Impaired immune signaling and changes in the lung microbiome precede secondary bacterial pneumonia in COVID-19

Impaired immune signaling and changes in the lung microbiome precede secondary bacterial pneumonia in COVID-19 | 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 Article Impaired immune signaling and changes in the lung microbiome precede secondary bacterial pneumonia in COVID-19 Alexandra Tsitsiklis, Beth Zha, Ashley Byrne, Catherine DeVoe, and 30 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-380803/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 Secondary bacterial infections, including ventilator-associated pneumonia (VAP), lead to worse clinical outcomes and increased mortality following viral respiratory infections including in patients with coronavirus disease 2019 (COVID-19). Using a combination of tracheal aspirate bulk and single-cell RNA sequencing (scRNA-seq) we assessed lower respiratory tract immune responses and microbiome dynamics in 28 COVID-19 patients, 15 of whom developed VAP, and eight critically ill uninfected controls. Two days before VAP onset we observed a transcriptional signature of bacterial infection. Two weeks prior to VAP onset, following intubation, we observed a striking impairment in immune signaling in COVID-19 patients who developed VAP. Longitudinal metatranscriptomic analysis revealed disruption of lung microbiome community composition in patients with VAP, providing a connection between dysregulated immune signaling and outgrowth of opportunistic pathogens. These findings suggest that COVID-19 patients who develop VAP have impaired antibacterial immune defense detectable weeks before secondary infection onset. Virology Infectious Diseases Immunology COVID-19 SARS-CoV-2 secondary bacterial pneumonia VAP metagenomics 41 scRNA-seq Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Full Text Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the manuscript can be downloaded and accessed as a PDF. Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryAppendixCOMETConsortiumMemberlist.xlsx Appendix SupplementaryMaterialsNC3.31.2021.pdf Supplementary Materials DATAS1BulkRNAseqDEgenes.xlsx Data S1 DATAS2BulkRNAseqFGSEA.xlsx Data S2 DATAS3BulkRNAseqIPAUpstreamRegCytokines.xlsx Data S3 DATAS4singlecellRNAseqDEgenes.xlsx Data S4 DATAS5scRNAseqIPACannonicalPath.xlsx Data S5 DATAS6scRNAseqIPAUpstreamRegCytokines.xlsx Data S6 DATAS7PatientMetadata.xlsx Data S7 nrreportingsummaryNCOMMS2112534.pdf Reporting Summary nrsoftwarepolicyNCOMMS2112534.pdf Software Policy checklist Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-380803","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":22652756,"identity":"61ddca42-ae17-43af-9231-20fda356db87","order_by":0,"name":"Alexandra Tsitsiklis","email":"","orcid":"https://orcid.org/0000-0003-3467-1171","institution":"University of California, San Francisco","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Alexandra","middleName":"","lastName":"Tsitsiklis","suffix":""},{"id":22652757,"identity":"9195c283-04a8-434e-91cf-4f9d9d22fcba","order_by":1,"name":"Beth 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Francisco","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Carolyn","middleName":"","lastName":"Calfee","suffix":""},{"id":22652789,"identity":"c9807cc2-3f17-428c-830a-dee878bab6b5","order_by":33,"name":"Charles Langelier","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIie3QMUvDQBTA8fcIOFWyHqj0K6Q4Kl39Ch3zCJiluBS6OPiKoEuh6235CimuDi8c6CJxDXSpCM7p1qFCcw5SxBwdBe8/PJLwfnAXAJ/vDxbqZiDbx4AF4Ny+LgEO2omqvglacmlt5CTRYIc0w+xBjm7fa+T+VfdiMpHV4yvNZg2rx6adHD9FEXIy6r0UXOiPBekqAdSlg6g4ipEDmmti05EF5SqU4PDORdJakG9onr2x2UhJeWgg+HSRwbDHyIYy1UwQoRwSCNBBVDUcAfEz5R3iYirJqb1LMS3TVhLq9KFeba4puzemXkv/pPljuFyPz1rJV7E9oex8kF/XftblvdZ8Pp/vP7YF8Nxi5JeT2+IAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-6708-4646","institution":"University of California, San Francisco","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Charles","middleName":"","lastName":"Langelier","suffix":""}],"badges":[],"createdAt":"2021-03-31 20:32:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-380803/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-380803/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":8368859,"identity":"3cc55621-efaf-4797-992c-ce3f50fcf5a7","added_by":"auto","created_at":"2021-04-23 13:43:52","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":196950,"visible":true,"origin":"","legend":"Study flowchart.