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Background: Kinesin-related gene diversity among strains and species of Leishmania may impact the sensitivity and specificity of serodiagnostic tests for visceral leishmaniasis (VL)., Methods: In this study, we report on the recombinant expression of this novel Iranian Leishmania infantum (MCAN14/47) homologue of rK39 (Li-rK39), in L. tarentolae. The diagnostic potential of the Li-rK39 antigen was evaluated in an ELISA, using sera from 100 VL patients, 190 healthy endemic controls, 46 non-endemic healthy controls and 47 patients with other infections., Results: The results showed a sensitivity of 96% and a specificity of 93.8%. A commercial rK39 immunochromatographic test (ICT) was 90% sensitive and 100% specific on the same cohort., Conclusions: Here, we show that the K39 gene from an Iranian L. infantum isolate is heterozygous as compared to the sequence of the Brazilian L. infantum (former L. chagasi), whose antigen is incorporated in most rK39-based immunochromatographic tests. Therefore, Li-rK39 has the potential to be used as an alternative for VL diagnosis in Iran.

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]

Background: Leishmaniasis is a rare infectious disease observed in subtropical and tropical areas. This disease that demonstrates different clinical characteristics is caused by intracellular Leishmania protozoan. One of the important countries facing the incidence of this infectious disease is Iran. Recently, human immunodeficiency virus–Leishmania coinfection has been indicated in Iran. Case presentation: In the present case report, we show an atypical case of severe visceral leishmaniasis in a 52-year-old Iranian-Arab male with positive human immunodeficiency virus status. Leishmaniasis was detected by node biopsy and subsequently histopathology evaluations and confirmed by molecular methods. Conclusions: The current study was the first report of an atypical case of a patient with Leishmania–human immunodeficiency virus coinfection in southwestern Iran, which was not responsive to the treatment. Therefore, the health authorities should be aware of these reports, which require permanent clinical follow-up of the patients as well as effective treatments. [ABSTRACT FROM AUTHOR]

Background: In recent years, cases of human visceral leishmaniasis (HVL) have been reported in some districts of Golestan Province, northeastern Iran, particularly in rural areas. Recent epidemiological evidence in Leishmania infantum endemic regions of in Iran indicates approximately 50%–80% of seropositive dogs are asymptomatic for Leishmania infection. Objectives: The goal in this study was to determine Leishmania species infecting domestic dogs in Golestan Province, Iran. Methods: Between 2015 and 2016, blood samples were obtained from 100 domestic dogs in rural regions of Golestan Province, northeastern Iran. All samples were tested for anti‐Leishmania antibodies using a direct agglutination test (DAT), and for Leishmania spp. kinetoplast DNA (kDNA) using PCR. Results: Seven (7%) dogs were antibody positive and 25 dogs (25%) were Leishmania spp. DNA positives by PCR positive for leishmaniasis. Four of the seven (71%) antibody‐positive dogs and 19 of the 25 (76%) PCR‐positive dogs were asymptomatic. The rate of infection detected by PCR was significantly higher in male dogs (21/75, 28%) than that in female dogs (4/25, 16%). The ITS1 PCR‐RFLP assay identified the presence of L. infantum, L. tropica or Crithidia spp. in the 25 PCR‐positive samples. Conclusions: The high proportion of asymptomatic dogs in the study areas represent they act as potential reservoirs in the transmission cycle of Leishmania spp. and also Crithidia fasciculata as an emerging agent for the first time. Moreover, our data showed that PCR is a more reliable assay than DAT for detecting Leishmania spp. infection among asymptomatic dogs. [ABSTRACT FROM AUTHOR]

Background: The magnitude of the health problems caused by leishmaniasis has been a major driving factor behind the development and implementation of leishmaniasis control programs by the national authorities in Iran, with a priority for health and environmental management. Such programs are not achievable unless all of the factors leading to the infection, including the parasite's life-cycle, vectors and reservoirs, are recognized. So far in Iran, humans and rodents have been considered the principal reservoirs of Leishmania tropica and Leishmania major, respectively, both associated with cutaneous leishmaniasis (CL), with domestic dogs considered to be the main reservoir for Leishmania infantum, associated with visceral leishmaniasis (VL). The role of other mammals in maintaining the Leishmania parasite has remained unclear. This study aimed to investigate Leishmania infection among livestock in endemic areas of VL and CL in Fars province, southern Iran, using serological and molecular methods. Methods: Blood samples from 181 clinically healthy livestock, including 49 sheep, 114 goats, 16 cattle and two donkeys, were screened to detect Leishmania DNA and anti-Leishmania antibodies using qPCR (quantitative PCR) and the direct agglutination test (DAT), respectively. Four qPCR-positive samples were amplified using the internal transcribed spacer one (ITS1) primers in conventional PCR and sent for directional sequencing. Results: Of the 181 livestock tested, 51 (28.2%) were infected with Leishmania, using serological and molecular methods. Anti-Leishmania antibodies were detected in 70 (38.7%) (95% confidence interval [CI]: 31.5–46.2) and Leishmania DNA in 93 (51.4%) (95% CI: 43.9–58.9) livestock. The identified Leishmania spp. were L. infantum and L. major. Conclusions: The findings of the present study show a relatively high prevalence of Leishmania infection among livestock in endemic areas of the disease, in Fars province, southern Iran. Given the large population of this group of animals and the fact that they live in the vicinity of the main reservoirs of the disease and vectors, it seems that sand flies regularly bite these animals. Further studies are needed to determine the role of livestock in the parasite's life-cycle and the epidemiology of Leishmania infection. [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]

