Anti- Leishmania chagasi immunoglobulin G3 detected by flow cytometry for early cure assessment in American visceral leishmaniasis

• Published in: Journal of immunological methods 2010-08, Vol.360 (1), p.76-83
• Main Authors: Gomes, Izabelle Teixeira, Carvalho, Sílvio Fernando Guimarães, Rocha, Roberta Dias Rodrigues, Peruhype-Magalhães, Vanessa, Dietze, Reynaldo, Martins-Filho, Olindo de Assis, Lemos, Elenice Moreira
• Format: Article
• Language: English
• Subjects: Amphotericin B - therapeutic use; Antigens, Protozoan - immunology; Biological and medical sciences; Biomarkers - blood; Brazil; Cell Separation; Child; Cure assessment; Female; Flow Cytometry; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Humans; IgG3; Immunoglobulin G - blood; Leishmania - chemistry; Leishmania - immunology; Leishmania chagasi; Leishmaniasis, Visceral - blood; Leishmaniasis, Visceral - diagnosis; Leishmaniasis, Visceral - drug therapy; Leishmaniasis, Visceral - immunology; Male; Molecular immunology; Prognosis; Techniques; Treatment Outcome; Visceral leishmaniasis
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2010.06.011

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.