The Origin and Spread of Drug Resistant Malaria in South America Public
Griffing, Sean Michael (2010)
Published
Abstract
The goal of this dissertation was to show how malaria control
influenced South American
Plasmodium falciparum population structure. A total of 565
Plasmodium falciparum samples
were obtained from from Brazil (eight sites), Peru (eight sites),
and Venezuela (one site).
Bolivian isolates previously sequenced for some resistant genes
were also included (8 samples).
We sequenced the Plasmodium falciparum chloroquine
resistance gene ( pfcrt), the Plasmodium
falciparum multidrug resistance gene ( pfmdr1),
dehydrofolate reductase ( dhfr, associated with
pyrimethamine resistance) and dihydropteoroate synthase (
dhps associated with sulphadoxine
resistance). We further characterized 56 microsatellites markers
around these genes and 12
neutral microsatellites located on 7 other chromosomes. For
Venezuela, we observed that the
chloroquine (CQ) and sulfadoxine pyrimethamine (SP) resistance were
fixed and linked in
multidrug resistant genotypes. Mefloquine resistance may have
evolved through pfmdr1 copy
number amplification, a first for South America. Tests suggested
the population was
bottlenecked. In Peru, P. falciparum populations were
restricted to five clonal lineages, after
years of low malaria incidence, during malaria epidemics in the
1990s, distinctive in South
America. One clonet was found on the coast and one western Amazon
site, indicating the Andes
were a major gene flow barrier. In the Amazon, there were four
clonets distributed in varying
proportions at different sites. Drug pressure influenced the
selection and expansion of clonal
lineages. Among isolates collected from the Peruvian Central Amazon
during 2006-7, there was
evidence for clonet outcrossing, contrary to our hypothesis that
clonal propagation would
continue. The shift from SP to artesunate combination therapy in
2001 influenced this
breakdown, favoring the emergence of two major hybrid clonets. In
Brazil, most parasites were
moderately CQ and SP resistant in the early 1980s and highly
resistant in the 1990s. We
suggested that human migration within the Brazilian Amazon led to
extensive admixture and
outcrossing between parasite clonal lineages and populations had
bottlenecked. We combined our
molecular data with a historical review of malaria control and
resistance to determine the
relationships between the parasite populations from different
countries and to examine how CQ
and SP resistance may have spread throughout South America.
Table of Contents
TABLE OF CONTENTS
CHAPTER 1 GENERAL
BACKGROUND..................................................................................1
CHAPTER 2 THE EARLY HISTORY OF SOUTH AMERICAN
MALARIA.....................................27
CHAPTER 3 THE HISTORY OF MALARIA IN
PERU...............................................................33
CHAPTER 4 THE HISTORY OF MALARIA IN
VENEZUELA.....................................................57
CHAPTER 5 THE HISTORY OF MALARIA IN
BRAZIL............................................................78
CHAPTER 6 PFMDR1 AMPLIFICATION AND FIXATION OF CHLOROQUINE
RESISTANT
PFCRT ALLELES IN VENEZUELA
.....................................................................................111
CHAPTER 7 SOUTH AMERICAN PLASMODIUM FALCIPARUM: SUCCESSFUL
SURVIVORS
AND RECENT
INTRODUCTIONS......................................................................................143
CHAPTER 8 EVIDENCE FOR RECOMBINATION BETWEEN PLASMODIUM
FALCIPARUM
CLONETS IN THE PERUVIAN AMAZON AFTER A MULTIYEAR
EPIDEMIC............................198
CHAPTER 9 HISTORICAL SHIFTS IN BRAZILIAN P. FALCIPARUM
POPULATION
STRUCTURE AND DRUG RESISTANCE
MARKERS..............................................................227
CHAPTER 10
CONCLUSIONS..........................................................................................257
LIST OF TABLES AND FIGURES
Figure 1.1 The lifecycle of
Plasmodium.............................................................................22
Figure 1.2 The predicted structure of
pfcrt.......................................................................23
Figure 1.3 The predicted structure of
pfmdr1...................................................................24
Figure 1.4 The active site of
dhfr......................................................................................25
Figure 1.5 The predicted structure of
dhps.......................................................................26
Figure 2.1 The distribution of malaria in the New World,
1970........................................32
Figure 3.1 The mosquito species of Peru,
1995...............................................................52
Figure 3.2 Malaria incidence in Peru,
1990-2001.............................................................53
Figure 3.3 Loreto and the city of
Iquitos.........................................................................54
Figure 3.4 Outbreaks of P. falciparum malaria in the Loreto
region in 1993.....................55
Figure 3.5 Initial P. falciparum malaria treatment schemesin
Loreto by district, 1998......56
Figure 4.1 The historical and modern distritubion of malaria in
Venezuela......................76
Figure 4.2 The distribution of malaria in Venezuela,
1979-1981......................................77
Figure 5.1 Portions of Brazil with the highest malaria transmission
in the early 1990s.110
Figure 6.1 The copy number of pfmdr1 in Sifonties,
Venezuela......................................134
Figure 6.2 Variations in He around pfmdr1 and
pfcrt......................................................135
Figure 6.3 Pairwise linkage disequilibrium between microsatellite
loci on different
chromosomes.................................................................................................................136
Table 6.1. pfmdr1 and pfcrt
methods..............................................................................137
Table 6.2. Frequency of pfcrt and pfmdr1 genotypes
and number of alleles (A) and expected
heterozygosity (He) per microsatellite
