Soluble ST2 as a biomarker for predicting severe adverse events among pediatric patients with Mycoplasma pneumoniae pneumonia

Fangying Cheng; Tingting Li; Lei Zhang; Menghua Xu; Luxi Chen; Zhicheng Ye; Jin Xu

doi:10.1371/journal.pone.0347651

. 2026 Apr 17;21(4):e0347651. doi: 10.1371/journal.pone.0347651

Soluble ST2 as a biomarker for predicting severe adverse events among pediatric patients with Mycoplasma pneumoniae pneumonia

Fangying Cheng ^1,^#, Tingting Li ^1,^#, Lei Zhang ¹, Menghua Xu ¹, Luxi Chen ¹, Zhicheng Ye ^1,^*, Jin Xu ^1,^*

Editor: Shivkumar Gopalakrishnan²

PMCID: PMC13089705 PMID: 41996444

Abstract

Aim

Mycoplasma pneumoniae (MP) is a leading cause of pneumonia in children. Early identification of patients at high risk is critical for improving outcomes. This study aimed to evaluate the association of soluble ST2 (sST2) with in-hospital adverse events in pediatric MP pneumonia (MPP).

Methods

We retrospectively analyzed 147 children with MPP admitted to the Children’s Hospital of Fudan University, Shanghai, China, between 01/04/2023 and 31/05/2024. Demographic, clinical, and laboratory data were collected, including sST2, inflammatory markers (CRP, PCT, IL-6), and blood cell counts. Severe adverse events were defined as in-hospital death, ICU admission, diagnosis of sepsis or use of extracorporeal membrane oxygenation.

Results

Twelve patients experienced severe adverse events and had significantly higher sST2 levels. ROC analysis showed that sST2 predicted severe adverse events (AUC = 0.944, 95% CI 0.894–0.975, P < 0.001), with an optimal cut-off of 114.18 ng/mL (sensitivity 91.7%, specificity 94.8%). The association remained significant after adjusting for age, sex, PCT, and IL-6. In addition, admission sST2 levels were significantly higher in severe MPP cases, those with co-infections and those with pulmonary complications and/or extrapulmonary complications during hospitalization. sST2 correlated positively with hospital length of stay and preadmission fever duration. They also correlated positively with neutrophil counts, neutrophil to lymphocyte ratio, PCT, CRP and IL-6 but negatively with lymphocyte counts. Conclusions.

sST2 was associated with in-hospital adverse events and it showed better performance in predicting severe adverse events than other inflammatory biomarkers. The potential of sST2 as a prognostic biomarker for MPP warrants further investigation.

Introduction

Mycoplasma pneumoniae (MP) is a major cause of community-acquired pneumonia (CAP) among children in China, especially those over 5 years old [1]. Mycoplasma pneumoniae pneumonia (MPP) typically presents with sore throat, coryza, intermittent irritating cough, often accompanied by headache, fever, and myalgia. The infection occurs endemically with an epidemic peak every few years. After the COVID-19 pandemic, China experienced a notable surge of MP infections from 2023 to 2024. This epidemic was associated with more severe clinical manifestations and a high prevalence of macrolide-resistant M. pneumoniae (MRMP), which complicated treatment [2,3]. These trends highlight the importance of early identification of children at risk of severe or rapidly progressing disease to enable timely intervention.

Currently, physicians often rely on clinical judgement based on overall patient assessment to distinguish high-risk patients. Previous studies have reported mixed results regarding the accuracy of this approach as well as substantial inter-physician variability [4,5].Thus, clinical decision aids incorporating objective parameters have been proposed. However, existing tools show only moderate accuracy in pediatric respiratory infections and require further validation [6–8]. Blood biomarkers may represent a potential alternative for assessing severity and predicting prognosis.

Soluble ST2 (sST2) is a circulating form of the ST2 receptor. It competes with membrane-bound ST2 for binding to interleukin-33 (IL-33). The IL-33/ST2 pathway modulates immune response and inflammation [9]. Immune dysregulation, including T-cell activation and cytokine-driven inflammation, is believed to contribute to MPP progression [10,11]. Elevated sST2 may reflect the degree of immune activation and inflammation in MPP. Consequently, elevated sST2 may be associated with disease severity and, in turn, adverse events. Some studies in adults have linked sST2 to prognosis in community-acquired pneumonia [12,13]. Few studies have evaluated sST2 in pediatric MPP. The relationships between sST2 levels and clinical outcomes in MPP remain unclear.

Inflammatory biomarkers such as procalcitonin (PCT), C-reactive protein (CRP), and interleukin-6 (IL-6), as well as immune cell counts such as white blood cell counts (WBC), neutrophil counts (NEU), and lymphocyte counts (LYM) are widely used laboratory tests for pneumonia patients. Studies have shown that CRP, IL-6 and neutrophil-to-lymphocyte ratio (NLR) are associated with severe MPP or refractory MPP (RMPP) in children [14–17]. How sST2 compares with conventional inflammatory markers in children with MPP remains unknown. Therefore, in this study, we aimed to evaluate the association of sST2 with in-hospital adverse events and clinical characteristics in pediatric MPP patients, and to assess its potential as a prognostic biomarker in comparison with conventional inflammatory markers.

Materials and methods

Subjects

This study retrospectively analyzed 147 children diagnosed with Mycoplasma pneumoniae pneumonia (MPP) who were admitted to the Children’s Hospital of Fudan University in Shanghai, China, between April 2023 and May 2024. The retrospective data analysis was performed from June 2024 to January 2025. The inclusion criteria were: (1) MPP diagnosis, (2) age between 1 month and 18 years, (3) hospital stay > 24 hours, and (4) available sST2 measurements at admission. The exclusion criteria were patients with (1) congenital heart diseases, (2) autoimmune diseases, (3) cancer, (4) active asthma or pulmonary tuberculosis, and (5) incomplete medical records, because sST2 levels may be elevated in these conditions, while patients with cancer and pulmonary tuberculosis were excluded because of their significant impact on prognosis.

