Evaluating the impact of larviciding with Bti and community education and mobilization as supplementary integrated vector management interventions for malaria control in Kenya and Ethiopia.

BACKGROUND: Malaria prevention in Africa is mainly through the use of long-lasting insecticide treated nets (LLINs). The objective of the study was to assess the effect of supplementing LLINs with either larviciding with Bacillus thuringiensis israelensis (Bti) or community education and mobilization (CEM), or with both interventions in the context of integrated vector management (IVM). METHODS: The study involved a factorial, cluster-randomized, controlled trial conducted in Malindi and Nyabondo sites in Kenya and Tolay site in Ethiopia, to assess the impact of the following four intervention options on mosquitoes and malaria prevalence: LLINs only (arm 1); LLINs and Bti (arm 2); LLINs and CEM (arm 3); and, LLINs combined with Bti and CEM (arm 4). Between January 2013 and December 2015, CDC light traps were used to sample adult mosquitoes during the second, third and fourth quarter of each year in 10 houses in each of 16 villages at each of the three study sites. Larvae were sampled once a fortnight from potential mosquito-breeding habitats using standard plastic dippers. Cross-sectional malaria parasite prevalence surveys were conducted involving a total of 11,846 primary school children during the 3-year period, including 4800 children in Tolay, 3000 in Malindi and 4046 in Nyabondo study sites. RESULTS: Baseline relative indoor anopheline density was 0.11, 0.05 and 0.02 mosquitoes per house per night in Malindi, Tolay and Nyabondo sites, respectively. Nyabondo had the highest recorded overall average malaria prevalence among school children at 32.4%, followed by Malindi with 5.7% and Tolay 1.7%. There was no significant reduction in adult anopheline density at each of the three sites, which could be attributed to adding of the supplementary interventions to the usage of LLINs. Malaria prevalence was significantly reduced by 50% in Tolay when using LLINs coupled with application of Bti, community education and mobilization. The two other sites did not reveal significant reduction of prevalence as a result of combining LLINs with any of the other supplementary interventions. CONCLUSION: Combining LLINs with larviciding with Bti and CEM further reduced malaria infection in a low prevalence setting in Ethiopia, but not at sites with relatively higher prevalence in Kenya. More research is necessary at the selected sites in Kenya to periodically determine the suite of vector control interventions and broader disease management strategies, which when integrated would further reduce adult anopheline populations and malaria prevalence beyond what is achieved with LLINs.

Shifts in malaria vector species composition and transmission dynamics along the Kenyan coast over the past 20 years.

BACKGROUND: Over the past 20 years, numerous studies have investigated the ecology and behaviour of malaria vectors and Plasmodium falciparum malaria transmission on the coast of Kenya. Substantial progress has been made to control vector populations and reduce high malaria prevalence and severe disease. The goal of this paper was to examine trends over the past 20 years in Anopheles species composition, density, blood-feeding behaviour, and P. falciparum sporozoite transmission along the coast of Kenya. METHODS: Using data collected from 1990 to 2010, vector density, species composition, blood-feeding patterns, and malaria transmission intensity was examined along the Kenyan coast. Mosquitoes were identified to species, based on morphological characteristics and DNA extracted from Anopheles gambiae for amplification. Using negative binomial generalized estimating equations, mosquito abundance over the period were modelled while adjusting for season. A multiple logistic regression model was used to analyse the sporozoite rates. RESULTS: Results show that in some areas along the Kenyan coast, Anopheles arabiensis and Anopheles merus have replaced An. gambiae sensu stricto (s.s.) and Anopheles funestus as the major mosquito species. Further, there has been a shift from human to animal feeding for both An. gambiae sensu lato (s.l.) (99% to 16%) and An. funestus (100% to 3%), and P. falciparum sporozoite rates have significantly declined over the last 20 years, with the lowest sporozoite rates being observed in 2007 (0.19%) and 2008 (0.34%). There has been, on average, a significant reduction in the abundance of An. gambiae s.l. over the years (IRR = 0.94, 95% CI 0.90-0.98), with the density standing at low levels of an average 0.006 mosquitoes/house in the year 2010. CONCLUSION: Reductions in the densities of the major malaria vectors and a shift from human to animal feeding have contributed to the decreased burden of malaria along the Kenyan coast. Vector species composition remains heterogeneous but in many areas An. arabiensis has replaced An. gambiae as the major malaria vector. This has important implications for malaria epidemiology and control given that this vector predominately rests and feeds on humans outdoors. Strategies for vector control need to continue focusing on tools for protecting residents inside houses but additionally employ outdoor control tools because these are essential for further reducing the levels of malaria transmission.

Larval density dependence in Anopheles gambiae s.s., the major African vector of malaria.

Anopheles gambiae sensu stricto is the most important vector of malaria in Africa although relatively little is known about the density-dependent processes determining its population size. Mosquito larval density was manipulated under semi-natural conditions using artificial larval breeding sites placed in the field in coastal Kenya; two experiments were conducted: one manipulating the density of a single cohort of larvae across a range of densities and the other employing fewer densities but with the treatments crossed with four treatments manipulating predator access. In the first experiment, larval survival, development rate and the size of the adult mosquito all decreased with larval density (controlling for block effects between 23% and 31% of the variance in the data could be explained by density). In the second experiment, the effects of predator manipulation were not significant, but again we observed strong density dependence in larval survival (explaining 30% of the variance). The results are compared with laboratory studies of A. gambiae larval competition and the few other studies conducted in the field, and the consequences for malaria control are discussed.

