Prevalence and genetic variants of G6PD deficiency among malaria suspected patients in Shele and Lante Health Center, Southern Ethiopia

doi:10.21203/rs.3.rs-1637812/v1

Download PDF

Research Article

Prevalence and genetic variants of G6PD deficiency among malaria suspected patients in Shele and Lante Health Center, Southern Ethiopia

https://doi.org/10.21203/rs.3.rs-1637812/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background

The glucose-6 phosphate dehydrogenase (G6PD) enzyme is a cytoplasmic enzyme for all human cells, involves in the pentose phosphate pathway of metabolic reaction. It protects red blood cells from cellular oxidative damage by detoxifying free radicals. Primaquine can cause acute hemolytic anemia while administered for the treatment of malaria patients with G6PD deficiency. It is the first-line drug against the pre-erythrocyte stage of P. vivax and the gametocyte stage of P. falciparum. The study aims to determine the G6PD deficiency among malaria-suspected patients.

Method

A health facility-based cross-sectional study was conducted for a total of 858 self-presented malaria suspected patients in Shele and Lante health centers. A structured questionnaire was used to collect the socio-demographic and clinical information of the study participants. Blood samples were collected for onsite G6PD activity measurement, DBS preparation, and malaria microscopy. The G6PD enzyme activity was measured by the CareSTART G6PD biosensor analyzer. Data was entered and analyzed by using SPSS version 20.

Results

Among 858 study participants, 49.3% (423) were males. The median age and interquartile range were 26 and 21 years respectively. The median of the G6PD enzyme level was 5.9, and the interquartile range of the G6PD enzyme level was 4.2 respectively. Of the study participants, 14.3%, 9.3%, and 4.1% were infected with P. falciparum, P. vivax, and mixed parasites respectively. The phenotypic G6PD deficiency rate was 4.8% (41/858).The common G6PD mutations identified in the study population were A376G (0.35%), G267 + 119C/T (1.05%), and G1116A (0.35%). Additional mutations such as A376T (A◊T) and G1116T (G◊T) were identified for the first time in Ethiopia.

Conclusion

The prevalence rate of G6PD deficiency was modest. However, the clinicians have to consider hemolysis risk during primaquine treatment in the study area. G267 + 119C/T was the most frequent variant, although A376G and G1116A were also identified.

G6PDd

Hemoglobin

Plasmodium vivax

Plasmodium falciparum

Primaquine

The glucose-6 phosphate dehydrogenase (G6PD) enzyme is a cytoplasmic enzyme in the pentose phosphate pathway of all human cells in the body and critically appears in red blood cells (RBCs). It prevents cellular oxidative damage to red blood cells by detoxifying free radicals [1–3]. Glucose-6 phosphate dehydrogenase (G6PD) deficiency is the most common enzymatic disorder of red blood cells affecting approximately 400 million people worldwide. Especially, it is high in Africa, Asia, the middle east, Latin America, the Caribbean, and Mediterranean countries [4, 2, 5–8]. The total prevalence rate of G6PD deficiency in Ethiopia is estimated at about 1–3% [9]. However, it showed a wide variation among the highlands and the lowland areas. It ranges from 1% in the highland parts of the country to 14% in the lowland areas like the Gambela and the Afar regions [10, 8].

G6PD deficiency (G6PDd) is caused by the coding gene's mutation, ultimately resulting in free radical-mediated oxidative damage to red blood cells and causing acute hemolysis. This is more common in hemizygous males than females since it’s X-linked enzymopathy. Females can be either homozygous or heterozygous. Heterozygous females can have a mixed population of RBCs with normal and deficient G6PD enzyme activity [7, 11].

Based on the biochemical and physicochemical characteristics of G6PD, more than 400 variants have been described to date, of which approximately 186 variants are related to G6PD deficiency [12]. Of these, most of the variants are single nucleotide substitutions, while the remaining have either more than one nucleotide substitution, deletions, and/or mutations of introns [4]. In Africa, the common genotyped variants were G6PD B⁺ (wild type), G6PD A⁺ (376), G6PD A⁻ (202), and Mediterranean variant (563) [13, 14, 7, 11]. Of these variants, A⁺ is a non-deficient G6PD-type variant, while A⁻ and Mediterranean variants are known deficient variants. In Ethiopia, an estimated prevalence rate of A⁺ (A376G) and A⁻(G202A) variants accounted for approximately 6–17% and 3.5%, respectively [14, 15].

G6PD deficient individuals appear normal and clinically silent until exposed to free-radical elements, e.g., primaquine. It causes drug-induced hemolysis during treatment for malaria parasites. Primaquine is the only available drug to treat the dormant liver stage (hypnozoite form, pre-erythrocytic phase) of P. vivax and P. falciparum gametocytes stage [16].

Ethiopia is a country in which 68% of the population is at risk of malaria and P. vivax and P. falciparum coexist, and account for about 40% and 60% of the malaria cases respectively [14, 17]. Moreover, most of the P. vivax infection episodes are occurred by relapsing and this, in turn, is due to the administration of non-primaquine treatment [18, 19].

