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Feb 2, 1983 - A. MITTAL,5 P. S. SESHADRI,2 H. K. PRASAD,1t M. SATHISH,' AND INDIRA NATHI* ..... Nath, I., H. K. Prasad, M. Sathish, Sreevatsa, K. V..

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 1983, 00664804/83/100579-07$02.00/0 Copyright © 1983, American Society for Microbiology

579-585

p.

Vol. 24, No. 4

Radiometric Macrophage Culture Assay for Rapid Evaluation of Antileprosy Activity of Rifampin A. MITTAL,5 P. S. SESHADRI,2 H. K. PRASAD,1t M. SATHISH,' AND INDIRA NATHI* Department of Pathology, All-India Institute of Medical Sciences, New Delhi 110029,1 and Central Leprosy Training Research Institute, Chingleput 603001,2 India

Received 2 February 1983/Accepted 26 July 1983

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.

In recent years, our laboratory has developed a rapid in vitro radiometric assay for the evaluation of the viability of Mycobacterium leprae. Over a 2- to 3-week period, 150 human-derived M. leprae strains maintained in mouse peritoneal and human monocyte-derived macrophage cultures showed significant uptake of [3H]thymidine as compared with parallel cultures of heatkilled bacilli obtained from the same skin biopsy (16, 20). That the incorporation of the radiolabel was in the bacilli and not in the host cells was shown by DNase experiments (8, 20). This assay has been effectively used to study the effect of dapsone (4,4'-diaminodiphenyl sulfone) and to identify dapsone-resistant strains (14). Moreover, immunologically mediated inhibition of [3H]thymidine incorporation was observed in M. leprae resident macrophage cultures in the presence of antigen-induced lymphokines derived from responder tuberculoid leprosy patients (17). This rapid in vitro assay compared well with the commonly used mouse footpad model (15, 18, 19, 21). Concordant results were obtained when biopsies obtained from clinically suspected dapsone-resistant patients were studied in the macrophage assay and mouse foot model in two independent centers (14). In the present study, the inhibitory effects of rifampin were investigated for 31 human-derived and 1 armadillo-derived M. leprae strain maintained in human and murine macrophage cul-

tures. A sharp increase in the inhibition of [3H]thymidine was observed in drug-treated cultures from 3 to 20 ng of rifampin per ml. Thereupon, the overall inhibition showed a gradual incremental increase up to 100 ng/ml, followed by a decline at 200 ng/ml.

t Present address: National Hansen's Disease Center, Carville, LA 70721.

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MATERIALS AND METHODS Patients. Thirty-one patients with bacilliferous lepromatous leprosy attending the clinics of the Central Leprosy Training and Research Institute, Chingleput, India, from July 1981 to May 1982 were selected for this study. Routine excision skin biopsies were undertaken under sterile conditions and sent by air freight and kept on ice as described earlier (16, 20). Extraction of M. leprae. In brief, M. leprae bacilli were extracted under sterile conditions by tissue homogenization of biopsies after the removal of the epidermis and subcutaneous fat (16). The homogenate was spun at 40 x g for 5 min to remove coarse tissue fragments. The supernatant was subsequently centrifuged at 800 x g for a further 30 min to recover the M. leprae. The pellet containing the bacilli was washed once with normal saline and subsequently suspended in RPMI 1640 (GIBCO Biocult, Irvine, Scotland). Bacilli were counted by the method of Shepard and McRae (22). Before inoculation in cultures, M. leprae extracts were checked for the absence of cultivable contaminants by plating on nutrient agar for 48 h and on Lowenstein-Jensen medium for 8 weeks. None of the extracts used showed growth of contaminants. One milliliter of medium containing 106 M. leprae was inoculated into each Leighton tube within 48 to 96 h after the removal of the biopsy. Control cultures were inoculated with heat-killed bacilli of the corresponding

