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Purpose: Visceral leishmaniasis (VL) is a systemic and parasitic disease that is usually fatal if left untreated. VL is endemic in different parts of Iran and is caused mainly by Leishmania infantum. This study aimed to recognition immunoreactive proteins in amastigote-like and promastigote stages of L. infantum (Iranian strain) by antibodies present in the sera of VL patients. Methods: Total protein extract from amastigote-like and promastigote cells was separated by two-dimensional electrophoresis (2DE). To detect the immunoreactive proteins, 2DE immunoblotting method was performed using different pools of VL patients' sera. Results: Approximately 390 and 430 protein spots could be separated in 2DE profiles of L. infantum amastigote-like and promastigote stages, respectively. In immunoblotting method, approximately 295 and 135 immunoreactive proteins of amastigotes-like reacted with high antibody titer serum pool and low antibody titer serum pool, respectively. Approximately 120 and 85 immunoreactive proteins of promastigote extract were recognized using the high antibody titer sera pool and low antibody titer sera, respectively. Conclusion: The present study has recognized a number of antigenic diversity proteins based on the molecular weight and pH in amastigote-like and promastigote stages of L. infantum. These results provide us a new concept for further analysis development in the field of diagnosis biomarkers and vaccine targets. [ABSTRACT FROM AUTHOR]

Objective: Phlebotomine sand flies are the primary vectors of leishmaniasis; visceral form of disease is transmitted mainly by species belonging to Larroussius and Adlerius subgenus. Species identification of some females in Larroussius subgenus is not easy due to the high similarity. Accurate species identification enables control operations to target only main vectors and increase understanding of ecological needs, bionomic characteristics, and behavioral traits. The purpose of current study was to use two approaches based on internal and external morphological characters to identify the wild caught of female specimens belonged to Larroussius subgenus and investigate Leishmania infection. Methods: Totally, 128 specimens belonging to Larroussius' subgenus were collected from a VL focus in northwestern Iran, species differentiation was done by two approaches proposed in literature: (1) characters of pharyngeal armature, number of Spermathecal segment, length of spermathecal neck, palpal and ascoid formulae; (2) blindly on the basis of the shape of the base of spermathecal duct. Their potential infection to Leishmania was investigated by kDNA-Nested-PCR. Results: The results of species identification were consistent based on two methods. Among three identified species, Phlebotomus perfiliewi was found to be the dominant species, followed by Ph. neglectus and Ph. tobbi. Two specimens of Ph. perfiliewi were found to be infected by Leishmania infantum, which emphasizes the role of this species in VL transmission in study area. Conclusion: It is suggested that combination of the characters used here be considered for species identification of female of Larroussius subgenus to take advantage of maximum characters, especially where species present sympatrically. [ABSTRACT FROM AUTHOR]

Mediterranean type of visceral leishmaniasis (VL) is a zoonotic parasitic infection. Some provinces of Iran are endemic for VL while other parts are considered as sporadic areas. This study aimed to assess a combination of recombinant K26 and rK39 antigens as well as crude antigen (CA), derived from an Iranian strain of L. infantum, compared to direct agglutination test (DAT) for the detection of VL in humans and domestic dogs as animal reservoir hosts of the disease. A combination of rK26 and rK39 antigens and also CA was evaluated using indirect ELISA on serum samples of 171 VL confirmed humans (n = 84) and domestic dogs (n = 87) as well as 176 healthy humans (n = 86) and domestic dogs (n = 90). Moreover, 36 serum samples of humans (n = 20) and canines (n = 16) with other potentially infectious diseases were collected and tested for finding cross- reactivity. The results of ELISA were compared to DAT, currently considered as gold standard for the serodiagnosis of VL. The sensitivity and specificity, positive predictive and negative predictive values were calculated compared to DAT. The positive sera had previously shown a positive DAT titer ≥ 1:800 for humans and ≥ 1:80 for dogs. Analysis was done by MedCalc and SPSS softwares. Using the combination of rK26 and rK39 in ELISA, a sensitivity of 95.2% and a specificity of 93.0% % were found in human sera at a 1:800 (cut-off) titer when DAT-confirmed cases were compared with healthy controls; a sensitivity of 98.9% and specificity of 96.7%% were found at a 1:80 (cut-off) titer compared with DAT. A good degree of agreement was found between the combined rK39 and rK26-ELISA with DAT in human (0.882) and dog serum samples (0.955) by kappa analysis (p < 0.05). The ELISA using the CA test showed 75% sensitivity in human and 93.1% in dog serum samples as well as 53.5% specificity in human and 83.3% in dog,s sera, respectively. The combination of rK26 and rK39 recombinant antigen prepared from Iranian strain of Leishmania infantum showed high accuracy for the serodiagnosis of VL in human and domestic dogs. Further extended field trial with a larger sample size is recommended. [ABSTRACT FROM AUTHOR]

