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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.

Several sub-Saharan African countries rely on irrigation for food production. This study examined the impact of environmental modifications resulting from irrigation on the ecology of aquatic stages of malaria vectors in a semi-arid region of western Kenya. Mosquito larvae were collected from irrigated and non-irrigated ecosystems during seasonal cross-sectional and monthly longitudinal studies to assess habitat availability, stability, and productivity of anophelines in temporary, semipermanent, and permanent habitats during the dry and wet seasons. The duration of habitat stability was also compared between selected habitats. Emergence traps were used to determine the daily production of female adult mosquitoes from different habitat types. Malaria vectors were morphologically identified and sibling species subjected to molecular analysis. Data was statistically compared between the two ecosystems. After aggregating the data, the overall malaria vector productivity for habitats in the two ecosystems was estimated. Immatures of the malaria vector (Anopheles arabiensis) Patton (Diptera: Culicidae) comprised 98.3% of the Anopheles in both the irrigated and non-irrigated habitats. The irrigated ecosystem had the most habitats, higher larval densities, and produced 85.8% of emerged adult females. These results showed that irrigation provided conditions that increased habitat availability, stability, and diversity, consequently increasing the An. arabiensis production and potential risk of malaria transmission throughout the year. The irrigated ecosystems increased the number of habitats suitable for Anopheles breeding by about 3-fold compared to non-irrigated ecosystems. These results suggest that water management in the irrigation systems of western Kenya would serve as an effective method for malaria vector control.

Gravid Anopheles malaria vectors depend on both chemical and physical (including microbial) cues for selection of preferred habitats for oviposition. This study focused on assessing the effects of bacterial composition and habitat metabolites on malaria vector larval availability in irrigated and non-irrigated potential larval sources. Water samples were collected from larval positive and negative habitats in the irrigated and non- irrigated areas of Homa Bay county. Bacteria cultured from the water samples were subjected to Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) for species identification. DNA was extracted from the colonies and polymerase chain reaction (PCR) and sequencing done. Finally, the metabolite composition of larval positive and negative habitats was determined. MALDI-TOF MS results revealed that Bacillus was the only genera identified from larval sources in the non-irrigated zone. In the irrigated area, Shigella was the dominant genera (47%) while Escherichia coli was the abundant species (13/51). Of the sequenced isolates, 65% were Bacillus. Larvicidal isolates Brevibacillus brevis, Bacillus subtilis, and Exiguobacterium profundum were isolated and grouped with Bacillus mojavensis, Bacillus tequilensis, Bacillus stercoris, and Brevibacillus agri. Irrigated areas with larvae had reduced crude fat (0.01%) and protein content (0.13%) in comparison to those without larvae. In irrigated and non- irrigated areas, larval presence was evident in habitats with high total chlorophyll content (1.12 μg/g vs 0.81μg/g and 3.37 μg/g vs 0.82). Aquatic habitats with larvae in both irrigated and non-irrigated areas exhibited higher sugar concentration than habitats without larvae; however, when compared, non-irrigated areas with larvae had higher sugar concentration than similar habitats in irrigated areas. In addition, substantial concentrations of Manganese, Calcium, and Copper were found in aquatic habitats containing larvae in both irrigated and non-irrigated areas. These results allow for prospective examination as potential larvicidal or adulticidal agents and could be considered when designing potential vector control interventions.

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.

BackgroundMalaria in western Kenya is currently characterized by sustained high Plasmodial transmission and infection resurgence, despite positive responses in some areas following intensified malaria control interventions since 2006. This study aimed to evaluate long-term changes in malaria transmission profiles and to assess patterns of asymptomatic malaria infections in school children aged 5-15 years at three sites in western Kenya with heterogeneous malaria transmission and simultaneous malaria control interventions.MethodsThe study was conducted from 2018 to 2019 and is based on data taken every third year from 2005 to 2014 during a longitudinal parasitological and mosquito adult surveillance and malaria control programme that was initiated in 2002 in the villages of Kombewa, Iguhu, and Marani. Plasmodium spp. infections were determined using microscopy. Mosquito samples were identified to species and host blood meal source and sporozoite infections were assayed using polymerase chain reaction.ResultsPlasmodium falciparum was the only malaria parasite evaluated during this study (2018-2019). Asymptomatic malaria parasite prevalence in school children decreased in all sites from 2005 to 2008. However, since 2011, parasite prevalence has resurged by > 40% in Kombewa and Marani. Malaria vector densities showed similar reductions from 2005 to 2008 in all sites, rose steadily until 2014, and decreased again. Overall, Kombewa had a higher risk of infection compared to Iguhu (χ2 = 552.52, df = 1, P

BackgroundIrrigated 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.MethodsWe 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.ResultsWe 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.ConclusionsOur findings collectively suggest that irrigation may sustain and enhance Plasmodium transmission and affects intervention effectiveness.

