Highlighting the interplay of microRNAs from Leishmania parasites and infected-host cells.

Leishmania parasites, the causative agents of leishmaniasis, are protozoan parasites with the ability to modify the signalling pathway and cell responses of their infected host cells. These parasite strategies alter the host cell environment and conditions favouring their replication, survival and pathogenesis. Since microRNAs (miRNAs) are able to post-transcriptionally regulate gene expression processes, these biomolecules can exert critical roles in controlling Leishmania-host cell interplay. Therefore, the identification of relevant miRNAs differentially expressed in Leishmania parasites as well as in infected cells, which affect the host fitness, could be critical to understand the infection biology, pathogenicity and immune response against these parasites. Accordingly, the current review aims to address the differentially expressed miRNAs in both, the parasite and infected host cells and how these biomolecules change cell signalling and host immune responses during infection. A deep understanding of these processes could provide novel guidelines and therapeutic strategies for managing and treating leishmaniasis.

Echinococcus granulosus sensu stricto G1 is the predominant genotype in human and livestock isolates from Turkey and Iran, based on mitochondrial nad5 gene differentiation.

BACKGROUND: Different genotypes of Echinococcus granulosus sensu stricto (s.s.) isolated from livestock and humans have been identified based on cox1 and nad1 genomic fragments. The present study was performed to differentiate the G1/G3 genotypes of Echinococcus granulosus (s.s.) isolated from humans and livestock (sheep and cattle) from Azerbaijan in northwestern Iran, Fars Province in southern Iran, and Van province in Eastern Turkey, using the nad5 gene fragment as a suitable marker to distinguish these two genotypes. METHODS: A total of 60 pathologically confirmed human hydatid cysts and 90 hydatid cyst samples from livestock were collected from Turkey and Iran. PCR was performed on all of the samples, targeting the nad5 gene. Based on PCR product quality, host type, and the geographical area where the samples were obtained, 36 of the samples were sequenced and were used in the phylogenetic analysis. RESULTS: Out of 36 evaluated samples, 26 (72.2%) samples belonged to G1, and 10 (27.8%) samples belonged to the G3 genotype. Out of 21 samples from Turkey, 16 (76.2%) were G1 and 5 (23.8%) were G3, while out of 15 samples from Iran, 10 (66.7%) were G1 and 5 (33.3%) were the G3 genotype. None of the samples isolated from humans in Iran or from sheep in Turkey were G3. Overall, between the two countries, 18.18% of E. granulosus isolates in cattle, 41.66% of isolates in sheep, and 23.07% of human samples were identified as G3, and the others as the G1 genotype. The G3 genotype was not detected in human samples from Iran or sheep samples from Turkey. CONCLUSION: The findings of the study revealed that the G1 genotype of E. granulosus s.s. is the predominant genotype in humans and livestock, both in Turkey and Iran. The ratio of the E. granulosus s.s. G1 to G3 genotype was 3.2 in Turkey and 2 in Iran. The study also further confirmed that the nad5 gene properly differentiated the G1/G3 isolates of E. granulosus from both humans and livestock.

Investigating the Prevalence of Intestinal Parasites in Immunocompromised Patients in Bushehr Province, Southwest Iran: A Conventional and Molecular Study

Objective: The aim of this study was to determine the status of intestinal parasitic infections in immunocompromised patients in Bushehr province, southwest Iran by conventional and molecular methods. Methods: A total of 201 stool samples were collected from kidney transplant recipients, AIDS patients and patients under chemotherapy. Samples were collected from healthy people as the control group. The specimens were tested using various conventional methods. Polymerase chain reaction (PCR) testing was performed on samples identified as positive for Coccidia by direct microscopic examination. Results: Approximately 32.45% were infected with at least one type of intestinal parasite. The highest (46.8%) and lowest rates of infection (24%) were observed in AIDS and chemotherapy patients, respectively, while the infection rate of the control group was 16%. Isospora spp. and Cryptosporidium spp. were observed in all patient groups, and Sarcocystis spp. sporocysts were detected in one of the transplant recipients. All identified coccidia were confirmed by PCR. There was a significant relationship between the rate of intestinal parasite infection and certain variables. Conclusion: Given the potential risk of certain intestinal parasites in people with immune deficiency, it is recommended that diagnosis of parasitic infections in such patients be based on specific parasitological methods. Thus, it is advisable that physicians refer them to a parasitology laboratory prior to drug administration.

