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VOLUME 73, NUMBER 3 SEPTEMBER 2005

Special Grantors Listed in Contents

INTERNATIONALJOURNAL OF LEPROSYAnd Other Mycobacterial Diseases

Official Organ of theINTERNATIONAL LEPROSY ASSOCIATION

(Association Internationale contre la Lèpre)(Asociación Internacional de la Lepra)

Images from the History of Leprosy - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Original ArticlesThomas H. Rea and Robert S. Jerskey. Clinical and Histologic Variations Among Thirty Patients with Lu-

cio’s Phenomenon and Pure and Primitive Diffuse Lepromatosis (Latapi’s Lepromatosis) - - - - - - - - - - - - - - - - - - - - - - - - -

Nand Lal Sharma, Vikram K. Mahajan, Vikas C. Sharma, Sandip Sarin, and Ramesh Chander Sharma.Erythema Nodosum Leprosum and HIV Infection: A Therapeutic Experience - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Ramanuj Lahiri, Baljit Randhawa, and James L. Krahenbuhl. Effects of Purification and Fluorescent Stain-ing on Viability of Mycobacterium leprae - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Case ReportsTarun Narang, Sunil Dogra, and Inderjeet Kaur. Borderline Tuberculoid Leprosy with Type 1 Reaction in an

HIV Patient—A Phenomenon of Immune Reconstitution - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Tarun Narang, Sunil Dogra, and Inderjeet Kaur. Co-localization of Pityriasis Versicolor and BT Hansen’sDisease - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

CommentaryOttenhoff, Tom H.M., and Klein, Michèl R. Leprosy Bacillus Triggers the Wrong Cells - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

EditorialsRao, P. Narasimha. Leprosy Program in India at the Crossroads - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Ji, Baohong. Comments on WHO/AFRO’s “Post-Elimination” Strategy Paper: A New Bottle with Old Wine ofthe “Final Push” - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

CorrespondencePremkumar, Ramaswamy, Rajan, Pichaimuthu, and Daniel, Ebenezer. Quantitative Measurement of

Sensory Impairment in Referral Centers - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Rada, Elsa María, Zambrano, Edgar A., Aranzazu, Nacarid, and Convit, Jacinto. Serologic Recognitionof Low Molecular Weight Mycobacterial Protein Fractions in Lepromatous Patients with Type II Reactions(ENL) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Santos, Mônica Nunes Souza, Ferreira, Luis Carlos de Lima, and Talhari, Sinésio. PaucibacillaryTreatment for Large Tuberculoid Lesions of Leprosy? - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Kumarasinghe, S. Prasad W., and Kumarasinghe, M. P. Reply to the Editor - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Ganapati, R., and Pai, V. V. Has the Term “Elimination” Outlived It’s Utility? - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

News and Notes - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

U.S.-Japan Meeting, 2004 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Special Grantors - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

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INTERNATIONAL JOURNAL OF LEPROSYand Other Mycobacterial Diseases

CONTENTSVolume 73, Number 3, September 2005

INTERNATIONAL JOURNAL OF LEPROSY Volume 73, Number 3Printed in the U.S.A.

(ISSN 0148-916X)

INTERNATIONAL JOURNAL OF LEPROSY

and Other Mycobacterial Diseases


Images from the History of LeprosyKalaupapa, Hawaii.Music was an integral part of peoples’ lives at Kalaupapa, Hawaii. In 1901, the Kalawao

Choir posed alongside Father Damien’s Church. This image was electronically reproducedfrom an original black and white photograph.

Photo courtesy of IDEA.

167

1 Received for publication on May 9, 2005. Accepted for publication on Aug 27, 20052 T. H. Rea, M.D., Emeritus Professor, Division of Dermatology, Keck School of Medicine, University of

Southern California, and Attending Physician, Los Angeles County/University of Southern California MedicalCenter, Los Angeles, California. R. S. Jerskey, LOTR, Prevention of Impairment and Disability Consultant,National Hansen’s Disease Program.

Correspondence and reprint requests to Dr. T. H. Rea, M.D., Division of Dermatology, Keck School ofMedicine, University of Southern California, and Attending Physician, Los Angeles County/University ofSouthern California Medical Center, Los Angeles, California. Division of Dermatology, Room 8440, LAC/USCMedical Center, 1200 N. State St., Los Angeles, CA 90033, USA. e-mail: [email protected].


(ISSN 0148-916X)

INTERNATIONAL JOURNAL OF LEPROSY

and Other Mycobacterial Diseases


Clinical and Histologic Variations Among

Thirty Patients with Lucio’s Phenomenon and

Pure and Primitive Diffuse Lepromatosis

(Latapi’s Lepromatosis)1

Thomas H. Rea and Robert S. Jerskey2

ABSTRACTThe clinical and histologic experience with 30 patients who had Lucio’s phenomenon,

and pure and primitive diffuse lepromatosis (Latapi’s lepromatosis) has been reviewed. Theunanticipated clinical findings were a male to female ratio of nearly 1:1, a 21 month mediantime of onset of erythema nodosum leprosum (Type 2 reaction) after starting antibacterialtreatment, and an absence of a stocking-glove pattern of anesthesia in 7 patients. The onlyunanticipated histologic finding was a lepromatous-granulomatous vasculitis, occurring incomparatively large vessels, or in vessels made large by pathologic changes, located near thedermal-subcutaneous interface. This finding was present in 6 of the 22 patients with histo-logic material available for review. In 2 of these 6 this vasculitis was identified before theonset of Lucio’s phenomenon. With one conspicuous exception, the onset of treatment witha microbicidal agent was associated with a cessation of new lesions of Lucio’s phenomenonwithin one week. Long-term morbidity, other than Type 2 reaction, was found in 22 of the25 patients followed for more than 1.3 years. Usually this was the consequence of Latapi’slepromatosis, specifically venous insufficiency and/or loss of protective sensation, and onlyrarely from Lucio’s phenomenon, specifically scar formation. Briefly summarized are theseven patients who had had a skin biopsy before the onset of Lucio’s phenomenon, as wellas the two patients who were considered to be atypical. Criteria for the diagnosis of Latapi’slepromatosis, in the absence of Lucio’s phenomenon, are also considered.

RÉSUMÉCet article s’est attaché à faire la revue de l’expérience clinique et histopathologique de

30 patients atteints de phénomène de Lucio et/ou de lèpre lépromateuse pure et primitive-

169

170 International Journal of Leprosy 2005

ment diffuse, encore appelée lèpre lépromateuse de Latapi. Les données cliniques sur-prenantes furent un ratio homme-femme de presque 1:1, un temps médian de 21 mois entrela mise en ouvre d’un traitement antibactérien et le déclanchement d’un érythème noueuxlépreux (réaction de type 2), et l’absence d’une anesthésie distribuée en bas-résille chez 7patients. La lésion histologique non anticipée a été la découverte d’une vasculite léproma-teuse et granulomateuse atteignant des vaisseaux de diamètre relativement élevé ou bien desvaisseaux élargis par les changements histopathologiques, situés prés de l’interfacederme/hypoderme. Cette lésion était présente chez 6 des 22 patients qui présentaient dumatériel histologique pour une revue. Chez 2 de ces 6 individus, cette vasculite fut identifiéeavant le déclenchement du phénomène de Lucio. Lors de phénomène de Lucio et à l’excep-tion d’un cas plutôt remarquable, l’apparition de nouvelles lésions a été interrompue dans lasemaine qui a suivi la mise en place d’un traitement avec un agent bactéricide. Une morbid-ité à long terme, autre que les réactions de type 2, fut trouvée chez 22 des 25 patients qui ontété suivis pendant plus de 1,3 ans. Le plus souvent, cette morbidité était la conséquence dela lèpre lépromateuse de Latapi, spécifiquement l’insuffisance veineuse et/ou la perte de sen-sibilité protectrice, et seulement rarement les conséquence du phénomène de Lucio, spéci-fiquement l’apparition de cicatrices. Brièvement résumés sont les 7 patients qui ont eu unebiopsie cutanée avant l’apparition d’un phénomène de Lucio, ainsi que les 2 patients quifurent considérés comme atypiques. Les critères pour le diagnostic de lèpre lépromateuse deLatapi, en l’absence de phénomène de Lucio, sont également présentés.

RESUMENSe hizo una revisión de los datos clínicos e histológicos de 30 pacientes que habían tenido

el fenómeno de Lucio y lepromatosis difusa pura y primitiva (lepromatosis de Lucio). Loshallazgos clínicos no anticipados fueron: una relación masculino: femenino casi de 1:1, untiempo promedio de aparición de eritema nodoso leproso (reacción de tipo 2) de 21 mesesdespués del inicio del tratamiento antibacteriano, y ausencia del patrón de anestesia “media-guante” (stocking-glove) en 7 pacientes. El único hallazgo histológico no anticipado fue unavasculitis lepromatosa-granulomatosa, presente en vasos comparativamente grandes o envasos agrandados por los cambios patológicos, localizados cerca de la interfase dermo-subcutánea. Este hallazgo estuvo presente en 6 de 22 pacientes con material accesible pararevisión. En 2 de estos 6 pacientes la vasculitis fue identificada antes de la aparición delfenómeno de Lucio. Con una sola excepción, el tratamiento con un agente microbicida es-tuvo asociado con la suspensión, en la primera semana, de nuevas lesiones del fenómeno deLucio. La morbilidad crónica, diferente a la reacción de tipo 2, se encontró en 22 de 25 pa-cientes seguidos por más de 1.3 años.

Usualmente la insuficiencia venosa y/o la pérdida de sensación protectora fueron conse-cuencia de la lepromatosis de Lucio y sólo raramente del fenómeno de Lucio en cuyo casola consecuencia más frecuente fue la formación de cicatriz.

Se describen brevemente los casos de los 7 pacientes que habían tenido una biopsia depiel antes de la aparición del fenómeno de Lucio, y de dos pacientes considerados comoatípicos. También se discuten los criterios para el diagnóstico de la lepromatosis de Latapien ausencia de fenómeno de Lucio.

Since reporting 10 patients with Lucio’sphenomenon (25), seen in this institutionfrom 1969 through 1977, a further 20 hadbeen observed by the end of 2004. Theprimary purpose of this report is to describethe kinds and the extent of the clinical andhistologic findings in all these 30 patientswith Lucio’s phenomenon as well as in theunderlying diffuse lepromatosis. In addi-tion, this report presents data on long-termfollow-up, and details information concern-ing patients who had had a skin biopsyprior to the onset of Lucio’s phenomenon.

Also, a higher incidence of a lepromatous-granulomatous vasculitis (L-GV) wasfound in patients with Lucio’s phenomenonthan was present in those with erythema no-dosum leprosum (Type 2 reaction) or non-reactional lepromatous leprosy.

Concerning history and nomenclature, in1852 Lucio and Alvarado (16) reported anecrotic skin reaction that occurred in lep-rosy, as judged by the concomitant changesof peripheral neuropathy, eyebrow loss, andnasal involvement. These authors also de-scribed the absence of the nodular, dermal

73, 3 Rea and Jerskey: Lucio’s Phenomenon and Latapi’s Lepromatosis 171

lesions expected in leprosy, as well as theassociated fatal termination. Latapi andZamora (15) established that the necrosiswas a result of vascular involvement, andthat the absence of dermal nodules was apart of a diffuse lepromatous infiltrate,which they described in considerable detail.Also, they reported a much better prognosiswith dapsone therapy. Latapi and Zamoracalled the necrotic skin reaction “Lucio’sphenomenon” or “erythema necroticans”and the diffuse, non-nodular lepromatousinfiltration “pure and primitive diffuse lep-romatosis.” For the latter expression, thesynonym “Latapi’s lepromatosis” is pro-posed, and will be used hereafter herein.This gives an appropriate and briefeponymic recognition of Latapi’s importantcontributions. Also, having “Lucio” in twoclosely related eponymic terms, i.e., “Lucioleprosy” and “Lucio’s phenomenon,” oftenis a needless source of confusion.

The initial report of Lucio and Alvaradowas virtually forgotten in 50 years (15). Inthe over 50 years following the report ofLatapi and Zamora (15), both Lucio’s phe-nomenon and the underlying Latapi’s lepro-matosis have been recognized in diverseethnic groups, and in a wide geographicdistribution, including, but not limited to,Louisiana (8), Hawaii (3), Brazil (1, 9, 31),Greece (11), the Near East (30), India (29),Singapore (2), Indonesia (13), and Polynesia(6). Apparently the condition remains rareexcept in Mexicans, Costa Ricans (28), andCubans (18), where its incidence is aptly de-scribed as “not common.”

This retrospective study is somewhat in-complete because of the institutional policyof “deep” storage of charts, and the North-ridge earthquake of January 1994 trashedthe room holding both histologic glassslides and paraffin blocks. The materialavailable was considered sufficient to illus-trate a range of clinical and histologic find-ings, the responses to treatment, and thelong-term morbidity encountered.

MATERIALS AND METHODSThe subjects were patients in the

Hansen’s Disease Clinic or the Dermatol-ogy Clinic of the Los Angeles County-University of Southern California MedicalCenter. Included in this study were all pa-tients who had one or more characteristic

lesions of Lucio’s phenomenon, i.e., ser-rated hemorrhagic infarcts usually arisingin crops, and at least one characteristic signof Latapi’s lepromatosis (see below) or oflepromatous leprosy, but no lepromatousnodules. The diagnosis of Latapi’s lepro-matosis was made after the fact of Lucio’sphenomenon. No criteria for exclusionwere established.

The data base for the clinical informationin this study was compiled from fivesources: the available charts, data ab-stracted from charts for previous publica-tions, clinical photographs obtained beforestarting treatment, patients currently beingfollowed in clinic, and available histologicspecimens. The summary of the histologicchanges was compiled from the materialavailable for review.

RESULTSThe results will be presented in two ways.

First will be 9 brief case reports. Followingthe case reports, the available data on all pa-tients will be summarized in narrative form.

CASE REPORTSThe initial 7 case reports concern those

patients who had had skin biopsies takenprior to the development of Lucio’s phe-nomenon. Five of these biopsy specimenshad been seen by one of us (THR), and 3 ofthese 5 were available for review. Amongthese 7 patients, 4 had a diagnosis of lep-rosy established and treatment initiated be-fore the onset of Lucio’s phenomenon(Cases 2, 3, 6, and 7), whereas 3 had had abiopsy but the diagnosis of leprosy was notmade until the onset of Lucio’s phenome-non (Cases 1, 4, and 5). The remaining 2case reports (Cases 8 and 9) concern thosewho were considered to be atypical.

Patients with skin biopsies prior toLucio’s phenomenon

Case 1. An 18 year old woman soughtcare because of numbness of the hands andfeet of several months duration. Neurol-ogists interpreted her findings to be a pe-ripheral neuropathy secondary to a systemicdisease. Consultation with many medicalspecialties could identify no systemic ill-ness. After 18 months, a skin biopsy takenfrom an area of diminished sensory percep-tion, but otherwise clinically normal skin,

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was interpreted as “normal skin.” Threeyears after her initial presentation crops ofinfarcts of Lucio’s phenomenon quickly ledto the correct diagnosis. A Fite stain thendone on the “normal skin” material demon-strated acid-fast bacilli (AFB) and globiwithin nerves and endothelial cells (slidenot available for review).

Comment: this case illustrates the diffi-culty of making a diagnosis of leprosy inthe absence of a sign more readily associ-ated with leprosy.

Case 2. A 22 year old woman presentedto the obstetrical service in labor with anincidental complaint of eyebrow alopecia,progressing from medial to lateral, of 2 months duration. Because rhinitis wasalso present, leprosy was considered. Abiopsy of clinically normal skin showedAFB and globi in some endothelial cellsand perivascular macrophages (slide notavailable for review). No AFB were foundin placental endothelium. One month afterinitiating dapsone monotherapy, erythemanodosum leprosum (ENL) developed,which was managed with thalidomide.Eight months later the patient was lost tofollow-up, only to return after a 13 monthsabsence with the necrotic lesions of Lucio’sphenomenon and ulcers (biopsy not avail-able for review).

Comment: This case confirms the possi-bility of considering a diagnosis of Latapi’slepromatosis without the infarcts of Lucio’sphenomenon, as suggested by others (9).Also, this is the only patient seen in this se-ries who had ENL prior to having Lucio’sphenomenon. Because of the absence ofAFB in placental endothelial cells, but theirabundance in cutaneous endothelium, thepredilection of AFB for endothelial cells islikely to be organ specific.

Case 3. A 45 year old woman presentedwith eyebrow alopecia, rhinitis, and palpa-ble but not visible, indurated lesions in thesubcutis. Skin biopsy showed, in addition toan extensive lepromatous infiltrate, an AFB-positive, lepromatous-granulomatous vas-culitis (L-GV) in the subcutis, characterizedby endothelial proliferation, macrophagesdissecting between the smooth muscle cellsof the media, and macrophages infiltratingthe adventitia (Fig. 1a, b). Also present wasan aneurysmal lepromatous-granulomatousout-pouching originating in a subcuticular

vessel (Fig. 1c), as well as signs of new ves-sel formation. Our working diagnoses werelepromatous leprosy and a reactional stateof unknown type. She was treated withdaily dapsone and rifampin, as well as pred-nisone and thalidomide. The unknown reac-tional state was managed with decreasingdoses of thalidomide and prednisone, thelatter being discontinued after 5 months.Seven months after initiating treatment, and2 months after stopping prednisone, but stilltaking thalidomide 100 mg daily, the pa-tient developed Lucio’s phenomenon (Fig.1d). Her new Lucio infarcts ceased within 2weeks upon resuming prednisone at 40 mgdaily; the dose being tapered slowly overthe ensuing 5 months.

Comment: This patient made us aware ofinvolvement in larger vessels, or vesselsmade large by pathologic changes, than pre-viously recognized by us. Also, the previ-ous suggestion that viable bacilli might be asine qua non for Lucio’s phenomenon (25)appeared to be incorrect. In retrospect, whatwas called a reactional state of unknowntype was perhaps the L-GV.

Case 4. A 37 year old woman sought carebecause of what was described in her chartas nodular lesions on the arms and legs sug-gestive of erythema nodosum. The report ofa skin biopsy at that time noted inflamma-tion and was interpreted as suggesting ery-thema nodosum. The patient was treatedwith a saturated solution of potassium io-dide. When seen 2 years later for anotherproblem, it was noted that the erythema no-dosum had resolved. After an additional 2years, crops of Lucio’s infarcts developed.Of the 2 biopsy specimens obtained at thistime, both showed characteristic changes ofthe Lucio’s phenomenon, and the onewhich was available for review also demon-strated subcutaneous vessels with endothe-lial proliferation and lumen occlusion (Fig.2a). Also, the biopsy taken four years be-fore the onset of Lucio’s phenomenon wasavailable for review, showing a focal lobu-lar panniculitis consisting of macrophagesand, in the dermis, an arcuate, perivascularinfiltrate of lymphocytes and foamy macro-phages (Virchow cells) (Fig. 2b, c). The lat-ter was interpretable as evidence of a lepro-matous change. The tissue blocks were notavailable.

Comment: Two explanations for the sug-

FIG. 1. From Case 3. a) A low power view of a biopsy from a palpable, but not visible, subcutaneous lesion,on the right arm, which shows in the lower right corner an enlarged vessel in the subcutis with a conspicuous in-ternal elastic lamina. The lumen is occluded, and adventitial involvement is apparent. Heavy infiltration of thedermis is evident. 1.25× objective. b) A high power view of the same vessel as shown in “a.” The now less con-spicuous internal elastic lamina is indicated by wide arrows, but is readily seen in color. The lumen is occluded



gested “erythema nodosum” appear possi-ble. A leprologic hypothesis is the develop-ment of mild, neutrophil-free, transientENL in a patient who had yet to expressother clinical manifestations of leprosy. Analternative explanation is that of a leprosy-unrelated inflammatory disorder occurringon a lepromatous background.

Case 5. A 28 year old man sought carebecause of the development of palpable, butnot visible, nodules in the subcutaneous tis-sue of his legs. The clinical impression wasa vasculitis. A skin biopsy was interpretedas “nodular vasculitis,” the pattern consistingof endothelial proliferation with occlusionof the lumen, as well as a dense infiltrate ofmacrophages in the adventitia (Fig. 3a). Hewas begun on an anti-inflammatory regi-men, which included methotrexate andprednisolone. Five months later he devel-oped an acute, febrile illness, diagnosed asa Salmonella non-typhoid bacteremia, sec-ondary to the ingestion of snake powders.He responded well to intravenousciprofloxacin, and was discharged onciprofloxacin 500 mg twice daily orally,methotrexate 5 mg twice daily, and pred-nisone 80 mg every other day in the morn-ing. One day after discharge he abruptly de-veloped extensive and widely distributedinfarcts of Lucio’s phenomenon. These in-volved about one-third of his body surfacearea, most extensive on the legs (Fig. 3b),and arms, but present also on his ears, face(Fig. 3c), trunk, scrotum, and the urethralmeatus. This patient was promptly readmit-ted, and 1 day later his right hand was af-fected by apparently complete arterial oc-clusion. Circulation was restored by promptintervention, but deep necrosis eventuallyresulted in the loss of the 5th right distalmetacarpal head and the right 5th finger.His left patella was also lost, as a conse-quence of extensive tissue necrosis over theleft knee. A Fite stain on the tissue initiallyinterpreted as “nodular vasculitis” was pos-

itive for AFB in endothelial cells and ad-ventitial macrophages.

Comment: This one patient recapitulatesthree prior case reports. 1) Leprosy maymimic nodular vasculitis (34). 2) Occlusionof large muscular arteries may occur in as-sociation with Lucio’s phenomenon (8). 3)Lucio’s phenomenon may occur in a settingof convalescence from serious infection,i.e., in 2 cases of erysipelas managed withpenicillin (1). (These 2 patients, and Case 5,received antibiotics ineffective against M.leprae.) New infarcts ceased when rifampinwas begun. This patient was the only onewhose prognosis was poor when first seen.

Case 6. A 37 year old woman presentedto another clinic because of eyebrow alope-cia and rhinitis. A skin biopsy confirmed theimpression of lepromatous leprosy (slidenot available for review). She took 100 mgof dapsone daily for 18 years. At age 68, 13years after stopping dapsone, she presentedto this clinic with Lucio’s phenomenon, oc-curring intermittently for one month.

Case 7. A 15 year old boy presented to adermatology clinic in Mexico City becauseof eruptive telangiectasias and eyebrowalopecia. A skin biopsy (not available forreview) established a diagnosis of leprosy.Dapsone treatment was initiated at that timebut he took it infrequently. At age 21 hepresented to our clinic because of leg ulcersand skin infarcts of 6 months duration;telangiectasias were prominent at that time.Dapsone was resumed, but he was seen in-frequently. At age 35 he made 2 visits to ourclinic because of leg ulcers. Two skin biop-sies performed at that time were negativefor AFB (not available for review).

The atypical patientsCase 8. This 45 year old woman satisfied

entry criteria for this study on clinicalgrounds because she had one 8 mm charac-teristic infarct, a perforated nasal septum,total alopecia of eye lashes and eye lids,

←by endothelial proliferation. The lepromatous infiltrate is dissecting between smooth muscle bundles, shown bythin arrows, and is infiltrating the adventitia. 20× objective. c) Elsewhere in the same specimen an aneurysmalchange in a vessel is shown by the relatively dark internal elastic lamina. 10× objective. d) A high power view ofa re-epithelized Lucio’s phenomenon lesion which shows, from outside inward, the old stratum corneum, the nowshrunken infarcted epidermis with a hint of cellular ghosts and persistent melanin, a very thin new stratumcorneum, and new granular cell, prickle cell and basal cell layers. The dermal repair is not complete. Five oc-cluded or congested small vessels are evident. 40× objective. (Images available in color in the electronic edition,www.leprosy-ila.org)

FIG. 2. From Case 4. a) From a Lucio’s phenomenon lesion, a vessel in the subcutis with prominent en-dothelial proliferation, and adventitial infiltration. 40× objective. b) A low power view of an earlier biopsy ob-tained from a red dermal nodule 4 years before the onset of Lucio’s phenomenon, showing epidermal thickening,a superficial infiltrate in the upper dermis, and an arcuate infiltrate in the mid dermis. 10× objective. c) An oil-immersion view from the arcuate infiltrate showing 2 nucleated Virchow cells and the cytoplasm of a 3rd.Lymphocytes are evident, but no neutrophils were found. 90× objective.


several stellate scars, and 3 large leg ulcers,each approximately 10 cm in diameter.Biopsy showed a heavy infiltrate of macro-phages, AFB and globi in some endothelialcells, but no endothelial proliferation. Sheis considered to be atypical because of a“peau d’orange” appearance to the skin ofher forehead and cheeks, as well as beadingon corneal nerves (15). Also, she is the onlypatient in this series with just one infarct,and one of two without endothelialproliferation.

Comment: At present it is unknown ifany of these atypical findings, or a combi-nation thereof, would constitute valid crite-ria for exclusion.

Case 9. A 24 year old man presented tothis clinic with typical Lucio’s phenome-non, and giving a history of similar clinicalfindings, as well as intermittent dapsone usefor 4 years. A lesional biopsy did not showepidermal necrosis (probably exfoliatedduring processing), or congestion of super-ficial vessels, or extravasation of erythro-cytes , but did demonstrate AFB in prolifer-ating as well as in non-proliferating en-dothelial cells, globi in endothelial cells,and passive congestion of deep dermal ves-sels. Also present were larger vessels withlumens occluded from endothelial prolifer-ation, and macrophage infiltration of the ad-ventitia, but no infiltration of the media(Fig. 4a). New lesions ceased to form 4weeks after starting dapsone monotherapy.To this point, on balance, the patient wasconsidered to be typical. Eight years later,in the setting of seemingly good compli-ance with dapsone monotherapy, new Lu-cio’s phenomenon-like infarcts developedon the trunk and extremities. With theworking diagnosis of a relapsing Lucio’sphenomenon, and an inference of dapsoneresistance, dapsone was discontinued, beingreplaced by daily rifampin and dailyminocycline, and the new lesions ceased af-ter 8 weeks. Biopsy now showed epidermalnecrosis, passive congestion of superficialvessels, and endothelial proliferation indeep dermal vessels, but stains for AFBwere repeatedly negative (slides not avail-able for review).

Comment: No support for dapsone resis-tance was found. A self-limited process ofunknown type, unrelated to leprosy, butmimicking Lucio’s phenomenon must beconsidered.

SUMMARY OF ALL CASES

Demographic dataSixteen men and 14 women satisfied in-

clusion criteria. Twenty-five were born inMexico, 2 in Cuba and 3 in the UnitedStates, each of the latter 3 also havingresided in known leprosy-endemic areas ofMexico for more than 5 years. Age at thetime of diagnosis of Lucio phenomenon,leprosy was known in 29 and ranged in du-ration from 15 to 71 years, with a median of34 and an average of 33.7 years.

First sign or symptom of leprosy, andtime to diagnosis

The first sign or symptom attributed toleprosy was alopecia in 16 (eyebrows in 15,extremities in 1), leg ulcers in 4, sensoryimpairment in the hands and/or feet in 3,nasal symptoms in 2 (1 with nose bleeds, 1with congestion), eruptive telangiectasias in2, and infarcts of Lucio’s phenomenon in 2,but was not recorded in 1. The elapsed timefrom the initial sign or symptom to the di-agnosis of leprosy ranged from a minimumof 2 months to a maximum of 10 years,with a median of 3 years and an average of4.1 years.

Mode of presentationOf the 28 patients presenting to our clinic

with Lucio’s phenomenon, the presentingcomplaint was leg ulcers in 14 (duration 2months to 7 years, median 8 months and av-erage 19.7 months). The other 14 com-plained of the infarcts of Lucio’s phenome-non, usually occurring in intermittent crops(duration 5 days to 10 years, median 4months, and average 4.1 months). Both in-farcts and ulcers were characteristicallypresent at presentation, excepting Case 5,who had no ulcers at presentation. The in-farcts were hemorrhagic, slightly indurated,not tender, but sometimes painful. Sharplymarginated, irregularly serrated borderswere characteristic, such that an observerviewing the infarct, whether large or small,from “normal” skin would see a concavity(Fig. 3b, c). Early, pre-hemorrhagic lesionswere seen in only one patient, being slightlyindurated, light blue in color, and having anerythematous halo. (Previously publishedphotomicrographs of this specimen are figs.4 and 5 in Rea and Levan (25), and figs. 3and 4 in Quismorio, et al. (23)) The least


number of infarcts recorded was one, inCase 8. In all other patients the lesions weremultiple, arising in crops, most commonlyon the legs, but less frequently on the thighsand forearms. In one patient, Case 5, the in-farcts were numerous (over 100), widelydistributed, and clearly placed his life inperil. In another patient, 24 infarcts werecounted. And in yet another patient the in-farcts were small, and present only belowthe ankles. Infarcts on the arms resolvedrapidly, behaving more like erosions thanulcers. In 2 patients the infarcts becamebullous. Infarctions in organs other than theskin were not evident.

