| Abstract|| |
Background: Malaria placed a huge burden on human life and has been reported to be a key health problem affecting developing countries. This study was designed to assay for glucose-6-phosphate dehydrogenase (G6PD) status and malaria parasite density of individuals with sickle cell gene in University of Calabar Teaching Hospital, Calabar. Subjects and Methods: The methemoglobin method was used to determine the G6PD status. Thick blood films were used to ascertain the malaria parasite density while hemoglobin genotype was determined using cellulose acetate paper electrophoresis with tris ethylenediaminetetracetic acid borate buffer (pH 8.6). Thirty hemoglobin SS (HbSS) and 30 hemoglobin AS (HbAS) individuals were recruited for the study while 30 hemoglobin AA (HbAA) individuals were recruited as control. Results: The study showed a high frequency of G6PD deficiency (17.78%) in the study area while G6PD deficiency was significantly (P < 0.05) higher in HbAA individuals (33.33%) when compared to HbSS (10.00%) and HbAS (10.00%) individuals. The prevalence of malaria parasitemia and parasite density was comparable in the three hemoglobin variants. The distribution of malaria parasitemia and parasite density in both gender among the various hemoglobin variants showed no association (P > 0.05). G6PD deficiency distribution in both gender were found to be comparable (P > 0.05). The distribution of malaria parasitemia in the various hemoglobin variants in the G6PD-deficient individuals showed no significant difference (P > 0.5). However, the parasite density of the HbAS (3100 ± 1828.48 μL) and HbSS (2400 ± 1687.06 μL) were significantly lower than that of HbAA (4040 ± 1529.44 μL). Conclusion: The result of this study supports the hypothesis that inheriting the G6PD deficiency gene and sickle cell gene (both in homozygous and heterozygous form) reduces the severity of malaria parasite infection and hence protects against severe acute malaria while having less effect on infection.
Keywords: Glucose-6-phosphate dehydrogenase, hemoglobin, hemoglobin SS, sickle cell, malaria parasite, G6PD
|How to cite this article:|
Okafor IM, Okoroiwu HU, Ekechi CA. Hemoglobin S and glucose-6-phosphate dehydrogenase deficiency coinheritance in AS and SS individuals in malaria-endemic region: A study in Calabar, Nigeria. J Global Infect Dis 2019;11:118-22
|How to cite this URL:|
Okafor IM, Okoroiwu HU, Ekechi CA. Hemoglobin S and glucose-6-phosphate dehydrogenase deficiency coinheritance in AS and SS individuals in malaria-endemic region: A study in Calabar, Nigeria. J Global Infect Dis [serial online] 2019 [cited 2019 Dec 14];11:118-22. Available from: http://www.jgid.org/text.asp?2019/11/3/118/265396
| Introduction|| |
Malaria, a tropical disease, is caused by the protozoa of the genus plasmodium species and is transmitted by the female anopheles mosquito., Severe malaria is a multisystem disorder which may arise from multiple poorly understood processes including acute hemolysis of infected and uninfected red blood cells (RBCs) and dyserythropoiesis as well as through the interaction of malaria infection with other parasitic infections and with nutritional deficiency., Immune processes and genetic traits have contributed in reducing the profligacy of the malaria parasite, and a wide range of genetic polymorphism has been developed to modify individual response to this lethal disease. The high frequency of genetic defect such as the genes for glucose-6-phosphate dehydrogenase (G6PD) deficiency and sickle cell hemoglobin (HbS) in malaria-endemic regions is believed to be due to their advantage against severe malaria infection, especially HbS when in heterozygous form (hemoglobin AS [HbAS]).
G6PD in human is an X-linked enzyme which plays an important role in the generation of reduced nicotinamide adenine phosphate, which is the only source of reducing power in RBC, where it is required to maintain the equilibrium and in particular to detoxify hydrogen peroxide and other compounds through reduced glutathione (GSH). GSH also maintains hemoglobin and other red cells' protein in a reduced active form and possibly enhances the ability of the cells to withstand oxidative damage, particularly during infections and exposure to oxidant drugs. Patients with G6PD deficiency develop hemolytic anemia during acute malaria infection and when treated with certain therapeutic agents such as antimalarial, antipyretics, and antibiotics which have oxidant properties. Increased oxidative stress in G6PD deficiency cell is well documented.
