Bull. Org. mond. Sant# 1972, 46, 821-826Bull. Wld Hlth Org.

A test for the determination of competency inclearing bacilli in leprosy patients *

J. CONVIT,1 J. L. AVILA,2 M. GOIHMAN,3 & M. E. PINARDI4

A skin test has been developed to determine the degree ofcompetency in clearing bacillifrom the tissues ofpatients suffering from various forms of leprosy. The test involves theintradermal injection of a suspension of killed Mycobacterium leprae. The response ofleprosy patients to the injection of other mycobacterial antigens, one prepared fromM. Iepraemurium and another from an atypical mycobacterium from a hamster, was alsoinvestigated in order to study the isopathic phenomenon. Since lepromatous patients reactnegatively in tests with standard Mitsuda antigen, a concentration of 640 x 106 M. lepraeper ml was used to produce macroscopic responses. The results of the test can be applied todetermine the duration of consolidation treatment for lepromatous and indeterminatebacteriologically negative patients after regular treatment has ended. The test can also beused to indicate which Mitsuda-negative contacts should be given preventive treatment, andmight be used to identify a given mycobacterium as M. leprae.

The different forms of leprosy are a consequenceof cell-mediated immune mechanisms. This is shownto be so by the structure of the lesions, which arecharacterized by the various cell types of the granu-loma, with macrophages in different stages of differ-entiation towards epitheloid cells (i.e., maturity), bythe degree of infiltration of lymphoid cells, and bythe competency of the organism in clearing Myco-bacterium leprae from the granuloma, the latter pointbeing crucial.One of the most outstanding differences between

the polar forms of leprosy (lepromatous and tuber-culoid) are the characteristics of the macrophage,which in the tuberculoid form of leprosy undergoescomplete differentiation towards an epithelioid celltype; in the lepromatous form of the disease thisdifferentiation does not occur. The degree of com-petency in clearing M. leprae from the cells is high in

* This work was carried out at the WHO InternationalReference Centre for the Histopathological Identificationand Classification of Leprosy, Divisi6n de DermatologiaSanitaria, Ministerio de Sanidad y Asistencia Social, Caracas,Venezuela. Requests for reprints should be addressed toDr J. Convit.

Director; 'Chief of the Biochemical Laboratory;'Chief of the Immunology Laboratory; ' Assistant Professorof Research; National Institute of Dermatology, Caracas,Venezuela.

the tuberculoid form but nonexistent in the lepro-matous form of leprosy.

Recently, Patterson & Youmans (1970) haveshown that the growth of virulent M. tuberculosiswithin normal macrophages was inhibited only whenthese macrophages were cultured in the presence oflymphocytes from immunized animals. Possibly thesame mechanism operates in leprosy.

In the last few years some experiments have beenmade with the purpose of studying in vitro theactivity of macrophages in the different forms ofleprosy, and in Mitsuda-positive and Mitsuda-nega-tive contacts (Barbieri & Correa, 1967; Beiguelman,1967). These authors have reported that macro-phages from patients with tuberculoid leprosy andfrom Mitsuda-positive contacts are capable of digest-ing intracellular M. leprae, while those from lepro-matous patients and Mitsuda-negative contacts donot have this capacity. Godal et al. (1971), andDrutz & Cline (unpublished data) have not beenable to confirm these results.

In order to determine the competency of the macro-phages in clearing bacilli from leprosy patients, par-ticularly in relation to M. leprae, we have studied thegranuloma produced by the intradermal inoculationof a killed suspension of M. leprae. We were spe-cially interested in the cellular components of the

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J. CONVIT ET AL.

granuloma and in the influence that these cellularcomponents have on M. leprae.

Since the lepromatous patient has a negative re-sponse to the intradermal injection of standard Mit-suda antigen (concentration: 160 x 106 acid-fast ba-cilli/ml) and considering that a macroscopic reactionin this form of leprosy might facilitate the study ofthe fate of the bacilli, it was decided to increase theconcentration of M. leprae in the antigen. Afterseveral concentrations had been tried it was con-cluded that an intradermal inoculation of 0.1 ml ofan antigen having a concentration of 640 x 106M. leprae per ml produced a constant and reprodu-cible macroscopic granuloma in all the lepromatouspatients tested. The reaction was characterized by anodule that appeared about 15 days after the inocu-lation. The diameter of the nodule ranged from5 mm to 8 mm.

