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Abstract Leishmaniasis is a disease of poverty that imposes a devastating medical, social, and economic burden on over 1 billion people nationwide. To date, no in-depth study to analyze the major global challenges and needs assessment has been carried out. This investigation aimed to explore a comprehensive narrative review of leishmaniasis’s main challenges and initially highlight obstacles that might impede the implementation of control measures. Also, we propose a specific list of priorities for needs assessment. The presence of socioeconomic factors, multiple clinical and epidemiological forms, various Leishmania species, the complexity of the life cycle, the absence of effective drugs and vaccines, and the lack of efficient vector and reservoir control make this organism unique and sophisticated in playing a tangled role to react tricky with its surrounding environments, despite extensive efforts and implementation of all-inclusive former control measures. These facts indicate that the previous strategic plans, financial support, and basic infrastructures connected to leishmaniasis surveillance are still insufficient. Strengthening the leishmaniasis framework in a context of accelerated programmatic action and intensification of cross-cutting activities along with other neglected tropical diseases (NTDs) is confidently expected to result in greater effectiveness, cost–benefit, and fruitful management. Sensitive diagnostics, effective therapeutics, and efficacious vaccines are vital to accelerating advancement toward elimination, and reducing morbidity/mortality and program costs. Collective actions devoted by all sectors and policy-makers can hopefully overcome technical and operational barriers to guarantee that effective and coordinated implementation plans are sustained to meet the road map for NTDs 2021- 2030 goals.

Background: Leishmaniasis is an important public health parasitic infection, which is endemic in many parts of the world, including Iran. We aimed to investigate genetic diversity and phylogenetic relationship among different Leishmania isolates using multilocus sequence typing (MLST). Methods: Totally, 41 isolates collected either from patients referred to Leishmaniasis Diagnostics and Treatment Center at Tehran University of Medical Sciences, Tehran, Iran or from animals during 2019-2021, were subjected to the study. They included L. major and L. tropica from human, L. infantum from canine, and L. turanica from rodents from different endemic foci of Iran analyzed using MLST including gp63, g6pdh, lack, nagt, and hsp70 genes. Results: A total of 5010 bps was analyzed from each isolate. The three targets, nagt, lack, and g6pdh, generated better topology comparing to the other genes. In the 44 isolates, 22 haplotypes (STs) were identified. Leishmania tropica contained the highest number of haplotypes (n=12) comparing to L. major (n=8), L. infantum (n=1) and L. turanica (n=1). All five genomic loci caused separation of Iranian Leishmania species at the species level, indicating conservation of these genes in the Leishmania parasite. Conclusion: The highest number of haplotypes belonged to L. tropica, indicating that the genetic diversity of this species is higher than that of L. major. It was further confirmed that the MLST is a suitable method to examine genetic variation of Leishmania parasites with respect to evolutionary and epidemiological studies.

Abstract Background Cutaneous Leishmaniasis (CL) is a parasitic disease with diverse outcomes. Clinical diversity is influenced by various factors such as Leishmania species and host genetic background. The role of Leishmania RNA virus (LRV), as an endosymbiont, is suggested to not only affect the pathogenesis of Leishmania, but also impact host immune responses. This study aimed to investigate the influence of LRV2 on the expression of a number of virulence factors (VFs) of Leishmania and pro-inflammatory biomarkers. Materials and methods Sample were obtained from CL patients from Golestan province. Leishmania species were identified by PCR (LIN 4, 17), and the presence of LRV2 was checked using the semi-nested PCR (RdRp gene). Human monocyte cell line (THP-1) was treated with three isolates of L. major with LRV2 and one isolate of L. major without LRV2. The treatments with four isolates were administered for the time points: zero, 12, 24, 36, and 48 h after co-infection. The expression levels of Leishmania VFs genes including GP63, HSP83, and MPI, as well as pro-inflammatory biomarkers genes including NLRP3, IL18, and IL1β, were measured using quantitative real-time PCR. Results The expression of GP63, HSP83, and MPI revealed up-regulation in LRV2 + isolates compared to LRV2- isolates. The expression of the pro-inflammatory biomarkers including NLRP3, IL1β, and IL18 genes in LRV2- were higher than LRV2 + isolates. Conclusion This finding suggests that LRV2 + may have a probable effect on the Leishmania VFs and pro-inflammatory biomarkers in the human macrophage model.

Abstract Cutaneous leishmaniasis (CL) is a very common parasitic infection in subtropical areas worldwide. Throughout decades, there have been challenges in vaccine design and vaccination against CL. The present study introduced novel T-cell-based vaccine candidates containing IFN-γ Inducing epitopic fragments from Leishmania major (L. major) glycoprotein 46 (gp46), cathepsin L-like and B-like proteases, histone H2A, glucose-regulated protein 78 (grp78) and stress-inducible protein 1 (STI-1). For this aim, top-ranked human leukocyte antigen (HLA)-specific, IFN-γ Inducing, antigenic, CD4 + and CD8 + binders were highlighted. Four vaccine candidates were generated using different spacers (AAY, GPGPG, GDGDG) and adjuvants (RS-09 peptide, human IFN-γ, a combination of both, Mycobacterium tuberculosis Resuscitation promoting factor E (RpfE)). Based on the immune simulation profile, those with RS-09 peptide (Leish-App) and RpfE (Leish-Rpf) elicited robust immune responses and their tertiary structure were further refined. Also, molecular docking of the selected vaccine models with the human toll-like receptor 4 showed proper interactions, particularly for Leish-App, for which molecular dynamics simulations showed a stable connection with TLR-4. Upon codon optimization, both models were finally ligated into the pET28a( +) vector. In conclusion, two potent multi-epitope vaccine candidates were designed against CL and evaluated using comprehensive in silico methods, while further wet experiments are, also, recommended.

Currently, no safe vaccine against leishmaniasis is available. So far, different control strategies against numerous reservoir hosts and biological vectors have not been environment-friendly and feasible. Hence, employing medicinal components and conventional drugs could be a promising approach to developing novel therapeutic alternatives. This study aimed to explore diallyl sulfide (DAS), a dynamic constituent of garlic, alone and in a mixture with meglumine antimoniate (MAT as standard drug) using in vitro and animal model experiments against Leishmania major stages. The binding affinity of DAS and four major defense elements of the immune system (iNOS, IFN-ɣ, IL-12, and TNF-α) was used to predict the predominant binding mode for molecular docking configurations. Herein, we conducted a broad range of experiments to monitor and assess DAS and MAT potential treatment outcomes. DAS, combined with MAT, displayed no cytotoxicity and employed a powerful anti-leishmanial activity, notably against the clinical stage. The function mechanism involved immunomodulation through the induction of Th1 cytokine phenotypes, triggering a high apoptotic profile, reactive oxygen species (ROS) production, and antioxidant enzymes. This combination significantly decreased cutaneous lesion diameter and parasite load in BALB/c mice. The histopathological findings performed the infiltration of inflammatory cells associated with T-lymphocytes, particularly CD4+ phenotypes, as determined by biochemical markers in alleviating the amastigote stage and improving the pathological changes in L. major infected BALB/c mice. Therefore, DAS and MAT deserve further advanced therapeutic development and should be considered as possible candidates for treating volunteer cases with cutaneous leishmaniasis in designing an upcoming clinical trial.

Background: Recent studies have shown an increasing number of patients with cutaneous leishmaniasis (CL) who do not respond to pentavalent antimonials as the first line of treatment for CL. Nanocarriers such as extracellular vesicles (EVs) are efficient vehicles that might be used as drug delivery systems for the treatment of diseases. Therefore, we aimed to isolate and characterize the EVs of Leishmania major, load them with Amphotericin B (AmB), and investigate the toxicity and efficacy of the prepared drug form. Methods: The EVs of L. major were isolated, characterized, and loaded with amphotericin B (AmB), and the EVs-Amphotericin B (EVs-AmB) form was synthesized. Relevant in vitro and in vivo methods were performed to evaluate the toxicity and efficacy of EVs-AmB compared to the control. Results: The anti-leishmanial activity of the EVs-AmB showed a higher percentage inhibition (PI%) (P = 0.023) compared to the AmB at different concentrations and time points. Obtained data showed a significant increase in the lesion size and parasite load in the lesion, PBS, and EVs mice groups in comparison with EVs-AmB, AmB, and Glucantime groups (P < 0.05), EVs-AmB had a significant decrease in lesion sizes in comparison with AmB (P < 0.05). Results showed that EVs-AmB decreased its toxicity to the kidneys and liver (P < 0.05). Conclusion: EVs-AmB improved the efficacy of AmB in mouse skin lesions and reduced hepatorenal toxicity. Furthermore, EVs could be a promising nanoplatform for the delivery of AmB in CL caused by L. major.

