Recurrent parasitemias with artemisinin partial resistance mutations during the 2024 Ethiopia malaria resurgence: a case series

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Brhane, Adugna Abera, Mahlet Belachew, and 18 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9094035/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background Ethiopia experienced a marked resurgence of malaria in 2024. Artemisinin-based combination therapies (ACTs) are first-line treatment for uncomplicated Plasmodium falciparum malaria and threatened by the emergence of artemisinin partial resistance (ART-R), associated with mutations in the P. falciparum kelch13 ( k13 ) gene, that could undermine treatment efficacy and accelerate transmission. Methods and material: We used the national Public Health Emergency Management (PHEM) surveillance system to characterize malaria resurgence and further investigate antimalarial drug resistance markers among cases of recurrent clinical malaria in three selected resurgence sites in central Ethiopia. Results Parasite isolates from 15 patients with confirmed clinical recurrent P. falciparum malaria were genotyped for molecular markers associated with drug resistance, including mutations in k13, pfcrt , pfmdr1 , pfdhfr , and pfdhps using PfSMARRTer multiplex amplicon sequencing in Addis Ababa. Clinical presentation and treatment history were reviewed alongside genotyping results. Three patients (3/15, 20%) with confirmed recurrence were infected by parasites carrying the WHO-candidate ART-R molecular marker K13 P441L. Markers of resistance to other antimalarial drugs were largely fixed in the population. These cases occurred in the context of increasing malaria incidence, with evidence of clonal expansion or dominance of a related lineage. The findings indicate the presence of ACT resistance-associated markers within genetically heterogeneous parasite populations. Conclusion The current study documents cases of recurrent parasitemia caused by P. falciparum with K13 P441L during the malaria resurgence in the Oromia region. The detection of multiple independent resistance markers suggests ongoing drug pressure on first-line treatments. These findings underscore the need for strengthened molecular surveillance integrated with routine case monitoring to inform treatment policy and support malaria control and elimination efforts in Ethiopia. Ethiopia Malaria ACT-resistance ART-R PfSMARRTer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Historically, Ethiopia has faced past malaria resurgence, including a major surge in cases in 2023-24, when over 10 million cases were reported compared to around 1 million cases in 2019 [1, 2]. The resurgence has led to an uptick in malaria cases and irregular outbreaks in areas once considered malaria-free, including highland regions, as well as an increase in asymptomatic cases [3, 4]. This poses a significant challenge to established malaria control measures and has set back progress made over the past decades [4–6]. Since 2004, artemisinin-based combination therapy (ACT), primarily artemether–lumefantrine (AL), has been the first-line treatment for uncomplicated P.falciparum malaria in Ethiopia. Although most therapeutic efficacy studies reported high cure rates [7–9], artemisinin partial resistance (ART-R) has become a major concern [6, 10–14], suggesting the need for further molecular surveillance of antimalarial drug resistance markers [11]. Artemisinin based combination therapy (ACT) combines a fast-acting artemisinin derivative with a longer acting partner drug to ensure rapid parasite clearance and sustained post-treatment suppression. The artemisinin component is rapidly converted to its active metabolite dihydroartemisinin (DHA) and has a short half-life (approximately 1–2 hours), leading to a marked reduction in parasite biomass within the first 48–72 hours. In contrast, the partner drug has a longer elimination half-life, often persisting for days to weeks after treatment completion. During this elimination phase, parasites may be exposed to declining, subtherapeutic drug concentrations, creating a selective window that favors parasites with reduced susceptibility to the partner drug. Consequently, recurrent parasitemia occurring within the first few weeks after ACT is more consistent with recrudescence or reinfection involving parasites with decreased partner-drug sensitivity, particularly in moderate- to high-transmission settings[15]. In response to the malaria resurgence, this study investigated patients who were returning after previous treatment for malaria to the Gelana Primary Hospital and Tore Health Center between August 10 and September 17, 2024, in West Guji, Gelana Woreda, Oromia regional state, Ethiopia. We enrolled Patients who were seeking treatment for malaria-like illness within 6 weeks of treatment [16]. First, we confirmed the resurgence of malaria in this region using national surveillance data from 2019 to 2024. Samples from enrolled participants were then screened by real-time PCR and samples positive for Plasmodium falciparum underwent amplicon deep sequencing using PfSMARRTer to characterize antimalarial resistance polymorphisms and parasite diversity. The resulting molecular resistance profiles were linked with patient clinical and treatment data to explore the relationship between molecular resistance markers and clinical outcomes. Methods Malaria case data 2019–2024 The Ethiopian Public Health Institute (EPHI) has surveillance infrastructure, which allows it to obtain epidemiologic data from sources using both an active surveillance and a passive surveillance (weekly report) system. The surveillance unit of the Public Health Emergency Management (PHEM) department at EPHI collects data on epidemic-prone diseases from the health facility level, which is aggregated by catchment, district, zone, and region. Weekly clinical malaria surveillance data used in this study were obtained from the PHEM surveillance unit spanning January 2019 to December 2024. Study population, data collection and analysis Patients who re-visited the selected health facilities after prior treatment with suspected malaria and who had a positive blood smear within 42 days after their initial treatment during the study period were enrolled. Demographic, clinical, and laboratory test data were collected from all consented participants by trained health workers at the Outpatient Department (OPD) and/or laboratory in each study health facility. Eligible patients' medical record numbers (MRN) were identified. After routine malaria diagnosis by blood film an additional 2–3 ml of venous blood, stored at 2–8 0 C and transported to the EPHI laboratory for further investigation. A structured questionnaire was used for data collection including demographic characteristics, malaria diagnosis and treatment history, additional data from the registries were collected using MRN. Data collectors were trained using a pre-tested questionnaire to ensure data quality. Real-time PCR [17]was used to determine species of infection. In brief, pan- Plasmodium specific primers and probe sets targeting the small subunit ribosomal RNA (18S rRNA) along with primers and probes specific for the var gene acidic terminal sequence (ATS) of P. falciparum . The samples were further investigated with multiplexed primers for P. falciparum (varATS) and Plasmodium vivax 's small subunit ribosomal RNA (P.v18S rRNA) (Belachew et al, 2021). DNA samples confirmed with P. falciparum mono-infection and with a cycle threshold (Ct) value below 30 were further sequenced using PFSMARRTer V13 [18] on the NextSeq 550 Illumina platform at EPHI. Data analysis Data were analyzed using the R software (v4.5.1). Descriptive analysis was done to describe the study variables and outcomes. Trend analysis specific to the Oromia Region was conducted from the weekly epidemiological malaria data from 2019 to 2024. The mean plus two standard deviations of the baseline data was analyzed to calculate the statistical threshold for each epidemiological week. The diagnostic performance of the blood film was determined for identification of P. falciparum and P. vivax by considering real time PCR as the reference method. The Cohen’s Kappa coefficient was computed to measure agreement between microscopy and real time PCR classifications [19]. Sequencing data was processed by SeekDeep v3.0.1 ( https://github.com/bailey-lab/SeekDeep ). The prevalence of molecular markers associated with antimalarial drug resistance was calculated as the proportion of successfully sequenced infections harboring the mutant allele among the total number of genotyped samples. The R v4.5.2, tidyverse, ggplot2 package was used for visualization. Complexity of infection (COI) was estimated using sample allele frequency data across polymorphic loci included in the sequencing panel [20]. Genetic relatedness among parasite isolates was estimated using the hmmIBD method[21]. Parasite relatedness networks were constructed and visualized using the igraph package in R. Ethical clearance This study obtained approval from the scientific and review committee of the EPHI. Permission was obtained from EPHI to use the national EPI-week-based malaria data. Data were collected from consented patients as per the Helsinki principles. Results Malaria epidemiology in Oromia Region Analysis of PHEM weekly surveillance data from 2019–2024 demonstrates a steadily increasing malaria burden, with reported cases rising from 75,854 in 2019 to 4,489,228 in 2024 (Fig. 1 ). Analysis of trends in 2024 highlight a sustained resurgence throughout 2024, during which malaria incidence exceeded the outbreak threshold in every epidemiologic week (Fig. 1 ). The epidemic curve showed two distinct transmission peaks: an initial rise between epidemiologic weeks 20–30, followed by a larger and more prolonged peak between weeks 35–43. Socio-demography and clinical characteristics of the study participants Samples were collected from 34 study participants seeking repeated treatment. Complete clinical and socio-demographic data were available for 31 (92%) (Table 1 ). A microscopic parasite species identification at health facility showed that a total of 27 patients were diagnosed with p. falciparum during the second visit compared with 23 patients during the first visit, of whom 20 were consistently diagnosed with p. falciparum at both visits (Table 1 ). Among the 31 participants, 18 (58.1%) were male (Table 2 ). The median age was 25 years (IQR = 20) (Table 2 ). The mean number of days between the initial treatment and second visit to the health facility was 15.65 (SD = 7.45; range, 6–26) for P. falciparum and 15.75 (SD = 7.48; range, 6–27) for P. vivax cases. Almost half 17 (54.84%) of the patients visited the health facilities for the second time within the first two weeks after completion of the initial treatment. The majority of the patients 29(93.55%) were treated with artemether-lumefantrine (AL) and single-dose primaquine 27(87.1%) during the second course of the treatment (Table 2 ). 