Glucose-6-phosphate Dehydrogenase Deficient Individuals Research Articles

Novel Humanized G6PD Deficient Mice Reveal Mechanisms of Increased Tolerance to Exercise As Gleaned By Multi-Omics in Models of Critical Speed

INTRODUCTION: Glucose-6-phosphate-dehydrogenase (G6PD) deficiency is the most common enzymatic deficiency in humans, affecting ~500 million people or ~6% of mankind. G6PD is the rate limiting enzyme of the pentose phosphate pathway (PPP), the main antioxidant pathway in mature red blood cells (RBCs). Activation of the PPP generates NADPH, an essential reducing equivalent for antioxidant metabolism that protects against intra- and extra-vascular hemolysis in response to oxidant stress. Impaired G6PD activity makes RBCs susceptible to hemolysis induced by oxidant challenges. Exercise-induced oxidant stress is a well-established concept in the field of physiology. Individuals with G6PD deficiency are recommended to refrain from strenuous exercise, as they are deemed to be at increased risk for hemolytic events in response to exercise-induced oxidant stress. While biochemically sound, this rationale has never been tested mechanistically, owing to ethical and technical limitations. As such, knowledge is limited regarding molecular impacts of exercise activity in G6PD deficient individuals. METHODS: Here we generated novel humanized mouse models carrying either (i) human canonical G6PD (normal activity), or two of the most common G6PD variants (ii) African (V68M - < 10% residual enzymatic activity) or (iii) Mediterranean (S188F - <1% residual enzymatic activity). We leveraged these mouse models to assess critical speed (CS), a functional measurement of the speed-to-duration relationship. To expand the molecular resolution of genetically associated adaptations to exercise, CS tests were combined with measurements of hemodynamics - including cardiac output, imaging (e.g., scanning electron microscopy of RBCs before and after exercise) and multi-omics (mass spectrometry-based metabolomics, lipidomics and proteomics) characterization of plasma, RBCs, and multiple tissue matrices including liver, heart, kidney, lung, spleen, and muscles (gastrocnemius and soleus). RESULTS: First of all, we confirmed a significant decrease in G6PD activity in RBCs from mice carrying the African and Mediterranean variants ( Figure 1). Unexpectedly, G6PD deficient mice showed ~10% faster CS compared to mice carrying the canonical human G6PD (p<0.0001 and p<0.02, Figure 1). While scanning electron microscopy showed differences before and after exercise, with accumulation of reversibly modified morphologies across all groups after the run, no significant differences were observed across groups. No significant changes in hemolysis markers were observed in plasma samples after exercise, neither at the metabolic (e.g., heme catabolites) or proteomics level (e.g., hemoglobin chains, RBC-derived proteins). Within hemodynamics, the hG6PD Med- mice are showing a higher efficiency in their cardiac function through cardiac function (p<0.005 - Figure 1). Pathway analysis of metabolomics data generated from RBC and plasma fractions showed significant decreases in arginine metabolism, increased glycolysis, and elevated Krebs cycle intermediates, in addition to expected differences in PPP. Specifically, in hG6PD A- RBC acylcarnitines accumulate after running. In hG6PD Med-, there are clear left ventricular differences highlighted in energy metabolism ( Figure 1). Multiomics analyses identified five major tissue clusters between heart, muscle, liver, kidney, and spleen, with G6PD function having the most significant metabolic impact on liver and soleus. RBC proteomics results highlighted deceased ferroportin (key iron transporter) in both the deficient groups, with markedly significant changes in the hG6PD Med- (p<0.0001). CONCLUSION: Novel humanized G6PD deficient mice maintain faster CS compared to mice. Omics approaches afford the opportunity to identify metabolic correlates to measurements of performance in relevant animal models, such as the CS model, which offer expanded mechanistic understanding of molecular bases of CS and the associated impact of G6PD deficiency. Our results not only challenge the assumption that exercise-induced oxidant stress predisposes the ~500M individuals with G6PD deficiency worldwide to increased hemolytic propensity, but rather indicate a metabolic advantage of functional polymorphisms to G6PD with respect to exercise performance, an observation that could contribute explaining the prevalence of such mutation in human populations.

Mechanisms of Primaquine Induced Hemolysis in a Novel Humanized Murine Model of Mediterranean G6PD Deficiency

