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Review 2: "Evidence of Artemisinin Partial Resistance in North-Western Tanzania: Clinical and Drug Resistance Markers Study"

Overall, reviewers highlight that this study has important findings and significantly contributes to molecular surveillance efforts in Africa. However, they expressed concern about the study’s analytical methodology and the overall conclusions drawn from the data.

Published onMar 08, 2024
Review 2: "Evidence of Artemisinin Partial Resistance in North-Western Tanzania: Clinical and Drug Resistance Markers Study"
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Evidence of artemisinin partial resistance in North-western Tanzania: clinical and drug resistance markers study
Evidence of artemisinin partial resistance in North-western Tanzania: clinical and drug resistance markers study

Abstract Background Artemisinin-based combination therapies (ACTs) are the recommended antimalarial drugs for the treatment of uncomplicated malaria. The recent emergence of artemisinin partial resistance (ART-R) in Rwanda, Uganda and Eritrea is of great concern. In Tanzania, a nationwide molecular malaria surveillance in 2021 showed a high prevalence of the Kelch13 (K13) 561H mutation in Plasmodium falciparum from the north-western region, close to the border with Rwanda and Uganda. This study was conducted in 2022 to evaluate the efficacy of artemether-lumefantrine (AL) and artesunate-amodiaquine (ASAQ) for the treatment of uncomplicated falciparum malaria and to confirm the presence of ART-R in Tanzania.Methods This single-arm study evaluated the efficacy of AL and ASAQ in eligible children aged six months to 10 years at Bukangara Dispensary in Karagwe District, Kagera Region. Clinical and parasitological responses were monitored for 28 days according to standard WHO protocol. Mutations in K13 gene and extended haplotypes with these mutations were analysed using Sanger and whole genome sequencing data, respectively.Findings 176 children (88 in each AL and ASAQ group) were enrolled and all achieved the defined outcomes. PCR-corrected adequate clinical and parasitological response (ACPR) was 98.3% (95% CI: 90.8-100) and 100.0% (95% CI: 95.8-100) for AL and ASAQ, respectively. Parasitaemia on day 3 was observed in 11/88 (12.5%) and 17/88 (19.3%) in the AL and ASAQ groups, respectively. The half-life of parasitaemia was significantly higher (>6.5 hrs) in patients with parasitaemia on day 3 and/or mutations in K13 gene at enrolment. Most patients with parasitaemia on day 3 (8/11 = 72.7% in the AL group and 10/17 = 58.8% in the ASAQ group) had 561H mutation at enrolment. The parasites with K13 mutations were not similar to those from south-east Asia and Rwanda, but had the same core haplotype of a new 561H haplotype reported in Kagera in 2021.Interpretation These findings confirm the presence of ART-R in Tanzania. A context-specific strategy to respond to artemisinin partial resistance is urgently needed. Although both AL and ASAQ showed high efficacy, increased vigilance for reduced efficacy of these ACTs and detection of ART-R in other parts of the country is critical.Funding Bill and Melinda Gates Foundation to the World Health Organization (WHO, OPP 1209843) and the National Institute for Medical Research (NIMR, Inv. No. 002202), and US National Institute for Health (R01AI156267 to JAB, DSI and JJJ, and K24AI134990 to JJJ).Research in context Evidence before this study Artemisinin partial resistance (ART-R) is defined as delayed clearance after treatment with an artemisinin combination therapy (ACT) or artesunate monotherapy of a parasite strain carrying a validated marker of ART-R. At present, 13 different Kelch13 (K13) mutations have been validated as markers of ART-R. ART-R is confirmed in an area if a quality-controlled study using an ACT or artesunate monotherapy, finds more than 5% of patients have parasites with validated K13 mutations and delayed clearance as evidenced by either persistent parasitemia detected by microscopy on day 3 or a parasite clearance half-life of ≥5 hours. ART-R was first reported from Cambodia in 2008 and later from several countries in Southeast Asia. Published articles up to December 2023 were searched in PubMed with the terms; “artemisini n”, “artemisinin partial resistance”, “artemisinin-based combination therapies”, “Kelch 13” in combination with “Africa” or “Tanzania”. The publications confirmed the emergence of ART-R associated with mutations in K13: 561H in Rwanda, A675V and C469Y in Uganda and R622I in Eritrea. All these studies showed a high cure rate of the tested ACTs. The R622I mutant was not reported from Southeast Asia but is circulating in the Horn of Africa (Eritrea, Ethiopia, Sudan and Somalia). In Tanzania, a nationwide malaria molecular surveilla nce launched in January 2021 showed a high prevalence of 561H mutation in the north-western region of Kagera, close to the border with Rwanda and Uganda.Added value of this study The study documented delayed parasite clearance associated with pre-treatment validated K13 561H mutation. It confirms and provides evidence for the first-time of ART-R in Kagera region, north-western Tanzania, an area close to the border with Rwanda and Uganda. This makes Tanzania the fourth country in Africa with confirmed ART-R. The study documents presence of K13 mutation associated with ART-R suggesting that partial resistance to artemisinins is rapidly evolving and can still be found in more areas of Africa. Parasites with K13 mutations were not similar to those from south-east Asia and Rwanda, but had the same core haplotype of a new 561H haplotype reported in Kagera in 2021.The findings of this study furthermore show that both AL and ASAQ are highly effective.Implications of all the available evidence The emergence of confirmed ART-R in Africa, so far in four countries (Rwanda, Uganda, Eritrea and Tanzania), poses a serious threat to malaria control in Africa, which accounts for more than 95% of the global malaria burden. The current evidence of ART-R in Kagera region calls for an urgent response, including the development of a context-specific strategy based on the recently launched WHO strategy to respond to antimalarial drug resistance in Africa. The fact that ART-R has been confirmed in Kagera region, an area bordering Rwanda and Uganda, where resistance also has been reported, also calls for cross-border collaboration to harmonize strategies to combat this threat in the Great Lakes region of Africa. Nationwide studies on molecular markers in Tanzania, which revealed a high prevalence of K13 validated mutatio ns in the Kagera region, guided where to conduct the current study. This suggests that molecular marker surveillance could play an important role in conducting targeted antimalarial drug efficacy studies and confirming ART-R in other parts of Tanzania and beyond.

