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PLoS One. 2014 Nov 7;9(11):e112356. doi: 10.1371/journal.pone.0112356. eCollection 2014.

Sensitive real-time PCR detection of pathogenic Leptospira spp. and a comparison of nucleic acid amplification methods for the diagnosis of leptospirosis.

Author information

1
Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, California, United States of America.
2
Laboratório de Zoonoses Bacterianas, Centro de Referência Nacional para Leptospirose, Coleção de Leptospira, WHO/PAHO Centro Colaborador para Leptospirose, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil.
3
Department of Pathology, Stanford University School of Medicine, Stanford, California, United States of America.
4
Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, California, United States of America; Department of Pathology, Stanford University School of Medicine, Stanford, California, United States of America.

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.

PMID:
25379890
PMCID:
PMC4224423
DOI:
10.1371/journal.pone.0112356
[Indexed for MEDLINE]
Free PMC Article

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