Over a third of the world’s population lives at risk of P. vivax infection
[14, 52, 54]. Limited evidence underpins estimates of clinical cases, but these have been estimated at 70-400 million annually
[55, 56], including potentially severe illness and death
[5, 24, 57, 58]. In the context of malaria elimination, therapy must target all infections, including asymptomatic and submicroscopic blood-stage infections, dormant liver-stage hypnozoites as well as clinical cases
[59, 60]. One of the many consequences of a half-century of neglect of P. vivax has been the failure to address the primaquine toxicity problem with G6PDd and thereby solve the real problem of lack of access to primaquine for almost all malaria patients. No non-toxic therapeutic alternatives exist, and existing G6PDd diagnostics are largely impractical in point-of-care settings
[4, 6, 21, 45, 56].
Fear of harm also leads to avoidance of primaquine as a P. falciparum transmission-blocking agent. The single transmission-blocking 0.75 mg/kg dose, consistently inconsequential in G6PD A- volunteers, reliably caused 20-30% drops in haematocrit in healthy Europeans with G6PD Mediterranean. This risk, acknowledged at least 30 years ago, was only acted upon in 2012 with the WHO offering a recommendation for a lower dose of primaquine (0.25 mg/kg) gametocytocide alongside P. falciparum blood schizontocidal treatment with no requirement for prior G6PDd testing
[10, 11, 61–63]. Nonetheless, many nations, in particular in Africa where there has been an almost complete lack of experience with primaquine
 and where G6PDd prevalence is commonly greater than 10%
, still resist use of this drug. An understanding of the therapeutic risks from primaquine in relation to the G6PD variants predominant in each region will contribute to the body of evidence on which to base policy.
Access to safe therapy either requires an alternative non-haemolytic drug or a practical means to identify G6PDd P. vivax malaria patients in remote, impoverished sites. Neither is likely in the short-term and improved use of primaquine promises immediately applicable and useful prospects
. This aim demands refined knowledge of the spatial epidemiology and primaquine-sensitivity phenotypes of G6PD variants. Development of primaquine treatment strategy could exploit, for example, the total dose effect (whereby therapeutic efficacy is dependent upon the total drug dose irrespective of the regimen duration
[64–66]), to tailor population/region-specific dosing parameterized according to the highest dose safely tolerated by individuals with the most vulnerable local G6PD variant.
G6PD variants in Africa
The G6PDd phenotype has long been considered homogenous across African populations, with much of the pre-molecular era literature reporting G6PD A- from electrophoresis assays across the continent
. The early investigations of “primaquine sensitivity” among G6PD A- volunteers found primaquine-induced haemolysis to be mild and self-limiting
[1, 30, 64, 67–69]. Nevertheless, the haemolytic susceptibility of this “mild” variant has been observed through severe reactions requiring transfusion
, as well as through the failure of the Lapdap (chlorproguanil-dapsone) anti-malarial trials
[71, 72], and as haemolysis induced by the ingestion of fava beans
, a pathology previously thought to only be triggered by more severe variants
. Haemolysis associated with G6PD A-, while perhaps less severe than with other variants in some circumstances or settings, ought not be universally characterized as “mild” as this risks leading to misplaced confidence in a relatively cavalier application of the drug.
Molecular analyses of G6PD variants among Africans revealed a diversity of SNPs within the umbrella of the G6PD A- phenotype
. The data, here mapped spatially, indicated a transition of SNP predominance from west (G6PD A-
polymorphism) to east (G6PD A-
widespread and G6PD A-
apparently absent). However, expansion of the relatively small number of surveys (n = 20) may reveal more complex distributions. Furthermore, the true diversity of G6PD variants cannot be discerned from the assembled surveys; it is not possible to know what proportion of genetic diversity is reflected in the map (Figure
3) because the diagnoses are limited to assessing the presence or absence of a limited number of anticipated SNPs, without any prior identification of phenotypically deficient hosts (as in map series 1). Full gene sequencing is the only reliable way of identifying all gene variants in the population. Genetic diversity within the umbrella G6PD A- phenotype may explain the spectrum of expression levels observed
: inheritance of a single SNP may not be sufficient to explain the observed phenotype. If these patterns of co-occurring SNP diversity encoding variable G6PD A- phenotypes are widely observed, a haplotype-based approach which allows for the co-inheritance of multiple SNPs may be required if the use of molecular diagnostics is to the scaled-up, as seems to be increasingly common
. Despite its obvious limitations to field-based applications, a gene-wide haplotype perspective would also allow increased compatibility with the rapidly expanding human genome sequencing efforts which are cataloguing human genetic diversity
Although demand for primaquine has historically been low across Africa
, it was Ethiopia that reported the highest number of P. vivax cases globally in 2011 (665,813 cases;
). G6PDd prevalence has been estimated at 1.0% in Ethiopia (50% CI: 0.7-1.5)
, though no molecular information exists to indicate which variants may be responsible. Furthermore, beyond these areas with acknowledged endemic P. vivax, the status of transmission in human populations previously considered refractory to infection
 is being brought into question by several lines of evidence
[80–86]. The use of primaquine for P. falciparum control may also increase following the new WHO gametocytocide guidelines
. The elimination agenda includes African nations, with most located at the fringes of entrenched holo-endemic transmission
[87, 88], and the pressure to apply primaquine both as a hypnozoitocide for P. vivax and Plasmodium ovale, and as a gametocytocide for P. falciparum will increase with further progress towards zero transmission. A more detailed and targeted understanding of African G6PD epidemiology is necessary beyond the opportunistic surveys presented in this paper.
