An Evaluation of the Direct Agglutination Test for Chagas' Disease

Abstract

In the direct agglutination (DA) test, nonspecific agglutination with Trypanosoma cmzi antigen suspensions has been controlled by treating the organisms with trypsin, formalin, and filtration. The resultant antigen suspension containing treated T. cruzi epimastigotes is a sensitive and stable preparation for use in the DA test. Normal sera reacted at low dilutions of 1:2 to 1:64, whereas 93% of the sera from active cases of Chagas' disease were reactive at dilutions of 1:512 or greater. Less sensitivity was observed when sera from chronic cases were tested. Twenty-five per cent of the sera were reactive at dilutions of 1:512 or greater, 33% showed a titer of 128 to 256, and 42% showed a titer of 64 or less. Slide and tube agglutination techniques, for both live and killed Trypanosoma cruzi organisms, were described as far back as 30 years ago (Packchanian, 1940; Senekjie, 1943; Senekjie and Lewis, 1944). In addition to being used in the diagnosis of Chagas' disease, agglutination techniques have also been used in studies comparing antigenic differences between various strains of T. cruzi (Hauschka et al., 1950; Nussenzweig et al., 1963). Although these techniques are relatively simple to perform, they have not proved to be suitable for routine diagnosis due to spontaneous agglutination which often occurs in the antigen preparations and nonspecific agglutination which occurs with some normal human serum at rather high dilutions (Hauschka et al., 1950). Furthermore, since tests with killed organisms were generally not satisfactory, living organisms had to be readily available. For these reasons, agglutination tests for the diagnosis of Chagas' disease have not been widely used. Recently, Vattuone and Yanovsky (1971) described an agglutination test with a suspension of enzyme-treated, formalin-fixed T. cruzi epimastigotes as antigen. This antigen suspension proved to be sensitive and stable in the direct agglutination (DA) test. In studies comparing the results of the DA test with results of other standard serologic procedures, these workers showed that the DA test and the indirect immunofluorescence (IFA) test were the most sensitive in detecting antibodies to T. cruzi infections in the acute Received for publication 14 May 1973. tages of the disease. In contrast, most serologic tests employed for the diagnosis of Chagas' disease detect antibody in the sera of patients with chronic but not acute infections (Vattuone et al., 1973). Further evaluation of the use of the enzyme-treated, fixed T. cruzi organisms in the DA test is presented. MATERIALS AND METHODS Serologic test procedures I. Direct agglutination (DA) test A. Inoculum cultivation (Maekelt, 1960): Cultures of T. cruzi, Corpus Christi strain, were grown in 125-ml Erlenmeyer flasks containing a diphasic medium. The solid phase consists of brain heart infusion agar, 37.0 g; dextrose, 10.0 g; agar, 20.0 g; and distilled water, 1,000 ml. After dissolving the various ingredients, the medium is dispensed in 25-ml amounts and sterilized by autoclaving. After the medium has cooled to approximately 46 C, 2 ml of fresh defibrinated rabbit blood is added, mixed well with the agar base, and allowed to solidify. Five ml of overlay (0.95% sterile saline) is then added to each flask, and the medium is tested for sterility by incubation at 37 C for 18 hr. Each flask is inoculated with the overlay from a tube of 7to 10-day-old stock culture. The flasks are incubated at 25 C until peak growth is reached, usually in 7 to 10 days. The overlay from 4 flasks is pooled and diluted to 100 ml with 0.95% sterile saline. This amount is enough to inoculate 20 flasks for antigen production. B. Antigen cultivation (Maekelt, 1960): The same (Maekelt's) medium without blood is used for antigen production. Five ml of inoculum containing approximately 3,000,000 T. cruzi organisms per ml is added to each flask. The flasks are incubated at 25 C until peak growth is reached, usually 7 to 10 days. Overlay from all uncon-