Comparison of a SYBR Green I-Based Assay with a Histidine-Rich Protein II Enzyme-Linked Immunosorbent Assay for In Vitro Antimalarial Drug Efficacy Testing and Application to Clinical Isolates

Abstract

In vitro drug susceptibility testing with the malaria parasite has been used to assess the antimalarial activities of new compounds and to monitor drug resistance in field isolates. We investigated the validity of a SYBR green I fluorescent-based assay under various culture conditions and compared the assay results to those of previously published histidine-rich protein II (HRPII) enzyme-linked immunosorbent assay (ELISA) methods. Reference strains of Plasmodium falciparum were cultured in vitro by using standard conditions in complete medium with and without phenol red before they were dispensed into 96-well plates predosed with chloroquine, mefloquine, or quinine. Following incubation, the culture supernatants were divided and the 50% inhibitory concentrations (IC 50 s) were determined by using a SYBR green I-based method and the HRPII capture ELISA method. There were no significant differences in IC 50 values when phenol red was included in the medium. The IC 50 s and the IC 90 s of the antimalarials tested by both methods were similar or identical for each of the reference strains. Fresh clinical isolates of P. falciparum collected from imported cases of malaria in Lyon, France, were tested for in vitro resistance to chloroquine and mefloquine by using the validated SYBR green I and HRPII ELISA methods. The SYBR green I-based method was able to calculate IC 50 and IC 90 values similar or identical to those calculated by the HRPII assay with fresh clinical samples without removal of white blood cells. The SYBR green I-based method for determination of drug sensitivity levels produced results comparable to those produced by other methods, showing that this method can be used routinely to conduct surveillance for drug resistance in P. falciparum with fresh or cultured parasites.Currently, in vivo drug efficacy studies are the "gold standard" for assessment of drug resistance in malaria parasites (20). While such studies are routinely conducted on a small scale, the cost associated with in vivo drug studies with the malaria parasite prohibits sustained surveillance at the required level of vigilance. Molecular marker determination has been used to identify the single-nucleotide polymorphisms commonly associated with drug resistance in malaria parasites; however, the presence of molecular markers does not always correlate with in vivo drug resistance (25). Other molecular biology methodologies have been developed to assess the flow of drug resistance markers by using known microsatellite markers, but this technology requires specialized equipment and can be quite costly (10, 26). Although microsatellite differentiation is quite powerful and informative, it would be difficult to conduct microsatellite differentiation in the field in real time.A quick and very cost-effective alternative means to determination of the level of resistance is in vitro drug susceptibility testing (4,17). Unlike the in vivo test for drug resistance, the in vitro assay is designed to assess the parasites' responses to drugs without interf...

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

“…Our experiments do not necessarily confirm earlier reports based on similar IC 50 and IC 90 values measured in field samples using the SYBR Green I and the HRP2 assays, suggesting a similar sensitivity for both assays. 8 The high background readings obtained with whole blood seem to considerably increase the variation of the background noise thereby significantly reducing the sensitivity of the SYBR Green I assay. Although high starting parasitemias around 0.75% as used in Ref.…”

“…12 The results obtained by Bacon and others, who used a mean starting parasitemia of 0.75% for their comparison of the SYBR Green I and the HRP2 assay, confirm the good performance of the SYBR Green I assay at higher parasite densities. 8 Particularly under field conditions, where parasite densities tend to be low, a lack in sensitivity can preclude the testing of samples that may otherwise provide valuable information.…”

Advances in Antimalarial Drug Evaluation and New Targets for Antimalarials

“…The currently used fluorophores are Hoechst, DAPI, SYBRGreen I, PICO green and YOYO, the two former being less used because they exhibit excitation and emission properties not appropriate for current fluorescence plate readers and consumables, which is not the case for the latter. Protocols have been optimized and they propose one-step assays applicable to high-throughput screening and as sensitive as the isotopic and the immunocapture assays on laboratory strains or clinical isolates (Bacon et al, 2007, Baniecki et al, 2007, Bennett et al, 2004. The assays are cost-effective, requiring only a spectrofluorometer, and dyes are readily available worldwide.…”

Antimalarial Drug Sensitivity Profile of Western Kenya Plasmodium falciparum Field Isolates Determined by a SYBR Green I in vitro Assay and Molecular Analysis

“…A SYBR Green I-based in vitro IC 50 drug sensitivity assay, described earlier, 6,7,9 was used to test each P. falciparum field isolate against a panel of six conventional antimalarials supplied as chloroquine diphosphate (CQ), mefloquine hydrochloride (MQ), quinine sulfate hydrate (QN), artemisinin (AR), amodiaquine hydrochloride (AQ), and doxycycline hyclate (DX). Because all test drugs, except AR (pure base), were provided as a salt and prepared by weight:volume convention, we used salt formulation weights (except AR) to calculate IC 50 values.…”

“…Parasite replication inhibition was quantified and the IC 50 for each drug calculated by an equation generating a sigmoidal concentration-response curve (variable slope), with log transformed drug concentrations on the X axis and relative fluorescent units (RFUs) on the Y axis (Graphpad Prism for Windows, version 4.0; Graphpad Software, Inc., San Diego, CA). 6,7 Assessing possible sources of SYBR Green I background signal. In experiment set 1, to measure the intrinsic effect of each drug alone on SYBR Green I RFU, in comparison with complete assay wells, we plated each drug over the standard 10 dilutions in complete RPMI 1640 media alone ("drug only" wells), or in complete RPMI 1640 media containing 1% P. falciparum parasitemia and 32,000 peripheral blood mononuclear cells (PBMC)/well ("complete assay" wells), respectively.…”