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Background: Leishmania tropica is a genetically divergent species. Amplification of entire internal transcribed spacer (ITS) region of L. tropica isolates obtained from Bam district, one of the well known focus of anthroponotic cutaneous leishmaniasis )ACL( in Iran, revealed a double-band pattern in agarose gel electrophoresis. This study explains how this pattern occurs. Methods: Twenty seven L. tropica smear preparations were collected from Bam district, south east Iran, and eight L. major and one L. infantum smear preparations were gathered from Shiraz, south west Iran. Furthermore one L. major and one L. infantum cultured standard strains were tested using entire ITS-PCR to survey their electrophoretic pattern. The ITS sequences of L. tropica, L. major, and L. infantum already deposited in GenBank were analyzed. Analysis of GenBank sequences of L. tropica revealed two groups of sequences based on length size, one group having a 100 bp gap. Therefore, a new reverse primer namely LITS-MG was designed to exclude this gap in PCR products. Results: Whole ITS fragment amplification resulted in a double-band pattern in all L. tropica cases, while a sharp single band was observed for L. infantum and L. major isolates. This result was corresponding to the result obtained from in silico analysis of GenBank sequences. Use of LITS-MG pri-mer was expectedly resulted in a single band including ITS1, 5.8s and partial ITS2 product for L. tropica which is appropriate for following molecular studies such as sequencing or restriction analysis. Conclusion: Sequences analysis of GenBank L. tropica sequences and following practical laboratory tests revealed at least two alleles in L. tropica which were confirmed in Bam isolates. This especial double-band pattern is because of a 100 bp fragment difference within ITS-rDNA alleles. [ABSTRACT FROM AUTHOR]

Malaria is a major problem in tropical and sub-tropical countries, with high morbidity and mortality. Splenectomy makes patients more susceptible to serious bacterial and parasitic infections. We report for the first time in Iran a fatal case of Plasmodium vivax malaria, confirmed by microscopic and molecular (Semi-nested multiplex PCR) tests in a patient who had undergone splenectomy due to hemolytic anemia. [ABSTRACT FROM AUTHOR]

Background: Cutaneous leishmaniasis constitutes a major public health problem in many parts of the world including Iran. The primary objective of this study was to identify Leishmania species in endemic districts of Kerman Province, south-eastern Iran. Methods: This study was conducted by random sampling as cross- sectional descriptive between 2008 and 2010. Overall, 203 skin scraping smears were taken from the patients. Nested -PCR was performed to amplify variable minicircle fragments of Leishmania kDNA. Results: Bam was the most infected district (71.1%), followed by Kerman (14.7%), Jiroft (5.4%), Baft (2.7%), Sirjan (1.6%), Shahr-e Babak (1.5%) and others (3.0%). L. tropica was the most common species identified (194 cases, 95.6%), while L. major was found in only 9 cases (4.4%). Of 203 identified patients, all species in Bam (l07 cases), Kerman (32 cases), Jiroft (l6 cases) and Shahr-e- Babak (l1 cases) were detected as L. tropica, whereas infected subjects in Baft and Sirjan showed L. tropica or L. major. Characterization of Leishmania species resulted in generation of 750 bp and 560 bp fragments, corresponding to those of L. tropica and L. major, respectively. Conclusion: L. tropica is the main species (95.6%) caused ACL in endemic areas of Kerman Province; however L. major is present in low level (4.4%). [ABSTRACT FROM AUTHOR]

Background: Visceral leishmaniasis (kala-azar) is an endemic disease in some areas of Iran. A cross- sectional study was conducted for sero-epidemiological survey of visceral leishmaniasis (VL) in Baft district from Kerman Province, southeast of Iran. Methods: Blood samples were collected from children up to 12 years old and 10% of adult population from Baft villages with a multi-stage randomized cluster sampling. In addition, blood samples were collected from 30 domestic dogs from the same areas. All the collected blood samples were tested by direct agglutination test (DAT) for the detection of anti-Leishmania antibodies in both human and dog using the cut-off value of ⩾1:3200 and ⩾ 1:320, respectively. Parasitological, molecular, and pathological were performed on infected dogs. Chi-square and Fisher exact tests were used to compare sero-prevalence values. Results: From 1476 collected human serum samples, 23 (1.55%) showed anti-Leishmania antibodies at titers of 1:800 and 1:1600 whereas 14 (0.95%) showed anti-Leishmania infantum antibodies at titers of ⩾ 1:3200. No statistically significant difference was found between male (1.18 %) and female (0.69%) sero-prevalence (P=0.330). Children of 5-8 years showed the highest sero-prevalence rate (3.22%). Seven out of 30 domestic dogs (23%) showed anti-Leishmania antibodies at titers ⩾1:320. Leishmania infantum was identified in five infected dogs by nested — PCR assay. Conclusion: It seems that visceral leishmaniasis is being endemic in southern villages of Baft district, southeast of Iran. [ABSTRACT FROM AUTHOR]

