First Evaluation of Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency in Vivax Malaria Endemic Regions in the Republic of Korea

Background

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect and affects more than 400 million people worldwide. This deficiency is believed to protect against malaria because its global distribution is similar. However, this genetic disorder may be associated with potential hemolytic anemia after treatment with anti-malarials, primaquine or other 8-aminoquinolines. Although primaquine is used for malaria prevention, no study has previously investigated the prevalence of G6PD variants and G6PD deficiency in the Republic of Korea (ROK).

Methods

Two commercialized test kits (Trinity G-6-PDH and CareStart G6PD test) were used for G6PD deficiency screening. The seven common G6PD variants were investigated by DiaPlexC kit in blood samples obtained living in vivax malaria endemic regions in the ROK.

Results

Of 1,044 blood samples tested using the CareStart G6PD test, none were positive for G6PD deficiency. However, a slightly elevated level of G6PD activity was observed in 14 of 1,031 samples tested with the Trinity G-6-PDH test. Forty-nine of the 298 samples with non-specific amplification by DiaPlexC kit were confirmed by sequencing to be negative for the G6PD variants.

Conclusions

No G6PD deficiency was observed using phenotypic- or genetic-based tests in individuals residing in vivax malaria endemic regions in the ROK. Because massive chemoprophylaxis using primaquine has been performed in the ROK military to kill hypnozoites responsible for relapse and latent stage vivax malaria, further regular monitoring is essential for the safe administration of primaquine.     

Prevalence of Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency among Malaria Patients in Southern Thailand: 8 Years Retrospective Study

Erythrocytes deficient in glucose-6-phosphate dehydrogenase (G6PD) is more susceptible to oxidative damage from free radical derived compounds. The hemolysis triggered by oxidative agents such as primaquine (PQ) is used for the radical treatment of hypnozoites of P. vivax. Testing of G6PD screening before malaria treatment is not a common practice in Thailand, which poses patients at risk of hemolysis. This retrospective study aimed to investigate the prevalence of G6PD in malaria patients who live in Southern Thailand. Eight hundred eighty-one malaria patients were collected for 8-year from 2012 to 2019, including 785 (89.1%) of P. vivax, 61 (6.9%) of P. falciparum, 27 (3.1%) of P. knowlesi, and 8 (0.9%) of mixed infections. The DiaPlexC genotyping kit (Asian type) and PCR-RFLP were employed to determine the G6PD variants. The result showed that 5 different types of G6PD variants were identified in 26 cases (2.9%); 12/26 (46.2%) had Mahidol (487G>A) and 11/26 (42.3%) had Viangchan (871G>A) variants, while the rest had Kaiping (1388G>A), Union (1360C>T), and Mediterranean (563C>T) variants. G6PD Songklanagarind (196T>A) variant was not found in the study. Our result did not show a significant difference in the malaria parasite densities in patients between G6PD-deficient and G6PD-normal groups. According to our findings, testing G6PD deficiency and monitoring the potential PQ toxicity in patients who receive PQ are highly recommended.     

African Glucose-6-Phosphate Dehydrogenase Alleles Associated with Protection from Severe Malaria in Heterozygous Females in Tanzania

X-linked Glucose-6-phosphate dehydrogenase (G6PD) A- deficiency is prevalent in sub-Saharan Africa populations, and has been associated with protection from severe malaria. Whether females and/or males are protected by G6PD deficiency is uncertain, due in part to G6PD and malaria phenotypic complexity and misclassification. Almost all large association studies have genotyped a limited number of G6PD SNPs (e.g. G6PD202 / G6PD376), and this approach has been too blunt to capture the complete epidemiological picture. Here we have identified 68 G6PD polymorphisms and analysed 29 of these (i.e. those with a minor allele frequency greater than 1%) in 983 severe malaria cases and controls in Tanzania. We establish, across a number of SNPs including G6PD376, that only female heterozygotes are protected from severe malaria. Haplotype analysis reveals the G6PD locus to be under balancing selection, suggesting a mechanism of protection relying on alleles at modest frequency and avoiding fixation, where protection provided by G6PD deficiency against severe malaria is offset by increased risk of life-threatening complications. Our study also demonstrates that the much-needed large-scale studies of severe malaria and G6PD enzymatic function across African populations require the identification and analysis of the full repertoire of G6PD genetic markers.     