11(91.67%) patients with P. Vivax infection were treated with AL due to misdiagnosis by microscopy. Table 1 Results of microscopic identification of parasite species during the subsequent visit at two health facilities in Gelana Woreda, Oromia Region, Ethiopia (n = 31). Initial Diagnosis Second visit Diagnosis Mixed Pf. Pv. Total Pf. 2 (8.7%) 20 (87.0%) 1 (4.3%) 23 Pv. 0 6 (85.71%) 1 (14.29%) 7 Mixed 0 1 (100%) 0 1 Total 2 27 2 31 Table 2 Socio-demographic and clinical profile of malaria patients confirmed by PCR from Gelana Woreda, Oromia Region, Ethiopia (n = 31). Variables Category Pf. (n = 14) Pv. (n = 12) Neg (n = 3) Mix (n = 2) Total Sex Male 7 (50%) 8(66.67%) 2(66.67%) 1(50%) 18(58.1%) Female 7 (50%) 4(33.3%) 1(33.33%) 1(50%) 13(41.94%) Total 14 12 3 2 31 Duration between initial and second visit < 14 8(57.14%) 7(58.33%) 2(66.67%) 0 17(54.84%) 14–21 1(7.14%) 0 (0%) 1(33.33%) 1(50%) 3(9.68%) 21 and above 5(35.71%) 5(41.67%) 0 1(50%) 11(35.48%) Total 14 12 3 2 31 Treatment given on second visit AL 14(100%) 11(91.67%) 3(100%) 1(50%) 29(93.55%) No AL 0 1(8.33%) 0 1(50%) 2(6.45%) Total 14 12 3 2 31 Full dose Primaquine 1(7.14%) 2(16.67%) 0 1(50%) 4(12.9%) Single dose Primaquine 13(92.86%) 10(83.33%) 3(100%) 1(50%) 27(87.1%) Total 14 12 3 2 31 First symptom Back pain 0 0 1(33.33%) 0 1(3.23%) Chills and rigor 2(14.29%) 3(25%) 0 0 5(16.13%) Fever 8(57.14%) 5(41.67%) 0 0 13(41.94%) Headache 4(28.57%) 3(25%) 2(66.67%) 1(50%) 10(32.26%) Shivering 0 1(8.33%) 0 1(50%) 2(6.45%) Total 14 12 3 2 31* Pf.= P. falciparum , Pv. = P. vivax , AL = artemether-lumefantrine *Two cases, one P. falciparum and one P. vivax , were excluded from further analysis due to incomplete clinical and demographic profile. Microscopy and real-time PCR result agreement Microscopy results from the health facility reported 30 (88.2%), 2 (5.9%) and 2 (5.9%) of P. falciparum , P. vivax , and mixed species infections, respectively. Out of 34 samples, real-time PCR identified P. falciparum in 15 (44.1%), P. vivax in 13 (38.2%), mixed in 2 (5.9%), and no malaria parasite in 4 (11.8%) samples. Table 3 Microscopy and real-time PCR test results of the microscopy-confirmed P. falciparum samples collected from Gelana Woreda, Oromia Region, Ethiopia (n = 26) Result real time PCR Total Pf. Pv. Microscopy Pf. 14 11 25 Pv. 0 1 1 Total 14 12 26 * Pf.= P. falciparum , Pv. = P. Vivax *Two real-time positive cases, one P. falciparum and one P. vivax , were excluded from further analysis due to incomplete clinical and demographic profile. From the 26 samples classified as P. falciparum by microscopy, 14 (53.8%) were confirmed as P. falciparum by PCR, while the remaining 11 (42.3%) were identified as P. vivax . The result from microscopy performance showed 100% (14/14) sensitivity for detecting P. falciparum , but much lower specificity at 8.3% (1/2) for P. falciparum with PCR as the gold standard. Sensitivity and specificity for P. vivax was 8.3% (1/12) and 100% (14/14), respectively. The overall species-level agreement between microscopy and PCR was low, with a Cohen’s Kappa of 0.09 (95% CI: − 0.08 to 0.26). Quality of amplicon sequencing of P. falciparum infections Amplicon sequencing of 24 P. falciparum loci across 15 clinical isolates generated uniformly high coverage. Median read depth per amplicon was 8,124× (interquartile range: 4,210 15,630×), with more than 92% of locus-sample combinations exceeding 1,000× coverage. This depth enabled reliable detection of minor haplotypes at frequencies as low as 1%. Seventeen low coverage outliers, marked in red (Fig. 2 ), corresponded to samples with low parasitemia, as confirmed by quantitative PCR. Cases of participants returning with symptomatic malaria and genetic markers of ART-R Analysis of samples from 15 participants revealed the presence of the WHO-candidate ART-R marker k13 P441L mutation in three cases, detailed below. Case 1 A 10-year-old female patient presented to the outpatient department (OPD) with a chief complaint of severe headache and high-grade fever on 25 August 2024. The patient was diagnosed positive for mixed malaria parasite infection ( P. falciparum and P. vivax ) by microscopic investigation of peripheral blood film. The patient was critically ill and subsequently admitted and given initial treatment of intravenous artesunate followed by Artemether/Lumefantrine and a full dose of primaquine for mixed infection in accordance with the national first-line antimalarial treatment guideline. The patient returned to the health facility with recurrent symptoms fourteen days (on 8 September 2024) after the completion of the initial P. falciparum treatment course. Repeated blood film microscopy test confirmed P. falciparum . A blood sample was collected and sent to the National Reference Laboratory for further molecular investigation. Case 2 A 43-year-old male patient who presented with severe back pain confirmed for P. falciparum malaria parasite on 10 August 2024. Following the national malaria treatment protocol, the patient was treated with a full course of artemether-lumefantrine followed by single dose primaquine. Eight days after completion of the initial treatment course, the patient returned to the same health facility on 22 August 2024 with recurrent symptoms, and the microscopic investigation revealed the presence of P. falciparum . Case 3 A two-year-old female patient presented with high-grade fever and chills was confirmed for P. falciparum after blood film microscopic examination on 29 July 2024. The patient was treated with a complete course of artemether-lumefantrine followed by single dose primaquine. The patient returned to the same health facility with recurrent symptoms on August 16, 2024, fifteen days after the completion of the initial treatment course. The repeated microscopy investigation detected P. falciparum . Prevalence of Non-ACT and Partner Drug Resistance Mutations Amplicon sequencing showed that molecular markers for many antimalarials were common in the sequenced isolates including pfcrt (PfcRT_76T) , pfmdr1 (N86, 184F, D1246) , pfdhfr (51I, 59R,108N) , and pfdhps (540E). The pfdhps 581G and k13 441L were detected in three samples each 3/15 (20%). Within-sample allele frequencies of each mutation are shown in Fig. 3 , showing that in most cases, samples contained a single allele at these loci. A sextuple mutant haplotype combining pfcrt 76T, the pfmdr1 NFD triple, the pfdhfr IRN triple, and pfdhps 437G/540E occurred in almost all the samples, except three missing data for pfcrt 76T Fig. 3 . Complexity of Infection (COI) The majority of P. falciparum isolates were monoclonal 10/15(66%). Ten isolates exhibited a COI of 1, while a smaller proportion showed higher COI values, including COI = 2 (n = 2), COI = 3 (n = 1), and COI = 4 (n = 2) (Fig. 4 ) . Genetic diversity and relatedness Haplotype clustering at 99% nucleotide identity revealed pronounced variation in polyclonality (Fig. 4 ). The ama1 locus displayed the highest diversity, with up to four haplotypes detected in sample PHEM-6 and a mean complexity of infection (COI) of 1.9 ± 0.8 across all samples (Fig. 4 ). The heome loci cluster exhibited intermediate diversity, with mean COI values ranging from 1.6 to 1.8. In contrast, drug-resistance loci ( pfcrt, pfmdr1, pfdhfr, pfdhps, pfk13 ) were monoclonal (COI = 1) in nearly all instances, and 66% of isolates (10 of 15) showed COI = 1 across all resistance markers (Fig. 4 ). Mean COI was significantly higher in neutral and antigenic loci than in mutated genes (Wilcoxon rank-sum test, P < 0.001). Although the sample size is limited, Fig. 5 highlights notable genetic clustering among parasites carrying the 441L mutation. At an IBD threshold of 0.50 (indicative of sibling-level relatedness), 441L mutant parasites form distinct high-relatedness clusters, consistent with recent clonal expansion and focal transmission amplification. At a lower threshold (IBD = 0.25), these mutants remain genetically connected to a broader parasite population, suggesting ongoing gene flow within the study area. Together, these patterns support the interpretation that the increase in 441L prevalence is likely driven by local expansion of established parasite lineages rather than repeated introductions of genetically divergent strains. Discussion Ethiopia experienced a marked resurgence of malaria during 2024, with Oromia Region contributing substantially to the national increase in cases. In this investigation of recurrent clinical malaria cases from Gelana Woreda, we combined surveillance data, clinical characterization, and targeted amplicon sequencing to examine whether antimalarial drug resistance may be occurring during early recurrence. Our findings demonstrate four key observations: (a) sustained resurgence throughout 2024 in the study region; (b) low agreement between routine microscopy and molecular species identification; (c) the presence of WHO-validated pfk13 P441L mutations among recurrent Plasmodium falciparum infections and (d) there are high levels of molecular markers of antimalarial resistance in these recurrent parasitemias. The discordance between microscopy and qPCR species identification was substantial, with very low agreement (κ = 0.09). Although microscopy remains the cornerstone of malaria diagnosis in Ethiopia, this level of misclassification—particularly between P. falciparum and P. vivax —has important implications in a resurgence setting. Species misidentification may lead to suboptimal treatment selection, inappropriate use of primaquine, and inaccurate surveillance data [22, 23]. These findings reinforce the need to strengthen diagnostic quality assurance systems and consider strategic incorporation of molecular confirmation in sentinel or outbreak settings. Among the 15 qPCR-confirmed P. falciparum infections that underwent deep amplicon sequencing, three harbored the WHO-validated pfk13 P441L mutation [24]. These three cases returned with recurrent parasitemia 8–15 days after completion of artemether–lumefantrine (AL) treatment. Although we cannot definitively distinguish recrudescence from reinfection in this small cohort, the temporal proximity of recurrence and the presence of validated artemisinin partial resistance (ART-R) markers are notable. These findings align with recent reports documenting geographically heterogeneous emergence of pfk13 mutations in Ethiopia, including R622I, P574L, A675V, and P441L [9, 11, 12]. The P441L mutation has been reported from geographical proximate areas in low prevalence 1–2% in Oromia and Central Ethiopia regions [9, 11]. A recent work on malaria resurgence in Ethiopia reported 441L mutations in 9 of 22 samples in Dila, Central Ethiopia (In press). Given the limited number of samples, IBD sharing supports the previous reports that malaria resurgence in the area may not be independent, implicating a sustained transmission of similar lineage may be due expansion of pre-existing parasite lineages[9]. In addition to artemisinin resistance markers, we observed near-fixation of mutations in pfcrt , pfmdr1 , pfdhfr , and pfdhps . The dominance of the sextuple mutant haplotype (including pfcrt 76T, pfmdr1 NFD, pfdhfr IRN, and pfdhps 437G/540E) reflects longstanding antifolate and chloroquine selection pressure in Ethiopia [11, 12]. Of particular relevance is the high prevalence of the pfmdr1 N86 allele, previously associated with altered lumefantrine tolerance, which was present at 100% frequency within all parasitemias genotyped. Fortunately, ACT therapeutic efficacy studies in Ethiopia have continued to report PCR-corrected cure rates above 95%. However, the molecular landscape suggests important changes that need continued