Quantification of glucose-6-phosphate dehydrogenase activity by spectrophotometry: A systematic review and meta-analysis

Abstract

Background: The radical cure of Plasmodium vivax and P. ovale requires treatment with primaquine or tafenoquine to clear dormant liver stages. Either drug can induce haemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency, necessitating screening. The reference diagnostic method for G6PD activity is ultraviolet (UV) spectrophotometry; however, a universal G6PD activity threshold above which these drugs can be safely administered is not yet defined. Our study aimed to quantify assay-based variation in G6PD spectrophotometry and to explore the diagnostic implications of applying a universal threshold.

Methods and findings: Individual-level data were pooled from studies that used G6PD spectrophotometry. Studies were identified via PubMed search (25 April 2018) and unpublished contributions from contacted authors (PROSPERO: CRD42019121414). Studies were excluded if they assessed only individuals with known haematological conditions, were family studies, or had insufficient details. Studies of malaria patients were included but analysed separately. Included studies were assessed for risk of bias using an adapted form of the Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) tool. Repeatability and intra- and interlaboratory variability in G6PD activity measurements were compared between studies and pooled across the dataset. A universal threshold for G6PD deficiency was derived, and its diagnostic performance was compared to site-specific thresholds. Study participants (n = 15,811) were aged between 0 and 86 years, and 44.4% (7,083) were women. Median (range) activity of G6PD normal (G6PDn) control samples was 10.0 U/g Hb (6.3-14.0) for the Trinity assay and 8.3 U/g Hb (6.8-15.6) for the Randox assay. G6PD activity distributions varied significantly between studies. For the 13 studies that used the Trinity assay, the adjusted male median (AMM; a standardised metric of 100% G6PD activity) varied from 5.7 to 12.6 U/g Hb (p < 0.001). Assay precision varied between laboratories, as assessed by variance in control measurements (from 0.1 to 1.5 U/g Hb; p < 0.001) and study-wise mean coefficient of variation (CV) of replicate measures (from 1.6% to 14.9%; p < 0.001). A universal threshold of 100% G6PD activity was defined as 9.4 U/g Hb, yielding diagnostic thresholds of 6.6 U/g Hb (70% activity) and 2.8 U/g Hb (30% activity). These thresholds diagnosed individuals with less than 30% G6PD activity with study-wise sensitivity from 89% (95% CI: 81%-94%) to 100% (95% CI: 96%-100%) and specificity from 96% (95% CI: 89%-99%) to 100% (100%-100%). However, when considering intermediate deficiency (<70% G6PD activity), sensitivity fell to a minimum of 64% (95% CI: 52%-75%) and specificity to 35% (95% CI: 24%-46%). Our ability to identify underlying factors associated with study-level heterogeneity was limited by the lack of availability of covariate data and diverse study contexts and methodologies.

Conclusions: Our findings indicate that there is substantial variation in G6PD measurements by spectrophotometry between sites. This is likely due to variability in laboratory methods, with possible contribution of unmeasured population factors. While an assay-specific, universal quantitative threshold offers robust diagnosis at the 30% level, inter-study variability impedes performance of universal thresholds at the 70% level. Caution is advised in comparing findings based on absolute G6PD activity measurements across studies. Novel handheld quantitative G6PD diagnostics may allow greater standardisation in the future.

Fig 1. PRISMA flow diagram depicting the literature search and data-screening procedures.

G6PD, glucose-6-phosphate dehydrogenase; RBC, red blood cell.

Fig 2. Repeatability of G6PD spectrophotometry.

Inter-replicate agreement in five studies enrolling 2,204 participants. (A) Absolute G6PD activity (U/g Hb) of replicate measures. Each point represents a single G6PD activity measurement, with measurements from the same individual connected by vertical lines for clarity. (B) Relative difference (CV; %) between replicate measures of G6PD activity. Five points with CV greater than 50% are shown as ‘50+’ for clarity (CV = 51, 61, 98, 110, 140). For both panels, the assay used is labelled for each study and the study-wise mean (range) inter-replicate difference is shown. Note: One study, Ley et al., 2018, provided measurements from the same samples using both Randox (n = 92) and Trinity (n = 100). CV, coefficient of variation; G6PD, glucose-6-phosphate dehydrogenase.

Fig 3. Inter-study variation in control sample measurements.

Quality control data from nine studies are shown (n = 1,509). The three leftmost panels contain measurements of deficient, intermediate, and normal controls from studies using the Trinity assay; panels on the right depict deficient and normal control measurements using the Randox assay. The median and interquartile range for each category are superimposed in black. Points are coloured to indicate studies conducted in the same laboratory. A square-root transformation is applied to the y-axis to depict variation in deficient samples more clearly. Note: One study, Ley et al., 2018, provided measurements from the same samples using both Randox (n = 92) and Trinity (n = 100). G6PD, glucose-6-phosphate dehydrogenase.

Fig 4. Inter-study variation in G6PD activity measured using the Trinity assay.

The distribution of G6PD activity measurements is shown for males enrolled in representative studies using the Trinity G6PD assay. Participants with (n = 2,694) and without malaria (n = 3,516) are shown separately. The AMM (black text), 70% threshold (blue), and 30% threshold (red) are labelled for each study (U/g Hb). Point colours indicate the country of origin of each study. Filled points represent G6PDn individuals (>30% study AMM) and hollow points indicate G6PDd individuals (<30% study AMM). Note: Roh et al., 2016, included 63 males <1 year of age, who may have had elevated G6PD activity [31,32]. AMM, adjusted male median; G6PD, glucose-6-phosphate dehydrogenase; G6PDd, G6PD deficient; G6PDn, G6PD normal.

Fig 5. Universal diagnostic classifications by G6PD activity—both males and females, using pooled AMM.

Relative G6PD activity is shown (x-axis, normalised to site-specific AMM) for individuals falling into each diagnostic category, as depicted by coloured bars (false normal [FN], red; false deficient [FD], orange; true normal [TN], light grey; true deficient [TD], black). Diagnoses are shown at both the 30% threshold (left; 17 FN, 21 FD, 6,926 TN, 556 TD) and 70% threshold (right; 139 FN, 258 FD, 5,983 TN, 1,140 TD). All individuals tested with Trinity without malaria infection are shown, except 69 individuals with G6PD activity >200% local AMM (n = 7,451). AMM, adjusted male median; G6PD, glucose-6-phosphate dehydrogenase.