Nasopharyngeal myiasis due to Cephalopina titillator in Southeastern Iran: a prevalence, histopathological, and molecular assessment.

The Cephalopina titillator is one of the most important causative agents of nasal myiasis in camels. This study aimed to explore the prevalence, histopathological effects, and molecular identification of C. titillator infestation in camels of Kerman province, South-Eastern Iran, between 2019 and 2021. The larvae were placed in 10% formalin for histopathological evaluation and species identification. Pieces of larval abdominal segments of C. titillator were selected for extraction of DNA. Partial mitochondrial CO1 genes were sequenced for final analysis. Out of the 870 camels examined, 339 (38.9%) were infested with larval stages of C. titillator. There was a significant difference between age and infection rate (P = 0.001), while no association between males and females (P = 0.074) was found. The infection rate was significantly higher in the winter (P < 0.001) than in the other seasons. In this study, different lesions depending on duration, locations, and the depth of larval adhesion notably degeneration changes, necrosis, and ulceration were observed. Also, in chronic cases, granulation tissue reactions were organized. Cephalopina titillator was confirmed by PCR sequencing analysis using mitochondrial CO1 region. A 582 bp nucleotide sequence was deposited in GenBank under the MW136151 accession number. Phylogenetic analysis of CO1 produced a single uniform sister clade to MZ209004 and MW167083 records from China and Iraq, respectively. The high prevalence of C. titillator in camels in this region and other areas of Iran declares that the country is in an endemic status and displays the existence of the potential risk for camels.

Poor adherence is a major barrier to the proper treatment of cutaneous leishmaniasis: A case-control field assessment in Iran.

Leishmaniasis is an overlooked, poverty-stricken, and complex disease with growing social and public health problems. In general, leishmaniasis is a curable disease; however, there is an expansion of unresponsive cases to treatment in cutaneous leishmaniasis (CL). One of the effective and ignored determinants in the treatment outcome of CL is poor treatment adherence (PTA). PTA is an overlooked and widespread phenomenon to proper Leishmania treatment. This study aimed to explore the effect of poor adherence in unresponsiveness to treatment in patients with anthroponotic CL (ACL) by comparing conventional statistical modalities and machine learning analyses in Iran. Overall, 190 cases consisting of 50 unresponsive patients (case group), and 140 responsive patients (control group) with ACL were randomly selected. The data collecting form that included 25 queries (Q) was recorded for each case and analyzed by R software and genetic algorithm (GA) approaches. Complex treatment regimens (Q11), cultural and lay views about the disease and therapy (Q8), life stress, hopelessness and negative feelings (Q22), adverse effects of treatment (Q13), and long duration of the lesion (Q12) were the most prevalent significant variables that inhibited effective treatment adherence by the two methods, in decreasing order of significance. In the inherent algorithm approach, similar to the statistical approach, the most significant feature was complex treatment regimens (Q11). Providing essential knowledge about ACL and treatment of patients with chronic diseases and patients with misconceptions about chemical drugs are important issues directly related to the disease's unresponsiveness. Furthermore, early detection of patients to prevent the long duration of the disease and the process of treatment, efforts to minimize side effects of treatment, induction of positive thinking, and giving hope to patients with stress and anxiety by medical staff, and family can help patients adhere to the treatment.

Baseline susceptibility of a wild strain of main vectors of leishmaniasis to WHO-recommended insecticides in southeastern Iran.