Effect of predation on Anopheles larvae by five sympatric insect families in coastal Kenya.

BACKGROUND & OBJECTIVES: The use of insecticides to eliminate mosquito larvae from ground pools may disrupt atural predator-induced control of mosquito larvae. Detrimental effects on predators may be directly from toxicity or by eliminating prey organisms. Identifying the principal predators responsible for mosquito suppression is needed to select non-target indicator species for insecticide studies. In this study, we sought to determine trophic level interactions between predators and immature stages of Anopheles gambiae Giles mosquitoes under experimental conditions in the coastal region of Kenya. METHODS: To identify effective predation pattern, a series of prey choice experiments was conducted. The relative abilities of five common species of aquatic insects found in the malaria-endemic coastal region of Kenya were assessed in a series of experiments. Experiments were conducted in semi-field conditions at Jaribuni, near the sites of insect collection. RESULTS: In single predator experiments, notonectids consumed most of the mosquito larvae; hydrometrids consumed about half of the mosquito larvae in treatments. Veliids and gerrids had significant, but small effects on larval survivorship. Dytiscids did not have a significant effect on mosquito larvae survivorship. In a two-predator experiment, notonectids significantly decreased survivorship of dytiscids without a change in suppressive effects on mosquito larvae. Of the five common predators evaluated, notonectids were clearly the most voracious consumers of mosquito larvae. The predation pressure on mosquito larvae was not affected by the addition of additional prey items, consisting of small dytiscid beetles. The importance of this notonectid species in coastal Kenya suggests that it would be a valuable non-target indicator species for insecticide studies. Hydrometrids were also efficient at consuming mosquito larvae. INTERPRETATION & CONCLUSION: Of the five common predators from the Kenyan coast evaluated in this study, notonectids were the most voracious consumers of immature mosquitoes. Their predation pressure on mosquito larvae was not affected by the addition of additional prey items, consisting of small dytiscid beetles.

Towards an integrated vector management approach for sustainable control of schistosomiasis and malaria in Mwea, Kirinyaga County, Kenya: Baseline epidemiological and vector results.

Background: Vector control is an important approach in the control of most parasitic and vector-borne diseases including malaria, and schistosomiasis. Distribution of these two infections often overlaps and in such areas it's more economically viable to employ an integrated approach in the control of their vectors which largely shares the same breeding ecosystem. We carried out a baseline epidemiological and vector surveys for malaria and schistosomiasis in Mwea, Kirinyaga County, in preparation for the upscaling of integrated vector management (IVM) for the two diseases. Methods: This was a repeated cross sectional survey, where mosquito and snails were sampled during dry and wet seasons in three different ecological zones, Kiamaciri, Thiba and Murinduko to identify possible breeding sites. Mosquito larvae were collected using standard dippers, adults using CDC miniature light traps while snail vectors were sampled using standard snail scoops in different breeding habitats. A total of 1200 pupils from 12 primary schools were tested for malaria using rapid diagnostic tests (Malaria Pf/PAN Ag combo). Stool samples were processed using the Kato Katz technique for intestinal schistosomiasis. Results: The overall prevalence of intestinal schistosomiasis was 9.08 % (95 % CI: 07.00-11.00), with Kiamaciri zone recording the highest prevalence at 19 % (95%CI: 15.00-23.00) and Murinduko zone the least at 0.17 % (95%CI: 0.00-0.01). Majority of the infections were of light intensity 78.9 % (95%CI: 70.04-86.13). There was no positive malaria case detected in this study. Of the 3208 adult mosquitoes sampled during the dry season, 20.6 % (95 % CI: 19.25-22.08) were Anopheles gambiae s.l while 79.4 % (95 % CI: 77.92-80.75) were culicines. During the wet season, 3378 adult mosquitoes were collected, of which 14.7 % (95 % CI: 13.56-15.98) were Anopheles gambiae s.l and 85.3 % (95 % CI: 84.02-86.44) culicines. Overall, 4085 mosquito larvae were collected during the two seasons, of which, 57.3 % and 42.7 % were anopheles and culicine respectively. Majority of the larvae (85.1 % (95%CI: 84.01-86.10) were collected during the wet season, with only 14.9 % (95%CI: 14.10-16.00) being collected during the dry season. A total of 2292 fresh water vector snails were collected with a majority (69.6 % (95%CI: 68.00-71.10) being Biomphalaria pffeiferi responsible for transmission of intestinal schistosomiasis. Conclusion: This study demonstrates that intestinal schistosomiasis is prevalent in Kiamaciri and Thiba zones, and points to the possibility of active transmission of schistosomiasis in Murinduko zone. Malaria vectors were predominantly observed in all sites despite there being no malaria positive case. Culex quinquefaciatus responsible for the spread of several arboviruses was also observed. The presence of these vectors may lead to future disease outbreaks in the area if concerted control initiatives are not undertaken. The disease vectors shared the same breeding sites and thus its economical and feasible to adopt an integrated vector management approach in control efforts for these disease in the study area.

