Genetic markers associated with the widespread insecticide resistance in malaria vector Anopheles funestus populations across Tanzania.

BACKGROUND: Anopheles funestus is a leading vector of malaria in most parts of East and Southern Africa, yet its ecology and responses to vector control remain poorly understood compared with other vectors such as Anopheles gambiae and Anopheles arabiensis. This study presents the first large-scale survey of the genetic and phenotypic expression of insecticide resistance in An. funestus populations in Tanzania. METHODS: We performed insecticide susceptibility bioassays on An. funestus mosquitoes in nine regions with moderate-to-high malaria prevalence in Tanzania, followed by genotyping for resistance-associated mutations (CYP6P9a, CYP6P9b, L119F-GSTe2) and structural variants (SV4.3 kb, SV6.5 kb). Generalized linear models were used to assess relationships between genetic markers and phenotypic resistance. An interactive R Shiny tool was created to visualize the data and support evidence-based interventions. RESULTS: Pyrethroid resistance was universal but reversible by piperonyl-butoxide (PBO). However, carbamate resistance was observed in only five of the nine districts, and dichloro-diphenyl-trichloroethane (DDT) resistance was found only in the Kilombero valley, south-eastern Tanzania. Conversely, there was universal susceptibility to the organophosphate pirimiphos-methyl in all sites. Genetic markers of resistance had distinct geographical patterns, with CYP6P9a-R and CYP6P9b-R alleles, and the SV6.5 kb structural variant absent or undetectable in the north-west but prevalent in all other sites, while SV4.3 kb was prevalent in the north-western and western regions but absent elsewhere. Emergent L119F-GSTe2, associated with deltamethrin resistance, was detected in heterozygous form in districts bordering Mozambique, Malawi and the Democratic Republic of Congo. The resistance landscape was most complex in western Tanzania, in Tanganyika district, where all five genetic markers were detected. There was a notable south-to-north spread of resistance genes, especially CYP6P9a-R, though this appears to be interrupted, possibly by the Rift Valley. CONCLUSIONS: This study underscores the need to expand resistance monitoring to include An. funestus alongside other vector species, and to screen for both the genetic and phenotypic signatures of resistance. The findings can be visualized online via an interactive user interface and could inform data-driven decision-making for resistance management and vector control. Since this was the first large-scale survey of resistance in Tanzania's An. funestus, we recommend regular updates with greater geographical and temporal coverage.

Dynamics of malaria vector composition and Plasmodium falciparum infection in mainland Tanzania: 2017-2021 data from the national malaria vector entomological surveillance.

BACKGROUND: In 2015, Tanzania National Malaria Control Programme (NMCP) established a longitudinal malaria vector entomological surveillance (MVES). The MVES is aimed at a periodical assessment of malaria vector composition and abundance, feeding and resting behaviours, and Plasmodium falciparum infection in different malaria epidemiological strata to guide the NMCP on the deployment of appropriate malaria vector interventions. This work details the dynamics of malaria vector composition and transmission in different malaria epidemiological strata. METHODS: The MVES was conducted from 32 sentinel district councils across the country. Mosquitoes were collected by the trained community members and supervised by the NMCP and research institutions. Three consecutive night catches (indoor collection with CDC light trap and indoor/outdoor collection using bucket traps) were conducted monthly in three different households selected randomly from two to three wards within each district council. Collected mosquitoes were sorted and morphologically identified in the field. Thereafter, the samples were sent to the laboratory for molecular characterization using qPCR for species identification and detection of P. falciparum infections (sporozoites). ELISA technique was deployed for blood meal analysis from samples of blood-fed mosquitoes to determine the blood meal indices (BMI). RESULTS: A total of 63,226 mosquitoes were collected in 32 district councils from January 2017 to December 2021. Out of which, 39,279 (62%), 20,983 (33%) and 2964 (5%) were morphologically identified as Anopheles gambiae sensu lato (s.l.), Anopheles funestus s.l., and as other Anopheles species, respectively. Out of 28,795 laboratory amplified mosquitoes, 13,645 (47%) were confirmed to be Anopheles arabiensis, 9904 (34%) as An. funestus sensu stricto (s.s.), and 5193 (19%) as An. gambiae s.s. The combined average entomological inoculation rates (EIR) were 0.46 (95% CI 0.028-0.928) for An. gambiae s.s., 0.836 (95% CI 0.138-1.559) for An. arabiensis, and 0.58 (95% CI 0.165-0.971) for An. funestus s.s. with variations across different malaria transmission strata. Anopheles funestus s.s. and An. arabiensis were predominant in the Lake and South-Eastern zones, respectively, mostly in high malaria transmission areas. Monthly mosquito densities displayed seasonal patterns, with two peaks following the rainy seasons, varying slightly across species and district councils. CONCLUSION: Anopheles arabiensis remains the predominant vector species followed by An. funestus s.s. in the country. Therefore, strengthening integrated vector management including larval source management is recommended to address outdoor transmission by An. arabiensis to interrupt transmission particularly where EIR is greater than the required elimination threshold of less than one (< 1) to substantially reduce the prevalence of malaria infection.

Efficacy of 3D screens for sustainable mosquito control: a semi-field experimental hut evaluation in northeastern Tanzania.

