The SYBR Green I Malaria Drug Sensitivity Assay: Performance in Low Parasitemia Samples

Abstract

INTRODUCTIONFast and reliable in vitro growth assessment of malaria parasites is the key to the assessment of in vitro drug susceptibility of malaria parasites, and to high throughput screening of newly developed drugs and drug combinations. Although there are several highly sensitive assays available today, the time and effort necessary to test a single microtiter plate remains substantial. Our goal was to assess the value of the SYBR Green I dye for drug susceptibility testing in Plasmodium falciparum cultures, both, under controlled, simulated field conditions and under optimal laboratory conditions.Recently, several new in vitro flourescene assays have been reported. 1 -4 The assays detect the presence of malaria DNA in infected erythrocytes as a measure of parasite growth and propagation and parasite growth inhibition by antimalarial drugs .Although fast and relatively inexpensive, growth assessment using nucleic acid stains has inherent limitations because these stains are not specific for malaria DNA. The SYBR Green I binds to any double-stranded DNA, including the DNA inherently present in whole blood samples, thereby resulting in high background readings.SYBR Green I has been used in molecular biology as a substitute for ethidium bromide for several years. It is an asymmetrical cyanine dye, binding to double stranded DNA, preferring G and C base pairs.2 When intercalated into DNA, it is highly fluorescent, absorbing light at a wavelength between 390 and 505 nm, with a peak at 497 nm and a secondary peak near 254 nm. It emits light at 505 to 615 nm, with a peak at 520 nm. MATERIALS AND METHODSA series of experiments were conducted to evaluate the usefulness of SYBR Green I for in vitro drug susceptibility testing of malaria parasites, not only at the previously tested higher, 4 but also at lower parasite densities. For all tests 20 µL of a 10× concentration of SYBR Green I plus 80 µL either undiluted or, if postincubation parasitemia exceeded ~3.5% infected red blood cells (IRBC), diluted (1:2) sample was used. The dye was obtained as 10,000X stock in DMSO (Sigma-Aldrich GesmbH, Vienna, Austria) and diluted in sterilized distilled water. The enzymelinked immunosorbent assay (ELISA) assays were done after dilution of the samples in distilled water as previously described. 6 Background fluorescence of the medium was analyzed by comparing regular RPMI 1640 medium containing HEPES, gentamycin, and phenol red to RPMI medium with HEPES and gentamycine, but without phenol red. The latter medium was then measured with and without addition of gentamycine.Following initial testing the phenol red-free medium containing HEPES and gentamycine was used as standard medium. To assess the influence of uninfected erythrocytes on the measurements, these were tested against the standard medium at different hematocrit levels. In addition, whole blood collected in heparinized tubes and fresh erythrocyte concentrate, both washed three times in RPMI medium, were analyzed. Two-fold serial dilutions of infected blood, with and w...

Assessment of the Worldwide Antimalarial Resistance Network Standardized Procedure for In Vitro Malaria Drug Sensitivity Testing Using SYBR Green Assay for Field Samples with Various Initial Parasitemia Levels

“…This indicates that the WWARN assay is robust and high quality within this range of parasitemia, which directly correlates to the consistent IC 50 s determined and the high success rate of the assay within the range of 0.075% to 0.6% initial field sample parasitemia. Further, a wider RFU range of Ͼ10-fold difference was attained in a comparison of signal from untreated wells and background from highest-drug (kill)-concentration wells, contrary to previously reported RFU signal ranges (12,16). This allowed for easy fitting of dose-response curves and accurate depiction of IC 50 s across parasitemia levels.…”

“…This is troublesome, as Chaorattanakawee et al (15) has reported that about 49% of Cambodian and 22% of Kenyan natural infections had parasitemia levels of Ͻ0.2%, depicting large variations in parasite density across the different transmission regions (15). However, with the limit of detection of 0.20% in white blood cell (WBC)-free culture and 0.55% in whole blood (16), the original SYBR green assay technique is inapplicable to screening of most of the isolates found in these regions by immediate ex vivo technique (i.e., patient to assay plate without the need for in vitro culture before assaying). As the SYBR green I drug sensitivity assay based on the WWARN procedure continues to be widely adopted by field-based laboratories, we examined this assay for its sensitivity and ability to accurately determine IC 50 s in samples with a wide range of initial parasitemia levels.…”

“…The technique has been utilized for in vitro and ex vivo profiling of field isolate susceptibility to antimalarials (12)(13)(14). However, there are conflicting published data on its performance in isolates with low parasitemia and high hematocrit levels (15)(16)(17). For example, both Chaorattanakawee et al (15) and Vossen et al (16) have demonstrated that the SYBR green assay may falter in its ability to accurately determine antimalarial drug sensitivity for samples from P. falciparum laboratory strains and field isolates possessing low parasitemia levels.…”

Assessment of SYBR Green I Dye-Based Fluorescence Assay for Screening Antimalarial Activity of Cationic Peptides and DNA Intercalating Agents

“…The SYBR green I (SG) dye-based fluorescence assay is a recently developed high-throughput screening method which has been reported to be as sensitive as the [ 3 H]hypoxanthine incorporation assay (8, 10). It has been extensively validated and compared with other known methods and is commonly used for high-throughput antimalarial drug screening (9,(11)(12)(13).…”

“…The SYBR green I (SG) dye-based fluorescence assay is a recently developed high-throughput screening method which has been reported to be as sensitive as the [ 3 H]hypoxanthine incorporation assay (8, 10). It has been extensively validated and compared with other known methods and is commonly used for high-throughput antimalarial drug screening (9,(11)(12)(13).Peptides and DNA intercalating agents have been widely tested for their antimalarial activity (14-17). Some cell-penetrating peptides (CPPs), e.g., TP10 (18), and a number of DNA intercalators have been shown to have antimalarial activity (16,17).…”