Mosquito species abundance and diversity in Malindi, Kenya and their potential implication in pathogen transmission.

Mosquitoes (Diptera: Culicidae) are important vectors of human disease-causing pathogens. Mosquitoes are found both in rural and urban areas. Deteriorating infrastructure, poor access to health, water and sanitation services, increasing population density, and widespread poverty contribute to conditions that modify the environment, which directly influences the risk of disease within the urban and peri-urban ecosystem. The objective of this study was to evaluate the mosquito vector abundance and diversity in urban, peri-urban, and rural strata in Malindi along the Kenya coast. The study was conducted in the coastal district of Malindi between January and December 2005. Three strata were selected which were described as urban, peri-urban, and rural. Sampling was done during the wet and dry seasons. Sampling in the wet season was done in the months of April and June to cover the long rainy season and in November and December to cover the short rainy season, while the dry season was between January and March and September and October. Adult mosquito collection was done using Pyrethrum Spray Collection (PSC) and Centers for Disease Control and Prevention (CDC) light traps inside houses and specimens were identified morphologically. In the three strata (urban, peri-urban, and rural), 78.5% of the total mosquito (n = 7,775) were collected using PSC while 18.1% (n = 1,795) were collected using the CDC light traps. Using oviposition traps, mosquito eggs were collected and reared in the insectary which yielded 329 adults of which 83.8% (n = 276) were Aedes aegypti and 16.2% (n = 53) were Culex quinquefasciatus. The mosquito distribution in the three sites varied significantly in each collection site. Anopheles gambiae, Anopheles funestus and Anopheles coustani were predominant in the rural stratum while C. quinquefasciatus was mostly found in urban and peri-urban strata. However, using PSC and CDC light trap collection techniques, A. aegypti was only found in urban strata. In the three strata, mosquitoes were mainly found in high numbers during the wet season. Further, A. gambiae, C. quinquefasciatus, and A. aegypti mosquitoes were found occurring together inside the houses. This in turn exposes the inhabitants to an array of mosquito-borne diseases including malaria, bancroftian filariasis, and arboviruses (dengue fever, Yellow fever, Rift Valley fever, Chikungunya fever, and West Nile Virus). In conclusion, our findings provide useful information for the design of integrated mosquito and disease control programs in East African environments.

Plasmodium falciparum transmission and aridity: a Kenyan experience from the dry lands of Baringo and its implications for Anopheles arabiensis control.

BACKGROUND: The ecology of malaria vectors particularly in semi-arid areas of Africa is poorly understood. Accurate knowledge on this subject will boost current efforts to reduce the burden of malaria in sub-Saharan Africa. The objective of this study was to describe the dynamics of malaria transmission in two model semi-arid sites (Kamarimar and Tirion) in Baringo in Kenya. METHODS: Adult mosquitoes were collected indoors by pyrethrum spray collections (PSC) and outdoors by Centers for Disease Control (CDC) light traps and identified to species by morphological characteristics. Sibling species of Anopheles gambiae complex were further characterized by rDNA. PCR and enzyme-linked immuno-sorbent assays (ELISA) were used to test for Plasmodium falciparum circumsporozoite proteins and host blood meal sources respectively. RESULTS: Anopheles arabiensis was not only the most dominant mosquito species in both study sites but also the only sibling species of An. gambiae s.l. present in the area. Other species identified in the study area were Anopheles funestus, Anopheles pharoensis and Anopheles coustani. For Kamarimar but not Tirion, the human blood index (HBI) for light trap samples was significantly higher than for PSC samples (Kamarimar, 0.63 and 0.11, Tirion, 0.48 and 0.43). The HBI for light trap samples was significantly higher in Kamarimar than in Tirion while that of PSC samples was significantly higher in Tirion than in Kamarimar. Entomological inoculation rates (EIR) were only detected for one month in Kamarimar and 3 months in Tirion. The number of houses in a homestead, number of people sleeping in the house, quality of the house, presence or absence of domestic animals, and distance to the animal shelter and the nearest larval habitat were significant predictors of An. arabiensis occurrence. CONCLUSION: Malaria transmission in the study area is seasonal with An. arabiensis as the dominant vector. The fact this species feeds readily on humans and domestic animals suggest that zooprophylaxis may be a plausible malaria control strategy in semi-arid areas of Africa. The results also suggest that certain household characteristics may increase the risk of malaria transmission.

Wide-scale application of Bti/Bs biolarvicide in different aquatic habitat types in urban and peri-urban Malindi, Kenya.

