Topical Treatment of Cutaneous Leishmaniasis

Joseph EI-On, Ph.D., Rita Livshin, M.D., Zvi Even-Paz, M.D. , David Hamburger, Ph.D., and Louis Weinrauch, M. D . Department of Microbio logy and Immunology, Facult y of Hea lth Sciences, Dcn G urion University of the N cgev UE-O), Deer Sheva; Departmcnt of Dcrmatology, Hadassah Universi ty H ospital, Hebrcw University Had assa h Mcdical School (R L, ZE-P, LW), j erusalem; Teva Pharm aceutica l In dustries Ltd. (01-1 ), j erusa lem, Israel

Six ty- seven patients, 19 fem :ll es and 48 m ales, 4-66 years develo pments did not affect the clinical he:ding process o ld , sufferin g fro m lesions o f cutaneous leishmaniasis which was generall y completed in a period of 10-30 d ays were treated to pica ll y with an o intme nt comprising 15% after termination of trea tment. In addition, 94% of the paro m o mycin sulfate and 12% m eth ylbenzethonium chl o treated lesio n s healed with little o r no scarring. N o adverse ride in white soft pa raffin (P-ointment, U .K . patent clinica l or laboratory side effe cts w ere observed except fo r GBll7237A). After 10 d ays of treatment, twice daily, the a burning sensation at the site of trea tment. Parasites iso_ lesio ns in 72% of the treated pati ents were free of parasites, lated fro m patients w ho failed to respond to topical treat_ 15% becam e free with in an additional 20 d ays, witho ut m ent were found to be susceptible to PR-MBC I in both further trea tment, and 13% failed to respond. Pig m entation in vitro infected m acrophages and in vivo in experimenta lly develo ped in 18% of the trea ted lesio n s and inA ammatio n infected BALE/c mice. J In vest Dennato/ 87:284-288, 1986 of varying degree was associated with t he treatm ent. T hese

eishmania sis is a disorder produced by Aa gelbted pro prove to be a major antileishmani al th erapeutic advance against tozoa of the genus L eis/lll1 allia. Depending on the ca us both cutaneO Ll S and mucocutaneous leishmaniasis [1 0, 111. ative species, th e disease Ill ay m anifest itse lf as a sys Although C L is purely a skin disease, very fe w studies have tellli c, a destructive mucocutaneous, or a purely been made to devel op a simple topical application for its treat cutaneous chronic in fection. AII 3 forms are translilitted ment. Local treatment with imidazole derivatives was found to Lby species of sa ndAi es belong in g to the genus Phlevolo/III.IS (Old be in effective in curing CL ca used by either L. lIIajor or L. lIIexicana Worl d) or the genus LlIlz o/llia (New World). a/lla ZO Il ClIsis in both ex perimental anim.als and humans 11 21. H ow_ L eishlllania //"Iajor, one of the eti ologic agents of cutaneous lei sh ever, a chJ orpro l11a zin e-containing ointment was recentl y u sed mani as is (Cl) in the O ld World , produces in humans a se lf successfull y in hea lin g diffuse CL due to L. (/clhiop ica in hUlllan

