Bronchoalveolar lavage combined with metagenomic assessment of children hospitalized with Mycoplasma pneumoniae pneumonia

Bronchoalveolar lavage combined with metagenomic assessment of children hospitalized with Mycoplasma pneumoniae pneumonia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Bronchoalveolar lavage combined with metagenomic assessment of children hospitalized with Mycoplasma pneumoniae pneumonia Yun Pan, Dingxiang Lai, Haitao Lv, Yahua Zhang, Huiqing Xu, Qinsong Ye, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4833361/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: The purpose of this study was to investigate the clinical value of bronchoalveolar lavage (BAL) combined with metagenomic assessment for children hospitalized with Mycoplasma pneumoniae pneumonia (MPP). Methods: This study included children hospitalized with MPP at The Affiliated Women and Children's Hospital of Ningbo University from May 2023 to August 2023. The patients were divided into a moderate M . pneumoniae pneumonia (MMPP) group and a severe M. pneumoniae pneumonia (SMPP) group according to the results of the community-acquired pneumonia (CAP) assessment at admission. All the patients underwent fibre bronchoscopic lavage within 3–7 days after macrolide treatment. Patient throat swab (TS) and bronchoalveolar lavage fluid (BALF) samples were collected for metagenomic next-generation sequencing (mNGS) analysis. The patients were divided into a macrolide-sensitive M. pneumoniae (MSMP) group and a macrolide-resistant M. pneumoniae (MRMP) group according to the presence of macrolide resistance genes in 23S rRNA domain V. The respiratory microbial community diversity was also analysed. Differentially abundant species prediction in TS and BALF samples between the MSMP group and the MRMP group was performed. The maximum-likelihood phylogenetic trees of some patients were also analysed. Results: During the 4-month study, TS and BALF samples were collected from 60 children aged 2-12 years, with a median age of 7 years . On the basis of the CAP assessment results at admission, 48 patients were included in the MMPP group, and 12 patients were included in the SMPP group. The total number of febrile days, D-dimer levels, and procalcitonin (PCT) levels were increased in the SMPP group. On the basis of the mNGS results, these patients were divided into 48 cases of MSMP and 10 cases of MRMP (all cases were A2063G positive). The MP detection rate in TS samples was 92% (55/60), and that in BALF samples was 97% (58/60). The Shannon index of the MRMP group was lower than that of the MSMP group in the BALF samples (p<0.01). The abundance of Actinomyces naeslundii and the relative abundance of MP had optimal predictive performance for MRMP in TS and BALF samples. Five strains (MRMP) from MP12, MP20, MP23, MP48, and MP57 exhibited the closest relationships with the strain M. pneumoniae -15-885, which was isolated in Seoul in 2015. One strain (MSMP) from MP05 was most closely related to the strain M. pneumoniae -E57, which was isolated in Egypt in 2009. Conclusion: MRMP is not the main cause of SMPP-coinfection is. In terms of relative abundance, in MRMP patients, MP is dominant in BALF specimens. TS specimens cannot replace BALF samples for determining the aetiology of MPP. A. naeslundii and the relative abundance of MP are optimal biomarkers for predicting whether a case is MRMP in TS samples and BALF samples, respectively. The majority of the MRMP strains from this study are closely related to the strain isolated in Seoul in 2015. Bronchoalveolar lavage metagenomic Mycoplasma pneumoniae pneumonia Community-acquired pneumonia macrolide resistance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Community-acquired pneumonia (CAP) is a prevalent cause of hospitalization in children[1], with Mycoplasma pneumoniae (MP) being a significant pathogen, accounting for 10–40% of CAP cases in school-aged children[2, 3]. Over the past three decades, conjugate vaccines targeting Streptococcus pneumoniae and Haemophilus influenzae type b have reduced the number of cases caused by these former main causes of CAP. However, a safe vaccine against MP infection has not yet been developed, despite the recent discovery of a new target—community-acquired respiratory distress syndrome toxin (CARDS TX), which is released by MP[4]. The incidence and aetiological spectrum of CAP have changed substantially due to the above issues. The introduction of nonpharmaceutical interventions (NPIs) against COVID-19 in early 2020 led to the disappearance of almost all respiratory pathogens, and the reappearance of MP did not occur till the autumn of 2023[5], and it appeared earlier in China. M. pneumoniae pneumonia (MPP) is characterized by nonspecific clinical manifestations such as cough, fever, and chest radiographic changes. The “gold standard” for determining the aetiology of MPP is the detection of MP in samples taken directly from the lungs via bronchoalveolar lavage fluid (BALF), pleural fluid sampling, or lung biopsy or aspiration. Culture of MP is expensive and time-consuming because of the slow growth of this species in vitro[6, 7], and the sensitivity of this method is approximately 60% that of PCR[8]. Taxonomic profiling via metagenomic next-generation sequencing (mNGS) techniques has higher sensitivity and resolution than conventional culture approaches, providing a better understanding of microbial compositions. Since 2000, the emergence of more macrolide-resistant M. pneumoniae (MRMP) strains[9] and coinfection with other pathogens ( S. pneumoniae , H. parainfluenzae , EB virus) have led to an increasing number of refractory M. pneumoniae pneumonia (RMPP) cases[10]. BAL is not used only to obtain BALF samples. In this study, we used it as a treatment for children hospitalized with MPP, especially severe M. pneumoniae pneumonia (SMPP) and MRMP patients. Although BAL was previously considered an invasive examination method, with the development of fiberoptic bronchoscopy technology and the increase in physician experience, the amount of injury can be minimized. Moreover, BALF samples can be subjected to mNGS to determine the aetiology of MPP. The latest guidelines for childhood CAP issued by the Infectious Diseases Society of America (IDSA)[11] and the British Thoracic Society (BTS)[12] were issued more than a decade ago. During this period, new insights into the causes and treatment of childhood MPP have been obtained, and the guidelines need to be updated. Methods Patients and Samples This study included children hospitalized with MPP at The Affiliated Women and Children's Hospital of Ningbo University from May 2023 to August 2023. In this study, patients were enrolled on the basis of abnormal chest X-ray imaging results, including chest infiltration, consolidation, and pleural cavity effusion. Serological tests for MP igM- or igG-positive or MP-positive samples were performed via PCR in nasopharyngeal aspirates. The patients were divided into the MMPP group and the SMPP group according to the results of the admission CAP assessment, with the following evaluation criteria: 1) severely worsened general condition; 2) altered mental status; 3) capillary refill time >2 sec; 4) dyspnoea, apnoea, or cyanosis; 5) SpO 2 <90%; and 6) lack of feeding, vomiting, or signs of dehydration. All the patients underwent fibre bronchoscopic lavage within 3–7 days after macrolide treatment. The patients’ throat swab (TS) and BALF samples were collected for mNGS analysis. These patients were divided into the MSMP and MRMP groups according to whether they had macrolide resistance genes in 23S rRNA domain V. The current study was approved by the IRB of The Affiliated Women and Children's Hospital of Ningbo University (No. EC2023-015). mNGS Analysis For preprocessing of specimens, TS and BALF samples were homogenized via dithiothreitol (DTT) treatment. The cells were removed via centrifugation to reduce the background levels of host nucleic acids. The HostZERO Microbial DNA Kit (Zymo Research, USA) was used to extract total DNA according to the manufacturer's instructions. The yield of the extracted DNA was quantified via a Quant-iT-dsDNA HS assay kit and a Qubit 3.0 fluorometer (Thermo Scientific, USA). The DNA molecules were fragmented via enzymatic shearing (~200 bp), and the Nextera XT DNA Library Preparation Kit (Illumina, USA) was subsequently used to construct the library. A certain number of PCR cycles (5-7) were applied to generate a 1 μg macrogenomic library. The quality of the library was evaluated via a 2100 Bioanalyzer with high-sensitivity DNA analysis (Agilent Technologies, USA). Metagenome shotgun sequencing in single-end 75-bp mode was performed via the NextSeq 500/550 High Output Kit (92 cycles) on an Illumina NextSeq 550 sequencer. Samples serving as nontemplate controls (NTCs) were also sequenced to assess contamination during wet-laboratory experiments. Bioinformatics Analysis of Species-Level Abundance Profiles The raw sequencing data were first subjected to quality control by trimming adapter sequences and removing low-quality reads via Trimmomatic v0.36 for tail trimming and reading. To remove duplicate reads that were considered technical artefacts by PCR, deduplication was performed via an in-house Perl script. Microbial reads were classified via Kraken v2.0.9-beta and a custom k-mer database constructed from 51,543 genomes from approximately 27,000 species in the NCBI assembly database. To estimate species-level abundances, the Bayesian algorithm implemented in Bracken v2.2 further estimated the number of reads in the Kraken taxonomy report. On the basis of the information for each batch of NTC samples, microbial taxa that may have been present as contaminants during the experiment were removed from the final map. The estimated relative abundance percentage of each bacterial, viral (excluding bacteriophages), and fungal species was calculated separately. To investigate the pathogenic signals in the sputum microbiome, we proposed an empirical rule to assess the detection limit for the pathogenic risk of individual organisms through