monitoring [12]. Continued reliance on AL as first-line therapy for P. falciparum , particularly during periods of intense transmission, may further select for parasites with reduced partner-drug susceptibility, potentially accelerating the clinical impact of emerging artemisinin resistance, as has been seen with the emergence of the PX1 PIN haplotype in Uganda [25]. This study provides, to our knowledge, the first molecular documentation of artemisinin partial resistance markers among clinically recurrent malaria cases in Ethiopia. Malaria incidence reportedly declined in 2025, suggesting that resurgence dynamics are likely multifactorial and not solely attributable to drug resistance. However, the detection of multiple independent ART-R markers, both here and in other studies [9, 11, 12], in a resurgence setting signals ongoing evolutionary pressure and underscores the need for strengthened surveillance. Several limitations should be considered. The sample size was small, and sites were purposefully selected based on recurrent presentations, limiting generalizability. We did not have initial parasitemia to genotype. We did not perform whole genome sequencing or pharmacokinetic analyses to definitively distinguish recrudescence from reinfection or to assess drug exposure. In addition, diagnostic discrepancies between microscopy and qPCR may have influenced case classification. Nonetheless, the integration of epidemiologic, clinical, and high-depth amplicon sequencing data provides a coherent signal that resistance-associated polymorphisms are present among recurrent cases in this resurgence context. In this case series from a district experiencing malaria resurgence, we identified circulating ART-R–associated mutations within genetically related Plasmodium falciparum parasite populations, in an area with the absence of clear evidence of artemisinin-based combination therapy (ACT) clinical failure. Although standard treatment outcomes remain largely preserved at the population level, the genomic findings raise concern for emerging resistance within the region. The findings of this work underscore the importance of integrating molecular surveillance with routine case detection, strengthening diagnostic and laboratory capacity, and conducting timely therapeutic efficacy studies to contextualize genomic signals. Proactive genomic monitoring during periods of resurgence may enable malaria control programs to identify early changes in parasite susceptibility and adjust treatment policy before widespread clinical failure becomes apparent. Abbreviations CT: Artemisinin-based Combination Therapy AL: Artemether Lumefantrine ART-R: Artemisinin Partial Resistance K13: Kelch 13 propeller domain pfk13: P. falciparum kelch 13 gene pfcrt : P. falciparum chloroquine resistance transporter pfmdr1 : P. falciparum multidrug resistance 1 pfdhfr : P. falciparum dihydrofolate reductase pfdhps : P. falciparum dihydropteroate synthase rPCR: realtimePolymerase Chain Reaction PfSMARRTer: Plasmodium falciparum Streamlined Multiplex Antimalarial Resistance and Relatedness NGS: Next-Generation Sequencing SNP: Single Nucleotide Polymorphism MOI: Multiplicity of Infection COI: Complexity of Infection PHEM: Public health emergency management EPHI: Ethiopian public Health institute Declarations Ethics approval: This study obtained approval from the scientific and review committee of the EPHI. Permission was obtained from EPHI to use the national EPI-week-based malaria data. Data were collected from consented patients as per the Helsinki principles. Competing interests: Authors declare that there is no competing interest. Jonathan B Par reports non-financial support from Abbott Laboratories and past research support from Gilead Sciences and consulting for Zymeron Corp, outside the scope of this work, Funding: This work is funded by Global fund through the Ethiopian Ministry of Health. The genomic work was supported by the US National Institutes of Health (R01AI177791). Author Contribution DG, BG and AA conceived the study. DG, NT, MH, Bk and MK led patient recruitment field data and sample collection. AA, MB, HS, AG performed laboratory work. MG and AF led genetic data analysis. DG, XX and AAB wrote the first draft of the manuscript. JJ, JB and JP critically reviewed the manuscript. All authors edited and approved the manuscript. Acknowledgements: Patients and data collectors are largely acknowledged. Data Availability Data is available from the corresponding author with a modest request. References 1. Venkatesan, P., WHO world malaria report 2024. Lancet Microbe, 2025: p. 101073. 2. GBD., Burden of 375 diseases and injuries, risk-attributable burden of 88 risk factors, and healthy life expectancy in 204 countries and territories, including 660 subnational locations, 1990–2023: a systematic analysis for the Global Burden of Disease Study 2023. Lancet, 2025. 406 (10513): p. 1873–1922. 3. Jalilian, A., et al., Waning success: a 2013–2022 spatial and temporal trend analysis of malaria in Ethiopia. Infect Dis Poverty, 2024. 13 (1): p. 93. 4. Tamiru, A., et al., Prevalence of asymptomatic malaria and associated factors in Ethiopia: Systematic review and meta-analysis. SAGE Open Med, 2022. 10 : p. 20503121221088085. 5. Ewnetu, Y. and W. Lemma, Highland Malaria Transmission Dynamics in Space and Time Before Pre-elimination Era, Northwest Ethiopia. J Epidemiol Glob Health, 2022. 12 (3): p. 362–371. 6. Yutura, G., et al., Prevalence of malaria and associated risk factors among household members in South Ethiopia: a multi-site cross-sectional study. Malar J, 2024. 23 (1): p. 143. 7. Abamecha, A., et al., Efficacy and safety of artemether-lumefantrine for treatment of uncomplicated Plasmodium falciparum malaria in Ethiopia: a systematic review and meta-analysis. Malar J, 2021. 20 (1): p. 213. 8. Hwang, J., et al., In vivo efficacy of artemether-lumefantrine against uncomplicated Plasmodium falciparum malaria in Central Ethiopia. Malar J, 2011. 10 : p. 209. 9. Assefa, A., A.A. Fola, and G. Tasew, Emergence of Plasmodium falciparum strains with artemisinin partial resistance in East Africa and the Horn of Africa: is there a need to panic? Malar J, 2024. 23 (1): p. 34. 10. Conrad, M.D., et al., Evolution of Partial Resistance to Artemisinins in Malaria Parasites in Uganda. N Engl J Med, 2023. 389 (8): p. 722–732. 11. Brhane, B.G., et al., Rising prevalence of Plasmodium falciparum Artemisinin partial resistance mutations in Ethiopia. Commun Med (Lond), 2025. 5 (1): p. 297. 12. Fola, A.A., et al., Plasmodium falciparum resistant to artemisinin and diagnostics have emerged in Ethiopia. Nat Microbiol, 2023. 8 (10): p. 1911–1919. 13. Hailemeskel, E., et al., Prevalence of Plasmodium falciparum Pfcrt and Pfmdr1 alleles in settings with different levels of Plasmodium vivax co-endemicity in Ethiopia. Int J Parasitol Drugs Drug Resist, 2019. 11 : p. 8–12. 14. Heuchert, A., et al., Molecular markers of anti-malarial drug resistance in southwest Ethiopia over time: regional surveillance from 2006 to 2013. Malar J, 2015. 14 : p. 208. 15. White, N.J., Pharmacokinetic and Pharmacodynamic Considerations in Antimalarial Dose Optimization. Antimicrobial Agents and Chemotherapy, 2013. 57 (12): p. 5792–5807. 16. World Health, O., Guidelines for the Treatment of Malaria . 3rd ed. 2015, Geneva: World Health Organization. 17. Belachew, M., et al., Evaluating performance of multiplex real time PCR for the diagnosis of malaria at elimination targeted low transmission settings of Ethiopia. Malaria Journal, 2022. 21 (1): p. 9. 18. Juliano, J. and J. Sadler, Pf-SMARRTer: Plasmodium falciparum Streamlined Multiplex Antimalarial Resistance and Relatedness Testing v1.13 . 2025, protocols.io. 19. Landis, J.R. and G.G. Koch, The measurement of observer agreement for categorical data. Biometrics, 1977. 33 (1): p. 159 − 74. 20. Chang, H.-H., et al., THE REAL McCOIL: A method for the concurrent estimation of the complexity of infection and SNP allele frequency for malaria parasites. PLOS Computational Biology, 2017. 13 (1): p. e1005348. 21. Schaffner, S.F., et al., hmmIBD: software to infer pairwise identity by descent between haploid genotypes. Malaria Journal, 2018. 17 (1): p. 196. 22. Abebe, A., et al., Significant number of Plasmodium vivax mono-infections by PCR misidentified as mixed infections (P. vivax/P. falciparum) by microscopy and rapid diagnostic tests: malaria diagnostic challenges in Ethiopia. Malaria Journal, 2023. 22 (1): p. 201. 23. Deora, N., et al., A systematic review and meta-analysis on sub-microscopic Plasmodium infections in India: Different perspectives and global challenges. Lancet Reg Health Southeast Asia, 2022. 2 : p. 100012. 24. World Health, O., Compendium of molecular markers for antimalarial drug resistance . 2025, World Health Organization: Geneva. 25. Niaré, K., et al., A novel locus associated with decreased susceptibility of Plasmodium falciparum to lumefantrine and dihydroartemisinin has emerged and spread in Uganda. bioRxiv, 2025: p. 2025.07.30.667738. Annex 1 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9094035","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Case Report","associatedPublications":[],"authors":[{"id":607042149,"identity":"4e487505-a71a-4eae-ae1c-abbbfe88b451","order_by":0,"name":"Dessalegn Geleta","email":"data:image/png;base64,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","orcid":"","institution":"Ethiopian Public Health Institute","correspondingAuthor":true,"prefix":"","firstName":"Dessalegn","middleName":"","lastName":"Geleta","suffix":""},{"id":607042150,"identity":"9a4ac893-c0a3-4607-b84d-04f661077f06","order_by":1,"name":"Bokretsion G. Brhane","email":"","orcid":"","institution":"Ethiopian Public Health Institute","correspondingAuthor":false,"prefix":"","firstName":"Bokretsion","middleName":"G.","lastName":"Brhane","suffix":""},{"id":607042151,"identity":"39dede52-e84a-4714-8f3c-e0a18be0a355","order_by":2,"name":"Adugna Abera","email":"","orcid":"","institution":"Ethiopian Public Health Institute","correspondingAuthor":false,"prefix":"","firstName":"Adugna","middleName":"","lastName":"Abera","suffix":""},{"id":607042152,"identity":"45f1859e-e457-460b-8d45-7b8a01db2dcf","order_by":3,"name":"Mahlet Belachew","email":"","orcid":"","institution":"Ethiopian Public Health Institute","correspondingAuthor":false,"prefix":"","firstName":"Mahlet","middleName":"","lastName":"Belachew","suffix":""},{"id":607042153,"identity":"2ab5485a-86ec-4cd5-a2e5-c6e995323b4b","order_by":4,"name":"Heven Sime","email":"","orcid":"","institution":"Ethiopian Public Health Institute","correspondingAuthor":false,"prefix":"","firstName":"Heven","middleName":"","lastName":"Sime","suffix":""},{"id":607042154,"identity":"ef2f4b6a-56b8-4e43-8a6e-1bcfb1bdd802","order_by":5,"name":"Atsbeha Gebreegziaxher","email":"","orcid":"","institution":"Ethiopian Public Health Institute","correspondingAuthor":false,"prefix":"","firstName":"Atsbeha","middleName":"","lastName":"Gebreegziaxher","suffix":""},{"id":607042155,"identity":"423379b6-db1d-456c-a221-5343444f8d49","order_by":6,"name":"Melak Getu","email":"","orcid":"","institution":"Ethiopian Public Health Institute","correspondingAuthor":false,"prefix":"","firstName":"Melak","middleName":"","lastName":"Getu","suffix":""},{"id":607042156,"identity":"7e07b14c-4da5-4323-a7c3-d9410784dd61","order_by":7,"name":"Abraham