Lynette Isabella Ochola-Oyier

Background: Antimalarial drug resistance (AMDR) continues to threaten malaria control and elimination, underscoring the need for timely and reliable malaria molecular surveillance. Data generation through therapeutic efficacy studies (TES) and independent surveys is critical, yet limited guidance exists to support policymakers and National Malaria Control Programs (NMCPs) in selecting appropriate laboratory techniques for different surveillance objectives and operational contexts. This descriptive technical review aimed to assess the currently used laboratory techniques based on their technical and operational characteristics, identify knowledge gaps and provide decision support for Malaria Molecular Surveillance (MMS) of Plasmodium falciparum . Methods: The literature search was conducted in PubMed and supplemented by citation searching. Following title and abstract screening, data were extracted on predefined performance, operational, and technical characteristics. A non-exclusionary assessment of reporting quality was also performed. The results were reviewed by field experts, who added additional information, based on the findings. Results: Thirty-nine studies published between 1998 and 2025 met the inclusion criteria. Performance varied between PCR-based and sequencing-based techniques, with analytical sensitivity ranging from 1–1000 parasites/µL for PCR-based techniques, and 1–2000 parasites/µL for sequencing-based techniques. Clinical sensitivity and specificity were high across all techniques, though highest for sequencing-based methods. PCR-based techniques provided rapid results and required less infrastructure for the targeted detection of known SNPs but could not identify novel markers. Sequencing-based techniques offered high multiplexing potential and could often detect minority variants and discover novel markers. However, they required greater capital investment, more extensive training, and substantial bioinformatics capacity. Cost data were highly variable and often incompletely reported, highlighting a knowledge gap. We developed a structured decision-support framework, illustrated through three hypothetical country scenarios reflecting differing surveillance needs, laboratory capacities, and resource constraints. These examples demonstrate that no single method is universally optimal and that technique selection should be tailored to specific surveillance objectives, epidemiological context, existing infrastructure, budget, and long-term capacity-building goals. Conclusion: This review provides an updated overview of available techniques and practical guidance to support stakeholders in selecting accurate, feasible, and sustainable approaches for monitoring AMDR through MMS, while also highlighting key knowledge gaps.

Background: A multicomponent vaccine is increasingly recognized as a necessary tool to control and eliminate malaria globally. However, genetic diversity in Plasmodium falciparum poses a significant challenge, particularly for blood-stage antigens associated with protective immunity. Individuals in endemic regions acquire clinical immunity without exposure to all antigenic variants, suggesting that a deeper understanding of antigen diversity could inform more effective vaccine strategies. Methods: The genetic diversity of the polymorphic gene locus for merozoite surface protein 2 was assessed with nested polymerase chain reaction and capillary electrophoresis fragment sizing in P falciparum isolates from 34 countries across sub-Saharan Africa. High fidelity long-read sequencing was used to investigate sequence diversity within msp2 variants. Results: We successfully genotyped msp2 from 2761 P falciparum–positive samples using capillary electrophoresis, identifying 412 distinct msp2 size variants. Several msp2 size variants were consistently overrepresented across geographical regions, transmission intensities, and time points. These variants comprised multiple unique sequences, of which several were geographically and temporally widespread. Nucleotide and amino acid sequences showed high diversity without geographical clustering. Conclusions: This study provides a comprehensive assessment of msp2 diversity across sub-Saharan Africa. The widespread prevalence of certain variants of this highly polymorphic antigen suggests possible structural constraints or selective advantages for the parasite. Maintenance of specific gene lengths and sequences across diverse settings highlights their potential as targets for next-generation, multicomponent malaria vaccines.

