Infants delivered prematurely are at an increased risk of severe respiratory disease,1–3 2 4 5–9 2 7 , 10 RSV ) infection, including a longer duration of hospitalization, an increased rate of admission to the intensive care unit (ICU) and a greater requirement for mechanical ventilation compared with term infants.11 RSV -associated infections are higher in children <24 months of age (including those <6 months of age) who were born <36 wGA compared with children at lower risk for infection (children born full term or without bronchopulmonary dysplasia or congenital heart disease).12
Prospective studies of the role of viral pathogens on the burden of severe respiratory disease in infants are scarce, particularly in developing countries. In addition, it is still controversial whether coinfection by multiple respiratory viruses may increase the risk of developing severe lower respiratory tract infection (LRTI) compared with infection by a single virus.13–18 RSV infection on prematurely born infants.
Recent studies in different geographic locations have shown the importance of the role of mixed viral infections on respiratory disease severity.13 , 15 , 19 RSV coinfections with disease severity. A number of epidemiologic assessments on the burden of acute viral respiratory disease in preterm infants have been performed,20–24 13 , 14 , 25–28
MATERIALS AND METHODS
Study Design
This was a prospective epidemiologic study conducted between January 2008 and December 2010 to determine the incidence of severe LRTI among children born ≤35 wGA at 3 sites (public hospitals) in southern Brazil (Curitiba, Porto Alegre and Ribeirão Preto). Following birth, preterm children were identified by the study nurses and enrolled throughout the hospitalization period up to 72 hours after hospital discharge and were followed for 1 year after enrollment. Subjects were seen monthly for the first 6 months and bimonthly until 1 year of follow up. Parents were instructed to contact the study nurses whenever children presented a series of defined respiratory symptoms. Whenever the presence of cough or fever or any signs of respiratory distress were noted, children were seen by a study pediatrician. LRTIs were diagnosed based on symptoms noted during physical examination and included respiratory rate, retractions, wheezing and/or crackles on auscultation, and, whenever present, radiologic signs were compatible with viral bronchiolitis or pneumonia. Severe LRTI was defined as an event that required hospitalization because of these signs and symptoms. Many of these admissions were associated with oxygen saturation levels <93%.
Nasal washes from subjects with an LRTI were collected by trained personnel (study nurses) and transported to the laboratory at 4°C. Aliquots were then prepared and stored at −70°C. Samples were then tested for respiratory viruses using polymerase chain reaction (PCR) using TaqMan (Life Technologies, Grand Island, NY) assays (see Table, Supplemental Digital Content 1, https://links.lww.com/INF/B871 48-58 There was only 1 specimen collected per event per subject. Subjects with LRTI not requiring hospitalization were followed weekly by telephone until parents informed study nurses that their children were healthy again. Hospitalized children with LRTI were followed daily during their hospitalization stay and weekly by telephone after hospital discharge until illness resolution. Children had blood drawn for determination of serum levels of RSV -specific IgG titers at enrollment. RSV antibody titers were assessed using enzyme-linked immunosorbent assay, according to standard methodology, which was performed at the central laboratory in Ribeirão Preto.
The study protocol was approved by the institutional review boards/independent ethics committees for each site; written informed consent was obtained from legal representatives of each subject.
Participants
Infants born ≤35 wGA and who were <6 months of age at screening were included. Subjects were born in or transferred to 1 of the participating hospitals and were eligible to receive follow-up medical care at the participating site. Legal representatives of included subjects had to be able to provide information during the follow-up period. Subjects were excluded if they had experienced RSV infection or if they had received palivizumab or other RSV -specific immunoglobulin products before the start of the study.
Assessments
The primary outcome was occurrence of severe LRTI (an LRTI requiring hospitalization) associated with RSV infection within 1-year of follow up. Secondary outcomes were occurrence of LRTIs associated with detection of other viruses or with personal (clinical history associated with prematurity, including ICU stay and oxygen use) or familial risk factors (including socioeconomic status and history of asthma) for severe LRTI in preterm infants. Additionally, the study enabled the description of the seasonality of RSV infection in the South and Southeast regions of Brazil.