\nTwo patient cohorts were studied. Cohort 1 consisted of COVID-19 patients from the COVID\nMultiphenotyping for Effective Therapies (COMET) / Immunophenotyping Assessment in a\nCOVID-19 Cohort (IMPACC) studies (described in Methods). Cohort 2 consisted of critically ill\nintubated control patients from a prior prospective cohort study led by our research group 18. The\n“early” samples were the first available tracheal aspirate specimens after intubation. For COVID-\n19 patients who developed VAP, the “late” samples were obtained a median of two days before\nVAP onset. Timing of sample collection with respect to VAP versus No-VAP groups was matched\nat “early” and “late” time points. Controls included eight critically ill, mechanically ventilated\npatients without LRTI. All COVID-19 patients included in the primary bulk analysis were also\nincluded in the longitudinal host expression and microbiome analyses. Abbreviations:\nVAP=ventilator-associated pneumonia; TA=tracheal aspirate; QC=quality control; sc or scRNAseq=\nsingle cell RNA sequencing; PNA=pneumonia; CDC=United States Centers for Disease\nControl and Prevention.","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-380803/v1/5c5ff9fd4320688a15538ba4.jpg"},{"id":8369128,"identity":"5cd6b1be-5651-4686-ab88-d7022538f0e6","added_by":"auto","created_at":"2021-04-23 13:46:52","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":120640,"visible":true,"origin":"","legend":"COVID-19 VAP is associated with a lower respiratory tract transcriptional\nsignature of bacterial infection 2 days before VAP onset.\nA) Heatmap of the top 50 differentially expressed genes by adjusted P-value between COVID-19\npatients who developed VAP (yellow) versus those who did not (red) at the “late” time-point, 2\ndays before the onset of VAP, from bulk RNA-seq. B) Gene set enrichment analysis (GSEA) at\nthe “late” time-point based on differential gene expression analyses. GSEA results were\nconsidered significant with an adjusted P-value \u003c0.05. C) Ingenuity Pathway Analysis (IPA) of\nupstream cytokines at the “late” time-point based on differential gene expression analyses. IPA\nresults were considered significant with a Z-score absolute value \u003e2 and overlap P-value \u003c0.05.\n*Denotes cytokines with an overlap P-value \u003c 0.1. All pathways and cytokines are shown in\nSupplementary data files 2 and 3.","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-380803/v1/4bf8ad3183cb27f7974e5693.jpg"},{"id":8369294,"identity":"1290b480-4d50-4f64-a1e5-41a32db65820","added_by":"auto","created_at":"2021-04-23 13:49:52","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":146656,"visible":true,"origin":"","legend":"COVID-19 patients who develop VAP have attenuated immune signaling in the\nlower respiratory tract two weeks before onset of secondary bacterial pneumonia.\nA) Heatmap of the top 50 differentially expressed genes by adjusted P-value between COVID-19\npatients who developed VAP (blue) versus those who did not (green) at the “early” time-point from\nbulk RNA-seq. B) Gene set enrichment analysis at the “early” time-point based on differential\ngene expression analyses. GSEA results were considered significant with an adjusted P-value\n\u003c0.05. C) Expression of GSEA pathways at the “early” time-point with respect to a baseline of\nuninfected, intubated controls. Pathways were selected from the GSEA results if they had an\nadjusted P-value \u003c0.05 in at least one of the comparisons (VAP vs controls or No-VAP vs\ncontrols). Pathways with an adjusted P-value \u003c0.05 when compared to controls are indicated by\ncircles with a black outline. D) Ingenuity Pathway Analysis (IPA) of upstream cytokines at the\n“early” time-point based on differential gene expression analyses. IPA results were considered\nsignificant with a Z-score absolute value \u003e2 and overlap P-value \u003c0.05. *Denotes cytokines with\nan overlap P-value \u003c0.1. All pathways and cytokines are shown in Supplementary data files 2 and\n3.","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-380803/v1/539fd08a3b2a1453fc9af850.jpg"},{"id":8368853,"identity":"9e1a0900-28b3-475e-9a5c-94ad9c54ba79","added_by":"auto","created_at":"2021-04-23 13:43:52","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":116966,"visible":true,"origin":"","legend":"scRNA-seq demonstrates that COVID-19 VAP is associated with early impaired\nanti-bacterial immune signaling in lower respiratory tract monocytes, macrophages and\nneutrophils.