Anopheles stephensi is an invasive malaria vector that has been rapidly spreading within Africa since it was first identified in Djibouti in 2012. As of 2025, it has been identified in eight other countries: Ethiopia, Sudan, Somaliland, Eritrea, Nigeria, Kenya, Ghana and Niger. We collected mosquitoes from an urban slaughterhouse in Kisumu City in western Kenya in mid-2022 for bloodmeal analyses. 19.6% (11/56) of the amplicon sequence variants (ASVs) assigned using the basic local alignment search tool (BLAST) returned matches for mosquito species, one of which was An. stephensi, from one pool of five mosquitoes that had been morphologically classified as An. gambiae s.l. Further analysis of the mitochondrial cytochrome c oxidase 1 (cox1) gene later confirmed the presence of An. stephensi, which were distributed across two well-supported clades. Some haplotypes grouped with others from northern Kenya (Wajir and Marsabit), while others clustered with haplotypes from Ethiopia and Sudan. This serendipitous detection of An. stephensi is the first report of the species South of the Equator. Therefore, we recommend continued entomological and malaria case surveillance in this area to evaluate how shifting vector dynamics and vector-livestock interactions may impact future control initiatives.

Matrix-assisted laser desorption-ionisation time of flight mass spectrometry (MALDI-TOF MS) is a powerful analytical method that has been used extensively to identify sample ions of complex mixtures, and biological samples such as proteins, tissues and microorganisms. MALDI-TOF MS has revolutionised clinical microbiology with accurate, rapid, and inexpensive species-level identification of microbes. MALDI-TOF MS technology generates spectral signatures and matches them to a library of similar organisms using bioinformatics pattern matching. The use of MALDI-TOF MS for entomological samples has been explored by multiple groups with proven efficacy at differentiating between closely related species, as well as detecting pathogens in different vectors. The low cost per sample processing, rapid turnaround and robustness are attractive for surveillance of vector control programs. Libraries are built in-house for institutional usage, although a multi-user platform with sharing of spectra and data would be attractive. Only a few studies have strived to make their libraries publicly available. Here, we outline a stepwise approach for creating an in-house MALDI-TOF MS library and subsequent query, using malaria vector species identification as a case study for entomological samples. A protocol and video of the methodology are also shared. Moreover, the libraries related to this publication have been deposited in public repository (https://doi.org/10.7910/DVN/VYQFNO37) for anyone with MALDI-TOF MS equipment to adapt.

BACKGROUND:Characterizing malaria epidemiology at the local level requires understanding the diverse malaria vector species driving transmission, including both primary and secondary vectors. Effective mosquito surveillance and accurate species identification are critical; however, due to the associated cost and complexity, most surveillance strategies mainly focus on the primary malaria vectors. There is a need for cost-effective methods that can reliably identify both primary and secondary vectors as their role in transmission becomes increasingly important while reaching towards elimination. This study aimed to evaluate the use of MALDI-TOF MS as a sustainable tool for identifying secondary malaria vector. METHODS:Adult mosquitoes were collected in Kenya and Mozambique and morphologically identified. Secondary malaria vectors were considered as any Anopheline that did not pertain to Anopheles gambiae sensu lato (s.l.). or Anopheles funestus sensu lato (s.l.). At KEMRI Wellcome Trust Research Programme, MALDI TOF MS spectra were obtained from individual cephalothoraxes. Library creation and querying were guided by confirmatory species identification using Sanger sequencing of a subset of mosquitoes, targeting the Internal Transcribed Spacer 2 (ITS2) region of nuclear ribosomal DNA and the mitochondrial Cytochrome c Oxidase Subunit I (COI) gene. The libraries were then applied for the identification of other secondary malaria vectors. RESULTS:Species identification of secondary malaria vectors using MALDI-TOF MS showed high concordance with Sanger sequencing with an overall accuracy of 91% and a kappa value of 0.87. The technique demonstrated a sensitivity and specificity of 100% for most species, except for distinguishing between Anopheles cf. coustani 2 NFL-2015 and Anopheles ziemanni. In Kenya, the Anopheles species identified were Anopheles cf. coustani 2 NFL-2015 (19), Anopheles pretoriensis (6), Anopheles rufipes (8), Anopheles ziemanni (8), Anopheles coustani (2), and Anopheles pharoensis (1). In Mozambique, the identified species comprised: An. cf. coustani 2 NFL-2015 (10), An. pretoriensis (2), An. ziemanni (7), An. coustani (28), and An. pharoensis (4). CONCLUSION:The results provide evidence that MALDI-TOF can identify secondary malaria vectors from Eastern and Southeastern African regions. This technique was as efficient as DNA sequencing in identifying mosquito species. Indeed, except for An. cf coustani 2NFL-2015 and An. ziemanni, an exact species identification was obtained for all individual mosquitoes. These findings highlight the potential of MALDI-TOF MS for monitoring malaria vectors.

