Neutrophil extracellular traps as a predictive biomarker for severe Mycoplasma pneumonia in children: a clinical observational study

Abstract

Objective

This study aims to evaluate the association between NETs levels and SMPP severity.

Methods

From January 2023 to August 2024, we selected children hospitalized with mycoplasma pneumonia (MPP). They were divided into two groups: a non-severe group (MPP group, n = 100) and a severe group (SMPP group, n = 50). ELISA was used to detect the concentration of NETs in the serum of both groups. Bronchoalveolar lavage fluid (BALF) was collected from the affected and unaffected lungs of children in the SMPP group, and ELISA was used to measure the concentration of NETs in BALF.

Results

There was a statistically significant difference in the concentration of NETs in the serum between the MPP and SMPP groups (P < 0.01). The concentration of NETs in the BALF of the affected lung in the SMPP group was higher than that in the intact lung (P < 0.001). The concentration of NETs in the serum of the SMPP group was higher than that in BALF (P < 0.001). The area under the receiver operating characteristic curve (ROC ) for serum NETs predicting SMPP was 0.820 (95% CI 0.760–0.884, P < 0.001), with a sensitivity of 0.821 and specificity of 0.845, and a cutoff value of 18.50 ng/mL. The ROC AUC for predicting SMPP using serum NETs combined with C-reactive protein (CRP) and lactate dehydrogenase (LDH) was 0.894 (95% CI 0.845–0.942, P < 0.001), with a sensitivity of 0.883 and specificity of 0.820.

Conclusion

Overactivated NETs in children with MPP may be related to the occurrence of SMPP.

Introduction

Mycoplasma pneumoniae pneumonia (MPP) is a common type of pneumonia acquired in the community among children [1, 2]. In the past two years, with the end of the COVID-19 pandemic, the incidence of MPP and the proportion of drug-resistant Mycoplasma strains among outpatient children have rebounded from initially low levels [3]. The sharp rise in outpatient children requires clinicians to quickly diagnose the disease and assess its severity. Studies [4]indicate that as resistance to macrolide antibiotics has developed, the incidence of severe Mycoplasma pneumoniae pneumonia (SMPP) has increased annually. Children with SMPP are also at a higher risk of various extrapulmonary complications, which can lead to multiple organ dysfunction syndrome and pose a threat to their lives [5]. Thus, clinicians must explore better diagnostic and classification methods to prevent misdiagnosis and misinterpretation of disease severity, which will help reduce drug misuse and the development of resistance.Previous studies have demonstrated that neutrophils contribute to the body’s antibacterial mechanisms by forming neutrophil extracellular traps (NETs) [6]. However, recent research suggests that this mechanism also plays a role in various inflammatory responses and is significant in infectious and autoimmune diseases, as well as tumor metastasis.Therefore, this clinical study aims to assess the correlation between circulating NETs levels and disease severity, providing a foundation for future mechanistic investigations.

Objects and methods

Research subjects

The study subjects were children with MPP hospitalized from January 2023 to August 2024, divided into a non-severe group (MPP group, n = 100) and a severe group (SMPP group, n = 50). BALF samples were not collected from the MPP group; the SMPP group had a total of 10 BALF samples collected, with imaging examinations showing unilateral lung involvement, and BALF samples were collected from both the affected and unaffected lungs. This research was approved by the Medical Ethics Committee of The Second Affiliated Hospital of Zhejiang University Linping campus, and the guardians of the enrolled children signed informed consent forms.

Inclusion and exclusion criteria

Inclusion Criteria: ① Aged 0–14 years; ② Meets the diagnostic criteria for MPP and SMPP (refer to the “Expert Consensus on the Diagnosis and Treatment of Mycoplasma Pneumonia in Children (2023 Edition)”); ③ Duration of illness within 14 days; ④ Negative blood culture, with no clear evidence of viral infection.

Exclusion Criteria: ① Children with a history of bronchial asthma and chronic obstructive pulmonary disease; ② Children with serious underlying diseases such as hematological diseases, severe congenital heart disease, severe liver and kidney dysfunction, congenital bronchopulmonary dysplasia, genetic metabolic diseases, and immunodeficiency diseases; ③ Children who have been on long-term treatment with glucocorticoids, immunomodulators, or immunosuppressants.

Method

Record the gender and age of the enrolled children. After the children were admitted, collect 2 mL of fasting venous blood, and performed bronchoalveolar lavage according to the principle of first the healthy side and then the affected side, collecting 5 mL of BALF each, centrifuge, and separate the supernatant for storage at -80 ℃ for later testing.

Used an automatic blood cell analyzer to detect routine blood tests and C-reactive protein (CRP), recording white blood cell count, neutrophil ratio, and CRP concentration; used an automatic latex agglutination immunoturbidimetric method to detect D-dimer, recording its concentration; used the ELISA method to detect lactate dehydrogenase (LDH), recording its level. Used ELISA to detect the content of NETs in serum and BALF, and strictly followed the kit instructions for specific operational steps, measuring and recording the absorbance of NETs.

