Molecular Characterisation of Plasmodium falciparum in Children with Uncomplicated Malaria in Homa-Bay, Kenya; Two Decades Post-Adoption of Artemisinin-Based Combination Therapies

Molecular Characterisation of Plasmodium falciparum in Children with Uncomplicated Malaria in Homa-Bay, Kenya; Two Decades Post-Adoption of Artemisinin-Based Combination Therapies | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Molecular Characterisation of Plasmodium falciparum in Children with Uncomplicated Malaria in Homa-Bay, Kenya; Two Decades Post-Adoption of Artemisinin-Based Combination Therapies Florence T. Akinyi, Joyce B Oluokun, Kabir Gorden, John Openibo, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6897552/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract BACKGROUND The emergence and spread of P. falciparum parasites with decreased susceptibility to Artemisinin-based combination therapies (ACTs) is causing global concern. Active surveillance of the emergence of resistance in malaria-endemic areas is important for efficient management of the infection. Slow parasite clearance following treatment with artemisinin derivatives is associated with single-nucleotide polymorphism in the propeller domain of the Plasmodium falciparum kelch13 ( pfk13 ) gene. This study investigated the polymorphism in the Pfk 13 gene and parasite population diversity of Plasmodium falciparum in children with uncomplicated malaria in Homa-Bay County, Kenya, two decades post-adoption of Artemisinin-based combination treatment. METHODS This study assessed polymorphisms on the pfk13 gene and parasite population diversity in 86 PCR-confirmed Plasmodium falciparum positive samples obtained from children between six months and fifteen years old. The parasite diversity was determined by nested PCR amplification of msp1 and msp2 genes and direct PCR amplification of the polyα microsatellite locus followed by capillary electrophoresis. Sanger sequencing was carried out on samples with successful amplification of the Pfk13 gene to determine polymorphisms. The sequenced data were analyzed by Geneious Prime software (version 2024.0.7). RESULTS The study did not report validated or candidate mutations as classified by WHO. However, the previously described mutations P667S and P667L were detected on Pfk13 gene. Additionally, this study reported polyclonal infection in 86% of the samples and a parasite diversity of 0.91. CONCLUSION In conclusion, this study demonstrated the circulation of P. falciparum parasites with mutant alleles on the kelch13 gene in Homa-Bay County, Kenya. The study also showed a high frequency of polyclonal infections and high parasite genetic diversity, suggesting a high transmission rate. The high polyclonal infection and diversity show that malaria transmission is still high in this region; hence, the control measures need to be heightened. The observed polymorphisms on Pfk13 need close monitoring, especially since the P667S mutation has been reported to cause artemisinin resistance in Rwanda, and the effect of mutation P667L is yet to be known. Malaria Resistance kelch13 Kenya Transmission Polymorphisms Figures Figure 1 INTRODUCTION Malaria is a significant public health concern with high morbidity and mortality in Sub-Saharan Africa. In 2022, 249 million cases and 608,000 deaths were reported globally. Kenya recorded approximately 3.5 million malaria cases and 1.4% of global deaths reported in 2022 (WHO, 2023). Chemotherapy is one of the control measures for this disease. The recommended first-line treatment for uncomplicated malaria by the WHO is the artemisinin-based combination therapies (ACTs), which combine an artemisinin derivative with a long-acting partner drug. It was introduced following parasite resistance to chloroquine and sulfadoxine-pyrimethamine (Ecker et al., 2012 ; Artimovich et al., 2015 ). Artemether/Lumefantrine was recommended as the first-line treatment for uncomplicated malaria in Kenya in 2002. However, the actual implementation started in 2006 (Lawford et al., 2011 ). Since then, the drug has been effective, but the emergence of Plasmodium falciparum parasites with decreased susceptibility to artemisinin threatens its efficacy. Artemisinin resistance arose in Southeast Asia's Greater Mekong Subregion (GMS) and has spread to other malaria-endemic regions like Eastern Africa (Noedl et al., 2008 ; Dondorp et al ., 2009). Four Eastern African countries, namely Rwanda, Uganda, the United Republic of Tanzania, and Eritrea have been declared by WHO to have partial artemisinin resistance (WHO, 2023; Straimer et al ., 2021; Conrad et al., 2023 ; Ishengoma et al., 2024 ; Rosenthal et al., 2024 ). This is alarming since there is a possibility of resistance in Kenya due to the proximity and frequent border movements between the countries. The slow parasite clearance is associated with single nucleotide polymorphism (SNP) in the propeller domain of the pfk13 gene (Shafik et al., 2022 ). Mutations on codons F446I, N458Y, C469Y, M476I, Y493H, R539T, I543T, P553L, R561H, P574L, R622I, A675V and C580Y are validated markers for artemisinin resistance (WHO, 2022). Periodic surveillance of the resistance markers is needed to effectively track the emergence of resistance. The Plasmodium falciparum genetic diversity influences the spread and maintenance of resistant strains. Multiple parasite genotypes in polyclonal infections increase the frequency of spread of mutations associated with drug resistance compared to monoclonal infections, as they have more infective parasites. Additionally, the drug may eliminate the sensitive strains in polyclonal infections, but the resistant strains remain and proliferate (Roh et al., 2019 ; Oyedeji et al., 2020 ). The parasite genetic diversity can be determined by genotyping the polymorphic regions of antigenic markers like merozoite surface protein 1 (block 2), merozoite surface protein 2 (block 3), and glutamate-rich protein and the neutral microsatellite loci (Ajogbasile et al., 2021 ). However, WHO recently recommended genotyping using msp1 and msp2 genes with one or two highly polymorphic microsatellites. The latter can be poly alpha (poly-α), TA1 , or Plasmodium falciparum protein kinase 2 ( PfK2 ) (WHO,2021). Despite WHO recommendations for active surveillance of the artemisinin resistance markers to effectively track the resistance, only a small number of molecular epidemiological surveillance of these markers are available in Homa-Bay County. A study on asymptomatic and low-density Plasmodium falciparum infections in Ngodhe Island in Homa-Bay County did not report any mutation in the Pfk13 gene related to reduced susceptibility to artemisinin. However, different variants on the Pfmdr1 gene were reported to be associated with reduced susceptibility to the ACT partner drug lumefantrine (Osborne et al., 2023 ). Homa-Bay County is part of the Lake endemic region with the highest malaria prevalence and stable transmission throughout the year. Periodic surveillance of the resistant markers is essential (Maniga et al., 2023 ). Therefore, this study investigated the polymorphism in the Pfk 13 gene and parasite population diversity of Plasmodium falciparum in children with uncomplicated malaria in Homa-Bay County, Kenya, two decades post-adoption of Artemisinin-based combination treatment. The outcome of this study informs malaria control policy in this county. In this study, the Pfk13 mutations, P667S, P667L, and T478T, were detected. MATERIALS AND METHODS Study Site The study was a cross-sectional study conducted among children who presented at Homa-Bay County Teaching and Referral Hospital (HBCTRH), Kenya with common symptoms of malaria between October 2023 and January 2024. The County has a population of 1,131,950 and occupies a landmass of 3,154.7 km 2 , Latitude: 0° 40' 60.00"N and Longitude: 34° 27' 0.00" E. It forms part of the lake endemic regions with Kenya's highest malaria prevalence and transmission (Maniga et al ., 2022). Study Population The study population was children aged six (6) months to 15 years who presented with common symptoms of uncomplicated malaria at HBCTRH. Inclusion and Exclusion criteria The inclusion criteria for blood collection included symptoms of uncomplicated malaria, such as fever (temperature > 37°C) in children of six (6) months to 15 years old, absence of concomitant infection, and willingness to participate in the study. Participants diagnosed with severe malaria, participants above 15 years old, those with concomitant infections, and those unwilling to provide informed consent were excluded from the study Sample Collection Demographic information was collected from 150 participants recruited into the study. From each participant, 2 mL of venous blood was collected into EDTA tubes for malaria diagnosis using thick blood smears. Additionally, dried blood spots (DBS) were prepared from the EDTA blood of 100 participants that tested positive for malaria. The blood was spotted onto Whatman filter paper (Cytiva, Marlborough, United States), air-dried for approximately 3 hours, and stored in zip-lock plastic bags containing desiccant at room temperature until laboratory analysis. Extraction of Parasite Genomic DNA from the Dried Blood Spot A QIAmp DNA mini kit (QIAGEN, Hilden, Germany) was used to extract the parasite genomic DNA from the DBS filter paper according to the manufacturer’s instructions. DNA was stored at -20 o C until further use. PCR confirmation of Plasmodium falciparum infection The Plasmodium species in each sample were determined using nested PCR amplification of the 18S rRNA gene adapted from Singh et al . (1999). Both primary and secondary PCR reactions were carried out in 20 µL total volume consisting of 4 µL master mix 5X FIREPol® Master Mix (Solis BioDyne, Tartu, Estonia), 1.0 µM forward and reverse primers each, and 2 µL of template DNA for the primary PCR. One microliter of primary PCR product was used as template for the secondary PCR. Details of the primer sequence and cycling conditions are shown in Supplemental Table 1. Genomic DNA from Plasmodium falciparum strain 3D7 was used as a positive control. PCR amplification of msp1, msp2 genes, and Poly alpha microsatellite locus Samples positive for P. falciparum by PCR were selected for msp1 and msp2 genes and Poly alpha microsatellite locus analysis. Nested PCR amplification of msp1 and msp2 genes was performed using non-family-specific primer pairs corresponding to conserved sequences spanning the polymorphic regions of each antigenic marker previously described by Segun et al. ( 2021 ). PCR amplification of msp1 and msp2 gene was carried out in a 20 µL total reaction volume using 4 µL master mix 5X FIREPol® Master Mix (Solis BioDyne, Tartu, Estonia),1 µM of forward and reverse primers, and 4 µL DNA template for primary reaction, and 2 µL DNA template for the secondary reaction using cycling conditions and primer sequences