Multiple Anopheles species complicate downstream analysis and decision-making in a malaria pre-elimination area in southern Mozambique.

BACKGROUND: Different anopheline species (even within a species group/complex) can differ in their feeding and resting behaviours, which impact both malaria transmission patterns as well as the efficacy of vector control interventions. While morphological identification of sampled specimens is an important first step towards understanding species diversity and abundance, misidentification can result in the implementation of less effective vector control measures, and consequently smaller reductions in the number of local malaria cases. Focusing on southern Mozambique, a malaria pre-elimination area where malaria remains persistent, the aims of this preliminary study were to use molecular identification (CO1 and ITS2 barcoding) to (1) validate the results from the morphological identification (with a particular focus on Anopheles pharoensis and Anopheles squamosus), and (2) have a closer look at the Anopheles coustani group (which includes Anopheles tenebrosus and Anopheles ziemanni). METHODS: Female anopheline mosquitoes (n = 81) were identified morphologically and subsequently sequenced at the ribosomal DNA internal transcribed spacer region 2 (ITS2) and/or cytochrome oxidase subunit 1 (CO1) loci towards species determination. RESULTS: Out of the 62 specimens that were identified morphologically to species, 4 (6.5%) were misidentified. Regarding the An. coustani group, morphological identification showed that several members are present in southern Mozambique, including An. coustani sensu lato (s.l.), An. ziemanni and An. tenebrosus. However, based on both ITS2 and CO1 sequences, the exact species remains unknown for the latter two members until voucher sequences are available for comparison. CONCLUSION: The reason(s) for morphological misidentification of anopheline mosquitoes need to be mitigated. This is usually related to both the capacity (i.e. training) of the microscopist to identify anopheline species, and the information provided in the dichotomous identification key. As the An. coustani complex contributes to (residual) malaria transmission in sub-Saharan Africa, it may play a role in the observed persistent malaria in southern Mozambique. A better baseline characterizing of the local anophelines species diversity and behaviours will allow us to improve entomological surveillance strategies, better understand the impact of vector control on each local vector species, and identify new approaches to target those vector species.

Key considerations, target product profiles, and research gaps in the application of infrared spectroscopy and artificial intelligence for malaria surveillance and diagnosis.

Studies on the applications of infrared (IR) spectroscopy and machine learning (ML) in public health have increased greatly in recent years. These technologies show enormous potential for measuring key parameters of malaria, a disease that still causes about 250 million cases and 620,000 deaths, annually. Multiple studies have demonstrated that the combination of IR spectroscopy and machine learning (ML) can yield accurate predictions of epidemiologically relevant parameters of malaria in both laboratory and field surveys. Proven applications now include determining the age, species, and blood-feeding histories of mosquito vectors as well as detecting malaria parasite infections in both humans and mosquitoes. As the World Health Organization encourages malaria-endemic countries to improve their surveillance-response strategies, it is crucial to consider whether IR and ML techniques are likely to meet the relevant feasibility and cost-effectiveness requirements-and how best they can be deployed. This paper reviews current applications of IR spectroscopy and ML approaches for investigating malaria indicators in both field surveys and laboratory settings, and identifies key research gaps relevant to these applications. Additionally, the article suggests initial target product profiles (TPPs) that should be considered when developing or testing these technologies for use in low-income settings.

'We spray and walk away': wall modifications decrease the impact of indoor residual spray campaigns through reductions in post-spray coverage.

Malaria prevalence has significantly reduced since 2000, largely due to the scale-up of vector control interventions, mainly indoor residual spraying (IRS) and long-lasting insecticide-treated nets (LLINs). Given their success, these tools remain the frontline interventions in the fight against malaria. Their effectiveness relies on three key ingredients: the intervention, the mosquito vector and the end-user. Regarding the intervention, factors such as the insecticide active ingredient(s) used and the durability and/or bio-efficacy of the tool over time are critical. For the vectors, these factors include biting and resting behaviours and the susceptibility to insecticides. Finally, the end-users need to accept and properly use the intervention. Whilst human attitude and behaviour towards LLINs are well-documented both during and after distribution, only initial coverage is monitored for IRS and in a few geographic settings the residual efficacy of the used product. Here, the historical evidence on end-users modifying their wall surfaces post-spraying is presented, a behaviour that has the potential to reduce actual IRS coverage, effectiveness and impact, as fewer people are truly protected. Therefore, clear guidelines on how to monitor IRS acceptability and/or coverage, both before, during and after spraying, are urgently needed as part of the Monitoring and Evaluation of malaria programmes.

