INTERNATIONAL JOURNAL OF LEPROSY^ Volume 53, Number 3

Printed in the U.S.A.

Comparison of Radiometric Macrophage Assayand the Mouse Foot Pad Infection for the

Evaluation of Mycobacterium lepraeSensitivity/Resistance to Dapsonel

M. Sathish, R. J. W. Rees, P. S. Seshadri, and Indira Nath 2

The in vitro uptakes of various radiola-beled compounds by Mycobacterium lepraemaintained alone ( 3 ' 4 ' 17 ) or within cells(5, 9

• I I. 14 ) have been reported in recent years.

These studies, lasting 1-2 weeks, give pre-cise measurements of Al. leprae viability andhave potential for screening antileprosydrugs and for diagnosing drug resistance.Our laboratory has developed a rapid in vi-tro, radiometric assay of Al. leprae withinresident macrophage (MO) cultures forevaluating bacillary viability ( 6 ' 11 ' 14 ), sen-sitivity to dapsone (DDS, 4,4'-diaminodi-phenyl sulfone) (9 ), rifampin ( 7 ), and lym-phokine activity ( 12 ). Over a two-weekperiod of continual pulsing with high spe-cific activity 3 H-thymidinc, human andmurine MO cultures containing freshly ex-tracted ("live") M. leprae showed a signif-icant uptake of 3 H-thymidine (p 0.05 to- 0.001) as compared to control culturescontaining heat-killed bacilli from the sameskin biopsy. The specificity of the uptakeand the location of the radiolabel in Al. lep-rae and not in the host macrophage wasshown by autoradiography ( 8 ), and DNaseexperiments ( 14 ).

We had earlier reported that seven M.Ieprae strains derived from lepromatous pa-tients and suspected of dapsone resistanceshowed concordant results in the in vitro

' Received for publication on 27 November 1984;accepted for publication on 21 March 1985.

2 M. Sathish, M.Sc.; I. Nath, M.D., M.N.A.M.S.,M.R.C.Path., Department of Pathology, All India In-stitute of Medical Sciences, New Delhi 110029, India.R. J. W. Rees, F.R.C.P., F.R.C.Path., National Insti-tute for Medical Research, Mill Hill, London NW7IAA, England.* P. S. Seshadri, B.Sc. M.B.B.S., CentralLeprosy Training and Research Institute, Chingleput603001, India.

Reprint requests to Dr. Indira Nath.* Current address: Division of Communicable Dis-

eases, Clinical Research Centre, Harrow, London HAI3UJ, England.

assay and the conventional mouse foot padinfection ("). To evaluate the radiometricmethod further, a collaborative study wasundertaken between three independent cen-ters.

Skin biopsies from lepromatous patientsclinically suspected of drug resistance at theCentral Leprosy Training and Research In-stitute (CLTRI), Chingleput, India, werecoded and shipped by air to the All IndiaInstitute of Medical Sciences (AIIMS), NewDelhi, for testing in the MO assay and theNational Institute for Medical Research(NIMR), Mill Hill, London, for the mousefoot pad test. Results obtained in the in vitroassay were conveyed to the clinicians in 3-4 weeks time and compared with the mousefoot pad results a year later.

MATERIALS AND METHODSPatients

Twenty-three lepromatous leprosy pa-tients attending the clinics at the CLTRIfrom 1979 to 1981 were included in thestudy. They were clinically suspected ofdapsone resistance on the basis of the de-velopment of new lesions and an increasein the bacterial index (BI) and/or the mor-phological index (MI) while on therapy. Ex-cision biopsies of active skin lesions weretaken at CLTRI on a Monday, coded anddivided equally, and shipped by air on wetice the same day. One half of each biopsywas sent to AIIMS and the other half toNIMR, reaching their destinations on Mon-day and Tuesday, respectively. Table 1 givesthe clinical details of the 13 patients forwhom results on both assays from the twocenters are available.

Radiometric macrophage assayExtraction of M. leprae. Al. leprae were

extracted from the biopsies within 24 hr of

378

53, 3^Sathish, et al.: Dapsone Sensitivity of M. leprae^379

receipt of the biopsies, and the bacilli wereinoculated into macrophages within 24 hrof extraction. After the removal of the epi-dermis and the adipose tissue, the bacilliwere extracted in glass homogenizers inRPMI 1640 (GIBCO-Biocult, Irvine, Scot-land), buffered with 20 mM N-2-hydroxy-ethyl piperazine-N'-2-ethane-sulfonic acid(HEPES, GIBCO) according to the methodof Rees ( 13 ), and counted as described byShepard and McRae ('').