Data analyses and statistics

Statistical analysis was performed using SPSS 26.0 and GraphPad Prism 8.0. Normally distributed continuous data were expressed as mean ± standard deviation, and comparisons between two groups were conducted using independent samples t-test. Comparisons between serum and BALF within groups were performed using paired t-test. Non-normally distributed continuous data were expressed as M (P25, P75), and comparisons between groups were conducted using the Wilcoxon rank-sum test. Categorical data were expressed as n (%), and comparisons between groups were conducted using the χ2 test. The relationship between BALF and serum NETs in the SMPP group was analyzed using Pearson correlation analysis. Indicators with statistical significance were selected based on univariate analysis for multivariate logistic regression analysis. Receiver operating characteristic (ROC) curve analysis was used to evaluate the value of serum NETs concentration and combined peripheral blood routine indicators in predicting the diagnosis of SMPP, and DeLong test was used to compare the predictive performance of each ROC curve. A two-tailed P < 0.05 was considered statistically significant.

Result

There were no statistically significant differences in age, BMI, PLR, NLR, PCT and gender between the MPP group and the SMPP group (P > 0.05), however, the white blood cell count, neutrophil ratio, CRP, D-dimer, and LDH levels in the SMPP group were all higher than those in the MPP group (P < 0.05),Table 1.

Table 1.

Baseline characteristics of the studied population

	SMPP(n = 50)	MPP(n = 100)	χ 2/t/Z	P
Sex(male,%)	25(50)	49(49)	0.24	0.650
Age, years	7.22 ± 2.55	7.93 ± 3.67	-0.57	0.562
WBC, (*109/L)	12.33 ± 2.63	6.28 ± 2.51	3.52	0.001
neutrophil ratio(%) CRP(mg/L) D–dimer(mg/dl) LDH(U/L) BMI PCT(ng/ml) NLR PLR	74(64,80) 28.42(16.58,42.53) 1.16(0.67,2.75) 385.70 ± 108.60 24.58 ± 6.23 0.12 ± 0.02 2.7 ± 0.6 85.4 ± 12.6	65(54,72) 10.25(7.26,14.55) 0.54(0.34,0.74) 321.20 ± 85.40 25.24 ± 5.62 0.11 ± 0.03 2.4 ± 0.5 81.6 ± 15.4	4.26 6.28 4.89 3.85 -0.38 1.01 1.75 0.79	0.001 0.001 0.001 0.001 0.610 0.320 0.106 0.443

Data are presented as mean ± standard deviation, n (%), or median (interquartile range)

NLR: neutrophil—lymphocyte ratio; PLR; platelet lymphocyte ratio

The serum NETs concentration in the SMPP group was higher than that in the MPP group (t = 54.60, P < 0.001); the NETs concentration in the BALF of the affected lung in the SMPP group was higher than that in the healthy lung BALF (t = 2.80, P < 0.001). Figure 1.

Fig. 1.

Comparison of NETs in serum and BALF between the SMPP and MPP

A logistic regression analysis was conducted, which excluded WBC counts and neutrophil ratio, due to a P-value greater than 0.05. However, the logistic regression showed that the CRP, LDH and serum NETs were independent risk factors for SMPP. Therefore, these factors were included as predictive indicators (P < 0.05), Table 2.

Table 2.

Logistic regression analysis of the influencing factors of SMPP

Factors	β	SE	Wald χ 2	OR	95% CI	P
CRP	0.16	0.06	6.10	1.24	1.03–1.32	0.001
LDH	0.02	0.01	5.29	1.02	1.01–1.03	0.001
Serum Nets	0.87	0.26	11.69	2.71	1.84–3.46	0.001

The sensitivity, specificity, area under the curve (AUC), and critical points for predicting SMPP using serum NETs, CRP, and LDH were presented in Table 3; Fig. 2. The area under the receiver operating characteristic curve (ROC AUC) for serum NETs predicting SMPP was 0.820 (95% CI 0.760–0.884, P < 0.001), with a sensitivity of 0.821 and specificity of 0.845, and a cutoff value of 18.50 ng/mL. The ROC AUC for predicting SMPP using serum NETs combined with C-reactive protein (CRP) and lactate dehydrogenase (LDH) was 0.894 (95% CI 0.845–0.942, P < 0.001), with a sensitivity of 0.883 and specificity of 0.820.

Table 3.

ROC curve of serum nets, CRP and LDH

Factors	AUC	95% CI	Sensitivity	Specificity	Cut off	P
Serum Nets	0.820	0.760–0.884	0.821	0.845	18.5	0.001
CRP	0.680	0.586–0.780	0.620	0.742	10.8	0.001
LDH	0.740	0.642–0.795	0.688	0.726	312.5	0.001
Nets + LDH + CRP	0.894	0.845–0.942	0.883	0.820	/	0.001

Fig. 2.