hea ling les ion fo ll owed by long-las ting immunity to reinfecti on 1 13J. 1"1] . The introducti on of the paras ite into the skin is fo ll owed by In a previous study, an ointment containing paromomycin and the development of a pap ul ar lesion w hi ch may enlarge and ul methylbenzeth onium chlo rid e was fou nd to be hi ghly effective ce rate and persist for up to 18 months. Spontaneous resolution in curing CL in experimental animals [14]. All mice infected w ith of a large lesion often lea ves an un sightl y sca r. Both sexes and L. lIIajor LRC-LJ37 were cured of the cutaneous disease after local all ages are susceptible. treatment applied twice dail y for 6-10 days. The paras ites were C utaneous leishmani as is is of clinica l importance beca use of its tota ll y eliminated from th e lesions, which rapidly healed. The chroni city and its potential for local des tructi on and disfigure present study describes the effi cacy of similar topica l treatlllent ment. N o sa ti sfactory systemicall y ad ministered drug against th e in humans against CL ca used by L. I/Iajor. disease has yet been developed; reli ance is on a few drugs w hi ch are sometimes in effecti ve and may be associated with side effec ts. The need for effi cacious, more easil y administered, and less toxic MATERIALS AND METHODS antiicishmania l agents is required. Rifampicin was the first orall y Patients, Treatment, and Evaluation of Responses Sixty_ effective antileishmanial agent introduced to clini ca l medi cin e 12-4]. seven patients of both sexes and different ages with ea rl y and Other oral medica ti on, sLi ch as metronidazole 15-7] and co-tri advanced infecti on were trea ted (Table I). The number of lesions moxazole [8,91 showed controversial effi cacy against the disease. per patient was '/-39. In most cases only one, th e largest and The development of the new antifungal agent ketoconazole may most active les ion , was trea ted while either one or more other untreated lesions on the sa me patient se rved as control. The le siollS were trea ted with an ointment containing 15% paromo m ycin sulphate (PR) and 12% mcthylbenzethonium chl oride (MBCI) in white soft paraffi n (P-ointmcnt), twice daily fOr a M anuscript received October 25, 1985; ~cceptcd for publi ca ti o n March peri od o f 1° day s. All ointment preparations were manufactured 3, 1986. and supplied by Teva Pharmaceutical Industries Ltd. , Jerusalem. RcpriIH requcsts to: j oseph E I-On, Ph.D. , DcpartmcntofMicro bi o logy In se veral cases th e treatment was extended for another 10 da and Immunology, Fa culty or Hea lth Sciences, Ben G urion Uni vcrsit y of and in one patient th e trea tment was given for a period of 2 the N egev, l3 ec r -S h cv~, 84 105 Israel. Abbreviatio ns: days. Treated lesions were generally left un covered, but in a few C L: cutancous Ici shman.ia sis cases the lesions were covered by adhes ive tape (Dermiciear) to LRC: Leis/lIl/fIIl ia I{cfcrcncc Ccnter prevent cl othing or dirt contamin ation. Between treatments the M 13 I: Ill cthylbcnzethonium chl orid e lesions were w iped clean. PR: paromomycin sul phate The res ults obtained were defined as follows: (1) rapidl y ef-

284 VOL. 87. NO.2 AUGUST 1986 TOPIC AL T ltEATMENT OF CUTANEOUS LE ISHMANIAS IS 285

Table I. Res ults of Topical T rea tment w ith P-Ointment on C utaneous Leish n1Jniasis

Description of Patients Da ys from Appearance of Lesion ro O nset of Age (years) Treatment No. of Lesions per Patient Effect of Treatment 5ex N o. Percent Average ± 5D Ran ge Average ± 5D Ran ge Average ± 5D Range

Rapidly effective M 34 50.7 24.34 ± 8.6 19- 55 86.09 ± 43.9 30-240 6.H7 ± 5.6 1- 19 F 14 20.8 23.23 ± 14.0 19-66 120.38 ± 77.3 30- 270 5.7 1 ± 3.5 1- 12 Less effective M 6 8.9 29.83 ± 12.2 19-4·<) 78.33 ± 58.4 60-180 10. 16 ± 9.8 3- 39 F 4 5.9 29.75 ± 20.2 19-60 100.00 ± 34.6 60-120 8.50 ± 8. 1 1-20 Ineffecti ve M 8 11. 9 21.1 5 ± 10.3 2- 36 96.25 ± 38.9 60- 180 8.25 ± 8.9 1- 29 F I 1.5 25.00 ± 0.0 25 250.00 ± 0.0 250 2.00 ± 0.0 2

