Subjects and data collection
The study was conducted in children (<16 years) with bronchopneumonia admitted to the pediatric department of our hospital from January to December 2018.They were divided into two groups:children with MPP and children without MPP (NMPP) severed as controls. The diagnosis of MPP was based on the presence of (i) met CAP diagnostic criteria[9] issued by the American Thoracic Society and the Infectious Diseases Society of America in 2007 or has clinical symptoms and signs of pneumonia such as cough,auscultation,and lung infiltration on chest radiographs; and (ii) the antibody titer of anti-mycoplasma was 1:160 or greater, M. pneumoniae DNA was positive,or the microorganism was isolated by respiratory specimens culture. Exclusion criteria included patients with (i) recurrent respiratory tract infections ,congenital heart diseases, bronchopulmonary dysplasia,neurological disorders,hereditary metabolic diseases,pulmonary embolism, empyema,lung abscess,tuberculosis,and immunodeficiency; or (ii) history use of immunosuppressive agents, immunoglobulin and other drugs;or (iii) co-infected with other pathogens,such as typical bacterial pneumonia, viral pneumonia;or (iv) unknown clinical data. Our study was approved by the hospital’s Medical Ethics Review Committee, and informed consent was obtained from a parent or guardian of each participant.
Clinical assessment and infectious parameters
Upon admission, patients were subjected to detailed medical examination and demographic data such as name,gender,age and so on were collected. At the day of the study visit,sufficient fasting blood was collected by a forearm vein venipuncture through an aseptic technique and the specimen were immediately sent for laboratory measurements.The peripheral blood in EDTA-K2 anticoagulant tubes was used to detect white blood cell counts (WBC),C-reactive protein (CRP),procalcitonin (PCT),and that in non-anticoagulant tubes was centrifuged and the supernatant serum was collected for other biochemical examinations.
CRP levels were measured via the Ottoman 1000 automatic specific protein instant detection analyzer ( Upper Bio-tec Pharma Co., Ltd, Shanghai, China) and procalcitonin levels were assessed via electrochemiluminescence immunoassay on COBAS e601 analyzer (Roche Diagnostics, Basel, Switzerland).Other parameters,such as WBC and levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),gamma-glutamyl transferase(GGT), lactate dehydrogenase(LDH),total bilirubin(TBil),total protein(TP),albumin(ALB),prealbumin(PA),creatine kinase(CK),creatinine kinase MB isoenzyme(CK-MB), were also conducted via internationally accepted laboratory.
The specific IgM and IgG antibodies against M. pneumoniae were analyzed by passive particle agglutination using a commercial diagnostic kit (Fuji Rebio Inc., Tokyo, Japan) according to the manufacturer’s instructions.
Nasopharyngeal aspirates obtained within 24h after hospitalization were added 1 milliliter RNase free normal saline and stored at −80°C. The load of M. pneumoniae was measured by quantitative diagnostic kit (ZhiJiang Co., Ltd. Shanghai, China).Real-time fluorescence quantitative PCR was performed using a SLAN-96P detector (Hongshi Co., Ltd.,Shanghai, China) and the cycling conditions were as follows : 37 °C for 2 min, 94 °C for 2 min, followed by 40 cycles of 93 °C for 15 sec and 60 °C for 1 min.
Measurement of AGP
AGP were analyzed via immunoturbidimetric kits purchased from the Austria Dialab Company on module COBAS c702 analyzer (Roche Diagnostics, Basel, Switzerland). The methodologies had passed the performance verification, and the inspection operation, instrument maintenance, and quality control were strictly carried out in accordance with the requirements of the laboratory operating instructions.
Statistical methods
Data were expressed as (mean ±SD) or median (interquartile range (IQR)) for quantitative variables with normally or nonnormally distributed continuous variables as checked via Kolmogorov-Smirnov test,respectively. Comparisons of categorical variables between groups were analyzed by Chi-square test or Fisher’s exact test ,while differences of quantitative variables between groups were tested with Student T-test or Wilcoxon test, according to distributions. Multiple logistic regressions were used to evaluate predictors of MPP. The variables that proved significant predictors in logistic regression were studied further using receiver operating characteristic (ROC) curves’ analysis. The area under the curve (AUC) for each parameter was estimated and compared with respect to their diagnostic and prognostic capabilities in patients with MPP. All statistical analysis were performed by the statistical package for social science statistical software version 19.0. A 2-tailed P < 0.05 was considered statistically significant.