Incidence and consequences of damage to insecticide-treated mosquito nets in Kenya.

BACKGROUND: Efforts to improve the impact of long-lasting insecticidal nets (LLINs) should be informed by understanding of the causes of decay in effect. Holes in LLINs have been estimated to account for 7-11% of loss in effect on vectorial capacity for Plasmodium falciparum malaria in an analysis of repeated cross-sectional surveys of LLINs in Kenya. This does not account for the effect of holes as a cause of net attrition or non-use, which cannot be measured using only cross-sectional data. There is a need for estimates of how much these indirect effects of physical damage on use and attrition contribute to decay in effectiveness of LLINs. METHODS: Use, physical integrity, and survival were assessed in a cohort of 4514 LLINs followed for up to 4 years in Kenya. Flow diagrams were used to illustrate how the status of nets, in terms of categories of use, physical integrity, and attrition, changed between surveys carried out at 6-month intervals. A compartment model defined in terms of ordinary differential equations (ODEs) was used to estimate the transition rates between the categories. Effects of physical damage to LLINs on use and attrition were quantified by simulating counterfactuals in which there was no damage. RESULTS: Allowing for the direct effect of holes, the effect on use, and the effect on attrition, 18% of the impact on vectorial capacity was estimated to be lost because of damage. The estimated median lifetime of the LLINs was 2.9 years, but this was extended to 5.7 years in the counterfactual without physical damage. Nets that were in use were more likely to be in a damaged state than unused nets but use made little direct difference to LLIN lifetimes. Damage was reported as the reason for attrition for almost half of attrited nets, but the model estimated that almost all attrited nets had suffered some damage before attrition. CONCLUSIONS: Full quantification of the effects of damage will require measurement of the supply of new nets and of household stocks of unused nets, and also of their impacts on both net use and retention. The timing of mass distribution campaigns is less important than ensuring sufficient supply. In the Kenyan setting, nets acquired damage rapidly once use began and the damage led to rapid attrition. Increasing the robustness of nets could substantially increase their lifetime and impact but the impact of LLIN programmes on malaria transmission is ultimately limited by levels of use. Longitudinal analyses of net integrity data from different settings are needed to determine the importance of physical damage to nets as a driver of attrition and non-use, and the importance of frequent use as a cause of physical damage in different contexts.

Efficacy of extended release formulations of Natular™ (spinosad) against larvae and adults of Anopheles mosquitoes in western Kenya.

BACKGROUND: Larval source management is recommended as a supplementary vector control measure for the prevention of malaria. Among the concerns related to larviciding is the feasibility of implementation in tropical areas with large numbers of habitats and the need for frequent application. Formulated products of spinosad that are designed to be effective for several weeks may mitigate some of these concerns. METHODS: In a semi-field study, three formulations of spinosad (emulsifiable concentrate, extended release granules and tablet formulations) were tested in naturalistic habitats in comparison to an untreated control. Cohorts of third instar Anopheles gambiae (Diptera: Culicidae) were introduced into the habitats in screened cages every week up to four weeks after application and monitored for survivorship over three days. A small-scale field trial was then conducted in two villages. Two of the spinosad formulations were applied in one village over the course of 18 months. Immature mosquito populations were monitored with standard dippers in sentinel sites and adult populations were monitored by pyrethrum spray catches. RESULTS: In the semi-field study, the efficacy of the emulsifiable concentrate of spinosad waned 1 week after treatment. Mortality in habitats treated with the extended release granular formulation of spinosad was initially high but declined gradually over 4 weeks while mortality in habitats treated with the dispersable tablet formulation was low immediately after treatment but rose to 100% through four weeks. In the field study, immature and adult Anopheles mosquito populations were significantly lower in the intervention village compared to the control village during the larviciding period. Numbers of collected mosquitoes were lower in the intervention village compared to the control village during the post-intervention period but the difference was not statistically significant. CONCLUSIONS: The extended release granular formulation and the dispersible tablet formulations of spinosad are effective against larval Anopheles mosquitoes for up to four weeks and may be an effective tool as part of larval source management programmes for reducing adult mosquito density and malaria transmission.

Human Direct Skin Feeding Versus Membrane Feeding to Assess the Mosquitocidal Efficacy of High-Dose Ivermectin (IVERMAL Trial).

