Scientists from the US and Cambodia have developed a novel and rapid way to test whether the most common and lethal form of malaria is resistant to artemisinin, the key anti-malarial drug.
“In the race against time to stop the spread of artemisinin-resistant malaria, new diagnostic tools are urgently needed to identify and track resistant parasites. These simple in vitro and ex vivo ring-stage survival assays (RSAs) can clearly identify artemisinin-resistant, slow-clearing Plasmodium falciparum (the protozoa that causes malaria) parasites in people with malaria, and can deliver results much faster than the current clinical approach used to monitor response to drugs in patients”, explained study leader Didier Menard from the Institut Pasteur du Cambodge in Cambodia.
The findings of the study were published Wednesday in The Lancet Infectious Diseases journal.
More than 200 million people are infected with the malaria parasite P. falciparum, which kills between 655,000 and 1.2 million people every year. Antimalarial control efforts are mainly dependent on artemisinin-based combination treatments (called ACTs), having replaced older drugs that the malaria parasite developed resistance to. Should these regimens fail, no other drugs are ready for widespread use, and eliminating malaria in the near future might then prove impossible.
Until now, standard laboratory artemisinin susceptibility tests have been unable to distinguish the slow parasite clearance seen in people who are infected with drug-resistant parasites from normal, fast parasite clearance.
The emergence of P. falciparum resistance to artemisinin has been described by the World Health Organisation as an urgent public health concern, threatening the sustainability of the ongoing global effort to reduce the burden of malaria. In January 2011, WHO released the Global Plan for Artemisinin Resistance Containment (GPARC), calling on countries and global malaria partners to implement a five-pillar strategy to prevent and contain artemisinin resistance.
In this study, a team of researchers from the Cambodian National Malaria Centre, the Institut Pasteur, Phnom Penh and Paris, and the US National Institutes of Health set out to test whether a novel in vitro RSA could distinguish slow-clearing from fast-clearing parasites, investigate the in vitro response to dihydroartemisinin (DHA; the active metabolite of all artemisinins) of three different blood stages of parasites, and assess whether an ex-vivo RSA might detect artemisinin-resistant P falciparum infections.
Parasites from Pursat province, an area of artemisinin resistance in western Cambodia, and Preah Vihear and Ratanakiri, regions of artemisinin sensitivity in northern and eastern Cambodia were exposed to a clinically-relevant pulse of DHA and their survival measured 72 hours later.
“We were able to clearly see the difference in the clinical response to artemisinins between people infected with parasites that were drug-resistant or drug-susceptible in vitro,” said Menard. “Our observations confirm that artemisinin resistance is associated with the very early stages of parasite development in the blood.”
In the ex vivo RSA, parasite survival rate also significantly correlated with parasite clearance half-life (the time it takes for the drug to reduce the number of parasites in the blood by half), and the test accurately detected slow-clearing infections in the Preah Vihear and Ratanakiri provinces of Cambodia where they have not yet been described.
According to study co-leader Rick M Fairhurst from the US National Institutes of Health, “The in vitro RSA can be used to understand molecular mechanisms of artemisinin resistance, to investigate the mode of action of artemisinins, and to screen and identify next-generation antimalarial drugs that can effectively kill artemisinin-resistant parasite strains. On the other hand, the ex vivo RSA can be readily implemented in field-based settings to monitor the worsening of artemisinin resistance in Cambodia where it is highly prevalent and to map its spread to other regions of southeast Asia. Also, this simple test can be easily established at sentinel sites in sub-Saharan Africa, where the arrival or evolution of artemisinin-resistant P falciparum is expected to be particularly devastating.”
In a linked Comment, Carol Hopkins Sibley from the WorldWide Antimalarial Resistance Network (WWARN) remarked, “These assays will allow the rapid validation of candidate molecular markers by directly testing the correlation of proposed markers with the output from their survival assay. The in vitro test will also provide a platform for understanding the mechanism of the reduced artemisinin response. With these simple methods in place, rapid tracking of the geographical and temporal changes in artemisinin resistance will be feasible in many sites. This far more comprehensive information will allow policy makers to design effective responses to the threat of artemisinin failure, and prolong the useful therapeutic life of ACTs.”
In vitro (Latin: in glass) studies in experimental biology are those that are conducted using components of an organism that have been isolated from their usual biological surroundings in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments are commonly called "test tube experiments".
Ex vivo (Latin: "out of the living") means that which takes place outside an organism. In science, ex vivo refers to experimentation or measurements done in or on tissue in an artificial environment outside the organism with the minimum alteration of natural conditions. Ex vivo conditions allow experimentation under more controlled conditions than is possible in in vivo experiments (in the intact organism), at the expense of altering the "natural" environment.
* Antimalarial drug efficacy
Efficacious antimalarial medicines are critical to malaria control, and continuous monitoring of their efficacy is needed to inform treatment policies in malaria-endemic countries, and to ensure early detection of, and response to, drug resistance.
* Antimalarial drug resistance
To date, parasite resistance has been documented in three of the five malaria species known to affect humans: P. falciparum, P. vivax and P. malariae. Drug resistance results in a delayed or incomplete clearance of parasites from the patient’s blood. The problem of antimalarial drug resistance is compounded by cross resistance, in which resistance to one drug confers resistance to other drugs that belong to the same chemical family or which have similar modes of action. During the past decades, several highly efficacious antimalarials had to be removed from markets after the development of parasite resistance to them.
* Artemisinin-based combination therapies
Today, WHO recommends artemisinin-based combination therapies (ACTs) for the treatment of uncomplicated malaria caused by P. falciparum. ACTs have been integral to the remarkable recent successes in global malaria control, and there is broad consensus that protecting the efficacy of these medicine combinations is an urgent priority. However, P. falciparum resistance is now emerging to artemisinin, and has been detected in four countries of the Greater Mekong Subregion: in Cambodia, Myanmar, Thailand and Viet Nam. Containment activities are ongoing in all four countries as part of a multi-stakeholder effort.