Abstract

ARIAS MARCIALES, María Helena; RODRIGUEZ NOVOA, Yinneth Victoria  and  GARAVITO CARDENAS, Giovanny. Adaptation and optimization of a fluorometric reading method, in the in vitro pharmacological model of Plasmodium falciparum culture. Rev. colomb. cienc. quim. farm. [online]. 2016, vol.45, n.1, pp.127-146. ISSN 0034-7418.  https://doi.org/10.15446/rcciquifa.v45n1.58024.

The in vitro pharmacological model of P. falciparum culture is crucial in the initial screening for substances or plant extracts with possible antiplasmodial activity. The parasite density can be determined by varied methods, however have been described numerous advantages and disadvantages associated with each of them. The incubation time required for staining and the use of synchronous or asynchronous cultures were assessed for optimal settings; showing optimal time of 2 h, and lower limits of detection and quantification in asynchronous cultures. Employing the FCB2 and FCR3 strains, was evidenced a background noise of 12% and 38% respectively; linearity showed a good correlation, r² of 0.9644 (FCR3) and 0.9841 (FCB2) and a slope of 1761.8 and 852.4, respectively. It was evidenced agreement between the methods, fluorometric with SYBR Green I (SYBRG I) and microscopic with Giemsa, the mean difference was 0.00002% and 0.09109% respectively for FCR3 and FCB2, The limits of detection and quantification were 0.5% and 1.5% of parasitaemia. The Z factor was 0.376 with FCB2, whereas with FCR3 reached 0.702. The inhibitory concentration 50 (IC₅₀) against P. falciparum FCR3, generated by chloroquine (CQ), was 0.37 mcg/mL by microscopy and 0.35 mcg/mL by fluorometry. Our findings suggest that the SYBRG I fluorescence based assay, by using fluorometers commonly available in many laboratories, is precise, robust, fast and accurate; for the in vitro evaluation of substances or extracts with possible antiplasmodial activity.

Keywords : Antimalarials; Plasmodium falciparum; Sybr Green I; fluorescence spectrometry.

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