Development of an HRP2-based malaria drug sensitivity assay

Over the past decades the drug resistance of falciparum malaria, a disease that kills almost 2 million people every year, has become an issue of utmost importance. In vitro techniques have become indispensable tools for the assessment of drug-resistance. Currently used techniques have been in use for more than two decades and are associated with a number of drawbacks. In 2000 and 2001 the foundation for a novel antimalarial drug sensitivity assay was developed by Noedl et al. (Department of Specific Prophylaxis and Tropical Medicine, University of Vienna) in cooperation with leading tropical medicine research institutions in Thailand and the USA (USAMC- AFRIMS). The assay is based on the measurement of histidine rich protein II (HRP2) in drug exposed culture samples by a simple HRP2-ELISA assay. HRP2 is a protein produced by the malaria parasites (Plasmodium falciparum) in the course of their development. It is closely associated with parasite growth and propagation, and is therefore perfectly suited to reflect growth inhibition as a measure of drug-action. In the course of a pilot project the basis for the new assay was developed under optimal laboratory conditions. It was validated with a number of culture adapted parasite strains, which were taken from falciparum malaria patients, stored in liquid nitrogen, adapted to continuous culture, diluted with uninfected erythrocytes and tested in the assay. The assay was found to be extremely sensitive, easier to establish and perform than any previous technique, highly reproducible and may have the potential to revolutionize malaria in vitro drug susceptibility testing. This procedure, however, requires a well equipped tropical parasitology laboratory. A major issue for a future worldwide application of the new assay will therefore be the field-adaptation of the test system. It is consequently the aim of this study to develop and validate a field-adapted HRP2-based drug sensitivity assay for P. falciparum, which includes all the advantages of traditional in vitro assays while omitting most of their disadvantages. In addition analytical software specifically designed to evaluate data obtained from the new assay will be programmed and published through a project website together with all the information required to reproduce the assay (e.g. SOPs). First results obtained during the pilot phase of the study indicate that the new HRP2 assay may be perfectly suited to determine antimalarial drug resistance in a field setting. The results of this study are expected to lead to the availability of an innovative field-adapted malaria drug sensitivity assay, which may open up remarkable new perspectives for malaria drug resistance research.

With hundreds of millions of clinical cases and more than one million deaths every year, malaria remains one of the biggest threats to humanity even in the 21st century. Ever since the appearance of chloroquine-resistant malaria parasites after World War II antimalarial drug resistance has become one of the most important problems in controlling this deadly disease. Since then malaria has become resistant to almost all known treatments thereby requiring the constant development of new drugs. The objective of this project was to develop a highly sensitive and economical drug susceptibility assay for field deployment that would allow for an earlier detection of antimalarial drug resistance in malaria affected areas. Only an early detection of drug resistance allows for adequate countermeasures to be taken to limit the impact of drug resistance on the malaria patients. Based on previous basic research and development of drug sensitivity assays by H. Noedl using histidin-rich protein 2 (HRP2) measured in a simple enzyme-linked immunosorbent assay (ELISA) as an indicator of parasite growth under drug exposure we developed a novel field assay. As compared to previously used tests, such as the radioisotopic drug sensitivity assay, the new assay is more than 100 times more sensitive and therefore allows for testing of almost any parasitized blood sample without selecting for high parasite densities, freezing or preculturing of the blood samples. It is non-isotopic and also much more economical than the isotopic test and is therefore much better suited for use under field conditions and in malaria endemic countries. The research was done in close collaboration between the Medical University of Vienna (H. Noedl) and the Armed Forces Research Institute of Medical Sciences, Bangkok (a special activity of the Walter Reed Institute, Washington, DC; M. Fukuda and RS. Miller) as well as the Ministry of Public Health in Thailand and the International Centre for Diarrheal Disease Research in Bangladesh (ICDDR,B). All objectives of the project were achieved within the given time frame. The new assay is currently being employed in malaria research throughout the world (Europe, Africa, Asia, America, and Australia). In the process of validation the new assay has proven to be an extremely helpful tool not just for the detection of drug resistance but also in clinical trials and drug development. In retrospective we conclude that this project can therefore be seen as an outstanding success.