Knocking down the expression of the molecular motors, myosin A, C and F genes in Toxoplasma gondii to decrease the parasite virulence.

Toxoplasmosis is a serious parasitic infection and novel therapeutic options are highly demanded to effectively eliminate it. In current study, Toxoplasma gondii myosin A, C and F genes were knocked down using small interference RNA (siRNA) method and the parasite survival and virulence was evaluated in vitro and in vivo. The parasites were transfected with specific siRNA, virtually designed for myosin mRNAs, and co-cultured with human foreskin fibroblasts. The transfection rate and the viability of the transfected parasites were measured using flow cytometry and methyl thiazole tetrazolium (MTT) assays, respectively. Finally, the survival of BALB/c mice infected with siRNAs-transfected T. gondii was assessed. It was demonstrated that a transfection rate of 75.4% existed for siRNAs, resulting in 70% (P = 0.032), 80.6% (P = 0.017) and 85.5% (P = 0.013) gene suppression for myosin A, C and F in affected parasites, respectively, which was subsequently confirmed by Western blot analysis. Moreover, lower parasite viability was observed in those with knocked down myosin C with 80% (P = 0.0001), followed by 86.15% (P = 0.004) for myosin F and 92.3% (P = 0.083) for myosin A. Considerably higher mouse survival (about 40 h) was, also, demonstrated in mice challenged with myosin siRNA-transfected T. gondii, in comparison with control group challenged with wild-type parasites. In conclusion, myosin proteins knock down proposes a promising therapeutic strategy to combat toxoplasmosis.

Antibody response to glycan antigens of hydatid cyst fluid, laminated layer and protoscolex of Echinococcus granulosus.

Background: Hydatid disease is characterized by long-term growth of hydatid cysts in the human. The glycan antigens have an important role in the immunology of hydatid cyst. In this study immunological reaction of host sera to different glycan antigens of the cyst, has been investigated. Methods: The antibody responses were tested to glycoprotein and glycolipid of the laminated layer (LL), cyst fluid (CF) and protoscolex (PS) antigens of E. Granulosus using ELISA and western immunoblotting tests. Thin-layer chromatography and ß-elimination were used for glycan purification. Results: Both hydatid cyst and normal human sera reacted with hydatid cyst fluid, protoscolices, laminated layer, glycoprotein and glycolipid antigens. The most antigen-antibody reaction was related to CF and PS antigens, and LL antigens had the minimal reaction with the sera. Thin layer chromatography (TLC) of the antigens showed presence of many glycan bands in the laminated layer. Conclusion: The parasite may elaborate different glycan antigens in LL to evade host immune response.

Evaluation of expression variations in virulence-related genes of Leishmania major after several culture passages compared with Phlebotomus papatasi isolated promastigotes.

Cutaneous leishmaniasis (CL) is a prevalent infectious disease with considerable morbidity annually. Here, we aimed to investigate the likely variations in gene expression of glycoprotein63 (gp63), heat shock protein 70 (HSP70), histone, arginase, cysteine protease B (CPB), Leishmania homologue of receptors for activated C kinase (LACK), small hydrophilic endoplasmic reticulum-associated protein (SHERP) in metacyclic promastigotes of L. major isolated from Phlebotomus papatasi sand flies and promastigotes excessively cultured in culture medium. The parasites were collected from suspected CL cases in Pasteur Institute of Iran, cultured and inoculated into the female BALB/c mice (2×106 promastigotes). Sand flies were trapped in Qom province, fed with the blood of euthanized infected mice and subsequently dissected in order to isolate the midgut including stomodeal valve. The metacyclic promastigotes were isolated from Ph. papatasi (Pro-Ppap) using peanut agglutinin test (PNA), then continuously cultured in RPMI-1640 medium enriched with fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 mg/ml) to reach stationary phase (Pro-Stat). The gene expression was evaluated in both parasitic stages (Pro-Ppap and Pro-Stat) using qRT-PCR. Out results showed a significant increased gene expression at Pro-Ppap stage for gp63 (P = 0.002), SHERP (P = 0.001) and histone (P = 0.026) genes, in comparison with Pro-Stat stage. Noticeably, significant changes were, also, demonstrated in 10th to 15th passages [gp63 (P = 0.041), arginase (P = 0.016), LACK (P = 0.025)] and in 5th to 20th passage (SHERP) (P = 0.029). In conclusion, the findings of the present study seem to be essential in designing Leishmania studies, in particular regarding host-parasite interaction, immunization and infectivity studies.

