Bio-Membrane SELEX as a New Approach for Selecting ss-DNA Aptamers that Bind to the Hydatid Cyst Laminated Layer.

BACKGROUND: Hydatid cyst (HC) is the larval stage of the canine intestinal tapeworm (cestode), Echinococcus granulosus. In addition to the high global economic cost of livestock farming, the infection can lead to dangerous problems for human health. Therefore, research into new diagnosis and treatment approaches is valuable. This study is set out to explore aptamers that bind to HC antigens. METHODS: The similarity between HC genotype in sheep and humans was that sheep HCs were collected and used as a biological membrane for aptamer selection. Four Bio- Membrane SELEX rounds were conducted, and ssDNA aptamers were selected. Selected aptamers' affinity and specificity to the laminated layer antigens were evaluated using membrane staining by fluorescein primer as a probe. Biotinylated primer was used as a probe for aptahistochemistry and dot blot techniques. Subsequently, cloning and plasmid extraction was conducted. The affinity and specificity of sequenced aptamers were examined with the dot blot method. RESULTS: Selected aptamers reacted with HC wall in aptahistochemistry, aptahistofluorescent, and dot blot experiments. Following cloning and sequencing, 20 sequences were achieved. A strong reaction between HC total antigens and sequenced aptamers has emerged in the dot blot method. CONCLUSION: We propose a novel method to determine specific aptamers in this investigation. Bio-Membrane SELEX could be assumed as a practical and sensitive method for aptamer selection. Selected aptamers in this study possibly may be used for specific HC antigens detection.

Environmental Contamination of Different Areas of Isfahan Province of Iran with Toxocara spp. Eggs using Molecular Methods.

Background: Toxocariasis is a parasitic disease caused by the larval stage of Toxocara canis and Toxocara cati. Infective stage of this parasite for human develops on soil. So, in this work contamination of the soil of public environments in five geographical areas of Isfahan province of Iran has been investigated. Materials and Methods: In this descriptive study, 355 soil samples were collected from parks, children's playgrounds, student dormitories, and university environments, and examined by Flotation method. The samples were then inspected using microscopic and molecular methods. Results: From the 355 examined soil samples in 77 (21.69%), and 87 (24.50%) cases Toxocara eggs were detected by microscopic and molecular methods, respectively. In the molecular method, 31 (8.70%) cases of T. cati and 44 (12.39%) cases of T. canis were identified. Conclusion: Toxocara eggs were identified in all areas of Isfahan province, although contamination rate was higher in Fereydun Shahr and Semirum counties.

The prevalence of human trichuriasis in Asia: a systematic review and meta-analysis.

Trichuriasis is one of the most common soil-transmitted helminth (STH) infections, affecting populations globally. The condition is particularly prevalent in tropical and subtropical areas with low levels of sanitation and poor living conditions. The objective of this systematic review and meta-analysis was to evaluate the prevalence of Trichuris trichiura infection in Asia at the country and region level. Multiple databases/academic search engines (Web of Science, PubMed, ProQuest, Scopus, and Google Scholar) were searched for literature on T. trichiura prevalence in Asia published through January 2021. Pooled prevalence was determined using the meta-package in R (version 3.6.1). Out of 13,836 articles, 226 studies (5,439,500 individuals) from 26 countries met the inclusion criteria. Of the 226 studies, 151 were community-based studies that included individuals across the age spectrum, while 75 studies focused on school children (typically in the 5-16 years age range). The overall T. trichiura pooled prevalence was 15.3% (95% CI: 12.4-19.1%), with a pooled prevalence of 13.3% (95% CI: 10.0-17.1%) for the community studies and 20.9% (95% CI: 14.7-27.9%) for the studies only including school children. For studies including all age groups, individuals in the 1-15 years age group had the highest pooled prevalence at 23.4% (95% CI: 1.7-49.4%). There was a significant difference found in overall pooled prevalence by sex (p < 0.001) and community type (rural versus urban) (p < 0.001). Although prevalence appears to be decreasing, study findings suggest that T. trichiura infection continues to be a public health problem in Asia. Therefore, control programs focused on at-risk individuals in endemic areas are needed.

