Regulatory clinical trials are required to ensure the continued supply and deployment of effective antimalarial drugs. Patient follow-up in such trials typically lasts several weeks, as the drugs have long half-lives and new infections often occur during this period. "Molecular correction" is therefore used to distinguish drug failures from new infections. The current WHO-recommended method for molecular correction uses length-polymorphic alleles at highly diverse loci but is inherently poor at detecting low-density clones in polyclonal infections. This likely leads to substantial underestimates of failure rates, delaying the replacement of failing drugs with potentially lethal consequences. Deep-sequenced amplicons (AmpSeq) substantially increase the detectability of low-density clones and may offer a new "gold standard" for molecular correction. Pharmacological simulation of clinical trials was used to evaluate the suitability of AmpSeq for molecular correction. We investigated the impact of factors such as the number of amplicon loci analyzed, the informatics criteria used to distinguish genotyping "noise" from real low-density signals, the local epidemiology of malaria transmission, and the potential impact of genetic signals from gametocytes. AmpSeq greatly improved molecular correction and provided accurate drug failure rate estimates. The use of 3 to 5 amplicons was sufficient, and simple, nonstatistical criteria could be used to classify recurrent infections as drug failures or new infections. These results suggest AmpSeq is strongly placed to become the new standard for molecular correction in regulatory trials, with potential extension into routine surveillance once the requisite technical support becomes established.
Keywords: P. falciparum; PCR correction; Plasmodium falciparum; TES; drug resistance; drug trials; malaria; molecular correction.