Day 3 parasitemia and Plasmodium falciparum Kelch 13 mutations among uncomplicated malaria patients treated with artemether-lumefantrine in Adjumani district, Uganda

Artemisinin resistance threatens malaria control and elimination efforts globally. Recent studies have reported the emergence of Plasmodium falciparum parasites tolerant to artemisinin agents in sub-Saharan Africa, including Uganda. The current study assessed the day 3 parasite clearance and its correlation with P. falciparum K13 propeller gene (pfkelch13) mutations in P. falciparum parasites isolated from patients with uncomplicated malaria under artemether-lumefantrine (AL) treatment. This study enrolled 100 P. falciparum-positive patients to whom AL was prescribed between 09/September/2022 and 06/November/2022. Blood samples were collected in EDTA tubes before treatment initiation (day 0) and on day 3. Parasitemia was assessed by microscopy from blood smears and quantitative polymerase chain reaction (qPCR) from the DNA extracted. The day 0 parasite K13 gene was sequenced using Sanger sequencing. Sequence data were analysed using MEGA version 11 software. The data were analysed using STATA version 15, and the Mann‒Whitney U test was used to compare PCR parasite clearance on day 3 using the comparative CT value method and pfkelch13 mutations. The prevalence of day 3 parasitaemia was 24% (24/100) by microscopy and 63% (63/100) by qPCR from the AL-treated patients. P. falciparum K13-propeller gene polymorphism was detected in 18.8% (15/80) of the day 0 DNA samples. The K13 mutations found were C469Y, 12.5% (10/80); A675V, 2.5% (2/80); A569S, 1.25%, (1/80), A578S, 1.25%, (1/80) and; F491S, 1.25%, (1/80) a new allele not reported anywhere. The C469Y mutation, compared to the wild-type, was associated with delayed parasite clearance p = 0.0278, Hodges-Lehmann estimation 3.2108 on the log scale, (95%CI 1.7076, 4.4730). There was a high prevalence of day 3 P. falciparum among malaria patients treated using artemether-lumefantrine. We conclude the presence of the K13 mutation associated with artemisinin resistance by P. falciparum in Adjumani district, Uganda, necessitates regular surveillance of the effectiveness and efficacy of artemether-lumefantrine in the country.


Introduction
Malaria continues to pose a substantial global health challenge, with sub-Saharan Africa (SSA) bearing the highest burden of the disease's impact.In 2022, the World Health Organization (WHO) reported 249 million malaria cases worldwide, an increase of at least 5 million cases compared to the previous year, with the WHO African region accounting for 94% of the cases [1,2].Uganda contributes at least 5% of the worldwide malaria cases, most of which occur in rural areas, especially in Northern and Eastern Uganda.There have been extensive control and prevention interventions, such as the distribution of insecticide-treated bed nets (ITNs) covering at least 82% of households in Uganda, indoor residual spraying (IRS) in some regions being scaled up, and antimalarial medications.Despite these interventions, the area has witnessed stagnation in malaria elimination efforts and an increase in malaria cases in Northern Uganda, highlighting the persistent challenge of malaria, especially in resource-limited settings [3].
The current malaria treatment approach for uncomplicated Plasmodium falciparum infection involves using artemisinin-based combination therapies (ACTs).Uganda adopted artemether-lumefantrine (AL) as a first-line therapy for uncomplicated P. falciparum malaria, which has been crucial in reducing malaria mortalities for almost two decades [1,[4][5][6].These therapies combine the short-acting artemisinin derivative (artemether) with another long-acting antimalarial drug (lumefantrine), enhancing efficacy and reducing the risk of resistance development.In addition to case management by the ACTs, control measures such as ITNs, IRS, and malaria chemoprevention have been integral in reducing malaria transmission [7][8][9][10][11][12].
Despite these efforts, several challenges persist in the fight against malaria.Inadequate supply of antimalarial medicines, especially in remote areas, often results in stockouts of ACTs, the cornerstone of malaria treatment [13].Moreover, the proliferation of substandard antimalarial agents and self-medication pose a significant public health risk, undermining treatment effectiveness and contributing to artemisinin resistance development [14,15].Artemisinin resistance, characterised by Pfkelch13 (K13) gene mutations [16] and delayed parasite clearance, has emerged as a serious concern, and molecular markers of artemisinin resistance have been identified in many SSA countries [17,18].Recent studies in Uganda reported the emergence of artemisinin resistance in the Eastern and Northern regions [19][20][21], a threat that is becoming evident to the effectiveness of artemisinin combination therapy.
The widespread development of artemisinin resistance could reverse the gains made in malaria control and elimination, potentially leading to an increase in malaria morbidity and mortality.This may lead to a similar catastrophic event previously observed when chloroquine was withdrawn from malaria treatment due to widespread resistance [22,23].In Uganda, where healthcare systems are often overburdened with limited access to quality healthcare [24,25], the spread of artemisinin resistance could undermine decades of progress and investment in malaria control, and this could be worsened by the lack of effective alternatives to artemisinin antimalarial agents in malaria treatment.
This study assessed the prevalence of day 3 P. falciparum in patients with uncomplicated malaria treated with AL and the prevalence of K13-propeller gene mutations associated with artemisinin resistance.The study's findings provide context-specific evidence on artemisinin resistance among P. falciparum parasites in patients treated using artemether-lumefantrine in the West Nile region, Uganda.