Expanding agricultural irrigation efforts to enhance food security and socioeconomic development in sub-Saharan Africa may affect malaria transmission and socioeconomic variables that increase the risk of anemia in local communities. We compared the prevalence of anemia, Plasmodium falciparum infection, and indicators of socioeconomic status related to nutrition in communities in Homa Bay County, Kenya, where an agricultural irrigation scheme has been implemented, to that in nearby communities where there is no agricultural irrigation. Cross-sectional surveys conducted showed that anemia prevalence defined by WHO criteria (hemoglobin < 11 g/dL) was less in communities in the irrigated areas than in the non-irrigated areas during the wet season (38.9% and 51.5%, χ2 = 4.29, P = 0.001) and the dry season (25.2% and 34.1%, χ2 = 7.33, P = 0.007). In contrast, Plasmodium falciparum infection prevalence was greater during the wet season in irrigated areas than in non-irrigated areas (15.3% versus 7.8%, χ2 = 8.7, P = 0.003). There was, however, no difference during the dry season (infection prevalence, < 1.8%). Indicators of nutritional status pertinent to anemia pathogenesis such as weekly consumption of non-heme- and heme-containing foods and household income were greater in communities located within the irrigation scheme versus those outside the irrigation scheme (P < 0.0001). These data indicate that current agricultural irrigation schemes in malaria-endemic communities in this area have reduced the risk of anemia. Future studies should include diagnostic tests of iron deficiency, parasitic worm infections, and genetic hemoglobin disorders to inform public health interventions aimed at reducing community anemia burden.

Abstract 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.

BackgroundLong-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.MethodsA 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.ResultsPlasmodium 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

Background: Malaria elimination and eradication efforts can be advanced by including transmission-blocking or reducing vaccines (TBVs) alongside existing interventions. Key transmission-blocking vaccine candidates, such as Pfs230 domain one and Pfs48/45 domain 3, should be genetically stable to avoid developing ineffective vaccines due to antigenic polymorphisms. We evaluated genetic polymorphism and temporal stability of Pfs230 domain one and Pfs48/45 domain three in Plasmodium falciparum parasites from western Kenya. Methods: Dry blood spots on filter paper were collected from febrile malaria patients reporting to community health facilities in endemic areas of Homa Bay and Kisumu Counties and an epidemic-prone area of Kisii County in 2018 and 2019. Plasmodium speciation was performed using eluted DNA and real-time PCR. Amplification of the target domains of the two Pfs genes was performed on P. falciparum positive samples. We sequenced Pfs230 domain one on 156 clinical isolates and Pfs48/45 domain three on 118 clinical isolates to infer the levels of genetic variability, signatures of selection, genetic diversity indices and perform other evolutionary analyses. Results: Pfs230 domain one had low nucleotide diversity (π = 0.15 × 10-2) with slight variation per study site. Six polymorphic sites with nonsynonymous mutations and eight haplotypes were discovered. I539T was a novel variant, whereas G605S was nearing fixation. Pfs48/45 domain three had a low π (0.063 × 10-2), high conservation index, and three segregating sites, resulting in nonsynonymous mutation and four haplotypes. Some loci of Pfs230 D1 were in positive or negative linkage disequilibrium, had negative or positive selection signatures, and others (1813, 1955) and (1813, 1983) had a history of recombination. Mutated loci pairs in Pfs48/45 domain three had negative linkage disequilibrium, and some had negative and positive Tajima's D values with no history of recombination events. Conclusion: The two transmission blocking vaccine candidates have low nucleotide diversity, a small number of zone-specific variants, high nucleotide conservation index, and high frequency of rare alleles. With the near fixation a polymorphic site and the proximity of mutated codons to antibody binding epitopes, it will be necessary to continue monitoring sequence modifications of these domains when designing TBVs that include Pfs230 and Pfs48/45 antigens.

