Serologic Tests of IgG and IgM Antibodies and IgG Avidity for Diagnosis of Ocular Toxoplasmosis

This prospective study was aimed to detect acute and chronic ocular toxoplasmosis by comparison of anti-Toxoplasma gondii IgM and IgG antibody levels and IgG avidity test. One hundred and seventeen patients with ocular toxoplasmosis (OT) who referred to the Farabi Eye Hospital, Tehran, Iran were included in this study. Of the patients, 77 cases were positive for anti-T. gondii IgG, and 8 cases were positive for anti-T. gondii IgM. IgG avidity test revealed 11, 4, and 102 cases were low, intermediate, and high, respectively, and 6.8% and 9.4% of cases were positive for IgM and IgG avidity tests, respectively (P=0.632). Agreement (Kappa value) between paired tests IgG-IgM, IgG-IgG avidity, and IgM-IgG avidity was 0.080, 0.099, and 0.721, respectively (P < 0.05). This study showed that conventional serologic tests (IgM and IgG levels) and IgG avidity correlate well each other and can be used to differentiate recent infections from old OT. It seems that reactivated old infections rather than recently acquired infections are majority of Iranian OT patients.

Detection of acute toxoplasmosis: the genitally transmittable infection

Toxoplasma gondii is an obligate intracellular parasite that infects a broad range of warm- blooded animals including human. Tachyzoites of T.gondii invade the host cell, replicate and finally lead to the lysis of the cell. T. gondii is associated with congenital infection and it can cause encephalitis, or systemic infection in immunocompromised patients. It is important to know whether the infection is recently acquired or is chronic. Differentiation between acute and chronic infection has a dramatic impact, especially for the developing fetus. In this study, Toxoplasma gondii was detected in acute phase of infection in serum sample of a person who had been accidentally infected with tachyzoites of RH strain in the laboratory. Anti- T.gondii IgG antibody was prepared by rabbit immunization with soluble antigen of tachyzoites of RH strain. Capture- ELISA, immunoblotting and PCR were performed in the laboratory. Antigenemia and parasitemia was detected in serum sample of infected person by capture_ELISA, immunoblotting and PCR techniques respectively. Acute T.gondii infection could be detectable in a short period of time in the sera of infected person

Physical, antioxidant and antimicrobial characteristics of carboxymethyl cellulose edible film cooperated with clove essential oil

Carboxymethyl cellulose [CMC] is one of the most common cellulose derivatives. It has many applications such as edible films and coating. Antioxidant and antimicrobial of essential oils and their direct or indirect usage in foods have been investigated. This study focuses on the physical, chemical, antioxidant and antimicrobial characteristics of CMC edible film incorporated with clove essential oils [CEO]. In this experimental study, CMC edible films with or without clove essential oil were prepared by casting method and many characteristics such as thickness, water vapor permeability [WVP], tensile strength, elongation at break, optical characteristics, microstructure, antimicrobial and antioxidant properties of the films were assessed. Tensile strength values were higher when compared with those of control film [pure CMC film], especially in 1% EO concentration. Elongation at break value in 1% EO was higher than control film, but by increasing of EO portion, it decreased. Antioxidant properties and total phenolic compounds as expected increased in higher concentration of EO. Antimicrobial properties of the films showed that films incorporated with EO are effective against selected pathogen bacteria, especially in the higher concentration of EO. Some variations in the structure of various films were shown by scanning electron microscopy [SEM]. Additions of EO into CMC film disrupted condense structure of film and produced a heterogeneous structure. As antimicrobial and antioxidant properties of CEO retain when it used in CMC edible film, it could be beneficial in food packaging to retard of deterioration

Frequency of Toxoplasma gondii in HIV positive patients from West of Iran by ELISA and PCR

Toxoplasma gondii, the obligate intracellular parasite is life threatening in AIDS patients. Diagnosis of toxoplasmosis is based on serological methods especially increasing of IgM and IgG titers, but finding of parasite or its components [antigenemia] may be beneficial method in order to detection of acute toxoplasmosis in immunocompromised patients. Ninety-four serum samples from HIV positive patients were collected from Sanandaj, Kordistan west of Iran. These patients were lived in Sanandaj of whom 26 were prisoners infected with HIV virus in prison. Toxoplasma gondii antibodies were determined by IgG ELISA. T. gondii antigen was identified by capture- ELISA. PCR was performed on samples with T. gondii antigenemia. CD4+ T cells counts had been determined by flowcytometry and were obtained from records of each patient. Among the examined HIV seropositive individuals, 19.1% [18/94] and 5.3% [5/94] were positive for Toxoplasma-IgG and antigenemia, respectively. Besides, one of the samples was positively detected by PCR method. Mean age of participants was 37.9 +/- 9.5 year. Prevalence of IgG antibody and antgenemia was higher in age group of 40-50 years old. The Mean of CD4+ T cells counts of participants [total of HIV+ patients, IgG positive patients and patients with antigenemia] was 699.2 +/- 345.2, 655.1 +/- 237.9 and 620.2 +/- 215.1 respectively. Capture-ELISA and PCR could confirm the T. gondii acute infection in HIV positive patients. For precise diagnosis of acute toxoplasmosis in HIV positive patient, performance of more studies based on more sensitive types of PCR is suggested