Ethical statement
The protocol was reviewed, and ethical approval was obtained from the School of Biomedical Sciences Research and Ethics Committee at the College of Health Sciences, Makerere University (SBS-2022-157).Administrative clearance was obtained from Adjumani district authorities.Written informed consents were obtained from all participants before data collection, parental consents were obtained from participants aged 5-7 years, assent and parental consent were obtained from participants aged 8-17 years, and participants 18 years old and above consented to the study.

Study area
The study was conducted in the Adjumani district located in the West Nile region, northwest of Uganda.The region neighbours the Democratic Republic of Congo and South Sudan.The district is among the highest malaria endemic districts in the country, with malaria transmission throughout the year and a prevalence reported above 50% by microscopy during the country's malaria indicator survey [26].

Study design
This was a prospective longitudinal study which recruited malaria patients between 09/September/2022 and 06/November/2022 receiving artemether-lumefantrine (AL) 20 mg artemether/120 mg lumefantrine tablets, 3-day six-dose regimen prescribed according to patient weight for treatment of uncomplicated malaria at Adjumani District Hospital.Blood samples were collected from the patient on day 0 (before drug intake) and day 3 (after treatment).The study enrolled 100 purposively selected uncomplicated malaria patients aged five years and above with confirmed P. falciparum infection.A study purpose non-validated questionnaire was used to collect information on the socio-demographic characteristics of the patients, including age, gender, and previous history of malaria treatment.The study collected both patient socio-demographic and laboratory data.Plasmodium falciparum parasite infections among symptomatic malaria patients were determined using microscopy and qPCR from the College of Veterinary Medicine Animal Resources and Biosecurity, Pharmacology/Research Center for Tropical Diseases and Vector Control, and Central Diagnostic Laboratory.

Blood sample collection
Venous blood (2-3ml) was collected in an EDTA tube.Two blood samples were collected from each patient; the first was on day 0, before initiation of treatment, and the second was collected on day 3, after completion of artemether-lumefantrine therapy.

Microscopy
On days 0 and 3, thick and thin smears were prepared for detection and speciation of Plasmodium falciparum infection, respectively.The blood smears were stained with 10% Giemsa stain for 30 minutes, then examined using an Olympus microscope under 100X magnification with oil immersion to identify the parasite and determine the parasite density.Two independent microscopists/laboratory technologists examined the slides for P. falciparum confirmation and enumeration.

