Genetic analysis and molecular basis of G6PD deficiency among malaria patients in Thailand: implications for safe use of 8-aminoquinolines.

BACKGROUND: It was hypothesized that glucose-6-phosphate dehydrogenase (G6PD) deficiency confers a protective effect against malaria infection, however, safety concerns have been raised regarding haemolytic toxicity caused by radical cure with 8-aminoquinolines in G6PD-deficient individuals. Malaria elimination and control are also complicated by the high prevalence of G6PD deficiency in malaria-endemic areas. Hence, accurate identification of G6PD deficiency is required to identify those who are eligible for malaria treatment using 8-aminoquinolines. METHODS: The prevalence of G6PD deficiency among 408 Thai participants diagnosed with malaria by microscopy (71), and malaria-negative controls (337), was assessed using a phenotypic test based on water-soluble tetrazolium salts. High-resolution melting (HRM) curve analysis was developed from a previous study to enable the detection of 15 common missense, synonymous and intronic G6PD mutations in Asian populations. The identified mutations were subjected to biochemical and structural characterisation to understand the molecular mechanisms underlying enzyme deficiency. RESULTS: Based on phenotypic testing, the prevalence of G6PD deficiency (< 30% activity) was 6.13% (25/408) and intermediate deficiency (30-70% activity) was found in 15.20% (62/408) of participants. Several G6PD genotypes with newly discovered double missense variants were identified by HRM assays, including G6PD Gaohe + Viangchan, G6PD Valladolid + Viangchan and G6PD Canton + Viangchan. A significantly high frequency of synonymous (c.1311C>T) and intronic (c.1365-13T>C and c.486-34delT) mutations was detected with intermediate to normal enzyme activity. The double missense mutations were less catalytically active than their corresponding single missense mutations, resulting in severe enzyme deficiency. While the mutations had a minor effect on binding affinity, structural instability was a key contributor to the enzyme deficiency observed in G6PD-deficient individuals. CONCLUSIONS: With varying degrees of enzyme deficiency, G6PD genotyping can be used as a complement to phenotypic screening to identify those who are eligible for 8-aminoquinolines. The information gained from this study could be useful for management and treatment of malaria, as well as for the prevention of unanticipated reactions to certain medications and foods in the studied population.

Operational feasibility of Plasmodium vivax radical cure with tafenoquine or primaquine following point-of-care, quantitative glucose-6-phosphate dehydrogenase testing in the Brazilian Amazon: a real-life retrospective analysis.

BACKGROUND: To achieve malaria elimination, Brazil must implement Plasmodium vivax radical cure. We aimed to investigate the operational feasibility of point-of-care, quantitative, glucose-6-phosphate dehydrogenase (G6PD) testing followed by chloroquine plus tafenoquine or primaquine. METHODS: This non-interventional, observational study was done at 43 health facilities in Manaus (Amazonas State) and Porto Velho (Rondônia State), Brazil, implementing a new P vivax treatment algorithm incorporating point-of-care quantitative G6PD testing to identify G6PD status and single-dose tafenoquine (G6PD normal, aged ≥16 years, and not pregnant or breastfeeding) or primaquine (intermediate or normal G6PD, aged ≥6 months, not pregnant, or breastfeeding >1 month). Following training of health-care providers, we collated routine patient records from the malaria epidemiological surveillance system (SIVEP-Malaria) retrospectively for all consenting patients aged at least 6 months with parasitologically confirmed P vivax malaria mono-infection or P vivax plus P falciparum mixed infection, presenting between Sept 9, 2021, and Aug 31, 2022. The primary endpoint was the proportion of patients aged at least 16 years with P vivax mono-infection treated or not treated appropriately with tafenoquine in accordance with their G6PD status. The trial is registered with ClinicalTrials.gov, NCT05096702, and is completed. FINDINGS: Of 6075 patients enrolled, 6026 (99·2%) had P vivax mono-infection, 2685 (44·6%) of whom were administered tafenoquine. G6PD status was identified in 2685 (100%) of 2685 patients treated with tafenoquine. The proportion of patients aged at least 16 years with P vivax mono-infection who were treated or not treated appropriately with tafenoquine in accordance with their G6PD status was 99·7% (95% CI 99·4-99·8; 4664/4680). INTERPRETATION: Quantitative G6PD testing before tafenoquine administration was operationally feasible, with high adherence to the treatment algorithm, supporting deployment throughout the Brazilian health system. FUNDING: Brazilian Ministry of Health, Municipal and State Health Secretariats; Fiocruz; Medicines for Malaria Venture; Bill & Melinda Gates Foundation; Newcrest Mining; and the UK Government. TRANSLATION: For the Portuguese translation of the abstract see Supplementary Materials section.

