Evaluation of commercial serodiagnostic kits for toxoplasmosis.

To evaluate commercially available diagnostic kits for human immunoglobulin G to Toxoplasma gondii, we purchased three enzyme immunoassay (EIA) (Cordia-T, Toxo Bio-EnzaBead, and Toxoelisa), two indirect hemagglutination (IHA) (TPM-Test and ToxHAtest), one fluoroimmunoassay (Toxoplasma-G FIAX), and one latex agglutination (Eiken Toxotest-MT) kit from U.S. suppliers. A total of 100 serum specimens, including 27 that were negative (less than 1:16) and 73 that were positive for the various titers in the Toxoplasma indirect immunofluorescence (IIF) test, were tested once with each kit; serum samples with discrepant results were retested. Qualitatively, results obtained with the Toxo Bio-EnzaBead EIA, the TMP-Test IHA, and FIAX, and the Toxotest-MT latex agglutination kits agreed exactly with those of IIF. Although all IIF-positive serum samples were detected by the Cordia-T and the Toxoelisa EIAs, four samples determined to be negative by IIF were identified as positive with the Cordia-T kit, and six negative samples by IIF were determined to be positive with the Toxoelisa kit. Results of the ToxoHAtest IHA kit were extremely difficult to read. Quantitatively, the seven kits were difficult to compare because the expression of results was not standardized. Of the four kits that gave positive results in titers, Toxoplasma-G FIAX had the closest agreement with IIF, as determined by the Spearman rank correlation coefficient (0.9168), followed by the Eiken Toxotest-MT (0.8293), the Toxo Bio-EnzaBead (0.7553), and the TPM-Test (0.7206) kits.

Toxoplasma antibodies and retinochoroiditis in the Marshall Islands and their association with exposure to radioactive fallout.

Nearly universal serologic evidence of Toxoplasma gondii infection was found to have occurred by adulthood in 517 Marshallese tested in 1981-1982. The prevalence and incidence of retinal lesions compatible with toxoplasmosis were 3.9% and 273 cases/year/100,000 seropositive persons, respectively, thus indicating a significant public health problem. Seronegativity was significantly more common in a subgroup of Marshallese that had received 110-190 rads of total-body gamma radiation as a consequence of accidental exposure to radioactive fallout in 1954. Despite this finding there was no evidence of an increase in clinically significant lesions in exposed persons.

Toxocara canis infestation. Clinical and epidemiological associations with seropositivity in kindergarten children.

We studied a cohort of 333 children in kindergarten to determine the prevalence of seropositivity to Toxocara canis, and to detect and measure chronic health effects that might be attributable to past infection. We found that 23.1% of the children had serologic evidence of infection (antibody titer greater than or equal to 1:32), assayed by means of an enzyme-linked immunosorbent assay. Black children were more frequently infested than white children, as were children of parents who did not graduate from high school. In a subsample of seropositive and seronegative children, we found associations between seropositivity and both pica and puppy ownership; we did not find differences in the symptoms and signs that occur in toxocaral visceral larva migrans nor differences in measures of growth and nutrition. No child had ocular toxocariasis although 31.8% (106) of the children had antibody titers greater than or equal to 1:16. In a population in which approximately 20 to 30% of the children show serologic evidence of Toxocara infestation, care must be taken in differentiating toxocariasis-like ophthalmic lesions, due to the potential for the coincidental occurrence of retinoblastoma in a child who is seropositive for the Toxocara parasite.

An oocyst-transmitted outbreak of toxoplasmosis: patterns of immunoglobulin G and M over one year.

Sera from 32 patients who became ill after jungle combat training were tested for antibodies to Toxoplasma gondii using the indirect immunofluorescence test. Swift rises of both IgG and IgM antibodies occurred within 2 weeks of infection. Reduction in IgM titers, due to competitive suppression by IgG antibody, occurred in most but not all cases. Suppression of IgM reaction by IgG antibody could be prevented by adsorption of serum with Staphylococcus aureus containing protein A. Antibody of the IgM class could be detected at greater than or equal 1:256 level in many sera at 6-month and 1-year intervals after exposure. In groups with exposures such as were experienced in this study, the presence of IgM antibody titers in single serum specimens cannot be used to indicate recent exposure. Both IgG and IgM antibody may rise together to high levels very rapidly after infection; IgM did not precede IgG antibody in our 32 subjects.

