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Clinical Epidemiology of Bocavirus Rhinovirus Two Polyomaviruses and Four Coronaviruses in HIV Infected and HIV Uninfected South African Children Marta C Nunes 1 2 Zachary Kuschner 3 Zelda Rabede 2 Richard Madimabe 1 2 Nadia Van Niekerk 1 2 Jackie Moloi 4 Locadiah Kuwanda 1 2 John W Rossen 5 Keith P Klugman 2 6 Peter V Adrian 1 2 Shabir A Madhi 1 2 7 1Department of Science and Technology National Research Foundation Vaccine Preventable Diseases Johannesburg South Africa 2Medical Research Council Respiratory and Meningeal Pathogens Research Unit Faculty of Health Sciences University of the Witwatersrand Johannesburg South Africa 3Stony Brook School of Medicine Stony Brook University Stony Brook New York United States of America 4Molecular and Immunology Division BioMe rieux Johannesburg South Africa 5Department of Medical Microbiology University Medical Center Groningen University of Groningen Groningen The Netherlands 6Hubert Department of Global Health Rollins School of Public Health and Division of Infectious Diseases Emory University School of Medicine Atlanta Georgia United States of America 7National Institute for Communicable Diseases a division of National Health Laboratory Service Sandringham South Africa Abstract Background Advances in molecular diagnostics have implicated newly discovered respiratory viruses in the pathogenesis of pneumonia We aimed to determine the prevalence and clinical characteristics of human bocavirus hBoV human rhinovirus hRV polyomavirus WU WUPyV and KI KIPyV and human coronaviruses CoV OC43 NL63 HKU1 and 229E among children hospitalized with lower respiratory tract infections LRTI Methods Multiplex real time reverse transcriptase polymerase chain reaction was undertaken on archived nasopharyngeal aspirates from HIV infected and uninfected children 2 years age hospitalized for LRTI who had been previously investigated for respiratory syncytial virus human metapneumovirus parainfluenza I III adenovirus and influenza A B Results At least one of these viruses were identified in 274 53 0 of 517 and in 509 54 0 of 943 LRTI episodes in HIV infected and uninfected children respectively Human rhinovirus was the most prevalent in HIV infected 31 7 and uninfected children 32 0 followed by CoV OC43 12 2 and hBoV 9 5 in HIV infected and by hBoV 13 3 and WUPyV 11 9 in HIV uninfected children Polyomavirus KI 8 9 vs 4 8 p 0 002 and CoV OC43 12 2 vs 3 6 p 0 001 were more prevalent in HIV infected than uninfected children Combined with previously tested viruses respiratory viruses were identified in 60 9 of HIV infected and 78 3 of HIV uninfected children The newly tested viruses were detected at high frequency in association with other respiratory viruses including previously investigated viruses 22 8 in HIV infected and 28 5 in HIV uninfected children Conclusions We established that combined with previously investigated viruses at least one respiratory virus was identified in the majority of HIV infected and HIV uninfected children hospitalized for LRTI The high frequency of viral co infections illustrates the complexities in attributing causality to specific viruses in the aetiology of LRTI and may indicate a synergetic role of viral co infections in the pathogenesis of childhood LRTI Citation Nunes MC Kuschner Z Rabede Z Madimabe R Van Niekerk N et al 2014 Clinical Epidemiology of Bocavirus Rhinovirus Two Polyomaviruses and Four Coronaviruses in HIV Infected and HIV Uninfected South African Children PLoS ONE 9 2 e86448 doi 10 1371 journal pone 0086448 Editor Amit Kapoor Columbia University United States of America Received August 8 2013 Accepted December 10 2013 Published February 3 2014 Copyright C223 2014 Nunes 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 Funding This work is based upon research supported in part by the South African Research Chairs Initiative of the Department of Science and Technology DST