fective: no parasites were detected in the treated les ion at th e cnd RESULTS of 10 days of treatment; (2) less rapidl y effective: para si tes werc still present in the treated lesion at the cnd ofl 0 days oftrea tl11 cnt, T he effcct of topica l treatment w ith P-ointmcnt as determined but disa ppeared w ithin the nex t 20 days; (3) in effe cti ve: paras ites by smcar and cul tu re techniques in 67 pati ents is summarized in still present in the lesions at least 1 month after the end of tre:J[ Table I. After 10 da ys of treatment, rapid clearing was obtained in 48 (7 1.6%) patients. Less rapid clearing was demonstrated in ment. 10 (14.9%) and no effe ct was obscrved in 9 (13.4%) of the treated Diagnosis and Laboratory Examinations In all cases the di patients. In the rapidl y clearing group, parasites were totall y elim agnosis was based on positive smea r and/ or cultures . La rge les ions in ated from the lesions by the end of the treatment and the les ions were examined at several sites. Fo r smear and culture preparations were clinicall y hea led within an add itional 10-30 days. In mos t the lesion was first cleaned with 70% eth ano l. Materi al aspirated cases, at the end of the treatment parasites were eliminated only with a fine Pasteur pipette through a sm all in cision made at the fro lll the treated les ions, w hile the untreated les ion (s) of the sa me margin of the les ion with a sterile surgica l blade was stain ed with patient still contained livin g parasites. Age, sex, size of the les ion, Giemsa and cultured in N . N .N . blood-agar medium. In multiple and duration sin ce infection, as well as the presence of crusts and infecti on 1-4 lesions of the sa me patient were subjected to par bacteri al contamination did not influence the response to treat asitologic evaluation and used as contro l. T he cul ture was in cu ment. Comparison of trea ted and untreated lesions of the same bated at 28°C and the development of li vin g promas ti gotes was pati cnt in a " rapidly clea red " group, who have more than I lesion determined. C ultures were considered negative only after 20 days and only 1 of them has been treated w hile the others were left w itho ut growth. Protozoal examinations were performed prior untreated and used as control, is given in Fig 1. T he resu lts ob to, at the end of, and every 2 weeks after termination of treatment. tai ned indicated that the rate of protozoal cl ea rance in the "rapidly The parasites were identified as Leishlllallia lII ajor (formerl y L. cleared" group was signifi ca ntl y fas ter for treated lesions than for tropica lII ajor) by the isolated paras ites excreted factor (EF) sero control lesions of the sa me patients during the first 20 days after typing '1 5] and characterization of enzymes by electrophoresis termination oftre;Jtment. Furthermore, w hi le 100% of the treatcd [16]. lesions were clea r of parasites 20 days after te rmination of treat Routine laboratory exa minations co mprisin g blood sedimen ment, 100% clearance was obtai ned w ith the untrea ted control tation rate, differential bl ood coun t, blood biochemistry including les ions of these patients onl y after 60 days. T he sign test fo r 2 li ver fu nction tests, and urinalysis