Prevalence and clinical features of respiratory syncytial virus in children hospitalized for community-acquired pneumonia in northern Brazil

  • Letícia Martins Lamarão1,

    Affiliated with

    • Francisco Luzio Ramos2,

      Affiliated with

      • Wyller Alencar Mello2,

        Affiliated with

        • Mirleide Cordeiro Santos2,

          Affiliated with

          • Luana Soares Barbagelata2,

            Affiliated with

            • Maria Cleonice Aguiar Justino2,

              Affiliated with

              • Alexandre Ferreira da Silva3,

                Affiliated with

                • AnaJudithPiresGarcia Quaresma2,

                  Affiliated with

                  • Veronilce Borges da Silva2,

                    Affiliated with

                    • Rommel Rodríguez Burbano1Email author and

                      Affiliated with

                      • Alexandre Costa Linhares2

                        Affiliated with

                        BMC Infectious Diseases201212:119

                        DOI: 10.1186/1471-2334-12-119

                        Received: 27 March 2011

                        Accepted: 23 February 2012

                        Published: 16 May 2012

                        Abstract

                        Background

                        Childhood pneumonia and bronchiolitis is a leading cause of illness and death in young children worldwide with Respiratory Syncytial Virus (RSV) as the main viral cause. RSV has been associated with annual respiratory disease outbreaks and bacterial co-infection has also been reported. This study is the first RSV epidemiological study in young children hospitalized with community-acquired pneumonia (CAP) in Belém city, Pará (Northern Brazil).

                        Methods

                        With the objective of determining the prevalence of RSV infection and evaluating the patients’ clinical and epidemiological features, we conducted a prospective study across eight hospitals from November 2006 to October 2007. In this study, 1,050 nasopharyngeal aspirate samples were obtained from hospitalized children up to the age of three years with CAP, and tested for RSV antigen by direct immunofluorescence assay and by Reverse Transcription Polymerase Chain Reaction (RT-PCR) for RSV Group identification.

                        Results

                        RSV infection was detected in 243 (23.1%) children. The mean age of the RSV-positive group was lower than the RSV-negative group (12.1 months vs 15.5 months, p<0.001) whereas gender distribution was similar. The RSV-positive group showed lower means of C-reactive protein (CRP) in comparison to the RSV-negative group (15.3 vs 24.0 mg/dL, p<0.05). Radiological findings showed that 54.2% of RSV-positive group and 50.3% of RSV-negative group had interstitial infiltrate. Bacterial infection was identified predominantly in the RSV-positive group (10% vs 4.5%, p<0.05). Rhinorrhea and nasal obstruction were predominantly observed in the RSV-positive group. A co-circulation of RSV Groups A and B was identified, with a predominance of Group B (209/227). Multivariate analysis revealed that age under 1 year (p<0.015), CRP levels under 48 mg/dL (p<0.001) and bacterial co-infection (p<0.032) were independently associated with the presence of RSV and, in the analyze of symptoms, nasal obstruction were independently associated with RSV-positive group (p<0.001).

                        Conclusion

                        The present study highlights the relevance of RSV infection in hospitalized cases of CAP in our region; our findings warrant the conduct of further investigations which can help design strategies for controlling the disease.

                        Background

                        Globally, RSV is the most common cause of childhood acute lower respiratory infection and is responsible for annual outbreaks worldwide [14]. RSV infection usually results in upper respiratory tract illness characterized by profuse rhinorhea, however 25 – 40% of children experiencing infections in their first year of life may develop severe respiratory disease requiring hospitalization [1, 5, 6]. This may result in long-term respiratory disorders such as abnormal pulmonary function, asthma, recurrent cough, and bronchitis [7, 8]. RSV has two Groups, A and B, which are distinguished largely by antigenic and genetic characteristics. During epidemics, either Group A or B may predominate, or both Groups may circulate concurrently [7, 9]. Evidence for RSV infection has been found in every geographic area studied and the predominant occurrence changes according to the region’s climates. In temperate countries RSV outbreaks coincide with winter and in tropical climates the pattern varying with most literature associating RSV with rainy season [1013].

                        Pneumonia is among the main causes of illness and death in the younger children throughout the world [1416]. There is the need for a better assessment of the epidemiology of viral CAP in developing countries where RSV infections substantially account for epidemics and are associated with a more severe clinical presentation of pneumonia [4]. Some retrospective studies investigated the occurrence of bacterial coinfection in children hospitalized with severe RSV infection and found the incidence of pulmonary bacterial coinfection to vary between 17.5 and 44% [15, 17, 18].

