Neutrophil extracellular traps formation: effect of Leishmania major promastigotes and salivary gland homogenates of Phlebotomus papatasi in human neutrophil culture.

BACKGROUND: Leishmaniasis as a neglected tropical disease (NTD) is caused by the inoculation of Leishmania parasites via the bite of phlebotomine sand flies. After an infected bite, a series of innate and adaptive immune responses occurs, among which neutrophils can be mentioned as the initiators. Among the multiple functions of these fighting cells, neutrophil extracellular traps (NETs) were studied in the presence of Leishmania major promastigotes and salivary gland homogenates (SGH) of Phlebotomus papatasi alone, and in combination to mimic natural conditions of transmission. MATERIAL & METHODS: The effect of L. major and SGH on NETs formation was studied in three different groups: neutrophils + SGH (NS), neutrophils + L. major (NL), neutrophils + L. major + SGH (NLS) along with negative and positive controls in 2, 4 and 6 h post-incubation. Different microscopic methods were used to visualize NETs comprising: fluorescence microscopy by Acridine Orange/ Ethidium Bromide staining, optical microscopy by Giemsa staining and scanning electron microscopy. In addition, the expression level of three different genes NE, MPO and MMP9 was evaluated by Real-Time PCR. RESULTS: All three microscopical methods revealed similar results, as in NS group, chromatin extrusion as a sign of NETosis, was not very evident in each three time points; but, in NL and especially NLS group, more NETosis was observed and the interaction between neutrophils and promastigotes in NL and also with saliva in NLS group, gradually increased over times. Real-time reveals that, the expression of MPO, NE and MMP9 genes increased during 2 and 4 h after exposure, and then decreased at 6 h in most groups. CONCLUSION: Hence, it was determined that the simultaneous presence of parasite and saliva in NLS group has a greater impact on the formation of NETs compared to NL and NS groups.

Design and evaluation of loop-mediated isothermal amplification for rapid detection of Enterocytozoon bieneusi.

Enterocytozoon bieneusi is one of the most prevalent microsporidia species, responsible for more than 90% of human and animal microsporidiosis. Microsporidia species, particularly E. bieneusi, are frequently reported from waterborne and foodborne outbreaks. Therefore, early detection is crucial in clinics and outbreak investigations. This study aimed to design a loop-mediated isothermal amplification (LAMP) for rapid detection of E. bieneusi. Total DNA was extracted from 30 E. bieneusi -positive samples, which had been confirmed with nested PCR. LAMP primers were designed based on the identical fragment of small subunit ribosomal RNA (SSU rRNA) gene. LAMP reactions were performed at 63 °C for 60 min. The sensitivity and specificity of the assay were analyzed and the results of amplification were compared to real-time PCR. Our results showed that the LAMP assay successfully amplified 25/30 (83.3%) samples. The specificity results indicated no false positive with other microorganisms. Furthermore, the LAMP method exhibited a sensitivity (limit of detection, LoD) as low as 34 ag/µL of total DNA. Compared to the LAMP assay, real-time PCR was able to detect all 30 nested PCR-positive samples. Our findings showed that the LAMP assay was able to detect 83.3% of E. bieneusi-positive samples. Although the current assay was not able to detect all nested PCR-positive samples, the lack of need for specific instruments, rapid processes, and high specificity makes LAMP assay a suitable tool for screening.

Assemblage characterization of Giardia duodenalis in South Khorasan province, eastern Iran, using HRM real-time PCR method.

BACKGROUND: Giardia duodenalis is a common parasitic protozoan causing gastrointestinal illness in humans worldwide. The genetic diversity of G. duodenalis is reflected through the identification of different assemblages. In this study, we aimed to determine the assemblages of G. duodenalis in eastern Iran using nested-PCR and high-resolution melting (HRM) real-time PCR methods. METHODS: A total of 58 positive G. duodenalis, which were isolated from 1800 subjects, referred to medical center laboratories in South Khorasan province, eastern Iran, from April 2020 to March 2022, were included in this study. DNA was extracted and HRM real-time PCR was performed for assemblage characterization. RESULTS: HRM real-time PCR successfully characterized all samples. Accordingly, out of 58 positive samples, 53 (91.36%) and 5 (8.62%) were identified as assemblage A and B, respectively. CONCLUSIONS: Our findings showed that HRM real-time PCR was able to characterize the assemblages of G. duodenalis. In addition, our results suggest high prevalence of assemblage A in eastern region of Iran.

