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Background: In Iran, both forms of cutaneous (CL) and visceral leishmaniasis (VL) have been reported; so the accurate species identification of the parasite(s) and the analysis of genetic diversity are necessary. Methods: The smears were collected from lesions samples of 654 patients with CL, who attended local health centers in 12 provinces of Iran during 2013–2015. The smears were checked for the presence of amastigotes by light microscopy. DNA of 648 Leishmania isolates, amplified by targeting a partial sequence of ITS (18S rRNA–ITS1–5.8S rRNA–ITS2) gene. Twenty-five of all the amplicons were sequenced and analyzed with restriction fragment length polymorphism (RFLP) using the Taq1 enzyme. Results: All the smears were positive microscopically. The PCR-RFLP analysis revealed that 176 (27%) CL patients were infected with L. tropica and, 478 (73%) with L. major. The dominant species in all over Iran is L. major. The sequencing results of all CL patients and RFLP analysis confirmed each other. Based on our phylogenetic tree, 25 ITS DNA sequences were grouped into two clusters representing L. major and L. tropica species. Phylogenetic tree derived from the ITS sequences supports a clear divergence between L. major from the other species. Conclusion: Discrimination of Iranian Leishmania isolates using ITS gene gives us this opportunity to detect, identify, and construct the phylogenetic relationship of Iranian isolates.

Background: Domestic dogs have been implicated as the main reservoir host of Mediterranean type of visceral leishmaniasis (VL) that is endemic in some parts of Iran. This study was performed about role of dogs in canine VL (CVL) epidemiology in Jiroft District, south of Kerman Province, southeastern Iran. Methods: Totally, 165 dogs including 100 stray and 65 sheepdogs were randomly selected. After complete clinical examination blood sample was taken from each dog. All the collected samples were examined following the serum separation by direct agglutination test (DAT) for detection of anti-Leishmania infantum antibodies. The titers of ≥1:320 were defined as positive. Results: Overall, of 165 serum samples, 13 samples (7.9%) were positive by DAT at titers of ≥1:320. The seroprevalence was 11% among the stray dogs and 3% among the sheepdogs. There was no significant difference between stray and sheepdogs in CVL infection. The highest seroprevalence rate (14.3%) was found in seven-year old dogs. Conclusion: The present finding indicates the role of stray and sheepdogs in CVL epidemiology in this area. Further investigations are needed to evaluate the status of VL infection in human subjects in this area.

Background: To compare three molecular methods, PCR-RFLP for internal transcribed spacer, PCR sequencing and high resolution melting analysis shown reliable sensitivity and specificity for detecting Leishmania tropica as a model for cutaneous leishmaniasis (CL) as the perspective overview for scientific and economic approaches. Methods: This study was carried out between 2015 and 2016 in Leishmaniasis Research Center in Kerman University of Medical Sciences, Kerman, Iran. The positives smears (n=50) were obtained from patients referred from the health clinics in a major anthroponotic CL (ACL) focus, southeastern Iran. Only smear preparations with the same grade were selected according to the method described by the WHO for future PCR assays. Results: All three molecular methods had capability to identify positive samples at species level with the same specificity and sensitivity. However, these techniques were different in simplicity, consuming time, and cost effectiveness. Although additional enzymatic process in PCR-RFLP provided good resolution to find Leishmania species but this would cause time and cost increases. Conclusion: HRM (high resolution melting) is a relatively new technique that allows direct characterization of PCR amplicons in a closed system with more simplicity, cost effectiveness and time-consuming compared with other PCR-based assays for epidemiological or clinical identification purposes.

