Insecticidal and repellent activities of pyrethroids to the three major pyrethroid-resistant malaria vectors in western Kenya

Background: The dramatic success of insecticide treated nets (ITNs) and long-lasting insecticidal nets (LLINs) in African countries has been countered by the rapid development of pyrethroid resistance in vector mosquitoes over the past decade. One advantage of the use of pyrethroids in ITNs is their excito-repellency. Use of the excito-repellency of pyrethroids might be biorational, since such repellency will not induce or delay the development of any physiological resistance. However, little is known about the relationship between the mode of insecticide resistance and excito-repellency in pyrethroid-resistant mosquitoes. Methods. Differences in the reactions of 3 major malaria vectors in western Kenya to pyrethroids were compared in laboratory tests. Adult susceptibility tests were performed using World Health Organization (WHO) test tube kits for F1 progenies of field-collected An. gambiae s.s., An. arabiensis, and An. funestus s.s., and laboratory colonies of An. gambiae s.s. and An. arabiensis. The contact repellency to pyrethroids or permethrin-impregnated LLINs (OlysetR Nets) was evaluated with a simple choice test modified by WHO test tubes and with the test modified by the WHO cone bioassay test. Results: Field-collected An. gambiae s.s., An. arabiensis, and An. funestus s.s. showed high resistance to both permethrin and deltamethrin. The allelic frequency of the point mutation in the voltage-gated sodium channel (L1014S) in An. gambiae s.s. was 99.3-100%, while no point mutations were detected in the other 2 species. The frequency of takeoffs from the pyrethroid-treated surface and the flying times without contacting the surface increased significantly in pyrethroid-susceptible An. gambiae s.s. and An. arabiensis colonies and wild An. arabiensis and An. funestus s.s. colonies, while there was no significant increase in the frequency of takeoffs or flying time in the An. gambiae s.s. wild colony. Conclusion: A different repellent reaction was observed in the field-collected An. gambiae s.s. than in An. arabiensis and An. funestus s.s. It might be that resistant mosquitoes governed by knockdown resistance (kdr) loose repellency to pyrethroids, whereas those lacking kdr maintain high repellency irrespective of their possessing metabolic resistance factors to pyrethroids. Further genetic evaluation is required for the demonstration of the above hypothesis

Preventive effect of permethrin-impregnated long-lasting insecticidal nets on the blood feeding of three major pyrethroid-resistant malaria vectors in western Kenya

Background: Since the World Health Organization (WHO) adopted the use of long-lasting insecticidal nets (LLINs) as a principal strategy for effective malaria prevention and control, pyrethroids have been the only class of insecticides used for LLINs. The dramatic success of insecticide-treated nets (ITNs) and LLINs in African countries, however, has been threatened by the rapid development of pyrethroid resistance in vector mosquitoes. ITNs and LLINs are still used as effective self-protection measures, but there have been few studies on the effectiveness of ITNs and LLINs in areas where vector mosquitoes are pyrethroid-resistant. Methods. To investigate the behavioral pattern of mosquitoes in the houses where LLINs were used, indoor mosquito trappings of Anopheles gambiae s.s., An. arabiensis, and An. funestus s.s. were performed with Centers for Disease Control and Prevention (CDC) miniature light trap equipped with a collection bottle rotator at 2-hour intervals between 4:00 pm and 8:00 am. The trapped female mosquitoes were identified and classified as unfed, blood fed, and gravid. The abdominal contents of fed female mosquitoes were used for DNA extractions to identify the blood source. Results: A large proportion of human blood feeding of An. arabiensis and An. funestus s.s. (but not An. gambiae s.s.) took place during the time people were active outside LLINs. However, during the hours when people were beneath LLINs, these provided protective efficacy as indicated by reduced human blood feeding rates. Conclusion: LLINs provided effective protection against pyrethroid-resistant malaria vector populations during bedtime hours. However, protection of LLINs was insufficient during the hours when people were active outside of the bed nets. Such limitation of LLINs will need to be intensively addressed in African countries in the near future

Long-Lasting Insecticidal Nets Incorporating Piperonyl Butoxide Reduce the Risk of Malaria in Children in Western Kenya: A Cluster Randomized Controlled Trial

