Tools for mass screening of G6PD deficiency: validation of the WST8/1-methoxy-PMS enzymatic assay in Uganda

Abstract

BackgroundThe distribution of the enzymopathy glucose-6-phosphate dehydrogenase (G6PD) deficiency is linked to areas of high malaria endemicity due to its association with protection from disease. G6PD deficiency is also identified as the cause of severe haemolysis following administration of the anti-malarial drug primaquine and further use of this drug will likely require identification of G6PD deficiency on a population level. Current conventional methods for G6PD screening have various disadvantages for field use.MethodsThe WST8/1-methoxy PMS method, recently adapted for field use, was validated using a gold standard enzymatic assay (R&D Diagnostics Ltd ®) in a study involving 235 children under five years of age, who were recruited by random selection from a cohort study in Tororo, Uganda. Blood spots were collected by finger-prick onto filter paper at routine visits, and G6PD activity was determined by both tests. Performance of the WST8/1-methoxy PMS test under various temperature, light, and storage conditions was evaluated.ResultsThe WST8/1-methoxy PMS assay was found to have 72% sensitivity and 98% specificity when compared to the commercial enzymatic assay and the AUC was 0.904, suggesting good agreement. Misclassifications were at borderline values of G6PD activity between mild and normal levels, or related to outlier haemoglobin values (<8.0 gHb/dl or >14 gHb/dl) associated with ongoing anaemia or recent haemolytic crises. Although severe G6PD deficiency was not found in the area, the test enabled identification of low G6PD activity. The assay was found to be highly robust for field use; showing less light sensitivity, good performance over a wide temperature range, and good capacity for medium-to-long term storage.ConclusionsThe WST8/1-methoxy PMS assay was comparable to the currently used standard enzymatic test, and offers advantages in terms of cost, storage, portability and use in resource-limited settings. Such features make this test a potential key tool for deployment in the field for point of care assessment prior to primaquine administration in malaria-endemic areas. As with other G6PD tests, outlier haemoglobin levels may confound G6PD level estimation.

Diagnosis and Treatment of Plasmodium vivax Malaria

“…Dried blood blots collected in the field and kept refrigerated are returned to the laboratory for G6PD activity determination in a 96-well spectrophotometric assay format. 34,35 The results correlate well with other standard techniques. Clinical trials.…”

PCR-Based Allelic Discrimination for Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency in Ugandan Umbilical Cord Blood

“…These frequencies nearly followed Hardy-Weinberg equilibrium, where we expect about 8 (17.6%) homozygous females would be 376 GG. The overall incidence of G6PD A- (G202A/A376G) was 6%, which is consistent with a recent report by De Niz et al where enzyme-based fluorescent spot screening was performed on 235 Ugandan children and a prevalence of moderate deficiency of 7.4% was found [11] .…”

Screening for Glucose-6-Phosphate Dehydrogenase Deficiency Using Three Detection Methods: A Cross-Sectional Survey in Southwestern Uganda

“…G6PD deficiency is an X-linked trait and the most common enzymopathy in the world, with over 180 known alleles associated with varying phenotypic severity. [3][4][5] The complexity of this disorder presents a diagnostic challenge, making it difficult to define the degree of deficiency in an individual, particularly in heterozygous females who exhibit variable X-chromosome inactivation. In addition, the correlation between the degree of deficiency and one's risk of clinically significant hemolysis remains unclear.…”