Presented by The New York Academy of Sciences and The Johns Hopkins Bloomberg School of Public Health
The Forever War: Malaria Against the World
Posted March 08, 2012
In spite of the greater-than-a-century-long battle scientists and clinicians have waged to control or eradicate malaria, this disease remains a leading cause of illness and death in underdeveloped regions of the world.
This battle against malaria—its theaters, its successes, its failures, and its future—was the focus of The Forever War: Malaria versus the World symposium at the New York Academy of Sciences on November 16, 2011. The conference was presented by the Johns Hopkins Bloomberg School of Public Health (JHBSPH) and the New York Academy of Sciences in honor of the 10th anniversary of the founding of the Johns Hopkins Malaria Research Institute (JHMRI). The symposium featured presentations from the world's leading physicians, public health experts, and scientists who combat malaria head-on in the disease's most endemic areas or on a molecular level in laboratories all over the globe. Mayor Michael Bloomberg was also in attendance not only to congratulate the Malaria Research Institute on its anniversary, but also to commend the Institute's members for their efforts to combat this devastating disease.
During the symposium, attendees learned about the Plasmodium parasite—the organism that causes malaria—and about its complex life cycle. Speakers delved into a discussion of the progress of efforts to thwart the disease in the lab, on the ground, and in policy. On the lab front, speakers revealed the latest biological discoveries aimed to unravel the genetic basis of drug resistance, the biochemical processes that result in parasite infection in the human host, and the mosquito's immune response to the parasite. On the clinical front, attendees heard about the latest progress to carry out vector and disease control strategies on the ground and about the fight against pockets of disease resurgence. From a public health perspective, presenters looked at the current global fight through an historical lens, the policy-driven progress in controlling the disease and the on-going battle to secure enough support—financial and otherwise—to fund available vector- and disease-control strategies or to facilitate the development of new ones.
Use the tabs above to find a meeting report and multimedia from this event.
Presentations available from:
Peter Agre, MD (Johns Hopkins Malaria Research Institute)
Mayor Michael Bloomberg (The City of New York)
George Dimopoulos, PhD, MBA (Johns Hopkins Bloomberg School of Public Health)
Diane E. Griffin, MD, PhD (Johns Hopkins Bloomberg School of Public Health)
Michael J. Klag, MD, MPH (Johns Hopkins Bloomberg School of Public Health)
Sungano Mharakurwa, PhD, MSc (Johns Hopkins Center for Global Health)
Jeffrey D. Sachs, PhD (The Earth Institute, Columbia University)
Photini Sinnis, MD (New York University Langone Medical Center)
David Smith, PhD (University of Florida)
Alfred Sommer, MD, MHS (Johns Hopkins Bloomberg School of Public Health)
Phil E. Thuma, MD (Malaria Institute at Macha)
- 00:011. Introduction; Plasmodium in the mosquito
- 02:292. Targeting plasmodium with the mosquito's immune system
- 05:583. Making a genetically modified plasmodium resistant mosquito
- 11:064. Introducing the modified mosquito in nature
- 14:565. Using gut bacteria to block malaria
- 21:106. Other plasmodium inhibiting bacteria; Chromobacterium
- 28:157. The research program; Acknowledgements and conclusio
Websites, Books & Journal Articles
Sachs J, Malaney P. The economic and social burden of malaria. Nature 2002;415(6872):680-5.
Image of malaria-causing parasite available from the Centers for Disease Control and Prevention image library and can be found at: http://www.dpd.cdc.gov/dpdx/default.htm (ID # 3405).
For more information of the life cycle of the Plasmodium parasite as well as malaria signs, symptoms, treatments, and more, see the Centers for Disease Control and Prevention malaria website, the World Health Organization's malaria page, and the Roll Back Malaria Partnership.
Cirimotich CM, Dong Y, Clayton AM, et al. Natural microbe-mediated refractoriness to Plasmodium infection in Anopheles gambiae. Science 2011;332(6031):855-8.
Dong Y, Das S, Cirimotich CM, et al. Engineered Anopheles immunity to Plasmodium infection. PLoS Pathogens 2011;7(12):e1002458.
Garver LS, Dong Y, Dimopoulos G. Caspar controls resistance to Plasmodium falciparum in diverse anopheline species. PLoS Pathog. 2009;5(3):e1000335.
Sinkins SP, Gould F. Gene drive systems for insect disease vectors. Nat. Rev. Genet. 2006;7(6):427-435.
Basco LK, Ramiliarisoa O, Le Bras J. In vitro activity of pyrimethamine, cycloguanil, and other antimalarial drugs against African isolates and clones of Plasmodium falciparum. Am. J. Trop. Med. Hyg. 1994;50(2):193-9.
Mharakurwa S, Kumwenda T, Mkulama MAP, et al. Malaria antifolate resistance with contrasting Plasmodium falciparum dihydrofolate reductase (DHFR) polymorphisms in humans and Anopheles mosquitoes. Proc. Natl. Acad. Sci. USA 2011;108(46):18796-801.
Schweitzer BI, Dicker AP, Bertino JR. Dihydrofolate reductase as a therapeutic target. FASEB J. 1990;4(8):2441-52.
Jeffrey D. Sachs
Heffernan JM, Smith RJ, Wahl LM. Perspectives on the basic reproductive ratio. J. R. Soc. Interface 2005;2(4):281-293.
Sachs JD, Chambers RG. The new global war on malaria. In: Realizing the Right to Health Volume 3. Swiss Human Rights Book. Zurich: Rüffer & Rub; 2009.
Teklehaimanot A, McCord GC, Sachs JD. Scaling up malaria control in Africa: an economic and epidemiological assessment. Am. J. Trop. Med. Hyg. 2007;77(6 Suppl):138-144.
White Johansson E, Newby H, Carvajal L, et al. A decade of partnerships and results. Geneva: World Health Organization; 2011.
Agnandji ST, Lell B, Soulanoudjingar SS, et al. First results of phase 3 trial of RTS,S/AS01 malaria vaccine in African children. N. Engl. J. Med. 2011;365(20):1863-1875.
Coppi A, Pinzon-Ortiz C, Hutter C, et al. The Plasmodium circumsporozoite protein is proteolytically processed during cell invasion. J. Exp. Med. 2005;201(1):27-33.
Coppi A, Tewari R, Bishop JR, et al. Heparan sulfate proteoglycans provide a signal to Plasmodium sporozoites to stop migrating and productively invade host cells. Cell Host & Microbe 2007;2(5):316-327.
Coppi A, Natarajan R, Pradel G, et al. The malaria circumsporozoite protein has two functional domains, each with distinct roles as sporozoites journey from mosquito to mammalian host. J. Exp. Med. 2011;208(2):341-356.
Kappe SHI, Buscaglia CA, Bergman LW, et al. Apicomplexan gliding motility and host cell invasion: overhauling the motor model. Trends Parasitol. 2004;20(1):13-16.
Mwakingwe A, Ting LM, Hochman S, et al. Noninvasive real-time monitoring of liver-stage development of bioluminescent Plasmodium parasites. J. Infect. Dis. 2009;200(9):1470-1478.
Plassmeyer ML, Reiter K, Shimp RL Jr, et al. Structure of the Plasmodium falciparum circumsporozoite protein, a leading malaria vaccine candidate. J. Biol. Chem. 2009;284(39):26951-26963.
Sinnis P, Coppi A. A long and winding road: the Plasmodium sporozoite's journey in the mammalian host. Parasitol Int. 2007;56(3):171-178.
Sultan AA, Thathy V, Frevert U, et al. TRAP is necessary for gliding motility and infectivity of Plasmodium sporozoites. Cell 1997;90(3):511-522.
Vanderberg JP, Frevert U. Intravital microscopy demonstrating antibody-mediated immobilisation of Plasmodium berghei sporozoites injected into skin by mosquitoes. Int. J. Parasitol. 2004;34(9):991-996.
David L. Smith
Belani A, Ali A, Kaneko A, et al. Malaria Elimination in Zanzibar: A Feasibility Assessment, Univ. Cal. San Fran. 2009.
Doull JA, Kramer M. The First World Health Assembly. Public Health Rep. 1948, Oct 22;63(43):1379-1403.
Gething PW, Smith DL, Patil AP, et al. Climate change and the global malaria recession. Nature 2010;465(7296):342-5.
Livadas GA, Georgopoulos G. Development of resistance to DDT by Anopheles sacharovi in Greece. Bull. World Health Organ. 1953;8(4):497-511.
Nájera JA. Malaria control: achievements, problems and strategies. Parassitologia 2001;43(1-2):1-89.
Needham CA, Canning R. Global Disease Eradication: the Race for the Last Child. 2003, ASM Press, Washington, DC.