BACKGROUND: In Iran, both cutaneous leishmaniases (CL) and visceral leishmaniases (VL) are endemic, recording one of the 10 highest CL prevalence in the world. Parasites are transmitted by the bite of infected Phlebotomus sand fly females. Several sand fly species have been identified as vectors in the studied region of Kerman province. Residual spraying to control adult sand flies, is the only way to decrease the spreading of the diseases but, following control treatment against malaria vectors in endemic areas in Iran, resistance or tolerance to insecticides emerged in some sand fly species. The objective of this study was to survey insecticides susceptibility levels of 3 vector species in wild sand fly populations in different foci of the diseases in Kerman province. Ph. sergenti, and Ph. papatasi respectively vectors of anthroponotic and zoonotic cutaneous leishmaniases and for the first time Ph. alexandri one of the anthroponotic visceral leishmaniases vector were tested against: deltamethrin 0.05%, malathion 5%, dichloro-diphenyl-trichloroethane (DDT) 4%. MATERIALS AND METHODS: In leishmaniases endemic areas species specific sand fly sites were selected in Kerman province, and specimens were collected by manual aspirators at different time intervals during the spring and summer 2019. All the susceptibility tests were performed according to the WHO tube test recommended procedure. RESULTS: Twenty five blood-fed female sand flies from the region's prevalent species were used in each pooled test replicates. All wild specimens died within 60 min of exposure to DDT 4%, malathion 5%, and deltamethrin 0.05%, but the mortality rate for Ph. papatasi exposed to malathion and DDT was 91.6% and 66.6%, respectively. CONCLUSION: According to current study results, Ph. sergenti and Ph. alexandri are highly susceptible to all the evaluated insecticides in the study areas. However, Ph. papatasi was susceptible to deltamethrin (100% mortality), possibly resistant or tolerant to malathion (91.6% mortality), and confirmed to be resistant to DDT (66.6% mortality).

In vitro and in vivo therapeutic potentials of 6-gingerol in combination with amphotericin B for treatment of Leishmania major infection: Powerful synergistic and multifunctional effects.

The ongoing conventional drugs for leishmaniasis treatment are insufficient. The present study aimed to assess 6-gingerol alone and in combination with amphotericin B on Leishmania major stages using experimental and in vivo murine models. Here, arrays of experimental approaches were designed to monitor and evaluate the 6-gingerol potential therapeutic outcomes. The binding affinity of 6-gingerol and IFN-γ was the basis for docking conformations. 6-Gingerol combined with amphotericin B represented a safe mixture, extremely leishmanicidal, a potent antioxidant, induced a remarkable apoptotic index, significantly increased the expression of the Th1-related cytokines (IL-12p40, IFN-γ, and TNF- α), iNOS, and transcription factors (STAT1, c-Fos, and Elk-1). In contrast, the expression of the Th2-related cytokines was significantly downregulated (p < 0.001). This combination was also potent when the lesion appearance was evaluated following three weeks of treatment. The histopathological and immunohistochemical patterns of the murine model represented clusters of CD4+ and CD8+ T lymphocytes which compressed and deteriorated the macrophages harboring Leishman bodies. The primary mode of action of 6-gingerol and amphotericin B involved broad mechanistic insights providing a coherent basis for further clinical study as a potential drug candidate for CL. In conclusion, 6-gingerol with amphotericin B synergistically exerted anti-leishmanial activity in vitro and in vivo and potentiated macrophages' leishmanicidal activity, modulated Th1- and Th2-related phenotypes improved the histopathological changes in the BALB/c mice infected with L. major. They elevated the leukocyte infiltration into the lesions. Therefore, this combination should be considered for treating volunteer patients with CL in clinical studies.

Dirofilariosis caused by Dirofilaria immitis in the south of Kerman province, Iran.

BACKGROUND: Dirofilariosis due to Dirofilaria immitis is endemic in various areas of Iran. Domestic dogs are the main reservoirs and represent a major potential infection source for the vector and humans. OBJECTIVE: This study aimed to explore the prevalence of dirofilariosis due to D.immitis and its public health importance in domestic dogs in the Jiroft district, south of Kerman province, Iran, by serological and parasitological methods. METHODS: This descriptive study was carried out as a cross-sectional investigation. A questionnaire was completed for 100 domestic dogs from May 2017 to February 2018 and recorded their age, sex, and clinical features. Also, we used enzyme-linked immunosorbent assay (ELISA) to identify antigens of heartworms in the bloodstream, with 98% sensitivity and 100% specificity, and parasitological techniques (Knott's test) to detect microfilariae in canine blood in Jiroft district, south of Kerman province, Iran. RESULTS: Overall, 10 (10%) and 4 (4%) domestic dogs were infected as confirmed by ELISA and modified Knott's tests, respectively. The rate of occult infections in the ELISA test than Knott's test was 60%. No significant difference was found between dirofilariosis and gender. In contrast, there was a significant difference between dirofilariosis infection and age (P < 0.05). CONCLUSION: The present findings could help understand the epidemiological aspects of D. immitis for future control programs and take appropriate preventive and therapeutic strategies against the disease.

Immunopathology of anthroponotic cutaneous leishmaniasis and incidental diagnostic tool of metastatic granuloma: A case-control study.