\nA) UMAP of single cell RNA-seq data from patients that do or do not develop VAP at the “early”\ntime-point, annotated by cell type. B) Cell type proportions in single cell RNA-seq from VAP and\nNo-VAP patients at the “early” time-point. Bars represent the median with IQR. Statistical\nsignificance was determined by Mann-Whitney tests. None of the cell types were significantly\ndifferent with a p-value \u003c0.05. The p-values for each cell type are as follows: B cells: 0.073;\nNeutrophils: 0.28; T/NK cells: 0.21; Secretory: 0.46; Ciliated: 0.94, and Mono/Mac: 0.81. C)\nVolcano plot displaying the differentially expressed genes between VAP and No-VAP patients in\nmonocytes and macrophages. D) Ingenuity Pathway Analysis (IPA) of key canonical pathways\nand upstream cytokines based on differential gene expression analysis in monocytes and\nmacrophages of patients who develop VAP versus those who do not, with adjusted p-values \u003c\n0.05. Only significant pathways (IPA Z-score of \u003e2 or \u003c-2 and overlap p-value \u003c0.05) are shown.\nE) Volcano plot displaying the differentially expressed genes between VAP and No-VAP patients\nin neutrophils. F) IPA of canonical pathways and upstream cytokines based on differential gene\nexpression analysis in neutrophils of patients who develop VAP versus those who do not, with\nadjusted p-values \u003c 0.05. Only significant pathways (IPA Z-score of \u003e2 or \u003c-2 and overlap p-value\n\u003c0.05) are shown. All pathways and cytokines are shown in Supplementary data files 5 and 6.","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-380803/v1/3eee3ee3b7e43b1bd2c089e6.jpg"},{"id":8369293,"identity":"092946b7-4a17-43eb-9880-d76f06a19e5e","added_by":"auto","created_at":"2021-04-23 13:49:52","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":132016,"visible":true,"origin":"","legend":"Temporal dynamics of the host response to VAP\nA) Heatmap of the top 50 differentially expressed genes by adjusted P-value between COVID-19\npatients who developed VAP at the “early” time-point (blue) versus the “late” time-point (yellow)\nfrom bulk RNA-seq. B) Gene set enrichment analysis (GSEA) based on differential gene\nexpression of VAP patients at the “early” vs “late” time-point from bulk RNA-seq. GSEA results\nwere considered significant with an adjusted P-value \u003c0.05. C) Ingenuity Pathway Analysis (IPA)\nof upstream cytokines based on differential gene expression analyses of VAP patients at the\n“early” vs “late” time-point from bulk RNA-seq. IPA results were considered significant with a Zscore\nabsolute value \u003e2 and overlap P-value \u003c0.05. (D-E) Ingenuity Pathway Analysis (IPA) of\nkey canonical pathways based on differential gene expression analysis in monocytes and\nmacrophages (D) or neutrophils (E) from scRNA-seq of patients who develop VAP versus those\nwho do not, with adjusted p-values \u003c 0.05. Only significant pathways (IPA Z-score of \u003e2 or \u003c-2\nand overlap p-value \u003c0.05) are shown. All pathways and cytokines are shown in Supplementary\ndata files 2, 3, 5, and 6. (F-I) Longitudinal analysis of selected pathway expression in VAP (blue)\nversus No-VAP (green) patients from bulk RNA-seq samples taken from time of intubation to\nonset of VAP for all patients. Pathway Z-scores were calculated by averaging Z-scores for the top\n20 leading edge genes of each pathway, determined by the results of GSEA comparing VAP\nversus No-VAP patients at the “early” time-point. Multiple Z-scores per patient at a given time\ninterval were averaged so that each patient corresponds to one datapoint at each interval.\nSamples from day 21+ after intubation are not shown due to a lack of these later time-points in\nthe No-VAP group. VAP onset in these patients ranged from 10-39 days post intubation. Selected\npathways are innate immune system (F), neutrophil degranulation (G), cytokine signaling (H), and\nadaptive immune system (I). Box plots represent the median and range. Statistical significance\nwas determined by two-way ANOVA, and interaction p-values are shown.","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-380803/v1/60d5e6b126d7c2fce2d987ce.jpg"},{"id":8369139,"identity":"427fc7ef-f0b7-4a1a-8071-e3899f924c2f","added_by":"auto","created_at":"2021-04-23 13:46:52","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":68246,"visible":true,"origin":"","legend":"Lung microbiome community collapse precedes VAP in COVID-19 patients.\n(A) SARS-CoV-2 viral load (reads per million sequenced, rpM) over time by days since intubation\nin patients who develop VAP vs those who do not. For plotting purposes, log(rpM+1) was used to\navoid negative values. Lung microbiome (B) bacterial diversity (Shannon’s Index) and (C)\nb-diversity (Bray Curtis Index, NMDS scaling) in COVID-19 patients with relation to VAP\ndevelopment over time by days since intubation. Box plots represent the median and range (AC).\nStatistical significance was determined by two-way ANOVA. P-values \u003c0.05 were considered\nsignificant.","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-380803/v1/2fb251233765f3a0a9736412.jpg"},{"id":8369516,"identity":"f3676bcc-b8ee-4d60-9b8b-d325f9c2bd02","added_by":"auto","created_at":"2021-04-23 13:52:52","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":87396,"visible":true,"origin":"","legend":"Mechanistic hypothesis of secondary bacterial pneumonia susceptibility in\npatients with COVID-19.