INTRODUCTION MATERIAL AND METHODS

466

RANDOMIZED, CONTROLLED, DOUBLE-BLIND TRIAL OF TOPICAL TREATMENT OF

CUTANEOUS LEISHMANIASIS WITH PAROMOMYCIN PLUS

METHYLBENZETHONIUM CHLORIDE OINTMENT IN GUATEMALA

Center for Health Studies, Universidad del Valle de Guatemala, Guatemala; Liverpool School of Tropical Medicine, United Kingdom

Abstract. A double-blind, randomized trial was undertaken in Guatemala to determine the therapeutic efficacy of an ointment for the treatment of cutaneous leishmaniasis that contained 15% paromomycin and 12% methylbenzeth-onium chloride and that was applied twice a day for 20 days. The treatment group included 35 patients, and the placebo group included 33 patients. The initial clinical response rate (13 weeks after completing the treatment) was 91.4% in the treatment group and 39.4% in the placebo group. The final clinical response rate at the 12-month follow-up examination was 85.7% (31 of 35) in the treatment grofollow-up and 39.4% (13 of 33) in the placebo grofollow-up (Pⱕ 0.001). In general, the treatment was well tolerated and was never interrupted because of adverse effects. The number of adverse effects reported in the placebo group was lower than in the treatment group (16 events versus 30 events). All adverse effects reported by patients disappeared within 1 week of completing the treatment. Our findings show that the combination of paromomycin with methylbenzethonium chloride for 20 days is a good alternative for anti-monial treatments of cutaneous leishmaniasis in Guatemala.

INTRODUCTION

More than half a century after the introduction of anti-monials into the chemotherapy of leishmaniasis, pentavalent antimonials—either sodium stibogluconate or meglumine antimonate—remain the drugs of choice in the treatment of all forms of leishmaniasis. They are widely used despite their toxicity, difficulty of administration, high cost, and the promising good clinical response showed by some other che-motherapeutic agents.1–5 _{The development of new drugs for}

the treatment of leishmaniasis has been impeded by the lack of a simple and rapid drug evaluation system, applicable to the various Leishmania species infecting humans. In addi-tion, leishmaniasis, like most of the protozoan diseases, is largely a problem of developing countries and therefore of-fers little commercial incentives for pharmaceutical compa-nies to develop cheap and effective antileishmanial drugs.4

The development of a compound used for the topical treat-ment of cutaneous leishmaniasis (CL) is an attractive option for overcoming the problems of antimonials. One line of research has focused on paromomycin (PR) ointment in dif-ferent combinations. Paromomycin is an aminocyclitol ami-noglycoside antibiotic used for the treatment of leishmania-sis. Diverse preparations of topical PR have been tested on CL in animals and humans. Randomized, placebo-controlled trials that used 15% PR plus 10% urea topically applied during 2 weeks, for example, have been conducted in Tu-nisia,6 _Iran,7_{and Honduras.}8

The topical application of PR (15%) plus methylbenzeth-onium chloride (MBCL; 12%) in a soft paraffin-based oint-ment was effective against Leishmania major in both exper-imental animals and in humans.9–11_{In a study of 52 patients}

with CL in Ecuador, it was reported that after 20 applications of PR ointment over 10 or 20 days, 90% of lesion were cured at 100 days after treatment and 85% at the 1-year follow-up examination, but only 9% of the lesions of untreated patients healed spontaneously after 50 days.12 _{However, this}

con-trolled trial under field conditions did not include randomi-zation or placebo treatment. In Colombia, the application of PR ointment in combination with systematic meglumine

an-timonate treatment did not show an additional benefit when compared with meglumine antimonate alone.13

In order to shed light on the question whether PR ointment is an efficacious alternative treatment for CL, a double-blind, randomized trial was undertaken in Guatemala that used an ointment containing 15% PR and 12% MBCL applied twice a day for 20 days.

MATERIAL AND METHODS

Patients. The study population was selected from patients

attending the leishmaniasis clinic of the Universidad del Val-le de Guatemala. Persons were eligibVal-le for the study if they met the following criteria: either male or female sex, aged 10–60 years, and parasitologically confirmed CL. Further, all patients had to provide written informed consent to par-ticipate in the study, and they had to be available for follow-up examinations for 12 months. People were excluded from the study for any of the following reasons: ⬎ 4 lesions or an active lesion measuring⬎ 5 cm in diameter; previous use of antimony-containing drugs; serious concomitant medical problems; and evidence of mucosal involvement of leish-maniasis.

