Genetic Diversity and Phylogenetic Analysis of the Iranian Leishmania Parasites Based on HSP70 Gene PCR-RFLP and Sequence Analysis

Despite the broad distribution of leishmaniasis among Iranians and animals across the country, little is known about the genetic characteristics of the causative agents. Applying both HSP70 PCR-RFLP and sequence analyses, this study aimed to evaluate the genetic diversity and phylogenetic relationships among Leishmania spp. isolated from Iranian endemic foci and available reference strains. A total of 36 Leishmania isolates from almost all districts across the country were genetically analyzed for the HSP70 gene using both PCR-RFLP and sequence analysis. The original HSP70 gene sequences were aligned along with homologous Leishmania sequences retrieved from NCBI, and subjected to the phylogenetic analysis. Basic parameters of genetic diversity were also estimated. The HSP70 PCR-RFLP presented 3 different electrophoretic patterns, with no further intraspecific variation, corresponding to 3 Leishmania species available in the country, L. tropica, L. major, and L. infantum. Phylogenetic analyses presented 5 major clades, corresponding to 5 species complexes. Iranian lineages, including L. major, L. tropica, and L. infantum, were distributed among 3 complexes L. major, L. tropica, and L. donovani. However, within the L. major and L. donovani species complexes, the HSP70 phylogeny was not able to distinguish clearly between the L. major and L. turanica isolates, and between the L. infantum, L. donovani, and L. chagasi isolates, respectively. Our results indicated that both HSP70 PCR-RFLP and sequence analyses are medically applicable tools for identification of Leishmania species in Iranian patients. However, the reduced genetic diversity of the target gene makes it inevitable that its phylogeny only resolves the major groups, namely, the species complexes.

Inter-and intraspecific variations of leishmania strains isolated from patients with cutaneous and visceral leishmaniases in pars province, south of Iran

Background: Cutaneous and visceral leishmaniases are present in Fars Province in the south of Iran. The current study aimed to evaluate the inter- and intragenic diversities of Leishmania species isolated from patients with leishmaniasis in Fars Province, using PCR-based analyses and DNA sequencing of the N-acetylglucosamine-1-phosphate transferase [nagf] gene

Methods: Clinical samples were taken from the skin lesions of 120 individuals with clinical suspicion of cutaneous leishmaniasis [CL] referred to the major health centers of Shiraz. Along with microscopic examination, a part of each sample was used for in vitro cultivation. DNA was extracted from the cultured parasites and the nagt gene was PCR-amplified. For RFLP analysis, the PCR product of the nagt gene was digested with the Accl restriction enzyme. Moreover, the PCR products of 23 isolates were sequenced and analyzed, using MEGA5

Results: From the 120 patients with clinical suspicion of CL, 110[91.7%] cases were found to be positive by direct microscopy while 77[64.1%] of the cultures were positive. Digestion of the PCR product with the Accl restriction enzyme detected L. major in 57 out of the 77 [74.1%] and Z. tropica, in 20 out of the 77 [25.9%] cases with CL. Phylogenetic analysis grouped the Leishmania isolates into 3 main clades, representing L. major, L. infantum, and L. rop/ca,encompassing 2, 2, and 2 haplotypes, respectively. Within the clades, the L. tropica intraspecies divergence was more pronounced in L. major

Conclusion: The findings of this study demonstrated that the causative agent of CL in Fars Province was mainly L. major and that there was considerable heterogeneity between the Leishmania species and also within the L. major isolates

[Cutaneous leishmaniasis in suspected refereed patients to health centers of varamin and determination of sand flies species during 2012-2013]

Leis mania is one of the most important diseases in Iran, with high prevalence in some part of country including, Tehran province this study was aimed to investigate the cutaneous lesions of patients studies was also referred to different 1aporatory of Health center of Vermin [a city In Tehran province], sand flies species m selected area. In this cross sectional study demographic data was collected by special questionnaire. Smears of suspected patients to stained and examined by microscope Sand flies were collected by sticky traps CDC light haps and aspirators, female specimens mere mounted and identified by diagnostic keys at species level Leis mania parasites were observed m 56 820 of cases. Disease was more common among male [64%] the majority of patients [3094] aged between 25-39 year old. Most lesions [50%] observed in hands and legs of patients, 76% of affected people had lust one lesion Half of them had a history of travel to endemic areas. More than 2500 sand flies captured during this study; among them 1100 female specimens were mounted and identified The prevalent spicies was Sergentomyia spp [65/9%], the second was papatasi [33/20%]. It should be mentioned that Ph sergenti, Ph alexandri and Ph caucasicus group also were identified in lower percentage According to current results and the status of the studied region including it seems that [Agricultural Activities animal husbandry and migration] cutaneous leishmaniasis could be a health threatening problem

