TRANSACTIONSOFTHEROYALSOCIETYOFTROPICALMEDICINEANDHYGIENE(1996)90,578-581 Total and specific anti-Trypanosoma cruzi immunoglobulin E in pericardial fluid samples from patients with chronic Chagas disease Jose Roberto Mineol, Ademir Rocha2, Julia Maria Costa-Cruz3, Arnaldo Moreira da Silva’, Deise Aparecida de Oliveira Silva”, Maria do Roskio de Fgtima Gongalves-Pires 3, Edison Reis Lopes4 and Edmund0 Chapadeiro4 Laboratories of ‘Immunology, 2Anatomopathology and 3Parasitology, Universidade Federal de Uberkkdia, Uberlindia, Minas Gerais, Brazil; 4Division of Anatomopathology and Legal Medicine, Faculdade de Medicina do Tri&gulo Mineiro, Minas Gerais State, Brazil Abstract Levels of total and specific anti-Typanosoma cruzi immunoglobulin E (IgE) were determined by immu- noenzymatic assay among 101 samples of pericardial fluid from patients who had died in one trypanosomi- asis endemic area in central Brazil. These samples were divided into 6 groups. Group I, 17 samples from patients with the cardiac form of Chagas disease; group II, 11 samples from patients with the digestive form of Chagas disease, presenting megaoesophagus and/or megacolon; group III, 41 samples from patients with the indeterminate form of Chagas disease; group IV, 4 samples from patients with both cardiac and diges- tive forms of Chagas disease; group V, 5 samples from patients who suddenly died and were seropositive for T. cruzi antibodies; group VI, 23 samples, used as a control group, which came from patients seronegative for T. cruzi antibodies. Significantly high levels of total IgE were observed in groups I, II, III, IV and V when compared with group VI (mean concentrations 708-1157 iu/mL compared with 394 iu/mL). In groups I-V, 32 samples (41%) had specific anti-T. cruzi IgE antibodies. The individual percentage positivity rates in these groups were 64.7% (group I), 454% (group II), 34.1% (group III), nil (group IV), and 40.0% (group V). A significant correlation between total IgE and specific anti-T. cruzi IgE was observed only in the samples from patients with the cardiac form of Chagas disease (group I). Keywords: American trypanosomiasis, Chagas disease, Typanosoma cmzi, immunoglobulin E, pericardial fluid Introduction Typanosoma cruzi causes Chagas disease in humans and it is estimated that approximately 35 million people living in rural areas of Central and South America are at risk of infection and between 10 and 12 million are actu- ally infected (TAKLE & SNARY, 1993). The humoral and cellular immune responses as well as the resistance to in- fection with bloodstream forms of T. cruzi and the im- munopathology of chronic Chagas disease have been widely studied in human and animal models (YOSHIDA et al., 1993; MOTRAN et al., 1994; NORRIS et al., 1994; GRUPPI et al., 1995). Sera from individuals infected with T. cruzi, unlike sera from individuals with other proto- zoal infections, do not contain high levels of immuno- globulin. However, T. cruzi infection in Balb/C mice in- duced a reversible polyisotypic hypergammaglobulin- aemia (EL-B• UHDIDI et al, 1994). Post-mortem serologi- cal and anatomopathological studies of Chagas disease have been done in order to increase knowledge of its dif- ferent clinical forms (LOPES et al., 1978; ROCHA et al., 1987; SAGUA et al., 1994). Immunoglobulin (Ig) profiles are similar in serum and pericardial fluid samples, as has been confirmed by STUCHL~KOVA et al. (1966) and REIS (1984). So far, only the isotypes IgG, IgM and IgA have been studied in pericardial fluid samples from patients who have died with the cardiac form of Chagas disease. Higher levels of IgG were found in the cardiac form, and higher levels of IgM in patients with Chagas disease who suddenly died, compared with the levels in uninfected individuals (REIS, 1984). As there has been no study of IgE in pericardial fluid samples from individuals who have died with chronic Chagas disease, and as this immunoglobulin may be in- volved in the mechanisms of chagasic myocardiopathy and/or sudden death of these patients, we decided to in- vestigate the presence of total and specific anti-T. cruzi IgE antibodies in pericardial fluid. Materials and Methods Pericardial fluid samples Samples were obtained from 101 individuals, from the Division of Anatomopathology in the Hospital das Clinicas, Universidade Federal de Uberlandia, Brazil. Author for correspondence: J. R. Mineo, Laboratbrio de Imu- nologia, Universidade Federal de Uberllndia, Av. Parh 1720, Campus Umuarama, Uberllndia, MG 38400-902, Brazil. Based on the results of the indirect immunofluorescence test for T. cruzi antibodies (CAMARGO, 1966), the pericar- dial fluid samples were divided into 6 groups: group I, 17 samples from patients with the cardiac form of Cha- gas disease; group II, 11 samples from patients with the digestive form of Chagas disease, presenting megaoeso- phagus and/or megacolon; group III, 41 samples from patients with the indeterminate form of Chagas disease; group IV, 4 samples from patients with both cardiac and digestive forms of Chagas disease; group V, 5 samples from patients who died suddenly and were seropositive for T. cruzi antibodies> and group VI, 23 samples, used as a control group, obtained from patients seronegative for T. cruzi antibodies. All samples were collected during necropsy at Hospital de Clinicas and transported to the Laboratory of Immunology, where they were centri- fuged at 1000 g and kept at -20°C until used. Immunoenzymatic testfor total IgE A double antibody immunoenzymatic test, as described by KEMENY &Rx~~~~s(1987),wasused,withsomemodi- fications. Briefly, polyvinyl chloride enzyme-linked immu- noassay (ELISA) microtitre plates (HemobagB, Brazil) were coated overnight at 4°C with 10 PgjrnL of sheep IgG anti- human IgE in 0.1 M sodium carbonate buffer (pH 9.6). The plates were washed 3 times with phosphate-buffered saline containing 0.05% Tween 208 and incubated for 1 h at 37°C with duplicate undiluted pericardial fluid samples and with samples containing 500 iu/mL of standard human IgE anti- body (Behring, Germany). As controls, the samples were also incubated with uncoated plates in the same conditions. The second incubation step was carried out with rabbit an- tibody to human IgE, labelled with peroxidase, according to WILSON & NAKANE (1978). After incubation for 1 h at 37°C and another 3 washes, the plates were incubated with substrate solution consisting of Hz02 and o-phenyle- nediamine in 0.1 M citrateNqHPO4 buffer (pH 5.5) for 15 min at room temperature. The reaction was stopped with 2 N HzS04 and the absorbance was read at 492 nm in a mi- crowell reader system (Organon Teknika, Belgium). Immunoenzymatic testfor speczjic anti-T. cruzi IgE An ELISA for IgE antibodies against T. cruzi was car- ried out according to ENGVALL & PERLMANN (1972), with some modifications. Briefly, soluble extract of epi- mastigote forms of T. cruzi, obtained as described by