Tungiasis, one of the oldest and most neglected tropical diseases endemic to sub-Saharan Africa and the Americas, is caused by the female parasitic flea, Tunga penetrans . The flea burrows into the skin, leading to acute and chronic inflammation, often exacerbated by bacterial superinfection. Despite its significant public health impact, genomic studies on T. penetrans are scarce. Here, we present the first complete mitochondrial genome of T. penetrans , comprising 17,279 base pairs and encoding 13 protein-coding genes, 22 transfer RNAs and 2 ribosomal RNAs. Phylogenetic analysis of the cox2 gene revealed a divergent basal lineage from Brazil, supporting a South American origin of T. penetrans and highlights genetic differentiation within the Americas. Clustering of the Ecuadorian and African isolates further suggests historical connections, likely linked to transatlantic maritime trade. The tree highlights a strong South American origin and evidence of migration and diversification, facilitated by human and animal movement. Phylogenetic analysis of the complete genomes relying on the protein-coding genes of other fleas revealed that T. penetrans is closely related to Dorcadia ioffi , a semi-sessile flea of goats and sheep in China. This mitochondrial genome provides a critical resource for future studies on molecular epidemiology, evolutionary history, and control of tungiasis. ### Competing Interest Statement The authors have declared no competing interest. Royal Society, https://ror.org/03wnrjx87, FLR\R1\190497, FCG\R1\211043

Malaria remains a major threat during humanitarian crises, necessitating targeted vector control strategies informed by local vector dynamics. Between May and July 2023, we conducted larval surveys in refugee settlements across Dadaab, Kakuma, and Kalobeyei (Kenya), collecting Anopheles larvae. Genotyping of 728 specimens revealed spatial variations in species composition. Overall, Anopheles arabiensis was the dominant species (59%, n=426), followed by Anopheles coluzzii (35%, n=252), and Anopheles rufipes (1%, n=7). In Dadaab, An. arabiensis was overwhelmingly dominant (94%, n=352/374). In contrast, the Kakuma/Kalobeyei complex was characterized by the co-occurrence of An. coluzzii (72%, n=252/350) and An. arabiensis (22%, n=74/350), with An. rufipes exclusively found in Kalobeyei (7%, n=6/89) ([Figure 2B][1]). Notably, no members of the Anopheles funestus group or Anopheles stephensi were detected. However, approximately 5% of the larvae across the sites could not be resolved molecularly. High frequencies of the L1014F kdr mutation, a pyrethroid resistance marker, were detected in An. coluzzii (Kakuma: 50%; Kalobeyei: 63%) and An. arabiensis (Kakuma: 10%; Kalobeyei: 30%) populations in Turkana County. Interestingly, no kdr mutations were observed in the An. arabiensis population from Dadaab. These findings highlight significant spatial diversity in vector species composition and resistance profiles, with An. coluzzii emerging as a dominant, pyrethroid-resistant vector in the Kakuma/Kalobeyei complex. The results underscore the urgent need for targeted interventions, including resistance monitoring and alternative insecticide-based strategies, to mitigate malaria transmission risks in fragile, humanitarian settings. Further studies are warranted to address unidentified larval species and seasonal transmission dynamics. ### Competing Interest Statement The authors have declared no competing interest. The Royal Society FLAIR fellowship, UK, FLR\R1\190497, FCG\R1\211043 Africa CDC PGI and ASLM, Pathogen genomics sub-Award Wellcome Trust, 212176 [1]: #F2

