Introduction
RSV is a major pathogen that often causes outbreaks during the cold season [
1]. By 2 years of age, more than 90% of children have serological evidence of RSV infection [
2]. RSV is commonly present in young infants and children with bronchiolitis, which is a lower respiratory tract infection (LRTI) with small airway obstruction, and can rarely progresses to pneumonia, respiratory failure, apnea, and death [
3]. RSV causes approximately 33 million LRT illnesses, 3 million hospitalizations, and up to 199,000 childhood deaths worldwide, with RSV bronchiolitis accounting for the largest proportion [
4,
5]. Many countries have updated their bronchiolitis guidelines [
6‐
9] to define early-life severe RSV bronchiolitis and to prevent its potential long-term poor prognosis, including recurrent episodes of wheezing and asthma after recovery from bronchiolitis or pneumonia [
10,
11].
Despite intensive research, safe and effective vaccines for preventing RSV bronchiolitis remain elusive [
12]. Currently, only one approved antiviral treatment for RSV is available; however, its use is limited by questionable efficacy, side effects, cost, and it is not accessible in our country China [
13]. The widespread administration of prophylactic drugs emphasizes the need for active surveillance of RSV subtypes and the timely detection of viral mutants. Only two subtypes (RSV-A and RSV-B) are prevalent, and the clinical impact of viral factors associated with RSV remains controversial. Some studies have reported no differences between the two subtypes [
14,
15], while others have shown that RSV-A has a worse [
15,
16] or better prognosis than RSV-B [
17,
18]. In the absence of vaccine coverage and due to the inaccessibility of antiviral medicines, understanding the differences in clinical symptoms severity and outcomes caused by different subtypes could help select appropriate therapeutic regimens and formulate vaccine development strategies. In this study, we aimed to investigate the subtype-specific severity of RSV infection in paediatric bronchiolitis and identify factors that may be associated with disease severity.
Methods
Ethics approval
This study was approved by the Medical Research Ethics Committee of the Children’s Hospital of Hebei (CHH) in accordance with the principles of the Declaration of Helsinki and the Code of Ethics of the World Medical Association. As this was a retrospective study that poses no risk of harm to the subjects, and all patients were de-identified, informed consent was waived by the committee.
Study subjects
Children (aged < 2 years) admitted to our hospital between June 2018 and January 2019 with a discharge diagnosis of RSV bronchiolitis were included in this study. Demographic data, clinical characteristics, underlying diseases, laboratory test results and treatment outcomes of these patients were retrieved and retrospectively analyzed. Patients who underwent repeat testing within 14 days were excluded from the study.
Pathogen detection
A multiplex PCR-based platform (i.e., GenomeLab system, Beckman countler, USA) was used to simultaneously detect RSV and 10 other pathogens: influenza A virus, influenza B virus, adenovirus, parainfluenza virus, human rhinovirus, human metapneumovirus, human bocavirus, human coronavirus,
Chlamydia pneumoniae, and
Mycoplasma pneumoniae. Multiplex PCR was performed as previously described [
19]. RSV-positive samples were tested for RSV-A and RSV-B using a real-time RT-PCR (RT-qPCR) assay (Liferiver Biotechnology Co. Ltd) according to the manufacturer’s recommendations. Bacterial and fungal cultures from induced sputum (IS) and blood samples were isolated according to the protocols developed in our diagnostic laboratory.
To obtain an IS sample, a sterile negative-pressure suction catheter was used by a skilled nurse to stimulate the throat and induce coughing. Evidence of bacterial co-infection was proven using blood cultures from sterile sites or IS from non-sterile sites. For positively induced sputum, if the clinician judged that it was clinically significant and an appropriate antibiotic treatment was administered, we also regarded it as a bacterial co-infection.
Disease severity and complications
Disease diagnosis was performed according to the “Clinical Practice Guideline: The Diagnosis, Management, and Prevention of Bronchiolitis” [
8]. Meanwhile, according to the Chinese guidelines on bronchiolitis, severe disease was defined as the presence of one or more of the following manifestations: (1) increasing irritability and/or lethargy, fatigue; (2) marked increase in respiratory rate; (3) marked chest wall retractions, marked tracheal tugging; (4) marked nasal flaring; (5) O
2 saturation less than 88% (in room air) and hypoxemia, which may not be corrected by O
2; (6) increasingly frequent or prolonged apnea; or (7) reluctance or inability to feed [
7]. Complications in the cardiovascular system involvement included heart failure, abnormal cardiac enzyme profiles, and right heart failure; while those in the gastrointestinal system included diarrhea, vomiting, and gastrointestinal bleeding.
Treatment and outcomes
All patients were treated according to the “Clinical Practice Guidelines: The Diagnosis, Management, and Prevention of Bronchiolitis” [
8]. The outcomes recorded included recovery and discharge, transfer to a community hospital, or death.
Statistical analyses
Normality of the data was determined using the Shapiro-Wilk test. Non-normal data are presented as medians (first and third quantiles) and analyzed using the Mann-Whitney test. Categorical data were analyzed using the chi-squared or Fisher’s exact tests. Logistic regression analysis was performed to select the variables associated with severe RSV bronchiolitis. Statistical analyses were performed using SPSS version 23.0. A p <.05 was considered statistically significant.
