Non-Coding RNAs Potentially Involved in Pyrethroid Resistance of Anopheles funestus Population in Western Kenya.

Backgrounds: The resurgence of Anopheles funestus, a dominant vector of human malaria in western Kenya was partly attributed to insecticide resistance. However, evidence on the molecular basis of pyrethroid resistance in western Kenya is limited. Noncoding RNAs (ncRNAs) form a vast class of RNAs that do not code for proteins and are ubiquitous in the insect genome. Here, we demonstrated that multiple ncRNAs could play a potential role in An. funestusresistance to pyrethroid in western Kenya. Materials and Methods: Anopheles funestus mosquitoes were sampled by aspiration methods in Bungoma, Teso, Siaya, Port Victoria and Kombewa in western Kenya. The F1 progenies were exposed to deltamethrin (0.05%), permethrin (0.75%), DDT (4%) and pirimiphos-methyl (0.25%) following WHO test guidelines. A synergist assay using piperonyl butoxide (PBO) (4%) was conducted to determine cytochrome P450s' role in pyrethroid resistance. RNA-seq was conducted on a combined pool of specimens that were resistant and unexposed, and the results were compared with those of the FANG susceptible strain. This approach aimed to uncover the molecular mechanisms underlying pyrethroid resistance. Results: Pyrethroid resistance was observed in all the sites with an average mortality rate of 57.6%. Port Victoria had the highest level of resistance to permethrin (MR=53%) and deltamethrin (MR=11%) pyrethroids. Teso had the lowest level of resistance to permethrin (MR=70%) and deltamethrin (MR=87%). Resistance to DDT was observed only in Kombewa (MR=89%) and Port Victoria (MR=85%). A full susceptibility to P-methyl (0.25%) was observed in all the sites. PBO synergist assay revealed high susceptibility (>98%) to the pyrethroids in all the sites except for Port Victoria (MR=96%, n=100). Whole transcriptomic analysis showed that most of the gene families associated with pyrethroid resistance comprised non-coding RNAs (67%), followed by imipenemase (10%), cytochrome P450s (6%), cuticular proteins (5%), olfactory proteins (4%), glutathione S-transferases (3%), UDP-glycosyltransferases (2%), ATP-binding cassettes (2%) and carboxylesterases(1%). Conclusions: This study unveils the molecular basis of insecticide resistance in An. funestus in western Kenya, highlighting for the first time the potential role of non-coding RNAs in pyrethroid resistance. Targeting non-coding RNAs for intervention development could help in insecticide resistance management.

Genetic diversity, haplotype analysis, and prevalence of Hepatitis B virus MHR mutations among isolates from Kenyan blood donors.

BACKGROUND: The rapid spread of HBV has resulted in the emergence of new variants. These viral genotypes and variants, in addition to carcinogenic risk, can be key predictors of therapy response and outcomes. As a result, a better knowledge of these emerging HBV traits will aid in the development of a treatment for HBV infection. However, many Sub-Saharan African nations, including Kenya, have insufficient molecular data on HBV strains circulating locally. This study conducted a population-genetics analysis to evaluate the genetic diversity of HBV among Kenyan blood donors. In addition, within the same cohort, the incidence and features of immune-associated escape mutations and stop-codons in Hepatitis B surface antigen (HBsAg) were determined. METHODS: In September 2015 to October 2016, 194 serum samples were obtained from HBsAg-positive blood donors residing in eleven different Kenyan counties: Kisumu, Machakos, Uasin Gishu, Nairobi, Nakuru, Embu, Garissa, Kisii, Mombasa, Nyeri, and Turkana. For the HBV surface (S) gene, HBV DNA was isolated, amplified, and sequenced. The sequences obtained were utilized to investigate the genetic and haplotype diversity within the S genes. RESULTS: Among the blood donors, 74.74% were male, and the overall mean age was 25.36 years. HBV genotype A1 (88.14%) was the most common, followed by genotype D (10.82%), genotype C (0.52%), and HBV genotype E (0.52%). The phylogenetic analysis revealed twelve major clades, with cluster III comprising solely of 68 blood donor isolates (68/194-35.05%). A high haplotype diversity (Hd = 0.94) and low nucleotide diversity (π = 0.02) were observed. Kisumu county had high number of haplotypes (22), but low haplotype (gene) diversity (Hd = 0.90). Generally, a total of 90 haplotypes with some consisting of more than one sequence were observed. The gene exhibited negative values for Tajima's D (-2.04, p<0.05) and Fu's Fs (-88.84). Several mutations were found in 139 isolates, either within or outside the Major Hydrophilic Area (MHR). There were 29 mutations found, with 37.9% of them situated inside the "a" determinant. The most common mutations in this research were T143M and K122R. Escape mutations linked to diagnostic failure, vaccination and immunoglobulin treatment evasion were also discovered. Also, one stop-codon, W163STP, inside the MHR, was found in one sample from genotype A. CONCLUSION: In Kenya, HBV/A1 is still the most common genotype. Despite limited genetic and nucleotide diversity, haplotype network analysis revealed haplotype variance among HBV genotypes from Kenyan blood donors. The virological properties of immune escape, which may be the source of viral replication endurance, were discovered in the viral strains studied and included immune-escape mutations and stop-codon. The discovery of HBsAg mutations in MHR in all isolates highlighted the need of monitoring MHR mutations in Kenya.

