Applications of the research of IgG antibodies alive anti-forms promastigotas of Leishmania (V.) braziliensis, for Flow Cytometry, in clinical studies of leishmaniose cutaneous located
In this study we described the development of a new flow cytometry-based methodology to detected anti-live Leishmania (V.) braziliensis promastigotes IgG antibodies (FC-ALPA-IgG) for the diagnosis and monitoring of cure of patients treated for localized cutaneous leishmaniasis-LCL. As a first step, we standardized, the methodology which allowed for the discrimination between patients with LCL(L+) and those individuals without clinical manifestation of LCL(L -), based on their sera IgG reactivity under the following conditions: a) 500.000 live promastigotes obtained after 10 days of in vitro culture; b) sera samples diluted 1:1,024; c) secondary anti-IgG human-FITC antibody diluted at 1:400; d) incubations at 37ºC for 30 min and e) use of the percentage of positive fluorescent parasite PPFP=60% as the cut-off between the positive and negative results. In a second step, we have comparatively analyzed the performances of FC-ALPA-IgG and indirect immunofluorescence assay-IFA. The analysis of the statistical indexes expressed in percentile (sensitivity, specificity, predictive values, probability of disease after negative test, false-positive rate, accuracy and Youden?s J index) and the likelihood ratio-LR demonstrated that the best performance of FC-ALPA-IgG when compared to IFA, was on the identification of clinically active LCL cases. Analysis of LR indicated that the IFA titers do not have any diagnostic value whereas the AAPV-IgG, when considering the cut-off PPFPgt;60%, contribute to significantly to the confirmation of LCL diagnosis. On the other hand, AAPV-IgG with PPFP£60% practically excluded cases of LCL. Additionally, we have prepared simulations tests to identify clinical situations that may provide false negative and false positive results in the sample population using this methodology. The time of lesion development in LCL, visceral leishmaniasis-VL and Chagas?disease-CD were identified as clinical situations that can interfere in the performance of FC-ALPA-IgG for the diagnosis of LCL. In these cases both false-negative and false-positive results were obtained. Considering the lower performance of FC-ALPA-IgG to identify LCL when applied in a population that includes VL and CD, we proposed the quantification of IgG subclasses as a tool to increase specificity to the original FC-ALPA-IgG test. The combined analysis of FC-ALPA-IgG1/IgG2 was shown to be useful to differentiate further the positive AAPV-IgG test when applied in a population that includes VL and CD. The use of the PPFP= 60% for ALPA-IgG1 and of 50% for ALPA-IgG2 as an the detection limit for the assay, allowed to clarify the diagnosis of 93% of previously positive or negative FC-ALPA-IgG results which belonged to individuals without clinical manifestation of LCL with negative results on the combined FC-ALPA-IgG1/IgG2 tests and confirm 100% of active cases of LCL as positive using the combined methodology. The LR analysis demonstrated that a positive FC-ALPA-IgG1/IgG2 practically confirms the clinical LCL diagnosis, while a negative result practically excludes the diagnosis of the disease. Finally, the usefulness of FC-ALPA-IgG was demonstrated for its applicability as early and late cure criteria after LCL treatment. In this context, it was observed that 71% of the patients without recurrences 2 years after the end of the treatment were earlier identified (30d-PT), with decrease in the PPFP reactivity, superior to 10%, at 1:4,096 serum dilution.
Advisor:Olindo Assis Martins Filho
School:Faculdades Oswaldo Cruz
Source Type:Master's Thesis
Date of Publication:03/21/2005