Detection of anti-leishmania (Leishmania) chagasi immunoglobulin G by flow cytometry for cure assessment following chemotherapeutic treatment of American visceral leishmaniasis

Abstract

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 (DeltaPPFP) 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, DeltaPPFP was >25% in 60% and 57.1% of treated patients, whereas at 6 mAT, a DeltaPPFP 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.

FIG. 1.

Representative flow cytometry charts used to determine the anti-L. chagasi FC-ALPA-IgG and FC-AFPA-IgG reactivities in serum samples. Promastigotes were first selected on FSC × SSC dot plots. Live and fixed parasites were found to assume similar and characteristic FSC × SSC dot plot distributions (top panel). The relative FL1/FITC fluorescence intensity was quantified by first establishing a maximum value of reactivity to the internal control (FITC-conjugated anti-human IgG), setting up a marker, M1 (left bottom histogram). This marker was maintained to determine the reactivity in all data analyses performed on the experimental batch. The reactivity of IgG for serum samples from VL patients before and after treatment was reported as the PPFP, which represents the frequency of parasite shift toward higher fluorescence intensity, across the M1 (middle and right bottom histograms, respectively).

FIG. 2.

Anti-L. chagasi FC-ALPA-IgG (left panels) and FC-AFPA-IgG (right panels) reactivities in serum samples from VL patients BT (•) and 12 mAT (▵). The results are expressed as mean PPFP (top graphs) and individual PPFP values (bottom graphs) from 1:2,000 to 1:128,000 serum dilutions. The patterned rectangles represent the selected serum dilutions of the higher segregation range between BT and 12 mAT (1:4,000 for FC-ALPA-IgG and 1:32,000 for FC-AFPA-IgG).

FIG. 3.

Anti-L. chagasi FC-ALPA-IgG (left panel) and FC-AFPA-IgG (right panel) reactivities of individual serum samples from VL patients BT (•), 2 mAT (○), 6 mAT (□), and 12 mAT (▵). The results are expressed as PPFP for individual samples, at serum dilutions of 1:4,000 for FC-ALPA-IgG and 1:32,000 for FC-AFPA-IgG (top panels). New interpretation parameters to monitor IgG reactivities early after chemotherapy are expressed as PPFP for individual samples, at serum dilutions of 1:8,000 for FC-ALPA-IgG and 1:64,000 for FC-AFPA-IgG (bottom panels). The dotted line represents the cutoff between negative and positive results. Specificity and sensitivity indices are provided on the graphs.

FIG. 4.

Differential anti-L. chagasi IgG reactivities of paired samples (ΔPPFP) detected by FC-ALPA-IgG (left panels) and FC-AFPA-IgG (right panels) and defined as the variation between PPFP at 2 mAT (○) and 6 mAT (□) and PPFP values BT (•). The results are expressed as ΔPPFP for each pair of samples. The rectangle identifies the serum dilution that yielded the higher differential reactivity at 2 mAT (1:16,000 for FC-ALPA-IgG and 1:128,000 for FC-AFPA-IgG) and at 6 mAT (1:8,000 for FC-ALPA-IgG and 1:64,000 for FC-AFPA-IgG). Data analysis was performed after the establishment of a gray zone (the patterned rectangle in the bottom panels) corresponding to the first quartile of the ΔPPFP range (cutoff of 25%).