Ulcers were common on the legs, but oc-casionally on the thighs. Ulcers of recentonset appeared to be the direct sequelae ofthe infarcts, being ovoid and irregular inshape, not exceeding 5 cm in greatest diam-eter. If of long standing, ulcers were roundin shape and up to 10 cm in diameter, per-haps being complicated by neglect and un-suitable topical therapy.

In two patients the onset of Lucio’s phe-nomenon was associated with pregnancy,each ending with a normal delivery. In Case5, the onset occurred while the patient wasconvalescing from a Salmonella bac-teremia. No other potentially precipitatingevents could be identified.

Signs of Latapi’s lepromatosis at thetime of presentation

Absent nodules. The absence of leproma-tous dermal nodules was noted in all 30 pa-tients, with the possible exception of Case8, who had a “peau d’ orange” prominenceto the follicular orifices on the face, pre-sumably the result of infiltration or edema,but no dermal nodules in the conventionalsense.

Diffuse infiltration. Signs of diffuse cuta-neous infiltration were not mentioned aseither present or absent in 8 patients. One ormore signs of diffuse infiltration wererecorded as present in 22 patients. Widen-ing of the nasal root was described in 9 (orretrievable from clinical photographs).Diffuse infiltration in the hands was notedin 9 patients, being variously described as“swelling,” or “non-pitting edema,” of thebacks of the hands, in association with“fusiform fingers.” Changes in the cheeksof the face were variously described in 4 as“red plaques, poorly marginated,” “in-

durated erythema,” or “a cyanotic edema.”“Full” or “swollen” ear lobes were stated to be present in 7 patients. A “duskyswelling” of the feet was described in 1.(Swollen ear lobes and fusiform fingers aresigns of diffuse infiltration not uncom-monly found in patients with ordinary lep-romatous leprosy.)

Subcutaneous plaques. Not visible, non-tender, subcutaneous plaques were foundon the arms or legs in 8 patients, but werenot mentioned as absent in any of the 20other charts available for review.

Telangiectasias. Telangiectasias were de-scribed as eruptive in 3 patients; these per-sisted in 2, but treatment-associated remis-sion occurred in 1. Persistent telangiectaticmats, occurring on the face or upper chest,were noted as present in 7, (masqueradingas spider angiomas, but having no centralarteriole, and upon expression, filling fromthe periphery).

Ordinary changes of lepromatousleprosy at time of presentation

Alopecia. Alopecia of the eyebrows wasstated to be complete in 20, partial in 7, andwas not noted as present or absent in 3.Rhinitis. Rhinitis was stated to be present in25, and was not mentioned as present or ab-sent in 5. Of the 25 with recognized rhinitis,the nasal septum was recorded as perforatedin 9, as intact in 8, but was not mentioned asperforated or intact in 8. Stocking-glove pat-tern sensory impairment (S-GPSI). S-GPSIis a withering away of the sensations medi-ated by the type C sensory fibers, beginningdistally and proceeding proximally. S-GPSIwas recorded as present to some degree in20, as absent in 7, not mentioned as presentor absent in 3. Motor impairment. Motorimpairment was present in only three: twopatients with ulnar nerve involvement andone with common peroneal nerve involve-ment. The motor changes were dispropor-tionately few compared with the magnitudeof the sensory loss.

Routine laboratory findings at presentation

A mild anemia, normochromic and nor-mocytic, was common. The average leuko-cyte count (normal range 3.7–11.6 ×1000/mm3) was 6.5 and the median was6.3, among the 23 initial counts availablefor review. The highest count, 11.9, was


FIG. 3. From Case 5. a) In the center of the field is the largest of several vessels showing endothelial prolif-eration in the specimen from a palpable, but not visible, subcutaneous lesion obtained from the left thigh, 5months before the onset of Lucio’s phenomenon. 20× objective. b) Several lesions of various sizes on the face.Most show the characteristic serrated margins. c) The left knee with adjoining thigh and leg. The skin of the an-terior portion is largely necrotic, but the serrations still evident, although farther apart than in “4b.” Smaller le-sions are evident posteriorly.


seen was in Case 5; the lowest was 3.1. Twocounts were above and 2 were below thenormal limits. Among 19 patients thecardiolipin-based serologic test for syphiliswas reactive or weakly reactive in 15 in 3;of the 15 who were reactive, a Treponema-specific test was positive in 3. Hyperglobu-linemia was common; in 18 patients themean value was 4.8 gm/dl (normal3.0–4.0), the median 5, the high 6.0 and thelow 3.2. Serum albumin values were nor-mal in 7, ranging from 3.8 to 4.6 gm/dl,clearly low in 3, ranging from 2.9 to 3.1,and extreme in 1, being 1.8 in the one pa-tient with extensive infarcts, Case 5.

Response to treatmentCessation of new infarcts. Of the 28 pa-

tients who presented to the clinic with Lu-cio’s phenomenon, 19 were begun on dap-sone alone. Ten of these 19 had no new in-farcts after one week of follow-up. Theremaining 9 continued to develop new in-farcts for up to 5 months after starting treat-ment, 2 of which appeared to worsen beforethey improved. In one of the latter, new le-sions ceased at 6 weeks, in association withthe addition of daily rifampin.

Of the 7 previously untreated patientswith Lucio’s phenomenon who were startedon a daily microbicidal agent (5 with ri-fampin, 1 with clarithromycin and 1 withminocycline) no new infarcts were seen af-ter 7 days. (In these 7, a second daily agentwas added within 2 weeks, so that all werereceiving daily rifampin and daily clar-ithromycin or minocycline or dapsone.)

In two patients, followed for less than 4weeks, no judgement was made as to treat-ment response.

Of the two patients who developed Lucio’sphenomenon after initiating anti-microbialtreatment in our clinic, the one, Case 2, whodeveloped the reaction after discontinuingdapsone, responded without new lesions af-ter resumption of dapsone. The other, Case3, who developed Lucio’s phenomenon re-action after 7 months of daily dapsone andrifampin is a glaring exception to the usuallyfavorable outcome of microbicidal treat-ment. Her response to increased daily dosesof prednisone was good, with new lesionsceasing within 2 weeks, and no recurrencesin association with a slow tapering of theprednisone over 5 months.

Healing of ulcers. The ulcers usuallyhealed within 4 months, in 3 patients takingas long as 8 months, the length of time be-ing roughly proportional to ulcer size.

Follow up dataAs of December 31, 2004, 14 patients

were still being followed. Of the remainder,15 had been lost, and 1 was deceased after20 years of follow up. The length of followup has been as brief as less than 1 monthand as long as 35 years, mean 12.9 and me-dian 10. Five were followed for 1.3 years orless.

Erythema nodosum leprosumThirteen of the 30 patients, 7 women and

6 men, were known to have developedENL. In Case 2 ENL definitely precededthe onset of Lucio’s phenomenon. AlsoCase 4, who might have had mild ENL4 years prior to developing Lucio’s phe-nomenon, did develop typical ENL 37months after initiation of treatment withdaily rifampin and dapsone. ExceptingCase 2, the time of onset of the ENL rangedfrom 1 to 41 months after treatment wasstarted in our clinic, median 21 months, av-erage 22.1. Apart from this long mediantime of onset after initiation of treatment,the ENL in these patients did not differfrom the ENL seen in ordinary lepromatousleprosy. No patient had lesions of ENL atthe time of having new lesions of Lucio’sphenomenon.

Long term morbidityExcluding from analysis the 5 patients

followed for 1.3 years or less, some degreeof long-term morbidity has been experi-enced by 22 of the remaining 25 patients.Apart from ENL, the long-term morbidityobserved in these patients arose from threemechanisms. Two of these mechanisms, S-GPSI and venous insufficiency, appear to be a part of Latapi’s lepromatosis. Thethird mechanism producing long-term mor-bidity, scar formation, was the direct conse-quence of Lucio’s phenomenon. Secondaryto S-GPSI, 11 have experienced ongoingproblems with pathologic plantar callositiesand/or ulcers. Also secondary to S-GPSI, 5have physical impairment in the hands, in-cluding fissures, ulcerations, and bone re-sorption. Morbidity from muscle weakness


or contracture was rare, occurring only inCase 5, and consisted of soft tissue contrac-ture of the fascia and tendinous muscle inthe left knee complex. Recurrent leg ulcersfrom venous insufficiency have been an on-going problem for 10; 4 of these also hav-ing trophic problems in their feet. Scar for-mation led to hand and leg disabilities inone patient, Case 5.

Of the 14 who continue to be followed, 7have a disability grade 1 or 2 according tothe WHO grading system; 3 with a grade 1disability and 4 with a grade 2 disability.Those with grade 2 disability include onewith bilateral finger resorption, one withunilateral finger resorption, one with a re-solving plantar ulceration, and one (Case 5)with right hand partial amputation due to is-chemic changes.

Histologic changesHistologic material obtained from lesions

of Lucio’s phenomenon was available forreview in 22 patients. In 15 both H&E andadequately preserved Fite stained materialwas available, in 5 H&E only, and in 2 ade-quately preserved Fite stained tissue only.The common source of variation amongspecimens was the age of the lesion sampled.Most of the features have been described indetail and illustrated elsewhere (25, 26).

In all 22 patients the histologic materialdemonstrated foamy macrophages in thedermis in association with a few lympho-cytes. The volume of the dermis occupiedby the macrophages was small in 16 speci-mens, ranging from 2–10%, with a medianvalue of 5%. In the remaining 6, the volumeoccupied was larger, ranging from15–40%, with a median of 23%. In con-trast, in biopsy specimens obtained fromthe indurated, but not visible subcutaneousplaques present before the Lucio phenome-non in Cases 3 and 5, the volume of the in-filtrate occupied approximately 70 and 80%of the dermis, respectively. In their Lucio’sphenomenon lesions the volume of the der-mis occupied by macrophages was approx-imately 5–10% in both patients.

Epidermal necrosis was present in 18 andabsent in 2 but could not be evaluated in 2because of absent or insufficient epidermis.In the recent lesions, the necrotic epidermiswas of normal thickness but manifested theabsence of staining of nuclei, nuclear

ghosts, and early regeneration at the periph-ery, which was an epithelial tongue dissect-ing between the necrotic epidermis and thedermis. In older lesions, a new keratinizingepidermis was well developed, the necroticepidermis now located above the new stra-tum corneum, and identifiable by com-pacted nuclear ghosts in the former pricklecell layer and melanin in the former basalcell layer (Fig. 1d). Similarly, necrosis ofeccrine ducts and/or coils was present tosome degree in 16 and absent in 6. In theoldest lesions, the necrotic epidermis wasevidently exfoliated in the processing, andnecrotic eccrine structures had been ab-sorbed.

Intense passive congestion of vessels waspresent in 16 and absent in 4, but could notbe evaluated in 2. This was usually con-fined to the superficial dermis, but was pres-ent in the deep dermis or subcutis in 3. Ex-travasation of erythrocytes was present in12 and absent in 8, but could not be evalu-ated in 2.

In the medium sized vessels of the dermisand subcutis, endothelial proliferation wasidentified in 20, but could not present in 2.The proliferation ranged from mild to se-vere, frequently producing luminal occlu-sion, and was associated with thrombosis in7. Inflammatory cells with in these vesselswere few in number, suggesting that theterm “vasculosis” would be better than“vasculitis.” The generally sparse inflam-matory infiltrate was primarily lympho-cytic. Neutrophils or neutrophilic dust wereidentified in 6 of 22 specimens, but werenot associated with vascular changes, butinstead were infiltrating necrotic areas in 5,and in the subcutis in 1. Fibrinoid changewas present in 1 specimen.

Subcutaneous tissue was present in spec-imens from 18 patients. The area occupiedby the subcutis was estimated to be 20% orless than that of the dermis in 5, 20–50% ofthat of the dermis in 6, and 50% or more ofthat of the dermis in 7, respectively. All 18specimens had some degree of a lobularpanniculitis. This was considered to be fo-cal in 9, involving an estimated 15% or lessof the panniculus, moderate in 5, involving20–40%, and extensive in 4, involving50–80%. The nature of the infiltrate variedconsiderably, 8 with leprotic macrophagesand only a few lymphocytes, 9 with an ob-


vious mixture of leprotic macrophages andlymphocytes, and 1 with leprotic macro-phages and neutrophils, the latter infiltrat-ing between the lipocytes, apparently ignor-ing the vessels.

Concerning specimens with adequatelypreserved Fite stains, AFB and globi werefound in macrophages in the dermis in all17 specimens, as well as in the macro-phages in the subcutis of the 15 specimensso endowed. In all 15 specimens with bothendothelial proliferation and a Fite stain,bacilli in some of the proliferating endothe-lial cells were readily identified in all but 1,often with globi. Similarly, in all 17 speci-mens with a Fite stain, bacilli could befound in some non-proliferating endothelialcells. Bacilli were most difficult to find inendothelial cells in Case 3, probably theconsequence of 7 months of continuous an-timicrobial treatment

Histologic changes in large vessels, orvessels made large by pathologic changes,which we have chosen to call L-GV, werepresent in a total of 6 of the 22 (27%) pa-tients with Lucio’s phenomenon. In 2,Cases 3 and 5, the large vessel changeswere observed only in non-necrotic speci-mens, obtained before the onset of Lucio’sphenomenon. The fully developed L-GVconsisted of endothelial proliferation,macrophages infiltrating the media, andmacrophages infiltrating the adventitia.This fully developed change was found inCase 3, (Fig. 1a–c), and in lesions from 2other Lucio’s phenomenon patients. In oneof these latter 2, the changes were active,(Fig. 4b, and in the other the changes wereconsidered to be regressing (Fig. 4c). In-complete expression of the L-GV consistedof endothelial proliferation with a variabledegree of adventitial infiltration, as exem-plified by Fig. 4a, also found in Figs. 2a and 3a. A similar L-GV was found in 6 of70 (9%) of histologic specimens obtainedfrom lesions of ENL (Fig. 4d), and as anincipient change in 3 of 51 (6%) non-reactional lepromatous patients (data notshown). These incipient changes werefound in comparatively large subcutaneousvessels located near the dermis, and con-sisted of foci of endothelial proliferation inwhich AFB were identified, whereas nonewere found in non-proliferating endothe-lium. In Lucio-Latapi disease, ENL, and

non-reactional lepromatous leprosy, thevessels involved with L-GV were located inthe subcutis, or what was subcutis prior tolepromatous infiltration or connective tissueproliferation. The L-GV involved vesselswere largest in the Lucio-Latapi patients,smallest in the non-reactional lepromatousmaterial, and of intermediate size in speci-mens of ENL.

DISCUSSIONThe findings in the additional 20 patients

with Lucio’s phenomenon are in good ac-cord with the initial report of 10 such pa-tients (25) from this clinic. Hence the 30 pa-tients have been taken together in this re-port. The additional 20 patients and thefollow-up information add detail to thevariations in the clinical picture without al-tering its broad outlines. The one exceptionto this accord is the finding of large vesselinvolvement in the subcutis, where it wasnot found in a previous report (26). This fail-ure was not due to a lack of looking, butwas probably the result of inadequate sam-pling of the subcutis with the 4mm in diam-eter punch biopsy instruments, then in rou-tine use. This contrasts to the more gener-ous amounts, and greater depths, obtainedwith the 6mm punches, in common use inour clinic for the past 2 decades, as exem-plified by Fig. 1a.

What is being called L-GV is not a newpattern. For example, this pattern has beenpreviously observed in non-reactional lepro-matous specimens (7), as well as in specimensof ENL (17, 27). Also it has been observed andillustrated in Lucio’s phenomenon (10), andwas alluded to by Latapi and Zamora (15).The pattern is similar to, if not the same as,that of the “leprous phlebitis” reported byMukherjee and his associates (19, 20).

L-GV occurs in larger vessels, or in ves-sels made large by pathologic changes, pri-marily in the subcutis. L-GV, and what isinterpreted as its variants, was present in 4of the specimens from 22 Lucio’s phenom-enon patients with material available for re-view, and in 2 of the 3 pre-Lucio’s phenom-enon specimens available for review. How-ever, the importance of L-GV to thepathogenesis of Lucio infarcts, if any, is notknown.

An argument can be made to support thehypothesis that the L-GV is not important


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FIG. 4 (a). From Case 9, a Lucio’s phenomenon lesion. An obliquely sectioned, subcutaneous vessel show-ing extensive endothelial proliferation, and heavy adventitial infiltration, but little disturbance in the smooth mus-cle bundles. 20× objective. b) A panvasculitic vessel from the subcutis of a Lucio’s phenomenon lesion showingintimal proliferation, a chaotic infiltration of macrophages among the smooth muscle bundles, and infiltration inthe adventitia. c) A high power view of a large subcutaneous vessel which shows modest endothelial change,


to the pathogenesis of Lucio’s phenome-non. Because the pattern of L-GV also hasbeen seen in lesions of ENL (17, 27) (Fig. 4d),as well as in non-reactional lepromatousleprosy (7), and because it has similarities tothe leprous phlebitis reported by others (19,

20) it is not specific for either Latapi’s lepro-matosis or the Lucio reaction. Also, becauseits presence in Cases 3 and 5 were not di-rectly associated with necrotic lesions, it isnot a change necessarily leading to necrosis.In this context L-GV is most easily inter-preted as part of the vascular changesknown to be associated with leprosy, fromlarge muscular arteries, to arterioles, to cap-illaries, to venules, and on to large veins.Such changes have been reported by, amongothers, Fite (12), Coruh and McDougall (7),Kaur, et al. (14), Bansai, et al. (5), andMukherjee and his associates (19, 20).

An argument can be made to support thecontrary hypothesis of importance for L-GVin pathogenesis. Anoxia is a final cause oftissue necrosis, however diverse the respon-sible mechanisms. In patients with Lucio-Latapi disease, anatomical changes with thepotential of leading to anoxia have beendemonstrated at three levels of the vasculartree. Best described is the endothelial pro-liferation with lumen occlusion, with orwithout thrombosis formation, occurring inthe mid-sized vessels in the dermis of thenecrotic lesions (25, 26, 31), as confirmed in thepresent study. Also, swelling and parasitiza-tion, with lumen occlusion, of capillary en-dothelium has been found by electron mi-croscopy in 3 of 3 specimens of clinicallynormal skin (33), i.e., Latapi’s lepromatosis,from patients with Lucio’s phenomenon,and could well be widespread. The subcu-ticular L-GV found in the present study inboth Lucio’s phenomenon and Latapi’s lep-romatosis is a third anatomical change thatcould contribute to anoxia; this change, iffocal, could also be more prevalent than hasbeen observed. All of these changes actingsynchronously might result in an ischemia

sufficient to produce necrosis, whereas anyone by itself would be less likely do so. Inaddition, the circulating immune complexesassociated with Lucio’s phenomenon (23)might interact with the anatomical changesin ways that lead to necrosis.

Perhaps the changes of the L-GV are bet-ter developed Latapi’s lepromatosis, be-cause this difficult to diagnose lepromatouscondition may give more time for bacterialproliferation and for a granulomatous vas-culitis to develop.

Three retrospective findings were not an-ticipated, but emerged only when the com-paratively large numbers of patients re-ported here were gathered together. 1) Thenear parity of the genders is in contrast tothe expected male preponderance, 2 to 1, inlepromatous disease (21). 2) The absence ofS-GPSI in 7 patients was not anticipated tobe this common in Latapi’s lepromatosis.3) The median time of onset of ENL, 21months after initiating treatment, appears todiffer considerably, and perhaps signifi-cantly, from the 12 months median time ob-served in this clinic (unpublished data).Good explanations for these three unex-pected findings are not available.

Clinically, the ischemic infarcts of Lu-cio’s phenomenon were of a uniform char-acter, varying in size and extent, but alwaysof the same kind, or, in other words, amonomorphic response. Accompanying ul-cers, erosions and bullae, were clearly sec-ondary to the infarct.

The report by Diogenes, et al. (9) and ourexperience with Case 2, raises the possibil-ity of the diagnosis of Latapi’s lepromatosisin the absence of Lucio’s phenomenon.Usually a diagnosis of Latapi’s lepromato-sis is made after the fact of Lucio’s phe-nomenon. Which findings or combinationof findings might be considered as criteriafor a diagnosis of Latapi’s lepromatosis inthe absence of Lucio’s phenomenon?

Either of two findings could be regardedas a sine qua none for this diagnosis. Dif-


←spotty infiltration in the wall, and involvement of the adventitia. The process appears to be old and regressing.This impression is supported by the absence of a defined muscular layer, and by the several lines of “hash marks”at different levels in the wall, suggesting repeated reduplication of the internal elastic lamina, (block not avail-able for definitive staining). 40× objective. d) A high power view of a subcutaneous vessel in an ENL lesion, giv-ing an exemplary demonstration of the features of lepromatous-granulomatous vasculitis (L-GV), that is, en-dothelial proliferation, infiltration of macrophages between smooth muscle bundles, and adventitial infiltration.20× objective.

fuse non-nodular infiltration is one; theother is heavy endothelial parasitization byM. leprae. Neither finding is specific forLatapi’s lepromatosis, but the presence ofdermal nodules or the absence of endothe-lial parasitization virtually excludes thepossibility of Latapi’s lepromatosis.

Three other distinct findings could be re-garded as highly suggestive of Latapi’s lep-romatosis. These are 1) telangiectasias, ei-ther eruptive or as mats on the face andchest, 2) palpable but not visible subcuta-neous plaques, and 3) as manifestations ofdiffuse infiltration, widening of the nasalroot, poorly defined induration of the facialcheeks, with or without erythema, andswelling of the backs of the hands.

In the presence of the two “sine quanon’s” other common changes could be re-garded as supportive of a diagnosis of Lat-api’s lepromatosis. These include completeeyebrow and or eyelash alopecia, nasal sep-tum perforation, and significant S-GPSIwith little motor change.

In lepromatous patients who presentwithout nodular change and who haveheavy parasitization of endothelial cells, (inour experience those who present withspontaneous ENL (24)), findings whichpoint away from a diagnosis of Latapi’s lep-romatosis include normal eyebrows andeyelashes, mild or absent rhinitis, and ocu-lar involvement.

Five biopsy specimens obtained beforethe onset of Lucio’s phenomenon were ex-amined by one of us, three being availablefor review. In all four with a Fite stain, en-dothelial parasitization by M. leprae wasevident. As demonstrated by hematoxylinand eosin, three histologic patterns wereidentified, and each pattern could be relatedto the clinical findings at the biopsy site. InCases 1 and 2, where the specimen chosenwas from clinically normal skin, the infil-trate was scant, and with little if any otherinflammatory change, aptly described as“apparently normal.” In Case 4, with theclinical findings of erythematous nodules, adistinct lymphocytic infiltrate was associ-ated with foamy macrophages. In cases 3and 5, the specimens being obtained fromnot visible, non-tender, indurated subcuta-neous plaques, well developed vascularchanges were associated with a heavy infil-trate of macrophages.

Two of the 3 pre-Lucio specimens avail-able for review, from cases 3 and 5, demon-strated endothelial proliferation with lumenocclusion, and were obtained 7 and 5months, respectively, before the onset onLucio’s phenomenon. In the third specimen,obtained 4 years before the onset of the Lu-cio’s phenomenon, no such vascular changewas evident.

The most conspicuous disagreement inthe literature concerning Lucio’s phenome-non is in regard to its histologic pattern,leukocytoclastic vasculitis (LCV): LCV yes(2, 18, 31) or LCV no (10, 22, 25)? The review ofour biopsy material is in accord with ourprevious conclusion that the histologic pat-tern is not that of LCV (25). The most likelyexplanation for the disagreement is differ-ing criteria for what constitutes LCV. An-other possibility is the intellectual difficultyin dissociating or uncoupling the idea of aputatively immune complex disorder ofskin (23) from the histologic pattern of LCV.The histologic pattern called LCV is thatfound in “palpable purpura” and is the samewhatever disease may be producing the le-sions of “palpable purpura.” The lesions of“palpable purpura” were not found in anyof these 30 patients.

Clinically, a variety of septic infarcts andthrombotic syndromes (4) may mimic theinfarct of Lucio’s phenomenon, beinghemorrhagic and having serrated borders.In addition, one recent case report givesstrong evidence that other vasculitic condi-tions may closely mimic Lucio’s phenome-non. Tang and Yosipovitch (32), in their re-port of an acute Churg-Strauss syndrome,have in their clinical photograph (their fig.1) illustrated changes perfectly consistentwith the serrated, hemorrhagic infarcts ofthe Lucio reaction. In the same report is aphotomicrograph (their fig. 2) which showsextravasation of erythrocytes, and in our in-terpretation, congestion of superficial ves-sels, and a necrotic epidermis, identified inthe legend as “scale crust,” a pattern look-ing much like our Fig. 1d. Viewed in thisperspective, the second episode of infarc-tions in Case 9, would be best regarded asbeing a Lucio’s phenomenon-like tissue re-sponse of unknown cause, not a true Lu-cio’s phenomenon.

Comfort may be taken from this series,where by the time of development of


Lucio’s phenomenon, overt clinical signs oflepromatous leprosy, without exception,were present. Conversely, anxiety may arisefrom this series, where, in some cases, thediagnosis of leprosy was not made until theLucio phenomenon occurred, even thoughpreceding characteristic signs and symp-toms, although seen and heard by physi-cians, were not interpreted as suggestingthe possibility of leprosy.

Acknowledgment. Many physicians have pro-vided help in making this report possible. For access toclinical data and histologic material on some of the pa-tients, the authors are indebted to Drs. Lewis Bowman,Randy Burke, and Keith Carlson. Dr. Nancy Warnergave expert help with photomicrographs. Dr John T.Crissey provided valuable advice and printed the pho-tographs in “black and white.” Dr. Claudia Renn trans-lated reference number 1.

REFERENCES1. ALMEIDA, H. L., JANNKE, H. A., RIVITTI, E. A., MI-

CARETTA, S., and CASTRO, S. N. PostinfektiosesLucio-phanomen bei diffuser lepra. Hautarzt 51(2000) 945–949.

2. ANG, P., YONG-KWANG, T., SEE-KET, N., andCHEW-SWEEN, S. Fatal Lucio’s phenomenon in 2patients with previously undiagnosed leprosy. J.Am. Acad. Dermatol. 48 (2003) 958–961.

3. ARNOLD, H. L., and SLOAN, N. R. Lucio’s spottedleprosy (diffuse lepromatous leprosy of Mexico):report of a case in Hawaii. Int. J. Lepr. Other My-cobact. Dis. 19 (1951) 23–27.

4. BAKOS, L., CORREA, C., BERGMANN, C.,BONAMIGO, R. R., and MULLER, L. F. B. An-tiphospholipid antibodies throbotic syndrome mis-diagnosed as Lucio’s phenomenon. Int. J. Lepr.Other Mycobact. Dis. 64 (1996) 320–323.

5. BANSAI, R., KAUR, S., KUMAR, B., SHARMA, V. K.,KATARIYA, S., CHAKRAVARTI, R. N., and BUSHAR-NAMATH, S. R. Venous involvement in leprosy: avenogrophic and histopathological correlation, Int.J. Lepr. Other Mycobact. Dis. 55 (1987) 499–506.

6. BERNADAT, J. P., FAUCHER, J. F., and HUERRE, M.Diffuse lepromatous leprosy disclosed by cuta-neous vasculitids. Ann. Dermatol. Venereol.. 123(1996) 21–23.

7. CORUH, G., and MCDOUGALL, A. C. Untreatedlepromatous leprosy: histopathological finding, incutaneous blood vessels. Int. J. Lepr. Other My-cobact. Dis. 43 (1979) 500–511.

