Background
Malaria is a deadly disease caused by five
Plasmodium species with
Plasmodium falciparum being the most lethal.
Plasmodium falciparum caused an estimated 438,000 deaths globally in 2015 [
1]. The heaviest burden of malaria, according to the World Health Organization (WHO), is in Africa where an estimated 395,000 malaria deaths was recorded in 2015 [
1].
G6PD deficiency (G6PDd) is one of the most common human genetic enzyme defects [
2], affecting over 400 million people. Although this enzymopathy is globally distributed, it is more prevalent in the tropics and sub-tropics, especially in malaria-prone countries [
3]. This X-linked genetic condition is characterized by reduced G6PD enzyme activity, which can remain asymptomatic. Red blood cells obtain reduced glutathione (GSH) solely from the G6PD/reduced nicotinamide adenine dinucleotide phosphate (NADH) pathway. The deficiency makes red cells more susceptible to oxidative haemolysis that can be triggered by certain drugs, such as primaquine (PQ) and other 8-amino quinolone drugs [
4]. Glucose-6-phosphate dehydrogenase (G6PD) and a number of other human genetic traits including sickle cell anaemia and related haemoglobinopathies are predominantly found in populations living in malaria endemic countries and have been suggested to provide the host protection from severe forms of malaria [
5‐
8] and asymptomatic malaria [
9]. A number of genetic traits that protect the host against severe forms of malaria have recently been found to promote the development of the sexual transmissible stages of the parasite, and individuals with these traits serve as reservoirs for malaria transmission [
10] or alter the acquisition of anti-parasite antibodies [
11].
G6PD deficiency can be determined using a number of approaches, including, quantitative tests, which measure precise enzyme activity, qualitative tests which classify enzyme activity as normal or deficient and genetic tests, which identify gene mutations [
12‐
19].
Over 400 G6PD variants have been identified [
4] and the polymorphisms are predominantly defined to specific geographic locations [
20]. In sub-Saharan Africa, the predominant G6PD variants are B, A and A-, with frequencies greater than 1 %. The G6PD B variant has the 376A cDNA sequence and is classified as possessing normal enzyme activity. The non-deficient G6PD A variant has been shown to possess about 85 % of the activity of the normal enzyme and is classified as possessing normal activity carries a cDNA mutation A376G, which translates into amino acid N126D. The sub-Saharan G6PD A- variants carry the G6PD A backbone with an additional single mutation, the most common A- variant has the A376G/G202A cDNA mutation. G6PD A- been suggested to possess 10 % of normal enzyme activity in their red blood cells though their white blood cells maintain 100 % normal enzyme activity [
21] Additional A- variants peculiar to sub-Saharan Africa include the A376G/A543T, A376G/G680T and A376G/T968C [
19].
Global efforts to achieve malaria elimination have lead the WHO to recommend PQ, the only WHO certified anti-gametocyte drug, to be incorporated into malaria treatment regimens in selected countries to reduce malaria transmission [
22]. Although low-dose PQ has been found to be safe for use in G6PDd individuals [
23,
24], a number of other reports have found PQ to cause dangerous side effects in G6PDd individuals [
25,
26] making it necessary to monitor the prevalence of G6PD as well as determine possible effects this trait has on malaria in Ghanaians.
Discussion
In order to identify any possible influence of G6PD deficiency on asymptomatic malaria carriage, this study was conducted over the off peak malaria season, from February through to May 2015, when symptomatic
P. falciparum carriage is low. Some previous studies have identified relationships between G6PD deficiency and protection from severe malaria [
2,
31] as well as asymptomatic status [
9]. In Ghana, however, most studies have concentrating on pregnant women due to the national sulfadoxine-pyrimethamine intermittent preventative treatment in pregnant women (SP-IPTp) policy [
32‐
35]. Very few studies have focused on asymptomatic malaria patients or simultaneously carried out genotypic and phenotypic analysis of G6PD on the same individual [
30,
36] as such, this study set out to find a possible correlation of G6PD with asymptomatic malaria in Ghana.
Asymptomatic parasite carriage estimated by PCR was very high, averaging about 40 % over the 4 months of the study (Fig.
2a). An average of 26 % of the children were identified as asymptomatic for malaria by microscopy for the first 2 months (February to April). The asymptomatic status of the children who tested positive for
P. falciparum was confirmed by the fact that none of the children had a fever of 37.5 °C and above or any other physical symptoms of malaria during any of the four blood draws. Such high levels of asymptomatic
P. falciparum carriage suggests that the children have developed immunity to malaria and serve as reservoirs for the parasite, which is likely to be a channel for intense malaria transmission when the mosquitoes start breeding during the rainy season.
