Epidemiology and associations with climatic conditions of Mycoplasma pneumoniae and Chlamydophila pneumoniae infections among Chinese children hospitalized with acute respiratory infections

From 1 January 2006 to 31 December 2006, 1598 consecutive children with ARTIs admitted to Children’s Hospital affiliated with Soochow University were enrolled and evaluated prospectively. These children were hospitalized because of prolonged fever (>3 d), severe symptoms of cough, wheeze, tachypnea, and chest retractions. The clinical outcome and diagnosis for all children were obtained after discharge from the hospital. The discharge diagnosis was based on standard clinical criteria made by attending physicians. Upper respiratory tract infection was diagnosed if a child had nasal obstruction, nasal discharge, fever, or sore throat. Lower respiratory tract infection was diagnosed when wheeze, tachypnea, chest retractions, abnormal auscultatory findings, and radiologic evidence of a lower respiratory tract infection were present. Children were excluded from the study if they had proven chronic lung disease, immunodeficiency, congenital heart disease, or bronchopulmonary dysplasia. The Institutional Review Boards of Suzhou University approved the study protocol, and the parents or legal guardians of each child gave informed written consent.

Nasopharyngeal aspirate (NPA) samples were obtained from each patient within 24 hours of admission using a sterile plastic catheter introduced into the lower part of the pharynx via the nasal cavity. The samples were immediately transported to the Laboratory of Molecular Biology of our hospital for detection of M. pneumoniae and C. pneumoniae using PCR. Seven common respiratory virus (respiratory syncytial virus, influenza A and B, parainfluenza 1, 2, 3 and adenovirus ) using direct immunofluorescence and human metapneumovirus using RT-PCR described previously [10]. Blood samples were also obtained at admission and immediately sent to the Department of Biochemical Laboratory for routine blood, C-reactive protein, humoral and cell immunity, and alanine transaminase tests.

Each NPA sample was diluted in 2 mL of normal saline before centrifugation at 500 × g for 10 minutes. The resultant cell pellet was resuspended and then centrifuged at 12 000 × g for 5 minutes, followed by extraction of DNA from a 400-μL sample using DNA-EZ Reagents (Sangon Biotech, USA) in accordance with the manufacturer’s instructions. A final 200 μL of DNA was eluted and divided into 2 aliquots for PCR and stored at −20°C.

Real-time PCR was performed to identify the P1 adhesion protein gene of M. pneumoniae, as described previously [11]. The forward and reverse primers were 5’-CCA ACC AAA CAA CAA CGT TCA-3’ and 5’-ACC TTG ACT GGA GGC CGT TA-3’, respectively, and the probe sequence was 5’-TCA ACT CGA ATA ACG GTG ACT TCT TAC CAC TG-3’. The fluorescent reporter dye at the 5’ end was 6-carboxyfluorescein (FAM) and the quencher at the 3’ end was 6-carboxytetramethylrhodamine (TAMRA). The PCR reactions consisted of a 21-μL PCR mast mixture (Shenyou Biotechnology, Shanghai, China) including primers and probes combined with 3 μL of the sample DNA and 1 U GoTaq DNA Polymerase (Promgea, Wisconsin, USA). Real-time PCR was performed using an iCycler iQ5 real-time PCR detection system (Bio-Rad, Hercules, CA, USA) and cycling conditions were: 2 min at 37°C; 10 min at 94°C, and 40 cycles of 10 s at 94°C, 30 s at 55°C, and 40 s at 72°C.

Quantification curves were plotted using several concentrations of control plasmids containing the target gene.

A different aliquot of extracted DNA from the NPA sample was used for detecting the gene of the major outer membrane protein of C. pneumoniae by nested touchdown PCR, as described previously [12]. The external and internal primers were: Cpex-F 5’-TTA CAA GCC TTG CCT GTA GG-3’; Cpex-R 5’-GCG ATC CCA AAT GTT TAA GGC-3’; Cpin-F 5’-TTA TTA ATT GAT GGT ACA AT A-3’; and Cpin-R 5’ATC TAC GGC AGT AGT ATA GTT-3’. Amplifications were performed in a thermal cycler (GeneAmp PCR System 9600, Applied Biosystems). For the first amplification, 0.4 μM of each CPex-F and Cpex-R primer and 2 U GoTaq DNA polymerase (Promgea, Madison, WI, USA) were included in these touchdown PCR reactions. The annealing temperature was lowered 1°C every 2 cycles, from 65°C until touching down at 55°C, at which temperature 20 more cycles were performed. The denaturation and extension temperature were constant at 94°C and 72°C, respectively. One microliter of the products of the first round of amplification by external primers were added into the PCR reactions of the second round amplification using internal primers. The second PCR cycling conditions were: 2 min at 95°C; and 30 cycles of 1 min at 94°C, 1 min at 50°C, and 1 min at 72°C. The nested PCR products were separated via 1.5% agarose gel electrophoresis and visualized using ethidium bromide staining.