G6PD Deficiency at Sumba in Eastern Indonesia Is Prevalent,Diverse and Severe: Implications for Primaquine Therapy against Relapsing Vivax Malaria

Safe treatment of Plasmodium vivax requires diagnosis of both the infection and status of erythrocytic glucose-6-phosphate dehydrogenase (G6PD) activity because hypnozoitocidal therapy against relapse requires primaquine, which causes a mild to severe acute hemolytic anemia in G6PD deficient patients. Many national malaria control programs recommend primaquine therapy without G6PD screening but with monitoring due to a broad lack of G6PD deficiency screening capacity. The degree of risk in doing so hinges upon the level of residual G6PD activity among the variants present in any given area. We conducted studies on Sumba Island in eastern Indonesia in order to assess the potential threat posed by primaquine therapy without G6PD screening. We sampled 2,033 residents of three separate districts in western Sumba for quantitative G6PD activity and 104 (5.1%) were phenotypically deficient (<4.6U/gHb; median normal 10U/gHb). The villages were in two distinct ecosystems, coastal and inland. A positive correlation occurred between the prevalence of malaria and G6PD deficiency: 5.9% coastal versus inland 0.2% for malaria (P<0.001), and 6.7% and 3.1% for G6PD deficiency (P<0.001) at coastal and inland sites, respectively. The dominant genotypes of G6PD deficiency were Vanua Lava, Viangchan, and Chatham, accounting for 98.5% of the 70 samples genotyped. Subjects expressing the dominant genotypes all had less than 10% of normal enzyme activities and were thus considered severe variants. Blind administration of anti-relapse primaquine therapy at Sumba would likely impose risk of serious harm.     

Evaluation of the Diagnostic Accuracy of CareStart G6PD Deficiency Rapid Diagnostic Test (RDT) in a Malaria Endemic Area in Ghana,Africa

Background

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most widespread enzyme defect that can result in red cell breakdown under oxidative stress when exposed to certain medicines including antimalarials. We evaluated the diagnostic accuracy of CareStart G6PD deficiency Rapid Diagnostic Test (RDT) as a point-of-care tool for screening G6PD deficiency.

Methods

A cross-sectional study was conducted among 206 randomly selected and consented participants from a group with known G6PD deficiency status between February 2013 and June 2013. A maximum of 1.6ml of capillary blood samples were used for G6PD deficiency screening using CareStart G6PD RDT and Trinity qualitative with Trinity quantitative methods as the “gold standard”. Samples were also screened for the presence of malaria parasites. Data entry and analysis were done using Microsoft Access 2010 and Stata Software version 12. Kintampo Health Research Centre Institutional Ethics Committee granted ethical approval.

Results

The sensitivity (SE) and specificity (SP) of CareStart G6PD deficiency RDT was 100% and 72.1% compared to Trinity quantitative method respectively and was 98.9% and 96.2% compared to Trinity qualitative method. Malaria infection status had no significant (P=0.199) change on the performance of the G6PD RDT test kit compared to the “gold standard”.

Conclusions

The outcome of this study suggests that the diagnostic performance of the CareStart G6PD deficiency RDT kit was high and it is acceptable at determining the G6PD deficiency status in a high malaria endemic area in Ghana. The RDT kit presents as an attractive tool for point-of-care G6PD deficiency for rapid testing in areas with high temperatures and less expertise. The CareStart G6PD deficiency RDT kit could be used to screen malaria patients before administration of the fixed dose primaquine with artemisinin-based combination therapy.     

