Optimization of a New Cell-Based Fluorescence Assay for U.S. Army Global Malaria Surveillance Efforts in Support of the Warfighter
Abstract
High-throughput in vitro anti-malarial drug screens have traditionally incorporated the use of radioactive substrates to measure the effect test compounds have on parasitic growth. Several alternative growth inhibition screening assays using fluorescent nucleic acid intercalating dyes have been recently published. In this study we evaluated the malaria SYBR Green I-based fluorescence (MSF) assay, described by Smilkstein et al., for its use in laboratory research and in support of the U.S. Army malaria drug resistance program and the Global Emerging Infection Surveillance and Response System (GEIS) objectives. We expanded upon Smilkstein's initial characterization and validation of the MSF assay to fit our program-specific drug screening needs by testing various culture conditions commonly used for resistance screening. Plasmodium falciparum strains D6 and W2 were treated with a panel of known anti-malarial drugs and their respective IC50s were determined using the MSF assay. The results were then compared to our IC50 data generated using our standard [3H]hypoxanthine incorporation assay. Assay conditions that could potentially affect MSF assay readout, including assay length, starting parasite density and hematocrit levels, microtiter plate selection, and different culture medium components, were also examined. The IC50 values from the MSF assay showed the expected pattern of drug resistance for both parasitic strains tested when compared to the values from the [3H]hypoxanthine incorporation assay. One possible limitation of the MSF assay for some drug resistance applications is due to a significant edge effect observed, which could influence IC50 calculation. The MSF assay was easily amended for use with our robotic plate and handling equipment. Compared to our gold standard radioactive assay, the MSF assay is more cost-effective, simple, and less hazardous, while still allowing for accurate high throughput, automated drug screening.
Document Details
- Document Type
- Technical Report
- Publication Date
- Nov 01, 2006
- Accession Number
- ADA481676
Entities
People
- Jacob D. Johnson
- Lucia Gerena
- Miriam Lopez-sanchez
- Norma E. Roncal
- Norman C. Waters
- Richard A. Dennull
Organizations
- Walter Reed Army Institute of Research