Evaluation of the DN-Mini (miniaturized double net) trap for sampling host-seeking Anopheles mosquitoes in malaria-endemic villages of southern Tanzania.

BACKGROUND: Surveillance of malaria vectors is crucial for assessing the transmission risk and impact of control measures. Human landing catches (HLC) directly estimate the biting rates but raise ethical concerns due to the exposure of volunteers to mosquito-borne pathogens. A common alternative is the CDC-light trap, which is effective for catching host-seeking mosquitoes indoors but not outdoors. New, exposure-free methods are needed for sampling mosquitoes indoors and outdoors in ways that reflect their natural risk profiles. The aim of this study was therefore to evaluate the efficacy of the miniaturized double net trap (DN-Mini) for sampling host-seeking mosquitoes in south-eastern Tanzania, where malaria transmission is dominated by Anopheles funestus. METHODS: Adult mosquitoes were collected from 222 randomly selected houses across three villages (74 per village) in Ulanga district, south-eastern Tanzania, using the DN-Mini traps, CDC-Light traps, and Prokopack aspirators. First, we compared CDC-light and DN-Mini traps for collecting indoor host-seeking mosquitoes, while Prokopack aspirators were used for indoor-resting mosquitoes. Second, we deployed the DN-Mini and Prokopack aspirators to collect host-seeking and resting mosquitoes indoors and outdoors. Generalized linear mixed models (GLMM) with a negative binomial distribution were used to compare the effectiveness of the traps for catching different mosquito species. RESULTS: The DN-Mini was 1.53 times more efficient in collecting An. funestus indoors (RR = 1.53, 95% CI: 1.190-1.98) compared to the CDC-Light trap. However, for Anopheles arabiensis, the DN-Mini caught only 0.32 times as many mosquitoes indoors as the CDC-Light traps (RR = 0.32, 95% CI: 0.183-0.567). Both An. funestus and An. arabiensis were found to be more abundant indoors than outdoors when collected using the DN-Mini trap. Similarly, the Prokopack aspirator was greater indoors than outdoors for both An. funestus and An. arabiensis. CONCLUSION: The DN-Mini outperformed the CDC-light trap in sampling the dominant malaria vector, An. funestus species, but was less effective in capturing An. arabiensis, and for both vector species, the biting risk was greater indoors than outdoors when measured using the DN-Mini trap. These findings highlight the importance of selecting appropriate trapping methods based on mosquito species and behaviors.

A statistical calibration tool for methods used to sample outdoor-biting mosquitoes.

BACKGROUND: Improved methods for sampling outdoor-biting mosquitoes are urgently needed to improve surveillance of vector-borne diseases. Such tools could potentially replace the human landing catch (HLC), which, despite being the most direct option for measuring human exposures, raises significant ethical and logistical concerns. Several alternatives are under development, but detailed evaluation still requires common frameworks for calibration relative to HLC. The aim of this study was to develop and validate a statistical framework for predicting human-biting rates from different exposure-free alternatives. METHODS: We obtained mosquito abundance data (Anopheles arabiensis, Anopheles funestus and Culex spp.) from a year-long Tanzanian study comparing six outdoor traps [Suna Trap (SUN), BG Sentinel (BGS), M-Trap (MTR), M-Trap + CDC (MTRC), Ifakara Tent Trap-C (ITT-C) and Mosquito Magnet-X Trap (MMX)] and HLC. Generalised linear models were developed within a Bayesian framework to investigate associations between the traps and HLC, taking intra- and inter-specific density dependence into account. The best model was used to create a calibration tool for predicting HLC-equivalents. RESULTS: For An. arabiensis, SUN catches had the strongest correlation with HLC (R2 = 19.4), followed by BGS (R2 = 17.2) and MTRC (R2 = 13.1) catches. The least correlated catch was MMX (R2 = 2.5). For An. funestus, BGS had the strongest correlation with the HLC (R2 = 53.4), followed by MTRC (R2 = 37.4) and MTR (R2 = 37.4). For Culex mosquitoes, the traps most highly correlated with the HLC were MTR (R2 = 45.4) and MTRC (R2 = 44.2). Density dependence, both between and within species, influenced the performance of only BGS traps. An interactive Shiny App calibration tool was developed for this and similar applications. CONCLUSION: We successfully developed a calibration tool to assess the performance of different traps for assessing outdoor-biting risk, and established a valuable framework for estimating human exposures based on the trap catches. The performance of candidate traps varied between mosquito taxa; thus, there was no single optimum. Although all the traps tested underestimated the HLC-derived exposures, it was possible to mathematically define their representativeness of the true biting risk, with or without density dependence. The results of this study emphasise the need to aim for a consistent and representative sampling approach, as opposed to simply seeking traps that catch the most mosquitoes.

