Molecular Surveillance for Malaria Drug Resistant Genotypes in South America

Kumar V. Udhayakumar, Ph.DMalaria Branch

CDC, Atlanta, USA

Talk Outline

Training Background Peru data Venezuelan data Brazilian data (Marinete Povoa)

Diagnostics

Training

Ms. Nancy Arrospide VelascoInstituto Nacional de Salud (INS),

PeruDates: 02/06/2008 to 04/30/2008

Dr. Marinete M. Povoa, PhD Ms. Giselle Maria Rachid Viana, MScInstituo Evandro Chagas/SVS/MSSecao de ParasitologiaLaboratorio de Pesquisa Basica em

MalariaBrazilDates: 07/072008 to 08/01/2008

Training

Dr. Malti R. Adhin, Ph.D (Week in August, 2008)

Mergiory Y. Bracho Garrido (Jan to Feb 2009)

Medisch Wentenschappelijk InstitutInstitute of Bioedical SciencesAdek Universiteit, Suriname

Mr. Cesar Bedoya, M.Sc. (week in 2008)

Area de Biotecnologia, Subproceso Virologia

Instituto Nacional de Higiene y Medicina Tropical

Guayaquil – EcuadorSean Griffing from CDC visited last

week to plan for lab training in Ecuador

Molecular Markers of Drug Resistance

Chloroquine (CQ)—Pfcrt Mefloquine (MQ)—Pfmdr-1

copy number variation

Sulfadoxine—Pfdhps Pyrimethamine—Pfdhfr Artesunate/Artemisinin—no

good markers

pfcrt 72 73 74 75 76

Ancestral (W.T.)

C V M N K

South America S V M N TSouth America C V M N T

South America

C V M E T

pfmdr1 86 184 1034

1042

1246

Ancestral (W.T.) N Y S N D

South America N F C D Y

pfcrt & pfmdr1 SNPs

pfcrt K76T is critical mutation for CQ resistance

Copy number increase is correlated with MQ resistance.

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

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)

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

pfcrt

pfcrt

pfcrt

pfcrt

Study Results

1

2

3

Peru

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?

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 CoastCentral Amazon

Western Amazon

Eastern Amazon

Pfcrt Genotypes in Peru

pfcrt

PacificCVMNT

W. AmazonCVMNT

C/E AmazonCVMNT

& 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

Pfdhfr Genotypes in Peru

dhfr

PacificOnly 108N

W. AmazonOnly 108N

C/E. AmazonTriple 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

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

PacificNo dhps

mutationsW. AmazonSilent dhps mutation

C. AmazonTriple

mutant dhps

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

48.9

2.2

46.7

78

5.8

16.2

0

20

40

60

80

100

Single (S108N) Double Triple

dhfr mutations

19

97

19

97

19

97

20

05

20

05

20

05

0 0

46.7

2.4 0

13

0

20

40

60

80

100

Single Double Triple

dhps mutations

19

97

19

97

19

9720

05

20

05

20

05

Highly resistant triple mutant dhfr and dhps genotypes declined after SP removal

dhps dhfr

Zhou et al., Antimicrob. Agents and Chemo. 2008, 52:739-741

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

Pfmdr1 and SERCA genotypesIn 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 1999769 mutation reported in French Guyana

as associated with artesunate resistance was not found

Venezuela

Malaria treatment in PeruBackground 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

Copy Number Variation in pfMDR1

• Mefloquine resistance has been linked to duplications of pfmdr1: More copies means more MQ resistance1-4

• 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.2Price et al., 1999, Antimicrob. Agents Chemother.3Pickard et al., 2003, Antimicrob. Agents Chemother.4 Price et al., 2004 Lancet

Copy Number Variation in pfmdr1 from Venezuelan Parasites

Pfmdr1 copy number & Mefloquine

79

11

0

10

20

30

40

50

60

70

80

90

100

Single Multi copy

Per

cen

tag

e

0.000.501.001.502.002.503.003.504.004.505.00

Z15

Z19

Z25

Z33

Z35

Z44

Z62

Z70

Z76

Z77

Z80

Sample Name

Nu

mb

er o

f p

fmd

r1 c

op

ies

Pfmdr1 copy number & Mefloquine

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

Brazil

INSTITUTO EVANDRO CHAGAS-IEC/SVS/MS

MARINETE MARINS POVOA

GISELLE MARIA RACHID VIANA

LABORATÓRIO DE PESQUISAS BÁSICAS EM MALÁRIA

JULY 2008

THE SPREAD OF DRUG RESISTANCE Plasmodium falciparum IN BRAZIL

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

BRAZIL

PARA STATE

Parauapebas

Maraba

Novo Repartimento

Tucurui

Tailandia

AMAPA STATE

RONDONIA STATE

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

Pfcrt ANALYSES

• Sequencing - presence of mutations in pfcrt (K76T)

• Microsatellites – frequency and evolutionary history

PfCRT (39) - 11 microsatellites

04 microssatelites (11Kb): - 5KB

- 4.3 KB1 KB6 KB

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)

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

Thanks!

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

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.

Summary-Brazil

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

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?

Future Directions

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

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 reactivityHRP-2 and HRP-3 deletion:

Total loss of RDT reactivity

A Symposium Proposal During ASTMH Meeting in 2009

Molecular Surveillance for Drug Resistance in South America

AMI participants?

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

Thank you