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VOLUME 73, NUMBER 3 SEPTEMBER 2005

Special Grantors Listed in Contents

INTERNATIONAL

JOURNAL OF LEPROSY

And Other Mycobacterial Diseases

Official Organ of theINTERNATIONAL LEPROSY ASSOCIATION

(Association Internationale contre la Lpre)

(Asociacin Internacional de la Lepra)

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

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

cios Phenomenon and Pure and Primitive Diffuse Lepromatosis (Latapis 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 PatientA Phenomenon of Immune Reconstitution - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

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

Disease - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

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

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

Ji, Baohong. Comments on WHO/AFROs Post-Elimination Strategy Paper: A New Bottle with Old Wine ofthe Final Push - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Correspondence

Premkumar, Ramaswamy, Rajan, Pichaimuthu, and Daniel, Ebenezer. Quantitative Measurement ofSensory Impairment in Referral Centers - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Rada, Elsa Mara, 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, Mnica Nunes Souza, Ferreira, Luis Carlos de Lima, and Talhari, Sinsio. 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 Its Utility? - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

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

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

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

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INTERNATIONAL JOURNAL OF LEPROSY

and Other Mycobacterial Diseases

CONTENTS

Volume 73, Number 3, September 2005

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

(ISSN 0148-916X)

INTERNATIONALJOURNAL OF LEPROSY



Images from the History of Leprosy

Kalaupapa, Hawaii.

Music was an integral part of peoples lives at Kalaupapa, Hawaii. In 1901, the KalawaoChoir posed alongside Father Damiens Church. This image was electronically reproducedfrom an original black and white photograph.

Photo courtesy of IDEA.

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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 Hansens 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)

INTERNATIONALJOURNAL OF LEPROSY



Clinical and Histologic Variations Among

Thirty Patients with Lucios Phenomenon and

Pure and Primitive Diffuse Lepromatosis

(Latapis Lepromatosis)1

Thomas H. Rea and Robert S. Jerskey2

ABSTRACTThe clinical and histologic experience with 30 patients who had Lucios phenomenon,

and pure and primitive diffuse lepromatosis (Latapis lepromatosis) has been reviewed. Theunanticipated clinical findings were a male to female ratio of nearly 1:1, a 21 month median

time 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 in

comparatively 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 Lucios phenomenon. With one conspicuous exception, the onset of treatment witha microbicidal agent was associated with a cessation of new lesions of Lucios 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 Latapislepromatosis, specifically venous insufficiency and/or loss of protective sensation, and onlyrarely from Lucios phenomenon, specifically scar formation. Briefly summarized are the

seven patients who had had a skin biopsy before the onset of Lucios phenomenon, as wellas the two patients who were considered to be atypical. Criteria for the diagnosis of Latapislepromatosis, in the absence of Lucios phenomenon, are also considered.

RSUMCet article sest attach faire la revue de lexprience clinique et histopathologique de

30 patients atteints de phnomne de Lucio et/ou de lpre lpromateuse pure et primitive-

169

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170 International Journal of Leprosy 2005

ment diffuse, encore appele lpre lpromateuse de Latapi. Les donnes cliniques sur-prenantes furent un ratio homme-femme de presque 1:1, un temps mdian de 21 mois entrela mise en ouvre dun traitement antibactrien et le dclanchement dun rythme noueuxlpreux (raction de type 2), et labsence dune anesthsie distribue en bas-rsille chez 7patients. La lsion histologique non anticipe a t la dcouverte dune vasculite lproma-teuse et granulomateuse atteignant des vaisseaux de diamtre relativement lev ou bien des

vaisseaux largis par les changements histopathologiques, situs prs de linterfacederme/hypoderme. Cette lsion tait prsente chez 6 des 22 patients qui prsentaient dumatriel histologique pour une revue. Chez 2 de ces 6 individus, cette vasculite fut identifie

