A Novel Dna-Based Microfluorimetric Method to Evaluate Antimalarial Drug Activity

Corbett, Yolanda; Herrera, Liuris; González, José; Cubilla, Luis; Capson, Todd L.; Coley, Phyllis D.; Kursar, Thomas A.; Romero, Luz; Ortega-Barrı́a, Eduardo

doi:10.4269/ajtmh.2004.70.119

Cited by 141 publications

(173 citation statements)

References 17 publications

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“…Although high starting parasitemias around 0.75% as used in Ref. 4 are likely to give similar IC values with the SYBR Green I assay as compared with other assays, geometric mean parasite densities as high as this are rarely encountered under field conditions in malaria-endemic countries, e.g., Ref. 9.…”

Section: Discussionmentioning

confidence: 99%

“…A 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.…”

Section: Methodsmentioning

confidence: 99%

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

Vossen¹,

Pferschy²,

Chiba³

et al. 2010

The American Society of Tropical Medicine and Hygiene

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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...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Vossen¹,

Pferschy²,

Chiba³

et al. 2010

The American Society of Tropical Medicine and Hygiene

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“…Plasmodium falciparum F32 (Indochine, clone W2, chloroquine sensitive strain) was cultivated in glucoseenriched RPMI 1640 medium, supplemented with 10% human serum. Viability was measured by fluorochrome (PicoGreen ® ) intercalation and read in a micrometric fluorometer according to Corbett et al (2004).…”

Section: Antiprotozoal Activitymentioning

confidence: 99%

Chemical composition of essential oils of Piper jacquemontianum and Piper variabile from Guatemala and bioactivity of the dichloromethane and methanol extracts

Cruz¹,

Cáceres²,

Alvarez³

et al. 2011

Rev. bras. farmacogn.

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Abstract:The essential oils from two native species from Guatemala were studied for their chemical composition and the dichloromethane and methanol extracts for their biological activity. A GC-MS analysis of the essential oil from Piper jacquemontianum Kunth, Piperaceae, showed 34 constituents, consisting mainly of linalool (69.4%), while Piper variabile C. DC. essential oil had 36 constituents, camphor (28.4%), camphene (16.6%) and limonene (13.9%) being the major components. Dichloromethane extracts of both species were cytotoxic against MCF-7, H-460 and SF-268 cell lines (<7 µg/mL). Dichloromethane extract of P. jacquemontianum was slightly active against bacteria (0.5 mg/mL), was active against promastigotes of Leishmania (20.4-61.0 µg/mL), and epimastigotes of Trypanosoma cruzi (51.9 µg/mL). The methanol extract of P. variabile showed antimalarial activity against Plasmodium falciparum F32 (4.5 µg/mL), and the dichloromethane extract against Leishmania (55.8-76.3 µg/mL) and T. cruzi (45.8 µg/mL). None of the extracts from the two species was active against Aedes aegypti larvae and Artemia salina nauplii.

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“…While the latter method could in principle be automated, it is not well suited for HTS, as it requires radioactive materials that pose safety and disposal problems and has multiple steps that are technically demanding. Recently, new nonradioactive screens have emerged, using DNA stains as a reporter to measure parasite growth (1,5,16,17). The use of DNA stains to detect parasite DNA has greatly aided the ease of drug susceptibility testing.…”

mentioning

confidence: 99%

High-Throughput Plasmodium falciparum Growth Assay for Malaria Drug Discovery

Baniecki

Wirth

Clardy

2007

Antimicrob Agents Chemother

151

144

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New therapeutic agents for the treatment of malaria, the world's most deadly parasitic disease, are urgently needed. Malaria afflicts 300 to 500 million people and results in 1 to 2 million deaths annually, and more than 85% of all malaria-related mortality involves young children and pregnant women in sub-Saharan Africa. The emergence of multidrug-resistant parasites, especially in Plasmodium falciparum, has eroded the efficacy of almost all currently available therapeutic agents. The discovery of new drugs, including drugs with novel cellular targets, could be accelerated with a whole-organism high-throughput screen (HTS) of structurally diverse small-molecule libraries. The standard whole-organism screen is based on incorporation of [ 3 H]hypoxanthine and has liabilities, such as limited throughput, high cost, multiple labor-intensive steps, and disposal of radioactive waste. Recently, screens have been reported that do not use radioactive incorporation, but their reporter signal is not robust enough for HTS. We report a P. falciparum growth assay that is technically simple, robust, and compatible with the automation necessary for HTS. The assay monitors DNA content by addition of the fluorescent dye 4 ,6-diamidino-2-phenylindole (DAPI) as a reporter of blood-stage parasite growth. This DAPI P. falciparum growth assay was used to measure the 50% inhibitory concentrations (IC 50 s) of a diverse set of known antimalarials. The resultant IC 50 s compared favorably with those obtained in the [ 3 H]hypoxanthine incorporation assay. Over 79,000 small molecules have been tested for antiplasmodial activity using the DAPI P. falciparum growth assay, and 181 small molecules were identified as highly active against multidrug-resistant parasites.

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A Novel Dna-Based Microfluorimetric Method to Evaluate Antimalarial Drug Activity

Cited by 141 publications

References 17 publications

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

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

Chemical composition of essential oils of Piper jacquemontianum and Piper variabile from Guatemala and bioactivity of the dichloromethane and methanol extracts

High-Throughput Plasmodium falciparum Growth Assay for Malaria Drug Discovery

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