title: Efficient Detection of Pathogenic Leptospires Using 16S Ribosomal RNA.
abstract: Pathogenic Leptospira species cause a prevalent yet neglected zoonotic disease with mild to life-threatening complications in a variety of susceptible animals and humans. Diagnosis of leptospirosis, which primarily relies on antiquated serotyping methods, is particularly challenging due to presentation of non-specific symptoms shared by other febrile illnesses, often leading to misdiagnosis. Initiation of antimicrobial therapy during early infection to prevent more serious complications of disseminated infection is often not performed because of a lack of efficient diagnostic tests. Here we report that specific regions of leptospiral 16S ribosomal RNA molecules constitute a novel and efficient diagnostic target for PCR-based detection of pathogenic Leptospira serovars. Our diagnostic test using spiked human blood was at least 100-fold more sensitive than corresponding leptospiral DNA-based quantitative PCR assays, targeting the same 16S nucleotide sequence in the RNA and DNA molecules. The sensitivity and specificity of our RNA assay against laboratory-confirmed human leptospirosis clinical samples were 64% and 100%, respectively, which was superior then an established parallel DNA detection assay. Remarkably, we discovered that 16S transcripts remain appreciably stable ex vivo, including untreated and stored human blood samples, further highlighting their use for clinical detection of L. interrogans. Together, these studies underscore a novel utility of RNA targets, specifically 16S rRNA, for development of PCR-based modalities for diagnosis of human leptospirosis, and also may serve as paradigm for detection of additional bacterial pathogens for which early diagnosis is warranted.
authors.affiliation: Department of Veterinary Medicine and Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America.
authors.forename: Brian T
authors.initials: BT
authors.lastname: Backstedt
authors.affiliation: Department of Veterinary Medicine and Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America.
authors.forename: Ozlem
authors.initials: O
authors.lastname: Buyuktanir
authors.affiliation: Department of Epidemiology of Microbial Diseases, Yale University School of Public Health, New Haven, Connecticut, United States of America; Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Brazil.
authors.forename: Janet
authors.initials: J
authors.lastname: Lindow
authors.affiliation: Department of Epidemiology of Microbial Diseases, Yale University School of Public Health, New Haven, Connecticut, United States of America.
authors.forename: Elsio A
authors.initials: EA
authors.lastname: Wunder
authors.affiliation: Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Brazil.
authors.forename: Mitermayer G
authors.initials: MG
authors.lastname: Reis
authors.affiliation: L2 Diagnostics, New Haven, Connecticut, United States of America.
authors.forename: Sahar
authors.initials: S
authors.lastname: Usmani-Brown
authors.affiliation: L2 Diagnostics, New Haven, Connecticut, United States of America.
authors.forename: Michel
authors.initials: M
authors.lastname: Ledizet
authors.affiliation: Department of Epidemiology of Microbial Diseases, Yale University School of Public Health, New Haven, Connecticut, United States of America; Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Brazil.
authors.forename: Albert
authors.initials: A
authors.lastname: Ko
authors.affiliation: Department of Veterinary Medicine and Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America.
authors.forename: Utpal
authors.initials: U
authors.lastname: Pal
mesh_terms: D000818:Animals; D006224:Cricetinae; D004195:Disease Models, Animal; D006801:Humans; D007919:Leptospira / Q000235:genetics*; D007922:Leptospirosis / Q000175:diagnosis / Q000382:microbiology*; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S