BackgroundMalaria 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.MethodsActive 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.ResultsOf 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

BackgroundLeading transmission-blocking vaccine candidates such as Plasmodium falciparum surface protein 25 (Pfs25 gene) may undergo antigenic alterations which may render them ineffective or allele-specific. This study examines the level of genetic diversity, signature of selection and drivers of Pfs25 polymorphisms of parasites population in regions of western Kenya with varying malaria transmission intensities.MethodsDry blood spots (DBS) were collected in 2018 and 2019 from febrile outpatients with malaria at health facilities in malaria-endemic areas of Homa Bay, Kisumu (Chulaimbo) and the epidemic-prone highland area of Kisii. Parasites DNA were extracted from DBS using Chelex method. Species identification was performed using real-time PCR. The 460 base pairs (domains 1-4) of the Pfs25 were amplified and sequenced for a total of 180 P. falciparum-infected blood samples.ResultsNine of ten polymorphic sites were identified for the first time. Overall, Pfs25 exhibited low nucleotide diversity (0.04×10-2) and low mutation frequencies (1.3% to 7.7%). Chulaimbo had the highest frequency (15.4%) of mutated sites followed by Kisii (6.7%) and Homa Bay (5.1%). Neutrality tests of Pfs25 variations showed significant negative values of Tajima's D (-2.15, p

BackgroundMalaria control in Kenya is based on case management and vector control using long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS). However, the development of insecticide resistance compromises the effectiveness of insecticide-based vector control programs. The use of pesticides for agricultural purposes has been implicated as one of the sources driving the selection of resistance. The current study was undertaken to assess the status and mechanism of insecticide resistance in malaria vectors in irrigated and non-irrigated areas with varying agrochemical use in western Kenya.MethodsThe study was carried out in 2018-2019 in Homa Bay County, western Kenya. The bioassay was performed on adults reared from larvae collected from irrigated and non-irrigated fields in order to assess the susceptibility of malaria vectors to different classes of insecticides following the standard WHO guidelines. Characterization of knockdown resistance (kdr) and acetylcholinesterase-inhibiting enzyme/angiotensin-converting enzyme (Ace-1) mutations within Anopheles gambiae s.l. species was performed using the polymerase chain reaction (PCR) method. To determine the agricultural and public health insecticide usage pattern, a questionnaire was administered to farmers, households, and veterinary officers in the study area.ResultsAnopheles arabiensis was the predominant species in the irrigated (100%, n = 154) area and the dominant species in the non-irrigated areas (97.5%, n = 162), the rest being An. gambiae sensu stricto. In 2018, Anopheles arabiensis in the irrigated region were susceptible to all insecticides tested, while in the non-irrigated region reduced mortality was observed (84%) against deltamethrin. In 2019, phenotypic mortality was decreased (97.8-84% to 83.3-78.2%). In contrast, high mortality from malathion (100%), DDT (98.98%), and piperonyl butoxide (PBO)-deltamethrin (100%) was observed. Molecular analysis of the vectors from the irrigated and non-irrigated areas revealed low levels of leucine-serine/phenylalanine substitution at position 1014 (L1014S/L1014F), with mutation frequencies of 1-16%, and low-frequency mutation in the Ace-1R gene (0.7%). In addition to very high coverage of LLINs impregnated with pyrethroids and IRS with organophosphate insecticides, pyrethroids were the predominant chemical class of pesticides used for crop and animal protection.ConclusionAnopheles arabiensis from irrigated areas showed increased phenotypic resistance, and the intensive use of pesticides for crop protection in this region may have contributed to the selection of resistance genes observed. The susceptibility of these malaria vectors to organophosphates and PBO synergists in pyrethroids offers a promising future for IRS and insecticide-treated net-based vector control interventions. These findings emphasize the need for integrated vector control strategies, with particular attention to agricultural practices to mitigate mosquito resistance to insecticides.

