Clinico-Molecular Detection of Human Parvovirus B19 in Post-Exchange Transfusion, Encephalitis, and Ewing’s Sarcoma: From Development to Utilization of a Molecular Detection Assay in a Tertiary Hospital Setup

Clinico-Molecular Detection of Human Parvovirus B19 in Post-Exchange Transfusion, Encephalitis, and Ewing’s Sarcoma: From Development to Utilization of a Molecular Detection Assay in a Tertiary Hospital Setup | 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 Clinico-Molecular Detection of Human Parvovirus B19 in Post-Exchange Transfusion, Encephalitis, and Ewing’s Sarcoma: From Development to Utilization of a Molecular Detection Assay in a Tertiary Hospital Setup Kashmi Sharma, Rohan Shrivastava, Somesh Mishra, Poonam Parihar, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9404388/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 Human parvovirus B19 (HB19V) infection poses significant clinical challenges in resource-limited settings, particularly regarding blood safety surveillance and diagnosis of associated syndromes. Limited access to validated diagnostic assays restricts clinical management and transfusion safety protocols. Objective To develop and validate an in-house duplex real-time PCR assay for HB19V detection and evaluate its clinical utility in a resource-limited setting. Methods A duplex real-time PCR assay targeting the conserved NS1 gene of HB19V with RNase P as endogenous control was developed. Analytical validation assessed amplification efficiency, limit of detection (LOD), precision (intra- and inter-assay coefficients of variation), and cross-reactivity. Clinical validation compared qualitative agreement with a CE-certified commercial kit using Cohen's kappa statistic. Positive samples underwent Sanger sequencing for genotype characterization. Clinical specimens from suspected HB19V infections were tested and results correlated with patient presentations. Results The assay demonstrated 98.82% amplification efficiency with LOD of 6 copies per reaction. Precision metrics showed excellent reproducibility (CoV <1% intra-assay, 0.95). Clinical application identified three significant cases: post-transfusion HB19V in neonatal bilirubin encephalopathy, HB19V consideration in severe anaemia with ventriculitis, and active infection in paediatric Ewing sarcoma with chemotherapy-induced cytopenias requiring IVIG therapy. Genotyping revealed genotypes 1a and 3, confirming regional viral diversity. Conclusion This validated in-house duplex real-time PCR assay provides a cost-effective, locally sustainable diagnostic tool for HB19V detection. It addresses critical gaps in blood safety surveillance and expands diagnostic capacity for HB19V-associated syndromes in resource-limited settings, while highlighting the necessity of contextual clinical interpretation for optimal patient management. Human Parvovirus B19 Differential Diagnosis Blood Transfusion Figures Figure 1 Figure 2 Figure 3 Full Text Additional Declarations No competing interests reported. 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-9404388","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":626746267,"identity":"77c3e3f4-55e8-48fa-919c-f622e372f44d","order_by":0,"name":"Kashmi Sharma","email":"","orcid":"","institution":"All India Institute of Medical Sciences Bhopal","correspondingAuthor":false,"prefix":"","firstName":"Kashmi","middleName":"","lastName":"Sharma","suffix":""},{"id":626746268,"identity":"2022178f-0697-43f9-8d6f-313ce2c1131a","order_by":1,"name":"Rohan Shrivastava","email":"","orcid":"","institution":"All India Institute of Medical Sciences Bhopal","correspondingAuthor":false,"prefix":"","firstName":"Rohan","middleName":"","lastName":"Shrivastava","suffix":""},{"id":626746269,"identity":"d99e8675-41cd-47e1-87a3-493cf81f9e46","order_by":2,"name":"Somesh Mishra","email":"","orcid":"","institution":"All India Institute of Medical Sciences Bhopal","correspondingAuthor":false,"prefix":"","firstName":"Somesh","middleName":"","lastName":"Mishra","suffix":""},{"id":626746270,"identity":"62ac0c7c-ae4a-4a0c-8476-13e438fbc04d","order_by":3,"name":"Poonam Parihar","email":"","orcid":"","institution":"All India Institute of Medical Sciences Bhopal","correspondingAuthor":false,"prefix":"","firstName":"Poonam","middleName":"","lastName":"Parihar","suffix":""},{"id":626746271,"identity":"41ce0fe9-a0df-4345-b0c4-7437c5197451","order_by":4,"name":"Debasis Biswas","email":"","orcid":"","institution":"All India Institute of Medical Sciences