DNA extraction
The parasite DNA was extracted using a Qiagen DNA mini kit (QIAamp DNA Blood Mini Kit-250, Germany), following the manufacturer's instructions.Elution was performed in 50 μL elution buffer, and the DNA concentration was determined (25-40 ng/μl) using NanoDrop (NanoDrope Lite Plus, thermo scientific) before PCR amplification.

Plasmodium falciparum qPCR
Quantitative PCR (qPCR) was performed using a real-time PCR system (QuantStudio TM 5) to detect and quantify the Plasmodium falciparum in duplicate.Primers and probes were based on the 18S rRNA sequence [27] synthesised from Inqaba Biotec Laboratory, South Africa.The amplification followed a method in a previous study [28].The CT values were recorded and used to calculate parasite clearance through fold change, 2 −ΔΔCT by a comparative CT value method [29].The method considered two data points of CT values at day 0 and day 3 for individuals that remained positive on day 3.
Specific primers were used for the qPCR (Table 1); the probe was labelled with 5 0 FAM (6-carboxyfluorescein) and BHQ-1 TM (Black Hole Quencher) as the reporter and quencher, respectively.20 μl reaction volume was prepared, composed of 0.8 μl of each forward and reverse primer, 10 μl of qPCR master mix (Luna Universal Probe qPCR Master Mix NEB, New England BioLabs Inc), 0.4 μl of the probes, 20 ng of the DNA template and nuclease-free water (variable).The PCR conditions involved one cycle at 94˚C for 5 min of initial denaturation, followed by 40 cycles of denaturation at 94˚C for 30 s, annealing at 54˚C for 90 s and extension at 68˚C for 90 s, and a final extension at 68˚C for 90 s.

P. falciparum K13 allele
Conventional nested PCR was performed (SimpliAmp Thermal Cycler, applied biosystem) to amplify approximately 826 bp fragment that extend from nucleotides position 1329-to-2178 (codons 445-to-727) within the propeller domain of the K13 gene using the specific primers [16] Table 1.

P. falciparum K13 gene amplification
The first round PCR was performed in a 25 μl reaction volume of 12.5 μl OneTaq 2X Master Mix (NEB, New England BioLabs Inc), 1 μl of each forward and reverse primer and 2.5 ng of the DNA template and nuclease-free water (variable).The PCR was run with the following conditions: 94˚C for 5 m initial denaturation, 40 cycles at 94˚C for 30 s, annealing at 54˚C for 90 s, and extension at 68˚C for 90 s.The final extension was performed at 68˚C for 10 minutes, then kept at 4˚C, which was set to infinite.The amplicon from the first round of PCR was used

PCR Primers Sequence
Nested PCR K131/K13_PCR-F https://doi.org/10.1371/journal.pone.0305064.t001 as a template for the second round.This was run in a 25 μL reaction mixture volume; it involved the same master mix preparation as the first run, but the second primer sets were used with 1 μl of the first PCR product/amplicon.The PCR product was visualised using gel electrophoresis run at 100 V for 30 minutes in 1.5% agarose stained with 0.05 μg/mL Acridine orange (SafeView Classic).

Amplicon preparation and cleaning
The nested PCR products were cleaned using ExoSAP.The Exo/SAP master mix was prepared by adding 50 μL of exonuclease I (Catalogue No. NEB M0293) 20 U/μl and 200 μl of shrimp alkaline phosphatase (Catalogue No. NEB M0371) 1 U/μl to a 0.6 ml microcentrifuge tube.Then, 2.5 μl of the ExoSAP mixture was mixed with 10 μL of the amplicon/PCR product and incubated at 37˚C for 15 min; the reaction was stopped by heating the mixture at 80˚C for 15 min.