Larval control is a major component in mosquito control programs. This study evaluated the wide-scale application of Bti/Bs biolarvicide (Bacillus thuringiensis var. israelensis [Bti] and Bacillus sphaericus [Bs]) in different aquatic habitats in urban and peri-urban Malindi, Kenya. This study was done from June 2006 to December 2007. The urban and peri-urban area of Malindi town was mapped and categorized in grid cells of 1 km(2). A total of 16 1-km(2) cells were selected based on presence Community Based Organization dealing with malaria control within the cells. Each of the 16 1-km(2) cells was thoroughly searched for the presence of potential larval habitats. All habitats, whether positive or negative for larvae, were treated and rechecked 24 h (1 day), 6 days, and 10 days later for the efficacy of Bti/Bs. Weekly larval sampling was done to determine the mosquito larval dynamics in the aquatic habitats during the study period. Morphological identification of the mosquito larvae showed that Anopheles gambiae s.l. Giles was the most predominant species of the Anopheles and while in the culicines, Cx. quinquefasciatus Say was the predominant species. Anopheles larvae were all eliminated in habitats within a day post-application. For culicine larvae, 38.1% (n=8) of the habitat types responded within day 1 post-treatment and all the larvae were killed, they turned negative during the days of follow-up. Another 38.1% (n=8) of the habitat types had culicine larvae but turned negative by day 6, while three habitats (14.3%) had larvae by 6th day but turned negative by 10th day. However during this Bti/Bs application studies, two habitat types, house drainage and cesspits (9.5%), remained positive during the follow-up although the mosquito larvae were significantly reduced. Both early and late instars of Anopheles larvae immediately responded to Bti/Bs application and reached 100% mortality. The early and late instars of culicine responded to the Bti/Bs application but not as fast as the Anopheles larval instars. The early instars Culex, responded with 90.8% mortality at day 1 post-treatment, and the mortality was 99.9% at day 10. Similarly, the late instars Culex followed the same trend and exhibited same mortalities. The weekly sampling in the aquatic habitats showed that there was a 36.3% mosquito larval reduction in the aquatic habitats over the 18-months study period. In conclusion, Bti/Bs biolarvicide are useful in reducing the mosquito larval densities in a wide range of habitats which have a direct impact of adult mosquito populations.

Insecticide resistance status in Anopheles gambiae (s.l.) in coastal Kenya.

BACKGROUND: The rapid and widespread evolution of insecticide resistance has emerged as one of the major challenges facing malaria control programs in sub-Saharan Africa. Understanding the insecticide resistance status of mosquito populations and the underlying mechanisms of insecticide resistance can inform the development of effective and site-specific strategies for resistance prevention and management. The aim of this study was to investigate the insecticide resistance status of Anopheles gambiae (s.l.) mosquitoes from coastal Kenya. METHODS: Anopheles gambiae (s.l.) larvae sampled from eight study sites were reared to adulthood in the insectary, and 3- to 5-day-old non-blood-fed females were tested for susceptibility to permethrin, deltamethrin, dichlorodiphenyltrichloroethane (DDT), fenitrothion and bendiocarb using the standard World Health Organization protocol. PCR amplification of rDNA intergenic spacers was used to identify sibling species of the An. gambiae complex. The An. gambiae (s.l.) females were further genotyped for the presence of the L1014S and L1014F knockdown resistance (kdr) mutations by real-time PCR. RESULTS: Anopheles arabiensis was the dominant species, accounting for 95.2% of the total collection, followed by An. gambiae (s.s.), accounting for 4.8%. Anopheles gambiae (s.l.) mosquitoes were resistant to deltamethrin, permethrin and fenitrothion but not to bendiocarb and DDT. The L1014S kdr point mutation was detected only in An. gambiae (s.s.), at a low allelic frequency of 3.33%, and the 1014F kdr mutation was not detected in either An. gambiae (s.s.) or An. arabiensis. CONCLUSION: The findings of this study demonstrate phenotypic resistance to pyrethroids and organophosphates and a low level of the L1014S kdr point mutation that may partly be responsible for resistance to pyrethroids. This knowledge may inform the development of insecticide resistance management strategies along the Kenyan Coast.

Urban Ecology of Arboviral Mosquito Vectors Along the Kenyan Coast.

The purpose of this study was to determine the ecology of the common arboviral mosquito vectors in Mombasa, Kilifi and Malindi urban areas of coastal Kenya. Mosquito larvae were collected using standard dippers and pipettes. Egg survivorship in dry soil was evaluated by collecting soil samples from dry potential larval developmental sites, re-hydrating them for hatching and rearing of the eventual larvae to adults. Adult mosquitoes were collected with CDC light traps and BG-Sentinel traps. All blood-fed females were tested for bloodmeal origin. Mosquitoes were screened for arboviruses using RT-qPCR. Overall, the predominant species were Culex quinquefasciatus (Say) 72.4% (n = 2,364) and Aedes aegypti (L.), 25.7%, (n = 838). A total of 415 larval developmental sites were identified indoors (n = 317) and outdoors (n = 98). The most productive larval developmental sites, both indoors and outdoors, were assorted small containers, water tanks, drainages, drums, and jerricans. Overall, 62% (n = 18) of the soil samples collected were positive for larvae which were used as a proxy to measure the presence of eggs. The mosquitoes fed on humans (29.8%) and chickens (3.7%). Of 259 mosquitoes tested for viral infection, 11.6% were positive for Flavivirus only. The most productive larval developmental sites for arboviral vectors indoors were small containers, water tanks, jerricans, and drums whereas small containers, water tanks, drainage channels, buckets, tires, and water troughs were the productive larval developmental sites outdoors.

Distribution of the main malaria vectors in Kenya.