Background and Purpose: Gastroenteritis and the clinical profile caused by Microsporidia, an opportunistic pathogen, may be severe in immunocompromised individuals, especially in AIDS patients. Conventionally, it is necessary to detect the small infective spores in stained smears. However, due to the limitations of the microscopy-based methods, several DNA-based methods such as polymerase chain reaction (PCR) have recently been developed to enhance diagnosis sensitivity. Therefore, we sought to evaluate the rate of infection in immunocompromised patients as compared with immunocompetent patients in Kerman, Iran. Materials and Methods: We collected stool samples of 199 human subjects (116 males and 83 females), aged 1 to 69 years old. They were divided into immunocompromised (i.e., AIDS [n=72] and cancer-positive patients [n=59]) and immunocompetent (n=68) groups. We comparatively examined the fecal materials using the microscopy and PCR methods. Results: The overall prevalence rate of Microsporidia infection was 10.05% (20/199). Entrocytozoon bieneusi was the only species within the Microsporidia genus that was identified in 14.5% (19/131) of the immunocompromised patients and 1.47% (1/68) of the immunocompetent individuals. Conclusion: Considering the higher prevalence rate of microsporidiosis in patients with immunodeficiency (10.03%), we suggest performing sensitive and specific techniques such as PCR for the detection of these parasites in immunocompromised patients.

Background: Different background of immunity responses determine resistance or susceptibility of certain mouse strains to Leishmania major infection. Some have been well known previously. Antimicrobial peptides (AMPs) such as cathelicidins and defensins are unique fragments of innate immunity system with well-known effects against the invasion pathogens. Despite their outstanding roles and being of extensive cases of cutaneous leishmaniasis (CL) caused by L. major, they have been less studied in Leishmania fields. The aim of present study was to determine whether these components play a role in the protection of skin against Leishmania infections. Methods: The animal model of Leishmania infection was established by the subcutaneous inoculation of 5×106(parasites/ml) from the stationary phase of L. major promastigotes to BALB/c and C57BL/6 mice from January 2016 to August 2016 in Kerman Province, southeast of Iran. After 1, 3 and 7 d of post-infection (PI), the samples needed for detecting of the mRNA levels of mouse beta-defensin (mBD)-1, mBD2, mBD3, mBD4, mBD6, cathlin-related antimicrobial peptide (CRAMP), interleukin (IL)-10, IL-12 and parasite load were taken under standard methods. Results: The findings related to cytokines profiles in BALB/c (­IL-10, ¯IL-12) and C57BL/6 mouse strains (¯IL-10, ­IL-12) demonstrated that immunity system has been accurately activated during CL caused by L. Major parasites. We also observed a significant up-regulation of all aforementioned AMPs genes in BALB/c mice at selected times compared to another strain. Conclusion: CL occurred in BALB/c mice in spite of the fact that the expression of AMPs was higher than the other strain. AMPs genes are well expressed to provide defense against the parasites that have increased and escaped from immunity system but cannot create an absolute protection.

Visceral leishmaniasis (VL) is endemic in Iran and is caused predominantly by Leishmania infantum, but L. tropica is emerging as an important cause. We studied the intra-species population structure of Leishmania spp. causing VL in southwest Iran by sequence analysis of the internal transcribed spacer (ITS) 1 of DNA samples from 29 bone marrow aspiration smears. L. infantum (n = 25) and L. tropica (n = 4) were identified, consisting of 10 and three ITS1 sequence types (STs), respectively. Compared to GenBank ITS1 STs, our L. infantum parasites displayed high heterogeneity but less heterogeneity compared than northwest Iranian isolates. VL affects mostly nomadic populations in southwest Iran, and their mobility may explain partly the L. infantum heterogeneity. The VL causing L. tropica was also genetically heterogeneous but genetically indistinguishable from L. tropica strains causing anthroponotic cutaneous leishmaniasis from southwest Iran.