Safety and tolerability of elubaquine (bulaquine, CDRI 80/53) for treatment of Plasmidium vivax malaria in Thailand

We conducted a study to compare the safety and tolerability of anti-relapse drugs elubaquine and primaquine against Plasmodium vivax malaria. After standard therapy with chloroquine, 30 mg/kg given over 3 days, 141 patients with P. vivax infection were randomized to receive primaquine or elubaquine. The 2 treatment regimens were primaquine 30 mg once daily for 7 days (group A, n = 71), and elubaquine 25 mg once daily for 7 days (group B, n = 70). All patients cleared parasitemia within 7 days after chloroquine treatment. Among patients treated with primaquine, one patient relapsed on day 26; no relapse occurred with elubaquine treatement. Both drugs were well tolerated. Adverse effects occurred only in patients with G6PD deficiency who were treated with primaquine (group A, n = 4), whose mean hematocrit fell significantly on days 7, 8 and 9 (P = 0.015, 0.027, and 0.048, respectively). No significant change in hematocrit was observed in patients with G6PD deficiency who were treated with elubaquine (group B, n = 3) or in patients with normal G6PD. In conclusion, elubaquine, as anti-relapse therapy for P. vivax malaria, was as safe and well tolerated as primaquine and did not cause clinically significant hemolysis.     

Primaquine Administration after Falciparum Malaria Treatment in Malaria Hypoendemic Areas with High Incidence of Falciparum and Vivax Mixed Infection: Pros and Cons

Mixed infections of Plasmodium falciparum and Plasmodium vivax is high (~30%) in some malaria hypoendemic areas where the patients present with P. falciparum malaria diagnosed by microscopy. Conventional treatment of P. falciparum with concurrent chloroquine and 14 days of primaquine for all falciparum malaria patients may be useful in areas where mixed falciparum and vivax infections are high and common and also with mild or moderate G6PD deficiency in the population even with or without subpatent vivax mixed infection. It will be possibly cost-effective to reduce subsequent vivax illness if the patients have mixed vivax infection. Further study to prove this hypothesis may be warranted.     

Single Low Dose Primaquine (0.25mg/kg) Does Not Cause Clinically Significant Haemolysis in G6PD Deficient Subjects

Background

Primaquine is the only drug consistently effective against mature gametocytes of Plasmodium falciparum. The transmission blocking dose of primaquine previously recommended was 0.75mg/kg (adult dose 45mg) but its deployment was limited because of concerns over haemolytic effects in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. G6PD deficiency is an inherited X-linked enzymatic defect that affects an estimated 400 million people around the world with high frequencies (15–20%) in populations living in malarious areas. To reduce transmission in low transmission settings and facilitate elimination of P. falciparum, the World Health Organization now recommends adding a single dose of 0.25mg/kg (adult dose 15mg) to Artemisinin-based Combination Therapies (ACTs) without G6PD testing. Direct evidence of the safety of this low dose is lacking. Adverse events and haemoglobin variations after this treatment were assessed in both G6PD normal and deficient subjects in the context of targeted malaria elimination in a malaria endemic area on the North-Western Myanmar-Thailand border where prevalence of G6PD deficiency (Mahidol variant) approximates 15%.

Methods and Findings

The tolerability and safety of primaquine (single dose 0.25 mg base/kg) combined with dihydroartemisinin-piperaquine (DHA-PPQ) given three times at monthly intervals was assessed in 819 subjects. Haemoglobin concentrations were estimated over the six months preceding the ACT + primaquine rounds of mass drug administration. G6PD deficiency was assessed with a phenotypic test and genotyping was performed in male subjects with deficient phenotypes and in all females. Fractional haemoglobin changes in relation to G6PD phenotype and genotype and primaquine round were assessed using linear mixed-effects models. No adverse events related to primaquine were reported during the trial. Mean fractional haemoglobin changes after each primaquine treatment in G6PD deficient subjects (-5.0%, -4.2% and -4.7%) were greater than in G6PD normal subjects (0.3%, -0.8 and -1.7%) but were clinically insignificant. Fractional drops in haemoglobin concentration larger than 25% following single dose primaquine were observed in 1.8% of the population but were asymptomatic.