The age structure of a mosquito population helps estimate the proportion of vectors capable of transmitting malaria. Many malaria transmission models rely on mosquito longevity as key parameter. However, these are rarely measured in the field due to lack of a reliable and scalable age-grading method. An accurate method could improve predictions of malaria risk and the impact assessment of interventions. This study aimed to investigate the use of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) for malaria vector age-grading using insectary-reared and wild-caught mosquitoes. Anopheles gambiae s.s. mosquitoes were reared in the insectary to different known physiological and chronological ages to evaluate if MALDI-TOF MS could be used to distinguish between different age groups. Wild mosquitoes were collected from Mozambique and Kenya and dissected to determine their parity status. Reference spectra were obtained from mosquito’s cephalothorax and used to create predictive databases which were validated using independent samples. MALDI-TOF MS identified the physiological and chronological age of insectary-reared mosquitoes with 94.52% and 77% accuracy respectively. Field-collected mosquitoes were primarily An. funestus s.s. and An. gambiae s.s. Parity prediction accuracy was between 81% and 87%. MALDI-TOF MS was able to distinguish and differentiate mosquitoes based on their age structure (chronological and physiological) and parity status.

BACKGROUND:The use of MALDI-TOF MS for mosquito identification and surveillance is routinely used in developed countries as an affordable alternative to molecular methods. However, in low- and middle-income countries (LMIC) where mosquito-borne diseases carry the greatest burden, the method is not commonly employed. Using the Kenyan national malaria program (NMCP) as a case study, we compared the costs of current methods used for malaria vector surveillance to those that would be incurred if MALDI-TOF MS were used instead. METHODS:A deterministic decision tree analytic model was developed to systematically calculate the costs associated with materials and labour, and time-to-results for two workflows, i.e., current molecular methods versus MALDI-TOF MS. The analysis assumed an annual sample size of 15,000 mosquitoes (representing the average number of mosquitoes analysed annually by the Kenyan NMCP) processed at a local laboratory in Kenya. FINDINGS:We estimate that if the Kenyan national entomological surveillance program shifted sample processing completely to MALDI-TOF MS, it would result in 74.48% net time saving, up to 84% on material costs and 77% on labour costs, resulting in an overall direct cost savings of 83%. INTERPRETATION:Adoption of MALDI-TOF MS for malaria vector surveillance can result in substantial time and cost savings. The ease of performance, the rapid turn-around time, and the modest cost per sample may bring a paradigm shift in routine entomological surveillance in Africa.

BACKGROUND:Long-lasting insecticidal nets are the primary malaria vector control measure in coastal Kenya. In 2018, phenotypic resistance to pyrethroids and low frequency of L1014S kdr mutation were reported in the Anopheles gambiae complex. Since then, additional pyrethroid-treated nets were distributed in 2021. The objectives of this study were to determine the insecticide resistance profiles of An. gambiae and Anopheles funestus from Kwale County and evaluate potential resistance mechanisms. METHODS:From July 2023 to May 2024, adult and larval collections of An. funestus and An. gambiae mosquitoes were done with the aim of conducting insecticide susceptibility bioassays using WHO protocol for permethrin, deltamethrin, bendiocarb, DDT and pirimiphos-methyl. Species found resistant to pyrethroids were subjected to synergism testing by pre-exposure to piperonyl-butoxide (PBO). This was followed by genotyping of resistance-associated mutations in An. funestus (CYP6P9a, CYP6P9b, GSTe2-L119F and 6.5kb S.V) and An. gambiae (kdr L1014S and L1014F). Sibling species identification was done using PCR. The association between genetic markers and phenotypic resistance was explored using logistic regression. RESULTS:A total of 1826 An. gambiae and 715 An. funestus were used in insecticide susceptibility bioassays. Both An. gambiae and An. funestus were resistant to permethrin (mortality, 58.7% and 57.1, respectively) and deltamethrin (mortality 51% and 76%, respectively), but susceptible to DDT, bendiocarb and Pirimiphos-methyl. Pre-exposure to PBO increased susceptibility to deltamethrin in both species. Both kdr west and east were detected in Anopheles arabiensis (L1014S freq = 0.083, L1014F freq = 0.063) and Anopheles quadriannulatus (L1014S freq = 0.074, L1014F freq = 0.043) at low frequencies. Anopheles funestus sensu stricto and Anopheles rivolurum had the presence of CYP6Pa, CYP6Pb, 6.5kb S.V and GSTe2-L119F, with low allele frequencies. There were no significant associations between the genotypes and phenotypic profile. CONCLUSIONS:Malaria vectors in Kwale are resistant to pyrethroids. PBO fully restored susceptibility, indicating this resistance could be caused by metabolic mechanism. The presence of kdr and metabolic resistance alleles suggests a recent selection on Anopheles mosquitoes. Pyrethroid-only nets may not fully ensure community protection against malaria in coastal Kenya due to resistance. Operational failure remains uncertain, requiring further studies. Net distribution programs should consider pyrethroid-PBO nets to enhance malaria control effectiveness.

