Two new tests distinguish drug-resistant from drug-susceptible parasites

An international collaboration among scientists at the Institut Pasteur and the Ministry of Health in Cambodia, the Institut Pasteur in France, and the National Institute of Allergy & Infectious Diseases (NIAID) in the United States has produced two laboratory and field-based tests to monitor the spread of artemisinin-resistant malaria parasites.

The World Health Organization (WHO) reported an estimated 219 million malaria cases and 660,000 deaths worldwide in 2010.[1] For many years artemisinin-based combination therapies (ACTs) have been highly effective in reducing malaria mortality rates in multiple countries. Since 2009 the efficacy of ACTs has diminished in southeast Asia as P. falciparum has emerged with signs of reduced susceptibility to artemisinins in western Cambodia, western Thailand, southern Myanmar and southern Vietnam.[2]

The two tests were developed using Cambodian P. falciparum parasites adapted to culture in the laboratory (in-vitro RSA^0-3h) or obtained directly from Cambodian patients with malaria (ex-vivo RSA). Both tests briefly expose parasites to a high level of artemisinin that is comparable to dosing levels found in patient blood samples. The parasite survival rate is then measured after 3 days.

The first test (ex-vivo RSA) usedblood samples from patients in western, northern and eastern Cambodia – where parasites differ greatly in artemisinin response – and demonstrated that the simple test results correlate with clinical data. The results highlight in particular that parasites from slow-clearing infections survive this artemisinin pulse much better than those from fast-clearing infections.

Didier Menard, from the Institut Pasteur du Cambodge and co-author of the paper released by The Lancet Infectious Diseases [3] says, ‘This new ex-vivo RSA accurately identifies artemisinin-resistant infections where they have not been previously described. The findings suggest that resistance has spread or independently emerged in northern and eastern Cambodia’; Rick Fairhurst, from the NIAID and co-author, goes on to highlight that ‘the ex-vivo RSA is simple to use, provides reliable results, and can be adapted for different field settings where equipment and conditions may not support more elaborate or reliable analysis, but where surveillance is critical.’

The ex-vivo RSA, which is expected to save time and money, can now be used to map the geographical and temporal spread of artemisinin resistance in real-time in Cambodia, across Asia, and into Africa where the spread or emergence of resistance is likely to develop.

The second test (in-vitro RSA^0-3h) can provide a platform for understanding the mechanism of reduced artemisinin responses, but is more complicated and time-consuming. This test requires that specially-trained technicians first adapt parasites from patients in the clinic to cultures in the laboratory, and then synchronise them at the 0-3 hour stage of development before drug exposure.

The in-vitro test has enabled these researchers to identify more precisely the stage of parasite development where artemisinin resistance occurs, and has confirmed that only the youngest parasites are resistant to artemisinin’s mode of action. Results from the RSA^0-3h can help to clarify the molecular basis of artemisinin resistance. In the future, this assay could also be used to screen new drugs and validate candidate molecular markers by providing robust phenotypes for genome-wide association studies.

Thanks to this team of researchers and their latest results, it is expected that future studies using these novel approaches will provide the tools and evidence needed to enable policy makers to develop strategies to fight against the spread of artemisinin-resistant parasites, and prolong the useful therapeutic life of ACTs.

You can download and share the Standard Operating Procedure (SOP) entitled: Ring-stage Survival Assays (RSA) to evaluate the in-vitro and ex-vivo susceptibility of Plasmodium falciparum to artemisinins.

The study was led by Rick Fairhurst, M.D., Ph.D., of the National Institute of Allergy & Infectious Diseases, NIH, and Didier Menard, Ph.D., of the Institut Pasteur du Cambodge. 

Find out more about the Institut Pasteur in Paris and Cambodia, the NIAID and the NIH .