Background: Ongoing antimalarial drug resistance surveillance is essential to guide effective treatment strategies. Historically, resistance to chloroquine and sulfadoxine-pyrimethamine (SP) has been associated with well-characterized mutations in the chloroquine resistance transporter ( Pfcrt ; K76T) and antifolate pathway genes, including dihydrofolate reductase ( Pfdhfr ; N51I, C59R, S108N) and dihydropteroate synthase ( Pfdhps; A437G, K540E, A581G). Since the introduction of artemisinin-based combination therapies (ACTs), 13 mutations in the kelch 13 (PfK13) propeller domain have emerged as World Health Organization (WHO) validated markers of partial artemisinin resistance. This study aimed to characterize temporal trends in both established, Pfcrt and Pfk13, and less well-described potential markers, cysteine desulfurase ( Pfnfs) and Pfcoronin , using febrile malaria samples collected across diverse regions of Kenya between 2013 and 2022. Methods: The temporal trend of these markers of resistance were assessed by screening archived P. falciparum -positive dried blood spots (DBS). A total of 1750 DBS samples were collected from therapeutic efficacy studies (TES) conducted across distinct malaria transmission settings in Kenya, including coastal Kenya (Kwale 2013, n = 350; 2018, n = 150), the lake endemic region of Western Kenya (Kisumu 2015, n = 314; Busia 2016, n = 334), and the highland epidemic region of western Kenya (Kisii 2017, n = 314). Additional samples were obtained from an hrp2 study conducted in Kisii in 2022, ( n = 288). Parasite genomic DNA was extracted using the Chelex-saponin method and confirmed by a Pf 18S real-time polymerase chain reaction (RT-PCR). Pfk13, Pfcrt, Pfnfs , and Pfcoronin PCR amplicons were sequenced using capillary electrophoresis, Illumina Miseq or Oxford Nanopore (GridION) platforms. Results: The prevalence of Pfcrt mutations declined over time and no WHO-validated Pfk13 mutations associated with artemisinin resistance were detected. However, synonymous substitutions at WHO-validated codons C469C and P553P were identified. In the Pfcoronin gene, nonsynonymous mutations distinct from those reported in West Africa were observed at high frequencies (> 75%). Notably, the Pfnfs -K65Q mutation, previously associated with reduced lumefantrine sensitivity in West Africa, was detected in more than 80% of the samples. Conclusions: Our findings reveal no WHO-validated k13 mutations up until 2022 and confirm previous findings of a reduction in the Pfcrt resistance genotypes over time. This study underscores the importance of continued molecular surveillance and suggests that resistance may evolve through different pathways in East compared with West Africa and Southeast Asia. Graphical

Two licensed vaccines block Plasmodium falciparum malaria sporozoites through anticircumsporozoite protein antibodies. In animal models, intradermal (ID) sporozoites are more readily blocked than intravenous sporozoites. We hypothesized that this complicates human studies, where infectious mosquito bites deliver a mixture of ID and venous sporozoites. Here, to test whether vaccine efficacy varies by route of inoculation, we undertook a phase 2b open, randomized controlled trial, recruiting healthy volunteers in Kenya for randomization to the circumsporozoite protein-based R21/Matrix-M vaccine ( n = 38), thrombospondin-related adhesive protein fused to a multi-epitope string (ME-TRAP)-based vaccines ( n = 24) or to control ( n = 18). We enrolled 37 of these volunteers to controlled human malaria infection (CHMI) using ID or direct venous injection (DVI) of sporozoites, with PCR monitoring of parasitemia. Systemic and local postvaccination adverse events and systemic CHMI-related events were detected in 4.8%, 12.9% and 72.9% of volunteers, respectively, most commonly fever, headache and fatigue. No serious or severe adverse events were seen. Seven of 8 (88%) control volunteers and 11 of 12 (92%) ME-TRAP vaccinated volunteers, but none of the 12 R21 vaccinated volunteers receiving ID challenge met the prespecified treatment criteria for the primary endpoint. However, five of five R21 vaccinated volunteers receiving DVI sporozoites met the primary endpoint ( P < 0.0005 by log rank across all groups). Secondary efficacy outcomes were similar; that is, R21 vaccinated volunteers receiving ID challenge did not meet any secondary endpoints, whereas volunteers for other groups met secondary endpoints between days 7 and 21 ( P < 0.0005, P = 0.0001, P = 0.0007 and P < 0.0005 by log rank for time to parasitemia >20, >500, >1,000 and >10,000, respectively). R21/Matrix-M was highly protective against CHMI using ID. inoculation of sporozoites, but not against DVI sporozoites. CHMI is used in clinical development to select efficacious vaccines and to define correlates of efficacy. Correlates of efficacy for antibodies to sporozoites should also be assessed by separate DVI and ID challenges. The study was registered with ClinicalTrials.gov ( NCT03947190 ) and PACTR ( PACTR202108505632810 ).

