Dhorda, M. | bioRxiv Science

bioRxiv2019

Development of copy number assays for detection and surveillance of piperaquine resistance-associated plasmepsin 2/3 copy number variation in Plasmodium falciparum

Long regarded as an epicenter of drug-resistant malaria, Southeast Asia continues to provide new challenges to the control of Plasmodium falciparum malaria. Recently, resistance to the artemisinin combination therapy partner drug piperaquine has been observed in multiple locations across Southeast Asia. Genetic studies have identified a single nucleotide polymorphism as well as a copy number variation molecular marker that associate with clinical and in vitro resistance. The copy number polymorphism is a duplication of a region containing members of the plasmepsin multi-gene family of proteases. To accurately and quickly determine the presence of copy number variation in the plasmepsin 2/3 duplication in field isolates, we developed a quantitative PCR assay using TaqMan probes. We validated copy number estimates using a separate SYBR green-based quantitative PCR assay as well as a novel breakpoint assay to detect the hybrid gene product. Field samples from 2012 - 2015 across 3 sites in Cambodia were tested using DNA extracted from dried blood spots and whole blood to monitor the extent of plasmepsin 2/3 gene duplications, as well as pfmdr1. We found high concordance across all methods of copy number detection. For samples derived from dried blood spots we found a greater than 80% success rate in each assay, with more recent samples performing better. We found evidence of extensive plasmepsin 2/3 copy number amplifications in Pursat (94%, 2015) and Preah Vihear (87%, 2014), and lower levels in Ratanakiri (16%, 2014) in eastern Cambodia. We also see evidence of a shift from two copies of plasmepsin 2/3 in Pursat 2013 to three copies in 2014-15 (25% to 64%). Pfmdr1 duplications are absent from all samples in 2014 from Preah Vihear and Ratanakiri and 2015 from Pursat. This study shows increasing levels of plasmepsin 2/3 gene amplifications across Cambodia from 2012 - 2015 and a complete reversion of pfmdr1 mutant parasites in all study locations. The multiplex TaqMan assay is a robust tool for monitoring both plasmepsin and pfmdr1 copy number variations in field isolates, and the SYBR-green and breakpoint assays are useful for monitoring plasmepsin 2/3 duplications.

genetics↗

bioRxiv2019

Evolution and expansion of multidrug resistant malaria in Southeast Asia: a genomic epidemiology study

BackgroundA multidrug resistant co-lineage of Plasmodium falciparum malaria, named KEL1/PLA1, spread across Cambodia c.2008-2013, causing high treatment failure rates to the frontline combination therapy dihydroartemisinin-piperaquine. Here, we report on the evolution and spread of KEL1/PLA1 in subsequent years.\n\nMethodsWe analysed whole genome sequencing data from 1,673 P. falciparum clinical samples collected in 2008-2018 from northeast Thailand, Laos, Cambodia and Vietnam. By investigating genome-wide relatedness between parasites, we inferred patterns of shared ancestry in the KEL1/PLA1 population.\n\nFindingsKEL1/PLA1 spread rapidly from 2015 into all of the surveyed countries and now exceeds 80% of the P. falciparum population in several regions. These parasites maintained a high level of genetic relatedness reflecting their common origin. However, several genetic subgroups have recently emerged within this co-lineage with diverse geographical distributions. Some of these emerging KEL1/PLA1 subgroups carry recent mutations in the chloroquine resistance transporter (crt) gene, which arise on a specific genetic background comprising multiple genomic regions.\n\nInterpretationAfter emerging and circulating for several years within Cambodia, the P. falciparum KEL1/PLA1 co-lineage diversified into multiple subgroups and acquired new genetic features including novel crt mutations. These subgroups have rapidly spread into neighbouring countries, suggesting enhanced fitness. These findings highlight the urgent need for elimination of this increasingly drug-resistant parasite co-lineage, and the importance of genetic surveillance in accelerating elimination efforts.\n\nFundingWellcome Trust, Bill & Melinda Gates Foundation, UK Medical Research Council, UK Department for International Development.\n\nResearch in context O_TEXTBOXEvidence before this studyThis study updates our previous work describing the emergence and spread of a multidrug resistant P. falciparum co-lineage (KEL1/PLA1) within Cambodia up to 2013. Since then, a regional genetic surveillance project, GenRe-Mekong, has reported that markers of dihydroartemisinin-piperaquine (DHA-PPQ) resistance have increased in frequency in neighbouring countries. A PubMed search (terms: \"artemisinin\", \"piperaquine\", \"resistance\", \"southeast asia\") for articles listed since our previous study (from 30/10/2017 to 05/01/2019) yielded 28 results, including reports of a recent sharp decline in DHA-PPQ clinical efficacy in Vietnam; the spread of genetic markers of DHA-PPQ resistance into neighbouring countries by Imwong and colleagues; and multiple reports associating mutations in the crt gene with piperaquine resistance, including newly emerging crt variants in Southeast Asia.\n\nAdded value of this studyWe analysed P. falciparum whole genomes collected up to early 2018 from Eastern Southeast Asia (Cambodia and surrounding regions), describing the fine-scale epidemiology of multiple KEL1/PLA1 genetic subgroups that have spread out from Cambodia since 2015 and taken over indigenous parasite populations in northeastern Thailand, southern and central Vietnam and parts of southern Laos. Several newly emerging crt mutations accompanied the spread and expansion of KEL1/PLA1 subgroups, suggesting an active proliferation of biologically fit, multidrug resistant parasites.\n\nImplications of all the available evidenceThe problem of P. falciparum multidrug resistance has dramatically worsened in Eastern Southeast Asia since previous reports. KEL1/PLA1 has diversified and spread widely across Eastern Southeast Asia since 2015, becoming the predominant parasite group in several regions. This may have been fuelled by continued parasite exposure to DHA-PPQ, resulting in sustained selection after KEL1/PLA1 became established. Continued drug pressure enabled the acquisition of further mutations, resulting in higher levels of resistance. These data demonstrate the value of pathogen genetic surveillance and the urgent need to eliminate these dangerous parasites.\n\nC_TEXTBOX

epidemiology↗