Evaluation of In Vitro Cytotoxic and Apoptotic Effects of Miltefosine on the Toxoplasma gondii RH Strain.

Background: We aimed to investigate the cytotoxic and apoptotic effects of miltefosine on Toxoplasma gondii RH strain by various techniques. Methods: The study was conducted at the Department of Parasitology and Mycology, Urmia University of Medical Sciences, Iran in 2020. Four groups of five BALB/c mice were selected. The cytotoxicity test was conducted by adding miltefosine to T. gondii tachyzoites; control tachyzoites received PBS and MTT assay was done on each suspension. For evaluating the Th1-type immune responses, the serum levels of IFN-γ and nitric oxide (NO) were assessed in mice after injecting tachyzoites and miltefosine, respectively. The flow cytometry technique was performed on T. gondii tachyzoites challenged with IC50 and IC90 doses of miltefosine and unchallenged cells. DNA fragments in T. gondii tachyzoites were detected by Terminal dUTPnick-end labeling (TUNEL) method. Results: Overall, 256, 64, 32, and 16 µg concentrations of miltefosine, respectively could kill more than 50% of viable T. gondii tachyzoites. The infected mice group, treated with miltefosine, significantly produced more IFN-γ relative to other groups (P< 0.001). Moreover, a significant difference was found in inducible NO synthase between the experimental and control groups (P<0.05). The flow cytometry results demonstrated a concentration-dependent apoptosis rate in tachyzoites incubated with miltefosine, though the necrosis rate was non-significant. DNA fragmentation analysis indicated oligonucleotides (18-200 bp) in tachyzoites treated with 11µg of miltefosine for 24, 48 and 72 h. However, this pattern was not observed in untreated control microorganisms. Conclusion: Miltefosine could be a favorable candidate for use as a new treatment for toxoplasmosis.

Seroprevalence of Fasciola sp. and Toxoplasma gondii Infections in Rural and Urban Inhabitants of Jolfa County, Northwest Iran.

Fascioliasis and toxoplasmosis are the two important zoonotic diseases that are endemic in Iran and share some common transmission routes. The present study is aimed at determining the seroprevalence of human fascioliasis and toxoplasmosis in rural and urban areas of Jolfa County, Northwest Iran. In a cross-sectional study, 600 human sera were collected randomly from humans living in Jolfa County including three cities and 13 villages from 2017 to 2018. Anti-Toxoplasma IgG and anti-Fasciola sp. IgG tests have been performed using the enzyme-linked immunosorbent assay. Four (0.7%) out of 600 human sera showed positive levels of anti-Fasciola IgG. Three out of four seropositive humans were from an urban area, and one (25%) was from rural inhabitants. Considering T. gondii infection, 45% of studied human sera were seropositive for anti-T. gondii IgG. In conclusion, this is the first study reporting Fasciola seropositivity in the area. Based on the findings, human fascioliasis is present in the studied area, Northwest Iran, granted in low prevalence. Considering T. gondii seropositivity, the prevalence is high, yet close to the reports from other regions in the province.

Cytotoxic and Immunomodulatory Activity of Curcumin and Chitosan on Experimental Toxoplasmosis.