BACKGROUND: Cutaneous leishmaniasis (CL) is a neglected disease with important public health concerns in many parts of the world including Iran. OBJECTIVES: We aimed to explore the histological changes and immunohistochemical quantification of inflammatory cells and their role in the immunopathology of acute, chronic non-lupoid, and chronic lupoid skin lesions in anthroponotic CL (ACL). METHODS: In this study, skin biopsies of 53 patients with ACL were taken. Samples were studied by light microscopy and immunohistochemistry to quantify the immune and inflammatory cells. RESULTS: Of the 53 skin lesions, 38 were acute, nine chronic non-lupoid and six chronic lupoid. CD68+ macrophages were the most common cells. CD3+ T-lymphocytes were present as diffuse and focal dermal infiltrates and CD8+ cytotoxic T-lymphocytes were the dominant lymphocyte type, constituting more than 50% of the lymphocyte population. CD4+ T-lymphocytes in chronic non-lupoid (10.57 ± 2.37%) and chronic lupoid (14.40 ± 1.28%) lesions were more than those observed in the acute form (8.61 ± 1.31%), but the differences were not statistically significant. CD20+ B-lymphocytes constituted a small percentage of inflammatory cell infiltrates. CD1a + Langerhans cells showed progressively higher percentages from acute to chronic non-lupoid to chronic lupoid lesions. The differences were statistically significant (P < 0.05) between acute and chronic lupoid lesions. CD68+ macrophages were the most common cells and CD8+ T lymphocytes remained the predominant T-lymphocytes in acute, chronic non-lupoid, and chronic lupoid lesions, suggesting their central role in the pathogenesis and possible healing of CL. CONCLUSION: Focusing on the deep dermis, periadnexal and/or peripheral margins or even papillary tip of inflammatory sites of sandfly bites, we sometimes find granuloma inside lymphatic vessels (lymphangiectatic metastatic granuloma) or even infected macrophages with engulfed Leishman bodies faraway. Knowledge of the histopathological and immunohistochemical findings for various forms of ACL is essential in improving clinical and medical strategies and crucial for proper prophylactic and therapeutic plans.

Antileishmanial activity and immune modulatory effects of benzoxonium chloride and its entrapped forms in niosome on Leishmania tropica.

Benzoxonium chloride is an anti-infective agent that is used as anti-septic drugs for disinfection of the mucus membrane, skin surface and anti-bacterial, and it is also found to be effective against cutaneous leishmaniasis. The present study aims to evaluate the leishmanicidal activity of benzoxonium chloride and niosomal forms against Leishmania tropica stages. Benzoxonium chloride niosomes were prepared by the thin film hydration method and evaluated for morphology, particle size and release study and encapsulation efficiency. This study measured the cytotoxicity, leishmanicidal activity against promastigote and intra macrophage amastigote, apoptosis, and mRNA transcripts by quantitative real time PCR (qPCR) of free solution and niosomal-encapsulated benzoxonium chloride. Span/Tween 60 niosomal formulation of benzoxonium chloride showed superior physical stability and high encapsulation efficiency (96%) than the other forms. Release from the formulations showed that the Span/Tween 60 containing drug had a milder gradient so that 10% of the drug was not released after 4 h. The benzoxonium chloride and niosomal forms inhibited the in vitro growth of promastigote and amastigote forms of L. tropica after 48 h of incubation and represented IC50 values of 90.7 ± 2.7 and 25.4 ± 0.6 µg/ mL, respectively. The rate of apoptosis in niosomal formulations was approximately equal to the positive control (meglumine antimoniate) at the same concentration. Also, an increase in the concentration of this drug reduced the expression of IL-10, but increased the expression of IL-12. The niosomal formulations provided improved anti-leishmanial activities of benzoxonium chloride and played an immunomodulatory role as the mode of action in the treatment of anthroponotic CL.

Toxico-pathological effects of meglumine antimoniate on human umbilical vein endothelial cells.

