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

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.

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). 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. Competing Interest Statement The authors have declared no competing interest. Clinical Trial ACTRN12622001243707 Funding Statement 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). Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethical clearance was obtained from the Medical Research Coordinating Committee of the National Institute for Medical Research and WHO ethics review committee. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Footnotes ↵ǂ co-senior author Data Availability Once the publication process is completed, the clinical, laboratory and parasite sequencing data which have been shared with WHO will be available upon request to the corresponding author. The data include de-identified patient clinical data, a data dictionary and sWGS data. Requests to conduct analyses that are outside the scope of this publication will be reviewed by the core study team (DSI, CIM, MW, CR, JB and JJJ) to determine if the proposed use of the data is scientifically and ethically appropriate and falls within the permits of the institutions that provided ethical approval for the study. Any requests to reanalyse the data presented in this paper will not need to be reviewed by the MRCC of NIMR. The data have been shared with WHO and will be uploaded on its database. Abbreviations - ACPR - Adequate clinical and parasitological response - ACT - Artemisinin based combination therapy - AE - Adverse event - AL - Artemether- lumefantrine - ASAQ - Artesunate-amodiaquine - DBS - Dried blood spots on filter papers - ETF - Early treatment failure - glurp - Glutamate rich protein gene - K13 - Kelch propeller domain located on Chromosome 13 of P. falciparum - LCF - Late clinical failure - LPF - Late parasitological failure - MRCC - Medical Research Coordinating Committee - msp1 - Merozoite surface protein 1 gene - msp2 - Merozoite surface protein 2 gene - NIMR - National Institute for Medical Research - NMCP - National Malaria Control Programme - PCR - Polymerase chain reaction - Pfmdr1 - Plasmodium falciparum multidrug resistance 1 gene - SAE - Serious adverse event - WHO - World Health Organization