The R3 peptide ELISA showed 92.4% sensitivity and 100% specificity for chronic chagasic sera in comparison with sera from individuals infected withLeishmaniaand patients suffering IDC and EHS, sera which were below the cutoff value. observed in patients treated with antiparasitic drugs. Those results suggest that the presence of anti-R3 antibodies is a highly specific marker of Chagas’ disease and that R3 ELISA could be helpful in the diagnosis and monitoring of this disease. Chagas’ disease, which is caused by the protozoan parasiteTrypanosoma cruzi, affects several million people in Central and South America (3). Approximately 30% of infected persons develop symptoms of the disease in their lifetime, which include cardiomyopathy, neuropathies, and dilatation of colon or esophagus (27). The finding of a T-cell-rich inflammatory mononuclear cell infiltrate and the scarcity of parasites in heart lesions questioned the direct participation ofT. cruziin chronic Chagas’ cardiomyopathy (CCC) and suggested the possible involvement of autoimmunity (24), although this remains a hotly debated issue (13). Natural infections occur via the triatomid insect vector and have been almost abolished through vector control programs. Congenital transmission and transfusion of blood from infected donors have become the major routes of transmission of Chagas’ disease, and blood bank testing is now necessary in many countries. Diagnosis ofT. cruziinfection often requires a combination of some of the Ezatiostat commercially available tests (15). Traditional methods of parasite detection such as xenodiagnosis and hemocultures have low sensitivity and require long periods of time to carry out. Recently, PCR amplification of nuclear or kinetoplast DNA has been shown to be very sensitive (2,4,25,31). However, PCR is not yet feasible for blood bank testing in many of the areas where Chagas’ disease is endemic. At present, the best way of diagnosing an indeterminate or chronicT. cruziinfection is the serologic detection of antibodies directed against the parasite. Usually, two tests based on different methodologies are required, indirect immunofluorescence (IIF) and indirect hemagglutination (IHA), with the results confirmed by a third test, an enzyme-linked immunosorbent assay (ELISA) (15). Thus, specificity of Chagas’ disease diagnosis is still a problem. Previously, we isolated a human antigen recognized by chagasic sera, named Cha. The epitope of Cha recognized by chagasic sera was mapped to amimo acids 120 to 129 Ezatiostat (the R3 peptide) (11). We studied whether the R3 peptide of the Cha autoantigen could be used as a marker of the disease. For this, we studied the reactivity of chagasic sera, including sera from patients at Ezatiostat different clinical stages, against the R3 peptide in ELISA. In addition, we compared the R3 ELISA with other available tests. == MATERIALS AND METHODS == == Synthetic peptides. == Peptides R3 (MRQLDTNVERRALGEIQNV) from human Cha and S1 (STPSTPADSSAHSTPSTPV) fromT. cruzished acute-phase antigen were synthesized on an Applied Biosystems synthesizer model 431A. Peptides were purified by high-pressure liquid chromatography and checked for accuracy by mass spectrometry. == Human sera. == A total of 79 sera from patients with chronic Chagas’ disease from Venezuela and Argentina were tested. Of those, 50 were from patients at different clinical stages, including chronic patients treated with antiparasite drugs Ezatiostat (Radanil or Lampit). Sera from Argentina were obtained from the Servicio Nacional de Chagas Argentina, and sera from Venezuela were a gift from J. Sequ (Centro de Investigacin Clnica, Instituto Carlos III, Madrid). Nonchagasic patients included 10 healthy individuals from an area were Chagas’ disease is Capn1 endemic (EHS samples) (Servicio Nacional de Chagas, Argentina), 10 individuals infected with theLeishmaniaparasite whose antigens cross-react withT. cruzi(kindly supplied by C. Alonso, Centro de Biologa Molecular, Madrid), and 6 patients with nonchagasic cardiomyopathy with diagnosis of idiopathic dilated cardiomyopathy (IDC), a disease with similar cardiac symptoms as CCC (kindly supplied by Barbieri, Chagas Center and Regional Pathology, Santiago del Estero, Argentina). == ELISA. == ELISA with totalT. cruziantigens was performed in microtiter plates covered with soluble antigens following the directions of the manufacturer (T..