Leishmaniasis is one of the most important parasitic diseases after malaria. The standard treatment of leishmaniasis includes pentavalent antimonials (SbV); however, these drugs are associated with serious adverse effects. There have been very few studies pertaining to their side effects and mechanism of action in the fetus. This investigation examines the effects of meglumine antimoniate (MA) on the survival rate, angiogenesis and cellular apoptosis in the human umbilical vein endothelial cells (HUVECs). HUVECs were treated with varying doses of MA (100-800 µg/ml) for 24, 48 and 72 h and the survival rate was studied by colorimetric assay, flow cytometry, immunocytochemistry, migration (scratch) assay and tube formation assay. The results of quantitative real-time PCR (qPCR) studies indicated that the most important genes involved in presenting angiogenesis included VEGF and its receptors (Kdr and Flt-1), NP1 and Hif-1α genes including the anti-apoptotic gene of Bcl2, were significantly reduced compared to the control group (p < 0.05). In contrast, the most leading genes involved in the phenomenon of apoptosis were P53, Bax, Bak, Apaf-1 and caspases 3, 8 and 9, which were significantly up regulated compared to the control group (p < 0.05).

Anti-Leishmanial and Immunomodulatory Effects of Epigallocatechin 3-O-Gallate on Leishmania tropica: Apoptosis and Gene Expression Profiling.

BACKGROUND: Pentavalent antimonials such as meglumine antimoniate (MA, Glucantime), are the first-line treatment against leishmaniasis, but at present, they have basically lost their efficacy. This study was aimed to explore epigallocatechin 3-O-gallate (EGCG), alone or in combination with MA against Leishmania tropica stages. METHODS: All experiments were carried out in triplicate using colorimetric assay, macrophage model, flow cytometry and quantitative real-time PCR. This experimental study was carried out in 2017 in Leishmaniasis Research Center, Kerman University of Medical Sciences, Kerman, Iran. RESULTS: Promastigotes and amastigotes were more susceptible to EGCG than MA alone, but the effect was more profound when used in combination. EGCG exhibited high antioxidant level with a remarkable potential to induce apoptosis. Furthermore, the results showed that the level of gene expression pertaining to Th-1 was significantly up-regulated (P<0.001). CONCLUSION: EGCG demonstrated a potent anti-leishmanial effect alone and more enhanced lethal activity in combination. The principal mode of action entails the stimulation of a synergistic response and up-regulation of the immunomodulatory role towards Th-1 response against L. tropica.

Comparison between intralesional injection of zinc sulfate 2 % solution and intralesional meglumine antimoniate in the treatment of acute old world dry type cutaneous leishmaniasis: a randomized double-blind clinical trial.

Zinc sulfate (ZS) has been used for the treatment of acute cutaneous leishmaniasis (CL) in both forms of in vivo and in vitro recently. The aim of the present study was to compare the efficacy of intralesional injection of ZS 2 % solution with intralesional glucantime in the treatment of acute CL. In this double-blind randomized clinical trial, 80 cases with acute old world dry type CL were enrolled in the study. The treatment protocol in the first group consisted of intralesional injection of ZS 2 % vials once a week for 10 weeks or sooner in case of complete resolution of the lesions. In the second group, intralesional glucantime once a week for 10 weeks or sooner in case of complete resolution of the lesions were used. In both groups cryotherapy was performed once every other week for 10 weeks. In ZS versus second group, partial and complete clinical response was observed with fewer injections although this difference was not statistically significant. In addition, we found that the trend of treatment in second group was faster but again it was not significant [partial treatment: hazard ratio (HR) 1.4, 95 % CI 0.7-2.9; complete treatment: HR 1.3, 95 % CI 0.6-2.8]. The results of this study showed that the intralesional injection of ZS 2 % solution was as effective as glucantime on the healing of the acute old world dry type CL.

Prevalence of Trypanosoma evansi in camels using molecular and parasitological methods in the southeast of Iran, 2011.

Surra is caused by infection with the protozoal parasite, Trypanosoma evansi. This parasite was transmitted mechanically by biting flies which is widespread in camels in the world. The aim of this study is to determine the prevalence of T. evansi in camels in Rafsanjan, Kerman province, southeast of Iran. In this study, 95 suspected camels were randomly selected in 2011. Blood samples were taken from deep blood vessels. Thin and thick blood smears were prepared in laboratory. Blood smears were stained by Giemsa and studied under a light microscope. The positive blood samples were also used for further molecular analysis. Data were analyzed using SPSS 17.0 software and P ≤ 0.05 was considered as statistical difference. A total of 95 camels were examined for infection with T.evansi using parasitological and molecular methods. The overall prevalence of infection was 2.1 %. It was found that the frequency of infection was significantly higher (P < 0.05) in age group >6 years old than the corresponding younger camels. However, there was no significant difference when the gender was considered. PCR technique confirmed the two infected cases were T. evansi. Results of the present study indicated that surra is present in Rafsanjan county, Kerman province in an infection rate of 2.1 % in camels. To our knowledge, this is the first study reported from this province. Further investigations are needed to focus on vectors and to evaluate the risk factors.

