This prospective descriptive study was conducted on children presenting with acute bronchiolitis who were admitted to the Department of Respiratory Medicine in the Children's Hospital of Soochow University between January 1, 2017, and December 31, 2019. Acute bronchiolitis was characterized by age ≤2, cough, tachypnea, retraction, and expiratory wheezes, often accompanied by rales (11). B. pertussis was confirmed by polymerase chain reaction (PCR) assays (12). Patients requiring oxygen supply were considered with severe conditions. The exclusion criteria were as follows: (1) patients with incomplete clinical data; (2) patients with bronchopulmonary dysplasia, heredity metabolic diseases, neurological disorders, congenital heart disease, and immunodeficiency; and (3) patients with evidence suggesting that wheezing was caused by tuberculosis and non-infectious factors such as bronchial foreign bodies.

The study was approved by the ethics committees of Children's Hospital Soochow University (Approval No. 2016026). Informed consent was obtained from the parents of all children enrolled in this study.

Vaccination history was obtained by querying the “Suzhou Children's Vaccination Inquiry and Evaluation Platform.” A diphtheria, tetanus, and acellular pertussis combination vaccine was administered as a primary series at 3, 4, and 5 months, followed by a booster dose at 24 months in China. The vaccination status was regarded as ever-vaccinated if one to three doses were received.

Data regarding demographic, clinical, and laboratory characteristics were documented. Demographic and clinical characteristics included age, gender, length of hospital stay, and requirement of supplemental oxygen. Laboratory specimens were obtained including blood and nasopharyngeal aspirates (NPAs). NPAs were obtained during the first 24 h of hospitalization, using a sterile plastic catheter briefly inserted into the lower pharynx via the nasal cavity. The blood samples were taken immediately after hospitalization. The laboratory data of leukocyte count, percentages of lymphocytes and neutrophils, and detection of common viruses were collected.

B. pertussis DNA was detected in NPAs by real-time PCR assays. The primer sequence was synthesized by Shanghai Sangon Biotech Company. The pertussis PtxA-pr and IS481 gene sequences were used as specific primers (Table 1). The RT-PCR assay result was considered negative if the cycle threshold (CT) was ≥40. Specimens that tested positive by PCR for both insertion sequence IS481 (CT < 40) and ptxS1 (CT < 40) were considered positive for B. pertussis. If a specimen was PtxA-pr target negative with an IS481 assay CT < 35, it was also considered positive for B. pertussis.

Direct immunofluorescence was used to detect RSV; ADV; influenza virus A (IV-A) and B (IV-B); and parainfluenza virus 1 (PIV I), 2 (PIV II), and 3 (PIV III) using a D³ Ultra Respiratory Virus Screening and LD Kit (Diagnostic Hybrids, Athens, OH, USA). A positive result was defined as over five inclusion bodies analyzed under a fluorescence microscope. Mycoplasma pneumoniae (MP), HRV, HMPV, and HBoV were detected by a PCR (nucleic acid amplification fluorescent reagent kit, Ann Gene Co., Guangdong, China) according to the manufacturer's instructions.

Statistical analyses were conducted using SPSS 26.0 (IBM, SPSS, Chicago, IL, USA).

Data were shown as mean ± standard deviation and median and interquartile range. Quantitative variables among the three age groups were compared using one-way analysis of variance or the Kruskal–Wallis test when appropriate. Frequency distribution was compared by the chi-square test. A p value <0.05 was considered as a significant difference.

Of the total 1,092 patients admitted for bronchiolitis, one or more respiratory pathogens including virus and MP were detected in 1,057 of 1,092 patients (a positive rate of 96.8%) and B. pertussis was identified in 78 patients (7.1%, based on positive results by PCR). Of the 78 cases of bronchiolitis with B. pertussis infection, 47 (60.3%) were male and 31 (39.7%) were female. The male-to-female ratio was 1.52:1. The median age was 6.45 ± 4.94 months. The age distribution of patients is shown in Figure 1; 31 (39.7%) patients were aged ≤3 months, 20 (25.6%) patients were aged 4–6 months, 16 (20.5%) patients were aged 7–11 months, and 11 (14.1%) patients were aged ≥12 months.

