Journal of Clinical Virology 73 (2015) 77–80
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Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Adenovirus species C detection in children under four years of age with acute bronchiolitis or recurrent wheezing Rosângela Prendin Tórtora a,b , Maria Angélica Arpon Marandino Guimarães a,b,c,e,∗ , Leandro Magalhães de Souza a,b , Isabela Arruda Santos a,d , Rafael Brandão Varella e,f , Maria de Fátima Pombo March d , Antonio Jose Ledo Alves da Cunha a,d , Clemax Couto Sant’ Anna a,d,e a
Laboratório do Nucleo de Apoio a Crianc¸a e ao Adolescente do Instituto de Puericultura e Pediatria Martagão Gesteira, UFRJ, Brazil Laboratório de Virologia, Hospital Universitário Clementino Fraga Filho, UFRJ, Brazil c Depto de Medicina Preventiva, Faculdade de Medicina, UFRJ, Brazil d Depto de Pediatria, Faculdade de Medicina, UFRJ, Brazil e Programa de Pós graduac¸ão em Doenc¸as Infecciosas, Faculdade de Medicina, UFRJ, Brazil f Depto de Microbiologia, Instituto Biomédico, UFF, Brazil b
a r t i c l e
i n f o
Article history: Received 29 April 2015 Received in revised form 25 September 2015 Accepted 1 November 2015 Keywords: Human Adenovirus species C Real time PCR Acute bronchiolitis Recurrent wheezing Children under 48 months of age
a b s t r a c t Background: Lower respiratory tract viral infection is an important cause of morbidity and mortality in children worldwide. Among viral etiological agents the human Adenovirus (AdV) has been associated to mild or severe respiratory tract infection. Objective: To detect the presence of human Adenovirus (AdV) in children with acute bronchiolitis or recurrent wheezing, describing their clinical features and determining Adenovirus species and AdV association to Respiratory Syncytial Virus (RSV), Human Metapneumovirus (MPV) and Parainfluenza virus (PIV). Study design: A total of 155 children bellow 48 months of age with acute bronchiolitis or recurrent wheezing were investigated for the presence of AdV, RSV, MPV and PIV in nasopharyngeal aspirate, by real-time PCR method. Results: AdV, predominantly of species C, has been detected as the unique pathogen (AdVi) or in association to other pathogens (AdVa.), in 39/155 samples. Crackles were more frequent in children with AdV. RSVi was detected predominantly in children with acute bronchiolitis while AdVi and AdVa were detected more frequently in patients with recurrent wheezing. Conclusion: A small outbreak of AdV species C was observed in 2012 and 2013. AdV was detected more frequently in children with recurrent wheezing while RSVi was more frequent in infants with acute bronchiolitis. Published by Elsevier B.V.
1. Background Acute bronchiolitis and recurrent wheezing are observed in children under 5 years of age associated with Respiratory Syncytial Virus (RSV), Meta-pneumovirus (MPV), Adenovirus (AdV), Rhinovirus (RV) and Para-influenza Virus (PIV) infections. In children under 5 years of age, RSV is considered to be the most frequent
∗ Corresponding author at: Hospital Universitário Clementino Fraga Filho UFRJ Laboratório de Virologia 3◦ andar-Servic¸o de Patologia Clínica, Rua Professor Rodolpho Paulo Roco Cidade Universitária, CEP 21910590 Rio de Janeiro, RJ, Brazil. E-mail address: [email protected] (M.A.A.M. Guimarães). http://dx.doi.org/10.1016/j.jcv.2015.11.007 1386-6532/Published by Elsevier B.V.
etiologic agent of acute bronchiolitis, while AdV is frequently associated to pneumonia and recurrent wheezing [1–3]. AdV has been considered an important pathogen due to the possibility of respiratory tract sequels and evolution to death. Of the seven AdV species (A–G), AdV A–C and E, have been associated to respiratory tract infection, with AdV specie B type 7 being associated to high morbidity and mortality rate [3]. Brazilian epidemiological bulletins show AdV infection occurring year-round affecting predominantly children up to 4 years of age [4]. Different laboratorial methods have been employed for the screening of viral etiology in cases of lower respiratory tract infection. Although viral isolation in cell culture and direct antigen detection with monoclonal antibodies are highly specific,
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molecular methods have been the most widely used due to their practicality, specificity and sensitivity. 2. Objectives In this study we detected the presence of human Adenovirus (AdV) in children with acute bronchiolitis or recurrent wheezing, describing their clinical features and determining Adenovirus species classification and AdV association to Respiratory Syncytial Virus (RSV), Human Metapneumovirus (MPV) and Parainfluenza virus (PIV). 3. Study design A total of 155 children below 48 months of age, with acute bronchiolitis or recurrent wheezing, were included in a cross-sectional study, with a convenience sample in an open population. The study was conducted in a pediatric hospital in Rio de Janeiro (IPPMGUFRJ). Patients assisting the Hospital came from low income families with health coverage provided by the Brazilian health system. Children were hospitalized in the emergency service of the hospital between January 2010 and April 2013. Nasopharyngeal aspirate (NPA) was collected within the first 48 h after admission, to avoid cases of nosocomial infection. Variables such as age at admission, gender, the presence of household smokers, breastfeeding and atopic manifestations were observed. Also, clinical manifestations, radiological abnormalities, the use of oxygen supply or mechanical ventilation were included in the study. Disease severity was categorized as grade I (no need of oxygen supply but with feedings difficulties), grade II (need of oxygen supply but no need of mechanical ventilation) and grade III (need of mechanical ventilation). Children with cardiac disease, chronic pulmonary disease, immunodeficiency or prematurity were excluded from the study. The diagnosis of acute bronchiolitis and recurrent wheezing, made by the physicians involved in the study, had the same clinical criteria described by Bezerra et al. [1]. Acute brochiolitis was considered when the episode of wheezing occurred for the first time in children under 18 months of age. Recurrent wheezing was considered in children with two or more episodes of wheezing. The qualitative detection of viral DNA/cDNA was performed in NPA, by a real-time PCR (qPCR) in a SyberPower system (Applied Biosystems, Life Tech, USA). Clinical specimens were submitted to a nucleic acid extraction by the use of a commercial kit (RTP virus DNA/RNA Mini Kit, Invisorb Stratek, USA). A reverse transcription PCR was performed for viruses with RNA genomes (High Capacity cDNA Reverse transcription, commercial kit, Applied Biosystems, Life Tech, USA). The primers used in qPCR were the internal degenerated primers described by Allard et al. [5] for AdV, Kaida et al.
