Performance of European laboratories testing serum samples forToxoplasma gondii

One hundred twenty-nine European laboratories participated in a collaborative, multicentre study designed to evaluate the overall reliability of different serological techniques for diagnosis ofToxoplasma gondii infection. Five freeze-dried reference sera were distributed to each laboratory, each of which analysed the sera with its routine methods. The enzyme-linked immunosorbent assay was the technique used most frequently, followed by the immunofluorescent antibody technique. Only nine laboratories performed the Sabin-Feldman dye test. In general, there was good concordance between qualitative results, but for sera with low concentrations ofToxoplasma gondii-specific IgG antibodies, some false-negative results were found. For specific IgM and IgA antibodies, the immunosorbent agglutination assay proved the most sensitive. The present study demonstrates the need for regular assessment of laboratory serodiagnosis ofToxoplasma gondii infection.     

Neospora spp. and Toxoplasma gondii antibodies in horses in the Czech Republic

During January 2007, blood samples were collected from 552 healthy horses from nine different regions of the Czech Republic. Sera were tested for serum antibodies to Neospora caninum by a competitive-inhibition enzyme-linked immunosorbent assay and confirmed by an indirect fluorescent antibody test. The same samples were tested for serum antibodies against Toxoplasma gondii by a latex agglutination test. In total, 131 of 552 (24%) horses reacted positively for Neospora antibodies in competitive-inhibition enzyme-linked immunosorbent assay; seven of them had ≥50% of inhibition. Samples were confirmed in indirect fluorescence test, and only two samples were positive with final titres 50 and 100, while others were negative. Antibodies against T. gondii were found in 125 (23%) horses. This is the first serologic survey for Neospora spp. antibodies performed on horses in the Czech Republic.     

Duration of specific immunoglobulin a antibody following acute toxoplasmosis as determined by enzyme immunoassay and immunosorbent agglutination assay

Modifications of an enzyme immunoassay (EIA) and an immunosorbent agglutination assay (ISAGA) for measuringToxoplasma gondii-specific IgM antibody were made to enable the measurement ofToxoplasma gondii-specific IgA antibody. It was shown that specific IgA could be measured by both assays but that the ISAGA was slightly more sensitive. IgA appears about two weeks after IgM and persists for 6 to 7 months. However, the IgA response varies considerably both in degree and duration, and demonstration of IgM antibody is at present the most suitable routine test for the diagnosis of recentToxoplasma gondii infection.     

A Standardized Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of Antibodies to Toxoplasma Gondii

An enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Toxoplasma gondii infections on single serum dilutions was developed.

This test system is a standardized kit designed to detect circulating specific antibodies to Toxoplasma gondii in human sera. It consists of Toxoplasma gondii soluble antigen-coated microtitration multiwell plates, specific immunoglobulin-enzyme conjugate and other required reagents.

In a clinical trial performed on sera from 1,035 clinically suspected toxoplasmosis cases, the Sabin Feldman Dye Test (SFDT) and this ELISA system agreed closely. Relative to the SFDT, the sensitivity and specificity of the latter was 98.0% and 97.6% respectively with a correlation coefficient of 0.97. In a further study of 121 sera, the Indirect Fluorescent Antibody Test (IFAT), the Indirect Haemagglutination Test (IHAT) and this ELISA procedure showed over 90% agreement, with correlation coefficients of 0.98 and 0.95 respectively. Within the working concentration of specific antibody to T. gondii in human serum, there was a linear relationship between the ELISA values and the WHO international standard for human anti-Toxoplasma serum.     

Prevalence of latent toxoplasmosis and serological diagnosis of active infection in HIV-positive patients

The seroprevalence of latentToxoplasma gondii infection was determined in a cohort of 715 HIV-positive patients followed up at an HIV outpatient clinic. Using indirect immunofluorescence and direct agglutination assays for detecting IgG, the prevalence of anti-Toxoplasma gondii antibodies was shown to be 50 %. During a four-year period, clinically apparent acute toxoplasmosis occurred in 47 patients (43 with cerebral, 3 with ocular and 1 with bone marrow toxoplasmosis) among the 360 patients positive for anti-Toxoplasma gondii IgG and in one patient (with cerebral toxoplasmosis) among the 355 patients who were serologically negative. A significant rise in IgG levels could be shown during acute toxoplasmosis episodes in only 30 % of patients, compared with 3 % of patients without active toxoplasmosis. During acute toxoplasmosis, IgM antibodies were detected in only two patients (6 %) by an immunosorbent agglutination assay and in one (3 %) by an enzymatic immunocapture assay. Specific IgA was detected by a non-enzymatic immunocapture assay in six patients (18 %) during acute episodes. The very high predictive value (99.7 %) of a negative IgG test remains the best serological parameter for excluding an acute episode of toxoplasmosis in HIV-positive patients.     