Background: Approximately half a billion individuals are genetically deficient in glucose 6-phosphate dehydrogenase (G6PD) due to amino acid variants generally thought to decrease enzyme half-life. Decreased G6PD activity limits the antioxidant capacity of red blood cells (RBCs). As such, certain drugs that induce oxidative stress can cause life-threatening hemolysis in G6PD deficient (G6DPd) individuals. Standard regiments of the only approved drugs that can cure liver phase Plasmodium vivax ( p. vivax) (i.e. primaquine and tafenoquine) are contraindicated in G6PDd patients due to risks of hemolysis. Effective treatment of G6PDd individuals infected with p. vivax requires a prolonged course of primaquine (8 weeks), leading to non-adherence, and subsequent reduced efficacy and increased risk of transmission. Despite decades of research and large drug screening programs, no non-hemolytic compound has been identified that can eradicate p. vivax. Lack of progress in this area has likely been hampered by an incomplete mechanistic understanding of primaquine induced hemolysis, in part due to the lack of an animal model that recapitulates G6PD instability. Methods: Recombinant non-deficient human hG6PD(ND) or the deficient Mediterranean variant (hG6PD(MED)) were expressed, purified, and subjected to thermal proteome profiling in combination with TMT10-assisted quantitative cross-linking proteomics. Mice were generated in which murine genomic G6PD is replaced with either genomic human hG6PD(ND) or hG6PD(MED). Hemolysis in humans is caused by primaquine metabolites (e.g. primaquine-5,6-orthoquinone (5,6-POQ)) and not primaquine itself. As such, RBCs from both strains were exposed to 5,6-POQ and then analyzed by 1) high resolution metabolomics and proteomics, 2) electron paramagnetic resonance (EPR) to measure superoxide, 3) methemoglobin (MetHb) formation, 4) light and electron microscopy, 5) in vitro hemolysis, and 6) in vivo hemolysis (clearance) after infusion into animals. In vivo pulse chase biotinylation allowed visualization and identification of younger (1-6 days) RBCs vs. older (7-55 day) RBCs. Results: Recombinant hG6PD(MED) enzyme had a 2.6-fold lower specific activity compared to hG6PD(ND). Thermal gradient crosslinking proteomics identified specific amino acid spacing (less than 26 angstroms) that was higher in hG6PD(ND) [82-95 and 91-432] vs. higher in hG6PD(MED) [89-205, 97-429, and 429-497]. Analysis of mice demonstrated that hG6PD(MED) RBCs had 5% of G6PD activity as compared to hG6PD(ND) RBCs, with an RBC age dependent decrease in G6PD activity in hG6PD(MED) RBCs. Exposure to 5,6-POQ caused increased superoxide generation, increased MetHb formation, alterations to morphology, in vitro hemolysis, and in vivo hemolysis of hG6PD(MED) RBCs compared to hG6PD(ND) RBCs. However, younger hG6PD(MED) RBCs with higher G6PD activity were highly resistant to 5,6-POQ induced hemolysis (both in vivo and in vitro). All experiments were carried out a minimum of 3 times and all stated findings were statistically significant (p<0.05). Proteomic and metabolomic analysis (with combined samples from multiple experiments) demonstrated oxidation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), resulting in a block in glycolysis that increases dihydroxyacetone phosphate (DHAP) leading to cytotoxic methylglyoxal that broadly oxidizes protein lysines to carboxymethyllysine (CML) - the first step in advanced glycation end products (AGEs) known to facilitate RBC clearance. Discussion: We identify specific conformational changes caused by the hG6PD(MED) variant resulting in decreased specific activity and half-life, and confirm that G6PD activity decreases with RBC age, in a humanized mouse model. The resistance of young hG6PD(MED) RBCs to 5,6-POQ has immediate practical ramifications, in that it supports the rationale of clinical studies currently proposing new dose escalation primaquine regimens to induce low level early hemolysis, leading to reticulocytosis, and decreasing the mean age of circulating RBCs such that increased parricidal doses of primaquine can then be tolerated in G6PDd patients. The elucidation of the metabolic and proteomic lesion (under conditions confirmed to cause 5,6-POQ induced hemolysis of hG6PD(MED) but not hG6PD(ND) RBCs) provides a mechanistic landscape to guide development of non-hemolytic drugs to treat p. vivax.

Propensity score matched analysis and risk stratification of donors with G6PD deficiency in living donor liver transplantation.

Glucose 6 phosphate dehydrogenase (G6PD) deficiency (G6PDd) can trigger hemolysis following surgical stress. Differentiating G6PDd-related post-operative hemolytic episodes (PHE) and post-hepatectomy liver failure may be challenging especially in living donors where donor safety is paramount. We analysed outcomes of our cohort of G6PDd liver donors. G6PDd individuals with no evidence of hemolysis were considered as living donors if there was no alternative family donor. Outcomes of G6PDd donors undergoing left lateral/left lobe donation (Group LL) and right lobe donation (Group RL) were compared with non-G6PDd donors matched in a 1:3 ratio using propensity score matching. 59 G6PDd donors (5.8% of 1011) underwent living donor hepatectomy (LiDH) during the study period. LL-G6PDd donors (22.37%) had higher post-operative peak bilirubin level compared to matched controls, but no difference in morbidity or need for post-operative blood transfusion.RL-G6PDd donors (37.63%) had higher peak bilirubin level, morbidity (16.2% vs. 3.6%, p = 0.017) and more post-operative blood transfusion (21.6% vs. 6.4%, p = 0.023) as compared to matched non-G6PDd cohort. Four RL-G6PDd donors (10.8%) developed PHE. Low G6PD activity (15% vs. 40%, p = 0.034) and lower future liver remnant (FLR) (34.3% vs. 37.8%, p = 0.05) were identified as risk factors for PHE. We report the largest to-date series of G6PDd individuals undergoing LiDH and confirm the safety of LL donation in G6PDd. Our analysis identifies specific risk factors for PHE and suggests that right lobe LiDH be avoided in individuals with less than 25% G6PD activity when the FLR is less than 36%.