species-level abundance analysis. In short, the average and standard deviation values of relative abundance were calculated on the basis of the estimated values for each species in the sample. Strain-Level Phylogenetic Analysis Strain level analysis was performed on the basis of single-nucleotide variations (SNVs) via the StrainPhlAn v3.0 software package. Briefly, metagenomic reads per sample were first aligned to the marker gene database of MetaPhlAn v3.0. For the most abundant strains of each species, we reconstructed the labelled sample-specific consensus sequences and extracted species-specific markers from MetaFlAn to identify homologous sequences in the genomes of the reference isolates via BLASTN. On the basis of sequence alignment, the maximum likelihood phylogenetic tree was reconstructed via RAxML and then visualized via MEGA10. The genome sequences of the isolated strains were retrieved from the NCBI Assembly database and used for phylogenetic reconstruction together with the strains detected in the metagenomic samples. The MetaMLST v1.2.2 program was used to predict known and novel sequence types of the most abundant strains in the metagenomic samples. Owing to the complexity of determining the phylogeny of diploid fungi, an alternative method was used to construct a phylogenetic tree on the basis of multilocus sequence typing (MLST) analysis of seven loci. Seven MLST allele sequences were reconstructed from the metagenome via MetaMLST, and the relevant gene sequences in the isolate genome were processed via FastMLST. Multiple sequence alignment was performed via MUSCLE, and phylogenetic inference was performed via RAxML. Statistical Analysis of Community Diversity On the basis of the Chao1 index representing community richness, diversity analysis and t tests for two samples were conducted via the fossil v0.4.0 software package. To estimate the variation in sample community composition between groups, β diversity was measured by calculating the Bray‒Curtis distance matrix via Vegan v2.5-7. The similarity of within- and between-group rank differences was inferred via a nonparametric ANOSIM test with 1000 permutations. All the statistical analyses and visualizations were performed in R v4.1.0. Results Characteristics of Patients and Samples During the 4-month study, TS and BALF samples were collected from 60 children aged 2-12 years, with a median age of 7 years ( Figure 1 ). On the basis of the CAP assessment results at admission, 48 patients were included in the MMPP group, and 12 patients were included in the SMPP group. The clinical characteristics and laboratory test results of the MMPP and SMPP patients are listed in Table 1 . The total number of febrile days in the SMPP group was greater than that in the MMPP group. The D-dimer level in the SMPP group was higher than that in the MMPP group. The procalcitonin (PCT) level in the SMPP group was approximately twice that in the MMPP group, while the other characteristics were not significantly different. On the basis of the results of mNGS, these patients were divided into 48 cases of MSMP and 10 cases of MRMP (all cases were A2063G positive). Two cases were excluded because they were MP negative in both the TS and BALF samples. The clinical characteristics and laboratory test results of the MSPP and MRMP patients are listed in Table 2 . Community Structure of the TS and BALF Microbiota The MP detection rate in TS samples was 92% (55/60), and that in BALF samples was 97% (58/60) ( Figure 2A ). In addition to MP, the five most abundant species in the TS samples were Haemophilus parainfluenzae (85%), Epstein–Barr virus (28%), Candida albicans (18%), human herpesvirus 7 (15%), and Staphylococcus aureus (10%) ( Figure 2B ). In addition to MP, the five most abundant species in the BALF samples were H. parainfluenzae (28%), C. albicans (12%), Klebsiella pneumoniae (8%), Epstein–Barr virus (7%), and S. aureus (5%) ( Figure 2B ). To compare the community structure of the TS and BALF microbiota between MSMP and MRMP, the top 20 species for a single case are listed in Figure 2C and Figure 2E , respectively. The top 20 species for the total cases are listed in Figure 2D and Figure 2F . In terms of relative abundance, MP was dominant in the MRMP group among BALF specimens (Figure 2F) . To further explore the dominance of MP in the MRMP group, we compared the α diversity of community richness between MSMP and MRMP in TS and BALF samples. The α diversity analysis of community richness revealed no significant difference between the two groups in TS specimens ( Figure 3A ), whereas the Shannon index of the MRMP group was lower than that of the MSMP group in BALF specimens (p<0.01) ( Figure 3B ). The β diversity based on the sample distribution diagram (PC, MDS, NMDS) between the MSMP group and the MRMP group showed a similar distribution in both the TS samples ( Figure 3C ) and the BALF samples ( Figure 3D ). On the basis of the β-diversity ANOSIM test, the mean of the ranked dissimilarities between the MSMP group and the MRMP group was not significantly different in the TS samples ( Figure 3E ) and BALF samples ( Figure 3E ). Prediction of Differentially Abundant Species in TS and BALF Specimens between the MSMP and MRMP Groups In addition to comparing the composition of pathogenic microorganisms in the upper and lower respiratory tracts, we also attempted to use intergroup differences in species abundances to predict whether MRMP emerged. Linear discriminant analysis (LDA) effect size analysis of the differentially abundant species between the MSMP group and the MRMP group in the TS samples revealed three such species, namely, Prevotella oralis , Actinomyces naeslundii , and Corynebacterium durum ( Figure 4A ). These three species were combined to construct a ROC curve for the prediction of multiple biomarkers (AUC=0.692) ( Figure 4B ). When single species were used to construct a ROC curve, A. naeslundii exhibited the best predictive performance (AUC=0.78, 95% CI (0.63, 0.92), KW_P=0.007) ( Figure 4C ). LDA effect size analysis of the differentially abundant species between the MSMP group and MRMP group in the BALF samples revealed nearly 70 species ( Figure 4D ). These species were combined to construct a ROC curve for the prediction of multiple biomarkers (AUC=0.775) ( Figure 4E ). The relative abundance of M. pneumoniae in the BALF samples from the MSMP group and MRMP group was used as a biomarker to construct a ROC curve to predict whether it MRMP was present (AUC: 0.86, 95% CI (0.75, 0.97)) ( Figure 4F ).  Strain-Level Characteristics of Pathogens  Strain identification is highly important for better understanding microbial pathogenicity and the transmission of clinically relevant microorganisms. StrainPhlAn analysis reveals strain-level phylogenetic trees visualizing the relatedness between the metagenome-recovered strains and clinical bacterial isolates with publicly available genome sequences. M. pneumoniae strains were reconstructed from the metagenomic samples of six patients from the MRMP group (MP12, MP20, MP23, MP48, and MP57) and the MSMP group (MP05) ( Figure 5 ). Five strains, from MP12, MP20, MP23, MP48, and MP57, exhibited the closest relationships with the strain M. pneumoniae -15-885 (GCA_009941325.1), which was isolated in Seoul in 2015. One strain from MP05 was most closely related to the strain M. pneumoniae -E57 (GCF_002128005.1), which was isolated in Egypt in 2009. Discussion MP is an important pathogen causing CAP in school-aged (mainly 5–10 years old) children. It is transmitted mainly through the respiratory tract, and pandemics occur every 3 to 5 years[3]. The cases studied herein were from the recent outbreak in 2023. Currently, macrolides are the recommended first-line treatment for MP infection. In recent years, the widespread use of these drugs in patients with various types of infections has led to an increase in macrolide resistance[13, 14]. However, it remains unclear whether there is a definite connection between the severity of MPP and MRMP. We divided the patients into the MMPP and SMPP groups according to the results of the admission CAP assessment and found that MRMP was not the main cause of SMPP, the co-infection was ( Table 1 ). The increase in PCT in the SMPP group also suggested a greater correlation between coinfection and the severity of MPP. Although the total number of febrile days in the SMPP group was greater than that in the MMPP group, there was no difference in the total length of hospital stay between the two groups. The increase in the D-dimer level in the SMPP group indicated that patients with severe infection had a greater risk of thrombosis than those with moderate infection. Both SMPP patients and MRMP patients need effective drugs and treatments other than macrolides. Drugs such as tetracyclines or quinolones can be used as alternatives for treating MRMP, but they are not appropriate for children under 12 years of age due to osteotoxicity[15]. In this study, we attempted to use BAL to treat children hospitalized with MPP. On the other hand, the “gold standard” for determining the aetiology of MPP is the detection of MP in samples taken directly from the lungs via BAL. Therefore, we performed BAL on all 60 patients. BAL washed away most of the pathogens and removed the sputum blocked in the bronchial orifice. This enabled better absorption of drugs, especially drugs inhaled by atomization. In addition, MP grows very slowly, which is beneficial for reducing the possibility of recurrence after BAL. Hospital stays after BAL were similar between the MMPP group and the SMPP group ( Table 1 ). We were curious about whether TS samples can replace BALF samples for determining the aetiology of MPP, so both TS samples and BALF samples were collected. The results revealed that 3 of the BALF samples were MP positive, whereas the TS samples were MP negative (Figure 2A). Thus, TS samples cannot be used to replace BALF samples. The results of mNGS revealed that 10 cases of MRMP (17%) were detected among these patients, which was inconsistent with several Chinese studies (with values ranging from 60 