RAPID, EARLY AND SPECIFIC DIAGNOSIS OF TUBERCULOSIS AND OTHER MYCOBACTERIAL DISEASES IN BURUNDI

The potential usefulness of ELISA based serological tests to assist in rapid, early and specific diagnosis of tuberculosis was investigated. The materials were selected, based on published data and on our preli minary findings. Initially screening tests were performed using crude antigens such as Purified Protein Derivate (PPD) and a BCG-filtrate. U nfortunately, the results with these antigens were not promising. The specificity of both antigens using sera from 94 healthy controls was 6 4%. As a consequence of these findings, the crude antigens were exclud ed from further tests, and the study was continued with purified antig ens. The work focused on 2 purified proteins: Antigen 60 (A60), a lipo polysaccharide-protein complex, and P32, a stress protein produced in zinc deprived cultures, identified as Antigen 85 A in the BCG referenc e system, both isolated from Mycobacterium bovis BCG. The commercial A 60 based ELISA and our own P32 based ELISA were used to test a total o f 300 sera from HIV positive, negative and unscreened individuals, mai nly originating from Burundi. These sera were collected from clinical established cases of pulmonary TB, extrapulmonary TB, and patients wit h non-tuberculous tropical diseases such as salmonellosis, trypanosomi asis, malaria, etc. and healthy individuals. The A60 based ELISA had a sensitivity of 76.8 % for the proven cases of active pulmonary tuberc ulosis and 61.9 % for the extrapulmonary tuberculosis cases. No differ ence was shown between HIV positive and HIV negative patients. Specifi city reached 95.2 % for healthy individuals, but dropped to 68.1 % whe n persons with active non-tuberculous tropical diseases were included. Eighty-six percent of the pulmonary cases and 87.7 % of the extrapulm onary cases were detected by the ELISA-P32. These findings suggest tha t this test might be useful as a confirmatory test for the diagnosis o f extrapulmonary tuberculosis. Again no difference was noticed between HIV negative and positive patients. The main contraindication for the use of the ELISA-P32 for the diagnosis of tuberculosis is its low spe cificity: 70.2 % with sera from healthy controls and 22.2 % for hospit alised patients and persons with non-tuberculous tropical diseases. In a small recent prospective study 4 out of 10 HIV+ persons with no evi dence for TB yielded a positive result for the ELISA-P32. Two of them developed pulmonary tuberculosis within 6 months, whereas 2 P32-positi ves and 6 P32-negatives remained up to now without any manifestations of tuberculosis. The difference was not significant, but the number of cases was limited. To avoid false positive reactions, two specific li pid antigens isolated from M. tuberculosis were selected : Sulfolipid IV (SLIV), a diacyl trehalose sulfate, and Phenolic Glycolipid Tbl (PG LTb1), a glycolipid. Most of the testing was done with SLIV, where a s pecificity of 81.2 % was reached among control sera. When only 'health y' persons (HIV negatives and positives) were included, the specificit y increased to 87%. The test detected 75.0 % of the pulmonary TB cases and 52.0% of the tuberculous lymphadenitis cases. Our results are pro mising and further studies on the usefulness of the ELISA-P32, ELISA-S LIV and other antigens, in confirming active tuberculosis or predictin g reactivation in HIV + persons, should be pursued on a larger number of subjects. In view of the low specificity of the ELISA-P32 among pat ients with other diseases, research on the possible occurence of cross reactions with shared epitopes among other etiological agents is bein g studied in collaboration with the Erasmus Hospital in Brussels (Drow art and coworkers). Although a good diagnostic ELISA test (with both h igh sensitivity and specificity) for the detection of active tuberculo sis still needs to be developed, our findings and those of others woul d suggest that it is possible to improve or modify existing tests by i dentifying specific epitopes and careful application of these tests to high risk groups.