Clinical and epidemiological profiles including meteorological factors of low respiratory tract infection due to human rhinovirus in hospitalized children

From January 2013 to December 2015, children < 14 years old with LRTIs admitted to Department of Respiratory Disease in Children’s Hospital of Soochow University were prospectively enrolled in this study. After completing a full clinical record, nasopharyngeal aspirate (NPA) was performed on each child enrolled as described previously [16] and stored at 4 °C until sent to the clinical laboratory for further direct immunofluorescence assay and polymerase chain reactions (PCR) analysis. Pending the molecular diagnostic, all samples were stored at −80 °C after DNA or RNA extraction. LRTI (bronchiolitis, bronchitis, pneumonia and asthma exacerbation) was defined by the presence of signs and symptoms of an acute respiratory infection (cough, nasal discharge, with or without fever), lower respiratory signs (tachypnea, retractions, prolonged expiratory time, or crackles/wheezing on auscultation) and radiologic evidence of LRTI. Chest radiography was performed using standard equipment and radiographic techniques, and reviewed by the radiologists in digital format. Generally speaking, bronchiolitis presents bilateral hyperinflation with variable, patchy areas of atelectasis. Pneumonia presents increased shadowing or consolidation while increased lung markings shows in bronchitis or asthma exacerbation. Children with a history of chronic lung disease, underlying immunodeficiency states, or preexisting cardiac, renal, neurologic, or hepatic dysfunction, or bronchopulmonary malformation were excluded from the study. This study was conducted with the approval of the Institutional Human Ethical Committee of Children’s Hospital of Soochow University. An informed consent was obtained from all the subjects or guardians who participated in this study.

In present study, a sensitive quantitative PCR method targeted a 210-bp region from the conserved 5’ untranslated region (5’ UTR) was used to screen for HRV modified from a previous study [17]. The primers and probe sequences were HRV-F: 5’-TGGACAGGGTGTGAAGAGC -3’;HRV-R:5’-CAAAGTAGTCGGTCCCATCC-3’ ; HRV-probe: FAM-TCCTCCGGCCCCTGA ATG-TAMRA. Nucleic acids were extracted from NPA samples with TRIzol reagent (Life Technologies, Carlsbad, USA) following the protocol provided by the manufacturer. A final 200 μl of RNA was eluted and reverse transcription reactions were performed with M-MLV reverse transcriptase (Promega, Madison, USA) and random hexamers (Sangon Biotech, Shanghai, China) for cDNA synthesized at 37 °C for 60 min according to the manufacturer’s specifications. PCRs were performed in a volume of 25 μl containing 3 μl of sample DNA, 0.25 μl of 5 U/μl TaqMan (Promega, Madison, USA), 75% DEPC treated water 14.75 μl, buffer solution 2.5 μl, 25 mM MgSO₄ 2 μl, 10 mM dNTP 1 μl, forward, reverse primers (1.5 μmol/L) and probe (0.2 μmol/L) each 0.5 μl. The PCR conditions were as follows: reverse transcription, 30 min at 50 °C; polymerase activation, 15 min at 95 °C; and 45 cycles of 30 s at 95 °C and 60 s at 60 °C. The concentration of each detected sample was then calculated automatically according to standard curve.

From January 2014 to December 2014, positive HRV samples were used to determine HRV-C strain. According to analysis of HRV VP4/VP2 coding region sequences from GenBank, the conserved sequence (8–37 amino acid) from VP2 was confirmed to differentiate HRV-C from HRV-A and -B after comparison of 52 newly identified HRV-C subtypes using a phylogenetic tree. The degenerate primers were designed according to the conserved sequence. The degenerate primers sequences were HRV-F: 5’-GGNWWBTCYGATAGGCTHAA-3’;HRV-R:5’-TANBBDGGCCAYTCHCCRTANGC-3’(N = A,T, C or G, W = A or T, B = G, T or C, Y = C or T, H = A, T or C, D = G, A or T, R = A or G,V = G, A or C). Compared to positive control sample of HRV-51 strain, the optimal concentration of primers and melting temperature were obtained. Then all the screened HRV positive samples were amplified by real time PCR and HRV-C subtypes could be confirmed by comparing to high-resolution melting curve of HRV-C51.

Samples were equally analyzed for seven common viruses, including RSV, IV-A and IV-B, parainfluenza viruses 1, 2, 3 (PIV-1, 2, 3), and adenovirus (ADV) using direct immunofluorescence (DFA) with virus-specific fluorescence-labeled monoclonal antibodies (Diagnostic Hybrids, Athens, USA) and ultraviolet light microscopy. Human metapneumovirus (hMPV) were detected by reverse transcription PCR whereas human bocavirus (HBoV) and Mycoplasma pneumonia (MP) were detected by real time PCR respectively as previously described [14]. Briefly, for hMPV detection, primers were designed to specifically amplify the N gene (213 bps). The forward and reverse primers were hMPV-F: 5’-AACCGTGTACTAAGTGATGCACTC-3’ and hMPV-R: 5’-CATTGTTTGACCGGCCCCATAA-3’, respectively. For HBoV VP1 gene detection, the primers and probe sequences were HBoV-F: 5’-TGACATTCAACTACCAACAACCTG-3’;HBoV-R:5’-CAGATCCTTTTCCTCCTCCAATAC-3’ ;HBoV-probe: FAM-AGCACCACAAAACACCTCAGGGG-TAMRA. As for MP detection, another fluorescent real-time PCR was performed to identify the 16S rRNA gene of MP. The primers and probe sequences were MP-F: 5’-GCAAGGGTTCGTTATTTG-3’ and MP-R: 5’-CGCCTGCGCTTGCTTTAC-3’, and MP-probe: FAM-AGGTAATGGCTAGAGTTTGACTG-TAMRA. MP and HBoV positive samples were defined with a concentration of DNA >2.5 × 10³copies/ml to exclude the MP or HBoV colonization.

