Treatment of Cutaneous Leishmaniasis by Topical Application of Honey

Apitherapy has become the focus of interest as a form of alternative and preventive medicine for treatment of a number of clinical cases. Honey has been suggested as an effective healing agent in treatment of postoperative wounds [1]. In many cases honey has cleaned up wounds when conventional treatment was unsuccessful. Honey has a potent antibacterial activity and is very effective in clearing wounds infection by inhibiting a wide range of bacteria [2]. Commercial honey has been reported to accelerate wound healing when applied topically to experimentally induced wounds [3]. Wadi [4], was the first to investigate the biological activity of Sudanese honey on purulent wounds of hospitalized patients when applied topically, favorable results were obtained. Honey can enhance wound healing by promoting healthy granulation tissue growth and act as a barrier to prevent cross infection [4]. Antibacterial activity of honey was attributed to hydrogen peroxide naturally present in it [5].


Introduction
Apitherapy has become the focus of interest as a form of alternative and preventive medicine for treatment of a number of clinical cases. Honey has been suggested as an effective healing agent in treatment of postoperative wounds [1]. In many cases honey has cleaned up wounds when conventional treatment was unsuccessful. Honey has a potent antibacterial activity and is very effective in clearing wounds infection by inhibiting a wide range of bacteria [2]. Commercial honey has been reported to accelerate wound healing when applied topically to experimentally induced wounds [3]. Wadi [4], was the first to investigate the biological activity of Sudanese honey on purulent wounds of hospitalized patients when applied topically, favorable results were obtained. Honey can enhance wound healing by promoting healthy granulation tissue growth and act as a barrier to prevent cross infection [4]. Antibacterial activity of honey was attributed to hydrogen peroxide naturally present in it [5].
Cutaneous leishmaniasis is a zoonotic disease caused by protozoa of the genus Leishmania and is transmitted to humans by sand fly insects of the genus Phlebotomus. There are two important stages in the life cycle of Leishmania: amastigotes found in man and other non-human reservoir mammals and promastigotes found in the sand fly. In the vertebrate host, Cutaneous leishmaniasis is caused by the amastigote form, which infects the macrophage cells in which it multiplies by binary fission, causing death of the host cell. The lesion then becomes necrotic. The clinical outcome will depending on the parasite strain and the host immune response. The lesions are confined to the skin and to the mucous membrane A granulomatous response occurs and a necrotic ulcer forms at the bite site. Macrophages containing amastigotes, which may be killed by sensitized lymphocytes were detected in microscopic smear. The lesion may become chronic, usually accompanied by secondary bacterial infection [6]. Diagnosis is usually confirmed microscopically by demonstrating the presence of amastigotes in smear taken from the skin lesion or organs or may be demonstrated in hamsters inoculated with samples collected from patients. Sodium stibogluconate (pentostam), is the drug of choice given intramuscular in case of multiple lesions [6]. Recent clinical and laboratory research has addressed diagnosis by the polymerase chain reaction which seems to be approaching a gold standard status as the novel techniques offer considerable advantages [7 ]. Treatment in single lesion it may be injected into the margin of the ulcers. Leishmaniasis responds poorly to treatment and frequently relapses. Secondary infections add to the severity of lesions both in the skin and mucous membranes [6]. Zenia et al. [8] investigated the in vitro activity of honey dilutions against three species of Leishmania (the promastigote stage (. The result was compared with the effects of the same concentrations of sugars. Honey and sugar both had anti-leishmanial effects in vitro, but honey was found to be more effective than sugar. Mohammad et al. [9] tested honey as an adjuvant therapy with intralesional glucantime and reported that the effect of glutacantime in healing was actually decreased. parasite stocks and is available from Sigma Chemical, ( U.K ) in the form of powder. The recommended amount of powder was dissolved in 1L of deionized water and stirred with a magnetic stirrer until all the powder was dissolved, p H was adjusted to 7.4 by using 0.1 mL HCL. The medium was filtered through sterile filter using a 0.2 micrometer membrane filter. Then it was poured into pre-autoclaved sterile bottles and 10% V/V sterile fetal calf serum was added, 1% antibiotic-penicillin and streptomycin was added to prevent bacterial contamination. The medium was checked for sterility by incubation at 37ºC for 24 hours. 1 X 10 5 cells of leishmania major, ( isolated from patients ) was inoculated to the medium. Regular checks for parasite growth were made by observing the inoculated medium using microscopical objective lens( x 40 ). Incubation time was six days at 22 ºC.