BACKGROUND: A three-dimensional window screen (3D-Screen) has been developed to create a window double-screen trap (3D-WDST), effectively capturing and preventing the escape of mosquitoes. A 2015 laboratory study demonstrated the 3D-Screen's efficacy, capturing 92% of mosquitoes in a double-screen setup during wind tunnel assays. To further evaluate its effectiveness, phase II experimental hut trials were conducted in Muheza, Tanzania. METHODS: Three experimental hut trials were carried out between 2016 and 2017. Trial I tested two versions of the 3D-WDST in huts with open or closed eaves, with one version using a single 3D-Screen and the other using two 3D-Screens. Trial II examined the 3D-WDST with two 3D-Screens in huts with or without baffles, while Trial III compared handmade and machine-made 3D structures. Mosquito capturing efficacy of the 3D-WDST was measured by comparing the number of mosquitoes collected in the test hut to a control hut with standard exit traps. RESULTS: Trial I showed that the 3D-WDST with two 3D-Screens used in huts with open eaves achieved the highest mosquito-capturing efficacy. This treatment captured 33.11% (CI 7.40-58.81) of female anophelines relative to the total collected in this hut (3D-WDST and room collections) and 27.27% (CI 4.23-50.31) of female anophelines relative to the total collected in the control hut (exit traps, room, and verandahs collections). In Trial II, the two 3D-Screens version of the 3D-WDST captured 70.32% (CI 56.87-83.77) and 51.07% (CI 21.72-80.41) of female anophelines in huts with and without baffles, respectively. Compared to the control hut, the capturing efficacy for female anophelines was 138.6% (37.23-239.9) and 42.41% (14.77-70.05) for huts with and without baffles, respectively. Trial III demonstrated similar performance between hand- and machine-made 3D structures. CONCLUSIONS: The 3D-WDST proved effective in capturing malaria vectors under semi-field experimental hut conditions. Using 3D-Screens on both sides of the window openings was more effective than using a single-sided 3D-Screen. Additionally, both hand- and machine-made 3D structures exhibited equally effective performance, supporting the production of durable cones on an industrial scale for future large-scale studies evaluating the 3D-WDST at the community level.

Community evaluation of the physical and insecticidal durability of DuraNet® Plus, an alpha-cypermethrin and piperonyl butoxide incorporated mosquito net: protocol for a multi-country study in West, Central and East Africa.

BACKGROUND: Pyrethroid-PBO nets have demonstrated improved impact against clinical malaria transmitted by pyrethroid resistant mosquito vectors and are being scaled up across Africa. However very little is known about their physical and insecticidal durability under operational conditions. This study will investigate the attrition, fabric integrity, insecticide content and bioefficacy of DuraNet® Plus, a new WHO prequalified alphacypermethrin and PBO incorporated net developed by Shobikaa Impex Private Limited over 3 years of field use in communities in Benin, Cameroon and Tanzania. METHODS: The study will be conducted in parallel in selected villages in Zakpota District in Benin, Mbalmayo, District in Cameroon and Muheza District in Tanzania. In each country, ~ 1800 households will be recruited and randomised to receive DuraNet® Plus or DuraNet® (a WHO prequalified alphacypermethrin-only ITN). Follow up surveys will be performed at 1 month post distribution to investigate adverse events and subsequently every 6-12 months to assess ITN attrition and fabric integrity following standard WHO procedures. A second cohort of nets will be withdrawn every 6-12 months and assessed for alpha-cypermethrin and PBO content and for entomological activity in laboratory bioassays (cone bioassays and tunnel tests). Alpha-cypermethrin bioefficacy will be monitored using the susceptible Anopheles gambiae Kisumu strain in cone bioassays while PBO bioefficacy will be monitored using pyrethroid resistant strains with overexpressed P450 enzymes in tunnel tests to determine the proportion of efficacious nets (≥ 95% knockdown, ≥ 80% mortality or ≥ 90% blood feeding inhibition in tunnels) at each time point. Nets withdrawn at 12, 24 and 36 months from each country will also be tested in experimental hut trials against wild free-flying pyrethroid resistant Anopheles gambiae sl in Côvè Benin to investigate the superiority of DuraNet® Plus over DuraNet® at each time point under semi field conditions. CONCLUSION: This large-scale multi country trial will provide useful information on the durability of a pyrethroid-PBO net (DuraNet® Plus) in 3 different regions in sub-Saharan Africa. The methods proposed for bioefficacy testing could also contribute towards the development of new standardised guidelines for monitoring the insecticidal efficacy of pyrethroid-PBO nets under operational conditions.

Assessing the variability in experimental hut trials evaluating insecticide-treated nets against malaria vectors.

Experimental hut trials (EHTs) are used to evaluate indoor vector control interventions against malaria vectors in a controlled setting. The level of variability present in the assay will influence whether a given study is well powered to answer the research question being considered. We utilised disaggregated data from 15 previous EHTs to gain insight into the behaviour typically observed. Using simulations from generalised linear mixed models to obtain power estimates for EHTs, we show how factors such as the number of mosquitoes entering the huts each night and the magnitude of included random effects can influence study power. A wide variation in behaviour is observed in both the mean number of mosquitoes collected per hut per night (ranging from 1.6 to 32.5) and overdispersion in mosquito mortality. This variability in mortality is substantially greater than would be expected by chance and should be included in all statistical analyses to prevent false precision of results. We utilise both superiority and non-inferiority trials to illustrate our methodology, using mosquito mortality as the outcome of interest. The framework allows the measurement error of the assay to be reliably assessed and enables the identification of outlier results which could warrant further investigation. EHTs are increasingly playing an important role in the evaluation and regulation of indoor vector control interventions so it is important to ensure that these studies are adequately powered.

