Development and Validation of a Real-Time PCR for Detection of Pathogenic Leptospira Species in Clinical Materials 英文参考文献.docVIP

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Development and Validation of a Real-Time PCR for Detection of Pathogenic Leptospira Species in Clinical Materials 英文参考文献.doc

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Development and Validation of a Real-Time PCR for Detection of Pathogenic Leptospira Species in Clinical Materials 英文参考文献

DevelopmentandValidationofaReal-TimePCRfor DetectionofPathogenicLeptospiraSpeciesinClinical Materials AhmedAhmed1,MirjamF.M.Engelberts1,KimberlyR.Boer1,NiyazAhmed2,RudyA.Hartskeerl1* 1WHO/FAO/OIEandNationalCollaboratingCentreforReferenceandResearchonLeptospirosisandSectionofEpidemiology,DepartmentofBiomedicalResearch,Royal TropicalInstitute(KIT),Amsterdam,TheNetherlands,2PathogenBiologyLaboratory,SchoolofLifeSciences,UniversityofHyderabad,Hyderabad,India Abstract Availableserologicaldiagnosticsdonotallowtheconfirmationofclinicallysuspectedleptospirosisattheearlyacutephase ofillness.Severalconventionalandreal-timePCRsfortheearlydiagnosisofleptospirosishavebeendescribedbutthese have been incompletely evaluated. We developed a SYBR Green-based real-time PCR targeting secY and validated it accordingtointernationalguidelines.Todeterminetheanalyticalspecificity,DNAfrom56Leptospirastrainsbelongingto pathogenic, non-pathogenic and intermediate Leptospira spp. as well as 46 other micro-organisms was included in this study.AllthepathogenicLeptospiragaveapositivereaction.Wefoundnocross-reactionwithsaprophyticLeptospiraand othermicro-organisms,implyingahighanalyticalspecificity.TheanalyticalsensitivityofthePCRwasonecopyperreaction fromculturedhomologousstrainM20and1.2and1.5copyforheterologousstrains1342KandSarmin,respectively.In spikedserumbloodandkidneytissuethesensitivitywas10and20copiesforM20,15and30copiesfor1342Kand30 and50copiesforSarmin.Todeterminethediagnosticsensitivity(DSe)andspecificity(DSp),clinicalbloodsamplesfrom26 laboratory-confirmed and 107 negative patients suspected of leptospirosis were enrolled as a prospective consecutive cohort. Based on culture as the gold standard, we found a DSe and DSp of 100% and 93%, respectively. All eight PCR positivesamplesthathadanegativecultureseroconvertedlateron,implyingahigheractualDSp.Whenusingcultureand serology as the gold standard, the DSe was lower (89%) while the DSp was higher (100%). DSe was 100% in samples collecte