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]

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 mixed-breed shepherd dogs with no anti-Leishmania antibodies and no response to Leishmanin reagent were immunized with 2 doses of alumprecipitated autoclaved L. major (Alum-AML) while BCG and imiquimod (for skin pretreatment) were used as adjuvants. The productions of a few characteristic cytokines of Thelper 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-ß 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 FROM AUTHOR]

It has been documented that the genotypic traits in symptomatic and asymptomatic cases of visceral leishmaniasis (VL) may be different. The current study aimed to find out and compare the genotype and intraspecies diversity of Leishmania Internal Transcribed Spacer 1 (ITS1) from asymptomatic and symptomatic VL cases in southern Iran. Methods. Buffy coat samples from seven VL patients, with clinical signs and symptoms, and seven asymptomatic VL cases, were evaluated in this study. Samples of asymptomatic individuals were obtained from children living in a VL endemic area in southern Iran, while the samples of symptomatic subjects were obtained from patients admitted to hospitals with a diagnosis of VL. DNA was extracted from the buffy coats of the samples and PCR-amplified, targeting the ITS1of Leishmania. The PCR products were sequenced, and the consensus sequences were assembled and multiple-aligned with a set of Leishmania strains retrieved from the GenBank, using Clustal W. The phylogenetic tree was rooted, using MEGAX software, and the diversities based on haplotype and nucleotides, as well as the number of polymorphic sites, were measured using DnaSP v5.0 software. The results of ITS1 sequencing in 5 out of 7 asymptomatic VL cases showed 99.25% to 100% similarity with the Leishmania infantum ITS1 sequence (accessed number: MN648746), and one isolate was considered as just Leishmania sp. In one sample, 99.75% similarity was seen with the ITS1 sequence of Crithidia fasciculata. Of the symptomatic VL patients, the PCR product revealed a 340 bp band corresponding to L. infantum in all of the samples. By analyzing the ITS1 sequences, all seven sequences formed a clade somewhat different from other Leishmania species and considered as Leishmania sp. Haplotype and nucleotide diversity were much more prevalent in symptomatic cases where six haplotypes were seen in the ITS1 of Leishmania from symptomatic patients and only two haplotypes were observed in the samples from asymptomatic cases. The findings of the current study showed that the Leishmania ITS1 from symptomatic VL and asymptomatic cases has significant genetic differences. Besides, infection with Crithidia fasciculata was reported, for the first time, in an asymptomatic case, which deserves further study. [ABSTRACT FROM AUTHOR]

Background: Visceral and cutaneous leishmaniasis are common in some areas of Iran and consider as health problems. Phlebotomus alexandri has been incriminated as a suspected vector for the both form of leishmaniasis. Methods: This study was carried out in 4 western provinces of Iran. Sand flies were collected using sticky traps and light traps from indoor and outdoor resting places. Nested PCR was employed to detect Leishmania parasites among collected sand flies. Results: Seven hundred and twenty two P. alexandri females were collected and pooled in 179 batches. Results of nested PCR showed, out of 9 samples from East Azerbaijan Province, only one sample was infected by Leishmania infantum. Of 34 individual and pooled samples from Kermanshah Province, only one pooled sample was infected with Leishmania major and among 30 individual and pooled samples in Fars Province, five specimens were infected by L. major, L. infantum, Leishmania donovani and Leishmania tropica. Furthermore, out of 108 individual and pooled samples from Khuzestan Province, 10 samples showed infection with L. major and L. infantum. Conclusion: The results of this study showed that P. alexandri is more active in hot zones than in moderate zones and this species may be considered as a permissive species. [ABSTRACT FROM AUTHOR]