Real-time PCR for Respiratory Viruses
Nasopharyngeal washes were tested using real-time PCR for the presence of RSV A and B; human rhinovirus (HRV); human bocavirus (HBoV); human metapneumovirus (HMPV) A and B; human influenza virus A and B, including A(H1N1)pdm09; human parainfluenza virus (HPIV) 1 and 3; human adenovirus (HAdV) and human coronavirus (HCoV) OC43 and 229E. Total RNA was extracted from 250 μL nasopharyngeal washes using TRIzol (Life Technologies), and DNA was extracted from 200 μL nasopharyngeal washes with Wizard Genomic DNA purification kit (Promega, Madison, WI), both following the manufacturer’s protocols. Real-time PCR was done using the TaqMan strategy with specific primers and probes (data available on request) in a Thermocycler 7300 (Life Technologies) and with the internal control (the β-actin housekeeping gene) tested in all samples. Mixtures of 2 sets of primers and probes were used for RSV A and B, HCoV OC43 and 229E and HAdV and HBoV. A single pair of primers was used with 2 different probes to cover 2 non–cross-reactive subsets of HRV types. Reverse transcription, assembly of real-time PCR mixes, and cycling parameters were per a previously published protocol,29
RSV Antibody TestSerum IgG antibodies to RSV were assayed using the RSV Virotech kit (Sekisui Virotech GmbH, Rüsselsheim, Germany) following the manufacturer’s instructions.
Statistical Analysis
For numerical variables, such as demographics, hospital admissions and viral pathogens identified, the mean, standard deviation (SD), median, minimum and maximum values are provided. For nominal variables, the number and percentage were calculated for non-missing data. The Kaplan-Meier method was used to estimate the median time to first LRTI event. Univariate analyses were performed for comparing groups (severe vs. nonsevere LRTI and severe vs. no LRTI) according to predefined factors, using the χ2 test or the Student t test.
RESULTS
A total of 310 infants were originally enrolled in the study (Curitiba, 110; Porto Alegre, 116; Ribeirão Preto, 77); 7 were excluded because of protocol violations. No significant differences for the main outcome variables were noted among the 3 study sites. Of 303 subjects initially enrolled, 57 (18.8%) had their participation discontinued; 246 subjects completed the study. The most frequent reason for discontinuation was loss to follow up.
Subject Demographics
The mean ± SD weights at birth and enrollment were 1.6 ± 0.5 kg and 2.1 ± 0.3 kg, respectively (see Table, Supplemental Digital Content 1, https://links.lww.com/INF/B871
During follow up, 3 subjects died, 2 from causes unrelated to RSV . The third death occurred outside a hospital participating in the study. A review of the case indicated that death was because of pneumonia and sepsis and was associated with RSV infection, with RSV antigen detected using immunofluorescence at admission to the hospital at which the death occurred.
Most children were enrolled while still in the birth hospital; 88.8% had been previously admitted to the neonatal ICU, 25.4% had required mechanical ventilation and 65.7% had been on supplemental oxygen for a mean of 14.6 days. Approximately two-thirds of the infants had at least 1 parent or sibling (62%) with a confirmed history of asthma and nearly half (46.2%) had a smoker living in the same household.
Subjects With Severe and Nonsevere LRTIs
Of the 303 subjects included in the study, 176 (58.1%) experienced an LRTI at some point during the follow-up period (Table 1 ), 36.4% (64/176) of which were severe (requiring hospitalization). Therefore, the overall hospitalization rate was 21.1% (64/303). A total of 37.6% of subjects (114/303) had >1 LRTI during follow up, with a mean (SD) of 2.6 ± 2.2 LRTIs per subject. The mean time to the first LRTI was 6 ± 3.2 months. Among subjects with an LRTI, 162 (of 176) had samples tested using PCR for the identification of infecting viruses, and 27.8% of these subjects (45/162) experienced a severe LRTI. RSV was the most prevalent virus associated with severe LRTI (30/45 subjects, 66.7%) alone or as a coinfection with other viruses. Thus, the prevalence of severe RSV -associated LRTIs in the whole population was 9.9% (30/303). Almost half of these RSV infections had concomitant positive PCRs for other viruses. No statistically significant difference in time to first severe LRTI was observed among subjects with RSV -associated episodes (mean ± SD: 4.3 ± 2.8 months) compared with those not associated with RSV (mean ± SD: 5.5 ± 4.0 months).
TABLE 1: Infection Characteristics According to Number of Subjects (N = 303)
Subjects who developed severe LRTI during the study period were noted to have lower birth weight, lower level of maternal education, longer initial hospitalizations following birth and lower IgG levels when compared with patients who did not develop severe LRTI during the study period (see Table, Supplemental Digital Content 2, https://links.lww.com/INF/B872
Viral Pathogens Associated With LRTI Events
There were 461 detected LRTI events among the 303 subjects in the analysis population; samples from 93.7% (432/461) of these events were tested for viral identification using PCR (Table 2 ). Among these collected samples, 21.1% (91/432) were negative for all tested viruses. Of the 341 samples from LRTI events that tested positive for any virus, 48 were from severe events (events that required hospitalization). Coinfections were identified in approximately half of all samples that tested positive for any virus (161/341; 47.2%) and in half of all samples from severe LRTIs that tested positive for any virus (23/48; 47.9%).
