Sleeping sickness in Zaire: a nested polymerase chain reaction improves the identification of Trypanosoma (Trypanozoon) brucei gambiense by specific kinetoplast DNA probes

Abstract

Blood samples collected in the sleeping sickness focus of Boma, Zaire, from human patients and domestic animals were analysed by polymerase chain reaction (PCR) for the presence of trypanosome DNA. The comparison of PCR and miniature anion exchange centrifugation technique (m-AECT) results clearly shows that in domestic animals mixed infections (Trypanozoon/Trypanosoma [Nannomonas] congolense) are more frequently diagnosed by PCR than by m-AECT. Trypanozoon positive blood samples were further analysed for Trypanosoma (Trypanozoon) brucei gambiense. For that purpose amplified minicircle kinetoplast DNA (minicircle kDNA) was differentiated in gambiense and non-gambiense by hybridization with DNA probes. To analyse blood samples, especially those with low parasite numbers, the amplification step had to be improved by a nested PCR. Subsequent hybridization was done with kDNA probes generated by PCR from blood samples which had been obtained from a human patient infected with T.(T.) b. gambiense and a pig infected with Trypanozoon. The hybridization results clearly show that at least two genotypes of Trypanozoon parasites occur in the sleeping sickness focus of Boma, Baz-Zaire. One obviously corresponds to T.(T.) b. gambiense and was present in humans and two domestic animals (pig, dog). The other genotype seems to be associated with T.(T.) b. brucei and could be detected only in the blood of domestic animals. This is the first time that field samples could be analysed by a technique which facilitates the molecular identification of T.(T.) b. gambiense without prior cloning, propagation, and/or isolation of the parasites. Therefore, this technique seems to be a promising tool to elucidate the significance of the animal reservoir for the epidemiology of the gambiense sleeping sickness in Africa.