avant le dclenchement du phnomne de Lucio. Lors de phnomne de Lucio et lexcep-tion dun cas plutt remarquable, lapparition de nouvelles lsions a t interrompue dans lasemaine qui a suivi la mise en place dun traitement avec un agent bactricide. Une morbid-it long terme, autre que les ractions de type 2, fut trouve chez 22 des 25 patients qui ontt suivis pendant plus de 1,3 ans. Le plus souvent, cette morbidit tait la consquence dela lpre lpromateuse de Latapi, spcifiquement linsuffisance veineuse et/ou la perte de sen-sibilit protectrice, et seulement rarement les consquence du phnomne de Lucio, spci-fiquement lapparition de cicatrices. Brivement rsums sont les 7 patients qui ont eu une

biopsie cutane avant lapparition dun phnomne de Lucio, ainsi que les 2 patients quifurent considrs comme atypiques. Les critres pour le diagnostic de lpre lpromateuse deLatapi, en labsence de phnomne de Lucio, sont galement prsents.

RESUMENSe hizo una revisin de los datos clnicos e histolgicos de 30 pacientes que haban tenido

el fenmeno de Lucio y lepromatosis difusa pura y primitiva (lepromatosis de Lucio). Loshallazgos clnicos no anticipados fueron: una relacin masculino: femenino casi de 1:1, un

tiempo promedio de aparicin de eritema nodoso leproso (reaccin de tipo 2) de 21 mesesdespus del inicio del tratamiento antibacteriano, y ausencia del patrn de anestesia media-guante (stocking-glove) en 7 pacientes. El nico hallazgo histolgico no anticipado fue unavasculitis lepromatosa-granulomatosa, presente en vasos comparativamente grandes o envasos agrandados por los cambios patolgicos, localizados cerca de la interfase dermo-subcutnea. Este hallazgo estuvo presente en 6 de 22 pacientes con material accesible pararevisin. En 2 de estos 6 pacientes la vasculitis fue identificada antes de la aparicin delfenmeno de Lucio. Con una sola excepcin, el tratamiento con un agente microbicida es-tuvo asociado con la suspensin, en la primera semana, de nuevas lesiones del fenmeno de

Lucio. La morbilidad crnica, diferente a la reaccin de tipo 2, se encontr en 22 de 25 pa-cientes seguidos por ms de 1.3 aos.

Usualmente la insuficiencia venosa y/o la prdida de sensacin protectora fueron conse-cuencia de la lepromatosis de Lucio y slo raramente del fenmeno de Lucio en cuyo casola consecuencia ms frecuente fue la formacin de cicatriz.

Se describen brevemente los casos de los 7 pacientes que haban tenido una biopsia depiel antes de la aparicin del fenmeno de Lucio, y de dos pacientes considerados comoatpicos. Tambin se discuten los criterios para el diagnstico de la lepromatosis de Latapi

en ausencia de fenmeno de Lucio.

Since reporting 10 patients with Luciosphenomenon (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 Lucios phenomenon as well as in the

underlying 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 Lucios phenomenon.

Also, a higher incidence of a lepromatous-granulomatous vasculitis (L-GV) wasfound in patients with Lucios 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 a

necrotic 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

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73, 3 Rea and Jerskey: Lucios Phenomenon and Latapis 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 a

part 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 Luciosphenomenon or erythema necroticansand the diffuse, non-nodular lepromatousinfiltration pure and primitive diffuse lep-romatosis. For the latter expression, thesynonym Latapis lepromatosis is pro-posed, and will be used hereafter herein.This gives an appropriate and briefeponymic recognition of Latapis importantcontributions. Also, having Lucio in twoclosely related eponymic terms, i.e., Lucioleprosy and Lucios 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 Lucios phe-nomenon and the underlying Latapis lepro-matosis have been recognized in diverse

ethnic 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 policy

of 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

Hansens 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 Lucios phenomenon, i.e., ser-rated hemorrhagic infarcts usually arisingin crops, and at least one characteristic signof Latapis lepromatosis (see below) or oflepromatous leprosy, but no lepromatous

nodules. The diagnosis of Latapis lepro-matosis was made after the fact of Luciosphenomenon. 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 Lucios 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 Lucios phenomenon(Cases 2, 3, 6, and 7), whereas 3 had had abiopsy but the diagnosis of leprosy was notmade until the onset of Lucios 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 toLucios 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 systemic

disease. 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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TABLE1.

