Apparent isocitrate lyase activity in Leishmania amazonensis.

Early reports have demonstrated the occurrence of glyoxylate cycle enzymes in several Leishmania species. However, these results have been underestimated because genes for the two key enzymes of the cycle, isocitrate lyase (ICL) and malate synthase (MS), are not annotated in Leishmania genomes. We have re-examined this issue in promastigotes of Leishmania amazonensis. Enzyme activities were assayed spectrophotometrically in cellular extracts and characterized partially. A 40 kDa band displaying ICL activity was visualized on zymograms of the extracts. By immunoblotting with mouse antibodies against ICL from Bacillus stearothermophilus, a band of approximately 40 kDa was identified, coincident with the relative molecular mass of the activity band revealed on zymograms. Indirect immunofluorescence of intact promastigotes showed that the recognized antigen is distributed as a punctuated pattern, mainly distributed beneath the subpellicular microtubules, over a diffused cytoplasmic stain. These results clearly demonstrate the existence of an apparent ICL activity in L. amazonensis promastigotes, which is associated to a 40 kDa polypeptide and distributed both diffused and as punctuate aggregates in the cytoplasm. The relevance of this activity is discussed.

An inexpensive antigen for serodiagnosis of Chagas' disease.

UNLABELLED: In this prospective study we evaluated the performance characteristics of a specific and sensitive antigen preparation (AgA) used in an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-Trypanosoma cruzi antibodies in serum samples, for Chagas' disease diagnosis. The antigen production was achieved by combination of nutritional stress and autoclaving the parasites. Specificity and sensitivity were evaluated in two separate tests, using 152 sera from healthy individuals and 175 sera from Chagas' patients (70 by xenodiagnosis). Cross-reactivity was tested using 289 sera from patients who had a parasitological diagnosis of a disease known to induce antigenic responses towards T. cruzi. All of these sera were tested with our AgA-ELISA and with 3 commercial diagnosis kits. To evaluate the agreement of results between our AgA-ELISA and a "gold standard" test for Chagas, we tested 566 sera from an endemic area. RESULTS: sensitivity and specificity were 100%; cross-reactivity was the lowest compared with commercial kits. Overall agreement with the gold standard test was excellent (kappa = 0.92). AgA-ELISA exhibits levels of sensitivity, specificity and cross-reactivity comparable or superior to those shown, obtained with the commercial kits used in our country, while being at least 10 times less expensive. This balance between diagnostic accuracy and cost makes AgA-ELISA useful for blood bank screening in poor regions of the world suffering from Chagas' disease. Further validations of this antigenic formulation in other countries are necessary.

An inexpensive antigen for serodiagnosis of Chagas’ Disease

In this prospective study we evaluated the performance characteristics of a specific and sensitive antigen preparation (AgA) used in an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-Trypanosoma cruzi antibodies in serum samples, for Chagas’ disease diagnosis. The antigen production was achieved by combination of nutritional stress and autoclaving the parasites. Specificity and sensitivity were evaluated in two separate tests, using 152 sera from healthy individuals and 175 sera from Chagas’ patients (70 by xenodiagnosis). Cross-reactivity was tested using 289 sera from patients who had a parasitological diagnosis of a disease known to induce antigenic responses towards T. cruzi. All of these sera were tested with our AgA-ELISA and with 3 commercial diagnosis kits. To evaluate the agreement of results between our AgA-ELISA and a “gold standard” test for Chagas, we tested 566 sera from an endemic area. Results: sensitivity and specificity were 100 percent; cross-reactivity was the lowest compared with commercial kits. Overall agreement with the gold standard test was excellent (kappa=0.92). AgA-ELISA exhibits levels of sensitivity, specificity and cross-reactivity comparable or superior to those shown, obtained with the commercial kits used in our country, while being at least 10 times less expensive. This balance between diagnostic accuracy and cost makes AgA-ELISA useful for blood bank screening in poor regions of the world suffering from Chagas’ disease. Further validations of this antigenic formulation in other countries are necessary.