Prevalence and molecular heterogeneity of glucose-6-phosphate dehydrogenase (G6PD) deficiency in the Senoi Malaysian Orang Asli population.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked genetic disorder characterized by reduced G6PD enzyme levels in the blood. This condition is common in populations exposed to malaria; an acute febrile disease caused by Plasmodium parasites. G6PD-deficient individuals may suffer from acute hemolysis following the prescription of Primaquine, an antimalarial treatment. The population at risk for such a condition includes the Senoi group of Orang Asli, a remote indigenous community in Malaysia. This study aimed to elucidate the G6PD molecular heterogeneity in this subethnic group which is important for malaria elimination. A total of 662 blood samples (369 males and 293 females) from the Senoi subethnic group were screened for G6PD deficiency using a quantitative G6PD assay, OSMMR2000-D kit with Hb normalization. After excluding the family members, the overall prevalence of G6PD deficiency in the studied population was 15.2% (95% CI: 11-19%; 56 of 369), with males (30 of 172; 17.4%) outnumbering females (26 of 197; 13.2%). The adjusted male median (AMM), defined as 100% G6PD activity, was 11.8 IU/gHb. A total of 36 participants (9.6%; 26 male and 10 female) were deficient (<30% of AMM) and 20 participants (5.4%; 4 male and 16 female) were G6PD-intermediate (30-70% of AMM). A total of 87 samples were genotyped, of which 18 showed no mutation. Seven mutations were found among 69 genotyped samples; IVS11 T93C (47.1%; n = 41), rs1050757 (3'UTR +357A>G)(39.1%; n = 34), G6PD Viangchan (c.871G>A)(25.3%; n = 22), G6PD Union (c.1360C>T)(21.8%; n = 19), c.1311C>T(20.7%; n = 18), G6PD Kaiping (c.1388G>A)(8.0%; n = 7), and G6PD Coimbra (c.592C>T)(2.3%; n = 2). Our analysis revealed 27 hemizygote males, 18 heterozygote females, 7 homozygote females, and 2 compound heterozygote females. This study confirms the high prevalence of G6PD deficiency among the Senoi Malaysian Orang Asli, with a significant degree of molecular heterogeneity. More emphasis should be placed on screening for G6PD status and proper and safe use of Primaquine in the elimination of malaria among this indigenous population.

One-time ozone treatment improves the postharvest quality and antioxidant activity of Actinidia arguta fruit.

The major aim of this study was to check the effect of one-time ozonation on selected quality parameters and antioxidant status of Actinidia arguta fruit. For this purpose, A. arguta fruit was ozonated with gas at a concentration of 10 and 100 ppm, which was carried out successively for 5, 15 and 30 min. Next, the selected quality attributes, antioxidants level as well as NADPH and mitochondrial energy metabolism in mini-kiwi fruit after ozonation were analysed. Our research has shown that ozonation reduced the level of yeast and mould without affecting the content of soluble solids or acidity. In turn, ozonation clearly influenced the antioxidant activity and the redox status of the fruit. The ozonated fruit was characterised by a lower level of ROS due to the higher level of low molecular weight antioxidants, as well as the higher activity of superoxide dismutase and catalase. In addition, improved quality and antioxidant activity of the fruit were indirectly due to improved energy metabolism and NADPH level. The ozonated fruit showed a higher level of ATP, due to both higher activity of succinate dehydrogenase and higher availability of NADH. Moreover, the increased level of NAD+ and the activity of NAD+ kinase and glucose-6-phosphate dehydrogenase contributed to higher levels of NADPH in the fruit.

A novel point-of-care device for measuring glucose-6-phosphate dehydrogenase enzyme deficiency.

