Etiology and Clinical characteristics of bronchiolitis in Suzhou

We investigated the respiratory specimens and clinical data of 1012 children with bronchiolitis who were treated at the Hospital of University between November 2011 and December 2018. The nasopharyngeal aspirates were examined by direct immunofluorescence assay or polymerase chain reaction (PCR) to detect viruses and by PCR and enzyme-linked immunosorbent assay to detect Mycoplasma pneumoniae (MP). Of the 1012 with bronchiolitis, 842 were detected at least a 614 had single viral infections, 91 had MP infections, 70 (6.9%) had multiple viral infections, and 67 (6.6%) had mixed viral and MP infection. The most common pathogens detected were respiratory syncytial virus (RSV) (44.4%), MP (15.6%), and human rhinovirus (HRV) (14.4%). RSV was the most common pathogen detected in children less than 6 months. Coinfection was detected in 13.5% (137/1012) of the children, but it was less common in children less than 6 months. The age of children with single virus infection was the youngest. Children with single virus infection had a higher proportion of oxygen therapy compared with single MP infection.


Background
Bronchiolitis is a clinical syndrome commonly encountered in practice, particularly among infants and young children. However, the etiology and clinical impact of the condition remains elusive.

Methods
We investigated the respiratory specimens and clinical data of 1012 children with bronchiolitis who were treated at the Children's Hospital of Soochow University between November 2011 and December 2018. The nasopharyngeal aspirates were examined by direct immunofluorescence assay or polymerase chain reaction (PCR) to detect viruses and by PCR and enzyme-linked immunosorbent assay to detect Mycoplasma pneumoniae (MP).

Results
Of the 1012 children with bronchiolitis, 842 (83.2%) were detected at least a pathogen. 614 (60.7%) had single viral infections, 91 (9.0%) had MP infections, 70 (6.9%) had multiple viral infections, and 67 (6.6%) had mixed viral and MP infection. The most common pathogens detected were respiratory syncytial virus (RSV) (44.4%), MP (15.6%), and human rhinovirus (HRV) (14.4%). RSV was the most common pathogen detected in children less than 6 months. Coinfection was detected in 13.5% (137/1012) of the children, but it was less common in children less than 6 months. The age of children with single virus infection was the youngest. Children with single virus infection had a higher proportion of oxygen therapy compared with single MP infection.

Conclusions
The most common pathogen detected in children with bronchiolitis is RSV, followed by MP and HRV. Co-infections lead to prolonged illness and worsening of the symptoms 3 Background Bronchiolitis is an acute infection of the lower respiratory tract, particularly affecting the terminal and respiratory bronchioles, with the possibility of extending to the adjacent alveolar ducts and spaces. 1 For infants and young children, severe bronchiolitis is one of the most common reasons for hospitalization and clinical visits. Bronchiolitis is a wellknown clinical entity, which affects around 1-3% of all healthy children. 2 The mortality rate reported for this condition is reported to be approximately 2 per 100000 infants, with the rate being higher in developing countries than in developed countries. 3 However, the mortality rate of bronchiolitis in China has been reported to be 3.5%, 4 which is closer to that in developed countries rather than in the developing nations. 5,6 Patients with bronchiolitis are at a high risk of developing recurrent wheezing, although the risk factors and exact pathogenesis are yet to be clearly understood.
Viruses are known to be the most common pathogens causing bronchiolitis; viral bronchiolitis is clinically by acute inflammatory changes, edema and necrosis of the epithelial cells lining the small airways, excessive mucus production and bronchospasm.
The main pathogen implicated in bronchiolitis is respiratory syncytial virus (RSV), other viral pathogens that cause the disease include parainfluenza virus and adenovirus. 7,8 Furthermore, recent studies have determined that bacterial pathogens, particularly Mycoplasma pneumoniae (MP) and Chlamydophila pneumoniae (CP), are responsible for bronchiolitis in children under 2 years of age. 9− 11 However, the clinical relevance of the various pathogens involved in children still remains unclear.
In this study, we sought to evaluate the distribution of pathogens responsible for bronchiolitis in children ≤ 2 years of age and analyze the differences in the clinical features of bronchiolitis caused by different pathogenic agents.

