Electrochemical immunosensor based on carbon nanofibers and gold nanoparticles for detecting anti-Toxoplasma gondii IgG antibodies.

An electrochemical immunosensor based on carbon nanofibers (CNFs) and gold nanoparticles (AuNPs) was developed for detecting anti-Toxoplasma gondii antibodies  (anti-T. gondii) IgG in human serum. CNFs were produced using electrospinning and carbonization processes. Screen-printed carbon electrode (SPCE) surface was modified with CNFs and AuNPs which were electrodeposited onto the CNFs. Then, T. gondii antigen was immobilized onto the AuNPs/CNFs/SPCE. Afterward, anti-T. gondii IgG positive serum samples were coated on the modified electrode and assessed via adding anti-human IgG labeled with horseradish peroxidase (HRP) enzyme. The morphology of SPCE, CNFs, and AuNPs/CNFs/SPCE surface was characterized using field emission scanning electron microscopy (FESEM) equipped with energy dispersive spectroscopy (EDS). Characterization of CNFs was evaluated by Raman spectroscopy and X-ray diffraction (XRD). Electrochemical characterization of the immunosensor was verified using cyclic voltammetry (CV), and electrochemical response of modified electrode for anti-T. gondii IgG was detected via differential pulse voltammetry (DPV). This immunosensor was detected in the range 0-200 U mL-1 with a low detection limit (9 × 10-3 U mL-1). In addition, the proposed immunosensor was exhibited with high selectivity, strong stability, and acceptable reproducibility and repeatability. Furthermore, there was a strong correlation between results obtained via the designed immunosensor and enzyme-linked immunosorbent assay (ELISA) as gold standard. In conclusion, the developed immunosensor is a promising route for rapid and accurate clinical diagnosis of toxoplasmosis.

Utility of blood as the clinical specimen for the diagnosis of ocular toxoplasmosis using uracil DNA glycosylase-supplemented loop-mediated isothermal amplification and real-time polymerase chain reaction assays based on REP-529 sequence and B1 gene.

BACKGROUND: Ocular infection with Toxoplasma gondii is a major preventable cause of blindness, especially in young people. The aim of the present study was to assess detection rate of T. gondii DNA in blood samples of clinically diagnosed of ocular toxoplasmosis using uracil DNA glycosylase-supplemented loop-mediated isothermal amplification (UDG-LAMP) and real-time quantitative PCR (qPCR) based on REP-529 and B1. METHODS: One hundred and seventeen patients with clinically diagnosed ocular toxoplasmosis (OT) were participated in the study as well as 200 control patients. Peripheral blood samples were assessed using UDG-LAMP and qPCR techniques targeting REP-529 and B1. RESULTS: Detection limits of qPCR using REP-529 and B1 were estimated as 0.1 and 1 fg of T. gondii genomic DNA, respectively. The limits of detection for UDG-LAMP using REP-529 and B1 were 1 and 100 fg, respectively. In this study, 18 and 16 patients were positive in qPCR using REP-529 and B1, respectively. Based on the results of UDG-LAMP, 15 and 14 patients were positive using REP-529 and B1, respectively. Results of the study on patients with active ocular lesion showed that sensitivity of REP-529 and BI targets included 64 and 63%, respectively using qPCR. Sensitivity of 62 and 61%, were concluded from UDG-LAMP using REP-529 and B1 in the blood cases of active ocular lesion. qPCR was more sensitive than UDG-LAMP for the detection of Toxoplasma gondii DNA in peripheral blood samples of patients with clinically diagnosed toxoplasmic chorioretinitis. Furthermore, the REP-529 included a better detection rate for the diagnosis of ocular toxoplasmosis in blood samples, compared to that the B1 gene did. Moreover, the qPCR and UDG-LAMP specificity assessments have demonstrated no amplifications of DNAs extracted from other microorganisms based on REP-529 and B1. CONCLUSIONS: Data from the current study suggest that qPCR and UDG-LAMP based on the REP-529 are promising diagnostic methods for the diagnosis of ocular toxoplasmosis in blood samples of patients with active chorioretinal lesions.

Development of High Resolution Melting Analysis as a Diagnostic Tool for Molecular Detection of Toxoplasma Infection in Pregnant Women and HIV Positive Cases.

BACKGROUND: Toxoplasma gondii is an obligate intracellular protozoan with worldwide distribution. Diagnosis of toxoplasmosis is a very critical issue, especially in pregnant women and immunocompromised patients. The aim of this study was rapid detection of T. gondii DNA in peripheral blood samples (PBS) employing HRM technique and using RE gene. METHODS: Totally, 242 samples from pregnant women and human immunodeficiency virus (HIV) patients were collected from different hospitals and medical centers of Tehran during Oct 2017 to Dec 2018. High resolution melting analysis (HRM) using partial sequences of repetitive element (RE) gene was done and compared with ELISA test. RESULTS: Overall, 51 were positive for acute toxoplasmosis that among them, 12 and 20 reported as positive in pregnant women and HIV+ patients, respectively using HRM technique. Among 70 patients in chronic phase of disease, 10 and 3 samples were reported as positive for pregnant women and HIV+ patients respectively. From 121 negative control, 3 (4.62%) samples associated with HIV+ patients, showed positive real-time PCR and HRM analysis results. CONCLUSION: For the first time, HRM technique via employing RE gene was used for detection of T. gondii infection in PBS. This method is suitable, helpful and in parallel with serological methods for early diagnosis of acute as well as active form of toxoplasmosis in pregnant women and HIV+ patients. The use of techniques based on melt curve and through employing next-generation dyes for diagnosis of T. gondii would be accessible for patients in developing countries.