A Triplex Nested PCR for Diagnosis of Plasmodium, Babesia and Toxoplasma in Blood to Promote Safety of Blood Transfusion Samples.

Background: The risk of transmission of some infectious agents has always been an important life-threatening side effect of blood transfusion. The aim of this study was to develop a triplex nested Polymerase Chain Reaction (tnPCR) to assess the presence of protozoan parasites Plasmodium, Toxoplasma and Babesia in blood samples concurrently. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy of developed tnPCR method compared to gold standard methods were determined for all three parasite genera. Methods: After selecting the genus level specific primers and setting up of tnPCR, blood samples were collected partially from Isfahan Blood Transfusion Organization (IBTO). Some different samples from human and animal were tested by this method in comparison with the gold standard methods (microscopic method for Plasmodium, Babesia and ELISA for Toxoplasma) in 2021. Results: This tnPCR works well and the sensitivity, specificity, PPV, NPV and accuracy, of this molecular method were all 100% for Plasmodium spp., 93.33%, 99.16%, 93.33%, 99.16% and 99.25% for Babesia spp. and 100%,98.5%, 85.72%, 98.5% and 100% for Toxoplasma gondii respectively compared to standard methods. In average there were 100%, 99.22%, 95.24%, 99.5% and 99.75% contingency for all three parasites (α<0.05). The designed and provided method can detect one, two, or all three potentially dangerous pathogens simultaneously in one tube and one-step, in biological specimens as well as blood. Conclusion: The developed tnPCR worked well. It could be recommended for facilitating test, saving time, reducing the expense and cross contamination, subsequently the promotion of blood transfusion safety.

Misdiagnosis of Plasmodium vivax in a Case of Mixed Malaria, Lead to Wrong Anti-Cancer Chemotherapy, Splenectomy, and Partial Hepatectomy Due to Relapse: A Case Report.

Malaria is a multilateral parasitic infection, which causes wonderful mortality and morbidity worldwide. It sometimes accompanied a quaint appearance. An Iranian 50-year-old man was admitted to Omid, hospital, a specialized cancer hospital in Isfahan, Iran. Because of a 15-year persisted anemia due to misdiagnose of vivax malaria led him to three courses of anticancer chemotherapy and splenectomy. His blood smears were sent to the Department of Parasitology, School of Medicine, Isfahan University of Medical Sciences, Iran. Our findings from his history, file documents, clinical signs and symptoms, and parasitological and molecular assessments revealed an interesting case, which is reported.

Evaluation of Th17 immune responses of recombinant DNA vaccine encoding GRA14 and ROP13 genes against Toxoplasma gondii in BALB/c mice.

Toxoplasma gondii, a worldwide opportunistic parasite, causes serious diseases in both humans and fetuses with defective immune systems. The development of an effective vaccine is urgently required to prevent and control the spread of toxoplasmosis, caused by the apicomplexan parasite Toxoplasma gondii which is one of the most damaging zoonotic diseases of global importance. Plasmid DNA vaccination is a promising procedure for vaccine development and following the previous studies, pcROP13 + pcGRA14 cocktail DNA vaccine was evaluated for Th17 immune responses. Four groups of BALB/c mice were immunized intramuscularly three times at 2-week intervals. Subsequently, the production of anti- T. gondii antibodies and serum levels of cytokines IL-17, and IL-22 were evaluated against the RH strain of T. gondii. In addition, both the reactive oxygen species (ROS) and parasite load were assessed using ELISA and Q-PCR, respectively. The results of this study showed that high levels of IgG were found in mice immunized with cocktail DNA vaccine (p < 0.05). The cytokines level of Th17, IL-17, and IL-22, increased remarkably in the immunized mice (p < 0.05). Also, significant induction (p < 0.05) was observed in ROS. In addition, immunization with pcROP13 + GRA14 resulted in a considerable decrease in parasite load compared to the control groups (p < 0.05). Based on the results, the pcROP13 + GRA14 cocktail DNA vaccine induced Th17 related cytokines and decreased the parasite load in spleen and brain tissues. Hence, pcGRA14 + pcROP13 cocktails are suitable candidates for DNA-based vaccines and due to the development of protective immune responses against T. gondii infection, future studies may yield promising results using these antigens in vaccine design.