Investigating the Prevalence of Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency among Patients with Favism Symptoms in Kerman City, Southern Iran

Background and Aims: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is humans’ most common erythrocyte enzyme defect. About 400 million people are estimated to be affected by this disorder worldwide. Antimalarial drugs, especially primaquine, and other oxidative stress, can cause hemolytic complications in G6PD deficient individuals. This study aimed to evaluate the prevalence of G6PD deficiency in Kerman City in southern Iran.
 Materials and Methods: This descriptive cross-sectional study was conducted from 2016 to 2021. Blood samples were taken from all patients with symptoms of G6PD deficiency who were referred to a general hospital in Kerman City in southern Iran. The G6PD enzyme activity was measured qualitatively by fluorescent spot test.
 Results: A total of 6369 patients were included in this study. G6PD deficiency was seen in 424 (6.7%) subjects. Of 424 patients, 359 (84.7%) were severely G6PD deficient, and 65 (15.3%) patients exhibited partial deficiency. G6PD deficiency was seen in 324 (9.3%) males and 100 (3.4%) females (p < 0.001).
 Conclusion: The results of our study confirmed the existence of G6PD deficiency in a significant percentage of patients in Kerman City. Therefore, many people in this city are exposed to hemolytic complications if they use antimalarial drugs and other oxidative substances. According to our results, testing G6PD deficiency and monitoring the potential primaquine toxicity in patients who receive primaquine are highly recommended.

Comparison of various RBC indices and Glucose 6 phosphate dehydrogenase activity in patients with and without malaria

Introduction and Aim: Malaria is endemic in many parts of India. Glucose 6 phosphate dehydrogenase (G6PD) deficiency is known to protect against malaria. G6PD deficient individuals afflicted with malaria when treated with primaquine, the first line oxidant drug of malaria, encounter adverse to fatal complications due to acute precipitation of hemolytic anemia. There is a need to assess RBC indices in malaria, its implications in G6PD deficiency, and its acute manifestations. The aim of this study was to compare and correlate various RBC indices and G6PD activity in patients with and without malaria and to find a prevalence of G6PD deficiency in a tertiary care hospital. Materials and Methods: The present study was carried out by the Biochemistry Department of Kasturba Medical College, Manipal in 363 participants (with malaria and without malaria). Mann Whitney U test and Spearman’s Rank correlation were employed to assess group differences and correlation, respectively. Results: 218 cases of malaria in 365 days from a tertiary care hospital in South India is an alarming incidence and annuls the fact that the malaria prevalence is relatively low in South India. Complete blood counts and red blood indices did not show any statistically significant difference between the study groups. No statistically significant correlation was found between G6PD activity and RBC indices in the present study. Conclusion: No significant differences between hematological indicators and malaria with or without G6PD deficiency hint towards the necessity of G6PD tests for radical treatment of malaria as hematological indices are unable to predict the defective enzyme activity.

The integrity and stability of specimens under different storage conditions for glucose-6-phosphate dehydrogenase deficiency screening using WST-8

Accurate measurement of glucose-6-phosphate dehydrogenase (G6PD) activity is critical for malaria treatment as misclassification of G6PD deficiency could cause serious harm to patients. G6PD activity should be assessed in blood samples on the day of collection. Otherwise, specimens should be stored under suitable conditions to prevent loss of G6PD activity. Here, we assessed stability and integrity of G6PD testing in samples from normal controls, heterozygous females, and G6PD deficient individuals using water-soluble tetrazolium salts (WST-8) assay. Specimens were stored as ethylenediaminetetraacetic acid (EDTA) whole blood and dried blood spots (DBS) at various temperatures (37 °C, room temperature, 4 °C and -20 °C) and under different humidity conditions (with and without desiccant). G6PD normal samples were stable for up to 1 year when stored at -20 °C under controlled conditions, with 85% and 91% G6PD activity in EDTA whole blood and DBS in the presence of desiccant, respectively. Specimens from heterozygous females showed greater G6PD activity when stored as DBS, with 85% enzyme activity after 1 year of storage at -20 °C under controlled conditions in the presence of desiccant. G6PD deficient samples rapidly lost enzyme activity in all storage conditions tested. However, the reduction in G6PD enzyme activity in G6PD deficient samples did not interfere with G6PD classification. Samples stored under suitable conditions for G6PD testing will allow accurate measurement of enzyme activity, prevent misclassification of G6PD deficiency and enable safe and effective use of antimalarial drugs such as primaquine and tafenoquine.

Favism Induced Methemoglobinemia in G6DP Deficient Patients: Case Series and Review of Literature

