title: Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria.
abstract: Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs. One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures. SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status. While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele. In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs can inform control programs. This manuscript describes modifications to high resolution melting technology that further increase its sensitivity to identify polygenomic infections in patient samples.
authors.affiliation: Department of Organismic and Evolutionary Biology, Harvard University; Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health; rdaniels@broadinstitute.org.
authors.forename: Rachel
authors.initials: R
authors.lastname: Daniels
authors.affiliation: Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health.
authors.forename: Elizabeth J
authors.initials: EJ
authors.lastname: Hamilton
authors.affiliation: Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health.
authors.forename: Katelyn
authors.initials: K
authors.lastname: Durfee
authors.affiliation: Faculty of Medicine and Pharmacy, Cheikh Anta Diop University.
authors.forename: Daouda
authors.initials: D
authors.lastname: Ndiaye
authors.affiliation: Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health; Institute of Infectious Diseases, Broad Institute.
authors.forename: Dyann F
authors.initials: DF
authors.lastname: Wirth
authors.affiliation: Department of Organismic and Evolutionary Biology, Harvard University.
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Hartl
authors.affiliation: Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health; School of Nursing and Health Sciences, Simmons College.
authors.forename: Sarah K
authors.initials: SK
authors.lastname: Volkman
mesh_terms: D000483:Alleles; D016054:DNA, Protozoan / Q000097:blood / Q000235:genetics; D023281:Genomics / Q000379:methods; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D008288:Malaria; D016778:Malaria, Falciparum / Q000097:blood / Q000469:parasitology*; D010963:Plasmodium falciparum / Q000737:chemistry / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity
chemical_list: D016054:DNA, Protozoan