8. DERBES, V. J., SAMUELS, M., WILLIAMS, O. P., andWALSH, J. J. Diffuse leprosy: case in a Louisiananegro. Arch. Dermatol. 81 (1960) 210–224.

9. DIOGENES, M. J. N., DE MORALES, R. M., S. DE

TOME, G., DA CUNHA, M. B., and DE C. B. NETO,C. The Lucio-Alvarado-Latapi form of LeprosyLep. Rev. 72 (2001) 360–362.

10. DONNER, R. S., and SHIRELY, J. A. The “Luciophenomenon” in diffuse leprosy. Ann. Iutern.Med. 67 (1967) 831–836.

11. DROSOS, A. A., BRENNAN, P. J., ELISAF, M. S., STE-FANOU, S. G., PEPADIMITRIOU, C. S., and MOUT-SOPAOULOS, H.M. Specific antigen and antibodyto Mycobacterium leprae in the cryoprecipitate ofa patient with Lucio phenomenon. Rheumatol. Int.6 (1986) 93–94.

12. FITE, G. L. The vascular lesions of leprosy. Int. J.Lepr. Other Mycobact. Dis. 9 (1941) 193–202.

13. KAMP, H., LEIKER, D. L., and FRENKEN, J. H. Therelationship between the Lucio phenomenon andcutaneous allergic vasculitis (Ruiter). Int. J. Lepr.Other Mycobact. Dis. 30 (1962) 138–151.

14. KAUR, S., WAHI, P. L., CHAKRAVARTI, R. N., SODHI,J. S., VADHWA, M. B., and KHERA, A. S. Periph-eral vascular defect in leprosy. Int. J. Lepr. OtherMycobact. Dis. 44 (1976) 332–339.

15. LATAPI, F., and ZAMORA, A. C. The “spotted”leprosy of Lucio ( la lepra “manchada” de Lucio).Int. J. Lepr. Other Mycobact. Dis. 16 (1948)421–437.

16. LUCIO, R., and ALVARADO, Y. Opusculo sobre elmal de San Lazaro o elefanciasis de los Griegos.M. Murguia y Cia, Mexico, 1852, 53 pp.

17. MABALAY, M. C., HELWIG, E. B., TOLENTINO, J. G.,and BINFORD, C. H. The histopathology andhistochemistry of erythema nodosum leprosum.Int. J. Lepr. Other Mycobact. Dis. 33 (1965)28–49.

18. MOSCHELLA, S. L. The lepra reaction with necro-tizing skin lesians: A report of six cases. Arch.Dermatol. 95 (1967) 565–575.

19. MUKHERJEE, A., GIRDHAR, B. K., and DESIKAN, K.V. Leprous Phlebitis. Int. J. Lepr. Other My-cobact. Dis. 48 (1980) 48–50.

20. MUKHERJEE, A., GIRDHAR, B. K., MALVIYA, G. N.,RAMU, G., and DESIKAN, K. V. Involvement ofsubcutaneous veins in lepromatous leprosy. Int. J.Lepr. Other Mycobact. Dis. 51 (1983) 1–6.

21. NEWELL, K. W. An epidemiologist’s view of lep-rosy. Bull. WHO 37 (1966) 827–857.

22. PURSELEY, T. V., JACOBSEN, R. R., and APISARN-THANARAX, P. Lucio’s phenomenon. Arch. Der-matol. 116 (1980) 201–204.

23. QUISMORIO, F. P., REA, T., CHANDOR, S., LEVAN,N., and FRIEU, G. Lucio’s phenomenon: and im-mune complex deposition syndrome in leproma-tous leprosy. Clin. Immunol. Immunopathol. 9(1978) 184–193.

24. REA, T. H., and LEVAN, N. E. Erythema nodosumleprosum in a general hospital. Arch. Dermatol.111 (1975) 1575–1580.

25. REA, T. H., and LEVAN, N. E. Lucio’s phenome-non and diffuse non-nodular lepromatous leprosy.Arch. Dermatol. 114 (1978) 1023–1028.

26. REA, T. H., and RIDLEY, D. S. Lucio phenomenon:A comparative histologic study. Int. J. Lepr. 47(1979) 161–166.


27. RIDLEY, D. S., REA, T. H., and MCADAM, K. P. W.J. The histology of erythema nodosum leprosum.Variant forms in New Guineans and other ethicgroups. Lepr. Rev. 52 (198)1 65–78.

28. ROMERO, A., IBARRA, A. B., and FALLAS, M. Clini-cal study of lepromatous leprosy in Costa Rica. Int.J. Lepr. Other Mycobact. Dis. 17 (1949) 27–33.

29. SAOJI, V., and SALODHAR, A. Lucio leprosy withlucio phenonenon. Indian J. Lepr. 73 (2001)267–272.

30. SHESKIN, J. Diffuse lepromatosis of the Lucio-Alvarado-Latapi with Lucio phenomenon: first casein the Near East. Rev. Leprol. 13 (1982) 651–656.

31. SOUZA, C. S., ROSELINO, A. M., FIGUEIREDO, F.,and FOSS, N. T. Lucio’s phenomenon:clinical andtherapeutic aspects Int. J. Lepr. Other Mycobact.Dis. 68 (2000) 417–25.

32. TANG, M. B. Y., and YOSIPOVITCH, G. AcuteChurg-Strauss Syndrome in an asthmatic patientreceiving montelukast therapy. Arch. Dermatol.139 (2003) 715–717.

33. TURKEL, S. B., VAN HALE, H. M., and REA, T. H.Ultrastructure in leprosy. Int. J. Lepr. Other My-cobact. Dis. 50 (1982) 164–171.

34. WONG, W. S. Leprosy presenting as modular vas-culitis. (Letter). Brit. J. Rheumatol. 26 (1987) 398.


1 Received for publication on Dec. 13, 2004. Accepted for publication on May 20, 2005.2 N. L. Sharma, M.D.; V. K. Mahajan, M.D.; V. C. Sharma, M.D.; S. Sarin, M.B.B.S.; R. C. Sharma, M.D. De-

partment of Dermatology, Venereology & Leprosy, Indira Gandhi Medical College, Shimla, India.Reprint requests to: Dr. N . L . Sharma, Department of Dermatology, Venereology and Leprosy, Indira Gandhi

Medical College, Shimla 171001 (H.P.), India; e-mail: [email protected]

Erythema Nodosum Leprosum and HIV Infection:

A Therapeutic Experience1

Nand Lal Sharma, Vikram K. Mahajan, Vikas C. Sharma, Sandip Sarin, and Ramesh Chander Sharma2

ABSTRACTThe relationship between leprosy and HIV infection is not yet fully understood, as not

much is known about the natural history of the co-infected patients. The matter has becomemore confusing because of conflicting reports. Type-1 lepra reactions and neuritis appear tobe severe and more frequent among them. But erythema nodosum leprosum too is not as un-common among these patients as it was once thought.

Management of these co-infected patients is often difficult for want of clear-cut guide-lines on clinical care. We report here our experience of treating recurrent, severe erythemanodosum leprosum in a patient concurrently having leprosy and HIV infection. Early insti-tution of antiretroviral therapy appears to provide an edge in improving the therapeuticoutcome for him. It also suggests a direct and more complex interplay of HIV and Myco-bacterium leprae infection.

RÉSUMÉLa relation entre la lèpre et l’infection par le VIH n’est pas encore complètement com-

prise, comme peu de choses sont connues sur l’histoire des patients co-infectés. Le sujet estdevenu d’autant plus confus que des rapports contradictoires ont été publiés. Les réactionslépreuses de type 1 et les névrites seraient plus sévère et fréquentes parmi les patients co-infectés. Mais l’érythème noueux lépreux n’est pas aussi rare parmi ces patients que ce quiavait été originellement considéré.

La prise en charge clinique de ces patients co-infectés est souvent difficile, car ellemanque de recommandations claires pour le traitement. Nous rapportons ici notre expéri-ence à traiter un érythème noueux lépreux récurrent et sévère chez un patient souffrant con-comitamment de lèpre et d’infection par le VIH. La mise en ?uvre rapide d’une thérapieanti-rétrovirus semble avoir joué un rôle pivot dans l’amélioration du résultat thérapeutiquechez ce patient. Cela suggère une interaction directe et plus complexe entre les infections parMycobacterium leprae et le VIH.

RESUMENLa relación entre la lepra y la infección por el VIH no está completamente entendida,

como tampoco se conoce mucho acerca de la historia natural de los pacientes co-infectados.El tema ha llegado a ser todavía más confuso debido a la publicación de reportes conflic-tivos. Las reacciones tipo-1 de la lepra y la neuritis parecen ser graves y muy frecuentes en-tre los pacientes co-infectados pero el eritema nodoso leproso no es tan raro como antes secreía.

El manejo de estos pacientes co-infectados a menudo es difícil debido a la ausencia delineamientos claros sobre su tratamiento y cuidado clínico. Aquí, nosotros reportamos nues-tra experiencia sobre el tratamiento recurrente de eritema nodos leproso grave en un pacientecon lepra co-infectado por el VIH. La administración temprana de la terapia anti-retroviralfavoreció el resultado de la terapia general en el paciente. Se analiza el caso y se reconoceel interjuego directo y muy complejo entre el VIH y la infección por M. leprae.

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The relationship between leprosy andHIV infection remains obscure due to con-flicting reports appearing over a period oftime. By analogy with the development ofactive tuberculosis and other mycobacterialinfections among HIV positive patients, anincreased prevalence of leprosy was ex-pected, particularly towards lepromatousspectrum, and possibly also the prevalenceof erythema nodosum leprosum (ENL) re-action, in areas where both leprosy and HIVare endemic. Earlier literature is also repletewith reports on increased frequency ofType-1 lepra (reversal) reactions, severeneuritis, poor therapeutic outcome and re-lapses among HIV infected leprosy patients(5, 9, 23), without reports on ENL reaction.More recent data, however, show that HIVinfection has no significant effect on epi-demiology, clinical, histological, andimmunological spectrum of leprosy (4). Re-ports on ENL among HIV positive leprosypatients, too, have started to appear (13, 14).Studies on granuloma formation and im-mune patterns in co-infected patients revealno greater risk for development of multi-bacillary (MB) leprosy or ENL and, rathercontrary to expectations, a satisfactory re-sponse to antileprosy treatment has beenrecorded (13).

Due to lack of information on the naturalhistory of co-infected patients and the ab-sence of guidelines for the management ofsuch cases, it is often a challenge to the in-genuity of the treating clinician. We reporthere our experience treating recurrent, se-vere ENL in a leprosy patient concurrentlyhaving HIV infection without HIV-relatedclinical disease.

Case Report. A 30-year-old male washospitalized with recurrent episodes of mul-tiple, erythematous, painful, widely spreadcutaneous lesions of 4-month duration.Each episode was accompanied by fever,malaise, body aches, arthralgia, and ankleedema. Some of these lesions had also de-veloped into necrotic crusted ulcers. Antibi-otics and anti-inflammatory drugs wouldtemporarily improve his condition. He alsoreported promiscuous sexual behavior. Hewas febrile (102°F). Dermatological exam-ination showed diffuse infiltration, numer-ous, erythematous, tender papulo-nodularlesions involving the whole body except forpalms, soles, and scalp. Some of them

showed central necrotic sloughing andcrusting. Atrophic scars of previouslyhealed lesions were also noted. His conjuc-tivae were congested. Hair, nails, andoropharynx were normal. He had no signif-icant lymphadenopathy. Asymmetric, ten-der thickening of all peripheral nerve trunksalong with corresponding hypoesthesia wasnoted over hands and feet. Slit-skin smearexamination from 5 sites (World HealthOrganization, W.H.O.) showed 6+ BI. Oph-thalmic, CNS, CVS, pulmonary and ab-dominal examination revealed no abnor-mality and there was no historical orclinical evidence of opportunistic infec-tions. The erythrocyte sedimentation ratewas 50 mm in first hour and other labora-tory studies including complete bloodcounts, hepato-renal function tests, urinaly-sis, chest radiograph, VDRL and Tre-ponema pallidum haemagglutination testsshowed no abnormality. He was HIV posi-tive by ACON rapid card chromatographicimmunoassay (ACON Biotech HangzhouCo. Ltd., China), Capillus direct latex ag-glutination assay (Trinity Biotech U.S.A.,N.Y. 14702-1059) and Genedia HIV ELISAtest (Greencross Life Sciences Corp., Ko-rea). His CD4+ and CD8+ cell counts were798 and 1541 cells/microl, respectively, us-ing the Fluorescence Activated Cell Sortercounting system (Beckton Dickenson Im-munocytometry Systems, California, U.S.A.),and the CD4 : CD8 ratio was 0.52 (NormalRanges = 865 CD4+, 552 CD8+ cells/microland CD4 : CD8 ratio 1.7) (16). He did notconsent for biopsy and could not afford thecost of viral load studies.

Clinical progress. The clinical diagnosiswas lepromatous leprosy with recurrent,severe ENL and HIV infection, and the pa-tient was initially given W.H.O. MB multi-drug therapy (M.D.T.) along with pred-nisolone 60 mg/d, ibuprofen 400 mg t.i.d.and colchicine 0.5 mg b.i.d. Over the next 2weeks, healing of lesions and symptomaticimprovement occurred without recurrences.Subsequently, when the dose of pred-nisolone was tapered off to 40 mg/d, freshENL lesions, ulnar nerve neuritis (withoutsensory motor deterioration) and systemicsymptoms reappeared. He did not improvein spite of increasing the dose of pred-nisolone to 80 mg/d. At this juncturecolchicine was stopped and thalidomide 100

73, 3 Sharma, et al.: ENL and HIV 191

mg b.i.d. was added with the plan to taperoff prednisolone once the remission wasachieved. The ENL and nerve tendernessdecreased within a week and tapering ofprednisolone was started. All the while, thepatient continued to receive MB-M.D.T.,thalidomide 100 mg b.i.d. and other sup-portive treatment.

After 3 weeks of thalidomide therapy,while still on prednisolone (30 mg/d), hedeveloped dryness of mucosae and a gener-alized, pruritic, erythematous, diffuse (dis-crete at places) maculo-papular rash. Initialwithdrawal of MB-M.D.T. did not improvethe rash. The rash, however, subsided afterstopping thalidomide. The dose of pred-nisolone was increased to 60 mg/day imme-diately upon recurrence of ENL. The casewas reviewed and in view of apparentcushingoid features, as well as poor controlof ENL, it was decided to add anti-retroviraltreatment (ART) comprising stavudine 30mg, lamivudine 150 mg and nevirapine 200mg, all in twice daily doses (the only avail-able and affordable regimen), to the alreadyexisting regimen of MB-MDT and oralprednisolone.

His general condition improved, recur-rences of ENL stopped and dose of pred-nisolone could be reduced to 30 mg/d innext 10 days when the patient left the hos-pital on his own. On a subsequent visit aftera month, he was free of ENL, continuingMB-M.D.T. and ART but had stopped pred-nisolone. However, he was lost to furtherfollow-up.

DISCUSSIONAs yet there is conflicting and inadequate

information on the interactions of HIV andleprosy co-infection. HIV infection wasthought to decrease the risk of ENL untilrecently when reports of ENL among co-infected patients started to appear albeit in-frequently. This is contrary to increased fre-quency of Type-1 lepra reactions andneuritis in co-infected individuals. Nery, etal. (13) observed no enhanced risk of ENLamong their patients. In contrast, Gebre, etal. (6) recorded a definite higher risk (rela-tive risk: 5.2; 95% CI 1.7–15.9) of ENL re-actions in a prospective study comprising22 HIV positive leprosy patients. Our pa-tient had severe, recurrent ENL reactionwith necrotic lesions. Considering that lep-

rosy has a much longer incubation periodthe HIV infection in this patient appears asubsequent development. Possibly, likeother intercurrent infections, it acted as atrigger for ENL reaction. The ENL also ap-pears to be severe and recurrent in thisgroup of patients similar to Type-1 leprareactions.

Among HIV seropositive patients neuritisalways occurs in association with skin man-ifestations suggesting that nerve dysfunctionis due to Type-1 lepra reaction. HIV isneurotropic and may cause necrotizing vas-culitis of the nerves (15). Possibly the inter-action of neurotropicity of both Mycobacte-rium leprae and HIV may result inneuropathy that is severe and unresponsiveto steroid therapy (5). Vreeburg, et al. (24)also noted that though neuritis is equallycommon in both HIV positive and HIVnegative patients, the therapeutic outcomewith steroids was poorer in the HIV posi-tive group. Similarly, HIV-induced vascu-lopathy might aggravate immune complexmediated vasculitis/paniniculitis of ENLthat responds poorly to steroid therapy ashas been observed in our patient.

Thalidomide, 100–400 mg/d, is currentlythe recommended drug for recurrent, mod-erate to severe ENL reactions. Its use hasbeen associated with normalizing effects onTNF, IFN, and helper-suppressor T-cell ratio(20), decreases in dermal infiltration of poly-morphonuclear leukocytes and T-cells, anddown regulation of the expression ofICAM-1 and MHC-1 antigens on epidermalkeratinocytes (20). Its exact mechanism ofaction in ENL, however, remains unclear.Recently it has also been reported to pro-duce an anti-retroviral effect without anynegative effect on immunocompetence (21),possibly through inhibition of TNF produc-tion and by blocking TNF stimulated HIVreplication (18). Due to these properties itappears to be the drug of choice for treatingENL among HIV infected leprosy patients.However, in view of other reports of in-creased HIV viral counts caused by thisdrug it should be used with caution for thesepatients until further studies are available(17). Apart from its well known teratogeniceffects other common adverse reactions likeperipheral neuropathy, somnolence andconstipation limit its routine use. Hypersen-sitivity skin rash is uncommon and usually


appears 2 to 10 days after treatment andsubsides after its withdrawal. Thalidomidewas effective in our patient also. However,he developed a generalized cutaneous rashin spite of simultaneously receiving 30mg/d prednisolone. Curiously these patientsseem to tolerate the drug poorly and thisphenomenon has also been previously cor-related with lower numbers of pre-existingCD4+ cells (21).

The predominance of CD8+ cells in lep-romatous lesions as compared to predomi-nance of CD4+ cells in the typical granulo-matous response seen in tuberculoid lesions(13) and the presence of ICAM 1, HLA DR,TNF, IFN at lesional sites suggests no dif-ference in immune response in both HIVpositive and negative leprosy patients (19,

20). The tissue cellular immunity (CMI)against M. leprae appears well preserved ir-respective of low CD4 + cell counts in theperipheral blood of HIV infected patients orthe stage of HIV infection (13). The exactpathologic mechanism of ENL among theseco-infected patients is, however, not fullyunderstood. There is indirect evidence con-sistent with increased CD4+ lymphocytesactivity in ENL (11, 23). The relative lack ofENL (immune complex mediated) reactionas compared to reversal (cell mediated) re-actions among HIV positive lepromatouscases and no therapeutic effect of thalido-mide in reversal reactions, that acts throughsuppression of helper T-cells activity (12),suggests some kind of involvement ofCD4+ lymphocytes in ENL. Furthermore,the loss of lesional CD4+ cell function maynot be complete as is postulated in tubercu-loid leprosy lesions in HIV infected patients(19). It is also known that CD4+ lympho-cytes become depleted as the HIV diseaseprogresses and cytotoxic CD8+ lympho-cytes, including various subsets, increasesignificantly (6). Findings such as these mayeventually help to explain the occurrencesof ENL among these patients. Furthermore,in the early stages of HIV infection somedegree of immunocompetence is retainedand the occurrence of ENL in early HIV in-fection may not be unusual as has been thecase in our patient.

In our patient, despite an early HIV infec-tion and near normal CD4+/CD8+ counts(the low CD4+ : CD8+ ratio is apparently anartifact of high CD8+ counts), the severe

ENL reaction showed poor control evenwith higher doses of corticosteroids. Addi-tion of ART not only improved the thera-peutic response to lower doses of steroidsbut also helped in their complete withdrawalsubsequently. We make no attempt to spec-ulate about the mechanisms underlying ourobservation. However, clinicians must bearin mind the possibility of precipitatingType-1 lepra reactions during ART/HAARTdue to immune reconstitution (8).

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immunologic and virologic impact of glucocorti-coids on the chronic phase of HIV infection. BMCMed. 2 (2004) 17.

2. ANDRIEU, J. M., and LU, W. Viro-immunopatho-genesis of HIV disease: implications for therapy.Immunol. Today 16 (1995) 5–7.

3. BARNES, P. J. Anti-inflammatory actions of gluco-corticoids: molecular mechanisms. Clinical Sci.94 (1998) 557–572.

4. BELLIAPPA, A. D., BHAT, R. M., and MARTIS, J.Leprosy in type-1 reaction and diabetes mellitus ina patient with HIV infection. Int. J. Dermatol. 41(2002) 694–695.

5. BWIRE, R., and KAWUMA, H. J. S. Type 1 reactionsin leprosy, neuritis and steroid therapy: the impactof the human immunodeficiency virus. Trans. R.Soc. Trop. Med. Hyg. 88 (1994) 315–316.

6. GEBRE, S., SAUNDERSON, P., MESSELE, T., andBYASS, P. The effect of HIV status on the clinicalpicture of Leprosy : a prospective study inEthiopia. Lepr. Rev. 71 (2000) 338–343.

7. GROSSMAN, Z., MEIER–SCHELLERSHEIM, M.,SOUSA, A. E., VICTORINO, R. M., and PAUL, W. E.CD4+ T-cell depletion in HIV infection: are wecloser to understanding the cause? Nat. Med. 8(2002) 319–323.

8. LAWN, S. D., WOOD, C., and LOCKWOOD, D. N.Borderline tuberculoid leprosy: an immune recon-stitution phenomenon in a human immunodefi-ciency virus infected person. Clin. Infect. Dis. 36(2003) e5–6.

9. LIENHARTDT, C., KAMATE, B., JAMET, P.,TOUNKARA, A., FAYE, O. C., SOW, S. O., andBOBIN, P. Effect of HIV infection on leprosy: athree-year survey in Bamako, Mali. Int. J. Lepr.Other Mycobact. Dis. 64 (1996) 383–391.

10. LU, W., SALERNO-GONCALVES, R., YUAN, J.,SYLVIE, D., HAN, D. S., and ANDRIEU, J. M. Glu-cocorticoids rescue CD4+ T lymphocytes from ac-tivation-induced apoptosis triggered by HIV-1:implication for pathogenisis and therapy. AIDS 9(1995) 35–42.

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13. NERY, J. A. C., SAMPAIO, E. P., GALHARDO, M. C.G., PERISS, A. R. S., VIEIRA, L. M. M., SALLES, A.M., and SARNO, E. N. M. leprae–HIV co-infection: pattern of immune response in vivo andin vitro. Indian J. Lepr. 72 (2000) 155–167.

14. OLIVARES, L. M., PIZZARIELLO, G. E. A., BENE-TUCCI, J., FARINA, M. H., KIEN, C., and BTESH, A.Lepromatous leprosy and HIV infection. Int. J.Lepr. Other Mycobact. Dis. 62 (1994) 295–296.

15. PATKI, A. H. Some possible interactions of Myco-bacterium leprae and HIV in the peripheral nerves.Int J Lepr. Other Mycobact. Dis. 59 (1991)331–332.

16. PERRI, A. J., III, and HSU, S. A review of thalido-mide’s history and current dermatological applica-tions. Dermatol. Online J. 9(3) 5. htt://dermatology.cdlib.org/93/reviews/thalidomide/hsu.html.2/25/2004.

17. RADOMSKY, C. L., and LEVINE, N. Thalidomide.Dermatol. Clinics 19 (2001) 87–103.

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drourea, cyclosporin and thalidomide. Drugs 58(1999) 953–963.

19. SAMPAIO, E. P., CANESHI, J. R. T., NERY, J. A. C.,DUPPRE, N. C., PEREIRA, G. M. V., VIEIRA, L. M.M., MOREIRA, A. L., KAPLAN, G., and SARNO, E.N. Cellular immune response to Mycobacteriumleprae infection in HIV-infected individuals. In-fect. Immun. 63 (1995) 1848–1855.

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22. UPPAL, S. S., VERMA, S., and DHOT, P. S. Normalvalue of CD4 and CD8 lymphocyte subsets inhealthy Indian adults and the effect of sex, age,ethnicity, and smoking. Cytometry B. Clin. Cy-tom. 52 (2003) 32–36.

23. UYEMURA, K., DIXON, J. F. P., WONG, L., REA, T.H., and MODLIN, R. L. Effect of cyclosporin A inerythema nodosum leprosum. J. Immunol. 137(1986) 3620–3623.

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1 Received for publication on Feb. 28, 2005. Accepted for publication on May 15, 2005.2 R. Lahiri, Ph.D.; B. Randhawa, B.S.; and J. L. Krahenbuhl, Ph.D. Laboratory Research Branch, National

Hansen’s Disease Programs, Louisiana State University, Baton Rouge, 70803 U.S.A.Reprint requests to: James L. Krahenbuhl, Ph.D., Laboratory Research Branch, National Hansen’s Disease

Programs, Louisiana State University, Skip Bertman Drive, Baton Rouge, LA 70803, U.S.A. E-mail:[email protected]

Effects of Purification and Fluorescent Staining on

Viability of Mycobacterium leprae1

Ramanuj Lahiri, Baljit Randhawa, and James L. Krahenbuhl2

ABSTRACTOver the years, researchers have carried out experiments with Mycobacterium leprae ob-

tained from either human multibacillary lesions, or infected armadillo tissues, or infectedfootpad tissues of conventional mice as well as athymic nu/nu mice. In general, thesesources of leprosy bacilli are satisfactory for most biochemical and mouse footpad studies,but less than satisfactory for studies in cell biology and immunology where contaminatinghost tissues pose a serious problem. We examined the utility of a procedure for eliminatingmouse footpad tissue from M. leprae suspension using sodium hydroxide solution and itssubsequent effect on the viability of the organism by determining the rate of palmitic acidoxidation, bacterial membrane integrity, and growth in the mouse footpad. We found thattreating M. leprae suspension, obtained from infected nu/nu mouse footpad, with 0.1NNaOH for 3 min was sufficient to remove the majority of mouse tissue without adversely af-fecting the viability of the organism. This is a simple and rapid method to get suspensions ofnu/nu footpad-derived viable M. leprae essentially free of host tissues, which can be a re-search reagent for studying the host-pathogen relationship in leprosy. We also report here amethod for labeling M. leprae with the fluorescent dye PKH26, without compromising onthe viability of the organism. This method may be useful in intracellular trafficking studiesof M. leprae or in other cell biology studies that require tracking of the bacteria using fluo-rescent tag. We observed the staining to be stable in vitro over considerable lengths of timeand did not affect the viability of the bacteria.

RÉSUMÉDepuis des années, les chercheurs ont mené des expériences à partir de Mycobacterium

leprae issues de lésions multibacillaires humaines, de tissus infectés de tatous à neuf bandesou bien de tissu de coussinets plantaires de souris tant conventionnelles que nues athymiques(nu/nu). Ces sources de bacilles de Hansen sont en général satisfaisantes pour la plupart desétudes biochimiques et d’inoculation au coussinet plantaire de souris, mais pas satisfaisantespour les études de biologie cellulaire et d’immunologie, où les éléments contaminantsprovenant de l’hôte peuvent représenter un sérieux problème. Nous avons vérifié l’utilitéd’une procédure à base de soude pour éliminer les tissus plantaires de souris contaminant lessuspensions de M. leprae, en vérifiant son effet sur la viabilité de la bactérie par la détermi-nation du taux d’oxydation de l’acide palmitique, de l’intégrité de la membrane de la bac-térie et de la croissance dans le coussinet plantaire de souris. Nous avons trouvé que le traite-ment pendant 3 minutes avec 0.1 N NaOH, de suspensions de M. leprae obtenues à partir decoussinets plantaires de souris nu/nu, était suffisant pour enlever la majorité des tissus desouris sans pour autant affecter de façon adverse la viabilité de l’organisme. C’est une méth-ode simple et rapide, qui permet d’obtenir des suspensions viables de M. leprae à partir delépromes de souris nu/nu dévolues presque entièrement de tissus de l’hôte, représentant demeilleurs réactifs de recherche pour étudier la relation hôte-pathogène de la lèpre. Nous rap-portons également ici une méthode pour marquer les M. leprae avec le produit fluorescentPKH26, sans compromettre la viabilité du microorganisme. Cette méthode peut être utilepour étudier les mouvements et localisations intracellulaires de M. leprae ou bien des étudesde biologie cellulaire, où le marquage de la bactérie par un colorant fluorescent est requis,afin de pouvoir la suivre. Nous avons constaté que le marquage était stable in vitro pendantdes temps importants et n’affectait pas la viabilité de la bactérie.