Meteorological data for Suzhou, including daily mean temperature (°C), mean relative humidity (%), total month rainfall (mm), sum of sunshine (h), and mean wind velocity (m/s), were obtained from Suzhou Weather Bureau at longitude 120°6’ east and latitude 31°3’ north, which is located 8 km away from the hospital. Meteorological data were obtained hourly, and average daily values were calculated. Monthly means were calculated using the daily means for temperature, relative humidity, and wind velocity. Total rainfall and hours of sunshine were calculated as a total measurement for the month.

The continuous variables were compared using the Student t test or Mann–Whitney U test if the data were abnormal in distribution. Categorical data were analyzed using the Mentel-Haenszel or chi-squared (χ2). Correlations of incidence of HRV with meteorological factors were evaluated using Pearson’s or Spearman rank correlation. Because of colinearity between meteorological factors, associations between meteorological factors and HRV incidence were also analyzed using Linear Regression. P < 0.05 was considered to be statistically significant.

A total of 6196 NPA samples were available and 276 children were not enrolled because of a refusal to participate from their parent or presenting underlying disease. Of 6196 NPA samples, 3882 (62.7%) were positive for at least one pathogen and the most commonly identified pathogen was Mycoplasma pneumonia (MP) (22.1%, 1369/6196), followed by RSV (12.0%, 744/6196), HRV (11.4%, 707/6196), human bocavirus (HBoV) (5.7%, 354/6196), PIV-3 (5.1%, 315/6196), human metapneumovirus (hMPV) (3.7%, 230/6196), adenovirus (ADV) (1.1%, 69/6196), parainfluenza virus 1 (PIV-1) (0.9%, 55/6196), IV-A (0.5%, 32/6196), and parainfluenza virus 2 (PIV-2) (0.1%, 7/6196). Among 707 HRV-positive cases, 467 (66.1%) were infected only with HRV and 240 (33.9%) were co-infected with other pathogens. The most commonly co-detected pathogen with HRV was Mycoplasma pneumoniae, followed by HBoV and RSV (Table 1). Of all the 228 HRVs cases in 2014, HRV-C was detected in 85 cases and accounting for 37.3%.

Of the 707 HRV positive cases, the mean age was 23.2 months (range 1 to 140 months) and the ratio of male to female was 2.3:1 and slightly higher than 1.7:1 of non-HRV infected cases (P = 0.052). HRV affected mostly children aged 13–36 months with an incidence of 15.1% (203/1341). The HRV incidence in different age groups was as follows: 1–6 months (9.3%, 192/2065), 7–12 months (13.3%, 181/1357, 13–36 months (15.1%, 203/1341), 37–60 months (9.8%, 67/682), and > 60 months (8.5%, 64/749) and HRV incidence was significantly different between groups (χ2 = 40.31, P < 0.0001) as shown in Fig. 1. The demographic and clinical characteristics of cases with single HRV infection and co-infection are summarized and compared in Table 2. Children with co-infection presented more frequent fever, tachypnea and longer hospital stay compared to children infected with HRVs only (all P < 0.05). However, there was no difference of clinical characteristics between HRV-C infection and HRV-A/B infection (data not shown).

As shown in Fig. 2, HRVs infection occurred throughout the year during the study period, although a higher incidence was observed in summer and autumn seasons, especially in June 2015 (20.7%, 28/135). The HRV incidence was 14.7% (206/1400) in autumn, followed by 13.3% (203/1532) in summer, 11.8% (190/1612) in spring, and 6.5% (108/1652) in winter. The similar seasonality of HRV-C was shown in Fig. 3. The Suzhou area has a subtropical climate. The monthly mean temperature was 18.2 ± 9.2 (mean ± standard deviation) °C, relative humidity was 69.7 ± 7.4%, total rainfall was 100.5 ± 73.9 mm, sum of sunshine was 181.1 ± 65.8 h, and wind velocity was 1.8 ± 0.5 m/s. The monthly mean data for these meteorological variables over the course of this study are shown in Fig. 2. The associations of total HRVs, HRVs with or without coinfection or HRV-C activity with meteorological factors were performed using Spearman correlations and multivariate regression analysis. Total HRVs, HRVs without coinfection and HRV-C incidence was associated with mean temperature (all P < 0.05). No correlation was found between total HRVs, HRVs without coinfection, HRV-C incidence and other meteorological factors by multivariate regression analysis (Table 3). The incidence of HRVs with coinfection was not associated with all meteorological factors (all P > 0.05).