Molecular Detection of Plasmodium Infection among Anophelinae Mosquitoes and Differentiation of Biological Forms of Anopheles Stephensi Collected from Malarious Areas of Afghanistan and Iran.

Background: Updated information on the vectorial capacity of vectors is required in each malarious areas as well in Iran and its neighboring countries such as Afghanistan. The aims of this study were to investigate the potential infection of about 800 specimens collected from malarious areas of Afghanistan and Iran, and to differentiate biological forms of Anopheles stephensi. Method: Two molecular markers, 18S RNA gene subunit and AsteObp1 intron I, were used respectively for investigation Plasmodium infection and identifying the biological forms of An. stephensi. Results: Plasmodium infection was detected in 4 pools of Afghanistan specimens, including An. stephensi, collected from Nangarhar. Individually examination showed infection in 5 An. stephensi (infection rate: 1.25), to P. falciparum (2), P. vivax (2) and a mix infection. Out of five infected specimens, three were intermediate forms and two were mysorensis. No infection was found in specimens collected from Iran (Chabahar County), probably due to the active malaria control program in south-east of Iran. Conclusion: The key role of An. stephensi, as a known Asian malaria vector, was re-emphasized in Afghanistan by the results achieved here. The fauna of vectors and the pattern of biological forms of An. stephensi are similar in both countries that urge regional investigations to provide evidence-based and applied data for decision-maker in malaria control.

Successful Isolation of Leishmania RNA Virus (LRV) from Leishmania major in a Cutaneous Leishmaniasis Focus in Central Iran: An Update on Cases.

PURPOSE: Cutaneous leishmaniasis (CL) is a major vector-borne disease that affects people globally, including Iran. Different factors are associated with leishmaniasis pathogenicity; recently, a link of the possible relationship between Leishmania RNA Virus (LRV) and disease severity was proposed, especially in the New World leishmaniasis (NWL). This study was aimed to investigate the presence of LRV2 in Leishmania isolates in Aran o Bidgol, Isfahan province. METHODS: Samples were collected from 110 CL-suspected patients referred to the health center. In this study, we aimed to investigate CL cases (parasitologically and clinically), identify Leishmania species (by ITS1-PCR-RFLP), and finally detection of LRV2 (by RdRp-semi-nested PCR). RESULTS: Parasitological methods showed 60 positive cases, based on the HaeIII enzyme restriction profile, 59 cases were caused by L. major and 1 case by L. tropica. Our project is the first study on LRV2 isolation in Aran o Bidgol city and the LRV was successfully detected from a single L. major isolated in a women's hand lesion. Using BLAST, 94.8-100% similarity was observed in the RdRp sequence of current LRV isolate with those available in GenBank from Iran or overseas. CONCLUSION: L. major was the main cause of CL in Aran o Bidgol, although L. tropica is also present in a much lower proportion in the area. This is the first report on the presence of LRV2 in Aran o Bidgol and the fifth in Iran.

Association of Blastocystis ST6 with higher protease activity among symptomatic subjects.