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73, 3 Lahiri, et al.: Purification and Fluorescent Staining of M. leprae 195

Over one hundred thirty years after its dis-covery as the causative agent of leprosy, My-cobacterium leprae is yet to be cultured invitro. This obstacle has not stymied experi-mentation with leprosy bacilli of human ori-gin or from infected armadillos or the mousefootpad, although adequate numbers, purity,and questionable viability of M. leprae haveaffected experimental reproducibility andpresented additional obstacles to researchers.

In order to have weekly access to largenumbers of highly viable M. leprae, wemaintain several isolates in serial passagesin athymic nu/nu mice where growth in thefootpad routinely produces a few billion or-ganisms. We have adapted radiorespirome-try (RR) procedures to measure oxidationof radiolabeled palmitic acid (5) to compareviability of different suspensions of M. lep-rae as defined by metabolic activity. RRwas shown to correlate well with growth inthe mouse footpad (MFP) (22). Recently, wehave employed evaluation of the membraneintegrity of individual M. leprae in a sus-pension with LIVE/DEAD BacLight Bacte-rial Viability Staining (VS) Kit® as an addi-tional assay of bacterial viability on the as-sumption that the bacilli with damagedmembrane are dead (11). However, evennu/nu MFP derived M. leprae suffer fromone drawback and that is the presence ofcontaminating mouse tissue in the bacterial

suspension. While, this contaminating hosttissue does not affect subsequent passage toa new host or some in vitro investigations, itis absolutely not desirable in studies de-signed to observe immunological reactions,intracellular trafficking, and pathogenesisof the disease, which is markedly differentfrom that of tuberculosis. Slow speed cen-trifugation removes larger pieces of mousetissue from the suspension, but counter-staining of the acid-fast bacilli (AFB) givesclear evidence for unacceptable levels of re-maining mouse tissue in the suspension.

In this study, we examined the utility of aprocedure for eliminating mouse footpad tis-sue from M. leprae suspension using sodiumhydroxide (NaOH) solution and its subse-quent effect on the viability of the organismas defined by RR and VS. We also reporthere a method for labeling M. leprae withhighly aliphatic reporter molecules contain-ing fluorochrome groups, without compro-mising on the viability of the organism as de-fined by RR, VS and growth on the MFP.

METHODS AND MATERIALSNude mouse-derived M. leprae. Myco-

bacterium leprae (isolate Thai-53) is main-tained in serial passage in the footpads ofathymic nu/nu mice (Harlan, Indianapolis,Indiana, U.S.A.). Mice were inoculated onthe plantar surface of both hind feet with 5

RESUMENA lo largo del tiempo los investigadores han realizado experimentos con Mycobacterium

leprae obtenido de lesiones humanas multibacilares, de tejidos infectados de armadillo y delas almohadillas plantares de ratones convencionales y atímicos, nu/nu. En general, losbacilos de la lepra obtenidos de estas fuentes son satisfactorios para la mayoría de los estu-dios bioquímicos y para su inoculación en las almohadillas plantares del ratón, pero no sonsatisfactorios para estudios inmunológicos y de la biología celular donde los tejidos contam-inantes del huésped representan un verdadero problema. Nosotros examinamos la utilidad deun procedimiento para eliminar el tejido de la almohadilla plantar de una suspensión de M.leprae usando una solución de hidróxido de sodio, y estudiamos el efecto de este tratamientoen la viabilidad del organismo (por oxidación del ácido palmítico), en la integridad de sumembrana, y en su capacidad de replicación en la almohadilla plantar del ratón. Encontramosque el tratamiento de la suspensión de M. leprae obtenida de la almohadilla plantar de ratonesnu/nu con NaOH 0.1N por 3 min fue suficiente para remover la mayoría del tejido de ratónsin afectar sensiblemente la viabilidad del microorganismo. Este es un método simple yrápido para obtener suspensiones viables del microorganismo esencialmente libres de tejido,a partir de la almohadilla plantar del ratón desnudo, que pueden usarse para estudiar las rela-ciones entre el bacilo y el hospedero. También reportamos aquí un método para marcar M.leprae con el colorante fluorescente PKH26 que puede ser de utilidad en los estudios del trá-fico intracelular del microorganismo o en otros estudios de la biología celular donde se re-quiere localizar la bacteria usando una marca fluorescente. Observamos que la tinción es es-table in vitro por periodos largos de tiempo y que no afecta la viabilidad de la bacteria.


× 107 fresh, viable nu/nu-derived M. leprae.When the mouse footpads became moder-ately enlarged (at ~6 months), they wereharvested for intracellular M. leprae as de-scribed previously (22), washed by centrifu-gation (18,000 g for 30 min), resuspendedin either medium 7H12 or RPMI-1640(Gibco Invitrogen, Carlsbad, California,U.S.A.) + 10% (v/v) fetal calf serum [(FCS)Gibco Invitrogen, Carlsbad, California,U.S.A.], enumerated by direct count ac-cording to Shepard’s method (18) and heldovernight at 4°C, pending quality controltesting for contamination. The bacterial sus-pension was passed 3 to 4 times through a27G needle prior to counting in order to re-move clumps. Freshly harvested bacilliwere always employed in experiments(within 24 hr of harvest).

NaOH treatment of M. leprae. 1 × 109

fresh M. leprae were resuspended in 1.0 mlof the appropriate concentration of NaOH[0.1N–0.9N] (Sigma, St. Louis, Missouri,U.S.A.) and incubated for 3 min at roomtemperature, after which the bacteria werewashed (10,000 g for 5 min at 4°C) thrice in7H12 medium and finally resuspended inappropriate media. There was a 30 to 50%loss of bacteria in this process.

Scanning Electron Microscopy. Ten μlsuspension (1 × 109/ml) of 0.1N NaOHtreated or untreated M. leprae were spreadon poly-lysine coated plastic cover slips, airdried, fixed, and washed prior to 1% Osmiumtertraoxide treatment. Following which thecover slips were washed in deionized waterand then dehydrated by several changes of30% to 100% ethyl alcohol. After dehydra-tion the samples were subject to criticalpoint drying prior to sputter coating withgold and palladium. The samples were thenvisualized in a FEI Quanta 200 scanningelectron microscope

Radiorespirometry. 1 × 107 M. lepraewere inoculated into 1.0 ml of BACTEC7H12B media (Becton Dickinson, FranklinLakes, New Jersey, U.S.A.) containing 14C-palmitic acid in a loosely capped vialwhich, in turn, was inserted into a widemouth liquid scintillation vial lined with fil-ter paper impregnated with NaOH, 2,5-diphenyloxazole (Sigma, St. Louis, Mis-souri, U.S.A.) and Concentrate I (Kodak,Rochester, New York, U.S.A.) and incu-bated at 33°C. When read daily, captured

14CO2

determines the rate of 14C-palmiticacid oxidation (5). In the present study, onthe seventh day cumulative counts perminute (CPM) are reported.

Fluorescent staining for assessing bac-terial membrane integrity. The membraneintegrity of individual M. leprae in a sus-pension was evaluated with LIVE/DEADBacLight Bacterial Viability Staining (VS)Kit® (Molecular Probes, Eugene, Oregon,U.S.A.) as described previously (11). Briefly,M. leprae were washed (10,000 g for 5min) in normal saline and incubated for 15min at room temperature with 6 μM Syto9and 30 μM propidium iodide (PI). Afterstaining the bacteria were resuspended in10% (v/v) glycerol in normal saline, passedthrough 27G needle to dissociate clumps,and the percentage of dead and live bacteriain the suspension were enumerated by di-rect counting of fluorescent green and redbacilli using appropriate single bandpassfilter sets.

At least 200 individual bacteria or 10 mi-croscopic fields, whichever was more, werecounted to evaluate the percentage of bacte-ria having membrane damage in the suspen-sion.

Staining with PKH dyes. Freshly har-vested M. leprae treated with 0.1N NaOH(1 × 109) were resuspended in 1.0 ml of theprovided “diluent C” and then stained for 2min at room temperature with a 1:250 dilu-tion of either PKH26 (red) or PKH67(green) dye (Sigma, St. Louis, Missouri,U.S.A.). After 2 min the staining was haltedby adding an equal volume of FCS. Thesuspension was washed (10,000 g for 5min) thrice in appropriate medium. Thenumbers of bacteria were recounted follow-ing staining by Shepard’s direct countmethod (18).

Macrophage culture. Resident peri-toneal cells from Swiss mice were har-vested and allowed to adhere for at least 6hr at 37°C and 5% (v/v) CO

2, on plastic

cover slips in 24 well tissue culture plates(Corning, Corning, New York, U.S.A.) aspreviously described (14). After washing toremove non-adherent cells, the adherentcells were infected overnight at 33°C withPKH26 stained M. leprae at a multiplicityof infection of 20:1. At the end of the incu-bation extracellular M. leprae were re-moved by washing the cover slips.


Footpad growth of M. leprae. BALB/cmice, 5 in each group, were inoculated onthe plantar surface of both hind feet with 1 ×104 PKH26 stained or control M. leprae. At 3and 6 months both hind footpads were har-vested, processed and the number of AFBenumerated using Shepard’s technique.

Statistical analysis. The data are shownas means ± standard deviation (S.D.) from arepresentative of three to four experiments.The raw data were subjected to one-tailedor two-tailed Student’s t test to determinewhether the observed differences betweenthe means were significant. p <0.05 wastaken as significant.

RESULTSScanning electron microscopy of NaOH

treated M. leprae. To observe the effects ofNaOH treatment on the appearance of asuspension of nu/nu footpad derived M.leprae, suspensions were treated for 3 minwith 0.1N NaOH, washed and observedwith the S.E.M. The results (Fig. 1) showed

that treatment resulted in a marked clear-ance of mouse footpad tissues from the M.leprae suspension in comparison to un-treated controls. We did not observe any sig-nificant improvement in the quality of theM. leprae suspension, by scanning electronmicroscopy, following treatment with 0.1NNaOH for a longer period or with higherconcentrations of NaOH (data not shown).

Effects of NaOH treatment on meta-bolic activity of M. leprae. To determinethe effects of NaOH treatment on sustainedin vitro metabolic activity, M. leprae weretreated with 0.1N, 0.3N, 0.6N, or 0.9NNaOH for 3 min at room temperature,washed in 7H12 medium and prepared forRR. The seventh day RR data (Fig. 2)showed no significant differences in the cu-mulative oxidation of radiolabeled palmiticacid between the control and the NaOHtreated M. leprae. However, a significantfall in the RR was observed when the 0.6Nor higher concentration NaOH treatmentwas carried out for longer than 7 min (datanot shown).

Effects of NaOH treatment on mem-brane integrity of M. leprae. To assess theeffects of different NaOH treatments on themembrane integrity of M. leprae BacLightfluorescent staining was done. In this assayall the bacilli in the suspension stain withSyto9 (green), i.e. both those with intactmembranes as well as those with damagedmembranes but the bacilli having damaged

FIG. 1. Scanning electron photomicrograph (6200×)showing nu/nu mouse footpad derived M. leprae sus-pension before (a) and after (b) treatment with 0.1NNaOH for 3 min. Most of the mouse tissue had beenremoved by the treatment and subsequent washings.

FIG. 2. Effect of different concentrations of NaOHtreatment for 3 min on palmitic acid oxidation rate ofM. leprae. Bargraph showing the cumulative seventhday radiorespirometry counts ± S.D. The data are rep-resentative of five independent experiments. M. lepraewere obtained freshly from athymic (nu/nu) mousefootpads for each experiment.


membrane also stain with PI (red). This as-say assumes that all the bacteria havingdamaged membrane (staining red) are non-viable or dead. The data (Fig. 3) clearlyshowed that 3 min treatment with 0.1N,0.3N or 0.6N NaOH did not impart any sig-nificant membrane damage. However, treat-ment with 0.9N NaOH for the same dura-tion resulted in a significant decrease (p<0.0001) in the number of M. leprae havingintact membrane.

Staining of M. leprae with PKH26 dye.The staining of M. leprae with PKH26 dye

was done after treating the suspension ofbacilli with NaOH. We chose the 3 mintreatment with 0.1N NaOH as describedabove. A 1:250 dilution of PKH26 dye pro-vided bright red fluorescent bacteria thatmaintained solid fluorescence for at least 15 days when held in vitro in dark at 4°C(data not shown). Similar findings were ob-served when the green (PKH67) dye wasemployed. We used 3 different dilutions ofthe dye (1:250, 1:500 and 1:1000) and fol-lowed the manufacturer’s protocol forstaining M. leprae and found no detrimentaleffects of PKH26 staining on subsequentpalmitic acid metabolism as measured byRR (Fig. 4).

Uptake of PKH26 stained M. leprae bymouse peritoneal macrophages. To visu-alize intracellular fluorescent bacilli, adher-ent mouse peritoneal macrophages were in-fected in vitro with either PKH26 stained orunstained live M. leprae at a MOI of 20:1.We did not observe any difference in the up-

FIG. 3. Effect of different concentrations of NaOHtreatment for 3 min on membrane integrity of M. lep-rae. Bargraph showing the percentage live M. leprae ±S.D. as determined by the viability staining. The dataare representative of three independent experiments.M. leprae was obtained freshly from athymic (nu/nu)mouse footpads for each experiment.

FIG. 4. Effect of PKH26 staining on palmitic acidoxidation rate of M. leprae. Bargraph showing the cu-mulative seventh day radiorespirometry counts ± S.D.The data were representative of three independent ex-periments. M. leprae were obtained freshly fromathymic (nu/nu) mouse footpads for each experiment.

FIG. 5. Photomicrograph (400×) showing the up-take of unlabeled control (a) and PKH26 labeled (b)viable M.leprae by mouse peritoneal macrophages in-fected overnight at 33°C. Macrophages infected withunlabeled M. leprae (a) were fixed and stained by theKinyoun acid-fast technique.

take of PKH26 stained M. leprae when com-pared to that of unstained control (Fig. 5).

Growth of PKH26 stained M. leprae inmouse footpads. One × 104 PKH26 stainedor unstained M. leprae were used to infecteach hind footpad of BALB/c mice. Thefootpads were harvested at 3 and 6 monthsand the total number of AFB per footpadwere counted. The results (Fig. 6) indicateno significant difference between thegrowth kinetics of the PKH26 stained M.leprae (3.3 × 106 ± 1.3 × 106AFB/ footpadat 6 months) to that of the control (1.8 × 106

± 0.7 × 106AFB/ footpad at 6 months).

DISCUSSIONOver the years, researchers have carried

out experiments with M. leprae obtainedfrom a variety of sources, including thenodules or lesions from multibacillary(MB) patients, infected armadillo tissue,and infected footpads from conventionalmice as well as immunocompromisedneonatal thymectomized, lethally irradiated(NTLR) and nu/nu mice. In general, thesesources of leprosy bacilli have proved satis-factory for MFP studies but less than satis-factory for in vitro experiments.

M. leprae from infected human patientsare difficult to obtain and there is no controlof the investigator over the quality of thesebacilli. Human derived bacilli were ob-tained from untreated cases if viable organ-isms were required (4, 7), but the quality (vi-ability) of these bacilli was poor and humanbiopsies were an inconsistent source of or-ganisms as it would be unethical to with-hold treatment from an identified case ofMB leprosy solely to provide a source ofbacilli. The quality of bacilli obtained frompassage of M. leprae in the conventionalMFP model was more consistent than thatfrom human origin. However, though con-ventional MFP model yielded adequatenumbers of bacilli for a variety of addi-tional MFP studies (16), these organismswere unsatisfactory for most in vitro studiesas they were too few (maximum yield of ~1× 106 per footpad) and consisted largely offootpad tissue. A single infected armadillocan yield tens of billions of bacteria (10), butthe quality of bacilli in terms of viability re-mains poor (unpublished results). Hence,armadillo derived M. leprae are good forconducting certain biochemical studies but

not for in vitro or cell culture studies wherethe viability of the bacterial inoculum candictate the outcome of the experiments. Forthe latter kind of studies athymic nu/nufootpad-derived M. leprae is best, as asingle mouse can yield a few billion highlyviable bacteria.

The present report establishes a simplepurification method for removing host tis-sue from suspensions of M. leprae withoutaffecting the viability of the bacilli. Otherprocedures have been developed to purifytissue derived M. leprae on a large scale butthe effects of these treatments on viabilityof the bacilli was never determined. Ar-madillo infected liver, spleen and lymphnode tissue harbors billions of M. leprae(10)and a two phase method devised by Draperfor isolation of pure bacilli from large quan-tities of infected armadillo liver and spleenwas developed(17) and is used routinely forthe provision of the enormous numbers ofbacilli required to isolate and characterizecell wall and other M. leprae constituents(8). However, until very recently the in-fected tissues were irradiated with 2.5 × 106

rads(10), a dose that kills the bacilli(1) mak-ing the issue of viable, armadillo-derivedM. leprae moot. In recent years these tis-sues have not been irradiated but our stud-ies, employing both RR as a measure ofmetabolic activity and the BACTLIGHTstain for membrane integrity and MFP chal-lenge show that the viability of even un-irradiated armadillo-derived M. leprae isextremely low (unpublished results).

Another important consideration for theprovision of M. leprae as a research reagentis that armadillos are too expensive to


FIG. 6. Effect of PKH26 staining on the growth ofM. leprae in the mouse footpad.

maintain (1 to 2 years) as a source of bacillifor routine (weekly) experimentation. Ar-madillos are infected experimentally to pro-vide maximum numbers of M. leprae uponharvest, a goal we have found to be incon-sistent with providing highly viable organ-isms harvested during their log phase ofgrowth(11). M. leprae-infected athymicnu/nu mice on the other hand are readilyavailable, far less expensive than armadillosto maintain and the infected footpads of in-dividual mice can be harvested weekly toyield billions of bacilli in the crude ho-mogenates of the infected footpad tissues.

Our laboratory is committed to character-izing nu/nu-derived M. leprae as a researchresource and we have described their re-sponse to physical-chemical treatment in-cluding susceptibility to ionizing(1) andUV(21) radiation, effects of deep-freezestorage and incubation temperatures and re-sponse to various fixatives(22, 11).

Previously we employed a Percoll den-sity gradient separation of nu/nu derived M.leprae to yield pure suspensions of bacillithat were enriched for viability as definedby RR but losses of total bacilli were rou-tinely >90%, an unacceptable yield. Treat-ment with 0.1N NaOH has been routinelyemployed to purify M. leprae of host tissuefor studying the enzyme activity(13) and iso-lation of bacterial components(9). The pre-sent S.E.M. studies clearly show that brieftreatment of nu/nu mouse footpad derivedM. leprae with 0.1N NaOH eliminatesmouse footpad tissue providing a pure sus-pension of bacilli for potential in vitro useas a leprosy research reagent. But a purifiedreagent is only a partial fulfillment of theneeds of researchers; a pure and viablereagent is needed. For example, previousstudies from this laboratory have describedmarked differences in afferent and efferentfunction of M. leprae infected macro-phages, depending on whether infectionwas carried out with viable or non-viablebacilli(19).

The present study investigated the effectsof NaOH treatment on subsequent viabilityand shows that this method of purificationdoes not affect their viability as defined invitro by RR, a measure of metabolic ac-tivity that correlates well with growth inmouse footpad(22). To further characterizeviable M. leprae as a research reagent we

have recently adapted the VS procedure topermit evaluation of the viability of indi-vidual leprosy bacilli as defined by mem-brane integrity(11). Interestingly we foundthat a consistent, though not significant, in-crease in the cumulative seventh day RRcounts and percentage live in VS assay wasobserved after treatment of bacterial sus-pension with 0.1N and 0.3N NaOH. Thesefindings may be due to removal of somenon-viable bacteria from the suspension bythe NaOH treatment. The yield of bacteriaafter the NaOH treatment and subsequentwashings was routinely between 30 and50%.

Combining RR analysis with VS has al-lowed us to adjust our routine passage of M.leprae in the nu/nu mouse to maximize theviability of a harvested suspension and min-imize the duration of the infection(22, 11). Inthe infected nude mouse the footpad in-creases in size as bacillary numbers in-crease markedly and host cells becomegorged with M. leprae. Viability as mea-sured by RR correlated with MFP growthand was significantly correlated with timein tissue and the number of bacilli per gramof granuloma(22). Very large footpads withhigh numbers of M. leprae per gram of tis-sue yield less viable bacilli. Highest viabil-ity for nude mouse derived M. leprae is as-sociated with short to moderate periods invivo. Thus routine short term passage is thebest means to assure plentiful, viable stocksof M. leprae. The present study extends ourinterests in defining the properties of viableM. leprae as a research reagent.

Access to a reliable source of large num-bers of pure, viable M. leprae would be animportant research resource for today’s lep-rosy researcher especially those interestedin pursuing the cell biology of intracellularinfection with M. leprae and the unique re-lationship between the leprosy bacillus andits host cell. A major tool became availableto cell biologists a dozen or so years agowith the development of fluorescent trackerdyes which allowed the stable labeling ofmammalian cells(20). The technology wasbased on the incorporation of highlyaliphatic reporter molecules containing flu-orochrome groups into the lipid bilayers ofcytoplasmic membranes. A key feature ofthese dyes is their retention. Once incorpo-rated, they are trapped in the membrane by


virtue of their inherent insolubility in aque-ous solutions. In a variety of eukaryote cellsthese dyes have been shown to be stableand non-toxic, permitting tracking of adop-tively transferred cells in vivo without inter-fering with their function, for example cy-totoxicity(12). The tracking dyes do not in-terfere with doubling times of labeled cellsand the dye appears to be equally parti-tioned between daughter cells when a la-beled cell divides(6). Similar dyes havebeen employed as fluorescent trackers to la-bel prokaryote cells such as protozoa(15)and bacteria(3).

In the present studies, the short term ef-fects of staining with PKH26 tracker dye onthe metabolic activity of M. leprae was de-termined in vitro in axenic media. Thegrowth of stained bacteria in MFP was alsodetermined. Notably, all the PKH dye label-ing of bacilli reported in this study wasdone subsequent to NaOH treatment to re-move the contaminating mouse tissueswhich would also label with the PKH dye.We have also observed that M. leprae didnot label uniformly with PKH dye if mousetissue was present in the suspension. There-fore, these studies also demonstrated thatneither NaOH treatment nor PKH26 label-ing of M. leprae affected the viability of M.leprae as defined by RR, VS and growth inthe mouse footpad, the “gold standard”measurement of the ability of the leprosybacillus to survive and multiply in vivo. ThePKH labeled bacteria can be visualized in-side mouse peritoneal macrophages usingeither fluorescence or confocal microscope.It should be noted, that in cultures wherethe PKH26 stained M. leprae were main-tained in peritoneal macrophages for morethan 7 days there was slight diffusion of thePKH26 dye into the macrophage cytosoliccompartments (data not shown).

The NaOH treatment reported here is aneasy and fast method to obtain suspensionsof nu/nu MFP derived viable M. leprae es-sentially free of host tissue, a valuable re-search reagent required for studying thehost pathogen relationship in leprosy. Theother research reagent described here is thefluorescently labeled viable M. lepraewhich can be utilized in intracellular traf-ficking studies of M. leprae(2) or in othercell biology studies that require tracking ofthe bacteria using a fluorescent tag. We ob-

served the staining to be stable in vitro overa considerable length of time and did notaffect the viability of the bacteria. MFPstudies that will explore in more detail thePKH-staining characteristics of multiplyingM. leprae are underway.

Acknowledgement. The authors gratefully ac-knowledge the expertise of Mr. Greg McCormick inthe preparation of S.E.M. photos of M. leprae. Thesestudies were supported by a grant from The AmericanLeprosy Missions, Greenville, SC.

REFERENCES

1. ADAMS, L. B., SOILEAU, N. A., BATTISTA, J. R., andKRAHENBUHL, J. L. Inhibition of metabolism andgrowth of Mycobacterium leprae by gamma irra-diation. Int. J. Lepr. Other Mycobact. Dis. 68(2000) 1–10.

2. ALVES, L., DE MENDONCA LIMA, L., DA SILVA

MAEDA, E., CARVALHO, L., HOLY, J., SARNO, E. N.,PESSOLANI, M. C., and BARKER, L. P. Mycobacte-rium leprae infection of human Schwann cells de-pends on selective host kinases and pathogen-modulated endocytic pathways. FEMS Microbiol.Lett. 238 (2004) 429–437.

3. CHUNG, J. D., CONNER, S., and STEPHANOPOULOS,G. Flow cytometric study of differentiating cul-tures of Bacillus subtilis. Cytometry. 20 (1995)324–333.

4. DELVILLE, J. In vitro behavior of macrophagesfrom healthy persons against M. leprae and othermycobacteria. Int. J. Lepr. Other Mycobact. Dis.39 (1971) 329–339.

5. FRANZBLAU, S. G. Oxidation of palmitic acid byMycobacterium leprae in an axenic medium. J.Clin. Microbiol. 26 (1988) 18–21.

6. GIVAN, A. L., FISHER, J. L., WAUGH, M., ERNSTOFF,M. S., and WALLACE, P. K. A flow cytometricmethod to estimate the precursor frequencies ofcells proliferating in response to specific antigens.J. Immunol. Methods. 230 (1999) 99–112.

7. GODAL, T., and REES, R. J. Fate of Mycobacte-rium leprae in macrophages of patients with lep-romatous or tuberculoid leprosy. Int. J. Lepr. OtherMycobact. Dis. 38 (1970) 439–442.

8. HUNTER, S. W., and BRENNAN, P. J. A novel phe-nolic glycolipid from Mycobacterium leprae pos-sibly involved in immunogenicity and pathogenic-ity. J. Bacteriol. 147 (1981) 728–735.

9. HUNTER, S. W., STEWART, C., and BRENNAN, P. J.Purification of phenolic glycolipid I from ar-madillo and human sources. Int. J. Lepr. OtherMycobact. Dis. 53 (1985) 484–486.

10. JOB, C. K., DRAIN, V., TRUMAN, R., DEMING, A. T.,SANCHEZ, R. M., and HASTINGS, R. C. The patho-genesis of leprosy in the nine-banded armadilloand the significance of IgM antibodies to PGL-1.Indian. J. Lepr. 64 (1992) 137–151.


11. LAHIRI, R., RANDHAWA, B., and KRAHENBUHL, J. L.Application of a viability staining method for My-cobacterium leprae derived from the athymic(nu/nu) mouse foot pad. J. Med. Microbiol. 54(2005) 235–242.

12. LEE-MACARY, A. E., ROSS, E. L., DAVIES, D., LAY-LOR, R.,HONEYCHURCH, J., GLENNIE, M. J., SNARY,D., and WILKINSON, R. W. Development of anovel flow cytometric cell-mediated cytotoxicityassay using the fluorophores PKH-26 and TO-PRO-3 iodide. J. Immunol. Methods. 252 (2001)83–92.

13. PRABHAKARAN, K., HARRIS, E. B., and KIRCH-HEIMER, W. F. Glutamic acid decarboxylase inMycobacterium leprae. Arch. Microbiol. 134(1983) 320–323.

14. RAMASESH, N., ADAMS, L. B., FRANZBLAU, S. G.,and KRAHENBUHL, J. L. Effects of activatedmacrophages on Mycobacterium leprae. Infect.Immun. 59 (1991) 2864–2869.

15. SHAHABUDDIN, M., GAYLE, M., ZIELER, H., andLAUGHINGHOUSE, A. Plasmodium gallinaceum:fluorescent staining of zygotes and ookinetes tostudy malaria parasites in mosquito. Exp. Para-sitol. 88 (1998) 79–84.

16. SHEPARD, C. C. A brief review of experienceswith short-term clinical trials monitored bymouse-foot-pad-inoculation. Lepr. Rev. 52 (1981)299–308.

