients are lacking, particularly regarding infections due to fastidious or newly identified viruses, such as human metapneumovirus (hMPV) and human coronavirus (hCoV). Overlapping clinical presentations shared by different respiratory viruses make differential diagnoses difficult to perform based solely on the clinical parameters. 5 Moreover, effective antiviral agents are currently restricted to influenza virus infections. Hence, a better understanding of the epidemiology of adult viral RTIs would aid the future design of diagnostic strategies, infection control, and patient management. Among the various multiplex NATs, multilocus polymerase chain reaction coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) can simultaneously identify and subtype multiple respiratory viruses. [6] [7] [8] [9] Despite the diagnostic potential, the ability of PCR/ESI-MS to detect human enterovirus and   in respiratory samples from patients with RTIs has not been well evaluated. Previous PCR/ESI-MS studies in patients with RTIs did not include these 2 viruses in the diagnostic panels. [6] [7] [8] [9] Here, we expanded upon these previous studies utilizing PCR/ESI-MS for respiratory virus detection. We aimed to comprehensively investigate the epidemiology of adult viral RTIs using PCR/ESI-MS and compare the diagnostic performance between PCR/ESI-MS and conventional culture methods for identifying multiple, clinically relevant, respiratory viruses, including enterovirus and  . To conduct a comprehensive epidemiologic study that included patients with and without comorbidity, we enrolled adults (of at least 18 yr of age) with acute RTIs within 7 days of onset who were treated at a local outpatient clinic of YC hospital or the outpatient or emergency departments of National Cheng-Kung University Hospital (NCKUH), a university-affiliated medical center in southern Taiwan, between October 2012 and June 2013. Acute RTI was defined as the simultaneous occurrence of at least 1 respiratory symptom or sign (new or worsening cough, sputum production, sore throat, nasal congestion, rhinorrhea, dyspnea, wheezing, or injected tonsils) and at least 1 of the following symptoms: fever, chills, and cough. Lower RTI (LRTI) was defined as the presence of acute RTI and a new infiltrate on chest radiograph. For patients experiencing more than 1 episode of RTI, the most recent episode was counted as separate only if the patient fully recovered from the previous episode and there was a least a 3-week interval between the onset of the 2 episodes. Clinical, laboratory, and radiological data and the contact history of each patient were retrieved. Comorbidities were assessed in all patients based on the Charlson comorbidity index (CCI). 10 Steroid use was defined as the receipt of corticosteroid treatment (10 mg prednisolone or an equivalent daily dosage) for more than 2 weeks. An immunocompromised state was diagnosed if the patients met one of the following conditions: corticosteroid treatment, solid organ or hematopoietic stem cell recipient, or chemotherapy for an underlying malignancy during the past 6 months. Nasopharyngeal or throat swabs were obtained from all patients and collected in transport medium, as previously described. 11 for virus detection and identification by both virus isolation and PCR/ESI-MS. Clinical specimens were stored at 48C and transported to the study sites within 24 hours of collection. Throat swabs from 42 cases without respiratory infections during the month prior to enrollment were included as control samples for PCR/ESI-MS analysis, including 15 patients with exclusively bacterial infections (documented cases of bacteremia or urinary tract infection) who were admitted to NCKUH and 27 individuals without active infections. These subjects without active infections included 10 patients with stable chronic diseases followed up in NCKUH clinics and 17 healthy individuals whose medical information was collected using a clinical questionnaire. The study was approved by the Institutional Review Board (B-ER-101-031) of the study hospital, and all patients provided informed consent. Respiratory specimens were inoculated onto appropriate tissue cultures (Madin-Darby canine kidney, MRC-5, A549, and rhabdomyosarcoma) to isolate human influenza virus, parainfluenza virus, genus Enterovirus, cytomegalovirus (CMV), adenovirus, respiratory syncytial virus (RSV), herpes simplex viruses 1 and 2 (HSV-1 and -2), and varicella zoster virus (VZV). The isolation and identification of viruses were performed using a previously described method 11 and enteroviruses were identified by a immunofluorescence assay using a Chemicon Pan EV mix that cross-reacts with   (Light Diagnostics, Chemicon [Millipore], MA). 