Impact of transmission intensity on the accuracy of genotyping to distinguish recrudescence from new infection in antimalarial clinical trials

Abstract

Antimalarial clinical trials use genotyping techniques to distinguish new infection from recrudescence. In areas of high transmission, the accuracy of genotyping may be compromised due to the high number of infecting parasite strains. We compared the accuracies of genotyping methods, using up to six genotyping markers, to assign outcomes for two large antimalarial trials performed in areas of Africa with different transmission intensities. We then estimated the probability of genotyping misclassification and its effect on trial results. At a moderate-transmission site, three genotyping markers were sufficient to generate accurate estimates of treatment failure. At a high-transmission site, even with six markers, estimates of treatment failure were 20% for amodiaquine plus artesunate and 17% for artemether-lumefantrine, regimens expected to be highly efficacious. Of the observed treatment failures for these two regimens, we estimated that at least 45% and 35%, respectively, were new infections misclassified as recrudescences. Increasing the number of genotyping markers improved the ability to distinguish new infection from recrudescence at a moderate-transmission site, but using six markers appeared inadequate at a high-transmission site. Genotyping-adjusted estimates of treatment failure from high-transmission sites may represent substantial overestimates of the true risk of treatment failure.

FIG. 1.

Allele frequency distributions for msp2, msp1, and four microsatellite markers at both study sites. The numbers on the x axis represent allele sizes. The solid bars represent frequencies in Bobo-Dioulasso and the open bars frequencies in Tororo.

FIG. 2.

Genotyping using markers sequentially. Subjects with recurrent parasitemia were genotyped in a stepwise fashion in the order shown. For each step in the algorithm, a subject was classified as having a new infection if genotyping was successful and none of the alleles present in the pretreatment sample persisted in the recurrent-parasitemia sample. All other subjects were genotyped with the next marker. Subjects not classified as having a new infection after being genotyped with all six markers were classified as having recrudescence, as long as genotyping was successful for at least one marker.

FIG. 3.

Estimates of the risk of treatment failure at each point in the stepwise genotyping algorithm. The x axis represents the number of genotyping markers used, where zero represents results prior to genotyping. Genotyping markers were applied sequentially in the order shown in Fig. 2.