Differentiation between Trypanosoma cruzi and Trypanosoma rangeli using heat-shock protein 70 polymorphisms.
OBJECTIVE
Differential diagnosis of infection with Trypanosoma cruzi or T. rangeli is relevant for epidemiological studies and clinical practice as both species infect humans, but only T. cruzi causes Chagas' disease. Their common antigen determinants complicate the distinction between both species, while current PCR assays used for differentiation show some drawbacks. We developed and validated a generic PCR discriminating the species by restriction fragment length polymorphism (RFLP) analysis and a duplex PCR specifically amplifying a differently sized fragment of both species.


METHODS
The assays are based upon a partial region of the heat-shock protein 70 gene (hsp70). The analytical sensitivity and specificity were determined for both PCRs. The assays were analytically evaluated on a panel of six T. cruzi, one T. cruzi marinkellei and four T. rangeli strains, various other infectious pathogens, a panel of spiked samples of T. cruzi, and artificially mixed infections of T. cruzi and T. rangeli. Finally, the tools were applied on 36 additional isolates of Trypanosoma species.


RESULTS
The detection limit of the PCRs was between 0.05 and 0.5 parasite genomes, and 1-10 parasites spiked in 200 μl blood. In artificial mixtures, PCR-RFLP picked up both species in ratios up to 10(2) and duplex PCR up to 10(4) . In the 36 isolates tested, both single and mixed infections were identified. All assays were shown to be specific.


CONCLUSIONS
Our PCRs show high potential for the differential diagnosis of T. cruzi and T. rangeli, which in view of their sensitivity can aid in the confirmation of infection with these parasites in vectors, reservoirs and clinical samples.
Parasitology Department, Institute of Tropical Medicine Pedro Kouri, La Havana, Cuba.
Jorge
J
Fraga
Aymé
A
Fernandez-Calienes
Ana Margarita
AM
Montalvo
Ilse
I
Maes
Jean-Claude
JC
Dujardin
Gert
G
Van der Auwera
D014355:Chagas Disease / Q000175:diagnosis / Q000469:parasitology; D003937:Diagnosis, Differential; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012150:Polymorphism, Restriction Fragment Length; D014349:Trypanosoma cruzi / Q000235:genetics*; D057509:Trypanosoma rangeli / Q000235:genetics*; D014352:Trypanosomiasis / Q000175:diagnosis / Q000469:parasitology*
D018840:HSP70 Heat-Shock Proteins
Trypanosoma cruzi; Trypanosoma rangeli; differentiation; hsp70; restriction fragment length polymorphism