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Serological and Molecular Diagnosis of Toxoplasma gondii Infections in Thalassemia Patients.

ABSTRACT: Background:This study aimed to the serological and molecular diagnosis of Toxoplasma gondii infections and related risk factors in patients with thalassemia major and healthy controls. Methods:This case-control study was performed in Shahrekord University of Medical Sciences, Shahrekord, west of Iran from Jan 2014 to Jan 2015. Overall, 235 patients with thalassemia major and 235 healthy controls were enrolled. Assessment of anti-Toxoplasma antibodies in sera samples was performed using commercial ELISA kits. In order to the molecular investigate of T. gondii in blood samples, a relatively new molecular assay, LAMP technique based on Toxoplasma SAG1 gene was conducted for the first time. The specificity of LAMP outer primers for the T. gondii detection was confirmed by sequencing the purified PCR product. Results:51.9% of thalassemia patients and 34.8% of healthy controls were positive for anti-Toxoplasma IgG antibodies, which the difference was statistically significant (P<0.01). In terms of anti-Toxoplasma IgM antibody, 3.4% of thalassemia patients and 2.1% of healthy individuals were positive, which the difference was not statistically significant (P=1). Based on SAG1-LAMP, 9.78% of the thalassemia patients and 5.95% of healthy controls were positive for T. gondii DNA, which the difference was not statistically significant (P?0.230). Conclusion:Thalassemia patients, probably due to repeated blood transfusion and consequently, immune deficiency, are at risk of transmitting Toxoplasma infection more than healthy people. Therefore, screening of Toxoplasma infection in blood transfusion centers may be effective in the prevention of toxoplasmosis in these patients.

SUBMITTER: Hanifehpour H

PROVIDER: S-EPMC6511596 | biostudies-literature | 2019 Jan-Mar

REPOSITORIES: biostudies-literature

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Serological and Molecular Diagnosis of <i>Toxoplasma gondii</i> Infections in Thalassemia Patients.

Hanifehpour Hooman H Samsam Shariat Seyed Kamal SK Ghafari Mohammad Saleh MS Kheirandish Farnaz F Saber Vafa V Fallahi Shirzad S

Iranian journal of parasitology 20190101 1

<h4>Background</h4>This study aimed to the serological and molecular diagnosis of <i>Toxoplasma gondii</i> infections and related risk factors in patients with thalassemia major and healthy controls.<h4>Methods</h4>This case-control study was performed in Shahrekord University of Medical Sciences, Shahrekord, west of Iran from Jan 2014 to Jan 2015. Overall, 235 patients with thalassemia major and 235 healthy controls were enrolled. Assessment of anti-<i>Toxoplasma</i> antibodies in sera samples ...[more]

PMID: 31123465

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Project description:BackgroundLittle is known about the epidemiology of Toxoplasma gondii and Neospora caninum infections in alpacas (Vicugna pacos) and llamas (Lama glama) outside South America. The study aimed to estimate the seroprevalence of T. gondii and N. caninum infections in South American camelids (SAC) in Switzerland, to optimize serological tests for SAC and to identify risk factors, which may favour infection.MethodsA total of 571 sera from 132 Swiss farms (374 alpacas and 197 llamas, mean 4.3 animals/farm) were obtained. Four commercial enzyme-linked immunosorbent assays (ELISA) for detecting antibodies against T. gondii (ID Screen® Toxoplasmosis Indirect (TOXO-MS)) or N. caninum (i.e. ID Screen® Neospora caninum Indirect Multi-species (NCS-MS); ID Screen® Neospora caninum Competition (NCC) and ID Screen® Neospora caninum Indirect (NCS)) were first assessed for their use on SAC comparing their results with those in immunoblot, and optimizing cut-offs. Subsequently, two kits (TOXO-MS and NCS-MS) were selected for seroprevalence estimation. Additionally, a risk factor analysis for infection was performed on 41 farms, which agreed to participate in a web-based survey.ResultsThree kits (TOXO-MS, NCS-MS and NCC) showed almost perfect agreement (kappa > 0.901) with immunoblot results when the cut-offs were optimized, and one kit (NCS) proved not to be useful for detecting N. caninum seropositive SAC. By TOXO-MS ELISA, 82.3% (308/374) of the alpacas and 84.8% (167/197) of the llamas were seropositive for T. gondii, and 131/132 (99.2%) farms had seropositive animals. By NCS-MS ELISA, 3.5% (13/374) of the alpacas and 2.5% (5/197) of the llamas evidenced antibodies against N. caninum, and 9.1% (12/132) of the farms had seropositive animals. The variables "age" and "female sex" were identified as risk factors for T. gondii infection and "absence of cats in the farm during the last two years" as a protective factor. No risk or protective factors for N. caninum infection could be identified.ConclusionsThis nationwide cross-sectional study demonstrated for the first time the presence of antibodies against T. gondii and N. caninum in the Swiss SAC population, highlighting a high seroprevalence for T. gondii, the presence of cats as a risk factor and suggesting that SAC meat might represent an additional infection source for humans.

