Howes G6PDDef AdvParasit

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+)
2013 Elsevier Ltd.
Advances in Parasitology, Volume 81
ISSN 0065-308X, http://dx.doi.org/10.1016/B978-0-12-407826-0.00004-7 All rights reserved.
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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).
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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
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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.
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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.
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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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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.
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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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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
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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
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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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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.
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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
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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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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.
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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
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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
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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
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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
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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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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.
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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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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
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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
192
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 FOUR

G6PD Deficiency: Global

7. E volutionary Drivers of the Distribution of G6PD Deficiency

P rimaquine and its Metabolites

8.2. F actors Affecting Haemolytic Risk

G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy

primaquine-induced haemolysis to determine safe and effective therapeutic strategies

The widespread prevalence of G6PD deficiency across populations in

G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy

2.2. The Path to Primaquine

Figure 4.1 Chemical structure of key 8-aminoquinolines: pamaquine, primaquine and

G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy

evaluation of therapies against relapse (Comfort, 2009). They leveraged a

2.3. Primaquine Tolerability and Safety

G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy

concentrations was observed. In one experiment, G6PD deficient subjects

G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy

and unexplored approaches to mitigating 8-aminoquinoline toxicity. The

3.1. G6PD Genetics and Inheritance

G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy

3.2. The G6PD Enzyme

G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy

This in turn maintains the oxidisedreduced glutathione (GSSG2GSH)

3.4. Clinical Manifestations of G6PD Deficiency

G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy

Table 4.1 G6PD Variant Types and Their Key Characteristics

Severe and chronic: CNSHA

Asymptomatic until triggered:

In the present context of P. vivax therapy, the generally asymptomatic

4. DIAGNOSING G6PD DEFICIENCY

4.1. Phenotypic Diagnostic Tests

G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy

sample was deemed to be G6PD deficient. Developments to this method

4.2. Molecular Diagnostic Tests

G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy

screening or routine care in their current form. Even if these limitations

severity of haemolysis not conclusively established, is it likely that this test

G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy

5. MAPPING THE SPATIAL DISTRIBUTION OF G6PD

5.1. G6PD Deficiency Prevalence Mapping

Human Genes (Cavalli-Sforza etal., 1994), however the statistical mapping

G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy

this assumption. Populations were also assumed to be in HardyWeinberg

G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy

G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy

of model confidence, as areas rich in data and with more homogenous

5.2. G6PD Deficient Population Estimates

G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy

Although many of the highest frequencies of deficiency were predicted

5.3. G6PD Deficiency Mutation Mapping

6. SPATIAL CO-OCCURRENCE OF G6PD DEFICIENCY

Figure 4.6 Proportions of G6PD variants among phenotypically deficient community

G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy

6.1. G6PD Deficiency in Asia

G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy

the Mediterranean variant, which was identified in >70% of deficient

6.2. G6PD Deficiency in Asia-Pacific

6.3. G6PD Deficiency in the Americas

G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy

to 7% coincided with G6PD deficiency allele frequency national estimates

7. EVOLUTIONARY DRIVERS OF THE DISTRIBUTION

G6PD Deficiency: Global Distribution, Genetic Variants and Primaquine Therapy

7.1. Evidence of a Selective Advantage

protective mechanism. Although they found no significant difference in the

7. E volutionary Drivers of the Distribution of G6PD Deficiency

P rimaquine and its Metabolites

8.2. F actors Affecting Haemolytic Risk

2.2. The Path to Primaquine

2.3. Primaquine Tolerability and Safety

3.1. G6PD Genetics and Inheritance

3.2. The G6PD Enzyme

3.4. Clinical Manifestations of G6PD Deficiency

4. DIAGNOSING G6PD DEFICIENCY

4.1. Phenotypic Diagnostic Tests

4.2. Molecular Diagnostic Tests

5. MAPPING THE SPATIAL DISTRIBUTION OF G6PD

5.1. G6PD Deficiency Prevalence Mapping

5.2. G6PD Deficient Population Estimates

5.3. G6PD Deficiency Mutation Mapping

6. SPATIAL CO-OCCURRENCE OF G6PD DEFICIENCY

6.1. G6PD Deficiency in Asia

6.2. G6PD Deficiency in Asia-Pacific

6.3. G6PD Deficiency in the Americas

7. EVOLUTIONARY DRIVERS OF THE DISTRIBUTION

7.1. Evidence of a Selective Advantage

8. PRIMAQUINE, P. VIVAX AND G6PD DEFICIENCY

8.1. Mechanism of Primaquine-Induced Haemolysis

8.2. Factors Affecting Haemolytic Risk

8.3. Predicting Haemolytic Risk

9.2. Assessing Haemolytic Risk at the Level of the Individual