Anopheles funestus is playing an increasing role in malaria transmission in parts of sub-Saharan Africa, where An. gambiae s.s. has been effectively controlled by long-lasting insecticidal nets. We investigated vector population bionomics, insecticide resistance and malaria transmission dynamics in 86 study clusters in North-West Tanzania. An. funestus s.l. represented 94.5% (4740/5016) of all vectors and was responsible for the majority of malaria transmission (96.5%), with a sporozoite rate of 3.4% and average monthly entomological inoculation rate (EIR) of 4.57 per house. Micro-geographical heterogeneity in species composition, abundance and transmission was observed across the study district in relation to key ecological differences between northern and southern clusters, with significantly higher densities, proportions and EIR of An. funestus s.l. collected from the South. An. gambiae s.l. (5.5%) density, principally An. arabiensis (81.1%) and An. gambiae s.s. (18.9%), was much lower and closely correlated with seasonal rainfall. Both An. funestus s.l. and An. gambiae s.l. were similarly resistant to alpha-cypermethrin and permethrin. Overexpression of CYP9K1, CYP6P3, CYP6P4 and CYP6M2 and high L1014S-kdr mutation frequency were detected in An. gambiae s.s. populations. Study findings highlight the urgent need for novel vector control tools to tackle persistent malaria transmission in the Lake Region of Tanzania.

Background The spread of pyrethroid resistance in malaria vectors threatens the effectiveness of standard long-lasting insecticidal nets (LLIN). Synergist nets combine pyrethroid (Py) and piperonyl-butoxide (PBO) to enhance potency against resistance mediated by mono-oxygenase mechanisms. Our project assessed personal protection of the World Health Organization first-in-class PBO-Py LLIN (Olyset Plus) versus the standard LLIN (Olyset net) against pyrethroid-resistant Anopheles gambiae sensu lato (s.l.) and An. funestus in North-West Tanzania after 20 months of household use. Methods From a household survey, 39 standard Olyset net and 39 Olyset Plus houses were selected. The physical integrity and hole index (HI) of the nets were assessed, and resting mosquitoes were collected from inside nets and from room walls. The indoor abundance was estimated using CDC light traps and species identified using PCR. The bioefficacy of PBO and standard LLINs against wild Anopheles was assessed using 30-minute cylinder bioassays. Results Of 2397 Anopheles collected, 8.9% (n = 213) were resting inside standard Olyset nets, while none were found inside Olyset Plus nets (PBO-Py LLINs) of any HI category. Resting density of blood-fed mosquitoes was higher on walls of sleeping rooms with Olyset nets compared to Olyset Plus (0.62 vs 0.10, density ratio [DR]: 0.03, 95% CI 0.01–0.13, p < 0.001). Mosquitoes were found inside Olyset nets of all WHO HI categories, but more were collected inside the more damaged nets (HI ≥ 643) than in less damaged (HI 0–64) nets (DR: 6.4, 95% CI 1.1–36.0, p = 0.037). In bioassay, mortality of An. gambiae s.l. was higher with Olyset Plus than with Olyset nets for new nets (76.8% vs 27.5%) and nets used for 20 months (56.8% vs 12.8%); similar trends were observed with An. funestus. Conclusion The PBO-Py LLINs provided improved protection after 20 months of household use, as demonstrated by the higher bioassay mortality and absence of pyrethroid-resistant An. gambiae sensu stricto (s.s.) and An. funestus collected from inside Olyset Plus nets, irrespective of HI category, as compared to Olyset nets.

In response to growing concerns over the sustained effectiveness of pyrethroid-only based control tools, new products are being developed and evaluated. Some examples of these are dual-active ingredient (AI) insecticide-treated nets (ITNs) which contain secondary insecticides, or synergist ITNs which contain insecticide synergist, both in combination with a pyrethroid. These net types are often termed ‘next-generation’ insecticide-treated nets. Several of these new types of ITNs are being evaluated in large-scale randomized control trials (RCTs) and pilot deployment schemes at a country level. However, no methods for measuring the biological durability of the AIs or synergists on these products are currently recommended. In this publication, we describe a pipeline used to collate and interrogate several different methods to produce a singular ‘consensus standard operating procedure (SOP)’, for monitoring the biological durability of three new types of ITNs: pyrethroid + piperonyl butoxide (PBO), pyrethroid + pyriproxyfen (PPF), and pyrethroid + chlorfenapyr (CFP). This process, convened under the auspices of the Innovation to Impact programme, sought to align methodologies used for conducting durability monitoring activities of next-generation ITNs.

