K13 and the spread (or simultaneous emergence) of drug resistance in malaria parasites

We’ve mentioned this before, but the malaria and evolution story is complicated by multiple evolutionary tales:

• Humans adapt to parasites
• Parasites adapt to humans
• Mosquitoes adapt to both

Parasites may adapt to mosquitoes too – and humans have adaptations to mosquitoes…

Malaria parasites bursting from red blood cells.  From National Geographic, June 1986 - scanned and shared online by Centuron: http://imgur.com/a/nBJb6

This post is a story about parasites developing responses to some of the things we do to get rid of them.  Malaria parasites appear to have a real knack for survival, or at least the ones that survive and spread do.  Time and time again they have developed immune responses to our antimalarials.  Sometimes it happens quickly, sometimes it seems to take decades, but each time a new antimalarial is used, parasite strains emerge that are resistant to that antimalarial drug.

Southeast Asia appears to be a “special” place with regard to the evolution of antimalarial resistance.  For whatever reason, parasites that are resistant to new antimalarials always seem to be first documented here and then sometimes appear to subsequently spread globally.  (See Klein 2013 for a nice review of some theory around this problem (2)).  For example, chloroquine resistance in falciparum malaria seems to have independently emerged in both South America and Southeast Asia, but then seems to have spread globally from Southeast Asia (3).

Plasmodium falciparum parasites are almost globally resistant now to all antimalarials except for artemisinin.  In an attempt to keep these drugs effective, there has been a huge push to only use them in combination with other antimalarials.  The mechanisms of action of most antimalarials aren’t well understood, but the hope has been that by using different drugs, with different half-lifes, and probably different modes of action, then it will be much more difficult for parasites to develop resistance when compared to monotherapy (only using a single drug).

However, despite these efforts, artemisinin resistance has emerged in Southeast Asia (4).  It is not normally complete treatment failure at this point, but rather increased clearance times.  For example, while it would once take at most two days for parasites to be cleared from a patient’s blood stream after taking a dose of artemisinin, it now can take five.  Occasionally the treatment doesn’t work at all.   This is even occurring with artemisinin combination therapy.  Strains of parasites with “reduced sensitivity” have been found in Cambodia, in part of Vietnam, and along both sides of the Thailand-Myanmar border.

Some work has attempted to understand the genetics behind artemisinin resistance but many results, including a few I’ve been a part of, have contradicted each other.  However, one region on the parasite’s chromosome 13 keeps popping up in analyses.   Earlier this year, mutations in a particular gene (Kelch 13 (K13)-propeller) were identified as being potentially important in artemisinin resistance.  The function of this gene in the parasites isn’t well understood, but it is related to protein interactions.  And it isn’t a single point mutation that seems to confer resistance.  It appears that a wide variety of mutations, any of which are occurring in this gene, lead to parasites that are less sensitive to artemisinins – and this has now been confirmed both in vitro and in vivo.

The in vitro portion of this work began with a lab strain of falciparum malaria (3d7) which was intermittently exposed to artemisinins over a period of five years(5).  Doses of the drug were applied, then removed, then applied at higher proportions over this period of time.  Parasites from each dose cycle were sequenced so that the origin of mutations could be documented and so that mutations could be compared between case and control strains.  Ultimately the researchers narrowed their search down to a mutation in a single gene that corresponded to a point in time where some of the lab parasites seemed to no longer have strong, negative reactions to the antimalarial.

[It is important, I think, to remember that drug resistance isn’t usually an all or nothing type of trait, it is much more a trait of degree.  Even in situations where an antimalarial no longer works, it is likely that by increasing the dose of that antimalarial, there will be a point at which the parasites are still sensitive.  The problem is that it also becomes toxic to the human at some point.]

Next the researchers began looking at field isolates, across space and time, in Southeast Asia.   While they didn’t always find the same point mutations, they did find mutations in the same gene, in geographic areas where parasites are known to be less sensitive to artemisinins.  In areas where parasites still appear to be sensitive to the drug, they did not find mutations in this gene.  Furthermore, the prevalence of these mutations appears to have increased in certain regions (the ones that now have artemisinin resistance) over time.  

These findings are interesting I think for several reasons.
Here we have a gene in which mutations are somehow related to artemisinin resistance in malaria parasites.  But there isn’t a single mutation that leads to this resistance phenotype – rather it seems that just about any mutation(s) in this “gene” leads to resistance.  Does that make this a gene for resistance?

Another major finding, this time from a paper that came out in September 2014 (6), is that these mutations may not be spreading in the same way that other resistant strains (like chloroquine resistant falciparum malaria, for example) seem to have.  By analyzing the flanking regions of the K13 gene, analyzing patterns of linkage disequilibrium, the authors noted that several mutations in the K13 gene appear to have emerged independently and almost simultaneously both in Cambodia and along the Thailand-Myanmar border.