Background: Toxoplasma gondii is a pathogenic parasite with worldwide distribution. We investigated curcumin and chitosan in combination on the viability of T. gondii tachyzoites in silico, in vitro and in vivo. Methods: A 3D model was employed in Urmia University of Medical Sciences, Urmia, Iran in 2021 to study the interaction between curcumin and dihydrofolate reductase (DHFR). Ramachandran root-mean-square deviation and VERIFY3D validated the model. Cytotoxicity of curcumin and chitosan was evaluated by MTT viability assay. BALB/c mice infected with 104 Toxoplasma organisms were treated with curcumin, chitosan, and the combination of curcumin+chitosan. Serum levels of inducible NO synthetase (iNOs), interferon gamma (IFN-γ), interleukin (IL)-5, glutamate oxaloacetic transaminases(SGOT), and glutamic pyruvate transaminase (SGPT) were determined. Result: Curcumin-DHFR and curcumin-DHPS (dihydropteroate synthase) interactions and calculated enzyme energy indicated an excellent affinity for curcumin with DHFR, but not DHPS. MTT results of concurrent treatments demonstrated IC50 rates of 0.1, 0.05, and 0.01 mg/ml at 24, 48, and 72h, respectively. IFN-γ, IL-5 and iNOs levels in curcumin+chitosan treated mice were 1.71, 0.51, and 1.51 IU/L, while those of SGOT and SGPT were 76 and 84 IU/L, respectively. Conclusion: The combination of curcumin and chitosan increased survival time of infected mice by seven days. Curcumin and chitosan in combination regulated the immune system and reduced liver damage, potentially forming the basis of a new treatment for toxoplasmosis.

Anti-Leishmania major activity of Calotropis procera extract by increasing ROS production and upregulating TNF-α, IFN-γ and iNOS mRNA expression under in vitro conditions.

BACKGROUND: Leishmaniasis, caused by protozoan parasites of the genus Leishmania, is a neglected tropical disease with 700,000 to 1,000,000 global new cases annually. Adverse effects associated with expense, long-term treatment and drug resistance have made conventional therapies unfavorable, encouraging the search for alternative drugs based on plant products. In this study, the effect of Calotropis procera (Asclepiadaceae) extract against viability of promastigotes and amastigotes of Leishmania major was evaluated in vitro. METHODS: The extract from the leaves of C. procera seedlings was prepared using a methanol maceration method. The colorimetric cell viability 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the growth-inhibitory effect of the extract on promastigotes. The level of reactive oxygen species (ROS) in promastigote cultures was determined after treatment with the extract using the 2',7'-dichlorofluorescein diacetate (DCFH-DA) method and compared with untreated cultures (control). After exposure to the extract the expression levels of tumor necrosis factor-α (TNF-α), interferon gamma (IFN-γ) and inducible nitric oxide synthase (iNOS) genes were determined and compared to control in peripheral blood mononuclear cells (PBMCs) infected with L. major. RESULTS: Based on the MTT assay, the C. procera extract significantly reduced the proliferation of L. major promastigotes with IC50 values of 377.28 and 222.44 µg/mL for 24 and 72 h, respectively (p < 0.01). After treatment with 222.44 and 377.28 µg/mL of C. procera extract, ROS production in L. major promastigote cultures increased 1.2- to 1.65-fold and 2- to 4-fold compared to the control, respectively (p < 0.05). C. procera extract induced significant increases in gene expression of TNF-α (2.76-14.83 fold), IFN-γ (25.63-threefold) and iNOS (16.32-3.97 fold) in infected PBMCs compared to control (p < 0.01). CONCLUSIONS: On the basis of its anti-leishmanial activity, C. procera can be considered as a promising new plant source for the potential treatment of leishmaniasis.

Anti-toxoplasma and Cytotoxic Activities of Holothuria leucospilota Extract and TiO2NPs In vitro and In vivo.