Background Progress achieved by long-lasting insecticidal nets (LLINs) against malaria is threatened by widespread selection of pyrethroid resistance among vector populations. LLINs with non-pyrethroid insecticides are urgently needed. This study aims to assess the insecticide and textile durability of three classes of dual-active ingredient (A.I.) LLINs using techniques derived from established WHO LLIN testing methods to set new standards of evaluation. Methods A WHO Phase 3 active ingredients and textile durability study will be carried out within a cluster randomized controlled trial in 40 clusters in Misungwi district, Tanzania. The following treatments will be evaluated: (1) Interceptor®G2 combining chlorfenapyr and the pyrethroid alpha-cypermethrin, (2) Royal Guard® treated with pyriproxyfen and alpha-cypermethrin, (3) Olyset™ Plus which incorporates a synergist piperonyl butoxide and the pyrethroid permethrin, and (4) a reference standard alpha-cypermethrin only LLIN (Interceptor®). 750 nets will be followed in 5 clusters per intervention arm at 6, 12, 24 and 36 months post distribution for survivorship and hole index assessment. A second cohort of 1950 nets per net type will be identified in 10 clusters, of which 30 LLINs will be withdrawn for bio-efficacy and chemical analysis every 6 months up to 36 months and another 30 collected for experimental hut trials every year. Bio-efficacy will be assessed using cone bioassays and tunnel tests against susceptible and resistant laboratory strains of Anopheles gambiae sensu stricto. Efficacy of field-collected nets will be compared in six experimental huts. The main outcomes will be Anopheles mortality up to 72 h post exposure, blood feeding and egg maturation using ovary dissection to assess impact on fecundity. Conclusions Study findings will help develop bio-efficacy and physical durability criteria for partner A.I., in relation to the cRCT epidemiological and entomological outcomes, and refine preferred product characteristics of each class of LLIN. If suitable, the bioassay and hut outcomes will be fitted to transmission models to estimate correlation with cRCT outcomes. Trial registration number: NCT03554616.

Background Vector control through long-lasting insecticidal nets (LLINs) and focal indoor residual spraying (IRS) is a major component of the Tanzania national malaria control strategy. In mainland Tanzania, IRS has been conducted annually around Lake Victoria basin since 2007. Due to pyrethroid resistance in malaria vectors, use of pyrethroids for IRS was phased out and from 2014 to 2017 pirimiphos-methyl (Actellic® 300CS) was sprayed in regions of Kagera, Geita, Mwanza, and Mara. Entomological surveillance was conducted in 10 sprayed and 4 unsprayed sites to determine the impact of IRS on entomological indices related to malaria transmission risk. Methods WHO cone bioassays were conducted monthly on interior house walls to determine residual efficacy of pirimiphos-methyl CS. Indoor CDC light traps with or without bottle rotator were hung next to protected sleepers indoors and also set outdoors (unbaited) as a proxy measure for indoor and outdoor biting rate and time of biting. Prokopack aspirators were used indoors to capture resting malaria vectors. A sub-sample of Anopheles was tested by PCR to determine species identity and ELISA for sporozoite rate. Results Annual IRS with Actellic® 300CS from 2015 to 2017 was effective on sprayed walls for a mean of 7 months in cone bioassay. PCR of 2016 and 2017 samples showed vector populations were predominantly Anopheles arabiensis (58.1%, n = 4,403 IRS sites, 58%, n = 2,441 unsprayed sites). There was a greater proportion of Anopheles funestus sensu stricto in unsprayed sites (20.4%, n = 858) than in sprayed sites (7.9%, n = 595) and fewer Anopheles parensis (2%, n = 85 unsprayed, 7.8%, n = 591 sprayed). Biting peaks of Anopheles gambiae sensu lato (s.l.) followed periods of rainfall occurring between October and April, but were generally lower in sprayed sites than unsprayed. In most sprayed sites, An. gambiae s.l. indoor densities increased between January and February, i.e., 10–12 months after IRS. The predominant species An. arabiensis had a sporozoite rate in 2017 of 2.0% (95% CI 1.4–2.9) in unsprayed sites compared to 0.8% (95% CI 0.5–1.3) in sprayed sites (p = 0.003). Sporozoite rates were also lower for An. funestus collected in sprayed sites. Conclusion This study contributes to the understanding of malaria vector species composition, behaviour and transmission risk following IRS around Lake Victoria and can be used to guide malaria vector control strategies in Tanzania.

