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Molecular Surveillance for Malaria Drug Resistant Genotypes in South America Kumar V. Udhayakumar, Ph.D Malaria Branch CDC, Atlanta, USA.

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Presentation on theme: "Molecular Surveillance for Malaria Drug Resistant Genotypes in South America Kumar V. Udhayakumar, Ph.D Malaria Branch CDC, Atlanta, USA."— Presentation transcript:

1 Molecular Surveillance for Malaria Drug Resistant Genotypes in South America Kumar V. Udhayakumar, Ph.D Malaria Branch CDC, Atlanta, USA

2 Talk Outline  Training  Background  Peru data  Venezuelan data  Brazilian data (Marinete Povoa)  Diagnostics

3 Training Ms. Nancy Arrospide Velasco Instituto Nacional de Salud (INS), Peru Dates: 02/06/2008 to 04/30/2008 Dr. Marinete M. Povoa, PhD Ms. Giselle Maria Rachid Viana, MSc Instituo Evandro Chagas/SVS/MS Secao de Parasitologia Laboratorio de Pesquisa Basica em Malaria Brazil Dates: 07/072008 to 08/01/2008

4 Training Dr. Malti R. Adhin, Ph.D (Week in August, 2008) Mergiory Y. Bracho Garrido (Jan to Feb 2009) Medisch Wentenschappelijk Institut Institute of Bioedical Sciences Adek Universiteit, Suriname Mr. Cesar Bedoya, M.Sc. (week in 2008) Area de Biotecnologia, Subproceso Virologia Instituto Nacional de Higiene y Medicina Tropical Guayaquil – Ecuador Sean Griffing from CDC visited last week to plan for lab training in Ecuador

5 Molecular Markers of Drug Resistance  Chloroquine (CQ) — Pfcrt  Mefloquine (MQ)—Pfmdr-1 copy number variation  Sulfadoxine — Pfdhps  Pyrimethamine—Pfdhfr  Artesunate/Artemisinin—no good markers

6 pfcrt7273747576 Ancestral (W.T.) CVMNK South America S VMN T CVMN T CV M ET pfmdr186184103410421246 Ancestral (W.T.) NYSND South America N FCDY pfcrt & pfmdr1 SNPs  pfcrt K76T is critical mutation for CQ resistance  Copy number increase is correlated with MQ resistance.

7 DHFR & DHPS Resistance Markers  For DHFR: C50R, N51I, C59R, S108N, I164L  I51,N108,L164  R50,I51,N108  For DHPS: S436A, A437G, K540E, A581G, A613T  G437,E540,G581 ** * * * 5051 59 108 164 dhfr * * * * * 436437 540 581 613 dhps

8 Microsatellite Markers As Tools  Understanding origin and spread of drug resistance is aided by microsatellite typing  Microsatellites are tandem repeats of one to four nucleotides (ATATATATATATATA)

9 Haplotypes & Hitchhiking  A haplotype refers to a given set of microsatellites in a chromosomal region  if two chromosomal regions share ancestry, their haplotypes should appear similar VS pfcrt

10 Study Results 1 2 3

11 Peru

12 Questions  What are the molecular pattern of resistant genotypes in different parts of Peru?  What is the ancestral relationship between the resistant genotypes in different parts of Peru?  What happened to the resistant genotypes after ACT implementation?

13 Malaria treatment in Peru  Andes divide Peru into 2 major regions  Coast 1999: CQ resistant, SP sensitive 1999-2001: CQ to Artesunate-SP  Peruvian Amazon 1999: East, CQ and SP resistance 1999-2001: SP to Artesunate-Mefloquine 2000/2002: West, CQ resistant, SP sensitive  What is the molecular pattern of CQ and SP resistance in different regions? Iquitos P. falciparum in Peru Pacific Coast Central Amazon Western Amazon Eastern Amazon

14 Pfcrt Genotypes in Peru pfcrt Pacific CVMNT W. Amazon CVMNT C/E Amazon CVMNT & SVMNT  Coast: 100% CVMNT genotype  Western Amazon: 100% CVMNT genotype  Central and Eastern Amazon: 1999 2006 CVMNT: 46% 34% SVMNT: 54% 66% Bacon et al, AAC, In Press

15 Pfdhfr Genotypes in Peru dhfr Pacific Only 108N W. Amazon Only 108N C/E. Amazon Triple mutant dhfr  Coast: 24% 108N mutation  Western Amazon: 82% 108N mutation  Central & East Amazon 47% triple mutant 53% single mutants no wild type found  Migration of non-Peruvian Dhfr 50R 51I 108N in 2006

16 Pfdhps genotypes in Peru  Coast: No mutant genotypes  Western Amazon: Silent dhps mutant 540K  Central/Eastern Amazon 29% Triple mutant 23% Double mutant 48% Wild type dhps Pacific No dhps mutations W. Amazon Silent dhps mutation C. Amazon Triple mutant dhps

17 Malaria treatment in Peru  SP was removed from use in Iquitos in 2000  We compared reported resistant allele prevalence from 1997 with our samples from 2005/2006 1997 2005 1997 2005 Highly resistant triple mutant dhfr and dhps genotypes declined after SP removal dhpsdhfr Zhou et al., Antimicrob. Agents and Chemo. 2008, 52:739-741

18 Microsatellite Data  Coastal parasites are highly clonal  Coastal and Central Amazon parasites show different lineages of resistant genotypes. Western Amazon contains parasites from both coastal and Central Amazon  DHFR triple mutants in Peru has a common founder  DHPS triple mutant in Peru and other parts of S. America have common ancestor