BACKGROUND: An impressive treatment for toxoplasmosis is the combinatory use of sulfadiazine and pyrimethamine. However, both the drugs involve significant side effects and toxicity for the host. Therefore, the discovery of new anti-toxoplasma medications with high efficacy and less to no side effects is urgently needed. OBJECTIVE: This study aimed to evaluate the anti-toxoplasmic effects of Holothuria leucospilota (H. leucospilota) extract and TiO2NPs on the cell death of Toxoplasma gondii (T. gondii) tachyzoites in vitro and serum liver enzymes (AST, ALT, and ALP), and also to evaluate the immune response and production of IL-5, IFN-γ, and TNF-α in a mouse model. MATERIALS AND METHODS: The cytotoxicity of TiO2NPs and H. leucospilota extract against the tachyzoite of T. gondii was evaluated by the methyl thiazolyl tetrazolium (MTT) assay. The levels of serum TNF-α, IFN-γ, IL-5, and liver enzymes were measured, as well. All the groups were subjected to T. gondii, and the survival rate of experimental mice was evaluated. RESULTS: Our findings suggested in vivo and in vitro anti-toxoplasmic activity of TiO2NPs and H. leucospilota extract by inhibiting the proliferation and invasion of T. gondii tachyzoite. In addition, a significant increase in IFN-γ and TNF-α production was observed in mice treated with high doses of TiO2NPs and H. leucospilota extract. However, IL-5 levels decreased in TiO2NPs and H. leucospilota extract-treated mice. Our results also showed a highly significant increase (P < 0.05) in the levels of ALT, AST, and ALP in the groups injected with TiO2NPs and H. leucospilota extract, but not the control group. CONCLUSION: TiO2NPs and H. leucospilota extract have greater anti-toxoplasma effects in vitro and in vivo. These two compounds could be considered as a candidate for use against toxoplasmosis, both therapeutically and prophylactically.

The Effect of Curcumin on the Expression of INFγ, TNF-α, and iNOS Genes in PBMCs Infected with Leishmania major [MRHO/IR/75/ER].

BACKGROUND: Leishmaniasis, caused by the Leishmania parasite, is one of the most important tropical neglected diseases. The urgent search for effective, inexpensive, and preferably herbal anti-leishmanial agents, is needed. OBJECTIVE: Curcumin is a natural polyphenolic compound derived from turmeric that is well known for its antioxidant, anti-inflammatory, anti-tumor, and anti-cancer activity. METHODS: The present work evaluates the anti-leishmanial [Leishmania major] activity of curcumin. The infected PBMCs were treated with curcumin. The ROS level at 6, 12, 24 h and gene expression levels at 24, 48, and 72 h of PBMCs after treatment with curcumin were determined. RESULTS: Based on the results, the curcumin concentrations of 268 µM [24 h] and 181.2 µM [72 h] were defined as IC50 against L. major promastigotes. Treatment of L. major infected-peripheral blood mononuclear cells [PBMCs] with IC50 concentrations of curcumin, depending on exposure time, significantly induced the reactive oxygen species [ROS] generation and increased the expression levels of interferongamma [IFN-γ], tumor necrosis factor-alpha [TNF-α], and nitric oxide synthase [iNOS] genes. CONCLUSION: These findings suggest the potential of curcumin against Leishmaniasis.

Screening of Cystic Echinococcosis and Toxocariasis in Urmia Municipal Workers, Northwest Iran.

BACKGROUND: Echinococcus granulosus and Toxocara spp., the causative agents of hydatid cyst and toxocariasis in humans and animals, are zoonotic infections with medical and veterinary significance respectively. AIM: Herein, we aimed to investigate the seroprevalence of toxocariasis and cystic echinococcosis in Urmia municipal workers (gardeners and sweepers) in 2018 using ELISA assay. OBJECTIVE: There is no precise report on the seroprevalence of toxocariasis and cystic echinococcosis in Urmia municipal workers using ELISA assay. Therefore, this study found an opportunity to investigate this subject. METHODS: A total of 220 blood samples were obtained from municipal workers with an average age of 41.31 ± 9.42. Commercial anti-Toxocara canis and hydatid cyst ELISA kits were used to assess the seroprevalence rates. RESULTS: Approximately, 14.5% and 2.3% of individuals were seropositive for toxocariasis and hydatidosis, respectively. Consuming unboiled milk and/or raw/uncooked meat and rural conditions are risk factors for toxocariasis. Regarding hydatidosis, consuming unwashed vegetables was the only statistically significant risk factor. Toxocara infection (p = 0.01), but not hydatid cyst (p = 0.05), is more prevalent among sweepers than municipal gardeners. CONCLUSION: Due to the occupational condition of municipal workers, they are exposed to many infectious agents; thus, more researches should be done on these populations.