Report on the malaria conference in Equatorial Africa, Kampala, Uganda, 27November – 9 December 1950. World Health Organization Tech. Rep. Ser. 1951 Apr;38:1-72.
Smith DL, Cohen JM, Moonen B, et al. Infectious disease. Solving the Sisyphean problem of malaria in Zanzibar. Science 2011;332(6036):1384-1385.
Tatem AJ, Smith DL, Gething PW, et al. Ranking of elimination feasibility between malaria-endemic countries. Lancet 2010;376(9752):1579-91.
Philip E. Thuma
Kent RJ, Thuma PE, Mharakurwa S, Norris DE. Seasonality, blood feeding behavior, and transmission of Plasmodium falciparum by Anopheles arabiensis after an extended drought in southern Zambia. Am. J. Trop. Med. Hyg. 2007;76(2):267-274.
Mharakurwa S, Thuma PE, Norris DE, et al. Malaria epidemiology and control in Southern Africa. Acta. Trop. 2011.
Moss WJ, Norris DE, Mharakurwa S, et al. Challenges and prospects for malaria elimination in the Southern Africa region. Acta. Trop. 2011.
Sutcliffe CG, Kobayashi T, Hamapumbu H, et al. Changing individual-level risk factors for malaria with declining transmission in southern Zambia: a cross-sectional study. Malar. J. 2011;10:324.
Sutcliffe CG, Kobayashi T, Hamapumbu H, et al. Reduced risk of malaria parasitemia following household screening and treatment: a cross-sectional and longitudinal cohort study. PLoS ONE 2012;7(2):e31396.
Thuma PE, van Dijk J, Bucala R, et al. Distinct clinical and immunologic profiles in severe malarial anemia and cerebral malaria in Zambia. J. Infect. Dis. 2011;203(2):211-219.
Jeffrey D. Sachs, PhD
The Earth Institute, Columbia University
e-mail | website | publications
Jeffrey D. Sachs is the Director of The Earth Institute, Quetelet Professor of Sustainable Development, and Professor of Health Policy and Management at Columbia University. He is Special Advisor to United Nations Secretary-General Ban Ki-moon. From 2002 to 2006, he was Director of the UN Millennium Project and Special Advisor to United Nations Secretary-General Kofi Annan on the Millennium Development Goals. Sachs is also President and Co-Founder of Millennium Promise Alliance. For more than 20 years Sachs has been in the forefront of the challenges of economic development, poverty alleviation, and enlightened globalization, promoting policies to help all parts of the world to benefit from expanding economic opportunities and wellbeing. He is author of hundreds of scholarly articles and many books, including the New York Times bestsellers Common Wealth: Economics for a Crowded Planet (Penguin, 2008) and The End of Poverty (Penguin, 2005).
Peter Agre, MD
Johns Hopkins Malaria Research Institute
e-mail | website | publications
A native Minnesotan, Peter Agre studied chemistry at Augsburg College (BA 1970) and medicine at Johns Hopkins (MD 1974). Following Internal Medicine Residency at Case Western Reserve University Hospitals of Cleveland and Hematology–Oncology Fellowship at the University of North Carolina at Chapel Hill, Agre joined the faculty at the Johns Hopkins School of Medicine where his laboratory became widely recognized for discovering the aquaporins, a family of water channel proteins found throughout nature and responsible for numerous physiological processes as well as multiple clinical disorders. Following a term as Vice Chancellor at Duke Medical Center, Agre joined the Johns Hopkins Bloomberg School of Public Health in 2008, where he is University Professor and Director of the Malaria Research Institute and Program Director of the NIH International Center of Excellence in Malaria Research for Zambia and Zimbabwe.
Agre shared the 2003 Nobel Prize in Chemistry with Roderick MacKinnon "for discoveries concerning channels in cell membranes." Agre is a member of the National Academy of Sciences and the Institute of Medicine; he is past-Chair and member of the Committee on Human Rights of the National Academies. From 2009–11, Agre served as President and Chair of the Board of Directors of the American Association for the Advancement of Sciences and led scientific diplomacy visits to Cuba, Democratic Republic of Korea (North Korea), and Myanmar (Burma).
The City of New York
Michael R. Bloomberg is the 108th Mayor of the City of New York. Born in Boston and raised in a middle class home in Medford, Massachusetts, Michael Bloomberg attended Johns Hopkins University. After college, he went on to receive an MBA from Harvard Business School. In 1966 he was hired by a Wall Street firm, Salomon Brothers, for an entry-level job. He quickly rose through the ranks at Salomon, overseeing equity trading and sales before heading up the firm's information systems. When Salomon was acquired in 1981, he launched a small startup company called Bloomberg LP. Today, Bloomberg LP is a global media company that has over 310,000 subscribers to its financial news and information service. As his company grew, Michael Bloomberg started directing more of his attention to philanthropy, donating his time and resources to many different causes. He has sat on the boards of numerous charitable, cultural, and educational institutions, including Johns Hopkins University, where, as chairman of the board, he helped build the Bloomberg School of Public Health into one of the world's leading institutions of public health research and training. In 2001 he ran for mayor of the City of New York and, in a major upset, won the election. Since then Mayor Bloomberg has focused his efforts on reforming public education, spurring economic growth and launching new programs that encourage entrepreneurship, combat poverty, and help people acquire the skills they need to build careers. His passion for public health has led to ambitious new health strategies that have become national models, including a ban on smoking in all indoor workplaces, as well as at parks and beaches. He has also created a far-reaching plan to fight climate change and promote sustainable development on an unprecedented scale.
George Dimopoulos, PhD, MBA
Johns Hopkins Bloomberg School of Public Health
e-mail | website | publications
George Dimopoulos received his BSc in microbiology at Stockholm University and his PhD from the University of Crete where he studied population biology and gene expression of malaria-transmitting mosquitoes. He was a Marie Curie postdoctoral fellow with Prof Fotis Kafatos at the European Molecular Biology Laboratory where he studied mosquito immunity to Plasmodium. He became a Senior Lecturer at Imperial College London in 2001 and was recruited as an Assistant Professor at the Johns Hopkins University Malaria Research Institute in 2003. Dimopoulos also received his MBA in 2008 from Johns Hopkins Carey Business School. Dimopoulos's research focuses on the innate immune system of mosquitoes that transmit malaria parasites and dengue virus. He is a recipient of the Ellison Medical Foundation Young Investigator Award and an editorial board member of a variety of scientific journals.
Diane E. Griffin, MD, PhD
Johns Hopkins Bloomberg School of Public Health
e-mail | website | publications
Diane Griffin earned her BA at Augustana College in Rock Island, Ill and her MD and PhD at Stanford University School of Medicine. She received clinical training in Internal Medicine at Stanford University Hospital and postdoctoral training in infectious diseases and virology at Johns Hopkins University School of Medicine. Her research interests are in the area of pathogenesis of viral diseases with a particular focus on measles and alphavirus encephalitis. These studies address issues related to virulence and the role of immune responses in protection from infection and in clearance of infection. She is currently Distinguished University Service Professor and Alfred and Jill Sommer Chair of the W. Harry Feinstone Department of Molecular Microbiology and Immunology at the Johns Hopkins Bloomberg School of Public Health. She is an editor for Field's Virology, has published more than 300 scientific papers, serves on several editorial and advisory boards and has been President of the American Society for Microbiology, the American Society for Virology and the Association of Microbiology and Immunology Chairs. She is a fellow of the American Association for the Advancement of Science and member of the National Academy of Sciences, American Academy of Microbiology and the Institute of Medicine.
Michael J. Klag, MD, MPH
Johns Hopkins Bloomberg School of Public Health
website | publications
Michael Klag is Dean of the Johns Hopkins Bloomberg School of Public Health, the oldest and largest independent graduate school of public health in the world. He is chair of the Association of Schools of Public Health and chair of the NIH Advisory Board on Clinical Research. Klag is a world-renowned kidney disease epidemiologist whose scientific contributions have been in the prevention and epidemiology of kidney disease, hypertension, and cardiovascular disease. He was one of the earliest investigators to apply epidemiologic methods to the study of kidney disease. He directs one of the longest running longitudinal studies in existence, the Precursors Study, which began in 1946. Klag is the author of over 200 publications and was the Editor-in-Chief of the Johns Hopkins Family Health Book. He earned his medical degree at the University of Pennsylvania and his MPH degree from the Johns Hopkins School of Hygiene and Public Health. For eight years, he was Director of the Division of General Internal Medicine and was the first Vice Dean for Clinical Investigation at the Johns Hopkins School of Medicine.