Unspecific Reactivity of IgM Directed Against the Low-Molecular-Weight Antigen of<Emphasis Type="Italic"> Toxoplasma gondii</Emphasis>

During routine serological survey, eight patients (5 pregnant women, 3 grafted patients) were positive for Toxoplasma gondii-specific IgM by enzyme-linked immunoassay but negative by a simultaneously performed immunosorbent agglutination assay. No clinical or biological symptoms of toxoplasmosis were observed later, despite the absence of treatment. Only one IgM-reactive band, which corresponded to the low-molecular-weight antigen of Toxoplasma gondii, was observed by Western blotting of these patients' sera. Dot blotting of lipid extracts of Toxoplasma gondii demonstrated that this reactivity was directed against sphingolipids or ceramides. This IgM positivity, which is unrelated to acute toxoplasmosis, raises strong concerns about the possibility of misleading results of this test in the diagnosis of toxoplasmosis in humans.     

Serodiagnosis of acute toxoplasmosis using a recombinant form of the dense granule antigen GRA6 in an enzyme-linked immunosorbent assay

We developed an indirect enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of acute toxoplasmosis that used the recombinant granule antigen GRA6-GST as diagnostic antigen for the detection of IgG antibodies to Toxoplasma gondii in human sera. A total of 431 sera obtained from 336 patients with acute and chronic toxoplasmosis and from patients who were not infected with T. gondii were tested. Sera from patients with acute T. gondii infection, chronic infection, and no infection showed different absorbance values. For discrimination between the presence and the absence of acute toxoplasmosis the assay reached a specificity of 99.6%. Only one of the sera without significant anti-T. gondii. IgM antibodies showed a positive reaction to rGRA6-GST. The assay showed good intra- and interassay reproducibility (CV 6%/14%). We included a glutathione S-transferase (GST)-IgG enzyme immunoassay as a control assay in this study. Only 7 (4%) of 159 random sample sera reacted positively with GST. Received: 22 November 1997 / Accepted: 26 March 1998     

Estimation of the avidity of immunoglobulin G for routine diagnosis of chronicToxoplasma gondii infection in pregnant women

Present serological methods differentiate poorly between acute and chronic toxoplasmosis in pregnant women, particularly when immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies toToxoplasma gondii are present simultaneously. In the present study, a simple test for discriminating between high-avidity antibodies, which are usually present in chronic infections, and low-avidity antibodies, typical of acute infection, was evaluated. Sera were evaluated forToxoplasma gondii antibodies using a commercial enzyme immunoassay, but a duplicate well was washed in 6M urea to disrupt lowavidity complexes. Results are expressed as the percentage of antibodies resisting elution by urea. Equivocal sera (n=493) containing both IgG and IgMToxoplasma gondii antibodies from 309 pregnant women whose status as chronically or acutely infected had been independently determined using standard methods were evaluated for antibody avidity. A value of >35% elution-resistant antibodies was always associated with chronic infection and could absolutely exclude a recent (<3 months) infectious incident. Values of <35% require repeat testing four weeks later to confirm the patient's status, since a proportion of individuals with chronic toxoplasmosis maintain low-avidity antibodies over long periods. This inexpensive, simple method can provide reassurance to clearly chronically infected individuals and avoids the need for repeated testing in these cases.     

Prevalence of <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> antibodies in cats in maiduguri,northeastern nigeria

Cats play an important role in the spread of Toxoplasma gondii because they are the only animals that excrete resistant oocysts into the environment. Prevalence of T. gondii antibodies in cats in Maiduguri, Northeastern Nigeria was determined using the latex agglutination test (LAT). Thirty eight cats (36.2%) were seropositive using a cut off point of 1:64 with antibody titer ranging from 1:64 to 1:1024. Seroprevalence was higher in older animals and stray cats. Age and ownership status of cats were identified as risk factors but not gender or area of capture.     

Toxoplasma gondii surface antigen-1 in sera of HIV-infected patients as an indicator of reactivated toxoplasmosis

The major surface antigen from the proliferative form ofToxoplasma gondii (P-30 or SAG-1) was chosen as a target for exploration ofToxoplasma gondii reactivation in sera from immunocompromised patients. Samples were obtained from 37 HIV-infected subjects with lymphocyte levels of CD4+ <200/mm³. The prevalence of IgG antibodies toToxoplasma gondii was 64.9 %. Ten patients had clinical symptoms of reactivated toxoplasmosis; eight of these hadToxoplasma encephalitis. The SAG-1 epitopes were found as circulating antigen in five cases with an immunocapture enzyme immunoassay (EIA). The EIA was improved with an IgG1 monoclonal antibody to SAG-1 and a streptavidinbiotin amplification. The sensitivity, specificity and positive predictive value were 30, 92 and 60 %, respectively. The SAG-1 levels were compared with different biological parameters such as HIV p24 antigen, ₂ microglobulin, CD4+ cell count and IgG antibodies toToxoplasma gondii. The levels of SAG-1 in these patients were significantly higher than those in the 75 healthy control persons with or without a chronicToxoplasma gondii infection. Therefore, SAG-1 may be involved as a marker of reactivated toxoplasmosis in HIV-infected patients.     