Hemoglobinopathies are the most common monogenic disease and mutation. There are hundreds of hemoglobin variants identified, of which only three: HbS, HbC, and HbE have reached polymorphic frequencies. HbS is due to point mutation in the genetic code in which thymine replaces adenine in the deoxyribonucleic acid encoding the beta globin gene; consequently, valine replaces glutamate at the 6th position in the beta globin product. The gene for HbS is distributed widely throughout sub-Saharan Africa and countries with African immigration where a carrier frequency ranges from 5% to 40% or more., This study was aimed at investigating the effect of inheritance of G6PD deficiency and hemoglobin SS (HbSS) in malaria parasitaemia and severity.
| Subjects and Methods|| |
Subjects and sample collection
This study was approved by Health Research Ethical Committee of the University of Calabar Teaching Hospital (UCTH). Informed consent was obtained from the participants. Thirty known sickle cell patients were recruited into the study from hematology day care unit of the UCTH, Calabar, Nigeria. They comprised of 13 females and 17 males all within the age range of 5–30 years of age and were not having any crisis and were in hospital for routine check. Sixty apparently healthy individuals comprising of 30 HbAS (used as part of the test individuals) and 30 hemoglobin AA (HbAA) individuals (used as control) from Calabar metropolis were also recruited. Three milliliters of venous blood was collected by venipuncture from each individual into ethylenediaminetetraacetic acid container. The methemoglobin reduction method by Brewer et al. was used to screen for G6PD deficiency. Hemoglobin electrophoresis was done using cellulose acetate electrophoresis at pH 8.6. For the laboratory diagnosis of malaria parasite infection, thick blood films were prepared for each individual and stained using Giemsa staining method as described by Cheesbrough. Malaria parasites were counted against white blood cells (WBCs). A minimum of 1000 WBCs were counted, and the number of malaria parasites counted per white cells were recorded. The parasite density was then converted to parasite per milliliter of blood according to the formula below:
Data generated in this study were analyzed using SPSS version 20 (IBM Corp., Armonk, NY, USA). Categorical variables were analyzed using frequency, percentages, and Chi-square while continuous variables were analyzed using descriptive statistics, t-test, and ANOVA. Chi-square was used to assess association among variables, while t-test and ANOVA were used to assess differences between two and multiple means, respectively. Alpha value was set at 0.05.
| Results|| |
The G6PD deficiency distribution among the HbAA, HbAS, and HbSS individuals were 33.33% (n = 10), 10.00% (n = 3), and 10.00% (n = 3), respectively. The distribution was statistically significant (P< 0.05). Malaria parasitemia in this study was found to be 17.78% (n = 16). Among these, 8 (26.67%), 5 (16.67%), and 3 (10.00%) were recorded in HbAA, HbAS, and HbSS variants, respectively. The malaria prevalence was comparable in the different hemoglobin variants (P > 0.05). The mean parasite density of the hemoglobin variants were comparable (P > 0.05) with HbAA, HbAS, and HbSS having mean values of 3725 ± 1888.1 μL, 4320 ± 2057.6 μL, and 2360 ± 981.4 μL, respectively [Table 1].
|Table 1: Distribution of glucose-6-phosphate dehydrogenase deficiency, malaria parasitemia, and parasite density among the studied population|
Click here to view
Gender distribution of malaria parasitemia and parasite density in the studied population showed comparable values for the males and the females (P > 0.05). Approximately 16.67% (n = 5%), 16.6% (n = 5), and 6.67% (n = 2) represented males with malaria parasitemia while 10.0% (n = 3), nil, and 3.33% (n = 1), respectively, represented females with malaria parasitemia. The mean parasite density of the male and female HbAA individuals were 4064 ± 1487 μL and 3168 ± 3011 μL, respectively, while the HbAS had mean values of 4320 ± 2057.6 μL and nil, respectively, for male and female individuals. The HbSS individuals had malaria parasite densities of 1740 ± 754 μL and 1374 ± 1309 μL for the male and female individuals, respectively [Table 2].
|Table 2: Frequency of malaria parasitemia and malaria parasite density of all individuals among the hemoglobin variants based on gender|
Click here to view
[Table 3] shows the gender distribution of G6PD-deficient individuals in the studied population. Approximately 21.56% (n = 11) of the males were G6PD deficient while 12.82% (n = 5) of the females were G6PD deficient. The distribution of G6PD deficiency was found not to be associated with gender (P > 0.05).
