294^ International Journal of Leprosy^ 1993

erythematous, 1-2 cm, firm, tender, sub-cutaneous nodules and pustules present pre-dominantly on the extensor aspects of thearms and legs, and a few crusted punched-out ulcers discharging seropurulent mate-rial. There was neither thickening nor ten-derness of any of the peripheral cutaneousnerves. No significant lymphadenopathy waspresent, and a systemic examination waswithin normal limits.

Ziehl-Neelsen staining of a pus smear re-vealed AFB in clumps, and a slit-skin smearexamination from the earlobes and eye-brows showed a bacterial index (BI) of 5+.The morphological index (MI) from the pusand slit-skin smear was 5% and 25%, re-spectively. Hematological investigations re-vealed a normal hemogram with a raisedESR (64 mm in first hr, Westergren). Hisrenal and liver function tests were withinnormal limits, and examinations of urineand stool showed no abnormalities.

A skin biopsy showed a foamy macro-phage granuloma throughout the dermiswith neutrophilic, leukocytoclastic vascu-litis. Ziehl-Neelsen staining of tissue for AFBshowed fragmented and granular bacilli. Thepatient was treated with oral cephalexin 2g, prednisolone 20 mg, rifampin 600 mg,clofazimine 300 mg, and dapsone 100 mgdaily. All of the pustular lesions subsidedwithin 1 week, and all of the nodular lesionsflattened by more than 80% in 2 weeks. Af-ter 2 weeks, the cephalexin was stopped andthe prednisolone reduced to 10 mg daily,but he continued on other antileprosy drugsin the same dosages. The patient had a sim-ilar episode within an interval of 1 month,while he was still on the same treatment.This time he was treated with prednisolone30 mg. rifampin 600 mg, clofazimine 300

mg, and dapsone 100 mg daily. Pustularlesions subsided within a week, at whichtime he was put on thalidomide 300 mgdaily. The prednisolone was tapered offslowly over the next 2 weeks, and the tha-lidomide was withdrawn completely withinthe next 2 subsequent weeks. The patient isnow on the World Health Organization'sregimen for multibacillary leprosy, and hasnot had any episodes of ENL in the last 11/2years.

The purpose of this report is to highlightan unusual presentation of lepromatous lep-rosy, presenting as pustular erythema no-dosum leprosum.

—Kaushal K. Verma, M.D.Assistant Prolessor

— Ravinder K. Pandhi, M.D.Department of Dermato-VenereologyAll India Institute of Medical SciencesNew Delhi 110029, India

REFERENCESI. BROWNE, S. G. Localized bacilliferous skin lesions

appearing in patients with quiescent lepromatousleprosy. Int. J. Lepr. 34 (1966) 289-293.

2. ORTIZ, Y. and GINER, M. [Lucio's leprosy (diffuselepromatous leprosy). II. Recent advances: clinicaland laboratory data.] Dermatologia (Mexico City)22 (1978) 141-163.

3. PFALTZGRAFF, R. E. and BRYCESON, A. Clinical lep-rosy. In: Leprosy. Hastings, R. C., ed. New York:Churchill Livingstone, 1985, pp. 134-176.

4. RAMESH, V., SAXENA, U., MISHRA, R. S., MUKHER-JEE, A. and RAVI, S. Multibacillary leprosy pre-senting as a solitary skin lesion; report of three casesand its significance in control programs. Int. J. Lepr.59 (1991) 1-4.

5. WADE, H. W. The histoid variety of lepromatousleprosy. Int. J. Lepr. 31 (1963) 129-142.

Attempts to Grow Mycobacterium leprae in a Mediumwith Palmitic Acid as the Substrate

To THE EDITOR:To use a multifactorial medium under

microaerophilic conditions has been pro-posed by Kato (4 ) in cultivation trials forMycobacterium leprae. Recently, Ishaque

( 3 ) investigated the effects of various knowngas mixtures on the growth of M. leprae. Anoptimal growth, although limited, on bothsolid and liquid media was obtained whenthe cultures were incubated under a gas mix-