Since we were interested also in the response oflepromatous patients to other mycobacteria, twoother mycobacterial antigens were prepared underexactly the same conditions as the M. leprae antigen.

MATERIAL AND METHODS

The antigens were prepared in buffered saline withphenol at a concentration of 0.4%. The mycobacte-ria used were M. leprae, M. lepraemurium, and amycobacterium obtained from lesions produced inthe hamster by the inoculation of material from aborderline patient (Convit et al., 1962). All theantigens were prepared by grinding the tissue in TenBroeck grinders, counting the number of bacteria inthe suspension, using the method developed byShepard (1968), and sterilizing in an autoclave for20 minutes at 120°C. All three suspensions werediluted to a concentration of 640 x 106 acid-fast bac-teria per ml.The patients who received injections of these anti-

gens were 40 lepromatous patients divided into twogroups: (1) 20 patients whose lesions had completelydisappeared and who were bacteriologically negativeafter several years of treatment with dapsone, and(2) 20 patients with active lesions who were under-going sulfone treatment. All the patients were nega-tive to the test with " standard" lepromin. Alsoselected were 5 tuberculoid, 2 indeterminate, 2 bor-derline patients, and 2 Mitsuda-negative contacts.The 2 indeterminate patients had a weakly positiveMitsuda reaction; one of them had had the diseasefor several years and a few bacilli were presentin his hypochromic spots; the other was a recent

case with a single hypochromic spot and was bac-teriologically negative. The borderline patients hadalmost no active lesions and they both had a discretepositive Mitsuda reaction about 4 mm in diameter.The lepromatous, tuberculoid, and borderline pa-

tients were inoculated with 0.1 ml of the M. lepraeand the M. lepraemurium antigens on the volarsurface of the right forearm, and with 0.1 ml of thehamster mycobacteria antigen on the volar surfaceof the left forearm. The indeterminate patients andthe contacts were given only 0.1 ml of the M. lepraeantigen on the volar surface of the right forearm.The results ofthe tests were recorded after 48 hours

and 1, 2, 3, and 4 weeks. After the fourth week, abiopsy sample was taken from each injection sitewith a 6-mm punch. The biopsy samples were fixedin 10% neutral formol, cut into 6-,um sections, andstained with haematoxylin-eosin and with a stain(Fite-Faraco) for acid-fast bacteria. The origin ofthe sections was not disclosed to the observer. Thenumber of acid-fast bacteria in the granuloma wasestimated according to the logarithmic index (Ridley& Hilson, 1967), 8-10 sections being examined foreach test.

In the lepromatous patients, control biopsy sam-ples were taken from areas adjacent to the intra-dermal test sites and sections were prepared in thesame way.

RESULTS

The test biopsies produced the following results.A nodule 5-10 mm in diameter appeared at the sitewhere M. leprae antigen was injected in all thelepromatous patients. The other two antigens pro-duced erythematous nodules approximately 20 mmin diameter, some of them with a central necrosis.The borderline patients reacted to M. leprae antigenwith a nodule about 12 mm in diameter, and to theother two antigens with nodules 22 mm in diameterhaving a central necrosis. The indeterminate patientsand the Mitsuda-negative contacts, who were givenonly M. leprae antigen, produced nodules varyingfrom 6 to 8 mm in diameter.The nodules started to appear 2 weeks after the

injection. At 30 days, when the biopsy specimenswere taken, the dimensions were those stated above.The results of histopathological examinations are asfollows.Lepromatous groupM. leprae antigen. A granuloma, which was si-

tuated partially or totally in the dermis, was present,being formed by macrophage or fibroblast type cells

822

Fig. 1. CCB test for M. Ieprae in a lepromatous patient at 30 days. The granulomais formed of macrophages and giant cells of the foreign-body type. Haematoxylinand eosin stain; x 16.

Fig. 2. CCB test for M. Ieprae in an LL patient showing a negative result at 30 days.Abundant acid-fast bacilli are seen inside macrophages. Fite-Faraco stain; x 100.

Fig. 3. CCB test in an LL patient showing a negativeresult at 30 days. A giant cell of the foreign-bodytype is seen to contain abundant acid-fast bacilli.Fite-Faraco stain; x 100.