were done prior to and on related sa mples indicated that thc ratc of protozoal clearance of completion of trea tment. the les ions that responded to trea tment was signifi can tl y faste r than that of the control les ions. O intment comprising 15% PR Determination of Paronlomycin Coneentration in Serum and 1% MBCl fa iled to cure C L infcction after 10-20 da ys of The microbiologic inhibition zone method, described by Arret treatmcnt. In the " in effective" group, hea lin g of the lesions was et al [17], was adopted for meas uring paromo m ycin concentration achi eved only w ithin 3-1 8 months after termin ation of treatment. in the serum of patients treated with P-ointment. T he zones of inhibition produced in a Petri dish covered w ith agar medium Histologic and Laboratory ExaUlinations Generall y the and seeded w ith a Staphylococc/ls cpiderlllidis ATCC 12228 culture clinical appearance of th e les ion during and at the cnd of treatment around a 100-fLl sa mple of serum to be assayed were eva luated. was worse than at the start. Various degrees of inflammation, Sera were coll ected 1, 4, and 24 h after term in ation of 10 days of depending on the lesion size and the hos t response, were asso treatment. ciated w ith this treatment. Pigmcntation developed in 17.6% of the treated les ions and several weeks to I yea r were required for Resistance and Susceptibility of Parasites to Paro its regress ion. However, all these developments did not influence momycin Pron'las ti gotes cultured f!'Om lesions w hi ch re the hea lin g process w hi ch was generall y completed within a pe sponded poorly to topica l treatment were used to infect C3H ri od of 10-30 da ys after termination of treatment (Fig 2). It was mouse peritonea l m acrophages in culture at 37°C and also the notable that only 6% of the treated les ions resolved w ith marked base of the tai l of BALB/c mice as previously described [1 4,18]. scarring as compared with 94% of the untreated control lesions In the in vitro study, the infected macrophages were exposed to (Table 11 ). Routine laboratory examinations indicated no adverse paromomycin at 100 ILg/m l for 4 days and the effect on the side effects. A burning sensa ti on at the site ofrrea tment someti mcs intracellular am astigotes was examined microscopicall y in Giemsa occurred during the fi rst few ointment appli ca ti ons. stained preparations. In the in vivo stud y, the base of th e ta il of Bioassay using Srap hylococC/is epidenlliridis, a test scnsitive to 0.1 the mice was inoculated with 2.5 x 10" promas ti gotes. Approx p,g/ ml of paromomycin, fa iled to detect the drug in the sera of imately 45 days after infection the les ions that had developed at treated patients, 1, 4, and 24 h after drug ad ministrati on. the site of infection w ere treated w ith P-ointment, twice daily for a period of 10 days. T he presence of viable parasites in the treated Microbial Susceptibility and Clinical Response to Paro lesions was monitored microscopicall y in both smears and cul momycin Four of the 9 pati ents w ho responded poorl y to tures as previously described [14] . treatment ("ineffective") were highl y reactive to the infecti on, 286 EL-ON ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

