Advanced Parasitology-The Epidemiology of Plasmodium Vivax

SERIES EDITORS
D. ROLLINSON
S. I. HAY
Department of Zoology,
The Natural History Museum, London, UK
d.rollinson@nhm.ac.uk
Spatial Epidemiology and Ecology Group,

Tinbergen Building, Department of
Zoology, University of Oxford, South Parks
Road, Oxford, UK
simon.hay@zoo.ox.ac.uk
EDITORIAL BOARD
M. G. BASEZ
R. E. SINDEN
Reader in Parasite Epidemiology,

Department of Infectious Disease
Epidemiology Faculty of Medicine
(St Marys campus), Imperial College,
London, London, UK
Immunology and Infection Section,

Department of Biological Sciences,
Sir Alexander Fleming Building, Imperial
College of Science, Technology and
Medicine, London, UK
S. BROOKER
D. L. SMITH
Wellcome Trust Research Fellow and

Professor, London School of Hygiene and
Tropical Medicine, Faculty of Infectious
and Tropical, Diseases, London, UK
Johns Hopkins Malaria Research

Institute & Department of Epidemiology,
Johns Hopkins Bloomberg School of Public
Health, Baltimore, MD, USA
R. B. GASSER
R. C. A. THOMPSON
Department of Veterinary Science,

The University of Melbourne, Parkville,
Victoria, Australia
Head, WHO Collaborating Centre for

the Molecular Epidemiology of Parasitic
Infections, Principal Investigator,
Environmental Biotechnology CRC
(EBCRC), School of Veterinary and
Biomedical Sciences, Murdoch University,
Murdoch, WA, Australia
N. HALL
School of Biological Sciences,
Biosciences Building, University of
Liverpool, Liverpool, UK
R. C. OLIVEIRA
Centro de Pesquisas Rene Rachou/
CPqRR-A FIOCRUZ em Minas Gerais,
Rene Rachou Research Center/CPqRR The Oswaldo Cruz Foundation in the State
of Minas Gerais-Brazil, Brazil
X. N. ZHOU
Professor, Director, National Institute of
Parasitic Diseases, Chinese Center for
Disease Control and Prevention, Shanghai,
Peoples Republic of China
VOLUME EIGHTY ONE
Advances in
PARASITOLOGY
Edited by
S. I. HAY
Spatial Epidemiology and Ecology Group
Tinbergen Building, Department of Zoology,
University of Oxford, South Parks Road,
Oxford, UK
RIC PRICE
Centre of Tropical Medicine,
University of Oxford,
Oxford, UK
J. KEVIN BAIRD
Eijkman-Oxford Clinical Research Unit
Jalan Diponegoro No. 69
Jakarta, Indonesia
AMSTERDAM BOSTON HEIDELBERG LONDON

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CONTRIBUTORS
Myriam Arevalo-Herrera
Caucaseco Research Center, Cali, Colombia
J. Kevin Baird
Eijkman-Oxford Clinical Research Unit, Jakarta, Indonesia; Centre for Tropical Medicine,
Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK
John W. Barnwell
Department of Medicine, Division of Infectious Diseases, Emory University School
of Medicine, Emory University, Atlanta, Georgia, USA; Centers for Disease Control
and Prevention, Malaria Branch, Division of Parasitic Diseases and Malaria, Atlanta,
Georgia, USA
Katherine E. Battle
Department of Zoology, University of Oxford, Oxford, UK
Jane M. Carlton
Center for Genomics and Systems Biology, Department of Biology, New York University,
New York, NY, USA
William E. Collins
Institutional Association: Malaria Branch, Division of Parasitic Diseases, Centers for
Disease Control and Prevention, Atlanta, Georgia
Aparup Das
Evolutionary Genomics and Bioinformatics Laboratory, Division of Genomics and
Bioinformatics, National Institute of Malaria Research (ICMR), Dwarka, New Delhi,
India
Ananias A. Escalante
Center for Evolutionary Medicine and Informatics, The Biodesign Institute, Arizona State
University, Tempe, AZ, USA
Marcelo U. Ferreira
Departamento de Parasitologia, Instituto de Cincias Biomdicas, Universidade de So
Paulo, So Paulo (SP), Brasil
Mary R. Galinski
Department of Medicine, Division of Infectious Diseases, Emory University School
of Medicine, Emory University, Atlanta, Georgia, USA; Emory Vaccine Center, Yerkes
National Primate Research Center, Atlanta, Georgia, USA
Simon I. Hay
Rosalind E. Howes
ix
Contributors
Christopher L. King
Center of Global Health & Diseases (CGHD), Case Western Reserve University, V
eterans
Affairs Medical Center, Cleveland, OH, USA
Odile Mercereau-Puijalon
Institut Pasteur, Centre National de la Recherche Scientifique Unit de Recherche
Associe, Unit dImmunologie Molculaire des Parasites, Paris, France
Esmeralda V.S. Meyer
Emory Vaccine Center, Yerkes National Primate Research Center, Atlanta, Georgia, USA
Ivo Mueller
Walter + Eliza Hall Institute, Infection & Immunity Division, Parkville, Victoria, Australia;
Barcelona Centre for International Health Research (CRESIB, Hospital Clnic-Universitat
de Barcelona), Barcelona, Spain
Jean-Louis Prignon
Inserm, UMR-S 945, Paris, France; Facult de Mdecine Piti-Salptrire, Universit
Pierre et Marie Curie-Paris 6, CHU Piti-Salptrire, Paris, France; Facult de Mdecine
Paris 5, Universit Ren Descartes-Paris 5, CHU Necker-Enfants Malades, Paris, France
Ari W. Satyagraha
Eijkman Institute for Molecular Biology, Jakarta, Indonesia
Georges Snounou
Inserm, UMR-S 945, Paris, France; Facult de Mdecine Piti-Salptrire, Universit
Pierre et Marie Curie-Paris 6, CHU Piti-Salptrire, Paris, France
Takafumi Tsuboi
Cell-Free Science and Technology Research Center and Venture Business Laboratory,
Ehime University, Matsuyama, Ehime, Japan
Peter A. Zimmerman
Center for Global Health & Diseases, Case Western Reserve University, Cleveland, Ohio,
USA
PREFACE
The epidemiology of Plasmodium vivax: history, hiatus and hubris forms is a
two volume special issue of Advances in Parasitology on the epidemiology of
P. vivax.The aim of the review collection is to present a contemporary summary of what is known about P. vivax, with the challenge set to the authors
to (1) retrieve what has been lost from history, (2) summarize objectively
the current state of knowledge including the reasons for the hiatus in interest and; (3) identify research gaps/directions/priorities to gently temper the
prevailing hubris with respect to control and elimination.
Part A (volume 80) was published in December 2012. It was composed
of six chapters and dealt principally with the most practical dimensions
of vivax malaria, describing the epidemiology, clinical consequences, treatment, and control strategies shaped by the biology of the parasite. Part B
(volume 81) is published here and brings together a further six chapters that
investigate more fully aspects of the parasite life cycle, innate and inherited
aspects that confer host resistance to P. vivax infection, the epidemiological
importance of G6PD deficiency, what is known about the genome of
P. vivax and finally the lessons that can still be learned from the interpretation of the old neurosyphilis literature.
Chapter 1 by Mary R. Galinski and colleagues looks in detail at the parasites life cycle, how it is adapted to its life history challenges and how this differentiates it from P. falciparum.They also explore the research challenges that
remain in combining non-human primate models with new technologies
to potentially provide further insights and epidemiological understanding
of the biology of this parasite. Chapter 2, by Peter A. Zimmerman and colleagues, reviews fascinating new complexities to what is canonically taught
about red blood cell polymorphism (predominantly of the Duffy gene) and
susceptibility to P. vivax infection at the individual and population levels.
Chapter 3 led by Ivo Mueller reviews the natural acquisition of immunity
to P. vivax. Epidemiological observations are synthesised and used to support
the premise that a multi-stage P. vivax vaccine may be feasible. Chapter 4 by
Rosalind E. Howes reviews the geography of G6PD deficiency. The global
distribution of its prevalence and genetic variants are discussed along with
the implications that this has for the potential risk of haemolysis triggered
by primaquine therapy in different parts of the world. Chapter 5, written by
Jane M. Carlton and colleagues, considers the genomics, population genetics
xi
xii
Preface
and evolutionary history of P. vivax. New insights from detailed genomic

investigations of P. vivax across India are synthesised, as well as, research
avenues opening as a result of next generation sequencing technologies,
discussed. In the final Chapter 6, Georges Snounou and Jean-Louis Prignon conclude these volumes by reviewing the epidemiological insights
gained from a thorough analysis of the malaria therapy for neurosyphilis
literature. They outline further some of the insights that might be gained in
relation to the current challenges of P. vivax epidemiology, immunology and
pathology by a deeper engagement with this literature.
It is perhaps worth noting that the enormous literature summarized in
these two volumes evidences a rich history that we are unwise to forget.
Moreover, that the authorship have perhaps helped turn the corner on the
research hiatus of P. vivax. Conversely, and perhaps predictably, it also reveals
there is much still to learn and therefore that we must approach with some
hubris the immediate challenges of P. vivax control and future challenges of
its elimination. Finally we take this opportunity to thank all the authors
for their considerable time and energy devoted to putting these chapters
together. We hope that these two volumes lower the bar for a new cohort
of malariologists inspired by future challenges in the epidemiology and
control of P. vivax.
Simon I. Hay, Ric N. Price and J. Kevin Baird
CHAPTER ONE
Plasmodium vivax: Modern

Strategies to Study a Persistent
Parasites Life Cycle
Mary R. Galinski*,,1, Esmeralda V. S. Meyer, John W. Barnwell*,
*Department of Medicine, Division of Infectious Diseases, Emory University School of Medicine,

Emory University, Atlanta, Georgia, USA
Emory Vaccine Center,Yerkes National Primate Research Center, Atlanta, Georgia, USA
Centers for Disease Control and Prevention, Malaria Branch, Division of Parasitic Diseases and
Malaria, Atlanta, Georgia, USA
1Corresponding author: E-mail: Mary.Galinski@emory.edu
Contents
1. Introduction
2. T he General Life Cycle of Plasmodium vivax and Other Primate Malaria
Parasite Species
2.1. T he Hypnozoite: An Alternative Life Style for Liver-stage Development
2.2. T he Reticulocyte as a Host Cell: An Environmental Safety Program for P. vivax
2.3. F ast and Furious: The Sexual Life Strategies of P. vivax
3. In Vitro and Ex Vivo Models for Examining P. vivax Biology
4. N
eotropical Non-Human Primate Models (New World Monkeys) for Investigating
the Varied Biology of vivax Malaria
5. T he Relapsing Malaria Parasites of Southern Asian Macaque Monkeys as
Models for P. vivax Biology
6. F rom Genomics to Systems Biology: The Bigger Picture Puzzles
7. C
onclusions
Acknowledgements
References
2
3
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20
20
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Abstract
Plasmodium vivax has unique attributes to support its survival in varying ecologies
and climates. These include hypnozoite forms in the liver, an invasion preference
for reticulocytes, caveolavesicle complex structures in the infected erythrocyte
membrane and rapidly forming and circulating gametocytes. These characteristics make this species very different from P. falciparum. Plasmodium cynomolgi
and other related simian species have identical biology and can serve as informative models of P. vivax infections. Plasmodium vivax and its model parasites
can be grown in non-human primates (NHP), and in short-term ex vivo cultures.
2013 Elsevier Ltd.

Advances in Parasitology, Volume 81
ISSN 0065-308X, http://dx.doi.org/10.1016/B978-0-12-407826-0.00001-1 All rights reserved.
Mary R. Galinski et al.
For P. vivax, in the absence of invitro culture systems, these models remain highly
relevant side by side with human clinical studies. While post-genomic technologies
allow for greater exploration of P. vivax-infected blood samples from humans, these
come with restrictions. Two advantages of NHP models are that infections can be
experimentally tailored to address hypotheses, including genetic manipulation.
Also, systems biology approaches can capitalise on computational biology combined with set experimental infection periods and protocols, which may include
multiple sampling times, different types of samples, and the broad use of omics
technologies. Opportunities for research on vivax malaria are increasing with the
use of existing and new methodological strategies in combination with modern
technologies.
1. INTRODUCTION

Plasmodium vivax has been neglected as a disease of major global
importance. Recently, expanded efforts have been made to bring more
widespread attention to this disease and to overcome perceptions that
there are insurmountable barriers to advancing research and basic
knowledge on vivax malaria (Carlton et al., 2011; Galinski and Barnwell, 2008; Lacerda etal., 2012; Mueller etal., 2009; Price etal., 2011).
In fact, research and methodological strategies are in place to move
forward using the most modern technologies available, and advances are
being made. Basic vivax malaria research is benefiting from the incorporation of exvivo samples from humans, non-human pr imate (NHP)
experimentation and invitro analyses. In addition, an increased focus on
the epidemiology of P. vivax, with an increased attention to interactions
with other species, and greater consideration of the ecological factors
that affect this parasites range is apparent (Gething etal., 2012). Various
clinical, epidemiological and biological attributes associated with vivax
malaria have also gained the attention of mathematical modellers and
computational biologists who wish to apply currently available knowledge on the host and vector interactions with this parasite to understand
transmission and the influence of current control interventions and drug
treatments on those dynamics (Aguas etal., 2012; Chamchod and Beier,
2012; Gething etal., 2011; Mueller etal., 2009; Price etal., 2011; White,
2011). However, to improve modelling efforts and control strategies,
interventions or drug therapies will benefit from a better understanding of the biological attributes that afford P. vivax and its sibling species
life strategies that enable it to persist when confronted with control
methods implemented by its human host.
Plasmodium vivax: Modern Strategies to Study a Persistent Parasites Life Cycle
2. T
HE GENERAL LIFE CYCLE OF PLASMODIUM VIVAX
AND OTHER PRIMATE MALARIA PARASITE SPECIES

In general terms, the life cycle of P. vivax is like that of all of the
other primate malaria species in that it requires an invertebrate and a vertebrate host for survival and perpetuation; a female mosquito of a susceptible
Anopheles species and a primate, whether human or NHP (Fig. 1.1). When
the female mosquito bites, or more precisely, probes the dermis with her
proboscis looking for a vessel to obtain her blood meal, she releases salivary
fluid and along with it a few sporozoites from her salivary glands. In the
dermal tissues, the sporozoites are motile and capable of penetrating small
blood vessels, and beginning to stimulate a host immune response (Guilbride
etal., 2012; Sinnis and Zavala, 2008). In the circulating blood, they are swept
into the liver sinusoid vessels where they penetrate through the professional
phagocytes known as Kpffer cells into the Space of Disse to begin the exoerythrocytic or liver-stage cycle of growth (reviewed in Baer etal., 2007b;
Frevert etal., 2008; Meis etal., 1983; Pradel and Frevert, 2001). Once there,
the sporozoite then penetrates a hepatocyte, rounds up and differentiates
into a small trophozoite (4 in diameter) growing in size over the next
few days eventually differentiating into a multinucleated schizont in 5 days.
By 6 or 7 days, of primary growth and development, a fully mature schizont
4060 in diameter has differentiated into thousands of individual invasive
single nucleated merozoites surrounded by a parasitophorous membrane
capsule. As reported for rodent malaria experimental model systems (not
yet investigated in primate malarias), the plasma membrane of an infected
hepatocyte breaks down, and blebs of the parasitophorous membrane full of
merozoites called merosomes break off and flow into the circulation of the
liver sinusoid vessels (Prudencio etal., 2006; Thiberge etal., 2007). These
merosomes are carried into the faster flowing general blood circulation and
break apart releasing the imprisoned merozoites (Baer etal., 2007a), which
then attach to and invade red blood cells (RBCs) to start the erythrocytic
cycle of infection, also known as the blood-stage cycle.
The newly invaded merozoite immediately differentiates into an erythrocytic trophozoite and begins remodelling the anucleate RBC to provide
a suitable environment for it to grow larger over a period of 48 hours
feeding upon the haemoglobin of the parasitised RBC. Thirty-eight or 40
h into this cycle of growth, the nucleus divides in two to create a schizont
and over the next 8 or so hours continues to divide by schizogony to form
Figure 1.1 Schematic of the life cycle of Plasmodium vivax and comparable sibling
simian species, depicted to represent the unique biological features of these species
in the life cycle of primate malaria species and the importance of clinical and experimental interventions. The monkey figure represents neotropical NHP models of P. vivax
or macaque NHP models of P. cynomolgi and other simian parasite species that serve
as surrogates for P. vivax. The purple and green icons indicate where natural events and
experimental manipulations can take place. The green mosquito icons refer to the natural inoculation of sporozoites through biting and the purple mosquito icons refer to
the natural biting and infection of Anopheles ssp. mosquitoes by drawing in gametocyte-infected blood. The green medical symbol and syringe denoting the inoculation of
sporozoites into the human and NHP hosts, respectively, refer to the possibility of challenging these hosts after immunisation with a vaccine candidate to determine if protection can be induced. The purple medical symbol and syringe denote the collection
of blood for testing involving human and NHPs, respectively. The purple syringe also
signifies the specific collection of blood containing gametocytes from NHP to artificially
feed and infect Anopheles mosquitoes, to support experiments on the transmission of
sporozoites or transmission blocking vaccines. The unique biological features of P. vivax
and comparable species depicted are the hypnozoite, the preferential invasion of merozoite into reticulocytes, the production of CVCs, represented as a mottled appearance of
the infected RBCs, and the early and rapid development and circulation of gametocytes.
Red arrows refer to processes relating to these features, in need of special research
emphasis: 1) what is the make-up of hypnozoites and how are they activated, 2) what
are the similarities and differences in primary and relapsing liver-stage schizonts and
is their biology with merosome release in the blood stream comparable to other Plasmodium species, 3) what critical factors are required for reticulocyte host cell selection,
invasion and growth in these cells, and 4) what factors determine the development and
circulation of gametocytes, potentially permitting transmission from the early stages
of a blood-stage infection. (For interpretation of the references to colour in this figure
legend, the reader is referred to the online version of this book).
1216 or more differentiated merozoites in the case of P. vivax. When the

host cell membrane ruptures after 48h of parasite intracellular growth, the
merozoites are released and invade new RBCs to begin the entire asexual
blood cycle again. The merozoites in some schizonts are preprogrammed to
differentiate into sexual-stage male or female micro- and macro-gametocytes,
respectively, and not into asexual trophozoites upon entry into a new RBC
(Bruce etal., 1990; Reininger etal., 2012; Silvestrini etal., 2000).
The insect or sexual stage of the life cycle begins when a feeding female
Anopheles mosquito takes up some of these circulating male and female
gametocytes in her blood meal. In the midgut of the blood-engorged mosquito,
the gametocytes lose their RBC membrane outer cover and become sexually
stimulated. The nucleus of the microgamete fractures into eight nuclei and
eight flagellating bodies are formed (Carter and Nijhout, 1977; Prasad etal.,
2011). When one penetrates a macrogamete, a diploid zygote is formed that
over a period of 2436 h metamorphoses into an ookinete, another tissue
invasive parasite stage.The ookinete penetrates through the mosquito midgut
lining to lodge under the basal membrane where it transforms into an oocyst
that undergoes multiple nuclear divisions to form a capsule of several thousand
elongated sporozoites. At maturity, the capsule breaks open, releasing the
thousands of sporozoites into the haemocoel of the mosquito, which then
migrate to and penetrate the salivary glands to lodge in the glandular spaces
waiting until the mosquito probes dermal tissue seeking a blood meal. As the
mosquito probes, she releases salivary fluid and along with it several dozen up
to a few hundred sporozoites into the dermis of the skin, basically coming
full circle from where we started this story and completing the life cycle of a
primate malaria parasite (reviewed in Ejigiri and Sinnis, 2009).
Plasmodium vivax and its three sibling species in Southeast Asian macaque
monkeys, P. cynomolgi, P. simiovale and P. fieldi, form a group of malaria parasites that have evolved a set of distinctive biological features in their life
cycles that set them apart from the other most often studied of the human
malaria parasite species, P. falciparum (reviewed in Carlton et al., 2008b;
Galinski and Barnwell, 2012, 2008).While there is much more to be understood regarding the evolutionary advantages of these life cycle features for
the parasite, both our present knowledge about their biology and intuitive
reasoning suggest that these parasites have evolved these characteristics as
life strategies to evade and persist in hostile environments whether it be
within their hosts or ecological.
In particular, these parasites have dormant hypnozoite forms in the liver that
are unique to the P. vivax clade of relapsing malaria parasites (Cogswell, 1992;
Cogswell etal., 1991; Krotoski etal., 1982b, 1986).The distantly related human
malaria parasite, P. ovale, has also been known to produce relapse infections
and thus suspected of forming hypnozoites, which may be a good example of
convergent evolution. However, this species and its possible relapses are much
less studied (Richter etal., 2010).The adjunctive liver cycle of the hypnozoite,
as explained further below, enables recurring blood-stage infections, termed
relapses, to occur weeks or months after a primary infection in the blood has
taken place (reviewed in White, 2011).
A second life strategy that P. vivax and the other relapsing malaria parasite
species as well have evolved is to preferentially select reticulocytes as host
cells (Galinski etal., 1992; Kitchen, 1938; Kosaisavee etal., 2011; Li and
Han, 2012), which has been hypothesised to be the source of the benign
status of P. vivax because it limits parasite levels in the blood. However,
as explained below, this probably is not the only reason this young RBC
population provides a selective advantage for P. vivax and its cousin species.
Another distinctive characteristic for P. vivax and other relapsing malarias,
which perhaps is also correlated to the invasion of reticulocytes, is that in
Giemsa-stained blood smears the infected erythrocytes are recognised by a
multitude of reddish dots known as Schffners stippling.These dots actually
represent numerous elaborate flask-shaped and tubular membranous
structures, known as caveola-vesicle complexes (CVCs), which are created
by the parasite and positioned all along the inside of the host cell membrane and open to the exterior (Aikawa et al., 1975; Akinyi et al., 2012;
Barnwell etal., 1990).
A fourth life cycle difference from P. falciparum and likely the other species
of the Laverania subgenus is that P. vivax gametocytes develop quickly and
circulate early in an acute infection, a condition that would allow transmission prior to patients feeling severely ill and seeking treatment (Bousema and
Drakeley, 2011). Each of these biological features is discussed in more
detail in the context of Fig. 1.1, depicting the life cycle of P. vivax, summarising key points that are relevant for understanding and designing
experiments using available technologies, human clinical samples or NHP
models.
2.1. T
he Hypnozoite: An Alternative Life Style for Liver-stage
Development
When a sporozoite of a relapsing malaria parasite enters the liver, it will differentiate into an early small liver trophozoite of about 4.0 or 5.0 in size
and then it may enter either of two very different developmental pathways.
First there can be immediate growth in the host liver cell all the way through
to schizogony and the production of thousands of merozoites that will initiate the erythrocytic cycle. This is the primary pathway for primate Plasmodium species including most strains of P. vivax detailed above. Alternatively,
the small trophozoites become dormant and may remain in this quiescent
metabolic state for weeks, months or up to 2 years in a hepatocyte. Whether
the route taken is a predetermined genetic or epigenetic trait programmed
in the mosquito, a factor of the hepatocyte environment or a combination of
both is unknown as is almost everything else about P. vivax liver-stage parasites
and their relationship with the hepatic host cell. The small dormant forms of
P. vivax (and P. ovale, and the simian species P. cynomolgi, P. simiovale and P. fieldi),
termed hypnozoites, remain a black box for researchers to decipher. At about
45, the hypnozoite is a fraction of the size of a growing primary liver-stage
schizont (4060) (Krotoski etal., 1982b, 1986), and its make-up and what
reactivates it for growth is entirely unknown.
What is known is that P. vivax hypnozoites remain dormant in the liver
for a varied range of times depending to some extent on geographical distribution but more so on what appears to be different strains of the parasite
(Garnham etal., 1975;White, 2011). In the Northern latitudes the so-called
temperate strains, also known as hibernans types, were characterised early
on by their delayed relapse patterns of blood-stage infections. These strains
exhibited a pattern of a first relapse that would occur 8, 9 or 12 months
after being infected with sporozoites. In these cases, there may or may not
be an initial primary parasitaemia following within 2 or 3 weeks after sporozoite infection, which results from some liver-stage trophozoites going
directly through the primary cycle of development. The lack of an early
primary cycle of exo-erythrocytic development indicates that the sporozoites from the temperate strains mostly differentiate into hypnozoites. The
other basic type that is designated as tropical strains have early and frequent relapse patterns at regular intervals of a few weeks initially over 1824
months and almost always with an initial primary blood-stage infection. It
is clear that the temperate form is a life-cycle strategy suited to climates
with long winters and exists to infect mosquitoes emerging in late spring
and early summer (reviewed in Galinski and Barnwell, 2012; White, 2011).
It is not clear, though, what the selective advantage is for the early and
frequent relapse of tropical strains is except perhaps to thwart an emerging
host immune response. Most relapse parasites in individual patients with
tropical strains, even in low transmission conditions, have been shown to
be genetically heterogeneous reflecting past and present infections with
different genotypes of P. vivax (Chen etal., 2007; Imwong etal., 2007). Much
work remains to be done, to fully understand the origins and mechanisms
of dormancy and activation of hypnozoites.
2.2. T
he Reticulocyte as a Host Cell: An Environmental Safety
Program for P. vivax
For P. vivax and the few species of primate Plasmodium dependent on reticulocyte host cells, an efficient process must ensue, enabling these organisms
with the ability to effectively find and latch onto the limited number of
young RBCs amidst a virtual sea of more mature erythrocytes. Generally,
reticulocytes only make up 0.52.0% of the erythrocytes in circulation.
A few proteins have so far been identified and characterised as being
required during the invasion process of the reticulocyte by the merozoite.
The reticulocyte-binding proteins have been defined as critical proteins
that select these host cells in the circulation (Galinski and Barnwell, 1996;
Galinski etal., 1992, 2000).The parasites Duffy Binding Protein has also been
recognised for several decades as a critical RBC adhesin (Chitnis and Miller,
1994;Wertheimer and Barnwell, 1989); only recently have P. vivax infections
been associated with individuals with Duffy-negative RBC phenotypes
(Cavasini etal., 2007; Menard etal., 2010; Ryan etal., 2006). Other proteins in
common with P. falciparum and other Plasmodium species are believed to have
comparable roles across species, as elaborated in Mueller et al. (Chapter 3).
But, critically, the reticulocyte must be targeted prior to the release of
other internally localised adhesins in the merozoite apical organelles, which
would otherwise permit the abortive binding of merozoites to more mature
cells, which likely do not support the continued growth and propagation of
P. vivax (Galinski and Barnwell, 1996; Galinski etal., 1992).
As introduced above, P. vivax trophozoites (and related simian model
parasites, like P. cynomolgi) grow in the reticulocyte and early during this
growth phase the parasitised erythrocyte develops elaborate CVCs all along
the surface of the infected host cell (Aikawa etal., 1975), each appearing in
3D analyses to have its own signature with a different number and length
of tubules and associated vesicles (Akinyi etal., 2012); these structures and
isolated vesicles in the cytoplasm suggest a biogenesis mechanism that may
involve the incremental fusion of such vesicles. These structures include
dozens of parasite-encoded proteins (Barnwell etal., 1990) Udagama et al.,
1988.What metabolic functions may be served by the CVC are only speculative at this time but based on early experiments (Aikawa etal., 1975) and
3D images of hollow openings to the surface (Akinyi etal., 2012) uptake
and perhaps release of metabolites is suggested.The need for these structures
in P. vivax and in each of the other relapsing, reticulocyte-preferring species and the apparent interconnectedness with the host cell and extracellular environment of the host may explain why the field has faced challenges
growing these parasites in long-term invitro culture.We would argue that the
parasite does not only require reticulocytes but also other unknown critical
factors for its development in the reticulocyte that are not currently supplied
by or adequate in current culture media. Ongoing research shows that the
CVCs can release vesicles akin to exosomes as an active mechanism as well as
upon rupture of the infected RBC (Dluzewski etal, unpublished data).
Restricting an infection to these young RBCs, which typically represent 0.52% of the circulating RBCs, may limit the rise in parasitaemia, but
this strategy may also enable P. vivax to alter the infected RBCs (iRBCs) to
remain or become more flexible and able to circulate through small capillary vessels and more likely to survive passage through the sinusoidal vasculature of the spleen (Handayani etal., 2009). This is the opposite strategy of
P. falciparum that invades mature erythrocytes and increases the rigidity of its
host cell such that it must display cytoadherence ligands, which are variant
antigens, at the surface of infected erythrocytes. This allows the parasitised
erythrocyte to sequester in small vessels by adhesion to endothelium and
thus avoid passage through and destruction in the spleen when the intracellular parasite matures. Plasmodium vivax does not have var genes encoding
variant antigens like P. falciparum (Baruch et al., 1995; del Portillo et al.,
2001; Smith etal., 1995; Su etal., 1995) and because of the fluidity/flexibility of its host cell does not need to sequester to avoid spleen passage. To
what degree the evolution of the CVC structures and their placement all
along the iRBC membrane (Aikawa etal., 1975; Akinyi etal., 2012; Barnwell etal., 1990) impacts this cellular flexibility remains to be investigated.
The recently reported but limited adhesive potential of P. vivax iRBCs
(Carvalho et al., 2010) compared to the strong cytoadherence and deep
vascular sequestration of P. falciparum iRBCs does not impact the hypothesis
that selecting and further altering reticulocytes by P. vivax and cousin species also permit the parasitised RBC to circulate through the spleen and
survive.
2.3. Fast and Furious: The Sexual Life Strategies of P. vivax

Gametocyte development for P. falciparum compared to P. vivax gametocyte
development is slow and complex occurring over a period of about 1011
days (Carter and Miller, 1979).The first stage recognisable as gametocytes in
P. falciparum are small round compact forms with hemozoin pigment granules in infected erythrocytes, which soon disappear from the circulation.
10
These sexual-stage parasites are sequestered in the spleen and bone marrow
sinusoids as they mature towards the typical stage V sausage-shaped crescents
that reappear in the blood and are infective for mosquitoes (Smalley etal.,
1980). In sharp contrast, while not studied nearly as much as P. falciparum
gametocytes, especially in the more recent time period, P. vivax gametocytes
at maturity are large, round and fill an enlarged RBC with prominent Schffners stippling and a large nucleus in pink (male) and blue (female) staining
cytoplasm. P. vivax gametocytes are known to develop early in an acute primary infection, within 5 days of the clinical onset. In fact, gametocytes are
known to be produced earlier, perhaps, within 8 days after mosquito inoculation before they can be seen by light microscopy (3060 gametocytes per
microliter) as mosquitoes can become infected at this time (Boyd et al.,
1936; Boyd and Andstratman-Thomaws, 1934). Although not well studied,
P. vivax gametocyte development requires probably about 48h and they do
not remain more than 3 days after differentiation towards sexual maturity.
However, gametocyte densities become greater as blood-stage infections
progress, seeming to come in waves at 5-day intervals and the production of gametocytes continues as the infection progresses on into chronicity becoming asymptomatic or more mildly symptomatic. Plasmodium vivax
gametocytes, which can be efficiently infective to mosquitoes throughout
this time, comparatively seem to be more infective towards susceptible mosquito species than P. falciparum, likely attributable to intrinsic attributes of
both the parasite and the mosquito species (Bousema and Drakeley, 2011;
Boyd etal., 1935). Nevertheless, this ability to form infective gametocytes
early and continuously, in addition to the periodic renewal of blood infections (and gametocyte propagation) by reactivated hypnozoites, makes
P. vivax transmission fast, effective and persistent. Like the asexual stages, the
P. vivax-gametocyte-infected erythrocyte, which continues to circulate in
the blood and not become immobilised in a tissue site as P. falciparum gametocytes do, remains highly flexible and capable of passing through small capillaries or splenic sinusoidal vessels despite containing a large parasite body.
Although we have only discussed P. vivax gametocytes above, the gametocyte biology of P. cynomolgi, or any of the other relapsing malaria parasite
species, parallels that of P. vivax.
There are many Anopheline mosquito species in the tropical, subtropical and, most illustratively, in the temperate latitudes quite far north
that can act as efficient and effective transmitters of P. vivax; more so than
P. falciparum. In fact, at temperatures less than optimal for the continued
development of P. falciparum sporozoites (<16C), P. vivax sporozoites will
11
continue to develop, albeit at a slower pace, in their mosquito hosts, which

likely accounts for P. vivax transmission above a 16 C isotherm but not
P. falciparum.The biological attributes of sporozoite development over a large
temperature range in an array of susceptible mosquito vector species that
over winter combined with delayed relapse genotypes/phenotypes allow
the successful transmission of P. vivax as opposed to the other three human
malaria species, whether it be P. falciparum, P. malariae, or that other human
relapsing parasite, P. ovale, in the more northern latitudes.
3. IN VITRO AND EX VIVO MODELS FOR EXAMINING

P. VIVAX BIOLOGY

Scientists since Bass and Johns (1912) first reported their attempts
at the in vitro propagation of both P. falciparum and P. vivax blood stages
have been attempting to cultivate these two parasite species in a continuous in vitro cycle in test tubes, Leighton tubes, tissue culture flasks and a
variety of other vessels. It was not until 1976 that Trager and Jensen (1976)
and Haynes etal. (1976) reported the continuous invitro development of
P. falciparum blood stages. To date, there is no comparable long-term invitro
continuous culture system to propagate P. vivax-infected RBCs. There have
been various attempts, reported and unreported, over the past 30 or so years
to culture P. vivax-infected RBCs long-term, in vitro, similarly to methods shown to be successful for P. falciparum. However, most attempts have
been no more successful than those of Bass and Johns (1912) reported 100
years ago! The most successful was the attempt by Golenda et al. (1997)
when they cultivated P. vivax Chesson strain parasites for six or seven generations with approximately a two-fold multiplication rate by the addition
of fresh blood highly enriched with reticulocytes every 48h and the use
of McCoys tissue culture medium. This has not been successfully repeated
since. Nonetheless, attempts continue presently with efforts largely focused
on the routine addition of fresh reticulocytes, either from umbilical cord
blood enriched with reticulocytes or produced invitro from erythroid stem
cell cultures (Borlon etal., 2012; Golenda etal., 1997; Grimberg etal., 2012;
Lanners, 1992; Mons, 1990; Mons etal., 1988a, 1988b; Noulin etal., 2012;
Panichakul etal., 2007; Udomsangpetch etal., 2008).
Just as a reliable robust culture system still does not exist for the bloodstage forms of P. vivax, this is also the case for the most closely related simian
malaria species, P. cynomolgi. There has been one publication from 30 years
ago demonstrating the culture of the Berok strain of this species for up to
12
3 weeks invitro in rhesus monkey RBC (Nguyen-Dinh etal., 1981). Our

unpublished results support these claims, but this is the same time limit we
have achieved so far in our attempts to repeat these results. After 3 weeks,
the cultured parasites no longer thrive in vitro despite the routine addition of fresh reticulocyte-enriched target cells. All signs are that the parasite
requires from its host, as per the classic definition of parasitism, factors that
are not sufficiently supplied invitro with the addition of rhesus monkey or
human serum to medium or fresh reticulocytes. Whether further research
will determine what needs to be added to the culture to allow continued
robust propagation beyond 3 weeks remains to be seen. A systems biology
approach may aid this endeavour and those trying to culture P. vivax long
term. Clearly, this same limitation of the current invitro environment for
P. cynomolgi also applies to P. vivax culture investigations and there is a need
that goes beyond just the addition of reticulocytes, which will require systematic investigation of the growth requirements of these parasites and their
metabolites. Metabolomics comparing invivo and invitro-derived samples
and environments may provide the data to solve this problem.
Regardless, in the absence of long-term cultures, reliable short-term
exvivo blood-stage cultures for growth of developmental stages and merozoite invasion assays have been established since 1989 for P. vivax using
strains derived from neotropical New World monkey species infected with
this parasite (Barnwell etal., 1989).These cultures and assays have served the
purpose of obtaining parasite samples for basic research relevant for discovering diagnostics, vaccine development and drug testing as well as investigation of merozoite biology and for genetic transformation of P. vivax (Pfahler
etal., 2006). Similar short-term cultures also exist for P. cynomolgi-infected
blood obtained from infected rhesus monkeys (Nguyen-Dinh etal., 1981).
Other investigators have tried to develop short-term cultures of P. vivax
from ex vivo clinical specimens for invasion assays or drug susceptibility
studies but these have not been especially robust (Grimberg et al., 2007;
Russell etal., 2003). More recently, Bruce Russell and collaborators have
developed a more robust and reliable system to do short-term invitro culture of P. vivax from exvivo clinical samples from patients using umbilical
cord blood RBCs, which can be cryo-preserved and could prove useful
in invasion assays for vaccine studies and growth assays for drug discovery
(Russell etal., 2011).
We and other investigators are routinely using such methods to mature
parasites to the stage of interest for biological and genetic experiments and
to carry out short-term merozoite invasion experiments. Through these
13
assays and collaborations, an increasing number of investigators are managing to circumvent the barriers posed by the lack of long-term cultures. In
effect, and perhaps as a fortuitous bonus, these investigations are working
with parasites that more closely mimic the parasites invivo than do the parasites after long-term adaptation to culture environment. It is worth noting
that ex vivo and invitro comparisons in P. knowlesi blood-stage gene expression were striking, with hundreds of differentially expressed genes (Lapp
etal, unpublished data). It will be useful to examine similar data sets in a
rigorous manner for P. falciparum, from invitro and exvivo derived parasites
samples of the same strain, with Aotus or Saimiri monkeys as hosts.
Investigating the liver stages of P. vivax is even more challenging than
for studies on the blood stages. Sampling of the liver by biopsy for exvivo
studies even in NHP cannot be done and long-term in vitro hepatocyte cultures that mimic the host liver environment are not yet a reality. Short-term liver-stage in vitro assays for P. vivax are possible using
primary human hepatocytes or hepatoma cell lines (Hollingdale et al.,
1985; Mazier et al., 1984). However, high-throughput for drug testing
and discovery investigations require a reliable and, ideally, an aseptic, sustainable source of P. vivax sporozoites. This is quite difficult to come by
for P. vivax.
Some P. vivax liver-stage and vaccine research has relied in the past on
the availability of chimpanzee infections for P. vivax-gametocyte-infected
blood to get highly infected Anopheles mosquitoes by membrane feeding
to produce millions of sporozoites (Collins et al., 2009). Although strict
limitations have since placed these higher primates off limits from conducting most forms of infectious disease research including malaria (Altevogt
etal., 2012), New World monkey infections remain an option, though less
ideal, for obtaining P. vivax sporozoites. However, these animals are in limited supply and with even greater limitations on the availability of honed
expertise and experience for conducting research on P. vivax in these animals (Galinski and Barnwell, 2012). The ideal situation would be to have a
robust reliable invitro culture system, with the added benefit of the continuous production of mosquito infective gametocytes. This added requirement
of a (nonexistent) P. vivax culture system, however, is another tall order, as
gametocytogenesis tends to turn off in the majority of P. falciparum invitro
cultures, which is also the case for P. knowlesi cultures. Still, the development
of P. cynomolgi liver-stage parasites including hypnozoites in primary Macaca
fascicularis hepatocytes maintained invitro for medium throughput assays for
drug discovery remains one backup as high numbers of sporozoites can be
14
obtained from mosquitoes fed on infected rhesus monkeys (Dembele etal.,

2011). However, this system still is limited for the study of hypnozoites
because of its short-term nature due to the limited longevity of primary
hepatocytes invitro and the routine unavailability of aseptic conditions for
obtaining sporozoites, which result in the microbial flora of mosquitoes
contaminating such cultures.
Early models for in vitro study of P. vivax hypnozoites using primary
human liver cells (Mazier et al., 1984), or hepatoma cell lines such as
HepG2-A2 cells (Hollingdale etal., 1985), while capable of producing both
primary schizonts and small forms that appear to be hypnozoites, did not
provide sufficient support (long-term host cell viability, enough invasive
sporozoites in the cells, appropriate metabolism, etc.) for testing of antihypnozoite malarial drugs or truly capable of investigating hypnozoite biology. Production of aseptic P. vivax sporozoites is now feasible for infecting
invitro cultures of HepG2 hepatoma cells and it may be possible to study
hypnozoite biology in this system as well as drug screening, though, with
limitations that come with the difficulties of sourcing P. vivax sporozoites
and the lack of hepatocyte physiology in hepatoma cells (Chattopadhyay
etal., 2010).
4. NEOTROPICAL NON-HUMAN PRIMATE MODELS

(NEW WORLD MONKEYS) FOR INVESTIGATING
THE VARIED BIOLOGY OF VIVAX MALARIA

Prior to 1966, any investigations of P. vivax biology had to garner data from naturally acquired clinical cases, clinical infections induced
by the bites of infected mosquitoes or infected blood for the treatment
of tertiary neurosyphilis, or experimental infections induced in prison
and armed forces volunteers (Mueller et al., Chapter 3). Numerous
attempts to adapt P. vivax to Old World primates of the Cercopithecidae
family had failed miserably and limited use had been made of chimpanzees, which P. vivax can infect. However, as described below, these
primates contain their own fantastic array of malaria parasites species,
some of which can serve as honest, reliable models for P. vivax. However, in 1966, Young and colleagues reported on the adaptation of
P. vivax to small New World monkeys in Panama and could show that
these productive infections could be transmitted to human volunteers
through mosquitoes and back again to these neotropical primates (Young,
1966). Later that same year, Deane etal. (1966) described the infection
15
of squirrel monkeys in Brazil (Deane etal., 1966). From that time forward, continuing for the next 46 years right up to today, a large number of P. vivax isolates of varied biological characteristics ranging from
the frequent early relapsing Chesson strain to the late relapsing North
Korean strain have been adapted to grow in neotropical primate species,
primarily of the genus Aotus (owl or night monkeys) and Saimiri (squirrel monkeys). These NHP adapted strains have been used in studies to
test schizonticidal antimalarial drugs, in malaria vaccine studies for both
pre-erythrocytic and erythrocytic candidate antigens as well as transmission blocking vaccines and for studies of P. vivax biology and genetics in
both the invertebrate mosquito vector and the primate vertebrate host
(Arevalo-Herrera etal., 2011; Galinski and Barnwell, 2012). Today, these
models remain as highly relevant as they did in 1966, especially since we
still cannot culture P. vivax continuously invitro in any practical manner.
One overriding advantage of these neotropical NHP models for P. vivax
is that experimental research designs can be devised to address specific
questions about the biology of this parasite within certain limitations of
these models.
5. T
HE RELAPSING MALARIA PARASITES OF
SOUTHERN ASIAN MACAQUE MONKEYS AS
MODELS FOR P. VIVAX BIOLOGY

In the forests of South and Southeast Asia, the varied species of
the wonderfully adaptive macaque monkeys, such as Macaca fascicularis,
M. nemestrina, M. radiate, M. sinica and other species harbour a varied group
of at least seven or eight and probably more species of primate malaria
(Coatney, 1971). They are transmitted by forest mosquitoes primarily of the
Leucosphyrus group of Anopheles spp, but some can be transmitted by other
anopheline species that transmit human malaria in Southern Asia. These
simian malaria parasite species present with different phenotypic characteristics that can quite remarkably mimic characteristics of the human species
of malaria parasites.
However, more remarkably, genetic studies have shown that within this
group there is a clade of three parasite species that are not only nearly phenotypically identical to P. vivax but also are genetically close, so much so
they must be considered as sibling species of P. vivax (Escalante etal., 2005;
Mu etal., 2005). All four of these species (P. vivax, P. cynomolgi, P. simiovale
and P. fieldi) share the characteristics of 1) hypnozoites that cause relapse
16
parasitaemia, 2) a preference for invading reticulocytes and 3) the production of CVCs in the membrane of infected erythrocytes. Sometime in the
past 70,000 years or so, the ancestor of P. vivax must have jumped from the
monkeys dwelling in the forests our ancestors were trekking through on
the way to Australia and China. It is perhaps of some interest that P. cynomolgi has two Duffy Binding Protein genes, a major ligand that determines
invasion of RBC by merozoites, whereas P. vivax has only one dbp gene
(Carlton etal., 2008a; Tachibana etal., 2012). Is it possible that P. vivax lost
one of its dbp genes and in doing so lost the ability to invade macaque
RBCs? Plasmodium cynomolgi, on the other hand, with two dbp genes also
is capable of infecting humans. An alternative scenario is that the ancestor
of P. vivax first infected our Homo erectus ancestors, also present in Asia for
1.5 million years until about 50,000 BC, who then passed it on to their
Homo sapien cousins.
Plasmodium cynomolgi is the best studied of these NHP-relapsing malaria
species and because of its genetic relationship along with almost identical biological traits in regards to P. vivax offers much as a model for the
human species. In fact, the exo-erythrocytic stages of primate malarias, as
well as, the hypnozoite stage were first discovered in P. cynomolgi-infected
rhesus monkeys (Krotoski et al., 1980; Shortt and Garnham, 1948) prior
to being identified in P. vivax-infected humans and chimpanzees (Krotoski
etal., 1982a, 1982c; Shortt etal., 1948). P. cynomolgi, because of its frequent
relapse patterns exhibited by the hypnozoite stage in the livers of macaques
has been used in many studies for screening 8-aminoquinolines related to
primaquine and other compounds for the discovery of liver-stage drugs
that will provide radical cure of vivax-like malaria (Bray etal., 1985; Deye
etal., 2012; Jiang etal., 1988; Schmidt, 1983). Deye etal. (2012) recently
utilised the P. cynomolgi model to screen antimalarial drug combinations for
preventing relapses.
The P. cynomolgi model will be an invaluable aid for dissecting the biology
of the hypnozoite.The fact that P. cynomolgi can be genetically transformed, as
shown by the genomic integration and expression of the red fluorescent protein
gene (Akinyi etal., 2012) also bodes well for the use of this primate malaria
model in being able to experimentally manipulate and follow hypnozoites
in the livers of experimentally infected macaque monkeys. Additionally, the
development of these imaging tools and purification methods will allow
the study of the biology and metabolic make-up of the developing primary
trophozoites and schizonts in infected hepatocytes, in addition to the hypnozoite forms. The study of P. cynomolgi, with as many as 14 archived strains,
17
provides the opportunity for expedited progress in this area, compared to the
comparably difficult experiments based on experimental P. vivax infections
in small New World monkeys (Aotus or Saimiri species) (reviewed in Galinski
and Barnwell, 2012).
Plasmodium simiovale has been much less studied since its discovery
in 1965 (Dissanaike, 1965). However, hypnozoites have been identified
in this species in rhesus monkeys and studies with mosquito inoculations show that it can have relapses lasting over nearly 2 years that can
be early and frequent or occur late but then with several recurrences at
short intervals (Cogswell etal., 1991; Collins and Contacos, 1979, 1974;
Collins etal., 1972). In summary, P. cynomolgi and P. simiovale have similar
biology to each other and P. vivax and can serve as excellent models of P.
vivax infection. This is an important advantage that has enabled and will
continue to enable the deeper study of P. vivax, especially in the absence
of long-term invitro culture systems.
6. FROM GENOMICS TO SYSTEMS BIOLOGY: THE

BIGGER PICTURE PUZZLES

With the advent of genomics, some 20 years ago, scientists aimed to
glean what they could from analysis of DNA sequences, and then through
comparative genomics.The first P. falciparum genome was published in 2002,
P. vivax and P. knowlesi in 2008 and P. cynomolgi just recently (Carlton etal.,
2008a; Gardner etal., 2002; Pain etal., 2008; T
achibana etal., 2012); these
type of strains have been followed by the availability of sequences from many
other P. falciparum genomes and a number of P. vivax genomes, especially
since NextGen sequencing has amplified what is possible and brought down
the timeframe and cost (Chan etal., 2012). Functional genomics ushered
in the omics era, with emphasis moving from invitro to exvivo transcriptomes and proteomes for P. falciparum (Daily etal., 2007; Dharia etal., 2010;
Kuss et al., 2012; LaCount et al., 2005; Lasonder et al., 2002, 2008) and
recently initial P. vivax transcriptomes (Bozdech etal., 2008; Westenberger
etal., 2010) and proteomes (Acharya etal., 2009, 2011; Roobsoong etal.,
2010; Roobsoong etal., 2011) based on clinical samples.We have been complementing such data with parasites generated in NHP infections initiated
from P. vivax adapted and simian malaria parasite cryo-preserved stocks, from
which we can obtain multiple biological replicates of stage-specific samples
(Lapp etal., unpublished data). As in P. falciparum, the transcriptome alone
cannot precisely predict the expression and timing of protein products; thus,
18
such complimentary work is important. Such studies are also helping groups
focus on what genes and proteins are unique to P. vivax (and its comparable
simian species) and can represent its unique biology. Others have also begun
to investigate the serum proteome of P. vivax-infected individuals, in attempts
to associate differentially expressed proteins with pathogenic features of
vivax malaria and the immune response (Ray etal., 2012). Metabolomics
high-throughput technologies have been developing in parallel as critical
tools in this progression of post-genomics inquiry (reviewed in Kafsack and
Llinas, 2010). Such powerful methods are setting new sights, standards and
expectations (Jones etal., 2012) for driving in depth investigations into the
biology, biochemistry, immunology and pathogenesis of malaria parasitism.
Importantly, the malaria research field is rapidly advancing from the use of
isolated omics technologies and the study of either the host or parasite,
to highly integrated systems biology approaches designed to understand the
hostparasite interactions and dynamics (reviewed in Le Roch etal., 2012).
It is clear that increased efforts in vivax malaria research will rapidly lead
to enormous amounts of heterogeneous information from a wide range of
fields, reaching from molecular biology and omics to physiology, immunology, pathogenesis and toxicology. These data will be too voluminous
to permit optimal analysis with intuitive means and therefore suggest the
utilisation of tools that are at the core of the emerging field of systems
biology (Voit, 2012). A generic strategy might begin with machine learning techniques capable of discerning static or even dynamic patterns in the
very large data sets expected from malaria research laboratories. These patterns are first to be interpreted in terms of simple and complex correlation
structures, which in turn will suggest hypotheses regarding the functional
networks governing the disease and, in particular, the interactions between
pathogen and host, including host cell invasion mechanisms and parasite
evasion strategies. These functional interaction networks are most certainly
regulated very tightly, probably by both the host and the parasite, and must
be expected to change significantly over short and long time horizons.
These dynamic and regulatory features imply that the initial static networks
must be morphed into fully dynamic systems models. These models will
typically be formulated first as differential equations, but may eventually
have to account for stochastic effects, such as environmental insults, and
discrete interventions, such as treatment regimens and reinfection events.
Some techniques for these analyses are yet to be created, but given the
rapid advances in systems biology, there is justified expectation that the
malaria community will have access to effective means for the integration of
19
information into adequate dynamical models within the near future. Given
the enormous capacity of computational methods, thousands of model settings can be investigated quite rapidly, and their interpretation will allow the
laboratory researcher and clinician to prescreen experiments and develop
testable hypotheses. Once such models have been constructed and validated;
they will not only capture details of the infection process but also allow the
simulation of new diagnostics and treatments, possibly even in a personalised manner (Voit and Brigham, 2008).
For vivax malaria research, systems biology approaches may prove to
be the best way forward for investigating the unique biology, immunology and pathogenesis associated with P. vivax infections, and NHP experimental models provide ready tools for exploration at a depth not possible
with human clinical research. Systems biology approaches can be particularly revealing, especially compared to traditional approaches for studying
P. vivax, which in contrast have been painstakingly slow. Fortunately, scientific
methods keep advancing from the old fashion one-gene at a time approach
to a growing number of omic technologies, and now the opportunity
to combine the use of omics to achieve a more complete understanding
of how a system functions. Questions and hypotheses generated from the
field can now be posed to computational biologists who can literally create and model thousands of experimental scenarios to inform the experimental design of actual studies. Plasmodium vivax research, whether through
human clinical sampling or NHP in vivo and ex vivo studies, can harness
these capabilities and start to maximise research output. The time has come
where scientists can dissect the intricacies of the hostpathogen relationship, with all its complex interactions with host cells and factors, to better
understand host cell invasion mechanisms, parasite evasion strategies, the
immune response and ultimately what factors result in successful parasitism
versus death of the parasite or the host. Co-infection dynamics can also be
explored with modelling perspectives to ultimately improve upon the treatment for malaria and other infectious diseases.
With the sensitivity of metabolomics in identifying thousands of metabolites, and the power of computations involving available metadata, it is
reasonable to start to imagine what cultural/social/nutritional and geographical/environmental factors may be potentially associated with the
severity of disease and possible stress factors that may also help predict the
relapse of hypnozoites. Its particular biological features enable the persistence of this parasite to the extent that this organism is also gaining the
interest of ecologists.
20
7. CONCLUSIONS

The rapid pace of science and technology is making inroads that are
bringing P. vivax research to the forefront, and no longer leaving it in the
dust behind P. falciparum. The potential for a comprehensive understanding
of this organism, hostpathogen interactions, and the global nature of this
disease is at an all-time high.
ACKNOWLEDGEMENTS
The authors acknowledge the financial support of the National Institutes of Health,
National Institutes of Allergy and Infectious Diseases, Grant # R01AI24710 and Contract
# HHSN272201200031C, as well as the National Center for Research Resources
P51RR000165 and the Office of Research Infrastructure Programs/OD P51OD011132.
The authors also acknowledge the scientists from the Malaria HostPathogen Interaction
Center (MaHPIC) for their inspiration and contributions (http://mahpic.emory.edu).
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CHAPTER TWO
Red Blood Cell Polymorphism

and Susceptibility to
Plasmodium vivax
Peter A. Zimmerman*,1, Marcelo U. Ferreira, Rosalind E. Howes,
Odile Mercereau-Puijalon$
*Center for Global Health & Diseases, Case Western Reserve University, Cleveland, Ohio, USA
Departamento de Parasitologia, Instituto de Cincias Biomdicas, Universidade de So Paulo, So Paulo
(SP), Brasil
$Institut Pasteur, Centre National de la Recherche Scientifique Unit de Recherche Associe, Unit
dImmunologie Molculaire des Parasites, Paris, France
1Corresponding author: E-mail: paz@case.edu / Phone 1-216-368-0508
Contents
1. Introduction
2. T he Era of Great Biological Discovery
2.1. C
ell Biology and the Germ Theory
2.2. M
alariotherapy and African-Based Resistance to P. vivax
2.3. H
uman Variation and Blood Groups
3. R
esistance to P. vivax and Insights on Malaria Red Cell Invasion
3.1. S erological Recognition of Duffy (Fy) Blood Group Polymorphism
3.2. T he Genetic Resistance Factor to P. vivax Duffy Negativity
3.3. T he Molecular and Cellular Basis of Duffy Blood Group Polymorphism
3.4. T he Duffy Binding Protein
4. E volving Perspectives on Resistance to P. vivax
4.1. F urther Influence of Duffy Polymorphism on Resistance to P. vivax Malaria
4.2. D
uffy-Independent Red Cell Invasion by P. vivax
4.3. G
lobal Distribution of Duffy Polymorphism and the Population
at Risk of P. vivax Malaria
4.4. A
ssociation of Non-Duffy Gene Polymorphisms with P. vivax Resistance
4.4.1. G
6PD Deficiency
4.4.2. Haemoglobinopathies
4.4.3. Southeast Asian Ovalocytosis
5. C
onclusions and Future Directions
Acknowledgements
References
2013 Elsevier Ltd.