Background: Visceral leishmaniasis (VL) is one of the most important neglected tropical diseases. The zoonotic form of VL is endemic in some areas of Iran. We aimed to determine the status of VL identified in humans and canines in different parts of Iran from 2013 to 2022., Method: A national representative cross-sectional study was conducted in 10 provinces of Iran, including the national leishmaniasis reference lab. We employed the direct agglutination test (DAT) as a reliable serological method to detect anti- Leishmania infantum antibodies in humans and animal reservoir hosts. Additionally, a narrative literature review was conducted to identify relevant studies on VL seroprevalence in Iran from 2013 to 2023., Results: The results of 21281 human and 5610 canine serum samples from 2013 to 2022 are reported. Altogether, 448 (2.1%, 95%CI: 2.0-2.3) human serum samples showed anti- L. infantum antibody levels of ≥1:3200. Of these samples, 13716 (64.5%) were collected actively, which showed a seroprevalence of 0.6% (95% CI: 0.5-0.8) and 7565 (35.5%) were collected passively, which showed a seroprevalence of 4.8% (95%CI: 4.3-5.3). Overall, 1035 (20.1%, 95%CI: 19.0-21.2) of 5160 domestic dogs ( Canis familiaris ) samples showed anti- L. infantum antibody levels of ≥1:320. Northwest (2.8%) and northeast (0.96%) regions had the highest human VL seroprevalence, while northwest (21.5%) and south (14.4%) regions had the highest canine VL seroprevalence., Conclusion: Zoonotic VL, an endemic parasitic disease, is still present in several different distinct areas across Iran. While human VL cases have shown a declining trend over the last decade, the prevalence of canine VL remains significant., Competing Interests: Conflict of interest The authors have no conflicts of interest to declare for this study., (© 2023 Mohebali et al. Published by Tehran University of Medical Sciences.)

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., Competing Interests: Conflict of interests The authors declare that there is no conflict of interests., (Copyright© Iranian Society of Parasitology & Tehran University of Medical Sciences.)

Background: This study was aimed to silencing the Nucleoside transporter 3 (NT3) permease nucleobases involved in the salvage pathway of Leishmania in order to disrupt purine nucleotide uptake in the parasite and consequently, destruction of the parasite., Methods: Overall, 502 bp fragment of the NT3 gene sequence was designed to produce an antisense transcript upon entry of the vector into the parasite. The NT3 construct was transfected into L. major promastigotes and NT3 gene expression was studied in vivo and in vitro conditions., Results: Relative expression NT3 gene in transgenic Leishmania was decreased in tenth day. The percentages and the number of amastigotes infected macrophages with transgenic L. major were less than infected macrophages with wild-type strain. Our results in two groups of BALB/c female mice (wild-type strain and mutant, n=4 each group) were showed that size and number of ulcers in BALB/c mice infected with transgenic Leishmania promastigotes were less than the BALB/c mice infected with wild-type parasites., Conclusion: The results indicate the use of antisense RNA reduces of NT3 gene expression in L. major . More studies are required to obtain a new approach for treating Leishmania infection., Competing Interests: Conflict of interest The authors have declared no conflict of interest.

Background: Cutaneous leishmaniasis (CL) is associated with a broad and complex clinical spectrum of diseases. The objectives of this study were to assess the clinical features and identification of the causative agents of CL in a well-known focus of anthroponotic CL (ACL) caused by Leishmania tropica , southeast Iran., Methods: This study was performed randomly as a descriptive cross-sectional survey to evaluate 2000 CL patients by active and passive case-detection approaches in Kerman Province from 1994 to 2014. The ACL patients were confirmed by direct smear and 600 cases by one or a combination of intrinsic methods., Results: Children aged <10 yr old were the most infected patients ( P <0.001). The majority of the CL lesions were located in hands (46.3%), face (34.1%), legs (14.3%), and other parts of the body (5.3%). The mean number of lesions was 1.5 and most of the patients had single lesion (65%).Typical clinical lesions included papule (36.8%), followed by ulcerated nodule (20.7%), plaque (18.4%), and ulcerated plaque (18.5%). While among atypical clinical features, leishmaniasis recidivans (LR) (4.7%) and leishmanid (0.3%) were the dominant forms, followed by diffuse, disseminated, sporotrichoid, and erysipeloid types, 0.1% each, and then lymphedematous, lymphadenic, hyperkeratotic, paronychial, and mutilating types, 0.05% each. Based on various intrinsic methods the parasites isolated from the lesions were characterized as L. tropica., Conclusion: ACL due to L. tropica presents numerous cases of localized form and diverse uncommon clinical presentations, which mimic other disease conditions. Therefore, physicians should be aware of such manifestations for selecting appropriate treatment modality., Competing Interests: Conflict of Interests The authors declare that there is no conflict of interests.