Once again the implications are quite interesting, if also scary.
One is that the evolutionary response seems less rare and unique if it can happen independently and simultaneously in different regions.  Does this mean that combination therapy is not working the way we hoped it would?

Another is more directly related to public health.  Right now there are several small scale elimination attempts occurring throughout Southeast Asia.  In fact, I’m working with one of the teams doing this (briefly discussed here).  Our hope is that we can wipe out resistant strains before they spread (via mosquitoes or humans) to other regions – perhaps especially Africa.  If resistance is likely to evolve anywhere that artemisinins are being used, we may not be able to halt this spread.  I would argue that our intentions to eliminate malaria in targeted subregions are worthwhile regardless.  But, it is a bit scary nevertheless.  

*** My opinions are my own!  This post and my opinions do not necessarily reflect those of Shoklo Malaria Research Unit, Mahidol Oxford Tropical Medicine Research Unit, or the Wellcome Trust.  

1. Network MGE. Reappraisal of known malaria resistance loci in a large multicenter study. Nat Genet. 2014;46(11):1197–205.

2. Klein EY. Antimalarial drug resistance: a review of the biology and strategies to delay emergence and spread. Int J Antimicrob Agents [Internet]. Elsevier B.V.; 2013 Feb 7 [cited 2013 Mar 8];41(4):311–7. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23394809

3. Payne D. Spread of chloroquine resistance in Plasmodium falciparum. Parasitol Today [Internet]. 1987 Aug;3(8):241–6. Available from: http://www.ncbi.nlm.nih.gov/pubmed/15463062

4. Dondorp A, Nosten F, Yi P. Artemisinin resistance in Plasmodium falciparum malaria. New Engl J Med J … [Internet]. 2009 [cited 2013 Nov 17];455–67. Available from: http://www.nejm.org/doi/full/10.1056/nejmoa0808859

5. Ariey F, Witkowski B, Amaratunga C, Beghain J, Langlois A-C, Khim N, et al. A molecular marker of artemisinin- resistant Plasmodium falciparum malaria. Nature. 2014;505(7481):50–5.

6. Takala-harrison S, Jacob CG, Arze C, Cummings MP, Silva JC, Khanthavong M, et al. Independent Emergence of Artemisinin Resistance Mutations Among Plasmodium falciparum in Southeast Asia. J Infect Dis. 2014;491:1–10.

Why less malaria can be more of a problem

Most people who are interested in malaria are concerned with regions with really high transmission.  These are areas, such as sub-Saharan Africa, where malaria prevalence is high year round and where malaria related mortality is highest.  Sadly, that mortality generally seems to disproportionately affect children.  It’s no wonder then, that these are the areas that most people think about.  

Huge amounts of research money goes toward potential vaccines, toward genome sequencing of parasites, laboratory and field based science, and much of it is focused on the malaria situation in sub-Saharan Africa.  I’ve already questioned whether this is a wise use for a limited amount of money and that by changing socio-economic factors we would probably get more bang for our buck.   

Here I am also going to argue that for what most of us in tropical medicine, epidemiology, and disease ecology do, areas of low transmission are more important.  And while I can make this argument from a few different vantage points, today I’m going to base it off the fact that drug resistant malaria (see Note 1) seems to recurrently arise in areas of low transmission, only to spread to places like Africa where the implications of treatment failure at the population level are most severe (White, 2004).   

First, what do I mean by low or high transmission?  

In the malaria system there are always at least two hosts, humans and mosquitoes.  Humans get infected by infectious mosquitoes that have in turn been infected by infectious humans.  In areas of high transmission, people are bitten by infected and infectious mosquitoes more frequently in comparison to people in low transmission areas.  For example, in parts of sub-Saharan Africa people may be bitten by infectious mosquitoes more than 100 times in a single year.  In areas of low transmission people might only be bitten by an infectious mosquito once a year (Bousema & Drakeley, 2011). 

There are several ways to break the transmission cycle between humans and mosquitoes.  Some efforts are based on preventing mosquitoes from feeding on human blood (e.g. mosquito nets).  Others are based on treating infected humans with antimalarials; which should both cure the infected persons and halt the spread of parasites within the infected to others.  Suffice it to say that at least a huge component of modern malaria control efforts depends on the use of antimalarials.  Drug resistance is therefore a very large problem, and it is a problem with a relatively long history.  

