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.

Burma in my back yard

I’ve just recently begun my final stretch of field work for my PhD.  The last several years I’ve been travelling in the summers to the Thai-Myanmar border to do field work concerning malaria epidemiology and human demography.  This time I’ve moved my family, my wife Amber and my son Salem, to the field with me, and we’ll be staying until at least March of next year (2014).  We’ve settled into a cheap rent house in a town named Mae Tan in Tha Song Yang District, Tak Province and have spent the last several days trying to get it set up to our liking.  In fact, I’m writing this from my new home over the weekend with the hope that I’ll be able to post it while visiting the malaria clinic down the street (where I have internet access).

My new stove and the view out back of our rent house

From my kitchen window I can look out back over the Moei river, which marks the international border between Myanmar and Thailand.  Down one end of my block is an international border crossing and down the other is the malaria clinic.  While I’ve been working on my Thai, it does me little good in this neighborhood since most of my neighbors are Karen and they seem to know very little Thai.  

Across the Moei River from Tha Song Yang

Across the river there appears to be a simple mosque.  We can’t really see it, but there are several wooden, thatch roof structures and we hear the call to prayer several times during the day.  The river here is sandwiched by mountains on both sides and the resulting canyon makes a perfect echo chamber for carrying the sounds of the Imam’s song.  I don’t know whether the moslem adherents there/here are mostly Karen or if they are Rohingya who are attempting to flee persecution from the other side of Myanmar.  I do know that some of the Rohingya have been making their way to the refugee camps, one of which (Mae La, with about 50,000 people living in it) is a few miles south of here.  

A river ferry transporting someone across a very high Moei River

Over the last several days I’ve been struck by some interesting realities, a few that are related to research areas that I hope to investigate while here.  The most obvious to my family and me has been the openness of households…

Mosquitoes are most apparent at dusk and dawn, but if they get into your dwelling they’ll feed during the night too.  My toes and feet have been pretty thoroughly gnawed.  I brought a mosquito net for my son’s bed, and we’ve all been using some repellant from Mahidol University which appears to be made from lemongrass, but I’ve yet to find a net that will work for our own bed.  Furthermore, almost every house has openings that aren’t covered.  The tops of the walls in our bathroom and kitchen areas have ventilation holes large enough for me to stick my arm through – a feature which actually makes the humid bathroom and kitchen bearable in the humid heat.  

Such openings also make it quite easy for mosquitoes and other creatures to move into the house at will.  Last night we were awakened by a Tokay gecko, probably close to a foot long, that appears to have worked his way in as well.  If you’ve never heard a Tokay gecko’s call before, it isn’t something that is easy to ignore.  My son and I decided to name him Figaro, being that he so loves to sing.   

There has been some question about whether or not malaria transmission here occurs within households or outdoors, perhaps in agricultural fields.  For one species, Plasmodium vivax, infections appear to cluster in younger children.  I take this as potential evidence of exposure in or around the household, with older household members probably still being infected but having some acquired tolerance for vivax infections.  

Last night as I sat out front of my house, drinking a sweaty Chang beer, listening to the call to prayer and the rain that hasn’t stopped now for close to 3 days, I looked down the street and saw almost every household with doors and windows wide open, families sitting inside enjoying each other’s company.  If transmission is occurring within households, it’s going to be near impossible to completely stop.  I’m fortunate to have a relatively well built structure to stay in while living here and I can’t keep the mosquitoes out.  It is more than a little overwhelming to think about the options for the countless others who aren’t as well off as a poor graduate student.  

  

But even if exposure occurs outside of the household, and I think that for Plasmodium falciparum it does, it will be at least as hard to halt transmission.  Children cannot stay inside their beds or houses forever, it is healthy that they run and play outdoors.  Parents need to tend their agricultural fields, to hunt and fish, so that they can put food on the table.  

Another thing that has become blatantly clear is the effect of the rain on everyday life.  As I write this I believe it has been raining almost non-stop for about 2 ½ days.  The rains don’t make life come to a stand-still, but they do make things more difficult and uncomfortable.  The market was still open early this morning.  The steady stream of Karen migrants walking down our street continues.  A few hours ago I walked down to the border patrol check station, where migrants check in, and found empty, closed buildings (it is Sunday here) and a few boat drivers ferrying people to and from Myanmar from the Thai side of the border.  One jokingly asked if I’d like a ride across the river (and as exciting as that might be, it’s probably a bad idea).  When the Karen here were still at war with the Burmese military, fighting mainly occurred during the dry season – it is too difficult to traverse the countryside during the wet season.  

I frequently find heterogeneities in life to be fascinating fodder for research and theory.  Many of the statistical and mathematical models that population scientists, demographers, and epidemiologists use assume homogeneity in whatever processes we are studying: random mating, random mixing, random contacts, etc.  I think we all know that these things aren’t realities, but frequently that fact gets lost in the simple assumptions that are made, sometimes out of necessity, in research.  

Here I find some interesting heterogeneities in the rainy season.  While I complain that it has basically rained for several days straight, at times the rain slows.  During those times, traffic on the streets picks up.  This isn’t surprising, wading through puddles of muddy water isn’t fun.  Though the countless motorcycle drivers around here are quite adept at driving one-handed while holding an umbrella in the other hand, I imagine they’d much rather the rain stop altogether as well.  

And with the rains here comes malaria.  Tak Province consistently reports the highest number of malaria cases (both vivax and falciparum) of any province in the Kingdom, and within Tak Province, Tha Song Yang District has the heaviest malaria burden.  Cases here are highly seasonal, with only a very few in the dry season.  However, the trickle of infected people at the local malaria clinic isn’t a steady flow throughout the rainy season.  I once fancied a Poisson process of arrivals at the clinic, but this too doesn’t appear to be the pattern.  What is a pattern, however, is that after a rain, the clinic is quite busy.  (The clinic is closed on weekends and so Monday morning is generally a busy time too).  Does this mean that no one feels sick while it’s raining??  Probably not.  Also, it takes approximately 2 weeks from the time of infection until a person actually feels classic malaria symptoms.  So rain can lead to increased mosquito vector populations but infections that have occurred in the last day or two aren’t likely to be symptomatic so quickly.  No, it appears that people who are sick would rather not wade through muddy water, potentially have to illegally cross an international border, and then make their way down to the waiting area of a clinic during a torrential downpour.  

I think this makes perfect sense, but it doesn’t wind up in any model of seasonality in malaria infections that I’ve ever seen.  Rains = more mosquitoes = more malaria.  But rains also mean that people are likely to at least momentarily delay seeking treatment.  Infected people who wait longer before seeking treatment are important in malaria epidemiology because they spend longer amounts of time being infectious – potentially acting as so-called “super spreaders” (individuals who spread infectious disease at higher rates than do others).