BACKGROUND: Blastocystis sp. is an anaerobic intestinal protozoan parasite of humans and a wide range of animals worldwide. In the current study the correlation between the cysteine protease activity of clinical samples of Blastocystis sp. ST1-3 and 6 with the levels of pro-inflammatory cytokines was evaluated. METHODS: Stool samples were collected from subjects with or without clinical symptoms. All samples were cultivated in DMEM medium. The bacteria were eliminated or reduced in Blastocystis sp. positive samples subtypes 1-3 and 6 by a variety of antibiotics and consecutive sub-cultures. To prepare parasite lysate, 1 × 105 Blastocystis sp. from each isolate were harvested and lysed using freeze-thaw. Protease activity of each isolate was measured and the gene expression of pro-inflammatory biomarkers in HT-29 cell line sensed by isolates was investigated using quantitative Real-time PCR. RESULTS: Protease activity assay showed inter- and intra-subtype variations among subtypes regarding the presence of symptoms, while the protease activity of symptomatic isolates was higher than asymptomatic isolates. The highest and lowest levels of protease activity were seen in ST6 and ST2, respectively. However, patterns of the expression of pro-inflammatory biomarkers in HT-29 cell line was different regarding the presence of symptoms and time points. There was no significant correlation between protease activity of different subtypes with the expression levels of pro-inflammatory biomarkers. CONCLUSIONS: Our study indicated a higher protease activity among isolates from symptomatic compared to asymptomatic subjects, suggesting functional role for proteases in clinical symptoms due to Blastocystis sp. The lack of correlation between the levels of expression of pro-inflammatory biomarkers with subtypes regarding the presence of clinical symptoms proposes the importance of host-related factors in presentation of clinical symptoms.

Immunodiagnosis and molecular validation of Toxoplasma gondii-recombinant dense granular (GRA) 5 protein for the detection of toxoplasmosis in hemodialysis patients.

Toxoplasmosis causes serious complications in immunocompromised and pregnant women. Serological tests for the detection of toxoplasmosis are often designed from parasitic tachyzoites antigens. The process of producing these antigens is very difficult. The purpose of this study was evaluation of T. gondii-rGRA5 for the immunodiagnosis and molecular detection of Toxoplasma infection using enzyme-linked immunosorbent assay (ELISA) and LAMP methods in hemodialysis patients. The GRA5 gene was successfully expressed and purified by affinity chromatography assay and evaluated by western blot. Then it was used to design an ELISA assay. A total of 260 samples were tested for anti-Toxoplasma IgG and IgM antibodies using a commercial ELISA kit and designed ELISA kit. Finally, the LAMP method was used to evaluate the precision and reliability of the results obtained by commercial and designed ELISA kits. The consistency of the results of two methods was analyzed using the Kappa coefficient of agreement. The rGRA5 revealed higher immunoreactivity with 1:100 dilution of sera from toxoplasmosis patients. The specificity and sensitivity of the assay were 93% and 96%, respectively. According to the Kappa coefficient, there was a substantial correlation between the results of ELISA and LAMP based on rGRA5 (≈98%, p < 0.001). Also it showed that rGRA5 protein can be used as an antigenic protein for designing sero-diagnostic tests to identify Toxoplasma infection especially in hemodialysis patients.

Novel somatic variants in CTNNA1 gene in Iranian patients with diffuse gastric cancer.

AIM: we aimed to evaluate somatic mutations of CTNNA1 in DGC patients. BACKGROUND: Diffuse gastric cancer (DGC) is a major type of gastric cancer where most cases are sporadic diffuse gastric cancer (SDGC). It has been shown that mutations in CTNNA1 are responsible for some cases of hereditary diffuse gastric cancer (HDGC). METHODS: In the present work, 48 formalin-fixed paraffin-embedded tissues, including samples of 38 SDGC and 10 HDGC patients were examined through Sanger sequencing approach on PCR products amplified from 18 exons and boundaries of intron/exon of CTNNA1 gene. RESULTS: We revealed 9 novel somatic mutations in CTNNA1 gene in patients with HDGC and SDGC, from which one variant was intronic. Eight patients had at least one disease-causing mutation (16.6%). Most of the patients were in the III stage of cancer (50%). Except for one patient, histological type of the rest of mutation-harboring patients was signet ring cell carcinoma, and only one HDGC patient had CTNNA1 mutation. CONCLUSION: Our study showed several novel variants in the CTNNA1 gene in Iranian sporadic and hereditary DGC patients, and implies that the CTNNA1 gene mutations could be involved in the pathogenesis of DGC, either hereditary or in sporadic cases.