Abstract Background Treatment of cutaneous leishmaniasis (CL) remains a major challenge for the public health and medical community. It has been claimed that natural compounds derived from fly larvae have anti-leishmania properties against some species of Leishmania. The present study aimed at assessing the in vitro effects of larval products of Lucilia sericata against the promastigote and intracellular amastigote forms of Leishmania major. Also, the therapeutic effect of larval products on lesions induced by L. major infection was evaluated in BALB/c mice models. Methods Parasite specimens and macrophage cells were exposed to varying concentrations of larval products for 24–120 h. Lesion progression and parasite load were investigated in the models to assess the therapeutic effects of the products. Results The larval products displayed more potent cytotoxicity against L. major promastigotes. The IC50 values for larval saliva and hemolymph were 100.6 and 37.96 ug/ml, respectively. The IC50 of glucantime was 9.480 ug/ml. Also, the saliva and hemolymph of L. sericata exhibited higher cytotoxicity against the promastigotes of L. major but were less toxic to the macrophage cells. Treatment with leishmanicidal agents derived from larvae of L. sericata decreased the infection rate and the number of amastigotes per infected host cell at all concentrations. Lesion size was significantly (F (7, 38) = 8.54, P

Abstract Leishmaniasis is one of the common diseases transmitted by sand flies in tropical and subtropical regions of the world. Currently, antimonial derivatives are the first line of treatment. Some of the members of the ATP-binding cassette (ABC) family of Leishmania are shown to be associated with no response to treatment. In this study, we evaluated ABCI4, ABCG2, ABCC7, ABCB4, and ABCC3 genes expression in Leishmania isolated from patients with non-healing cutaneous leishmaniasis and treatment response isolates. We selected 17 clinical isolates including 8 treatment failure and 9 treatment response samples from September 2020 to March 2021. The isolates were obtained from patients of Health Center Laboratory of Varzaneh, Isfahan, Iran with cutaneous leishmaniasis. The diagnosis was performed using microscopic observation. The samples were directly collected from the lesions. The expression profiling of genes was assessed using SYBR Green real-time PCR that was analyzed with delta-delta Ct. All treatment failure clinical isolates were L. major. Gene expression analysis in treatment failure isolates showed that the ABC transported genes had a different pattern in each isolate. Treatment failure has been reported for cutaneous leishmaniasis worldwide. Knowledge of the molecular mechanisms of treatment failure could solve this problem. ABC transporter genes are considered controversial over the mechanisms of treatment failure outcomes. In this study, we showed that ABC transporter genes could be considered one of the important mechanisms.

Treatment of Cutaneous leishmaniasis (CL) is based on using antimoniate derivatives; patients' compliance for systemic injections is low due to the pain and systemic complications. In this randomized open trial, the efficacy of intra-lesional (IL) injections of meglumine antimoniate (MA) once a week vs. twice a week in the treatment of Anthrpoponothic CL caused by L. tropica was studied. Eligible volunteer patients were selected according to inclusion/exclusion criteria. The included patients were randomly allocated to receive IL-MA injections once a week or twice a week. The primary outcome was set as complete healing of the lesion(s), and defined as complete re-epithelialization and absence of induration in the lesions. A total of 180 parasitologicaly proven CL patients caused by L. tropica were recruited, 90 patients were treated with weekly IL-MA and 90 patients received IL-MA twice a week. The complete cure was 87.9% vs. 89.2% in the group received weekly and twice a week IL-MA injections, respectively (P = 0.808). Patients' compliance was acceptable and side effects were limited to a few local allergic reactions to MA. Median time to healing was significantly shorter in patients who received IL-MA twice a week (median ± SE) 37±3.8, (CI: 29.6-44.4) days compared to whom received IL-MA once a week 60±2.3, (CI: 55.6-64.5) days (P< 0.001), however the number of injections was higher in group who received IL-MA twice a week (12 vs. 9 injections). In conclusion, the rate of cure in the group of CL patients with IL-MA twice a week was not significantly different from the group who received IL-MA once a week shorten, but the duration of healing was shorter in the group who received IL-MA twice a week while the group received more injections so is recommended to use IL-MA once a week due to the fact the compliance is acceptable with limited side effects. Clinical Trial Registration: IRCT20081130001475N13; https://en.irct.ir/.

Background: Recovery from cutaneous leishmaniasis (CL) leads to protection against further lesion development. In contrast, vaccination using killed parasites does not induce enough protection; the reason(s) is not currently known but might be related to different immune response induced against live versus killed parasites. In this study, Th1/Th2 cyto-kine profiles of CL patients were evaluated against live versus killed Leishmania major. Methods: In this study peripheral blood mononuclear cells (PBMC) of the volunteers with active CL lesion (CL), history of CL (HCL) and healthy volunteers were cultured and stimulated with live or killed Leishmania major, the superna-tants were collected and levels of IFN-γ, IL-5 and IL-10 were titrated using ELISA method. Results: The results showed that IFN-γ levels in CL patients (p< 0.001) and HCL volunteers (p< 0.005) are signifi-cantly higher when stimulated with live than stimulated with killed L. major. IFN-γ production in PBMC volunteers with CL and HCL stimulated with live or heat-killed L. major was significantly (p< 0.001) higher than in unstimulated ones. The level of IL-5 in CL patients (p< 0.005) and HCL volunteers (p< 0.001) are significantly lower when stimulated with live than killed L. major. There was no significant difference between the levels of IL-10 in PBMC stimulated with either live or killed L. major. Conclusion: It is concluded that using live Leishmania induces a stronger Th1 type of immune response which justify using leishmanization as a control measure against CL.

Abstract Background Leishmaniasis is a major parasitic disease worldwide, except in Australia and Antarctica, and it poses a significant public health problem. Due to the absence of safe and effective vaccines and drugs, researchers have begun an extensive search for new drugs. The aim of the current study was to investigate the in vitro leishmanicidal activity of larval saliva and hemolymph of Lucilia sericata on Leishmania tropica. Methods The effects of different concentrations of larval products on promastigotes and intracellular amastigotes of L. tropica were investigated using the mouse cell line J774A.1 and peritoneal macrophages as host cells. The 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and direct observation and counting method were used to assess the inhibitory effects and cell cytotoxicity of the larval products. The effects of larval products on the amastigote form of L. tropica were quantitatively estimated by calculating the rate of macrophage infection, number of amastigotes per infected macrophage cell, parasite load and survival index. Results The 50% cytotoxicity concentration (CC50) value of both larval saliva and hemolymph was 750 µg/ml, and the 50% inhibitory concentration (IC50) values were 134 µg/ml and 60 µg/ml for larval saliva and larval hemolymph, respectively. The IC50 for Glucantime, used a positive control, was (11.65 µg/ml). Statistically significant differences in viability percentages of promastigotes were observed for different doses of both larval saliva and hemolymph when compared with the negative control (p ≤ 0.0001). Microscopic evaluation of the amastigote forms revealed that treatment with 150 µg/ml larval hemolymph and 450 µg/ml larval saliva significantly decreased the rate of macrophage infection and the number of amastigotes per infected macrophage cell. Conclusion Larval saliva and hemolymph of L. sericata have acceptable leishmanicidal properties against L. tropica.