Ali","email":"","orcid":"","institution":"Ethiopian Public Health Institute","correspondingAuthor":false,"prefix":"","firstName":"Abraham","middleName":"","lastName":"Ali","suffix":""},{"id":607042157,"identity":"3248449f-8b49-41d0-9ec6-043ae3f9ce9b","order_by":8,"name":"Neamin Tesfaye","email":"","orcid":"","institution":"Ethiopian Public Health Institute","correspondingAuthor":false,"prefix":"","firstName":"Neamin","middleName":"","lastName":"Tesfaye","suffix":""},{"id":607042158,"identity":"858c9707-b2d3-476b-8c1e-c779d0de6ee7","order_by":9,"name":"Medhanye Habtetsion","email":"","orcid":"","institution":"Ethiopian Public Health Institute","correspondingAuthor":false,"prefix":"","firstName":"Medhanye","middleName":"","lastName":"Habtetsion","suffix":""},{"id":607042159,"identity":"fb3d63d9-703f-4a86-b1a4-77580855f9f1","order_by":10,"name":"Belayneh Kokobe","email":"","orcid":"","institution":"Ethiopian Public Health Institute","correspondingAuthor":false,"prefix":"","firstName":"Belayneh","middleName":"","lastName":"Kokobe","suffix":""},{"id":607042160,"identity":"7e09cb37-0971-4650-89b7-7d606c45b6f6","order_by":11,"name":"Mandefro Kebede","email":"","orcid":"","institution":"Ethiopian Public Health Institute","correspondingAuthor":false,"prefix":"","firstName":"Mandefro","middleName":"","lastName":"Kebede","suffix":""},{"id":607042161,"identity":"44015595-1ce5-4e5b-801c-342c832a1fe1","order_by":12,"name":"Zemene Worku","email":"","orcid":"","institution":"Ethiopian Public Health Institute","correspondingAuthor":false,"prefix":"","firstName":"Zemene","middleName":"","lastName":"Worku","suffix":""},{"id":607042162,"identity":"1318a59e-4ccf-4ccc-8300-d5b88623a4c5","order_by":13,"name":"Geremew Tasew","email":"","orcid":"","institution":"Ethiopian Public Health Institute","correspondingAuthor":false,"prefix":"","firstName":"Geremew","middleName":"","lastName":"Tasew","suffix":""},{"id":607042163,"identity":"04587104-e093-4877-af57-8430a4161d61","order_by":14,"name":"Gemechu Tadesse","email":"","orcid":"","institution":"Ethiopian Public Health Institute","correspondingAuthor":false,"prefix":"","firstName":"Gemechu","middleName":"","lastName":"Tadesse","suffix":""},{"id":607042164,"identity":"520d3640-c611-4d27-a07f-3704e563c835","order_by":15,"name":"Getachew Tollera","email":"","orcid":"","institution":"Ethiopian Public Health Institute","correspondingAuthor":false,"prefix":"","firstName":"Getachew","middleName":"","lastName":"Tollera","suffix":""},{"id":607042165,"identity":"3123e518-1c08-4e5e-8f80-ad8db7035123","order_by":16,"name":"Abebe A. Fola","email":"","orcid":"","institution":"Brown University","correspondingAuthor":false,"prefix":"","firstName":"Abebe","middleName":"A.","lastName":"Fola","suffix":""},{"id":607042166,"identity":"4ab80311-aab3-4269-ac22-b1537723cf8b","order_by":17,"name":"Jeffrey A. Bailey","email":"","orcid":"","institution":"Brown University","correspondingAuthor":false,"prefix":"","firstName":"Jeffrey","middleName":"A.","lastName":"Bailey","suffix":""},{"id":607042168,"identity":"a5be776b-bbfd-4943-8ad7-995704952eb0","order_by":18,"name":"Jonathan J. Juliano","email":"","orcid":"","institution":"Institute for Global Health and Infectious Diseases","correspondingAuthor":false,"prefix":"","firstName":"Jonathan","middleName":"J.","lastName":"Juliano","suffix":""},{"id":607042172,"identity":"8ba35e6c-82b6-4c3b-bd3d-39c686b127c2","order_by":19,"name":"Jonathan B. Parr","email":"","orcid":"","institution":"Institute for Global Health and Infectious Diseases","correspondingAuthor":false,"prefix":"","firstName":"Jonathan","middleName":"B.","lastName":"Parr","suffix":""},{"id":607042173,"identity":"10284d03-9e87-41c9-a0da-9a29ff4f49a9","order_by":20,"name":"Melkamu Abte","email":"","orcid":"","institution":"Ethiopian Public Health Institute","correspondingAuthor":false,"prefix":"","firstName":"Melkamu","middleName":"","lastName":"Abte","suffix":""},{"id":607042175,"identity":"d9efe920-d58f-4b3f-8ebd-81b9dc41cc20","order_by":21,"name":"Ashenafi Assefa","email":"","orcid":"","institution":"Ethiopian Public Health Institute","correspondingAuthor":false,"prefix":"","firstName":"Ashenafi","middleName":"","lastName":"Assefa","suffix":""}],"badges":[],"createdAt":"2026-03-11 12:32:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9094035/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9094035/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104888597,"identity":"84594738-3fef-4ec2-a4e9-e0b2354001e5","added_by":"auto","created_at":"2026-03-18 10:17:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":75294,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWeekly confirmed malaria case trend during 2019 to 2024 in Oromia Regional State, Ethiopia.\u003c/strong\u003e The weekly case counts for the region are shown by each colored line, with the resurgence year (2024) in pink. The dashed red line represents the outbreak threshold, defined as a five-year weekly baseline mean plus two times standard deviation.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9094035/v1/db5011ba7e7a41f1d408f87f.png"},{"id":104888595,"identity":"bd70b167-d59f-411a-861d-d7d7742aef52","added_by":"auto","created_at":"2026-03-18 10:17:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":92571,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAmplicon read depth distribution per sample among 15 falciparum samples.\u003c/strong\u003e Box plots of the distribution of read depth across loci in a sample is shown. Read depth is shown on log₁₀ scale. Red points indicate outliers.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9094035/v1/5c5c6fcde0e7f002a2188050.png"},{"id":104888596,"identity":"fa88fa20-5944-4b3b-8310-8a3841e817b0","added_by":"auto","created_at":"2026-03-18 10:17:19","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":90154,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWithin-sample drug-resistance mutation frequencies for 15 falciparum infections. \u003c/strong\u003eStacked bar plot illustrating mutant (red) and wild type otherwise, \u003cem\u003epfcrt\u003c/em\u003e has three missing data points. When detected, all antimalarial resistance polymorphisms occurred at 100% within sample frequency, except \u003cem\u003edhps\u003c/em\u003e A581G and \u003cem\u003ek13\u003c/em\u003e P441L. Three samples fail sequencing for\u003cem\u003e pfcrt \u003c/em\u003e76T.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9094035/v1/5a71c6cfe4c162eb6d0b7534.jpg"},{"id":104888599,"identity":"325b10a5-c338-486b-80df-fab8b6e28eca","added_by":"auto","created_at":"2026-03-18 10:17:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":24910,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution of the Complexity of Infection (COI) among fifteen \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. falciparum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003esamples.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9094035/v1/7b7743de875a0fc950bd5284.png"},{"id":104888598,"identity":"dbce1ad5-d46b-46e7-8e80-d793e09ad9bf","added_by":"auto","created_at":"2026-03-18 10:17:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":81744,"visible":true,"origin":"","legend":"\u003cp\u003eGenetic relatedness among samples based on estimated Identity By Descent (eIBD) analysis. Each dot represents an individual sample, and connecting lines indicate sample pairs with IBD values at or above the defined threshold, reflecting close genetic relatedness. The IBD value denotes the proportion of the genome shared identically by descent between two parasite isolates. Notably, samples PHEM1, PHEM6, and PHEM13 harbor the 441L mutation; however, in PHEM6 the mutation is present at a minor allele fraction of 24.3%, suggesting within-host heterogeneity rather than a fully fixed variant.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9094035/v1/022fc70d6b7b13ca5c4907c1.png"},{"id":105034032,"identity":"5e4bd139-23a9-4afd-a8f3-6e74fcab9d4e","added_by":"auto","created_at":"2026-03-20 07:22:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1593018,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9094035/v1/d194831b-3ae2-45e9-beb2-ac2fcd70ba10.pdf"},{"id":104888600,"identity":"7df65719-1d66-4a47-afa3-015db00eb2c2","added_by":"auto","created_at":"2026-03-18 10:17:19","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":259228,"visible":true,"origin":"","legend":"","description":"","filename":"Annex.docx","url":"https://assets-eu.researchsquare.com/files/rs-9094035/v1/c4b2b398294b38cff26c5a90.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Recurrent parasitemias with artemisinin partial resistance mutations during the 2024 Ethiopia malaria resurgence: a case series","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHistorically, Ethiopia has faced past malaria resurgence, including a major surge in cases in 2023-24, when over 10\u0026nbsp;million cases were reported compared to around 1\u0026nbsp;million cases in 2019 [1, 2]. The resurgence has led to an uptick in malaria cases and irregular outbreaks in areas once considered malaria-free, including highland regions, as well as an increase in asymptomatic cases [3, 4]. This poses a significant challenge to established malaria control measures and has set back progress made over the past decades [4\u0026ndash;6].\u003c/p\u003e \u003cp\u003eSince 2004, artemisinin-based combination therapy (ACT), primarily artemether\u0026ndash;lumefantrine (AL), has been the first-line treatment for uncomplicated \u003cem\u003eP.falciparum\u003c/em\u003e malaria in Ethiopia. Although most therapeutic efficacy studies reported high cure rates [7\u0026ndash;9], artemisinin partial resistance (ART-R) has become a major concern [6, 10\u0026ndash;14], suggesting the need for further molecular surveillance of antimalarial drug resistance markers [11].\u003c/p\u003e \u003cp\u003eArtemisinin based combination therapy (ACT) combines a fast-acting artemisinin derivative with a longer acting partner drug to ensure rapid parasite clearance and sustained post-treatment suppression. The artemisinin component is rapidly converted to its active metabolite dihydroartemisinin (DHA) and has a short half-life (approximately 1\u0026ndash;2 hours), leading to a marked reduction in parasite biomass within the first 48\u0026ndash;72 hours. In contrast, the partner drug has a longer elimination half-life, often persisting for days to weeks after treatment completion. During this elimination phase, parasites may be exposed to declining, subtherapeutic drug concentrations, creating a selective window that favors parasites with reduced susceptibility to the partner drug. Consequently, recurrent parasitemia occurring within the first few weeks after ACT is more consistent with recrudescence or reinfection involving parasites with decreased partner-drug sensitivity, particularly in moderate- to high-transmission settings[15].