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strain. The absolute numbers of bacilli extracted from the excision biopsies ranged from 3 x 107 to 4 x 108. Macrophage cultures. Macrophage cultures were set up as described earlier (16, 20). In brief, adherent mononuclear cells from peripheral blood of normal volunteers and peritoneal resident macrophages from BALB/c mice were cultured in Leighton tubes containing RPMI 1640 supplemented with 50% autologous plasma and 30o fetal calf serum (GIBCO Biocult), respectively. After incubation at 37°C for 16 to 18 h, the nonadherent cells were removed, and RPMI 1640 supplemented with 50% AB serum and 30% fetal calf serum was added to human and murine cultures, respectively. Macrophages showed morphological differentiation by 5 to 6 days in human macrophage cultures and 24 to 48 h in the murine macrophage cultures. M. leprae was inoculated at this stage. No differences were observed in the number of macrophages with intracellular bacilli or in the phagocytic index of parallel cultures with live as compared with killed bacilli. Overall, 60 to 90% of macrophages in various experiments had bacilli, and the phagocytic index varied from 0.8 to 2.5. After the removal of unphagocytosed bacilli at 18 h, the medium was replaced and supplemented with 100 ,ul of medium containing 1 ,Ci of [methyl-3H]thymidine (Amersham Corp., Arlington Heights, Ill.) (specific activity, 42 Ci/mmol) and 0.25, 0.5, 1, 3, 10, 20, 40, 50, 100, and 200 ng of rifampin. The medium, radiolabel, and drugs were replaced every 4 to 5 days. After 14 days of incubation, the cultures were harvested on fiber glass disks. Macrophages were first stripped with a rubber policeman after treatment with 100 pJl of 12 mM Xylocaine (Astra Pharmaceutical Products, Worcester, Mass.). Then they were harvested by using a vacuum filtration manifold (VFM-1; Amicon Corp., Lexington, Mass.) onto glass fiber disks and serially washed with saline containing cold thymidine (1 mg/ml), twice each with 5% trichloroacetic acid and methanol. The dried disks were placed in scintillation mixture and counted in a Beta Scintillation Counter (LKB 1215 Rackbeta II). With the same batch of macrophages, five replicates of each of the following were put up: (i) macrophage cultures alone, (ii) macrophages with freshly extracted live M. leprae, (iii) macrophages plus heat-killed M. leprae of the same strain, and (iv) macrophages with freshly extracted live M. leprae and various concentrations of rifampin. The mean counts per minute (cpm) ± standard deviation of three to five replicate cultures were calculated. The percent inhibition in the presence of rifampin was calculated as follows: percent inhibition = 100 - percent incorporation, where percent incorporation = {[mean cpm of macrophage cultures + live M. leprae + rifampin) - (mean cpm of macrophage cultures + autoclaved M. leprae)]/[(mean cpm of macrophage cultures + live M. leprae) - (mean cpm of macrophage cultures + autoclaved M. leprae)]} x 100. The P value was calculated by the Mann-Whitney U test. Rifampin. Pure rifampin powder was a kind gift from S. P. Tripathi, Tuberculosis Research Centre, Madras, India. The powder was dissolved in ethylene glycol at a concentration of 1 mg/ml, subsequently diluted in RPMI 1640 to 1 pug/ml, sterilized by passage through 22-,um Millipore disks, and kept at 4°C. Work-

ANTIMICROB. AGENTS CHEMOTHER. ing dilutions were made fresh before addition. Autoradiography. Cultures were initiated as described above except that 5 x 106 live or killed M. leprae were inoculated. The harvested macrophages were cytocentrifuged (Cytospin-2; Shandon, Cheshire, U.K.) at 1,000 rpm for 5 min. The slides were fixed in a freshly prepared methanol-acetic acid mixture (3:1) and coated with a film of 2-dimethyl-POPOP (1,4-bis[5-phenyloxazolyl]benzene) (Sigma Chemical Co., St. Louis, Mo.) and 5 g of PPO (2,5-diphenyloxazole) (Sigma Chemical Co.) dissolved in 1 liter of toluene. The slides were then dipped in NTB-2 emulsion (Eastman Kodak, Rochester, N.Y.) and stored at -20°C in the dark. After 7 days, the slides were treated with Kodak D-19B developer, dried, and stained with Ziehl-Neelsen solution.

