Genetic Diversity and Phylogenetic Study of Leishmania Species in Iran by Multilocus Sequence Typing.

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 multi-locus 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.

Insights into the trypanothione system in antimony-resistant and sensitive Leishmania tropica clinical isolates.

Pentavalent antimonials are the mainstay treatment against different clinical forms of leishmaniasis. The emergence of resistant isolates in endemic areas has led to treatment failure. Unraveling the underlying resistance mechanism would assist in improving the treatment strategies against resistant isolates. This study aimed to investigate the RNA expression level of glutathione synthetase (GS), Spermidine synthetase (SpS), trypanothione synthetase (TryS) genes involved in trypanothione synthesis, and thiol-dependent reductase (TDR) implicated in drug reduction, in antimony-sensitive and -resistant Leishmania tropica isolates. We investigated 11 antimony-resistant and 11 antimony-sensitive L. tropica clinical isolates from ACL patients. Drug sensitivity of amastigotes was determined in mouse macrophage cell line J774A.1. The RNA expression level in the promastigote forms was analyzed by quantitative real-time PCR. The results revealed a significant increase in the average expression of GS, SpS, and TrpS genes by 2.19, 1.56, and 2.33-fold in resistant isolates compared to sensitive ones. The average expression of TDR was 1.24-fold higher in resistant isolates, which was insignificant. The highest correlation coefficient between inhibitory concentration (IC50) values and gene expression belonged to the TryS, GS, SpS, and TDR genes. Moreover, the intracellular thiol content was increased 2.17-fold in resistant isolates compared to sensitive ones and positively correlated with IC50 values. Our findings suggest that overexpression of trypanothione biosynthesis genes and increased thiol content might play a key role in the antimony resistance of L. tropica clinical isolates. In addition, the diversity of gene expression in the trypanothione system and thiol content among L. tropica clinical isolates highlighted the phenotypic heterogeneity of antimony resistance among the parasite population.

A Geomedical Survey: Is There an Association Between Climatic Conditions and Leishmania Species Distribution in Iran During the Years 1999-2021?

PURPOSE: Iran is among the high-risk leishmaniasis regions in the world. WHO recommends the use of GIS as an ideal tool for healthcare authorities to predict the evolution of a disease, delimit the risk of outbreaks and identify critical areas. The aim of this research is to find the association between the main species of Leishmania (L. major, L. tropica, L. infantum) dispersion and climatic variables in Iran. METHODS: All molecular-based reports of leishmaniasis from Iran between 1999 and 2021 were gathered from reliable medical sources. Meteorological data (air and soil temperatures, annual rainfall and humidity) of the country along the study period were obtained from the Iranian Climatological Research Centre. The data concerning species distribution and climatic conditions during this period were moved to a base-map through raster layers using ArcGIS 10.4.1 software. The relationship between parasitological and climatic models was examined using ANOVA. RESULTS: High risk area maps, based on the cut-off thresholds, were generated for Leishmania major, L. tropica and L. infantum. According to the molecular-based reports, the L. major distribution was significantly related to all climatic variables, while L. tropica was merely related to rainfall and humidity, and the L. infantum distribution was significantly associated with rainfall, soil and air temperatures. CONCLUSION: The association between climatic conditions and Leishmania species distribution in Iran has been confirmed. Consequently, both, the relationship between climatic conditions and the geographical distribution of Leishmania species, and the use of GIS to better understand the spatial epidemiology of leishmaniasis, have been reaffirmed.

Recognition of Immunoreactive Proteins in Leishmania infantum Amastigote-Like and Promastigote Using Sera of Visceral Leishmaniasis Patients: a Preliminary Study.

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.

The effects of Leishmania RNA virus 2 (LRV2) on the virulence factors of L. major and pro-inflammatory biomarkers: an in vitro study on human monocyte cell line (THP-1).

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 IL1ß, 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, IL1ß, 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.

Visceral Leishmaniasis in Iran: An Update on Epidemiological Features from 2013 to 2022.

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.

