Topical Therapy of Acute Cutaneous Leishmaniasis Using Zinc Sulphate Solution 25 % versus Podophyllin Solution 25 %

Background: Zinc sulphate as intralesional and oral therapy was used as a successful therapy in treatment of cutaneous leishmaniasis while 25% topical podophyllin is now commonly used in the treatment of cutaneous leishmaniasis. Objective: To treat acute cutaneous leishmaniasis using topical zinc sulphate solution 25% to be compared with topical podophyllin solution 25%. Patients and Methods: This is a single, blind, interventional, comparative study done in the Department of Dermatology, Baghdad Teaching Hospital, Baghdad, Iraq during the period from January 2015 to June 2015. Forty patients with a total 88 lesions of acute cutaneous leishmaniasis were enrolled in this study; 54 (61.36%) were dry lesions and 34 (38.6%) were ulcerative type. The duration of lesions ranged from 2 11 (7.7 + 2.483) weeks. The size of the lesions ranged from 0.3 to 8 (2.13 ± 1.53) cm. There were 21 females and 19 males, female to male ratio (1.1:1) and their ages ranged from 1 to 60 (25.92 ± 16.59) years. Diagnosis was confirmed by tissue smear and histopathology. Lesions were divided into two groups with matching types and sizes, and scored according to Sharquie modified Leishmania score to assess the objective response to the topical or systemic therapy and then during follow up responses were graded into mild, moderate, marked response and complete clearance. Group A treated with topical 25% podophyllin solution once weekly for a maximum of 6 weeks, while group B was treated with topical 25% zinc sulphate solution twice daily for 6 weeks. Follow up was every 2 weeks during therapy, and once monthly for 3 months after therapy. Results: A total of 40 patients with clinical diagnosis of acute cutaneous leishmaniasis were included in this study, with a total of 88 lesions. Fifteen (37.50%) patients had How to cite this paper: Sharquie, K.E., Noaimi, A.A., Sharara, Z.A.A., Saleh, B.A. and Al-Salam, W.S. (2017) Topical Therapy of Acute Cutaneous Leishmaniasis Using Zinc Sulphate Solution 25% versus Podophyllin Solution 25%. Journal of Cosmetics, Dermatological Sciences and Applications, 7, 258-274. https://doi.org/10.4236/jcdsa.2017.73024 Received: June 3, 2017 Accepted: September 26, 2017 Published: September 30, 2017 Copyright © 2017 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/


