Molecular surveillance of Kelch 13 polymorphisms in Plasmodium falciparum isolates from Kenya and Ethiopia.

BACKGROUND: Timely molecular surveillance of Plasmodium falciparum kelch 13 (k13) gene mutations is essential for monitoring the emergence and stemming the spread of artemisinin resistance. Widespread artemisinin resistance, as observed in Southeast Asia, would reverse significant gains that have been made against the malaria burden in Africa. The purpose of this study was to assess the prevalence of k13 polymorphisms in western Kenya and Ethiopia at sites representing varying transmission intensities between 2018 and 2022. METHODS: Dried blood spot samples collected through ongoing passive surveillance and malaria epidemiological studies, respectively, were investigated. The k13 gene was genotyped in P. falciparum isolates with high parasitaemia: 775 isolates from four sites in western Kenya (Homa Bay, Kakamega, Kisii, and Kombewa) and 319 isolates from five sites across Ethiopia (Arjo, Awash, Gambella, Dire Dawa, and Semera). DNA sequence variation and neutrality were analysed within each study site where mutant alleles were detected. RESULTS: Sixteen Kelch13 haplotypes were detected in this study. Prevalence of nonsynonymous k13 mutations was low in both western Kenya (25/783, 3.19%) and Ethiopia (5/319, 1.57%) across the study period. Two WHO-validated mutations were detected: A675V in three isolates from Kenya and R622I in four isolates from Ethiopia. Seventeen samples from Kenya carried synonymous mutations (2.17%). No synonymous mutations were detected in Ethiopia. Genetic variation analyses and tests of neutrality further suggest an excess of low frequency polymorphisms in each study site. Fu and Li's F test statistic in Semera was 0.48 (P > 0.05), suggesting potential population selection of R622I, which appeared at a relatively high frequency (3/22, 13.04%). CONCLUSIONS: This study presents an updated report on the low frequency of k13 mutations in western Kenya and Ethiopia. The WHO-validated R622I mutation, which has previously only been reported along the north-west border of Ethiopia, appeared in four isolates collected from eastern Ethiopia. The rapid expansion of R622I across Ethiopia signals the need for enhanced monitoring of the spread of drug-resistant P. falciparum parasites in East Africa. Although ACT remains currently efficacious in the study areas, continued surveillance is necessary to detect early indicators of artemisinin partial resistance.

Epidemiology of Plasmodium vivax in Duffy negatives and Duffy positives from community and health centre collections in Ethiopia.

BACKGROUND: Malaria remains a significant cause of morbidity and mortality in Ethiopia with an estimated 3.8 million cases in 2021 and 61% of the population living in areas at risk of malaria transmission. Throughout the country Plasmodium vivax and Plasmodium falciparum are co-endemic, and Duffy expression is highly heterogeneous. The public health significance of Duffy negativity in relation to P. vivax malaria in Ethiopia, however, remains unclear. This study seeks to explore the prevalence and rates of P. vivax malaria infection across Duffy phenotypes in clinical and community settings. METHODS: A total of 9580 and 4667 subjects from community and health facilities from a malaria endemic site and an epidemic-prone site in western Ethiopia were enrolled and examined for P. vivax infection and Duffy expression from February 2018 to April 2021. Association between Duffy expression, P. vivax and P. falciparum infections were examined for samples collected from asymptomatic community volunteers and symptomatic subjects from health centres. RESULTS: Infection rate of P. vivax among Duffy positives was 2-22 fold higher than Duffy negatives in asymptomatic volunteers from the community. Parasite positivity rate was 10-50 fold higher in Duffy positives than Duffy negatives among samples collected from febrile patients attending health centres and mixed P. vivax and P. falciparum infections were significantly more common than P. vivax mono infections among Duffy negative individuals. Plasmodium vivax parasitaemia measured by 18sRNA parasite gene copy number was similar between Duffy positives and Duffy negatives. CONCLUSIONS: Duffy negativity does not offer complete protection against infection by P. vivax, and cases of P. vivax in Duffy negatives are widespread in Ethiopia, being found in asymptomatic volunteers from communities and in febrile patients from health centres. These findings offer evidence for consideration when developing control and intervention strategies in areas of endemic P. vivax and Duffy heterogeneity.

Malaria transmission heterogeneity in different eco-epidemiological areas of western Kenya: a region-wide observational and risk classification study for adaptive intervention planning.

BACKGROUND: Understanding of malaria ecology is a prerequisite for designing locally adapted control strategies in resource-limited settings. The aim of this study was to utilize the spatial heterogeneity in malaria transmission for the designing of adaptive interventions. METHODS: Field collections of clinical malaria incidence, asymptomatic Plasmodium infection, and malaria vector data were conducted from 108 randomly selected clusters which covered different landscape settings including irrigated farming, seasonal flooding area, lowland dryland farming, and highlands in western Kenya. Spatial heterogeneity of malaria was analyzed and classified into different eco-epidemiological zones. RESULTS: There was strong heterogeneity and detected hot/cold spots in clinical malaria incidence, Plasmodium prevalence, and vector abundance. The study area was classified into four zones based on clinical malaria incidence, parasite prevalence, vector density, and altitude. The two irrigated zones have either the highest malaria incidence, parasite prevalence, or the highest malaria vector density; the highlands have the lowest vector density and parasite prevalence; and the dryland and flooding area have the average clinical malaria incidence, parasite prevalence and vector density. Different zones have different vector species, species compositions and predominant species. Both indoor and outdoor transmission may have contributed to the malaria transmission in the area. Anopheles gambiae sensu stricto (s.s.), Anopheles arabiensis, Anopheles funestus s.s., and Anopheles leesoni had similar human blood index and malaria parasite sporozoite rate. CONCLUSION: The multi-transmission-indicator-based eco-epidemiological zone classifications will be helpful for making decisions on locally adapted malaria interventions.

