The ongoing battle against drug resistant malaria

Malaria occurs in more than 90 countries worldwide. On average it kills one child every minute and around half a million people every year. As a result, efforts to control the disease are a major global health priority.

Since 2000, deaths resulting from malaria have fallen globally by 47 per cent

Since 2000, funding for malaria control has increased and huge progress has been made, including improved management of the mosquito vector and the deployment of effective antimalarial drugs that work by killing the malaria parasite. Consequently, death rates have fallen globally by 47 per cent. However, the development and spread of drug resistant malaria parasites is proving to be one of the greatest challenges to malaria control today. Plasmodium falciparum, the most deadly species of malaria parasite, has developed resistance to nearly all antimalarial drugs currently in use.

What is drug resistance?

Drug resistance is the reduction in the effectiveness of a drug that has been designed to kill or inhibit a particular pathogen. It can arise as the result of one or more mutations in the genome of the pathogen that give it the advantage of being able to evade the effects of a drug.

Often drug resistance emerges gradually, starting initially with a handful of drug resistant pathogens that survive exposure to a drug while all the drug-sensitive pathogens die. Having virtually nothing to stop them, neither drugs nor competition from drug-sensitive pathogens, the drug-resistant pathogens can multiply and their population grows.

How does drug resistance come about?

Malaria parasites are genetically very diverse and their genomes are changing (mutating) all the time. Occasionally, a genetic change can be beneficial, for example, by helping the parasite to hide from our immune system or by making the parasite resistant to a particular drug.

When inside the mosquito, the male and female forms of the parasite can pass these mutations to the next generation of parasites when they fuse and multiply. The resistant parasites will then enter another human when the mosquito bites and injects the parasites into their blood.

When drug resistant parasites are present, the drugs either have no effect or are very slow to work.

When working effectively, antimalarial drugs can clear all the malaria parasites from an individual’s body in a matter of days. However, when drug resistant parasites are present, the drugs either have no effect or are very slow to work, potentially leaving some parasites behind. This is called delayed parasite clearance. To rid an individual of malaria, every single parasite needs to be removed from the body, if not, malaria could more easily reoccur.

Looking back – the history of drug resistance

Malaria parasites have affected humans for thousands of years. During that time, humans have evolved mechanisms to protect themselves from the parasites and the parasites have evolved means of avoiding these defences.

With the introduction of the first drugs to tackle malaria, a new evolutionary battle was started…

However, with the introduction of the first drugs to tackle malaria, a new evolutionary battle was started: the parasites’ struggle to evade antimalarial drugs. The first recorded use of an antimalarial drug was quinine in 1632 and although it took the parasites a while (almost 300 years!), quinine-resistant parasites eventually emerged in 1910. Since then there have been several major drugs developed, but each time the malaria parasites have developed resistance:

• The drug chloroquine was introduced in 1945 with resistant parasites cropping up 12 years later.
• Sulfadoxine pyrimethamine was used from 1967 and resistant parasites were found in the same year!
• Mefloquine was given from 1977 but resistance was first recorded in 1982.

Scientists, doctors and patients have now been battling with drug resistant malaria for over a century.

Drug resistance déjà vu

Interestingly, drug resistance often emerges in the same place: the Greater Mekong Subregion in South East Asia.

Although it isn’t known for sure, there are several theories as to why resistance may emerge in this region. One theory is that, compared to Africa, malaria infections are much less common in South East Asia. This means that drug resistant malaria parasites face much less competition from other malaria parasites, and are therefore more likely to survive and reproduce.

Another theory is that because many people in Africa develop natural immunity to malaria, only a small fraction of all the people infected are actually treated with antimalarial drugs. This translates into a much lower drug pressure on the parasite when compared to South East Asia where antimalarial drugs are more widely distributed.

Survival of the fittest

We call it drug resistance; the malaria parasite calls it surviving!

Perhaps surprisingly, for a malaria parasite a drug resistance mutation isn’t necessarily very good for their biological fitness. Imagine all the drug resistant parasites are wearing a big, heavy suit of armour. They are protected from the effects of the drug, but in terms of speed and agility they are less able to compete for food with parasites that are not drug resistant. This means that they can only thrive in the presence of an antimalarial drug when the fast, but drug-sensitive, parasites will be killed. We call it drug resistance; the malaria parasite calls it surviving!