Background: Cutaneous leishmaniasis by different species of Leishmania. Pentavalen antimonials as a first line drug for treatment of cutaneous leishmaniasis have several limitations and side effects. Natural products are more considered due to their less toxicity and cost, more efficient, safety and readily available antileishmania agents. Objective: The aim of this study was to explore the effect of hydroalcoholic extract of Glycyrrhiza glabra (licorice) roots and its main component glycyrrhizic acid on promastigote and amastigote of L. major. Methods: Different dosages of hydroalcoholic extract of licorice root, glycyrrhizic acid and Glucantime with promastigote of L. major were incubated at 26 °C for 24 and 48 hours then the percentages of alive promastigotes were measured by MTT assay. The antiamastigote effects of these drugs was examined by microscopic counting of the number of amastigotes in macrophages 24 hours after treating the parasite infected macrophages with them. Promastigote and infected macrophages without any treatment was used as negative controls. Results: The IC50 values of hydroalcoholic extracts of licorice root, glycyrrhizic acid and Glucantime on promastigote of L. major was 1250 ± 0.018, 3000 ± 0.017 and 50 ± 0.043 μg/ml after 24 hours, and 1000 ± 0.016, 3000 ± 0.017 and 25 ± 0.009 μg/ml after 48 hours, respectively. The IC50 values of the licorice root extract, glycyrrhizic acid and Glucantime on amostigate was 500, 1000 and 25 μg/ml, respectively. Conclusion: Hydroalcoholic extract of licorice root and glycyrrhizic acid had cytotoxic effects on promastigotes and amastigotes of L. major.

Background: Treatment of leishmaniasis is a challenge due to problems such as high price and dose of the drug, drug resistance and side effects. Objective: The study aims to introduce plants compounds, which their antileishmanial effects were approved in vivo conditions. Methods: This study as a review article was performed by searching the keywords of “medicinal plants with in vivo antileishmanial effects, nanocarrier, clinical trials, and mechanism of action” in the well-known databases to 2018. In this study, 14 medicinal plant compounds with antileishmanial effects were reviewed and mechanism of action and their in vivo therapeutic effect were evaluated. Results: It was found that while some of these compounds had low antileishmanial effects, their efficacy against leishmaniasis was significantly increased through loading into nanocarriers. Conclusion: This study indicated that active component of medicinal plants especially along with nano carriers can be of interest for the treatment of Leishmania.

Abstract Objective Leishmaniasis is caused by different Leishmania spp. Treatment failure (TF) of cutaneous leishmaniasis (CL) is a serious issue that may be due to various reasons, previous studies suggested Leishmania RNA virus (LRV) as a potential cause of TF. Two variant groups of LRV1 and LRV2 are reported. In this study, the presence of LRV1/LRV2 was compared in TF with treatment response (TR) isolates of L. major. Clinical isolates of 15 TF and 15 TR were collected from CL patients referred to the Health Centers of Isfahan. Genomic DNA was extracted to identify Leishmania spp. using ITS1-PCR–RFLP. Identification of LRV1/LRV2 was performed using SYBR Green Real-Time PCR. The statistical analysis to test relationship between the treatment response with Glucantime and the presence of LRV were performed using SPSS 16.0 with Fisher’s Exact test. P value of less than 0.05 was considered significant. Results ITS1-PCR–RFLP results showed that every isolate was identified as L. major. The results showed no LRV1 in any of the samples but 7 TR isolates and 2 TF isolates showed positive for LRV2. Statistical analysis showed no significant difference between the presence of LRV2 and response to Glucantime (p-value = 0.1086). Therefore, other mechanisms might be responsible for TF.

Objective: To investigate Phlebotomus (P.) sergenti Parrot, 1917 (Diptera: Psychodidae) salivary gland antigens and their immune response in human. Methods: Human volunteers were exposed to sand flies’ bites in the laboratory, and following each exposure the size of induration was recorded. The mean protein concentration of salivary gland lysate and specific anti-P. sergenti saliva IgG was measured. Sand fly salivary proteins were separated by SDS-PAGE and their immunoreactivity was examined by Western blotting assays. Results: Individuals exposed to P. sergenti salivary gland lysate for 8 months showed both antibody and delayed type hypersensitivity responses, although exposure for one month did not provoke any immune responses. The trend of antibody fluctuated during the exposure time and dropped by the end of antigen loading. The mean protein content was (0.36±0.08) μg in each pair salivary glands. Salivary gland lysate showed 11 to 12 major protein bands and 3 to 6 of them were immunoreactive. Conclusions: Our study showed that the salivary gland components of P. sergenti provoked both cellular and humoral immune responses in human. Furthermore, there are some mmunogenic proteins in P. sergenti saliva which could be subjected for further investigation as vector-based vaccine candidate/s against anthroponotic cutaneous leishmaniasis.

Background: Currently, there is no topical treatment available for any form of cutaneous leishmaniasis (CL) in most of the endemic areas. The aim of the current study was to develop a topical nano-liposomal Amphotericin B (AmB) for the treatment of CL. Methodology/principal findings: Liposomes containing 0.1, 0.2 and 0.4% AmB (Lip-AmB) were formulated and characterized for the size, entrapment efficiency, long term stability, and skin penetration properties using Franz diffusion cells. Liposomes diameters were around 100 nm with no change during more than 20 months’ storage either at 4 °C or at room temperature. Franz diffusion cells studies showed that almost 4% of the applied formulations penetrated across the skin and the highest skin retention (73.92%) observed with Lip-AmB 0.4%. The median effective doses (ED50), the doses of AmB required to kill 50% of L. major amastigotes were 0.151, 0.151, and 0.0856 (μg/mL) in Lip-AmB 0.1, 0.2, 0.4%, respectively. Lip-AmB 0.4% caused 80% reduction in fluorescence intensity of GFP+ L. tropica infected macrophages at 5 μg/mL of AmB concentration. Topical Lip-AmB was applied twice a day for 4 weeks to the skin of BALB/c mice to treat lesions caused by L. major. Results showed the superiority of Lip-AmB 0.4% compared to Lip-AmB 0.2 and 0.1%. The parasite was completely cleared from the skin site of infection and spleens at week 8 and 12 post-infection in mice treated with Lip-AmB 0.4%. The results suggest that topical Lip-AmB 0.4% may be a useful tool in the treatment of CL and merits further investigation. Keywords: Nano-liposomal amphotericin B, Cutaneous leishmaniasis, Topical treatment, Leishmania tropica, BALB/C mice

Objective(s): To design a multivalent DNA vaccine encoding the most immunogenic regions of the Leishmania major antigens including TSA (Thiol-specific antioxidant protein), LmSTI1 (Leishmania major stress-inducible protein1), LACK (Leishmania homologue of receptors for activated C Kinase), and KMP11 (kinetoplastid membrane protein-11) on BALB/c mice.Materials and Methods: The chimeric construct was generated including the most immunogenic epitopes containing a combination of domains and oligopeptides of the aforementioned genes. The construct was cloned into pcDNA 3.1 plasmid and named “pleish-dom.” Following intramuscular injection of mice, the capability of the vector pleish-dom alone and with pIL-12 (expressing murine IL-12) to raise protective cytokines and parasite burden was evaluated in the BALB/c mice as a susceptible animal model against L. major.Results: The immunized mice with pleish-dom/pIL-12 showed the highest and the lowest levels of interferon-gamma (IFN-γ) and interleukin-10 (IL-10), as well as the lowest leishmanin skin test (LST) reactions, were found through 8 weeks post-infection.Conclusion: Although the obtained DNA vaccine from the immunogenic chimeric protein of L. major antigens was able to induce a high level of IFN-γ, it partially protected mice against L. major. However co-administration with pIL-12 led to shift immune response to Th1 phenotype, granuloma formation, and lowered parasite burden, and consequently distinct protection was found.

Background and Objectives: Cutaneous leishmaniasis (CL) treatment is a challenging issue, although numerous modalities have been introduced as candidate treatment for CL yet only antimonial agents are commonly used to treat CL, a different form of amphotericin B is used to treat visceral form of leishmaniasis but the efficacy against CL is not high. There are a few reliable clinical trials on CL, the main reason is the nature of the disease which required a well design protocol to evaluate the efficacy of any candidate treatment against CL. In this study, a protocol was developed and used to evaluate a topical formulation of a nano-liposomal form of amphotericin B in addition to glucantime to treat CL caused by L. tropica. Materials and Methods: This study is a phase 3, randomized, double-blind, placebo-controlled trial to evaluate the efficacy and safety of topical nano-liposomal amphotericin B (SinaAmpholeish 0.4%) in combination with intralesional injections of meglumine antimoniate in the treatment of ACL caused by L. tropica. Overall, 130 patients, aged 12-60 years, with a diagnosis of ACL caused by L. tropica are recruited and treated according to the protocol. Results: A total of 130 patients with CL lesion will be recruited and double- blind randomly treated with received intralesional injections of Glucantime weekly or Glucantime plus SinaAmpholeish for 4 weeks. Conclusion: The results of this study showed that the protocol works well and the treatment was tolerated by both groups of patients.

