Temperature, mosquito feeding status and mosquito density influence the measured bio-efficacy of insecticide-treated nets in cone assays.

BACKGROUND: The WHO cone bioassay is routinely used to evaluate the bioefficacy of insecticide-treated nets (ITNs) for product pre-qualification and confirmation of continued ITN performance during operational monitoring. Despite its standardized nature, variability is often observed between tests. We investigated the influence of temperature in the testing environment, mosquito feeding status and mosquito density on cone bioassay results. METHODS: Cone bioassays were conducted on MAGNet (alphacypermethrin) and Veeralin (alphacypermethrin and piperonyl butoxide (PBO)) ITNs, using laboratory-reared pyrethroid-resistant Anopheles funestus sensu stricto (FUMOZ strain) mosquitoes. Three experiments were conducted using standard cone bioassays following WHO-recommended test parameters, with one variable changed in each bioassay: (i) environmental temperature during exposure: 22-23 °C, 26-27 °C, 29-30 °C and 32-33 °C; (ii) feeding regimen before exposure: sugar starved for 6 h, blood-fed or sugar-fed; and (iii) mosquito density per cone: 5, 10, 15 and 20 mosquitoes. For each test, 15 net samples per treatment arm were tested with four cones per sample (N = 60). Mortality after 24, 48 and 72 h post-exposure to ITNs was recorded. RESULTS: There was a notable influence of temperature, feeding status and mosquito density on An. funestus mortality for both types of ITNs. Mortality at 24 h post-exposure was significantly higher at 32-33 °C than at 26-27 °C for both the MAGNet [19.33% vs 7%; odds ratio (OR): 3.96, 95% confidence interval (CI): 1.99-7.87, P < 0.001] and Veeralin (91% vs 47.33%; OR: 22.20, 95% CI: 11.45-43.05, P < 0.001) ITNs. Mosquito feeding status influenced the observed mortality. Relative to sugar-fed mosquitoes, The MAGNet ITNs induced higher mortality among blood-fed mosquitoes (7% vs 3%; OR: 2.23, 95% CI: 0.94-5.27, P = 0.068) and significantly higher mortality among starved mosquitoes (8% vs 3%, OR: 2.88, 95% CI: 1.25-6.63, P = 0.013); in comparison, the Veeralin ITNs showed significantly lower mortality among blood-fed mosquitoes (43% vs 57%; OR: 0.56, 95% CI: 0.38-0.81, P = 0.002) and no difference for starved mosquitoes (58% vs 57%; OR: 1.05, 95% CI: 0.72-1.51, P = 0.816). Mortality significantly increased with increasing mosquito density for both the MAGNet (e.g. 5 vs 10 mosquitoes: 7% vs 12%; OR: 1.81, 95% CI: 1.03-3.20, P = 0.040) and Veeralin (e.g. 5 vs 10 mosquitoes: 58% vs 71%; OR 2.06, 95% CI: 1.24-3.42, P = 0.005) ITNs. CONCLUSIONS: The results of this study highlight that the testing parameters temperature, feeding status and mosquito density significantly influence the mortality measured in cone bioassays. Careful adherence to testing parameters outlined in WHO ITN testing guidelines will likely improve the repeatability of studies within and between product testing facilities.

Time of exposure and assessment influence the mortality induced by insecticides against metabolic resistant mosquitoes.

