Presence of the knockdown resistance mutation, Vgsc-1014F in Anopheles gambiae and An. arabiensis in western Kenya.

INTRODUCTION: The voltage gated sodium channel mutation Vgsc-1014S (kdr-east) was first reported in Kenya in 2000 and has since been observed to occur at high frequencies in the local Anopheles gambiae s.s. POPULATION: The mutation Vgsc-1014F has never been reported from An. gambiae Complex complex mosquitoes in Kenya. FINDINGS: Molecularly confirmed An. gambiae s.s. (hereafter An. gambiae) and An. arabiensis collected from 4 different parts of western Kenya were genotyped for kdr from 2011 to 2013. Vgsc-1014F was observed to have emerged, apparently, simultaneously in both An. gambiae and An. arabiensis in 2012. A portion of the samples were submitted for sequencing in order to confirm the Vgsc-1014F genotyping results. The resulting sequence data were deposited in GenBank (Accession numbers: KR867642-KR867651, KT758295-KT758303). A single Vgsc-1014F haplotype was observed suggesting, a common origin in both species. CONCLUSION: This is the first report of Vgsc-1014F in Kenya. Based on our samples, the mutation is present in low frequencies in both An. gambiae and An. arabiensis. It is important that we start monitoring relative frequencies of the two kdr genes so that we can determine their relative importance in an area of high insecticide treated net ownership.

Pyrethroid susceptibility of malaria vectors in four Districts of western Kenya.

BACKGROUND: Increasing pyrethroid resistance in malaria vectors has been reported in western Kenya where long lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) are the mainstays of vector control. To ensure the sustainability of insecticide-based malaria vector control, monitoring programs need to be implemented. This study was designed to investigate the extent and distribution of pyrethroid resistance in 4 Districts of western Kenya (Nyando, Rachuonyo, Bondo and Teso). All four Districts have received LLINs while Nyando and Rachuonyo Districts have had IRS campaigns for 3-5 years using pyrethroids. This study is part of a programme aimed at determining the impact of insecticide resistance on malaria epidemiology. METHODS: Three day old adult mosquitoes from larval samples collected in the field, were used for bioassays using the WHO tube bioassay, and mortality recorded 24 hours post exposure. Resistance level was assigned based on the 2013 WHO guidelines where populations with <90% mortality were considered resistant. Once exposed, samples were identified to species using PCR. RESULTS: An. arabiensis comprised at least 94% of all An. gambiae s.l. in Bondo, Rachuonyo and Nyando. Teso was a marked contrast case with 77% of all samples being An. gambiae s.s. Mortality to insecticides varied widely between clusters even in one District with mortality to deltamethrin ranging from 45-100%, while to permethrin the range was 30-100%. Mortality to deltamethrin in Teso District was < 90% in 4 of 6 clusters tested in An arabiensis and <90% in An. gambiae s.s in 5 of 6 clusters tested. To permethrin, mortality ranged between 5.9-95%, with <90% mortality in 9 of 13 and 8 of 13 in An. arabiensis and An. gambiae s.s. respectively. Cluster specific mortality of An. arabiensis between permethin and deltamethrin were not correlated (Z = 2.9505, P = 0.2483). CONCLUSION: High levels of pyrethroid resistance were observed in western Kenya. This resistance does not seem to be associated with either species or location. Insecticide resistance can vary within small geographical areas and such heterogeneity may make it possible to evaluate the impact of resistance on malaria and mosquito parameters within similar eco-epidemiological zones.

Comparative transcriptome analyses of deltamethrin-resistant and -susceptible Anopheles gambiae mosquitoes from Kenya by RNA-Seq.