In line with the Ethiopian national malaria elimination plan since 2016 primaquine came to be the choice of the drug against the pre-erythrocyte stage of P. vivax and, the gametocyte stage of P. falciparum which is the key point of malaria transmission interruption [20]. Although the drug treatment can safely block the transmission of P. falciparum and P. vivax [21], the risk of developing drug-induced hemolysis is a strong challenge. Patients who had RBC’s G6PD enzyme activity < 30% of the normal G6PD activities had hemolysis in common after administration of primaquine. Unfortunately, it is high in P. vivax-infected patients because of a higher dose of primaquine (0.25–0.5 mg for 14 days) than in P. falciparum (a single dose of 0.25 mg) [22, 16, 20, 23, 11].

Therefore, determining the prevalence of G6PDd is important for safe case management using primaquine. This study aims to determine the prevalence of G6PDd among malaria suspected patients in Shele and Lante health center, Southern Ethiopia.

Study area

Arba Minch Zuria is a woreda in the Southern Nations, Nationalities, and Peoples' Region of Ethiopia. It is a part of the Gamo Zone located in the Great Rift Valley (Fig. 1). Arba Minch Zuria is bordered by Chencha in the north, Dirashe special woreda in the south, Bonke in the west, and Guji Zone in the east. This woreda also includes two lakes and their islands, Lake Abaya found in Lante kebele, Lake Chamo found in Kanchama, Ochole, Ganta, and Zeyise kebeles, and yegizer dildiy in between as part of the Nechisar National Park. The city of Arba Minch is bounded by Arba Minch Zuria. This is the most resourceful woreda and the range of most tourists. Banana, avocado, and mango were the widely cultivable fruits in the areas.

Shele health center is located in 5.89894^o N and 37.43443^o E while Lante health center is located in 6.13739^o N and 37.64495^o E, with altitude ranges from 1191m to 1285m above sea level. Mean annual temperature ranges from 16^oc to 37^oc. The transmission of malaria parasites is highly seasonal and unstable and P. falciparum is the dominant one [24].

Based on the 2007 Census conducted by the CSA, this woreda has a total population of 164,529, of whom 82,199 were men and 82,330 were women (CSA, 2007). A total of 858 study participants, 501 from shele health center and 357 from Lante health center were enrolled in the study. An estimated male to female ratio was almost one to one.

A health facility-based cross-sectional study was conducted for 858 study participants who had the signs and symptoms of malaria and who sought malaria parasite diagnosis in Shele and Lante health center, never received antimalaria medications in the last 60 days were included in the study during the study period from February 2021 until April 2021.

During the study, a pre-validated questionnaire was interviewed each study participant to record demographic data, history of previous malaria infection, and signs and symptoms of malaria.

Laboratory Diagnosis

The study was performed by measuring the amount of G6PD enzyme activities and molecular characterization of the G6PD gene. Capillary and venous blood was obtained from each study participant; capillary blood was used to perform a rapid diagnostic test (RDT) and blood film, whereas venous blood (3–4 ml) was used to perform dry blood spot (DBS) and measure G6PD enzyme activities. Blood film and DBS samples were examined to identify malaria species and perform molecular analysis respectively. CareSTART PF/PV (HRP2/3/ PLDH; Lot No MV19861) malaria RDT was used to support the microscopic detection of malaria parasites. G6PD enzyme activities were determined by using a quantitative rapid CareSTART G6PD biosensor machine (sensitivity = 92–100%, and specificity = 92–94% against spectrophotometric method) [25]. G6PD genetic characterization was performed at the University of North Carolina (UNC) at Charlotte, USA.

G6PD phenotypic measurements

The G6PD enzyme level was measured for 858 clinical samples following the manufacturer’s instructions. The diagnosis of the G6PD enzyme is based on the estimation of enzyme activity by quantitative spectrophotometric analysis of the rate of NADPH production from NADP [26]. The G6PD enzyme level was normalized by the concentration of hemoglobin (i.e., unit of G6PD enzyme per gram of hemoglobin, U/g Hb). G6PD normal and deficient individuals were categorized into three classes in females and two classes in males on the basis of the WHO classification[27]. The adjusted male median (AMM) G6PD activity was calculated after excluding samples with less than 10% of the overall male median activity. For females, G6PD enzyme activity < 30%, and > 80% of the AMM enzyme activity were homozygote deficient and normal respectively while 30–80% of the AMM enzyme activity were intermediate. For males, G6PD enzyme activities < 30% of the AMM activity were hemizygotes deficient while G6PD enzyme activities > 30% of the AMM activity were hemizygotes normal as show in Table 1 [14].

Table 1

Classification of G6PD enzyme activities among males and females in Shele, and Lante health centers from Feb 2021 to April 2021
Male	Deficient	< 30% of the AMM	< 1.8
	Normal	> 30% of the AMM	> 1.8
Female	Deficient	< 30% of the AMM	< 1.8
	Intermediate	30–80% of the AMM	1.8–4.8
	Normal	> 80% of the AMM	> 4.8

Examination of Malaria Parasite

Capillary blood samples were smeared, dried, fixed (thin films only were fixed with absolute methanol), and stained with Giemsa working solution (1:10) to examine malaria parasites, and in parallel, RDT was performed based on the manufacturer’s instruction. The microscopic examination of the blood film yielded the malaria parasite in terms of species and stage, and the parasitic load was calculated against 200 WBCs while it was double-checked by an accredited malaria expert in the Adama malaria center.