Topical treatment. The PR ointment, paromomycin

sul-fate 15% plus MBCL 12%, and the placebo ointment tubes, which contained only soft paraffin, were prepared and ran-domly numbered by Teva Pharmaceutical Industries, Petach Tikva, Israel, and provided to the investigators by Dr. F. Modabber (Tropical Diseases Research/World Health Orga-nization [TDR/WHO]). Both active and placebo ointment were supplied in tubes of 14 g; they were identical in ap-pearance and were marked only by a consecutive number. Neither the researchers nor the patients knew if the active substance or the placebo were in the tube. The codes iden-tifying the contents of each tube were kept in Geneva by the TDR/WHO representative (F. M.) until the study was com-pleted. The ointment was applied topically twice a day for 20 days across the lesions in 2 different directions at 90 degrees to each other. Patients were instructed to wash their

TABLE1

Pretreatment characteristics of 76 patients with cutaneous leishman-iasis by treatment group

Characteristics

Paromomycin and methylbenzethonium

chloride Placebo

No. of patients

Mean age of patients in years (standard error)

No. of lesion per patient Mean area of ulcers (cm2₎

Mean duration of lesions (days) 38 22.3 (1.8) 1.1 (0.05) 1.1 (0.3) 101.2 (19.9) 38 20.3 (0.8) 1.3 (0.1) 1.3 (0.3) 105.1 (13.8) Ethnic group Indian Ladinos* 47.4 52.5 42.1 57.9 Localization of lesions (%) Arms Legs Thorax Other 63.1 15.8 10.5 10.6 65.8 18.4 7.9 7.9 * Ethnic mixture of local indigenous people with immigrants.

lesions with soap and water before applying the ointment. After the applications, the lesion was left uncovered. The amount of ointment used during each application varied de-pending on the lesion size; however, a 14-g tube per patient was enough to treat all the patients.

Patient evaluation. Confirmation of the CL infection was

based on either observation of amastigotes in thin smears obtained from the edge of the ulcer and stained with Giemsa stain, or by culture of promastigotes from aspirates taken through the skin adjacent to the ulcer, following previously described methods.14_{Only patients with positive cultures or}

clearly distinguishable amastigotes entered the study. As re-ported in previous studies, most of the patients (75%) we studied are infected with L. braziliensis, and the rest (25%) are infected with L. mexicana15–17_{; therefore, no}

character-ization of Leishmania parasites was performed. Surprisingly, despite the high incidence of L. braziliensis infections, very few cases of mucocutaneous leishmaniasis were found in the study area (Arana B, unpublished data).

All patients were evaluated at the end of Weeks 1, 2, 3, 4, 6, 9, 13, 26, and 52 after therapy. The treatment response was classified as follows:

1. Initial clinical response: a patient whose lesions had completely reepithelialized and who had no evidence of in-flammation or indurations by the 13-week follow-up exam-ination.

2. Final clinical response: a patient who had an initial clinical response and had no disease reactivation during the follow-up period between 13 and 52 weeks.

3. Reactivation: the appearance of a lesion within or at the border of a previously healed lesion.

4. Treatment failure: an increase in lesion size of⬎ 100% compared with the size at the first day of treatment; lack of a clinical response by the 13-week follow-up examination; or reactivation of a lesion. Patients who experienced treat-ment failure were removed from the study and treated with meglumine antimonate (20 mg antimony per kilogram of body weight per day, administered intravenously, for 20 days).

Statistical analysis. Comparison of response rates over

the first 13 weeks was performed by Cox’s log rank test. Comparison of the final response rates and rates of adverse effects were performed by the chi-square test or Fisher’s exact test (Epi Info version 6.04b, Centers for Disease Con-trol and Prevention, Atlanta, GA). All values are 2-tailed, and P⬍ 0.05 was considered statistically significant.

Ethical considerations. The Ethical Review Committee

of the Universidad del Valle de Guatemala approved the study. Informed consent was obtained from all adult partic-ipants and from parents or legal guardians of minors.

RESULTS

Patient characteristics. A total of 76 patients were

en-rolled. Table 1 shows that randomization successfully allo-cated subjects with similar characteristics into the treatment and control groups. All 76 patients had their diagnoses con-firmed by smear, culture, or both; 36 (47%) of 76 were pos-itive for both culture and smear, 34 (45%) had only pospos-itive smears for Leishmania amastigotes, and 6 (8%) had only a

positive Leishmania cultures. The 76 patients had a total of 91 lesions.

