Identification of Glucose-6 Phosphate Dehydrogenase Deficient Patients Using Attenuated Total Reflection Fourier Transform Infrared Spectroscopy Using Partial Least Squares Discriminant Analysis in Aqueous Blood Samples.

Glucose-6 phosphate dehydrogenase (G6PD) deficiency is an X-linked blood disease that affects 400 million people globally and is especially prevalent in malaria-endemic regions. A significant portion of carriers are asymptomatic and undiagnosed posing complications in the eradication of malaria as it restricts the types of drugs used for malaria treatment. A simple and accurate diagnosis of the deficiency is vital in the eradication of malaria. In this study, we investigate the potential of attenuated total reflection Fourier transform infrared spectroscopy (ATR FT-IR) as a diagnostic technique for G6PD deficiency. Venous blood samples were collected in lithium heparin anticoagulant tubes from G6PD partial and fully deficient volunteers, n = 17, and normal volunteers, n = 59, in Khon Kaen, Thailand. Spectra of aqueous and dry samples were acquired of whole blood, plasma, and red blood cells, and modeled using partial least squares discriminant analysis (PLS-DA). PLS-DA modeling resulted in a sensitivity of 0.800 and specificity of 0.800 correctly classifying fully deficient participants as well as a majority of partially deficient females who are often misdiagnosed as normal by current screening methods. The viability of utilizing aqueous samples has always been hindered by the variability of hydration in the sample, but by employing multicurve curve resolution-alternating least squares to subtract water from each sample we are able to produce high-quality spectra with minimized water contributions. The approach shows proof of principle that ATR FT-IR combined with multivariate data analysis could become a frontline screening tool for G6PD deficiency by improving tailored drug treatments and ultimately saving lives.

Prevalence and Molecular Characterization of Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency in Females from Previously Malaria Endemic Regions in Northeastern Thailand and Identification of a Novel G6PD Variant.

INTRODUCTION: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common X-linked enzymopathy, highly prevalent in the areas where malaria is or has been endemic. Prevalence of G6PD deficiency and characterization of G6PD variants in females from previously malaria-endemic areas of northeastern Thailand remain unstudied. METHODS: Prevalence of G6PD deficiency was determined by a fluorescent spot test (FST), quantitative G6PD activity assay, and multiplex allele-specific (AS)- and restriction fragment length polymorphic (RFLP)-PCR developed for detection of common G6PD variants in the Thai population. RESULTS: Prevalence of G6PD deficiency in female samples (n = 355) was 18% by FST, 29.6% by quantitation of G6PD activity, and 28.1% by PCR-based genotyping. The most common variant was G6PD Viangchan (54%), followed by G6PD Canton (11%) and G6PD Union (11%); in addition, a novel heterozygous variant, G6PD Khon Kaen (c.305T>C, p.F102S), was identified. The majority of heterozygotes expressed G6PD activity within the intermediate deficiency range (30-70% median of normal enzyme activity). CONCLUSION: High prevalence of G6PD deficiency was present in females from northeastern Thailand, the majority being due to heterozygosity of G6PD variants. The findings will have a bearing on the inclusion of primaquine in antimalarial-based policies for malaria elimination in populations with a high prevalence of G6PD deficiency.

Relationship among glucose-6-phosphate dehydrogenase (G-6-PD) activity, G-6-PD variants and reticulocytosis in neonates of northeast Thailand.

BACKGROUND: Misdiagnosis of G-6-PD deficiency in neonates, at risk of severe hemolytic episodes, extreme hyperbilirubinemia, and bilirubin encephalopathy, could possibly occur due to presence of reticulocytes, which contain higher amounts of G-6-PD than mature erythrocytes. G-6-PD mutations in the population might also affect G-6-PD activity. This study evaluated the relationship among G-6-PD activity, G-6-PD variants and reticulocytosis in northeastern Thai neonates. METHODS: Blood samples obtained from routine fluorescence spot test examination for G-6-PD deficiency were analyzed using a quantitative enzymatic assay and for common G-6-PD mutations by restriction fragment length polymorphism (RFLP)-PCR. Correlation between G-6-PD activity and percent reticulocytosis was determined. RESULTS: Among 106G-6-PD-deficient (G-6PD activity<7.0U/g Hb) neonates, no significant association is observed between G-6PD activity and percent reticulocytosis (r=0.125, p-value=0.201), but there is a weak correlation in G-6-PD-normal neonates (r=0.377, p-value=0.014). There is a high frequency of G-6-PD Viangchan in male hemizygous and female heterozygous G-6-PD-deficient and G-6-PD-normal neonates. CONCLUSIONS: A high reticulocytosis does not bias measurements of enzyme activity in G-6-PD-deficient neonates. Also, G-6-PD activity varies among female heterozygous neonates, and G-6-PD mutation analysis provides a reliable method to detect G-6-PD deficiency.