Background: Recent global evidences showed that asymptomatic blood donor carriers of Leishmania infection will appear as a threat for blood transfusions recipients in endemic areas. As yet, there is no appropriate diagnostic procedure for detecting infection of blood donors in blood banks. Subjects and Methods: The present study was aimed to apply various current diagnostic tests among blood donors in an endemic area of visceral leishmaniasis (VL), Ardabil Province, northwestern Iran. Blood samples were gathered from 860 blood donors in endemic areas of the province between 2017 and 2018, at eight blood donation centers. These samples was assessed using microculture, serological (DAT and rK39-ICT) and molecular based (conventional kDNA-PCR and HRM-PCR) tests. Results: Of 860 eligible donors, 24 (2.8%) were seropositive for VL by DAT, and 388 (45%) were positive by kDNA-PCR. Moreover, 19 (19/860) were positive for both of them. Out of 19 subjects, 5.3% (1/19) was positive by rK39-ICT, 10.5% (2/19), and 79% (15/19) were detected positive in microculture and HRM-PCR methods, respectively. Nineteen donors were followed up for 2 years, of which 16 (84.2%) had a serological conversion, and 4 (21%) were positive by kDNA-PCR. The sensitivity of kDNA-PCR, and HRM-PCR procedures in detecting Leishmania parasite was found to be 98.7%, and 79%, respectively. Conclusions: Our findings justify the use of kDNA-PCR as a convenient and sensitive tool for screening subjects with leishmanial latent infection in blood banks at least in endemic regions. In these areas, however, a PCR-based test should be used to validate Leishmania infection among seropositive donors. [ABSTRACT FROM AUTHOR]

Purpose: Leishmaniasis comprises various clinical forms mainly including cutaneous, muco-cutaneous, and visceral leishmaniasis; caused by Leishmania species. Antimoniate is the first-line treatment but some cases showed no response to treatment in the worldwide. In this study, mitogen-activated protein kinase (MAPK) and aquaglyceroporin 1 (AQP1) gene expressions were assessed in treatment failure clinical isolates of Leishmania major. Also, molecular and phylogenic analyses of the mentioned isolates were performed. Methods: Samples were obtained from the patients with suspicious CL referred to the laboratory of Diagnosis Center, Gorgan Province, Iran, from October 2016 to December 2019. Detection and identification of the parasite was performed. The genes expressions of MAPK1 and AQP1 were done using SYBR Green real-time PCR. The AQP1 gene from the isolates with treatment failure was sequenced and analyzed using BLAST and multiple alignments. The phylogenic analysis was done using MEGA7. The statistical analysis was done using SPSS 16.0 by non-parametric Mann–Whitney U test. Results: All clinical isolates were detected L. major. The mean AQP1 and MAPK1 gene expressions in treatment failure isolates were 58.71 and 6.139 fold less than the ones in treatment response isolates, respectively. Based on the AQP1 gene sequence, a nucleotide change of aspartic acid with asparagine at the site 234 was observed. Phylogenic tree analysis showed three groups with the minimum dissimilarity of 0.008 between TF isolates with the standard L. major strains. Conclusion: We showed that MAPK1 and AQP1 may have critical roles in response to antimoniate in clinical isolates L. major in this study. [ABSTRACT FROM AUTHOR]