17. SHEPARD, C. C., DRAPER, P., REES, R. J., andLOWE, C. Effect of purification steps on the im-munogenicity of Mycobacterium leprae. Br. J.Exp. Pathol. 61 (1980) 376–379.

18. SHEPARD, C. C., and MCRAE, D. H. A method forcounting acid-fast bacteria. Int. J. Lepr. Other My-cobact. Dis. 36 (1968) 78–82.

19. SIBLEY, L. D., and KRAHENBUHL, J. L. Inductionof unresponsiveness to gamma interferon inmacrophages infected with Mycobacterium lep-rae. Infect. Immun. 56 (1988) 1912–1919.

20. TEARE, G. F., HORAN, P. K., SLEZAK, S. E., SMITH,C., and HAY, J. B. Long-term tracking of lympho-cytes in vivo: the migration of PKH-labeled lym-phocytes. Cell. Immunol. 134 (1991) 157–170.

21. TRUMAN, R. W., and GILLIS, T. P. The effect of ul-traviolet light radiation on Mycobacterium leprae.Int. J. Lepr. Other Mycobact. Dis. 68 (2000) 11–17.

22. TRUMAN, R. W., and KRAHENBUHL, J. L. ViableM. leprae as a research reagent. Int. J. Lepr. OtherMycobact. Dis. 69 (2001) 1–12.


1 Received for publication Feb. 25, 2005. Acceptedfor publication on June 2, 2005.

2 T. Narang, M.D.; S. Dogra, M.D., D.N.B.,M.N.A.M.S.; and I. Kaur, M.D., M.N.A.M.S., Depart-ment of Dermatology, Venereology and Leprology,Postgraduate Institute of Medical Education and Re-search, Chandigarh, India.

Reprint requests to: Dr. Inderjeet Kaur, AdditionalProfessor, Dept. of Dermatology, Venereology andLeprology, PGIMER, Chandigarh-160 012, India; E-mail: [email protected]

CASE REPORTS

Borderline Tuberculoid Leprosy with Type 1

Reaction in an HIV Patient—A Phenomenon of

Immune Reconstitution1

Tarun Narang, Sunil Dogra, and Inderjeet Kaur2

203


(ISSN 0148-916X)

The course of leprosy in patients withHIV infection has been a controversial issuefor a long time. It is still a matter of debatewhether the HIV status of an individual hasany impact on the natural history of leprosyand response to anti-leprosy treatment.Though various effects on immune systemcan be expected in a case of co-existent lep-rosy with HIV infection, epidemiologicalstudies failed to establish any such bearingin the clinical course of leprosy. A WorldHealth Organization (W.H.O.) meeting in1993 concluded that there is no convincingevidence for an association between HIVand leprosy (14). A case control study heldin South India among leprosy patients alsoconfirmed these findings (11). Individualswith profound immunosuppression due toHIV infection may have active coinfectionsthat are subclinical because of the lack ofhost inflammatory responses. Reconstitu-tion of the immune system during the initialmonths of antiretroviral treatment, how-ever, may result in the development of overtclinical manifestations of these coinfections(1, 3), as restoration of CD4+ T lymphocytespermits inflammatory responses to bemounted (8, 9). Immune reconstitution or im-mune restoration phenomenon is now a

well-recognized complication of highly ac-tive antiretroviral treatment (HAART) andhas been described in individuals infectedwith Mycobacterium tuberculosis, nontu-berculous mycobacteria, cytomegalovirus,and hepatitis B and C viruses (1, 3).

CASE REPORTA 28-year-old married man was referred

to our department for evaluation of erythe-matous plaque on his left thigh, observed 5weeks back. He had become aware of hisHIV status 4 months back when he was be-ing evaluated for prolonged fever and in-tractable diarrhoea. His wife was alsoseropositive. At the time of presentation thepatient had advanced immunosuppression,with a blood CD4+ lymphocyte count of125/µL and a plasma virus load of 150,000HIV-1 RNA copies/mL. He was started onHAART (zidovudine, lamivudine, andefavirenz) and after 2 months of triple drugtherapy, the patient noticed an erythematousplaque on his left thigh. Over the next week,the lesion enlarged, became swollen andsmaller lesions appeared in its periphery(The Figure) along with symptoms of painand paraesthesias in the left leg. Clinical ex-amination revealed well-demarcated, erythe-matous, edematous tender plaque with com-plete loss of sensation. Left lateral poplitealnerve was thickened and tender. A clinicaldiagnosis of borderline tuberculoid (BT) lep-rosy with reversal (type 1) reaction wasmade. Skin biopsy confirmed the diagnosis,showing noncaseating granulomas, markednerve destruction, and no acid-fast bacilli(AFB). At the time that the cutaneous lesionsdeveloped, the patient’s plasma viral loadhad declined to 1750 HIV-1 RNA


copies/mL, and his CD4+ lymphocyte counthad increased to 280/µL.

The patient was started on W.H.O. multi-drug therapy (M.D.T.) PBR. He was alsostarted on prednisolone 40 mg once dailyfor the reversal reaction, which was gradu-ally tapered and stopped in 12 weeks. Heresponded favorably to the treatment.

DISCUSSIONHIV has generally not been found to have

a significant impact on the clinical course oftreated and untreated leprosy. However, ithas been reported that the neuritis in co-in-fected people can be more severe and thereversal reaction may be more frequent af-ter therapy. In endemic areas with HIV dis-ease and leprosy, there does not appear tobe a greater incidence of leprosy amongHIV patients. It may be because of the veryslow proliferation of the bacilli or the pro-longed incubation period, or perhaps a par-ticular cellular mechanism involved in itspathogenesis.

HIV-infected patients responding toHAART can show a diverse spectrum ofsymptoms caused by immune reconstitutionand subsequent inflammatory reactions.The pathogenesis of this phenomenon,called immune restoration disease (IRD)/Immune reconstitution inflammatory syn-drome (IRIS) is unclear. IRIS is an unusualinflammatory reaction to an opportunisticinfection that occurs in a HIV-positive pa-tient with profound immunosuppressionduring the reconstitution of the immune sys-tem in the initial months of HAART (2). Avariety of manifestations of IRIS have beendescribed, most prominently including My-cobacterium avium complex lymphadenitis,paradoxical exacerbations of pulmonaryand CNS Mycobacterium tuberculosis in-fection (8), paradoxical exacerbations ofCryptococcus neoformans meningitis, her-pes zoster and cytomegalovirus uveitis (12).Reactions in leprosy, especially the type1/reversal reaction, should be recognized asan IRIS-associated manifestation with apossibility of atypical presentation.

Our patient may well have harbored la-tent infection with Mycobacterium lepraefor many years or had ill defined lesionswhich escaped his attention. He developedclinically apparent leprosy within a span oftwo months, and the timing of the presenta-

tion was related to his immune status. Thetemporal association between the develop-ment of skin lesions and the HAART-induced changes in plasma HIV-1 load andCD4+ lymphocyte count strongly suggeststhat leprosy manifested clinically as a resultof immune reconstitution. Immune recon-stitution either resulted in the developmentof active leprosy per se or triggered the re-versal reaction leading to presentation ofpreviously unrecognized disease. Indeed,many patients with BT leprosy present onlywhen a reversal reaction develops. The on-set of immune reconstitution phenomenaoften occurs within 1 to 6 months ofHAART, even prior to substantial increasein the blood CD4+ T lymphocyte count (1, 3).

Studies have shown that HIV-1 infection isnot a risk factor for leprosy (4, 6). Although ashift in the spectrum of leprosy from the tu-berculoid to the lepromatous form might beexpected, studies have shown that HIV-1co-infection does not alter either the clinicalor histological spectrum of the disease (6, 9).The development of bordeline tuberculoid(BT) leprosy in this patient (indicatingstrong cell-mediated immunity), after in-crease in CD4+ T lymphocyte counts is,therefore, consistent with previous observa-tions (2, 5). However, the presentation of lep-rosy as an immune reconstitution phenome-non does suggest that HIV-1 associatedimmunosuppression masked the patient’sdisease before the start of HAART. Thereare few case reports of this phenomenon inleprosy (2, 5), and it is possible that the inci-dence of clinically overt leprosy may be de-creased among HIV-infected individualswho are profoundly immunosuppressed.

Type 1 (reversal) reactions occur most

THE FIGURE. Well defined plaque with satellite le-sions on left thigh.

73, 3 Narang, et al.: Type 1 Leprosy Reaction and HIV 205

frequently among patients with BT leprosy,causing acute inflammation in cutaneous le-sions and nerves harboring M. leprae anti-gens. Such reactions result from an increasein cell-mediated immunity and typically oc-cur during the early stages of leprosy treat-ment. Paradoxically, such reactions are ob-served more frequently among those withHIV-1 coinfection (13).

The increasing availability of HAART inareas where both HIV and leprosy areprevalent may well reveal latent leprosycases as a result of an IRIS in patients start-ing antiretroviral treatment. Differentiationof IRIS from an opportunistic infection isimportant because IRIS indicates a success-ful, albeit undesirable, effect of HAART. Itis also important to differentiate it fromdrug toxicity to avoid unnecessary cessa-tion of HAART.

REFERENCES1. BEHRENS, G. M., MEYER, D., STOLL, M., and

SCHMIDT, R. E. Immune reconstitution syndromesin human immuno-deficiency virus infection fol-lowing effective antiretroviral therapy. Immuno-biol. 202 (2000) 186–193.

2. COUPPIE, P., ABEL, S., VOINCHET, H., ROUSSEL, M.,HELENON, R., HUERRE, M., SAINTE-MARIE, D., andCABIE, A. Immune reconstitution inflammatorysyndrome associated with HIV and leprosy. Arch.Dermatol. 140 (2004) 997–1000.

3. DESIMONE, J. A., POMERANTZ, R. J. and BABINCHAK,T. J. Inflammatory reactions in HIV-1 infected per-sons after initiation of highly active antiretroviraltherapy. Ann. Intern. Med. 133 (2000) 447–454.

4. KAWAKA, H. J., BWIRE, R., and ADATU-ENGWAU, F.Leprosy and infection with the human immunode-ficiency virus in Uganda: a case-control study. Int.J. Lepr. Other. Mycobact. Dis. 62 (1994) 521–526.

5. LAWN, S. D., WOOD, C., and LOCKWOOD, D. N.Borderline tuberculoid leprosy: an immune recon-

stitution phenomenon in a human immunodefi-ciency virus infected person. Clin. Infect. Dis. 36(2003) e5–e6.

6. LIENHARDT, C., KAMATE, B., JAMET, P., TOUNKARA,A., FAYE, O. C., SOW, S. O., and BOBIN, P. Effectof HIV infection on leprosy: a three-year survey inBamako, Mali. Int. J. Lepr. Other. Mycobact. Dis.64 (1996) 383–391.

7. LUCAS, S. Human immunodeficiency virus andleprosy. Lepr. Rev. 64 (1993) 97–103.

8. MASAHIRO, N., ASHKIN, D., HOLLENDER, E., andPITCHENIK, A. E. Paradoxical worsening of tuber-culosis following antiretroviral therapy in patientswith AIDS. Am. J. Respir. Crit. Care. Med. 158(1998) 157–161.

9. RACE, E. M., ADELAN-MITTY, J., KRIEGEL,G. R.,BARLAM, T. F., REIMANN, K. A., LETVIN, N. L., andJAPOUR, A. J. Focal mycobacterial lymphadenitisfollowing initiation of protease inhibitor therapyin patients with advanced HIV disease. Lancet 351(1998) 252–255.

10. SAMPAIO, E. P., CANESHI, J. R. T., NERY, J. A., DUP-PRE, N. C., PEREIRA, G. M., VIEIRA, L. M., MOR-EIRA, A. L., KAPLAN, G., AND SARNO, E. N. Cellu-lar immune response to Mycobacterium leprae inhuman immunodeficiency virus infected individu-als. Infect. Immun. 63 (1995) 1848–1854.

11. SEKAR, B., JAYASHEELA, M., CHATTOPADHYAYA, D.,ANANDAN, D., RATHINAVEL, L., VASANTHI, B.,SUBRAMANIAN, M. and RAO, P. S. Prevalence ofHIV infection and high risk characteristics amongleprosy patients of South India : A case controlstudy. Int. J. Lepr. Other Mycoabct. Dis. 62 (1994)527–531.

12. WHITCUP, S. M. Cytomegalovirus retinitis in theera of highly active antiretroviral therapy. JAMA283 (2000) 653–657.

13. WIRE, R., and KAWAKA, H. J. Type 1 reactions inleprosy, neuritis and steroid therapy: the impact ofthe human immunodeficiency virus. Trans. R. Soc.Trop. Med. Hyg. 88 (1994) 315–316.

14. World Health Organization. Report of a meetingof HIV infection in leprosy. Geneva, 1–2 May1993. W.H.O./CTD/ Lepr/93.3.

Pityriasis versicolor is a common super-ficial fungal infection considered in clinicaldifferential diagnosis of leprosy. Studieshave reported a higher incidence of Pityria-sis versicolor in leprosy patients when com-pared to the general population (4), but thereare no reports of co-localization of thesetwo infections. We describe a 24-year-oldman with P. versicolor lesions localized tothe plaque of borderline tuberculoid (BT)leprosy.

A 24-year-old male patient from Bihar,India was diagnosed with BT Hansen andstarted on multi-drug therapy (M.D.T.)(MBR), rifampicin 600mg and clofazimine300mg once monthly supervised and clo-fazimine 50mg with dapsone 100mg daily.Two months after starting M.D.T., he devel-oped type 1 reaction and was started onprednisolone 40 mg once daily, which wastapered after the reaction subsided. He alsodeveloped xerosis and ichthyosis after start-ing M.D.T. (clofazimine induced) and wasusing coconut oil liberally on the lesions.Four weeks after the initiation of steroids hepresented with hypopigmented itchy lesionsappearing over the pre-existing leprosy le-sions on the chest. On examination, he hadmultiple oval to round hypopigmentedscaly macular lesions on the large patchesof BT disease on his chest and upper back.The lesions were also present in the vicinityof the patch (Fig. 1). However, other lesionsof leprosy on the face, arms and lower back

did not show any such change. The diagno-sis of pityriasis versicolor was confirmed bypotassium hydroxide (KOH) examinationshowing numerous short thick hyphae withclusters of spores. He was given flucona-zole 400 mg single dose following whichthe versicolor lesions cleared in a month(Fig. 2).

P. versicolor distribution as normal florais related to sebaceous gland density, andthus the scalp, face, central chest, and backbear the highest number of fungi (1, 3). Highsebum levels, excessive sweating, warmclimate, application of oil, malnutrition, ad-ministration of systemic steroids, immuno-suppressants, and antibiotics are some ofthe factors that facilitate rapid growth offungus (2).

Leprosy is characterized by partial orcomplete destruction of skin appendagesincluding sebaceous glands, so that co-localization of lesions of leprosy and P. ver-sicolor is a clinical paradox. Ideally, this

Co-localization of Pityriasis Versicolor

and BT Hansen’s Disease1

Tarun Narang, Sunil Dogra, and Inderjeet Kaur2


(ISSN 0148-916X)

1 Received for publication on April 4, 2004. Ac-cepted for publication on July 14, 2005.

2 T. Narang, M.D.; S. Dogra, M.D., D.N.B.,M.N.A.M.S.; and I. Kaur, M.D., M.N.A.M.S., Depart-ment of Dermatology, Venereology and Leprology,Postgraduate Institute of Medical Education and Re-search, Chandigarh, India.

Reprint requests to: Dr. Inderjeet Kaur, AdditionalProfessor, Dept. of Dermatology, Venereology and Lep-rology, PGIMER, Chandigarh-160 012, India; E-mail:[email protected]

FIG. 1. Multiple oval hypopigmented scaly macu-lar lesions of P. versicolor over the patch of leprosy.

206

73, 3 Narang, et al.: Co-localization of Ptyriasis Versicolor and BT Hansen’s 207

should inhibit the growth of malassezia be-cause most of the species of malassezia areobligatory lipophilic, except M. pachyder-matis (1), which is mainly found in domes-tic animals like dogs—causing otitis ex-terna—but can occasionally infect humans(2). However, culture was not done in ourpatient to isolate the species.

In our case, administration of systemicsteroids could be the major factor that pro-moted the development of P. versicolor.However, preferential localization of versi-color lesions to patches of leprosy is per-plexing.

REFERENCES

1. HAY, R. J., and MOORE, M. Mycology. In: Text-book of Dermatology, 6th edn. Oxford: BlackwellScience; 1998. pp. 1277–1376.

2. KWON-CHUNG, K. J., and BENNETT, J. E. Infec-tions caused by Malassezia species. In: MedicalMycology. Philadelphia: Lea and Febiger, 1992.pp. 70–182.

3. MIDGLEY, G., GUEHO, E., and GUILLO, J. Diseasescaused by Malassezia species. In: Topley and Wil-son’s Microbiology and Microbial Infections(Medical Mycology), 9th edn. London: Arnold,1998. pp. 201–214. 1992. pp. 70–182

4. SINGH, M., KAUR, S., KUMAR, B., KAUR, I., andSHARMA, V. K. The associated diseases with lep-rosy. Indian J. Lepr. 59 (1987) 315–321.

FIG. 2. Lesions of P. versicolor cleared after anti-fungal therapy.

COMMENTARY

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In a recent issue of Nature Medicine,Krutzik, et al. report a novel finding on therole of macrophages and dendritic cells inleprosy. In lepromatous leprosy patients’blood and lesions, triggering of dendriticcells was impaired, suggesting a defect inthe initiation of adaptive immunity.

INTRODUCTIONDendritic cells (DC) and macrophages

(Mf) play major roles in innate immunityand provide a first line of defense againstinvading pathogens like Mycobacteriumleprae. Mf and particularly DC also play akey role in the onset of subsequent adaptiveimmunity by triggering pathogen specific T-cell and B-cell responses, and by the for-mation of immunological memory such thatthe immune system will be able to remem-ber previous encounters with pathogenslater in life. Besides representing key cellsof the immune system in combatting bac-terial infections, however, Mf and DC,paradoxically also provide a necessary safe-haven for so-called intracellular bacteria, ofwhich mycobacteria in general and M. lep-rae in particular are prime examples: M. lep-rae is widely believed to be unable to dwelloutside phagocytes, and exploits these other-wise hostile immune cells to survive andreplicate in the human body. Thus, changesin the volatile equilibrium between host andpathogen will dictate whether the host (“im-munity”) or the pathogen (“immune es-cape”) is favored.

Infection of Mf and DC by bacteria is me-diated via a series of cell-surface receptors,including Toll-like Receptors (TLR), theMannose Receptor and the Dendritic cell(DC)-specific surface receptor DC-SIGN(DC-specific ICAM-3–grabbing noninte-grin). These receptors interact with specificbiochemical structures on the surface of bac-teria and trigger phagocytosis, anti-microbialactivity and release of cytokines by the in-fected cell1–3. DC-SIGN ordinarily interacts

with cell surface molecules called ICAM-2and ICAM-3 that are expressed on a varietyof host cells. Interactions between DC-SIGNand ICAM-2 on endothelial cells inducetethering and rolling of immature DCs andthus promotes extravasation of these cellsfrom the blood to inflammatory foci.

Mycobacterial products can trigger TLR-family members and induce immature DCsto differentiate into mature DCs, cells thatare specialised in superior induction of Tcell mediated immunity. Mature DCs re-lease inflammatory cytokines, highly effi-ciently present captured antigens to naïve Tcells and drive their differentiation and acti-vation into effector and memory T cells.Presentation of antigens to T cells isachieved through surface molecules calledMHC class-I and class-II molecules, as wellas MHC class-I-like CD1 molecules. MHCclass-I and class-II molecules present shortprotein fragments of pathogens, whereasCD1 molecules present complementarycomponents such as bacterial lipids.

DC are present in very low numbers inthe blood (<1%), and have therefore beendifficult to study. Most studies on DC havetherefore used human blood monocytes thatwere differentiated in vitro into DC-likecells using the cytokines IL-4 and GM-CSF. This technique allows acquisition ofmuch higher cell numbers that are easier towork with. These cells have a DC-like phe-notype, express both DC-SIGN and CD1b(DC-SIGN+CD1b+), and are able to activateT cells potently.

Summary of the Krutzik, et al.(4) studyTo their surprise, however, Krutzik, et al.

now report that this cell type is not ob-served in vivo in the lymphoid tissues theyanalyzed. Importantly, they find that stimu-lation of cells via their TLR—as a mimic ofbacterial infection-induced differentiationof blood monocytes mostly into either DC-SIGN+CD1b– or DC-SIGN–CD1b+ cells,

73, 3 Commentary 209

whereas only small percentages of doublepositive cells were seen, the predominantcell type induced by the widely used IL-4/GM-CSF combination. Thus, DC-SIGNand CD1b molecules were expressedmainly on different cell types.

Elegant in vitro studies further revealedthat the DC-SIGN+CD1b– cells carried typi-cal Mf markers. The TLR induced expres-sion of DC-SIGN was particularly promi-nent following exposure to the mycobacte-rial 19-kDa lipopeptide that binds toTLR2/1, and was mediated by the innate cy-tokine IL-15. These Mf were able to bindand phagocytose mycobacteria via DC-SIGN, and secreted high levels of inflamma-tory cytokines which are necessary to acti-vate innate and adaptive immunity. In con-trast, TLR induction of DC-SIGN–CD1b+

cells was dependent on GM-CSF. Thesecells resembled DCs and were substantiallymore capable of activating T cells than DC-SIGN+CD1b– cells. DC-SIGN–CD1b+ cellslacked mature DC markers such as CD83,suggesting they had not fully matured yet.Finally, the latter cells were less able to bindBCG compared to the DC-SIGN+CD1b– Mflike cells.

The findings were further extended byexamining the expression of these new celltypes in leprosy. Much like healthy donors,tuberculoid leprosy patients’ monocytesyielded both DC-SIGN+CD1b– Mf-like andDC-SIGN–CD1b+ DC-like cells followingTLR activation. A striking finding was thatlepromatous patients only yielded Mf butnot DC like cells. Such a defect, however,was not observed when the above men-tioned IL-4/GM-CSF combination wasused to generate monocyte derived “classi-cal” DCs in vitro, ruling out a general de-fect in their capacity to generate DC-SIGN–CD1b+ DC-like cells at all. Also, nor-mal levels of DC-SIGN–CD1b+ DC-likecells were seen in lepromatous patients un-dergoing reversal reactions. More interest-ingly, the same cell type distribution wasseen in tuberculoid, lepromatous, and reac-tional lesions. Also in situ, tuberculoid le-sions contained both DC-SIGN–CD1b+ DC-like cells and DC-SIGN+CD1b– Mf-likecells, whereas lepromatous lesions lackedthe latter and mostly contained the formersubset. In addition, the Mf cells could bedemonstrated to contain M. leprae material

in lepromatous but not tuberculoid lesions.Thus, since lepromatous patients lack (lo-cal) DCs the implication of these findingsmay be that they are unable to induce andactivate proper T cell reponses to eradicateM. leprae.

Questions and discussionThe surprising findings by Krutzik, et al.

obviously need confirmation and extensionin other systems, but are certainly new andprovocative. The finding that DC-SIGN+

cells belong mostly to the Mf- but not DC-class is even highly provocative. Neverthe-less, some caution may be warranted inoverinterpreting this data to indicate thatmany DC-studies in the past have beenperformed on cells that hardly or not at allexist in vivo. Before such conclusions canbe drawn more work is clearly needed. Asmall subset of cells in the Krutzik, et al.study actually is double-positive (DC-SIGN+CD1b+), both in vitro and in vivo, butmay simply be a minor population thatcould be selectively expanded by IL-4/GM-CSF. It should be pointed out also that vari-ous DC and Mf subsets exist (5,6), and thatthere may even be a continuum of phago-cyte types, each with its own level of plas-ticity. This would even further allow thesecells to adapt to various conditions and ac-quire different phenotypes depending onthe precise (cytokine-) environment (5). Themicro environment in leprosy skin lesionsor tonsils may not be ideal to favor “doublepositive DCs,” but this does not excludetheir existence or relevance in the humanimmune system. Of interest, also other Mflike subsets (CD16+DC-SIGN–; unfortu-nately, it is not indicated whether these cellswere CD1b+) were found in lepromatous le-sions, pointing to the existence of a morecomplex local repertoire of phagocytic cellsin leprosy lesions.

The sample size of patients and lesionsstudied by Krutzik, et al. seems rather small(the numbers of samples studied are not al-ways clearly indicated in the manuscript) sothat it is as yet uncertain to what extent thefindings in the individuals analysed can begeneralised to human leprosy per se.

The mechanisms behind the impairmentof lepromatous patients’ cells in inducingDC like cells remains unexplained. It is im-portant to resolve this, as this may provide


novel therapeutic angles. The question iswhat phenotype would result when cellswould have been stimulated more physio-logically with M. leprae instead of unre-lated TLR stimuli, but no data are reportedon this issue. Furthermore, a general im-pairment in DC function in lepromatousleprosy is not easily reconciled with thecharacteristic and rather specific defect inT-cell responsiveness to antigens of M. lep-rae in lepromatous leprosy: general DC de-fects would be expected to lead to moregeneral defect in T-cell responses, but thisis not typically the case in lepromatousleprosy.

It also remains unknown if the defect inlocal DC like cells in lepromatous leprosylesions is permanent or not: is this defectdisease-activity dependent, or is it rather apermanent characteristic of lepromatousleprosy susceptible individuals? And if so,what are the host (genetic) factors that drivethis defect?

Whatever the issues to be resolved, thestudy by Krutzik, et al. sheds new light onMf and DC in innate and adaptive immuneresponses in general and in leprosy in par-ticular. Therapies to activate and expandDCs in lepromatous patients may help tocontrol disseminating infection.

—Tom H. M. Ottenhoff, M.D., Ph.D.,Michèl R. Klein

Dept. Immunohematology and Blood Transfusion

Leiden University Medical Center, Leiden, The Netherlands

Correspondence to: Tom H. M. Ottenhoff,M.D., Ph.D., Dept. Immunohematologyand Blood Transfusion, Leiden UniversityMedical Center, Albinusdreef 2 2333 ZALeiden, The Netherlands,E-mail: [email protected]

REFERENCES1. TAILLEUX, L., SCHWARTZ, O., HERRMANN, J. L.,

PIVERT, E., JACKSON, M., AMARA, A., LEGRES, L.,DREHER, D., NICOD, L. P., GLUCKMAN, J. C., LA-GRANGE, P. H., GICQUEL, B., and NEYROLLES, O.DC-SIGN is the major Mycobacterium tuberculo-sis receptor on human dendritic cells. J. Exp. Med.197(1) (2003) 121–127.

2. GEIJTENBEEK, T. B., VAN VLIET, S. J., KOPPEL, E.A., SANCHEZ-HERNANDEZ, M., VANDENBROUCKE-GRAULS, C. M., APPELMELK, B., and VAN KOOYK,Y. Mycobacteria target DC-SIGN to suppressdendritic cell function. J. Exp. Med. 6;197(1)(2003) 7–17.

3. KAUFMANN, S. H., and SCHAIBLE, U. E. A danger-ous liaison between two major killers: Mycobacte-rium tuberculosis and HIV target dendritic cellsthrough DC-SIGN. J Exp Med. 197(1) (2003)1–5.

4. KRUTZIK, S. R., TAN, B., LI, H., OCHOA, M. T.,LIU, P. T., SHARFSTEIN, S. E., GRAEBER, T. G.,SIELING, P. A., LIU, Y. J., REA, T. H., BLOOM, B. R.,and MODLIN, R. L. TLR activation triggers therapid differentiation of monocytes into macro-phages and dendritic cells. Nat Med. 11(6) (2005)653–660.

5. Verreck, F. A. W., de Boer, T., Langenberg, D. M.L., Hoeve, M. A., Kramer, M., Vaisberg, E.,Kastelein, R., Kolk, A., de Waal-Malefyt, R., andOttenhoff,T. H. M. Human IL-23-producing type-1 macrophages promote but IL-10-producingtype-2 macrophages subvert immunity to(myco)bacteria. Proc. Natl. Acad. Sci. USA 101(2004) 4560–4565.