                        To our knowledge, this is the first RSV epidemiological study in children hospitalized with CAP in Belém city, Pará, Northern Brazil, to assess the epidemiological, clinical, and laboratory features of RSV infections among infants and young children. Moreover we sought to characterize the circulating RSV Groups in our region.

                        Methods

                        Study population

                        Patients’ clinical and epidemiological data were obtained during a cross-sectional study comprising eight hospitals carried out between November 2006 and October 2007, in Belém, Pará, located in the Northern tropical area of Brazil. We investigated for RSV infection in hospitalized children with CAP. This condition was as defined by medical practitioners of the project based on (A), (B) and (C). (A) Two clinical findings: cough, history of fever, pleuritic pain, crackles or bronchial breath sounds; (B) Chest radiographic findings consistent with pneumonia (focal airspace consolidation, patchy increased interstitial markings), and (C) patients admitted with less than 48 hours. The last condition was used to exclude patients had been infected with pneumonia after being admitted to the hospital. Patients were included in this study if the following criteria were met: children up to the three years old, hospitalized, diagnosed with CAP and with a signed consent form obtained from parents or legal guardians at enrolment. This study was approved by the Ethics and Research Committee of Evandro Chagas Institute (IEC) in the context of a large prospective study that investigates the etiology of CAP in different countries.

                        Sample collection

                        Nasopharyngeal aspirate samples were collected by vacuum suction through a plastic catheter and refrigerated at 4°C until transported on ice to the Respiratory Virus Laboratory at IEC. At the institute, the samples were processed within two hours for RSV antigen detection using the Direct Immunofluorescence Assay (DFA). RSV-positive samples were subsequently subjected to RT-PCR for the detection of the RSV Group.

                        Demographic data and clinical symptoms were also obtained. A questionnaire was filled by a trained technician including age, onset of symptoms, gender, blood bacterial culture, chest radiography, C-Reactive Protein (CRP) levels, and signs or symptoms of cough, rhinorrhea, fever, nasal obstruction, vomiting and diarrhea were analyzed at the time of hospitalization.

                        Direct immunofluorescence assay (DFA)

                        DFA was carried out using specific monoclonal antibodies for the detection and identification of RSV in direct respiratory specimen cell preparation with Light DiagnosticsTM Respiratory Syncytial Virus DFA kit, cat. 3125 (Chemicon® International, Inc. Temecula, CA), in accordance with the manufacturer’s instructions.

                        Reverse transcriptase-polymerase chain reaction (RT-PCR)

                        RT-PCR was performed to detect RSV RNA sequence and identify RSV Groups in samples that were positive by DFA. Primers were adopted from Canducci et al. [19]. Total RNA was extracted directly from supernatant specimens with the QIAamp® Viral RNA Mini Kit, cat. 52904 (Qiagen) and the amplification and detection were performed with SuperScriptTM One-step RT-PCR and the Platinum Taq® comercial Kit, cat. 10928–034 (Invitrogen Life Technologies, CA, USA). The primers used for group determination were generated against the F regions of the RSV genome. Positive control and water as a negative control were run together in each RT-PCR assay to validate the amplification process and to exclude the presence of contaminants.

                        C-reactive protein (CRP)

                        CRP levels were determined from serum samples of patients and stored up to 4 days at about 4°C, until the completion of qualitative and semi-quantitative survey, by agglutination of latex particles using the Serolatex PCR kit, cat. 56 (Labtest Diagnóstica S.A., Brazil), in accordance with the manufacturer’s instructions.

                        Radiographs

                        The radiological assessment was performed by three independent physicians blinded to the patient’s condition, with reading of the radiographs standardized for all. Radiological typical for CAP included interstitial infiltrate, alveolar infiltrate, and lobar pneumonia.

                        Statistical analyses

                        Patients were divided into two groups according to DFA results and the analysis was performed using Statistical Package for the Social Sciences (SPSS, version 17.0, Inc., Chicago, IL), for Windows. Fisher’s exact test was used adjunctively if the expected values were less than 5. The student’s t test was applied to compare means.