[6] for MPV and PIV and Hu et al. [7], for RSV. Thermal cycling conditions for the qPCR were those described by Kaida et al. [6]. For AdV hexon segment sequencing, AdV-positive samples were submitted again to a PCR method as described by Allard et al. [5]. PCR products were purified by GE Healthcare commercial kit and the fragments (sense and anti-sense) were sequenced using the Big DyeTM terminator sequencing chemistry (Applied Biosystems Inc., Foster City, CA). Sequence alignments were conducted in ClustalW with default settings. Phylogenetic analysis was carried out using MEGA 5.2 by the maximum likelihood (ML) algorithm using the Kimura 2 parameters distance. Statistical significance of clades was measured by 2000 bootstrap replicates. Previously published AdV species C (JX173085.1), B (KC570906.1) and D (JF799911.1) sequences were used for the alignment in order to infer the phylogeny of the AdV samples. Statistical analysis of the observed results has been processed by the SAS statistical software (version 6.11, SAS Institute Inc., Cary, North Carolina). A descriptive analysis of the categorical variables was done through measures of frequency and percentages. Continuous variables were presented as minimum and maximum values, averages, medians and standard deviations. The associations of the observed variables were made by the use of chi-square test or Fisher exact test (p < 0.05).
4. Results Of the 155 collected NPA, 94 (60.6%) were positive for any of the viruses under investigation. AdV was detected in 10/155 (6.4%) samples as the unique pathogen (AdVi) and in 29/155 (18.7%) associated with other viruses (AdVa). RSV (25/29) was the most frequent virus associated to AdV, followed by MPV (16/29) and PIV (1/29). AdV was detected from 2010 to 2013 with an increased frequency in 2012 and 2013. RSV was detected throughout the studied period and was the most frequent viral pathogen. MPV was not detected in 2010 and PIV was detected only in 2012. The clinical features observed in the different groups of patients (AdVi, RSVi, MPVi and AdVa), are summarized in Table 1. All patients had disease severity grade I or II and there was no difference in the frequency of dyspnea, fever, running nose, nasal congestion, vomiting and/or diarrhea, in the different groups of infected patients (AdVi, RSVi, MPVi and AdVa,). Although cough was more frequent in patients with AdV infections (AdVi and AdVa) such difference did not reach statistical significance. Regarding pulmonary auscultation, patients with AdVa presented crackles in a higher proportion when compared to patients with RSVi (p = 0.001) and MPVi (p = 0.04). The most frequent chest radiography findings were hyperinflation (increased rib cage size and lowered
Table 1 Clinical features of children with AdV, RSV, hMPV and PIV (n = 94). Clinical features
Virus (n/%) AdVa(n = 29) (a) n/%
AdVi (n = 10) (b) n/%
RSVi (n = 46) (c) n/%
hMPVi (n= 9) (d) n/%
p value
Dyspnea Fever Cough Runny nose Nasal congestion Presence of crackles
29/100 22/76 27/93 17/58 17/58 18/62
10/100 7/70 10/100 5/50 5/50 4/40
45/98 26/56 41/89 28/61 26/56 11/24
9/100 5/55 7/77 4/44 7/78 2/22
Household smokers Administration of O2 Acute bronchiolitis Recurrent wheezing
17/59 22/75.8 12/22.6 17/41.5
2/20 7/70 03/5.7 07/17.1
21/45 29/63 33/62.3 13/31.7
4/44 6/66.7 5/9.4 4/9.7
– NS NS NS NS 0.001a/c 0.042a/d 0.04a/b NS – –
a = Associated to other virus; i = detected with no other virus; NS = no significance. AdV = Adenovirus, RSV = Respiratory Syncytial Virus, hMPV = Meta-pneumovirus, PIV = Para-influenza Virus.
