RESEARCH ARTICLE Human coronavirus alone or in co infection with rhinovirus C is a risk factor for severe respiratory disease and admission to the pediatric intensive care unit A one year study in Southeast Brazil Alessandra K Matsuno ID 1 Talita B Gagliardi 2 Flavia E Paula 2 Luciano K S Luna 2 Bruna L S Jesus 2 Renato T Stein 3 Davi C Aragon ID 1 Ana P C P Carlotti 1 Eurico Arruda 2 1 Department of Pediatrics Ribeir o Preto Medical School University of S o Paulo Ribeir o Preto Brazil 2 Department of Cell Biology and Virology Research Center Ribeir o Preto Medical School University of Sao Paulo Ribeir o Preto Brazil 3 Department of Pediatrics Pontif cia Universidade Cato lica do Rio Grande do Sul School of Medicine Porto Alegre Brazil These authors contributed equally to this work These authors also contributed equally to this work matsuno fmrp usp br Abstract Objective We aimed to assess the profile of respiratory viruses in young children hospitalized for acute lower respiratory tract infection ALRI and its association with disease severity defined as need for pediatric intensive care unit PICU admission Design Prospective observational cohort study Setting A tertiary care university hospital in Brazil Patients Children younger than three years attending the pediatric emergency room with ALRI who were admitted to the hospital Interventions None PLOS ONE https doi org 10 1371 journal pone 0217744 June 3 2019 1 9 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation Matsuno AK Gagliardi TB Paula FE Luna LKS Jesus BLS Stein RT et al 2019 Human coronavirus alone or in co infection with rhinovirus C is a risk factor for severe respiratory disease and admission to the pediatric intensive care unit A one year study in Southeast Brazil PLoS ONE 14 6 e0217744 https doi org 10 1371 journal pone 0217744 Editor Raymond J Pickles University of North Carolina at Chapel Hill UNITED STATES Received October 2 2018 Accepted May 19 2019 Published June 3 2019 Copyright 2019 Matsuno et al This is an open access article distributed under the terms of the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original author and source are credited Data Availability Statement All relevant data are within the manuscript and its Supporting Information files Funding AKM and EAN supported mainly by an academic grant from Abbott Laborato rios do Brasil Ltda based on an unrestricted investigator generated proposal EAN Additional funding was provided by a grant from the Sao Paulo State Research Foundation FAPESP and by FAEPA Measurements and main results Nasopharyngeal aspirates were collected from patients from June 1 st 2008 to May 31 st 2009within the first 48 hours of hospitalization Nasopharyngeal aspirates were tested for 17humanrespiratory viruses by molecular and immunofluorescence based assays Simple and multiple log binomial regression models were constructed to assess associations of virus type with a need for PICU admission Age prematurity the presence of an underlying disease and congenital heart disease were covariates Nasopharyngeal aspirates were pos itive for at least one virus in 236 patients Rhinoviruses were detected in 85 6 of samples with a preponderance of rhinovirus C RV C 61 9 Respiratory syncytial virus was detected in 59 8 and human coronavirus HCoV in 11 of the samples Co detections of two to five viruses were found in 78 of the patients The detection of HCoV alone adjusted relative risk RR 2 18 95 CI 1 15 4 15 or in co infection with RV C adjusted RR 2 37 95 CI 1 23 4 58 was independently associated with PICU admission Conclusions The detection of HCoV alone or in co infection with RV C was independently associated with PICU admission in young children hospitalized for ALRI Introduction Acute lower respiratory tract infections ALRI are responsible for the death of approximately 160 000 neonates and over 760 000 infants annually 1 Respiratory syncytial virus RSV and rhinovirus RV are the most frequent causes of acute respiratory infections in children 2 4 Primary RSV infections may lead to severe bronchiolitis and pneumonia 5 RV usually causes rhinopharyngitis 6 but may also cause illness of the lower respiratory tract and asthma exacer bations and has been associated with severe ALRI in young children 7 8 A few studies have investigated the viral profile in children attending the pediatric emergency room ER with ALRI and its association with disease severity 9 12 but finding vary Moreover the role of viral co infections in illness severity has been controversial 10 12 13 15 In young children who presented to an outpatient clinic or ER in Japan with acute respiratory illness RSV RV parainfluenza viruses PIV and human metapneumovirus HMPV were the most prevalent viruses Similarly to what was found in a previous study in Brazil 4 detection of RSV alone or in co detections was associated with increased disease severity in children in Japan 9 In Seat tle USA RV was the most frequent