Conclusions

The single low dose (0.25mg/kg) of primaquine is clinically well tolerated and can be used safely without prior G6PD testing in populations with high prevalence of G6PD deficiency. The present evidence supports a broader use of low dose primaquine without G6PD testing for the treatment and elimination of falciparum malaria.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01872702","term_id":"NCT01872702"}}NCT01872702     

Characterization of G6PD Genotypes and Phenotypes on the Northwestern Thailand-Myanmar Border

Mutations in the glucose-6-phosphate dehydrogenase (G6PD) gene result in red blood cells with increased susceptibility to oxidative damage. Significant haemolysis can be caused by primaquine and other 8-aminoquinoline antimalarials used for the radical treatment of Plasmodium vivax malaria. The distribution and phenotypes of mutations causing G6PD deficiency in the male population of migrants and refugees in a malaria endemic region on the Thailand-Myanmar border were characterized. Blood samples for G6PD fluorescent spot test (FST), G6PD genotyping, and malaria testing were taken from 504 unrelated males of Karen and Burman ethnicities presenting to the outpatient clinics. The overall frequency of G6PD deficiency by the FST was 13.7%. Among the deficient subjects, almost 90% had the Mahidol variant (487G>A) genotype. The remaining subjects had Chinese-4 (392G>T), Viangchan (871G>A), Açores (595A>G), Seattle (844G>C) and Mediterranean (563C>T) variants. Quantification of G6PD activity was performed using a modification of the standard spectrophotometric assay on a subset of 24 samples with Mahidol, Viangchan, Seattle and Chinese-4 mutations; all samples showed a residual enzymatic activity below 10% of normal and were diagnosed correctly by the FST. Further studies are needed to characterise the haemolytic risk of using 8-aminoquinolines in patients with these genotypes.     

Prevalence and Molecular Characterization of Glucose-6-Phosphate Dehydrogenase Deficiency at the China-Myanmar Border

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked hereditary disease that predisposes red blood cells to oxidative damage. G6PD deficiency is particularly prevalent in historically malaria-endemic areas. Use of primaquine for malaria treatment may result in severe hemolysis in G6PD deficient patients. In this study, we systematically evaluated the prevalence of G6PD deficiency in the Kachin (Jingpo) ethnic group along the China-Myanmar border and determined the underlying G6PD genotypes. We surveyed G6PD deficiency in 1770 adult individuals (671 males and 1099 females) of the Kachin ethnicity using a G6PD fluorescent spot test. The overall prevalence of G6PD deficiency in the study population was 29.6% (523/1770), among which 27.9% and 30.6% were males and females, respectively. From these G6PD deficient samples, 198 unrelated individuals (147 females and 51 males) were selected for genotyping at 11 known G6PD single nucleotide polymorphisms (SNPs) in Southeast Asia (ten in exons and one in intron 11) using a multiplex SNaPshot assay. Mutations with known association to a deficient phenotype were detected in 43.9% (87/198) of cases, intronic and synonymous mutations were detected alone in 34.8% (69/198) cases and no mutation were found in 21.2% (42/198) cases. Five non-synonymous mutations, Mahidol 487G>A, Kaiping 1388G>A, Canton 1376G>T, Chinese 4 392G>T, and Viangchan 871G>A were detected. Of the 87 cases with known deficient mutations, the Mahidol variant was the most common (89.7%; 78/87), followed by the Kaiping (8.0%; 7/87) and the Viangchan (2.2%; 2/87) variants. The Canton and Chinese 4 variants were found in 1.1% of these 87 cases. Among them, two females carried the Mahidol/Viangchan and Mahidol/Kaiping double mutations, respectively. Interestingly, the silent SNPs 1311C>T and IVS11nt93T>C both occurred in the same 95 subjects with frequencies at 56.4% and 23.5% in tested females and males, respectively (P<0.05). It is noteworthy that 24 subjects carrying the Mahidol mutation and two carrying the Kaiping mutation also carried the 1311C>T/IVS11nt93T>C SNPs. Further studies are needed to determine the enzyme levels of the G6PD deficient people and presence of additional G6PD mutations in the study population.     