Household modifications after the indoor residual spraying (IRS) campaign in Mozambique reduce the actual spray coverage and efficacy.

Indoor residual spraying of insecticides (IRS) is a key malaria vector control strategy. Whilst human attitude towards IRS is monitored before or shortly after implementation, human activities leading to the modification of insecticide-treated walls post-IRS are not. This could inadvertently reduce the protective effects of IRS. We monitored the extent of modifications to the sprayed indoor wall surfaces by household owners for six months post-IRS campaigns in two districts targeted for malaria elimination in southern Mozambique. In parallel, we assessed building of any additional rooms onto compounds, and mosquito net use. We quantified the contribution of wall modifications, added rooms, prolonged spray campaigns, and product residual efficacies on actual IRS coverage and relative mosquito bite reduction, using a mechanistic approach. Household owners continually modified insecticide-treated walls and added rooms onto compounds. Household surveys in southern Mozambique showed frequent modification of indoor walls (0-17.2% of households modified rooms monthly) and/or added rooms (0-16.2% of households added rooms monthly). Actual IRS coverage reduced from an assumed 97% to just 39% in Matutuine, but only from 96% to 91% in Boane, translating to 43% and 5.8% estimated increases in relative daily mosquito bites per person. Integrating post-IRS knowledge, attitude, and practice (KAP) surveys into programmatic evaluations to capture these modification and construction trends can help improve IRS program efficiency and product assessment.

Scaling up of tsetse control to eliminate Gambian sleeping sickness in northern Uganda.

BACKGROUND: Tsetse flies (Glossina) transmit Trypanosoma brucei gambiense which causes Gambian human African trypanosomiasis (gHAT) in Central and West Africa. Several countries use Tiny Targets, comprising insecticide-treated panels of material which attract and kill tsetse, as part of their national programmes to eliminate gHAT. We studied how the scale and arrangement of target deployment affected the efficacy of control. METHODOLOGY AND PRINCIPAL FINDINGS: Between 2012 and 2016, Tiny Targets were deployed biannually along the larger rivers of Arua, Maracha, Koboko and Yumbe districts in North West Uganda with the aim of reducing the abundance of tsetse to interrupt transmission. The extent of these deployments increased from ~250 km2 in 2012 to ~1600 km2 in 2015. The impact of Tiny Targets on tsetse populations was assessed by analysing catches of tsetse from a network of monitoring traps; sub-samples of captured tsetse were dissected to estimate their age and infection status. In addition, the condition of 780 targets (~195/district) was assessed for up to six months after deployment. In each district, mean daily catches of tsetse (G. fuscipes fuscipes) from monitoring traps declined significantly by >80% following the deployment of targets. The reduction was apparent for several kilometres on adjacent lengths of the same river but not in other rivers a kilometre or so away. Expansion of the operational area did not always produce higher levels of suppression or detectable change in the age structure or infection rates of the population, perhaps due to the failure to treat the smaller streams and/or invasion from adjacent untreated areas. The median effective life of a Tiny Target was 61 (41.8-80.2, 95% CI) days. CONCLUSIONS: Scaling-up of tsetse control reduced the population of tsetse by >80% across the intervention area. Even better control might be achievable by tackling invasion of flies from infested areas within and outside the current intervention area. This might involve deploying more targets, especially along smaller rivers, and extending the effective life of Tiny Targets.

The realized efficacy of indoor residual spraying campaigns falls quickly below the recommended WHO threshold when coverage, pace of spraying and residual efficacy on different wall types are considered.