Background: We aimed to investigate the potential effects of BCG and imiquimod on improvement of current experimental L. major vaccine against dogs in an endemic area of Zoonotic visceral leishmaniasis (ZVL) in Iran. Methods: During 2012 till 2014, seven mixedbreed shepherd dogs with no anti-Leishmania antibodies and no response to Leishmanin reagent were immunized with 2 doses of alum-precipitated autoclaved L. major (Alum-AML) while BCG and imiquimod (for skin pre-treatment) were used as adjuvants. The productions of a few characteristic cytokines of T-helper immune responses and the development of delayed-type hypersensitivity (DTH) of the immunized animals were then evaluated, up to 300 days. Blood samples were collected at 0, 30, 80 and 300 d post-vaccination and the concentrations of IFN-γ, IL10, IL-12 and TGF-β cytokines secreted from PBMCs at these time-points were quantified by ELISA. DTH was evaluated by Leishmanin skin test (LST). Results: Although a similar LST conversion was observed at all time-points, the cytokine measurement results indicated significantly higher levels of IFN-γ at day 80 and elevated levels of IL-10 at days 80 and 300, post-vaccination. Moreover, a significantly higher IFN-γ/IL-10 ratio was observed at day 30 post-vaccination compared to the other time-points. Conclusion: Although a Th1-like response could be observed at day 30 post-vaccination, the development of cytokine profiles was inclined toward mixed Th1 and Th2 responses at days 80 and 300 post-vaccination. This situation may indicate the requirement of an additional boosting by this Alum-AML formula, in order to induce long-lasting protection against ZVL.

Abstract Background Lupoid cutaneous leishmaniasis (LCL) is known as a rare but serious complication of anthroponotic cutaneous leishmaniasis (ACL) resistant to conventional treatments. Sodium chlorosum, a pro-oxidative preparation of pharmaceutical sodium chlorite (NaClO2), has been successfully used for the treatment of Old World cutaneous leishmaniasis lesions (OWCL) and of some LCL cases in Afghanistan. This clinical trial study aimed to evaluate the effect of a last resort therapy with topical 0.09% sodium chlorosum on LCL in Iran. Methods Twenty Iranian patients (12 women and 8 men) with LCL refractory to treatment were included in this salvage study. A magistral preparation of sodium chlorosum (10 mM NaClO2 in amphiphilic basic cream) was applied twice daily to the lesions for 6 weeks and continued up to 12 weeks in patients who showed a clinical response within the first 6 weeks. Responders were followed up for a maximum of 1 year. Lesions were photographed during weekly visits. Disappearance of erythema and indurated lesions were rated as complete clinical response. Results Patients with a mean age of 28.6 (±24.3) and with an ACL proven lesion history of 3.8 (±1.4) years were treated for an average of 7.9 (±1.8) weeks. At the end of the treatment period (12th week), a complete response was observed in 9 of 20 patients (45%). During the one-year follow-up period, LCL lesions recurred in 4 of these 9 patients (with one patient showing only a tiny lesion) and one case lost to follow up whereas the other four remained completely lesion-free. Mild temporary side-effects such as erythema and itching were seen in 4 of 20 patients (20%). Conclusions Topical sodium chlorosum showed promising therapeutic results and can be considered as safe, painless, and relatively effective treatment for LCL, an ethical prerequisite for a two-armed controlled trial. Trial registration This study was registered in Iranian registry of clinical trials on 2019-02-02 with registration number IRCT20190114042356N1.

Cutaneous leishmaniasis (CL) in Iran could be considered as an emerging disease that is rapidly increasing and expanding its traditional geographical range to new foci. Sixteen registered emerging epidemics have occurred since 1998 in different provinces. Various risk factors, including agricultural development, earthquake, movement to endemic areas, construction of buildings near colonies of rodents, sleeping outside, cross-border movements, and poor sanitation, play crucial roles in the expansion of the disease. The mentioned risk factors can lead to the gradual or sudden emergence of new CL epidemics, and long-lasting endemic foci can also erupt into epidemics. This paper reviews the emerging epidemics published between 1998 and 2019 in Iran with particular emphasis on the operational aspects of control and related risk factors caused by anthroponotic CL (ACL) and zoonotic CL (ZCL). The competent surveillance system should be extended to all high-risk areas to facilitate controlling the emerging epidemics of ACL and ZCL in the affected areas.

Background: Gene manipulation strategies including gene knockout and editing are becoming more sophisticated in terms of mechanism of action, efficacy and ease of use. In classical molecular methods of gene knockout, homologous arms are designed for induction of crossing over event in double strand DNA. Recently, CRISPR/Cas9 system has been emerged as a precise and powerful tool for gene targeting. In this effort, we aimed to generate a CRISPR/Cas9-based vector specific for targeting genes in Leishmania parasites. Methods: U6 and DHFR promoters and neomycin-resistance gene were amplified from genome of L. major (MHRO/IR/75/ER) and pEGFP-N1, respectively. U6 promoter was cloned in pX330 vector which is named as pX330-U6. DHFR promoter and neo resistance gene sequence fragments were fused using a combination of SOE (Splicing by overlap extension)-PCR and T/A cloning techniques. To generate pX-leish, fused fragments su-bcloned into the pX330-U6. Two sgRNAs were designed to target the gp63 gene and cloned in pX-leish. Results: The pX-leish vector was designed for simultaneous expression of cas9 and G418 resistance proteins along with a self-cleaving 2A peptide for efficient separation of the two proteins. In this study pX-leish was designed with 3 features: 1) Compatible promoters with Leishmania parasites. 2) Insertion of antibiotic selection marker 3) Designing an all-in-one vector containing all components required for CRISPR/Cas9 system. Conclusion: This modified system would be valuable in genome manipulation studies in Leishmania for vaccine research in future.

Background: Whole killed Leishmania vaccine reached phase III clinical trials but failed to display significant efficacy in human mainly due to limited Th1 inducer adjuvant. Liposomes consisting of 1, 2-dioleoyl-3trimethylammonium-propane (DOTAP) bearing an inherent adjuvanticity and 1, 2-dioleoyl-L-α-glycero-3-phosphatidylethanolamine (DOPE) is well known to intensify the efficacy of positively charged liposomes. Methods: Soluble Leishmania major antigens (SLA) encapsulated in cationic liposomes using lipid film method in 2016). BALB/c mice were immunized subcutaneously (SC), three times in a 2-wk interval, with Lip (DOTAP)-SLA+, Lip (DOTAP/DOPE)-SLA+, Lip (DOTAP/DOPE/CHO)-SLA+, Lip (DOTAP/DOPE/CHO), Lip (DOPE/CHO), SLA or HEPES buffer. At week 2 after the last booster injection, immunized mice have challenged SC in the footpad with L. major parasites. To investigate the rate of protection and the type of immune response generated in mice, lesions development was assessed, IL-4 and IFN-γ levels with the ratio of IgG2a/IgG1 isotype were studied to describe the type of generated immune response. Results: Mice immunized with all liposomal form of SLA showed smaller footpad swelling and lower parasite burden in the spleen and footpad compared to the group of mice received buffer. However, these formulations did not show protection against leishmaniosis because of a generated mixed Th1/Th2 response in mice characterized by high production of IFN-γ and IL4 and a high titer of IgG1 and IgG2a antibody. Conclusion: Immunization with Lip (DOTAP/DOPE/CHO)-SLA+ was not an appropriate strategy to protect mice against leishmaniosis.