The dynamics of deltamethrin resistance evolution in Aedes albopictus has an impact on fitness and dengue virus type-2 vectorial capacity.

BACKGROUND: Worldwide invasion and expansion of Aedes albopictus, an important vector of dengue, chikungunya, and Zika viruses, has become a serious concern in global public health. Chemical insecticides are the primary means currently available to control the mosquito populations. However, long-term and large-scale use of insecticides has selected for resistance in the mosquito that is accompanied by a genetic load that impacts fitness. RESULTS: A number of laboratory strains representing different resistance mechanisms were isolated and identified from laboratory-derived, deltamethrin-resistant Ae. albopictus recovered in previous work. Resistance levels and fitness costs of the strains were evaluated and compared to characterize the evolution of the resistance genotypes and phenotypes. The heterozygous F1534S mutation (1534F/S) in the voltage gated sodium channel (vgsc) gene product (VGSC), first detected in early stages of resistance evolution, not only confers high-level resistance, but also produces no significant fitness costs, leading to the rapid spread of resistance in the population. This is followed by the increase in frequency of homozygous F1534S (1534S/S) mosquitoes that have significant fitness disadvantages, prompting the emergence of an unlinked I1532T mutation with fewer side effects and a mating advantage better adapted to the selection and reproductive pressures imposed in the experiments. Metabolic resistance with no significant fitness cost and mediating a high-tolerance resistance phenotype may play a dominant role in the subsequent evolution of resistance. The different resistant strains had similar vector competence for dengue virus type-2 (DENV-2). Furthermore, a comparative analysis of vectorial capacity revealed that increased survival due to deltamethrin resistance balanced the negative fitness cost effects and contributed to the risk of dengue virus (DENV) transmission by resistant populations. The progressive evolution of resistance results in mosquitoes with both target-site insensitivity and metabolic resistance with lower fitness costs, which further leads to resistant populations with both high resistance levels and vectorial capacity. CONCLUSIONS: This study reveals a possible mechanism for the evolution of deltamethrin resistance in Aedes albopictus. These findings will help guide practical strategies for insecticide use, resistance management and the prevention and control of mosquito-borne disease.

Implementing landscape genetics in molecular epidemiology to determine drivers of vector-borne disease: A malaria case study.

This study employs landscape genetics to investigate the environmental drivers of a deadly vector-borne disease, malaria caused by Plasmodium falciparum, in a more spatially comprehensive manner than any previous work. With 1804 samples from 44 sites collected in western Kenya in 2012 and 2013, we performed resistance surface analysis to show that Lake Victoria acts as a barrier to transmission between areas north and south of the Winam Gulf. In addition, Mantel correlograms clearly showed significant correlations between genetic and geographic distance over short distances (less than 70 km). In both cases, we used an identity-by-state measure of relatedness tailored to find highly related individual parasites in order to focus on recent gene flow that is more relevant to disease transmission. To supplement these results, we performed conventional population genetics analyses, including Bayesian clustering methods and spatial ordination techniques. These analyses revealed some differentiation on the basis of geography and elevation and a cluster of genetic similarity in the lowlands north of the Winam Gulf of Lake Victoria. Taken as a whole, these results indicate low overall genetic differentiation in the Lake Victoria region, but with some separation of parasite populations north and south of the Winam Gulf that is explained by the presence of the lake as a geographic barrier to gene flow. We recommend similar landscape genetics analyses in future molecular epidemiology studies of vector-borne diseases to extend and contextualize the results of traditional population genetics.

International border malaria transmission in the Ethiopian district of Lare, Gambella region: implications for malaria spread into South Sudan.

