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.Antimalarial drug resistance has become an issue of utmost importance for the control of falciparum malaria worldwide. Individual treatment regimens as well as malaria control strategies therefore need to be based on profound knowledge of drug sensitivity. There are basically two approaches to the assessment of the antimalarial drug susceptibility of Plasmodium falciparum: in vivo and in vitro assays. The most commonly used in vitro assays, the isotopic assay, the World Health Organization (WHO) microtest, and a number of parasite lactate dehydrogenase-based assays, have their advantages but also have a number of known drawbacks (1,7,8,12,14,24,26). It was the aim of this study to develop an in vitro assay that is easy to establish and to standardize, that has a high sensitivity, and that requires little technical equipment, similar to the WHO test, but that is also as reproducible and that can be performed as fast as the isotopic assay.Histidine-rich protein II (HRP2) is a naturally occurring histidine-and alanine-rich protein localized in several cell compartments including the cytoplasm of P. falciparum. Recent studies have implicated HRP2 as an important factor in the detoxification of heme (11,16,17,21). HRP2 was identified in all P. falciparum strains regardless of knob phenotype and was recovered from plasma and culture supernatants as a secreted water-soluble protein (20). It is found as concentrated pack...

<i>In vitro</i> evaluation of antiplasmodial activity of extracts of <i>Acanthospermum hispidum</i> dc (Asteraceae) and <i>Ficus thonningii</i> blume (moraceae), two plants used in traditional medicine in the republic of Congo

“…Chloroquine disphosphate (AppliChem GmbH, Germany) was used as standard antiplasmodial product with an initial concentration of 100 ng/ml. The sensitivity of laboratory established Plasmodium falciparum strains to Acanthospermum hispidum and Ficus thonningii extracts was measured using histidin rich protein-2 (HRP2) double site sandwich enzyme-linked immunosorbent assay (ELISA) as described by Noedl et al (2002). Briefly, Plasmodium falciparum cultures (0.05% parasitemia and 1.5% hematocrit) were incubated for 72h in the presence of different concentrations of plant extracts and the control drug.…”

Susceptibilidad in vitro de aislamientos colombianos de Plasmodium falciparum a diferentes antipalúdicos

“…Los métodos tradicionalmente usados para evaluar la susceptibilidad in vitro de P. falciparum a los antipalúdicos son el micrométodo de Rieckmann y el método radioisotópico (14); frente a ellos, la prueba HRP2 es más fácil de ejecutar y tiene sensibilidad similar a la mostrada por los métodos tradicionales (15)(16)(17). En el presente estudio, se halló una elevada y significativa correlación entre las CI 50 obtenidas con HRP-2 y el micrométodo de Rieckmann (r=0,897; r 2 =0,805; p<0,01) y, además, la clasificación del fenotipo sensible/resistente fue 100% concordante para todos los tratamientos.…”

“…El método HRP-2 se basa en la detección de la proteína rica en histidina II de P. falciparum, mediante un ensayo inmunoenzimático (ELISA), como una medida del crecimiento de los parásitos; es fácil de ejecutar y no requiere equipos ni material muy especializados, además, tiene sensibilidad similar al método microscópico y es altamente reproducible como el método radioisotópico (15)(16)(17).…”

Intraerythrocytic Plasmodium falciparum Growth in Serum-Free Medium with an Emphasis on Growth-Promoting Factors

“…Parasite lactate dehydrogenase levels have also been used to assess the growth of malarial parasites (Asahi, et al, 2005;Makler and Hinrichs, 1993;Noedl et al, 2003). ELISA-based assays can provide measures of parasite growth by quantifying biomolecules produced during parasite development, such as histidine-rich protein 2 or parasite lactate dehydrogenase, by double-site sandwich ELISA (Druihe, et al, 2001;Noedl et al, 2002;Noedl et al, 2003). ELISA-based tests are rapid and easy to perform, and are also well suited for the screening of large number of drugs.…”