This study was a retrospective analysis of real-time PCR for diagnostic testing of M. pneumoniae infection by antigen detection. Patients hospitalized with clinically and radiologically confirmed lower respiratory tract infections (n=1429) were identified at the Children's Hospital Affiliated to Soochow University database during an outbreak of MP between January 2012 and February 2013. Both serum and NPAs were obtained from these patients. Therefore, patients without stored serum samples or results of NPAs were excluded from our study. Paired serum samples taken on admission and at least seven days after the first collection were available for 733 patients (51.3%).

Nasopharyngeal swabs were obtained within 24h of admission. The specimens were centrifuged and were stored at −80°C until tested. A quantitative diagnostic kit (DaAn Gene Co., Ltd., Guangzhou, China) for M. pneumoniae DNA was used to determine the load of MP, as previously reported.10 The method is based on the TaqMan PCR technology, and the target is 16S rRNA gene specific for MP genome. Briefly, 1mL of nasopharyngeal aspirates diluted with 4% NaOH was centrifuged at 12,000rpm for five min. The sediment was collected, washed twice with 0.9% NaCl, blended with 50μL of DNA extraction solution, incubated at 100°C for 10min, and centrifuged at 12,000rpm for five min. Real-time PCR was performed on the resulting supernatant with 2μL, and PCR mix with 43μL (supplied with the kits) using the DA 7600 real-time PCR system (Applied Biosystems, California, USA) as follows: 93°C for 2min, 10 cycles of 93°C for 45s and 55°C for 60s, followed by 30 cycles of 93°C for 30s and 55°C for 45s. All nasopharyngeal swabs were tested for antigen detection by immunofluorescence for seven common viruses (RSV, adenovirus, influenza viruses A and B, and parainfluenza viruses 1, 2 and 3).

Detection of serum M. pneumoniae-specific antibody was performed using enzyme-linked immunosorbent assay (Virion-Serion, Germany). The assay was considered positive if IgM≥1.1 or if there was ≥4-fold rise in IgG titer.8

Diagnosis of MP was based on serology or PCR findings. A significant rise in M. pneumoniae IgG titer or the presence of IgM antibodies was used as criteria for current M. pneumoniae infection. Likewise, DNA detection by real-time PCR was also considered M. pneumoniae infection.

Data compiled from a retrospective chart review of all study patients were extracted through the use of specially prepared data forms without knowledge of serologic status or PCR results. The information extracted included demographics, clinical features and laboratory data.

We used n (%) for categorical variables and median (IQR) for continuous variables with non-normal distribution or mean (SD) for those with normal distribution. We assessed differences in categorical variables with the χ2 test. We calculated 95% CI for differences in medians with an exact test. Logistic regression analysis was performed to identify clinical characteristics associated with different bacterial loads. SPSS (version 17.0) software was used for all statistical analysis.

Among 1429 patients with lower respiratory tract infections, 511 (35.8%) were diagnosed with MP based on serology or PCR findings. Of the 511 patients with MP, 304 (59.5%), the mean age was 35 months (range, one month to 156 months). The age distribution of the patients is shown in Fig. 1. The remaining 918 patients who were not diagnosed with MP did not differ significantly from patients with MP in terms of sex and age at disease onset.

Fig. 1.

Age distribution of our patients.

(0.06MB).

Of the 511 patients with MP, PCR for M. pneumoniae was positive in 330 (64.6%) nasopharyngeal samples. Genomal bacterial load in NPAs ranged from <2500 to >2.5×107copies/mL of sampled material. According to the distribution of genomal bacterial loads (Fig. 2), patients were classified into two groups: high bacterial load group (>107copies/mL) (n=165) and low bacterial load (<107copies/mL) (n=165).

Fig. 2.

Distribution of different bacterial loads in our patients.

(0.06MB).

The genomal bacterial load increased significantly with age (p<0.001). Fewer Children younger than one year of age had less high bacterial load (17.7%) than those older than five years (53.8%) [p<0.001] (Fig. 3).

Fig. 3.

Different bacterial loads individuals categorized according to age group.

(0.06MB).

Seasonal distribution of MP according to genomal bacterial load is shown in Fig. 4.

Fig. 4.

Monthly occurrence of total episodes of Mycoplasma pneumoniae pneumonia and cases with different bacterial loads during the outbreaks in 2012.

(0.07MB).

Outbreaks began in summer months and peaked during August and September (73.5% of all MP cases). The frequency of patients with high bacterial load also increased during summer and peaked in August and September. From January to March 18 (15%) MP patients had high bacterial load, compared to 52 (27.5%) from April to June, 137 (43.3%) from July to September, and 96 (38.7%) from October to December.

Clinical characteristics according to PCR status are summarized in Table 1. To assess the independent association of evaluated parameters, mean age, fever, and wheezing at presentation were tested by logistic regression analysis. Patients with high bacterial load were significantly older (p<0.01) and had significantly higher fever (p=0.01). Wheezing at presentation was associated with low bacterial load (p=0.03).

Out of 330 PCR positive children, 179 patients had paired serum samples. No difference was found in the interval between onset of symptoms and the time of obtaining the first serum sample or second serum sample. 93 (56.4%) patients with high bacterial load 46 (27.8%) had more initial positive IgM compared to patients with low bacterial load (p<0.001). Geometric mean titers of initial IgG and IgM of patients with high bacterial load were not significantly different from those of patients with low load. In patients with low bacterial load, geometric mean titers of second IgG significantly increased compared with the initial IgG in patients more than five years old. In patients with high bacterial load, geometric mean titers of second IgG significantly increased in younger patients (Fig. 5). Patients with high bacterial load also had geometric mean titers of second IgM significantly increased in younger patients compared with patients with low bacterial load.

Fig. 5.

Correlation between initial and second antibody titers among patients with different bacterial loads. Filled circles indicate geometric mean titers (GMTs) of initial antibody titers and unfilled circles indicate GMTs of second antibody titers in each age group and bars indicate 95% confidence intervals.

(0.1MB).

Co-infections were detected in 204 patients with MP (102 with virus and 102 with bacteria). Co-infection with viruses was significantly more frequent among patients with low bacterial load than those with high bacterial load (24.2% vs. 8.5%; p<0.001). Bacterial co-infections were also more frequently detected among patients with low bacterial load than in those with high bacterial load (22.4% vs. 12.1%; p=0.01).