Patients were diagnosed with MPP according to the Chinese guideline for the diagnosis and treatment of childhood Mycoplasma pneumoniae pneumonia (2023). The diagnostic criteria were as follows [1,2]: (1) evidence of acute respiratory tract infections, accompanied by chest radiography-proven pneumonia; (2) a fourfold or greater increase in MP antibody titers of paired sera, or > 1:160 titer of single serum MP antibody, or MP-DNA(+) or MP-RNA(+).

The sST2 assay was a newly introduced test during the study period and was performed at the attending physician’s discretion. During this period, 3,794 patients with MPP were hospitalized, of whom 205 had sST2 results available. Of these, 58 patients were excluded due to the following reasons: age or hospital stay length (n = 4), sST2 assay performed after 24 hours after admission (n = 19), congenital heart diseases (n = 6), autoimmune diseases (n = 4), cancer (n = 3), and ative bronchial asthma or pulmonary tuberculosis (n = 22). Finally, 147 patients were included. Complete medical records were available for included patients (S1 Fig).

Clinical data collection

Data were obtained from medical records, including demographic characteristics, identified pathogens, physician-diagnosed conditions, ICU admissions, and in-hospital deaths. Severe adverse events and pulmonary and extrapulmonary complications were classified according to predefined criteria. Severe MPP was defined as MPP with any of the following criteria according to the Chinese guideline for the diagnosis and treatment of childhood Mycoplasma pneumoniae pneumonia (2023) [1,2]: (1) high fever (> 39°C) for more than 5 days or fever for more than 7 days without a declining trend in peak temperature; (2) hypoxemia (maintained an SaO₂ < 92% on room air); (3) increasing respiratory and pulse rates with clinical evidence of respiratory distress and exhaustion with or without a elevated PaCO₂; (4) signs of intrapulmonary infection, such as moderate to large pleural effusion, large area of pulmonary consolidation, plastic bronchitis, pulmonary embolism, necrotizing pneumonia, and acute asthma exacerbations; and (5) signs of extrapulmonary complications, such as meningoencephalitis, ascending (i.e., Guillain-Barré) paralysis, myopericarditis, erythema multiforme, autoimmune hemolytic anemia, hemophagocytic syndrome, or disseminated intravascular coagulation. Severe adverse events were defined as in-hospital death, ICU admission, diagnosis of sepsis or use of extracorporeal membrane oxygenation (ECMO). Pulmonary complications included acute respiratory distress syndrome, respiratory failure, necrotizing pneumonia, pleural effusion, plastic bronchitis, pulmonary embolism and pulmonary atelectasis. Extrapulmonary complications included complications of nervous system, circulatory system, blood, skin and mucous, liver, kidney, muscles, and pancreas (S1 File).

Laboratory tests

Laboratory test results including sST2, CRP, PCT, IL-6, WBC, LYM, NEU and NLR at admission were collected from medical records. All tests were performed using venous blood samples collected at admission. sST2 and IL-6 were measured by fluorescent immunoassays on Pylon 3D immune analyzer from ET Healthcare Inc. (Suzhou, China) using EDTA-anticoagulated plasma. PCT was measured by electrochemiluminescence assays using the instruments and reagents from Roche Diagnostics GmbH (Mannheim, Germany) with serum. CRP was measured by turbidimetric immunoassays using the instruments and reagents from Shanghai Upper Bio-Tech Pharma Ltd. (Shanghai, China) with EDTA-anticoagulated plasma. Complete blood counts were acquired by an XN-2000 analyzer using reagents from Sysmex Corp.(Kobe, Japan) with EDTA-anticoagulated whole blood. All the assay kits and analyzers were approved by the China National Medical Products Administration (NMPA) as in-vitro diagnostic products.

Study variables

The outcome variable of this study was severe adverse events, defined as in-hospital death, ICU admission, diagnosis of sepsis or use of ECMO (S1 File). The primary predictor variable was sST2, and the covariates included demographic variables (age, sex) as well as other blood biomarkers (CRP, PCT, IL-6, WBC, LYM, NEU and NLR).

Statistical analysis

Continuous variables were presented as median (interquartile range) and compared by Mann-Whitney U test or Kruskal-Wallis test. A post hoc Conover test was conducted after a significant Kruskal-Wallis test. Categorical variables were expressed as numbers (%) and compared using Fisher’s exact test. Spearman correlation was used for correlation analyses between sST2 and other parameters. The optimal cutoff for sST2 was determined by maximizing Youden’s index from receiver operating characteristic (ROC) curve analysis. The areas under the ROC curve (AUC) were calculated to compare the predictive performance of biomarkers for severe adverse events. Multivariable logistic regression assessed the independent prognostic value of sST2 after adjustment for age and sex or other biomarkers. All statistical tests were two-tailed, and a value of P < 0.05 was considered statistically significant. Statistical analyses were performed using MedCalc Statistical Software version 22.009 (MedCalc Ltd., Ostend, Belgium). All analyses were performed on complete observations given that there were no missing values present in the data.

Ethics statement

This study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the Children’s Hospital of Fudan University (Ethics No. 2024−129). We confirm that all methods were performed in accordance with the relevant guidelines and regulations. This study is a retrospective analysis, only involving the statistical analysis of patient data, and all patient information has been anonymized and does not contain sensitive data; therefore, the requirement for informed consent was waived by the ethics committee.

Results

Patient characteristics

147 children (77 females, 52.4%) hospitalized with MPP were included, of whom the median age was 6 years, and median hospital stay was 7 days. Severe adverse events occurred in 12 patients (8.2%), including 1 death, 10 ICU admissions, 9 patients diagnosed with sepsis and 4 patients requiring ECMO during their hospital stay. The demographic data, clinical parameters and laboratory results at admission of all the study subjects were presented in Table 1 and compared between patients with and without severe adverse events. The two groups had similar sex distribution and pre-admission fever duration. However, in the group with severe adverse events, patients were significantly younger and had lower body weight. There were also significantly higher proportions of severe MPP patients and patients with pulmonary and extrapulmonary complications in the group of severe adverse events, although the proportions of RMPP/MRMP and co-infection with other pathogens were similar between the two groups. The group with severe adverse events had a significantly longer hospital stay.