Project description:BackgroundFree-ranging chickens are often infected with Toxoplasma gondii and seroconvert upon infection. This indicates environmental contamination with T. gondii.MethodsHere, we established a bead-based multiplex assay (BBMA) using the Luminex technology for the detection of T. gondii infections in chickens. Recombinant biotinylated T. gondii surface antigen 1 (TgSAG1bio) bound to streptavidin-conjugated magnetic Luminex beads served as antigen. Serum antibodies were detected by a fluorophore-coupled secondary antibody. Beads of differing color codes were conjugated with anti-chicken IgY or chicken serum albumin and served for each sample as an internal positive or negative control, respectively. The assay was validated with sera from experimentally and naturally infected chickens. The results were compared to those from reference methods, including other serological tests, PCRs and bioassay in mice.ResultsIn experimentally infected chickens, the vast majority (98.5%, n = 65/66) of birds tested seropositive in the BBMA. This included all chickens positive by magnetic-capture PCR (100%, n = 45/45). Most, but not all inoculated and TgSAG1bio-BBMA-positive chickens were also positive in two previously established TgSAG1-ELISAs (TgSAG1-ELISASL, n = 61/65; or TgSAG1-ELISASH, n = 60/65), or positive in an immunofluorescence assay (IFAT, n = 64/65) and in a modified agglutination test (MAT, n = 61/65). All non-inoculated control animals (n = 28/28, 100%) tested negative. In naturally exposed chickens, the TgSAG1bio-BBMA showed a high sensitivity (98.5%; 95% confidence interval, CI: 90.7-99.9%) and specificity (100%; 95% CI: 85.0-100%) relative to a reference standard established using ELISA, IFAT and MAT. Almost all naturally exposed chickens that were positive in bioassay or by PCR tested positive in the TgSAG1bio-BBMA (93.5%; 95% CI: 77.1-98.9%), while all bioassay- or PCR-negative chickens remained negative (100%; 95% CI: 85.0-100%).ConclusionsThe TgSAG1bio-BBMA represents a suitable method for the detection of T. gondii infections in chickens with high sensitivity and specificity, which is comparable or even superior to other tests. Since assays based on this methodology allow for the simultaneous analysis of a single biological sample with respect to multiple analytes, the described assay may represent a component in future multiplex assays for broad serological monitoring of poultry and other farm animals for various pathogens.

Project description:BackgroundToxoplasmosis caused by Toxoplasma gondii is a serious disease threatening human and animal health. People can be infected with T. gondii by ingesting raw pork contaminated with cysts or oocysts. Serological test is a sensitive and specific method usually used for large-scale diagnosis of T. gondii infection in humans and animals (such as pigs). Commercial pig Toxoplasma antibody ELISA diagnostic kits are expensive, which limits their use; moreover, the wide antigen composition used in these diagnostic kits is still unclear and difficult to standardize. The multiepitope peptide antigen is a novel diagnostic marker, and it has potential to be developed into more accurate and inexpensive diagnostic kits.MethodsThe synthetic multiepitope antigen (MAG) cDNA encoding a protein with epitopes from five T. gondii-dominant antigens (SAG1, GRA1, ROP2, GRA4, and MIC3) was designed, synthesized, and expressed in Escherichia coli BL21 (DE3) strain. The recombinant protein was detected through western blot with pig anti-T. gondii-positive and -negative serum, and then IgG enzyme-linked immunosorbent assay (ELISA) named MAG-ELISA was designed. The MAG-ELISA was evaluated in terms of specificity, sensitivity, and stability. The MAG-ELISA was also compared with a commercial PrioCHECK® Toxoplasma Ab porcine ELISA (PrioCHECK ELISA). Finally, the trend of pig anti-T. gondii IgG levels after artificial infection with RH tachyzoites was evaluated using MAG-ELISA and two other ELISA methods (rMIC3-ELISA and PrioCHECK ELISA).ResultsMAG antigen could be specifically recognized by pig anti-T. gondii-positive but not -negative serum. MAG-ELISA showed high diagnostic performance in terms of specificity (88.6%) and sensitivity (79.1%). MAG-ELISA could be used for detecting anti-T. gondii IgG in the early stage of T. gondii infection in pigs (at least 7 days after artificial infection).ConclusionsOur results suggest that MAG antigen can be applied to specifically recognize anti-T. gondii IgG in pig, and MAG-ELISA has the potential for large-scale screening tests of T. gondii infection in pig farms and intensive industries.

Dataset Information

Serological and Molecular Diagnosis of Toxoplasma gondii Infections in Thalassemia Patients.

Publications

Serological and Molecular Diagnosis of <i>Toxoplasma gondii</i> Infections in Thalassemia Patients.

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