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Standardization and validation of real time PCR assays for the diagnosis of histoplasmosis using three molecular targets in an animal model

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dc.creatorLópez, Luisa F.spa
dc.creatorMuñoz, César O.spa
dc.creatorCáceres, Diego H.spa
dc.creatorTobón, Ángela M.spa
dc.creatorLoparev, Vladimirspa
dc.creatorClay, Oliverspa
dc.creatorChiller, Tomspa
dc.creatorLitvintseva, Anastasiaspa
dc.creatorGade, Lalithaspa
dc.creatorGonzález, Ángelspa
dc.creatorGómez, Beatriz L.spa
dc.date.accessioned2020-05-25T23:58:14Z
dc.date.available2020-05-25T23:58:14Z
dc.date.created2017spa
dc.description.abstractHistoplasmosis is considered one of the most important endemic and systemic mycoses worldwide. Until now few molecular techniques have been developed for its diagnosis. The aim of this study was to develop and evaluate three real time PCR (qPCR) protocols for different protein-coding genes (100-kDa, H and M antigens) using an animal model. Fresh and formalin-fixed and paraffin-embedded (FFPE) lung tissues from BALB/c mice inoculated i.n. with 2.5x106 Histoplasma capsulatum yeast or PBS were obtained at 1, 2, 3, 4, 8, 12 and 16 weeks post-infection. A collection of DNA from cultures representing different clades of H. capsulatum (30 strains) and other medically relevant pathogens (36 strains of related fungi and Mycobacterium tuberculosis) were used to analyze sensitivity and specificity. Analytical sensitivity and specificity were 100% when DNAs from the different strains were tested. The highest fungal burden occurred at first week post-infection and complete fungal clearance was observed after the third week; similar results were obtained when the presence of H. capsulatum yeast cells was demonstrated in histopathological analysis. In the first week post-infection, all fresh and FFPE lung tissues from H. capsulatum-infected animals were positive for the qPCR protocols tested except for the M antigen protocol, which gave variable results when fresh lung tissue samples were analyzed. In the second week, all qPCR protocols showed variable results for both fresh and FFPE tissues. Samples from the infected mice at the remaining times post-infection and uninfected mice (controls) were negative for all protocols. Good agreement was observed between CFUs, histopathological analysis and qPCR results for the 100-kDa and H antigen protocols. We successfully standardized and validated three qPCR assays for detecting H. capsulatum DNA in fresh and FFPE tissues, and conclude that the 100-kDa and H antigen molecular assays are promising tests for diagnosing this mycosis. © This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.eng
dc.format.mimetypeapplication/pdf
dc.identifier.doihttps://doi.org/10.1371/journal.pone.0190311
dc.identifier.issn19326203
dc.identifier.urihttps://repository.urosario.edu.co/handle/10336/22827
dc.language.isoengspa
dc.publisherPublic Library of Sciencespa
dc.relation.citationIssueNo. 12
dc.relation.citationTitlePLoS ONE
dc.relation.citationVolumeVol. 12
dc.relation.ispartofPLoS ONE, ISSN:19326203, Vol.12, No.12 (2017)spa
dc.relation.urihttps://www.scopus.com/inward/record.uri?eid=2-s2.0-85039851390&doi=10.1371%2fjournal.pone.0190311&partnerID=40&md5=f282e3a7043a347cbcb9caceb2129aa8spa
dc.rights.accesRightsinfo:eu-repo/semantics/openAccess
dc.rights.accesoAbierto (Texto Completo)spa
dc.source.instnameinstname:Universidad del Rosariospa
dc.source.reponamereponame:Repositorio Institucional EdocURspa
dc.subject.keywordAntigenspa
dc.subject.keywordAnimaleng
dc.subject.keywordH antigenspa
dc.subject.keywordM antigenspa
dc.subject.keywordUnclassified drugspa
dc.subject.keywordFungal dnaspa
dc.subject.keywordAjellomyces capsulatusspa
dc.subject.keywordAnimal cellspa
dc.subject.keywordAnimal experimentspa
dc.subject.keywordArticlespa
dc.subject.keywordBacterial strainspa
dc.subject.keywordCell countspa
dc.subject.keywordColony forming unitspa
dc.subject.keywordControlled studyspa
dc.subject.keywordDiagnostic accuracyspa
dc.subject.keywordFungal strainspa
dc.subject.keywordFungus identificationspa
dc.subject.keywordHistopathologyspa
dc.subject.keywordHistoplasmosisspa
dc.subject.keywordHumanspa
dc.subject.keywordMousespa
dc.subject.keywordMycobacterium tuberculosisspa
dc.subject.keywordNonhumanspa
dc.subject.keywordPathogen clearancespa
dc.subject.keywordReal time polymerase chain reactionspa
dc.subject.keywordSensitivity and specificityspa
dc.subject.keywordValidityspa
dc.subject.keywordYeast cellspa
dc.subject.keywordAnimalspa
dc.subject.keywordBagg albino mousespa
dc.subject.keywordDisease modelspa
dc.subject.keywordFungal genespa
dc.subject.keywordGeneticsspa
dc.subject.keywordHistoplasmaspa
dc.subject.keywordHistoplasmosisspa
dc.subject.keywordIsolation and purificationspa
dc.subject.keywordLungspa
dc.subject.keywordMicrobiologyspa
dc.subject.keywordProceduresspa
dc.subject.keywordReal time polymerase chain reactionspa
dc.subject.keywordValidation studyspa
dc.subject.keywordAnimalsspa
dc.subject.keywordDisease modelseng
dc.subject.keywordDna fungaleng
dc.subject.keywordGenes fungaleng
dc.subject.keywordHistoplasmaspa
dc.subject.keywordHistoplasmosisspa
dc.subject.keywordLungspa
dc.subject.keywordMicespa
dc.subject.keywordMice, inbred balb cspa
dc.subject.keywordMycobacterium tuberculosisspa
dc.subject.keywordReal-time polymerase chain reactionspa
dc.subject.keywordSensitivity and specificityspa
dc.titleStandardization and validation of real time PCR assays for the diagnosis of histoplasmosis using three molecular targets in an animal modelspa
dc.typearticleeng
dc.type.hasVersioninfo:eu-repo/semantics/publishedVersion
dc.type.spaArtículospa
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