A Comparison of Three Quantitative Methods to Estimate G6PD Activity in the Chittagong Hill Tracts, Bangladesh

Background
Glucose-6-phosphate-dehydrogenase-deficiency (G6PDd) is a major risk factor for primaquine-induced haemolysis. There is a need for improved point-of-care and laboratory-based G6PD diagnostics to unsure safe use of primaquine.
Methods
G6PD activities of participants in a cross-sectional survey in Bangladesh were assessed using two novel quantitative assays, the modified WST-8 test and the CareStart™ G6PD Biosensor (Access Bio), The results were compared with a gold standard UV spectrophotometry assay (Randox). The handheld CareStart™ Hb instrument (Access Bio) is designed to be a companion instrument to the CareStart™ G6PD biosensor, and its performance was compared to the well-validated HemoCue™ method. All quantitative G6PD results were normalized with the HemoCue™ result.
Results
A total of 1002 individuals were enrolled. The adjusted male median (AMM) derived by spectrophotometry was 7.03 U/g Hb (interquartile range (IQR): 5.38–8.69), by WST-8 was 7.03 U/g Hb (IQR: 5.22–8.16) and by Biosensor was 8.61 U/g Hb (IQR: 6.71–10.08). The AMM between spectrophotometry and WST-8 did not differ (p = 1.0) but differed significantly between spectrophotometry and Biosensor (p<0.01). Both, WST-8 and Biosensor were correlated with spectrophotometry (rs = 0.5 and rs = 0.4, both p<0.001). The mean difference in G6PD activity was -0.12 U/g Hb (95% limit of agreement (95% LoA): -5.45 to 5.20) between spectrophotometry and WST-8 and -1.74U/g Hb (95% LoA: -7.63 to 4.23) between spectrophotometry and Biosensor. The WST-8 identified 55.1% (49/89) and the Biosensor 19.1% (17/89) of individuals with G6PD activity <30% by spectrophotometry. Areas under the ROC curve did not differ significantly for the WST-8 and Biosensor irrespective of the cut-off activity applied (all p>0.05). Sensitivity and specificity for detecting G6PD activity <30% was 0.55 (95% confidence interval (95%CI): 0.44–0.66) and 0.98 (95%CI: 0.97–0.99) respectively for the WST-8 and 0.19 (95%CI: 0.12–0.29) and 0.99 (95%CI: 0.98–0.99) respectively for the Biosensor. Hb concentrations measured by HemoCue™ and CareStart™ Hb were strongly correlated (rs = 0.8, p<0.001, mean difference = 0.09 g Hb/dL, 95% LoA: -2.15 to 2.34).
Conclusion
WST-8 and the CareStart™ G6PD Biosensor represent advances in G6PD diagnostics in resource poor settings, but will require further development before clinical deployment. The CareStart™ Hb instrument produced a precise measure of haemoglobin concentration.

背景
葡萄糖-6-磷酸脱氢酶缺乏症（Glucose-6-phosphate-dehydrogenase-deficiency, G6PDd）是伯氨喹诱导溶血的主要危险因素。目前亟需优化床旁检测与实验室级别的G6PD诊断手段，以保障伯氨喹的安全使用。

方法
本研究对孟加拉国一项横断面调查中的受试者进行G6PD活性检测，采用两种新型定量检测方法：改良WST-8试验与CareStart™ G6PD生物传感器（Access Bio公司），并以金标准紫外分光光度法（Randox公司）作为参照进行结果比对。手持设备CareStart™ Hb检测仪为CareStart™ G6PD生物传感器的配套设备，本研究将其检测性能与经过充分验证的HemoCue™方法进行对比。所有定量G6PD检测结果均以HemoCue™的检测结果进行标准化校正。

结果
本研究共纳入1002名受试者。分光光度法测得的校正男性中位数（adjusted male median, AMM）为7.03 U/g Hb（四分位间距（interquartile range, IQR）：5.38~8.69）；WST-8法测得的AMM为7.03 U/g Hb（IQR：5.22~8.16）；生物传感器法测得的AMM为8.61 U/g Hb（IQR：6.71~10.08）。分光光度法与WST-8法的AMM无显著差异（p=1.0），而分光光度法与生物传感器法的AMM差异具有统计学意义（p<0.01）。WST-8法与生物传感器法的检测结果均与分光光度法呈显著相关（斯皮尔曼相关系数rs=0.5和rs=0.4，均p<0.001）。分光光度法与WST-8法的G6PD活性平均差值为-0.12 U/g Hb（95%一致性界限（95% limit of agreement, 95% LoA）：-5.45~5.20）；分光光度法与生物传感器法的平均差值为-1.74 U/g Hb（95% LoA：-7.63~4.23）。以分光光度法检测结果为参照，G6PD活性<30%的受试者中，WST-8法检出55.1%（49/89），生物传感器法仅检出19.1%（17/89）。无论采用何种临界值，WST-8法与生物传感器法的受试者工作特征曲线（ROC）下面积均无显著差异（所有p>0.05）。针对G6PD活性<30%的检测，WST-8法的灵敏度为0.55（95%置信区间（95% confidence interval, 95%CI）：0.44~0.66），特异度为0.98（95%CI：0.97~0.99）；生物传感器法的灵敏度为0.19（95%CI：0.12~0.29），特异度为0.99（95%CI：0.98~0.99）。HemoCue™与CareStart™ Hb检测仪测得的血红蛋白浓度呈强相关（rs=0.8，p<0.001，平均差值为0.09 g Hb/dL，95% LoA：-2.15~2.34）。

结论
WST-8法与CareStart™ G6PD生物传感器为资源匮乏环境下的G6PD诊断提供了新的技术选择，但仍需进一步优化后方可应用于临床。CareStart™ Hb检测仪可精准测定血红蛋白浓度。