BACKGROUND: Despite notable progress in the control and prevention of malaria in the Horn of Africa, the disease continues to cause significant morbidity and mortality in various regions of Ethiopia, and elsewhere in the region. The transmission of malaria is affected by genetic, sociocultural, and ecological factors. Lare is an Ethiopian district adjacent to the Ethio-South Sudan border, in Gambella region. The region currently has the highest prevalence of malaria in Ethiopia. This study assesses the burden and spatiotemporal patterns of disease transmission, including the effect of climatic factors on the occurrence of malaria, across an international border crossing. This understanding can assist in crafting informed programmatic and policy decisions for interventions. METHODS: This study was conducted in Lare district, Southwest Ethiopia, a temperate zone. A retrospective descriptive analysis was conducted using clinical service data collected between 2011 and 2021 from the 9 health facilities of the district. Both clinically diagnosed patients and those identified using microscopy and rapid diagnostic testing (RDT) were included in the study. Additionally, climate data was incorporated into analyses. Examples of analyses include malaria burden, positivity rate, incidence, species frequency, and an ANOVA to assess inter-annual case number and meteorological factor variation. RESULTS: Between 2011 and 2021, a total of 96,616 suspected malaria cases were tested by microscopy or RDT, and 39,428 (40.8%) of these cases were reported as positive. There were 1276 patients admitted with 22 deaths recorded. There were further more significant fluctuations in positivity rates across years, the highest being 74.5% in 2021. Incidence varied from 18.0% in 2011 to 151.6% in 2016. The malaria parasite species most detected was Plasmodium falciparum, followed by a smaller proportion of Plasmodium vivax. The greatest proportions of P. falciparum cases were observed in 2018 and 2019, at 97.4% and 97.0% prevalence, respectively. There was significant seasonal variation in case number, the highest observed in July through September of each year. Climatic conditions of annual rainfall, temperature and humidity favored the increment of malaria cases from June until October. CONCLUSION: The study shows that the burden, i.e. morbidity and mortality (with fluctuating patterns) of malaria are still significant public health problems and can pose serious consequences in the district. This has implication for cross-border malaria transmission risk due to considerable border crossings. The predominant cause of the disease is P. falciparum, which causes severe complications in patients. The district has to prepare to deal with such complications for better patient care and outcomes.

First report of Anopheles stephensi from southern Ethiopia.

BACKGROUND: Anopheles stephensi is an emerging exotic invasive urban malaria vector in East Africa. The World Health Organization recently announced an initiative to take concerted actions to limit this vector's expansion by strengthening surveillance and control in invaded and potentially receptive territories in Africa. This study sought to determine the invasion of An. stephensi in southern Ethiopia. METHODS: A targeted entomological survey, both larvae and adult, was conducted in Hawassa City, southern Ethiopia between November 2022 and February 2023. Anopheles larvae were reared to adults for species identification. CDC light traps and BG Pro traps were used indoors and outdoors overnight at selected houses to collect adult mosquitoes in the study area. Prokopack aspirator was employed to sample indoor resting mosquitoes in the morning. Adults of An. stephensi was identified using morphological keys and then confirmed by PCR. RESULTS: Larvae of An. stephensi were found in 28 (16.6%) of the 169 potential mosquito breeding sites surveyed. Out of 548 adult female Anopheles mosquitoes reared from larvae, 234 (42.7%) were identified as An. stephensi morphologically. A total of 449 female anophelines were caught, of which 53 (12.0%) were An. stephensi. Other anopheline species collected in the study area included Anopheles gambiae sensu lato (s.l.), Anopheles pharoensis, Anopheles coustani, and Anopheles demeilloni. CONCLUSION: This study confirmed the presence of An. stephensi in southern Ethiopia. The presence of both larval and adult stages of this mosquito attests that this species established sympatric colonization with native vector species such as An. gambiae (s.l.) in southern Ethiopia. The findings warrant further investigation on the ecology, behaviour, population genetics, and role of An. stephensi in malaria transmission in Ethiopia.

Community case management of malaria in Western Kenya: performance of community health volunteers in active malaria case surveillance.

BACKGROUND: In western Kenya, not all malaria cases are reported as stipulated in the community case management of malaria (CCMm) strategy. This underreporting affects the equity distribution of malaria commodities and the evaluation of interventions. The current study aimed to evaluate the effectiveness of community health volunteers' active case detection and management of malaria in western Kenya. METHODS: Cross-sectional active case detection (ACD) of malaria survey was carried out between May and August 2021 in three eco-epidemiologically distinct zones in Kisumu, western Kenya: Kano Plains, Lowland lakeshore and Highland Plateau. The CHVs conducted biweekly ACD of malaria household visits to interview and examine residents for febrile illness. The Community Health Volunteers (CHVs) performance during the ACD of malaria was observed and interviews done using structured questionnaires. RESULTS: Of the total 28,800 surveyed, 2597 (9%) had fever and associated malaria symptoms. Eco-epidemiological zones, gender, age group, axillary body temperature, bed net use, travel history, and survey month all had a significant association with malaria febrile illness (p < 0.05). The qualification of the CHV had a significant influence on the quality of their service. The number of health trainings received by the CHVs was significantly related to the correctness of using job aid (χ2 = 6.261, df = 1, p = 0.012) and safety procedures during the ACD activity (χ2 = 4.114, df = 1, p = 0.043). Male CHVs were more likely than female CHVs to correctly refer RDT-negative febrile residents to a health facility for further treatment (OR = 3.94, 95% CI = 1.85-5.44, p < 0.0001). Most of RDT-negative febrile residents who were correctly referred to the health facility came from the clusters with a CHV having 10 years of experience or more (OR = 1.29, 95% CI = 1.05-1.57, p = 0.016). Febrile residents in clusters managed by CHVs with more than 10 years of experience (OR = 1.82, 95% CI = 1.43-2.31, p < 0.0001), who had a secondary education (OR = 1.53, 95% CI = 1.27-1.85, p < 0.0001), and were over the age of 50 (OR = 1.44, 95% CI = 1.18-1.76, p < 0.0001), were more likely to seek malaria treatment in public hospitals. All RDT positive febrile residents were given anti-malarial by the CHVs, and RDT negatives were referred to the nearest health facility for further treatment. CONCLUSIONS: The CHV's years of experience, education level, and age had a significant influence on their service quality. Understanding the qualifications of CHVs can assist healthcare systems and policymakers in designing effective interventions that assist CHVs in providing high-quality services to their communities.