Trends of insecticide resistance monitoring in mainland Tanzania, 2004-2020.

BACKGROUND: Insecticide resistance is a serious threat to the continued effectiveness of insecticide-based malaria vector control measures, such as long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS). This paper describes trends and dynamics of insecticide resistance and its underlying mechanisms from annual resistance monitoring surveys on Anopheles gambiae sensu lato (s.l.) populations conducted across mainland Tanzania from 2004 to 2020. METHODS: The World Health Organization (WHO) standard protocols were used to assess susceptibility of the wild female An. gambiae s.l. mosquitoes to insecticides, with mosquitoes exposed to diagnostic concentrations of permethrin, deltamethrin, lambdacyhalothrin, bendiocarb, and pirimiphos-methyl. WHO test papers at 5× and 10× the diagnostic concentrations were used to assess the intensity of resistance to pyrethroids; synergist tests using piperonyl butoxide (PBO) were carried out in sites where mosquitoes were found to be resistant to pyrethroids. To estimate insecticide resistance trends from 2004 to 2020, percentage mortalities from each site and time point were aggregated and regression analysis of mortality versus the Julian dates of bioassays was performed. RESULTS: Percentage of sites with pyrethroid resistance increased from 0% in 2004 to more than 80% in the 2020, suggesting resistance has been spreading geographically. Results indicate a strong negative association (p = 0.0001) between pyrethroids susceptibility status and survey year. The regression model shows that by 2020 over 40% of An. gambiae mosquitoes survived exposure to pyrethroids at their respective diagnostic doses. A decreasing trend of An. gambiae susceptibility to bendiocarb was observed over time, but this was not statistically significant (p = 0.8413). Anopheles gambiae exhibited high level of susceptibility to the pirimiphos-methyl in sampled sites. CONCLUSIONS: Anopheles gambiae Tanzania's major malaria vector, is now resistant to pyrethroids across the country with resistance increasing in prevalence and intensity and has been spreading geographically. This calls for urgent action for efficient malaria vector control tools to sustain the gains obtained in malaria control. Strengthening insecticide resistance monitoring is important for its management through evidence generation for effective malaria vector control decision.

Multi-centre discriminating concentration determination of broflanilide and potential for cross-resistance to other public health insecticides in Anopheles vector populations.

Novel insecticides are urgently needed to control insecticide-resistant populations of Anopheles malaria vectors. Broflanilide acts as a non-competitive antagonist of the gamma-aminobutyric acid receptor and has shown prolonged effectiveness as an indoor residual spraying product (VECTRON T500) in experimental hut trials against pyrethroid-resistant vector populations. This multi-centre study expanded upon initial discriminating concentration testing of broflanilide, using six Anopheles insectary colonies (An. gambiae Kisumu KCMUCo, An. gambiae Kisumu NIMR, An. arabiensis KGB, An. arabiensis SENN, An. coluzzii N'Gousso and An. stephensi SK), representing major malaria vector species, to facilitate prospective susceptibility monitoring of this new insecticide; and investigated the potential for cross-resistance to broflanilide via the A296S mutation associated with dieldrin resistance (rdl). Across all vector species tested, the discriminating concentration for broflanilide ranged between LC99 × 2 = 1.126-54.00 µg/ml or LC95 × 3 = 0.7437-17.82 µg/ml. Lower concentrations of broflanilide were required to induce complete mortality of An. arabiensis SENN (dieldrin-resistant), compared to its susceptible counterpart, An. arabiensis KGB, and there was no association between the presence of the rdl mechanism of resistance and survival in broflanilide bioassays, demonstrating a lack of cross-resistance to broflanilide. Study findings provide a benchmark for broflanilide susceptibility monitoring as part of ongoing VECTRON T500 community trials in Tanzania and Benin.

Rift Valley fever seropositivity in humans and domestic ruminants and associated risk factors in Sengerema, Ilala, and Rufiji districts, Tanzania.

OBJECTIVES: Data on Rift Valley fever virus (RVFV) prevalence in urban settings and pastoral areas of Tanzania are scarce. We performed a cross-sectional study of RVFV seroprevalence and determinants in humans and animals from Ilala, Rufiji, and Sengerema districts of Tanzania. METHODS: Blood samples from the study participants were tested for anti-RVFV immunoglobulin G (IgG) antibodies using an enzyme-linked immunosorbent assay. Logistic regression was used to determine association between exposure risk practices and RVFV seropositivity. RESULTS: The study involved 664 humans, 361 cattle, 394 goats, and 242 sheep. The overall anti-RVFV IgG seroprevalence in humans and animals was 2.1% (95% confidence interval [CI] 0.01-0.04) and 9.5% (n = 95, 95% CI 0.08-0.12), respectively. Seroprevalence in humans in Rufiji, Ilala, and Sengerema was 3.0% (n = 225, 95% CI 0.01-0.06), 1.8% (n = 230, 95% CI-0.005- 0.04), and 1.4% (n = 209, 95% CI 0.01-0.04), respectively (P >0.05). Seroprevalence in animals in Sengerema, Rufiji, and Ilala was 12.1% (n = 40, 95% CI 0.09-0.16), 11.1% (n = 37, 95% CI 0.08-0.15), and 5.4% (n = 18, 95% CI 0.03-0.08), respectively (P = 0.006). Handling of carcasses increased the odds of RVFV seropositivity 12-fold (odds ratio 11.84, 95% CI 1.97-71.16). CONCLUSION: The study confirms previous occurrence of RVFV in multiple species in the study districts. Animal handling practices appear to be essential determinants of seropositivity.