Extreme hyperbilirubinemia can cause bilirubin neurotoxicity. Infants with glucose-6-phosphate dehydrogenase (G6PD) deficiency can develop hemolysis and thus are at high risk. We evaluated a device that quantitatively measures G6PD activity kinetically using digital microfluidics (DMF). Intra- and inter-instrument and -day imprecision (CVs) were first assessed. G6PD activity in 86 samples was then measured and compared between DMF and 2 reference methods. Overall DMF reproducibility was 3.8% over 5 days by 2 operators on 2 instruments. Mean intra- and inter-instrument variabilities were 3.6% and 3.9%, respectively (n = 28), with a user variability of 4.3%. Mean G6PD activity was 6.40±4.62 and 6.37±4.62 U/g hemoglobin for DMF and Reference Methods 1 (n = 46) and 12.15±3.86 and 11.48±1.55 for DMF and 2 (n = 40), respectively, and strongly correlated (r = 0.95 and 0.95) with mean biases of +0.04±2.90 and +0.67±1.55 for methods 1 and 2, respectively. The novel device could be used for early newborn G6PD screening.

Glucose-6-phosphate dehydrogenase deficiency in Tunisian jaundiced neonates.

BACKGROUND AND OBJECTIVES: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzymopathy worldwide associated with hemolysis as well as neonatal jaundice, kernicterus, and even death. The goal of this study is to determinate the prevalence of G6PD deficiency in icteric neonates and to investigate its biochemical, hematological and molecular characteristics. PATIENTS AND METHODS: This cross sectional study was carried out on 154 icteric newborns admitted to the Bechir Hamza Children's Hospital in Tunisia. Laboratory evaluations included complete blood count, total serum bilirubin level (TSB), and erythrocyte G6PD activity. The G6PD activity was determined using a quantitative assay, which allowed us to divide the total population into two groups: normal and deficient population. The common G6PD Tunisian mutations (GdA - and GdMed) were determined using the amplification refractory mutation system (ARMS-PCR) method. RESULTS: The prevalence of G6PD deficiency among total population (154 icteric newborns) was 18.83%. Male neonates showed a higher incidence of G6PD deficiency of 11.03% compared to females (7.79%). There was no statistical difference between the two groups (normal and deficient), in relation to the sex, peak TSB level, age at peak TSB, hemoglobin level, and hematocrit. However, there was a significant difference in gestational age. In the deficient group, 48.28% neonates presented the peak TSB level between 3 to 7 days and 55% of the cases show a peak TSB level greater than 250 µmol/L. The G6PD G202A variant was found in 41.37% of cases. CONCLUSION: This study shows a higher prevalence of G6PD deficiency in icteric newborns of Tunisia (18.83%). This emphasizes the necessity of neonatal screening for G6PD deficiency to prevent the exposure of these newborns to known hemolytic agents and, subsequently, to prevent kernicterus or other serious complications.

Controlled reversible assembly of gold nanoparticles as a new colorimetric and sensitive detection of glucose-6-phosphate dehydrogenase deficiency.

Recently, several studies have examined possible applications of nanoparticles for the development of electronic and optical sensors. The plasmon absorbance of gold nanoparticles has been used extensively to study biomolecular processes, including nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate-dependent enzymatic reactions. In this report, we describe the development of gold nanoparticles as a new colorimetric and sensitive detection method of glucose-6-phosphate dehydrogenase deficiency by means of controlled reversible assembly of gold nanoparticles. 3-nm polyvinylpyrrolidone/N,N'-dimethylaminopyridine-stabilized gold nanoparticles were synthesized, characterized and applied for an in vitro activity assay of 11 recombinant human glucose-6-phosphate dehydrogenase variants. Differences in the activity of the glucose-6-phosphate dehydrogenase variants from different deficiency classes were readily detected using the synthesized gold nanoparticles. The developed method can be easily distinguished with color change by naked eye for the detection of glucose-6-phosphate dehydrogenase deficiency. Moreover, we are the first to propose the segregation mechanism of polyvinylpyrrolidone/N,N'-dimethylaminopyridine-stabilized gold nanoparticles by reduced nicotinamide adenine dinucleotide phosphate. The method enables visual detection of glucose-6-phosphate dehydrogenase deficiency, which could be further developed for diagnostic testing of glucose-6-phosphate dehydrogenase deficiency.

Malaria radical cure opportunity assessment in India: Discussing opportunities through stakeholder convening workshop and recommendation for improved access to malaria treatment.