Introduction: The aim of the study was to evaluate the protective effect of Hedera helix (ivy) leaf extract on paracetamol-induced oxidative stress and renal toxicity in mouse model. Methods: Sixty male Swiss albino mice were randomly divided into six equal groups. Group A: as the control group, received NaCl 0.9%. Group B: received a single (i.p.) injection of paracetamol (600 mg/kg). Pretreatment groups: included T1 (50 mg/kg), T2 (100 mg/kg), T3 (200 mg/kg), and T4 (300 mg/kg) which were treated with ivy leaf extract in different doses. The mice were sacrificed under mild anesthesia and the blood samples were collected to titrate the levels of serum blood urea nitrogen (BUN), creatinine and, uric acid. Kidneys were removed for histopathological examination. Results: Paracetamol administration significantly increased the BUN, creatinine and uric acid levels (P < 0.05). Treatment with Ivy leaf extract resulted in a significant reduction in serum creatinine, uric acid and BUN concentrations in comparison with the paracetamol group. Ivy leaf extract treatment similarly reduced histological alteration induced by paracetamol. Conclusion: This study can be used as prove of reading that ivy leaf extract might be used to prevent renal damage induced by paracetamol.

Background and Objectives: The outcome of Leishmania infection mainly depends upon the Leishmania species which causes the disease and the generation of the type of host immune response, the healing process and protection in leishmaniasis depends upon induction of Th1 response. In this study, the Th1/Th2 cytokine profile in cutaneous leishmaniasis (CL) is evaluated. Materials and Methods: This study was carried out in leishmaniasis clinic of CRTSDL, TUMS, during March 2018 to March 2019. Peripheral blood mononuclear cells (PBMC) of volunteers with active healing and non-healing lesion (s) of cutaneous leishmaniasis (CL), volunteers with and without history of CL were cultured and stimulated with Soluble Leishmania antigen (SLA). The supernatants were collected and the levels of IFN-γ, IL-5 and IL-10 were titrated using ELISA method. Results: The results showed a significantly higher levels of IFN-γ in volunteers with active CL healing form (p

Background: In a new approach, computational methods are used to design and evaluate the vaccine. The aim of the current study was to develop a computational tool to predict epitope candidate vaccines to be tested in experimental models. Methods: This study was conducted in the School of Allied Medical Sciences, and Center for Research and Training in Skin Diseases and Leprosy, Tehran University of Medical Sciences, Tehran, Iran in 2018. The random forest which is a classifier method was used to design computer-based tool to predict immunogenic peptides. Data was used to check the collected information from the IEDB, UniProt, and AAindex database. Overall, 1,264 collected data were used and divided into three parts; 70% of the data was used to train, 15% to validate and 15% to test the model. Five-fold cross-validation was used to find optimal hyper parameters of the model. Common performance metrics were used to evaluate the developed model. Results: Twenty seven features were identified as more important using RF predictor model and were used to predict the class of peptides. The RF model improves the performance of predictor model in comparison with the other predictor models (AUC±SE: 0.925±0.029). Using the developed RF model helps to identify the most likely epitopes for further experimental studies. Conclusion: The current developed random forest model is able to more accurately predict the immunogenic peptides of intracellular parasites.

Introduction: In this study, we aimed to evaluate the protective effects of Descurainia sophia seed extract on paracetamol-induced oxidative stress and acute liver injury in mice. Methods: Sixty male Swiss albino mice were divided into 6 groups, group I: as control group received NaCl 9%. Group II: as paracetamol group received paracetamol intraperitoneally (i.p.) (500 mg/kg). Groups III to VI as treatment groups received different doses of D. sophia seed extract (T1 = 50, T2 = 100, T3 = 200 and T4 = 300 mg/kg, respectively). Twenty-four hours after the paracetamol administration, the mice were sacrificed under mild anesthesia and their blood samples were collected to estimate serum alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin and malondialdehyde (MDA) levels. Their livers were also removed for histopathological examinations. Results: Pretreatment of mice with D. sophia seed extract, significantly prevented the paracetamol induced elevation in the levels of serum ALT, AST and ALP, total bilirubin and MDA (P < 0.05). The results of histopathologic studies were consistent with the above findings. Conclusion: Descurainia sophia seed extract has a protective effect against paracetamol induced oxidative damage and acute hepatotoxicity in mice.

Background & objective: Pruritus is the most frequent chronic dermal complication of sulfur mustard (SM), which negatively influences the quality of life. Exact pathophysiology of SM-induced itching is unknown. The current study aimed at evaluating the possible association between SM-induced itching and the serum levels of matrix metalloproteinase (MMP)-9 and their endogenous inhibitors, and serum levels of soluble forms of selectins (sL-, sP-, and sE-selectins) as adhesion molecules involved in the development of different inflammatory reactions. Methods: Serum levels of MMP-9, MMP-9/ tissue inhibitors of metalloproteinases (TIMPs), and selectins were measured by the enzyme-linked immunosorbent assay (ELISA), and compared between the groups (n=368) with and without itching, and matched control groups (n=126). Results: Serum levels of MMP-9 were significantly higher in the SM exposed group with itching, compared with that of the group without itching (medians: 894 and 624 pg/mL respectively; P-value =0.034). There was no relationship between the serum levels of MMP-9/TIMP-1, MMP-9/TIMP-2, MMP-9/TIMP-4, and itching in the patients exposed to SM. Median serum levels of sE- and sL-selectins in the exposed group with itching were higher than those of the exposed group without itching. These differences were statistically insignificant (P-values =0.084 and 0.095, respectively). Conclusion: According to the results of the current study, the increased serum levels of MMP-9 and selectins 20 years after exposure may play role in the pathogenesis and persistence of SM-induced itching in the exposed individuals.

Objective(s): Leishmaniasis is a complex parasitic disease that represents a major public health problem. Despite numerous attempts over the past decades, yet there is no effective vaccine against human leishmaniasis probably due to the lack of suitable adjuvants. In this study, a first generation liposomal-based Leishmania vaccine was developed using soluble Leishmania major antigens (SLA) and á, Ü-trehalose6, 6'-dibehenat (TDB) as an immunostimulatory adjuvant. In this liposome structure, the cationic lipid Dimethyldioctadecylammonium (DDA) provides intrinsic adjuvant activity and cholesterol was added as a membrane stabilizer. Liposomes containing SLA were prepared.Materials and Methods: BALB/c mice were subcutaneously (sc) immunized with Lip (DDA/TDB/CHOL)-SLA+, Lip (DDA/TDB)-SLA+, Lip (DDA)-SLA+, Lip (DDA/CHOL)-SLA+, SLA or Tris-HCl buffer. Immunization was done every two weeks for three weeks. The immunized mice were then challenged sc in the left footpad with 1×106 stationary phase L. major promastigotes (50 ìl), at 2 weeks after last booster injection.Results: mice immunized with any of the liposomal formulations containing SLA (Lip-SLA+), substantially increased footpad swelling and parasite loads of foot and spleen with no significant difference compared to Tris-HCl buffer or SLA alone. Lip-SLA+ formulations induced a mixed Th1/Th2 immune response characterized by IFN-ã and IL-4 production as well as high levels of IgG1 anti-Leishmania antibody. Conclusion: immunization with liposomes containing DDA and/or TDB in combination with SLA induces a mixed Th1/Th2 immune response and is not an appropriate strategy for preferential induction of a Th1 response and protection against leishmaniasis.

Background and Objectives: The surrogate marker (s) of cure and protection in intracellular pathogens is not yet well defined. The aim of this study was to compare the cytokine profile using whole blood cells (WBC) vs. peripheral blood mononuclear cells (PBMC) in healthy and cutaneous leishmaniasis (CL) volunteers. Materials and Methods: In this study, WBC and PBMC of the volunteers with history of CL (HCL), Active lesion (ACL) and healthy volunteers were collected. The WBC and PBMC were cultured and stimulated with either PHA or soluble Leishmania antigens (SLA), after 72 hours, the supernatants were collected and the levels of IFN-γ, IL-5 and IL-10 were titrated using ELISA method. Results: The mean ± SD of cytokines using WBC and PBMC in cutaneous leishmaniasis volunteers stimulated with phytohemagglutin (PHA) or SLA are as follow, PHA, IFN-γ=2295±995 vs. 2339±1115, IL-10=853±309 vs. 1330±966, and IL-5=299±136 vs. 352+156, SLA, IFN-γ, 931±824 vs. 825±532, IL-10, 233±78 vs. 408±381, and IL-5, 185±59 vs. 217±76, respectively. There was no significant difference between the IFN-γ, IL-5 and IL-10 levels using WBC vs. PBMC. There was a strong correlation between the cytokine profiles using WBC and PBMC in cutaneous leishmaniasis volunteers. Conclusion: There was no significant difference between IFN-γ, IL-10, IL-5 levels in whole blood and PBMC of volunteers with active lesion or history of CL. Whole-blood culture which is easier, cheaper and more convenient could be used instead of PBMC to evaluate the cytokine profile in field conditions.

