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Antimicrobial Agents and Chemotherapy, June 2007, p. 1926-1933, Vol. 51, No. 6
0066-4804/07/$08.00+0     doi:10.1128/AAC.01607-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Assessment and Continued Validation of the Malaria SYBR Green I-Based Fluorescence Assay for Use in Malaria Drug Screening{triangledown}

Jacob D. Johnson,* Richard A. Dennull, Lucia Gerena, Miriam Lopez-Sanchez, Norma E. Roncal, and Norman C. Waters

Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, Maryland 20910

Received 22 December 2006/ Returned for modification 14 February 2007/ Accepted 12 March 2007

Several new fluorescence malaria in vitro drug susceptibility microtiter plate assays that detect the presence of malarial DNA in infected erythrocytes have recently been reported, in contrast to traditional isotopic screens that involve radioactive substrate incorporation to measure in vitro malaria growth inhibition. We have assessed and further characterized the malaria SYBR Green I-based fluorescence (MSF) assay for its ability to monitor drug resistance. In order to use the MSF assay as a drug screen, all assay conditions must be thoroughly examined. In this study we expanded upon the capabilities of this assay by including antibiotics and antifolates in the drug panel and testing folic acid-free growth conditions. To do this, we evaluated a more expansive panel of antimalarials in combination with various drug assay culture conditions commonly used in drug sensitivity screening for their activity against Plasmodium falciparum strains D6 and W2. The detection and quantitation limits of the MSF assay were 0.04 to 0.08% and ~0.5% parasitemia, respectively. The MSF assay quality was significantly robust, displaying a Z' range of 0.73 to 0.95. The 50% inhibitory concentrations for each drug and culture condition combination were determined by using the MSF assay. Compared to the standard [3H]hypoxanthine assay, the MSF assay displayed the expected parasite drug resistance patterns with a high degree of global and phenotypic correlation (r2 ≥ 0.9238), regardless of which culture condition combination was used. In conclusion, the MSF assay allows for reliable one-plate high-throughput, automated malaria in vitro susceptibility testing without the expense, time consumption, and hazard of other screening assays.

* Corresponding author. Mailing address: Principal Investigator, Department of Parasitology, Division of Experimental Therapeutics, Walter Reed Army Institute of Research, 503 Robert Grant Ave., Silver Spring, MD 20910. Phone: (301) 319-9755. Fax: (301) 319-9954. E-mail: jacob.d.johnson{at}us.army.mil

{triangledown} Published ahead of print on 19 March 2007.

Antimicrobial Agents and Chemotherapy, June 2007, p. 1926-1933, Vol. 51, No. 6
0066-4804/07/$08.00+0     doi:10.1128/AAC.01607-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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