G6PD genotyping

A total of thirteen samples, i.e., ten G6PD deficient (six males and four females) and randomly selected three intermediate females were directed to molecular G6PD characterization. Four PCR assays were conducted to determine the G6PD gene mutations of exon 3–11. PCR amplification was conducted in a 20µl reaction mixture containing 2µl of genomic DNA (~ 50ng/µl), 10µl of 2×Maxima SYBR Green PCR Master Mix (Thermo Fisher), and 0.3uM of each forward and reverse primer. For each PCR assay, water was used in a separate reaction as a negative control. Amplifications were done through an initial denaturation at 94°C for 3 min, followed by 38 cycles at 94°C for 30 sec, 58°C for 30 sec and 72°C for 60 sec, with a final 6 min extension at 72°C [14]. Then, the PCR products were run by gel electrophoresis with 1.5% agarose gel at 120 Volt for two hours and sequenced on an ABI 3730xl DNA analyzer based on the protocols. Sequences were analyzed using BioEdit. All sequences were aligned to the NCBI reference sequence to check the specificity of the PCR products. Amplified PCR products with poor sequencing quality or exhibited singleton mutations were sequenced repeatedly.

Data processing and statistical analysis

All laboratory and clinical data were recorded in the questionnaire and copied into SPSS V.20 during the study period. Descriptive statistics were used to describe the frequency of each variable. A binary logistic regression model and independent t-test were performed to assess factors associated with G6PD deficiency and genetic variants among study participants. The phenotypic classifications of the G6PD enzyme were performed based on the WHO classification by calculating the adjusted male median (AMM). P-value < 0.05 was considered statistically significant.

Socio-demographic characteristics of study participants

A total of 858 malaria suspected patients were included in the study. Of these, 49.3% (423) were males. The median age of the study participants was 26, and the interquartile range was 21 years. A mean hemoglobin value was 12.13 (95% CI: 11.98, 12.27) g/dl. Of the study participants, 80.4% were above the age of 14, and small proportions of the participants were below the age of five (1.7%). A total of 79.6% (357) of the participants had a history of malaria infection (Table 2).

Among the study participants, most of them had fever and headaches while the least had abdominal discomfort. This was the same for malaria-positive cases in that almost all malaria-positive cases had fever and headache whereas abdominal discomfort (12%) and fatigue (32%) were not typically appeared in positive cases. This finding also revealed that muscle and joint pain, vomiting (anorexia), shaking, and chills appeared almost equally in positive and negative cases

Table 2

Sociodemographic and clinical characteristics of study participants from February 2021 to April 2021 in Shele and Lante health center, Southern Ethiopia
Socio-demographic characteristics N = 858		N (%)
Sex
	Males	423 (49.3)
	Females	435 (50.7)
Age group
	< 5 years old	15 (1.7)
	5–14 years old	153 (17.8)
	> 15 years old	690 (690)
Place of residence
	Urban	389 (45.3)
	Rural	466 (54.3)
History of malaria infection
	Yes	683 (79.6)
	No	172 (20.3)
Clinical signs and symptoms
Headache N = 858
	Yes	806 (93.9)
	No	52 (6.1)
Fatigue N = 858
	Yes	302 (35.2)
	No	556 (64.8)
Muscle and Joint pain N = 856 (99.8)
	Yes	457 (53.3)
	No	399 (46.5)
Shaking and chills N = 858
	Yes	383 (44.6)
	No	457 (55.4)
Sweating N = 858
	Yes	325 (37.9)
	No	533 (62.1)
Anorexia N = 858
	Yes	265 (30.9)
	No	593 (69.1)
Abdominal discomfort N = 858
	Yes	157 (18.3)
	No	701 (81.7)

All study participants were examined for malaria parasites with careStart PF/PV malaria RDT and microscopy in Shele and Lante health center routinely while only 853 (99.4%) study participants were examined by malaria experts in Adama malaria center. The result was shown disagreement between RDT, routine microscopy, and expert microscopy, i.e., P. falciparum detection rate was 22.1%, 22.4%, and 14.3% while P. vivax detection rate was 11.7%, 11.3%, and 9.3% among RDT, Routine microscopy, and expert microscopy respectively. There was high disagreement in P. falciparum and mixed parasites detection rate between expert microscopy, and routine microscopy as well as malaria RDT results. Even though there is an absolute difference in numbers, there was no statistically significant difference between the two methods (P = 0.07). Negative results were almost agreed in the three dimensions. Similarly, the malaria RDT result, and the routine microscopic result was also concordant (Fig. 2).

The median and interquartile range of the G6PD enzyme level was 5.9, and 4.2 respectively. The AMM of participants was 6.0 after excluding < 10% of the median activity. Based on this, values less than 0.6 G6PD enzyme level were excluded and only 415 male participants were analyzed for their respective AMM results.