R.P. Tórtora et al. / Journal of Clinical Virology 73 (2015) 77–80
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5. Discussion
Fig. 1. Phylogenetic tree based on ML algorithm of human AdV sequences (N = 21) represented with the year of detection. AdV species C, B and D (outgroup) retrieved from the NCBI databank were included in the analysis to establish a phylogenetic relationship with AdV sequences from the study.
diaphragm), interstitial infiltrate and atelectasis. The frequency of household smokers was greater in the group of children with AdVa in comparison to children with AdVi (p = 0.04). Concerning disease severity and viral co-infection (AdVa), there was no statistical difference in the frequency of children showing disease severity grade II, with co-infection (AdVa, 75.9%) and monoinfection (AdVi, RSVi, MPVi, 65%, p = 0.2). We observed that 31/39 (79.5%) children of 3 month of age or younger (≤3 m) and 33/22 (60%, p = 0.036) of older children (>3 m), showed disease severity grade II (60%, p = 0.036). Concerning only children infected by RSVi, we observed that 19/25 (76%) children of ≤3 m and 10/11 (47.6%, p = 0.046) of >3 m showed disease severity grade II. On the other hand, children infected by AdVi, AdVa or MPVi with disease severity grade II, showed no predominant age group. Concerning clinical diagnosis and age groups, acute bronchiolitis was detected predominantly in children of 6 months of age or younger (≤6 m) and recurrent wheezing, in children older than 6 m (>6 m) (p < 0.0001). Acute bronchiolitis was diagnosed in 53/94 (56.4%) children and recurrent wheezing in 41/94 (43.6%) children. In relation to the virus etiology, AdVa (17/29) and AdVi (7/10) were more frequently detected in children with recurrent wheezing while RSVi was more frequently detected in children with acute bronchiolitis (33/46). The analysis of the association between clinical diagnosis and viral infection, based on patient’s age, showed a high correlation (Pearson 0.870; p < 0.0001) between age and the clinical diagnosis of acute bronchiolitis and recurrent wheezing, due to a multicollinearity effect, but showed no interaction effect in relation to the presence or absence of a particular virus. Of the 39 AdV-positive samples, 21 were successfully sequenced. The phylogenetic analysis indicated that 20 were species C AdV and one was species B AdV (Fig. 1). The clustering of the samples in the phylogenetic tree was not related to the year of AdV detection or any specific clinical feature.
In this study virus detection occurred in nearly 61% (94/155) of the children. Of the 94 children, 29 (31%) had two or more viruses detected in NPA. This high frequency of virus detection was similar to the findings of other authors who used molecular methods to show the presence of viruses in NPA [1,2,8,9]. Regarding the frequency of AdV, our results showed similarity to other studies in 2010 (6.5%) and 2011 (8.1%), but in 2012 and 2013 we observed an increase in the frequency of AdV that reached 35% and 24%, respectively, suggesting a small outbreak also reported by the epidemiological bulletins of 2012 and 2013, published by the Ministry of Health, in Brazil [4]. In our study there was a predominance of specie C AdV (20/21). Infections by species C AdV affects more than 80% of people early in life and is found in sporadic form or in endemic outbreaks, although epidemic outbreaks have also been reported [9,10]. There are controversies on the fact that viral co-detection increases the severity of respiratory disease. Semple et al. [11] and Richard et al. [12] reported an association between disease severity and viral co-detection. Differently Canducci et al. [13] and Brand et al. [14] did not report an association between viral co-infection and disease severity. In our results we observed no children with disease severity grade III, and the number of children with disease severity grade II and co-infection (76%) although higher than the number of children with mono-infection (65.6%, p = 0.2), was not statistically significant. In studies that look for the role of co-infection in disease severity one can we find patients with comorbidities (prematurity, cardiac congenital disease, chronic pulmonary disease, immunodeficiency) included in the study protocol, what may contribute to the controversy seen in the conclusion of different authors, concerning the importance of co-infection in disease severity. Studies that include patients with comorbidities may cause bias in the analysis of data, since patients with comorbidities tend to have bad prognosis. In our protocol the exclusion of patients with comorbidities may have contributed to the absence of patients with disease severity grade III, but facilitated the verification of virus effect in host. RSVi was detected in 33/53 (62.3%) of children with acute bronchiolitis and predominated in the first 3 months of age (25/53). These results are in accordance to the findings of other authors, that reported a predominance of acute bronchiolitis and RSV infection, in the first 3 months of age [1–3,11–14]. Concerning all kinds of viral infection (AdVa, AdVi, RSVi, MPVi) and age groups, we observed that the frequency of children of ≤3 m with disease severity grade II (79.5%) was greater than children of >3 (60%, p = 0.038). If we consider only children with RSVi infection and disease severity grade II, also, the number of children of ≤3 m (76%) was greater than the number of children of >3 m (48%, p = 0.046). These results suggest a greater morbidity of RSV infection in children of ≤3 m, and it is not surprising, since it has been described