Abstract Background Malaria in western Kenya is currently characterized by sustained high Plasmodial transmission and infection resurgence, despite positive responses in some areas following intensified malaria control interventions since 2006. This study aimed to evaluate long-term changes in malaria transmission profiles and to assess patterns of asymptomatic malaria infections in school children aged 5–15 years at three sites in western Kenya with heterogeneous malaria transmission and simultaneous malaria control interventions. Methods The study was conducted from 2018 to 2019 and is based on data taken every third year from 2005 to 2014 during a longitudinal parasitological and mosquito adult surveillance and malaria control programme that was initiated in 2002 in the villages of Kombewa, Iguhu, and Marani. Plasmodium spp. infections were determined using microscopy. Mosquito samples were identified to species and host blood meal source and sporozoite infections were assayed using polymerase chain reaction. Results Plasmodium falciparum was the only malaria parasite evaluated during this study (2018–2019). Asymptomatic malaria parasite prevalence in school children decreased in all sites from 2005 to 2008. However, since 2011, parasite prevalence has resurged by > 40% in Kombewa and Marani. Malaria vector densities showed similar reductions from 2005 to 2008 in all sites, rose steadily until 2014, and decreased again. Overall, Kombewa had a higher risk of infection compared to Iguhu (χ 2 = 552.52, df = 1, P

Abstract 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

Abstract 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

Gravid Anopheles malaria vectors depend on both chemical and physical (including microbial) cues for selection of preferred habitats for oviposition. This study focused on assessing the effects of bacterial composition and habitat metabolites on malaria vector larval availability in irrigated and non-irrigated potential larval sources. Water samples were collected from larval positive and negative habitats in the irrigated and non- irrigated areas of Homa Bay county. Bacteria cultured from the water samples were subjected to Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) for species identification. DNA was extracted from the colonies and polymerase chain reaction (PCR) and sequencing done. Finally, the metabolite composition of larval positive and negative habitats was determined. MALDI-TOF MS results revealed that Bacillus was the only genera identified from larval sources in the non-irrigated zone. In the irrigated area, Shigella was the dominant genera (47%) while Escherichia coli was the abundant species (13/51). Of the sequenced isolates, 65% were Bacillus. Larvicidal isolates Brevibacillus brevis, Bacillus subtilis, and Exiguobacterium profundum were isolated and grouped with Bacillus mojavensis, Bacillus tequilensis, Bacillus stercoris, and Brevibacillus agri. Irrigated areas with larvae had reduced crude fat (0.01%) and protein content (0.13%) in comparison to those without larvae. In irrigated and non- irrigated areas, larval presence was evident in habitats with high total chlorophyll content (1.12 μg/g vs 0.81μg/g and 3.37 μg/g vs 0.82). Aquatic habitats with larvae in both irrigated and non-irrigated areas exhibited higher sugar concentration than habitats without larvae; however, when compared, non-irrigated areas with larvae had higher sugar concentration than similar habitats in irrigated areas. In addition, substantial concentrations of Manganese, Calcium, and Copper were found in aquatic habitats containing larvae in both irrigated and non-irrigated areas. These results allow for prospective examination as potential larvicidal or adulticidal agents and could be considered when designing potential vector control interventions.

Abstract Background The gold standard for diagnosing Plasmodium falciparum infection is microscopic examination of Giemsa-stained peripheral blood smears. The effectiveness of this procedure for infection surveillance and malaria control may be limited by a relatively high parasitaemia detection threshold. Persons with microscopically undetectable infections may go untreated, contributing to ongoing transmission to mosquito vectors. The purpose of this study was to determine the magnitude and determinants of undiagnosed submicroscopic P. falciparum infections in a rural area of western Kenya. Methods A health facility-based survey was conducted, and 367 patients seeking treatment for symptoms consistent with uncomplicated malaria in Homa Bay County were enrolled. The frequency of submicroscopic P. falciparum infection was measured by comparing the prevalence of infection based on light microscopic inspection of thick blood smears versus real-time polymerase chain reaction (RT-PCR) targeting P. falciparum 18S rRNA gene. Long-lasting insecticidal net (LLIN) use, participation in nocturnal outdoor activities, and gender were considered as potential determinants of submicroscopic infections. Results Microscopic inspection of blood smears was positive for asexual P. falciparum parasites in 14.7% (54/367) of cases. All of these samples were confirmed by RT-PCR. 35.8% (112/313) of blood smear negative cases were positive by RT-PCR, i.e., submicroscopic infection, resulting in an overall prevalence by RT-PCR alone of 45.2% compared to 14.7% for blood smear alone. Females had a higher prevalence of submicroscopic infections (35.6% or 72 out of 202 individuals, 95% CI 28.9–42.3) compared to males (24.2%, 40 of 165 individuals, 95% CI 17.6–30.8). The risk of submicroscopic infections in LLIN users was about half that of non-LLIN users (OR = 0.59). There was no difference in the prevalence of submicroscopic infections of study participants who were active in nocturnal outdoor activities versus those who were not active (OR = 0.91). Patients who participated in nocturnal outdoor activities and use LLINs while indoors had a slightly higher risk of submicroscopic infection than those who did not use LLINs (OR = 1.48). Conclusion Microscopic inspection of blood smears from persons with malaria symptoms for asexual stage P. falciparum should be supplemented by more sensitive diagnostic tests in order to reduce ongoing transmission of P. falciparum parasites to local mosquito vectors.