G6PD variants in West Asia
The G6PD Mediterranean variant, appearing predominant across southwest Asia and exceedingly common in much of India (especially western regions), is known to be highly sensitive to AHA induced by primaquine. This wide distribution and dominance of G6PD Mediterranean presents a significant hurdle to the malaria elimination programmes in West Asia, which include Azerbaijan, Georgia, Iran, Iraq, Kyrgyzstan, Saudi Arabia, Tajikistan, Turkey, and Uzbekistan
. These nations must consider the relatively high risk of serious harm caused by unintentional dosing of G6PDd patients in need of malaria therapy. Implementation of G6PDd screening prior to primaquine radical cure therapy would greatly mitigate such risks and accelerate progress to malaria elimination in this region.
Although G6PD Mediterranean was common across Indian populations, two other variants were also frequently reported which were indigenous to populations from this subcontinent: G6PD Kalyan-Kerala and Orissa. Little is known of the primaquine sensitivity phenotypes of these two variants. Since almost half of the global population at risk of P. vivax malaria occurs in this single nation
[52, 79, 90] studies characterizing such sensitivity would ultimately prove very useful in planning towards elimination.
G6PD in East Asia and the West Pacific
The maps of eastern Asia and the western Pacific present the most complex picture of G6PD variants globally, coinciding with regions of heterogeneous and high P. vivax endemicity
. Almost all the common variants of public health concern globally were reported from this region. Reasons for this genetic diversity are unclear, but it is interesting that P. falciparum parasites (postulated to be selective agents of G6PDd
) have been found to show a greater degree of population structure with lower genetic relatedness between populations in Asia than across Africa
. As well as this overall diversity, the structure of G6PD variant heterogeneity with multiple variants co-occurring is starkly different from other areas where single variants predominate.
Despite large variant diversity across this region where many countries now target elimination (thus require primaquine therapy)
, only one truly Asian variant has been examined in relation to AHA sensitivity to primaquine. G6PD Mahidol, common across Myanmar and parts of Thailand (Figure
6), exhibits residual G6PD enzyme activity ranging between 5-32%
. A handful of small primaquine sensitivity studies examining this variant have been conducted in Thailand, reporting very mild to moderate sensitivity to both 14-day and eight weekly primaquine regimens
[40, 92, 93], as well as to single-dose regimens of P. falciparum transmission blocking therapy
. However, the G6PD Mahidol primaquine sensitivity phenotype must not be assumed to be representative of the region. As emphasized by the WHO Expert Review Group on primaquine in 2012
[62, 63], far greater evidence-based understanding of these phenotypes among the many common Asian variants is urgently needed. The identity and distribution of variants as sensitive as G6PD Mediterranean in the Asia-Pacific region remains unexplored and unknown. This lack of understanding threatens patients and programmes with a more aggressive roll-out of primaquine therapy. In populations of such high heterogeneity, a dual approach of phenotypic screening followed by variant analysis of residual enzyme activity may be required to identify such risks.
The danger of under-diagnosing deficiency by using molecular identification alone (i e, the data informing map series 2) is illustrated by comparison of the two types of maps (Figure
2C and Additional file
5). If only a selected subset of perceived “common” variants are used in population screening surveys, a proportion of deficient individuals may be missed and put at risk from primaquine therapy: only those variants that are looked for can be identified. The maps presented here also repeatedly highlight considerable proportions of “Other” G6PD variants. These correspond to an unknown haemolytic risk, and hint towards an ever greater diversity of variants. Only full gene sequencing will allow full characterization of the diversity in this polymorphic gene.