specified in Supplemental Table 2. The PCR amplicons were further resolved on 2% gel electrophoresis to confirm the amplification and determine the number of clones per sample. A direct PCR, according to CDC protocol, was used to amplify the poly alpha microsatellite locus. The 15 µL reaction mixture consisting of 2 µL genomic DNA, 0.6 µL of 10 µM primers, and 7.5 µL 2XPlatinum hot start master mix (Invitrogen, Carlsbad, California) was used (CDC, 2021). Details of primer sequence and cycling conditions are shown in Supplemental Table 2. Genomic DNA 3D7 clone was used as the positive control. The amplicons were further analyzed using capillary electrophoresis. The fragment analysis was performed using the Genetic Analyzer 3500XL series (Applied Biosystems). PCR amplification of the Pfk13 gene and targeted sequencing Nested PCR was used to amplify the propeller domain of the Pfk13 gene in samples successfully amplified for msp1 and msp2 genes to determine SNPs present in the parasites according to the method previously described (Ajogbasile et al., 2021 ). In the primary amplification, a 20 µL reaction mixture consisting of 1.5 µL genomic DNA, 1 µM of each primer, and 4 µL 5X FIREPol® Master Mix (Solis BioDyne, Tartu, Estonia). For the secondary amplification, 1 µL of the primary amplicon was utilized as the template, and each reaction contained 1 µM primers. The specific primers and cycling conditions are in Supplemental Table 3. The amplification of the Pfk13 gene was confirmed by 2% gel electrophoresis. The amplicons from PCR positive reaction (64 samples) were sequenced by the Sanger method using an Applied Biosystems 3500 XL series Genetic Analyser at ACEGID, Redeemer’s University, Ede, Osun State, Nigeria Data analysis Data were double entered serially using patients’ codes and analyzed using IBM SPSS (version 20.01) and Microsoft Excel. Crosstab descriptive statistics was carried out using SPSS, while Excel was used to draw graphs. The parasite diversity analysis of msp1 and msp2 was determined by counting the number of bands on the gel electrogram and for Poly alpha; GeneMapper version 6 software was used for normalization across runs and automatic determination of allele length and peak heights in samples containing multiple alleles per locus. The highest peak was identified as the predominant allele. Multiple alleles at a given locus were considered if minor peaks observed were ≥ 20% of the height of the predominant allele. A peak was considered an allele if it had a size of 114–201 and a difference of 5bps from the nearby peak. The results were either monoclonal (having one clone per sample) or polyclonal (more than one clone per sample). Expected heterozygosity ( H e ), defined as the probability that two randomly selected clones from a population will carry distinct alleles at the polyα locus, was used as a measure of genetic diversity using a formula, H e = [n/ (n − 1)] [1 − Σp i 2 ], where n and p i represent the number of isolates analysed and frequency of the i th allele in the population. Expected heterozygosity ( H e ) values range between 0 and 1 for low and high genetic diversity, respectively. To identify the polymorphism in the propeller domain of the Pfk13 gene, Sequencing data was analysed using Geneious Prime software (version 2024.0.7). Forward and reverse nucleotide sequences were imported into the software and aligned using Geneious alignment. Consensus sequences were generated for all the samples. The consensus sequence was mapped against the reference nucleotide sequence of the Pfk13 gene obtained from the PlasmoDB database (PF3D7_1343700 sequence region spanning region 1,724,817–1,726,997 bp of chromosome 13). Sequences with nucleotide change were translated into amino acids to determine the type of mutation. RESULTS Demographic characteristics A total of 100 participants were screened for malaria using thick Giemsa-stained microscopy. The female and male distribution was 56% (56/100) and 44% (44/100) respectively. The median age of the children was 5 years (Range: 0.5–15 years) with 46% (46/100) of all participants being aged < 5 years. Prevalence of P. falciparum infection among study participants All 100 samples positive for malaria by microscopy were included in a P. falciparum confirmatory PCR test, and 86% (86/100) were confirmed positive for P. falciparum infection, and 14% (14/100) were negative for P. falciparum infection. (Supplemental Fig. 1) Plasmodium falciparum population structure Plasmodium falciparum speciation positive samples (86) were analysed by nested PCR to determine the parasite population structure. Out of these, 93.0% (80/86) were successfully amplified for msp1 gene producing amplicons of between 400–1000bp DNA fragment sizes on 2% agarose gel electrophoresis. The msp2 gene was successfully amplified in 90.7% (78/86) samples producing amplicons between 200–500 bp DNA fragment sizes and 100% (86/86) amplified for poly alpha with peak sizes ranging from 129bp-183bp by capillary electrophoresis (Supplemental Fig. 2,3,4). The results were either monoclonal or polyclonal. Monoclonal and polyclonal infections were detected in 16.3% (14/86) and 76.7% (66/86) of the isolates, respectively, for the msp1 gene. All the polyclonal infections for msp 1 gene had 2 clones per sample. For the msp2 gene, 38.4% (33/86) and 52.3% (45/86) of samples had monoclonal and polyclonal infections, respectively. However, 26.7% (23/86) had a monoclonal infection (defined by a single peak in a sample), and 73.3% (63/86) had a polyclonal infection (defined by two or more peaks) for poly alpha (Fig. 1 ). The polyclonal infections in msp2 and poly alpha had clones ranging from two to five per sample, with two clones having the highest frequency for both msp2 (73.3%) and poly alpha (38.1%). The overall population of polyclonal infection was 86%. The expected heterozygosity (He) was 0.91. Polymorphisms on the Pfk13 gene in the isolates Nested PCR was successful in 64 isolates (Supplemental Fig. 5) and were included in Sanger sequencing to detect polymorphisms on the PfK13 gene. A snapshot of the Sanger chromatogram indicating a nucleotide change is shown in (Supplemental Figs. 6&7). No WHO-validated or candidate resistance markers were detected in this study. However, two non-synonymous and one synonymous mutation were detected in the 4/64 DNA sequences sequenced (Table 1 ). The mutations P667S (1), P667L (2), and T478T (1) were detected in this study. The prevalence of parasites with mutations on the Pfk13 gene in this study was 6.25% (4/64), while 93.75% (60/64) did not have any mutations when compared to the Pfk13 sequence of 3D7 downloaded from PlasmoDB (Fig. 2 ). The mutation P667L occurred in the same nucleotide position (2000) in the 2 samples with this mutation Table 1 Detailed profile of the Pfk13 polymorphisms detected among the study population S/N K13 amino acid locus Age of patients (Yrs) Number of samples with the mutation Nucleotide Locus Reference allele Mutant Allele Type of mutation Countries previously observed in 1 P667S 5 1 2905 C T Non-synonymous Rwanda (Straimer et al ., 2021), Kenya (Kakamega County) (Jeang et al., 2024 ) 2 P667L 2 2 2000 C T Non-synonymous Southeast Asia, South Asia (MalariaGEN Plasmodium falciparum Community Project, 2016 ) 3 T478T 2 1 2314 C G Synonymous Kenya, Côte d'Ivoire (Kamau et al ., 2014), Senegal (Gaye et al ., 2020) DISCUSSION Artemisinin-based combination therapies (ACTs) are the first-line treatment for uncomplicated malaria. However, polymorphisms in the Pfk13 gene are associated with slow parasite clearance after treatment with ACTs. This study investigated the prevalence of single nucleotide polymorphism in the Pfk 13 gene and parasite population diversity using the msp1 and msp2 genes and the poly alpha microsatellite. Although all samples were positive for P. falciparum by microscopy, PCR did not confirm P. falciparum in 14 samples. This may be due to human error in the microscopy, as the PCR is expected to be more sensitive (Wu et al., 2015 ). It is also possible that it was non- falciparum malaria; species differentiation is difficult in thick blood films and will require a higher level of microscopy expertise (Fitri et al .,2022). The malaria infections in this study were shown to be 86% polyclonal infection. This aligns with the study by Gatei et al. ( 2015 ), which reported an overall polyclonal infection of ≥ 80% in Siaya County, Kenya. A study carried out on asymptomatic children in Homa-Bay County reported a prevalence of 79.69% polyclonal infection (Touray et al., 2020 ). The polyclonality could be due to high transmission intensity in this region; individuals are more likely to be bitten by multiple infected mosquitoes carrying different parasite strains (Agaba et al ., 2021). Another possible explanation could be high asymptomatic infections, which harbor multiple parasite strains without showing symptoms and are hence reservoir and contributing to the spread of diverse strains. Since this is a malaria-endemic region, frequent Plasmodium exposures lead to strong immune modulation, reducing clinical symptoms (Omondi et al ., 2022; Mwesigwa et al ., 2024). High P. falciparum genetic diversity has remained unchanged in Kenya even after introducing the ACTs in the country (Chebon et al., 2016 ; Nderu et al ., 2019). In this study, a high genetic diversity of 0.91 was reported. This is consistent with other studies in the country (Gatei et al., 2015 ; Ingasia et al., 2016 ; Mulenge et al., 2016 ; Kimenyi et al., 2022 ). The high P. falciparum genetic diversity reported here could be due to high levels of transmission leading to extensive gene flow and recombination (Maniga et al ., 2022; Mulenge et al., 2016 ). This study reported two non-synonymous mutations (P667L and P667S) and one synonymous mutation (T478T) on the Pfk13 gene. None of the WHO-validated or candidate markers for partial artemisinin resistance were reported in this study. However, the non-synonymous mutation P667S reported in this study has earlier been reported in Rwanda to cause a delayed parasite clearance time of ˃5hours and positive parasitemia count on a smear on the third day of treatment, meeting the criteria for clinical artemisinin resistance (Straimer et al ., 2021; WHO, 2022). Interestingly, the same mutation has been reported in Kakamega County, Kenya, during the passive surveillance and epidemiological studies on Pfk13 polymorphism between 2018 and 2022 in Western Kenya (Homa-Bay, Kisumu, Kisii, and Kakamega Counties) (Jeang et al., 2024 ). The mutation may be due to geographical migration, considering the daily interactions between these two counties (Homa-Bay and Kakamega) (Jeang et al., 2024 ). Although this mutation is yet to be implicated in artemisinin resistance in Kenya, there is a need for continuous surveillance of the mutation, considering its proximity to Rwanda, the effect of this mutation may be