Laboratory and semi-field evaluation of the efficacy of Bacillus thuringiensis var. israelensis (Bactivec®) and Bacillus sphaericus (Griselesf®) for control of mosquito vectors in northeastern Tanzania.

Bacterial larvicides Bacillus thuringiensis var. israelensis (Bti) and Bacillus sphaericus (Bs) have been used extensively for mosquito control. However, their efficacy varies greatly mainly due to factors related to target mosquitoes, larval habitat conditions, and inherent larvicide properties. We evaluated the efficacy of Bti (Bactivec®) and Bs (Griselesf®) for control of Anopheles gambiae complex, Culex quinquefasciatus and Aedes aegypti larvae under laboratory and semi-field conditions in northeastern Tanzania. Laboratory bioassays were conducted with five to six different concentrations of Bti and Bs, replicated four times and the experiment repeated on three different days. Larvae mortality was recorded at 24 or 48 h after the application of larvicide and subjected to Probit analysis. Laboratory bioassays were followed by semi-field trials to establish initial and residual activity of Bti and Bs. Semi-field trials were conducted in artificial larval habitats in the open sunlit ground and in "mosquito spheres". These artificial larval habitats were colonized with mosquito larvae, treated with Bti and Bs, and the impact of treatments on mosquito larvae was monitored daily. Lethal concentration values that caused 50% and 95% mortalities of test larvae (LC50 and LC95) showed that An. gambiae complex and Cx. quinquefasciatus tested were highly susceptible to Bti and Bs under laboratory conditions. Likewise, larvae of Ae. aegypti were highly susceptible to Bti, with LC95 value as low as 0.052 mg/l. However, Ae. aegypti larvae were not susceptible to Bs under practical doses of laboratory settings. In semi-field trials, all treatment dosages for Bti provided 91.0-100% larval mortality within 24 h whereas Bs resulted in 96.8-100% larval mortality within the same time-frame. Bs had a more prolonged residual activity, with pupal reductions range of 55.7-100% for 9 days at all application rates while the corresponding pupal reduction with Bti was 15.4-100% for 5 days. Due to the low residual activity of Bti and Bs tested, weekly application at a maximum label rate would be appropriate to reduce mosquito larvae in natural larval habitats. Based on laboratory findings, Bs product tested would not be recommended for use in the control of Ae. aegypti.

Large-scale (Phase III) evaluation of broflanilide 50WP (VECTRON™ T500) for indoor residual spraying for malaria vector control in Northeast Tanzania: study protocol for a two-arm, non-inferiority, cluster-randomised community trial.

BACKGROUND: Indoor residual spraying (IRS) is a major method of malaria vector control across sub-Saharan Africa. Effective control is being undermined by the rapid spread of insecticide resistance. There is major investment in development of new insecticides for IRS that possess novel modes of action, long residual activity, low mammalian toxicity and minimal cross-resistance. VECTRON™ T500, a new IRS product containing the active ingredient broflanilide as a 50% wettable powder (WP), has been shown to be efficacious against pyrethroid susceptible and resistant vector species on mud and concrete substrates in experimental hut (Phase II) trials. METHODS: A two-arm non-inferiority cluster randomized controlled trial (Phase III) will be undertaken in Muheza District, Tanga Region, Tanzania. VECTRON™ T500 will be compared to the IRS product Fludora® Fusion (clothianidin 50% WP + deltamethrin 6.25% WP). The predominant malaria vectors in the study area are pyrethroid-resistant Anopheles gambiae s.s., An. arabiensis and An. funestus s.s. Sixteen village clusters will be pair-matched on baseline vector densities and allocated to reference and intervention arms. Consenting households in the intervention arm will be sprayed with VECTRON™ T500 and those in the reference arm will be sprayed with Fludora® Fusion. Each month, CDC light traps will collect mosquitoes to estimate changes in vector density, indoor biting, sporozoite and entomological inoculation rates (EIR). Susceptibility to IRS active ingredients will be assessed using World Health Organisation (WHO) bottle bioassays. Target site and metabolic resistance mechanisms will be characterised among Anopheles field populations from both trial arms. Residual efficacy of both IRS products will be monitored for 12 months post intervention. Questionnaire and focus group discussions will explore factors that influence adherence, adverse effects and benefits of IRS. DISCUSSION: This protocol describes a large-scale non-inferiority evaluation of a novel IRS product to reduce the density and EIR of pyrethroid-resistant Anopheles vectors. If VECTRON™ T500 proves non-inferior to Fludora® Fusion, it will be considered as an additional vector control product for malaria prevention and insecticide resistance management. TRIAL REGISTRATION: ClinicalTrials.gov, NCT05150808, registered on 26 November 2021. Retrospectively registered.

Optimising the deployment of vector control tools against malaria: a data-informed modelling study.