by other authors [12–14], concerning disease severity in children of ≤3 m with RSV respiratory tract infection. The high correlation between age and the clinical diagnosis of acute bronchiolitis and recurrent wheezing should be related to the definition of these two clinical entities that characterize bronchiolitis as the first episode of wheezing, in children under 18 months of age, while the diagnosis of recurrent wheezing is considered in children with two or more episodes of wheezing, demanding at least few months to occur. The exclusion of any interaction effect in relation to the presence or absence of a particular virus should be the result of virus characteristics like, number of sorotypes, presence of latent infection, for example, or to the absence of immunological protection concerning the host. In our results AdVa and AdVi were detected, more frequently, in children with recurrent wheezing (17/29 and 7/10, respectively). To
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our knowledge, there are no studies demonstrating the role of AdV in recurrent wheezing, as has been suggested in asthma [10]. Also, experiments in guinea pig suggest an increase in lung inflammatory response, due to AdV [15]. In brief, we observed a small outbreak of AdV species C, detected in 2012 and 2013. AdVa and AdVi were detected more frequently in children with recurrent wheezing while RSVi was more frequent in infants with acute bronchiolitis. Concerning disease severity, we observed no statistical significance in the number of patients with co-infection (AdVa) and mono-infection (AdVi, RSVi, and MPVi). As our study used a convenience sample, in a specific Hospital, its results cannot be extrapolated to the general population. Funding This work was supported by the Research Foundation of the State of Rio de Janeiro (FAPERJ). Competing interests The authors declare no conflict of interest concerning the submitted paper. Ethical approval The study was approved by the Ethics Committee of IPPMG and by the National Research Ethics Office of Brazil. Each child had a written informed consent that was obtained from the parents before the enrolment. Children whose parents refused to sign the written informed consent were excluded from the study. Acknowledgment Juliana Pina Metzner, MD for the collection of samples and Dr Marcelo Land, PhD for the statistical revision. References [1] P.G.M. Bezerra, M.C.A. Britto, J.B. Correia, et al., Viral and atypical bacterial detection in acute respiratory infection in children under five years, PLoS One 18 (2011) e18928.
[2] L.F. Costa, J. Yokosawa, O.C. Mantese, et al., Respiratory viruses in children younger than five years old with acute respiratory disease from 2001 to 2004 in Uberlandia, MG, Brazil, Mem. Inst. Oswaldo Cruz 101 (2006) 301–306. [3] S.M. Straliotto, M.M. Siqueira, V. Machado, Respiratory viruses in the pediatric intensive care unit: frequency and clinical aspects, Mem. Inst. Oswaldo Cruz 99 (2004) 883–887. [4] Brasil, Ministério da Saúde, Secretaria de Vigilância e Saúde. Boletim Epidemiológico Influenza monitoramento até semana 52 em 2012 e semana 29 em 2013 (Brazil, Ministry of Health. Influenza, Epidemiological Buletin: Monitoring until Epidemiological Week 52 in 2012 and Week 29 in 2013). BE SUS 44 (14–15) (2013) http://portalsaude.saude.gov.br/index.php/oministerio/principal/leia-mais-o-ministerio/197-secretaria-svs/11955boletins-epidemiologicos-arquivos. [5] A. Allard, B. Albinsson, G.J. Wadell, Rapid typing of human Adenoviruses by a general PCR combined with restriction endonuclease analysis, J. Clin. Microbiol. 39 (2001) 498–505. [6] A. Kaida, H. Kubo, M. Shiomi, Evaluation of real-time RT-PCR compared with conventional RT-PCR for detecting human metapneumovirus RNA from clinical specimens, Jpn. J. Infect. Dis. 61 (2008) 461–464. [7] A. Hu, M. Colella, J.S. Tam, Simultaneous detection, subgrouping, and quantitation of respiratory syncytial virus A and B by real-time PCR, J Clin. Microbiol. 41 (2003) 149–154. [8] J.Y. Hong, H.J. Lee, P.A. Piedra, et al., Lower respiratory tract infections due to Adenovirus in hospitalized Korean children: epidemiology, clinical features, and prognosis, Clin. Infect. Dis. 32 (2001) 1423–1429. [9] F.A. Moura, J.R.L. Mesquita, S.A.R. Portes, Antigenic and genomic characterization of Adenovirus associated to respiratory infections in children living in Northest Brazil, Mem. Inst. Oswaldo Cruz 102 (2007) 937–941. [10] C.T. Garnett, D. Erdman, W. Xu, et al., Prevalence and quantitation of species C Adenovirus DNA in human mucosal lymphocytes, J. Virol. 76 (2002) 10608–10616. [11] M. Semple, A. Cowell, W. Dove, et al., Dual infection of infants by human metapneumovirus and human Respiratory Syncytial Virus is strongly associated with severe bronchiolitis, J. Infect. Dis. 191 (2005) 382–386. [12] N. Richard, F. Komurian-Pradel, E. Javouhey, et al., The impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis, Pediatr. Infect. Dis. J. 27 (2008) 213–217. [13] F. Canducci, M. Debiaggi, M. Sampaolo, et al., Two-year prospective study of single infections and co-infections by Respiratory Syncytial Virus and viruses identified recently in infants with acute respiratory disease, J. Med. Virol. 80 (2008) 716–723. [14] H.K. Brand, R. Groot, J.M.D. Galama, M.L. Brouwer, et al., Infection with multiple viruses is not associated with increased disease severity in children with bronchiolitis, Pediatr. Pulmonol. 47 (2012) 393–400. [15] T.Z. Vitalis, A. Kern, H. Croome, The effect of latent Adenovirus 5 infection on cigarette smoke-induced lung inflammation, Eur. Respir. J. 11 (1998) 664–669.
