A Leishmania minicircle DNA footprint assay for sensitive detection and rapid speciation of clinical isolates

Abstract

Background: Diversity in clinical outcome, due to different species of Leishmania, and its presence in asymptomatic blood donors in endemic areas warrant development of methods that are sensitive and can rapidly identify infecting species.

Study design and methods: The kinetoplast minicircle DNA is known to have heterogeneity in sequence and is present in many thousands of copies in Leishmania. Fluorescence-based polymerase chain reaction (PCR) was used to amplify minicircle DNA from six Leishmania species from different geographic locations. The sequences were then used to construct a phylogenetic tree. Speciation of 46 blinded parasite clinical isolates from various geographic regions was validated using the assay.

Results: Analysis displayed a distinct cluster for each species or strain. Forty-three of 46 isolates were correctly assigned to the same species identified by isoenzyme electrophoresis. The three untyped isolates were all either new species or samples from a unique geographic region. The minicircles of the three isolates formed new clusters in the tree analysis. Using minicircle DNA as PCR target, the sensitivity of the parasite detection in the spiked blood samples was five parasites per mL.

Conclusion: Increased sensitivity and speciation without the need for parasite culture will be useful for diagnosis and treatment in clinical settings.