Therefore, participants with G6PD enzyme levels less than 1.8 were 41 (4.8%) and were considered as deficient. Males were accounted for about 4.3% (18) while G6PD deficient and intermediate females had accounted for about 5.3% (23) and 29% (126) respectively. Based on the expert microscopic result, of 41 G6PD deficient participants, 19 were negative while 22 were positive for malaria parasites. Of these, only two participants had shown severe anemia exhibiting below 5g/dl of hemoglobin, of which one was pregnant and P. falciparum-infected with high parasitemia (> 2000 parasite/µl of WBCs), and the other was negative for malaria parasites.

Assessment of risk Factors for G6PD deficiency

Almost all G6PD deficient (89.5%) were above the age of 14 years old. However, no significant association was observed with G6PD deficiency despite the remarkable difference in frequency. This finding also revealed that P. falciparum infection and G6PD deficiency had no significant association although they had a high chance of being deficient than non-P. falciparum participants (AOR, 1.5; CI, 0.33–6.87; P = 0.6) (Table 3). This is also the same with all malaria-positive cases (AOR, 2.6; CI, 0.47–14.26; P = 0.3) and for P. vivax positive cases (P > 0.05). Regarding the place of residence, G6PD deficiency was four times highly likely to occur in a rural area than compared the urban territory (P = 0.002) (Table 3).

Of the study participants, 27.9% (239) were positive for malaria parasites which were diagnosed with microscopy. Of these, the expert microscopic result revealed that 26.7% of positive cases had corresponding parasite load count. This was first categorized into < 200, 201–2000, and > 2000 parasite/µl of 200 WBCs. Then, the corresponding parasite load of positive cases was compared between G6PD deficient and normal. The correlation coefficient between the G6PD enzyme level and the parasite load was 0.08 (P = 0.2), the correlation was statistically not significant. This was also confirmed by logistics regression output (P = 0.5, 95% CI, 0.132–2.822). However, the overall parasite load proportion of G6PD normal versus G6PD deficient had a statistically significant difference (P < 0.05) (STATA V.14).

Table 3

Association of factors with G6PD deficiency among malaria suspected patients from February 2021 to April 2021 in Shele and Lante health center, Southern Ethiopia
Variables	Categories	G6PD Deficiency
Variables	Categories	COR (95% CL)	P-value	AOR (95% CL)	P-value
P. falciparum Positivity	Negative	1		1
P. falciparum Positivity	P. falciparum	3.4 (1.765–6.440)	< 0.001	1.5 (0.33–6.87)	0.6
Malaria positivity	Negative	1		1
Malaria positivity	Positive	3.2 (1.685–5.980)	< 0.001	2.6 (0.47–14.26)	0.3
P. vivax positivity	Negative	1		1
P. vivax positivity	P. vivax	1.9 (0.870–4.034)	0.1	0.9 (0.25–3.78)	0.9
Sex	Female	1		1
Sex	Male	0.8 (0.423–1.498)	0.5	0.9 (0.42–1.71)	0.7
Age group	> 14	1		1
	< 5	0	1	0
	5–14	0.5 (0.166–1.350)	0.16	0.3 (0.06–1.13)	0.07
Place of residence	Urban	1		1
Place of residence	Rural	0.2 (0.66 − 0.442)	< 0.001	4.5 (1.7-12.04)	0.002
History of infection	No	1		1
History of infection	Yes	1.7 (0.652–4.410)	0.28	0.8 (0.29–2.27)	0.7
Treatment complication	No	1		1
Treatment complication	Yes	3.6 (1.440–9.213)	0.006	2.7 (1.002–7.33)	0.05

Genotype characteristics of Glucose-6 Phosphate Dehydrogenase

A total of thirteen, six male, and seven female participants were recruited for molecular characterization (Table 4). i.e., G6PD A⁺ (A◊G & A◊T) at nucleotide position of 376 was detected in 3 (23.1%) of the study participants. Of these, A◊T was shown in 2 (15.4%) of the phenotypically deficient participants and the female sex only. Interestingly, this was the first time to been isolated in our country. G267 + 119C/T and G1116A were also detected in nine (69.2%) and three (23.1%) of the study participants respectively. No mutation was detected for G6PD A⁻ (G202A) and Mediterranean type (C563T) among all participants (Table 4).

Of the study participants, five of them had shown genotypic polymorphism while five of the study participants had mutation due to single nucleotide substitution. Among participants with polymorphic mutation, one homozygous participant who was > 14 years old had a triple mutation at three nucleotide positions (A376T, G267 + 119C, and G1116A) while the remaining four participants had double mutations.

Three of the phenotypically deficient study participants had not shown any mutation in their G6PD gene. Among the individuals who did not have mutations, none of them were phenotypically intermediate. The mean of non-mutant individuals’ phenotypic G6PD enzyme level was 0.87 (0.1–1.4) while the average mean of mutant genes of individuals was 1.79 (0.1-5.0) (mean diff. =0.9, 95% CI; -0.954-2.801, P = 0.3). However, it was not significantly associated.