This study analyzes the distribution, genetic diversity, and spread of Anopheles stephensi in Kenya following initial detection in December 2022. A total of 114 larval and 33 adult An. stephensi samples were confirmed in 7 of 18 surveyed counties majorly along transportation routes. Genetic analyses revealed three distinct genetic compositions with different levels of genetic diversity, suggesting multiple introductions into the country. The genetic composition of mosquitoes in most counties resembled southern Ethiopian populations, while those from Turkana showed a unique haplotype. A species distribution model predicts a more extensive range than currently observed, with low precipitation and minimal seasonal temperature variations as key factors influencing distribution. Challenges in adult sampling were noted, with larval sampling revealing co-occurrence with native Anopheles species. The findings have implications for surveillance and control strategies, emphasizing the need for continued monitoring, refined sampling techniques to inform bionomics, and cross-border collaboration.

Trichuris trichiura remains a major global public health concern, particularly in low-resource settings where standard anthelmintic regimens are limited. This study evaluated the diagnostic performance of real-time PCR (qPCR) compared to the Kato-Katz (KK) method in assessing the efficacy of a fixed-dose combination (FDC) of albendazole and ivermectin versus albendazole for the treatment of T. trichiura. The study was embedded within the ALIVE clinical trial (NCT05124691), a phase 2/3 trial conducted in Kenya, Mozambique, and Ethiopia. Stool samples were collected at baseline and 21 ± 7 days post-treatment, with KK performed on fresh samples and qPCR on ethanol-preserved aliquots. In total 534 participants were selected based on positive KK and qPCR at baseline and complete data post-treatment. The primary endpoint was cure rate (CR) by KK and qPCR; secondary endpoints included egg reduction rate (ERR) and cycle threshold (Ct) value incrementation rate (CtIR). Additionally, machine learning algorithms were used to predict infection intensity from qPCR Ct-values and demographic variables. qPCR confirmed the superior efficacy of FDC compared to albendazole as previously shown by KK, but discrepancies were observed in CRs between qPCR and KK, particularly lower qPCR CRs for FDC×1 and FDC×3. Concordance between stool egg counts and Ct-value decreased post-treatment, likely due to reduced KK sensitivity in low-intensity infections. ERR and CtIR showed parallel patterns of efficacy across treatment arms. Machine learning models showed good performance for predicting baseline infection intensity. While not interchangeable, qPCR complements KK and enhances the precision of drug efficacy evaluation in helminth clinical trials.

This study analyzes the distribution, genetic diversity, and spread of An. stephensi in Kenya following initial detection in December 2022. A total of 114 larval and 33 adult An. stephensi samples were confirmed in 7 of 18 surveyed counties majorly along transportation routes. Genetic analyses revealed three distinct genetic compositions with different levels of genetic diversity, suggesting multiple introductions into the country. The genetic composition of mosquitoes in most counties resembled southern Ethiopian populations, while those from Turkana showed a unique haplotype. A species distribution model predicts a more extensive range than currently observed, with low precipitation and minimal seasonal temperature variations as key factors influencing distribution. Challenges in adult sampling were noted, with larval sampling revealing co-occurrence with native Anopheles species. The findings have implications for surveillance and control strategies, emphasizing the need for continued monitoring, refined sampling techniques to inform bionomics, and cross-border collaboration.

