Centre for Molecular, Environmental, Genetic & Analytic Epidemiology, Melbourne School of Population and Global Health, University of Melbourne, Melbourne, Australia.
PLoS One. 2013 Jul 24;8(7):e69505. doi: 10.1371/journal.pone.0069505. Print 2013.
The analysis of in vitro anti-malarial drug susceptibility testing is vulnerable to the effects of different statistical approaches and selection biases. These confounding factors were assessed with respect to pfmdr1 gene mutation and amplification in 490 clinical isolates. Two statistical approaches for estimating the drug concentration associated with 50% effect (EC50 ) were compared: the commonly used standard two-stage (STS) method, and nonlinear mixed-effects modelling. The in vitro concentration-effect relationships for, chloroquine, mefloquine, lumefantrine and artesunate, were derived from clinical isolates obtained from patients on the western border of Thailand. All isolates were genotyped for polymorphisms in the pfmdr1 gene. The EC50 estimates were similar for the two statistical approaches but 15-28% of isolates in the STS method had a high coefficient of variation (>15%) for individual estimates of EC50 and these isolates had EC50 values that were 32 to 66% higher than isolates derived with more precision. In total 41% (202/490) of isolates had amplification of pfmdr1 and single nucleotide polymorphisms were found in 50 (10%). Pfmdr1 amplification was associated with an increase in EC50 for mefloquine (139% relative increase in EC50 for 2 copies, 188% for 3+ copies), lumefantrine (82% and 75% for 2 and 3+ copies respectively) and artesunate (63% and 127% for 2 and 3+ copies respectively). In contrast pfmdr1 mutation at codons 86 or 1042 were associated with an increase in chloroquine EC50 (44-48%). Sample size calculations showed that to demonstrate an EC50 shift of 50% or more with 80% power if the prevalence was 10% would require 430 isolates and 245 isolates if the prevalence was 20%. In conclusion, although nonlinear mixed-effects modelling did not demonstrate any major advantage for determining estimates of anti-malarial drug susceptibility, the method includes all isolates, thereby, potentially improving confirmation of candidate molecular markers of anti-malarial drug susceptibility.
体外抗疟药物敏感性测试的分析容易受到不同统计方法和选择偏差的影响。在 490 例临床分离株中，评估了 pfmdr1 基因突变和扩增对这些混杂因素的影响。比较了两种估计药物浓度与 50%效果相关的统计方法（EC50）：常用的标准两阶段（STS）方法和非线性混合效应建模。来自泰国西部边境的患者的临床分离株中，获得了氯喹、甲氟喹、青蒿琥酯和青蒿素的体外浓度-效应关系。所有分离株均进行 pfmdr1 基因多态性基因分型。两种统计方法的 EC50 估计值相似，但 STS 方法中有 15-28%的分离株对 EC50 的个体估计值具有高变异系数（>15%），这些分离株的 EC50 值比更精确的分离株高 32-66%。共有 41%（202/490）的分离株扩增 pfmdr1，发现 50 个（10%）分离株存在单核苷酸多态性。pfmdr1 扩增与甲氟喹 EC50 增加相关（2 个拷贝的 EC50 相对增加 139%，3 个以上拷贝的 EC50 相对增加 188%），青蒿琥酯（2 个和 3 个以上拷贝的 EC50 分别增加 82%和 75%）和青蒿素（2 个和 3 个以上拷贝的 EC50 分别增加 63%和 127%）。相比之下，pfmdr1 密码子 86 或 1042 突变与氯喹 EC50 增加相关（44-48%）。样本量计算表明，如果流行率为 10%，则需要 430 个分离株，流行率为 20%则需要 245 个分离株，才能以 80%的功效证明 EC50 变化 50%或更多。总之，虽然非线性混合效应建模在确定抗疟药物敏感性估计值方面没有显示出任何主要优势，但该方法包括所有分离株，从而有可能提高候选抗疟药物敏感性分子标记物的确认。