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Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Adenovirus species C detection in children under four years of age with acute bronchiolitis or recurrent wheezing Rosângela Prendin Tórtora a,b , Maria Angélica Arpon Marandino Guimarães a,b,c,e,∗ , Leandro Magalhães de Souza a,b , Isabela Arruda Santos a,d , Rafael Brandão Varella e,f , Maria de Fátima Pombo March d , Antonio Jose Ledo Alves da Cunha a,d , Clemax Couto Sant’ Anna a,d,e a
Laboratório do Nucleo de Apoio a Crianc¸a e ao Adolescente do Instituto de Puericultura e Pediatria Martagão Gesteira, UFRJ, Brazil Laboratório de Virologia, Hospital Universitário Clementino Fraga Filho, UFRJ, Brazil c Depto de Medicina Preventiva, Faculdade de Medicina, UFRJ, Brazil d Depto de Pediatria, Faculdade de Medicina, UFRJ, Brazil e Programa de Pós graduac¸ão em Doenc¸as Infecciosas, Faculdade de Medicina, UFRJ, Brazil f Depto de Microbiologia, Instituto Biomédico, UFF, Brazil b
a r t i c l e
i n f o
Article history: Received 29 April 2015 Received in revised form 25 September 2015 Accepted 1 November 2015 Keywords: Human Adenovirus species C Real time PCR Acute bronchiolitis Recurrent wheezing Children under 48 months of age
a b s t r a c t Background: Lower respiratory tract viral infection is an important cause of morbidity and mortality in children worldwide. Among viral etiological agents the human Adenovirus (AdV) has been associated to mild or severe respiratory tract infection. Objective: To detect the presence of human Adenovirus (AdV) in children with acute bronchiolitis or recurrent wheezing, describing their clinical features and determining Adenovirus species and AdV association to Respiratory Syncytial Virus (RSV), Human Metapneumovirus (MPV) and Parainfluenza virus (PIV). Study design: A total of 155 children bellow 48 months of age with acute bronchiolitis or recurrent wheezing were investigated for the presence of AdV, RSV, MPV and PIV in nasopharyngeal aspirate, by real-time PCR method. Results: AdV, predominantly of species C, has been detected as the unique pathogen (AdVi) or in association to other pathogens (AdVa.), in 39/155 samples. Crackles were more frequent in children with AdV. RSVi was detected predominantly in children with acute bronchiolitis while AdVi and AdVa were detected more frequently in patients with recurrent wheezing. Conclusion: A small outbreak of AdV species C was observed in 2012 and 2013. AdV was detected more frequently in children with recurrent wheezing while RSVi was more frequent in infants with acute bronchiolitis. Published by Elsevier B.V.
1. Background Acute bronchiolitis and recurrent wheezing are observed in children under 5 years of age associated with Respiratory Syncytial Virus (RSV), Meta-pneumovirus (MPV), Adenovirus (AdV), Rhinovirus (RV) and Para-influenza Virus (PIV) infections. In children under 5 years of age, RSV is considered to be the most frequent
∗ Corresponding author at: Hospital Universitário Clementino Fraga Filho UFRJ Laboratório de Virologia 3◦ andar-Servic¸o de Patologia Clínica, Rua Professor Rodolpho Paulo Roco Cidade Universitária, CEP 21910590 Rio de Janeiro, RJ, Brazil. E-mail address: [email protected] (M.A.A.M. Guimarães). http://dx.doi.org/10.1016/j.jcv.2015.11.007 1386-6532/Published by Elsevier B.V.
etiologic agent of acute bronchiolitis, while AdV is frequently associated to pneumonia and recurrent wheezing [1–3]. AdV has been considered an important pathogen due to the possibility of respiratory tract sequels and evolution to death. Of the seven AdV species (A–G), AdV A–C and E, have been associated to respiratory tract infection, with AdV specie B type 7 being associated to high morbidity and mortality rate [3]. Brazilian epidemiological bulletins show AdV infection occurring year-round affecting predominantly children up to 4 years of age [4]. Different laboratorial methods have been employed for the screening of viral etiology in cases of lower respiratory tract infection. Although viral isolation in cell culture and direct antigen detection with monoclonal antibodies are highly specific,
78
R.P. Tórtora et al. / Journal of Clinical Virology 73 (2015) 77–80
molecular methods have been the most widely used due to their practicality, specificity and sensitivity. 2. Objectives In this study we detected the presence of human Adenovirus (AdV) in children with acute bronchiolitis or recurrent wheezing, describing their clinical features and determining Adenovirus species classification and AdV association to Respiratory Syncytial Virus (RSV), Human Metapneumovirus (MPV) and Parainfluenza virus (PIV). 3. Study design A total of 155 children below 48 months of age, with acute bronchiolitis or recurrent wheezing, were included in a cross-sectional study, with a convenience sample in an open population. The study was conducted in a pediatric hospital in Rio de Janeiro (IPPMGUFRJ). Patients assisting the Hospital came from low income families with health coverage provided by the Brazilian health system. Children were hospitalized in the emergency service of the hospital between January 2010 and April 2013. Nasopharyngeal aspirate (NPA) was collected within the first 48 h after admission, to avoid cases of nosocomial infection. Variables such as age at admission, gender, the presence of household smokers, breastfeeding and atopic manifestations were observed. Also, clinical manifestations, radiological abnormalities, the use of oxygen supply or mechanical ventilation were included in the study. Disease severity was categorized as grade I (no need of oxygen supply but with feedings difficulties), grade II (need of oxygen supply but no need of mechanical ventilation) and grade III (need of mechanical ventilation). Children with cardiac disease, chronic pulmonary disease, immunodeficiency or prematurity were excluded from the study. The diagnosis of acute bronchiolitis and recurrent wheezing, made by the physicians involved in the study, had the same clinical criteria described by Bezerra et al. [1]. Acute brochiolitis was considered when the episode of wheezing occurred for the first time in children under 18 months of age. Recurrent wheezing was considered in children with two or more episodes of wheezing. The qualitative detection of viral DNA/cDNA was performed in NPA, by a real-time PCR (qPCR) in a SyberPower system (Applied Biosystems, Life Tech, USA). Clinical specimens were submitted to a nucleic acid extraction by the use of a commercial kit (RTP virus DNA/RNA Mini Kit, Invisorb Stratek, USA). A reverse transcription PCR was performed for viruses with RNA genomes (High Capacity cDNA Reverse transcription, commercial kit, Applied Biosystems, Life Tech, USA). The primers used in qPCR were the internal degenerated primers described by Allard et al. [5] for AdV, Kaida et al.