|Table 3: Gender distribution of glucose-6-phosphate dehydrogenase-deficient individuals in all the individuals|
Click here to view
The gender distribution of G6PD deficiency among the various hemoglobin variants was found to be comparable in all groups. Distribution of G6PD among the various hemoglobin variants was found not to be associated with gender (P > 0.05) [Table 4].
|Table 4: Distribution of glucose-6-phosphate dehydrogenase deficiency among hemoglobin variants based on gender|
Click here to view
[Table 5] shows the prevalence of malaria parasitemia and malaria parasite density of G6PD-deficient individuals according to the various hemoglobin variants. Approximately 50.0% (n = 5), 66.7% (n = 2), and 66.7% (n = 2) of the HbAA, HbAS, and HbSS G6PD-deficient individuals, respectively, had malaria parasitemia. However, this distribution is not statistically significant (P > 0.05). The mean parasite density of the HbAS (3100 ± 1828.48 μL) and HbSS (2400±1687.06 μL) were found to be significantly lower than that of HbAA (4040 ± 1529.44 μL) (P< 0.05).
|Table 5: Malaria parasitemia and malaria parasite density of glucose-6-phosphate dehydrogenase-deficient individuals in different hemoglobin variants|
Click here to view
| Discussion|| |
G6PD is very significant to red cell survival. Its deficiency deprives the red cell of the reducing power necessary for protection against oxidation. The malaria hypothesis maintains that the average people without G6PD deficiency and sickle cell gene died of malaria at higher frequency.
This study had a malaria parasitemia prevalence of 17.78%. This value was quite lower than earlier report of 80% by Orok et al. This large margin may be due to seasonal variation in malaria prevalence.
This study recorded 17.78% prevalence of G6PD deficiency among the studied population. This value is lower than 37.6% reported in previous study in Sokoto, Nigeria. In contrast, lower values were documented in developed countries such as America and in Europe. G6PD deficiency has been attributed to genetic adaptation to malaria in malaria-endemic regions, and seems to corroborate the malaria protection hypothesis and also the role malaria plays in the distribution of G6PD gene in most malaria-endemic areas in the world. Although the electrophoretic mobility was not carried out to ascertain the G6PD variants, the common African variant G6PD A - was assumed.
More so, the HbAS and HbSS hemoglobin variants had the least prevalence of malaria parasitemia. However, this variation was not statistically significant. This trend is consistent with earlier study by Bougouma et al. and Carnevale et al. Their result showed that incidence and premunition of malaria is comparable among individuals of different hemoglobinopathies going by the malaria prevalence and parasite density. Conversely, some studies, have reported protective effect of abnormal hemoglobin against clinical and subclinical malaria. Some researchers have proposed; decreased RBC invasion/poor growth under high oxygen tension, accelerated acquisition of antibodies specific for Plasmodium falciparum erythrocyte membrane protein-1 and other variant surface antigens as mechanisms of this protection.
The effect of G6PD deficiency on individuals with sickle cell trait and sickle cell anemia is controversial. Several reports have emphasized increased frequency of G6PD deficiency in patients with sickle cell disease. In these reports, there are contradictory views on the issue of the protective effect of the enzyme deficiency on the clinical manifestation of sickle cell anemia. Some reports claim that the combination is beneficial in sickle cell trait while others have found no beneficial or adverse relationship. In the present study, coinheritance of G6PD deficiency with HbAS and HbSS was both 10%. This finding is similar to 7% reported in previous study in Ghana. Coinheritance of both the G6PD deficiency and the sickle cell gene has been reported to confer a better protection against malaria., In this study, G6PD deficiency was associated with reduction in the risk of severe malaria for both G6PD-deficient HbAS and HbSS individuals as compared to HbAA individuals who were also G6PD deficient as seen in the significantly lower parasite density despite the fact that it did not show any association in preventing malaria parasitemia/incidence of malaria parasitemia. This observation is in agreement with the report of Awah and Uzoegwu. Malaria parasite density provides information on the severity of infection. The G6PD-deficient parasitized erythrocytes may have been phagocytized earlier thereby destroying the malaria parasite, hence keeping the parasite load low. This finding supports the premunition hypothesis of the protective effect of coinheritance of G6PD and HbS gene in malaria-endemic region. Premunition is not a sterile type of immunity, but it ensures that maximum parasite load is kept at low level.