locus...................................................................138
Supplementary Table 6.3. List of PCR primers used for
microsatellite amplification around pfcrt
and
pfmdr1.....................................................................................................................139
Supplementary Table 6.4. Microsatellite haplotypes for
pfmdr1 and pfcrt.......................141
Supplementary Table 6.5 pfcrt
haplotypes......................................................................142
Figure 7.1. Collection
Sites.............................................................................................172
Figure 7.2. Clonets and Collection
Sites.........................................................................173
Figure 7.3. Hypothesized Spread of Clonets Across
Peru...............................................174
Figure 7.4. Network diagram for pfcrt close microsatellite
marker...................................175
Figure 7.5. Network diagram for pfmdr1 close microsatellite
markers.............................176
Figure 7.6. Network diagram for dhfr close microsatellite
markers..................................177
Figure 7.7. Network diagram for dhps close microsatellite
markers................................178
Table 7.1. Pairwise Fst by Collection
Site........................................................................179
Table 7.2. AMOVA
Results...............................................................................................180
Table 7.3. Pairwise Fst by
Clonet....................................................................................181
Table 7.4. Pairwise Linkage Disequilibrium in
Clonets.....................................................182
Table 7.5. Multilocus Linkage Disequlibrium and
Clonets................................................183
Table 7.6. Bottleneck results for 11
markers...................................................................184
Table 7.7. Common dhfr genotypes and microsatellite markers
from study isolates.......185
Table 7.8. Common dhps genotypes and microsatellite markers
from study isolates......186
Table 7.9. Common pfcrt genotypes and microsatellite markers
from study isolates......188
Table 7.10. Common pfmdr1 genotypes and microsatellite
markers from study isolates.190
Table 7.11. Microsatellite Heterozygosity around
dhfr.....................................................192
Table 7.12. Microsatellite Heterozygosity around
dhps....................................................193
Table 7.13. Microsatellite Heterozygosity around
pfcrt....................................................195
Table 7.14. Heterozygosity around
pfmdr1......................................................................196
Figure 8.1: A network diagram of Iquitos in comparison to
earlier clonets from neutral
markers...........................................................................................................................217
Figure 8.2: A network diagram of Iquitos in comparison to earlier
clonets from neutral markers
and pseudo
markers........................................................................................................218
Figure 8.3: Microsatellite allele frequency distributions for
clonets A and C in Padre Cocha ...219
Figure 8.4: Allele frequency distributions for Padre Cocha and
Iquitos............................220
Table 8.1: Drug resistance allele haplotypes seen in Iquitos in
2006-2007.....................221
Table 8.2: He around
dhfr................................................................................................222
Table 8.3: He around
dhps...............................................................................................223
Table 8.4: He around
pfcrt...............................................................................................224
Table 8.5: He around
pfmdr1...........................................................................................225
Figure 9.1 dhfr triple mutant He changes over time
(50/51/108).....................................238
Figure 9.2 dhfr triple mutant He changes over time
(51/108/164)...................................239
Figure 9.3 He around dhps from multiple periods across
Brazil........................................240
Figure 9.4 He around pfcrt from multiple periods across
Brazil.........................................241
Figure 9.5 He around pfmdr1 from multiple periods across
Brazil.....................................242
Figure 9.6 Network diagram of Brazilian data from the
1980s.........................................243
Figure 9.7 Network diagram of Brazilian data from the late
1990s..................................244
Figure 9.8 Network diagram of all Brazilian data regardless of
date................................245
Table 9.1 Samples used in this
study...............................................................................246
Table 9.2 Temporal and geographic distribution of dhfr
alleles in Brazil...........................247
Table 9.3 Triple mutant dhfr
51/108/16............................................................................248
Table 9.4 Triple mutant dhfr
51/108.................................................................................249
Table 9.5 Triple mutant dhfr
50/51/108............................................................................250
Table 9.6 Pairwise FST of different sites in
Brazil..............................................................251
Table 9.7 Tests for
bottlenecks........................................................................................252
Figure 10.1 Network diagram of Venezuelan isolates......................................................267
Figure 10.2 Network diagram of all dissertation data that falls between 1998 and 2003...268
Figure 10.3 Network diagram of historical data from Brazil with
all data outside of the
Country............................................................................................................................270
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