The high prevalence (23 %) of samples, of children who were found to be parasite positive by microscopy more than once during the 4 monthly visits (Fig.
2b) could explain the sustained transmission of malaria immediately after the rainy season begins as there is a possible continuous production of gametocytes from these asexual parasites. The superiority of PCR over microscopy, the gold standard for the detection and diagnosis of malaria was confirmed in this study as the number of children found to be
P. falciparum parasite free over the entire study period reduced from 50.6 % as observed by microscopy to 20 % after PCR analysis (Fig.
2b).
This study identified only the 376G/202A G6PDA- variant, which has so far been the only G6PD variant identified in Ghana [
9,
30,
36]. The prevalence of G6PDd, of % (7/170) A- males and 1.8 % (3/170) A-/A- females identified in the study population (Table
1) is lower than previously published for Ghana [
30]. The difference could be due to the use of a study population inhabiting the southern coast of Ghana as a previous report has shown that the prevalence of G6PDd in both males and females is higher in Kintampo, which is to the north of Kumasi than in Kumasi, which is in the middle belt of Ghana [
30]. Forty four percent of G6PD A- and A-/A- children possed normal enzyme activity (Table
2) which is higher than that perviously reported in a study conducted in six African countrie including Ghana, where 10 % of G6PD A- males and 24 % G6PD A-/A- females possessed normal enzyme activity [
30].
A previous study conducted in Ghana found G6PDd children to be about 1.5 times more likely to be parasitaemic based on microscopic evaluation of thin and thick blood films than non-deficient children [
37], suggesting that the G6PD deficiency was associated with marginal susceptibility to clinical malaria in a child under 5 years of age. This study involved slightly older children who did not display such an association of G6PDd and malaria. PCR detectable parasite carriage but not parasite carriage by microscopy was significantly (p = 0.038) associated with the G6PD normal genotype (Table
3, Additional file
1), which slightly supports previous reports that suggests the G6PD trait offers some protection from malaria [
38,
39]. G6PD deficient children also had a lower tendency to carry sub-microscopic
P. falciparum parasites more than once during the 4 month period (Fig.
3a) although some G6PDd deficient children carried microscopically detectable parasites three out of the four sample visits (Fig.
3b).
Table 3
Summary of linear regression analysis
(Constant) | | 0.032 | | 0.009 | | 0.103 |
G6PD (P) | −0.03 | 0.707 | −0.011 | 0.89 | 0.126 | 0.112 |
G6PD (G) | 0.166 | 0.038 | 0.02 | 0.8 | 0.133 | 0.093 |
Genotyping G6PD by RFLP analysis identifies only the known G6PD genotypes, leaving the possibility of identifying novel mutations, which could possibly impact enzyme activity (28). A sample can be misclassified as normal due to the absence of particular mutations characterized despite carrying mutations that were not analysed. The six children who did not carry an A- allele but were identified as having reduced G6PD enzyme activity (Table
2) could have possessed other deficiency causing mutations that were not characterized possibly because they have not been assigned a genotype [
40]. A study conducted in the Gambia on 1437 children between 5 and 14 years found half (50 %) the study population to carry the G6PD A376G (B) variant and 39 % to carry mutations that have not been assigned a genotype [
41]. Heterozygous G6PD deficient females are able to randomly inactivate one of their two X-chromosomes and as such exhibit either normal or deficient G6PD enzyme activity [
42,
43]. As such a little over half (5/9) of the heterozygous deficient children exhibited normal G6PD enzyme activity.
The CareStart™ G6PD RDT classified 44.4 % (4/9) of A- and A-/A- deficient children as normal (to possess normal G6PD enzyme activity). This could be due to inaccuracies and the level of subjectivity of the RDT kit readout [
13,
14,
16,
44]. Some studies have found people with the same G6PD cDNA mutation to exhibit different enzyme activities due to possible differences in the G6PD acetylator in the deficient persons [
45] as well as the possible contribution of a number of superimposed genetic deficiencies [
46] and blood sugar levels [
47].
Authors’ contributions
LEA was the Principal Investigator who conceived and designed the study and supervised data collection as well as sample and data analysis; AO collected the samples; JA, AO, FKA and RAT performed molecular assays; LEA performed statistical analysis; LEA wrote the paper. All authors have read and approved the final manuscript.