[6] for MPV and PIV and Hu et al. [7], for RSV. Thermal cycling conditions for the qPCR were those described by Kaida et al. [6]. For AdV hexon segment sequencing, AdV-positive samples were submitted again to a PCR method as described by Allard et al. [5]. PCR products were purified by GE Healthcare commercial kit and the fragments (sense and anti-sense) were sequenced using the Big DyeTM terminator sequencing chemistry (Applied Biosystems Inc., Foster City, CA). Sequence alignments were conducted in ClustalW with default settings. Phylogenetic analysis was carried out using MEGA 5.2 by the maximum likelihood (ML) algorithm using the Kimura 2 parameters distance. Statistical significance of clades was measured by 2000 bootstrap replicates. Previously published AdV species C (JX173085.1), B (KC570906.1) and D (JF799911.1) sequences were used for the alignment in order to infer the phylogeny of the AdV samples. Statistical analysis of the observed results has been processed by the SAS statistical software (version 6.11, SAS Institute Inc., Cary, North Carolina). A descriptive analysis of the categorical variables was done through measures of frequency and percentages. Continuous variables were presented as minimum and maximum values, averages, medians and standard deviations. The associations of the observed variables were made by the use of chi-square test or Fisher exact test (p < 0.05).
4. Results Of the 155 collected NPA, 94 (60.6%) were positive for any of the viruses under investigation. AdV was detected in 10/155 (6.4%) samples as the unique pathogen (AdVi) and in 29/155 (18.7%) associated with other viruses (AdVa). RSV (25/29) was the most frequent virus associated to AdV, followed by MPV (16/29) and PIV (1/29). AdV was detected from 2010 to 2013 with an increased frequency in 2012 and 2013. RSV was detected throughout the studied period and was the most frequent viral pathogen. MPV was not detected in 2010 and PIV was detected only in 2012. The clinical features observed in the different groups of patients (AdVi, RSVi, MPVi and AdVa), are summarized in Table 1. All patients had disease severity grade I or II and there was no difference in the frequency of dyspnea, fever, running nose, nasal congestion, vomiting and/or diarrhea, in the different groups of infected patients (AdVi, RSVi, MPVi and AdVa,). Although cough was more frequent in patients with AdV infections (AdVi and AdVa) such difference did not reach statistical significance. Regarding pulmonary auscultation, patients with AdVa presented crackles in a higher proportion when compared to patients with RSVi (p = 0.001) and MPVi (p = 0.04). The most frequent chest radiography findings were hyperinflation (increased rib cage size and lowered
Table 1 Clinical features of children with AdV, RSV, hMPV and PIV (n = 94). Clinical features
Virus (n/%) AdVa(n = 29) (a) n/%
AdVi (n = 10) (b) n/%
RSVi (n = 46) (c) n/%
hMPVi (n= 9) (d) n/%
p value
Dyspnea Fever Cough Runny nose Nasal congestion Presence of crackles
29/100 22/76 27/93 17/58 17/58 18/62
10/100 7/70 10/100 5/50 5/50 4/40
45/98 26/56 41/89 28/61 26/56 11/24
9/100 5/55 7/77 4/44 7/78 2/22
Household smokers Administration of O2 Acute bronchiolitis Recurrent wheezing
17/59 22/75.8 12/22.6 17/41.5
2/20 7/70 03/5.7 07/17.1
21/45 29/63 33/62.3 13/31.7
4/44 6/66.7 5/9.4 4/9.7
– NS NS NS NS 0.001a/c 0.042a/d 0.04a/b NS – –
a = Associated to other virus; i = detected with no other virus; NS = no significance. AdV = Adenovirus, RSV = Respiratory Syncytial Virus, hMPV = Meta-pneumovirus, PIV = Para-influenza Virus.