BACKGROUND: Ivermectin is being considered for mass drug administration for malaria, due to its ability to kill mosquitoes feeding on recently treated individuals. In a recent trial, 3-day courses of 300 and 600 mcg/kg/day were shown to kill Anopheles mosquitoes for at least 28 days post-treatment when fed patients' venous blood using membrane feeding assays. Direct skin feeding on humans may lead to higher mosquito mortality, as ivermectin capillary concentrations are higher. We compared mosquito mortality following direct skin and membrane feeding. METHODS: We conducted a mosquito feeding study, nested within a randomized, double-blind, placebo-controlled trial of 141 adults with uncomplicated malaria in Kenya, comparing 3 days of ivermectin 300 mcg/kg/day, ivermectin 600 mcg/kg/day, or placebo, all co-administered with 3 days of dihydroartemisinin-piperaquine. On post-treatment day 7, direct skin and membrane feeding assays were conducted using laboratory-reared Anopheles gambiae sensu stricto. Mosquito survival was assessed daily for 28 days post-feeding. RESULTS: Between July 20, 2015, and May 7, 2016, 69 of 141 patients participated in both direct skin and membrane feeding (placebo, n = 23; 300 mcg/kg/day, n = 24; 600 mcg/kg/day, n = 22). The 14-day post-feeding mortality for mosquitoes fed 7 days post-treatment on blood from pooled patients in both ivermectin arms was similar with direct skin feeding (mosquitoes observed, n = 2941) versus membrane feeding (mosquitoes observed, n = 7380): cumulative mortality (risk ratio 0.99, 95% confidence interval [CI] 0.95-1.03, P = .69) and survival time (hazard ratio 0.96, 95% CI 0.91-1.02, P = .19). Results were consistent by sex, by body mass index, and across the range of ivermectin capillary concentrations studied (0.72-73.9 ng/mL). CONCLUSIONS: Direct skin feeding and membrane feeding on day 7 resulted in similar mosquitocidal effects of ivermectin across a wide range of drug concentrations, suggesting that the mosquitocidal effects seen with membrane feeding accurately reflect those of natural biting. Membrane feeding, which is more patient friendly and ethically acceptable, can likely reliably be used to assess ivermectin's mosquitocidal efficacy. CLINICAL TRIALS REGISTRATION: NCT02511353.

Infant and child mortality in relation to malaria transmission in KEMRI/CDC HDSS, Western Kenya: validation of verbal autopsy.

BACKGROUND: Malaria transmission reduction is a goal of many malaria control programmes. Little is known of how much mortality can be reduced by specific reductions in transmission. Verbal autopsy (VA) is widely used for estimating malaria specific mortality rates, but does not reliably distinguish malaria from other febrile illnesses. Overall malaria attributable mortality includes both direct and indirect deaths. It is unclear what proportion of the deaths averted by reducing malaria transmission are classified as malaria in VA. METHODS: Both all-cause, and cause-specific mortality reported by VA for children under 5 years of age, were assembled from the KEMRI/CDC health and demographic surveillance system in Siaya county, rural Western Kenya for the years 2002-2004. These were linked to household-specific estimates of the Plasmodium falciparum entomological inoculation rate (EIR) based on high resolution spatio-temporal geostatistical modelling of entomological data. All-cause and malaria specific mortality (by VA), were analysed in relation to EIR, insecticide-treated net use (ITN), socioeconomic status (SES) and parameters describing space-time correlation. Time at risk for each child was analysed using Bayesian geostatistical Cox proportional hazard models, with time-dependent covariates. The outputs were used to estimate the diagnostic performance of VA in measuring mortality that can be attributed to malaria exposure. RESULTS: The overall under-five mortality rate was 80 per 1000 person-years during the study period. Eighty-one percent of the total deaths were assigned causes of death by VA, with malaria assigned as the main cause of death except in the neonatal period. Although no trend was observed in malaria-specific mortality assessed by VA, ITN use was associated with reduced all-cause mortality in infants (hazard ratio 0.15, 95% CI 0.02, 0.63) and the EIR was strongly associated with both all-cause and malaria-specific mortality. 48.2% of the deaths could be attributed to malaria by analysing the exposure-response relationship, though only 20.5% of VAs assigned malaria as the cause and the sensitivity of VAs was estimated to be only 26%. Although VAs assigned some deaths to malaria even in areas where there was estimated to be no exposure, the specificity of the VAs was estimated to be 85%. CONCLUSION: Interventions that reduce P. falciparum transmission intensity will not only significantly reduce malaria-diagnosed mortality, but also mortality assigned to other causes in under-5 year old children in endemic areas. In this setting, the VA tool based on clinician review substantially underestimates the number of deaths that could be averted by reducing malaria exposure in childhood, but has a reasonably high specificity. This suggests that malaria transmission-reducing interventions such as ITNs can potentially reduce overall child mortality by as much as twice the total direct malaria burden estimated from VAs.

Insecticide-Treated Nets and Protection against Insecticide-Resistant Malaria Vectors in Western Kenya.

Insecticide resistance might reduce the efficacy of malaria vector control. In 2013 and 2014, malaria vectors from 50 villages, of varying pyrethroid resistance, in western Kenya were assayed for resistance to deltamethrin. Long-lasting insecticide-treated nets (LLIN) were distributed to households at universal coverage. Children were recruited into 2 cohorts, cleared of malaria-causing parasites, and tested every 2 weeks for reinfection. Infection incidence rates for the 2 cohorts were 2.2 (95% CI 1.9-2.5) infections/person-year and 2.8 (95% CI 2.5-3.0) infections/person-year. LLIN users had lower infection rates than non-LLIN users in both low-resistance (rate ratio 0.61, 95% CI 0.42-0.88) and high-resistance (rate ratio 0.55, 95% CI 0.35-0.87) villages (p = 0.63). The association between insecticide resistance and infection incidence was not significant (p = 0.99). Although the incidence of infection was high among net users, LLINs provided significant protection (p = 0.01) against infection with malaria parasite regardless of vector insecticide resistance.

