Background
Despite some reduction in the global burden of malaria in recent years[
1], the disease still stands a grave public health concern. Apart from multiple environmental and parasite factors that complicate human-
Plasmodium interactions, human genetic background confers additional complexity in the fight against malaria[
2]. A high degree of variation exists between human populations and individuals with respect to malaria incidence, parasite density and disease severity and reactions to anti-malarials[
3]. Although in some studies glucose-6-phosphate dehydrogenase deficiency (G6PDd) is associated with protection from severe malaria[
4,
5] deficient individuals are reported to suffer from haemolytic anaemia when treated with primaquine[
6], which is the only available drug against
Plasmodium falciparum gametocytes[
7] and a radical cure of
Plasmodium vivax[
8]. Further individuals with G6DPd are reported to suffer from haemolysis when treated with tafenoquine, another anti-malarial with proposed indication of treating hypnozoites of
P. vivax and
Plasmodium ovale[
9].
G6PDd is one of the most common human enzymopathies which is in a very high frequency in tropical and subtropical malaria endemic regions[
10,
11]. In sub-Saharan Africa (SSA), the prevalence of G6PDd can reach up to 35% although organized reports are lacking and different allelic variants are unevenly distributed in the region[
12]. However, with progressive improvements in detection methods past G6PDd prevalence figures may need continuous revision. Population screening is recommended in regions where G6PDd prevalence is ≥3–5% to decide on the use of primaquine/tafenoquine and minimize the risk of primaquine/tefenoquine-induced complications in malaria elimination schemes[
13]. In Ethiopia, past population-based G6PDd studies are limited and the practice of G6PDd screening in healthcare settings is also missing. As a result, vital information on G6PDd prevalence is lacking in the country, especially in malaria endemic foci. This study, therefore, was aimed at bridging the gap by diagnosing G6PDd among malaria suspects attending Gambella hospital, southwest Ethiopia, using a qualitative rapid G6PDd screening kit.
Results
Overall, 510 febrile patients were prescribed for malaria blood smear during the study period. Fourteen individuals who declined to participate and 47 who had received anti-malarial treatment within the past 48 hours prior to sample collection were excluded. Thus blood samples from 449 patients, 210 males and 239 females, were analysed. The mean age was 16 years, median 15 and range 1–90 years; and mean haemoglobin (Hb) was 14.02 g/dl and median 13.3 g/dl, with no significant difference between the sexes. Overall, 266(59.2%) individuals were malaria smear positive with 257(96.6%)
P. falciparum mono, 7(2.6%)
P. vivax mono and 2(0.75%)
P. falciparum/P. vivax mixed infections. Thirty three (7.3%) individuals were deficient for G6PD enzyme activity (Table
1). Although the prevalence of G6PDd was slightly higher among males (8.6%) than females (6.3%), the difference was not statistically significant (
p = 0.354). In a univariate logistic analysis, the odds of being G6PD-deficient were 1.4 times higher for males [95% CI 0.678-2.853] than for females but the difference was not statistically significant (
p > 0.05). However, the occurrence of G6PDd varied between ethnic groups. A greater proportion of the
Nuer were G6PD-deficient (14.3%) compared to the ‘highlanders’ combined, who were all non-deficient except a single
Kambata. The 12.0% G6PDd prevalence among the
Anuak was also substantial compared to the almost nil ‘highlanders’. The differences were statistically significant in both cases (
p < 0.0001). The odds of being G6PD-deficient were 4.9 [95% CI 0.635-38.00] and 3.9 [95% CI 0.481-31.418] times higher for the
Nuer and
Anuak ethnic groups compared to the ‘highlanders’, respectively (
p < 0.0001) (Table
2). When the G6PDd prevalence for the
Nuer and
Anuak was compared from within the difference was also significant.