Introduction
Cutaneous leishmaniasis (CL) results from the parasitization of skin macrophages and generally is manifested as a papule that enlarges to a nodule that often ulcerates.Diverse leishmania species cause cutaneous leishmaniasis.In Iraq cutaneous leishmaniasis is mainly caused by L. major (60%) and L. tropica (40%) [1].
Many important strides have been made in treatment and control of cutaneous leishmaniasis.However, there are still approximately 1.5 million new cases of CL each year worldwide, with the bulk reported from Afghanistan, Iran, Iraq, Algeria, Saudi Arabia, Pakistan [2].According to the World Health Organization (WHO); leishmaniasis is endemic in 88 countries, with a total of 350 million people at risk, it is believed that worldwide 12 million people are currently infected by leishmaniasis [3].CL was endemic disease in Iraq since ancient times and from time to time outbreak and\or epidemic occurs.On 2014 an outbreak occurred in Iraq when hundreds of patients had been seen since October 2014 till April 2015 due to floods that occurred after heavy rains also by the role of terrorist who opened the gates of dams (Fallujah dam) on Euphrates river that resulted in floods in many towns and villages leading to spread of rodents the reservoir of leishmaniasis.
Most CL lesions are self limiting but spontaneous cure may take several months or even years.In spite of this, treatment is justified in a variety of cases, namely early lesions, multiple lesions, lesions involving cosmetically sensitive sites, and patients with significant immunosuppression.In addition psychological impact of the disease cannot be ignored.The aim of therapy is namely clinical healing and disappearance of parasites.The disease still presents a therapeutic challenge in several parts of the world.Unfortunately to date there is no safe, However, these have the disadvantage of both toxicity and clinical resistance in certain regions.Other treatment options given systemically have found favor in some studies [4]; such as imidazole compound, allopurinol, rifampicin, dapsone, zinc sulfate [5] and chloroquine [6].Local infiltration of drug have the advantage of direct effect on parasite due to chemical and osmotic mechanisms such as intralesional sodium stibogluconate antimony [7] and intralesional hypertonic sodium chloride solution (7%) [8], while intralesional zinc sulphate 2% didn't act as osmotic effect, when zinc sulphate was added to axenic amastigotes of both species of Leishmania it caused immediate death.There was no change in the shape of either forms with zinc sulphate, in contrast to 7% hypertonic saline that caused parasite shrinkage beside sudden paralysis [9].Physical methods have also been tried with success such as microwave thermotherapy [10], CO 2 laser [11], Cryotherapy [12].For simple cutaneous lesions which are few in number and where there is no risk of disfigurement or joint mobility restriction, topical application or local treatment would be a valuable option and often justified and many have been used like; azoles compound like; topical ketoconazole [13], clorimazole cream and miconazole cream [14].Other compounds like; topical paramomycine [15] and recently topical podophyllin with 85% cure rate [16].Topical therapy is an attractive choice as it is less toxic than systemic ones, and non painful as compared to intralesional therapies.Zinc sulphate had been used successfully in treatment of cutaneous leishmaniasis both systemically and intralesionally in treatment of CL with an encouraging cure rates [5] [8].Accordingly the present work was arranged to evaluate topical zinc sulphate 25% as an alternative therapy for CL and to be compared with effective topical therapy like 25% podphyllin.

Patients and Methods
This is a single blinded, interventional, comparative study done in the Department of Dermatology, Baghdad Teaching Hospital, Baghdad, Iraq, from January 2014 to June 2014.
A full history was taken from each patient regarding the followings: age, address, duration of the lesions and their number, recurrence of the lesion, history of previous therapy, also family history, past medical history, obstetric history regarding the females in reproductive period and past drug history.
A total of 48 patients with typical acute CL, had been enrolled in this study.