Frequencyofclinic

alsignsofLatapislepromatosis.

Numberof

Number(%)

Number(%)

N

umber(%)

patients

notrecorded

Comment

evaluated

positive

Negative

orsought

I.Clinical

sinequanoneforLatapisLeproma

tosis

A.Diffu

senon-nodularLepromatousleprosy

30

30(100)

0

0

peaudorangech

angeinCase#8

II.Highlys

uggestiveofLatapislepromatosis

A.Telangectasias

28

10(3)

0

18(69)

Maybemorecom

mon

1.e

ruptive

3

2.m

atsonfaceandtrunk

7

B.Notvisiblesubcutaneousplaques

28

8(29)

0

20(71)

Maybemorecom

mon

C.Signsofdiffuseinfiltration

28

22(79)

0

6(21)

Maybemorecom

mon

1.n

on-marginatedindurationofcheeks

4

2.w

ideningofnasalroot

9

3.swollenbacksofhands

9

4.d

uskyswellingoffeet

1

III.Consiste

ntwithLatapislepromatosis

A.Alop

eciaofeyebrows

30

27(90)

0

3(10)

Perhapsuniversa

l

1.total

20(67)

2.p

artial

7(23)

B.Rhin

itis

30

25(85)

0

3(10)

Perhapsuniversa

l

C.Septalperforation

25

9(36)

8(32)

8(32)

D.S-GP

SIwithlittlemotorchange

30

20(67)

7(23)

3(10)


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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) Ahigh 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


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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. An

alternative 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 Lucios 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 prompt

intervention, 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) Occlusion

of large muscular arteries may occur in as-sociation with Lucios phenomenon (8). 3)Lucios 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 Lucios 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 for

review) 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 by

thin arrows, and is infiltrating the adventitia. 20 objective. c) Elsewhere in the same specimen an aneurysmal

change in a vessel is shown by the relatively dark internal elastic lamina. 10

objective. d) A high power view ofa re-epithelized Lucios 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)

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FIG. 2. From Case 4. a) From a Lucios 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 Lucios 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.


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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. She

is considered to be atypical because of apeau dorange 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 Lucios 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-cios phenomenon-like infarcts developed

on the trunk and extremities. With theworking diagnosis of a relapsing Luciosphenomenon, 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 Lucios phenomenon must beconsidered.

SUMMARY OF ALL CASES

Demographic dataSixteen men and 14 women satisfied in-

clusion criteria. Twenty-five were born in

Mexico, 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 Lucios 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 of

4.1 years.

Mode of presentationOf the 28 patients presenting to our clinic

with Lucios 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 Lucios phenome-non, usually occurring in intermittent crops(duration 5 days to 10 years, median 4

months, 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 lesions

were 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


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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 Lucios phe-nomenon was associated with pregnancy,each ending with a normal delivery. In Case5, the onset occurred while the patient was

convalescing from a Salmonella bac-teremia. No other potentially precipitatingevents could be identified.

Signs of Latapis 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 noted

in 9 patients, being variously described asswelling, or non-pitting edema, of thebacks of the hands, in association withfusiform fingers. Changes in the cheeksof the face were variously described in 4 asred plaques, poorly marginated, in-

durated erythema, or a cyanotic edema.Full or swollen ear lobes were statedto be present in 7 patients. A duskyswelling of the feet was described in 1.(Swollen ear lobes and fusiform fingers are

signs 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 in

20, 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 findingsat presentation

A mild anemia, normochromic and nor-mocytic, was common. The average leuko-cyte count (normal range 3.711.6 1000/mm3) was 6.5 and the median was6.3, among the 23 initial counts availablefor review. The highest count, 11.9, was


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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 Lucios 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.


