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

Background The incidence of severe acute respiratory tract infections in children caused by Mycoplasma pneumoniae (syn. Schizoplasma pneumoniae) and Chlamydophila pneumoniae (formerly Chlamydia pneumoniae) varies greatly from year to year and place to place around the world. This study investigated the epidemiology of M. pneumoniae and C. pneumoniae infections among children hospitalized with acute respiratory infections in Suzhou, China in the year 2006, and associations between incidence rates and climatic conditions. Methods Nasopharyngeal aspirates obtained from 1598 patients (aged 26.4 ± 28.3 months; range, 1 month to 13 years) were analyzed with real-time PCR and ELISA. Meteorological data were obtained from the weather bureau. Results About 18.5% of patients were infected with M. pneumoniae and, C. pneumoniae, or both. Isolated M. pneumoniae infection was positively correlated with increasing age (χ2 = 34.76, P < 0.0001). Incidence of M. pneumoniae infection was seasonal with a peak in summer (P < 0.0001) and minimum in winter (P = 0.0001), whereas C. pneumoniae infection was low only in autumn (P = 0.02). Monthly mean temperature was strongly correlated with the incidence of M. pneumoniae infection (r = 0.825, P = 0.001). Conclusions M. pneumoniae and C. pneumoniae are important infectious agents in hospitalized children with acute respiratory tract infections. M. pneumoniae infection showed a strong direct correlation with environmental temperature.


Introduction
The atypical pathogens Mycoplasma pneumoniae (syn. Schizoplasma pneumoniae), Chlamydophila pneumoniae (Chlamydia pneumoniae), and Legionella pneumophila cause mild, moderate, or severe acute respiratory tract infections (ARTIs) in children, although Legionella pneumophila infection is more common in adults. These infections occur worldwide [1][2][3]. Cyclical outbreaks of M. pneumoniae infections can be expected on average every 3-7 years, but at any given time may account for as many as 40% of community-acquired pneumonia cases [3]. The prevalence of C. pneumoniae in children with ARTIs varies from 0 to 44% [4].
Studies of possible associations between the epidemiology of atypical pathogens and meteorological conditions (e.g., temperature, humidity, rainfall, amount of solar radiation and wind velocity) are few. However, it was recently reported that the community incidence of pneumonia due to M. pneumoniae increased weekly by 16.9% for every 1°C increase in the average temperature, and by 4.1% for every 1% increase in relative humidity [5]. Community-acquired M. pneumoniae or C. pneumoniae infections affect mainly preschool-and school-aged children and young adults. Few studies have reported the frequency of M. pneumoniae and C. pneumoniae infections in infants [6].
Clinically it is difficult to distinguish M. pneumoniae from C. pneumoniae infections and hence laboratory tests are essential in identifying these pathogens. Serological detections, although commonly used, are complicated by false negative results in the early acute phase of infection, and the difficulty in obtaining convalescent serum during hospital stays of one week or less. Polymerase chain reaction (PCR) is suitable for rapid diagnosis of these infections, even when the colonization rate is only 1-2% of the population [7][8][9]. Combining both PCR and serology seems to be a more reliable diagnostic approach.
The incidence rate of childhood ARTIs due to these pathogens is very different from one country to another [2][3][4][5][6]. Our purpose was to use PCR to identify and determine the percentage of ARTIs that were due to M. pneumoniae and C. pneumoniae, especially in infants, and investigate the relatedness between their epidemiology and meteorological conditions in Suzhou, Jiangsu Province, China. In addition we compared the clinical characteristics of M. pneumoniae and C. pneumoniae infections, host immune state, radiographic appearance, and tidal breathing measurements.

Study population
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.

Sample collection
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.

DNA extraction
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.

Detection of M. pneumoniae gene by real-time PCR
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.

Detection of C. pneumoniae gene by nested-PCR
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.
Serology testing for M. pneumoniae and C. pneumoniae 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.
Diagnostic criteria for M. pneumoniae and C. pneumoniae acute infection 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.

Tidal breathing measurements
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.

Meteorological data
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.

Statistical analyses
Categorical data were analyzed using the Cochran-Mantel-Haenszel statistic or the chi-squared (χ 2 ) 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).
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  (Table 2).

Seasonality and correlations with meteorological conditions
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.

Clinical manifestations
There was no significant difference in terms of clinical manifestations among the groups except for fever   (χ 2 = 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.

