Gel versus capillary electrophoresis genotyping for categorizing treatment outcomes in two anti-malarial trials in Uganda

Abstract

Background: Molecular 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.

Methods: Samples 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.

Results: Capillary 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.

Conclusions: Genotyping 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.

Figure 1

Genotyping using markers sequentially at both study sites. Subjects with recurrent parasitaemia in Kanungu and Apac had samples genotyped in a stepwise fashion in the order shown. A new infection was defined as no alleles matching in samples taken on the day of treatment and the day of recurrent parasitaemia. A subject not classified as a new infection by one marker was then genotyped sequentially with the next marker. Those not classified as having a new infection after being genotyped by all three markers were classified as having a recrudescence, as long as genotyping was successful for at least one marker.

Figure 2

Allele sizes measured by capillary electrophoresis at both study sites. The y axis represents the probability of an allele of the given size matching another by chance. As described in the methods, this is calculated as the proportion of all allele measurements from all Day 0 samples that fall within the specified match criteria (within 1 base in size for msp1 and msp2, greater for glurp alleles 880 bases or larger, see methods for more detail).