Anopheles larval habitats seasonality and environmental factors affecting larval abundance and distribution in Arjo-Didessa sugar cane plantation, Ethiopia.

BACKGROUND: Water resource development projects are essential for increasing agricultural productivity and ensuring food security. However, these activities require the modification of pre-existing environmental settings, which may alter mosquito larval habitat availability and seasonality. The intensive utilization of current adult vector control tools results in insecticide resistance among the main vectors. When coupled with behavioural resistances, a shift in malaria vector feeding and resting behaviours could compromise the effectiveness of the current adult vector control strategies. Thus, it is important to look for new or alternative vector control interventions for immatures to complement adult control by focusing on different larval habitats and their seasonal availability. Thus, this study investigated larval habitat seasonality and seasonal larval abundance and distribution in irrigated sugar cane plantation settings in Ethiopia. METHODS: Anopheles mosquito larval habitats were surveyed and visited twice a month for a period of 14 months. Anopheline larvae and pupae were collected, reared, and fed finely ground fish food. Adults were provided with sucrose solution and kept under standard conditions. Female Anopheles mosquitoes were identified morphologically and using a species-specific PCR assay. Environmental parameters, which include habitats' physico-chemical characteristics, were assessed. Larval habitat diversity and larval abundance and distribution were determined across different seasons. RESULTS: The study revealed that Anopheles gambiae sensu lato (s.l.) was the most predominant 4197(57%) vector species, followed by Anopheles coustani complex 2388 (32.8%). Molecular analysis of sub-samples of An. gambiae s.l. resulted in Anopheles arabiensis (77.9%) and Anopheles amharicus (21.5%), and the remaining 1.1% (n = 7) sub-samples were not amplified. Physico-chemical parameters such as temperature (t = 2.22, p = 0.028), conductivity (t = 3.21, p = 0.002), dissolved oxygen (t = 7.96, p = 0.001), nitrate ion (t = 2.51, p = 0.013), and ammonium ion (t = 2.26, p = 0.025) showed a significant and direct association with mosquito larval abundance. Furthermore, mosquito larval abundance was correlated with distance to the nearest houses (r = - 0.42, p = 0.001), exposure to sunlight (r = 0.34, p = 0.001), during long and short rainy season animal hoof prints, truck tires/road puddles and rain pools were negatively correlated (r = - 0.22, p = 0.01) and types of habitat (r = - 0.20, p = 0.01). Significant habitat type productivity were observed in man-made pools (t = 3.881, P = 0.01163), rain pools, animal hoof prints, (t = - 4.332, P = 0.00749 in both short and long rainy season, whereas, during dry seasons habitat type productivity almost similar and have no significance difference. CONCLUSION: The study found that different larval habitats had variable productivity in different seasons, and that physical and physicochemical features like ammonium and nitrate, as well as the distance between larval habitats and households, are related to larval production. As a result, vector control should take into account the seasonality of Anopheles larval habitat as well as the impact of pesticide application on larval source management.

Effect of predators on Anopheles arabiensis and Anopheles funestus larval survivorship in Homa Bay County Western Kenya.

BACKGROUND: The rise of insecticide resistance against malaria vectors in sub-Saharan Africa has resulted in the need to consider other methods of vector control. The potential use of biological methods, including larvivorous fish, Bacillus thuringiensis israelensis (Bti) and plant shading, is sustainable and environmentally friendly options. This study examined the survivorship of Anopheles arabiensis and Anopheles funestus larvae and habitat productivity in four permanent habitat types in Homa Bay county, western Kenya. METHODS: Predator densities were studied in a laboratory setup while habitat productivity and larval survivorship was studied in field setup. RESULTS: Fish were observed as the most efficient predator (75.8% larval reduction rate) followed by water boatman (69%), and dragonfly nymph (69.5%) in predation rates. Lower predation rates were observed in backswimmers (31%), water beetles (14.9%), water spiders (12.2%), mayflies (7.3%), and tadpoles (6.9%). Increase in predator density in the field setup resulted in decreased Culex larval density. Larval and pupa age-specific distribution was determined and their survivorship curves constructed. Combined larvae (Stage I-IV) to pupa mortality was over 97% for An. arabiensis and 100% for An. funestus. The highest larval stage survival rate was from larval stages I to II and the lowest from larval stage IV to pupa. Stage-specific life tables indicated high mortality rates at every developmental stage, especially at the larval stage II and III. CONCLUSION: Determination of the efficiency of various larval predators and habitat productivity will help with the correct identification of productive habitats and selection of complementary vector control methods through environmental management and/or predator introduction (for instance fish) in the habitats.

Asymptomatic and submicroscopic malaria infections in sugar cane and rice development areas of Ethiopia.