Malaria causes more than 300 million clinical cases and 665,000 deaths each year, and the majority of the mortality and morbidity occurs in sub-Saharan Africa. Due to the lack of effective vaccines and wide-spread resistance to antimalarial drugs, mosquito control is the primary method of malaria prevention and control. Currently, malaria vector control relies on the use of insecticides, primarily pyrethroids. The extensive use of insecticides has imposed strong selection pressures for resistance in the mosquito populations. Consequently, resistance to pyrethroids in Anopheles gambiae, the main malaria vector in sub-Saharan Africa, has become a major obstacle for malaria control. A key element of resistance management is the identification of resistance mechanisms and subsequent development of reliable resistance monitoring tools. Field-derived An. gambiae from Western Kenya were phenotyped as deltamethrin-resistant or -susceptible by the standard WHO tube test, and their expression profile compared by RNA-seq. Based on the current annotation of the An. gambiae genome, a total of 1,093 transcripts were detected as significantly differentially accumulated between deltamethrin-resistant and -susceptible mosquitoes. These transcripts are distributed over the entire genome, with a large number mapping in QTLs previously linked to pyrethorid resistance, and correspond to heat-shock proteins, metabolic and transport functions, signal transduction activities, cytoskeleton and others. The detected differences in transcript accumulation levels between resistant and susceptible mosquitoes reflect transcripts directly or indirectly correlated with pyrethroid resistance. RNA-seq data also were used to perform a de-novo Cufflinks assembly of the An. gambiae genome.

Evidence for a useful life of more than three years for a polyester-based long-lasting insecticidal mosquito net in Western Uganda.

BACKGROUND: Long-lasting insecticidal nets (LLIN) are now standard for the prevention of malaria. However, only products with recommendation for public use from the World Health Organization should be used and this evaluation includes the assessment of net effectiveness after three years of field use. Results for one of the polyester-based products, Interceptor is presented. METHODS: In five villages, 190 LLIN and 90 nets conventionally treated with the insecticide alpha-cypermethrin at 25 mg/m2 were distributed randomly and used by the families. Following a baseline household survey a net survey was carried out every six months to capture use, washing habits and physical condition of the nets. Randomly selected nets were collected after 6, 12, 24, 36 and 42 months and tested for remaining insecticide content and ability to knock-down and kill malaria transmitting mosquitoes. RESULTS: During the three and a half years of observation only 16 nets were lost to follow-up resulting in an estimated attrition rate of 12% after three and 20/% after 3.5 years. Nets were used regularly and washed on average 1.5 times per year. After three and a half years 29% of the nets were still in good condition while 13% were seriously torn with no difference between the LLIN and control nets. The conventionally treated nets quickly lost insecticide and after 24 months only 7% of the original dose remained (1.6 mg/m2). Baseline median concentration of alpha-cypermethrin for LLIN was 194.5 mg/m2 or 97% of the target dose with between and within net variation of 11% and 4% respectively (relative standard deviation). On the LLIN 73.8 mg/m2 alpha-cypermethrin remained after three years of use and 56.2 mg/m2 after three and a half and 94% and 81% of the LLIN still had > 15 mg/m2 left respectively. Optimal effectiveness in bio-assays (≥ 95% 60 minute knock-down or ≥ 80% 24 hour mortality) was found in 83% of the sampled LLIN after three and 71% after three and a half years. CONCLUSIONS: Under conditions in Western Uganda the tested long-lasting insecticidal net Interceptor fulfilled the criteria for phase III of WHO evaluations and, based on preliminary criteria of the useful life, this product is estimated to last on average between three and four years.

Spatial and temporal variation in the kdr allele L1014S in Anopheles gambiae s.s. and phenotypic variability in susceptibility to insecticides in Western Kenya.