Abstract Background Malaria control in Kenya is based on case management and vector control using long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS). However, the development of insecticide resistance compromises the effectiveness of insecticide-based vector control programs. The use of pesticides for agricultural purposes has been implicated as one of the sources driving the selection of resistance. The current study was undertaken to assess the status and mechanism of insecticide resistance in malaria vectors in irrigated and non-irrigated areas with varying agrochemical use in western Kenya. Methods The study was carried out in 2018–2019 in Homa Bay County, western Kenya. The bioassay was performed on adults reared from larvae collected from irrigated and non-irrigated fields in order to assess the susceptibility of malaria vectors to different classes of insecticides following the standard WHO guidelines. Characterization of knockdown resistance (kdr) and acetylcholinesterase-inhibiting enzyme/angiotensin-converting enzyme (Ace-1) mutations within Anopheles gambiae s.l. species was performed using the polymerase chain reaction (PCR) method. To determine the agricultural and public health insecticide usage pattern, a questionnaire was administered to farmers, households, and veterinary officers in the study area. Results Anopheles arabiensis was the predominant species in the irrigated (100%, n = 154) area and the dominant species in the non-irrigated areas (97.5%, n = 162), the rest being An. gambiae sensu stricto. In 2018, Anopheles arabiensis in the irrigated region were susceptible to all insecticides tested, while in the non-irrigated region reduced mortality was observed (84%) against deltamethrin. In 2019, phenotypic mortality was decreased (97.8–84% to 83.3–78.2%). In contrast, high mortality from malathion (100%), DDT (98.98%), and piperonyl butoxide (PBO)-deltamethrin (100%) was observed. Molecular analysis of the vectors from the irrigated and non-irrigated areas revealed low levels of leucine-serine/phenylalanine substitution at position 1014 (L1014S/L1014F), with mutation frequencies of 1–16%, and low-frequency mutation in the Ace-1R gene (0.7%). In addition to very high coverage of LLINs impregnated with pyrethroids and IRS with organophosphate insecticides, pyrethroids were the predominant chemical class of pesticides used for crop and animal protection. Conclusion Anopheles arabiensis from irrigated areas showed increased phenotypic resistance, and the intensive use of pesticides for crop protection in this region may have contributed to the selection of resistance genes observed. The susceptibility of these malaria vectors to organophosphates and PBO synergists in pyrethroids offers a promising future for IRS and insecticide-treated net-based vector control interventions. These findings emphasize the need for integrated vector control strategies, with particular attention to agricultural practices to mitigate mosquito resistance to insecticides. Graphic abstract

Background: Malaria elimination and eradication efforts can be advanced by including transmission-blocking or reducing vaccines (TBVs) alongside existing interventions. Key transmission-blocking vaccine candidates, such as Pfs230 domain one and Pfs48/45 domain 3, should be genetically stable to avoid developing ineffective vaccines due to antigenic polymorphisms. We evaluated genetic polymorphism and temporal stability of Pfs230 domain one and Pfs48/45 domain three in Plasmodium falciparum parasites from western Kenya.Methods: Dry blood spots on filter paper were collected from febrile malaria patients reporting to community health facilities in endemic areas of Homa Bay and Kisumu Counties and an epidemic-prone area of Kisii County in 2018 and 2019. Plasmodium speciation was performed using eluted DNA and real-time PCR. Amplification of the target domains of the two Pfs genes was performed on P. falciparum positive samples. We sequenced Pfs230 domain one on 156 clinical isolates and Pfs48/45 domain three on 118 clinical isolates to infer the levels of genetic variability, signatures of selection, genetic diversity indices and perform other evolutionary analyses.Results:Pfs230 domain one had low nucleotide diversity (π = 0.15 × 10–2) with slight variation per study site. Six polymorphic sites with nonsynonymous mutations and eight haplotypes were discovered. I539T was a novel variant, whereas G605S was nearing fixation. Pfs48/45 domain three had a low π (0.063 × 10–2), high conservation index, and three segregating sites, resulting in nonsynonymous mutation and four haplotypes. Some loci of Pfs230 D1 were in positive or negative linkage disequilibrium, had negative or positive selection signatures, and others (1813, 1955) and (1813, 1983) had a history of recombination. Mutated loci pairs in Pfs48/45 domain three had negative linkage disequilibrium, and some had negative and positive Tajima’s D values with no history of recombination events.Conclusion: The two transmission blocking vaccine candidates have low nucleotide diversity, a small number of zone-specific variants, high nucleotide conservation index, and high frequency of rare alleles. With the near fixation a polymorphic site and the proximity of mutated codons to antibody binding epitopes, it will be necessary to continue monitoring sequence modifications of these domains when designing TBVs that include Pfs230 and Pfs48/45 antigens.

