Stress-Inflammation Dysregulation and Biomarker Dynamics in Pediatric Mycoplasma Pneumoniae Pneumonia

Summary

Elevated LDH and D-dimer levels in pediatric Mycoplasma pneumoniae pneumonia were linked to systemic inflammation, psychological stress, and prognostic outcomes. High MP-DNA loads predicted longer hospitalization. Findings highlight LDH and D-dimer as valuable biomarkers for early risk stratification, enabling improved clinical management and identification of high-risk pediatric patients.

Abstract

Mycoplasma pneumoniae pneumonia (MPP) is a common pediatric infection frequently accompanied by systemic inflammation and psychological stress; however, its potential bidirectional relationship remains poorly defined. We retrospectively analyzed 120 hospitalized children aged 5-12 years using the Child Stress Questionnaire (CSQ), inflammatory mediators (CRP, IL-6), and three clinical biomarkers (LDH, D-dimer, MP-DNA load). Elevated LDH and D-dimer were significantly associated with higher CSQ scores (r = 0.67, p < 0.01) and more severe inflammatory responses. Children with prolonged illness (>7 days) and high MP-DNA load had markedly higher stress scores (mean 38 vs. 25, p < 0.01). LDH ≥ 450 U/L independently predicted extended hospitalization (OR = 2.1, 95% CI: 1.2-3.7), and ROC analysis demonstrated strong discriminative power for severe complications (AUC = 0.89, 95% CI: 0.83-0.95; sensitivity = 82%, specificity = 81%). These findings support a bidirectional link between psychological stress and immune dysregulation in pediatric MPP and highlight LDH and D-dimer as practical biomarkers for early identification of high-risk children.

Introduction

Mycoplasma pneumoniae (MP) is among the most common causes of community-acquired pneumonia in children, accounting for a substantial proportion of respiratory hospitalizations worldwide^1,2. Beyond pulmonary involvement, MP infection frequently elicits systemic immune responses that may result in extrapulmonary complications and delayed recovery³. In pediatric populations, immune function is closely linked to psychological well-being, as acute illness and hospitalization can trigger chronic stress responses that disrupt endocrine-immune balance^4,5. Activation of the hypothalamic-pituitary-adrenal (HPA) axis may either suppress immune defenses or amplify proinflammatory signaling, suggesting that inflammation and psychological stress are mutually reinforcing processes. Additional evidence also highlights the mediating role of the gut-brain axis, where stress-induced microbiome alterations and increased intestinal permeability can promote systemic inflammation^6,7, partly explaining the disproportionate hepatic or cardiac symptoms sometimes observed in children with MP pneumonia^8,9.

Clinically, certain patients experience slower recovery or deterioration despite timely treatment, often showing persistent MP-DNA in respiratory samples together with elevated markers of tissue damage or coagulation^10,11 and heightened stress scores. These patterns raise the possibility of a self-sustaining loop between stress and inflammation. However, current clinical guidelines remain largely pathogen-focused, with little integration of psychological and inflammatory dynamics into decision-making¹². Conventional markers such as C-reactive protein (CRP) and interleukin-6 (IL-6) are informative but nonspecific ^3,14.

By contrast, lactate dehydrogenase (LDH) and D-dimer are rapidly measurable, clinically accessible, and mechanistically relevant: LDH reflects tissue injury and hypoxia, while D-dimer signals coagulation imbalance and vascular stress^15,16. Prior observations indicate that LDH levels ≥ 450 U/L and D-dimer levels > 0.5 mg/L represent clinically relevant thresholds associated with prolonged hospitalization, underscoring their prognostic utility¹⁷. Similarly, MP-DNA load, though primarily diagnostic, may provide direct insight into pathogen burden and its relationship with host responses¹⁸. Compared with conventional markers such as CRP and IL-6, LDH and D-dimer provide mechanistically specific and clinically interpretable signals, while MP-DNA load offers a direct measure of pathogen burden, thus complementing and extending existing diagnostic tools¹⁹.

This study was therefore designed to examine whether LDH, D-dimer, and MP-DNA load can reliably predict disease severity in pediatric MP pneumonia and to explore how these biomarkers relate to psychological stress, with the goal of improving risk stratification and clinical management in this population.

Protocol

This study was approved by the Institutional Ethics Committee of Anhui Provincial Children's Hospital. Written informed consent was obtained from the parents or legal guardians of all participants prior to data collection. All procedures involving patient samples were conducted in accordance with institutional biosafety regulations and internationally recognized ethical standards. The equipment and software used are listed in the Table of Materials.

1. Study design

This retrospective cohort study enrolled children aged 5-12 years with confirmed Mycoplasma pneumoniae (MP) pneumonia between June 2021 and December 2024 at a tertiary pediatric center. Diagnosis was confirmed by PCR detection of MP-DNA from throat swabs or bronchoalveolar lavage fluid (BALF)^20,21. Children with known immunodeficiency, chronic pulmonary disease (e.g., asthma), or incomplete clinical records were excluded²².

2. Clinical data collection

Axillary temperature was recorded twice daily (07:00 and 19:00), with additional fever episodes documented as they occurred. Baseline peripheral oxygen saturation was assessed using a pulse oximeter. Venous blood samples were collected within 24 h of admission for inflammatory biomarker analysis^23,24. Whole blood and serum were stored at 2-8 °C and processed within 2 h of collection; serum/plasma aliquots were then frozen at −80 °C until analysis.