The monthly distribution of B. pertussis infection is shown in Figures 2A,B. Bronchiolitis could occur throughout the year, and the peak incidence was in winter. The most common pathogen of bronchiolitis was RSV (534/1092, 48.9%), and the peak incidence was in December. MP was detected in 159/1,092 (14.6%) children with bronchiolitis, and the peak incidence was in September. Differing from the above two pathogens, the peak incidence of B. pertussis infection was in May, with a total of 10 (19.2%, 10/52) patients reported, and no patients were infected in October and December.

Overall, one or more respiratory pathogens including virus and MP were detected in 1,057 of 1,092 patients. The most commonly detected pathogens in patients with bronchiolitis were as follows: RSV (48.9%), HRV (25.9%), HMPV (13.0%), MP (14.6%), HBoV (12.1%), B. pertussis (7.1%), PIV III (7.0%), ADV (1.1%), and PIV I (1.1%).

Of the 78 B. pertussis–infected patients, B. pertussis was the sole pathogen detected in 33 (42.3%) patients. The remaining 45 patients (57.7%) were coinfected with other respiratory pathogens, most frequently with HRV (n = 28, 35.9%), followed by MP (n = 9, 11.4%), HBoV (n = 6, 7.7%), PIV III (n = 4, 5.1%), RSV (n = 3, 3.9%), IV-A (n = 3, 3.9%), and HMPV (n = 2, 2.6%) (Figure 3).

In the present study, 33 patients with B. pertussis–only infection, 438 patients with RSV-only infection, 87 patients with MP-only infection, and 534 patients infected with other pathogens were analyzed. In unadjusted comparisons, children with B. pertussis–only infection were similar to children with RSV-only infection in age, but the number of children with age ≤3 months who were only infected with pertussis was less than that of children with RSV-only infection (Table 2). Children with B. pertussis–only infection were significantly more likely to have vomiting (36.4%), cyanosis (12.1%), leukocyte count >15 × 10⁹ (57.6%), longer duration of symptoms before admission (media day, 14.0), and longer hospital stay (media day, 9.0) compared with those with RSV-only infection (17.8%, 2.7%, 7.5%, media 5.0, and 8.0 days, respectively; p < 0.05 for all comparisons). Patients with B. pertussis–only infection requiring supplement oxygen were fewer than patients with RSV-only infection (6.1 vs. 34.9%; p < 0.05).

In unadjusted comparisons, among 120 patients with B. pertussis and MP infections excluding co-detection with other pathogen types, children with B. pertussis–only infection were younger than children with MP-only infection (median 3.9 vs. 5.8 months, respectively) (Table 2). Children with B. pertussis–only infection were significantly more likely to have dyspnea (6.1%), rhinorrhea (15%), vomiting (15%), cyanosis (4%), and leukocyte count >15 × 10⁹/L (57.6%) compared with those with MP-only infection (0.0, 24.1, 10.3, 0.0, and 6.9%, respectively; p < 0.05 for all comparisons). Children with B. pertussis–only infection had a higher number of leukocyte and higher percentage of lymphocyte compared with children with MP-only infection. Children with B. pertussis–only infection had a longer duration of hospital stay (median 9.0 days) than those with MP-only infection (median 8.0 days); however, no significant difference was observed in the duration of symptoms before admission.

B. pertussis–positive patients were divided into three age groups to assess the difference among different age groups (Table 3). A total of 31 patients aged ≤3 months, 36 patients aged 4–11 months, and 11 patients aged ≥12 months were analyzed. Patients aged ≤3 months had a longer duration of hospital stay than others (p < 0.05). The common clinic characteristics among the 78 confirmed patients were paroxysmal cough 92.3% (72/78), whoops 15.5% (12/78), post-tussive vomiting 38.5% (30/78), and cyanosis 12.8% (10/78). Patients with cyanosis aged ≤3 months were more compared with older ones (p < 0.05); the others exhibited no difference among three age groups (p > 0.05). Patients aged ≤3 months requiring supplemental oxygen were more compared with older ones (p < 0.05). The gender ratio exhibited no significant difference among the three groups (p < 0.05). Coinfection among the three age groups was also compared, which showed no difference (p > 0.05). Although, patients aged ≥12 months had a higher number of leukocytes, and higher percentages of neutrophils and lymphocytes, no significant difference was observed among the three groups (p > 0.05).

Patients with B. pertussis–only infection were younger and had a high incidence of paroxysmal cough compared with patients with coinfection (p < 0.05). However, patients with coinfection had an increased demand for oxygen and showed more crackles in lungs (p < 0.05) (Table 4).