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

RR:C19 Evidence Scale rating by reviewer:

• Potentially informative. The main claims made are not strongly justified by the methods and data, but may yield some insight. The results and conclusions of the study may resemble those from the hypothetical ideal study, but there is substantial room for doubt. Decision-makers should consider this evidence only with a thorough understanding of its weaknesses, alongside other evidence and theory. Decision-makers should not consider this actionable, unless the weaknesses are clearly understood and there is other theory and evidence to further support it.

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Review: This study assessed Day 3 parasite carriage in 100 malaria-symptomatic falciparum-positive individuals from Adjmani District, Uganda, who were treated with artemether-lumefantrine as well as the prevalence of mutations in the Plasmodium falciparum kelch13 gene. Microscopic examination of blood smears revealed that 24% of the patients were parasitaemic on Day 3, which increased to 64% when a highly sensitive PCR assay was used. Of the 100 parasite isolates collected before treatment was initiated, the kelch13 gene was successfully amplified and sequenced from 80 isolates.  While the majority of the sequences were wildtype, single nucleotide polymorphisms (SNPs) were detected in 15 isolates, with the previously reported C469Y mutation present in 10 of these 15 mutant isolates.  A novel F491S mutation was detected in a single isolate. Nine of the 64 patients with detectable parasites on Day 3 carried kelch13 SNPs, eight with the C469Y mutation, and one with the A578S mutation. Based on these findings the authors concluded that artemisinin-resistance parasites were most likely present in the Adjimani District, with the C469Y associated with delayed parasite clearance, and recommended regular therapeutic efficacy monitoring.  

Unfortunately, the data presented are only suggestive of these conclusions. As the researchers did not confirm if the infections detected on Day 0 were the same infections detected on Day 3, it is possible that the parasites detected on Day 3 were a consequence of either a new infection or the emergence of a low-frequency infection not initially detected on Day 0, both of which are possibilities given the high transmission intensity in the study area. The omission of this analysis weakens the strength of the conclusion of increased Day 3 parasite carriage due to artemisinin-resistance. 

In addition, and possibly more importantly, the researchers did not confirm whether the kelch13 mutations present on Day 0 were the same as the ones present on Day 3. If the parasites detected on Day 3 were a consequence of the emergence of a low-frequency infection that was not initially detected on Day 0, then it is possible that different kelch13 mutations were present on Day 3, again weakening the conclusion that the C496Y mutation is driving observed delayed parasite clearance. 

Very little detail is provided on how study participants were selected, why children under the age of 5 were excluded from the study, and how a malaria infection was confirmed. As the study was conducted in a high transmission area, it is likely that many children under the age of 5  as well as pregnant women were infected with malaria. Therefore, it would have been interesting to see if these high-risk population groups had higher rates of Day 3 parasite carriage and/or carried different kelch13 mutations.

Patient adherence and antimalarial drug quality were not assessed, therefore, they cannot be ruled out as contributing factors to the observed delayed parasite clearance.