Table 4

Frequency of G6PD genetic mutation at Exon three to eleven among malaria suspected patients from February 2021 to April 2021 in Shele and Lante Health Center, Southern Ethiopia
Target Genes N = 13			Frequency (%)
A376G	N (%)		10 (76.9)
	Wild Type (%)		7 (53.8)
	Mutant	A→G (%)	1 (7.7)
	Mutant	A→T (%)	2 (15.4)
G202A	N (%)		9 (69.2)
	Wild Type (%)		9 (69.2)
	Mutant	G→A (%)	0
G267 + 119C/T	N (%)		11 (84.6)
	Wild Type (%)		2 (15.4)
	Mutant	G→C (%)	9 (69.2)
Chrx:154535443 G→C	N (%)		12 (92.3)
	Wild Type (%)		11 (84.6)
	Mutant (%)		1 (7.7)
485 + 37 (G→T)	N (%)		10 (76.9)
	Wild Type (%)		10 (76.9)
	Mutant (%)		0
G1116A	N (%)		12 (92.3)
	Wild Type (%)		9 (69.2)
	Mutant	G→A (%)	2 (15.4)
	Mutant	G→T (%)	1 (7.7)
C563T	N (%)		11 (84.6)
	Wild Type (%)		11 (84.6)
	Mutant	C→T (%)	0
Polymorphism [N = 5/13 (38.5%)
Triple mutation	A376G G267 + 119C/T 2G1116A67 + 119C/TG1116A	A→T G→C G→A G→C G→T	1 (7.7)
Double mutation	G267 + 119C/T G1116A	A→T G→C G→A G→C G→T	4 (30.8)
	G267 + 119C/T Chrx:154535443	G→C G→C
	A376G G267 + 119C/T	A→T G→C
	A376G G267 + 119C/T	A→G G→C
Among the sequenced samples, five were positive for malaria parasites (four P. falciparum and one P. vivax). The binary logistic regression revealed no significant association between G6PD mutation and malaria positive cases even if the odds of having mutation increase by 1.3 times in positive cases than in malaria negative cases (COR, 1.3; 95% CI, 0.09–20.1; P = 0.8).

Regarding the phenotypic status association with genotype status, there was no statistically significant association, since the P-value (Fisher’s exact test, 0.53) is larger than that of the conventional significance level (P > 0.05). Similarly, we were also analyzing the potential factors and whether there was a significant implication for the gene mutation (Table 5). However, there was no significant association.

Table 5

Association of factors with G6PD genetic mutation among malaria suspected patients from February 2021 to April 2021 in Shele and Lante Health Center, Southern Ethiopia
Variables	Categories		Genotype Status
			COR (95% CL)	P-value	Remark
Malaria positivity cases	Negative	1		0.84
	Positive	1.33 (0.09–20.11)
Sex	Male	1		0.43
	Female	0.3 (0.02–5.03)
Phenotypic status	Intermediate	1		0.53	Fisher exact test
	Deficient	0.7 (0.47–1.05)
History of malaria	Yes	1		0.49	Fisher exact test
	No	1.5 (0.95–2.38)
Place of residence	Urban	1		0.19	Fisher exact test
	rural	0.6 (0.30–1.09)

This study revealed that almost all G6PD deficient participants were above the age of fourteen. This was the same as the study performed by Nguetse and his colleagues (P = 0.29). Likewise, a study performed in Saudi Arabia also revealed that G6PD deficiency was increased as age getting increased [28, 6].

This research also revealed that males and females had an equally likely chance of being G6PD deficient (AOR, 0.8, 95% CI, 0.423–1.498, P = 0.5). This was in concordant with a study performed in Southwest Ethiopia (AOR, 1.4, P > 0.05) [29]. Contrarily, a study performed in Saudi Arabia and Egypt showed that males had a high chance of being G6PD deficient than females. This is true that since it is an X-linked disorder, males can have the chance of receiving the deficient gene during the inheritance of a single X-chromosome copy [28, 30, 8].

All malaria-positive cases had an insignificant association with G6PD deficiency despite the high odds value (AOR, 2.6; 95% CI, 0.47–14.26; P = 0.3). Similarly, research performed by Getasew Shitaye and his colleagues revealed a negative association of G6PD deficiency with malaria parasites (P = 0.9) [15]. By far, the natural selection pressure exerted by malaria parasites led to rising in G6PD deficiency [31, 6]. This disagreement could be due to the methodological difference.

This study revealed that the prevalence rate of Glucose-6 Phosphate Dehydrogenase deficiency was 4.8% using a quantitative rapid point-of-care CareStart biosensor machine. Since we were searching, other studies were not found there, and we didn’t get the local prevalence rate of G6PD deficiency. However, a study performed in Southwest Ethiopia around Gambela (average altitude of 300–500 meters above sea level) revealed a 7.3% prevalence rate of G6PD deficiency [8]. Thus, the altitude difference and malaria endemicity pattern of both areas, we suggested that the G6PD deficient prevalence rate might be different. Similarly, a review paper performed in Southwest Ethiopia stated that the G6PD deficiency prevalence was above 1–3% [9]. Relatively, broader parts of the country were included by Shitaye and his colleagues and revealed an overall prevalence rate of 1.4% G6PD deficiency [15].