Malaria vectors have acquired an enzyme that metabolizes pyrethroids. To tackle this problem, we evaluated long-lasting insecticidal nets incorporating piperonyl butoxide (PBO-LLINs) with a community-based cluster randomized control trial in western Kenya. The primary endpoints were anopheline density and Plasmodium falciparum polymerase chain reaction (PCR)-positive prevalence (PCRpfPR) of children aged 7 months to 10 years. Four clusters were randomly selected for each of the treatment and control arms (eight clusters in total) from 12 clusters, and PBO-LLINs and standard LLINs were distributed in February 2011 to 982 and 1,028 houses for treatment and control arms, respectively. Entomological surveys targeted 20 houses in each cluster, and epidemiological surveys targeted 150 children. Cluster-level permutation tests evaluated the effectiveness using the fitted values from individual level regression models adjusted for baseline. Bootstrapping estimated 95% confidence intervals (CIs). The medians of anophelines per house were 1.4 (interquartile range [IQR]: 2.3) and 3.4 (IQR: 3.7) in the intervention and control arms after 3 months, and 0.4 (IQR: 0.2) and 1.6 (IQR: 0.5) after 10 months, respectively. The differences were –2.5 (95% CI: –6.4 to –0.6) and –1.3 (95% CI: –2.0 to –0.7), respectively. The datasets of 861 and 775 children were analyzed in two epidemiological surveys. The median PCRpfPRs were 25% (IQR: 11%) in the intervention arm and 52% (IQR: 11%) in the control arm after 5 months and 33% (IQR: 11%) and 45% (IQR: 5%) after 12 months. The PCRpfPR ratios were 0.67 (95% CI: 0.38, 0.91) and 0.74 (95% CI: 0.53, 0.90), respectively. We confirmed the superiority of PBO-LLINs

A preliminary study on designing a cluster randomized control trial of two new mosquito nets to prevent malaria parasite infection

Background: Although long-lasting insecticidal nets (LLINs) are the most effective tool for preventing malaria parasite transmission, the nets have some limitations. For example, the increase of LLIN use has induced the rapid expansion of mosquito insecticide resistance.More than two persons often share one net, which increases the infection risk. To overcome these problems, two new mosquito nets were developed, one incorporating piperonyl butoxide and another covering ceilings and open eaves. We designed a cluster randomized controlled trial (cRCT) to evaluate these nets based on the information provided in the present preliminary study. Results: Nearly 75% of the anopheline population in the study area in western Kenya was Anopheles gambiae s. l., and the remaining was Anopheles funestus s. l. More female anophelines were recorded in the western part of the study area. The number of anophelines increased with rainfall. We planned to have 80% power to detect a 50% reduction in female anophelines between the control group and each intervention group. The between-cluster coefficient of variance was 0.192. As the number of clusters was limited to 4 due to the size of the study area, the estimated cluster size was 7 spray catches with an alpha of 0.05. Of 1619 children tested, 626 (48%) were Plasmodium falciparum positive using a rapid diagnostic test (RDT). The prevalence was higher in the northwestern part of the study area. The number of children who slept under bed nets was 929 (71%). The P. falciparum RDT-positive prevalence (RDTpfPR) of net users was 45%, and that of non-users was 55% (OR 0.73; 95% CI 0.56, 0.95). Using 45% RDTpfPR of net users, we expected each intervention to reduce prevalence by 50%. The intracluster correlation coefficient was 0.053. With 80% power and an alpha of 0.05, the estimated cluster size was 116 children. Based on the distribution of children, we modified the boundaries of the clusters and established 300-m buffer zones along the boundaries to minimize a spillover effect. Conclusions: The cRCT study design is feasible.As the number of clusters is limited, we will apply a two-stage procedure with the baseline data to evaluate each intervention

Push by a net, pull by a cow: can zooprophylaxis enhance the impact of insecticide treated bed nets on malaria control?

