Molecular genotyping to distinguish between recrudescents and new infections in treatment trials of Plasmodium falciparum malaria conducted in Sub‐Saharan Africa: adjustment of parasitological outcomes and assessment of genotyping effectiveness

Abstract

SummaryMolecular genotyping of baseline and post-treatment recurrent Plasmodium falciparum is recommended to distinguish recrudescent from new infections. However, genotyping performance and adjustment of treatment outcomes have not been evaluated in large field trials. Parasitological outcomes were assessed in nine double-blinded trials of uncomplicated P. falciparum malaria in African children treated with artesunate/placebo plus standard monotherapies. Day 28 failure rates were adjusted by stepwise genotyping the P. falciparum glutamate rich protein (glurp), merozoite surface protein 1 (msp1) and 2 (msp2). We calculated overall and laboratory genotyping performance and compared unadjusted (crude) and PCR-adjusted outcomes. 3455 (93.6%) of 3691 enrolled patients were evaluable by Day 28. 767 (22%) had post-Day 14 recurrent parasitemias of which 686 could be genotyped: 246 were recrudescences, 286 new infections and 154 unresolved. The overall and laboratory genotyping performance were 69 (12-100)% and 78 (50-100)%, respectively. The mean Day 28 crude parasitological failure rate was 44 (range 3-87)%. PCR-adjusted rates were 36 (range 2-86)% if unresolved infections were counted as failures or 33 (range 2-86)% if excluded from analysis. The overall difference between crude Day 28 and Day 14 failure rates was 22% (95% CI 20.3, 24.6) but decreased to 14% (95% CI 12.1, 16.3) if unresolved infections are counted as failures, or to 11% (95% CI 9.8, 16.3) if unresolved infections are excluded from the analysis. Genotyping refined treatment outcomes but diligence is needed in sample collection and analysis to improve its performance. Our findings support the WHO recommendation of PCR genotyping in malaria clinical trials and suggest that stepwise genotyping of only two loci (msp2 and msp1 or glurp) can reliably discriminate recrudescences from new infections.

Impact of Transmission Intensity on the Accuracy of Genotyping To Distinguish Recrudescence from New Infection in Antimalarial Clinical Trials

“…To decrease the probability of a match occurring by chance, multiple genotyping markers can be used sequentially in a stepwise approach (15). In this approach, starting with the first genotyping marker, any subject with no alleles in common between pretreatment and recurrent-parasitemia samples is classified as having a new infection, and no further genotyping is necessary.…”

“…The majority of antimalarial trials are now performed in Africa, where transmission is much higher, but with few exceptions (14), the same genotyping methods are used without a reassessment of the increased likelihood of genotypes matching by chance (6), and it is commonly stated that the probability of genotypes matching by chance is low (7,15). In this study, we replaced GLURP, which has less diversity than msp1 (5), with four microsatellite markers, one of which (TA40) has diversity similar to that of msp1.…”

Selection of Plasmodium falciparum Multidrug Resistance Gene 1 Alleles in Asexual Stages and Gametocytes by Artemether-Lumefantrine in Nigerian Children with Uncomplicated Falciparum Malaria

“…The higher rate of recrudescence of infections after treatment with AL observed in our study could be explained in a number of ways. First, we did not use stepwise genotyping of two highly polymorphic loci (msp-1 and msp-2), as proposed by Mugittu and colleagues (22), to distinguish between treatment failures and new infections. Thus, our use of a single genetic marker (msp-2) to establish the PCR-adjusted cure rate might have resulted in an underestimation of the efficacy of AL.…”

Treatment with Coartem (Artemether-Lumefantrine) in Papua New Guinea

“…If the first positive sample contained both, alleles present in the treatment sample and new alleles, the outcome was considered recrudescent. As suggested by Mugittu and others, 9 we used a stepwise approach to discriminate recrudescent from new infections. All msp2 treatment failures were further genotyped for the polymorphic marker gene, merozoite surface protein 1 ( msp1 ).…”