BACKGROUND: A detailed knowledge of the distribution of the main Anopheles malaria vectors in Kenya should guide national vector control strategies. However, contemporary spatial distributions of the locally dominant Anopheles vectors including Anopheles gambiae, Anopheles arabiensis, Anopheles merus, Anopheles funestus, Anopheles pharoensis and Anopheles nili are lacking. The methods and approaches used to assemble contemporary available data on the present distribution of the dominant malaria vectors in Kenya are presented here. METHOD: Primary empirical data from published and unpublished sources were identified for the period 1990 to 2009. Details recorded for each source included the first author, year of publication, report type, survey location name, month and year of survey, the main Anopheles species reported as present and the sampling and identification methods used. Survey locations were geo-positioned using national digital place name archives and on-line geo-referencing resources. The geo-located species-presence data were displayed and described administratively, using first-level administrative units (province), and biologically, based on the predicted spatial margins of Plasmodium falciparum transmission intensity in Kenya for the year 2009. Each geo-located survey site was assigned an urban or rural classification and attributed an altitude value. RESULTS: A total of 498 spatially unique descriptions of Anopheles vector species across Kenya sampled between 1990 and 2009 were identified, 53% were obtained from published sources and further communications with authors. More than half (54%) of the sites surveyed were investigated since 2005. A total of 174 sites reported the presence of An. gambiae complex without identification of sibling species. Anopheles arabiensis and An. funestus were the most widely reported at 244 and 265 spatially unique sites respectively with the former showing the most ubiquitous distribution nationally. Anopheles gambiae, An. arabiensis, An. funestus and An. pharoensis were reported at sites located in all the transmission intensity classes with more reports of An. gambiae in the highest transmission intensity areas than the very low transmission areas. CONCLUSION: A contemporary, spatially defined database of the main malaria vectors in Kenya provides a baseline for future compilations of data and helps identify areas where information is currently lacking. The data collated here are published alongside this paper where it may help guide future sampling location decisions, help with the planning of vector control suites nationally and encourage broader research inquiry into vector species niche modeling.

Anopheles larval abundance and diversity in three rice agro-village complexes Mwea irrigation scheme, central Kenya.

BACKGROUND: The diversity and abundance of Anopheles larvae has significant influence on the resulting adult mosquito population and hence the dynamics of malaria transmission. Studies were conducted to examine larval habitat dynamics and ecological factors affecting survivorship of aquatic stages of malaria vectors in three agro-ecological settings in Mwea, Kenya. METHODS: Three villages were selected based on rice husbandry and water management practices. Aquatic habitats in the 3 villages representing planned rice cultivation (Mbui Njeru), unplanned rice cultivation (Kiamachiri) and non-irrigated (Murinduko) agro-ecosystems were sampled every 2 weeks to generate stage-specific estimates of mosquito larval densities, relative abundance and diversity. Records of distance to the nearest homestead, vegetation coverage, surface debris, turbidity, habitat stability, habitat type, rice growth stage, number of rice tillers and percent Azolla cover were taken for each habitat. RESULTS: Captures of early, late instars and pupae accounted for 78.2%, 10.9% and 10.8% of the total Anopheles immatures sampled (n = 29,252), respectively. There were significant differences in larval abundance between 3 agro-ecosystems. The village with 'planned' rice cultivation had relatively lower Anopheles larval densities compared to the villages where 'unplanned' or non-irrigated. Similarly, species composition and richness was higher in the two villages with either 'unplanned' or limited rice cultivation, an indication of the importance of land use patterns on diversity of larval habitat types. Rice fields and associated canals were the most productive habitat types while water pools and puddles were important for short periods during the rainy season. Multiple logistic regression analysis showed that presence of other invertebrates, percentage Azolla cover, distance to nearest homestead, depth and water turbidity were the best predictors for Anopheles mosquito larval abundance. CONCLUSION: These results suggest that agricultural practices have significant influence on mosquito species diversity and abundance and that certain habitat characteristics favor production of malaria vectors. These factors should be considered when implementing larval control strategies which should be targeted based on habitat productivity and water management.

Expression of metallothionein and alpha-tubulin in heavy metal-tolerant Anopheles gambiae sensu stricto (Diptera: Culicidae).

Anopheles mosquitoes have been shown to adapt to heavy metals in their natural habitats. In this study we explored the possibility of using Anopheles gambiae sensu stricto as bio-reporters for environmental heavy metal pollution through expressions of their metal-responsive metallothionein and alpha-tubulin genes. The study was undertaken with third instar larvae after selection by cadmium, copper, or lead at LC(30) through five successive generations. Expression levels were determined in the 5th generation by semi-quantitative RT-PCR on the experimental and control populations. The data were analyzed using one-way ANOVA. The highest metallothionein (F(3,11)=4.574, P=0.038) and alpha-tubulin (F(3,11)=12.961, P=0.002) responses were observed in cadmium-tolerant treatments. There was significantly higher expression of metallothionein in cadmium or copper treatments relative to the control (P=0.012), and in cadmium than in lead treatments (P=0.044). Expressions of alpha-tubulin were significantly higher in cadmium than in control treatments (P=0.008). These results demonstrate the capacity of An. gambiae s.s. to develop tolerance to increased levels of heavy metal challenge. The results also confirm the potential of heavy metal-responsive genes in mosquitoes as possible bio-indicators of heavy metal environmental pollution. How the tolerance and expressions relate to An. gambiae s.s. fitness and vectorial capacity in the environment remains to be elucidated.