Background: Mass insecticide treated bed net (ITN) deployment, and its associated coverage of populations at risk, had "pushed" a decline in malaria transmission. However, it is unknown whether malaria control is being enhanced by zooprophylaxis, i.e., mosquitoes diverted to feed on hosts different from humans, a phenomenon that could further reduce malaria entomological transmission risk in areas where livestock herding is common. Methods. Between May and July 2009, we collected mosquitoes in 104 houses from three neighboring villages with high ITN coverage (over 80%), along Lake Victoria. We also performed a census of livestock in the area and georeferenced tethering points for all herds, as well as, mosquito larval habitats. Bloodmeal contents from sampled mosquitoes were analyzed, and each mosquito was individually tested for malaria sporozoite infections. We then evaluated the association of human density, ITN use, livestock abundance and larval habitats with mosquito abundance, bloodfeeding on humans and malaria sporozoite rate using generalized linear mixed effects models. Results: We collected a total of 8123 mosquitoes, of which 1664 were Anopheles spp. malaria vectors over 295 household spray catches. We found that vector household abundance was mainly driven by the number of householders (P < 0.05), goats/sheep tethered around the house (P < 0.05) and ITNs, which halved mosquito abundance (P < 0.05). In general, similar patterns were observed for Anopheles arabiensis, but not An. gambiae s.s. and An. funestus s.s., whose density did not increase with the presence of livestock animals. Feeding on humans significantly increased in all species with the number of householders (P < 0.05), and only significantly decreased for An. arabiensis in the presence of cattle (P < 0.05). Only 26 Anopheles spp. vectors had malaria sporozoites with the sporozoite rate significantly decreasing as the proportion of cattle feeding mosquitoes increased (P < 0.05). Conclusion: Our data suggest that cattle, in settings with large ITN coverage, have the potential to drive an unexpected "push-pull" malaria control system, where An. arabiensis mosquitoes "pushed" out of human contact by ITNs are likely being further "pulled" by cattle

Impacts of insecticide treated bed nets on Anopheles gambiae s.l. populations in Mbita district and Suba district, Western Kenya

Background: Abundance and species composition of sympatric malaria vector species are the important factors governing transmission intensity. A widespread insecticidal bed net coverage may replace endophagic species with exophagic species. However, unique local environments also influence a vector population. This study examined the impacts of insecticidal bed nets on An. gambiae s.l populations in Mbita District and Suba District. Methods. The species compositions of An. gambiae s.l. larvae were compared between 1997, 2009 and 2010 and between geographical areas. The abundance and species composition of An. gambiae s.l. females resting indoors were compared between 1999, 2008 and 2010 and between geographical areas. Bed net coverage was also examined temporally and spatially, and its relationships with vector abundance and species composition were examined. Results: The relative abundance of An. gambiae s.s. larvae was 31.4% in 1997, decreasing to 7.5% in 2008 and 0.3% in 2010. The density of indoor resting An. gambiae s.l. females decreased by nearly 95%, and the relative abundance of An. gambiae s.s. females decreased from 90.6% to 60.7% and 72.4% in 2008 and 2010, respectively. However, the species composition of indoor resting An. gambiae s.l. females changed little in the island villages, and An. gambiae s.s. remained dominant in the western part of the study area. The density of house resting females was negatively associated with the number of bed nets in a retrospective analysis, but the effect of bed nets on species composition was not significant in both retrospective and cross-sectional analyses. Conclusion: An increase in bed net coverage does not necessarily replace endophilic species with exophilic species. The effect of bed nets on An. gambiae s.l. populations varies spatially, and locally unique environments are likely to influence the species composition

Malaria Vectors in Lake Victoria and Adjacent Habitats in Western Kenya

The prevalence of malaria among the residents of the Lake Victoria basin remains high. The environment associated with the lake may maintain a high number of malaria vectors. Lake habitats including water hyacinths have been suspected to be the source of vectors. This study investigated whether malaria vectors breed in the lake habitats and adjacent backwater pools. Anopheline larvae were collected within the littoral zone of the lake and adjacent pools located along approximately 24.3 km of the lakeshore in western Kenya, and their breeding sites characterized. Three primary vector species, Anopheles arabiensis, Anopheles gambiae s.s. and Anopheles funestus s.s., and three potential vectors, were found in the lake habitats. Unexpectedly, An. arabiensis was the most dominant vector species in the lake sampling sites. Its habitats were uncovered or covered with short grass. A potential secondary malaria vector, Anopheles rivulorum, dominated the water hyacinths in the lake. Most breeding sites in the lake were limited to areas that were surrounded by tall emergent plants, including trees, and those not exposed to waves. Nearly half of adjacent habitats were lagoons that were separated from the lake by sand bars. Lagoons contained a variety of microhabitats. Anopheles arabiensis dominated open habitats, whereas An. funestus s.s. was found mainly in vegetated habitats in lagoons. The current study confirmed that several breeding sites are associated with Lake Victoria. Given that Lake Victoria is the second largest lake in the world, the lake related habitats must be extensive; therefore, making targeted vector control difficult. Further exploration is necessary to estimate the effects of lake associated habitats on malaria transmission so as to inform a rational decision-making process for vector control