to 92%)[16-20]. We speculate that this may be related to the fact that we included only hospitalized patients. There was no significant difference in clinical manifestations between the MSMP group and the MRMP group ( Table 2 ). Another difference between the TS samples and BALF samples was that there were many colonizing bacteria in the TS samples, which also increased the relative abundance of MP in the BALF samples compared with that in the TS samples ( Figure 2D, Figure 2F ). On the basis of the α diversity analysis of community richness between the MSMP group and the MRMP group in the BALF samples, it can be inferred that MP was the dominant strain in the MRMP group ( Figure 3B ). However, the results of the β diversity analysis of community richness between the MSMP group and the MRMP group were not statistically significant. These findings indicate that the compositions of the microbiota in the MSMP group and MRMP group were similar. To further investigate the dominance of MP in MRMP, we used the relative abundance of MP as a biomarker to predict whether a case was MRMP. The results showed that this biomarker had good predictive performance ( Figure 4D ). Interestingly, in the TS samples, although the LDA score of A. naeslundii was not the highest, its predictive performance as a biomarker was the best ( Figure 4C ). Finally, we performed sequence alignment on strains to trace their origins. The results revealed that five strains (MRMP) were most closely related to the strain isolated in Seoul in 2015, and one strain (MSMP) was most closely related to the strain isolated in Egypt in 2009 ( Figure 5 ). In summary, this study provides new insights into the diagnosis and treatment of children hospitalized with MPP. The combination of BAL and metagenomic assessment can not only accelerate the recovery of patients but also provide evidence for epidemiological investigations of MPP. However, this study also had several limitations; for example, the sample size was not very large, and the mNGS detection results did not include RNA viruses. Conclusion MRMP is not the main cause of SMPP-coinfection is. In terms of relative abundance, in MRMP patients, MP was dominant in BALF specimens. TS specimens cannot replace BALF samples for determining the aetiology of MPP. A. naeslundii and the relative abundance of MP are optimal biomarkers for predicting whether a case is MRMP in TS samples and BALF samples, respectively. A majority of the MRMP strains from this study are closely related to the strain isolated in Seoul in 2015. Abbreviations BAL: Bronchoalveolar lavage MP: Mycoplasma pneumoniae MPP: Mycoplasma pneumoniae pneumonia MMPP: moderate M . pneumoniae pneumonia SMPP: severe M. pneumoniae pneumonia CAP: community-acquired pneumonia TS: throat swab BALF: bronchoalveolar lavage fluid mNGS: metagenomic next-generation sequencing MSMP: macrolide-sensitive M. pneumoniae MRMP: macrolide-resistant M. pneumoniae PCT: procalcitonin CARDS TX: community-acquired respiratory distress syndrome toxin NPIs: nonpharmaceutical interventions IDSA: Infectious Diseases Society of America BTS: the British Thoracic Society DTT: dithiothreitol NTCs: nontemplate controls SNVs: single-nucleotide variations MLST: multilocus sequence typing LDA: Linear discriminant analysis Declarations Ethics approval and consent to participate The current study was approved by the IRB of The Affiliated Women and Children's Hospital of Ningbo University (No. EC2023-015) and signed informed consent was obtained from the parents of each patient. Consent for publication Not applicable. Availability of data and materials Sequence data that support the findings of this study have been deposited in NCBI SRA database with the primary accession code PRJNA1138771 ( https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA1138771 ) Competing interests The authors declare no competing interests. Funding This study was supported by grants from Medical and Health project of Zhejiang province(2023KY1118), Zhejiang Province Traditional Chinese Medicine Science and Technology Planning Project (2024ZL960), Ningbo Key R&D Programme (2023Z178), Ningbo Top Medical and Health Research Program (2022020405). Authors' contributions  YP and JSZ initiated the study. YP, DXL, and HBL drafted the manuscript. HTL, YHZ, HQX, QSY, LLH, YBX, and YZ collected the samples. YP and DXL analyzed the data. JSZ and HBL provided funding support and assisted in the data analysis. All authors read and approved the final manuscript. Acknowledgements We thank all the participants and the editors of Springer Nature Author Services team for their language editing work on the manuscript. Authors' information 1 The Central Laboratory of Birth Defects Prevention and Control, The Affiliated Women and Children's Hospital of Ningbo University, Ningbo, China; 2 Ningbo Key Laboratory for the Prevention and Treatment of Embryogenic Diseases, The Affiliated Women and Children's Hospital of Ningbo University, Ningbo, China; 3 Ningbo Key Laboratory of Genomic Medicine and Birth Defects Prevention, The Affiliated Women and Children's Hospital of Ningbo University, Ningbo, China 4 Department of Pediatric, Ningbo Zhenhai Dist Lianhua Hospital, Ningbo, China; 5 Pediatrics Internal Medicine，The Affiliated Women and Children's Hospital of Ningbo University, Ningbo, China References Jain S, Williams DJ, Arnold SR, Ampofo K, Bramley AM, Reed C, Stockmann C, Anderson EJ, Grijalva CG, Self WH et al : Community-acquired pneumonia requiring hospitalization among U.S. children . N Engl J Med 2015, 372 (9):835-845. 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Tables Table 1 Clinical characteristics and laboratory tests of MMPP and SMPP patients Characteristic MMPP (N=48) SMPP (N=12) P value Male/female 19/29 5/7 0.895 Age (years) 7 (5.1-8.5) 6.8 (5.3-8) 0.993 Hospital stays (days) 9 (7-11) 10 (8-12) 0.315 Hospital stays after BAL (days) 3 (2-5) 4.5 (2.3-7.3) 0.165 Total febrile days (days) 8 (6.3-10) 10 (8-12) 0.007 Chest X-ray Patchy high-density lesion/consolidation [n (%)] 4 (8.3%) 3 (25%) 0.108 Laboratory tests C-reactive protein (mg/L) 13.8 (5.9-32.8) 24.3 (10.2-43.2) 0.114 D-dimer (μg/L) 216 (168-298) 578 (509-1877) <0.0001 PCT (ng/mL) 0.123 (0.068-0.199) 0.209 (0.123-0.431) 0.031 Coinfection [n (%)] 21 (44%) 8 (67%) 0.155 A2063G variation, No. [n (%)] 10 (21%) 0 (0%) 0.083 Note: Data are No. (%) of patients or median (interquartile range) Moderate mycoplasma pneumoniae pneumonia (MMPP); severe mycoplasma pneumoniae pneumonia (SMPP); procalcitonin. (PCT) Table 2 Clinical characteristics and laboratory tests of MSPP and MRMP Characteristic MSMP (N=48) MRMP (N=10) P value Male/female 20/28 4/6 0.922 Age (years) 6.7 (5.1-8) 8.5 (6-10) 0.056 Hospital stays (days) 8.5 (7-11) 10 (8-12) 0.127 Hospital stays after BAL (days) 3 (2-5) 3.5 (3-5.3) 0.340 Total febrile days (days) 8 (7-10) 8.5 (7.8-9.3) 0.952 Chest X-ray Patchy high-density lesion/consolidation [n (%)] 5 (10%) 2 (20%) 0.397 Laboratory tests C-reactive protein (mg/L) 14.4 (8.3-33) 15 (1.4-55.4) 0.968 D-Dimer (μg/L) 273 (187-509) 201 (178-593) 0.463 PCT (ng/mL) 0.125 (0.072-0.225) 0.185 (0.058-0.264) 0.714 Coinfection [n (%)] 25 (52%) 4 (40%) 0.487 Note: Data are No. (%) of patients or median (interquartile range) Macrolide-sensitive mycoplasma pneumoniae (MSMP); Macrolide-resistant mycoplasma pneumoniae (MRMP); procalcitonin (PCT) Additional Declarations No competing interests reported. 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Hospital","correspondingAuthor":false,"prefix":"","firstName":"Dingxiang","middleName":"","lastName":"Lai","suffix":""},{"id":345275382,"identity":"5ed4f860-acfb-4459-b50a-52bf0ffa7758","order_by":2,"name":"Haitao Lv","email":"","orcid":"","institution":"Pediatrics Internal Medicine，The Affiliated Women and Children's Hospital of Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Haitao","middleName":"","lastName":"Lv","suffix":""},{"id":345275383,"identity":"f2b15cb1-73f0-4fcc-8006-cd6883ec552e","order_by":3,"name":"Yahua Zhang","email":"","orcid":"","institution":"Pediatrics Internal Medicine，The Affiliated Women and Children's Hospital of Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Yahua","middleName":"","lastName":"Zhang","suffix":""},{"id":345275384,"identity":"bca4143b-7b2c-41a7-b885-2553acccf381","order_by":4,"name":"Huiqing Xu","email":"","orcid":"","institution":"Pediatrics Internal Medicine，The Affiliated Women and Children's Hospital of Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Huiqing","middleName":"","lastName":"Xu","suffix":""},{"id":345275385,"identity":"e677e05e-11c3-46a6-bb9f-f01c2c8f86ff","order_by":5,"name":"Qinsong Ye","email":"","orcid":"","institution":"Pediatrics Internal Medicine，The Affiliated Women and Children's Hospital of Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Qinsong","middleName":"","lastName":"Ye","suffix":""},{"id":345275386,"identity":"de64493e-b4f2-4f63-8ba9-2ee6f585325d","order_by":6,"name":"Lingling Hua","email":"","orcid":"","institution":"Pediatrics Internal Medicine，The Affiliated Women and Children's Hospital of Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Lingling","middleName":"","lastName":"Hua","suffix":""},{"id":345275387,"identity":"faa61dc4-c24c-43f9-9b94-1eb6230c67d9","order_by":7,"name":"Yuebo Xu","email":"","orcid":"","institution":"Pediatrics Internal Medicine，The Affiliated Women and Children's Hospital of Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Yuebo","middleName":"","lastName":"Xu","suffix":""},{"id":345275388,"identity":"716886b0-b459-4dfe-8b56-2c0f6c2793e1","order_by":8,"name":"Ying Zhou","email":"","orcid":"","institution":"The Affiliated Women and Children's Hospital of Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Zhou","suffix":""},{"id":345275389,"identity":"7a4b4fa6-df7f-41fa-a290-bfdd834caf69","order_by":9,"name":"Jishan Zheng","email":"","orcid":"","institution":"Pediatrics Internal Medicine，The Affiliated Women and Children's Hospital of Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Jishan","middleName":"","lastName":"Zheng","suffix":""},{"id":345275390,"identity":"e642efa2-c46f-4bbe-a65d-e4196a2d2848","order_by":10,"name":"Haibo Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIie3QMQrCMACF4VcCcYl2jSCeISAUB8GrNAidFBw7CHaQdhA9i6OjReiU7jrZnkDddDNFnBRTN4f8cz6SF8Bm+8NahAEyAoanc1n44cxM6ItgNyaiUFkNAk2cJ6HtckFqkEYzvZTbgWZ5FsqIwk2WvuFhrRGXKgBx1sFBbjvgKt+Ytggu4/08JvAOUlEIPjGS3k0TMApvKjWsQ7zqFnDGPNQm/WqL4HTEfZUx4xbXVb3jXf+Y4CS93sJZ101W38lb7LfjNpvNZvvYA+aeQLxDD/QuAAAAAElFTkSuQmCC","orcid":"","institution":"The Affiliated Women and Children's Hospital of Ningbo University","correspondingAuthor":true,"prefix":"","firstName":"Haibo","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-07-31 07:45:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4833361/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4833361/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":64009488,"identity":"ac9c2914-e18d-4a5f-ac73-734f7520e150","added_by":"auto","created_at":"2024-09-04 23:16:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":196963,"visible":true,"origin":"","legend":"\u003cp\u003eFlow chart of this study.