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Peter A. Zimmerman et al.
Abstract
Resistance to Plasmodium vivax blood-stage infection has been widely recognised to
result from absence of the Duffy (Fy) blood group from the surface of red blood cells
(RBCs) in individuals of African descent. Interestingly, recent studies from different malariaendemic regions have begun to reveal new perspectives on the association between
Duffy gene polymorphism and P. vivax malaria. In Papua New Guinea and the Americas,
heterozygous carriers of a Duffy-negative allele are less susceptible to P. vivax infection
than Duffy-positive homozygotes. In Brazil, studies show that the Fya antigen, compared
to Fyb, is associated with lower binding to the P. vivax Duffy-binding protein and reduced
susceptibility to vivax malaria. Additionally, it is interesting that numerous studies have
now shown that P. vivax can infect RBCs and cause clinical disease in Duffy-negative
people. This suggests that the relationship between P. vivax and the Duffy antigen is more
complex than customarily described. Evidence of P. vivax Duffy-independent red cell invasion indicates that the parasite must be evolving alternative red cell invasion pathways.
In this chapter, we review the evidence for P. vivax Duffy-dependent and Duffyindependent red cell invasion. We also consider the influence of further host gene
polymorphism associated with malaria endemicity on susceptibility to vivax malaria.
The interaction between the parasite and the RBC has significant potential to influence the effectiveness of P. vivax-specific vaccines and drug treatments. Ultimately, the
relationships between red cell polymorphisms and P. vivax blood-stage infection will
influence our estimates on the population at risk and efforts to eliminate vivax malaria.
1. INTRODUCTION

The image of Plasmodium knowlesi pulling its way into erythrocytes of
Macaca mulatta is iconic in malaria research (Fig. 2.1) and sets the stage for reviewing the mechanisms of human resistance to Plasmodium vivax. Aikawa and colleagues described the events underlying erythrocyte invasion to involve an initial
attachment to the red cell membrane by the parasites apical end, invagination of
the red cell membrane around the merozoite and sealing of the erythrocyte on
completion of invasion (Aikawa etal., 1978). As brilliantly shown through their
electron micrographs, evidence of a tight junction formed through molecular
interactions between the parasite and host continues to inspire malaria research.
Identifying specific molecules involved in the formation and gliding motility
of this junction is central to unravelling the mechanism of Plasmodium species
invasion of the red cell. Understanding how to inhibit, disrupt or block this intimate parasitehost interaction potentially leads to strategies for a vaccine against
blood-stage infection, malaria morbidity and mortality.
In this chapter, we rely on a wide range of clinical, field and laboratory
findings to illustrate our evolving understanding of the factors that influence
resistance to P. vivax malaria and the selective barrier that has confronted
this parasite. Reviewing this work according to a general chronological
Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax
29
Figure 2.1 Plasmodium knowlesi invasion of Macaca mulatta red blood cells.
A P. knowlesi merozoite has commenced the invasion process through formation of gliding junctions involving merozoite and red cell membranes. This enables invagination
of the erythrocyte membrane and movement of the parasite into the parasitophorous
vacuole. R, rhoptry; M, micronemes; J, gliding junction; PV, parasitophorous vacuole; E,
erythrocyte. (Figure from unpublished data, Hisashi Fujioka)
time frame will remind readers how our understanding of P. vivax infection
and malaria has developed over the past 95 years. This approach also seeks
to emphasise how medical and basic research scientists have applied available experimental strategies in collaborations across multiple generations to
solve the important puzzle as to how malaria parasites infect red blood cells
(RBCs) and cause a disease that has had significant impact on human health
and the evolution of our genome.
2. T
HE ERA OF GREAT BIOLOGICAL DISCOVERY
2.1. Cell Biology and the Germ Theory
The late 1800s to the early 1900s was a revolutionary time period that began
the integration of medicine and the sciences. Of paramount importance to
this chapter is the germ theory that proposed that microorganisms were the
cause of many diseases. Pasteurs experimental evidence showing that microorganisms in nutrient broth did not arise through spontaneous generation
30
(1860s) significantly demystified the relationship between disease and the

microbial world, and Kochs series of objective criteria provided a formal test
to link specific microbes to specific diseases (1890). Following this lead, in
1880, Laveran first linked human malaria to infection of RBCs by plasmodia
(Laveran, 1880) (P. falciparum (Welch 1897), P. vivax and P. malariae (Grassi and
Feletti, 1890) as well as P. ovale (James, 1929)). During this same time, the medical discipline of psychiatry was coming to understand that infection with the
spirochaete bacterium Treponema pallidum, caused syphilis (Schaudinn, 1905;
Schaudinn and Hoffman, 1905) and that the resulting disease could advance
to cause numerous visceral forms and overlapping clinical outcomes (Merrit
etal., 1946). Primary infections (marked by the appearance of a chancre at
the site of the infection) would heal in 2 to 6 weeks without leaving a scar.
Secondary stages would lead to clinical symptoms in approximately half of all
cases, producing skin lesions, rash or other generalised inflammatory symptoms. Following a period of latency that could last for years, approximately
one-quarter of patients would go on to develop tertiary stages (paretic and
tabetic neurosyphilis; general paralysis of the insane (Brandt, 1985)). Despite
an improved understanding of the relationship between the microbe and
the natural history of the disease, treatment of syphilis remained based on
administration of mercury by mouth, injection, dermatologic application or
exposure to its vapours to purge the humour through salivation or sweating.
Understandably, optimism greeted Paul Erlichs development of the arsenicals
salvarsan (1910) and neosalvarsan as potential magic bullets against syphilis;
however, these treatments still exposed patients to significant risk and still
failed to ward off or cure neurosyphilis (Stokes and Shaffer, 1924; Arnold,
1984; Jolliffe, 1993). Patients with neurosyphilis were difficult to manage,
became completely demented and unable to care for themselves, dying most
often in insane asylums (Brown, 2000). Accurate syphilis prevalence data is
difficult to come by as the disease carried significant social stigma; however,
population estimates in Europe and the United States suggested that 15% of
the general population had the disease (Stokes, 1918). With at least 1030%
of syphilis patients requiring long-term palliative care, mental institutions
were being pushed beyond their effective operating capacities (Stokes, 1918;
Chernin, 1984; Hook and Marra, 1992). As a result, there was considerable
interest in developing more effective treatments for neurosyphilis.
Sporadic reports had been published that mentally ill syphilitic patients
who experienced bouts of fever showed signs of recovery or remission
(Brown, 2000), and in 1876, Rosenblum reported that approximately
50% of psychiatric patients were cured after an attack of recurrent fever
31
(Chernin, 1984). It was also noted that observation of neurosyphilis was

uncommon in malaria-endemic regions of Africa (Merrit etal., 1946). In
an 1887 review, the Viennese psychiatrist Julius Wagner-Jauregg noted 163
incidents of psychoses remitting following typhoid, intermittent fevers or
erysipelas. While findings of this nature seemed to encourage treatment of
paretic patients by artificially inducing fever, the dangers of experimental
treatment of human beings through exposure to agents such as tuberculin
or injection of malarial parasites made physicians reluctant to perform these
procedures for fear of legal repercussions (Brown, 2000).
Wagner-Jaureggs first treatments of neurosyphilis patients with malariotherapy occurred in 1917, when a shell-shocked soldier from the Macedonian Front was admitted to the hospital in Vienna. Coincidently, this
patient was experiencing malaria fevers and chills. With blood from this
patient, Wagner-Jauregg was able to induce fevers in a small number of
paretic patients (Withrow, 1990). While improvements were observed in
this first series of malaria-treated patients, complications associated with
malaria tropica (falciparum malaria) soon became apparent1. After establishing a steady supply of benign tertian malaria (vivax malaria), WagnerJauregg reported in 1921 that 25% of his first 200 patients were able to
return to work (Brown, 2000). While malariotherapy was not without risk,
the success reported by these early trials quickly led to widespread practice throughout Europe and treatment of paretic patients with malariotherapy was first attempted in the United States in 1922. In the United
States, equally positive results as experienced in Europe were observed
following treatment with tertian malaria as Paul OLeary and colleagues
described in a first report on malariotherapy at the Mayo Clinic (Minnesota)
1 Human malaria is caused by five parasite species (Plasmodium falciparum, P. vivax, P. malariae, P. ovale
and recently P. knowlesi in Malaysia (Cox-Singh and Singh, 2008)). Tertian malaria (recurring fevers at
approximately 48-hour intervals) caused by P. vivax has been classified as benign, while disease associated with P. falciparum has been classified as malignant. While P. vivax was preferred over P. falciparum
by practitioners of malariotherapy (Becker, 1949, Chernin, 1984, Withrow, 1990), the literature notes
that induced malaria was not without risks for patients. Furthermore, in a note on The Nomenclature of Malaria by Bruce (1903), it is noted I have left out the commonly used terms, simple, benign,
malignant, pernicious, as they are misleading. The so-called simple tertian may often be more severe
than the so-called malignant tertian (Bruce, 1903). General differences between vivax and falciparum
malaria have been described frequently (Zimmerman etal., 2004, Price etal., 2007). Plasmodium
vivax shows a selective preference for infecting young red blood cells (reticulocytes) (Kitchen, 1938,
Garnham, 1966), where P. falciparum infects a wider range of red cells (Kitchen, 1939); as reticulocytes
comprise less than 1% of the circulating red blood cells, this preference may constrain P. vivax parasitaemia. P. vivax and P. ovale are noted to produce dormant liver stages, termed hypnozoites (Krotoski
etal., 1982, Krotoski, 1989). Plasmodium falciparum and P. malariae do not produce hypnozoites. Hypnozoites can be reactivated after clearance of primary infection to cause relapses weeks to years later.
32
(OLeary etal., 1926). Because of the impact of malaria treatment on neurosyphilis, Wagner-Jauregg was awarded the Nobel Prize in Medicine in
1927 (Withrow, 1990).
2.2. Malariotherapy and African-Based Resistance to P. vivax

Treatment of neurosyphilis in the United States significantly expanded the
practice of malariotherapy. In the application of malariotherapy to AfricanAmericans in particular, publications documented that certain individuals
were observed to exhibit notable immunity to infection by P. vivax. Early
evidence of this clinical observation was noted in published discussions of
papers presented to the Dermatology and Syphilology section of the American Medical Association.
May 19, 1927; Washington, D.C.
Dr. Watson W. Eldridge, Jr., St. Elizabeths Hospital, Washington, D.C. I inoculated
the first patient in December, 1922, and we have treated approximately 275 cases
since...I should like to know whether, in the group in which the malaria failed to take,
reinoculations were successful. I have had several inoculation failures, principally
among coloured males.
(OLeary, 1927)
Dr. Paul A. OLeary, Mayo Clinic, Rochester, Minn. - ...We have reinoculated as many as
six times those patients who have not developed chills and fever and have not been
successful in obtaining a take.
(OLeary, 1927)
Resistance to vivax malaria was seen to be the primary disadvantage that

would prevent malariotherapy from being used as a routine treatment of
neurosyphilis. When carefully controlled studies were performed in the
context of malariotherapy, details showed that African-Americans and
Africans consistently displayed significantly higher levels of resistance to
P. vivax strains from numerous geographic origins and inoculation doses
compared to Caucasians (Young etal., 1955). Additionally, because AfricanAmericans from nonmalarious regions of the United States were as refractory to P. vivax infection as those from malarious regions, this resistance was
suggested to be natural rather than acquired (Boyd and Stratman-Thomas,
1933; Becker etal., 1946;Young etal., 1946;Young etal., 1955; Bray, 1958).
Of further interest, to determine if a P. vivax infection once established in
an African-American patient would acquire characteristics enabling more
successful infection of resistant individuals, Young etal. used blood from a
P. vivax-infected African-American to inoculate two resistant individuals of
the same race (Young etal., 1955). Despite receiving inocula three to seven
33
times higher than that routinely causing blood-stage infection of Caucasian

patients, neither of the resistant individuals developed blood-stage parasitaemia. From these results it was concluded that the P. vivax strain would
not be transformed to acquire characteristics that would enable subsequent
infection of resistant individuals (Young et al., 1955). Interestingly, it was
also reported that African-Americans displayed resistance to P. knowlesi and
P. cynomolgi in addition to P. vivax. From their studies with patients at the
Manhattan State Hospital, Milam and Coggeshall reported that AfricanAmerican patients experienced significantly milder P. knowlesi infections
compared to Caucasian patients as measured by a delayed time to first bloodstage parasitaemia and shorter duration of blood-stage infection (Milam and
Coggeshall, 1938). Following their report of accidental human infections
with P. cynomolgi (Eyles etal., 1960; Eyles, 1963), Beye etal. wanted to further test whether non-human primates could act as reservoirs of malaria.
Their follow-up study included 7 African-American and 13 Caucasian volunteers from the US Penitentiary in Atlanta. An overall summary of their
results showed that blood-stage parasites were not observed in any of the
African-American study participants, but 12 of the 13 Caucasian patients
did exhibit blood-stage parasitaemia (Beye etal., 1961). As P. knowlesi and
P. cynomolgi were observed to have difficulties similar to P. vivax for causing
malaria in African-American patients and study participants, results have
suggested that these parasite species may infect the human RBC by similar
invasion pathways.
In 1947, Butler and Sapero published an interesting exceptional report
on resistance to P. vivax among African-Americans following natural exposure to P. vivax in the South Pacific. The authors wrote that, With the onset
of the present war in the Pacific and the arrival of negro [sic] troops on
highly malarious bases in the South Pacific (Melanesia), it was hoped that
the negro [sic] might be spared the ravages of Pacific vivax malaria because
of his racial tolerance to the United States strains (Butler and Sapero, 1947).
The authors study design indicated that the surveyed population included
several thousand troops and that 28% were African-American (2035 years
of age; primarily from the Carolinas and Georgia), and virtually no malaria
was noted among the study group during training procedures in the United
States. Significant exposure to Anopheles farauti was noted once the troops
were deployed in the South Pacific and while suppressive atabrine was provided, a sizable incidence of initial and recurrent malaria attacks indicated
that this prophylactic treatment was not taken regularly. Plasmodium species
infections were calculated monthly for primary and recurrent attacks.While
these infections were not differentiated according to racial groups, the study
34
observations suggested that this was not a serious omission.The results from
the study showed that 90.5% of all re-admissions were due to P. vivax and
that 41.5% of the re-admissions occurred in the African-American group.
The authors conceded that if the entire 9.5% of the non-vivax re-admissions occurred in the African-American group, 32% of African-American
re-admissions would have correlated with P. vivax infection (Butler and
Sapero, 1947). In contrast to reports from malariotherapy trials, the conservative evaluation of this study population suggested that African-American
troops were highly susceptible to blood-stage infection by Pacific strains of
P. vivax when naturally exposed to the parasite.
2.3. Human Variation and Blood Groups

A theoretical synthesis similar to the one occurring in the cell biology and
infectious disease world also gained momentum in genetics and evolution at the dawn of the twentieth century. At this time, Darwins On the
Origin of Species had stimulated wide-ranging controversy throughout the
scientific community. Although Mendels re-discovered work with garden
peas provided a foundation for understanding the dynamics of heredity,
opposing factions debating the role of natural selection in evolution took
his observations to argue that evolution resulted from successive leaps or
gradual change; the role of mutation in natural selection of new species
was at first questioned (Mayr and Provine, 1981). The population biologists
Fisher, Wright and Haldane promoted ideas that genes worked together to
bring about genetic variation in populations. From this population-based
perspective, genetic variations that optimised fitness would be transmitted
from one generation to the next (Mayr and Provine, 1981). The Malaria
Hypothesis, first proposed by Haldane, is a well-known but counterintuitive twist of population biology and selection theory. This hypothesis proposes that otherwise harmful mutations (e.g. the thalassaemias) are
transmitted at higher frequencies in some populations because they confer
selective advantages against plasmodia and balance susceptibility to malaria
(Haldane, 1949).
Practical implications regarding the influence of mutation on phenotype, heritability and human population biology came to light in studies
on blood transfusion, where many of the first human genetic polymorphisms were identified through serological cross-reactivity, recognising
variations in blood group antigens (Race and Sanger, 1950; Mourant
et al., 1976). Karl Landsteiner made the first observations that serum
from healthy humans had an agglutinating effect on the blood corpuscles
35
of other humans, leading to the identification of the ABO blood group

system in 1901 (Landsteiner, 1901). Blood group systems enabled early
human geneticists to test heritability of these polymorphic traits, provided
consistent opportunity to test population genetic hypotheses and allowed
them to make some of their first observations regarding genetic similarities and differences between races and ethnicities. At present, there are 32
blood group systems inclusive of over 600 specific antigens, encoded by
42 genes; overall polymorphism is captured among 1312 alleles (Table
2.1). Cross-reacting antibodies recognise extracellular epitopes of proteins imbedded in the RBC membrane. Malarial parasites would naturally
encounter these epitopes when contacting the red cell prior to bloodstage infection. Therefore, it is not surprising that a number of these
blood group proteins have been implicated in influencing susceptibility
to malaria (Table 2.1).
3. RESISTANCE TO P. VIVAX AND INSIGHTS ON

MALARIA RED CELL INVASION
Investigator Profile: An Interview with Louis Miller, M.D., with Vicki Glaser.
(Glaser, 2004)
[In the early 1970s] we knew that invasion was very specific each type of
Plasmodium goes to a specific host but we did not know why. At that time I was
working on a monkey parasite, P. knowlesi The parasite would only invade certain
types of RBCs [in tissue culture], such as human or monkey cells, and it would not
invade others such as mouse As host red cell specificity was likely based on surface
molecules that act as receptors, I began to study red cellsnull for various blood
groups in the hope that one would be the receptor for P. knowlesi invasion of human
red cells. I found that P. knowlesi was not able to invade Duffy blood group negative
red cells. I went to the library that night, and I knew right away that I had discovered
the missing factor for the resistance of West Africans to P. vivax.
(Louis Miller)
3.1. S
erological Recognition of Duffy (Fy) Blood Group
Polymorphism
The Duffy blood group antigen (Fya) was first observed in 1950 on erythrocytes using allo-antisera found in a multiply transfused haemophiliac
(named by permission of the patient) at the time a haemolytic transfusion
reaction was observed (Cutbush etal., 1950).The expected Fyb antisera was
discovered in Berlin shortly thereafter (Ikin etal., 1951); surveys of European
36
Table 2.1 Blood Group Systems

No.
1
2
No. of
Antigens
System
Symbol
ABO
MNS
4
46
ABO
MNS
P
Rh
Lutheran
Kell
Lewis
Duffy
Kidd
Diego
Yt (Cartwright)
Xg
Scianna
Dombrock
Colton
Landsteiner-Wiener
Chido/Rodgers
1
50
19
31
6
6
3
21
2
2
7
6
3
3
9
P1
RH
LU
KEL
LE
FY
JK
DI
YT
XG
SC
DO
CO
LW
CH/RG
Gene Name(s)
ABO
GYPA, GYPB,
GYPE
RHD, RHCE
LU
KEL
FUT3
DARC
SLC14A1
SLC4A1
ACHE
XG, MIC2
ERMAP
ART4
AQP1
ICAM4
C4A, C4B
Chromosomal
Location
CD
Malaria
Discovered
9q34.2
4q31.21
CD235
Yes
Yes
1900
1927
22q11.2-qter
1p36.11
19q13.32
7q34
19p13.3
1q23.2
18q12.3
17q21.31
7q22.1
Xp22.33
1p34.2
12p12.3
7pl4.3
19p13.2
6p21.3
CD240
CD239
CD238
CD234
CD233
CD99
CD297
CD242
Yes
1927
1940
1945
1946
1946
1950
1951
1953
1956
1962
1962
1965
1967
1942
1962
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
System Name
H
Kx (McLeod syndrome)
Gerbich
Cromer
Knops
Indian
Ok
Raph
John Milton Hagen
I
Globoside
Gill
Rh-associated glycoprotein
1
1
8
15
9
4
1
1
5
1
1
1
3
H
XK
GE
CROM
KN
IN
OK
RAPH
JMH
I
GLOB
GIL
RHAG
FUT1
XK
GYPC
CD55
CR1
CD44
BSG
CD151
SEMA7A
GCNT2
B3GALT3
AQP3
RHAG
19q13.33
Xp21.1
2q14.3
1q32.2
1q32.2
11p13
19p13.3
11p15.5
15q24.1
6p24.2
3q26.1
9p13.3
6p21-qter
CD173
CD236
CD55
CD35
CD44
CD147
CD151
CD108
CD241
Yes
Yes
1952
1977
1960
1965
1970
1973
1979
1978
As of March 5, 2012, there are 32 blood group systems, 42 genes and 1312 alleles. For the latest figures, please consult the following website. http://www.ncbi.nlm.
nih.gov/projects/gv/rbc/xslcgi.fcgi?cmd=bgmut/summary.
18
19
20
21
22
23
24
25
26
27
28
29
30
37
38
populations suggested frequencies for the co-dominantly expressed Fya and

Fyb antigens of 35% and 65%, respectively (Cutbush etal., 1950; Ikin etal.,
1951). Upon screening, a series of blood samples from African-American
donors to the Knickerbocker Blood Bank of New York City, Sanger etal.
observed that 68% of the samples did not react with either the Fya or Fyb
antisera (Sanger etal., 1955). Additional analysis of Nigerian families showed
that the null phenotype was inherited in Mendelian manner and provided
an opportunity to investigate Fya copy number. In an earlier study, Race
et al. found that the antiserum Pri reliably distinguished between single
and double donors for Fya (Race etal., 1953). Results obtained from tests
on three African-Americans who were phenotypically Fy(a+b+) and two
who were Fy(a+b) all suggested that Fya was observed to be present in
single-dose quantity, as compared to double-dose quantities observed in
Fy(a+b) Europeans (Sanger et al., 1955) (Duffy blood group nomenclature is summarised at the conclusion of Section 3.3; Table 2.2). These
observations suggested that those of African ancestry possessed either a different antigen, Fyc, or they did not express the Duffy antigen and carried a
Duffy-null allele, Fy0. Blood group researchers hypothesised that identification of an Fyc antigen would be forthcoming as a result of large numbers of
blood transfusions involving African donor and Caucasian recipient pairs,
or through their attempts to stimulate anti-Fyc reactivity through injection of Fy(ab) red cells into European volunteers (Sanger etal., 1955).
The failure to discover the Fyc antigen and therefore the possibility of a Duffynull phenotype was not unanticipated. A comparable observation had been
made previously in the MNSs system (Table 2.1), where an occasional African blood sample reacted with neither the anti-S nor the anti-s antisera
(Sanger etal., 1955).
3.2. T
he Genetic Resistance Factor to P. vivax Duffy
Negativity
The impressive distribution of the Fy(ab) phenotype in diverse African populations has been a fascination to population geneticists, evolutionary biologists and infectious disease physicians and biologists. Based
on the overlapping distribution of the Fy(ab) phenotype, the very low
prevalence of P. vivax in African populations (Bray, 1957) and the desire to
identify receptors used by malarial parasites to invade human erythrocytes
(Butcher etal., 1973; Miller and Dvorak, 1973), Louis Miller and colleagues
at the US National Institutes of Health performed a series of studies in
the mid-1970s to determine if red cells deficient for any of the human
39
blood group systems would resist infection using newly developed abilities
to culture malarial parasites in the laboratory (Butcher and Cohen, 1971).
In their studies, Miller and colleagues first observed that the non-human
primate malarial parasite P. knowlesi was not able to infect erythrocytes from
Fy(ab) African-Americans invitro (Miller etal., 1975), while the parasite easily infected erythrocytes from Fy(a+b), Fy(a+b+) and Fy(ab+)
donors. From these results, Miller etal. hypothesised that Fy(ab) would
explain the absence of P. vivax from West Africa where blood group system
surveys were reporting that Fy(ab) frequency was nearly 100% (Mourant
etal., 1976). Millers invitro findings led directly to a study testing the invivo
susceptibility of Duffy-negative and Duffy-positive individuals to P. vivax
blood-stage infection (Miller etal., 1976). In this study, 17 consenting prisoner volunteers were first characterized for their Duffy blood group phenotype serologically. P. vivax-infected mosquitoes were then allowed to take
blood meals, first from Fy(ab) African-Americans, and then following
interruption, were allowed to continue feeding on Fy(a+b), Fy(a+b+) and
Fy(ab+) Caucasian and African-Americans. Results showed that none of
the five Fy(ab) study subjects developed blood-stage parasitaemia despite
evaluation of daily blood smears for 90180 days, while all 12 of the Duffypositive individuals developed blood-stage infection within 15 days (Miller
etal., 1976).
While this study showed strong evidence that P. vivax required the Duffy
blood group antigen to be present on the erythrocyte surface to invade the
cell successfully and continue its life cycle, no information beyond basic
susceptibility to blood-stage infection was produced. Studies at this point of
the investigation neither tested for differences in susceptibility based on Fya
vs. Fyb, nor were they on individuals who were heterozygous for the Duffynegative allele, expressing a single gene dose of the Duffy blood group
protein. Further comparisons regarding P. vivax susceptibility among these
Duffy phenotypes could have provided important insight towards understanding the relative selective differences among the Fya, Fyb and Duffynegative alleles.
In an attempt to explain how the frequency of Duffy blood group negativity had risen to 100% corresponding with the absence of P. vivax from
vast regions of malaria-endemic West Africa, Miller and colleagues offered
the following hypotheses. Although P. vivax infection rarely causes death,
it may decrease survival in African children with malnutrition and other
endemic diseases as the frequency of the Duffy-negative gene increased
in the population, the number of susceptible persons decreased below a
40
critical level, and P. vivax disappeared from the region (Miller etal., 1976).
This hypothesis suggests that the Duffy-negative phenotype increased the
fitness of human populations against vivax malaria and would have led to
the evolution of a human host population in which P. vivax was not able to
reproduce with enough success to maintain its life cycle. This, and queries
about other pathogens that may interact with the Duffy antigen have fuelled
the debate surrounding the relationship between P. vivax and evolution of
the Duffy-negative phenotype for decades. (Livingstone, 1984; Carter, 2003;
Kwiatkowski, 2005; Rosenberg, 2007).
3.3. T
he Molecular and Cellular Basis of Duffy Blood Group
Polymorphism
As identification of Fyc was not forthcoming, understanding the molecular
differences responsible for this and the other Duffy blood group polymorphisms would rely on the advance of molecular biology. Methodical progress towards cloning the Duffy gene can be marked through attempts to
purify the Duffy protein (Moore etal., 1982; Hadley etal., 1984; Chaudhuri
et al., 1989) and identification of a series of Duffy epitopes: Fy3 (Albrey
etal., 1971), Fy4 (Behzad etal., 1973), Fy5 (Colledge etal., 1973) and Fy6
(Nichols etal., 1987) and their respective antisera. These antisera have been
used to illustrate that the Duffy protein was characterized by a number
of different epitopes. It is therefore important to note that all these epitopes were absent from Fy(ab) African individuals. Beginning in 1988,
Chaudhuri and colleagues at the New York Blood Centre described a series
of experiments using the murine anti-Fy6 monoclonal antibody to affinity purify Duffy antigens from solubilised erythrocytes (Chaudhuri etal.,
1989). Chaudhuri etal. further studied these Duffy peptides by amino acid
sequencing and synthesis of a DNA probe to identify a gene-specific cDNA
molecule (Chaudhuri et al., 1993). Through this process, they identified
a 338 codon open reading frame (ORF) sequence exhibiting significant
homology to the human interleukin 8 receptor, predicting seven transmembrane segments, an extracellular amino terminus, three extracellular loop
domains, three intracellular loop domains and a carboxy-terminal cytoplasmic tail (Fig. 2.2) (Chaudhuri etal., 1993). Further studies on the genomic
organisation of the Duffy gene sequence confirmed early predictions that
the gene locus was present in a peri-centromeric region of human chromosome 1 (1q22-23) (Donahue etal., 1968; Dracopoli etal., 1991; Mathew
et al., 1994). While the role of the Duffy blood group antigen is potentially of great interest in allergy (Vergara etal., 2008), cardiovascular disease
41
(Reich etal., 2009), cancer biology (Shen etal., 2006) and HIV-AIDS (He
etal., 2008), we will not cover these topics here. Additional details related
to Duffy antigen chemokine receptor biology (Pruenster etal., 2009) are
provided in the legend to Fig. 2.2.
Specific analysis of Duffy cDNA molecules (5-RACE) produced evidence that the gene was composed of two exons (Iwamoto etal., 1996).
Exon 1 was observed to encode seven amino acids, MGNCLHR; exon
2 was found to encode 338 amino acids. It was subsequently shown that
the primary transcript of the Duffy gene was composed of codons 17
from exon 1 joined to codons 10338 from exon 2 encoding a protein of
336 amino acids; this splice variant is expressed in erythroid lineage cells.
Additional studies characterizing the Duffy gene were also successful in
identifying a single nucleotide polymorphism (SNP) in codon 42 associated
with the Fya (GGT; encodes glycine) and Fyb (GAT; encodes aspartic acid)
antigens (Chaudhuri etal., 1995; Iwamoto etal., 1995; Tournamille etal.,
1995b). Then, after acknowledging no additional polymorphism compared
to the FY*B allele, suggesting that Africans carried no important disruption in the Duffy ORF (Chaudhuri etal., 1995; Tournamille etal., 1995a),
Tournamille etal. discovered a T to C SNP 33 nucleotides upstream from
the primary transcription starting position (33) in the Duffy gene promoter (originally positioned at nucleotide 46) (Tournamille etal., 1995a),
resulting in an FY*BES allele (ES=erythroid silent). Duffy-negative Africans were homozygous for this polymorphism that was shown to occur in
a tissue-specific GATA1 transcription-factor-binding motif. Invitro assays
showed that this polymorphism blocked gene expression in erythroid lineage cells but did not block expression in non-erythroid cells. Results of
this study provided the molecular genetic explanation for erythrocyte Duffy
negativity.
Since the identification of the Fya, Fyb and Duffy-negative SNPs, addi
tional less-common variants have been identified to provide a more complete
description of Duffy genotype and serological phenotype polymorphisms.
Following identification of the FY*BES allele, Zimmerman etal. sought to
determine if the Duffy gene in Papua New Guineans living in P. vivaxendemic regions would be characterized by accumulation of any functional
polymorphism. A survey of the Duffy gene promoter and ORF polymorphisms identified above revealed that the same promoter SNP found on the
African FY*BES allele was observed on the resident Papua New Guinea
(PNG) FY*A allele (suggests FY*AES) (Zimmerman et al., 1999). Flow
cytometry comparing anti-Fy6 (Nichols etal., 1987) antibody binding to
42
Figure 2.2 The Duffy antigen. The diagram illustrates the primary structure of the
236-amino-acid 3646-kDa Duffy antigen with seven predicted transmembrane domains
and extracellular and intracellular domains. Amino acids comprising the Fy6 and Fy3
antibody-binding domains are marked by brackets. Amino acid sequence polymorphisms are identified at residues 42 (G vs. D; Fya vs. Fyb), 89 (R vs. C; Fyb vs. Fybweak) and
100 (A vs. T) and the two premature termination codons (W vs. X) at residue positions 96
and 134. Glycosylation sites are identified at amino acid residues N16 and N27. Disulfide
bonds occurring between C129 (extracellular loop 2) and C195 (extracellular loop 3)
and between C51 (amino terminal head) and C276 (extracellular loop 3) are predicted
to contribute to further tertiary structure within the cell membrane as depicted in the
inset. Amino acids predicted to comprise the P. vivax binding region are identified in
red (Chitnis etal., 1996). Duffy Antigen Function The Duffy antigen receptor for chemokines (DARC) is a silent 7-transmembrane receptor. This results from the absence
of a DRYLAIV amino acid motif in the second intracellular loop needed to couple with
G-proteins that initiate intracellular signalling cascades (Murphy, 1996). Duffy is one of a
few chemokine receptors that bind to inflammatory chemokines, categorised by structural features into two different groups, (amino acid motif -CC-) and (amino acid
motif CXC-). On erythrocytes, the Duffy antigen is proposed to act as a sink that binds
to excess chemokines and limits inflammation (Darbonne et al., 1991). Reciprocally,
Duffy binding of chemokines prevents their diffusion into organs and peripheral tissue
43
erythrocytes from six PNG homozygous wild-type individuals and six PNG
heterozygous individuals showed that individuals with two erythroid-functional alleles expressed approximately twice the amount of the Fya antigen
compared to individuals with one erythroid-functional allele. Since the time
that FY*AES was reported in PNG, this allele has also been found in Tunisia
(Sellami etal., 2008). Before describing additional polymorphism, the Duffynegative phenotype has been observed in association with a 14-nucleotide
deletion in the Duffy coding sequence (Mallinson etal., 1995).
Further variation in Duffy serology, originally described by Chown etal.,
was observed as a result of low Fyb expression levels (termed Fyx) (Chown
etal., 1965), observed primarily in Caucasian families. Again, application of
molecular genetic strategies enabled identification of nucleotide sequence
changes associated with this serological phenotype. First, descriptions of the
mutation underlying the Fyx, or Fybweak, variant identified polymorphism
in codon 89 of the FY*B allele, changing the amino acid sequence from
arginine (codon CGC) to cysteine (codon TGC) in Fyb and Fybweak antigens (Olsson etal., 1998; Parasol etal., 1998; Tournamille etal., 1998). This
polymorphism has been observed in association with an alanine to threonine amino acid substitution at codon 100 (GCA to TCA), and an additional alanine to serine substitution at codon 49 (GCA to TCA) (Castilho
2004).The substitution these FY*X alleles all share is arginine to cysteine at
codon 89; to date, this polymorphism has not been observed on the FY*A
allele. The Fyx polymorphism occurs within the first intracellular loop of
the Duffy protein and is associated with reduced cell surface expression of
Duffy (Olsson etal., 1998; Tournamille etal., 1998). The frequency of the
space and in this way acts as a reservoir of chemokines in the circulating blood (Fukuma
etal., 2003). Duffy is also expressed on a variety of non-erythroid cells including venular
endothelial cells; in this context recent studies suggest two potential roles for Duffy.
On venular endothelial cells, Duffy has been proposed to act as a chemokine interceptor (internalisation receptor) by internalising and scavenging chemokines (Nibbs etal.,
2003). Alternatively, Pruenster etal. have shown that Duffy acts to mediate chemokine
transcytosis (Pruenster etal., 2009). In their invitro system, Duffy-mediated chemokine
transcytosis led to apical retention of intact chemokines and leukocyte migration across
Duffy-expressing endothelial cell monolayers. How these complex roles of the Duffy
antigen are regulated and influence human health remains to be determined. (Originally
published in Zimmerman 2004. The enigma of vivax malaria and erythrocyte Duffy-negativity,
in: Dronamraju, K.R., (Ed.), Infectious Disease and Host-Pathogen Evolution.Cambridge University Press, New York, pp 141172.) (Reproduced with permission from Cambridge University Press and Krishna R. Dronamraju). (For interpretation of the references to colour in
this figure legend, the reader is referred to the online version of this book.)
44
FY*Bweak allele is approximately 2% in Caucasians (Chown et al., 1965;

Olsson etal., 1998). Finally, weak expression of Fyb has been reported in
association with deletion of a C nucleotide residue, between 76 and 74,
of the Duffy gene promoter in an Sp1 regulatory site (Moulds etal., 1998).
Overall, flow cytometry studies testing the association between Duffy
promoter and Fybweak polymorphisms have demonstrated consistent relationships. Relative levels of erythroid expression have shown that heterozygous carriers of a Duffy-negative allele express approximately 50% the
level of the Duffy antigen on their red cells compared to the red cells from
individuals homozygous for Duffy-positive alleles. The FY*X allele is associated with approximately 10% of the expression compared to the FY*A
and FY*B alleles (Tournamille etal., 1998). The overall Duffy phenotype
is dependent on both promoter and coding region SNPs. Expression phenotypes relative to the 15 different genotypes possible from the five known
Duffy alleles (FY*A, FY*B, FY*X, FY*AES, FY*BES) are summarised in
Table 2.2. Additional observations that Duffy expression is highest on reticulocytes (Woolley etal., 2000; Woolley etal., 2005) may contribute to the
observation of preferential invasion of these immature red cells by P. vivax
(Kitchen, 1938).
Comparative sequence analyses of Duffy gene orthologues in non-human
primates have shown that the FY*B allele is the ancestral state (Palatnik
and Rowe, 1984; Chaudhuri etal., 1995; Li etal., 1997; Tournamille etal.,
2004; Demogines et al., 2012; Oliveira et al., 2012). The Duffy-negative
allele FY*BES bears clear signatures of strong and recent positive selection in African populations (Hamblin and Di Rienzo, 2000; Hamblin etal.,
2002). Interestingly, the FY*A allele predicted to have arisen after FY*BES
(Li etal., 1997) has also been characterized by levels of polymorphism lower
than would be expected by a neutral model of evolution in a sample of Chinese individuals (Hamblin etal., 2002). Phylogenetic comparisons of this
nature for the FY*X and FY*AES alleles have not yet been performed. That
P. vivax would be considered to be the agent behind the selection observed
in human-specific alleles is curious given the long-held considerations that
P. vivax infection is rarely lethal in humans. As a growing number of clinical research studies are establishing connections between P. vivax and severe
malaria and malaria mortality (e.g. (Genton etal., 2008; Tjitra etal., 2008;
Anstey etal., 2009; Baird, 2009; Kochar etal., 2009)), further consideration
is necessary to determine how vivax malaria exerts its pressure as an agent
of natural selection (Anstey etal. discuss details of clinical vivax malaria in
Chapter 3 in Volume 80 of this special issue).
45
Table 2.2 Working Guidelines for Duffy* blood group nomenclature

Phenotype
Allele
Antigen
Genotype
Serologic
Expression$
FY*A
FY*B
FY*X
FY*AES
FY*BES
Fya
Fyb
Fybweak
FY*A/FY*A
FY*A/FY*AES
FY*A/FY*BES
FY*B/FY*B
FY*B/FY*X
FY*B/FY*AES
FY*B/FY*BES
FY*X/FY*X
FY*X/FY*AES
FY*X/FY*BES
FY*A/FY*B
FY*A/FY*X
FY*AES/FY*AES
FY*AES/FY*BES
FY*BES/FY*BES
Fya+/b
Fya/b+
Fya/b+weak
Fya+/Fyb+
Fya/Fyb
2Fya, 0Fyb
1Fya, 0Fyb
1Fya, 0Fyb
0Fya, 2Fyb
0Fya, 1.1Fyb
0Fya, 1Fyb
0Fya, 1Fyb
0Fya, 0.2Fyb
0Fya, 0.1Fyb
0Fya, 0.1Fyb
1Fya, 1Fyb
1Fya, 0.1Fyb
0Fya, 0Fyb
0Fya, 0Fyb
0Fya, 0Fyb
*Alternate gene name=Duffy antigen/receptor for chemokines (DARC)

Consistent with International Society of Blood Transfusion blood group terminology (web address
provided below) www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blooct-
group-terminology/blood-group-terminology/#c581
ES, erythrocyte silent attributed to the T to C transition at nucleotide 33 in the Duffy gene promoter
$Expression phenotypes based on composite of flow cytometry and chemokine binding. (Mnard
et al., 2010)
3.4. The Duffy Binding Protein

Several studies have now described the parasite ligand interacting with
the Duffy blood group antigen. P. knowlesi Duffy binding proteins (Haynes
etal., 1988; Adams etal., 1990) and P. vivax DBP (PvDBP) (Wertheimer and
Barnwell, 1989; Fang etal., 1991) have molecular weights of approximately
140kD; P. knowlesi (Pk) binds to the Duffy antigen (Chitnis and Miller,
1994). A 330-amino acid cysteine-rich region of the DBP is predicted to be
the protein domain responsible for binding to Duffy-positive human RBCs
(Ranjan and Chitnis, 1999). The DBP is expressed in the micronemes and
on the surface of P. knowlesi, and by homology, P. vivax merozoites. A number of structural features of the DBPs are shared with erythrocyte binding proteins of other malarial parasites. As such, these proteins have come
to be known as Duffy-binding-like erythrocyte binding proteins (DBLEBP) (Adams etal., 1992) Shared features among EBPs include two cysteine-rich regions (Region II containing 12 conserved cysteine residues;
Region VI containing 8 conserved cysteine residues), a highly polymorphic
46
region (Regions III to V) and numerous aromatic amino acids (tryptophan,

phenylalanine and tyrosine) (Adams et al., 1992). Because of the overall
significance of EBPs in blood-stage infection, it is important to consider
further the interaction between the PvDBP and the Duffy antigen.
Despite the difficulties in growing P. vivax in culture, a number of invitro
studies have built a body of information on the importance of DBPDuffy
antigen interaction to invasion of the RBC. As noted above, while P. knowlesi
merozoites would successfully contact and reorient their apical surfaces in
apposition to the membrane of both Duffy-positive and Duffy-negative
erythrocytes, the tight junction between merozoite and erythrocyte membranes did not form with Duffy-negative cells (Miller et al., 1979). These
observations have suggested that some aspect of the parasites invasion mechanism failed to engage in the absence of the Duffy antigen. Experiments
more specifically focused on the P. vivax DBP have demonstrated competitive interference between recombinant PvDBP expressed on COS cells and
the Duffy receptor on donor erythrocytes using a 35-amino-acid peptide
(amino acid 842) from the receptors NH2-terminal domain, the monoclonal antibody anti-Fy6 and the chemokine MGSA (CXCL1) (Chitnis
and Miller, 1994; Chitnis et al., 1996). These same studies have also demonstrated that sulphation of tyrosine 41 is critical for optimal DBP binding
to the Duffy receptor in vitro (Choe etal., 2005). Further studies employing this COS-cell-binding affinity assay showed that Duffy-negative
heterozygous, compared to homozygous positive, erythrocytes exhibit consistently lower affinity for PvDBP transfected cells (Michon et al., 2001).
Additionally, elevated levels of amino acid sequence polymorphism in the
DBP binding region, as well as antibody responses from people living in
P. vivax-endemic regions recognising DBP, suggest that this molecule may be
under selective pressure (Tsuboi etal., 1994; Ampudia etal., 1996; Fraser etal.,
1997; Michon etal., 1998; Cole-Tobian etal., 2002) by the human immune
system. A number of years later, Singh etal. have now shown that P. knowlesi
merozoites of Pk knockout strain are not able to form a junction with or
invade Duffy-positive human RBCs (Singh etal., 2005).That the Pk knockout continues to successfully invade rhesus erythrocytes suggests that the P.
knowlesi and proteins bind other receptors and enable Duffy-independent
red cell invasion (Chitnis and Miller, 1994; Ranjan and Chitnis, 1999).
Interrogation of the parasite ligandhost receptor relationship has since
been examined through studies to characterize more specifically the interaction of PvDBP with the Duffy antigen. Recombinant chimeric proteins
have been used to localise PvDBP-Duffy binding to a 170-amino-acid
47
segment of the parasite ligand between cysteines 4 and 7 (Ranjan and Chitnis, 1999). Further studies have gone on to suggest that there are discontinuous epitopes within this segment that are predicted to be important for
Duffy antigen interaction (VanBuskirk etal., 2004; Hans etal., 2005) and
that receptor binding residues and polymorphic residues under immune
pressure map to opposing surfaces of PvDBP (Singh et al., 2006). More
recent studies have shown that both patient-derived and polyclonal rabbit
antibodies specific for PvDBP are able to inhibit P. vivax invasion of human
RBCs invitro (Grimberg etal., 2007; Russell etal., 2011).
4. EVOLVING PERSPECTIVES ON RESISTANCE

TO P. VIVAX

Molecular and cell biology technologies have provided powerful
strategies for cloning and expressing proteins from malarial parasites to
study their interactions with human red cells invitro (Adams etal., 1992).
Molecular diagnostic methods for interrogating genetic polymorphisms
and diagnosing infection have transformed strategies for studying the epidemiology of malaria (Greenwood, 2002). Over the past 20 years, application of these methods has significantly influenced our perspectives on the
frequency and distribution of P. vivax as well as the role played by genetic
polymorphism and the interaction between P. vivax and the human RBC.
4.1. F
urther Influence of Duffy Polymorphism on Resistance
to P. vivax Malaria
Discovery of the FY*AES allele in PNG (Zimmerman et al., 1999) provided a new opportunity to evaluate the association between a Duffy-negative allele and susceptibility to P. vivax infection and disease. Although no
individual has been identified to be homozygous for the FY*AES allele in
PNG, the opportunity was provided to determine if there was any selective
advantage associated with being a heterozygous carrier of a Duffy-negative
allele. In these studies, Kasehagen and colleagues performed cross-sectional
malaria prevalence surveys in the same PNG communities where the
FY*AES allele had been identified.These Wosera villages, north of the PNG
Central Ranges, are highly endemic for all four human malarial parasite
species (Genton etal., 1995a; Kasehagen etal., 2006; Lin etal., 2010). Plasmodium species infection status was evaluated by conventional blood smear
light microscopy and semi-quantitative polymerase chain reaction (PCR)based strategies. In both unmatched and matched (adjusted for age, sex
48
and village of residence) longitudinal cohort analyses, results showed that

Duffy-negative heterozygotes (FY*A/*AES) were partially protected from
P. vivax blood-stage infection compared to those homozygous for wild-type
alleles (FY*A/*A) (Kasehagen et al., 2007). In these same study cohorts,
there were no differences in susceptibility to P. falciparum infection between
FY*A/*AES and FY*A/*A study participants (Kasehagen etal., 2007).
Additional analyses evaluated parasitaemia by a semi-quantitative PCR
assay between FY*A/*AES and FY*A/*A study participants, in age group
categories less than and greater than 15 years. Among FY*A/*AES children under 15 years of age, the mean P. vivax fluorescent signal intensity
(corresponds with parasitaemia (McNamara etal., 2006)) was significantly
lower among FY*A/*AES (mean=2.37, log10 transformed) compared to
FY*A/*A children (mean=2.96) (MannWhitney U:P=0.023). Interestingly, this was similar to the difference in parasitaemia observed between
FY*A/*A individuals older vs. younger than 15 years of age (mean fluorescent signal intensity 2.23 vs. 2.81, respectively; MannWhitney
U:P<0.0001). This suggested that the difference in parasitaemia attributed
to the difference in genotype (FY*A/*AES and FY*A/*A) was similar to
the difference that would otherwise be attributed to acquired immunity
of older individuals. Similar to results from the longitudinal cohort studies,
no association was observed between susceptibility to P. falciparum parasitaemia and the Duffy genotype. The findings from this study provided
the first evidence that Duffy-negative heterozygosity reduced erythrocyte
susceptibility to P. vivax infection (Kasehagen etal., 2007). Reduced susceptibility to P. vivax was not associated with an increased susceptibility
to P. falciparum malaria as may have been predicted by studies that previously reported reduced severity of falciparum malaria conferred by exposure to P. vivax. Similarly, cross-sectional studies in the Amazon Basin of
Brazil (Cavasini et al., 2007; Sousa et al., 2007) and Rio Grande do Sul
(Albuquerque etal., 2010) have shown significantly reduced prevalence of
P. vivax infection among Duffy-negative heterozygotes (either FY*A/*BES
or FY*B/*BES) when compared with subjects from the same endemic areas
with the FY*A/*A, FY*B/*B or FY*A/*B genotypes. These results suggest that Duffy-negative heterozygosity confers significant protection from
vivax malaria and may provide some insight regarding a selective advantage
that led to the FY*BES allele reaching genetic fixation in Africa.
The frequency of the FY*X allele at 2% in Caucasian populations may
limit opportunities to perform epidemiological studies to determine if the
Fybweak antigen is associated with reduced risk of P. vivax malaria. In contrast,
49
the FY*A allele is widely distributed in Southeast Asia (Howes etal., 2011),
where P. vivax is proposed to have evolved from origins as a parasite of Old
World monkeys (Carter, 2003; Escalante etal., 2005; Culleton etal., 2011),
and in South America, where P. vivax is the predominant malaria parasite in
human infections (Oliveira-Ferreira etal., 2010;Arevalo-Herrera etal., 2012).
Invitro studies have shown that the P. knowlesi DBP interacts with stronger affinity to the Fyb compared to the Fya antigen. After observing similar
results with the PvDBP (4050% decreased binding to Fya+b vs. Fyab+
erythrocytes (King etal., 2011); P<0.0001), King etal. performed a cohort
study in the Brazilian Amazon to determine if the invitro results translated to
invivo protection from clinical vivax malaria in association with the FY*A
compared to the FY*B allele (King etal., 2011). Study participants (n=400;
574 years of age) lived along the Iquiri River where the annual incidence
rates of P. vivax and P. falciparum malaria during the 14-month study period
were 0.31 and 0.17, respectively (124 cases of P. vivax, 66 cases of P. falciparum, 31 cases of P. vivax+P. falciparum malaria). Overall, when compared
to the FY*A/*B genotype (n=140), individuals with the FY*A/*BES and
FY*A/*A genotypes experienced 80% (n=35; risk ratio, 0.204 (95% confidence interval (CI), 0.090.87)) and 29% (n=52; risk ratio, 0.715 (95% CI,
0.311.21)) reduced risk of clinical vivax malaria, respectively. Consistent
with stronger affinity between PvDBP and the Fyb antigen, individuals with
the FY*B/*BES and FY*B/*B genotypes experienced 220270% increased
risk of clinical vivax malaria, respectively, when compared to FY*A/*B
(FY*B/*BES: n=76; risk ratio, 2.17 (95% CI, 0.914.77); FY*B/*B: n=87;
risk ratio, 2.70 (95% CI, 1.365.49). As in the studies performed by Kasehagen etal., there was no association between the FY genotype and risk
for P. falciparum in the multivariate analysis (overall risk ratio 1.08, 95% CI
0.872.38, P=0.42). While it will be helpful to see if additional epidemiological studies corroborate these findings, results from Cavasini etal. are of
interest (Cavasini etal., 2007). In their studies, FY*A/*BES was observed
less frequently among 312 P. vivax patients (10.9%) than in 330 healthy
blood donors (Brazilian blood bank; 18.8%). Results from King etal. would
suggest that like the FY*BES allele, the frequency of FY*A has increased
in frequency (reaching genetic fixation in many populations) to improve
human fitness against P. vivax malaria (King etal., 2011).
4.2. Duffy-Independent Red Cell Invasion by P. vivax

With consistent observation of resistance to P. vivax malaria associated with
Duffy negativity, reduced susceptibility to P. vivax associated with lower
50
Duffy expression and/or reduced affinity between the Duffy antigen and
the parasite invasion ligand, it is understandable that the Duffy antigen has
come to be regarded as an essential receptor for P. vivax red cell invasion. It
has therefore been of keen interest that an increasing number of studies have
reported P. vivax PCR positivity in Duffy-negative people. These studies
have included P. vivax in Duffy-negative people in the Nyanza Province of
Western Kenya (Ryan etal., 2006) and the Amazon Basin (Brazil) (Cavasini
etal., 2007). However, another large-scale PCR-based survey covering nine
different African countries detected only one P. vivax-positive person in
over 2500 samples, and this individual was Duffy positive (Culleton et al.,
2008). With a history of clinical reports of P. vivax malaria occurring in
Europeans returning from holiday or business travel to Africa (PhillipsHoward etal., 1990; Gautret etal., 2001; Mendis etal., 2001; Muhlberger
etal., 2004; Guerra etal., 2010), new questions have arisen with regard to
the Duffy-negative P. vivax resistance factor (Rosenberg, 2007).
In an effort to understand the epidemiology of malaria throughout
Madagascar, Mnard and colleagues initiated a series of blood sample
collections in 2006 from the countrys four major malaria-transmission
regions (Mnard et al., 2010). These surveys provided new insight into
the basic prevalence of Plasmodium species infections and drug resistance.
They also opened the opportunity to investigate the intersection of
malaria infection in a human population characterized by unique origins
and admixture.
The peopling of Madagascar is recent in human history and is suggested to have been initiated by sea-faring people of Indonesia or Malaysia
(Nias Island of western Sumatra or Borneo, respectively) with evidence
that founding individuals arrived 2300 years before present (Burney etal.,
2004). Upon Bantu migration from Africa (Tanzania and Mozambique)
during the second and third centuries and new waves of Malayo-Indonesian
immigration from the eighth century onwards, significant cultural assimilation and genetic admixture has occurred. Malaria is likely to have been
transported to Madagascar through the earliest human settlers more than
2000 years ago. It is more difficult to predict when during the first millennium of human settlement the human population numbers and density
became favourable to support endemic transmission of the four common
species of human malaria parasites that are observed in Madagascar today.
In this setting, surveys of school-aged children revealed P. vivax PCR positivity in 8.8% of asymptomatic Duffy-negative children (n=476) (Mnard
etal., 2010). During surveys to assess invivo efficacy of drugs recommended
51
by the Madagascar Ministry of Health to treat malarial illness, nine Duffynegative people were identified who had PCR-confirmed, mono-infection
P. vivax malaria (4.9% of 183 participants). Given the unusual prevalence of
vivax malaria in people considered to be resistant to this disease, Mnard
etal. took additional steps to validate this finding by microscopy to provide
the first evidence to confirm Duffy-independent blood-stage infection and
development by P. vivax (Fig. 2.3) (Mnard etal., 2010). Microscopy results
included the observation of sexual-stage gametocytes necessary to continue
the parasite life cycle through mosquito transmission. Consistent with observations reported by many blood group laboratories, flow cytometry analysis
of erythrocytes from Malagasy study participants who had experienced clinical P. vivax malaria showed that Duffy-negative genotype and phenotype
Figure 2.3 Standard Giemsa-stained thin smear preparations of P. vivax infection and
development in human Duffy-negative erythrocytes. Panels A and B originated from
a 4-year-old female, genotyped as Duffy negative (FY*BES/*BES), who presented at the
Tsiroanomandidy health center with fever (37.8 C), headache and sweating without
previous anti-malarial treatment. Standard blood smear diagnosis revealed a mixed
infection with P. vivax (parasitaemia=3040 parasitised red blood cells [pRBC]/l) and
P. falciparum (parasitaemia=980 pRBC/l). PCR-based Plasmodium species diagnosis confirmed the blood smear result; P. malariae and P. ovale were not detected. (A) a
P. vivax early-stage trophozoite with condensed chromatin, enlarged erythrocyte volume, Schffner stippling and irregular ring-shaped cytoplasm. (B) a P. vivax gametocyte
lavender parasite, larger pink chromatin mass and brown pigment scattered throughout
the cytoplasm are characteristics of microgametocytes (male). Panel C originated from a
12- year-old Duffy-negative (FY*BES/*BES) male, who presented at the Miandrivazo health
centre with fever (37.5C) and shivering without previous anti-malarial treatment. Standard blood smear diagnosis and light microscopy revealed infection with only P. vivax
(parasitaemia=3000 pRBC/l). PCR-based Plasmodium species diagnosis confirmed this
blood smear result; P. falciparum, P. malariae and P. ovale were not detected. The parasite featured shows evidence of a P. vivax gametocyte large blue parasite, smaller pink
chromatin mass and brown pigment scattered throughout the cytoplasm are characteristics of macrogametocytes (female). (Adapted from Mnard etal, 2010. Plasmodium
vivax clinical malaria is commonly observed in Duffy-negative Malagasy people. Proc. Natl.
Acad. Sci. U. S. A. 107, 59675971). (For interpretation of the references to colour in this
figure legend, the reader is referred to the online version of this book.)
52
were 100% concordant. Additionally, DNA sequence analysis of the Duffy

gene confirmed that the Duffy-negative allele identified in Madagascar was
identical to the FY*BES allele observed in West Africa (included>2550bp
of the genes proximal promoter and full coding sequence).
Population-level observations from Mnard etal. provide further insight
regarding the unique parasitehost relationships discovered in Madagascar
(Mnard et al., 2010). In the communities with the highest frequencies
of Duffy negativity, there was little to no prevalence of P. vivax infection
detected in either Duffy-positive or Duffy-negative people. In contrast,
with an increase in the frequency of Duffy positivity, P. vivax prevalence
increased and was not significantly different between Duffy-positive and
Duffy-negative people (Fig. 2.4; 2 results: Miandrivazo P=0.733; Maevatanana P=0.278; Tsiroanomandidy P=0.09). These results suggest that
high population levels of Duffy negativity may act similarly to herd immunity to reduce transmission and consequently protect Duffy-positives from
vivax malaria. In populations with higher frequencies of Duffy positivity,
opportunities for the parasite to attempt invasion of the Duffy-negative
RBCs are more frequently available as antibody reactivity against P. vivax
merozoites, PvDBP and PvMSP-1 indicate that liver-stage infection (and
therefore hypnozoite formation) commonly occurs in Duffy-negative
people (Spencer et al., 1978; Michon et al., 1998; Herrera et al., 2005;
Culleton et al., 2009). It is important to note that a survey of clinical
malaria from this Madagascar study did suggest that Duffy negativity was
associated with protection from malarial illness. Finally, it is of interest that
genotyping results from six unlinked P. vivax-specific microsatellites suggested that multiple P. vivax strains were present in the blood samples from
Duffy-negative infections.
With additional reports of P. vivax PCR-positive Duffy-negative people
from Equatorial Guinea (Rubio etal., 1999; Mendes etal., 2011), Gabon
(Mendes et al., 2011) and Mauritania (Wurtz et al., 2011), evidence of
Duffy-independent P. vivax blood-stage infection has been observed across
geographically distant communities of the Duffy-negative population of
sub-Saharan Africa. It will be important to continue surveillance of P. vivax
strains capable of Duffy-independent red cell invasion because of implications regarding the potential that P. vivax may compound the burden of clinical malaria in Africa and complicate P. vivax-specific vaccine development
and malaria elimination.
53
Figure 2.4 Frequency distribution of P. vivax infections and clinical cases identified in
Duffy-positive and Duffy-negative Malagasy people. Pie graphs show the prevalence
of Duffy-positive (dark/light green) and Duffy-negative (red/pink quadrants) phenotypes in the eight Madagascar study sites. Prevalence of P. vivax infection observed in
the survey of school-aged children is shown in red and dark green; population subsets not infected with P. vivax are pink and light green. Study sites identified by a red
star indicate that clinical vivax malaria was observed in Duffy-negative individuals. A
green star indicates that vivax malaria was observed in Duffy-positive individuals only
(Ejeda). In Ihosy, clinical malaria was observed in one individual with a mixed P. vivax/P.
falciparum infection. P. vivax malaria was not observed in Andapa and Farafangana
(black star). Malaria transmission strata are identified as tropical (lightest grey), subdesert (light grey), equatorial (middle grey) and highlands (dark grey). (Adapted from
Mnard et al, 2010. Plasmodium vivax clinical malaria is commonly observed in Duffynegative Malagasy people. Proc. Natl. Acad. Sci. U. S. A. 107, 59675971). (For interpretation of the references to colour in this figure legend, the reader is referred to the online
version of this book.)
54
4.3. G
lobal Distribution of Duffy Polymorphism and the
Population at Risk of P. vivax Malaria
Much like the maps used to illustrate an overlap between malaria endemicity
and the distribution of the sickle cell allele (Piel et al., 2010), - and
-thalassaemias (Weatherall and Clegg, 2001) and glucose-6-phosphate
dehydrogenase (G6PD) deficiency (Howes etal., 2012), population surveys
of Duffy blood group variants have been used to map the polymorphisms
spatial distribution. These maps provide an important overview of the ongoing relationship between P. vivax and human malaria. Recently, Howes
etal. have collated a comprehensive geographically referenced database of
available Duffy phenotype and genotype survey data to refine the global
cartography of the common Duffy variants (FY*A, FY*B and FY*BES)
(Howes etal., 2011). Results of this effort are summarised in Fig. 2.5. (For
information on source data for this study, see Howes etal. Supplemental
Information for 320 references). Recalling that non-human primate studies
provide evidence that FY*B (green) is the ancestral allele in the hominid
lineage, this map suggests that the FY*BES (red to orange) originated in
Africa and has spread across a vast geographical and ethnic landscape. The
map also indicates that FY*A (blue) has reached genetic fixation in regions
of the world where the non-human malaria parasite ancestors originated
and dispersed (noted above). From these potential Asian origins, FY*A
has admixed with FY*B and also spread into the Americas. Areas of the
map appearing in different shades of grey identify regions where populations are characterized by heterogeneous frequencies of FY*A, FY*B and
FY*BES ranging from 2050%. In these latter regions, P. vivax is likely to
have been exposed to a range of RBC phenotypes with varying contact
affinities between PvDBP and the Duffy receptor, including heterozygous
and homozygous Duffy negativity. In the struggle to survive, it seems reasonable to hypothesise that P. vivax strains have optimised effective contact
of the apical invasion mechanism across a gradient of Duffy polymorphism,
which now includes absence of this receptor, to engage the moving junction needed for successful red cell invasion.
With increasing evidence that P. vivax is not restricted to a Duffydependent invasion pathway, it is important to consider how this might
affect the estimation of the population at risk of P. vivax malaria (PvPAR).
To date, Malaria Atlas Project estimates of the PvPAR have applied a biological exclusion criterion based on 100% protection from P. vivax infection by Duffy negativity (Guerra etal., 2010). With potential that a model
55
Figure 2.5 Global frequencies of the FY alleles. Areas predominated by a single allele (frequency50%) are represented by a colour gradient
(blue, FY*A; green, FY*B; red/yellow, FY*BES). Areas of allelic heterogeneity where no single allele predominates, but two or more alleles each
have frequencies20%, are shown in grey-scale: palest for heterogeneity between the silent FY*BES allele and either FY*A or FY*B (when coinherited, these do not generate new phenotypes), and darkest being co-occurrence of all three alleles (and correspondingly the greatest
genotypic and phenotypic diversity). Overall percentage surface area of each class is listed in the legend. The probability distribution based
on a Bayesian model is summarised as a single statistic: in this case, the median value, as this corresponds best to the input dataset, as previously described (Howes etal., 2011). Median values of the predictions were generated for each allele frequency at a 1010 km resolution on
a global grid with GIS software (ArcMap 9.3; ESRI). (For interpretation of the references to colour in this figure legend, the reader is referred
to the online version of this book.)
56
based on this conservative perspective may significantly underestimate the