Background:Protein analysis using matrix-assisted laser desorption/ionisation time-of-flight mass-spectrometry (MALDI-TOF MS) represents a promising tool for entomological surveillance. In this study we tested the discriminative power of this tool for measuring species and blood meal source of main Afrotropical malaria vectors on the Kenyan coast. Methods:Mosquito collections were conducted along the coastal region of Kenya. MALDI-TOF MS spectra were obtained from each individual mosquito's cephalothorax as well as the abdomens of blood-engorged mosquitoes. The same mosquitoes were also processed using gold standard tests: polymerase chain reaction (PCR) for species identification and enzyme linked immunosorbent assay (ELISA) for blood meal source identification. Results:Of the 2,332 mosquitoes subjected to MALDI-TOF MS, 85% (1,971/2,332) were considered for database creation and validation. There was an overall accuracy of 97.5% in the identification of members of the An. gambiae ( An. gambiae, 100%; An. arabiensis, 91.9%; An. merus, 97.5%; and An. quadriannulatus, 90.2%) and An. funestus ( An. funestus, 94.2%; An. rivulorum, 99.4%; and An. leesoni, 94.1%) complexes. Furthermore, MALDI-TOF MS also provided accurate (94.5% accuracy) identification of blood host sources across all mosquito species. Conclusions:This study provides further evidence of the discriminative power of MALDI-TOF MS to identify sibling species and blood meal source of Afrotropical malaria vectors, further supporting its utility in entomological surveillance. The low cost per sample (<0.2USD) and high throughput nature of the method represents a cost-effective alternative to molecular methods and could enable programs to increase the number of samples analysed and therefore improve the data generated from surveillance activities.

Abstract Background Lymphatic filariasis (LF) is an infectious neglected tropical disease caused by mosquito-borne nematodes such as Wuchereria bancrofti, Brugia malayi, and Brugia timori. Globally, LF affects 51 million people, with approximately 863 million at risk in 47 countries. In Kenya, filariasis is endemic along the entire coastal strip, and more recently, at the Kenya–Ugandan border. The World Health Organization (WHO) recommends mass drug administration to reduce disease transmission and morbidity. Monitoring the effectiveness of such interventions relies on robust surveillance, achieved through microscopic examination of microfilariae in nighttime blood, detection of circulating filarial antigens (CFA), and molecular xenomonitoring. We focused on molecular xenomonitoring along the Kenyan coast due to its noninvasive nature and the opportunity to identify new vectors. Methods In 2022, mosquitoes were collected from Kilifi, Kwale, and Taita-Taveta counties located within the LF endemic region in Kenya. Subsequently, genomic deoxyribonucleic acid (gDNA) was extracted from these mosquitoes for speciation and analysis of Wuchereria bancrofti infection rates. The impact of sociodemographic and household attributes on infection rates was assessed using generalized estimating equations. Results A total of 18,121 mosquitoes belonging to Culicinae (63.0%, n = 11,414) and Anophelinae (37.0%, n = 6707) subfamilies were collected. Morphological identification revealed that Anopheline mosquitoes were dominated by An. funestus (45.4%, n = 3045) and An. gambiae (42.8%, n = 2873). Wuchereria bancrofti infection rates were highest in Kilifi (35.4%; 95% CI 28.0–43.3%, n = 57/161) and lowest in Taita Taveta (5.3%; 95% CI 3.3–8.0%, n = 22/412). The major vectors incriminated are An. rivulorum, An. funestus sensu stricto, and An. arabiensis. Mosquitoes of the An. funestus complex were significantly associated with LF transmission (OR 18.0; 95% CI 1.80–180; p = 0.014). Additionally, a higher risk of transmission was observed outdoors (OR 1.74; 95% CI 1.08–2.82; p = 0.024) and in homesteads that owned livestock (OR 2.00; 95% CI 1.09–3.66; p = 0.025). Conclusions In this study, we identified An. funestus s.l. sibling species, An. rivulorum and An. funestus s.s., as the primary vectors of lymphatic filariasis along the Kenyan coast. These findings also highlight that a significant portion of disease transmission potentially occurs outdoors where indoor-based vector control tools, including long-lasting insecticidal nets and indoor residual spray, may not be effective. Therefore, control measures targeting outdoor resting mosquitoes such as zooprophylaxis, larval source management, and attractive sugar baits may have potential for LF transmission reduction. Graphical Abstract