No country in the world can be more con-cerned about leprosy than India, as Indiarepresents nearly 76% of the global burdenof the disease (1); India alone represents87% of prevalencea and 90% of new de-tected cases in the South East Asian Region.This clearly indicates that India is a keycountry in efforts to eliminate leprosy. (2)

The year 2005, which is the extendeddate for global elimination of leprosy, hasbrought new challenges to Indian leprosyworkers and administrators. In their effortsto reach targets many steps are being takenby leprosy authorities of the Government ofIndia, most of them with the support of theinternational agencies and NGOs. The Gov-ernment of India, in its efforts to eliminateleprosy through its National leprosy elimi-nation program (NLEP), has constantlybeen in consultation with organizationssuch as the World Health Organization(WHO), Global Alliance for Elimination ofLeprosy (GAEL), and Non Governmentalorganizations (NGOs) such as the Interna-tional Federation of Anti-Leprosy Associa-tions (ILEP), and others.

India being the seventh largest and sec-ond most populated country in the world,the organization of leprosy services reach-ing to many distant parts of India was anenormous task. The WHO MDT program,which was initially introduced in India in1983, could only be extended to all parts ofthe country by the end of 1995. (3) In the re-gion of Jharkhand, for example, which ishighly endemic for leprosy, MDT was in-

troduced in only 1994–1995. (4) Thus, sincethe WHO in 1991 had declared its intentionto reach the global elimination target by2000, program mangers were already plan-ning for the elimination of leprosy even asthe complete coverage of leprosy in allparts of India had barely been achieved, andinfrastructure had just been put in place. Asnoted, however, this goal was extended to2005 as WHO observed that 12 countrieswould not be able to achieve the elimina-tion target by the year 2000.

Meanwhile, the MDT therapy in itselfhas undergone significant modificationswith respect to the duration of therapy andalso to the criteria for inclusion of patientsinto MDT-PB and MDT-MB groups. (5)Various methodologies were adopted in theleprosy program with the belief that theywould benefit the patient and at the sametime bring about rapid reduction in theprevalence rates of leprosy in India alongwith the rest of the world. Two such ex-amples were the introduction the of single-dose ROM (rifampicin, ofloxacin andminocycline) therapy for single skin lesionleprosy (SSL –PB) (6) and initiation of theLeprosy Elimination Campaigns (LECs) allover India.

The rationale for ROM therapy was al-ways controversial as it was based on asingle multi-centric double blind field trialstudy whose results actually showed thatROM therapy was marginally less effectivethan MDT-PB in treating SSL-PB patients.(7) These results notwithstanding, ROMwas introduced in India probably becausesingle skin lesions comprise a significantlyhigh proportion (up to 60%) of leprosy pa-tients in this part of the world. (5) For rea-sons not detailed, ROM therapy was dis-

EDITORIALEditorial opinions expressed are those of the writers.

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a The author has followed the WHO convention inusing “prevalence” and “incidence” of leprosy to referto the total number of registered cases, and the numberof new cases, respectively. Ed.


continued five years after its introduction.However, the names of the patients who re-ceived ROM-single day therapy were re-moved from registers, as they were consid-ered to have completed their treatment.

During the same time, Modified LECs(MLECs) were being conducted all over In-dia and the state machinery participatedwith enthusiasm. There have been fiveMLECs between 1997 and 2004. Four na-tionwide MLECs have been conducted inthe country as special efforts towards earlydetection of leprosy cases and their prompttreatment with MDT. The Fifth MLEC wasconducted in eight high priority States dur-ing 2003–04.

In September 2003, with only one andhalf years to go to meet the deadline of lep-rosy elimination in India by 2005, the Gov-ernment of India and the WHO organized ameeting in Goa of health secretaries of In-dia’s major leprosy-endemic states. It wasin part a follow-up to a meeting held inTokyo in June, 2002. Several importantNGOs also participated in this meeting. TheGoa meeting recommended that the sevenstates in India where the prevalence rate ofleprosy was between 1–2/10,000 wouldwork hard to achieve the elimination targetby March, 2005. These endemic states andunion territories were advised carry out theStrategic Plan of Action discussed duringthe meeting in specified areas during thenext one-year period. (8)

After the Goa recommendations, furthermeetings were held at various state head-quarters and leprosy directorates of India toencourage these program officers to reachthe elimination target by March, 2005. Todo this, new instructions were given by thehealth authorities of Andhra Pradesh to thefield staff. These instructions are called the“Kathmandu recommendations.” They are:

1. Stop all active search for case detec-tion.

2. Do not register cases before reconfir-mation by experienced staff.

3. Declare patients as released from treat-ment (RFT) and delete the names of thesepatients from registers as soon as they re-ceive the last pulse of treatment.

4. Do not register single lesion cases fornow.

The first three instructions were throughofficial documents and office orders to the

field workers.9 The last instruction was averbal instruction; such verbal instructionswere not limited to the state of AndhraPradesh but were also given in other statesof India.

Let us examine these directives. First, thefirst directive ‘to stop all active case detec-tion’ is endorsed on the website of theWHO representative of India (2), whichstates that ‘at present, the emphasis for de-tection is based on routine voluntary report-ing, with no more routine active case detec-tion’. The WHO document containing theplan for leprosy work for the period2006–2010 proposes the use of case detec-tion as the main indicator to monitorprogress. (10) It states that the importantcomponent of the leprosy control programis timely detection of new cases, but it rec-ommends that case-finding should mainlybe focused on promoting self-reporting.

With falling prevalence rates and the goalof elimination in sight, the focus of the lep-rosy program has shifted from an activesearch for new cases to voluntary reporting.However, there are some who feel that lep-rosy elimination cannot be accomplishedwithout full geographic and population cov-erage and without intensified effort to treatall patients. (11) It is true that intensifyingcase detection may lead to over-registrationand over-reporting of cases, but this shouldnot mean that we do away with activesearching for new cases. Although WHOevaluators observed that the number of newcases detected in LECs included a signifi-cant proportion of wrong diagnoses, re-registration, and ‘non-existent patients’b inprograms (12), not all evaluators foundLECs to be the cause of over diagnosis orre-registration. Some evaluation teams ac-tually diagnosed additional new casesmissed by the LEC teams. (13)

A balance needs to be struck between de-tecting all hidden cases and avoiding re-registration and wrong diagnoses. Intensiveactive case detection conducted throughMLECs for the discovery of new casesproved to be one of the most successfulhealth care interventions undertaken in In-dia in recent years, particularly in the statesof Bihar and Orissa. (14) It is only fair to say

bSee also Editorial, Int. J. Leprosy 72: 501, 2004. Ed.

73, 3 Editorial 213

that in a vast and diverse country like India,a combination of strategies and methods isrequired to reach varied target groups. (15)

The second guideline, which is re-validation by an experienced health workerwithin one month of the diagnosis of lep-rosy could serve to delay the inclusion ofnew patients into the registers and hence tohelp keep the monthly new case detectionrate and prevalence rate within limits untilthe elimination goal is achieved.

The third guideline also serves a similarpurpose, by deleting the patient from caseregisters and advancing the RFT date by amonth. In such cases the last month’s ther-apy becomes accompanied MDT.

The fourth and important verbal instruc-tion was confirmed with various healthworkers of Hyderabad, Andhra Pradesh. Is-suance of similar verbal instructions waspersonally confirmed with health workersof one other state (Delhi) which is in thenorthern part of India, while AndhraPradesh is in south India. It is not unreason-able to assume that such instructions maynot be limited to these two states.

Single-lesion leprosy cases have alwayshad a special place in leprosy. The percent-age of single-lesion disease among leprosypatients in India is quite high. (7) Althoughsingle lesion leprosy is considered pau-cibacillary, multibacillary leprosy may alsopresent as a single lesion. (16, 17)

Generally it is believed that single-lesionleprosy cases have no transmission poten-tial and are not of great significance fromthe public health point of view, as a highpercentage of these case show a tendencyfor self-healing. (18) However, large num-bers of single-lesion cases detected repre-sent an exposure of the population to areservoir of infection which may contributeto the number of new cases and hence can-not be ignored. On the whole it is believedthat at least a proportion of single-lesionleprosy will, without treatment, progress tomulti-lesion leprosy. (19)

Some workers wanted single-lesion casesto be excluded from the number of leprosypatients when calculating new case detec-tion rates, arguing that they do not con-tribute to the spread of the disease. (20)However, many studies have suggested thatuntreated MB patients do not represent thesole source of infection and that household

contacts of PB patients have also beenshown to be at a greater risk of developingthe disease than non-contacts, although therisk is smaller than that of contacts of MBpatients. (21) It is unwise and unethical toexclude single-lesion leprosy cases fromthe registers as new cases and to deny ther-apy. However, such non-registration/non-inclusion of single-lesion cases will sub-stantially help the program mangers tobring down the number of new cases andthus assist in reaching the elimination targetin time.

What about the present leprosy statisticsof Andhra Pradesh? The reported tentativeaverage prevalence rate of leprosy inAndhra Pradesh state (with 22 districts) inmid 2004 (22) was 1.78/10,000, with 10 dis-tricts having rates of 1–2/10,000, 9 districtshaving rates of 2–3/10,000, and 2 districtswith rates of 3–5/10,000. In the epidemio-logical indicators of new leprosy cases pre-pared for Andhra Pradesh up to March2005, the percentage of child cases was19.8% and of MB cases was 28.2%. Sched-uled castes (SC) and Scheduled tribes (ST),who are the under-privileged of the societyand live in areas with difficult access, con-stitute 33.5% of all new cases. A large pro-portion of children among the newly de-tected patients is a sign of active and recenttransmission of infection, (21) especiallywhen there is no active search or campaignfor case detection. The large number of lep-rosy cases being detected among SC andST populations indicates that focusedhealth and communication campaigns arerequired to improve access to informationand health services of these populations,particularly to those in remote areas.

In the neighboring state of Tamil Nadu,the leprosy prevalence rate was 1.4/10,000in the year 2004. (23) On May 15th of 2005the health ministry of Tamil Nadu has de-clared (24) that the present prevalence rate inTamil Nadu is only 0.85/10,000, whichmeans that it has already reached the elimi-nation target. As other states are also en-couraged to reach the targets, it is bound tohappen sooner rather than later. It has al-ready been reported in media that foursouthern states (Tamil Nadu, AndhraPradesh, Kerala and Karnataka) havereached the elimination target of a preva-lence rate of <1/10,000 population by May

of this year. (25) This was substantiated by a‘news and notes’ report of a regional con-ference on leprosy held at Chennai, pub-lished by the Indian Journal of Leprosy. (26)

It will not be out of context here to con-sider the experience of the NationalMalaria Control Program of India, whichwas initiated in 1953—a story of failure.Initially it made rapid gains so that by1961, the annual number of new cases reg-istered was only 50,000. However, a resur-gence of malaria was reported from 1962onwards. By 1976, 6.4 million new caseswere reported. Presently, the annual inci-dence is around 2 million. (27) Some of theimportant causes detailed for the failure ofthe National Malaria Eradication Programof India (28) were as follows: diversion ofthe work force, promoting newer prioritieswhen greater effort was needed to root outthe last pockets of endamicity, entrustingwork to multi-purpose and basic healthworkers who were ill prepared for the taskand, above all, laxity in national commit-ment and determination. It was also men-tioned that the third world countries did notfully understand the epidemiological ‘rulesof the game’. In short, the present resur-gence of malaria is due to the relaxation ofeffort.

Similar indicators already exist in presentleprosy program of India. Added to theseare newer national priorities such as HIVand a resurgence of tuberculosis. Dilutionand relaxation in the efforts of the NLEPhas already set in.

What is being presented here is commonknowledge in India, and the government or-ders cited were circulated openly and werenot privileged information. Most of theNGO`s participating in NLEP in Indiawould also be aware of these directives andfigures, as they work very closely with thecentral and state governments of India andare participate in the national and interna-tional meetings and consultations. Thecredit for decreasing the leprosy prevalenceand the efficacy of leprosy control programin India should be shared equally by GOI,WHO and NGOs. However, for reasons un-known, there seems to be a great hurry onthe part of everyone involved with NLEP inIndia to reach the elimination target by theend of 2005 and to get on with a vision ofleprosy beyond 2006.

—P. Narasimha Rao, Assistant ProfessorD.V.S. Pratap, Professor and Head

Department of Dermatology and Leprosy,Gandhi Medical College,Secunderabad, Andhra Pradesh, India

Correspondence to: Dr. Rao, B-48, incometax colony, Mehdipatnam, Hyderabad – 500028 India.E-mail: [email protected]

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2. WORLD HEALTH ORGANIZATION REPRESENTATIVE TO

INDIA. http://www.whoindia.org/CDS/CD/Leprosy/Leprosy.htm accessed on 23rd June, 2005

3. RAO, C. K. Implementation of WHO MDT in In-dia 1982–2001. In: Multi drug Therapy AgainstLeprosy, Development and Its Implementation overthe Past 25 Years. WHO: Geneva, 2004: 92–105.

4. PANDA, B. K. Leprosy elimination in Jharkahand.Indian J. Lepr. 76 (2004) 137–138.

5. RAO, P. N. Recent advances in the control pro-grams and therapy of leprosy. Indian J. Dermatol.Venereol. Leprol. 70 (2004) 269–76

6. Modified Guidelines on MDT Regimen to be Fol-lowed under NLEP. Directorate General (Lep),Nirman Bhavan, New Delhi, 1997

7. LOCKWOOD, D. N. Rifampicin/minocycline andofloxacin (ROM) for single lesions—what is theevidence? Lepr. Rev. 68 (1997) 299–300

8. Eliminating leprosy: health secretaries meet inGoa to speed up progress: Bulletin of the LeprosyElimination Alliance 3 (July–Dec. 2004) 9–10

9. R C LETTER 25976/lep.V/2005 dated 3-2-05. Officeof the directorate of Health, Andhra Pradesh., Hy-derabad

10. Global Strategy for Further Reducing the LeprosyBurden and Sustaining Leprosy Control Activities(Plan period: 2006–2010). World Health Organ-ization, 2005. WHO/CDS/CPE/CEE/2005.53

11. NOORDEEN, S. K. LEA Bulletin July–Dec 2004http://lepelall.com/fromeditordesk.htm accessedon 23rd June 2005.

12. Report on the Sixth Meeting of the WHO Techni-cal Advisory Group on the Elimination of Lep-rosy. Geneva, 9 and 10 February, 2004.

13. SEKAR B., KOTHANDAPANI, G., PRABHAKAR RAO,T., and KRISHNAMURTHY, P. Evaluation of themodified leprosy elimination campaign in a highleprosy endemic district of Jharkhand. Indian J.Lepr. 75 (2003) 233–242

14. DHARMSHAKTU, N. S., BARKAKATY, B. N., PAT-NAIK, P. K., and ARIF, M. A. Progress towardselimination of leprosy as a public health problemin India and role of modified leprosy eliminationcampaign. Lepr. Rev. 70 (1999) 430–439.


15. MUKHERJI, S., PRIYADARSHI, M., and SINGH, S.Communication In public health programs: TheLeprosy Project in India. March, 2005. The Inter-national Bank for Reconstruction and Develop-ment/The World Bank, 1818 H Street, NW Wash-ington, DC 20433.

16. JOB, C. K., KOHKONEN, M. E., JACOBSON, R. R.,and HASTINGS, R. C. Single lesion sub polar lep-rosy and its possible mode of origin. Int. J. Lepr.Other Mycobact. Dis. 57 (1989) 12–19.

17. RAMESH, V., SAXENA, U., MISRA, R. S., MUKHERGEE,A., and RAVI, S. Multibacillary leprosy presentingas a solitary skin lesion; report of three cases andits significance in control programmes. Int. J.Lepr. Other Mycobact. Dis. 59 (1991) 1–4.

18. BROWNE, S. G. Self healing leprosy—report of2749 patients. Lepr. Rev. 45 (1974) 104–111.

19. PONNIGHAUS, J. M. Diagnosis and management ofsingle lesions in leprosy. Lepr. Rev. 67 (1996)89–94

20. REVANKAR, C. R., GANDEWAR, K. L., and GANAPATI,R. Interpretation of data on monolesion leprosycase v/s total new case detection rate. Lepr. Rev.66 (1995) 324–325

21. Epidemiology and Control: Report of the Inter-national Leprosy Association Technical Forum,Paris, France. Indian J. Lepr. 74 (2002) S63–S74.

22. Leprosy in India—the scene in different states in2004. Bulletin of the Leprosy Elimination Al-liance 3 (July–Dec. 2004) 3–8.

23. Health and Family Welfare Department, Govern-ment of Tamil Nadu http://www.tnhealth.org/dphdblep.htm accessed on 23rd June, 05

24. State Committed to Eradication of Leprosy: Min-ister, The Hindu, May 16th, 2005 http://www.hindu.com/2005/05/16/stories/2005051612560500.htmaccessed on 24th June, 2005

25. CURRENT TOPICS. Deccan Chronicle, Chennai,May 17th 05; www.nipponfoundation.or.jp/eng/current/2005708/20057081.html. Accessed on23rd June 05

26. NEWS AND NOTES. Indian J. Lepr. 77 (2005) 97–99.27. MALARIA SITUATION (1998–2001) http://www.

whoindia.org/CDS/CD/RBM/rbm_annexure_01.htmRoll Back Malaria accessed on 24th June, 05

28. PARK, J. E., and PARK, K. Text Book of Preventiveand Social Medicine, 10th edn. Banarasidas BanotPublishers: Jabalpur, India. 1985; 316–329.

73, 3 Editorial 215

We are told that after 2005 we will entera new, “post-elimination” era, during whichleprosy cases will be rare. It is understand-able that a new era requires a new strategy,which is why WHO/AFRO is circulating astrategy paper, which will be discussed atthe AFRO annual meeting on leprosy, to beheld from 27th to 29th June 2005 in Braz-zaville, by the leprosy program managers ofthe WHO African region, and by the repre-sentatives of international non-governmentalorganizations. Nevertheless, the strategy isnot really new; in fact, it does not differsubstantially from the “Final Push”strategy. It remains “elimination”-oriented,and the quality of leprosy services contin-ues to be ignored.

MY COMMENTS1. The WHO/AFRO strategy misses a

golden opportunity to re-define the prioritiesof leprosy control programs.

During the “elimination” era, the onlypriority was achieving the elimination tar-get by bringing down the prevalence rate atany price. Hence, a number of simplifiedtechniques for diagnosis and treatment ofleprosy were implemented intensively,without paying adequate attention to qualitycontrol. At the same time, many essentialactivities — e.g., prevention of disability —were completely neglected in the field, pri-marily because these activities were unre-lated to the prevalence. As a consequence,the quality of leprosy services was poor andachievement of the final goal of leprosycontrol was jeopardized. Now, in the “post-elimination” era, because political pressureto achieve the elimination target is dimin-ished, the leprosy programs could, and

should re-define the priorities of the activi-ties, by focusing on quality of diagnosis andtreatment and prevention of disability. Al-though the title of the strategy paper in-cludes the phrase “to maintain the quality ofleprosy services,” the relevant paragraphsin the text are extremely sloppy and vague,and fail to suggest concrete actions (see5.2.2 and 5.2.3). “Prevention of disabilities”is mentioned only once (p. 3), but withoutdetails; one might therefore wonder how se-riously the strategy deals with the issue ofquality.

2. The strategy paper stubbornly up-holds the poorly-justified technical policiesthat have already damaged the quality ofleprosy services during the “elimination”era.

• The strategy paper over-estimates thesensitivity with which leprosy can bediagnosed using only clinical criteria,and under-estimates the important roleof the skin-smear (see 5.2.1) in the di-agnosis of smear-positive MB leprosypatients and relapsed MB patients, whorepresent the major sources of leprosyinfection in the community. Apparently,the authors of the strategy paper do notunderstand that a significant proportionof smear-positive MB patients (espe-cially those close to the lepromatousend of the spectrum), and the great ma-jority of relapsed MB patients cannotbe diagnosed without skin-smears; thestrategy paper therefore fails to recom-mend re-introduction of skin-smearservice in the field.

• Supervised administration (or directlyobserved treatment) of the monthly

EDITORIALEditorial opinions expressed are those of the writers.

Comments on WHO/AFRO’s “Post-Elimination” Strategy

Paper: A New Bottle with Old Wine of the “Final Push”


(ISSN 0148-916X)

216

component of MDT regimens is an im-portant element of the multi-drug ther-apy for leprosy, which ensures that thepatients take the right drugs, in the rightdoses, at the right intervals. However,the strategy paper continues to ignorethe supervised administration of themonthly component of MDT regimensby promoting “flexible MDT,” espe-cially so-called “accompanied MDT”or “self-supervision” (which is, in fact,no supervision) (see 5.2.8). The recom-mendation that the patient who has re-ceived the total amount of MDT drugsat the beginning of treatment and “whois not seen in a health facility at the endof his treatment should be consideredas having no concern on his conditionand being cured” (original phrase in5.2.8) is ridiculous; it is virtually thesame as declaring that the patient iscured at the time he receives the totalamount of MDT drugs.

• Relapse and emergence of drug resis-tance are the most serious outcomes ofpoor treatment in any large-scale treat-ment campaign including MDT for lep-rosy, and all efforts should therefore bemade to prevent or reduce their occur-rence. Surprisingly, the strategy paperomits any mention of detection and pre-vention of relapse after MDT and emer-gence of rifampicin-resistant leprosy, asif these phenomena have not been en-countered and will not occur; such ablindly optimistic attitude is an invita-tion to disaster.

• As already mentioned, “prevention ofdisability” is grossly neglected.

MY RECOMMENDATIONS1. The strategy paper should be thor-

oughly revised. Involvement in the revisionof the program managers and representa-tives of NGOs and scientific community ishighly desirable.

2. The priority of leprosy control activityshould focus on quality and sustainabilityof leprosy services, especially in the areasof diagnosis, treatment and prevention ofdisability.

3. The efficiency of integration and de-centralisation should be reviewed.

4. The potential role of general healthworkers in case-finding and case-

management should be reviewed and, pos-sibly, revised. When leprosy cases becomerare, it would be more logical that generalhealth workers at the most peripheral levelbe responsible only to detect suspectedcases; the diagnosis of leprosy will be vali-dated or confirmed by more experiencedworkers from either the district or the refer-ral center.

5. Serious efforts should be made to in-crease the number and improve the qualityof the referral centers; ideally, each en-demic district will have one. The role ofthese centers should be defined in detail.

6. Training of health workers should bean important component of the strategy.With support from NGOs and other part-ners, AFRO should provide assistance totrain the trainers for each of the nationalleprosy programs. At the country level, ba-sic training must be provided to those work-ers responsible for the leprosy program atthe national, intermediate and district lev-els, to make certain that they are able tomanage the program and deal with patientsindependently; for those workers at themost peripheral level, training is still neces-sary but needs only to be task-oriented.

7. The recommendation that leprosy mightbe diagnosed by the presence of anaestheticskin lesions alone is problematic, becauseabout 30 per cent of leprosy lesions are non-anaesthetic, and most of these are observedin smear-positive MB cases. To improve thequality of diagnosis, instead of relying upona single criterion, leprosy should be diag-nosed by presence of one or more of thethree cardinal signs (anaesthetic skin le-sions, thickened peripheral nerves, andacid-fast bacilli in the skin-smear or biopsyspecimen). Diagnosis of leprosy willmainly be the responsibility of health work-ers who have received better training andhave access to skin-smears, presumably atthe district level or at the referral centers.

8. The skin-smear service must be rein-troduced in the field, beginning in leprosyendemic areas; the skin-smear service maybe associated or combined with the labora-tory facilities of the tuberculosis program.

9. To improve the quality of MDT treat-ment, adherence of patients to treatmentshould never be compromised; therefore,supervised administration of the monthlycomponent of MDT must be ensured. The

73, 3 Editorial 217

supervisor could be one of the staff in thehealth facility; for those patients who mayhave difficulty to visit the health facilityonce monthly, the supervisor could be acommunity health worker or a trained localcommunity member. In general, membersof the patient’s family should not serve astreatment supervisor.

10. To detect relapse after MDT and theemergence of rifampicin-resistance, post-MDT surveillance should be reintroduced,and skin-smear positive MB patients shouldbe examined both clinically and microscop-ically (skin-smears) once yearly for as longas 7 years after completion of MDT. AFROshould identify the facilities that are capableof testing the rifampicin-susceptibility of therelapsed strains detected by the programs.

11. Because the prevention of disability

has been neglected for too long, the na-tional leprosy program should make specialefforts to initiate this activity, includingtraining of health workers, health educationof the patients and the community, identifi-cation and upgrading of the referral centers,supplying medications, and providing so-cial and financial support to the patientswhen necessary.

12. Community participation in leprosycontrol activities should be encouraged, es-pecially in the areas of case-finding, case-holding, prevention of disability and socialrehabilitation.

—Baohong Ji, M.D.

Association Française Raoul Follereau,Paris, France


TO THE EDITOR:

In 1998, we had suggested the quantitativemethod of sensory assessment of face andtesting sites for the limbs (4). The followingimprovement to the original method may berequired and this is based on our clinical ex-perience in using this technique in a referralcenter with specialists and time available.

The 10 sites for testing sensation on theface, hands, and feet are unchanged. Wesuggest two changes with the Semmes-Weinstein monofilaments as a result of therecent understanding of the normal sensa-tion of the hands and feet. Similarly, themethod to score sensory nerve status is alsoaltered. This is because the clinicians ex-pressed that the norms for muscle gradingare: “zero” indicating flaccidity, and a max-imum score of 5 given for normal muscula-ture; this was reversed in our quantitativesensory testing. In order to have a unifor-mity between sensory and muscle testing,we recommend the changes depicted in thefollowing assessment form. In the revisedform, 0 to 4 sensory grading system is fol-lowed for the hands. For the foot, 0 to 3grading system is used because their sen-sory function is less than that of hands,which have to manipulate objects and re-quire well developed sensory nerve end-ings. For the face, a 0 to 3 grade sensorythreshold scale was used with the interpre-tations suggested by Premkumar, et al. (4).

The interpretations presented for the footand hand are also based on the following

previous scientific studies. Krotoski pub-lished the details on interpretation for thehands (1). Similarly, Birke, et al. interpreted10 g filament as the level of protective sensa-tion in leprosy patients (2). Kets, et al. studydemonstrated that the touch sensibilitymonofilament threshold screening in healthyNepalese population were 0.2 g for handsand 2 g for feet (3). Since all of the SouthAsian population is likely to be similar tothat of Nepalese, we had taken the interpre-tation of this study and made a small modifi-cation to Krotoski’s hand sensory battery byremoving 0.05 to 0.07 g filament as an in-strument to test normal sensation. In theoriginal neurological mappings by Weinsteindemonstrated the higher sensitivity in theface; the mean threshold of males to be 0.02g; females, 0.018 g (5). Despite the abovework in neurology, in the facial sensation as-sessment we suggest using a filament thatgives a force of 0.05 to 0.07 g. It will behigher than the threshold for the face andwill avoid false negative responses for thefollowing reason: The lowest sensory thresh-old in normal individuals quoted in the We-instein article is in the laboratory situation,which cannot be duplicated in clinics. There-fore, the next higher threshold may be re-quired to increase the test sensitivity.

We are also aware that more studies areneeded to answer the following researchquestions arising from this work. For in-stance, the lack of testing the corneal sensa-tion to an extent limits the usefulness oftesting facial sensation. Since this study

CORRESPONDENCE

This department is for the publication of informal communications that are of interestbecause they are informative and stimulating, and for the discussion of controversialmatters. The mandate of the JOURNAL is to disseminate information relating to leprosy inparticular and also other mycobacterial diseases. Dissident comment or interpretation onpublished research is of course valid, but personality attacks on individuals would seemunnecessary. Political comments, valid or not, also are unwelcome. They might result ininterference with the distribution of the JOURNAL and thus interfere with its prime purpose.

Quantitative Measurement of Sensory

Impairment in Referral Centers1

219


(ISSN 0148-916X)

THE FIGURE.


73, 3 Correspondence 221

confines in using the instrument of S-W fil-aments in testing only the skin in the limbsand face, and not the cornea, it is beyondthe scope of this work. In the previous workon facial sensory testing, the authors hy-pothesized that the corneal sensation as-sesses only the ophthalmic branch of thetrigeminal nerve (4). The other two branchesof the nerve usually go unexamined. Facialsensory testing, we have suggested, willgive quantitative sensory information for allthree branches of the trigeminal nerve.Hence, the specific research question wouldbe whether testing the facial sensationaround the eyes could indicate corneal in-sensitivity?