global PvPAR, we were interested in considering how reduced Duffy-
negative exclusion would alter this metric in global and regional estimates.
Figure 2.6 shows that even if Duffy-negative protection were reduced to
50%, the overall global burden of P. vivax infection would remain heavily
Figure 2.6 Estimated change in the P. vivax population at risk before (A) and after (B)
relaxing the level of resistance conferred by Duffy negativity from 100% to 50%, respectively. Overall increases in the percentage of PvPAR (C). The population-at-risk exclusions
are based on the methods developed by Guerra etal. in 2010 (Guerra etal., 2010) and
subsequently refined by Gething etal. To define the P. vivax population at risk (PvPAR),
Gething et al. imposed several layers of exclusion from the countries with endemic
P. vivax transmission (95 countries). Annual parasite incidence (API) data were used to
refine the sub-national levels of transmission along administrative boundaries, classifying areas into unstable (<0.1 cases per 1000 population per year), stable transmission
(0.1 cases per 1000 population per year) and malaria free (Guerra etal., 2010). Temperature exclusion based on minimum requirements for parasite sporogony modelled in
relation to vector lifespan (Gething etal., 2011). Aridity mask to exclude areas too dry to
sustain transmission by restricting vector survival and availability of ovipositioning sites.
The aridity mask was derived from the bare ground areas in the GlobCover land cover
imagery (Guerra etal., 2008). Medical intelligence was used to further exclude malariafree urban areas (modulated with knowledge of the local Anopheles vectors) (Guerra
etal., 2010). All these methods have been described in greater detail by Gething etal.
(2012) and in Chapter 1 in Volume 80 of this special issue. Extensive data collection was
necessary for the API exclusions, and individuals who contributed data to this process
are acknowledged on the Malaria Atlas Project website (MAP: www.map.ox.ac.uk/). (For
a colour version of this figure, the reader is referred to the online version of this book.)
57
focused in Asia. Not surprisingly, the most significant changes in PvPAR

would be observed in the Africa, Saudi Arabia and Yemen region (Africa+)
with a projected fivefold increase. Interestingly, this increased PvPAR in
Africa would surpass the estimated risk in the Americas by threefold.
4.4. A
ssociation of Non-Duffy Gene Polymorphisms with
P. vivax Resistance
Recently, a number of groups have begun to consider the possibility that
non-Duffy human gene polymorphisms associated with protection against
falciparum malaria may also affect susceptibility to infection and disease
attributable to P. vivax.
4.4.1. G6PD Deficiency
Among the RBC variants considered to date, G6PD deficiency is important
to examine for a combination of reasons.The enzyme catalyses the first reaction in the pentose phosphate pathway leading to the formation of NADPH
needed by cells to counter oxidative stress (Cappellini and Fiorelli, 2008).
The enzyme deficiency was discovered because of its association with haemolytic anaemia following administration of primaquine (Beutler, 1994), the
only clinically validated medication against P. vivax hypnozoites (Wells etal.,
2010). Because of the widespread distribution of G6PD deficiency in malariaendemic regions, administration of primaquine and therefore, elimination of
P. vivax is problematic. The gene (13 exons distributed over 18.5kb; 140
mutations (Cappellini and Fiorelli, 2008; Minucci etal., 2012)) is located in
the telomeric region of the human X chromosome and is therefore characterized by classical X-linked inheritance patterns, hemizygosity in males and
X-inactivation in females (Beutler, 1996). The most common West African
G6PD variant, G6PD A- (ValMet, codon 68; moderate (1060%) activity
variant), has been associated with significant reduction in the risk of severe
falciparum malaria in male hemizygotes (Ruwende etal., 1995; Guindo etal.,
2007) and in heterozygous females (Ruwende etal., 1995). More recently,
Louicharoen etal. found that the G6PD-Mahidol487A mutation (GlySer,
codon 163; moderate (1060%) activity variant) was associated with reduced
P. vivax, but not P. falciparum, parasite density (Louicharoen et al., 2009).
Whether the G6PD-Mahidol487A mutation reduces the severity of clinical
vivax malaria was not reported. Leslie etal. have more recently reported that
the G6PD-Mediterranean type (Med; SerPhe, codon 188; severely deficient (110% activity)) is associated with protection from clinical vivax in a
case-control study of Afghan refugees living in Pakistan (Leslie etal., 2010).
58
Howes et al. further discuss details regarding the complexities of G6PD

deficiency and vivax malaria in Chapter 4 of this Volume.
4.4.2. Haemoglobinopathies
To date, very few studies have been performed to investigate associations
between the major haemoglobinopathies and P. vivax (Taylor etal., 2012).
Increased susceptibility to P. vivax infection has been observed in association
with -thalassaemia (/) in Vanuatu (Williams etal., 1996) and PNG
(Allen etal., 1997) and with HbE -thalassaemia in Sri Lanka (ODonnell
etal., 2009). It is suggested that -thalassaemia may increase susceptibility
to P. vivax infection because of higher overall red cell turnover increasing
reticulocytaemia (Weatherall and Clegg, 2001). As P. vivax shows a strong
preference for infecting reticulocytes (Kitchen, 1938), increased reticulocyte
counts associated with the thalassaemias would produce more target cells for
the merozoites to infect. In one additional study in India, reduced susceptibility to P. vivax infection was reported in association with HbE (Kar etal.,
1992). Although red cell remodelling and pathogenesis is markedly different
among human malaria species parasites, the association of reduced susceptibility to P. vivax with HbE may be more consistent with observations from
falciparum malaria studies where -thalassaemia confers protection against
malaria. This protection is proposed to occur because erythrocytes bind
higher levels of antibody from sera of malaria-exposed individuals (Luzzi
etal., 1991) and were observed to be more readily phagocytised by blood
monocytes (Yuthavong etal., 1988) compared with normal red cells.
Interestingly, the concept that -thalassaemia can increase susceptibility
to P. vivax infection in young children while being associated with decreased
susceptibility to P. falciparum has led to a hypothesis that the predilection to
P. vivax may lead to cross-immunity between parasite species, which protects against falciparum malaria later in life. This hypothesis has stirred a
lively debate regarding benefits and risks of cross-species infections (Bruce
and Day, 2003; Snounou, 2004; Zimmerman etal., 2004). While this debate
will continue, it must be acknowledged that P. vivax is responsible for causing severe illness and death (Baird, 2009) and that mixed infections with
P. vivax and P. falciparum can be more severe than mono-infections by these
same two species (Tjitra etal., 2008).
4.4.3. Southeast Asian Ovalocytosis
More consistent with P. vivax and the Duffy receptor interactions, there is
significant interest in the influence of mutations contributing to Southeast
59
Asian ovalocytosis (SAO) and protection against vivax malaria.Two proteins

associated with red cell ovalocytosis in Melanesians include the solute carrier family 4, anion exchanger, member 1 (SLC4A1, also known as erythrocyte membrane protein band 3) and glycophorin C (GYPC). SLC4A1 is
expressed in the red cell membrane and functions as a chloride/bicarbonate
exchanger involved in CO2 transport from tissues to lungs. Three domains
of the protein are structurally and functionally distinct. First, the 40kDa
N-terminal cytoplasmic domain (400 amino acids) contains an 11-aminoacid segment (residues 175185) that acts as an attachment site for the red
cell skeleton by binding ankyrin (Chang and Low, 2003; Stefanovic etal.,
2007). Second, a hydrophobic, polytopic transmembrane domain (481
amino acids; 14 membrane-spanning segments) carries out anion exchange
(Abdalla etal., 1980). Third, the cytoplasmic tail at the extreme C-terminus of the membrane domain (41 amino acids) binds carbonic anhydrase
II. Association with glycophorin A (GYPA) promotes the correct folding
and translocation of the SLC4A1 protein. This protein is predominantly
dimeric but forms tetramers in the presence of ankyrin (Alper, 2009). A
number of single amino acid substitutions in SLC4A1 give rise to the Diego
blood group system (Poole, 2000) and a 27 base pair deletion removing 9
amino acids (SLC4A127; codons 400408) near the first transmembrane
region of the protein leads to SAO (Jarolim etal., 1991; Mgone etal., 1996;
Mgone etal., 1998). GYPC is a physiologically important monomer (128
amino acids, apparent 35kDa, integral membrane sialoglycoprotein) (Cartron etal., 1993) that interacts with the peripheral membrane protein 4.1
to mediate attachment of the submembranous cytoskeleton to the erythrocyte membrane. Deletion of GYPC exon 3 (GYPCDex3) results in the
Gerbich-negative blood group phenotype (Colin etal., 1989; High etal.,
1989) and has also been associated with ovalocytosis in PNG in the absence
of SLC4A127 (Patel etal., 2001).
Invitro studies have shown that SAO compared to normal RBCs show
resistance to both P. falciparum and P. knowlesi (Kidson etal., 1981; Hadley
et al., 1983). Although trypsin treatment had previously been shown to
render resistant Duffy-negative red cells susceptible to P. knowlesi infection
(Mason et al., 1977; Miller et al., 1979), this same experimental strategy
was not successful with SAO cells (Hadley et al., 1983). The mechanism
of resistance was suggested to be increased rigidity of the RBC membrane
(Mohandas et al., 1984). Both SAO and Gerbich negativity have been
shown to reduce the severity of falciparum malaria (Cattani et al., 1987;
Serjeantson, 1989; Genton et al., 1995b; Allen et al., 1999). These overall
60
invitro and invivo observations prompted the investigation of the impact of

SAO on susceptibility to P. vivax malaria in PNG.
It is important to note that P. vivax malaria in PNG is observed to peak by
approximately 3 years of age (Kasehagen etal., 2006; Mueller etal., 2009) and
evidence of significant protection from clinical symptoms associated with P.
vivax is observed in young school-aged children (Michon etal., 2007). The
impact of SAO was studied through multiple childhood cohorts. Results from
this study showed that in a cohort of infants 321 months of age SAO was
associated with a 55% reduction in the risk of clinical P. vivax episodes with
parasitaemia greater than 500 infected cells/l (adjusted IRR (incidence rate
ratio)=0.54; CI95 (0.34, 0.59), P<0.0001). Additionally, in a treatment-time
to re-infection cohort of 514 year olds, SAO children experienced a 52%
reduction in P. vivax re-infection diagnosed by light microscopy (CI95 (23,
87), P=0.014) (Rosanas-Urgell etal., 2012). Further studies showed that
while Duffy antigen expression was not significantly different on SAO
compared to normal erythrocytes, high-level PvDBP-specific binding
inhibitory antibodies (>90% binding inhibition) were observed significantly more often in sera from SAO than non-SAO children (SAO, 22.2%;
non-SAO, 6.7%; P=0.008)(Rosanas-Urgell etal., 2012). Consistent with
invitro observations, interactions leading to reorientation and apical contact of the P. vivax merozoite are likely to occur invivo. Results indicating
that PvDBP-specific binding inhibitory antibodies were more common
in SAO children suggest that PvDBP exposure to the immune system is
somehow different in SAO compared to non-SAO children and stimulates
production of higher quality antibody recognition of this important parasite
invasion ligand.
5. CONCLUSIONS AND FUTURE DIRECTIONS

Very early experience with malariotherapy in the United States
revealed that high-level resistance to blood-stage infection with P. vivax
was observed in many, but not all, African Americans (Fig. 2.7). Although
not considered at that time, observations of syphilologists of the 1920s
initiated efforts to explain this curious occurrence and launched investigations that have revealed the mechanisms that malarial parasites use to infect
the RBC. The first critical breakthroughs identifying components of these
red cell invasion mechanisms were made by Louis Miller and colleagues in
the mid-1970s when the Duffy blood group antigen was identified as the
receptor for P. knowlesi and P. vivax.
61
Figure 2.7 Summary of major events providing insight on resistance to P. vivax.

(For a colour version of this figure, the reader is referred to the online version of this book.)
As in other fields of biomedical research, invention of the PCR by

Kerry Mullis in 1983 transformed many aspects of malaria research. The
first published application of PCR demonstrated how sickle cell anaemia
could be diagnosed by amplification of a small segment of the -globin
gene (Saiki et al., 1985). Amplification of malarial parasites from human
blood samples was performed in the early 1990s to diagnose species with
greater sensitivity than conventional blood smear methods (Barker et al.,
1992; Barker etal., 1994) and to identify P. falciparum strains that were carrying mutations associated with drug resistance (Zolg etal., 1989). As powerful high-throughput multiplex assays for diagnosing malarial infections have
become routinely available, perspectives on species complexity of malarial
infections have changed significantly. PCR applications have improved our
understanding of the epidemiology of vivax malaria, in particular, where
blood smear diagnosis is hampered by lower sensitivity, and in differentiating P. vivax from P. ovale (which share numerous morphological similarities).
Reliable diagnoses of P. vivax have brought into question preconceptions
that Africans are always fully resistant to P. vivax erythrocyte infection and
that the parasite is absent from sub-Saharan Africa. PCR diagnostic and
genotyping strategies have therefore played significant roles in identifying
P. vivax infections in Duffy-negative people (Ryan et al., 2006; Cavasini
etal., 2007; Mnard etal., 2010; Mendes etal., 2011;Wurtz etal., 2011) and
62
in the performance of the field-based studies that have identified new vivax
malaria resistance factors (Cavasini etal., 2007; Kasehagen etal., 2007; Sousa
etal., 2007; Louicharoen etal., 2009; Albuquerque etal., 2010; King etal.,
2011; Rosanas-Urgell etal., 2012).
Do these recent observations suggest that P. vivax is now evolving new
capacity to infect human RBCs or has this parasite always had this capacity? Species naturally evolve, particularly when confronted with a selective
barrier that threatens their ability to reproduce Duffy negativity clearly
represents this kind of barrier for P. vivax. Evidence suggests that the parasites human red cell invasion mechanism has not been restricted to the
Duffy antigen. Clinical observations for decades have reported that Duffypositive Caucasians return from travels to Duffy-negative Africa with P.
vivax infections (Phillips-Howard etal., 1990; Gautret etal., 2001; Mendis
etal., 2001; Muhlberger etal., 2004; Guerra etal., 2010), and records of
African or African-American individuals infected with P. vivax (albeit lacking Duffy phenotype data) have appeared periodically (Butler and Sapero,
1947; Hankey etal., 1953; Bray, 1958). The alternative explanation for the
sudden increase in P. vivax-positive Duffy-negative people is that molecular
diagnostic methods now provide clinicians and researchers with tools that
are more sensitive than the parasitological methods previously employed.To
understand the true epidemiology of vivax malaria in Africa, future population studies should include surveillance for P. vivax in molecular diagnostic
assays routinely.
With confirmation that P. vivax can infect Duffy-negative red cells, it is
important to know how the parasite is progressing through the critical steps
leading to reticulocyte invasion (Fig. 2.8).What then are the components of
the P. vivax Duffy-independent invasion mechanism? The electron microscopy that has so vividly captured P. knowlesi interactions with Duffy-positive
and Duffy-negative red cells has shown that the parasite is able to reorient
its apical end in apposition to the red cell membrane of both Duffy-positive
and Duffy-negative cells. However, absence of the Duffy antigen limits further junction formation that sets in motion the events required to complete
invasion. In the absence of the Duffy antigen, what red cell protein(s) enable
the junction formation needed for further downstream events what is the
new invasion receptor? The Duffy antigen has been shown to reside in a
cluster of other red cell membrane proteins as part of a protein 4.1R multiprotein complex (Mohandas and Gallagher, 2008). This complex includes
Band 3 (link to SAO), glycophorin C and other blood group proteins (Kell,
reticulocyte binding homologues (Rh), XK). One wonders, if through its
63
Figure 2.8 Overview of P. vivax merozoite interaction with the human red blood
cell. Initial attachment occurs between any part of the merozoite (blue) and erythrocyte (red). The merozoite reorients, positioning its apical end for attachment to
the red cell membrane. A junction forms between the apical end of the merozoite and the erythrocyte membrane of Duffy-positive cells (first call-out box). In
contrast, P. knowlesi electron microscopy has shown thin filaments between the
merozoite apical end and the Duffy-negative red cell membrane; however, the
merozoite is not drawn into contact with the red cell and the junction fails to form.
This has implied that junction formation fails to occur between P. vivax and the
Duffy-negative red cell membrane as well (second call-out box). Once a durable
junction has formed between the merozoite and the red cell, micronemes (green)
and rhopteries (dark blue) release their contents, the red cell membrane invaginates and the merozoite moves into the parasitophorous vacuole (third call-out
box). Movement of the gliding junction is complete once the merozoite is engulfed
within the parasitophorous vacuole and the orifice at the red cell membrane is
sealed (fourth call-out box). (For interpretation of the references to colour in this
figure legend, the reader is referred to the online version of this book.)
64
dependence on interaction with the Duffy antigen, whether the parasites

DBP has gained or optimised a molecular connection with other members
of the 4.1R complex.
From the parasites vantage point, an equally important piece to the
Duffy-independent puzzle must be identified. Unlike P. knowlesi, the DBP
in P. vivax is found only as a single-copy gene (Carlton et al., 2008). It
is possible that new PvDBP polymorphism could enable this protein to
interact with alternative receptors; however, studies performed to date
have not identified PvDBP variants that are able to bind Duffy-negative
cells. Recent cell biology studies indicate that parasite proteins released in
succession from the micronemes, rhopteries and dense granules are integral to parasite-host cell recognition, attachment and junction motility
(Carruthers and Sibley, 1997). While AMA1 and RON protein interactions are critical components of the moving junction (Richard etal., 2010;
Srinivasan et al., 2011), it is clear that some parasite-host interaction is
required to secure initial contact by the parasites apical end before the
junction can be formed. As a number of reticulocyte binding proteins have
been localised to the micronemes (Meyer etal., 2009), these proteins would
appear to be the likely alternative ligands if PvDBP has been rendered
irrelevant in a Duffy-independent invasion mechanism. In P. falciparum evidence exists for functional redundancy of erythrocyte binding antigen and
Rh, so that inhibition of one pathway is compensated for by the functioning of others (Stubbs etal., 2005; Triglia etal., 2009). Once an invasion pathway can no longer be used because the receptor is absent or the
gene for the dominant ligand has been deleted, P. falciparum is able to use
alternative pathways by re-deploying the expressed suite of ligands (Baum
etal., 2005) or by differential gene expression of PfRh genes (Stubbs etal.,
2005). It is possible that vivax parasites are able to use alternative invasion
pathways that remain cryptic in the presence of the Duffy receptor and
become operational in its absence.
Answers to the questions prompted by Duffy-independent infection by
P. vivax are of critical importance to development of a vivax-specific vaccine.
The challenge confronting this mission is a familiar one: P. vivax is reluctant
to grow in laboratory cultures and this precludes many of the experimental approaches that have been so effective in studying red cell invasion by
P. falciparum (Cowman and Crabb, 2006). As cellular invasion mechanisms
are highly conserved across apicomplexa, new strategies available through
parasite genomics and proteomics will be important to mine. It may also be
65
important to keep in mind the important insights gained through studies on

P. knowlesi (Aikawa etal., 1978).
ACKNOWLEDGEMENTS
We wish to acknowledge the contributions of thousands of study volunteers who have contributed to the studies reviewed here. We are grateful for the contributions of Peter Gething
for sharing methodologies and unpublished data related to adjusted PvPAR predictions,
Hisashi Fujioka, Didier Mnard, Arsne Ratsimbasoa, Yves Colin and Christiane Bouchier
for developing critical data for this review, and Samantha Zimmerman for graphic design.
We thank Louis Miller and David Serre for helpful comments that improved the clarity of
this manuscript.
PAZ has been supported through the US National Institutes of Health (NIH) (AI46919,
AI089686, AI093922) and the Fogarty International Center (TW007377, TW007872).
MUF is supported through the US National Institutes of Health (AI 075416) and the
Fundao de Amparo Pesquisa do Estado de So Paulo, FAPESP (03/09719-6, 05/51988-0,
and 07/51199-0). REH was funded by a Wellcome Trust Biomedical Resources Grant
(#085406). OMP has been supported through the 7th European Framework Program
(FP7/2007-2013, contract 242095, Evimalar) and the Agence Nationale de la Recherche
(contract ANR-07-MIME-021-0).
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CHAPTER THREE
Natural Acquisition of
Immunity to Plasmodium vivax:
Epidemiological Observations
and Potential Targets
Ivo Mueller*, Mary R. Galinski**, Takafumi Tsuboi, Myriam
Arevalo-Herrera, William E. Collins$, Christopher L. King
*Walter + Eliza Hall Institute, Infection & Immunity Division, Parkville,Victoria, Australia; Barcelona Centre
for International Health Research (CRESIB, Hospital Clnic-Universitat de Barcelona), Barcelona, Spain
**Department of Medicine, Division of Infectious Diseases, Emory University School of Medicine;
Emory Vaccine Center and Yerkes National Primate Research Center, Emory University, Atlanta,
Georgia, USA
Cell-Free Science and Technology Research Center and Venture Business Laboratory, Ehime University,
Matsuyama, Ehime, Japan
Caucaseco Research Center, Cali, Colombia
$Institutional Association:
Malaria Branch, Division of Parasitic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia
Center of Global Health & Diseases (CGHD), Case Western Reserve University,Veterans Affairs Medical
Center, Cleveland, OH, USA
Contents
1. O
verview of Naturally Acquired Immunity to Malaria
2. D
ifferential Acquisition of Immunity to P. vivax and P. falciparum Under
Natural Exposure
3. A
cquisition of Immunity in Experimental Infections Lessons from Malaria
Therapy Patients and Irradiated Sporozoites
4. U
nique Biological Characteristics of P. vivax that Contribute to NAI
4.1. R
elapses
4.2. F orce of Blood-Stage Infection
4.3. T he Infected Red Blood Cell Membrane and Variant Surface Antigens
4.4. C
ritical Red Cell Invasion Ligands
4.5. Immune Regulation
5. E ffector Mechanisms for Blood-Stage Immunity
6. T argets of Blood-Stage Immunity
6.1. P
vMSP1
6.2. P
vMSP3
6.3. P
vMSP9
6.4. P
vAMA1
6.5. P
vRBPs
6.6. P
vDBP
6.7. R
ole of T-Cell Immune Responses in Acquired Immunity
6.8. Immunological Memory and Duration of Clinical Immunity
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7. Immune Responses to Malaria Pre-Erythrocytic Stages

8. S exual Stage Parasites and Transmission-Blocking Immunity
8.1. E vidences of Naturally Acquired TBI of P. falciparum
8.2. E vidences of Naturally Acquired TBI of P. vivax
8.3. T owards the Discovery of the Target Molecules against TBI of P. vivax
9. C
onclusions
10. F uture Directions
References
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Abstract
Population studies show that individuals acquire immunity to Plasmodium vivax more
quickly than Plasmodium falciparum irrespective of overall transmission intensity,
resulting in the peak burden of P. vivax malaria in younger age groups. Similarly, actively
induced P. vivax infections in malaria therapy patients resulted in faster and generally
more strain-transcending acquisition of immunity than P. falciparum infections. The
mechanisms behind the more rapid acquisition of immunity to P. vivax are poorly
understood. Natural acquired immune responses to P. vivax target both pre-erythrocytic and blood-stage antigens and include humoral and cellular components. To date,
only a few studies have investigated the association of these immune responses with
protection, with most studies focussing on a few merozoite antigens (such as the Pv
Duffy binding protein (PvDBP), the Pv reticulocyte binding proteins (PvRBPs), or the Pv
merozoite surface proteins (PvMSP1, 3 & 9)) or the circumsporozoite protein (PvCSP).
Naturally acquired transmission-blocking (TB) immunity (TBI) was also found in several
populations. Although limited, these data support the premise that developing a
multi-stage P. vivax vaccine may be feasible and is worth pursuing.
1. OVERVIEW OF NATURALLY ACQUIRED IMMUNITY

TO MALARIA

A major controlling force that determines the incidence and prevalence of malaria infection and disease in endemic areas is the parasitological
and clinical immunity collectively referred to as naturally acquired immunity (NAI). Generally, NAI determines not only the age-specific incidence
and prevalence of P. falciparum (Pf) and P. vivax (Pv) infections but also the
expression of pathological processes that underlie the clinical manifestations
of infection. Improved understanding of the development and maintenance
of NAI and the ability to cope with the severe manifestations of malaria are
now particularly important, since effective public health interventions that
reduce transmission are being deployed.
The microbiologist Robert Koch in 1899/1900 first outlined the basic elements of NAI to malaria during an expedition to the Dutch East Indies (Indonesia) and German New Guinea (now Papua New Guinea (PNG)) (Koch,
1900a, 1900b, 1900c, 1900d). By studying malaria infections in both indigenous
Natural Acquisition of Immunity to Plasmodium vivax
79
and immigrant clinical cases and asymptomatic individuals, he determined that

(1) in endemic areas, malaria is most apparent in young children and the parasites may be completely absent from the blood of adults; (2) individuals who are
constantly exposed to infection develop immunity to malaria with immigrants
from non-endemic areas, achieving an unmistakable degree of immunity just
as is the case in the children of natives after 34 years of exposure; (3) in people
who have experienced numerous previous malaria attacks, infections will often
present only with mild or no symptoms at all; and (4) adults from an island
where he only detected Plasmodium malariae became readily infected and ill
with P. falciparum malaria upon migrating to a highly endemic area of mainland
New Guinea, leading him to hypothesise that immunity against one Plasmodium species offers little protection against the other species. He summarised
his observations as follows:One may assume an anti-malarial immunity, which
under ordinary conditions is acquired only in 46 years and after repeated
attacks, to be conferred artificially and in a short time. Seeing, however, that as
yet we have no idea how to obtain the toxins necessary for the immunizing
process, chances of progress in this direction are small(Koch, 1900c).
Based on Robert Kochs seminal observations and many subsequent
studies, it is accepted that NAI is the capacity of the host to respond more
effectively during the second and subsequent exposures to a pathogen, as
compared to the primary exposure.The quality and longevity of this adaptive
response is highly variable, with some individuals acquiring long-term protection following a limited number of exposures, whereas repeated or ongoing
exposure being needed to generate and sustain protective immunity in other
cases. Both situations occur in malarial infection depending on the host, parasite, age, type of prior exposure and transmission intensity (related to parasite
diversity). An emerging consensus indicates that the essential features of NAI
are multifactorial, being primarily directed to blood-stage infection. In P. vivax
malaria, the development of NAI is achieved by, or dependent on, exposure
to both primary blood-stage infections and relapse infections in the blood.
Although the human immune system will attack almost at any stage in
the parasite life cycle in the human host, from sporozoite entry to the gamete fertilisation process in the mosquito midgut (Hollingdale etal., 1984),
NAI appears to be primarily target blood-stage infections. With respect
to P. vivax NAI is manifested as protection against high-density parasitaemia and uncomplicated clinical disease (fever, malaise and anaemia). This
immunity has the following primary characteristics: (1) the highest degree
of NAI occurs in adult residents of holoendemic areas, since this immunity
is presumed to depend on cumulative exposure to multiple parasite infections (and relapses) over time, yielding a sufficiently diverse repertoire of
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Ivo Mueller et al.
strain-specific immune responses of sufficient magnitude. (2) It is generally thought that a significant degree of continuous or regular antigenic
exposure, a condition referred to as premunition, is required, and in some
circumstances NAI is greatly diminished on cessation of infection (transmission). (3) NAI is relatively species specific (with respect to P. vivax versus
P. falciparum), and relatively strain specific (Collins etal., 2004a), although
strain-specific immunity to P. vivax is more difficult to determine than with
P. falciparum, because of the potential for diverse parasites resulting from the
relapse of dormant liver stages representing different strains. However, there
can be some degree of cross-protection among strains (Collins etal., 2004a).
NAI also develops to severe malaria-related illness (manifestations that result
in organ dysfunction, or severe anaemia <5gm/dl), which appears to be
acquired quickly and persists for a long time. This phenotype has been well
studied for falciparum malaria, but remains poorly understood for vivax
malaria. Premunition does not seem to be important in maintaining NAI
to this phenotype. It seems to be more important to maintaining NAI to
uncomplicated clinical malaria.
NAI can also result in a reduction in transmission in sexual stages to mosquitos and/or interferes with the development of gametes in the mosquito
midgut. There is still relatively little information available at the genomic
and post-genomic levels for P. vivax, but such information is becoming
available (Acharya etal., 2011; Bozdech etal., 2008; Carlton etal., 2008;
Chen et al., 2010; Dharia et al., 2010), allowing for deeper enquiry into
these questions and mechanisms of NAI to P. vivax.While there are naturally
acquired immune responses against pre-erythrocytic antigens, it is unlikely
that natural exposure to sporozoites is sufficiently high to induce protection
against pre-erythrocytic infection.
Broadly defined, premunition implies the maintenance of high levels of
malaria-specific antibodies (Abs) and a high frequency of malaria-specific T
cells, as a result of frequent or continual priming to malarial infections.The role
of premunition in maintaining NAI too is controversial; the precise duration
of time required for loss of NAI is unknown, as are the effects of age and variable transmission conditions. It is unclear whether continuous antigenic exposure impairs the ability to generate long-term immunologic memory and/or
whether protection is partially maintained by activating innate immune mechanisms. Alternatively, repeated antigen exposure may be necessary to sustain
high levels of cross-reactive Abs to multiple parasite stage to be fully protective.
Both these hypotheses are testable and remain to be fully explored.
NAI to P. vivax has been noted without premunition, such as with individuals treated for syphilis with malaria therapy (Boyd, 1947; Ciuca etal.,
81
1934; Collins etal., 2004a), and in low P. vivax transmission areas, such as
in the Solomon Islands (Harris etal., 2010) and Amazon (Alves etal., 2002;
Branch etal., 2005).Thus, NAI may develop with comparatively much lower
exposure than previously appreciated. This may occur because of restricted
parasite diversity. In low- or seasonal transmission settings, NAI to P. vivax has
not been adequately explored, and it may involve mechanisms of immune
protection distinct from those occurring in high-transmission areas.With the
recent wide introduction of malaria control measures, a better understanding of NAI in intermediate- and low-transmission conditions is timely and
important to understand how elimination efforts, including interventions
such as universal deployment of long-lasting insecticide-treated nets (LLINs),
may affect the health of local communities. On this note, it is important to
recognise that LLINs can help to prevent initial infections, but not relapses.
Absolute invitro correlates of protection to malaria are unknown. Specific biomarkers of protection to malaria have been proposed; however, there
is a lack of consensus as to what these biomarkers are. Potential biomarkers
for P. falciparum include elevated levels of serum Abs directed at merozoite
antigens (particularly invasion ligands) and/or variant surface antigen (VSA)
on infected erythrocytes (IEs). Potential biomarkers for P. vivax are not known,
given the limited investigation in this area to date, although there is some
evidence that functional Abs to PvDBP (Cole-Tobian etal., 2009; King etal.,
2008) and PvMSP1 (Nogueira etal., 2006) correlate with protection. These
immune responses represent associations rather than causal relations of protective immunity, but they are valuable if they can predict the level of NAI in
populations before and after the implementation of malaria control measures.
2. DIFFERENTIAL ACQUISITION OF IMMUNITY TO

P. VIVAX AND P. FALCIPARUM UNDER NATURAL
EXPOSURE

In the New Guinea island, which is highly endemic for both P. falciparum and P. vivax (Muller etal., 2003), P. vivax is the predominate source of
malarial infections and disease in children younger than 2years (Senn etal.,
2012). The incidence of P. vivax malaria started to rapidly decrease from the
second year of life, whereas P. falciparum incidence continued to increase
until the fourth year in observational cohort studies in children aged 14
(Lin etal., 2010) and 514 years (Michon etal., 2007; Fig. 3.1A) By 5years
of age most children had acquired an almost complete immunity to clinical P. vivax yet remained at considerable risk of P. falciparum illness despite a
similar burden of P. vivax blood-stage infections (Fig. 3.1B).
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Ivo Mueller et al.
Figure 3.1 Evidence for rapid acquisition of immunity to P. vivax under natural exposure
in areas with different transmission intensities. (A) Incidence of malaria in a cohort of PNG
children aged 14years (Lin etal., 2010). (B) Time to first PCR-positive infection and clinical
episode in PNG children aged 514years (Michon etal., 2007). (C) Prevalence of Plasmodium spp. infection among patients attending a rural PNG health centre and corresponding incidence of malaria-attributable fevers (lower panel) (Muller etal., 2009). (D) Incidence
of malaria in a cohort in Thailand (light bars: P. falciparum, dark bars: P. vivax) (Phimpraphi
etal., 2008). (E) Prevalence of infection by LM and PCR in a cross-sectional population
survey in East Sepik Province, PNG (Mueller etal., 2009). (F) Incidence of malaria in Sri
Lanka (Mendis etal., 2001). (For colour version of this figure, the reader is referred to the
online version of this book).
83
As a consequence of this remarkable difference in the rate of acquisition

of clinical immunity, the incidence of P. vivax-attributable febrile illness in
routine health surveillance is highest in children 1.01.9 years old, whereas
that of P. falciparum peaks in children aged 2.03.9years (Fig. 3.1A) (Muller
etal., 2009). Although P. vivax is a very significant source of severe illness
in infants (Poespoprodjo etal., 2009), the proportion of P. vivax infection
presenting with severe symptoms decreases rapidly with age (Genton etal.,
2008; Tjitra etal., 2008) and in children older than 1 year the incidence
of severe P. vivax illness is significantly lower than that due to P. falciparum
(Manning etal., 2012).
Similar differences are found in the age-specific prevalence of P. vivax
and P. falciparum infections: the prevalence of P. vivax by light microscopy
(LM) tends to reach its peaks in children aged 4.06.9years (24%), while
P. falciparum prevalence is highest in children aged 7.09.9 years (48%)
(Mueller etal., 2009). In adults, LM-positive P. vivax infections are less frequently detected (<10% prevalence) than P. falciparum infections (approximately 20%). When diagnosed by polymerase chain reaction (PCR), the
prevalence of both P. vivax and P. falciparum increased, the peak prevalence
shifted into older age groups (Pv: 7.09.9years: 51%, Pf: 10.019.9years:
74%) and infections were commonly detected even in adults (Fig. 3.1E).
Interestingly, in longitudinal cohorts of children aged 14 and 514years,
the incidence of newly acquired, genetically distinct, P. vivax blood-stage
infections (i.e. the molecular force-of-infection (molFOI)) was substantially
higher than that of P. falciparum (Koepfli etal., submitted for publication;
Michon etal., 2007; Mueller etal., 2012) despite a comparable (Michon
etal., 2007) or higher entomological inoculation (Benet etal., 2004; Hii
etal., 2001) rates for P. falciparum. P. falciparum molFOI increased from 1 to 4
years (Mueller etal., 2012) and remained constant thereafter (Michon etal.,
2007), whereas P. vivax molFOI did not change with increasing age (Koepfli
etal., submitted for publication; Michon etal., 2007).
Together these epidemiological observations indicate that in New
Guinea children malarial immunity is acquired much more rapidly to
P. vivax than P. falciparum. After 5 years of continuous exposure, children can
acquire an almost complete clinical immunity to P. vivax, which is characterized by their control of blood-stage parasite densities (often to a level
only detected by PCR) rather than the acquisition of a significant immunity against infection per se.
This difference in the rate of natural acquisition of immunity between
P. vivax and P. falciparum was not only found in the highly endemic areas
84
Ivo Mueller et al.
of New Guinea but also in other co-endemic areas of the world, including those with substantially lower transmission levels. In longitudinal studies carried out on the Western border of Thailand (Fig. 3.1D, (Lawpoolsri
etal., 2010; Phimpraphi etal., 2008)), in Sri Lanka (Fig. 3.1F, (Mendis etal.,
2001)) and in Vanuatu (Maitland et al., 1996), the incidence of P. vivax
malaria also decreased significantly faster with age than that due to P. falciparum, whereas in a Brazilian cohort among Amazonian settlers the risk of P.
vivax malaria started decreasing after 56 years of residence in the endemic
area compared to 89yrs for P. falciparum (da Silva-Nunes etal., 2008). This
is also not a recent phenomenon, as it was well known in the 1930s in areas
such as Greece (Balfour, 1935) and Puerto Rico (Earle, 1939) that P. vivax
malaria was a disease that predominantly affected children and P. falciparum
affected adolescents and adults.
The age at first exposure may be an important modulator of the natural
acquisition of malarial immunity. Studies involving non-immune migrants
to Indonesian New Guinea who were exposed for the first time to heavy
transmission indicated that adults acquired clinical immunity to malaria
after relatively few P. falciparum infections, in contrast to children, who
remained susceptible after comparable exposure (Baird etal., 1991, 2003).
This relationship was not observed with P. vivax infections; adults did not
acquire clinical immunity any more quickly than children (Baird, 1995).
This suggests there may be fundamental differences in how NAI develops
to Pv compared to Pf.
3. ACQUISITION OF IMMUNITY IN EXPERIMENTAL

INFECTIONS LESSONS FROM MALARIA
THERAPY PATIENTS AND IRRADIATED
SPOROZOITES

Prior to the confirmation in 1947 that penicillin could cure syphilis,
the primary treatment for neurosyphilis was fevers induced with malarial
infections. Plasmodium vivax was the preferred treatment over P. falciparum
because it could produce sustained fevers without requiring treatment.
Because malaria therapy did not cure syphilis, but just delayed the progression of disease, individuals were often repeatedly infected. With support from the Rockefeller Foundation in the 1930s and early 1940s, a
malaria research centre based at the Florida State Hospital in Tallahassee,
Florida, undertook a remarkable series of experiments involving neurosyphilis patients to study P. vivax biology and immunity (see (Boyd, 1947)).
85
Similar studies were being performed in the South Carolina State Hospital
(Collins and Jeffery, 1999; Collins etal., 2003, Collins etal., 2004a, 2004b)
and in hospitals in Europe (Ciuca etal., 1934). These experimental studies
were designed to study the natural history of Pv infections in humans. Individuals were infected either intravenously with Pv-IEs or by infected mosquitoes (to mimic natural infection). Malarial parasites were first detected
in malaria-nave subjects by day 11 or 12 following a mosquito inoculation
of P. vivax sporozoites. The onset of fevers occurred almost simultaneously
with the presence of parasites in the peripheral blood, with levels as low
as 10 parasites/l. Peak P. vivax parasitaemias generally occurred 57 days
after the onset of symptoms, with parasite densities rarely exceeding 12,000
parasites/l. Peak parasitemias coincided with highest fevers. As parasitaemia declined, fevers abated slightly, yet they persisted over the next 20 or
more days.There was a rough correlation with parasitaemia levels and fevers
yet parasites could persist in the peripheral blood at low densities for 23
months in the absence of fevers indicating some tolerance or immunity
to disease independent of infection. In some subjects with natural infections, relapse would occur after clearance of blood-stage parasites and these
induced much more attenuated clinical disease and lower parasitemias.
A similar pattern of infection was observed when blood-stage parasites were
directly inoculated, but the pre-patent period was shorter and depended on
the inoculum. Relapses, as expected, did not occur under this experimental
protocol.
About 23 months after clinical symptoms had disappeared (although
low levels of parasitaemia often persisted), the subjects were experimentally reinoculated either by mosquito infection or intravenous blood-stage
challenge with the same parasite strain. Up to two-thirds of the individuals were completely immune to clinical malaria upon rechallenge. By the
third exposure with the same strain, almost 100% of the individuals were
clinically immune (Boyd, 1947; Ciuca etal., 1934).This acquisition of clinical immunity with repeated infection of the same P. vivax parasite strain
occurred more rapidly than observed with Pf, although patients often had
to be treated for Pf when they became severely ill.These experimental studies support epidemiological studies described above (Gunewardena etal.,
1994; Lin etal., 2010; Maitland etal., 1996; Michon etal., 2007) that NAI
develops more rapidly to Pv compared to Pf. Clinical immunity persisted
for many years to the same strain. This was shown in some patients who
cleared their parasitemias and were again infected 3.5 years later and again
3.5 years after that with the same strain and retained solid clinical immunity,
86
Ivo Mueller et al.
although they developed low-level parasitemias. One subject had an interval

of 11 years between Pv infections and still retained solid clinical immunity. It
is unknown whether submicroscopic parasitemias persisted over this period
(relapsing parasites rarely occur past 2 years following initial infection) and
whether they were naturally exposed to P. vivax since the parasite was still
endemic at the time in Eastern Europe and the United States. Thus these
malaria therapy studies cannot exclude whether any persisting low-grade
infection(s) (premunition) accounted for the observed long-term immunity.
Since subjects quickly developed clinical immunity to the same parasite
strain, they were then infected with different P. vivax strains with the aim to
continue fever treatments. It became quickly apparent that immunity was
strain specific. In a series of patients treated between 1940 and 1963 at the
South Carolina State Hospital (see (Collins and Jeffery, 1999a, 1999b, 1999c,
1999d; Collins etal., 2004a, 2004b) for details), there were 36 patients with
primary infections with the St. Elizabeth strain of P. vivax followed by reinfection with P. vivax. Of these, 14 were re-infected with the homologous strain of the parasite (Group 1) and 22 patients were re-infected with
heterologous strains (17 with the South Pacific Chesson strain, Group 2).
During a secondary infection with the homologous St. Elizabeth strain
the geometric mean maximum parasite count was reduced from 9101/l to
998/l (P<0.001) and the geometric mean daily parasite count for the first
20 days was reduced from 924 to 16/l/day (P<0.001). This resulted in an
80% reduction in the incidence of fever of or above 101F (2.98 vs. 0.60/
person/week, rate ratio=0.20. P=0.002) and in a 91% reduction in fevers
of or above 104F (1.92 vs. 0.18/person/week, rate ratio=0.09. P=0.003).
These results show the rapid development of strain-specific immunity even
after a single primary infection. By contrast, only partial protection was
observed against the heterologous strain. Even though the maximum parasitaemia in 22 pairs of patients re-infected with heterologous parasites was
comparable to the primary infection (8460/l and 9196/l), the geometric
mean daily parasite count for the first 20 days of infection was significantly
lower (847/l/day vs. 336/l/day, P=0.002) and the incidence of fevers of
or above 101F and 104F were reduced by 48% (2.08 vs. 1.07/person/
week, rate ratio=0.52, P 0.007) and 55% (1.24 vs. 0.57/person/week, rate
ratio=0.45, P=0.004), respectively (Collins et al., 2004a). Similar results
were found in many different studies involving experimental malaria therapy patients (reviewed in (McGregor and Wilson, 1988; Taliaferro, 1949)).
For example, in a very large series of Roumanian patients (with likely prior
natural exposure), Ciuca etal. (1934) found that protection against P. vivax
87
parasitaemia rose from 34 to 100% during five P. vivax inoculations but

only from 22 to 97% during 10 homologous P. falciparum infections. Thus a
single P. vivax infection was sufficient to induce a strong clinical protection
during homologous re-infections and a partial protection during heterologous re-infections. However, no protection was found when patients initially infected with P. vivax were re-infected with P. falciparum (Collins and
Jeffery, 1999a), indicating no evidence for cross-species protection. A more
detailed description of experimental P. vivax infections in humans is found
in Chapter X [Snounou].
Experimental infections produced by inoculation with malaria- IEs
compared to those induced by bites of infected mosquitos induced similar
patterns of immunity. This indicates that the NAI induced was directed to
blood-stage infection. To assess whether pre-erythrocytic stage immunity
could develop and control the onset of a blood-stage infection, five volunteers were vaccinated with irradiated P. vivax sporozoites in the 1970s
(Clyde, 1990; Rieckmann, 1990). One volunteer exposed to 1979 mosquito
bites remained protected (i.e. no blood-stage parasites were detected) for
up to 9 months when challenged by a dose of six infective bites but not
when challenged with 12 infective bites. A second volunteer, who was vaccinated with both irradiated P. vivax (539 mosquito bites) and P. falciparum
sporozoites, was also protected for 8 months against both homologous and
heterologous infections, but this person was re-infected when re-challenged
at 11 months (Clyde, 1990). No protection was observed in the other three
volunteers exposed to fewer than 200 irradiated infected mosquitoes.
A similar dose-dependent protection was observed in two trials with irradiated P. vivax sporozoites in Saimiri monkeys (Collins etal., 1992) and Aotus
monkeys (Jordan-Villegas etal., 2011). These data indicate that very high
numbers of sporozoites acquired at a single time are required to induce sterile pre-erythrocytic immunity. Such doses are unlikely to be reached under
natural exposure, although the cumulative number of sporozoites over
months and years may reach similar levels in areas of high malaria transmission. This trickle effect of sporozoite exposure on pre-erythrocytic stage
immunity is unknown. It is difficult to extrapolate infections with irradiated sporozoite to natural infection since irradiated sporozites arrest development and can persist in the liver, which may induce a different type of
immune response compared to natural exposure (Scheller and Azad, 1995).
Table 3.1 summarises the primary findings from these extensive studies
of malaria infections in thousands of patients. There are some important
limitations to these human studies, however. It is uncertain whether the
88
Ivo Mueller et al.
Table 3.1 Features of NAI Based on Artificially Induced Infections in Humans
Immunity is directed against blood-stage infection no evidence of

pre-erythrocytic stage immunity.
A substantial degree of clinical immunity develops after a single untreated
infection and complete clinical immunity after two or three infections by the
same strain.
Immunity is strain specific with substantial cross-protection among strains.
Immunity is species specific; Pv does not seem to protect against Pf. It is
unclear whether the reverse is true.
Clinical immunity develops to P. vivax more rapidly than to P. falciparum.
Solid clinical immunity can persist for years. It is unknown whether this
requires relapsing blood-stage infection or persisting low-grade infections, but
this seems unlikely.
Exposure to large numbers of irradiated sporozoites could elicit pre-
erythrocyte stage immunity, but is not of long duration (<1 year). An
equally large exposure to sporozoites is unlikely under natural conditions.
strains used were really just one strain. In addition, submicroscopic parasitaemia could not be detected since only blood smears were used at the
time. Today, genetic methods can distinguish the presence of individual or
multiple strains or submicroscopic levels of P. vivax in the blood.
4. UNIQUE BIOLOGICAL CHARACTERISTICS OF

P. VIVAX THAT CONTRIBUTE TO NAI

Individuals exposed to a comparable number of infections and
clinical episodes of P. vivax and P. falciparum acquire clinical immunity to
P. vivax more rapidly than P. falciparum (Lin etal., 2010; Maitland etal., 1996;
Michon et al., 2007). Pv has several important differences in its biology
compared to Pf, which may help to explain the differences in the acquisition
of immunity to each of these species.
4.1. Relapses
In contrast to P. falciparum, P. vivax develops latent stages in the liver referred
to as hypnozoites. The hypnozoites can become activated to initiate one
or more blood-stage infections up to 2 years after an initial inoculation of
sporozoites by a mosquito bite (White, 2011) (and Chapter X: white).Thus,
P. vivax blood-stage immunity can be boosted even when malaria transmission
89
is low or absent. Initial relapses appear to be of the same original infecting

strain(s) (Imwong etal., 2012), which may contribute to the rapid and persistent NAI observed in each of the malaria therapy studies or development
of NAI in low-transmission areas (Craig and Kain, 1996). As individuals
acquire additional sporozoite inoculations, a diverse population of hypnozoites (and resulting relapsing parasite strains) may develop (Chen et al.,
2007; Imwong etal., 2007). NAI may develop quickly in PNG and other
areas of the South Pacific because of the frequent periodicity of relapsing
parasites, 3045 days following the primary infection, compared to P. vivax
strains from more temperate areas (White, 2011) (and Chapter X: white).
Thus in Pv endemic areas where Pv strains have longer relapsing periodicity
NAI would be predicted to develop more slowly. As children presenting
with clinical P. vivax are treated for both blood- and liver-stage infections
(i.e. with primaquine) to also eliminate relapsing parasites, it would also be
predicted that NAI may be significantly delayed if relapsing parasites are
important in boosting blood-stage immunity. Longitudinal cohort studies
can potentially help to distinguish and characterize the acquisition of NAI
in groups of individuals over time with more or less frequent relapsing patterns and with or without receiving primaquine therapy. In a recent cohort
in children aged 15 years in PNG, relapses contributed at least 50% of the
burden of P. vivax infections (Betuela etal., 2012).As a result, the number of
new P. vivax blood-stage infections is 23 times higher than that for P. falciparum (Mueller etal., 2012), despite similar or higher P. falciparum sporozoite
rates (Benet etal., 2004; Hii etal., 2001).
4.2. Force of Blood-Stage Infection

The force of blood-stage infection may also be higher for P. vivax than
P. falciparum as shown in the previous section in studies in PNG, resulting
in more frequent exposure to novel genotypes (Koepfli etal., submitted for
publication; Mueller etal., 2012). This may arise because effective population of P. vivax is often larger in a community (Ord etal., 2008) and because
of activation of genetically distinct hypnozoities (Chen etal., 2007; Imwong
etal., 2007, 2012).
4.3. T
he Infected Red Blood Cell Membrane and Variant
Surface Antigens
During intraerythrocytic development, both P. vivax and P. falciparum express
highly polymorphic antigens on the IE surface (Bernabeu etal., 2012; del
Portillo etal., 2001; Gunewardena etal., 1994; Leech etal., 1984; Marsh and
90
Ivo Mueller et al.
Howard, 1986) In the case of Pf they undergo clonal antigenic variation,

and acquired Abs against these antigens typically demonstrate a high degree
of strain specificity (Biggs etal., 1991). The predominant Pf VSA is erythrocyte membrane protein 1 (PfEMP1), which is encoded by a large family of
highly variable var genes (Baruch, 1999; Smith etal., 2001; Su etal., 1995)
and causes the adhesion of trophozoite and schizont IE to endothelial cell
receptors. One member from a repertoire of about 60 encoded proteins is
typically expressed at a time, in association with electron-dense parasiteinduced knob protrusions on the surface of the IEs (reviewed in (Scherf
etal., 2008)). Antigenic diversity and variation of surface antigens facilitates
the development of repeated infections over time, as new infections appear
to exploit gaps in the repertoire of variant-specific Abs. Prospective studies in children provide strong evidence that surface antigens are targets of
protective immunity by showing that Abs are associated with a reduced risk
of malaria and studies suggest that increasing exposure leads to a broad repertoire of Abs that provides protection against different variants (Bull etal.,
1998; Giha etal., 2000; Mackintosh etal., 2008). Abs to these IE surface
antigens are thought to confer protection by inhibiting vascular adhesion
and sequestration of IEs and by opsonising IEs for phagocytic clearance.
Thus NAI to VSA requires repeated exposure.
The role of predicted VSA at the surface of Pv IE is less clear but is
exemplified by the larger vir gene family (Bernabeu etal., 2012; Carlton
et al., 2008; del Portillo et al., 2001). There are 346 vir genes in the Pv
genome (Sal I strain) of varying sizes and about 160 have domains consistent with the VIR proteins targeted to the erythrocyte surface. This large
diversity suggests multiple functions of these proteins including, and perhaps predominantly, host immune evasion mechanisms. The Pv VIR proteins are much smaller than PfEMP1, lack comparable adhesive domains
and are not clonally expressed (Fernandez-Becerra et al., 2005). VIR
proteins are expressed earlier than PfEMP1 during the intraerythrocytic
cycle and the timing of their expression is not consistent among isolates
(Bozdech et al., 2008) although these observations may be complicated
by the difficulty of growing synchronised parasites in vitro. Recent evidence suggests that certain VIR proteins that are expressed on the surface
of IEs facilitate the adherence of P. vivax IE to endothelial cells possibly
via ICAM-1 (Bernabeu et al., 2012; Carvalho et al., 2010), which may
facilitate their sequestration in the lung (Anstey et al., 2009), and it has
also been speculated that they may facilitate escape from spleen clearance
(Fernandez-Becerra etal., 2009).
91
By immunofluorescent assay (IFA), schizont stages of Pv were shown

to be recognised by individuals with prior Pv infection and this reactivity could be isolate specific (Mendis et al., 1988). Moreover, individuals
with more than three documented Pv infections compared to individuals with fewer known Pv infections had a greater number and breadth
of Pv isolates suggesting acquired Ab responses to Pv IEs (Mendis et al.,
1988). It appears that some of the Ab responses are directed to VIR proteins
on the Pv-infected red blood cell (RBC) surface (Bernabeu et al., 2012;
Carvalho et al., 2010; del Portillo et al., 2001; Fernandez-Becerra et al.,
2005, 2009); however, their role in NAI is unclear. The potential role of
VIR proteins in NAI may be better understood in longitudinal cohort
studies by studying the degree and breadth of Ab responses to Pv isolates.
Although continuous invitro cultures of Pv are not a reality today, shortterm cultures (Grimberg et al., 2007; Nichols et al., 1987; Russell et al.,
2011; Udomsangpetch etal., 2007, 2008; Wertheimer and Barnwell, 1989)
using fresh isolates or frozen Pv could examine the breadth and strength of
Ab recognition surface of Pv-infected schizonts using flow cytometry from
Pv-exposed individuals with and without NAI to Pv.
A large number of proteins have been identified in association with the
P. vivax IE membranes and these structures may also be targets of the host
immune response (Barnwell et al., 1990). In contrast to rigid, the knobby
and sticky Pf-infected red cell membrane, Pv remodels the reticulocyte host
cell membrane to produce numerous caveolavesicle complexes (CVCs)
all along the surface of the infected cell (Aikawa, 1971; Aikawa etal., 1975;
Akinyi etal., 2012; Suwanarusk etal., 2004) that may increase erythrocyte
membrane deformability. Where the VIR proteins may be positioned relative to these abundant surface-exposed CVC structures is not known. The
function of CVCs may be targets of host immune responses. For example, the recently described Plasmodium helical interspersed subtelomeric
(PHIST) protein known as PvHIST/CVC-8195 (Table 3.2) is a dominant
component of the CVCs (Akinyi et al., 2012; Barnwell et al., 1990). As
such, it may stimulate NAI. Alternately, its release upon rupture of infected
RBCs may be pro-inflammatory and enhance virulence of the infection
(Akinyi etal., 2012).
4.4. Critical Red Cell Invasion Ligands