Using pastoralist community knowledge to locate and treat dry-season mosquito breeding habitats with pyriproxyfen to control Anopheles gambiae s.l. and Anopheles funestus s.l. in rural Tanzania.

Fundamentally, larviciding with pyriproxyfen (PPF) has potential to complement Long Lasting Insecticide Nets (LLINs) and indoor residual sprays (IRS) in settings where resistance to pyrethroids and residual malaria transmission exist. In this study, we evaluated the field effectiveness of larviciding using PPF to reduce dry season productivity of mosquito breeding habitats that were located by pastoralists within the study area. Using pastoralist knowledge, dry season breeding habitats in Mofu village rural Tanzania were located and monitored for larval productivity for a period of 8 months before PPF intervention. During the intervention, six out of twelve breeding habitats were treated with Sumilarv 0.5G PPF granules. The impact of deposited PPF was monitored by recording emergence inhibition of larvae collected from treated habitats compared to the appropriate control group for a period of three months and half post-intervention. During baseline, the average proportion (+SD) of adult emerged was similar between two clusters, with (0.89 + 0.22) for the control cluster and (0.93 + 0.16) for the treatment cluster of breeding habitats. Following treatment with PPF, the average proportion (+SD) of adult emerged in the treated breeding habitats was significantly low (0.096 + 0.22) compared to adults that emerged from larvae in the untreated habitats (0.99 + 0.22) (p < 0.0001). Of all emerged adults, approximately 94% were An. gambiae s.l. and the remaining 6% were An. funestus s.l. This is the first study demonstrating the usefulness of engaging pastoralist community to locate and identify hard to find mosquito breeding habitats. Reduced productivity of the targeted habitats with PPF offers prospect of implementing PPF larviciding in dry season when habitats are few and permanent to control mosquito population in rural settings.

Habitat characteristics and insecticide susceptibility of Aedes aegypti in the Ifakara area, south-eastern Tanzania.

BACKGROUND: Aedes-borne diseases such as dengue and chikungunya constitute constant threats globally. In Tanzania, these diseases are transmitted by Aedes aegypti, which is widely distributed in urban areas, but whose ecology remains poorly understood in small towns and rural settings. METHODS: A survey of Ae. aegypti aquatic habitats was conducted in and around Ifakara, a fast-growing town in south-eastern Tanzania. The study area was divided into 200 × 200 m search grids, and habitats containing immature Aedes were characterized. Field-collected Ae. aegypti were tested for susceptibility to common public health insecticides (deltamethrin, permethrin, bendiocarb and pirimiphos-methyl) in the dry and rainy seasons. RESULTS: Of 1515 and 1933 aquatic habitats examined in the dry and rainy seasons, 286 and 283 contained Aedes immatures, respectively (container index, CI: 18.9-14.6%). In the 2315 and 2832 houses visited in the dry and rainy seasons, 114 and 186 houses had at least one Aedes-positive habitat, respectively (house index, HI: 4.9-6.6%). The main habitat types included: (i) used vehicle tires and discarded containers; (ii) flowerpots and clay pots; and (iii) holes made by residents on trunks of coconut trees when harvesting the coconuts. Used tires had highest overall abundance of Ae. aegypti immatures, while coconut tree-holes had highest densities per habitat. Aedes aegypti adults were susceptible to all tested insecticides in both seasons, except bendiocarb, against which resistance was observed in the rainy season. CONCLUSIONS: To our knowledge, this is the first study on ecology and insecticide susceptibility of Ae. aegypti in Ifakara area, and will provide a basis for future studies on its pathogen transmission activities and control. The high infestation levels observed indicate significant risk of Aedes-borne diseases, requiring immediate action to prevent potential outbreaks in the area. While used tires, discarded containers and flowerpots are key habitats for Ae. aegypti, this study also identified coconut harvesting as an important risk factor, and the associated tree-holes as potential targets for Aedes control. Since Ae. aegypti mosquitoes in the area are still susceptible to most insecticides, effective control could be achieved by combining environmental management, preferably involving communities, habitat removal and insecticide spraying.