The Association of Human Leucocyte Antigen (HLA) Class I and II Genes with Cutaneous and Visceral Leishmaniasis in Iranian Patients: A Preliminary Case-Control Study.

Background: Leishmaniasis is currently considered a re-emerging or emerging infection based on the geographic region. The outcome of leishmaniasis vastly depends on Leishmania-host interaction. This preliminary study aimed to show the association of human leukocyte antigen (HLA) class I and II genes with healed and non-healed cutaneous leishmaniasis (CL), and symptomatic and asymptomatic visceral leishmaniasis (VL) compared with control groups in Iran. Methods: Ninety-five people, including 31 patients versus 64 individuals in the control group, were enrolled. Among them, 20 patients had confirmed CL based on amastigote observation, 10 had improved CL and 10 non-healed CL. Eleven patients were suffering from confirmed VL based on direct agglutination test (Five asymptomatic and six symptomatic VL cases). Besides, they were residents in an endemic area of VL in the northwest of Iran. To select a control group, it was ensured that they had no history of leishmaniasis. Peripheral blood samples were collected from each patient. After DNA extraction, HLA typing was conducted using polymerase chain reaction - sequence-specific priming (PCR-SSP). Subsequently, data were statistically analyzed by SPSS. Results: There was a statistical relationship between the presence of HLA-A26 and CL, healed CL and the existence of the B38 allele, C1 allele and symptomatic VL, as well as B1.4 allele and asymptomatic VL (P<0.05). Conclusion: This primary finding indicates that several HLA genes have a potential role in the susceptibility of Iranian people to CL and VL.

Proteomics investigation of human sera for determination of postoperative indicators of pulmonary cystic echinococcosis.

BACKGROUND: Cystic echinococcosis (CE)/hydatidosis is an important zoonotic parasitic disease caused by the larval stage of Echinococcus granulosus. The disease is a major health problem all over the world. Finding specific and sensitive biomarkers for follow-up of CE in patients after surgery is essential. Using proteomics methods, the present study aimed to evaluate post-surgical treatment by finding probable biomarker/s in the serum of human lungs CE. METHODS: A total of 24 human sera were tested. These sera included eight confirmed lung/s CE patients sera before surgery (BS), eight sera 12 months post-surgery (12MPS) as well as eight control sera from healthy people. Proteomics methods including 2DE and LC-MS/MS were performed on the specimens followed by bioinformatics analysis. Differentially expressed proteins (DEP) were detected and, separately integrated with protein-protein interaction (PPI) data to construct the PPI network. RESULTS: A total of 171 protein spots were detected in three groups including BS, 12MPS, and control groups; of which a total of 106 DEP have been expressed based on fold changes > = 2 and p-value < 0.05. More analysis was performed and a total of 10 protein spots were selected for identification by mass spectrometry showing the following proteins: APOA1, BGN, SPP2, EAF1, ACOXL, MRPL55, MCTP2, SEPTIN1, B4GALNT1, and ZNF843. Based on centrality parameters of the PPI network (degree and betweenness) five Hub-bottlenecks proteins with significant centrality values were found including APOA1, BGN, SPP2, EAF1, and ACOXL. CONCLUSION: This study showed five proteins as hub-bottleneck proteins; of which APOA1 was more prominent. It can be concluded that a change in expression of this protein in patients' sera could be used as an indicator tool for the achievement of lungs CE surgical therapy.

Effect of Leishmania RNA virus 2 on virulence factors and cytokines gene expression in a human macrophage infected with Leishmania major: A preliminary study.