and National Research Foundation NRF in Vaccine Preventable Diseases Additional funding support was received from the National Health Laboratory Service Research Fund and Medical Research Council Respiratory and Meningeal Pathogens Research Unit Any opinion findings and conclusions or recommendations expressed in this material are those of the author s and therefore the NRF and DST do not accept any liability with regard thereto MCN had financial support from the University of the Witwatersrand The funders had no role in study design data collection and analysis decision to publish or preparation of the manuscript Competing Interests Jackie Moloi is an employee of BioMe rieux South Africa She is employed as an application specialist for the Molecular and Immunology Division She does own any shares or stock in BioMe rieux South Africa This does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials The other authors have declared that no competing interests exist E mail shabirm nicd ac za Introduction Pneumonia is a leading cause of mortality in children under 5 years age worldwide including in HIV infected children 1 3 The aetiology of childhood pneumonia may include infection with bacteria and or respiratory viruses Although respiratory viruses are more frequently identified than bacteria in children with pneumonia this may be biased by lack of availability of sensitive and specific tests for diagnosing bacterial causes of pneumonia 4 Furthermore respiratory viral infections may heighten the PLOS ONE www plosone org 1 February 2014 Volume 9 Issue 2 e86448 susceptibility to developing a super imposed bacterial infection resulting in severe pneumonia 5 6 Traditionally respiratory viruses that have been associated with lower respiratory tract infections LRTI include respiratory syncytial virus RSV parainfluenza viruses I III PIV I III influenza viruses A B and adenovirus Two human coronaviruses CoV OC43 CoV OC43 and 229E CoV 229E were initially identified as causes of upper respiratory tract infections URTI in the 1960s using classical culture methods 7 8 More recently advances in molecular diagnostics have resulted in the discovery of other respiratory viruses which have also been associated with LRTI Included among these are human metapneumovirus hMPV 9 human bocavirus hBoV 10 human coronavirus NL63 CoV NL63 11 and HKU1 CoV HKU1 12 and WU and KI polyomaviruses WUPyV KIPyV 13 15 Also human rhinovi rus hRV which was previously mainly associated with mild URTI has increasingly been implicated in having a role in the pathogenesis of LRTI and asthma exacerbations 16 17 Due to impaired humoral and cell mediated immunity HIV infection in children has been described as a risk factor for severe illness and mortality caused by respiratory viral associated LRTI such as RSV hMPV and influenza virus 18 19 There are however limited data on the role of other respiratory viruses including the more recently discovered viruses which occur as single or co infecting pathogens in HIV infected children hospi talized with LRTI and of these studies most have small sample sizes 20 22 The aim of this study was to identify the prevalence of hBoV hRV WUPyV KIPyV CoV OC43 CoV NL63 CoV HKU1 and CoV 229E among HIV infected and uninfected children who were hospitalized for LRTI using real time reverse transcrip tase polymerase chain reaction RT PCR The study cohort had been previously investigated for RSV influenza A B PIV I III and adenovirus by immunofluorescence assay and hMPV by nested PCR as described 5 23 Methods Ethics Statement The main 9 valent pneumococcal conjugated vaccine PCV9 efficacy trial and subsequent retrospective analysis of study participants were approved by the Human Research Ethics Committee Medical of the University of the Witwatersrand The main study did not have a clinical trials number since it was undertaken prior to registration of clinical trials having been made mandatory Signed informed consent