Indoor residual spraying (IRS) has been and remains an important malaria control intervention in southern Mozambique, South Africa and Eswatini. A better understanding of the effectiveness of IRS campaigns is critical to guide future elimination efforts. We analyze the three IRS campaigns conducted during a malaria elimination demonstration project in southern Mozambique, the "Magude project", and propose a new method to calculate the efficacy of IRS campaigns adjusting for IRS coverage, pace of house spraying and IRS residual efficacy on different wall types. Anopheles funestus sensu lato (s.l.) and An. gambiae s.l. were susceptible to pirimiphos-methyl and DDT. Anopheles funestus s.l. was resistant to pyrethroids, with 24h post-exposure mortality being lower for An. funestus sensu stricto (s.s.) than for An. parensis (collected indoors). The percentage of structures sprayed was above 90% and percentage of people covered above 86% in all three IRS campaigns. The percentage of households sprayed was above 83% in 2015 and 2016, but not assessed in 2017. Mosquito mortality 24h post-exposure stayed above 80% for 196 days after the 2016 IRS campaign and 222 days after the 2017 campaign and was 1.5 months longer on mud walls than on cement walls. This was extended by up to two months when 120h post-exposure mortality was considered. The district-level realized IRS efficacy was 113 days after the 2016 campaign. While the coverage of IRS campaigns in Magude were high, IRS protection did not remain optimal for the entire high malaria transmissions season. The use of a longer-lasting IRS product could have further supported the interruption of malaria transmission in the district. To better estimate the protection afforded by IRS campaigns, National Malaria Control Programs and partners are encouraged to adjust the calculation of IRS efficacy for IRS coverage, pace of house spraying during the campaign and IRS efficacy on different wall types combined with wall type distribution in the sprayed area.

Sub-lethal aquatic doses of pyriproxyfen may increase pyrethroid resistance in malaria mosquitoes.

BACKGROUND: Pyriproxyfen (PPF), an insect growth hormone mimic is widely used as a larvicide and in some second-generation bed nets, where it is combined with pyrethroids to improve impact. It has also been evaluated as a candidate for auto-dissemination by adult mosquitoes to control Aedes and Anopheles species. We examined whether PPF added to larval habitats of pyrethroid-resistant malaria vectors can modulate levels of resistance among emergent adult mosquitoes. METHODOLOGY: Third-instar larvae of pyrethroid-resistant Anopheles arabiensis (both laboratory-reared and field-collected) were reared in different PPF concentrations, between 1×10-9 milligrams active ingredient per litre of water (mgAI/L) and 1×10-4 mgAI/L, or no PPF at all. Emergent adults escaping these sub-lethal exposures were tested using WHO-standard susceptibility assays on pyrethroids (0.75% permethrin and 0.05% deltamethrin), carbamates (0.1% bendiocarb) and organochlorides (4% DDT). Biochemical basis of pyrethroid resistance was investigated by pre-exposure to 4% PBO. Bio-efficacies of long-lasting insecticide-treated nets, Olyset® and PermaNet 2.0 were also examined against adult mosquitoes with or without previous aquatic exposure to PPF. RESULTS: Addition of sub-lethal doses of PPF to larval habitats of pyrethroid-resistant An. arabiensis, consistently resulted in significantly reduced mortalities of emergent adults when exposed to pyrethroids, but not to bendiocarb or DDT. Mortality rates after exposure to Olyset® nets, but not PermaNet 2.0 were also reduced following aquatic exposures to PPF. Pre-exposure to PBO followed by permethrin or deltamethrin resulted in significant increases in mortality, compared to either insecticide alone. CONCLUSIONS: Partially-resistant mosquitoes exposed to sub-lethal aquatic concentrations of PPF may become more resistant to pyrethroids than they already are without such pre-exposures. Studies should be conducted to examine whether field applications of PPF, either by larviciding or other means actually exacerbates pyrethroid-resistance in areas where signs of such resistance already exist in wild the vector populations. The studies should also investigate mechanisms underlying such magnification of resistance, and how this may impact the potential of PPF-based interventions in areas with pyrethroid resistance.

