Assessment of malaria in vitro drug combination screening and mixed-strain infections using the malaria Sybr green I-based fluorescence assay

Abstract

Several drug development strategies, including optimization of new antimalarial drug combinations, have been used to counter malaria drug resistance. We evaluated the malaria Sybr green I-based fluorescence (MSF) assay for its use in in vitro drug combination sensitivity assays. Drug combinations of previously published synergistic (atovaquone and proguanil), indifferent (chloroquine and azithromycin), and antagonistic (chloroquine and atovaquone) antimalarial drug interactions were tested against Plasmodium falciparum strains D6 and W2 using the MSF assay. Fifty percent inhibitory concentrations (IC(50)s) were calculated for individual drugs and in fixed ratio combinations relative to their individual IC(50)s. Subsequent isobologram analysis and fractional inhibitory concentration determinations demonstrated the expected drug interaction pattern for each combination tested. Furthermore, we explored the ability of the MSF assay to examine mixed parasite population dynamics, which are commonly seen in malaria patient isolates. Specifically, the capacity of the MSF assay to discern between single and mixed parasite populations was determined. To simulate mixed infections in vitro, fixed ratios of D6 and W2 strains were cocultured with antimalarial drugs and IC(50)s were determined using the MSF assay. Dichotomous concentration curves indicated that the sensitive and resistant parasites composing the genetically heterogeneous population were detectable. Biphasic analysis was performed to obtain subpopulation IC(50)s for comparison to those obtained for the individual malaria strains alone. In conclusion, the MSF assay allows for reliable antimalarial drug combination screening and provides an important method to discern between homogenous and heterogeneous parasite populations.

FIG. 1.

Isobologram analysis of IC₅₀s of single and combination drugs at fixed concentrations. ΣFIC₅₀ values at fixed drug concentrations are shown in inset tables. Graphs display the following interactions: synergism (A), indifference (B and C), antagonism (D). ND, not determined. Data are from a representative of three to five experiments.

FIG. 2.

In vitro susceptibilities of mixed population cultures against chloroquine. IC₅₀ curves are shown for 100% D6 (A) and 100% W2 (E), 90% D6 and 10% W2 (B), 10% D6 and 90% W2 (F), 75% D6 and 25% W2 (C), 25% D6 and 75% W2 (G), 50% D6 and 50% W2 (D). Data are from a representative of four to six experiments.

FIG. 3.

In vitro susceptibilities of mixed population cultures against mefloquine (A) and artemisinin (B). Data are from a representative of four to six experiments.