Plasmodium falciparum K13 Sequencing
The nested PCR products were sequenced using the Nimagen BrilliantDye TM Terminator Cycle Sequencing kit V3.1, BRD3-100/1000, according to the manufacturer's instructions at the Inqaba Biotec laboratory in South Africa, a commercial sequencing company.The labelled product was then cleaned with a ZR-96 DNA sequencing clean-up Kit (Catalogue No. D4053).
The cleaned product was injected into an Applied Biosystems ABI 3730XL Genetic Analyser with a 50 cm array using POP7.Sequence chromatogram analysis was performed using FinchTV analysis software.

Data management and analysis
The K13 sequence qualities were assessed, and then BLAST against the NCBI database to confirm the identity of the gene sequence.After exporting the sequence into the Bioedit sequence alignment editor, manual editing was performed, and the sequence was exported into MEGA11 software for polymorphism detection using the sequence PF3D7_1343700 from the NCBI database as the reference sequence.Sequence alignment was performed using the Clus-talW algorithm, and a phylogenetic tree was drawn using MEGA11 Tree Explorer to identify divergence in the P. falciparum K13 genes and identify the mutations.Data were entered in Excel and analysed in STATA version 17; CT value fold change from the qPCR data was logtransformed before correlation.The relationship between the CT value fold change related to parasite clearance and K13 mutation was determined through a nonparametric test (Mann-Whitney U test) with a 95% confidence interval and level of significance set at p<0.05.

Characteristics of the study participants
The study included 100 symptomatic uncomplicated malaria patients, of which the majority, 65% (65/100), were females.The mean age of the study participants was 17±10 years, ranging from 5 to 46 years.Parasitemia/μl geometric mean by microscopy was 3868, IQR 11104 for all patients.

