The Spread and Mechanisms of CQ and SP Resistance
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Distribution Agreement In presenting this thesis or dissertation as a partial fulfillment of the requirements for an advanced degree from Emory University, I hereby grant to Emory University and its agents the non- exclusive license to archive, make accessible, and display my thesis or dissertation in whole or in part in all forms of media, now or hereafter known, including display on the world wide web. I understand that I may select some access restrictions as part of the online submission of this thesis or dissertation. I retain all ownership rights to the copyright of the thesis or dissertation. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. Signature: Sean Michael Griffing_ __________________ Date The Origin and Spread of Drug Resistant Malaria in South America By Sean Michael Griffing Doctor of Philosophy Graduate Division of Biological and Biomedical Sciences Population Biology, Ecology, and Evolution _________________________________________ [Advisor‘s signature] Venkatachalam Udhayakumar Advisor _________________________________________ [Member‘s signature] Michael E. Zwick Co-mentor _________________________________________ [Member‘s signature] Ananias A. Escalante Committee Member _________________________________________ [Member‘s signature] Mary Galinski Committee Member _________________________________________ [Member‘s signature] John Gimnig Committee Member Accepted: _________________________________________ Lisa A. Tedesco, Ph.D. Dean of the James T. Laney School of Graduate Studies ___________________ Date The Origin and Spread of Drug Resistant Malaria in South America By Sean M. Griffing B.A., Oberlin College, 1999 Certificate in Science Journalism, University of California, Santa Cruz, 2002 Advisor: Udhayakumar Venkatachalam, Ph.D., Madurai-Kamaraj University, 1986 An abstract of A dissertation submitted to the Faculty of the James T. Laney School of Graduate Studies of Emory University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Graduate Division of Biological and Biomedical Sciences in Population Biology, Ecology, and Evolution 2010 Abstract The Origin and Spread of Drug Resistant Malaria in South America By Sean Michael Griffing 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. The Origin and Spread of Drug Resistant Malaria in South America By Sean M. Griffing B.A., Oberlin College, 1999 Certificate in Science Journalism, University of California, Santa Cruz, 2002 Advisor: Udhayakumar Venkatachalam, Ph.D., Madurai-Kamaraj University, 1986 A dissertation submitted to the Faculty of the James T. Laney School of Graduate Studies of Emory University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Graduate Division of Biological and Biomedical Sciences in Population Biology, Ecology, and Evolution 2010 Acknowledgments I give my sincere thanks to my advisor, Dr. Venkatachalam Udhayakumar, who couldn‘t have been a more helpful, supportive, and patient mentor. I thank my dissertation committee for generously contributed their time and effort to my studies. I thank our international collaborators in the Amazon Malaria Initiative; without the help and support of Dr. Nancy Arrospide, Dr. David Bacon, Dr. Marinete Marins Povoa and Dr Giselle M. Rachid Viana, Dr. Wilmer Marquiño Quezada, and Dr. Leopoldo Villegas, my work would have been impossible. I thank Dr. Christopher Plowe for contributing Bolivian samples. I thank the the staff at the US Medical Research Center Detachment in Peru including Stella Chenet, Carmen Lucas, Carola Salas, Valeria Soberon, Lorena Tapia, and Pablo Tsukayama. I thank Emory University, the National Science Foundation, and the Atlanta Research and Education Foundation for their support. At the CDC, I thank Dr. Md Tauqeer Alam, Ira Goldman, Dr. Tonya Mixson-Hayden, Dr. Andrea M. McCollum, Amanda Poe, Sankar Sridaran, Luke Syphard, Dr. Sumiti Vinayak, Cecilia Nelson, and Pauline Abdallah for their advice, assistance, and support. I thank the faculty and staff of University and the Program in Population Biology, Ecology, and Evolution for allowing for me to pursue my PhD. I appreciate the emotional support and intellectual input of my fellow students, particularly Omar Cornejo, Karoun Bagamian, Lynn Huynh, and Kristin Harper. I thank the scientists and educators that encouraged my studies including Dr. Yolanda Cruz, Dr. Ronald DiCenzo, Dr. William Fuchsman, and Janene Brunson, Dr. Alan Masters, and Dr. Peter Marra (who was my first scientific mentor and can‘t be thanked enough). I hope that others that I have neglected to mention here will still understand my appreciation. Finally, I thank my mother and father for contributions to long to list here. While my extended family has also contributed to my upbringing, I would like to particularly acknowledge my great-grandfather, Diógenes Escalante Ugarte, who serves as an example of public service and international collaboration for the betterment of humanity. TABLE OF CONTENTS CHAPTER 1 GENERAL BACKGROUND……………...……………………………….……1 2 THE EARLY HISTORY OF SOUTH AMERICAN MALARIA.………………27 3 THE HISTORY OF MALARIA IN PERU………………..……………….……33 4 THE HISTORY OF MALARIA IN VENEZUELA………………………….….57 5 THE HISTORY OF MALARIA IN BRAZIL ………………..………………….78 6 PFMDR1 AMPLIFICATION AND FIXATION OF CHLOROQUINE RESISTANT PFCRT ALLELES IN VENEZUELA ………………………………………111 7 SOUTH AMERICAN PLASMODIUM FALCIPARUM: SUCCESSFUL SURVIVORS AND RECENT INTRODUCTIONS………………………………………………………….143 8 EVIDENCE FOR RECOMBINATION BETWEEN PLASMODIUM FALCIPARUM CLONETS IN THE PERUVIAN AMAZON AFTER A MULTIYEAR EPIDEMIC ……………………………...………………………………………………..198 9 HISTORICAL SHIFTS IN BRAZILIAN P. FALCIPARUM POPULATION STRUCTURE AND DRUG RESISTANCE MARKERS.…………………………………………...……………………..227 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