Performance of molecular inversion probe DR23K and Paragon MAD4HatTeR Amplicon sequencing panels for detection of Plasmodium falciparum mutations associated with antimalarial drug resistance.

Abstract

Background: Molecular surveillance of drug-resistant Plasmodium falciparum is crucial for malaria control in endemic regions. Two targeted-resequencing tools, the Molecular Inversion Probe (MIP) drug resistance panel DR23K and the Multiplexed Amplicons for Drugs, Diagnostics, Diversity, and Differentiation using High-Throughput Targeted Resequencing (MAD⁴HatTeR) panel, are widely used to detect resistance genotypes. However, comparisons of their performance for genotyping drug resistance polymorphisms in malaria parasites and their comparative utility for other use cases is lacking.

Methods: To compare the performance of DR23K and MAD⁴HatTeR in terms of sequencing depth, sensitivity to minor alleles, and precision, each platform was used to evaluate SNP alleles and microhaplotypes in double- and triple-strain mixtures of well-characterized laboratory parasites at densities of 10, 100, 1000, and 10,000 parasites/μL. In addition, 67 Ugandan field samples collected in 2022 were genotyped using each platform to assess performance and concordance.

Results: Across the four parasite densities of 10, 100, 1000, and 10,000 parasites/μL, MAD⁴HatTeR exhibited superior sequencing depth (mean reads per locus: 144, 992, 1153, and 1300) compared to DR23K (mean unique molecular identifiers [UMIs] per locus: 1, 4, 49, and 364). For SNP detection, MAD⁴HatTeR achieved 100% sensitivity at 2% within-sample allele frequency (WSAF) at 1000 and 10,000 parasites/μL, whereas DR23K achieved 100% sensitivity at 40% and 5% WSAF at these densities, respectively. Microhaplotype sensitivity was lower for both assays; MAD4HatTeR reached 69% sensitivity at 10 parasites/μL when WSAF was ≥ 10%, increasing to 100% sensitivity at 2% WSAF and 100 parasites/μL. DR23K had < 50% sensitivity at 10 and 100 parasites/μL. In field samples, which commonly contain polyclonal infections, high concordance was observed between the two methods for all SNPs (94%, 1848/1969) and polymorphic SNPs (88%, 898/1019). All discrepancies were attributed to varied detection of minority alleles in mixed genotype infections.

Conclusions: MAD⁴HatTeR demonstrated higher sensitivity than DR23K, particularly at low parasite densities. Both assays showed strong concordance for genotyping key resistance mutations in field samples, supporting their reliability. These findings suggest MAD⁴HatTeR as the preferred assay for low-density parasite studies and microhaplotype analysis, while DR23K may be appropriate for specific applications with high-parasite density samples, where detection of minority alleles is not prioritized, or when more comprehensive genome coverage is required.