virus detected in children less than three years old present ing to the ER with ALRI and 52 of them required hospital admission 10 In addition RV viral load and co detection with RSV was associated with more severe disease in that study 10 In children younger than two years old who presented to a pediatric ER with ALRI in Malaysia RSV and RV were the most frequently detected viruses and although RSV was associated with a history of wheezing virus detection was not associated with need for hospitalization 11 In The Netherlands the most frequent viruses detected in children presenting to the ER or outpa tient clinic with ALRI were RSV RV and human coronavirus HCoV and RSV detection cor related with longer duration of oxygen therapy 12 However there was no association between the virus species and hospital length of stay and virus co detections were not associ ated with disease outcome 12 In the present study a comprehensive polymerase chain reac tion PCR panel of primers and probes was used to detect respiratory viruses enabling virus Human coronavirus alone or in coinfection with rhinovirus C is a risk factor for severe respiratory disease PLOS ONE https doi org 10 1371 journal pone 0217744 June 3 2019 2 9 Fundac o de Apoio ao Ensino Pesquisa e Assist ncia in English would be Foundation of Support to Teaching Research and Assistance of the University of Sao Paulo Hospital of Ribeir o Preto EA has long standing scholarship support from the Brazilian Research Council CNPq The funder had no role in study design data collection and analysis decision to publish or preparation of the manuscript Competing interests AKM and EAN were supported by an academic grant from Abbott Laborato rios do Brasil Ltda based on an unrestricted investigator generated proposal This does not alter our adherence to PLOS ONE policies on sharing data and materials as detailed online in the guide for authors https journals plos org plosone s competing interests identification to the species level in respiratory samples collected from children younger than three years with ALRI seen at the pediatric ER and subsequently admitted to hospital Associa tion of different respiratory viruses with disease severity defined as need for admission to the pediatric intensive care unit PICU was assessed Material and methods Patients and samples This was a prospective cohort study conducted in a tertiary care university hospital in Brazil The study was approved by the Research Ethics Committee of the Clinical Hospital School of Medicine of Ribeir o Preto University of S o Paulo a tertiary care facility in the city of Ribeir o Preto state of Sao Paulo Brazil A written informed consent was obtained from parents guardians protocol number HCRP A07 020 All consecutive children younger than three years attending the pediatric emergency room with ALRI who were admitted to hospital from June 1 st 2008 to May 31 st 2009were eligible for the study ALRI was defined by the pres ence of cough tachypnea respiratory distress with prolonged expiratory time and wheezing or crackles on auscultation Patients with a diagnosis of bacterial pneumonia as indicated by clinical presentation and chest X ray findings or a positive blood culture were excluded from analysis Demographic clinical and outcome data were collected from patients health records Need for PICU admission was considered the main indicator of disease severity Nasopharyngeal aspirates were collected from patients within the first 48 hours of hospitalization as previously described 16 and they were split into aliquots two 250 L aliquots mixed with 750 L of TRI zol Invitrogen Thermo Fisher Scientific MA USA to DNA RNA extraction and two 500 L backup aliquots mixed 1 1 with 500 L of viral transport medium which consists of minimal essential medium with Eagle s salts plus 20 fetal bovine serum 15 glycerol and 1 antibi otic antimycotic solution GIBco Thermo All backup aliquots were stored at 70 C until analysis Rapid screening for RSV Nasopharyngeal aspirates were routinely screened for RSV by eithera rapid chromatographic immunoassay for RSV antigen Directigen EZ RSV Test Becton Dickinson and Company Franklin Lakes NJ USA or indirect immunofluorescence IF assay Directigen EZ RSV was performed on 250 L of nasopharyngeal aspirates following the manufacturer s protocol Indi rect IF assay was performed with RSV specific monoclonal antibody MAb 858 4 Millipore MA USA diluted 1 100 in phosphate buffered saline GIBco Thermo revealed with Alexa Fluor 488 labeled donkey anti mouse IgG Life Technologies Carlsbad CA USA diluted 1 200 in phosphate buffered saline Slide preparation and IF protocols are available at http dx doi org 10 17504 protocols io w8rfhv6 and http dx doi org 10 17504 protocols io w8ufhww Detection of RNA and DNA respiratory viruses by real time PCR TRIzol aliquots from each sample were used to do DNA and RNA extractions Total RNA was extracted following manufacturer s protocol with