Genotyping and phylogenetic analysis of hydatid cysts isolated from livestock in Bushehr province, Iran.

Hydatid cyst is one of the parasitic zoonoses caused by infection with the larval stage of Echinococcus granulosus tapeworm. The spread of this parasite is global and is of great importance in terms of public health. To date, ten different species of this parasite have been identified that differ in characteristics such as life cycle, epidemiology and pathogenesis. The purpose of this study was to determine the genotype and phylogenetic relationship of hydatid cysts isolated from livestock of Bushehr province, Iran. About 62 samples of hepatic and pulmonary hydatid cysts were collected from slaughtered animals. DNA extracted by phenol-chloroform method was amplified by PCR using primers specific for the cox1 gene. The PCR products of 50 samples were sequenced and analyzed using BioEdit software and compared with sequences in the GenBank. The phylogenetic tree was drawn using Neighbor Joining tree-NJ method, and its reliability was evaluated. Sequencing results showed that out of 50 sequenced samples, 43 samples had the genotype of Echinococcus granulosus and 7 samples had the genotype of Taenia hydatigena. By drawing a phylogenetic tree, all 43 hydatid cyst samples belonged to G1 strain. The predominance of G1 strain of hydatid cyst in livestock of Bushehr province shows the main role of this genotype in establishing the life cycle of parasite in this region and if the genotype of the parasite in dogs and humans is determined, then these findings can be used to disrupt the life cycle of the parasite and reduce the human infections.

Potential therapeutic targets shared between leishmaniasis and cancer.

The association of leishmaniasis and malignancies in human and animal models has been highlighted in recent years. The misdiagnosis of coexistence of leishmaniasis and cancer and the use of common drugs in the treatment of such diseases prompt us to further survey the molecular biology of Leishmania parasites and cancer cells. The information regarding common expressed proteins, as possible therapeutic targets, in Leishmania parasites and cancer cells is scarce. Therefore, the current study reviews proteins, and investigates the regulation and functions of several key proteins in Leishmania parasites and cancer cells. The up- and down-regulations of such proteins were mostly related to survival, development, pathogenicity, metabolic pathways and vital signalling in Leishmania parasites and cancer cells. The presence of common expressed proteins in Leishmania parasites and cancer cells reveals valuable information regarding the possible shared mechanisms of pathogenicity and opportunities for therapeutic targeting in leishmaniasis and cancers in the future.

Comparison of various methods for DNA extraction from human isolated paraffin-embedded hydatid cyst samples.

Successful molecular research with reliable results depends on achieving significant and uniform amounts of genomic DNA from the parasite as the first and most basic step. Therefore, selection of an appropriate method that minimizes damage to the DNA of the parasite, is very important. In this study, we are going to describe a method that can extract DNA from human isolated paraffin-embedded hydatid cysts with a high quality and quantity. Formalin fixed and Paraffin-embedded hydatid cyst samples isolated from human lung and archived in the pathology laboratory were used for this purpose. Several sections of the paraffin blocks were prepared with 5 micron thickness and DNA were extracted by three different methods including; modified boiling, commercial kit and the method described by Larissa A. Pikor et al. The obtained DNA were evaluated by Nanodrop in terms of the yield of DNA and possible contaminations. To compare the quality of DNA prepared, cox1 region was amplified using specific primers. It was found that the DNA extracted by modified boiling had the lowest rate of contamination and the best electrophoretic band on the gel, compared to other two performed methods. Considering the findings of this study, this simple and high throughput DNA extraction method with high yield and quality can be recommended for extraction of DNA from formalin fixed and paraffin-embedded hydatid cysts.

Diagnostic performance of Echinococcus granulosus protoscolices antigens in the serodiagnosis of human cystic echinococcosis.

The development of suitable serological tests for the diagnosis of CE is still necessary. This study aimed to evaluate the efficacy of ELISA in the diagnosis of human cystic echinococcosis (CE), using parasite protoscolices antigens. Liver hydatid cysts were isolated from sheep infected with hydatid cysts and the protoscolices were isolated from the hydatid cyst fluid. Protoscolices crude antigen was prepared by mechanical disruption, plus freeze-thawing and sonication methods. Thirty sera samples of confirmed hydatid cyst patients, 30 samples of healthy individuals, and 30 samples of people with other infections were collected and the samples were evaluated in an ELISA system, using the crude protoscolices antigen. The sera samples were also simultaneously evaluated by antigen B-ELISA. The estimated value of sensitivity and specificity for the ELISA, using the crude protoscolices antigens, was 93.3% (95% CI: 76.4-98.8%) and 90% (95% CI: 78.8-95.8%), respectively. These values were 86.6 (95% CI: 68.3-95.6) and 91 (95% CI: 80.81-96.9) for the antigen-B based ELISA. Antigens prepared from protoscolices of hydatid cyst are suitable candidates for the serologic diagnosis of human CE. Further studies are needed to identify a single specific antigen among the protoscolices antigens to improve the diagnostic performance of these antigens.