Sungano Mharakurwa, PhD, MSc
Johns Hopkins Center for Global Health
e-mail | website | publications
Sungano Mharakurwa earned his BSc from the University of Zimbabwe in 1991. He was trained for MSc. (medical parasitology) at the London School of Hygiene and Tropical Medicine, awarded in 1993. Mharakurwa undertook his PhD studies at Oxford University, completing in 2001. He was a Beit Memorial postdoctoral fellow at the Peter Medawar Building for Pathogen Research, Oxford University, under leadership of geneticist Karen Day. Mharakurwa joined the Johns Hopkins Malaria Research Institute for posting at the Malaria Institute at Macha field research station during its inception in November 2003. He is currently Malaria Institute at Macha Scientific Director and Johns Hopkins Bloomberg School of Public Health Senior Research Associate. Mharakurwa and others were the first to show proof of concept for the detection of malaria infection using human saliva samples in lieu of blood. His research interests include malaria non-invasive diagnosis and strategies for containment of drug resistance.
Photini Sinnis, MD
New York University Langone Medical Center
e-mail | website | publications
Photini Sinnis received a BA from Swarthmore College and attended medical school at Dartmouth. Following this she did postdoctoral training in the laboratory of Victor Nussenzweig at New York University where she studied the molecular events leading to hepatocyte recognition by malaria sporozoites. She joined the faculty at NYU School of Medicine where she continued her work on the pre-erythrocytic stages of the malaria parasite. This Fall, Sinnis relocated to the Johns Hopkins School of Public Health where she is an Associate Professor. She serves on the editorial boards of PLoS Neglected Tropical Diseases and PLoS ONE.
David Smith, PhD
University of Florida
e-mail | website | publications
David L. Smith earned BS and MS degrees in mathematics from Brigham Young University in Provo, Utah where he was also elected President of the Honors Student Council. He attended graduate school at Princeton University where he earned his MA and PhD in Ecology and Evolutionary Biology working with Simon A. Levin. He is currently Professor of the Department of Epidemiology (JHBSPH), member of the JHMRI, and Senior Fellow and Associate Director for Research at the Center for Disease Dynamics, Economics & Policy. He has worked extensively on the epidemiology and control of malaria, strategic planning for malaria elimination, and the evolution of resistance to antimalarial drugs and antibiotics, and he has also published on the spatio-temporal dynamics or bioeconomics of human and avian flu, cholera, rabies, hospital-acquired infections, agricultural antibiotic use, and MRSA. Smith is currently a member of the Malaria Atlas Project (MAP), the Malaria Elimination Group (MEG), the Malaria Eradication Research Agenda (MalERA), Extending the Cure (ETC), the Global Antibiotic Resistance Partnership (GARP), and The Human Mobility Mapping Project (THUMMP). He recently collaborated with the Zanzibar Minister of Health and the Zanzibar Malaria Control Progamme to write Malaria Elimination in Zanzibar, a Feasibility Assessment. Methodology from the report was recently adopted by the Global Malaria Programme at the W.H.O. He serves on the editorial boards of Advances in Parasitology and Malaria Journal.
Alfred Sommer, MD, MHS
Johns Hopkins Bloomberg School of Public Health
e-mail | website | publications
Alfred Sommer is a Gilman Scholar and University Distinguished Service Professor at Johns Hopkins University; Johns Hopkins Professor of Epidemiology, Ophthalmology, and International Health; and Dean Emeritus of the Bloomberg School of Public Health. He received his MD from Harvard Medical School (1967) and his Master of Health Science in Epidemiology from the Johns Hopkins School of Public Health (1973). He has published 5 books and over 300 scientific articles; has received numerous awards including the Albert Lasker Award for Clinical Research, the Warren Alpert Foundation Prize, and the Duke Elder International Gold Medal for Contributions to Ophthalmology; has delivered over 30 named lectureships, including the Jackson Memorial Lecture (American Academy of Ophthalmology), Duke Elder Oration (Royal College of Ophthalmologists), De Schweinitz Lecture (College of Physicians, Philadelphia), Dohlman Lecture (Harvard Medical School), Doyne Lecture (Oxford Ophthalmologic Congress), and the Kimura Lecture (University of California, San Francisco), among others; and is a member of both the National Academy of Sciences and its Institute of Medicine. His current research interests include child survival and blindness prevention strategies, micronutrient interventions, and the interface between public health and clinical medicine.
Philip E. Thuma, MD
Malaria Institute at Macha
e-mail | website | publications
Phil Thuma completed his undergraduate degree in Chemistry at Messiah College, Grantham, Pennsylvania and his medical degree at Temple University Medical School in Philadelphia. He subsequently completed a pediatric residency at Johns Hopkins Hospital, including an additional year as Chief Resident in Pediatrics. Other than a seven year period as a faculty member at Penn State University Hershey Medical Center in the 90's, he has spent most of his life and medical career working in rural Zambia as a medical doctor and researcher, concentrating on malaria as it presents in children. This work has led to over 45 publications in peer reviewed journals. In 2003 he began collaborating with the Johns Hopkins Malaria Research Institute to develop a malaria research field site at Macha Hospital in southern Zambia, and continues to work with the Johns Hopkins Bloomberg School of Public Health as a Senior Research Associate. Over the last 8 years these efforts have witnessed a marked decrease in malaria prevalence in the area around the hospital. Thuma is active at the national level in Zambia serving on various committees, including the National Health Ethics Research Committee.
Malaria afflicts 10% of the world's population and is responsible for the deaths of 1 million people, 85% of whom are children under 5 years of the age, every year. That means that malaria claims the lives of 2,000 children a day (Sachs J and Melaney P. Nature, 2002).
In humans, malaria is caused by infection by the parasitic species of the genus Plasmodium. These parasites include P. vivax, P. malariae, P. ovale, and P. falciparum. The last, P. falciparum, causes the most lethal form of the disease. The parasite's vector—an organism that transmits the disease—is a female Anopheles mosquito. The mosquito becomes infected with the parasite when it takes a blood meal from an infected human host, only to transmit the parasite when it next takes a blood meal from an uninfected human.
On a cellular level the parasite has a complex life cycle and exists in different stages in both the vector mosquito and in the infected human. The ability of the parasite to evade detection during stages of its life cycle has further complicated treatment protocols. However, as many presenters discovered, the complex life cycle has also offered a plethora of targets where the parasite is vulnerable to attack.
Plasmodium life cycle in the mosquito (Sporogonic cycle)
When an uninfected mosquito bites an infected human the mosquito ingests, into its midgut, red blood cells (erythrocytes) that harbor the Plasmodium parasites (see C in above figure). The parasites are in the gametocyte stage—a stage of the life cycle where they exist as haploid germ cells that develop into mature, male or female gametes. A complex development and fertilization process occurs, and the fertilized macrogametocyte develops into an ookinete (C10).
Further development occurs as the ookinete crosses the mosquito's midgut region and enters the basal lamina, where it develops into an oocyst. At this stage the mosquito mounts an immune response, allowing it to transmit but not be adversely affected by the parasite. Genetic recombination can occur at this stage in the parasite's development. The mosquitos' immune response was the focus of the presentation by George Dimopolous, Director of the Parasitology Core Faculty of the Bloomberg School of Public Health, while the importance of genetic recombination in the parasite and the evolution of parasitic drug resistance was a focus of the presentation by Sungano Mharakurwa, scientific director of the Malaria Institute at Macha, Zambia.
After the parasite, now an oocyst, has left the mosquito's gut, sporogony occurs. Sporogony is a 1-2 week process (approximately the duration of the mosquito's lifetime) in which the oocyst grows, matures, and forms sporozoites. The sporozoite is the form of the parasite that is transmitted from the mosquito to the human host. Once released from the oocyst sporozoites migrate to the salivary glands of the mosquito, and from there are injected into the skin (dermis) of any human from which the mosquito takes a blood meal (C10 to C1).
Plasmodium life cycle in the human (Erythrocyte cycle)
In the human the sporozoites leave the dermis, enter the blood circulation, and travel to the liver. This migration process was the focus of the presentation by Photini Sinnis, a physician-scientist at New York University. The sporozoites then enter and infect the liver cells or hepatocytes (see A2). The sporozoite in the hepatocyte differentiates into a schizont, a sort of receptacle that contains many merozoites—the form of the parasite that infects red blood cells (A3–A4). Some Plasmodium (P. vivax and P. ovale) species do not produce merozoites, but rather produce hypnozoites, a sort-of pre-merozoite that allows the disease to lay dormant for months to a few years. Once merozoites are released they enter the blood stream and infect red blood cells (RBCs) (B5), which starts the second stage of the parasite's life cycle, called the "blood or erythrocyte stage." This is the stage during which the clinical symptoms of malaria infection become apparent. Once a merozoite is released, it can either amplify itself to release more merozoites by infecting other RBCs (see B5–B6), or it can differentiate into male and female gametocytes (B7). These gametocytes can then infect a mosquito, thus propagating the disease.