Inhibition of anti-inflammatory cytokines, IL-10 and TGF-ß, in Leishmania major infected macrophage by miRNAs: A new therapeutic modality against leishmaniasis.

Leishmania major (L. major) applies several mechanisms to escape the immune system. Interleukin-10 (IL-10) and Transforming Growth Factor (TGF-ß) downregulate nitric oxide synthase (iNOS) leading to the survival of Leishmania within macrophages. The miRNAs are known as the modulators of the immune system. The present study was conducted to assess the effect of synthetic miR-340 mimic on cytokines (IL-10 and TGF-ß1) involved in L. major infected macrophages. The miRNAs targeting of IL-10 and TGF-ß1 was predicted using bioinformatic tools. Relative expression of predicted miRNA, IL-10, and TGF-ß1 was measured by RT-qPCR before and after synthetic miRNA mimic transfection. Concentration of IL-10 and TGF-ß was measured in posttreatment condition using ELISA method. Also, infectivity was assessed by Giemsa staining. mmu-miR-340 received the highest score for targeting cytokines. The expression of miR-340 was downregulated in L. major infected macrophages. By contrast, expression of IL-10 and TGF-ß1 was upregulated in infected macrophages. After miRNA transfection, TGF-ß1 and IL-10 were both downregulated and interestingly, the combination of miR-340 and miR-27a had a stronger effect on the downregulation of target genes. This research revealed that transfection of infected macrophages with miR-340 alone or in combination with miR-27a mimic can reduce macrophage infectivity and might be introduced as a novel therapeutic agent for cutaneous leishmaniasis.

A Nuclear Magnetic Resonance-Based Metabolomic Study to Identify Metabolite Differences between Iranian Isolates of Leishmania major and Leishmania tropica.

Background: Cutaneous leishmaniasis caused by Leishmania species (L. spp) is one of the most important parasitic diseases in humans. To gain information on the metabolite variations and biochemical pathways between L. spp, we used the comparative metabolome of metacyclic promastigotes in the Iranian isolates of L. major and L. tropica by proton nuclear magnetic resonance (1H-NMR). Methods: L. tropica and L. major were collected from three areas of Iran, namely Gonbad, Mashhad, and Bam, between 2017 and 2018, and were cultured. The metacyclic promastigote of each species was separated, and cell metabolites were extracted. 1H-NMR spectroscopy was applied, and the data were processed using ProMatab in MATLAB (version 7.8.0.347). Multivariate statistical analyses, including the principal component analysis and the orthogonal projections to latent structures discriminant analysis, were performed to identify the discriminative metabolites between the two L. spp. Metabolites with variable influences in projection values of more than one and a P value of less than 0.05 were marked as significant differences. Results: A set of metabolites were detected, and 24 significantly differentially expressed metabolites were found between the metacyclic forms of L. major and L. tropica isolates. The top differential metabolites were methionine, aspartate, betaine, and acetylcarnitine, which were increased more in L. tropica than L. major (P<0.005), whereas asparagine, 3-hydroxybutyrate, L-proline, and kynurenine were increased significantly in L. major (P<0.01). The significantly altered metabolites were involved in eight metabolic pathways. Conclusion: Metabolomics, as an invaluable technique, yielded significant metabolites, and their biochemical pathways related to the metacyclic promastigotes of L. major and L. tropica. The findings offer greater insights into parasite biology and how pathogens adapt to their hosts.

Comparative Expression Profile Analysis of Apoptosis-Related miRNA and Its Target Gene in Leishmania major Infected Macrophages.