Table 1. Patient characteristics.

Characteristics¹	Overall (N = 147)	Severe Adverse Events		P-value²
Characteristics¹	Overall (N = 147)	No (N = 135)	Yes (N = 12)	P-value²
Sex				0.135
Female	77 (52.4%)	68 (50.4%)	9 (75.0%)
Male	70 (47.6%)	67 (49.6%)	3 (25.0%)
Age (years)	6.0 (4.0, 9.0)	6.0 (4.0, 9.0)	4.0 (1.8, 6.0)	0.047
Weight (kg)	23.0 (16.5, 31.0)	23.2 (17.0, 31.0)	15.50 (12.8, 20.0)	0.018
Severe MP	46 (31.3%)	34 (25.2%)	12 (100.0%)	<0.001
RMPP/MRMP	51 (34.7%)	47 (34.8%)	4 (33.3%)	0.999
Co-infections	83 (56.5%)	74 (54.8%)	9 (75.0%)	0.231
Bacterial infection	21 (14.3%)	18 (13.3%)	3 (25.0%)	0.38
Viral infection	72 (49.0%)	63 (46. 7%)	9 (75.0%)	0.074
Fungal infection	5 (3.4%)	3 (2.2%)	2 (16. 7%)	0.053
Complications				<0.001
None	99 (67.4%)	99 (73.3%)	0 (0.0%)
Extrapulmonary	14 (9.5%)	14 (10.4%)	0 (0.0%)
Pulmonary	24 (16.3%)	19 (14.1%)	5 (41. 7%)
Pulmonary and extrapulmonary	10 (6.8%)	3 (2.2%)	7 (58.3%)
Pulmonary complications	34 (23.1%)	22 (16.3%)	12 (100.0%)	<0.001
ARDS	2 (1.4%)	0 (0.0%)	2 (16. 7%)	0.006
Respiratory failure	11 (7.5%)	2 (1.5%)	9 (75.0%)	<0.001
Pulmonary necrosis	3 (2.04%)	1 (0.7%)	2 (16.7%)	0.018
Pleural effusion	11 (7.5%)	9 (6.7%)	2 (16.7%)	0.222
Plastic bronchitis	2 (1.4%)	2 (1.5%)	0 (0.0%)	0.999
Pulmonary embolism	1 (0.7%)	1 (0.7%)	0 (0.0%)	0.999
Pulmonary consolidation	54 (36.7%)	52 (38.5%)	2 (16. 7%)	0.211
Pulmonary atelectasis	11 (7.5%)	10 (7.4%)	1 (8.3%)	0.999
Extrapulmonary complications	24 (16.33%)	17 (12.59%)	7 (58.33%)	<0.001
Nervous system	4 (2.7%)	1 (0.7%)	3 (25.0%)	0.002
Circulatory system	10 (6.8%)	7 (5.2%)	3 (25.0%)	0.036
Blood system	5 (3.4%)	3 (2.2%)	2 (16.7%)	0.053
Skin and mucous	5 (3.4%)	5 (3.7%)	0 (0.0%)	0.999
Other systems³	5 (3.4%)	2 (1.5%)	3 (25.0%)	0.004
Fever duration before hospital admission (days)	7.0 (5.0, 9.0)	7.0 (5.0, 9.0)	8.0 (5.0, 10.5)	0.415
Hospital stay (days)	7.0 (5.0, 9.0)	6.0 (5.0, 9.0)	17.5 (11.0, 37.0)	<0.001
ST2 (ng/mL)	39.3 (16.5, 84.6)	34.0 (15.7, 68.0)	170.7 (132.5, 246.3)	<0.001
WBC (10^9/L)	7.5 (5.6, 10. 6)	7.5 (5.6, 10.4)	9.0 (5.3, 15.1)	0.408
NEU (10^9/L)	4.6 (3.1, 6.6)	4.46 (3.1, 6.4)	6.92 (3.8, 12.9)	0.073
LYM (10^9/L)	2.0 (1.5, 2.8)	2.1 (1.6, 3.2)	1.5 (1.2, 1.8)	0.01
NLR	2.1 (1.2, 3.7)	2.0 (1.2, 3.4)	5.7 (2.1, 8.1)	0.004
CRP (mg/L)	10.9 (2.3, 24.0)	7.4 (2.0, 22.0)	48.8 (18.5, 58.6)	<0.001
PCT (ng/mL)	0.09 (0.04, 0.20)	0.08 (0.04, 0.17)	0.85 (0.23, 1.80)	<0.001
IL-6 (pg/mL)	11.0 (4.2, 27.4)	9.7 (3.6, 21.6)	40.1 (28.3, 65.1)	<0.001

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1 n (%); Median (Q1, Q3) ²Fisher’s exact test; Mann-Whitney U test ³Complications in liver, kidney, muscles, or pancreas.

Correlation of sST2 with other laboratory biomarkers and clinical parameters

sST2 levels were correlated positively with NEU (rho = 0.165, P = 0.045), NLR (rho = 0.384, P < 0.001), PCT (rho = 0.386, P < 0.001), CRP (rho = 0.378, P < 0.001) and IL-6 (rho = 0.211, P = 0.010) and negatively correlated with LYM (rho = −0.317, P < 0.001) (S2 Fig). We also found sST2 at admission was positively correlated with hospital length of stay (rho = 0.632, P < 0.001) and pre-admission fever duration (rho = 0.198, P = 0.017) (S3 Fig).