21 PATIENTS 25 PATIENTS 13 PATIENTS 11 PATIENT S g PAT IENTS 5 PATIENTS

~ . f:: . g .; g- . , . ~: o...... »: ~ . .. : ;:~;!=:~:~;~t ~:1-,,;=-I-----l~: : ~~ .~ ~ £~ :f:: T:EAT~:TREATED

1-10 11-20 21-30 31-40 41-50 51-60 DAYS AFTER TERMINATION OF TREATMENT

Figure 1. Percentage of parasite clearance from a si ngle treated and sin gle untreated les ion on each patient from a group of 25 patients wi th CL designated ·'rapid hea lin g" after treatment wi th P-ointment. Drop-out of patient attenda nce fell from 25 to 5 patients during th e time of exam i nation.

with well-developed granulomata at the inoculation sites. In these cases only partial clinical improvem ent was observed and the parasites were detectable even after 28 days (1 case) of treatment. The other 5 patients suffered from 1-11 sim pie cutaneous lesions located on the nose, arm s, hands, legs, elbow, neck, and chin. Generally lesions on the tip of the nose responded slowly and poorl y to topical treatment with P-ointment. Studies in vivo in experimental animals showed that parasites isolated fr om ' ''in effectively'' treated patients were as susceptible to parom omycin as those from patients successfull y responding to trea tment. Total eliminati on of intracellular am as tigotes was achieved in vitro with all the 4 isolates studied within 4 days of exposure to 100 IL g/ml PR, and all the infected BALB/c mice were completel y cured of the cutaneous disease aft er application of the ointment twice dail y for a period of 10 days. N o relapse of the disease was noted in these mice over the next 60 days. DISC USSION In the present study of the efficacy o f a new topical trea tment of CL, the instructi ons of the WHO report [1 9] were foll owed in most cases. Apart from 2 patients who had previously been un successfully treated by intralesional infiltration of Pentostam, none of the pati ents participating in this study had received any antl leishmanial treatment prior to the start of the topical treatment. Systemic drug trea tment of CL is limited to only a few com pounds, of which th e pentavalent antimonial compounds, Pen tostam and Glu ca ntime, given either i. v. or i.m ., are considered the first drugs of choice [20]. Although generall y well tolerated and associated with only minor side effects, these drugs should be given under hospital ca re. Drugs that have the advantage of bein g given o ralJ y, such as ketoconazole and rifampicin, have features which m ay sometimes limit their clinica l usefuln ess and the results in leishmanias is are vari able. In the study described, almost 72% of the 67 patients treated with P-ointment were clear of paras ites after 10 days of treatment and another 15% cleared within an additional 20 days. After the end o f the treatment, clinical improvem ent occurred rapidly with most patients becoming free of inflammati on w ithin 10-30 days . N either the number of lesions nor the duration of the disease correlated with the response rate. Furthermore, treatment of 1 Figure 2. A patient with C L before and after treatment with P-ointmenr. les ion affected the hea ling of other untreated lesions of the sa me The lesion was trea ted twice daily for 10 da ys. The ",,/libel'S are days after patient showing total clea rance of these lesions w ithin 60 days infection, whereas th e ,,,,,,,bers wi/hi" pnl'OIlIheses are days after termination after termination of treatment. During this period all the patients of treatment. in the " in effective" group remained infected. In this group com plete hea li ng of the lesions was achieved onl y 4-16 months after termination of treatment. lesion is a result of immune response activation. (Paromomycin In human leishmaniasis the host reaction is considered to be concentrati on in serum at therapeutic dose as determined by oth predominantly ce ll mediated and humoral fac tor apparently plays ers is 5-12 IL g/ml [21 ,22].) This hypothesis is further supported onl y a secondary role [1] . The fact that the parom omycin level by our previous study with BALBlc mice [14] . These mice are was undetectable in patients' sera indicates a limited penetration highly susceptible to infection with L. major and suffer from of the drug and suggests that the rapid healing of the untreated visceral and lethal disease [23]. In these mice trea tment of a leish- VOL. 87. NO.2 AUGUST 1986 TOPIC AL TREATMENT OF C UTANEOUS LEISHMANIASIS 287

