1.1 Including, excluding, and grouping criteria
In this study, the diagnosis of MPP met the following criteria: 1) fever, coughing, and other respiratory tract infection symptoms; 2) chest radiographic examination with bronchial pneumonia, interstitial pneumonia, segmental or lobar pneumonia, and even pleural effusion; and 3) a single serum anti- MP IgM antibody titers of ≥1:160 at the acute phase following admission (in those with no history of respiratory infections in the past 3 months) and a positive PCR test for MP. Patients with any of the following criteria [12-13] would be diagnosed as severe cases: 1) tachypnea or tachycardia (Tachypnea was defined as a respiratory rate of >40/m for children aged 1-5 years, and 30/m for children aged>5 years. Tachycardia was defined as >140 bpm for children ages 1-3 years, >120 bpm for children aged 3-5, >118 bpm for children aged 5-10 years, and >100 bpm for children aged >10 years of age.) with or without nasal flare, moaning, three concave sign, cyanosis; 2) hypoxemia (SaO2≤92%); 3) refractory MPP; 4) multilobar involvement or involvement area ≥2/3 on chest radiographs; 5) pleural effusion (>300ml) , severe atelectasis, pulmonary necrosis and pulmonary abscess. 6) Other severe complications (central nervous system infection, heart failure, myocarditis, gastrointestinal hemorrhage, obvious electrolyte/ acid-base balance disorder). Patients having infections within 3 months, or suffering from known coexisting chronic, progressive or oncological illnesses, or receiving corticosteroids or immunosuppressive agents within 3 months, or with immune hypofunction or immune related diseases, or allergic diseases or suspected allergic diseases (including allergic rhinitis and atopic dermatitis) or asthma were excluded from the research.
All the patients were divided into severe cases and mild cases by the severity of the diseases. The whole patients could also be divided into MP single infection group and MP mixed infection group according to the infection types as well as low MP DNA loads (＜105copies/ml) and high MP DNA loads (≥105copies/ml) according to the MP DNA loads.
1.2 Data collection
Data including age, gender, clinical signs and symptoms, laboratory and radiological findings were collected from patients during Jan 1st and Dec 31st of the year 2017. All chest radiographs and computer tomography were reviewed by two experienced radiologists and they agreed on the conclusion.
1.3 MP DNA extraction, detection and quantification
MP DNAs from BALFs were extracted using QIAamp DNA MINI kit (Qiagen, Hilden Germany). The target gene for detecting MP by PCR was a segment of gene p1 adhesion with 150 bp (P1-178: CAATGCCATCAACCCGCGCTTAACC,P1-331: CGTGGTTTGTTGACTGCCACTGCCG). The PCR conditions were: 30 cycles of 94°C for 30 s, 62°C for 30 s and 72°C for 30 s. MP DNA was quantified using Mycoplasma pneumoniae DNA Fluorescence Diagnostic Kit (Shengxiang Biotechnology Co. Ltd, Hunan Province, China) with ABI PRISM 7500 instrument (Applied Biosystems TM, Foster City, California, United States). The experiments were strictly conducted in accordance with the manufacturer’s instructions.
1.4 Collection of BALFs
Bronchoscopy was performed within 3 days after hospital admission for patients with MPP and immediately after hospital admission to remove the foreign bodies in bronchus from children in control. Flexible fiber optic bronchoscopy and bronchoalveolar lavage were performed following the guidelines described previously . BALFs were collected from these children and stored in -80 ℃ freezer.
1.5 The assay of IL-6s and IL-27s
IL-6s and IL-27s in BALFs from the children were measured by sandwich enzyme-linked immunosorbent assay (ELISA) with commercial reagent kits (Abcan Company, USA). The experiments were strictly conducted in accordance with the manufacturer’s instructions.
1.6 Statistical analysis
Statistical analyses were performed using SPSS21.0 Statistical package. Continuous variables were reported as mean ± standard deviation. ANOVA were used to compare means of multiple groups. LSD test were used for the inter-comparison of 2 means in the multiple groups. The ages and genders between MPP patients and control were compared with t test and x2 test, respectively. MP DNA loads, IL-6s and IL-27s were shown and compared by box plots. P﹤0.05 was considered to indicate a statistically significant difference.