BackgroundLeading transmission-blocking vaccine candidates such as Plasmodium falciparum surface protein 25 (Pfs25 gene) may undergo antigenic alterations which may render them ineffective or allele-specific. This study examines the level of genetic diversity, signature of selection and drivers of Pfs25 polymorphisms of parasites population in regions of western Kenya with varying malaria transmission intensities.MethodsDry blood spots (DBS) were collected in 2018 and 2019 from febrile outpatients with malaria at health facilities in malaria-endemic areas of Homa Bay, Kisumu (Chulaimbo) and the epidemic-prone highland area of Kisii. Parasites DNA were extracted from DBS using Chelex method. Species identification was performed using real-time PCR. The 460 base pairs (domains 1-4) of the Pfs25 were amplified and sequenced for a total of 180 P. falciparum-infected blood samples.ResultsNine of ten polymorphic sites were identified for the first time. Overall, Pfs25 exhibited low nucleotide diversity (0.04×10-2) and low mutation frequencies (1.3% to 7.7%). Chulaimbo had the highest frequency (15.4%) of mutated sites followed by Kisii (6.7%) and Homa Bay (5.1%). Neutrality tests of Pfs25 variations showed significant negative values of Tajima's D (-2.15, pConclusionGiven the low level of nucleotide diversity, it is unlikely that a Pfs25 antigen-based vaccine would be affected by antigenic variations. However, continued monitoring of Pfs25 immunogenic domain 3 for possible variants that might impact vaccine antibody binding is warranted.

Desde hace tiempo, muchos especialistas señalan que la clave para incorporar la sostenibilidad a una propuesta, de manera económicamente viable, es considerar los objetivos y criterios sostenibles desde los primeros pasos del proyecto, porque es cuando la relación entre la efectividad de las estrategias planteadas y el coste de implementarlas es más beneficiosa. El principal objetivo de este trabajo ha sido el desarrollo de una herramienta, denominada Green Improvement Tool v1.7, que simplifica al máximo las cuestiones relativas a la sostenibilidad, dando solución a las necesidades de sus potenciales usuarios y reuniendo al mismo tiempo varias características: claridad en sus términos y objetivos (para evitar confusiones y fallos de implementación), viabilidad técnica y económica en su aplicación, flexibilidad suficiente para adaptarse a situaciones y contextos diversos, capacidad de promover el uso de diferentes tecnologías y, ante todo, estimular el interés de dichos usuarios en un proceso de mejora continua. Los ensayos realizados en casos de estudio reales, durante el proceso de validación, indican que la herramienta permite anticipar desde las fases iniciales, con una fiabilidad media superior al 95%, más del 80% de los resultados al final del proceso. Abstract Many experts have long pointed out that the key to incorporating sustainability into a proposal in an economically viable way is to consider sustainable objectives and criteria from the very first steps of the project because the relationship between the effectiveness of the strategies proposed and The cost of implementing them is more beneficial. The main objective of this work has been the development of a tool, called Green Improvement Tool v1.7, which simplifies as much as possible the questions related to sustainability, giving solution to the needs of its potential users and bringing together several characteristics: Clarity in terms and objectives (to avoid confusion and failure to implement), technical and economic feasibility in its application, sufficient flexibility to adapt to different situations and contexts, ability to promote the use of different technologies and, above all, to stimulate interest of these users in a process of continuous improvement. During the validation process, the tests carried out in real cases indicate that the tool allows more than 80% of the results at the end of the process to be anticipated from the initial phases, with an average reliability of more than 95%.