Initial clinical response. All patients but 4 received their

treatment without interruption. The 4 patients who did not finish their treatment, and 4 additional patients who were lost after their 21-day clinical evaluation were not included in the final analysis. Out of the 68 patients who completed their evaluation at the 13-week examination, 35 belonged to the treatment group (PR-MBCL ointment) and 33 to the pla-cebo group.

The initial clinical response rate was 91.4% (32 of 35) in the treatment group and 39.4% (13 of 33) in the placebo group. The cure rate in both treatment groups at the different control times is shown in Figure 1. Among the 3 patients who failed to respond to therapy in the treatment group, 2 (5.7%) showed a reduction in the size of lesion, and only one (2.9%) showed no clinical improvement at all. In the 20 failures to respond to therapy in the placebo group, 2 patients (6.1%) showed a reduction in lesion size, 3 (9.1%) showed no improvement in the lesions, and 15 (45.4%) showed in-creased lesion size, including 5 patients who had to be re-moved from the study before Week 13 because the area of their lesion had more than doubled.

Final clinical response. Between Weeks 13 and 52, only

one patient (3.1%) of the 32 in the treatment group, and none of the 13 patients in the placebo group, with healed clinical lesions at the 13-week follow-up examination experienced reactivation of the lesion. The reactivation occurred 6 months after the beginning of the treatment. As a result, the final clinical response at the 12-month follow-up examina-tion was 85.7% (31 of 35) in the treatment group and 39.4% (13 of 33) in the placebo group (P⫽ 0.000025). All patients in both treatment groups who failed to respond to therapy were treated with meglumine antimonate at 20 mg/kg once a day parenterally for 10 days. All patients were cured with only one course of treatment.

Adverse effects. In general, the treatment was well

tol-erated and had never to be interrupted because of adverse effects. Of the 35 patients who received PR-MBCL oint-ment, 22 (62.8%) reported a total of 30 adverse effects. These effects included local pruritus (46.7%), sensation of

FIGURE1. Percentage of patients who responded to treatment with paromomycin (PR) ointment or placebo by week of follow-up exami-nation

burning (30%), local pain (20%), and local edema (3.3%). All adverse effects reported by patients disappeared within 1 week after finishing the treatment.

No statistically significant difference was found between the number of patients reporting adverse effects in the treat-ment and placebo group (22 of 35 versus 14 of 33; P ⫽ 0.14). However, the number of adverse effects reported by the placebo group was lower (16 events) than the number of adverse effects in the treatment group (30 events).

DISCUSSION

Topical treatment of CL is attractive compared with the systemic treatment with antimonials because of the easy ap-plication, particularly in remote areas (Behrend M, unpub-lished data) and the reduced cost. Likewise, the treatment with orally administered antileishmanial drugs, which have been tested successfully in Guatemala,17_{are expensive and}

the treatment course prolonged, which make them, from a public health services perspective, not a good choice. The main question with topical treatments is related to its ther-apeutic efficacy and its potential adverse effects. The con-trolled trials on therapeutic efficacy of topical CL treatment with PR ointment are summarized in Table 2. They all used PR mixed with either urea (10%) or MBCL (in concentra-tions of 5 or 12%) together or not with meglumine antimon-ate. The therapeutic effect was measured either against a placebo group6–8_{or a quasiplacebo group (i.e., without}

treat-ment12_{) or against a group treated with meglumine}

antimon-ate.13,18 _{There was a wide variation in the study protocols}

used, in the determination of cure rates, and in the patho-genicity of the Leishmania parasites, so a comparison of the therapeutic efficacy is difficult. However, the following pat-tern seems to emerge: urea (10%) in addition to PR seems to have little or no therapeutic efficacy. The poor results with PR when combined with urea emphasize the antileishmanial properties of MBCL shown by El-On and Messer19_{and the}

synergetic anti leishmanial effect of MBCL and PR.20

Ad-ditionally, PR as an antibiotic may reduce secondary infec-tions and promote healing. Only in a recent controlled trial in Ecuador has the combination of PR with urea showed a similar cure rate of CL (70% in 30 patients) as PR-MBCL (78.2% in 23 patients) and meglumine antimonate (88.6% in 35 patients) after 1 year of observation; however, the treat-ment period was very long: twice a day for at least 12 weeks.21

The addition of MBCL to PR apparently has a high ther-apeutic efficacy if applied long enough—that is, for ⬎ 15 days. This was reconfirmed by our study in Guatemala, which showed a therapeutic efficacy of 54% (cure rate of treatment group minus cure rate in placebo group divided by cure rate in treatment group). In other words, the prob-ability of cure with the PR-MBCL in 20 days was 2.17 times higher than in the placebo group. This result is no different from the cure rates obtained in the same endemic area, where a 90% cure rate has been obtained when meglumine anti-monate is used at doses of 20 mg/kg for 10–20 days22_(P⫽