Protecting migratory farmers in rural Tanzania using eave ribbons treated with the spatial mosquito repellent, transfluthrin.

BACKGROUND: Many subsistence farmers in rural southeastern Tanzania regularly relocate to distant farms in river valleys to tend to crops for several weeks or months each year. While there, they live in makeshift semi-open structures, usually far from organized health systems and where insecticide-treated nets (ITNs) do not provide adequate protection. This study evaluated the potential of a recently developed technology, eave ribbons treated with the spatial repellent transfluthrin, for protecting migratory rice farmers in rural southeastern Tanzania against indoor-biting and outdoor-biting mosquitoes. METHODS: In the first test, eave ribbons (0.1 m × 24 m each) treated with 1.5% transfluthrin solution were compared to untreated ribbons in 24 randomly selected huts in three migratory communities over 48 nights. Host-seeking mosquitoes indoors and outdoors were monitored nightly (18.00-07.00 h) using CDC light traps and CO2-baited BG malaria traps, respectively. The second test compared efficacies of eave ribbons treated with 1.5% or 2.5% transfluthrin in 12 huts over 21 nights. Finally, 286 farmers were interviewed to assess perceptions about eave ribbons, and their willingness to pay for them. RESULTS: In the two experiments, when treated eave ribbons were applied, the reduction in indoor densities ranged from 56 to 77% for Anopheles arabiensis, 36 to 60% for Anopheles funestus, 72 to 84% for Culex, and 80 to 98% for Mansonia compared to untreated ribbons. Reduction in outdoor densities was 38 to 77% against An. arabiensis, 36 to 64% against An. funestus, 63 to 88% against Culex, and 47 to 98% against Mansonia. There was no difference in protection between the two transfluthrin doses. In the survey, 58% of participants perceived the ribbons to be effective in reducing mosquito bites. Ninety per cent were willing to pay for the ribbons, the majority of whom were willing to pay but less than US$2.17 (5000 TZS), one-third of the current prototype cost. CONCLUSIONS: Transfluthrin-treated eave ribbons can protect migratory rice farmers, living in semi-open makeshift houses in remote farms, against indoor-biting and outdoor-biting mosquitoes. The technology is acceptable to users and could potentially complement ITNs. Further studies should investigate durability and epidemiological impact of eave ribbons, and the opportunities for improving affordability to users.

Using a miniaturized double-net trap (DN-Mini) to assess relationships between indoor-outdoor biting preferences and physiological ages of two malaria vectors, Anopheles arabiensis and Anopheles funestus.

BACKGROUND: Effective malaria surveillance requires detailed assessments of mosquitoes biting indoors, where interventions such as insecticide-treated nets work best, and outdoors, where other interventions may be required. Such assessments often involve volunteers exposing their legs to attract mosquitoes [i.e., human landing catches (HLC)], a procedure with significant safety and ethical concerns. Here, an exposure-free, miniaturized, double-net trap (DN-Mini) is used to assess relationships between indoor-outdoor biting preferences of malaria vectors, Anopheles arabiensis and Anopheles funestus, and their physiological ages (approximated by parity and insemination states). METHODS: The DN-Mini is made of UV-resistant netting on a wooden frame and PVC base. At 100 cm × 60 cm × 180 cm, it fits indoors and outdoors. It has a protective inner chamber where a volunteer sits and collects host-seeking mosquitoes entrapped in an outer chamber. Experiments were conducted in eight Tanzanian villages using DN-Mini to: (a) estimate nightly biting and hourly biting proportions of mosquitoes indoors and outdoors; (b) compare these proportions to previous estimates by HLC in same villages; and, (c) compare distribution of parous (proxy for potentially infectious) and inseminated mosquitoes indoors and outdoors. RESULTS: More than twice as many An. arabiensis were caught outdoors as indoors (p < 0.001), while An. funestus catches were marginally higher indoors than outdoors (p = 0.201). Anopheles arabiensis caught outdoors also had higher parity and insemination proportions than those indoors (p < 0.001), while An. funestus indoors had higher parity and insemination than those outdoors (p = 0.04). Observations of indoor-biting and outdoor-biting proportions, hourly biting patterns and overall species diversities as measured by DN-Mini, matched previous HLC estimates. CONCLUSIONS: Malaria vectors that are behaviourally adapted to bite humans outdoors also have their older, potentially infectious sub-populations concentrated outdoors, while those adapted to bite indoors have their older sub-populations concentrated indoors. Here, potentially infectious An. arabiensis more likely bite outdoors than indoors, while potentially infectious An. funestus more likely bite indoors. These observations validate previous evidence that even outdoor-biting mosquitoes regularly enter houses when young. They also demonstrate efficacy of DN-Mini for measuring indoor-outdoor biting behaviours of mosquitoes, their hourly biting patterns and epidemiologically relevant parameters, e.g., parity and insemination status, without exposure to volunteers. The trap is easy-to-use, easy-to-manufacture and affordable (prototypes cost ~ 100 US$/unit).