                        Multivariate analysis was performed using logistic regression models. Variables with a p value <0.1 in univariate analysis were entered in the multivariate analysis. The level of significance was set at <0.05. The both analyses (1 and 2) including pneumonia positive and negative for RSV as the dependent variable. (1) was a comparison of pneumonia by RSV positive and negative group included the following independents variables: age, gender, CRP levels and bacterial culture; (2) was a comparison of pneumonia positive and negative for RSV and the signs and symptoms reported at hospitalization included the following variables: cough, rhinorrhea, fever, nasal obstruction, vomiting and diarrhea.

                        Results

                        The inclusion criteria were met by 1,214 patients with CAP during the period of study, of whom 1,050 (86.49%) had consent from parents or legal guardians to participate. The differences in distribution of demographic characteristics and admission diagnoses were not statistically significant for those who declined relative to those who participate in the study (data not shown). RSV DFA results were available for all 1,050 patients included in the study. The prevalence of RSV infection was 23.1% (243/1,050 patients). Demographic and clinical features of RSV- positive and RSV-negative children are shown in Table 1. In terms of age, the average of RSV-positive group (12.1 months) was lower than that of the RSV-negative group (15.5 months) (p<0.001). There was no statistically significant difference between the groups in relation to gender (51.8% male and 48.2% female). Among the patients who had CRP levels analyzed (810/1.050, 77.1%), the RSV-positive group showed a lower mean level in comparison to the RSV-negative group (p<0.001). The chest radiological findings have shown that 54.2% of RSV-positive and 50.3% of RSV-negative patients developed interstitial infiltrate. Bacterial culture were available for 46.7% of study participants (490/1.050). Although the 90.0% of RSV-positive patients yielded negative bacteriological culture, bacterial co-infection was identified in this group with 10.0% of culture growth, while the RSV-negative group showed only 4.5% growth (p<0.05) (Table 1).
                        Table 1

                        Epidemiologic, clinical, and laboratory characteristics of RSV-positive and RSV-negative children hospitalized for community-acquired pneumonia in Belém, Para, Brazil

                        Characteristics

                        RSV- positive 243 (23.1%)

                        RSV-negative 807 (76.9%)

                         

                        Groups (n)

                         
                         

                        n

                        %

                        n

                        %

                         p 

                        A

                        B

                         p 

                        Age (years)

                        0 - 1

                        152/241

                        63.1

                        340/805

                        42.1

                        <0.001

                        10/18

                        135/207

                        0.282

                        Gender

                        Male

                        126/243

                        51.8

                        449/807

                        55.6

                        0.113

                        9/18

                        108/207

                        0.542

                        C-Reactive protein

                        ≤48 mg/dL

                        155/178 87.0

                        469/632 74.2

                        <0.001

                        9/13

                        133/151

                        <0.001

                        Radiographs

                        Interstitial infiltrate

                        117

                        54.2

                        366

                        50.3

                        0.085

                        9

                        102

                        0.235

                        Alveolar infiltrate

                        49

                        22.7

                        164

                        22.5

                         

                        1

                        45

                         

                        Lobar pneumonia

                        50

                        23.1

                        198

                        27.2

                         

                        5

                        39

                         

                        Bacterial culture

                        Positive

                        11 Δ /109

                        10.0

                        17*/381

                        04.5

                        0.043

                        1/8

                        10/92

                        0.342

                        Δ S. epidermidis (3 patients), A. baumannii (2), S. pneumoniae (1), Com. acidovorans (1), K. pneumoniae (1), Gram Positive (1), Candida spp (1), other fungi (1).

                        * S. pneumoniae (4 patients), S. epidermidis (3), Tetrads (3), Gram Positive (3), K. pneumoniae (2), S. viridians (1), A. baumannii (1).

                        It was possible to determine the RSV Group in 227 (93.4%) out of 243 RSV-positive samples. RSV Group B infections predominated RSV Group A (209 vs 18 patients, respectively). Group B infection was associated with a lower age than Group A (11.0 vs 13.0 months; p<0.03). With regards to the CRP levels, Group B infection showed a lower CRP mean when compared to Group A (11.0 vs 19.0 mg/dL, p<0.05). Gender, radiological pattern, bacterial culture and the onset of symptoms denoted a similar distribution in both Groups.