Table 1
Prevalences of G6PDd among outpatients of Gambella hospital, southwest Ethiopia by malaria status, sex and ethnic group, 2013
Normal | Male | 192 | 117 (60.9) | 75 (39.0) | 38 (19.7) | 59 (30.7) | 22 (11.4) | 21 (10.9) | 14 (7.3) | 16 (8.3) | 11 (5.7) | 11 (5.7) |
| Female | 224 | 119 (53.1) | 105 (46.8) | 42 (18.7) | 67 (29.9) | 24 (10.7) | 20 (8.9) | 27 (12.0) | 10 (4.4) | 18 (8.0) | 16 (7.1) |
Deficient | Male | 18 | 17 (94.4) | 1 (5.5) | 7 (14.5) | 10 (55.5) | 00 (0.0) | 00 (0.0) | 00 (0.0) | 00 (0.0) | 1 (5.5) | 00 (0.0) |
| Female | 15 | 13 (86.7) | 2 (13.3) | 4 (26.6) | 11 (73.3) | 00 (0.0) | 00 (0.0) | 00 (0.0) | 00 (0.0) | 00 (0.0) | 00 (0.0) |
Total | | 449 | 266 (59.2) | 183 (40.8) | 91 (20.2) | 147 (32.7) | 46 (10.2) | 41 (9.1) | 41 (9.1) | 26 (5.8) | 30 (6.6) | 27 (6.0) |
p-value | 0.354 | | 0.000 | | 0.000 | | | | | | | |
Table 2
Logistic regression analysis of sex and ethnic group for G6PDd among malaria suspect outpatients in Gambella hospital, southwest Ethiopia, 2013
Gender
|
Female | 1 | | 1 | |
Male | 1.4000 (0.678-2.853) | 0.354 | 1.459 (0.669-2.944) | 0.371 |
Ethnic group
|
Nuer
| 4.9 (0.635-38.00) | 0.000 | 4.6 (0.589-35.79) | 0.000 |
Anuak
| 3.9 (0.481-31.418) | 0.063 | 3.7 (0.454-30.08) | 0.063 |
Amhara
| 0.00 | 0.059 | 0.00 | 0.059 |
Oromo
| 0.00 | 0.044 | 0.00 | 0.044 |
Tigre
| 0.00 | 0.139 | 0.00 | 0.139 |
Dawuro
| 0.00 | 0.131 | 0.00 | 0.131 |
Hadya
| 0.00 | 0.059 | 0.00 | 0.059 |
Kambata
| 1 | | 1 | |
While G6PD-deficient males who were malaria smear positive were 94.4% (17/18), females of the same category were 87.6% (13/15). Thirty (90.9%) out of the 33 G6PD-deficient participants were malaria smear positive which was significantly higher than the incidence of malaria among normal individuals for the enzyme (
p < 0.0001). However, the mean parasite density of G6DP-deficient individuals (1.64 (±1.55) was significantly lower than that of G6DP-normal individuals (2.97 (±1.22) (
p < 0.0001). Similarly, the mean Hb concentration in G6DP-deficient individuals (14.15 (±3.50) was significantly higher than that of normal ones (12.44 (±2.70) (
p = 0.006). Further, the mean HCT of G6PD-deficient individuals (37.86 (±8.609) was significantly higher than that of normal participants (43.13 (±10.54) (
p = 0.005). But the mean values of MCV, MCH and MCHC were not significantly different between the two groups (
p > 0.05) (Table
3).
Table 3
Mean ± SD parasitaemia, Hb, HCT and RBC indices in relation to G6PD status among malaria suspected outpatients in Gambella hospital, southwest Ethiopia, 2013
Normal | 2.97 ± 1.22 | 12.44 ± 2.70 | 37.86 ± 8.609 | 85.51 ± 6.68 | 26.87 ± 5.58 | 31.98 ± 2.50 |
Deficient | 1.64 ± 1.55 | 14.15 ± 3.50 | 43.13 ± 10.54 | 85.75 ± 5.84 | 28.12 ± 4.92 | 32.43 ± 1.43 |
p-value | 0.000 | 0.006 | 0.005 | 0.834 | 0.201 | 0.149 |
Seven (21.2%), 1 (3.0%), 9 (27.2%) and 7 (21.2%) individuals were macrocytic, microcytic, hypochromic and microcytic-hypochromic, respectively, among the G6PD-deficient (Table
4). From the non-deficient; 31 (7.4%), 4 (0.96%) 78 (18.7%) and 29 (6.7%) were macrocytic, microcytic, hypochromic and microcytic-hypochromic respectively. The differences between the two groups for the respective parameters were significant.