Patients with severe MPP and MPP patients with co-infection by other respiratory tract pathogens had significantly higher sST2 levels (severe MPP vs non-severe MPP: 71.91 [30.76–140.12] vs 26.2 [15.15–54.69], P < 0.001; MPP patient with co-infection vs those without: 48.46 [23.23–94.17] vs 23.47 [13.18–52.87] ng/mL, P < 0.001) (Fig 1). However, sST2 levels were similar between patients with or without RMPP/MRMP (45.97 [17.00–87.01] vs 34.39 [13.90–79.26] ng/mL, P = 0.379) (S4 Fig). We also found patients with pulmonary complications and/or extrapulmonary complications during hospitalization had significantly higher sST2 levels at admission compared to those without complications (77.84 [29.56–147.16] vs 26.68 [14.53–54.54] ng/mL, P < 0.001) (Fig 2A). Pairwise comparisons showed that patients with both pulmonary and extrapulmonary complications had the highest sST2 (170.15 [140.12–245.95] ng/mL) followed by those with only pulmonary complications (58.80 [28.79–91.93] ng/mL), while sST2 levels were similar in those without complications (26.68 [14.53–54.54] ng/mL) and patients with only extrapulmonary complications (41.78 [14.32–100.05] ng/mL) (Fig 2B).

Associations of sST2 with severe adverse events during hospital stay

Patients with severe adverse events had significantly higher sST2 levels than those without severe adverse events (170.69 [132.53, 246.26] vs 34.03 [15.69, 67.99] ng/mL, P < 0.001) (Fig 3A). Patients with severe adverse events also had higher CRP, PCT and IL-6, lower LYM and higher NLR (all P < 0.05, Table 1). Meanwhile, WBC and NEU did not differ significantly between the two groups.

ROC analysis showed that sST2 predicted patients with severe adverse events (AUC = 0.944, 95% CI 0.894–0.975, P < 0.001), with an optimal cut-off of 114.18 ng/mL (sensitivity 91.7%, specificity 94.8%) determined by Youden’s index. The AUC of sST2 was higher than that of PCT (0.944 vs 0.900, P = 0.427) and that of IL-6 (0.944 vs 0.858, P = 0.101) without statistically significant differences, but the AUC of sST2 was significantly higher than those of CRP (AUC: 0.944 vs 0.822, P = 0.027), LYM (AUC: 0.944 vs 0.724, P < 0.001) and NLR (AUC: 0.944 vs 0.750, P = 0.025) (Fig 3B). Logistic regression analysis showed that increasing sST2 was associated with increased risk of severe adverse events (unadjusted OR = 1.023 per 1 ng/mL increase, 95% CI 1.012–1.033, P < 0.001). The association remained significant after adjusting for age and sex (adjusted OR= 1.023, 95% CI 1.022–1.034, P < 0.001) or PCT and IL-6 (adjusted OR= 1.025, 95% CI 1.013–1.037, P < 0.001) (Table 2).

Table 2. Multivariant logistic regression of ST2 on prediction of severe adverse events.

	Odds ratio	95% CI	P-value
Single variant model
sST2	1.023	1.012 to 1.033	< 0.001
Multivariant model 1
sST2	1.023	1.012 to 1.034	< 0.001
Age	0.896	0.697 to 1.152	0.371
Sex = “Male”	0.346	0.059 to 2.040	0.452
Multivariant model 2
sST2	1.025	1.013 to 1.037	< 0.001
IL-6	1.006	0.997 to 1.016	0.181
PCT	0.869	0.607 to 1.246	0.445

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Discussion

The prolonged implementation of non-pharmaceutical interventions to tackle the COVID-19 pandemic between 2020 and 2022 led to a significant “immunity debt”. Consequently, MP caused a prolonged non-seasonal epidemic during 2023 and 2024 across mainland China [18]. In this retrospective study, we evaluated sST2 as a prognostic biomarker in children diagnosed with MPP, and found that higher sST2 levels were associated with severe MPP, co-infections, and complications. More importantly, patients who experienced severe adverse events during hospitalization had significantly higher sST2 levels at admission. ROC analysis demonstrated that sST2 showed good predictive performance for these events. The prognostic value of sST2 remained significant after adjusting for age, sex, or other biomarkers. These findings support the potential of sST2 as a prognostic biomarker for risk stratification in pediatric MPP patients. Similarly, in adult CAP, sST2 has been shown to predict clinical stability, in-hospital mortality, and ICU admission, and improve the predictive performance of CURB-65 or PSI scores [12,13]. In children with severe pneumonia, sST2 at admission was associated with a higher 6-month readmission rate [19]. In children with SARS-CoV-2 related Multisystem Inflammatory Syndrome in Children (MIS-C), sST2 was associated with ICU admission [20]. It should be noted that sST2 is not a disease-specific marker, therefore it should be interpreted as a prognostic indicator rather than a diagnostic marker. To our knowledge, this is the first study to suggest sST2 as a predictor of short-term prognosis in pediatric MPP.

Previous studies have demonstrated that conventional inflammatory biomarkers are useful in risk stratification of children with pneumonia, including MPP. A previous study in pediatric CAP patients found that CRP and PCT may be useful in predicting the most severe outcomes, although they have limited use in discriminating non-severe from severe disease in children [21]. However, only 7.3% of cases in that study involved MPP. The levels of inflammatory biomarkers varied between CAP of different pathogens [22]. Studies in MPP found that lactate dehydrogenase has clinical value in predicting MP necrotizing pneumonia [23], and NLR and CRP were independent risk factors for severe MPP in children [24]. However, few studies have investigated the value of inflammatory biomarkers in predicting severe adverse events in pediatric MPP. We found that sST2 positively correlated with other inflammatory biomarkers and it showed better performance in predicting severe adverse events in this cohort based on AUC comparisons. The prognostic value of sST2 may be explained by the mechanism of sST2 involvement in modulating immune regulation and inflammation.

sST2 is a decoy receptor of IL-33, and the IL-33/ST2 axis plays a role in immune regulation [24]. IL-33 binding to membrane-bound ST2 enhances Th2 response. Notably, Th1 and Th17 cells both express sST2, and increased sST2 production promotes the Th1/Th17 response and reduces Th2/Treg response through sequestering IL-33 [25]. In addition, an in vitro study showed proinflammatory cytokines such as IL-1β and TNF-α also increase sST2 production in human lung epithelial cells [26]. High sST2 levels may reflect severe immune response and inflammation. Excessive immune activation is thought to drive severe pulmonary and extrapulmonary manifestations in MPP [18]. This may partly explain the association between sST2 and disease severity, complications, and severe adverse events. IL-33, as an alarmin, is released early during cell stress and damage, and it can activate the production of sST2 in mast cells or T cells [26]. The early production after the immune response may explain the advantage of sST2 at admission over other inflammatory biomarkers in risk stratification. Additionally, recent data from adults with COVID-19 and children with severe pneumonia also suggest that sST2 is a dual cardio-inflammatory biomarker related to both myocardial injury and severity of inflammation and has prognostic value [20,27]. In accordance, in our cohort, those with circulatory complications (i.e., abnormal cardiac biomarkers, tachycardia, septic shock, and myocarditis) had significantly higher sST2 levels compared to the rest (107.15 [91.88–178.97] vs. 34.75 [15.74–72.21] ng/mL, P < 0.001). This may also contribute to its predictive performance on prognosis. Further, the association of sST2 and severe adverse events also indicated that the IL-33/ST2 axis could represent a potential therapeutic target for MPP. Additional studies are required to determine whether modulation of this pathway could influence disease progression.