Lymphatic filariasis (LF) is an infectious neglected tropical disease caused by a mosquito-borne nematode and is a major cause of disability. In 2022, it was estimated that 51 million people were infected with LF. In Kenya filariasis is endemic along the entire coastal strip. The main vectors are Anopheles funestus and Anopheles gambiae in rural areas and Culex quinquefaciatus mosquitoes in urban areas. In 2022, mosquitoes were collected from Kilifi, Kwale and Taita-Taveta counties which are located within the LF endemic region in Kenya. Subsequently, genomic Deoxyribonucleic acid (DNA) was then extracted from these mosquitoes for speciation and analysis of W. bancrofti infection rates. The impact of socio-demographic and household attributes on infection rates were assessed using generalized estimating equations. A total of 18,121 mosquitoes belonging to Culex ( n = 11,414 ) and Anopheles (n = 6,707) genera were collected. Morphological identification revealed that Anopheline mosquito were dominated by An. funestus (n = 3,045) and An. gambiae (n = 2,873). Wuchereria bancrofti infection rates were highest in Kilifi (35.4%; 95% CI 28%-43.3%, n = 57/161) and lowest in Taita Taveta (5.3%; 95% CI 3.3%-8.0%, n = 22/412). The major vectors incriminated are An. rivulorum, An. funestus sensu stricto and An. arabiensis . The risk of W. bancrofti infection was significantly higher in An. funestus complex (OR = 18.0; 95% CI 1.80-180; p = 0.014) compared to An. gambiae (OR = 1.54; 95% CI 0.16-15.10; p = 0.7). Additionally, higher risk was observed in outdoor resting mosquitoes (OR = 1.72; 95% CI 1.06-2.78; p = 0.027) and in homesteads that owned livestock (OR = 2.05; 95% CI 1.11-3.73; p = 0.021). Bednet (OR = 0.39; 95% CI 0.12-1.32; p = 0.13) and poultry ownership (OR = 0.52; 95% CI 0.30-0.89, p = 0.018) seems to provide protection. Anopheles funestus complex emerged as the primary vectors of lymphatic filariasis along the Kenyan coast. These findings also highlight that a significant portion of disease transmission potentially occurs outdoors. Therefore, control measures targeting outdoor resting mosquitoes such as zooprophylaxis, larval source management and attractive sugar baits may have potential for LF transmission reduction. Author summary Lymphatic filariasis (LF) in the African continent is mainly caused by a mosquito-borne nematode: Wuchereria bancrofti . In urban areas transmission is mainly by Culex quinquefaciatus whereas in rural areas it is dominated by Anopheles funestus and Anopheles gambiae mosquitoes. We investigated the vectorial systems for LF in rural coastal Kenya and factors associated with the risk of diseases transmission in the region. We identified An. funestus sensu lato sibling species An. rivulorum and An. funestus sensu stricto as the dominant vectors of lymphatic filariasis along the Kenyan coast. We also show that a higher proportion of transmission is likely to take place outdoors necessitating the implementation of vector control strategies that target exophilic mosquitoes such as zooprophylaxis and larval source management. Factors associated with transmission of LF include ownership of livestock and houses made of natural materials such as thatched roof and mud walls. Bednet and poulty ownership were associated with protection. We also highlight the importance of molecular xenomonitoring in the surveillance of lymphatic filariasis, because of its’ non-invasive nature and potential for incriminating new vectors of lymphatic filariasis. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement Yes ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study was approved by the KEMRI Scientific and Ethics Review Unit (SERU) with the protocol number: KEMRI/SERU/CGMR-C/024/3148. Verbal informed consent was obtained from the household heads before metadata and mosquito collection. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Most of the dataset used for analysis is available in the manuscript. We withheld the geo-data which may predispose individual homesteads to a high risk of identifiability. However, they are under the custodianship of the KEMRI-Wellcome Trust Data Governance Committee and are accessible upon request addressed to that committee.

Background The EDCTP_STOP project is a multicentric clinical trial (ALIVE trial ct.gov: NCT05124691) that aims to interrupt the transmission of soil-transmitted helminths using novel treatment regimens. While cure rate measured by microscopy is the primary efficacy outcome, limitations in sensitivity after successful treatment pose a challenge. Nucleic acid amplification tests are a promising alternative. One objective in the EDCTP_STOP project is to assess real-time polymerase chain reaction (qPCR) as a secondary efficacy outcome, which necessitates implementing an external quality assessment scheme (EQAS). Methods The Helminth External Molecular Quality Assessment Scheme (HEMQAS), provided by the Dutch Foundation for Quality Assessment in Medical Laboratories (SKML), was implemented in the study. The sample distribution consists of blinded ethanol-preserved stool samples to assess DNA extraction, and purified DNA samples in stabilizing buffer to assess the amplification technique. Four consortium partners participated in the 2022 assessment. LUMC scored 99% (91/92 targets correctly identified). KEMRI scored 74% (68/92 targets). CISM scored 99% (75/76) and ULE scored 100% (62/62 targets). Results For stool samples, the outcomes demonstrated that ineffective DNA extraction caused multiple false negative outcomes, particularly for Trichuris trichiura. Pipetting-error during DNA extraction may explain false positive outcomes. For DNA samples, false negative outcomes most likely resulted from handling errors. Systematic errors such as qPCR channels used to detect targets may account for false positive outcomes as spectral overlap in a multiplex qPCR may cause incorrect data interpretation. The use of validated positive control DNA elucidated which primer and probe pairs required optimization. Conclusion These outcomes facilitated targeted molecular optimization per trial site prior to testing trial samples. Participating in an EQAS facilitates capacity building by identifying training and laboratory validation needs, and ensures reliable reproducible results.