Applications and limitations of Centers for Disease Control and Prevention miniature light traps for measuring biting densities of African malaria vector populations: a pooled-analysis of 13 comparisons with human landing catches.

BACKGROUND: Measurement of densities of host-seeking malaria vectors is important for estimating levels of disease transmission, for appropriately allocating interventions, and for quantifying their impact. The gold standard for estimating mosquito-human contact rates is the human landing catch (HLC), where human volunteers catch mosquitoes that land on their exposed body parts. This approach necessitates exposure to potentially infectious mosquitoes, and is very labour intensive. There are several safer and less labour-intensive methods, with Centers for Disease Control light traps (LT) placed indoors near occupied bed nets being the most widely used. METHODS: This paper presents analyses of 13 studies with paired mosquito collections of LT and HLC to evaluate these methods for their consistency in sampling indoor-feeding mosquitoes belonging to the two major taxa of malaria vectors across Africa, the Anopheles gambiae sensu lato complex and the Anopheles funestus s.l. group. Both overall and study-specific sampling efficiencies of LT compared with HLC were computed, and regression methods that allow for the substantial variations in mosquito counts made by either method were used to test whether the sampling efficacy varies with mosquito density. RESULTS: Generally, LT were able to collect similar numbers of mosquitoes to the HLC indoors, although the relative sampling efficacy, measured by the ratio of LT:HLC varied considerably between studies. The overall best estimate for An. gambiae s.l. was 1.06 (95% credible interval: 0.68-1.64) and for An. funestus s.l. was 1.37 (0.70-2.68). Local calibration exercises are not reproducible, since only in a few studies did LT sample proportionally to HLC, and there was no geographical pattern or consistent trend with average density in the tendency for LT to either under- or over-sample. CONCLUSIONS: LT are a crude tool at best, but are relatively easy to deploy on a large scale. Spatial and temporal variation in mosquito densities and human malaria transmission exposure span several orders of magnitude, compared to which the inconsistencies of LT are relatively small. LT, therefore, remain an invaluable and safe alternative to HLC for measuring indoor malaria transmission exposure in Africa.

Design of a study to determine the impact of insecticide resistance on malaria vector control: a multi-country investigation.

BACKGROUND: Progress in reducing the malaria disease burden through the substantial scale up of insecticide-based vector control in recent years could be reversed by the widespread emergence of insecticide resistance. The impact of insecticide resistance on the protective effectiveness of insecticide-treated nets (ITN) and indoor residual spraying (IRS) is not known. A multi-country study was undertaken in Sudan, Kenya, India, Cameroon and Benin to quantify the potential loss of epidemiological effectiveness of ITNs and IRS due to decreased susceptibility of malaria vectors to insecticides. The design of the study is described in this paper. METHODS: Malaria disease incidence rates by active case detection in cohorts of children, and indicators of insecticide resistance in local vectors were monitored in each of approximately 300 separate locations (clusters) with high coverage of malaria vector control over multiple malaria seasons. Phenotypic and genotypic resistance was assessed annually. In two countries, Sudan and India, clusters were randomly assigned to receive universal coverage of ITNs only, or universal coverage of ITNs combined with high coverage of IRS. Association between malaria incidence and insecticide resistance, and protective effectiveness of vector control methods and insecticide resistance were estimated, respectively. RESULTS: Cohorts have been set up in all five countries, and phenotypic resistance data have been collected in all clusters. In Sudan, Kenya, Cameroon and Benin data collection is due to be completed in 2015. In India data collection will be completed in 2016. DISCUSSION: The paper discusses challenges faced in the design and execution of the study, the analysis plan, the strengths and weaknesses, and the possible alternatives to the chosen study design.

Standardizing operational vector sampling techniques for measuring malaria transmission intensity: evaluation of six mosquito collection methods in western Kenya.