the same in Kenya. The non-synonymous mutation P667L in two samples (3.2%) reported in this study had earlier been reported in Southeast and South Asia in a study conducted by the MalariaGEN Plasmodium falciparum Community Project ( 2016 ), where the frequency of 2/ 1599 and 1/75 samples were reported, respectively, suggesting a less frequent mutation. Although the role this mutation plays in resistance to artemisinin is still unknown; it is important to monitor this mutation as it might have a different effect in the two continents due to different parasite genetic backgrounds. Most non-synonymous African mutations similar to those in Southeast Asia have arisen independently (Ariey et al., 2014 ). Therefore, the mutation in the current study might not be related to the one reported in Southeast Asia. It is also possible that this mutation may be due to geographical migration, considering the pattern with chloroquine resistance, where resistance emerged in Southeast Asia and spread into Africa through Kenya (Peters, 1970 ; Borrmann et al., 2011 ; Beshir et al., 2013 ; Ashley et al., 2014 ; Miotto et al., 2015 ). To the best of our knowledge, this is the first report on the non-synonymous mutation P667L in Africa. All the mutations reported in this study occurred in participants of five years old and below. This should be monitored since mutations sometimes lead to drug resistance or affect parasite fitness (Behrens et al., 2024 ). Children below five years of age are the population at most risk of malaria due to a lack of well-developed immunity; hence, they may not be able to clear the parasite in case of high parasite fitness cost, as opposed to children above five with better-developed immunity to malaria following several infections. Thus, children below the age of 5 years may aid in transmitting the mutant alleles as they experience more clinical episodes of malaria infection (Umaru and Uyaiabasi, 2015 ). Additionally, all the non-synonymous mutations in this study occurred in polyclonal infections. This could have negative or positive implications in the fight against malaria. Polyclonal infection has more infective parasites, which can aid in transmitting the mutant alleles (Oyedeji et al., 2020 ). It is also possible that drug-susceptible parasites may exist in parallel to mutant parasites - (potentially resistant); hence, ACT may remain active as the mutant parasites circulates within the environment. Although the efficacy of the ACT used for treatment was not determined in the enrolled children, the data provided in this study are more of epidemiological surveillance of the malaria parasite drug resistant markers and the genetic diversity for insight into the circulating parasite and transmission pattern. In addition, the non-family specific msp1/2 genes were used for the parasite diversity hence the most circulating alleles could not be determined. However, the diversity was equally determined, and the implications of more polyclonal infection was clearly observed, hence the need for continuous surveillance and monitoring of circulating parasite. Conclusion In conclusion, this study reports polymorphism in the Pfk13 gene of P. falciparum parasites isolated from children in Homa-Bay County, Kenya. The study also showed a high frequency of polyclonal infections and high parasite genetic diversity, indicating a high regional transmission rate. Active monitoring of the mutant alleles in the Pfk13 gene and heightened malaria control strategies are recommended. Declarations Ethical Approval and consent to participate This study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of Redeemer’s University Ethics Committee (RUN/REC/2023/101), Homa-Bay County Ministry of Health (MOH/RA/VOL.VI (021)), and the research permit obtained from the National Commission for Science, Technology & Innovation (NACOSTI/P/23/31605). Written informed consent was obtained from parents/guardians for children before enrolment into this study, and child assent was obtained from children between 12 and 15 years. Acknowledgment We thank the parents/guardians of the children and the children for participating in the study. We also thank the medical officers at the recruiting facility for assisting with patient recruitment and sample collection. Our gratitude goes to the Homa-Bay County health office for allowing us to collect the samples. Finally, we are grateful to Institute of Genomics and Global health (formerly ACEGID), Redeemer’s University, Nigeria in the ACE Impact project for funding this project. Availability of data and materials: All raw data is available as supplementary materials. Authors contribution: Florence T. Akinyi: Study design, Laboratory analysis, Bioinformatics and Data Analysis, Manuscript Drafting, and Review. Oluokun B. Joyce: Laboratory analysis and Manuscript Review Kabir Gorden: Data analysis and Manuscript Review Gouton D. Clemence: Laboratory analysis and manuscript review Openibo John: Laboratory analysis and Manuscript Review Oloo O. Edwin: Sample Collection Mitesser Vera: Bioinformatics analysis and Manuscript Review Happi T. Christian: Funding acquisition, lab Resources and Manuscript review Folarin A. Onikepe: Conceptualization and Study Design, Proposal Review, coordinating logistics for Lab work, Project administration, Resources, Supervision, Validation, Visualization, Manuscript review & editing and Mentorship. All authors read and approved the manuscript. Funding : This research was funded by the World Bank ACE IMPACT grant (worldbank.org) (ACE-019 to CTH). FTA was funded through the ACE IMPACT graduate fellowship for MSc. Potential conflicts of interest: All authors declare no conflict. References Ajogbasile FV, Kayode AT, Oluniyi PE, Akano KO, Uwanibe JN, Adegboyega BB, Philip C, John OG, Eromon PJ, Emechebe G, Finimo F. Genetic diversity and population structure of Plasmodium falciparum in Nigeria: insights from microsatellite loci analysis. Malar J. 2021;20(1):1–9. Ariey F, Witkowski B, Amaratunga C, Beghain J, Langlois AC, Khim N, Kim S, Duru V, Bouchier C, Ma L, Lim P. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature. 2014;505(7481):50–5. Artimovich E, Schneider K, Taylor TE, Kublin JG, Dzinjalamala FK, Escalante AA, Plowe CV, Laufer MK, Takala-Harrison S. Persistence of sulfadoxine-pyrimethamine resistance despite reduction of drug pressure in Malawi. J Infect Dis. 2015;212(5):694–701. Ashley EA, Dhorda M, Fairhurst RM, Amaratunga C, Lim P, Suon S, Sreng S, Anderson JM, Mao S, Sam B, Sopha C. Spread of artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med. 2014;371(5):411–23. Behrens HM, Schmidt S, Henshall IG, López-Barona P, Peigney D, Sabitzki R, May J, Maïga-Ascofaré O, Spielmann T. Impact of different mutations on Kelch13 protein levels, ART resistance, and fitness cost in Plasmodium falciparum parasites. Mbio. 2024;6(15):1–25. Beshir KB, Sutherland CJ, Sawa P, Drakeley CJ, Okell L, Mweresa CK, Omar SA, Shekalaghe SA, Kaur H, Ndaro A, Chilongola J. Residual Plasmodium falciparum parasitemia in Kenyan children after artemisinin-combination therapy is associated with increased transmission to mosquitoes and parasite recurrence. J Infect Dis. 2013;208(12):2017–24. Borrmann S, Sasi P, Mwai L, Bashraheil M, Abdallah A, Muriithi S, Frühauf H, Schaub B, Pfeil J, Peshu J, Hanpithakpong W. Declining responsiveness of Plasmodium falciparum infections to artemisinin-based combination treatments on the Kenyan coast. PLoS ONE. 2011;6(11):1–10. Chebon LJ, Ngalah BS, Ingasia LA, Juma DW, Muiruri P, Cheruiyot J, Opot B, Mbuba E, Imbuga M, Akala HM, Bulimo W. Genetically determined response to artemisinin treatment in western Kenyan Plasmodium falciparum parasites. PLoS ONE. 2016;11(9):1–19. Cochran WG. Sampling techniques. Wiley; 1977. Conrad MD, Asua V, Garg S, Giesbrecht D, Niaré K, Smith S, Namuganga JF, Katairo T, Legac J, Crudale RM, Tumwebaze PK. Evolution of partial resistance to artemisinins in malaria parasites in Uganda. N Engl J Med. 2023;389(8):722–32. Dondorp AM, Yeung S, White L, Nguon C, Day NP, Socheat D, Von Seidlein L. Artemisinin resistance: current status and scenarios for containment. Nat Rev Microbiol. 2010;8(4):272–80. Ecker A, Lehane AM, Clain J, Fidock DA. PfCRT and its role in antimalarial drug resistance. Trends Parasitol. 2012;28(11):504–14. Fitri LE, Widaningrum T, Endharti AT, Prabowo MH, Winaris N, Nugraha RYB. Malaria diagnostic update: From conventional to advanced method. J Clin Lab Anal. 2022;36(4):1–14. Gatei W, Gimnig JE, Hawley W, Ter Kuile F, Odero C, Iriemenam NC, Shah MP, Howard PP, Omosun YO, Terlouw DJ, Nahlen B. Genetic diversity of Plasmodium falciparum parasite by microsatellite markers after scale-up of insecticide-treated bed nets in western Kenya. Malar J. 2015;14:1–12. Ingasia LA, Cheruiyot J, Okoth SA, Andagalu B, Kamau E. Genetic variability and population structure of Plasmodium falciparum parasite populations from different malaria ecological regions of Kenya. Infect Genet Evol. 2016;39:372–80. Ishengoma DS, Mandara CI, Madebe RA, Warsame M, Ngasala B, Kabanywanyi AM, Mahende MK, Kamugisha E, Kavishe RA, Muro F, Mandike R. Microsatellites reveal high polymorphism and high potential for use in anti-malarial efficacy studies in areas with different transmission intensities in mainland Tanzania. Malar J. 2024;23(1):1–15. Jeang B, Zhong D, Lee MC, Atieli H, Yewhalaw D, Yan G. Molecular surveillance of Kelch 13 polymorphisms in Plasmodium falciparum isolates from Kenya and Ethiopia. Malar J. 2024;23(1):1–11. Kimenyi KM, Wamae K, Ngoi JM, de Laurent ZR, Ndwiga L, Osoti V, Obiero G, Abdi AI, Bejon P, Ochola-Oyier LI. (2022). Maintenance of high temporal Plasmodium falciparum genetic diversity and complexity of infection in asymptomatic and symptomatic infections in Kilifi, Kenya from 2007 to 2018. Malaria Journal , 21 (1), p.192. Kishoyian G, Njagi EN, Orinda GO, Kimani FT, Thiongo K, Matoke-Muhia D. Efficacy of artemisinin–lumefantrine for treatment of uncomplicated malaria after more than a decade of its use in Kenya. Epidemiol Infect. 2021;149(27):1–10. Larrañaga N, Mejía RE, Hormaza JI, Montoya A, Soto A, Fontecha GA. Genetic structure of Plasmodium falciparum populations across the Honduras-Nicaragua border. Malar J. 2013;12:1–10. Lawford H, Zurovac D, O'Reilly L, Hoibak S, Cowley A, Munga S, Vulule J, Juma E, Snow RW, Allan R. Adherence to prescribed artemisinin-based combination therapy in Garissa and Bunyala districts, Kenya. Malar J. 2011;10:1–8. MalariaGEN Plasmodium falciparum Community Project. Genomic epidemiology of artemisinin-resistant malaria. Elife. 2016;5:1–29. Maniga JN, Samuel MA, John O, Rael M, Muchiri JN, Bwogo P, Martin O, Sankarapandian V, Wilberforce M, Albert O, Onkoba SK. Novel Plasmodium falciparum k13 gene polymorphisms from Kisii County, Kenya during an era of artemisinin-based combination therapy deployment. Malar J. 2023;22(1):1–13. Miotto O, Amato R, Ashley EA, MacInnis B, Almagro-Garcia J, Amaratunga C, Lim P, Mead D, Oyola SO, Dhorda M, Imwong M. Genetic architecture of artemisinin-resistant Plasmodium falciparum. Nat Genet. 