Animal inoculation
A group of 50 adult male and female hamsters were shaved at the base of the tail and were cleaned by alcohol swabs. The medium was examined microscopically for promastigotes. Positive culture was centrifuged at 3000 rpm for 3 minutes, the supernatant layer was discarded. An aliquot of 0.1 Ml from the culture deposit was inoculated into the hamsters-tail base-intradermally. The hamsters were checked for lesion at the site of inoculation weekly. Impression smears were taken from detected lesion. The smears were then fixed with methanol and stained with Giemsa's. Biopsy was smeared on slide and allowed to dry. The slide was immersed in methanol for 1-3 minutes for fixation. Diluted Gimesa's stain (1:10) in buffered saline (p H 7.2) was used for 20 minutes. The slide was washed by distilled water and examined under oil immersion objective lens x100.

Biopsy technique
A small piece of skin was removed from the opened lesion of the infected hamsters by a sharp sterile blade near the edge of the lesion, in which amastigotes were present. The biopsy was transferred to 10% formalin in sterile universal containers for diagnosis. The material was fixed and sectioned for standard histopathological processing. The fixed smear material was processed for staining with Giemsa's stain.

Tissue processing
The following steps for tissue processing was; dehydration and embedding in paraffin wax. Automated tissue processing was performed.

Topical dressing of the infected lesion by honey
Infected hamsters were divided randomly into two groups, one group of 25 hamsters were treated with topical dressing of natural raw honey. The other 25 hamsters group served as control and were treated with pentostam injection. The animals were examined daily. All opened lesion of the 25 hamsters -test group -was covered by honey sample K obtained from Yemen-(Sidr). Honey was applied topically to the infected lesion by soaking gauze in honey and covered by plasters. Each animal was kept separately, to prevent licking by other animals. Animals were dressed daily by topical application of honey, lesion size was measured daily in cm. The control group was injected every other day by with 0.1ml intramuscular pentostam and the lesion was cleaned daily by saline and alcohol and covered by sterile gauze. The lesions were measured daily.

Results
After two weeks all inoculated hamsters developed different types of lesions ranging from nodules to opened ulcerated lesions Figures 1  and 2. The size of the lesion ranged from 0.5 to 2cm.  Smears were taken from all lesions and stained with Giemsa's stain and examined microscopically for amastigotes stage. The smears showed intracellular amastigotes, inflammatory cells, lymphocytes, histocytes and plasma cells. Amastigotes were found intracellular as well as extracellular due to the cell ruptured and a biopsy from the lesion was taken to make a definitive diagnosis. The group of hamsters treated daily by honey application on sterile clean gauze, showed a good response to treatment after one week. Lesion appears bright red with early epithelization tissue. Necrotic tissue was replaced with healthy granulation tissue Figures 3 and 4.