some adaptations http dx doi org 10 17504 protocols io w8vfhw6 and DNA enriched fractions were used for DNA purification using the Wizard Genomic DNA purification kit Promega Madison WI USA as per manu facturer s protocol One microgram of total RNA was used in a reverse transcription reaction carried out with Multiscribe reverse transcriptase Applied Biosystems Thermo primed Human coronavirus alone or in coinfection with rhinovirus C is a risk factor for severe respiratory disease PLOS ONE https doi org 10 1371 journal pone 0217744 June 3 2019 3 9with random hexamers and following manufacturer s protocol This random cDNA was used in TaqMan real time PCR assays to detect RSV A and B HMPV A and B human influenza viruses FLU A and B and PIV 1 and 3 Human beta actin housekeeping gene was used as internal controls in all assays All PCR assays were performed using only one set of primers probe per reaction except for RSV and HMPV detection which were tested in duplex format Human bocavirus HBoV and human adenovirus HAdV were detected by qPCR in a single format assay http dx doi org 10 17504 protocols io w8yfhxw HCoV detection was per formed by a nested RT PCR method that uses primers targeting the RNA dependent RNA polymerasegene 17 Rhinovirus RV detection wasperformed by a two step PCR method http dx doi org 10 17504 protocols io w83fhyn 18 19 All PCR reactions were performed on a Thermocycler 7300 Applied Biosystems Thermo using published primers and probes sequences 20 All real time PCR plates included appro priate negative controls matched to every step of the testing of nasopharyngeal aspirates Nega tive controls were total RNA or DNA extracted from uninfected Hela cells and ultrapure water treated in the same way as clinical samples Statistical analysis The analysis was made using SAS 9 4 SAS STAT User s Guide Version 9 4 Cary NC SAS Institute Inc 2013 Data were expressed as median range or number Patients were grouped according to the need for PICU admission Continuous variables between groups were compared by Mann Whitney U test and categorical variables by Fisher s exact test Sim ple and multiple log binomial regression models were constructed to assess associations of virus type with need for PICU admission Relative risks RR and 95 confidence intervals 95 CI were obtained after adjusting log binomial regression models Initially simple log binomial regression models were fitted resulting in crude relative risks Subsequently the adjustment of multiple log binomial regression models considering age prematurity the pres ence of an underlying disease and congenital heart disease as covariates resulted in adjusted relative risks 21 A 5 significance level was considered in all analysis Results Over the study period nasopharyngeal aspirates were collected from 279 children seen at the pediatric emergency room with ALRI Thestudy population comprised 236 patients who had at least one respiratory virus detected in respiratory specimens All patients were hospitalized Twenty five percent of patients n 60 had comorbidities The most common underlying dis eases were neurological impairment n 17 congenital anomalies n 13 and chronic lung disease n 11 Forty seven patients 19 9 were admitted to the PICU 25 of them 53 2 received invasive mechanical ventilation for a median time of 7 days range 1 to 99 days Length of PICU stay ranged from 1 to 254 days median 9 5 days Demographic data were not significantly different between patients admitted to the PICU compared with those who did not need PICU admission However use of systemic antibiotics and the presence of underly ing diseases and congenital heart disease were more frequent in patients admitted to the PICU and they also had a longer hospital length of stay Table 1 The most frequently detected virus was RV 85 6 followed by RSV 59 8 HBoV 23 7 HMPV 17 8 HCoV 11 4 HAdV 10 6 PIV 10 2 and FLU 8 5 Co detections were found in 182 78 patients S1 Table The results of multiple log binomial regression analyses showed thatthe detection of HCoV alone adjusted relative risk RR 2 18 95 CI 1 15 4 15 or in co infection with RV C adjusted RR 2 37 95 CI 1 23 4 58 was independently associated with PICU admission S2 Table Human coronavirus alone or in coinfection with rhinovirus C is a risk factor for severe respiratory disease PLOS ONE https doi org 10 1371 journal pone 0217744 June 3 2019 4 9Eight patients 3 4 died Their median age was 3 5 months range 2 3 9 3 months six 75 were female Six patients 75 who died had comorbidities hydrocephalus n 3 Pompe disease n 1 Down syndrome n 1 and pulmonary hypoplasia n 1 Three patients had a single virus type detected in their respiratory samples RV C in two patients and RV A in one patient two patients had dual viral co detection RV C RSV B and RV C HCoV OC43 and three patients had triple viral co detection RSV A FLU A HBoV RV C RSV A HMPV A and RV C HMPV A FLU B The causes of death were respi ratory insufficiency n 4 cardiogenic