Diagnosis of human trypanosomiasis, due to Trypanosoma brucei gambiense in central Africa, by the polymerase chain reaction

The nuclear DNA of Trypanosoma congolense contains a family of highly conserved 369 base pair (bp) repeats. The sequences of three cloned copies of these repeats were determined. An unrelated family of 177 bp repeats has previously been shown to occur in the nuclear DNA of Trypanosoma brucei brucei (Sloof et al. 1983a). Oligonucleotides were synthesized which prime the specific amplification of each of these repetitive DNAs by the polymerase chain reaction (PCR). Amplification of 10% of the DNA in a single parasite of T. congolense or T. brucei spp. produced sufficient amplified product to be visible as a band in an agarose gel stained with ethidium bromide. This level of detection, which does not depend on the use of radioactivity, is about 100 times more sensitive than previous detection methods based on radioactive DNA probes. The oligonucleotides did not prime the amplification of DNA sequences in other trypanosome species nor in Leishmania, mouse or human DNAs. Amplification of DNA from the blood of animals infected with T. congolense and/or T. brucei spp. permitted the identification of parasite levels far below that detectable by microscopic inspection. Since PCR amplification can be conducted on a large number of samples simultaneously, it is ideally suited for large-scale studies on the prevalence of African trypanosomes in both mammalian blood and insect vectors.

Diagnosis of Gambian sleeping sickness is problematic because of the very low levels of parasitaemia encountered in the field. A PCR method developed for the sensitive detection of Trypanosoma brucei was used to diagnose parasitologically negative suspects in a recent survey in Cameroon. Individuals were screened in two foci (Mbam and Fontem), firstly with the card agglutination test for trypanosomiasis (CATT) as a primary serological test, together with palpation and puncture of enlarged cervical lymph glands. Any suspects found positive by CATT (CATT+) and any clinical suspects were then subjected to several parasitological tests (examination of thick blood films and use of hematocrit centrifugation, mini-anion-exchange chromatography and a commercial kit for in-vitro isolation). Overall, 43 of the 1703 subjects screened in the Mbam focus were CATT+ and three (two of whom were CATT+) had enlarged glands. In Fontem, 56 of the 1210 subjects screened were CATT+, 78 (24 of whom were CATT+) had enlarged glands and two (both CATT+) had trypanosomes in their gland juice. However, all the suspected cases of sleeping sickness, including the two gland-positives, gave negative results in the secondary, parasitological tests. Blood samples from 28 suspects from Mbam and 30 from Fontem were selected for PCR analysis on the basis of high CATT response or clinical grounds. For each suspect, DNA was prepared from 0.5 ml blood by phenol extraction or differential lysis and then amplified by PCR using specific primers for T. brucei ssp. Four samples from Mbam and nine from Fontem, including the two gland-positives, were found positive by PCR. Compared with the other parasitological techniques, therefore, PCR was the most sensitive diagnostic method in this study, with an estimated sensitivity of 25 trypanosomes/ml blood. Although PCR analysis is too expensive for routine diagnosis, it could be very useful in determining which sleeping-suspects should be closely followed up.

We have developed a sensitive and specific method to identify Trypanosoma brucei ssp. using PCR to amplify conserved expression-site-associated gene 6 and 7 DNA target sequences. Amplification of 10% of the DNA in a single trypanosome produced sufficient PCR product to be visible as a band in an agarose gel stained with ethidium bromide. We analysed 59 blood samples of serologically positive cases of sleeping sickness by PCR, and directed parasitological examination of tissue fluids. The PCR test detected 87% of the parasitologically positive cases, with a specificity of 97%. In 5 cases, the parasite was demonstrated by the PCR test 4-6 months prior to parasitological detection. This result shows the potential of the assay in early diagnosis of actual T. b. gambiense infections in apparently aparasitaemic sleeping sickness patients.