Discussion
Cutaneous leishmaniasis is one of the important endemic diseases in tropical and subtropical courtiers. It is a long lasting skin disease and the ulcer might be at high risk of secondary infection. Honey is a well known antibacterial agent that help healing wounds and ulcers [11]. The findings of the present study showed that healing of cutaneous leishmaniasis of hamsters treated with honey dressing was achieved within 4 weeks. Ulcers appeared bright red with healthy granulation  Healing of 80 % hamsters treated with honey were achieved within 4 weeks. Healing was assessed by disappearance of amastigotes in microscopic smear and ulcer size was decrease Figure 9.
Futhermore, formation of healthy granulation tissue and cleanliness of ulcers from bacterial infection confirmed the healing process. In healing lesion smears showed giant and lymphocytes cells. The other 25 hamsters served as control group: treated with pentostam injection showed poor response to the treatment, 48 % of hamsters were healed. Ulcers remaing infected with amastigotes, lesions appeared with pus and necrotic tissue, Figures 5 and 6. It took prolonged time to heal up to 16 weeks. Animals treated with daily application of honey responded within short period as compared with those treated with pentostam injection Table 1. Healing was judged by absence of amastigotes and cleanliness of ulcer from bacterial and fungal infection. Growth of healthy granulation tissue was obtained.
Complete healing of the lesions was assessed by absence of tissue free from amastigotes, bacteria and fungus, while the control group treated with pentostam injection took prolonged time to heal of 14-16 weeks. The lesions appeared infected with pus and necrotic tissue. Amastigotes were detected. The course of the healing process may take more than a year [6]. Honey has been suggested as an effective treatment for different kinds of infected ulcers as it was proved as antibacterial agent that inhibit a wide range of micro-organisms [10]. In speculating about the mechanisms of honey as destructive agent for amastigotes, it is reasonable to assume that Hygroscopic nature of honey, plus it is acidic properties (low p H 3.6) may inhibiting   Weeks Honey group -lesion size Control group -pentostam injection -Lesion size intracellular amastigotes. Honey is a saturated solution of sugars, with very low water content [5]. This condition is inhibitory to the most of microorganisms. Bactericidal and fungicidal effect of honey will most probably prevent secondary infection of the leishmanial ulcers. Also the low p H of honey may help to create and maintain optimal conditions for fibroblast activity which requires mild acidic wound condition [11]. Compatibility of these findings were confirmed previously by Wadi [4] who proved that honey accelerated healthy granulation tissue of 12 hospitalized patients. Recent findings reported by Zenia et al. [8] which proved that honey has an antileishmanial activity when tested in vitro. However, the combination of honey and glucatime was found ineffective in controlling cutaneous leishmaniasis when compared with glucantime alone [8]. Furthermore the pentamidine the drug of choice for the treatment of cutaneous leishmaniasis is considered the most effective drug but with side effects [11]. Leishmaniasis responded poorly to treatment and frequently relapses. Secondary bacterial infection of the ulcer, when present should be eliminated by treatment with appropriate antibacterial agents. Supportive treatment and good nursing care are needed in most cases, particularly those with complications. Treatment should be given until the aspirate or smear are free of parasites for at least two weeks. If relapses occur, the drug should be given for twice as long as the first treatment [6]. Alternatively, honey can be suggested as an effective treatment for cutaneous leishmaniasis concerning the toxic effects of drugs. Additional advantages with the use of honey it is low cost effectiveness as compared with the drugs.
Honey has been suggested as an effective agent for different kinds of infected ulcers, as honey was proved as antibacterial agent that inhibit a wide range of micro-organisms [1]. Honey cleans the infected wounds (i.e: diabetic wounds, ulcers, burns and gangrene) by inhibiting a numbers of Gram-positive and Gram -negative bacteria and fungi, so this might prevent secondary infection in leishmanisis, obviously honey help the healing process. Edema was subsided and necrotic tissue was replaced by healthy granulation tissue [4]. The hygroscopic properties of honey, may inhibiting amastigotes in macrophage cells. However, there has been tendency for healing process as honey gave favorable results when applied topically on infected wounds and ulcers [4] Bactericidal effect of honey prevented secondary infection of the leishmanial ulcers. Based on the above premise honey was thought as clearing and effective agent against amastigote. These results are in agreement with the previous findings by Zeina et al. [8], who found that honey was effective in vitro against promastigotes. Although the low pH of honey may help to create and maintain optimal conditions for fibroblast activity (migration, proliferation and organization of collagen which require mild acidic wound condition), [12]. Healing of cutaneous leishmaniasis was maintaned when the lesion was free from parasite and bacterial infection. this particular important steps in healing process. Anti -inflammatory effects of honey was attributed to the physical properties of honey, due to it is viscosity, honey provides a protective barrier which prevent cross infection. Additional to the anti -inflammatory effects, low hydrogen peroxide naturally present in honey promotes anabolic metabolism, giving cell growth. Tonks et al. [13], reported that honey stimulates cytotoxine release from monocytes, which gives rise to activation of tissue repair. Additional to these effects promoting tissue growth, honey provides vitamins, minerals, amino acids and sugars for the growing cells. Thus honey was found more effective than antileishmanial drugs.
Topical dressing of cutaneous leishmaniasis ulcers with honey is very effective and helps in the treatment in a short term as compared with pentostam treatment. Honey is a natural product can be used easily without side effect with low cost. Further studies to test the efficacy of honey in cutaneous leishmaniasis is needed.