\u003c/p\u003e \u003cp\u003eIn response to the malaria resurgence, this study investigated patients who were returning after previous treatment for malaria to the Gelana Primary Hospital and Tore Health Center between August 10 and September 17, 2024, in West Guji, Gelana Woreda, Oromia regional state, Ethiopia. We enrolled Patients who were seeking treatment for malaria-like illness within 6 weeks of treatment [16]. First, we confirmed the resurgence of malaria in this region using national surveillance data from 2019 to 2024. Samples from enrolled participants were then screened by real-time PCR and samples positive for \u003cem\u003ePlasmodium falciparum\u003c/em\u003e underwent amplicon deep sequencing using \u003cem\u003ePfSMARRTer\u003c/em\u003e to characterize antimalarial resistance polymorphisms and parasite diversity. The resulting molecular resistance profiles were linked with patient clinical and treatment data to explore the relationship between molecular resistance markers and clinical outcomes.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMalaria case data 2019\u0026ndash;2024\u003c/h2\u003e \u003cp\u003eThe Ethiopian Public Health Institute (EPHI) has surveillance infrastructure, which allows it to obtain epidemiologic data from sources using both an active surveillance and a passive surveillance (weekly report) system. The surveillance unit of the Public Health Emergency Management (PHEM) department at EPHI collects data on epidemic-prone diseases from the health facility level, which is aggregated by catchment, district, zone, and region. Weekly clinical malaria surveillance data used in this study were obtained from the PHEM surveillance unit spanning January 2019 to December 2024.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStudy population, data collection and analysis\u003c/h3\u003e\n\u003cp\u003ePatients who re-visited the selected health facilities after prior treatment with suspected malaria and who had a positive blood smear within 42 days after their initial treatment during the study period were enrolled.\u003c/p\u003e \u003cp\u003eDemographic, clinical, and laboratory test data were collected from all consented participants by trained health workers at the Outpatient Department (OPD) and/or laboratory in each study health facility. Eligible patients' medical record numbers (MRN) were identified. After routine malaria diagnosis by blood film an additional 2\u0026ndash;3 ml of venous blood, stored at 2\u0026ndash;8 \u003csup\u003e0\u003c/sup\u003eC and transported to the EPHI laboratory for further investigation.\u003c/p\u003e \u003cp\u003eA structured questionnaire was used for data collection including demographic characteristics, malaria diagnosis and treatment history, additional data from the registries were collected using MRN. Data collectors were trained using a pre-tested questionnaire to ensure data quality.\u003c/p\u003e \u003cp\u003eReal-time PCR [17]was used to determine species of infection. In brief, pan-\u003cem\u003ePlasmodium\u003c/em\u003e specific primers and probe sets targeting the small subunit ribosomal RNA (18S rRNA) along with primers and probes specific for the var gene acidic terminal sequence (ATS) of \u003cem\u003eP. falciparum\u003c/em\u003e. The samples were further investigated with multiplexed primers for \u003cem\u003eP. falciparum\u003c/em\u003e (varATS) and Plasmodium \u003cem\u003evivax\u003c/em\u003e's small subunit ribosomal RNA (P.v18S rRNA) (Belachew et al, 2021). DNA samples confirmed with \u003cem\u003eP. falciparum\u003c/em\u003e mono-infection and with a cycle threshold (Ct) value below 30 were further sequenced using PFSMARRTer V13 [18] on the NextSeq 550 Illumina platform at EPHI.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using the R software (v4.5.1). Descriptive analysis was done to describe the study variables and outcomes. Trend analysis specific to the Oromia Region was conducted from the weekly epidemiological malaria data from 2019 to 2024. The mean plus two standard deviations of the baseline data was analyzed to calculate the statistical threshold for each epidemiological week. The diagnostic performance of the blood film was determined for identification of \u003cem\u003eP. falciparum\u003c/em\u003e and \u003cem\u003eP. vivax\u003c/em\u003e by considering real time PCR as the reference method. The Cohen\u0026rsquo;s Kappa coefficient was computed to measure agreement between microscopy and real time PCR classifications [19].\u003c/p\u003e \u003cp\u003eSequencing data was processed by SeekDeep v3.0.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/bailey-lab/SeekDeep\u003c/span\u003e\u003cspan address=\"https://github.com/bailey-lab/SeekDeep\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e The prevalence of molecular markers associated with antimalarial drug resistance was calculated as the proportion of successfully sequenced infections harboring the mutant allele among the total number of genotyped samples. The R v4.5.2, \u003cem\u003etidyverse, ggplot2\u003c/em\u003e package was used for visualization. Complexity of infection (COI) was estimated using sample allele frequency data across polymorphic loci included in the sequencing panel [20]. Genetic relatedness among parasite isolates was estimated using the \u003cem\u003ehmmIBD\u003c/em\u003e method[21]. Parasite relatedness networks were constructed and visualized using the \u003cem\u003eigraph\u003c/em\u003e package in R.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEthical clearance\u003c/h3\u003e\n\u003cp\u003eThis study obtained approval from the scientific and review committee of the EPHI. Permission was obtained from EPHI to use the national EPI-week-based malaria data. Data were collected from consented patients as per the Helsinki principles.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMalaria epidemiology in Oromia Region\u003c/h2\u003e \u003cp\u003eAnalysis of PHEM weekly surveillance data from 2019\u0026ndash;2024 demonstrates a steadily increasing malaria burden, with reported cases rising from 75,854 in 2019 to 4,489,228 in 2024 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Analysis of trends in 2024 highlight a sustained resurgence throughout 2024, during which malaria incidence exceeded the outbreak threshold in every epidemiologic week (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The epidemic curve showed two distinct transmission peaks: an initial rise between epidemiologic weeks 20\u0026ndash;30, followed by a larger and more prolonged peak between weeks 35\u0026ndash;43.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSocio-demography and clinical characteristics of the study participants\u003c/h3\u003e\n\u003cp\u003eSamples were collected from 34 study participants seeking repeated treatment. Complete clinical and socio-demographic data were available for 31 (92%) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A microscopic parasite species identification at health facility showed that a total of 27 patients were diagnosed with \u003cem\u003ep. falciparum\u003c/em\u003e during the second visit compared with 23 patients during the first visit, of whom 20 were consistently diagnosed with \u003cem\u003ep. falciparum\u003c/em\u003e at both visits (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong the 31 participants, 18 (58.1%) were male (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The median age was 25 years (IQR\u0026thinsp;=\u0026thinsp;20) (Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The mean number of days between the initial treatment and second visit to the health facility was 15.65 (SD\u0026thinsp;=\u0026thinsp;7.45; range, 6\u0026ndash;26) for \u003cem\u003eP. falciparum\u003c/em\u003e and 15.75 (SD\u0026thinsp;=\u0026thinsp;7.48; range, 6\u0026ndash;27) for \u003cem\u003eP. vivax\u003c/em\u003e cases. Almost half 17 (54.84%) of the patients visited the health facilities for the second time within the first two weeks after completion of the initial treatment. The majority of the patients 29(93.55%) were treated with artemether-lumefantrine (AL) and single-dose primaquine 27(87.1%) during the second course of the treatment (Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). 11(91.67%) patients with \u003cem\u003eP. Vivax\u003c/em\u003e infection were treated with AL due to misdiagnosis by microscopy.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eResults of microscopic identification of parasite species during the subsequent visit at two health facilities in Gelana Woreda, Oromia Region, Ethiopia (n\u0026thinsp;=\u0026thinsp;31).\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eInitial Diagnosis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eSecond visit Diagnosis\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMixed\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePf.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePv.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePf.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (8.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20 (87.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 (4.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePv.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6 (85.71%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 (14.29%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMixed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSocio-demographic and clinical profile of malaria patients confirmed by PCR from Gelana Woreda, Oromia Region, Ethiopia (n\u0026thinsp;=\u0026thinsp;31).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCategory\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePf. (n\u0026thinsp;=\u0026thinsp;14)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePv. (n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNeg (n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMix (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7 (50%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8(66.67%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2(66.67%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1(50%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e18(58.1%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7 (50%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4(33.