Specificity of low anti-Toxoplasma IgG titers with IMx and AxSYM Toxo IgG assays.

Low antibody titers (3 to 6 IU/mL) detected by IMx and AxSYM Toxo IgG assays in serum samples from 264 pregnant women were confirmed by the dye test and a high-sensitivity agglutination test in, respectively, 98.5% and 95.6% of cases, attesting a premunition of these patients.

Prenatal diagnosis of congenital toxoplasmosis in 261 pregnancies.

Two hundred and sixty-one pregnant women underwent prenatal screening by cordocentesis and/or amniocentesis between 1987 and 1994. The following tests were used: (i) detection of anti-Toxoplasma gondii IgM, IgA, and IgE antibodies by immunocapture and the comparative immunological profile method based on enzyme-linked immunofiltration assay of fetal blood and (ii) direct detection of the parasite in cell culture and by mouse inoculation with fetal blood (FB) and/or amniotic fluid (AF). Of the 31 cases of congenital toxoplasmosis, 24 (77 per cent) were detected prenatally. Overall, the FB and AF inoculation methods were the most effective (50 per cent sensitivity with FB inoculation to mice and/or cell culture and 74 per cent with AF). However, antibody detection in FB was the only positive test in three cases. Of 18 surviving children diagnosed prenatally, only one developed chorioretinitis (9 months of age). Seven newborns (23 per cent) with negative prenatal tests were diagnosed by postnatal laboratory monitoring, but none of these children developed clinical toxoplasmosis. There may have been more false negatives, as only 48 per cent of unaffected children were followed up for at least 12 months. All the tests had a specificity of 100 per cent. Fetal blood sampling has considerable value but also carries some risks and is currently being abandoned in favour of amniocentesis alone with gene amplification and mouse inoculation.

Detection of Toxoplasma gondii in immunodeficient subjects by gene amplification: influence of therapeutics.

Polymerase chain reaction (PCR) technology was used to detect Toxoplasma gondii DNA in 253 immunodeficient subjects, 179 of whom were infected with the human immunodeficiency virus (HIV). The incidence of toxoplasmosis was 12.3% (22/179) in the HIV-infected subjects and 2.7% (2/74) in the remainder. The sensitivity of the PCR during episodes of toxoplasmosis in HIV-infected subjects not on antiparasitic treatment was 86.6% on peripheral blood and 60% on cerebrospinal fluid (CSF), but was only 25% and 16.7%, respectively, in subjects receiving specific treatment or prophylaxis against Pneumocystis carinii. Among the HIV-seronegative population, six patients undergoing anticancer chemotherapy were PCR positive on bronchoalveolar lavage fluid but did not develop pulmonary toxoplasmosis, suggesting transient carriage.

Automated reading and processing of quantitative IgG, IgM, IgA, and IgE isotypic agglutination results in microplates. Development and application in parasitology-mycology.

Microplate agglutination techniques represent a simple and commonly used approach for the quantitative or qualitative isotypic analysis of specific antibodies. However, they require optical reading by the investigator and are thus prone to an important degree of variability. In order to solve some of the problems associated with the variability of optical readings, we have used an automatic reader scanning each of the 96 wells of a standard microplate in 32 different locations. The inherent advantages of the automatic reader were further maximized by coupling it to a dedicated computer running customized software designed to process data coming on-line from the spectrophotometer. This approach has been applied to the diagnosis of human toxoplasmosis and candidosis. Suspensions of Toxoplasma gondii tachyzoites or of sensitised erythrocytes were used for the determination of IgG antibodies or the quantification of IgM, IgA, or IgE specific isotypes. This procedure allows the simple and reproducible collection of objective results. Moreover, it permits a reduction in cut-off values and direct interpretation of results with automatic conversion of scores into titer, units, index, or into any other scale appropriate for standardization purposes.

Value of specific immunoglobulin A detection by two immunocapture assays in the diagnosis of toxoplasmosis.