The logistics regression output between the G6PD enzyme level and the parasite load was P = 0.2, the association was statistically not significant. This is the same with Lo and her colleagues and contrarily with Tsegaye and his colleagues (P < 0.0001) [14, 8]. G6PD deficiency is associated with hypo-parasitemia [26, 32–34]. Male hemizygotes and female homozygotes had a high protective effect against severe malaria (P = 0.0006), and cerebral malaria (P = 0.0005) [35]. Male hemizygotes and female heterozygotes have a reduced risk of cerebral malaria [36].

Of the participants, the common mutation, 376 (A◊G and A◊T) was detected in 3 (0.35%) of the participants. G267 + 119C/T (G◊C) was detected in 9 (1.05%) while G1116A (G◊A & G◊T) was detected in 3 (0.35%) of the participants. In Southwest Ethiopia, a Nationwide study revealed 6.1% and 8.9% of A376G genomic mutation respectively while almost correlated result with this study was found in G267 + 119C/T and G1116A gene mutations which were 1.2% [10, 14]. Especially, in this research almost included participants had G267 + 119C/T gene mutation (69.2%) than other variants. Of these, three of the participants were heterozygous while homozygous and hemizygous were equally three in each participant. One mutation in G1116T was also heterozygous while surprisingly, one heterozygous participant had a double mutation in her G6PD gene.

This study also revealed new nucleotide substitution at the position of 376 (A◊T) and 1116 (G◊T) in Ethiopia. 376 (A◊T) was first isolated in Mexico (126 Asn◊Tyr) named San Luis Potosi [37]. This finding didn’t show G6PD A⁻ (G202A) and Mediterranean (C543T) variants among sequenced samples. However, G6PD A⁻ was a common variant in Africa [38] and in Ethiopia, 3.5% of G6PD A⁻ was detected around Southwest Ethiopia [14] while no mutation was detected for the Mediterranean variant in Ethiopia.

Likewise, this finding was searching the associated factors between genomic mutation and gender, phenotypic G6PD result, and malaria positivity which resulted in a non-significant association (Table 5). This was also the same with Lo and her colleagues [14]. Thus, the number of samples has highly influenced this analysis at large so we recommend performing genomic analysis with increased sample size.

Conclusion and recommendation

This finding revealed a modest prevalence rate of G6PD deficiency rate. In a place where the prevalence rate of male G6PD deficiency is greater than 3–5%, mass screening should be performed before administration [39]. In case of this finding, the clinicians have to consider the risk of hemolysis during primaquine treatment in the study area. Genotypically, almost all included study participants had G267 + 119C/T G6PD gene mutation, and rarely A376G and G1116A had been shown in the study area. Genotypic analysis with broader samples could be important to identify the distribution of dominant variants in the study site. G6PD genotypic mutation was not significantly associated with phenotypic G6PD enzyme level. Since genetic mutation is a gradual and recurring situation, continuous assessment of a given population could be mandatory.

DBS

Dry Blood Spot

G6PD

Glucose-6-phosphate dehydrogenase

Haemoglobin

NADPH

Nicotinamide Adenine Nucleotide Phosphate

RBCs

Red Blood Cells

WHO

World Health Organization.

Ethics approval and consent to participate

Ethical approval was sought from the IRB of Akililu Lemma Institute of Pathobiology, Addis Ababa University, and Ethiopian Public Health Institute after presenting the study proposal. Written informed consent was obtained from participants ages greater than eighteen years old and oral assent from all participants aged less than eighteen years old. All collected information was kept confidential and records were coded. No personal identifiers were kept in the database or used to report findings. The data was restricted using password locked and personal computers other than the principal investigator while the hard copies were locked in the cardboard.

Conflict of interest

The authors declare that they have no conflicts of interest.

Availability of data and materials

All data generated in this study are included in this published article.

Funding

The funding agents for this paper were the Global Fund through the Ministry of Health-Ethiopia, Addis Ababa University, Akililu Lemma Institute of Pathobiology, Access Bio Inc. Company, and Ethiopian Public Health Institute.

Authors' Contributions

MTN, SD, LG, and SMF conceived the research idea and participated in the design of the study; MTN, EK, SD, TT, DN, BG, and MY participated in the collection of samples; EL, DK, LW, MTN, TTS, and AA participated in laboratory diagnosis and analysis of the data; MTN, SD, LG, SMF, TTS, and AA wrote the paper; LG, SD, SMF, HA, EL, DN, and GT participated in data interpretation and revised the manuscript. All authors read and approved the final manuscript.

Acknowledgment

First, we would like to thank Almighty God for giving me the strength to face and succeed in my life challenges. Secondly, we would like to provide my heart full thanks to my brother, Mr. Afework who is polite and cooperative and he converted the consent form, information sheet, and questionnaire into the Gamogna version. Moreover, we would like to thank Malaria and the Neglected Tropical Disease Research Team for their unlimited support in the thesis write-up.