Gender distribution in G6PD deficiency is comparable in both males and females, though the proportion being higher in males (21.56%) than females (12.82%), showing male preponderance. However, this variation was not statistically significant (P = 0.366). This finding is in consonance with previous studies,, while same is in contrast with the findings of Jelani et al. Some authors have argued that considering the fact that the abnormal gene responsible for G6PD deficiency is located on the X chromosome, and males are hemizygous while females are dizygous for X chromosomes, that the probability of finding two genes of G6PD mutation on chromosome X is lower in females.
| Conclusion|| |
We conclude that G6PD coexistence with hemoglobin S gene (HBAS and HbSS) does not offer protective role in incidence of malaria infection, but, however, helps to adapt to low severity of the infection via low parasite density (premunition). However, there is still need for a collaborative study between scientists involving larger sectors of the population to be able to shed more light on the unresolved aspects of coinheritance of these two red cell genetic abnormalities and their interaction, especially in malaria-endemic regions.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Tsuji M, Rodrigues EG, Nussenzweig S. Progress toward a malaria vaccine: Efficient induction of protective anti-malaria immunity. Biol Chem 2001;382:553-70.
Okafor IM, Akpan PA, Usanga. EA. Prevalence and types of anaemia in malaria infected pregnant women attending antenatal clinic in university of Calabar teaching hospital, Calabar, Nigeria. J Nat Sci Res 2012;2 (7):73-79.
Newton CR, Warn PA, Winstanley PA, Peshu N, Snow RW, Pasvol G, et al.
Severe anaemia in children living in a malaria endemic area of Kenya. Trop Med Int Health 1997;2:165-78.
Okafor IM, Mbah M, Usanga EA. The impact of anaemia and malaria parasite infection in pregnant women. Nigerian perspective. IOSR J Dent Med Sci 2012;1: 34-8.
Flint J, Harding RM, Biyce AJ, Cleg JB. The Population Genetics of the Hemoglobinopathies. London: BaillereTindall, W. B. Saunders; 1998. p. 1-51.
Allison AC. The discovery of resistance to malaria of sickle cell heterozygotes. Biochem Mol Biol Educ 2002;30:279-87.
Mason PJ, Bautista JM, Gilsanz F. G6PD deficiency: The genotype-phenotype association. Blood Rev 2007;21:267-83.
Liu TZ, Lin TF, Hung IJ, Wei JS, Chiu DT. Enhanced susceptibility of erythrocytes deficient in glucose-6-phosphate dehydrogenase to alloxan/glutathione-induced decrease in red cell deformability. Life Sci 1994;55:PL55-60.
Weatherall DJ, Clegg JB. Genetic variability in response to infection: Malaria and after. Genes Immun 2002;3:331-7.
Ashley-Koch A, Yang Q, Olney RS. Sickle hemoglobin (HbS) allele and sickle cell disease: A huge review. Am J Epidemiol 2000;151:839-45.
Brewer GJ, Tarlov AR, Alving AS. The methemoglobin reduction test for primaquine-type sensitivity of erythrocytes. A simplified procedure for detecting a specific hypersusceptibility to drug hemolysis. JAMA 1962;180:386-8.
Cheesbrough M. District Laboratory Practice in Tropical Countries Part 2. Cambridge: University Press; 2000. p. 271-340.
World Health Organization. World Malaria Report. Geneva, Switzerland: World Health Organization; 2013. p. 32-42.
Beutler E. G6PD: Population genetics and clinical manifestations. Blood Rev 1996;10:45-52.
Orok DA, Usang AI, Ikpan OO, Duke EE, Eyo EE, Edadi UE, et al
. Prevalence of malaria and typhoid fever co-infection among febrile patients attending college of health technology medical centre in Calabar, Cross River state, Nigeria. Int J Curr Microbiol Appl Sci 2016;5:825-35.
Jelani I, Garba N, Zakariyya A. Isah SV. Co-inheritance of Glucose-6-phosphate dehydrogenase deficiency and sickle cell trait in Sokoto metropolis. Asian J Med Health 2017;2:1-6.
Nkhoma ET, Poole C, Vannappagari V, Hall SA, Beutler E. The global prevalence of glucose-6-phosphate dehydrogenase deficiency: A systematic review and meta-analysis. Blood Cells Mol Dis 2009;42:267-78.
Troye-Blomberg M, Perlmann P, Mincheva Nilsson L, Perlmann H. Immune regulation of protection and pathogenesis in Plasmodium falciparum
malaria. Parassitologia 1999;41:131-8.
