Comparison of viral and epidemiological profiles of hospitalized children with severe acute respiratory infection in Beijing and Shanghai, China

The study protocol was approved by the hospitals’ Ethics Committees and the Chinese Center for Disease Control and Prevention. Participants received a document entitled “Written Informed Consent” describing the study’s purpose and their right to keep information confidential. Written consent was obtained from all of the participants or their guardians.

SARI surveillance was conducted in Beijing Children’s Hospital in Beijing and the Children’s Hospital of Fudan University in Shanghai, China between May 2008 and March 2014. All of the patients included in the study were younger than 14 years of age and were diagnosed with SARI according to the World Health Organization (WHO) case definition of a history of symptoms for ≤72 h [4, 5, 15]. Eligibility and classification of the clinical syndromes of SARI were determined from each individual’s original record of medical history and examination. The criteria for inclusion of hospitalized patients in this study were: sudden onset of fever > 38 °C and cough or sore throat and difficulty breathing (dyspnea, oxygen saturation < 90%). To reduce or avoid the inclusion of bacterial causes in children with SARI, additional criteria were a normal or low leukocyte count, or indrawing of the lower chest wall.

We collected 700 nasopharyngeal aspirates (NPAs) between May 2008 and March 2014. There were 259 NPAs from inpatients admitted to Beijing Children’s Hospital, the largest pediatric hospital in northern China, and 441 from children admitted to the Children’s Hospital of Fudan University, the largest pediatric hospital in southeast China. It should be noted that several of the SARI patients in our study resided in areas around Beijing and Shanghai. Informed consent was obtained from the parents of all of the participants before samples were collected. NPAs were collected on the day of admission, placed in a viral transport medium, and stored at − 70 °C prior to analysis. Basic demographic and clinical data were obtained from a questionnaire completed on admission.

Nucleic acid was extracted from the samples using QIAamp MiniElute Virus Spin kits (Qiagen, Mississauga, Ontario, Canada) following the manufacturer’s protocol. cDNA was synthesized from 10 μL RNA eluted using the Promega Reverse Transcription System with random hexamer primers and avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI, USA) as described previously [15, 16].

All of the specimens were screened for Flu A/B, PIV1–3, RSV, PIC (RV/EV), and ADV using three validated multiple-nested polymerase chain reaction (PCR) assays [16, 17]. The assays were performed with two rounds under the following conditions. The first round consisted of 94 °C for 5 min; then 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, 35 cycles; and then 72 °C for 5 min. The second round consisted of 94 °C for 5 min; then 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 1 min, 25 cycles; and then 72 °C for 5 min. The HCoV (including HCoV-OC43, HCoV-229E, HCoV-NL63, and HCoV-HKU1) and HMPV were detected by nested one-step RT-PCR, as described previously [15‐17]. The nested one-step RT-PCR was performed in two rounds as follows. The first round consisted of 50 °C for 30 min and then 95 °C for 15 min; then 94 °C for 40 s, 52 °C for 40 s, and 72 °C for 40 s for 35 cycles; and then 72 °C for 5 min. The second round consisted of 94 °C for 5 min; then 94 °C for 40 s, 55 °C for 40 s, and 72 °C for 40 s for 25 cycles; and then 72 °C for 5 min. HBoV was detected using a nested PCR method as described previously [6]. The nested PCR programs were performed in two cycles as follows. The first round consisted of 94 °C for 5 min; then 94 °C for 45 s, 55 °C for 45 s, and 72 °C for 1 min for 35 cycles; and then 72 °C for 5 min. The second round consisted of 94 °C for 5 min; 94 °C for 45 s, 55 °C for 45 s, and 72 °C for 1 min for 25 cycles; and then 72 °C for 5 min. All of the detection assays were validated and optimized to ensure reproducibility, specificity, and sensitivity. Furthermore, all of the positive PCR products were confirmed by sequencing.