Introduction: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a well-known cause of hemolysis. It has a notable prevalence in African, Asian, and Mediterranean countries. Favism is a common trigger of oxidative stress in G6PD deficient people, which can lead to hemolysis. Additionally, fava bean ingestion can cause methemoglobinemia (MethHgb), an abnormal variation in the Hgb in which the ferrous (Fe2+) iron in heme is oxidized to the ferric (Fe3+) state. It is rare to have both G6PD deficiency and MethHgb secondary to favism at the same time. The first-line treatment for MethHgb is methylene blue. However, in G6PD deficient patients, it can potentiate hemolysis. Methods: We reviewed the literature using PubMed and Google scholar and found 6 cases of MethHgb secondary to favism in patients with G6PD deficiency. We also analyzed two cases which are still unpublished, making a total of 8 cases. Results: All 8 cases were male with median age of 18 years (1 - 56). 1 patient had a prior diagnosis of G6PD deficiency while 7 were newly diagnosed. Median Hgb was 8.3gm/dL (4.6 - 12.5) and median MetHgb was 7.8 % (3.5 - 35). 1 patient received methylene blue, and 4 received Vitamin C. All of the patients recovered and were discharged [Table 1]. Unpublished case 1: A 56-year-old male presented with MethHgb and hemolytic anemia, secondary to fava bean ingestion. Hgb on admission and lowest recorded were 9.9 and 6.5 gm/dL, respectively. He had an SPO2 of 70% on room air and 101.2 % on ABG. Methylene blue administration worsened the hemolysis as he was G6PD deficient but not diagnosed before. He got better with discontinuation of methylene blue and Vitamin C and was discharged on day 5. Unpublished case 2: A 43-year-old male, known case of G6PD deficiency presented with MethHgb and hemolytic anemia, secondary to fava bean ingestion. Hgb on admission and lowest recorded were 12.5 and 7.4 gm/dL, respectively. He had an SPO2 of 82% on room air and 100 % on ABG. He received IV vitamin C and recovered and was discharged on day 4. Discussion: Methemoglobinemia is usually acquired, secondary to oxidative stress in the body, but can rarely be congenital. Enzyme systems such as NADH methemoglobin reductase, NADPH methemoglobin reductase, ascorbic acid, and glutathione reductase systems keep a check on the accumulation of methemoglobin in the blood. However, these mechanisms can be insufficient to counter the conversion of Hgb to MethHgb, consequently promoting an oxidative state in the body. It can be due to the overproduction of methemoglobin (secondary to exposure to certain drugs, chemicals, or food items, but can sometimes be hereditary) or under conversion to Hgb due to unavailable enzyme mechanisms. One of the causes of the inability to counteract methemoglobin can be secondary to G6PD deficiency. Patients with MetHgb have a low oxygen saturation (SPO2) on pulse oximeters but a falsely high SPO2 on arterial blood gasses (ABG). The treatment depends on symptoms and the level of MethHgb. The first step is to remove any possible precipitator if present. Symptomatic patients (and asymptomatic with a level of methemoglobin >30 %) are treated with methylene blue (1-2mg/kg), which is reduced to leuko-methylene blue via NADPH dependent methemoglobin reductase. This, in turn, reduces methemoglobin back to Hgb, correcting the abnormality [Figure 1]. Rarely, patients can present with co-occurrence of MethHgb and G6PD deficiency. In such cases, caution is required while giving methylene blue as they do not have sufficient levels of NADPH to reduce it. Otherwise, a cascade of oxidative hemolysis ensues secondary to underlying G6PD deficiency, resulting in a vicious cycle of further methemoglobinemia. The most frequent cause of this co-occurrence is the ingestion of fava beans, which can simultaneously induce MethHgb and potentiate G6PD deficiency. One of our patients had a history of favism without developing any symptoms. Only this time, he ate fava beans in a larger amount, leading to hemolysis and MethHgb. Conclusion: Favism is a rare cause of the co-occurrence of methemoglobinemia and hemolysis in G6PD deficient individuals. It is vital to identify G6PD deficiency in patients presenting with MethHgb, as the initiation of methylene blue in such individuals can result in a cascade of oxidative hemolysis. A history of fava beans ingestion without any symptoms does not rule out G6PD deficiency, as it is proportional to the number of beans ingested. Disclosures No relevant conflicts of interest to declare.

Prevalence and spectrum of mutations causing G6PD deficiency in Indian populations

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common human erythroenzymopathy affecting around 10% of the world population. India is endemic for malaria and antimalarial drugs are known to induce haemolysis in G6PD deficient individuals. Here we report the prevalence as well as the molecular diversity of G6PD deficiency in geographical regions of India. A total of 20,896 individuals (11,838 males and 9058 females) were screened by DPIP dye decolorisation method followed by quantitation of G6PD enzyme activity on the suspected samples. Molecular analysis was undertaken in a total of 350 G6PD deficient individuals by PCR-RFLP and DNA sequencing. A structural characteristic of the novel variant was deduced by using DynaMut web-server. The prevalence rate of G6PD deficiency varied between 0.8 and 6.3% with an overall prevalence of 1.9%. A total of twelve mutations were identified. Of the total deleterious alleles detected G6PD Orissa (56.5%) was found to be the most predominant variant followed by G6PD Mediterranean (23.6%). G6PD Mediterranean, G6PD Kaiping and G6PD Mahidol were found to be severely deficient variant and 14.1% of them showed undetectable activity. A novel mutation c.544C➔G (R182G) in exon 6 was identified in one tribal male where substitution of arginine by glycine, likely causes the alteration in the alpha helix leading to disruption of secondary structure of the protein. There are large differences in the distribution of G6PD causal variants between Indian states, and this may have implications for the treatment in the malaria endemic areas.

Wide range of G6PD activities found among ethnic groups of the Chittagong Hill Tracts, Bangladesh.