Evaluation of RE and B1 Genes as Targets for Detection of Toxoplasma gondii by Nested PCR in Blood Samples of Patients with Ocular Toxoplasmosis.

INTRODUCTION: To evaluate B1 and RE genes as targets to detect Toxoplasma gondii, nested PCR is used in blood samples of patients with ocular toxoplasmosis. MATERIALS AND METHODS: Following the measurement of IgG and IgM antibodies using indirect ELISA, IgG avidity and assessment of blood samples by nested PCR, the agreement between various test results was studied. RESULTS: From 117 patients, 77 (65.81%) were found to be positive for IgG anti-Toxoplasma antibody, 12 cases were positive for both IgG and IgM, and 1 patient was positive for IgM only. The detection limit for the RE-nested PCR assay was one T. gondii tachyzoite, whereas the limit for B1-nested PCR was five tachyzoites. Nested PCR results showed higher agreement with IgM test results than IgG test results. CONCLUSION: The results of this study showed that nested PCR of peripheral blood is a useful and non-invasive method for detection of T. gondii in patients with OT, especially in case of recently acquired infections, and RE targeted assay is more sensitive than B1 targeted assay for this purpose.

Detection of Toxoplasma gondii in Acute and Chronic Phases of Infection in Immunocompromised Patients and Pregnant Women with Real-time PCR Assay Using TaqMan Fluorescent Probe.

BACKGROUND: Toxoplasma gondii, cause severe medical complications in infants and immune-compromised individuals. As using early, sensitive and rapid technique has major in diagnosis of toxoplasmosis, the present study was aimed to detect parasite by using from repetitive element (RE) and B1genes, in blood samples of seropositive immuno-compromised patients and pregnant women. METHODS: A total of 110 peripheral blood samples were collected from seropositive cases with anti-T. gondii antibodies, including immunocompromised patients and pregnant women. DNA was extracted by a commercial kit and subjected to TaqMan probe-based real-time PCR assay by using primers and probes specific for RE and B1 genes, separately. The data were analyzed by Kappa test and SPSS-22 software. RESULTS: In the pregnant women, 17 (68%) and 14 (56%) samples from 25 IgM+/ IgG+ cases and, 7 (25%) and 6 (21.4%) samples from 28 IgG+/IgM- cases were positive by RE and B1 real time PCR, respectively. Likewise, in immunocompromised group, 20 (66.6%) and 17 (56.6%) samples from 30 IgM+/ IgG+ cases and 2 (7.4%) and 2 (7.4%) samples from 27 IgG+/ IgM- cases were positive by RE and B1 real time PCR, respectively. CONCLUSION: Probe-based real time PCR assay is a quantitative approach for early diagnosis of T. gondii infection in clinical samples. Moreover, this method can be more appropriate in diagnosis of acute and reactivated toxoplasmosis. In addition our results indicated that RE gene is more sensitive than B1 gene.

Toxoplasma gondii: sexual transmission in mice.

This study was performed to evaluate sexual transmission of Toxoplasma gondii in mice. RH strain tachyzoites were intraperitoneally inoculated into 10 Balb/C male mice and after 48 h, their semen were collected from epididymis and examined by giemsa staining and PCR. Twenty Balb/C female mice mated with four infected male mice four times and any mating time was 48 h whilst 20 female control mice mated with four uninfected male mice for 8 days. Female mate choice was assessed using a three-chambered cage. Four female mice were placed in a central chamber and in one side of it, two infected male mice were kept and in other side, two naïve male mice were placed. Due each quarter, every of the female movement was reported and then the female was replaced to middle chamber. Besides on the detection of DNA and whole parasite in semen, no abortion and death was seen in female mice. Pregnancy was seen only 4 out of 20 female mice which mated with infected males while 17 pregnancies were seen from 20 control female mice (P value = 0.0001). No statistical significant was seen in female mate choice between naïve male (45 movement) and infected male (36 movement). This study showed that toxoplasmosis could not transmit to female mice and their offspring due to mating and the parasite had not effect on female mate choice. It seems that infected male mice cannot entirely mate with females due to reduction of male weapon and body size, physiological vigor and energy.

A planning model for expansion and stagnation of higher education in Iran.

Iran universities of medical sciences have experienced a period of expansion in past decades. Now previous concerns are alleviated, and the former quantity-based policy has given a way to a more quality-seeking attitude. In this study, we developed a planning model for expansion and stagnation of higher education in Iranian universities of medical sciences based on workforce requirements of the country and capabilities of the universities. The plan provided an objectively documented base for the authorities to decide on developmental limits of universities. We devised guidelines for justifying existing programs within universities, assigning new undergraduate and postgraduate programs to universities, voluntary request of universities to cancel a program, and their request to offer new programs for the first time in the country, based on three factors: university educational status, each university-program educational status and the nation's need for each discipline. Related councils of the Ministry of Health and Medical Education legitimately approved the plan and guidelines. In this article, we introduced the methodology of developing the plan, described it and its related guidelines and discussed challenges and limitations we encountered in design and application phases.