Subjects
We conducted a retrospective analysis of the data of 1012 children who were admitted to the Children's Hospital of Soochow University for the management of bronchiolitis between November 2011 and December 2018. Children's Hospital of Soochow University is a tertiary referral center at Jiangsu Province, East China. It has over 1000 beds and 50,000 inpatients annually. The inclusion criteria for this study were children aged between 1 month and 2 years, occurrence of first episode of wheezing, and clinical evidence of bronchiolitis (tachypnoea, wheeze, prolonged expiratory phase, and crackles on auscultation). This study protocol was approved by the Medical Ethics Committee of Soochow University.

Specimen Collection
Within 24 hours of admission, nasopharyngeal aspiration was performed to collect specimens from all patients. For aspiration, a suction catheter was used introduced through the nose and advanced into the lower portion of the pharynx, up to a distance of 7-9 cm. Nasopharyngeal aspirate of 2 mL was then collected and sent for histopathological analysis within 30 min of collection. The retrieved sample was centrifuged (500 × g, 10 min) and suspended in 2 mL saline and separated into 2 aliquots for direct immunofluorescence assay (DFA) and polymerase chain reaction (PCR) to identify pathogens.

Viral Detection
A quantitative diagnostic kit for MP DNA was performed to identify the 16 s rRNA gene of MP extracted from nasopharyngeal specimens. DFA was performed to detect RSV, influenza virus (IV), parainfluenza virus (PIV), and adenovirus (ADV). The assay kits were obtained from Chemicon (USA), and all staining procedures were performed according to the manufacturer's instructions. Immunofluorescence studies were then conducted (Leica 020-518.500, Germany).RNA was extracted from the specimens using Trizol reagent (Invitrogen, USA), followed by cDNA synthesis by reverse transcription. The cyclic temperature settings were 94 °C for 30 s; 55 °C for 30 s; followed by 68 °C for 30 s; finally, amplification was performed over 45 cycles at 68 °C for 7 min. For detection of human metapneumovirus (hMPV) and rhinoviruses (HRV) fluorescent real-time PCR (BIO-RAD iCycler) was performed.

Data Collection
The medical records of the patients were reviewed and data regarding the following parameters were recorded: (1) demographic and clinical characteristics, including age, gender, and duration of symptoms prior to admission; (2) results of viral diagnostic tests performed in nasopharyngeal aspirates; (3) results of blood tests for inflammatory indices, including white blood cell (WBC) count, percentage of neutrophils, serum C-reactive protein (CRP) levels.

Statistical Analysis
All statistical analyses in this study were performed using the Statistical Package for the Social Sciences (version 25.0). Frequency distributions and rates were used for descriptive analyses. Parameters regarding the patient's demographic data and baseline characteristics were analyzed using means (SD) or medians (25th -75th percentiles).
Normal distribution was met by the data, as confirmed by using t-test variance analysis.
For parameters without the non-normal distribution of data, the Wilcoxon rank sum test and Kruskal-Wallis H test was used for comparison between two groups and multiple groups, respectively. P value of < 0.05 was considered to indicate statistical significance.

Etiology
The most common pathogens detected were RSV (44. with MP. Among patients aged ≤ 6 months, 12% had co-infection, with 6.5% having multiple viral infections and 5.5% having coinfection of a virus and MP. Among patients between 6 months and ≤ 1 years of age, 22.8% had co-infection, with 11.4% having mixed viral-viral infections and 11.4% having viral-MP infections. In the patients aged 1 to 2 years, 24.3% had co-infection, with 11.3% and 14.0% having mixed viral-viral and viral-MP co-infections, respectively. The probability of co-infection in children of age ≤ 6 months was significantly lower than in children of age between 6 months and ≤ 2 years (all P < 0.002).   Table 2).