Effect of wall type, delayed mortality and mosquito age on the residual efficacy of a clothianidin-based indoor residual spray formulation (SumiShield™ 50WG) in southern Mozambique.

Indoor residual spraying (IRS) is one of the main malaria vector control strategies in Mozambique alongside the distribution of insecticide treated nets. As part of the national insecticide resistance management strategy, Mozambique introduced SumiShield™ 50WG, a third generation IRS product, in 2018. Its residual efficacy was assessed in southern Mozambique during the 2018-2019 malaria season. Using a susceptible Anopheles arabiensis strain, residual efficacy was assessed on two different wall surfaces, cement and mud-plastered walls, using standard WHO (World Health Organization) cone bioassay tests at three different heights. Female mosquitoes of two age groups (2-5 and 13-26 day old) were exposed for 30 minutes, after which mortality was observed 24h, 48h, 72h, and 96h and 120h post-exposure to assess (delayed) mortality. Lethal times (LT) 90, LT50 and LT10 were estimated using Bayesian models. Mortality 24h post exposure was consistently below 80%, the current WHO threshold value for effective IRS, in both young and old mosquitoes, regardless of wall surface type. Considering delayed mortality, residual efficacies (mosquito mortality equal or greater than 80%) ranged from 1.5 to ≥12.5 months, with the duration depending on mortality time post exposure, wall type and mosquito age. Looking at mortality 72h after exposure, residual efficacy was between 6.5 and 9.5 months, depending on wall type and mosquito age. The LT50 and LT10 (i.e. 90% of the mosquitoes survive exposure to the insecticides) values were consistently higher for older mosquitoes (except for LT10 values for 48h and 72h post-exposure mortality) and ranged from 0.9 to 5.8 months and 0.2 to 7.8 months for LT50 and LT10, respectively. The present study highlights the need for assessing mosquito mortality beyond the currently recommended 24h post exposure. Failure to do so may lead to underestimation of the residual efficacy of IRS products, as delayed mortality will lead to a further reduction in mosquito vector populations and potentially negatively impact disease transmission. Monitoring residual efficacy on relevant wall surfaces, including old mosquitoes that are ultimately responsible for malaria transmission, and assessing delayed mortalities are critical to provide accurate and actionable data to guide vector control programmes.

Evidence of the absence of human African trypanosomiasis in two northern districts of Uganda: Analyses of cattle, pigs and tsetse flies for the presence of Trypanosoma brucei gambiense.

BACKGROUND: Large-scale control of sleeping sickness has led to a decline in the number of cases of Gambian human African trypanosomiasis (g-HAT) to <2000/year. However, achieving complete and lasting interruption of transmission may be difficult because animals may act as reservoir hosts for T. b. gambiense. Our study aims to update our understanding of T. b. gambiense in local vectors and domestic animals of N.W. Uganda. METHODS: We collected blood from 2896 cattle and 400 pigs and In addition, 6664 tsetse underwent microscopical examination for the presence of trypanosomes. Trypanosoma species were identified in tsetse from a subsample of 2184 using PCR. Primers specific for T. brucei s.l. and for T. brucei sub-species were used to screen cattle, pig and tsetse samples. RESULTS: In total, 39/2,088 (1.9%; 95% CI = 1.9-2.5) cattle, 25/400 (6.3%; 95% CI = 4.1-9.1) pigs and 40/2,184 (1.8%; 95% CI = 1.3-2.5) tsetse, were positive for T. brucei s.l.. Of these samples 24 cattle (61.5%), 15 pig (60%) and 25 tsetse (62.5%) samples had sufficient DNA to be screened using the T. brucei sub-species PCR. Further analysis found no cattle or pigs positive for T. b. gambiense, however, 17/40 of the tsetse samples produced a band suggestive of T. b. gambiense. When three of these 17 PCR products were sequenced the sequences were markedly different to T. b. gambiense, indicating that these flies were not infected with T. b. gambiense. CONCLUSION: The lack of T. b. gambiense positives in cattle, pigs and tsetse accords with the low prevalence of g-HAT in the human population. We found no evidence that livestock are acting as reservoir hosts. However, this study highlights the limitations of current methods of detecting and identifying T. b. gambiense which relies on a single copy-gene to discriminate between the different sub-species of T. brucei s.l.

