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PubMed1981

Reverse enzyme immunoassay for detection of specific anti-Toxoplasma immunoglobulin M antibodies.

A reverse enzyme immunoassay (R-EIA) is described, in which polystyrene muplates are sensitized with anti-immunoglobulin M (IgM) (mu chain) antibodies and then sequentially allowed to react with patient's serum, peroxidase-labeled Toxoplasma gondii soluble antigen, and substrate. Measurement of activity of the solid-phase bound enzyme conjugate was done by colorimetric reading of the final developed color and kinetically by the initial rate of color development. This R-EIA allowed full resolution between absorbance values of a group of 36 sera which presented positive results in the Toxoplasma IgM immunofluorescence test and the remaining groups, which consisted of 39 normal individuals, 22 rheumatoid factor-positive sera, 8 Waldenstrom's macroglobulinemic sera, 3 infectious mononucleosis samples, and 6 high-titered IgG anti-T. gondii sera. No interference of rheumatoid factor IgM or inhibition by high-titered specific IgG was detected, even in the false IgM immunofluorescence-positive rheumatoid factor samples. Likewise, false-negative IgM immunofluorescence samples gave positive R-EIA even without adsorption with Staphylococcus aureus protein A. The possibility of direct tagging of the antigen with the enzyme eliminates the need for using antigen and anti-antigen conjugates as separate layers, therefore eliminating one step in the assay.

Animals↗

PubMed1982

Evaluation of an enzyme-linked immunosorbent assay specific for human immunoglobulin G as a Screening test for detecting anti-toxoplasma antibodies.

An evaluation of the enzyme-linked immunosorbent assay (ELISA) as a screening test for immunoglobulin G class-specific anti-Toxoplasma antibodies was conducted. Results for 191 serum specimens assayed by the ELISA, indirect hemagglutination (IHA), and methylene blue dye tests indicated that good qualitative agreement exists among these tests. High ELISA titers relative to methylene blue dye titers may be attributed in part to the presence of rheumatoid factor. ELISA results were found to be reproducible. A parallel study of 1,760 sera assayed by ELISA and IHA was performed with 3.0-mm-diameter serum filter paper spots as a source of specimen. When a 1:64 titer and 40 IU were used as threshold values for IHA and the ELISA, respectively, 4.8% of the specimens tested were positive in both tests. An additional 2% of these specimens were ELISA positive and IHA negative, whereas 2% were ELISA negative and IHA positive. The total number of positive specimens by either test (7%) closely agreed with previously reported results. In view of these data, the simplicity of the test, and relative economic considerations, the ELISA may be considered an alternative method for screening for anti-Toxoplasma antibodies.

Enzyme-Linked Immunosorbent Assay↗

PubMed1987

Evaluation of a rapid screening immunoassay for antibodies to Toxoplasma gondii.

A total of 349 human serum samples were examined for anti-Toxoplasma antibodies by the Murex Single Use Diagnostic System (SUDS) qualitative screening test, indirect hemagglutination assay (IHA), and indirect immunofluorescence assay (IFA). Concordant results with SUDS and IHA were obtained for 91.9% of serum samples; 8.9% were SUDS+ and IHA-; none were SUDS- and IHA+. Comparison of the SUDS assay with IFA showed a concordance of 95.3%, with a sensitivity of 97.5% and a specificity of 94.7%. Moreover, the positive and negative predictive values were 84.9 and 99.2%, respectively, when results of the SUDS assay and IFA were compared. The SUDS assay is a rapid, simple test requiring no instrumentation and can be performed on 50 microliters of serum, features which make this an excellent screening test for detecting anti-Toxoplasma antibodies, particularly in outpatient settings.

Animals↗

PubMed1993

Diagnosis of Toxoplasma parasitemia in patients with AIDS by gene detection after amplification with polymerase chain reaction.

We sought evidence of toxoplasma parasitemia among 37 people with active or dormant Toxoplasma gondii infection or no evidence of infection. DNA was extracted from erythrocyte-free portions of blood samples, and the T. gondii B1 gene was amplified by the polymerase chain reaction. Evidence of T. gondii parasitemia was found in six patients with severe immunosuppression from AIDS and clinical evidence suggestive of or compatible with toxoplasmosis. Results were negative for patients unlikely to have active toxoplasmosis. Gene detection after amplification with the polymerase chain reaction is a promising test for detection of parasitemia, and parasitemia should be tested for in patients with AIDS and unexplained fever or central nervous system abnormalities.

AIDS-Related Opportunistic Infections↗

PubMed1997

False-positive results in immunoglobulin M (IgM) toxoplasma antibody tests and importance of confirmatory testing: the Platelia Toxo IgM test.

