A semi-automated method for counting fluorescent malaria oocysts increases the throughput of transmission blocking studies
A semi-automated method for counting fluorescent malaria oocysts increases the throughput of transmission blocking studies
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DOI：
10.1186/1475-2875-9-35
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发表时间：
2010-01-29
期刊：
影响因子：
3
通讯作者：
Sinden, Robert E.
中科院分区：
文献类型：
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作者：
Delves, Michael J.;Sinden, Robert E.
背景：疟疾传播现已被认为是干预的关键目标。按蚊属中肠中疟原虫卵囊负荷的评估。对于许多研究传播的测定很重要。然而，目前的检测方法非常耗时、需要手动操作，而且明显会受到观察者之间差异的影响。 方法：本报告介绍了一种方法的开发，该方法可使用表达 GFP 的伯氏疟原虫和为 ImageJ 定制的宏来快速、准确和一致地确定蚊子中肠的卵囊负荷。通过用寄生虫感染的血液进行配子体膜供给，计数宏被优化并被发现适合目的。手动和使用自动宏对解剖的中肠进行计数，然后进行比较。然后，通过使用宏的优化设置来确定两种抗疟疾化合物硫酸脱氢表雄酮和苯芴醇的传播阻断功效，并与同一实验的手动确定分析进行比较，从而验证宏的优化设置。结果：手动和宏计数的一致性非常高（R-2 = 0.973）并且可重复。手动和自动分析之间估计的传播阻断功效高度一致，表明硫酸脱氢表雄酮几乎没有或没有传播阻断潜力，而本芴醇强烈抑制孢子生殖。结论：认识到吞吐量可能增加五倍，从而减少人员成本，并且使用这种方法可能不存在操作者/实验室间的差异，这种计数宏可能对疟疾界有益。
Background: Malaria transmission is now recognized as a key target for intervention. Evaluation of the Plasmodium oocyst burden in the midguts of Anopheles spp. is important for many of assays investigating transmission. However, current assays are very time-consuming, manually demanding and patently subject to observer-observer variation.Methods: This report presents the development of a method to rapidly, accurately and consistently determine oocyst burdens on mosquito midguts using GFP-expressing Plasmodium berghei and a custom-written macro for ImageJ. The counting macro was optimized and found to be fit-for-purpose by performing gametocyte membrane feeds with parasite infected blood. Dissected midguts were counted both manually and using the automated macro, then compared. The optimized settings for the macro were then validated by using it to determine the transmission blocking efficacies of two anti-malarial compounds dehydroepiandrosterone sulphate and lumefantrine, in comparison to manually determined analysis of the same experiment.Results: Concurrence of manual and macro counts was very high (R-2 = 0.973) and reproducible. Estimated transmission blocking efficacies between manual and automated analysis were highly concordant, indicating that dehydroepiandrosterone sulphate has little or no transmission blocking potential, whilst lumefantrine strongly inhibits sporogony.Conclusion: Recognizing a potential five-fold increase in throughput, the resulting reduction in personnel costs, and the absence of inter-operator/laboratory variation possible with this approach, this counting macro may be a benefit to the malaria community.