India contributes to over 40% of the global Plasmodium vivax disease burden, and P. vivax contributes to approximately one-third of all malaria in India. Government of India has set goals to eliminate malaria by 2030. Doing so will require scaling up existing and new strategies, treatments and diagnostic tools. Access to appropriate diagnosis and treatment for P. vivax malaria is currently limited, and it is unclear how new tools will be rolled out. To support the government in its malaria elimination efforts, the current challenges associated with access to best clinical management of vivax malaria must be understood and mitigated to effectively deploy new tools and scale up existing solutions. The recent Food and Drug Administration (US-FDA) as well as Therapeutics Goods Administration (Australian TGA) approval of tafenoquine, developed by GSK GlaxoSmithKline and Medicines for Malaria Venture (MMV) as a new single-dose radical cure treatment for P. vivax malaria, and the commercial availability of new point-of-care glucose-6-phosphate dehydrogenase (G6PD) tests provide new opportunities to improve clinical management of vivax malaria in India. This report discusses the background, objectives, implementation strategies, and next steps that came out of the Stakeholder Workshop on Malaria Radical Cure in New Delhi, India on 4 February 2019. The focus was to understand the risks and opportunities associated with access to best clinical practices for managing vivax malaria in India. A key outcome was to propose a framework for articulating and segmenting important investment opportunities for improving access to best clinical practices for P. vivax radical cure in India.

Evaluation of quantitative biosensor for glucose-6-phosphate dehydrogenase activity detection.

Neonatal jaundice is a common and severe disease in premature infants with Glucose-6-Phosphate Dehydrogenase (G-6-PD) deficiency. The World Health Organization (WHO) has recommended screening for G-6-PD deficiency in newborns for early recognition as well as to prevent unwanted outcomes in a timely manner. The present study aimed to assess a point-of-care, careSTARTTM G6PD biosensor as a quantitative method for the diagnosis of G-6-PD deficiency. Factors influencing the evaluation of G-6-PD enzyme activity were examined in 40 adults, including ethylenediaminetetraacetic acid (EDTA) anticoagulant, hematocrit concentration, storage temperature and time. Analytic performance of the careSTARTTM G6PD biosensor was evaluated in 216 newborns and compared with fluorescent spot test (FST) and standard quantitative G-6-PD enzyme activity (SGT) assay. The results of factors affecting the G-6-PD enzyme activity showed that the activity determined from finger-prick was not statistically different from venous blood (p = 0.152). The G-6-PD value was highly dependent on the hematocrit and rose with increasing hematocrit concentration. Its activity was stable at 4°C for 3 days. Reliability analysis between the careSTARTTM G6PD biosensor and SGT assay showed a strong correlation with a Pearson's correlation coefficient of 0.82 and perfect agreement by intraclass correlation coefficient (ICC) of 0.90. Analysis of the area under the Receiver Operating Curve (AUC) illustrated that the careSTARTTM G6PD biosensor had 100% sensitivity, 96% specificity, 73% positive predictive value (PPV), 100% negative predictive value (NPV) and 97% accuracy at 30% of residual activity. While the diagnostic ability for identifying G-6-PD deficiency had 78% sensitivity, 89% specificity, 56% positive predictive value (PPV), 96% negative predictive value (NPV) and 88% accuracy when stratified by gender. The careSTARTTM G6PD biosensor is an attractive option as a point-of-care quantitative method for G-6-PD activity detection. Quantification of G-6-PD enzyme activity in newborns is the most effective approach for the management of G-6-PD deficiency to prevent severe jaundice and acute hemolysis.

Self-coalescing flows in microfluidics for pulse-shaped delivery of reagents.

Microfluidic systems can deliver portable point-of-care diagnostics without the need for external equipment or specialist operators, by integrating all reagents and manipulations required for a particular assay in one device1. A key approach is to deposit picogram quantities of dried reagents in microchannels with micrometre precision using specialized inkjet plotters2-5. This means that reagents can be stored for long periods of time and reconstituted spontaneously when adding a liquid sample. But it is challenging to carry out complex operations using multiple reagents, because shear flow enhances their dispersion and they tend to accumulate at moving liquid fronts, resulting in poor spatiotemporal control over the concentration profile of the reconstituted reagents6. One solution is to limit the rate of release of reagents into the liquid7-10. However, this requires the fine-tuning of different reagents, conditions and targeted operations, and cannot readily produce the complex, time-dependent multireagent concentration pulses required for sophisticated on-chip assays. Here we report and characterize a capillary flow phenomenon that we term self-coalescence, which is seen when a confined liquid with a stretched air-liquid interface is forced to 'zip' back onto itself in a microfluidic channel, thereby allowing reagent reconstitution with minimal dispersion. We provide a comprehensive framework that captures the physical underpinning of this effect. We also fabricate scalable, compact and passive microfluidic structures-'self-coalescence modules', or SCMs-that exploit and control this phenomenon in order to dissolve dried reagent deposits in aqueous solutions with precise spatiotemporal control. We show that SCMs can reconstitute multiple reagents so that they either undergo local reactions or are sequentially delivered in a flow of liquid. SCMs are easily fabricated in different materials, readily configured to enable different reagent manipulations, and readily combined with other microfluidic technologies, so should prove useful for assays, diagnostics, high-throughput screening and other technologies requiring efficient preparation and manipulation of small volumes of complex solutions.