Genotype Characteristics of Giardia duodenalis in Patients Using High Resolution Melting Analysis Technique in Khorramabad, Iran.

BACKGROUND: We aimed at genotyping and evaluating the predominance of G. duodenalis assemblages isolated from patients referred to medical laboratories in Khorramabad, Iran from Nov 2015 to Sep 2016. Hence, the development of a cost-effective HRM approach to determine genotypes of G. duodenalis based on the triosephosphate isomerase (tpi) gene was examined and the genotyping results with and without diarrhea was compared. METHODS: Seventy G. duodenalis positive fecal samples were collected. A microscopic confirmation for the presence of Giardia spp. was performed, cysts of 70 Giardia spp. positive specimens were concentrated using sucrose flotation technique and sucrose solution PCR amplification was performed on 69 of 70 (98.5%) samples, and High Resolution Melting (HRM) analysis was performed using a software. RESULTS: The results showed two distinct genotypes (assemblages A and B) of G. duodenalis but infections with mixture of both assemblages were not detected. The genotypes of G. duodenalis showed that the sub assemblage AI, BIII and BIV were present in a proportion of 68.1%, 20.3% and 11.6% respectively in samples. Assemblage AI was significantly (P<0.05) more frequently found in patients with diarrhea. CONCLUSION: The sub-assemblage AI, BIII, and BIV are more zoonotic potential. According to the comparison of the results of this study with the results of previous studies in this area and around of it, as well as the way people live and keep pets. This pattern established in Khorramabad city. HRM can be an ideal technique to detect and genotyping of G. duodenalis in clinical samples.

Prevalence of Toxocara and Toxascaris infection among human and animals in Iran with meta-analysis approach.

BACKGROUND: Toxocariasis is a worldwide zoonotic parasitic disease caused by species of Toxocara and Toxascaris, common in dogs and cats. Herein, a meta-analysis was contrived to assess the prevalence of Toxocara/Toxascaris in carnivore and human hosts in different regions of Iran from April 1969 to June 2019. METHODS: The available online articles of English (PubMed, Science Direct, Scopus, and Ovid) and Persian (SID, Iran Medex, Magiran, and Iran Doc) databases and also the articles that presented in held parasitology congresses of Iran were involved. RESULTS: The weighted prevalence of Toxocara/Toxascaris in dogs (Canis familiaris) and cats (Felis catus) was 24.2% (95% CI: 18.0-31.0%) and 32.6% (95% CI: 22.6-43.4%), respectively. Also, pooled prevalence in jackal (Canis aureus) and red fox (Vulpes vulpes) was 23.3% (95% CI: 7.7-43.2%) and 69.4% (95% CI: 60.3-77.8%), correspondingly. Weighted mean prevalence of human cases with overall 28 records was 9.3% (95% CI: 6.3-13.1%). The weighted prevalence of Toxocara canis, Toxocara cati, and Toxascaris leonina was represented as 13.8% (95% CI: 9.8-18.3%), 28.5% (95% CI: 20-37.7%) and 14.3% (95% CI: 8.1-22.0%), respectively. CONCLUSION: Our meta-analysis results illustrate a considerable prevalence rate of Toxocara/Toxascaris, particularly in cats and dogs of northern parts of Iran. The presence of suitable animal hosts, optimum climate and close contact of humans and animals would have been the reason for higher seroprevalence rates of human cases in our region. Given the significance clinical outcomes of human Toxocara/Toxascaris, necessary measures should be taken.

A new effective antiplasmodial compound: Nanoformulated pyrimethamine.

