Abstract

BackgroundThe 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.MethodsThree 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.ResultsThe 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.ConclusionsThe 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.

Highlights

  • The emergence and spread of anti-malarial resistance continues to hinder malaria control

  • Cases of delayed parasite clearance following treatment with an artemisinin-based combination therapy (ACT) have been reported in the Greater Mekong sub-region. This represent a major threat to the ability to control and treat malaria, since this is the current first line treatment for uncomplicated P. falciparum infections [3, 5]. This anti-malarial resistance arises from the development and subsequent expansion of Single Nucleotide Polymorphisms (SNPs) in specific parasite genes [6, 7]

  • The Kompetitive Allele Specific Polymerase Chain Reaction (PCR) (KASP) showed positive results in all the expected samples except one for the SNP of Pfmdr1, one more in the case of Pfcytb SNP and 3 for P. falciparum Kelch13 gene (PfK13) SNP. In all cases this lack of results was related to low parasitaemia; in the first two cases, where the samples were not amplified, presented a parasitaemia, quantified by real-time PCR, lower than 0.05 parasites/μl, being less than 1 parasites/μl for the PfK13

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Summary

Introduction

The emergence and spread of anti-malarial resistance continues to hinder malaria control. Cases of delayed parasite clearance following treatment with an artemisinin-based combination therapy (ACT) have been reported in the Greater Mekong sub-region This represent a major threat to the ability to control and treat malaria, since this is the current first line treatment for uncomplicated P. falciparum infections [3, 5]. This anti-malarial resistance arises from the development and subsequent expansion of Single Nucleotide Polymorphisms (SNPs) in specific parasite genes [6, 7]. Generation sequencing genotyping is an emerging method of genotyping SNPs that is increasingly being adopted for both diagnosis and research [8]

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