Isoenzyme and molecular approach for authenticating and monitoring of animal cell lines.

Authentication of cell lines is of paramount importance to validate the results from their use in biomedical research. Although isoenzyme polymorphism is the standard method, molecular methods based on mitochondrial DNA (mtDNA) have been developed to replace it. The aim of this study was the improvement of our isoenzyme electrophoretic analysis and the validation of one molecular technique targeted at mtDNA for the authentication of our animal cell lines. The combined method of cellular lysing through osmotic shock, followed by freezing-thawing in N2 to obtain isoenzyme extracts, and with 42 × 106 cells maintained the best efficiency. The superior electrophoretic conditions were PAGE run at 200 V. All cell lines had isoenzymatic mobility corresponding to their species to lactate dehydrogenase, malate-dehydrogenase, and glucose-6-phosphate dehydrogenase isoenzymes, and could be distinguished from each other. Two molecular techniques based on mtDNA were tested, one on the cytochrome b gene and other on cytochrome c oxidase I subunit gene. Due to difficulties in distinguishing all cell lines using only one these techniques, we merged the primers of two methods in such a way that there was a sufficient differentiation of all DNA fragments. The sequencing of these PCR products was also performed to validate these data.

Prevalence of G6PD Viangchan variant in malaria endemic areas in Lao PDR: an implication for malaria elimination by 2030.

BACKGROUND: Primaquine is effective against the latent liver stage of Plasmodium vivax. Eliminating the latent liver stage of P. vivax is one of the necessary conditions to achieve the goal of malaria elimination in Lao People's Democratic Republic (PDR) by 2030. However, people with glucose-6-phosphate dehydrogenase (G6PD) deficiency are at risk of haemolysis when ingesting primaquine. The aim of this study was to detect the prevalence of the G6PD Viangchan variant, which is said to be common in Lao PDR and which can result in severe haemolysis in patients exposed to primaquine. METHODS: Blood samples were collected from villagers in three malaria endemic provinces: Champasak and Savannakhet in the south, and Phongsaly in the north. Each blood sample was semi-quantitatively assayed for G6PD enzyme activity using the G6PD Assay Kit-WST Lyophilized (DOJINDO Laboratories, Japan). Blood samples that were found to be G6PD deficient were sequenced to detect G6PD Viangchan mutation. RESULTS: In total, 2043 blood samples were collected from Phongsaly (n = 426, 20.9%), Savannakhet (n = 924, 45.2%), and Champasak (n = 693, 33.9%) provinces in Lao PDR from 2016 to 2017. Of these, 964 (47.2%) were taken from male villagers and 1079 (52.8%) were taken from female villagers. G6PD Viangchan mutation was not detected in Phongsaly province in this study. In Savannakhet province, 48 of the 924 samples (45 males, 3 females) had the G6PD Viangchan mutation (n = 48, 5.2%). In Champasak province, 42 of the 693 samples (18 males, 24 females) had the G6PD Viangchan mutation (n = 42, 6.1%). CONCLUSIONS: G6PD Viangchan variant, which can cause severe haemolysis in the carrier when exposed to primaquine, was detected among 6.1% of the villagers in Champasak and 5.2% in Savannakhet but not in Phongsaly in this study. G6PD Viangchan variant might be common in the south of Laos but not so in the north. In the north, other G6PD deficiency variants might be more prevalent. However, in order not to overlook anyone and ensure a safe primaquine therapy for people living in malaria endemic areas in Lao PDR, G6PD testing is necessary.

Molecular characterization and mapping of glucose-6-phosphate dehydrogenase (G6PD) mutations in the Greater Mekong Subregion.