Visceral Leishmaniasis in Rural Areas of Alborz Province of Iran and Implication to Health Policy

Visceral leishmaniasis (VL) or kala-azar mainly affects children in endemic areas. This study was conducted to determine the seroprevalence of VL using direct agglutination test (DAT) in children living in rural districts of Alborz Province located 30 km from Tehran capital city of Iran. Multi-stage cluster random sampling was applied. Blood samples were randomly collected from 1,007 children under 10 years of age in the clusters. A total of 37 (3.7%) of the studied population showed anti-Leishmania infantum antibodies with titers of > or =1:800. There was a significant association between positive sera and various parts of the rural areas of Alborz Province (P or =1:3,200 indicated kala-azar clinical features and treated with anti-leishmaniasis drugs in pediatric hospital. The findings of this study indicated that Leishmania infection is prevalent in rural areas of Alborz Province. Therefore, it is necessary to increase the awareness and alertness among physicians and public health managers, particularly in high-risk rural areas of the province in Iran.

rate of plasmodium vivax infectivity within gloucose-6-phosphate dehydrogenase [G6PD] deficient individuals in hor-mozgan province, Iran

One of the most important enzymatic disorders that interact with malaria is deficiency of G6PD [Gloucose-6-phosphate dehydrogenase]. This enzyme protects red blood cells from hydrogen peroxide and other oxidative damages. Distribution of this enzyme deficiency usually accompanies with low level distribution of malaria disease in most malarious areas. So this hypothesis may be considered that the G6PD deficiency could be protective against malaria. Totally 160 samples were taken from vivax malaria infected and non-infected individuals. Preparing blood smears and quantitative test for G6PD deficiency were employed for all of the samples. To ensure accuracy of the malaria in negative samples besides using microscopical examination, semi-nested multiplex PCR was also performed for the two groups. In microscopical examination 36 and 124 samples were vivax malaria positive and negative respectively. Out of 36 P.vivax positive cases 3 [8.3%] cases were detected to be G6PD deficient versus 30 [24.2%] cases out of 124 P. vivax negative cases. The results showed a significant differentiation between P. vivax positive and P. vivax negative cases in the rate of G6PD deficiency [3/36 in positive cases versus 30/124 in negative cases] [P<0.05]. vivax malaria positive individuals with G6PD deficiency showed too mild symptoms of Malaria or even asymptomatic

efficacy of residual chlorine content on the control of Legionella spp. in hospital water systems

Outbreaks of legionellosis may be a side effect of institution-water treatment. However, the long-term outcomes and the predictive factors of Legionella prevalence in such systems have still not been fully studied. This study was therefore conducted to investigate the prevalence of Legionella spp. and to evaluate the role of bacteriological water quality parameters on its prevalence and removal in hospital water systems. A total of 45 samples were collected from distinct sites at seven hospitals in Tehran, Iran. The prevalence of this bacterium was assayed through a sensitive and specific technique for DNA detection using PCR. Multivariable stepwise regression analysis was used to explore the independent effects of the baseline factors on the incidence of Legionella. Two positive samples were also identified for species by DNA sequencing. Legionella were detected in 31.1% of samples. Showerheads and cold-water taps were the most and the least contaminated sources with 55.3 and 9 percent positive samples, respectively. Total mean of residual chlorine was 0.38 mg/L, with the peak value of 1.7 mg/L. Legionella detection was proportional to the residual chlorine content of water and the results indicated that residual chlorine content is a critical factor in the incidence and proliferation of Legionella [r=-0.33]. The prevalence of Legionella also coincided with the prevalence of HPC and amoeba cysts. The high positive rate of Legionella colonization shows that hospital-acquired legionellosis might be under diagnosed in studied hospitals. Further, Legionella colonization is independent of the type of water, system characteristics and of preventive maintenance measures

Clinical features and laboratory findings of visceral leishmaniasis in children referred to children medical center hospital, Tehran, Iran during 2004-2011