BACKGROUND: Malaria vector control in Africa depends upon effective insecticides in bed nets and indoor residual sprays. This study investigated the extent of insecticide resistance in Anopheles gambiae s.l., Anopheles gambiae s.s. and Anopheles arabiensis in western Kenya where ownership of insecticide-treated bed nets has risen steadily from the late 1990s to 2010. Temporal and spatial variation in the frequency of a knock down resistance (kdr) allele in A. gambiae s.s. was quantified, as was variation in phenotypic resistance among geographic populations of A. gambiae s.l. METHODS: To investigate temporal variation in kdr frequency, individual specimens of A. gambiae s.s. from two sentinel sites were genotyped using RT-PCR from 1996-2010. Spatial variation in kdr frequency, species composition, and resistance status were investigated in additional populations of A. gambiae s.l. sampled in western Kenya in 2009 and 2010. Specimens were genotyped for kdr as above and identified to species via conventional PCR. Field-collected larvae were reared to adulthood and tested for insecticide resistance using WHO bioassays. RESULTS: Anopheles gambiae s.s. showed a dramatic increase in kdr frequency from 1996 - 2010, coincident with the scale up of insecticide-treated nets. By 2009-2010, the kdr L1014S allele was nearly fixed in the A. gambiae s.s. population, but was absent in A. arabiensis. Near Lake Victoria, A. arabiensis was dominant in samples, while at sites north of the lake A. gambiae s.s was more common but declined relative to A. arabiensis from 2009 to 2010. Bioassays demonstrated that A. gambiae s.s. had moderate phenotypic levels of resistance to DDT, permethrin and deltamethrin while A. arabiensis was susceptible to all insecticides tested. CONCLUSIONS: The kdr L1014S allele has approached fixation in A. gambiae s.s. populations of western Kenya, and these same populations exhibit varying degrees of phenotypic resistance to DDT and pyrethroid insecticides. The near absence of A. gambiae s.s. from populations along the lakeshore and the apparent decline in other populations suggest that insecticide-treated nets remain effective against this mosquito despite the increase in kdr allele frequency. The persistence of A. arabiensis, despite little or no detectable insecticide resistance, is likely due to behavioural traits such as outdoor feeding and/or feeding on non-human hosts by which this species avoids interaction with insecticide-treated nets.

The effect of repeated washing of long-lasting insecticide-treated nets (LLINs) on the feeding success and survival rates of Anopheles gambiae.

BACKGROUND: Insecticide-treated nets protect users from mosquito bites, thereby preventing transmissions of mosquito borne pathogens. Repeated washing of nets removes insecticide on the netting rendering them ineffective within a short period. Long-lasting insecticide-treated nets (LLINs) offer longer time protection against such bites because they are more wash resistant, and are preferred to conventionally treated nets. However, there is limited information on the effect of repeated washing of LLINs on the feeding success and survival of wild malaria vectors. METHODS: The current study evaluated the effect of repeated washing of four brands of LLINs on the feeding success and survival rates of Anopheles gambiae sl reared from wild strains. In this study, two- to five-day old F1s, reared from gravid mosquitoes collected from an area with a high coverage of LLINs were offered blood meals through protective barriers of the above LLINs. Mosquitoes were exposed for a period of 10 minutes each time. Nets were tested unwashed and subsequently after every 5th through wash 15. After exposure mosquitoes were sorted out according to their feeding status. They were then held under normal laboratory conditions for 24 hours and mortality was scored in both fed and unfed. RESULTS: It was observed that mosquitoes did not feed through a barrier of unwashed LLINs. However, the feeding success and survival rates increased with successive number of washes and were also net brand dependant. After 15 washes, 49% of vectors succeeded to feed through a protective barrier of PermaNet 2.0 and 50% of the fed died after 24 hrs while after the same number of washes 60% of vectors succeeded to feed through Olyset brand of LLINs and all of them survived. In general, more mosquitoes survived after feeding through Olyset compared to the other four brands that were evaluated. When efficacy of individual LLINs was compared by a t-test analysis to a conventionally treated net, the results were not significantly different statistically for Olyset (p = 0.239) and NetProtect (TNT) (p = 0.135). However, the results were highly significant when comparison was made with PermaNet and Interceptor (BASF); p values 0.015 and 0.025 respectively. CONCLUSION: The result of this study shows that repeated washing of LLINs at short time intervals using local washing methods may render them infective within a short time in preventing local vectors from feeding.

Wash durability and optimal drying regimen of four brands of long-lasting insecticide-treated nets after repeated washing under tropical conditions.