Background: Despite the broad distribution of leishmaniasis in Iran, there is a little genetic information about the causative agents and epidemiological status of the disease. Genetic diversity of the parasite is suggested to be one of the factors, which influences the clinical manifestations of the disease. In this study, we investigated the genetic variations, population structure, and evolutionary history of Leishmania species from endemic foci of Iran. Methods: Fifty-two isolates from humans, canines, and rodents from different endemic foci of Iran were used to sequence the N-acetyl glucosamine-1-phosphate transferase (Nagt) gene. Phylogenetic and structure analyses were performed to investigate inter- and intra-species diversity of the Leishmania isolates. Results: In total, 10 haplotypes including L. major (n = 6), L. tropica (n = 2), L. infantum (n = 1) and L. turanica (n = 1) were identified across 52 isolates. Haplotype diversity (Hd) ranged from zero for L. infantum and L. turanica to 0.78 ± 0.136 for L. major. This study identified population structure of Leishmania isolates from different geographical regions of Iran. The results of the phylogenetic tree showed 4 distinct clades for each species of Leishmania. In addition, the highest intraspecies diversity was observed among L. major isolates. No correlation was observed between species and geographic distribution of haplotypes. Conclusions: Leishmania isolates were identified at the species level using the Nagt gene, low variation within species indicates conservation of this gene in Leishmania. The results provide knowledge into the evolutionary history of Iranian Leishmania isolates. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In the past few years, the incidence of human visceral leishmaniasis (HVL) has increased in many districts of Fars Province, southwest of Iran, particularly, among communities of nomadic tribes. Recent epidemiological reports in Leishmania infantum endemic regions of Iran indicate that more than 50-70% of seropositive dogs are asymptomatic for Leishmania infection. Between 2004 and 2006, blood samples were collected from 110 domestic dogs from nomadic and rural areas. Each of these samples was tested for anti- Leishmania antibodies, in direct agglutination tests (DATs), and for L. infantum kinetoplast deoxyribonucleic acid (kDNA), in polymerase chain reaction (PCR)-based assays. Of the 110 dogs, 5.5% (6/110) were found seropositive and 23% (25/110) PCR-positive. Four of the six seropositive (67%) and 22 of the 25 PCR-positive (88%) were asymptomatic. The rate of infection in dogs from nomadic communities was higher (27%) than dogs from rural areas (18%). Since positivity in the PCR-based assay indicated the presence of L. infantum amastigotes in the peripheral blood of 23% of the subjects, it is clear that these asymptomatic dogs (88%) are quite common in the study areas and probably act as reservoirs in the transmission of Leishmania parasites, to humans and to other dogs, by sandflies. Moreover, our study showed that application of PCR to buffy coat samples gave a better estimate of the real rate of infection in asymptomatic dogs than DAT. [ABSTRACT FROM AUTHOR]

Abstract  We report a case of Mediterranean visceral leishmaniasis (MVL) associated with acute lymphoblastic leukemia (ALL) from the South of Iran. The patient, a 12-year-old girl, was known to be a case of ALL and had bone pain and prolonged fever on referral. A trephine biopsy revealed a hypocellular marrow with many amastigotes. Moreover, by specific polymerase chain reaction (PCR) on peripheral blood, a 145 bp band corresponding to kDNA from the genus Leishmania was detected and the species was identified as Leishmania infantum using nested PCR. The patient was treated successfully with two courses of amphotericin B plus IFN-γ. To our knowledge this is first report of MVL/ALL from Iran and possibly the world. [ABSTRACT FROM AUTHOR]

A 4-year-old male German shepherd with weight loss, alopecia, skin thickness, cachexia, black diarrhea, abnormal locomotion, and deformed nails was presented to the Veterinary Clinic of Shiraz University. The dog had not responded to antibiotic therapy, vitamin therapy, deworming, and ectoparasite treatment. All joints were intact by radiography. Leukocyte count was markedly raised. The concentrations of triglyceride, cholesterol, and albumin in serum were high. The result of a direct agglutination test for the diagnosis of visceral leishmaniasis was positive (1:320 titer), and a dipstick test (rk39) confirmed visceral leishmaniasis in this case. No parasite was seen in touch smears and culture of spleen or in liver biopsy and bone marrow aspirate. Additionally, the result of polymerase chain reaction using specific primers (RV1, RV2) was negative. [ABSTRACT FROM AUTHOR]

In 2004, a 27-year-old man from Sirjan in Kerman Province (southeastern Iran) presented to Nemazee hospital with high-grade fever, chills, wet cough, abdominal pain, and mild splenomegaly. Anti- Brucella antibodies and anti-leishmanial antibodies were both shown to be present. Leishmania dipstick (rK39) was positive, but no amastigote was observed in the bone marrow. Leishmania kDNA corresponding to L. infantum was detected by nested-polymerase chain reaction (PCR) on liver biopsy and whole blood. These results confirmed co-infection of Leishmania infantum and Brucella spp. [ABSTRACT FROM AUTHOR]