Aplicação da citometria de fluxo no estudo da resposta imune de pacientes com Leishmaniose tegumentar americana

Abstract

The American tegumentary leishmaniasis (ATL) is an infectious, non-contagious disease with an average of 1.3 million annual cases. Brazil shows the highest prevalence in number of reported cases in the Americas, where the specific agent is the species L. (V.) braziliensis. The diagnosis is commonly performed by an association of clinical, epidemiological and laboratorial tests. The immunological approaches which are most commonly used in the routine are the indirect immunofluorescence (IFI) and the enzyme-linked immunosorbent assay (ELISA), however, they may present limitations. Therefore, alternative methods have been studied, and one of them is flow cytometry (FC). Thus, the aim of this work is to evaluate, by flow cytometry, the role of the humoral response in the development of cutaneous lesions of patients with the active disease before treatment (BT), and in those healed after therapeutic intervention (PT). Sera samples were inactivated. For the FC assays, promastigote forms of L. (V.) braziliensis obtained by the reference strain were expanded in Schneider’s medium until they reached exponential phase. After washing protocols, the concentration of the parasites was adjusted and they were fixed with 1% paraphormaldehyde. For the IFI and ELISA assays, the total antigen of L. (V.) braziliensis were used. In FC and IFI assays, the IgG antibody was conjugated to a fluorophore and in ELISA, to an enzyme. Regarding IFI results, from the evaluated sera, 92.85% (13/14) were positive on BT group, 61.54% (8/13) were positive 1 year PT, 70% (7/10) 2 years PT and 50% (5/10) 5 years PT were positive. In ELISA, 92.8% (13/14) of BT patients were positive; and 53.8% (7/13) 1 year PT, 88.8% (8/9) 2 years PT and 100% (5/5) 5 years PT (5/5) remained positive for ATL. In FC, 86% (12/14) of BT patients were positive and 77% (10/13), 80% (8/10) and 70% (7/10) of the patients 1, 2 and 5 PT, respectively, were negative for the assay. Based on the ROC curves analysis to compare the performance of the 3 tests, it was observed that the area under the curve (AUC) of IFI (AUC=0.879; IC95%= 0.754-0.954) was lower that FC’s (AUC= 0.890; IC95%=0.767-0.961), meaning it had a lower performance. Comparing ELISA and FC, it was observed that the AUC of ELISA (AUC=0.808; IC95%= 0.652-0.915) has differed from the one observed for FC (AUC= 0.896; IC95%= 0.758-0.970), where again, FC has shown a superior performance. As for the results of the IgG isotypes’ (IgG1, IgG2 and IgG3) standardization, it was observed that the best dilution for IgG1 was 1:400; for IgG2, 1:100; and for IgG3, 1:200. On the analysis to verify IgG1 applicability, it was seen that BT 63.2% of the patients were positive, and 1PT, 17.7%; 2PT, 72.8% and 5PT, 12.55% of the patients were positive. Therefore, we believe that FC can be used as a diagnostic tool for ATL since it is positive in the presence of the disease, being superior to the two conventional methods used in the laboratory routine. Also, the use of the IgG1 isotype has the ability to contribute as a more sensitive and specific diagnostic method when compared to the classical ones.