Research at the Johns Hopkins Malaria Research Institute uses a multi-pronged approach to combat the disease. In the laboratory scientists are targeting the parasite at every step in its life cycle to find new treatments. On the clinical front physicians are fighting the disease by treating symptomatic and asymptomatic disease carriers. Successful clinical treatment protocols such as the "Macha model," based on the successes of a clinic in Macha, Zambia, were presented as ways to combat the disease on a local level. Presenters also addressed pockets of resurgence and the economic constraints on effective intervention.
Ellis Rubinstein, The New York Academy of Sciences
Michael J. Klag, MD, MPH, The Johns Hopkins Bloomberg School of Public Health
Michael R. Bloomberg, Honorable Mayor of New York City
Diane Griffin, MD, Ph.D., The Johns Hopkins Bloomberg School of Public Health
Alfred Sommer, MD, MHS, The Johns Hopkins Bloomberg School of Public Health
Peter Agre, MD, The Malaria Research Institute at the Johns Hopkins Bloomberg School of Public Health
Philip E. Thuma, MD, Malaria Institute at Macha, Zambia
- A screen of children in local villages in Zambia revealed a large subset of children had malaria, but did not show symptoms.
- Treating the asymptomatic malaria carriers as well as the symptomatic carriers can reduce the spread of malaria.
- The "Macha model" for combating malaria locally may be extended to other parts of sub-Saharan Africa.
The Motivation for change: Founding the Malaria Research Institute
Ellis Rubinstein, President and CEO of the New York Academy of Sciences, began the symposium by praising the "marriage" of Johns Hopkins School of Public Health (JHSPH) and the Bloomberg philanthropic organization, and by noting the achievements of their offspring, as it were, the Johns Hopkins Malaria Research Institute (JHMRI). He highlighted the strength of multi-lateral partnerships created to address global health challenges.
An introduction from Michael J. Klag, Dean of the Johns Hopkins Bloomberg School of Public Health followed. He commemorated the tenth anniversary of the establishment of the school's Malaria Research Institute (JHMPI) as well as the second joint Academy–JHBSPH symposium focused on malaria.
The Honorable Mayor of New York City Michael R. Bloomberg then addressed symposium attendees. Mayor Bloomberg's commitment to "saving lives," as Klag described it, is united with the Institute's ambition "to save lives millions at a time," with a particular focus on public health issues in the fight to defeat malaria. Mayor Bloomberg referred to malaria as "one of the consummate public health challenges in the world, a challenge that time and time again has defied our best efforts." Though, he remarked, "we still have a very long way to go to get [to a solution] ... this Institute is making ground-breaking and incredibly encouraging progress. The work of the Malaria Institute is designed to attack the disease along the broadest possible front. It marries lab research and public health outreach, ... [an approach] we think has the only hope of success." The Mayor's and others' commitment of funds to these efforts enabled the establishment of a new research facility at the Johns Hopkins Bloomberg School of Public Health (JHBSPH), its Malaria Research Institute.
The establishment of a research institute focused on eradicating malaria became a goal of the JHBSPH in 2001. Certainly other diseases deserved attention as well, "but at the time and to this day," noted Diane Griffin, Professor and Chair in Molecular Microbiology and Immunology at JHBSPH, "malaria remains a leading cause of mortality, especially in impoverished areas, with most deaths occurring in children." Furthermore, Griffin pointed out, in 2001 "there was a need to increase basic research in this area as funding for malaria research lagged far behind research on other diseases, such as HIV/AIDS, and increasing insecticide and parasitic drug resistance posed a significant public health threat."
It was in this spirit that the symposium began. It continued with presentations focused on the combination of public health outreach and bench research focused on "targeting every aspect of the life cycle of this complex organism" as characterized by Alfred Sommer, former Dean of Johns Hopkins Bloomberg School of Public Health and co-founder of the Malaria Research Institute. The goal of combating the disease in the clinic and on a molecular level is the common thread that guides scientists at JHBSPH to "change the way malaria is being evaluated, prevented and treated in Africa," stated Peter Agre, Nobel laureate and Director of the Malaria Research Institute at JHBSPH.
The war from the trenches: one battle is won in Macha
Battling malaria on the ground in areas where the disease is endemic, such as Macha in southern Zambia, has been a "grass-roots" combined effort of the staff of the Malaria Institute at Macha Hospital and the community, said Philip E. Thuma, Director of the Malaria Institute at Macha, which was established in 2003 by a grant from JHBSPH.
Macha hospital serves a population of 160,000 people. In 1998, before Thuma's efforts took effect, the Zambian National Health Strategic Plan (1998–2000) reported that malaria was a greater burden to the country than other diseases. Thuma remarked that "in the late 1980s and 1990s it was not unusual for us to see two to three children die everyday from malaria at our hospital." Inpatient data from the pediatric children's ward of Macha Hospital confirmed there was an average of nearly 1,400 cases a year of malaria from 2000 to 2003.
Thuma reported that in Macha cerebral malaria and severe anemia caused by malaria were not uncommon. In fact, the cases of malaria paralleled the number of blood transfusions, suggesting malaria was a driving force for severe anemia in the area. In an effort to combat the disease and the high mortality rate and also to reduce the resource load associated with continual blood transfusions (and the associated risks to the patients) Thuma began to study disease patterns in local villages in the Macha area. Specifically, he sought to find out the number of times in a season that a child contracts malaria.
In 2000 Thuma screened a small set of children in five villages for malaria. Of the children screened, 38% had Plasmodium parasites in their blood, but did not show any signs of malarial disease (asymptomatic) and 3% had full-blown malaria symptoms. From these results Thuma noticed "there was a huge group in our population who have asymptomatic parasitemia and yet never come for treatment, because they are not sick."
In 2003 health care workers at the Macha Malaria Institute screened larger group of children, from 10 villages, than were screened in the previous study in 2000. The health care workers found this time that 43% of the children had Plasmodium infection, and were visually asymptomatic. The results again suggested to Thuma that it was possible that "asymptomatic malaria carriers are potential reservoirs" to infect mosquitoes that go on to "transmit (the disease) to someone else who may become ill with malaria." Motivated by these data, the researchers resolved to "treat the asymptomatic parasite carriers" in Macha (Kent RJ, Thuma PE, Mharakurwa S et al. Am. J. Trop. Med. Hyg., 2007).
To do this Thuma and his team of health care workers and volunteer medical students reached out to the community to educate the people about the disease, its symptoms, and its treatment, and then they performed a census of malaria in the area. To determine who was infected with the parasite (with or without symptoms) a smear was used. The smear had a 1-day turnaround time, and the team treated all those infected with the parasite, whether they were symptomatic or not, with artemisinin combination therapy (ACT), newly released by the Zambian government. Artemisinins are a drug used to treat Plasmodium infection. Artemisinins kill not only the form of the parasite that causes the disease and its symptoms (during the blood stage), but also the parasite in its gametocyte stage. The gametocyte stage is the reproductive stage of the parasite where the host (human) remains asymptomatic, but the disease can still be transmitted from humans to mosquitoes, and then to other humans.
Later, the development of rapid diagnostic tests (RDTs), such as an assay that tests blood samples for the Plasmodium antigen histidine-rich protein II (HRP2), allowed for even faster diagnosis (15 minutes) and the ability to begin treating patients in the same day they were tested.
Using this new therapy, RDTs, and the combined efforts of hospital staff and the community, Thuma's team was able to dramatically alter the malaria situation in the Macha region. At the symposium he presented some statistics that illustrate the degree of importance of the "Macha model" of treating aymptomatic malaria carriers in southern Zambia: Over the last decade the number of malaria cases reported annually dropped from 1,500 per year in 1999 to 50 per year by 2010. The change continues, Thuma explained: "In 2010 we had 50 cases, so far in this year we've had 12." He continued, "We would like to believe that this approach of treating asymptomatic malaria carriers is utterly doable," but, he emphasized, "it does take a cooperative community, and we'd like to believe that the 'Macha Model' is reproducible for other parts of sub-Saharan Africa."
Sungano Mharakurwa, PhD, MSc, Johns Hopkins Center for Global Health
- Further research is needed to explain observed regional declines or resurgences in infection rates.
- P. falciparum exhibits host-dependent antifolate resistance polymorphisms causing pyrimethamine and cycloguanil resistance.
- Surveillance of drug resistance polymorphisms should be done in humans and in mosquitoes.