Table 4
Prevalence of various anaemia types and G6PD status among malaria suspect outpatients in Gambella hospital, southwest Ethiopia, 2013/2014
Normal | 31 (7.4) | 4 (0.96) | 78 (18.7) | 29 (6.7) |
Deficient | 7 (21.2) | 1 (3.0) | 9 (27.2) | 7 (21.2) |
p- value | 0.000 | 0.316 | 0.000 | 0.000 |
Discussion
In this study,
P. falciparum, P. vivax and mixed infections accounted for 96.6
, 2.6 and 0.8% of malaria cases, respectively, indicating the overwhelming occurrence of
P. falciparum in the study population. This result is comparable to a previous report in the area[
21] which was 88.9% for
P. falciparum and 11.1% for
P. vivax cases. However, the overall malaria prevalence reported by these authors was much lower (13.5%) than that of the current study (59.2%). On the other hand, results from elsewhere in Ethiopia attribute only 60–70% of the cases to
P. falciparum and the rest to
P. vivax[
22,
23]. The high malaria prevalence in this study shows that the disease is a year-round problem in Gambella despite the undergoing control activities and the fact that the survey was conducted outside the peak transmission season. This may suggest that either the control strategies in use are ineffective and/or some specific local predisposing factors to malaria are impacting. Malaria prevalence did not significantly correlate with participant age or sex but ethnic group. The
Nuer and
Anuak were more frequently infected compared to the ‘highlanders’ combined. Environmental and differences in exposure to infectious mosquito bites, among other things, may account for this apparent difference.
The prevalence of G6PDd was 7.3% in this study. This figure is relatively lower compared to the world health organization (WHO) report for SSA which is as high as 35%[
12]. The prevalence of G6PDd significantly varies among different countries and regions or even localities for several reasons[
24,
25]. For instance, in southern European countries, such as Greece[
26] and Italy[
27], G6PDd prevalence ranges from 1–30 and 0–3% respectively. A study in India reported G6PDd prevalence of 27.5% in males and 12.8% in females[
28]. G6PDd prevalence in Yemen was 7.1%[
29] and in the neighbouring Saudi Arabia it was 9.05%[
30]. It is widely believed that isolated populations have higher G6PDd cases[
26,
31].
Although the frequency of G6PDd was higher in males (4.0%) than in females (3.3%) in the present study, the difference was not statistically significant. Since the gene of the enzyme is on the X-chromosome and males are hemizygous, a single allele of the gene would suffice to express the deficient phenotype. In light of this, a higher frequency of the deficient phenotype is expected in males than in females. Since females inherit two copies of the X-chromosome and therefore have two populations of red blood cells (RBCs), each expressing one of the two G6PD alleles they carry (homozygous females), two deficient alleles are required to render them G6PD-deficient phenotypically. From this point of view, the present study seems to be contrary to what is theoretically expected. However, diagnosing heterozygote females that express two different RBC populations (normal and deficient) to the enzymatic activity level is highly challenging, as the deficiency can be masked by cells expressing normal activity. G6PDd is relatively reliably detected in males[
32,
33] and diagnostics for heterozygous females are altogether more complex and uncertain. That is perhaps why G6PDd in females is underreported in many epidemiological studies. Indeed there are studies that reported a narrow variation of G6PDd prevalence between boys and girls with a
p-value just slightly below the 0.05 level[
34].