BACKGROUND: Water resource development projects, such as dams and irrigation schemes, have a positive impact on food security and poverty reduction. However, such projects could increase prevalence of vector borne disease, such as malaria. This study investigate the impact of different agroecosystems and prevalence of malaria infection in Southwest Ethiopia. METHODS: Two cross-sectional surveys were conducted in the dry and wet seasons in irrigated and non-irrigated clusters of Arjo sugarcane and Gambella rice development areas of Ethiopia in 2019. A total of 4464 and 2176 study participants from 1449 households in Arjo and 546 households in Gambella enrolled in the study and blood samples were collected, respectively. All blood samples were microscopically examined and a subset of microscopy negative blood samples (n = 2244) were analysed by qPCR. Mixed effect logistic regression and generalized estimating equation were used to determine microscopic and submicroscopic malaria infection and the associated risk factors, respectively. RESULTS: Prevalence by microscopy was 2.0% (88/4464) in Arjo and 6.1% (133/2176) in Gambella. In Gambella, prevalence was significantly higher in irrigated clusters (10.4% vs 3.6%) than in non-irrigated clusters (p < 0.001), but no difference was found in Arjo (2.0% vs 2.0%; p = 0.993). On the other hand, of the 1713 and 531 samples analysed by qPCR from Arjo and Gambella the presence of submicroscopic infection was 1.2% and 12.8%, respectively. Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale were identified by qPCR in both sites. Irrigation was a risk factor for submicroscopic infection in both Arjo and Gambella. Irrigation, being a migrant worker, outdoor job, < 6 months length of stay in the area were risk factors for microscopic infection in Gambella. Moreover, school-age children and length of stay in the area for 1-3 years were significant predictors for submicroscopic malaria in Gambella. However, no ITN utilization was a predictor for both submicroscopic and microscopic infection in Arjo. Season was also a risk factor for microscopic infection in Arjo. CONCLUSION: The study highlighted the potential importance of different irrigation practices impacting on submicroscopic malaria transmission. Moreover, microscopic and submicroscopic infections coupled with population movement may contribute to residual malaria transmission and could hinder malaria control and elimination programmes in the country. Therefore, strengthening malaria surveillance and control by using highly sensitive diagnostic tools to detect low-density parasites, screening migrant workers upon arrival and departure, ensuring adequate coverage and proper utilization of vector control tools, and health education for at-risk groups residing or working in such development corridors is needed.

Irrigation-Induced Environmental Changes Sustain Malaria Transmission and Compromise Intervention Effectiveness.

BACKGROUND: Irrigated agriculture enhances food security, but it potentially promotes mosquito-borne disease transmission and affects vector intervention effectiveness. This study was conducted in the irrigated and nonirrigated areas of rural Homa Bay and Kisumu Counties, Kenya. METHODS: We performed cross-sectional and longitudinal surveys to determine Plasmodium infection prevalence, clinical malaria incidence, molecular force of infection (molFOI), and multiplicity of infection. We examined the impact of irrigation on the effectiveness of the new interventions. RESULTS: We found that irrigation was associated with >2-fold higher Plasmodium infection prevalence and 3-fold higher clinical malaria incidence compared to the nonirrigated area. Residents in the irrigated area experienced persistent, low-density parasite infections and higher molFOI. Addition of indoor residual spraying was effective in reducing malaria burden, but the reduction was more pronounced in the nonirrigated area than in the irrigated area. CONCLUSIONS: Our findings collectively suggest that irrigation may sustain and enhance Plasmodium transmission and affects intervention effectiveness.

Mobile phone handover data for measuring and analysing human population mobility in Western Ethiopia: implication for malaria disease epidemiology and elimination efforts.

BACKGROUND: Human mobility behaviour modelling plays an essential role in the understanding and control of the spread of contagious diseases by limiting the contact among individuals, predicting the spatio-temporal evolution of an epidemic and inferring migration patterns. It informs programmatic and policy decisions for effective and efficient intervention. The objective of this research is to assess the human mobility pattern and analyse its implication for malaria disease epidemiology. METHODS: In this study, human mobility patterns in Benishangul-Gumuz and Gambella regions in Western Ethiopia were explored based on a cellular network mobility parameter (e.g., handover rate) via real world data. Anonymized data were retrieved for mobile active users with mobility related information. The data came from anonymous traffic records collected from all the study areas. For each cell, the necessary mobility parameter data per hour, week and month were collected. A scale factor was computed to change the mobility parameter value to the human mobility pattern. Finally, the relative human mobility probability for each scenario was estimated. MapInfo and Matlab softwares were used for visualization and analysis purposes. Hourly travel patterns in the study settings were compared with hourly malaria mosquito vector feeding behaviour. RESULTS: Heterogeneous human movement patterns were observed in the two regions with some areas showing typically high human mobility. Furthermore, the number of people entering into the two study regions was high during the highest malaria transmission season. Two peaks of hourly human movement, 8:00 to 9:00 and 16:00 to 18:00, emerged in Benishangul-Gumuz region while 8:00 to 10:00 and 16:00 to 18:00 were the peak hourly human mobility time periods in Gambella region. The high human movement in the night especially before midnight in the two regions may increase the risk of getting mosquito bite particularly by early biters depending on malaria linked human behaviour of the population. CONCLUSIONS: High human mobility was observed both within and outside the two regions. The population influx and efflux in these two regions is considerably high. This may specifically challenge the transition from malaria control to elimination. The daily mobility pattern is worth considering in the context of malaria transmission. In line with this malaria related behavioural patterns of humans need to be properly addressed.