There are limitations of this study. First, the sample size was relatively small, with only 12 severe adverse events. This increased the risk of model overfitting and precluded robust adjustments for multiple confounders simultaneously. Second, the validated clinical severity scores (e.g., pediatric early warning scores) or oxygenation indices are absent in our cohort. This prevented direct comparison of the prognostic performance of sST2 against established clinical assessment tools to determine its incremental value. Furthermore, as a single center retrospective study conducted during a unique post-pandemic epidemic, the cohort may have selection bias and the findings may reflect a unique epidemiological context, potentially limiting the generalizability. In the future, we plan to conduct a multi-center prospective study with a larger study population and comprehensive clinical data to validate the clinical value of sST2 in children with pneumonia.

Conclusions

In this retrospective study of pediatric MPP patients, we found that sST2’s association with disease severity and it showed better performance in predicting severe adverse events than other inflammatory biomarkers. sST2 is a potential a prognostic biomarker in children with MPP which warrants further investigation.

Supporting information

S1 File. Definitions and classifications of severe adverse events and complications.

(PDF)

pone.0347651.s001.pdf^{(148.7KB, pdf)}

S1 Fig. Flow diagram of study election process.

(PDF)

pone.0347651.s002.pdf^{(95KB, pdf)}

S2 Fig. Associations of ST2 with other biomarkers.

(PDF)

pone.0347651.s003.pdf^{(102.2KB, pdf)}

S3 Fig. Associations of ST2 with days of hospital stay and days of pre-admission fever.

(PDF)

pone.0347651.s004.pdf^{(88.9KB, pdf)}

S4 Fig. Comparison of ST2 levels in patients with and without RMPP/MRMP.

(PDF)

pone.0347651.s005.pdf^{(80.9KB, pdf)}

S1 Data. The pediatric MPP dataset in EXCEL file format.

(XLSX)

pone.0347651.s006.xlsx^{(37KB, xlsx)}

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

The author(s) received no specific funding for this work.

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PLoS One. doi: 10.1371/journal.pone.0347651.r001

Decision Letter 0

Shivkumar Gopalakrishnan

20 Jan 2026

Dear Dr. YE,

Please submit your revised manuscript by Mar 06 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Shivkumar Gopalakrishnan, MD

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Additional Editor Comments :

Dear Dr ZHICHENG YE,

Your article is well conceived and holds promise. However, there are a few uncertainties which demand clarification.

1. Severe adverse events were defined as in-hospital death, ICU admission, diagnosis of sepsis or use of extracorporeal membrane oxygenation. Please specify the criteria used to define these, and what about the need for vent support, RRT, etc.

2. In some patients, sepsis might be mild without MODS, others severe sepsis with MODS. Was there any subgroup analysis performed and why if not?

3. You have mentioned that sST2 levels were significantly higher in severe MPP cases and those with co-infections. If co-infections were detected, what is the specificity for its association with MPP? Sst2 could have been elevated due to other infections as well. How do you explain the uncertainty?

4. You have mentioned that sST2 levels correlated positively with neutrophil count, neutrophil to lymphocyte ratio, PCT, CRP and IL-6- comparative analysis. Was sST2 more specific, more sensitive, cost effective, etc. What is the distinct advantage of doing Sst2, compared to performing NLR, ANC, PCT, CRP?

5. Lines 49-50: Studies reported mixing findings on accuracy of this method to predict patients’ outcome and inter-physician variations [4,5]- Grammar error needs to be corrected.

6. What is authenticity of definition of severe MPP? Your aim was to evaluate a biomarker whether it predicts severity. If only 4 parameters are assessed, there is a large gap of knowledge about those patients who were oxygen dependent, MODS but did not require organ support, those left with complications, destroyed lungs, etc.

7. Lines 83-84: The diagnosis of SMPP is made in MPP patients with either of the following criteria [2]- Grammar error needs to be corrected.

8. Line 140: media hospital stay was 7 days- grammar error

9. Line 142: ECOM- spelling error needs to be corrected.

10. The study design is ambiguous- criteria for severe MPP is delineated, however, the parameter Sst2 was evaluated only for 12 patients who had fit into 4 separate set of “adverse events”. These adverse events are overlapping over the defined criteria. It is unclear about sst2 levels among the actual population of severe MPP patients who qualified based on set criteria [2]. The study is actually assessing predictive capacity of sst2 to identify severe adverse events among MPP rather than severity of MPP.

11. Line 229: Consequently, MP infection undergone a prolonged non-seasonal epidemic, which emerged in April 2023- grammar error needs correction.

12. Title: Soluble ST2 is a potential biomarker for risk stratification of pediatric patients with mycoplasma pneumoniae pneumonia- better stated as sst2 as a biomarker for predicting severe adverse events among pediatric patients with MPP.

Thank you, with regards, Dr SHIVKUMAR GOPALAKRISHNAN, MD.,

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Partly

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: I Don't Know

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: The study explores the association of sST2 with in-hospital adverse events and clinical characteristics in pediatric MPP patients, and assesses its potential as a prognostic biomarker in comparison with conventional inflammatory markers. The work is sound but requires major revisions before publication.

Methods

A brief description of the study setting will be appropriate. Clarify if it's routine to have sST2 as a baseline investigation for MPP in the hospital.