BACKGROUND: Increasing metabolic resistance in malaria vector mosquitoes resulted in the development of insecticide-treated nets (ITNs) with active ingredients (AI) that target them. Bioassays that accurately measure the mortality induced by these AIs on ITNs are needed. Mosquito metabolic enzyme expression follows a circadian rhythm. Thus, this study assessed (i) influence of the time of day of mosquito exposure and (ii) timing of assessment of mortality post exposure (24 and 72 h) to ITNs against vectors that are susceptible to pyrethroids and those with metabolic and knockdown resistance mechanisms. METHODS: Two cone bioassay experiments were conducted following World Health Organization (WHO) guidelines. Firstly, on ITNs incorporated with 2 g AI/kg of deltamethrin (DM) alone, or combined with 8 g AI/kg piperonyl butoxide (PBO) synergist, during the day (9:00-14:00 h) and repeated in the evening (18:00-20:00 h). This was followed by a confirmatory experiment during the afternoon (12:00-14:00 h) and repeated in the night (22:00-24:00 h) using mosquitoes unexposed or pre-exposed to PBO for 1 h before exposure to DM ITNs. Each net piece was tested with a minimum of eight cones per time (N = 24). The outcome was mortality after 24 h (M24) or 72 h (M72) of holding. RESULTS: The cone bioassays performed using metabolic resistant mosquitoes during the evening showed significantly lower M24 than those performed in the day for DM: odds ratio (OR) 0.14 [95% confidence interval (CI) 0.06-0.30, p < 0.0001] and DM PBO [OR 0.29 (95% CI 0.18-0.49, p < 0.0001). M72 was higher than M24 for metabolic resistant mosquitoes exposed to DM [OR 1.44 (95% CI 1.09-1.88), p = 0.009] and DM PBO [OR 1.82 (95% CI 1.42-2.34), p < 0.0001]. An influence of hour of experiment and time of assessment was not observed for mosquitoes that had knockdown resistance or that were pyrethroid-susceptible. CONCLUSIONS: Time of day of experiment and hour of assessment of delayed mortality after exposure of mosquitoes are important considerations in evaluating insecticides that interact with mosquito metabolism to counter metabolic resistant mosquitoes. This is important when evaluating field-aged ITNs that may have lower concentrations of AI.

Life expectancy of Anopheles funestus is double that of Anopheles arabiensis in southeast Tanzania based on mark-release-recapture method.

Anopheles arabiensis and Anopheles funestus sensu stricto mosquitoes are major East African malaria vectors. Understanding their dispersal and population structure is critical for developing effective malaria control tools. Three mark-release-recapture (MRR) experiments were conducted for 51 nights to assess daily survival and flight range of An. arabiensis and An. funestus mosquitoes in south-eastern, Tanzania. Mosquitoes were marked with a fluorescent dye as they emerged from breeding sites via a self-marking device. Mosquitoes were collected indoors and outdoors using human landing catches (HLC) and Centers for Disease Control and Prevention light traps (CDC-LT). In total, 4210 An. arabiensis and An. funestus were collected with 316 (7.5%) marked and recaptured (MR). Daily mean MR was 6.8, standard deviation (SD ± 7.6) for An. arabiensis and 8.9 (SD ± 8.3) for An. funestus. Probability of daily survival was 0.76 for An. arabiensis and 0.86 for An. funestus translating into average life expectancy of 3.6 days for An. arabiensis and 6.5 days for An. funestus. Dispersal distance was 654 m for An. arabiensis and 510 m for An. funestus. An. funestus life expectancy was substantially longer than that of An. arabiensis. The MRR method described here could be routinely utilized when evaluating the impact of new vector control tools on mosquito survival.

The MTego trap: a potential tool for monitoring malaria and arbovirus vectors.

