Predictive value of chemokines (CCL 2) in bronchoalveolar lavage fluid for refractory mycoplasma pneumonia in children

Children diagnosed with acute MPP and requiring bronchoscopy or treatment according to the Guideline of pediatric flexible bronchoscopy in China (2018 version) [20] in the pediatrics department and PICU of Yellow River Sanmenxia Hospital and Huanghe Sanmenxia Hospital and The First Clinical College of Xinxiang Medical University from October 2021 to February 2023 were selected as the study group. The treatment was conducted in accordance with the Guideline for diagnosis and treatment of community-acquired pneumonia in Children (2019 version) [4]. Based on the response to treatment and development of the disease, the study group was divided into the non-refractory mycoplasma pneumoniae pneumonia (NRMPP) group and the RMPP group according to the diagnostic criteria for RMPP in the Expert consensus on diagnosis and treatment of mycoplasma pneumoniae pneumonia in children (2015) [1]. In addition, 11 children with bronchial foreign bodies in the same period were selected as the control group (meeting the ethical requirements). Clinical data of 87 children with MPP were collected, including 16 cases with mixed infections, 5 cases with congenital underlying diseases, and 4 cases with incomplete clinical data. According to the inclusion and exclusion criteria, 62 study subjects (51 patients in the study group and 11 patients in the control group) were finally included. This study was approved by the Medical Ethics Committee of our hospital, with the ethics number 2,019,064, and with informed consent from the guardians.

The resulting data were analyzed using the SPSS 26.0 software. Qualitative data is expressed by the rate or composition ratio (%), and comparisons between groups were performed using the chi-square test. Quantitative data were described by the mean ± standard deviation (‾x ± s) or median, interquartile range M (IQR) according to the data distribution, and comparisons between groups were performed using the independent sample T-test or the non-parametric rank-sum test. The assessment value of CCL2 level in BALF for RMPP was evaluated using the receiver operating characteristic (ROC) curve. The risk factors for RMPP were analyzed with logistic regression model. And a prediction model was established based on the results. Significance level α = 0.05.

Age and height of the two groups were fit with the normal distribution, and body weight and BMI did not meet the normal distribution. Genders were compared using the chi-square test, and non-parametric rank sum test was used for comparison of other items between the two groups. The study group included 22 boys (43.1%) and 29 girls (56.9%); and the control group had 4 boys (36.4%) and 7 girls (63.6%). There was no significant difference in gender and BMI between the study group and the control group (P > 0. 05). CCL2 level in BALF, NE%, CRP, D-dimer, and LDH in the study group were significantly higher than those in the control group, with statistically significant differences (P < 0.05). Table 2; Fig. 1A.

In the RMPP group, CCL2 level in BALF, age, NE%, CRP, D-dimer, and LDH in the RMPP group were significantly higher than those in the NRMPP group, and MONO and MONO% were lower than those in the NRMPP group, with statistically significant differences (P < 0.05). There was no statistically significant difference in the remaining clinical data between the two groups (P > 0.05). There was no significant difference in the number of lobes invaded between the RMPP group and the NRMPP group with continuous variable (P > 0.05). Table 3; Fig. 1B.

All the study subjects included in this study were eventually cured and discharged from hospital without death or disability.

In recent years, the incidence of RMPP has been increasing and the treatment is difficult. Current studies generally believe that excessive inflammatory response plays a key role in the pathogenesis of RMPP, and the early prediction of RMPP has become a focus of research. Ding Y et al. [23] have found that High Mobility Group Box 1 level is increased in the peripheral blood of children with RMPP and is a good biomarker of RMPP. Fu et al. [24] found that the level of autotaxin was significantly increased in the serum and BALF of children with RMPP, which can be used as a predictor of children RMPP. Zhang Y et al. [25] found that IL-10 and IFN-γ may be good predictors for RMPP in school-age children. Studies have proposed that cytokines can be used as new therapeutic targets to reduce the damage caused by excessive inflammatory response to the body [26]. Most of these biomarkers (including CCL2) are still under study or are not widely available in clinical practice. This study investigated the predictive value of chemokine CCL2 in BALF for RMPP in children.

In this study, by measuring CCL2 level in BALF in the study group and the control group, we found that CCL2 level in BALF was significantly higher in MPP children than that in the tracheal foreign body group, and CCL2 level in BALF in the RMPP group was significantly higher than that in the NRMPP group. This is consistent with the report of Yang M and others [22]. They measured 13 cytokines in BALF in 88 children with MPP and 26 children with foreign body inhalation (FB) with the Luminex system, and found higher CCL2 level in BALF in mild MPP children than in the FB group and higher CCL2 level in BALF in severe MPP children than in the mild MPP group, with statistically significant differences (P < 0.05). The study of Chen X et al. [27] found that CCL2 in BALF in MPP children was higher than that in bronchial foreign body group, and serum CCL2 of MPP children was not significantly different from healthy controls, suggesting that CCL2 in BALF could reflect the disease status more than serum CCL2.