was obtained from the parent legal guardians of all the study participants as part of the main trial The Ethics Committee did not require additional consent for this study Study population This was a retrospective study of children who participated in a phase III trial in South Africa which investigated the efficacy of a PCV9 as described 5 24 Briefly 39836 children recruited from March 1998 to October 2000 were randomized 1 1 to receive 3 doses of either PCV9 or placebo Vaccination occurred at a mean of 6 661 2 6standard deviation 11 262 5 and 15 963 9 weeks of age 24 Hospital based surveillance for all cause hospitaliza tion was undertaken at Chris Hani Baragwanath Hospital the only public hospital in the study community Hospitalized children had their signs and symptoms recorded and underwent HIV testing according to the study protocol 24 Nasopharyngeal aspirates NPA were obtained for respiratory viral studies from children hospitalized with LRTI 5 and archived from January 2000 onward In the present study only NPA collected from 1 st February 2000 to 31 st January 2002 from children less than 2 years old were analysed If a child had recurrent LRTI hospitalizations only NPA collected at least 28 days apart were included in the analysis Blood cultures were performed with the use of an automated blood culture system BacT Alert Organon Teknika Viral testing Total nucleic acids were extracted from archived NPA using a NucliSENS easyMAG platform bioMerieux and eluted in a final volume of 60 ml of elution buffer 25 RNA was reverse transcribed with High Capacity cDNA Reverse Transcriptase Invitrogen Life Technologies and primed with oligo dT primers Invitrogen Life Technologies Real time PCR was done in an ABI 7500 RT PCR system Applied Biosystems Life Technolo gies reactions were performed in 20 ml using TaqMan Universal PCR Master Mix Applied Biosystems Life Technologies and the primers and probes listed in Table S1 Five duplex RT PCR reactions targeting the 8 respiratory viruses were developed Internal controls the human genes ribonucleoprotein and glyceraldehyde 3 phosphate dehydroge nase or the spiked viruses lambda and Newcastle Disease Virus were included to check the efficiency of the extraction step and to detect the presence of PCR inhibitors Positive controls were included in each experiment Study specific definitions The clinical definitions used in this study are the same as previously described 5 Briefly LRTI was defined as any episode with clinical diagnosis of pneumonia or bronchiolitis done by a study physician Children with LRTI were categorized as having clinical pneumonia if they had evidence of alveolar consolidation on chest x ray CXR AC or if they fulfilled the clinical diagnosis of LRTI without wheeze on chest auscultation but had rales and or bronchial breathing Children were categorized as having bronchiolitis in the presence of wheezing on chest auscultation and in the absence of documented CXR AC or bronchial breathing on chest wall auscultation A clinical diagnosis of WHO severe very severe pneumonia was made if the child had a cough 14 days in duration and lower chest wall in drawing and or any of the following signs and symptoms of severe pneumonia feeding difficulties convulsions central cyanosis or encephalopathy The previously developed respiratory index of severity in children RISC score was used to compare disease severity between single and multiple viral infections 26 Statistical analysis Demographic clinical and laboratory characteristics at admis sion were compared between HIV infected and uninfected children chi square or Fisher s exact tests were used to compare the distribution of categorical variables and mean or median of continuous variables were compared by two tailed Student t test or Mann Whitney test respectively Viral prevalence was com pared between HIV groups and clinical and laboratory charac teristics were compared between episodes with single