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

Abstract

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 ϳ0.5% 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 [ 3 H]hypoxanthine assay, the MSF assay displayed the expected parasite drug resistance patterns with a high degree of global and phenotypic correlation (r 2 > 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.

Plasmodium falciparum phosphoethanolamine methyltransferase is essential for malaria transmission

“…Gametocyte morphology was determined by light microscopy following Giemsa staining. Immunofluorescence, luciferase, and SYBR Green assays were performed as described (8,14,20).…”

Quantitative dissection of clone-specific growth rates in cultured malaria parasites

“…For example, fluorescent DNA stains such as SYBR Green I and 4',6-diamidino-2-phenylindole (DAPI) are amenable to high-throughput inference of parasite growth inhibition in the presence of antimalarial drugs and diverse small molecules (Baniecki et al, 2007;Johnson et al, 2007). The SYBR green method measures parasite density almost instantaneously (Bennett et al, 2004) and gives similar inhibition curves and IC 50 values for drug tests compared with standard radioisotope methods (Smilkstein et al, 2004).…”

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

“…A SYBR Green I-based in vitro IC 50 drug sensitivity assay, described earlier, 6,7,9 was used to test each P. falciparum field isolate against a panel of six conventional antimalarials supplied as chloroquine diphosphate (CQ), mefloquine hydrochloride (MQ), quinine sulfate hydrate (QN), artemisinin (AR), amodiaquine hydrochloride (AQ), and doxycycline hyclate (DX). Because all test drugs, except AR (pure base), were provided as a salt and prepared by weight:volume convention, we used salt formulation weights (except AR) to calculate IC 50 values.…”

“…5 Quinine sulfate hydrate generally parallels CQ for D6 and W2 IC 50 trends. 7 The D6 and W2 clones were obtained from frozen stocks and culture adapted for SYBR Green I IC 50 assays.…”