Estandarización de la técnica de aglutinación directa para el inmunodiagnóstico de la enfermedad de Chagas / Standardization of a direct agglutination test for the immunodiagnosis of Chagas disease

Introducción. La enfermedad de Chagas es causada por el parásito Trypanosoma cruzi y su diagnóstico inmunológico se basa principalmente en la detección de anticuerpos contra T. cruzi mediante pruebas tales como ELISA, inmunofluorescencia indirecta (IFI) y hemaglutinación indirecta (HAI). Esta última tiene el inconveniente de requerir la preparación de eritrocitos de carnero, difíciles de obtener y de poca duración. Sin embargo, existen pruebas alternativas, como la técnica de aglutinación directa. Objetivo. Estandarizar la técnica de aglutinación directa para el diagnóstico de la enfermedad de Chagas. Materiales y métodos. Se prepararon parásitos epimastigotes de T. cruzi mediante dos protocolos, con tratamiento con tripsina y sin él. Los parásitos se colorearon, y se determinaron las condiciones óptimas de concentración parasitaria y diluciones de suero. Se utilizaron sueros de pacientes con enfermedad de Chagas, de individuos sanos y con otras parasitosis. Resultados. La concentración parasitaria óptima fue de 500 x10 6 parásitos/ml, utilizando parásitos coloreados y sin tratamiento con tripsina. Las diluciones de suero óptimas fueron de 1/25, 1/50 y1/100, y el punto de corte, la dilución de 1/50. La técnica estandarizada mostró índices diagnósticos de sensibilidad de 94,3 % (IC 95% 79,5-99,0) y de especificidad de 96,3 % (IC 95% 88,8-99,0); se encontró reacción cruzada en tres sueros de individuos con leishmaniasis visceral, con valores pronósticos positivo y negativo de 91,7 % (IC 95% 76,4-97,8) y de 97,5 % (IC 95% 90,4-99,6), respectivamente. Se compararon los resultados con los obtenidos por HAI, ELISA e IFI y la concordancia fue de 96 % con un índice kappa de 0,90 (IC 95% 0,81-0,99). Conclusión. La técnica de aglutinación directa estandarizada podría ser útil para el inmunodiagnóstico de la enfermedad de Chagas.

Introduction: Chagas´ disease is caused by the parasite Trypanosoma cruzi and its immunological diagnosis is mainly based on the detection of antibodies against T. cruzi using tests such as the ELISA, the indirect fluorescence antibody test (IFAT) and the indirect hemagglutination test (IHAT). The main disadvantage of the IHAT is the need to prepare sheep erythrocytes, whose availability is limited and they have a short duration once prepared. However, there are alternative tests, such as the direct agglutination test (DAT). Objective: To standardize the direct agglutination test for the diagnosis of Chagas disease. Materials and methods: Trypanosoma cruzi epimastigotes were prepared using two protocols, with and without trypsin treatment. The parasites were stained and optimal conditions for parasitic concentration and serum dilutions were determined. We evaluated the technique using sera from patients with Chagas disease, from healthy individuals and from individuals with other parasitic diseases. Results: The optimal parasitic concentration was 500 x 10 6 parasites/ml using stained parasites without trypsin treatment. The optimal serum dilutions were 1/25, 1/50 y 1/100 and the cut-off point was the 1/50 dilution. The diagnostic indices for the standardized technique were as follows: Sensitivity, 94.3% (95% CI: 79.5-99.0) and specificity, 96.3% (95% CI: 88.8-99.0), with positive and negative predictive values ?? of 91.7% (95% CI: 76.4-97.8) and 97.5% (95% CI: 90.4-99.6), respectively. Cross-reaction was observed only in three sera from individuals with visceral leishmaniasis. The results were compared with those obtained by IHA, ELISA, and IFA, and the concordance rate was 96% and the kappa index, 0.90 (95% CI: 0.81-0.99). Conclusion: The standardized direct agglutination test could be useful for immunodiagnosis of Chagas disease.

Diagnóstico serológico de la enfermedad de Chagas: autosuficiencia y concordancia interlaboratorios / Serologic diagnosis of Chagas disease: Self sufficiency and interlaboratory concordance

El dato fundamental en el diagnóstico de la tripanosomiasis americana (enfermedad de Chagas), en su fase crónica, es el estudio serológico, ya que es difícil la demostración del parásito en circulación o en los tejidos. Una seria limitación en el diagnóstico serológico se relaciona con la estandarización de las diferentes técnicas accesibles, y esto depende considerablemente de la calidad de los antígenos usados para el inmunodiagnóstico. En México no se ha abordado este problema. Los laboratorios del Instituto Nacional de Cardiología y del Instituto Nacional de Diagnóstico y Referencia Epidemiológicos, compararon sus técnicas de inmunodiagnóstico: inmunofluorescencia indirecta, hemaglutinación y ensayo inmunoenzimático en fase sólida (ELISA), con cepas de T. cruzi aisladas en México. La concordancia interlaboratorios fue de 0.8 (Indice Kappa) y la sensibilidad, especificidad y valor predictivo positivo y negativo de las pruebas, aseguran resultados confiables en el inmunodiagnóstico de la enfermedad de Chagas