Discussion
Bronchiolitis is the most frequent disease affecting children of age < 2 years, and it is a leading cause of hospital admissions in this age group. In this study, we conducted a retrospective investigation of 842 children hospitalized with bronchiolitis in order to identify the distribution of pathogens and co-infection. We found that 83.7% of the children had a single pathogen infection, whereas 16.3% had co-infections.
The most common pathogens identify in our study were RSV (53.5%), followed by MP (18.8%) and HRV (17.3%). A longitudinal prospective investigation conducted in the USA revealed that RSV was the causative pathogen for bronchiolitis in 76% of infants; this percentage is much higher than that observed in our study; however HRV was isolated in 18% of the their cases, which is similar to the rate of HRV infection noted in our study. 12 In an 11-year study in Spain, 62.7% of the patients (children ≤ 2 years of age with acute bronchiolitis) had RSV infections, which is a percentage slightly higher than that noted in our study. 13 In a similar retrospective Slovenian study of children under 2 years with bronchiolitis, RSV (57.5%), HRV (25.6%), and HBoV (18.4%) were identified as the most common pathogenic viruses; 14 their results were similar to ours in the case of RSV but higher in the case of HRV. Further, in our study, MP was isolated in 18.8% of our patients, which is similar to the percentage (17.2%) reported in a previous study. 15 The current study indicated that a single viral infection (72.9%) was most common type of infection in children under 2 years of age with bronchiolitis. We noted that RSV was the most common virus isolated, especially in infants under 6 months of age. We also noted that the percentage of RSV infection gradually decreased with age, which suggests that younger infants are more vulnerable to RSV disease; this is consistent with the findings of previous studies. 16,17 A retrospective cohort study indicated that the reduced exposure of pregnant women to RSV epidemic contributed to more severe RSV-induced bronchiolitis in children under 6 months of age. 18 Therefore, RSV-induced bronchiolitis is common in 6 months age; this may be associated with the circulation of antibodies that are not associated with RSV infection during pregnancy. Further investigations are necessary to identify specific susceptibility factors.
One hundred and thirty seven (16.3%) of our patients had infection due to two pathogens.
Interestingly, the distribution of multiple viral coinfection and viral-bacterial infection was similar. The probability of co-infection in children ≤ 6 months old was significantly lower than that in children aged between 6 months and ≤ 2 years of age. A prospective study from Turkey identified that the rate of coinfection among children with acute bronchiolitis was 34.2%, which is higher than the percentage observed in our study. 19 Similarly, a study from Israel showed that the rate of co-infectin in infants with acute bronchiolitis was nearly 40%, which is also higher than that in our study. 20 A UK study reported an even higher percentage of 46%. 21 The discrepancies in the rate of co-infection in bronchiolitis may be attributed to differences in the pathogen detection methods and the type of pathogens isolated.
The impact of co-infection on the severity of bronchiolitis still remains questionable. A comprehensive review in London revealed that multiple viral infection was associated with the admission of infants to the pediatric intensive care unit for the management of severe bronchiolitis. 22 In contrast, some studies have shown that there is no correlation between the presence of multiple co-infections and severity of bronchiolitis. 23 A Taiwanese study also reported that different viral pathogens do not give rise to different clinical characteristics among children with bronchiolitis. 24 However, a Brazilian study revealed that both co-infection and RSV load influenced the prognosis of acute bronchiolitis in infants. 25 Our findings in this study indicated that the duration of symptoms and duration of hospitalization in cases of simple virus infection were significantly less than those observed in case of combined viral and MP infection. Further, simple virus infection was least likely to induce fever. Thus, we believe that co-infection can aggravate the disease.
Further studies are necessary to confirm these associations.
Some of the most common reasons for admission due to bronchiolitis are hypoxia, requirement for supplemental oxygen, poor feeding, and respiratory disease.  Availability of data and materials The manuscript detailing where the data supporting the findings in this study can be found if requested.
Author's contributions JHT and JFW wrote the main manuscript text. WJJ and LH conceptualized and designed the study, drafted the initial manuscript, and approved the final manuscript. WJ and YDY carried out the initial analyses, reviewed and revised the manuscript, and approved the final manuscript. MJW and XJS did the microbiological detection. All authors read and approved the final manuscript.
Ethics approval and consent to participate The study was approved by the Medical Ethics Committee of Children's Hospital of Soochow University. The parents of all study participants gave written informed consent before study enrollment.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests.