A protocol to improve genotyping of problematic microsatellite loci of Trypanosoma brucei gambiense from body fluids.

Microsatellite genotyping of Trypanosoma brucei gambiense, the causative agent of human African trypanosomiasis or sleeping sickness, and population genetics tools, are useful for inferring population parameters such as population size and dispersal. Amplifying parasite DNA directly from body fluids (i.e., blood, lymph or cerebrospinal fluid) allows avoiding costly and tedious isolation phases. It is however associated to increased frequencies of amplification failures (allelic dropouts and/or null alleles) at some loci. In this paper, we present a study focused on three T. brucei gambiense microsatellite loci suspected to present amplification problems when amplified from body fluids sampled in Guinean sleeping sickness foci. We checked for the real nature of blank and apparent homozygous genotypes of parasite DNA directly amplified from body fluids and tested the effect of three different DNA quantities of trypanosomes. Our results show that some initially blank and homozygous genotypes happen to be actual heterozygous genotypes. In Guinea, lymph from the cervical nymph nodes, known to contain the highest concentrations of parasites, appeared to provide the best amplification results. Simply repeating the PCR may be enough to retrieve the correct genotype, but we also show that increasing initial DNA content provides better results while undertaking first amplification. We finally propose an optimal protocol for amplifying trypanosome's DNA directly from body fluids that should be adapted to local characteristics and/or constraints.