Evaluation of a simple polytetrafluoroethylene (PTFE)-based membrane for blood-feeding of malaria and dengue fever vectors in the laboratory.

BACKGROUND: Controlled blood-feeding is essential for maintaining laboratory colonies of disease-transmitting mosquitoes and investigating pathogen transmission. We evaluated a low-cost artificial feeding (AF) method, as an alternative to direct human feeding (DHF), commonly used in mosquito laboratories. METHODS: We applied thinly-stretched pieces of polytetrafluoroethylene (PTFE) membranes cut from locally available seal tape (i.e. plumbers tape, commonly used for sealing pipe threads in gasworks or waterworks). Approximately 4 ml of bovine blood was placed on the bottom surfaces of inverted Styrofoam cups and then the PTFE membranes were thinly stretched over the surfaces. The cups were filled with boiled water to keep the blood warm (~37 °C), and held over netting cages containing 3-4 day-old inseminated adults of female Aedes aegypti, Anopheles gambiae (s.s.) or Anopheles arabiensis. Blood-feeding success, fecundity and survival of mosquitoes maintained by this system were compared against DHF. RESULTS: Aedes aegypti achieved 100% feeding success on both AF and DHF, and also similar fecundity rates (13.1 ± 1.7 and 12.8 ± 1.0 eggs/mosquito respectively; P > 0.05). An. arabiensis had slightly lower feeding success on AF (85.83 ± 16.28%) than DHF (98.83 ± 2.29%) though these were not statistically different (P > 0.05), and also comparable fecundity between AF (8.82 ± 7.02) and DHF (8.02 ± 5.81). Similarly, for An. gambiae (s.s.), we observed a marginal difference in feeding success between AF (86.00 ± 10.86%) and DHF (98.92 ± 2.65%), but similar fecundity by either method. Compared to DHF, mosquitoes fed using AF survived a similar number of days [Hazard Ratios (HR) for Ae. aegypti = 0.99 (0.75-1.34), P > 0.05; An. arabiensis = 0.96 (0.75-1.22), P > 0.05; and An. gambiae (s.s.) = 1.03 (0.79-1.35), P > 0.05]. CONCLUSIONS: Mosquitoes fed via this simple AF method had similar feeding success, fecundity and longevity. The method could potentially be used for laboratory colonization of mosquitoes, where DHF is unfeasible. If improved (e.g. minimizing temperature fluctuations), the approach could possibly also support studies where vectors are artificially infected with blood-borne pathogens.

Field evaluation of the BG-Malaria trap for monitoring malaria vectors in rural Tanzanian villages.