Cutaneous leishmaniasis (CL) is one of the most important infectious parasitic diseases in the world caused by the Leishmania parasite. In recent decades, the presence of a virus from the Totiviridae family has been proven in some Leishmania species. Although the existence of LRV2 in the Old world Leishmania species has been confirmed, almost no studies have been done to determine the potential impact of LRV2 on the immunopathogenicity of the Leishmania parasite. In this preliminary study, we measured the expression of target genes, including Glycoprotein 63 (gp63), Heat Shock Protein 70 (hsp70), Cysteine Protease b (cpb), Interleukin 1 beta (IL-1ß), IL8 and IL-12 in LRV2 positive Leishmania major strain (LRV2+L. major) and LRV2 negative L. major strain (LRV2-L. major). We exposed THP-1, a human leukemia monocytic cell line, to promastigotes of both strains. After the initial infection, RNA was extracted at different time points, and the relative gene expression was determined using a real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Findings showed that the presence of LRV2 in L. major was able to increase the expression of gp63, hsp70, and cpb genes; also, we observed lower levels of expression in cytokine genes of IL-1ß, IL-8, IL-12 in the presence of LRV2+, which are critical factors in the host's immune response against leishmaniasis. These changes could suggest that the presence of LRV2 in L. major parasite may change the outcome of the disease and increase the probability of Leishmania survival; nevertheless, further studies are needed to confirm our results.

Assessment of genetic markers for multilocus sequence typing (MLST) of Fasciola isolates from Iran.

BACKGROUND: Several markers have been described to characterise the population structure and genetic diversity of Fasciola species (Fasciola hepatica (F. hepatica) and Fasciola gigantica (F. gigantica). However, sequence analysis of a single genomic locus cannot provide sufficient resolution for the genetic diversity of the Fasciola parasite whose genomes are ∼1.3 GB in size. OBJECTIVES: To gain a better understanding of the gene diversity of Fasciola isolates from western Iran and to identify the most informative markers as candidates for epidemiological studies, five housekeeping genes were evaluated using a multilocus sequence typing (MLST) approach. METHODS: MLST analysis was developed based on five genes (ND1, Pepck, Pold, Cyt b and HSP70) after genomic DNA extraction, amplification and sequencing. Nucleotide diversity and phylogeny analysis were conducted on both concatenated MLST loci and each individual locus. A median joining haplotype network was created to examine the haplotypes relationship among Fasciola isolates. RESULTS: Thirty-three Fasciola isolates (19 F. hepatica and 14 F. gigantica) were included in the study. A total of 2971 bp was analysed for each isolate and 31 sequence types (STs) were identified among the 33 isolates (19 for F. hepatica and 14 for F. gigantica isolates). The STs produced 44 and 42 polymorphic sites and 17 and 14 haplotypes for F. hepatica and F. gigantica, respectively. Haplotype diversity was 0.982 ± 0.026 and 1.000 ± 0.027 and nucleotide diversity was 0.00200 and 0.00353 ± 0.00088 for F. hepatica and F. gigantica, respectively. There was a high degree of genetic diversity with a Simpson's index of diversity of 0.98 and 1 for F. hepatica and F. gigantica, respectively. While HSP70 and Pold haplotypes from Fasciola species were separated by one to three mutational steps, the haplotype networks of ND1 and Cyt b were more complex and numerous mutational steps were found, likely due to recombination. CONCLUSIONS: Although HSP70 and Pold genes from F. gigantica were invariant over the entire region of sequence coverage, MLST was useful for investigating the phylogenetic relationship of Fasciola species. The present study also provided insight into markers more suitable for phylogenetic studies and the genetic structure of Fasciola parasites.

Amphotericin B-Loaded Extracellular Vesicles Derived from Leishmania major Enhancing Cutaneous Leishmaniasis Treatment through In Vitro and In Vivo Studies.

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.

In Vitro Study on Four Types of Commercial Lectins on Leishmania infantum, L. major and L. tropica with Stage-Specific Binding and Leishmania Species Identification.

