Diagnostic Value of a Rec-ELISA Using Toxoplasma gondii Recombinant SporoSAG,BAG1, and GRA1 Proteins in Murine Models Infected Orally with Tissue Cysts and Oocysts

Toxoplasma gondii causes congenital toxoplasmosis in newborns resulting with fetal anomalies. Determining the initiation time of infection is very important for pregnant women and current serological assays have drawbacks in distinguishing the recently acute toxoplasmosis. Diagnosis of recently acute infection may be improved by using stage specific antigens in serological assays. In the present study, the diagnostic value of sporozoite specific SporoSAG, bradyzoite specific BAG1 proteins and GRA1 protein expressed by all forms of the parasite have been evaluated ELISA using sera systematically collected from mice administered orally with tissue cyst and oocysts. The anti-SporoSAG IgM antibodies in sera obtained from mice infected with oocysts peaked significantly at days 1, 10, and 15 (P<0.01). The anti-BAG1 IgM antibodies in sera obtained from mice infected with tissue cysts peaked significantly at days 15, 40, and 120 (P<0.05). The anti-GRA1 IgM antibodies in sera obtained from mice infected with oocysts peaked significantly at days 2, 10, and 40 (P<0.01). The anti-GRA1 IgM antibodies in sera obtained from mice infected with tissue cysts peaked significantly only at day 40 (P<0.05). The anti-SporoSAG, anti-BAG1, and anti-GRA1 IgG titers of mice showed significant increases at day 40 (P<0.05) and decrement started for only anti-GRA1 IgG at day 120. The presence of anti-SporoSAG IgM and IgG antibodies can be interpreted as recently acute infection between days 10–40 because IgM decreases at day 40. Similarly, presence of anti-BAG1 IgM and absence of IgG can be evaluated as a recently acute infection that occurred 40 days before because IgG peaks at day 40. A peak in anti-GRA1 antibody level at first testing and reduction in consecutive sample can be considered as an infection approximately around day 40 or prior. Overall, recombinant SporoSAG, BAG1 and GRA1 proteins can be accepted as valuable diagnostic markers of recently acute toxoplasmosis.     

Can peroxygenase and microperoxidase substitute cytochrome P450 in biosensors

Aromatic peroxygenase (APO) from the basidiomycetous mushroom Agrocybe aegerita (AaeAPO) and microperoxidases (MPs) obtained from cytochrome c exhibit a broad substrate spectrum including hydroxylation of selected aromatic substrates, demethylation and epoxidation by means of hydrogen peroxide. It overlaps with that of cytochrome P450 (P450), making MPs and APOs to alternate recognition elements in biosensors for the detection of typical P450 substrates. Here, we discuss recently developed approaches using microperoxidases and peroxygenases in view of their potential to supplement P450 enzymes as recognition elements in biosensors for aromatic compounds. Starting as early as the 1970s, the direct electron transfer between electrodes and the heme group of heme peptides called microperoxidases has been used as a model of oxidoreductases. These MP-modified electrodes are used as hydrogen peroxide detectors based on the catalytic current generated by electrically contacted microperoxidase molecules. A similar catalytic reaction has been obtained for the electrode-immobilised heme protein AaeAPO. However, up to now, no MP-based sensors for substrates have been described. In this review, we present biosensors which indicate 4-nitrophenol, aniline, naphthalene and p-aminophenol based on the peroxide-dependent substrate conversion by electrode-immobilised MP and AaeAPO. In these enzyme electrodes, the signal is generated by the conversion of all substrates, thus representing in complex media an overall parameter. The performance of these sensors and their further development are discussed in comparison with P450-based electrodes.     

Toxoplasma gondii RH Ankara: production of evolving tachyzoites using a novel cell culture method

Nitric oxide (NO) and NO-derived reactive nitrogen species (RNS) are present in the food vacuole (FV) of Plasmodium falciparum trophozoites. The product of PFL1555w, a putative cytochrome b₅, localizes in the FV membrane, similar to what was previously observed for the product of PF13_0353, a putative cytochrome b₅ reductase. These two gene products may contribute to NO generation by denitrification chemistry from nitrate and/or nitrite present in the erythrocyte cytosol. The possible coordination of NO to heme species present in the food vacuole was probed by resonance Raman spectroscopy. The spectroscopic data revealed that in situ generated NO interacts with heme inside the intact FVs to form ferrous heme nitrosyl complexes that influence intra-vacuolar heme solubility. The formation of heme nitrosyl complexes within the FV is a previously unrecognized factor that could affect the equilibrium between soluble and crystallized heme within the FV in vivo.     

Determining Toxoplasma high-risk autologous and allogeneic hematopoietic stem cell transplantation patients by systematic pre-transplant PCR screening of stem cell originated buffy coat

The diagnosis of Toxoplasma infection or disease in hematopoietic stem cell transplantation (HSCT) patients is achieved mainly by PCR screening; however screening did not find wide field of use in practice due to costly expenditures of PCR. This study aimed to determine patients at high risk of Toxoplasma infection or disease before transplantation by stem cell originated buffy coat PCR and subsequently to screen them. Buffy coats collected from 12 autologous and 18 allogeneic HSCT patients' donors were investigated by PCR before transplantation. After transplantation, blood and sera collected at fixed time intervals were screened by two PCR methods and serological assays. Screening results first time assessed a toxoplasmosis incidence level as 25% in autologous HSCT patients and increased incidence level in allogeneic HSCT patients to 22%. Importantly, buffy coat PCR was first time performed before transplantation, to determine the risk of toxoplasmosis. Buffy coat PCR results showed that four patients were at high risk of toxoplasmosis before transplantation. After transplantation, these patients experienced toxoplasmosis. In conclusion, for the determination of patients at risk of toxoplasmosis, clinicians should consider buffy coat PCR in combination with serology before transplantation. After transplantation, PCR screening can be initiated in high risk patients upon clinical suspicion.     