Background: The present study aimed to evaluate the effect of methotrexate (MTX) alone and in combination with meglumine antimoniate (MA, Glucantime) against sensitive and MA-resistant Leishmania tropica stages in vitro., Methods: The present study was carried out in 2014 in Leishmaniasis Research Center at School of Medicine, Kerman University of Medical sciences, Kerman, Iran. The effects of MTX alone and along with MA on promastigote and amastigote stages of sensitive (SS) and MA-resistant (RS) L. tropica strains have been evaluated using a colorimetric MTT assay and in a macrophage model, respectively. In addition, the inhibitory effect of MTX on the Leishmania invasion of murine macrophages was assessed in promastigotes of both strains of L. tropica . Sensitive and MA resistant L. tropica are referred to those isolates that are responsive or non-responsive to one or two courses of treatment by MA systemically and/or intralesionally, respectively., Results: The findings of OD and IC 50 showed that MTX plus MA (SS: 16.1 μg/ml, RS: 39.8 μg/ml) had a higher anti-leishmanial effect than MA (SS: 52.2 μg/ml, RS: 170 μg/ml) or MTX alone (SS: 22.2 μg/ml, RS: 51.4 μg/ml) on promastigotes of both strains of L. tropica. The MTX plus MA caused a significant decrease ( P< 0.05) in the mean infection rate (MIR) and the mean number of amastigotes in each macrophage compared with positive control. Infectivity of promastigotes is significantly ( P< 0.05) reduced when it was preincubated with MTX., Conclusion: This study indicated high potency and a synergistic effect of MTX on MA in inhibiting the growth rateof promastigote and amastigote stages of sensitive and meglumine antimoniate-resistant L. tropica . Further works are needed to evaluate the anti-leishmanial effects of MTX on L. tropica using a clinical setting., Competing Interests: Conflict of Interests The authors declare that there is no conflict of interest in this study.

Background: Visceral leishmaniasis (VL) has strong links with poverty, substantial medical and veterinary impacts. This review aimed to focus in studies published during 1994-2016 on VL in southeastern Iran., Methods: The present review is based on expert knowledge and historical studies published during the past 23 yr (1994-2016) on VL in southeastern Iran. In addition, related literature found in PubMed by using the keywords such as visceral leishmaniasis, kala-azar, and Leishmania infantum are included., Results: Overall, 118 children aged 4.2 yr were detected as infected with human VL (HVL). The majority of the cases were from Orzoieh district (37.1%) in southwest of Kerman Province, followed by Sirjan (15.7%), Jiroft (14.8%), Kahnuj (9.3%) and to lesser extent from other areas. The male to female ratio was 1.7. The three most frequent clinical features were represented by fever (100.0%), anemia (95.0%) and splenomegaly (91.5%). Altogether, 42.0% of the VL cases developed secondary bacterial infections, the overall case-fatality rate was 3.4%, and majorities (88.0%) of the VL patients were undernourished. Overall, 733 dogs and wild canines were examined by different techniques with various seroprevalence ranges., Conclusion: In southeastern Iran, VL is endemic in Orzoieh district in Kerman Province. While the dogs are implicated as the main domestic reservoir of VL, wide range of wild canines can serve as a secondary potential reservoir host.

Background: Leishmaniasis represents a major public health concern in tropical and sub-tropical countries. At present, there is no efficacious vaccine against the disease and new control methods are needed. One way to access this important goal is to knock out genes of specific macromolecules to evaluate the effect of deletion on the growth, multiplication, pathogenesis and immunity of the parasite. The aim of this study was to design and clone molecular constructs to knock out N-acetylglucosamine phosphatidylinositol de-N-acetylase ( GPI12 ) gene in Leishmania major ., Methods: For designing and making molecular constructs, we used pLEXSY-neo2 and pLEXSY-hyg2 vectors. The molecular constructs were cloned in E. coli strain Top10. The molecular constructs were transfected by electroporation into L. major in two stages., Results: The molecular constructs were confirmed by Colony PCR and sequencing. The recombinant strains were isolated by selective antibiotics, after which they were confirmed by PCR, Southern and Western blots., Conclusion: Recombinant parasites were created and examined for subsequent study. With the use of molecular constructs, it was possible to remove and study gene GPI12 and to achieve a live recombinant Leishmania parasite that maintained the original form of the antigenic parasites. This achievement can be used as an experimental model for vaccine development studies. Further investigations are essential to check this model in a suitable host.

