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Review 4: "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 4: "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 authors have hypothesized that the northern part of Uganda presents a hotspot for the emergence and spread of artemisinin resistant P. falciparum in Uganda. They have supported his hypothesis by recruiting 100 participants, collecting blood from them on day 0 before treatment and day 3 after the completion of treatment. They went further in determining the parasite clearance rate/fold change reduction using both microscopy and qPCR, and further sequenced 80 P. falciparum isolates from both days 0 and 3 P. falciparum isolates. The authors observed high prevalence of parasitaemia on day three both by microscopy (24%) and qPCR (63%), and showed associated with the reduced parasite clearance and mutations in the P. falciparum kelch13 propeller domain gene.

Introduction:

  • Line 2 -53 (L52-53): It would enrich the manuscript if the authors provide the last updated prevalence rate of malaria in the different regions of Uganda – norther, eastern. This will help the readers to appreciate the dynamics of malaria in Uganda and buttress the point the authors made in the next two sentences following this.

Materials and Methods – Study design:

  • L107: The study was a cross-sectional study and not a longitudinal one, the authors should please correct this.

  • Plasmodium falciparum qPCR

  • L145 to “respectively” in L147 should go to L140 just immediately after South Africa. This provides a well synchronised write up with that paragraph.

  • L147: The authors cannot commence a sentence with a figure (i.e 20 µl). It should be changed to word.

  • L153 and L160: These two sub-title should be merged to read “P. falciparum K13 allele and gene amplification”

Results:

  • Prevalence of Pfkelch13 Single Nucleotide polymorphism.

  • L218: “……but all microscopy negative (20/20)”. How many samples are microscopy negative? If 20, then adding it (20) to the three qPCR positive samples that were unsequenced would give 25. The authors should please check the figures very well.

Discussion:

  • Although the authors stated the study limitations, however, the none discrimination between the parasite present on day 0 from that on day 3 pose a huge caveat in the interpretation of the data viz-a-viz if the infections, (as well as the mutations) observed on day 3 is re-infection or recrudescence. Since, it seems likely that the laboratory where the authors are  are well placed to carry out this aspect of the study (distinguishing between new infect from recrudescence), I would suggest that the authors do carry this out.

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