Eight patients with a total of 10 lesions were defaulted for unknown reasons.So the number of patients that completed the study were 40 patients with a total of 88 lesions; 54 (61.36%) were dry type and 34 (38.6%)ulcerative type.The duration of lesions ranged from 2 -11 weeks with mean ± SD of (7.7 ± 2.483 ) weeks.
The size of the lesions ranged from 0.3 to 8 cm with mean ± SD of (2.13 ± 1.53) cm.J. Cosmetics, Dermatological Sciences and Applications There were twenty one females and 19 males, female to male ratio (1.1:1) and their ages ranged from 1 to 60 years with mean ± SD of (25.92 ± 16.59) years.
Skin examination was performed including the site, size and type of the lesions; and regional lymphadenopathy.
Patients with the following criteria were excluded from this study: Pregnancy, chronic diseases, immunosuppressed like diabetes mellitus, peripheral neuropathy, poor peripheral circulation, and prolonged corticosteroid therapy, lesions with surface area more than 10 cm 2 or lesions close to eyes, patients who received anti-leishmanial treatment either local or systemic, if the lesions of more than 12 weeks duration were excluded because the possibility of self healing during follow-up period.Patients with re-infestation were also excluded.
After full interrogation and explanation to each patient about the nature of disease including its course, complications and prognosis, treatment modalities and their complications, a formal consent was taken from each patient.
Serial photos were taken in the same place in each visit, with fixed illumination and distance, using Samsung® Galaxy S3 mobile camera with 8 Megapixels.
Also, the ethical approval was granted by the Scientific Committee of the Scientific Council of Dermatology-Iraqi Board for Medical Specializations.In all patients, the diagnosis was established by history and clinical examination, and confirmed by dental broach smear and 3 mm punch biopsy for histopathological examination.
Smear was done by cleaning the lesion with 70% alcohol then using a dental broach.A sample was taken from more than one area of the lesion, smeared onto a clean glass slide, and stained with Leishman stain.Then a microscopical examination of the smear was done to demonstrate the amastigotes inside or outside the macrophages.
According to the therapy, the lesions had been divided into two groups with matching of types and sizes of lesions.Statistically, there was no significant difference between the two treated groups, regarding the number of the ulcerated and the non ulcerated lesions in each group, where, χ 2 = 3.761 and P value = 0.52 (Table 3).
Group A: Treated by 25% topical podophyllin in tincture benzoin: Twenty patients with a total of 39 lesions; 20 (51.28%) lesions were dry and 19 (48.72%) lesions were ulcerative, all treated with 25% topical podophyllin in tincture benzoin that was prepared by dissolving 25 g of podophyllin resin powder, purchased from Merck Company, Germany, in 100 ml of tincture benzoine and was kept sealed in dark place within dark glass container in room temperature.The solution was applied with cotton-tipped applicator.For the non ulcerated type, the lesion was covered with thin layer of solution, while for ulcerated type; the solution was applied to the margin of the ulcer.The amount of solution that was used in each session did not exceed 0.5 milliliter and the surface area treated did not exceed 10 square centimeters, in order to minimize the local and the systemic side effects of the drug.The solution was allowed to dry in approximately 2 minutes and patients were instructed to wash off it after 6 hours.The solution was applied once weekly for maximum 6 weeks.
Group B: Treated by topical 25% zinc sulphate: Twenty patients with a total 49 lesions; 35 (71.43%) lesions were dry and 14 (28.57%)lesions were ulcerative; all were treated with twice daily application of topical 25% zinc sulphate that was prepared by dissolving 25 g of zinc sulphate powder (heptahydrate purified)-ZnSO 4 .7H 2 O, M.W. 287.54, purchased from THOMAS BAKER Company, India, in 100 ml of distilled water and kept in dark glass container in room temperature.The solution was applied with a cotton tipped applicator and not to wash off, this was done twice daily for a maximum 6 weeks.

