Chagas disease: Performance analysis of immunodiagnostic tests anti-Trypanosoma cruzi in blood donors with inconclusive screening results

ABSTRACT Background: The screening of Trypanosoma cruzi-infected blood donors using two serological techniques frequently leads to conflicting results. This fact prompted us to evaluate the diagnostic performance of four "in-house" immunodiagnostic tests and two commercially available enzyme-linked immunosorbent assays (ELISAs). Material and Methods: One hundred and seventy-nine blood donors, whose screening for Chagas disease was doubtful, underwent three in-house ELISAs, one in-house immunoblotting test (TESA-blot), and two commercial ELISAs (bioMérieux and Wiener) in an attempt to define the presence or absence of infection. Simultaneously, 29 donors with previous positive results from three conventional serological tests and 30 donors with constant negative results were evaluated. Results: The ELISA-Wiener showed the highest rate in sensitivity (98.92%) and the ELISA-bioMérieux, the highest specificity (99.45%), followed by the TESA-blot, which showed superior performance, with lower false-negative (2.18%) and false-positive (1.12%) rates. In series, the combination composed of the TESA-blot and ELISA-bioMérieux showed slightly superior performance, with trifunctional protein deficiency (TFP) = 0.01%. Conclusion: Our study confirms the high sensitivity and specificity of commercial kits. To confirm the presence or absence of T. cruzi infection, the combination of TESA-blot and ELISA-bioMérieux may be suggested as the best alternative. Individually, the TESA-blot performed the closest to the gold standard; however, it is not commercially available.

Biological parameters and molecular markers of clone CL brener - the reference organism of the Trypanosoma cruzi genome project

Clone CL Brener is the reference organism used in the Trypanosma cruzi Genome Project. Some biological paramenters of CL Brener were determined: (a) the doubling time of epimastigote forms cultured in liver infusion-tryptose (LIT) medium at 28ºC is 58ñ13 hr; (b) differentiation of epimastigotes to metacyclic trypomastigotes is obtained by incubation in LIT-20 per cent Grace's medium; (c) trypomastigotes infect mammalian cultured cells and perform the complete intracellular cycle at 33 and 37ºC; (c) blood forms are highly infective to mice; (e) blood forms are susceptible to nifurtimox and benznidazole. The molecular typing of CL Brener has been determined: (a) isoenzymatic profiles are characteristic of zymodeme ZB; (b) PCR amplification of a 24 alpha ribosomal RNA sequence indicates it belongs to T. cruzi lineage 1; (c) schizodeme, randomly amplified polymorphic DNA (RAPD) and DNA fingerprinting analyses were performed.

Trypanosoma cruzi defined antigens in the serological evaluation of an outbreak of acute Chagas disease in Brazil (Catolé do Rocha, Paraíba)

Immunoglobbulin G and M humoral response to recombinant protein B13 and glycoconjugate LPPG Trypanosoma cruzi defined antigens was evaluated by ELISA in 18 patients in the acute phase of Chagas disease, who were contaminated on the same occasion. LPPG showed 100 por cento positivity detecting both IgM and IgG antibodies, while positivity of 55-65 por cento was observed for B13. An epimastigote alkaline extract (EPI) also showed high sensitivity for acute IgM (100 por cento) and IgG (90 por cento) antibodies. However LPPG had better discriminatory reactivity since with EPI two patients showed negative IgG and several other sera presented OD values for IgG and IgM antibodies very close to the cutoff. Thus, it is suggested that detection of Igm antibodies by LPPG may be used for diagnosis of the acute phase of Chagas disease. An intense decline of IgG and IgM antibodies to three antigens was observed in response to anti-T. cruzi chemoterapy in all acute phase patients. After treatment, six (30 por cento) individuals maintained IgG positivity to EPI, LPPG, and B13 with lower reactivity than that measured at the acute phase. For comparison, serology of a group of 22 patients in the chronic phase of Chagas disease and also submitted to chemotherapy was determined. Positive IgM antibodies to EPI, LPPG abd B13 were detected in only 5-9 por cento cases. In all chronic-phase patients IgG antibodies highly reactive to the three antigens were present and no significant decrease resulted after benznidazole administration. These observations reinforce previous reports that treatment in the acute phase may reduce or eliminate the parasite.

Trypanosoma cruzi: serum antibody reactivity to the parasite antigens in susceptible and resistant mice

The specific antibody responses were compared among susceptible (A/Sn), moderately susceptible (Balb/c) and resistant (C57 BL/lOJ) mice infected with Trypanosoma cruzi (Y strain). Sera obtained during the second week of infection recognized a surface trypomastigote antigen of apparent Mr 80 kDa while displaying complex reactivity to surface epimastigote antigens. Complex trypomastigote antigens recognition was detected around the middle of the third week of infection. No major differences were observed along the infection, among the three strains of mice, neither in the patterns of surface antigen recognition by sera, nor in the titres of antibodies against blood trypomastigotes (lytic antibodies), tissue culture trypomastigotes or epimastigotes. On immunoblot analysis, however, IgG of the resistant strain displayed the most complex array of specificities against both trypo and epimastigote antigens, followed by the susceptible strain. IgM antibodies exhibited a more restricted antigen reactivity, in the three mouse strains studied. Balb/c sera (IgG and IgM) showed the least complex patterns of reactivity to antigens in the range of 30 kDa to 80 kDa. The onset of reactivity in the serum to trypomastigote surface antigens was also dependent on the parasite load to which the experimental animal was subjected

Chaga's disease: IgA, IgM and IgG antibodies to T. cruzi amastigote, trypomastigote and epimastigote antigens in acute and in different chronic forms of the disease

Com o intúito de se aperfeiçoar o diagnóstico sorológico das diferentes formas clínicas da doença de Chagas, foram estudados antígenos de formas amastigotas e tripomastigota, obtidas de camundongos imunossuprimidos infectados com cepa Y de T. cruzi, em comparaçäo com o de epimastigota convencionalmente utilizado. Um total de 506 amostras de soro de pacientes chagásicos com formas aguda e crônicas (indeterminada, cardíaca e digestiva), de indivíduos com infecçöes näo relacionadas e de indivíduos sadios foi analisado por reaçäo de imunofluorescência, para detecçäo de anticorpos IgG, IgM e IgA. O antígeno de amastigota apresentou os mais altos índices de eficiência relativa em testes de IF IgG (0,946), IF IgM (0,871) e IF IgA (0,914), mostrando ser mais conveniente para a finalidade proposta. Anticorpos anti-amastigota apresentaram médias geométricas de títulos mais altas que anti-tripomastigota e anti-epimastigota. Anticorpos IgG anti-corpos IgG anti-amastigota foram encontrados em todas as formas clínicas da doença de Chagas, e anticorpos IgA foram encontrados predominantemente em formas crônicas digestivas e em formas agudas, além de anticorpos IgM nestas últimas formas. Portanto, testes com antígeno amastigotas poderiam ser úteis para a triagem de indivíduos chagásicos em bancos de sangue. Aspectos práticos e econômicos na obtençäo de amastigotas, conforme descrito neste trabalho favorecem seu uso em países em desenvolvimento, já que o antígeno obtido por meio de outras fontes requer uma infraestrutura mais complexa, equipamentos e pessoal especializados