Culture-Independent Detection and Identification of Leptospira Serovars

Abstract

Pathogenic Leptospira, the causative agents of leptospirosis, comprise >200 serotypes (called serovars). Most have a restricted reservoir-host range, and some, e.g., serovar Copenhageni, are cosmopolitan and of public health importance owing to their propensity to produce severe, fatal disease in humans. Available serotyping approaches--such as multi-locus sequence typing, core genome sequence typing, pulsed-field gel electrophoresis, and the cross-agglutination absorption test--are tedious and expensive, and require isolation of the organisms in culture media--a protracted and incredibly inefficient process-- precluding their use in prospective studies or outbreak investigations. The unavailability of culture-independent assays capable of distinguishing Leptospira serotypes remains a crucial gap in the field. Here, we have developed a simple yet specific real-time qPCR assay--targeting a Leptospira-unique gene encoding a putative polysaccharide flippase--that provides intra-species, serotype-defining (i.e., epidemiologically useful) information, and improves upon the sensitivity of preferred lipL32-based qPCR-based diagnostic tests. The assay, dubbed RAgI ("rage one"), is rapid and affordable, and reliably and specifically detects group I pathogenic Leptospira in culture, serum and urine, with no detectable off-target amplification--even of the genetically related but low virulence group II pathogenic (formerly "intermediate") or non-pathogenic Leptospira. It retained 100% diagnostic specificity when tested against difficult sample types, including field-collected dog urine-samples and environmental samples containing varied and complex microbial species-consortia. And holds considerable promise in the clinical setting, and for routine epidemiological and environmental surveillance studies.