2015;47(3):226–34. Mohd Abd Razak MR, Sastu UR, Norahmad NA, Abdul-Karim A, Muhammad A, Muniandy PK, Jelip J, Rundi C, Imwong M, Mudin RN, Abdullah NR. Genetic diversity of Plasmodium falciparum populations in malaria declining areas of Sabah, East Malaysia. PLoS ONE. 2016;11(3):1–22. Mulenge FM, Hunja CW, Magiri E, Culleton R, Kaneko A, Aman RA. (2016). Genetic diversity and population structure of Plasmodium falciparum in Lake Victoria Islands, a region of intense transmission. The American Journal of Tropical Medicine and Hygiene , 95 (5), p.1077. Musyoka KB, Kiiru JN, Aluvaala E, Omondi P, Chege WK, Judah T, Kiboi D, Nganga JK, Kimani FT. Prevalence of mutations in Plasmodium falciparum genes associated with resistance to different antimalarial drugs in Nyando, Kisumu County in Kenya. Infect Genet Evol. 2020;78:1–9. Noedl H, Se Y, Schaecher K, Smith BL, Socheat D, Fukuda MM. Evidence of artemisinin-resistant malaria in western Cambodia. N Engl J Med. 2008;359(24):2619–20. Osborne A, Phelan JE, Kaneko A, Kagaya W, Chan C, Ngara M, Kongere J, Kita K, Gitaka J, Campino S, Clark TG. Drug resistance profiling of asymptomatic and low-density Plasmodium falciparum malaria infections on Ngodhe island, Kenya, using custom dual-indexing next-generation sequencing. Sci Rep. 2023;13(1):1–10. Ototo EN, Zhou G, Kamau L, Mbugi JP, Wanjala CL, Machani M, Atieli H, Githeko AK, Yan G. Age-specific Plasmodium parasite profile in pre and post ITN intervention period at a highland site in western Kenya. Malar J. 2017;16:1–6. Oyebola MK, Idowu ET, Nyang H, Olukosi YA, Otubanjo OA, Nwakanma DC, Awolola ST, Amambua-Ngwa A. Microsatellite markers reveal low levels of population sub-structuring of Plasmodium falciparum in southwestern Nigeria. Malar J. 2014;13:1–8. Oyedeji SI, Bassi PU, Oyedeji SA, Ojurongbe O, Awobode HO. Genetic diversity and complexity of Plasmodium falciparum infections in the microenvironment among siblings of the same household in North-Central Nigeria. Malar J. 2020;19:1–10. Peters W. (1970). Chemotherapy and drug resistance in malaria. Roh ME, Tessema SK, Murphy M, Nhlabathi N, Mkhonta N, Vilakati S, Greenhouse B. High genetic diversity of Plasmodium falciparum in the low-transmission setting of the Kingdom of Eswatini. J Infect Dis. 2019;220(8):1346–54. Rosenthal PJ, Asua V, Bailey JA, Conrad MD, Ishengoma DS, Kamya MR, Rasmussen C, Tadesse FG, Uwimana A, Fidock DA. The emergence of artemisinin partial resistance in Africa: how do we respond? Lancet Infect Dis. 2024;591(24):1–10. Segun OP, Obafemi AM, Abdul-Rahman AA. Evaluation of pfmdr-1 Polymorphisms and Parasites’ Population Diversity in Children with Acute Uncomplicated Malaria 5 Years Post-Adoption of Artemisinin-Based Combination Therapies. Asian J Res Infect Dis. 2021;7(3):14–26. Shafik SH, Richards SN, Corry B, Martin RE. The mechanistic basis for multidrug resistance and collateral drug sensitivity conferred to the malaria parasite by polymorphisms in PfMDR1 and PfCRT. PLoS Biol. 2022;20(5):1–40. ‌Straimer J, Gandhi P, Renner KC, Schmitt EK. High prevalence of Plasmodium falciparum K13 mutations in Rwanda is associated with slow parasite clearance after treatment with artemether-lumefantrine. J Infect Dis. 2022;225(8):1411–4. Touray AO, Mobegi VA, Wamunyokoli F, Herren JK. Diversity and Multiplicity of P. falciparum infections among asymptomatic school children in Mbita, Western Kenya. Sci Rep. 2020;10(1):1–8. Umaru ML, Uyaiabasi GN. Prevalence of malaria in patients attending the general hospital Makarfi, Makarfi Kaduna–State, North-Western Nigeria. Am J Infect Dis Microbiol. 2015;3(1):1–5. Wamae K, Okanda D, Ndwiga L, Osoti V, Kimenyi KM, Abdi AI, Bejon P, Sutherland C, Ochola-Oyier LI. No evidence of Plasmodium falciparum k13 artemisinin resistance-conferring mutations over a 24-year analysis in coastal Kenya but a near complete reversion to chloroquine-sensitive parasites. Antimicrob Agents Chemother. 2019;63(12):10–1128. World Health Organization. (2022). Artemisinin partial resistance. World Health Organization. (2023). WHO guidelines for malaria, 14 March 2023 (No. WHO/UCN/GMP /2023.01 ). Wu L, van den Hoogen LL, Slater H, Walker PG, Ghani AC, Drakeley CJ, Okell LC. Comparison of diagnostics for the detection of asymptomatic Plasmodium falciparum infections to inform control and elimination strategies. Nature. 2015;528(7580):86–93. Additional Declarations No competing interests reported. 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Akinyi","email":"","orcid":"","institution":"Redeemer’s University","correspondingAuthor":false,"prefix":"","firstName":"Florence","middleName":"T.","lastName":"Akinyi","suffix":""},{"id":475680541,"identity":"3e4da6dc-a377-4208-ae2d-74b5280fd21d","order_by":1,"name":"Joyce B Oluokun","email":"","orcid":"","institution":"Redeemer’s University","correspondingAuthor":false,"prefix":"","firstName":"Joyce","middleName":"B","lastName":"Oluokun","suffix":""},{"id":475680543,"identity":"f07f006b-31db-4032-9959-2c2b2110fbda","order_by":2,"name":"Kabir Gorden","email":"","orcid":"","institution":"Redeemer’s University","correspondingAuthor":false,"prefix":"","firstName":"Kabir","middleName":"","lastName":"Gorden","suffix":""},{"id":475680545,"identity":"26728583-f945-48bc-b28d-649558a9f226","order_by":3,"name":"John Openibo","email":"","orcid":"","institution":"Institute of Genomics and Global Health","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"","lastName":"Openibo","suffix":""},{"id":475680548,"identity":"75b82005-6cbb-4d77-9ac4-86ff01724dcc","order_by":4,"name":"Clemence D. 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In 2022, 249\u0026nbsp;million cases and 608,000 deaths were reported globally. Kenya recorded approximately 3.5\u0026nbsp;million malaria cases and 1.4% of global deaths reported in 2022 (WHO, 2023). Chemotherapy is one of the control measures for this disease. The recommended first-line treatment for uncomplicated malaria by the WHO is the artemisinin-based combination therapies (ACTs), which combine an artemisinin derivative with a long-acting partner drug. It was introduced following parasite resistance to chloroquine and sulfadoxine-pyrimethamine (Ecker et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Artimovich et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Artemether/Lumefantrine was recommended as the first-line treatment for uncomplicated malaria in Kenya in 2002. However, the actual implementation started in 2006 (Lawford et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Since then, the drug has been effective, but the emergence of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e parasites with decreased susceptibility to artemisinin threatens its efficacy. Artemisinin resistance arose in Southeast Asia's Greater Mekong Subregion (GMS) and has spread to other malaria-endemic regions like Eastern Africa (Noedl et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Dondorp \u003cem\u003eet al\u003c/em\u003e., 2009). Four Eastern African countries, namely Rwanda, Uganda, the United Republic of Tanzania, and Eritrea have been declared by WHO to have partial artemisinin resistance (WHO, 2023; Straimer \u003cem\u003eet al\u003c/em\u003e., 2021; Conrad et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Ishengoma et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Rosenthal et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This is alarming since there is a possibility of resistance in Kenya due to the proximity and frequent border movements between the countries. The slow parasite clearance is associated with single nucleotide polymorphism (SNP) in the propeller domain of the \u003cem\u003epfk13\u003c/em\u003e gene (Shafik et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Mutations on codons F446I, N458Y, C469Y, M476I, Y493H, R539T, I543T, P553L, R561H, P574L, R622I, A675V and C580Y are validated markers for artemisinin resistance (WHO, 2022). Periodic surveillance of the resistance markers is needed to effectively track the emergence of resistance.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ePlasmodium falciparum\u003c/em\u003e genetic diversity influences the spread and maintenance of resistant strains. Multiple parasite genotypes in polyclonal infections increase the frequency of spread of mutations associated with drug resistance compared to monoclonal infections, as they have more infective parasites. Additionally, the drug may eliminate the sensitive strains in polyclonal infections, but the resistant strains remain and proliferate (Roh et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Oyedeji et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The parasite genetic diversity can be determined by genotyping the polymorphic regions of antigenic markers like merozoite surface protein 1 (block 2), merozoite surface protein 2 (block 3), and glutamate-rich protein and the neutral microsatellite loci (Ajogbasile et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, WHO recently recommended genotyping using \u003cem\u003emsp1\u003c/em\u003e and \u003cem\u003emsp2\u003c/em\u003e genes with one or two highly polymorphic microsatellites. The latter can be poly alpha (poly-α), \u003cem\u003eTA1\u003c/em\u003e, or \u003cem\u003ePlasmodium falciparum\u003c/em\u003e protein kinase 2 (\u003cem\u003ePfK2\u003c/em\u003e) (WHO,2021).\u003c/p\u003e \u003cp\u003eDespite WHO recommendations for active surveillance of the artemisinin resistance markers to effectively track the resistance, only a small number of molecular epidemiological surveillance of these markers are available in Homa-Bay County. A study on asymptomatic and low-density \u003cem\u003ePlasmodium falciparum\u003c/em\u003e infections in Ngodhe Island in Homa-Bay County did not report any mutation in the \u003cem\u003ePfk13\u003c/em\u003e gene related to reduced susceptibility to artemisinin. However, different variants on the \u003cem\u003ePfmdr1\u003c/em\u003e gene were reported to be associated with reduced susceptibility to the ACT partner drug lumefantrine (Osborne et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Homa-Bay County is part of the Lake endemic region with the highest malaria prevalence and stable transmission throughout the year. Periodic surveillance of the resistant markers is essential (Maniga et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, this study investigated the polymorphism in the \u003cem\u003ePfk\u003c/em\u003e13 gene and parasite population diversity of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e in children with uncomplicated malaria in Homa-Bay County, Kenya, two decades post-adoption of Artemisinin-based combination treatment. The outcome of this study informs malaria control policy in this county. In this study, the \u003cem\u003ePfk13\u003c/em\u003e mutations, P667S, P667L, and T478T, were detected.