BACKGROUND: Concern that insecticide resistant mosquitoes are threatening malaria control has driven the development of new types of insecticide treated nets (ITNs) and indoor residual spraying (IRS) of insecticide. Malaria control programmes have a choice of vector control interventions although it is unclear which controls should be used to combat the disease. The study aimed at producing a framework to easily compare the public health impact and cost-effectiveness of different malaria prevention measures currently in widespread use. METHODS: We used published data from experimental hut trials conducted across Africa to characterise the entomological effect of pyrethroid-only ITNs versus ITNs combining a pyrethroid insecticide with the synergist piperonyl butoxide (PBO). We use these estimates to parameterise a dynamic mathematical model of Plasmodium falciparum malaria which is validated for two sites by comparing simulated results to empirical data from randomised control trials (RCTs) in Tanzania and Uganda. We extrapolated model simulations for a series of potential scenarios likely across the sub-Saharan African region and include results in an online tool (Malaria INtervention Tool [MINT]) that aims to identify optimum vector control intervention packages for scenarios with varying budget, price, entomological and epidemiological factors. FINDINGS: Our model indicates that switching from pyrethroid-only to pyrethroid-PBO ITNs could averted up to twice as many cases, although the additional benefit is highly variable and depends on the setting conditions. We project that annual delivery of long-lasting, non-pyrethroid IRS would prevent substantially more cases over 3-years, while pyrethroid-PBO ITNs tend to be the most cost-effective intervention per case averted. The model was able to predict prevalence and efficacy against prevalence in both RCTs for the intervention types tested. MINT is applicable to regions of sub-Saharan Africa with endemic malaria and provides users with a method of designing intervention packages given their setting and budget. INTERPRETATION: The most cost-effective vector control package will vary locally. Models able to recreate results of RCTs can be used to extrapolate outcomes elsewhere to support evidence-based decision making for investment in vector control. FUNDING: Medical Research Council, IVCC, Wellcome Trust. TRANSLATION: For the French translation of the abstract see Supplementary Materials section.

Bio-efficacy and wash-fastness of a lambda-cyhalothrin long-lasting insecticide treatment kit (ICON® Maxx) against mosquitoes on various polymer materials.

BACKGROUND: Long-lasting efficacy of insecticide-treated nets is a balance between adhesion, retention and migration of insecticide to the surface of netting fibres. ICON® Maxx is a twin-sachet 'home-treatment kit' of pyrethroid plus binding agent, recommended by the World Health Organization (WHO) for long-lasting, wash-fast treatment of polyester nets. While knitted polyester netting is widely used, fine woven polyethylene netting is increasingly available and nets made of cotton and nylon are common in Africa and Asia. It is important to investigate whether ICON Maxx is able to fulfill the WHO criteria of long-lasting treatment on a range of domestic fabrics to widen the scope for malaria protection. METHODS: This study was a controlled comparison of the bio-efficacy and wash-fastness of lambda-cyhalothrin CS, with or without binder, on nets made of cotton, polyethylene, nylon, dyed and undyed polyester. Evaluation compared an array of bioassays: WHO cone and cylinder, median time to knockdown and WHO tunnel tests using Anopheles mosquitoes. Chemical assay revealed further insight. RESULTS: ICON Maxx treated polyethylene and polyester netting met the WHO cone and tunnel test bio-efficacy criteria for LLIN after 20 standardized washes. Although nylon and cotton netting failed to meet the WHO cone and cylinder criteria, both materials passed the WHO tunnel test criterion of 80% mortality after 20 washes. All materials treated with standard lambda-cyhalothrin CS without binder failed to meet any of the WHO bio-efficacy criteria within 5 washes. CONCLUSION: The bio-efficacy of ICON Maxx against mosquitoes on netting washed up to 20 times demonstrated wash durability on a range of synthetic polymer and natural fibres: polyester, polyethylene, nylon and cotton. This raises the prospect of making insecticide-binder kits into an effective approach for turning untreated nets, curtains, military clothing, blankets-and tents and tarpaulins as used in disasters and humanitarian emergencies-into effective malaria prevention products. It may provide a solution to the problem of reduced LLIN coverage between campaigns by converting commercially sourced untreated nets into LLINs through community or home treatment. It may also open the door to binding of non-pyrethroid insecticides to nets and textiles for control of pyrethroid resistant vectors.

Effectiveness of a long-lasting insecticide treatment kit (ICON® Maxx) for polyester nets over three years of household use: a WHO phase III trial in Tanzania.

