SLEEPING SICKNESS IN ZAIRE - A NESTED POLYMERASE CHAIN-REACTION IMPROVES THE IDENTIFICATION OF TRYPANOSOMA (TRYPANOZOON) BRUCEI GAMBIENSE BY SPECIFIC KINETOPLAST DNA PROBES

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 comparis on of PCR and miniature anion exchange centrifugation technique (m-AEC T) results clearly shows that in domestic animals mixed infections (Tr ypanozoon/Trypanosoma [Nannomonas] congolense) are more frequently dia gnosed by PCR than by m-AECT. Trypanozoon positive blood samples were further analysed for Trypanosoma (Trypanozoon) brucei gambiense. For t hat purpose amplified minicircle kinetoplast DNA (minicircle kDNA) was differentiated in gambiense and non-gambiense by hybridization with D NA probes. To analyse blood samples, especially those with low parasit e numbers, the amplification step had to be improved by a nested PCR. Subsequent hybridization was done with kDNA probes generated by PCR fr om blood samples which had been obtained from a human patient infected with T. (T.) b. gambiense and a pig infected with Trypanozoon. The hy bridization results clearly show that at least two genotypes of Trypan ozoon parasites occur in the sleeping sickness focus of Boma, Bas-Zair e. One obviously corresponds to T. (T.) b. gambiense and was present i n 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 samp les could be analysed by a technique which facilitates the molecular i dentification of T. (T.) b. gambiense without prior cloning, propagati on, and/or isolation of the parasites. Therefore, this technique seems to be a promising tool to elucidate the significance of the animal re servoir for the epidemiology of the gambiense sleeping sickness in Afr ica.