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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-cios 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 Lucios 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 Luciosphenomenon 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 Lucios 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 Lucios phenomenon. AlsoCase 4, who might have had mild ENL4 years prior to developing Lucios 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 Luciosphenomenon.

Long term morbidityExcluding from analysis the 5 patients

followed for 1.3 years or less, some degree

of 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, appearto be a part of Latapis lepromatosis. Thethird mechanism producing long-term mor-bidity, scar formation, was the direct conse-quence of Lucios phenomenon. Secondaryto S-GPSI, 11 have experienced ongoing

problems 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


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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 Lucios 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 among

specimens 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 210%, with a medianvalue of 5%. In the remaining 6, the volumeoccupied was larger, ranging from

1540%, 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 Luciosphenomenon lesions the volume of the der-mis occupied by macrophages was approx-imately 510% in both patients.

Epidermal necrosis was present in 18 and

absent 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 necrotic

epidermis 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 thanvasculitis. 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, 2050% 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 less

of the panniculus, moderate in 5, involving2040%, and extensive in 4, involving5080%. The nature of the infiltrate variedconsiderably, 8 with leprotic macrophagesand only a few lymphocytes, 9 with an ob-


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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 Lucios phenomenon. In 2,Cases 3 and 5, the large vessel changeswere observed only in non-necrotic speci-

mens, obtained before the onset of Luciosphenomenon. 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. 1ac), and in lesions from 2other Lucios 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. 2aand 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 subcutaneous

vessels 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 vessels

were 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 Lucios 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 Lucios 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 Lucios phenom-enon patients with material available for re-view, and in 2 of the 3 pre-Lucios 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 Lucios 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 Lucios phenomenon lesion showing

intimal 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,


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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 6578.

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) 2733.29. SAOJI, V., and SALODHAR, A. Lucio leprosy withlucio phenonenon. Indian J. Lepr. 73 (2001)267272.

30. SHESKIN, J. Diffuse lepromatosis of the Lucio-Alvarado-Latapi with Lucio phenomenon: first casein the Near East. Rev. Leprol. 13 (1982) 651656.

31. SOUZA, C. S., ROSELINO, A. M., FIGUEIREDO, F.,and FOSS, N. T. Lucios phenomenon:clinical andtherapeutic aspects Int. J. Lepr. Other Mycobact.Dis. 68 (2000) 41725.

32. TANG, M. B. Y., and YOSIPOVITCH, G. AcuteChurg-Strauss Syndrome in an asthmatic patient

receiving montelukast therapy. Arch. Dermatol.139 (2003) 715717.

33. TURKEL, S. B., VAN HALE, H. M., and REA, T. H.

Ultrastructure in leprosy. Int. J. Lepr. Other My-cobact. Dis. 50 (1982) 164171.

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


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1 Received for publication on Dec. 13, 2004. Accepted for publication on May 20, 2005.2N. 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 andMyco-bacterium leprae infection.

RSUMLa relation entre la lpre et linfection par le VIH nest pas encore compltement com-

prise, comme peu de choses sont connues sur lhistoire des patients co-infects. Le sujet estdevenu dautant plus confus que des rapports contradictoires ont t publis. Les ractions

lpreuses de type 1 et les nvrites seraient plus svre et frquentes parmi les patients co-infects. Mais lrythme noueux lpreux nest pas aussi rare parmi ces patients que ce quiavait t originellement considr.

La prise en charge clinique de ces patients co-infects est souvent difficile, car elle

manque de recommandations claires pour le traitement. Nous rapportons ici notre expri-ence traiter un rythme noueux lpreux rcurrent et svre chez un patient souffrant con-comitamment de lpre et dinfection par le VIH. La mise en ?uvre rapide dune thrapieanti-rtrovirus semble avoir jou un rle pivot dans lamlioration du rsultat thrapeutiquechez ce patient. Cela suggre une interaction directe et plus complexe entre les infections par

Mycobacterium leprae et le VIH.