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4833361/v1/ab1dee5d677bddaf09060080.png"},{"id":64008878,"identity":"2ce0d00a-e8f5-4d4e-8ccf-c91e555c7c3d","added_by":"auto","created_at":"2024-09-04 23:08:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":454353,"visible":true,"origin":"","legend":"\u003cp\u003eBacterial community structure and diversity of the throat swab (TS) and bronchoalveolar lavage fluid (BALF) microbiota from the children hospitalized with \u003cem\u003eMycoplasma pneumoniae\u003c/em\u003e pneumonia. (A) Bar chart of pathogenic microorganisms detected in TS and BALF specimens, including the top 30 microorganisms. (B) The relative abundance of pathogenic microorganisms detected in TS and BALF specimens, including the top 30 microorganisms. (C) Percent stacked bar plot for species-level taxonomic profiling of the bacterial microbial communities for individual cases in TS specimens. Labels indicate the top 20 abundant species. The samples are grouped by MSMP (NEG means A2063G negative) and MRMP (POS means A2063G positive). (D) Percent stacked bar plot for species-level taxonomic profiling of the bacterial microbial communities for total cases in TS specimens. (E) Percent stacked bar plot for species-level taxonomic profiling of the bacterial microbial communities for individual cases in BALF specimens. (F) Percent stacked bar plot for species-level taxonomic profiling of the bacterial microbial communities for total cases in BALF specimens.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4833361/v1/30f9a2c5a3864ff1c577d864.png"},{"id":64008875,"identity":"4ac47bbb-bb8a-44e0-82ec-0991dca45e8e","added_by":"auto","created_at":"2024-09-04 23:08:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":337051,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of MSMP (NEG means A2063G negative) and MRMP (POS means A2063G positive) in terms of bacterial community α diversity and β diversity in throat swab (TS) and bronchoalveolar lavage fluid (BALF) specimens. (A) Shannon index between the MSMP and MRMP groups in TS specimens. (B) Shannon index between the MSMP and MRMP groups in BALF specimens. (C) Dimensionality reduction analysis specimen distribution diagram (PC, MDS, NMDS) between the MSMP and MRMP groups in TS specimens. (D) Dimensionality reduction analysis specimen distribution diagram (PC, MDS, NMDS) between the MSMP and MRMP groups in BALF specimens. (E) ANOSIM of microbial community distribution in TS specimens from the MSMP and MRMP groups. (F) ANOSIM of microbial community distribution in BALF specimens from the MSMP and MRMP groups.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4833361/v1/57f9f240c08087eee1a76d23.png"},{"id":64008877,"identity":"afc22071-6960-4c4e-bf3c-1b875aec832f","added_by":"auto","created_at":"2024-09-04 23:08:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":197173,"visible":true,"origin":"","legend":"\u003cp\u003eDifferentially abundant species prediction in TS and BALF specimens between the MSMP (NEG means A2063G negative) and MRMP (POS means A2063G positive) groups. (A) Linear discriminant analysis (LDA) effect size analysis of differentially abundant species between the MSMP and MRMP groups in TS specimens. LDA score=2. (B) Three differentially abundant species of TS specimens from the MSMP and MRMP groups were used to predict MRMP. (C) \u003cem\u003eActinomyces naeslundii\u003c/em\u003e was used to predict MRMP (AUC:0.78, 95% CI (0.63, 0.92)). (D) Linear discriminant analysis (LDA) effect size analysis of differentially abundant species between the MSMP and MRMP groups in BALF specimens. LDA score=2. (E) All differentially abundant species of BALF specimens from the MSMP and MRMP groups were used to predict MRMP. (F) The relative abundance of \u003cem\u003eMycoplasma pneumoniae\u003c/em\u003e in BALF specimens from the MSMP and MRMP groups was used to predict MRMP (AUC:0.86, 95% CI (0.75, 0.97)).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4833361/v1/738b71661f8773ecb3b3dcc8.png"},{"id":64008876,"identity":"acd7c925-de77-44fb-9f5a-e05cb3dd7871","added_by":"auto","created_at":"2024-09-04 23:08:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":128777,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum-likelihood phylogenetic trees of six strains. \u0026nbsp;Trees were built according to the StrainPhlAn analysis. Five MRMP cases were excluded from the multilocus sequence typing (MLST) tree due to its low sequencing depth and failure to reconstruct the MLST locus sequences.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4833361/v1/fa8c160e67a2227493cfc721.png"},{"id":79064311,"identity":"e9cf3fef-c346-4253-bad0-f88e9fb43b06","added_by":"auto","created_at":"2025-03-24 03:46:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3014524,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4833361/v1/0afbc707-6071-4ebd-b0bf-276acd5097d7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bronchoalveolar lavage combined with metagenomic assessment of children hospitalized with Mycoplasma pneumoniae pneumonia","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCommunity-acquired pneumonia (CAP) is a prevalent cause of hospitalization in children[1], with \u003cem\u003eMycoplasma pneumoniae\u003c/em\u003e (MP) being a significant pathogen, accounting for 10\u0026ndash;40% of CAP cases in school-aged children[2, 3]. Over the past three decades, conjugate vaccines targeting \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e and \u003cem\u003eHaemophilus influenzae\u003c/em\u003e type b have reduced the number of cases caused by these former main causes of CAP. However, a safe vaccine against MP infection has not yet been developed, despite the recent discovery of a new target\u0026mdash;community-acquired respiratory distress syndrome toxin (CARDS TX), which is released by MP[4].\u0026nbsp;The incidence and aetiological spectrum of CAP have changed substantially due to the above issues. The introduction of nonpharmaceutical interventions (NPIs) against COVID-19 in early 2020 led to the disappearance of almost all respiratory pathogens, and the reappearance of MP did not occur till the autumn of 2023[5], and it appeared earlier in China.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eM. pneumoniae\u003c/em\u003e pneumonia (MPP) is characterized by nonspecific clinical manifestations such as cough, fever, and chest radiographic changes. The \u0026ldquo;gold standard\u0026rdquo; for determining the aetiology of MPP is the detection of MP in samples taken directly from the lungs via bronchoalveolar lavage fluid (BALF), pleural fluid sampling, or lung biopsy or aspiration. Culture of MP is expensive and time-consuming because of the slow growth of this species in vitro[6, 7], and the sensitivity of this method is approximately 60% that of PCR[8]. Taxonomic profiling via metagenomic next-generation sequencing (mNGS) techniques has higher sensitivity and resolution than conventional culture approaches, providing a better understanding of microbial compositions. Since 2000, the emergence of more macrolide-resistant \u003cem\u003eM. pneumoniae\u003c/em\u003e (MRMP) strains[9]\u0026nbsp;and coinfection with other pathogens (\u003cem\u003eS. pneumoniae\u003c/em\u003e, \u003cem\u003eH. parainfluenzae\u003c/em\u003e, EB virus) have led to an increasing number of refractory \u003cem\u003eM.\u003c/em\u003e \u003cem\u003epneumoniae\u003c/em\u003e pneumonia (RMPP) cases[10].\u003c/p\u003e\n\u003cp\u003eBAL is not used only to obtain BALF samples. In this study, we used it as a treatment for children hospitalized with MPP, especially severe \u003cem\u003eM. pneumoniae\u003c/em\u003e pneumonia (SMPP) and MRMP patients. Although BAL was previously considered an invasive examination method, with the development of fiberoptic bronchoscopy technology and the increase in physician experience, the amount of injury can be minimized. Moreover, BALF samples can be subjected to mNGS to determine the aetiology of MPP. The latest guidelines for childhood CAP issued by the Infectious Diseases Society of America (IDSA)[11]\u0026nbsp;and the British Thoracic Society (BTS)[12]\u0026nbsp;were issued more than a decade ago. During this period, new insights into the causes and treatment of childhood MPP have been obtained, and the guidelines need to be updated.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003ePatients and Samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study included children hospitalized with MPP at The Affiliated Women and Children\u0026apos;s Hospital of Ningbo University from May 2023 to August 2023. In this study, patients were enrolled on the basis of\u0026nbsp;abnormal chest X-ray imaging results, including chest infiltration, consolidation, and pleural cavity effusion. Serological tests for MP igM- or igG-positive or MP-positive samples were performed via PCR in nasopharyngeal\u0026nbsp;aspirates. The patients were divided into the MMPP group and the SMPP group according to the results of the admission CAP assessment, with the following evaluation criteria: 1) severely worsened general condition; 2) altered mental status; 3) capillary refill time \u0026gt;2 sec; 4) dyspnoea, apnoea, or cyanosis; 5) SpO\u003csub\u003e2\u003c/sub\u003e\u0026lt;90%; and 6) lack of feeding, vomiting, or signs of dehydration. All the patients underwent fibre bronchoscopic lavage within 3\u0026ndash;7 days after macrolide treatment. The patients\u0026rsquo; throat swab (TS) and BALF samples were collected for mNGS analysis. These patients were divided into the MSMP and MRMP groups according to whether they had macrolide resistance genes in 23S rRNA domain V. The current study was approved by the IRB of The Affiliated Women and Children\u0026apos;s Hospital of Ningbo University (No. EC2023-015).