Although tests for detection of immunoglobulin M (IgM) toxoplasma antibodies have been reported to have a high degree of accuracy, it is well recognized by investigators in the United States and Europe that false-positive results may occur with many of these tests, at times to an alarming degree. Unfortunately, this information is not well documented in the literature. Studies on various toxoplasma IgM test kits are frequently flawed. The investigators often use reference tests which have not previously been carefully evaluated as well as sera that were not appropriate to answer the question of how often false-positive results might occur. We recently had the unique opportunity to evaluate the accuracy of the Platelia Toxo IgM test in 575 serum samples obtained during an outbreak of toxoplasmosis which occurred in 1995 in the Capital Regional District of British Columbia, Canada. When compared with results obtained in a reference IgM enzyme-linked immunosorbent assay (ELISA), the Platelia Toxo IgM test had a sensitivity of 99.4%, specificity of 49.2%, positive predictive value of 51.9%, negative predictive value of 99.3%, and an overall agreement of 67.0%. In an attempt to resolve discrepancies between these two tests, a serological profile (Sabin-Feldman dye test, IgA and IgE antibody tests, differential agglutination [AC/HS] test, and IgG avidity method) was performed. Of 153 serum samples that were positive in the Platelia Toxo IgM test and negative in the IgM ELISA, 71 (46.4%) were negative in the Sabin-Feldman dye test. Of the serum samples that were positive in the dye test, 77 (93.9%) had a serological profile most compatible with an infection acquired in the distant past. These results reveal high numbers of false-positive results in the Platelia Toxo IgM test and highlight the importance of appropriate evaluation of commercial tests that are currently being marked. Our results also emphasize the importance of confirmatory testing to determine whether the results of an IgM antibody test reflect the likelihood of a recently acquired infection.

Animals↗

PubMed1999

Value of PCR for detection of Toxoplasma gondii in aqueous humor and blood samples from immunocompetent patients with ocular toxoplasmosis.

Toxoplasma gondii infection is an important cause of chorioretinitis in the United States and Europe. Most cases of Toxoplasma chorioretinitis result from congenital infection. Patients are often asymptomatic during life, with a peak incidence of symptomatic illness in the second and third decades of life. Diagnosis is mainly supported by ophthalmological examination and a good response to installed therapy. However, establishment of a diagnosis by ophthalmological examination alone can be difficult in some cases. To determine the diagnostic value of PCR for the detection of T. gondii, 56 blood and 56 aqueous humor samples from 56 immunocompetent patients were examined. Fifteen patients with a diagnosis of ocular toxoplasmosis had increased serum anti-T. gondii immunoglobulin G levels but were negative for anti-T. gondii immunoglobulin M (group 1), and 41 patients were used as controls (group 2). Samples were taken before antiparasitic therapy was initiated, and only one blood sample and one aqueous humor sample were obtained for each patient. Single nested PCRs and Southern blot hybridization were performed with DNA extracted from these samples. The results obtained showed sensitivity and specificity values of 53. 3 and 83%, respectively. Interestingly, among all patients with ocular toxoplasmosis, a positive PCR result with the aqueous humor sample was accompanied by a positive PCR result with the blood sample. This result suggests that ocular toxoplasmosis should not be considered a local event, as PCR testing of blood samples from patients with ocular toxoplasmosis yielded the same result as PCR testing of aqueous humor samples. PCR testing may be useful for discriminating between ocular toxoplasmosis and other ocular diseases, and also can avoid the problems associated with ocular puncture.

Adolescent↗

PubMed2000

Detection of immunoglobulin M antibodies to P35 antigen of Toxoplasma gondii for serodiagnosis of recently acquired infection in pregnant women.

We examined the efficiency of detection of immunoglobulin M (IgM) antibodies to a 35-kDa antigen (P35) of Toxoplasma gondii for serodiagnosis of acute infection in pregnant women. A double-sandwich enzyme-linked immunosorbent assay (ELISA) with recombinant P35 antigen (P35-IgM-ELISA) was used for this purpose. On the basis of the clinical history and the combination of results from the toxoplasma serological profile (Sabin-Feldman dye test, conventional IgM and IgA ELISAs, and the differential agglutination test), the patients were classified into three groups: group I, status suggestive of recently acquired infection; group II, status suggestive of infection acquired in the distant past; group III, status suggestive of persisting IgM antibodies. Eighteen (90.0%) of 20 serum samples from group I patients were positive by the P35-IgM-ELISA, whereas none of the 33 serum samples from group II patients were positive. Only 4 (25.0%) of 16 serum samples from group III patients were positive by the P35-IgM-ELISA, whereas all these serum samples were positive by the conventional IgM ELISA. These results indicate that demonstration of IgM antibodies against P35 by the P35-IgM-ELISA is more specific for the acute stage of the infection than demonstration of IgM antibodies by the ELISA that uses a whole-lysate antigen preparation. Studies with sera obtained from four pregnant women who seroconverted (IgG and IgM antibodies) during pregnancy revealed that two of them became negative by the P35-IgM-ELISA between 4 and 6 months after seroconversion, whereas the conventional IgM ELISA titers remained highly positive. The P35-IgM-ELISA appears to be useful for differentiating recently acquired infection from those acquired in the distant past in pregnant women.

