Journal of Medical Virology 87:2027–2032 (2015)
The Role of Rhinovirus in Children Hospitalized for Acute Respiratory Disease, Santa Fe, Argentina Juan Manuel Rudi,1* Fabiana Molina,2 Rocıo Dıaz,2 Virginia Bonet,2 Lucila Ortellao,3 mez,1 Judith Pierini,3 Raquel Cociglio,2 and Gabriela Kusznierz1 Diego Cantarutti,3 Alejandra Go 1
National Institute of Respiratory Diseases “Emilio Coni”, National Administration of Laboratories and Health Institutes, Santa Fe, Argentina 2 Childrens Hospital “Dr. O. Alassia”, Santa Fe, Argentina 3 Department of Pediatrics, Hospital “J. B. Iturraspe”, Santa Fe, Argentina
Human rhinoviruses (HRVs) were historically considered upper airway pathogens. However, they have recently been proven to cause infections in the lower respiratory tract, resulting in hospitalization of children with pneumonia, bronchiolitis, and chronic pulmonary obstruction. In this report, HRV frequency and seasonality are described together with patient clinical-epidemiological aspects. From a total of 452 surveyed samples, the HRV nucleic acids was detected in 172 (38.1%) and found in every month of the study year. 60% of inpatients with acute respiratory infection (ARI) associated with HRV were under 6 months of age and 31% had a clinical history, being preterm birth and recurrentwheezing the prevailing conditions. The most frequent discharge diagnoses were pneumonia (35.2%), bronchiolitis (32.4%), and bronchitis (12.4%). Fifteen point nine percent of patients required admission into intensive care units. The results obtained in this study demonstrated the ́ association between HRV and children hospitalizations caused by ARI. J. Med. Virol. 87:2027–2032, 2015. © 2015 Wiley Periodicals, Inc.
population and account for considerable morbidity and mortality [Tregoning and Schwarze, 2010; Bicer et al., 2013]. Human rhinoviruses (HRVs) belong to the order Picornavirales, family Picornaviridae, genus Enterovirus, and species Rhinovirus. They were first isolated in 1956 and were historically considered upper airway pathogens. However, they have recently been proven to cause infections in the lower respiratory tract, resulting in hospitalization of children with pneumonia, bronchiolitis, and chronic pulmonary obstruction. They are also associated with patients who have recurring wheezing episodes and €ranta et al., 1997; Arola asthma exacerbations [Pitka et al., 1988; Fields et al., 2001; Papadopoulos and Johnston, 2001; Papadopoulos et al., 2002; Hayden, 2004; Calvo Rey et al., 2006; Maffey et al., 2008; Costa et al., 2009; Gern, 2010; Jackson and Lemanske, 2010; Meerhoff et al., 2010; Nascimento et al., 2010; de Paula et al., 2011; Fujitsuka et al., 2011; Hara et al., 2011]. In this report, HRV frequency and seasonality are described together with clinical-epidemiological characteristics of ARI hospitalized pediatric patients detected with HRV.
KEY WORDS:
A retrospective study was conducted involving patients who were hospitalized in Santa Fe, Argentina, from March 2010 to February 2011.Patients
acute respiratory infection; pneumonia; pediatric population; rhinovirus; nested RT-PCR
INTRODUCTION Acute respiratory infections (ARI) represent one of the major sources of medical consultation and are placed among the main causes of mortality worldwide [Ministry of Health, Department of Epidemiology, 2011]. In the year 2000, 1.9 million children died because of ARI across the world. Viral respiratory infections have a massive impact on the pediatric C 2015 WILEY PERIODICALS, INC.
MATERIALS AND METHODS Design and Participants
This work was performed at National Institute of Respiratory Diseases “Emilio Coni”, Respiratory Viruses, Santa Fe, Argentina Correspondence to: Juan Manuel Rudi, National Institute of Respiratory Diseases “Emilio Coni”, Blas Parera 8260, 3000 Santa Fe, Argentina. E-mail: [email protected] Accepted 7 May 2015 DOI 10.1002/jmv.24266 Published online 26 May 2015 in Wiley Online Library (wileyonlinelibrary.com).
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younger than 15 years with an ARI diagnosis at the time of admission were included. Viral Diagnosis A total of 2,020 nasopharyngeal aspirates (NPAs) were obtained from patients with ARI, who underwent antigen detection for respiratory syncytial virus (RSV), adenovirus, parainfluenza, and influenza A and B through indirect immunofluorescence (IIF). Influenza A and B detection was also achieved by real time RT-PCR. A total of 452 IIF-confirmed negative NPAs, randomly collected during all months of the study period, were analyzed for HRV, using a RT-PCR developed by Steininger et al. [2001]; which amplifies a 93 bp fragment located at the end of the 50 noncoding region (NCR) of the HRV genome and conserved among all serotypes of the virus.