Before inoculation into cultures, the ex-tracted bacilli were screened for contami-nating organisms by plating on NutrientAgar and LOwenstein-Jensen (11) medium.The Nutrient Agar plates and LJ slants wereincubated at 37°C for 48 hr and 8 weeks,respectively. None of the strains reportedin the study grew on either medium.

Macrophage cultures. The details of themacrophage assay have been published ear-lier (9. ' ' 4). a) In brief, human macrophageswere derived from glass-adherent mono-cytes of heparinized (preservative-free hep-arin, 10 u/ml; Upjohn and Co., Kalamazoo,Michigan, U.S.A.) peripheral blood of nor-mal subjects. Equal volumes of leukocyte-rich plasma and RPMI 1640 were mixedand dispensed in 1.0 ml aliquots in Leightontubes and incubated at 37°C for 16-18 hr.Subsequently, the nonadherent cells wereremoved by washing with prewarmedHanks' balanced salt solution (HBSS, GIB-CO). The adherent cells were cultured for5-7 days in RPMI 1640 enriched with 50%AB serum to permit differentiation. b)Mouse macrophages were obtained fromperitoneal resident cells of a close bred strainof Swiss white mice. Equal volumes of peri-toneal resident cells and RPMI 1640 en-riched with 30% fetal calf serum (FCS, GIB-CO) were mixed, dispensed into Leightontubes in 1 ml aliquots, and incubated at37°C. After 16-18 hr, the nonadherent cellswere removed and the adherent cells werecultured in 30% FCS-enriched RPMI for48-72 hr to permit differentiation of mac-rophages.

One million Al. leprae were inoculatedinto macrophage cultures and incubatedat 37°C for 18 hr. The non-phagocytosedM. leprae were removed, and the mediumreplaced with 50% AB serum and 30%FCS-enriched RPMI for human and mousemacrophages, respectively. One of 3 H-

thymidine (methyl 3 H-thymidine, specificactivity 51 Ci/mmol; Amersham Interna-tional, U.K.) and 3 ng/ml and 10 ng/ml ofdapsone were added at this stage. The cul-tures were incubated for 14 days. The me-dium, drugs and the radiolabel were re-placed every 7 days and 5 days for humanand mouse macrophage cultures, respec-tively. The adherent macrophages were re-moved by incubating the cultures for 45-60 min at 37°C in the presence of 12 mMxylocaine (Astra-IDL, Bombay, India). Thecells were transferred to glass-fiber paperdiscs (GF/C; Whatman Corp., Clifton, NewJersey, U.S.A.) using a vacuum filtrationmanifold (VFM-1; Amicon Corp., Lexing-ton, Massachusetts, U.S.A.) and seriallywashed with cold saline containing 1 mg/ml of unlabeled thymidine (Sigma ChemicalCo., St. Louis, Missouri, U.S.A.), 5% tri-chloroacetic acid (BDH, Poole, London,U.K.) and cold methanol (Ranbaxy, NewDelhi, India). The dried discs were countedfor radioactivity in a Rackbeta II 1215(LKB-Bromma, Finland) liquid scintilla-tion (3 counter using a toluene-based scin-tillation fluid.

Five replicates each of macrophage cul-tures were put up with a) heat-killed AI.leprae, b) freshly extracted ("live") Al. lep-rae, c) "live" M. leprae + 3 ng/ml, and d)live If. leprae + 10 ng/ml of dapsone. Per-cent inhibition of 3 H-thymidine uptake ateach concentration of the drug was calcu-lated as (1 — mean cpm of cultures in pres-ence of "live" Al. leprae and drug/mean cpmof cultures in presence of live Al. lepraealone) x 100.

Statistical analysis was done using Mann-Whitney's two-tailed U test ( 16 )

Mouse foot pad assayThe biopsies still on wet ice reached

NIMR, London, on Tuesday and Al. lepraefrom the biopsies were inoculated into miceon Thursday or Friday (i.e., 3 or 4 daysfollowing excision from the patient, com-pared to 2 days for the radiometric mac-rophage assay).