3. Bronchoscopy and sample collection

Flexible bronchoscopy with lavage was performed under sedation using a pediatric fiberoptic bronchoscope. Oxygen saturation and heart rate were continuously monitored during the procedure²⁵. Suction pressure was maintained at 100-120 mmHg with a 2.0 mm working channel. Three to five milliliters of sterile saline were instilled per segment, and lavage quality was considered adequate when the recovery volume reached at least 50% of the instilled saline. BALF was immediately placed on ice, centrifuged at 1,500 g for 10 min to remove cells and debris, and supernatants were stored at −80 °C until MP-DNA testing and cytokine measurements.

4. Inflammatory biomarker assays

CRP was analyzed using particle-enhanced. IL-6 was quantified by chemiluminescent immunoassay. LDH activity was determined spectrophotometrically by pyruvate-to-lactate conversion²⁶. D-dimer was measured using latex-enhanced immunoturbidimetry. Manufacturer-recommended incubation times, calibration, and internal quality controls were strictly followed; external quality assessment was performed according to the laboratory's accreditation schedule. Units were reported as mg/L (CRP), pg/mL (IL-6), U/L (LDH), and mg/L FEU (D-dimer), and MP-DNA as copies/µL.

5. Psychometric assessment

Psychological stress was assessed using the parent-reported Child Stress Questionnaire within 72 h of admission. The total score range was 0-80, and scores ≥30 were interpreted as moderate-to-severe stress. Trained nurses provided standardized instructions and item-level examples to minimize recall bias, and incomplete questionnaires were re-checked on the same day to ensure data integrity.

6. Safety considerations

All blood and BALF samples were handled in a certified Class II biosafety cabinet with appropriate personal protective equipment (gloves, gown, mask/respirator, and eye protection). Sharps were discarded into puncture-resistant containers. Liquid biohazard waste was pre-treated with 10% bleach for 30 min before disposal. Surfaces were decontaminated after each session, and all procedures complied with institutional and national regulations (e.g., WHO Laboratory Biosafety Manual; BMBL).

7. Statistical analysis

Analyses were conducted using IBM SPSS Statistics. Data normality was tested with the Shapiro-Wilk test. Continuous variables were expressed as mean ± standard deviation or median with interquartile range, while categorical variables were summarized as counts and percentages (Table 1). Associations between CSQ scores, inflammatory mediators, and clinical indicators were examined using Pearson or Spearman correlations. Logistic regression was used to evaluate predictors of prolonged hospitalization (>7 days) and severe pneumonia, defined as lung consolidation, pleural effusion, atelectasis, or respiratory failure²⁷. ROC curves were constructed to evaluate model discrimination, reporting AUC, sensitivity, specificity, and 95% CI. Ten-fold cross-validation was applied to assess robustness. Missing data (<5%) were handled by median imputation. Biomarker values were z-score standardized prior to modeling. Statistical significance was set at p < 0.05 (two-sided) (Table 2).

8. Visual outcomes and reproducibility checkpoints

Adequate BALF was defined as ≥50% recovery of instilled saline with clear, non-bloody appearance. Acceptable CSQ administration was defined as completion within 72 h of admission with ≤1 missing item per domain. Assay runs required passing internal QC per manufacturer targets before patient results were released.

This protocol provides a reproducible framework for evaluating the interplay between psychological stress, inflammatory mediators, and clinical biomarkers in pediatric Mycoplasma pneumoniae pneumonia and establishes practical checkpoints (BALF quality, CSQ timing, assay QC) to support reliable implementation across centers.

Results

The overall study design and workflow are summarized in Figure 1. LDH demonstrated superior accuracy for predicting severe complications (AUC = 0.89, 95% CI: 0.83-0.95) compared with CRP (AUC = 0.76) and IL-6 (AUC = 0.72). The optimal cutoff for LDH was 450 U/L, yielding a sensitivity of 82% and a specificity of 81%. Logistic regression confirmed that LDH ≥ 450 U/L was independently associated with prolonged hospitalization (OR = 2.1, 95% CI: 1.2-3.7). Persis...

Discussion

Mycoplasma pneumoniae (MP) pneumonia in children is a heterogeneous disease, with immunoinflammatory dysregulation acting as a central determinant of severity and recovery. This dysregulation arises from interactions between host immune responses and MP virulence factors, shaping both disease course and treatment responses. Clinical biomarkers, therefore, represent indispensable tools for patient stratification, treatment guidance, and prognosis.

CRP serves as a frontline marker of ac...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Child Stress Questionnaire (CSQ)	Not specified		Psychometric assessment of stress
CRP assay kit	Siemens Healthineers	11537224 (Atellica CH hCRP2, 720 tests)	Measurement of C-reactive protein
D-dimer assay kit	Diagnostica Stago	00515 (STAÂ- Liatest D-Di kit)	Measurement of plasma D-dimer levels
IBM SPSS Statistics version 28.0	IBM Corp., Armonk, NY, USA		Data analysis
IL-6 assay kit	Roche Diagnostics	09015612190 (cobas e 801 pack); 09015612500 (100 tests)	Measurement of interleukin-6
LDH assay kit	Not specified	MAK066 (MilliporeSigma LDH Activity Assay Kit)	Measurement of lactate dehydrogenase activity
Pediatric flexible bronchoscope (e.g., BF-P190)	Olympus	BF-P190 (model/order no.)	Bronchoalveolar lavage collection
Pulse oximeter	Not specified	Not provided	Measurement of oxygen saturation