                        We described the signs and symptoms in Table 2 for RSV-positive and RSV-negative groups. Approximately 98% of RSV-positive children had a cough at admission but no statistically significant difference was observed when compared to the RSV-negative group (96.1%; p>0.05). Five clinical parameters showed significantly different rates (p<0.05) when comparing both groups: fever, vomiting and diarrhea were detected predominantly in the RSV-negative group (80.2% vs 72.4%, 12.2% vs 4.9% and 8.1% vs 4.1%, respectively), while rhinorrhea and nasal obstruction were predominantly observed in RSV-positive group (78.2% vs 71.5% and 59.2% vs 32.8%, respectively, both comparisons yielding a p<0.05). Patients infected with either A or B Groups did not significantly differ in terms of signs and symptoms.
                        Table 2

                        Signs and symptoms reported at hospitalization in RSV-positive and RSV-negative patients with community-acquired pneumonia and detected RSV Groups

                         

                        RSV-positive 243 (23.1%)

                        RSV-negative 807 (76.9%)

                         

                        Groups

                         
                         

                        n

                        %

                        n

                        %

                         p 

                        A

                        B

                         p 

                        Cough

                        238

                        97.9

                        714

                        96.1

                        0.056

                        100%

                        98.5%

                        0.779

                        Rhinorrhea

                        190

                        78.2

                        531

                        71.5

                        0.002

                        83.3%

                        77.8%

                        0.443

                        Fever

                        176

                        72.4

                        596

                        80.2

                        0.009

                        83.3%

                        71.8%

                        0.323

                        Nasal obstruction

                        144

                        59.2

                        244

                        32.8

                        <0.001

                        66.6%

                        58.7%

                        0.359

                        Vomiting

                        12

                        04.9

                        91

                        12.2

                        <0.001

                        0

                        4.8%

                        0.428

                        Diarrhea

                        10

                        04.1

                        60

                        08.1

                        0.023

                        0

                        14.8%

                        0.428

                        Figure 1 shows that RSV infection was detected from January to July 2007, with higher prevalence rates observed from April (48.6%) to June 2007 (52.4%).
                        http://static-content.springer.com/image/art%3A10.1186%2F1471-2334-12-119/MediaObjects/12879_2011_1958_Fig1_HTML.jpg
                        Figure 1

                        Monthly prevalence of Respiratory Syncytial Virus (RSV) in children up to three years old, hospitalized with community-acquired pneumonia (CAP), from November 2006 to October 2007.

                        The Multivariate Analysis of Risk Factors for RSV CAP showed in analyze (1) the age under 1 year (OR 1.36; 95% CI; 1.06–1.74; p<0.015), CRP levels under 48 mg/dL (OR 3.49; 95% CI; 1.80–6.77; p<0.001) and bacterial co-infection (OR 2.57; 95% CI; 1.08–6.09; p<0.032) were independently associated with the presence of RSV as opposed to RSV-negative group. In analyze (2) only nasal obstruction was independently associated with presence of RSV (OR 3.07; 95% CI; 2.24–4.21; p<0.001) (Table 3).
                        Table 3

                        The Multivariate Analysis of Risk Factors for RSV infection in patients hospitalized with community-acquired pneumonia

                        Analyzes

                         p 

                        OR

                        95% CI

                            

                        Lower

                        Upper

                        Signs and symptoms

                            
                         

                        Fever

                        0.109

                        0.754

                        0.535

                        1.065

                         

                        Rhinorrhea

                        0.798

                        0.952

                        0.654

                        1.386

                        (1)

                        Nasal obstruction

                        <0.001

                        3.075

                        2.244

                        4.215

                         

                        Cough

                        0.107

                        2.428

                        0.825

                        7.146

                         

                        Diarrhea

                        0.646

                        0.836

                        0.390

                        1.794

                         

                        Vomiting

                        0.078

                        0.551

                        0.283

                        1.069

                        Characteristics

                            
                         

                        CRP <48 mg/dL

                        <0.001

                        3.498

                        1.807

                        6.772

                        (2)

                        Bacterial co-infection

                        0.032

                        2.570

                        1.081

                        6.094

                         

                        Age <1 year

                        0.015

                        1.361

                        1.062

                        1.745

                         

                        Male gender

                        0.476

                        1.186

                        0.742

                        1.895

                        (1) (2) pneumonia positive and negative for RSV as the dependent variable.