Background: Culex quinquefasciatus (Diptera: Culicidae) is an important vector for many human diseases. The aim of this study was to evaluate the susceptibility level of larval and adult stages of Cu. quinquefasciatus to different groups of WHO recommended insecticides for vector control. Methods: Larval stages of the Culex mosquitoes were collected from their natural habitats in Rafsanjan County at Kerman Province, southeast of Iran in 2016. Insecticide susceptibility status of adult female Cx. quinquefasciatus against DDT (4%), deltamethrin (0.05%), malathion 5%, and bendiocarb (0.1%) were determined using WHO stand­ard insecticide susceptibility test. Additional test was carried out to determine the susceptibility status of larvae of Cx. quinquefasciatus to temephos. Bioassay data were analyzed by Probit program. Results: Cx. quinquefasciatus adults showed resistance to all four groups of the tested insecticides according to the WHO criteria for resistance evaluation. The lethal concentrations for 50% mortality (LC50) and 90% mortality (LC90) of temephos against Cx. quinquefasciatus larvae were 0.18mg/l and 0.78mg/l, respectively. This finding also con­firms resistance to temephos based on the WHO recommended instructions for resistance evaluation. Conclusion: Resistance to all groups of the tested insecticides should be considered for future vector control investi­gations in the study area.

Background: Dirofilariasis is a serious and potentially deadly condition in dogs and one of the zoonotic filarial infections, which inadvertently affects the humans. The objectives of this study were to determine the seroprevalence and the molecular identity of dirofilariasis in Kerman Province, southeastern Iran between Jul and Aug 2013. Methods: A hundred and forty-nine domestic dogs were randomly selected and five ml blood samples were taken from each dog. One ml of anticoagulant (EDTA) was used for each test in the parasitological study (modified Knott´s test) and sera samples were examined, using ELISA kit to detect Dirofilaria immitis antigen. Extracted DNA of all positive blood samples was used for molecular characterization and sequencing. Results: Four (2.7%) domestic dogs of the total 149 domestic dogs were infected with micofilariae of D. immitis, while the serological study showed 8 (5.4%) domestic dogs were infected with D. immitis. No signifi­cant difference, however, was found between dirofilariasis infec­tion and gender. On the other hand, a signifi­cant difference was observed between dirofilariasis infec­tion and age (P

Background: Leishmaniasis is important vector-borne parasitic disease worldwide, caused by the genus Leishmania. The objective of the current study was to identify genetic polymorphism in L. major, one of the species causing cutaneous leishmaniasis (CL), isolated from southeastern Iran, using Permissively Primed Intergenic Polymorphic-Polymerase Chain Reaction (PPIP-PCR) method. Methods: Overall, 340 patients with suspected CL were examined. They referred to the Central Laboratory in Chabahar, Iran during Apr 2013 to Feb 2014. Microscopic examination of Giemsa-stained slides from lesions as well as aspirates cultured in Novy- Mac Neal-Nicolle (NNN) Media was employed in order to diagnose CL in these patients. Our analyses detected 86 suspected subjects as having CL from which 35 isolates were cultured successfully. PPIP-PCR method was performed on extracted genomic DNA from selected isolates in order to determine the genetic polymorphism among L. major isolates. Results: The electrophoresis patterns demonstrated two genetic profiles including A or A1 patterns between all samples tested. Frequency of A and A1 sub-types were 33 (94.3%) and two (5.7%), respectively. Conclusion: Both host and parasite factors may contribute to the clinical profile of human leishmaniasis in the endemic foci of the disease. Here we showed that genetic variations pertaining to the Leishmania parasites might determine, in part, the clinical outcomes of human leishmaniasis.

