Abstract
Sera from patients chronically infected with Trypanosoma cruzi display antibodies that bind to epitopes of living trypomastigotes, known as lytic antibodies (LA), and are detected by a complement-mediated lysis test. Conventional serology antibodies (CSA) are also present in sera from patients with chronic infections but, in contrast to LA, are unable to recognize viable trypomastigotes. The presence of LA has been used as an important element in the criterion of cure in human Chagas' disease. Using flow cytometry technology, we introduced a new and sensitive immunomethod for the detection of anti-live trypomastigote membrane-bound antibodies. On the basis of serological tests (LA and CSA detection) and parasitological assays such as hemoculture (HE), patients were classified into the following groups: chronically infected untreated patients (NT) and treated not-cured patients (TNC), with positive HE and both LA and CSA in their sera; "dissociated" HE-negative patients (DIS), in whom LA was not detected whereas CSA were present; a group of cured HE-negative patients (CUR), who were both LA and CSA negative; and, as control, a group of non-chagasic individuals (NC). Sera from these patients were assayed by incubation with live bloodstream trypomastigotes, which were subsequently exposed to fluorescein isothiocyanate-conjugated anti-human immunoglobulin G. The parasites were then fixed, run in the cytometer, and identified on basis of their size and granularity gain adjustments. On the basis of experience with the complement-mediated lysis test, a level of 20% of parasites being fluorescein isothiocyanate fluorescence positive was used as a cutoff between effective and ineffective treatments. With this criterion, our results indicated that sera from NT and TNC patients were antibody positive whereas all sera from DIS, CUR, and NC patients did not contain membrane-bound antibodies. This new approach is a tool to easily identify anti-live T. cruzi membrane-bound antibodies that can be used to monitor the efficacy of Chagas' disease treatment.
Full Text
The Full Text of this article is available as a PDF (205.7 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Almeida I. C., Ferguson M. A., Schenkman S., Travassos L. R. Lytic anti-alpha-galactosyl antibodies from patients with chronic Chagas' disease recognize novel O-linked oligosaccharides on mucin-like glycosyl-phosphatidylinositol-anchored glycoproteins of Trypanosoma cruzi. Biochem J. 1994 Dec 15;304(Pt 3):793–802. doi: 10.1042/bj3040793. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Almeida I. C., Krautz G. M., Krettli A. U., Travassos L. R. Glycoconjugates of Trypanosoma cruzi: a 74 kD antigen of trypomastigotes specifically reacts with lytic anti-alpha-galactosyl antibodies from patients with chronic Chagas disease. J Clin Lab Anal. 1993;7(6):307–316. doi: 10.1002/jcla.1860070603. [DOI] [PubMed] [Google Scholar]
- Andrade S. G., Freitas L. A., Peyrol S., Pimentel A. R., Sadigursky M. Trypanosoma cruzi antigens detected by immunoelectron microscopy in the spleen of mice serologically positive but parasitologically cured by chemotherapy. (Preliminary report). Rev Soc Bras Med Trop. 1988 Jan-Mar;21(1):41–42. doi: 10.1590/s0037-86821988000100010. [DOI] [PubMed] [Google Scholar]
- Cançado J. R. Forma aguda da doença de Chagas no Brasil. AMB Rev Assoc Med Bras. 1980 Aug;26(8):285–288. [PubMed] [Google Scholar]
- Chiari E., Dias J. C., Lana M., Chiari C. A. Hemocultures for the parasitological diagnosis of human chronic Chagas' disease. Rev Soc Bras Med Trop. 1989 Jan-Mar;22(1):19–23. doi: 10.1590/s0037-86821989000100004. [DOI] [PubMed] [Google Scholar]
- Galvao L. M., Nunes R. M., Cançado J. R., Brener Z., Krettli A. U. Lytic antibody titre as a means of assessing cure after treatment of Chagas disease: a 10 years follow-up study. Trans R Soc Trop Med Hyg. 1993 Mar-Apr;87(2):220–223. doi: 10.1016/0035-9203(93)90501-g. [DOI] [PubMed] [Google Scholar]
- Gazzinelli R. T., Galvão L. M., Cardoso J. E., Cançado J. R., Krettli A. U., Brener Z., Gazzinelli G. Anti-Trypanosoma cruzi and anti-laminin antibodies in chagasic patients after specific treatment. J Clin Microbiol. 1988 Sep;26(9):1795–1800. doi: 10.1128/jcm.26.9.1795-1800.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gazzinelli R. T., Morato M. J., Nunes R. M., Cançado J. R., Brener Z., Gazzinelli G. Idiotype stimulation of T lymphocytes from Trypanosoma cruzi-infected patients. J Immunol. 1988 May 1;140(9):3167–3172. [PubMed] [Google Scholar]
- Krettli A. U., Brener Z. Resistance against Trypanosoma cruzi associated to anti-living trypomastigote antibodies. J Immunol. 1982 May;128(5):2009–2012. [PubMed] [Google Scholar]
- Krettli A. U., Cançado J. R., Brener Z. Effect of specific chemotherapy on the levels of lytic antibodies in Chagas's disease. Trans R Soc Trop Med Hyg. 1982;76(3):334–340. doi: 10.1016/0035-9203(82)90184-5. [DOI] [PubMed] [Google Scholar]
- Krettli A. U., Weisz-Carrington P., Nussenzweig R. S. Membrane-bound antibodies to bloodstream Trypanosoma cruzi in mice: strain differences in susceptibility to complement-mediated lysis. Clin Exp Immunol. 1979 Sep;37(3):416–423. [PMC free article] [PubMed] [Google Scholar]
- Martins M. S., Hudson L., Krettli A. U., Cançado J. R., Brener Z. Human and mouse sera recognize the same polypeptide associated with immunological resistance to Trypanosoma cruzi infection. Clin Exp Immunol. 1985 Aug;61(2):343–350. [PMC free article] [PubMed] [Google Scholar]
- Norris K. A., Harth G., So M. Purification of a Trypanosoma cruzi membrane glycoprotein which elicits lytic antibodies. Infect Immun. 1989 Aug;57(8):2372–2377. doi: 10.1128/iai.57.8.2372-2377.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]