                        Discussion

                        The detection of RSV in 23.1% of the samples in our study denoted a prevalence rate similar to those observed in other studies on the occurrence of lower respiratory tract infection, ranging from 23% to 61% [6, 20, 21]. The possible limitation of the study was the use of a less sensitive method (IF) as compared to RT-PCR that has shown improved sensitivity in the detection of RSV infection [14, 22]. In fact there is differential detection related to viral load. Infants usually have severe RSV disease associated with higher viral load hence a better chance of detection compared to the older counterparts leading to a differential misclassification (BIAS) [23]. However, the large number of samples analyzed in this study minimizes the bias and did not alter the statistical significance of our results.

                        RSV has been referred in the literature as the main agent responsible for bronchiolitis and pneumonia during the first year of life. According to our study, 62.5% of children were younger than one year old, suggesting that illnesses caused by RSV can be severe in this age group, thus requiring hospitalization with prompt and effective medical intervention [5, 24].

                        Gender was not identified as a significant risk factor for RSV infection in our study in agreement with other published studies [21, 25]. Nevertheless, some findings in the literature have shown a male predominance, as reported by D’Elia et al. [5], particularly between 0 and 2 months.

                        Evaluation of CRP is very useful for clinicians because it may help differentiate between bacterial and viral etiologies. Shin et al. [26] used levels of CRP (≥1.87 mg/dL) as criteria to rule out serious bacterial infection in infants from self-limiting viral illness in febrile infants younger than three months. Diniz et al. [25] also found a statistically significant difference between nosocomial viral lower respiratory tract infection and levels of CRP less than or equal to 40 mg/L. In our study, the RSV-positive group showed a lower CRP mean level when compared to the RSV-negative group. Thus, low CRP levels may suggest a viral infection and, depending upon the clinical and radiological findings, enables the suspension of antibiotic therapy and helps considerably in the reduction of the hospital stay.

                        The radiological findings in our study have shown that 54.2% of RSV-positive patients developed interstitial infiltrate. Diniz et al. [25] found a significant correlation between nosocomial viral lower respiratory tract infection and interstitial infiltrate and it was observed that all patients with confirmed bacterial, fungal, or mixed infection presented alveolar infiltrate.

                        We found a predominance of RSV infection without bacterial co-infection in our study, in agreement with Duttweiler et al. [27] who found that concomitant bacterial sepsis was a rare event in 127 hospitalized RSV infected infants. However, when we compared the groups, positive culture was predominantly observed in the RSV-positive group than the RSV-negative group (10.0% vs 4.5%, p<0.028). Thorburn et al. [15] reported on pulmonary bacterial co-infection in children with severe RSV bronchiolitis showing that 40% of children with severe RSV infection were infected with bacteria in their lower airways. Unfortunately, we were unable to demonstrate if these infections would be either secondary or concurrent to viral infection.

                        It was possible to determine the RSV Group in 227 (93.4%) samples, and 6.6% were untypable probably due to problems in processing samples or RNA extraction. This study reports predominance of RSV Group B infection in children hospitalized with CAP, which is not unusual in other regions [9, 28]. Unfortunately, our data presents some limitation since we could collect samples for only one year and annual RSV Group epidemic may change. Suwanjutha et al. [28] identified in the first year of study the predominance of Group B, in contrast to the second year when Group A was more predominant.

                        Due to the high number of RSV Group B infection, comparison with.Group A (only 18 children) epidemiological data is not conclusive. However, in our findings, Group B was found among children with a mean age lower than that for Group A (11.0 vs 13.0; p<0.03), a finding similar to that of Papadopoulos et al. [29] who reported a predominance of RSV B infection in the youngest children, but the reasons are still unknown. Mlinaric-Galinovic et al. [9] and Oliveira et al. [8] did not find age differences between Groups, even though Mlinaric-Galinovic et al. [9] found that Group B infections occurred more frequently in males less than 12 months of age than in females. Oliveira et al. [8] on the other hand, found that the 57.8% of RSV A-infected children were male. Unfortunately, there is no plausible explanation for this variation.

                        We were able to determine that the CRP mean level in Group B was lower than Group A (13.0 vs 19.0; p<0.05) but the reasons for this remain unknown. Further research with an adequate number of samples needs to be conducted in an attempt to better understand RSV Groups in cases of CAP and their association with the epidemiological data of patients.