OBJECTIVES: The aim of this study was to evaluate the efficacy of pyrimethamine-loaded poloxamer 407 nanomicelles on Plasmodium berghei strain NICD in vivo. METHODS: Pyrimethamine-loaded nanomicelles were prepared and their zeta potential, particle size and polydispersity index were measured. For antiplasmodial assessment, 54 mice were randomly divided into six groups. Four groups were infected intraperitoneally with P. berghei, whereas the two remaining groups did not receive the parasite (negative controls). Three of the P. berghei-infected groups received treatment with either pyrimethamine-loaded nanomicelles (2 mg/kg), pyrimethamine (2 mg/kg) or empty nanomicelles (2 mg/kg); the fourth group remained untreated (positive control). The parasitaemia rate, survival rate and histopathological changes in the liver, spleen and kidneys were examined and were compared with the negative and positive control groups. RESULTS: The mean parasitaemia rate differed significantly between the nanoformulated pyrimethamine group and each of the other groups (P<0.05). Moreover, the survival rate of mice in the nanoformulated pyrimethamine group (7/9; 78%) was significantly higher compared with each of the other groups (P<0.01). The main histopathological changes, including hepatic necrosis in the liver, lymphoid hypoplasia in the spleen, and tubular nephrosis and perivascular and interstitial lymphocytic infiltration in the kidneys, were considerably lower in the nanoformulated pyrimethamine group than in the pyrimethamine and positive control groups. CONCLUSION: Pyrimethamine-loaded nanomicelles showed potent antimalarial activity and can be considered as a potential candidate for further examination of their suitability as an antimalarial drug.

Induction of Apoptosis in Toxoplasma gondii Infected Hela Cells by Cisplatin and Sodium Azide and Isolation of Apoptotic Bodies and Potential Use for Vaccination against Toxoplasma gondii.

BACKGROUND: Toxoplasma gondii can infect a wide range of mammalians, especially humans. It controls several intracellular signals for the inhibition of apoptosis. This study aimed to investigate the apoptogenic effect of cisplatin and sodium azide on T. gondii infected HeLa cells and isolate apoptotic bodies (blebs) as a potent stimulator of the immune system. METHODS: The cytotoxic properties of cisplatin and sodium azide (NaN3) on HeLa cells were evaluated by MTT assay. Moreover, the apoptogenic activity of cisplatin and NaN3 was studied using flow cytometry (Annexin V/PI double staining) and scanning electron microscopy (SEM). Finally, apoptotic bodies were separated by centrifugation. RESULTS: MTT assay data showed that the survival rate of cells treated with different concentration of NaN3 was significantly reduced, compared to negative control groups. Concerning cisplatin, only concentration of 20 µM had not a significant impact on the cell viability; however, the other concentration of cisplatin significantly reduced cell viability, compared to negative control groups. The level of early apoptosis in uninfected HeLa cells was higher compared to infected HeLa cells treated with cisplatin and NaN3. Finally, apoptotic bodies were separated from T. gondii infected HeLa cells treated with cisplatin. CONCLUSION: Apoptosis was induced in both uninfected and infected HeLa cells with T. gondii and apoptotic bodies were isolated from infected cells. Therefore, further studies on apoptotic bodies are required in order to find a proper candidate for vaccine preparation against T. gondii infections.

Synthesis and antileishmanial activity of antimony (V) complexes of hydroxypyranone and hydroxypyridinone ligands.

A novel series of antimony (V) complexes with the hydroxypyranone and hydroxypyridinone ligands were synthesized and characterized by 1HNMR, FT-IR and electron spin ionization mass spectroscopic (ESI-MS) techniques. The synthesis process involved protection of hydroxyl group followed by the reaction of the intermediate with primary amines and finally deprotection. All compounds were evaluated for in vitro activities against the amastigote and promastigote forms of Leishmania major. Most of the synthesized compounds exhibited good antileishmanial activity against both forms of L. major. IC50 values of the most active compounds; 9d, 9d and 9e, after 24, 48 and 72 h against amastigote model were 15, 12.5 and 5.5 µg/mL, respectively. 9e, 11 and 9e inhibited the promastigote form of parasite after 24, 48 and 72 h with IC50 values of 10, 2 and 1 µg/mL, respectively.

Chitosan-titanium dioxide-glucantime nanoassemblies effects on promastigote and amastigote of Leishmania major.