Chen L, Zhang C, Wang Y, Li Y, Han Q, Yang H, et al., Data mining and pathway analysis of glucose-6-phosphate dehydrogenase with natural language processing. Molecular Medicine Reports, 2017. 16(2): p. 1900–10.
Gómez-Manzo S, Marcial-Quino J, Vanoye-Carlo A, Serrano-Posada H, Ortega-Cuellar D, González-Valdez A, et al., Glucose-6-phosphate dehydrogenase: Update and analysis of new mutations around the world. International journal of molecular sciences, 2016. 17(12): p. 2069.
Ruwende C and Hill A, Glucose-6-phosphate dehydrogenase deficiency and malaria. Journal of molecular medicine, 1998. 76(8): p. 581–88.
Gómez-Manzo S, Marcial-Quino J, Vanoye-Carlo A, Serrano-Posada H, González-Valdez A, Martínez-Rosas V, et al., Functional and biochemical characterization of three recombinant human glucose-6-phosphate dehydrogenase mutants: Zacatecas, vanua-lava and viangchan. International journal of molecular sciences, 2016. 17(5): p. 787.
Monteiro WM, Val FF, Siqueira AM, Franca GP, Sampaio VS, Melo GC, et al., G6pd deficiency in latin america: Systematic review on prevalence and variants. MemÃ³rias do Instituto Oswaldo Cruz, 2014. 109(5): p. 553–68.
Nguetse CN, Meyer CG, Adegnika AA, Agbenyega T, Ogutu BR, Kremsner PG, et al., Glucose-6-phosphate dehydrogenase deficiency and reduced haemoglobin levels in african children with severe malaria. Malar J, 2016 15(1): p. 1–8.
Okebe J, Amambua-Ngwa A, Parr J, Nishimura S, Daswani M, Takem EN, et al., The prevalence of glucose-6-phosphate dehydrogenase deficiency in gambian school children. Malar J, 2014. 13(1): p. 148.
Tsegaye A, Golassa L, Mamo H, and Erko B, Glucose-6-phosphate dehydrogenase deficiency among malaria suspects attending gambella hospital, southwest ethiopia. Malar J, 2014. 13(1): p. 438.
Recht J, Ashley EA, and White NJ, Use of primaquine and glucose-6-phosphate dehydrogenase deficiency testing: Divergent policies and practices in malaria endemic countries. PLoS neglected tropical diseases, 2018. 12(4): p. e0006230.
Assefa A, Ali A, Deressa W, Tsegaye W, Abebe G, Sime H, et al., Glucose-6-phosphate dehydrogenase (g6pd) deficiency in ethiopia: Absence of common african and mediterranean allelic variants in a nationwide study. Malaria journal, 2018. 17(1): p. 1–7.
Valencia ScH, Ocampo InD, Arce-Plata MI, Recht J, and ArÃ©valo-Herrera M, Glucose-6-phosphate dehydrogenase deficiency prevalence and genetic variants in malaria endemic areas of colombia. Malar J, 2016. 15(1): p. 291.
Lee J, Im Kim T, Kang J-M, Jun H, Lê HG, Thái TL, et al., Prevalence of glucose-6-phosphate dehydrogenase (g6pd) deficiency among malaria patients in upper myanmar. BMC infectious diseases, 2018. 18(1): p. 1–7.
Bwayo D, Kaddumukasa M, Ddungu H, and Kironde F, Prevalence of glucose-6-phosphate dehydrogenase deficiency and its association with plasmodium falciparum infection among children in iganga distric in uganda. BMC Research Notes, 2014. 7(1): p. 372.
Lo E, Zhong D, Raya B, Pestana K, Koepfli C, Lee M-C, et al., Prevalence and distribution of g6pd deficiency: Implication for the use of primaquine in malaria treatment in ethiopia. Malaria journal, 2019. 18(1): p. 1–10.
Shitaye G, Gadisa E, Grignard L, Shumie G, Chali W, Menberu T, et al., Low and heterogeneous prevalence of glucose-6-phosphate dehydrogenase deficiency in different settings in ethiopia using phenotyping and genotyping approaches. Malar J, 2018. 17(1): p. 281.
Haeussler K, Berneburg I, Jortzik E, Hahn J, Rahbari M, Schulz N, et al., Glucose 6-phosphate dehydrogenase 6-phosphogluconolactonase: Characterization of the plasmodium vivax enzyme and inhibitor studies. Malaria journal, 2019. 18(1): p. 22.
Yenew C, Mulatu S, and Alamneh A, The trend of malaria cases, positivity rate, and determinant factors in the amhara regional state, ethiopia: A mixed method. Case Reports in Infectious Diseases, 2021. 2021.
Baird K, Origins and implications of neglect of g6pd deficiency and primaquine toxicity in plasmodium vivax malaria. Pathogens and Global Health, 2015. 109(3): p. 93–106.
Organization WH, Control and elimination of plasmodium vivax malaria: A technical brief. 2015: World Health Organization.
Nega D, Abebe A, Abera A, Gidey B, G/Tsadik A, and Tasew G, Comprehensive competency assessment of malaria microscopists and laboratory diagnostic service capacity in districts stratified for malaria elimination in ethiopia. Plos one, 2020. 15(6): p. e0235151.
