Poster Presenter

Quantification Of Parasitemia In Leishmania Donovani-Infected Hamsters By Real-Time PCR
Brian Vesely, Anuradha Srivastava Azliyati Azizan, Mark Sweat, and Dennis Kyle
USA

Visceral leishmaniasis affects over a half million individuals in 88 countries each year including the Middle East. And is caused by the intracellular parasite Leishmania donovani new drugs are urgently required to treat this devastating disease. Preclinical evaluation of new drugs is modeled in hamsters infected with L. donovani, although measurement of parasite loads is difficult and time consuming. In the current study a real-time PCR assay has been optimized and validated against two other methods to estimate the parasite load within the liver of infected hamsters. Parasite quantification has been optimized in both naïve strains and antimony resistant L. donovani.

Methods Reverse and forward primers were used that amplify a 120-bp target template of the kinetoplast DNA. Serial ten-fold dilutions of parasites were either assayed directly or spiked into un-infected liver homogenates. Quantification of the parasite load in infected-hamster livers was analyzed by a SYBR Green-based PCR assay and compared to microscopic and growth dilution assays.

Results Standard curves were generated from parasite cultures and parasite-spiked tissue. L. donovani DNA was readily detected in spiked homogenates and no products were observed with un-infected tissue and non-template control samples. The real time PCR assay was able to detect as few as 2 parasites in 2 mg of liver. In addition quantitative differences in parasite load were detected at low levels where microscopic confirmation was not possible.

Conclusion: The real time PCR method is sensitive and specific for the quantification of the parasite load within L. donovani-infected hamsters, which will accelerate the evaluation of new drugs in the definitive preclinical model of disease.