shock n 2 and septic shock n 2 Of note the patient who had the longest hospital stay 254 days and also the longest duration of mechani cal ventilation 99 days had pulmonary hypoplasia a severe underlying condition and he ulti mately died of sepsis Discussion In this study viruses were detected in 85 of young children attending the emergency room with ALRI and subsequently admitted to the hospital RV especially RV C was the most fre quently detected virus followed by RSV Twenty percent of patients were admitted to the PICU Comorbidities and congenital heart disease were more frequent in patients who were admitted to the PICU In addition the detection of HCoV alone or in co infection with RV C was independently associated with PICU admission Similarly to our data RV was also the most commonly detected virus in children younger than 3 years presenting to the Seattle Children s Hospital pediatric ER with a symptomatic respiratory tract infection and the majority of them had lower respiratory tract infection and required hospitalization 10 RV was also the most prevalent virus detected in children aged two weeks to 5 years admitted to a hospital with ALRI in Germany exceeding the frequency of RSV 22 Moreover RV C has been reported as the most frequent RV species and has been associated with severe disease in children less than 3 years old 8 22 Viral co detection was found in approximately three quarters of our study population with RV RSV as the most frequent duo However we observed that viral co detection per se was not a risk factor for PICU admission In keeping with this a recent systematic review and meta analysis showed that respiratory viral coinfection was not associated with need for hospi talization intensive care admission or length of stay in children 15 In addition the number of detected viruses has not been associated with illness severity 14 Indeed surprisingly an Table 1 Demographic and clinical data Characteristic All n 236 PICU admission n 47 No PICU admission n 189 Age months 5 2 0 2 35 3 6 0 2 35 5 9 0 3 34 Age 6 months 125 53 29 61 7 96 50 8 Weight kg 6 2 21 5 2 2 1 14 6 2 2 21 Male gender 89 38 18 38 3 71 37 6 Prematurity 43 18 2 7 14 9 36 19 Underlying disease 60 25 21 44 7 39 20 6 Congenital heart disease 23 9 7 9 19 14 7 4 Use of systemic antibiotics 153 64 8 37 78 7 116 61 4 Length of hospital stay days 8 1 254 18 3 254 7 1 210 Data are expressed as median range or n PICU pediatric intensive care unit P 0 05 for comparison between PICU admission and No PICU admission groups https doi org 10 1371 journal pone 0217744 t001 Human coronavirus alone or in coinfection with rhinovirus C is a risk factor for severe respiratory disease PLOS ONE https doi org 10 1371 journal pone 0217744 June 3 2019 5 9inverse correlation between the clinical severity score and the number of viruses detected has been observed 23 Nevertheless a previous study performed in Southeast Brazil showed that coinfections especially involving RSV were associated with increased severity 13 In the present study RSV in co detection with other viruses was not associated with increased disease severity HCoV was detected in 11 of children admitted to hospital with ALRI in our study with a predominance of OC43 40 7 and 229E 33 types Although human coronaviruses most frequently cause common colds they may cause severe respiratory diseases such as severe acute respiratory syndrome 24 We observed that one third of children with HCoV infectio nand 41 of patients withHCoV in co infection withRV C were admitted to the PICU Fur thermore we found that the detection of HCoV alone or in co infection with RV C was an independent risk factor for PICU admission Similar to our results HCoV was detected in 8 2 of hospitalized children aged 3 2 3 9 years with respiratory tract infection in New York OC43 was the most prevalent type 40 1 and 11 of patients with HCoVinfectionneeded PICU admission Additionally the presence of chronic complex underlying conditions including cardiovascular genetic and respiratory diseases was associated with increased dis ease severity 25 which is corroborated by our data Almost two thirds of our children were treated with systemic antibiotics This high fre quency of antibiotic use is similar to that reported in developed countries 26 27 Empiric antimicrobial therapy driven by unfavorable clinical conditions in the face of probable bacte rial infections is likely to be initiated in the absence of immediate laboratory confirmation of virus detection In the present study the only rapid diagnostic test for respiratory virus availa bleearly at hospital admission was the RSV rapid antigen detection test Therefore the devel opment of clinically relevant rapid tests for respiratory viruses is needed and shall help antimicrobialstewardship programs Since the present study was done in a single health center this raises concern that epidemi ologic data may not be generalizable While this limitation is ack