The battle goes on in Nchelenge and Mutasa: Surprises from the field
Sugano Mharakurwa, Scientific Director of the Malaria Institute at Macha Hospital and Senior Research Associate at JHBSPH presented the goals for his future work on the application of the 'Macha model' in Mutasa and Nchelenge, as well as his work from the bench studying patterns of drug resistance on a molecular level. Before going on to discuss his recent "surprises from field," Mharakurwa made two key observations about fighting the disease on the ground and applying the model to regions with contrasting epidemiological results. First, although he is optimistic about the recorded declines in malaria, he cautioned against attributing all of that success to human intervention. In some regions, he suggested, there is an unidentified helping hand—another factor contributing to the decline—that predated interventions in some areas. This factor must be identified to prevent malaria from rebounding out of control. Second, Mharakurwa mentioned that in the field he has observed there is also an unexplained heterogeneity in the incidences of malaria [malaria is declining in one area and rising in another], which he called "alarming." He further cautioned that the cause of discrepancy must be elucidated to gain a better understanding of resurgence patterns of malaria.
Fighting the resurgence
Mharakurwa seeks to identify the causes of unexplained declines or regional spikes in malaria and the heterogeneity from region to region, by working in other areas of Africa in addition to Macha. Specifically, he will be focused in northern Zambia, in a region called Nchelenge, and in eastern Zimbabwe, in a region called Mutasa. In Nchelenge, which borders the Democratic Republic of Congo, the number of cases of malaria has remained unchanged, in spite of a 65% reduction in reported cases in the rest of the country, achieved using the artemisinin combination therapies. In Mutasa there has been a recent localized resurgence in the number of recorded malaria cases, despite previously successful long-term control. Commenting on the disheartening recurrence of the disease in Mutasa, Mharakurwa noted, "Now that we have a wonder drug it seems malaria was better in the days of chloroquine."
He mentioned that there is a factor that is further complicating the battle to quash the resurgence in Mutasa. He suggests that there may be a potential new vector at play, rather than immigration of cases from neighboring areas. Some researchers have postulated that an as yet unidentified mosquito vector that feeds during the day, as opposed to at night, might be infecting local populations. The daytime feeding habits of this "new" vector would render bed nets and some other precautions useless. "We need to find out what sort of vector we are dealing with," said Mharakurwa. He quoted one local nurse aide as saying, "In this area it bites you when you are out in the field, [and] by the time you go back to sleep under your treated net or in your sprayed house you've already caught malaria."
Tracking drug resistance mutations
Not only is Mharakurwa studying the disease patterns of the parasite at clinics in Zambia and Zimbabwe, but also at the bench. Keeping the public health and clinical implications in mind, he then shifted the focus of his talk to recent molecular discoveries he and colleagues made while searching for resistance polymorphisms—mutations associated with drug resistance—in the parasites, while the parasites are in the mosquito.
The Plasmodium genus of parasites has a tremendous capability to develop drug resistance, which has caused significant problems for treatment protocols. Traditionally, scientists have focused on the resistance that the parasites present in the human host. However, Mharakurwa challenges this notion and suggests we should also be looking into the resistance developed while the parasite lives in its true host—its vector, not the intermediate human host—the mosquito. "This is where the sexual reproduction of the parasite takes place, and therefore where genetic recombination occurs," he elaborated.
Some parasites have successfully developed resistance against crucial anti-malaria drugs, putting increased pressure on scientists to understand how these resistance mechanisms operate. Unfortunately, Mharakurwa explained, some parasite populations have been able to develop resistance to DHFR inhibitors—pyrimethamine and cycloguanil—through a mechanism called antifolate resistance. Malaria-causing parasites need folate to conduct important precursor steps to DNA replication. DHFR inhibitors, a class of anti-malaria drugs, exploit this dependence by targeting dihydrofolate reductase (DHFR), a key enzyme in the tetrahydrofolate biosynthesis pathway. Whereas humans can salvage folate from dietary sources, the parasite must synthesize it, thus selective toxicity for the inhibitors is higher in the parasite. To circumvent some of the resistance problem, however, DHFR inhibitors are used in combination with dihydropteroate synthetase inhibitors, which attack another step in parasite folate biosynthesis.
To understand the DHFR inhibitor resistance Mharakurwa and colleagues searched for resistance polymorphisms present in the parasite during the two-week period when the parasite undergoes sexual reproduction, which occurs inside the mosquito. In this case he studied the parasite in the A. arabiensis mosquito. These mutations were then compared to the resistance polymorphisms present in the parasite during the blood stage—inside the human host. The results revealed something interesting.
The results are shown in the figure below. The prevalence of the mutation is given on the x-axis in the figure as a percentage, the number of people or mosquitoes with the mutation as function of the total number of people with submicroscopic parasitemia or infected mosquitoes. Cycloguanil-resistance is depicted in the yellow regions and pyrimethamine-resistance in blue. The figure on the left (A) is the prevalence of the mutation in the mosquito midgut and the figure on the right (B) is the prevalence of the mutation in the human blood smear negative samples (patients whose blood showed only submicroscopic parasitemia). The data suggest that cycloguanil resistance was more prevalent in the midgut of mosquitoes. A result Mharakurwa mentioned that was, "extremely unexpected as it [cycloguanil] had not been used in this population." In the human phase cycloguanil-resistant mutants (yellow) were detected at a low frequency in the blood smear negative panels, and pyrimethamine-resistant mutants (blue) were significantly more prevalent. In smear-positive samples, routinely used as the sentinel group for tracking drug resistance, no cycloguanil resistance mutants were detectable. Overall the results of Mharakurwa and his colleagues's research suggest cycloguanil resistance polymorphisms are present in the sexual reproduction phase of the parasite (inside the mosquito) and this resistance is only beginning to emerge in parasites infecting humans. The implication is that surveillance for drug resistance should be conducted in both stages of the parasite's life cycle, in the human and in the mosquito.
On the molecular level this resistance occurs as single point mutations in the amino acid codons of DHFR, including codon 108, where a key amino acid residue has been mutated from a serine to an asparagine in the case of pyrimethamine-resistant mutants and to a threonine in cycloguanil-resistant mutants. This single amino acid modification causes the disruption of a key hydrogen bond between the hydroxy group of serine 108 and the pyrophosphate group of the NADPH binding site in the active site. In the case of the S108T mutant (cycloguanil-resistant) the mutation enhances DHFR activity, and in the S108N mutant the alteration reduces the activity of DHFR (Mharakurwa S, Kumwenda T, Mkulama MA et al. Proc. Natl. Acad. Sci. USA, 2011).
Photini Sinnis, Johns Hopkins Bloomberg School of Public Health
- Circumsporozoite Protein (CSP) mediates movement of sporozoites out of the skin to the liver.
- A cleaved form of CSP is responsible for the sporozoites' recognizing hepatocytes and stopping of their sporozoite journey at the liver.
- CSP is cleaved by a sporozoite cysteine protease of the papain family. The protease is inhibited by E-64, suggesting protease inhibitors may provide novel antimalarials.
The parasites' journey from the skin to the liver and beyond
The focus of the symposium then shifted from the clinical fight on the ground to the fight that is being waged on a molecular and organismal level. The laboratory battle, as described at the symposium, focuses on using aspects of the parasite's lifecycle against itself. Sporozoites are the form of the parasites that are transmitted from the mosquito to the human host. It is during this stage that the clinical symptoms of malaria are difficult to detect. There are few in vitro models to study this stage, and typically mouse models are employed instead.
Photini Sinnis of the Johns Hopkins Bloomberg School of Public Health became interested in the molecular interactions that guide the journey of the sporozoite from the skin to the liver in mouse models of malaria. She and her colleagues quickly found that the motility of the parasite is crucial to successful infection of the host.
When an infected mosquito bites a human it injects sporozoites into the dermis (skin). From the dermis the sporozoites must move into the blood stream (perhaps crossing an endothelial barrier), and from the blood stream the sporozoites invade the liver cells (hepatocytes), where they begin to multiply. Approximately 25% of the sporozoites make it to the liver, and a single sporozoite can give rise to approximately 5000 parasites within a single hepatocyte. From there the parasites then invade the red blood cells (RBCs), at which point the blood stage begins, the clinical signs of the disease begin to show, and more and more RBCs are infected. In the case of the P. falciparum infection the blood stage can be reached anywhere from 6 to 14 days after infection.
Plasmodium species (Apicomplexa protist) are propelled using a form of motion called gliding motility—substrate-dependent movement using surface proteins. This movement is essential for the parasites to reach the liver and thereby cause infection (Sultan AA, Thathy V, Frevert U et al. Cell, 1997). After observing that the sporozoite stopped moving when it encountered the hepatocyte, Sinnis sought to identify the molecular process that causes the sporozoite to recognize that cell type.
Stop. Who goes there?
The sporozoite stops at the hepatocyte under the direction of its major surface protein, circumsporozoite protein (CSP). CSP forms a dense coat on the surface of the sporozoite. The overall structure of CSP is remains elusive, but the protein's important components are known via homology. CSP is essential for sporozoite function and the invasion of the hepatocyte (Sinnis P and Coppi A. Parasitol Int, 2007).