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1(33.33%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1(50%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13(41.94%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e14\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e31\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eDuration between initial and second visit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8(57.14%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7(58.33%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2(66.67%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e17(54.84%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14\u0026ndash;21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1(7.14%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1(33.33%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1(50%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3(9.68%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21 and above\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5(35.71%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5(41.67%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1(50%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e11(35.48%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e14\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e31\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eTreatment given on second visit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14(100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11(91.67%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3(100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1(50%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e29(93.55%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo AL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1(8.33%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1(50%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2(6.45%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e14\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e31\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFull dose Primaquine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1(7.14%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2(16.67%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1(50%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4(12.9%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSingle dose Primaquine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13(92.86%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10(83.33%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3(100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1(50%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e27(87.1%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e14\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e31\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eFirst symptom\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBack pain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1(33.33%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1(3.23%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChills and rigor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2(14.29%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3(25%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5(16.13%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFever\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8(57.14%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5(41.67%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13(41.94%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHeadache\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4(28.57%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3(25%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2(66.67%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1(50%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10(32.26%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eShivering\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1(8.33%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1(50%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2(6.45%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e14\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e31*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ePf.= \u003cem\u003eP. falciparum\u003c/em\u003e, Pv. =\u003cem\u003eP. vivax\u003c/em\u003e, AL\u0026thinsp;\u003cem\u003e=\u003c/em\u003e\u0026thinsp;artemether-lumefantrine *Two cases, one \u003cem\u003eP. falciparum\u003c/em\u003e and one \u003cem\u003eP. vivax\u003c/em\u003e, were excluded from further analysis due to incomplete clinical and demographic profile.\u003c/p\u003e\n\u003ch3\u003eMicroscopy and real-time PCR result agreement\u003c/h3\u003e\n\u003cp\u003eMicroscopy results from the health facility reported 30 (88.2%), 2 (5.9%) and 2 (5.9%) of \u003cem\u003eP. falciparum\u003c/em\u003e, \u003cem\u003eP. vivax\u003c/em\u003e, and mixed species infections, respectively. Out of 34 samples, real-time PCR identified \u003cem\u003eP. falciparum\u003c/em\u003e in 15 (44.1%), \u003cem\u003eP. vivax\u003c/em\u003e in 13 (38.2%), mixed in 2 (5.9%), and no malaria parasite in 4 (11.8%) samples.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMicroscopy and real-time PCR test results of the microscopy-confirmed \u003cem\u003eP. falciparum\u003c/em\u003e samples collected from Gelana Woreda, Oromia Region, Ethiopia (n\u0026thinsp;=\u0026thinsp;26)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e \u003cp\u003eResult\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003ereal time PCR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePf.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePv.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eMicroscopy\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePf.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePv.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ePf.= \u003cem\u003eP. falciparum\u003c/em\u003e, Pv. =\u003cem\u003eP. Vivax\u003c/em\u003e *Two real-time positive cases, one \u003cem\u003eP. falciparum\u003c/em\u003e and one \u003cem\u003eP. vivax\u003c/em\u003e, were excluded from further analysis due to incomplete clinical and demographic profile.\u003c/p\u003e \u003cp\u003eFrom the 26 samples classified as \u003cem\u003eP. falciparum\u003c/em\u003e by microscopy, 14 (53.8%) were confirmed as \u003cem\u003eP. falciparum\u003c/em\u003e by PCR, while the remaining 11 (42.3%) were identified as \u003cem\u003eP. vivax\u003c/em\u003e. The result from microscopy performance showed 100% (14/14) sensitivity for detecting \u003cem\u003eP. falciparum\u003c/em\u003e, but much lower specificity at 8.3% (1/2) for \u003cem\u003eP. falciparum\u003c/em\u003e with PCR as the gold standard. Sensitivity and specificity for \u003cem\u003eP. vivax\u003c/em\u003e was 8.3% (1/12) and 100% (14/14), respectively. The overall species-level agreement between microscopy and PCR was low, with a Cohen\u0026rsquo;s Kappa of 0.09 (95% CI: \u0026minus;\u0026thinsp;0.08 to 0.26).\u003c/p\u003e \u003cp\u003e \u003cb\u003eQuality of amplicon sequencing of\u003c/b\u003e \u003cb\u003eP. falciparum\u003c/b\u003e \u003cb\u003einfections\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAmplicon sequencing of 24 \u003cem\u003eP. falciparum\u003c/em\u003e loci across 15 clinical isolates generated uniformly high coverage. Median read depth per amplicon was 8,124\u0026times; (interquartile range: 4,210 15,630\u0026times;), with more than 92% of locus-sample combinations exceeding 1,000\u0026times; coverage. This depth enabled reliable detection of minor haplotypes at frequencies as low as 1%. Seventeen low coverage outliers, marked in red (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), corresponded to samples with low parasitemia, as confirmed by quantitative PCR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCases of participants returning with symptomatic malaria and genetic markers of ART-R\u003c/h2\u003e \u003cp\u003eAnalysis of samples from 15 participants revealed the presence of the WHO-candidate ART-R marker \u003cem\u003ek13\u003c/em\u003e P441L mutation in three cases, detailed below.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCase 1\u003c/strong\u003e \u003cp\u003eA 10-year-old female patient presented to the outpatient department (OPD) with a chief complaint of severe headache and high-grade fever on 25 August 2024. The patient was diagnosed positive for mixed malaria parasite infection (\u003cem\u003eP. falciparum and P. vivax\u003c/em\u003e) by microscopic investigation of peripheral blood film. The patient was critically ill and subsequently admitted and given initial treatment of intravenous artesunate followed by Artemether/Lumefantrine and a full dose of primaquine for mixed infection in accordance with the national first-line antimalarial treatment guideline. The patient returned to the health facility with recurrent symptoms fourteen days (on 8 September 2024) after the completion of the initial \u003cem\u003eP. falciparum\u003c/em\u003e treatment course. Repeated blood film microscopy test confirmed \u003cem\u003eP. falciparum\u003c/em\u003e. A blood sample was collected and sent to the National Reference Laboratory for further molecular investigation.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCase 2\u003c/strong\u003e \u003cp\u003eA 43-year-old male patient who presented with severe back pain confirmed for \u003cem\u003eP. falciparum\u003c/em\u003e malaria parasite on 10 August 2024. Following the national malaria treatment protocol, the patient was treated with a full course of artemether-lumefantrine followed by single dose primaquine. Eight days after completion of the initial treatment course, the patient returned to the same health facility on 22 August 2024 with recurrent symptoms, and the microscopic investigation revealed the presence of \u003cem\u003eP. falciparum\u003c/em\u003e.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCase 3\u003c/strong\u003e \u003cp\u003eA two-year-old female patient presented with high-grade fever and chills was confirmed for \u003cem\u003eP. falciparum\u003c/em\u003e after blood film microscopic examination on 29 July 2024. The patient was treated with a complete course of artemether-lumefantrine followed by single dose primaquine. The patient returned to the same health facility with recurrent symptoms on August 16, 2024, fifteen days after the completion of the initial treatment course. The repeated microscopy investigation detected \u003cem\u003eP. falciparum\u003c/em\u003e.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePrevalence of Non-ACT and Partner Drug Resistance Mutations\u003c/h2\u003e \u003cp\u003eAmplicon sequencing showed that molecular markers for many antimalarials were common in the sequenced isolates including \u003cem\u003epfcrt (PfcRT_76T)\u003c/em\u003e, \u003cem\u003epfmdr1 (N86, 184F, D1246)\u003c/em\u003e, \u003cem\u003epfdhfr (51I, 59R,108N)\u003c/em\u003e, and \u003cem\u003epfdhps\u003c/em\u003e (540E). The \u003cem\u003epfdhps\u003c/em\u003e 581G and k13 441L were detected in three samples each 3/15 (20%). Within-sample allele frequencies of each mutation are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, showing that in most cases, samples contained a single allele at these loci. A sextuple mutant haplotype combining \u003cem\u003epfcrt\u003c/em\u003e 76T, the \u003cem\u003epfmdr1\u003c/em\u003e NFD triple, the \u003cem\u003epfdhfr\u003c/em\u003e IRN triple, and \u003cem\u003epfdhps\u003c/em\u003e 437G/540E occurred in almost all the samples, except three missing data for \u003cem\u003epfcrt\u003c/em\u003e 76T Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eComplexity of Infection (COI)\u003c/h2\u003e \u003cp\u003eThe majority of \u003cem\u003eP. falciparum\u003c/em\u003e isolates were monoclonal 10/15(66%). Ten isolates exhibited a COI of 1, while a smaller proportion showed higher COI values, including COI\u0026thinsp;=\u0026thinsp;2 (n\u0026thinsp;=\u0026thinsp;2), COI\u0026thinsp;=\u0026thinsp;3 (n\u0026thinsp;=\u0026thinsp;1), and COI\u0026thinsp;=\u0026thinsp;4 (n\u0026thinsp;=\u0026thinsp;2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eGenetic diversity and relatedness\u003c/h2\u003e \u003cp\u003eHaplotype clustering at 99% nucleotide identity revealed pronounced variation in polyclonality (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The ama1 locus displayed the highest diversity, with up to four haplotypes detected in sample PHEM-6 and a mean complexity of infection (COI) of 1.