In contrast to Pf, Pv selectively invades reticulocytes and requires the
Duffy antigen, a chemokine receptor on erythrocytes, for successful
invasion (Miller etal., 1976) (although exceptions occur (Menard etal.,
92
Table 3.2 P. vivax blood-stage antigens that elicite host immune responses and/or correlated with protection
Protection in Inhibition of
Ab response Correlate with
non-human
Pv invasion/
increase with protection
Antigen
primates
growth invitro age/exposure longitudinal studies References
Merozoite surface proteins
MSP119
MSP1 N-terminal
MSP1 C-terminal
+/
MSP3.10 (N-terminal)
MSP9.10 (N-terminal)
+
+
+
+
(Barbedo etal., 2007; Braga etal., 2002; Han

etal., 2004; Soares and Rodrigues, 2002;
Soares etal., 1997, 1999; Zeyrek etal., 2008)
(Nogueira etal., 2006)
(Collins etal., 1999; Dutta etal., 2005;
Nogueira etal., 2006; Sierra etal., 2003;
Yang etal., 1999)
(Lima-Junior etal., 2008)
Apical membrane protein

AMA1
Erythrocyte-binding ligands
PvDBPII (total)
+
(Rodrigues etal., 2005; Seth etal., 2010)
+
+
(Arevalo-Herrera et al., 2006, Ceravelo et al.,

2005, Cole-Tobian et al., 2002, 2009)
(Grimberg et al, 2007, King et al 2008)
(Tran etal., 2005)
+
(Akinyi etal., 2012)
Ivo Mueller et al.
PvDBPII (binding
inhibitory)
Reticulocyte-binding proteins
RBP1
RBP2
Variant surface antigens
VIR
Caveolavesicle complex
(CVC) proteins
PvPHIST-8195
93
2010)). This is reminiscent of the requirement of the chemokine receptor, CCR5, for HIV invasion of T cells. There is a single known parasite
ligand for the Duffy antigen called PvDBP (Adams etal., 1992; Chitnis
etal., 1996). PvRBPs and possibly other molecules target invasion of the
immature erythrocytes (Barnwell and Galinski, 1995; Galinski and Barnwell, 1996; Galinski et al., 1992). Although there are likely many more
molecules yet to be discovered that are critical for Pv invasion of erythrocytes, some individuals may develop NAI by targeting immune responses
to these critical and perhaps non-redundant pathways. This might contribute to rapid development of NAI compared to Pf with more redundant invasions pathways.
4.5. Immune Regulation

The induction of potent immunosuppressive networks can impair the development of adaptive and innate immunity. Malaria infections often develop
asymptomatic parasitemia, occasionally with high parasite burdens. Thus
infection in the absence of inflammation (lack of costimulatory or other
signals) promotes immune tolerance. It has been shown that patients with
severe falciparum malaria induce a potent set of regulatory T cells whose
numbers and suppressive function increase with parasite burden (Hansen
and Schofield, 2010; Jangpatarapongsa et al., 2008; Minigo et al., 2009;
Scholzen etal., 2009, 2010; Walther etal., 2009). Other immunoregulatory
networks likely exist with heavy infections such as development of atypical
and potentially suppressive B cells (Pierce, 2009; Weiss etal., 2009) and suppression of antigen-presenting cell functions akin to endotoxin tolerance
(Boutlis etal., 2006). Immune tolerance can also result from in utero exposure to malaria or their soluble products (King etal., 2002; Mackroth etal.,
2011; Malhotra et al., 2008, 2009; Metenou et al., 2007) that can persist
into early childhood (Malhotra etal., 2009). Malaria in pregnancy is much
less common and if present, is less likely to cause altered placental dysfunction for Pv compared to Pf (Rogerson etal., 2007; ter Kuile and Rogerson,
2008). Thus prenatal exposure to Pv antigens are less likely to occur compared to Pf; however, prenatal exposure to Pv remains poorly studied.
5. EFFECTOR MECHANISMS FOR BLOOD-STAGE

IMMUNITY

Adoptive transfer of serum from avian (Manwell and Goldstein,
1940), murine (Parashar et al., 1977), non-human primate (Coggeshall
94
Ivo Mueller et al.
and Kumm, 1937) and human falciparum malarias with NAI (Cohen
et al., 1961; McGregor, 1964) into nave animals or humans protects
against clinical malaria or significantly attenuates the severity and burden of malaria (Cohen etal., 1961; McGregor, 1964). Such experiments
have established that Abs are critical for NAI to blood-stage malarial
infection. Interestingly, passive transfer of 500ml of blood from neurosyphilis subjects with solid clinical immunity to Pv failed to induce any
protection against Pv disease in nave subjects (Boyd, 1947) either at the
time of infection or early in the course of clinical disease. The relative
amount of transferred serum was similar to that observed with successful
passive transfer of immunity observed to Plasmodium knowlesi in rhesus
macaques (Coggeshall and Kumm, 1937) but with about half the amount
of total Abs observed with Pf in humans (Cohen etal., 1961). Humoral
immunity is likely important for NAI to Pv but cellular immunity may
contribute to Pv protection more than appreciated. For example, it has
been proposed that Pv IE escape splenic clearance by increasing their
deformability, enabling passage through adjacent endothelial cells into
the venous sinus lumen, thus avoiding destruction by splenic macrophages (Fernandez-Becerra etal., 2009). If this is the case, then immune
mechanisms that may impair Pv IE deformability would facilitate their
clearance by the spleen. Another proposed hypothesis is that VIR proteins on the surface of Pv IE facilitate adherence in the spleen, preventing
phagocytosis by splenic macrophages. Since NAI to Pv is strain specific,
this suggests that targets of NAI are likely to be highly variable among
strains.
6. T
ARGETS OF BLOOD-STAGE IMMUNITY

Although much can be inferred from the basic biology of Plasmodium merozoites (Fig. 3.2), there are many gaps in our knowledge of the
biology of P. vivax merozoites. For example, why they selective invade
host reticulocytes and details of the growth and development of the various blood-stage developmental forms (reviewed in Galinski etal., 2005).
In addition, only a relatively small group of merozoite proteins have so
far been identified with specific localisations and confirmed functional
roles. It is important to remain cognizant of the fact that there are a large
number of hypothetical proteins and that many are likely to have critical
biological functions and be important for the development of NAI. Moreover, the specific proteins, precise hostparasite interactions, and order of
95
Figure 3.2 Potential targets and mechanisms for blood-stage immunity. Experimental
systems based on different species of Plasmodium have shown that there are multiple
points in the erythrocyte invasion process that could be targets of the host immune
response. The exact molecules involved and sequence of events remain to be fully
defined, and these may differ among species. Free merozoites, prior to invasion of RBCs,
are susceptible to host immune responses. The various MSPs that cover the merozoite
could function in the initial recognition of RBC host cells and be the target of opsonising Abs than can trigger cell-mediated merozoite killing or directly interfere with initial
adherence of the merozoite to the RBC. The merozoite quickly reorients, and through
sequential predicted interactions of the RBPs and the DBP, the parasites apical pole is
brought into close apposition to the RBC membrane. Next, RON proteins are released
from rhoptry organelles near the apical end of the merozoite and presumably inserted
into the merozoite membrane. With the interaction of apical membrane protein (AMA1)
a junction forms with the RBC membrane that generates an actin-dependent process
that facilitates the entry of the merozoite into the RBC. A parasitophorous vacuole
forms in the process. As P. vivax parasites grow, the IE membrane is modified by the
development of numerous CVCs and proteins that may facilitate some level of adherence to the vascular endothelium. All these alterations represent potential targets of
opsonising Abs and iRBC clearance. Since the Pv membrane is permeable to Abs (Lyon
and Haynes, 1986) and proteinases are essential for the release of merozoites from the IE
(Salmon etal., 2001), it is conceivable that acquired immune responses may also target
these proteinases. (For colour version of this figure, the reader is referred to the online
version of this book).
molecular events can differ among the species of Plasmodium, and among
strains within a species.
In this section, key highlights of the current known targets of P. vivax
blood-stage immune responses are presented. Several recent review articles
also summarise what is known about the biology, immunology and vaccine progress pertinent to each of these proteins (Arevalo-Herrera etal.,
96
Ivo Mueller et al.
2010; Galinski and Barnwell, 2008; Mueller et al., 2009). Post-genomic

research and modern technologies have the potential to enable the rapid
great expansion of our understanding of the biology of P. vivax bloodstage parasites and stimulate a greater understanding of NAI and the relative importance of immune responses to a much broader panel of antigens.
Evaluating the global individual and population-based immune response
using P. vivax blood-stage protein arrays is one way forward to grasp a
more complete picture (Chen etal., 2010; Trieu etal., 2011). The ultimate
challenge will be to decipher which of these immune responses can be
confidently and reliably associated with protection of individuals and not
simply represent active immune stimulation and the possible masking of
protective responses. Protective immune responses are likely to be composed of functional Abs of high affinity and memory T cells that target
critical portions of molecules involved in essential biological function of
the parasite. The parasite is likely to have evolved mechanisms to impair
development of protective immune responses such as antigenic variation,
poor immunogenicity, masking of critical regions by non-essential immunogenic regions or controlled expression of these regions only at essential points in the parasite invasion or development. Therefore, a detailed
understanding of the structural biology and function of these molecules
are essential. So far, no robust correlates of NAI have been identified for
Pf, although the levels and breadth of Ab reactivity to different variants
of PfEMP1 suggest an association with NAI (Bull etal., 1998; Giha etal.,
2000; Mackintosh et al., 2008; Marsh et al., 1989). Likewise, confirming reliable immune correlates of NAI for Pv will continue to pose a
challenge.
Plasmodium vivax merozoite proteins that have been investigated with
some consistency over the past 20 years include the PvRBPs, the PvDBP,
apical membrane protein (PvAMA1) and several PvMSPs.
Merozoites can initially attach to RBCs at any point on their surface
and then must reorient prior to invasion so that the apical pole of the
parasite is juxtaposed to the RBC surface (Galinski and Barnwell, 1996;
Srinivasan, P., et al., 2011 also reviewed in Chapter X and see Fig. 3.2). At
least a dozen distinctive MSP (including MSP3 which constitutes a family of proteins) have been reported to cover the surface of merozoites and
can contribute to the merozoites reversible adhesive properties. They can
be abundantly expressed and as a group these proteins have been considered important vaccine candidates based on their location and extensive
immune response and vaccination studies carried out for over 30 years.
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Plasmodium vivax research on MSPs has so far focused predominantly on

PvMSP1, PvMSP3 and PvMSP9.
6.1. PvMSP1
This is the most abundant and best-studied malaria blood-stage antigen.
The known biological and immunological processes relating to MSP1
have derived from studies of Pf and Pk. Whether these observations can
be extrapolated to PvMSP1 is uncertain, yet its biology is likely to be
similar among Plasmodium spp. MSP-1 undergoes two successive proteolytic cleavage events (Blackman etal., 1994). The second processing event
occurs immediately before merozoite invasion of RBCs, resulting in the
cleavage of the p42 fragment into p33 and p19 sub-fragments (Gerold
etal., 1996). The p19 molecule remains attached to the merozoite surface
through a glycosylphosphatidylinisotol (GPI) anchor and is composed of
two epidermal growth factor-like domains (Morgan etal., 1999), which
may have a role in the invading complex. Experimental data suggests that
PvMSP1 has a similar biology and function as PfMSP1 (del Portillo etal.,
1991; Han et al., 2004). Antibodies specific to PfMSP1, particularly the
C-terminal region (42- and 19-kD sub-fragments) have been shown to
block parasite invasion invitro (Egan etal., 1999), induce protective immunity in animal models and functional Abs to PfMSP1 correlate with protection in human studies (John etal., 2004). With respect to Pv, multiple
studies have shown older or more heavily exposed individuals who are
more likely to have NAI to show greater humoral and cellular reactivity to PvMSP1 (Bang etal., 2011; Collins etal., 1999; Egan etal., 1999;
Galinski etal., 1999, 2001; John etal., 2004; Singh etal., 2009;ValderramaAguirre et al., 2005) and human Abs specific to PvMSP119 correlated
with protection against clinical P. vivax malaria in a longitudinal study
(Nogueira et al., 2006). However, vaccination of non-human primates
with PvMSP119 failed to consistently induce protection. Immunisation
of Aotus monkeys with a polymorphic N-terminal region of PvMSP1
afforded only partial protection to challenge with Pv (Valderrama-Aguirre
et al., 2005), while immunisation with PvMSP119 failed to induce substantial protection in splenectomised Saimiri boliviensis monkeys (Collins
et al., 1999). Yet, immunisation with recombinant MSP142 and MSP119
from Plasmodium cynomolgi, a simian malaria species closely related to Pv,
in its natural host, Macaca sinica, was highly protective to challenge infection (160) suggesting that protection may be host specific or production of
properly refolded PvMSP119 may be more difficult than other Plasmodium
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Ivo Mueller et al.
spp. Thus the role of PvMSP1 in NAI and the value of PvMSP119 as a
vaccine candidate remain uncertain.
6.2. PvMSP3
Plasmodium vivax has a family of msp3 genes (Galinski etal., 1999, 2001)
with 11 members reported for the initially sequenced strain, Sal I (Carlton
etal., 2008). These gene family members are positioned head to tail on
chromosome 10 and gene expression studies demonstrate that all but the
final member are expressed during a blood-stage infection. These data are
similar to earlier studies showing the presence of an msp3 gene family in
P. falciparum on chromosome 10 and the expression of all members (Singh
etal., 2009). The first PfMSP3 described has been advancing as a viable
vaccine candidate to clinical trials (Bang etal., 2011; Belard etal., 2011;
Sirima etal., 2011). PvMSP3s can be dramatically diverse, as demonstrated
for the PvMSP3 and PvMSP3 members, showing numerous point
mutations as well as insertions and deletions (Rayner etal., 2002, 2004a).
Basic immunological enquiry to understand the role that PvMSP3s may
have in NAI has so far been focused on PvMSP3 (Lima-Junior et al.,
2011, 2012). In summary, Lima-Junior etal. (2011) have reported the identification of a set of B-cell epitopes from the central -helical region of
PvMSP3 that are widely recognised in Rondonia State, in the Brazilian
Amazon. Levels of total IgG and specifically subclass (IgG1 and IgG3) Abs
were associated with increased exposure to the parasite Lima-Junior etal.,
2011. This group has also begun to investigate the human leukocyte antigen (HLA) types associated with such responses (Lima-Junior etal., 2012).
Others have confirmed the natural acquisition of Abs to a different central
domain called PvMSP3359798, noting a predominance of IgG1 Abs followed by IgG2 and an association with anaemia (Mourao et al., 2012).
A pre-clinical vaccine study showed partial efficacy upon immunisation
with PvMSP3 and PvMSP3 near full-length recombinant proteins
using Freunds adjuvant in S. boliviensis monkeys (Barnwell and Galinski,
unpublished data); however, no efficacy was achieved with these proteins
in a subsequent trial using Montanide 720 as an adjuvant (Jiang, Barnwell,
Galinski etal., unpublished data). PvMSP3, and have been renamed
as PvMSP3.10, PvMSP3.3 and PvMSP3.1, respectively, based on their
chromosomal positioning among all 11 gene family members. The relative
importance of the different members of the PvMSP3 family needs further
investigation, with regard to the biology of the parasite, diversity and NAI
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and to discern which components of this family may warrant further study
as vaccine candidates. The function of these proteins is not known. Each
member is characterized by an NLRNG motif near the signal peptide,
a centrally located alpha helical coiled-coil domain and the absence of
a membrane-anchoring mechanism (Carlton etal., 2008; Galinski etal.,
1999, 2001).The expression pattern of each protein is not precisely identical and one member is in fact uniquely expressed at the apical pole of the
merozoite (Jiang etal submitted).
6.3. PvMSP9
Similar to PvMSP3 family members (Carlton et al., 2008; Galinski et al.,
1999, 2001), PvMSP9 associates with the surface of the merozoite but
does not have an anchoring mechanism, i.e. hydrophobic transmembrane
domain or GPI anchor (Barnwell etal., 1999; V
argas-Serrato etal., 2002).
It has a homologue in P. falciparum known as p101 or ABRA (Kushwaha
etal., 2000) and the simian malaria species P. knowlesi and P. cynomolgi (Barnwell etal., 1999; V
argas-Serrato etal., 2002). Short-term cultures have been
used to show that a P. vivax monoclonal Ab (mAb) and polyclonal antisera
raised against the native P. cynomologi protein can inhibit merozoite invasion
of reticulocytes, raising its profile as a possible vaccine candidate (Barnwell
etal., 1999; V
argas-Serrato etal., 2002). NAI studies have been ongoing in
Brazil and PNG, two epidemiologically distinctive regions with regard to
malaria transmission (Lima-Junior etal., 2008, 2011, 2012). In the Brazilian Amazon, Lima-Junior and colleagues have studied humoral and cellular immune responses in a cross-sectional study involving individuals from
three Rondonia communities with different exposure histories. Abs to the
conserved N-terminal region and a C-terminal repeat region were higher
in residents who had established in the area for the longest period of time,
and the responses correlated with age. IgG1 and IgG2 subclass responses
predominated and interferon (IFN)- and interleukin (IL)-4 responses were
generated against PvMSP9 peptides (Lima-Junior et al., 2008). Promiscuous T-cell epitopes in PvMSP9 were shown to induce the production of
these cytokines in the individuals from this study, with diverse HLA-DR
backgrounds, which could prove to be advantageous for vaccine development (Lima-Junior etal., 2010). HLA-DRB1*04 carriers, frequent in the
native Amazon population, produced higher levels of PvMSP9 Abs compared to PvMSP1 and PvMSP3 and the time of exposure correlated with
these responses (Lima-Junior etal., 2012). A more recent prospective study
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in PNG concluded that Abs to the N-terminal region of PvMSP9 were

significantly associated with a reduced risk of P. vivax malaria (unpublished
observations). Continued research is underway in these endemic regions,
aiming for a more comprehensive understanding of NAI to PvMSP9, other
proteins and P. vivax overall.
6.4. PvAMA1
Investigations on the NAI to Apical Membrane Antigen-1 of P. vivax
(PvAMA1) have followed in the footsteps of PfAMA1, which has been
advancing as a vaccine candidate (Dutta et al., 2009; Remarque et al.,
2008a, 2008b, 2012; Sheehy etal., 2012). This interesting protein localises
to the microneme organelles of the merozoite and then becomes translocated to the surface where it binds to the moving junction protein
complex at the interface of the host erythrocyte target cell through interaction with the RON2 protein (Hossain et al., 2012; Lamarque et al.,
2011; Srinivasan etal., 2011). Antibodies directed to PvAMA1 may function to prevent its adhesive interactions and pathogenic properties (see
below). Lacerda Bueno et al have been investigating NAI to PvAMA1
in Manaus and Cuiab, Brazil, and have identified a linear B-cell epitope in Domain II that is highly antigenic in natural infections. IgG and
subclass Abs that correspond to this region and the whole protein were
analysed (Bueno etal., 2011). The genetic diversity and immune response
to PvAMA1 have also been studied in Sri Lanka (Dias etal., 2011), with
a predominance of IgG1 and IgG3 responses (Wickramarachchi et al.,
2006). Most individuals studied developed Ab responses to linear and
conformational epitopes of Domain II. Extensive polymorphism was
reported in this suggesting this region of PvAMA1 is under immune
selection (Dias et al., 2011). Similar results were reported from genetic
studies in India and Thailand, with the immunological implications of
varying selective forces and functional constraints (Putaporntip et al.,
2009; Thakur et al., 2008). However, polymorphism was not detected
in the Domain II loop region that has been shown to be conserved and
predicted to be functionally important in PfAMA1 (Chesne-Seck et al.,
2005, Pizarro et al., 2005). Both B- and T-cell epitopes have been identified in this region of the molecule (Lal et al., 1996, Bueno et al., 2011).
NAI to Domain II was also found to stand out in earlier immunogenicity
studies from Brazil (Mufalo etal., 2008). Interestingly PvAMA1 itself is
pro-inflammatory which may contribute to its strong immunogenicity
(Bueno etal., 2008, 2009).
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6.5. PvRBPs
PvRBPs may be the first, or among the first apically located proteins, to
bind in an irreversible manner to reticulocyte target cells, in essence performing the critical functions of host cell section and commitment (Galinski and Barnwell, 1996; Galinski etal., 1992). At the time of their discovery,
two members of the PvRBP family (PvRBP1 and PvRBP2) were identified and shown to adhere to reticulocytes in invitro erythrocyte adhesion
assays (Galinski etal., 1992). It has been proposed that the PvRBPs select
the immature RBCs as host cells and then trigger the release of the PvDBPs
from their micronemal location in time for the critical junction formation
step that precedes entry of the merozoite into the target host cell (Singh
et al., 2005). The PvRBPs are large proteins of approximately 330 kDa.
Related reticulocyte binding-like (RBL) proteins have been identified in
P. falciparum (Rayner etal., 2000; Triglia etal., 2001) (reviewed in (Gaur and
Chitnis, 2011)), P. knowlesi (Meyer etal., 2009) and other non-human primate (P. cynomolgi, Plasmodium reichenowi) and rodent malaria model systems
(Keen etal., 1994; Iyer etal., 2007; Okenu etal., 2005; Rayner etal., 2004b,
2004c).
The pvrbp2 gene was found to be much more diverse than pvrbp1 in
an initial survey of parasite isolates originating from Brazil and Thailand
(Rayner et al., 2005). Polymorphisms within pvrbp1 were most abundant
in the N-terminal region of this protein, within the location of a predicted RBC-binding domain (Rosas-Acosta, 1998; Urquiza etal., 2002); a
similar binding region has been identified in P. falciparum (Gao etal., 2008;
Gaur etal., 2007; Rodriguez etal., 2008; Triglia etal., 2011) and P. knowlesi
(Semenya etal., 2012) RBL homologues.
Although regarded as adhesion proteins that could potentially be inhibited by Abs, there have only been a few studies geared at understanding NAI
to PvRBP1 and PvRBP2. Segments spanning these proteins have been
expressed as recombinant proteins and immune responses to PvRBP1 studied in comparison to PvDBP-II (Region II) in Brazil (Tran etal., 2005).
Ab responses were predominantly cytophilic IgGs and significantly correlated with exposure, and the highest levels of anti-PvRBP1 IgG were
to the N-terminal region that is predicted to contain the binding domain
and focal increased polymorphisms (Tran et al., 2005). As noted below
with regard to the PvDBP-II-binding region, diversity of the N-terminal
region of RvRBP1 may similarly result from immune pressure on this proteins binding domain. It is also noteworthy that more rapid and robust
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Ab responses were detected in this study for PvDBP-RII; however, the

evidence suggests that anti-PvRBP1 Abs may last longer in the absence of
repeat vivax infections (Tran etal., 2005).
The P. vivax genome (Sal I strain) project reported the existence of additional pvrbp genes (Carlton et al., 2008). Continued sequence analysis of
these genes (as many as 10 reported) from Sal I and other monkey-adapted
P. vivax isolates indicates that several of them represent gene fragments or
incomplete reading frames (unpublished data). Recent investigations of four
P. vivax isolates from Thailand confirmed the presence of multiple pvrbp
pseudogenes and a high level of diversity in the pvrbp2a and pvrbp2b members of this gene family, suggesting immune pressure on these particular
family members (Kosaisavee et al., 2012). Continued investigations are
important to help predict which of these proteins, and which regions of
them, may be most critical for the biology of the parasite and relevant for
the development of NAI.
6.6. PvDBP
Plasmodium vivax DBP is a microneme protein associated with the decisive irreversible step of junction formation between the merozoite and the
host erythrocyte receptors during invasion, giving DBP priority as a prime
target for vaccine-induced Ab-mediated immunity against asexual stages
of the parasite (Fig. 3.2) (Adams etal., 1990; Chitnis, 2001). The receptor
for DBP is the Duffy antigen receptor for chemokines or Fy blood group
antigen, which is the non-transducing chemokine receptor on the erythrocyte surface (Horuk etal., 1993; Miller etal., 1975, 1976). Fy is a minor
blood group antigen that has two immunologically distinct alleles referred
to as FY*A and FY*B resulting from a single point mutation. Historically,
the vital need of the DBP-Fy interaction is evident from the absence of
P. vivax from regions of Africa with a high prevalence of Fy negativity
(Guerra etal., 2010). Recent additional evidence of the importance of the
DBP-Fy interaction was the demonstration of the protective effect against
clinical vivax malaria by the FY*A allele. Individuals with the Fy a+b
phenotype demonstrated a 3080% reduced risk of clinical vivax, but not
falciparum malaria, in a prospective cohort study in the Brazilian Amazon
(King etal., 2011). The major finding is the FY*A allele, which is predominant in Southeast Asian and many American populations, that confers a
selective advantage against vivax malaria.
Naturally acquired Abs to DBP are prevalent in residents of areas
where vivax malaria is endemic and these Abs can inhibit DBPII binding
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and merozoite invasion of human erythrocytes (Grimberg etal., 2007).

Individuals tend to show significant quantitative and qualitative differences in their anti-DBPII serological responses that overall increase with
age and exposure (Chootong etal., 2010; Cole-Tobian etal., 2002; V
anBuskirk etal., 2004; Xainli etal., 2003). Interestingly, about 810% of P.
vivax exposed individuals acquire high titre, strain-transcending broadly
neutralising Abs (Chootong etal., 2010; King etal., 2008) that are associated with 50% reduction in the risk for P. vivax infection (King etal.,
2008) and disease (unpublished data) based on longitudinal cohort studies.
Usually, the initial Ab response is not broadly protective, while strain transcendent immunity develops only after repeated exposure (Cole-Tobian
etal., 2002; Xainli etal., 2003). Vaccine-induced anti-DBP Abs partially
inhibit erythrocyte binding (Devi etal., 2007; Grimberg etal., 2007;VanBuskirk etal., 2004) and invasion of host erythrocytes and induce partial
protection in monkeys (Arevalo-Herrera etal., 2005). Epitope targets of
neutralising Abs mapped onto DBPII crystal structure suggest a functional mechanism of anti-DBP immunity that blocks DBP dimerisation
to inhibit merozoite invasion (Batchelor etal., 2011). However, PvDBPII is highly polymorphic presumably as an immune evasion mechanism
responsible for strain-specific immunity. Strain-transcending neutralising
immunity is achieved when Abs target functionally conserved epitopes
thereby blocking DBP dimerisation and inhibiting invasion. The challenge in developing PvDBPII-based vaccine is the induction of straintranscending Abs. Of interest, invitro studies suggest that naturally acquired
and artificially induced Abs block binding of recombinant PvDBPII to
erythrocytes expressing Fya+a+ (associated with protection) more effectively than to Fy b+b+ erythrocytes. Thus, Fy polymorphism may be
an important variable suggesting that vaccine efficacy to PvDBPII-based
vaccine may be more effective in people with Fya+a+ compared to Fy
b+b+ genotype (King et al., 2011). Other erythrocyte polymorphisms
such as Southeast Asian ovalocytosis may also affect the acquisition of
PvDBPII-blocking Abs under natural exposure (Rosanas-Urgell et al.,
2012).
6.7. Role of T-Cell Immune Responses in Acquired Immunity

Although an increasing number of studies have begun to look at T-cell
responses to P. vivax antigens, there is no study that has correlated certain
P. vivax-specific T-cell responses with NAI in longitudinal cohort studies.
CD4+ T cells provide help to B cells and regulate inflammatory responses
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to acute malaria infections, e.g. by induction of natural and adaptive

regulatory T cells (i.e. increased IL-10 production). T cells play important
roles in parasite elimination by activation of natural killer (NK) cells and
monocytes that facilitate parasite elimination by direct killing or phagocytosis of opsonised merozoites and IEs in the peripheral circulation and
particularly the spleen. P. vivax-specific CD4+ T cells have been shown
to activate NK cells and T cells involved in innate and intermediate
immune mechanisms that facilitate removal of malaria-infected RBCs
(Artavanis-Tsakonas et al., 2003; DOmbrain et al., 2008), possibly by
granzyme B release from NK cells causing erythrocytosis (Bottger etal.,
2012). Cytokines involved in NK activation include IL-12, IL-15, IL-18
and IL-2. Thus antigen-specific CD4+ memory T cells that secrete IL-2
(i.e. (T helper) Th1-type) can enhance NK cell activation along with
pro-inflammatory mediators, i.e. molecules that engage Toll-like and
innate receptors on monocytes to produce IL-12. NK cells themselves
are potent producers of IFN- and tumour necrosis factor (TNF)-
that can further amplify NK-cell-mediated and macrophage-mediated
parasite elimination. Recently, an expanded absolute number of IL-17secreting CD4+ memory T cells have been associated with P. vivax infection (Bueno etal., 2012). This is another important effector CD4+ T cell
subset that further amplifies pro-inflammatory cytokine networks and
facilitates recruitment of neutrophils and monocytes. Overall, subjects
with putative immunity to Pv have been shown to have increased frequencies of memory CD4+ T cells (Jangpatarapongsa etal., 2006), suggesting a possible role in NAI; however, these were not assessed for their
antigen specificity or pattern of cytokine secretion. This Pv antigenspecific effector CD4+ T-cell expansion appears to be reciprocally
regulated by an increase of CD4+(FoxP3+) natural regulatory T cells to
modulate the inflammatory response. Following an acute P. vivax infection
there is expansion of CD4+(FoxP3+) natural regulatory T cells (Jangpatarapongsa etal., 2008) whose numbers positively correlated with P. vivax
parasite load (Bueno et al., 2010; Goncalves et al., 2010). Interestingly,
acute P. vivax infections induce threefold higher serum levels of IL-10
compared to acute P. falciparum (Goncalves etal., 2010), consistent with
the more pro-inflammatory nature of P. vivax (lower pyrogenic threshhold of Pv compared to Pf) (Bueno etal., 2010). Overall, these studies
would predict that P. vivax antigens that correlate with NAI will show an
increased ratio of P. vivax antigen-specific Th1- and Th17-specific T cells
relative to regulatory T cells.
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6.8. I mmunological Memory and Duration of Clinical

Immunity
As noted above, NAI to Pf takes longer to acquire compared to Pv in
highly endemic areas, requiring multiple and often persistent infections
and when individuals move away from an endemic area their immunity to severe malaria appears to persist yet immunity to uncomplicated
malaria wanes after several years in a non-malaria endemic area (Castelli
et al., 1999; Jelinek et al., 2002; Matteelli et al., 1999; Struik and Riley,
2004). One interpretation of these data is that malaria induces only shortterm memory. The observations mostly arise from studies in sub-Saharan
Africa where malaria transmission is intense. This loss of NAI is often
associated with the rapid decline or loss in blood-stage specific Abs (Langhorne etal., 2008) that may result from memory B cell (MBC) anergy or
exhaustion (Pierce, 2009). Blood-stage Pf may suppress generation and
maintenance of malaria MBC and long-lived plasma cells by virtue of
their high Ag loads that elicit systemic pro-inflammatory responses, e.g.
increased TNF- and IFN- (Hemmer et al., 1991; Karunaweera et al.,
1992; Rudin et al., 1997), which are eventually downregulated (Weiss
etal., 2010) (possibly explaining, in part, why many malaria infected children in endemic areas are asymptomatic).Whether this is also true for Pv is
unknown. Highly immunogenic P. vivax-specific Abs such as AMA1 persist
for years after known malaria infection (Braga etal., 1998; Bueno etal.,
2009); however, Abs to less-immunogenic antigens such as the PvDBP
can be transient (Ceravolo et al., 2005, 2008, 2009). Memory T and B
cells to both P. vivax pre-erythrocytic stage antigens have also been shown
to persist for years in areas of low transmission (e.g. Brazil, Thailand)
(Bilsborough etal., 1993; Wipasa etal., 2010; Zevering etal., 1994) where
individuals are frequently observed with asymptomatic parasitaemia
(Alves etal., 2002; Branch etal., 2005; V
inetz and Gilman, 2002) indicating development of NAI. Thus, it appears that long-term immunological
memory frequently develops in areas of low P. vivax transmission associated
with NAI. Whether the presence of Pv-specific Abs and T and B cells are
associated with long-term protection against clinical P. vivax infections is
unknown and difficult to assess because of infrequent P. vivax infection
in low-transmissions settings. In areas of higher P. vivax transmission such
as in PNG, the persistence of immunological memory and association of
NAI has not yet been rigorously investigated. The recent interventions
to reduce both Pf and Pv transmission in PNG afford the opportunity to
examine this in detail.
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7. IMMUNE RESPONSES TO MALARIA

PRE-ERYTHROCYTIC STAGES

In humans, malarial infection is initiated by the bite of an infected
Anopheles mosquito that injects sporozoites into the hosts bloodstream.
Although mosquitoes from highly endemic areas are estimated to carry up
to 104 sporozoites in their salivary glands, it has been calculated that during
a blood meal an infected mosquito inoculates a median number of only 15
parasites into the host (Rosenberg etal., 1990).
After inoculation, the sporozoites move through dermal cells and enter
into the bloodstream and within 1-hours time, they go in the liver and
subsequently invade hepatocytes, where they differentiate into mature
liver schizonts. However, it is uncertain as to how many of the infective
forms actually reach the liver and successfully develop into mature schizonts, remain in the skin (Sidjanski and Vanderberg, 1997) or are removed
by the lymph node via lymphatic circulation, where antigen-specific T-cell
responses are initiated (Amino etal., 2008; Mota etal., 2001). It has been
recently shown that malarial parasites successfully develop to schizonts in
the skin (Guilbride etal., 2012; V
oza etal., 2012), but it is not yet clear if this
affects blood infection and malaria immunity (Hafalla etal., 2011).
It has been demonstrated that hepatocyte invasion is mediated through
a specific interaction between hepatocyte receptors and binding domains
present in at least two sporozoite surface proteins (SSPs) (Frevert et al.,
2008), the circumsporozoite (CS) protein (Cerami et al., 1992) and the
SSP2/TRAP (Hedstrom etal., 1990; Sultan etal., 1997), thus initiating parasite entry into the liver cell. This interaction occurs through the formation
of ionic bonds between the negatively charged chains of heparan sulphate
proteoglycan molecules present on the basolateral domain of hepatocytes
and the positively charged and hydrophobic residues of the region II-plus
of the CS and SSP2/TRAP proteins (Frevert etal., 1993; Gantt etal., 1997;
Sinnis etal., 1994).
Once inside the cell, the parasite undergoes asexual division or exoerythrocytic schizogony. Maturation from malaria trophozoites to schizonts
and subsequent merozoite release is a process that can take as little as 2 days
in rodent malarias to as long as 58 days in human malaria species. Plasmodium vivax schizonts each produce approximately 10,000 daughter parasites
with P. falciparum resulting in an estimated 40,000 merozoites per liver schizont (Wernsdorfer and McGregor, 1988). In P. vivax and Plasmodium ovale,
107
liver parasites may transform into hypnozoites, a latent form that can persist
in the liver for several years after initial infection, thus producing relapsing malaria episodes. Hypnozoites were first described in 1985 by Krotoski (Krotoski, 1985), who revealed the presence of uninucleated bodies of
4.56.6 nm inside liver parenchyma cells. The periodicity of the relapses
appears to depend on the geographic origin of the parasite strain; parasites
from tropical areas emerge more quickly than those from temperate zones
(Shute etal., 1976).
At the pre-erythrocytic stages, i.e. sporozoite and liver stages, immune
responses against Plasmodium parasites are mediated by both humoral and
cellular mechanisms (Fig. 3.3). Abs play a central role in protection through
different mechanisms including blockage of sporozoite invasion of liver cells
by specific neutralising Abs that induce what is known as the circumsporozoite precipitation (CSP) reaction (Vanderberg et al., 1969). This reaction has been consistently shown to be induced by Abs specific to the CS
protein which is abundantly expressed on the sporozoite surface. Abs are
also able to induce opsonisation (Schwenk etal., 2003) and Ab-dependent
T-cell-mediated cytotoxicity (Mazier etal., 1990), as well as inhibition of
intra-hepatic development (Hollingdale etal., 1984; Mellouk etal., 1990).
Cell-mediated mechanisms appear to be particularly important for
malaria pre-erythrocytic immunity. First, innate mechanisms involve both
monocytes/macrophages, dendritic cells and polymorphonuclear leukocytes that have the capacity to eliminate pre-erythrocytic parasites by
phagocytosis (Ferrante et al., 1990; Hafalla et al., 2011). As mentioned
above, it has been demonstrated that macrophages in the presence of
hyperimmune sera can efficiently phagocytise and eliminate sporozoites
(Danforth etal., 1980). In addition, infiltration of cells such as macrophages
and neutrophils is noted around intra-hepatic parasite forms (Faure etal.,
1995; Khan and Vanderberg, 1992). However, the protective role of Kpffer
cells (KC), which are liver-specific tissue macrophages, is as yet not clear
(Hafalla etal., 2011). Some invivo studies suggest that these cells contribute to the sporozoite invasion process (Baer etal., 2007; Meis etal., 1982),
may also modulate cytokine profile and induce apoptosis in KC (Klotz and
Frevert, 2008), while other studies indicate that in vivo depletion of KC
increases the number of intra-hepatic forms (Vreden etal., 1993). Second,
cytolytic activity mediated by antigen-specific CD4+ and CD8+ T cells is
believed to play a prominent role in liver-stage malaria defence mechanisms.
Peripheral blood lymphocytes of human immune donors from endemic areas
recognise multiple Th (Tse, Radtke etal. 2011) and cytotoxic lymphocyte
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Figure 3.3 Immune response against malaria pre-erythrocytic stages (sporozoites and
liver stages). DC, dendritic cells; Th, CD4+ T-helper cells; Tc, CD8+ T-cytotoxic cells; MHC,
major histompatibility complex; NK, natural killer cells; IFN-, Interferon-gamma; NO,
nitric oxide; ROI, reactive oxygen intermediates. (For colour version of this figure, the
reader is referred to the online version of this book).
109
(CTL) epitopes both in sporozoite and liver-stage antigens in a genetically restricted manner (Aidoo etal., 1995; Malik etal., 1991; Nardin and
Nussenzweig, 1993). The role of these two lymphocyte subsets has been
actively studied particularly in the rodent system (Del Giudice etal., 1986;
Nardin and Nussenzweig, 1993; Romero et al., 1989; Weiss et al., 1988).
Because of technical limitations, the protective role ofT-cells (Th and CTL)
in humans has been more difficult to document unequivocally.
In rodents, it has been shown that in vivo depletion of CD8+ T
lymphocytes completely abolishes malaria immunity (Schofield et al.,
1987; Weiss et al., 1988). In addition, passive transfer of cytotoxic T-cell
clones recognising the Plasmodium berghei CS protein completely protects mice against sporozoite challenge (Romero et al., 1989). Furthermore, murine CD8+ T cells have been shown to contribute to protective
immune mechanisms against malaria liver stages (Overstreet etal., 2008;
Renia, 2008; Rodrigues etal., 1991; Weiss etal., 1988). Likewise, passive
transfer and cell subset depletion studies have indicated the protective role
of CD4+ T cells clones in rodents. Passive transfer of cytolytic CD4+
T cell clones has shown to induce protection in a parasite-stage-specific
manner (Tsuji etal., 1990) regardless of the Th1 or Th2 phenotype (Renia
et al., 1993; Takita-Sonoda et al., 1996). Moreover, a number of soluble
immune mediators have been shown to be capable of blocking liver
schizogony, e.g. cytokines IL-12 (Sedegah et al., 1994), IFN- (Ferreira
etal., 1986; Mellouk etal., 1987; Schofield etal., 1987; Tsuji etal., 1995),
TNF- (Korner et al., 2010; Nussler et al., 1991), IL-1 (Mellouk et al.,
1987) and IL-6 (Nussler etal., 1991; Pied etal., 1991). Additionally, reactive oxygen and nitrogen intermediates, O2 (Allison and Eugui, 1983)
and nitric oxide (Mellouk etal., 1994; Nussler etal., 1991, 1993; Seguin
etal., 1994), respectively, significantly contribute to protection. Other proteins described as participating in the immune responses are hemopexin,
1-anti-trypsin, 2-macroglobulin (Pied etal., 1995) and C-reactive protein (Pied etal., 1989). Several studies have indicated an important protective role for IFN- in both human and non-human primates, either as a
critical immune mediator or as a valuable surrogate marker of protection
(Arevalo-Herrera et al., 2010, 2011; Doolan and Martinez-Alier, 2006;
Herrera etal., 2007; John etal., 2004; Jordan-Villegas etal., 2011; Perlaza
etal., 2008, 2011).
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Ivo Mueller et al.
In humans, most work has involved P. falciparum where CD4+ T cells

have been shown to be correlated with protection from natural infection
and disease in West Africa (Reece etal., 2004). CS-specific cytolytic CD4+
T cells derived from individuals vaccinated with irradiated sporozoites
(Moreno etal., 1991) or PfCS peptides (Calvo-Calle etal., 2005) and CD8+
T cells appear to contribute significantly to protection at the pre-erythrocytic level (Bettiol etal., 2010; Doolan and Martinez-Alier, 2006; Tsuji and
Zavala, 2003). In P. falciparum, several CD4+ and CD8+ T-cell epitopes have
been identified in pre-erythrocytic antigens, i.e. CS, LSA-1, LSA-3, TRAP,
using classical techniques (Cockburn etal., 2010; Joshi etal., 2000; Overstreet etal., 2008). Furthermore, immunisation of both Aotus monkeys and
chimpanzees with the P. falciparum LSA-3 liver-stage antigen formulated in
different adjuvants has been shown to induce significant IFN-? production
in protected as compared to non-protected monkeys (Perlaza etal., 2008).
The absence of P. vivax parasite cultivation methods has limited studies
on this Plasmodium species. This is reflected in the number of P. vivax parasite antigens identified thus far. Blood samples of individuals from malariaendemic areas or from volunteers participating in P. vivax vaccine studies in
Colombia have been used to characterize immune responses to naturally and
artificially acquired P. vivax CS (Arevalo-Herrera etal., 2002; Fleischhauer
etal., 1999; Herrera etal., 1994). Studies have shown that similar CS epitopes are recognized by individuals from endemic areas and by non-human
primates and human volunteers immunised with long synthetic peptides
(Arevalo-Herrera etal., 2011; Herrera etal., 2005).Volunteers participating
in phase I vaccine clinical trials mounted significant CD4+ and CD8+ T
cell responses with high levels of IFN-? upon vaccination (Herrera etal.,
2007, 2011), together with production of Abs capable of efficiently blocking parasite invasion invitro. A fragment of another pre-erythrocytic antigen
PvTRAP (amino acids 209256) containing the region II motif involved in
parasite binding to hepatocytes has been shown to induce partial protection
of Aotus primates against experimental P. vivax sporozoite challenge (Castellanos etal., 2007). Although not considered pre-erythrocytic per se, MSP-1
and possibly other malaria antigens expressed in hepatic schizonts are likely
to be involved in pre-erythrocytic immunity (Suhrbier etal., 1989).
In recent years with the advent of genomic and proteomic technologies,
significant progress has been achieved in the discovery of human parasite
antigens associated with the induction of immune responses to target T- and
B-cell epitopes of malaria antigens (Doolan, 2011). Using sera from naturally and experimentally malaria-exposed individuals, 5% of the P. falciparum
111
genome was screened using protein microarrays (Doolan et al., 2008). A

similar strategy has been used to analyse P. vivax gene sequences (Dharia
etal., 2010).
In conclusion, a number of studies have suggested the importance of the
induction of protective Ab- and T-cell-specific responses against the preerythrocytic forms of malaria parasite. In terms of the prevention and/or
reduction of the clinical manifestation of infection, both Abs and cytokines,
particularly IFN-, play a critical role in protection. Nonetheless, more
studies are warranted for better understanding of the immune mechanisms
involved in protection against malaria. Development and use of additional
innovative genomic approaches to vaccine design are also needed.
8. SEXUAL STAGE PARASITES AND TRANSMISSIONBLOCKING IMMUNITY

The target stages of the TBI are the sexual stages of the Plasmodium
life cycle. The sexual stage is initiated in the vertebrate host bloodstream
as male and female gametocytes. After the ingestion of the infected blood
by Anopheles mosquito, male and female gametes emerge and fertilise to
form zygotes in the mosquito midgut. The zygotes transform into motile
ookinetes, which traverse the epithelium of the mosquito midgut to further
develop into oocysts (Tsuboi etal., 2003).
TBI has initially been reported with sexual stage parasites in non-human
malaria models (Carter and Chen, 1976; Gwadz, 1976; Gwadz and Green,
1978; Mendis and Targett, 1979), and is mainly mediated by Abs that prevent
fertilisation and destroy the gametes and newly fertilised zygotes by acting
against the surface proteins of the sexual stage parasites within the midgut
of a blood-fed mosquito. After the ingestion of the blood, Abs against the
gametes act to prevent their fertilisation and formation of ookinetes (Carter,
2001).
Individuals in malaria endemic areas that have been repeatedly exposed
to the parasite infection acquire specific immune responses against bloodstage parasite capable of controlling parasite burden and the clinical symptoms. In addition, sera from individuals in malaria endemic areas are able to
block malaria transmission (Arevalo-Herrera etal., 2005; Gamage-Mendis
etal., 1992; Mendis etal., 1987). While ingesting the blood meal from the
host, the mosquito also ingests host Abs specific to sexual stage antigens.
These Abs interfere with the development of the parasite life cycle in the
mosquito midgut (Peiris etal., 1988).
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Ivo Mueller et al.
8.1. Evidences of Naturally Acquired TBI of P. falciparum

TBI appears to depend on the malaria transmission intensity, suggesting that the generation of TBI requires prolonged exposure to multiple
malaria inoculations (Bousema etal., 2010; Premawansa etal., 1994). TBI
also correlates with the period of exposure to gametocytes and the number
of gametocytes developed during the infections; sera of individuals with
high Ab concentrations are more efficient at blocking parasite transmission to the mosquito (Boudin et al., 2004; Bousema et al., 2011). Sera
of humans exposed to P. falciparum contain IgG Abs that recognise sexual-stage antigens on the surface of gametocytes and gametes. Pfs230 and
Pfs48/45 were identified as antigens responsible for the TBI (Carter, 2001;
Healer etal., 1999; Roeffen etal., 1994). The levels of Abs against Pfs230
and Pfs48/45 could be boosted by exposure to gametocytes in further
infections (Bousema et al., 2010). Recently, Sutherland has summarised
the efforts to identify novel sexual-stage antigens conferring TBI against
P. falciparum (Sutherland, 2009).
8.2. Evidences of Naturally Acquired TBI of P. vivax

There is a big difference in the timing of the gametocyte appearance between
P. falciparum and P. vivax. In the case of P. falciparum, gametocytes appear a
few weeks after the onset of febrile illness. In contrast, gametocytes appear
almost at the same time as the asexual parasitaemia in P. vivax (Bousema
and Drakeley, 2011). Although previous studies on malaria TBI have mostly
focused on P. falciparum and have been carried out in Africa, so far, only a
small number of studies have been taken up to understand the TBI against
P. vivax in both Asia and Latin America.
Pioneering work conducted on P. vivax in Sri Lanka has demonstrated
the development of TBI as a result of naturally acquired malaria infection.
The sera of approximately 48% of patients significantly suppressed gametocyte infectivity (Gamage-Mendis etal., 1992; Peiris etal., 1988).The efforts
(Premawansa etal., 1990; Snewin etal., 1995) hitherto to identify the definitive target antigens that can elicit TBI against P. vivax by production of
anti-gamete mAbs and screening genomic expression library of P. vivax by
the TB mAbs were unsuccessful.
There are only a few studies in Thailand that suggest that serum factors play a key role in the ability of P. vivax gametocytes to infect mosquitoes. Sattabongkot etal. (2003) compared the infectivity of P. vivax-infected
blood containing the patients own sera with that of blood in which the
113
patients sera had been replaced with malaria-nave sera. They found that
the patients own sera significantly reduced mosquito infection rates as well
as oocyst density. Although they did not specifically identify host factors
that were responsible for reductions in mosquito infectivity, it seems likely
that TB Abs may cause a reduction in infectivity. All patients evaluated in
this study were symptomatic adults, suggesting that TB Ab levels may have
been relatively low. In addition, Coleman et al. (2004) reported that the
blood obtained from individuals in a highly endemic village in western
Thailand exhibited reduced infection in mosquitoes.
Naturally acquired TBI to P. vivax was also studied in patients from the
southern coast of Mexico. Anopheles albimanus mosquitoes were fed with
patients IEs in the presence of autologous or malaria-nave serum. Patients
from both primary and secondary infection had TBI, although the quality
and quantity of the blocking activity was significantly higher in the secondary infection groups (Ramsey etal., 1996).
P. vivax TB activity was also assessed in sera from acutely infected patients
from a malaria-endemic area in Colombia. The reduction in the number of
oocysts in An. albimanus mosquitoes artificially fed with blood from these
patients were measured in comparison with parasitised erythrocytes mixed
with malaria-nave serum as negative control. One-third (36.4%) showed
full TB activity (90% inhibition) when mixed with autologous sera and
29.6% showed partial activity (5089%).The TB activity correlated with Ab
titre by an immunofluorescent Ab test and decreased with the serial dilution
of the sera (Arevalo-Herrera etal., 2005).
8.3. T
owards the Discovery of the Target Molecules against
TBI of P. vivax
Although a few definitive antigens that elicit TBI against P. falciparum have
been identified (Bousema etal., 2010, 2011), candidate antigens that induce
TBI against P. vivax have not been identified yet (Tsuboi et al., 2003).
Recently, we have established an efficient recombinant malaria protein
expression system using the wheat germ cell-free system (Tsuboi et al.,
2008, 2010a, 2010b). Using this protein expression system, we were able
to express 89 blood-stage proteins of P. vivax as a protein array, screen them
by the human immune sera and successfully identify novel blood-stage
antigens (Chen etal., 2010). Similarly, in the near future, we will express
gametocyte-stage proteins of P. vivax as a protein array to screen them by
the serum samples having TBI, to discover novel TB vaccine candidates
against P. vivax.
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Ivo Mueller et al.
9. CONCLUSIONS

Although we have only just begun to understand some of the major
processes involved in NAI to P. vivax, the results to date support the premise
that developing a multistage P. vivax vaccine may be feasible and is worth
pursuing.The more rapid development of NAI to P. vivax even indicates that
a highly efficacious P. vivax vaccine may be easier to achieve that a vaccine
to P. falciparum. This might be especially true if there are fewer redundant
pathways for erythrocyte invasion. A pre-erythrocytic-stage vaccine may be
of particular value because each infective mosquito bite has the potential to
generate multiple blood-stage infections because some sporozoites became
latent. An ideal P. vivax vaccine should target not only blood stage but also
pre-erythrocitic and transmission stages. While the rationale for continued
P. vivax vaccine development efforts is strong, a lot of work remains to identify
the optimal combination of antigens to be included in such a vaccine.
While the lack of continuous long-term P. vivax culture greatly complicates both the screening and development of novel vaccine candidate,
important insights can be gained by conducting further in-depth studies of
the acquisition of immunity in naturally exposed population that combine
appropriate epidemiological design, state-of-the-art immunology and novel
genomic and proteomic technologies. In addition, a more thorough use of
the existing non-human primate models as well as experimental infection
in human volunteers will be essential for advancing our understanding of
the basic processes and specific antigens involved in the establishment of
NAI to P. vivax.
10. FUTURE DIRECTIONS

1. C
ompare Ab responses from putatively immune and susceptible individuals using protein arrays representing Pv blood-stage antigens in
longitudinal cohort studies with the aim to identify new targets of
blood-stage immune responses.
2. B
etter characterize surface-expressed proteins on Pv-IEs and relate how
these proteins are involved in stimulating NAI, and, potentially, immune
evasion strategies.
3. S tudy the role of host cellular immunity and protection against bloodstage Pv infection, including the role of opsonizsng Abs and the spleen.
115
4. C
ontinue to define the biology of molecules involved in the invasion of
and release from erythrocytes.
5. Identify the conserved binding motifs in critical Pv invasion molecules
such as RBPs and DBPs as potential blood-stage antigens.
6. D
etermine the interaction of erythrocyte polymorphisms related to susceptibility to Pv infection (e.g. Duffy antigen polymorphisms or Southeast Asian ovalocytosis) and how these modify development of NAI.
7. C
haracterize the pre-erythrocytic-stage antigens and immune responses
in humans associated with protection following immunisation with irradiated sporozoites.
8. T
o further identify antigens associated with protection against TB NAI,
especially molecules expressed on gametocytes in the peripheral circulation. Immunisation with such molecules would facilitate natural boosting
by blood-stage infections to sustain high Ab levels required for TBI.
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CHAPTER FOUR
G6PD Deficiency: Global

Distribution, Genetic Variants
and Primaquine Therapy
Rosalind E. Howes*,1, Katherine E. Battle*, Ari W. Satyagraha,
J. Kevin Baird,, Simon I. Hay*
*Department of Zoology, University of Oxford, Oxford, UK
Eijkman Institute for Molecular Biology, Jakarta, Indonesia
Eijkman-Oxford Clinical Research Unit, Jakarta, Indonesia
Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford,
Oxford, UK
1Corresponding author: E-mail: rosalind.howes@zoo.ox.ac.uk
Contents
1. Introduction
2. H
istorical Overview
2.1. F avism
2.2. T he Path to Primaquine
2.3. P
rimaquine Tolerability and Safety
3. G
lucose-6-Phosphate Dehydrogenase Deficiency: The Enzyme and Its Gene
3.1. G
6PD Genetics and Inheritance
3.2. T he G6PD Enzyme
3.3. T he Pentose Phosphate Pathway as an Anti-Oxidative Defence
3.4. C
linical Manifestations of G6PD Deficiency
4. D
iagnosing G6PD Deficiency
4.1. P
henotypic Diagnostic Tests
4.2. M
olecular Diagnostic Tests
4.3. T he Case for a New Diagnostic for Safe P. vivax Radical Cure
5. M
apping the Spatial Distribution of G6PD Deficiency
5.1. G
6PD Deficiency Prevalence Mapping
5.1.1. G
enerating a Map: the Evidence-Base
5.1.2. Generating a Map: the G6PD Mapping Model
5.1.3. G6PD Deficiency Prevalence Map: an Overview
5.2. G
6PD Deficient Population Estimates
5.3. G
6PD Deficiency Mutation Mapping
6. S patial Co-occurrence of G6PD Deficiency with P. vivax Endemicity
6.1. G
6PD Deficiency in Asia
6.2. G
6PD Deficiency in Asia-Pacific
6.3. G
6PD Deficiency in the Americas
6.4. G
6PD Deficiency in Africa, Yemen and Saudi Arabia (Africa+)
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7. E volutionary Drivers of the Distribution of G6PD Deficiency

7.1. E vidence of a Selective Advantage
7.1.1. E pidemiological Evidence
7.1.2. Invitro Evidence
7.1.3. Case-Control Invivo Evidence
7.2. N
eglect of the Selective Role of P. vivax as a Driver of G6PD Deficiency
8. P
rimaquine, P. vivax and G6PD Deficiency
8.1. M
echanism of Primaquine-Induced Haemolysis
8.1.1.
8.1.2.
8.1.3.
8.1.4.
8.1.5.
P rimaquine and its Metabolites

A Role for Oxidative Stress
A Role for Methaemoglobin
A Role for Altered Redox Equilibrium
Significance of Primaquine-Induced Haemolysis
8.2. F actors Affecting Haemolytic Risk