calculated. Mantel–Haenszel x2 test was used to compare discrete variables and Mann–Whitney test was applied to compare continuous variables. A P-value of 1,000 per cubic millimeter) was present in 72.7% (Table I). Among patients from whom data was available, two showed microbiologic evidence of a secondary bacterial infection. The pathogens identified were Streptococcus pneumoniae and Klebsiella sp. In an attempt to evaluate the rhinovirus recovery time, the time elapsing between the onset of the respiratory disease and the sampling was associated. In 51 cases (37.5%), the elapsed time was less than 72 hr, in 79 cases (58.1%) between 4 and 14 days elapsed, and in 6 cases (4.4%) the HRV genome was successfully detected after 15 days from the onset of symptoms. The median time elapsed until samples were taken was 5.6 days (Range: 1–23 days). Radiographic Findings Of the 137 patients who underwent chest radiography, the most frequent radiographic findings included air trapping (75.2%) and interstitial pattern (67.9%) (Table I). DISCUSSION This study has produced fresh knowledge about the epidemiology and clinical status of ARI ascribable to HRV in children by associating the virus to hospitalizations caused by moderate and severe respiratory disease. The choice of amplification technique was based on the results obtained by other authors [Steininger et al., 2001], who designed primers that allowed amplify a small fragment of the 50 NCR of the HRV genome, highly conserved among different serotypes of the virus. These authors were able to detect most HRV serotypes hitherto known with a high sensitivity and specificity, since no enterovirus was amplified. While the total negative to other respiratory virus samples were not analyzed, a statistically significant sample size was calculated to infer this result to the overall population, although it is true that positivity obtained for HRV may be underestimated by not having studied co-infections with other respiratory viruses. Several research studies have measured HRV frequency in ARI hospitalized children, ranging from 16 to 57.8%. The 38.1% value from this study is within this range of values, considering PCR-tested samples as a denominator [Calvo Rey et al., 2006; Miller et al., 2007; Fry et al., 2011; Iwane et al., 2011; Vidaurreta et al., 2011; Albuquerque et al., 2012; Huijskens et al., 2012; Marcone et al., 2012; Pi~ nero Fern andez et el., 2012; Bicer et al., 2013]. Rhinoviruses circulated throughout the year, though the months revealing a higher circulation J. Med. Virol. DOI 10.1002/jmv
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were March 2010 to May 2010, August 2010, October 2010, November 2010, and February 2011, with positivity percentages ranging between 39 and 51%, also coinciding with RSV low circulation. The months with the largest circulation of RSV (June 2010 and July 2010; data not shown) match those months where HRV was detected lessfrequently. Our findings are in line with observations in previous studies [Calvo Rey et al., 2006; Miller et al., 2007; Fry et al., 2011; Vidaurreta et al., 2011; Albuquerque et al., 2012; Bicer et al., 2013].Differences are likely to exist in HRV seasonality depending on the year being surveyed. It is thus important to extend the study period for at least 3 years to be able to observe possible yearly differences. In assessing the relation between virus prevalence and patient age, it has been observed that 60% of infected children were under 6 months of age, their median age being 4 months. Children over 2 years of age accounted for only 14% of the whole of inpatients with ARI associated to HRV. This is in line with HRV greater prevalence over the first months of life as manifested in previous publications [Miller et al., 2007; Savon-Valdes et al., 2008; Meerhoff et al., 2010; de Paula el al., 2011; Fry et al., 2011; Iwane el al., 2011]. The majority of hospitalized patients (70%) were children with no prior clinical history. However, in the patient group that did have a history, the most frequent conditions were preterm birth and recurrent wheezing. Recently, results from a study in the city of Buenos Aires have revealed that rhinovirus has been the respiratory infection predominant pathogen among preterm infants [Miller et al., 2012]. Furthermore, wheezing is a frequent manifestation of low respiratory tract infection in infants and young children, being viral infection the most common cause [Maffey et al., 2008; Gern, 2010; Fujitsuka et al., 2011; Smuts et al., 2011]. Results from various studies have suggested HRV could be associated with wheezing symptoms. Maffey et al. [2008]; in the city of Buenos Aires, traced HRV in respiratory samples from children with recurrent wheezing, with a frequency of 23% and after RSV, while in Africa [Smuts et al., 2011] and Japan [Fujitsuka et al., 2011] it has been found in 58 and 31%, respectively. Numerous studies on asthma exacerbation etiology [Matthew et al., 2009; Gern, 2010; Albuquerque et al., 2012] have pointed out that HRV is the most widely detected agent among this patient group. Miller et al. [2007] found out that children with asthma have a larger amount of rhinovirus-related hospitalizations than those without a wheezing or asthma history. In this study, only 2% of the cases—all older than 5 years of age—have had asthma diagnosis, in contrast with 28% reported in Miller’s study. While rhinoviruses have been traditionally associated with common cold alone, new developments in diagnosis techniques helped detect its presence in patients with lower respiratory tract infections. In the year 2000, Papadopoulos et al. [2000] investigated J. Med. Virol. DOI 10.1002/jmv
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in vitro lower respiratory infection through exposure of human bronchial epithelial cells to rhinovirus and in vivo in normal and asthmatic human volunteers. The infection was confirmed in both models, inducing inflammatory response in normal and asthmatic subjects alike. In the study by Kusel et al. [2006]; the pathogenic role of rhinovirus associated with lower respiratory tract infections in the first year of life was recognized. Costa et al. [2009] found a 25% prevalence of rhinovirus in bronchoalveolar lavages of immunocompetent and immunocompromised patients. In line with these studies, pneumonia was the most frequent discharge diagnosis in our study, and it was observed in 35% of patients, agreeing with data presented by Bicer et al.[2013]. No other viral pathogens were detected in the cases studied and two cases alone had sepsis due to Streptococcus pneumoniae and Klebsiella. On the other hand, bronchiolitis was the main cause of hospital admission in children under 2 years of age [Papadopoulos et al., 2000,2002; Nascimento et al., 2010]. Recent research on bronchiolitis etiology has reported RSV and HRV as the most important etiological agents in hospitalized children, with detection rates of 39 and 13%, respectively, [Nascimento et al., 2010; Pi~ nero Fern andez et al., 2012]. In this series, a high proportion of bronchiolitis was also observed (32%), while in the study by Calvo Rey et al. [2006] it was 21% of inpatients. These findings are at odds with those observed in other studies, where pneumonia was diagnosed in 8–16% of patients [Calvo Rey et al., 2006; Miller et al., 2007; de Paula et al., 2011; Fry et al., 2011] and bronchiolitis in 11–13% of cases [Miller et al., 2007; de Paula et al., 2011]. Conversely, in the study by Savon-Valdes et al. [2006], a high proportion of bronchiolitis has been observed (74%). Fever has been one of the most frequent clinical manifestations, in line with other published studies [Papadopoulos et al., 2002; Calvo Rey et al., 2006; Smuts et al., 2011; Vidaurreta el al, 2011; Marcone et al., 2012; Maffey et al., 2014]. The findings obtained in this work support recent studies [Papadopoulos et al., 2002; Bicer et al., 2013] that have demonstrated HRV to be associated with severe illness, as 15% of patients had severe symptoms leading up to admission in intensive care units (ICUs), though no fatal cases were recorded. 