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Site\u003c/h2\u003e \u003cp\u003eThe study was a cross-sectional study conducted among children who presented at Homa-Bay County Teaching and Referral Hospital (HBCTRH), Kenya with common symptoms of malaria between October 2023 and January 2024. The County has a population of 1,131,950 and occupies a landmass of 3,154.7 km\u003csup\u003e2\u003c/sup\u003e, Latitude: 0\u0026deg; 40' 60.00\"N and Longitude: 34\u0026deg; 27' 0.00\" E. It forms part of the lake endemic regions with Kenya's highest malaria prevalence and transmission (Maniga \u003cem\u003eet al\u003c/em\u003e., 2022).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStudy Population\u003c/h3\u003e\n\u003cp\u003eThe study population was children aged six (6) months to 15 years who presented with common symptoms of uncomplicated malaria at HBCTRH.\u003c/p\u003e\n\u003ch3\u003eInclusion and Exclusion criteria\u003c/h3\u003e\n\u003cp\u003eThe inclusion criteria for blood collection included symptoms of uncomplicated malaria, such as fever (temperature\u0026thinsp;\u0026gt;\u0026thinsp;37\u0026deg;C) in children of six (6) months to 15 years old, absence of concomitant infection, and willingness to participate in the study. Participants diagnosed with severe malaria, participants above 15 years old, those with concomitant infections, and those unwilling to provide informed consent were excluded from the study\u003c/p\u003e\n\u003ch3\u003eSample Collection\u003c/h3\u003e\n\u003cp\u003eDemographic information was collected from 150 participants recruited into the study. From each participant, 2 mL of venous blood was collected into EDTA tubes for malaria diagnosis using thick blood smears. Additionally, dried blood spots (DBS) were prepared from the EDTA blood of 100 participants that tested positive for malaria. The blood was spotted onto Whatman filter paper (Cytiva, Marlborough, United States), air-dried for approximately 3 hours, and stored in zip-lock plastic bags containing desiccant at room temperature until laboratory analysis.\u003c/p\u003e\n\u003ch3\u003eExtraction of Parasite Genomic DNA from the Dried Blood Spot\u003c/h3\u003e\n\u003cp\u003eA QIAmp DNA mini kit (QIAGEN, Hilden, Germany) was used to extract the parasite genomic DNA from the DBS filter paper according to the manufacturer\u0026rsquo;s instructions. DNA was stored at -20\u003csup\u003eo\u003c/sup\u003eC until further use.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePCR confirmation of\u003c/b\u003e \u003cb\u003ePlasmodium falciparum\u003c/b\u003e \u003cb\u003einfection\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ePlasmodium\u003c/em\u003e species in each sample were determined using nested PCR amplification of the 18S rRNA gene adapted from Singh \u003cem\u003eet al\u003c/em\u003e. (1999). Both primary and secondary PCR reactions were carried out in 20 \u0026micro;L total volume consisting of 4 \u0026micro;L master mix 5X FIREPol\u0026reg; Master Mix (Solis BioDyne, Tartu, Estonia), 1.0 \u0026micro;M forward and reverse primers each, and 2 \u0026micro;L of template DNA for the primary PCR. One microliter of primary PCR product was used as template for the secondary PCR. Details of the primer sequence and cycling conditions are shown in Supplemental Table\u0026nbsp;1. Genomic DNA from \u003cem\u003ePlasmodium falciparum\u003c/em\u003e strain 3D7 was used as a positive control.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePCR amplification of\u003c/b\u003e \u003cb\u003emsp1, msp2\u003c/b\u003e \u003cb\u003egenes, and Poly alpha microsatellite locus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSamples positive for \u003cem\u003eP. falciparum\u003c/em\u003e by PCR were selected for \u003cem\u003emsp1\u003c/em\u003e and \u003cem\u003emsp2\u003c/em\u003e genes and Poly alpha microsatellite locus analysis. Nested PCR amplification of \u003cem\u003emsp1\u003c/em\u003e and \u003cem\u003emsp2\u003c/em\u003e genes was performed using non-family-specific primer pairs corresponding to conserved sequences spanning the polymorphic regions of each antigenic marker previously described by Segun et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). PCR amplification of \u003cem\u003emsp1\u003c/em\u003e and \u003cem\u003emsp2\u003c/em\u003e gene was carried out in a 20 \u0026micro;L total reaction volume using 4 \u0026micro;L master mix 5X FIREPol\u0026reg; Master Mix (Solis BioDyne, Tartu, Estonia),1 \u0026micro;M of forward and reverse primers, and 4 \u0026micro;L DNA template for primary reaction, and 2 \u0026micro;L DNA template for the secondary reaction using cycling conditions and primer sequences specified in Supplemental Table\u0026nbsp;2. The PCR amplicons were further resolved on 2% gel electrophoresis to confirm the amplification and determine the number of clones per sample.\u003c/p\u003e \u003cp\u003eA direct PCR, according to CDC protocol, was used to amplify the poly alpha microsatellite locus. The 15 \u0026micro;L reaction mixture consisting of 2 \u0026micro;L genomic DNA, 0.6 \u0026micro;L of 10 \u0026micro;M primers, and 7.5 \u0026micro;L 2XPlatinum hot start master mix (Invitrogen, Carlsbad, California) was used (CDC, 2021). Details of primer sequence and cycling conditions are shown in Supplemental Table\u0026nbsp;2. Genomic DNA 3D7 clone was used as the positive control. The amplicons were further analyzed using capillary electrophoresis. The fragment analysis was performed using the Genetic Analyzer 3500XL series (Applied Biosystems).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePCR amplification of the\u003c/b\u003e \u003cb\u003ePfk13\u003c/b\u003e \u003cb\u003egene and targeted sequencing\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNested PCR was used to amplify the propeller domain of the \u003cem\u003ePfk13\u003c/em\u003e gene in samples successfully amplified for \u003cem\u003emsp1\u003c/em\u003e and \u003cem\u003emsp2\u003c/em\u003e genes to determine SNPs present in the parasites according to the method previously described (Ajogbasile et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In the primary amplification, a 20 \u0026micro;L reaction mixture consisting of 1.5 \u0026micro;L genomic DNA, 1 \u0026micro;M of each primer, and 4 \u0026micro;L 5X FIREPol\u0026reg; Master Mix (Solis BioDyne, Tartu, Estonia). For the secondary amplification, 1 \u0026micro;L of the primary amplicon was utilized as the template, and each reaction contained 1 \u0026micro;M primers. The specific primers and cycling conditions are in Supplemental Table\u0026nbsp;3. The amplification of the \u003cem\u003ePfk13\u003c/em\u003e gene was confirmed by 2% gel electrophoresis. The amplicons from PCR positive reaction (64 samples) were sequenced by the Sanger method using an Applied Biosystems 3500 XL series Genetic Analyser at ACEGID, Redeemer\u0026rsquo;s University, Ede, Osun State, Nigeria\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eData were double entered serially using patients\u0026rsquo; codes and analyzed using IBM SPSS (version 20.01) and Microsoft Excel. Crosstab descriptive statistics was carried out using SPSS, while Excel was used to draw graphs. The parasite diversity analysis of \u003cem\u003emsp1\u003c/em\u003e and \u003cem\u003emsp2\u003c/em\u003e was determined by counting the number of bands on the gel electrogram and for Poly alpha; GeneMapper version 6 software was used for normalization across runs and automatic determination of allele length and peak heights in samples containing multiple alleles per locus. The highest peak was identified as the predominant allele. Multiple alleles at a given locus were considered if minor peaks observed were \u0026ge;\u0026thinsp;20% of the height of the predominant allele. A peak was considered an allele if it had a size of 114\u0026ndash;201 and a difference of 5bps from the nearby peak. The results were either monoclonal (having one clone per sample) or polyclonal (more than one clone per sample). Expected heterozygosity (\u003cem\u003eH\u003c/em\u003e\u003csub\u003e\u003cem\u003ee\u003c/em\u003e\u003c/sub\u003e), defined as the probability that two randomly selected clones from a population will carry distinct alleles at the polyα locus, was used as a measure of genetic diversity using a formula, \u003cem\u003eH\u003c/em\u003e\u003csub\u003e\u003cem\u003ee\u003c/em\u003e\u003c/sub\u003e = [n/ (n\u0026thinsp;\u0026minus;\u0026thinsp;1)] [1\u0026thinsp;\u0026minus;\u0026thinsp;Σp\u003csub\u003ei\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e], where n and p\u003csub\u003ei\u003c/sub\u003e represent the number of isolates analysed and frequency of the i\u003csup\u003eth\u003c/sup\u003e allele in the population. Expected heterozygosity (\u003cem\u003eH\u003c/em\u003e\u003csub\u003e\u003cem\u003ee\u003c/em\u003e\u003c/sub\u003e) values range between 0 and 1 for low and high genetic diversity, respectively. To identify the polymorphism in the propeller domain of the \u003cem\u003ePfk13\u003c/em\u003e gene, Sequencing data was analysed using Geneious Prime software (version 2024.0.7). Forward and reverse nucleotide sequences were imported into the software and aligned using Geneious alignment. Consensus sequences were generated for all the samples. The consensus sequence was mapped against the reference nucleotide sequence of the \u003cem\u003ePfk13\u003c/em\u003e gene obtained from the PlasmoDB database (PF3D7_1343700 sequence region spanning region 1,724,817\u0026ndash;1,726,997 bp of chromosome 13). Sequences with nucleotide change were translated into amino acids to determine the type of mutation.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eDemographic characteristics\u003c/h2\u003e \u003cp\u003eA total of 100 participants were screened for malaria using thick Giemsa-stained microscopy. The female and male distribution was 56% (56/100) and 44% (44/100) respectively. The median age of the children was 5 years (Range: 0.5\u0026ndash;15 years) with 46% (46/100) of all participants being aged\u0026thinsp;\u0026lt;\u0026thinsp;5 years.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePrevalence of\u003c/b\u003e \u003cb\u003eP. falciparum\u003c/b\u003e \u003cb\u003einfection among study participants\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAll 100 samples positive for malaria by microscopy were included in a \u003cem\u003eP. falciparum\u003c/em\u003e confirmatory PCR test, and 86% (86/100) were confirmed positive for \u003cem\u003eP. falciparum\u003c/em\u003e infection, and 14% (14/100) were negative for \u003cem\u003eP. falciparum\u003c/em\u003e infection. (Supplemental Fig.