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.

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.

Endemic amebiasis in an extended family.

Two cousins from a large Spanish-American family were simultaneously diagnosed as having amebic liver abscesses. Survey of 183 extended-family members revealed that 45.7% of 162 had a positive amebiasis indirect hemagglutination test and 12.6% of 111 had cysts or trophozoites of Entamoeba histolytica demonstrated in a single stool examination. A total of five family members had had liver abscesses; two deaths had occurred. In a random sample survey of the remainder of the community, only one person (0.3%) had a positive serologic test. Within the extended family, person-to-person appeared to be the predominant mode of transmission. Water supplies were not contaminated. Both community and extended family homes had the same source of water. Type and source of food supply were not correlated with infection and there was no evidence to implicate an infected food handler. Clustering of seropositivity occurred in homes without indoor toilets. Homes of the extended family were more crowded and significantly fewer of them had indoor toilets. Endemic foci of amebiasis continue to exist in the United States. Follow-up family and other close contacts of persons with amebiasis will frequently identify other cases.

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.

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.

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.

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.

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.

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.

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.

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.

Evaluation of some of the parameters of the enzyme-linked immunospecific assay.

In an attempt to standardize the procedure for the enzyme-linked immunospecific assay, several parameters were examined. It was determined that horseradish peroxidase was the enzyme of choice and that several brands of polystyrene tubes and plates could be used. The test was considerably shortened by omitting long incubation steps previously used for eliminating background fixation of conjugate. The reproducibility of the procedure proved to be excellent, but reactivity curves indicated that use of a single dilution is not adequate for quantitative tests. A "standard" procedure was proposed.

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.

Effect of age and sex on the acquisition of immunity to toxoplasmosis in cats.

The effects of age and sex of the cat on oocyst shedding, multiplication of Toxoplasma gondii in tissues of cats, and acquisition of immunity were investigated after oral inoculation of cats with Toxoplasma cysts. Twenty-five cats varying in age from 1 week to 39 months were killed 7-97 days after inoculation with T. gondii. Homogenates of brain, heart, mesenteric lymph nodes, retina, and blood from these cats were inoculated into mice to test for Toxoplasma infectivity. Toxoplasma was isolated more frequently and in higher titers in mice receiving inocula from cats of the youngest age group (1 week old). Toxoplasma gondii was isolated from tissues of only 2 of 21 cats older than 2 months (at the time of inoculation), although all of the animals shed oocysts within 1 week after ingesting the parasites. The number of oocysts shed varied among littermates of the same sex and between sexes. Generally, cats younger than 12 months shed more oocysts than older cats. The number of oocysts shed by older cats varied considerably; males generally shed more oocysts than the females. However, the numbers of cats examined were too small for statistical comparison. Nevertheless, the observations suggest that cats older than 12 months should not be used in experiments where numbers of oocysts shed is critical.