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emNGS Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003c/strong\u003eFor preprocessing of specimens, TS and BALF samples were homogenized via dithiothreitol (DTT) treatment. The cells were removed via centrifugation to reduce the background levels of host nucleic acids. The HostZERO Microbial DNA Kit (Zymo Research, USA) was used to extract total DNA according to the manufacturer\u0026apos;s instructions. The yield of the extracted DNA was quantified via a Quant-iT-dsDNA HS assay kit and a Qubit 3.0 fluorometer (Thermo Scientific, USA). The DNA molecules were fragmented via enzymatic shearing (~200 bp), and the Nextera XT DNA Library Preparation Kit (Illumina, USA) was subsequently used to construct the library. A certain number of PCR cycles (5-7) were applied to generate a 1 \u0026mu;g macrogenomic library. The quality of the library was evaluated via a 2100 Bioanalyzer with high-sensitivity DNA analysis (Agilent Technologies, USA). Metagenome shotgun sequencing in single-end 75-bp mode was performed via the NextSeq 500/550 High Output Kit (92 cycles) on an Illumina NextSeq 550 sequencer. Samples serving as nontemplate controls (NTCs) were also sequenced to assess contamination during wet-laboratory experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003c/strong\u003e\u003cstrong\u003eBioinformatics Analysis\u0026nbsp;of Species-Level Abundance\u0026nbsp;Profiles\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw sequencing data were first subjected to quality control by trimming adapter sequences and removing low-quality reads via Trimmomatic v0.36 for tail trimming and reading. To remove duplicate reads that were considered technical artefacts by PCR, deduplication was performed via an in-house Perl script. Microbial reads were classified via Kraken v2.0.9-beta and a custom k-mer database constructed from 51,543 genomes from approximately 27,000 species in the NCBI assembly database. To estimate species-level abundances, the Bayesian algorithm implemented in Bracken v2.2 further estimated the number of reads in the Kraken taxonomy report. On the basis of the information for each batch of NTC samples, microbial taxa that may have been present as contaminants during the experiment were removed from the final map. The estimated relative abundance percentage of each bacterial, viral (excluding bacteriophages), and fungal species was calculated separately.\u003c/p\u003e\n\u003cp\u003eTo investigate the pathogenic signals in the sputum microbiome, we proposed an empirical rule to assess the detection limit for the pathogenic risk of individual organisms through species-level abundance analysis. In short, the average and standard deviation values of relative abundance were calculated on the basis of the estimated values for each species in the sample.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStrain-Level Phylogenetic Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStrain level analysis was performed on the basis of single-nucleotide variations (SNVs) via the StrainPhlAn v3.0 software package. Briefly, metagenomic reads per sample were first aligned to the marker gene database of MetaPhlAn v3.0. For the most abundant strains of each species, we reconstructed the labelled sample-specific consensus sequences and extracted species-specific markers from MetaFlAn to identify homologous sequences in the genomes of the reference isolates via BLASTN. On the basis of sequence alignment, the maximum likelihood phylogenetic tree was reconstructed via RAxML and then visualized via MEGA10. The genome sequences of the isolated strains were retrieved from the NCBI Assembly database and used for phylogenetic reconstruction together with the strains detected in the metagenomic samples. The MetaMLST v1.2.2 program was used to predict known and novel sequence types of the most abundant strains in the metagenomic samples. Owing to the complexity of determining the phylogeny of diploid fungi, an alternative method was used to construct a phylogenetic tree on the basis of multilocus sequence typing (MLST) analysis of seven loci. Seven MLST allele sequences were reconstructed from the metagenome via MetaMLST, and the relevant gene sequences in the isolate genome were processed via FastMLST. Multiple sequence alignment was performed via MUSCLE, and phylogenetic inference was performed via RAxML.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003c/strong\u003e\u003cstrong\u003eStatistical Analysis of Community Diversity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOn the basis of the Chao1 index representing community richness, diversity analysis and t tests for two samples were conducted via the fossil v0.4.0 software package. To estimate the variation in sample community composition between groups, \u0026beta; diversity was measured by calculating the Bray‒Curtis distance matrix via Vegan v2.5-7. The similarity of within- and between-group rank differences was inferred via a nonparametric ANOSIM test with 1000 permutations. All the statistical analyses and visualizations were performed in R v4.1.0.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCharacteristics of Patients and Samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003c/strong\u003eDuring the 4-month study, TS and BALF samples were collected from 60 children aged 2-12 years, with a median age of 7 years (\u003cstrong\u003eFigure 1\u003c/strong\u003e). On the basis of the CAP assessment results at admission, 48 patients were included in the MMPP group, and 12 patients were included in the SMPP group.\u0026nbsp;The clinical characteristics and laboratory test results of the MMPP and SMPP patients are listed in \u003cstrong\u003eTable 1\u003c/strong\u003e.\u0026nbsp;The total number of febrile days in the SMPP group was greater than that in the MMPP group. The D-dimer level in the SMPP group was higher than that in the MMPP group. The procalcitonin (PCT) level in the SMPP group was approximately twice that in the MMPP group, while the other characteristics were not significantly different. On the basis of the results of mNGS, these patients were divided into 48 cases of MSMP and 10 cases of MRMP (all cases were A2063G positive). Two cases were excluded because they were MP negative in both the TS and BALF samples. The clinical characteristics and laboratory test results of the MSPP and MRMP patients are listed in \u003cstrong\u003eTable 2\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCommunity Structure of the TS and BALF Microbiota\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe MP detection rate in TS samples was 92% (55/60), and that in BALF samples was 97% (58/60) (\u003cstrong\u003eFigure 2A\u003c/strong\u003e). In addition to MP, the five most abundant species in the TS samples were \u003cem\u003eHaemophilus parainfluenzae\u003c/em\u003e (85%), Epstein\u0026ndash;Barr virus (28%), \u003cem\u003eCandida albicans\u003c/em\u003e (18%), human herpesvirus 7 (15%), and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (10%) (\u003cstrong\u003eFigure 2B\u003c/strong\u003e). In addition to MP,\u0026nbsp;the five most abundant species in the BALF samples were \u003cem\u003eH. parainfluenzae\u003c/em\u003e (28%), \u003cem\u003eC. albicans\u003c/em\u003e (12%), \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (8%), Epstein\u0026ndash;Barr virus (7%), and \u003cem\u003eS. aureus\u003c/em\u003e (5%) (\u003cstrong\u003eFigure 2B\u003c/strong\u003e). To compare the community structure of the TS and BALF microbiota between MSMP and MRMP, the top 20 species for a single case are listed in\u003cstrong\u003e\u0026nbsp;Figure 2C\u003c/strong\u003e and \u003cstrong\u003eFigure 2E\u003c/strong\u003e, respectively. The top 20 species for the total cases are listed in \u003cstrong\u003eFigure 2D\u003c/strong\u003e and \u003cstrong\u003eFigure 2F\u003c/strong\u003e. In terms of relative abundance, MP was dominant in the MRMP group among BALF specimens\u003cstrong\u003e\u0026nbsp;(Figure 2F)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; To further explore the dominance of MP in the MRMP group, we compared the \u0026alpha; diversity of community richness between MSMP and MRMP in TS and BALF samples. The \u0026alpha; diversity analysis of community richness revealed no significant difference between the two groups in TS specimens (\u003cstrong\u003eFigure 3A\u003c/strong\u003e), whereas the Shannon index of the MRMP group was lower than that of the MSMP group in BALF specimens (p\u0026lt;0.01) (\u003cstrong\u003eFigure 3B\u003c/strong\u003e). The \u0026beta; diversity based on the sample distribution diagram (PC, MDS, NMDS) between the MSMP group and the MRMP group showed a similar distribution in both the TS samples (\u003cstrong\u003eFigure 3C\u003c/strong\u003e) and the BALF samples (\u003cstrong\u003eFigure 3D\u003c/strong\u003e). On the basis of the \u0026beta;-diversity ANOSIM test, the mean of the ranked dissimilarities between the MSMP group and the MRMP group was not significantly different in the TS samples (\u003cstrong\u003eFigure 3E\u003c/strong\u003e) and BALF samples (\u003cstrong\u003eFigure 3E\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrediction of Differentially Abundant Species in TS and BALF Specimens between the MSMP and MRMP Groups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn addition to comparing the composition of pathogenic microorganisms in the upper and lower respiratory tracts, we also attempted to use intergroup differences in species abundances to predict whether MRMP emerged. Linear discriminant analysis (LDA) effect size analysis of the differentially abundant species between the MSMP group and the MRMP group in the TS samples revealed three such species, namely, \u003cem\u003ePrevotella oralis\u003c/em\u003e, \u003cem\u003eActinomyces naeslundii\u003c/em\u003e, and \u003cem\u003eCorynebacterium durum\u003c/em\u003e (\u003cstrong\u003eFigure 4A\u003c/strong\u003e). These three species were combined to construct a ROC curve for the prediction of multiple biomarkers (AUC=0.692) (\u003cstrong\u003eFigure 4B\u003c/strong\u003e). When single species were used to construct a ROC curve, \u003cem\u003eA. naeslundii\u003c/em\u003e exhibited the best predictive performance (AUC=0.78, 95% CI (0.63, 0.92), KW_P=0.007) (\u003cstrong\u003eFigure 4C\u003c/strong\u003e). LDA effect size analysis of the differentially abundant species between the MSMP group and MRMP group in the BALF samples revealed nearly 70 species (\u003cstrong\u003eFigure 4D\u003c/strong\u003e). These species were combined to construct a ROC curve for the prediction of multiple biomarkers (AUC=0.775) (\u003cstrong\u003eFigure 4E\u003c/strong\u003e).