title: Anti-Leishmania chagasi immunoglobulin G3 detected by flow cytometry for early cure assessment in American visceral leishmaniasis.
abstract: We have previously reported a novel flow cytometric based methodology to access the reactivity of seric anti-live (FC-ALPA) and fixed (FC-AFPA) L. chagasi IgG antibodies applicable for cure assessment after specific therapy of VL. Both, FC-ALPA-IgG and FC-AFPA-IgG are promising targets to be used for early cure assessment. However, our finding suggested that further refinements were still required to improve the performance of FC-AFPA IgG for early cure assessment in VL. In the present investigation, we have established and evaluated the performance of FC-AFPA-IgG1/IgG2/IgG3/IgG4 aiming to increase the performance index of the previously reported for FC-AFPA-IgG. The data was expressed as percentage of fluorescent positive parasites after incubation of pre-fixed L. chagasi promastigotes with the test sera samples and addition of second-step FITC-labeled anti-human IgG subclasses conjugates. The analysis of anti-L. chagasi IgG reactivity in polled sera samples from VL patients demonstrated that, before the etiological treatment, the IgG subclass profile was characterized by IgG1>IgG3 with the absence of IgG2 and IgG4 at the specific sera dilution tested. Following the establishment of specific PPFP cut-off-edges to segregate negative and positive results (PPFP of 50% for FC-AFPA-IgG1 and PPFP of 40% for FC-AFPA-IgG3), the analysis of IgG1 and IgG3 reactivity demonstrated good performance for early cure assessment in VL. The analysis of individual samples indicated that despite at 2 mAT, most treated VL patients (81%) still displayed positive results in FC-AFPA-IGg1 analysis, an increased fraction of treated patients (76%) presented negative in FC-AFPA-IgG1 analysis at 6 mAT. Interestingly, the data from FC-AFPA-IgG3 demonstrated an outstanding performance of this method to early cure assessment in VL with increased frequency of treated patients displaying negative results at 2 mAT (90.5%) as well as at 6 mAT (95.2%). The analysis of likelihood ratio (LR) further confirmed the remarkable performance of FC-AFPA-IgG3 as an early complementary biomarker useful to monitor the post-therapeutic cure in human VL.
authors.affiliation: Laboratório de Leishmanioses, Núcleo de Doenças Infecciosas, Universidade Federal do Espírito Santo, Vitória, Espírito Santo, Brazil.
authors.forename: Izabelle Teixeira
authors.initials: IT
authors.lastname: Gomes
authors.forename: Sílvio Fernando Guimarães
authors.initials: SF
authors.lastname: Carvalho
authors.forename: Roberta Dias Rodrigues
authors.initials: RD
authors.lastname: Rocha
authors.forename: Vanessa
authors.initials: V
authors.lastname: Peruhype-Magalhães
authors.forename: Reynaldo
authors.initials: R
authors.lastname: Dietze
authors.forename: Olindo de Assis
authors.initials: OA
authors.lastname: Martins-Filho
authors.forename: Elenice Moreira
authors.initials: EM
authors.lastname: Lemos
mesh_terms: D000666:Amphotericin B / Q000627:therapeutic use*; D000953:Antigens, Protozoan / Q000276:immunology*; D015415:Biomarkers / Q000097:blood; D001938:Brazil; D002469:Cell Separation; D002648:Child; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D007891:Leishmania / Q000737:chemistry / Q000276:immunology*; D007898:Leishmaniasis, Visceral / Q000097:blood / Q000175:diagnosis* / Q000188:drug therapy* / Q000276:immunology; D008297:Male; D011379:Prognosis; D016896:Treatment Outcome
chemical_list: D000953:Antigens, Protozoan; D015415:Biomarkers; D007074:Immunoglobulin G; D000666:Amphotericin B