BACKGROUND: Operational vector sampling methods lack standardization, making quantitative comparisons of malaria transmission across different settings difficult. Human landing catch (HLC) is considered the research gold standard for measuring human-mosquito contact, but is unsuitable for large-scale sampling. This study assessed mosquito catch rates of CDC light trap (CDC-LT), Ifakara tent trap (ITT), window exit trap (WET), pot resting trap (PRT), and box resting trap (BRT) relative to HLC in western Kenya to 1) identify appropriate methods for operational sampling in this region, and 2) contribute to a larger, overarching project comparing standardized evaluations of vector trapping methods across multiple countries. METHODS: Mosquitoes were collected from June to July 2009 in four districts: Rarieda, Kisumu West, Nyando, and Rachuonyo. In each district, all trapping methods were rotated 10 times through three houses in a 3 × 3 Latin Square design. Anophelines were identified by morphology and females classified as fed or non-fed. Anopheles gambiae s.l. were further identified as Anopheles gambiae s.s. or Anopheles arabiensis by PCR. Relative catch rates were estimated by negative binomial regression. RESULTS: When data were pooled across all four districts, catch rates (relative to HLC indoor) for An. gambiae s.l (95.6% An. arabiensis, 4.4% An. gambiae s.s) were high for HLC outdoor (RR = 1.01), CDC-LT (RR = 1.18), and ITT (RR = 1.39); moderate for WET (RR = 0.52) and PRT outdoor (RR = 0.32); and low for all remaining types of resting traps (PRT indoor, BRT indoor, and BRT outdoor; RR < 0.08 for all). For Anopheles funestus, relative catch rates were high for ITT (RR = 1.21); moderate for HLC outdoor (RR = 0.47), CDC-LT (RR = 0.69), and WET (RR = 0.49); and low for all resting traps (RR < 0.02 for all). At finer geographic scales, however, efficacy of each trap type varied from district to district. CONCLUSIONS: ITT, CDC-LT, and WET appear to be effective methods for large-scale vector sampling in western Kenya. Ultimately, choice of collection method for operational surveillance should be driven by trap efficacy and scalability, rather than fine-scale precision with respect to HLC. When compared with recent, similar trap evaluations in Tanzania and Zambia, these data suggest that traps which actively lure host-seeking females will be most useful for surveillance in the face of declining vector densities.

The efficacy of long-lasting nets with declining physical integrity may be compromised in areas with high levels of pyrethroid resistance.

BACKGROUND: Long-lasting insecticide-treated mosquito nets (LLINs) are a primary malaria prevention strategy in sub-Saharan Africa. However, emergence of insecticide resistance threatens the effectiveness of LLINs. METHODS: Cross-sectional surveys of LLINs were conducted in houses of seven and four villages in Gem and Bungoma Districts in western Kenya, respectively. Condition (number and area of holes in the nets), number and species of mosquitoes resting inside them, and insecticidal activity of nets were quantified. Mosquitoes collected inside nets were allowed to lay eggs and progeny tested for susceptibility to deltamethrin and permethrin, pyrethoids commonly deployed in LLINs in western Kenya. RESULTS: In Gem, 83.3% of nets were less than three years old and 32.4% had at least one hole of any size; while in Bungoma, 92% were less than three years old and 48% had at least one hole. No anopheline and five Culex spp. mosquitoes were found resting inside nets in Gem regardless of the number and size of holes, while 552 Anopheles gambiae s.l., five Anopheles funestus s.l. and 137 Culex spp. were in nets in Bungoma. The number of mosquitoes resting inside nets increased with hole areas >50 cm in Bungoma. In WHO resistance assays, f1 offspring of samples collected in nets in Bungoma were 94 and 65% resistant to deltamethrin and permethrin, respectively. Nets from Bungoma retained strong activity against a susceptible laboratory strain, but not against f1 offspring of field-collected An. gambiae s.s. All An. gambiae s.s. samples collected in nets were homozygous for the kdr genotype L1014S. CONCLUSIONS: In areas with pyrethroid resistant vectors, LLINs with modest hole areas permit mosquito entry and feeding, providing little protection against the vectors. LLIN formulations develop large holes within three years of use, diminishing their presupposed lifetime effectiveness.

Bioefficacy and durability of Olyset® Plus, a permethrin and piperonyl butoxide-treated insecticidal net in a 3-year long trial in Kenya.

BACKGROUND: Long-lasting insecticide nets (LLINs) are a core malaria intervention. LLINs should retain efficacy against mosquito vectors for a minimum of three years. Efficacy and durability of Olyset® Plus, a permethrin and piperonyl butoxide (PBO) treated LLIN, was evaluated versus permethrin treated Olyset® Net. In the absence of WHO guidelines of how to evaluate PBO nets, and considering the manufacturer's product claim, Olyset® Plus was evaluated as a pyrethroid LLIN. METHODS: This was a household randomized controlled trial in a malaria endemic rice cultivation zone of Kirinyaga County, Kenya between 2014 and 2017. Cone bioassays and tunnel tests were done against Anopheles gambiae Kisumu. The chemical content, fabric integrity and LLIN survivorship were monitored. Comparisons between nets were tested for significance using the Chi-square test. Exact binomial distribution with 95% confidence intervals (95% CI) was used for percentages. The WHO efficacy criteria used were ≥ 95% knockdown and/or ≥ 80% mortality rate in cone bioassays and ≥ 80% mortality and/or ≥ 90% blood-feeding inhibition in tunnel tests. RESULTS: At 36 months, Olyset® Plus lost 52% permethrin and 87% PBO content; Olyset® Net lost 24% permethrin. Over 80% of Olyset® Plus and Olyset® Net passed the WHO efficacy criteria for LLINs up to 18 and 12 months, respectively. At month 36, 91.2% Olyset® Plus and 86.4% Olyset® Net survived, while 72% and 63% developed at least one hole. The proportionate Hole Index (pHI) values representing nets in good, serviceable and torn condition were 49.6%, 27.1% and 23.2%, respectively for Olyset® Plus, and 44.9%, 32.8% and 22.2%, respectively for Olyset® Net but were not significantly different. CONCLUSIONS: Olyset® Plus retained efficacy above or close to the WHO efficacy criteria for about 2 years than Olyset® Net (1-1.5 years). Both nets did not meet the 3-year WHO efficacy criteria, and showed little attrition, comparable physical durability and survivorship, with 50% of Olyset® Plus having good and serviceable condition after 3 years. Better community education on appropriate use and upkeep of LLINs is essential to ensure effectiveness of LLIN based malaria interventions.