There is also a research question relatedto the testing sites: whether further reduc-tion in the number of testing points would

be more beneficial than the 25 sites we pro-posed? Our suggestion for further testingsites reduction is to 10; for example, twoeach for facial, great auricular, ulnar, me-dian and posterior tibial. A further scrutinyis also needed into the validity of the facialsensory loss and its interpretation to func-tion that we have suggested in our previouswork (4), in a larger population.

Method used to score sensory nerves sup-plying face, hand and feet

Ten testing sites have been selected foreach hand, foot and face. Three testingpoints have been identified for each trigemi-nal and 2 for each great auricular nerve: 4 forulnar, 6 for median and 10 for posterior tib-ial. If the patient feels 0.05 g filaments in theface and 0.2 g in hands or 2 g filaments in

KEY FOR GRADING

FACENot Felt Felt Interpretation Score

0.05 g Normal superficial sensation 30.05 g 0.2 g Normal superficial sensation diminished 20.2 g 2.0 g Loss of normal superficial sensation—deep sensation intact 12.0 g Total loss of pressure sensation 0

FOOTNot Felt Felt Interpretation Score

2 g Normal superficial sensation 32 g 10 g Normal superficial sensation lost—protective sensation intact 2

10 g 300 g Protective sensation lost—deep pressure sensation intact 1300 g Total loss of pressure sensation 0

HANDNot Felt Felt Interpretation Score

0.2 g Normal superficial sensation 40.2 g 2 g Normal superficial sensation diminished 32 g 4 g Superficial sensation lost—protective sensation intact 24 g 300 g Protective sensation lost—deep pressure sensation intact 1

300 g Total loss of pressure sensation 0

SUMMARY TABLE.

Number of Maximum Score Maximum ScoreBody Nerves Sites Tested For Each Nerve For EachPart (per nerve) Right Left Body Part

Face Trigeminal 3 /9 /9 /18Auricular 2 /6 /6 /12

Hands Ulnar 4 /16 /16/40

Median 6 /24 /24Foot Posterior Tibial 10 /30 /30 /30

“Zero” score indicates maximum sensory loss. The denominator indicates normal sensation.Source: Int.. J. Lepr. Other Mycobact. Dis. 66 (1998) 348–355 with revisions in 2004.


Serologic Recognition of Low Molecular Weight

Mycobacterial Protein Fractions in Lepromatous

Patients with Type II Reactions (ENL)

TO THE EDITOR:

Hansen’s disease is a mycobacterial infec-tion that produces physical disabilities. Theprogression of the disease is slow and indo-lent but in some cases there are changes inthe immunological status with the develop-ment of acute episodes represented by reac-tional states. Many of these reactionalepisodes occur after treatment has been final-ized and, therefore, it is important to clarify

whether they constitute relapses. We wishedto determine if specific patterns of serologicrecognition of mycobacterial proteins wereassociated with Type 2 reactional states inlepromatous patients. Serum samples weretaken from 12 adult patients, mean age of 43± 16 yrs, with a predominance of males(80% M and 20% F), who were undergoinga Type 2 reactional episode (erythema, no-dosum leprosum, ENL). These sera were di-vided in two groups of six sera each: sera

feet on each point, three score is given to thatsite for face and foot. Four score is given tothat site in hand as four filaments are usedfor the palmar surface: two for not feelingthat filament in face and foot and so forth.

Thus, total loss of sensation at a pointwill be scored as zero for the face, hand,and feet; i.e., since there are 3 testing pointsfor trigeminal, the maximum sensory lossper this nerve is scored, as 0 + 0 + 0, whichis 0. Normal sensation will be scored as 3 +3 + 3 = 9. The maximum score for normalsensation of the following nerves are statedbelow and these are indicated as denomina-tors in the first Table.

Nerves Maximum score per intact nerve

R. Trigeminal 9L.Trigeminal 9R.Great auricular 6L.Great auricular 6R.Ulnar 16L.Ulnar 16R.Median 24R.Median 24R.Posterior tibial 30L.Posterior tibial 30

—Ramaswamy Premkumar, Ph.D.,Pichaimuthu Rajan, BOT,

Ebenezer Daniel, MS, MPH

Schieffelin Leprosy Research and TrainingCentre, Karigiri - 632106, Tamil Nadu, India.

REFERENCES1. BELL-KROTOSKI, J. A. Sensibility testing: current

concepts. In: Rehabilitation of the Hand: Surgeryand Therapy, 4th edn. St. Louis: Mosby-YearBook, Inc., 1995. pp. 109–128.

2. BIRKE, J. A., and SIMS, D. S. Plantar sensorythreshold in the ulcerative foot. Lepr. Rev. 57(1986) 261–267.

3. KETS, C. M., VAN LEERDAM, M. E., VAN BRAKEL,W. H., DEVILLE, W., and BERTELSMANN, F. W.Reference values for touch sensibility thresholdsin healthy Nepalese volunteers. Lepr. Rev. 67(1996) 28–38.

4. PREMKUMAR, R., DANIEL, E., SUNEETHA, S., andYOVAN, P. Quantitative assessment of facial sen-sation in leprosy. Int. J. Lepr. Other Mycobact.Dis. 66 (1998) 348–355.

5. WEINSTEIN, S. Intensive and extensive aspects oftactile sensitivity as a function of body part, sexand laterality. In: The Skin Senses. Springfield, IL:Charles C. Thomas Publishers, 1968. pp. 193–218.


from Group I were antibody-positive to phe-nolic glycolipid (PGL-I), the other six(Group II) were negative. ENL reactionswere characterized using histopathologicalcriteria, including the presence of undiffer-entiated macrophages and relatively abun-dant PMNs, with or without acid-fast bacilli.The group of six patients that gave negativereactions for antibodies to PGL-I (Group II)had completed multidrug therapy; they pre-sented an average of six episodes of ENL. Ofthe six patients in Group I with detectableantibodies to PGL-I, two were still beingtreated. ENL reactions were less frequent inGroup I (average 4 episodes).

Soluble component fractions were ob-tained by an electroelution technique fromMycobacterium leprae soluble extract(MLSA) and Mycobacterium bovis solubleextract (MbSA) (5, 6). The soluble extractswere obtained by rupturing purified bacilliwith the French Press (2). The extracts con-tain cytosol proteins as well as proteinsfreed from the cell walls. Insoluble materialwas eliminated by centrifugation. Proteinconcentration was determined by the BCAmethod (7).

Starting with a 10% SDS-PAGE prepara-tive gel under dissociating and denaturingconditions, 1 mg of MLSA and MbSA wasresolved in polypeptides of different mobil-ities (see The Figure), which were fraction-ated by electroelution in a mini BIORAD®

65-1256 electroelutor, according to the in-structions provided by the manufacturer.

Twelve electroeluted fractions were ob-tained for both the MLSA and the MbSAantigens. ELISA tests were used to evaluate

activity with the pooled sera, using IgG an-tibodies specific for the Fc gamma chain(Sigma A0170) as the second antibody (4).

A clear difference in recognition wasseen between the two groups of sera stud-ied. In the ELISA tests with both MLSAand MbSA electroeluted fractions, we sawan immunodominant recognition of pro-teins with a relative mobility of 30 kDa,corresponding to Fraction 9 (see TheTable). There was also serologic recogni-

THE FIGURE. OE, original extract (M. bovis). F1,F2. different electro-eluted fractions MW: molecularweight standards. (A, Coomassie brillant blue stain; B,Western blot with LL serum.) The typical ladder ofeluted fractions corresponding to their molecularweights is shown, with successively smaller proteinsfrom F1 to F12.

THE TABLE.

Patients Fractions MbSA

group F6 F7 F8 F9 F10 F11 F12

GI <0.125 <0.125 0.241 ± 0.001 0.470 ± 0.01 0.256 ± 0.01 <0.125 <0.125GII <0.125 <0.125 <0.125 0.296 ± 0.003 <0.125 <0.125 <0.125

Patients Fractions MLSA

group F6 F7 F8 F9 F10 F11 F12

GI <0.125 <0.125 0.257 ± 0.004 0.501 ± 0.001 <0.125 0.313 ± 0.03 <0.125GII <0.125 <0.125 0.178 ± 0.025 0.483 ± 0.01 <0.125 <0.125 <0.125

The results are expressed as optical density (O.D.) at 492 nm. To establish the criterion of positivity, the valueresulting from mean OD plus 3 times the standard deviation of 12 healthy subjects was used as the cut-off point(0.125 O.D. units).

tion of low molecular weight MLSA pro-teins (less than 30 kDa) in patients in groupI which was not observed in Group II.

In preliminary studies we previously re-ported a clear difference between the IgGantibody levels directed towards solublemycobacterial proteins (Mycobacterium bo-vis MbSA and Mycobacterium lepraeMLSA) in an ENL active group (n = 4) ascompared with the non-active group (n = 4)(3). In the ENL active patients we foundIgG antibody levels towards MbSA andMLSA of 0.535 ± 0.24 and 0.731 ± 0.32, re-spectively, as compared with the non-activepatients, whose values towards the same to-tal proteins were zero. In this study usingthe electroelution technique we were able todemonstrate the immunodominant antigensfound in patients in an ENL reactional state.

Many authors have shown a decrease ofIgM antibodies directed towards phenolicglycolipid (PGL-I), which is an M. lepraestructural component (1) in these reactionalpatients. To examine this, we separated thereactional patients in two groups, accordingto their PGL-I positivity. IgM antibodiesagainst native PGL-I were measured in anenzyme linked immunosorbent assay usingthe method described previously (8).

In addition to the immunodominantrecognition towards proteins with a 30 kDarelative mobility, both with MbSA andMLSA, we also saw that the recognition inGroup I involves a larger number of proteinfractions, including low molecular weightproteins (<30 kDa), compared to the pa-tients in Group II.

We have recently increased the numberof multibacillary patients (n = 70), andthere have been no significant differences inthe Mycobacterium leprae 30 kDa proteinantibodies between patients who had TypeII reactions and those who did not. In thislarger group of 70 multibacillary patients,nine presented ENL reactions and the other61 did not. Of the nine with ENL, eight(89%) gave positive reactions to the 30 kDaprotein, average optical density 0.8816. Ofthe 61 remaining patients, 42 (69%) gavepositive reactions to the 30 kDa protein, av-erage OD 0.5885. This difference was notstatistically significant, p = 0.42, but the ob-servation suggests a trend toward strongerreactivity in patients with ENL. The sera ofnewly diagnosed multibacillary patients re-

acted with other peptides of both higher andlower molecular weights. In this populationof 70 patients, 62.6% were in treatment andpresented bacillary indices of less than 2+.Reactivity was strongly associated withbacillary load. Reactivity to the 10 kDa pro-tein of M. leprae was lower in treated pa-tients than in new cases (unpublished data).

In conclusion, both patients who hadENL as well as those who did not re-sponded to the 30 kDa peptide of M. leprae,but the reactions tended to be stronger inthe former group. Additional more detailedstudies will be necessary to detect a clearmarker for ENL, using individual proteinsof the 85B complex or specific peptide se-quences of other proteins that might dis-criminate between patients with or withoutreactional phenomena.

Acknowledgment. This research was supportedby grant number S1-2001000859 from Fondo Na-cional Ciencias y Tecnología (FONACIT) Caracas,Venezuela.

—Elsa María Rada, M.Sc.,

Laboratorio Leprología y Patología Experimental

Instituto de Biomedicina, Caracas, Venezuela

—Edgar A. Zambrano, Ph.D.,

Laboratorio de Bioquímica de Parásitos,Instituto de Biomedicina,Caracas, Venezuela

—Nacarid Aranzazu, M.D.,

Clinical Section M.D., Instituto de Biomedicina

Caracas, Venezuela

—Jacinto Convit, M.D.,

Director, Instituto de BiomedicinaCaracas, Venezuela

REFERENCES1. BRENNAN, P. J., and BARROW, W. W. Evidence for

species-specific lipid antigens in M. leprae. Int. J.Lepr. Other Mycobact. Dis. 48 (1980) 382–387.

2. CONVIT, J., ARANZAZU, N., ULRICH, M., PINARDI,M. E., REYES, O., and ALVARADO, J. Immunother-apy with a mixture of Mycobacterium leprae and



BCG in differents forms of leprosy and Mitsuda-negative contacts. Int. J. Lepr. Other Mycobact.Dis. 50 (1982) 415–424.

3. RADA, E., ARANZAZU, N., and CONVIT, J. Im-munological reactions to mycobacterial proteins inthe spectrum of leprosy. Int. J. Lepr. Other My-cobact. Dis. 65 (1997) 497–500.

4. RADA, E., SANTAELLA, C., ARANZAZU, N., andCONVIT, J. Detection of antibodies toward se-creted Mycobacterial antigen 85 in untreated lep-rosy patients´sera. Int. J. Lepr. Other Mycobact.Dis. 67 (1999) 168–170

5. RADA, E., ARANZAZU, N., ULRICH, M., and CONVIT,J. Serologic response to mycobacterial proteins inHansen´s patients during multidrug treatment. Int.J. Lepr. Other Mycobact. Dis. 67 (1999) 414–421.

6. RADA, E., ARANZAZU, N., and CONVIT, J. Estudioserológico frente a diferentes proteínas micobacte-riales en pacientes con ENL. [Serological studywith different mycobacterial proteins in patientswith ENL.] (Resumen) Memorias de XLIX Con-vención Anual de Asovac, 1999. Maracay.Venezuela, p. 260.

7. SMITH, P. K. Measurement of protein using bicin-chonic acid. Anal. Biochem. 150 (1985) 76–85.

8. ULRICH, M., SMITH, P., SAMPSON, C., ZÚÑIGA, M.,CENTENO, M., GARCÍA, V., MANRIQUE, X., SAL-GADO, A., and CONVIT, J. Ig M antibodies to nativephenolic glycolipid-I in contacts of leprosy pa-tients in Venezuela; epidemiological observationsand a prospective study of the risk of leprosy. Int. J.Lepr. Other Mycobact. Dis. 59 (1991) 405–415.

Paucibacillary Treatment for

Large Tuberculoid Lesions of Leprosy?

TO THE EDITOR:

Under the title “Should large lesions of lep-rosy be considered as multibacillary for treat-ment purposes even if the total number of le-sions is less than five?” [Int. J. Lepr. 72 (2004)173–174], Kumarasinghe and Kumarasinghecalled attention to an interesting aspect re-garding the treatment of big size tuberculoidlesions or borderline-tuberculoid lesions ac-cording to Ridley & Jopling classification.

Their arguments for the treatment of pa-tients with large plaques are valid but therecommendation since the beginning ofMulti-drug Therapy - M.D.T./World HealthOrganization/82 (2) was to treat patientsupon a positive or negative bacilloscopy.The size or the number of lesions were notto be taken into account. Millions of pa-tients have been treated since, with a re-lapse rate of less than 1%. We present a pa-tient classified and treated as PB leprosywith a large plaque and five smaller lesions.

Patient. N.R.L, 45 years old, registeredat the Fundação de Medicina Tropical doAmazonas, Manaus, Brazil.

The patient presented a large plaque le-sion on the chest (Fig. 1) and five smallerlesions on the face, arm and posterior partof the trunk. No enlargement of the ulnarnerve or of other peripheral nerves could befound. The patient was clinically classifiedas reactional borderline tuberculoid leprosy.

The histopathology (Hematoxylin-Eosin)showed a granulomatous lesion with lym-phocytes, histiocytes and giant cells (Fig.2). The Wade stain was negative for acid-fast bacilli. The patient was classified asborderline tuberculoid (BT) leprosy.

Paucibacillary M.D.T. according to theW.H.O. plus 60mg prednisone per day wasstarted in March 2003. M.D.T. was stoppedafter 6 months of regular treatment and cor-tisone was slowly tapered off after 3 to 4months.

All the lesions regressed leaving a hy-popigmented area (Fig. 3). During the lastclinical reexamination (10/11/04) no re-lapse was found. A new histopathology ofthe edge of the lesion showed a regressiveinfiltrate (Fig. 4).

COMMENTSThe W.H.O. recommendation to treat lep-

rosy was based on bacilloscopy for manyyears on the bacilloscopy results, and theefficacy of M.D.T. has been the sameworldwide: Less than 1% of relapses. TheW.H.O. (3) recommendation to treat leprosypatients according to the number of lesionswas mainly an operational decision to im-plement M.D.T. in the field. There was norecommendation related to the size of thelesion.

We think that a patient with a negativebacilloscopy and a histopathology consistent

with a tuberculoid granuloma with scarce orno bacilli in the Wade or Fite-Faraco stainmust be classified as paucibacillary leprosy.We agree with Kumarsinghe and Kumars-inghe (1) that “. . . the larger the lesions ofleprosy, the higher the number of bacilli thatcause the pathology. . . .,” but in such a le-sion the clinical aspect is roughly the samewith defined edges and very well establishedborders between the lesion and the normalskin. Besides the relatively uniform clinicalaspect as observed in our patient, the skinsmears and the histopathology were thesame in repeated biopsies with the numberof bacilli scarce or negative. We could notfind any information in the literature to sub-stantiate the statement of the authors, that tu-berculoid leprosy could evolve to the lepro-matous pole over several years with the low-ering of patient´s cellular immunity (1).

We have been treating patients with mul-

tiple (more than 5) lesions as PB leprosywhen the clinical aspect of the lesions andthe histopathology showed a picture of tu-berculoid leprosy.

We agree with Kumarasinghe and Ku-marasinghe (1) that the W.H.O. recommen-dation is “particularly important in areaswhere treatment is initiated without anybacteriological and histopathological con-firmation . . .” However, in referral centersand in universities with good laboratorysupport the present WHO guidelines to treatas PB leprosy or MB leprosy should not befollowed. It seems there are no scientificdata to justify a formal recommendation totreat leprosy according to the number orsize of the lesions.

Acknowledgement. To Dr. Gotfried Schmer ofthe State University of Washington.


FIGS. 1–4. 1. Borderline tuberculoid leprosy. Large infiltrated plaque with well defined edges. 2. Hematoxylin-Eosin—presence of granulomatous infiltrate with epithelioid cells, giant cells and lymphocytes. 3. After nearlytwo years. Regression of the plaque, leaving a residual hipochromic lesion. 4. Regression of the infiltrate.


—Mônica Nunes Souza Santos—Luis Carlos de Lima Ferreira

—Sinésio Talhari (1)

Fundação de Medicina Tropical do Amazonas

Departments of Pathology

REFERENCES

1. KUMARASINGHE, S. P. W., and KUMARASINGHE, M.P. Should large lesions of leprosy be consideredas “multibacillary” for treatment purposes even ifthe total number of lesions is less than five? Int. J.Lepr. Other Mycobact. Dis. 72 (2004) 173–174.

2. Quimioterapia de la lepra para los programas delucha. W.H.O./LEP 675 (1982).

3. W.H.O. The Final Push Strategy to EliminateLeprosy as a Public Health Problem: Questionsand Answers, 2nd edn. Geneva: World Health Or-ganization,

Drs. Kumarasinghe Reply: Should Large Lesions of Leprosy Be Considered as “Multibacillary” for Treatment Purposes Even If the Total Number of

Lesions Is Less Than Five?

TO THE EDITOR:

We thank Souza Santos, et al. for their in-terest in our article (5). While agreeing withsome points made by them, it seems thatthey have misunderstood some of the pointswe made.

First, our recommendation of “consider-ing large lesions of leprosy as multibacil-lary” was not aimed for teaching hospitalsettings where microbiological and histo-pathological facilities and good clinical ex-pertise are available, but for field settings,in areas where treatment is initiated withoutany investigations, purely based on thenumber of hypopigmented lesions. At theteaching hospitals and tertiary care centerswe also treat patients after considering thesmear results and skin biopsy results, in ad-dition to the clinical picture.

It is well known that even a single lesioncan be multibacillary (3, 4, 6). The rationaleof total number of lesions as the only crite-rion for deciding on the treatment type, aswell as for scientific analyses has beenquestioned (8). However, in a retrospective study carried out in India, Gift, et al. have

found that World Health Organization(W.H.O.) operational classification is a sat-isfactory method for deciding on the formof treatment (2). In this analysis, they havetaken the smear examination as the goldstandard for evaluating the sensitivity andspecificity of the W.H.O. operational clas-sification. However, where the larger le-sions (>10 cm) were present they havefound that the specificity was 91.2% al-though the sensitivity was low. As only4.9% of smear positive cases have hadrecords of the size of the lesion, that studyappears to be inadequate to evaluate the va-lidity of the size of lesions as an additionalparameter.

Our recommendation for treatment oflarge plaque leprosy with three drugs forone year (“multibacillary treatment”) isbased on the observation of more relaspsesin this group of patients who have beentreated with two drugs for six months. Inanother study conducted in Sri Lanka it wasshown that several patients with large le-sions (>10cm) of leprosy were smear posi-tive although the total number of thepatches was less than five (1).

1 Reprint requests to: Sinésio Talhari, Av. PedroTeixeira, 25, Manaus, Am, Brazil. E-mail: [email protected]

We do not dispute that many cases ofpaucibacillary leprosy have less than 5patches. Although the authors agree on theW.H.O. operational classification based onthe number of patches, the case describedby the authors, with a large plaque of lep-rosy plus 5 other lesions on the face, wouldhave been classified as “multibacillary”, ifthe microbiological investigations were notdone, going by the visual classification rec-ommended by the W.H.O. It is known thatsome cases of leprosy may improve evenwith dapsone monotherapy (as was thepractice before the advent of multi-drugtherapy, M.D.T.), or single dose multidrugtherapy. It would be interesting to see thelong term outcome of the case presented bythe authors. Even though a smear was neg-ative, in the case presented by the authors,we would have not have been comfortablein administering paucibacillary treatmentonly for 6 months, in a patient with such ex-tensive lesions. Cell mediated immunity isof paramount importance in the pathogene-sis of leprosy (7). It is clear that patientsprogress in the leprosy spectrum towardsthe lepromatous pole when the immunity ofthe host is unable to overcome the infectionby lepra bacilli. In cases of subpolar lepro-matous leprosy some areas with typical hy-popigmented semianaesthetic lesions canoften be seen while other smear positive le-sions coexist in the same patient. Clearlynot all cases of multibacillary leprosy startas polar lepromatous leprosy. In our state-ment in the article we did not imply that“polar tuberculoid leprosy” would down-grade to “polar lepromatous leprosy” whichare generally immunologically stable.

We agree with the authors that a largerscale study would be helpful to resolve theissue whether larger lesions due to leprosyshould be treated with the “multibacillarydrug regime” at least for one year.

A representative lesion should be micro-biologically and histopathologically evalu-ated whenever possible, and the findingsshould be evaluated in conjunction with theclinical features before commencing ontreatment. The current W.H.O. operationalclassification; while being useful in thecommunity perspective, appears to be anover-simplification in some situations. Thesearch for any additional features to finetune the parameters should be continued.

—S. Prasad W. Kumarasinghe 1

Senior Consultant Dermatologist, National Skin Centre, Singapore

—M. P. Kumarasinghe

Senior Consultant Pathologist,Singapore General Hospital, Singapore

REFERENCES1. ARIYAWANSA, D., SATGURUNATHAN, K., and

WASALAARACHCHI, K. Should the size of the lesionbe considered as an additional factor in the clinicaldiagnosis of leprosy? 3rd South Asian Regional As-sociation of Dermatology Conference, Colombo, SriLanka, 2003.

2. GIFT, N., JOSEPH, G., and RICHARD, J. Validity of theW.H.O. operational classification and value of otherclinical signs in the classification of leprosy. Int. J.Lepr. Other Mycobact. Dis. 72 (2004) 278–283.

3. JOB, C. K., KAHKOHEN, M. E., JACOBSON, R. R., andHASTINGS, R. C. Single lesion subpolar lepromatousleprosy and its possible mode of origin. Int J Lepr.Other Mycobact. Dis. 57 (1989) 12–19.

4. KAR, B. K., BELIAPPA, P. R., EBENIZER, G., and JOB,C. K. Single lesion borderline lepromatous leprosy.Int. J. Lepr. Other Mycobact. Dis. 72 (2004) 45–47.

5. KUMARASINGHE, S. P..W., and KUMARASINGHE, M.P. Should large lesions of leprosy be considered asmultibacillary for treatment of treatment purposeseven if the total number of lesions is less than five?Int. J. Lepr. Other Mycobact. Dis. 72 (2004)173–174.

6. MISRA, R. S., RAVIS, S., IYERHGAR, B., and NASH, I.A histopathological and immunological profile of asingle lesion lepromatous leprosy. Int J Lepr. OtherMycobact. Dis. 59 (1991) 645–648.

7. RIDLEY, D. S. Pathogenesis of leprosy and relateddiseases. London: Wright, 1988. p. 55–58.

8. SCOLLARD, D. M. Classification of leprosy: a fullcolor spectrum, or black and white? Int. J. Lepr.Other Mycobact. Dis. 72 (2004) 166–168.


1 Reprint requests to: S. Prasad W. Kumarasinghe.MBBS, MD, FCCP, FAMS, Senior consultant Derma-tologist, 1 Mandalay Road, National Skin Centre, Sin-gapore. E-mail: [email protected]


TO THE EDITOR:

Please permit us to make some more ob-servations (arising from our combined ex-perience of over 60 years in leprosy reliefwork) particularly relevant to India, whichcontributes 77% of active cases to theglobal pool of active leprosy cases. One to1.5 million out of 2 to 3 million leprosy-disabled in the world are reported to live inIndia.

Dr. Yo Yuasa, who was the President ofthe International Leprosy Association fortwo terms, exhorted everyone to work to-wards a “World Without Leprosy” at the In-ternational Leprosy Congress, Beijing in1998. He defined this state as “a worldwithout leprosy-related problems, bothmedical and social, emphasizing the pointthat it is not the disease per se but its relatedproblems, mostly social but some medical,which require attention.”

This slogan was, however, pooh-poohedby the World Health Organization (W.H.O.)and the W.H.O.-influenced governmentsand the “program managers,” who were ob-sessed with the term “Elimination.” The tar-get year was 2000, which is now revised to2005, when the mean prevalence rate of 1case per 10,000 is expected to be reached.Unfortunately by then, the world will alsobe free from the so-called “Leprologists.”The enormous funds still needed to do jus-tice to the clinical problems related to lep-rosy and the rehabilitation of patients wouldhave dried up. The “pool” of leprosy pa-tients with reaction, neuritis and its seque-lae, and those needing rehabilitation con-tributing to the “disease burden” in thecommunity will far out number the activecases needing multi-drug therapy (M.D.T.)As yet there is no evidence of the muchtalked about secondary level and tertiary

level “Referral Centers” easily accessible topatients living in areas deprived of even ba-sic health services, where the primaryhealth centers with which leprosy is “inte-grated.” Most patients and the healthproviders are not even aware of the tech-nology to prevent the adverse progressionof complications and palliative care of irre-versible disabilities, let alone the concept of“Community-Based Rehabilitation.”

It is strange that the same public healthspecialists who talk about “Elimination”have now started fighting for “HumanRights” of leprosy patients without even at-tempting to formulate a mass-based strategyfor addressing the clinical problems of pa-tients “released from control.”

Perhaps they are waiting to celebrate theeventful day of 31 December 2005 to an-nounce their “Victory over Leprosy” beforethinking of planning the secondary and ter-tiary level referral centers! It is time that thepeople, patients, and particularly the donorsare made aware that this victory is by nomeans a victory over all leprosy-relatedproblems, as enshrined in the definition of“World Without Leprosy.” The donors aremade to believe that with the magic word“Elimination,” the disease is already on theverge of being wiped out.

Has not the jargon “Elimination” of lep-rosy outlived its utility? Though it is ratherlate, should we not devise a more patient-friendly term for “Elimination” that trulyreflects the sincere attempt at the eradica-tion of all ills afflicting the persons whohave contracted specially the progressiveforms of the disease?

—Dr. R. Ganapati,—Dr. V. V. Pai

Bombay Leprosy Project

Has the Term “Elimination” Outlived Its Utility?

