Continued Validation Of Sybr Green-1-Based Fluorescence Assay For The Routine Screening Of Plasmodium Falciparum Susceptibility To Anti Malarial` Drugs In Ghana.

ABSTRACT This study describes the validation of a sybr green-1 based in vitro test of susceptibility of Plasmodium falciparum to antimalarial drugs. The assay was evaluated by determining fluorescence development at various ranges of parasitaemia of 3D7 clones incubated with sybr green in the dark. The flourescence was measured with a fluorimeter after every 1hr time interval of incubation. The effect of haemoglobin on the sybr green fluorescence was also investigated with salmon sperm DNA. The relationship between the levels of parasitemia and the sybr green fluorescence units determined for varying periods of incubation of parasite with the sybr green dye in the dark showed a well-correlated, linear relationship (r 2 =0.9962). A decreasing trend in Sybr green fluorescence with increasing haemoglobin at fixed salmon sperm DNA (1000ng/ml) was observed in this study. The quantification limit (QL) as determined by the assay was 0.5%. A z’ - value of ≥ 4.7 for a test well was also determined, which was well within a quality assay acceptable range. A comparison of this fluorescence assay and a standard Giemsa stain microscopy method showed similar effective concentrations of known antimalarial drugs that resulted in a 50% reduction in the observed parasite counts (IC50) after 72hrs of incubating the parasites with each drug. A positive correlation (r2 =0.1182, P=0.1378, CI; -2.563 to 17.10), also suggest a strong antiplasmodial activity of chloroquine by both methods likewise for artesunate (r2 =0.0098, P=0.6772, CI; -21.38 to 14.21). The sybr green-1 based assay is an easy to perform, sensitive and suited for screening of large numbers of samples. The sybr green assay proved successful for cultured 3D7 strains at 7.8% to 0.004% levels of parasitaemia and 0.2ng/µl to 0.003ng/µl of extracted DNA as well as its applicability to clinical isolates. The Sybr green assay is an easy to perform alternative, due to the substantial amount of time spent in reading slides making the microscopy method somehow a cumbersome procedure. Based on the results, it is therefore recommended that x the Sybr green assay is a reliable tool without time consumption and hazards of other screening assays and can be used for malaria drug susceptibility monitoring on the field.