Real-life implementation of a G6PD deficiency screening qualitative test into routine vivax malaria diagnostic units in the Brazilian Amazon (SAFEPRIM study)

BackgroundGlucose-6-phosphate dehydrogenase (G6PD) deficiency greatly hinders Plasmodium vivax malaria radical cure and further elimination due to 8-aminoquinolines-associated hemolysis. Although the deleterious health effects of primaquine in G6PD deficient individuals have been known for over 50 years, G6PD testing is not routinely performed before primaquine treatment in most P. vivax endemic areas.Method/Principal findingsThe qualitative CareStart G6PD screening test was implemented in 12 malaria treatment units (MTUs) in the municipality of Rio Preto da Eva, Western Brazilian Amazon, a malaria endemic area, between February 2019 and early January 2020. Training materials were developed and validated; evaluations were conducted on the effectiveness of training health care professionals (HCPs) to perform the test, the interpretation and reliability of routine testing performed by HCPs, and perceptions of HCPs and patients. Most HCPs were unaware of G6PD deficiency and primaquine-related adverse effects. Most of 110 HCPs trained (86/110, 78%) were able to correctly perform the G6PD test after a single 4-hour training session. The test performed by HCPs during implementation showed 100.0% (4/4) sensitivity and 68.1% (62/91) specificity in identifying G6PD deficient patients as compared to a point-of-care quantitative test (Standard G6PD).Conclusions/SignificanceG6PD screening using the qualitative CareStart G6PD test performed by HCPs in MTUs of an endemic area showed high sensitivity and concerning low specificity. The amount of false G6PD deficiency detected led to substantial loss of opportunities for radical cure.     

Characterization of glucose-6-phosphate dehydrogenase deficiency and identification of a novel haplotype 487G>A/IVS5-612(G>C) in the Achang population of Southwestern China

The prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency and its gene mutations were studied in the Achang population from Lianghe County in Southwestern China. We found that 7.31% (19 of 260) males and 4.35% (10 of 230) females had G6PD deficiency. The molecular analysis of G6PD gene exons 2–13 was performed by a PCR-DHPLC-Sequencing or PCR-Sequencing. Sixteen independent subjects with G6PD Mahidol (487G>A) and the new polymorphism IVS5-612 (G>C), which combined into a novel haplotype, were identified accounting for 84.2% (16/19). And 100% Achang G6PD Mahidol were linked to the IVS5-612 C. The percentage of G6PD Mahidol in the Achang group is close to that in the Myanmar population (91.3% 73/80), which implies that there are some gene flows between Achang and Myanmar populations. Interestingly, G6PD Canton (1376G>T) and G6PD Kaiping (1388G>A), which were the most common G6PD variants from other ethnic groups in China, were not found in this Achang group, suggesting that there are different G6PD mutation profiles in the Achang group and other ethnic groups in China. Our findings appear to be the first documented report on the G6PD genetics of the AChang people, which will provide important clues to the Achang ethnic group origin and will help prevention and treatment of malaria in this area.     