8.2.1.
8.2.2.
8.2.3.
8.2.4.
ose Dependency
D
Variant Dependency
Red Blood Cell Age Dependency
Sex Dependency
8.3. P
redicting Haemolytic Risk
9. T owards a Risk Framework for P. vivax Relapse Treatment
9.1. A
ssessing National-Level Haemolytic Risk of
Primaquine Therapy
9.1.1. P roposed Framework for Ranking National-Level Risk from G6PD Deficiency
9.1.2. Important Limitations to Predicting National-Level Haemolytic Risk
9.2. A
ssessing Haemolytic Risk at the Level of the Individual
10. C
onclusions
Acknowledgements
References
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Abstract
Glucose-6-phosphate dehydrogenase (G6PD) is a potentially pathogenic inherited
enzyme abnormality and, similar to other human red blood cell polymorphisms, is
particularly prevalent in historically malaria endemic countries. The spatial extent
of Plasmodium vivax malaria overlaps widely with that of G6PD deficiency; unfortunately the only drug licensed for the radical cure and relapse prevention of P. vivax,
primaquine, can trigger severe haemolytic anaemia in G6PD deficient individuals. This
chapter reviews the past and current data on this unique pharmacogenetic association, which is becoming increasingly important as several nations now consider strategies to eliminate malaria transmission rather than control its clinical burden. G6PD
deficiency is a highly variable disorder, in terms of spatial heterogeneity in prevalence and molecular variants, as well as its interactions with P. vivax and primaquine.
Consideration of factors including aspects of basic physiology, diagnosis, and clinical
triggers of primaquine-induced haemolysis is required to assess the risks and benefits
of applying primaquine in various geographic and demographic settings. Given that
haemolytically toxic antirelapse drugs will likely be the only therapeutic options for the
coming decade, it is clear that we need to understand in depth G6PD deficiency and
G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy
135
primaquine-induced haemolysis to determine safe and effective therapeutic strategies

to overcome this hurdle and achieve malaria elimination.
1. INTRODUCTION

Glucose-6-phosphate dehydrogenase (G6PD) is a ubiquitously expre
ssed enzyme that has a housekeeping role in all cells, and is particularly
critical to the integrity and functioning of red blood cells (RBCs). The
G6PD gene has many mutant alleles which entail a decrease in enzyme
activity, expressing the G6PD deficient phenotype. This trait is widespread
in many human populations in whom several of the underlying mutant
alleles are present at variable polymorphic frequencies.
G6PD deficiency selectively affects RBCs for two reasons. First, most
known mutations cause a decreased stability of the enzyme, and since these
cells do not have the ability to synthesise proteins, the enzyme level decreases
as cells age during their 120days lifespan in circulation. Second, RBCs are
exquisitely susceptible to oxidative stress from exogenous oxidizing agents
in the blood as well as the oxygen radicals continuously generated as haemoglobin cycles between its deoxygenated and oxygenated forms. When
G6PD activity is deficient, they have a diminished ability to withstand stress,
and therefore risk destruction (haemolysis).
Fortunately, the large majority of G6PD deficient subjects have no clinical manifestations and the condition remains asymptomatic until they are
exposed to a haemolytic trigger. For centuries, the most common known
trigger of haemolysis has been fava beans, and favism remains a public health
problem in areas where these are a common food item and G6PD deficiency
is prevalent. However, a haemolysing trigger of great contemporary public
health significance is the antimalarial primaquine, a key drug for malaria
control as the only licenced treatment against (i) the relapsing liver stages of
Plasmodium vivax hypnozoites which become dormant in infected hepatocytes and subsequently reactivate blood-stage infections, and (ii) the sexual
blood stages of all species of Plasmodium. Since its introduction, primaquine
has emerged as a major drug trigger of haemolysis in G6PD deficient individuals, making this a paradigm of pharmacogenetics. This chapter focuses
on the use of primaquine as a hypnozoitocide in P. vivax malaria. The complex problem of its use as a gametocytocide in Plasmodium falciparum malaria
is not further considered here: detailed reviews of the effectiveness and safety
of single-dose transmission-blocking primaquine have recently been carried
out for the WHO Primaquine Evidence Review group (Recht etal., 2012)
and in a Cochrane Review (Graves etal., 2012).
136
R. E. Howes et al.
The widespread prevalence of G6PD deficiency across populations in

malaria endemic areas has hindered the use of this drug, despite its uniquely
useful range of therapeutic properties. One aim of studying G6PD deficiency is to increase access to primaquine. Optimising primaquine use
involves delivery of the drug in such a way as to maintain its therapeutic
activity against parasites whilst reducing risk for G6PD deficient individuals.
Here, we review the knowledge base and interactions between the different
component parts of this relationship (the human G6PD gene, the parasite
and the drug). All of these elements must be considered when weighing the
risks and benefits of putting this valuable drug to work. We examine these
factors in the historical context of their development the discovery of
G6PD deficiency having been tied to the early research into primaquine, and
consider the important knowledge gaps which remain despite six decades
of continuous use of this drug. Extensive work was conducted in the midtwentieth century to improve the chemotherapeutic options for treating
P. vivax, particularly against its relapsing form. This chapter opens by revisiting those early experiments, asserting why G6PD deficiency seems to be
an unavoidably major hurdle in hypnozoitocidal therapeutics. We examine
what is known about G6PD the enzyme, its mutations and population
genetics. We then consider how this problem is being or could be managed
in the context of contemporary targets for malaria elimination. This leads
us to suggest steps to be taken to move forward into a new era when G6PD
deficiency no longer seriously impedes treatment of P. vivax infection in
individual patients and when primaquine can be used to its full potential as
an essential tool for eliminating reservoirs of P. vivax.
2. HISTORICAL OVERVIEW
2.1. Favism
Awareness of the symptoms associated with G6PD deficiency was well
established long before the underlying mechanisms were understood
(Beutler, 2008). The earliest suspected reports of G6PD deficiency are from
Pythagoras forbidding his students to eat fava beans (Vicia faba). His strong
aversion to these commonly eaten beans must mean that favism had already
been recognised as a dangerous disease; and since G6PD deficiency is common in Greece, it is possible that he or some of his followers may have suffered
from favism, the haemolytic condition triggered by ingesting these beans
(Simoons, 1998). In more recent times there has been a vast literature on
favism (Fermi and Martinetti, 1905; Luisada, 1940; Meloni etal., 1983). Fava
137
beans are unique among other beans because they contain high concentrations of two glucosides, vicine and divicine; and their respective aglycones,
convicine and isouramil, are powerful triggers of oxidative stress that causes
the characteristic haemolytic attacks (Chevion etal., 1982; Luzzatto, 2009).
2.2. The Path to Primaquine

Although methylene blue was the first synthetic compound used to successfully treat acute malaria, it was never developed or distributed. The
first such drug, structurally derived from methylene blue, was the 8-aminoquinoline pamaquine (Fig. 4.1) which became widely used, and quickly
feared. While Mhlens (1926) was correct in asserting in 1926 that his
laboratorys production of pamaquine (marketed as Plasmochin) was of
huge importance and would have immeasurable effect on malarial countries
(trans.), his clinical trials (conducted among malaria-infected individuals of
European origin) did not support his assurances about its safety. The only
side-effects reported in his initial paper were some cases of cyanosis in lips,
gums, tongues and fingernails, which he did not consider prohibitive to
the drugs widespread use (in retrospect, this was probably cyanosis
caused by methaemoglobinaemia). At least 250 case reports of toxic reactions to pamaquine were subsequently published; these were cases of
acute haemolytic anaemia (AHA), some resulting in death (Beutler, 1959;
Hardgrove and Applebaum, 1946). In 1938, the League of Nations recommended against use of this drug.
In 1941, no synthetic drug effectively competed with quinine for
the treatment of malaria, and quinine could not protect against relapsing
malaria. The Dutch operated a global cartel on the trade of quinine, with
95% of production coming from cinchona tree plantations on Java in the
Figure 4.1 Chemical structure of key 8-aminoquinolines: pamaquine, primaquine and

tafenoquine.
138
R. E. Howes et al.
Netherlands East Indies. The fall of those holdings to the Imperial Japanese armed forces in early 1942 forced the Allies to use the few inferior
synthetic drugs available, principally atabrine (also called mepacrine or
quinacrine) and pamaquine (Elyazar etal., 2011). The embattled Americans holding out on the Bataan peninsula and Corregidor Island near
Manila (January to April 1942) suffered terribly from malaria. CondonRall (1992) encapsulated the significance of this, stating that the medical
disaster that developed among the US troops in the Philippines was a
symptom as much as a cause of the American general military defeat. In the
region of New Guinea, 1598 American soldiers died of wounds sustained
in battle, whereas 6292 perished with a diagnosis of malaria (Joy, 1999).
Similar figures were reported among Australian forces, who suffered 21,600
malaria casualties during the same campaigns. At Guadalcanal in the Solomon Islands during 1942, the US Army Americal division suffered malaria
attack rates of 1.3/personyear (despite atabrine prophylaxis). More telling, however, was what happened to this division when they were evacuated to nonmalarious Fiji for rest and recuperation: the malaria attack rate
was 3.7/personyear, virtually all of it relapses of P. vivax (Downs etal.,
1947). The US Navy estimated that 79% of the 113,774 recorded cases of
malaria were relapses (Joy, 1999).
Despite the great demand for antirelapse therapy, in 1943 the US Surgeon General withdrew pamaquine for prevention of relapses (Office of the
Surgeon General, 1943) due to its toxicity, which was highly significant in
some individuals. Acute haemolytic attacks could be triggered by the use
of daily pamaquine dosing (30mg1), with associated jaundice, dark urine
and weakness due to severe anaemia (Earle etal., 1948). Furthermore, when
used with atabrine, its plasma levels increased 10-fold causing serious toxicity problems (Baird, 2011). This unexpected drugdrug interaction effectively removed the only therapeutic option to the serious threat of malaria
relapse. The US government responded to this problem by launching one
of the largest biomedical research endeavours up to that time. Created in
1943, the Board for the Coordination of Malaria Studies oversaw basic
and clinical research on over 14,000 compounds for antimalarial activity
(Condon-Rall, 1994). Beginning in 1944, academic clinical investigators
and US armed forces clinicians set up the capacity to study induced malaria
in volunteers at a number of prisons in the United States (Coatney
et al., 1948), and the penitentiary at Stateville, Illinois specialised in the
1 All
doses in this review are subscribed for adults weighing 4070kg, mean of 60kg.
139
evaluation of therapies against relapse (Comfort, 2009). They leveraged a

strain of P. vivax taken from an American soldier infected in New Guinea
in 1944, called the Chesson strain (Alving etal., 1948; Craige etal., 1947).
In the context of the clinical trials, this strain offered the advantage of relatively frequent, rapid, and multiple relapses compared with Korean or North
American strains, though also required higher drug dosages to achieve
wholly efficacious radical cure.
A Stateville Penitentiary study protocol from 1948 states that approximately 500 volunteers were involved (Alving etal., 1948) although a recent
review of the overall project suggested that thousands of inmates were
ultimately inoculated and included in the experiments (Comfort, 2009).
The very modern and controlled prison environment provided a convenient setting for running multiple complex protocols involving many
different compounds (Alving et al., 1948), especially 8-aminoquinolines
(Craige etal., 1948; Earle etal., 1948; Jones etal., 1948). The stage was
thus set for careful clinical observation of the AHA induced by this class
of compounds. The vetting of 24 candidate 8-aminoquinolines, a family
of drugs whose antirelapse efficacy had been demonstrated by pamaquine,
for safety, tolerability, and efficacy in subjects not sensitive to haemolysis
had been completed by about 1949. Subsequently, the best candidate, primaquine (Edgcomb etal., 1950; Elderfield etal., 1955), was widely used in
American soldiers in the Korean War (19501953). It is thus important to
understand that the 8-aminoquinolines were not evaluated for optimum
safety in G6PD deficient subjects. Instead, the US Army programme later
strived to evaluate safety of primaquine alone in vulnerable subjects.
Studies were conducted with pamaquine to investigate the predisposing
conditions which made some individuals particularly vulnerable to haemolysis. These suggested that pamaquine sensitivity was racially correlated,
being more common in subjects of African origin (6 of 76) than Caucasians
(1 of 87) (Earle etal., 1948). They also found that haemolysis was unlikely
to be associated with the plasma pamaquine levels. These observations led
investigators to suspect a predisposing factor in certain individuals, triggered
by the drug acting as a precipitating factor (Earle et al., 1948); although
they found race to be the most significant predictor of haemolysis when
considered alongside a range of haematological and exogenous factors, its
genetic basis remained only a possibility. Indeed, it required 10 more years
of investigation to zero in on that cause. Carson etal. (1956), working at
the Stateville Penitentiary, described G6PD deficiency as the basis of primaquine sensitivity in 1956.
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R. E. Howes et al.
The newly qualified clinician, Ernest Beutler (19282008), went to Stateville and participated in the ground-breaking work characterising G6PD
deficiency. The remainder of his prolific and distinguished professional life
substantially advanced understanding of this important disorder (Beutler,
2009).
A Chicago University database archives approximately 150 publications
arising from the Stateville Penitentiary Malaria Treatment Trials (http://
www.lib.uchicago.edu/e/collections/sci/malaria.html). In this next section, we review those studies relating to G6PD deficiency and tolerance to
effective primaquine dosing. While these studies have been held as a prime
example of unethical human experimentation (Harcourt, 2011), their legacy
still forms the foundation of P. vivax radical cure today.
2.3. Primaquine Tolerability and Safety

The early experimental clinical studies in nondeficient individuals established that a total primaquine dose of approximately 200mg was needed
to achieve P. vivax radical cure (Alving et al., 1953; Coatney et al.,
1953; Edgcomb et al., 1950). The next step was establishing an efficacious dosing regimen with acceptable safety profiles. Most et al. (1946)
described a 14-day regimen of quinine and pamaquine very early in the
8-aminoquinoline clinical development programme, which they considered to be the optimum compromise between safety (3- to 7-day dosing
came with high risk of intolerability or toxicity) and practicality (dosing up to 21 days risked poor compliance). The 14 days of daily 15 mg
dosing was subsequently applied to all candidate 8-aminoquinolines for
the simple reason that their therapeutic indexes were essentially similar to
pamaquine. The course of haemolysis in G6PD deficient subjects, with the
onset of symptoms after the third dose, permitted withdrawal of the treatment after relatively little exposure to the drug. Higher daily doses over a
shorter duration, although equally efficacious, were considered too risky
for use in unscreened patients.The 14-day regimen emerged before G6PD
deficiency was known as the basis of the 8-aminoquinolines most severe
toxicity. The compromise thus effectively included unscreened G6PD deficient patients. Indeed, the US Army would not screen its troops for G6PD
deficiency until 2005.They considered the recommended dosing regimens
not threatening, largely because withdrawal of therapy after low exposures
to drug was at least possible, and even 14 days of treatment did not appear
seriously harmful among the African American troops exposed to that
dosage.
141
Primaquine sensitivity was nonetheless a major concern during the development of primaquine (Editorial, 1952, 1955) (though did not preclude its
licencing by the US Food and Drug Association [FDA] in 1952) and extensive studies were undertaken at Stateville to understand this problem. Primaquine toxicity was investigated among both African American (Hockwald
etal., 1952) and CaucasianAmerican prisoner volunteers (Clayman etal.,
1952), comparing the toxicity of primaquine with pamaquine, and assessing
the influence of co-administration with quinine. Follow-on experiments
were carried out on sensitive individuals who had undergone haemolysis to investigate the severity and toxicity of multiple-drug treatments and
regimens on the same individuals. Although all cases of severe haemolysis
recovered without transfusion after cessation of treatment, the higher dose
of 30mg primaquine daily was deemed too dangerous for administration
without close supervision: of 110 African American volunteers given 30mg
primaquine daily over 14days, five developed severe anaemia with severity
comparable with that following equivalent dosage of pamaquine, and there
were 17 cases of mild anaemia. Reducing the schedule to 15mg daily doses
did not trigger any cases of severe anaemia, thus this was deemed a safe daily
dose; nevertheless, 12 patients still developed mild anaemia. The relatively
safe 15mg dose among this population was corroborated by other largescale studies (Alving etal., 1952; Hockwald etal., 1952). Parallel toxicity
studies were conducted among Caucasian volunteers. As would be expected,
abdominal complaints among others were also reported for high-dose regimens (60, 120, 240mg daily); no symptoms were reported with 15mg daily
regimens (n=699 Caucasian volunteers at Stateville Penitentiary), and only
mild side-effects noted with the 30 mg dose. Severe haemolysis was not
encountered among this group of patients, even at doses as high as 240mg
daily this clearly contrasts with the threshold of haemolytic susceptibility
in the African American volunteers.
Haemolysis in primaquine sensitive African American subjects was
noticed to be self-limiting (Dern etal., 1954a). Radiochromium labelling
(51Cr) used to mark sensitive and nonsensitive RBCs showed that cells
maintained the same haemolytic predisposition to risk regardless of the status of the host they were transfused into (Dern etal., 1954b). Time-series
data then characterised the course of the haemolysis and found a self-limiting pattern: in spite of continued drug administration throughout the initial
acute haemolysis (which lasted about a week, at which point up to half the
original cell population had haemolysed with the 30 mg/day dosage), a
marked recovery and then re-establishment of equilibrium of haemoglobin
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R. E. Howes et al.
concentrations was observed. In one experiment, G6PD deficient subjects

were given 30mg primaquine daily for 4months (Kellermeyer etal., 1961).
Following trough haematocrits around day 710, these levels returned to
normal within a week or so despite continued daily dosing with primaquine. This cycle of haemoglobin levels mirrored the percentage of reticulocytes in the blood (Dern et al., 1954a). 59Fe-labelling studies of RBCs
demonstrated that only older RBCs (6376 days old vs. 821 days old)
were susceptible this age-dependent phenomenon led the investigators
to astutely surmise the involvement of an enzyme deficiency (Beutler etal.,
1954).
The self-limiting nature of primaquine-induced haemolysis in African American volunteers, however, cannot be generalised to all G6PD
deficient patients globally. Differences were noted between individuals
originating from different areas. Studies of a severe G6PD variant (Mediterranean variant) in the 1960s showed even the youngest reticulocytes to
be vulnerable to primaquine (Piomelli etal., 1968). In other words, continued daily dosing would result in progressively steeper losses of RBC
and, presumably, death if not discontinued. The existence of these highly
vulnerable variants, in contrast to the relatively nonthreatening African
type (A- variant), imposes risk of fatal outcomes with the unbridled application of primaquine among populations where the character of locally
prevalent G6PD variants is not known. The safety of primaquine hinges
on G6PD status and primaquine sensitivity phenotype of the variant
involved.
The impediments imposed by G6PD deficiency and primaquine toxicity in these patients still severely limit the effectiveness of this singularly
important drug. Growing acknowledgement of this problem today, especially in the context of emergent malaria elimination strategies, has recently
activated research endeavours on this old and persistent problem.The disappointing search for superior alternative drugs, both in scope and outcome,
that essentially ended around 1980 is detailed in Chapter 4 of Volume 80.
Only one plausible successor to primaquine exists today, tafenoquine (Fig.
4.1), a GlaxoSmithKline (GSK) - Medicines for Malaria Venture (MMV)
partnership drug currently in Phase IIb/III trials originally discovered and
developed by the US Army (Crockett and Kain, 2007; Shanks etal., 2001).
However, also being an 8-aminoquinoline, tafenoquine presents similar
haemolytic challenges as primaquine to G6PD deficient individuals, and
this complicates the path to licencing: tafenoquine has already been in
development since the 1980s. Chapter 4,Volume 80 also lays out alternative
143
and unexplored approaches to mitigating 8-aminoquinoline toxicity. The

safe exclusion of patients from harm caused by this drug by the diagnosis
of G6PD deficiency currently requires technical capacities nearly wholly
absent where most malaria patients live, although work is ongoing to
develop a practical point-of-care kit. Greater understanding of the geographic distribution of G6PD deficiency in relation to endemic malaria
will inform decisions on primaquine therapeutics policy and practice. The
remainder of this chapter details the science and technology underpinning
all of these important endeavours.
3. GLUCOSE-6-PHOSPHATE DEHYDROGENASE
DEFICIENCY: THE ENZYME AND ITS GENE

The G6PD enzyme plays a critical role in maintaining RBC integrity
through catalysing a key step in the cells metabolic production of reducing
equivalents that maintain reductionoxidation (redox) equilibrium of the
cytoplasm. This protects the cell from oxidative attack by radicals derived
from oxygen and organic compounds such as drugs and their metabolites.
In spite of its vital function, the G6PD enzyme is highly variable, both
biochemically and genetically. Detailed reviews of G6PD genetics, biochemistry and clinical characteristics have been previously published (Beutler, 1994, 1996; Cappellini and Fiorelli, 2008; Luzzatto, 2006, 2009, 2010;
Mason etal., 2007; Mehta etal., 2000; WHO Working Group, 1989).
3.1. G6PD Genetics and Inheritance

The advent of molecular diagnostics following the successful mapping of
the G6PD genes 13 exons (Martini etal., 1986) which span 18.5kb, and
the genes cloning and sequencing in 1986 (Persico etal., 1986; Takizawa
etal., 1986) started to uncover the genetic basis to the enzymes great variability (Vulliamy etal., 1988) (Fig. 4.2). This Mendelian X-linked gene is
one of the most highly polymorphic of the human genome with at least
186 mutations having been described (Minucci etal., 2012). That said, not
all mutations are polymorphic and of public health significance, but many
instead appear only sporadically within populations: almost half (66 of 140
mutations reviewed in 2005 by Mason and V
ulliamy) are associated with the
most severe clinical phenotypes and are very rare.
Most mutations are single point substitutions (121 of 140 (Beutler and
Vulliamy, 2002; Mason and Vulliamy, 2005)) leading to amino acid substitutions. The absence of more severe mutations reflects the enzymes
144
R. E. Howes et al.
Figure 4.2 Diversity of mutations in the G6PD gene and enzyme. Panel A shows the
distribution of common mutations along the G6PD gene coding sequence. Exons are
shown as open numbered boxes. Open circles are mutations causing Class II and III variants; filled circles are Class I variants; filled squares are small deletions; the cross represents a nonsense mutation; f shows a splice site mutation. (Figure from Cappellini
and Fiorelli (2008), reprinted with permission from Elsevier; figure originally modified from
Luzzatto and Notaro (2001)). Panel B shows the distribution of amino acid substitutions
across the enzymes tetrameric structure (each identical monomer subunit is labelled
AD), numbered according to the affected amino acids. The diamonds indicate polymorphic or sporadic mutations, and their colour shows the associated clinical phenotype. The grey shadowed areas cover the two dimer interfaces. Across this region, a
molecule of structural NADP per monomer is buried which stabilises the monomers
and the associations between them. Each mutation is shown in only one monomer, but
would be present in all four. (Figure from Mason etal. (2007), reprinted with permission
from Elsevier). The positions of a few common mutations (A-, Mediterranean, Seattle,
Union) are shown both in the gene (Panel A) and the enzyme (Panel B). (For a colour
version of this figure, the reader is referred to the online version of this book).
145
housekeeping function which requires some residual activity for cell survival. Knockout studies in mice found G6PD-null mutations to be lethal
(Longo et al., 2002) and a high degree of evolutionary conservation
of certain regions of the gene was identified by comparing the position
of mutations across 42 different organisms, pinpointing certain regions of
the gene as highly conserved, and hence essential for enzyme function and
cell survival (Notaro etal., 2000). All known mutations have been found
to affect the coding regions of the gene and none described in the regulatory regions (Beutler and Vulliamy, 2002; Fig. 4.2), suggesting that reduced
enzyme activity levels are associated with enzyme instability, rather than
deficiencies in gene expression.
The G6PD genes position on the X chromosome has important implications for its population genetics. Unlike in males, for whom the G6PD
phenotype was early-on observed to be binary with individuals being
either deficient or nondeficient depending upon which allele was inherited
(Beutler etal., 1955), the genes X-linked inheritance means that deficiency
in females is more complex. Females inherit two copies of the X chromosome and therefore have two populations of RBCs, each expressing one of
the two G6PD alleles they carry. If females inherit two identical alleles (both
either normal or deficient), their phenotype and clinical symptoms will be
identical to those of hemizygous males. Heterozygous females, however,
inherit one wild-type and one deficient allele but display a mosaic effect
of expression as only one X chromosome is expressed in each cell. One
population of cells will express the normal allele and the other population
the deficiency (Beutler etal., 1962). The ratio of normal to deficient cells
is variable, due to the phenomenon of Lyonization (Lyon, 1961). Lyonization is a random process and the resulting proportions of normal and deficient cells may deviate significantly from the expected 50:50 ratio (Beutler,
1994), leading some heterozygotes to have virtually normal expression,
and others with expression levels comparable with female homozygotes
(i.e. entirely deficient). Heterozygotes may therefore express a spectrum of
phenotypes; making appropriate diagnoses with standard binary methods
much harder than for deficient males, as many heterozygotes will be phenotypically normal. At the population level, G6PD deficiency is more commonly expressed in males, though in populations with high frequencies
of deficiency, homozygotic inheritance can be common, and the prevalence of affected heterozygotes may also be of public health concern. More
details about the population genetics of the G6PD gene are discussed by
Hedrick (2011).
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R. E. Howes et al.
3.2. The G6PD Enzyme

The G6PD enzyme consists of either dimer or tetramer forms of a protein
subunit consisting of 514 amino acids. Each subunit binds to an NADP+
molecule for its structural stability, which are positioned close to the interface
where the two subunits of each dimer bind (Au etal., 2000; Fig. 4.2). The
majority of mutations disrupt the enzyme structural stability and thus reduce
its overall activity. T
he effect of each mutation on enzyme structure and function depends on the location of the substituted amino acid. For example, many
of the most severe mutations map to exon 10 (Mehta etal., 2000) which
encodes the binding interface of the subunits and therefore disrupt its quaternary structure and stability. These mutations cause the most severe clinical
symptoms and as such do not reach polymorphic frequencies; instead they
usually result from independent spontaneous mutations (Fiorelli etal., 2000).
Mutations which do not cause such severe reductions in enzyme activity
are widely distributed across the genes coding region and throughout the
enzyme structure (Fig. 4.2), and have been found to reduce the efficacy of
protein folding, for example (Gomez-Gallego et al. (1996)). The residual
enzyme activity of G6PD variants ranges from <1% to 100%.
As with all enzymes, G6PD activity decreases with cell age: it is estimated that in normal blood, reticulocytes have about five times higher
activity levels than the oldest 10% of RBCs (Luzzatto, 2006). The oldest
cells are therefore most vulnerable to oxidative stress. In individuals with
intrinsically reduced G6PD enzyme activity due to genetic mutations, the
ageing process is effectively sped up, with larger proportions of cells having
lower enzyme levels and being at increased risk of oxidative damage. This
has implications for the clinical severity of the mutations, as discussed below.
The properties of these enzyme variants correspond to a broad spectrum of
enzyme biochemical phenotypes, i.e. electrophoretic properties, heat stability and enzyme kinetics. WHO guidelines (WHO Working Group, 1989)
for standardised biochemical characterisation of the enzyme led to 387
variants of G6PD being described by 1990 (Beutler, 1990), though many of
these would later prove to be genetic duplicates.
Few variants have been fully characterised, but a handful of those which
are common enough to be of major public health significance have been
well researched. The residual enzyme activity of these variants affects cell
primaquine susceptibility. Though it is widely accepted, these must be
inversely correlated; little evidence informs that presumption (Baird and
Surjadjaja, 2011). Three such variants have formed the basis to current drug
recommendations, which are further discussed in Section 8.2 (p. 179).
147
All the earliest evidence about the haemolytic risk of G6PD deficiency pertained to the African A- variant (G202A/A376G), due to the
racial background of the primaquine sensitive patients studied in the 1950s
Stateville primaquine experiments (Section 2, p. 136). Although rare as a
genetic variant for having a double-point mutation, this type of deficiency
is very common among individuals of sub-Saharan African origin. The Avariant characteristically expresses residual enzyme activity about 10% of
normal levels (Beutler, 1991). It was studies with this variant which led
to the discovery of G6PD deficiency (Carson et al., 1956). The Mahidol
variant (G487A) is the predominant allele among many G6PD deficient
populations of Myanmar and is also common among Thais (Section 5.3,
p. 163 and Fig. 4.6A). Enzyme activity is reduced to 532% of normal levels
(Louicharoen etal., 2009). Finally, the Mediterranean variant (C563T) was
originally known for its association with the clinical pathology of favism,
and causes some of the most severely deficient phenotypes (Beutler and
Duparc, 2007). This variant usually expresses <1% enzyme activity, with
undetectable enzyme levels in older erythrocytes (Piomelli et al., 1968).
Despite expressing such low levels of enzyme activity, carriers of this mutation are nevertheless asymptomatic until exposed to haemolytic triggers
(Beutler, 1991) (Section 3.4, p. 148).
3.3. T
he Pentose Phosphate Pathway as an Anti-Oxidative
Defence
G6PD enzyme activity is necessary for RBC survival as it catalyses the
only metabolic pathway capable of generating reducing power to these cells
lacking mitochondria (Pandolfi etal., 1995). Reducing power, supplied in
the form of NADPH, is necessary as an electron donor, i.e. chemical reduction, for detoxifying oxidative challenges to cells. The metabolic reactions
concerned are part of the pentose phosphate pathway (PPP, also called the
hexose monophosphate shunt), the first and rate-limiting step of which is
catalysed by the G6PD enzyme: the oxidation of glucose-6-phosphate into
6-phosphoglucono--lactone, which simultaneously reduces NADP to
NADPH. The electron of NADPH passes to abundant glutathione dimers
(GSSG) via another enzyme, glutathione reductase. Reduced glutathione
monomers (GSH) represent the primary defence against hydrogen peroxides, organic peroxidises, and free radicals. When G6PD functions normally,
the drain of electrons from the NADPH pool caused by oxidative challenge
within the cell prompts the PPP to accelerate according to need, i.e. maintaining an NADPNADPH equilibrium that strongly favours NADPH.
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This in turn maintains the oxidisedreduced glutathione (GSSG2GSH)

equilibrium strongly in the direction of the reduced state, i.e. 1:500 at steady
state (Greene, 1993).
However, in cells that have a mutant and defective G6PD gene, the PPP
may, depending upon the extent of the enzyme activity defect, function at
near-maximum rate even at steady-state redox equilibrium. When oxidative challenge occurs and the equilibrium of NADP to NADPH shifts to
the oxidised direction, the PPP is intrinsically unable to accelerate rapidly
enough to force the equilibrium in favour of NADPH. This effectively
stymies the flow of electrons to GSH, and that equilibrium shifts in favour
of GSSG.The oxidants consuming these reducing equivalents, in turn, overwhelm the ability of the cell to provide them and damage may then occur.
Visible evidence of such occurs in the form of Heinz bodies in the RBC
membrane that attend acute primaquine-induced haemolytic anaemia
(Greene, 1993). Heinz bodies cause the membrane to become rigid, and
thus decrease the cells lifespans. The mechanism of primaquine-induced
haemolysis remains uncertain, but will be discussed in more detail later in
this chapter (Section 8.1, p. 175).
3.4. Clinical Manifestations of G6PD Deficiency

In discussing the clinical manifestations, it is important to note that the
majority of G6PD deficient individuals are asymptomatic most of the time.
The public health importance of this condition comes from the sheer numbers affected and at potential risk of developing clinical symptoms: 400 million globally (Cappellini and Fiorelli, 2008), or 350 million within malaria
endemic countries (Howes etal., 2012).
Symptoms are induced when cells are exposed to exogenous oxidative
stresses against which they cannot defend themselves. The severity of the
clinical symptoms and the subsequent treatment required depends upon the
degree of enzyme deficiency (which is variant-dependent), the nature and
total dose of the oxidative agent, the time course of exposure, the presence
of additional oxidative stresses and pre-existing factors such as age, haemoglobin concentration and concurrent infection (Cappellini and Fiorelli,
2008). The relative contribution of each to determining the severity of the
response is not fully known but is further discussed in Section 8.2, p. 179).
The most clinically serious symptom of G6PD deficiency is neonatal
jaundice (NNJ), which peaks 2 to 3days after birth (Luzzatto, 2010). This
is highly variable in severity but can lead to kernicterus (Beutler, 2008) and
permanent neurological damage or death if left untreated (Doxiadis and
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Valaes, 1964; Luzzatto, 2006). Not all neonates with NNJ are G6PD deficient, but this congenital condition greatly increases the risks, and in some
countries is the most common cause of NNJ (Luzzatto, 2010).
AHA is the most common manifestation of the deficiency, and may be
triggered by a range of exogenous agents causing intravascular haemolysis and jaundice, and may include haemoglobinuria (dark urine) (Luzzatto,
2009). The most severe outcome of AHA is acute renal failure (Cappellini and Fiorelli, 2008). The longest-known of these triggers are fava beans:
favism can be very severe or even life-threatening if left untreated without transfusion (Beutler, 2008; Luisada, 1941); favism is most common in
children. Infection is another important trigger of AHA (Burka etal., 1966),
with severe pathology having been previously attributed to hepatitis viruses
A and B, cytomegalovirus, pneumonia, and typhoid fever (Cappellini and
Fiorelli, 2008). Finally, a number of haemolysis-inducing drugs have also
been identified as triggers of AHA (Youngster etal., 2010); in the present
context of P. vivax malaria, the most pertinent is primaquine.
The exceptions to G6PD deficiency being asymptomatic until triggered by certain exogenous triggers are those sporadically emerging, highly
unstable variants expressing very low residual enzyme activity.These variants
never reach polymorphic frequencies due to their severe pathology, which
is characterised as chronic nonspherocytic haemolytic anaemia (CNSHA).
While individuals with these mutations make up only a very small minority of the population affected by G6PD deficiency (almost always males),
they are the most clinically severe and may be transfusion-dependent
(Luzzatto, 2010). In addition to susceptibility to all the aforementioned triggers of AHA, the very low residual enzyme levels mean that cells cannot
even protect themselves against oxygen radicals continuously generated by
the on-going process of haemoglobin de-oxygenation. CNSHA is therefore
a lifelong condition, with haemolysis ongoing even in steady state.
Based on these pathologies, G6PD alleles can be categorised into three
types: (1) those sporadic severe variants associated with chronic symptoms,
(2) polymorphic types which are typically asymptomatic but susceptible to
trigger-induced acute haemolytic episodes, and (3) those with normal activity (Table 4.1). Previous classifications have included additional subdivisions
of the polymorphic variants into mild and severe types (WHO Working Group, 1989; Yoshida etal., 1971). However, as suggested by Luzzatto,
the distinctions between these further classes are blurred and are no longer
useful (Luzzatto, 2009). As such, we distinguish only three variant types
(Table 4.1).
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Table 4.1 G6PD Variant Types and Their Key Characteristics

Residual
Type Enzyme Activity Prevalence
Clinical Significance
<10%
150%
Sporadic, never
polymorphic
Polymorphic
Severe and chronic: CNSHA
Asymptomatic until triggered:

risk of NNJ, AHA, favism
Normal (>50%) Polymorphic (wild-type) None
In the present context of P. vivax therapy, the generally asymptomatic

polymorphic variants vulnerable to AHA (type 2 variants) are the primary
threat to safe therapy. These variants are the subject of this review. Primaquine-induced haemolysis is further discussed later in this chapter (Section 8.1, p. 175), but can require transfusion even after relatively low doses
(Shekalaghe etal., 2010). Identifying this risk is therefore essential.
4. DIAGNOSING G6PD DEFICIENCY

Given the absence of a universally safe drug and the potential severity
of primaquine-induced AHA, widespread safe radical treatment of P. vivax is
contingent upon reliable diagnosis of G6PD deficiency.There are two types
of tests for diagnosing G6PD deficiency: biochemical enzyme activity tests
and molecular DNA-based methods.These are suited to different situations,
depending upon the type of diagnosis required and the laboratory capacities available. We discuss here those currently available and consider their
limitations in respect to the heterogeneity of this condition, then discuss
on-going developments towards improving these methods.
4.1. Phenotypic Diagnostic Tests

Most tests for G6PD deficiency consider the biochemical phenotype
qualitative or quantitative measures of residual enzyme activity. These tend
to use dyes or fluorescence markers serving as direct or proxy indicators
of enzyme activity representing the rate of NADP reduction to NADPH
(Beutler, 1994); qualitative assessments generally allow classification as normal, intermediate, or deficient, while quantitative tests employ spectrophotometry to determine exact measures of enzyme activity. One of the earliest
screening methods was developed by Motulsky and CampbellKraut, using
the rate of brilliant cresyl blue decolourisation: if decolourisation had not
occurred within a predetermined timeframe (commonly 180 min), the
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sample was deemed to be G6PD deficient. Developments to this method

have included Brewers methaemoglobin (metHb) reduction test (Brewer
et al., 1962), Bernsteins DPIP method (2,6-dicholorophenol indophenol
dye test (Bernstein, 1962)), Beutlers fluorescent spot test (FST) (Beutler and
Mitchell, 1968; which is the WHO recommended method (Beutler etal.,
1979)), and most recently the WST-8/1-methoxy phenazine methosulfate
(PMS) method (Tantular and Kawamoto, 2003) created to facilitate field use
of the diagnostic.
Applying these diagnostic tests in large-scale population screening is
typically feasible in the context of research endeavours. However, such testing is very often impracticable for routine care in those settings where most
malaria patients live. Most methods still depend on a cold chain and specialised laboratory equipment. Even the FST, perhaps the most widely used
test, requires cold storage of reagents, micropipetters, a water bath and a UV
light source. Further practical limitations include the time-delay on obtaining the results, which may take several hours, and the difficulties of reading
the results naked eye judgements have been found to be subjective with
some of the colour changes, which are also influenced by room temperature and humidity. Furthermore, although standardised protocols for using
the most common methods were published in 1979, tests are invariably
modified by users (for example, in terms of the cut-off times imposed) and
adapted to local conditions and survey constraints. This diagnostic variation, therefore, hinders comparative analyses between surveys by adding a
level of uncertainty into the results. Furthermore, anaemia is an important
potentially confounding factor increasing the probability of false-positive
diagnoses. This condition reduces the overall number of RBCs, and therefore the level of G6PD enzyme per volume of blood. Conversely, increased
proportions of reticulocytes following malaria infection can lead to false
negatives as reticulocytes have the highest G6PD activity levels. The influence of malaria parasites on qualitative tests has not been evaluated.
The most important limitation to these qualitative tests, however, is their
relatively poor ability to diagnose female G6PD deficient heterozygotes. As
previously explained (Section 3.2, p. 146), heterozygotes express a mosaic
of two RBC populations: cells expressing the normal G6PD gene and cells
with the deficiency. The deficient cells carry exactly the same haemolytic
risk as those of homo- or hemizygotic individuals. Heterozygote diagnostic
outcome is dependent upon: (i) the diagnostic tests threshold for determining deficiency (the longer the delay, the larger the proportion of samples that will appear normal); (ii) the mutation and the level of residual
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enzyme activity expressed; and (iii) the ratio of normal to deficient cells
determined by Lyonization, as previously described. These issues present
serious problems to heterozygote diagnosis, as the normal enzyme expression in one population of cells can mask serious deficiency in others. A
heterozygote may have, for example, 70% of her RBCs expressing very
low enzyme activity, and therefore exquisitely sensitive to primaquine and
vulnerable to harm, but she may test as normal.
Although some biochemical tests have been found to be better suited
to detecting heterozygosity, including G6PD/6-phosphogluconate-
dehydrogenase (6PDG) and G6PD/pyruvate kinase (PK) ratio analysis, and
the cytochemical G6PD straining assay (Minucci et al., 2009; Peters and
Van Noorden, 2009), these are highly technically challenging and therefore
impractical for large-scale, field-based population surveys, and rarely used.
Molecular methods, on the other hand, provide an unambiguous diagnosis
of genetic heterozygotes. A recently described flow cytometric assay (Shah
etal., 2012) appears much more practical, albeit still limited to the setting of
relatively sophisticated laboratories.
Haunting all of these methods is the important question of what represents an acceptable level of enzyme activity with respect to risk of primaquine-induced haemolysis. In other words, at what level of residual activity
does each test classify patients as normal, and does this genuinely exclude all
at risk of harm? This is the issue of sensitivity. Specificity poses the converse
problem by excluding patients who could safely receive effective treatment
of their infection. A clinically appropriate sensitivity/specificity balance
must be incorporated into the diagnostics. Unfortunately, very little evidence regarding primaquine sensitivity phenotypes informs this decision
across the broad spectrum of mutant enzymes.
4.2. Molecular Diagnostic Tests

A seemingly more clear-cut diagnostic approach examines the gene itself.
Molecular methods use variant-specific primers to identify the presence or
absence of specific mutations. These direct methods overcome uncertainties
associated with variable enzyme activity cut-offs, anaemia, reagent breakdown and subjective classifications. Molecular diagnoses allow insight into
the severity of the condition for those mutations for which residual enzyme
level phenotypes and primaquine sensitivity phenotypes are known. Female
heterozygotes will not be dangerously misclassified as G6PD normal.
Irrespective of these benefits, the high-end laboratory requirements
and time lags for results leave these methods impracticable for population
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screening or routine care in their current form. Even if these limitations

could be overcome, deeper limitations remain. Although heterozygosity can
be diagnosed, the state of Lyonization, and thus the phenotype remains
uncertain, potentially putting individuals with these intermediate genotypes
at risk. For instance, a study in Tanzania reported a case of severe haemolysis
(defined as <5g/dl) in a heterozygote A- variant female child following a
single dose of primaquine (45mg dose) (Shekalaghe etal., 2010). Further,
although the enzyme activity of three major variants has been characterised
(Mediterranean, A-, Mahidol (Baird and Surjadjaja, 2011)), the clinical character of the vast majority remain unknown; in these cases, molecular diagnoses cannot inform clinical severity. Even in the well-studied A- genotype,
enzyme activity assays in Uganda identified surprising variation between
individuals of the same genotypes (Johnson etal., 2009). Finally, molecular
methods look for specific mutations, which is usually only a subset of all
described mutations, and hence cannot reliably diagnose all cases of potentially clinically significant deficiency. For example, the Tanzanian study previously referred to used primers to identify the common A (A376G) and
A- (G202A) mutations, but then reported instances of haemolysis in apparent
wild-type B individuals (Shekalaghe etal., 2010). It seems plausible, given
greater heterogeneity in the pool of G6PD deficient variants reported from
individuals in The Gambia (Clark etal., 2009) and Senegal (De Araujo etal.,
2006), that diversity in other populations of sub-Saharan Africa has also
been underestimated and that some of the Tanzanian wild-type individuals
who haemolysed were in fact deficient, but that the primers employed were
not comprehensive enough to identify all cases of phenotypic deficiency.
4.3. T
he Case for a New Diagnostic for Safe P. vivax
Radical Cure
Given the limitations to existing diagnostic methods, it is evident that
there is currently no well-adapted point-of-care G6PD test for use prior
to primaquine treatment in the field. A new, practical and standardised kit
is required to ensure safe use of primaquine this will require the test not
only to identify an enzyme deficiency, but to give a binary assessment of
whether a given dosage of primaquine can or cannot be safely administered. An important prerequisite step will be determining the position of
this binary cut-off against the spectrum of haemolytic outcomes, which are
known to range from negligible to highly severe.
Although the mechanisms whereby primaquine triggers haemolysis
remain uncertain (Section 8.1, p. 175), and the factors determining the
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severity of haemolysis not conclusively established, is it likely that this test

will assess residual enzyme activity levels. Given this, environmental conditions are of major concern as these will strongly influence the diagnostic
outcome as enzyme activity is heavily dependent on ambient temperature
which fluctuates significantly over diurnal and seasonal cycles. Total RBC
count as influenced by anaemia and the blood volume used will also
impact the diagnosis. In terms of practicality, the test needs to be inexpensive, provide a result rapidly, and be easily used and interpreted with
minimal training (Asia Pacific Malaria Elimination Network (APMEN),
2012). Although a binary qualitative test would be easiest to implement, the
heterogeneity of this disorder may require quantitative diagnoses; this consideration is particularly relevant to diagnosing heterozygotes.
Two visual, qualitative rapid diagnostic test (RDT) kits have been
described. The BinaxNOW G6PD assay (Binax, Inc., Maine, USA) was
found to have sensitivity and specificity of 0.98 and 0.98, respectively with
a cut-off of 4.0U/g Hb (Tinley etal., 2010)2; this accuracy may not be sufficient for mass screening; furthermore, at around $25 per test, the cost of
the test would be prohibitively high for mass screening or routine clinical
use where malaria is endemic. However, this tests major shortcoming is that
it must be used within a temperature range of 1825C, and is thus further
unsuited to most field-based settings where P. vivax is prevalent. The CareStart G6PD screening test (AccessBio, New Jersey, USA), another qualitative phenotypic test, is similar to a malaria RDT in appearance and use
(Kim etal., 2011), and has been demonstrated to be robust after prolonged
storage at high temperatures (up to 45C for 90days). Using a cut-off of
3.5U/g Hb with ethylenediaminetetraacetic acid (EDTA) blood samples,
the test had sensitivity of 0.68 and specificity of 1 (n=903). The test was
able to identify deficient cases up to 2.7U/g Hb, corresponding to 22%
residual enzyme activity (Kim etal., 2011). However, 13 of 903 individuals
with very low G6PD activity (<2U/g Hb) were misdiagnosed as G6PD
normal; these false negative cases would therefore be at high risk of harm if
administered primaquine. This evaluation study followed the manufacturer
recommendations to classify any test showing even slight colour change
as normal. In practice a far more conservative approach would be taken
which would increase this tests sensitivity and decrease its specificity. The
discrepancy between the thresholds used by these two binary tests (4.0U/g
2 These sensitivity and specificity values were from heparinized blood samples; EDTA blood samples
had 0.98 sensitivity and 0.97 specificity. Samples included 50 individuals with G6PD activity 4.0U/g
Hb (Trinity Assay) and 196 individuals with G6PD activity >4.0U/g Hb.
155
Hb for BinaxNOW vs. 2.7U/g Hb for AccessBio) demonstrates the uncertainty around what exactly determines an intolerance to primaquine and an
acceptable level of haemolysis. These basic questions need to be answered
before an apparent arbitrary threshold is set. So, while these tests show tantalizing promise, further development is required for their widespread use
to enable more aggressive application of primaquine with the confidence
of patients safety.
5. MAPPING THE SPATIAL DISTRIBUTION OF G6PD

DEFICIENCY

Maps provide an important evidence-base for assessing disease burden, for public health decision-making and for efficient resource-allocation
through optimal targeting of interventions (Cromley, 2003; Hay and Snow,
2006; McLafferty, 2003), not least in relation to disorders as spatially and
genetically heterogeneous as G6PD deficiency.The prevalence of G6PD deficiency has been mapped among populations in malaria endemic countries
(Howes etal., 2012) to allow identification of where this disorder may be a
problem for malaria treatment options. From this modelled map, sex-specific
population estimates of deficient individuals were derived and aggregated to
national and regional scales. Reported occurrences of the underlying G6PD
gene variants have also been assembled (Howes et al., in preparation). The
characteristics of the underlying variants are what will determine the severity of potential primaquine-induced haemolysis; while the prevalence map
indicates how common the deficient phenotype is. Assembly of the evidencebases of both types of population data, and the methodological steps involved
in generating the maps are summarised here. The following Section 6
(p. 165) then explores in more detail what these maps indicate about the spatial
characteristics of G6PD deficiency in relation to the endemicity of P. vivax.
5.1. G6PD Deficiency Prevalence Mapping

Various attempts to represent the spatial distribution of G6PD deficiency
prevalence have been made. The most recent WHO map dates from 1989,
and presented only national-level summaries of available data (Luzzatto and
Notaro, 2001; WHO Working Group, 1989). A similar national-level map
published by Nhkoma etal. in 2009 updated this effort, but still presented
only national summaries, masking any subnational spatial variation in prevalence. Subnational variation was possible to discern from the impressive compilation of human gene maps in Cavalli-Sforzas History and Geography of
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Human Genes (Cavalli-Sforza etal., 1994), however the statistical mapping

methods used were rudimentary, and modern geostatistics have advanced
significantly since their publication. A main limitation to all of these maps is
that they give no measure of uncertainty in their predictions, either at the
national-level or in a spatially specific manner. Modern geostatistics allow
more comprehensive approaches to mapping, particularly in respect to summarising relative confidence in the predictions (Patil etal., 2011).
We focus here on the recently developed G6PD deficiency map by
Howes et al. 2012, an effort of the Malaria Atlas Project (MAP), which
attempted to address some of the limiting factors associated with existing maps. This map was intended to represent the prevalence of clinically
significant deficiency, as diagnosed by phenotypic tests, in malaria endemic
countries.The evidence-base of surveys and all maps are available for download from the MAP website (www.map.ox.ac.uk).
5.1.1. Generating a Map: the Evidence-Base
A total of 1734 spatially unique surveys were included in the map evidencebase which met four criteria: (i) Community representativeness: all potentially biased samples were excluded: patients (including malaria cases), all
related individuals, and all samples which selected individuals according to
ethnicity; (ii) Spatial specificity: surveys had to be geographically specific
and possible to geoposition accurately; (iii) Gender specificity: to allow the
model to represent the X-linked inheritance mechanism of the G6PD gene,
data had to be reported according to sex; (iv) Phenotypic diagnosis: only
surveys which had used phenotypic diagnostic methods were included.
5.1.2. Generating a Map: the G6PD Mapping Model
Modelling a continuous map of the prevalence of G6PD deficiency had
to account for several difficulties: heterogeneity in the data set (both in
terms of variable G6PD deficiency prevalence found at nearby locations,
and in terms of the uneven distribution of the surveys across the map),
the relative reliability of the data set (from highly ranging sample sizes),
and the difficulties of predicting deficiency in heterozygotes due to the
genes X-linked inheritance (Section 4.1, p. 150). A final requirement of the
model was that predictions were supported by uncertainty metrics (Patil
etal., 2011). A Bayesian geostatistical model was developed to cope with
separate input data according to sex and the different outputs required. The
core assumption of geostatistics is that of spatial-autocorrelation: namely
that populations closer in space would be more similar than populations
further apart. While there were some exceptions, the raw data supported
157
this assumption. Populations were also assumed to be in HardyWeinberg

equilibrium (Hardy, 1908; Weinberg, 1908).
5.1.3. G6PD Deficiency Prevalence Map: an Overview
G6PD deficiency is widespread across malaria endemic regions (Fig. 4.3).
The modelled prevalence map of G6PD deficiency represents the allele frequency of phenotypic deficiency, equivalent to the prevalence of deficiency
in males. At the continental scale, frequency of the deficiency is highest
among the populations of sub-Saharan Africa, where prevalence peaks
Figure 4.3 The prevalence of G6PD deficiency in malaria endemic countries. The prevalence is the allele frequency, which corresponds to the frequency of deficiency in males.
Panels AD correspond to Asia, Asia-Pacific, the Americas, and Africa+ regions, respectively. (The figure is adapted from Howes etal. (2012)). (For a colour version of this figure,
the reader is referred to the online version of this book).
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R. E. Howes et al.
Figure 4.3contd
159
around 30% in several areas, but is also absent from parts of southern Africa
and communities in the Horn of Africa. Prevalence of G6PD deficiency is
less common across the Americas, being concentrated among populations
in coastal regions.While prevalence is generally lower among Asian populations than sub-Saharan Africans, the condition is widespread across Asia and
particularly patchy and heterogenous in some areas.
The associated uncertainty map of the predictions is shown in Fig. 4.4;
and the allele frequency map must be considered alongside these metrics
Figure 4.4 Uncertainty in the prevalence map. Uncertainty is quantified by the interquartile range of the model prediction and is closely associated with the proximity of
population surveys, which are shown by the black dots. Panels AD correspond to Asia,
Asia-Pacific, the Americas, and Africa+ regions, respectively. (The figure is adapted from
Howes etal. (2012)). (For a colour version of this figure, the reader is referred to the online
version of this book).
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R. E. Howes et al.
Figure 4.4contd
161
of model confidence, as areas rich in data and with more homogenous

frequencies of G6PD deficiency will be easier to model than areas where
surveys are scarcely distributed or where available data indicated the underlying prevalence of deficiency to be heterogeneous. The G6PD deficiency
prevalence maps prediction confidence is quantified in the uncertainty
map by the interquartile range (IQR, the 50% confidence interval) around
the model prediction. Thus, a smaller IQR is indicative of a more reliable prediction. Areas of greatest uncertainty are those which would most
benefit from new population surveys. Some potential sources of variability,
however, are not accounted for by this measure, such as the underlying
heterogeneity in the local population (which will not be represented if no
surveys are available from that region) and the variation introduced by the
diagnostic methods used. These limitations are discussed in greater detail in
the original publication of this map (Howes etal., 2012).
Regional prevalence of the deficiency is discussed in detail in relation to
P. vivax endemicity in Section 6 (p. 164). Geographic information systems
(GIS) grids and high-resolution images of these maps, as well as the input
evidence-base of surveys are freely available via the MAP website (www.
map.ox.ac.uk).
5.2. G6PD Deficient Population Estimates

Estimates of the population of G6PD deficient individuals needed to account
for high-resolution patterns of population density as well as short-scale variation in G6PD deficiency prevalence to ensure that population density was
reflected in the overall prevalence estimate. Howes and colleagues (2012)
derived these population estimates in a Bayesian framework so as to generate
uncertainty metrics around the population estimates (Patil etal., 2011). The
aggregated national-level numbers of G6PD deficient individuals are mapped
in Fig. 4.5. An overall allele frequency of deficiency of 8.0% (IQR: 7.48.8)
was predicted across all malaria endemic countries.This corresponded to 220
million affected males (IQR: 203241) and an estimated 133 million females
(IQR: 122148).The model results indicate that a median estimate of 26% of
expected genetic heterozygotes were diagnosed as being phenotypically deficient based on the raw survey data. Within the subset of 35 countries targeting malaria elimination, where primaquine treatment would be particularly
beneficial, overall prevalence was lower with a predicted allele frequency of
5.3% (IQR: 4.46.7), meaning that in 2010 an estimated 61 million males
(IQR: 5177) and 35 million females (IQR: 2946) were predicted to be
phenotypically G6PD deficient in countries eliminating malaria.
162
Figure 4.5 National allele frequency of G6PD deficiency. (Figure from Howes etal. (2012)).
R. E. Howes et al.
163
Although many of the highest frequencies of deficiency were predicted

from sub-Saharan Africa, the very high population densities across Asia
meant that the overall population burden was largely focussed there. China
and India, for instance, were estimated to be home to 41.3% of all G6PDdeficient males across the whole malaria endemic region globally. The high
allele frequencies seen in Africa meant that 28.0% of the overall deficient
population was in sub-Saharan Africa, while only 4.5% of the global population burden was in the Americas, and 67.5% across the whole of Asia.
National-level estimates for all malaria endemic countries were provided in
the original publication (Howes etal., 2012).
5.3. G6PD Deficiency Mutation Mapping

To understand the clinical characteristics of G6PD deficiency in different
regions, it is also necessary to map the underlying G6PD mutations. A simple map of key genetic variants was published in 2001 (Luzzatto and Notaro,
2001), and a new database of G6PD variants has since been assembled to
update this effort (Howes etal., 2012; Howes etal., in preparation). Surveys
were collated which provided measures of the proportions of each variant
among G6PD deficient individuals in different areas. Exclusion of population samples from hospitalised or exclusively symptomatic patients avoided
a bias towards higher proportions of the more severe variants, which might
otherwise have been preferentially identified.
Striking patterns emerged across malaria endemic regions (Fig. 4.6).
Genetic heterogeneity was found to be relatively low across populations
of the Americas and West Asia, where the A- and Mediterranean variants predominated, respectively. Further east, genetic diversity increased
among Indian populations where the Orissa variant, barely reported
outside India, predominated among certain communities in east, central India. Populations in countries east of India carried a completely
different set of mutations, and genetic diversity of the G6PD gene was
far greater. Although a handful of variants were found to be more commonly reported from specific populations (such as the Mahidol variant
across Myanmar and Thailand; Viangchan variant across Mekong region;
Kaiping variant among Chinese populations and the Vanua Lava variant
in central and eastern Indonesia), genetic diversity was high among these
populations. The proportion of Unidentified variants was also greatest among Asian populations, emphasising the inadequacy of molecular methods for diagnosing phenotypic deficiency: a limited number of
primers cannot reliably identify all possible cases of deficiency.
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6. SPATIAL CO-OCCURRENCE OF G6PD DEFICIENCY

WITH P. VIVAX ENDEMICITY

We now discuss the spatial epidemiology of G6PD deficiency, its
prevalence and genetic variants, in relation to its public health significance
in the context of P. vivax transmission. The geographical limits of P. vivax
Figure 4.6 Proportions of G6PD variants among phenotypically deficient community

samples. Panels A to D correspond to Asia, Asia-Pacific, the Americas and the Africa+
regions. Pie charts show the local proportions of each variant, sized according to the survey sample sizes and plotted on a logarithmic scale. The individuals from whom these data
originate are known to be phenotypically deficient. All samples are therefore deficient,
and the Other/Unidentified category represents G6PD variants which were too rarely
reported to be individually represented in the map, or which could not be identified. (For a
colour version of this figure, the reader is referred to the online version of this book).
Figure 4.6contd
165
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R. E. Howes et al.
Figure 4.7 Global endemicity of Plasmodium vivax endemicity. Assembly of this map
is described in Chapter 1 of Volume 80. (Figure from Gething etal. (2012)). (For a colour
transmission and its endemicity within those limits have been described in
detail in the opening chapter of volume 80 and are shown in Fig. 4.7. The
map shows P. vivax endemicity, as quantified by community parasite rate in
the 199year age range (PvPR199), with grey areas representing unstable
transmission where <1 case per 10,000 population occurs per year (Chapter
1 of V
olume 80; and Gething etal., 2012).
6.1. G6PD Deficiency in Asia