BACKGROUND: Cutaneous Leishmaniasis (CL) is an emerging uncontrollable and neglected infectious disease worldwide including Iran. The aim of this study was to investigate the expression profile of apoptosis-related miRNA and its target gene in macrophages. METHODS: This study was carried out in the Department of Medical Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran from January 2016 to November 2018. Applying literature reviews, bioinformatics software, and microarray expression analysis, we selected miRNA-24-3p interfering in apoptosis pathway. The expression profile of this miRNA and target gene were investigated in Leishmania major (MRHO/IR/75/ER)-infected primary and RAW 264.7 macrophages (IBRC-C10072) compared with non-infected macrophages (control group) using quantitative Real-time PCR. RESULTS: Results of bioinformatics analysis showed that miR-24-3p as anti-apoptotic miRNA inhibits pro-apoptotic genes (Caspases 3 and 7). Microarray expression data presented in Gene Expression Omnibus (GEO) revealed a significant difference in the expression level of selected miRNA and its target gene between two groups. QRT-PCR results showed that the expression of miR-24-3p was upregulated in L. major infectioned macrophages that approved the results of bioinformatics and microarray analysis. CONCLUSION: Parasite can alter miRNAs expression pattern in the host cells to establish infection and its survival. Alteration in miRNAs levels likely plays an important role in regulating macrophage functions following L. major infection. These results could highlight current understanding and new insights concerning the gene expression in macrophages during leishmaniasis and will help to development of novel strategies for control and treatment of CL.

Anti-Leishmanial Effects of Chitosan-Polyethylene Oxide Nanofibers Containing Berberine: An Applied Model for Leishmania Wound Dressing.

BACKGROUND: Leishmaniasis is the most important parasitic disease in Iran and is the third highest rate of rural cutaneous leishmaniasis in the world. Chitosan-polyethylene oxide nanocomposite fibers can be a suitable replacement for ordinary bandages. For the first time, in the absence of any published reports, the present in vitro study aimed to evaluate the anti-leishmanial effects of chitosan (CS)-polyethylene oxide (PEO)-berberine nanofibers on Leishmania major. METHODS: The present experimental study was conducted in 2018 in Tehran, Iran. The CS-PEO nanofibers containing berberine, as a natural anti-parasitic agent, were prepared using the electrospinning technique. Biocompatibility and fibroblast proliferation on nanofibers were investigated. In addition, the anti-leishmanial activity of CS-PEO nanofibers in both the promastigote and amastigote stages of Leishmania major was evaluated after parasite vital staining and MTT assay and compared to a control group. Statistical analysis was performed using SPSS software (version 18.0). Statistically significant differences were determined using the one-way ANOVA. The Duncan and Dunnett post hoc tests were used for within-group comparisons. P<0.05 was considered statistically significant. RESULTS: The results showed that nanofiber scaffolds with a mean diameter of 77.5±19.5 nm were perfect, regular, bead-free, and non-toxic, on which fibroblast cells grew well and proliferated. In addition, the optical density indicated that berberine 20% (w/v) significantly prevented promastigotes growth (IC50=0.24 µg/mL) and amastigotes death (IC50=0.91 µg/mL) compared with other concentrations and the control group. CONCLUSION: The study on the cytotoxic effects showed that CS-PEO-berberine nanofibers had strong lethal effects on Leishmania major in promastigote and amastigote stages in vitro. Further studies are required to investigate the effects of this nanofiber on leishmanial ulcers in laboratory animals and clinical cases.

Chitosan-based nano-scaffolds as antileishmanial wound dressing in BALB/c mice treatment: Characterization and design of tissue regeneration.