Kar BC, Agrawal KC, Panda A. Sickle haemoglobin, G-6PD deficiency and malaria in Western Orissa. J Assoc Physicians India 1990;38:555-7.
el-Hazmi MA, Warsy AS. Interaction between glucose-6-phosphate dehydrogenase deficiency and sickle cell gene in Saudi Arabia. Trop Geogr Med 1987;39:32-5.
Beutler E. G6PD deficiency. Blood 1994;84:3613-36.
Bougouma EC, Tiono AB, Ouédraogo A, Soulama I, Diarra A, Yaro JB, et al.
Haemoglobin variants and Plasmodium falciparum
malaria in children under five years of age living in a high and seasonal malaria transmission area of Burkina Faso. Malar J 2012;11:154.
Carnevale P, Bosseno MF, Lallemant M, Feingold J, Lissouba P, Molinier M, et al. Plasmodium falciparum
malaria and sickle cell gene in the popular republic of Congo. I. Relationship between parasitemia and sicke cell trait in Djoumouna (region of Brazzaville) (author's transl). Ann Genet 1981;24:100-4.
Modiano D, Luoni G, Sirima BS, Simporé J, Verra F, Konaté A, et al.
Haemoglobin C protects against clinical Plasmodium falciparum
malaria. Nature 2001;414:305-8.
Agarwal A, Guindo A, Cissoko Y, Taylor JG, Coulibaly D, Koné A, et al.
Hemoglobin C associated with protection from severe malaria in the Dogon of Mali, a West African population with a low prevalence of hemoglobin S. Blood 2000;96:2358-63.
Friedman MJ. Erythrocytic mechanism of sickle cell resistance to malaria. Proc Natl Acad Sci U S A 1978;75:1994-7.
Marsh K, Otoo L, Hayes RJ, Carson DC, Greenwood BM. Antibodies to blood stage antigens of Plasmodium falciparum
in rural Gambians and their relation to protection against infection. Trans R Soc Trop Med Hyg 1989;83:293-303.
Awah FM, Uzoegwu PN. Influence of sickle cell heterozygous status and glucose-6-phosphate dehydrogenase deficiency on clinic – Haematologucal profile of Plasmodium faciparum
- infected children. Biokemistri 2006;18:89-97.
Ouattara AK, Yameogo P, Diarra B, Obiri-Yeboah D, Yonli A, Compaore TR, et al.
Molecular heterogeneity of glucose-6-phosphate dehydrogenase deficiency in Burkina Faso: G-6-PD betica selma and Santamaria in people with symptomatic malaria in Ouagadougou. Mediterr J Hematol Infect Dis 2016;8:e2016029.
Adu P, Simpong DL, Takyi G, Ephraim RK. Glucose-6-phosphate dehydrogenase deficiency and sickle cell trait among prospective blood donors: A cross-sectional study in Berekum, Ghana. Adv Hematol 2016;2016:7302912.
KhooK K. Glucose-6-phosphate dehydrogenase and malaria. Australas Med J 2010;3:422-5.
World Health Organization. Malaria Parasite Counting. Malaria Microscopy Standard Operation Procedure. Ver. 1. Geneva: World Health Organization; 2016. p. 1-5.
Obi RK, Okangba CC, Nwanebu FC, Ndubuisi UU, Orji NM. Premunition in Plasmodium falciparum
malaria. Afr J Biotechnol 2010;9:1397-401.
Sanpavat S, Nuchprayoon I, Kittikalayawong A, Ungbumnet W. The value of methemoglobin reduction test as a screening test for neonatal glucose 6-phosphate dehydrogenase deficiency. J Med Assoc Thai 2001;84 Suppl 1:S91-8.
Ferreira A, Marguti I, Bechmann I, Jeney V, Chora A, Palha NR, et al.
Sickle hemoglobin confers tolerance to Plasmodium
infection. Cell 2011;145:398-409.
Mohammad AM, Ardatl KO, Bajakian KM. Sickle cell disease in Bahrain: Coexistence and interaction with glucose-6-phosphate dehydrogenase (G6PD) deficiency. J Trop Pediatr 1998;44:70-2.
Dr. Ifeyinwa M Okafor
Department of Medical Laboratory Science, Hematology Unit, College of Medical Sciences, University of Calabar, Calabar
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]