The majority of the global population at risk of P. vivax transmission is
on the Asian continent (Gething et al., 2012), defined as stretching from
Turkey, south to Vietnam and east to the Peoples Republic of Korea.
The prevalence and genetic variants of G6PD deficiency among these
populations were found to be heterogeneous, as was the transmission endemicity of P. vivax. While P. vivax endemicity is generally very low in Asian
countries west of India, being either at no risk or having small patches of
low endemicity in areas of unstable transmission, several of these countries
had relatively high prevalence of G6PD deficiency compared with other
parts of Asia. For instance, the national allele frequency of deficiency across
Iran was predicted to be 11.8% (IQR: 9.914.1), and Azerbaijan had a
national estimate of 10.2% (IQR: 8.911.7); both countries had only
limited areas of very low/unstable P. vivax transmission. However, G6PD
prevalence model uncertainty was high in this region, peaking (up to 37%
IQR) in an area devoid of data across southern Pakistan. This high uncertainty emphasises the need for additional community surveys to support
mapping of G6PD deficiency in this apparently high prevalence region.
The underlying G6PD mutation among these populations was reportedly
167
the Mediterranean variant, which was identified in >70% of deficient

individuals in all surveys between Turkey and Pakistan (Fig. 4.6A).
Further east, G6PD deficiency was present but at relatively low prevalence
(25%) across much of the Indian subcontinent, increasing in the eastern states
of Chhattisgarh, Orissa and Jharkhand where prevalence reached 525%; the
common variants in this area were the Mediterranean, Kerala-Kalyan and
Orissa variants, the latter being highly restricted in its spatial extent to east
India. These areas coincided with high P. vivax endemicity, which reached
>7% PvPR199 in the neighbouring province of Andhra Pradesh. Another
high prevalence area of G6PD deficiency in Asia (approximately 20%) was
around the northern LaoThai border, an area largely P. vivax free, with only
small patches of unstable transmission. Lower G6PD deficiency frequencies
were predicted along coastal Myanmar (23%), an area of high (>7%) P. vivax
endemicity. High frequencies of the two diseases, however, occurred simultaneously in southern Thailand where endemicity was >7% and G6PD deficiency prevalence 59%. Prevalence of both disorders was therefore variable
with often highly focal P. vivax transmission across this region.
In terms of the G6PD genetic mutations, there was a stark change in
variants and greatly increased diversity across countries east of India. G6PD
Mahidol was either universal or very common among communities in Myanmar, remaining prevalent among Thai populations, in whom the Viangchan
was also frequently reported. No single variant predominated among Chinese
populations, instead the Kaiping, Canton and Gaohe variants were all common. A high proportion of samples were also rare or could not be identified
using standard molecular primers, an indicator of high genetic heterogeneity.
6.2. G6PD Deficiency in Asia-Pacific

Some of the largest continuous regions of high P. vivax endemicity globally were predicted across the Asia-Pacific region, where endemicity reached
>7% across much of Papua New Guinea, the Solomon Islands andVanuatu.
Coinciding with this, G6PD deficiency was also prevalent on these islands,
peaking at 23% prevalence on the southern reaches of Santa Isabel and Guadalcanal islands (where the Union G6PD variant was the main cause of
deficiency, Fig. 4.6B). Both diseases were highly heterogeneous across Indonesia, by far the most populous nation in this region. Both G6PD deficiency
prevalence and P. vivax endemicity were particularly high across the central
Nusa Tenggara islands, such as on Flores where endemicity was over 7% and
G6PD deficiency approximately 10%. A dearth of G6PD population surveys
on Sulawesi introduced relatively high uncertainty to the map; in contrast,
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numerous parasite rate surveys indicated that P. vivax transmission was mostly
unstable in this area. Additional G6PD surveys would be particularly valuable
among the south-eastern populations of Sulawesi, where endemicity reached
5%, and G6PD deficiency was predicted at 8% due to the high frequencies
observed on nearby islands. G6PD deficiency was heterogeneous across the
region. Relatively high genetic diversity was reported with multiple variants
commonly co-existing in high proportions alongside important frequencies
of unidentified variants. For instance across Papua New Guinea, frequencies
of 1% were found along the southern coast which rose to 15% along the
East Sepik northern coast. The Vanua Lava G6PD variant was commonly
reported from populations in both Indonesia and Papua New Guinea, but
was not reported from anywhere outside this region.
A neonatal screening programme for G6PD deficiency exists across the
Philippines which contributed high density of prevalence data (n = 636
data points) indicating a spatially variable national prevalence of 2 to 3%
(population-adjusted national allele frequency estimate is 2.5% [IQR: 2.4
to 2.5] (Howes etal., 2012)). Across the Philippines, stable P. vivax transmission was only found on islands at the northern and southern ends of the
country, peaking in northern Luzon at around 5% endemicity, where G6PD
deficiency ranged in prevalence between 1 and 4%.
6.3. G6PD Deficiency in the Americas

The lowest predictions of G6PD deficiency prevalence globally were across
the Americas, where 40.8% of the land area had median prevalence predictions of 1%. Prevalence ranged from 0% across parts of Mexico, Peru, Bolivia
and Argentina, to a national allele frequency prediction of 8.6% in Venezuela.
Surveys were relatively scarce across large parts of the continent, particularly in
Venezuela which led to high uncertainty around the national allele frequency
estimate (IQR: 4.018.0). Plasmodium vivax endemicity in this area of high
G6PD deficiency prevalence was patchy, with areas of 3 to 4% PvPR199 interspersed with large expanses of unstable transmission. Deficiency prevalence
was also predicted to rise in the central and southern coastal provinces of Brazil, such as around the cities of So Paulo and Porto Alegre where P. vivax was
absent.The A- variant of G6PD was predominant, causing >80% of deficiency
cases across coastal communities of Brazil and Central America, and explaining
62% of deficient cases in a survey in central Mexico (Fig. 4.6C).
Plasmodium vivax endemicity peaked in two areas of the Americas:
Honduras and Nicaragua in Central America, and Amazonas province in
northwest Brazil (Fig. 4.7); areas where G6PD deficiency prevalence was
relatively low compared with other parts of the continent. Parasite rates of 6
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to 7% coincided with G6PD deficiency allele frequency national estimates

of 2.9% (IQR: 1.55.8) in Honduras and 1.5% (IQR: 0.63.6) in Nicaragua. Very few G6PD surveys were found from Amazonian communities,
but parts of this region where P. vivax endemicity exceeded 7% were predicted to have G6PD deficiency prevalence of up to 3%. Additional G6PD
deficiency surveys in both these areas of high P. vivax endemicity would be
valuable, particularly focussed in areas of high population density.
6.4. G
6PD Deficiency in Africa, Yemen and
Saudi Arabia (Africa+)
Plasmodium vivax epidemiology in the Africa+ region differs starkly from other
malaria endemic areas due to the high prevalence of Duffy negativity among
these populations (Howes etal., 2011) which depresses transmission to unstable levels across most of the continent (Chapter 2 of this volume). Therefore,
in spite of its high prevalence, G6PD deficiency in Africa+ has relatively little
bearing on the global picture of P. vivax therapy. The only two areas of stable
P. vivax transmission in Africa+ are Ethiopia and close surrounds, and Madagascar. G6PD deficiency prevalence in the Horn of Africa is low, with national
allele frequency in Ethiopia estimated at 1% (IQR: 0.71.5); though stable, the
coincident parasite endemicity is equivalently low, with most areas being at 1%
PvPR199, interspersed with patches of 2% endemicity.The highest single prediction of G6PD deficiency prevalence globally is on the Persian Gulf coast of
Saudi Arabia where P. vivax is absent. Plasmodium vivax transmission is negligible
across this whole peninsula, with only a narrow strip of unstable transmission
along the west coast of Saudi Arabia and Yemen. Endemicity on Madagascar is
more significant, reaching 2 to 3% PvPR199 in the inland and central coastal
regions. Only a single-community G6PD survey was available so although the
national allele frequency estimate is high at 19.4%, additional surveys would be
needed to reduce uncertainty in this estimate (IQR: 11.530.3).
Although G6PD deficiency does not present a hurdle to P. vivax radical
cure in most parts of Africa+, the main potential application of primaquine
across this region is for blocking transmission of P. falciparum (Eziefula et al.,
2012; Bousema and Drakeley, 2011). Prevalence estimates of G6PD deficiency in Africa+ are the highest globally, with 14 countries predicted to have
national allele frequencies >15%, all across sub-Saharan Africa from Ghana
(19.6% [IQR: 14.227.0]) in the west across to Mozambique in the east
(21.1% [IQR: 14.729.8]). Several areas were predicted to have particularly
high prevalence (approximately 30%), including the coastal areas of West
Africa (Ghana to Nigeria), the mouth of the Congo river (western Congo,
Democratic Republic of Congo and Angola) and west Sudan. Subnational
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heterogeneity was important in some areas, for example ranging from 30%
around Ibadan to 2% in northwest Nigeria. Prevalence of the deficiency
decreased at the continental extremities: in the western Sahel, southern
Africa and the Horn of Africa. Prediction uncertainty across the continent
was heterogeneous, being very high in areas lacking data such as central
Africa between the Democratic Republic of Congo and Madagascar, and the
SudanChad border. Additional G6PD community surveys are imperative to
reduce the maps high uncertainty, and caution with primaquine administration must reflect the high prevalence of the condition.
There is a notable absence of G6PD variant surveys from Africa (Fig.
4.6D), which may be in part associated with the presumption of low G6PD
genetic heterogeneity among those populations. As a consequence, many of
the community G6PD surveys conducted do not use phenotypic diagnostics
to identify deficient individuals and instead use only molecular methods to
detect a narrow range of variants. These data cannot therefore inform the
relative prevalence of variants among deficient individuals. Available surveys
indicated that the A- variant was predominant across deficient individuals in
sub-Saharan Africa (Burkina Faso and the Comores), with the exception of a
Sudanese study which identified a greater diversity, including the Mediterranean variant (Saha and Samuel, 1991).The Mediterranean variant was common (>50%) in two investigations of deficient individuals in Saudi Arabia.
7. EVOLUTIONARY DRIVERS OF THE DISTRIBUTION

OF G6PD DEFICIENCY

The widespread distribution and frequent high prevalence of G6PD
deficiencya genetic disorder associated with important clinical costs
presents an evolutionary paradox. The spatial overlap between G6PD deficiency and the precontrol distribution of malaria (Lysenkos map of precontrol
malaria is reprinted in Chapter 1 of Volume 80) was first remarked upon by
Motulsky (1960) and Allison (1960) shortly after the description of G6PD
deficiency and led Allison and Clyde (1961) to propose Haldanes Malaria
Hypothesis (Haldane, 1949) as an explanation for the natural selection of this
deleterious condition. This idea, which had recently been substantiated by
empirical evidence for the sickle-cell mutation (Allison, 1954), implied that
the deleterious G6PD deficiency condition carried, at least in some genotypes (homo- or heterozygotes), a selective survival advantage over normal
enzyme levels against malaria morbidity or mortality: a possible case of balancing selection (Haldane, 1949). This has attracted much attention over the
past half century with evidence from epidemiological observations, in vitro
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laboratory findings studies and invivo clinical studies strongly supporting the
hypothesis that this common genetic trait has been selected for by malaria
through conferring some degree of resistance against the severity of the
infectious disease. A number of excellent reviews of this body of work have
been published (Greene, 1993; Hedrick, 2011; Kwiatkowski, 2005; Luzzatto,
1979, 2004; Ruwende and Hill, 1998; Tripathy and Reddy, 2007).
7.1. Evidence of a Selective Advantage

7.1.1. Epidemiological Evidence
The most convincing epidemiological evidence of selection by malaria is the
sheer diversity of variants of the G6PD gene, many of which have reached
polymorphic frequencies in genetically isolated populations suggesting the
independent selection of each variant: an apparent case of convergent evolution by a common agent of selection, perhaps. Given that all polymorphic
variants are found in historically malaria endemic regions, it would appear
that these two diseases, infectious and congenital, are somehow associated.
Micro-mapping studies across altitudinal or climatic gradients of malaria
endemicity also suggest that the prevalence of this disorder is the result of
selection by malaria, rather than random drift or selection (Luzzatto, 1979;
Ruwende and Hill, 1998). A recent study from Sumba island in Indonesia
provides an impressive example of G6PD deficiency frequencies correlating with a strong malaria endemicity gradient over short spatial distances
(Satyagraha, unpublished data). G6PD deficiency prevalence ranged from
2.2 to 3.8% in Central Sumba where malaria endemicity was low, to as high
as 11% in highly endemic malaria hotspots to the west and southwest of the
island where P. falciparum was found year-round together with seasonable P.
vivax endemicity. While these observations provide no evidence of causality,
the spatial association between these two diseases is evident.
7.1.2. Invitro Evidence
Invitro studies have demonstrated unambiguously that parasitaemia is less
successful in G6PD deficient cells than in wild-type cells (Cappellini and
Fiorelli, 2008; Roth et al., 1983). The clearest demonstration of this was
by Luzzatto et al. (1969), who compared parasitaemia in both cell types
in heterozygotes (studying heterozygotes overcame the potentially confounding effect of different levels of acquired immunity which would exist
between different individuals). Cells with normal enzyme levels were 280
times more likely to be infected than deficient cells. Cappadoro etal. (1998)
examined P. falciparum intracellular development and identified selective
early-stage phagocytosis of infected G6PD deficient cells as a possible
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protective mechanism. Although they found no significant difference in the

growth and development of P. falciparum parasites between normal and deficient cells (Mediterranean variant), infected deficient cells were 2.3 times
more intensely phagocytosed when parasites reached the early stages of
the schizogonic developmental cycle, early in the erythrocytic stages of
infection.
7.1.3. Case-Control Invivo Evidence
Large-scale case-control studies look for a protective effect against malaria at
the population level. Specifically, this invivo evidence is drawn on to identify
which G6PD deficient genotypes the selective advantage is conferred upon:
hemi- and homozygotes, or heterozygotes? While studies all concur in identifying a selective advantage associated with G6PD deficiency, the particular
genotypes benefitting vary between studies, with data seemingly supporting
all scenarios. For instance, a study in southwest Nigeria reported significantly lower parasitaemia in heterozygous females but not hemizygous males
(Bienzle et al., 1972). Subsequent large case-control studies used clinical
symptoms rather than parasitaemia as the indicator of protection, and found
robust evidence for a protective role of the G6PD A- variant (G202A) in
hemizygous males and homozygous females in sub-Saharan Africa (Guindo
etal., 2007; Ruwende etal., 1995); the protection extended to heterozygous
femals remained contentious. Ruwende etal. (1995) surveyed children in The
Gambia and Kenya and found a 46% reduction in risk from severe malaria
in A- heterozygotes, similar to the 58% protection estimated against severe
malaria in hemizygotes. This study compared mild or severe malaria against
community controls who were asymptomatic or parasite-free. A comparable
case-control study in most respects was subsequently conducted in Malian
children (Guindo etal., 2007), except that the control samples used were of
uncomplicated malaria cases, rather than asymptomatic/malaria-free cases, a
reflection of the local hyperendemic malaria transmission. This study found
no protection conferred by this same A- mutation against heterozygous
females (n=221), in spite of a very similar positive result for hemizygotes.
As well as the different indicators of parasitic protection (parasitaemia vs.
clinical symptoms) and the different control groups (asymptomatic/malaria
free vs. mild parasitaemia), further difficulty in comparing case-control
studies may be introduced by the method used to diagnose the enzyme
deficiency. Johnson etal. (2009) demonstrated in Uganda how differences
between phenotypic and genotypic diagnostics can affect the apparent susceptibility to malaria of different G6PD statuses, with significant protection
173
from uncomplicated malaria only identified in phenotypically deficient

females.
7.2. N
eglect of the Selective Role of P. vivax as a Driver of
G6PD Deficiency
Most early studies have focussed on the protective role of G6PD deficiency
on malaria in Africa, and thus considered only a narrow representation of
the G6PD genes overall genetic variation and clearly neglected a potential
role for P. vivax, despite this parasite having a wider transmission range than P.
falciparum (Guerra etal., 2010) and causing significant morbidity and mortality (Chapter 3 of V
olume 80 and Price etal., 2007). An important life cycle
difference between these parasites is P. vivaxs preference for infecting reticulocytes (Anstey et al., 2009; Kitchen, 1938), which could confer a much
greater fitness cost on the host by hindering regeneration of the erythrocyte
pool. From the host perspective, G6PD enzyme activity levels in reticulocytes are at their highest. If a deficiency in enzyme activity can convey a
protective advantage against severe clinical symptoms, then the deficiency
would need to be particularly severe to be expressed in the reticulocyte
stages. In theory, therefore, P. vivax could be exerting much stronger selection
pressures on the host than P. falciparum, selecting more severe variants of the
G6PD gene; the generally asymptomatic nature of these mutations would
mean that the fitness cost of severe deficiency would not always be felt.
Indeed, G6PD Mediterranean, one of the most severe polymorphic variants
(<1% residual activity), has been found to offer significant protection against
symptomatic P. vivax among male and female Afghan refugees in Pakistan
(Leslie etal., 2010). Males and homozygous females were more significantly
protected than heterozygotes in whom only weak protection was found. This
study offered no comparison with protection against P. falciparum, however, as it
accounted for only 5% of infections in the study area. Another study, in an area
of co-endemic P. falciparum and P. vivax malaria in Thailand (Louicharoen etal.,
2009) found evidence of P. vivax having been the selective agent of the G6PD
Mahidol variant, which is common across parts of southeast Asia (Section 6.1,
p. 166). In this very comprehensive study, an evolutionary approach using extensive single-nucleotide analysis (SNP) around the G6PD gene locus showed
high homogeneity between haplotypes of the Mahidol mutation (G487A),
indicating that the mutation had undergone recent and strong positive selection, thus suggestive of conferring a strong advantage to human survival. Clinical studies indicated that this survival advantage was conferred as protection
against P. vivax parasitaemia, having no effect on P. falciparum parasitaemia.
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Moving forward, a number of fascinating questions remain unanswered.

For instance, the large number of G6PD mutations which have reached
polymorphic frequencies is of interest, as is the apparent lower diversity among African populations than others. Estimates of the ages of some
of these mutations propose relatively recent origins, which may explain
part of this diversity. Estimates range from 1000 to 6357 years for the Avariant (Sabeti etal., 2002; Slatkin, 2008; Tishkoff etal., 2001), 3330years for
the Mediterranean variant (Tishkoff etal., 2001), and 1575years for the Mahidol variant (Louicharoen etal., 2009). Further study of the ages of a range of
variants would allow a comprehensive picture of the evolutionary history of
this condition, and its association with the spread of human Plasmodium infections. Studies should consider both the role of P. vivax as well as P. falciparum to
allow the relative selection pressure of the two parasites to be determined with
respect to specific variants. The role of P. vivax as a selective agent of human
polymorphisms is further discussed in Chapter 2 of this volume, particularly in
reference to the protective role of the Duffy negativity blood group.
8. PRIMAQUINE, P. VIVAX AND G6PD DEFICIENCY

Primaquine has a vital and unique role in the malaria elimination
toolkit, fulfilling three critical functions: first, it is the only licenced radical
cure of P. vivax; second, primaquine is the only drug active against mature,
infectious P. falciparum gametocytes making it vital for blocking transmission; and third, in areas of emerging drug resistance, primaquine is being
used in containment programmes to prevent the spread of artemisinin resistant P. falciparum strains (WHO, 2011b). These invaluable properties make
understanding the triangle of interplaying aspects determining primaquineinduced haemolytic risk crucial: the human enzyme, the drug and the parasite. The relationship between P. vivax and primaquine is considered in
detail in Chapter 4 of V
olume 80, and not revisited in detail again here.
We consider here means of safely administering the 200mg total dose of
primaquine required for P. vivax radical cure (Alving et al., 1953; Baird
and Hoffman, 2004; Baird and Rieckmann, 2003; Coatney et al., 1953;
Edgcomb etal., 1950).
First, we review the molecular mechanisms by which primaquine triggers haemolysis, second how haemolytic risk varies according to primaquine dosing regimens, and third, how different G6PD variants modulate
haemolytic risk and severity. Understanding primaquines pharmacological
properties and biochemical effects on the cell is necessary for the development of safer regimens, and alternative safer drugs.
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8.1. Mechanism of Primaquine-Induced Haemolysis

It is well established that primaquine-induced haemolysis does not occur in
individuals with normal levels of G6PD activity (Baird etal., 2001; Bunnag
etal., 1994; Edgcomb etal., 1950). Furthermore, haemolytic risk is greatest
in the oldest RBCs, corresponding to an increasing risk as enzyme activity decays over time (Beutler, 1994; Beutler etal., 1954). These indications
suggest that primaquine-induced haemolysis is directly associated with the
consequences of reduced G6PD enzyme activity. However, the mechanism
by which this occurs remains uncertain, and the instability and diversity of
primaquine metabolites make studying this system exceedingly difficult.
Despite having been in circulation since the 1950s when clinical use of
primaquine began, relatively little work has been done to elucidate the precise molecular events leading to primaquine-induced AHA. Understanding
these may be critical in rationally disassociating the haemolysing toxicity of
the drug from its broad-acting therapeutic properties (Pybus etal., 2012) in
developing superior therapies.
Although the focus here of primaquine side-effects is the haemolytic
risk to G6PD deficient individuals, it should also be noted that primaquine
can cause a number of other side-effects, previously reviewed (Baird and
Hoffman, 2004; Hill etal., 2006). For instance, abdominal pain is a common
dose-dependent side-effect (Edgcomb etal., 1950; Hill etal., 2006), which
can be prevented through simply taking the pills with food (Clayman etal.,
1952). Primaquine also routinely causes a relatively mild, although occasionally symptomatic methaemoglobinaemia (typically about 6% for as long
as dosing lasts), further discussed below.
8.1.1. Primaquine and its Metabolites
Primaquine is rapidly excreted, with an elimination half-life of about 4h
(Carson et al., 1981; Greaves et al., 1980), and metabolises into a complex array of a dozen or so distinct moieties. One metabolite is carboxyprimaquine, which has been detected in plasma within 30min of dosing,
reaching plasma concentrations 10-fold higher than primaquine (Mihaly
etal., 1985). Carboxy-primaquine, however, appears physiologically inert,
both with respect to toxicity and therapeutic activity. On the other hand,
some moieties are highly unstable and oxidatively volatile. For instance,
5-hydroxy-6-methoxy-8-aminoquinoline (5H6MQ) was 2500 times more
potent than primaquine in stimulating the PPP in normal RBCs (Baird
etal., 1986b). It is likely to be one or several metabolic products of primaquine which is the active agent against the parasites, rather than the parent
compound itself (Beutler, 1969; Carson etal., 1981; Fletcher etal., 1988).
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The molecular events triggering haemolysis remain unproven, though

several mechanisms of action have been proposed, including the build-up of
methaemoglobin (metHb) causing damage to the cell membrane, and oxidative damage provoked by primaquine metabolites. The relative significance of
these pathways remains unclear. Invitro haemotoxicity assays have demonstrated
that it is cytochrome P450-linked pathways which metabolise the breakdown
of primaquine by redox reactions into its haemotoxic metabolites, including
the formation of metHb and the generation of reactive oxygen intermediates
(Ganesan etal., 2009), in a dose-dependent response (Ganesan etal., 2012); the
assay found no haemotoxic effect of primaquine when exposed to cells in the
absence of these cytochromes. Further study with isoenzyme activity screening and steady state kinetic data has identified two cytochrome P450 enzymes
(MAO-A and 2D6) to be strongly involved in primaquine metabolism, and
reported that the metabolites generated by these enzymes made it likely that
the drugs toxicity and efficacy were enabled by the same single-cytochrome
pathway, making it unlikely that these two properties of the drug could be
separated (Pybus etal., 2012). Further study is required to support these findings and determine the relative influence of inhibitors and inducers of these
specific cytochrome enzymes to test their individual effect on the drugs efficacy and toxicity. Similarly, while a number of primaquine metabolites have
been identified, none have been definitively associated with activity against the
Plasmodium parasite (Baird and Hoffman, 2004; Myint etal., 2011).
8.1.2. A Role for Oxidative Stress
Oxidative stress caused by primaquine metabolites has been long-held as
the favoured hypothesis for explaining the drugs toxicity. Reduced levels of
G6PD enzyme activity leave the cell with diminished anti-oxidant reserves,
namely NADPH and reduced glutathione (Flanagan etal., 1958), due to the
constrained rate of the PPP, as already discussed. Possible oxidative agents
which have been proposed include free radicals, such as activated oxygen,
and hydroxylated metabolites of primaquine which auto-oxidise into quinoneimine products, superoxides, hydroxyl radicals and hydrogen peroxide
(Brueckner et al., 2001; Fletcher et al., 1988); with oxidative metabolites
generated by the cytochrome P450 enzymes previously described (Ganesan
etal., 2009, 2012; Pybus etal., 2012). Oxidised glutathione, which accumulates during oxidative stress, has been found to be a strong intracellular
mediator activating membrane cation channels (Koliwad etal., 1996). The
opening of these Ca2+-permeable channels can trigger cell death (apoptosis) during oxidative stress (Lang etal., 2003, 2006), although a recent study
177
by Ganesan etal. (2012) did not find this mechanism to be triggered by

the primaquine-induced haemolytic pathway. Another suggested mechanism whereby depletion of reduced glutathione can lead to cell death was
described by Bowman and colleagues, who demonstrated that primaquineassociated oxidative stress (triggered in their experiments by exposure to
5-hydroxyprimaquine) induced oxidative injury to the erythrocyte cytoskeleton (Bowman etal., 2005b), accelerating the process of cell phagocytosis (Bowman etal., 2005a).
8.1.3. A Role for Methaemoglobin
The role of metHb accumulation has been proposed to be highly significant,
with oxidised primaquine derivatives, such as quinones or iminoquinones
(including 5-hydroxyprimaquine) found strongly associated with the formation of metHb (Link etal., 1985). Oxidative stress from primaquine can
lead to the oxidation of haemoglobin iron (Fe2+ Fe3+) and the buildup of metHb. This resulting condition, methaemoglobinaemia, is usually
asymptomatic and self-limiting if only low levels accumulate (Brueckner
etal., 2001; Fernando etal., 2011). At higher proportions, however, methaemoglobinaemia can result in tissue hypoxia, hypoxemia and cyanosis due
to metHbs low affinity for oxygen (Hill et al., 2006; Percy et al., 2005).
However, exacerbated methaemoglobinaemia is not a feature of clinical
acute haemolytic anaemia in G6PD deficient patients.
8.1.4. A Role for Altered Redox Equilibrium
Other experimental evidence points away from damage mediated through
oxidative degradation of the RBC cytosol or membrane. Baird and colleagues observed that the increased PPP activity stimulated by primaquine
metabolites occurred independently of glutathione redox activity. They
observed that when sodium nitrite was applied to the system, glutathione
redox stimulated the PPP. When the dose of sodium nitrite exceeded the
ability of the PPP to maintain steady state redox equilibrium, a proteolytic
system that degraded irreversibly oxidised proteins became active (Baird
etal., 1986b). In contrast, doses of primaquine metabolites that also saturated PPP activity came with no such proteolytic activity, and the drugs
were not mediating oxidative damage like sodium nitrite. It appeared that
the drugs themselves drove NADPH depletion and the PPP response to
that disturbed equilibrium (Baird et al., 1986a). In other words, redox
equilibrium between a reduced and oxidised species of primaquine would,
in a G6PD deficient cell, strongly favour the oxidised species, without
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necessarily prompting a broad oxidative degradation of cytosol proteins

(Brueckner etal., 2001). The oxidised species could be the agent of haemolysis. Brueckner et al. (2001) proposed a mechanism similar to that
demonstrated for the well-characterised molecular events in phenylhydrazine-induced haemolysis in rats. They postulated that the covalent linkage
of a metabolite to the haem moiety of haemoglobin could force the molecule from its globin fold by simple hydrophobic force into the lipid bilayer
of the RBC membrane.
Any mechanism of haemolysis must ultimately be reconciled with what is
highly likely to be a very brief and quantitatively insubstantial oxidative challenge to the RBC by primaquine.The daily 15 or 30mg dose may be appreciated as an exceedingly small quantity of molecules relative to its distribution
in tissue, rapid metabolism to a dominant and inert species, and very rapid
elimination. The oxidative challenge must be very insubstantial and fleeting
compared with, for example, nitrite poisoning. Although a mild methaemoglobinaemia typically occurs with normal primaquine dosing (CarmonaFonseca etal., 2009), it does not appear linked to depletion of GSH via a
generalised oxidative attack of RBC proteins.The damage being done, whatever it may be, seems irreversible during the intervals between dosing. The
damage appears cumulative across doses, with haemolysis not commencing in
earnest until after the third or fourth dose. These features appear incompatible with a general oxidative stress and would point to an irreversible capture
of the harmful primaquine species, or the damage done by it, in the RBC. An
accumulation of displaced haem molecules in the RBC membrane, manifest
as Heinz bodies, is compatible with all of these features.
8.1.5. Significance of Primaquine-Induced Haemolysis
Whatever the mechanism, be it the build-up of metHb, direct oxidative
damage, the balance of redox equilibrium, or any other pathway, the physiological damage caused to the cells leads to intravascular haemolysis, making acute haemolytic anaemia the main clinical symptom. Freely circulating
haemoglobin from the haemolysed cells causes the most severe and potentially lethal conditions, including haemoglobinuria and acute renal failure
(Burgoine etal., 2010).
Understanding the mechanisms by which primaquine induces
haemolysis is critical to developing safer administration of primaquine. In the same way that knowledge of the dose-dependency effect
of primaquine (described in the next section) has allowed treatment
regimens to be extended over a number of weeks to reduce their toxicity, further manipulations to improve safe dosing schedules would
179
likely be possible given a better understanding of its mode of action.

For example, primaquine interaction with co-administered schizonticides has been suggested to affect its toxicity (Myint et al., 2011).
If confirmed, this could prove a relatively straightforward solution towards
safer therapy. Improvements to diagnostic methods by ensuring that their
target corresponds to the predisposing factor to haemolysis might also be
possible with an improved understanding of the predictors of haemolytic
susceptibility. Another main motivation for studying the biochemical processes of primaquine action is pharmaceutical: the potential for disassociating
the drugs therapeutic properties from its toxicity. If feasible, this could lead
to safe and effective killing of hypnozoites in P. vivax therapy and control.
8.2. Factors Affecting Haemolytic Risk

While the biochemical mechanisms of primaquine-induced haemolysis remain uncertain, it is clear that the risk and severity of haemolysis
is highly variable, affected by both exogenous and endogenous factors
(Beutler, 1994). Generalisations about haemolytic risk and its clinical severity are frequently cited based on the binary categorisations of
mild and severe enzyme deficiency (Hill et al., 2006; WHO, 2010,
2011a). In reality, a spectrum of clinical severity exists, ranging from
asymptomatic to lethal, which is determined by several factors including the primaquine dose and the genetic and biochemical determinants
of enzyme activity levels. Many studies investigating haemolytic risk
aggregated all phenotypically deficient individuals, sometimes specifying mild or severe deficiency (Hill etal., 2006; Recht etal., 2012);
here we consider reports relating to specific genetic variants. The primaquine sensitivity phenotypes of three have been investigated in detail,
and we discuss these here.
8.2.1. Dose Dependency
The dose dependency of the haemolytic effect of primaquine was noted
from the first clinical investigations into primaquine, with observations
that toxicity appeared to be cumulative: in some instances symptoms began
late in the course of drug administration and continued for several days after its
discontinuance (Edgcomb et al., 1950). The pharmacokinetics of primaquine, however, are unaffected by dose, and its efficacy is contingent upon the total dose administered, rather than the total amount per
dose or the timescale over which it is administered (Mihaly etal., 1985).
A meta-analysis by Schmidt and colleagues of the early data (19461975)
on dosing regimens in Rhesus monkeys found that the success of radical
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cure is determined by the overall total primaquine dose, independent from

the timescale over which it is administered: 1-, 3-, 7- and 14-day regimens
were not found to differ in their therapeutic success (Schmidt etal., 1977).
This pharmacokinetic property has the advantageous benefit of permitting
extended dosing schedules which reduce the risks of haemolysis without
impacting on its effectiveness.
8.2.2. Variant Dependency
The diversity of G6PD genetic variants leads to a spectrum of residual activity levels and associated clinical severity (Section 3, p. 143). We discuss here
how drug regimens which can be tolerated by some G6PD deficient individuals were developed in relation to studies on certain key G6PD variants,
and as a corollary, how haemolytic risk is variant-dependent.
(a) A- variant. The early Stateville studies into primaquine sensitivity
were all based on the A- variant, due to the origins of the individual studied.
The vulnerability presented by this variant to primaquine-induced haemolysis was recently re-affirmed by a Brazilian study which resulted in
severe haemolysis in three G6PD deficient P. vivax patients treated with
30mg daily doses of primaquine over 5 or 7days (Silva etal., 2004). The
Stateville studies found that daily dosing of the 30mg regimen led to haemolytic symptoms in affected individuals appearing on the second or third
day, peaking 47 days after starting treatment, continuing for just over a
week overall (Clyde, 1981; Hill etal., 2006; Hockwald etal., 1952). Haemolysis ended within a few days of ceasing treatment, after which haemoglobin levels recovered; recovery even occurred with continued treatment
as haemolysis was compensated by erythropoiesis and the regeneration of
reticulocytes which have inherently higher G6PD levels.
The drugs total dose effect has allowed regimens with an acceptable
level of toxicity for G6PD deficient individuals to be developed. Studies
by Alving etal. found that intermittent weekly 45mg doses of primaquine over 8weeks was effective (treating 90% of P. vivax Chesson strain
infections; n=40) and did not produce any clinically significant haemolysis
(the number of G6PD deficient individuals was not specified) (Alving etal.,
1960). Subsequently, Brewer and Zarafonetis (1967), studying individuals of
African origin, confirmed that twice-weekly administration of 45mg primaquine triggered more haemolysis than once-weekly dosing in deficient
individuals; no haemolysis was reported from individuals with no deficiency.
A review by Clyde (1981) determined that daily 15mg primaquine doses
for 14days would be safely tolerated, with any side-effects remaining largely
181
asymptomatic; but recommended the extended 8-weekly 45 mg dosing

schedule. As such, current WHO recommendations for adults with mild
G6PD deficiency are for 8-weekly 45mg doses of primaquine (Hill etal.,
2006; WHO, 2010, 2011a). As well as averting the severity of haemolysis,
another benefit of extending the dosing schedule is the opportunity for
affected G6PD deficient patients to discontinue treatment before the severity of haemolysis becomes too serious. In these mild cases, haemolysis is
self-limiting and ends a few days after stopping treatment.
However, a review by the American Centers for Disease Control and
Prevention (CDC) recommends great caution when administering primaquine to individuals with any degree of G6PD deficiency, and emphasises
that very careful risk assessments and strict medical supervision are essential
(Hill et al., 2006). The authors note that this weekly dosing schedule for
G6PD deficient individuals has not been approved by the FDA. Indeed, case
reports of severe adverse effects to these safe regimens exist, including even
severe reactions in individuals once deemed to be at especially low risk. For
instance, a heterozygote with the A- variant (G202A mutation) was reported
to suffer a clinically severe haemoglobin drop to <5g/dl following a single
45 mg dose of primaquine (Shekalaghe etal., 2010). A possible reason for
this unexpected outcome may stem from a misdiagnosis, as only one locus in
the gene was tested in the mutation analysis and other more severe variants
would not have been identified.
Recent studies of primaquine in individuals with the A- variant are few
(Eziefula et al., 2012) and focus on the drugs application to P. falciparum
gametocyte clearance single dose of 0.75mg/kg primaquine in combination with artemisinin combination therapy (ACT) (Shekalaghe etal., 2007,
2010). However, a clinical trial in several African countries of individuals
with the A- variant with another drug-trigger of AHA in G6PD deficient
individuals, dapsone, resulted in high rates of haemolysis, with 10.9% of
hemi- and homozygotes requiring transfusion following 150mg/day dosing over 3days.3 Authors analysing these outcomes conclude that, contrary
to current perception, the A- variant cannot be considered mild (Pamba
etal., 2012).
(b) Mahidol variant.This is an important variant among populations from
Myanmar and Thailand, and is reported to cause residual activity to drop to
3
Direct comparison on the haemolysing effect of dapsone in relation to primaquine was investigated
during the Stateville trials, when a G6PD deficient African male was exposed on different occasions to
daily doses of 100mg dapsone for 21days, and 30mg of daily primaquine doses for 18days (Degowin
etal., 1966). The haemolytic effect was less marked with dapsone.
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532% of normal levels (Louicharoen etal., 2009). A study investigating the

effects of primaquine has been conducted in a relatively small number of P.
vivax-positive G6PD deficient individuals (n=22) in Thailand who were
given 15mg primaquine for 14days with standard chloroquine treatment
(Buchachart et al., 2001). No serious adverse effects were subsequently
reported during the 28-day follow-up period, though a 24.5% (13.9)
drop in haematocrit was observed in the G6PD deficient patients with an
equivalent only 1.2% (14.4) drop in G6PD normal individuals. No patient
required transfusion.This study concludes that standard primaquine therapy
would be safe in Thailand, even for those G6PD deficient (Buchachart etal.,
2001). Although these individuals were only diagnosed as deficient phenotypically, the authors reported the Mahidol variant predominant in the study
area. Polymerase chain reaction (PCR) diagnosed individuals from a similar study in Thailand were all diagnosed as carrying Mahidol variant; no
serious adverse effects were reported from this study of 14-day primaquine
therapy (Takeuchi et al., 2010). A similar attitude is presented from an
economic perspective by Wilairatana etal. (2010) who determined that in
the absence of widely available G6PD testing at the malaria clinic level,
only patients who develop black urine or anaemia should be tested for
G6PD deficiency, and that mild-moderate deficient cases should be prescribed the 8-weekly dose. A study from Myanmar where 22 G6PD deficient individuals were given the 8-weekly 45mg primaquine dosage also
reported no severe adverse effects (Myat Phone etal., 1994), and similarly
concludes that weekly dosing is safe for G6PD deficient individuals in
Myanmar. The authors of these studies therefore encourage blind primaquine therapy in this region, where G6PD deficiency allele frequency is
high: estimated to be 13.6% (IQR: 11.915.5) in Thailand and 6.1% (IQR:
4.19.3) across Myanmar (Howes et al., 2012). The WHO specifies that
primaquine should be used for P. vivax radical cure in this region, but that
prior G6PD screening is necessary (WHO, 2010). Given the difficulties of
field-based G6PD diagnosis, it is uncertain whether clinicians actually risk
administering primaquine (John etal., 2012). The evidence-base for these
recommendations, however, is relatively meagre (Recht etal., 2012). The
heterogeneity of local variants reported from this region (Fig. 4.6A) is also
an important reason to exercise great caution, even if a handful of studies
have reported no serious adverse reactions to primaquine, these studies
may not have included patients with severe G6PD variants. For instance, a
more severe variant,Viangchan, is also common in Thailand, and along the
Cambodian and Laos borders.
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In spite of these risks, the use of primaquine in mass drug administration has
been investigated in Cambodia for P. falciparum transmission control.Very low
dosing (9mg/10days for 6months; n=6040) administered alongside an ACT
without G6PD testing (despite local prevalence being 18.6% in males) was
not found to cause any severe adverse effects (Song etal., 2010). Importantly,
however, no active monitoring was in place to detect these.This very low dosing, therefore, was deemed safe for P. falciparum transmission control; however,
this dosing bears little relation to P. vivax radical cure, and would not be logistically feasible as a standard therapy due to its very resource-intensive dosing.
(c) Mediterranean variant. Originally known for its association with the
clinical pathology of favism, the Mediterranean variant causes one of the
most severely deficient phenotypes, reducing enzyme activity to <1% of
normal levels (Beutler and Duparc, 2007). Serious haemolysis is caused by
15 mg daily or 45 mg weekly courses of primaquine (Clyde, 1981) and,
unlike with the A- variant, haemolysis in G6PD Mediterranean individuals
is not self-limiting. A review by Clyde concluded that individuals affected
by the Mediterranean variant should be administered supervised weekly
doses of 30mg for 15weeks (Clyde, 1981). WHO guidelines today, however, state that no primaquine should be given to individuals with so severe
a deficiency (WHO, 2010, 2011a).
8.2.3. Red Blood Cell Age Dependency
Senescent RBCs are most likely to succumb to haemolytic challenges
(Beutler etal., 1954).Wild-type erythrocytes appear to have a large surplus
of potential G6PD activity, allowing the PPP to be significantly upregulated when exposed to oxidative stress (Salvador and Savageau, 2003).
This enzyme activity decays naturally with RBC age (as erythrocytes lack
nuclei and therefore have no mechanism for regenerating enzyme levels),
correlating with susceptibility to haemolytic risk. This decay was found
to be exponential, with a half-life of 62days for wild-type enzyme and
13days for A- enzyme (Piomelli etal., 1968). The more severe variant,
Mediterranean, had such a rapid decline that no enzyme activity could
be detected in mature erythrocytes. Cells with the A- genetic variant
were demonstrated to be insensitive to primaquine when 821days old,
but were rapidly destroyed 55days later (corresponding to slightly older
than half their normal lifespans) when re-exposed to primaquine (Beutler
etal., 1954). In wild-type individuals natural enzyme decay does not reach
levels which put the individual at clinical risk; in G6PD deficient cells,
however, the ageing process being so much more marked than in normal
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R. E. Howes et al.
cells, means that even moderately deficient cells will have enzyme activity levels that drop to clinically at-risk levels. If erythropoiesis can replace
haemolysed cells, then the clinical effect will be negligible and the haemolysis self-limiting once all susceptible cells have been destroyed (Alving
etal., 1960; Dern etal., 1954a).
8.2.4. Sex Dependency
Haemolytic risk is also sex-dependent. Although primaquines fundamental pharmacokinetics are not affected by gender (Cuong etal., 2006;
Elmes etal., 2006), the majority of affected females are heterogeneous and
therefore present less commonly with severe symptoms than males. In areas
of high prevalence, however, homozygote inheritance of deficiency can be
common (Howes et al., 2012), and heterozygous females can also suffer
severe haemolysis (Pamba etal., 2012; Shekalaghe etal., 2010). The population of RBCs carrying the deficiency are at equal haemolytic risk as
homozygous or hemizygous cells. The relative proportion of wild-type and
deficient cells will have a major influence in determining the overall clinical
severity of haemolytic stress at the individual level. Interactions with other
genetic blood disorders may also exacerbate the effects of the deficiency in
females (Chopra, 1968).
8.3. Predicting Haemolytic Risk

The core motivation for understanding G6PD deficiency in the context
of P. vivax control is in being able to ascertain haemolytic risk and prevent adverse drug events from primaquine therapy. However, while much
research has been done and the complexity of interacting factors has been
clearly demonstrated, key knowledge gaps still hinder reliable predictions
of haemolytic risk.The main assumption made when assessing haemolytic
risk is that enzyme activity levels can be a reliable indicator. Although this
would appear to be generally true, exceptions exist, such as an Iranian
boy with 19.5% residual activity requiring a transfusion after a single
45 mg dose of primaquine (Ziai et al., 1967), and a heterozygote Tanzanian experiencing severe adverse reaction also following a single dose
(Shekalaghe et al., 2010); a detailed record of adverse drug events has
been compiled (Recht et al., 2012). While these cases are exceptions,
from a clinical perspective, interpretation of diagnostic outcomes must
allow for the uncertainties in these relationships. The practical implications of these considerations and difficulties in assessing haemolytic risk
are discussed in the next section.
185
9. T
OWARDS A RISK FRAMEWORK FOR P. VIVAX
RELAPSE TREATMENT

In this final section, we consider how the current state of understanding about G6PD deficiency and primaquine may be practically applied
to promoting safe radical cure of P. vivax. G6PD deficiency is widespread,
predicted in all malaria endemic countries, with an overall estimated allele
frequency of 8.0% (IQR: 7.48.8), as discussed in Section 5.2 (p. 161).
Given its potential clinical severity, primaquine cannot be administered
without careful prior assessment of risk. This haemolytic risk may be considered at two scales: (i) large regional scales for public health perspectives,
and (ii) directly by clinicians in relation to individual-level treatment decisions. Once risk has been satisfactorily judged, primaquine can be administered, or withheld, accordingly. Important limitations hinder risk assessment
at both scales, however, and we discuss necessary developments towards
overcoming these and improving safe access to P. vivax radical cure.
9.1. A
ssessing National-Level Haemolytic Risk of
Primaquine Therapy
Knowledge of the spatial characteristics of G6PD deficiency can be coupled with information about clinical phenotypes to allow comparisons
of haemolytic risk between regions at scales of public health significance.
It is important to note that assessment of risk at such large spatial scales
cannot inform risk at the level of the individual, and can never replace
the need for careful oversight of primaquine therapy by clinicians (Hill
etal., 2006), even in areas considered to be at low risk from a public health
perspective.
A simple national-level framework assessing large-scale risk has been
proposed (Howes etal., 2012), but important limitations to the data informing this analysis make it only a coarse-scaled and crude framework: one
which must be refined as our understanding of clinical risk improves.
9.1.1. P
roposed Framework for Ranking National-Level Risk from
G6PD Deficiency
Current WHO treatment guidelines consider haemolytic risk to differ between mild and severe classes of deficiency. Mild to moderate cases of deficiency may be treated with 8 weekly 45mg doses, while
severely deficient individuals should not be administered any primaquine
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R. E. Howes et al.
(WHO, 2010, 2011a). Consequently, relative haemolytic risk from primaquine due to G6PD deficiency at the population level may be considered
to be contingent upon two factors: (i) the overall prevalence of deficiency
within that population, and (ii) the relative composition of mild and severe
genetic G6PD variants reported from that population.The simple framework
suggested by Howes etal. (2012) scores the national prevalence of deficiency
based on the national allele frequency estimates described in Section 5.2
(p. 161), and assigns variant severity scores using a database of documented
reports of G6PD variants to assess the relative proportion of Class II and Class
III variants nationally (WHO-endorsed subdivisions of the Type 2 category
of variants; Table 4.1 and WHO Working Group, 1989). An overall risk score
was obtained for each country by multiplying the prevalence score by the variant severity score to give six categories of risk across a spectrum from rare and
mild G6PD deficiency to common and severe G6PD deficiency (Fig. 4.8).
Overall, this simple risk analysis ranked the highest level of G6PD
deficiency risk as being in the Asia and AsiaPacific regions where severe
variants were reported and population prevalence of deficiency was common (>1% allele frequency). High prevalence of deficiency caused by predominantly Class III variants across sub-Saharan African countries led to
moderate levels of risk in this region. Risk scores in the Americas ranged
from low to moderate. National-level scores are mapped in Fig. 4.8; further
details about the methodology employed are given in the original publication (Howes etal., 2012).
9.1.2. I mportant Limitations to Predicting National-Level Haemolytic
Risk
Both the underlying assumptions and the value of the described risk
stratification are questionable. It is nonetheless useful to attempt to do so
with regard to identifying weaknesses and the knowledge gaps that cause
them. The analysis main assumption is that the severity of haemolysis
can be predicted from the genetic variant and that haemolytic severity is
adequately represented by the subjective categorisations of Classes II and
III. Further, this assumes that primaquine sensitivity phenotypes inversely
correlate with residual enzyme activity (categorised here as Classes II and
III). Given that the mechanism of primaquine-induced haemolysis remains
uncertain (Section 8.1, p. 175) only isolated case reports exist to support
this assumption. Although the data from the three variants described in
Section 8.2 (p. 179) would appear to support this correlation, the evidence underpinning this relationship across all variants is not robust, and
187
extrapolating risk to all variants based on their WHO classifications is

therefore very uncertain. Furthermore, the evidence used to determine
the classifications of variants into their corresponding WHO Class (tabulated by Beutler, 1993; Kwok etal., 2002; Luzzatto etal., 2001; Minucci
et al., 2012; UCL Bioinformatics Group website; Vulliamy et al., 1997;
Yoshida etal., 1971) is weak in many cases, being determined from small
numbers of samples with variable laboratory techniques (Recht et al.,
2012). Given this uncertainty in the risk analysis, only a very coarse classification with three scores of variant severity was used. Finally, the data set
informing the national classifications of variant severity is often poor, with
data reported from only 54 of 90 malaria endemic countries, meaning that
severity scores had to be inferred for many of them.
If a robust understanding of the relationship between genetic variants
and their primaquine sensitivity phenotypes could be incorporated in this
analysis with a more complete background picture of the spatial heterogeneity of the G6PD variants and their prevalence, the conclusions drawn
would be much more valuable. Given these gaps in our current understanding, the main robust predictor of haemolysis is the phenotypic deficiency
in G6PD enzyme activity. The modelled prevalence map may therefore be
the most detailed, reliable, and appropriate risk assessment of overall G6PD
deficiency-associated harm be it moderate or severe which is relevant
to informing public health policy for P. vivax radical cure.
9.2. Assessing Haemolytic Risk at the Level of the Individual

Given the numerous testing kits which have been developed to diagnose
G6PD deficiency (Section 4, p. 150), assessing haemolytic risk at the level
of the individual ought to be straightforward. However, logistical constraints hinder the widespread use of these diagnostic methods in the rural
communities where P. vivax is most common and point-of-care testing is
most needed. Further, it would appear that many of the available diagnostic methods have not been calibrated to any predetermined and clinically
relevant measures of risk, and are seemingly arbitrary in their classifications
of deficiency. For instance, the threshold for distinguishing deficient from
nondeficient cases ranges from 10% to 60% residual enzyme activity. These
assessments of haemolytic risk also assume that enzyme activity is a suitable
indicator of primaquine-induced haemolysis.Their poor suitability to diagnosing deficiency in females (Section 4.1, p. 150) is a further hindrance to
their suitability for discerning haemolytic risk. No molecular methods are
currently suited to field-based settings.
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R. E. Howes et al.
Although WHO treatment recommendations distinguish mild from

severe deficiency, no details are provided to indicate what these categories
correspond to in terms of enzyme activity or associated degree of haemolysis. Given the difficulties with diagnosing these severity types, and the
largely unknown clinical risk which mild deficient individuals would be
exposed to, it has been argued that any G6PD deficient phenotype, regardless of severity, should suffice as a contraindication for primaquine therapy
(Baird and Surjadjaja, 2011). Distinguishing the degree of severity is not
possible with most diagnostic tests, including the binary point-of-care tests
currently in development (Section 4.3, p. 153). Attempting to account for
the severity of deficiency in a framework determining safe primaquine
therapy may therefore be an unnecessary complication at this stage, and
instead it may be more appropriate to re-assign the WHO treatment guidelines with binary options, removing the additional subjective mild/severe
distinction. Anecdotally, the importance of simplicity and great caution in
determining drug guidelines is well illustrated by a recent case report of a
Burmese P. vivax patient, undiagnosed as G6PD deficient, misunderstanding his primaquine regimen guidelines due to linguistic barriers, and taking
almost his full course of primaquine (165mg) in one go, resulting in severe
haemoglobinuria (Burgoine etal., 2010). Risk assessment with a potentially
harmful drug must be especially cautious.
Until the evidence underpinning the mechanism of haemolytic risk (Section 8, p. 174) allows predictability of the haemolytic outcome of different
variants with specific primaquine dosages, simple and safe guidelines based on
practicable diagnoses appear the most appropriate end-points of haemolytic
risk assessments. A better understanding of the mechanism of drug-induced
Figure 4.8 National risk index from G6PD deficiency. Two aspects of G6PD deficiency
epidemiology were used to define the national risk of G6PD deficiency: (1) the national
prevalence of deficiency was stratified into three classes (1%; >110%; >10%) based on
the national allele frequency estimates described in Section 5.2 (Fig. 4.5); (2) the severity
of local variants was classified at the national level based on a literature search of reports
of occurrences of G6PD variants. Variants were classified into mild and severe types,
in accordance with the WHO-endorsed classification (WHO Working Group, 1989). The
relative proportion of reported Class II and Class III variants was used to categorise the
severity of variants nationally (Class III variants only; minority of Class II variants, 1/3;
Class II variants common, >1/3), as shown in Panel A. The two scores were multiplied to
given an overall risk score (Panel B and C). Uncertainty in the two scores was scored and
ranked between countries (Panel D and E). (Figure from Howes etal. (2012)). (For a colour
189
190
R. E. Howes et al.
haemolysis might allow diagnostic methods to be engineered as predictors of

haemolysis, rather than a direct indicator of enzyme activity (which may not
prove to be the only determinant of haemolytic risk). The heterogeneity of
genetic variants (Fig. 4.6A,B), particularly across areas where P. vivax radical
cure is most relevant (Fig. 4.7), supports the potential benefits of improving
the diagnostic resolution and tailoring treatments to different variants. However, until the underlying haemolytic mechanisms can be understood, the
short-term primary goal must be refinement of binary diagnostics.
For individuals diagnosed as G6PD normal, a different set of considerations apply to optimising primaquine treatment options. For instance,
there is pressure to reduce treatment duration from 14days to a 7-day regimen with equivalent efficacy so as to improve compliance (Fernando etal.,
2011; Krudsood et al., 2008; Schmidt et al., 1977; Takeuchi et al., 2010).
Prerequisite to promoting this higher dosage schedule is a high sensitivity
diagnostic of haemolytic risk for G6PD deficiency.
10. CONCLUSIONS

The aim of studying G6PD deficiency in the context of P. vivax
therapy and malaria elimination is to support safe use of 8-aminoquinoline
drugs for radical cure that will enable access to effective, life-saving therapy.
At this pivotal time in malaria control, when important progress is being
made and vital funding cuts imposed (Garrett, 2012), it is more imperative
than ever to maximise efficient use of available tools. As discussed in Chapter
6 of V
olume 80, the relapsing P. vivax hypnozoite reservoir makes this parasite life-form the major challenge to patient health and malaria elimination
programmes. The only licenced drug active against these parasites is primaquine. However, widespread G6PD deficiency (estimated allele frequency
of 5.3% [IQR: 4.46.7] in declared malaria eliminating countries, Howes
et al. (2012)) and poor associated diagnostics result in primaquine being
under-used.While great caution must be taken in dealing with a potentially
haemolysing drug, informed caution may increase access to this drug.
Although a good deal is known about the molecular characteristics of
the G6PD enzyme and its clinical manifestations as favism and NNJ, for
instance, the disorders interactions with P. vivax and primaquine remain
much less well understood. This is likely to be a symptom of the higher
priority which has been placed in recent decades on therapy against the
asexual blood stages of P. falciparum (Baird, 2010), to the neglect of most
other therapeutic targets, such as P. vivax radical cure. As such, a recurring
191
theme emerging from many sections of this review is a lack of data, tools
and understanding. Prioritising these gaps to increase access to safe primaquine can be considered in three steps, though fundamental to all of
these is an understanding of how residual enzyme activity levels and genetic
variants interact with the underlying mechanisms triggering haemolysis,
thereby providing evidence that can guide rationally developed and practical solutions to the problem.
1. Improving point-of-care assessment of haemolytic risk through development of a highly sensitive, practical diagnostic which can be considered a conservative indicator of tolerance of the standard 14-day
regimen. It may be necessary to develop separate methods to adequately
diagnose deficient males and females (Peters and Van Noorden, 2009;
Shah etal., 2012). Adequate diagnostics would greatly increase access to
primaquine and could be available in the near future.
2. Extending access to primaquine by identifying dosing regimens of
reduced toxicity for G6PD deficient individuals by leveraging unexplored synergies with other drugs.
3. Developing alternative therapies (likely non-8-aminoquinolines) which
present no risk to G6PD deficient individuals.
The operational inadequacy and potentially mortal threat posed by primaquine due to G6PD deficiency, renders it unfit for purpose in endemic
zones in its current form.The dawning realization that acute P. vivax malaria
is associated with significant burdens of severe illness and death in endemic
zones and no longer misclassified as benign (Price etal., 2007) may finally
crystalise the determination of the scientific community to address this
60-year-old problem. There may be no higher priority for malaria research
than the triangular G6PD-primaquine-P. vivax problem.
If elimination is to be the focus, perspectives on future malaria therapy need
to shift away from treatment of symptomatic parasitaemia towards comprehensive treatment for all parasites and multiple life stages (Baird, 2012). Given their
unique therapeutic action, the importance of overcoming the dangers of the
8-aminoquinolines cannot be over-emphasised towards meeting this target.
ACKNOWLEDGEMENTS
The authors are particularly grateful to Lucio Luzzatto and Pete Zimmerman for valuable comments on the manuscript, and to Jennie Charlton and David Pigott for proofreading. This work was supported by a Wellcome Trust Biomedical Resources Grant
(#085406), which funded R.E.H.; S.I.H. is funded by a Senior Research Fellowship from
the Wellcome Trust (#095066) that supports K.E.B. also. A.W.S. is supported by grant
#107-13 from the Asia Pacific Malaria Elimination Network (APMEN). J.K.B. is supported
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R. E. Howes et al.
by grant #B9RJIXO of the Wellcome Trust. This work forms part of the output of the
Malaria Atlas Project (MAP, http://www.map.ox.ac.uk/), principally funded by the
Wellcome Trust, UK.
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CHAPTER FIVE
Genomics, Population Genetics

and Evolutionary History of
Plasmodium vivax
Jane M. Carlton*,1, Aparup Das, Ananias A. Escalante
*Center for Genomics and Systems Biology, Department of Biology, New York University,
New York, NY, USA
Evolutionary Genomics and Bioinformatics Laboratory, Division of Genomics and Bioinformatics,
National Institute of Malaria Research (ICMR), Dwarka, New Delhi, India
Center for Evolutionary Medicine and Informatics, The Biodesign Institute, Arizona State University,
Tempe, AZ, USA
1Corresponding author: E-mail: jane.carlton@nyu.edu
Contents
1. T he Importance of Studying Plasmodium Diversity
2. T he Evolutionary History of P. vivax
3. T he P. vivax Genome and Comparative Genomics
4. P . vivax Global Genetic Diversity and Population Structure
5. P . vivax Population Genetics in India
6. C
onclusion
Acknowledgements
References
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204
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213
215
218
218
219
Abstract
Plasmodium vivax is part of a highly diverse clade that includes several Plasmodium
species found in nonhuman primates from Southeast Asia. The diversity of primate
malarias in Asia is staggering; nevertheless, their origin was relatively recent in the
evolution of Plasmodium. We discuss how humans acquired the lineage leading to
P. vivax from a nonhuman primate determined by the complex geological processes
that took place in Southeast Asia during the last few million years. We conclude that
widespread population genomic investigations are needed in order to understand
the demographic processes involved in the expansion of P. vivax in the human
populations. India represents one of the few countries with widespread vivax
malaria. Earlier studies have indicated high genetic polymorphism at antigenic loci
and no evidence for geographic structuring. However, new studies using genetic
markers in selectively neutral genetic regions indicate that Indian P. vivax presents
complex evolutionary history but possesses features consistent with being part of
the ancestral distribution range of this species. Such studies are possible due to the
availability of the first P. vivax genome sequences. Next generation sequencing technologies are now paving the way for the sequencing of more P. vivax genomes that
will dramatically increase our understanding of the unique biology of this species.
2013 Elsevier Ltd.
203
204
Jane M. Carlton et al.
1. THE IMPORTANCE OF STUDYING PLASMODIUM