Assessment to Response of Therapy
Each lesion was assessed in the first visit through detecting the color of erythema and its diameter, measuring the size of each lesion was carried out by palpation and marking the induration and measuring its diameter with a caliber in centimeters unit.For rounded regular lesions one diameter was taken, while in irregular lesions the two maximum diameters (length and width) were taken to calculate the mean, in ulcerative lesion the diameter of ulcer was measured also.
Sharquie has modified his previous score to become more sensitive and specific; so scoring was done using "Sharquie modified leishmania score to assess the objective response to topical or systemic therapy" [17] (Table 1).

Follow-Up
Patients in both groups were followed up every 2 weeks during the treatment period that continued for six weeks, and then monthly for three months after

Statistical Analysis
Statistical analysis was done using SPSS 20; chi square were used to compare between grades of responses in each group, paired T test was used to compare between means of score in each visit for particular group, P value less than 0.05 considered significant.

Results
A total of 40 patients with acute CL, proved clinically and confirmed parasitologically were included in this study, with a total of 88 lesions; 54 (61.36%) of which were dry and 34 (38.6%)lesions were ulcerative type.Fifteen (37.50%) patients had single lesion and 25 (62.50%)patients had multiple lesions.Family history of CL was positive in 42.5% of patients.
The most common sites of body that frequently affected were the upper extremities with 51 lesion (57.95%), then the lower extremities with 26 (29.54%), while in the trunk there were only 4 lesions (4.54%) and neck with 5 lesions (5.68%) lastly the face with 2 lesions (2.27%).
1) The therapeutic response from the first visit until 6 weeks after treatment in each group were as follow: (Table 2).
Group A: a total of twenty patients with 39 lesions, 20 (51.28%) lesions were dry type and 19 (48.72%) lesions were ulcerative.
The baseline first visit the mean score of response ± SD was (13.74 ± 2.087).Two weeks after starting therapy the mean score of response ± SD was (10.56 ± 2.17), this was statistically significant when compared to the first visit (p value < 0.0001).
Four weeks after starting therapy; mean score of response + SD was (7.36 ± 2.518) by comparing the means with the first visit; it was statistically significant, p value < 0.0001.
Six weeks after starting therapy; mean score of response ± SD was (4.41 ± 2.314) which was statistically significant when compared to the first visit, (p value < 0.0001).The baseline first visit the mean score of response ± SD was (12.84 ± 2.12).
Two weeks after starting therapy; the mean score of response ± SD was (10.2 ± 1.75); this was statistically significant when compared to the first visit (p value < 0.0001).
Four weeks after starting therapy; mean score of response ± SD was (6.84 ± 2.35) by comparing the means with the first visit; it was statistically significant, p value < 0.0001.
Six weeks after starting therapy; mean score of response + SD was (4.47 ± 2.72) which was statistically significant when compared to the first visit, (p value < 0.0001) (Table 2).
Using ANOVA test to compare between the means of scores of both groups the affectivity (F = 128) in group B, and (F = 88) in group A. So treatment in group B is more effective (Table 2, Figure 1).
2) The percentage of grades of responses in each visit during therapy were as follow: Group A: Two weeks after starting the therapy: • Mild response in 25 (64.10%)lesions.
• No lesion showed marked or complete response.
Four weeks after starting the therapy: • Mild response in 5 (12.80%) lesions.
At the end of six weeks after starting the therapy (Table 3):   • No lesion with mild response was detected.
So the total cure rate (both marked and complete responses) was seen in 32 (82%) lesions.
In all clinical cured lesions, there were no or minimum scarring at the site of lesions, but post-inflammatory hyper pigmentation was noted in all treated lesions that disappear over time.No recurrence of lesions was noticed in any patient.There were no serious local or systemic side effects (Figure 2 and Figure 3).

Group B
Two weeks after starting the therapy: • Marked response; in 16 (32.70%)lesions.
At the end of 6 weeks after starting treatment (Table 3): • Mild response was detected in 4 (8.20%)lesions.
So the cure rate in this group (both marked and complete response) was achieved in 36 (73.4%) lesions.
After clinical resolution, post-inflammatory hyperpigmentation that disappear over time and minimal scarring were noticed in all lesions but no recurrence of lesion in any patients (Figure 4 and Figure 5).There were no significant side effects in this group apart from mild burning J. Cosmetics, Dermatological Sciences and Applications  sensation that occurred in one patient with ulcerative lesions.

3) Comparison between groups:
By comparing the percentages of grade of responses between the two groups in each visit: • Two weeks after starting therapy χ 2 = 0.896, P value = 0.344, no statistical significant difference.• Four weeks after therapy χ 2 = 0.528 and P value = 0.913, no statistical significant difference.
• Six weeks after starting therapy there were no statistical significant difference between the cure rates of both groups where χ 2 = 6.094 and P value = 0.107 (Table 3).In regards to lesions with complete clearance there were 16 (41%) lesions showed this response in group A, while 11 (22.4%)lesions in group B showed complete clearance which was not statistically significant difference, (P value = 0.06).

4) Comparison between dry and ulcerative lesions responses:
This study revealed that there was no statistical difference between the ulcerative lesions and dry lesions responses to each therapy; where, in group A; 16 (80%) out of 20 dry lesions had clinical cure 6 weeks after starting the therapy; and 16 (84.%)out of 19 ulcerative lesions had a clinical cure 6 weeks after starting the therapy, x 2 = 0.117, p value = 0.943.Also in group B; 24 (68.6%)out of 35 dry lesions showed clinical cure, and 12 (85.7%)out of 14 ulcerative lesions showed clinical cure at the end of six weeks therapy, x 2 = 6.739, p value = 0.081; statistically not significant.