OBJECTIVES: Rapid healing of cutaneous leishmaniasis as one of the most important parasitic diseases leads to the decrease of scars and prevention of a great threat to the looks of the affected people. Today, the use of nano-scaffolds is rapidly increasing in tissue engineering and regenerative medicine with structures similar to the target tissue. Chitosan (CS) is a bioactive polymer with antimicrobial and accelerating features of healing wounds, which is commonly used in biomedicine. This study aimed to investigate the effects of CS/polyethylene oxide (PEO)/berberine (BBR) nanofibers on the experimental ulcers of Leishmania major in BALB/c mice. MATERIALS AND METHODS: CS/PEO/BBR nanofibers were prepared by the electrospinning method, and their morphology was examined by SEM, TEM, and AFM. Then, water absorption, stability, biocompatibility, porosity, and drug release from nano-scaffolds were explored. Afterward, 28 BALB/c mice infected with the parasite were randomly divided into control and experimental groups, and their wounds were dressed with the produced nano-scaffolds. Finally, the effect of nanobandage on the animals was investigated by macroscopic, histopathologic, and in vivo imaging examinations. RESULTS: The prepared nanofibers were completely uniform, cylindrical, bead-free, and biocompatible with an average diameter of 94±12 nm and had appropriate drug release. In addition, the reduced skin ulcer diameter (P=0.000), parasite burden (P=0.003), changes in the epidermis (P=0.023), and dermis (P=0.032) indicated significantly strong effectiveness of the produced nano-scaffolds against leishmania ulcers. CONCLUSION: Studies showed that CS/PEO/BBR nanofibers have a positive effect on the rapid healing of leishmania ulcers. Future studies should focus on other chronic ulcers treatment.

Designing a Recombinant Multi-Epitope Antigen of Echinococcus granulosus to Diagnose Human Cystic Echinococcosis.

BACKGROUND: Cystic echinococcosis can cause severe disease and probable death in humans. Epitopes of its antigens play a key role in the sensitivity and specificity of immunodiagnostic tests. METHODS: Epitope prediction software programs predict the most antigenic linear B-cell epitopes of AgB (8 kD), Ag5, and Ag95. Six such epitopes were predicted and connected by "Gly-Ser" linker and synthesized. The purity of the concentrated recombinant multi-epitope protein was assessed by 15% SDS-PAGE. Overall, 186 serum samples were collected from the Loghman Hakim Hospital and different laboratories, Tehran, Iran, from July 2016 to February 2017. Patients infected with hepatic hydatid cysts, patients infected by other parasites and viruses, and healthy individuals were used to detect the anti-CE IgG using recombinant multi-epitope protein. RESULTS: Forty-one samples out of 43 cases of hydatidosis were diagnosed correctly as positive, and two were negative. In addition, six negative cases of healthy individual group were diagnosed as positive and negative with rMEP-ELISA and the commercial kit, respectively. Therefore, these six samples were considered as false positive using our method. In addition, a diagnostic sensitivity of 95.3% (95% CI, 84.19% to 99.43%) and a specificity of 95.0% (95% CI, 89.43% to 98.14%) were obtained using optimum cutoff value (0.20). The sensitivity and specificity of the commercial kit was 100%. CONCLUSION: Our findings showed high diagnostic accuracy of the ELISA test using the developed recombinant protein, which encourages the use of this recombinant multi-epitope protein for rapid serological diagnosis of hydatidosis.

Peptide-Based Monoclonal Antibody Production Against SAG1 (P30) Protein of Toxoplasma gondii.

Toxoplasma gondii is an intracellular protozoan parasite that can infect a wide range of warm-blooded animals. Humans as an intermediate host are infected by ingesting infectious oocytes or tissue cysts, or passing through the placenta in pregnant women. The aim of this study is producing monoclonal antibodies against a synthetic peptide from (surface antigen 1 [SAG1] or P30) protein of T. gondii. A synthetic peptide from SAG1 (P30) protein was conjugated to Keyhole Limpet Hemocyanin (KLH (and then used for immunization of two BALB/c mice. The produced antibody was purified by affinity chromatography and its specific interaction with the immunized peptide was then determined by enzyme-linked immunosorbent assay (ELISA). Immunoreactivity of the antibody was also tested by Western blot in T. gondii cell lysate. The results show that the produced antibody has excellent reactivity with the immunizing peptide and also detects a single band of 30 kDa, which corresponds to SAG1 protein. This antibody can be used as a tool in different applications in T. gondii research areas, including diagnosis, therapy, and infection inhibition.