Von Seidlein L, Auburn S, Espino F, Shanks D, Cheng Q, McCarthy J, et al., Review of key knowledge gaps in glucose-6-phosphate dehydrogenase deficiency detection with regard to the safe clinical deployment of 8-aminoquinoline treatment regimens: A workshop report. Malaria journal, 2013. 12(1): p. 112.
Ghimire P, Singh N, Ortega L, Rijal KR, Adhikari B, Thakur GD, et al., Glucose-6-phosphate dehydrogenase deficiency in people living in malaria endemic districts of nepal. Malaria journal. 16(1): p. 214.
Pal S, Bansil P, Bancone G, Hrutkay S, Kahn M, Gornsawun G, et al., Evaluation of a novel quantitative test for glucose-6-phosphate dehydrogenase deficiency: Bringing quantitative testing for glucose-6-phosphate dehydrogenase deficiency closer to the patient. The American journal of tropical medicine and hygiene, 2019. 100(1): p. 213–21.
Abossie A, Yohanes T, Nedu A, Tafesse W, and Damitie M, Prevalence of malaria and associated risk factors among febrile children under five years: A cross-sectional study in arba minch zuria district, south ethiopia. Infect Drug Resist, 2020. 13: p. 363–72.
Bancone G, Gornsawun G, Chu CS, Porn P, Pal S, Bansil P, et al., Validation of the quantitative point-of-care carestart biosensor for assessment of g6pd activity in venous blood. PLoS One, 2018. 13(5): p. e0196716.
Cappellini MD and Fiorelli G, Glucose-6-phosphate dehydrogenase deficiency. Lancet, 2008. 371(9606): p. 64–74.
Organization WH, In vitro diagnostics medical devices to identify glucose-6-phosphate dehydrogenase (g6pd) activity. 2016.
Albagshi MH, Alomran S, Sloma S, Albagshi M, Alsuweel A, and AlKhalaf H, Prevalence of glucose-6-phosphate dehydrogenase deficiency among children in eastern saudi arabia. Cureus, 2020. 12(10): p. e11235.
Tsegaye A, Golassa L, Mamo H, and Erko B, Glucose-6-phosphate dehydrogenase deficiency among malaria suspects attending gambella hospital, southwest ethiopia. Malaria journal. 13(1): p. 438.
Elella SA, Tawfik M, Barseem N, and Moustafa W, Prevalence of glucose-6-phosphate dehydrogenase deficiency in neonates in egypt. Annals of Saudi medicine, 2017. 37(5): p. 362–65.
DePina AJ, Pires CM, Andrade AJB, Dia AK, Moreira AL, Ferreira MCM, et al., The prevalence of glucose-6-phosphate dehydrogenase deficiency in the cape verdean population in the context of malaria elimination. PLoS One, 2020. 15(3): p. e0229574.
Luzzatto L, Nannelli C, and Notaro R, Glucose-6-phosphate dehydrogenase deficiency. Hematology/Oncology Clinics, 2016. 30(2): p. 373–93.
Mbanefo EC, Ahmed AM, Titouna A, Elmaraezy A, Trang NTH, Long NP, et al., Association of glucose-6-phosphate dehydrogenase deficiency and malaria: A systematic review and meta-analysis. Scientific reports, 2017. 7: p. 45963.
Ruwende C and Hill A, Glucose-6-phosphate dehydrogenase deficiency and malaria. J Mol Med (Berl), 1998. 76(8): p. 581–8.
Shah SS, Rockett KA, Jallow M, Sisay-Joof F, Bojang KA, Pinder M, et al., Heterogeneous alleles comprising g6pd deficiency trait in west africa exert contrasting effects on two major clinical presentations of severe malaria. Malaria journal, 2016. 15(1): p. 1–8.
Network MGE and Epidemiology MG, Reappraisal of known malaria resistance loci in a large multicenter study. Nat Genet, 2014. 46(11): p. 1197–204.
Vaca G, Arámbula E, Monsalvo A, Medina C, Nuñez C, Sandoval L, et al., Glucose-6-phosphate dehydrogenase (g-6-pd) mutations in mexico: Four new g-6-pd variants. Blood Cells, Molecules, and Diseases, 2003. 31(1): p. 112–20.
Carter R and Mendis KN, Evolutionary and historical aspects of the burden of malaria. Clinical microbiology reviews, 2002. 15(4): p. 564–94.
Organization WH, Glucose-6-phosphate dehydrogenase deficiency. Who working group. Bull World Health Organ, 1989. 67(6): p. 601–11.

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

Prevalence and genetic variants of G6PD deficiency among malaria suspected patients in Shele and Lante Health Center, Southern Ethiopia

Status:

Version 1

Abstract

Background

Method

Results

Conclusion

Figures

Background

Method

Study area

Laboratory Diagnosis

G6PD phenotypic measurements

Examination of Malaria Parasite

G6PD genotyping

Data processing and statistical analysis

Results

Socio-demographic characteristics of study participants

Assessment of risk Factors for G6PD deficiency

Genotype characteristics of Glucose-6 Phosphate Dehydrogenase

Discussion

Abbreviations

Declarations

Ethics approval and consent to participate

References

Additional Declarations

Status:

Version 1