Health care provider practices in diagnosis and treatment of malaria in rural communities in Kisumu County, Kenya.

BACKGROUND: Accurate malaria diagnosis and appropriate treatment at local health facilities are critical to reducing morbidity and human reservoir of infectious gametocytes. The current study assessed the accuracy of malaria diagnosis and treatment practices in three health care facilities in rural western Kenya. METHODS: The accuracy of malaria detection and treatment recommended compliance was monitored in two public and one private hospital from November 2019 through March 2020. Blood smears from febrile patients were examined by hospital laboratory technicians and re-examined by an expert microscopists thereafter subjected to real-time polymerase chain reaction (RT-PCR) for quality assurance. In addition, blood smears from patients diagnosed with malaria rapid diagnostic tests (RDT) and presumptively treated with anti-malarial were re-examined by an expert microscopist. RESULTS: A total of 1131 febrile outpatients were assessed for slide positivity (936), RDT (126) and presumptive diagnosis (69). The overall positivity rate for Plasmodium falciparum was 28% (257/936). The odds of slide positivity was higher in public hospitals, 30% (186/624, OR:1.44, 95% CI = 1.05-1.98, p < 0.05) than the private hospital 23% (71/312, OR:0.69, 95% CI = 0.51-0.95, p < 0.05). Anti-malarial treatment was dispensed more at public hospitals (95.2%, 177/186) than the private hospital (78.9%, 56/71, p < 0.0001). Inappropriate anti-malarial treatment, i.e. artemether-lumefantrine given to blood smear negative patients was higher at public hospitals (14.6%, 64/438) than the private hospital (7.1%, 17/241) (p = 0.004). RDT was the most sensitive (73.8%, 95% CI = 39.5-57.4) and specific (89.2%, 95% CI = 78.5-95.2) followed by hospital microscopy (sensitivity 47.6%, 95% CI = 38.2-57.1) and specificity (86.7%, 95% CI = 80.8-91.0). Presumptive diagnosis had the lowest sensitivity (25.7%, 95% CI = 13.1-43.6) and specificity (75.0%, 95% CI = 50.6-90.4). RDT had the highest non-treatment of negatives [98.3% (57/58)] while hospital microscopy had the lowest [77.3% (116/150)]. Health facilities misdiagnosis was at 27.9% (77/276). PCR confirmed 5.2% (4/23) of the 77 misdiagnosed cases as false positive and 68.5% (37/54) as false negative. CONCLUSIONS: The disparity in malaria diagnosis at health facilities with many slide positives reported as negatives and high presumptive treatment of slide negative cases, necessitates augmenting microscopic with RDTs and calls for Ministry of Health strengthening supportive infrastructure to be in compliance with treatment guidelines of Test, Treat, and Track to improve malaria case management.

Molecular characterization and genotype distribution of thioester-containing protein 1 gene in Anopheles gambiae mosquitoes in western Kenya.

BACKGROUND: Evolutionary pressures lead to the selection of efficient malaria vectors either resistant or susceptible to Plasmodium parasites. These forces may favour the introduction of species genotypes that adapt to new breeding habitats, potentially having an impact on malaria transmission. Thioester-containing protein 1 (TEP1) of Anopheles gambiae complex plays an important role in innate immune defenses against parasites. This study aims to characterize the distribution pattern of TEP1 polymorphisms among populations of An. gambiae sensu lato (s.l.) in western Kenya. METHODS: Anopheles gambiae adult and larvae were collected using pyrethrum spray catches (PSC) and plastic dippers respectively from Homa Bay, Kakamega, Bungoma, and Kisumu counties between 2017 and 2020. Collected adults and larvae reared to the adult stage were morphologically identified and then identified to sibling species by PCR. TEP1 alleles were determined in 627 anopheles mosquitoes using restriction fragment length polymorphisms-polymerase chain reaction (RFLP-PCR) and to validate the TEP1 genotyping results, a representative sample of the alleles was sequenced. RESULTS: Two TEP1 alleles (TEP1*S1 and TEP1*R2) and three corresponding genotypes (*S1/S1, *R2/S1, and *R2/R2) were identified. TEP1*S1 and TEP1*R2 with their corresponding genotypes, homozygous *S1/S1 and heterozygous *R2/S1 were widely distributed across all sites with allele frequencies of approximately 80% and 20%, respectively both in Anopheles gambiae and Anopheles arabiensis. There was no significant difference detected among the populations and between the two mosquito species in TEP1 allele frequency and genotype frequency. The overall low levels in population structure (FST = 0.019) across all sites corresponded to an effective migration index (Nm = 12.571) and low Nei's genetic distance values (< 0.500) among the subpopulation. The comparative fixation index values revealed minimal genetic differentiation between species and high levels of gene flow among populations. CONCLUSION: Genotyping TEP1 has identified two common TEP1 alleles (TEP1*S1 and TEP1*R2) and three corresponding genotypes (*S1/S1, *R2/S1, and *R2/R2) in An. gambiae s.l. The TEP1 allele genetic diversity and population structure are low in western Kenya.

