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Review 1: "Day 3 Parasitemia and Plasmodium Falciparum Kelch 13 Mutations among Uncomplicated Malaria Patients Treated with Artemether-lumefantrine in Adjumani District, Uganda"

The reviewers overall state that the manuscript is potentially informative, suggesting a need to continuously evaluate the effectiveness of artemether in this context.

Published onMay 28, 2024
Review 1: "Day 3 Parasitemia and Plasmodium Falciparum Kelch 13 Mutations among Uncomplicated Malaria Patients Treated with Artemether-lumefantrine in Adjumani District, Uganda"
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Day 3 parasitemia and Plasmodium falciparum Kelch 13 mutations among uncomplicated malaria patients treated with artemether-lumefantrine in Adjumani district, Uganda
Day 3 parasitemia and Plasmodium falciparum Kelch 13 mutations among uncomplicated malaria patients treated with artemether-lumefantrine in Adjumani district, Uganda
Description

Abstract Artemisinin resistance threatens malaria control and elimination efforts globally. Recent studies have reported the emergence of Plasmodium falciparum parasites tolerant to artemisinin agents in sub-Saharan Africa, including Uganda. The current study assessed the day 3 parasite clearance and its correlation with P. falciparum K13 propeller gene (pfkelch13) mutations in P. falciparum parasites isolated from patients with uncomplicated malaria under artemether-lumefantrine (AL) treatment. This study enrolled 100 P. falciparum-positive patients to whom AL was prescribed between 09/September/2022 and 06/November/2022. Blood samples were collected in EDTA tubes before treatment initiation (day 0) and on day 3. Parasitemia was assessed by microscopy from blood smears and quantitative polymerase chain reaction (qPCR) from the DNA extracted. The day 0 parasite K13 gene was sequenced using Sanger sequencing. Sequence data were analysed using MEGA version 11 software. The data were analysed using STATA version 15, and the Mann‒Whitney U test was used to compare PCR parasite clearance on day 3 using the comparative CT value method and pfkelch13 mutations.The prevalence of day 3 parasitaemia was 24% (24/100) by microscopy and 63% (63/100) by qPCR from the AL-treated patients. P. falciparum K13-propeller gene polymorphism was detected in 18.8% (15/80) of the day 0 DNA samples. The K13 mutations found were C469Y, 12.5% (10/80); A675V, 2.5% (2/80); A569S, 1.25%, (1/80), A578S, 1.25%, (1/80) and; F491S, 1.25%, (1/80) a new allele not reported anywhere. The C469Y mutation, compared to the wild-type, was associated with delayed parasite clearance p=0.0278, Hodges-Lehmann estimation 3.2108 on the log scale, (95%CI 1.7076, 4.4730).There was a high prevalence of day 3 P. falciparum among malaria patients treated using artemether-lumefantrine. We conclude that the K13 mutation associated with artemisinin resistance by P. falciparum is present in Adjumani district, Uganda. This necessitates regular surveillance of the effectiveness and efficacy of artemether-lumefantrine in the country.

RR:C19 Evidence Scale rating by reviewer:

  • Strong. The main study claims are very well-justified by the data and analytic methods used. There is little room for doubt that the study produced has very similar results and conclusions as compared with the hypothetical ideal study. The study’s main claims should be considered conclusive and actionable without reservation.

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Review: The study by Angwe et al. examined the status of the kelch13 artemisinin-resistance gene in Plasmodium-infected patient samples collected over a two-month period in northwest Uganda. Patients were treated with the first-line treatment of artemether-lumefantrine, and samples collected on day 0 prior to treatment and day 3. Notably, the authors identified a relatively high proportion of day 3-positive samples, and further go on to document several kelch13 mutations in a subset of these sample, of which C469Y was the most prevalent. This latter observation is consistent with other reports of the relatively high prevalence of this SNP in the region. Collectively the study serves to further highlight the potential risk of reduced efficacy of ACTs in Africa, and the potential of known and novel mutations in kelch13 to mediate this.

The authors may want to consider clarifying their text to address these minor points below:

  • Clarify if the PCR for kelch13 sequencing was performed on day 0 or day 3 samples.

  • Add a definition for day 3 positivity. For microscopy, would this be a single observed parasite across 100 fields?

  • Line 284 – The authors note that the study by Stokes et al did not link the C469Y mutation to resistance. However, I believe their observation was more nuanced - introduction of the C469Y mutation into the Dd2 strain did cause a modest but significant increase in IC50, although this was not seen when the same mutation was edited into a Ugandan isolate. Nonetheless it provides further evidence of the relevance of this mutation to the resistance phenotype.

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