TABLE 2: Infection Characteristics According to LRTI Event (N = 461)
The viruses that were identified using PCR are presented in Figure 1 and Table 3 (along with a complete listing of all viruses identified in single and coinfections, data available on request). RSV was the most prevalent viral agent in the overall analysis (143/432; 33.1%) and among those with positive tests (143/341; 41.9%). Interestingly, 57.3% (82/143) of the RSV infections were coinfections, approximately half of which were with HRV (39/82; 47.6%). Of any coinfection involving RSV + HRV (39/432; 9.0%), nearly half also included infection with yet another virus (19/39; 48.7%). Among 18 LRTI events in which 3 or more viruses were identified, the combination of RSV + HRV was identified in 8. The frequency of HRV detections was similar to that of RSV for all LRTIs (127/432; 29.4%), whereas HBoV, HMPV (subtypes A and B) and influenza appeared in substantial, albeit lower, numbers (Fig. 1 and Table 3 ).
TABLE 3: Viral Pathogens Associated With LRTI Events Identified Using PCR
FIGURE 1: Viral pathogens identified using PCR in LRTI events. A) Percentage of positive PCR results from nasal washes from subjects (all LRTI events; n = 432). B) Percentage of positive PCR results from nasal washes from subjects (severe LRTI events, n = 56; nonsevere LRTI events, n = 301). FLU, human influenza virus (subtypes A and B); H1N1, influenza virus; HCoV, human coronavirus (OC43 and 229E); HMPV, human metapneumovirus (subtypes A and B); HPIV, human parainfluenza virus (subtypes 1 and 3); RSV , respiratory syncytial virus (includes subtypes A and B).
Severe LRTI events were largely associated with RSV (34/56; 60.7%; Fig. 1B and Table 3 ), with half of these events being single virus detections (17/34, 50.0%) and half being coinfections. HRV was the most common coinfecting virus with RSV in severe LRTI events (10/17; 58.8%). Significantly longer hospital stays (mean ± SD) were associated with LRTI events in which RSV and another coinfecting virus were identified in the same sample collection (14.3 ± 7.7 days) compared with LRTI events in which RSV was the sole virus identified (7.5 ± 4.3 days; P = 0.012 for the difference between these 2 groups). Events associated with any single virus other than RSV necessitated a mean hospital stay of 6.47 ± 5.64 days, which was not statistically significantly different when compared with RSV -only associated LRTI events necessitating hospitalization.
Seasonality of RSV Infection
The identification of RSV infection in subjects with an LRTI evaluated at the 3 centers indicates that infections occur in all months (see Figure, Supplemental Digital Content 3, https://links.lww.com/INF/B873 RSV infections varied with periods of higher frequency observed throughout the year for 3 consecutive years (2008–2010). The peak incidence generally occurred from April to July, with additional smaller peaks occurring in November 2009 and September 2010.
DISCUSSION
In this study conducted at 3 sites in Brazil, RSV (33.1% of LRTI events) and HRV (29.4% of LRTI events) were the respiratory viruses most frequently identified in preterm subjects born <35 wGA and <1 year of age who experienced an LRTI. More importantly, in LRTI events that were severe (events requiring a hospital admission), RSV was identified at a frequency more than twice that of HRV. The hospitalization rate for severe LRTI associated with RSV infection was 9.9%. Although other respiratory viruses, including HRV, HBoV, HMPV and influenza, were present in >10% of LRTI events, their association with severe LRTIs was well below that of RSV .
The association of RSV with serious respiratory infection has been documented in a number of geographic locations,17 , 30–34 13 , 14 , 25–28 RSV infection, particularly in the children who are most susceptible to the development of serious respiratory disease.