BACKGROUND: ICON® Maxx (Syngenta) is an insecticide treatment kit of pyrethroid and binding agent for long-lasting treatment of mosquito nets. Interim recommendation for use on nets was granted by the World Health Organization (WHO) after successful evaluation in experimental huts following multiple washes. A full WHO recommendation is contingent upon demonstration of continued bio-efficacy after 3 years of use. METHODS: A household-randomized prospective study design was used to assess ICON Maxx-treated nets over 3 years in north-eastern Tanzania. Conventional treated nets (with lambda-cyhalothrin, but without binder) served as a positive control. At 6-monthly intervals, cross-sectional household surveys monitored net use and physical integrity, while cone and tunnel tests assessed insecticidal efficacy. Pyrethroid content was determined after 12 and 36 months. A parallel cohort of nets was monitored annually for evidence of net deterioration and attrition. RESULTS: After 12 months' use, 97% of ICON Maxx-treated nets but only 67% of CTN passed the WHO efficacy threshold for insecticidal durability (> 80% mortality in cone or tunnel or 90% feeding inhibition in tunnel). After 24- and 36-months use, 67% and 26% of ICON Maxx treated nets met the cone criteria, respectively, and over 90% met the combined cone and tunnel criteria. Lambda-cyhalothrin content after 36 months was 17% (15.8 ± 4.3 mg/m2) of initial content. ICON Maxx nets were used year-round and washed approximately 4 times per year. In cross-sectional survey after 36 months the average number of holes was 20 and hole index was 740 cm2 per net. Cohort nets had fewer holes and smaller hole index than cross-sectional nets. However, only 15% (40/264) of cohort nets were not lost to follow-up or not worn out after 36 months. CONCLUSIONS: Because more than 80% of nets met the WHO efficacy criteria after 36 months use, ICON Maxx was granted WHO full recommendation. Cross-sectional and cohort surveys were complementary and gave a fuller understanding of net durability. To improve net usage and retention, stronger incentives and health messaging should be introduced in WHO LLIN longitudinal trials. Untreated polyester nets may be made long-lastingly insecticidal in Africa through simple household treatment using ICON Maxx pyrethroid-binder kits.

Efficacy of interceptor® G2, a long-lasting insecticide mixture net treated with chlorfenapyr and alpha-cypermethrin against Anopheles funestus: experimental hut trials in north-eastern Tanzania.

BACKGROUND: The effectiveness of long-lasting insecticidal nets (LLIN), the primary method for preventing malaria in Africa, is compromised by evolution and spread of pyrethroid resistance. Further gains require new insecticides with novel modes of action. Chlorfenapyr is a pyrrole insecticide that disrupts mitochrondrial function and confers no cross-resistance to neurotoxic insecticides. Interceptor® G2 LN (IG2) is an insecticide-mixture LLIN, which combines wash-resistant formulations of chlorfenapyr and the pyrethroid alpha-cypermethrin. The objective was to determine IG2 efficacy under controlled household-like conditions for personal protection and control of wild, pyrethroid-resistant Anopheles funestus mosquitoes. METHODS: Experimental hut trials tested IG2 efficacy against two positive controls-a chlorfenapyr-treated net and a standard alpha-cypermethrin LLIN, Interceptor LN (IG1)-consistent with World Health Organization (WHO) evaluation guidelines. Mosquito mortality, blood-feeding inhibition, personal protection, repellency and insecticide-induced exiting were recorded after zero and 20 washing cycles. The trial was repeated and analysed using multivariate and meta-analysis. RESULTS: In the two trials held in NE Tanzania, An. funestus mortality was 2.27 (risk ratio 95% CI 1.13-4.56) times greater with unwashed Interceptor G2 than with unwashed Interceptor LN (p = 0.012). There was no significant loss in mortality with IG2 between 0 and 20 washes (1.04, 95% CI 0.83-1.30, p = 0.73). Comparison with chlorfenapyr treated net indicated that most mortality was induced by the chlorfenapyr component of IG2 (0.96, CI 0.74-1.23), while comparison with Interceptor LN indicated blood-feeding was inhibited by the pyrethroid component of IG2 (IG2: 0.70, CI 0.44-1.11 vs IG1: 0.61, CI 0.39-0.97). Both insecticide components contributed to exiting from the huts but the contributions were heterogeneous between trials (heterogeneity Q = 36, P = 0.02). WHO susceptibility tests with pyrethroid papers recorded 44% survival in An. funestus. CONCLUSIONS: The high mortality recorded by IG2 against pyrethroid-resistant An. funestus provides first field evidence of high efficacy against this primary, anthropophilic, malaria vector.

Systematic review of the entomological impact of insecticide-treated nets evaluated using experimental hut trials in Africa.

Resistance of anopheline mosquitoes to pyrethroid insecticides is spreading rapidly across sub-Saharan Africa, diminishing the efficacy of insecticide-treated nets (ITNs) - the primary tool for preventing malaria. The entomological efficacy of indoor vector control interventions can be measured in experimental hut trials (EHTs), where hut structures resemble local housing, but allow the collection of mosquitoes that entered, exited, blood-fed and/or died. There is a need to understand how the spread of resistance changes ITN efficacy and to elucidate factors influencing EHT results, including differences in experimental hut design, to support the development of novel vector control tools. A comprehensive database of EHTs was compiled following a systematic review to identify all known trials investigating ITNs or indoor residual spraying across sub-Saharan Africa. This analysis focuses on EHTs investigating ITNs and uses Bayesian statistical models to characterise the complex interaction between ITNs and mosquitoes, the between-study variability, and the impact of pyrethroid resistance. As resistance rises, the entomological efficacy of ITNs declines. They induce less mortality and are less likely to deter mosquitoes from entering huts. Despite this, ITNs continue to offer considerable personal protection by reducing mosquito feeding until resistance reaches high levels. There are clear associations between the different entomological impacts of ITNs, though there is still substantial variability between studies, some of which can be accounted for by hut design. The relationship between EHT outcomes and the level of resistance (as measured by discriminating dose bioassays) is highly uncertain. The meta-analyses show that EHTs are an important reproducible assay for capturing the complex entomological efficacy of ITNs on blood-feeding mosquitoes. The impact of pyrethroid resistance on these measures appears broadly consistent across a wide geographical area once hut design is accounted for, suggesting results can be extrapolated beyond the sites where the trials were conducted. Further work is needed to understand factors influencing EHT outcomes and how the relationship between outcomes and resistance varies when different methods are used to assess the level of resistance in wild mosquito populations. This will allow more precise estimates of the efficacy of these important vector control tools.