BACKGROUND: Odour-baited traps are useful for vector surveillance and control. However, most existing traps have shown inconsistent recapture rates across different mosquito species, necessitating the need for more effective and efficient traps. The MTego trap with integrated thermal stimuli has been developed as an alternative trap. This study was undertaken to determine and compare the efficacy of the MTego trap to that of the Biogents (BG) modular BG-Pro (BGP) trap for sampling different mosquito species in a semi-field system. METHODS: Fully balanced Latin square design experiments (no-choice and dual choice) were conducted in semi-field chambers using laboratory-reared female Anopheles gambiae sensu stricto, Anopheles funestus, Anopheles arabiensis, Culex quinquefasciatus and Aedes aegypti. There were 16 replicates, and 50 mosquitoes of each species were released in each chamber per replicate. The evaluated traps were as follows: the MTego trap baited with PM6 (MT-PM6), the MTego trap baited with BG-Lure (BGL) (MT-BGL), and the BGP trap baited with BG-Lure (BGP-BGL). RESULTS: In the no-choice test, the MT-BGL and BGP-BGL traps captured a similar proportion of An. gambiae (31% vs 29%, P-value = 0.519) and An. funestus (32% vs 33%, P = 0.520). The MT-PM6 and BGP-BGL traps showed no significant difference in capturing Ae. aegypti (33% vs 31%, P = 0.324). However, the BGP-BGL caught more An. arabiensis and Cx. quinquefasciatus mosquitoes than the other traps (P < 0.0001). In the dual-choice test of MT-PM6 vs BGP-BGL, similar proportions of An. funestus (25% vs 27%, P = 0.473) and Ae. aegypti (29% vs 25%, P = 0.264) were captured in the traps, while the BGP-BGL captured more An. gambiae, An. arabiensis and Cx. quinquefasciatus mosquitoes than the MT-PM6 (P < 0.0001). CONCLUSIONS: This study demonstrated that the MTego trap has potential as a tool that can be used interchangeably with the BGP trap for sampling anthropophilic mosquitoes including African malaria vectors An. gambiae and An. funestus and the principal arbovirus vector Ae. aegypti.

Multi-country evaluation of the durability of pyrethroid plus piperonyl-butoxide insecticide-treated nets: study protocol.

BACKGROUND: Mass distributions of long-lasting insecticidal nets (LLINs) have contributed to large reductions in the malaria burden. However, this success is in jeopardy due in part to the increasing pyrethroid-resistant mosquito population as well as low LLINs coverage in various areas because the lifespan of LLINs is often shorter than the interval between replenishment campaigns. New insecticide-treated nets (ITNs) containing pyrethroid and piperonyl-butoxide (PBO) have shown a greater reduction in the incidence of malaria than pyrethroid LLINs in areas with pyrethroid-resistant mosquitoes. However, the durability (attrition, bio-efficacy, physical integrity and chemical retainment) of pyrethroid-PBO ITNs under operational settings has not been fully characterized. This study will measure the durability of pyrethroid-PBO ITNs to assess whether they meet the World Health Organization (WHO) three years of operational performance criteria required to be categorized as "long-lasting". METHODS: A prospective household randomized controlled trial will be conducted simultaneously in Tanzania, India and Côte d'Ivoire to estimate the field durability of three pyrethroid-PBO ITNs (Veeralin®, Tsara® Boost, and Olyset® Plus) compared to a pyrethroid LLIN: MAGNet®. Durability monitoring will be conducted up to 36 months post-distribution and median survival in months will be calculated. The proportion of ITNs: (1) lost (attrition), (2) physical integrity, (3) resistance to damage score, (4) meeting WHO bio-efficacy (≥ 95% knockdown after 1 h or ≥ 80% mortality after 24 h for WHO cone bioassay, or ≥ 90% blood-feeding inhibition or ≥ 80% mortality after 24 h for WHO Tunnel tests) criteria against laboratory-reared resistant and susceptible mosquitoes, and insecticidal persistence over time will be estimated. The non-inferiority of Veeralin® and Tsara® Boost to the first-in-class, Olyset® Plus will additionally be assessed for mortality, and the equivalence of 20 times washed ITNs compared to field aged ITNs will be assessed for mortality and blood-feeding inhibition endpoints in the Ifakara Ambient Chamber Test, Tanzania. CONCLUSION: This will be the first large-scale prospective household randomized controlled trial of pyrethroid-PBO ITNs in three different countries in East Africa, West Africa and South Asia, simultaneously. The study will generate information on the replenishment intervals for PBO nets.

Insecticide-treated eave nets and window screens for malaria control in Chalinze district, Tanzania: a study protocol for a household randomised control trial.