56.5% of critical patients had complications, such as sepsis and acute imminent respiratory failure. In the study by Bicer et al. [2013]; 36% of HRV-infected children had moderate to severe illness, while in RSV-infected children the proportion was 33%. In Vietnam, HRV was associated with an outbreak of severe respiratory infection in 12 children that were hospitalized and developed acute respiratory distress syndrome, 7 of whom died [Hai et al., 2012]. The median duration of hospital stay was 6 days. This period has proven similar to what has been reported in previously published studies [Maffey et al., 2008; Marcone et al., 2012]. Supplemental
Rhinovirus in Hospitalized Children
oxygen was given to a high proportion of patients (85%), as opposed to mechanical ventilation that was only necessary in slightly more than 10% of cases. Those values are higher than those observed by other authors [Miller et al., 2007; Iwane et al., 2011]. Age under 3 months, presence of immunodeficiency, congenital cardiopathy, chronic pulmonary disease, preterm birth, and malnutrition are risk factors for the development of severe low ARI, according to the Argentine Pediatric Society Consensus. In this study, age and presence of medical history did not represent a risk factor for severe ARI development, although 60% of cases were under 6 months and 40% of severe patients were recorded as having some type of medical history. In addition, over 30% of patients in the study group showed at least one ARI-caused hospitalization prior to this study, which was not a risk factor either. There are many factors that can have a bearing on the severity of the disease, related to both the host and the virus. As to HRV-related factors, the presence of co-infections with other respiratory viruses [Tregoning and Schwarze, 2010; de Paula et al., 2011] or the HRV species involved [Franco et al., 2012] could be cited, but neither has been assessed in this study. Although there are controversial results regarding association between the different species and clinical symptoms [Fry et al., 2011; Franco et al., 2012], some literature has proven the most prevalent role of HRVCs in lower ARI, associating them with severe illness. In their study, Iwane et al. [2011] have observed an association between HRVA and HRVC-infected patients and severe illness development, needing longer hospital stays, ICU and mechanical ventilation, as opposed to patients infected with HRVB. Sixty six percent of confirmed cases were treated with ATBs. Incidentally, it must be considered that this study was retrospective and virological diagnosis was negative for the rest of the respiratory viruses. This highlights the importance of considering early diagnosis of other respiratory viruses involved, to contribute to the clinical management of patients with ARI. Elsewhere, HRV has been identified in asymptomatic patients, detection prevalence varying from 12 to 28% [Jartti et al., 2004; Wright et al., 2004; Jansen et al., 2011]. This may be suggesting either an asymptomatic colonization or a long-lasting virus elimination after a respiratory infection, detecting HRV RNA even 5 or 6 weeks after the respiratory infection [Jartti et al., 2004]. Consequently, clinical relevance of hospitalized patient rhinovirus detection can be difficult to interpret. In this study, in 26% of patients the virus was detected 7 days after the onset of respiratory symptoms. In all this cases, sampling was made within 48 hr since hospitalization, as recommended by national guidelines. The strength of this study is the important volume of samples assessed for HRV detection and the high proportion of positivity obtained in relation to other
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studies. This enabled us to carry out an exhaustive analysis of clinical data, though this analysis has some limitations. First of all, there were no controls in whom HRV was measured. One of the core problems in case control studies is the selection of an appropriate control group, because it must be ascertained whether this group had not previously (5 or 6 weeks before) suffered from catarrh in the upper airways. It was therefore decided not to include any control group in our study. Nonetheless, detection studies were made for other known respiratory viruses, and confirmed negative population was analyzed, suggesting rhinovirus could be the ARI-causing agent. Admittedly, having used PCR for influenza alone and IIF for the rest of viruses as a screening technique, which is less sensitive than molecular techniques, could be a constraint in this study. Another constraint in this study is the limited geographical region where the study was conducted and that the study period is confined to one year, and the seasonal situation observed might not be a reflection of other years. This was a first test locally with the intention to extend the study to other regions and for longer periods. Regardless of such constraints, and in view of the results obtained, HRV participation in ARI has been demonstrated. ACKNOWLEDGMENTS We gratefully acknowledge the technical assistance rendered by Mr. Gonzalo Vidal in the detection of respiratory viruses through IIF. REFERENCES Albuquerque MCM, Varella RB, Santos N. 2012. Acute respiratory viral infections in children in Rio de Janeiro and Teres opolis, Brazil. Rev Inst Med Trop Sao Paulo 54:249–255. Arola M, Ziegler T, Ruuskanen J. 1988. Rhinovirus in acute otitis media. J Pediatr 113:693–695. Bicer S, Giray T, C€ ol D, Erdag GC, Vitrinel A, G€ urol Y, Celik G, € 2013. Virological and clinical characterKaspar C, K€ uk€ uk O. izations of respiratory infections in hospitalized children. Ital J Pediat 39:1–10. Calvo Rey C, Garcıa Garcıa ML, Casas Flecha I, S anchez Mateos MF, Rodrigo Garcıa G, de Cea Crespo JM, P erez-Bre~ na P. 2006. Papel del rinovirus en las infecciones respiratorias en niıos hospitalizados. An Pediatr 65:205–210. Costa C, Bergallo M, Sidoti F, Terlizzi ME, Astegiano S, Botto S, Elia M, Cavallo R. 2009. What role for human rhinoviruses in the lower respiratory tract? New Microbiol 32:115–117. Couch RB 2001. Rhinoviruses. In: Fields BN, Howley PM, Griffin DE, Lamb RA, Martin MA, Roizman B, Straus SE, Knipe DM, editors. Fields virology. 4th edition. Philadelphia: Lippincott Williams & Wilkins Publishers, pp 631–648. de Paula NT, Carneiro BM, Yokosawa J, Ramos Oliveira e Freitas G, de Mattos Oliveira TF, Faria Costa L, Lopez da Silveira H, Oliveira Queir oz DA. 2011. Human rhinovirus in the lower respiratory tract infections of young children and the possible involvement of a secondary respiratory viral agent. Mem Inst Oswaldo Cruz 106:316–321. Franco D, Delfraro A, Abrego L, Cano M, Castillo C, Castillo M, Castillo J, Pascale J, Arbiza J. 2012. High genetic diversity and predominance of rhinovirus A and C from Panamanian hospitalized children under five years with respiratory infections. Virol J 9:1–6.
