1. MULTIPLE POPULATIONS OF ARTEMISININ-RESISTANT PLASMODIUM FALCIPARUM IN CAMBODIA MIOTTO ET. AL. 2013 Presented by Josie Benson

2. Malaria Global Disease >1 million deaths annually Eukaryote Protozoan parasite Replicates inside the Anopheles mosquito Caused 5 Plasmodium species P falciparum and P knowlesi More severe P vivax, P ovale, and P malariae

4. Prevention and Treatment Prevention Strategies Control of mosquito vectors Vaccine Chemotherapy Combination therapy (limits resistance development) Drugs: quinine sulfate + doxycycline or clindamycin or pyrimethamine-sulfadoxine Artemisinins Approved 2009 New standard

5. Question Why has drug resistance typically emerged in Western Cambodia? Need to discover genetic factors for resistance ID markers to monitor spread Does genetic epidemiology of the population explain why drug resistance originates in Western Cambodia?

6. Methods To Start: Patterns in genome variation 825 samples 10 locations Illumina Genome Analyzer 86,158 coding SNPs Could be “genotyped with confidence” Sequenom platform To double-check specific SNPs 99.4% concordance

7. Question 1: Population Structure? Methods: Neighbor-joining tree Pairwise distance matrix Results: Distinct W. Cambodian Clade Population structure that could be analyzed

8. Question 1: Results cont. Principal Component Analysis Showed variation within Western Cambodia

9. Question 1 results cont. Population structure Can’t be explained by geographical distance 

11. Question 2: Different Resistance Levels Between Subpopulations? Methods: Parasite clearance rates after artesunate treatment In vivo is standard for surveillance of resistance No reliable in vitro methods

12. Question 2: Results Clearance half-life vs subpopulation types Artemisinin resistant, longer half-lives

13. Question 3: Is Differentiation Due to Founder Effects? Founder Effects: reduced genetic diversity when a population has a small number of common ancestors Methods: Fixation Index (genome-wide estimates) between sub-populations Showed higher levels of differentiation within Cambodia than on different continents How?

14. Question 3: Results

15. Question 3: Other evidence for founder effects KH2, KH3, KH4 had fewer low-frequency alleles than core group (KH1) and others in SE and W Asia

16. Question 3: Other evidence for founder effects Compared SNPs w/ high differentiation to SNPs w/ low differentiation Similar dN/dS ratios Differentiation not result of positive selection

17. Question 3: Other evidence for founder effects KH2, KH3, KH4: reduced haplotype diversity Higher levels linkage disequilibrium Sometimes, single haplotype across most of a chromosomal region Only in non- core groups (KH2, KH3, KH4)

18. Question 4: Genetic Markers of Subpopulations? Variance found within gene that affects chloroquine and quinine resistance Also region of positive selection Transporter genes Could be implicated in causation of resistance Reservoirs for resistance-conferring alleles

19. Results Summary Unusual population structure in Western Cambodia Multiple distinct sympatric populations High genetic differentiation Founder effects and recent population expansion Skewed allele frequency spectra High haplotype homozygosity

20. Question 5/6: What Caused Founder Effects? What is Biological Relevance? Assumptions: Drug pressure  potential for resistant variants But mutations have little effect alone Fitness cost associated with resistance conference Postulate Recombination can sometimes produce highly-resistant variants w/ high fitness Inbred progeny at selective advantage Outcrossing: risk of losing “winning combination”

21. Questions 5/6 Cont. Hypothesis: Founder effects in West Cambodia represent recent expansion of artemisinin-resistant lines If correct: Multigenic artemisinin-resistant more likely Favoring propogation once emerged: Low transmission intensity (favors inbreeding). Physical isolation

22. Importance and Implications Helps explain why drug-resistance develops in Western Cambodia Could be due to high usage of anti-malarials in 50’s and 60’s Artemisinin resistance could be merely the most recent event in a long chain of events Major WHO Objective: Stop spread of resistant parasites Multiple subpopulations  multiple forms of resistance? Defining genetic markers  understanding geographic distribution Can eliminate major points of transmission

23. References http://emedicine.medscape.com/article/221134- treatment#aw2aab6b6b2 http://emedicine.medscape.com/article/221134- treatment#aw2aab6b6b2 http://en.wikipedia.org/wiki/Linkage_disequilibrium http://bio.classes.ucsc.edu/bio107/Class%20pdfs/W05_lecture 15.pdf http://bio.classes.ucsc.edu/bio107/Class%20pdfs/W05_lecture 15.pdf http://blogs.discovermagazine.com/gnxp/2007/01/basic- concepts-linkage-disequilibrium/#.UoMPqpETFuY http://blogs.discovermagazine.com/gnxp/2007/01/basic- concepts-linkage-disequilibrium/#.UoMPqpETFuY http://www.npr.org/blogs/health/2012/06/15/155103946/know- the-enemy-scientists-use-genetics-to-get-ahead-of-malaria http://www.npr.org/blogs/health/2012/06/15/155103946/know- the-enemy-scientists-use-genetics-to-get-ahead-of-malaria http://www.mayoclinic.com/health/malaria/DS00475/DSECTIO N=treatments-and-drugs http://www.mayoclinic.com/health/malaria/DS00475/DSECTIO N=treatments-and-drugs http://www.webmd.com/drugs/condition-225- Plasmodium+Falciparum+Malaria.aspx?diseaseid=225&diseas ename=Plasmodium+Falciparum+Malaria&source=0 http://www.webmd.com/drugs/condition-225- Plasmodium+Falciparum+Malaria.aspx?diseaseid=225&diseas ename=Plasmodium+Falciparum+Malaria&source=0

24. Questions?