RESULTS M. leprae strains derived from 31 bacilliferous lepromatous patients and one infected armadillo liver were maintained in human and murine macrophages. Of the human-derived strains, 5 were obtained from recently diagnosed patients, and 26 were individuals treated for various lengths of time and clinically suspected of resistance to dapsone. None of the patients was known to have received rifampin. It was not possible to assay all of the drug concentrations on each of the M. leprae strains. Thus, experiments were designed to test low (0.25 to 3 ng/ml per culture), middle (5 to 40 ng/ml), and high (50 to 200 nglml) concentrations of rifampin on individual M. leprae strains maintained under similar culture conditions. Of the 31 humanderived M. leprae strains tested, 2 failed to incorporate [3H]thymidine. Three experiments were ignored because of stripping of some macrophages during the culture period. Thus, results were expressed for 26 human-derived strains, of which 20 were tested in murine macrophage cultures and 6 were tested in human monocytederived macrophage cultures. All of the 26 human-derived strains and 1 armadillo-derived strain reported in the study showed a significant level of [3H]thymidine incorporation in cultures with live, freshly extracted M. leprae as compared with control cultures with heat-killed bacilli obtained from the same biopsy (P of £0.05 to -0.001; Fig. 1) and had an incorporation index ranging from 2.6 to 14.5. In agreement with our earlier reports (16, 20), it was observed that the M. leprae strains tested in different batches of macrophages showed various levels of [3H]thymidine uptake. This was noted in the control macrophages with heat-killed bacilli as well as in the parallel cultures containing live M. leprae. The localization of the radiolabel preferentially in the leprosy bacillus was further confirmed by autoradiography; 2 to 5% of the live bacilli had two to four silver grains arranged linearly over the individual rods. Bacilli having a single

VOL. 24, 1983

RIFAMPIN ANTILEPROSY ACTIVITY

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FIG. 1. Incorporation of [3H]thymidine by human-derived M. leprae strains, of which 20 were maintained in murine macrophage cultures (A) and 6 in monocyte-derived human macrophage cultures (B) (mean cpm ± standard error of five replicate cultures). Numbers along the abscissa refer to M. leprae strains. All of the strains showed significant incorporation of [3H]thymidine (P S 0.05) in cultures of live bacilli (l) as compared with heatkilled bacilli of the same strain ([I and i).

grain were ignored. An occasional globus of M. leprae had 8 to 10 grains (Fig. 2). Effect of rifampin on [3H]thymidine incorporation in M. leprae resident macrophage cultures. Morphology, adherence, and phagocytic proper-

a

ties of macrophages were similar in M. leprae resident cultures maintained over a 3-week period with and without rifampin. The effect of various concentrations of rifampin on individual M. leprae strains was expressed as percent

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FIG. 2. Presence of one to three discrete grains (arrows) on individual leprosy bacilli (a, b, and c) and a cluster of grains on a globus of bacilli (d) maintained in murine macrophage cultures pulsed with [3H]thymidine. Note the absence of grains on the nuclei of cells and on the majority of bacilli (cytocentrifuge preparation stained with Ziehl-Neelsen solution; x500).

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ANTIMICROB. AGENTS CHEMOTHER.

inhibition of [3H]thymidine uptake in cultures containing M. leprae with the drug as compared with parallel cultures without the drug. In general, it was observed that >50% inhibition gave P values of -0.05 to -0.005 by the Mann-Whitney U test. Table 1 shows representative control experiments where no significant differences in [3H]thymidine incorporation were observed between cultures of live bacilli exposed to the diluent in concentrations equivalent to those used in low (3 ng/ml) and high (100 ng/ml) dose ranges of rifampin and cultures of heat-killed bacilli in the presence and absence of the drug. The results obtained on individual M. leprae strains tested in the presence of low, middle, and high concentrations of rifampin are shown in Table 2 and Fig. 3. In the presence of -1 ng of rifampin per ml, bacilli maintained in human (Table 2) and murine (Fig. 3) macrophages showed various effects. At 3 ng/ml, consistent and significant (P s0.01) inhibition of [3H]thymidine uptake was observed in all of the M. leprae strains tested (Table 2 and Fig. 3A). An increase in the level of inhibition was observed in six strains tested with 5 to 40 ng of rifampin per ml (Fig. 3B). A further increase in drug concentration showed marginal effects in three of the five strains tested (Fig. 3C). At 200 ng/ml, some strains showed a decline in inhibition (Fig. 3C and D). One armadillo- and two human-derived M. leprae strains were maintained in murine macro-