Cloning of 1183 bp fragment from rhoptry protein I [ROPI] gene of toxoplasma gondii [RH] in expression prokaryote plasmid PET32a

Toxoplasma gondii is an obligatory intracellular protozoan. Considering to high prevalence of this disease the best way to reduce the raised loses is prevention of human and animal infection, rapid diagnosis, differentiation between acute and chronic disease. Rhoptry protein 1 of Toxoplasma gondii is an excretory-secretory antigen that exists in the most stages of life cycle. According to specifications of excretory-secretory antigen that seems this antigen is a suitable candidate to produce recombinant vaccine and diagnostic kit. The main object of the present work was cloning rhoptry protein 1 [ROP1] gene of Toxoplasma gondii [RH] in a cloning vector for further production of rhoptry proteins. Genomic DNA was extracted by phenol-chloroform method. The ROP1 fragment was amplified by PCR. This product was approved by sequencing and was cloned between the EcoR1 and Sal1 sites of the pTZ57R/T vector. Then transformed into Escherichia coli DH5 alpha strain and screened by IPTG and X-Gal. After isolating of this gene from pTZ57R/T, it was subcloned into pET32a plasmid. The plasmid was purified and approved by electrophoresis, enzyme restriction and PCR. After isolating of this gene from pTZ57R/T, it was subcloned into pET32a plasmid. After enzyme restriction and electrophoresis a fragment about 1183bp was separated from pET32a. Recombinant plasmid of ROP1 gene was constructed and ready for future study. That seems the antigen is a suitable candidate to produce recombinant vaccine and diagnostic kit

In vitro and in vivo potential of RH strain of toxoplasma gondii [type I] in tissue cyst forming

Based on recent studies, there are controversial reports on the capacity of tissue cyst forming of Toxoplasma gondii RH strain. In this study, the capacity was evaluated by in vivo and in vitro experiments. RH strain was subcutaneously inoculated to ten Wistar rats. After one month, their blood, brain, tongue and diaphragm were collected and evaluated by MAT, PCR, pathological and bioassay methods. The parasite was cultivated in the cell monolayer. To change to bradyzoite, the media pH was altered to 6.8. Biological aspect of the bradyzoites was evaluated by incubation in acidic pepsin and it's inoculation in ten BALB/c mice. All rats showed antibodies to Toxoplasma at titers >/= 1:320 but no DNA and tissue cyst were detected in the tissues. Following intraperitoneal inoculation of rats' brain homogenate into BALB/c mice, no infection was established in none of the animals. During presence of cell culture, in acid media for a 3-5 days period, cyst-like structures were noticed when they were stained with PAS. The visible bradyzoites in the cysts that were incubated in acid pepsin medium were not able to kill any mice. This study confirmed that Iranian RH strain has lost the potential of tissue cyst forming in rats and bradyzoites cultivated in cell culture lost their resistance to acidic condition, so this strain can be a candidate for future vaccine researches

Real-Time RT-PCR on SAG1 and BAG1 Gene Expression during Stage Conversion in Immunosuppressed Mice Infected with Toxoplasma gondii Tehran Strain

Toxoplasmic encephalitis is caused by reactivation of bradyzoites to rapidly dividing tachyzoites of the apicomplexan parasite Toxoplasma gondii in immunocompromised hosts. Diagnosis of this life-threatening disease is problematic, because it is difficult to discriminate between these 2 stages. Toxoplasma PCR assays using gDNA as a template have been unable to discriminate between an increase or decrease in SAG1 and BAG1 expression between the active tachyzoite stage and the latent bradyzoite stage. In the present study, real-time RT-PCR assay was used to detect the expression of bradyzoite (BAG1)- and tachyzoite-specific genes (SAG1) during bradyzoite/tachyzoite stage conversion in mice infected with T. gondii Tehran strain after dexamethasone sodium phosphate (DXM) administration. The conversion reaction was observed in the lungs and brain tissues of experimental mice, indicated by SAG1 expression at day 6 after DXM administration, and continued until day 14. Bradyzoites were also detected in both organs throughout the study; however, it decreased at day 14 significantly. It is suggested that during the reactivation period, bradyzoites not only escape from the cysts and reinvade neighboring cells as tachyzoites, but also converted to new bradyzoites. In summary, the real-time RT-PCR assay provided a reliable, fast, and quantitative way of detecting T. gondii reactivation in an animal model. Thus, this method may be useful for diagnosing stage conversion in clinical specimens of immunocompromised patients (HIV or transplant patients) for early identification of tachyzoite-bradyzoite stage conversion.

