Should Deep-Sequenced Amplicons Become the New Gold Standard for Analyzing Malaria Drug Clinical Trials?

Abstract

Background. 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-recommend 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. Methods. 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 analysed, 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. Results. AmpSeq greatly improved molecular correction and provided accurate drug failure rate estimates. The use of 3 to 5 amplicons was sufficient, and simple, non-statistical, criteria could be used to classify recurrent infections as drug failures or new infections. Conclusions. These results suggest AmpSeq is strongly placed to become the new standard for molecular correction in regulatory trials, with its potential extension into routine surveillance once the requisite technical support becomes established.

Persistent and multiclonal malaria parasite dynamics despite extended artemether-lumefantrine treatment in children

“…Notably, most persistent clones were not detected beyond 14 days, potentially reflecting the sustained impact of lumefantrine given its half-life of 3–4 days. A notable limitation to amplicon sequencing is the limit of detection, which is comparable to other nested PCR methods (~ 1,000 parasites/mL), and the practical problem of distinguishing extremely low density clones from sequencing artifacts 14 , 19 . As a consequence, sequencing is unable to reliably describe the clonal dynamics of very low density infections, such as may be present in the early post-treatment period, or immediately following parasite emergence from the liver.…”

Bridging the Gap from Molecular Surveillance to Programmatic Decisions for Malaria Control and Elimination

“…22,23 The recommended WHO protocol for molecular monitoring during clinical trials was recently amended to 21 Nevertheless, sampling at a single time point can also result in missed clones because of parasite sequestration and fluctuation in parasite densities. 24,25 Furthermore, different data analysis algorithms have been proposed, [26][27][28][29] and a consensus still needs to emerge about how to validate them.…”

Amplicon deep sequencing of five highly polymorphic markers of Plasmodium falciparum reveals high parasite genetic diversity and moderate population structure in Ethiopia

“…Genetic diversity of Plasmodium falciparum populations and multiplicity of infection (MOI) in humans might vary according to transmission intensity in different geographical regions [4][5][6][7][8][9]. The study of genetic diversity helps researchers to understand the distribution, dynamics, and genetic structure of the parasite population [10][11][12][13][14].…”