See the published version of this article at Malaria Journal.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
KASP: a genotyping method to rapid identification of resistance in Plasmodium falciparum
Jose M Rubio
Instituto de Salud Carlos III
Corresponding Author
ORCiD: https://orcid.org/0000-0002-1903-6711
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published version at Malaria Journal.
Background
The emergence and spread of antimalarial 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 antimalarials. This antimalarial resistance arises from the development and subsequent expansion of Single Nucleotide Polymorfisms (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 programs. 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 antimalarial treatments have been analyzed 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 analyzed 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 analyzed.
Conclusions
The KASP assays developed in our 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.
Keywords
Plasmodium falciparum, KASP, resistance, SNPs, K13, MDR, Cytb
Figure 1
Figure 2
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
Version 2
Posted 28 Dec, 2020
Published
On 06 Jan, 2021
No community comments so far
Editorial decision: Accept
On 09 Dec, 2020
Editor assigned
On 03 Dec, 2020
Submission checks complete
On 03 Dec, 2020
Editor invited
On 03 Dec, 2020
No comments provided
Editorial decision: Major Revision
On 01 Nov, 2020
Review #1 received
Received 30 Aug, 2020
Reviewer #2 agreed
On 11 Aug, 2020
Reviewer #1 agreed
On 10 Aug, 2020
Reviewers invited
Invitations sent on 04 Aug, 2020
Editor assigned
On 22 Jul, 2020
Editor invited
On 21 Jul, 2020
Submission checks complete
On 18 Jul, 2020
First submitted
On 15 Jul, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Infectious Diseases
Learn more about our company.
This preprint is under consideration at Malaria Journal. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
KASP: a genotyping method to rapid identification of resistance in Plasmodium falciparum
Jose M Rubio
Instituto de Salud Carlos III
Corresponding Author
ORCiD: https://orcid.org/0000-0002-1903-6711
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
Version 2
Posted 28 Dec, 2020
Published
On 06 Jan, 2021
No community comments so far
Editorial decision: Accept
On 09 Dec, 2020
Editor assigned
On 03 Dec, 2020
Submission checks complete
On 03 Dec, 2020
Editor invited
On 03 Dec, 2020
No comments provided
Editorial decision: Major Revision
On 01 Nov, 2020
Review #1 received
Received 30 Aug, 2020
Reviewer #2 agreed
On 11 Aug, 2020
Reviewer #1 agreed
On 10 Aug, 2020
Reviewers invited
Invitations sent on 04 Aug, 2020
Editor assigned
On 22 Jul, 2020
Editor invited
On 21 Jul, 2020
Submission checks complete
On 18 Jul, 2020
First submitted
On 15 Jul, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Infectious Diseases
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published version at Malaria Journal.
Background
The emergence and spread of antimalarial 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 antimalarials. This antimalarial resistance arises from the development and subsequent expansion of Single Nucleotide Polymorfisms (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 programs. 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 antimalarial treatments have been analyzed 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 analyzed 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 analyzed.
Conclusions
The KASP assays developed in our 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.
Figure 1
Figure 2