Este estudio fue realizado para evaluar las características de sensibilidad y especificidad de una formulación antigénica (AgA), producida a bajo costo, para detectar anticuerpos dirigidos a Trypanosoma cruzi, en muestras de suero de pacientes con enfermedad de Chagas. El AgA fue producido por el efecto combinado de estrés nutricional y autoclave de los parásitos. La especificidad y sensibilidad fueron evaluadas en dos estudios separados, con 152 sueros de individuos sanos y 175 de pacientes Chagásicos. La reactividad cruzada con 289 sueros de pacientes con diagnóstico parasitológico de enfermedades con anticuerpos que reaccionan con antígenos de T. cruzi. Estos sueros fueron evaluados con AgA-ELISA y con tres estuches comerciales. 566 muestras de suero provenientes de un área endémica, fueron empleadas para estudiar la concordancia entre nuestro diagnostico y una prueba designada por nosotros como patrón oro estándar. Resultados: la sensibilidad y especificidad fue de 100 por ciento. El AgA presento el más bajo porcentaje de reactividad cruzada, respecto a los estuches comerciales evaluados. La concordancia con la prueba patrón oro, en Venezuela, fue excelente (kappa=0,92). En conclusión, Aga-ELISA, presentó niveles de sensibilidad, especificidad y de reactividad cruzada, comparables o superiores a los obtenidos por los tres estuches comerciales mas empleados en el país, pero con costos de producción al menos 10 veces menor. Este balance conveniente, favorece su potencial uso para el despiste en los bancos de sangre de los países pobres y endémicos para la enfermedad de Chagas. Futuras validaciones de esta formulación en otros países es necesaria.

Serological diagnosis of Chagas disease: evaluation and characterisation of a low cost antigen with high sensitivity and specificity.

In spite of evident progress in the serology of Chagas disease, the requirement for new diagnostic antigens persists. We have evaluated different antigens obtained from Trypanosoma cruzi grown in medium rich in nutrients or under nutrient stress, autoclaved or sonicated and fractionated by differential centrifugation. The resulting antigens were evaluated for diagnosis of Chagas disease using ELISA. Immunofluorescence of the parasites demonstrated that nutrient stress induced changes in the distribution and density of antigens recognised by a pool of sera from experimentally infected mice. When evaluated using ELISA, it was evident that most fractions had good sensitivity but poor specificity. Surprisingly, the best specificity and sensitivity was observed with parasites cultured under nutrient stress and autoclaved. Furthermore this antigen had low cross reactivity with sera from other parasitic diseases, Leishmaniasis in particular. Western blot analysis demonstrated that autoclaving seems to non-specifically eliminate cross-reactive antigens. In conclusion, autoclaving epimastigotes of T. cruzi, after nutrient stress, allowed us to obtain an antigen that could be used in the serological diagnosis of Chagas disease.

Serological diagnosis of Chagas disease: evaluation and characterisation of a low cost antigen with high sensitivity and specificity

In spite of evident progress in the serology of Chagas disease, the requirement for new diagnostic antigens persists. We have evaluated different antigens obtained from Trypanosoma cruzi grown in medium rich in nutrients or under nutrient stress, autoclaved or sonicated and fractionated by differential centrifugation. The resulting antigens were evaluated for diagnosis of Chagas disease using ELISA. Immunofluorescence of the parasites demonstrated that nutrient stress induced changes in the distribution and density of antigens recognised by a pool of sera from experimentally infected mice. When evaluated using ELISA, it was evident that most fractions had good sensitivity but poor specificity. Surprisingly, the best specificity and sensitivity was observed with parasites cultured under nutrient stress and autoclaved. Furthermore this antigen had low cross reactivity with sera from other parasitic diseases, Leishmaniasis in particular. Western blot analysis demonstrated that autoclaving seems to non-specifically eliminate cross-reactive antigens. In conclusion, autoclaving epimastigotes of T. cruzi, after nutrient stress, allowed us to obtain an antigen that could be used in the serological diagnosis of Chagas disease.