CCR7+ central and CCR7- effector memory CD4+ T cells in human cutaneous leishmaniasis.

PURPOSE: The profile of central (=T(CM)) and effector (=T(EM)) memory CD4(+) T cell subsets and the possible role as surrogate markers of protection is studied in the volunteers with history of cutaneous leishmaniasis (HCL). METHODS: Profile of T cell subsets based on CCR7/CD45RA expressions and phenotypic changes after soluble Leishmania antigen (SLA) stimulation were analyzed. Then, sorted CD4(+)CD45RO(-)CD45RA(+) naïve T, CD4(+)CD45RO(+)CD45RA(-)CCR7(-) T(EM,) CD4(+)CD45RO(+)CD45RA(-)CCR7(+) T(CM) subsets were cultured with SLA for proliferation, cytokine production and intracellular cytokine assays. RESULTS: In the HCL and control volunteers, the mean frequencies of CD4(+)CD45RA(+)CCR7(+) naïve T cells and CD4(+)CD45RA(-)CCR7(-) T(EM) cells were higher than the other subsets before culture. Frequency of naïve T cells and CD4(+)CD45RA(-)CCR7(+) T(CM) cells was significantly decreased (P=0.01 for naïve T and P<0.05 for T(CM) cells) and frequency of T(EM) cells was significantly increased after SLA stimulation compared to before culture (P<0.001). By CFSE labeling, CD4(+)CD45RO(+)CD45RA(-)CCR7(+) T(CM) cells showed more proliferation potential than CD4(+)CD45RO(+)CD45RA(-)CCR7(-) T(EM) cells. Stimulation of the T(EM) cells in HCL volunteers induced a significantly higher IFN-γ production (P=0.04) with higher number of intracellular IFN-γ positive cells (P=0.032) than the same cells from controls. A significantly higher number of T(CM) cells produced IL-2 in HCL volunteers compared with controls (P<0.05). Most of the intracellular IFN-γ positive T(EM) cells were proliferating CFSE-dim populations (P<0.05). CONCLUSIONS: A combination of Leishmania-reactive IFN-γ producing CD4(+)CD45RO(+)CD45RA(-)CCR7(-) T(EM) and Leishmania-reactive IL-2 producing CD4(+)CD45RO(+)CD45RA(-)CCR7(+) T(CM) are identified in individuals with history of CL which might play a role in protective recall immune response against Leishmania infection.

Spatial outline of malaria transmission in Iran.

OBJECTIVE: To conduct for modeling spatial distribution of malaria transmission in Iran. METHODS: Records of all malaria cases from the period 2008-2010 in Iran were retrieved for malaria control department, MOH&ME. Metrological data including annual rainfall, maximum and minimum temperature, relative humidity, altitude, demographic, districts border shapefiles, and NDVI images received from Iranian Climatologic Research Center. Data arranged in ArcGIS. RESULTS: 99.65% of malaria transmission cases were focused in southeast part of Iran. These transmissions had statistically correlation with altitude (650 m), maximum (30 °C), minimum (20 °C) and average temperature (25.3 °C). Statistical correlation and overall relationship between NDVI (118.81), relative humidity (⩾45%) and rainfall in southeast area was defined and explained in this study. CONCLUSIONS: According to ecological condition and mentioned cut-off points, predictive map was generated using cokriging method.

Phenotyping of circulating CD8⁺ T cell subsets in human cutaneous leishmaniasis.

Recovery from CL is usually accompanied with long-lasting protection and induction of strong immune response. The phenotypes, generation and maintenance of central (=T(CM)) and effector (=T(EM)) memory T cell subsets in human leishmaniasis are not well known. Profile of T cell subsets were analyzed on peripheral CD8⁺ T cells from volunteers with history of cutaneous leishmaniasis (HCL). In HCL and control groups, mean frequencies of CCR7⁺CD45RA⁺CD8⁺ naïve and CCR7⁻CD45RA⁻CD8⁺ T(EM) cells were higher than other subsets before culture, but after stimulation with soluble Leishmania antigen, the frequency of naïve T cells was significantly decreased and the frequency of T(EM) cells was significantly increased. T(EM) phenotype composed the highest portion of proliferating Carboxy Fluorescein diacetate Succinimidyl Ester (CFSE)-dim population which was significantly higher in HCL volunteers than in control group. Stimulation of isolated CD8⁺ memory T cells, but not naïve T cells, from HCL volunteers induced a significantly higher IFN-γ production compared with that of healthy controls. Intracellular IFN-γ staining provided the same result. Memory population is shown to be responsible for Leishmania-induced IFN-γ production. Leishmania-reactive proliferating T(EM) cells were identified as the most frequent subset which may play a role in recall immune response and protection against Leishmania infection.