Abstract
BackgroundBacteria 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 FindingsFor 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/SignificanceThe 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.
Highlights
Leptospirosis is a potentially fatal systemic illness resulting from infection with spirochetes of the genus Leptospira [1,2]
Extracted nucleic acids from Staphylococcal isolates and cultured strains of S. enterica subsp. arizonae, S. enterica serovar Typhi, T. denticola, and B. burgdorferi produced no amplification when tested in the pathogenic real-time PCR (rtPCR)
We describe the development of an rtPCR for the detection of pathogenic Leptospira species and present the results from a comparison of molecular diagnostics for Leptospira using samples from 65 suspected leptospirosis cases
Summary
Leptospirosis is a potentially fatal systemic illness resulting from infection with spirochetes of the genus Leptospira [1,2]. A wide range of clinical manifestations can occur following human infection with Leptospira, spanning asymptomatic infection to severe disease, multi-system organ failure and death [1,2,4,5,6]. It is estimated that 873,000 severe infections occur annually, with 49,000 deaths [3]. The non-specific disease presentation and limitations in available diagnostics for Leptospira likely render these disease estimates inaccurate [1,2,7,8]. Bacteria of the genus Leptospira, the causative agents of leptospirosis, are categorized into pathogenic and non-pathogenic species. 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
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