The proportion of Plasmodium vivax malaria among all malarias is increasing worldwide. Treatment with 8-aminoquinolines remain the only radical cure. However, 8-aminoquinolines can cause severe hemolysis in glucose-6-phosphate dehydrogenase (G6PD) deficient patients. The population of the multi-ethnic Chittagong Hill Tracts (CHT) carry the highest malaria burden within Bangladesh. As in many countries the national treatment guidelines recommend 8-aminoquinoline based radical cure without routine G6PD deficiency (G6PDd) testing to guide treatment. Aim of this study was to determine the need for routine testing within a multi-ethnic population by assessing the prevalence of G6PDd among the local population. Participants from 11 ethnicities were randomly selected and malaria status was assessed by microscopy, rapid diagnostic test (RDT) and polymerase chain reaction (PCR). G6PD status was determined by spectrophotometry and G6PD genotyping. The adjusted male median (AMM) was defined as 100% G6PD activity, participants were categorized as G6PD deficient (<30% activity), G6PD intermediate (30% to 70% activity) or G6PD normal (>70% activity). Median G6PD activities between ethnicities were compared and the association between G6PD activity and malaria status was assessed. 1002 participants were enrolled and tested for malaria. G6PD activity was measured by spectrophotometry in 999 participants and host G6PD genotyping undertaken in 323 participants. Seven participants (0.7%) had peripheral parasitaemia detected by microscopy or RDT and 42 by PCR (4.2%). Among 106 participants (32.8%) with confirmed genotype, 99 (93.4%) had the Mahidol variant. The AMM was 7.03U/gHb with 90 (9.0%) G6PD deficient participants and 133 (13.3%) with intermediate G6PD activity. Median G6PD activity differed significantly between ethnicities (p<0.001), proportions of G6PD deficient individuals ranged from 2% to 26% but did not differ between participants with and without malaria. The high G6PDd prevalence and significant variation between ethnicities suggest routine G6PDd testing to guide 8-aminoquinoline based radical in the CHT and comparable settings.

Geographical Accessibility to Glucose-6-Phosphate Dioxygenase Deficiency Point-of-Care Testing for Antenatal Care in Ghana.

Background: Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency screening test is essential for malaria treatment, control, and elimination programs. G6PD deficient individuals are at high risk of severe hemolysis when given anti-malarial drugs such as primaquine, quinine, other sulphonamide-containing medicines, and chloroquine, which has recently been shown to be potent for the treatment of coronavirus disease (COVID-19). We evaluated the geographical accessibility to POC testing for G6PD deficiency in Ghana, a malaria-endemic country. Methods: We obtained the geographic information of 100 randomly sampled clinics previously included in a cross-sectional survey. We also obtained the geolocated data of all public hospitals providing G6PD deficiency testing services in the region. Using ArcGIS 10.5, we quantified geographical access to G6PD deficiency screening test and identified clinics as well as visualize locations with poor access for targeted improvement. The travel time was estimated using an assumed speed of 20 km per hour. Findings: Of the 100 clinics, 58% were Community-based Health Planning and Services facilities, and 42% were sub-district health centers. The majority (92%) were Ghana Health Service facilities, and the remaining 8% were Christian Health Association of Ghana facilities. Access to G6PD deficiency screening test was varied across the districts, and G6PD deficiency screening test was available in all eight public hospitals. This implies that the health facility-to-population ratio for G6PD deficiency testing service was approximately 1:159,210 (8/1,273,677) population. The spatial analysis quantified the current mean distance to a G6PD deficiency testing service from all locations in the region to be 34 ± 14 km, and travel time (68 ± 27 min). The estimated mean distance from a clinic to a district hospital for G6PD deficiency testing services was 15 ± 11 km, and travel time (46 ± 33 min). Conclusion: Access to POC testing for G6PD deficiency in Ghana was poor. Given the challenges associated with G6PD deficiency, it would be essential to improve access to G6PD deficiency POC testing to facilitate administration of sulphadoxine-pyrimethamine to pregnant women, full implementation of the malaria control program in Ghana, and treatment of COVID-19 patients with chloroquine in malaria-endemic countries. To enable the World Health Organization include appropriate G6PD POC diagnostic tests in its list of essential in-vitro diagnostics for use in resource-limited settings, we recommend a wider evaluation of available POC diagnostic tests for G6PD deficiency, particularly in malaria-endemic countries.

G6PD A- is the major cause of G6PD deficiency among the Siddis of Karnataka, India

Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human erythroenzymopathy affecting more than 400 million people worldwide. G6PD deficiency was reported in India more than 50 years ago and the prevalence rate varies from 5.7% to 27.9% in different caste and tribal groups.Aim: To study the prevalence of, and the mutations causing, G6PD deficiency among the Siddis of Karnataka.Subjects and methods: A total of 755 individuals were screened using the DPIP dye decolorisation method and the deficiency was further confirmed by quantitative assay. Molecular characterisation was performed by PCR-RFLP method and DNA sequencing. Biochemical characterisation was performed as per WHO criteria.Results: Of the 755 individuals, 71 individuals (9.4%) were found to be G6PD deficient with an enzyme activity ranging from 0.02 to 3.83 IU/gm Hb. Mutational analysis could be performed on 49 G6PD deficient individuals and 45 (91.8%) of them showed the presence of the G6PD A- variant while the remaining 4 (8.2%) had the G6PD Kerala-Kalyan mutation. Microsatellite analysis in G6PD A- individuals showed the presence of 166/195 bp, AC/CTT alleles.Conclusions: G6PD deficiencies among the Siddis are predominantly due to G6PD A- mutation. Furthermore, biochemical parameters and the microsatellite repeat markers in the Siddi A- chromosome confirmed they are African descendants with Indian admixture.