Background: Sensitive and glucantime (MA) resistance Leishmania tropica are referred to those isolates, which are responsive, or non-responsive to one or two full courses of treatment by MA systematically and/or intra-lesionally, respectively. In this study, we evaluated the antileishmanial activity of biogenic selenium nanoparticles (Se NPs) alone and in combination with MA against sensitive and glucantime-resistant L. tropica on in vitro model., Methods: The Se NPs were synthesized by employing the Bacillus sp. MSh-1. The antileishmanial effects of Se NPs alone and in combination with MA on promastigote and amastigote stages of sensitive and glucantime-resistant L. tropica strains have been investigated using a colorimetric MTT assay and in a macrophage model. In addition hemolytic activity in type O+ human red blood cells and infectivity rate of the promastigotes before and after treatment with the Se NPs was evaluated., Results: In the promastigote stage, various concentrations of Se NPs significantly inhibited (P<0.05) the growth of promastigotes of both strains in a dose-dependent manner. Similarly, Se NPs especially in combination with MA significantly reduced the mean number of amastigotes of both strains in each macrophage. Se NPs showed no hemolytic effect on human RBCs at low concentrations. Moreover, infection rate of macrophages by promastigotes significantly (P<0.05) was reduced when promastigotes pre-treated with Se NPs., Conclusion: The findings of this study suggest a first step in the search of Se NPs as a new antileishmanial agent. Further experiments are needed to investigate antileishmanial effects of biogenic Se NPs on L. tropica using a clinical setting.

Background: Canine visceral leishmaniasis (CVL) is a systemic disease with a high mortality rate, caused by a diphasic protozoan parasite, Leishmania infantum/chagasi in the world. The objective of the present study was to determine the presence of CVL in the city and suburbs of Kerman, using a range of serological, histopathological and molecular methods., Methods: Blood samples were taken from 80 clinically symptomatic stray dogs All the collected blood samples were tested by direct agglutination test (DAT) to detect the anti-Leishmania antibodies in dogs, using a cut-off value of ≥1:320. Pathological specimens including spleen, liver and lymph nodes were prepared for paraffin blocks, sectioning, staining and final microscopic examination in the pathology laboratory. PCR amplification of kDNA from 9 samples of DAT positive stray dogs was studied., Results: The anti-Leishmania antibody was detected in 9 dogs (11.25 %) of the total 80 studied dogs. No significant difference was found between VL infection and gender. In contrast, there was a significant difference between seropositivity and age (P<0.05). Pathological samples showed changes including hyperplasia of infected macrophages and inflammatory cells that occupied sinusoids and splenic cords. Among the samples which was characterized by PCR, only one specimen revealed to be mixed infection between L. infantum and L. tropica., Conclusion: The results revealed a high prevalence of L. infantum infection in stray dogs in Kerman. This kind of information is needed for implementation of future control programs.

Background: Leishmaniasis has been identified as a major public health problem in tropical and sub-tropical countries. The present study was aimed to investigate antileishmanial effects of various extracts of Berberis vulgaris also its active compoenent, berberine against Leishmania tropica and L. infantum species on in vitro experiments., Methods: In this study in vitro antileishmanial activity of various extracts of B. vulgaris also its active compoenent, berberine against promastigote and amastigote stages of L. tropica and L. infantum was evaluated, using MTT assay and in a macrophage model, respectively. Furthermore, infectivity rate and cytotoxicity effects of B. vulgaris and berberine in murine macrophage cells were investigated., Results: The findings of optical density (OD) and IC50 indicated that B. vulgaris particulary berberine significantly (P<0.05) inhibited the growth rate of promastigote stage of L.tropica and L.infantum in comparison to meglumine antimoniate (MA). In addition, B. vulgaris and berberine significantly (P<0.05) decreased the mean number of amastigotes in each macrophage as compared with positive control. In the evaluation of cytotoxicity effects, it could be observed that berberine as compared with B. vulgaris exhibited more cytotoxicity against murine macrophages. Results also showed that when parasites were pre-incubated with B. vulgaris their ability to infect murine macrophages was significantly decreased., Conclusion: B.vulgaris particularly berberine exhibited potent in vitro leishmanicidal effects against L. tropica and L.infantum. Further works are required to evaluate the antileishmanial effects of B.vulgaris on Leishmania species using clinical settings.

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.