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      KASP: a genotyping method to rapid identification of resistance in Plasmodium falciparum

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          Abstract

          Background

          The emergence and spread of anti-malarial resistance continues to hinder malaria control. Plasmodium falciparum, the species that causes most human malaria cases and most deaths, has shown resistance to almost all known anti-malarials. This anti-malarial resistance arises from the development and subsequent expansion of Single Nucleotide Polymorphisms (SNPs) in specific parasite genes. A quick and cheap tool for the detection of drug resistance can be crucial and very useful for use in hospitals and in malaria control programmes. It has been demonstrated in different contexts that genotyping by Kompetitive Allele Specific PCR (KASP), is a simple, fast and economical method that allows a high-precision biallelic characterization of SNPs, hence its possible utility in the study of resistance in P. falciparum.

          Methods

          Three SNPs involved in most cases of resistance to the most widespread anti-malarial treatments have been analysed by PCR plus sequencing and by KASP (C580Y of the Kelch13 gene, Y86N of the Pfmdr1 gene and M133I of the Pfcytb gene). A total of 113 P. falciparum positive samples and 24 negative samples, previously analysed by PCR and sequencing, were selected for this assay. Likewise, the samples were genotyped for the MSP-1 and MSP-2 genes, and the Multiplicity of Infection (MOI) and parasitaemia were measured to observe their possible influence on the KASP method.

          Results

          The KASP results showed the same expected mutations and wild type genotypes as the reference method, with few exceptions that correlated with very low parasitaemia samples. In addition, two cases of heterozygotes that had not been detected by sequencing were found. No correlation was found between the MOI or parasitaemia and the KASP values of the sample. The reproducibility of the technique shows no oscillations between repetitions in any of the three SNPs analysed.

          Conclusions

          The KASP assays developed in this study were efficient and versatile for the determination of the Plasmodium genotypes related to resistance. The method is simple, fast, reproducible with low cost in personnel, material and equipment and scalable, being able to core KASP arrays, including numerous SNPs, to complete the main pattern of mutations associated to P. falciparum resistance.

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          Most cited references25

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          BioEdit - a user friendly biological sequence alignment editor and analysis program for Windows 95/98/NT

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            A molecular marker of artemisinin-resistant Plasmodium falciparum malaria.

            Plasmodium falciparum resistance to artemisinin derivatives in southeast Asia threatens malaria control and elimination activities worldwide. To monitor the spread of artemisinin resistance, a molecular marker is urgently needed. Here, using whole-genome sequencing of an artemisinin-resistant parasite line from Africa and clinical parasite isolates from Cambodia, we associate mutations in the PF3D7_1343700 kelch propeller domain ('K13-propeller') with artemisinin resistance in vitro and in vivo. Mutant K13-propeller alleles cluster in Cambodian provinces where resistance is prevalent, and the increasing frequency of a dominant mutant K13-propeller allele correlates with the recent spread of resistance in western Cambodia. Strong correlations between the presence of a mutant allele, in vitro parasite survival rates and in vivo parasite clearance rates indicate that K13-propeller mutations are important determinants of artemisinin resistance. K13-propeller polymorphism constitutes a useful molecular marker for large-scale surveillance efforts to contain artemisinin resistance in the Greater Mekong Subregion and prevent its global spread.
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              Single nucleotide polymorphism genotyping using Kompetitive Allele Specific PCR (KASP): overview of the technology and its application in crop improvement

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                Author and article information

                Contributors
                jmrubio@isciii.es
                Journal
                Malar J
                Malar J
                Malaria Journal
                BioMed Central (London )
                1475-2875
                6 January 2021
                6 January 2021
                2021
                : 20
                : 16
                Affiliations
                [1 ]GRID grid.413448.e, ISNI 0000 0000 9314 1427, Malaria & Parasitic Emerging Diseases Laboratory, , National Microbiology Center, Instituto de Salud Carlos III, ; Madrid, Spain
                [2 ]GRID grid.411171.3, ISNI 0000 0004 0425 3881, Department of Clinical Microbiology, , Hospital Universitario, ; 12 de Octubre, Madrid, Spain
                [3 ]GRID grid.414676.6, ISNI 0000 0001 0687 2000, Parasitology Unit, Infectious Disease Research Centre, Institute for Medical Research, , National Institute of Health, ; Setia Alam, Selangor, Malaysia
                Author information
                http://orcid.org/0000-0002-1903-6711
                Article
                3544
                10.1186/s12936-020-03544-7
                7789257
                33407529
                346ae12e-1add-4e14-bd05-cf28c640a1b0
                © The Author(s) 2021

                Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

                History
                : 15 July 2020
                : 11 December 2020
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100004587, Instituto de Salud Carlos III;
                Award ID: PI17CIII/00035
                Award ID: PI17/01791
                Award ID: CM17CIII/00012
                Award Recipient :
                Categories
                Research
                Custom metadata
                © The Author(s) 2021

                Infectious disease & Microbiology
                plasmodium falciparum,kasp,resistance,snps,k13,mdr,cytb
                Infectious disease & Microbiology
                plasmodium falciparum, kasp, resistance, snps, k13, mdr, cytb

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