Critical Evaluation of Molecular Monitoring in Malaria Drug Efficacy Trials and Pitfalls of Length-Polymorphic Markers.
Estimation of drug efficacy in antimalarial drug trials requires parasite genotyping to distinguish new infections from treatment failures. When using length-polymorphic molecular markers, preferential amplification of short fragments can compromise detection of coinfections, potentially leading to misclassification of treatment outcome. We quantified minority clone detectability and competition among msp1, msp2, and glurp amplicons using mixtures of Plasmodium falciparum strains and investigated the impact of template competition on genotyping outcomes in 44 paired field samples. Substantial amplification bias was detected for all three markers, with shorter fragments outperforming larger fragments. The strongest template competition was observed for the marker glurp Detection of glurp fragments in multiclonal infections was severely compromised. Eight of 44 sample pairs were identified as new infections by all three markers. Ten pairs were defined as new infections based on one marker alone, seven of which were defined by the questionable marker glurp The impact of size-dependent template competition on genotyping outcomes therefore calls for necessary amendments to the current WHO recommendations for PCR correction of malaria drug trial endpoints. Accuracy of genotyping outcomes could be improved by separate amplification reactions per allelic family and basing results on markers msp1 and msp2 first, with glurp only used to resolve discordant results.
Swiss Tropical and Public Health Institute, Basel, Switzerland.|University of Basel, Basel, Switzerland.
Camilla
C
Messerli
Swiss Tropical and Public Health Institute, Basel, Switzerland.|University of Basel, Basel, Switzerland.
Natalie E
NE
Hofmann
Swiss Tropical and Public Health Institute, Basel, Switzerland.|University of Basel, Basel, Switzerland.
Hans-Peter
0000-0001-8326-4834
HP
Beck
Swiss Tropical and Public Health Institute, Basel, Switzerland ingrid.felger@unibas.ch.|University of Basel, Basel, Switzerland.
Ingrid
0000-0003-1255-2606
I
Felger
D000953:Antigens, Protozoan / Q000235:genetics; D000962:Antimalarials / Q000494:pharmacology*; D005838:Genotype; D020066:Merozoite Surface Protein 1 / Q000235:genetics; D010963:Plasmodium falciparum / Q000187:drug effects / Q000235:genetics; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic / Q000235:genetics*; D015800:Protozoan Proteins / Q000235:genetics*
D000953:Antigens, Protozoan; D000962:Antimalarials; D020066:Merozoite Surface Protein 1; D015800:Protozoan Proteins; C065974:merozoite surface protein 2, Plasmodium; C072492:glutamate-rich protein, Plasmodium
PCR; Plasmodium falciparum; amplification bias; drug trial; genotyping; glurp; msp1; msp2; template competition