Lower respiratory tract infections (LRTI) are a common cause of hospital admission in pediatric patients,1–3 especially under two years of age.4–6 Different viruses have been identified as etiologic agents of LRTI [respiratory syncytial virus (RSV), adenovirus (ADV), influenza virus A (FLUA) and B (FLUB), human rhinovirus (HRV), parainfluenza1 (PIV1), 2 (PIV2), 3 (PIV3)].7–10 RSV remains the most important viral pathogen in infancy both as a single agent and in coinfection.4,6

In the last two decades, the emergence of polymerase chain reaction (PCR) based technology has substantially increased the sensitivity for viral diagnosis, detecting a larger number of viruses, including more than one virus in the same respiratory secretion sample.2,11,12 Such assays identify virus not growing in standard tissue culture,2 and have identified at least four new viruses associated with LRTI: metapneumovirus (MPV),13 coronavirus (COV),14–16 bocavirus (HBOV)17 and parainfluenza 4 (PIV4).18,19

In children hospitalized with severe LRTI, viral coinfection detected by PCR shows a prevalence ranging between 14% and 44%.20 Whether viral coinfection has a significant clinical impact is yet to be defined.6,11,21–24 A recent meta-analysis found divergent outcomes dependent mainly on age (adult patients included), comorbidities, seasonality, geographical region and methods of detection employed.25

The primary objective of this study was to compare severity of presentation and short-term clinical evolution between single virus infection and viral coinfection in children admitted for LRTI. We additionally intended to compare the amount of health resources used between groups.

We hypothesized that patients with viral coinfection would have a more severe disease and a higher need for diagnostic and therapeutic health care resources.

This was a single-center prospective observational study enrolling sequential patients admitted for LRTI, from October 2012 to September 2015, at the Department of Pediatrics of a tertiary care, University affiliated Hospital. This Department receives previously healthy children attending the emergency department (ED) from the geographic area of the hospital and from other local hospitals for chronic and/or severe disease. During the study period, a total of 119,682 children were seen at the ED, from which 3.3% were admitted as inpatients.

Children aged less than two years old with the diagnosis of LRTI were enrolled. The discharge diagnosis was made by the attending physician, who also collected the clinical data by fulfilling a standardized written form, created specifically for the study, at patient's admission and discharge. We analyzed patient's demography (gender, age), comorbidities or associated chronic conditions (prematurity, very low birth weight, respiratory, cardiac, neurologic or genetic), clinical and environmental data (breast-feeding, passive smoke exposure, day care attendance), duration of symptoms and signs related to the acute respiratory infection, and physical examination on admission. Furthermore, we compared the amount of health resources used, namely diagnostic tests performed [including blood analysis, bacteriological cultures of respiratory specimens, chest X-ray (CXR)], treatments prescribed (bronchodilators, antibiotics, oxygen delivery and ventilatory support) and hospital length of stay (LOS) and intensive care unit (ICU) admission.

Respiratory secretions, nasopharyngeal aspirate (NPA) or a nasopharyngeal swab, were obtained within 72h of the child's admission. The strains were sent to the Microbiology Laboratory where the viral and bacteriological studies were carried out. Viral identification was performed through qualitative reading of a real-time PCR technique (Anyplex™ IIRV16 Seegene Inc, till early December 2015 and Allplex Respiratory Pannel I, II e III Seegene Inc, afterwards), both allowing for the simultaneous identification of 16 viruses: ADV, FLUA, FLUB, PIV1, 2, 3 and 4, HRV, RSVA and RSVB, HBOV, COV 229E, NL63 and OC43, MPV and enterovirus (HEV), the second kit with further differentiation of influenza A(H1N1)pdm09 and A(H3).

Viral coinfection was defined when multiple simultaneous viral pathogens were detected from the same respiratory sample and bacterial coinfection or secondary bacterial pulmonary infection based on positive bacteria cultures. Bacterial diagnostic tests were performed only if requested by attending physicians. Strains isolated from products of the respiratory system (expectoration, bronchial secretions) came as samples from a swab, based on the presence of epithelial cells, neutrophils and the morphology of the predominant bacteria. For all types of samples, macroscopically distinct colonies were isolated in pure culture.26 Coinfection was admitted based on microbiological definitions because of the overlap between symptoms and clinical diagnosis, mainly of bronchiolitis and pneumonia.27

Disease severity was analyzed through hospital LOS in days, ICU admission and need for ventilatory support, both invasive and non-invasive.

In this single-center study we found a high rate of respiratory virus detection (>85%). RSV A/B were the most frequently found viruses followed by HRV, either as single virus or coinfection, both accounting for over half of single viral identification.

