Anti-Leishmania chagasi immunoglobulin G3 detected by flow cytometry for early cure assessment in American visceral leishmaniasis.
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.
Gomes
Izabelle Teixeira
IT
Laboratório de Leishmanioses, Núcleo de Doenças Infecciosas, Universidade Federal do Espírito Santo, Vitória, Espírito Santo, Brazil.
Carvalho
Sílvio Fernando Guimarães
SF
Rocha
Roberta Dias Rodrigues
RD
Peruhype-Magalhães
Vanessa
V
Dietze
Reynaldo
R
Martins-Filho
Olindo de Assis
OA
Lemos
Elenice Moreira
EM
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
D000953:Antigens, Protozoan; D015415:Biomarkers; D007074:Immunoglobulin G; D000666:Amphotericin B