Comparative study of three immunoassays for detection of immunoglobulin M antibodies againstToxoplasma gondii

Immunoglobulin M antibodies against a major serological antigen (M_r 6 kD) were detected in human sera by indirect ELISA, antibody capture ELISA, and immunoblot test. In contrast to indirect ELISA, the immunoblot test gave no false positive reactions, not even with those sera containing a high level of rheumatoid factor. However, the immunoblot test gave false negative results with sera which gave positive results in both ELISA tests. The antibody capture ELISA gave no false negative reactions. All positive sera except one reacted specifically withToxoplasma gondii antigens.     

Seroprevalence of Toxoplasma gondii and Neospora caninum in dogs from Korea

Toxoplasma gondii and Neospora caninum are closely related protozoan parasites, they share many common hosts, and can cause neurological diseases in dogs. Dogs can have close contacts with humans and livestock and therefore they can act as reservoirs of these parasites. The aim of this study was to survey the seroprevalence of antibodies against T. gondii and N. caninum and their co-infection rate in dogs in Korea. In total, sera from 553 domestic dogs were collected from different breeds, sexes, and ages of dogs from nine provinces across the country of Korea during 2006 and 2007. The presence of antibodies against T. gondii and N. caninum was analyzed using the latex agglutination test (LAT) with a cut-off value of 1:32, and the indirect fluorescent antibody test (IFAT) using a serum titer of 1:100. In the total dog population, 71 (12.8%) dogs were positive for anti-T. gondii antibodies and only 20 (3.6%) were positive for anti-N. caninum antibodies. Relatively higher seropositive frequencies of antibodies against T. gondii (20.1%) and N. caninum (4.9%) were detected in the dog population from the Gyeonggi. A higher proportion of animals seropositive for anti-T. gondii antibodies was found in stray dog populations as compared to household dog populations: 18.5% (59/319) vs 5.1% (12/234), respectively. The Chi-square tests revealed significant differences in the seropositive frequencies of antibodies against T. gondii between stray and household dogs in the total population (p<0.0001), and in dogs from the Gyeonggi (p<0.01). No significant differences were observed for the presence of antibodies against T. gondii or N. caninum when compared across the sex or age (p>0.05). The first serological survey on antibodies against both T. gondii and N. caninum parasites across the entire country showed that co-infection was not common in these canine populations with a seropositive level of 0.72%. The significantly higher positive frequency of T. gondii antibodies in stray dogs in both, Gyeonggi and in the total dog populations suggests that further investigation on the seroprevalence of parasites should focus on stray dogs.     

Onset of ocular complications in congenital toxoplasmosis associated with immunoglobulin M antibodies toToxoplasma gondii

Four patients with congenital toxoplasmosis serologically diagnosed by the Sabin-Feldman test (SFT) and the IgM-indirect fluorescent antibody test (IgM-IFAT) in the first year of life presented with eye disease between the age of 21 months and ten years. Repeated serological testing revealed increasing levels of specific antibodies as measured by the SFT. IgM antibodies toToxoplasma gondii were detected in all four patients by the immunosorbent agglutination assay, in two by the IgM-IFAT and in three by the IgM-indirect haemagglutination test. Findings suggest that specific IgM antibodies reappear at the time of reactivation of congenital toxoplasmosis later in life, or possibly persist for an extraordinary long period (up to ten years).     

Recognition of recombinantToxoplasma gondii antigens by human sera in an ELISA

We developed an enzyme-linked immunosorbent assay (ELISA) that uses one of two recombinant polypeptides, termed H4/GST and H11/GST, as diagnostic antigens for the detection of antibodies toToxoplasma gondii in human sera. A total of 59 sera from humans with acute toxoplasmosis, 194 sera from patients with chronic toxoplasmosis, and 151 sera from subjects who were not infected withT. gondii were examined. In all, 68% of the sera from humans with acute toxoplasmosis reacted positively with one or both recombinantT. gondii antigens. By contrast, only 14% of those from patients with chronic toxoplasmosis recognized H4/GST or H11/GST. None of the sera from humans who were not infected withT. gondii, including patients with echinococcosis, entamoebosis, toxocarosis, trichinellosis, glandular fever, or rheumatoid arthritis, recognized H4/GST or H11/GST.This publication is dedicated to Professor J. Eckert (Zürich) on the occasion of his 60th birthday     