\u0026nbsp;1)\u003c/p\u003e \u003cp\u003e \u003cb\u003ePlasmodium falciparum\u003c/b\u003e \u003cb\u003epopulation structure\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003ePlasmodium falciparum\u003c/em\u003e speciation positive samples (86) were analysed by nested PCR to determine the parasite population structure. Out of these, 93.0% (80/86) were successfully amplified for \u003cem\u003emsp1\u003c/em\u003e gene producing amplicons of between 400\u0026ndash;1000bp DNA fragment sizes on 2% agarose gel electrophoresis. The \u003cem\u003emsp2\u003c/em\u003e gene was successfully amplified in 90.7% (78/86) samples producing amplicons between 200\u0026ndash;500 bp DNA fragment sizes and 100% (86/86) amplified for poly alpha with peak sizes ranging from 129bp-183bp by capillary electrophoresis (Supplemental Fig.\u0026nbsp;2,3,4). The results were either monoclonal or polyclonal. Monoclonal and polyclonal infections were detected in 16.3% (14/86) and 76.7% (66/86) of the isolates, respectively, for the \u003cem\u003emsp1\u003c/em\u003e gene. All the polyclonal infections for \u003cem\u003emsp\u003c/em\u003e1 gene had 2 clones per sample. For the \u003cem\u003emsp2\u003c/em\u003e gene, 38.4% (33/86) and 52.3% (45/86) of samples had monoclonal and polyclonal infections, respectively. However, 26.7% (23/86) had a monoclonal infection (defined by a single peak in a sample), and 73.3% (63/86) had a polyclonal infection (defined by two or more peaks) for poly alpha (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The polyclonal infections in \u003cem\u003emsp2\u003c/em\u003e and poly alpha had clones ranging from two to five per sample, with two clones having the highest frequency for both \u003cem\u003emsp2\u003c/em\u003e (73.3%) and poly alpha (38.1%). The overall population of polyclonal infection was 86%. The expected heterozygosity (He) was 0.91.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePolymorphisms on\u003c/b\u003e \u003cb\u003ethe Pfk13\u003c/b\u003e \u003cb\u003egene in the isolates\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNested PCR was successful in 64 isolates (Supplemental Fig.\u0026nbsp;5) and were included in Sanger sequencing to detect polymorphisms on the \u003cem\u003ePfK13\u003c/em\u003e gene. A snapshot of the Sanger chromatogram indicating a nucleotide change is shown in (Supplemental Figs.\u0026nbsp;6\u0026amp;7). No WHO-validated or candidate resistance markers were detected in this study. However, two non-synonymous and one synonymous mutation were detected in the 4/64 DNA sequences sequenced (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The mutations P667S (1), P667L (2), and T478T (1) were detected in this study. The prevalence of parasites with mutations on the \u003cem\u003ePfk13\u003c/em\u003e gene in this study was 6.25% (4/64), while 93.75% (60/64) did not have any mutations when compared to the \u003cem\u003ePfk13\u003c/em\u003e sequence of 3D7 downloaded from PlasmoDB (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The mutation P667L occurred in the same nucleotide position (2000) in the 2 samples with this mutation\u003c/p\u003e \u003cp\u003e \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\u003eDetailed profile of the \u003cem\u003ePfk13\u003c/em\u003e polymorphisms detected among the study population\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\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=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" 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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS/N\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eK13 amino acid locus\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAge of patients (Yrs)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNumber of samples with the mutation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNucleotide Locus\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eReference allele\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMutant Allele\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eType of mutation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCountries previously observed in\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP667S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2905\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNon-synonymous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eRwanda (Straimer \u003cem\u003eet al\u003c/em\u003e., 2021), Kenya (Kakamega County) (Jeang et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP667L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNon-synonymous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eSoutheast Asia, South Asia (MalariaGEN Plasmodium falciparum Community Project, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT478T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2314\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSynonymous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eKenya, C\u0026ocirc;te d'Ivoire\u0026nbsp;(Kamau \u003cem\u003eet al\u003c/em\u003e., 2014), Senegal (Gaye \u003cem\u003eet al\u003c/em\u003e., 2020)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eArtemisinin-based combination therapies (ACTs) are the first-line treatment for uncomplicated malaria. However, polymorphisms in the \u003cem\u003ePfk13\u003c/em\u003e gene are associated with slow parasite clearance after treatment with ACTs. This study investigated the prevalence of single nucleotide polymorphism in the \u003cem\u003ePfk\u003c/em\u003e13 gene and parasite population diversity using \u003cem\u003ethe msp1\u003c/em\u003e and \u003cem\u003emsp2\u003c/em\u003e genes and the poly alpha microsatellite.\u003c/p\u003e \u003cp\u003eAlthough all samples were positive for \u003cem\u003eP. falciparum\u003c/em\u003e by microscopy, PCR did not confirm \u003cem\u003eP. falciparum\u003c/em\u003e in 14 samples. This may be due to human error in the microscopy, as the PCR is expected to be more sensitive (Wu et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). It is also possible that it was non-\u003cem\u003efalciparum\u003c/em\u003e malaria; species differentiation is difficult in thick blood films and will require a higher level of microscopy expertise (Fitri \u003cem\u003eet al\u003c/em\u003e.,2022).\u003c/p\u003e \u003cp\u003eThe malaria infections in this study were shown to be 86% polyclonal infection. This aligns with the study by Gatei et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), which reported an overall polyclonal infection of \u0026ge;\u0026thinsp;80% in Siaya County, Kenya. A study carried out on asymptomatic children in Homa-Bay County reported a prevalence of 79.69% polyclonal infection (Touray et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The polyclonality could be due to high transmission intensity in this region; individuals are more likely to be bitten by multiple infected mosquitoes carrying different parasite strains (Agaba \u003cem\u003eet al\u003c/em\u003e., 2021). Another possible explanation could be high asymptomatic infections, which harbor multiple parasite strains without showing symptoms and are hence reservoir and contributing to the spread of diverse strains. Since this is a malaria-endemic region, frequent \u003cem\u003ePlasmodium\u003c/em\u003e exposures lead to strong immune modulation, reducing clinical symptoms (Omondi \u003cem\u003eet al\u003c/em\u003e., 2022; Mwesigwa \u003cem\u003eet al\u003c/em\u003e., 2024).\u003c/p\u003e \u003cp\u003eHigh \u003cem\u003eP. falciparum\u003c/em\u003e genetic diversity has remained unchanged in Kenya even after introducing the ACTs in the country (Chebon et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Nderu \u003cem\u003eet al\u003c/em\u003e., 2019). In this study, a high genetic diversity of 0.91 was reported. This is consistent with other studies in the country (Gatei et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Ingasia et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Mulenge et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kimenyi et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The high \u003cem\u003eP. falciparum\u003c/em\u003e genetic diversity reported here could be due to high levels of transmission leading to extensive gene flow and recombination (Maniga \u003cem\u003eet al\u003c/em\u003e., 2022; Mulenge et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study reported two non-synonymous mutations (P667L and P667S) and one synonymous mutation (T478T) on the \u003cem\u003ePfk13\u003c/em\u003e gene. None of the WHO-validated or candidate markers for partial artemisinin resistance were reported in this study. However, the non-synonymous mutation P667S reported in this study has earlier been reported in Rwanda to cause a delayed parasite clearance time of ˃5hours and positive parasitemia count on a smear on the third day of treatment, meeting the criteria for clinical artemisinin resistance (Straimer \u003cem\u003eet al\u003c/em\u003e., 2021; WHO, 2022). Interestingly, the same mutation has been reported in Kakamega County, Kenya, during the passive surveillance and epidemiological studies on \u003cem\u003ePfk13\u003c/em\u003e polymorphism between 2018 and 2022 in Western Kenya (Homa-Bay, Kisumu, Kisii, and Kakamega Counties) (Jeang et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The mutation may be due to geographical migration, considering the daily interactions between these two counties (Homa-Bay and Kakamega) (Jeang et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Although this mutation is yet to be implicated in artemisinin resistance in Kenya, there is a need for continuous surveillance of the mutation, considering its proximity to Rwanda, the effect of this mutation may be the same in Kenya.\u003c/p\u003e \u003cp\u003eThe non-synonymous mutation P667L in two samples (3.2%) reported in this study had earlier been reported in Southeast and South Asia in a study conducted by the MalariaGEN Plasmodium falciparum Community Project (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), where the frequency of 2/ 1599 and 1/75 samples were reported, respectively, suggesting a less frequent mutation. Although the role this mutation plays in resistance to artemisinin is still unknown; it is important to monitor this mutation as it might have a different effect in the two continents due to different parasite genetic backgrounds. Most non-synonymous African mutations similar to those in Southeast Asia have arisen independently (Ariey et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Therefore, the mutation in the current study might not be related to the one reported in Southeast Asia. It is also possible that this mutation may be due to geographical migration, considering the pattern with chloroquine resistance, where resistance emerged in Southeast Asia and spread into Africa through Kenya (Peters, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1970\u003c/span\u003e; Borrmann et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Beshir et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Ashley et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Miotto et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). To the best of our knowledge, this is the first report on the non-synonymous mutation P667L in Africa.