\u0026nbsp;The relative abundance of \u003cem\u003eM. pneumoniae\u003c/em\u003e in the BALF samples from the MSMP group and MRMP group was used as a biomarker to construct a ROC curve to predict whether it MRMP was present (AUC: 0.86, 95% CI (0.75, 0.97)) (\u003cstrong\u003eFigure 4F\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStrain-Level Characteristics of Pathogens\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003c/strong\u003eStrain identification is highly important for better understanding microbial pathogenicity and the transmission of clinically relevant microorganisms. StrainPhlAn analysis reveals strain-level phylogenetic trees visualizing the relatedness between the metagenome-recovered strains and clinical bacterial isolates with publicly available genome sequences. \u003cem\u003eM. pneumoniae\u003c/em\u003e strains were reconstructed from the metagenomic samples of six patients from the MRMP group (MP12, MP20, MP23, MP48, and MP57) and the MSMP group (MP05) (\u003cstrong\u003eFigure 5\u003c/strong\u003e). Five strains, from MP12, MP20, MP23, MP48, and MP57, exhibited the closest relationships with the strain \u003cem\u003eM. pneumoniae\u003c/em\u003e-15-885 (GCA_009941325.1), which was isolated in Seoul in 2015. One strain from MP05 was most closely related to the strain \u003cem\u003eM. pneumoniae\u003c/em\u003e-E57 (GCF_002128005.1), which was isolated in Egypt in 2009.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMP is an important pathogen causing CAP in school-aged (mainly 5\u0026ndash;10 years old) children. It is transmitted mainly through the respiratory tract, and pandemics occur every 3 to 5 years[3]. The cases studied herein were from the recent outbreak in 2023. Currently, macrolides are the recommended first-line treatment for MP infection. In recent years, the widespread use of these drugs in patients with various types of infections has led to an increase in macrolide resistance[13, 14]. However, it remains unclear whether there is a definite connection between the severity of MPP and MRMP. We divided the patients into the MMPP and SMPP groups according to the results of the admission CAP assessment and found that MRMP was not the main cause of SMPP, the co-infection was (\u003cstrong\u003eTable 1\u003c/strong\u003e). The increase in PCT in the SMPP group also suggested a greater correlation between coinfection and the severity of MPP. Although the total number of febrile days in the SMPP group was greater than that in the MMPP group, there was no difference in the total length of hospital stay between the two groups. The increase in the D-dimer level in the SMPP group indicated that patients with severe infection had a greater risk of thrombosis than those with moderate infection.\u003c/p\u003e\n\u003cp\u003eBoth SMPP patients and MRMP patients need effective drugs and treatments other than macrolides. Drugs such as tetracyclines or quinolones can be used as alternatives for treating MRMP, but they are not appropriate for children under 12 years of age due to osteotoxicity[15]. In this study, we attempted to use BAL to treat children hospitalized with MPP. On the other hand, the \u0026ldquo;gold standard\u0026rdquo; for determining the aetiology of MPP is the detection of MP in samples taken directly from the lungs via BAL. Therefore, we performed BAL on all 60 patients. BAL washed away most of the pathogens and removed the sputum blocked in the bronchial orifice. This enabled better absorption of drugs, especially drugs inhaled by atomization. In addition, MP grows very slowly, which is beneficial for reducing the possibility of recurrence after BAL. Hospital stays after BAL were similar between the MMPP group and the SMPP group (\u003cstrong\u003eTable 1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;We were curious about whether TS samples can replace BALF samples for determining the aetiology of MPP, so both TS samples and BALF samples were collected. The results revealed that 3 of the BALF samples were MP positive, whereas the TS samples were MP negative (Figure 2A). Thus, TS samples cannot be used to replace BALF samples. The results of mNGS revealed that 10 cases of MRMP (17%) were detected among these patients, which was inconsistent with several Chinese studies (with values ranging from 60 to 92%)[16-20]. We speculate that this may be related to the fact that we included only hospitalized patients. There was no significant difference in clinical manifestations between the MSMP group and the MRMP group (\u003cstrong\u003eTable 2\u003c/strong\u003e). Another difference between the TS samples and BALF samples was that there were many colonizing bacteria in the TS samples, which also increased the relative abundance of MP in the BALF samples compared with that in the TS samples (\u003cstrong\u003eFigure 2D, Figure 2F\u003c/strong\u003e). On the basis of the \u0026alpha; diversity analysis of community richness between the MSMP group and the MRMP group in the BALF samples, it can be inferred that MP was the dominant strain in the MRMP group (\u003cstrong\u003eFigure 3B\u003c/strong\u003e). However, the results of the \u0026beta; diversity analysis of community richness between the MSMP group and the MRMP group were not statistically significant. These findings indicate that the compositions of the microbiota in the MSMP group and MRMP group were similar.\u003c/p\u003e\n\u003cp\u003eTo further investigate the dominance of MP in MRMP, we used the relative abundance of MP as a biomarker to predict whether a case was MRMP. The results showed that this biomarker had good predictive performance (\u003cstrong\u003eFigure 4D\u003c/strong\u003e). Interestingly, in the TS samples, although the LDA score of \u003cem\u003eA. naeslundii\u0026nbsp;\u003c/em\u003ewas not the highest, its predictive performance as a biomarker was the best (\u003cstrong\u003eFigure 4C\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eFinally, we performed sequence alignment on strains to trace their origins. The results revealed that five strains (MRMP) were most closely related to the strain isolated in Seoul in 2015, and one strain (MSMP) was most closely related to the strain isolated in Egypt in 2009 (\u003cstrong\u003eFigure 5\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eIn summary, this study provides new insights into the diagnosis and treatment of children hospitalized with MPP. The combination of BAL and metagenomic assessment can not only accelerate the recovery of patients but also provide evidence for epidemiological investigations of MPP. However, this study also had several limitations; for example, the sample size was not very large, and the mNGS detection results did not include RNA viruses.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eMRMP is not the main cause of SMPP-coinfection is. In terms of relative abundance, in MRMP patients, MP was dominant in BALF specimens. TS specimens cannot replace BALF samples for determining the aetiology of MPP. \u003cem\u003eA. naeslundii\u003c/em\u003e and the relative abundance of MP are optimal biomarkers for predicting whether a case is MRMP in TS samples and BALF samples, respectively. A majority of the MRMP strains from this study are closely related to the strain isolated in Seoul in 2015.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBAL:\u0026nbsp;Bronchoalveolar lavage\u003c/p\u003e\n\u003cp\u003eMP:\u003cem\u003e\u0026nbsp;Mycoplasma pneumoniae\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMPP:\u003cem\u003e\u0026nbsp;Mycoplasma pneumoniae\u003c/em\u003e pneumonia\u003c/p\u003e\n\u003cp\u003eMMPP: moderate \u003cem\u003eM\u003c/em\u003e.\u003cem\u003e\u0026nbsp;pneumoniae\u003c/em\u003e pneumonia\u003c/p\u003e\n\u003cp\u003eSMPP: severe \u003cem\u003eM. pneumoniae\u003c/em\u003e pneumonia\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCAP: community-acquired pneumonia\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTS:\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ethroat swab\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBALF: bronchoalveolar lavage fluid\u003c/p\u003e\n\u003cp\u003emNGS: metagenomic next-generation sequencing\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMSMP:\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003emacrolide-sensitive \u003cem\u003eM. pneumoniae\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMRMP: macrolide-resistant \u003cem\u003eM. pneumoniae\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePCT: procalcitonin\u003c/p\u003e\n\u003cp\u003eCARDS TX:\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ecommunity-acquired respiratory distress syndrome toxin\u003c/p\u003e\n\u003cp\u003eNPIs:\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003enonpharmaceutical interventions\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIDSA: Infectious Diseases Society of America\u003c/p\u003e\n\u003cp\u003eBTS: the British Thoracic Society\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDTT: dithiothreitol\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNTCs: nontemplate controls\u003c/p\u003e\n\u003cp\u003eSNVs: single-nucleotide variations\u003c/p\u003e\n\u003cp\u003eMLST: multilocus sequence typing\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLDA: Linear discriminant analysis\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe current study was approved by the IRB of The Affiliated Women and Children\u0026apos;s Hospital of Ningbo University (No. EC2023-015) and signed informed consent was obtained from the parents of each patient.