Knocking down of the DHFR-TS gene in Toxoplasma gondii using siRNA and assessing the subsequences on toxoplasmosis in mice.

Toxoplasma gondii (T. gondii), an obligatory intracellular parasite, is the etiologic agent of toxoplasmosis. Dihydrofolate reductase-thymidylate synthase (DHFR-TS) is one of the most important enzymes in toxoplasma folic acid cycle. Due to the emergence of resistance in RH strain of T. gondii against pyrimethamine that acts via DHFR-TS inhibition and also the crucial role of small interference RNA (siRNA) technology in gene silencing, we aimed to use siRNA to knock down DHFR-TS gene expression in T. gondii as a therapeutic target against toxoplasmosis in a mouse model. Based on the DHFR-TS gene sequence, siRNA was designed. The siRNAs were transfected into the parasites by electroporation. Total RNA was extracted using RNX-Plus kit. The viability of parasite was assessed by methylthiazole tetrazolium (MTT). The survival time of mice challenged with siRNA-treated T.gondii were compared to the control group infected with the same amount of wild-type tachyzoites. The viability of siRNA-embedded parasites was 70.7% (29.3% decreased) compared to the wild-type parasite as control (P = 0.0001). The transcription level of siRNA-transfected parasites was reduced to 17.4% (82.6% inhibition) (P = 0.016). The in vivo assessment showed that the mean survival time of the mice inoculated with modified parasites was increased about 2 days after the death of all mice in the control group. The designed siRNAs in the current study were able to silence the DHFR-TS gene efficiently. This silencing led to a decrease in viability of the parasites and an increase in the survival time of the parasites-treated mice.

Antileishmanial activity of prenylated coumarins isolated from Ferulago angulata and Prangos asperula.

Leishmaniasis has a wide spectrum of signs and symptoms due to infection to numbers of Leishmania species and makes enormous mortality and morbidity. There are clues of antileishmanial effects of prenylated coumarins. Apiaceae family is one of the most important sources of coumarins. Air-dried aerial parts of Ferulago angulata and fruits of Prangos asperula were extracted with n-hexane, using a soxhlet apparatus. The solvents were evaporated under reduced pressure. Column chromatography and crystallization process resulted to isolation of three prenylated coumarins. (1)H-nuclear magnetic resonance, electron ionization Mass and Infrared spectra were used for elucidation of isolated compounds. Leishmanicidal activity of isolated coumarins was assessed on Leishmania major strain (MRHO/IR/75/ER) for the first time. Suberosin epoxide and suberosin were isolated from aerial parts of F. angulata and osthol was extracted from grounded fruits of P. asperula. Osthol showed a significant antileishmanial effect on promastigotes in early hours of exposure with IC50 of 14.40 µg/mL but suberosin epoxide showed only a weak antileishmanial activity. IC50 of osthol and suberosin epoxide after 48 h were 10.79 and 54.0 µg/mL, respectively. Suberosin showed no remarkable effect in these concentrations. This is the first report on the pharmacological activity of suberosin epoxide. Substantial difference between efficacies of two isomers, osthol and suberosin remarks the importance of prenyl substituent location on C-8.

Do mutations in Plasmodium falciparum dihydropteroate synthase and dihydrofolate reductase confer resistance to sulfadoxine-pyrimethamine in Iran?

The key codons of dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr) genes implicated in sulfadoxine-pyrimethamine (SP) resistance were determined by allele-specific polymerase chain reaction in Plasmodium falciparum isolates collected in 1999 from 35 Iranian patients treated with SP. Seven isolates had Glu-540 dhps allele but 5 of these isolates were characterized to possess wild-type dhfr alleles. Seven additional isolates were polyclonal with mixed Lys- and Glu-540. The key dhfr mutation associated with pyrimethamine resistance, Asn-108, was found in 4 isolates. In one patient the presence of Lys- and Glu-540 in dhps and Asn-108 and Arg-59 in dhfr was associated with treatment failure. However, more studies are needed to determine whether clinical response to SP and mutations in these genes are correlated in Iran.