Serologic evaluation of cattle inoculated with Toxoplasma gondii: comparison of Sabin-Feldman dye test and other agglutination tests.

Six cows and 6 calves were each inoculated with 100 or 100,000 Toxoplasma gondii oocysts. Serum samples were analyzed, using the Sabin-Feldman dye test (DT), indirect hemagglutination test, latex agglutination test, and the modified direct agglutination test (MAT). Antibody titers in cows were lower than in calves. In the cows, DT titers increased briefly during the first month after inoculation, after which the titers were negative; however, T gondii was isolated from the tissues of 4 cows. Indirect hemagglutination and latex agglutination titers were generally less than 1:256. The MAT titers increased to 1:1,024 during the first month after inoculation. In 5 of the 6 cows, the MAT titers persisted. The 6th cow had a preinoculation MAT titer of 1:2,000 for 3 to 6 months. Therefore, the DT was not useful in serologic surveys for T gondii in cattle; the MAT was the most sensitive test and may be useful in the diagnosis of T gondii infection in cattle.

Computer-assisted multiple categorization of absorbance values in ELISA through pictorial emulation of 96-well plates.

A simple algorithm is proposed by which multiple categorization of absorbance values from ELISA plates is performed under a microcomputer control. The printed output is a pictorial emulation of a 96-well plate with the color intensities represented for each reaction. Although the method is presented as a colorimeter computer interfaced system, a provision for manual entry of absorbance values via keyboard is also included. Simulation is based solely on the magnitude of absorbance values. Therefore, it is possible to utilize any enzyme/substrate combination within the range of filters of the colorimeter. We have tested the present system for titration of anti-malarial antibodies in human serum and for the screening of mouse hybridoma culture supernatants.

Toxocara canis infection: clinical and epidemiological associations with seropositivity in kindergarten children.

To determine epidemiological and clinical associations with Toxocara canis seropositivity, we studied 333 (87%) children of a cohort of 383 five- to seven-year-olds. The prevalence of seropositivity (antibody titer to T canis, greater than or equal to 1:32) was 23.1%. Black children were more frequently seropositive than were white children, as were children of parents who did not graduate from high school. In a sample of seropositive and seronegative children, seropositivity was associated with both a history of pica and puppy ownership, but not with a greater frequency of symptoms and signs that occur in visceral larva migrans or with poor growth. No child had evidence of ocular toxocariasis on retinal examination. For the whole sample, poor reading achievement, marked distractibility, and lower intelligence were associated with seropositivity, but by using multiple regression analysis, we found that these associations may be attributable to confounding variables.

Microvolume, kinetic-dependent enzyme-linked immunosorbent assay for amoeba antibodies.

We describe a microvolume enzyme-linked immunosorbent assay based on enzyme rate kinetics. Antigens from Entamoeba histolytica were adsorbed in wells of disposable polystyrene strips containing 12 flat-bottom wells. After exposure to the serum of a patient and peroxidase-labeled anti-human immunoglobulin G, the rate of color change in specific substrate was determined by eight sequential readings of individual wells over a 2-min period with a microcomputer-controlled model MR-600 automated plate reader. The changes in absorbance readings were converted to slope values for each well by the microcomputer. Thus, 12 samples were read, and results were printed in ca. 3.5 min. Assay conditions are described and data are presented to show that this assay is quantitative for antibody and antigen concentration with a single-tube (well) dilution.

Evaluation of serologic tests for Pneumocystis carinii antibody and antigenemia in patients with acquired immunodeficiency syndrome.