BACKGROUND: The current study was undertaken to determine the optimal wash-drying regimen and the effects of different washing procedures on the efficacy, and durability of four brands of newly introduced long-lasting insecticide-treated nets (LLINs) under tropical conditions. METHODS: In the current study, the following four LLINs were tested: Olyset, PermaNet 2.0, BASF and TNT. Nets were divided into three sets; one set was washed by hand rubbing and air-dried either hanging or spread on the ground in direct sunlight or hanging or spread on the ground under the shade. A second set was washed using the WHO protocol (machine) and the third set was washed by beating the nets on rocks. The biological activities of the nets were assessed by a three-minute bioassay cone test and the residual insecticide contents were determined using high performance liquid chromatography (HPLC) procedure. RESULTS: Nets that were dried hanging under the shade retained more insecticide, 62.5% and recorded higher mortality compared to nets which were dried lying on the ground in direct sunlight 58.8%, nets dried under the shade spread on the ground 56.3%, and 57.8% for nets dried hanging in direct sunlight. It was also observed that nets washed by the standard WHO protocol, retained more insecticide and were more effective in killing mosquitoes compared to nets washed by local methods of hand rubbing and beating on rocks. There were significant differences between drying regimens (p < 0.0001) and between washing procedures (p < 0.001) respectively. However, the effect of net type was statistically insignificant. The statistical differences on individual nets were also compared, for PermaNet and TNT there were no significant differences observed between the four drying regimens (p = 0.7944 and 0.4703) respectively). For BASF and Olyset, the differences were significant (p < 0.001 and p > 0.0001). CONCLUSION: The results of this study suggest that washing and drying regimen influence the insecticidal activity of LLINs. The standard WHOPES washing protocol underestimates the amount of insecticide washed from LLINs compared to the abrasive washing procedures that are used in the field. This suggests that there is need to educate net users to adopt a more gentle washing procedure while handling LLINs. The education should accompany net distribution campaigns.

Long-term field performance of a polyester-based long-lasting insecticidal mosquito net in rural Uganda.

BACKGROUND: In order to evaluate whether criteria for LLIN field performance (phase III) set by the WHO Pesticide Evaluation Scheme are met, first and second generations of one of these products, PermaNet, a polyester net using the coating technology were tested. METHODS: A randomized, double blinded study design was used comparing LLIN to conventionally treated nets and following LLIN for three years under regular household use in rural conditions. Primary outcome measures were deltamethrin residue and bioassay performance (60 minute knock-down and 24 hour mortality after a three minute exposure) using a strain of Anopheles gambiae s.s. sensitive to pyrethroid insecticides. RESULTS: Baseline concentration of deltamethrin was within targets for all net types but was rapidly lost in conventionally treated nets and first generation PermaNet with median of 0.7 and 2.5 mg/m2 after six months respectively. In contrast, second generation PermaNet retained insecticide well and had 41.5% of baseline dose after 36 months (28.7 mg/m2). Similarly, vector mortality and knockdown dropped to 18% and 70% respectively for first generation LLIN after six months but remained high (88.5% and 97.8% respectively) for second generation PermaNet(R) after 36 months of follow up at which time 90.0% of nets had either a knockdown rate > or = 95% or mortality rate > or = 80%. CONCLUSION: Second generation PermaNet showed excellent results after three years of field use and fulfilled the WHOPES criteria for LLIN. Loss of insecticide on LLIN using coating technology under field conditions was far more influenced by factors associated with handling rather than washing.

Topography and malaria transmission heterogeneity in western Kenya highlands: prospects for focal vector control.

BACKGROUND: Recent resurgence of malaria in the highlands of Western Kenya has called for a more comprehensive understanding of the previously neglected complex highland vector ecology. Besides other drivers of malaria epidemiology, topography is likely to have a major effect on spatial vector and parasite distribution. The aim of this study was to determine the effects of topography on malaria spatial vector distribution and parasite prevalence. METHODOLOGY: Indoor resting adult malaria vectors and blood parasites were collected in three villages along a 4 km transect originating from the valley bottom and ending at the hilltop for 13 months. Members of the Anopheles gambiae complex were identified by PCR. Blood parasites were collected from children 6-13 years old and densities categorized by site of home location and age of the children. RESULTS: Ninety eight percent (98%) of An. gambiae s.s. and (99%) Anopheles funestus were collected in houses located at the edge of the valley bottom, whereas 1% of An. gambiae s.s. were collected at mid hill and at the hilltop respectively. No An. funestus were collected at the hilltop. Malaria prevalence was 68% at the valley bottom, 40.2% at mid hill and 26.7% at the hilltop. Children aged six years and living at the edge of the valley bottom had an annual geometric mean number of 66.1 trophozoites for every 200 white blood cells, while those living at mid-hill had a mean of 84.8, and those living at hilltop had 199.5 trophozoites. CONCLUSION: Malaria transmission in this area is mainly confined to the valley bottom. Effective vector control could be targeted at the foci. However, the few vectors observed at mid-hill maintained a relatively high prevalence rate. The higher variability in blood parasite densities and their low correlation with age in children living at the hilltop suggests a lower stability of transmission than at the mid-hill and valley bottom.