Production and Evaluation of Toxoplasma gondii Recombinant GRA7 for Serodiagnosis of Human Infections

The precise diagnosis of the acute toxoplasmosis in pregnant women and immunocompromsied patients has critical importance. Most of the commercially available assays use the whole Toxoplasma soluble extract as the antigen. However, the assays currently available for the detection of specific anti-Toxoplasma antibodies may vary in their abilities to detect serum immunoglobulins, due to the lack of a purified standardized antigen. The aim of this study was production and evaluation of the usefulness of the recombinant Toxoplasma gondii GRA7 antigen for the serodiagnosis of Toxoplasma gondii IgM and IgG by ELISA. A total of 70 T. gondii IgM positive sera, 74 T. gondii IgG positive sera, and 60 sera from subjects who were not infected with T. gondii were examined. These sera were shown different absorbance values in ELISA test. To control the specificity of the rGRA7 other parasitic diseases, for example, echinococcosis, malaria, leishmaniasis, fascioliasis, and strongyloidiasis were tested of which none showed positive results. Sensitivity and specificity of the generated recombinant IgG ELISA in comparison with commercial ELISA (com ELISA) were 89% and 90%, and the sensitivity and specificity of the generated recombinant IgM ELISA were 96% and 90%, respectively. The results obtained here show that this antigen is useful for diagnostic purposes.

IgG Avidity ELISA Test for Diagnosis of Acute Toxoplasmosis in Humans

Serum samples, 100 in the total number, were collected from different laboratories in Tehran, Iran and tested for anti-Toxoplasma specific IgG and IgM antibodies using indirect immunofluorescent antibody test (IFAT). Using the IgG (chronic) and IgM (acute) positive samples, the IgG avidity test was performed by ELISA in duplicate rows of 96-well microtiter plates. One row was washed with 6 M urea and the other with PBS (pH 7.2), then the avidity index (AI) was calculated. Sixteen out of 18 (88.9%) sera with acute toxoplasmosis showed low avidity levels (AI60). Six sera had borderline ranges of AI. The results showed that the IgG avidity test by ELISA could distinguish the acute and chronic stages of toxoplasmosis in humans.

IgG Avidity ELISA Test for Diagnosis of Acute Toxoplasmosis in Humans

Serum samples, 100 in the total number, were collected from different laboratories in Tehran, Iran and tested for anti-Toxoplasma specific IgG and IgM antibodies using indirect immunofluorescent antibody test (IFAT). Using the IgG (chronic) and IgM (acute) positive samples, the IgG avidity test was performed by ELISA in duplicate rows of 96-well microtiter plates. One row was washed with 6 M urea and the other with PBS (pH 7.2), then the avidity index (AI) was calculated. Sixteen out of 18 (88.9%) sera with acute toxoplasmosis showed low avidity levels (AI60). Six sera had borderline ranges of AI. The results showed that the IgG avidity test by ELISA could distinguish the acute and chronic stages of toxoplasmosis in humans.

Effect of protein and DNA components of toxoplasma gondii on IL-12 production from dendritic cells and T cell proliferation

The aim of the present study was to investigate the effect of protein and DNA components of Toxoplasma gondii on maturation of dendritic cells and their efficiency in IL-12 production and proliferation of T cells. for DC generation, Bone marrow cells were cultured in the presence of GM- CSF and IL-4 for 5 days. Tumor lysate and protein or DNA components of Toxoplasma gondii were added to the culture media and incubated for another 2 days. LPS was added as control for DC maturation. Proliferation of T cells were determined by MLR and IL-1 production was measured by ELISA kit. Maturation of dendritic cell were determined by flowcytometry. DCs treatment with protein components of Toxoplasma gondii caused a significant increase in IL- 1 2 production and proliferation of T cells [P<0.001]. Different compositions of microbial body like protein and DNA components of Toxoplasma gondii can cause augmentation of antigen presentation capacity of DC and their IL-12 production capability. Among these components the protein was more effective as compared to DNA

Detection of toxoplasma gondii antigens in sera from experimentally infected mice

Detection of Toxoplasma antigen in serum of mice by Immunoblotting. Serum samples isolated from Balb/C mice experimentally infected with T. gondii, RH strain. IgG isolated from rabbits that were immunized with T. gondii Immunoblotting was performed to detect T. gondii antigens in sera of mice. School of Public Health. Tehran University of Medical Sciences. Serum samples from mice experimentally infected with T. gondii RH strain. The value of Immunoblotting in diagnosis of toxoplasmosis in acute stage of infection. The antigen bands detected in serum sample of mice were experimentally infected with T. gondii tachyzoite in immunoblotting. Six bands demonstrated on seventh post infection day six bands were identified. Similarly on sixth day four bands, on day five three bands and on fourth post infection day two bands were identified. No band was detected in control group sera. Immunoblotting is a sensitive method for diagnosis of acute stage of toxoplasmosis