Visceral leishmaniasis [VL] is one of the most important parasitic diseases endemic in northwestern and southern areas of Iran. The aim of the present study was to review the records of children hospitalized with VL in order to characterize the clinical features of children as well as laboratory finding in Children Medical Center Hospital, Tehran, Iran. The medical records of all children with a final diagnosis of VL were reviewed from 2004 to 2011. Demographic, clinical information, laboratory finding and treatment were considered. A total number of 34 children with confirmed VL through 2004-2011 were included in the study. The most prevalent sign and symptoms were fever [97.1%], pallor and weakness [97.1%], appetite loss [61.8%], splenomegaly [97.1%] and hepatomegaly [88.2%]. The most frequent laboratory abnormalities were hematological including anemia [97.1%], thrombocytopenia [91.2%] and leukopenia [67.6%]. Direct agglutination test [DAT] was performed in 23 cases and all of them showed anti-Leishmania antibodies with titers of >/= 1: 3200. In addition, 90% of patients had positive rK39 results. Identification of Ieishmania in the aspirates of the bone marrow was found in 83.3% of patients. Regional surveillance system in order to monitoring of leishmaniasis trends as well as detection of new emerging foci is recommended

Imported lymphatic filariasis in an indian immigrant to Iran

Lymphatic filariasis [LF], a nematode disease transmitted by arthropod vectors, is repeatedly reported in immigrant population. This disease is not endemic in Iran; however, different species of mosquitoes, capable of transmission of parasite mi-crofilaria, are distributed in the country. Hereby, incidental detection of an imported case of LF due to Wuchereria bancrofti in an Indian worker in Iran is reported. Identification of the case was performed based on morphological and morphomet-rical characteristics of microfilaria and PCR sequencing

Downregulation of calcineurin gene is associated with glucan-time[registered sign] resiatance in leishmania infantum

Pentavalent antimonials are the first line drugs for the treatment of leishmaniasis. Unresponsiveness of Leishmania spp. to antimonial drugs is a serious problem in some endemic areas. Investigations on molecular mechanisms involved in drug resistance are essential for monitoring and managing of the disease. Cal-cineurin is an essential protein phosphatase for number of signal transduction pathways in eukaryotic cells and it has a mediated role in apoptosis. This study aimed to determine of biomarker[s] in Glucantime[registered sign] resiatance strain of L. infan-tum. We used cDNA amplified fragment length polymorphism [cDNA-AFLP] and real time-RT PCR assays to compare gene expression profiles at the mRNA levels in resistant and susceptible L. infantum field isolates. The cDNA-AFLP results showed downlegulation of calcineurin in resis-tant isolate in comparison with susceptible one. Significant downregulation of cal-cineurin [0.42 fold] [P<0.05] was found in resistant isolate compared to susceptible one by Real time-RT PCR. This is the first report of calcineurin implication in Glucantime[registered sign] drug resistance of field [natural] isolate of L. infantum. Downregulation of calcineurin could protect parasites from antimonial-induced apoptosis

Overexpression of Ubiquitin and Amino Acid Permease Genes in Association with Antimony Resistance in Leishmania tropica Field Isolates

The mainstay therapy against leishmaniasis is still pentavalent antimonial drugs; however, the rate of antimony resistance is increasing in endemic regions such as Iran. Understanding the molecular basis of resistance to antimonials could be helpful to improve treatment strategies. This study aimed to recognize genes involved in antimony resistance of Leishmania tropica field isolates. Sensitive and resistant L. tropica parasites were isolated from anthroponotic cutaneous leishmaniasis patients and drug susceptibility of parasites to meglumine antimoniate (Glucantime(R)) was confirmed using in vitro assay. Then, complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) and real-time reverse transcriptase-PCR (RT-PCR) approaches were utilized on mRNAs from resistant and sensitive L. tropica isolates. We identified 2 known genes, ubiquitin implicated in protein degradation and amino acid permease (AAP3) involved in arginine uptake. Also, we identified 1 gene encoding hypothetical protein. Real-time RT-PCR revealed a significant upregulation of ubiquitin (2.54-fold), and AAP3 (2.86-fold) (P<0.05) in a resistant isolate compared to a sensitive one. Our results suggest that overexpression of ubiquitin and AAP3 could potentially implicated in natural antimony resistance.

effect of otostegia persica in combination with chloroquine on chloroquine-sensitive and chloroquine-resistant strains of plasmodium berghei using in-vivo fixed ratios method