The CSP protein contains four domains: a lipid anchor domain (black, above), a thrombospondin repeat (TSR) domain, a central repeat region, and an N-terminal region. The TSR domain is a cell adhesive domain that shares homology with a type 1 thrombospondin repeat—a "sticky" domain found in thrombospondin (red). The central repeat domain is an immuno-dominant region against which mice and people make antibodies (black). Based on molecular modeling, the N-terminal domain (green) is thought to be helical in structure.
CSP is a major component of the malaria vaccine RTS,S (Mosquirix), a subunit vaccine that is in Phase III clinical trials. This vaccine is composed of part of the CSP, the repeats and part of the C-terminal TSR domain, fused to the hepatitis B surface antigen which enables particle formation. In addition it is formulated with a powerful adjuvant that is used to boost the immune response. RTS,S contains a component (region) from CSP, a hepatitis B surface antigen, and an adjuvant compound used to boost the immune response. The vaccine has been shown to prevent malaria infection in 47% of the children inoculated, for up to 15 months (Agnandji ST, Lell B, Soulanoudjigar SS et al. N. Engl. J. Med., 2011).
Odd man out?
Sinnis found that during the sporozoite's journey to the liver part of the CSP protein is removed or "clipped," by a sporozoite protease—a protein that hydrolyzes other proteins by breaking specific amide bonds in that protein. This particular papain family cysteine protease removed the N-terminal region of CSP at a highly conserved five amino acid sequence. Inhibition of this protease with E-64, a broadly active cysteine protease inhibitor, in mice inoculated with the sporozoites, drastically reduced the amount of parasite RNA in the liver to an almost undetectable level. E-64 had no effect on the sporozoite's journey to the liver, it only prevented the "clipping" of CSP, which suggests that this cleavage process is necessary for hepatocyte invasion (Coppi A, Pinzon-Ortiz C, Hutter C et al. J. Exp. Med., 2005).
Further studies suggested that an interaction between CSP and the hepatocyte triggers this proteolytic cleavage. Sinnis found the recognition process was guided by an interaction with heparan sulfate proteoglycans (HSPG)—negatively charged polysaccharides that are linked to the cell surface proteins or to extracellular matrix proteins in animal cells—on the surface of the hepatocyte. The HSPGs of the liver differ from those of other cells in that they are more negatively charged—contain a higher density of sulfates—than other cells. The recognition process between CSP and HSPG also caused the sporozoite to stop its journey at the liver (Coppi A, Tewari R, Bishop JR et al. Cell Host Microbe, 2007).
TSR hidden down under
To decipher the points at which CSP and its downstream, cleaved form are expressed, and by extension to elucidate their potential roles in hepatocyte invasion, Sinnis and colleagues generated antibodies to different regions of CSP. Interestingly, antibodies against the TSR (cell adhesive) domain were unable to bind to, and recognize, the surface of the sporozoites taken directly from the salivary glands of mosquitoes. However, antibodies to the N-terminal region of the CSP bound to the surface of these sporozoites. After the sporozoites reached the hepatocytes the N-terminal antibodies no longer recognized to the sporozoites while the TSR antibodies did. Taken together the results suggest that N-terminal helical domain of CSP masks the TSR domain, and that the TSR domain is not accessible until after cysteine protease cleavage of the N-terminal domain (Coppi A, Natarajan R, Pradel G et al. J. Exp. Med., 2011).
Sinnis also found that mutant parasites that express only the cleaved form of CSP on their cell surface are able to invade hepatocytes, but when these mutants are introduced into the dermis they are unable to leave that location and journey to the liver. To explain these phenomena, Sinnis proposed a model wherein the full-length CSP is presented on the surface of the sporozoite as it journeys from the dermis to the liver, during which transit the N-terminal portion of the protein masks the TSR domain. Once the sporozoites reach the liver they recognize the highly negatively charged HSPG and secrete the protease that clips the CSP, revealing the TSR domain. This cleavage process effectively signals the parasite to stop its journey.
Sinnis and colleagues are now actively trying to identify the protease, "because this could be a good drug target," she explained. Further, they seek to improve upon RTS,S whose results are "extremely promising," but she expressed some reservation, "we're not there yet." She suggested that expanding the region of CSP that RTS,S expresses to include the "highly conserved five amino acid sequence" that contains the N-terminal cleavage sight could be even more promising.
George Dimopoulos, Johns Hopkins Bloomberg School of Public Health
- Genetic modification of mosquitoes to turn on the gene that codes for an immune activator, Rel2, after a blood meal are resistant to the human malaria parasite.
- Gut bacteria of mosquitoes in Macha, Zambia, such as Enterobacter (Esp_Z) and Chromobacterium (Csp_P), can prevent Plasmodium infection in the mosquito.
- Chromobacterium (Csp_P), can prevent Plasmodium and dengue virus infection by production of a secondary metabolite, which is of pharmacological interest.
- "Mosquitoes may have favored the colonization of their gut with bacteria exerting anti-pathogen activity."
Turning the mosquito against the parasite
In an effort to combat the malaria parasite, scientists have genetically engineered Anopheles mosquitoes that are completely resistant to the malaria parasite, so the mosquito can no longer transmit the disease to humans. In effect the scientists have turned the vector against the parasite. George Dimopoulos, of the Johns Hopkins Bloomberg School of Public Health and his colleagues have spearheaded the efforts to transform the mosquito to help scientists fight the disease. Specifically, he genetically manipulated the mosquito's immune system so that it recognizes and responds to the parasite [at the merozoite stage] before the parasite becomes transmissible [the sporozoite stage]. Then in a multi-pronged approach Dimopoulos and his colleagues identified several strains of the mosquitoes symbiotic gut bacteria that have strong anti-malarial properties that can be employed to fight the parasite as well.
When a mosquito becomes infected with the Plasmodium parasites the mosquito's immune system recognizes the parasite and its IMD immune signaling pathway is activated. When the IMD pathway is active it produces proteins and factors, such as Rel2, that ultimately kill the parasite (Garver LS, Dong Y, and Dimopoulos G. PLoS Pathog., 2009). Dimopoulos presented a timecourse model for the activity of the mosquito's immune system in killing the parasite, which is shown in the figure below. As demonstrated in the figure, in wild-type (WT) mosquitoes the immune system becomes fully active 18 hours after a blood meal, a point at which the parasites have already begun to invade the midgut epithelium. Unfortunately, not all the parasites are killed, and the remaining will go on to develop into sporozoites that the mosquito can still transmit to humans.
Dimopoulos aimed to genetically engineer mosquitoes that could kill the Plasmodium parasite completely before they can transmit the disease. His approach was to modify the mosquitoes' DNA such that their immune systems would act earlier (in under 18 hours) and more powerfully. To do this Dimopoulos and colleagues fused Rel2, the gene that codes for the immune activator Rel2, to a 'new' promoter—a part of the DNA that determines when its associated gene(s) will be active. In wild-type mosquitoes the promoter activates transcription of the Rel2 gene when the mosquito becomes infected, which is too late to break transmission. In the mutant mosquitoes, the gene was fused with a promoter that ordinarily activates a gene encoding an enzyme that digests blood. As a result, transcription of Rel2 is activated when the mosquito feeds on blood, which is the first stage of the process. A second gene was also inserted with the fused Rel2 gene that allowed the transformed mosquitoes to fluoresce under UV light. The researchers then created three genetically modified lines that expressed the modified Rel2 in either the gut tissue (indicated by "Cp" or red coloring in the image below), in the fat body (Vg, green), or in both (Hyb, yellow). Further, each of these lines had a different fluorescence phenotype.
The most active mutants were the mutants with Rel2 expressed in the midgut (Cp) and in both the midgut and fatbody (Hyb). These mosquitoes were capable of mounting an immune response a great deal earlier than WT mosquitoes, as early as three hours after taking a blood meal. These mosquitoes mounted their response before the parasite reached the midgut epithelium and before it had the chance to develop into oocytes and then sporozoites. Thus, the genetically modified mosquitoes are now completely resistant to the Plasmodium parasite, and most importantly, they can no longer transmit the disease to humans. Further evidence to support this observation came from samples taken from the guts of the mutant mosquitoes, which show that the Hyb mosquito has significantly fewer parasites both than the Cp and Vg transformants and than the wild-type mosquitoes.
The next phase of the process will be to find ways to introduce these transgenic mosquitoes into nature. Dimopoulos knows that introduction of these mosquitoes is "not possible following the classical laws of Mendelian genetics" (because of the slightly lower "fitness" of the transgenic mosquitoes). However, he suggests it may be possible to introduce the transgenic mosquitoes by using a "genetic drive system." A genetic drive system is a population replacement strategy that uses different mechanisms to spread a gene of interest to all offspring of a cross between wild-type and mutant organism (Sinkins SP and Gould F. Nat. Rev. Genet., 2006).