The diagnosis of Toxoplasma gondii infection is currently based on immunological tests, but tests for IgG and IgM antibodies alone are often insufficient to assess the risk of active disease, especially during pregnancy and in immunodeficient subjects. The supplementary diagnostic value of testing for antitoxoplasmic IgA in cases of acute, chronic, congenital and reactivated toxoplasmosis, relative to classical immunological tests, was evaluated using two immunocapture tests, one based on tachyzoite agglutination and the other on an immunoenzymatic complex recognizing the membrane protein P30 of Toxoplasma gondii. A total of 4,541 sera from 395 uninfected subjects, 468 immunized subjects with chronic infection, 117 subjects with acute infection and 403 children, 103 of whom had congenital toxoplasmosis, was tested. Specific IgA tests were negative in the nonimmune population, but tests for this immunoglobulin subtype became positive very rapidly during primary infection, and IgA disappeared more rapidly than IgM. In the children infected in utero, specific IgA was detected more frequently than IgM. In contrast, in a population of HIV-seropositive subjects with clinical toxoplasmosis, tests for IgA were poorly sensitive. The two tests for specific IgA produced similar results, except in the early stages of primary infection, in which immunoenzymatic testing for anti-P30 IgA was less sensitive than the agglutination method.

Rapid detection of toxoplasmic nucleic acid by enzyme-linked immunofiltration-assay after membrane transfer.

We report the use of enzyme-linked immunofiltration-assay (ELIFA) for rapid detection of Toxoplasma gondii DNA after transfer to nitrocellulose or nylon membranes. Saturation, specific labeling with a nonradioactive oligonucleotide probe and washing steps are included in an active filtration process, which is controled by microcomputer regulating reagent flow. This method provides excellent specificity, reproducibility and repeatability. Recycling in a closed circuit or by reversing the direction of flow provides the advantage of reducing the required volume of costly reagents (e.g. probes). ELIFA does not require lengthy membrane manipulation, and hybridization as well as stringency and revelation steps can be automated. The procedure can be carried out in less than 2 h, which is a major time saving compared to conventional solid-phase molecular hybridization techniques.

[The risks of pyrimethamine-sulfadoxine combination in the prenatal treatment of toxoplasmosis]. / Les risques de l'association pyriméthamine-sulfadoxine dans le traitement anténatal de la toxoplasmose.

Some of the alternative treatments to avoid termination of pregnancy in cases where the fetus is affected by toxoplasmosis is to treat it as soon as the diagnosis has been made. The authors who already have experience of using pyrimethamine with sulfadoxoine (Fansidar) in the post-natal treatment of congenital infection, thought after reviewing the literature that this association of drugs would be harmless if applied during pregnancy. The principal risk that arises in the fetus is the teratogenicity of each of the components of pyrimethamine and sulfadoxine and also their associations. In animals pyrimethamine can increase the frequency of cleft palates probably because of its antifolinic action but there is no formal proof that it is teratogenic in human beings. Furthermore, the theoretical risk of karnicerus in the new born using the Sulfonamide has not been demonstrated. In the mother the main but rare risk (1 in 75,000) seems to be for the production of severe skin lesions such as Lyell and Stevens-Johnson which could be brought about by sulfonamides, but not particularly sulfadoxine.

[Value and limitations of anti-mannan antibodies research by co-immunoelectrodiffusion in the diagnosis of deep candidiasis]. / Intérêt et limites de la recherche des anticorps anti-mannane par co-immunoélectrodiffusion dans le diagnostic des candidoses profondes.

Several groups have evaluated detection of antibodies against Candida, with somewhat conflicting results. In this study, co-counterimmunoelectrodiffusion was used to detect antimannan antibodies specific of components of the Candida membrane. Study patients were divided into two groups according to whether their history for Candida infection was negative (population A, n = 102) or positive (population B). Different antigen levels were used in order to differentiate low and high antimannan antibody levels. Among the 102 sera in population A, 42 were positive for antimannan antibodies; the antimannan antibody titer was low in 40 cases and high in 2 cases. In population B (53 patients), antimannan antibodies were found in 97 of the 98 sera studied; titers were high in 95 cases. Use of an antigen level that detects only high titers of antimannan antibodies thus provides a sensitive and specific tool for the diagnosis of deep candidiasis. The simplicity and rapidity of this test are particularly valuable in situations where emergency treatment is needed.

Enzyme-linked immuno-filtration assay applied to accelerated immunodetection of gel transferred proteins on immobilizing matrices.