Background Malaria remains one of the most important infectious diseases in sub-Saharan Africa, responsible for approximately 228 million cases and 602,000 deaths in 2020. In this region, malaria transmission is driven mainly by mosquitoes of the Anopheles gambiae and, more recently, Anopheles funestus complex. The gains made in malaria control are threatened by insecticide resistance and behavioural plasticity among these vectors. This, therefore, calls for the development of alternative approaches such as malaria transmission-blocking vaccines or gene drive systems. The thioester-containing protein 1 ( TEP1 ) gene, which mediates the killing of Plasmodium falciparum in the mosquito midgut, has recently been identified as a promising target for gene drive systems. Here we investigated the frequency and distribution of TEP1 alleles in wild-caught malaria vectors on the Kenyan coast. Methods Mosquitoes were collected using CDC light traps both indoors and outdoors from 20 houses in Garithe village, along the Kenyan coast. The mosquitoes were dissected, and the different parts were used to determine their species, blood meal source, and sporozoite status. The data were analysed and visualised using the R (v 4.0.1) and STATA (v 17.0). Results A total of 18,802 mosquitoes were collected, consisting of 77.8% ( n = 14,631) Culex spp., 21.4% ( n = 4026) An. gambiae sensu lato, 0.4% ( n = 67) An. funestus , and 0.4% ( n = 78) other Anopheles ( An. coustani , An. pharoensis , and An. pretoriensis ). Mosquitoes collected were predominantly exophilic, with the outdoor catches being higher across all the species: Culex spp. 93% (IRR = 11.6, 95% Cl [5.9–22.9] P < 0.001), An. gambiae s.l. 92% (IRR = 7.2, 95% Cl [3.6–14.5]; P < 0.001), An. funestus 91% (IRR = 10.3, 95% Cl [3.3–32.3]; P < 0.001). A subset of randomly selected An. gambiae s.l. ( n = 518) was identified by polymerase chain reaction (PCR), among which 77.2% were An. merus , 22% were An. arabiensis , and the rest were not identified. We were also keen on identifying and describing the TEP1 genotypes of these mosquitoes, especially the *R3/R3 allele that was identified recently in the study area. We identified the following genotypes among An. merus : *R2/R2 , *R3/R3 , *R3/S2 , *S1/S1 , and *S2/S2 . Among An. arabiensis , we identified *R2/R2 , *S1/S1 , and *S2/S2 . Tests on haplotype diversity showed that the most diverse allele was TEP1*S1 , followed by TEP1*R2 . Tajima’s D values were positive for TEP1*S1 , indicating that there is a balancing selection, negative for TEP1*R2 , indicating there is a recent selective sweep, and as for TEP1*R3 , there was no evidence of selection. Phylogenetic analysis showed two distinct clades: refractory and susceptible alleles. Conclusions We find that the malaria vectors An. gambiae s.l. and An. funestus are predominantly exophilic. TEP1 genotyping for An. merus revealed five allelic combinations, namely *R2/R2 , *R3/R3 , *R3/S2 , *S1/S1 and *S2/S2 , while in An. arabiensis we only identified three allelic combinations: *R2/R2 , *S1/S1 , and *S2/S2. The TEP1*R3 allele was restricted to only An. merus among these sympatric mosquito species, and we find that there is no evidence of recombination or selection in this allele. Graphical Abstract

