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Clinical and Vaccine Immunology, May 2007, p. 569-576, Vol. 14, No. 5
1071-412X/07/$08.00+0     doi:10.1128/CVI.00354-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Detection of Anti-Leishmania (Leishmania) chagasi Immunoglobulin G by Flow Cytometry for Cure Assessment following Chemotherapeutic Treatment of American Visceral Leishmaniasis

Núcleo de Doenças Infecciosas, Universidade Federal do Espírito Santo, Vitória, Espírito Santo,¹ Centro de Pesquisas René Rachou, FIOCRUZ, Belo Horizonte, Minas Gerais,² Universidade Estadual de Montes Claros, Montes Claros, Minas Gerais, Brazil³

Received 26 September 2006/ Returned for modification 2 January 2007/ Accepted 5 March 2007

The residual serological reactivity observed in patients cured of visceral leishmaniasis (VL) represents the major factor underlying the low efficiency of most anti-Leishmania serological approaches to assess posttherapeutic cure in VL. Herein, we have described a detuned flow cytometry-based methodology to detect anti-live (FC-ALPA-immunoglobulin G [IgG]) and anti-fixed (FC-AFPA-IgG) L. chagasi promastigote IgG, along the titration curve (1:2,000 to 1:128,000), as a tool to assess late (12 months after treatment [12 mAT]) and early (2 and 6 mAT) posttherapeutic cure of pediatric American visceral leishmaniasis. Reactivities were reported as the percentage of positive fluorescent parasite (PPFP), using a PPFP of 50% as a cutoff to segregate positive and negative results. Our data demonstrated that both FC-ALPA-IgG at 1:4,000 and FC-ALPA-IgG at 1:32,000 are useful for late cure assessment in VL, with 100% specificity and outstanding likelihood ratio indices. Cure assessment at 6 mAT also showed promising performance indices, identifying 81% and 71.4% of the treated patients with negative results. However, new interpretation parameters were necessary to monitor cure at 2 mAT. We then introduced the differential PPFP (PPFP) of 25% as a new cutoff for early cure assessment at specific serum dilutions to analyze IgG reactivity by FC-ALPA-IgG and FC-AFPA-IgG. Our data demonstrated that at 2 mAT, PPFP was >25% in 60% and 57.1% of treated patients, whereas at 6 mAT, a PPFP of >25% was observed in 100% and 95.2% of samples assayed by FC-ALPA-IgG and FC-AFPA-IgG, respectively. Together, our findings showed the potential of both FC-ALPA-IgG and FC-AFPA-IgG regarding their applicability to detect differential serological reactivity and further contribution to posttherapeutic cure assessment in VL.

Published ahead of print on 14 March 2007.