Geospatial distribution and determinants of child mortality in rural western Kenya 2002-2005.

OBJECTIVE: To describe local geospatial variation and geospatial risk factors for child mortality in rural western Kenya. METHODS: We calculated under-5 mortality rates (U5MR) in 217 villages in a Health and Demographic Surveillance System (HDSS) area in western Kenya from 1 May 2002 through 31 December 2005. U5MRs by village were mapped. Geographical positioning system coordinates of residences at the time of death and distances to nearby locations were calculated. Multivariable Poisson regression accounting for clustering at the compound level was used to evaluate the association of geospatial factors and mortality for infants and children aged 1-4 years. RESULTS: Among 54 057 children, the overall U5MR was 56.5 per 1000 person-years and varied by village from 21 to 177 per 1000 person-years. High mortality villages occurred in clusters by location and remained in the highest mortality quintile over several years. In multivariable analysis, controlling for maternal age and education as well as household crowding, higher infant mortality was associated with living closer to streams and further from public transport roads. For children 1-4 years, living at middle elevations (1280-1332 metres), living within lower population densities areas, and living in the northern section of the HDSS were associated with higher mortality. CONCLUSIONS: Childhood mortality was significantly higher in some villages. Several geospatial factors were associated with mortality, which might indicate variability in access to health care or exposure and transmission of infectious diseases. These results are useful in prioritising areas for further study and implementing directed public health interventions.

Reduced mosquito survival in metal-roof houses may contribute to a decline in malaria transmission in sub-Saharan Africa.

In The Gambia, metal-roof houses were hotter during the day than thatched-roof houses. After 24 h, the mortality of Anopheles gambiae, the principal African malaria vector, was 38% higher in metal-roof houses than thatched ones. During the day, mosquitoes in metal-roof houses moved from the hot roof to cooler places near the floor, where the temperature was still high, reaching 35 °C. In laboratory studies, at 35 °C few mosquitoes survived 10 days, the minimum period required for malaria parasite development. Analysis of epidemiological data showed there was less malaria and lower vector survival rates in Gambian villages with a higher proportion of metal roofs. Our findings are consistent with the hypothesis that the indoor climate of metal-roof houses, with higher temperatures and lower humidity, reduces survivorship of indoor-resting mosquitoes and may have contributed to the observed reduction in malaria burden in parts of sub-Saharan Africa.

Pharmacokinetics-Pharmacodynamics of High-Dose Ivermectin with Dihydroartemisinin-Piperaquine on Mosquitocidal Activity and QT-Prolongation (IVERMAL).

High-dose ivermectin, co-administered for 3 days with dihydroartemisinin-piperaquine (DP), killed mosquitoes feeding on individuals for at least 28 days posttreatment in a recent trial (IVERMAL), whereas 7 days was predicted pretrial. The current study assessed the relationship between ivermectin blood concentrations and the observed mosquitocidal effects against Anopheles gambiae s.s. Three days of ivermectin 0, 300, or 600 mcg/kg/day plus DP was randomly assigned to 141 adults with uncomplicated malaria in Kenya. During 28 days of follow-up, 1,393 venous and 335 paired capillary plasma samples, 850 mosquito-cluster mortality rates, and 524 QTcF-intervals were collected. Using pharmacokinetic/pharmacodynamic (PK/PD) modeling, we show a consistent correlation between predicted ivermectin concentrations and observed mosquitocidal-effects throughout the 28-day study duration, without invoking an unidentified mosquitocidal metabolite or drug-drug interaction. Ivermectin had no effect on piperaquine's PKs or QTcF-prolongation. The PK/PD model can be used to design new treatment regimens with predicted mosquitocidal effect. This methodology could be used to evaluate effectiveness of other endectocides.

Safety and mosquitocidal efficacy of high-dose ivermectin when co-administered with dihydroartemisinin-piperaquine in Kenyan adults with uncomplicated malaria (IVERMAL): a randomised, double-blind, placebo-controlled trial.