BG-Malaria (BGM) trap is a simple adaptation of the widely-used BG-Sentinel trap (BGS). It is proven to be highly effective for trapping the Brazilian malaria vector, Anopheles darlingi, in field conditions, and the African vector, Anopheles arabiensis, under controlled semi-field environments, but has not been field-tested in Africa. Here, we validated the BGM for field sampling of malaria vectors in south-eastern Tanzania. Using a series of Latin-Square experiments conducted nightly (6pm-7am) in rural villages, we compared mosquito catches between BGM, BGS and human landing catches (HLC). We also compared BGMs baited with different attractants (Ifakara-blend, Mbita-blend, BG-Lure and CO2). Lastly, we tested BGMs baited with Ifakara-blend from three odour-dispensing methods (BG-Cartridge, BG-Sachet and Nylon strips). One-tenth of the field-collected female Anopheles gambiae s.l. and Anopheles funestus were dissected to assess parity. BGM captured more An. gambiae s.l. than BGS (p < 0.001), but HLC caught more than either trap (p < 0.001). However, BGM captured more An. funestus than HLC. Proportions of parous An. gambiae s.l. and An. funestus consistently exceeded 50%, with no significant difference between methods. While the dominant species caught by HLC was An. gambiae s.l. (56.0%), followed by Culex spp. (33.1%) and Mansonia spp. (6.0%), the BGM caught mostly Culex (81.6%), followed by An. gambiae s.l. (10.6%) and Mansonia (5.8%). The attractant-baited BGMs were all significantly superior to un-baited controls (p < 0.001), although no difference was found between the specific attractants. The BG-Sachet was the most efficient dispenser for capturing An. gambiae s.l. (14.5(2.75-42.50) mosquitoes/trap/night), followed by BG-Cartridge (7.5(1.75-26.25)). The BGM caught more mosquitoes than BGS in field-settings, but sampled similar species diversity and physiological states as BGS. The physiological states of malaria vectors caught in BGM and BGS were similar to those naturally attempting to bite humans (HLC). The BGM was most efficient when baited with Ifakara blend, dispensed from BG-Sachet. We conclude that though BGM traps have potential for field-sampling of host-seeking African malaria vectors with representative physiological states, both BGM and BGS predominantly caught more culicines than Anopheles, compared to HLC, which caught mostly An. gambiae s.l.

Semi-field assessment of the BG-Malaria trap for monitoring the African malaria vector, Anopheles arabiensis.

Odour-baited technologies are increasingly considered for effective monitoring of mosquito populations and for the evaluation of vector control interventions. The BG-Malaria trap (BGM), which is an upside-down variant of the widely used BG-Sentinel trap (BGS), has been demonstrated to be effective to sample the Brazilian malaria vector, Anopheles darlingi. We evaluated the BGM as an improved method for sampling the African malaria vectors, Anopheles arabiensis. Experiments were conducted inside a large semi-field cage to compare trapping efficiencies of BGM and BGS traps, both baited with the synthetic attractant, Ifakara blend, supplemented with CO2. We then compared BGMs baited with either of four synthetic mosquito lures, Ifakara blend, Mbita blend, BG-lure or CO2, and an unbaited BGM. Lastly, we compared BGMs baited with the Ifakara blend dispensed via either nylon strips, BG cartridges (attractant-infused microcapsules encased in cylindrical plastic cartridge) or BG sachets (attractant-infused microcapsules encased in plastic sachets). All tests were conducted between 6P.M. and 7A.M., with 200-600 laboratory-reared An. arabiensis released nightly in the test chamber. The median number of An. arabiensis caught by the BGM per night was 83, IQR:(73.5-97.75), demonstrating clear superiority over BGS (median catch = 32.5 (25.25-37.5)). Compared to unbaited controls, BGMs baited with Mbita blend caught most mosquitoes (45 (29.5-70.25)), followed by BGMs baited with CO2 (42.5 (27.5-64)), Ifakara blend (31 (9.25-41.25)) and BG lure (16 (4-22)). BGM caught 51 (29.5-72.25) mosquitoes/night, when the attractants were dispensed using BG-Cartridges, compared to BG-Sachet (29.5 (24.75-40.5)), and nylon strips (27 (19.25-38.25)), in all cases being significantly superior to unbaited controls (p < 000.1). The findings demonstrate potential of the BGM as a sampling tool for African malaria vectors over the standard BGS trap. Its efficacy can be optimized by selecting appropriate odour baits and odour-dispensing systems.

Using Stable Isotopes of Carbon and Nitrogen to Mark Wild Populations of Anopheles and Aedes Mosquitoes in South-Eastern Tanzania.