Al. leprae from each biopsy were extract-ed following homogenization and countedas described above for the radiometric as-say, but in 0.1% w/v bovine serum albumin/saline instead of RPM I. For each assay, 20female CBA mice were inoculated with

380^

International Journal of Leprosy^ 1985

TABLE 1. Clinical details of lepromatous leprosy patients suspected of dapsone resis-lance.

No. Age Sex Current therapy(dapsone daily)

Past therapy(dapsone 2-8 yrs) ENL' BP' ME

13 17 F 100 mg, 2 yr Irregular, 10-25 mg Nil 2.00 <116 50 M Irregular, 50 mg, 10 yr 100-300 mg biweekly Nil 3.00 <120 42 M 50 mg, 5 yr 1-5 mg daily Nil 2.50 <126 32 M 50 mg + thiacetazone, 100

mg + INH, 200 mg, 2 yrIrregular, 50-150 mg bi-

weekly + hydnocarpusoil & Solapson

Frequent, thalido-mide & steroids

2.83 4

27 23 M 50 mg, 6 yr Irregular, 1 biweekly— 10mg daily

Frequent, thalido-mide & steroids

3.33 <I

28 51 M 50 mg, 2 yr Discharged as negative af-ter Solapson; active 19yrs later

Nil 3.00 1.6

29 30 F 100 mg, 5 yr 5 biweekly, 60 mg daily Nil 3.50 <I30 26 M 100 mg, 1 yr Irregular Nil 2.83 033 20 M 100 mg, 1 mo Interrupted, 10-70 mg

dailyNil 3.00 22.1

37 24 M 50 mg, 2 yr Irregular Nil 3.50 138 32 M 100 mg, 1 yr Very irregular, 10-75 mg

dailyFrequent, thalido-

mide4.00 0.6

39 28 M 50 mg + thiacetazone Irregular 3.75-50 mg daily Nil 2.00 <I40 37 F 100 mg, 2 yr Irregular, 15-25 mg daily Mild, steroids 2.33 0

ENL = erythema nodosum leprosum.BI = bacterial index.MI = morphological index.

1.0 x 10 4 acid-fast bacilli into both hindfoot pads. The mice were randomized intogroups of 5 mice each fed normal diet (groupI), and diet mixed with dapsone 10 -4 %(group II), 10 -3 % (group III), and 10 -2 %w/w (group IV).

The results are expressed as the numberof positive foot pads (i.e., a foot pad harvest

5 times the inoculum) per total numberof foot pads harvested per group of mice.The schedule for harvesting of foot pads wasmodified to reduce the work load withoutaffecting the results. At 6 months, a controlmouse (untreated, group I) was harvestedand if found to be positive for bacilli, 2mice receiving the lowest concentration ofdapsone (10 -4 %, group II) were harvested.If these mice were found to be negative,indicating the presence of dapsone-sensitivebacilli, all remaining group II mice wereharvested, and all remaining groups of micewere discarded. If multiplication was ob-served, then 2 mice from the groups III andIV, receiving the next higher concentrationsof dapsone, were serially harvested. If groupIV, receiving the highest dose of dapsone(10 -2 %), had positive foot pads, indicatingthereby the presence of fully resistant ba-cilli, then the groups receiving lower con-

centrations were not evaluated further.When control mice were negative at 6months, further sampling of foot pads wasdone at 9 and 12 months in the same man-ner as described above.

RESULTSTwenty-three lepromatous patients were

clinically suspected of dapsone resistancebased on the development of new lesionsand an increase of BI and/or MI while ontherapy. Excision skin biopsies from thesepatients were taken on Monday and con-currently tested in the macrophage (MO)assay in New Delhi on Wednesday and inthe mouse foot pad in London on Thursdayor Friday. Of the 23 biopsies shared, 2 werediscarded due to insufficient bacilli; 2 x 10 7

and 4 x 10 5 bacilli were required for the invitro and in vivo assays, respectively. Four

leprae strains failed to grow in the mousefoot pad although they showed incorpora-tion of 3 H-thymidine in the MO cultures.Four more strains showed multiplication inthe mouse foot pad but failed to incorporatethe radiolabel. It should be stressed that allof the biopsies were treated in a similarmanner in both laboratories. Delays enroute, insufficient bacilli, or experimental

dapsone in diet

10 3^10

Dapsone (ng/ml)

3^1010 -4

=50'<50<50

NH:150'<50

50cNH°NH

NH

Control

+ h

Conclusion'

SRR

Conclusion

SRPRPR

53, 3^Sathish, et al.: Dapsone Sensitivity of M. leprae^381

TABLE 2. Criteria for assessing dapsone resistance and sensitivity.