BACKGROUND: Ivermectin is being considered for mass drug administration for malaria due to its ability to kill mosquitoes feeding on recently treated individuals. However, standard, single doses of 150-200 µg/kg used for onchocerciasis and lymphatic filariasis have a short-lived mosquitocidal effect (<7 days). Because ivermectin is well tolerated up to 2000 µg/kg, we aimed to establish the safety, tolerability, and mosquitocidal efficacy of 3 day courses of high-dose ivermectin, co-administered with a standard malaria treatment. METHODS: We did a randomised, double-blind, placebo-controlled, superiority trial at the Jaramogi Oginga Odinga Teaching and Referral Hospital (Kisumu, Kenya). Adults (aged 18-50 years) were eligible if they had confirmed symptomatic uncomplicated Plasmodium falciparum malaria and agreed to the follow-up schedule. Participants were randomly assigned (1:1:1) using sealed envelopes, stratified by sex and body-mass index (men: <21 vs ≥21 kg/m2; women: <23 vs ≥23 kg/m2), with permuted blocks of three, to receive 3 days of ivermectin 300 µg/kg per day, ivermectin 600 µg/kg per day, or placebo, all co-administered with 3 days of dihydroartemisinin-piperaquine. Blood of patients taken on post-treatment days 0, 2 + 4 h, 7, 10, 14, 21, and 28 was fed to laboratory-reared Anopheles gambiae sensu stricto mosquitoes, and mosquito survival was assessed daily for 28 days after feeding. The primary outcome was 14-day cumulative mortality of mosquitoes fed 7 days after ivermectin treatment (from participants who received at least one dose of study medication). The study is registered with ClinicalTrials.gov, number NCT02511353. FINDINGS: Between July 20, 2015, and May 7, 2016, 741 adults with malaria were assessed for eligibility, of whom 141 were randomly assigned to receive ivermectin 600 µg/kg per day (n=47), ivermectin 300 µg/kg per day (n=48), or placebo (n=46). 128 patients (91%) attended the primary outcome visit 7 days post treatment. Compared with placebo, ivermectin was associated with higher 14 day post-feeding mosquito mortality when fed on blood taken 7 days post treatment (ivermectin 600 µg/kg per day risk ratio [RR] 2·26, 95% CI 1·93-2·65, p<0·0001; hazard ratio [HR] 6·32, 4·61-8·67, p<0·0001; ivermectin 300 µg/kg per day RR 2·18, 1·86-2·57, p<0·0001; HR 4·21, 3·06-5·79, p<0·0001). Mosquito mortality remained significantly increased 28 days post treatment (ivermectin 600 µg/kg per day RR 1·23, 1·01-1·50, p=0·0374; and ivermectin 300 µg/kg per day 1·21, 1·01-1·44, p=0·0337). Five (11%) of 45 patients receiving ivermectin 600 µg/kg per day, two (4%) of 48 patients receiving ivermectin 300 µg/kg per day, and none of 46 patients receiving placebo had one or more treatment-related adverse events. INTERPRETATION: Ivermectin at both doses assessed was well tolerated and reduced mosquito survival for at least 28 days after treatment. Ivermectin 300 µg/kg per day for 3 days provided a good balance between efficacy and tolerability, and this drug shows promise as a potential new tool for malaria elimination. FUNDING: Malaria Eradication Scientific Alliance (MESA) and US Centers for Disease Control and Prevention (CDC).

Implications of insecticide resistance for malaria vector control with long-lasting insecticidal nets: a WHO-coordinated, prospective, international, observational cohort study.

BACKGROUND: Scale-up of insecticide-based interventions has averted more than 500 million malaria cases since 2000. Increasing insecticide resistance could herald a rebound in disease and mortality. We aimed to investigate whether insecticide resistance was associated with loss of effectiveness of long-lasting insecticidal nets and increased malaria disease burden. METHODS: This WHO-coordinated, prospective, observational cohort study was done at 279 clusters (villages or groups of villages in which phenotypic resistance was measurable) in Benin, Cameroon, India, Kenya, and Sudan. Pyrethroid long-lasting insecticidal nets were the principal form of malaria vector control in all study areas; in Sudan this approach was supplemented by indoor residual spraying. Cohorts of children from randomly selected households in each cluster were recruited and followed up by community health workers to measure incidence of clinical malaria and prevalence of infection. Mosquitoes were assessed for susceptibility to pyrethroids using the standard WHO bioassay test. Country-specific results were combined using meta-analysis. FINDINGS: Between June 2, 2012, and Nov 4, 2016, 40 000 children were enrolled and assessed for clinical incidence during 1·4 million follow-up visits. 80 000 mosquitoes were assessed for insecticide resistance. Long-lasting insecticidal net users had lower infection prevalence (adjusted odds ratio [OR] 0·63, 95% CI 0·51-0·78) and disease incidence (adjusted rate ratio [RR] 0·62, 0·41-0·94) than did non-users across a range of resistance levels. We found no evidence of an association between insecticide resistance and infection prevalence (adjusted OR 0·86, 0·70-1·06) or incidence (adjusted RR 0·89, 0·72-1·10). Users of nets, although significantly better protected than non-users, were nevertheless subject to high malaria infection risk (ranging from an average incidence in net users of 0·023, [95% CI 0·016-0·033] per person-year in India, to 0·80 [0·65-0·97] per person year in Kenya; and an average infection prevalence in net users of 0·8% [0·5-1·3] in India to an average infection prevalence of 50·8% [43·4-58·2] in Benin). INTERPRETATION: Irrespective of resistance, populations in malaria endemic areas should continue to use long-lasting insecticidal nets to reduce their risk of infection. As nets provide only partial protection, the development of additional vector control tools should be prioritised to reduce the unacceptably high malaria burden. FUNDING: Bill & Melinda Gates Foundation, UK Medical Research Council, and UK Department for International Development.