Seizure following acute hemolysis caused by Glucose-6-phosphate dehydrogenase Deficiency

Background: Storage of platelet concentrates (PCs) at room temperature (20-24°C) limits its storage time to 5 Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common inherited enzyme deficiency of the human red blood cells . Most of G6PD deficient individuals are asymptomatic, but acute hemolytic anemia may be presented with nausea, vomiting, abdominal pain, headache, jaundice, pallor, discoloration of the urine, chills, and fever. Seizure is reported as a rare symptom, as well. The present study aimed to investigate seizure following acute hemolysis caused by Glucose-6-phosphate dehydrogenase deficiency.&#x0D; Material and Methods: This analytic cross-sectional study was conducted on all consecutive patients aged 1-12 years with G6PD deficiency hospitalized for hemolysis in 17 Shahrivar children hospital, Rasht, Iran, in 2016. Demographic characteristics and other variables such as place of inhabitants, type of drinking water, history of seizure in the patients and family, cause of hemolysis, hemoglobin level and hemoglobinuria on admission, and infection history prior to hemolysis were recorded. Data were analyzed by Mann-Whitney U test and Fischer Exact Test. P-value &lt; 0.05 indicated statistical significance and data were assessed by SPSS (version 20).&#x0D; Results: The youngest patient was one year old and the oldest was 11 years old. Most of them were males (68.9%). Out of 244 patients, 8 ones (3.3%) experienced seizure. There was a significant correlation between seizure occurrence and family history of seizure (p=0.03) as well as fava bean consumption (p=0.019) as the causes of hemolysis; but not with infection as the cause of hemolysis, hemoglobin or hemoglobinuria level on admission, types of drinking water, place of living, and gender. Methemoglobinemia was considered as the main cause of the seizure.&#x0D; Conclusion: Although the rate of seizure was not so high (3.3%), it seems that seizure can be a critical and potentially life-threatening complication in these patients. Environmental factors may also play a role in the pathogenesis of the seizure in these patients.

A Case Report of Haemolysis in a G6PD Deficient Child due to Acetaminophen Overdose Resulted from Parental Unawareness

Although acetaminophen is considered a low-risk analgesic and antipyretic drug in Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency patients, cases of haemolysis following acetaminophen overdose have been published in the literature. An eleven-month-old boy was admitted to the paediatrics department of the hospital with the complaints of haematuria and yellowish discolouration of the eyes since one day. He had been previously diagnosed as G6PD deficiency. Unknowingly the mother of the child simultaneously given three different brands of acetaminophen prescribed by different paediatricians at various times resulted in an overdose of acetaminophen. On admission, the child had haemoglobin of 4 g/dl. The laboratory results were suggestive of haemolytic process. Direct Coombs test was negative. Acetaminophen overdose was the most likely cause of the haemolysis. The child was managed conservatively and no further episodes of haematuria was observed. Naranjo and WHO causality assessments were done, indicating a probable relationship between the patient’s symptoms and the use of acetaminophen. Acetaminophen overdose induced haemolysis due to G6PD deficiency is an unusual complication which is generally reported in children. The majority of G6PD deficient individuals remains asymptomatic until a trigger (during certain infections or fava beans or certain drugs) induces haemolysis. We document a case report of acetaminophen overdose induced haemolysis in a baby with G6PD deficiency and conclude that there is a need for parental education about the drugs that can trigger haemolysis in G6PD deficient children and also about the different brands of the same drug available in the market. Key words: Acetaminophen, Paracetamol, NSAIDs, G6PD deficiency, Haemolysis, Anaemia.

The impact of using primaquine without prior G6PD testing: a case series describing the obstacles to the medical management of haemolysis

Radical cure of Plasmodium vivax malaria in glucose-6-phosphate dehydrogenase (G6PD) deficient individuals employs weekly primaquine dosing. This is the only recommended regimen for this patient sub-group. If national malaria programs mandate daily primaquine dosing (the recommended regimen for G6PD normal individuals), then G6PD testing before prescription is necessary to avoid iatrogenic haemolysis in G6PD deficient individuals. In this case series, two P. vivax infected patients with unknown G6PD status from two different countries were prescribed primaquine as per national malaria program guidelines. During treatment both patients presented to the clinic with symptoms of anaemia after taking primaquine incorrectly. The clinical management of the iatrogenic severe haemolysis that occurred in these patients demonstrates the various adverse effects primaquine can cause, that other common medical treatments also have haemolytic potential, and how the diagnosis of G6PD deficiency can be elusive during acute haemolysis. Health care providers should provide careful instructions about primaquine dosing, be watchful for haemolysis, and have a high index of suspicion for G6PD deficiency in the presence of haemolysis if the G6PD status is previously unknown.

