Optimisation of template preparation and laboratory evaluation of the Loopamp™ Trypanosoma brucei kit for detection of parasite DNA in blood.

The Loopamp™ Trypanosoma brucei Detection Kit is the latest addition of molecular techniques for amplification of parasite DNA in biological materials. We have evaluated the kit on a number of preparations of crude templates from the blood of experimentally infected rodents, to provide the best option that can be extrapolated to resource-poor healthcare settings where human African trypanosomiasis (HAT) is endemic. We used rodent blood spiked with T. b. brucei at various serial dilutions to test whole blood, that was concentrated by differential lysis of red blood cells (RBCs) followed by centrifugation, or buffy coat samples recovered from whole blood after centrifugation. We also tested crude templates produced after lysis of blood with sodium dodecyl sulphate (SDS) or Triton X, and storage for up to 28 days at room temperature after spotting on filter paper or glass slides. Concentration by RBC lysis provided the highest analytical sensitivity (0.04 trypanosomes/ml), closely followed by the much cheaper SDS at 0.1 trypanosomes/ml sensitivity. We also monitored the persistence of DNA in lysed blood dried onto filter papers by testing them weekly with the LAMP kit and by PCR for the 177bp repeats characteristic of the T. brucei subspecies. At a concentration of 100 trypanosomes/ml, signals indicating presence of parasite DNA could be detected up to week 10, while at 10 trypanosomes/ml detection of signals was limited to week 4. Thus, an ordinary filter paper provides a convenient medium for preservation of trypanosome DNA at ambient conditions for use with the LAMP kit in the short run. Lysis of samples with SDS enhanced sensitivity by facilitating parasite DNA availability. This opens the avenue to incorporate LAMP in routine algorithms for HAT diagnosis and surveillance, as well as for monitoring elimination programs.