For example, chloroquine was a wonder drug for malaria treatment, it was easy to manufacture, cheap, and compared to previous antimalarials it seemed to have fewer negative side effects.  However, it wasn’t long after it had been rolled out into the global scene that people in the field began to notice decreased sensitivity in parasites to chloroquine, almost simultaneously in parts of Southeast Asia and South America in the late 1950s and early 1960s (Payne, 1987).  The move was slow for chloroquine resistant parasites, but they eventually did make the passage to Africa.  Perhaps especially in Southeast Asia this story has been repeated over and over again (Parker et al., 2012), with each new antimalarial losing its efficacy shortly after its widespread use and subsequently parasites with the drug resistant mutation (or mutations) spread throughout the world (Anderson & Roper, 2005).  

I believe that I’m relatively safe in stating that there is a growing consensus among malariologists that drug resistant strains continue to emerge in certain areas and not in others because of the level of transmission intensity in those areas (Klein, 2013).  Take Thailand, for example: This is a nation that has very little malaria in the central plains regions and highly seasonal malaria along its international borders with Cambodia and Myanmar.  Those border sites appear to consistently be centers for the emergence of drug resistant parasites strains, most recently with decreased sensitivity to artemisinin (Noedl et al., 2008; Phyo et al., 2012).  

But what is it about low transmission areas that make them primed for drug resistant strains to pop up?  

Here there is certainly room for debate.  Several things must happen in order for drug resistant parasites to become prevalent enough to be a public health problem.  First there must be mutations that arise which confer some sort of resistance to antimalarial drugs.  However, some mutations are likely to be harmful for the parasite, even if offering some protection against antimalarials.  Therefore the mutation must not be so harmful as to provide an overall disadvantage in comparison to its advantage in the presence of drugs.  Then, that mutation (or mutations) must spread and be retained throughout the population.  

One potential explanation is that in low transmission areas people are unlikely to have developed immunity to malaria (White, 2004).  When they are infected they have high parasite densities in their blood and they are more likely to take antimalarials; meaning that more parasites are exposed to more antimalarials, therefore leading to more chances for resistance to emerge.  Then, parasites with resistant strains don’t have to survive their host’s immune responses, and are likely to have higher parasite densities, meaning that they are also more likely to be passed on to mosquitoes and ultimately other human hosts.  

Population genetics likely also plays an important role.  In areas with really high transmission, a single infected person is likely to be carrying multiple parasite strains at the same time.  Mosquitoes are also likely to be infected by multiple strains, as they have probably both fed off of individuals with more than one strain and potentially have fed on more than one infected individual.  Malaria parasites have quite complicated life cycles, undergoing several different life stages within human and mosquito hosts.  They reproduce asexually inside humans until some of them split off and become ready for sexual reproduction, basically developing into either males or females.  These sexual parasites are then picked up by the mosquito where they undergo sexual reproduction.  Therefore genetic recombination only occurs within the mosquito.  This has strong implications for drug resistance which is conferred through multiple genes.  Genetic recombination means that such mutant combinations can be lost through sexual recombination.  However, in areas of low transmission, such as in Southeast Asia, many parasites are actually reproducing with themselves; the mosquito isn’t picking up sexual parasites from multiple strains but instead from a single strain (Anderson et al., 2000).  Recombination still occurs, but it is occurring with a single parasite strain, meaning the gene combinations will not be lost.

Intrahost competition between parasites is probably also important.  There is evidence that, within individuals who are infected by multiple strains of parasites, not all parasite strains do equally as well.  Some appear to be more aggressive and to propagate themselves at higher levels than other strains.  In areas with high transmission, a parasite strain with a drug resistance mutation may still need to out-compete other parasite strains within the host (Klein, 2013).  Since a mutation isn’t likely to confer absolute resistance to antimalarials, and since it might actually be slightly harmful to the parasite strain, it is possible that aggressive parasite strains (even without drug resistance mutations) can out-compete those with such mutations.  Conversely, in low transmission areas this is less likely to be the case.   

What does all of this mean for the big picture though?   The obvious implication related to the drug resistant strains that keep emerging in Southeast Asia and then spreading throughout the world is only part of this story.  

Currently there are increased efforts for malaria control and, in some situations, even efforts directed toward malaria elimination.  But there are no easy fixes to the malaria problem.  That is, in some areas, malaria transmission will be greatly reduced but will continue to persist at very low levels of transmission.  Most likely, even in areas where complete eradication is achieved, there will be periods of time where malaria persists at low transmission levels.  The paradox, therefore, is that as the malaria situation improves, as transmission is reduced and less people become sick or even die, the situation with regard to effective antimalarials might simultaneously worsen.  