Linking human behaviours and malaria vector biting risk in south-eastern Tanzania.

To accelerate malaria elimination in areas where core interventions such as insecticide-treated nets (ITNs) are already widely used, it is crucial to consider additional factors associated with persistent transmission. Qualitative data on human behaviours and perceptions regarding malaria risk was triangulated with quantitative data on Anopheles mosquito bites occurring indoors and outdoors in south-eastern Tanzania communities where ITNS are already used but lower level malaria transmission persists. Each night (18:00h-07:00h), trained residents recorded human activities indoors, in peri-domestic outdoor areas, and in communal gatherings. Host-seeking mosquitoes were repeatedly collected indoors and outdoors hourly, using miniaturized exposure-free double net traps (DN-Mini) occupied by volunteers. In-depth interviews were conducted with household representatives to explore perceptions on persistent malaria and its control. Higher proportions of people stayed outdoors than indoors in early-evening and early-morning hours, resulting in higher exposures outdoors than indoors during these times. However, exposure during late-night hours (22:00h-05:00h) occurred mostly indoors. Some of the popular activities that kept people outdoors included cooking, eating, relaxing and playing. All households had at least one bed net, and 83.9% of people had access to ITNs. Average ITN use was 96.3%, preventing most indoor exposure. Participants recorgnized the importance of ITNs but also noted that the nets were not perfect. No complementary interventions were reported being used widely. Most people believed transmission happens after midnight. We conclude that insecticide-treated nets, where properly used, can still prevent most indoor exposures, but significant risk continues unabated before bedtime, outdoors and at communal gatherings. Such exposure is greatest for rural and low-income households. There is therefore an urgent need for complementary interventions, particularly those targeting outdoor-biting and are applicable for all people including the marginalised populations such as migratory farmers and fishermen. Besides, the differences in community understanding of ongoing transmission, and feedback on imperfections of ITNs should be considered when updating malaria-related communication and interventions.

Swarms of the malaria vector Anopheles funestus in Tanzania.

BACKGROUND: Anopheles funestus mosquitoes currently contribute more than 85% of ongoing malaria transmission events in south-eastern Tanzania, even though they occur in lower densities than other vectors, such as Anopheles arabiensis. Unfortunately, the species ecology is minimally understood, partly because of difficulties in laboratory colonization. This study describes the first observations of An. funestus swarms in Tanzania, possibly heralding new opportunities for control. METHOD: Using systematic searches by community-based volunteers and expert entomologists, An. funestus swarms were identified in two villages in Ulanga and Kilombero districts in south-eastern Tanzania, starting June 2018. Swarms were characterized by size, height, start- and end-times, presence of copulation and associated environmental features. Samples of male mosquitoes from the swarms were examined for sexual maturity by observing genitalia rotation, species identity using polymerase chain reaction and wing sizes. RESULTS: 581 An. funestus (98.1% males (n = 570) and 1.9% (n = 11) females) and 9 Anopheles gambiae sensu lato (s.l.) males were sampled using sweep nets from the 81 confirmed swarms in two villages (Ikwambi in Kilombero district and Tulizamoyo in Ulanga district). Six copulation events were observed in the swarms. Mean density (95% CL) of An. funestus caught/swarm/village/evening was 6.6 (5.9-7.2) in Tulizamoyo and 10.8 (5.8-15.8) in Ikwambi. 87.7% (n = 71) of the swarms were found in Tulizamoyo, while 12.3% (n = 10) were in Ikwambi. Mean height of swarms was 1.7 m (0.9-2.5 m), while mean duration was 12.9 (7.9-17.9) minutes. The PCR analysis confirmed that 100% of all An. funestus s.l. samples processed were An. funestus sensu stricto. Mean wing length of An. funestus males was 2.47 mm (2.0-2.8 mm), but there was no difference between swarming males and indoor-resting males. Most swarms (95.0%) occurred above bare ground, sometime on front lawns near human dwellings, and repeatedly in the same locations. CONCLUSION: This study has demonstrated occurrence of An. funestus swarms for the first time in Tanzania. Further investigations could identify new opportunities for improved control of this dominant malaria vector, possibly by targeting the swarms.