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2032 Fry AM, Lu X, Olsen SJ, Chittaganpitch M, Sawatwong P, Chantra S, Baggett HC, Erdman D. 2011. Human rhinovirus infections in rural Thailand: Epidemiological evidence for rhinovirus as both pathogen and bystander. PloS ONE 6:17780. Fujitsuka A, Tsukagoshi H, Arakawa M, Goto-Sugai K, Ryo A, Okayama Y, Mizuta K, Nishina A, Yoshizumi M, Kaburagi Y, Noda M, Tashiro M, Okabe N, Mori M, Yokota S, Kimura H. 2011. A molecular epidemiological study of respiratory viruses detected in Japanese children with acute wheezing illness. BMC Infect Dis 11:168–177. Gern JE 2010. The ABCs of rhinoviruses, wheezing, and asthma. J Virol 84:7418–7426. Hai LT, Bich VTN, Ngai LK, Diep NTN, Phuc PH, Hung VP, Taylor WR, Horby P, Liem NT, Wertheim HFL. 2012. Fatal respiratory infections associated with rhinovirus outbreak. Vietnam Emerg Infect Dis 18:1886–1888. Hara K, Yahara K, Gotoh K, Nakazono Y, Kashiwagi T, Imamura Y, Hamada N, Khositsakulchai W, Sanchai T, Khantawa B, Tharavichitkul P, Maneekarn N, Sirisanthana T, Wanatabe H. 2011. Clinical study concerning the relationship between community-acquired pneumonia and viral infection in Northern Thailand. Intern Med 50:991–998. Hayden FG. 2004. Rhinovirus and the lower respiratory tract. Rev Med Virol 14:17–31. Huijskens EG, Biesmans RC, Buiting AG, Obihara CC, Rossen JW. 2012. Diagnostic value of respiratory virus detection in symptomatic children using real-time PCR. Virol J 9:1–7. Iwane MK, Prill MM, Lu X, Miller EK, Edwards KM, Hall CB, Griffin MR, Staat MA, Anderson LJ, Williams JV, Weinberg GA, Ali A, Szilagyi PG, Zhu Y, Erdman DD. 2011. Human rhinovirus species associated with hospitalizations for acute respiratory illness in young US children. J Infect Dis 204:1702–1710. Jackson DJ, Lemanske RF, Jr. 2010. The role of respiratory virus infections in childhood asthma inception. Immunol Allergy Clin North Am 30:513–522. Jansen RR, Wieringa J, Koekkoek SM. 2011. Frequent detection of respiratory viruses without symptoms: toward defining clinically relevant cutoff values. J Clin Microbiol 49:2631–2636. Jartti T, Lehtinen P, Vuorinen T, Koskenvuo M, Ruuskanen O. 2004. Persistence of rhinovirus and enterovirus RNA after acute respiratory illness in children. J Med Virol 72:695–699. Kusel MMH, de Klerk NH, Holt PG, Kebadze T, Johnston SL, Sly PD. 2006. Role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: A birth cohort study. Pediatr Infect Dis J 25:680–686. Maffey A, Venialgo C, Barrero PR, Fuse VA, M arques MA, Saia M, Villalba A, Teper AM, Mistchenko AS. 2008. Nuevos virus respiratorios en ni~ nos de 2 meses a 3 a~ nos con sibilancias recurrentes. Arch Argent Pediatr 106:302–309. Marcone DN, Videla C, Ricarte C, Carballal G, Vidaurreta S, Echavarrıa M. 2012. Rhinovirus detection by real-time PCR in children with acute respiratory infection in Buenos Aires, Argentina. Rev Argent Microbiol 44:259–265. Matthew J, Pinto Pereira LM, Pappas TE, Swenson CA, Grindle KA, Roberg KA, Lemansje RF, Jr, Lee W, Gern JE. 2009. Distribution and seasonality of rhinovirus and other respiratory viruses in a cross-section of asthmatic children in Trinidad, West Indies. Ital J Pediatr 35:16–25. Meerhoff TJ, Houben ML, Coenjaerts FEJ, Kimpen JLL, Hofland RW, Schellevis F, Bont LJ. 2010. Detection of multiple respiratory pathogens during primary respiratory infection: nasal swab
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Rudi et al. versus nasopharyngeal aspirate using real-time polymerase chain reaction. Eur J Clin Microbiol Infect Dis 29:365–371. Miller EK, Lu X, Erdman DD, Poehling KA, Zhu Y, Griffin MR, Hartert TV, Anderson LJ, Weinberg GA, Hall CB, Iwane MK, Edwards KM. 2007. Rhinovirus-associated hospitalizations in young children. J Infect Dis 195:773–781. Miller EK, Bugna J, Libster R, Shepherd BE, Scalzo PM, Acosta Pl, Hijano D, Reynoso N, Batalle JP, Coviello S, Klein MI, Bauer G, Benitez A, Kleeberger SR, Polack FP. 2012. Human rhinoviruses in severe respiratory disease in very low birth weight infants. Pediatrics 129:60–67. Ministry of Health, Department of Epidemiology. 2011. Integrated approach to acute respiratory infections. Guide to the health team No. 6. 