Abstracts Category : Other

In situ detection, molecular epidemiology, and improvement of molecular tools for the understanding and diagnosis of infections caused by spirochetes

by Mencias Pablo Rojas

Pathogenic spirochetes are the causative agents of some re-emerging diseases with global significance such as human intestinal spirochetosis (HIS), syphilis, Lyme disease and leptospirosis. Limitations in isolation and cultivation of these microorganisms lead to under- and misdiagnosis of bacterial infections. Fluorescence in situ hybridization (FISH), a technique designed to simultaneously visualize and identify microorganisms in biopsy samples, is an accurate microscopy tool for the diagnosis and understanding of pathogenicity in several infections. FISH, in combination with other molecular methods, was applied for the diagnosis of HIS and the identification of Treponema pallidum, the causative agent of syphilis. Brachyspira spp., the infectious agent of HIS, is a slow growing and fastidious anaerobe spirochete that colonizes the human colonic mucosa. Current routine clinical diagnosis of HIS involve histopathology analysis without molecular identification of the causative agent. Genus-specific FISH, in combination with 16S rRNA gene PCR and sequencing were applied to 149 intestinal biopsies from 91 patients, previously diagnosed with HIS by histopathology. The genus Brachyspira was identified in 77 patients, 53 of which cases could be assigned to the B. aalborgi lineage, 23 to B. pilosicoli, one mixed infection and 14 FISH and PCR negative cases. Four out of the 14 negative cases showed colonization of the intestinal epithelium by rods and cocci only, suggesting a misdiagnosis by histopathology. Syphilis is a multi-systemic disease characterized by its atypical clinical manifestations and complex diagnosis. Its causative agent, Treponema pallidum, disseminates through the body via the circulatory system and has not yet been successfully cultured in vitro. Although serology is currently used for the identification of different stages of infection, the results may be difficult to interpret due to an overlap of the signs and symptoms. A species-specific FISH probe assay, designed to target the 16S rRNA gene, was used to show the presence of T. pallidum in four different tissue samples: lymph node tissue of an HIV patient, preputial tissue, oral mucosa, and tonsils. Additionally, recombinant strains of Borrelia burgdorferi, the causative agent of Lyme disease, were used to understand the binding role of candidate adhesion protein Tp0751 (pallilysin) from T. pallidum as a factor promoting bacterial dissemination in animal models. Four different strains of B. burgdorferi, were used to assess the function of Tp0751 in in vitro and in vivo experiments. With the use of video tracking microscopy, the binding interaction of spirochetes was quantified in different conditions and showed that Tp0751 is an adhesin capable of restoring bacteria-vascular interaction that may play a role in bacterial dissemination. This dissertation explores the benefits of microscopy techniques for the understanding of infections caused by spirochetes. FISH facilitates the accurate diagnosis of HIS and provides insights about the