Review 1: "Evidence of Artemisinin Partial Resistance in North-Western Tanzania: Clinical and Drug Resistance Markers Study"

Frances Rocamora

doi:doi:10.1162/2e3983f5.65e494e8

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.

by Frances Rocamora

Published onMar 08, 2024

Evidence of artemisinin partial resistance in North-western Tanzania: clinical and drug resistance markers study

by Deus S. Ishengoma, Celine I. Mandara, Catherine Bakari, Abebe A. Fola, Rashid A. Madebe, Misago D. Seth, Filbert Francis, Creyton Buguzi, Ramadhan Moshi, Issa Garimo, Samwel Lazaro, Abdallah Lusasi, Sijenunu Aaron, Frank Chacky, Ally Mohamed, Ritha J. A. Njau, Jovin Kitau, Charlotte Rasmussen, Jeffrey A. Bailey, Jonathan J. Juliano, and Marian Warsame

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Published on Feb 01, 2024
www.medrxiv.org

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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.

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Review: The results in the manuscript are highly valuable and contribute significantly to molecular surveillance efforts in Africa, especially being the first to document the presence of ART-R relevant haplotypes in Tanzania. I appreciate the authors’ detailed summary of clinical variables and outcomes of all subjects enrolled in the study. It is also well-noted that the authors discuss the distinction between SEA haplotypes compared to K13 markers that have been detected in Africa, which are consistent with what they observe in their study.

On the other hand, I also found some numerical descriptions confusing and didn’t seem to match what was shown in the data tables. I would like to see statistical testing when comparing between populations (i.e. half-life, prevalence of K13 mutation among patients with Day 3 parasitemia, etc). I also encourage the authors to utilize visual/graphical presentations of data when possible—rather than just relying on tables—especially if it will help demonstrate the study’s conclusions more clearly. While the manuscript is extremely informative, I find it a little too vaguely descriptive; I believe that with more specific statistical analyses, the authors can glean more insight and stronger conclusions from this clinically relevant dataset.

Specific Comments:

The authors noted that 49 (mutant) and 34 (wildtype) samples were subjected sWGA and high-throughput sequencing. I assume the haplotypes shown in Figure 2 were constructed from a few of these samples. Is there any other type of genomic analysis performed to compare these two populations?
Were there any other novel mutations found in the study that are associated with delayed clearance? Or any other mutation in K13 (not 561H?)
Figure 2 shows selected samples with the TZ2 haplotype, is this representative of all samples sequenced? Further, is this haplotype observed to be significantly overrepresented in infections with longer half-life or day 3 parasitemia?
The text states “delayed parasite clearance at day 3 was observed in 12.5% and 19.3% of the AL- and ASAQ-treated groups, respectively”, but I don’t think this is noted in Table 2 (outcomes). It’s only described in the text.
The text also states that “delayed parasite clearance was significantly associated with the K13 561H mutation (≥58.8%)”; is the enrichment of K13 561H mutants in the delayed parasite clearance group statistically significant? The number of subjects in this category is not clear based on Table 5.
Some questions for Table 3:
- The header says: “Patients with day 3 parasitaemia and k-13 mutations”, does this only include patients with BOTH day 3 parasitemia and K13 mutations?
- Perhaps the authors can include a graphical representation of the spread of half-life with respect to K13 mutation status?
- Also, is the overlap between parasites with day 3 parasitemia and K13 mutations statistically significant?
I found Table 5 a little confusing as well:
- When it says “wildtype” does that mean no mutations in the K13 gene or just no 561H mutation?
- DF = Day of Failure. The AL treatment group says n=31 in the DF group, does this mean 31/88 subjects in the AL group had Day 3 parasitemia? If this is the case, the table also shows that more or about an equal number of DF subjects were wild-type vs. K13 561H mutant. This does not agree with the statement that the mutation is associated with ART-R (at least based on this table).
- How many of the DF samples are the same subjects in the Day 0 subjects that were counted? Were there subjects who changed K13 mutation status between Day 0 and Day of failure?
- Perhaps this table should be restructured to Failed Treatment (i.e Day 3-positive?) vs. Successful Treatment (i.e Day3-negative?) and which of each population was mutant vs. wild-type (like an expanded 2x2 table)
- Is the prevalence of any K13 mutation (including 561H) significantly associated with longer parasite half-life and/or treatment failure

Review 1: "Evidence of Artemisinin Partial Resistance in North-Western Tanzania: Clinical and Drug Resistance Markers Study"

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