In this study, by comparing clinical data and CCL 2 in BALF between the RMPP group and the NRMPP group, we found that CCL2 level in BALF, age, NE%, CRP, D-dimer, and LDH of the RMPP group were significantly higher than those of the NRMPP group, and MONO and MONO% were lower than those in the NRMPP group, with statistically significant differences (P < 0.05). A systematic review and Meta-analysis by Huang W et al. [28] concluded that CRP, LDH, elevated neutrophil percentage and lung consolidation were predictors of RMPP, which is consistent with our study. A study by Shen F et al. [12] found CRP, LDH and D-dimer as predictors of RMPP and developed an RMPP prediction model that can visualize and accurately quantify the risk of developing RMPP with nomograms, early identify RMPP and assist in the selection of appropriate treatment options. Plasma D-dimer (D-D) is a degradation product of cross-linked fibrin. D-D value increases when acute coagulation occurs [29] and it can be used as molecular markers of hypercoagulability. It is also a specific marker of the fibrinolytic system and can be used as one of the indicators to monitor inflammation and severe infections. The elevated plasma D-D level in MPP patients may be related to the vascular endothelial cell damage caused by the release of various inflammatory mediators from the inflammatory cells in children with MPP [30]. Huang X et al. [31] found significantly higher serum D-D level in RMPP patients, indicating excessive inflammatory response and long-lasting vascular endothelial damage in this patient population. Elevated serum D-D level can be used as an early predictor of the occurrence of RMPP and complications (pleural effusion and liver injury). In this study, we found that the median age of RMPP patients was higher than that of NRMPP patients (7.0 (6.1,9.0) vs. 5.3 (3.1,7.0)), with statistically significant difference (P < 0.01), which is consistent with previous reports [32]. The immune system of older children is relatively more mature and may be more likely to produce excessive immune response [33]. However, there are also different study findings. Zhai YY et al. [34] analyzed the clinical data of 20 children with RMPP and found that there was no significant difference in age between the refractory MPP group and the general MPP group. However, there were statistically significant differences in the duration of fever, CRP, ESR, LDH, PLT, WBC, D-dimer, and intrapulmonary and extrapulmonary complications (P < 0.05). Several studies have found a clear correlation between duration of fever and the occurrence of RMPP [12, 31], which is consistent with the results of this study.

CCL2 belongs to pro-inflammatory chemokines according to functions and belongs to monocyte/macrophage chemokines according to the cell types with different chemotactic effects. It plays an important role in chemotaxis and activation of monocytes to the site of inflammation. CCL2 is not only a specific parameter of MPP, it is usually an anti-inflammatory value. Betakova T et al. [26] summarized the data of induced cytokines and chemokines in the serum of patients infected with human and avian influenza virus, and determined that they may play a role in the pathogenesis. Chemokines CCL-2, CXCL-8, CXCL-9 and CXCL-10 are related to the pathogenicity and mortality of avian influenza virus. Khalil BA [35] and others described the role of chemokines and their receptors in the pathogenesis of COVID-19, emphasizing the most prominent chemokines related to the progression of COVID-19, including CCL2, CXCL10 and CXCL8, and their importance as potential biomarkers, which helps to determine targeted treatment options for chemokines. Lee YC et al. [36] included 42 children with MPP. Plasma samples were collected on admission and after one to two weeks of treatment with antibiotics or methylprednisolone to detect chemokine levels. M1-related chemokines (CCL2, CCL8 and CXCL10) decreased and M2-related chemokines (CCL17 and CCL22) increased in plasma of MPP children. In this study, CCL2 level in BALF was increased in children with MPP and significantly increased in children with RMPP. The assessment value of CCL2 level in BALF on RMPP was analyzed with ROC curve. The AUC of CCL2 was 0.94 and the cutoff value was 0.645 ng/ml. The AUC of CRP was 0.816 with cutoff value of 56.2 mg/L. The AUC of LDH was 0.910 with cutoff value of 397 U/L. The results showed that the AUC and cut-off value of CCL2 were significantly greater than those of CRP and LDH, and the difference was statistically significant, which indicated that CCL2 had higher accuracy in the diagnosis of RMPP. CRP and LDH in serum reflect the general state and are affected by many factors. CCL2 in bronchoalveolar lavage fluid is a local inflammatory index, which has better sensitivity and specificity, and is more stable in predicting RNMPP. Studies suggest that excessive immune response is involved in the formation of RMPP, and CCL2 is involved in the immune response. Hue Erika et al. [37] found in an animal experiment that the level of cytokines in the bronchoalveolar lavage fluid (BALF) of the affected side was significantly higher than that of the healthy side. It has a higher predictive value than CRP and LDH in predicting the occurrence of RMPP. Variables that were statistically significant in univariate analysis and those with significant impact on the outcome were included in the Logistic regression model, and CCL2 and duration of fever were selected to make a nomogram to identify the risk of RMPP. CCL2 showed good predictive value, which may be one of the biomarkers for predicting RMPP.

This study also has some limitations. BALF was not easily available due to ethical requirements, and children with bronchial foreign bodies in the same period were selected as the control group in this study. The number of patients in the control group was small due to the young age of tracheal foreign body. This study is a single-center study. Multi-center joint studies can be conducted in the future. The level of CCL2 in BALF measured in this study is lower than that reported abroad, which may be related to the difference in immune status of children in different regions. In addition, some children have been given drug treatment before the bronchoscopy lavage, which may have an impact on the measured value. In conclusion, this study has preliminarily demonstrated the good potential of CCL2 as a predictor of RMPP, and more high-quality studies are needed for subsequent validation.

The present study provides the following conclusions: Elevated levels of CCL2 were observed in the BALF of children with MPP, particularly in the RMPP group compared to the NRMPP group. However, it is important to note that while the findings suggest a potential of CCL2 as a biomarker for predicting RMPP, further research is essential to validate and generalize these findings. Therefore, our study lays the foundation for future investigations into the utility of CCL2 and other predictive methods for RMPP, underscoring the importance of further research to comprehensively assess its clinical applicability.