Sensitive Real-Time PCR Detection of Pathogenic Leptospira spp. and a Comparison of Nucleic Acid Amplification Methods for the Diagnosis of Leptospirosis

• Type of Material: Article
• Authors: Jesse Waggoner, Emory University; Ilana Balassiano, Instituto Oswaldo Cruz; Janaki Abeynayake, Stanford University; Malaya K. Sahoo, Stanford University; Alisha Mohamed-Hadley, Stanford University; Yuanyuan Liu, Stanford University; Juliana Magalhães Vital-Brazil, Instituto Oswaldo Cruz; Benjamin A. Pinsky, Stanford University
• Language: English
• Date: 2014-11-07
• Publisher: Public Library of Science
• Publication Version: Final Published Version
• Copyright Statement: © 2014 Waggoner et al.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1371/journal.pone.0112356
• Title of Journal or Parent Work: PLoS ONE
• ISSN: 1932-6203
• Volume: 9
• Issue: 11
• Start Page: e112356
• End Page: e112356
• Grant/Funding Information: Salary support was provided by the NIH (grant 2T32AI007502-16A1 to JJW).; This research was supported by the National Institutes of Health (NIH) (grant RC4 TW008781-01) and the Thrasher Research Fund Award (11979).
• Keywords: VALIDATION; Nucleic acids; TESTS; Polymerase chain reaction; DNA extraction; Multidisciplinary Sciences; Bacterial pathogens; IDENTIFICATION; Leptospirosis; QUANTITATIVE PCR; CULTURE; TOOL; Leptospira interrogans; Science & Technology; Leptospira; Sequence alignment; ASSAY
• Research Categories: Health Sciences, Pathology; Biology, Genetics

Abstract

Background: Bacteria of the genus Leptospira, the causative agents of leptospirosis, are categorized into pathogenic and non-pathogenic species. However, the benefit of using a clinical diagnostic that is specific for pathogenic species remains unclear. In this study, we present the development of a real-time PCR (rtPCR) for the detection of pathogenic Leptospira (the pathogenic rtPCR), and we perform a comparison of the pathogenic rtPCR with a published assay that detects all Leptospira species [the undifferentiated febrile illness (UFI) assay] and a reference 16S Leptospira rtPCR, which was originally designed to detect pathogenic species. Methodology/Principal Findings: For the pathogenic rtPCR, a new hydrolysis probe was designed for use with primers from the UFI assay, which targets the 16S gene. The pathogenic rtPCR detected Leptospira DNA in 37/37 cultured isolates from 5 pathogenic and one intermediate species. Two strains of the non-pathogenic L. biflexa produced no signal. Clinical samples from 65 patients with suspected leptospirosis were then tested using the pathogenic rtPCR and a reference Leptospira 16S rtPCR. All 65 samples had tested positive for Leptospira using the UFI assay; 62 (95.4%) samples tested positive using the pathogenic rtPCR (p = 0.24). Only 24 (36.9%) samples tested positive in the reference 16S rtPCR (p,0.0001 for comparison with the pathogenic rtPCR and UFI assays). Amplicon sequencing confirmed the detection of pathogenic Leptospira species in 49/50 cases, including 3 cases that were only detected using the UFI assay. Conclusions/Significance: The pathogenic rtPCR displayed similar sensitivity to the UFI assay when testing clinical specimens with no difference in specificity. Both assays proved significantly more sensitive than a real-time molecular test used for comparison. Future studies are needed to investigate the clinical and epidemiologic significance of more sensitive Leptospira detection using these tests.