Field evaluation of Veeralin, an alpha-cypermethrin + PBO long-lasting insecticidal net, against natural populations of Anopheles funestus in experimental huts in Muheza, Tanzania.

The success of long-lasting insecticidal nets (LLIN) as the primary method for preventing malaria is threatened by pyrethroid resistance in Anopheles vectors. New generation long-lasting nets incorporating PBO synergist (piperonyl butoxide) with pyrethroid are designed to control insecticide-resistant mosquitoes. The efficacy of Veeralin® PBO LLINs was evaluated in experimental huts against wild free-flying pyrethroid-resistant Anopheles funestus (s.l.). Mosquito mortality, blood-feeding inhibition and personal protection were compared between untreated nets, standard LLINs and PBO/pyrethroid combination nets. Blood-feeding rates recorded with 20-times washed Veeralin were not significantly different from those with 20-times washed PermaNet 3.0 LLIN, a WHO Pre-Qualification Team (PQT) approved PBO/pyrethroid LLIN. This provides evidence that Veeralin LLIN provides similar blood-feeding inhibition to the standard approved LLIN and thus meets WHO PQT criteria for blood-feeding. Results show significantly higher mortality for Veeralin PBO LLINs against pyrethroid-resistant Anopheles funestus (s.l.) compared to DuraNet, a WHO PQT approved standard pyrethroid-only LLIN, both when unwashed and washed 20 times. The improved efficacy over a standard pyrethroid-only LLIN can be attributed to the effect of PBO in the Veeralin LLIN, hence meeting the Vector Control Advisory Group (VCAG) criteria for a resistance breaking LLIN.

Data quality of the routine health management information system at the primary healthcare facility and district levels in Tanzania.

BACKGROUND: Effective planning for disease prevention and control requires accurate, adequately-analysed, interpreted and communicated data. In recent years, efforts have been put in strengthening health management information systems (HMIS) in Sub-Saharan Africa to improve data accessibility to decision-makers. This study assessed the quality of routine HMIS data at primary healthcare facility (HF) and district levels in Tanzania. METHODS: This cross-sectional study involved reviews of documents, information systems and databases, and collection of primary data from facility-level registers, tally sheets and monthly summary reports. Thirty-four indicators from Outpatient, Inpatient, Antenatal care, Family Planning, Post-natal care, Labour and Delivery, and Provider-Initiated Testing and Counselling service areas were assessed. Indicator records were tracked and compared across the process of data collection, compilation and submission to the district office. Copies of monthly report forms submitted by facilities to the district were also reviewed. The availability and utilization of HMIS tools were assessed, while completeness and data accuracy levels were quantified for each phase of the reporting system. RESULTS: A total of 115 HFs (including hospitals, health centres, dispensaries) in 11 districts were involved. Registers (availability rate = 91.1%; interquartile range (IQR) 66.7-100%) and report forms (86.9%; IQR 62.2-100%) were the most utilized tools. There was a limited use of tally-sheets (77.8%; IQR 35.6-100%). Tools availability at the dispensary was 91.1%, health centre 82.2% and hospital 77.8%, and was low in urban districts. The availability rate at the district level was 65% (IQR 48-75%). Wrongly filled or empty cells in registers and poor adherence to the coding procedures were observed. Reports were highly over-represented in comparison to registers' records, with large differences observed at the HF phase of the reporting system. The OPD and IPD areas indicated the highest levels of mismatch between data source and district office. Indicators with large number of clients, multiple variables, disease categorization, or those linked with dispensing medicine performed poorly. CONCLUSION: There are high variations in the tool utilisation and data accuracy at facility and district levels. The routine HMIS is weak and data at district level inaccurately reflects what is available at the source. These results highlight the need to design tailored and inter-service strategies for improving data quality.

Chikungunya Outbreak in the Republic of the Congo, 2019-Epidemiological, Virological and Entomological Findings of a South-North Multidisciplinary Taskforce Investigation.

The Republic of Congo (RoC) declared a chikungunya (CHIK) outbreak on 9 February 2019. We conducted a ONE-Human-Animal HEALTH epidemiological, virological and entomological investigation. Methods: We collected national surveillance and epidemiological data. CHIK diagnosis was based on RT-PCR and CHIKV-specific antibodies. Full CHIKV genome sequences were obtained by Sanger and MinION approaches and Bayesian tree phylogenetic analysis was performed. Mosquito larvae and 215 adult mosquitoes were collected in different villages of Kouilou and Pointe-Noire districts and estimates of Aedes (Ae.) mosquitos' CHIKV-infectious bites obtained. We found two new CHIKV sequences of the East/Central/South African (ECSA) lineage, clustering with the recent enzootic sub-clade 2, showing the A226V mutation. The RoC 2019 CHIKV strain has two novel mutations, E2-T126M and E2-H351N. Phylogenetic suggests a common origin from 2016 Angola strain, from which it diverged around 1989 (95% HPD 1985-1994). The infectious bite pattern was similar for 2017, 2018 and early 2019. One Ae. albopictus pool was RT-PCR positive. The 2019 RoC CHIKV strain seems to be recently introduced or be endemic in sylvatic cycle. Distinct from the contemporary Indian CHIKV isolates and in contrast to the original Central-African strains (transmitted by Ae. aegypti), it carries the A226V mutation, indicating an independent adaptive mutation in response to vector replacement (Ae. albopictus vs Ae. aegypti).