Identification of field caught Anopheles gambiae s.s. and Anopheles arabiensis by TaqMan single nucleotide polymorphism genotyping.

BACKGROUND: Identification of Anopheles gambiae s.s. and Anopheles arabiensis from field-collected Anopheles gambiae s.l. is often necessary in basic and applied research, and in operational control programmes. The currently accepted method involves use of standard polymerase chain reaction amplification of ribosomal DNA (rDNA) from the 3' 28S to 5' intergenic spacer region of the genome, and visual confirmation of amplicons of predicted size on agarose gels, after electrophoresis. This report describes development and evaluation of an automated, quantitative PCR method based upon TaqMan single nucleotide polymorphism (SNP) genotyping. METHODS: Standard PCR, and TaqMan SNP genotyping with newly designed primers and fluorophore-labeled probes hybridizing to sequences of complementary rDNA specific for either An. gambiae s.s. or An. arabiensis, were conducted in three experiments involving field-collected An. gambiae s.l. from western Kenya, and defined laboratory strains. DNA extraction was from a single leg, sonicated for five minutes in buffer in wells of 96-well PCR plates. RESULTS: TaqMan SNP genotyping showed a reaction success rate, sensitivity, and species specificity comparable to that of standard PCR. In an extensive field study, only 29 of 3,041 (0.95%) were determined to be hybrids by TaqMan (i.e., having rDNA sequences from both species), however, all but one were An. arabiensis by standard PCR, suggesting an acceptably low (ca. 1%) error rate for TaqMan genotyping in mistakenly identifying species hybrids. CONCLUSION: TaqMan SNP genotyping proved to be a sensitive and rapid method for identification of An. gambiae s.l. and An. arabiensis, with a high success rate, specific results, and congruence with the standard PCR method.

Quantifying the intensity of permethrin insecticide resistance in Anopheles mosquitoes in western Kenya.

BACKGROUND: The development and spread of resistance among local vectors to the major classes of insecticides used in Long-Lasting Insecticidal Nets (LLINs) and Indoor Residual Spraying (IRS) poses a major challenge to malaria vector control programs worldwide. The main methods of evaluating insecticide resistance in malaria vectors are the WHO tube bioassay and CDC bottle assays, with their weakness being determination of resistance at a fixed dose for variable populations. The CDC bottle assay using different insecticide dosages has proved applicable in ascertaining the intensity of resistance. METHODS: We determined the status and intensity of permethrin resistance and investigated the efficacy of commonly used LLINs (PermaNet® 2.0, PermaNet® 3.0 and Olyset®) against 3-5 day-old adult female Anopheles mosquitoes from four sub-counties; Teso, Bondo, Rachuonyo and Nyando in western Kenya. Knockdown was assessed to 4 doses of permethrin; 1× (21.5 µg/ml), 2× (43 µg/ml), 5× (107.5 µg/ml) and 10× (215 µg/ml) using CDC bottle assays. RESULTS: Mortality for 0.75% permethrin ranged from 23.5% to 96.1% in the WHO tube assay. Intensity of permethrin resistance was highest in Barkanyango Bondo, with 84% knockdown at the 30 min diagnostic time when exposed to the 10× dose. When exposed to the LLINs, mortality ranged between- 0-39% for Olyset®, 12-88% for PermaNet® 2.0 and 26-89% for PermaNet® 3.0. The efficacy of nets was reduced in Bondo and Teso. Results from this study show that there was confirmed resistance in all the sites; however, intensity assays were able to differentiate Bondo and Teso as the sites with the highest levels of resistance, which coincidentally were the two sub-counties with reduced net efficacy. CONCLUSIONS: There was a reduced efficacy of nets in areas with high resistance portraying that at certain intensities of resistance, vector control using LLINs may be compromised. It is necessary to incorporate intensity assays in order to determine the extent of threat that resistance poses to malaria control.

Efficacy and Safety of High-Dose Ivermectin for Reducing Malaria Transmission (IVERMAL): Protocol for a Double-Blind, Randomized, Placebo-Controlled, Dose-Finding Trial in Western Kenya.

