Diagnostic performances of the fluorescent spot test for G6PD deficiency in newborns along the Thailand-Myanmar border: A cohort study

Abstract

Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an inherited enzymatic disorder associated with severe neonatal hyperbilirubinemia and acute haemolysis after exposure to certain drugs or infections. The disorder can be diagnosed phenotypically with a fluorescent spot test (FST), which is a simple test that requires training and basic laboratory equipment. This study aimed to assess the diagnostic performances of the FST used on umbilical cord blood by locally-trained staff and to compare test results of the neonates at birth with the results after one month of age. Methods: We conducted a cohort study on newborns at the Shoklo Malaria Research Unit, along the Thai-Myanmar border between January 2015 and May 2016. The FST was performed at birth on the umbilical cord blood by locally-trained staff and quality controlled by specialised technicians at the central laboratory. The FST was repeated after one month of age. Genotyping for common local G6PD mutations was carried out for all discrepant results. Results: FST was performed on 1521 umbilical cord blood samples. Quality control and genotyping revealed 10 misdiagnoses. After quality control, 10.7% of the males (84/786) and 1.2% of the females (9/735) were phenotypically G6PD deficient at birth. The FST repeated at one month of age or later diagnosed 8 additional G6PD deficient infants who were phenotypically normal at birth. Conclusions: This study shows the short-comings of the G6PD FST in neonatal routine screening and highlights the importance of training and quality control. A more conservative interpretation of the FST in male newborns could increase the diagnostic performances. Quantitative point-of-care tests might show higher sensitivity and specificity for diagnosis of G6PD deficiency on umbilical cord blood and should be investigated.

Evaluating the performance of automated UV enzymatic assay for screening of glucose 6‐phosphate dehydrogenase deficiency

“…Since male and female distribution patterns of blood G6PD activity were different, normal G6PD activity reference values for males and females were reported separately. It was shown that bimodal distribution of male G6PD activity was affected by obvious enzymatic deficient values of mutated hemizygous males, whereas normal distribution of female G6PD activity showing wide range of enzymatic activities resulted from lyonization in heterozygous females . After minimizing the impact of severe G6PD deficient activity (below 10% of median value) on the reference normal value by excluding 5 males from spectrophotometric data set, the standardized normal reference values of female and AMM G6PD activity from spectrophotometric and automated UV enzymatic method of a study population were established.…”

“…It was shown that bimodal distribution of male G6PD activity was affected by obvious enzymatic deficient values of mutated hemizygous males, whereas normal distribution of female G6PD activity showing wide range of enzymatic activities resulted from lyonization in heterozygous females. 3 After minimizing the impact of severe G6PD deficient activity (below 10% of median value) on the reference normal value by excluding 5 males from spectrophotometric data set, the standardized normal reference values of female and AMM G6PD activity from spectrophotometric and automated UV enzymatic method of a study population were established. The normal reference values of females from both spectrometric and automated UV enzymatic assays are slightly higher than that of males, which was consistent with a previous report.…”

“…G6PD deficiency is the most common inherited enzymopathy, affecting more than 400 million people worldwide . Mosaicism of X‐chromosome inactivation in heterozygous females results in population‐based large distribution of enzymatic activities . Individuals with G6PD deficiency are asymptomatic and prone to acute haemolytic anaemia, haemoglobinuria and favism after exposure to triggers, such as high levels of oxidative free radicals, infections, fava bean and antimalarial drugs …”

“…2 Mosaicism of X-chromosome inactivation in heterozygous females results in population-based large distribution of enzymatic activities. 3 Individuals with G6PD deficiency are asymptomatic and prone to acute haemolytic anaemia, haemoglobinuria and favism after exposure to triggers, such as high levels of oxidative free radicals, infections, fava bean and antimalarial drugs. 1 G6PD deficiency is associated with neonatal hyperbilirubinemia and jaundice, commonly found in Mediterranean and Asian populations.…”

“…However, its power in discrimination between intermediate (30%-60% of normal G6PD activity levels) and normal is limited. 3,7 The gold standard for the determination of G6PD activity is based on a quantitative measurement of the amount of NADPH by UV spectrophotometer or flow cytometer, which make it possible to resolutely distinguish intermediate from normal activity levels. 7,8 Nonetheless, performing these assays is manual and time-consuming and requires experienced hands.…”