Background: We aimed to verify the susceptibility of Leishmania infantum, L. major and L. tropica, to commercial lectins in order to identify the three Leishmania species. Methods: The degree of agglutination was determined both macroscopically and microscopically and was scored negative (-) to positive (from 1+- 4+) based on their percentage of agglutination. Results: Jacalin and UEA-1 were capable of agglutination of L. infantum isolates in both logarithmic and stationary phases at a concentration of 1000 µg/ml (100%). L. tropica isolates showed agglutination with the lectin UEA-1 in both logarithmic and stationary phases (62.5% and 87.5%). L. major and L. tropica showed 75% agglutination with lectin Jacalin in both logarithmic and stationary phases. L. tropica isolates showed 25% agglutination with the lectin WGA in the logarithmic phase. L. infantum, L. major and L. tropica isolates showed 25, 12.5 and 37.5% agglutination in the stationary phase, however, did not show agglutination in logarithmic phases. L. major isolates showed 12.5% agglutination with the lectin PHA in the stationary phase, however, were incapable of agglutination with the L. tropica and L. infantum in both logarithmic and stationary phases. Conclusion: Despite the fact, that JCA and I-UEA lectins were not able to completely separate L. infantum, L. major and L. tropica. WGA lectin and PHA lectin can help in separating the species of Leishmania parasites.

Development of an Indirect Fluorescent Antibody (IFA) Assay for the Detection of Leishmania RNA Virus 2 (LRV2) in Leishmania Parasites.

Background: Detection of Leishmania RNA virus (LRV) in Old World Leishmania species and their possible role in the disease prognosis requires sensitive and specific methods, preferably independent of the viral genome. We aimed to develop an indirect immunofluorescence antibody (IFA) assay to detect LRV in the Old World Leishmania parasites. Methods: Clinical samples were collected from 86 cutaneous leishmaniasis (CL) patients in different endemic areas of CL in Iran, during 2017-2019. For antibody preparation, the viruses were obtained from sediment of an LRV-infected L. major culture-using freeze and thaw cycles followed by gradient cesium chloride centrifugation. The purified viruses were used to immunize a male 3-4 months rabbit. Various dilutions of the LRV-immunized rabbit's serum and a conjugated antibody were deployed to detect LRV in 48 isolates by IFA assay. Results: LRV virus was detected in four of the 48 CL cases using IFA method. Amplification of a partial fragment of RNA-dependent RNA polymerase (RdRp) gene from the isolates confirmed the IFA results. In phylogeny, the generated RdRp sequences from four isolates were grouped with the other Old World LRVs, but separate from L. aethiopica LRVs, which appeared as a highly supported distinct clade. Conclusion: Further optimization of this approach to detect the LRV directly in lesion scrapings can make it a more reliable tool for field studies and disclosing the virus's possible role in disseminating and unusual clinical features.

Identification and phylogenic analysis of Leishmania species among patients and reservoir hosts based on N-acetylglucosamine-1-phosphate transferase gene in Central Iran.

Leishmaniosis, a vector-born disease that infects humans and other vertebrates, is the result of infection with Leishmania species belong to the family Trypanosomatidae. The present study was performed to determine the status of cutaneous leishmaniosis in Isfahan province. Samples were taken from the margin of skin ulcers of patients with suspected CL referred to the medical health centers in Isfahan province. Also, ear and snout samples were taken from the rodents. In total, 85 parasitologically positive samples were subjected to the PCR-RFLP method based on the nagt gene for identification of Leishmania species, also 11 samples were subjected to sequencing and phylogenetic analysis. For all positive samples, a 1450-1460 bp band of the nagt gene was amplified in PCR method. The digestion pattern of ACC1 enzyme in 79 of patients indicated L. major and in one sample was similar to L. tropica. Four rodent reservoirs distingue as L. major and one sample as L. turanica. Phylogenetic analysis confirmed the species identification and three haplotypes were reported. The results of the current study showed that L. major is the predominant species of Leishmania parasites in Isfahan province and the main reservoir of CL is Rhombomys opimus. Also, the nagt gene is a useful and practical marker for determining different species of Leishmania parasites as well as their phylogenetic analysis.

Leishmania RNA virus 2 (LRV2) exacerbates dermal lesions caused by Leishmania major and comparatively unresponsive to meglumine antimoniate treatment.