\u003c/p\u003e \u003cp\u003eAll the mutations reported in this study occurred in participants of five years old and below. This should be monitored since mutations sometimes lead to drug resistance or affect parasite fitness (Behrens et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Children below five years of age are the population at most risk of malaria due to a lack of well-developed immunity; hence, they may not be able to clear the parasite in case of high parasite fitness cost, as opposed to children above five with better-developed immunity to malaria following several infections. Thus, children below the age of 5 years may aid in transmitting the mutant alleles as they experience more clinical episodes of malaria infection (Umaru and Uyaiabasi, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Additionally, all the non-synonymous mutations in this study occurred in polyclonal infections. This could have negative or positive implications in the fight against malaria. Polyclonal infection has more infective parasites, which can aid in transmitting the mutant alleles (Oyedeji et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It is also possible that drug-susceptible parasites may exist in parallel to mutant parasites - (potentially resistant); hence, ACT may remain active as the mutant parasites circulates within the environment.\u003c/p\u003e \u003cp\u003eAlthough the efficacy of the ACT used for treatment was not determined in the enrolled children, the data provided in this study are more of epidemiological surveillance of the malaria parasite drug resistant markers and the genetic diversity for insight into the circulating parasite and transmission pattern. In addition, the non-family specific msp1/2 genes were used for the parasite diversity hence the most circulating alleles could not be determined. However, the diversity was equally determined, and the implications of more polyclonal infection was clearly observed, hence the need for continuous surveillance and monitoring of circulating parasite.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this study reports polymorphism in the \u003cem\u003ePfk13\u003c/em\u003e gene of \u003cem\u003eP. falciparum\u003c/em\u003e parasites isolated from children in Homa-Bay County, Kenya. The study also showed a high frequency of polyclonal infections and high parasite genetic diversity, indicating a high regional transmission rate. Active monitoring of the mutant alleles in the \u003cem\u003ePfk13\u003c/em\u003e gene and heightened malaria control strategies are recommended.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of Redeemer’s University Ethics Committee (RUN/REC/2023/101), Homa-Bay County Ministry of Health (MOH/RA/VOL.VI (021)), and the research permit obtained from the National Commission for Science, Technology \u0026amp; Innovation (NACOSTI/P/23/31605). Written informed consent was obtained from parents/guardians for children before enrolment into this study, and child assent was obtained from children between 12 and 15 years.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the parents/guardians of the children and the children for participating in the study. We also thank the medical officers at the recruiting facility for assisting with patient recruitment and sample collection. Our gratitude goes to the Homa-Bay County health office for allowing us to collect the samples. Finally, we are grateful to Institute of Genomics and Global health (formerly ACEGID), Redeemer’s University, Nigeria in the ACE Impact project for funding this project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003eAll raw data is available as supplementary materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contribution:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFlorence T. Akinyi: Study design, Laboratory analysis, Bioinformatics and Data Analysis, Manuscript Drafting, and Review.\u003c/p\u003e\n\u003cp\u003eOluokun B. Joyce: Laboratory analysis and Manuscript Review\u003c/p\u003e\n\u003cp\u003eKabir Gorden: Data analysis and Manuscript Review\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGouton D. Clemence: Laboratory analysis and manuscript review\u003c/p\u003e\n\u003cp\u003eOpenibo John: Laboratory analysis and Manuscript Review\u003c/p\u003e\n\u003cp\u003eOloo O. Edwin: Sample Collection\u003c/p\u003e\n\u003cp\u003eMitesser Vera: Bioinformatics analysis and Manuscript Review\u003c/p\u003e\n\u003cp\u003eHappi T. Christian: Funding acquisition, lab Resources and Manuscript review\u003c/p\u003e\n\u003cp\u003eFolarin A. Onikepe: Conceptualization and Study Design, Proposal Review, coordinating logistics for Lab work, Project administration, Resources, Supervision, Validation, Visualization, Manuscript review \u0026amp; editing and Mentorship.\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: This research was funded by the World Bank ACE IMPACT grant (worldbank.org) (ACE-019 to CTH). FTA was funded through the ACE IMPACT graduate fellowship for MSc. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePotential conflicts of interest:\u0026nbsp;\u003c/strong\u003eAll authors declare no conflict.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAjogbasile FV, Kayode AT, Oluniyi PE, Akano KO, Uwanibe JN, Adegboyega BB, Philip C, John OG, Eromon PJ, Emechebe G, Finimo F. Genetic diversity and population structure of Plasmodium falciparum in Nigeria: insights from microsatellite loci analysis. Malar J. 2021;20(1):1\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAriey F, Witkowski B, Amaratunga C, Beghain J, Langlois AC, Khim N, Kim S, Duru V, Bouchier C, Ma L, Lim P. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature. 2014;505(7481):50\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArtimovich E, Schneider K, Taylor TE, Kublin JG, Dzinjalamala FK, Escalante AA, Plowe CV, Laufer MK, Takala-Harrison S. Persistence of sulfadoxine-pyrimethamine resistance despite reduction of drug pressure in Malawi. J Infect Dis. 2015;212(5):694\u0026ndash;701.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAshley EA, Dhorda M, Fairhurst RM, Amaratunga C, Lim P, Suon S, Sreng S, Anderson JM, Mao S, Sam B, Sopha C. Spread of artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med. 2014;371(5):411\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBehrens HM, Schmidt S, Henshall IG, L\u0026oacute;pez-Barona P, Peigney D, Sabitzki R, May J, Ma\u0026iuml;ga-Ascofar\u0026eacute; O, Spielmann T. Impact of different mutations on Kelch13 protein levels, ART resistance, and fitness cost in Plasmodium falciparum parasites. Mbio. 2024;6(15):1\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeshir KB, Sutherland CJ, Sawa P, Drakeley CJ, Okell L, Mweresa CK, Omar SA, Shekalaghe SA, Kaur H, Ndaro A, Chilongola J. Residual Plasmodium falciparum parasitemia in Kenyan children after artemisinin-combination therapy is associated with increased transmission to mosquitoes and parasite recurrence. J Infect Dis. 2013;208(12):2017\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBorrmann S, Sasi P, Mwai L, Bashraheil M, Abdallah A, Muriithi S, Fr\u0026uuml;hauf H, Schaub B, Pfeil J, Peshu J, Hanpithakpong W. Declining responsiveness of Plasmodium falciparum infections to artemisinin-based combination treatments on the Kenyan coast. PLoS ONE. 2011;6(11):1\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChebon LJ, Ngalah BS, Ingasia LA, Juma DW, Muiruri P, Cheruiyot J, Opot B, Mbuba E, Imbuga M, Akala HM, Bulimo W. Genetically determined response to artemisinin treatment in western Kenyan Plasmodium falciparum parasites. PLoS ONE. 2016;11(9):1\u0026ndash;19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCochran WG. Sampling techniques. Wiley; 1977.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eConrad MD, Asua V, Garg S, Giesbrecht D, Niar\u0026eacute; K, Smith S, Namuganga JF, Katairo T, Legac J, Crudale RM, Tumwebaze PK. Evolution of partial resistance to artemisinins in malaria parasites in Uganda. N Engl J Med. 2023;389(8):722\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDondorp AM, Yeung S, White L, Nguon C, Day NP, Socheat D, Von Seidlein L. Artemisinin resistance: current status and scenarios for containment. Nat Rev Microbiol. 2010;8(4):272\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEcker A, Lehane AM, Clain J, Fidock DA. PfCRT and its role in antimalarial drug resistance. Trends Parasitol. 2012;28(11):504\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFitri LE, Widaningrum T, Endharti AT, Prabowo MH, Winaris N, Nugraha RYB. Malaria diagnostic update: From conventional to advanced method. J Clin Lab Anal. 2022;36(4):1\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGatei W, Gimnig JE, Hawley W, Ter Kuile F, Odero C, Iriemenam NC, Shah MP, Howard PP, Omosun YO, Terlouw DJ, Nahlen B. Genetic diversity of Plasmodium falciparum parasite by microsatellite markers after scale-up of insecticide-treated bed nets in western Kenya. Malar J. 2015;14:1\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIngasia LA, Cheruiyot J, Okoth SA, Andagalu B, Kamau E. Genetic variability and population structure of Plasmodium falciparum parasite populations from different malaria ecological regions of Kenya. Infect Genet Evol. 2016;39:372\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIshengoma DS, Mandara CI, Madebe RA, Warsame M, Ngasala B, Kabanywanyi AM, Mahende MK, Kamugisha E, Kavishe RA, Muro F, Mandike R. Microsatellites reveal high polymorphism and high potential for use in anti-malarial efficacy studies in areas with different transmission intensities in mainland Tanzania. Malar J. 2024;23(1):1\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJeang B, Zhong D, Lee MC, Atieli H, Yewhalaw D, Yan G. Molecular surveillance of Kelch 13 polymorphisms in Plasmodium falciparum isolates from Kenya and Ethiopia. Malar J. 2024;23(1):1\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKimenyi KM, Wamae K, Ngoi JM, de Laurent ZR, Ndwiga L, Osoti V, Obiero G, Abdi AI, Bejon P, Ochola-Oyier LI. (2022). Maintenance of high temporal Plasmodium falciparum genetic diversity and complexity of infection in asymptomatic and symptomatic infections in Kilifi, Kenya from 2007 to 2018. \u003cem\u003eMalaria Journal\u003c/em\u003e, \u003cem\u003e21\u003c/em\u003e(1), p.192.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKishoyian G, Njagi EN, Orinda GO, Kimani FT, Thiongo K, Matoke-Muhia D. Efficacy of artemisinin\u0026ndash;lumefantrine for treatment of uncomplicated malaria after more than a decade of its use in Kenya. Epidemiol Infect. 