Asymptomatic and submicroscopic Plasmodium infections in an area before and during integrated vector control in Homa Bay, western Kenya.

BACKGROUND: Long-lasting insecticidal nets (LLINs) have been the primary vector control strategy until indoor residual spraying (IRS) was added in Homa Bay and Migori Counties in western Kenya. The objective of this study was to evaluate the impact of LLINs integrated with IRS on the prevalence of asymptomatic and submicroscopic Plasmodium infections in Homa Bay County. METHODS: A two-stage cluster sampling procedure was employed to enroll study participants aged ≥ 6 months old. Four consecutive community cross-sectional surveys for Plasmodium infection were conducted in residents of Homa Bay county, Kenya. Prior to the start of the study, all study households received LLINs, which were distributed between June 2017 and March 2018. The first (February 2018) and second (June 2018) surveys were conducted before and after the first round of IRS (Feb-Mar 2018), while the third (February 2019) and fourth (June 2019) surveys were conducted before and after the second application of IRS (February-March 2019). Finger-prick blood samples were obtained to prepare thick and thin smears for microscopic determination and qPCR diagnosis of Plasmodium genus. RESULTS: Plasmodium spp. infection prevalence by microscopy was 18.5% (113/610) before IRS, 14.2% (105/737) and 3.3% (24/720) after the first round of IRS and 1.3% (11/849) after the second round of IRS (p < 0.0001). Submicroscopic (blood smear negative, qPCR positive) parasitaemia reduced from 18.9% (115/610) before IRS to 5.4% (46/849) after IRS (p < 0.0001). However, the proportion of PCR positive infections that were submicroscopic increased from 50.4% (115/228) to 80.7% (46/57) over the study period (p < 0.0001). Similarly, while the absolute number and proportions of microscopy positives which were asymptomatic decreased from 12% (73/610) to 1.2% (9/849) (p < 0.0001), the relative proportion increased. Geometric mean density of P. falciparum parasitaemia decreased over the 2-year study period (p < 0.0001). CONCLUSIONS: These data suggest that two annual rounds of IRS integrated with LLINs significantly reduced the prevalence of Plasmodium parasitaemia, while the proportion of asymptomatic and submicroscopic infections increased. To reduce cryptic P. falciparum transmission and improve malaria control, strategies aimed at reducing the number of asymptomatic and submicroscopic infections should be considered.

Clinical malaria incidence and health seeking pattern in geographically heterogeneous landscape of western Kenya.

BACKGROUND: Malaria remains a public health problem in Kenya despite sustained interventions deployed by the government. One of the major impediments to effective malaria control is a lack of accurate diagnosis and effective treatment. This study was conducted to assess clinical malaria incidence and treatment seeking profiles of febrile cases in western Kenya. METHODS: Active case detection of malaria was carried out in three eco-epidemiologically distinct zones topologically characterized as lakeshore, hillside, and highland plateau in Kisumu County, western Kenya, from March 2020 to March 2021. Community Health Volunteers (CHVs) conducted biweekly visits to residents in their households to interview and examine for febrile illness. A febrile case was defined as an individual having fever (axillary temperature ≥ 37.5 °C) during examination or complaints of fever and other nonspecific malaria related symptoms 1-2 days before examination. Prior to the biweekly malaria testing by the CHVs, the participants' treatment seeking methods were based on their behaviors in response to febrile illness. In suspected malaria cases, finger-prick blood samples were taken and tested for malaria parasites with ultra-sensitive Alere® malaria rapid diagnostic tests (RDT) and subjected to real-time polymerase chain reaction (RT-PCR) for quality control examination. RESULTS: Of the total 5838 residents interviewed, 2205 residents had high temperature or reported febrile illness in the previous two days before the visit. Clinical malaria incidence (cases/1000people/month) was highest in the lakeshore zone (24.3), followed by the hillside (18.7) and the highland plateau zone (10.3). Clinical malaria incidence showed significant difference across gender (χ2 = 7.57; df = 2, p = 0.0227) and age group (χ2 = 58.34; df = 4, p < 0.0001). Treatment seeking patterns of malaria febrile cases showed significant difference with doing nothing (48.7%) and purchasing antimalarials from drug shops (38.1%) being the most common health-seeking pattern among the 2205 febrile residents (χ2 = 21.875; df = 4, p < 0.0001). Caregivers of 802 school-aged children aged 5-14 years with fever primarily sought treatment from drug shops (28.9%) and public hospitals (14.0%), with significant lower proportions of children receiving treatment from traditional medication (2.9%) and private hospital (4.4%) (p < 0.0001). There was no significant difference in care givers' treatment seeking patterns for feverish children under the age of five (p = 0.086). Residents with clinical malaria cases in the lakeshore and hillside zones sought treatment primarily from public hospitals (61.9%, 60/97) traditional medication (51.1%, 23/45) respectively (p < 0.0001). However, there was no significant difference in the treatment seeking patterns of highland plateau residents with clinical malaria (p = 0.431).The main factors associated with the decision to seek treatment were the travel distance to the health facility, the severity of the disease, confidence in the treatment, and affordability. CONCLUSION: Clinical malaria incidence remains highest in the Lakeshore (24.3cases/1000 people/month) despite high LLINs coverage (90%). The travel distance to the health facility, severity of disease and affordability were mainly associated with 80% of residents either self-medicating or doing nothing to alleviate their illness. The findings of this study suggest that the Ministry of Health should strengthen community case management of malaria by providing supportive supervision of community health volunteers to advocate for community awareness, early diagnosis, and treatment of malaria.