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.

Malaria diagnosis in rural healthcare facilities and treatment-seeking behavior in malaria endemic settings in western Kenya.

Accurate malaria diagnosis and timely treatment are requirements for effective management of the disease. However, treatment efficacy may be significantly reduced in resource-constrained healthcare facilities with poorly equipped laboratories and frequent drug and rapid diagnostic test kit (RDT) stock-outs. Furthermore, patient may avoid seeking treatment from such facilities. The study's goal was to determine treatment-seeking behavior, malaria diagnosis and treatment quality, and likely treatment-seeking determinants in the local population. Passive case detection, which targeted all patients with suspected malaria cases, was conducted in ten public healthcare facilities over a three-month period. Monthly malaria cases, methods of diagnosis and antimalarial drug availability were assessed. A household-based survey was also carried out. Structured questionnaires were used to collect knowledge, attitude and practice (KAP) data from household heads. Malaria knowledge, treatment seeking behavior, and predictors of malaria treatment-seeking were all determined. Three of the seven dispensaries lacked a laboratory to conduct microscopy- diagnosis. These three dispensaries also experienced frequent RDT stock-outs, which resulted in depending on clinical signs as diagnosis for malaria. The majority of local residents with fever (50.3%) purchased antimalarial drugs from a chemist. About 37% of fever patients sought treatment at healthcare facility while the remaining 12.7% did not treat their fevers. In irrigated areas, 45.5% (46/64) of fever patients sought treatment at healthcare facilities, compared to 25% (18/64) in non-irrigated areas (p = 0.009). Most children aged below 5 who had fever (77.7%) were taken to healthcare facility for treatment compared to 31.4% of children aged 5-14 years or 20.9% of adults (0.0001). Predictors of treatment seeking included access to healthcare facility (OR = 16.23, 95% CI: 2.74-96.12), and ability to pay hospital bills (OR = 10.6, 95% CI: 1.97-57). Other factors that influenced health-seeking behavior included the severity of symptoms, the age of the patient and knowledge of malaria symptoms.

Effects of bacterial composition and aquatic habitat metabolites on malaria vector larval availability in irrigated and non-irrigated sites of Homa Bay county, western Kenya.

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.

Genetic Diversity and Population Structure of Anopheles funestus in Western Kenya Based on Mitochondrial DNA Marker COII.

The mitochondrial marker, COII, was employed to assess the genetic structure and diversity of Anopheles funestus, a very important malaria vector in Africa that adapt and colonize different ecological niches in western Kenya. Mosquitoes were collected using mechanical aspirators in four areas (Bungoma, Port Victoria, Kombewa, and Migori) in western Kenya. Following morphological identification, PCR was used to confirm the species. The COII gene was amplified, sequenced, and analyzed to determine genetic diversity and population structure. A total of 126 (Port Victoria-38, Migori-38, Bungoma-22, and Kombewa-28) sequences of COII were used for population genetic analysis. Anopheles funestus had a high haplotype diversity (Hd = 0.97 to 0.98) but low nucleotide diversity (Π = 0.004 to 0.005). The neutrality test revealed negative Tajima's D and Fs values indicating an excess of low-frequency variation. This could be attributed to either population expansion or negative selection pressure across all the populations. No genetic or structural differentiation (Fst = -0.01) and a high level of gene flow (Gamma St, Nm = 17.99 to 35.22) were observed among the populations. Population expansion suggests the high adaptability of this species to various ecological requirements, hence sustaining its vectorial capacity and malaria transmission.

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.

Sero-prevalence of hepatitis B virus and compliance with hepatitis B vaccination schedules among outpatient clinic attendees in Nairobi.