2021;149(27):1\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLarra\u0026ntilde;aga N, Mej\u0026iacute;a RE, Hormaza JI, Montoya A, Soto A, Fontecha GA. Genetic structure of Plasmodium falciparum populations across the Honduras-Nicaragua border. Malar J. 2013;12:1\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLawford H, Zurovac D, O'Reilly L, Hoibak S, Cowley A, Munga S, Vulule J, Juma E, Snow RW, Allan R. Adherence to prescribed artemisinin-based combination therapy in Garissa and Bunyala districts, Kenya. Malar J. 2011;10:1\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalariaGEN Plasmodium falciparum Community Project. Genomic epidemiology of artemisinin-resistant malaria. Elife. 2016;5:1\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eManiga JN, Samuel MA, John O, Rael M, Muchiri JN, Bwogo P, Martin O, Sankarapandian V, Wilberforce M, Albert O, Onkoba SK. Novel Plasmodium falciparum k13 gene polymorphisms from Kisii County, Kenya during an era of artemisinin-based combination therapy deployment. Malar J. 2023;22(1):1\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiotto O, Amato R, Ashley EA, MacInnis B, Almagro-Garcia J, Amaratunga C, Lim P, Mead D, Oyola SO, Dhorda M, Imwong M. Genetic architecture of artemisinin-resistant Plasmodium falciparum. Nat Genet. 2015;47(3):226\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohd Abd Razak MR, Sastu UR, Norahmad NA, Abdul-Karim A, Muhammad A, Muniandy PK, Jelip J, Rundi C, Imwong M, Mudin RN, Abdullah NR. Genetic diversity of Plasmodium falciparum populations in malaria declining areas of Sabah, East Malaysia. PLoS ONE. 2016;11(3):1\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMulenge FM, Hunja CW, Magiri E, Culleton R, Kaneko A, Aman RA. (2016). Genetic diversity and population structure of Plasmodium falciparum in Lake Victoria Islands, a region of intense transmission. \u003cem\u003eThe American Journal of Tropical Medicine and Hygiene\u003c/em\u003e, \u003cem\u003e95\u003c/em\u003e(5), p.1077.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMusyoka KB, Kiiru JN, Aluvaala E, Omondi P, Chege WK, Judah T, Kiboi D, Nganga JK, Kimani FT. Prevalence of mutations in Plasmodium falciparum genes associated with resistance to different antimalarial drugs in Nyando, Kisumu County in Kenya. Infect Genet Evol. 2020;78:1\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNoedl H, Se Y, Schaecher K, Smith BL, Socheat D, Fukuda MM. Evidence of artemisinin-resistant malaria in western Cambodia. N Engl J Med. 2008;359(24):2619\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOsborne A, Phelan JE, Kaneko A, Kagaya W, Chan C, Ngara M, Kongere J, Kita K, Gitaka J, Campino S, Clark TG. Drug resistance profiling of asymptomatic and low-density Plasmodium falciparum malaria infections on Ngodhe island, Kenya, using custom dual-indexing next-generation sequencing. Sci Rep. 2023;13(1):1\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOtoto EN, Zhou G, Kamau L, Mbugi JP, Wanjala CL, Machani M, Atieli H, Githeko AK, Yan G. Age-specific Plasmodium parasite profile in pre and post ITN intervention period at a highland site in western Kenya. Malar J. 2017;16:1\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOyebola MK, Idowu ET, Nyang H, Olukosi YA, Otubanjo OA, Nwakanma DC, Awolola ST, Amambua-Ngwa A. Microsatellite markers reveal low levels of population sub-structuring of Plasmodium falciparum in southwestern Nigeria. Malar J. 2014;13:1\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOyedeji SI, Bassi PU, Oyedeji SA, Ojurongbe O, Awobode HO. Genetic diversity and complexity of Plasmodium falciparum infections in the microenvironment among siblings of the same household in North-Central Nigeria. Malar J. 2020;19:1\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeters W. (1970). Chemotherapy and drug resistance in malaria.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoh ME, Tessema SK, Murphy M, Nhlabathi N, Mkhonta N, Vilakati S, Greenhouse B. High genetic diversity of Plasmodium falciparum in the low-transmission setting of the Kingdom of Eswatini. J Infect Dis. 2019;220(8):1346\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosenthal PJ, Asua V, Bailey JA, Conrad MD, Ishengoma DS, Kamya MR, Rasmussen C, Tadesse FG, Uwimana A, Fidock DA. The emergence of artemisinin partial resistance in Africa: how do we respond? Lancet Infect Dis. 2024;591(24):1\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSegun OP, Obafemi AM, Abdul-Rahman AA. Evaluation of pfmdr-1 Polymorphisms and Parasites\u0026rsquo; Population Diversity in Children with Acute Uncomplicated Malaria 5 Years Post-Adoption of Artemisinin-Based Combination Therapies. Asian J Res Infect Dis. 2021;7(3):14\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShafik SH, Richards SN, Corry B, Martin RE. The mechanistic basis for multidrug resistance and collateral drug sensitivity conferred to the malaria parasite by polymorphisms in PfMDR1 and PfCRT. PLoS Biol. 2022;20(5):1\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u0026zwnj;Straimer J, Gandhi P, Renner KC, Schmitt EK. High prevalence of Plasmodium falciparum K13 mutations in Rwanda is associated with slow parasite clearance after treatment with artemether-lumefantrine. J Infect Dis. 2022;225(8):1411\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTouray AO, Mobegi VA, Wamunyokoli F, Herren JK. Diversity and Multiplicity of P. falciparum infections among asymptomatic school children in Mbita, Western Kenya. Sci Rep. 2020;10(1):1\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUmaru ML, Uyaiabasi GN. Prevalence of malaria in patients attending the general hospital Makarfi, Makarfi Kaduna\u0026ndash;State, North-Western Nigeria. Am J Infect Dis Microbiol. 2015;3(1):1\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWamae K, Okanda D, Ndwiga L, Osoti V, Kimenyi KM, Abdi AI, Bejon P, Sutherland C, Ochola-Oyier LI. No evidence of Plasmodium falciparum k13 artemisinin resistance-conferring mutations over a 24-year analysis in coastal Kenya but a near complete reversion to chloroquine-sensitive parasites. Antimicrob Agents Chemother. 2019;63(12):10\u0026ndash;1128.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWorld Health Organization. (2022). Artemisinin partial resistance.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWorld Health Organization. (2023). \u003cem\u003eWHO guidelines for malaria, 14 March 2023\u003c/em\u003e (No. WHO/UCN/GMP\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e/2023.01\u003c/span\u003e\u003cspan address=\"http:///2023.01\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu L, van den Hoogen LL, Slater H, Walker PG, Ghani AC, Drakeley CJ, Okell LC. Comparison of diagnostics for the detection of asymptomatic Plasmodium falciparum infections to inform control and elimination strategies. Nature. 2015;528(7580):86\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Malaria, Resistance, kelch13, Kenya, Transmission, Polymorphisms","lastPublishedDoi":"10.21203/rs.3.rs-6897552/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6897552/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBACKGROUND\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe emergence and spread of \u003cem\u003eP. falciparum\u003c/em\u003e parasites with decreased susceptibility to Artemisinin-based combination therapies (ACTs) is causing global concern. Active surveillance of the emergence of resistance in malaria-endemic areas is important for efficient management of the infection. Slow parasite clearance following treatment with artemisinin derivatives is associated with single-nucleotide polymorphism in the propeller domain of the \u003cem\u003ePlasmodium falciparum\u003c/em\u003e \u003cem\u003ekelch13 \u003c/em\u003e(\u003cem\u003epfk13\u003c/em\u003e) gene. This study investigated the polymorphism in the \u003cem\u003ePfk\u003c/em\u003e13 gene and parasite population diversity of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e in children with uncomplicated malaria in Homa-Bay County, Kenya, two decades post-adoption of Artemisinin-based combination treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMETHODS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study assessed polymorphisms on the \u003cem\u003epfk13\u003c/em\u003e gene and parasite population diversity in 86 PCR-confirmed\u003cem\u003e Plasmodium falciparum \u003c/em\u003epositive samples obtained from children between six months and fifteen years old. The parasite diversity was determined by nested PCR amplification of \u003cem\u003emsp1\u003c/em\u003e and \u003cem\u003emsp2\u003c/em\u003e genes and direct PCR amplification of the polyα microsatellite locus followed by capillary electrophoresis. Sanger sequencing was carried out on samples with successful amplification of the \u003cem\u003ePfk13\u003c/em\u003e gene to determine polymorphisms. The sequenced data were analyzed by Geneious Prime software (version 2024.0.7).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRESULTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study did not report validated or candidate mutations as classified by WHO. However, the previously described mutations P667S and P667L were detected on \u003cem\u003ePfk13\u003c/em\u003e gene. Additionally, this study reported polyclonal infection in 86% of the samples and a parasite diversity of 0.91.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONCLUSION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn conclusion, this study demonstrated the circulation of \u003cem\u003eP. falciparum\u003c/em\u003e parasites with mutant alleles on the \u003cem\u003ekelch13\u003c/em\u003egene in Homa-Bay County, Kenya. The study also showed a high frequency of polyclonal infections and high parasite genetic diversity, suggesting a high transmission rate. The high polyclonal infection and diversity show that malaria transmission is still high in this region; hence, the control measures need to be heightened. The observed polymorphisms on \u003cem\u003ePfk13\u003c/em\u003e need close monitoring, especially since the P667S mutation has been reported to cause artemisinin resistance in Rwanda, and the effect of mutation P667L is yet to be known.\u003c/p\u003e","manuscriptTitle":"Molecular Characterisation of Plasmodium falciparum in Children with Uncomplicated Malaria in Homa-Bay, Kenya; Two Decades Post-Adoption of Artemisinin-Based Combination Therapies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-29 14:34:55","doi":"10.21203/rs.3.rs-6897552/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1b027ff4-060d-4d80-8711-58735e6e7008","owner":[],"postedDate":"June 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-30T20:08:37+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-29 14:34:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6897552","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6897552","identity":"rs-6897552","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}