DETECTION OF TRYPANOSOMA-BRUCEI-GAMBIENSE, IN SEROLOGICALLY POSITIVE BUT APARASITAEMIC SLEEPING-SICKNESS SUSPECTS IN CAMEROON, BY PCR

Diagnosis of Gambian sleeping sickness is problematic because of the v ery low levels of parasitaemia encountered in the field. A PCR method developed for the sensitive detection of Trypanosoma brucei was used t o diagnose parasitologically negative suspects in a recent survey in C ameroon. Individuals were screened in two foci (Mbam and Fontem), firs tly with the card agglutination test for trypanosomiasis (CATT) as a p rimary serological test, together with palpation and puncture of enlar ged cervical lymph glands. Any suspects found positive by CATT (CATT+) and any clinical suspects were then subjected to several parasitologi cal tests (examination of thick blood films and use of haematocrit cen trifugation, mini-anion-exchange chromatography and a commercial kit f or in-vitro isolation). Overall, 43 of the 1703 subjects screened in t he Mbam focus were CATT+ and three (two of whom were CATT+) had enlarg ed 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 t rypanosomes in their gland juice. However, all the suspected cases of sleeping sickness, including the two gland-positives, gave negative re sults in the secondary, parasitological tests. Blood samples from 28 s uspects 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 mi blood by phenol extraction or different ial lysis and then amplified by PCR using specific primers for T. bruc ei 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 diag nostic method in this study, with an estimated sensitivity of 25 trypa nosomes/ml blood. Although PCR analysis is too expensive for routine d iagnosis, it could be very useful in determining which sleeping-suspec ts should be closely followed up.