DIVERSITY

As parasite diversity is one of the fundamental factors governing
malaria transmission and immunity knowledge of parasite population
genetic structure is necessary to understand the epidemiology, distribution
and transmission dynamics of natural malaria parasite populations. This is
essential because population genetic structure can predict how fast phenotypes of interest, such as novel antigenic variants, particular relapsing
patterns, or drug resistance, arise and spread in natural populations (Brito
and Ferreira, 2011). Plasmodium vivax is a major public health challenge for
Central and South America, the Middle East, Central, South and Southeast Asia, Oceania and East Africa, where 2.85 billion people are currently
at risk of infection and 7080 million clinical cases are reported each year
(Guerra etal., 2010; Muller etal., 2009). The emergence of drug-resistant
strains and severe (sometimes fatal) disease (Kochar et al., 2009) challenges the traditional view of vivax malaria as a benign infection (Price
etal., 2009). Much about the biology, epidemiology and pathogenesis of
P. vivax that is essential for control programs remains unknown, although
it is now understood that global P. vivax populations are highly genetically diverse, and that this diversity varies greatly according to geographic
regions. It has thus become apparent that in order to achieve malaria
control and elimination targets, population genetic surveys are vital to
map the diversity and structure of local populations and to estimate the
likelihood of success and measure the outcome of malaria intervention
methods (Arnott etal., 2012).
2. THE EVOLUTIONARY HISTORY OF P. VIVAX

Early inferences about the origin of P. vivax were based on limited
evidence. An Asian origin was proposed due to the diversity of primate
malarias in Southeast Asia and their phenotypic similarity to human malaria
parasite species including P. vivax (Coatney et al., 1971). Alternatively, an
African origin was proposed based on the fixation of the FY*BES allele (ES
is an abbreviation of erythrocyte silent or Duffy negative) from the Duffy
blood group that provides resistance to vivax malaria (Miller etal., 1976),
and the existence of Plasmodium schwetzi, a putatively vivax-like parasite in
chimpanzees (Carter, 2003; Livingstone, 1984).
Genomics, Population Genetics and Evolutionary History of Plasmodium vivax
205
Nowadays, molecular phylogenies support an Asian origin for P. vivax

as the result of a host switch from a nonhuman primate (Cornejo and
Escalante, 2006; Escalante etal., 2005; Mu etal., 2005). Figure 5.1 depicts a
Bayesian phylogeny that includes only those Plasmodium lineages found in
Asian primates.The phylogeny includes many species found in Cercopithecidae (mostly macaques), P. vivax, the gibbon parasite Plasmodium hylobati,
and a recently rediscovered lineage of Plasmodium in orangutan [9]. As the
phylogeny shows, there is evidence of several host switches including one
that allowed the colonization of humans by the P. vivax lineage (Escalante
etal., 2005; Mu etal., 2005). Indeed, the diversity of nonhuman primate
malarias in Southeast Asia appears to be the result of an explosive adaptive radiation that took place between 4 to 8 million years ago (Mu etal.,
2005; Pacheco etal., 2011, 2012). It is worth noting that the Asian origin
model for P. vivax is not based on the number of Plasmodium species parasitic to primates in Southeast Asia (Coatney etal., 1971), but on the fact
that malaria parasites found in Cercopithecidae are basal in a monophyletic
group that includes P. vivax (Escalante etal., 2005). The closest known species related to P. vivax is Plasmodium cynomolgi, and these two parasites could
have diverged as early as 1.3Mya ago depending upon the molecular clock
method used and the assumptions applied (Pacheco etal., 2011, 2012).
Whereas phylogenetic evidence supports an Asian origin as the most parsimonious explanation, it is worth noting that the fixation of the FY*BES allele
in Africa does not preclude a more recent introduction of P. vivax from Asia.
Natural selection, in order to act, requires population sizes that are more likely
in agriculture-based societies (Hedrick, 2011).This seems to be supported by
the time estimates for the fixation of the FY*BES allele, timeframes that are all
consistent with a relatively late introduction of P. vivax into Africa (Hamblin
et al., 2002; Seixas et al., 2002). Regarding P. schwetzi, some considered it
related to Plasmodium ovale (Coatney etal., 1971), an assertion consistent with
recent findings of ovale-like parasites in chimpanzees (Duval et al., 2009).
P. schwetzi could simply be P. vivax found in chimpanzees (Krief etal., 2010),
since these parasites are identical to Asian isolates suggesting a recent introduction from that part of the world rather than a new species.Thus, all available evidence so far supports, or is consistent with, an Asian origin for P. vivax.
Nevertheless, the model of an Asian origin for P. vivax is still a work
in progress. New data could still change our perspective on the origin
of this parasite. Part of the problem is that phylogenetic studies are still
missing data from most Southeast Asian apes (Pacheco et al., 2012). A
possibility that should be systematically explored is whether ancestors of
206
Figure 5.1 Phylogenetic tree of P. vivax and closely related taxa from Southeast Asia.
Bayesian phylogenetic tree using complete mitochondrial genomes for parasites of
Southeast Asian primates, using parasites found in African Cercopithecidae as outgroups (P. gonderi and Plasmodium sp). This phylogenetic tree was inferred under the
general time reversible+gamma model (GTR+) model with MrBayes (Ronquist and
Huelsenbeck, 2003). The search was performed with 6,000,000 Markov chain Monte
Carlo steps and discarded 50% as a burn-in. Sampling was performed every 100 generations. Values above branches are posterior probabilities. Branch length is representative
of number of nucleotide substitutions per site, as indicated by the scale bar. Maximum
likelihood analyses yield comparable results. (For colour version of this figure, the reader
is referred to the online version of this book).
207
Southeast Asian apes could have introduced nonhuman primate parasites

into Asia. Although unlikely, such an event is still possible given that orangutans and gibbons are naturally infected by many species of malaria parasite
[1] and the fact that there was a host switch from Asian apes into Asian Cercopithecidae as evidenced by the relative position of Plasmodium inui in the
phylogeny (Fig. 5.1). Thus, an ape-driven introduction of primate malarias
into Asia cannot be completely ruled out without a comprehensive survey
of ape malarias.
The extraordinary biological diversity of these nonhuman primate
malarias closely related to P. vivax offers an invaluable resource to understand the genetic basis of the adaptations that allowed this human parasite to
successfully thrive in humans. It also provides a framework to better understand the extant population variation of P. vivax and to ascertain the genetic
bases of important biologic characteristics of this very successful group.
3. THE P. VIVAX GENOME AND COMPARATIVE

GENOMICS

P. vivax -omics has had a slow and chequered history, primarily due
to problems with obtaining sufficient material for whole genome sequencing, but also due to a lack of resources exacerbated by the perceived lesser
importance of vivax malaria (Carlton etal., 2011). However, there are now
five completely assembled and annotated reference genomes (Table 5.1),
more than is available for Plasmodium falciparum, providing a solid resource
for the malaria community. In addition, the genomes of several closely related
monkey malaria parasites are becoming available (Table 5.2), broadening the
usefulness of the P. vivax genomes and enabling comparative genomics of
the monkey malaria clade. Below we provide a historical overview of the
P. vivax whole genome sequencing projects, describe main features of the
P. vivax genome and some of the comparative genomics studies between different members of the monkey malaria clade, and finally provide a brief summary of the P. vivax transcriptome and proteome studies undertaken so far.
Some of the difficulties of sequencing P. vivax genomes are exemplified
by the first genome to be sequenced, that of the strain Salvador I isolated
from a patient from El Salvador. Initial attempts to obtain sufficient genetic
material from P. vivax-infected patients for sequencing were unsuccessful, so
an alternative source had to be pursued: the Salvador I strain of the parasite
that had been adapted to growth in New World monkeys. P. vivax is unable
to grow in Old World monkeys such as macaques, and instead must be
208
Table 5.1 Whole Genome Sequences of P. vivax, with their Country of Isolation,
Source and Data Type Indicated
P. vivax strain Country
Source
Data
References
Salvador I
El Salvador
India VII,
North
Korean,
Mauritania
I, Brazil I
IQ07
India
Peru
MonkeyReference
adapted lab
assembly
strain
and
annotation
MonkeyReference
assembly
adapted lab
and
strains
annotation
Carlton etal.
(2008)
Patient isolate Unassembled
Dharia etal.
(2010)
Bright etal.
(2012))
SA-94, SA-95, Peru

Patient isolates Unassembled
SA-96,
SA-97,
SA-98
Belem, M08, Brazil,
MonkeyUnassembled
M09, C08,
Madagascar,
adapted lab
C15, C127
Cambodia
strain, and
five patient
isolates
26 isolates
Thailand & six Patient isolates Unassembled
travellers
Neafsey etal.
(2012)
Chan etal.
(2012)
S. Auburn
etal.,
unpublished
cultivated in small, expensive Aotus and Saimiri monkeys found in Central

and South America.This adaptation to growth in a nonhuman host can take
many months, but fortunately a large number of such adapted strains are
available as part of the research resources of the Centers for Disease Control and Prevention (http://www.cdc.gov/malaria/tools_for_tomorrow/
research_resources.html), and now made available to researchers through
the malaria repository MR4 (http://www.mr4.org/).
However, no sooner were the problems with obtaining sufficient material
overcome, then issues with funding the sequencing project arose. Contrary
to popular belief, there was never a designated fund for sequencing the first
P. vivax genome; instead, U.S. Department of Defence and National Institutes of Health funds remaining from the first P. falciparum genome project
published in 2002 (Gardner et al., 2002) were reassigned. However,
these funds were depleted by 2003, and it was not until the Burroughs Wellcome
Fund rescued the project by supporting the generation of
209
Table 5.2 Whole Genome Sequences of Other Members of the Asian Old World
Monkey Malaria Clade, with their Country of Isolation and Data Type Indicated
Plasmodium Species
and Strain
Country
Data
Genome References
Plasmodium
knowlesi
H
Plasmodium
cynomolgi
Berok
Plasmodium
cynomolgi
B
Plasmodium
cynomolgi
Cambodian
Plasmodium inui
OS
Malaysia
Reference assembly
and annotation
Pain etal. (2008)
Malaysia
Reference assembly
and annotation
Tachibana etal.
(2012)
Malaysia
Reference assembly
and annotation
Tachibana etal.
(2012)
Cambodia Reference assembly

and annotation
Tachibana etal.
(2012)
India
Reference assembly
and annotation
Plasmodium gonderi
Africa
Reference assembly
and annotation
Plasmodium coatneyi
Malaysia
Reference assembly
and annotation
J. Carlton, J. Barnwell,
A. Escalante,
unpublished
A. Escalante,
unpublished
A. Escalante,
unpublished
additional parasite material that the project was restarted in late 2004. The
first P. vivax reference genome was finally published six years after the first
P. falciparum genome. In 2012, four more P. vivax reference strains were
sequenced (Table 5.1), once again using patient isolates adapted to growth in
New World monkeys (Neafsey etal., 2012). These reference genomes from
Brazil, Mauritania, India and North Korea were sequenced using next generation sequencing technology, and have been fully assembled and annotated.
There are now five reference genomes available to the vivax research community, and biological material for each of these strains is available through MR4.
An extraordinary finding from the analysis of these P. vivax genomes
is that the species exhibits almost twice as much genetic diversity than
P. falciparum (Neafsey etal., 2012). Analysis of two different types of mutation
markers (SNPs and microsatellites) and in different sequence classes (intergenic, intronic, coding etc.) indicated a globally higher genetic diversity in
P. vivax than P. falciparum, pointing to a distinct history of global colonization
compared with P. falciparum. Additional analyses also suggested a capacity for
210
greater functional variation in the global population of P. vivax. Of course,

the implications for this as regards control and eventual elimination of vivax
malaria are enormous, and these studies although still preliminary serve as a
warning that P. vivax is a very different beast from P. falciparum.
The complete P. vivax genomes have begun to be supplemented by
several unassembled and unannotated SNP-discovery sequences from
Peru, Madagascar, Cambodia and Thailand that have started the trend of
direct sequencing from patient infections (Table 5.1). These projects are
pioneering the DNA extraction methods and bioinformatic analyses that
are required to sequence parasites directly from human blood tasks complicated by the small quantities of P. vivax genetic material, mixed genotype
infections, and obtaining high quality sequence data from the highly [AT]rich subtelomeric regions of P. vivax chromosomes. In addition, reference
genomes of other members of the monkey malaria clade are becoming
available.Three strains of the monkey malaria species P. cynomolgi, the closest
living relative to P. vivax, were recently sequenced (Tachibana etal., 2012),
joining a reference genome of the more distantly related zoonosis Plasmodium knowlesi sequenced several years ago (Pain etal., 2008), and reference
genomes of P. inui, Plasmodium coatneyi and Plasmodium gonderi that are also
underway (Table 5.2).
What does the genome of P. vivax tell us about the biology of the parasite?
Briefly, since this has been covered in several reviews (see for example (Carlton
etal., in press)) as well as the original genome paper (Carlton etal., 2008), comparing the P. vivax 27Mb genome with the genome of three other sequenced
malaria parasites (P. falciparum, P. knowlesi and the rodent malaria species Plasmodium yoelii yoelii), the Plasmodium genomes were found to be similar in size
(2327Mb), number of genes (5000), gene structure (>50% contain introns),
but differ dramatically in [GC] content, repeat structure and DNA complexity. T
he overall basic predicted metabolic pathways and transporter proteins in
both P. vivax and P. falciparum were found to be very similar, including those
metabolic pathways predicted in the apicoplast such as Type II fatty acid biosynthesis, the isopentenyl diphosphate pathway, and a glyoxalase pathway, which
can potentially be mined for drug targets. Interestingly, many of the same vaccine candidate genes were found in P. vivax as in P. falciparum, where there are
1520 P. falciparum genes actively being studied as potential vaccine candidates
versus only two in P. vivax). A major difference between Plasmodium genomes
is the presence of species-specific gene families, and expansion of some gene
families in certain lineages. In the P. vivax genome, the largest is the extremely
divergent vir gene family. Members of the msp3 and msp7 are also massively
211
amplified in P. vivax, although their functions are unclear. Many more genes
coding for reticulocyte binding proteins (rbp) involved in invasion were found
in P. vivax than had originally been thought to exist, suggesting that invasion of
red blood cells by P. vivax may be more complex than previously envisioned.
Several P. vivax-specific gene families were identified, including the pvtrag gene
family that contains 36 members and localises to caveolae, part of the tubovesicular structure found in infected red blood cells. Proteins of P. vivax orthologs
implicated in drug resistance in P. falciparum were analysed using modelling and
protein structure analysis, and using these data several predictions were made,
namely that P. vivax should be more susceptible to artemisinin than P. falciparum
because of certain predicted binding residues, and that should P. vivax develop
resistance to atovaquone (used in combination therapy), the same mutations in
the cytochrome B gene may be implicated as in P. falciparum. Finally, on a chromosome scale, kilobase stretches of the P. vivax chromosomes were found to be
syntenic with other Plasmodium spp. chromosomes. From these synteny maps, it
was possible to determine that the Plasmodium common ancestor probably had
a P. vivax chromosome karyotype; and the evolutionary events that occurred
between the time of this ancestral parasite form and the current chromosome
forms of the existing species could be inferred.
More recently, a comparison of the P. vivax Salvador I genome with
three P. cynomolgi genomes and P. knowlesi has provided further insight into
the monkey malaria clade (Tachibana etal., 2012). Interestingly, despite the
close phylogenetic relationship between P. vivax and P. cynomolgi (Fig. 5.1),
the latter was found to have some P. knowlesi-specific features, such as (1)
intrachromosomal telomeric sequences (GGGTT[T/C]A) discovered in the
P. knowlesi genome (Pain etal., 2008) but absent in P. vivax; (2) an example
of a member of the P. cynomolgi cyir gene family containing a 56-amino acid
region highly similar to the extracellular domain of a molecule involved in
the regulation of T-cell function in primates (so-called molecular mimicry as
first identified in P. knowlesi); and (3) two genes similar to P. knowlesi SICAvar
genes that are expressed on the surface of schizont-infected macaque erythrocytes and are involved in antigenic variation. The genomes of P. cynomolgi,
P. vivax and P. knowlesi can almost be completely characterised by variations
in the copy number of multigene families, with copy number of multigene
families being generally greater in the P. cynomolgi/P. vivax lineage than in
P. knowlesi, suggesting repeated gene duplication in the ancestral lineage of
P. cynomolgi/P. vivax or potentially repeated gene deletion in the P. knowlesi
lineage. Finally, an analysis of orthologs between P. vivax and P. cynomolgi
revealed an extraordinary 83 genes under possible accelerated evolution but
212
3739 genes under possible purifying selection, indicating that the genome
of P. cynomolgi is highly conserved in single-copy genes when compared
with P. vivax, and emphasising the value of P. cynomolgi as a biomedical and
evolutionary model for studying P. vivax.
One disappointment from these various genome sequencing projects
of P. vivax and P. cynomolgi has been the lack of identification of a definitive hypnozoite developmental pathway or genetic determinant(s). Several
candidate genes have been proposed, for example genes annotated as dormancy-related or with the upstream ApiAP2 motifs necessary for sporozoite-specific transcription. However, the role of these genes in the relapse
phenotype is speculative, and what is really required is hypnozoite biological material with which to do transcriptome and proteome analyses.
While whole genome sequencing of P. vivax isolates is gradually
gathering steam, functional genomic studies of P. vivax have been limited. The first microarray (a 60-mer long oligoarray with 5700 probes)
developed from the Salvador I reference genome sequence was used to
describe the 48-h intraerythrocytic cycle of three P. vivax isolates from
Thailand (Bozdech etal., 2008). Strain-specific patterns of expression for
genes predicted to encode proteins associated with virulence and host
pathogen interactions were identified, and a comparison with P. falciparum intraerythrocytic expression revealed differences in the expression
of genes involved in certain cellular functions. A second array developed
using the Affymetrix platform and consisting of 4.2 million 25-bp probes
covering 5419 P. vivax genes was used to generate transcriptome data from
eight P. vivax patients from Peru (Westenberger etal., 2010), including a
sample enriched for zygotes, ookinetes and gametes, and a sporozoite sample from mosquitoes fed on an infected chimpanzee. Interestingly, large
differences in the expression profiles of asexual samples were found, with
the most pronounced differences observed in genes involved in glycolysis.
The orthologs of some genes shown to be upregulated in P. falciparum sporozoites (Le Roch etal., 2004) were found to be downregulated in P. vivax
sporozoites, and vice versa. A number of uncharacterised genes showed
substantial up-regulation of at least threefold in ookinetes. Interestingly,
gene expression of the vir gene family, was lower than for other genes,
perhaps as a consequence of genetic differences between the reference
genome Salvador I on which the array was designed, and the Peruvian
samples.
Apart from a proteomics study that used P. vivax parasites from patients
in India and identified 150 proteins expressed in the asexual blood stages
213
(Acharya etal., 2011), no large-scale proteomics studies have been published

in P. vivax. Obviously the lack of biological material is a major confounding
factor, and it is to be hoped that with the awarding of several grants from
the Bill and Melinda Gates Foundation to develop invitro culture methods
this situation will be rectified.
4. P. VIVAX GLOBAL GENETIC DIVERSITY AND

POPULATION STRUCTURE

Although Plasmodium genomics is advancing our knowledge of the
organisms complex biology, population-based investigations are still needed
to explore the extent of the parasites genetic variation, how the observed
variation is geographically distributed, and how such diversity affects the
development/deployment of new interventions aimed to control and elimination (e.g. vaccines and antimalarial drugs). Unfortunately, several circumstances limit population genetic investigations of P. vivax. The lack of
suitable invitro culturing methods for P. vivax, as mentioned above, imposes
the use of field specimens or adapted monkey strains on population studies.
While population genomic investigations are desirable in many contexts
(e.g. linkage analyses looking for gene associations to specific phenotypes),
the high frequency of multiple infections in field isolates requires well-
conceived studies in order to generate high-quality data usable by population
genetic approaches that require defined lineages or haplotypes. In addition,
population genomics demands equipment and expertise that make such
approaches difficult to scale-up for supporting routine decision-making processes required by regional or national malaria elimination efforts. However,
for many molecular epidemiologic applications, target gene approaches or
more traditional multilocus analyses are not only sufficient but more appropriate; moreover, they are within reach of most malaria-endemic countries.
Target gene approaches and/or multilocus investigations are still needed
to deal with the prevalence of multiple infections in field specimens. In
practical terms, direct sequencing of polymerase chain reaction products
can generate false consensus that affects estimates of population genetic
parameters. Multilocus approaches (e.g. microsatellites) have the same problem since lineages cannot be properly identified in parts of the processed
samples. Those problems could make molecular population studies particularly expensive and laborious for endemic countries.
Nevertheless, there have been several population-based studies using
target genes including merozoite surface protein 3 (MSP-3) (Mascorro
214
etal., 2005; Schousboe etal., 2011; Zhong etal., 2011); apical membrane
antigen 1 (AMA1) (Putaporntip etal., 2009), circumsporozoite surface protein (CSP) (Chenet etal., 2012; Hernandez-Martinez etal., 2011; Imwong
et al., 2005); MSP-1 (Imwong et al., 2005; Pacheco et al., 2007); Duffy
receptor binding domain (Ju etal., 2012; Premaratne etal., 2011); transmission blocking antigens Pvs25 and Pvs28 (Feng etal., 2011) and dihydrofolate reductase (DHFR) (Imwong etal., 2003). For most of those genes,
genetic diversity was of interest because they are considered vaccine candidates or confer resistance to certain antimalarial drugs such as antifolates.
MSP-3, however, is used as a target for genotyping given its extraordinary
diversity; unfortunately, high diversity does not necessarily make it a good
tool. Restriction fragment length polymorphism-based essays are not easily
interpretable in genetic terms beyond this seems the same or these two
are different; so given the plethora of other markers that are reproducible,
it seems inappropriate to invest in this particular approach unless it involves
sequencing. However, regardless of the fact that many of these studies have
been geographically biased (with a few exceptions), all studies indicate a
strong geographic structure. The same pattern emerges from microsatellite
markers (Gunawardena etal., 2010; Imwong etal., 2007).
Nowadays, the clearest picture of the global population genetic structure and the recent history of P. vivax populations come from the studies
of mitochondrial genomes (Cornejo and Escalante, 2006; Jongwutiwes
etal., 2005; Miao etal., 2012; Mu etal., 2005). The combined data set
includes 390 sequences and 125 haplotypes from several populations
across the P. vivax geographic distribution (Jongwutiwes et al., 2005;
Miao etal., 2012; Mu etal., 2005). The original published studies overlap
in their estimates of TMRCA (the Time to the Most Recent Common
Ancestor): Mu et al., 2005 concluded that P. vivax originated between
53,000 and 265,000 years ago, Jongwutiwes et al., 2005 estimated an
origin between 217,000 and 304,000 years ago. However, there were
differences in their sampling efforts and in the genetic patterns emerging from different geographic regions (Cornejo and Escalante, 2006)
so the combined data sets gave older times, 346,000464,000 Mya for
the entire sample. Those estimates were supported using other methods
and expanding the samples from other Asian regions, which yielded a
TMRCA of 346,000452,000years ago (Miao etal., 2012). It is important to emphasise that the information available on the geographic origin
of the isolates is still poor and the sampling has been opportunistic rather
than systematic. Thus, it is not possible to separate the effects of hidden
215
population substructure due to inappropriate sampling from the scenario

of different local dynamics. Those problems could affect our TMRCA
estimates. Nevertheless, even if we cut these estimates by half they are
still consistent with an early origin of P. vivax and its introduction into
Africa at a later time. Recent phylogeographic investigations that include
human and chimpanzee P. vivax from Africa actually show that those
lineages are identical to those found in Asia (Culleton etal., 2011; Krief
etal., 2010). However, the sampling from Africa (including the Middle
East) and India is still poor.
Another important finding derived from sequencing mtDNA is the
extensive number of unique haplotypes in several regions of the world,
e.g. Melanesia (30 haplotypes out of 73 isolates) and Southeast Asia (24
haplotypes out of 56 samples). In fact, these two regions form separated
blocks that are connected by haplotypes from the Americas (Cornejo and
Escalante, 2006; Miao etal., 2012).This pattern is consistent with an ancient
population expansion (Miao etal., 2012). Even further, the excess of rare
mutations within some (but not all) of the subpopulations suggest that different demographic processes took place within each of the proposed subpopulations. However, an alternative explanation is that this excess of rare
mutations is the result of poorly sampled subpopulations that were pooled
together (Cornejo and Escalante, 2006).
Information derived from the mtDNA studies will provide a framework to population genomics investigations in the context of defining
areas that require special attention. Although population genomics actually
could unveil complex demographic processes easier, given the richness of
the data, it is not immune to sampling bias. Thus, there is much to learn
from the mtDNA studies regarding the events that need to be taken into
account by population genomic investigations. In addition, it is important
to highlight that the integration of broad population genomic studies (e.g.
global genetic diversity or linkage analyses looking for gene candidates)
with localregional gene target or multilocus investigations, will provide
most needed information about how the parasite populations disperse and
change at the different spatialtemporal scales required to coordinate elimination programs.
5. P. VIVAX POPULATION GENETICS IN INDIA

The extensive polymorphism found in loci associated with P. vivax
adaptive traits, such as those coding for surface antigens and drug resistance
216
(Cui et al., 2003), reflects the combined effects of the parasites population history and selective constraints imposed by host immunity and
drug usage (Escalante etal., 2004). Thus, these loci are less informative for
determining P. vivax population structure, highlighting the need for neutral and nearly neutral markers which would allow for unbiased estimates
of genetic variation, population structure and gene flow in natural parasite
populations. Moreover, such kinds of inference can be drawn better from a
country setting with variable P. vivax malaria epidemiology, such as exhibited in India.
India is one of a handful of countries known to have widespread P. vivax
infections. P. vivax used to be the principal cause of malaria in India, with
few cases of P. falciparum infection. However, in recent years, P. falciparum has
increased in India and the rates of infection for these two parasite species
have equalised (Das et al., 2012; Singh et al., 2009). At present, although
there are distinct geographic patches with high or low endemicity of both
malaria species, P. vivax is widespread and present over almost the whole
country (Joshi etal., 2008). Such epidemiological features increase the complexity of P. vivax and have burdened the country with high morbidity and
socioeconomic losses.
A majority of P. vivax population genetic studies in India have employed
antigenic genes, such as AMA1 (Rajesh etal., 2007; Thakur etal., 2008a),
MSP1 (Kim et al., 2006; Thakur et al., 2008b), MSP3 (Kim et al., 2006;
Prajapati etal., 2010), and CSP (Kim etal., 2006), and these studies generally reported high genetic polymorphism at these loci. However, since these
markers are antigenic genes of P. vivax that are subjected to natural selection,
population genetic structure (which is mainly influenced by demography)
could not be well understood from these studies. In order to overcome the
limitations imposed by these genetic markers, 12 multilocus, putatively neutral markers in noncoding regions of the nuclear genome were developed
recently (Gupta etal., 2010). These were used to infer population genetic
structure and demographic history of Indian P. vivax isolates using representative samples from low and high endemic regions of India in total 10 different populations. Genetic diversity was shown to vary across all 12 neutral
markers in Indian populations, and in addition, introns appeared to contain
less diversity than intergenic regions, which might be an intrinsic property
of P. vivax (Feng etal., 2003; Gupta etal., 2010). Furthermore, since the 12
markers came from a contiguous region of one P. vivax chromosome, it was
possible to infer changes in genetic diversity across the region. A significant
drop in nucleotide diversity was observed at two loci, which may represent
217
interesting regions containing genes necessary for parasite survival and/or

reproduction (Gupta etal., 2012).
How does the population genetics of P. vivax in India fit into the global
population structure of the species? A few samples of Indian P. vivax have
been included in global studies using microsatellites as genetic markers
(Imwong etal., 2006, 2007). In general, relatively less genetic differentiation
between Indian P. vivax population samples was observed based on the presence of common alleles/genotypes (Joshi etal., 1989, 2007). Using these
observations, Indian P. vivax subpopulations were generally considered less
or loosely structured (Joshi etal., 2008). Using SNP markers, Gupta etal.,
2012 found that Indian P. vivax exhibits higher overall nucleotide diversity
than other global populations and the diversity was maintained within population samples but moderate amount of genetic differentiation could be
seen between population samples (Gupta etal., 2012). In particular, Bayesian genetic cluster analysis (implemented in the program STRUCTURE
(Pritchard etal., 2000)) grouped 10 population samples into three clusters
based on the proportion of the genetic ancestries in each population. However, the pattern of clustering does not correlate with sampling locations in
India, confirming the fact that Indian P. vivax are loosely structured (Joshi
et al., 2008). Furthermore, analyses of past demographic events indicated
reduction of population size in a majority of population samples, but when
isolates from all the 10 regions were considered as a single population, the
data fit a demographic equilibrium model (Gupta etal., 2012).The observed
population genetic features of P. vivax point to a complex evolutionary history for this parasite in India; however, it seems likely that Indian P. vivax is
part of the ancestral distribution range of this species.
Interestingly, the reduction in population size of P. vivax in specific
Indian areas has been found to be correlated with a higher prevalence of
the FY*A allele of the human Duffy gene (known to confer some degree of
resistance to vivax malaria (King etal., 2011)), and with a lower prevalence
of P. vivax (specifically in the northeast, east and central Indian regions)
than in other Indian regions (Chittoria etal., 2012). The distribution of the
FY*A allele also correlated with population coordinates in India and was
clinaly variable, indicating malaria-influenced evolution of protection in
Indians through adaptive evolution of the Duffy gene by natural selection
(Chittoria etal., 2012). Such genetic, environmental and epidemiological
correlations underscore the need for P. vivax population genomics and associated human genetic studies if we are to understand evolution of host
parasite interactions in India.
218
6. CONCLUSION

P. vivax has a broad geographic distribution with an extraordinary phenotypic diversity exhibited in its life cycle (e.g. periodicity and
morphology (Coatney et al., 1971)). Unfortunately, there are serious
gaps in our basic biological knowledge concerning the parasite that will
delay its elimination (Mueller et al., 2009). One such gap is our limited understanding of the genetic diversity observed from extant P. vivax
populations. Despite stalwart efforts, we are just starting to understand
the diversity and evolutionary history of P. vivax. For example, newly
available whole genome data from four P. vivax genomes shows that it
has high levels of genetic polymorphism, gene duplication events, and
repetitive tandem repeats in its proteins (Neafsey etal., 2012). Whether
such genetic polymorphism is maintained by positive selection or is
the result of complex demographic processes is a matter that requires
further investigation. Whole genome sequencing of isolates directly
from patients paves the way for such deeper analysis of genetic variation
worldwide.
The fact that this human malaria parasite originated as part of a complex chain of evolutionary events involving host switches and a rapid species radiation with astonishing phenotypic differences, provides researchers
with an extraordinary resource to better understand the origin of molecular
adaptations in malaria parasites. This opens unique opportunities to both
evolutionary biologists interested in understanding the dynamic of host
parasite interactions and biomedical researchers working on vivax malaria
pathogenesis. Understanding the key molecular adaptations that allow this
parasite to flourish in the human host will provide new targets for intervention. However, whereas population genomics will provide the framework to ascertain the extent of this parasite genetic diversity, well-defined
field investigations are still urgently needed in under to understand the
fine details of this parasites epidemiology and ecology. Such local and/or
regional information is essential for deploying and evaluating intervention
strategies.
ACKNOWLEDGEMENTS
J.M.C. is supported by NIH/National Institute of Allergy and Infectious Diseases award
U19AI089676, and by an NIH/Fogarty International Center Global Infectious Disease
research training grant D43TW007884. A.D. is supported by the Indian Council of Medical
219
Research and the Department of Biotechnology, Government of India, New Delhi. A.A.E.
is supported by NIH/National Institute of General Medicine award R01GM080586. The
content is solely the responsibility of the authors and does not necessarily represent the official views of the FIC or NIH. This paper bears the NIMR publication committee clearance
number 27/2012.We thank Dr Steven Sullivan for critical reading and editing of this chapter.
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14191425.
CHAPTER SIX
Malariotherapy Insanity at the

Service of Malariology
Georges Snounou*,,1, Jean-Louis Prignon*,,
*Inserm, UMR-S
945, Paris, France

de Mdecine Piti-Salptrire, Universit Pierre et Marie Curie-Paris 6, CHU Piti-Salptrire,
Paris, France
Facult de Mdecine Paris 5, Universit Ren Descartes-Paris 5, CHU Necker-Enfants Malades, Paris, France
1Corresponding author: E-mail: georges.snounou@upmc.fr
Facult
Contents
1.
2.
3.
4.
Introduction
T he Era of Malariotherapy
T he Practice of Malariotherapy
M
alariotherapy and Malariology
4.1. S tate of Malariology in the Early 1920s
4.2. L imitations to Malariological Observations
4.3. T he Malariologists of Malariotherapy
5. M
alariotherapys Major Contributions to Malariology
5.1. T he Demise of the Unicity Theory
5.2. T he Diversity of Malarial Parasites
5.3. T he Characteristics of Naturally Acquired Immunity
5.4. T he Natural Course of the Malaria Infection
5.5. T he Hidden Cycle
5.6. E stablishment of Laboratory-Bred Anopheline Colonies
5.7. T he Dynamics of Transmission to Anophelines
6. L essons from Malariotherapy: Caveats and Current Relevance
7. C
onclusions
References
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Abstract
From the early 1920s until the advent of penicillin in the mid 1940s, a clinical course of
malaria was the only effective treatment of general paresis, a common manifestation of
tertiary syphilis that was nearly always fatal. For a number of reasons, Plasmodium vivax
became the parasite species most often employed for what became known as malariotherapy. This provided an opportunity, probably unique in the annals of medicine, to
observe and investigate the biology, immunology and clinical evolution of a dangerous human pathogen in its natural host. There is little doubt that the lessons learned
from these studies influenced the malaria research and control agendas. It is equally
true that over the last 40 years, the insights afforded by malariotherapy have remained
2013 Elsevier Ltd.
223
224
Georges Snounou and Jean-Louis Prignon
largely undisturbed on the dusty shelves of institutional libraries. In this chapter,

we broadly review the published data derived from malariotherapy, and discuss its
relevance to current challenges of P. vivax epidemiology, immunology and pathology.
1. INTRODUCTION

Towards the close of the nineteenth Century, malaria, tuberculosis
and syphilis were the three infectious diseases most feared by humankind.
At that time all three were globally distributed. Syphilis, a sexually transmitted disease whose dissemination was only checked by morality, was the least
prevalent. The highly contagious tuberculosis thrived on poverty especially
in densely populated areas. There was no effective cure for either disease.
Malaria, though recorded in all climes, was primarily a disease of warm
countries. Its prevalence was such that only few would remain free of infection while residing in the humid tropical and subtropical areas. Malaria
could be cured by quinine, a remedy that emerged from Spanish-colonised
South America in the middle of the seventeenth century. However, quinine
supplies were limited and restricted to a minority who could afford it.
The discovery of the causative agents of malaria (Plasmodium) in 1880 by
Charles Louis Alphonse Laveran, that of tuberculosis (Mycobacterium tuberculosis) in 1882 by Robert Koch, and that of syphilis (Treponema pallidum) in
1905 by Fritz Richard Schaudinn and Erich Hoffmann, opened the way for
scientific investigations aimed at finding a means to cure, prevent and eventually eliminate these diseases. Research was intense and it was conducted independently on each of these three biologically and clinically distinct pathogens.
There was, however, one extraordinary period where the paths of malaria
and syphilis scientists and clinicians converged, namely the 40 or so years during which induced malaria became the standard treatment of neurosyphilis.
Malariotherapy, as it became known, is remarkable for many reasons. From a
medical point of view, it is a unique example of one pathogen being used to
reverse the pathology caused by another. Curiously, few if any of the syphilologists or the malariologists switched specialities. Whereas malariotherapy
added little to the knowledge of the treponemal infection, it provided the
bulk of our knowledge on the natural course of clinical malaria and substantial
insights into the biology of the life cycle of the malaria parasites of humans.
Given the unique value of the observations to malariology, it is regrettable that
the publications on the subject are, to say the least, poorly known or explored.
The bulk of the relevant observations were published between 1920
and 1950, often in journals that are now difficult to access, and most are
Malariotherapy Insanity at the Service of Malariology
225
not indexed in the usual databases. Relatively few books were devoted to
malariotherapy and its techniques (Gerstmann, 1928; Kupper, 1939; Leroy
and Mdakovith, 1931; Rudolf, 1927; Shute and Maryon, 1966); and it is
likely that others escaped our notice. Chapters dealing with this treatment
appeared in many of the syphilology books of that time. To date we have
gathered about 600 primary publications and we estimate that at least half as
many remain to be uncovered. In this article, we wish to provide a historical
perspective of the era of malariotherapy, and to present an overview of the
rich advances it afforded malariology. We will finally discuss their relevance
to current questions, in particular those related to P. vivax, the parasite that
was most often used for malariotherapy.
2. THE ERA OF MALARIOTHERAPY

The syphilitic origin of the general paralysis of the insane (GPI), first
described in 1798 by Haslam in an inmate of Bethlehem Hospital in London, was suggested in 1822 by Bayle in Paris (Anonymous, 1922). The 300
years gap between the introduction and spread of syphilis in Europe and the
hypothesis of Bayle is not only due to the fact that GPI develops in some but
not all syphilitics, but also that it does so only after many years of latency that
follow the relatively closely spaced localised primary and systemic secondary
episodes of the infection. Moreover, in tertiary syphilis, T. pallidum can affect
different organs, though most often the ascending aorta or the central nervous
system. In those that develop neurosyphilis, a third remains clinically silent
while the remainder progress to primary optic atrophy, tabes dorsalis or GPI
(also known as progressive paralysis or dementia paralytica). Patients in who
GPI declares, deteriorate inexorably and few survive beyond 4 to 5 years after
onset, though temporary remissions occur in some. The symptoms, which
include partial paralysis, loss of memory and reason, delusions, hallucinations,
and aphasia, remove the patients from normal society into the care of mental
institutions. In the early decades of the twentieth century, patients with GPI
accounted for 1020% of the inmates in mental institutions. The fact that
persons who had syphilis had to live for years under the palpable threat to
develop GPI, from which they were then condemned to a slow descent into
wretchedness before death, amply justified the view that GPI was the most
terrible of all human diseases. By the turn of the twentieth century, GPI
patients, their families and the clinicians who cared for them were prepared
to endure heroic treatments, principally injections of toxic formulations of
mercury, bismuth or arsenic, in the hope of bettering their fate.
226
The discovery of malariotherapy was primarily due to the singlemindedness of one person, the Austrian psychiatrist Julius Wagner-Jauregg
(18571940) (Fig. 6.1), and indirectly to the First World Wars campaign
in the Balkans. The details are best related by Wagner-Jauregg (1946) in a
monograph he composed in 1935 but that remained unpublished until it
was translated by Walter Bruetsch in 1946. In the nineteenth century, many
psychiatrists espoused the view that a bout of fever can improve mental disorders, a notion prevalent since Hippocratic times. In 1887,Wagner-Jauregg
proposed to infect patients by erysipelas or by malaria to obtain such febrile
episodes. After trying erysipelas unsuccessfully (no fever was obtained), he
employed tuberculin but this was stopped in the face of objections from
the medical community. Wagner-Jauregg was eventually presented with an
opportunity to try malaria when a P. vivax-infected soldier returning from
the Balkans in 1917 was admitted by mistake to Wagner-Jaureggs psychiatric ward in Vienna; a gap of 30 years between conception and execution. Nine paretics were infected in that summer (Fig. 6.1). They were then
followed up for 1year before the results were published. Of the nine, six
had improved significantly, an outcome so encouraging that by 1922 more
than 200 GPI cases had been treated (Wagner-Jauregg, 1922). Of these, an
astounding 50 patients were effectively cured (including three of the nine
originally inoculated in 1917), in that they resumed their erstwhile life and
activities. Many more had partial remission or saw the progression of their
symptoms halted. Unlike his earlier publications, Wagner-Jauregg published
these results in an American journal in English rather than in German or
Austrian journal in German.Thus, the world at large was finally made aware
of the success of Wagner-Jaureggs treatment.
Given GPIs dire prognosis, this was a momentous discovery. Malariotherapy was immediately tried elsewhere with a rate of success (Delgado,
1922; Grant, 1923; McAlister, 1923; Moll, 1924; OLeary et al., 1926;
Worster-Drought and Beccle, 1923) that ensured that it became the most
favoured standard treatment of GPI. Many tens of thousands were treated
with malaria (often supplemented with the traditional heavy metal-based
therapies) and generally, nearly half could be expected to resume normal
life or improve significantly, while only one in five failed to respond (Austin
et al., 1992; Chernin, 1984). It was not surprising that Wagner-Jauregg
was promptly rewarded with the Nobel Prize in 1927. The dominance of
malariotherapy waned after the arrival of the antibiotic penicillin in the late
1940s, though its practice persisted in some institutions until the late 1970s
(Maurois etal., 1979; Ungureanu etal., 1976).
Figure 6.1 The proponent of malariotherapy, Wagner-Jauregg, kindly acceded to a

request by Shute to re-enact the first inoculations of P. vivax into patients suffering from
neurosyphilis. The photograph, dated December 1937(4?), has been reproduced from
the preface of Shute and Maryons book in which they distilled the wealth of practical knowledge gained over many decades of malaria investigations, principally at the
Horton Mental Hospital (Shute and Maryon, 1966). Yorke (England) was one of the first
malariologists outside Germany who exploited malariotherapy to study malaria parasites. James (England), Ciuca (Romania) and Boyd and Young (United States) were the
first directors of the most important, productive and longest running malariology units
associated with the practice of malariotherapy.
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3. THE PRACTICE OF MALARIOTHERAPY

For the syphilologists, malariotherapy was most effective when the
patient experienced at least 815 malarial distinct paroxysms after which
the malarial infection was terminated. Generally, improvements in the clinical signs related to GPI clinical were only observed weeks or months following the malarial episode. In patients where no such improvements could
be noted, further courses of induced malaria are attempted. The likelihood
of cure and the magnitude of any remission significantly increased when
malariotherapy was administered early in the course of GPI.
For the clinician, the Hippocratic primum non nocere was pre-eminent.
First, in a nonimmune person, malarial paroxysms, irrespective of the
infecting Plasmodium species, constitute a serious physical and physiological
assault. Thus, only those paretics who were in relative good health and of
sufficiently strong constitution were selected for malariotherapy. They were
then monitored closely during their malarial infection and any sign of clinical severity, hyperpyrexia, or parasite burdens exceeding 100,000 parasites
per microlitre of blood precipitated immediate administration of quinine,
the only antimalarial available at that time. Depending on the condition of
the patient, the dose of quinine was varied to effect rapid eradication of the
parasites or to dampen the infection and alleviate symptoms temporarily. Of
the three species known at that time P. vivax was the species most employed
for malariotherapy, principally because it was relatively benign and was more
sensitive to quinine than Plasmodium malariae. The use of Plasmodium falciparum was generally avoided because of the risk of sudden and unpredictable shift to life-threatening clinical severity, though it was used to treat
persons found to be insusceptible to infection with P. vivax.
Second, some methods of inducing the malaria infection were not
without danger. The inoculation of infected blood was the most practical; typically, a few millilitres of infected blood were injected subcutaneously or intramuscularly. Some workers preferred intravenous inoculation
because it reduced the prepatent period to a few days (as opposed to 1
to 2weeks), but this carried the risk of introducing blood clots into the
recipients circulation. The major disadvantage of using infected blood
was the potential to transfer unwanted pathogens from donor to recipient. Given that the donors were predominantly GPI patients undergoing malariotherapy, there was justifiable unease that the recipient could
become infected with other infectious agents or be re-infected with
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T. pallidum, the pathoge responsible for initiating malariotherapy in the

first place. In some centres malariotherapy was initiated by the bite of
infected mosquitoes, but for many the expenditure needed to maintain
anopheline colonies were prohibitive.
Finally, from a public health standpoint, it was important that the malaria
infections induced in the inmates should not spread unintentionally to others in the wards, or to those outside the walls of the institution where
malariotherapy was provided. This concern and those above were raised
early in the era of malariotherapy, and in some countries official guidelines
for the employment of malaria, such as those drafted in 1924 in the United
Kingdom (Dickinson, 1924), were drawn.
4. MALARIOTHERAPY AND MALARIOLOGY

4.1. State of Malariology in the Early 1920s
Intermittent fevers (the ague, or malaria) had long been recognised and
equally feared by the common inhabitant and the military, and thwarted
many an effort to expand into and colonise vast tracts of the world. The
discovery of the causative agent in 1880 and then of the method of their
transmission in 1898 (independently by Ronald Ross and Patrick Manson,
and the Italian workers Giovanni Battista Grassi, Amico Bignami and
Giuseppe Bastianelli) were followed by a plethora of important epidemiological, clinical and biological observations. The wealth of knowledge
thereby acquired sustained the hope, for the first time in history, that the
strategies to control and eventually eradicate malaria were at hand. By the
1920s it had become clear that the impeccable logic of an all-out attack
on mosquitoes, or of quinine mass administration (championed by Ross
and Koch, respectively), was all too frequently ineffective at implementation. It became increasingly clear that much needed to be learnt about
the natural history of malaria if effective control measures were to be
devised. The only experimental models known then were avian malarias,
but their relevance to human malaria was dubious. On rare occasions,
experimental infections of a small number of healthy volunteers were
conducted to provide a definitive answer (for example Blacklock and
Adler, 1922; Mitzmain, 1916c). For the most part malariologists relied
on clinical and epidemiological observations. However, it was often difficult to draw any firm conclusions because the subjects (or mosquitoes)
were selected opportunistically and mainly without knowledge of their
prior malaria history. The practice of malariotherapy provided a unique
230
opportunity for malariologists to decipher behaviour of the parasite in its

hosts under scientifically controlled conditions.
4.2. Limitations to Malariological Observations

Whereas it is true that large numbers of paretics were infected with malaria
parasites in a large number of widely situated institutions, the opportunities
to derive new knowledge on the malaria parasites were limited by practical
and ethical considerations.
First, malariotherapy was a major and potentially dangerous clinical procedure that was primarily administered by syphilologists in specialised wards
where the presence of a malariologist was considered surplus to requirement. It was not too difficult to procure parasites, generally from locally
infected patients, from persons who acquired it in the tropics, or from other
clinics. Usually, a relatively mild P. vivax highly susceptible to quinine was
selected, and was then maintained from one patient to the other. Malariotherapy patients were closely monitored (Sweeney, 1929), but the detailed
data collected were principally used for the day-to-day management of the
malaria infection (history of the parasite strain, dose and mode of inoculation, prepatent period, parasitaemia, temperature charts, clinical signs, haematological changes, etc.). This information was often lost to malariologists.
It appeared only infrequently and parsimoniously in the publications reporting treatment outcome.Thus, observations of interest and value to malariology were only made in very few of the institutions where malariotherapy
was practiced.
Second, the type of experiments that a malariologist might have
wished to conduct was ethically restricted. The selection of the patient
for malariotherapy and the decision to terminate treatment were the preserve of the clinicians and/or the syphilologists in charge. Malariologists
selected post hoc those patients whose induced fever history best served
to probe a particular aspect of malaria, or they waited until one suitable
for this purpose became available. The ethics of malariotherapy and its
exploitation by malariologists might be considered somewhat dubious
when viewed through the prism of the ethical rules that regulate todays
experimentation on human subjects. This point was discussed cogently
in an introduction to a series of articles that analysed retrospectively data
acquired during the malariotherapy era (Collins and Jeffery, 1999a, 1999b,
1999c, 1999d). It was clearly concluded that there was no evidence that
the patients were exploited or that the standards for informed consent of
the day were violated (Weijer, 1999).
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4.3. The Malariologists of Malariotherapy

The malariotherapy clinics to which malariologists were associated for sustained periods were few. Nonetheless, it was this tiny group of malariologists who provided the lion share of the new knowledge on malaria, which
many considered to have vastly exceeded all that was learnt in the 40years
since Laverans discovery of Plasmodium.
In England, malariotherapy was adopted very soon after the publication of Wagner-Jauregg (1922). Starting in June 1922, Warrington Yorke of
the Liverpool School of Tropical Medicine and Hygiene (Fig. 6.1) was the
first to derive malariological knowledge from observations on GPI patients
inoculated with malaria at the County Mental Hospital of Whittingham.
Although his work in this domain did not extend beyond a few years, it
clearly demonstrated the value of malariotherapy to malariology (Yorke,
1925, 1926; Y
orke and Macfie, 1924a, 1924b;Yorke and Wright, 1926).
The officials, who administer the Acts relating to the care of mental
patients in England, took the view that there were serious ethical objections to inducing malarial attacks by the direct inoculation of blood from
patient to patient. Malariotherapy for GPI patients and those suffering
other mental diseases was to be initiated by the bites of infected mosquitoes. Sydney Price James (Fig. 6.1), an eminent malariologist at the Ministry of Health, was charged with producing infected mosquitoes that could
then be dispatched to mental hospitals elsewhere in the world. Thus, the
Mott Clinic for Malaria Therapy was born at the Horton Mental Hospital.
Directed first by James and then by John Alexander Sinton, Gordon Covell
and finally Percy Cyril Claude Garnham, the Mott Clinic remained active
for 40years (19231973) and 17,000 patients underwent malariotherapy
in its wards. The success and fame of this clinic was in no small part due
to technical prowess and the biological insights of Percy George Shute
(Fig. 6.1) and his companion Marjorie E. Maryon, both of who worked
there for more than 40years. The contribution of the Horton workers to
malariology is inestimable as is evident by a selection of their published
observations (Covell, 1956; Covell and Nicol, 1951; Covell etal., 1949a,
1949b; Garnham etal., 1975; James, 1926, 1931, 1934; James and Ciuca,
1938; James and Shute, 1926; James etal., 1927, 1932a, 1932b, 1936; Shute,
1940, 1946, 1951, 1958; Shortt and Garnham, 1948; Shute and Covell,
1967; Shute and Maryon, 1948, 1963, 1968; Shortt et al., 1948a, 1976;
Sinton, 1938, 1939, 1940a, 1940b; Sinton etal., 1939a, 1939b; Ungureanu
etal., 1976).
232
In Romania, Mihael Ciuca (Fig. 6.1) initiated in 1926 an impressive series

of investigations into malaria parasites in a number of malariotherapy clinics
that continued for 50years. The breadth and significance of these studies is
illustrated by a selection of their published observations (Ballif etal., 1963;
Ciuca et al., 1928, 1930, 1934, 1937a, 1937b, 1937c, 1943, 1947a, 1947b,
1962, 1964a, 1964b; Lupascu etal., 1967, 1968; Ungureanu etal., 1976).
In the United States of America, two centres for malariotherapy boasted
an associated malariology unit. The first, the National Institute of Healths
Williams Malaria Research Laboratory, was based at the South Carolina
Mental Hospital at Colombia, South Carolina. The malariological research
was initiated by Bruce Mayne, a malaria expert at the United States Public
Health Service, and was considerably expanded by Martin Dunaway Young
(Fig. 6.1), who joined this laboratory in the late 1930s as a junior zoologist,
becoming its director from 1937 to 1961.The data generated from this unit
are of the highest interest (Coatney and Young, 1942; Coatney etal., 1950a,
1950b; Cooper etal., 1950; Ehrman etal., 1945; Mayne, 1932a, 1932b, 1933,
1937; Mayne and Young, 1938; Y
oung, 1944; Young and Burgess, 1947;
Young and Eyles, 1949; Y
oung etal., 1940a, 1940b, 1945, 1947). In the late
fifties, Geoffrey M. Jeffery, who also worked at Milledgeville State Hospital,
Georgia, and William E. Collins joined the team, and expanded the malariological observations (Eyles and Jeffery, 1949; Jeffery, 1952, 1954b, 1956,
1958, 1960; Jeffery etal., 1950, 1952, 1954a, 1956, 1963; Jeffery and Eyles,
1955; Young etal., 1955). These two outstanding scientists are likely to be
among the last, if not the only, malariologists still alive who had participated in administering malariotherapy to neurosyphilitics. Moreover, they
still regularly publish remarkably informative retrospective analyses of the
data gathered from the records that were kept from the patients that were
treated (Collins and Jeffery, 1999a, 1999b, 1999c, 1999d, 2002, 2003, 2005,
2007; Collins etal., 2003, 2004a, 2004b, 2009, 2012; Diebner etal., 2000;
Jeffery, 1966; McKenzie etal., 2001, 2002a, 2002b, 2007; Molineaux etal.,
2001, 2002; Simpson etal., 2002).
The second, the Station for Malaria Research, funded by the Rockefeller Foundation, was based in Tallahassee, Florida in association with the
Florida State Hospital. The considerable scope and the excellence of the
research carried out by Mark Frederick Boyd (Fig. 6.1) and his colleagues,
most notably Stuart F. Kitchen, can be appreciated in their prolific publications (Boyd, 1932, 1935, 1938, 1939, 1940a, 1940b, 1940c, 1942a, 1942b,
1944, 1946, 1947, 1949a, 1949b; Boyd and Coggeshall, 1938; Boyd and
Kitchen, 1936a, 1936b, 1936c, 1937a, 1937b, 1937c, 1937d, 1938a, 1938b,
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1938c, 1939, 1943, 1944; Boyd and Matthews, 1939; Boyd and StratmanThomas, 1932, 1933a, 1933b, 1933c, 1933d, 1934a, 1934b, 1934c; Boyd
etal., 1934, 1936a, 1936b, 1936c, 1938a, 1938b, 1939, 1941; Kitchen, 1938,
1949c; Putnam etal., 1947).
5. MALARIOTHERAPYS MAJOR CONTRIBUTIONS

TO MALARIOLOGY

As we delved into the malariotherapy literature, it became increasingly evident that a review embracing all the myriad observations, the novel
insights to which they led, and their impact on the field of malariology,
would in equal measure overwhelm both authors and readers. We have
opted to highlight the more important malariological advances consequent
to the practice malariotherapy, many of which could not have been made
otherwise. We limited ourselves to the biological aspects of the infection,
leaving discussion of equally important observations on various aspects of
chemotherapy including drug discovery to another occasion. We sincerely
hope that this will encourage the reader to peruse the original articles,
many of which are now available electronically, in order to appreciate the
full extent of these fascinating and unique observations.
5.1. The Demise of the Unicity Theory

It is an oft forgotten fact that malariologists in the early 1920s were still debating whether malaria was due to one or more species of Plasmodium. Laveran,
the discoverer of Plasmodium, proposed that there was only one species of
malaria parasite, but that it was capable of changing its morphology and clinical course depending on factors related to the state of the person it infects
(Laveran, 1890). This notion, termed the unicity theory, was contradicted by
the careful observations of Italian researchers in the late 1880s of parasites
in untreated malaria, which led them to conclude that there were three distinct species (P. falciparum, P. vivax, P. malariae). Laveran steadfastly maintained
his view to his last days in 1922; demonstrating that a professorship at the
Institut Pasteur, a fellowship of the Royal Society, and a Nobel Prize can
confer plausibility but not validity to a theory. The observations from malariotherapy, where each of the three Plasmodium species invariably maintained
their distinctive biological and morphological features in both vertebrate and
insect hosts, laid the unicity theory to rest. This became the standard test to
validate a species, for example Plasmodium ovale (James etal., 1932a) a parasite
first described in 1922 that many argued was merely a P. vivax variant, or to
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disprove its existence as was the case for P. falciparum quotidianum, an aestivoautumnal parasite with a 24-h periodicity (James etal., 1932b).
5.2. The Diversity of Malarial Parasites

