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The antileprosy effect of rifampin was evaluated by a newly developed rapid in vitro assay wherein 31 human-derived strains and 1 armadillo-derived strain of Mycobacterium leprae were maintained for 2 and 3 weeks, respectively, in murine and human macrophages in the presence of [3H]thymidine. Of these strains, 27 showed significant incorporation of the radiolabel in cultures of live bacilli as compared with control cultures of heat-killed bacilli of the same strain. Consistent and significant inhibition of [3H]thymidine uptake was observed in M. leprae resident cultures with 3 to 200 ng of rifampin per ml as compared with similar cultures without the drug. In general, an increase in percent inhibition was seen from 3 to 20 ng/ml, with marginal increases at 40, 50, and 100 ng/ml. M. leprae strains appear to be remarkably susceptible to this drug in the in vitro assay.

Studies were undertaken in three independent centers to compare the newer, in vitro radiometric macrophage (M phi) assay with the conventional mouse foot pad infection for the diagnosis of dapsone resistance. Results obtained on 12 bacilliferous patients showed good concordance in both assays. One strain diagnosed as sensitive in the M phi assay was found to be resistant in the mouse foot pad. Three Mycobacterium leprae strains considered to be partially resistant in the mouse infection behaved as resistant strains in the M phi cultures. Attention is drawn to a rapid in vitro method for the identification of drug-resistant bacilli in leprosy patients.

The proportions of dapsone resistant strains of M. leprae in cases from the field and in patient institutionalized for treatment were compared. Identification of dapsone resistance was done by mouse-foot-pad experiments carried out in the Laboratory in Central Leprosy Teaching and Research Institute, Chengalpattu. A higher percentage of resistance was detected among institutional patients as compared to field cases. The distribution of M.I. ranges of M. leprae in dapsone resistant subjects was not different from that in dapsone sensitive cases.

Mycobacterium leprae cells extracted from the skin biopsies of 14 bacilliferous lepromatous patients were maintained in human-murine macrophage cultures for 3 weeks in the presence of [3H]thymidine and DDS (4,4'-diaminodiphenyl sulfone). All cultures except one containing freshly extracted viable bacilli showed significant incorporation of [3H]thymidine as compared to control cultures containing heat-killed bacilli of the corresponding strain. Six susceptible strains of M. leprae obtained from untreated, freshly diagnosed patients showed significant inhibition of the uptake of the radiolabel in the presence of 3 and 10 ng of DDS per ml per culture. Eight strains of M. leprae obtained from patients clinically suspected of DDS resistance were tested in a similar manner. These strains were also concurrently inoculated in the footpads of mice given orally 10(-2), 10(-3), and 10(-4) g of DDS per 100 g of body weight for 9 months. Concordant results were obtained by both methods: five strains were found to be resistant, one was susceptible, and one was partially resistant. Strain VIII did not incorporate [3H]thymidine in the macrophage cultures and proved to be resistant in the mouse footpad. The macrophage culture system provides a sensitive, rapid screening method for the early diagnosis of DDS resistance.

A case of borderline lepromatous leprosy with a history of 5 years duration of disease, was first seen in 1971 and treated with graded doses of dapsone in the OP Clinic of the Institute. He became inactive and bacteriologically negative in 3 years. While continuing on dapsone therapy he relapsed into active dapsone resistant leprosy 3 1/2 years later. He was admitted in the hospital and given Rifampicin 600 mg. daily for 15 days along with dapsone 100 mg. daily. He again became inactive and bacteriologically negative within 3 years. 3 years later under regular dapsone therapy he relapsed again for the second time into active BT leprosy, but remained bacteriologically negative. He was given 3 drug regimen subsequently and became clinically inactive within 15 months.

This paper describes a microculture rapid assay using radiolabeling and mouse macrophages to determine the viability and the drug susceptibility or resistance of Mycobacterium leprae. Comparison of M. leprae resident macrophage cultures maintained in 96-well flat-bottomed plates showed results for viability and susceptibility or resistance to dapsone that were similar to results for concurrent cultures in Leighton tubes with greater numbers of bacilli and macrophages.

The Central Leprosy Teaching and Research Institute (C.L.T.R.I.) Chengalpattu, took up studies on dapsone resistance in M. leprae from 1974. From 1978, the study was further strengthened by a project under THELEP (TDR) for eliciting information on the efficacy of certain drug regimens. The Thelep studies were to be conducted only on the dapsone sensitive untreated cases and, therefore, directed towards the detection of primary resistance, while the non-THELEP institutional studies were concentrated on secondary dapsone resistance problem. These two studies together detected 99 cases of dapsone resistance in the patients who attended CLTRI Hospital during 1974-81; 23 of them, were of primary origin, 16, 6 and 1 showing mild (RI), moderate (RII), and high (RIII) grades respectively. Of the remaining 76 cases of secondary resistance, 7 and 69 were of RII and RIII grades respectively. The need for vertical and horizontal monitoring of the drug resistance problem has been pointed out.

Indication of an enhancing effect of Rifampicin on the urinary excretion of DDs has already been reported from this institution. Further observations on this aspect in 25 cases of lepromatous leprosy treated with a 15 day schedule of Rifampicin at a dose of 600 mg daily along with DDS are presented. The earlier findings that Rifampicin in the initial phase of administration enhance the urinary output of DDS are confirmed. An estimation of Rifampicin Creatinine levels in urine done concurrently also showed a quicker elimination of the drug in the earlier phase compared to later phases of the drug administration. As part of the study of drug interactions, the influence of Clofazimine administration on DDS metabolism was also studied. The findings indicate that Clofazimine does not exert any influence on DDs excretion by leprosy patients. The findings and their implications are discussed.