Now there are only a limited number of drugs which can be used against malaria. The frontline treatment is currently artemisinin-based combination therapy.

Artemisinin-based combination therapy

Artemisinin-based combination therapy (ACT) has been integral to the recent successes in global malaria control. The main idea behind ACT was to provide an inexpensive, short-course treatment that would also help protect against the development of drug resistance. On paper, ACT should work perfectly. Artemisinin is a very fast acting drug which means that within 12 hours of starting treatment around half of the parasites in the body are removed. Artemisinin is combined with a partner drug that usually works more slowly, hammering the remaining malaria parasites until they are all dead.

One example of an effective ACT partner drug is piperaquine. First synthesised in the 1960s piperaquine was originally used alone as a malaria preventative and treatment in China. However, with the emergence of piperaquine-resistant Plasmodium falciparum in the 1980s, its use declined until it was deemed suitable for use in combination with artemisinin. This is due to its low cost, high efficacy and minimal side effects. A partner drug, like piperaquine, provides a counter punch so that if malaria parasites develop resistance to artemisinin, they will still be knocked out by the partner drug.

Is history repeating itself?

In 2009, researchers reported concerns that artemisinin was taking longer to clear parasites from patients infected with Plasmodium falciparum along the Thailand-Cambodia border — a worrying sign of emerging drug resistance. Since then, researchers have reported slow parasite clearance in four countries in the Greater Mekong Subregion and in some locations they’ve even seen treatment failures. This puts additional pressure on the partner drugs to kill the parasites, which may lead to resistance to these drugs too.

It is likely there would be a reversal in the recent declines seen in malaria mortality rates…

What’s more, if artemisinin resistance were to arise in Africa or emerge independently elsewhere, as has happened with other antimalarial drugs, the public health consequences would be catastrophic. It is likely there would be a reversal in the recent declines seen in malaria mortality rates and the number of deaths due to malaria would start to increase.

In response to this threat, the World Health Organization (WHO) launched an emergency plan of action to tackle artemisinin resistance in the Greater Mekong Subregion covering 2013-2015. They proposed an immediate and coordinated increase in efforts to tackle malaria in Cambodia, Laos, Myanmar, Thailand and Vietnam.

The WHO’s goal is to remove malaria completely from Greater Mekong Subregion countries by 2030.

Currently the WHO’s goal is to initiate elimination activities by 2020 in order to remove malaria completely from Greater Mekong Subregion countries by 2030.

But how are we going to stop the spread of drug resistance if we haven’t been able to in the past? Well, now we have one more weapon in our arsenal that we didn’t have before – genome sequencing!

Genomics vs. malaria – the fight is on

To develop an effective strategy to combat malaria once and for all it is crucial to understand the genetic factors that determine how drug resistance emerges and spreads.

At the time that artemisinin resistance was first discovered in early 2009, no one knew which genetic changes were responsible, and pinpointing those changes proved more challenging than expected.

However, faster and cheaper genome sequencing techniques have enabled us to learn a lot more about the underlying genetic changes responsible. Scientists have now compared thousands of parasite genomes from different areas of Africa and South East Asia to identify the genetic variations that could lead to drug resistance. By finding these genetic changes scientists are hoping that they may eventually be able to track and then prevent the spread of artemisinin resistance.

Clues on chromosome 13

In 2012, and then again in 2013, a couple of genome-wide association studies (GWAS) looking at the P. falciparum genome pointed towards two regions next to each other on chromosome 13 as potential sites of the mutations associated with artemisinin resistance. However, they needed to find out for sure if these mutations were directly involved in resistance. A year or so later, a collaboration led by scientists at the Institut Pasteur in Paris came up with an experiment that pointed them in the right direction.

With DNA sequencing scientists can study the genomes of resistant parasites and compare them to the genomes of non-resistant parasites.

Over a five year period, the scientists grew and nurtured a strain of Plasmodium parasite that they knew did not have any resistance to artemisinin. Every so often during this period they gave the colony of parasites a small amount of artemisinin. They hypothesised that sooner or later an artemisinin resistant parasite would emerge because of the selection pressure of the drug (the pressure to adapt in order to survive!). Sure enough, after four years of exposure to the drug, artemisinin resistant parasites were seen. With DNA sequencing they were then able to study the genome of the resistant parasites and compare them to the genome of the original, non-resistant strain of Plasmodium.