2nd edition. Buenos Aires, Argentina. Nascimento MS, Vieira de Souza A, Vada de Souza Ferreira A, Rodrigues JC, Abramovici S, Ferreira da Silva Filho LV. 2010. High rate of viral identification and coinfections in infants with acute bronchiolitis. Clinics 65:1133–1137. Papadopoulos NG, Johnston SL. 2001. The rhinovirus-not such an innocent?. Q J Med 94:1–3. Papadopoulos NG, Bates PJ, Bardin PG, Papi A, Leir SH, Fraenkel PG, Meyer J, Lackie PM, Sandreson G, Holgate ST, Johnston SL. 2000. Rhinoviruses infect the lower airways. J Infect Dis 181:1875–1884. Papadopoulos NG, Moustaki M, Tsolia M, Bossios A, Astra E, Prezerakou A, Gourgiotis D, Kafetzis D. 2002. Association of rhinovirus infection with increased disease severity in acute bronchiolitis. Am J Respir Crit Care Med 165:1285–1289. Pi~ nero Fern andez JA, Alfayete Miguel ez S, Menasalvas Ruiz A, Salvador Garcıa C, Moreno Doc on A, S anchez-Solıs de Querol M. 2012. Caracter~ nsticas epidemiol agicas, cl~ nnicas y terap euticas de lactantes hospitalizados por bronquiolitis. An Pediatr 77: 391–396. Pitk€ aranta A, Arruda E, Malmberg H, Hayden FG. 1997. Detection of rhinovirus in sinus brushings of patients with acute community-acquired sinusitis by Reverse Transcription-PCR. J Clin Microbiol 35:1791–1793. Savon-Vald es C, Vald es-Ramırez O, Acosta-Herrera B, Gonz alezMu~ noz G, Pi~ n on Ramos A, Gonz alez-B aez G, GoyenecheaHern andez A. 2008. Infecci on por rinovirus en ni eos hospitalizados menores de un a eo. Cuba 2006. Rev Biomed 19:1 22–123. Smuts HE, Workman LJ, Zar HJ. 2011. Human rhinovirus infection in young african children with acute wheezing. BMC Infect Dis 11:65–72. Steininger C, Aberle SW, Popow-Kraupp T. 2001. Early detection of acute rhinovirus infections by a rapid reverse transcription-PCR assay. J Clin Microbiol 39:129–133. Tregoning JS, Schwarze J. 2010. Respiratory viral infections in infants: causes, clinical symptoms, virology and immunology. Clin Microbiol Rev 23:74–98. Vidaurreta SM, Marcone DN, Ellis A, Ekstrom J, Cukier D, Videla C, Carballal G, Echavarrıa M. 2011. Infecci on respiratoria aguda viral en ni~ nos menores de 5 a~ nos. Estudio epidemiol ogico en dos centros de Buenos Aires. Argentina. Arch Argent Pediatr 109:296–304. Wright PF, Deatly AM, Karron RA, Belshe RB, Shi JR, Gruber WC, Zhu Y, Randolph VB. 2004. Comparison of results of detection of rhinovirus by PCR and viral culture in human nasal wash specimens from subjects with and without clinical symptoms of respiratory illness. J Clin Microbiol 45:2126–2129.
The Role of Rhinovirus in Children Hospitalized for Acute Respiratory Disease, Santa Fe, Argentina Juan Manuel Rudi,1* Fabiana Molina,2 Rocıo Dıaz,2 Virginia Bonet,2 Lucila Ortellao,3 mez,1 Judith Pierini,3 Raquel Cociglio,2 and Gabriela Kusznierz1 Diego Cantarutti,3 Alejandra Go 1
National Institute of Respiratory Diseases “Emilio Coni”, National Administration of Laboratories and Health Institutes, Santa Fe, Argentina 2 Childrens Hospital “Dr. O. Alassia”, Santa Fe, Argentina 3 Department of Pediatrics, Hospital “J. B. Iturraspe”, Santa Fe, Argentina
Human rhinoviruses (HRVs) were historically considered upper airway pathogens. However, they have recently been proven to cause infections in the lower respiratory tract, resulting in hospitalization of children with pneumonia, bronchiolitis, and chronic pulmonary obstruction. In this report, HRV frequency and seasonality are described together with patient clinical-epidemiological aspects. From a total of 452 surveyed samples, the HRV nucleic acids was detected in 172 (38.1%) and found in every month of the study year. 60% of inpatients with acute respiratory infection (ARI) associated with HRV were under 6 months of age and 31% had a clinical history, being preterm birth and recurrentwheezing the prevailing conditions. The most frequent discharge diagnoses were pneumonia (35.2%), bronchiolitis (32.4%), and bronchitis (12.4%). Fifteen point nine percent of patients required admission into intensive care units. The results obtained in this study demonstrated the ́ association between HRV and children hospitalizations caused by ARI. J. Med. Virol. 87:2027–2032, 2015. © 2015 Wiley Periodicals, Inc.
