Histidine-rich protein II: a novel approach to malaria drug sensitivity testing

Abstract

The production of histidine-rich protein II (HRP2), a histidine- and alanine-rich protein produced by Plasmodium falciparum, is closely associated with the development and proliferation of the parasite and therefore is perfectly suited to reflect growth inhibition as a measure of drug susceptibility. It was the aim of the present study to develop a malaria drug sensitivity assay based on the measurement of HRP2 in a simple enzyme-linked immunosorbent assay (ELISA). The new test proved to be as reliable as traditional in vitro assays, while it was considerably easier to establish and perform. Parasites are incubated at an initial level of parasitemia of 0.01 to 0.1% on microculture plates predosed with ascending concentrations of antimalarial drugs. After incubation for 48 to 72 h, the samples are freeze-thawed and transferred to ELISA plates. The complete ELISA takes about 2.5 h to perform, may be carried out with commercially available test kits, and requires relatively little technical equipment. In correlation analysis, the results closely paralleled those obtained by the isotopic assay (R = 0.892; P < 0.0001) and World Health Organization schizont maturation tests (R = 0.959; P < 0.0001). The novel HRP2 drug susceptibility assay proved to be very sensitive, simple to establish, and highly reproducible. It can be used for a wide range of applications, from epidemiological studies to the screening of new drugs, and may have the potential to replace traditional in vitro techniques. Standard operating procedures, updated information, and analytical software are available from http://malaria.farch.net.

FIG. 1.

Activities of mefloquine (MEF; R² = 0.9974), quinine (QNN; R² = 0.9951), chloroquine (CHL; R² = 0.9903), and artesunate (ARS; R² = 0.9977) against all P. falciparum strains tested (n = 20) by the new HRP2 drug susceptibility assay.

FIG. 2.

Activities of mefloquine (MEF; R² = 0.9990), quinine (QNN; R² = 0.9892), chloroquine (CHL; R² = 0.9891), and artesunate (ARS; R² = 0.9970) against all P. falciparum strains tested (n = 20) by a modified WHO schizont maturation (morphology) assay.

FIG. 3.

Activities of mefloquine (MEF; R² = 0.9985), quinine (QNN; R² = 0.9927), chloroquine (CHL; R² = 0.9926), and artesunate (ARS; R² = 0.9933) against all P. falciparum strains tested (n = 20) by the isotopic assay.

FIG. 4.

Scatter plot for the association of individual IC₅₀s (in nanomolar) for mefloquine (squares), quinine (diamonds), chloroquine (inverted triangles), and artesunate (triangles) determined by the HRP2 drug susceptibility assay and a modified WHO schizont maturation (morphology) assay (n = 20; R = 0.959; P < 0.001).

FIG. 5.

Scatter plot for the association of individual IC₅₀s (in nanomolar) for mefloquine (squares), quinine (diamonds), chloroquine (inverted triangles), and artesunate (triangles) determined by the HRP2 drug susceptibility assay and the isotopic assay (n = 20; R = 0.892; P < 0.001).

FIG. 6.

Bland-Altman plot of the difference in log IC₅₀s for mefloquine, quinine, chloroquine, and artesunate determined by the HRP2 drug susceptibility assay and a modified WHO schizont maturation (morphology) assay plotted against their mean values. The mean difference in IC₅₀s on the log scale was 0.112 (limits of agreement, −0.370 and 0.594).

FIG. 7.

Bland-Altman plot of the difference in log IC₅₀s for mefloquine, quinine, chloroquine, and artesunate determined by HRP2 drug susceptibility assay and the isotopic assay plotted against their mean values. The mean difference in IC₅₀s on the log scale was 0.024 (limits of agreement, −0.538 and 0.578).