A comprehensive review of cutaneous leishmaniasis in kerman province, southeastern iran-narrative review article.

BACKGROUND: Cutaneous leishmaniasis (CL) remains a serious public health concern in Kerman Province, eastern Iran. This study was aimed to conduct a comprehensive review and highlights various aspects of CL in the province of Kerman. METHODS: This article mainly focuses on the studies published in the past 26 years on CL in the province. Current data for the present status were obtained through the provincial health system. RESULTS: Bam was the most infected district (63.6%), followed by Kerman (24.7%) and other districts to a less extent. Leishmania tropica is the major causative agent (95.5%) of CL in Kerman province, however, L. major accounts for 4.5% of the total cases. Bam, Kerman and southern districts of Kerman province were purely anthroponotic CL (ACL), while Rafsanjan, Baft, and Sirjan showed both ACL and zoonotic CL (ZCL). In contrast, Orzoieh district was merely endemic to ZCL type. Phlebotomus sergenti was the main vector in ACL foci while Ph. papatasi was the major vector in the ZCL district of Orzoieh. Localized CL was the most prevalent form (80%) of the disease, while leishmaniasis recidivans was the most uncommon clinical manifestation (18.7%). CONCLUSION: Due to recent rises in CL disease both in regard of increases in incidence rate and expansion of the disease to new foci, and presence of various risk factors in the province, control measures and health strategies should have high priorities to help treat the existing cases and prevent the expansion of the disease to new areas.

A survey of reservoir hosts in two foci of cutaneous leishmaniasis in Kerman province, southeast of Iran.

In the Old World, cutaneous leishmaniasis (CL) is zoonoses and natural vertebrate hosts of CL parasites are mammals. This study was carried out on natural infection rates of Leishmania parasites in reservoir hosts in one new focus of zoonotic cutaneous leishmaniasis (ZCL) and in suspected reservoir in an old focus of ACL in Iran. The sampling of rodents using Sherman traps was carried out and PCR technique was used for detection and identification of Leishmania species in Bahreman district, Kerman province, southeast of Iran. In addition, the smears were taken from suspicious lesions in stray dogs in the city of Kerman, the center of Kerman province. Simultaneously, pieces of lesion (1 × 1×1 cm) were taken for further histopathological examination. Overall, 25 rodents were collected and identified, including Meriones libycus and Rhombomys opimus. Amastigotes were observed in 33 % of the R. opimus by microscopic examination and indentified as Leishmania major by PCR technique. Four suspicious dogs out of 391 stray dogs showed no Leishmania species. To the best of our knowledge, this is the first isolation and identification of L. major from R. opimus in Kerman province, where ZCL has been present in recent years. Therefore, R. opimus is considered as the main animal reservoir host in Bahreman ZCL focus. In ACL focus such as the city of Kerman, dogs had no role in CL infection as reservoir host.

A prospective cohort study of cutaneous leishmaniasis risk and opium addiction in south eastern Iran.

Opium addiction and cutaneous leishmaniasis (CL) are endemic in different parts of Iran, particularly in Bam, where a massive earthquake occurred. This study was designed to compare the incidence rate and severity of CL cases among opium addicted and non-addicted individuals in south-eastern Iran. This study was carried out as a prospective cohort by active house-to-house visits of 1,481 habitants in Bam. CL cases were confirmed by smear and identification of Leishmania species was performed using nested-PCR. The data was analyzed by χ(2) and t-tests, using SPSS software and also Kaplan-Meier survival curve and long-rank test in Stata 11.2 and P<0.05 was considered as significant. A total of 904 individuals consisting of 226 opium addicted and 678 non-addicted individuals were followed-up for a period of seven years. The two cohorts were similar in terms of age, sex and place of residency. A similar pattern of incidence was observed among the two cohort groups. In contrast, the severity of CL in terms of the number, duration and the size of the lesions in opium addicted individuals was significantly (P<0.001) higher than non-opium addicted individuals. In conclusion, the present findings indicate that there is no relationship between the incidence of CL and opium addiction.