61, 2^ Correspondence^ 295

ture containing 2.5% 0, and 10% CO,. Nosubstrate has yet been identified which canserve as a source of energy and carbon forthe multiplication of M. leprae. Franzblau(') reported evolution of CO, during the in-cubation of M. leprae with palmitic acid.Wheeler, et al. ( 8 ) have indicated that Al.leprae requires an exogenous source of fattyacid. Ishaque ( 2 ) has provided direct evi-dence for the oxidation of palmitic acid byAl. leprae. Based on the effect of classicalinhibitors of the electron transport chain,participation of the cytochrome system hasbeen suggested strongly during the oxida-tion of palmitic acid by M. leprae. Thesestudies suggest that palmitic acid possiblycould serve as an oxidizable substrate forthe growth of M. leprae. Attempts were thusmade to cultivate Al. leprae using an opti-mal gas mixture, i.e., 2.5% 0, and 10% CO,,in a medium containing palmitic acid as asubstrate.

During these studies, M. leprae were iso-lated from the foot pad lesions of nude mice(athymic) which previously had been in-fected with human leprosy bacilli. A bacilla-ry suspension was prepared to contain 5 x10 8 bacilli per ml. Both liquid and solidmedia were used. The liquid medium con-tained (NH,), SO, 0.2 g; KH, PO 4 , 1.0 g;glycerol 2.5 g; MgSO 4 • 7H„0, 0.2 g; sodiumthioglycolate, 0.4 g; hemin 0.0002 g; sodiumpalmitic acid, 0.002 g and 100 ml of water.The solid medium, in addition, contained200 ml of egg yolk.

Several tubes containing 9 ml of the liq-uid medium were inoculated with 1 ml ofthe bacillary suspension. To inoculate thesolid medium, foot pad lesions were cut intosmall pieces and a paste was prepared bymincing the tissues in a 2% NaOH solutionusing a mortar and pestle. Several tubescontaining the solid medium were inocu-lated with 0.3 ml of the bacillary paste. Theinoculated tubes were placed in an anaer-obic jar. The jar was closed, excess air wasremoved from the jar, and the cultures wereincubated at 34°C in a gas mixture contain-ing 2.5% 0,, 10% CO„ and 87.5% N,. Someinoculated tubes containing liquid and solidmedia without palmitic acid were also in-cubated under the same gas mixture. Thejars were opened every 2 weeks for obser-vation, and the cultures were reincubated.In parallel, some inoculated tubes contain-

ing liquid and solid media with and withoutpalmitic acid were incubated as such, i.e.,under normal air at 34°C.

The multiplication was evaluated bycounting the acid-fast bacilli (AFB). To countthe bacilli in the liquid medium, 1-ml ali-quots were removed at the time of inocu-lation (0 hr) and at various time intervals,and the final volume was made to 10 mlwith sterile 0.1% skim milk. In order to countAFB from the solid medium, the entiregrowth from the surface of the medium wasremoved, homogenized in about 50 ml ster-ile distilled water, and the final volume wasmade to 100 ml in 0.1% skim milk. Alldilutions made in 0.1% skim milk to countthe AFB were sonicated for 30 sec just be-fore staining. This technique resulted in sin-gle cells, and no clumping of the bacilli wasobserved in the stained preparations.

Acid-alcohol-fast bacilli (AAFB) werestained and counted by the method of Shep-ard and McRae ( 7 ). In tubes incubated undera gas mixture containing 2.5% 0, no growthwas observed during the first 2-3 weeks ofincubation. Thereafter, growth was ob-served and after 16 weeks of incubation, asixfold increase in the number of AAFB wasobtained. The number of AAFB remainedthe same up to 20 weeks and then slowlydeclined. No multiplication of AAFB oc-curred in a liquid or solid medium withoutpalmitate when incubated under air or thegas mixture. However, it is interesting tonote that sixfold increases in AAFB wereobserved when the cultures were incubatedunder air for 12 weeks in the liquid mediumor solid medium which contained palmiticacid.