Background: Antimalarial drug resistance is a major obstacle to sustainable malaria control. Here we use amplicon sequencing to describe molecular markers of drug resistance in Plasmodium falciparum parasites from Kilifi county in the coastal region of Kenya over a 25-year period. Methods: We performed P. falciparum amplicon sequencing on 1162 malaria-infected blood samples collected between 1994 and 2018 to identify markers of antimalarial drug resistance in the Pfcrt, Pfdhfr, Pfdhps, Pfmdr1, Pfexo, Pfkelch13, plasmepsin 2/3, Pfarps10, Pffd, and Pfmdr2 genes. We further interrogated parasite population structure using a genetic barcode of 101 drug resistance-unrelated single nucleotide polymorphisms (SNPs) distributed across the genomes of 1245 P. falciparum parasites. Results: Two major changes occurred in the parasite population over the 25 years studied. In 1994, approximately 75% of parasites carried the marker of chloroquine resistance, CVIET. This increased to 100% in 1999 and then declined steadily, reaching 6.7% in 2018. Conversely, the quintuple mutation form of sulfadoxine-pyrimethamine resistance increased from 16.7% in 1994 to 83.6% in 2018. Several non-synonymous mutations were identified in the Kelch13 gene, although none of them are currently associated with artemisinin resistance. We observed a temporal increase in the Pfmdr1 NFD haplotype associated with lumefantrine resistance, but observed no evidence of piperaquine resistance. SNPs in other parts of the genome showed no significant temporal changes despite the marked changes in drug resistance loci over this period. Conclusions: We identified substantial changes in molecular markers of P. falciparum drug resistance over 25 years in coastal Kenya, but no associated changes in the parasite population structure.

Abstract Background Malaria remains one of the most important infectious diseases in Sub-Saharan Africa, responsible for approximately 228 million cases and 602,000 deaths in 2020. Malaria transmission is mainly driven by mosquitoes of the Anopheles gambiae and more recently Anopheles funestus complex. The gains made in malaria control are threatened by insecticide resistance and behavioural plasticity among these vectors. This, therefore, calls for the development of alternative approaches such as malaria transmission-blocking using gene drive systems that can lead to population replacement of infection-susceptible mosquitoes with mosquitoes that are refractory to Plasmodium spp. infection. One such gene that can be utilised is the Thioester-containing protein 1 (TEP1) gene which mediates the killing of Plasmodium falciparum in the mosquito midgut. Here we investigated the frequencies and distribution of TEP1 alleles in wild-caught malaria vectors along the Kenyan coast. Methods Mosquitoes were collected using CDC light traps both indoors and outdoors from 20 houses in Garithe village, along the Kenyan coast. The mosquitoes were dissected, and the different parts were used to determine their species, blood-meal source, and sporozoite status. The data was analysed and visualised using the R (v 4.0.1) and STATA (v 17.0). Results A total of 18,802 mosquitoes were collected consisting of 77.8% (n = 14,631) Culex spp , 21.4% (n = 4,026) An. gambiae s.l , 0.4% (n = 67) An. funestus and 0.4% (n = 78) other Anopheles ( An. coustanii, An. pharoensis and An. pretoriensis ). A subset of randomly selected An. gambiae s.l (n = 518) was identified by polymerase chain reaction (PCR), of these 77.2% were An. merus , 22% were An. arabiensis and the rest were not detected. Mosquitoes collected were predominantly exophilic with the outdoor catches being higher across all the species: Culex spp 93% (IRR = 11.6, 95% Cl [5.9–22.9] p < 0.001), An. gambiae s.l 92% (IRR = 7.2, 95% Cl [3.6–14.5]; p < 0.001), An. funestus 91% (IRR = 10.3, 95% Cl [3.3–32.3]; p < 0.001). We identified the following genotypes among An. merus : *R2/R2, *R3/R3, *R3/S2, *S1/S1 and *S2/S2 . Among An. arabiensis , we identified *R2/R2, *S1/S1, *S2/S2 . Tests on haplotype diversity showed that the most diverse allele was TEP1*S1 followed by TEP1*R2 . Tajima’s D values were positive for TEP1*S1 indicating that there is a balancing selection, negative for TEP1*R2 indicating there is a recent selective sweep and as for TEP1*R3 there was no evidence of selection. Phylogenetic analysis showed two distinct clades exist: Refractory and susceptible alleles. Conclusion We find that the malaria vectors An. gambiae s.l and An. funestus are predominantly exophilic. TEP1 genotyping for An. merus revealed 5 allelic combinations: *R2/R2, *R3/R3, *R3/S2, *S1/S1 and *S2/S2 while in An. arabiensis , we only identified 3 allelic combinations: *R2/R2, *S1/S1, *S2/S2. The TEP1 *R3 allele was restricted to only An. merus among these sympatric mosquito species and we find that there is no evidence of recombination or selection in this allele.