and multiple viral detection using multivariate logistic regression models adjusted for study intervention arm i e whether received PCV9 or placebo year of sampling detection of a virus previously tested and age The results are presented as adjusted odds ratios aOR with 95 confidence intervals 95 CI and p values A p value 0 05 was considered significant Analyses were performed using STATA version 11 0 Statacorp Texas USA Respiratory Viruses in Hospitalized Children PLOS ONE www plosone org 2 February 2014 Volume 9 Issue 2 e86448 Results Study population During the follow up period included in this analysis there were a total of 2147 hospitalizations for LRTI including 2094 97 5 in which NPA had been collected Of the initial collected samples 69 7 were available for RT PCR analysis The same proportion of samples were available for RT PCR from PCV9 recipients 69 2 and placebo recipients 70 2 p 0 621 from HIV infected 78 2 vs 73 1 p 0 130 and HIV uninfected children 65 5 vs 68 6 p 0 218 Table 1 A lower proportion of samples was however available for RT PCR analysis 65 9 1000 1518 from the first period February 2000 January 2001 compared to the second period February 2001 January 2002 79 9 460 576 p 0 001 of the study Children from whom NPA were available for RT PCR testing compared to those in whom samples were unavailable were older median age 10 vs 8 months p 0 001 were 1 3 fold less likely to have tested positive for one of the previously tested respiratory viruses 33 3 vs 42 3 p 0 001 had a higher prevalence of cyanosis 11 4 vs 8 1 p 0 025 higher evidence of CXR AC 26 6 vs 22 1 p 0 041 higher C reactive protein CRP levels median 15 vs 12 mg l p 0 003 and higher procalcitonin PCT concentration median 0 26 vs 0 15 ng ml p 0 006 Table S2 There were no other demographic clinical or laboratory differences observed between the LRTI episodes with NPA available versus unavailable for RT PCR A total of 517 NPA samples from HIV infected children were analysed by RT PCR including 45 0 from PCV9 recipients and 55 0 from placebo recipients Among HIV uninfected children 943 specimens were available for RT PCR analysis including 49 5 from PCV9 recipients and 50 5 from placebo recipients On admission HIV infected children compared to HIV uninfected were younger median age 9 vs 11 months p 0 001 more frequently presented with cyanosis 23 8 vs 4 6 p 0 001 had lower mean oxygen saturation 89 8 vs 92 2 p 0 001 had a higher median respiratory rate 54 vs 48 breaths per minute p 0 001 were more likely to present as pneumonia 88 8 vs 49 6 p 0 001 than bronchiolitis had higher CRP 18 vs 14 mg l p 0 007 and PCT levels 0 47 vs 0 17 ng ml p 0 001 had a longer hospital stay 4 vs 1 median days p 0 001 had a higher case fatality rate 17 8 vs 0 95 p 0 001 and more frequently had bacteraemia 7 8 vs 2 7 p 0 001 Table 2 Of the 1460 specimens analysed all but 13 1 from HIV infected and 12 from HIV uninfected children were previously tested for hMPV by nested PCR 23 and 1458 were tested by an immunofluorescence assay for RSV which if found negative were further tested for influenza A B PIV I III and adenovirus as described 5 Prevalence of newly tested respiratory viruses Among HIV infected children the RT PCR viral panel was positive in 274 53 0 LRTI episodes for at least one of the newly tested viruses Table 3 The prevalence of any of the newly tested respiratory viruses was similar between PCV9 and placebo recipients in HIV infected children except for WUPyV 11 2 vs 6 3 p 0 047 respectively and hBoV 12 5 vs 7 0 p 0 034 respectively Table S3 In HIV infected children hRV was the most frequently detected virus 31 7 followed by CoV OC43 12 2 hBoV 9 5 KIPyV 8 9 WUPyV 8 5 CoV NL63 1 7 and CoV HKU1 1 4 Table 3 The newly tested viruses were frequently identified as co infecting viruses among HIV infected children including 49 4 of LRTI episodes associated with hRV Table 4 The most common viral co infections with hRV included KIPyV 14 