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ABSTRACT: Background
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. Efficient methods for extracting and amplifying k13 from dried blood spots (DBS) would facilitate resistance surveillance.Methods
Methods 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.Results
The 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.Conclusions
The optimized DBS protocol should facilitate drug resistance surveillance, especially in low-transmission settings where clinical malaria infections with high parasite densities are rare.
SUBMITTER: Zainabadi K
PROVIDER: S-EPMC6558694 | biostudies-literature | 2019 Jun
REPOSITORIES: biostudies-literature
Zainabadi Kayvan K Nyunt Myaing M MM Plowe Christopher V CV
Malaria journal 20190611 1
<h4>Background</h4>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. Efficient methods for extracting and amplifying k13 from ...[more]