Development of standard protocols for sampling, preparation and storage of bio-clinical materials for trypanosoma, leishmania and plasmodium research

Abstract

According to WHO, Human African Trypanosomiasis (HAT) or sleeping sickness, Human-Visceral Leishmaniasis (VL) and, Malaria all caused by protozoan parasites, are severely neglected diseases. The prevention and control of these diseases remain a major challenge to scientists. Molecular techniques continue to be exploited as a major tool in the prevention and control of parasitic diseases. However, the feasibility and dynamics of using these techniques directly in the field remain a major challenge. This is mainly due to the technicalities involved in molecular methods and the logistical constraints that prevail in the field situations. Therefore, the preservation of samples drawn from the endemic areas (field) to be sent for analysis several hours, days or weeks later to established laboratory setups remains an important pre-analytical task. The aim of this study was to develop a standard protocol for sample preparation, extraction and storage of bio-clinical materials for Leishmania, Trypanosoma and Plasmodium research. Seven different protocols were evaluated. Each protocol had two sets of blood samples; one set spiked with parasites and another set of plain blood samples (non-spiked). These samples were preserved in different media (either buffer or filter paper) and stored under different temperatures. DNA and RNA extraction was carried out at seven time points (days 0 to week 10) and analysis done by the use of quantitative and qualitative PCR and NASBA assays. The results obtained at each time point for the seven protocols were compared along the storage duration. A newly developed buffer, the L3 buffer proved to be very reliable in short term and long term preservation of parasite RNA and DNA in the spiked samples. It is envisaged to be ideal for utilization in field situations where bio-clinical samples for molecular work require preservation.