Acute Disease↗

PubMed2004

Comparison of two widely used PCR primer systems for detection of toxoplasma in amniotic fluid, blood, and tissues.

The PCR diagnosis of toxoplasmosis suffers from lack of standardization. Interlaboratory comparative studies of PCR methods have been performed, but intralaboratory comparisons are scarce. Here, we optimized and compared the technical performances of two PCR primer systems widely used for Toxoplasma detection. The differences between the two methods were visible only at low parasite concentrations (< or = 1 Toxoplasma genome per reaction tube). Nevertheless, when clinical samples were tested, both methods significantly differed in their technical sensitivities and specificities. Only one method appeared adequate for samples containing blood or tissue.

Amniotic Fluid↗

PubMed1998

Isolation of developmentally regulated genes from Toxoplasma gondii by a gene trap with the positive and negative selectable marker hypoxanthine-xanthine-guanine phosphoribosyltransferase.

Within its intermediate host, Toxoplasma gondii switches between two forms: a rapidly replicating tachyzoite and an encysted bradyzoite. Bradyzoites persist within the host throughout its life, hidden from antimicrobial agents and the immune system. The signals that mediate switching are poorly understood. A gene trap was employed to isolate genes whose expression is up-regulated early in the switching of bradyzoites via the negative and positive selectable marker hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT). T. gondii was transfected with promoterless HXGPRT and negatively selected with 6-thioxanthine to inhibit the growth of tachyzoites expressing HXGPRT. The surviving tachyzoites were then induced for in vitro bradyzoite formation and treated with mycophenolic acid and xanthine to positively select for parasites in which the construct had integrated downstream of a bradyzoite-specific gene. Strains were checked for their ability to differentiate by using Dolichos biflorus agglutinin (a bradyzoite-specific lectin) and a monoclonal antibody against P36 (a bradyzoite-specific surface antigen). After differentiation, all gene-trapped clones had Dolichos immunofluorescence and all but one expressed P36. The sequences flanking the insertion site of this P36-negative strain were homologous to the Toxoplasma family of surface antigens, strongly suggesting that P36 is encoded by the disruptive gene. Genetic mapping and complementation of the P36-negative strain further indicated that the disrupted gene is P36. Reverse transcriptase PCR and S1 nuclease digestion were used to compare mRNA levels during the tachyzoite and bradyzoite stages. The presumptive P36 gene does not appear to regulate its mRNA levels between the two stages, indicating a posttranscriptional mechanism of regulation for early bradyzoite-specific genes.

Amino Acid Sequence↗

PubMed2026

Genome-resolved analysis reveals disruption of gut microbial vitamin B and K2 biosynthesis during Toxoplasma gondii infection in mice.