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequence data that support the findings of this study have been deposited in NCBI SRA database with the primary accession code PRJNA1138771 ( https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA1138771 )\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from Medical and Health project of Zhejiang province(2023KY1118), Zhejiang Province Traditional Chinese Medicine Science and Technology Planning Project (2024ZL960), Ningbo Key R\u0026amp;D Programme (2023Z178), Ningbo Top Medical and Health Research Program (2022020405).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u003c/em\u003e\u003c/strong\u003eYP and JSZ initiated the study. YP, DXL, and HBL drafted the manuscript. HTL, YHZ, HQX, QSY, LLH, YBX, and YZ collected the samples. YP and DXL analyzed the data. JSZ and HBL provided funding support and assisted in the data analysis. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp skip=\"true\"\u003eWe thank all the participants and the editors of Springer Nature Author Services team for their language editing work on the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eThe Central Laboratory of Birth Defects Prevention and Control, The Affiliated Women and Children\u0026apos;s Hospital of Ningbo University, Ningbo, China;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eNingbo Key Laboratory for the Prevention and Treatment of Embryogenic Diseases, The Affiliated Women and Children\u0026apos;s Hospital of Ningbo University, Ningbo, China;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u003c/sup\u003eNingbo Key Laboratory of Genomic Medicine and Birth Defects Prevention, The Affiliated Women and Children\u0026apos;s Hospital of Ningbo University, Ningbo, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e4\u003c/sup\u003eDepartment of Pediatric, Ningbo Zhenhai Dist Lianhua Hospital, Ningbo, China;\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e5\u003c/sup\u003ePediatrics Internal Medicine，The Affiliated Women and Children\u0026apos;s Hospital of Ningbo University, Ningbo, China\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJain S, Williams DJ, Arnold SR, Ampofo K, Bramley AM, Reed C, Stockmann C, Anderson EJ, Grijalva CG, Self WH\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eCommunity-acquired pneumonia requiring hospitalization among U.S. children\u003c/strong\u003e. \u003cem\u003eN Engl J Med \u003c/em\u003e2015, \u003cstrong\u003e372\u003c/strong\u003e(9):835-845.\u003c/li\u003e\n\u003cli\u003eZhu Z, Zhang T, Guo W, Ling Y, Tian J, Xu Y: \u003cstrong\u003eClinical characteristics of refractory 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\u003cstrong\u003eSurveillance of macrolide-resistant Mycoplasma pneumoniae in Beijing, China, from 2008 to 2012\u003c/strong\u003e. \u003cem\u003eAntimicrob Agents Chemother \u003c/em\u003e2013, \u003cstrong\u003e57\u003c/strong\u003e(3):1521-1523.\u003c/li\u003e\n\u003cli\u003eMa Z, Zheng Y, Deng J, Ma X, Liu H: \u003cstrong\u003eCharacterization of macrolide resistance of Mycoplasma pneumoniae in children in Shenzhen, China\u003c/strong\u003e. \u003cem\u003ePediatr Pulmonol \u003c/em\u003e2014, \u003cstrong\u003e49\u003c/strong\u003e(7):695-700.\u003c/li\u003e\n\u003cli\u003eLiu Y, Ye X, Zhang H, Xu X, Li W, Zhu D, Wang M: \u003cstrong\u003eCharacterization of macrolide resistance in Mycoplasma pneumoniae isolated from children in Shanghai, China\u003c/strong\u003e. \u003cem\u003eDiagn Microbiol Infect Dis \u003c/em\u003e2010, \u003cstrong\u003e67\u003c/strong\u003e(4):355-358.\u003c/li\u003e\n\u003cli\u003eXin D, Mi Z, Han X, Qin L, Li J, Wei T, Chen X, Ma S, Hou A, Li G\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eMolecular mechanisms of macrolide resistance in clinical isolates of Mycoplasma pneumoniae from China\u003c/strong\u003e. \u003cem\u003eAntimicrob Agents Chemother \u003c/em\u003e2009, \u003cstrong\u003e53\u003c/strong\u003e(5):2158-2159.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u0026nbsp;\u003c/strong\u003eClinical characteristics and laboratory tests of MMPP and SMPP patients\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.10569105691057%\" valign=\"top\"\u003e\n \u003cp\u003eCharacteristic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.15447154471545%\" valign=\"top\"\u003e\n \u003cp\u003eMMPP (N=48)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.15447154471545%\" valign=\"top\"\u003e\n \u003cp\u003eSMPP (N=12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.585365853658537%\" valign=\"top\"\u003e\n \u003cp\u003eP value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.10569105691057%\" valign=\"top\"\u003e\n \u003cp\u003eMale/female\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.15447154471545%\" valign=\"top\"\u003e\n \u003cp\u003e19/29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.15447154471545%\" valign=\"top\"\u003e\n \u003cp\u003e5/7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.585365853658537%\" valign=\"top\"\u003e\n \u003cp\u003e0.895\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.10569105691057%\" valign=\"top\"\u003e\n \u003cp\u003eAge (years)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.15447154471545%\" valign=\"top\"\u003e\n \u003cp\u003e7 (5.1-8.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.15447154471545%\" valign=\"top\"\u003e\n \u003cp\u003e6.8 (5.3-8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.585365853658537%\" valign=\"top\"\u003e\n \u003cp\u003e0.993\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.10569105691057%\" valign=\"top\"\u003e\n \u003cp\u003eHospital stays (days)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.15447154471545%\" valign=\"top\"\u003e\n \u003cp\u003e9 (7-11)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.15447154471545%\" valign=\"top\"\u003e\n \u003cp\u003e10 (8-12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.585365853658537%\" valign=\"top\"\u003e\n \u003cp\u003e0.315\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.10569105691057%\" valign=\"top\"\u003e\n \u003cp\u003eHospital stays after BAL (days)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.15447154471545%\" valign=\"top\"\u003e\n \u003cp\u003e3 (2-5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.15447154471545%\" valign=\"top\"\u003e\n \u003cp\u003e4.5 (2.3-7.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.585365853658537%\" valign=\"top\"\u003e\n \u003cp\u003e0.165\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.10569105691057%\" valign=\"top\"\u003e\n \u003cp\u003eTotal febrile days (days)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.15447154471545%\" valign=\"top\"\u003e\n \u003cp\u003e8 (6.3-10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.15447154471545%\" valign=\"top\"\u003e\n \u003cp\u003e10 (8-12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.585365853658537%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.007\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.10569105691057%\" valign=\"top\"\u003e\n \u003cp\u003eChest X-ray\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.15447154471545%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.15447154471545%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.585365853658537%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.10569105691057%\" valign=\"top\"\u003e\n \u003cp\u003ePatchy high-density lesion/consolidation [n (%)]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.15447154471545%\" valign=\"top\"\u003e\n \u003cp\u003e4 (8.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.15447154471545%\" valign=\"top\"\u003e\n \u003cp\u003e3 (25%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.585365853658537%\" valign=\"top\"\u003e\n \u003cp\u003e0.108\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.10569105691057%\" valign=\"top\"\u003e\n \u003cp\u003eLaboratory tests\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.15447154471545%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.15447154471545%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.585365853658537%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.10569105691057%\" valign=\"top\"\u003e\n \u003cp\u003eC-reactive protein (mg/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.15447154471545%\" valign=\"top\"\u003e\n \u003cp\u003e13.8 (5.9-32.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.15447154471545%\" valign=\"top\"\u003e\n \u003cp\u003e24.3 (10.2-43.