Genetic Diversity of Echinococcus granulosus Isolated from Humans: A Comparative Study in Two Cystic Echinococcosis Endemic Areas, Turkey and Iran.

Cystic echinococcosis (CE) is one of the most important zoonotic parasitic diseases caused by the larval stage of Echinococcus granulosus. Based on molecular studies and DNA sequencing, E. granulosus has been classified into 10 different genotypes (G1 to G10). Two neighboring countries, Turkey and Iran, are considered the two main foci of CE in the Middle East. The current study is aimed at examining the genotype diversity of E. granulosus isolated from human clinical samples in Turkey and Iran. Surgically removed human hydatid cysts were collected from East Azerbaijan and Fars provinces in Iran and Van province in Turkey. After extracting DNA, performing PCR, targeting the cox1 gene, the PCR products were purified from the gel and were sequenced from both directions. The sequences were aligned and compared, using BioEdit and also the BLAST program of GenBank. The maximum likelihood tree was constructed based on the Tamura-Nei model, using the MEGAX software. Phylogenetic analysis showed that the human isolated samples were classified into two major clades: G1 (from Iran and Turkey) and G3 (5 samples from northwestern Iran and one sample from Turkey). The mean and degree of genetic divergence (K2P) between the two major clades, G1 and G3, were 0.2% and 0.7 ± 0.4%, respectively. The findings of the current study revealed that the sheep strain (G1) and the less important strain G3 have major roles in the transmission cycle of CE in two neighboring countries, Iran and Turkey. Therefore, it is necessary to interpose the life cycle of this parasite and reduce the disease burden in livestock and humans by adopting common regional preventive and control policies.

Molecular Identification of Species Caused Cutaneous Leishmaniasis in Southern Zone of Iran.

BACKGROUND: Leishmania major and Leishmania tropica are two main species causing cutaneous leishmaniasis (CL) in Iran. Recently, Crithidia spp. has also been reported in the wound of patients with CL. In this study, we determined the species causing CL in the southern of Iran and the role of Crithidia spp. in creating skin ulcers. METHODS: In this cross-sectional study from Apr to Sep 2016, 66 patients with CL referred to Diagnostic Lab of Leishmaniasis, Valfajr Health Center, Shiraz, Iran, were selected. After DNA extraction from the Giemsa stained smears, all samples were amplified in two separate steps using specific primers, firstly, to differentiate Leishmania species and then to identify Crithidia spp. RESULTS: Two species L. major and L. tropica were responsible for 60 and 6 cases, respectively. Moreover, in two patients, mixed infection with Crithidia was confirmed. In mix infection cases, the morphology of the cutaneous ulcers was not different from the wounds of other patients. CONCLUSION: Leishmania major is responsible for the most common CL in southern Iran. In addition, in two patients with L. major and L. tropica, mix infection with Crithidia was confirmed. The potential role of Crithidia as the main factor for CL and the probability of this parasite to have synergistic effects on Leishmania, as a hypothesis, requires more comprehensive researches on the ambiguity of this protozoon.

Comparative Genotyping of Echinococcus granulosus Infecting Livestock in Turkey and Iran