The prevalence of G6PDd significantly varied between ethnic groups in Gambella area; the highest frequency being among the indigenous
Nuer and
Anuak ethnic groups. This must be taken into account in planning primaquine/tafenoquine use to eliminate malaria especially in the rural areas of Gambella where the natives exclusively reside. A similar study in Nigeria recorded some variation in G6PDd with respect to ethnic affiliation though the effect was not significant possibly due to the relatively small number of
non-Yoruba children in the study, as the authors suggested[
35]. In a study from India the G6PDd prevalence ranged from 0 to 27% and a clear variation was noted between different castes, tribes and ethnic groups[
36].
The significantly higher occurrence of malaria smear positivity among G6PD-deficient individuals, compared to the non-deficient, observed in this study suggests lack of correlation between G6PDd and protection from clinical malaria. But the significantly lower mean parasite density, higher Hb and HCT values among the deficient shows the protective role of G6PDd against severe malaria. In fact, the literature is not concordant regarding the role of G6PDd in malaria. G6PDd was associated with protection against severe malaria in males but not in heterozygous females[
37]. Contrarily, others reported the protection of heterozygous females but not the hemi/homozygous with a more severe degree of G6PDd[
38]. Still there is evidence for protection from symptomatic malaria for both hemi/homozygous and heterozygous individuals[
39]. G6PDd is weakly associated with asymptomatic malaria infection[
40,
41]. High malaria parasitaemia and mortality was observed among G6PD-deficient patients in Iran[
42]. In another recent study the odds of malaria infection was not significantly lower in children with the deficient phenotype[
34].
In vitro studies are equally conflicting[
43,
44]. Since G6PD expression is vital for survival of
Plasmodium inside the host cell the parasite may synthesize its own G6PD within deficient RBCs[
45]. This may partially explain why G6PDd is not associated with protection from malaria at least in some settings including the present one. The method of G6PDd assessment can possibly affect the association studies of malaria susceptibility due to G6PDd[
46].
It is well-known that G6PDd contributes to macrocytic, microcytic and hypochromic anaemia. In microchromic anaemia the RBC are usually also hypochromic. Further, rapid RBC turnover from G6PDd haemolysis can trigger mild macrocytosis. This may explain the significantly higher proportion of G6PD-deficent individuals for these different types of anaemia compared to the non-deficient in the present study. However, the results should be interpreted with caution as there are several other causes of macrocytic and other types of anaemia. Multiple factors like nutrition, infection, medication, haemoglobinopathies and overall health status may influence G6PD activity[
47]. Since the MCV and MCH values for microcytic (MCV <80 fL)
, macrocytic (MCV >96 fL) and hypochromic (MCH <27 pg) anaemia are comparable especially with that of thalassaemia[
48], it may be misleading to conclude that the asymptomatic G6PDd is responsible for the observation in this study. Although little specific information is available concerning haemoglobinopathies in general, Gambella is part of the global thalassaemia map[
49]. Thus, future studies are warranted to clearly understand the role of asymptomatic G6PDd on haematological parameters and malaria in relation to haemoglobinopathies in the study area. Though rapid qualitative tests such as the CareStart™ G6PDd screening tests seem more feasible for mass screening in remote endemic areas genotyping G6PD and various haemoglobinopathies is also necessary to explore the local variants. Overall, comprehensive understanding of the relationship between primaquine/tafenoquine dose and risk of G6PDd-related haemolysis is required to allow deploying or withholding these drugs in future malaria control or eradication plans in the study area and beyond.
Acknowledgments
The authors are grateful to study patients, staff of Gambella hospital and Regional Health Bureau for their kind support. Dr Hyeonsuk Kim, Research and Development Director, Access Bio Inc., USA and Mr Seokje Park, President, Access Bio Inc., Ethiopia are thanked for providing the CareStart™ G6PDd screening kit. The study was financially supported by a seed money obtained from the Medical Research Council (UK - G0600718), and a thematic research fund from Office of Vice President for Research and Technology Transfer, Addis Ababa University.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.
The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
AT, LG, HM and BE conceived the research idea and participated in the design of the study. AT performed the field work, acquired and analysed the data. HM interpreted the data and prepared the draft manuscript. LG and BE participated in data interpretation and revised the manuscript. All authors have read and approved the final version of the manuscript.