The purpose of the present study was to design nanoassemblies of chitosan-titanium dioxide (TiO2) nanoparticles (NPs) loaded with glucantime for using their synergistic effects and enhancing the toxic effects of glucantime on Leishmania parasites. The nanoassemblies were prepared by electrostatic interactions and optimized by a response surface central composite design. The effects of glucantime, chitosan and TiO2 NPs amounts were studied on the particle size, zeta potential, loading efficiency, and release efficiency of drug from nanoassemblies. The conjugation of TiO2/chitosan-glucantime was verified by UV spectroscopy and changes in surface charge of NPs. The anti-promastigots effect of glucantime loaded in TiO2/chitosan nanoassemblies was studied by tripan blue dye test and their anti-amastigotes effect by counting the average number of parasites per infected J774 macrophages in 100 cells. The optimized formulation obtained by using 12.5mg glucantime, 25mg chitosan and 6mg TiO2 NPs. Although TiO2 NPs alone were effective more than negative control in reduction of promastigots and amastigotes but they didn't show significant difference compared with free glucantime (p>0.05). However, at the concentration of 50µg/mL and after 72h exposure nanoassemblies decreased the proliferation of L. major promastigotes and amastigotes 13 and 4-fold, respectively compared with glucantime alone.

Comparison and evaluation of four methods for extracting DNA from Giardia duodenalis cysts for PCR targeting the tpi gene.

Giardia duodenalis is an intestinal flagellated protozoan and the common cause of gastrointestinal diseases in human. This parasite can be seen in two different forms in its life cycle including as cyst and trophozoite. Due to presence of resistant cyst wall, DNA extraction inhibitors along with artifact in stool specimens, this study was performed aiming to evaluate four methods for DNA extraction from G. duodenalis cysts. Seventy G. duodenalis positive stool specimens that were confirmed by light microscope were included in this study. All stool samples were concentrated using four layered discontinuous sucrose flotation technique (0.5, 0.75, 1, and 1.5 M) and single-layered sucrose solution (0.85 M). The isolated cysts were then subjected to DNA extraction by four methods. To remove the artifacts, the extracted DNA were evaluated by PCR. The results of the present study showed the high level of optical density (OD) in the method I (P < 0.01) with the following steps; Giardia cysts plus crushed cover glass were vortexed. Then, the samples were boiled and then followed by freeze-thaw cycles, yet this method yielded the lowest concentration. Furthermore, the highest concentration were observed in the method II (P <0.01) with the following steps; Giardia cysts plus crushed cover glass and TAE buffer were mixed and then shaken, followed by boiling. Based on the results of the present study, using crushed cover glass, boiling and freeze-thaw cycles can be effective in destruction of G. duodenalis cyst wall and have enough efficiency for extracting DNA from G. duodenalis cysts.

Rapid Identification of Echinococcus granulosus and E. canadensis Using High-Resolution Melting (HRM) Analysis by Focusing on a Single Nucleotide Polymorphism.

High-resolution melting (HRM) is a reliable and sensitive scanning method to detect variation in DNA sequences. We used this method to better understand the epidemiology and transmission of Echinococcus granulosus. We tested the use of HRM to discriminate the genotypes of E. granulosus and E. canadensis. One hundred forty-one hydatid cysts were collected from slaughtered animals in different parts of Isfahan-Iran in 2013. After DNA extraction, the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was amplified using PCR coupled with the HRM curve. The result of HRM analysis using partial the sequences of cox1 gene revealed that 93, 35, and 2 isolates were identified as G1, G3, and G6 genotypes, respectively. A single nucleotide polymorphism (SNP) was found in locus 9867 of the cox1 gene. This is a critical locus for the differentiation between the G6 and G7 genotypes. In the phylogenic tree, the sample with a SNP was located between the G6 and G7 genotypes, which suggest that this isolate has a G6/G7 genotype. The HRM analysis developed in the present study provides a powerful technique for molecular and epidemiological studies on echinococcosis in humans and animals.

Prevalence of Intestinal Parasitic Infection Among Inhabitants and Tribes of Chelgerd, Iran, 2008-2009.

