KASP: a genotyping method to rapid identification of resistance in Plasmodium falciparum
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 th...
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| Format: | Article |
| Language: | English |
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BMC
2021-01-01
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| Series: | Malaria Journal |
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| Online Access: | https://doi.org/10.1186/s12936-020-03544-7 |
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| author | Ana Alvarez-Fernandez María J. Bernal Isabel Fradejas Alexandra Martin Ramírez Noor Azian Md Yusuf Marta Lanza Shamilah Hisam Ana Pérez de Ayala José M. Rubio |
| author_facet | Ana Alvarez-Fernandez María J. Bernal Isabel Fradejas Alexandra Martin Ramírez Noor Azian Md Yusuf Marta Lanza Shamilah Hisam Ana Pérez de Ayala José M. Rubio |
| author_sort | Ana Alvarez-Fernandez |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-17T22:29:56Z |
| format | Article |
| id | doaj.art-dea477b7b63349e883bcd864bece77ea |
| institution | Directory Open Access Journal |
| issn | 1475-2875 |
| language | English |
| last_indexed | 2024-12-17T22:29:56Z |
| publishDate | 2021-01-01 |
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| series | Malaria Journal |
| spelling | doaj.art-dea477b7b63349e883bcd864bece77ea2022-12-21T21:30:13ZengBMCMalaria Journal1475-28752021-01-012011810.1186/s12936-020-03544-7KASP: a genotyping method to rapid identification of resistance in Plasmodium falciparumAna Alvarez-Fernandez0María J. Bernal1Isabel Fradejas2Alexandra Martin Ramírez3Noor Azian Md Yusuf4Marta Lanza5Shamilah Hisam6Ana Pérez de Ayala7José M. Rubio8Malaria & Parasitic Emerging Diseases Laboratory, National Microbiology Center, Instituto de Salud Carlos IIIMalaria & Parasitic Emerging Diseases Laboratory, National Microbiology Center, Instituto de Salud Carlos IIIDepartment of Clinical Microbiology, Hospital UniversitarioMalaria & Parasitic Emerging Diseases Laboratory, National Microbiology Center, Instituto de Salud Carlos IIIParasitology Unit, Infectious Disease Research Centre, Institute for Medical Research, National Institute of HealthMalaria & Parasitic Emerging Diseases Laboratory, National Microbiology Center, Instituto de Salud Carlos IIIParasitology Unit, Infectious Disease Research Centre, Institute for Medical Research, National Institute of HealthDepartment of Clinical Microbiology, Hospital UniversitarioMalaria & Parasitic Emerging Diseases Laboratory, National Microbiology Center, Instituto de Salud Carlos IIIAbstract 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.https://doi.org/10.1186/s12936-020-03544-7Plasmodium falciparumKASPResistanceSNPsK13MDR |
| spellingShingle | Ana Alvarez-Fernandez María J. Bernal Isabel Fradejas Alexandra Martin Ramírez Noor Azian Md Yusuf Marta Lanza Shamilah Hisam Ana Pérez de Ayala José M. Rubio KASP: a genotyping method to rapid identification of resistance in Plasmodium falciparum Malaria Journal Plasmodium falciparum KASP Resistance SNPs K13 MDR |
| title | KASP: a genotyping method to rapid identification of resistance in Plasmodium falciparum |
| title_full | KASP: a genotyping method to rapid identification of resistance in Plasmodium falciparum |
| title_fullStr | KASP: a genotyping method to rapid identification of resistance in Plasmodium falciparum |
| title_full_unstemmed | KASP: a genotyping method to rapid identification of resistance in Plasmodium falciparum |
| title_short | KASP: a genotyping method to rapid identification of resistance in Plasmodium falciparum |
| title_sort | kasp a genotyping method to rapid identification of resistance in plasmodium falciparum |
| topic | Plasmodium falciparum KASP Resistance SNPs K13 MDR |
| url | https://doi.org/10.1186/s12936-020-03544-7 |
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