Burden of malaria, impact of interventions and climate variability in Western Ethiopia: an area with large irrigation based farming.

BACKGROUND: Land use change has increasingly been expanding throughout the world in the past decades. It can have profound effects on the spatial and temporal distribution of vector borne diseases like malaria through ecological and habitat change. Understanding malaria disease occurrence and the impact of prevention interventions under this intense environmental modification is important for effective and efficient malaria control strategy. METHODS: A descriptive ecological study was conducted by reviewing health service records at Abobo district health office. The records were reviewed to extract data on malaria morbidity, mortality, and prevention and control methods. Moreover, Meteorological data were obtained from Gambella region Meteorology Service Center and National Meteorology Authority head office. Univariate, bivariate and multivariate analysis techniques were used to analyze the data. RESULTS: For the twelve-year time period, the mean annual total malaria case count in the district was 7369.58. The peak monthly malaria incidence was about 57 cases per 1000 people. Only in 2009 and 2015 that zero death due to malaria was recorded over the past 12 years. Fluctuating pattern of impatient malaria cases occurrence was seen over the past twelve years with an average number of 225.5 inpatient cases. The data showed that there is a high burden of malaria in the district. Plasmodium falciparum (Pf) was a predominant parasite species in the district with the maximum percentage of about 90. There was no statistically significant association between season and total malaria case number (F3,8: 1.982, P:0.195). However, the inter-annual total case count difference was statistically significant (F11,132: 36.305, p < 0001). Total malaria case count had shown two months lagged carry on effect. Moreover, 3 months lagged humidity had significant positive effect on total malaria cases. Malaria prevention interventions and meteorological factors showed statistically significant association with total malaria cases. CONCLUSION: Malaria was and will remain to be a major public health problem in the area. The social and economic impact of the disease on the local community is clearly pronounced as it is the leading cause of health facility visit and admission including the mortality associated with it. Scale up of effective interventions is quite important. Continuous monitoring of the performance of the vector control tools needs to be done.

Malaria vector bionomics and transmission in irrigated and non-irrigated sites in western Kenya.

Irrigation not only helps to improve food security but also creates numerous water bodies for mosquito production. This study assessed the effect of irrigation on malaria vector bionomics and transmission in a semi-arid site with ongoing malaria vector control program. The effectiveness of CDC light traps in the surveillance of malaria vectors was also evaluated relative to the human landing catches (HLCs) method. Adult mosquitoes were sampled in two study sites representing irrigated and non-irrigated agroecosystems in western Kenya using a variety of trapping methods. The mosquito samples were identified to species and assayed for host blood meal source and Plasmodium spp. sporozoite infection using polymerase chain reaction. Anopheles arabiensis was the dominant malaria vector in the two study sites and occurred in significantly higher densities in irrigated study site compared to the non-irrigated study site. The difference in indoor resting density of An. arabiensis during the dry and wet seasons was not significant. Other species, including An. funestus, An. coustani, and An. pharoensis, were collected. The An. funestus indoor resting density was 0.23 in irrigated study site while almost none of this species was collected in the non-irrigated study site. The human blood index (HBI) for An. arabiensis in the irrigated study site was 3.44% and significantly higher than 0.00% for the non-irrigated study site. In the irrigated study site, the HBI of An. arabiensis was 3.90% and 5.20% indoor and outdoor, respectively. The HBI of An. funestus was 49.43% and significantly higher compared to 3.44% for An. arabiensis in the irrigated study site. The annual entomologic inoculation rate for An. arabiensis in the irrigated study site was 0.41 and 0.30 infective bites/person/year indoor and outdoor, respectively, whereas no transmission was observed in the non-irrigated study site. The CDC light trap performed consistently with HLC in terms of vector density. These findings demonstrate that irrigated agriculture may increase the risk of malaria transmission in irrigated areas compared to the non-irrigated areas and highlight the need to complement the existing malaria vector interventions with novel tools targeting the larvae and both indoor and outdoor biting vector populations.

Microgeographic Epidemiology of Malaria Parasites in an Irrigated Area of Western Kenya by Deep Amplicon Sequencing.

To improve food security, investments in irrigated agriculture are anticipated to increase throughout Africa. However, the extent to which environmental changes from water resource development will impact malaria epidemiology remains unclear. This study was designed to compare the sensitivity of molecular markers used in deep amplicon sequencing for evaluating malaria transmission intensities and to assess malaria transmission intensity at various proximities to an irrigation scheme. Compared to ama1, csp, and msp1 amplicons, cpmp required the smallest sample size to detect differences in infection complexity between transmission risk zones. Transmission intensity was highest within 5 km of the irrigation scheme by polymerase chain reaction positivity rate, infection complexity, and linkage disequilibrium. The irrigated area provided a source of parasite infections for the surrounding 2- to 10-km area. This study highlights the suitability of the cpmp amplicon as a measure for transmission intensities and the impact of irrigation on microgeographic epidemiology of malaria parasites.