Laboratory findings
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).  [18] reported an epidemic of M. pneumoniae infection in Hong Kong with a rate of 15% to 20% that occurred from 1986 to 1988, which was similar to our study.

Discussion
Apparent seasonality of M. pneumoniae infection was observed in this study, with a peak level in summer and the highest rate in July. When the disease is endemic, seasonality may not be a factor, but when it is an epidemic more cases occur in the summer or early autumn [3,16]. We observed a similar trend in this study. Onozuka et al. [5] found a strong correlation between temperature and relative humidity and the number of M. pneumoniae   pneumonia cases in hospitalized children younger than 15 years in Japan, while another study in Germany did not show any correlation [19]. However, we presume that climatic conditions in our area are similar to Japan, and different from the northwestern region of Germany where the range of temperatures across the seasons is not as great.
Regarding C. pneumoniae infections, the 6.0% rate in children with ARTIs found in the present study is similar to the 6.7% (4/112) found in a study performed by Kurz et al. [20] in Vienna, Austria, and lower than the 9.3% reported by Schmidt et al. [21] for Greifswald, Germany. No seasonality or correlation with climatic conditions was found in our study. Studies conducted in various countries have shown that M. pneumoniae and C. pneumoniae infections are most common in schoolaged children, followed by children from 1 to 5 years old, but rare in infants [16,[21][22][23][24]. In contrast, we found that M. pneumoniae (10.8%) and C. pneumoniae (5.9%) infections are also common in infants. The high population density in China, which increases exposure and the chance of infection, may be an important influence in acquiring these infections.
The use of PCR in the present study provided rapid and specific diagnosis of M. pneumoniae and C. pneumoniae infections. A recent retrospective study comparing the diagnostic value of PCR to the indirect particle agglutination antibody test for M. pneumoniae infection in children from NPA samples found that PCR provided a more rapid diagnosis, particularly in young children (P = 0.003), immunocompromised patients (P = 0.019), and during the early stage of the disease [25]. When PCR is combined with serological tests the yield increases further [26].
In the present study, the clinical manifestations and laboratory findings did not differ between M. pneumoniae and C. pneumoniae. Cough and fever were the most common symptoms. Fever was noticed more frequently in children with co-infection, although co-infection did not increase the severity of clinical manifestations. The rate of wheezing in M. pneumoniae or C. pneumoniae infections was higher than that reported by Esposito et al. [27], and the rates of dyspnea and tachypnea were lower. The reason for this may be that the present study included upper respiratory infection, bronchitis, and asthma exacerbation within the definition of ARTIs. *Chest X-rays and tidal lung function were only performed on children with LRTI. CD cluster of differentiation, LRTI lower respiratory tract infection.
The 8.8% (26/295) incidence of M. pneumoniae or C. pneumoniae infections with asthma exacerbation in this study indicates a possible role of infection by these atypical pathogens in asthma exacerbation. Recently, it was reported that in asthma M. pneumoniae and C. pneumoniae appeared to be involved more with asthma persistence than exacerbation [28].
In previous studies, no significant difference was found between M. pneumoniae and C. pneumoniae infections with regard to white blood cell count, C-reactive protein, percentage of neutrophils, platelets, or radiographic data [16,27,29]. To the best of our knowledge, this study is the first to analyze the humoral and cell immunity status and tidal lung function of children with M. pneumoniae and C. pneumoniae infections. Although no significant difference in immunity status existed between the patients infected with the two pathogens, some studies showed a depressed humoral and cellular immunity in children with M. pneumoniae infection compared to healthy children [30,31]. Because of the difficulty in distinguishing M. pneumoniae infection from C. pneumoniae on the basis of clinical manifestations or laboratory findings, PCR tests are very useful [32].
The limitations of our study include a probable bias, as the study was conducted only in hospitalized children and not outpatients. In addition, a study period of only one year is relatively short to assess the epidemiology of M. pneumoniae and C. pneumoniae infection.

Conclusion
M. pneumoniae infections in children demonstrated a definite seasonality, a strong correlation with the temperature, and a weak correlation with wind velocity. Consistent with former studies, ours indicates that M. pneumoniae and C. pneumoniae are important in children with ARTIs who are younger than 5 years old, and especially in infants. Clinical manifestations and laboratory findings were informative, but real-time PCR and nested-PCR provide adequate rapid and specific diagnosis of M. pneumoniae and C. pneumoniae infections.