A Small-Scale Field Trial of Pyriproxyfen-Impregnated Bed Nets against Pyrethroid-Resistant Anopheles gambiae s.s. in Western Kenya

Pyrethroid resistance is becoming a major problem for vector control programs, because at present, there are few suitable chemical substitutes for pyrethroids, as when used on bed nets the insecticide must have low mammalian toxicity as well as high activity to mosquitoes. Pyriproxyfen (PPF) is one of the most active chemicals among the juvenile hormone mimic (JHM) group. Sterilizing mosquitoes by using PPF could be a potential control measure for pyrethroid-resistant malaria vectors. We investigated the sterilizing effects of two types of PPF-impregnated bed nets - a 1% PPF-impregnated net and a 1% PPF +2% permethrin-impregnated net (Olyset Duo) - to pyrethroid-resistant wild population of Anopheles gambiae s.s. in western Kenya. High mortality of blood-fed mosquitos was observed 3 days post-collection, in the houses where PPF-impregnated nets were used, indicating the effect of PPF on the longevity of mosquitos that came in contact with the net. Reduction in the number of ovipositing females, number of eggs, and number of progeny per female were also observed in the houses in which both Olyset Duo and PPF-impregnated nets were used. This is the first field study showing the high sterilizing efficacy of PPF against wild pyrethroid-resistant An. gambiae s.s. population. In addition, we recognized the necessity of combined use of permethrin with PPF, in order to reduce the risk of mosquito bites and provide a level of personal protection. Further studies on wild pyrethroid-resistant mosquito populations such as An. arabiensis and An. funestus s.s. would provide more information on the practical use of the PPF-impregnated bed nets

Multimodal pyrethroid resistance in malaria vectors, Anopheles gambiae s.s., Anopheles arabiensis, and Anopheles funestus s.s. in western Kenya.

Anopheles gambiae s.s., Anopheles arabiensis, and Anopheles funestus s.s. are the most important species for malaria transmission. Pyrethroid resistance of these vector mosquitoes is one of the main obstacles against effective vector control. The objective of the present study was to monitor the pyrethroid susceptibility in the 3 major malaria vectors in a highly malaria endemic area in western Kenya and to elucidate the mechanisms of pyrethroid resistance in these species. Gembe East and West, Mbita Division, and 4 main western islands in the Suba district of the Nyanza province in western Kenya were used as the study area. Larval and adult collection and bioassay were conducted, as well as the detection of point mutation in the voltage-gated sodium channel (1014L) by using direct DNA sequencing. A high level of pyrethroid resistance caused by the high frequency of point mutations (L1014S) was detected in An. gambiae s.s. In contrast, P450-related pyrethroid resistance seemed to be widespread in both An. arabiensis and An. funestus s.s. Not a single L1014S mutation was detected in these 2 species. A lack of cross-resistance between DDT and permethrin was also found in An. arabiensis and An. funestus s.s., while An. gambiae s.s. was resistant to both insecticides. It is noteworthy that the above species in the same area are found to be resistant to pyrethroids by their unique resistance mechanisms. Furthermore, it is interesting that 2 different resistance mechanisms have developed in the 2 sibling species in the same area individually. The cross resistance between permethrin and DDT in An. gambiae s.s. may be attributed to the high frequency of kdr mutation, which might be selected by the frequent exposure to ITNs. Similarly, the metabolic pyrethroid resistance in An. arabiensis and An. funestus s.s. is thought to develop without strong selection by DDT

Distribution of water hyacinth mats and lagoons.

<p>Distribution of water hyacinth mats and lagoons.</p