Objective: Echinococcus granulosus contains a complex of different strains that represent diversity in the pattern of the life cycle and also their host types. So far 10 genotypes of this parasite have been identified, using molecular methods. The current study aimed to evaluate and compare the genotypic diversity of E. granulosus metacestodes from livestock of Turkey and Iran. Methods: A total of 90 livestock liver and lung organs infected with hydatid cyst from industrial slaughterhouses of Bonab Province in the East Azerbaijan Province in Iran (60 samples, including 30 sheep and 30 cattle) and Van Province in Turkey (30 samples, including 15 sheep and 15 cattle) were collected. DNA was extracted from the protoscolices or germinal layers and polymerase chain reaction (PCR) were utilized, targeting the partial mitochondrial cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase 1 (nad1) genes. PCR products were isolated from the electrophoresis gels and sequenced. The sequences were compared with each other, as well as with those related available sequences in the GenBank, using the BioEdit software and the BLAST algorithm. Finally, the phylogenetic trees were constructed by comparing sequences of cox1 and nad1 fragments, using the MEGA7 software and the maximum likelihood method. Results: All samples sequenced from Iran corresponded to the genotype G1 (100%). Among the samples from Turkey, 15 samples (78.9%) were identified as G1 while only one sample (5.3%) corresponded to the genotype G3 and 3 isolates (15.8%) were defined as genotypes G1/G3. Five distinct haplotypes were determined within the examined isolates from sheep and cattle in both countries and all isolates clustered in one group. Phylogenetic analysis revealed that the intra-species genetic variations were 0.0-0.6% and 0.0-1.4% for cox1 and nad1, respectively. Conclusion: The dominant genotype of E. granulosus sensu stricto of livestock in both countries was the G1 (sheep strain) genotype. Our findings indicate that the sheep-dog cycle is the leading cycle of E. granulosus in these two areas. Hence, adopting regional common policies and bilateral cooperation helps to control the disease in livestock as well as in human in these two regions. Further study is required to compare the genetic diversity of human isolates of E. granulosus in these two countries.

Preparation of meglumine antimonate loaded albumin nanoparticles and evaluation of its anti-leishmanial activity: an in vitro assay.

Cutaneous leishmaniasis is still a health problem worldwide, especially in tropical and subtropical areas. Currently, pentavalent antimony compounds are used to treat leishmaniasis. These compounds cause various side effects in the body. Therefore, there is a need to discover new drugs with less toxicity and more therapeutic effects. In this study, we encapsulated the meglumine antimonate into the albumin as a drug carrier and evaluated the anti-leishmanial effect of the prepared nanoparticles. The precipitation method was used for this purpose by applying different concentrations of glutaraldehyde and N-(3-Dimethylaminopropyl)-N-ethyl carbodiimide hydro chloride Ethyl (DEC) and then, field emission test was performed using Scanning Electron Microscopy for evaluating the morphology and size particles. The cytotoxicity and inhibitory of drugs were evaluated on J774 macrophages and Leishmania major promastigotes, respectively. Nanodrugs prepared using glutaraldehyde (10 µl/ml) and DEC (13 mg/ml) had the smallest and largest size, respectively. The highest anti-leishmanial activity was observed in the drugs prepared with glutaraldehyde (10 µl/ml). Also this nanodrug had the lowest cytotoxicity against macrophages. Given that meglumine antimonate loaded albumin nanoparticles prepared with glutaraldehyde (10 µg/ml), can improve the anti-leishmanial effects of this old drug, it can be a good option as a drug delivery system.

DNA extraction from hydatid cyst protoscolices: Comparison of five different methods.

AIM: The current study aimed to find out a simple, practical and high throughput DNA isolation method for extraction of DNA from hydatid cyst samples. MATERIALS AND METHODS: Cattle and sheep isolate of hydatid cysts were obtained from the slaughterhouse, and hydatid fluid and protoscolices were collected in a sterile condition. Protoscolices were washed, 3 times with phosphate buffered saline, and DNA was extracted by different methods including manual extraction with freeze/thawing and phenol-chloroform, Triton X-100 extraction, and by a commercial kit (YTA, Yekta Tajhiz Azma, Iran) with three different modifications in the kit's manufacturer instructions. The obtained DNA from the different methods was evaluated by Nanodrop in terms of the yield of DNA and carbohydrates or protein contaminations. To compare the quality of the extracted DNA, two pieces of the mitochondrial genome of Echinococcus granulosus, cox1, and nad1, were polymerase chain reaction (PCR)-amplified, using each of the DNA prepared by different methods. Electrophoresis of PCR products was carried out on the agarose gel. RESULTS: The DNA extracted by manual method, using phenol/chloroform, had the highest yield, yet with the highest level of protein and carbohydrate contamination. The DNA extracted using two-step incubations, initially at 60°C for 2 h and then overnight at 37°C, was the most purified DNA with the lowest rate of contamination. CONCLUSION: Findings of the study demonstrated that modification in the currently available commercially DNA extraction kit resulted in the development of a high throughput DNA isolation method. This method can be recommended for the extraction of DNA from hydatid cysts, especially the cattle isolate where the extraction of DNA in these samples are usually problematic.