Bhopal","correspondingAuthor":false,"prefix":"","firstName":"Debasis","middleName":"","lastName":"Biswas","suffix":""},{"id":626746272,"identity":"6ee6b86a-248c-432c-aabc-f11e3d5d4a9c","order_by":5,"name":"Narendra Kumar Chaudhary","email":"","orcid":"","institution":"All India Institute of Medical Sciences Bhopal","correspondingAuthor":false,"prefix":"","firstName":"Narendra","middleName":"Kumar","lastName":"Chaudhary","suffix":""},{"id":626746273,"identity":"5a02a05b-6bc6-4bb9-b7eb-11e50d8d8b10","order_by":6,"name":"Poorva Gohiya","email":"","orcid":"","institution":"Gandhi Medical college, Bhopal","correspondingAuthor":false,"prefix":"","firstName":"Poorva","middleName":"","lastName":"Gohiya","suffix":""},{"id":626746274,"identity":"950efd7e-4a88-44a5-9be4-f44c4a5265e7","order_by":7,"name":"Mahendra Jain","email":"","orcid":"","institution":"All India Institute of Medical Sciences Bhopal","correspondingAuthor":false,"prefix":"","firstName":"Mahendra","middleName":"","lastName":"Jain","suffix":""},{"id":626746275,"identity":"24db0315-43b5-4621-b6c9-f242ddbdd198","order_by":8,"name":"Ram K. 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Setup","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"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":"Human Parvovirus B19, Differential Diagnosis, Blood Transfusion","lastPublishedDoi":"10.21203/rs.3.rs-9404388/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9404388/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman parvovirus B19 (HB19V) infection poses significant clinical challenges in resource-limited settings, particularly regarding blood safety surveillance and diagnosis of associated syndromes. Limited access to validated diagnostic assays restricts clinical management and transfusion safety protocols.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjective\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo develop and validate an in-house duplex real-time PCR assay for HB19V detection and evaluate its clinical utility in a resource-limited setting.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA duplex real-time PCR assay targeting the conserved NS1 gene of HB19V with RNase P as endogenous control was developed. Analytical validation assessed amplification efficiency, limit of detection (LOD), precision (intra- and inter-assay coefficients of variation), and cross-reactivity. Clinical validation compared qualitative agreement with a CE-certified commercial kit using Cohen's kappa statistic. Positive samples underwent Sanger sequencing for genotype characterization. Clinical specimens from suspected HB19V infections were tested and results correlated with patient presentations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe assay demonstrated 98.82% amplification efficiency with LOD of 6 copies per reaction. Precision metrics showed excellent reproducibility (CoV \u0026lt;1% intra-assay, \u0026lt;5% inter-assay) with no cross-reactivity. Qualitative agreement with the commercial reference was 100% (Cohen's kappa \u0026gt;0.95). Clinical application identified three significant cases: post-transfusion HB19V in neonatal bilirubin encephalopathy, HB19V consideration in severe anaemia with ventriculitis, and active infection in paediatric Ewing sarcoma with chemotherapy-induced cytopenias requiring IVIG therapy. Genotyping revealed genotypes 1a and 3, confirming regional viral diversity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis validated in-house duplex real-time PCR assay provides a cost-effective, locally sustainable diagnostic tool for HB19V detection. It addresses critical gaps in blood safety surveillance and expands diagnostic capacity for HB19V-associated syndromes in resource-limited settings, while highlighting the necessity of contextual clinical interpretation for optimal patient management. \u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Clinico-Molecular Detection of Human Parvovirus B19 in Post-Exchange Transfusion, Encephalitis, and Ewing’s Sarcoma: From Development to Utilization of a Molecular Detection Assay in a Tertiary Hospital Setup","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-23 09:33:57","doi":"10.21203/rs.3.rs-9404388/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":"2a67ce1e-63b1-45f8-a91d-d7e19a8439c6","owner":[],"postedDate":"April 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-23T10:09:13+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-23 09:33:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9404388","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9404388","identity":"rs-9404388","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}