population and account for considerable morbidity and mortality [Tregoning and Schwarze, 2010; Bicer et al., 2013]. Human rhinoviruses (HRVs) belong to the order Picornavirales, family Picornaviridae, genus Enterovirus, and species Rhinovirus. They were first isolated in 1956 and were historically considered upper airway pathogens. However, they have recently been proven to cause infections in the lower respiratory tract, resulting in hospitalization of children with pneumonia, bronchiolitis, and chronic pulmonary obstruction. They are also associated with patients who have recurring wheezing episodes and €ranta et al., 1997; Arola asthma exacerbations [Pitka et al., 1988; Fields et al., 2001; Papadopoulos and Johnston, 2001; Papadopoulos et al., 2002; Hayden, 2004; Calvo Rey et al., 2006; Maffey et al., 2008; Costa et al., 2009; Gern, 2010; Jackson and Lemanske, 2010; Meerhoff et al., 2010; Nascimento et al., 2010; de Paula et al., 2011; Fujitsuka et al., 2011; Hara et al., 2011]. In this report, HRV frequency and seasonality are described together with clinical-epidemiological characteristics of ARI hospitalized pediatric patients detected with HRV.
KEY WORDS:
A retrospective study was conducted involving patients who were hospitalized in Santa Fe, Argentina, from March 2010 to February 2011.Patients
acute respiratory infection; pneumonia; pediatric population; rhinovirus; nested RT-PCR
INTRODUCTION Acute respiratory infections (ARI) represent one of the major sources of medical consultation and are placed among the main causes of mortality worldwide [Ministry of Health, Department of Epidemiology, 2011]. In the year 2000, 1.9 million children died because of ARI across the world. Viral respiratory infections have a massive impact on the pediatric C 2015 WILEY PERIODICALS, INC.
MATERIALS AND METHODS Design and Participants
This work was performed at National Institute of Respiratory Diseases “Emilio Coni”, Respiratory Viruses, Santa Fe, Argentina Correspondence to: Juan Manuel Rudi, National Institute of Respiratory Diseases “Emilio Coni”, Blas Parera 8260, 3000 Santa Fe, Argentina. E-mail: [email protected] Accepted 7 May 2015 DOI 10.1002/jmv.24266 Published online 26 May 2015 in Wiley Online Library (wileyonlinelibrary.com).
2028
Rudi et al.
younger than 15 years with an ARI diagnosis at the time of admission were included. Viral Diagnosis A total of 2,020 nasopharyngeal aspirates (NPAs) were obtained from patients with ARI, who underwent antigen detection for respiratory syncytial virus (RSV), adenovirus, parainfluenza, and influenza A and B through indirect immunofluorescence (IIF). Influenza A and B detection was also achieved by real time RT-PCR. A total of 452 IIF-confirmed negative NPAs, randomly collected during all months of the study period, were analyzed for HRV, using a RT-PCR developed by Steininger et al. [2001]; which amplifies a 93 bp fragment located at the end of the 50 noncoding region (NCR) of the HRV genome and conserved among all serotypes of the virus.
calculated. Mantel–Haenszel x2 test was used to compare discrete variables and Mann–Whitney test was applied to compare continuous variables. A P-value of 1,000 per cubic millimeter) was present in 72.7% (Table I). Among patients from whom data was available, two showed microbiologic evidence of a secondary bacterial infection. The pathogens identified were Streptococcus pneumoniae and Klebsiella sp. In an attempt to evaluate the rhinovirus recovery time, the time elapsing between the onset of the respiratory disease and the sampling was associated. In 51 cases (37.5%), the elapsed time was less than 72 hr, in 79 cases (58.1%) between 4 and 14 days elapsed, and in 6 cases (4.4%) the HRV genome was successfully detected after 15 days from the onset of symptoms. The median time elapsed until samples were taken was 5.6 days (Range: 1–23 days). Radiographic Findings Of the 137 patients who underwent chest radiography, the most frequent radiographic findings included air trapping (75.2%) and interstitial pattern (67.9%) (Table I). DISCUSSION This study has produced fresh knowledge about the epidemiology and clinical status of ARI ascribable to HRV in children by associating the virus to hospitalizations caused by moderate and severe respiratory disease. The choice of amplification technique was based on the results obtained by other authors [Steininger et al., 2001], who designed primers that allowed amplify a small fragment of the 50 NCR of the HRV genome, highly conserved among different serotypes of the virus. These authors were able to detect most HRV serotypes hitherto known with a high sensitivity and specificity, since no enterovirus was amplified. While the total negative to other respiratory virus samples were not analyzed, a statistically significant sample size was calculated to infer this result to the overall population, although it is true that positivity obtained for HRV may be underestimated by not having studied co-infections with other respiratory viruses. Several research studies have measured HRV frequency in ARI hospitalized children, ranging from 16 to 57.8%. The 38.1% value from this study is within this range of values, considering PCR-tested samples as a denominator [Calvo Rey et al., 2006; Miller et al., 2007; Fry et al., 2011; Iwane et al., 2011; Vidaurreta et al., 2011; Albuquerque et al., 2012; Huijskens et al., 2012; Marcone et al., 2012; Pi~ nero Fern andez et el., 2012; Bicer et al., 2013]. Rhinoviruses circulated throughout the year, though the months revealing a higher circulation J. Med. Virol. DOI 10.1002/jmv
2030