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 across all samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The heome loci cluster exhibited intermediate diversity, with mean COI values ranging from 1.6 to 1.8. In contrast, drug-resistance loci (\u003cem\u003epfcrt, pfmdr1, pfdhfr, pfdhps, pfk13\u003c/em\u003e) were monoclonal (COI\u0026thinsp;=\u0026thinsp;1) in nearly all instances, and 66% of isolates (10 of 15) showed COI\u0026thinsp;=\u0026thinsp;1 across all resistance markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Mean COI was significantly higher in neutral and antigenic loci than in mutated genes (Wilcoxon rank-sum test, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlthough the sample size is limited, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e highlights notable genetic clustering among parasites carrying the 441L mutation. At an IBD threshold of 0.50 (indicative of sibling-level relatedness), 441L mutant parasites form distinct high-relatedness clusters, consistent with recent clonal expansion and focal transmission amplification. At a lower threshold (IBD\u0026thinsp;=\u0026thinsp;0.25), these mutants remain genetically connected to a broader parasite population, suggesting ongoing gene flow within the study area.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTogether, these patterns support the interpretation that the increase in 441L prevalence is likely driven by local expansion of established parasite lineages rather than repeated introductions of genetically divergent strains.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eEthiopia experienced a marked resurgence of malaria during 2024, with Oromia Region contributing substantially to the national increase in cases. In this investigation of recurrent clinical malaria cases from Gelana Woreda, we combined surveillance data, clinical characterization, and targeted amplicon sequencing to examine whether antimalarial drug resistance may be occurring during early recurrence. Our findings demonstrate four key observations: (a) sustained resurgence throughout 2024 in the study region; (b) low agreement between routine microscopy and molecular species identification; (c) the presence of WHO-validated \u003cem\u003epfk13\u003c/em\u003e P441L mutations among recurrent \u003cem\u003ePlasmodium falciparum\u003c/em\u003e infections and (d) there are high levels of molecular markers of antimalarial resistance in these recurrent parasitemias.\u003c/p\u003e \u003cp\u003e The discordance between microscopy and qPCR species identification was substantial, with very low agreement (κ\u0026thinsp;=\u0026thinsp;0.09). Although microscopy remains the cornerstone of malaria diagnosis in Ethiopia, this level of misclassification\u0026mdash;particularly between \u003cem\u003eP. falciparum\u003c/em\u003e and \u003cem\u003eP. vivax\u003c/em\u003e\u0026mdash;has important implications in a resurgence setting. Species misidentification may lead to suboptimal treatment selection, inappropriate use of primaquine, and inaccurate surveillance data [22, 23]. These findings reinforce the need to strengthen diagnostic quality assurance systems and consider strategic incorporation of molecular confirmation in sentinel or outbreak settings.\u003c/p\u003e \u003cp\u003eAmong the 15 qPCR-confirmed \u003cem\u003eP. falciparum\u003c/em\u003e infections that underwent deep amplicon sequencing, three harbored the WHO-validated \u003cem\u003epfk13\u003c/em\u003e P441L mutation [24]. These three cases returned with recurrent parasitemia 8\u0026ndash;15 days after completion of artemether\u0026ndash;lumefantrine (AL) treatment. Although we cannot definitively distinguish recrudescence from reinfection in this small cohort, the temporal proximity of recurrence and the presence of validated artemisinin partial resistance (ART-R) markers are notable. These findings align with recent reports documenting geographically heterogeneous emergence of \u003cem\u003epfk13\u003c/em\u003e mutations in Ethiopia, including R622I, P574L, A675V, and P441L [9, 11, 12]. The P441L mutation has been reported from geographical proximate areas in low prevalence 1\u0026ndash;2% in Oromia and Central Ethiopia regions [9, 11]. A recent work on malaria resurgence in Ethiopia reported 441L mutations in 9 of 22 samples in Dila, Central Ethiopia (In press). Given the limited number of samples, IBD sharing supports the previous reports that malaria resurgence in the area may not be independent, implicating a sustained transmission of similar lineage may be due expansion of pre-existing parasite lineages[9].\u003c/p\u003e \u003cp\u003eIn addition to artemisinin resistance markers, we observed near-fixation of mutations in \u003cem\u003epfcrt\u003c/em\u003e, \u003cem\u003epfmdr1\u003c/em\u003e, \u003cem\u003epfdhfr\u003c/em\u003e, and \u003cem\u003epfdhps\u003c/em\u003e. The dominance of the sextuple mutant haplotype (including \u003cem\u003epfcrt\u003c/em\u003e 76T, \u003cem\u003epfmdr1\u003c/em\u003e NFD, \u003cem\u003epfdhfr\u003c/em\u003e IRN, and \u003cem\u003epfdhps\u003c/em\u003e 437G/540E) reflects longstanding antifolate and chloroquine selection pressure in Ethiopia [11, 12]. Of particular relevance is the high prevalence of the \u003cem\u003epfmdr1\u003c/em\u003e N86 allele, previously associated with altered lumefantrine tolerance, which was present at 100% frequency within all parasitemias genotyped.\u003c/p\u003e \u003cp\u003eFortunately, ACT therapeutic efficacy studies in Ethiopia have continued to report PCR-corrected cure rates above 95%. However, the molecular landscape suggests important changes that need continued monitoring [12]. Continued reliance on AL as first-line therapy for \u003cem\u003eP. falciparum\u003c/em\u003e, particularly during periods of intense transmission, may further select for parasites with reduced partner-drug susceptibility, potentially accelerating the clinical impact of emerging artemisinin resistance, as has been seen with the emergence of the PX1 PIN haplotype in Uganda [25].\u003c/p\u003e \u003cp\u003eThis study provides, to our knowledge, the first molecular documentation of artemisinin partial resistance markers among clinically recurrent malaria cases in Ethiopia. Malaria incidence reportedly declined in 2025, suggesting that resurgence dynamics are likely multifactorial and not solely attributable to drug resistance. However, the detection of multiple independent ART-R markers, both here and in other studies [9, 11, 12], in a resurgence setting signals ongoing evolutionary pressure and underscores the need for strengthened surveillance.\u003c/p\u003e \u003cp\u003eSeveral limitations should be considered. The sample size was small, and sites were purposefully selected based on recurrent presentations, limiting generalizability. We did not have initial parasitemia to genotype. We did not perform whole genome sequencing or pharmacokinetic analyses to definitively distinguish recrudescence from reinfection or to assess drug exposure. In addition, diagnostic discrepancies between microscopy and qPCR may have influenced case classification. Nonetheless, the integration of epidemiologic, clinical, and high-depth amplicon sequencing data provides a coherent signal that resistance-associated polymorphisms are present among recurrent cases in this resurgence context.\u003c/p\u003e \u003cp\u003eIn this case series from a district experiencing malaria resurgence, we identified circulating ART-R\u0026ndash;associated mutations within genetically related \u003cem\u003ePlasmodium falciparum\u003c/em\u003e parasite populations, in an area with the absence of clear evidence of artemisinin-based combination therapy (ACT) clinical failure. Although standard treatment outcomes remain largely preserved at the population level, the genomic findings raise concern for emerging resistance within the region. The findings of this work underscore the importance of integrating molecular surveillance with routine case detection, strengthening diagnostic and laboratory capacity, and conducting timely therapeutic efficacy studies to contextualize genomic signals. Proactive genomic monitoring during periods of resurgence may enable malaria control programs to identify early changes in parasite susceptibility and adjust treatment policy before widespread clinical failure becomes apparent.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCT: Artemisinin-based Combination Therapy\u003cbr\u003e\u0026nbsp;AL: Artemether Lumefantrine\u003c/p\u003e\n\u003cp\u003eART-R: Artemisinin Partial Resistance\u0026nbsp;\u003cbr\u003e\u0026nbsp;K13: Kelch 13 propeller domain\u003cbr\u003epfk13:\u0026nbsp;\u003cem\u003eP. falciparum\u003c/em\u003e kelch 13 gene\u003cbr\u003e\u003cem\u003epfcrt\u003c/em\u003e :\u0026nbsp;\u003cem\u003eP. falciparum\u003c/em\u003e chloroquine resistance transporter\u003cbr\u003e\u003cem\u003epfmdr1\u003c/em\u003e:\u0026nbsp;\u003cem\u003eP. falciparum\u003c/em\u003e multidrug resistance 1\u003cbr\u003e\u003cem\u003epfdhfr\u003c/em\u003e :\u0026nbsp;\u003cem\u003eP. falciparum\u003c/em\u003e dihydrofolate reductase\u003cbr\u003e\u003cem\u003epfdhps\u003c/em\u003e : \u003cem\u003eP. falciparum\u003c/em\u003e dihydropteroate synthase\u003c/p\u003e\n\u003cp\u003erPCR: realtimePolymerase Chain Reaction\u003c/p\u003e\n\u003cp\u003ePfSMARRTer:\u0026nbsp;\u003cem\u003ePlasmodium falciparum\u003c/em\u003e Streamlined Multiplex Antimalarial Resistance and Relatedness\u003cbr\u003e\u0026nbsp;NGS: Next-Generation Sequencing\u003cbr\u003e\u0026nbsp;SNP: Single Nucleotide Polymorphism\u003c/p\u003e\n\u003cp\u003eMOI: Multiplicity of Infection\u003cbr\u003e\u0026nbsp;COI: Complexity of Infection\u003cbr\u003e\u0026nbsp;PHEM: Public health emergency management\u003c/p\u003e\n\u003cp\u003eEPHI: Ethiopian public Health institute\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval:\u003c/strong\u003e \u003cp\u003eThis study obtained approval from the scientific and review committee of the EPHI. Permission was obtained from EPHI to use the national EPI-week-based malaria data. Data were collected from consented patients as per the Helsinki principles.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests:\u003c/h2\u003e \u003cp\u003eAuthors declare that there is no competing interest. Jonathan B Par reports non-financial support from Abbott Laboratories and past research support from Gilead Sciences and consulting for Zymeron Corp, outside the scope of this work,\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis work is funded by Global fund through the Ethiopian Ministry of Health. The genomic work was supported by the US National Institutes of Health (R01AI177791).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eDG, BG and AA conceived the study. DG, NT, MH, Bk and MK led patient recruitment field data and sample collection. AA, MB, HS, AG performed laboratory work. MG and AF led genetic data analysis. DG, XX and AAB wrote the first draft of the manuscript. JJ, JB and JP critically reviewed the manuscript. All authors edited and approved the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements:\u003c/h2\u003e \u003cp\u003ePatients and data collectors are largely acknowledged.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is available from the corresponding author with a modest request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e1. Venkatesan, P., \u003cem\u003eWHO world malaria report 2024.\u003c/em\u003e Lancet Microbe, 2025: p. 101073.