11, 12 Virus Detection and Identification by PCR/ESI-MS Total nucleic acids were extracted from 700 mL of swab samples using a nucleic acid autoextractor (MagNA Pure Compact Instrument, Mannheim, Germany), and the eluate was stored at À808C until analysis. During the analyses, the extracted nucleic acids were added to both a PLEX-ID Respiratory Virus assay plate and a PLEX-ID Broad Viral I assay plate (PLEX-ID, Abbott Laboratories, Abbott Park, Illinois). The PLEX-ID Respiratory Virus assay detects human adenovirus, hCoV, hMPV, influenza A and B, parainfluenza types 1 to 3, and RSV, 6 whereas the PLEX-ID Broad Viral I assay detects human adenovirus, enterovirus,  , BK and JC polyomavirus, parvovirus B19, HSV-1 and -2, VZV, Epstein-Barr virus (EBV), CMV, and human herpesvirus (HHV)-8. 13, 14 In this study, respiratory viruses refer to adenovirus, hCoV, hMPV, influenza, parainfluenza, RSV, enterovirus, and  . Nucleic acid amplification and analyses of PCR products were conducted using the PCR/ESI-MS platform (PLEX-ID, Abbott Laboratories) following the manufacturer's instructions, with test turnaround time from sample to result within 6 to 8 hours. 8, 13 The PCR/ESI-MS analyses included automated PCR desalting, ESI-MS signal acquisition, spectral analysis, and data reporting. Organism identification was based on the total mass and base compositions of the PCR amplicons compared with those in the molecular signature database established by the PLEX-ID manufacturer. 6, 8, 13, 14 Samples in which PCR/ESI-MS results disagreed with culture results at the species level were reexamined by a second molecular method. For enteroviruses,   was differentiated from enterovirus using a conventional PCR sequencing analysis with the previously described primers (Rhinovirus s1 and as) and a BLAST search. 15 

All analyses were performed with the Statistical Package for the Social Sciences version 17.0 (SPSS Inc, Chicago, IL). Continuous variables were expressed as mean values AE standard deviations and were compared using the analysis of variance test. Categorical variables were compared using the Fisher exact test or x 2 test. All biologically plausible variables with a P value 0.10 in the univariate analysis were considered for inclusion in the logistic regression model for the multivariate analysis. A P value less than 0.05 was considered statistically significant, and all tests were 2-tailed. During the 9-month study period, a total of 267 episodes of acute RTIs from 263 patients were recorded, including 96 episodes at a local clinic and 171 episodes at NCKUH (19 outpatient and 152 in the emergency departments). For convenience, each episode was counted as 1 case. Overall, 123 (46.1%) cases were male patients, and 152 (56.9%), 60 (22.5%), and 55 (20.6%) patients were 18 to 39, 40 to 59, and !60 years of age, respectively. Two-hundred and twelve (79.4%) patients presented with upper RTIs (URTIs), and 55 (20.6%) cases presented with LRTIs. Compared with patients attending the local clinic, patients attending the medical care center were older and had more comorbidities ( Table 1 ). The detailed demographic data of the 267 RTI cases and 42 control cases are presented in Table 1 . All 267 respiratory samples from each RTI case were examined for viruses by both virus isolation and PCR/ESI-MS, and the results are presented in Table 2 . For virus isolation, respiratory viruses were detected in 63 (23.6%) cases, including influenza A (48 cases, 18.0%), enterovirus (13, 4.9%), and parainfluenza virus (2, 0.7%), and no coinfection was detected. Virus isolation identified additional parainfluenza type 3 and enterovirus infections that were not found by PCR/ESI-MS in 2 samples. By PCR/ESI-MS, respiratory viruses were detected in 126 cases (47.2%). Influenza A (65 cases, 24.3%) was the most frequently identified virus, among which 36 (13.5%) cases were subtyped as pandemic H1N1/09 virus, 28 (10.5%) cases as seasonal H3N2 virus, and 1 case as influenza A matching both pandemic H1N1and seasonal H3N2. Genus Enterovirus (34, 12.7%) was the second-most frequently detected virus, including   (25, 9 .4%), enterovirus (8, 3.0%), and 1 culturenegative case matching for both   and enterovirus. hCoV (13, 4 .9%), hMPV (10, 3.7%), adenovirus (6, 2.2%), RSV (6, 2.2%), and parainfluenza (4, 1.5%) were detected in small proportions of cases. Simultaneous detection of more than 1 respiratory virus was observed in 11 (4.1%) patients, and   (5 cases) was most likely to be codetected with another respiratory virus ( Table 2 ). Of note, 4 cultivated viruses identified as enterovirus because of reactivity with the Chemicon Pan EV mix were characterized as   by PCR/ESI-MS. Further PCR-sequencing analysis of the 4 clinical specimens confirmed the existence of  es but not enteroviruses. PCR/ESI-MS identified additional respiratory viruses in 65 culture-negative samples, mostly   (21 samples), and a second respiratory virus in 3 culture-positive influenza A samples. Overall, the positive detection rates for any respiratory virus by culture, PCR/ESI-MS, and both methods were 23.6%, 47.2%, and 47.9% (128/267), respectively. Of 61 specimens positive by both methods, PCR/ESI-MS and culture methods reached levels of agreement of 100% at the species level for influenza and parainfluenza and 100% at the genus level for the genus Enterovirus.

Question: What was the most frequent coinfection?
rhinovirus