The identification of genetic markers has revolutionized the assessment of antimalarial drug resistance. Tracking the molecular markers of resistance emerged as a valuable tool over 60 years ago, following the identification of sulfadoxine-pyrimethamine (SP) genetic resistance markers, dhfr and dhps. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) guidelines were used. PubMed/MEDLINE, Embase, Scopus, Google Scholar and Web of Science were systematically searched to identify studies on antimalarial drug resistance markers in Kenya published in English between 01-Jan-1995 and 31-Oct-2024. The national analysis showed a regional shift in the timelines from the mutant to wild-type crt genotype and similarly from the mutant (CVIET) to wild-type (CVMNK) microhaplotype, with the Coast occurring earlier in 2002, while Western Kenya the change occurred later in 2008. MDR1 codons 86 and 1246, also genetic markers of chloroquine resistance have shown a full reversion to the wildtype, that was rising since 1994 in the Coast and 2003 in Western Kenya. By the time drug policy changed to SP in 1999 the dhps mutant genotype was already rising from 1996 in the Coast and 1998 from Western Kenya, while the dhfr codon 108 shift to mutant occurred as early as 1988 in the Coastal parasite populations. The World Health Organization-validated k13 mutations were first described, P553L, in 2006 in Kisumu. This aggregation of data across Kenya demonstrates the utility of this scoping review. The compilation and standardization of over 100 studies provides a high-level, structured overview of when and where resistance markers have been surveyed. This establishes a foundational national repository to support strategic surveillance planning by the Kenya NMCP.