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e2. GBD., \u003cem\u003eBurden of 375 diseases and injuries, risk-attributable burden of 88 risk factors, and healthy life expectancy in 204 countries and territories, including 660 subnational locations, 1990\u0026ndash;2023: a systematic analysis for the Global Burden of Disease Study 2023.\u003c/em\u003e Lancet, 2025. \u003cb\u003e406\u003c/b\u003e(10513): p. 1873\u0026ndash;1922.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e3. Jalilian, A., et al., \u003cem\u003eWaning success: a 2013\u0026ndash;2022 spatial and temporal trend analysis of malaria in Ethiopia.\u003c/em\u003e Infect Dis Poverty, 2024. \u003cb\u003e13\u003c/b\u003e(1): p. 93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e4. Tamiru, A., et al., \u003cem\u003ePrevalence of asymptomatic malaria and associated factors in Ethiopia: Systematic review and meta-analysis.\u003c/em\u003e SAGE Open Med, 2022. \u003cb\u003e10\u003c/b\u003e: p. 20503121221088085.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e5. Ewnetu, Y. and W. Lemma, \u003cem\u003eHighland Malaria Transmission Dynamics in Space and Time Before Pre-elimination Era, Northwest Ethiopia.\u003c/em\u003e J Epidemiol Glob Health, 2022. \u003cb\u003e12\u003c/b\u003e(3): p. 362\u0026ndash;371.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e6. Yutura, G., et al., \u003cem\u003ePrevalence of malaria and associated risk factors among household members in South Ethiopia: a multi-site cross-sectional study.\u003c/em\u003e Malar J, 2024. \u003cb\u003e23\u003c/b\u003e(1): p. 143.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e7. Abamecha, A., et al., \u003cem\u003eEfficacy and safety of artemether-lumefantrine for treatment of uncomplicated Plasmodium falciparum malaria in Ethiopia: a systematic review and meta-analysis.\u003c/em\u003e Malar J, 2021. \u003cb\u003e20\u003c/b\u003e(1): p. 213.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e8. Hwang, J., et al., \u003cem\u003eIn vivo efficacy of artemether-lumefantrine against uncomplicated Plasmodium falciparum malaria in Central Ethiopia.\u003c/em\u003e Malar J, 2011. \u003cb\u003e10\u003c/b\u003e: p. 209.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e9. Assefa, A., A.A. Fola, and G. Tasew, \u003cem\u003eEmergence of Plasmodium falciparum strains with artemisinin partial resistance in East Africa and the Horn of Africa: is there a need to panic?\u003c/em\u003e Malar J, 2024. \u003cb\u003e23\u003c/b\u003e(1): p. 34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e10. Conrad, M.D., et al., \u003cem\u003eEvolution of Partial Resistance to Artemisinins in Malaria Parasites in Uganda.\u003c/em\u003e N Engl J Med, 2023. \u003cb\u003e389\u003c/b\u003e(8): p. 722\u0026ndash;732.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e11. Brhane, B.G., et al., \u003cem\u003eRising prevalence of Plasmodium falciparum Artemisinin partial resistance mutations in Ethiopia.\u003c/em\u003e Commun Med (Lond), 2025. \u003cb\u003e5\u003c/b\u003e(1): p. 297.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e12. Fola, A.A., et al., \u003cem\u003ePlasmodium falciparum resistant to artemisinin and diagnostics have emerged in Ethiopia.\u003c/em\u003e Nat Microbiol, 2023. \u003cb\u003e8\u003c/b\u003e(10): p. 1911\u0026ndash;1919.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e13. Hailemeskel, E., et al., \u003cem\u003ePrevalence of Plasmodium falciparum Pfcrt and Pfmdr1 alleles in settings with different levels of Plasmodium vivax co-endemicity in Ethiopia.\u003c/em\u003e Int J Parasitol Drugs Drug Resist, 2019. \u003cb\u003e11\u003c/b\u003e: p. 8\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e14. Heuchert, A., et al., \u003cem\u003eMolecular markers of anti-malarial drug resistance in southwest Ethiopia over time: regional surveillance from 2006 to 2013.\u003c/em\u003e Malar J, 2015. \u003cb\u003e14\u003c/b\u003e: p. 208.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e15. White, N.J., \u003cem\u003ePharmacokinetic and Pharmacodynamic Considerations in Antimalarial Dose Optimization.\u003c/em\u003e Antimicrobial Agents and Chemotherapy, 2013. \u003cb\u003e57\u003c/b\u003e(12): p. 5792\u0026ndash;5807.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e16. World Health, O., \u003cem\u003eGuidelines for the Treatment of Malaria\u003c/em\u003e. 3rd ed. 2015, Geneva: World Health Organization.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e17. Belachew, M., et al., \u003cem\u003eEvaluating performance of multiplex real time PCR for the diagnosis of malaria at elimination targeted low transmission settings of Ethiopia.\u003c/em\u003e Malaria Journal, 2022. \u003cb\u003e21\u003c/b\u003e(1): p. 9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e18. Juliano, J. and J. Sadler, \u003cem\u003ePf-SMARRTer: Plasmodium falciparum Streamlined Multiplex Antimalarial Resistance and Relatedness Testing v1.13\u003c/em\u003e. 2025, protocols.io.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e19. Landis, J.R. and G.G. Koch, \u003cem\u003eThe measurement of observer agreement for categorical data.\u003c/em\u003e Biometrics, 1977. \u003cb\u003e33\u003c/b\u003e(1): p. 159\u0026thinsp;\u0026minus;\u0026thinsp;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e20. Chang, H.-H., et al., \u003cem\u003eTHE REAL McCOIL: A method for the concurrent estimation of the complexity of infection and SNP allele frequency for malaria parasites.\u003c/em\u003e PLOS Computational Biology, 2017. \u003cb\u003e13\u003c/b\u003e(1): p. e1005348.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e21. Schaffner, S.F., et al., \u003cem\u003ehmmIBD: software to infer pairwise identity by descent between haploid genotypes.\u003c/em\u003e Malaria Journal, 2018. \u003cb\u003e17\u003c/b\u003e(1): p. 196.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e22. Abebe, A., et al., \u003cem\u003eSignificant number of Plasmodium vivax mono-infections by PCR misidentified as mixed infections (P. vivax/P. falciparum) by microscopy and rapid diagnostic tests: malaria diagnostic challenges in Ethiopia.\u003c/em\u003e Malaria Journal, 2023. \u003cb\u003e22\u003c/b\u003e(1): p. 201.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e23. Deora, N., et al., \u003cem\u003eA systematic review and meta-analysis on sub-microscopic Plasmodium infections in India: Different perspectives and global challenges.\u003c/em\u003e Lancet Reg Health Southeast Asia, 2022. \u003cb\u003e2\u003c/b\u003e: p. 100012.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e24. World Health, O., \u003cem\u003eCompendium of molecular markers for antimalarial drug resistance\u003c/em\u003e. 2025, World Health Organization: Geneva.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e25. Niar\u0026eacute;, K., et al., \u003cem\u003eA novel locus associated with decreased susceptibility of \u0026amp;lt;em\u0026amp;gt;Plasmodium falciparum\u0026amp;lt;/em\u0026amp;gt; to lumefantrine and dihydroartemisinin has emerged and spread in Uganda.\u003c/em\u003e bioRxiv, 2025: p. 2025.07.30.667738.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnnex 1\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Ethiopia, Malaria, ACT-resistance, ART-R, PfSMARRTer","lastPublishedDoi":"10.21203/rs.3.rs-9094035/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9094035/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eEthiopia experienced a marked resurgence of malaria in 2024. Artemisinin-based combination therapies (ACTs) are first-line treatment for uncomplicated \u003cem\u003ePlasmodium falciparum\u003c/em\u003e malaria and threatened by the emergence of artemisinin partial resistance (ART-R), associated with mutations in the \u003cem\u003eP. falciparum kelch13\u003c/em\u003e (\u003cem\u003ek13\u003c/em\u003e) gene, that could undermine treatment efficacy and accelerate transmission.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods and material:\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe used the national Public Health Emergency Management (PHEM) surveillance system to characterize malaria resurgence and further investigate antimalarial drug resistance markers among cases of recurrent clinical malaria in three selected resurgence sites in central Ethiopia.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eParasite isolates from 15 patients with confirmed clinical recurrent \u003cem\u003eP. falciparum\u003c/em\u003e malaria were genotyped for molecular markers associated with drug resistance, including mutations in \u003cem\u003ek13, pfcrt\u003c/em\u003e, \u003cem\u003epfmdr1\u003c/em\u003e, \u003cem\u003epfdhfr\u003c/em\u003e, and \u003cem\u003epfdhps\u003c/em\u003e using PfSMARRTer multiplex amplicon sequencing in Addis Ababa. Clinical presentation and treatment history were reviewed alongside genotyping results. Three patients (3/15, 20%) with confirmed recurrence were infected by parasites carrying the WHO-candidate ART-R molecular marker K13 P441L. Markers of resistance to other antimalarial drugs were largely fixed in the population. These cases occurred in the context of increasing malaria incidence, with evidence of clonal expansion or dominance of a related lineage. The findings indicate the presence of ACT resistance-associated markers within genetically heterogeneous parasite populations.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe current study documents cases of recurrent parasitemia caused by \u003cem\u003eP. falciparum\u003c/em\u003e with K13 P441L during the malaria resurgence in the Oromia region. The detection of multiple independent resistance markers suggests ongoing drug pressure on first-line treatments. These findings underscore the need for strengthened molecular surveillance integrated with routine case monitoring to inform treatment policy and support malaria control and elimination efforts in Ethiopia.\u003c/p\u003e","manuscriptTitle":"Recurrent parasitemias with artemisinin partial resistance mutations during the 2024 Ethiopia malaria resurgence: a case series","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-18 10:17:14","doi":"10.21203/rs.3.rs-9094035/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-06T15:38:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-06T15:09:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-27T11:58:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"251365517410819317961749149282018061668","date":"2026-03-21T04:36:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"265686644186189995639943573037971000097","date":"2026-03-19T16:59:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"26793827084010348882010747179863232342","date":"2026-03-19T09:01:57+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-16T15:31:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-12T18:25:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-12T18:24:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"Malaria Journal","date":"2026-03-11T12:17:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b7e47108-68b1-440f-b0c3-a21640cad5eb","owner":[],"postedDate":"March 18th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-12T19:38:14+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-18 10:17:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9094035","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9094035","identity":"rs-9094035","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}