Mycoplasma pneumoniae (MP) is one of the principal pathogens responsible for community-acquired pneumonia (CAP) in children (1). MP spreads predominantly through respiratory droplets and exhibits cyclical outbreaks every 3–7 years, particularly in densely populated regions (2). While Mycoplasma pneumoniae pneumonia (MPP) is generally a self-limiting condition, severe cases can lead to significant complications, both within and outside the pulmonary system. These complications include necrotizing pneumonia, bronchiolitis obliterans, and acute respiratory distress syndrome, which can be life-threatening in severe cases (3, 4).

Co-infection with other pathogens in children with MPP is common, with reported co-infection rates ranging from 30% to 60% (5, 6). However, most previous studies were single-center or limited to certain pathogen types, providing insufficient comprehensive evidence regarding the clinical impact of co-infections on pediatric MPP severity.

In this prospective observational multicenter study, we utilized targeted next-generation sequencing (tNGS) to comprehensively detect respiratory co-pathogens among children hospitalized with MPP across different regions of China. By correlating pathogen profiles with clinical severity and inflammatory responses, we aimed to clarify how co-infections affect pediatric MPP outcomes. Additionally, receiver operating characteristic (ROC) curve analysis was conducted to determine whether inflammatory biomarkers could accurately predict multi-pathogen co-detection, thereby supporting early identification and optimized management strategies for Severe Mycoplasma pneumoniae pneumonia.

Previous studies have demonstrated that co-infections with viral and bacterial pathogens frequently occur in pediatric MPP, with reported co-infection rates generally ranging from 30% to 60% (5, 6, 10). Some studies have reported even higher rates, exceeding 88% (11). Clinically, pediatric MPP cases complicated by co-infections typically present with more severe and diverse symptoms, resulting in prolonged hospital stays (6, 10, 12). These findings highlight that co-infection with multiple respiratory pathogens significantly contributes to the severity and clinical complexity of MPP.

In the current prospective observational multicenter study, additional pathogens were detected in 132 of 266 children (49.62%) with MPP, a rate consistent with prior reports (5, 6, 10). Furthermore, the multi-pathogen co-detection rate was significantly higher in the severe group than in the mild group (54.05% vs. 39.51%, χ² = 4.769; P = 0.033), suggesting that co-infections may be associated with increased disease severity in pediatric MPP.

Interestingly, subgroup analysis by region revealed no significant difference in multi-pathogen co-detection rates between severe and mild cases within each center (Tianjin, Shanghai, Wenzhou, Chengdu; all P > 0.05). This is likely attributable to limited statistical power due to smaller sample sizes at each center, combined with the relatively short duration of the study (one winter month). Additionally, similar respiratory pathogens may have concurrently circulated across these regions during the study period, potentially obscuring regional variations. Future larger-scale studies conducted across multiple seasons are warranted to clarify potential geographic differences in co-infection patterns.

Retrospective analyses of 396 children with MPP identified S. pneumoniae, H. influenzae, and S. aureus as the most common bacterial co-infections, while viral co-infections commonly involved human bocavirus (HBoV), RhV, and respiratory syncytial virus (RSV) (6). In the current study, RhV (9.4%), AdV (7.1%), and IAV (6.8%) were the predominant viral pathogens co-detected with MP, while H. influenzae (9.8%) and S. pneumoniae (6.0%) were the primary bacterial co-pathogens. Some studies identified S. pneumoniae as the most common bacterial co-infection (7); however, our study found H. influenzae to be predominant, possibly due to seasonal differences and limited sample size.

Indeed, regional variations were notable, with H. influenzae (13.33%) being the most common co-pathogen in Tianjin, AdV and IAV (12.65%) dominating in Shanghai, and RhV being prominent in Wenzhou (18.42%) and Chengdu (12.36%). Such differences underscore the importance of considering local epidemiology, environmental factors, and healthcare practices when developing diagnostic and therapeutic strategies.

Given the high frequency and complexity of pathogen co-infections in severe pediatric MPP, there is an urgent need for rapid and cost-effective multiplex diagnostic tools. Recent evidence highlighting neurological dysfunction associated with atypical pathogens such as ammonia-producing microorganisms further underscores the importance of comprehensive pathogen screening in clinical practice (13). Metagenomic next-generation sequencing (mNGS) is of high value in detecting novel, rare, and atypical pathogens (14), but its use in routine practice is limited by its high cost, the influence of human genomic material, and the inability to simultaneously detect both DNA and RNA pathogens in one run (15). Targeted NGS (tNGS), in contrast, uses panels of known pathogen sequences to screen clinical isolates. These panels can target multiple types of pathogens (bacteria, viruses, fungi, atypical agents) with high specificity and sensitivity, and provide faster turnaround times while sequencing directly from clinical specimens (16, 17). In our study, the tNGS panel covered 198 pathogens (over 95% of common respiratory pathogens) and cost roughly one-quarter that of mNGS, making it a practical approach for early and comprehensive pathogen detection in MPP.