Fig. 4. CCB test for M. Ieprae in a TL patient positiveat 30 days. The granuloma is formed of epithelioidcells and giant cells. Haematoxylin and eosin stain;x 16. When this material was stained with Fite-Faraco stain, no acid-fast bacilli were seen.

fS 14!%Y

Fig. 5. CCB test for M. Iepraemurium in an LL patient at 30 days. There is anodular lesion, the centre of which is formed of epithelioid cells, and there isperipheral lymphocytic infiltration. Haematoxylin and eosin stain; x 16.When this material was stained with Fite-Faraco stain, no acid-fast bacilliwere seen.

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TEST FOR COMPETENCY IN CLEARING BACILLI IN LEPROSY PATIENTS

with some giant cells similar to foreign body cells.In some cases, fibroblast type cells (spindle shape)predominated; other biopsies showed a discrete in-filtration of lymphoid type cells. The stain for acid-fast bacteria revealed large numbers of intracellularbacilli, isolated or in groups, with an average loga-rithmic index of 4.8.The other two antigens produced very similar

reactions: a granuloma with epithelioid and tuber-culoid nodules; an abundant infiltrate formed byepithelioid cells, giant cells, and lymphoid cells; fociof polymorphonuclear cells; and important oedema.The acid-fast staining was negative or showed a veryfew isolated bacilli.

Control biopsies. The lepromatous cases in group 1showed no histological lesions or acid-fast bacteria.The cases in group 2 showed discrete perivascularand nervous lesions containing acid-fast bacilli.

Tuberculoid patientsThe structure of the lesions showed that all three

antigens produced a very similar granuloma withfoci of necrosis, tuberculoid and epithelioid nodules,lymphocytic infiltrate, foci of polymorphonuclearcells, and oedema. The stain for acid-fast bacteriarevealed a few isolated bacilli in one patient only.

Borderline patients

M. leprae antigen. The lesions showed a granu-loma with epithelioid cells, giant cells, macrophages,lymphocytes, foci of polymorphonuclear cells, andoedema. The stain for acid-fast bacteria showedrelatively large numbers of bacilli distributed irregul-arly in the granuloma.The other two antigens gave rise to a structure

similar to that produced by the other forms ofleprosy previously described.

Indeterminate patients and Mitsuda- negative contactsThese subjects received an injection of M. leprae

antigen only. The granuloma that formed was ma-crophagic with epithelioid foci but with abundantintracellular acid-fast bacteria, isolated or in groups.

COMMENTS

All the lepromatous patients showed the sameresponse to the M. leprae antigen. This response wascharacterized by a macrophagic granuloma formedby cells that were not able to remove M. leprae andtherefore had a negative competency for clearingbacilli. This response was not modified either by

treatment for several years or by the fact that someof these patients were clinically and bacteriologicallynegative (group 1). We consider that this reactionshows that these patients, from an immunologicalpoint of view, are unable to remove or lyse M. keprae.This could be due to an inability to recognizeM. keprae as foreign material.The reaction of the lepromatous patients to the

other two antigens was completely different, showinga granuloma formed by cells capable of removingthe bacilli (positive competency in clearing bacilli).This indicates that there was a selective negativeresponse, elicited only by M. leprae. The structuralcharacteristics of the granuloma developed after in-oculation with M. lepraemurium and the hamstermycobacteria do not confirm the so-called " isopa-thic" reaction (Sagher et al., 1953). In the tuber-culoid patients, the reaction was more or lessidentical, with a strongly positive competency in theclearing test.The borderline patients showed a reaction that

seemed to be intermediate between that of the lepro-matous and the tuberculoid patients. The two inde-terminate patients and the Mitsuda-negative contactshad reactions very similar to those produced by thelepromatous patients.From the types of granuloma that developed, it is

considered that the granuloma produced by M. lepraein the lepromatous patient is the " inert granuloma "type, which in a number ofrespects corresponds to thekind of granuloma produced by foreign bodies. Thiscan be explained in part by assuming that M. lepraeis not toxic to the lepromatous macrophage. Thegranuloma produced by M. keprae in the tuberculoidpatient, and the granulomas produced by the othermycobacteria in the lepromatous patient, have theappearance of proliferative-type granulomas, whichcorrespond to delayed hypersensitivity granulomas(Epstein, 1967).The results seem to indicate that this test might

have a therapeutic application in lepromatous pa-tients who are clinically and bacteriologically nega-tive. It might be established that, as long as alepromatous patient has a negative competency inclearing bacilli, he should continue treatment. Inde-terminate cases could be managed in the same way.For contacts, the competency in clearing bacilli

(CCB) test might be considered to indicate whetherthese persons might possibly develop the leproma-tous form of the disease later. A contact withnegative competency should be given preventivetreatment.