Background: Leishmaniasis represents the important public health problem in the world. One of the main objectives of World Health organization is capacity building of managers and authorities who are involved with diseases control ac­tivities. Methods: The course was conducted in Esfahan Health Research and Training Center (E.H.R.T.C) in summer 2005 and 2009. The course carried out jointly by the Ministry of Health and Medical Education (MOH) of Iran, World Health Or­ganization-Eastern Mediterranean Regional Office (WHO-EMRO) and School of Public health, Tehran University of Med­ical Sciences (SPH-TUMS) and designed for medical officers, senior technicians and managers involved in leish­mania­sis control. Prior to initiate the course, pre-test evaluations including different subjects on leishmaniasis and its control were carried out. The examinations include multiple choice questions. The duration of the course was 3 weeks. A total of 206 contact hours were taught. The main subjects were Basic epidemiology, Leishmaniasis parasitology, Leishmaniasis en­tomology, control of vectors and reservoirs, principles of integrated pest management, Field work and Planning. Dif­ferent methods of teaching including lecture, laboratory, workshop, team work, field exercise and presen­tation were used. Requirement for achievement of the course was to have at least 60% of the total mark for awarding the diploma certificate. Results: A total of 45 participants from Iraq, Afghanistan and Iran graduated from this course. Conclusion: The course is providing the skill for managers, how to combat against disease in their country and is paral­lel to the policy of the leishmaniasis control for capacity building in endemic areas of their countries.

This study aimed to evaluate the efficacy of glucantime and amphotericin B (AmB) encapsulated in niosome against cutaneous leishmaniasis (CL) using in vitro and in vivo models. The niosomal formulations of the drugs alone and in combination were prepared and characterized. Subsequent to the examination of their cytotoxicity, their efficacy was evaluated using an in vitro MTT assay, macrophage model, flow cytometry, and gene expression profiling. For evaluation of therapeutic effect of niosomal combination on the lesion induced by Leishmania major in inbred BALB/c mice, the size of lesions and number of parasites in spleen was assessed. The niosomal formulations demonstrated significantly greater inhibitory effects compared with the non-niosomal forms when the IC50 was considered. The niosomal combination showed an increase in the apoptotic values and gene expression levels of IL-12 and metacaspase and a decrease in the levels of IL-10 with a dose-response effect. The niosomal combination was also effective in reducing the lesion size and splenic parasite burden in mice. Our findings indicated that there is a synergistic effect between AmB and glucantime in niosomal form in the inhibition of intracellular and extracellular forms of L. tropica. Additionally, the in vivo results on L. major suggest that topical niosomal formulation could be useful in the treatment of CL.

Background: We aimed to evaluate the safety of SinaAmpholeish in a double-blind, randomized, phase 1 clinical trial in healthy human volunteers. Methods: The study was carried out in DermaLab of Center for Research and Training in Skin Diseases and Leprosy, Tehran University of Medical Sciences, Tehran, Iran in 2012. A topical Nano-liposomal formulation of 0.4% Amphotericin B was developed against Leishmania under trade name of SinaAmpholeish. In this randomized, double-blind, right-left, comparative, phase I clinical trial, in 2 steps; 7 and 20 healthy volunteers were recruited and applied SinaAmpholeish on the right and its vehicle on the left volar side of forearm, twice a day for one week or 3 times a day for two weeks. Seven biophysical skin parameters were measured in standard conditions before and 2 wk after application. Results: There was no adverse effect when SinaAmpholeish and its vehicle were used twice a day for seven days. However, when were used 3 times a day for two weeks, both SinaAmpholeish and its vehicle induced severe local skin reactions in 2 volunteers leading to discontinuation of application. Mild and temporary local reactions were observed in about half of the application sides and there was no significant difference between SinaAmpholeish and its vehicle. Conclusion: The new formulation is safe and worth to be tested in further phase 2 clinical trial and since there was no adverse effect with twice a day application it was decided to use SinaAmpholeish twice a day in phase 2 clinical trial.

BackgroundThe control of cutaneous leishmaniasis (CL) is facilitated by knowledge of factors associated with the treatment failures in endemic countries. The aim of this evaluation was to identify the potential risk determinants which might affect the significance of demographic and clinical characteristics for the patients with anthroponotic CL (ACL) and the outcome of meglumine antimoniate (MA) (Glucantime) treatment.Methodology/principal findingsThis current was executed as a cohort spanning over a period of 5 years which centered in southeastern part of Iran. Altogether, 2,422 participants were evaluated and 1,391 eligible volunteer patients with ACL caused by Leishmania tropica were included. Overall, 1,116 (80.2%) patients received MA intraleisionally (IL), once a week for 12 weeks along with biweekly cryotherapy, while 275 (19.8%) patients received MA alone (20 mg/kg/day for 3 weeks) (intramuscular, IM). The treatment failure rate in ACL patients was 11% using IL combined with cryotherapy plus IM alone, whilst 9% and 18.5% by IL along with cryotherapy or IM alone, respectively. Multivariate logistic regression model predicted 5 major associated-risk determinants including male (odds ratio (OR) = 1.54, confidence interval (CI) = 1.079-2.22, p = 0.018), lesion on face (OR = 1.574, CI = 1.075-2.303, p = 0.02), multiple lesions (OR = 1.446, CI = 1.008-2.075, p = 0.045), poor treatment adherence (OR = 2.041, CI = 1.204-3.46, p = 0.008) and disease duration > 4 months (OR = 2.739, CI = 1.906-3.936, p≤0.001).Conclusions/significanceThe present study is the original and largest cohort of ACL patients who treated with MA. A comprehensive intervention and coordinated action by the health authorities and policy-makers are crucial to make sure that patients strictly follow medical instructions. Early detection and effective therapy < 4 months following the onset of the lesion is critical for successful treatment of the patients. Since a significant number of patients are still refractory to MA, reducing man-vector exposure and development of new effective alternative drugs are essential measures against ACL due to L. tropica.

An amendment to this paper has been published and can be accessed via the original article.

Objective(s):Development of new generation of vaccines against leishmaniasis is possible because long-term protection is usually seen after recovery from cutaneous leishmaniasis. ISCOMATRIX is particulate antigen delivery system composed of antigen, cholesterol, phospholipid and saponin. In this study, the role of ISCOMATRIX bilayer composition made by different phase transition temperature (Tc) to induce a type of immune response and protection against leishmaniasis was assessed. Materials and Methods:ISCOMATRIX formulations with different bilayer compositions consisting of EPC (Tc

Background & objectives: The mechanism of antimony resistance in Leishmania has been studied extensively, in connection with decreased influx and/or increased eflux of the drug. Aquaporin 1 (AQP1) protein has been shown to mediate the uptake of trivalent antimony. This study was aimed to find the expression level of AQP1 gene in resistant versus non-resistant clinical isolates of Leishmania major in Iranian patients. Methods: Clinical isolates were obtained from 16 considered patients referred to Navab Safavi Clinical Center, Isfahan, Iran from October 2014 to December 2015. After diagnosis of cutaneous leishmaniasis using microscopic observation, biopsy was performed from lesion(s) of each patient and stored inside RNAlater solution at -20΀C. Written informed consent was obtained from all the patients to participate in the study before recording their information and sampling based on Helsinki declaration. Each patient was treated with Glucantime and followed for three months. All sensitive and resistance isolates were considered and compared with AQP1 gene expression using real time PCR that was analyzed with delta-delta Ct. Results: Out of 16 clinical isolates, four patients were resistant and 12 were non-resistant. The AQP1 gene expression in resistant isolates was significantly higher than the one in response failure isolates (p = 0.001). Interpretation & conclusion: The significant over expression (0.5 fold) of AQP1 gene in resistant versus non- resistant isolates suggests different mechanism of drug resistance such as mutations. Mutations may change the physiological function of the Aquaporin 1 protein that might affect its expression level.