Laboratory wash resistance of long-lasting insecticidal nets.

Long-lasting insecticidal nets (LLINs) may eliminate the need for retreatment of mosquito nets used for the control of malaria and other vector-borne diseases. The efficacy of LLINs after repeated washing under laboratory conditions has been used to predict long-lasting efficacy under field conditions. We evaluated under laboratory conditions the wash resistance of two LLINs (PermaNet 1.0, Vestergaard-Frandsen, Denmark; Olyset, Sumitomo Chemical Co., Japan), two candidate LLINs (Dawa, Siamdutch Mosquito Netting Co., Thailand; Insector, Athanor, France) and a net treated with a process designed to increase its wash resistance and compared them with conventionally treated nets (deltamethrin, 25 mg/m(2)). Nets of all six types were washed using a standard protocol and tested weekly using WHO cone bioassays with Anopheles gambiae (Kisumu strain). The PermaNet 1.0 was the most wash resistant with >50% mosquito mortality in WHO cone bioassays after as many as 20 washes. The Dawa net also retained some activity after repeated washing but exhibited wide variation in insecticide retention and biological activity. The remaining nets lost >90% of their biological activity after six washes as measured by 24-h mortality of A. gambiae in WHO cone tests. After 20 washes, all nets lost >50% of their initial insecticide concentrations except for the Olyset net. After 20 washes, nets were heated for 4 h at 60 degrees C to determine whether biological activity could be restored by heat-assisted regeneration. Only the Olyset net was regenerated by heating, with average mosquito mortality and knockdown in WHO cone tests rising to >90% after heating for 4 h at 60 degrees C. However, regeneration of the biological activity of Olyset nets that had been washed three times did not occur at 30 degrees C or 35 degrees C after 12 weeks. The wash resistance of these LLINs corresponded well to their retention of biological activity observed in a field trial, suggesting that wash resistance may be a good predictor of the longevity of insecticidal activity of LLINs under field conditions.

Spatial distribution of anopheline larval habitats in Western Kenyan highlands: effects of land cover types and topography.

The distributions of anopheline larval habitats were aggregated in valley bottoms in Kenya in both the rainy and dry seasons, although the degree of aggregation was higher in the dry season than in the rainy season. Larvae of the Anopheles gambiae complex larvae were found more frequently in habitats in farmlands and pastures. However, An. funestus larvae were found more frequently in natural swamps and pastures. Canopy cover was the only variable significantly associated with the occurrence of the An. gambiae complex and An. funestus. The average canopy cover was significantly less in the habitats with the An. gambiae complex and An. funestus larvae than those without the anopheline larvae. Thus, land cover types and topographic features showed important effects on the distribution of anopheline larval habitats. These results suggest that clearing riparian forests would improve growing conditions of the An. gambiae complex and An. funestus larvae in Kenyan highlands.

Dynamics of the pyrethroid knockdown resistance allele in western Kenyan populations of Anopheles gambiae in response to insecticide-treated bed net trials.

Permethrin and DDT resistance in Anopheles gambiae s.s. associated with a leucine-serine knockdown resistance (kdr) mutation in the voltage-gated sodium channel gene was discovered recently in western Kenya where a large scale permethrin-impregnated bed net (ITN) program has been implemented. Collections of An. gambiae s.l. were made from intervention and control villages prior to and after onset of the program. The kdr genotypes were determined using allele-specific polymerase chain reaction diagnostic tests. In An. gambiae s.s., the frequency of the kdr mutation prior to ITN introduction was approximately 3-4% in western Kenya and zero in samples from the coast. After ITN introduction, the kdr mutation increased in ITN and neighboring villages from approximately 4% to approximately 8%, but remained unchanged in villages at least 20 km distant and was not detected in coastal Kenya. The identical leucine-serine mutation was found in a single An. arabiensis individual among 658 tested. The leucine-phenylalanine kdr mutation common in west African An. gambiae populations was not detected in An. gambiae s.l. from Kenya. Implications for the population structure and control of An. gambiae are discussed.