The morphology of the bacilli was wellmaintained. The bacilli did not grow onLowenstein-Jensen, Dubos, or egg-yolkmedia. The bacilli recovered from theliquid and solid media grown for 12-16weeks showed 3,4-dihydroxyphenylala-nine (DOPA) oxidase activity, a character-istic specific for M. leprae (6 ). Such bacillialso exhibited good endogenous respiration.These observations indicated that the ba-cilli were, in fact, viable. To further confirmthe viability and authenticity of Al. leprae,the hindfoot pads of two groups of five fe-male nude mice 6 weeks of age were infectedwith the bacillary suspensions (containing 1x 10 7 bacilli) recovered from the solid and

296^ International Journal of Leprosy^ 1993

liquid cultures grown for 12 and 16 weeks.In parallel, hindfoot pads of five nude micewere also infected with the suspension (con-taining 1 x 10' bacilli) prepared from footpad lesions of nude mice previously infectedwith M. leprae. It was observed that thehindfoot pads of all of the mice infectedwith the above-mentioned preparationswere slightly swollen after about 8 monthsof infection. Experience has shown that suchmice will develop full lepromatoid lesionson the food pads 13-16 months postinfec-tion.

An important aspect of this study is therole of palmitic acid for the growth of thebacilli in the synthetic medium in the pres-ence of air. The use of any gas mixture isquite tedious, time-consuming and labori-ous. The use of palmitic acid in the presenceof air for in vitro cultivation trials of Al.leprae eliminates the use of any gas mixture.

Recently, water-soluble palmitic acid hasbecome available ( 5 ), and further studies arcin progress to compare the role of insolubleand water-soluble palmitic acid in meta-bolic studies and in cultivation trials of Al.leprae.

— Mohammed Ishaque, Ph.D.ProfessorApplied Microbiology Research CentreInstitut A rinand-FrappierUniversity of QuebecC.P. 100, Laval, QuebecCanada, H7N 4Z3

Acknowledgment. This study was supported bygrants from Le Secours aux Lepreux, Canada, Inc., andthe Military and Hospitaller Order of Saint Lazarus ofJerusalem, Canada.

REFERENCES1. FRANZBLAU, S. G. Oxidation of palmitic acid by

Mycobacterium leprae. J. Clin. Microbiol. 26 (1988)18-21.

2. IsIIAQUE, M. Direct evidence for the oxidation ofpalmitic acid by host grown Mycobacterium leprae.Res. Microbiol. 140 (1989) 83-93.

3. ISHAQUE, M. Growth of Mycobacterium leprae un-der low oxygen tension. Microbios 64 (1990) 7-17.

4. KATO, L. A multifactorial culture medium withgrowth factors from leprosy-derived mycobacteriaproposed in cultivation trials for Mycobacteriumleprae. Int. J. Lepr. 54 (1986) 310-31 I .

5. KATO, L. Water soluble complexes of palmitic acidand palmitates for metabolic studies and cultivationtrials ofMycobacterium leprae. Int. J. Lepr. 60 (1992)105-107.

6. PRABHAKARAN, K. Oxidation of 3,4-dihydroxy-phenylalanine by M. leprae. Int. J. Lepr. 35 (1967)42-51.

7. SHEPARD, C. C. and McRAE, D. H. A method forcounting acid-fast bacteria. Int. J. Lepr. 36 (1968)78-82.

8. WHEELER, P. R., BULMER, K. and RATLEDGE, C.Enzymes for biosynthesis de novo and elongationof fatty acids in mycobacteria grown in host cells:is Mycobacterium leprae competent in fatty acidbiosynthesis? J. Gen. Microbiol. 136 (1990) 211—217.

Leprosy; Another Possible Source of Transmission

To THE EDITOR:Armadillos, sooty mangabey monkeys,

chimpanzees and other primates have beenexamined by various workers at varioustimes using clinical and experimental meth-ods ( 1 . 4 . I -13) for affliction with leprosy andfor their possible role in transmission. Ac-cording to Meyers, et al. (7),". . . there seemsample justification for undertaking, forth-with, carefully designed surveys for enzo-otic leprosy in some of the major endemicareas." These authors suggest that such sur-veys should be initiated in the natural hab-itats of the mangabey monkey and the chim-

panzees in West Africa. As a means ofunderstanding the behavior of Mycobacte-rium leprae, this is an excellent suggestion.However, any hypothesis regarding trans-mission of leprosy from nonhumans to hu-mans must, of necessity, take into consid-eration a "most common factor" whichshould be consistent with regard to time andplace. For example, sooty mangabey mon-keys, chimpanzees, and armadillos simplydo not exist in some of the most endemicareas of leprosy today. They could not havefigured in the transmission in Norway be-fore it disappeared completely, after making

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