Leishmania major (L. major) causes cutaneous leishmaniasis in the Old World. The infection mostly induces a localized lesion restricted to the sand fly bite. The costs and the side effects of current treatments render imperative the development of new therapies that are affordable and easy to administrate. Topical treatment would be the ideal option for the treatment of cutaneous leishmaniasis. MF29 is a 3-haloacetamidobenzoate that was shown in vitro to inhibit tubulin assembly in Leishmania. Here, we tested a topical cream formulated with MF29. BALB/c mice were infected in the ear dermis with L. major metacyclic promastigotes and once the lesion appeared, mice were treated with different concentrations of MF29 and compared to the control group treated with the cream used as the vehicle. We observed that topical application of MF29 reduced the progression of the infection while control groups developed an unhealing lesion that became necrotic. The treatment decreased the type 2 immune response. Comparison with SinaAmphoLeish, another topical treatment, revealed that MF29 treatment once a day was sufficient to control lesion development, while application SinaAmphoLeish needed applications twice daily. Collectively, our data suggest that MF-29 topical application could be a promising topical treatment for cutaneous leishmaniasis.

Background: Cutaneous leishmaniasis (CL) remains a serious public health concern in some parts of Iraq. The aims of this study to report of CL in some parts of Iraq, by different parasitological, cultural, and molecular methods and evaluate sex differences among infected patients. This is the first study conducted to characterize Leishmania species causing CL among Iraqi patients using the sequence analysis of Internal Transcribed Spacer1 (ITS1). Methods: A total of 700 cases of suspected CL who were referred to the Iraqi's clinics and health centers and they checked for Leishmania amastigote using Giemsa-stained , however the NovyMacneal Nicolle (NNN) culture led to the growth of promastigotes in all samples, then the samples were examined using PCR and RFLP-PCR methods. Results: The present study indicated that the prevalence of CL in eight Iraqi provinces as follow: Diwaniya 88(15.1%) , Wasit 85(14.5%),Najaf 79(13.6%), Thi-Qar 76(13.1%), Basrah 67(11.5%), Baghdad 65(11.2%), Diyala 63(10.8%) and Salah-Adin province which recorded the lower infection 60(10.3%), and it appeared 83.3% by using Giemsa-smeared in eight Iraqi provinces. The highest infection (100 %) appeared using PCR while the lowest infection (68%) appeared by culture on NNN media. The present study was revealed that the highest infection (60 %) caused by L.major than L.tropica (40 %) . Our results showed that 368 (52.6 %) of CL patients were had single lesion and 215 (30.7 %) had multiple lesions, and the ulcerative wet type lesions were present in 49.6% , while the nodule dry type lesions were present in 33.7 %. The overall prevalence of cutaneous leishmaniasis in the study area was very high (83.3%) having a statistical significant association with sex; males are more prone (56.4%) to Cutaneous Leishmaniasis (CL) as compared to females(43.6%). Conclusions: A clear and reliable bias toward males exists in some tropical diseases, such as leishmaniasis. Cutaneous leishmaniasis is a major health problem in Iraq and CL caused by many countries including Iraq. Health authorities should be aware of the fact that war and terrorist activities induce expansion of the disease and increase incidence rate in the situation that access to medical treatment is not easy especially in poor conditions of leishmaniasis endemic areas.

Background and Objective: Cutaneous leishmaniasis (CL) remains a serious public health concern in some parts of Iraq. The aims of this study to diagnose the Leishmania sp. causative agent of CL in some parts of Iraq, by different parasitological, cultural, and molecular methods. It was carried during the period October 2014 to February 2015. Materials and Methods: One hundred-sixty one of skin samples were examined by direct Giemsa-smeared, culture on NNN medium and Nested – PCR methods in different age groups. Results: The results of our study showed that 110 (68.3%) gave positives by Giemsa-smeared, 104 (64.6%) by culture and 67 (100%) by Nested – PCR. Our results appeared that there was slight gender predilection ; CL cases were more frequent in males (62.1%) than females (37.9%). Also, the type of infection showed that disease was in wet type 133(82.6%) more than in dry type 28 (17.4%). It was found that 98 (60.9%) of CL cases were in contact with rodents while 63(39.1%) of cases were not. Conclusions: CL disease is endemic in many parts of Iraq with high incidence and expanding to new foci that is considered a public health threat which needs special attention.

Background: Development of a topical treatment for cutaneous leishmaniasis (CL) is an important step in the im­provement of lesion management. Amphotericin B (AmB) is effective against Leishmania species but it is toxic, a Nano-liposomal form of AmB with a size of about 100nm (Lip-AmB) was developed and showed to be effective against Leishmania major, and Leishmania tropica in vitro and against L. major in vivo in animal model. This study was designed to check the irritancy Draize test in rabbits and was completed in the Center for Research and Training in Skin Diseases and Leprosy, TUMS, in 2012. Methods: Twenty rabbits in 3 steps were housed individually with artificial lighting (12/12h light/dark). SinaAm­pholeish cream or empty liposomes (prepared under GMP condition at Minoo Company, Tehran, Iran), was applied on a gauze patch and the patches were placed on the designated sites of the skin in the back of the rabbits. At 48 and 72h later, the erythema and oedema were checked, scored and recorded. Results: The erythema score in rabbits was 0.83+0.41 for the SinaAmpholeish and 0.5+0.55 for empty liposomes (P= 0.16). The average score for oedema was 0.67+0.52 for SinaAmpholeish and 0.33+0.52 for empty liposomes (P= 0.16). Conclusion: Based on skin irritancy reactions the topical formulation of SinaAmpholeish is safe and could be further checked in human trials.

Progress with the treatment of cutaneous leishmaniasis (CL) has been hampered by inconsistent methodologies used to assess treatment effects. A sizable number of trials conducted over the years has generated only weak evidence backing current treatment recommendations, as shown by systematic reviews on old-world and new-world CL (OWCL and NWCL).Using a previously published guidance paper on CL treatment trial methodology as the reference, consensus was sought on key parameters including core eligibility and outcome measures, among OWCL (7 countries, 10 trial sites) and NWCL (7 countries, 11 trial sites) during two separate meetings.Findings and level of consensus within and between OWCL and NWCL sites are presented and discussed. In addition, CL trial site characteristics and capacities are summarized.The consensus reached allows standardization of future clinical research across OWCL and NWCL sites. We encourage CL researchers to adopt and adapt as required the proposed parameters and outcomes in their future trials and provide feedback on their experience. The expertise afforded between the two sets of clinical sites provides the basis for a powerful consortium with potential for extensive, standardized assessment of interventions for CL and faster approval of candidate treatments.

Background: Establishment of sand flies laboratory colonies is essential to understand various biological aspects of Phlebotominae sand flies. The aims of the current study were to establish the colony of Phlebotomus sergenti Parrot (1917), the main vector of anthroponotic cutaneous leishmaniasis in old world, and to study biological parameters of this species. Methods: The sand flies were reared at 26–28 °C temperature, 14:10 (light: dark) photoperiod and 70–80% relative humidity. Larval diet was a composted mixture of rabbit faces and rabbit pellets which is prepared through a special process. First to fifth generations of P. sergenti were used to define biological parameters. Results: Results showed that, P. sergenti blood feeding percentage were 42% on chicken, 21% on BALB/c and 37% on golden hamster. Average time of blood digestion, egg incubation, 1st instar larva, pupa and adult emerging was recorded at 3.4, 8.7, 15, 33.3 and 41.2 days after blood feeding, respectively. Mean number of laid eggs was 55.1 and retained eggs were 35 per a female. Fecundity and production rate were 61.6%, and 42.2% respectively. Average longevity recorded at 15.2 days for females and 14.8 days for males. Conclusion: Colony of P. sergenti has been established for the first time in Iran. Average interval time from egg to adult of this species was 32.5 days. Chicken and golden hamster were recommended as a blood source for colony initiation and routine blood feeding, respectively.

