Histidine-Rich Protein II: a Novel Approach to Malaria Drug Sensitivity Testing

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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.