Discussion
In Iraq CL is caused by two species as proved by PCR; (40%) L. tropica and (60%) L. major [1].Although this disease is endemic but now the country is showing an epidemic state.CL is a self limited disease but may continue for several months and even years [5] [7], causing great psychological trauma to the J. Cosmetics, Dermatological Sciences and Applications patients, in addition it can cause severe disfiguring with bad cosmetic appearance especially when occurs in the face, hence it's therapy is mandatory.So the aim of the treatment is to shorten the duration of the disease and to prevent scarring.On review of literature; many topical and systemic therapies were reported to be effective in clearance of cutaneous leishmania lesions [4].
Topical therapy is an attractive choice and is indicated when there is few lesions and to increase the concentration of the drug at the lesional site, then to increase its effectiveness.Also to reduce the cost of therapy and to avoid the systemic side effects of the systemic drug [18].
While systemic treatment for CL is often advised when there are numerous and/or large lesions that can't be locally infiltrated.The current recommended systemic treatment which had been used since the 1950s is parentally administered pentavalent antimonial compounds.Although it is effective but they are expensive and may cause serious side effects [24].Other systemic drugs that were used in treatment of CL like oral zinc sulfate, [4] [5] amphotericin B [9], rifampicin [25], antifungal agents [26]- [31], levamisole [32], dapsone [33] [34].
So the aim of present work is to use topical zinc sulphate rather than intralesional, as a simple non invasive mode of therapy for the patients, and to be compared with well-established topical therapy like 25% topical podophyllin sol [16].Using Sharquie modified leishmania score, in the present study, to assess response to treatment, topical 25% podophyllin solution gave (82%) cure rate, while the cure rate using topical 25% zinc sulphate solution was (73.4%) and there was no statistical difference between the two therapies.
Zinc is an essential element that its concentration in skin could be increased eightfold by topical application of zinc sulphate, a concentration of 3% was optimal [35].Topical zinc sulphate had been used successfully in the treatment of a wide variety of skin disorders such as: plane warts [36], recurrent herpes simplex [37], superficial fungal infection [38], melasma [39], and others.Intralesional zinc sulphate had also been used effectively for basal cell carcinoma [40] and in treatment of CL with (97.8%) cure rate [8].Zinc acts as caustic agent that cause a sudden paralysis of parasite in vitro.As the addition of 2% zinc sulphate solution to promastigotes of either L. major or L. tropica caused immediate paralysis and death of promastigotes, moreover when zinc sulphate was added to axenic amastigotes of both species of leishmania it caused immediate death.However there was no change in the shape of either forms of parasite with the addition of zinc sulphate, in contrast, the addition of 7% hypertonic saline to this parasite caused its shrinkage and sudden paralysis [5].On the other hand the live parasite growth were affected and dropped in culture containing zinc sulphate compound as compared to the parasite population in control culture [41].Accordingly zinc sulphate which is a cytotoxic agent [42] probably interfere with enzymes of parasites rather than changing the osmotic pressure of leishmania parasite.At this stage, the exact mechanism of the anti-leishmanial effect of zinc is not well established but it can be only speculated.It is well known that zinc plays an important role in specific metalloenzyme systems concerned with nucleic acid and protein synthesis [38], furthermore zinc has been reported to inhibit DNA polymerase of herpes simplex virus [37].Therefore, we might suggest that site of action of zinc is on enzymes that are concerned with nucleic acid metabolism.However further investigation is needed to confirm the exact mechanism of action of zinc against leishmania.
Podophyllin, is an alcoholic plant extract obtained from dried rhizomes of common plants emodi or podophyllum peltatum [24].
The mechanism of action of podophyllin is well known and as follow; 1) Podophyllin is proved to reversibly bind to tubulin which is the protein subunits of the spindle microtubules and then prevent polymerazation of the tubulin into microtubules [31] [47] [48].The microtubules which located beneath the plasma membrane of leishmania parasite and serve as a cytoskeleton [49].
2) It blocks oxidation enzymes in tricarboxylic acid cycle.This tricarboxylic acid cycle is one of cycles that involve in the nutrition of leishmania parasite [47] [48].
3) Also, it can inhibit axonal transport, protein, and RNA and DNA synthesis and inhibit of mitochondrium activity [47] [48].All these mechanisms could lead to death of the parasite, followed by cure of the lesion.
Topical zinc sulphate is more effective when it is compared with other topical treatments like clotrimazole cream which gave cure rate reaching to (15.7%) while topical 2% ketoconazole cream was not found to be more effective than placebo [35].
Furthermore the cure rate of topical podophyllin and topical zinc in the present work was higher than topical trichloracitic acid (TCA), which gave (65%) success rate [23], and higher than 5% topical imiquimod plus intralesional meglumine antimony which achieved (40%) cure rate [4].The above combination beside being expensive they were irritant on application as compared to topical zinc sulphate and topical podophyllin when both were well tolerated and non costly.
Moreover it was noticed that the cure rates of topical podophyllin and topical J. Cosmetics, Dermatological Sciences and Applications zinc sulphate individually were higher or approximate to topical paramomycine responsiveness that had been reported from (16.7% to 78%) in the studies of a 2-weeks regimen and up to (80%) in 4-weeks regimen study.Topical paramomycine had burning side effect and severe inflammatory reaction; in addition this drug is costly and unavailable [4] [15].
Cryotherapy had been used in the treatment of CL with 67.8% cure rate and with many side effects especially ugly scarring and permanent hypopigmentation; in addition to that, it was a painful method [4] [12].
In regards to intralesional sodium stiboglugonate which gave a high cure rate reaching to (94%) with one or 2 injections, but this drug is costly, not available, difficult in storage [7].Also in comparison with 2% zinc sulphate solution that was given intralesionally at 10 days intervals, the cure rate reached to (97.8%) usually with one or 2 injections [8], both above two drugs were painful when given intralesionally especially in children and need to be mixed up with xylocain and their mechanism of action, as antileishmanial drugs, was presumably as a result of their metallic contents which probably induce tissue damage and, subsequently, severe inflammatory changes had occurred [8].
The cure rates in the present work are less than intralesional hypertonic sodium chloride 7% which is given every 7 -10 days interval for 6 weeks, as it reached (96%), but topical zinc is non painful topical method, that doesn't need recurrent hospital visits [19].
In the present work, all the clinical cured lesions healed with minimal scarring and this result was similar to that reported in other modalities of treatment e.g.intralesional pentavalent antimonial compounds [7] and intralesional 2% zinc sulphate [8], intralesional 7% hypertonic sodium chloride [8], topical paromomycin [4].Also there was no serious local or systemic side effects, apart from mild burning sensation that occurred in one patient in the zinc group.Post inflammatory hyperpgimentation was noticed in all patients in each group and disappear over time.These side effects were similar and sometimes less than other modalities [18] [20].