BACKGROUND: Plasmodium vivax malaria elimination can only be achieved by the deployment of 8-aminoquinolines (primaquine and tafenoquine) in combination with ACT to kill both blood and liver-stage parasites. However, primaquine and the other 8-aminoquinolines cause dose-dependent haemolysis in subjects with G6PD deficiency, an X-linked disorder of red blood cells that is very common in populations living in tropical and subtropical areas. In order to inform safer use of 8-aminoquinolines in the Greater Mekong Subregion, a multi-centre study was carried out to assess the prevalence of G6PD deficiency and to identify the main G6PD variants in samples collected in Cambodia, Lao PDR, Myanmar, Thailand and Vietnam. METHODS: Blood samples were collected in the five countries during National Malaria Surveys or during Population Surveys. During Population Surveys samples were characterized for G6PD phenotype using the Fluorescent Spot Test. Samples were then genotyped for a panel of G6PD mutations. RESULTS: G6PD deficiency was found to be common in the region with an overall mean prevalence of deficient or mutated hemizygous males of 14.0%, ranging from a mean 7.3% in Thailand, 8.1% in Lao PDR, 8.9% in Vietnam, 15.8% in Myanmar and 18.8% in Cambodia. Mahidol and Viangchan mutations were the most common and widespread variants found among the nine investigated. CONCLUSIONS: Owing to the high prevalence of G6PD deficiency in the Greater Mekong Subregion, strategies for vivax malaria elimination should include point-of-care G6PD testing (both qualitative and quantitative) to allow safe and wide treatment with 8-aminoquinolines.

Comparison of glucose-6 phosphate dehydrogenase status by fluorescent spot test and rapid diagnostic test in Lao PDR and Cambodia.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy worldwide. Primaquine is the only licensed drug that effectively removes Plasmodium vivax hypnozoites from the human host and prevents relapse. While well tolerated by most recipients, primaquine can cause haemolysis in G6PD deficient individuals and is, therefore, underused. Rapid diagnostic tests (RDTs) could permit ascertainment of G6PD status outside of laboratory settings and hence safe treatment in remote areas. The performance of the fluorescent spot test (Trinity, Ireland; FST) and a G6PD RDT (Carestart, USA) against spectrophotometry were assessed. METHODS: Participants were enrolled during cross-sectional surveys in Laos and by purposive sampling in Cambodia. FST and RDT were performed during village surveys and 3 mL of venous blood was collected for subsequent G6PD measurement by spectrophotometry. RESULTS: A total of 757 participants were enrolled in Laos and 505 in Cambodia. FST and RDT performed best at 30% cut-off activity and performed significantly better in Laos than in Cambodia. When defining intermediate results as G6PD deficient, the FST had a sensitivity of 100% (95%CI 90-100) and specificity of 90% (95%CI 87.7-92.2) in Laos and sensitivity of 98% (94.1-99.6) and specificity of 71% (95%CI 66-76) in Cambodia (p < 0.001). The RDT had sensitivity and specificity of 100% (95%CI 90-100) and 99% (95%CI 97-99) in Laos and sensitivity and specificity of 91% (86-96) and 93% (90-95) in Cambodia (p < 0.001). The RDT performed significantly better (all p < 0.05) than the FST when intermediate FST results were defined as G6PD deficient. CONCLUSION: The interpretation of RDT results requires some training but is a good alternative to the FST. Trial registration clinicaltrials.gov; NCT01872702; 06/27/2013; https://clinicaltrials.gov/ct2/show/NCT01872702.

Antioxidant Action of an Aqueous Extract of Royal Sun Medicinal Mushroom, Agaricus brasiliensis (Agaricomycetes), in Rats with Adjuvant-Induced Arthritis.