Children in this study were primarily those of a less affluent socioeconomic status, as evidenced by their birth and treatment in the participating public hospitals (known to provide free assistance to poorer children) and by the low level of maternal education. Therefore, it is of interest that the observed overall rates of detection of respiratory viruses in children participating in the current study are similar to what has been described among more affluent populations,35
Another key finding of our study is the important burden of coinfections, especially those associated with RSV . Coinfection of children with >1 virus is not uncommon in cases of LRTI.13–18 , 35 RSV and other respiratory viruses pose a greater clinical burden when compared with infection by RSV alone, as was evident by the significantly increased length of hospital stays associated with these coinfections. Similar observations regarding the importance of coinfections have been previously reported.13 , 15 , 19 , 36 RSV + HMPV was the most commonly detected coinfection. Children <6 months with RSV + HRV coinfection were significantly more likely to spend additional time in the hospital compared with children without a coinfection.13 RSV coinfection was significantly associated with moderate to severe nonpneumonic respiratory disease.15 19 RSV was the most commonly identified respiratory virus, both as a single infection and as part of coinfections.36 14 RSV with additional viruses was not associated with increased hospital or ICU admission in children <2 years.35 16
Although RSV is usually the most frequently identified virus in samples of patients with respiratory infections, a number of other viruses may be present either alone or through coinfected samples. The identity of these viruses likely depends on geographic and seasonal factors, among others. This study found that RSV was the most frequently identified virus in single infections and in coinfected samples. Similarly, HRV was the second most identified virus in both single and coinfected samples, as has been observed in studies involving children with bronchiolitis in southeastern Brazil35 15 RSV , HMPV and HAdV were the most frequently identified coinfecting viruses in other studies conducted in Brazil,13 , 14 19
The mechanisms associated with disease virulence in cases of viral coinfections are complex and, as of yet, far from clear. Direct interactions of viral genes or gene products, indirect interactions related to an affected host environment or immunologic interactions are likely to be at play, acting independently, or in conjunction with each other.37 , 38
A number of risk factors were found to be significantly associated with severe LRTI in this study. Among children who experienced an LRTI that was classified as severe or nonsevere, children with a severe LRTI had lower RSV -specific IgG titers at the time of study enrollment compared with children with a nonsevere LRTI. These data support earlier evidence suggesting that higher levels or avidity of maternal antibodies to RSV correlate with protection against RSV infection during the first few months of life.39–43 RSV , confirming previous findings of an association of higher maternal education with decreased risk for severity of infection.44 RSV was increased with exposure to household tobacco smoke, as has been noted previously,45 , 46 RSV infections that occurred during the 3 years of the study indicated that RSV infections occurred throughout the year at these study sites, although months in which higher RSV infection frequencies occurred were identified.
Limitations of the study included the number of enrolled subjects, which was slightly smaller than originally planned, and the lack of PCR analysis on a number of subjects who experienced an LRTI. In addition, the study enrolled subjects at only 3 sites in Brazil; therefore, the results reported here might not be representative of the entire country. Another limitation may be related to the detection of a coinfecting virus in the same patient sample via a concomitant PCR assay. Recent evidence indicates the possibility of a human “virome” detectable by molecular methods,29 , 47
Epidemiologic studies of RSV incidence are instructive exercises that are often valuable tools in the formulation of effective treatment practices. However, results from individual studies are influenced by demographic, economic, geographic, seasonal and study design considerations, which make meaningful comparison between studies challenging. Although data on local populations are usually the best guide for the development of regional health care policies, differences observed between studies may uncover factors that influence those disparate results. Moreover, the demonstration of common findings between studies, despite unavoidable methodologic variations, may indicate the importance of those results.
The data reported in this study confirm the association of RSV infection with severe LRTI in children born ≤35 wGA during their first year of life and provide additional data that suggest that antibody specific for RSV may be protective in young children. The finding that coinfection of RSV with other viruses increases the severity of LRTIs, as evidenced by longer mean hospital stays among these preterm infants, is quite important and provides even further relevance to policies that indicate adequate prophylaxis for susceptible infants for whom RSV infection may be accompanied by an increased risk for significant morbidity and mortality.
National guidelines for the prevention of severe RSV -related respiratory disease are in place in Brazil. Our findings reinforce the previously well-known burden of this virus, especially among susceptible populations, and the importance of disease prevention. Data from this study are also indicative of an even more significant impact of RSV on severe LRTIs when detected as part of a coinfection with other common respiratory pathogens.
ACKNOWLEDGMENTS
This study was sponsored by AbbVie. AbbVie contributed to the study design, research, analysis and interpretation of data and publication writing, reviewing and approval. The authors determined the final content. No payments were made to the authors for writing this manuscript or performing the study. Medical writing support was provided by Daniel E. McCallus, PhD, at Complete Publication Solutions, LLC; this support was funded by AbbVie Inc. The authors thank the BREVI Study Group for their participation in this study; Rodrigo Sini (Abbott) for assistance in the review of study data; study subinvestigators Taisa Paim (PUC-RS), Camila Parreira Schmidt, MD (PUC-RS), Márcia S. Freitas da Motta (HC FMRP USP), Gustavo Xavier Yamaguti (HCFMRP USP) and Juliana Castanheira (HC FMRP USP); study coordinators: Ana Lúcia Campelo Prestes (HCUFPR), Giovana dos Santos (PUC-RS), Andréia Tompsen (PUC-RS) and Mariluiza Shizue Iwamoto (HC FMRP USP); study coordinator Marisa Akiko Iwamoto (HC FMRP USP); study CRA Priscila Biegun (Abbott) and study clinical operations manager Karla Salomão (Abbott).
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