Low and heterogeneous prevalence of glucose-6-phosphate dehydrogenase deficiency in different settings in Ethiopia using phenotyping and genotyping approaches

Background8-Aminoquinolines such as primaquine clear mature Plasmodium falciparum gametocytes that are responsible for transmission from human to mosquitoes and bring radical cure in Plasmodium vivax by clearing dormant liver stages. Deployment of primaquine is thus of relevance for malaria elimination efforts but challenged by the widespread prevalence of glucose-6-phosphate dehydrogenase deficiency (G6PDd) in endemic countries since primaquine in G6PDd individuals may lead to acute haemolysis. In this study, the prevalence of G6PDd was investigated in different settings in Ethiopia using phenotyping and genotyping approaches.MethodsCommunity and school based cross-sectional surveys were conducted from October to December 2016 in four administrative regions (Gambela, Benishangul Gumuz, Oromia, and Amhara) in Ethiopia. Finger prick blood samples were collected for G6PD enzyme activity using the CareStart™ G6PD screening test and genotyping of 36 selected single nucleotide polymorphisms (SNPs) located in the G6PD gene and its flanking regions.ResultsOverall, the prevalence of phenotypic G6PDd was 1.4% (22/1609). For the first time in the Ethiopian population, the African variant (A−) was detected in 3.5% (7/199) of the limited set of genotyped samples, which were all phenotypically normal. Interestingly, all of these individuals had a variation at the rs2515904 locus. Strong geographical variation was observed for both phenotypic and genotypic G6PDd; three-quarters of the phenotypically G6PDd individuals were detected in Gambela.ConclusionA very low prevalence of G6PDd was detected in the present study populations. The presence of the A− variant alongside other G6PD mutants and the patchy distribution of G6PDd indicate that larger studies specifically designed to unravel the distribution of G6PDd at small geographical scale may be needed to tailor malaria elimination efforts in Ethiopia to the local context.

The gene spectrum of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Guangdong province, China

BackgroundG6PD deficiency presents a higher incidence rate in southern China. Many variants of G6PD resulted from point mutations in the G6PD gene, which lead to decrease of enzyme activity. The objective of this study was to analyze the genotype of G6PD deficiency in four regions of Guangdong province. MethodsGenotype of 1756 cases with G6PD deficiency was identified by reverse dot blotting (RDB). Unidentified Genotype of the samples was further ascertained by direct DNA sequencing. Results34 genotype were found in 1756 cases of G6PD deficiency, Canton (c.1376 G>T) and Kaiping (c.1388 G>A) were the most common variants, accounting for more than 63% of G6PD deficiency individuals, and the following mutations were Gaohe (c.95 A>G), Chinese-5 (c.1024 C>T) and Chinese-4 (c.392 G>T). Two rare mutations Orisa (c.131 C>G) and IVS-5 637/638 T del have been discovered in this study. In addition, c.1311 C>T/IVS-1193 T>C polymorphism had a relatively high frequency in the normal G6PD individuals. ConclusionsThis study provided detailed genotypes of G6PD deficiency in Guangdong, and would be valuable for diagnosis and research of G6PD deficiency in this area.

Safety of single low-dose primaquine in glucose-6-phosphate dehydrogenase deficient falciparum-infected African males: Two open-label, randomized, safety trials.

BackgroundPrimaquine (PQ) actively clears mature Plasmodium falciparum gametocytes but in glucose-6-phosphate dehydrogenase deficient (G6PDd) individuals can cause hemolysis. We assessed the safety of low-dose PQ in combination with artemether-lumefantrine (AL) or dihydroartemisinin-piperaquine (DP) in G6PDd African males with asymptomatic P. falciparum malaria.Methods and findingsIn Burkina Faso, G6PDd adult males were randomized to treatment with AL alone (n = 10) or with PQ at 0.25 (n = 20) or 0.40 mg/kg (n = 20) dosage; G6PD-normal males received AL plus 0.25 (n = 10) or 0.40 mg/kg (n = 10) PQ. In The Gambia, G6PDd adult males and boys received DP alone (n = 10) or with 0.25 mg/kg PQ (n = 20); G6PD-normal males received DP plus 0.25 (n = 10) or 0.40 mg/kg (n = 10) PQ. The primary study endpoint was change in hemoglobin concentration during the 28-day follow-up. Cytochrome P-450 isoenzyme 2D6 (CYP2D6) metabolizer status, gametocyte carriage, haptoglobin, lactate dehydrogenase levels and reticulocyte counts were also determined.In Burkina Faso, the mean maximum absolute change in hemoglobin was -2.13 g/dL (95% confidence interval [CI], -2.78, -1.49) in G6PDd individuals randomized to 0.25 PQ mg/kg and -2.29 g/dL (95% CI, -2.79, -1.79) in those receiving 0.40 PQ mg/kg. In The Gambia, the mean maximum absolute change in hemoglobin concentration was -1.83 g/dL (95% CI, -2.19, -1.47) in G6PDd individuals receiving 0.25 PQ mg/kg. After adjustment for baseline concentrations, hemoglobin reductions in G6PDd individuals in Burkina Faso were more pronounced compared to those in G6PD-normal individuals receiving the same PQ doses (P = 0.062 and P = 0.022, respectively). Hemoglobin levels normalized during follow-up. Abnormal haptoglobin and lactate dehydrogenase levels provided additional evidence of mild transient hemolysis post-PQ.ConclusionsSingle low-dose PQ in combination with AL and DP was associated with mild and transient reductions in hemoglobin. None of the study participants developed moderate or severe anemia; there were no severe adverse events. This indicates that single low-dose PQ is safe in G6PDd African males when used with artemisinin-based combination therapy.Trial registrationClinicaltrials.gov NCT02174900Clinicaltrials.gov NCT02654730

Safety of primaquine given to people with G6PD deficiency: systematic review of prospective studies