                        We have evaluated the signs and symptoms and approximately 98% of RSV-positive cases had cough at admission, but no statistically significant difference was observed in comparison with RSV-negative group (96.1%) (p>0.05). Rhinorrhea and nasal obstruction were predominantly observed in the RSV-positive group (p<0.05). Regarding the clinical signs and symptoms observed by Diniz et al. [25] in São Paulo city, in the preterm infants infected with RSV, wheezing, rhinorrhea, vomiting, and diarrhea were significantly more frequent while in our study vomiting and diarrhea were detected predominantly in RSV-negative group. In addition, the clinical symptoms do not predict the viral etiology because there are difficulties in establishing the general etiologic diagnoses of pneumonia by clinical profiles, which are quite varied in literature and depend on the infectious agent as well as the age and immune state of the host.

                        With regards to the seasonality, our study showed that RSV activity in hospitalized children during January to July 2007, with a peak during April to June, coincided with a heavy rainfall period in the region. Outbreaks occurring mainly during the months with low temperatures were reported in Uberlandia (Midwestern Brazil) by Costa et al. [30]. This seasonal pattern was also observed in other Brazilian settings where RSV occurrence is not uniform. This has been noted in several Brazilian geographic regions and even among different states in the same region [10]. In Campinas (São Paulo) [21] and in Vitória (Espírito Santo) [31], South-eastern region, the annual highest incidence of RSV-infections occurred between January and June, the same period of months observed in our study.

                        A greater understanding of the factors that determine RSV activity would make this timing even more precise. However, such studies are important as they are expected to delineate the clinical and epidemiological behavior of RSV in this age range and in this region. Even though our study presents a low prevalence of bacterial co-infection among the RSV-positive patients, monitoring RSV activity is necessary in order to restrict antibiotic use to the infants in real need and to provide better prophylactic therapies.

                        Conclusion

                        In conclusion, to our knowledge this is the first report of RSV infection in hospitalized children with CAP in our region. We highlighted that the RSV infection prevalence associated with CAP in Northern Brazil is similar to those found in other regions and other countries. However, according to current findings and those reported in the literature, it may be concluded that the lower mean of age as well as the lower levels of CRP are characteristics found in viral infections of these cases in Belém city. The signs and symptoms associated with RSV in our study were rhinorrhea and nasal obstruction, even though divergent in the literature, and the predominant RSV Group identified was B. Furthermore, the data provided by the study indicate that there is a need for continuous efforts in order to broaden our knowledge on the epidemiological aspects of RSV infection and CAP.

                        Abbreviations

                        RSV: 

                        Respiratory syncytial virus

                        CAP: 

                        community-acquired pneumonia

                        RT-PCR: 

                        Reverse Transcription Polymerase Chain Reaction

                        IEC: 

                        Evandro Chagas Institute

                        DFA: 

                        Direct Immunofluorescence Assay

                        CRP: 

                        C-Reactive Protein

                        CI: 

                        Confidence interval.

                        Declarations

                        Acknowledgements

                        This study has been supported by Instituto Evandro Chagas, Secretaria de Vigilância em Saúde, Ministério da Saúde and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

                        Authors’ Affiliations

                        (1)
                        Instituto de Ciências Biológicas, Universidade Federal do Pará
                        (2)
                        Instituto Evandro Chagas, Secretaria de Vigilância em Saúde, Ministério da Saúde
                        (3)
                        Hospital Universitário João de Barros Barreto, Universidade Federal do Pará

