Comparison of Two Genotyping Methods for Distinguishing Recrudescence from Reinfection in Antimalarial Drug Efficacy/Effectiveness Trials

Abstract

Genotyping of allelic variants of Plasmodium falciparum merozoite surface proteins 1 and 2 (msp-1 and msp-2), and the glutamate-rich protein is the gold standard for distinguishing reinfections from recrudescences in antimalarial drug trials. We compared performance of the recently developed 24-single-nucleotide polymorphism (SNP) Barcoding Assay against msp-1 and msp-2 genotyping in a cluster-randomized effectiveness trial of artemether-lumefantrine and dihydroartemisinin-piperaquine in Malawi. Rates of recrudescence and reinfection estimated by the two methods did not differ significantly (Fisher's exact test; P = 0.887 and P = 0.768, respectively). There was a strong agreement between the two methods in predicting treatment outcomes and resolving the genetic complexity of malaria infections in this setting. These results support the use of this SNP assay as an alternative method for correcting antimalarial efficacy/effectiveness data.

Figure 1.

Comparison of two genotyping methods. (A) Rates of reinfection and recrudescence estimated by the 24-single-nucleotide polymorphism (SNP) Barcoding Assay and merozoite surface proteins 1 and 2 (msp-1 and msp-2) genotyping. The number on top of each bar represents number of patients with a defined treatment outcome out of 109 patients evaluated. Rates of reinfection and recrudescence estimated by the two methods were similar (Fisher’s exact test; P = 0.887 and P = 0.768, respectively). (B) Agreement between methods in determining treatment outcomes, infection clonality, and multiplicity of infection. Figures on top of each bar are percentages of concordant samples out of all samples analyzed in square brackets. Multiplicity of infection was determined from SNP data of each sample using complexity of infection likelihood (COIL) and from msp-1 and msp-2 data as the highest number of alleles observed at the most diverse locus. In both A and B, error bars are binomial exact 95% confidence intervals.

Figure 2.

Resolution power of the 24-single-nucleotide polymorphism (SNP) Barcoding Assay inferred from SNP resampling. The gray line shows maximum haplotype diversity captured when all 24 SNPs are used to characterize diversity, whereas the black line indicates diversity identified when only SNPs with a high minor allele frequency (≥ 0.30) are used. Error bars are 95% confidence intervals for the mean number of parasite haplotypes identified. Diversity plateaus after 17 and 12 loci if all 24 SNPs and SNPs with high minor allele frequency are used to genotype samples, respectively, indicating the assay’s sufficient discriminatory power.