19 Pfmdr1 and SERCA genotypes In Central Amazon  Single copy mdr-1  SERCA- mutations at 402, 466, 630, 884, 1031, 1168 deletion of codon 884 increased to 68% in 2006 from 49% in 1999 769 mutation reported in French Guyana as associated with artesunate resistance was not found

20 Venezuela

21 Malaria treatment in Peru Background of Drug Resistance in Venezuela  CQ banned by National Program of Malaria Therapeutics in 1986  SP stopped in 1998, MQ monotherapy and ACT adopted  Our lab found that both CQ and SP resistant genotypes fixed in this population (McCollum, et al. 2007, Griffings et al., unpublished) Sifontes

22 Copy Number Variation in pfMDR1 Mefloquine resistance has been linked to duplications of pfmdr1: More copies means more MQ resistance 1-4 We used Real time PCR to test pfmdr1 copy number in 90 Venezuelan samples collected in between 2002-2004 All run at least in triplicate Samples with multiple copies of pfmdr1 were verified. 1 Wilson, et al., 1993, Mol. Biochem. Parasitol. 2 Price et al., 1999, Antimicrob. Agents Chemother. 3 Pickard et al., 2003, Antimicrob. Agents Chemother. 4 Price et al., 2004 Lancet Copy Number Variation in pfmdr1 from Venezuelan Parasites

23 Pfmdr1 copy number & Mefloquine


25 Summary From Venezuela (Sifonte)  Multidrug resistant genotypes are accumulating and no decline in resistant genotypes after policy change  Multiple copy pfmdr1 was found in 12% of samples.  Further testing for mdr-1 copy number increase in the region is warranted

26 Brazil


28 Molecular Epidemiology CDC collaboration Objectives: Molecular surveillance for tracking the origins and spread of drug resistant mutations in Brazil and other parts of South America. Sequencing and microsatellite studies for: PfCRT dhfr dhps Samples: 243 from different Brazilian Amazonia states collected in 80`s, 90`s, 2000`s


30 PARA STATE Parauapebas Maraba Novo Repartimento Tucurui Tailandia



33 C50R N51I The origin and spread of SP resistance in South America (DHFR) WT=C50, N51, S108, I164 N51I, S108N, I164L 1955-1960s? C50R, N51I, S108N 1955-1960s? Late 1950s 1960s 1970s Late 1970s

34 PERSPECTIVES Plasmodium falciparum: Pfcrt, dhfr and dhps genotyping microsatellite markers Serca and other new targets Plasmodium vivax: 1 – Microsatellite markers for differentiating parasites Other projects and collaboration- in vitro drug sensitivity

35 Pfcrt ANALYSES Sequencing - presence of mutations in pfcrt (K76T) Microsatellites – frequency and evolutionary history PfCRT (39) - 11 microsatellites 04 microssatelites (11Kb): - 5KB - 4.3 KB 1 KB 6 KB

36 PRELIMINAR RESULTS Mutation presence (all sample analyzed) Microsatellites: 1 - No detection of the chloroquine sensitive ancestral genotype- CVMNK 2 - The haplotype found was the chloroquine resistant SVMNT – StctVMNT SagtVMNT (MORE FREQUENT ALLELE)

37 Team: Laboratório de Pesquisas Básicas em Malária (Parasites and Vectors)

38 Thanks!

39 Summary-Peru  There is distinct pattern of drug resistant genotypes in Coastal, Western and Central Amazon.  Highly SP resistant genotypes declined after the removal of SP. This is a positive sign.  Migration of non-Peruvian dhfr triple mutant to Iquitos in 2006  Evolving pattern in SERCA- shift in the 884 codon deletion

40 Summary-Venezuela  Multi drug resistant genotypes are accumulating. Continuation of this trend will limit the choices of drugs for the effective treatment of P. falciparum in this region  12% of parasites showed increased mdr-1 copy number.  Increase in the multi copy mdr-1 gene will be of concern as this could reduce the efficacy of mefloquine.

41 Summary-Brazil  CQ resistant genotypes have reached fixation in some parts of Brazil and further studies in progress to determine other drug resistant genotypes.

42 Future Directions  What is the prevalence of mdr-1 copy number increase in AMI countries especially where mefloquine is being used?  What is the prevalence of different drug resistant genotypes in various regions at this time and how they are changing as new drugs are used for the treatment?  What is the genetic relationship between the resistant parasite populations in different regions?

43 Future Directions  How do we plan for a comprehensive molecular surveillance program to monitor drug resistant population of parasites in the region?

44 How good are HRP-2 based RDTs for the diagnosis of malaria in the Amazon?  Recent studies from CDC/WHO/Peru collaboration shows at elast two pattern of deletion in HRP-2 gene HRP-2 deletion: partial/total loss of RDT reactivity HRP-2 and HRP-3 deletion: Total loss of RDT reactivity

45 A Symposium Proposal During ASTMH Meeting in 2009 Molecular Surveillance for Drug Resistance in South America  AMI participants?

46 Acknowledgements  Andrea McCollum  Sean Griffing  Alexandre Macedo de Oliveira  Sankar Sridaran  Luke Syphard  Ananias Escalante  David Bacon and colleagues  Nancy Arrospide, INS  Wilmer Marquino  Trent Ruebush  Leopoldo Villegas  Marinete Marin Povoa  Giselle M. Rachid Viana  USAID  AMI  RAVREDA

47 Thank you

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