Sampling: Throw more light on sampling. How many confirmed MPP in the study period, and how many were excluded. Include a flow diagram (as per PLOS ONE guidelines) showing total confirmed cases of MPP recorded in the study period, how many folders were reviewed, how many excluded, and the final sample analyzed.

Clearly define and specify (primary versus secondary outcome variables, factor variables) all variables. Consider listing all variables, measurement methods, and units for clarity.

Clarify how missing data were handled.

Analysis and Results

The whole table 1 lacks clarity. Some entries do not correspond with the labelling of the columns or rows. Authors should clarify why Table 1 contains ‘severe adverse event’ in both rows and columns. Also, the decimals with excess zeroes are not necessary for frequencies in the table. Clearly specify which values represent mean (sd), median (iqr), or frequency (%), in the table. Define what constitutes ‘other infections’ in the table?

Format and standardize the table and ensure statistical reporting follows PLOS ONE’s reporting standards.

The question remains, what is/are your primary outcome measure (s) (end-point (s))? Severe MPP? Severe adverse events? Complications?

Consider fitting logistic regression for Severe MPP if it is considered a primary outcome.

Recommendation: Your introduction section should include a conceptual framework to clarify the multiple relationships under study. For instance, how presenting disease severity may be associated with high levels of sST2, how sST2 may in turn be related to in-hospital adverse events, and/or complications.

Discussion

The discussion requires focus and direction. Provide clearer comparisons with existing evidence, focusing more on the association between sST2 and the primary outcome(s).

Minor

Minor typographical and grammatical errors include wrong spellings and tenses (E.g. Line 62: Studies have shown and not ‘have showed’), missing letters; the whole manuscript requires proofreading before resubmission.

Reviewer #2: This study evaluates soluble ST2 (sST2) as a prognostic biomarker in pediatric Mycoplasma pneumoniae pneumonia and addresses a clinically important and timely question. The authors show that sST2 is strongly associated with disease severity, complications, and in-hospital adverse events, with superior predictive performance compared to several conventional inflammatory markers. The study is well conducted and biologically plausible; however, its retrospective single-center design, small number of severe adverse events, and lack of clinical severity indices limit generalizability and warrant cautious interpretation. Overall, the findings are promising and support the need for larger, prospective, multicenter studies to validate sST2 for clinical risk stratification.

Reviewer #3: Introduction

The Introduction is relevant but needs improvement

Makes Sentences more clear and simple ,

Avoid using terms like “clinical gestalt” and better to be replaced with standard language,

Prefer to use more neutral tone and avoid assertive, strong statements

The research gap is clearly defined; but could be strengthened by briefly stating what is unknown about sST2 in pediatric MPP.

Correction of minor language and formatting issues also needed

Materials and Methods

The Materials and Methods section is looks clear,

clarification of the study timeline should be done.

explain the reasons for exclusion criteria,

specify the source of diagnostic guidelines

details on the laboratory methods used should be provided especially information about sST2 measurement

make the ethics statement more concise and properly formatted.

The Clinical Data Collection

provide basic descriptions but lack essential methodological details.

provide more details about The data collection and extraction, how complications and adverse events were defined and classified.

Some outcomes like sepsis needs to be referenced to established criteria,

More clear categorization of the complications is needed. cardiac complications—such as myocarditis, pericarditis, arrhythmias, and heart failure—should be clearly highlighted, given some evidence linking soluble ST2 to cardiac injury in recent studies.

Laboratory Tests

Provide information on sample timing, assay characteristics, units, and data handling.

Both subsections require clearer definitions, standardized terminology

Correct grammatical and formatting issues

Results

The Results section contains valuable data but

Sentence needs to be more clear with correction of the language errors

Write the result with more direct and objective way

More clear and correct terminology should be used

Tables and figures need better organization, and more detailed sand clear tatistical reporting is needed

Discussion

Although the discussion is well-organized, but should be written in more clear, direct objective way aligning with the nature of this single-center, retrospective study.

Present any potential therapeutic or predictive outcomes as theoretical possibilities or areas for future investigation, not as current treatment protocols or guaranteed outcomes.

Conclusions avoid statement that indicate accuracy and superiority.

limitations should be extended to properly reflect design and analytical difficulties.

**********

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Reviewer #1: Yes: Muhyideen BashirMuhyideen BashirMuhyideen BashirMuhyideen Bashir

Reviewer #2: No

Reviewer #3: No

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pone.0347651.s007.pdf^{(1.1MB, pdf)}

PLoS One. 2026 Apr 17;21(4):e0347651. doi: 10.1371/journal.pone.0347651.r002

Author response to Decision Letter 1

25 Mar 2026

We have reformatted the manuscript according to the two style templates.

The ORCID iDs for the corresponding authors have been updated via submission system

（https://orcid.org/my-orcid?orcid=0009-0009-6967-6969，ORCID：0009-0009-6967-6969）.

For additional information about PLOS ONE ethical requirements for human subjects research, please refer to http://journals.plos.org/plosone/s/submission-guidelines#loc-human-subjects-research.

This study was approved by the Ethics Committee of the Children’s Hospital of Fudan University (Ethics No:2024-129), and need for consent was waived by the ethics committee. We have included an Ethics Statement subsection in the Materials and methods: “This study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the Children’s Hospital of Fudan University (Ethics No. 2024-129). We confirm that all methods were performed in accordance with the relevant guidelines and regulations. This study is a retrospective analysis, only involving the statistical analysis of patient data, and all patient information has been anonymized and does not contain sensitive data; therefore, the requirement for informed consent was waived by the ethics committee.”

The same text has been added to the “Ethics Statement” field of the submission form via the submission system.

As described above, the full ethics statement has been provided in the ‘Materials and methods-Ethics Statement’ and via the online submission system.

The supplementary figures have been removed from the manuscript file and a list of supplementary figure captions has been added in the manuscript after the reference list. A supporting document named S1-4_Fig.pdf is uploaded via the submission system.

6. Please ensure that you refer to Figure 2 in your text as, if accepted, production will need this reference to link the reader to the figure.

