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Probe-based metagenomic pathogen detection: advancing laboratory capacity for complex diagnosis

2025-10-14Journal article Open access
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dc.contributor.authorFerreira, Rita
dc.contributor.authorCoelho, Luís
dc.contributor.authorSantos, João Dourado
dc.contributor.authorSobral, Daniel
dc.contributor.authorIsidro, Joana
dc.contributor.authorMixão, Verónica
dc.contributor.authorPinto, Miguel
dc.contributor.authorNunes, Alexandra
dc.contributor.authorBorrego, Maria José
dc.contributor.authorLopo, Sílvia
dc.contributor.authorOleastro, Mónica
dc.contributor.authorSousa, Rita
dc.contributor.authorPalminha, Paula
dc.contributor.authorVeríssimo, Cristina
dc.contributor.authorGargaté, Maria João
dc.contributor.authorGuiomar, Raquel
dc.contributor.authorCordeiro, Rita
dc.contributor.authorMacedo, Rita
dc.contributor.authorBajanca-Lavado, Paula
dc.contributor.authorPaixão, Paulo
dc.contributor.authorDuarte, Sílvia
dc.contributor.authorVieira, Luís
dc.contributor.authorBorges, Vítor
dc.contributor.authorGomes, João Paulo
dc.date.accessioned2025-11-18T13:01:03Z
dc.date.available2025-11-18T13:01:03Z
dc.date.issued2025-10-14
dc.descriptionThis article is part of the Research Topic: Rapid and Efficient Analytical Technologies for Pathogen Detection
dc.description.abstractProbe-based pathogen enrichment, followed by NGS, is a promising tool for complex diagnosis, overcoming traditional challenges of shotgun metagenomics, namely small microbial/human genetic material ratio and demanding computational resources. Here, we assessed the combined detection performance of two Illumina probe-based panels, the Respiratory and the Urinary Pathogen ID panels (RPIP and UPIP), using 99 clinical samples of 15 different matrices (e.g., cerebrospinal fluid, plasma, serum, urine, swabs, biopsies, etc.) available from Portuguese National Reference Laboratories. This sample set involved 114 "PCR-positive hits" (Ct values range of 9.7-41.3; median of 28.4) for 52 non-redundant human pathogens. For a more detailed bioinformatics assessment, as a complement of the Illumina turnkey solution (Explify), we applied an extended version of our INSaFLU-TELEVIR(+) metagenomics pipeline. Whereas Explify analyses resulted in an initial detection frequency of 73.7% (84/114), the subsequent application of INSaFLU-TELEVIR(+), including taxonomic classification followed by confirmatory read mapping, enabled an overall detection proportion of 79.8% (91/114) of the PCR-positive hits. This translated into a detection rate increment from 54.3% (19/35) to 65.7% (23/35) for bacteria, and from 85.3% (58/68) to 89.7% (61/68) for viruses. The implemented workflow was also very satisfactory for samples with qPCR Ct values above 30, with an overall detection frequency of 71.8% (28/39) when compared with the 92.0% (46/50) observed for those with Ct ≤ 30. In summary, this study validated and established a pioneering approach at the Portuguese National Institute of Health to support clinicians in complex diagnosis, contributing to advance diagnostic capabilities toward a more informed clinical decision and potential improvement of infectious disease outcomes.eng
dc.description.sponsorshipThis study was co-funded by the European Union project “Sustainable use and integration of enhanced infrastructure into routine genome-based surveillance and outbreak investigation activities in Portugal” - GENEO (Project No. 101113460) on behalf of the EU4H programme (EU4H-2022-DGA-MS-IBA-01-02). This study was also co-financed through the DURABLE project. The DURABLE project has been co-funded by the European Union, under the EU Health Programme (EU4H), Project No. 101102733. Neither the European Union nor the granting authority can be held responsible for them. The acquisition of WGS-associated equipment used in this study was co-funded by the Health Emergency Preparedness and Response (HERA) grant “Grant/2021/PHF/23776” and the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020 – Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by the Portuguese Science and Technology Foundation (FCT). We also thank the Infraestrutura Nacional de Computação Distribuída (INCD) for providing computational resources for INSaFLU-TELEVIR testing. INCD was funded by FCT and FEDER under project 22153-01/SAICT/2016.
dc.identifier.citationFront Microbiol. 2025 Oct 14:16:1656831. doi: 10.3389/fmicb.2025.1656831. eCollection 2025
dc.identifier.doi10.3389/fmicb.2025.1656831
dc.identifier.eissn1664-302X
dc.identifier.pmid41164002
dc.identifier.urihttp://hdl.handle.net/10400.18/10621
dc.language.isoeng
dc.peerreviewedyes
dc.publisherFrontiers Media
dc.relation101113460
dc.relation101102733
dc.relation22153-01/SAICT/2016
dc.relation.hasversionhttps://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2025.1656831/full
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/
dc.subjectINSaFLU-TELEVIR
dc.subjectBioinformatics Analysis
dc.subjectmNGS
dc.subjectMetagenomics
dc.subjectNext-Generation Sequencing
dc.subjectPathogen Detection
dc.titleProbe-based metagenomic pathogen detection: advancing laboratory capacity for complex diagnosiseng
dc.typejournal article
dcterms.referenceswww.frontiersin.org/articles/10.3389/fmicb.2025.1656831/full#supplementary-material
dspace.entity.typePublication
oaire.citation.startPage1656831
oaire.citation.titleFrontiers in Microbiology
oaire.citation.volume16
oaire.versionhttp://purl.org/coar/version/c_970fb48d4fbd8a85

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