Discussions
RSV is the most common cause of bronchiolitis that leads to hospitalisation in young children [
20,
21]. RSV outbreaks are common in autumn and winter, and the severity and serotypes of RSV infection in a given country vary annually [
4]. In China, subtype A was predominant from 2011 to 2017 [
22]. After 2018, the prevalence of RSV-B exceeded that of subtype A in China [
23], Japan [
24] and India [
25]. The present study found that, subtype B was twice as prevalent as subtype A in young children with bronchiolitis. After COVID-19, RSV-A outbreak was predominant [
26,
27]. Nevertheless, the impact of molecular epidemiology on the clinical course of the disease remains controversial [
4,
28,
29]. Based on the bronchiolitis guidelines, we investigated disease severity and its correlation in our patients with RSV bronchiolitis. We found that: (1) a greater proportion of severely ill children had shorter fever duration and bacterial co-infections; (2) RSV subtypes were not associated with disease severity.
RSV can cause complications in other organ system in children [
30‐
32]. Our results showed that, 25.6% of children with RSV bronchiolitis developed cardiovascular complications, ranging from myocardial injury to heart failure. A previous study on cardiovascular involvement in hospitalized children with RSV infection reported that approximately 76.5% of otherwise healthy infants with RSV bronchiolitis showed sinoatrial blocks and transient rhythm alterations [
30]. A study conducted in Japan found that 50% (9/18) of children with RSV infection had myocardial damage, 38.8% had conduction system disturbances and 16.6% had tachycardia [
33]. Thorburn et al. reported that, 20% (7/34) of children with severe RSV bronchiolitis admitted to a tertiary pediatric-intensive unit in the UK had reduced right ventricular function [
34].
Furthermore, the extrapulmonary complications have been described also in the digestive, neurological, and other organ systems [
32]. In the present study, 23.1% (19/82) of patients had gastrointestinal complications. In the USA, a previous case series reported that four infants with severe RSV bronchiolitis developed necrotizing enterocolitis shortly after admission [
35]. In addition, 87 unique studies from 26 countries described a spectrum of RSV-associated severe acute neurological syndromes, including proven encephalitis, acute encephalopathy, complex seizures, hyponatremic seizures, and immune-mediated disorders [
36]. These data suggest that RSV-associated extrapulmonary complications are common in children and can lead to high morbidity and mortality. Thus, management of such complications should be a critical part of the therapeutic regimen.
Persistent fever can worsen infectious diseases, and some studies reported a higher mortality rate for fever lasting more than 5 days [
37]. On the contrary, it has also been reported that fever may induce the expression of heat shock proteins that protect host cells and regulate immune responses [
38]. In the present work, the median duration of fever in severely ill children was 0 days, which was significantly shorter than 1 day in patients with mild to moderate bronchiolitis. We found this relatively short fever course to be a protective factor against severe bronchiolitis. On the other hand, aggressive hypothermia treatment does not alleviate disease progression. Schulman et al. reported that aggressive fever suppression (administration of paracetamol above 38.5 °C and application of a cooling blanket above 39.5 °C) was associated with significantly higher mortality compared to permissive suppression (the same interventions above 40 °C, 15.9% versus 2.6%,
p =.06, Fisher’s exact test) [
39]. These data revealed that fever duration may serve as a disease severity marker, which requires further study.
Lymphopenia and its association with disease severity have been reported in children with RSV infections [
40,
41]. A histopathological study of children with fatal RSV infection showed that double-negative T (DNT) cells infiltrated the bronchial and pulmonary arterioles, and promoted the formation of fibrin, mucus, and edema [
42]. The number of circulating DNT cells in peripheral blood mononuclear cells (PBMCs) was lower in patients with chronic autoimmune diseases than in healthy controls [
43]. A reduced number of DNT cells may lead to a loss of immune regulation, thereby breaking immune tolerance and promoting pathogenesis [
44]. In the present study, a substantially lower percentage of DNT cells was observed in the severe group compared to that in the mild-to-moderate group, suggesting that a decrease in DNT cells may reflect the disease severity.
Bacterial co-infection with RSV in severely ill children with RSV has been described previously [
45,
46]. In a study aimed at comparing outcomes between RSV with and without bacterial co-infection in children without underlying diseases, Lin et al. showed that children with co-infections required more intensive care and a longer hospital stays [
45]. In another study, the presence of bacterial co-infection was significantly associated with the development of acute respiratory distress syndrome (OR = 1.9) in children with RSV infection [
47]. Similar to the findings of Ghazali et al. [
46], we observed a significantly higher rate of bacterial co-infections in the severely ill children. Because some studies only tested for RSV and did not consider other viral or bacterial infections, confounding effects may have been omitted or have led to ascertainment bias. Our results suggest that the early recognition of bacterial infections and prompt, effective antibiotic treatment of suspected severe cases are important for preventing disease progression.
Our study had several limitations. First, this was a retrospective study involving a single center. Second, the sample size was relatively small, including only 26 patients with severe disease. Third, not all patients with RSV bronchiolitis underwent RSV subtyping. Fourth, we observed that the duration of fever was significantly shorter in the severe group, which requires further investigation of the relationship between disease severity and fever. Furthermore, bronchiolitis was screened in children using a laboratory molecular diagnosis for RSV infection. Although some studies have shown that the vast majority of children with bronchiolitis are infected with RSV, bronchiolitis due to infection by other pathogens may have also been neglected.
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