BACKGROUND: Marking wild mosquitoes is important for understanding their ecology, behaviours and role in disease transmission. Traditional insect marking techniques include using fluorescent dyes, protein labels, radioactive labels and tags, but such techniques have various limitations; notably low marker retention and inability to mark wild mosquitoes at source. Stable isotopes are gaining wide spread use for non-invasive marking of arthropods, permitting greater understanding of mosquito dispersal and responses to interventions. We describe here a simple technique for marking naturally-breeding malaria and dengue vectors using stable isotopes of nitrogen (15N) and carbon (13C), and describe potential field applications. METHODS: We created man-made aquatic mosquito habitats and added either 15N-labelled potassium nitrate or 13C-labelled glucose, leaving non-adulterated habitats as controls. We then allowed wild mosquitoes to lay eggs in these habitats and monitored their development in situ. Pupae were collected promptly as they appeared and kept in netting cages. Emergent adults (in pools of ~4 mosquitoes/pool) and individually stored pupae were desiccated and analysed using Isotope Ratio Mass Spectrometry (IRMS). FINDINGS: Anopheles gambiae s.l and Aedes spp. from enriched 13C and enriched 15N larval habitats had significantly higher isotopic levels than controls (P = 0.005), and both isotopes produced sufficient distinction between marked and unmarked mosquitoes. Mean δ15N for enriched females and males were 275.6±65.1 and 248.0±54.6, while mean δ15N in controls were 2.1±0.1 and 3.9±1.7 respectively. Similarly, mean δ13C for enriched females and males were 36.08±5.28 and 38.5±6.86, compared to -4.3±0.2 and -7.9±3.6 in controls respectively. Mean δ15N and δ13C was significantly higher in any pool containing at least one enriched mosquito compared to pools with all unenriched mosquitoes, P<0.001. In all cases, there were variations in standardized isotopic ratios between mosquito species. CONCLUSION: Enrichment of semi-natural mosquito larval habitats with stable isotopes of nitrogen and carbon resulted in effective marking of Anopheles and Aedes mosquitoes colonizing these habitats. This approach can significantly enhance studies on mosquito eco-physiology, dispersal, pathogen transmission and responses to control measures.

Effect of wall type, delayed mortality and mosquito age on the residual efficacy of a clothianidin-based indoor residual spray formulation (SumiShield™ 50WG) in southern Mozambique

Indoor residual spraying (IRS) is one of the main malaria vector control strategies in Mozambique alongside the distribution of insecticide treated nets. As part of the national insecticide resistance management strategy, Mozambique introduced SumiShield™ 50WG, a third generation IRS product, in 2018. Its residual efficacy was assessed in southern Mozambique during the 2018­2019 malaria season. Using a susceptible Anopheles arabiensis strain, residual efficacy was assessed on two different wall surfaces, cement and mud-plastered walls, using standard WHO (World Health Organization) cone bioassay tests at three different heights. Female mosquitoes of two age groups (2­5 and 13­26 day old) were exposed for 30 minutes, after which mortality was observed 24h, 48h, 72h, and 96h and 120h post-exposure to assess (delayed) mortality. Lethal times (LT) 90, LT50 and LT10 were estimated using Bayesian models. Mortality 24h post exposure was consistently below 80%, the current WHO threshold value for effective IRS, in both young and old mosquitoes, regardless of wall surface type. Considering delayed mortality, residual efficacies (mosquito mortality equal or greater than 80%) ranged from 1.5 to ≥12.5 months, with the duration depending on mortality time post exposure, wall type and mosquito age. Looking at mortality 72h after exposure, residual efficacy was between 6.5 and 9.5 months, depending on wall type and mosquito age. The LT50 and LT10 (i.e. 90% of the mosquitoes survive exposure to the insecticides) values were consistently higher for older mosquitoes (except for LT10 values for 48h and 72h post-exposure mortality) and ranged from 0.9 to 5.8 months and 0.2 to 7.8 months for LT50 and LT10, respectively. The present study highlights the need for assessing mosquito mortality beyond the currently recommended 24h post exposure. Failure to do so may lead to underestimation of the residual efficacy of IRS products, as delayed mortality will lead to a further reduction in mosquito vector populations and potentially negatively impact disease transmission. Monitoring residual efficacy on relevant wall surfaces, including old mosquitoes that are ultimately responsible for malaria transmission, and assessing delayed mortalities are critical to provide accurate and actionable data to guide vector control programmes.