Validation of G6PD Point-of-Care Tests among Healthy Volunteers in Yangon,Myanmar

Primaquine and other 8-amnoquinoline based anti-malarials can cause haemolysis in subjects with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Correct diagnosis of G6PD status in patients is crucial for safe treatment of both relapsing stages of Plasmodium vivax and transmitting forms of Plasmodium falciparum. Lack of suitable point-of-care tests has hampered a much needed wide use of primaquine for malaria elimination. In this study we have assessed the performances of two qualitative tests, the fluorescent spot test (FST) and the G6PD CareStart test (CST), against the gold standard quantitative spectrophotometric assay in a population of 1000 random adult healthy volunteers living in Yangon, Myanmar. The prevalence of G6PD deficiency in the Bamar, Karen and in the whole sample set was 6.6% (10.1% in males), 9.2% (21.0% in males) and 6.8% (11.1% in males) respectively. The FST and CST showed comparable performances with sensitivity over 95% and specificity over 90%, however for cases with severe G6PD activity the FTS had improved performance. If used with a conservative interpretation of the signal, the CareStart test has the potential to be used in the field and, by allowing a wider use of primaquine, to help malaria elimination.     

Real-life quantitative G6PD screening in Plasmodium vivax patients in the Brazilian Amazon: A cost-effectiveness analysis

BackgroundAs quantitative glucose 6-phosphate dehydrogenase deficiency (G6PDd) screening tools are evaluated in operational studies, questions remain as to whether they are cost-effective. Here, a cost-effectiveness analysis (CEA) was performed to estimate the Incremental Cost-effectiveness Ratio (ICER) of the introduction of quantitative screening test to detect G6PDd among P. vivax carriers in two municipalities in the Brazilian Amazon.Methodology/Principal findingsThis cost-effectiveness analysis evaluated the use of the Standard G6PD quantitative screening test in vivax malaria treatment units in two municipalities of the Brazilian Amazon. Using the perspective of the Brazilian public health system, the analysis was performed for the outcome ‘PQ-associated hospitalization avoided’, based on a decision tree model. The results indicated that the G6PDd screening strategy compared with the routine strategy was highly cost-effective, with an ICER of US$495 per additional hospitalization avoided, which represented less than 8% of one Brazilian gross domestic product per capita (US$6,822). The uncertainties evaluated in the sensitivity analysis did not significantly affect the ICER identified in the base-case.Conclusions/SignificanceThis cost-effectiveness analysis showed the quantitative G6PD testing was effective in avoiding PQ-associated hospitalizations. The incorporation of G6PD screening is of paramount importance towards P. vivax malaria elimination in the Amazon to promote the safe use of primaquine and tafenoquine.     

Characterization of glucose-6-phosphate dehydrogenase deficiency and identification of a novel haplotype 487G>A/IVS5-612(G>C) in the Achang population of southwestern China

The prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency and its gene mutations were studied in the Achang population from Lianghe County in Southwestern China. We found that 7.31% (19 of 260) males and 4.35% (10 of 230) females had G6PD deficiency. The molecular analysis of G6PD gene exons 2―13 was performed by a PCR-DHPLC-Sequencing or PCR-Sequencing. Sixteen inde-pendent subjects with G6PD Mahidol (487G>A) and the new polymorphism IVS5-612 (G>C), which combined into a novel haplotype, were identified accounting for 84.2% (16/19). And 100% Achang G6PD Mahidol were linked to the IVS5-612 C. The percentage of G6PD Mahidol in the Achang group is close to that in the Myanmar population (91.3% 73/80), which implies that there are some gene flows between Achang and Myanmar populations. Interestingly, G6PD Canton (1376G>T) and G6PD Kaiping (1388G>A), which were the most common G6PD variants from other ethnic groups in China, were not found in this Achang group, suggesting that there are different G6PD mutation profiles in the Achang group and other ethnic groups in China. Our findings appear to be the first documented report on the G6PD genetics of the AChang people, which will provide important clues to the Achang ethnic group origin and will help prevention and treatment of malaria in this area.     

