Blood test to track emergence of drug resistance in malaria parasites

Researchers have developed a new method for analysing the DNA of malaria parasites straight from patient blood samples.

The technique will help identify hotspots of malaria parasite evolution and track the rise of malarial drug resistance faster and more efficiently than ever before, say the researchers from Oxford University and the Wellcome Trust Sanger Institute near Cambridge.

To prove the approach worked, the team analysed clinical samples from six countries and uncovered genetic differences between malaria parasites in Africa, Asia and Oceania. They have published their findings in the journal Nature.

Severe forms of malaria infection are caused by the parasite Plasmodium falciparum, which is spread by mosquitoes. Malaria infects over 200 million people and kills approximately 600,000 people every year, primarily children under the age of five in sub-Saharan Africa.

'One of the most striking features of P. falciparum is its ability to evolve and overcome antimalarial drugs,' says lead researcher Professor Dominic Kwiatkowski, who directs the Centre for Genomics and Global Health, a joint research project between Oxford University and the Sanger Institute.

'Chloroquine [once a standard drug against malaria] has become ineffective against malaria, and resistance to the other frontline drugs is emerging. If we want to control resistance, we first need to be able to monitor the genetic diversity of P. falciparum and identify hotspots of potential resistance as they occur.

'Rapid sequencing of parasite genomes from the blood of infected people is a powerful way of detecting changes in the parasite population, and potentially an important new surveillance tool for controlling malaria,' explains Professor Kwiatkowski.

The team developed a new technique to extract the malaria parasite DNA directly from patients' blood samples, removing as much human DNA from the sample as possible. The method overcomes the need to grow the parasite in the lab before sequencing, speeding the process and minimising errors.

The genome of the P. falciparum malaria parasite is particularly difficult to sequence because, unlike humans, large parts of the DNA sequence are repeated. As a result, the reconstruction of whole parasite genome DNA sequences is slow, expensive and error-prone using current methods.

To avoid these problems, the team created a list of single DNA letter changes, known as SNPs, which can be reliably identified in the gene-rich areas of the genome. These SNPs allow the variability in natural parasite populations to be mapped.

'We catalogued approximately 86,000 SNPs in the parasite genome that allow us to pinpoint differences between parasites around the world – a starting point for understanding how these populations adapt to changes in their environment,' says Dr Magnus Manske, co-first author from the Wellcome Trust Sanger Institute.

Dr Olivo Miotto from Oxford University and the Sanger Institute, the other co-first author, adds: 'Many malaria patients, especially in Africa, are continually infected by malaria parasites, and we have created a new tool for studying the genetic diversity within a single patient, and compare it to the diversity in their environment.'

The team used these techniques to analyse blood samples from patients in Burkina Faso, Cambodia, Kenya, Mali, Papua New Guinea and Thailand.

They found that a single infected person could harbour many genetically different malarial parasites, allowing the parasite populations to swap DNA to create new forms. This suggests the pace of parasite evolution can be affected by human factors as well as geography.

For example, blood samples taken from people in the neighbouring African countries of Burkina Faso and Mali, where there are very high levels of malaria transmission, showed strong intermingling of parasite genomes.

In contrast, Asian parasites collected on the Thai-Burmese border were not only different from those in Africa, but also distinct from those found near the Thai border with Cambodia. This lack of intermingling could be the result of effective malaria control in Thailand, combined with a history of restricted travel of people between Thailand and Cambodia.

Professor Nick White of Oxford University and Mahidol University in Thailand, who also took part in the study, says: 'The emergence and spread of antimalarial drug resistance is a major threat to current global initiatives to control and eliminate malaria. This research provides fundamental insights into the population structure and evolution of P. falciparum that are essential if we are to identify, map, and then contain spreading resistance.

'Working as a global community, we can now build on this technique to identify hotspots of antimalarial drug resistance around the world and contain them effectively.'

The research was funded by the Wellcome Trust, the Medical Research Council and the Howard Hughes Medical Institute.