Development and Evaluation of Modified Cryopreservation for Long-Term Storage of Blastocystis Subtypes 1-3 and 6.

BACKGROUND: Blastocystis is a protozoan parasite living in the intestine of humans and a wide range of animals. Although Blastocystis grows in several cultivation media, axenification and serial cultivations for long time are the main challenges of the researchers. Therefore, the long-term storage of subtypes/strains of Blastocystis using cryopreservation provides a suitable source of this parasite for the physiological, biochemical, and biological studies. METHODS: In the current study, seven xenic isolates including two separated isolates from ST1-3 and one isolate from ST6 were cryopreserved using a standard method with minor modifications. After 3 months, all isolates were recovered and cultivated in DMEM medium. RESULTS: The findings of the method showed all seven isolates were successfully recovered in DMEM medium. In addition, all isolates remained viable after several sub-cultures. CONCLUSIONS: It seems that cryopreservation is a simple method that can provide a suitable condition for the long-term storage of Blastocystis.

Construction of A Synthetic Gene Encoding the Multi-Epitope of Toxoplasma gondii and Demonstration of the Relevant Recombinant Protein Production: A Vaccine Candidate.

BACKGROUND: Toxoplasma gondii is a widely-distributed parasite all over the world whose attributed severe afflicting complications in human necessitate the development of serodiagnostic tests and vaccines for it. Immunological responses to monovalent vaccines and the application of diagnostic reagents including single antigens are not optimally effective. Bioinformatics approaches were used to introduce these epitopes, predict their immunogenicity and preliminarily evaluate their potential as an effective DNA vaccine and for serodiagnostic goals. MATERIALS AND METHODS: A 3D structure of proteins was predicted by I-TASSER server, and linear and conformational B cell and T cell epitopes were predicted using the online servers. Then, the predicted epitopes were constructed and called Toxoeb, and their expression in the prokaryotic and eukaryotic cells was demonstrated using SDS-PAGE. In the next step, Western blotting with pooled sera of mice infected with T. gondii was done. RESULTS: The current in silico analysis revealed that the B cell epitopes with high immunogenicity for GRA4 protein were located in the residues 34-71, and 230-266, for GRA14 in 308-387, for SAG1 in 182-195, 261-278, and for GRA7 in residues 101-120, 160-176. The T cell epitopes were selected in overlapping regions with the B cell epitopes. The immunogenic region for GRA4 are in the residues 245-253, 50-58, and 40-54, for GRA14 in 307-315, 351-359, and 308- 322, for SAG1 261-269, and 259-267, and for GRA7 in the residues 103-112, and 167-175. The results of the western blotting showed that the expressed protein had immunogenicity. CONCLUSION: Our constructed multi-epitope of T. gondii could be considered as a candidate for diagnostic and vaccination purposes.

Serological and molecular approaches in clinical diagnosis of ocular toxoplasmosis in Iran.

Background: Toxoplasma gondii (T. gondii) is the most common parasite that can lead to a disease called toxoplasmosis. In this study, serological and molecular complementary tests have been conducted to detect or diagnose this parasite. Methods: A total of 71 patients with clinical symptoms of ocular toxoplasmosis and 20 patients with other ocular infections were evaluated. Serum and buffy coat samples were collected and tested using enzyme-linked immunosorbent assay (ELISA) and nested polymerase chain reaction (nPCR) assessments. Superficial T. gondii B1 gene was evaluated in PCR. The ocular toxoplasmosis patients were followed-up 2 weeks after the first sampling and 4 weeks following the first laboratory testing. The main outcome measures were the efficiency of the diagnostic procedure and positive and negative predictive values (PPV and NPV). Results: Overall, of the samples, 69% were PCR+, IgG+, and IgM-, and 4.2% showed PCR+, IgG+, and IgM+. In the first follow-up, after 2 weeks, from the 41 referred patients, 29 (70%) showed PCR+, IgG+, and IgM-, which confirmed the results of the first sampling. In the second follow-up, 9 (47%) patients were PCR+, IgG+, and IgM-. A correlation was observed between the first referral and the follow-ups. Also, from 71 patients, diagnosed clinically as ocular toxoplasmosis, the disease was confirmed in 73.2% and 26.8% of those suffering from other ocular infections. Of the 20 control group samples, 55% showed PCR-, IgG+, and IgM-. The sensitivity, specificity, negative and positive predictive values, and negative and positive likelihoods were analyzed for IgG and IgM antibodies and for PCR using ELISA method. Conclusion: As the ophthalmologic signs of T. gondii may be mimicked by other infections, clinical methods may be complemented by laboratory approaches for a definite diagnosis. This would assist clinicians to achieve timely diagnosis and successful therapy and to control the infection.