0.95). In terms of cost, however, if we could prepare the ointment locally, it would cost US$6.00, as compared with

TABLE2

Controlled trials of topical treatments of cutaneous leishmaniasis

Refer-ence Leishmania strain Country Treatment or placebo groups* Cure rate (follow-up time) 7 L. major Iran PR 15%/10% urea twice a day for 14 days

Topical placebo (soft paraffin) twice a day for 14 days

68% (15 weeks) 68% (15 weeks) 6 L. major Tunisia PR 15%/10% urea twice a day for 14 days

Topical placebo (urea 10%) twice a day for 14 days

69.8% (15 weeks) 68.4% (15 weeks) 27 L. tropica Turkey PR 15%/MBCL 12% twice a day for 15 days

Ketoconazole 400 mg/day po for 30 days

37.5% (not reported) 0% (not reported) 10 L. major Israel PR 15%/MBCL 12% twice a day for 10 days

PR 15%/MBCL 12% twice a day for 20 days PR 15%/MBCL 5% twice a day for 10 days

Topical placebo (soft paraffin) twice a day for 10–20 days

86.2% (not reported) 93.1% (10 weeks) 66.6% (10 weeks) 18.1% (10 weeks) 28 L. major Sudan PR 15% twice a day for 10–20 days

Placebo ointment

31.3% (3 weeks) 13.3% (3 weeks) 18 L. braziliensis panamensis Colombia PR 15%/MBCL 5% twice a day for 10 days plus Sb for 7

days

PR 15%/MBCL 5%. Twice a day for 10 days plus Sb for 3 days

90% (52 weeks) 42% (52 weeks) 13 L. braziliensis panamensis Colombia PR 15%/MBCL 12% twice a day for 10 days plus Sb for 7

days

PR 15%/MBCL 12% twice a day for 10 days plus Sb for 3 days

Topical placebo twice a day for 10 days plus Sb for 7 days Sb for 20 days

58% (52 weeks) 20% (52 weeks) 53% (52 weeks) 84%

8 L. mexicana, L. chagasi Honduras PR 15%/urea 10% 3 times daily for 28 days Topical placebo 3 times daily for 28 days

1.8% (15 weeks) 1.8% (15 weeks) 12 L. braziliensis panamensis Ecuador PR 15%/MBCL 12% twice a day for 10 days

PR 15%/MBCL 12% once a day for 20 days Placebo (no treatment, not randomized)

85% (52 weeks) 85% (52 (weeks)

9% (7 weeks) * MBCL⫽ methylbenzethonium chloride; PR ⫽ paromomycin; Sb ⫽ meglumine antimonate.

the standard treatment with meglumine antimonate, which costs US$280 in Guatemala.

Our study on the socioeconomic consequences of CL in Venezuela has shown that the direct and indirect costs for the patients are high and the negative social consequences severe.23 _{This is why an efficacious treatment of this}

gen-erally self-limiting disease is important both for patients and health services. Operational research has shown that the top-ical treatment, with few well-defined exceptions, can be eas-ily applied by basic health staff (Behrend M, unpublished data), so this option should be pursued further.

In addition, topical treatment has no systemic effects and cannot therefore prevent relapses, or in some areas the de-velopment of the mucocutaneous forms of the disease. How-ever, the recurrence of cutaneous lesions and parasite per-sistence has been reported in patients treated for CL24_{; there}

are also reports describing the detection of leishmanial DNA by means of polymerase chain reaction in patients with vis-ceral leishmaniasis who received treatment with antimonials and were believed to be cured.25,26

Acknowledgments: We thank Dr. F. Modabber from TDR/WHO for obtaining the ointment and for taking care of the control of treatment codes. We also thank the patients, who accepted taking part in the controlled trial in order to contribute to improved treatment options for CL.

Financial support: This investigation received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR).

Authors’ addresses: Byron Arana, Carlos Mendoza, and Nidia Rizzo, Center for Health Studies, Universidad del Valle de Guatemala, Apartado Postal No. 082, Guatemala, Guatemala. Axel Kroeger,

Liv-erpool School of Tropical Medicine, Pembroke Place, LivLiv-erpool L3 5QA, United Kingdom.

Reprint requests: Byron A. Arana, Center for Health Studies, Univ-ersidad del Valle de Guatemala, Apartado Postal No. 082, Guate-mala, GuateGuate-mala, Telephone: 502-369-0791, Fax: 502-364-0354 (e-mail: [email protected]).

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