Previously I’ve argued that if we really want to fix the malaria problem we should look toward socio-economic factors.  Malaria remains a disease that mostly afflicts poor people in poor nations in the tropical world.  If we could increase economic wellbeing and improve sanitation, we would probably fix a large part of the problem, much like we did here in the U.S.A. over half a century ago.  Also, it might make more sense to dump the millions of dollars that are directed toward researching single diseases into funding primary health care for children in the industrializing world (which is the approach of groups such as Partners in Health).  That is, if children in poor nations received free health care regardless of the cause of their illness we will probably see huge improvements in global health.

But the situations that I describe above, where the conditions in low transmission areas lead to a type of perfect storm, are probably best addressed through epidemiological, ecological, and evolutionary approaches.  While I’m quite pessimistic about spending millions and millions of dollars on potential vaccines to save children in Africa from one (out of a lot) of pathogens, I do think there is a very important role for the life and biomedical sciences in malaria control.  Here are a few examples:

1.) Antimalarials will continue to be important, even if they are only used sparingly in areas of low transmission.  That means that evolutionary biologists will continue to have an important role in malaria control (Read, Day, & Huijben, 2011).  

2.) Another interesting factor that I completely avoided in this post has to do with areas with several different hosts (zoonosis and anthroponosis) – such as in Borneo where nonhuman primates carry malaria parasites that can also easily infect humans (Singh et al., 2004).  This type of situation can also lead to persistent low transmission and is perhaps best addressed by ecologists.  

3.) Finally, in areas with low, sporadic transmission the role of human migration in continued transmission is quite important; meaning that human demographers, geographers, and population ecologists are also important for addressing the malaria situation (Prothero, 1999; Tatem & Smith, 2010).  

Note 1: Drug resistance isn't really a binary trait.  It is probably more accurate to talk about levels of reduced sensitivity to drugs, but this is quite a mouthful.  Typically when I use the term drug resistance, I am talking about parasites that no longer respond well to antimalarials at the doses that are safe to give to humans.    

REFERENCES

Anderson, T. J. C., & Roper, C. (2005). The Origins and Spread of Antimalarial Drug Resistance: Lessons for Policy Makers. Acta Tropica, 94, 269–280.

Anderson, T. J., Haubold, B., Williams, J. T., Estrada-Franco, J. G., Richardson, L., Mollinedo, R., Bockarie, M., et al. (2000). Microsatellite markers reveal a spectrum of population structures in the malaria parasite Plasmodium falciparum. Molecular biology and evolution, 17(10), 1467–82. 

Bousema, T., & Drakeley, C. (2011). Epidemiology and infectivity of Plasmodium falciparum and Plasmodium vivax gametocytes in relation to malaria control and elimination. Clinical microbiology reviews, 24(2), 377–410. doi:10.1128/CMR.00051-10

Klein, E. Y. (2013). Antimalarial drug resistance: a review of the biology and strategies to delay emergence and spread. International journal of antimicrobial agents, 41(4), 311–317. 

Noedl, H., Se, Y., Schaecher, K., Smith, B., Socheat, D., & Fukuda, M. (2008). Evidence of artemisinin-resistant malaria in western Cambodia. N Engl J Med, 359(24), 2619–2620.

Parker, D., Lerdprom, R., Srisatjarak, W., Yan, G., Sattabongkot, J., Wood, J., Sirichaisinthop, J., et al. (2012). Longitudinal in vitro surveillance of Plasmodium falciparum sensitivity to common anti-malarials in Thailand between 1994 and 2010. Malaria journal, 11(1), 290. 

Payne, D. (1987). Spread of chloroquine resistance in Plasmodium falciparum. Parasitology today (Personal ed.), 3(8), 241–6. 

Phyo, A. P., Nkhoma, S., Stepniewska, K., Ashley, E. A., Nair, S., McGready, R., ler Moo, C., et al. (2012). Emergence of artemisinin-resistant malaria on the western border of Thailand: a longitudinal study. The Lancet, 12.

Prothero, R. M. (1999). Malaria, forests and people in Southeast Asia. Singapore Journal of Tropical Geography, 20(1), 76–85.

Read, A. F., Day, T., & Huijben, S. (2011). The evolution of drug resistance and the curious orthodoxy of aggressive chemotherapy. Proceedings of the National Academy of Sciences of the United States of America, 108 Suppl , 10871–7. 

Singh, B., Kim Sung, L., Matusop, A., Radhakrishnan, A., Shamsul, S. S. G., Cox-Singh, J., Thomas, A., et al. (2004). A large focus of naturally acquired Plasmodium knowlesi infections in human beings. Lancet, 363(9414), 1017–24. 

Tatem, A. J., & Smith, D. L. (2010). International population movements and regional Plasmodium falciparum malaria elimination strategies. Proceedings of the National Academy of Sciences, 107(27), 12222.

White, N. J. (2004). Antimalarial Drug Resistance. The Journal of Clinical Investigation, 113(8), 1084–1092.