Centre for Medical Parasitology, Department of International Health, Immunology and Microbiology, University of Copenhagen, 1356, Copenhagen K, Denmark.
Department of Infectious Diseases, Copenhagen University Hospital, 2200, Copenhagen N, Denmark.
Sci Rep. 2017 May 25;7(1):2398. doi: 10.1038/s41598-017-02724-x.
Genetic polymorphisms in P. falciparum can be used to indicate the parasite's susceptibility to antimalarial drugs as well as its geographical origin. Both of these factors are key to monitoring development and spread of antimalarial drug resistance. In this study, we combine multiplex PCR, custom designed dual indexing and Miseq sequencing for high throughput SNP-profiling of 457 malaria infections from Guinea-Bissau, at the cost of 10 USD per sample. By amplifying and sequencing 15 genetic fragments, we cover 20 resistance-conferring SNPs occurring in pfcrt, pfmdr1, pfdhfr, pfdhps, as well as the entire length of pfK13, and the mitochondrial barcode for parasite origin. SNPs of interest were sequenced with an average depth of 2,043 reads, and bases were called for the various SNP-positions with a p-value below 0.05, for 89.8-100% of samples. The SNP data indicates that artemisinin resistance-conferring SNPs in pfK13 are absent from the studied area of Guinea-Bissau, while the pfmdr1 86 N allele is found at a high prevalence. The mitochondrial barcodes are unanimous and accommodate a West African origin of the parasites. With this method, very reliable high throughput surveillance of antimalarial drug resistance becomes more affordable than ever before.
疟原虫的遗传多态性可用于指示寄生虫对抗疟药物的敏感性及其地理来源。这两个因素都是监测抗疟药物耐药性发展和传播的关键。在这项研究中，我们结合多重 PCR、定制的双重索引和 Miseq 测序，以 10 美元/样本的成本对来自几内亚比绍的 457 例疟疾感染进行高通量 SNP 分析。通过扩增和测序 15 个遗传片段，我们覆盖了 pfcrt、pfmdr1、pfdhfr、pfdhps 中发生的 20 个耐药相关 SNP 以及 pfK13 的全长和寄生虫起源的线粒体条码。有意义的 SNP 以平均深度 2043 个读数进行测序，并且对于各种 SNP 位置，只要 p 值低于 0.05，就会有 89.8-100%的样本进行碱基调用。SNP 数据表明，研究地区的几内亚比绍不存在 pfK13 中赋予青蒿素耐药性的 SNP，而 pfmdr1 86N 等位基因的出现率很高。线粒体条码是一致的，适应寄生虫的西非起源。通过这种方法，非常可靠的高通量抗疟药物耐药性监测比以往任何时候都更加负担得起。