Mouse foot pad^

Macrophage assay

Multiplication

To inhibition

S = sensitive, R = resistant, PR = partially resistant.h + = evidence of multiplication.

— = no evidence of multiplication.° NH = not harvested.

p value ranging from . .0.05 to ^ 0.005 by Mann-Whitney two-tailed U test.

variables were not considered to be thecauses for the above discrepancies.

Thus, Al. leprae strains from 13 patientswere available for comparison in both as-says. Table 1 gives the details of therapyand the BI and MI of bacilli in smears ofthe skin scrapings from these patients. Fol-lowing biopsy, all the patients were givenrifampin. Some received an initial singledose of 1200 mg, whereas others received600 mg of rifampin daily for 2 weeks fol-lowed subsequently by a monthly dose of600 mg of rifampin. Daily dapsone (100 mg)was given throughout. In addition, patients26, 27, and 38 received 100 mg of clofaza-mine daily. Follow up of one to three yearswas available on patients, 16, 20, 26, and39, all of whom showed clinical improve-ment and reduction in BI and MI while onthese regimens.

The criteria used for evaluating dapsonesensitivity/resistance are given in Table 2.The criteria for the MO assay were basedon an earlier study of 14 bacilliferous lep-rosy patients ( 9 ) where >50% inhibition ofradiolabel uptake in drug-treated cultureswas found to be statistically significant bythe Mann-Whitney two-tailed U test (p0.05 to 0.005). In the mouse assay, 5 x10 4 bacilli harvested per foot pad was con-sidered as positive multiplication. In sub-sequent tables, the results are expressed asnumber of positives over the total numberof foot pads tested at each concentration ofthe drug.

Table 3 shows the data obtained in theMO assay. Four and nine strains were testedin human and murine MO, respectively. All

13 Al. leprae strains showed significant levelsof 3 H-thymidine incorporation in cultureswith freshly extracted "live" M. leprae ascompared to parallel cultures with heat-killed bacilli (p 0.05 to 0.005). The in-hibition of 3 H-thymidine uptake in thepresence of 3 ng/ml and 10 ng/ml of dap-sone is shown in terms of mean cpm andpercent inhibition in Tables 3 and 4, re-spectively.

Concordant results were obtained for 12Al. leprae strains in the radiometric MO andmouse foot pad assays (Table 4). Strains 26and 33 were graded as sensitive since theydid not multiply in the presence of the low-est concentration of dapsone (10 -4 %) andshowed more than 50% inhibition of ra-diolabel uptake at both drug concentrationsin the MO assay. Eight strains showedgrowth in mice fed the highest concentra-tion of dapsone (10 -2 %) and were consid-ered fully resistant. Of these, seven showed<50% inhibition of the radiolabel uptakeat 3 ng/ml dapsone concentration and vari-able levels of inhibition at the higher con-centration of 10 ng/ml.

Strains 13, 28, and 30 were graded as par-tially resistant in the mouse foot pad infec-tion because the bacilli multiplied at thelowest (10 -4 %) but not at the higher (10 -3 %and 10 -2 %) dapsone concentrations in thediet. These strains behaved as resistantstrains in the MO assay.

Strain 37 showed discordant results in thetwo assays, behaving as fully resistant in themouse foot pad and showing sensitivity toboth dapsone concentrations in the MO as-say.

Strainno.a MO"

13162026272829303337383940

382^ International Journal of Leprosy^ 1985

TABLE 3. Incorporation qf 3II-thymidine in M. leprae strains derived front lepromatouspatients and maintained in macrophage (MO) cultures in the presence and absence qfdapsone.

'H-thymidine incorporation (mean cpm ± S.D.)