                        References

                        1. Nair H, Nokes DJ, Gessner BD, Dherani M, Madhi SA, Singleton RJ, O’Brien KL, Roca A, Wright PF, Bruce N, et al.: Global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis. Lancet 2010, 375:1545–1555.PubMedView Article
                        2. Buckley BC, Roylance D, Mitchell MP, Patel SM, Cannon HE, Dunn JD: Description of the outcomes of prior authorization of palivizumab for prevention of respiratory syncytial virus infection in a managed care organization. J Manag Care Pharm 2010, 16:15–22.PubMed
                        3. Figueiredo LTM: Viral pneumonia: epidemiological, clinical, pathophysiological and therapeutic aspects. J Bras Pneumol 2009, 35:899–906.PubMedView Article
                        4. Mathisen M, Strand TA, Sharma BN, Chandyo RK, Valentiner-Branth P, Basnet S, Adhikari RK, Hvidsten D, Shrestha PS, Sommerfelt H: Clinical presentation and severity of viral community-acquired pneumonia in young Nepalese children. Pediatr Infect Dis J 2010, 29:e1-e6.PubMedView Article
                        5. D´Elia C, Siqueira MM, Portes SA, Sant’Anna CC: Respiratory syncytial vírus – associated lower respiratory tract infections in hospitalized infants. Rev Soc Bras Med Trop 2005, 38:7–10.View Article
                        6. Pecchini R, Berezin EN, Felicio MC, Passos SD, de Souza MC, de Lima LR, Ueda M, Matsumoto TK, Durigon EL: Incidence and clinical characteristics of the infection by the respiratory syncytial virus in children admitted in Santa Casa de São Paulo Hospital. Braz J Infect Dis 2008, 12:476–479.PubMedView Article
                        7. Mohapatra SS, Boyapalle S: Epidemiologic, experimental, and clinical links between respiratory syncytial virus infection and asthma. Clin Microbiol Rev 2008, 21:495–504.PubMedView Article
                        8. Oliveira TFM, Freitas GRO, Ribeiro LZG, Yokosawa J, Siqueira MM, Portes SAR, Silveira HL, Calegari T, Costa LF, Mantese OC, et al.: Prevalence and clinical aspects of respiratory syncytial virus A and B groups in children seen at Hospital de Clínicas of Uberlândia, MG, Brazil. Mem Inst Oswaldo Cruz 2008, 103:417–422.PubMedView Article
                        9. Mlinaric-Galinovic G, Vojnovic G, Cepin-Bogovic J, Bace A, Bozikov J, Welliver RC, Wahn U, Cebalo L: Does the viral subtype influence the biennial cycle of respiratory syncytial virus? Virol J 2009, 6:133.PubMedView Article
                        10. Moura FEA, Nunes IFS, Silva GB, Siqueira MM: Respiratory syncytial virus infection in Northeastern Brazil: seasonal trends and general aspects. Am J Trop Med Hyg 2006, 74:165–167.PubMed
                        11. Goddard NL, Cooke MC, Gupta RK, Nguyen-Van-Tam JS: Timing of monoclonal antibody for seasonal RSV prophylaxis in the United Kingdom. Epidemiol Infect 2007, 135:159–162.PubMedView Article
                        12. Robertson SE, Roca A, Alonso P, Simoes EA, Kartasasmita CB, Olaleye DO, Odaibo GN, Collinson M, Venter M, Zhu Y, Wright PF: Respiratory syncytial virus infection: denominator-based studies in Indonesia, Mozambique, Nigeria and South Africa. Bull World Health Organ 2004, 82:914–922.PubMed
                        13. Hall CB, Weinberg GA, Iwane MK, Blumkin AK, Edwards KM, Staat MA, Auinger P, Griffin MR, Poehling KA, Erdman D, Grijalva CG, Zhu Y, Szilagyi P: The burden of respiratory syncytial virus infection in young children. N Engl J Med 2009, 360:588–598.PubMedView Article
                        14. Marcos MA, Esperatti M, Torres A: Viral pneumonia. Curr Opin Infect Dis 2009, 22:143–147.PubMedView Article
                        15. Thorburn K, Harigopal S, Reddy V, Taylor N, van Saene HK: High incidence of pulmonary bacterial co-infection in children with severe respiratory syncytial virus (RSV) bronchiolitis. Thorax 2006, 61:611–615.PubMedView Article
                        16. Zhang HY, Li ZM, Zhang GL, Diao TT, Cao CX, Sun HQ: Respiratory viruses in hospitalized children with acute lower respiratory tract infection in Harbin, China. Jpn J Infect Dis 2009, 62:458–460.PubMed
                        17. Kneyber MC, Blusse´van Oud-Alblas H, van Vliet M, Uiterwaal CS, Kimpen JL, van Vught AJ: Concurrent bacterial infection and prolonged mechanical ventilation in infants with respiratory syncytial virus lower respiratory tract infection. Intensive Care Med 2005, 31:680–685.PubMedView Article