were March 2010 to May 2010, August 2010, October 2010, November 2010, and February 2011, with positivity percentages ranging between 39 and 51%, also coinciding with RSV low circulation. The months with the largest circulation of RSV (June 2010 and July 2010; data not shown) match those months where HRV was detected lessfrequently. Our findings are in line with observations in previous studies [Calvo Rey et al., 2006; Miller et al., 2007; Fry et al., 2011; Vidaurreta et al., 2011; Albuquerque et al., 2012; Bicer et al., 2013].Differences are likely to exist in HRV seasonality depending on the year being surveyed. It is thus important to extend the study period for at least 3 years to be able to observe possible yearly differences. In assessing the relation between virus prevalence and patient age, it has been observed that 60% of infected children were under 6 months of age, their median age being 4 months. Children over 2 years of age accounted for only 14% of the whole of inpatients with ARI associated to HRV. This is in line with HRV greater prevalence over the first months of life as manifested in previous publications [Miller et al., 2007; Savon-Valdes et al., 2008; Meerhoff et al., 2010; de Paula el al., 2011; Fry et al., 2011; Iwane el al., 2011]. The majority of hospitalized patients (70%) were children with no prior clinical history. However, in the patient group that did have a history, the most frequent conditions were preterm birth and recurrent wheezing. Recently, results from a study in the city of Buenos Aires have revealed that rhinovirus has been the respiratory infection predominant pathogen among preterm infants [Miller et al., 2012]. Furthermore, wheezing is a frequent manifestation of low respiratory tract infection in infants and young children, being viral infection the most common cause [Maffey et al., 2008; Gern, 2010; Fujitsuka et al., 2011; Smuts et al., 2011]. Results from various studies have suggested HRV could be associated with wheezing symptoms. Maffey et al. [2008]; in the city of Buenos Aires, traced HRV in respiratory samples from children with recurrent wheezing, with a frequency of 23% and after RSV, while in Africa [Smuts et al., 2011] and Japan [Fujitsuka et al., 2011] it has been found in 58 and 31%, respectively. Numerous studies on asthma exacerbation etiology [Matthew et al., 2009; Gern, 2010; Albuquerque et al., 2012] have pointed out that HRV is the most widely detected agent among this patient group. Miller et al. [2007] found out that children with asthma have a larger amount of rhinovirus-related hospitalizations than those without a wheezing or asthma history. In this study, only 2% of the cases—all older than 5 years of age—have had asthma diagnosis, in contrast with 28% reported in Miller’s study. While rhinoviruses have been traditionally associated with common cold alone, new developments in diagnosis techniques helped detect its presence in patients with lower respiratory tract infections. In the year 2000, Papadopoulos et al. [2000] investigated J. Med. Virol. DOI 10.1002/jmv
Rudi et al.
in vitro lower respiratory infection through exposure of human bronchial epithelial cells to rhinovirus and in vivo in normal and asthmatic human volunteers. The infection was confirmed in both models, inducing inflammatory response in normal and asthmatic subjects alike. In the study by Kusel et al. [2006]; the pathogenic role of rhinovirus associated with lower respiratory tract infections in the first year of life was recognized. Costa et al. [2009] found a 25% prevalence of rhinovirus in bronchoalveolar lavages of immunocompetent and immunocompromised patients. In line with these studies, pneumonia was the most frequent discharge diagnosis in our study, and it was observed in 35% of patients, agreeing with data presented by Bicer et al.[2013]. No other viral pathogens were detected in the cases studied and two cases alone had sepsis due to Streptococcus pneumoniae and Klebsiella. On the other hand, bronchiolitis was the main cause of hospital admission in children under 2 years of age [Papadopoulos et al., 2000,2002; Nascimento et al., 2010]. Recent research on bronchiolitis etiology has reported RSV and HRV as the most important etiological agents in hospitalized children, with detection rates of 39 and 13%, respectively, [Nascimento et al., 2010; Pi~ nero Fern andez et al., 2012]. In this series, a high proportion of bronchiolitis was also observed (32%), while in the study by Calvo Rey et al. [2006] it was 21% of inpatients. These findings are at odds with those observed in other studies, where pneumonia was diagnosed in 8–16% of patients [Calvo Rey et al., 2006; Miller et al., 2007; de Paula et al., 2011; Fry et al., 2011] and bronchiolitis in 11–13% of cases [Miller et al., 2007; de Paula et al., 2011]. Conversely, in the study by Savon-Valdes et al. [2006], a high proportion of bronchiolitis has been observed (74%). Fever has been one of the most frequent clinical manifestations, in line with other published studies [Papadopoulos et al., 2002; Calvo Rey et al., 2006; Smuts et al., 2011; Vidaurreta el al, 2011; Marcone et al., 2012; Maffey et al., 2014]. The findings obtained in this work support recent studies [Papadopoulos et al., 2002; Bicer et al., 2013] that have demonstrated HRV to be associated with severe illness, as 15% of patients had severe symptoms leading up to admission in intensive care units (ICUs), though no fatal cases were recorded. 56.5% of critical patients had complications, such as sepsis and acute imminent respiratory failure. In the study by Bicer et al. [2013]; 36% of HRV-infected children had moderate to severe illness, while in RSV-infected children the proportion was 33%. In Vietnam, HRV was associated with an outbreak of severe respiratory infection in 12 children that were hospitalized and developed acute respiratory distress syndrome, 7 of whom died [Hai et al., 2012]. The median duration of hospital stay was 6 days. This period has proven similar to what has been reported in previously published studies [Maffey et al., 2008; Marcone et al., 2012]. Supplemental
Rhinovirus in Hospitalized Children
