Comparative analysis of targeted next-generation sequencing for Plasmodium falciparum drug resistance markers

Abstract

Well-defined molecular resistance markers are available for a range of antimalarial drugs, and molecular surveillance is increasingly important for monitoring antimalarial drug resistance. Different genotyping platforms are available, but these have not been compared in detail. We compared Targeted Amplicon Deep sequencing (TADs) using Ion Torrent PGM with Illumina MiSeq for the typing of antimalarial drug resistance genes. We developed and validated protocols to type the molecular resistance markers pfcrt, pfdhfr, pfdhps, pfmdr1, pfkelch, and pfcytochrome b, in Plasmodium falciparum for the Ion Torrent PGM and Illumina MiSeq sequencing platforms. With P. falciparum 3D7 and K1 as reference strains, whole blood samples (N = 20) and blood spots from Rapid Diagnostic Test (RDT) samples (N = 5) from patients with uncomplicated falciparum malaria from Ubon Ratchathani were assessed on both platforms and compared for coverage (average reads per amplicon), sequencing accuracy, variant accuracy, false positive rate, false negative rate, and alternative allele detection, with conventional Sanger sequencing as the reference method for SNP calling. Both whole blood and RDT samples could be successfully sequenced using the Ion Torrent PGM and Illumina MiSeq platforms. Coverage of reads per amplicon was higher with Illumina MiSeq (28,886 reads) than with Ion Torrent PGM (1754 reads). In laboratory generated artificial mixed infections, the two platforms could detect the minor allele down to 1% density at 500X coverage. SNPs calls from both platforms were in complete agreement with conventional Sanger sequencing. The methods can be multiplexed with up to 96 samples per run, which reduces cost by 86% compared to conventional Sanger sequencing. Both platforms, using the developed TAD protocols, provide an accurate method for molecular surveillance of drug resistance markers in P. falciparum, but Illumina MiSeq provides higher coverage than Ion Torrent PGM.

The many definitions of multiplicity of infection

“…e.g., in the context of monitoring anti-malarial drug resistance, point mutations were often obtained by pyro-sequencing cf. Zhou et al ( 40 ), or microsatellite markers typed by gel electrophoresis e.g., Anderson et al ( 41 ), while currently next-generation sequencing techniques e.g., Kunasol et al ( 42 ) and whole genome sequencing are increasingly being used e.g., Akoniyon et al ( 43 ). For the concepts here, loci and alleles must be specified.…”

Global trends in the burden of malaria: Contemporary diagnostic approaches, and treatment strategies

“…One of the distinguishing features of next-generation RDTs is their ability to differentiate between various Plasmodium species [76]. This capacity is of paramount importance, as different species may necessitate distinct treatment approaches due to variations in drug susceptibility [77]. These RDTs retain the advantages of simplicity and accessibility associated with traditional RDTs, making them suitable for point-of-care testing [78].…”

“…These RDTs retain the advantages of simplicity and accessibility associated with traditional RDTs, making them suitable for point-of-care testing [78]. They can be administered quickly and easily by healthcare workers in diverse clinical settings [77].…”

Drug resistance profiling of asymptomatic and low-density Plasmodium falciparum malaria infections on Ngodhe island, Kenya, using custom dual-indexing next-generation sequencing

“…Advancements in low-cost sequencing-based approaches that target molecular markers for drug resistance (e.g. amplicon sequencing) offer a new high throughput method of parasite surveillance, although have not been utilised to look specifically at low-density or asymptomatic infections 28 – 30 . Due to the high proportion of sub-microscopic and asymptomatic P. falciparum infections on Ngodhe island, no genetic information is available for the island’s parasite population, including drug resistance frequencies 7 .…”