Differentiation of Toxocara canis and Toxocara cati based on PCR-RFLP analyses of rDNA-ITS and mitochondrial cox1 and nad1 regions.

The definitive genetic identification of Toxocara species is currently based on PCR/sequencing. The objectives of the present study were to design and conduct an in silico polymerase chain reaction-restriction fragment length polymorphism method for identification of Toxocara species. In silico analyses using the DNASIS and NEBcutter softwares were performed with rDNA internal transcribed spacers, and mitochondrial cox1 and nad1 sequences obtained in our previous studies along with relevant sequences deposited in GenBank. Consequently, RFLP profiles were designed and all isolates of T. canis and T. cati collected from dogs and cats in different geographical areas of Iran were investigated with the RFLP method using some of the identified suitable enzymes. The findings of in silico analyses predicted that on the cox1 gene only the MboII enzyme is appropriate for PCR-RFLP to reliably distinguish the two species. No suitable enzyme for PCR-RFLP on the nad1 gene was identified that yields the same pattern for all isolates of a species. DNASIS software showed that there are 241 suitable restriction enzymes for the differentiation of T. canis from T. cati based on ITS sequences. RsaI, MvaI and SalI enzymes were selected to evaluate the reliability of the in silico PCR-RFLP. The sizes of restriction fragments obtained by PCR-RFLP of all samples consistently matched the expected RFLP patterns. The ITS sequences are usually conserved and the PCR-RFLP approach targeting the ITS sequence is recommended for the molecular differentiation of Toxocara species and can provide a reliable tool for identification purposes particularly at the larval and egg stages.

Seroprevalence and Molecular Evaluation of Toxoplasmosis in Patients Undergoing Chemotherapy for Malignancies in the Bushehr Province, Southwest Iran.

BACKGROUND: Toxoplasmosis is a life-threatening infection in organ transplant recipients, people receiving corticosteroid or radiation therapy, people with malignancies, and AIDS patients. OBJECTIVES: The current study aimed to determine the prevalence of toxoplasmosis in patients receiving chemotherapy for malignancies in the Bushehr province of southwest Iran. METHODS: Blood samples were taken from 86 patients who were continuously referred to the chemotherapy center in Bushehr province and evaluated by ELISA to determine anti-Toxoplasma IgG and IgM antibodies. Moreover, a blood buffy coat of each sample was assessed by polymerase chain reaction (PCR), targeting a 529 bp gene of T. gondii. PCR products of the positive samples were sequenced to determine the genotype of the parasite. RESULTS: Anti-Toxoplasma IgG antibodies were detected in the sera of 21 (24.4%) cases. All of the patients were negative for anti-Toxoplasma IgM antibodies. No statistically significant correlation was found between seropositivity to Toxoplasma and duration of chemotherapy or having contact with cats. PCR detected a 529 bp band of T. gondii in the buffy coats of two out of 86 (2.3%) cases. The sequence analysis demonstrated that both cases were 95% identical to type III (VEG strain) of T. gondii. CONCLUSIONS: Findings of this study demonstrated the presence of type III T. gondii in the buffy coats of patients undergoing chemotherapy. Given that toxoplasmosis is a life-threatening infection in immunocompromised patients, these patients should be screened for toxoplasmosis before and during chemotherapy to prevent acute toxoplasmosis.

The prevalence of intestinal parasites in hemodialysis patients in Bushehr, Iran.

Hemodialysis patients, due to a dysfunction of the immune response, are prone to a variety of opportunistic infections. Studies of intestinal parasitic infections in these patients are limited. Therefore, the present study was performed to determine the prevalence of these infections in patients on hemodialysis in Bushehr. In this cross-sectional study, fecal samples have been collected from all hemodialysis patients who were continuously referred from September 2011 to September 2012 to the dialysis center at Bushehr and tested using routine parasitological methods. From a total of 88 patients studied, 25 patients (28.4%) were infected with one or more intestinal parasites. Blastocystis hominis and Entamoeba coli with 13.6% and 6.7% prevalence had the highest prevalence among the patients, respectively. The age group 51-70 years had the highest rates of infection. Statistical analysis showed no relationship between sex and the risk of intestinal parasites. Seventeen percent of infected patients showed up with diarrhea and this relationship was statistically significant. Considering the high prevalence of intestinal parasitic infection among hemodialysis patients in Bushehr and also the high probability of infection in these patients, it is recommended that periodic examinations and screening patients during dialysis and before kidney transplantation should be a part of routine medical care.