The presence of specific IgM and IgG antibodies against M. pneumoniae were investigated in serum samples of patients using a commercial ELISA kit (Serion ELISA classic M. pneumoniae IgG/IgM, Institute Virion\Serion, Germany). IgA and IgG antibodies against C. pneumoniae were detected with Serion ELISA classic C. pneumoniae IgA/IgG Kits. Evidence of acute M. pneumoniae infection was defined as either a single positive serum IgM (cutoff 13 U/mL) or a 4-fold increase in IgG in convalescent serum obtained a week after admission. Acute C. pneumoniae infection was defined as either a single positive serum IgA (cutoff 3 U/mL) or a 4-fold increase in IgG in convalescent serum.

Acute infection due to M. pneumoniae or C. pneumoniae was confirmed when any of the following was determined: NPA samples proved positive via PCR, serum samples positive for IgM or IgA, respectively, or IgG increasing 4-fold from the acute to the convalescent phases.

Measurements of tidal breathing in most infants were performed during natural and quiet sleep as assessed by behavioral criteria [13]. Tidal breathing flow-volume loops were obtained and analyzed with a commercially available pediatric pulmonary function device (ECO Medics, V’max 26, Switzerland). Tidal breathing measurements included flow and volume signals, and were assessed by professional staff. Lung function was graded as normal, mild, moderate, severe, or extremely severe according to three-component score system which could reflect underlying obstructive airways disease [14, 15] as shown in Table 1.

The primary investigator obtained data regarding monthly mean temperatures, relative humidity, rainfall, solar radiation, and mean wind velocity from the local weather bureau for lat 31.19° N, long 120.37° E. Suzhou has a subtropical climate and the monthly data for meteorological variables were shown in Figure 1.

Categorical data were analyzed using the Cochran-Mantel-Haenszel statistic or the chi-squared (χ²) or Fisher’s exact tests. The continuous variables were compared using analysis of variance (ANOVA). The Kruskal-Wallace test was used if the data were abnormal in distribution or nonparametric. The associations between meteorological conditions and the prevalence of the 2 pathogens were evaluated using Spearman’s rank correlations. A P-value < 0.05 was considered statistically significant. All analyses were performed using the Statistical Package for SAS for windows, version 8.2 (SAS, USA).

Of the 1598 children admitted to our hospital for ARTIs, 957 (59.9%) were boys, and the ratio of boys to girls was 1.5:1. The mean age was 26.4 ± 28.3 months (range: 1 month to 13 years). Of all the children, 817 (51.1%) were aged < one year, 616 (38.5%) were 1–5 years, and 165 (10.3%) were >5 years old.

Twenty-three cases of M. pneumoniae infection and 6 cases of C. pneumoniae infection had co-infections with other viruses (data not shown), and cases of M. pneumoniae or C. pneumoniae coinfection with viruses were excluded in this study. ARTIs were caused by M. pneumoniae or C. pneumoniae in 295 (18.5%) of the patients. The mean age of these children was 33.1 ± 36.3 months. There were 171 boys, and the ratio of boys to girls was 1.4:1. Of the 295 infected with either or both of these pathogens, 199 (12.5%) were due to M. pneumoniae alone (mean age 35.8 ± 37.3 months), 81 (5.1%) were due to C. pneumoniae alone (mean age 27.3 ± 34.5 months), and 15 (5.1%) were co-infected (mean age 28.0 ± 29.7 months). The infection rate due to M. pneumoniae only was significantly and positively associated with increasing age (χ² = 34.76, P < 0.0001). With regard to demographic data, no significant difference in gender or prematurity was found among the 3 etiological groups (Table 2).

Both M. pneumoniae and C. pneumoniae infections occurred throughout the year. The M. pneumoniae infection rate reached a maximum in summer (P < 0.0001) and a minimum in winter (P = 0.0001), whereas the C. pneumoniae infection rate was lowest in autumn (P = 0.02; Table 3). The peak number of M. pneumoniae infections occurred in July (23.2% or 32/138) and were the lowest (4.3% or 5/117) in December. C. pneumoniae infections were also high in July (12.3% or 17/138; Figure 2).

Rates of M. pneumoniae infection in hospitalized children in Suzhou correlated strongly with monthly mean temperature, and weakly with monthly mean wind velocity (Table 3). There was no significant correlation between meteorological conditions and C. pneumoniae infections.

There was no significant difference in terms of clinical manifestations among the groups except for fever (χ² = 7.824, P = 0.02; Table 4). Children co-infected with M. pneumoniae and C. pneumoniae were found to have fever more frequently. There were no significant differences in length of hospital stay among the different groups. All patients were cured or improved.

There were no significant differences in laboratory findings (routine blood, C-reactive protein level, humoral and cell immunity, alanine transaminase, radiographic data, and tidal lung function test) among the 3 groups (Table 5).