Preferred resting surfaces of dominant malaria vectors inside different house types in rural south-eastern Tanzania.

BACKGROUND: Malaria control in Africa relies extensively on indoor residual spraying (IRS) and insecticide-treated nets (ITNs). IRS typically targets mosquitoes resting on walls, and in few cases, roofs and ceilings, using contact insecticides. Unfortunately, little attention is paid to where malaria vectors actually rest indoors, and how such knowledge could be used to improve IRS. This study investigated preferred resting surfaces of two major malaria vectors, Anopheles funestus and Anopheles arabiensis, inside four common house types in rural south-eastern Tanzania. METHODS: The assessment was done inside 80 houses including: 20 with thatched roofs and mud walls, 20 with thatched roofs and un-plastered brick walls, 20 with metal roofs and un-plastered brick walls, and 20 with metal roofs and plastered brick walls, across four villages. In each house, resting mosquitoes were sampled in mornings (6 a.m.-8 a.m.), evenings (6 p.m.-8 p.m.) and at night (11 p.m.-12.00 a.m.) using Prokopack aspirators from multiple surfaces (walls, undersides of roofs, floors, furniture, utensils, clothing, curtains and bed nets). RESULTS: Overall, only 26% of An. funestus and 18% of An. arabiensis were found on walls. In grass-thatched houses, 33-55% of An. funestus and 43-50% of An. arabiensis rested under roofs, while in metal-roofed houses, only 16-20% of An. funestus and 8-30% of An. arabiensis rested under roofs. Considering all data together, approximately 40% of mosquitoes rested on surfaces not typically targeted by IRS, i.e. floors, furniture, utensils, clothing and bed nets. These proportions were particularly high in metal-roofed houses (47-53% of An. funestus; 60-66% of An. arabiensis). CONCLUSION: While IRS typically uses contact insecticides to target adult mosquitoes on walls, and occasionally roofs and ceilings, significant proportions of vectors rest on surfaces not usually sprayed. This gap exceeds one-third of malaria mosquitoes in grass-thatched houses, and can reach two-thirds in metal-roofed houses. Where field operations exclude roofs during IRS, the gaps can be much greater. In conclusion, there is need for locally-obtained data on mosquito resting behaviours and how these influence the overall impact and costs of IRS. This study also emphasizes the need for alternative approaches, e.g. house screening, which broadly tackle mosquitoes beyond areas reachable by IRS and ITNs.

Bio-efficacy and wash resistance of MAGNet long-lasting insecticidal net against wild populations of Anopheles funestus in experimental huts in Muheza, Tanzania.

BACKGROUND: The decline in malaria cases and vectors is major milestone in fighting against malaria. The efficacy of MAGNet long-lasting insecticidal nets (MAGNet LLIN), an alpha-cypermethrin incorporated long-lasting net, with the target dose ± 25% of 5.8 g active ingredient (AI)/kg (4.35-7.25 g AI/kg) was evaluated in six veranda-trap experimental huts in Muheza, Tanzania against freely flying wild population of Anopheles funestus. METHODS: MAGNet LLINs were tested against wild, free-flying, host-seeking An. funestus mosquitoes over a period of 6 weeks (total of 36 nights in the huts). MAGNet LLIN efficacy was determined in terms of mosquito mortality, blood-feeding inhibition, deterrence, induced exiting, personal protection, and insecticidal killing over 20 washes according to WHO standardized procedures. Efficacy was compared with reference to a WHOPES recommended approved LLINs (DuraNet) and to a net conventionally treated (CTN) treated with alpha-cypermethrin at WHO-recommended dose and washed to just before cut-off point. The efficacy of MAGNet was evaluated in experimental huts against wild, free-flying, pyrethroid-resistant An. funestus. The WHO-susceptibility method was used to detect resistance in wild Anopheles exposed to 0.75% permethrin. Mosquito mortality, blood-feeding inhibition and personal protection were compared between untreated nets and standard LLINs. Blood-feeding rates were recorded and compared between the 20 times washed; blood-feeding rates between 20 times washed MAGNet LLIN and 20 times washed WHOPES-approved piperonyl butoxide (PBO)/pyrethroid were not statistically different (p > 0.05). RESULTS: The results have evidently shown that MAGNet LLIN provides similar blood-feeding inhibition, exophily, mortality, and deterrence to the standard approved LLIN, thus meeting the WHOPES criteria for blood feeding. The significantly high feeding inhibition and personal protection over pyrethroid-resistant An. funestus recorded by both unwashed and 20 times washed MAGNet compared to the unwashed DuraNet, the WHOPES-approved standard pyrethroid-only LLIN provides proof of MAGNet meeting Phase II WHOPES criteria for a LLIN. CONCLUSION: Based on this study, MAGNet has been shown to have a promising impact on protection when 20 times washed against a highly resistant population of An. funestus.