RR:C19 Evidence Scale rating by reviewer:

  • Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.


Review: Ishengoma et al. report on a therapeutic efficacy study of artemether-lumefantrine (AL) and artesunate-amodiaquine (ASAQ) conducted in 2022 in Kagera Region, Tanzania. They document Day 3 positivity rates above 10% in both study arms, and the presence of the R561H mutation in pfk13, associated with artemisinin resistance. Taken together, these data are compelling evidence of artemisinin partial resistance in this part of Tanzania. Moreover, the fact that molecular surveillance guided the choice of the site selection for this study is a laudable example of the potential impact of surveillance strategies that incorporate use of molecular markers of resistance.

While the strength of evidence behind the finding of artemisinin partial resistance is substantial, the study conclusions related to the overall drug efficacy are less supported by the data. The study has important deviations from the standard World Health Organization in vivo protocol, including inclusion of older children, an unusually low lower bound on parasite density (500 parasites/µL versus the typical 2000 parasites/µL), and administration of AL without food. The consequent biases introduced by these study procedures act in opposing directions, complicating interpretation of the final efficacy results.

Notably, the study used a deprecated methodology for molecular correction to distinguish cases of recrudescence versus reinfection. Current WHO best practices call for genotyping samples using msp1, msp2and a microsatellite, with fragment length measurement performed with capillary electrophoresis. Instead, the study investigators analyzed msp1, msp2 and glurp markers using gel electrophoresis, an outdated method known to have low sensitivity in detecting recrudescent infections in high-transmission settings. The choice of this older genotyping methodology is particularly notable when contrasted with the sophisticated methods used for analysis of molecular markers of resistance, including whole genome sequencing.

One way to sidestep biases introduced during molecular correction and complement complicated interpretation of corrected efficacy data is to analyze the uncorrected efficacies. By this metric, ASAQ is observed to have terrific performance, with a 97.7% efficacy rate. In contrast, AL has an efficacy of 64.8%. In practice, this means that a substantial proportion of patients will be retreated with AL very soon after their initial infection (nearly 2/3 of AL failures occurred on or before day 21), accelerating the selection of parasites with artemisinin partial resistance. While the conclusions acknowledge the high rate of reinfection in patients treated with AL, the authors' assertion that this can be attributed to high transmission and addressed through intensified malaria control measures disregards the risk of continued AL use in driving resistance. A stronger recommendation for a switch to ASAQ in this region is warranted and would be consistent with the WHO-supported strategy for mitigating artemisinin resistance.1

  1. WHO. Strategy to respond to antimalarial drug resistance in Africa, 18 Nov 2022.

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