Table II. Effect of Topical Treatment w ith P- Ointment on host susceptibility rather than parasite resistance is responsible for the Development of Scars and Pigm entation the variable results obtained with patients. It is possible that the immunologic state of the host affects not only the clinical pattern Treated Untreated of this disease [27], but also plays an important role in the effi cacy (total no. of lesions = 34) (tota l no. of les ions = 100) of the treatment [28]. Deep Superfi cial No Dee p Superfi cial No Diffuse C L, recurrent CL, and chronic CL are likely to occur Scars Scars Scars Scars Scars Sca rs in individuals w ith an impaired immune res ponse m echanism [28]. These diseases are difficult to treat and are often resistant to Number 2 10 22 94 4 2 of les ions conventional therapy [28 ]. H enriksen and Lende [1 3] described Percent 5.9 29.4 64.7 94 4 2 the effectiveness of chlorpromazine ointment in healing diffuse CL due to L. aelhiop ica and w e showed the efficacy ofP-ointment Twcnty-nine pa tients werc examined with lesion durations of 30 (3 patients). 60 in curing recurrent CL caused by L. Il'Opica [29]. The present (4 patients). 70 (II paticnts). 120 (8 paticnts). and 180 (3 paticnts) da ys. work extends these resul ts to simple CL ca used by L major. Areas that sti ll require clarification include optimum dosage and sched ule techniques. Altho ugh further data must accumulate before definite statements on the relative effi cacy of this treatment can manial lesion due to L. major ca used elimination of parasites only be m ade, it seems that this topical treatment m ay prove to have from the trea ted les ion and almost no effect was o bserved on the a promising future in the treatment of CL. untreated lesions of the same animal [14]. Furthermore, guinea pigs, a laboratory model for cutaneous leishmaniasis, infected in both ears with L. cllriellii were clear of para sites on the treated, The alllh ors Ihallk Blallen MO lllillo jiJr her lechllica l assislall ce . right, ea r after 10 days of trea tment with P-ointment [24]. A delayed effect was also o bserved on the untrea ted lesion on the left ear. Those becam e clear of paras ites 30 days after the last REFERENCES treatment of the right ea r. Such an effect was not observed in I. Zuckerm an A: Paras ito logica l review. Curren t status of th e im highly susceptible guinea pigs with well-developed lesions and munology of blood and ti ss ue protozoa. I. Leishlllallia. Exp Par metastases. In addition , treatment given at diffe rent times after asito l 38:370-400. 1975 infection neither sto pped nor abolished antibody production and 2. EI-Din Sa lem MM. Kandel E: Rifampicin in the treatment of cu delayed type hypersensitivity development, and their level 13 tan eous leishmanias is. J Kuwait Med Assoc 6: 159-1 66. 1972 , weeks after infection was higher in the treated animals as com 3. Iskandcr 10: Rifampicin in cuta neous leishmaniasis. J Int Med Res pared w ith the untreated, spontaneously cured guinea pigs. Sim 6:280- 285, 1978 ilar phenomena were also described by Bassiouny et al [25] in 4. Even-Paz Z, Weinrauch L, Livshin R. EI-On J, Greenblatt CL: Ri f patients suffering from multiple infection with L. //Iajo l' treated ampicin trea tment of cutaneous leishmani as is. Int J Dermatol by cryotherapy. T hese authors suggested that the local treatment 21 :11 0-112, 1982 causing damage to the parasites is fo ll owed by the release of 5. 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19. World Health Organi zation Report of the workshop on chemo 25. Bassiouny A, EI Mes had M, Tabat M , Kutty K, Metawaa B: C ry_ therapy of O ld World cutaneous leishmaniasis. TDR/Leish/CL- osurgery in cutaneous lei shmaniasis. Br J Dcrmatol 107:467-474 J ER/83.3, Jerusa lem, Israel, 1983 . 1982 ' 20. Kern P: Leishmaniasis. Antibiot C hemother 30:203-223, 1981 26. Guirges SY: N atural and experimental reinfection of man w ith Ori_ 21. Arustayan TA: Treatment with 111 0no m ycin of patients with acute ental sore. Ann Trop Med H yg 65 :1 97-205, 197 1 necrotizing leishmaniasis. Vestll Derl11atol Venereol 41:45-49, 1967 27. Preston PM, Dumondc DC: C linica l and experimcntalleishm3niasis 22. Eastwood J: Paromomycin: serum levels fo ll owing parenteral Immuno logy of Parasiti c In fection. Edited by S Cohen. E Sadun: administrati on. Antibio t C hemother 12:77-84, 1962 Oxford, Blackwell Scientific Plibliciti ons. 1976, pp 167-202 23. Djoko-Tamnou J , Leclerc C, Modabber F, C hedid L: Studies on 28. Strick HA , Borok M , Gasiorowski HC: Recurrent cutaneo us leis h_ visceral Leishlllll llia I/'op iw infectio n BALBlc mice. C lin Exp Im manias is. J Am Acad Dermatol 9:437-443. 1983 lllunoI46:493-498, 198 1 29. EI-O n J . Weinrallch L. Li vs hin R, Jacobs GP, Even-Paz Z: Topical 24. EI-On J , Witztllm A, Schnur LF: Protecti on of guin ea pigs against treatment of recurrent cutaneo us leishmaniasis w ith paro m ol11Y_ cutaneous leishmaniasis by combined infection and chemotherapy. cin-Illethylbellzethollium chloride ointment. Br Med J 291:704-705 Infect Im l11un , 5 1:704-706, 1986 1985 '