BACKGROUND: Hepatitis B is becoming a growing public health problem in Kenya. To combat the threat, HBV vaccination should be recommended, particularly for individuals who are not covered by the national immunization program. Vaccination provides sero-protection rates approaching 95% among healthy adults after completing the three-dose vaccination course, but decreases to 87% among those who receive only two doses, emphasizing the importance of completing the three-dose vaccination course. However, data on adult adherence to HBV multi-dose vaccines in Sub-Saharan Africa are limited, despite the fact that this information is critical for prevention. As a result, more research on HBV vaccine dose completion is required. The purpose of this study is to estimate the prevalence of hepatitis B virus infection among out-patient clinic attendees in Nairobi, Kenya, as well as to identify beneficiaries of free vaccination and barriers to completing the recommended vaccine doses. METHODS: Between July 30th and September 30th, 2015, 2644 outpatient clinic attendees aged ≥ 4 were recruited from three hospitals in Nairobi County, Kenya: Mama Lucy, Riruta, and Loco. Self-administered questionnaires were used to collect socio-demographic information, and blood samples were tested for hepatitis B surface antigen (HBsAg) using the KEMRI HEPCELL Rapid® (Hepatitis B Detection kit) test kit. Individuals who tested negative for HBsAg were given a free course of three doses of HBV vaccine. The vaccination register provided information on the number of doses administered. RESULTS: The average age of the study population was 31.4 years (range: 4-66), with females accounting for 59.2%. 1.82% (48/2644) of the participants tested positive for HBsAg. Among the 2596 individuals eligible for vaccination, 66% (1720/2596) received at least one dose, and 51.8% (1345/2596) received all three doses. Vaccination acceptance increased with age, with older patients more likely to return for subsequent dose (OR>1 for second and third dose). Unavailability and failure to contact client were cited as significant (p<0.0001) barrier to vaccination completion by 53.7% (666/1226, 95% CI 0.5-0.6) and 37% (454/1226, 95% CI 0.3-0.4) of respondents respectively. CONCLUSION: The prevalence of HBV infection among outpatient clinic attendees highlights the importance of expanding HBV immunization programs in Kenya. However, given the low vaccination completion rate, there is a need for public awareness of the vaccine's importance in preventing HBV and HBV-related complications.

Habitat Diversity, Stability, and Productivity of Malaria Vectors in Irrigated and Nonirrigated Ecosystems in Western Kenya.

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.

A prospective cohort study of Plasmodium falciparum malaria in three sites of Western Kenya.

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 (χ2 = 552.52, df = 1, P < 0.0001) and Marani (χ2 = 1127.99, df = 1, P < 0.0001). There was a significant difference in probability of non-infection during malaria episodes (log-rank test, χ2 = 617.59, df = 2, P < 0.0001) in the study sites, with Kombewa having the least median time of non-infection during malaria episodes. Gender bias toward males in infection was observed (χ2 = 27.17, df = 1, P < 0.0001). The annual entomological inoculation rates were 5.12, 3.65, and 0.50 infective bites/person/year at Kombewa, Iguhu, and Marani, respectively, during 2018 to 2019. CONCLUSIONS: Malaria prevalence in western Kenya remains high and has resurged in some sites despite continuous intervention efforts. Targeting malaria interventions to those with asymptomatic infections who serve as human reservoirs might decrease malaria transmission and prevent resurgences. Longitudinal monitoring enables detection of changes in parasitological and entomological profiles and provides core baseline data for the evaluation of vector interventions and guidance for future planning of malaria control.

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.

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.

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.

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.

Malaria epidemic and transmission foci in highland of Kisii, western Kenya.

Background: The vulnerable population within the malaria epidemic zone remains at risk of increased burden and fatality. This is because of unpreparedness and overstretching of healthcare capacity in the event of a full-fledged epidemic. The purpose of this study was to determine the prevalence of microscopic and submicroscopic infections, as well as map specific Plasmodium transmission foci, in the malaria epidemic-prone zone of Kisii highland. Methodology: Patients seeking malaria treatment at Eramba health facility in the epidemic-prone zone of Kisii highland were enrolled in the study. Malaria outpatient data for the entire month of May were also included in the analysis. Patients' finger prick blood smears were examined for microscopic infections, while a real-time polymerase chain reaction targeting the Plasmodium species 18S rRNA gene was used to detect the presence of submicroscopic infections on DNA extracted from dry blood spots. Results: Based on outpatient data, the malaria positivity rate was 20.7% (231/1115, 95% CI, 0.18-0.23). The positivity rate varied significantly by age group (χ2 = 75.05, df 2, p < 0.0001). Children under the age of five had the highest positivity rate (27.8%, 78/281), followed by children aged 5-15 years (19.4%, 69/356), and individuals aged 15 years and above (17.6%, 84/478). Out of the 102 patients recruited, the positivity rate by microscopy was 57.8% (59/102) and 72.5% (74/102) by RT-PCR. Most of the microscopic infections (40.7%, 24/59) were from Morara and Nyabikondo villages in Rioma and Kiomooncha sublocations, respectively. The submicroscopic prevalence was 14.7% (15/102) and was observed only in patients from high-infection villages in Rioma (15.8%, 9/57) and Kiomooncha (16.2%, 6/37) sublocations. Across gender and age groups, females (19.7%, 12/61) and patients aged 15 years and above (21.1%, 8/38) had high levels of submicroscopic infections. There were two mixed infections of P. falciparum/P. malariae and P. falciparum/P. ovale, both from patients residing in Kiomooncha sublocation. Conclusion: Plasmodium falciparum infections remained relatively high in the Marani subcounty. Infections were concentrated in two villages, which could serve as a target for future public health intervention, particularly during a malaria epidemic.