Heat-killed,11. leprae

Freshly extracted .11. leprae

Alone'+ Dapsone

3 ng/ml 10 ng/ml

524 ± 60 1100 ± 189 909 ± 238 1183 ± 50589 ± 10 618 ± 456 418 ± 176 116 ± 14"

161 ± 10 340 ± 141 278 ± 139 281 ± 99172 ± 73 926 ± 233 356 ± 78' 340 ± 57'395 ± 303 859 ± 469 512 ± 364 47 ± 7'

58 ± 18 528 ± 274 364 ± 331 310 ± 74'120 ± 68 395 ± 214 493 ± 217 371 ± 134312 ± 199 1701 ± 984 1534 ± 623 150 ± 123'

1607 ± 249 2610 ± 1009 1171 ± 737" 1090 ± 608'59 ± 29 324 ± 63 127 ± 29' 109 ± 58'96 ± 49 401 ± 28 349 ± 125 178 ± 28'

301 ± 120 742 ± 70 593 ± 340 414 ± 228"254 ± 38 806 ± 208 478 ± 110d 178 ± 46'

Strain numbers refer to patients whose clinical details are given in Table I.M = mouse macrophages, H = human macrophages.All values significantly higher than heat-killed M. leprae controls, at most p < 0.05, Mann-Whitney U test.Less than live Al. leprae alone, p < 0.05, Mann-Whitney U test.Less than live M. leprae alone, p < 0.01, Mann-Whitney U test.

Using the mouse foot pad infection as thereference point the present results a) con-firm our earlier findings ( 9 ), and would in-dicate that b) 50% inhibition of the ra-diolabel uptake at 3 ng/ml may be a realistic

criterion for the diagnosis of dapsone resis-tance since four strains considered fully re-sistant in the mouse showed significant in-hibition of 3 H-thymidinc incorporation atthe higher 10 ng/ml concentration; c) by the

TABLE 4. Coniparison of results obtained in mouse foot pad infection and the radio-metric macrophage (MO) assay.

Strainno.

Mouse foot pad(no. positive/total)

Macrophage assay(% inhibition)

Control% dapsone in diet Conclu- Dapsone ng/ml^Conclu-

l0 - ' 10-' 10 - = sion" 3 10^sionb

26 5/6 0/10 NHL NH S 62 6333 6/8 0/10 NH NH S 55 5837 10/10 NH NH 8/10 R 61 6620 6/6 NH NH 6/6 R 18 1729 6/6 NH NH 6/6 R —/4" 616 7/8 NH NH 6/6 R 23 8127 5/6 NH NH 6/8 R 40 9438 10/10 NH NH 8/10 R 13 5639 7/8 NH NH 6/6 R 21 4540 6/6 NH NH 6/6 R 41 7813 6/10 NH 5/10 0/10 PR 17 —7d28 6/8 2/10 0/8 NH PR 31 4130 8/10 NH 5/6 0/10 PR 10 92

a Positive mouse foot pad indicates the presence of ±5 x 10' acid-fast bacilli at the time of harvest.S = sensitive, R = resistant, PR = partially resistant.NH = not harvested.Negative values indicate lack of inhibition.

53,3^Sathish, et al.: Dapsone Sensitivity of M. leprae^383

present criteria, the partially resistant bacillias tested in the murine model may not beidentifiable in the in vitro MO assay.

DISCUSSIONThe present study undertaken in three in-

dependent centers was aimed at comparingthe newer in vitro, macrophage-based ra-diometric assay with the conventionalmouse foot pad infection for the diagnosisof dapsone resistance. The results on bacilliobtained from 13 lepromatous patientscompared well in the two assays. Except forstrain 37, which was sensitive to dapsonein the MO assay but resistant in the mouse,all other strains showed concordance forsensitivity/resistance to dapsone. Taken to-gether with our earlier collaborative study(where Central JALMA Institute, Agra, In-dia, undertook mouse foot pad studies), onlyone of a total of 20 strains tested showeddiscordant results in the two assays.

The present investigation confirms thevalidity of using 50% inhibition of the ra-diolabel in the drug-treated cultures as anend point for the diagnosis of drug resis-tance. However, it would appear that 10 ng/ml dapsone concentration in vitro may behigh. The lower concentrations had beenselected earlier ( 9 ) because a) all sensitiveAI. leprae strains tested showed inhibitionof 3 H-thymidine uptake at these concentra-tions, b) 10 ng/ml correlated with plasmalevels reported in mice fed 10 -4 % dapsone( I.2. and c) one partially resistant strainshowed differential uptake of the radiolabelat 3 ng/ml and 10 ng/ml.