oxygen was given to a high proportion of patients (85%), as opposed to mechanical ventilation that was only necessary in slightly more than 10% of cases. Those values are higher than those observed by other authors [Miller et al., 2007; Iwane et al., 2011]. Age under 3 months, presence of immunodeficiency, congenital cardiopathy, chronic pulmonary disease, preterm birth, and malnutrition are risk factors for the development of severe low ARI, according to the Argentine Pediatric Society Consensus. In this study, age and presence of medical history did not represent a risk factor for severe ARI development, although 60% of cases were under 6 months and 40% of severe patients were recorded as having some type of medical history. In addition, over 30% of patients in the study group showed at least one ARI-caused hospitalization prior to this study, which was not a risk factor either. There are many factors that can have a bearing on the severity of the disease, related to both the host and the virus. As to HRV-related factors, the presence of co-infections with other respiratory viruses [Tregoning and Schwarze, 2010; de Paula et al., 2011] or the HRV species involved [Franco et al., 2012] could be cited, but neither has been assessed in this study. Although there are controversial results regarding association between the different species and clinical symptoms [Fry et al., 2011; Franco et al., 2012], some literature has proven the most prevalent role of HRVCs in lower ARI, associating them with severe illness. In their study, Iwane et al. [2011] have observed an association between HRVA and HRVC-infected patients and severe illness development, needing longer hospital stays, ICU and mechanical ventilation, as opposed to patients infected with HRVB. Sixty six percent of confirmed cases were treated with ATBs. Incidentally, it must be considered that this study was retrospective and virological diagnosis was negative for the rest of the respiratory viruses. This highlights the importance of considering early diagnosis of other respiratory viruses involved, to contribute to the clinical management of patients with ARI. Elsewhere, HRV has been identified in asymptomatic patients, detection prevalence varying from 12 to 28% [Jartti et al., 2004; Wright et al., 2004; Jansen et al., 2011]. This may be suggesting either an asymptomatic colonization or a long-lasting virus elimination after a respiratory infection, detecting HRV RNA even 5 or 6 weeks after the respiratory infection [Jartti et al., 2004]. Consequently, clinical relevance of hospitalized patient rhinovirus detection can be difficult to interpret. In this study, in 26% of patients the virus was detected 7 days after the onset of respiratory symptoms. In all this cases, sampling was made within 48 hr since hospitalization, as recommended by national guidelines. The strength of this study is the important volume of samples assessed for HRV detection and the high proportion of positivity obtained in relation to other
2031
studies. This enabled us to carry out an exhaustive analysis of clinical data, though this analysis has some limitations. First of all, there were no controls in whom HRV was measured. One of the core problems in case control studies is the selection of an appropriate control group, because it must be ascertained whether this group had not previously (5 or 6 weeks before) suffered from catarrh in the upper airways. It was therefore decided not to include any control group in our study. Nonetheless, detection studies were made for other known respiratory viruses, and confirmed negative population was analyzed, suggesting rhinovirus could be the ARI-causing agent. Admittedly, having used PCR for influenza alone and IIF for the rest of viruses as a screening technique, which is less sensitive than molecular techniques, could be a constraint in this study. Another constraint in this study is the limited geographical region where the study was conducted and that the study period is confined to one year, and the seasonal situation observed might not be a reflection of other years. This was a first test locally with the intention to extend the study to other regions and for longer periods. Regardless of such constraints, and in view of the results obtained, HRV participation in ARI has been demonstrated. ACKNOWLEDGMENTS We gratefully acknowledge the technical assistance rendered by Mr. Gonzalo Vidal in the detection of respiratory viruses through IIF. REFERENCES Albuquerque MCM, Varella RB, Santos N. 2012. Acute respiratory viral infections in children in Rio de Janeiro and Teres opolis, Brazil. Rev Inst Med Trop Sao Paulo 54:249–255. Arola M, Ziegler T, Ruuskanen J. 1988. Rhinovirus in acute otitis media. J Pediatr 113:693–695. Bicer S, Giray T, C€ ol D, Erdag GC, Vitrinel A, G€ urol Y, Celik G, € 2013. Virological and clinical characterKaspar C, K€ uk€ uk O. izations of respiratory infections in hospitalized children. Ital J Pediat 39:1–10. Calvo Rey C, Garcıa Garcıa ML, Casas Flecha I, S anchez Mateos MF, Rodrigo Garcıa G, de Cea Crespo JM, P erez-Bre~ na P. 2006. Papel del rinovirus en las infecciones respiratorias en niıos hospitalizados. An Pediatr 65:205–210. Costa C, Bergallo M, Sidoti F, Terlizzi ME, Astegiano S, Botto S, Elia M, Cavallo R. 2009. What role for human rhinoviruses in the lower respiratory tract? New Microbiol 32:115–117. Couch RB 2001. Rhinoviruses. In: Fields BN, Howley PM, Griffin DE, Lamb RA, Martin MA, Roizman B, Straus SE, Knipe DM, editors. Fields virology. 4th edition. Philadelphia: Lippincott Williams & Wilkins Publishers, pp 631–648. de Paula NT, Carneiro BM, Yokosawa J, Ramos Oliveira e Freitas G, de Mattos Oliveira TF, Faria Costa L, Lopez da Silveira H, Oliveira Queir oz DA. 2011. Human rhinovirus in the lower respiratory tract infections of young children and the possible involvement of a secondary respiratory viral agent. Mem Inst Oswaldo Cruz 106:316–321. Franco D, Delfraro A, Abrego L, Cano M, Castillo C, Castillo M, Castillo J, Pascale J, Arbiza J. 2012. High genetic diversity and predominance of rhinovirus A and C from Panamanian hospitalized children under five years with respiratory infections. Virol J 9:1–6.
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