Studies on Mefenamic Acid Microparticles: Formulation, In Vitro Release, and In Situ Studies in Rats

In this study, we investigated the in vitro characteristics of mefenamic acid (MA) microparticles as well as their effects on DNA damage. MA-loaded chitosan and alginate beads were prepared by the ionotropic gelation process. Microsponges containing MA and Eudragit RS 100 were prepared by quasi-emulsion solvent diffusion method. The microparticles were characterized in terms of particle size, surface morphology, encapsulation efficiency, and in vitro release profiles. Most of the formulation variables manifested an influence on the physical characteristics of the microparticles at varying degrees. We also studied the effects of MA, MA-loaded microparticles, and three different polymers on rat brain cortex DNA damage. Our results showed that DNA damage was higher in MA-loaded Eudragit microsponges than MA-loaded biodegradable chitosan or alginate microparticles.     

Isolation of Toxoplasma gondii strains similar to Africa 1 genotype in Turkey

IntroductionToxoplasma gondii is a protozoon parasite that has a worldwide dissemination. It can cause serious clinical problems such as congenital toxoplasmosis, retinochoroiditis, and encephalitis. Currently, T. gondii genotypes are being associated with these clinical presentations which may help clinicians design their treatment strategy.Case reportsTwo T. gondii strains named Ankara and Ege-1 were isolated from newborns with congenital toxoplasmosis in Central and Western Anatolia, respectively. Ankara and Ege-1 strains were isolated from the cerebrospinal fluid of newborns. According to microsatellite analysis, Ankara and Ege-1 strains were sorted as Africa 1 genotype.ConclusionT. gondii strains isolated in Turkey were first time genotyped in this study. Africa 1 genotype has previously been isolated in immunosuppressed patients originating from sub-Saharan Africa. The reason of detecting a strain mainly detected in Africa can be associated with Turkey's specific geographical location. Turkey is like a bridge between Asia, Europe and Africa. Historically, Anatolia was on the Silk Road and other trading routes that ended in Europe. Thus, detecting Africa 1 strain in Anatolia can be anticipated. Consequently, strains detected mainly in Europe and Asia may also be detected in Anatolia and vice versa. Therefore, further studies are required to isolate more strains from Turkey.     

Enzyme electrode for aromatic compounds exploiting the catalytic activities of microperoxidase-11

Microperoxidase-11 (MP-11) which has been immobilised in a matrix of chitosan-embedded gold nanoparticles on the surface of a glassy carbon electrode catalyzes the conversion of aromatic substances. This peroxide-dependent catalysis of microperoxidase has been applied in an enzyme electrode for the first time to indicate aromatic compounds such as aniline, 4-fluoroaniline, catechol and p-aminophenol. The electrode signal is generated by the cathodic reduction of the quinone or quinoneimine which is formed in the presence of both MP-11 and peroxide from the substrate. The same sensor principle will be extended to aromatic drugs.     

Genetic Characterization of Toxoplasma gondii Isolates and Toxoplasmosis Seroprevalence in Stray Cats of ?zmir,Turkey

Currently, some Toxoplasma gondii genotypes are being associated with serious clinical presentations. A recent report showing the Africa 1 genotype in two local congenital toxoplasmosis cases acquired in Turkey formed the basis of this study because atypical Africa 1 genotype is most frequently detected in animals and patients from sub-Saharan Africa. Since stray cats are considered as the linkage between wild life and urban life in T. gondii transmission, the present study aimed to isolate and characterize T. gondii strains circulating in stray cats of İzmir (Western Turkey). A secondary objective was to determine toxoplasmosis seroprevalence in this cat population. Tissues obtained from 100 deceased stray cats were bioassayed and isolated strains were genotyped using 15 microsatellite markers. In addition, toxoplasmosis seroprevalence was analyzed in 1121 cat sera collected from several large veterinary clinics in İzmir. Among the 22 isolates, 19 were Type II (86.3%), two were Type III (9%) and one was Africa 1 genotype (4.5%). The overall seropositivity rates in cats were 42–48% and 33.4–34.4% according to IFA and ELISA, respectively. Seroprevalence in deceased cats was significantly higher than in healthy cats (P = 0.0033). Finding both the major clonal Type II lineage together with the Type III lineage also found in Middle East, and an atypical genotype, Africa 1 appears consistent with the specific geographic location of Turkey between three continents and raises the possibility of transportation of these strains between continents through trade routes or long distance migratory birds. In addition, the first large study of toxoplasma seroprevalence in a stray cat population was also reported. The relatively high seropositivity rates and the variety of T. gondii genotypes confirm the local stray cat population as a risk factor for human toxoplasmosis in İzmir.