Conclusion
In conclusion, topical zinc sulphate sol.25% gave 74.3% cure rate versus 82% cure rate in 25% podophyllin sol.Hence topical zinc sulphate therapy is noninvasive simple non-costly therapy that could be used alone or in combination with other topical or systemic therapy in the treatment of acute cutaneous leishmaniasis.

Disclosure
This study was an independent study and not funded by any drug companies.

K
. E. Sharquie et al.DOI: 10.4236/jcdsa.2017.73024260 J. Cosmetics, Dermatological Sciences and Applications simple, cheap and effective ambulatory treatment for CL.Pentavalent antimony compounds; still remain the mainstay of treatment in the majority of cases.

Figure 1 .
Figure 1.The decline in the means of scores during therapy.

Table 1 .
Sharquie's modified Leishmania score to assess the objective response to the topical or systemic therapy.In each visit the lesions were re-scored and response was graded accordingly, and to detect any local and systemic side effects.

Table 2 .
The mean and SD of total score of response within group A and group B for each visit.B: A total twenty patients with 49 lesions; 35 (71.43%) lesions were dry and 14 (28.57%)lesions were ulcerative.The response was as follow: P = P value.*Pairedt test was used to compare base line visit with other visits.Group

Table 3 .
The percentages of grades of responses in the treated lesions in each group 6 weeks after starting the therapy.