Intra-Species Diversity of Leishmania major and L. tropica from Clinical Isolates of Cutaneous Leishmaniasis in Southwest Iran Inferred by ITS1-rDNA.

BACKGROUND: Cutaneous leishmaniasis (CL) as a public health concern is increasingly circulating by causative agents of Leishmania tropica and L. major in Iran. As regard to recent treatment failure and controlling problems, the accurate elucidation of heterogeneity traits and taxonomic status of Leishmania spp. should be broadly addressed by policymakers. This study was designed to determine the genetic variability and molecular characterization of L. major and L. tropica from Iranian CL patients. METHODS: One hundred positive Giemsa-stained slides were taken from clinical isolates of CL at Pol-e-Dokhtar County, Southwest Iran, from May 2014 to Sep 2016. DNAs were directly extracted and amplified by targeting ribosomal internal transcribed spacer (ITS) gene following microscopic observation. To identify Leishmania spp. amplicons were digested by restriction enzyme HaeIII subsequent PCR-RFLP technique. To reconfirm, the isolates were directly sequenced to conduct diversity indices and phylogenetic analysis. RESULTS: Based upon the RFLP patterns, 84 and 16 isolates were explicitly identified to L. tropica and L. major respectively. No co-infection was found in clinical isolates. The high genetic diversity of L. tropica (Haplotype diversity 0.9) was characterized compared to L. major isolates (Hd 0.476). The intra-species diversity for L. tropica and L. major isolates corresponded to 3%-3.9% and 0%-0.4%, respectively. CONCLUSION: Findings indicate the L. tropica isolates with remarkable heterogeneity than L. major are predominantly circulating at Pol-e-Dokhtar County. Occurrence of high genetic variability of L. tropica may be noticed in probable treatment failure and/or emerging of new haplotypes; however, more studies are warranted from various geographic regions of Southwest Iran, using large sample size.

Comprehensive Study of Phenotypic and Growth Rate Features of Blastocystis Subtypes 1-3 and 6 in Symptomatic and Asymptomatic Subjects.

BACKGROUND: The present study aimed to assess the grouping of subtypes 1-3 and 6 of Blastocystis according to the size and generation time of the parasite among the symptomatic and asymptomatic subjects. METHODS: Blastocystis subtypes 1-3 and 6 isolated from symptomatic and asymptomatic subjects and were cultivated in DMEM medium. In order to assess inter- and intra-subtype variation in size, all the isolates were measured using morphometric criteria. Generation time was calculated using approximately 1×104 Blastocystis, which were cultivated in DMEM, every 24h for 4 days. RESULTS: All subtypes had 5 to 185 µm diameter range. The smallest size was attributed to ST1, followed by ST6 and ST2. ST3 showed the most variable size and phenotypes compared with the other three subtypes. Furthermore, amoeboid forms and parasite clumping were only seen in ST3-S (symptomatic subjects). Generation time analysis showed that the number of ST1 isolated from symptomatic and asymptomatic subjects peaked higher than the other subtypes. CONCLUSION: This is the first study discussing inter-intra-size, phenotype and generation time variations among 4 common subtypes of Blastocystis. Accordingly, ST3 was largest subtype and showed most diversities in both size and phenotype, while ST1 was smallest subtype with lowest intra-subtype variation.