Dramatic decreases of malaria transmission intensities in Ifakara, south-eastern Tanzania since early 2000s.

BACKGROUND: Ongoing epidemiological transitions across Africa are particularly evident in fast-growing towns, such as Ifakara in the Kilombero valley, south-eastern Tanzania. This town and its environs (population ~ 70,000) historically experienced moderate to high malaria transmission, mediated mostly by Anopheles gambiae and Anopheles funestus. In early 2000s, malaria transmission [Plasmodium falciparum entomological inoculation rate (PfEIR)] was estimated at ~ 30 infectious bites/person/year (ib/p/yr). This study assessed the PfEIR after 15 years, during which there had been rapid urbanization and expanded use of insecticide-treated nets (ITNs). METHODS: Randomly-selected 110 households were sampled across Ifakara town and four adjacent wards. Mosquitoes were trapped nightly or monthly (June.2015-May.2016) using CDC-light-traps indoors, Suna® traps outdoors and human landing catches (HLC) indoors and outdoors. All Anopheles mosquitoes were morphologically identified and analysed by ELISA for Plasmodium circumsporozoite proteins. Mosquito blood meals were identified using ELISA, and sub-samples of An. gambiae and An. funestus examined by PCR to distinguish morphologically-similar siblings. Insecticide resistance was assessed using WHO-susceptibility assays, and some Anopheles were dissected to examine ovariole tracheoles for parity. RESULTS: After 3572 trap-nights, one Plasmodium-infected Anopheles was found (an An. funestus caught outdoors in Katindiuka-ward by HLC), resulting in overall PfEIR of 0.102 ib/p/yr. Nearly 80% of malaria vectors were from Katindiuka and Mlabani wards. Anopheles gambiae densities were higher outdoors (64%) than indoors (36%), but no such difference was observed for An. funestus. All An. funestus and 75% of An. gambiae dissected were parous. Anopheles gambiae complex consisted entirely of Anopheles arabiensis, while An. funestus included 84.2% An. funestus s.s., 4.5% Anopheles rivulorum, 1.4% Anopheles leesoni and 9.9% with unamplified-DNA. Anopheles gambiae were susceptible to bendiocarb and malathion, but resistant to pyrethroids, DDT and pirimiphos-methyl. Most houses had brick walls and/or iron roofs (> 90%), and 52% had screened windows. CONCLUSION: Malaria transmission in Ifakara has decreased by > 99% since early-2000s, reaching levels nearly undetectable with current entomological methods. These declines are likely associated with ITNs use, urbanization and improved housing. Remaining risk is now mostly in peri-urban wards, but concerted efforts could further decrease local transmission. Parasitological surveys are required to assess actual prevalence, incidence and importation rates.

Topographic mapping of the interfaces between human and aquatic mosquito habitats to enable barrier targeting of interventions against malaria vectors.