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5. Discussion
Fig. 1. Phylogenetic tree based on ML algorithm of human AdV sequences (N = 21) represented with the year of detection. AdV species C, B and D (outgroup) retrieved from the NCBI databank were included in the analysis to establish a phylogenetic relationship with AdV sequences from the study.
diaphragm), interstitial infiltrate and atelectasis. The frequency of household smokers was greater in the group of children with AdVa in comparison to children with AdVi (p = 0.04). Concerning disease severity and viral co-infection (AdVa), there was no statistical difference in the frequency of children showing disease severity grade II, with co-infection (AdVa, 75.9%) and monoinfection (AdVi, RSVi, MPVi, 65%, p = 0.2). We observed that 31/39 (79.5%) children of 3 month of age or younger (≤3 m) and 33/22 (60%, p = 0.036) of older children (>3 m), showed disease severity grade II (60%, p = 0.036). Concerning only children infected by RSVi, we observed that 19/25 (76%) children of ≤3 m and 10/11 (47.6%, p = 0.046) of >3 m showed disease severity grade II. On the other hand, children infected by AdVi, AdVa or MPVi with disease severity grade II, showed no predominant age group. Concerning clinical diagnosis and age groups, acute bronchiolitis was detected predominantly in children of 6 months of age or younger (≤6 m) and recurrent wheezing, in children older than 6 m (>6 m) (p < 0.0001). Acute bronchiolitis was diagnosed in 53/94 (56.4%) children and recurrent wheezing in 41/94 (43.6%) children. In relation to the virus etiology, AdVa (17/29) and AdVi (7/10) were more frequently detected in children with recurrent wheezing while RSVi was more frequently detected in children with acute bronchiolitis (33/46). The analysis of the association between clinical diagnosis and viral infection, based on patient’s age, showed a high correlation (Pearson 0.870; p < 0.0001) between age and the clinical diagnosis of acute bronchiolitis and recurrent wheezing, due to a multicollinearity effect, but showed no interaction effect in relation to the presence or absence of a particular virus. Of the 39 AdV-positive samples, 21 were successfully sequenced. The phylogenetic analysis indicated that 20 were species C AdV and one was species B AdV (Fig. 1). The clustering of the samples in the phylogenetic tree was not related to the year of AdV detection or any specific clinical feature.
In this study virus detection occurred in nearly 61% (94/155) of the children. Of the 94 children, 29 (31%) had two or more viruses detected in NPA. This high frequency of virus detection was similar to the findings of other authors who used molecular methods to show the presence of viruses in NPA [1,2,8,9]. Regarding the frequency of AdV, our results showed similarity to other studies in 2010 (6.5%) and 2011 (8.1%), but in 2012 and 2013 we observed an increase in the frequency of AdV that reached 35% and 24%, respectively, suggesting a small outbreak also reported by the epidemiological bulletins of 2012 and 2013, published by the Ministry of Health, in Brazil [4]. In our study there was a predominance of specie C AdV (20/21). Infections by species C AdV affects more than 80% of people early in life and is found in sporadic form or in endemic outbreaks, although epidemic outbreaks have also been reported [9,10]. There are controversies on the fact that viral co-detection increases the severity of respiratory disease. Semple et al. [11] and Richard et al. [12] reported an association between disease severity and viral co-detection. Differently Canducci et al. [13] and Brand et al. [14] did not report an association between viral co-infection and disease severity. In our results we observed no children with disease severity grade III, and the number of children with disease severity grade II and co-infection (76%) although higher than the number of children with mono-infection (65.6%, p = 0.2), was not statistically significant. In studies that look for the role of co-infection in disease severity one can we find patients with comorbidities (prematurity, cardiac congenital disease, chronic pulmonary disease, immunodeficiency) included in the study protocol, what may contribute to the controversy seen in the conclusion of different authors, concerning the importance of co-infection in disease severity. Studies that include patients with comorbidities may cause bias in the analysis of data, since patients with comorbidities tend to have bad prognosis. In our protocol the exclusion of patients with comorbidities may have contributed to the absence of patients with disease severity grade III, but facilitated the verification of virus effect in host. RSVi was detected in 33/53 (62.3%) of children with acute bronchiolitis and predominated in the first 3 months of age (25/53). These results are in accordance to the findings of other authors, that reported a predominance of acute bronchiolitis and RSV infection, in the first 3 months of age [1–3,11–14]. Concerning all kinds of viral infection (AdVa, AdVi, RSVi, MPVi) and age groups, we observed that the frequency of children of ≤3 m with disease severity grade II (79.5%) was greater than children of >3 (60%, p = 0.038). If we consider only children with RSVi infection and disease severity grade II, also, the number of children of ≤3 m (76%) was greater than the number of children of >3 m (48%, p = 0.046). These results suggest a greater morbidity of RSV infection in children of ≤3 m, and it is not surprising, since it has been described by other authors [12–14], concerning