\nIndividual immune responses to SARS-CoV-2 infection drive a restructuring of the microbial\ncommunity and increase susceptibility to VAP. Those predisposed to VAP have increased type I\ninterferon responses and dysregulated antibacterial immune signaling characterized by impaired\nmacrophage, neutrophil and T cell activity, decreased TLR signaling and impaired activation of\nkey cytokines important for pathogen defense including IL-1, IL-6, IL-8, TNF, and IL-17. This state\nof suppressed immunity disrupts the lower respiratory tract microbiome, predisposing to\noutgrowth of bacterial pathogens and VAP.","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-380803/v1/9d45181d33b24bd79c78871f.jpg"},{"id":13624736,"identity":"306ac3cd-45db-472d-8c43-1a48b048e2a7","added_by":"auto","created_at":"2021-09-17 07:23:45","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2895344,"visible":true,"origin":"","legend":"Article File","description":"","filename":"ManuscriptNCOMMS21125344.17.21.pdf","url":"https://assets-eu.researchsquare.com/files/rs-380803/v1_covered.pdf"},{"id":8369742,"identity":"a16f4721-0fd1-4f05-a0f1-c8494ace95ab","added_by":"auto","created_at":"2021-04-23 13:55:57","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3361331,"visible":true,"origin":"","legend":"Article 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Summary","description":"","filename":"nrreportingsummaryNCOMMS2112534.pdf","url":"https://assets-eu.researchsquare.com/files/rs-380803/v1/1339ecc4c7582f8066f242f3.pdf"},{"id":8369296,"identity":"f3cf8c81-a591-4fc4-b53b-43812b867c1b","added_by":"auto","created_at":"2021-04-23 13:49:52","extension":"pdf","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":1316965,"visible":true,"origin":"","legend":"Software Policy checklist","description":"","filename":"nrsoftwarepolicyNCOMMS2112534.pdf","url":"https://assets-eu.researchsquare.com/files/rs-380803/v1/7f7bff38bb3f0a043ccfc90b.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Impaired immune signaling and changes in the lung microbiome precede secondary bacterial pneumonia in COVID-19","fulltext":[{"header":"Full Text","content":"\u003cp\u003eDue to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the manuscript can be downloaded and accessed as a PDF.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"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":"COVID-19, SARS-CoV-2, secondary bacterial pneumonia, VAP, metagenomics, 41 scRNA-seq","lastPublishedDoi":"10.21203/rs.3.rs-380803/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-380803/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Secondary bacterial infections, including ventilator-associated pneumonia (VAP), lead to worse clinical outcomes and increased mortality following viral respiratory infections including in patients with coronavirus disease 2019 (COVID-19). Using a combination of tracheal aspirate bulk and single-cell RNA sequencing (scRNA-seq) we assessed lower respiratory tract immune responses and microbiome dynamics in 28 COVID-19 patients, 15 of whom developed VAP, and eight critically ill uninfected controls. Two days before VAP onset we observed a transcriptional signature of bacterial infection. Two weeks prior to VAP onset, following intubation, we observed a striking impairment in immune signaling in COVID-19 patients who developed VAP. Longitudinal metatranscriptomic analysis revealed disruption of lung microbiome community composition in patients with VAP, providing a connection between dysregulated immune signaling and outgrowth of opportunistic pathogens. These findings suggest that COVID-19 patients who develop VAP have impaired antibacterial immune defense detectable weeks before secondary infection onset.","manuscriptTitle":"Impaired immune signaling and changes in the lung microbiome precede secondary bacterial pneumonia in COVID-19","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-04-23 13:43:50","doi":"10.21203/rs.3.rs-380803/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":"9a384e93-a3aa-480f-8459-d225ebb01c3b","owner":[],"postedDate":"April 23rd, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":3850766,"name":"Virology"},{"id":3850767,"name":"Infectious Diseases"},{"id":3850768,"name":"Immunology"}],"tags":[],"updatedAt":"2021-11-01T11:46:01+00:00","versionOfRecord":[],"versionCreatedAt":"2021-04-23 13:43:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-380803","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-380803","identity":"rs-380803","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}