Geophysical topographic metrics of local water accumulation potential are freely available and have long been known as high-resolution predictors of where aquatic habitats for immature Anopheles mosquitoes are most abundant, resulting in elevated densities of adult malaria vectors and human infection burden. Using existing entomological and epidemiological survey data, here we illustrate how topography can also be used to map out the interfaces between wet, unoccupied valleys and dry, densely populated uplands, where malaria vector densities and infection risk are focally exacerbated. These topographically identifiable geophysical boundaries experience disproportionately high vector densities and malaria transmission risk, because this is where Anopheles mosquitoes first encounter humans when they search for blood after emerging or ovipositing in the valleys. Geophysical topographic indicators accounted for 67% of variance for vector density but for only 43% for infection prevalence, so they could enable very selective targeting of interventions against the former but not the latter (targeting ratios of 5.7 versus 1.5 to 1, respectively). So, in addition to being useful for targeting larval source management to wet valleys, geophysical topographic indicators may also be used to selectively target adult Anopheles mosquitoes with insecticidal residual sprays, fencing, vapour emanators or space sprays to barrier areas along their fringes.

Fine scale mapping of malaria infection clusters by using routinely collected health facility data in urban Dar es Salaam, Tanzania.

This study investigated whether passively collected routine health facility data can be used for mapping spatial heterogeneities in malaria transmission at the level of local government housing cluster administrative units in Dar es Salaam, Tanzania. From June 2012 to January 2013, residential locations of patients tested for malaria at a public health facility were traced based on their local leaders' names and geo-referencing the point locations of these leaders' houses. Geographic information systems (GIS) were used to visualise the spatial distribution of malaria infection rates. Spatial scan statistics was deployed to detect spatial clustering of high infection rates. Among 2407 patients tested for malaria, 46.6% (1121) could be traced to their 411 different residential housing clusters. One small spatially aggregated cluster of neighbourhoods with high prevalence was identified. While the home residence housing cluster leader was unambiguously identified for 73.8% (240/325) of malaria-positive patients, only 42.3% (881/2082) of those with negative test results were successfully traced. It was concluded that recording simple points of reference during routine health facility visits can be used for mapping malaria infection burden on very fine geographic scales, potentially offering a feasible approach to rational geographic targeting of malaria control interventions. However, in order to tap the full potential of this approach, it would be necessary to optimise patient tracing success and eliminate biases by blinding personnel to test results.

Spatially aggregated clusters and scattered smaller loci of elevated malaria vector density and human infection prevalence in urban Dar es Salaam, Tanzania.

BACKGROUND: Malaria transmission, primarily mediated by Anopheles gambiae, persists in Dar es Salaam (DSM) despite high coverage with bed nets, mosquito-proofed housing and larviciding. New or improved vector control strategies are required to eliminate malaria from DSM, but these will only succeed if they are delivered to the minority of locations where residual transmission actually persists. Hotspots of spatially clustered locations with elevated malaria infection prevalence or vector densities were, therefore, mapped across the city in an attempt to provide a basis for targeting supplementary interventions. METHODS: Two phases of a city-wide population-weighted random sample of cross-sectional household surveys of malaria infections were complemented by two matching phases of geographically overlapping, high-resolution, longitudinal vector density surveys; spanning 2010-2013. Spatial autocorrelations were explored using Moran's I and hotspots were detected using flexible spatial scan statistics. RESULTS: Seven hotspots of spatially clustered elevated vector density and eight of malaria infection prevalence were detected over both phases. Only a third of vectors were collected in hotspots in phase 1 (30 %) and phase 2 (33 %). Malaria prevalence hotspots accounted for only half of malaria infections detected in phase 1 (55 %) and phase 2 (47 %). Three quarters (76 % in phase 1 and 74 % in phase 2) of survey locations with detectable vector populations were outside of hotspots. Similarly, more than half of locations with higher infection prevalence (>10 %) occurred outside of hotspots (51 % in phase 1 and 54 % in phase 2). Vector proliferation hazard (exposure to An. gambiae) and malaria infection risk were only very loosely associated with each other (Odds ratio (OR) [95 % Confidence Interval (CI)] = 1.56 [0.89, 1.78], P = 0.52)). CONCLUSION: Many small, scattered loci of local malaria transmission were haphazardly scattered across the city, so interventions targeting only currently identifiable spatially aggregated hotspots will have limited impact. Routine, spatially comprehensive, longitudinal entomological and parasitological surveillance systems, with sufficient sensitivity and spatial resolution to detect these scattered loci, are required to eliminate transmission from this typical African city. Intervention packages targeted to both loci and hotspots of transmission will need to suppress local vector proliferation, treat infected residents and provide vulnerable residents with supplementary protective measures against exposure.