In this study, we retrospectively collected the clinical data of patients with MMP who were admitted to Children’s hospital, Zhejiang University School of Medicine between January 1, 2017, and December 31, 2019. The criteria for enrolling patients were as follows: (1) signs and symptoms indicative of CAP, including fever, cough, abnormal lung auscultation and new infiltrate(s) on chest radiograph; (2) had both positive results for MP RNA polymerase chain reaction (PCR) tests and positive results for MP-IgM (Witmer et al., 2007; Waites et al., 2017). Exclusion criteria were the follows (Zhang et al., 2014; Jin et al., 2018): (1) had positive results for other pathogens; (2) had received corticosteroids, intravenous immunoglobulin (IVIG), and anticoagulation therapy before admission; (3) with underlying diseases, such as chronic cardiac and pulmonary disease, rheumatic diseases, and immunodeficiency; (4) had incomplete medical records.

Demographic and clinical information, radiological findings, laboratory data, such as blood routine test, C-reactive protein (CRP), lactate dehydrogenase (LDH), D-dimer, prealbumin (PAB), subpopulations of T lymphocytes, immunoglobulins, cytokines were retrospectively collected from all patients by reviewing their electronic medical records. During the hospitalization, clinical signs and symptoms of patients were obtained, including body temperature, respiratory rates, extra-pulmonary complications (Poddighe, 2018,) (Witmer et al., 2007; Zhou et al., 2014), and so on. All patients underwent chest radiography during the illness, confirming unequivocal focal or segmental infiltration with or without pleural effusion. Although patients had progressive symptoms, suspected complications, clinical deterioration, or persistent fever after appropriate antibiotic therapy, chest CT scans were performed. The large lesion was defined as the extent of infiltration on chest imaging more than one third of the lung (Uehara et al., 2011).

Severe MPP (SMPP) was defined as MPP with any one of the follows: (1) a poor general condition; (2) fastidium or dehydration; (3) disturbance of consciousness; (4) an increased respiratory rate (infants > 70 breaths/min and older children > 50 breaths/min); (5) dyspnea; (6) cyanosis; (7) extent of infiltration on chest X-ray ≥2/3 of one lung or multilobe involvement; (8) extra-pulmonary complications; (9) pleural effusion; (10) oxygen saturation in room air ≤92% (Subspecialty Group of Respiratory Diseases, 2013). Refractory MPP (RMPP) was diagnosed based on the presence of persistent fever and clinical, as well as radiological deterioration after azithromycin treatment for 7 days or longer (Tamura et al., 2008; Subspecialty Group of Respiratory Diseases, 2013). The indications for oxygen therapy, bronchoscopy, glucocorticoid, and mechanical ventilation were evaluated according to the guidelines for management of community-acquired pneumonia in children in China (Subspecialty Group of Respiratory Diseases, 2013).

Nasopharyngeal aspirate/swab specimens were routinely tested within 24 hours after admission. Peripheral blood samples were obtained on admission for detecting the laboratory data, and the abnormal values of D-dimer and inflammatory indicators, such as WBC, CRP, cytokines, and so on, were detected every 3 to 5 days thereafter.

Serum cytokines were routinely measured because this would be, to some extent, helpful for therapy decisions, and the costs were not expensive. We detected the concentrations of interleukin (IL)-2, IL-4, IL-6, IL-10, tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ) in serum by a CBA HumanTh1/Th2 Cytokine Kit II (BD Biosciences, San Diego, CA, USA). The value of D-dimer was determined via INNOVANCE D-dimer (SIEMENS, Marburg, Germany), the normal value was less than 0.55 mg/L. The MP IgM was determined via a specific Anti-MP ELISA assay (EUROIMMUN, Luebeck, Germany), the absorbance above 1.1 was determined positive. MP RNA detection was performed on an ABI 7500 detection system via SAT-MP Assay Kit (Rendu Biotechnology Co., Ltd, Shanghai, China). All steps were performed according to the manufacturer’s instructions and previous studies (Zhang et al., 2016; Li et al., 2017).

The study was approved by the ethics committee of the Children’s Hospital, Zhejiang University School of Medicine (2019-RIB-058). And the data from patients were collected anonymously.

SPSS 20.0 (IBM Corp., Armonk, NY, USA) was used for statistical analysis. Continuous data are shown as median (25th to 75th percentile). Categorical data were shown as number (%). The Kruskal-Wallis-H(K-W-H) method was used to compare differences in continuous variables among multiple groups. The Mann-Whitney U-test was used to compare differences in continuous variables between two groups. Pearson’s chi-square test was used to analyze differences between categorical variables. Spearman rank-correlation coefficients were used to describe the association between different variables and D-dimer. Statistical significance was defined as P<0.05.

