Continued Validation of Sybr Green-1-Based Fluorescence Assay for the Routine Screening of Plasmodium Falciparum Susceptibility to Anti Malarial Drugs in Ghana
Date
2013-03
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University of 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 (IC 50 ) after 72hrs of incubating the parasites
with each drug. A positive correlation (r 2 =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 (r 2 =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
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.
Description
Thesis (MPHIL)-University of Ghana, 2013