Impact of Agricultural Irrigation on Anemia in Western Kenya.

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.

Rare Alleles and Signatures of Selection on the Immunodominant Domains of Pfs230 and Pfs48/45 in Malaria Parasites From Western Kenya.

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.

Risk associations of submicroscopic malaria infection in lakeshore, plateau and highland areas of Kisumu County in western Kenya.

BACKGROUND: Persons with submicroscopic malaria infection are a major reservoir of gametocytes that sustain malaria transmission in sub-Saharan Africa. Despite recent decreases in the national malaria burden in Kenya due to vector control interventions, malaria transmission continues to be high in western regions of the country bordering Lake Victoria. The objective of this study was to advance knowledge of the topographical, demographic and behavioral risk factors associated with submicroscopic malaria infection in the Lake Victoria basin in Kisumu County. METHODS: Cross-sectional community surveys for malaria infection were undertaken in three eco-epidemiologically distinct zones in Nyakach sub-County, Kisumu. Adjacent regions were topologically characterized as lakeshore, hillside and highland plateau. Surveys were conducted during the 2019 and 2020 wet and dry seasons. Finger prick blood smears and dry blood spots (DBS) on filter paper were collected from 1,777 healthy volunteers for microscopic inspection and real time-PCR (RT-PCR) diagnosis of Plasmodium infection. Persons who were PCR positive but blood smear negative were considered to harbor submicroscopic infections. Topographical, demographic and behavioral risk factors were correlated with community prevalence of submicroscopic infections. RESULTS: Out of a total of 1,777 blood samples collected, 14.2% (253/1,777) were diagnosed as submicroscopic infections. Blood smear microscopy and RT-PCR, respectively, detected 3.7% (66/1,777) and 18% (319/1,777) infections. Blood smears results were exclusively positive for P. falciparum, whereas RT-PCR also detected P. malariae and P. ovale mono- and co-infections. Submicroscopic infection prevalence was associated with topographical variation (χ2 = 39.344, df = 2, p<0.0001). The highest prevalence was observed in the lakeshore zone (20.6%, n = 622) followed by the hillside (13.6%, n = 595) and highland plateau zones (7.9%, n = 560). Infection prevalence varied significantly according to season (χ2 = 17.374, df = 3, p<0.0001). The highest prevalence was observed in residents of the lakeshore zone in the 2019 dry season (29.9%, n = 167) and 2020 and 2019 rainy seasons (21.5%, n = 144 and 18.1%, n = 155, respectively). In both the rainy and dry seasons the likelihood of submicroscopic infection was higher in the lakeshore (AOR: 2.71, 95% CI = 1.85-3.95; p<0.0001) and hillside (AOR: 1.74, 95% CI = 1.17-2.61, p = 0.007) than in the highland plateau zones. Residence in the lakeshore zone (p<0.0001), male sex (p = 0.025), school age (p = 0.002), and living in mud houses (p = 0.044) increased the risk of submicroscopic malaria infection. Bed net use (p = 0.112) and occupation (p = 0.116) were not associated with submicroscopic infection prevalence. CONCLUSION: Topographic features of the local landscape and seasonality are major correlates of submicroscopic malaria infection in the Lake Victoria area of western Kenya. Diagnostic tests more sensitive than blood smear microscopy will allow for monitoring and targeting geographic sites where additional vector interventions are needed to reduce malaria transmission.

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.

Signatures of selection and drivers for novel mutation on transmission-blocking vaccine candidate Pfs25 gene in western Kenya.

BACKGROUND: Leading 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. METHODS: Dry 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. RESULTS: Nine 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<0.01) and Fu's F (-10.91, p<0.001) statistics tests. Three loci pairs (123, 372), (364, 428) and (390, 394) were detected to be under linkage disequilibrium and none had history of recombination. These results suggested that purifying selection and inbreeding might be the drivers of the observed variation in Pfs25. CONCLUSION: Given 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.