6 WUPyV 11 6 CoV OC43 and hBoV 11 0 each Table 4 Among the 486 children on whom blood culture was done bacteria were isolated on 38 7 8 occasions 20 52 6 of which were associated with concomitant detection of one of the newly tested viruses and 22 57 9 of any of the viruses In HIV uninfected children at least one newly tested virus was detected in 509 54 0 LRTI episodes with hRV also being the most common 32 0 followed by hBoV 13 3 WUPyV 11 9 KIPyV 4 8 CoV OC43 3 6 CoV NL63 2 6 CoV HKU1 1 6 and CoV 229E 0 42 Table 3 Comparing HIV uninfected PCV9 and placebo recipients differences in the prevalence of newly tested viruses were evident for KIPyV 2 1 vs 7 4 p 0 001 CoV HKU1 0 21 vs 2 9 p 0 001 CoV OC43 2 1 vs 5 0 p 0 017 and hRV 36 2 vs 27 9 p 0 007 Table S3 Of the 302 LRTI episodes in which hRV was identified 51 3 had at least one other virus detected including 13 6 with RSV 12 3 with WUPyV or hBoV and 2 0 N 6 with both WUPyV and hBoV Table 5 The prevalence of bacteraemia in HIV uninfected children among those with blood culture results was 2 7 N 24 881 of which 12 50 0 occurred in the presence of infection with one of the newly tested viruses and 15 62 5 in presence of any of the studied viruses By multivariate analysis adjusting for PCV vaccination status period of collection and age single infections with a newly tested virus were more frequent in HIV infected 30 2 than HIV uninfected children 25 5 adjusted odds ratio aOR 1 3 p 0 033 Table 3 Also HIV infected compared to HIV uninfected children had a higher prevalence of KIPyV aOR Table 1 Number of specimens analysed in the current study and total specimens collected Overall HIV infected HIV uninfected Overall PCV9 Placebo p value 1 Overall PCV9 Placebo p value 1 Overall PCV9 Placebo p value 1 Visits according to study criteria 2 2147 1038 1109 712 310 402 1435 728 707 Nasopharyngeal aspirate done 2094 97 5 1010 97 3 1084 97 7 0 508 687 96 5 297 95 8 390 97 0 0 385 1407 98 1 713 97 9 694 98 2 0 762 Viral RT PCR performed in this study 1460 69 7 699 69 2 761 70 2 0 621 517 75 3 232 78 2 285 73 1 0 130 943 67 0 467 65 5 476 68 6 0 218 1 Chi square test comparing 9 valent pneumococcal conjugated vaccine PCV9 and placebo arms 2 From the full database patients 2 years old with diagnosis of lower respiratory tract infections hospitalized between 1 February 2000 and 31 January 2002 only 1 visit per patient within 28 days doi 10 1371 journal pone 0086448 t001 Respiratory Viruses in Hospitalized Children PLOS ONE www plosone org 3 February 2014 Volume 9 Issue 2 e86448 Table 2 Demographic clinical and laboratory features of children hospitalized for lower respiratory tract infection and in whom samples were available for the current study Overall N 1460 PVC9 699 Placebo 761 HIV infected N 517 PCV9 232 Placebo 285 HIV uninfected N 943 PCV9 467 Placebo 476 p value 1 Demographic characteristics Overall 10 1 24 9 1 24 11 1 23 0 001Median age in months range PCV9 10 1 23 8 1 23 11 1 23 0 018 Placebo 10 1 24 9 1 24 11 1 23 0 009 p value 2 0 612 0 706 0 512 Overall 836 57 3 284 54 9 552 58 5 0 194Male 3 N PCV9 423 60 5 137 59 1 286 61 2 0 577 Placebo 413 54 3 147 51 6 266 55 9 0 249 p value 2 0 015 0 089 0 095 Clinical characteristics Overall 37 3 0 96 37 3 0 93 37 3 1 0 0 895Mean axillary PCV9 37 2 0 80 37 2 0 81 37 3 0 79 0 675 temperature 4 uC SD Placebo 37 4 1 1 37 5 1 0 37 4 1 1 0 768 p value 2 0 005 0 032 0 05 Overall 222 27 3 85 29 7 137 26 0 0 255Axillary temperature 38uC PCV9 89 23 3 29 23 0 60 23 4 0 927 N Placebo 133 30 9 56 35 0 77 28 4 0 153 p value 2 0 016 0 028 0 193 Overall 1054 72 6 369 71 9 685 73 0 0 654Fever 5 N PCV9 488 70 2 155 67 4 333 71 6 0 252 Placebo 566 74 9 214 75 6 352 74 4 0 713 p value 2 0 047 0 039 0 333 Overall 624 42 9 209 40 7 415 44 1 0 205Vomit 6 N PCV9 275 39 5 85 37 0 190 40 8 0 333 Placebo 349 46 0 124 43 7 225 47 4 0 321 p value 2 0 014 0 124 0 042 Overall 45 3 1 9 1 8 36 3 9 0 029Seizures 7 N PCV9 23 3 3 4 1 7 19 4 1