Blood Parasite Load as an Early Marker to Predict Treatment Response in Visceral Leishmaniasis in Eastern Africa

Abstract

Background: To expedite the development of new oral treatment regimens for visceral leishmaniasis (VL), there is a need for early markers to evaluate treatment response and predict long-term outcomes.

Methods: Data from 3 clinical trials were combined in this study, in which Eastern African VL patients received various antileishmanial therapies. Leishmania kinetoplast DNA was quantified in whole blood with real-time quantitative polymerase chain reaction (qPCR) before, during, and up to 6 months after treatment. The predictive performance of pharmacodynamic parameters for clinical relapse was evaluated using receiver-operating characteristic curves. Clinical trial simulations were performed to determine the power associated with the use of blood parasite load as a surrogate endpoint to predict clinical outcome at 6 months.

Results: The absolute parasite density on day 56 after start of treatment was found to be a highly sensitive predictor of relapse within 6 months of follow-up at a cutoff of 20 parasites/mL (area under the curve 0.92, specificity 0.91, sensitivity 0.89). Blood parasite loads correlated well with tissue parasite loads (ρ = 0.80) and with microscopy gradings of bone marrow and spleen aspirate smears. Clinical trial simulations indicated a > 80% power to detect a difference in cure rate between treatment regimens if this difference was high (> 50%) and when minimally 30 patients were included per regimen.

Conclusions: Blood Leishmania parasite load determined by qPCR is a promising early biomarker to predict relapse in VL patients. Once optimized, it might be useful in dose finding studies of new chemical entities.

Keywords: biomarker; parasitemia; pharmacodynamics; qPCR; visceral leishmaniasis.

Figure 1.

Median absolute parasite load of cured patients (red line) and relapsed patients (blue line) at baseline and days 14, 28, and 56, stratified per treatment arm. Error bars represent the interquartile range. Gray dashed lines represent end of treatment.

Figure 2.

ROC curves of absolute parasite load as predictor of clinical relapse on days 14, 28, and 56 of follow-up. AUC represents the integrated area under the ROC curve. Green line: day 14 (AUC 0.71); red line: day 28 (AUC 0.74); blue line: day 56 (AUC 0.92). Abbreviations: AUC, area under the curve; ROC, receiver operating characteristic.

Figure 3.

Correlation between log-transformed qPCR blood and tissue parasite load (matching ID/timepoint) determined in bone marrow aspirates (red), lymph nodes (green), and spleen aspirates (blue). Tissue samples include 4 drops of bone marrow aspirate (~200 µL), or the remainder in the needle of the spleen or lymph node aspiration. Data below the limit of quantification are shown as 1 p/mL. Linear regression line (solid line) is based on the combined data, excluding data below the limit of quantification: y = 1.5 + 0.97x. Abbreviations: ID, identification; qPCR, quantitative polymerase chain reaction.

Figure 4.

Correlation between log-transformed qPCR blood parasite load and grading of amastigotes in aspirate smears by microscopy, stratified by parasite load according to tissue source. Abbreviation: qPCR, quantitative polymerase chain reaction.

Figure 5.

Correlation between log-transformed qPCR tissue parasite load and grading of amastigotes in aspirate smears by microscopy, stratified by parasite load according to tissue source. Tissue samples include 4 drops of bone marrow aspirate (~200 µL), or the remainder in the needle of the spleen or lymph node aspiration. Abbreviation: qPCR, quantitative polymerase chain reaction.

Figure 6.

Predictive power of blood parasite load is shown for day 28 (left) and day 56 (right), with clinical cure defined as parasite load ≤ 20 p/mL. The difference in cure rate is the difference between the alternative treatment regimens (20%, 40%, 60%, or 80% cure rate) and the reference treatment regimen (90% cure rate). Sample size ranges from n = 10 to n = 50. Dotted horizontal line represents the 80% power cutoff.