Sero-epidemiology of Hydatidosis Among General Population of Jolfa County, Northwestern Iran Using IHA, ELISA and Western Blot (2017-2018).

BACKGROUND: Human hydatidosis is mostly a latent and neglected disease with known endemicity in Iran. AIMS: Due to the importance of this infection in the country and its latent nature, we aimed to evaluate the serological status of hydatid cyst in northwestern Iran. OBJECTIVES: Herein, we evaluated the serological status of hydatid cyst in urban and rural inhabitants of Jolfa county, northwestern Iran during 2017-2018. METHODS: In total, 1296 blood samples were obtained from human individuals and the presence of anti-E. granulosus antibodies was investigated using IHA, ELISA and WB. RESULTS: Based on results, 25 IHA positive person were detected in the examined population, however ELISA test showed 14 of 25 IHA positive patients as negative. Also, 269 IHA negative fellows were shown as negative by ELISA. WB analysis of sera from 25 IHA positive subjects revealed consistent results with the ELISA test, and the most reactive SHCF Ag was a 37 KDa protein. The age-standardized seroprevalence of hydatidosis among Jolfa's general population was 1.12% with 95%CI: 1.02-1.20. Moreover, there existed a significant association between keeping/- contact with dogs (P = 0.022) as well as vegetable consumption (P < 0.001) with ELISA positive test results. CONCLUSION: Along with such serological evidence in this region, we highly suggest physical examination and applying imaging techniques for suspected cases in the area for a better understanding of CE.

Prevalence of strongyloidiasis in the general population of the world: a systematic review and meta-analysis.

Strongyloides stercoralis is a neglected soil-transmitted helminth affects approximately 100-370 million people globally. The life cycle is unusual as only larvae can be found in stool specimens. Thecurrent review and meta-analysis represented the distribution of strongyloidiasis in general population of the world based on published papers. Five English databases (Science Direct, Scopus, PubMed, Web of Science, and Google Scholar(were explored for literature published before October 2019.Altogether 235 studies (862243 participants) was eligible. Regarding diagnostic method, the overall prevalence for studies performed microscopic, culture, immunological and molecular method was 1.47% (95% CI = 1.56%), 10.08% (95% CI = 8.99% - 11.16%), 23.88% (95% CI =  20.82% - 26.94%) and 9.3% (95% CI  =  7.2% - 11.3%), respectively. Based on microscopic methods, the highest prevalence was related to the Western Pacific region [9.47% (95% CI =  8.55% - 10.39%)]. According to the culture method, Western Pacific region [21.36% (95% CI  =  16.32% - 26.39%)] had the highest estimated pooled prevalence. In immunological studies, Eastern Mediterranean Region [40.72% (95% CI  =  36.74% - 44.70%)] had the highest seroprevalence.Also in molecular surveys, the highest prevalence was related to the African region [19.72% (95% CI  =  16.71% - 22.73%)]. The current study indicated that strongyloidiasis is still considered a health problem in many parts of the world. Thus a comprehensive control program and improvement of public health sectors are required.

Association of Toxoplasma gondii infection with cardiovascular diseases: a cross-sectional study among patients with heart failure diseases in Urmia, North-West of Iran.