American trypanosomiasis (Chagas'disease) is becoming a relatively common condition in North America. Diagnosis at the chronic stage depends on demonstration of specific antibodies in body fluids, since parasitologic or pathologic diagnosis is uncertain at this stage. Therefore, standardization of immunodiagnostic techniques is mandatory, and it depends on antigen quality. Locally prepared antigens and crude extracts obtained from Mexican isolates, -both from infected vector and human cases- were compared using three different immunodiagnostic assays -indirect immunofluorescence, hemagglutination and enzyme linked immunosorbant assay (ELISA)- at two different laboratories from the Instituto Nacional de Cardiología and the Instituto Nacional de Diagnóstico y Referencia Epidemiológicos. Concordance between laboratories reached a significant Kappa value (0.8) and sensitivity, specificity and predictive values of individual diagnostic assays were adequate to use these tests in clinical diagnoses. This is the first attempt to standardize immunodiagnostic techniques in Mexico.

Enzyme-linked immunosorbent assay for leptospirosis immunoglobulin M specific antibody using surface antigen from a pathogenic Leptospira: a comparison with indirect hemagglutination and microagglutination tests.

A search for a sensitive and specific test for human leptospirosis was made by enzyme-linked immunosorbent assay for immunoglobulin M specific antibody (IgM ELISA) using a surface antigen from L.interrogans serovar bataviae, L. interrogans serovar pyrogenes and L.interogans serovar icterohaemorrhagiae. The IgM ELISA tests using each of the three antigens were evaluated in 103 sera primarily positive by microagglutination test (MA). Optical density of these IgM ELISA tests showed good correlation. The IgM ELISA using antigen from serovar bataviae was compared with MA and indirect hemagglutination (IHA) in 20 sera primarily positive by IHA, and 103 sera primarily positive by MA. IgM ELISA and IHA using antigen prepared from serovar bataviae in 103 sera positive for MA had a sensitivity of 98.06 and 92.23 per cent respectively. In 20 sera primarily positive by IHA, IgM ELISA and MA showed sensitivity of 80 and 45 per cent respectively. The surface antigen used in IgM ELISA is broadly specific making IgM ELISA a sensitive and specific test for human leptospirosis. IHA agreed more with IgM ELISA in comparison to MA. As MA is not sensitive for early infection, IHA and IgM ELISA should be in routine use in general laboratories.

A comparison of passive haemagglutination test and immunofluorescent test for antibody to native deoxyribonucleic acid.

Antibody to double-stranded DNA is a specific marker for systemic lupus erythematosus. The recommended method for detection of this antibody is immunofluorescence. Haemagglutination was developed and the results of antibody detection were evaluated with those obtained by immunofluorescence. Human group O erythrocytes were treated with glutaraldehyde and coated with DNA from calf thymus. Testing in 169 active and inactive SLE sera, 59 sera were positive and 91 sera were negative by both methods. Five sera were negative by haemagglutination but positive by immunofluorescence. Fourteen sera with low haemagglutination titer were negative by immunofluorescence. The correlation between the results obtained by both methods were highly significance with contingency coefficient of 0.61 and correlation coefficient between the results of 78 sera positive by both or either method was 0.74 (p less than 0.001). Sixty-three sera from blood donors and seventy sera from pregnant women were negative by the two techniques. PHA is simpler, quicker and can be assayed in laboratories without the use of fluorescent microscope. It can be established as a very useful alternative test to immunofluorescence.

Enfermedad de Chagas: evaluación del serodiagnóstico en bancos de sangre de zonas endémicas en Chile / Serologic diagnosis for Chagas disease in blood banks

Blood transfusion is one mechanism leading to transmission of Chagas disease. An evaluation of the serologic method used to survery donnors in 55 blood banks located in endemic areas was attempted. From 47 responders, 29 (62% ) showed complete correlation with the standard used (34% ) showed only 50% correlation and 2 were not possible to evaluate. Most errors were due to an incorrect evaluation of the negative standard serum, whereas the positive one was correctly diagnosed as such in 96% of cases

Passive haemagglutination test for human neuroCysticercosis immunodiagnosis: I. Standardization and evaluation of the passive haemagglutination test for the detection of anti-Cysticercus cellulosae antibodies

Foi padronizada e avaliada a reaçäo de hemaglutinaçäo passiva (RHA) para pesquisa de anticorpos específicos, anti-Cysticercus cellulosae, no líquido cefalorraquiano (LCR). Foram utilizadas hemácias humanas. O Rh-formolizadas e sensibilizadas com extrato antigênico salino total de cisticercos, ainda pouco estudado. De 115 amostras estudadas de LCR de pacientes com neurocisticercose, 94 foram reagentes, resultando em 81,7% de sensibilidade, com intervalo de confiança de 95% de probabilidade (IC95%) abrangendo de 74,5% e 88,9%. Também foram ensaiadas 89 amostras de LCR de indivíduos do grupo controle, sendo täo reagentes em 94,4%, com IC95%, de 89,6% a 99,2%. Os valores preditivos positivo e negativo obtidos para a RHA foram, respectivamente, de 14% e 99,9%, considerando a prevalência média de neurocisticercose na América Latina de 0,1%. Os resultados indicam que a RHA como um método simples, altamente reprodutível e moderadamente sensível para a detecçäo de anticorpos específicos no LCR, porém apropiados para a triagem de infectados