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PCR-based genotyping of neotropical leishmania: taxonomical, clinical and eco-epidemiological applications

Tuesday, 4 October, 2005 - 16:00
Campus: Brussels Humanities, Sciences & Engineering campus
Faculty: Science and Bio-engineering Sciences
Ana Lineth Garcia Orellana
phd defence

American tegumentary leishmaniasis (ATL) is an endemic
anthropozoonosis undergoing expansion on the American continent. The
disease is caused by different Leishmania species, and this is associated
with different pathologies. In addition differences at intra-species level
can also be observed. Finally, ATL presents different patterns of
transmission with many vectors and reservoirs involved and these
patterns are currently changing (e.g.domestication). Therefore, the
detection and differentiation of the etiological agent of the infection is
relevant for clinical diagnosis, management of the patients and ecoepidemiology.
The aim of this work was to study the genetic
polymorphism of Neotropical Leishmania species at different loci, and to
identify those more relevant for taxonomical, clinical and ecoepidemiological
applications. First, we developed three PCR-RFLP assays
to investigate the genetic polymorphism based on major immunogens like
hsp70, H2B, and cpb. Together with previous assays based on non-coding
(rDNA ITS) and the encoding major antigen gp63, a broad sample of
natural isolates of Leishmania (Viannia) species was analyzed. The results
of this multilocus analysis (MLP) supported the current taxonomy in a
most robust way than multilocus enzyme electrophoresis (MLEE) analysis.
Genetic relationships between the five species were similar to those
evidenced by other methods and more interestingly MLP detected a high
level of polymorphism among strains analysed (up to 95% within
L.(V).braziliensis) demonstrating a particular relevance for the analysis of
intra-species polymorphism. However, our study did not demonstrate so
far an association between strains and clinical pleomorphism. Second, we
investigated the capacity of the developed hsp70 PCR-RFLP assay for
Leishmania detection and direct species typing in clinical samples. The
assay combined with capillary electrophoresis in a microchip device,
demonstrated high sensitivity and allowed in situ Leishmania species
identification. Third, to simplify clinical sample collection we developed a
comparative study of hsp70 PCR-RFLP performance in different skin
samples (biopsies, skin scrapings and syringe aspirates). The assay
allowed in situ characterization of the etiological agent of Leishmania
infections in non-invasive clinical samples like a dermal scraping with high
sensitivity (95%). Finally, we integrated entomology and molecular
parasitology to document the transmission pattern of a peri-domestic
focus situated in the Isiboro-Secure area of Bolivia, Amazonian lowlands.
The hsp70 PCR-RFLP assay demonstrated to be a useful tool to detect and
type neotropical Leishmania species directly in vectors contributing to
establish the domestication of the Leishmania life cycle in the endemic
suited area. The genetic polymorphism evidenced by antigen-genes
supports the current taxonomy for neotropical Leishmania species,
allowing to type most Leishmania species through simple PCR-RFLP
assays that can be applied for clinical and eco-epidemiological studies.