Malaria Parasite Molecular and Drug Sensitivity Dynamics in Kenya

1. Malaria Parasite Molecular and Drug Sensitivity Dynamics in Kenya Dr. Ben Andagalu Co-Director, Malaria Drug Resistance Laboratory, US Army Medical Research Directorate-Africa |KEMRI ben.andagalu@usamru-k.org UNCLASSIFIED

2. Disclaimer Material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the author, and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense. The investigators have adhered to the policies for protection of human subjects as prescribed in AR 70–25 UNCLASSIFIED

3. Treatment failure • Treatment failure is defined as the inability to clear malarial parasitemia or prevent recrudescence after administration of an antimalarial medicine, regardless of whether clinical symptoms are resolved1 • incorrect dosage • poor patient compliance • poor drug quality • drug interactions • drug resistance 1. WHO UNCLASSIFIED

4. Therapeutic efficacy UNCLASSIFIED

5. Therapeutic efficacy y=Therapeutic response = Drug factors, parasite factors, patient factors, other factors, etc. • Drug factors • Molecule • Drug quality • Parasite factors • Life cycle • Drug resistance mutations • Patient factors • Compliance • Metabolism • Other factors • Policies • Political environment UNCLASSIFIED

6. Drug Resistance • The ability of a parasite strain to survive and/or multiply despite the administration and absorption of a drug in doses equal to or higher than those usually recommended but within the limits of tolerance of the subject1 • The drug in question must “gain access to the parasite or the infected red blood cell for the duration of the time necessary for its normal action2 • requires demonstration of malaria parasitemia in a patient who has received an observed treatment dose of an antimalarial drug and simultaneous demonstration of adequate blood drug and metabolite concentrations using established laboratory methods • Multidrug resistance is resistance to more than two antimalarial compounds of different chemical classes • This term usually refers to P. falciparum resistance to chloroquine, sulfadoxine-pyrimethamine, and a third antimalarial compound 1. WHO definition, 1973 2. WHO definition, 1986. UNCLASSIFIED

7. Key drivers of antimalarial drug resistance • Unusual genetic structure of malaria parasites in regions known for antimalarial drug resistance • Counterfeit or substandard treatments • Unregulated antimalarial drug use • Poorly administered antimalarial drugs WWARN UNCLASSIFIED

8. Detection of antimalarial drug resistance • In vivo studies to assess the efficacy of drugs in patients • In vitro and/or ex vivo studies to evaluate parasite susceptibility to the drugs • Molecular assays to detect validated gene mutations and/or gene copy number changes that are associated with drug resistance • Case reports and passive detection • Animal model studies UNCLASSIFIED

9. Malaria drug resistance surveillance KSM KBW KCH KSI MRT MLD KEMRI#3628, WRAIR#2454 UNCLASSIFIED

10. Malaria drug resistance surveillance UNCLASSIFIED

11. Molecular assays UNCLASSIFIED

12. Sulfadoxine-pyrimethamine resistance • Pyrimethamine and sulfadoxine act synergistically to inhibit two enzymes important in the parasite's folate biosynthetic pathways, • Dihydrofolate reductase (DHFR) and • Dihydropteroate synthetase (DHPS) • Point mutations in the DHFR and DHPS genes confer resistance to pyrimethamine and sulfadoxine, respectively, with decreasing in vitro Plasmodium falciparum susceptibility related to the number of mutations in each gene • Pfdhfr mutations: A16V, N51I, C59R, S108N, I164L • Pfdhps mutations: A437G S436A/F/H, A581G, K540E and A613S/T • The Pfdhfr/Pfdhps N51I, C59R, S108N/A437G, K540E quintuple mutation has strongly been associated with clinical SP treatment failure UNCLASSIFIED

13. WHO 1996 - 2004 UNCLASSIFIED

14. Quinine resistance • The molecular mechanism by which quinine acts against P. falciparum is only partially understood • Quinine has been demonstrated to accumulate in the parasite’s digestive vacuole and can inhibit the detoxification of heme, an essential process within the parasite • Recent studies show that the genetic basis for resistance to quinine is complex, with multiple genes influencing susceptibility • Currently, three genes have been associated with altered quinine response: • Pfcrt ( P. falciparum chloroquine resistance transporter) • Pfmdr1(P. falciparum multidrug resistance transporter 1) • Pfnhe1 (P. falciparum sodium proton exchanger 1) • all of them encoding for transporter proteins UNCLASSIFIED

15. Chloroquine resistance • Chloroquine sensitive parasites accumulate much more chloroquine in the digestive vacuole than chloroquine resistant strains • The reduced chloroquine accumulation observed is associated with point mutations in the gene encoding for the P. falciparum chloroquine resistance transporter (PfCRT) protein - K76T or, in two single cases, K76N or K76I • Another mutation, S163R, restores the chloroquine sensitivity of CQR parasites UNCLASSIFIED

16. Global map of Chloroquine resistance n engl j med 371;5 UNCLASSIFIED

17. Artemisinin Resistance • Artemisinin resistance is defined as delayed parasite clearance • it represents a partial resistance that has affected only ring-stage parasites thus far • the majority of patients who have delayed parasite clearance are still able to clear their infections following treatment with an ACT with an effective partner drug or with an artesunate treatment lasting seven days • mechanism not clearly understood • there are two main proposed pathways for artemisinin resistance with the involvement of Kelch (K-13) mutations • a cell survival signaling pathway with PfPI3K • unfolded protein response pathway (UPR) UNCLASSIFIED

18. Artemisinin Resistance UNCLASSIFIED

19. Global map of Artemisinin resistance Phyo et al. http://dx.doi.org/10.5772/intechopen.76519 UNCLASSIFIED

20. Molecular Data List of targets and their resistance implications UNCLASSIFIED

21. Molecular Data UNCLASSIFIED

22. Molecular data UNCLASSIFIED

23. UNCLASSIFIED

24. In vitro/ex vivo studies UNCLASSIFIED

25. In vitro culture and sensitivity data UNCLASSIFIED

26. . Median IC50s for CQ, AQ, LU and MQ per Year during 2008–2011 Eyase et al. 2013 UNCLASSIFIED

27. IC50 trends for quinine UNCLASSIFIED

28. Clinical response

29. Clinical response UNCLASSIFIED

30. Malaria drug resistance surveillance Highlights • 2002-04, provided evidence of resistance to sulfadoxine/pyrimethamine combination • In 2008-10, MDR was first to demonstrate in-vitro and molecular resistance to mefloquine in field isolates • In 2010-12, confirmed in-vitro and molecular efficacy of doxycycline in Kenyan in field isolates. • 2013-14 identified and report non-adherence to treatment as the key cause of poor response to artemisinin combination therapy • 2014-16, MDR confirmed in vivo efficacy of artemisinin based combination therapies after successful completion of two back-to- back clinical trials UNCLASSIFIED

31. UNCLASSIFIED