Comparison of various sample preparation methods for PCR diagnosis of visceral leishmaniasis using peripheral blood

We have compared various sample preparation methods for the PCR diagnosis o f visceral leishmaniasis (VL) using peripheral blood samples and tested the influence of these protocols upon sensitivity. Four methods of lysis-DNA e xtraction were used with two types of blood samples: whole blood (WB) and b uffy coat (BC). Comparisons were first carried out with seeded samples at v arious parasite concentrations. lit high concentrations (greater than or eq ual to1,000 parasites/ml), there were no significant differences in PCR sen sitivity among the methods tested. At concentrations of less than or equal to 100 parasites/ml, proteinase K (PK)-based methods proved clearly superio r to guanidine-EDTA-based methods. Moreover, a 10-fold increase in sensitiv ity was observed for BC over that for WE. Thus, the best sensitivity was ob tained with the BC prepared,vith PK-based methods. With this combination, t he PCR reliably detected 10 parasites/ml but was inconsistent when the samp le contained 1 para site/ml of blood. The methods that yielded the highest sensitivities were evaluated with seven dogs and four human VL patients. Ag ain, the utilization of the BC prepared with PK-based methods gave the best results. The optimization of each step of the assay (sample preparation, D NA extraction, and PCR conditions) yielded a highly sensitive tool for the diagnosis of VL using patient blood, thus avoiding more invasive diagnostic procedures and allowing the detection of low parasitemia during posttherap eutic follow-up.