It is evident from the present study thatbacilli considered to be partially resistant todapsone in the mouse cannot be identifiedreliably in the radiometric MO assay usingthe present criteria. Whether or not a con-centration lower than 3 ng/ml of dapsonein the MO cultures would reveal these ba-cilli remains to be investigated. Al. lepraeresident in MO cultures seem to be sensitiveto lower concentrations of drugs than thesame bacilli in the infected mouse ( 8 ).

It was not possible to correlate either as-say with the clinical behavior of the patientssince they had been subsequently treatedwith rifampin, clofazamine, or both. Thefollow-up data available for some patientsshow clinical improvement and reductionin BI 1-3 years after the institution of ther-apy. As expected, most of the patients who

developed dapsone resistance had takendapsone irregularly or were given low dosesfor a prolonged period of time. It wouldappear that the level of dapsone resistancein bacilli from the patients seen at CLTRIis distressingly high, as evidenced by ourearlier report ( 9 ), the present study, and ourunpublished data on more than 100 patientstested by the MO assay.

The radiometric MO assay reported herehas the advantage of early diagnosis of re-sistance, making it possible for the clinicianto wait for the laboratory report before theintroduction of new therapy. Moreover,within a 2-3-week period a full drug-sen-sitivity profile of the patient's bacilli can bemade available with little extra work by thelaboratory. Time kinetics and dose re-sponses for rifampin ( 8 ) and clofazamine ( 7 )have been defined on human- and arma-dillo-derived A/. leprae. Moreover, thepresent constraint of the higher numbers ofbacilli required in the MO assay have beenovercome by the development of a micro-culture method using 96-well, flat-bottomculture plates ( 6 ). As compared to the re-quirement of at least 20 mice over a 9-12-month period, macrophages derived fromone mouse were found to be adequate forthe screening of one patient. Thus, the needfor large numbers of inbred mice may beavoided in leprosy-endemic countries wheremaintenance of animals in air-conditionedfacilities is a major problem.

SUMMARYStudies were undertaken in three inde-

pendent centers to compare the newer, invitro radiometric macrophage (MO) assaywith the conventional mouse foot pad in-fection for the diagnosis of dapsone resis-tance. Results obtained on 12 bacilliferouspatients showed good concordance in bothassays. One strain diagnosed as sensitive inthe MO assay was found to be resistant inthe mouse foot pad. Three ilycobacteriumleprae strains considered to be partially re-sistant in the mouse infection behaved asresistant strains in the MO cultures. Atten-tion is drawn to a rapid in vitro method forthe identification of drug-resistant bacilli inleprosy patients.

RESUMENSe hicieron estudios en 3 centros independientes para

comparar el recientemente disefiado ensayo radiome.-

38 4^ International Journal of Leprosy^ 1985

trico con macrOfagos (MO), con el metodo convencio-nal de infecciOn en la almohadilla plantar del ratOnusado para el diagnOstico de resistencia a la dapsona.Los resultados obtenidos en 12 pacientes baciliferos,mostraron buena concordancia en ambos ensayos. Unacepa diagnosticada como sensible en el ensayo MOresult() ser resistente en el ensayo de la almohadillaplantar. Tres cepas de Mycobacterium leprae consi-deradas como parcialmente resistentes por la infecciOnen el ration, se comportaron como resistentes en losensayos MO. Se enfatiza la utilidad del ensayo MO invitro para la rapida iclentilicaciOn de bacilos resistentesa drogas en pacientes con lepra.

RESUME.

Dans trois centres independants, on a compare lavaleur, pour le diagnostic de la resistance a la dapsone,de la methode recemment mise au point pour le dosageradiometrique des macrophages in vitro (MO), et ('ino-culation classique du coussinet plantaire de la souris.Les resultats obtenus chez 12 malades bacilliEeres ontmontre une bonne concordance des deux mOthodes.Une souche qui avait etc relevee comme sensible dansl'essai MO, a Ole trouvee resistante par la methode ducoussinet plantaire. Trois souches de ,Ifycobacteriumleprae, considerees comme partiellement resistantes par('inoculation f.1 la souris, se sont revelees resistantesdans les cultures de macrophages. On attire ('attentionsur l'interet d'une methode in vitro rapide pour ('iden-tification des bacilles resistant aux medicaments chezles malades de la lepre.

Acknowledgment. The investigation received finan-cial assistance from the Chemotherapy of Leprosy(THELEP) component of the UNDP/World Bank/WHO Special Programme for Research and Trainingin Tropical Diseases and the British Leprosy ReliefAssociation (LEPRA).

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