Detection of Leishmania by PCR-oligochromatography with the "Leishmania OligoC-Test"
Abstract number: P1871
Laurent T., Deborggraeve S., Espinoza D., Leclipteux T., Büscher P., Arevalo J., Mertens P.
Leishmaniasis is a vector born disease caused by protozoan of the Leishmania genus. At least 15 species can infect humans causing a broad spectrum of clinical manifestations like Visceral leishmaniasis (VL), cutaneous leishmanaisis (CL), muco-cutaneous leishmaniasis (MCL) and PKDL.
As no vaccine is available, the control of leishmaniasis relies on diagnosis and treatment of detected cases. Current diagnosis of VL is based on serology and is confirmed by microscopy (not very sensitive) or culture, which can take more than 4 days. Leishmanin Skin Test (LST), culture or microscopy of lesion biopsy are used for CL diagnosis. While quite sensitive, serology and LST cannot discriminate past and present infection. Moreover, in immunocompromised patients (eg. transplant, HIV/VL co-infection) the performance of serology is seriously affected.
PCR was shown to be a valuable tool for Leishmania diagnosis. Despite its high sensitivity, PCR is not routinely used in endemic countries mainly due to it's complexity, and the equipment costs that makes it unaffordable by laboratories with limited research facilities and resources. We have recently developed and evaluated the "Leishmania OligoC-Test", a rapid, easy-to-use and sensitive PCR-oligochromatography (PCR-OC) assay. Both PCR mix and detection device were designed to be used in limited facilities and resources settings.
The PCR mix contains an internal control (IC) allowing to highlight the presence of PCR inhibitors in the sample to validate any negative results, and dUTP to allow elimination of carry-over contaminations. The detection of PCR products (Leishmania amplicon and IC amplicon) is made by Oligochromatography, a one-step assay which combines hybridisation of specific probes coupled to colloidal gold and migration on a dipstick at 55°C. The detection takes only 10 minutes and is made in closed tube. All the 12 Leishmania species tested with this assay are detected.
Up to 10fg (+/- 0.05 parasite) of genomic DNA are detected and it was possible to detected as few as 10 parasites in spiked blood. No cross-reactivity was observed with the other pathogenic Trypanosoma and bacteria we have tested. An evaluation on skin biopsies from Peru showed that most of the microscopy positive samples are detected with our assay. Leishmania OligoC-Test is a rapid, sensitive and specific assay. Its easiness and the fact that only a water bath is needed for post-PCR detection render it a valuable tool for a broad use.
|Session name:||18th European Congress of Clinical Microbiology and Infectious Diseases|
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