The purpose of this study was to evaluate possible effects of the administration of an aqueous Agaricus brasiliensis extract on the oxidative state of the liver, brain, and plasma in adjuvant-induced arthritic rats, a model for human rheumatoid arthritis. Daily doses of 400 mg · kg-1 were administered by gavage for 23 days. This treatment produced significant diminutions in protein carbonylation and lipid damage in the liver, brain, and plasma. It also diminished the tissue reactive oxygen species and increased the antioxidant capacity of the plasma. Antioxidant defenses, which are diminished by arthritis, were improved by treatment with the A. brasiliensis extract, as revealed by preservation of the reduced glutathione and protein thiol levels and by the tendency of the activities of some antioxidant enzymes to normalize. The increased glucose-6-phosphate dehydrogenase activity was also almost normalized by the treatment. In addition, there were indications that A. brasiliensis can inhibit the initiation of structural events that can lead to hepatocyte necrosis. In conclusion, A. brasiliensis aqueous preparations can, in principle, be visualized as potential auxiliaries in the treatment of patients with rheumatoid arthritis as a result of their capacity to reduce oxidative stress. This effect was exerted in multiple organs, as can be judged from the results obtained in the liver, brain, and plasma. The continuous ingestion of A. brasiliensis as specific preparations or as a food supplement can possibly help to attenuate the systemic effects of rheumatoid arthritis and improve the quality of life of patients affected by this disease.

Newborn screening of glucose-6-phosphate dehydrogenase deficiency in Guangxi, China: determination of optimal cutoff value to identify heterozygous female neonates.

The aim of this study is to assess the disease incidence and mutation spectrum of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Guangxi, China, and to determine an optimal cutoff value to identify heterozygous female neonates. A total of 130, 635 neonates were screened from the year of 2013 to 2017. Neonates suspected for G6PD deficiency were further analyzed by quantitatively enzymatic assay and G6PD mutation analysis. The overall incidence of G6PD deficiency was 7.28%. A total of 14 G6PD mutations were identified, and different mutations lead to varying levels of G6PD enzymatic activities. The best cut-off value of G6PD activity in male subjects is 2.2 U/g Hb, same as conventional setting. In female population, however, the cut-off value is found to be 2.8 U/g Hb (sensitivity: 97.5%, specificity: 87.7%, AUC: 0.964) to best discriminate between normal and heterozygotes, and 1.6 U/g Hb (sensitivity: 82.2%, specificity: 85.9%, AUC: 0.871) between heterozygotes and deficient subjects. In conclusion, we have conducted a comprehensive newborn screening of G6PD deficiency in a large cohort of population from Guangxi, China, and first established a reliable cut-off value of G6PD activity to distinguish heterozygous females from either normal or deficient subjects.

Ultra-performance liquid chromatography-tandem mass spectrometry-based multiplex enzyme assay for six enzymes associated with hereditary hemolytic anemia.

Deficiencies in erythrocyte metabolic enzymes are associated with hereditary hemolytic anemia. Here, we report the development of a novel multiplex enzyme assay for six major enzymes, namely glucose-6-phosphate dehydrogenase, pyruvate kinase, pyrimidine 5'-nucleotidase, hexokinase, triosephosphate isomerase, and adenosine deaminase, deficiencies in which are implicated in erythrocyte enzymopathies. To overcome the drawbacks of traditional spectrophotometric enzyme assays, the present assay was based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The products of the six enzymes were directly measured by using ion pairing UPLC-MS/MS, and the precision, linearity, ion suppression, optimal sample amounts, and incubation times were evaluated. Eighty-three normal individuals and 13 patients with suspected enzymopathy were analyzed. The UPLC running time was within 5min. No ion suppression was observed at the retention time for the products or internal standards. We selected an optimal dilution factor and incubation time for each enzyme system. The intra- and inter-assay imprecision values (CVs) were 2.5-12.1% and 2.9-14.3%, respectively. The linearity of each system was good, with R2 values >0.97. Patient samples showed consistently lower enzyme activities than those from normal individuals. The present ion paring UPLC-MS/MS assay enables facile and reproducible multiplex evaluation of the activity of enzymes implicated in enzymopathy-associated hemolytic anemia.

The pentose phosphate pathway leads to enhanced succinic acid flux in biofilms of wild-type Actinobacillus succinogenes.

Increased pentose phosphate pathway flux, relative to total substrate uptake flux, is shown to enhance succinic acid (SA) yields under continuous, non-growth conditions of Actinobacillus succinogenes biofilms. Separate fermentations of glucose and xylose were conducted in a custom, continuous biofilm reactor at four different dilution rates. Glucose-6-phosphate dehydrogenase assays were performed on cell extracts derived from in situ removal of biofilm at each steady state. The results of the assays were coupled to a kinetic model that revealed an increase in oxidative pentose phosphate pathway (OPPP) flux relative to total substrate flux with increasing SA titre, for both substrates. Furthermore, applying metabolite concentration data to metabolic flux models that include the OPPP revealed similar flux relationships to those observed in the experimental kinetic analysis. A relative increase in OPPP flux produces additional reduction power that enables increased flux through the reductive branch of the TCA cycle, leading to increased SA yields, reduced by-product formation and complete closure of the overall redox balance.