Discussion
Artemether-lumefantrine (AL), the first-line treatment for uncomplicated malaria in Uganda, at the current standard fixed weight-based dose regimen, has been pivotal in reducing malaria- related morbidity and mortality.However, emerging P. falciparum K13 mutations associated with reduced P. falciparum susceptibility to AL threaten malaria control and elimination efforts in sub-Saharan Africa.Reduced susceptibility to AL that may be characterised by the persistence of the parasite on day 3 could be associated with partial artemisinin resistance.We observed a high prevalence of P. falciparum parasites by microscopy, 24% and quantitative polymerase chain reaction (qPCR), 63%, after three days of AL treatment.The day 3 microscopy positivity aligns with previous studies that have reported similar persistence of parasites beyond the standard three-day treatment period in Africa and elsewhere [30][31][32][33].Equally, our qPCR results that indicated a prevalence of 63% are consistent with a study by Tadele et al. [28], which reported a 60% PCR prevalence of day 3 P. falciparum parasites among patients treated with AL, which may not be sufficient to indicate the development of partial artemisinin resistance.However, the 24% prevalence by microscopy could be a pointer to the potential emergence of partial P. falciparum resistance against AL, the current cornerstone of malaria treatment.WHO advises that a day 3 parasitemia in patients on ACT treatment calls for routine monitoring to identify suspected artemisinin partial resistance in P. falciparum [34].
The persistence of P. falciparum parasites on day 3 raises a concern regarding the effectiveness of artemether and the efficacy of the AL regimen.A similar finding on the persistence of P. falciparum has been reported previously in AL-treated malaria patients in Uganda [30,[35][36][37].Whether the persistence is attributed to the development of resistance, especially the K13 mutation, or lack of adherence to AL has not been fully established.However, a previous study in Uganda has demonstrated moderate to high adherence to AL in treating uncomplicated malaria, whether taken under supervision or not [38].Day 3 positivity and residual P. falciparum are likely to cause an increase in malaria recurrence and transmission to female Anopheles mosquitoes [39], driving infection to people and jeopardising the gain in malaria control.
The study identified nonsynonymous mutations at codons 469, 675, 569, 578 and 491 of the K13 propeller gene.This mutation pattern is consistent with recent findings in Uganda [20,37,[40][41][42].The codons 469 and 675 mutations have been associated with artemisinin resistance in northern Uganda [19,21].This is consistent with our finding that most patients with C469Y-mutated P. falciparum had parasites that persisted after three days of AL treatment.Among the K13 mutations identified, patients carrying parasites with C469Y (5/10) and A578S (1/1) mutations remained microscopy-positive on day 3; similarly, qPCR results showed high day 3 positivity (8/10) for the C469Y.Unlike a study by Stoke et al. [43] that did not link the C469Y mutation with parasite resistance or delay in parasite clearance, in this study, C469Y was associated with low parasite fold reduction 14, 792 (median, IQR), compared to the wild-type, an indication of reduced P. falciparum clearance.This is consistent with the findings of Balikagala et al. and Tumwebaze et al. [19,21], which demonstrated delayed parasite clearance and resistance.A similar study in Pakistan reported a high association between C469Y and artemisinin resistance [44].Among the patients with P. falciparum that remained positive on day 3, we identified one patient (1/1) with A578S mutated parasite having a low parasite fold reduction of 1.6.The A578S mutation has been sporadically reported in sub-Saharan African countries [20,[45][46][47], and a study in Uganda reported some association between A578S and slow parasite clearance [48], contrary to studies elsewhere that could not associate it with artemisinin resistance [49,50].
The prevalence of mutation at codon 675 in the malaria patients was 2.5%, and none of the patients with parasites having the A675V mutation remained P. falciparum positive on day 3.Although the A675V mutation was previously associated with resistance in Northern Uganda [19], our current study could not link the few to the persistence of P. falciparum on day 3, possibly due to the low prevalence and likelihood of chance finding that they are not positive on day 3.Although the A675V mutation is a candidate mutation for artemisinin resistance, a study by Tumwebaze et al. and Stoke et al. [21,43] reported high parasite clearance among parasites with the A675V mutations.We also identified a mutation at codon position 491, which had previously not been identified anywhere.This finding shows the existence of a reservoir for K13 propeller gene polymorphisms, not only in Uganda but also globally in malariaendemic places [51,52].The diversity of K13 propeller gene mutations across regions further emphasises the need for continued surveillance and research to understand their implications in artemisinin resistance.
Our study had some limitations.Firstly, parasites detected on day 3 were not confirmed whether they were the same parasite on day 0 or a newly acquired infection.Secondly, we did not confirm adherence to treatment among malaria patients who participated in the study.Third, the parasite load on day 0 was not the same across all the patients; low and high P. falciparum infections were all considered; the study considered P. falciparum positive patients with no specific parasite load and as long as they were prescribed AL.Fourth, the study used two data points of day 0 and day 3 to determine the parasite clearance through fold changes; this makes the data less likely to precisely establish the linear correlation of the parasite clearance slope and unable to capture the fold change pattern of those who are negative on day 3. Lastly, the clinical outcome of the participants was not evaluated according to the WHO 28-day recommendation for an efficacy study.However, the limitations were unlikely to significantly affect the study's outcome in determining artemisinin effectiveness and identifying markers of artemisinin resistance.Future studies should consider using multiple data points when using the CT value method to assess parasite clearance in effectiveness and efficacy studies of artemisinin.

Conclusion
The day 3 prevalence of 24% P. falciparum by microscopy in the AL-treated patients could indicate the presence of partial resistance to artemisinin.There is an increase in the prevalence of K13 mutation associated with resistance, especially the C469Y, which is associated with a low-fold change in parasite reduction.There is a need to re-evaluate the effectiveness of artemisinin agents in malaria treatment and ensure continuous monitoring of their efficacy in Uganda, and alternative treatment approaches should be intensified.

Table 3 . Prevalence of K13 single-nucleotide polymorphisms in P. falciparum parasites among patients in Adjumani district. Mutations Wild-type amino acid Mutant amino acid Codon position Wild-type nucleotide Mutant nucleotide Mutation type
a Previously reported K13 allele ab Candidate for artemisinin resistance https://doi.org/10.1371/journal.pone.0305064.t003