Recent reports from East African countries indicate the emergence and spread of artemisinin partial resistance (ART-R), posing a significant threat to malaria control efforts in the region. The presence of critical Plasmodium falciparum kelch13 (k13) resistance markers, including C469Y , P553L and A675V , have been detected in Kenya, although their clinical significance remains unclear. This highlights an urgent need to closely monitor the prevalence of these mutations. A total of 24,227 dried blood spot (DBS) samples were collected from 82 primary schools across eight counties in Western Kenya during repeated cross-sectional surveys conducted in 2019 ( n = 8,111), 2022 ( n = 8,086), and 2023 ( n = 8,200). Initial screening was performed using a rapid diagnostic test (RDT), and DNA extraction was conducted on RDT-positive samples. These samples were further analyzed using a Pf 18s qPCR assay to quantify the Plasmodium falciparum DNA. Amplicons from malaria-positive samples were sequenced using a previously established amplicon deep sequencing pipeline to analyze mutations in Pfk13 . A total of 500, 920 and 1058 samples from 2019, 2022, and 2023, respectively were successfully processed, enabling a temporal assessment of the changes in k13 mutations in the region. Four mutations that have previously been associated with artemisinin resistance were found. The A675V mutation was the most prevalent, being found in all 8 counties. It was absent in 2019 and increased from 0.9% in 2022 to 5% in 2023. In contrast, the C469Y mutation declined from 4% in 2022 to 1% in 2023, maintaining a presence in 3 counties. The P553L mutation was only detected in 2022 in 1.2% of the samples across 5 counties. The R561H mutation was not detected in 2019 and 2022 but emerged at a low frequency (0.5%) in 2023 in 2 counties. Siaya and Kisumu were the only counties with all 4 validated mutations between 2022 and 2023. The rising prevalence and geographical presence of the A675V mutation and the new detection of R561H in 2023 highlights the critical need for robust molecular surveillance systems to track the frequency and geographic spread of resistance markers. School-based sampling presents a practical and scalable approach for molecular surveillance, providing early warning signals for potential resistance hotspots. Additionally, the detection of the four WHO validated Pfk13 artemisinin resistance mutations in Western Kenya underscores the urgency of conducting regular Therapeutic Efficacy Studies (TES) to assess the continued efficacy of frontline antimalarial treatments. Integrating molecular surveillance with TES will generate important data to inform national treatment policies and support the long-term effectiveness of malaria control strategies in Kenya.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to evolve, with mutations leading to the emergence of new variants. JN.1, a subvariant of omicron BA.2.86, has demonstrated marked immune escape and is now included in updated vaccine formulations. While reduced sensitivity has been reported for antibody assays using ancestral spike protein subunits to detect omicron-induced responses, the performance of full-length spike-based assays against omicron sublineages remains unclear. We aimed to compare the sensitivity of ELISA and Luminex assays using full-length spike proteins from the ancestral Wuhan strain and the JN.1 variant. Methods: Wuhan and JN.1 full-length spike protein constructs were designed and expressed in Expi293F mammalian cells. In-house ELISAs based on previously validated protocols were used to measure anti-spike IgG levels. Additionally, a Luminex-based assay for anti-spike antibody detection was developed and validated. Both assays were applied to the following sample groups: pre-pandemic samples (designated “gold standard negatives”); PCR confirmed 2020 positives (“gold standard wildtype positives”); PCR confirmed 2024 positives (“gold standard omicron positives”); 2022 vaccinated individuals with verbal confirmed infection (“gold standard hybrid positives”); and 2024 household samples (“unknowns”). Results: Wuhan spike protein showed a sensitivity of 100% (95% CI: 0.88–1.0) in detecting omicron-specific antibodies using gold standard omicron positives with JN.1 spike protein as a reference assay. Overall, across all samples, in ELISA, the Wuhan antigen had a sensitivity of 0.93 (95% CI: 0.89–0.95) and a specificity of 0.98 (95% CI: 0.94–0.99). The JN.1 antigen showed a sensitivity of 0.91 (95% CI: 0.87–0.94) and a specificity of 0.97 (95% CI: 0.93–0.99). In Luminex, sensitivity was 0.95 (95% CI: 0.91–0.97) for Wuhan and 0.94 (95% CI: 0.91–0.96) for JN.1. Specificity for both antigens in Luminex was 0.98 (95% CI: 0.94–0.99). Conclusions: Both ELISA and Luminex assays showed comparable sensitivity and specificity for both Wuhan and JN.1 antigens, indicating that mutations in the JN.1 variant do not significantly impact assay performance. This suggests preserved antigenic recognition across variants.