BackgroundHaemolysis risk with single dose or short course primaquine was evaluated in glucose-6-phosphate dehydrogenase (G6PD) deficient people.MethodsMajor electronic databases (to August 2016) were searched for single or short course 8-aminoquinolines (8-AQ) in (1) randomized comparisons against placebo in G6PD deficient people; and (2) observational comparisons in G6PD deficient compared to replete people. Two authors independently assessed eligibility, risk-of-bias, and extracted data.ResultsFive randomized controlled trials and four controlled observational cohorts were included. In G6PD deficient individuals, high-dose (0.75 mg/kg) PQ resulted in lower average haemoglobin levels at 7 days (mean difference [MD] −1.45 g/dl, 95% CI −2.17 to −0.74, 2 trials) and larger percentage fall from baseline to day 7 (MD −10.31%, 95% CI −17.69 to −2.92, 3 trials) compared to placebo. In G6PD deficient compared to replete people, average haemoglobin was lower at 7 days (MD −1.19 g/dl, 95% CI −1.94 to −0.44, 2 trials) and haemoglobin change from baseline to day 7 was greater (MD −9.10%, 95% CI −12.55 to −5.65, 5 trials). One small trial evaluated mid-range PQ dose (0.4–0.5 mg/kg) in G6PD deficient people, with no difference detected in average haemoglobin at day 7 compared to placebo. In one cohort comparing G6PD deficient and replete people there was a greater fall with G6PD deficiency (MD −4.99%, 95% CI −9.96 to −0.02). For low-dose PQ (0.1–0.25 mg/kg) in G6PD deficient people, haemoglobin change from baseline was similar to the placebo group (MD 1.72%, 95% CI −1.89 to 5.34, 2 trials). Comparing low dose PQ in G6PD deficient with replete people, the average haemoglobin was lower in the G6PD deficient group at 7 days (−0.57 g (95% CI −0.97 to −0.17, 1 trial)); although change from baseline was similar (MD −1.45%, 95% CI −5.69 to 2.78, 3 trials).ConclusionsFalls in average haemoglobin are less marked with the 0.1 to 0.25 mg/kg PQ than with the 0.75 mg/kg dose, and severe haemolytic events are not common. However, data were limited and the evidence GRADE was low or very low certainty.

G6PD deficiency alleles in a malaria-endemic region in the Western Brazilian Amazon

BackgroundPlasmodium vivax parasites are the predominant cause of malaria infections in the Brazilian Amazon. Infected individuals are treated with primaquine, which can induce haemolytic anaemia in glucose-6-phosphate dehydrogenase (G6PD)-deficient individuals and may lead to severe and fatal complications. This X-linked disorder is distributed globally and is caused by allelic variants with a geographical distribution that closely reflects populations exposed historically to endemic malaria. In Brazil, few studies have reported the frequency of G6PD deficiency (G6PDd) present in malaria-endemic areas. This is particularly important, as G6PDd screening is not currently performed before primaquine treatment. The aim of this study was to determine the prevalence of G6PDd in the region of Alto do Juruá, in the Western Brazilian Amazon, an area characterized by a high prevalence of P. vivax infection.MethodsFive-hundred and sixteen male volunteers were screened for G6PDd using the fluorescence spot test (Beutler test) and CareStart™ G6PD Biosensor system. Demographic and clinical-epidemiological data were acquired through an individual interview. To assess the genetic basis of G6PDd, 24 SNPs were genotyped using the Kompetitive Allele Specific PCR assay.ResultsTwenty-three (4.5%) individuals were G6PDd. No association was found between G6PDd and the number of malaria cases. An increased risk of reported haemolysis symptoms and blood transfusions was evident among the G6PDd individuals. Twenty-two individuals had the G6PDd A(−) variant and one the G6PD A(+) variant. The Mediterranean variant was not present. Apart from one polymorphism, almost all SNPs were monomorphic or with low frequencies (0–0.04%). No differences were detected among ethnic groups.ConclusionsThe data indicates that ~1/23 males from the Alto do Juruá could be G6PD deficient and at risk of haemolytic anaemia if treated with primaquine. G6PD A(−) is the most frequent deficiency allele in this population. These results concur with reported G6PDd in other regions in Brazil. Routine G6PDd screening to personalize primaquine administration should be considered, particularly as complete treatment of patients with vivax malaria using chloroquine and primaquine, is crucial for malaria elimination.

Rapid diagnostic test for G6PD deficiency in Plasmodium vivax-infected men: a budget impact analysis based in Brazilian Amazon.

The aim of this study was to estimate the incremental budget impact (IBI) of a rapid diagnostic test to detect G6PDd in male patients infected with Plasmodium vivax in the Brazilian Amazon, as compared with the routine protocol recommended in Brazil which does not include G6PDd testing. The budget impact analysis was performed from the perspective of the Brazilian health system, in the Brazilian Amazon for the years 2013, 2014 and 2015. The analysis used a decision model to compare two scenarios: the first consisting of the routine recommended in Brazil which does not include prior diagnosis of dG6PD, and the second based on the use of RDT CareStart™ G6PD (CS-G6PD) in all male subjects diagnosed with vivax malaria. The expected implementation of the diagnostic test was 30% in the first year, 70% the second year and 100% in the third year. The analysis identified negative IBIs which were progressively smaller in the 3 years evaluated. The sensitivity analysis showed that the uncertainties associated with the analytical model did not significantly affect the results. A strategy based on the use of CS-G6PD would result in better use of public resources in the Brazilian Amazon.