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826 J. CONVrr ET AL.

Finally, the test might be used to determinewhether a given mycobacterium is M. leprae. If anantigen produces in the lepromatous patient a macro-phagic granuloma unable to destroy its intra-cellular mycobacteria, it should be classified asM. leprae.

Since there are several granulomatous diseasesproduced by parasitic agents, the application of theCCB test with the corresponding antigen in each ofthese diseases might be a useful way to study theimmunological alterations that usually occur in thesediseases.

RgSUM9iE-PREUVE VISANT A DETERMINER LA CAPACITE D'ALIMINATION DES BACILLES

CHEZ DES MALADES ATTEINTS DE LEPRE

Les auteurs decrivent un test cutane destin6 a determinerdans quelle mesure les macrophages sont capables d'eli-miner ou de lyser Mycobacterium leprae chez des maladesatteints de 1epre lepromateuse, indetermin6e ou tuber-culoide, ainsi que chez des contacts Mitsuda-negatifs.Pour obtenir une reaction macroscopique dans les

cas de lepre lepromateuse ou indeterminee et chez lescontacts, on a utifise un antigene concentr6 renfermant640 x 106 Myco. leprae par millilitre. Injecte par voieintradermique a la dose de 0,1 ml, cet antigene a entramn6apres 15 jours la formation, a 1'endroit d'inoculation,d'un nodule dont le diametre variait de 5 a 8 mm.L'etude, apres biopsie, des cellules du granulome amontre qu'elles etaient incapables d'eliminer ou delyser Myco. leprae (test negatif).On a aussi 6tudie la reponse des malades lepromateux

,i d'autres mycobacteries, en leur injectant, a la memeconcentration que l'antigene Myco. leprae, une suspensionde Myco. lepraemuriumn et une suspension d'une myco-

bact6rie atypique isolee chez le hamster. Ces inoculationsont entraine la formation d'un granulome de type tuber-culoide, renfermant des macrophages capables d'eli-miner ou de d6truire ces mycobacteries.

I1 est propose de recourir a cette 6preuve dans les casde 1lpre lepromateuse chez lesquels I'affection a et6reconnue cliniquement et bacteriologiquement inactive.Les malades qui presentent un test n6gatif devraientcontinuer a etre trait6s jusqu'a ce que le test deviennepositif. Le traitement devrait 6galement etre poursuividans les cas de lepre indeterminee chez les malades atest negatif. Pour les contacts, les resultats du testdevraient indiquer l'opportunite d'un traitement preventif.Le test pourrait servir a identifier Myco. leprae. Toute

mycobacterie injectee ai un malade l6promateux et pro-voquant l'apparition d'un granulome dont les macro-phages se revelent inaptes a detruire le micro-organismeinocule devrait etre consideree comme 6tant un bacillelepreux.

REFERENCES

Barbieri, T. A. & Correa, W. M. (1967) Int. J. Leprosy,35, 377

Beiguelman, B. (1967) Bull. Wld Hlth Org., 37, 461Convit, J. et al. (1962) Int. J. Leprosy, 30, 239Epstein, W. L. (1967) Progr. Allergy, 11, 36Godal, T. et al. (1971) Clin. exp. Immunol., 8, 625-637Patterson, R. J. & Joumans, G. P. (1970) Infect. Immunol.,

1, 600

Ridley, D. S. & Hilson, C. R. F. (1967) Int. J. Leprosy,25,184

Sagher, F. et al. (1952) J. invest. Derm., 19, 499Sagher, F. et al. (1953) J. invest. Derm., 20, 243Sagher, F. et al. (1953) Int. J. Leprosy, 21, 459Shepard, C. C. & McRae, D. H. (1968) Int. J. Leprosy,

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