Objective(s): ISCOMATRIX vaccines have now been shown to induce strong antigen-specific cellular or humoral immune responses to a broad range of antigens of viral, bacterial, parasite or tumor. In the present study, we investigated the role of ISCOMATRIX charge in induction of a Th1 type of immune response and protection against Leishmania major infection in BALB/c mice. Materials and Methods: Positively and negatively charged ISCOMATRIX were prepared. BALB/C mice were immunized subcutaneously, three times with 2-week intervals, with different ISCOMATRIX formulations. Soluble Leishmania antigens (SLA) were mixed with ISCOMATRIX right before injection. The extent of protection and type of immune response were studied in different groups of mice. Results: The group of mice immunized with negatively charged ISCOMATRIX showed smaller footpad swelling upon challenge with L. major and the highest IgG2a production compared with positively charged one. The mice immunized with positively charged ISCOMATRIX showed the lowest splenic parasite burden compared to the other groups. Cytokine assay results indicated that the highest level of IFN- γ and IL-4 secretion was observed in the splenocytes of mice immunized with negatively charged ISCOMATRIX as compared to other groups. Conclusion: The results indicated that ISCOMATRIX formulations generate an immune response with mixed Th1/Th2 response that was not protective against challenge against L. major.

Background: Chemotherapy still relies on the use of pentavalent antimonials, amphotericin B, paromomycin, miltefosin, and allopurinol. In this study, we explained about the native plant that grows in different regions of Iran and used as anti-leishmanial in Iran and even many other countries. Methods: This narrative review covers all information about local herbal medicine in Iran that used in treatment of cutaneous leishmaniasis in all the worlds, published in local and international journals from 1996 to 2015 using various databases including PubMed, SID, Google Scholar, Scopus, and Science Direct. Results: Overall, 150 articles in databases were identified. Many local plants grown in some places of Iran were used to treat this endemic disease. Conclusion: The cutaneous leishmaniasis is also a major health problem in Iran, especially in Mashhad (Northeast of Iran). Therefore, many patients seek for herbal therapy that is cheaper and readily available. This review provides information regarding plant that exists in Iran and exhibiting effects on anti- Leishmania activity. Among the anti-leishmanial mentioned in this review, most have never been tested for cytotoxicity and very few have been tested for in vivo activity.

Background: Zoonotic cutaneous leishmaniasis (ZCL) is a neglected tropical disease prevailed in many rural areas of 17 out of 31 provinces in Iran. The main vector of the disease is Phlebotomus papatasi and the causative agent is Leishmania major in ZCL foci of Iran. In the current study we investigated the validity of accessory glands secre­tions as an indicator to recognize parous from nulliparous Ph. papatasi females under laboratory conditions. Methods: Over 235 laboratory-reared females of Ph. papatasi were dissected in 6 groups including: newly emerged, one hour, one day, two days, and three days after blood feeding and also after oviposition under stereo microscope for their parity in 2014–2015. Results: Transparent glands were compatible with nulliparous only in newly emerged sand flies. In sand flies dis­sected after oviposition, accessory glands were rather large as a result of oviposition though they were transparent. Conclusion: The accessory glands secretions could not be as an indicator for distinguishing parous from nulliparous of Ph. papatasi females.

Objective: Leishmaniasis is one of the most important parasitic infectious diseases in the world. Since last century, many efforts have been made to control and treat the disease, but appropriate vaccines, pesticides and medicines are not available or even eligible. The purpose of this study was to evaluate the effect of hydro-alcoholic extract of Seidlitzia rosmarinus on the lesions of experimental Cutaneous Leishmaniasis (CL) in Balb/c mice. Materials and Methods: The population study was 60 Ballb/c mice which divided to 6 groups, all infected with Leishmania major [MRHO/75/IR]. Soon after the ulcer started to appear in the early stage, a dose of provided herbal extract with 5, 10 and 15% concentration applied on each lesion. The surface area of the lesions measured during an interval of 10 days. Direct Giemsa stained smears prepared two and four weeks after treatment. Results: Increasing the mean size of the lesions was statistically significant compared to those in control group (p>0.001). Visceral Leishmaniasis (VL) developed in all of the mice including the control group that received Eucerine alone. Survival rate in group receiving 15% S. rosmarinus extracts showed significantly higher compared to mice in control group (p

Background: Cutaneous leishmaniasis (CL) is a public health problem in several endemic countries. Recent studies on mouse model and a few clinical experiments showed that the type of immune response generated at the site of infection and especially balance between regulatory and effector T-cells determines the outcome of the disease toward self-limiting or long-lasting lesions. The aim of this study was to evaluate the role of natural regulatory T-cells (nTregs) in early and late cutaneous lesions of human Leishmania major (L. major) infection. Methods: Skin biopsies were collected from parasitologically proven lesions of 28 patients with cutaneous leishmaniasis, divided into two groups of early and late lesions. The causative agents were identified to be L. major. Quantitative real-time reverse transcription polymerase chain reaction (PCR) and immunofluorescent staining of biopsies were used to assess the Foxp3 mRNA expression and frequency of nTregs in two groups. Mann-Whitney U test was used to determine the significance of deferences between the two groups. Findings: Mean relative expressions of Foxp3 mRNA were 0.53 ± 0.23 and 1.26 ± 0.99 in early and late lesions, respectively, which was significantly upper in chronic lesions (P = 0.007). Parallel results were obtained in tissue staining method. Conclusion: Increased in gene expression and protein staining of nTreg markers in chronic biopsy samples indicates a role for these cells in chronic L. major induced leishmaniasis and supports the effectiveness of regulatory T cell-based immunotherapy for treatment of chronic cutaneous leishmaniasis.

Background: Adaptive immune response is an important factor in the healing process and development of protection in cutaneous leishmaniasis (CL). Little information is available in human CL about the importance of the balance between effector and regulatory immune responses. Therefore, the aim of this study was to asses messenger ribonucleic acid (mRNA) expression of interleukin-10 (IL-10), IL-4, transforming growth factor-β1 (TGF-β1), interferon-g (IFN-g), and forkhead box P3 (Foxp3) (as a marker of regulatory T cells) in acute and chronic CL lesions caused by Leishmania major compared with normal skin samples. Materials and Methods: Thirty biopsies were obtained from CL patients with acute lesions (AL, n = 13), chronic lesions (CH, n = 11) and healthy volunteers (n = 6). Relative expressions of target genes were determined by means of reverse transcription real time polymerase chain reaction and were compared with the controls. Results: Expression of Foxp3, IL-4, and IFN-g were significantly more in CH than AL group of patients (Foxp3: Median 0.48, inter-quartile range 0.32-0.76 [arbitrary units] for AL, and 0.97 (0.75-1.30) for CH, P = 0.006; IFN-g: 45.98 (33.39-173.48) for AL, and 200.53 (97.49-361.76) for CH, P = 0.023; IL-4: 0.49 (0.34-2.16) for AL, and 2.14 (1.30-7.11) for CH, P = 0.021). Expression of TGF-β was not significantly different between groups. Conclusion: The results indicate that IL-4 secretion at the site of L. major infection rather than low IFN-g production might have a role in prolongation of disease. Despite a moderate increase of Foxp3 expression in chronic lesions, function of Tregs in persistent infection is not clear.

Background: The current study was designed to evaluate immune responses induced by DNA vaccines encoding 8-kDa subunit of antigen B (HydI) of Echinococcus granulosus and murine interleukin 12 (IL-12) as genetic adjuvants in BALB/c mice. Methods: Expression plasmid pcDNA3.1 containing HydI (pcHyd1) as vaccine along with the murine interleukin 12 (pcMIL12) as adjuvant were used. Thirty-five mice in the five experimental groups received PBS, empty pcDNA3.1, pcHydІ, pcMIL-12, and pcHydІ+ pcMIL-12 in days zero, 14th and 28th. Two weeks after the last immunization, evaluation of the immune response was performed by evaluating the proliferation of splenic lymphocytes, IFN-γ and IL-4, determination of IgG isotyping titer. Results: Mice that received the pcHydI+pcMIL12 exhibited higher levels of lymphocyte proliferation compared to mice that received the pcHydI alone (P