Impact of Insecticide Resistance on P. falciparum Vectors' Biting, Feeding, and Resting Behaviour in Selected Clusters in Teso North and South Subcounties in Busia County, Western Kenya.

INTRODUCTION: Behavioural resistance to insecticides restrains the efficacy of vector control tools against mosquito-transmitted diseases. The current study is aimed at determining the impact of insecticide resistance on major malaria vectors' biting, feeding, and resting behaviour in areas with and areas without insecticide resistance in Teso North and Teso South, Busia County, Western Kenya. METHODS: Mosquito larvae were sampled using a dipper, reared into 3-5-day-old female mosquitoes [4944] which were exposed to 0.75% permethrin and 0.05% deltamethrin using World Health Organization tube assay method. Blood meal, species identification, and kdr Eastgene PCRs were also performed on adult mosquitoes sampled using mosquito collection methods [3448]. Biting, feeding, resting, and exiting behaviours of field-collected mosquitoes from five selected clusters were analysed. RESULTS: The lowest Kdr genotypic frequency (SS) proportion was found in female Anophelines collected in Kengatunyi at 58% while Rwatama had the highest genotypic frequency at 93%, thus susceptible and resistant clusters, respectively. The peak hour for mosquito seeking a human bite was between 0300 and 0400 hrs in the resistant cluster and 0400-0500 hrs in the susceptible cluster. The heterozygous mosquitoes maintained the known 2100-2200 hrs peak hour. There was a higher proportion of homozygous susceptible vectors (86.4%) seeking humans indoor than outdoor bitters (78.3%). Mosquito blood meals of human origin were 60% and 87% in susceptible Kengatunyi and resistant Rwatama cluster, respectively. There was significant difference between homozygous-resistant vectors feeding on human blood compared to homozygous susceptible mosquitoes (p ≤ 0.05). The proportion of bovine blood was highest in the susceptible cluster. A higher proportion of homozygous-resistant anophelines were feeding and resting indoors. No heterozygous mosquito was found resting indoor while 4.2% of the mosquitoes were caught while exiting the house through the window. Discussion. A shift in resistant Anopheles gambiae sl highest peak hour of aggressiveness from 2100-2200 hrs to 0300-0400 hrs is a key change in its biting pattern. Due to the development of resistance, mosquitoes no longer have to compete against the time the human host enters into the formerly lethal chemical and or physical barrier in the form of long-lasting insecticide-treated net. No heterozygous LS mosquito rested indoors possibly due to disadvantages of heterozygosity which could have increased their fitness costs as well as energy costs in the presence of the insecticidal agents in the treated nets. Conclusions and recommendations. Out of bed biting by female mosquitoes and partial susceptibility may contribute to residual malaria transmission. Insecticide-resistant vectors have become more endophagic and anthropophillic. Hence, insecticidal nets, zooprophylaxis, and novel repellents are still useful chemical, biological, and physical barriers against human blood questing female mosquitoes. Further studies should be done on genetic changes in mosquitoes and their effects on changing mosquito behaviour.

Species Composition, Phenotypic and Genotypic Resistance Levels in Major Malaria Vectors in Teso North and Teso South Subcounties in Busia County, Western Kenya.

INTRODUCTION: Knockdown resistance (kdr) is strongly linked to pyrethroid insecticide resistance in Anopheles gambiae in Africa, which may have vital significance to the current increased use of pyrethroid-treated bed net programmes. The study is aimed at determining species composition, levels of insecticide resistance, and knockdown patterns in Anopheles gambiae sensu lato in areas with and areas without insecticide resistance in Teso North and Teso South subcounties, Western Kenya. MATERIALS AND METHODS: For WHO vulnerability tests, mosquito larvae were sampled using a dipper, reared into 3-5-day-old female mosquitoes (4944 at 100 mosquitoes per insecticide) which were exposed to 0.75% permethrin, 0.05% deltamethrin, and 0.1% bendiocarb using the WHO tube assay method. Species identification and kdr East gene PCRs were also performed on randomly selected mosquitoes from the collections; including adult mosquitoes (3448) sampled using standard collection methods. RESULTS: Anopheles gambiae sensu stricto were the majority in terms of species composition at 78.9%. Bendiocarb caused 100% mortality while deltamethrin had higher insecticidal effects (77%) on female mosquitoes than permethrin (71%). Susceptible Kengatunyi cluster had higher proportion of An. arabiensis (20.9%) than resistant Rwatama (10.7%). Kengatunyi mosquitoes exposed to deltamethrin had the highest KDT50 R of 8.2. Both Anopheles gambiae sensu stricto and Anopheles arabiensis had equal S allelic frequency of 0.84. Indoor resting mosquitoes had 100% mortality rate after 24 h since exposure. Overall SS genotypic frequency in Teso North and Teso South subcounties was 79.4% against 13.7% homozygous LL genotype and 6.9% heterozygous LS genotype. There was a significant difference (ρ < 0.05) in S allele frequencies between Kengatunyi (0.61) and Rwatama (0.95). Mosquito samples collected in 2013 had the highest S allelic frequency of 0.87. Discussion. Most likely, the higher the selection pressure exerted indoors by insecticidal nets, the higher were the resistance alleles. Use of pyrethroid impregnated nets and agrochemicals may have caused female mosquitoes to select for pyrethroid resistance. Different modes of action and chemical properties in different types of pyrethroids aggravated by a variety of edaphic and climatic factors may have caused different levels of susceptibility in both indoor and outdoor vectors to pyrethroids and carbamate. Species composition and populations in each collection method may have been influenced by insecticide resistance capacity in different species. Conclusions and Recommendations. Both phenotypic and genotypic insecticide resistance levels have been confirmed in Teso North and Teso South subcounties in Western Kenya. Insecticide resistance management practices in Kenya should be fast tracked and harmonized with agricultural sector agrochemical-based activities and legislation, and possibly switch to carbamate use in order to ease selection pressure on pyrethroids which are useable in insecticidal nets and indoor residual spray due to their low human toxicity. The implication of such high resistance levels in mosquitoes collected in Teso subcounties is that resistance is likely to persist and or even increase if monomolecules of permethrin and deltamethrin or both continue to be used in all net- and nonnet-based mosquito control purposes. Usage of mutually reinforcing piperonyl butoxide (PBO) that prohibits particular enzymes vital in metabolic activities inside mosquito systems and has been integrated into pyrethroid-LLINs to create pyrethroid-PBO nets is an extremely viable option.