UNLABELLED: Toxoplasma gondii infection remodels the gut microbiome, yet its impact on microbial vitamin biosynthetic potential and host redox metabolism remains unclear. Here, we integrated mouse gut metagenomes with publicly available metagenome-assembled genomes (MAGs) to construct a genome-resolved atlas of B-vitamin and vitamin K2 biosynthesis. From 45,697 MAGs, we curated 4,771 representative genomes, of which 2,682 met high-quality criteria (completeness ≥90%, contamination <5%). Functional annotation identified 229,717 vitamin-related genes corresponding to 177 Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologs across de novo pathways for eight B vitamins, thiamine (B1), riboflavin (B2), niacin (B3), pantothenate (B5), pyridoxine (B6), biotin (B7), folate (B9), cobalamin (B12), and vitamin K2. Among the high-quality genomes, 1,665 encoded complete de novo pathways for at least one vitamin, highlighting functional specialization and community-level complementarity. Transcripts per million-normalized metagenomic read counts revealed significant differences in KEGG ortholog abundances across six of the nine vitamin pathways. Reanalysis of metagenomic data from infected mice (acute, chronic, and control; n = 10 per group) revealed a stage-dependent reduction in α-diversity of vitamin biosynthesis pathways during acute infection, and a clear β-diversity separation from chronic and control groups. Core niacin biosynthesis genes (nadB, nadA, nadC) displayed phylum-specific redistribution, indicating selective remodeling of microbial NAD+ precursor production under infection-induced metabolic stress. These results suggest that T. gondii infection disrupts cooperative vitamin biosynthetic networks while specifically modulating niacin pathways linked to host NAD+ metabolism. IMPORTANCE: Gut microbes can synthesize essential vitamins, but how infection alters this function is poorly understood. By integrating mouse gut metagenomes with genome-resolved microbial data, we show that Toxoplasma gondii infection reshapes the vitamin biosynthetic potential of the gut microbiome in a stage-dependent manner. Acute infection reduces the diversity of vitamin biosynthesis pathways and shifts the taxonomic distribution of key niacin biosynthesis genes involved in microbial NAD+ precursor production. These findings identify vitamin metabolism, especially niacin-related pathways, as a sensitive functional axis of microbiome remodeling during infection. Our work links microbial taxonomic changes to functional metabolic consequences and suggests that microbiome-mediated regulation of NAD+-related metabolism may contribute to host redox adaptation during T. gondii infection.

B vitamins↗

PubMed1993

Detection of Toxoplasma gondii in aqueous humour by the polymerase chain reaction.

Ocular toxoplasmosis is the most frequent infectious cause of chorioretinal inflammation in immunocompetent patients. Nowadays, the biological diagnosis of ocular toxoplasmosis requires serological tests and anterior chamber puncture to detect the local production of specific antibodies. A new technique is described to detect Toxoplasma in aqueous humour by a polymerase chain reaction in which the target is a specific ribosomal DNA segment. Sixty eight patients (71 eyes) were included; 59 (83%) eyes were suspected of having ocular toxoplasmosis. Of these 59 eyes, 15 (25.4%) had characteristic fundus lesions with obvious intraocular inflammation signs and 44 (75%) had retinal scar of ocular toxoplasmosis without clinically detectable inflammation. Twelve (17%) eyes had uveitis of non-Toxoplasma origin and constituted the control group. The parasite was present in aqueous humour in 20 (33.8%) cases. No false positives were detected. The sensitivity of the test is reduced by the low numbers of the sample. The combination of this technique with Witmer-Desmonts coefficient increases the probability of making a biological diagnosis of ocular toxoplasmosis. The physiopathological value of this technique is emphasised and the presence of tachyzoites in the anterior chamber is suggested. This should be a very promising technique for the diagnosis of ocular toxoplasmosis.

Adolescent↗

PubMed1980

Enzyme-linked immunosorbent assay for quantitation of toxoplasma antibodies in human sera.

An enzyme-linked immunosorbent assay(ELISA) was developed for the detection of toxoplasma antibodies using a single serum dilution (1:800) in conjunction with a standard curve Antigen was prepared from Toxoplasma gondii cultivated in human cell cultures. A nearly linear relationship was found between the logarithms of the absorbance values of 120 human sera at a dilution of 1/800 and the titres as determined by an end point dilution ELISA. The reproducibility of the single dilution ELISA was excellent; the coefficients of variation for within-day and day-to-day tests were less than 15%. A close correlation was found between the results obtained with ELISA, indirect immunofluorescence (IF), and complement fixation (CF). The titres in ELISA were 20 to 40 times higher than in IF and 200 to 1000 times higher than in CF.

Antibodies↗

PubMed1985

Detection of specific IgG and IgM antibodies to Toxoplasma gondii with a commercially available enzyme immunoassay kit system.

A total of 138 serum samples submitted for toxoplasma serology have been examined by enzyme immunoassay using kits produced by Labsystems Oy for the detection of specific antibodies of the IgG and IgM class. Results were compared with the dye test, an indirect haemagglutination test, and an indirect immunofluorescence test for specific IgM. The enzyme immunoassay was less sensitive than the dye test, but by running both IgG and IgM enzyme immunoassays, 92.4% sensitivity was achieved. The specificity of the enzyme immunoassay was good, with only one dye test negative serum giving a positive (but weak) IgG enzyme immunoassay reaction. Thirty serum samples from patients with no evidence of exposure to Toxoplasma gondii gave negative results in the IgM enzyme immunoassay. Enzyme immunoassay results were expressed in enzyme immunoassay units, as a percentage value of a standard serum. This convention will be of value in the direct comparison of assay systems and in the application of quality control procedures.

