[Drug susceptibility test for Mycobacterium tuberculosis using non-radioactive substance, alpha-antigen].

BACTEC system is a reliable and rapid drug susceptibility test for mycobacteria and is widely accepted in Europe and in the United States of America. In Japan, it is impossible to introduce the BACTEC system in clinical laboratories because of strict regulations for the use of radioactive substances in Japan. To resolve this dilemma, we adopted alpha-antigen (alpha-antigen), a widely distributed secretory protein of mycobacteria, as the index substance replacing the radioactive substance 14C in BACTEC system. Alpha-antigen was detected by reverse passive latex agglutination (LA), using latex sensitized with rabbit anti-alpha-IgG. Susceptibility of 17 M. tuberculosis strains isolated from patients, grown on Ogawa egg medium and then cultured in 7H9 medium, and of 46 M. tuberculosis strains freshly isolated from patients by 7H9 medium of MB check system, was tested for four antituberculosis drugs, isoniazid (INH), rifampicin (RFP), streptomycin (SM) and ethambutol (EB). Ninety four percent of the control cultures were positive for alpha-antigen within 7 days after the inoculation. The MIC values of H37Rv strain in 7H9 medium determined by the method of Heifets were 0.05 micrograms/ml for INH, 0.03 micrograms/ml for RFP, 0.025 micrograms/ml for SM and 1.9 micrograms/ml for EB. In 17 strains from Ogawa egg medium, the results obtained from all but 4 strains for SM, 1 for INH, 1 for RFP and 7 for EB were concurrent with that obtained by the method using 1% Ogawa egg medium. No strains were determined to be resistant to any drug by the alpha-antigen method and be sensitive by the Ogawa medium method. In 46 strains cultured by the MB check system, the results of 42 strains for SM, 35 for INH, 39 for RFP and 36 for EB coincided with those determined by the Microtiter method. Among the strains determined to be resistant by Microtiter method, 1/2 for SM, 10/14 for INH, 4/17 for RFP and 8/10 for EB were determined to be sensitive by alpha-antigen LA method. The disagreement was seen mostly in strains which were determined to be resistant by the method using egg medium, while sensitive by the alpha-antigen LA method. The discrepancy might originate from the difference of critical concentration due to heat inactivation of the drugs and absorption in the egg medium. However, some instability was observed in latex agglutination and its cause should be examined further. This method of utilizing 7H9 medium for culture and alpha-antigen as the index of mycobacterial growth can be an expedient and economical drug susceptibility test because it does not use radioactive substance as in the case of BACTEC system.