RESUMENLa relacin entre la lepra y la infeccin por el VIH no est completamente entendida,

como tampoco se conoce mucho acerca de la historia natural de los pacientes co-infectados.El tema ha llegado a ser todava ms confuso debido a la publicacin 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 secrea.

El manejo de estos pacientes co-infectados a menudo es difcil debido a la ausencia delineamientos claros sobre su tratamiento y cuidado clnico. Aqu, nosotros reportamos nues-tra experiencia sobre el tratamiento recurrente de eritema nodos leproso grave en un pacientecon lepra co-infectado por el VIH. La administracin temprana de la terapia anti-retroviralfavoreci 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 infeccin porM. 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 mycobacterial

infections 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. He

was febrile (102F). 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 diagnosis

was 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 (without

sensory 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

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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 30

mg/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)againstM. 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 of

ENL (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 the

case 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 withdrawal

subsequently. 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).

REFERENCES1. ANDRIEU, J. M., and LU, W. Long term clinical,

immunologic and virologic impact of glucocorti-coids on the chronic phase of HIV infection. BMC

Med. 2 (2004) 17.

2. ANDRIEU, J. M., and LU, W. Viro-immunopatho-genesis of HIV disease: implications for therapy.Immunol. Today 16 (1995) 57.

3. BARNES, P. J. Anti-inflammatory actions of gluco-corticoids: molecular mechanisms. Clinical Sci.94 (1998) 557572.

4. BELLIAPPA, A. D., BHAT, R. M., and MARTIS, J.Leprosy in type-1 reaction and diabetes mellitus in

a patient with HIV infection. Int. J. Dermatol. 41(2002) 694695.

5. BWIRE, R., and KAWUMA, H. J. S. Type 1 reactionsin leprosy, neuritis and steroid therapy: the impact

of the human immunodeficiency virus. Trans. R.Soc. Trop. Med. Hyg. 88 (1994) 315316.6. GEBRE, S., SAUNDERSON, P., MESSELE, T., and

BYASS, P. The effect of HIV status on the clinicalpicture of Leprosy : a prospective study in

Ethiopia. Lepr. Rev. 71 (2000) 338343.7. GROSSMAN, Z., MEIERSCHELLERSHEIM, 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) 319323.

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) e56.

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) 383391.

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.

11. MILLER, R. A., SHEN, Y.-Y., REA, T. H., and HAR-

NISCH, J. P. Treatment of chronic erythema no-dosum leprosum with cyclosporin-A produced

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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

Hansens Disease Programs, Louisiana State University, Baton Rouge, 70803 U.S.A.Reprint requests to: James L. Krahenbuhl, Ph.D., Laboratory Research Branch, National Hansens 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 withMycobacterium 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 fromM. leprae suspension using sodium hydroxide solution and its

subsequent 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 that

treating 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 viableM. 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 labelingM. leprae with the fluorescent dye PKH26, without compromising onthe viability of the organism. This method may be useful in intracellular trafficking studies

ofM. 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.

RSUMDepuis des annes, les chercheurs ont men des expriences partir de Mycobacterium

leprae issues de lsions multibacillaires humaines, de tissus infects de tatous neuf bandes

ou bien de tissu de coussinets plantaires de souris tant conventionnelles que nues athymiques(nu/nu). Ces sources de bacilles de Hansen sont en gnral satisfaisantes pour la plupart destudes biochimiques et dinoculation au coussinet plantaire de souris, mais pas satisfaisantespour les tudes de biologie cellulaire et dimmunologie, o les lments contaminantsprovenant de lhte peuvent reprsenter un srieux problme. Nous avons vrifi lutilitdune procdure base de soude pour liminer les tissus plantaires de souris contaminant lessuspensions deM. leprae, en vrifiant son effet sur la viabilit de la bactrie par la dtermi-nation du taux doxydation de lacide palmitique, de lintgrit de la membrane de la bac-trie et de la croissance dans le coussinet plantaire de souris. Nous avons trouv que le traite-

