Transcript Molecular Surveillance for Malaria Drug Resistant Genotypes in South America Kumar V.

Molecular Surveillance for Malaria Drug
Resistant Genotypes in South America
Kumar V. Udhayakumar, Ph.D
Malaria Branch
CDC, Atlanta, USA
Talk Outline
 Training
 Background
 Peru data
 Venezuelan data
 Brazilian data (Marinete Povoa)
 Diagnostics
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
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
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 & pfmdr1 SNPs
pfcrt
72
C
73
V
74
M
75
N
76
K
Ancestral (W.T.)
South America
V
M
N
S
T
South America
C
V
M
N
T
C
V
M
E
T
South America
 pfcrt K76T is critical mutation for CQ resistance
pfmdr1
86 184 1034 1042 1246
Ancestral (W.T.) N
Y
S
N
D
South America
N
F
C
D
Y
 Copy number increase is correlated with MQ
resistance.
DHFR & DHPS Resistance Markers
 For DHFR:
C50R, N51I, C59R, S108N, I164L
 I51,N108,L164
dhfr
 R50,I51,N108
** * * *
5051 59 108 164
 For DHPS:
S436A, A437G, K540E, A581G, A613T
 G437,E540,G581
dhps
*
*
436437
** *
540 581 613
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
pfcrt
 if two chromosomal regions share ancestry,
their haplotypes should appear similar
pfcrt
pfcrt
VS
pfcrt
pfcrt
Study Results
2
3
1
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?
P. falciparum in Peru
Malaria
treatment
 Andes divide
Peru into
2 major
regions
 Coast
in Peru
Central Amazon
Pacific Coast
1999: CQ resistant, SP sensitive
1999-2001: CQ to Artesunate-SP
Iquitos
 Peruvian Amazon
1999: East, CQ and SP resistance
1999-2001: SP to Artesunate-Mefloquine
2000/2002: West,
Western Amazon
CQ resistant, SP sensitive
 What is the molecular pattern of
CQ and SP resistance in
different regions?
Eastern Amazon
Pfcrt Genotypes in Peru
 Coast: 100% CVMNT
genotype
pfcrt
C/E Amazon
CVMNT
& SVMNT
 Western Amazon: 100%
CVMNT genotype
 Central and Eastern
Amazon:
1999
2006
CVMNT: 46% 34%
SVMNT: 54% 66%
Bacon et al, AAC, In Press
Pacific
CVMNT
W. Amazon
CVMNT
Pfdhfr Genotypes in Peru
 Coast: 24% 108N
mutation
dhfr
C/E. Amazon
Triple mutant
dhfr
 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
Pacific
Only 108N
W. Amazon
Only 108N
Pfdhps genotypes in Peru
 Coast: No mutant
genotypes
dhps
C. Amazon
Triple
mutant dhps
 Western Amazon: Silent
dhps mutant 540K
 Central/Eastern Amazon
29% Triple mutant
23% Double mutant
48% Wild type
Pacific
No dhps
mutations
W. Amazon
Silent dhps
mutation
HighlyMalaria
resistanttreatment
triple mutant
dhfr
and
in Peru
dhps genotypes declined after SP removal
 SP was removed from use in Iquitos in 2000
 We compared reported resistant allele prevalence from
1997 with our samples from 2005/2006
0
0
2005
13
1997
2005
2005
2.4
1997
20
1997
40
46.7
40
20
48.9
46.7
2.2
0
0
2005
60
60
dhfr
1997
dhps
78
2005
80
1997
80
2005
100
1997
100
16.2
5.8
0
Single
Double
dhps mutations
Triple
Single (S108N)
Double
dhfr mutations
Zhou et al., Antimicrob. Agents and Chemo. 2008, 52:739-741
Triple
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 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
Venezuela
Background of Drug Resistance in
Malaria treatment in Peru
Venezuela
 CQ banned by National Program of Malaria
Therapeutics in 1986
 SP stopped in 1998, MQ monotherapy and Sifontes
ACT adopted
 Our lab found that both CQ and SP resistant
genotypes fixed in this
population
(McCollum, et al. 2007,
Griffings et al., unpublished)
CopyCopy
Number
Variation
pfmdr1 from
Number
Variation in
in pfMDR1
Venezuelan Parasites
• 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
Pfmdr1 copy number & Mefloquine
100
90
79
80
Percentage
70
60
50
40
30
20
11
10
0
Single
Multi copy
Sample Name
Z80
Z77
Z76
Z70
Z62
Z44
Z35
Z33
Z25
Z19
5.00
4.50
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00
Z15
Number of pfmdr1 copies
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
THE SPREAD OF DRUG RESISTANCE
Plasmodium falciparum IN BRAZIL
MARINETE MARINS POVOA
GISELLE MARIA RACHID VIANA
LABORATÓRIO DE PESQUISAS BÁSICAS EM MALÁRIA
JULY 2008
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
Tailandia
Tucurui
Novo Repartimento
Maraba
Parauapebas
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 histor
PfCRT (39) - 11 microsatellites
04 microssatelites (11Kb):
- 5KB
- 4.3 KB
1 KB
6 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 –
SagtVMNT
StctVMNT (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 reactivity
HRP-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






Nancy Arrospide, INS
Wilmer Marquino
Trent Ruebush
Leopoldo Villegas
Marinete Marin Povoa
Giselle M. Rachid Viana
 USAID
 AMI
 RAVREDA
 Andrea McCollum
 Sean Griffing
 Alexandre Macedo de
Oliveira
 Sankar Sridaran
 Luke Syphard
 Ananias Escalante
 David Bacon and
colleagues
Thank you