Background: Historically, chloroquine resistance emerged that was driven by mutations in the chloroquine resistance transporter ( Pfcrt) gene. This led to the global withdrawal of chloroquine in 1998 and its subsequent replacement with sulfadoxine-pyrimethamine, whose efficacy was compromised by a high prevalence of mutations in the dihydrofolate reductase and the dihydropteroate synthase genes by 2004. Consequently, artemisinin-based combination therapies (ACTs) were introduced in 2006. Since then, thirteen mutations in the kelch 13 (Pfk13) propeller domain have emerged and validated by the World Health Organization (WHO) as markers of partial artemisinin resistance. This study aimed to characterize temporal trends in both established, Pfcrt and Pfk13 and less well-described potential markers, cysteine desulfurase ( Pfnfs) and Pfcoronin , using febrile malaria samples collected across diverse regions of Kenya between 2013 and 2022. Methods: The temporal trend of these markers of resistance were assessed by screening archived P. falciparum positive dried blood spots (DBS). A total of 1,750 DBS samples collected from Therapeutic Efficacy Studies (TES) conducted in: Kwale (2013, n=350), Kisumu (2015, n=314), Busia (2016, n=334), Kisii (2017, n=314), Kwale (2018, n=150), and a hrp2 study conducted Kisii (2022, n=288). Parasite genomic DNA was extracted using the Chelex-saponin method and confirmed by a Pf 18S RT-PCR. Pfk13, Pfcrt, Pfnfs and Pfcoronin PCR amplicons were sequenced using capillary electrophoresis, Illumina Miseq or the Oxford Nanopore (GridION) platform. Results: The prevalence of Pfcrt mutations declined over time and no WHO validated Pfk13 mutations associated with artemisinin resistance were detected. However, synonymous substitutions at WHO-validated codons C469C and P553P were identified. In the PfCoronin gene, non-synonymous mutations distinct from those reported in West Africa were observed at high frequencies (>75%). Notably, the Pfnfs -K65Q mutation, previously associated with reduced lumefantrine sensitivity in West Africa, was detected in over 80% of samples. Our findings reveal differences in some antimalarial resistance genetic markers between observations made in The Gambia and Senegal (West Africa) and Kilifi (East Africa). Based on the convenient sample set, there were no WHO validated k13 mutations up until 2022, suggesting continued ACT efficacy in Kenya. This study underscores the importance of continued molecular surveillance and suggests that resistance may evolve through different pathways in East compared to West Africa and Southeast Asia.

The identification of genetic markers has revolutionised our assessment of antimalarial drug resistance. Tracking the molecular markers of resistance emerged as a valuable tool over 60 years ago, following the identification of sulphadoxine-pyrimethamine genetic resistance markers, dhfr and dhps . The Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) guidelines were used. PubMed/MEDLINE, Embase, Scopus, Google Scholar and Web of Science were systematically searched to identify studies on antimalarial drug resistance markers in Kenya published in English between 01-Jan-1995 and 31-Oct-2024. The national analysis showed a regional shift in the timelines from the mutant to wild-type crt genotype and similarly from the mutant (CVIET) to wild-type (CVMNK) microhaplotype, with the Coast occurring earlier in 2002, while Western Kenya the change occurred later in 2008. MDR1 codons 86 and 1246, also genetic markers of chloroquine resistance have shown a full reversion to the wildtype, that was rising since 1994 in the Coast and 2003 in Western Kenya. By the time drug policy changed to SP in 1999 the dhps mutant genotype was already rising from 1996 in the Coast and 1998 from Western Kenya, while the dhfr codon 108 shift to mutant occurred as early as 1988 in the Coastal parasite populations. Emerging WHO validated k13 mutations were first described, P553L, in 2006 in Kisumu. This aggregation of data across Kenya demonstrates the utility of this scoping review. The compilation and standardization of over 100 studies provides a high-level, structured overview of when and where resistance markers have been surveyed. This establishes a foundational national repository to support strategic surveillance planning by the Kenya NMCP.

Genetic diversity in Plasmodium falciparum poses a significant challenge to malaria control and elimination. This is particularly important for developing fully efficacious vaccines, which should include valuable blood stage antigens. Several antigen candidates are highly diverse and require further understanding. We surveyed the genetic diversity of the highly polymorphic merozoite surface protein 2 (MSP2) in 2761 P. falciparum isolates collected across Sub-Saharan Africa. Using PCR-based genotyping and long-read sequencing, we identified extensive diversity among msp2 size variants and sequences. Some size variants were more prevalent than others across different geographical regions, transmission intensities, and time points. These variants comprised multiple unique sequences, of which several were geographically and temporally widespread. Our study reveals greater msp2 sequence diversity than previously known, while also identifying interesting similarities in sequence and gene length across Sub-Saharan Africa. These findings support the further exploration of common msp2 variants in relation to parasite virulence and vaccine development.