ROC curves of serum NETs, CRP, LDH for prediction of SMPP

Discussion

MPP is a self-limiting infection primarily transmitted through the respiratory tract, with an incubation period typically ranging from 2 to 4 weeks. The disease is active year-round, and there’s evidence it can lead to endemic outbreaks, occurring every 2 to 3 years in most regions of China [7]. MP can cause infections of the upper and lower respiratory tracts, with most cases presenting non-specific clinical symptoms. Although most MPP cases are not severe, they may progress to severe pneumonia and refractory pneumonia, accompanied by pleural effusion, multiple organ dysfunction syndrome, and serious long-term sequelae, including obstructive bronchiolitis and bronchiectasis; in severe cases, SMPP patients may experience respiratory failure and hypoxemia, requiring life support techniques such as mechanical ventilation and extracorporeal membrane oxygenation, and critically ill patients could even die [8, 9]. In cases of high incidence of SMPP, early diagnosis and treatment are particularly crucial.

Previous studies [10, 11]have indicated that NETs are beneficial in the early infection processes of bacteria, viruses, and fungi, and with further research, NETs have also been found to be involved in the inflammatory processes of these diseases. Research on the mechanisms of alveolar and endothelial injury in COVID-19 patients suggests that IL-1β and NETs may form a feedforward loop that directly activates and damages endothelial cells in severe COVID-19 cases, weakening the integrity of the endothelial barrier [12]. Streptococcus pneumoniae can even achieve immune evasion of neutrophils by promoting the conversion of neutrophils to NETs [13]. Pseudomonas aeruginosa, as one of the common pathogens in lower respiratory tract infections, has also been found to have NETs in the sputum of patients with chronic infections, and NETs are associated with increased sputum viscosity and decreased lung function [14]. NETs have been increasingly recognized by researchers, and exploring their role in infectious lung diseases may provide new ideas for treatment.

The results of this study show that the levels of various factors in the peripheral blood of the SMPP group and the quality concentration of serum NETs are higher than those in the MPP group, suggesting that the worsening condition of MPP children may be related to the factors mentioned earlier. Further logistic regression analysis results indicate that NETs, CRP, and LDH all affect disease progression in MPP children. NETs are a special form of neutrophils and are the first inflammatory factors to reach the site of injury, capable of simultaneously regulating both innate and adaptive immune responses, and have strong bactericidal and pro-inflammatory effects [15]. Some studies have shown that NETs can activate plasmacytoid dendritic cells (pDC) to produce high levels of interferon-α (IFN-α) in a manner dependent on DNA and Toll-like receptor 9 (TLR9) [16]. NETs can also activate pyroptosis of alveolar macrophages by regulating the deubiquitination of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), exacerbating lung injury caused by sepsis [17]. This study aims to explore the role of NETs in SMPP, providing a reference for the study of the pathogenesis of SMPP.

It is well known that the most significant feature of the transition from MPP to SMPP is the further development of the inflammatory storm and the occurrence of various pulmonary and extrapulmonary complications. This study shows that CRP and LDH be related to the occurrence of SMPP in children, which is consistent with the 2023 MPP diagnosis and treatment guidelines that highlight the value of CRP and LDH in diagnosing disease severity [18]. Currently, we still don’t have a clear understanding of the mechanisms behind localized lung injury. The results of this study show that the quality concentration of NETs in the BALF of the affected lung in SMPP children is higher than that in the healthy lung, suggesting that the excessive activation of NETs might play a role in the localized lung injury of SMPP children. Research by Kang Yanmeng et al. suggests that there is a severe imbalance in the ratio of type 1 and type 2 helper T cells (Th) in the BALF of SMPP children, combined with Zheng et al.‘s report that NETs are involved in the Th2-mediated signaling transducer and activator of transcription 6 (STAT6) dependent high expression of complement C3, suggesting that the NETs-mediated Th2 STAT6-C3-NETs cascade might be a key pathway for localized lung injury in SMPP children [19].– [20].

Conclusion

In summary, overactive NETs in MPP might be linked to the development of SMPP and the localized lung injury in SMPP. Peripheral blood NETs, CRP, and LDH can serve as predictive factors for disease progression in MPP children, helping to shape treatment plans.

Limitations

While our findings demonstrate a strong association between NETs and SMPP severity, the underlying biological mechanisms—such as NETs-mediated inflammation or tissue damage—remain to be explored in future experimental studies.Besides, the sample size may affect the generalizability of our findings, future multicenter studies with larger sample sizes are needed to verify these results and improve their clinical applicability.

Abbreviations

CRP

C-reactive protein

WBC

White blood cell

ROC

Receiver operating characteristic curve

MPP

Mycoplasma pneumoniae pneumonia

Nets

Neutrophil extracellular traps

NLR

Neutrophil—lymphocyte ratio

PLR

Platelet lymphocyte ratio

Author contributions

Xiaoping Fan. wrote the main manuscript text and junsheng Jiang prepared figures and table, revised it. All authors reviewed the manuscript.”

Funding

This research was supported by Zhejiang medical and health science and technology project, (2024KY1463 and 2025ky1223).

Data availability

The datasets used and /or analysed during the current study available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1975 Helsinki declaration as revised in 2008.This research was approved by the Medical Ethics Committee of The Second Affiliated Hospital of Zhejiang University Linping campus, and informed consent to participate was obtained from the legal guardians of the participants.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.