Spatial distribution, blood feeding pattern, and role of Anopheles funestus complex in malaria transmission in central Kenya.

Studies were conducted to determine the role of sibling species of Anopheles funestus complex in malaria transmission in three agro-ecosystems in central Kenya. Mosquitoes were sampled indoors and outdoors, and rDNA PCR was successfully used to identify 340 specimens. Anopheles parensis (91.8%), A. funestus (6.8%), and Anopheles leesoni (1.5%) were the three sibling species identified. A. parensis was the dominant species at all study sites, while 22 of 23 A. funestus were collected in the non-irrigated study site. None of the 362 specimens tested was positive for Plasmodium falciparum circumsporozoite proteins by enzyme-linked immunosorbent assay. The most common blood-meal sources (mixed blood meals included) for A. parensis were goat (54.0%), human (47.6%), and bovine (39.7%), while the few A. funestus s.s. samples had fed mostly on humans. The human blood index (HBI) for A. parensis (mixed blood meals included) in the non-irrigated agro-ecosystem was 0.93 and significantly higher than 0.33 in planned rice agro-ecosystem. The few samples of A. funestus s.s. and A. funestus s.l. also showed a trend of higher HBI in the non-irrigated agro-ecosystem. We conclude that agricultural practices have significant influence on distribution and blood feeding behavior of A. funestus complex. Although none of the species was implicated with malaria transmission, these results may partly explain why non-irrigated agro-ecosystems are associated with higher risk of malaria transmission by this species compared to irrigated agro-ecosystems.

Seasonal mosquito larval abundance and composition in Kibwezi, lower eastern Kenya.

BACKGROUND & OBJECTIVES: Changes in weather patterns especially rainfall affects the distribution and densities of mosquitoes. The objective of this study was to describe mosquito aquatic habitats, to determine larval abundance, species composition, and habitat types found in Kasayani village of Kibwezi division. METHODS: A cross-sectional survey of mosquito larval habitats was conducted in Kasayani village in Kibwezi division to determine species composition, larval abundance, and habitat types found in this village. This survey was conducted during the rainy season in November and December 2006 and during the dry season in February and March 2007. Larvae were collected using the standard dipping technique and a total of 24 habitats were sampled. The primary habitats identified were water reservoir tanks, puddles, temporary pools, and tyre tracks. RESULTS: A total of 2660 mosquito larvae were collected of which 2140 (80.45%) were culicines, 503 (18.91%) were Anopheles and 17 (0.64%) were pupae. For culicines, 1787 (83.5%) were categorized as early instars and 353 (16.5%) were as late instars while in the Anopheles, 425 (84.49%) were classified as early instars and 78 (15.51%) were late instars. Morphological identification of the III and IV instar larvae by use of microscopy yielded 16.24% (n = 70) Anopheles gambiae complex, 1.16% (n=5) An. funestus, 0.70% (n=3) An. coustani, 42.46% (n=183) Culex quinquefasciatus, 6.26% (n = 27) Cx. duttoni, and 33.18% (n=143) Ae. aegypti. Puddles, tyre tracks and pools had highly turbid water while water reservoir tanks had clear water. Anopheles gambiae and Cx. quinquefasciatus were found in all habitat categories while Ae. aegypti were found only in water storage tanks. INTERPRETATION & CONCLUSION: The mosquito larval densities indicate that the inhabitants of this village are at risk of mosquito-borne diseases including malaria, which is one of the greatest causes of morbidity and mortality in this area. Furthermore, mosquito control measures targeting both the mosquito immatures and adults should be enhanced especially during the rainy season to ensure maximum protection of the inhabitants.