INTRODUCTION: Although a notable development in treating and controlling of parasitic infections in recent years has occurred but, these infections are still counted as important problems in many countries. AIM: The aim of this study is to determine the prevalence of intestinal parasitic infections in the inhabitant and tribe populations who were referred to central health care of Chelgerd, Iran. MATERIALS AND METHODS: This descriptive cross-sectional study was carried out from April 2008 to October 2009 in Chelgerd, Iran. A total of 655 samples of feces from inhabitants and tribes were collected and each sample was examined by Direct smear, formol- ethyl acetate concentration and Trichorom staining. RESULTS: Out of 655 stool samples, 367(56%) patients revealed at least one intestinal parasite (pathogenic /non-pathogenic protozoa/helminth), 233(67.7%) in tribes and 134(43%) in inhabitants. There was significant difference between infected inhabitants and infected tribes (p=0.001). Although the intestinal parasitic infections were more in female than male it was not statistically significant (p=0.52). There was no significant difference in various age groups. Common intestinal parasitic infections which were detected in both the populations were Giardia intestinalis (28.2%) and Blastocystis hominis (27.5%). CONCLUSIONS: We found that the prevalence of intestinal parasitic infections was higher in the tribe than inhabitant populations. Prevalence of intestinal protozoa infections was much higher than the helminthic infections. These findings reflect poor sanitary conditions in this region. They should be educated and provided better facilities to get rid of intestinal parasitic infections.

Frequency of Blood-tissue Parasitic Infections in Patients with Multiple Sclerosis, as Compared to their Family Members.

BACKGROUND: Multiple Sclerosis (MS) is a chronic demyelinating disease of the central nervous system which has been identifies more prevalent in economically developed countries than in the developing countries. Low prevalence of parasitic infections (which can activate immune response and prevent or modulate damage to host antigens) in these areas is among the possible responsible factors for such a difference. In this study we aimed to compare frequency of blood-tissue parasitic infections in patients with MS, as compared to their healthy family members. METHODS: This study was conducted on 50 relapsing remitting MS patients and 50 family members attending MS clinic at Alzahra Hospital. IgM and IgG anti-Toxoplasma gondii were measured. Given the high prevalence of cutaneous leishmaniasis in Isfahan, all the participants were also examined for protozoan leishmania microscopically. Furthermore malaria parasite was investigated. RESULTS: Eighteen patients and 24 healthy family members had positive test in IgG Toxoplasma gondii(P = 0.09). In both groups, there was no positive IgM Toxoplasma gondii. In investigating leishmania, only 3 participants in the case group and 2 in the control tested positive (P = 0.25). No case of malaria was found among the participants. CONCLUSION: Our results showed a mismatch with hygiene hypotheses examined. However, considering that the prevalence of parasites varies with time, and depends on numerous epidemiological factors; these results do not discredit the theory investigated.

Molecular identification of Leishmania isolates obtained from patients suspected as having cutaneous leishmaniasis referred to reference laboratories from Yazd province in central Iran.

BACKGROUND: Cutaneous leishmaniasis (CL) continues to be an increasing public health problem in Iran. The dominant etiologic agents of CL in the Old World are Leishmania major and Leishmania tropica. One of the important endemic foci of CL in Iran is Yazd. Recently, previous studies showed the equal prevalence of L. major and L. tropica as the agents of cutaneous leishmaniasis in this area. This prompted us to identify the genotype of L. major isolates obtained from patients with cutaneous leishmaniasis. MATERIALS AND METHODS: After completing a clinical/epidemiologic data questionnaire for 218 patients with suspected skin lesions, scraping samples were collected, and each specimen was examined using both direct microscopy and molecular assay of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: Results showed that of the 218 samples, Leishman body was observed in 77 by direct smear and 104 by PCR assay. Molecular assay indicated 50 cases as L. major, 52 cases as L. tropica, and two cases as unknown. Molecular characterization of L. major isolates showed four patterns, named LmA1, LmA2, LmA3, and LmA4. CONCLUSION: Our study is the first report for molecular characterization of L. major from one of the important central province of Iran that could affect the control strategies in this field.

Identification of genotypes of Giardia duodenalis human isolates in Isfahan, Iran, using polymerase chain reaction - Restriction Fragment Length polymorphism.

