Abstract
BackgroundMutational analysis of the Plasmodium falciparum kelch 13 (k13) gene is routinely performed to track the emergence and spread of artemisinin resistance. Surveillance of resistance markers has been impeded by the difficulty of extracting sufficient DNA from low parasite density infections common in low-transmission settings, such as Southeast Asia. This problem can be overcome by collecting large volumes of venous blood. Efficient methods for extracting and amplifying k13 from dried blood spots (DBS) would facilitate resistance surveillance.MethodsMethods for k13 amplification from standard Whatman 3MM DBS (stored for 14 days at 28 °C with 80% relative humidity) were optimized by systematically testing different extraction conditions. Conditions that improved parasite DNA recovery as assessed by quantitative polymerase chain reaction (PCR) of 18S rDNA were then tested for their impact on k13 PCR amplification.ResultsThe optimized protocol for amplification of k13 from DBS is markedly more sensitive than standard methods using commercial kits. Using this method, k13 was successfully amplified from laboratory-created DBS samples with parasite densities as low as 500 parasites/mL. Importantly, the method recovers both DNA and RNA, making it compatible with RNA-based ultrasensitive techniques currently in use.ConclusionsThe optimized DBS protocol should facilitate drug resistance surveillance, especially in low-transmission settings where clinical malaria infections with high parasite densities are rare.
Highlights
Mutational analysis of the Plasmodium falciparum kelch 13 (k13) gene is routinely performed to track the emergence and spread of artemisinin resistance
The emergence of multidrug resistance in Southeast Asia has led to high rates of treatment failure for first-line artemisinin-based combination therapy, raising the spectre of untreatable malaria in this region and threatening prospects for malaria elimination [1,2,3,4,5,6,7]
As the original k13 polymerase chain reaction (PCR) strategy was not altered [8], the improvement in sensitivity was achieved by optimizing the extraction efficiency from dried blood spots (DBS)
Summary
Mutational analysis of the Plasmodium falciparum kelch 13 (k13) gene is routinely performed to track the emergence and spread of artemisinin resistance. Surveillance of resistance markers has been impeded by the difficulty of extracting sufficient DNA from low parasite density infections common in low-transmission settings, such as Southeast Asia This problem can be overcome by collecting large volumes of venous blood. These new techniques achieve vastly improved sensitivity (with lower limits of detection in the 10 s of parasites/mL) either by collecting high-volume blood samples (i.e., venous blood) or by amplifying highly abundant targets such as 18S rRNA [13, 14] These methods have revealed a large reservoir of previously unrecognized ‘silent’ malaria in Southeast Asia that vastly outnumbers clinically apparent cases [14,15,16,17]
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