A formative study of disposal and re-use of old mosquito nets by communities in Malindi, Kenya.

BACKGROUND: About 30 million insecticide treated mosquito nets have been distributed in Kenya since 2001 and ownership is approaching full coverage. As a consequence of this achievement, Kenya is faced with the challenge of disposing old mosquito nets that are no longer in use. The study aimed at investigating ways of disposal and re-use of old and torn nets by end users. MATERIALS AND METHODS: A formative study was conducted in the former Malindi District, which is comprised of Malindi and Magarini sub-counties of Kilifi County in Coastal Kenya. A total of 6 Focus Group Discussions, 10 Key Informant Interviews and 9 transect walks/drives were undertaken. Data from the different sources were analysed separately and triangulated for similarities and differences. RESULTS: There were variations in disposal and re-use of old nets between urban and rural or peri-urban residents. In all settings, people adopted innovative and beneficial ways of re-using old, expired nets, and those that were damaged beyond repair. Common causes of damage were fire, children, domestic animals sharing the sleeping room and friction from the bed poles while hanging or tacking it in under a sleeping mat. Re-use was most prominent in farming activities (78%) and less to for use in mosquito control, like window screening (15%). The remaining 8% was related to making ropes, swings, footballs, goal posts and fishing nets. Advantageous texture and nature of the netting material, perceived economic benefit and lack of guidelines for disposal were the main reasons cited by residents for re-using old nets. CONCLUSIONS: It is important that re-use and disposal of old mosquito nets is distinguished from misuse of newly distributed mosquito nets. Alternative uses of old nets as opposed to misuse of new nets was found to be common in our study.

Exploring communities' and health workers' perceptions of indicators and drivers of malaria decline in Malindi, Kenya.

BACKGROUND: Since 2000, a decrease in malaria burden has been observed in most endemic countries. Declining infection rates and disease burden and reduction in asymptomatic carriers are the outcome of improved quality of care and related health system factors. These include improved case management through better diagnosis, implementation of highly effective antimalarial drugs and increased use of bednets. We studied communities' and health workers' perceptions of indicators and drivers in the context of decreasing malaria transmission in Malindi, Kenya. MATERIALS AND METHODS: A variety of qualitative methods that included participatory rural appraisal (PRA) tools such as community river of life and trend lines, focus group discussions (FGDs) and key informant interviews were used. Studies took place between November 2013 and April 2014. RESULTS: Providing residents with bednets contributed to malaria reduction, and increasing community awareness on the causes and symptoms of malaria and improved malaria treatment were also perceived to contribute to the decline of malaria. The study identified three perceived drivers to the reported decline in malaria: a) community health workers' enhanced awareness creation towards household owners regarding malaria-related activities through visitations and awareness sessions, b) Women involvement in Savings Internal Lending Community was perceived to have increased their financial base, thereby improving their decision-making power towards the care of their sick child(ren), c) Non Governmental Organizations (NGOs) and partners played a promoter part in health and general economic development initiatives. CONCLUSIONS: To achieve the goal of malaria elimination, collaboration between governmental and NGOs will be crucial when improving the financial base of women and enhancing participation of community health workers.

Host choice and multiple blood feeding behaviour of malaria vectors and other anophelines in Mwea rice scheme, Kenya.

BACKGROUND: Studies were conducted between April 2004 and February 2006 to determine the blood-feeding pattern of Anopheles mosquitoes in Mwea Kenya. METHODS: Samples were collected indoors by pyrethrum spay catch and outdoors by Centers for Disease Control light traps and processed for blood meal analysis by an Enzyme-linked Immunosorbent Assay. RESULTS: A total of 3,333 blood-fed Anopheles mosquitoes representing four Anopheles species were collected and 2,796 of the samples were assayed, with Anopheles arabiensis comprising 76.2% (n = 2,542) followed in decreasing order by Anopheles coustani 8.9% (n = 297), Anopheles pharoensis 8.2% (n = 272) and Anopheles funestus 6.7% (n = 222). All mosquito species had a high preference for bovine (range 56.3-71.4%) over human (range 1.1-23.9%) or goat (0.1-2.2%) blood meals. Some individuals from all the four species were found to contain mixed blood meals. The bovine blood index (BBI) for An. arabiensis was significantly higher for populations collected indoors (71.8%), than populations collected outdoors (41.3%), but the human blood index (HBI) did not differ significantly between the two populations. In contrast, BBI for indoor collected An. funestus (51.4%) was significantly lower than for outdoor collected populations (78.0%) and the HBI was significantly higher indoors (28.7%) than outdoors (2.4%). Anthropophily of An. funestus was lowest within the rice scheme, moderate in unplanned rice agro-ecosystem, and highest within the non-irrigated agro-ecosystem. Anthropophily of An. arabiensis was significantly higher in the non-irrigated agro-ecosystem than in the other agro-ecosystems. CONCLUSION: These findings suggest that rice cultivation has an effect on host choice by Anopheles mosquitoes. The study further indicate that zooprophylaxis may be a potential strategy for malaria control, but there is need to assess how domestic animals may influence arboviruses epidemiology before adapting the strategy.