ment pendant 3 minutes avec 0.1 N NaOH, de suspensions deM. leprae obtenues partir decoussinets plantaires de souris nu/nu, tait suffisant pour enlever la majorit des tissus de

souris sans pour autant affecter de faon adverse la viabilit de lorganisme. Cest une mth-ode simple et rapide, qui permet dobtenir des suspensions viables deM. leprae partir delpromes de souris nu/nu dvolues presque entirement de tissus de lhte, reprsentant demeilleurs ractifs de recherche pour tudier la relation hte-pathogne de la lpre. Nous rap-portons galement ici une mthode pour marquer les M. leprae avec le produit fluorescentPKH26, sans compromettre la viabilit du microorganisme. Cette mthode peut tre utilepour tudier les mouvements et localisations intracellulaires deM. leprae ou bien des tudes

de biologie cellulaire, o le marquage de la bactrie par un colorant fluorescent est requis,afin de pouvoir la suivre. Nous avons constat que le marquage tait stable in vitro pendantdes temps importants et naffectait pas la viabilit de la bactrie.

194


(ISSN 0148-916X)

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107 fresh, viable nu/nu-derivedM. leprae.When the mouse footpads became moder-ately enlarged (at ~6 months), they wereharvested for intracellularM. 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 Shepards method (18) and heldovernight at 4C, 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 ofM. leprae. 1 109freshM. leprae were resuspended in 1.0 mlof the appropriate concentration of NaOH[0.1N0.9N] (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 4C) thrice in7H12 medium and finally resuspended inappropriate media. There was a 30 to 50%

loss of bacteria in this process.Scanning Electron Microscopy. Ten l

suspension (1 109/ml) of 0.1N NaOHtreated or untreatedM. 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 critical

point 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.) containing14C-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 33C. 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 M Syto9and 30 M 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. The

numbers of bacteria were recounted follow-ing staining by Shepards direct countmethod (18).

Macrophage culture. Resident peri-toneal cells from Swiss mice were har-vested and allowed to adhere for at least 6hr at 37C 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 adherent

cells were infected overnight at 33C withPKH26 stainedM. 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.

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73, 3 Lahiri, et al.: Purification and Fluorescent Staining of M. leprae 197

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

Statistical analysis. The data are shown

as means standard deviation (S.D.) from arepresentative of three to four experiments.The raw data were subjected to one-tailedor two-tailed Students t test to determinewhether the observed differences betweenthe means were significant. p

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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

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

Growth of PKH26 stainedM. leprae inmouse footpads. One 104 PKH26 stainedor unstainedM. leprae were used to infect

each 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 of

bacilli. 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 armadillo

can 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 ofM. leprae withoutaffecting the viability of the bacilli. Otherprocedures have been developed to purify

tissue derivedM. 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 ofM. 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 characterize

cell 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-derived

M. 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 the

provision ofM. 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.

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maintain (1 to 2 years) as a source of bacillifor routine (weekly) experimentation. Ar-madillos are infected experimentally to pro-vide maximum numbers ofM. 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-derivedM. 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 derivedM.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 purifyM. 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 derived

M. 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 the

needs 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 purification

does 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 ofM.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 with

high numbers ofM. leprae per gram of tis-sue yield less viable bacilli. Highest viabil-ity for nude mouse derivedM. leprae is as-sociated with short to moderate periods invivo. Thus routine short term passage is thebest means to assure plentiful, viable stocksofM. leprae. The present study extends ourinterests in defining the properties of viable

M. leprae as a research reagent.Access to a reliable source of large num-

bers of pure, viableM. leprae would be an

important research resource for todays lep-rosy researcher especially those interestedin pursuing the cell biology of intracellularinfection withM. 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 highly

aliphatic 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


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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 ofM. 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 that

neither NaOH treatment nor PKH26 label-ing ofM. leprae affected the viability ofM.leprae as defined by RR, VS and growth inthe mouse footpad, the gold standardmeasurement 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 viableM. 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 the

fluorescently 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 multiplying

M. leprae are underway.Acknowledgement. The authors gratefully ac-

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

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