The idea that within a given species there exists races or varieties that breed true
and that are distinguishable from each other had long been accepted. This was
after all the bedrock of the Darwinian theory of evolution.Whereas it was a relatively simple matter to investigate diversity in multicellular free-living organisms where individuals or groups can be observed in isolation, it was particularly
challenging to observe unicellular parasitic pathogens in their hosts. Nonetheless, by the early 1920s it was clear to all observers (who did not subscribe to
the unicity theory) that the parasites within each of the Plasmodium species were
not homogeneous. The differences most frequently noted were based on the
morphology of the blood stages, and observations of peculiar forms prompted
some to propose new species, such as Plasmodium tenue and Plasmodium perniciosum, two parasites that resemble P. falciparum (Russell, 1928). Otherwise, clinical
observations indicated differential susceptibility to quinine for some strains and
suggested that others caused specific pathologies (such as blackwater fever or
hyperpyrexia). However, in the absence of experimental data, the nature of the
diversity in malaria parasites remained a matter of debate, often restricted to
opinion and speculation.
Malariotherapy provided the means to gather a wealth of experimental data,
which by the mid 1930s irrevocably established that within each species of
malaria parasite there are races or strains distinguishable by their natural parasitological and clinical course, infectivity, antigenic content, and susceptibility to drug
treatment. The data clearly did not encompass the rich diversity of the malaria
infection in the varied endemic zones, in that observations were predominantly
limited to infections of a specific subset of mostly malaria-nave subjects with
a rather small and select number of parasites strains. Nonetheless, the data were
sufficient to elaborate a conceptual framework of the malaria infection incorporating the diversity within each parasite species. In the next few sections, we will
briefly describe the salient discoveries stemming from malariotherapy that made
this possible.We strongly recommend, and gratefully acknowledge to have been
inspired by, an excellent recently published essay on the evolution of the notion
of strain for malaria parasites (McKenzie etal., 2008).
5.3. The Characteristics of Naturally Acquired Immunity

The fact that a form of immunity to malaria could be acquired had been
suspected long before the discovery of Plasmodium; but it was only with
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the advent of accurate microscopic diagnosis of malaria (and measurement

of spleen rates) that it became possible to investigate how this immunity is
acquired. In the absence of animal models, the investigations were limited to
epidemiological observations. Building on the pioneering observations of
Koch (1901) and Schffner (1938), Christophers conducted a seminal study
of hyperendemic malaria in Singhbhum District (Bihar State, India) and
one of his principal conclusions sums up the state of knowledge at that time:
These results show that there is a definite acquired immunity in malaria,
evidenced by a restricted numerical prevalence of parasites in the blood and
the ability of the individual to live infected, but free from sickness, under
conditions that to the new comer (or newly born) means intense, almost
continuous heavy infection. (Christophers, 1924).
The efficacy of malariotherapy relied on producing paroxysms, and it was
natural to attempt inducing a second malaria infection should a first malaria
episode fail to improve the mental state of the patient. It quickly became
apparent that this was easier said than done. The authors of one of the
first reports of reinoculation (conducted with the same strain 620months
after the first infection) were surprised that this failed to produce any clinical signs in 14 patients, though all developed a patent asexual parasitaemia
(Nicole and Steele, 1926). At the same time, the observations of Ciuca in
Romania confirmed and added to those of Yorke, when he demonstrated
that the immunity acquired to infection by one parasite species does not
protect against infection or disease by another (Ciuca et al., 1928). Back
in England, James added to these observations by showing that the solid
immunity acquired against a homologous strain is only partially efficient at
protecting against challenge with a heterologous strain (James, 1931). Across
the Atlantic, Boyd (1947) and others significantly added to the knowledge
of acquired immunity against malaria in humans. Thus a new dimension,
the immunological, was added to the concept of strains, races or varieties in malaria parasites. We will burden the reader but little more on this
topic, as immunity is discussed in another article of this series. We strongly
recommend a recent review where the contributions of induced malaria
in humans to the acquisition of immunity are discussed with insight and
elegance (McKenzie etal., 2008).
Briefly, the principal conclusions reached from years of induced infections
in neurosyphilitics were that the immunity acquired against one particular
strain can eventually protect against challenge with another strain, but the
magnitude of this protection (clinical and parasitological) is inversely proportional to the antigenic distance between these strains. On the other hand,
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even the most robust immunity induced against parasites of a given Plasmodium species offers little protection against challenge by parasites from another
species. Furthermore, acquired immunity is stage-specific and it wanes relatively quickly (a few years or less) in the absence of further exposure to the
parasite. Given these characteristics of acquired immunity in malaria, and
the recognition that each species comprises a large number of antigenically
distinct strains, most of the malariologists at that time were united in their
opinion that it is more than apparent that there is little reason to hope for
an effective vaccine for malaria (Yount and Coggeshall, 1949).
5.4. The Natural Course of the Malaria Infection

It could be argued that the core of our knowledge of the course of naturally
acquired malarial infection in humans and of the factors that govern the
complex interactions between the parasite and its vertebrate host could not
have been acquired without the observations made principally in mental
patients undergoing malariotherapy, and thereafter in healthy volunteers.
Indeed, as the number of neurosyphilitics dwindled after the introduction of penicillin, observations on the course of induced malaria continued
unabated in army and prisoner volunteers throughout World War II. The
fact that experimental infections of humans (often untreated but always very
closely monitored) were allowed until the late 1970s at the Atlanta Federal Penitentiary, United States of America, under the aegis of the National
Institute of Health (Neva, 1974), is in no small part due to a recognition of
the major contributions of malariotherapy. The insights into the biology of
Plasmodium parasites profoundly altered perceptions of malaria epidemiology and significantly improved the efficacy of control measures worldwide.
The practice of malariotherapy provided the malariologist, for the first
time, with a steady supply of human subjects who could be inoculated by
the bite of mosquitoes infected with one or other established strains of the
four species of malaria parasites. The sole aim was to obtain a clinical attack
that should be allowed to evolve, preferably unhindered, so as to procure the
patient with 1015 paroxysms, before curative administration of quinine. In
a few centres, some patients were left untreated after spontaneous resolution of the infection and monitored over the following months for renewed
malarial activity.
In this manner, myriad data were collected on the prepatent period
(the time to the first observation of parasites in the blood), the incubation
period (the time to the first clinical symptoms), the evolution of the asexual
and sexual parasitaemia, the magnitude and periodicity of paroxysms, the
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fever curves, the clinical manifestations, changes in parasite morphology, the

timing and frequency of recrudescences and relapses, and the duration of
the infection. Side-by-side comparisons of infections induced by different
parasite strains made it possible to define and investigate their biological and
clinical characteristics, thus contributing to define the concept of strain in
malaria (Boyd, 1940b; Ciuca etal., 1937b; James and Ciuca, 1938; Y
oung
etal., 1947). Excellent summaries have been prepared for each of the four
species (James etal., 1949; Kitchen, 1949a, 1949b, 1949c). The value of this
type of data has recently come to the fore in the elaboration of mathematical models of the malaria infection (Diebner etal., 2000; McKenzie etal.,
2001, 2002a, 2002b, 2007, 2008; Molineaux et al., 2001, 2002; Simpson
etal., 2002), and the malaria community is grateful to Collins and Jeffery
for collating hitherto unpublished data gathered from neurosyphilitics subjected to malariotherapy at the Carolina State Hospital between 1940 and
1963 (Collins and Jeffery, 1999a, 1999b, 1999c, 1999d, 2002; McKenzie
etal., 2001).
One of the more baffling observations made early after the introduction of malariotherapy in the United States was the refractoriness of many
persons of African origin to infection with P. vivax, as opposed to universal susceptibility of persons of other origins (Boyd and Stratman-Thomas,
1933d, 1934b).This was baffling to malariologists because at that time many
still considered P. ovale as a variant of P. vivax, and thus considered that the
Africans in their native continent were susceptible to P. vivax in general.
The basis for this absolute resistance was only uncovered in the mid 1970s
(Miller etal., 1976), when it was demonstrated that the Duffy blood group
was necessary for red blood cell invasion by P. vivax, whereas nearly all West
Africans and a majority of the population in subtropical Africa are Duffy
negative.
5.5. The Hidden Cycle

The relapses of P. vivax are now considered as the major obstacle to an
eventual extirpation of this parasite. A relapse is the appearance, in a person
bitten by an infected mosquito on a single occasion, of the blood stage parasites with or without clinical symptoms many weeks, months or years after
parasites from the primary episodes have been fully eliminated from the
blood. Such episodes, which are quite distinct from recurrences due to subcurative treatment, were noted early last century (Thayer, 1901), and posed a
major public health problem when it was feared that World War I and World
War II soldiers who fought in malarious areas would reintroduce P. vivax in
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countries from which it has disappeared. Relapse aetiology was elucidated

only in 1980 (Bray etal., 1981; Krotoski etal., 1980): quiescent liver stage
parasites, hypnozoites that mature to yield hepatic merozoites long after the
initial infective bite. This topic is the subject of another article of this series,
and the same author recently extensively and superbly reviewed it (White,
2011). The hypnozoite remains the more elusive and least investigated stage
of the Plasmodium parasites. Its discovery would have no doubt been indefinitely delayed had the hepatic stage of the malaria infection not been discovered 32years previously. We will limit ourselves to highlighting the role
that malariotherapy played in leading to the discovery of the generally very
short and discreet hepatic phase of the malaria life cycle.
Having spent nearly two decades observing malaria parasites exclusively
in blood, the minds of the malariologists at the turn of the nineteenth century readily accepted the notion that the newly discovered sporozoite, the
form injected by the mosquito, would naturally invade a red blood cell to
initiate the infection. Indeed, Schaudinns one-off observation of such an
event in a solution where P. vivax sporozoites were mixed with red blood
cells (Schaudinn, 1903) was taken as sufficient confirmatory proof. It was
only 20years later that this view was put in doubt by Yorke and Macfie. In
the course of early observations on malariotherapy patients, they demonstrated that quinine failed to prevent infection when it was administered as
a short course around the time of mosquito inoculation, but not when the
treatment is initiated a week or more later the mosquito bite (Yorke and
Macfie, 1924b). Yorke then showed that patients inoculated with infected
blood whose primary infection is curatively treated tend not to relapse,
whereas they do so frequently if they were initially inoculated by mosquito
bite (Yorke, 1925). James independently obtained similar results (James,
1926, 1931), and despite numerous attempts neither Yorke nor others could
observe a sporozoite invading and developing in a red blood cell.
The hunt was on to discover what happened to the sporozoite between its
deposition by the mosquito into the skin and the appearance of blood stage
parasites. Access to malariotherapy patients and infected mosquitoes was the
only means to investigate the biology of the sporozoite and the initial short
and clinically silent period of the infection. Remarkable experiments, in
particular those based on blood transfusions from a malariotherapy patient
bitten by an infected mosquito into other malaria nave patients (classic subinoculation experiments), were conducted by Boyd (Boyd, 1940a; Boyd and
Stratman-Thomas, 1934a, 1934c; Boyd and Kitchen, 1937a, 1939), Ciuca
etal. (1937a), and in volunteers by Fairley (1947). These provided crucial
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insights into the behaviour of the sporozoite pre-and postinoculation, and

allowed to determine the minimal duration of the tissue phase of the Plasmodium parasites in humans with some accuracy.
It was Shortt and Garnham who first identified the hepatocyte as the
site where pre-erythrocytic development occurs. This demonstration was
first reported using Plasmodium cynomolgi (Shortt et al., 1948b) a parasite
of macaques. Soon thereafter the work was repeated using P. vivax (Shortt
etal., 1948a) in a malariotherapy patient who consented to a liver biopsy
7days after inoculation with a large number of sporozoites. To our knowledge, the last observations on hepatic stages to be made in malariotherapy
patients were those of Shute and his Romanian colleagues (Shute et al.,
1976; Ungureanu et al., 1976) who wished to investigate the difference
between sporozoites (and relapses) from two P. vivax strains distinguished by
the duration of their prepatent periods and relapse patterns.
5.6. Establishment of Laboratory-Bred Anopheline Colonies

The requirement for mosquito-induced infections for malariotherapy was
initially met with locally caught anophelines (James and Shute, 1926; Y
orke
and Macfie, 1924b). However, as the number of malariotherapy patients
grew, a year-round supply of susceptible uninfected mosquitoes became
indispensable. Thus were established the first insectaries for mass rearing of
anophelines: Anopheles maculipennis (Shute, 1936), Anopheles quadrimaculatus
(Boyd etal., 1935), and thereafter other species. The availability of massreared anophelines, to quote Boyd et al. (1935), afford opportunities for
research on entomological, parasitological, and epidemiological problems
of malaria that have heretofore either been approached with difficulty or
not at all.Thus, a wealth of data accrued on the factors concerned with the
transmission of malaria from humans to mosquitoes and from mosquitoes
to humans (see below), and on anopheline biology, bionomics, and ecology.
This illuminated the nature of malaria endemicity, and significantly benefited the design and efficiency of malaria control measures.
5.7. The Dynamics of Transmission to Anophelines

In order that anopheline mosquitoes may be infected from malarial blood
it is necessary that the sexual forms of the parasite be present in sufficient
numbers, of proper maturity and suitable proportion of sexes.This sentence
in Deadericks (1909) book on malaria sums up the views prevalent prior
to malariotherapy on malaria transmission to mosquitoes. Indeed, there
were only scanty studies in which gametocyte dynamics (numbers as well
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as proportions of micro- and macro-gametocytes) were examined (Ross

and Thomson, 1910; Thomson, 1911). In only few studies were mosquitoes
(usually field-caught) applied to the patient with a view to investigate factors influencing infectivity to the insect (Mitzmain, 1916a, 1916b, 1916d).
However, the history of the infection was poorly known, if at all, the infecting parasites varied between patients some of who harboured mixed species
infections, and finally many had been or were under quinine treatment.
Thus, interpretation of the observations was fraught, and knowledge of the
dynamics of the transmission of malaria to Anopheles was to say the least
fragmentary.
It was important for the practice of malariotherapy to have access to a
constant supply of infected mosquitoes. Thus, the factors that impinged on
the efficiency of transmitting the parasite to anophelines were investigated
with great care. James, who was tasked with providing infected mosquitoes
to malariotherapy centres in Great Britain, was one of the first to tackle
this problem. His early observations on the transmission by wild caught
A. maculipennis of P. vivax (James, 1926), and of P. falciparum and P. malariae
(James, 1931; James etal., 1936), were rich in novel observations and unexpected conclusions, of which a selection is presented. Despite the fact that
many patients were infected with the same strain of P. vivax only a few
proved to be consistently good infectors to mosquitoes. Equally surprising was the fact that it was not the number of gametocytes present in the
blood that correlated with the proportion of infected mosquitos (i.e. where
sporozoites appeared in the salivary glands), but the presence and quality
of the microgametocyte (principally their ability and the time they take
to exflagellate). In the patients, gametocytes were rarely observed less than
2weeks after mosquito inoculation, and were generally not infective until
the 10th day after the first rise in fever. By contrast, gametocytes appeared
and were infective on the first day on which a relapse occurred. Boyd
reported broadly similar results for the transmission of the local (Florida)
P. vivax strains by wild-caught and then laboratory-bred A. quadrimaculatus (Boyd, 1942b; Boyd and Stratman-Thomas, 1932; Boyd and Kitchen,
1937b, 1938b; Boyd etal., 1936c).
The mosquitoes themselves were also found to differ in their ability to
transmit the infection. Observations of consistently refractory individuals
from the same batch of wild-caught mosquitoes woke entomologists to the
existence of cryptic species and contributed to explain the phenomenon
of anophelism without malaria, which perplexed malariologists early last
century. It was further demonstrated that mosquito species could differ not
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only in their susceptibility to different parasite species, but also to different

parasite strains of the same species. The classic example was the refractoriness of the European A. maculipennis to African strains of P. falciparum but
not to the strains indigenous to Italy (James et al., 1932b; Shute, 1940).
Boyd noted a similar phenomenon for nearctic and neotropical mosquito
species with respect to P. vivax and P. falciparum (Boyd etal., 1938a).
These and a wealth of other observations relevant to the transmission
of malaria parasites between its hosts have helped understand how subtle
variations in environmental conditions, mosquito bionomics and human
behaviour could lead to highly contrasting epidemiological pictures (Boyd,
1949a, 1949b). This in turn substantially influenced the manner in which
control measures were selected and deployed to the best effect.
6. LESSONS FROM MALARIOTHERAPY: CAVEATS

AND CURRENT RELEVANCE

Given the foregoing list of the more remarkable discoveries that the
practice of malariotherapy brought within the grasp of malariologists, the
reader might have gained the impression that an in depth analysis of all
the related material and publications would leave little to be learnt about
malaria infections and their parasites. Such is not the case. There are some
caveats that must be taken into consideration if one wishes to apply and/or
extrapolate the lessons from the malariotherapy era to some questions
relevant to todays efforts to control malaria.
First, the data on the malaria infection were nearly exclusively collected in neurosyphilitic adults generally of sound constitution and under
constant care, who for the most part had not been previously exposed to
malaria. This question, elegantly phrased in the title of an article 70years
ago, Are the data of therapeutic malaria applicable to conditions obtaining
in nature? (Winckel, 1941), remains one that some could formulate today.
There are indeed differences between the acquisition and eventual resolution of the malaria infection in malariotherapy patients and in endemic residents. However, we do not feel that these are sufficient to justify the view
that the malariological observations made in the course of malariotherapy
merely represent special cases with little connection or relevance to what
happens in nature.
In areas of high endemicity, it is the children who bear the brunt of symptomatic malaria. Moreover, as endemicity increases fewer and fewer persons
can be considered truly malaria nave, as many would have been exposed to
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the parasite or its antigens in utero, and to infection from birth onwards. For
some unknown reason, cases of congenital and/or juvenile tertiary syphilis
were poorly responsive to malariotherapy (Anonymous, 1936; OLeary and
Welsh, 1933; Wile and Hand, 1935), consequently reports of malariotherapy in children are rare. On the other hand, there is little to indicate that
the natural course of the malaria in children is fundamentally different from
that observed in adults. As for concurrent neurosyphilis, it could be argued
that it might have modified the course of the induced malaria infections in
a special way. However, this is unlikely to be the case because the biological and immunological observations made in the course of malariotherapy
were in all respects similar to those that were made later in healthy volunteers (also exclusively adults in good health). Furthermore, few if any of
the conclusions derived from the malariotherapy data are inconsistent with
field-based observations.
Second, the substantial malariological observations made in the various malariotherapy centres were derived from infections by a few selected
strains of parasites. The use of P. malariae was somewhat restricted because
it was relatively difficult to obtain and quite difficult to transmit by mosquito, but it was the parasite species favoured to infect African American
patients who were refractory to P. vivax. Induced P. falciparum infections
were restricted to the few centres where expert malariologists were in attendance, and even there it was used sparingly (Ciuca etal., 1943; James etal.,
1932b). Throughout the malariotherapy era, P. vivax was the parasite species
of choice. Nonetheless, the P. vivax strains that were used for malariotherapy
had been selected to fulfil the requirements for malariotherapy: primarily
to be of relative low virulence yet leading to the reliable production of a
sufficient number of paroxysms, to be highly susceptible to quinine, and
for some centres, to produce an adequate number of infective gametocytes.
Thus, the vast majority of observations made by the leading malariologists
were based on infection with only a few strains.Yorke based his observation
from a single strain isolated from a patient who acquired the infection in
India (Yorke and Macfie, 1924b). At Horton, it was the Madagascar strain
that was predominantly used for more than 40years (Covell, 1956; James,
1931; James etal., 1936; Shute, 1958). Although its origin is presumed to be
Madagascar, it was actually derived from an Indian seaman who developed
malaria on arrival to London from India, and whose last port of call was
Madagascar. The parasites recovered from his blood were equally likely to
be from a chronic infection or a relapse from an infection acquired in India.
The Madagascar strain was distributed very widely in Europe. In Romania
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Ciuca relied on two local strains, TBR102 and TBAp (Ciuca et al., 1937b,
1943) that he isolated prior to World War II. In the United States of America,Young mainly employed a local strain, St. Elizabeth (Coatney and Young,
1942; Young, 1944) and subsequently the Chesson strain that was isolated
from a soldier who became infected in Papua New Guinea (Ehrman etal.,
1945). Although Boyd derived a number of distinct strains from patients
who acquired P. vivax in Florida, the McCoy strain that had been isolated in
1931 was the most frequently inoculated.
It is unlikely that this eclectic collection of strains were representative of
the gamut of biological, immunological and clinical diversity that the global
P. vivax population encompassed then, or for that matter today. Indeed, it is
probable that many of the parasite strains prevalent in the temperate northern climes, such as the long incubation period P. vivax strains used by the
Dutch (Swellengrebel etal., 1929) and the Russian malariologists (Tiburskaja etal., 1968) became extinct as a result of the WHOs Global Eradication Campaign of the 1950s to 1960s. This is possibly also the case for the
Italian strains of P. falciparum that were shown to be particularly virulent and
tolerant to quinine in head-to-head comparison with P. falciparum strains
originating from India (James et al., 1932b). Finally, although the strains
that were used tended to remain phenotypically constant, they were not
necessarily genetically homogeneous, and it is possible that their genetic
heterogeneity might have varied over the numerous passages from human
to human or between humans and mosquitoes.
Third, much of the published data obtained through malariotherapy
would be considered to be inadequate as judged by the standards of clinical
trial designs and statistical analyses that would be deemed acceptable today.
This is by no means a criticism of the clinicians and malariologists who,
it must be stressed, generally carried out their work investigations to the
highest scientific standards of their time. Moreover, it is not obvious that
it would have been practical or ethically acceptable to conduct the type of
placebo-controlled, double-blind or other types of clinical trials that have
now become standard.
It is with these main caveats in mind that one should interpret, or extrapolate from, the malariological data obtained through the malariotherapy
era, and the subsequent series of experimental infections in human volunteers. The data should be approached with an open frame of mind. Most if
not all of the biological, clinical and immunological phenomena that were
revealed during the course of malariotherapy are obviously valid because they
were directly observed. However, the frequency of these phenomena under
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natural conditions and the manner and extent to which each contributes to
shape malaria epidemiology in a particular setting remain to be determined.
For example, it was conclusively demonstrated that in two lots of 20 or so
untreated or subcuratively African American patients who were inoculated
with either one or the other of two P. falciparum strains (the local SanteeCooper, and the Panamanian El Limon), the blood infection persisted for
a median of 222 or 279days, respectively, with a maximum observed duration of 480 and 503days in a few individuals (Eyles and Young, 1951; Jeffery
and Eyles, 1954). Clearly P. falciparum infections can last for nearly 2 years.
Nonetheless, many important questions need to be answered if these data
are to be useful to the epidemiologist or the malaria control person. Do all
P. falciparum strains have the potential to persist for many years? Does infection
longevity vary with the ethnic/genetic background or the immune or health
status of the human host? Do infectious gametocytes circulate during the
chronic phase, in which case for how long? The validity of many an elegant
mathematical model of malaria depends on the answers to such questions.
Finally, there are aspects of malaria that remained outside the practical
or ethical scope of the observations or the investigations made in the context of malariotherapy. This is the case for issues of clinical severity. With the
recent reinstatement of P. vivax to its rightful place as a harmful parasite that
deserves the attention of the malaria community (Baird, 2007; Price etal.,
2007), the dogma of its relative clinical mildness has been challenged (Anstey
et al., 2009; Baird, 2009). Unfortunately, it is quite difficult to derive data
from the malariotherapy literature that are robust enough to favour one or
other side of the argument. The mortality rates observed in malariotherapy patients varied between centres and with time. Average values around
10% were often reported in the early years of malariotherapy (Anonymous,
1929; Graham, 1925). By the mid 1930s, though some individual centres
still reported double-figure rates, it was less than 1% in others; the average
recorded mortality rates to 2.45.4% (Fong, 1937; Krauss, 1932; OLeary
and Welsh, 1933). This might reflect the accumulated expertise of medical
and nursing staff in caring for and monitoring patients subjected to malariotherapy, or a switch to parasite strains with reduced virulence. Furthermore, in those centres where only those patients deemed to be physically
capable of withstanding a clinical malaria infection were selected, the mortality rate was up to nine times lower that in those where no such selection
was made (Krauss, 1932). It is unfortunate that Krauss did not provide any
details as to the nature and origin of the parasite species and strains that were
used in the selected and unselected patients. The infecting dose and route of
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administration (intravenous, intramuscular, or by mosquito bite) are potential confounding factor when interpreting mortality rates (Hoch and Kusch,
1940; Kusch et al., 1936); however, the infecting dose is rarely provided.
Moreover, there does not seem to be a standard set of criteria for attributing
mortality directly to the P. vivax infection, except maybe the presence of high
numbers of parasites at the time of death. For these and other reasons, it is
rather complicated to derive an accurate estimate of the malaria infections
contribution to mortality from the malariotherapy publications. Nonetheless, it is undisputed that deaths consequent to infection with moderately virulent P. vivax strains occurred despite close medical supervision in physically
fit patients. A conservative case fatality rate of 0.1%, low as it might appear,
is sufficient to sustain the notion that P. vivax infections are a threat to life.
7. CONCLUSIONS

Prior to the introduction of malaria therapy, the description of a
typical malarial attack given in most text-books on tropical medicine was
based on a study of the disease in persons resident in, or recently returned
from a malarious country. Observations of induced malaria attacks in subjects who have never before been exposed to infection have shown that in
many respects these descriptions were incorrect. (Covell, 1956).
These are humbling words from an eminent malariologist of 30years
experience, many of which were spent investigating the epidemiology and
treatment of malaria in India. The observations of induced malaria revolutionised the way in which Covell (18871975) and the malariologists of
his generation perceived malaria. We feel confident in thinking that few of
todays generation malariologists are aware of the existence of these observations. Our hope in writing this review (with its unapologetically abridged
list of relevant references) is that it will encourage our colleagues to become
acquainted with this unique record of the natural history of malaria and its
parasites and inspire them to devise investigations to fill the many gaps that
are still there. We should warn potential readers that this literature could be
addictive.
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INDEX
Note: Page numbers with f denote figures; t tables.
A
A-variant
G6PD deficiency, 147, 168
in sub-Saharan Africa, 170, 172
variant dependency, 180
primaquine sensitivity, 180181
Abs. See Antibodies
Acute haemolytic anaemia (AHA), 137,
149
Altered redox equilibrium, 177178
AMA1. See Apical membrane antigen 1
8-aminoquinoline clinical development
programme, 140141
8-aminoquinoline pamaquine, 137, 137f
Anaemia, 151152
AHA. See Acute haemolytic anaemia
(AHA)
CNSHA. See Chronic nonspherocytic
haemolytic anaemia (CNSHA)
sickle cell anaemia, 6162
Anopheles farauti, 3334
Anopheles mosquito, 3
Anophelines
malariotherapy practice, 240
malarial infection, 106
mosquito species, 1011
mosquito-induced infections, 239
observations, 240241
sexual stage of, 5
transmission dynamics to, 239240
zygote of, 111
Antibodies (Abs), 80
cross-reacting antibodies, 3435
specific for PvDBP, 4647, 60
Antigen-specific T-cell responses, 106
Antimalarial drugs, 213214
Aotus (owl or night monkeys), 1415, 9798
Apical membrane antigen 1 (AMA1),
9495, 213214
Apical membrane antigen-1 of P. vivax
(PvAMA1), 100
Apicoplast, 210211
Artificially induced infections, naturally
acquired immunity features based
on, 88t
Asia Pacific Malaria Elimination Network
(APMEN), 154
Atabrine, 137138
B
Bayesian geostatistical model, 156157
Benign tertian malaria, 3132
BinaxNOW G6PD assay, 154155
Biological discovery era
cell biology and germ theory, 2930
neurosyphilis observation, 3031
treatments, 3132
human variation and blood groups
blood group systems, 36t37t
cell biology and infectious disease,
34
heritability and human population
biology, 3435
malariotherapy and African-based
resistance, 3233
natural exposure, 33
treatments, 32
Biomarkers, 81
Blood group systems, 3435, 36t37t
human variation and, 3435
Blood-stage cycle, 3
Blood-stage immunity effector mechanisms,
9394
merozoite, 9495
P. knowlesi, 9394
RBC and Pv membrane, 9395
Blood-stage immunity targets
immunological memory, 105
P. vivax merozoite proteins, 9697
Plasmodium merozoites, 95, 95f
protective immune responses, 96
257
258
Blood-stage immunity targets (Continued)
PvAMA1, 100
PvDBP, 102
PvDBPII-based vaccine, 102103
vaccine-induced anti-DBP Abs,
102103
PvMSP1, 9798
PvMSP3, 9899
PvMSP9, 99100
PvRBPs, 101
P. vivax genome, 102
PvDBP-II, 101102
pvrbp2 gene, 101
T-cell immune responses, 103104
Blood-stage infection, force of, 89
C
Carboxy-primaquine, 175
Care-Start G6PD screening test, 154155
Caveola-vesicle complexes (CVCs), 6, 91
CD4+ memory T cell, 103104
CD8+T cells, 109
Chemokine receptor, 9193
Chesson strain, 138139
Chimpanzee infections, 1314
Chronic nonspherocytic haemolytic
anaemia (CNSHA), 149, 150t
Circumsporozoite precipitation reaction
(CSP reaction), 107
Circumsporozoite protein (CS protein),
106, 213214
CNSHA. See Chronic nonspherocytic
haemolytic anaemia
Comparative genomics, and P. vivax
genome, 207213
Bill and Melinda Gates Foundation,
grants, 212213
difficulties of, 207208
whole genome sequences
of Asian Old World Monkey malaria
clade, 209t
of P. vivax, 208t
CS protein. See Circumsporozoite protein
CS-specific cytolytic CD4+T cells, 110
CSP reaction. See Circumsporozoite
precipitation reaction
CTL. See Cytotoxic lymphocyte
Index
CVCs. See Caveola-vesicle complexes

Cytoadherence ligands, 9
Cytokines, 103104
Cytotoxic lymphocyte (CTL), 107109
D
DBLEBP. See Duffy-binding-like erythrocyte binding proteins
DBP. See Duffy binding protein
Deletion of GYPC exon 3 (GYPCDex3),
5859
Differential acquisition of immunity
to P. falciparum, 81
of clinical immunity, 83
endemic areas, 8384
epidemiological observations, 83
modulator, 84
prevalence, 83
to P. vivax, 81
of clinical immunity, 83
endemic areas, 8384
epidemiological observations, 83
evidence for rapid acquisition of
immunity, 82f
modulator, 84
prevalence, 83
treatment
development, 8586
experimental infections, 87
NAI features, 8788, 88t
P. vivax, 8486
P. vivax parasitaemias, 8485
strain-specific immunity, 8687
Dose dependency, haemolytic effect of
primaquine, 179180
Duffy antigen
for blood group, 3538
primary structure, 42f43f
Duffy binding protein (DBP), 3538, 4546
antigen interaction, 46
genes, 1516
in P. cynomolgi, 1516
parasite ligandhost receptor relationship,
4647
of parasites, 8
PvDBP, 4546
Duffy blood group nomenclature, 45t
Index
Duffy polymorphism
FY*AES allele in Papua New Guinea,
4748
Fybweak antigen, 4849
global distribution of, 5457
P. knowlesi Duffy binding protein, 49
semi-quantitative PCR assay, 48
Duffy protein, 4041
Duffy-binding-like erythrocyte binding
proteins (DBLEBP), 4546
Duffy-independent blood-stage infection,
5052
Duffy-independent red cell invasion, 52
E
Erythrocyte membrane protein band 3. See
Solute carrier family 4, anion
exchanger, member 1 (SLC4A1)
Erythroid silent (ES), 41
Extensive polymorphism, 100, 215216
F
Falciparum malaria, 3132, 48
-thalassaemia, 58
immune regulation, 93
and SAO and Gerbich negativity, 5960
Favism, 136137, 149
FDA. See US Food and Drug Association
Fluorescent spot test (FST), 150151
FY alleles, global frequencies of, 55f
Fy a+b-phenotype, 102
Fy blood group antigen, 102
FY*A allele, 102
FY*B allele, 102
Fybweak polymorphisms, 44
Fyx polymorphism, 4344
G
G6PD deficiency. See Glucose-6-phosphate
dehydrogenase deficiency
G6PD enzyme, 143
cell age, 146
dimer bind, 146
haemolytic risk, 147
Mediterranean variant, 147
mutations, 146
PPP as anti-oxidative defence
259
enzyme activity defect, 148
NADPH, 147148
residual enzyme activity, 146
G6PD genetics and inheritance, 143
13 exons, 143
genes X-linked inheritance, 145
mutations, 143145, 144f
G6PD Mediterranean, 173
G6PD gene, diversity of mutations in, 144f
Gametocyte, 910
P. falciparum, gametocyte development,
910
P. vivax- gametocyte-infected erythrocyte, 910
P. vivax gametocytes, 6
General paralysis of the insane (GPI),
2930, 225
Genetic resistance factor
blood-stage infection, 39
distribution, 3839
Duffy blood group negativity, 3940
Germ theory, 2930
neurosyphilis observation, 3031
treatments, 3132
Global genetic diversity
mitochondrial genomes, 214215
MSP-3, 213214
mtDNA, 215
multilocus investigations, 213
population genetic investigations, 213
population genomic investigations, 213
target gene approaches, 213
Glucose-6-phosphate dehydrogenase
deficiency (G6PD deficiency), 54,
5758, 135
in Africa+, 169170
in America, 168169
antimalarial primaquine, 135
in Asia Pacific, 164f165f, 166168
clinical manifestations, 148, 150t
AHA, 149
CNSHA, 149
G6PD alleles, 149150
NNJ, 148149
P. vivax therapy, 150
symptoms, 148
in countries East of India, 167
260
Glucose-6-phosphate dehydrogenase
deficiency (G6PD deficiency)
(Continued)
diagnosis methods, 150
evidence of selective advantage
case-control invivo evidence, 172173
epidemiological evidence, 171
invitro evidence, 171172
evolutionary drivers of distribution,
170171
favism, 136137
in Indian subcontinent, 167
molecular diagnostic tests, 152
enzyme activity, 152153
high-end laboratory requirements,
152153
mutation mapping, 163
national allele frequency, 162f
neglecting P. vivax role, 174
G6PD Mediterranean, 173
protective role, 173
with P. vivax endemicity, 164170
P. vivax transmission, 164166, 166f
phenotypic diagnostic tests, 150151
anaemia, 151
heterozygotes, 151152
in large-scale population, 151
molecular methods, 152
screening methods, 150151
sensitivity, 152
in Philippines, 168
point-of-care G6PD test, unavailability,
153
haemolysis, 154
RDT, 154155
total RBC count, 154
population, estimates of, 161163
prevalence, 136
prevalence mapping, 155156
evidence-base, 156
mapping model, 156157
overview, 157159
primaquine, path to
8-aminoquinoline pamaquine, 137
atabrine, 137138
drugdrug interaction, 138139
efficacy, 140
Index
pamaquine, 137139
predisposing factor, 139
primaquine tolerability and safety
daily dosage, 142
14-day regimen, 140141
growing acknowledgement, 142143
haemolysis, 141142
primaquine sensitivity, 141
toxicity, 140141
in Saudi Arabia, 169
in sub-Saharan Africa, 170
in Yemen, 169
Glutathione dimers (GSSG), 147148
Glutathione monomers (GSH), 147148
Glycophorin A (GYPA), 5859
Glycophorin C (GYPC), 5859
Glycosylphosphatidylinisotol anchor,
9798
GPI. See General paralysis of the insane
GSH. See Glutathione monomers
GSSG. See Glutathione dimers
GYPA. See Glycophorin A
GYPC. See Glycophorin C
GYPCDex3. See Deletion of
GYPC exon 3
H
Haemoglobin, 141142
Haemoglobinopathies, 58
Haemolysis, 141142
drug-induced, 188190
primaquine-induced, 175179
Haemolytic risk
assessment
drug-induced haemolysis, 188190
higher dosage schedule, 190
at individual level, 187
national risk index, 189f
WHO treatment guidelines, 188
factors affecting, 179
dose dependency, 179180
red blood cell age dependency, 183184
sex dependency, 184
variant dependency, 180183
prediction, 184
Hepatocyte, 239
HepG2-A2 cells, 14
261
Index
Heterozygotes, 145
Hexose monophosphate shunt. See Pentose
phosphate pathway (PPP)
Hibernans types. See Temperate strains
Hidden cycle
hepatocyte, 239
patients and infected mosquitoes,
238239
relapses of P. vivax, 237238
Schaudinns one-off observation, 238
HLA. See Human leukocyte antigen
5H6MQ. See 5-hydroxy-6-methoxy-8aminoquinoline
Human leukocyte antigen (HLA), 9899
Human malaria species, 1011
Human relapsing parasite, 1011
5-hydroxy-6-methoxy-8-aminoquinoline
(5H6MQ), 175
Hypnozoites, 68, 8889, 106107
I
IE. See Infected erythrocyte
IFN-. See Interferon-
IL. See Interleukin
Immune responses, against malaria
Ab- and T-cell-specific responses, 111
CD8+T cells, 109
cell-mediated mechanisms, 107109
genomic and proteomic technologies,
110111
hepatocyte invasion, 106
hypnozoites, 106107
lymphocyte, 107109
at pre-erythrocytic stages, 107, 108f
sporozoites movement, 106
Immunological memory, 105
Invitro models, 11
liver-stage investigation, 13
P. vivax-infected RBC, 11
in rhesus monkey RBC, 1112
Invitro evidence, 171172
Invivo evidence, case-control, 172173
Infected erythrocyte (IE), 81
hemozoin pigment granules in, 910
Schffners stippling, 6
Infected RBC (iRBC), 4f, 9

and blood-stage immunity targets,
95105
P. vivax- infected, 11
PvHIST/CVC-8195 release in, 91
Infected red blood cell membrane, 8991
Interferon- (IFN-), 99100, 108f
Interleukin (IL)
human interleukin 8 receptor, 4041
IL-4 responses, against PvMSP9 peptides,
99100
Intermittent fevers, 3031, 229230
Interquartile range (IQR), 159161
in uncertainty quantification,
159f160f
IQR. See Interquartile range
iRBC. See Infected RBC
K
Knickerbocker Blood Bank of New York
City, 3538
Korean War (19501953), primaquine use,
139
Kpffer cells (KC), 3, 107109
L
Light microscopy (LM), 4748, 83
Long-lasting insecticide-treated nets
(LLINs), 8081
Lyonization, 145, 151153
M
mAb. See Monoclonal Ab
Macaca mulatta red blood cells, P. knowlesi
invasion of, 28, 29f
Macrogamete, 5
Mahidol variant, 181182
Malaria, 204, 224
causative agents, 224
immune response
Ab- and T-cell-specific responses, 111
CD8+T cells, 109
genomic and proteomic technologies,
110111
262
Malaria (Continued)
hypnozoites, 106107
lymphocyte, 107109
sporozoite movement, 106
infection
malariotherapy, practice, 236
myriad data, 236237
natural course, 236
with P. vivax, 237
malariotherapy, 224
Malaria Atlas Project (MAP), 156
G6PD mapping model, 156157
generation, 156
maps prediction confidence, 159161
prevalence map, 157159, 157f158f
regional prevalence, 161
uncertainty, 159161, 159f160f
Malaria Hypothesis, 34
Malaria red cell invasion, 35
Duffy binding protein
DBPDuffy antigen interaction, 46
parasite ligandhost receptor relationship, 4647
PvDBP, 4546
genetic resistance factor
Duffy blood group negativity, 3940
impressive distribution, 3839
molecular and cellular basis
comparative sequence analyses, 44
Duffy antigen, 42f43f
Duffy serology, 4344
Fybweak polymorphisms, 44
methodical progress, 4041
resident PNG, 4143
SNP, 41
working guidelines, 45t
serological recognition, 3538
Malaria tropica, 3132
See also Falciparum malaria
Malarial parasites, diversity of, 234
Malariotherapy, 3233, 224
and African-based resistance to P. vivax,
3234
and treatment of neurosyphilis, 32
Index
Caucasian patients vs. African-American

patients, 3233
caveats and relevance
acquisition and eventual resolution, 241
clinical severity, 244245
eclectic collection of strains, 243
grasp of malariologists, 241
high endemicity, 241242
through malariotherapy, 243
malariological observations, 242243
contributions to malariology, 233
hidden cycle, 237239
laboratory-bred anopheline colonies,
239
malaria infection natural course,
236237
malarial parasites diversity, 234
naturally acquired immunity
characteristics, 234236
transmission to anophelines dynamics,
239241
unicity theory, demise, 233234
in England, 231
GPI, 225
dire prognosis, 226
and malariology
observations, limitations to, 230
malariologists, 231233
malariology state, 229230
natural exposure, 33
in paretic patients, 3132
practice, 228
hippocratic primum non nocere, 228
using infected blood, 228229
public health standpoint, 229
proponent of, 227f
single-mindedness, 226
Station for Malaria Research, 232233
in United States of America, 232
MAP. See Malaria Atlas Project
MBC. See Memory B cell
Mediterranean variant, 147, 183
Memory B cell (MBC), 105
Mendelian X-linked gene, 143
Merozoite apical organelles, 8
Merozoite surface protein 3 (MSP-3),
213214
Mepacrine. See Atabrine
263
Index
Methaemoglobin (metHb), 150151, 177

Mitochondrial genomes, 214215
Molecular diagnostic methods
Duffy polymorphism and population
distribution
Duffy-negative protection, 56f
frequency distribution, 53f
FY alleles, 55f
G6PD deficiency, 54
PvPAR, 5457
Duffy polymorphism on resistance
FY*AES allele in PNG, 4748
Fybweak antigen, 4849
P. knowlesi DBP, 49
semi-quantitative PCR assay, 48
Duffy-independent red cell invasion, 52
Duffy-negative P. vivax resistance
factor, 4950
Duffy-positive or Duffy-negative
people, 52
Giemsa-stained thin smear preparations, 51f
malaria-transmission regions, 50
molecular and cell biology technologies,
47
non-Duffy gene polymorphisms, 57
G6PD deficiency, 5758
haemoglobinopathies, 58
Southeast Asian ovalocytosis, 5860
Molecular force-of-infection (molFOI), 83
Molecular mimicry, 211212
molFOI. See Molecular force-of-infection
Monkey malaria clade
comparative genomics, 207
P. knowlesi, 211212
reference genomes, 210
whole genome sequences, 209t
Monoclonal Ab (mAb), 99100
Mosquito-induced infections, 239
MSP-3. See Merozoite surface protein
3
mtDNA sequencing, 215
N
NAI. See Naturally acquired immunity
Natural killer cells (NK cells), 103104
Natural regulatory T cells, 103104
Naturally acquired immunity (NAI), 78

characteristics, 234236
to malaria, 7879
biomarkers, 81
blood-stage infections, 7980
premunition, 80
transmission in sexual stages, 80
to P. vivax, 8081
Neonatal jaundice (NNJ), 148149
Neurosyphilis, 225
primary cure, 8485
New World monkey species, 12, 1415
NHP experimentation. See Non-human
primate experimentation
NK cells. See Natural killer cells
NNJ. See Neonatal jaundice
Non-Duffy gene polymorphisms, 57
G6PD deficiency, 5758
haemoglobinopathies, 58
Southeast Asian ovalocytosis, 5860
Non-human primate experimentation
(NHP experimentation), 2
New World monkey species, 1415
O
Omics technology, 1718, 207
On the Origin of Species (Darwin), 34
Open reading frame (ORF), 4041
Oxidative stress, 176177
P
Pamaquine, 137138
chemical structure of, 137f
Parasitaemia, 48
blood-stage, 3233, 3839
and fever, 8485
and NAI, 7980
PCR. See Polymerase chain reaction
Pentose phosphate pathway (PPP),
147148
Heinz bodies, 148
PfEMP1. See PfVSA erythrocyte
membrane protein 1
PfMSP3, 9899
PfVSA erythrocyte membrane protein 1
(PfEMP1), 8990
PHIST protein. See Plasmodium helical
interspersed subtelomeric protein
264
Plasmodium cynomolgi (P. cynomolgi), 5, 1516,
205
Plasmodium diversity
global genetic diversity, 213215
importance of, 204
Plasmodium falciparum (P. falciparum), 5, 81,
207, 228
Plasmodium fieldi (P. fieldi), 5
Plasmodium helical interspersed subtelomeric protein (PHIST protein), 91
Plasmodium ovale (P. ovale), 56, 233234
Plasmodium perniciosum, 234
Plasmodium semiovale (P. semiovale), 5, 17
Plasmodium vivax (P. vivax), 2, 28, 81, 204. See
also Molecular diagnostic methods
biological characteristics, 88. See also
Naturally acquired immunity (NAI)
force of blood-stage infection, 89
immunosuppressive networks, 93
infected RBC membrane, 8990
predicted VSA role, 90
Pv infections, 91
PvHIST/CVC-8195, 91, 92t
red cell invasion ligands, 9193
relapses, 8889
variation of surface antigens, 8990
biological discovery era
cell biology and germ theory, 2932
human variation and blood groups,
3435
malariotherapy and African-based
resistance, 3234
endemicity, 166f
evolutionary history
Asian and African origins, 204
extraordinary biological diversity, 207
molecular phylogenies, 205
P. inui, 205207
P. schwetzi, 205
phylogenetic tree, 206f
exvivo models
aseptic P. vivax sporozoites production,
14
chimpanzee infections, 1314
from neotropical New World monkey
species, 12
in P. knowlesi blood-stage gene
expression, 1213
Index
gametocytes, 910
genome and comparative genomics
apicoplast, 210211
biological material, 212213
genome sequences, 208t209t, 212
molecular mimicry, 211212
Plasmodium coatneyi, 210
P. falciparum genome, 207209
P. gonderi, 210
using Sanger sequencing technology,
207208
SNPs and microsatellites, 209210
sporozoite-specific transcription, 212
from genomics to systems biology, 1718
functional interaction networks, 1819
machine learning techniques, 1819
metabolomics high-throughput
technologies, 1718
metabolomics sensitivity, 19
systems biology approaches, 19
global genetic diversity
mitochondrial genomes, 214215
MSP-3, 213214
mtDNA, 215
multilocus investigations, 213
population genetic investigations, 213
population genomic investigations,
213
target gene approaches, 213
life cycle, 3, 4f
blood-stage cycle, 3
growing stage, 35
infection stage, 6
for liver-stage development, 67
P. ovale, 56
penetrating stage, 5
in Southeast Asian macaque monkeys, 5
sporozoite infection, 78
malaria red cell invasion, 35
DBP, 4547
genetic resistance factor, 3840
molecular and cellular basis, 4044
serological recognition, 3538
merozoite interaction, 63f
neotropical NHP models, 1415
phylogenetic tree of, 206f
Plasmodium knowlesi invasion, 29f
population genetics
Index
adaptive traits, 215216

antigenic genes, 216217
human Duffy gene, 217
in India, 216
using microsatellites and SNP markers,
217
RBC, 2829
relapse treatment, haemolytic risk
assessment, 185
drug-induced haemolysis, 188190
haemolytic risk prediction, 186187
higher dosage schedule, 190
at individual level, 187
national level, 185
national risk index, , 189f
proposed framework, 185186
WHO treatment guidelines, 188
resistance to, 61f
reticulocyte host cells, 8
CVCs, 89
iRBCs, 9
parasite-encoded proteins, 89
RBCs, 9
reticulocyte-binding proteins, 8
sexual life strategies
Anopheline mosquito species, 1011
gametocyte, 910
sexual maturity, 910
Southern Asian macaque monkeys, 15
NHP-relapsing malaria species, 16
P. cynomolgi, 1516
P. simiovale, 17
P. vivax characteristics, 1516
specific antibodies, 105
invitro models, 11
liver-stage investigation, 13
P. vivax-infected RBC, 11
in rhesus monkey RBC, 1112
whole-genome sequences of, 208t209t
Plasmodium vivax DBP (PvDBP), 4546,
101102
Point-of-care G6PD test, requirements, 153
haemolysis severity, 154
RDT, 154155
total RBC count, 154
Polymerase chain reaction (PCR), 83
semi-quantitative, 4748
for target gene approaches, 213
265
Population genetics
adaptive traits, 215216
antigenic genes, 216217
human Duffy gene, 217
in India, 216
using microsatellites and SNP markers,
217
Population genomic investigations, 213
PPP. See Pentose phosphate pathway
Pre-erythrocytic stages, immune responses,
205, 224
Ab-specific responses, 111
CD8+T cells, 109
genomic technologies, 110111
hypnozoites, 106107
lymphocyte, 107109
proteomic technologies, 110111
sporozoite movement, 106
T-cell-specific responses, 111
Premunition, 80
Primaquine, 137, 174. See also
Glucose-6-phosphate dehydrogenase deficiency (G6PD deficiency)
primaquine-induced haemolysis, 175
altered redox equilibrium, 177178
methaemoglobin, 177
oxidative stress, 176177
primaquine and metabolites, 175176
Primaquine (Continued)
significance of, 178179
sensitivity, 180
therapy, 185
haemolytic risk prediction, 186187
ranking national-level risk, 185186
Pv antigen-specific effector, 103104
PvAMA1. See Apical membrane antigen-1
of P. vivax
PvDBP. See Plasmodium vivax DBP
PvHIST/CVC-8195, 91
PvMSP1, 9798
PvMSP3, 9899
PvMSP9, 99100
266
PvPAR, 5457
pvrbp2 gene, 101
PvRBPs, 9193, 101
P. vivax genome, 102
PvDBP-II, 101102
pvrbp2 gene, 101
Q
Quinacrine. See Atabrine
R
Rapid diagnostic test (RDT), 154155
Rapid acquisition of immunity, evidence
for, 82f
RBC. See Red blood cell
RBL proteins. See Reticulocyte bindinglike proteins
RDT. See Rapid diagnostic test
Red blood cell (RBC), 3, 2829, 35, 135
age dependency, 183184
Red cell invasion
Duffy independent, 4952
ligands, 9193
Red fluorescent protein gene, 1617
Relapse aetiology, 237238
Reponema pallidum, 2930
Reticulocyte, 89
Reticulocyte binding-like proteins (RBL
proteins), 101
S
Salvador I reference genome sequence, 213
Saimiri (squirrel monkeys), 1415
SAO. See Southeast Asian ovalocytosis
Schffners stippling, 6
Sex dependency, 184
Sexual stage parasites, 111
in malaria endemic areas, 111
P. falciparum TBI, 112
P. vivax TBI, 112
Anopheles albimanus mosquitoes, 113
gametocyte infectivity, 112
patients sera, 112113
TBI, 111
target molecules against, 113
Single nucleotide polymorphism (SNP), 41,
173
Index
SLC4A1. See Solute carrier family 4, anion

exchanger, member 1
SNP. See Single nucleotide polymorphism
Soluble immune mediators, 109
Solute carrier family 4, anion exchanger,
member 1 (SLC4A1), 5859
Southeast Asian ovalocytoses (SAOs),
5860
Southern Asian macaque monkeys, 15
NHP-relapsing malaria species, 16
P. cynomolgi, 1516
P. simiovale, 17
P. vivax characteristics, 1516
Spatial distribution mapping. See also
Glucose-6-phosphate dehydrogenase deficiency (G6PD deficiency)
population estimation, 161
IQR, 161
malaria endemic regions, 163,
164f165f
mutation mapping, 163
national allele frequency, 161, 162f
prevalence map, 155156
MAP, 156
national-level map, 155156
regional prevalence, 161
uncertainty in, 159161, 159f160f
Sporozoite infection, 78
Sporozoite surface protein (SSP), 106
Sporozoite-specific transcription, 213
SSP. See Sporozoite surface protein
SSP2/TRAP proteins, 106
Station for Malaria Research, 232233
Syphilis, 224
T
T helper type (Th1-type), 103104
Tafenoquine, 137
TBI. See Transmission blocking
immunity
Temperate strains, 78
Th1-type. See T helper type
Transmission blocking immunity (TBI),
111112
Tropical strains, 78
Trickle effect, 87
267
Index
U
Unicity theory, demise of, 233234
US Food and Drug Association (FDA), 141
V
Variant dependency, 180183
Variant surface antigen (VSA), 81
Vivax malaria. See Benign tertian malaria
W
West Africans and P. vivax
absence of, 3839
FY*BES allele observed in, 5052
resistance to, 2930
WHO
Global Eradication Campaign of the
1950s to 1960s
recommendations, 180181
for Mediterranean variants, 183
mild and severe deficiency, 188
on primaquine, 181182
re-assigning of, 188
Whole genome sequences

of Asian Old World monkey malaria
clade
of P. vivax, 208t, 212
Wild-type erythrocytes, 183184
X
X-linked inheritance
of G6PD gene, 145
mechanism of, 156
Y
Young children, P. vivax in
less than 2 years, 81
school-aged children, 60
Young RBCs, 8
restricting infection to, 9
Z
Zygote formation, 5, 111
studies, using Affymetrix platform, 212
and TBI action, 111

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Advanced Parasitology-The Epidemiology of Plasmodium Vivax

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SERIES EDITORS

Spatial Epidemiology and Ecology Group,

Reader in Parasite Epidemiology,

Immunology and Infection Section,

Wellcome Trust Research Fellow and

Johns Hopkins Malaria Research

Department of Veterinary Science,

Head, WHO Collaborating Centre for

VOLUME EIGHTY ONE

AMSTERDAM BOSTON HEIDELBERG LONDON

Academic Press is an imprint of Elsevier

and evolutionary history of P. vivax. New insights from detailed genomic

Plasmodium vivax: Modern

*Department of Medicine, Division of Infectious Diseases, Emory University School of Medicine,

2013 Elsevier Ltd.

Mary R. Galinski et al.

Plasmodium vivax: Modern Strategies to Study a Persistent Parasites Life Cycle

Mary R. Galinski et al.

Plasmodium vivax: Modern Strategies to Study a Persistent Parasites Life Cycle

1216 or more differentiated merozoites in the case of P. vivax. When the

Mary R. Galinski et al.

Plasmodium vivax: Modern Strategies to Study a Persistent Parasites Life Cycle

Mary R. Galinski et al.

Plasmodium vivax: Modern Strategies to Study a Persistent Parasites Life Cycle

2.3. Fast and Furious: The Sexual Life Strategies of P. vivax

Mary R. Galinski et al.

Plasmodium vivax: Modern Strategies to Study a Persistent Parasites Life Cycle

continue to develop, albeit at a slower pace, in their mosquito hosts, which

3. IN VITRO AND EX VIVO MODELS FOR EXAMINING

Mary R. Galinski et al.

3 weeks invitro in rhesus monkey RBC (Nguyen-Dinh etal., 1981). Our

Plasmodium vivax: Modern Strategies to Study a Persistent Parasites Life Cycle

Mary R. Galinski et al.

obtained from mosquitoes fed on infected rhesus monkeys (Dembele etal.,

4. NEOTROPICAL NON-HUMAN PRIMATE MODELS

Plasmodium vivax: Modern Strategies to Study a Persistent Parasites Life Cycle

Mary R. Galinski et al.

Plasmodium vivax: Modern Strategies to Study a Persistent Parasites Life Cycle

6. FROM GENOMICS TO SYSTEMS BIOLOGY: THE

Mary R. Galinski et al.

Plasmodium vivax: Modern Strategies to Study a Persistent Parasites Life Cycle

Mary R. Galinski et al.

Plasmodium vivax: Modern Strategies to Study a Persistent Parasites Life Cycle

Mary R. Galinski et al.

Coatney, G.R., 1971.The Primate Malarias. US Government Printing Office,Washington, DC.

Plasmodium vivax: Modern Strategies to Study a Persistent Parasites Life Cycle

Mary R. Galinski et al.

Plasmodium vivax: Modern Strategies to Study a Persistent Parasites Life Cycle

Mary R. Galinski et al.

Red Blood Cell Polymorphism

2013 Elsevier Ltd.

Peter A. Zimmerman et al.

Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax

Peter A. Zimmerman et al.

(1860s) significantly demystified the relationship between disease and the

Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax

(Chernin, 1984). It was also noted that observation of neurosyphilis was

Peter A. Zimmerman et al.

2.2. Malariotherapy and African-Based Resistance to P. vivax

Resistance to vivax malaria was seen to be the primary disadvantage that

Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax

times higher than that routinely causing blood-stage infection of Caucasian

Peter A. Zimmerman et al.

2.3. Human Variation and Blood Groups

2.3. Fast and Furious: The Sexual Life Strategies of P. vivax

3. IN VITRO AND EX VIVO MODELS FOR EXAMINING

4. NEOTROPICAL NON-HUMAN PRIMATE MODELS

6. FROM GENOMICS TO SYSTEMS BIOLOGY: THE

2.2. Malariotherapy and African-Based Resistance to P. vivax

2.3. Human Variation and Blood Groups

3. RESISTANCE TO P. VIVAX AND INSIGHTS ON

3.4. The Duffy Binding Protein

4. EVOLVING PERSPECTIVES ON RESISTANCE

4.2. Duffy-Independent Red Cell Invasion by P. vivax

5. CONCLUSIONS AND FUTURE DIRECTIONS

7. Immune Responses to Malaria Pre-Erythrocytic Stages

1. OVERVIEW OF NATURALLY ACQUIRED IMMUNITY