Malaria is one of the worldwide parasitic diseases which threaten the life of hundreds of millions of people at the malarious areas each year. The emergence of chloroquine-resistant strains of Plasmodium falciparum in most of the malarious areas has encountered the relevant countries with some difficulties about treating the acute cases of the disease particulary if the monotherapy regimen has been used. Because of many advantages for the combination therapy, the effectiveness of chloroquine [CQ] and Otostegia persica [OP], a medicinal plant in combination form, was tested against the chloroquine-sensitive and chloroquine-resistant strains of Plasmodium berghei in sourian mouse using in-vivo adapted fixed ratios method in this study. At the first step, ED[50]s [50% effective dose] of chloroquine and O. persica against both CQ-sensitive and CQ-resistant strains of P. berghei were calculated using in-vivo test in the mice. Ratios of 0, 10, 30, 50, 70, 90 and100% from each ED[50] were prepared and contrarily combined together to make the following fixed ratios of 0/100, 10/90, 30/70, 50/50, 70/30, 90/10, and 100/0 of CQ/OP and the parasites were exposed to the combined ratios. Determination of ED[50]s showed 1.1 mg/Kg and 2.4 mg/Kg of mouse body weight for chloroquine in CQ-sensitive and CQ-resistant strains respectively and 450 mg/Kg for O. persica in both strains. The results also showed that the combinations of "50% CQ + 50% OP", "30% CQ + 70% O.P" and "70% CQ + 30% OP" were more effective than other combinations against CQ-sensitive strain. The fixed ratio combinations of chloroquine and O. persica showed an additive in CQ-resistant strain. Toxicity consideration showed no toxic effect of the combinations on the mice. Otostegia persica potentiated the effectiveness of chloroquine against the chloroquine-sensitive strain of P. berghei but not on chloroquine-resistant P. berghei. Moreover, the greatly modified fixed ratios method in this study can be considered as useful methods for in-vivo combination tests in murine malaria parasites

Comparison of leishmanin skin test and direct smear for the diagnosis of cutaneous leishmaniasis

Cutaneous leishmaniasis [CL] is an endemic disease in some parts of Iran and it has high morbidity in some areas of the country. The disease is detected by parasitological examinations including direct microscopic and culture tests. This comparative study aimed to evaluate the relationship between positivity of the leishmanin skin test [LST], microscopically examination and clinical forms of CL for the diagnosis of human cutaneous leishmaniasis. This study was performed on 66 patients suspected to cutaneous leishmaniasis. CL cases evaluated by both microscopical examination and leishmanin skin test. In this study, 1 ml of leishmanin fluid [lot no 121/1, produced in Pasteur institute of Iran] was injected intradermally in forearms of all patients and indurations were measured after 72 hours. Induration of 5 mm and higher was considered as positive results. The collected data were statistically analyzed using the SPSS version 13.5. From 66 CL patients who were evaluated in this study, 30 [45.5%] of them had positive microscopically results while 28 [42/4%] of them had showed positive leishmanin skin test [>/=5mm diameter]. From 36 [54.5%] patients who had negative microscopical examination, only 6 [16/6%] of them had positive leishmanin skin test. The agreement between two tests was 87.9% by kappa analysis [p< 0.01]. In attention to the results of this study, it seems the LST would be used as an alternative diagnosis method when there is a strong clinical doubt to cutaneous leishmaniasis even there is no parasite in direct smear

Molecular analysis of A2-genes encoding stage-specific s antigen-like proteins among isolates from iranian cutaneous and visceral leishmaniasis

Leishmania can lead to a broad spectrum of diseases, collectively known as leishmaniasis. The A2 gene/ protein family could be one of the most eligible candidate factors of virulence in visceral leishmaniasis [VL]. The previous results confirmed that in Leishmania infantum, several A2 proteins are abundantly expressed by the amastigote, but not the promastigote stage. As there are no data available on the pattern of A2 gene / protein in Iranian Leishmania isolates of either cutaneous leishmaniasis [CL] or VL; the current study aimed to investigate molecular analysis of A2 gene in leishmania species among field isolates of Iran. An A2 gene was identified by sequencing of crude PCR products resulting from 20 samples of CL and 10 samples of VL isolates from Iranian patients. The results indicated the A2 gene in CL is only a single copy of 153 bp encoding a protein of 51 amino acids, as opposed to A2 of VL species with multi-copy genes of varying length. A2 sequences in Iranian L. major strains represented a homology with stage-specific S antigen-like protein [A2] of L major and L infantum. Moreover, A2 sequences in Iranian L. tropica strains have homology with A2 protein of L. major and L. tropica. It is concluded that A2 is an antigen candidate for vaccine development and diagnosis purposes and that A2 sequences are conserved among field isolates