Proliferation and bystander suppression induced by antigens of Trypanosoma cruzi: evaluation with a modification of the T cell blot technique

Se estudió tanto la respuesta proliferativa inducida por antígenos solubles de epimastigotes y tripomastigotes de T. cruzi, como su efecto regulador en la respuesta proliferativa a PPD. Se evaluaron tanto los extractos crudos del parásito como las bandas de “Western blots” de antígenos solubles de epimastigotes y tripomastigotes. La proliferación de las células mononucleares, en respuesta a los extractos crudos, fue mas intensa en los pacientes con cardiomiopatía chagásica (CDM) que en los pacientes sin evidencia de patología cardiaca (INF). La respuesta a los antígenos fraccionados fue menor, en frecuencia e intensidad, a la observada con los antígenos crudos. Las bandas de bajo peso molecular (17 a 30 kDa) de los antígenos solubles de epimastigotes dieron una respuesta mas alta en pacientes CDM y las de peso molecular intermedio (31 a 62 kDa). Este patrón se invirtió con las bandas de antígenos solubles de tripomastigotes. Ambos extractos solubles indujeron sobre regulación o baja regulación de la respuesta a PPD en un número variable de pacientes en ambos grupos. Con los antígenos semi purificados la intensidad de la baja regulación fue mas intensa en pacientes sin evidencia de enfermedad cardiaca, sin embargo la frecuencia fue mayor en los pacientes con cardiomiopatía chagásica (CDM). Seis bandas del Western blot de antígenos solubles de tripomastigotes (B1, 4-7, y 9) se asociaron con una baja regulación en 100 por ciento de los pacientes CDM. La sobre regulación observada con la mayoría de las bandas de la fracciones crudas fue significativamente mas alta, y mas frecuente en pacientes con patología cardíaca. La mayoría de las bandas de antígeno soluble de tripomastigotes (10/15; 66,6 por ciento) no indujeron sobre regulación en pacientes con cardiomiopatía. Estos datos sugieren que la diferencia en la clínica de los dos grupos pueden deberse a que los pacientes de cada grupo reconocen diferentes antígenos conjuntamente con variaciones en la regulación as...

Proliferation and bystander suppression induced by antigens of Trypanosoma cruzi. Evaluation with a modification of the T cell blot technique.

We have studied in vitro proliferation induced by soluble antigenic fractions of T. cruzi epimastigotes and trypomastigotes, as well as their regulatory effect on the proliferative response to PPD. Both crude extracts of the parasite as well as bands from Western blots of soluble epimastigotes and trypomastigotes antigens were tested. Crude extracts elicited higher proliferation in mononuclear cells from patients with chagasic cardiomyopathy (CDM) than in those from patients with no evidence of cardiac pathology (INF). Fractionated antigens induced a lower proliferative response, in intensity as well as in frequency, than the crude extracts. With the soluble antigenic fractions of epimastigotes, cells from CDM patients gave higher responses to low molecular weight (MW) b ands (17 to 30 kDa), and from INF patients, to bands of intermediate MW (31 to 62 kDa); this pattern was inverted with soluble antigenic fractions of trypomastigotes. The two crude preparations induced either up-regulation or down-regulation of the PPD response in variable numbers of patients from both groups. With fractionated antigens, down-regulation intensity was stronger in patients without evidence of heart disease, but frequency was greater in patients with Chagasic cardiomyopathy (CDM). Six bands of western-blot of soluble trypomastigote antigens (B1, 4-7, and 9) induced significant down-regulation in 100% of CDM patients. The up-regulation elicited by most bands of the antigenic fractions was significantly higher, and more frequent in patients without heart pathology. Most bands of soluble trypomastigote antigens (10/15; 66.6%) did not induce up-regulation in patients with cardiomyopathy. These data suggest that differences in the clinical status of the two groups may reflect the recognition of different groups of antigens together with variations in the nature of the regulatory response.

Sensitivity of Leishmania spp. to glibenclamide and 4-Aminopiridine: a tool for the study of drug resistance development

We have demonstrated that Leishmania spp. grown as promastigotes, are sensitive to the K+ channel inhibitors 4-aminopyridine and glibenclamide. Their host cells, the macrophages, are not affected by similar concentrations of the drugs. We have also initiated the molecular characterization of the mechanisms involved in the development of drug resistance to glibenclamide by the parasite. Therefore, we have selected experimentally and begun to characterize the Venezuelan Leishmania (Leishmania) strain, NR resistant to glibenclamide [NR(Gr)]. The analysis of genomic DNA evidenced the existence of a fragment which apparently is amplified in NR(Gr). The fragment recognized by the pgpA probe, related to the Leishmania P-glycoprotein family and which was originally isolated from L. tarentolae, showed a size polymorfism between the sensitive and the resistant strain. These results suggest that the development of resistance to glibenclamide in the strain NR(Gr) might be associated with the amplification of the ItpgpA or related gene(s).