From January 1, 2017, to December 31, 2019, a total of 356 patients admitted to our hospital for MPP were enrolled in the study. All patients had positive MP PCR tests and serological detection. According to the peak value of D-dimer (Huang et al., 2021), they were divided into three groups, the normal group (D-dimer <0.55 mg/L, n=106), the mild-moderately elevated group (D-dimer 0.55–5.5 mg/L, n=204) and the severely elevated group (more than 10 times higher than the normal range, D-dimer >5.5 mg/L, n=46). As shown in Table 1 , the median age in the normal group was 4.1 years (range, 2.1–6.1), younger than that in the mild-moderately elevated group 5.8 years (range, 4.3–7.0) and the severely elevated group 6.1 years (range, 4.6–8.0) (P<0.01), but no difference was found in gender distribution (P>0.05).

The most common symptoms of MPP were cough (100.0%) and fever (98.0%). Chest pain (1.7%) was rare, and 62 patients (17.4%) presented with wheezing. As shown in Table 1 , the higher incidence of fever and the lower incidence of wheezing were found in the mild-moderately or severely elevated group than those in the normal group (P<0.01). We also found that the total length of fever, the total length of antibiotic therapy, the length of stay, and the incidences of RMPP and SMPP were significantly different among the three groups (P<0.01). Interestingly, there were 14 patients (13.2%) with extra-pulmonary complications in the normal group, 72 patients (35.3%) in the mild-moderately elevated group, and 33 patients (71.7%) in the severely elevated group, which showed significant differences among these three groups (P<0.01). Moreover, the total length of fever, the total length of antibiotic therapy, the length of stay, and the incidence of extra-pulmonary complications, RMPP, SMPP were increased with the level of D-dimer (P<0.01).

Laboratory findings of patients on admission were shown in Table 2 . Besides D-dimer, the WBC, percentage of peripheral neutrophils (N%) and lymphocytes (L%), platelet (PLT), CRP, LDH, PAB, percentage of CD₄ ⁺ and CD₈ ⁺ T lymphocytes (CD₄ ⁺%, CD₈ ⁺%), IgA, IL-6, IL-10, TNF-α, IFN-γ also differed significantly among the three groups (P<0.01), but there were no statistically significant differences in hemoglobin, percentage of CD₃ ⁺ T lymphocytes (CD₃ ⁺%), IL-2, IL-4, IgG, IgM, and IgE (P>0.05). Furthermore, we found that N%, CRP, LDH, CD₈ ⁺%, IL-6, IL-10, and IFN-γ were significantly increased in the elevated group, especially in the severely elevated group than those in the normal group, which were in line with the levels of D-dimer; whereas the L% and PAB decreased with the levels of D-dimer.

As shown in Table 3 , we found that there were significant differences in radiographic features among the three groups (P<0.01). With the increase of D-dimer, the incidences of pleural effusion (26.4% vs. 64.2% vs. 93.5%, P<0.01), lobar atelectasis (3.8% vs. 33.3% vs. 63.0%, P<0.01), pulmonary consolidation (22.6% vs. 57.8% vs. 87.0%, P<0.01), large lesions (18.9% vs. 33.5% vs. 55.6%, P<0.01) were all significantly increased. As for necrotizing pneumonia, we found that it was likely to occur in the severely elevated group (23.9%, P<0.01), and there was no difference between the normal group and the mild-moderately elevated group (0.9% vs. 3.4%, P>0.05). However, none of the patients developed embolism in our study.

In addition to antibiotics, patients received other treatments depending on the disease severity, such as oxygen therapy, glucocorticoid, bronchoscopy, immunoglobulin, thoracentesis, or ICU admission (as shown in Table 4 ). In our study, we found that patients in the severely elevated group received a higher proportion of oxygen therapy, glucocorticoid, bronchoscopy, immunoglobulin, thoracentesis, and ICU admission when compared with the other two groups (P<0.01). Meanwhile, the usage rates of glucocorticoid, bronchoscopy and thoracentesis were likewise higher in the mild-moderately elevated group than those in the normal group (P<0.01). None of the patients needed mechanical ventilation. All the children recovered and were discharged from the hospital without death.

Using the Spearman correlation test, we analyzed the relationships between the level of D-dimer and different variables. As shown in Figure 1 , we found that the following variables showed significant positive correlations with the level of D-dimer (P<0.01): N%, CRP, LDH, CD₈ ⁺%, IL-6, IL-10, IFN-γ, age, length of fever, length of stay, and length of antibiotic therapy. Meanwhile, L%, PLT, PAB, CD₄ ⁺%, and TNF-α were negatively correlated with the level of D-dimer (P<0.01). Among these variables, N%, L%, CRP, LDH, IL-10, length of fever, length of stay, and length of antibiotic therapy had particularly strong correlations with D-dimer.