disease severity in children of ≤3 m with RSV respiratory tract infection. The high correlation between age and the clinical diagnosis of acute bronchiolitis and recurrent wheezing should be related to the definition of these two clinical entities that characterize bronchiolitis as the first episode of wheezing, in children under 18 months of age, while the diagnosis of recurrent wheezing is considered in children with two or more episodes of wheezing, demanding at least few months to occur. The exclusion of any interaction effect in relation to the presence or absence of a particular virus should be the result of virus characteristics like, number of sorotypes, presence of latent infection, for example, or to the absence of immunological protection concerning the host. In our results AdVa and AdVi were detected, more frequently, in children with recurrent wheezing (17/29 and 7/10, respectively). To
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our knowledge, there are no studies demonstrating the role of AdV in recurrent wheezing, as has been suggested in asthma [10]. Also, experiments in guinea pig suggest an increase in lung inflammatory response, due to AdV [15]. In brief, we observed a small outbreak of AdV species C, detected in 2012 and 2013. AdVa and AdVi were detected more frequently in children with recurrent wheezing while RSVi was more frequent in infants with acute bronchiolitis. Concerning disease severity, we observed no statistical significance in the number of patients with co-infection (AdVa) and mono-infection (AdVi, RSVi, and MPVi). As our study used a convenience sample, in a specific Hospital, its results cannot be extrapolated to the general population. Funding This work was supported by the Research Foundation of the State of Rio de Janeiro (FAPERJ). Competing interests The authors declare no conflict of interest concerning the submitted paper. Ethical approval The study was approved by the Ethics Committee of IPPMG and by the National Research Ethics Office of Brazil. Each child had a written informed consent that was obtained from the parents before the enrolment. Children whose parents refused to sign the written informed consent were excluded from the study. Acknowledgment Juliana Pina Metzner, MD for the collection of samples and Dr Marcelo Land, PhD for the statistical revision. References [1] P.G.M. Bezerra, M.C.A. Britto, J.B. Correia, et al., Viral and atypical bacterial detection in acute respiratory infection in children under five years, PLoS One 18 (2011) e18928.
[2] L.F. Costa, J. Yokosawa, O.C. Mantese, et al., Respiratory viruses in children younger than five years old with acute respiratory disease from 2001 to 2004 in Uberlandia, MG, Brazil, Mem. Inst. Oswaldo Cruz 101 (2006) 301–306. [3] S.M. Straliotto, M.M. Siqueira, V. Machado, Respiratory viruses in the pediatric intensive care unit: frequency and clinical aspects, Mem. Inst. Oswaldo Cruz 99 (2004) 883–887. [4] Brasil, Ministério da Saúde, Secretaria de Vigilância e Saúde. Boletim Epidemiológico Influenza monitoramento até semana 52 em 2012 e semana 29 em 2013 (Brazil, Ministry of Health. Influenza, Epidemiological Buletin: Monitoring until Epidemiological Week 52 in 2012 and Week 29 in 2013). BE SUS 44 (14–15) (2013) http://portalsaude.saude.gov.br/index.php/oministerio/principal/leia-mais-o-ministerio/197-secretaria-svs/11955boletins-epidemiologicos-arquivos. [5] A. Allard, B. Albinsson, G.J. Wadell, Rapid typing of human Adenoviruses by a general PCR combined with restriction endonuclease analysis, J. Clin. Microbiol. 39 (2001) 498–505. [6] A. Kaida, H. Kubo, M. Shiomi, Evaluation of real-time RT-PCR compared with conventional RT-PCR for detecting human metapneumovirus RNA from clinical specimens, Jpn. J. Infect. Dis. 61 (2008) 461–464. [7] A. Hu, M. Colella, J.S. Tam, Simultaneous detection, subgrouping, and quantitation of respiratory syncytial virus A and B by real-time PCR, J Clin. Microbiol. 41 (2003) 149–154. [8] J.Y. Hong, H.J. Lee, P.A. Piedra, et al., Lower respiratory tract infections due to Adenovirus in hospitalized Korean children: epidemiology, clinical features, and prognosis, Clin. Infect. Dis. 32 (2001) 1423–1429. [9] F.A. Moura, J.R.L. Mesquita, S.A.R. Portes, Antigenic and genomic characterization of Adenovirus associated to respiratory infections in children living in Northest Brazil, Mem. Inst. Oswaldo Cruz 102 (2007) 937–941. [10] C.T. Garnett, D. Erdman, W. Xu, et al., Prevalence and quantitation of species C Adenovirus DNA in human mucosal lymphocytes, J. Virol. 76 (2002) 10608–10616. [11] M. Semple, A. Cowell, W. Dove, et al., Dual infection of infants by human metapneumovirus and human Respiratory Syncytial Virus is strongly associated with severe bronchiolitis, J. Infect. Dis. 191 (2005) 382–386. [12] N. Richard, F. Komurian-Pradel, E. Javouhey, et al., The impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis, Pediatr. Infect. Dis. J. 27 (2008) 213–217. [13] F. Canducci, M. Debiaggi, M. Sampaolo, et al., Two-year prospective study of single infections and co-infections by Respiratory Syncytial Virus and viruses identified recently in infants with acute respiratory disease, J. Med. Virol. 80 (2008) 716–723. [14] H.K. Brand, R. Groot, J.M.D. Galama, M.L. Brouwer, et al., Infection with multiple viruses is not associated with increased disease severity in children with bronchiolitis, Pediatr. Pulmonol. 47 (2012) 393–400. [15] T.Z. Vitalis, A. Kern, H. Croome, The effect of latent Adenovirus 5 infection on cigarette smoke-induced lung inflammation, Eur. Respir. J. 11 (1998) 664–669.