Stabilization of glucose-6-phosphate dehydrogenase oligomers enhances catalytic activity and stability of clinical variants

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a genetic trait that can cause hemolytic anemia. To date, over 150 nonsynonymous mutations have been identified in G6PD, with pathogenic mutations clustering near the dimer and/or tetramer interface and the allosteric NADP⁺-binding site. Recently, our lab identified a small molecule that activates G6PD variants by stabilizing the allosteric NADP⁺ and dimer complex, suggesting therapeutics that target these regions may improve structural defects. Here, we elucidated the connection between allosteric NADP⁺ binding, oligomerization, and pathogenicity to determine whether oligomer stabilization can be used as a therapeutic strategy for G6PD deficiency (G6PD^def). We first solved the crystal structure for G6PD^K403Q, a mutant that mimics the physiological acetylation of wild-type G6PD in erythrocytes and demonstrated that loss of allosteric NADP⁺ binding induces conformational changes in the dimer. These structural changes prevent tetramerization, are unique to Class I variants (the most severe form of G6PD^def), and cause the deactivation and destabilization of G6PD. We also introduced nonnative cysteines at the oligomer interfaces and found that the tetramer complex is more catalytically active and stable than the dimer. Furthermore, stabilizing the dimer and tetramer improved protein stability in clinical variants, regardless of clinical classification, with tetramerization also improving the activity of G6PD^K403Q and Class I variants. These findings were validated using enzyme activity and thermostability assays, analytical size-exclusion chromatography (SEC), and SEC coupled with small-angle X-ray scattering (SEC-SAXS). Taken together, our findings suggest a potential therapeutic strategy for G6PD^def and provide a foundation for future drug discovery efforts.     

Molecular dynamics of G6PD variants from sub-Saharan Africa

Precision medicine uses genomic guidance to improve drug treatment safety and efficacy. Prior knowledge of genetic variant impact can enable such strategies, but current knowledge of African variants remains scarce. G6PD variants are linked to haemolytic adverse effects for a number of drugs commonly used in African populations. We have investigated a set of G6PD variants with structural bioinformatics techniques to further characterise variants with known effect, and gain insights into variants with unknown impact. We observed wide variations in patterns of root-mean-square deviation between wild-type and variant structures. Variants with known, highly deleterious impact show structural effects which may likely result in the destabilisation of the G6PD homodimer. The V68M and N126D variants (which are both common across African populations, and together form the A- haplotype) induce large conformational shifts in the catalytic NADP+ binding domain. We observed a greater impact for the haplotype than for each of the individual variants in these cases. A novel African variant (M207T) shows the potential to disrupt interactions within the protein core, urging further investigation. We explore how characterising the molecular impact of African G6PD variants can enable advanced strategies for precision medicine, as well as impact the use of novel therapeutics aiming to treat G6PD deficiency. This knowledge can assist in bridging current knowledge gaps, and aid to facilitate precision medicine applications in African populations.     

The baseline distribution of malaria in the initial phase of elimination in Sabang Municipality, Aceh Province, Indonesia

ABSTRACT: BACKGROUND: Sabang Municipality, in Aceh Province, Indonesia, plans to initiate a malaria elimination programme in 2013. A baseline survey of the distribution of malaria in the municipality was conducted to lay the foundations for an evidence-based programme and to assess the island's readiness to begin the elimination process. METHODS: The entire population of the municipality was screened for malaria infection and G6PD deficiency. Specimens collected included blood slides, blots and tubes for selected households. Results and Discussion Samples were collected from 16,229 residents. Microscopic examination of the blood smears revealed 12 malaria infections; 10 with Plasmodium falciparum and 2 with Plasmodium vivax. To confirm the parasite prevalence, polymerase chain reaction (PCR) diagnosis was performed on the entire positive cases by microscopy and randomized 10% of the microscopically negative blood samples. PCR revealed an additional 11 subjects with malaria; one P. falciparum infection from the village of Paya Keunekai, and nine P. vivax infections and one mixed P. falciparum/P. vivax infection from the village of Batee Shok. The overall slide positivity rate was 0.074% (CI 95%: 0.070 - 0.078) and PCR corrected prevalence 0,590% (CI 95%: 0.582 - 0.597). Analysis of 937 blood samples for G6PD deficiency revealed two subjects (0.2%) of deficient G6PD. Analysis of several genes of the parasite, such as Pfdhfr, Pfdhps, Pfmdr1, Pfcrt, Pfmsp1, Pfmsp2, Pvdhfr, Pvdhps, Pvmdr1 and host gene, such as G6PD gene revealed that both P. falciparum and P. vivax carried the mutation associated with chloroquine resistance. CONCLUSION: Malariometric and host genetic analysis indicated that there is a low prevalence of both malaria and G6PD deficiency in the population of Sabang Municipality. Nevertheless, malaria cases were clustered in three rural villages and efforts for malaria elimination in Sabang should be particularly focused on those three villages.     

