The Origin and Spread of Drug Resistant Malaria in South America Open Access

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

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Population Biology, Ecology & Evolution
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Genetics
Keyword	South America Plasmodium falciparum
Committee Chair / Thesis Advisor	Zwick, Michael, Emory University
Committee Members	Escalante, Ananias, Arizona State University John, Gimnig, Centers for Disease Control and Prevention Galinski, Mary R, Emory University Venkatachalam, Udhayakumar, Centers for Disease Control and Prevention

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