Antibiotics for insects
In addition to genetically modifying the mosquito, Dimopoulos and his colleagues were also able to use the mosquitoes' gut bacteria against the parasite. Interestingly, feeding mosquitoes antibiotics makes them more susceptible to malaria infection, suggesting that the gut bacteria act against the parasite, in a symbiotic sort of relationship with the mosquito.
Dimopoulos and his colleagues isolated and cultured an array of bacteria from the gut of mosquitoes collected in Macha, Zambia. In total they collected members of 32 different bacterial species, including members of the Staphylococcus, Enterobacter, Chromobacterium, and Escherichia genera (Cirimotich CM, Dong Y, Clayton AM et al. Science, 2011). The cultures were screened for their ability to block Plasmodium development in mosquitoes. After feeding the mosquitoes Plasmodium-infected blood mixed with the desired cultured bacteria the researchers determined the number of parasites in the gut of the mosquitoes. Of the four different bacteria isolates (Bpu, Asp, Ppu, Esp_Z) Esp_Z, an Enterobacter, had the most potent ability to block Plasmodium development in the mosquito gut. Esp_Z also showed potent antimalarial activity in transwell-culturing systems, suggesting the bacteria releases the antimalarial substance.
Another bacteria isolated from the mosquito gut, Chromobacterium (Csp_P), was found to have potent antimalarial activity. Csp_P produces a secondary metabolite that kills not only the parasite, but also the mosquito, if the mosquito is given a sufficient amount of the bacteria. This Chromobacterium also possessed potent anti-dengue (antiviral) and in vitro antibiotic activity—it secretes a substance that kills other bacteria. The identity of the secondary metabolite is currently being elucidated. Reflecting on the complexity of isolated natural projects, Dimopoulos noted, "This is why many times natural products for drug development are superior to synthetic smaller molecules; it is very difficult to produce this kind of complexity in the test tube."
Concluding his presentation, Dimopoulos hypothesized that "Mosquitoes may have favored the colonization of their gut with bacteria exerting anti-pathogen activity." Thus he is beginning his quest to study the midgut bacteria microflora of many more mosquitoes throughout the world in search of more chemotherapeutics.
David L. Smith, Johns Hopkins Malaria Research Institute
- Malaria eradication will be expensive and it will take decades, but it may be possible.
- The greatest threat to successful malaria control efforts is a decline of public interest, political will, and international financing.
- Eradication will depend on effective leadership.
Policy and the burden of malaria
David L. Smith, epidemiologist from JHMRI and JHBSPH at the Center for Disease Dynamics, Economics and Policy, began his presentation by looking at past malaria eradication efforts and their organization. Smith's presentation focused on the success and failures of these eradication efforts, and how to learn from them. He went on to attempt to dispel "myths held about past eradication efforts," and lastly he presented his and his colleagues work to provide a rank of elimination feasibility in countries with endemic malaria.
It is well known that malaria eradication efforts failed in 1969 in Africa, which shoulders 80% of the burden of the disease, although malaria was successfully eradicated and/or controlled in North America, Europe, the Caribbean, Asia, and parts of Latin America. Smith and his colleagues sought to identify the real reasons for this failure, and he started by questioning the co-occurrence of this failure with the abrupt decline in donations to the organization that operated the global malaria eradication program in 1964 (see Figure below, Nájera, 1999).
Smith briefly alluded to the smallpox eradication program, which was successful to such an extent that smallpox is no longer in circulation and vaccination is no longer necessary. He explained that "the whole eradication program for smallpox...has paid for itself some 400 times over” because it is no longer necessary to vaccinate anyone. "This is by far one of the most cost-effective programs." Smith sought reasons that malaria eradication efforts have achieved such different results. Malaria has so far continued to evade eradication, but Smith queried whether there were other factors contributing to the failure of eradication programs.
A historical view of vector control
Smith's presentation shifted then to a brief history of vector control efforts, which included a discussion of Fred Soper, an American epidemiologist, and Paul Herman Müller, the Swiss chemist who invented DDT. Fred Soper, supported by the Rockefeller Foundation, successfully eradicated the mosquito in Brazil after the worst malaria epidemic in the America's occurred there in 1938. He achieved success in just 22 months. Paul Herman Müller found that dichlorodiphenyltrichloroethane (DDT) was a potent insecticide, and the U.S. military subsequently used it during WWII. Soper then employed DDT in an "ambitious plan to eradicate malaria," as Smith put it, to control malaria in Sardinia, Italy. In a successful effort from 1947–1951 Soper was able to reduce the cases of malaria from the 75,000 reported in 1947 to just the 9 reported in 1951.
Smith then went on to describe a global shift in commitment to eradication efforts. In an ambitious plan that was agreed upon at First World Health Assembly in Geneva, Switzerland in 1948, which established the World Health Organization, and later at the Malaria Conference in Equatorial Africa, held in Kampala, Uganda in 1950, the official policy became that "malaria should be controlled by modern methods as soon as feasible, whatever the original degree of endemicity, and without awaiting the outcome of further experiments," (Doull JA and Kramer M. Public Health Rep., 1948). In spite of these stern declarations, just one decade later, in 1964, funding for the eradication efforts dropped abruptly to almost nothing, before the disease could be completely eradicated. Eradication programs depended on sustained and adequate funding for their success. Smith noted the relevance of this situation to today's efforts: "We find ourselves in a parallel situation... Malaria eradication is the goal as declared by the WHO, the Bill and Melinda Gates Foundation, the Roll Back Malaria Partnership... Since 2005 financing for malaria has gone up ... and things are happening that weren't happening before."
Smith then presented a success story from Zanzibar, Tanzania, which has experienced cycles of malaria epidemics resulting from failed control efforts. Previous 'eradication' efforts from 1958–1968 reduced the number of cases but numbers rebounded to epidemic levels by 1981–1988. Recent programs that have phased out failing drugs (chloroquinine) in favor of arteminisin combination therapies, insecticide-treated bed nets, and indoor spraying, have seen more success, reducing reported cases to 1960s levels (Smith DL, Cohen JM, Moonen B et al. Science, 2011). Steeled by recent reductions but wary of past failures, Zanzibar faced the option of aiming for complete eradication. This question, "Should Zanzibar aim for complete eradication?" was posed to Smith and his colleagues.
Smith and his team's general mandate was to provide advice about the options. Specifically he and his colleagues sought to answer the following questions: What combinations of interventions or combinations of coverage levels are required to achieve elimination? How long would elimination take? How much would elimination cost, and can it be sustained? The Zanzibar Malaria Control Program (ZMCP), which has organized and supported control efforts in the region, as well as the Zanzibar Ministry of Health and Social Welfare to whom these answers would also be reported, were keen to know the feasibility of achieving and sustaining malaria elimination in the region.
Fact or fiction
In answering these questions, to provide advice to the Health Minister, the team weighed two options, control versus elimination, and produced "Malaria Elimination in Zanzibar: A Feasibility Assessment." In the process of producing the report, Smith and colleagues serendipitously discovered that many myths held about malaria eradication may only be weakly—if at all—supported by evidence. The "myths" Smith discussed were:
- DDT was responsible for the failure of the global malaria eradication efforts. While Smith acknowledges DDT resistance did arise, and that it "slowed down the programs," he argued that DDT was a factor, but it was not the main reason for failure. Citing a 1953 paper by Livadas and Georgopoulos, Smith argued that DDT resistance was reported as early as 1951, long before the malaria eradication failure in 1969 (Livadas GA and Georgeopoulos G. Bull. World Health Organ., 1953). Smith further argued that this resistance was "accounted for" in the plan for the global malaria eradication program. He hints that there were other insecticides available and that perhaps the failure was instead a result of another factor—a lack of money.
- Malaria transmission cannot be interrupted in Africa. Studies such as the Garki project, which Smith described as, "a very well designed, large-scale, high-intervention coverage levels ... [with] randomized controls ...," are often cited as evidence to suggest that transmission cannot be interrupted. The Garki project involved vector control spraying and drug administration in northern Nigeria, and it lasted 18 months. Smith argues that the trial was too small, the villages were not isolated, and the time frame was too short (less than three to five years) to substantiate the study's claims. He cited Zanzibar as evidence that transmission can be interrupted.
- Failed eradication efforts are more harmful than untried eradication because people's immunity wanes during attempted eradication. His study suggested that other factors, mainly the failure to sustain funding, had contributed. Other examples include droughts in Sri Lanka, caused hot spots of mosquito populations that coincided with rises in the numbers of malaria cases after the supply of DDT and funding ran low. Weighing control versus eradication efforts for Zanzibar, the data actually showed that control, which had been the policy from 1969 until very recently, usually meant doing nothing. Thus, the choice is between control and eradication, neither of which has ever been fully achieved.