Prevalence of G6PD deficiency and Plasmodium falciparum parasites in asymptomatic school children living in southern Ghana.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked genetic disorder that results in impaired enzyme activity. Although G6PD deficiency is globally distributed it is more prevalent in malaria-endemic countries. Several mutations have been identified in the G6PD gene, which alter enzyme activity. The G6PD genotype predominantly found in sub-Saharan Africa is the G6PDB (G6PD376A) with (G6PD376G) and G6PDA- (G6PD376G/202A, G6PD376G/542T, G6PD376G/680T and G6PD376G/968C) occurring at lower frequencies. AIM: The aim of this study was to identify the prevalence of G6PD deficiency and asymptomatic Plasmodium falciparum carriage in children living in southern Ghana and determine whether G6PD deficiency influences asymptomatic carriage of P. falciparum parasites. METHODS: Blood samples were obtained once a month from 170 healthy Ghanaian school children aged between 5 and 12 years from Basic schools in two communities Obom and Abura with similar rainfall patterns and malaria peak seasons. G6PD enzyme activity was assessed using the qualitative G6PD RDT kit (AccessBIO). G6PD genotyping and asymptomatic parasite carriage was determined by PCR followed by restriction fragment length polymorphism (RFLP) of DNA extracted from dried blood spots. RESULTS: The only sub-Saharan G6PD A- allele detected was the A376G/G202A found in 12.4 % (21/170), of the children and distributed as 4.1 % (7/170) A-, 1.8 % (3/170) A-/A- homozygous deficient males and females and 6.5 % (11/170) A/A- and B/A- heterozygous deficient females. Phenotypically, 10.6 % (15/142) of the children were G6PD deficient. The asymptomatic carriage of P. falciparum by PCR was 50, 29.4, 38.2 and 38.8 % over the months of February through May 2015, respectively, and 28.8, 22.4, 25.9 and 5.9 % by microscopy during the same periods. CONCLUSIONS: G6PD deficiency was significantly associated with a lowered risk of PCR-estimated asymptomatic P. falciparum carriage in children during the off peak malaria season in Southern Ghana.

Sub-Saharan red cell antigen phenotypes and glucose-6-phosphate dehydrogenase deficiency variants in French Guiana.

BACKGROUND: The treatment of Plasmodium vivax infections requires the use of primaquine, which can lead to severe haemolysis in glucose-6-phosphate dehydrogenase (G6PD)-deficient individuals. However, most of the Latin American countries, which are still endemic for vivax malaria, lack information on the distribution of G6PD deficiency (G6PDd). No survey has been performed so far in French Guiana. Herein, 80 individuals of the French Guianan Noir Marron population were scrutinized for red cell surface antigens of six blood group systems (ABO, Rh, Kell, Kidd, Duffy and MNS) and G6PD genetic polymorphisms. First, the sub-Saharan origin of the red cell phenotypes was assessed in relation with the literature. Then, given that the main sub-Saharan G6PDd variants are expected to be encountered, only the G6PD sequences of exons 4, 5, 6 and 9 were screened. This work aims at appraising the G6PD gene variation in this population, and thus, contributing to the G6PD piecemeal information in Latin America. RESULTS: Ninety-seven percent (97 %) of the red cells are Fy(a- b-), either D+ C- E- c+ e+ or D+ C+ E- c+ e+ and 44 % exhibited the Fya-/Jkb-/S- combined phenotype. Noteworthy is the detection of the G6PD(Val68Met) variant characterized by c.202G > A transition, G6PD(Asn126Asp) variant characterized by c.376A>G transition and G6PD(Asp181Val) variant characterized by c.542A>T transversion of the G6PD gene in 22.5 % of the sample, characteristic of the A(-(202)), A and Santamaria G6PDd variants, respectively. CONCLUSIONS: French Guianan Noir Marron population represents a pool of Rh-D antigen positive, Duffy-negative and G6PD-deficient erythrocytes, the latter accounting for one in every eight persons. The present study provides the first community-based estimation of the frequency of G6PDd polymorphisms in French Guiana. These results contribute to the G6PD genetic background information puzzle in Latin America.