The rate of viral coinfections (35.5%) was similar to that described by others using similar viral identification techniques.11,23 The most frequent viruses involved were HRV (60.4%) and RSV A (47.8%), pathogens that were also predominant in single infections, as shown in previous studies.11 ADV was the third most commonly identified in viral coinfections (26.4%). Its frequency was higher than that referred in other studies.28,29 Viruses such as ADV, HBOV, HEV, COV OC43, COV NL63, PIV1, 2 and 3 and FLUA were more frequently seen as an agent of viral coinfection than as a single agent.5,28 Nascimento et al. found similar results in 2010.23

Nevertheless, a great variability exists regarding viral coinfection rates, with results as low as 11–17.5%6,30,31 and as high as 50–60%.29,32,33 A multicenter national study7 that used immunofluorescence and PCR techniques for viral detection described a much lower coinfection rate (13.3%) in children with acute bronchiolitis. Differences in viral coinfection rates may result from the population studied (infants versus older children and different proportions and types of underlying comorbidities) or from viral diagnostic techniques used. In our study, viral coinfection was more frequent in older children, in those exposed to nursery or with individual susceptibility factors such as recurrent wheezing and also in patients with pneumoniae as the discharge diagnose. Others have reported similar results.5,6

Although there are some discrepancies between reports, we found no association between viral coinfection and clinical disease severity.5,6 In our study clinical severity was not evaluated based on a severity score, however others who have used it also found no differences.6

Asner et al.,25 in a recent meta-analysis, did not find convincing evidence that patients admitted with viral coinfection had higher risk of developing more severe disease comparing with single virus infection. However, it should be highlighted that the overall quality of evidence of the studies included applying the GRADE framework ranged from low to moderate quality of evidence. The studies of Suryadevara et al.,1 Marguet et al., Nascimento et al.,23 and De Paulis et al.11 corroborated that viral coinfection does not seem to have an impact on the severity of disease. On the other hand, Martínez-Roig et al.32 reported an inverse relationship between the number of detected viruses in the NPA, the need for oxygen supply and the hospital LOS. In the aforementioned study of Calvo et al.,6 viral coinfection appeared to be associated with a greater hospital LOS, unlike what was observed in our study.

The prescription of short-acting bronchodilators, antibiotics and systemic corticosteroids was more frequent in the viral coinfection group. We must be cautious in interpreting these data as this was probably due to children's older age and more symptomatic presentation on admission rather than the presence of viral coinfection. Some of these clinical characteristics, such as fever, were also more frequent in patients with multiple viral infections in other studies.6

Virus–bacteria coinfection, although not systematically performed, was found in 27.9% of cases, and, as previously reported, was associated with more frequent collection of blood samples, CXR and antibiotics’ prescription.5,27,34,35

There are some strengths of our study, explicitly the prospective nature of the data collection, the considerable number of respiratory samples assessed, the use of a broad molecular assay for the detection of 16 different viral respiratory pathogens and the high positivity rate. Moreover, our sample proved to be homogeneous in terms of patient's age (81.7% with less than 12 months and a median age of 4 months). Additionally, the impact of underlying comorbidities/risk factors was also addressed. Finally, an additional strength was the time span of the study including cases from all year round. Some previous studies performed seasonality-based analysis losing the characterization of some LTRI associated viruses that occur in the rest of the year.7,36

This study also has some limitations that should be acknowledged. Firstly, the fact that it was designed to describe the epidemiology of viral LRTI in hospitalized pediatric patients. As such, it included only hospitalized children in an academic medical center, so these results may not be generalizable to community attendance and cannot be extrapolated to children with less severe disease. Secondly, the viral identification through real-time PCR in respiratory secretions does not necessarily mean acute infection. It is well known that this technique is able to detect virus in up to 5% of asymptomatic patients36,37 and we should also remember that the detection of more than one virus may be due to the presence of viral fragments persisting for up to 5–6 weeks after onset of symptoms.38 Therefore, the term “co-detection” is, in fact, more precise than coinfection, especially when molecular diagnostic techniques are used. However, this limitation is difficult to overcome, since most of these viruses are difficult to grow in cell culture and, consequently, clinical interpretation of virus detection in respiratory secretions in a child with LRTI is quite challenging.21,36–38 This may be another possible reason for the absence of differences between the single infections and coinfections with regard to the clinical outcomes. Quantitative PCR and its correlation with clinical symptoms might become a useful tool to clarify the actual role of multiple infections. Finally, another important limitation is the absence of a systematic search for bacteria in the respiratory specimens. The potential influence of a concomitant bacterial infection can be a relevant issue contributing to contradictory data in studies examining the role of the viral coinfection in determining the severity of the disease. Prospective longitudinal studies including serial respiratory sampling for viruses and bacteria may lead to a better understanding of the clinical significance of polymicrobial acute LRTI.

In summary, on the basis of this prospective, single-center, multiyear data, we found that 1 in 3 children admitted for LRTI had multiple virus infection. Our results suggest that there was an association of viral coinfection with demographical, environmental and clinical characteristics on admission. Most importantly, our data challenge current thinking as we found that this does not change the short-term outcomes nor the relevance for co-hosting patients on the basis of single identification virus by other less sensitive methods.39,40

Due to the burden of LRTI in children, we highlight the need for further prospective longitudinal studies, with well-defined short-term outcomes and systematic search for viruses and bacteria in respiratory samples, in order to better understand the clinical importance of polymicrobial respiratory infections in children and adjust severity risk factors and hospital resources.

The authors declare that they have no conflict of interest.