MP infection impairs cellular and humoral immunity, leading to an immunosuppressed state that facilitates secondary infections (18). Previous studies have reported elevated levels of inflammatory cytokines, such as IL-1β, IL-6, IL-8, IL-10, and TNF-α, in patients with MPP compared to healthy controls (19, 20). Consistent with these findings, our study showed significantly higher levels of IL-6, IL-10, and IFN-γ levels in severe cases compared to mild cases, with further elevations of IL-6 and IL-10 observed in patients with multi-pathogen co-infections. These results suggest that co-infections intensify inflammatory responses, thereby exacerbating disease severity.

IL-6 and IL-10 play key roles in inflammation and immune regulation, and their marked elevation may reflect more severe disease progression and greater tissue damage. Our further ROC analysis confirmed that IL-6 and IL-10, individually and especially in combination, exhibit high sensitivity (>80%, and approximately 94% combined) for detecting co-infections. Thus, combined IL-6 and IL-10 measurement might be clinically valuable for early identification of SMPP cases at risk of co-infection, facilitating timely intervention and improved patient outcomes.

In addition to cytokines demonstrating significant differences (e.g., IL-6, IL-10, and IFN-γ), several other cytokines, including IL-4, IL-8, and TNF-α, did not differ significantly between the mild and severe groups. This might indicate that these cytokines play a less direct role in the immunopathology of SMPP, or it may reflect sampling timing that did not align with their peak concentrations. Future studies examining the dynamic changes of these cytokines during the course of illness may clarify their roles in MPP progression.

Markers of infection and inflammation such as CRP, ESR, and LDH were also higher in our severe cases than in mild cases, in line with previous findings (18). MP infection activates exogenous and endogenous coagulation systems, leading to coagulation abnormalities and promoting thrombosis (21). Higher D-dimer levels existed in children with SMPP children compared to mild cases in the current study. This indicates a heightened state of coagulation, suggesting that potential thrombotic complications may contribute to increased disease severity and should be carefully considered in the management of SMPP.

In contrast to previous single-center studies or those limited to detecting specific pathogens, our multicenter approach utilizing tNGS provided comprehensive, accurate, and generalizable identification of respiratory co-pathogens in pediatric MPP. Furthermore, our ROC curve analyses of inflammatory biomarkers—particularly the combined use of IL-6 and IL-10—provided novel and valuable diagnostic insights, significantly improving the predictive accuracy for detecting severe cases complicated by co-infections. The integration of clinical characteristics and biomarker analysis further enhances diagnostic precision and holds potential for improving clinical decision-making and patient outcomes.

Several limitations should be noted. The relatively short duration of our study (one winter month) and moderate sample size (n = 266) may restrict the generalizability of pathogen prevalence across different seasons and geographic regions. Additionally, distinguishing colonization from infection remains challenging with current tNGS technology, particularly for pharyngeal swab samples, as uniform diagnostic standards are lacking. Although we mitigated this concern by employing BALF samples for severe cases and independent clinical assessments by experienced clinicians, differences in sampling methods remain a limitation. Specifically, pathogen concentrations likely varied between mild (pharyngeal swabs) and severe cases (BALF samples), potentially resulting in higher observed co-detection rates among severe patients. Furthermore, logistic regression analysis was not performed, thus limiting our ability to identify co-detected pathogens as independent predictors of SMPP.

Future studies with larger cohorts, extended durations, and spanning multiple seasons are essential for validating and generalizing our preliminary results. Uniform sampling methods or the concurrent collection of paired upper and lower respiratory tract samples should be considered to accurately explore pathogen co-detection rates and their relationship with disease severity. Further studies employing multivariate logistic regression analysis would clarify whether multi-pathogen co-detection independently predicts SMPP.

In conclusion, this prospective observational multicenter study demonstrated a high prevalence of multi-pathogen co-infections among pediatric patients with MPP, particularly in severe cases. The presence of these co-infections was associated with significantly elevated inflammatory markers, including WBC count, LDH, IL-6, and IL-10, indicating more intense inflammatory responses. Understanding the complex interactions between MP and co-detected pathogens, along with regional and seasonal variations, is critical for improving early diagnosis and targeted management strategies, ultimately enhancing clinical outcomes in pediatric MPP.