BACKGROUND: Innovative approaches are needed to complement existing tools for malaria elimination. Ivermectin is a broad spectrum antiparasitic endectocide clinically used for onchocerciasis and lymphatic filariasis control at single doses of 150 to 200 mcg/kg. It also shortens the lifespan of mosquitoes that feed on individuals recently treated with ivermectin. However, the effect after a 150 to 200 mcg/kg oral dose is short-lived (6 to 11 days). Modeling suggests higher doses, which prolong the mosquitocidal effects, are needed to make a significant contribution to malaria elimination. Ivermectin has a wide therapeutic index and previous studies have shown doses up to 2000 mcg/kg (ie, 10 times the US Food and Drug Administration approved dose) are well tolerated and safe; the highest dose used for onchocerciasis is a single dose of 800 mcg/kg. OBJECTIVE: The aim of this study is to determine the safety, tolerability, and efficacy of ivermectin doses of 0, 300, and 600 mcg/kg/day for 3 days, when provided with a standard 3-day course of the antimalarial dihydroartemisinin-piperaquine (DP), on mosquito survival. METHODS: This is a double-blind, randomized, placebo-controlled, parallel-group, 3-arm, dose-finding trial in adults with uncomplicated malaria. Monte Carlo simulations based on pharmacokinetic modeling were performed to determine the optimum dosing regimens to be tested. Modeling showed that a 3-day regimen of 600 mcg/kg/day achieved similar median (5 to 95 percentiles) maximum drug concentrations (Cmax) of ivermectin to a single of dose of 800 mcg/kg, while increasing the median time above the lethal concentration 50% (LC50, 16 ng/mL) from 1.9 days (1.0 to 5.7) to 6.8 (3.8 to 13.4) days. The 300 mcg/kg/day dose was chosen at 50% of the higher dose to allow evaluation of the dose response. Mosquito survival will be assessed daily up to 28 days in laboratory-reared Anopheles gambiae s.s. populations fed on patients' blood taken at days 0, 2 (Cmax), 7 (primary outcome), 10, 14, 21, and 28 after the start of treatment. Safety outcomes include QT-prolongation and mydriasis. The trial will be conducted in 6 health facilities in western Kenya and requires a sample size of 141 participants (47 per arm). Sub-studies include (1) rich pharmacokinetics and (2) direct skin versus membrane feeding assays. RESULTS: Recruitment started July 20, 2015. Data collection was completed July 2, 2016. Unblinding and analysis will commence once the database has been completed, cleaned, and locked. CONCLUSIONS: High-dose ivermectin, if found to be safe and well tolerated, might offer a promising new tool for malaria elimination.

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.

An online tool for mapping insecticide resistance in major Anopheles vectors of human malaria parasites and review of resistance status for the Afrotropical region.

BACKGROUND: Malaria control programmes across Africa and beyond are facing increasing insecticide resistance in the major anopheline vectors. In order to preserve or prolong the effectiveness of the main malaria vector interventions, up-to-date and easily accessible insecticide resistance data that are interpretable at operationally-relevant scales are critical. Herein we introduce and demonstrate the usefulness of an online mapping tool, IR Mapper. METHODS: A systematic search of published, peer-reviewed literature was performed and Anopheles insecticide susceptibility and resistance mechanisms data were extracted and added to a database after a two-level verification process. IR Mapper ( http://www.irmapper.com) was developed using the ArcGIS for JavaScript Application Programming Interface and ArcGIS Online platform for exploration and projection of these data. RESULTS: Literature searches yielded a total of 4,084 susceptibility data points for 1,505 populations, and 2,097 resistance mechanisms data points for 1,000 populations of Anopheles spp. tested via recommended WHO methods from 54 countries between 1954 and 2012. For the Afrotropical region, data were most abundant for populations of An. gambiae, and pyrethroids and DDT were more often used in susceptibility assays (51.1 and 26.8% of all reports, respectively) than carbamates and organophosphates. Between 2001 and 2012, there was a clear increase in prevalence and distribution of confirmed resistance of An. gambiae s.l. to pyrethroids (from 41 to 87% of the mosquito populations tested) and DDT (from 64 to 91%) throughout the Afrotropical region. Metabolic resistance mechanisms were detected in western and eastern African populations and the two kdr mutations (L1014S and L1014F) were widespread. For An. funestus s.l., relatively few populations were tested, although in 2010-2012 resistance was reported in 50% of 10 populations tested. Maps are provided to illustrate the use of IR Mapper and the distribution of insecticide resistance in malaria vectors in Africa. CONCLUSIONS: The increasing pyrethroid and DDT resistance in Anopheles in the Afrotropical region is alarming. Urgent attention should be afforded to testing An. funestus populations especially for metabolic resistance mechanisms. IR Mapper is a useful tool for investigating temporal and spatial trends in Anopheles resistance to support the pragmatic use of insecticidal interventions.