| บทคัดย่อ(English) |
A major concern in the therapy of malaria caused by ~iPlasmodium falciparum~i is the efficacy of antimalarial drugs. The quickening pace of antimalarial drug resistance in recent years has necessitated the development of new, user-friendly assays for antimalarial drug sensitivity testing to complement or replace techniques that have been in use for more than two decades. It was therefore the principal objective of this study to explore the extent to which ~iP. falciparum~i specific histidine-rich protein II (PfHRP2) levels correspond to parasite growth and its inhibition by antimalarial drugs, as well as to develop a novel ~iin vitro~i drug susceptibility assay based on HRP2. In addition analytical software for the evaluation of malaria drug sensitivity tests as well as a website dedicated to the new assay were developed. HRP2 levels in the cell medium mixture, cellular compartment, and supernatant of samples obtained from a 72 hour ~iP. falciparum~i culture were determined by a double-site sandwich ELISA to calculate HRP2 secretion and production profiles in drug-free as well as drug-exposed culture. To determine ~iin vitro~i drug susceptibility, parasite strains were exposed to ascending drug concentrations, cultured for 48 to 72 hours and tested in the ELISA. Parallel to the new HRP2 drug susceptibility assay, the drug sensitivity was assessed in a modified WHO schizont maturation and an isotopic [(3)H]-hypoxanthine uptake assay. Characteristic increases in the overall HRP2 levels were found in the later ring and the trophozoite stages. During the later schizont development, rupture, and reinvasion, however, the HRP2 levels remained fairly stable. Exposure to antimalarials inhibited the production of HRP2. Serial dilutions of antimalarials resulted in sigmoid dose-response curves similar to the ones obtained from traditional drug sensitivity assays. HRP2 therefore allows for an accurate estimation of parasite growth and its inhibition by antimalarial drugs. The novel HRP2 drug sensitivity assay proved to be extremely sensitive, rapid and simple to establish and perform. The complete ELISA takes about 2.5 hours to perform, may be carried out with commercially available test kits and requires little technical equipment. In correlation analysis the results closely paralleled those obtained from the isotopic assay (R = 0.892; ~iP~i < 0.0001) and from WHO schizont maturation tests (R = 0.959; ~iP~i < 0.0001). It may therefore be suitable 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. In addition to the assessment of the role of HRP2 in parasite growth and the development of an HRP2-based drug susceptibility assay, software for the analysis of drug sensitivity data was developed. It allows for straightforward processing of raw data obtained from any malaria ~iin vitro~i drug susceptibility assay, using a simple user interface, customisable polynomial analysis, and tabular as well as graphic output. Furthermore a website (httP://malaria.farch.net) was designed that provides direct access to standard operating procedures, analytical software, updated information on the new assay, as well as interactive features, such as a discussion forum. |