It was a typo and we have corrected it. Figure 2 is now referred to in ‘Results- Correlation between sST2 and other laboratory biomarkers and clinical parameters’ section.

It was a typo and we have corrected it. Table 2 is now referred to in ‘Results- Associations of sST2 with severe adverse events during hospital stay’ section.

8. Please include a copy of Table 3, which you refer to in your text on page 15.

It was a typo and it should be ‘Table 2’. We have corrected it.

A list of supplementary figure and file captions has been added after the reference list. The supplementary figures are cited as S1-S4 Fig in the manuscript. A supporting document containing these figures has been uploaded via the submission system (S1-4_Fig.pdf). Another supporting file (S1_File.pdf) has been added too.

We have revised the manuscript according to the editor and the reviewers’ feedback.

�Additional Editor Comments :

1.Severe adverse events were defined as in-hospital death, ICU admission, diagnosis of sepsis or use of extracorporeal membrane oxygenation. Please specify the criteria used to define these, and what about the need for vent support, RRT, etc.

When we designed this study, we conducted a literature search on prognostic studies of biomarkers in both adult and pediatric patients with pneumonia. We found some variations in defining severe short-term outcomes for pneumonia patients. However, most papers used combinations of death, ICU care, sepsis, septic shock, ECMO, mechanical ventilation, vasoactive infusions, chest drainage, respiratory failure, empyema, and ARDS (see, for example, References 4,8,13 and 21 in the manuscript) as outcome factors. For our study, we chose severe adverse events as a composite endpoint, including in-hospital death, ICU admission, diagnosis of sepsis, and use of ECMO—all of which represent death or life-threatening conditions.

1.In-hospital death is death that occurred during hospitalization.

2.In our hospital, a patient is typically admitted to the ICU if at least one of the following conditions is present: (1) FiO2 ≥ 0.6, SaO2 ≤ 0.92, (2) shock and/or impaired consciousness, (3) tachypnea and tachycardia with severe respiratory distress or signs of exhaustions, with or without elevated PaCO2, (4) recurrent apnea, or slow and irregular breathing, and (5) other conditions requiring further monitoring and treatment (e.g. renal failure, severe thrombocytopenia)

3.The definition of sepsis follows the Third International Consensus Definitions for sepsis as life-threatening organ dysfunction caused by a dysregulated host response to infection, where organ dysfunction can be identified as ≥ 2 points on the Phoenix Sepsis Score in children with suspected infection [1].

4.The use of ECMO is mainly indicated for patients requiring respiratory support due to: (1) PaO2/FiO2 < 60-80 mmHg due to severe respiratory failure; (2) failure of conventional ventilation and/or other rescue therapies; (3) high ventilator settings (e.g., mean airway pressure > 20 - 25 cm H₂O during conventional ventilation or > 30 cm H₂O during high-frequency ventilation, or signs of iatrogenic barotrauma). ECMO is also indicated for circulatory support in cases such as cardiogenic shock unresponsive to standard medication, with low SBP < 50 mmHg, urine volume < 1 ml / (kg·h), lactic acidosis, central venous oxygen saturation < 0.6, altered mental status due to low cardiac output, and refractory septic shock with an epinephrine dose > 1 μg / (kg·min) or vasoactive-inotropic score >100.

Regarding renal replacement therapy and ventilation support, we collected information from medical records. Three patients received renal replacement therapy, and these patients were all admitted to the ICU. 6 patients received ventilation support including ECMO (n=4) and invasive mechanical ventilation (n=2). All of these patients were admitted to the ICU. Therefore, all patients who received mechanical ventilation or renal replacement therapy were included in the severe adverse events group.

We reviewed all the patient data to confirm sepsis diagnosis. Based on the diagnostic criteria, nine patients met the definition for sepsis, and all were included in the severe adverse events group.

The manuscript has been revised accordingly. The definitions have been provided in the supporting document ‘S1 File’ cited in the manuscript.

[1] Schlapbach LJ, Watson RS, Sorce LR, Argent AC, Menon K, Hall MW, et al. International Consensus Criteria for Pediatric Sepsis and Septic Shock. JAMA. 2024;331(8):665-674. doi:10.1001/jama.2024.0179

2.In some patients, sepsis might be mild without MODS, others severe sepsis with MODS. Was there any subgroup analysis performed and why if not?

There are nine sepsis patients in our cohort, among whom eight had multiorgan dysfunction. The most common organ dysfunctions happened in respiration system (n = 9) and coagulation system (n = 8), followed by circulation system (n = 4) and renal system (n = 3). One patient had only respiratory failure and received invasive mechanical ventilation. Due to a small sample size of sepsis patients, we did not perform subgroup analysis.

3.You have mentioned that sST2 levels were significantly higher in severe MPP cases and those with co-infections. If co-infections were detected, what is the specificity for its association with MPP? Sst2 could have been elevated due to other infections as well. How do you explain the uncertainty?

In this study, we have demonstrated that sST2 levels were significantly higher in severe MPP, MPP with complications, and MPP with severe adverse events. We consider sST2 to be a prognostic marker, rather than a diagnostic marker. Due to the biological functions of sST2, high sST2 levels may reflect excessive immune response and severe inflammation.

sST2 is not specific to MPP. Previous studies (Reference 12,27) have shown that sST2 levels increase in pneumonia caused by other pathogens. We found that sST2 levels were higher in MPP with co-infections. A higher proportion of patients with co-infections were in the severe MPP group compared to the proportion of patients with MP infection alone, although this difference did not reach statistical significance (37.3% vs. 23.4%, P = 0.0723). We think this may partly explain the association of sST2 and co-infection.

Meanwhile, among the patients with only MP infection, sST2 levels in severe MPP patients were higher than non-severe MPP (30.8 [14.5, 75.4] vs 22.6 [12.8, 52.0] ng/mL, P=0.361), although it did not reach statistical significance, possibly due to a relatively small sample size. Further, among the patients with only MP infection, three patients had severe adverse events and their sST2 levels (49.2, 145.2, and 189.7 ng/mL respectively) were significantly higher than the rest (22.6 [12.8, 52.0] ng/mL), but the sample size is very small. These findings warrant further validation in a larger cohort.