PURPOSE: The present study investigated the possible role of Leishmania RNA virus 2 (LRV2) in the severity of dermal lesions and treatment failure due to Leishmania major. METHODS: The drug susceptibility of 14 clinical isolates of L.major, including resistant (n = 7) and sensitive (n = 7) isolates, was checked in the J774A.1 macrophage cell line. The presence of LRV2 among isolates was investigated by the RdRp gene and semi-nested PCR. Moreover, 1 × 106 sensitive L. major LRV2+ and LRV2- promastigotes were inoculated subcutaneously into the base tails of the 40 BALB/c mice divided into 4 groups (n = 10 in each group), including clinical LRV2+, clinical LRV2-, positive control LRV2+ and negative control LRV2-. The groups were infected with a unique isolate. The lesion size and parasite burden were evaluated. RESULTS: Sensitive and resistant isolates were determined by the drug susceptibility method. A higher presence of LRV2 was observed among MA-resistant isolates (6/7) compared with susceptible isolates (4/7), which was not statistically significant (P = 0.237). On the other hand, a comparison of the lesion sizes between the LRV2+ and LRV2- BALB/c mice groups revealed that the mean size of the lesion in the LRV2+ groups was significantly higher than the LRV2- (P = 0.034). In the same direction, there was an increased parasite burden in mice inoculated with LRV2+ groups compared with the LRV2- BALB/c mice groups (P = 0.002). CONCLUSIONS: Our findings showed that the presence of LRV2 could be one of the factors contributing to exacerbating CL. Although we found a higher presence of LRV2 in the resistant isolates, it seems that further investigations are recommended to determine the detailed association between lesions' aggravation and being comparatively unresponsive to treatment.

Successful Isolation of Leishmania RNA Virus (LRV) from Leishmania major in a Cutaneous Leishmaniasis Focus in Central Iran: An Update on Cases.

PURPOSE: Cutaneous leishmaniasis (CL) is a major vector-borne disease that affects people globally, including Iran. Different factors are associated with leishmaniasis pathogenicity; recently, a link of the possible relationship between Leishmania RNA Virus (LRV) and disease severity was proposed, especially in the New World leishmaniasis (NWL). This study was aimed to investigate the presence of LRV2 in Leishmania isolates in Aran o Bidgol, Isfahan province. METHODS: Samples were collected from 110 CL-suspected patients referred to the health center. In this study, we aimed to investigate CL cases (parasitologically and clinically), identify Leishmania species (by ITS1-PCR-RFLP), and finally detection of LRV2 (by RdRp-semi-nested PCR). RESULTS: Parasitological methods showed 60 positive cases, based on the HaeIII enzyme restriction profile, 59 cases were caused by L. major and 1 case by L. tropica. Our project is the first study on LRV2 isolation in Aran o Bidgol city and the LRV was successfully detected from a single L. major isolated in a women's hand lesion. Using BLAST, 94.8-100% similarity was observed in the RdRp sequence of current LRV isolate with those available in GenBank from Iran or overseas. CONCLUSION: L. major was the main cause of CL in Aran o Bidgol, although L. tropica is also present in a much lower proportion in the area. This is the first report on the presence of LRV2 in Aran o Bidgol and the fifth in Iran.

Validation of a mixture of rK26 and rK39 antigens from Iranian strain of Leishmania infantum to detect anti-Leishmania antibodies in human and reservoir hosts.

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.

Molecular identification of Leishmania RNA virus in cutaneous leishmaniasis patients and rodent reservoirs in Isfahan province, Iran.