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Background: Vector control through long lasting insecticidal nets and focal indoor residual spraying (IRS) is a major component of the Tanzania national malaria control strategy. In mainland Tanzania, IRS has been conducted annually around Lake Victoria basin since 2007. Due to pyrethroid resistance in malaria vectors, use of pyrethroids for IRS was phased out and from 2014 to 2017 pirimiphos-methyl (Actellic® 300CS) was sprayed in regions of Kagera, Geita, Mwanza and Mara. Entomological surveillance was conducted in ten sprayed and four unsprayed sites to determine the impact of IRS on entomological indices related to malaria transmission risk. Methods: WHO Cone bioassays were conducted monthly on interior house walls to determine residual efficacy of pirimiphos-methyl CS. Indoor CDC light traps with or without bottle rotator were hung next to protected sleepers indoors and also set outdoors (un-baited) as a proxy measure for indoor and outdoor biting rate and time of biting. Prokopack aspirators were used indoors to capture potentially resting malaria vector. A sub-sample of Anopheles were tested by PCR to determine species identity and ELISA for sporozoite rate. Results: Annual IRS with Actellic® 300CS from 2015 to 2017 was effective on sprayed walls for a mean of 7 months in cone bioassay. PCR of 2016 and 2017 samples showed vector populations were predominantly An. arabiensis (58.1%, n=4,403 IRS sites, 58%, n=2,441 unsprayed sites). There was a greater proportion of An. funestus s.s. in unsprayed sites (20.4%, n=858) than sprayed sites (7.9%, n=595) and fewer An. parensis (2%, n=85 unsprayed, 7.8%, n=591 sprayed). Biting peaks of An. gambiae s.l. followed periods of rainfall occurring between October and April, but were generally lower in sprayed sites than unsprayed. In most sprayed sites, An. gambiae s.l. indoor densities increased between January and February, i.e. 10-12 months after IRS. The predominant species An. arabiensis had a sporozoite rate in 2017 of 2.0% (95% CI: 1.4-2.9) in unsprayed sites compared to 0.8% (95% CI: 0.5-1.3) in sprayed sites (p=0.003). Sporozoite rates were also lower for An. funestus collected in sprayed sites. Conclusion: This study contributes to the understanding of malaria vector species composition, behavior and transmission risk following IRS around Lake Victoria and can be used to guide malaria vector control strategies in Tanzania.

Anopheles funestus is playing an increasing role in malaria transmission in parts of sub-Saharan Africa, where An. gambiae s.s. has been effectively controlled by long-lasting insecticidal nets. We investigated vector population bionomics, insecticide resistance and malaria transmission dynamics in 86 study clusters in North-West Tanzania. An. funestus s.l. represented 94.5% (4740/5016) of all vectors and was responsible for the majority of malaria transmission (96.5%), with a sporozoite rate of 3.4% and average monthly entomological inoculation rate (EIR) of 4.57 per house. Micro-geographical heterogeneity in species composition, abundance and transmission was observed across the study district in relation to key ecological differences between northern and southern clusters, with significantly higher densities, proportions and EIR of An. funestus s.l. collected from the south. An. gambiae s.l. (5.5%) density, principally An. arabiensis (81.1%) and An. gambiae s.s. (18.9%), was much lower and closely correlated with seasonal rainfall. Both An. funestus s.l. and An. gambiae s.l. were similarly resistant to alpha-cypermethrin and permethrin. Overexpression of CYP9K1, CYP6P3, CYP6P4 and CYP6M2 and high L1014F-kdr mutation frequency were detected in An. gambiae s.s. populations. Study findings highlight the urgent need for novel vector control tools to tackle persistent malaria transmission in the Lake Region of Tanzania.