                        18. Randolph AG, Reder L, Englund JA: Risk of bacterial infection in previously healthy respiratory syncytial virus-infected young children admitted to the intensive care unit. Pediatr Infect Dis J 2004, 23:990–994.PubMedView Article
                        19. Canducci F, Debiaggi M, Sampaolo M, Marinozzi MC, Berrè S, Terulla C, Gargantini G, Cambieri P, Romero E, Clementi M: 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 2008, 80:716–723.PubMedView Article
                        20. Calegari T, Queiroz DA, Yokosawa J, Silveira HL, Costa LF, Oliveira TF, Luiz LN, Oliveira RC, Diniz FC, Rossi LM, et al.: Clinical-epidemiological evaluation of respiratory syncytial virus infection in children attended in a public hospital in midwestern Brazil. Braz J Infect Dis 2005, 9:156–161.PubMedView Article
                        21. Riccetto AGL, Ribeiro JD, da Silva MTN, Almeida RS, Arns CW, Baracat ECE: Respiratory Syncytial Virus (RSV) in infants hospitalized for acute lower respiratory tract disease: incidence and associated risks. Braz J Infect Dis 2006, 10:357–361.PubMedView Article
                        22. Reis AD, Fink MCD, Machado CM, Paz JP, Oliveira RR, Tateno AF, Machado AF, Cardoso MR, Pannut CS: Comparison of direct immunofluorescence, conventional cell culture and polymerase chain reaction techniques for detecting respiratory syncytial virus. Rev Inst Med Trop S Paulo 2008, 50:37–40.PubMed
                        23. Munywoki PK, Hamid F, Mutunga M, Welch S, Cane P, Nokes DJ: Improved detection of respiratory viruses in pediatric outpatients with acute respiratory illness by real-time PCR using nasopharyngeal flocked swabs. J Clin Microbiol 2011, 49:3365–3367.PubMedView Article
                        24. Lee JT, Chang LY, Wang LC, Kao CL, Shao PL, Lu CY, Lee PI, Chen JM, Lee CY, Huang LM: Epidemiology of respiratory syncytial virus infection in northern Taiwan, 2001–2005 – seasonality, clinical characteristics, and disease burden. J Microbiol Immunol Infect 2007, 40:293–301.PubMed
                        25. Diniz EMA, Vieira RA, Ceccon MEJ, Ishida MA, Vaz FAC: Incidence of respiratory viruses in preterm infants submitted to mechanical ventilation. Rev Inst Med Trop 2005, 47:37–44.View Article
                        26. Shin SH, Choi CW, Lee J-A, Kim E-K, Choi EH, Kim H-S, Kim B, Choi J-H: Risk factors for serious bacterial infection in febrile young infants in a community referral hospital. J Korean Med Sci 2009, 24:844–848.PubMedView Article
                        27. Duttweiler L, Nadal D, Frey B: Pulmonary and systemic bacterial co-infections in severe RSV bronchiolitis. Arch Dis Child 2004, 89:1155–1157.PubMedView Article
                        28. Suwanjutha S, Sunakorn P, Chantarojanasiri T, Siritantikorn S, Nawanoparatkul S, Rattanadilok Na Bhuket T, Teeyapaiboonsilpa P, Preutthipan A, Sareebutr W, Puthavathana P: Respiratory syncytial virus-associated lower respiratory tract infection in under-5-year-old children in a rural community of central Thailand, a population-based study. J Med Assoc Thai 2002,85(suppl 4):S1111-S1119.PubMed
                        29. Papadopoulos NG, Gourgiotis D, Javadyan A, Bossios A, Kallergi K, Psarras S, Tsolia MN, Kafetzis D: Does respiratory syncytial virus subtype influences the severity of acute bronchiolitis in hospitalized infants? Respir Med 2004, 98:879–882.PubMedView Article
                        30. Costa LF, Yokosawa J, Mantese OC, Oliveira TFM, Silveira HL, Nepomuceno LL, Moreira LS, Dyonisio G, Rossi LMG, Oliveira RC, et al.: Respiratory viruses in children younger than five years old with acute respiratory disease from 2001 to 2004 in Uberlândia, MG, Brazil. Mem Inst Oswaldo Cruz 2006, 101:301–306.PubMedView Article
                        31. Checon RE, Siqueira MM, Lugon AK, Portes S, Dietze R: Short report: seasonal pattern of respiratory syncytial virus in a region with a tropical climate in southeastern Brazil. Am J Trop Med Hyg 2002, 67:490–491.PubMed
                        32. Pre-publication history

                          1. The pre-publication history for this paper can be accessed here:http://​www.​biomedcentral.​com/​1471-2334/​12/​119/​prepub

                        Copyright

                        © Lamarao et al.; licensee BioMed Central Ltd. 2012

                        This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.