We will conduct a prospective study to evaluate the role of sST2 in children of community acquired pneumonia caused by different pathogens including MP with a larger sample size in the future.

4.You have mentioned that sST2 levels correlated positively with neutrophil count, neutrophil to lymphocyte ratio, PCT, CRP and IL-6- comparative analysis. Was sST2 more specific, more sensitive, cost effective, etc. What is the distinct advantage of doing Sst2, compared to performing NLR, ANC, PCT, CRP?

In this study, we aimed to demonstrate the value of sST2 in predicting severe adverse events as a prognostic marker. Through comparisons of ROC AUCs, we found that sST2 had better performance in predicting severe adverse events in this cohort. Its AUC was significantly higher than those of CRP and NLR, and higher than those of PCT and IL-6 without statistical significance. Furthermore, in our cohort, sST2 was the only independent biomarker associated with severe adverse events in a multiple logistic regression analysis which included sST2, PCT and IL-6 as independent variables (see Results—Associations of sST2 with severe adverse events during hospital stay). Therefore, we think sST2 may predict severe adverse events more effectively than conventional inflammatory biomarkers. However, this retrospective study has several limitations as we have mentioned in our manuscript, therefore we plan to confirm these findings in a prospective study with a larger sample size.

5.Lines 49-50: Studies reported mixing findings on accuracy of this method to predict patients’ outcome and inter-physician variations [4,5]- Grammar error needs to be corrected.

We corrected it as “Previous studies have reported mixed results regarding the accuracy of this approach as well as substantial inter-physician variability”.

6.What is authenticity of definition of severe MPP? Your aim was to evaluate a biomarker whether it predicts severity. If only 4 parameters are assessed, there is a large gap of knowledge about those patients who were oxygen dependent, MODS but did not require organ support, those left with complications, destroyed lungs, etc.

The diagnosis for MPP and severe MPP were established in accordance with the Chinese guideline for the diagnosis and treatment of childhood Mycoplasma pneumoniae pneumonia (2023) (Reference 1, 2). This guideline was issued by the National Health Commission of the People's Republic of China.

The definition of severe MPP is provided in the ‘Materials and Method- Clinical data collection section’, as ‘MPP patients with any of the following criteria[1,2]: (1) high fever (> 39°C) for more than 5 days or fever for more than 7 days without a declining trend in peak temperature; (2) hypoxemia (maintained an SaO2 < 92% on room air); (3) increasing respiratory and pulse rates with clinical evidence of respiratory distress and exhaustion with or without a elevated PaCO2; (4) signs of intrapulmonary infection, such as moderate to large pleural effusion, large area of pulmonary consolidation, plastic bronchitis, pulmonary embolism, necrotizing pneumonia, and acute asthma exacerbations; and (5) signs of extrapulmonary complications, such as meningoencephalitis, ascending (i.e., Guillain-Barré) paralysis, myopericarditis, erythema multiforme, autoimmune hemolytic anemia, hemophagocytic syndrome, or disseminated intravascular coagulation’ According to the above definition, the MPP patients with complications, and destroyed lungs requiring oxygenations are included in severe MPP.

Although our primary aim is to evaluate the association of sST2 with severe adverse events, we evaluated its association with clinical characteristics, including severe MPP. We found that sST2 levels in the severe MPP patients were significantly higher (71.91 [30.76-140.12] vs 26.2 [15.15-54.69], P < 0.001). Among the severe MPP patients, sST2 levels were significantly higher in those with severe adverse events compared to those without (170.7 [132.5, 246.3] vs. 45.1 [18.0, 90.7] ng/mL, P < 0.001).

7.Lines 83-84: The diagnosis of SMPP is made in MPP patients with either of the following criteria [2]- Grammar error needs to be corrected.

We corrected it as “Severe MPP was defined as MPP with any of the following criteria”.

8.Line 140: media hospital stay was 7 days- grammar error

We corrected it as “median hospital stay”.

9.Line 142: ECOM- spelling error needs to be corrected.

We corrected it as “ECMO”.

10.The

Attachment

Submitted filename: 260325 Response to Reviewers.docx

pone.0347651.s009.docx^{(43.5KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0347651.r003

Decision Letter 1

Shivkumar Gopalakrishnan

30 Mar 2026

Dear Dr. YE,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Additional Editor Comments:

Dear Dr. YE,

Your article is well conceived and holds promise. However, there are a few uncertainties which demand clarification.

2. In some patients, sepsis might be mild without MODS, others severe sepsis with MODS. Was there any subgroup analysis performed and why if not?

5. Lines 49-50: Studies reported mixing findings on accuracy of this method to predict patients’ outcome and inter-physician variations [4,5]- Grammar error needs to be corrected.

7. Lines 83-84: The diagnosis of SMPP is made in MPP patients with either of the following criteria [2]- Grammar error needs to be corrected.

8. Line 140: media hospital stay was 7 days- grammar error

9. Line 142: ECOM- spelling error needs to be corrected.

11. Line 229: Consequently, MP infection undergone a prolonged non-seasonal epidemic, which emerged in April 2023- grammar error needs correction.

Thank you, with regards, Dr SHIVKUMAR GOPALAKRISHNAN, MD.,

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PLoS One. 2026 Apr 17;21(4):e0347651. doi: 10.1371/journal.pone.0347651.r004

Author response to Decision Letter 2

1 Apr 2026

PONE-D-25-53987

Soluble ST2 is a potential biomarker for risk stratification of pediatric patients with mycoplasma pneumoniae pneumonia

PLOS One

Dear Editor and Reviewers,

Thank you very much for your valuable comments and constructive suggestions on our manuscript. We have carefully gone over all the feedback and revised the manuscript accordingly. The replies to all the comments are provided in the uploaded response document. A revised manuscript has been uploaded too.

We appreciate all your time and efforts in helping us make this study and this manuscript better.

We look forward to hearing from you.

Best regards,

Zhicheng Ye

The responses to the editor and the reviewers are as the below in blue.

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