Leishmania RNA virus (LRV) is a double-strand RNA virus that was first detected in members of the Leishmania viannia in the New World. The present study aimed to investigate the presence of LRV in the Leishmania species isolated from cutaneous leishmaniasis (CL) patients and rodents as reservoirs in Isfahan province an old zoonotic CL focus, center of Iran. Totally, 85 samples were collected from CL patients (n = 80) and rodent reservoirs (n = 5) from different regions of Isfahan province. Species identification was determined using the PCR-RFLP method. Viral dsRNA was extracted and for observation of 5.3 kb dsRNA on an agarose gel. The presence of LRV was surveyed using the Semi-nested PCR method. For phylogenetic analyzes, 6 samples of 13 isolates were sequenced and a phylogenetic tree was drawn by MEGA7 version 7.0.26. Of 80 Leishmania isolates recovered from the patients with CL, 79 and only one were identified as L. major and L. tropica, respectively. Also, the PCR assays detected four L. major and one L. turanica in five assessed Rhombomys opimus as the rodent reservoirs. LRV was detected only in Leishmania species isolated from 13 species of 85 (15.3%) CL including (L. major, n = 12) and (L. tropica, n = 1). Phylogenetic analysis showed that they were belonged to LRV2 and had the highest similarity with Iranian reference LRV2 in GenBank. Our results showed that the LRV2 was present in cutaneous Leishmania species in Isfahan province is the most historical and touristic province of Iran. In the study LRV was not reported from rodent reservoirs, it may be due to the small sample size. Phylogenetic analysis of current sequences demonstrated that these isolates belong to the registered LRV2 of the Old World.

Leishmania spp. infection in Rhombomys opimus and Meriones libycus as main reservoirs of zoonotic cutaneous leishmaniasis in central parts of Iran: Progress and implications in health policy.

Rodents from the subfamily Gerbillinae are the principal reservoir of cutaneous leishmaniasis (CL) caused by Leishmania major in the center and northeast of Iran. This study was conducted to determine both naturally occurring Leishmania infection rates and the distribution of Leishmania species in the central parts of Iran during 2019-2020. In this regard, presence of Leishmania parasites were confirmed by microscopic examination and the species were identified by nested-PCR using the Internal Transcribed Spacer2- Ribosomal DNA (ITS2-rDNA). Finally, some samples were sequenced and, the blast analysis of L. major samples, showed a 92.45-100% homology to the L. major sequence. Of the 181 wild gerbils collected (Rhombomys opimus=157 and Meriones lybicus=24), 88 (48.6%) tested positive for Leishmania sp. by microscopic examination whereas 162 (89.5%) were positive by nested-PCR. Of the 162 infected gerbils, 103 showed single strain infections (30 L. major, 28 L. gerbilli and 45 L. turanica), 43 showed dual infections with only the non-human species (L. gerbilli and L. turanica), and 16 were mixed infections of L. major and L. turanica (n = 14) or L. gerbilli (n = 2). All single or mixed L. major infections were detected in gerbils from areas with reports of human CL during the last decade. These findings suggest that Rhombomys opimus and Meriones libycus have a potential role in the maintenance of human and non-human transmission of Leishmania species in the CL foci.

Overexpression of Iron Super Oxide Dismutases A/B Genes Are Associated with Antimony Resistance of Leishmania tropica Clinical Isolates.

Background: Pentavalent antimonial has been a drug of choice against leishmaniasis, despite the emergence of treatment failure. Identification of resistance markers is urgently needed to design new therapeutic strategies. Iron-Superoxide dismutases (Fe-SODs) are antioxidant enzymes contributing to detoxify reactive oxygen species to prevent a cell from oxidative stress. Since antimonial compounds induce oxidative stress, in this survey, the expression of SOD genes was investigated to identify their expression pattern in clinical resistant isolates. Methods: This cross-sectional survey was done in Mashhad City, northeast of Iran during 2014 to 2019. The RNA expression level of mitochondrial (SODA) and glycosomal (SODB) superoxide dismutase was investigated in 25 antimony responsive (n=15) and unresponsive (n=10) anthroponotic cutaneous leishmaniasis (ACL) patients. Total RNA extraction and cDNA synthesis, the qRT-PCR approach was utilized to investigate the relative RNA expression level. Results: The transcript level of SODs was over-expressed in the most resistant isolates. Gene expression analysis demonstrated the over-expression of SODA and B by a factor of 3.8 and 4.81, respectively, in resistance isolates vs. sensitive ones. Conclusion: Aberrant expression of SODA/B in unresponsive parasites could potentially implicate in detoxifying antimony-induced oxidative stress. Moreover, SODs might be considered as potential predictive markers of the response to antimonials in ACL patients in endemic areas.