The study included 700 NPAs from pediatric cases diagnosed with SARI: 259 from the Beijing Pediatric Research Institute of the Affiliated Beijing Children’s Hospital, Capital Medical University (age range, 1 month to 6 years 2 months), and 441 specimens from the Children’s Hospital of Fudan University in Shanghai (age range, 10 days to 14 years). The male-to-female ratio did not differ between hospital samples. We noticed that the difference in the gender ratio (M/F) which is in the range of 1.5–1.8:1.for two groups,the reason need to be clarified on why there are more infected males than females for respiratory infections. The patients were divided into five groups according to age: 0 to 6 months (M), 7 M to 1 year (Y), 1 to 2 Y, 3 to 5 Y, and more than 5 Y (> 5 Y). The mean and median ages in months of the Beijing group were younger than those in the Shanghai group, with more cases in Beijing in the 0–1 Y group and fewer cases in Beijing in the >5Y group. The pediatric SARI cases from Beijing and Shanghai shown different clinical manifestations. The main symptoms found in our study group were cough and fever(≥38 °C), followed by wheezing and diarrhea, runny nose and cyanosis was also recorded. The pediatric SARI cases from Beijing presented a higher frequency of bronchopneumonia and lower Pneumonia when compared to those from Shanghai. A summary of the demographic and clinical characteristics of the participants is presented in Table 1.

The viral infection profiles are shown in Table 2. Of the 700 NPAs, 547 (78.1%) tested positive for one or more viral pathogens. Single infections were found in 43.9% (307/700) of the cases, and co-infections were found in 34.3% (240/700). The percentage of cases with co-infections in the Beijing sample (47.5%) was significantly higher than that in the Shanghai group (26.5%; P = 0.0012). The most frequently detected respiratory virus was PIC (RV/EV), with a prevalence rate of 34.4% (241/700), followed by RSV and HBoV (28.3 and 19.1%, respectively). HCoV was detected in 75 patients (10.7%), ADV was detected in 96 (13.7%), PIV1–3 was detected in 55 (7.9%), Flu A/B was detected in 62 (8.9%), and HMPV was detected in 35 patients (5.0%). The comparison of the respiratory virus detection rates in Beijing and Shanghai revealed that the prevalence of RSV, HCoV, and HMPV were higher in Beijing than in Shanghai.

The distributions of viral infections in Beijing and Shanghai are shown in Fig. 1 according to age groups. All of the viruses were detected in all age groups; however, the prevalence in Beijing and Shanghai differed according to age group. The respiratory virus infection rate in children younger than 1 year old was higher in Beijing than in Shanghai (P <  0.05). All of the age-matched distribution patterns for RSV in Beijing were higher than those in Shanghai, and most age-matched distribution patterns in Beijing were higher than those in Shanghai for both HCoV (except for the > 5 Y group) and HMPV (except for the 1–2 Y group). However, the prevalence of PIV 1–3 in Shanghai for the 7 M–1 Y group was significantly higher than that in Beijing. In addition, the peak of individual viral infection differed between Beijing and Shanghai: Flu A/B peaked in the > 5 Y group in Beijing. The ADV infections peaked in the 1–2 Y group in Beijing and in the 7 M–1Y group in Shanghai. The HMPV infections peaked in the 0–6 M group in Beijing and in the > 5 Y group in Shanghai. Moreover, RV/EV, HBoV, and PIV1–3 peaked in the 7 M–1 Y group in Shanghai.

Comparisons of the seasonal patterns of respiratory viral pathogens in Beijing and Shanghai are shown in Fig. 2. PIC (RV/EV), HBoV, HCoV, and ADV caused infections throughout the year; thus, a seasonal distribution was not apparent in Beijing or Shanghai. RSV peaked in the spring and winter, and Flu A/B and HMPV peaked in the winter in both Beijing and Shanghai. PIV1–3 occurred mainly in the spring and summer in Beijing, whereas this virus was most prevalent in summer and autumn in Shanghai. Interestingly, no SARI cases linked to HMPV infection were detected in the summer in Beijing or Shanghai.