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

Background: The recent devastating earthquake of December 26 in Bam, 2003 created various risk factors; caused a sharp increase in incidence of anthroponotic cutaneous leishmaniasis (ACL) cases and reached to an epidemic proportion. The objective of this study was to evaluate the status of ACL cases five years before the earthquake compared to the cases occurred five years after the earthquake (1999–2008). Methods: Status of disease was assessed retrospectively for the five years before the earthquake and prospectively for the five years after the earthquake. Identification was confirmed by smear and polymerase chain reaction (PCR). Results: The mean annual incidence of ACL for the period from 1999 to 2003 was 1.9 per 1000 comparing to post earthquake period, which was 7.6 per 1000. Most of the infection was in individuals of 20 years (P< 0.0001) in post earthquake era. The anatomical distribution of lesions considerably changed during the two periods. Most of the cases were limited to three zones within the city prior to the earthquake, whereas it was spread throughout different zones after the earthquake. PCR indicated that the CL was due to Leishmania tropica in the city. Conclusion: The results strongly suggest that in natural disasters such as earthquakes various precipitating factors in favor of disease will be created, which in turn provide a suitable condition for propagation of the vector and the transmission of the parasite.

"nBackground: To clinically characterize the cutaneous leishmaniasis and identify the causative parasite species in Mirjaveh, an important geographical region across the border of Iran-Pakistan at Southeast of Iran."nMethods: A number of 116 patients during a year since March 2005 to April 2006, subjected to the study. Clinical infor­mation collected and scrapings were taken from cutaneous lesions and used for microscopic examination, NNN cultivation and kinetoplast DNA-PCR amplification."nResults: The cases comprised of 48 males and 68 females, 84 (72.4%) Iranians and 32 (27.6%) non-Iranians. They aged between 2 months to 68 years with the most affection of children, 0-10 years (55.2%). The patients presented a total of 248 active lesions with an average of 2.14. The ulcers distributed mostly on upper extremity (42.3%) then on face (32.7%), fol­lowed by lower extremity (20.6%) and other parts (4.4%). The majority of ulcers stated to be developed rapidly

Background: Cutaneous leishmaniasis (CL) caused by Leishmania tropica is endemic in Kerman, southeastern Iran. While dogs have long been implicated as the main domestic reservoirs of L. infantum, etiological agent of zoonotic visceral leishmaniasis (ZVL), they can also carry L. tropica infection. The objective of the present study was to determine molecular identity and to evaluate histopathological changes due to CL in dogs in a well-known focus of anthroponotic CL (ACL) in Kerman, southeastern Iran. Methods: This study was carried out in three prospective series from 1994 to 2013 on dogs. Tissue samples were taken from 471 stray dogs. Pathological specimens including skin, spleen, liver and lymph nodes were prepared for paraffin blocks, sectioning and staining for further histopathological examination. PCR amplification of kDNA was performed to identify the causative agent and sequencing. Overall, two out of 471 stray dogs were infected with L. tropica. Hyperplasia of red pulp by the proliferation of histiocytes, lymphocytes, plasma cells and cytoplasm of histiocytes collection of amastigotes was noted. Results: Based on the results of PCR products and sequencing analysis, the parasites isolated from the lesions of two dogs were characterized as L. tropica, corresponding to a band of 830 bp Conclusion: This finding revealed infection with L. tropica in stray dogs in the city and suburbs of Kerman. This information is essential for public health concerns and planning effective future control programs. The role of dogs as potentional reservoir in the epidemiology of ACL needs further investigation. Keywords: Leishmania tropica, Dog, Histopathology, Molecular, Epidemiology, Iran

Leishmania infantum is the most frequent cause of visceral leishmaniasis and L. tropica has been rarely linked to the disease in Iran. In this study, bone marrow aspirates were collected from 10 child patients, suspected with visceral leishmaniasis referred to the Pediatric Wards of Kerman Medical Hospitals, Kerman, Iran during 2002–2011. Leishmania species were identified by using nested PCR in all slides. The PCR samples from nine patients indicated L. infantum as principal causative agent of visceral leishmaniasis and one L. tropica as a minor species.

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 com-poenent, berberine against Leishmania tropica and L. infantum species on in vitro ex-periments. Methods: In this study in vitro antileishmanial activity of various extracts of B. vul-garis also its active compoenent, berberine against promastigote and amastigote stages of L. tropica and L. infantum was evaluated, using MTT assay and in a macro-phage 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