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.585365853658537%\" valign=\"top\"\u003e\n \u003cp\u003e0.114\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.10569105691057%\" valign=\"top\"\u003e\n \u003cp\u003eD-dimer (\u0026mu;g/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.15447154471545%\" valign=\"top\"\u003e\n \u003cp\u003e216 (168-298)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.15447154471545%\" valign=\"top\"\u003e\n \u003cp\u003e578 (509-1877)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.585365853658537%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.10569105691057%\" valign=\"top\"\u003e\n \u003cp\u003ePCT (ng/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.15447154471545%\" valign=\"top\"\u003e\n \u003cp\u003e0.123 (0.068-0.199)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.15447154471545%\" valign=\"top\"\u003e\n \u003cp\u003e0.209 (0.123-0.431)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.585365853658537%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.031\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.10569105691057%\" valign=\"top\"\u003e\n \u003cp\u003eCoinfection [n (%)]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.15447154471545%\" valign=\"top\"\u003e\n \u003cp\u003e21 (44%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.15447154471545%\" valign=\"top\"\u003e\n \u003cp\u003e8 (67%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.585365853658537%\" valign=\"top\"\u003e\n \u003cp\u003e0.155\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.10569105691057%\" valign=\"top\"\u003e\n \u003cp\u003eA2063G variation, No. [n (%)]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.15447154471545%\" valign=\"top\"\u003e\n \u003cp\u003e10 (21%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.15447154471545%\" valign=\"top\"\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.585365853658537%\" valign=\"top\"\u003e\n \u003cp\u003e0.083\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eNote: Data are No. (%) of patients or median (interquartile range)\u003c/p\u003e\n\u003cp\u003eModerate mycoplasma pneumoniae pneumonia (MMPP); severe mycoplasma pneumoniae pneumonia (SMPP); procalcitonin. (PCT)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u0026nbsp;\u003c/strong\u003eClinical characteristics and laboratory tests of MSPP and MRMP\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"41.02079395085066%\" valign=\"top\"\u003e\n \u003cp\u003eCharacteristic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003eMSMP (N=48)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003eMRMP (N=10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003eP value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"41.02079395085066%\" valign=\"top\"\u003e\n \u003cp\u003eMale/female\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e20/28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e4/6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e0.922\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"41.02079395085066%\" valign=\"top\"\u003e\n \u003cp\u003eAge (years)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e6.7 (5.1-8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e8.5 (6-10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e0.056\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"41.02079395085066%\" valign=\"top\"\u003e\n \u003cp\u003eHospital stays (days)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e8.5 (7-11)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e10 (8-12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e0.127\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"41.02079395085066%\" valign=\"top\"\u003e\n \u003cp\u003eHospital stays after BAL (days)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e3 (2-5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e3.5 (3-5.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e0.340\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"41.02079395085066%\" valign=\"top\"\u003e\n \u003cp\u003eTotal febrile days (days)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e8 (7-10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e8.5 (7.8-9.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e0.952\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"41.02079395085066%\" valign=\"top\"\u003e\n \u003cp\u003eChest X-ray\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"41.02079395085066%\" valign=\"top\"\u003e\n \u003cp\u003ePatchy high-density lesion/consolidation\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;[n (%)]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e5 (10%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e2 (20%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e0.397\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"41.02079395085066%\" valign=\"top\"\u003e\n \u003cp\u003eLaboratory tests\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"41.02079395085066%\" valign=\"top\"\u003e\n \u003cp\u003eC-reactive protein (mg/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e14.4 (8.3-33)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e15 (1.4-55.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e0.968\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"41.02079395085066%\" valign=\"top\"\u003e\n \u003cp\u003eD-Dimer (\u0026mu;g/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e273 (187-509)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e201 (178-593)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e0.463\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"41.02079395085066%\" valign=\"top\"\u003e\n \u003cp\u003ePCT (ng/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e0.125 (0.072-0.225)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e0.185 (0.058-0.264)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e0.714\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"41.02079395085066%\" valign=\"top\"\u003e\n \u003cp\u003eCoinfection [n (%)]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e25 (52%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e4 (40%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.659735349716446%\" valign=\"top\"\u003e\n \u003cp\u003e0.487\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote:\u0026nbsp;Data are No. (%) of patients or median (interquartile range)\u003c/p\u003e\n\u003cp\u003eMacrolide-sensitive mycoplasma pneumoniae (MSMP); Macrolide-resistant mycoplasma pneumoniae (MRMP); procalcitonin (PCT)\u003cstrong\u003e\u003c/strong\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bronchoalveolar lavage, metagenomic, Mycoplasma pneumoniae pneumonia, Community-acquired pneumonia, macrolide resistance","lastPublishedDoi":"10.21203/rs.3.rs-4833361/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4833361/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e The purpose of this study was to investigate the clinical value of bronchoalveolar lavage (BAL) combined with metagenomic assessment for children hospitalized with \u003cem\u003eMycoplasma pneumoniae\u003c/em\u003epneumonia (MPP).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e This study included children hospitalized with MPP at The Affiliated Women and Children's Hospital of Ningbo University from May 2023 to August 2023. The patients were divided into a moderate \u003cem\u003eM\u003c/em\u003e.\u003cem\u003e pneumoniae\u003c/em\u003e pneumonia (MMPP) group and a severe \u003cem\u003eM. pneumoniae\u003c/em\u003e pneumonia (SMPP) group according to the results of the community-acquired pneumonia (CAP) assessment at admission. All the patients \u0026nbsp;underwent fibre bronchoscopic lavage within 3–7 days after macrolide treatment. Patient throat swab (TS) and bronchoalveolar lavage fluid (BALF) samples were collected for metagenomic next-generation sequencing (mNGS) analysis. The patients were divided into a macrolide-sensitive \u003cem\u003eM. pneumoniae\u003c/em\u003e (MSMP) group and a macrolide-resistant \u003cem\u003eM. pneumoniae\u003c/em\u003e (MRMP) group according to the presence of macrolide resistance genes in 23S rRNA domain V. The respiratory microbial community diversity was also analysed. Differentially abundant species prediction in TS and BALF samples between the MSMP group and the MRMP group was performed. The maximum-likelihood phylogenetic trees of some patients were also analysed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eDuring the 4-month study, TS and BALF samples were collected from 60 children aged 2-12 years, with a median age of 7 years\u003cstrong\u003e.\u003c/strong\u003e On the basis of the CAP assessment results at admission, 48 patients were included in the MMPP group, and 12 patients were included in the SMPP group. The total number of febrile days, D-dimer levels, and procalcitonin (PCT) levels were increased in the SMPP group. On the basis of the mNGS results, these patients were divided into 48 cases of MSMP and 10 cases of MRMP (all cases were A2063G positive). The MP detection rate in TS samples was 92% (55/60), and that in BALF samples was 97% (58/60). The Shannon index of the MRMP group was lower than that of the MSMP group in the BALF samples (p\u0026lt;0.01). The abundance of \u003cem\u003eActinomyces naeslundii\u003c/em\u003e and the relative abundance of MP had optimal predictive performance for MRMP in TS and BALF samples. Five strains (MRMP) from MP12, MP20, MP23, MP48, and MP57 exhibited the \u0026nbsp;closest relationships with the strain \u003cem\u003eM. pneumoniae\u003c/em\u003e-15-885, which was isolated in Seoul in 2015. One strain (MSMP) from MP05 was most closely related to the strain \u003cem\u003eM. pneumoniae\u003c/em\u003e-E57, which was isolated in Egypt in 2009.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e MRMP is not the main cause of SMPP-coinfection is. In terms of relative abundance, in MRMP patients, MP is dominant in BALF specimens. TS specimens cannot replace BALF samples for determining the aetiology of MPP. \u003cem\u003eA. naeslundii \u003c/em\u003eand the relative abundance of MP are optimal biomarkers for predicting whether a case is MRMP in TS samples and BALF samples, respectively. The majority of the MRMP strains from this study are closely related to the strain isolated in Seoul in 2015.\u003c/p\u003e","manuscriptTitle":"Bronchoalveolar lavage combined with metagenomic assessment of children hospitalized with Mycoplasma pneumoniae pneumonia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-04 23:08:37","doi":"10.21203/rs.3.rs-4833361/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8254ee46-1911-45b7-a54b-9343f7eba909","owner":[],"postedDate":"September 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-24T03:38:42+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-04 23:08:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4833361","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4833361","identity":"rs-4833361","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}