Fluorescent Antibody Technique↗

PubMed1987

Public Health Laboratory Service enzyme linked immunosorbent assay for detecting Toxoplasma specific IgM antibody.

An enzyme linked immunosorbent assay (ELISA) based on the antibody class capture method for the detection of specific IgM against Toxoplasma gondii, using the microtitre plate format, was developed. Antigen binding was detected using a monoclonal antibody, CIE3, conjugated to horseradish peroxidase. Prior mixing of the conjugate and antigen improved the stability of these reagents as well as removing an incubation stage from the assay. The incubation time of less than four hours permits a rapid throughput of specimens. Using the assay, a total of 163 sera were examined in a three centre study and good agreement was found. Results were expressed as arbitrary enzyme immunoassay units (EIUs) against a freeze dried standard. Throughout the study the standard serum showed a coefficient of variation less than 10% across the microtitre plate. By measuring IgM titres in patients having toxoplasmic lymphadenopathy with a known date of onset, IgM class antibodies were shown to peak at two months, persisting for about six months. In addition, a case of laboratory acquired toxoplasmosis was monitored. Sera shown to contain rheumatoid factor and antinuclear factor did not give false positive results. This rapid, robust, and simplified assay is used by the Public Health Laboratory Service Toxoplasma Reference Units and will provide a standard with which other assays can be compared.

Animals↗

PubMed1987

Use of monoclonal antibodies in an ELISA to detect IgM class antibodies specific for Toxoplasma gondii.

Two monoclonal antibodies CH6 and C1E3 were used in an antibody class capture assay for the detection of IgM antibodies specific for Toxoplasma gondii. CH6 was used on the solid phase to capture human IgM. After a Toxoplasma gondii antigen had been added, specifically bound material was detected using C1E3 coupled to horseradish peroxidase. The assay was compared with an established system using polyclonal antisera at both the capture and antigen detection stages. A good correlation was found, with 97.3% (125 of 128) of sera giving the same classification in both assays. Three sera were positive only in the polyclonal system. No false positive results were found when 118 negative sera were examined. The two monoclonal antibodies provide a viable alternative to the use of polyclonal sera at the capture and antigen detection stages in the antibody class capture assay for the measurement of specific IgM against T gondii.

Animals↗

PubMed1989

Discrepant toxoplasma latex agglutination test results.

The analysis of 4450 toxoplasma serology results showed that 59 (1.3%) latex agglutination reactions were not confirmed in the dye test. These discrepant results were associated with an unspecified IgM antibody but not associated with kit batch variation, inactivation of sera, concurrent cytomegalovirus infection, or the presence of hepatitis B virus "e" antigen. The latex agglutination test is useful as a screen for toxoplasma infection but false positive reactions do occur. Patients at risk of severe toxoplasmosis should be investigated by additional tests.

Animals↗

PubMed1994

Use of three immunological techniques for the detection of Toxoplasma spIgA antibodies in acute toxoplasmosis.

AIMS: To assess the performance and efficacy of three immunological techniques for the detection of Toxoplasma specific IgA antibodies in acute toxoplasmosis. METHODS: The following techniques were used to examine 128 serum samples (51 cases of acute toxoplasmosis, 50 cases of heterologous infections, and 27 healthy controls): direct enzyme linked immunosorbent assay (ELISA), antibody capture ELISA, and antibody capture agglutination. RESULTS: Direct ELISA had a sensitivity of 98% and a specificity of 97%, antibody capture ELISA of 100% and 99%, respectively, and antibody capture agglutination had sensitivity and specificity of 100%. CONCLUSIONS: All three immunological techniques performed well with similar efficacy. Detection of Toxoplasma specific IgA antibodies is a useful diagnostic marker for acute toxoplasmosis.

Acute Disease↗

PubMed1992

Contact with pigs and cats associated with high prevalence of Toxoplasma antibodies among farmers.

Antibodies to Toxoplasma were measured by enzyme linked immunosorbent assay (ELISA) in sera from 159 abattoir workers, 142 pig farmers, and 106 grain or berry farmers. Farmers occupationally exposed to pigs had antibodies in 53 (37%) cases, abattoir workers in 40 (25%) cases, and farmers not exposed to pigs in 24 (23%) cases. In each group antibodies were more prevalent among those who had a cat or cats in the household. Controlling for age and cat contacts changed the prevalences less than one percent. The results indicate that pig farmers might have an occupational risk of toxoplasmosis. As the prevalence of antibodies among abattoir workers was about the same as among the referent farmers, it seems unlikely that infection from Toxoplasma could be acquired by mere handling of raw meat.

Agricultural Workers' Diseases↗