The 40th Anniversary Meeting of all ofthe panels of the US-Japan CooperativeMedical Sciences Program (USJCMSP)took place in Kyoto in December, 2004.The Joint Tuberculosis and Leprosy Panelsorganized two half-day sessions dedicatedto TB and leprosy, and co-sponsored twohalf-day sessions with other panels—onewith the AIDS Panel on TB/HIV interac-tions, and the other with the Acute Respira-tory Infections Panel on antibiotic resistance.The Joint Committee of the USJCMSPannounced that new guidelines for panel ac-tivities would be implemented in the comingyear. The Joint Tuberculosis and LeprosyPanels were encouraged to develop high pri-ority scientific programmatic goals, andidentify implementable research objectivesfor the next five years. Examples might in-clude TB vaccine and drug development,management of latent TB, and the develop-ment of molecular tools to better character-ize leprosy transmission and incidence. Astrong emphasis will be placed on strength-ening the research capacity for both dis-eases in high-burden countries in the PacificRim, including training activities and tech-nology transfer. The Joint TB and LeprosyPanels were also encouraged to developstrategies for interacting effectively withother relevant Panels in the coming years.

The 40th Annual US-Japan Conferenceon Tuberculosis and Leprosy will take placein Seattle from 28–30 July, 2005. The mainconference will be preceeded by a half-daysession co-sponsored with the ImmunologyBoard which will focus on the definition ofimmunological determinants of protectioninduced by TB vaccines. The final day ofthe conference will consist of a LeprosyWorkshop with invited speakers which willfocus on the immunology and pathology ofthe disease, animal models, and challengesin leprosy research.

Modulation of the TH1 response toMycobacterium leprae in experimentalleprosy

Introduction. In leprosy, a disease causedby the obligate intracellular pathogen, Myco-bacterium leprae, an array of symptoms arepresented which are largely determined bythe host’s response, ranging from a highlevel of cell mediated immunity (CMI) intuberculoid leprosy (TT) to absence of CMIin lepromatous leprosy (LL). Animal modelsfor leprosy are limited. Armadillos exhibit adisease spectrum similar to man, but they arerestrictively expensive and immunologicalreagents are scarce. The murine system,while well-characterized and armed with aplethora of immunological reagents, is es-sentially restricted to the foot pad for evalu-ating growth of M. leprae and exhibits lim-ited nerve involvement by the bacilli. Nev-ertheless, murine models for leprosy,especially with the introduction of geneknockout (KO) strains, have shown promisefor immunological studies of the leprosyspectrum. M. leprae-induced granuloma for-mation and maintenance depends heavilyupon T cell and macrophage (MΦ) popula-tions and their respective cytokines. Micedeficient in inducible nitric oxide synthase(iNOS), an important MΦ effector mecha-nism, have shown promise as a model forborderline tuberculoid leprosy in that intensegranuloma formation rapidly appears with-out exacerbating M. leprae growth. IL-12, akey regulatory cytokine of the immune sys-tem, induces the production of IFN-γ by Tcells and NK cells and promotes the devel-opment of a Th1 type cell mediated immuneresponse. IL-10 is generated by T cells andMΦ and is an inhibitor of IFN-γ production.

Methods. M. leprae infection was evalu-ated in iNOS KO (NOS2–/–), IL-10 KO(IL10–/–) and IL-12 KO (IL12–/–) mice usinglow dose (LD) and high dose (HD) infec-tion models. C57Bl/6 (B6) control mice and

NEWS and NOTESThis department furnishes information concerning institutions, organizations, and

individuals engaged in work on leprosy and other mycobacterial diseases, and makes noteof scientific meetings and other matters of interest.

230


(ISSN 0148-916X)

US-Japan Meeting, 2004

73, 3 News and Notes 231

KO mice were infected in both hind foot padswith 6 × 103 (LD) viable M. leprae andgrowth, lymph node cell profiles and histol-ogy were monitored for 18 months. B6 andKO mice were also inoculated with 3 × 107

(HD) viable M. leprae in both hind footpads,with or without treatment with iNOS in-hibitors such as LNil (L-N6-(1-iminoethyl)lysine hydrochloride) or aminoguanidine(AG). Foot pad induration, cell profiles, andcytokine expression were analyzed in thesefoot pads.

Results. In the LD model, growth of M.leprae was controlled in the NOS2–/– andIL10–/– mice, similarly to B6 control mice.In contrast, there was augmented growth ofthe bacilli in the IL12–/– mice. Flow cyto-metric analysis of the draining popliteallymph nodes showed a sharp decrease inthe level of T lymphocytes in B6 mice at 6months post infection which correspondswith the peak of M. leprae growth. A simi-lar decrease was seen in IL10–/– mice. Incontrast, this decrease was not seen inIL12–/– mice. In the HD model, a large gran-ulomatous response occurred in theNOS2–/– mice compared to B6 mice whichconsisted primarily of CD11b+ MΦ andCD4+ lymphocytes and, to a smaller extent,CD8+ cells. The level of CD4+ and CD8+

cells expressing activation markers was sig-nificantly higher in NOS2–/– mice than B6mice. Concomitant with foot pad indurationwas an augmented expression of IFNγ,TNFα, and IL-10 as well as MIP-1α, MIP-1β, and MCP-1. A similar induration oc-curred in M. leprae-infected B6 micetreated with L-NIL. Interestingly, the in-duration subsided if the iNOS inhibitor wasremoved; conversely, if the iNOS inhibitorwas added 1 month post infection, en-hanced induration ensued, thus emphasiz-ing the dynamic nature of the foot pad le-sion. Upon infection with HD M. leprae,IL10–/– mice also exhibited greater indura-tion than control mice. Like B6 and iNOS–/–

mice, the T lymphocyte infiltration was pri-marily CD4+. Addition of LNil to theIL10–/– diet resulted in greater induration ofthe M. leprae-infected foot pad than eitherindividual model of deficiency. If LNil ad-ministration began 1 month post infection,induration rapidly exceeded that of theIL10–/– mice and was similar to mice thatwere iNOS and IL10 deficient from the be-

ginning of infection. In IL12–/– mice, HDinfection with M. leprae induced little in-duration in the foot pad and the T lympho-cyte infiltration was equally CD4+ andCD8+.

Discussion. These findings suggest thatKO mice infected with M. leprae can pro-vide insight into the subtle nuances of cellmediated immunity toward M. leprae infec-tions as well as contribute to the overall un-derstanding of the various processes under-lying the broad host response to infectionand, in particular, the unstable nature of theborderline area of the leprosy spectrum.

—Deanna A. Hagge, Nashone A. Ray,Vilma Tulagan and Linda B. Adams

National Hansen’s Disease Programs, USA.

Initiative for Diagnostic and Epidemio-logical Assays for Leprosy (IDEAL)

In 1977, the World Health Organization(WHO) Expert Committee on Leprosy esti-mated the global number of leprosy cases tobe over 12 million. In 1981, WHO con-vened the Study Group on Chemotherapyfor Leprosy Control, which recommendedcombined-drug regimens based on super-vised intermittent administration of ri-fampicin for both multibacillary (MB) andpaucibacillary (PB) leprosy. Thanks to theimplementation of this multidrug therapy(MDT), substantial progress in leprosy con-trol has been achieved, and over 12 millioncases had been cured by 2002. Thus, theWHO Leprosy Programme set a target forthe elimination of leprosy (less than 1 caseper 10,000) by the year 2000. With the fail-ure to achieve this goal, more recently,WHO formed a Global Alliance for theElimination of Leprosy (GAEL) with theaim of reaching the elimination target (lessthan 1 case per 10,000) in all countries by2005. However, to date, there is no clear ev-idence of an impact of introduction of MDTon the rate of detection of new cases. Whileglobal prevalence has dropped from themillions in the 1970s to less than 650,000cases in 2002, new case detection has re-mained steady over the years at over700,000 per annum. Approaches to address


this problem are impeded by a lack of fun-damental knowledge about the epidemiol-ogy of leprosy, the sources of infection, itsprecise mode of transmission, and the im-portance of contact patterns.

The “Initiative for Diagnostic and Epi-demiological Assays for Leprosy” (IDEAL)resulted from two workshops under the aus-pices of the TDR program of WHO. Thefirst workshop, held in Geneva in Novem-ber 2002, identified two fields of leprosy re-search in which advances are needed in or-der to eliminate leprosy. These are:

1) Nerve damage and 2) Early diagnosisand transmission.

In October 2003 a second workshop wasorganized in Amsterdam, in which the re-search needs in the field of early diagnosisand transmission were further identified andmade explicit in a proposal for a compre-hensive leprosy research program. This re-search program aims at the application ofnew developments in the fields of molec-ular typing of M. leprae and specific anti-gen/epitope definition to field studies to-wards better understanding of the epidemi-ology and transmission of leprosy, and theimproved diagnosis of leprosy infection.The three main areas of research in this pro-gram are:

• Assays for molecular epidemiology• Immunology-based diagnostic assays• Field studies related to transmission

and diagnosis

An Interim Steering Committee (Drs.Brennan, Dockrell, Engers, Klatser, Oskam,Richardus) was appointed to coordinateefforts to obtain funding and to invite part-ners (both research institutes and field pro-grams) to join the consortium. The partnersand sites chosen all have a proven trackrecord in leprosy research, providing accessto sufficient leprosy patients and their con-tacts within a functional leprosy controlprogramme, with well-equipped laborato-ries and/or with experience in capacitybuilding and technology transfer. The spe-cific aims of the IDEAL research programare to:

1. Identify and develop M. leprae-specificproteins and peptides suitable for usein T cell assays, to enable specificimmune responses to be identified in

paucibacillary leprosy patients or con-tacts.

2. Dissect biomarkers identifying protec-tive and non-protective immune re-sponses in groups of leprosy contacts,that could be used to develop simpleassays to identify infected subjectswithout protective immunity in leprosy-endemic countries.

3. Identify and assess the full range ofpolymorphisms at short tandem repeat(STR) and single nucleotides in theM.leprae genome with sufficient ge-netic variability to define sources of in-fection, transmission patterns and dis-tinguish between new and reactivatedcases of clinical leprosy.

4. Apply these new tools in field settingswith different population characteris-tics and levels of endemicity.

5. Form a global platform for researchgroups so that research can be imple-mented in a coordinated manner, thusspeeding up the quest for solutions tothe issues of leprosy.

The research developments underlyingthese aspirations will be explained as alsowill the convening of the first meeting ofIDEAL patners in Addis Ababa, Ethopia,October 25–27, 2004 will be described.

—Patrick J. Brennan

Colorado State University

Regulation by clofazimine of cytokineproduction in M. leprae-infected macro-phages

Anti-mycobacterial drug, clofazimine orB663, has been used for the treatment of lep-rosy and some of the mycobacterial infec-tions for the purpose of killing of thecausative bacilli, moreover, the drug is re-ported to be useful in several immunologi-cally mediated skin disorders. The mecha-nism of action by clofazimine is still unclear,although several studies have suggested itsmodulatory effects on immune response. Inleprosy, clofazimine is also used for the sup-pression of leprosy reaction. To investigatethe mechanism of immunomodulation by


clofazimine, the macrophage, one of the im-mune cells which play a very important rolein leprosy as a host cell of M. leprae, wasstudied on the basis of the effect of the drugon cytokine production in response to M.leprae. By in vitro study it was found thatB663 enhanced TNF production in M.leprae-stimulated mouse macrophages,moreover, the drug suppressed IL-10 andPGE2 production in the cells. The suppres-sive effect on IL-10 production could be dueto the suppression of PGE2 production, sincePGE2 was required to induce IL-10 by ele-vation of intracellular cAMP level throughthe stimulation of adenylate cyclase. PGE2is a well-known inflammatory factor, there-fore, anti-inflammatory activity of the drugcould be due to the suppressive effect onPGE2 production. TNF is known as the co-activator of macrophages with IFN gamma,suggesting that B663 could enhance anti-bacterial activity of the host through the en-hancement of TNF.

—Yasuo Fukutomi*,Fumihiko Takeshita**,

Masanori Matsuoka*and Masahiko Makino*

*Leprosy Research Center,National Institute of Infectious Diseases,Tokyo, and**Yokohama City University School

of Medicine

Nerve damage by bacteria causing Bu-ruli ulcer—ultrastructure of mouse inoc-ulated with Mycobacterium ulcerans

Buruli ulcer is an intractable skin diseasecaused by Mycobacterium ulcerans. It isobserved in tropical area such as Africa andAustralia. Large, necrotizing, relativelypainless, deep skin ulcers are formedmainly in the extremities. Because ofchronic course and occasional complicationof severe deformities, socioeconomic hand-icap is a great problem.

Recent study demonstrated that PhenolicGlycolipid-I (PGL-I), a Mycobacteriumleprae-specific membranous antigen re-sponsible for Schwann cell invasion, is pre-sent in Buruli ulcer. Thus, we hypothesized

that not only M. leprae but also M. ulceransmay invade peripheral nerve.

MATERIALS AND METHODSBacterial suspension of M. ulcerans

colony 97-107 cultured at 32°C in 7H9 cul-ture medium (CFU = 1.3 × 106/ml, 25μl)was inoculated into the bilateral footpads offemale BALB/c mice. Local swelling andredness were observed at day 33 after theinoculation, and sequential histopathologi-cal examination was performed since then.

Perfusion fixation by 10% formalin wasdone, and hind limbs were histopathologi-cally examined by HE, acid-fast stainingand immunohistochemistry using anti-PGL-I antibody. Also in selected cases, per-fusion fixation by 2% glutaraldehyde wasdone, and hind limbs embedded in Epon,cut into 1μm were examined. When nervedamage is observed, electron microscopicexamination was performed.

RESULTSDay 33 after the inoculation of M. ul-

cerans: Dermal erosion and extensiveedema of subcutaneous tissue were associ-ated with infiltration of small number ofneutrophils and monocyte. Granuloma for-mation was absent. Small clusters of longacid-fast bacilli were noted mainly in thestroma and in the cytoplasm of monocytes(Fig. 1). Peripheral nerves were well pre-served even in the edematous lesion.

Day 55 after the inoculation of M. ulcer-ans: Remarkable deep skin ulcer and exten-sive subcutaneous edema were observed.Large number of acid-fast bacilli formedclusters in the edematous stroma. Manynerve bundles were well preserved, butsome showed vacuolar change of Schwanncells (Fig. 2), and others were invaded bynumerous acid-fast bacilli with massivenerve damage. Ultrastructurally, the bacilliwere mainly in the endoneurium, andSchwann cells were spare (Fig. 3). PGL-Iimmunohistochemistry was negative.

DISCUSSIONAmong the mycobacterial species, only

M. leprae is known to show neurotropismand causes nerve damage. In the previousstudies, mild degenerative change withthickening of Schwann cell basal laminaand vacuolar change of axons were reported


{Mwanatambwe, 2000}, but direct nerveinvasion by the acid-fast bacilli has notbeen found. Our study first demonstratedthat nerve bundles are damaged by numer-ous M. ulcerans. This finding raises a newpossibility of pathogenesis of “painless-ness” of Buruli ulcer.

—Masamichi Goto1,Hajime Saito2, Kazue Nakanaga3,

Norihisa Ishii3, Suguru Yonezawa1

1Kagoshima University,2Hiroshima Environment and Health

Association,3National Institute of Infectious Diseases

Leprosy Research Center

Chemotherapy and Drug Resistance inLeprosy

The chemotherapy of leprosy, which iscaused by Mycobacterium leprae, waslaunched in 1943 and dapsone was intro-duced as standard chemotherapy for leprosyin the 1950s. Between the 1960s and 1970s,other anti-leprosy agents such as clofaz-imine and rifampin were introduced due tothe emergence of dapsone resistance re-sulted from long-term monotherapy withdapsone. The first dapsone resistant casewas proved by mouse foot-pad method in1964. To conquer the increasingly world-wide spread of dapsone resistance and con-trol leprosy, the World Health Organizationrecommended multidrug therapy in 1981.Regimens included dapsone, rifampin andclofazimine in different doses and durationsfor multibacillary case (MB) and pau-cibacillary case (PB). Recently, ofloxacinand minocycline have been added for treat-ing single lesion paucibacillary case (SPB).

Dapsone targets dihydropteroate synthase

(DHPS) encoded by the folP and inhibitsfolic acid biosynthesis by acting as a com-petitive inhibitor of p-aminobenzoic acid(PABA). The target for rifampin is betasubunit of the RNA polymerase, encodedby the ropB, and transcription is inhibited.The mechanisms of antimicrobial activityfor clofazimine has not been fully eluci-dated, however, the drug appears preferenceto bind to GC-rich sequences of mycobacte-ria. Similarly, the mechanism of bacterici-dal activity of minocycline against M.lep-rae is unknown, but thought to inhibit pro-tein synthesis by blocking the binding ofaminoacyl transfer RNA to the messengerRNA. Ofloxacin, one of new quinolones, itis likely to inhibit DNA replication by bind-ing to A-subunits (GyrA) of DNA gyrase, atype II topoisomerase.

In spite of discovery of the genetic back-ground for understanding of drug resistancein dapsone, rifampin and ofloxacin, only alimited number of mutations responsible forresistance have been reported in M. lepraeto date since drug susceptibility of M.lepraeto anti-leprosy drug has been tested bymouse foot-pad method. Almost all muta-tions in relevant genes conferring resistanceto each drug were point mutations. Respon-sible point mutations, at 513 (1 case), 516(1 case), 526 (3 cases), 531 (24 cases) and533 (1 case) in the rpoB, were observedfrom 30 isolates and a 6-bp insertion wasshown between 514 and 515 in one isolate.No mutation was detected in rifmapin resis-tance determining region of 65 wild typeisolates. Meanwhile, mutations at 53 (8cases) and 55 (10 cases) in the folP weredetected in dapson resistant isolates and 12susceptible isolates showed no mutations inthe gene. Three isolates resistant toofloxacin harbored mutation at 91 in thegyrA.

The prevalence of drug resistance de-duced by the mutation detection among re-

FIGS. 1–3. 1. Day 33. Macrophage contains numerous bacilli. 2. Day 55. Vacuolar change of Schwann cells(*). 3. Nerve damage with endoneurial invasion of bacilli.


lapsed, intractable and new cases will bediscussed. A method for rapid detection ofresistant isolates and the significance of re-lapse by the persistence of susceptiblebacilli will be also considered.

—Masanori Matsuoka

National Institute of Infectious Diseases,Leprosy Research Center

Loop-Mediated Isothermal Amplifica-tion of the dnaA sequence for rapid de-tection of Mycobacterium leprae

For the establishment of differential diag-nosis of mycobacterial species, a part of thenucleotide sequences of the mycobacterialDnaA protein gene was determined by PCRbased sequencing. Clinically relevant 27mycobacterial species and 46 clinical iso-lates of Mycobacterium avium, M. intracel-lulare and M. kansasii were analyzed. Al-though dnaA partial sequences of M. tuber-culosis complex were identical to eachother, all of the nontuberculous mycobacte-rial (NTM) laboratory strains and clinicalisolates tested, were easily identified as therespective species. The partial dnaA se-quence similarity between M. avium and M.intracellulare was 78.3%, and that of M.kansasii and non-pathogenic M. gastri was83.6%. Rapidly growing groups of myco-bacteria were clearly separated from otherspecies in unrooted phylogenetic tree.Based on the amplified DNA sequences,species specific-primers were successfullydesigned for the target mycobacteriumspecies, M. avium, M. intracellulare, M.kansasii, and M. gastri. These resultsdemonstrate that the variable sequence inDnaA coding gene were species-specific andwere potent for the development of accurateand rapid diagnosis of Mycobacteriumspecies. To develop rapid and simple identi-fication method, we used loop-mediatedisothermal amplification (LAMP) for detec-tion of Mycobacterium leprae, M. kansasiiand M. gastri. LAMP method is a novel nu-cleic acid amplification method in whichreagent reacts under isothermal conditionswith high specificity, efficiency and rapid-ity. The whole procedure was quite simple,

starting with mixing of reagents in a singetube, followed by an isothermal reactionduring the reaction mixture is held at 63°C.The resulting amplicons are load to agarosegel electrophoresis. The only equipmentneeded for the amplification reaction is aheat block that furnishes a constant temper-ature. Species-specific primers for M. lep-rae were designed by targeting the dnaAgene and specificity were validated with 27mycobacteria species and 8 clinical isolatesof M. leprae. The assay had a detectionlimit 5pg of purified DNA with 60 min. in-cubation time. The sensitivity and reactiontime for LAMP methods to detect of M.kansasii and M. gastri purified DNA were10 pg, 30 min. and 1 ng, 60 min., respec-tively. The results demonstrate the variablesequence in dnaA gene was species-specific.Application on LAMP method was potentfor the rapid diagnosis method of mycobac-trerial species and especially useful in de-velopment country.

—Tetsu Mukai, Yuji Miyamoto, Masanori Matsuoka, Toshio Yamazaki,

Masahiko Makino

Leprosy Research Center, National Instituteof Infectious Diseases, Japan.

Polymorphism on the 5′′ Flanking Re-gion of IL12R2 Affects Establishment ofClinical Type of Leprosy.

The intensity of cell-mediated immune(CMI) responses in mycobacterial infection,determines individual differences in suscepti-bility to the diseases. These differences mightbe clarified from the viewpoint of T cell re-sponsiveness against IL-12 in patients withleprosy since the disease shows the wide clin-ical spectrum due to the effect of their inher-ited factors. Leprosy, a chronic diseasecaused by the infection of Mycobacteriumleprae (M. leprae), shows a wide spectrum ofclinical features. Tuberculoid type of leprosy(T-lep) patients show high level of CMI re-sponses against M. leprae, which results inthe resistance to infection, whereas leproma-tous type of leprosy (L-lep) patients showpoor CMI responses (instead, rich antibodyresponses) against the pathogen which results


in the progressive form of the disease. Re-cently, it was reported that the IL-12Rβ2 wasmore highly expressed in tuberculoid lesionscompared with lepromatous lesions, whereasIL-12Rβ1 expression was similar in both le-sions. Then, we analyzed the polymorphismson the 5′ flanking region of IL12RB2 to de-termine possible immunogenetical factorsthat affect CMI responses, by employing lep-rosy as model.

The polymorphisms were examined byusing direct sequencing technique to com-pare the allele frequencies between 129 L-lep patients and 46 T-lep patients. SeveralSNPs, including –1035A>G, –1023A>G,–650delG and –465A>G SNPs, were de-tected on the 5′ flanking region of IL12RB2.Frequency of haplotype 1 (–1035A, –1023A,–650G, –464A), which exhibited the high-est frequency in the general Japanese popu-lation, was significantly lower in L-lep pa-tients as compared with findings in T-lep pa-tients and healthy controls. Reporter geneassays using Jurkat T cells revealed that allhaplotypes carrying one or more SNPs ex-hibited lower transcriptional activity ascompared with haplotype 1. Moreover, itwas also elucidated that activated T cells de-rived from the donors carrying haplotype 1showed higher expression of IL-12Rβ2mRNA in the presence of IL-12 by employ-ing real-time PCR method.

These results suggest that SNPs on the 5′flanking region of IL12RB2 affect the ex-pression level of IL-12Rβ2 molecules,which may be implicated in individual dif-ferences in CMI responsiveness againstmycobacterial antigens, thereby leading tothe lepromatous and tuberculoid types ofleprosy.

—Hideki Ohyama1, Koretsugu Ogata2,Kazu Takeuchi3, Masako Namisato4,

Yasuo Fukutomi5, Motoharu Suzuki6,Yasushi Uemura6, Tohru Tsujimura1,Nobuyuki Terada1, Sho Matsushita6

1Hyogo College of Medicine, 2SHIMADZU Cooperation, 3Okayama University, 4National Kuryu Rakusen-En Sanatorium,5National Institute of Infectious Disease,6Saitama Medical School

Genotypic variation of M. leprae withina high endemic community.

Mycobacterium leprae is an obligate in-tracellular pathogen that is widely distrib-uted around the globe. There are no recog-nized patho-vars or sub-types and the bacil-lus exhibits little genetic diversity. The onlydocumented highly variable sequences areassociated with variable number tandem re-peat (VNTR) sequences distributed through-out the genome. We recently showed thatVNTR polymorphisms could be used effec-tively to discriminate geographically di-verse M. leprae strains used in the labora-tory. Similar polymorphisms have beenused with other bacteria to suggest phyloge-netic relationships among worldwide iso-lates and to examine the dissemination dy-namics of disease agents in populations.However, the sensitivity, specificity andpredicative value of VNTR polymorphismsfor describing these relationships among M.leprae are not yet known. To better under-stand these relationships we examined theutility for VNTR genotyping to discrimi-nate M. leprae strain types in high endemiccommunities.

Using a battery of 7 VNTR loci we ex-amined the genetic diversity of M. lepraestrains recovered from 58 unrelated leprosycases presenting in Karigiri, India over aten year period. The alleles for microsatel-lites (GAA (21), GTA (9), AT (17), and TA(18)) were determined by direct sequencingoff of PCR products using forward and re-verse primers. The alleles for minisatellites(12-5, 21-3, and 27-5) were determined byelectrophoresis of fluorescently labeledPCR products in agarose gels. The variousVNTR exhibited diversity in alleles rangingfrom 0.3 to 0.9 in patient samples andsuccessfully discriminated a total of 58different VNTR genotypes among the 58Karigiri cases tested. No epidemiologicallysignificant relationships were discerned. Tobetter understand the likelihood of chanceinfluencing the remarkable discriminationseen hereobserved, we used the samebattery of VNTR to examine M. lepraefrom multiple tissue samples recoveredfrom naturally infected wild nine-bandedarmadillos.

M. leprae is intensely transmitted withinarmadillo communities in Louisiana. Inci-


dence density rates exceeding 3.5 cases/1000 animal-days have been measured andthe disease may approximate an outbreaksituation for M. leprae among armadillos.We recovered a total of 8 naturally infectedwild armadillos from 2 Louisiana researchsites during a 3 month period in 2004. M.leprae were harvested and the genotype ofthe bacilli determined from each of 8 differ-ent tissue samples for each animal. VNTRalleles showed markedly less diversityamong armadillos than among the Karigiripatient samples. GAA remained the mosthighly diverse locus with other loci exhibit-ing greater stability. Analysis of M. lepraefrom the different tissues of each animalshowed nearly identical VNTR genotypes

and consistency in genotypes discerned foranimals from each location.

VNTR genotyping can have high dis-criminatory power for differentiating M.leprae with high specificity. Additionalstudies addressing the appropriate mixtureof alleles to be used and their combinedsensitivity, specificity and predicative valueare warranted.

—Richard W. Truman, James E. Adams,Gigi Ebenezer,* and Thomas P. Gillis

National Hansen’s Disease Programs, Baton Rouge, LA USA, and *Schieffelin Leprosy Research and Train-ing Center, Karigiri, Vellore, India

*Aide aux Lepreux Emmaus-Suisse, Spi-talgasse, CH-3011 Berne, Switzerland.

*American Leprosy Missions, One ALMWay, Greenville, South Carolina 29601,U.S.A.

*Amici dei Lebbrosi, Foundazione ItalianaRaoul Follereau, Via Borselli 4, 40135Bologna, Italy.

Damien-Dutton Society, 616 Bedford Ave-nue, Bellmore, New York 11710, U.S.A.

*Damien Foundation (DF/APD), 16 RueStevin, B-1040 Bruxelles, Belgium.

*Deutsches Aussatzigen-Hilfswerk e. V.,Postfach 9062, D-97090 Würzberg 11,Germany.

*Le Secours aux Lépreux (Canada), 1275Rue Hodge Bureau 12, Montreal H4N3H4, Canada

*Netherlands Leprosy Relief, Wibaut-straat 137K, 1097 DN Ansterdam, TheNetherlands.

*Pacific Leprosy Foundation, 115 Sher-borne Street, Bag 4730, Christchurch,New Zealand.

*Sasakawa Memorial Health Founda-tion, Senpaku Shinko Bldg., 1-15-16Toranomon, Minato-ku, Tokyo 105,Japan.

ACKNOWLEDGMENT

The Board of Directors of the INTERNATIONAL JOURNAL OF LEPROSY gratefully ac-knowledges the financial assistance from special grantors and sustaining memberswhich, with the special donations of certain members, has made possible the con-tinuation of publication of the JOURNAL directly by the International Leprosy Asso-ciation. Without this assistance the official organ of the ILS, so essential to leprosyworkers everywhere, could not be published.

SPECIAL GRANTORS

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(ISSN 0148-916X)

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