The emergence of anthroponotic cutaneous leishmaniasis following the earthquake in southern villages of bam district, southeastern iran, 2010.

BACKGROUND: The objective of this study was to assess the epidemiological characteristics of a new emerging focus of cutaneous leishmaniasis (CL) in southern villages of Bam District, southeastern Iran, 2010. METHODS: A house-to- house census survey of 5544 individuals were interviewed and physically examined for the presence of active lesions or scars. Diagnosis was confirmed by direct smears, cultures and identification by PCR. The data were entered into a computer and SPSS ver. 15. RESULTS: Overall, 1.2% of the inhabitants were infected, 0.5% active and 0.7% scars and females were more significantly infected (1.7%) than males (0.8%), (P= 0.003). All age groups were equally affected. Most of the lesions were on the face and majority had single lesion. Most of the cases appeared from 2006 to 2008 during the CL epidemic in the city of Bam. PCR indicated L. tropica as the causative agent. CONCLUSION: The presence of non-immune individuals along with suitable ecological conditions could induce a new emerging focus of ACL in villages.

Evaluation of a Possible Synergistic Effect of Meglumine Antimoniate with Paromomycin, Miltefosine or Allopurinol on in Vitro Susceptibility of Leishmania tropica Resistant Isolate.

BACKGROUND: Pentavalent antimonials are still the first choice treatment for leishmaniasis, but with low efficacy and resistance is emerging. In the present study, the effect of meglumine antimoniate (MA, Glucantime) combined with paromomycin, miltefosine or allopurinol on in vitro susceptibility of Leishmania tropica resistant isolate was evaluated. METHOD: The drugs were obtained from commercial sources and diluents of each drug in medium were prepared on the day of experiment. J774 A.1 murine macrophage cell lines were attached to the cultured on slide and incubated at 37 °C with 5% CO2 for 24 h. Then the stationary phase promastigotes were added to the cells and after 4 hrs of incubation different concentrations of MA, paromomycin, miltefosine or allopurinol were added and incubated for an additional of 72 h. Then the slides were dried and fixed with methanol, stained by Giemsa and studied under a light microscope. Drug activity was evaluated by assessing the macrophage infection rate and the number of amastigotes per infected macrophage was done by examining 100 macrophages. The experiment was done in triplicates. RESULT: Various concentrations of MA along with paromomycin, miltefosine or allopurinol significantly inhibited (P<0.01) the proliferation of L. tropica amastigote stage in the macrophage cell line as compared with MA alone or positive control. CONCLUSION: Combination of Glucantime with paromomycin, miltefosine or allopurinol showed a synergistic effect on the clinical isolate of L. tropica in vitro. Use of combination therapy is a new hope and a logical basis for therapy of the patients with cutaneous leishmaniasis. Further investigations are needed to evaluate the therapeutic effects of these drugs on the CL patients.

The effect of verapamil on in vitro susceptibility of promastigote and amastigote stages of Leishmania tropica to meglumine antimoniate.

Pentavalent antimonials are the standard treatment for cutaneous leishmaniasis (CL) with low efficacy and resistance is emerging. CL is increased significantly in respect to incidence rate and expanding to new foci. In the present study, the effect of verapamil on in vitro susceptibility of promastigote and amastigote stages of Leishmania tropica to meglumine antimoniate (MA, Glucantime) was evaluated using colorimetric assay (MTT) and in a macrophage model, respectively. Verapamil, as a calcium channel blocker, affects drug uptake by preventing of drug efflux from the cells. In promastigote form, several concentrations of MA with or without verapamil showed significant decrease (P < 0.05) in optical density. The overall mean IC50 value with combination of MA plus verapamil (IC50 = 116.03 µg/ml) was significantly less than MA (IC50 = 225.14 µg/ml) alone (P < 0.05) for promastigote stage. Similarly, the amastigote stage was more susceptible to treatment with MA plus verapamil to that of MA alone (P < 0.05). Analysis of overall effect of different concentrations of MA alone, compared with combination of MA plus verapamil by mean infection rate of amastigotes in each macrophage showed a significant difference (P < 0.05).These findings indicated some degree of synergistic effects between MA and verapamil on in vitro susceptibility of L. tropica to MA. Further works are required to evaluate this synergistic effect on animal model or volunteer human subjects.