They found several genetic changes in the resistant parasite genome but the most significant one occurred bang in the middle of the previously-identified regions on chromosome 13, in a gene called kelch13.

What does kelch13 do?

kelch13 is one of the most conserved genes in the Plasmodium genome. This means that it has remained pretty much the same for about 50 million years. This suggests that its function must be crucial for the survival of the Plasmodium parasite. As a result, although a mutation in this gene may make it resistant to artemisinin, it would also probably reduce the normal biological fitness of the malaria parasite, as if they had put on a heavy suit of armour.

Searching across the genome

To better understand how malaria parasites are developing drug resistance in the wild, researchers carried out a study of P. falciparum parasites sampled from more than 1,000 malaria patients in South East Asia and Africa. The researchers collected blood samples from patients before artemisinin-based combination therapy (ACT) was given to them and then every six hours during treatment with ACT to track how quickly the parasites were being cleared from the body.

There wasn’t a single mutation that caused artemisinin resistance but several mutations…

They then isolated parasite DNA from all of the blood samples and determined the parasites’ genome sequence. By comparing a large number of parasite genomes it enabled the researchers to study genetic differences between drug sensitive and drug resistant parasites. The results were quite unexpected.

In the drug resistant parasites they didn’t just find one mutation in kelch13, they found more than 20! This explains why it was so hard to pinpoint the exact mutation. There wasn’t a single mutation that caused artemisinin resistance but several mutations, each emerging at different times and places in the kelch13 gene.

Kelch13 and friends

When scientists looked beyond kelch13 at the rest of the genome there was another surprise in store. They discovered mutations in at least four other genes (fd, arps10, mdr2 and crt) that are also associated with artemisinin resistance. Whenever the kelch13 mutation was present in the genome of a resistant parasite, the other four mutations almost invariably seemed to be there too. Was this just a coincidence?

This Manhattan plot shows the results from a genome-wide association study (GWAS) investigating artemisinin resistance. Each dot represents a single letter change in the parasite genome and its location on a chromosome. The vertical height of the dot on the plot shows how strongly associated that genetic change is to artemisinin resistance. You can see a mutation in the kelch gene on chromosome 13 is most strongly associated with artemisinin resistance. You can also see that mutations in other genes, arps10, fd, mdr2 and crt, are also associated with artemisinin resistance.
Image credit: Genetic architecture of artemisinin-resistant Plasmodium falciparum, Nature Genetics 47, 226-234 (2015) doi:10.1038/ng.3189.

Although it is not yet known what the exact role of the four mutations might be in drug resistance, it might be that they provide an ideal environment for the kelch13 mutation to arise. They may, for example, compensate for the parasite’s reduced fitness due to artemisinin resistance. If we think back to drug resistance being like a heavy suit of armour, these four background mutations could be providing the resistant parasites with a horse that helps them maintain their mobility and compete more equally with susceptible parasites for food.

Finding this fixed set of four mutations in parasite populations could act as a warning sign.

Whatever their role might be, discovering these mutations has given scientists a useful tool for monitoring the spread of artemisinin resistance. Finding this fixed set of four mutations in parasite populations could act as a warning sign that these parasites are at high-risk of developing artemisinin resistance. This will enable researchers to target those areas with medicines or insecticides to help kill any artemisinin-resistant parasites before they take hold and spread any further.

Looking forward

Any intervention we carry out to control malaria has an impact. We can think of malaria control interventions as large-scale evolutionary experiments. By introducing a drug or a vaccine against a parasite, we are applying a selection pressure and encouraging those genetic changes that will enable the parasite to survive in the presence of a drug or vaccine.

Genomics is a powerful tool to help us observe the evolutionary impact of these interventions. We can then use this information to inform decisions about which methods to use in future. For example, if the malaria parasite starts to show signs of resistance to one drug, it is possible to switch to another drug or change the drug regimen.

Like spies in an enemy country, genomics can provide us with the intelligence to track drug resistance emerging in the malaria parasite. This gives us more time to plan our counterattack before drug resistance becomes more widespread.