Immunoblot assay using excreted-secreted antigens of Trypanosoma cruzi in serodiagnosis of congenital, acute, and chronic Chagas' disease

Abstract

Immunoblotting with trypomastigote excreted-secreted antigens (TESA blot) of Trypanosoma cruzi was evaluated as a method for diagnosis of chronic and acute phases as well as congenital (in newborn children) Chagas' disease. Serum samples from acute-phase and congenital infections were considered to be positive when they reacted with ladder-like bands of 130- to 200-kDa antigens, recognized by immunoglobulin M (IgM) and IgG antibodies, while IgG from chronic-phase sera recognized a broad band antigen of 150 to 160 kDa. Nonchagasic sera were not reactive to these antigens. The study was carried out on 512 patients, 111 of whom were nonchagasic but included cases of leishmaniasis or other pathologies, and 401 chagasic patients. The latter group comprised 361 chronic cases, 36 acute cases, and 4 congenital cases in newborn children. Among the chronic cases, 256 were from areas in which T. cruzi is endemic but which differed widely in the pathogenic expression of T. cruzi infection and in parasitemia levels. These patients at the same time showed a broad range of low, medium, and high reactivity to conventional enzyme-linked immunosorbent assays and indirect immunofluorescence serotests for Chagas' disease. For these reasons they may better represent the universe of chagasic patients than would a sample of highly reactive sera obtained from chagasic patients in a single area endemic for T. cruzi. All acute and congenital cases showed positivity in the IgM and IgG TESA blots, while chronic cases were 100% positive for IgG antibodies. In nonchagasic sera, including 30 cases of visceral and muco-cutaneous leishmaniasis, the specificity index was 1.000, and no cross-reactions were observed. The TESA blot thus seems to be useful as a sensitive and specific diagnostic assay in cases of suspected acute or congenital T. cruzi infection and as a general confirmatory test for conventional Chagas' disease serology.

An improved serodiagnostic test for Chagas' disease employing a mixture of Trypanosoma cruzi recombinant antigens

“…Because of the lack of a serologic gold standard for the diagnosis of the chronic phase of Chagas' disease, these sera were submitted to the TESA blot test, a confirmatory test for Chagas' disease. 26 Among the 451 serum samples, 445 were negative (98.7%) and 6 were positive (1.3%). The samples were separated in two groups: positive and negative to TESA blot (Table 3).…”

“…19,24,25 The sera from 277 nonchagasic seronegative individuals included those from endemic and nonendemic areas for Chagas' disease from South and Central America countries: (1) 147 healthy blood donors; (2) 50 individuals with unrelated diseases, as defined by the clinical, epidemiologic, and serologic diagnoses of their respective pathologies (1 individual infected with T. rangeli, 5 with toxoplasmosis, 4 with malaria, 4 with paracoccidioidomycosis, 5 with schistosomiasis, 8 with syphilis, 16 with connective tissue diseases and positive for antinuclear antibodies, and 7 with rheumatic fever); and (3) 80 individuals with active visceral or mucocutaneous leishmaniasis, 85 percent of whom had cross-reactions with T. cruzi antigens as assessed by conventional serology and negative by trypomastigote excreted-secreted antigens (TESA) blot test. 24 The second panel of serum samples (n = 451) was obtained from excluded donors from several blood banks of the state of Goiás, an endemic area for Chagas' disease in Brazil. The first screening of these sera was carried out in blood banks that used two commercial diagnostic as-says, IHA, IFA, and/or ELISA.…”

“…The in-house EAE-ELISA, Y strain, was carried out as previously described. 19,24 EAE-ELISA was considered positive at A 492nm Ն 0.20.…”

“…The TESA were obtained as previously described for the Y strain. 24 T. cruzi was maintained in LLC-MK2 cells at 37ЊC in 5 percent CO 2 on RPMI 1640 (Sigma Chemical Co.) containing 2 percent FCS. The supernatants of LLC-MK2-infected cells containing TESA were harvested 5 days after infection, centrifuged at 2800 ‫ן‬ g for 10 minutes at 4ЊC, filtered through a cellulose acetate membrane, and used fresh or stored at -70ЊC.…”

Reactivation of Chagas disease among heart transplant recipients in the United States, 2012‐2016

“…In 2014, the TESA immunoblot replaced the IFA. 27 For PCR testing, blood specimens collected in EDTA tubes were sent to CDC where DNA was extracted from whole blood and the buffy coat fraction. The extractions were performed on a QIAcube using the QIAamp blood mini DNA kit (QIAGEN, Valencia, CA).…”