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.

Antimalarial Drug Sensitivity Profile of Western Kenya Plasmodium falciparum Field Isolates Determined by a SYBR Green I in vitro Assay and Molecular Analysis

“…This might reflect greater biological variability in IEV P. falciparum isolates, including better preserved sub-populations of drug-resistant and sensitive parasites. 11,25,26 It may be useful to determine multiplicity of infection of each P. falciparum isolate, processed IEV, and culture adapted, along with comparative IEV and culture-adapted SYBR Green I assays.…”

Development and Evaluation of a Rapid Diagnostic Test for Plasmodium falciparum, P. vivax, and Mixed-Species Malaria Antigens

“…However, therapeutic studies have demonstrated a high prevalence (up to 30%) of infection with other malaria species during convalescence, which suggests co-infection. 1,4,5 The diagnosis of mixed-species malaria is critical for therapeutic decisions, including the selection, dose, and timing of anti-malarial drugs. Mistreatment of a single species, rather than multiple species, can have serious clinical consequences.…”

Validating a Firefly Luciferase-Based High-Throughput Screening Assay for Antimalarial Drug Discovery

“…8,9 Therefore, there is a pressing need for discovering and developing novel chemotherapeutic agents against malaria parasites. 1,6,10,11 Developing robust and reliable in vitro high-throughput screening (HTS) assays to evaluate the effect of a compound on the growth of malaria parasites is key to antimalarial drug discovery. Fluorescencebased assays such as SYBR Green I and DAPI are fast, relatively inexpensive, and as sensitive as standard radioactive assays and have been used in evaluating the antimalarial efficacy and drug screening.…”