Comparison of Various Sample Preparation Methods for PCR Diagnosis of Visceral Leishmaniasis Using Peripheral Blood

Abstract

We have compared various sample preparation methods for the PCR diagnosis of visceral leishmaniasis (VL) using peripheral blood samples and tested the influence of these protocols upon sensitivity. Four methods of lysis-DNA extraction were used with two types of blood samples: whole blood (WB) and buffy coat (BC). Comparisons were first carried out with seeded samples at various parasite concentrations. At high concentrations (≥1,000 parasites/ml), there were no significant differences in PCR sensitivity among the methods tested. At concentrations of ≤100 parasites/ml, proteinase K (PK)-based methods proved clearly superior to guanidine-EDTA-based methods. Moreover, a 10-fold increase in sensitivity was observed for BC over that for WB. Thus, the best sensitivity was obtained with the BC prepared with PK-based methods. With this combination, the PCR reliably detected 10 parasites/ml but was inconsistent when the sample contained 1 parasite/ml of blood. The methods that yielded the highest sensitivities were evaluated with seven dogs and four human VL patients. Again, the utilization of the BC prepared with PK-based methods gave the best results. The optimization of each step of the assay (sample preparation, DNA 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 posttherapeutic follow-up.