Sera from 68 patients with acquired immunodeficiency syndrome and 135 controls were used to evaluate the indirect immunofluorescence and enzyme-linked immunosorbent assays for detection of antibodies to Pneumocystis carinii and a counterimmunoelectrophoresis assay for detection of circulating Pneumocystis antigen. None of these assays was helpful in the diagnosis of P. carinii pneumonia. An improved assay for antigenemia is needed to differentiate between clinical and subclinical infection.

Evaluation of a commercial latex agglutination method for toxoplasmosis.

A commercial latex agglutination test for detecting immunoglobulin G antibody to Toxoplasma gondii, the Syn-Kit Toxotest MT, was evaluated in two laboratories against the Sabin-Feldman dye test, the indirect immunofluorescence test, and FIAX. The Toxotest MT was found easy to perform and required no special equipment or training. The latex test was more reactive than the reference procedures but compared favorably with them. Failure to properly detect positive sera in two early acute cases and in three other cases causes concern and suggests caution in using the test for suspected early cases. The Toxotest MT is useful for screening for immunoglobulin G toxoplasma antibody and for determining serologic status in pregnant women.

Diagnosis of acute acquired toxoplasmosis with the enzyme-labelled antigen reversed immunoassay for immunoglobulin M antibodies.

The reversed enzyme-labelled antigen immunoassay (R-EIA), based on the capture of serum immunoglobulin M antibodies (IgM) and subsequent addition of Toxoplasma gondii soluble antigen tagged with peroxidase and substrate, was evaluated comparatively with the IgM-indirect immunofluorescence test (IgM-IIF) for the detection of anti-toxoplasma IgM antibodies in sera from individuals with diagnosed acute acquired toxoplasmosis. Additional serum groups from normal healthy individuals and sera presenting possible nonspecific reactivities were also evaluated. Complete specificity of R-EIA was shown. There was no correlation between the magnitude of R-EIA results and IgM-IIF titers, but a positive (although not linear) correlation was found between R-EIA and the IgM-IIF titers obtained after adsorption of sera with Staphylococcus aureus protein A. Direct labelling of the antigen by a simple coupling technique facilitated the assay standardization and improved its signal-to-noise ratio.

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.

Prevalence of antibodies to Toxoplasma gondii among bobcats of West Virginia and Georgia.

Bobcats (Felis rufus) from West Virginia and Georgia were examined for Toxoplasma gondii serum antibodies and fecal oocysts. Of the 150 bobcats, 27 (18%) had antibody titers greater than or equal to 1:16, as detected by the indirect hemagglutination test. Oocysts typical of T gondii were not seen in any of the fecal specimens.

Infectious disease patterns in the Waorani, an isolated Amerindian population.

The Waorani Indians of eastern Ecuador provide a unique opportunity for studying exposure of an isolated human population to various infectious disease agents. Using serologic tests to determine antibody prevalence, skin test data, and stool examination for parasites, we have been able to construct a profile of infectious diseases which are endemic, and others which have been introduced into the Waorani population. These findings are compared with similar data reported from elsewhere in the Amazon. Serologic studies demonstrating the presence of antibody to measles and poliovirus type 3 after vaccination indicate that the Waorani respond normally to viral challenge with these agents. The question of genetic inability among aboriginal Amerindians to respond to viral agents is discussed. Finally, general recommendations are made regarding the future health care of the Waorani.

Serodiagnosis of human cysticercosis by microplate enzyme-linked immunospecific assay (ELISA).

The micro-ELISA described in this report is simple and can give objective results for the serodiagnosis of human cysticercosis. The visual assessment of results was satisfactory. The ELISA results showed a remarkably good correlation with IHA. It gave good reproudcibility. Cross-reactions were eliminated by using whole worm antigen instead of the cysticercus.

Titration of human serum antibodies to Toxoplasma gondii with a simple fluorometric assay.

A new technique, FIAX, has been evaluated for the serodiagnosis of toxoplasmosis. It is based on a "dipstick" principle, and a special fluorometer is used to perform the indirect immunofluorescence test. The procedure appears to be simple and rapid and merits consideration as a useful serological test for toxoplasmosis.