phages in the presence of the whole concentration range of rifampin used in this study (1 to 100 and 200 ng/ml per culture). A sharp, linear, incremental increase in percent inhibition of [3H]thymidine incorporation occurred from 1 to 20 ng of the drug per ml, followed by a plateau phase at up to 50 to 100 ng/ml and a decline at 200 ng/ml (Fig. 3D). Similar features were observed when the overall results obtained for all of the M. leprae strains were expressed as mean percent inhibition at each concentration of rifampin tested (Fig. 4). DISCUSSION The present study is complementary to our earlier report (14) on the utility of the radiolabeled macrophage culture method for the rapid evaluation of antileprosy drugs. Of the 31 M. leprae strains tested, 26 showed successful radiolabel uptake in cultures with live as compared with killed bacilli. This is an improvement on our earlier studies (16, 20) where only 60 to 70% of similar cultures had shown significant levels of [3H]thymidine incorporation. Part of the success in the present study is thought to be due to the introduction of a more standardized, semiautomated harvesting of cultures. Furthermore, autoradiographic evidence indicated the preferential localization of [3H]thymidine in M. leprae maintained within the macrophages. Only 2 to 5% of individual bacilli showed two or more silver grains, the majority of bacilli being negative or having an occasional single grain.

TABLE 1. Representative experiments showing negligible effects of diluent and drug on control macrophage cultures

Rifampin

concn Culture ml)

Live

(ng/

ob

0

4,697 ±± 1,749

5,324 + 2,442

3

1,096 ± 667 5,047 + 2,804

1,239 ± 196 5,075 ± 650

Diluent equivalent

b

639

180

100

772 ± 257

Diluent equivalent

4,867 ± 1,110

Killed

a

incorporation (mean cpm ± SD)' [3H]thymidine Expt 1 Expt 2

Five replicate cultures.

Killed M. leprae control.

0 1 3 5 10 20 40 50 100 200

389 424 427 376 331 342 383 336 326 350

± 41 17 62 ± 11 ± 66 ± 122 ± 10 ± 47 ± 63 ± 33

1,178 ± 351

2,086 ± 1,280 5,320 ± 1,426 620 603 630 682 642 631 630 648 673 637

± ± ± ± ± ± ± ±

89 157 167 50 65 74 109 29

± 39 ± 119

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RIFAMPIN ANTILEPROSY ACTIVITY

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100 200 1 510 25 50 100 CONCENTRATION OF RIFAMPIN (ng/mi)

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200

FIG. 3. Effect of various concentrations of rifampin on the [3H]thymidine incorporation of M. Ieprae strains maintained in murine peritoneal macrophages, expressed as percent inhibition. Negative values on the ordinate indicate the enhancement of radiolabel uptake in the presence of the drug. The strain numbers are indicated against each graph. (A) Effect of 1 and 3 ng of rifampin per ml on seven M. leprae strains. Significant inhibition was observed at the higher drug concentration (P < 0.05). (B) Effect of 5, 10, 20, and 40 ng of rifampin on six M. leprae strains. A graded increase in inhibition was noted for all strains tested. (C) Effect of 50, 100, and 200 ng of rifampin per ml on five M. leprae strains, of which three showed marginal changes with increasing concentrations of the drug. (D) Effect of 1 to 200 ng of rifampin per ml in two human-derived strains (0) and one armadilloderived strain (0) of M. leprae. A sharp linear increase in inhibition is observable from 1 to 20 ng/ml, followed by a plateau at up to 50 to 100 ng/ml. At the highest concentration, a decline in inhibition was evident.

Rifampin was tested in this system over a wide range of concentrations from