Toxoplasmosis is a disease caused by the protozoan parasite Toxoplasma gondii. Infection with T. gondii is one of the most common parasitic diseases in humans and other warm-blooded animals with global distribution and generally, one-third of human populations are estimated to be contaminated with this parasite. The prevalence of infection varies according to age, geographical location and dietary habits. The socioeconomic losses caused by the disease can be costly for the community. Acquired toxoplasmosis is potentially associated with schizophrenia, Alzheimer's disease, driving accidents, self-injury and suicide. Also, unusual strains of parasite that are genetically different from the rest (atypical strains) are responsible for several cases of lethal acquired parasites in people with safe immunity, which highlights the potential danger of this parasite in public health. As there is no comprehensive study on the association between toxoplasmosis and cardiovascular diseases in Iran, therefore, current study aimed at assessing the relationship between cardiovascular disease and toxoplasmosis among cardiac patients at the Seyyed al-Shohada specialist Cardiology Centre, Urmia, Iran. This study investigated the seropositivity rate for anti-Toxoplasma IgG antibodies by ELISA in patients with cardiovascular diseases. So, 375 patients with cardiovascular diseases and 336 healthy volunteers were selected for this investigation. The seropositivity rate of anti-Toxoplasma IgG antibodies was significantly higher in cardiovascular patients (63.73%) than in healthy volunteers (37.64%) (P<0.001). Also, a positive association was observed between anti-T. gondii IgG antibody seropositivity and cat contact (P≤0.001, OR: 5.178; 95% CI: 1.97-13.57), consumption of raw or undercooked meat (P≤0.001, OR: 0.3; 95% CI: 0.15-0.61), and consumption of not boiled milk (P≤0.001, OR: 0.26, 95% CI: 0.12-0.54). Our results indicate that T. gondii infection is associated with heart disease and suggest that heart disease might be related with a chronic infection. Risk factors associated with T. gondii exposure found in the present study may help design future prevention strategies against T. gondii infection.

Prevalence of fasciolosis in livestock and humans: A systematic review and meta-analysis in Iran.

Fasciolosis is a re-emerging disease of livestock and rarely human, being endemic in Iran. Herein, we aimed to quantitatively assess the human seropositivity and prevalence of animal fasciolosis in our country. English and Persian databases were searched for online literature. In total, 10 human seroprevalence studies and 49 animal investigations were obtained from January 1999 to March 2019. Only animal studies were included in meta-analysis. The highest human seroprevalence was detected in Guilan province (326/452; 50%). The pooled prevalence of the animal infections was calculated as 6.2% (95% CI = 5.8%-6.5%). In detail, the prevalence ranges in three hosts included as 4.2% (95% CI = 3.8%-4.5%) in sheep, 9% (95% CI = 8.0%-9.9%) in cattle and 3.1% (95% CI = 2.4%-3.7%) in goat. The highest prevalence rate was observed in the north, 11.8% (95% CI = 8.4%-15.1%), while the lowest prevalence was detected in the central Iran with 1.8% (95% CI = 1.3%-2.3%). Egger's regression test revealed no significant publication bias (P = 0.307). Also, there was no remarkable correlation between weighted prevalence and sample size (P = 0.249) as well as year of study (P = 0.172). These findings would be necessary for better preventive strategies in case of human and animal Fasciola infections as well as snail intermediate hosts.

In silico and in vitro comparative activity of green tea components against Leishmania infantum.

OBJECTIVES: Green tea contains a predominant set of polyphenolic compounds with biological activities. The aim of this study was to investigate the antileishmanial activities of the main components of green tea, including catechin, (-)-epicatechin, epicatechin gallate (ECG) and (-)-epigallocatechin 3-O-gallate (EGCG), against Leishmania infantum promastigotes. METHODS: Green tea ligands and the control drug pentamidine were docked using AutoDock 4.3 software into the active sites of trypanothione synthetase and arginase, which were modelled using homology modelling programs. The colorimetric MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was used to measure L. infantum promastigotes at different concentrations of green tea compounds in a concentration- and time-dependent manner. Results were expressed as 50% and 90% inhibitory concentrations (IC50 and IC90, respectively). RESULTS: In silico and in vitro assays showed that all of the green tea compounds have antileishmanial activity. EGCG and ECG were the most active compounds against L. infantum promastigotes, with IC50 values of 27.7µM and 75µM and IC90 values of 88.4µM and 188.7µM, respectively. Pentamidine displayed greater growth inhibition than all of the other tested compounds in a concentration- and time-dependent manner. CONCLUSION: In this study, in silico and docking results were in accordance with the in vitro activity of the compounds. Moreover, EGCG and ECG showed reasonable levels of selectivity for Leishmania.