- Climate change poses a major threat to malaria eradication. Based on complex mathematical models, Smith and colleagues found that this scenario is "unlikely." Local temperature may indeed affect local transmission, but the data do not support the idea of a "major threat to eradication" on a larger scale (Gething PW, Smith DL, Patil AP et al. Nature, 2010).
Ranking eradication feasibility
Smith then presented some seemingly controversial results from a paper he and his colleagues published in The Lancet in 2010. Using complex mathematical models, the team generated estimates of the relative technical difficulty of elimination of malaria in particular endemic countries and ranked these nations according to eradication feasibility. The models relate the operational feasibility of elimination to the technical feasibility of elimination in a particular country. Operational feasibility was defined by those factors that included the stability, effectiveness, and commitment of governance, the state of the countries' health infrastructure, and the population at risk. Technical feasibility was defined by those factors that are dependent on malaria stability in a region (endemic malaria transmission rates) and the rates of malaria importation that can result from human migration. These operational factors were defined as such because they were a common thread among 108 countries that have successfully eliminated malaria. A low operational feasibility ranking indicated a country with a great deal of organization or infrastructure. A high operational feasibility ranking indicated a less organized country with poor infrastructure and a larger population. A low technical feasibility ranking meant a small population, low transmission rates, with low levels of importation. By contrast, a high feasibility ranking meant high malaria transmission and high importation rates.
Controversy arose from the fact that some may look at the data and think twice about aiming for elimination in countries that have high rankings (low feasibility), such as regions in Africa and India, and only invest in elimination efforts in countries in the Americas or Asia. As Smith described, one critic of the rankings suggested that if maps like this were available during efforts to eradicate smallpox, they may have swayed policy makers from attempting eradication in regions where such efforts have ultimately succeeded. Smith then argued the key lies not in the overall ranking, but in effective leadership for eradication efforts on a local, regional, and global level (Tatem AJ, Smith DL, Gething PW et al. Lancet, 2010).
Overall, Smith explained, the model provides "the size obstacle we have to overcome, but it does not tell you anything about the likelihood of success ... ." In the end, Smith was not certain that complete eradication would succeed, but he advocates trying nonetheless. Smith was clear that eradication efforts will take a long time—on the order of decades—and it will be very expensive.
Jeffrey D. Sachs, The Earth Institute, Columbia University
- Different Anopheles mosquito species have differing behaviors, such as feeding time (day or night), breeding patterns, and biting propensity. These factors influence the spread of the disease and the choice of vector control methods.
- From 2000–2010 anti-malarial efforts reduced malaria-related deaths an estimated 40% from the rates projected by the Roll Back Malaria Partnership.
- Current control efforts must combine vector control and case management.
The successes of the WHO's malaria eradication campaign
Keynote speaker Jeffrey D. Sachs, director of The Earth Institute at Columbia University, highlighted the many successes achieved in the fight against this deadly disease: "We have had some tremendous breakthroughs in the last 10 years in effective control of malaria in Africa." His talk focused on those achievements and on the strategy aimed at controlling malaria until 2015. Sachs began his presentation by sharing a map of the world on which he pointed out changes in the distribution of malaria that have occurred over the last century. In the early 1900s malaria was a global epidemic that afflicted the U.S., Russia, and Western Europe, in addition to the regions of the world where it remains rampant. This distribution shifted after World War II, and today the map looks quite different, with the concentration of the disease in Sub-Saharan Africa, Asia, and South America.
Accounting for mosquito behavior
The parasite vector, the Anopheles mosquito has many different species throughout the world each having particular behaviors, breeding patterns, populations, and propensity to bite humans. Sachs presented a map (below) clearly showing that each region has a particular dominant vector. He noted that Sub-Saharan Africa happens to be the home of the highest concentration of Anopheles gambiae, A. funestus, and A. arabiensis. Mosquitoes of these species have the highest human biting index (HBI = 1.0), where HBI is the propensity of the mosquitoes to bite humans rather than other animals. By contrast, the dominant vector in India, A. culicifacies, has an HBI of 0.3 (Sachs JD and Chambers RG, Realizing the Right to Health, 2009). This difference means that just in terms of biting index, the probability of obtaining two consecutive (human) malaria infection/transmission bites from the same mosquito is 0.09 in India, a rate that is significantly less than of the probability associated with African dominant vectors.
Given these rates, it is no wonder controlling malaria in Africa has been particularly challenging. Attempts at breaking transmission in Africa have also been hampered by other features of the disease's vector. Sachs made an interesting point about basic reproduction number (BRN) of the mosquito, a number that, in population ecology, refers to the average number of offspring each mosquito would produce over its lifetime. He proposed that it would only be possible to interrupt epidemic malaria conditions if the BRN were less than 1. In Africa the major vectors all have BRNs around 300. Another way to look at that rate is, "if you reduce 95%–98% of the mosquito abundance you still have an epidemic condition, ... a BRN greater than 1, meaning that the disease will transmit within the population," clarified Sachs. Given that near-total elimination of the vector would be needed to prevent the disease from spreading, Sachs' message seemed to be that for the near future, 'control' should be the main strategy.
Looking forward: strategies for the near future
Keeping those limits in mind, Sachs shifted his talk to address the current control efforts and how they differ from those used in the WHO Global Eradication Campaign that failed in 1969. He remarked that, by contrast, current efforts combine transmission interruption using vector control methods (such as bed nets and indoor residual spraying) and case management (treatment of infected people).
On an economic note, Sachs and colleague Amir Attaran calculated that as of the end of the 20th century, "worldwide spending on malaria control, not research, but control was probably 80 million dollars total, maybe 100 million dollars. There was almost no control, in other words, for a disease that had perhaps a billion clinical cases and was killing at the time 800 thousand to 1 million children [annually]." Clearly, more was needed. Following the recommendations of the Millennium Development Goals and of the report of the Commission of Macroeconomics and Health, a global fund to fight Malaria, AIDS and TB, was established. Funding increased, and as Sachs recalls, "for me this was a true breakthrough, because it was the first time...we had funding to bring malaria under control." Later, malaria control policy began to advocate the use of first-line drugs, such as artemisinins, the use of combination therapies, the widespread distribution of free bed nets, and a renewed focus on reducing transmission.
Since then, much has changed on the malaria battlefront. The latest advancements in the treatment of malaria began with a shift in diagnosis and treatment from the central clinic to mobile units, staffed by village health care workers trained in malaria control. He suggests this became possible with the advent of rapid diagnostic tests (RDT) and the widespread use of mobile telephones. This successful combination has propelled the development of a localized information structure in which local, trained health care workers, supplied with a backpack containing medicines to treat patients, can use an RDT (instead of a blood smear) to confirm the malaria diagnosis and can then use the phone to contact a clinic or centralized information system for dosage and treatment protocols.
Thus modern malaria control has advanced to a stage where more responsibility has been given to the individual community. This situation, coupled with vector control, has contributed to the progress made against the disease over the last decade. Current statistics from Roll Back Malaria that suggest 300 million insecticide-treated nets (ITNs) have been distributed throughout Africa, and in some areas 80% of the population has an ITN. In addition, as of 2010, Roll Back Malaria reported that 27 million people live in homes sprayed during the indoor residual spraying (IRS) campaign. According Roll Back Malaria's recent counts, these vector control strategies in Africa, combined with the deployment of community health care workers (CHW), have reduced the number of malaria-induced deaths by an estimated 40% from projections (White Johansson E, Newby H, Carvajal L, et al. A decade of partnerships and results, 2011).
According to Sachs, the projected cost of scaling up this platform is $3 billion dollars a year (Teklehaimanot A, McCord GC, Sachs JD. Am. J. Trop. Med. Hyg., 2007). To put that into perspective Sachs said, "There is no finer bargain on the planet as far as I am concerned. That's three dollars per person in the rich world per year ... one Starbucks a year from each person in the rich world would pay for the bed nets, the IRS, the CHWs, the training, and the RDTs." Unfortunately, despite the tremendous increase in donor assistance since the start of the 21st century, the situation is not ideal and disbursements of committed funds are falling short. Sachs said, "... we are roughly running at the half the rate that we need... It is of course worse than that because we are in an aid crisis... "
Whatever the cost, the system works where it is implemented; "if [the global fund] continues to get funded, even today's tools can carry us a great way towards reducing mortality and morbidity by 2015," said an optimistic Sachs, though he cautions that complete eradication in Africa will be difficult using current tools. He looks to innovation for "new weapons such as a malaria vaccine, whether RTS,S, an attenuated sporozoite-based vaccine, or other targets." And finally, Sachs concluded, "I wouldn't say that malaria has won, ... nor have we won, the race does continue. We are in the lead right now if we continue...applying what we have and continuing with the momentum of scientific breakthrough, we can continue to drive this disease...to a smaller and smaller foothold on the planet. Maybe someday to actual eradication."