Quantitation of Antibodies to Toxoplasma gondii in Swine Sera by Enzyme-Linked Immunosorbent Assay

Abstract

An enzyme-linked immunosorbent assay (ELISA) was developed for quantitation of antibodies to Toxoplasma gondii in swine sera. Because a commercial anti-swine IgG conjugate was directed also against swine IgM, the conjugate was absorbed with the IgM fraction to eliminate the interference of naturally occurring IgM antibodies that appeared consistently in sera collected from slaughtered pigs at an abattoir. The ELISA values of 0.2 or more observed in most of the sera successfully decreased to less than 0.2 by the use of absorbed conjugate. An attempt to use a protein A conjugate has failed. Evaluation of this system by comparing it with the latex agglutination test provided a high significant correlation, indicating its usefulness for serodiagnosis of swine toxoplasmosis.     

Induction of tolerance toToxoplasma gondii in newborn mice by maternal antibody

To develop an animal model for analysing the suppressed immune response toToxoplasma gondii in newborn humans with congenital toxoplasmosis, newborn mice from chronically infected mothers were inoculated intraperitoneally with bradyzoites of an avirulent strain. The newborn mice with maternalToxoplasma antibodies showed a marked delay in the production ofToxoplasma antibodies when infected after birth. Many mice (11/13; 85%) developed a state of tolerance toT. gondii after disappearance of the maternal antibody, demonstrable by the absence ofToxoplasma antibody in their sera despite the fact that they were infected. The duration of tolerance differed between individuals, with two mice showing the longest tolerant state of 8 weeks. This murine model might be suitable for analysing the mechanism of suppressed immune response toT. gondii that has been observed in many human cases of congenital toxoplasmosis.     

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.     

Seroepidemiology of <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> in pet dogs and cats in Beijing,China

Sera from 534 pet dogs and 335 pet cats from Beijing (China) were tested for anti-Toxoplasma gondii antibodies using an enzyme-linked immunosorbent assay or the latex agglutination test. The seropositivity by year, season, sex and age was analysed. Overall, 128 dogs (24.0%) and 50 cats (14.9%) had antibodies to T. gondii. When analysed by season, the highest seroprevalence was found in spring for dogs (31.3%) and cats (25.1%), and the differences in seroprevalence by season was statistically significant in cats (P<0.01) but not in dogs. The seroprevalence in male dogs (23.7%) and cats (15.1%) were slightly higher than their female counterparts (18.0% in dogs and 12.3% in cats). There was no obvious pattern of seropositivity or significant difference in different age groups in dogs or cats; nonetheless, a high proportion of dogs at 4 years of age were positive to T. gondii (31.8%) while cats with relatively high seropositivity rates were at 1 or 3.4 years of age (13.14%).     

Seroprevalence and risk factors of Toxoplasma gondii infection in invasive raccoons (Procyon lotor) in Central Europe

Toxoplasma gondii is an obligate intracellular protozoan that causes toxoplasmosis in warm-blooded animals. Most mammals, including humans, can become intermediate host, resulting in subclinical infection or even death. Generally, there is limited information on the epidemiology of T. gondii of game species in Germany. As omnivores, raccoons, which are particularly widespread and abundant in Germany, are particularly exposed to infection the parasite. Here, we report the seroprevalence of T. gondii antibodies from 15 study sites located in Luxembourg and Germany. Using the indirect modified agglutination test (MAT), 170 (37.4%; 95% CI: 33.0–41.9) out of 454 raccoons were surveyed to be T. gondii seropositive. While values ranged from 19.0% to 53.3%, there was no significant difference in seroprevalence between study areas. Animal weight had a strong influence on the presence of T. gondii antibodies in raccoon sera, with heavier animals more likely to be seropositive. Our results show that T. gondii infection is widespread in central European raccoons, suggesting a high degree of ecosystem circulation of the parasite.

     

Evaluation of immunoblotting for the detection ofToxoplasma gondii immunoglobulin M antibodies

Immunoblot analysis was used to detect human IgM antibodies toToxoplasma gondii in 20 patients with recent toxoplasmosis, 30 immune individuals, 30 non-immune individuals, and 24 children less then two years old. Analysis of the IgM strips revealed that specific IgM antibodies detectable after a recentToxoplasma gondii infection react with the same antigens as the natural antibodies present in the sera of immune and non-immune individuals and in the sera of young children. These data indicate that immunoblotting is not useful as a reference method forToxoplasma gondii IgM detection, and suggest that improvement of the specificity of IgM detection will remain difficult.