Objectives: Leishmania parasites are the causative agents of leishmaniasis. The Visceral leishmaniasis (VL) form of the disease is fatal if not treated in most cases. This study examined the epidemiological aspects of VL in two southwest counties of Iran. Methods: This was a retrospective study of hospitalized patients with a laboratory confirmation of VL from Larestan and Ghiro-Karzin Counties. Results: For Larestan county, a decline in the incidence of VL has been observed from 2004 to 2015. Significantly more males (n = 14) than females (n = 6) were infected with VL in this county (p < 0.05), >95% of cases in children under 5 years of age. In Ghiro-Karzin county, the results were similar to a decline in VL infection from 2004 to 2015, and slightly more males (n = 14) than females (n = 11). Similarly, the majority of the patients infected with VL were children under 5 years old (88%). Conclusion: The results from this study indicate that although the incidence of VL infection has reduced over time, VL was more prevalent in boys under 5 years old, suggesting that more attention to controlling the parasite and its vector are required. [ABSTRACT FROM AUTHOR]

We describe a case of visceral leishmaniasis (VL) due to Leishmania tropica in a 50- year-old Iranian man lived in a VL-endemic area in southwest of Iran. The patient presented with a 3-month history of fever and splenomegaly. Clinical signs and serological findings were suggestive of VL. Spleen biopsy was taken from the patient and intracellular forms of Leishmania amastigotes was seen in Giemsa stained smears. The patient was treated with pentavalent antimonial compound with complete resolution of his systemic signs and symptoms. DNA was extracted from the microscopic slides of the spleen biopsy and the nagt (N-Acetylglucosamine-1- Phosphate Transferase) gene of Leishmania was PCR-amplified. Sequence analysis of the PCR product demonstrated that the case has 99% identity with those of available sequences of L. tropica. Intra-species variation within isolate was 0-0.1%; whereas, inter-species differences of the isolate with those of L. major and L. infantum was significantly higher. [ABSTRACT FROM AUTHOR]

Toxicity and drug resistance against pentavalent antimonials, medications of choice in treatment of leishmaniasis for more than 5 decades, have become important subjects globally. This study was a randomized, open labeled trial that was designed to determine efficacy and safety of IMOD as a novel herbal immunomodulator drug for treatment of canine visceral leishmaniasis (CVL). Twenty healthy mongrel dogs were infected with Iranian strain of L. Infantum amastigotes and randomly divided to 5 groups with four animals for each included on: I: negative control (non-infected) II: Glucantime® III: Glucantime® plus IMOD (immune-chemotherapy) IV: IMOD and V: positive control (non-treated). Physical examination, hematological, biochemical, serological, parasitological, pathological and imaging evaluations were performed pre-/postinterventions every month for 3 months. Comparing with control groups (I&V), immune-chemotherapy group (Glucantime® plus IMOD) showed significantly higher efficacy in resolving the clinical signs and hematobiochemistry factors. Based on our results, using IMOD in combination with meglumine antimoniate (Glucantime®) has significantly improved CVL than the latter drug alone. So, it seems this new herbal medicine is useful as adjuvant therapy for canine visceral leishmaniasis. [ABSTRACT FROM AUTHOR]

Abstract: Leishmania infantum is the causative agent of infantile visceral leishmaniasis (IVL) in the Mediterranean Basin and, based on isoenzyme typing of the parasite isolated from dogs; this parasite was considered to predominate in the all foci of IVL in Iran. However, based on PCR detection and sequencing of parasite Cysteine Protease B (CPB), only one out of seven sandfly infections in Phlebotomus perfiliewi transcaucasicus was found to be L. infantum in the current investigation. The six other infections were haplotypes of Leishmania donovani, the causative agent of anthroponotic visceral leishmaniasis (AVL) in West Africa and India. The deduced amino acid of the L. donovani haplotype was found to be novel and the shortest CPB protein reported within the Leishmania spp. Circulation of both L. donovani and L. infantum by P. perfiliewi transcaucasicus, in addition to previous data indicating its ability to circulate L. tropica, suggests that this species, like other vectors of VL, is a permissive vector. Finding L. donovani infecting P. perfiliewi transcaucasicus in the area demands extensive and intensive typing of natural Leishmania infections in epidemiological investigations in Iran and the Mediterranean Basin in general. [Copyright &y& Elsevier]

