Abstract
BackgroundMolecular genotyping is performed in anti-malarial trials to determine whether recurrent parasitaemia after therapy represents a recrudescence (treatment failure) or new infection. The use of capillary instead of agarose gel electrophoresis for genotyping offers technical advantages, but it is unclear whether capillary electrophoresis will result in improved classification of anti-malarial treatment outcomes.MethodsSamples were genotyped using both gel and capillary electrophoresis from randomized trials of artemether-lumefantrine (AL) vs. dihydroartemisinin-piperaquine (DP) performed in two areas of Uganda: Kanungu, where transmission is moderate, and Apac, where transmission is very high. Both gel and capillary methods evaluated polymorphic regions of the merozoite surface protein 1 and 2 and glutamine rich protein genes.ResultsCapillary electrophoresis detected more alleles and provided higher discriminatory power than agarose gel electrophoresis at both study sites. There was only moderate agreement between classification of outcomes with the two methods in Kanungu (kappa = 0.66) and poor agreement in Apac (kappa = 0.24). Overall efficacy results were similar when using gel vs. capillary methods in Kanungu (42-day risk of treatment failure for AL: 6.9% vs. 5.5%, p = 0.4; DP 2.4% vs. 2.9%, p = 0.5). However, the measured risk of recrudescence was significantly higher when using gel vs. capillary electrophoresis in Apac (risk of treatment failure for AL: 17.0% vs. 10.7%, p = 0.02; DP: 8.5% vs. 3.4%, p = 0.03). Risk differences between AL and DP were not significantly different whether gel or capillary methods were used.ConclusionsGenotyping with gel electrophoresis overestimates the risk of recrudescence in anti-malarial trials performed in areas of high transmission intensity. Capillary electrophoresis provides more accurate outcomes for such trials and should be performed when possible. In areas of moderate transmission, gel electrophoresis appears adequate to estimate comparative risks of treatment failure.
Highlights
Molecular genotyping is performed in anti-malarial trials to determine whether recurrent parasitaemia after therapy represents a recrudescence or new infection
When anti-malarial drug efficacy trials are performed in malaria endemic areas, molecular genotyping is required to determine whether recurrent parasitaemia after therapy represents a recrudescence or new infection [1]
Samples genotyped To directly compare the results of gel versus capillary electrophoresis, the samples from two drug efficacy trials were independently genotyped with each method, using the stepwise algorithm of assessing msp2, glurp, msp1 recommended by the World Health Organization (WHO) [6]
Summary
Molecular genotyping is performed in anti-malarial trials to determine whether recurrent parasitaemia after therapy represents a recrudescence (treatment failure) or new infection. When anti-malarial drug efficacy trials are performed in malaria endemic areas, molecular genotyping is required to determine whether recurrent parasitaemia after therapy represents a recrudescence (treatment failure) or new infection [1]. To discriminate P. falciparum strains, the sizes of amplified products of these genes are compared, routinely based on migration patterns with agarose-gel electrophoresis because of ease of use and low cost. Gel electrophoresis may not provide adequate discrimination of alleles, especially in high transmission settings, where high multiplicity of infection (MOI) can lead to a large number of genotyping misclassifications due either to missed detection of alleles or to alleles matching by chance [3,4]. Obtaining accurate estimates of the risk of treatment failure is important for guiding policy, as decisions regarding anti-malarial therapy are primarily based on these estimates
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