The enzyme-linked immunofiltration assay (ELIFA) has been used for the rapid detection of electrophoresed proteins transferred from gels to immobilizing matrices. By controlled filtration, ELIFA permits the saturation of nylon or nitrocellulose membranes and the immunodetection of blotted antigens to be carried out in 15 min. The method is simple, can be automated and requires no handling of membranes. It complements the well standardized steps of gel electrophoresis and semi-dry horizontal electroblotting which can themselves be carried out in less than an hour. The sensitivity is at least 1-5 ng. The same process can be extended to the accelerated characterization of glycoproteins using appropriate ligands, or to the identification of antigens in a variety of biological fluids.

[Should immunologic monitoring of toxoplasmosis seronegative pregnant women stop at delivery?]. / La surveillance immunologique d'une femme enceinte séronégative pour la toxoplasmose doit-elle s'arrêter à l'accouchement?

The authors report 2 cases of congenital toxoplasmosis fortuitously diagnosed in 2 newborn infants aged 12 and 35 days respectively whose mothers had no anti-Toxoplasma antibodies detectable at the time of birth. These cases prompted us to carry out, over an 18 months' period, a systematic postnatal control of all pregnant women who were still seronegative at the time of delivery. This enabled us to detect 4 cases of perinatal maternal infection with Toxoplasma contamination in 2 neonates. In view of these results, and in order not to miss any maternal infection at the very end of pregnancy, it seems advisable to complete the control of seronegative women by taking a last blood sample about 30 days after they have given birth.

[Comparative study of antitoxoplasmic IgG isotypes titrated by high sensitivity direct agglutination and indirect immunofluorescence: consequence of the choice of methods on the expression of results in international units]. / Etude comparée des isotypes antitoxoplasmiques IgG titrés par agglutination directe haute sensibilité et immunofluorescence indirecte: incidence du choix des méthodes sur l'expression des résultats en unités internationales.

The use of International Units per ml (IU/ml) to express antitoxoplasmic IgG antibody titers in the various diagnostic systems presently proposed, is misleading owing to discrepancies in the values found from one test to the other for a given serum. The authors compared the results of high sensitivity direct agglutination (HSDA) to those of indirect immunofluorescence (IIF) in two studies, systematic and longitudinal, dealing with 158 sera stratified for values ranging from 102,400 to 5 IU/ml. Discordances between the methods, which are greater for high values, prompt the use of low-titer sera for standardization. From the systematic study, a correlation table was established and proposed to convert the HSDA results into the theoretical corrected values close to those that would be obtained by IIF. Although this may be of interest in maintaining a coherent language, this table has its limits, particularly in acute episodes where the various antibody kinetics vary and amplify further the discrepancies. In such situations, it seems advisable for both clinicians and biologists to raise any equivocal kept going by what is termed as International Units. Consequently, if the titers obtained by one method cannot be correlated to those of the technique of reference (IIF, Dye test), on the basis of using I.U., it would be appropriate to express the results in units related to the method or kit used (e.g. U/ml/HSDA for high sensitivity direct agglutination). Finally, whatever the technique, it is still mandatory to conserve a significant threshold value of protective immunization, common and identical to those classically adopted (8-12 IU/ml).

Isotypic characterization of anti-Toxoplasma gondii antibodies in 18 cases of congenital toxoplasmic chorioretinitis.

A serological study was carried out by ELIFA (Enzyme Linked Immuno-Filtration Assay) for 50 children with congenital toxoplasmosis diagnosed by several parasitological and serological methods showing that the fetus had been infected by the parasite and had developed it's own specific immune response. At birth, anti-Toxoplasma gondii IgE antibodies were detected in the sera of 66% of the 18 children who had retinochoroiditis and in 32% of the 32 children without this complication. During the 4 months before or at the time of diagnosis of retinochoroiditis, specific IgE antibodies were detected in 70% of the 20 cases (2 children with 2 successive lesions); but during the 4 months following the discovery of ocular lesions, anti-Toxoplasma IgE antibodies were only detected in 30% of the 20 cases. Among all the 50 children, the prolonged detection of specific IgM + IgE association (for at least 4 months) was followed in 46% of cases by the appearance of chorioretinitis (predictive value).