• Thi-Qar province considers one of the mixed foci of cutaneous and visceral leishmaniasis, which is considered as widely health problem and is an uncontrolled disease. • The phylogenetic relationship and sequncing analysis of L. tropica isolates have not been studied before in the local area. • The nested PCR assay is considered a suitable method for identifying Leishmania parasites in clinical samples. The Kinetoplast DNA minicircle gene has showed high sensitivity and specificity to characterize the parasite. • The kDNA minicircle sequences analysis results have showed variation of the local isolates that decrease whenever approached from the Iranian border. This means that L.tropica may transmit from Iran. Cutaneous leishmaniasis (CL) is one of wobbling endemic disease in Iraq, that cause intracellular obligate protistan parasite returned to the genus Leishmania. This study is aimed to identify epidemiology of CL, detect the prevalence of Leishmania tropica and find the phylogenetic relationship. The current study was conducted in the main hospitals of Thi-Qar province-south of Iraq for period from November 2018 to October 2019. Nested-PCR was used to amplify kinetoplast minicircle fragments DNA. It was recorded 247 clinical cases with CL, the infections of males were higher than females, while infection rate appeared gradual reduction with age progress. Furthermore, the most CL infections were as single lesions and occurred in December. The infections of upper limbs were high when compared with other body regions. The molecular diagnosis showed L. tropica was more frequently. DNA sequences of kDNA gene of L. tropica showed confirmative genetic detection of local isolates using NCBI-Blast data and phylogenetic tree analysis after comparison with global recorded isolates. The local L. tropica isolates showed genetically closed related to NCBI-Blast L. tropica with accession number AB678350.1. Generally, the analysis of kDNA nitrogen bases sequences showed that all of samples were consistent with those recorded at the NCBI. The kDNA minicircle sequences analysis results showed mismatching of the local isolates decrease whenever approached from the Iranian border. In addition, genetic heterogeneity diagnosis is important for detection of therapy, control and epidemiological studies. [ABSTRACT FROM AUTHOR]

Over the last decade, the incidence of visceral leishmaniasis (VL) has increased in many districts of the province of Fars, in southern Iran. Recent epidemiological reports indicate that asymptomatic human infections with Leishmania infantum (the causative agent of VL throughout the Mediterranean basin) occur more frequently in Iran than was previously believed. Between 2004 and 2006, blood samples were collected from 802 apparently healthy subjects from communities, in the north-west and south-east of Fars province, where VL cases had been recorded. Each of these samples was tested for anti-Leishmania antibodies, in direct agglutination tests (DAT), and for L. infantum kinetoplast DNA, in PCR-based assays. Of the 426 subjects from north-western Fars, eight (1.9%) were found seropositive and 68 (16.0%) PCR-positive. The corresponding values for the 376 subjects from south-eastern Fars were lower, with five (1.3%) seropositive and 32 (8.5%) PCR-positive. Of the 100 PCR-positive subjects, 18 (18.0%) each lived in a household in which there had been a case of VL, and six (6.0%) had had VL themselves (in each case, more than a year before the blood sampling for the present study). Although 21 of the PCR-positives have now been followed-up for at least 18 months, none has developed symptomatic VL. Since positivity in the PCR-based assay probably indicated the presence of L. infantum amastigotes in the peripheral blood of 12.5% of the subjects, it is clear that asymptomatic human carriers of L. infantum are quite common in the study areas and probably act as reservoirs in the transmission of the parasite, to humans and to dogs, by sandflies. [ABSTRACT FROM AUTHOR]

Summary: Leishmania infantum is the causative agent of infantile visceral leishmaniasis (IVL) in the Mediterranean Basin and, based on isoenzyme typing of a few isolates from patients and domestic dogs, this parasite was considered to predominate in the Kaleybar focus of IVL in northwest Iran. However, in the current investigation only one out of five sandfly infections was found to be L. infantum, based on PCR detection and sequencing of parasite internal transcribed spacer (ITS) rDNA infecting Phlebotomus perfiliewi transcaucasicus. The four other infections were of haplotypes of L. tropica, the causative agent of anthroponotic cutaneous leishmaniasis in the Middle East and a parasite occasionally detected in the viscera of dogs and patients in Iran and elsewhere. The widespread distribution of L. tropica in the Kaleybar focus suggests that this parasite is not a transient introduction. Kaleybar has been used for a deltamethrin dog collar intervention to reduce the biting rates of the vectors of L. infantum and this has significantly reduced the incidence of Leishmania infections both in children and the domestic dog, the usual reservoir host of IVL. The implications of finding L. tropica widespread in the heart of the intervention area are discussed. Extensive and intensive typing of natural Leishmania infections is a characteristic of epidemiological investigations in the Neotropics and the current report indicates that this will also be necessary in some regions of the Old World. [Copyright &y& Elsevier]

This report describes the first case of visceral leishmaniasis (VL) resistant to pentavalent antimonials and also the first use of combinational therapy in Iran. The patient was a two-year old boy, from a non-endemic area for leishmaniasis in northern Iran, presenting with pentavalent antimonial resistant VL. Additional treatment with conventional and liposomal amphotericin B was not effective. A complete cure was achieved following a three week treatment with liposomal amphotericin B (5 mg/kg/day for 5 days, then on the 14th and 21st days), allopurinol (25 mg/day for 5 days, then on the 14th and 21st days) and interferon gamma (50 μg/m2 subcutaneously three times weekly). Our results suggest a need for further studies to identify resistant Leishmania species and their susceptibility to different treatment regimens. [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]