BACKGROUND: Giardia duodenalis is one of the most prevalent intestinal parasites of human. It also infects a wide range of mammals. Two genotype of G.duodenalis (A and B) were commonly reported among humans with different frequency of distribution in different geographical locations. This work was conducted to discriminate genotypes of Giardia duodenalis human isolates in Isfahan city using PCR- RFLP. This is the first molecular study on human isolates of G.duodenalis in the area. METHODS: Samples were collected from different health centers of Isfahan city during June 2011 and February 2012. From 175 Giardia positive stool samples 67 specimens were selected randomly. Cysts of Giardia positive samples were concentrated by flotation sucrose. Extraction of genomic DNA from trophozoite and cysts was performed using QIAamp Stool Mini kit with a modified protocol. PCR- RFLP method was used to amplify a fragment of 458bp at the glutamate dehydrogenase locus, and restriction enzymes BspLI and RsaI differentiated human genotypes A and B and their subgroups. RESULTS: PCR - RFLP assay of 67 isolates showed 40(59.7%) isolates as Genotype A group II, 23(34.32%) samples as Genotype B Group III and two (2.98%) sample as Genotype B group IV. Mixed genotype of (AII and B) was detected only in two isolates (2.98%). CONCLUSIONS: PCR - RFLP assay targeting gdh locus is a sensitive tool and discriminates genotypes, sub genotypes and mixed type of G.duodenalis. Results of our study suggest both anthroponotic and zoonotic origins for the infections respectively.

Frequency of Entamoeba histolytica and Entamoeba dispar prevalence among patients with gastrointestinal complaints in Chelgerd city, southwest of Iran(*).

BACKGROUND: Differentiation between Entamoeba histolytica and Entamoeba dispar is very important for both clinical therapy and epidemiological studies. Although these two species are morphologically identical, they have differences in genetic, chemical specifications and pathogenicity. This study was carried out to differentiate E. histolytica from E. dispar and also to find out frequency of the two species. METHODS: Fecal samples were collected three times from 655 patients with gastrointestinal complaints (47.3% male and 52.7% female), who were referred to the primary health care centers of Chelgerd, Chaharmahal and Bakhtiary province. Samples were examined microscopically with direct smear, formalin-ethyl-acetate concentration and trichrom staining methods to distinguish E. histolytica from E. dispar complex and differentiate them from non-pathogenic intestinal amoeba. Genomic DNA was extracted from microscopy positive isolates and polymerase chain reaction (PCR) was carried out to different the two morphologically identical Entamoeba isolates. RESULTS: Among the 655 recruited patients, eleven subjects with E. histolytica / E. dispar isolates (1.7%) were identified by microscopy methods. Ten of the positive isolates (90.9%) were identified as E. histolytica by PCR and one isolate (9.09 %) was positive for E. dispar. CONCLUSIONS: This study revealed that E. histolytica was more prevalent than E. dispar in the studied area. This result was different from the previously reported data in other parts of Iran.

Prevalence rate of Cryptosporidium infection in hemodialysis patients in Iran.

Cryptosporidium is one of the most common causes of diarrhea in the world, which can be severe and prolonged in immunocompromised patients. We compared the prevalence rate of Cryptosporidium infection in hemodialysis patients and 2 control groups (i.e., their healthy family members and normal population). Stool specimens of 104 adult outpatient chronic hemodialysis patients, their 91 healthy family members, and 140 healthy individuals were examined for the presence of Cryptosporidium oocysts by using a modified acid-fast staining method. Twelve (11.5%) dialysis patients were infected with Cryptosporidium. This was significantly higher than 4 (4.4%), and 5 (3.6%) cases in the 2 control groups, respectively (p < 0.05). There was no significant difference between the 2 control groups. The prevalence rate of Cryptosporidium infection did not correlate with patients' sex, age, duration of dialysis, history of kidney transplantation, or history of taking immunosuppressive drugs. However, it was significantly higher in diabetics vs. nondiabetics (19.4% vs. 8.3%, respectively, p < 0.05). Our results indicate that the prevalence rate of Cryptosporidium infection is considerably higher in dialysis patients than in the general population. Moreover, dialyzed diabetic patients had the highest rate of infection. As hemodialysis patients are candidates for renal transplantation, general preventive measures against acquiring Cryptosporidium infection must be considered.