Variation in Glucose-6-Phosphate Dehydrogenase activity following acute malaria

Primaquine and tafenoquine are the only licensed drugs with activity against Plasmodium vivax hypnozoites but cause haemolysis in patients with glucose–6–phosphate dehydrogenase (G6PD) deficiency. Malaria also causes haemolysis, leading to the replacement of older erythrocytes with low G6PD activity by reticulocytes and young erythrocytes with higher activity. Aim of this study was to assess the impact of acute malaria on G6PD activity. Selected patients with uncomplicated malaria were recruited in Bangladesh (n = 87), Indonesia (n = 75), and Ethiopia (n = 173); G6PD activity was measured at the initial presentation with malaria and a median of 176 days later (range 140 to 998) in the absence of malaria. Among selected participants (deficient participants preferentially enrolled in Bangladesh but not at other sites) G6PD activity fell between malaria and follow up by 79.1% (95%CI: 40.4 to 117.8) in 6 participants classified as deficient (<30% activity), 43.7% (95%CI: 34.2 to 53.1) in 39 individuals with intermediate activity (30% to <70%), and by 4.5% (95%CI: 1.4 to 7.6) in 290 G6PD normal (≥70%) participants. In Bangladesh and Indonesia G6PD activity was significantly higher during acute malaria than when the same individuals were retested during follow up (40.9% (95%CI: 33.4–48.1) and 7.4% (95%CI: 0.2 to 14.6) respectively), whereas in Ethiopia G6PD activity was 3.6% (95%CI: -1.0 to -6.1) lower during acute malaria. The change in G6PD activity was apparent in patients presenting with either P. vivax or P. falciparum infection. Overall, 66.7% (4/6) severely deficient participants and 87.2% (34/39) with intermediate deficiency had normal activities when presenting with malaria.These findings suggest that G6PD activity rises significantly and at clinically relevant levels during acute malaria. Prospective case-control studies are warranted to confirm the degree to which the predicted population attributable risks of drug induced haemolysis is lower than would be predicted from cross sectional surveys.     

Maintaining Specimen Integrity for G6PD Screening by Cytofluorometric Assays

Cytochemical staining remains an efficient way of identifying females who are heterozygous for the X chromosome-linked glucose-6-phosphate dehydrogenase (G6PD) gene. G6PD is highly polymorphic with certain alleles resulting in low intracellular G6PD activity in red blood cells. Low intracellular G6PD activity is associated with a risk of severe hemolysis when exposed to an oxidative stress such as fava beans, certain drugs and infections. Heterozygous females express the enzyme from both X-chromosome alleles resulting in two red blood cell populations each with G6PD enzyme characteristics representative of each allele; for example, normal and deficient. Cytochemical staining is the only way to determine the relative representation of each allele in red blood cells, a feature that is critical when assessing the risk for severe hemolysis when exposed to an oxidant such as the anti-malarial drug primaquine. This letter discusses red blood cell integrity with respect to the cytofluorometric assays for G6PD activity. An approach to making this test more robust is suggested. The approach makes this test more reliable and extends its use to a broader range of blood specimens.