BACKGROUND: Long-lasting insecticidal nets (LLINs) have contributed to the reduction of malaria in sub-Saharan Africa, including Tanzania. However, they rely on daily user behaviour and high coverage which is difficult to maintain. Also, insecticide resistance among malaria vector mosquitoes is contributing to reduced efficacy of control tools. To overcome these problems, we propose to evaluate a new tool for house modification, the insecticide-treated eave nets (ITENs) in combination with insecticide-treated window screens (ITWS) incorporated with dual active ingredient (dual AI) for the control of malaria. METHODS: Four hundred and fifty (450) households with intact walls, open eaves without screens or nets on the windows in Chalinze district will be eligible and recruited upon written informed consent. The households will be randomly allocated into two arms: one with ITENs and ITWS installed and the other without. Malaria parasite detection using a quantitative polymerase chain reaction (qPCR) will be conducted shortly after the long rain (June/July, 2022) as the primary outcome and shortly after the short rain (January/February, 2022) as the secondary outcome. Other secondary outcomes include clinical malaria cases, and density of malaria vectors and nuisance after the short rain and long rain. In addition, surveys will be conducted in households with ITENs and ITWS to estimate the intervention's cost during installation, adverse effects one month after installation, and presence, fabric integrity and user acceptance six and twelve months after installation. Bioefficacy and chemical content will be evaluated twelve months after installation. DISCUSSION: ITENs and ITWS have been shown in Kenya to reduce indoor mosquito density. However, it is not known if indoor mosquito density reduction translates into reduction of malaria cases. Data from the study will measure the potential public health value of an additional intervention for malaria control at the household level in areas of mosquito insecticide resistance that does not require daily adherence. TRIAL REGISTRATION: The study is registered on ClinicalTrials.gov .

Modified World Health Organization (WHO) Tunnel Test for Higher Throughput Evaluation of Insecticide-Treated Nets (ITNs) Considering the Effect of Alternative Hosts, Exposure Time, and Mosquito Density.

The standard World Health Organization (WHO) tunnel test is a reliable laboratory bioassay used for "free-flying" testing of insecticide-treated nets (ITNs) bio-efficacy where mosquitoes pass through a ITN sample to reach a live animal bait. Multiple parameters (i.e., bait, exposure time, and mosquito density) may affect the outcomes measured in tunnel tests. Therefore, a comparison was conducted of alternative hosts, exposure time, and lower mosquito density against the current gold standard test (100 mosquitoes, animal bait, and 12-h exposure) as outlined in the WHO ITN evaluation guideline. This was done with the aim to make the tunnel test cheaper and with higher throughput to meet the large sample sizes needed for bio-efficacy durability monitoring of chlorfenapyr ITNs that must be evaluated in "free-flying" bioassays. Methods: A series of experiments were conducted in the WHO tunnel test to evaluate the impact of the following factors on bio-efficacy endpoints of mosquito mortality at 24-h (M24) and 72-h (M72) and blood-feeding success (BFS): (1) baits (rabbit, membrane, human arm); (2) exposure time in the tunnel (1 h vs. 12 h); and (3) mosquito density (50 vs. 100). Finally, an alternative bioassay using a membrane with 50 mosquitoes (membrane-50) was compared to the gold standard bioassay (rabbit with 100 mosquitoes, rabbit-100). Pyrethroid-resistant Anopheles arabiensis and pyrethroid susceptible Anopheles gambiae were used to evaluate Interceptor® and Interceptor® G2 ITNs. Results: Using a human arm as bait gave a very different BFS, which impacted measurements of M24 and M72. The same trends in M24, M72 and BFS were observed for both Interceptor® ITN and Interceptor® G2 unwashed and washed 20 times measured using the gold standard WHO tunnel test (rabbit-100) or rabbit with 50 mosquitoes (rabbit-50). M24, M72 and BFS were not statistically different when either 50 or 100 mosquitoes were used with rabbit bait in the tunnel bioassay for either the susceptible or resistant strains. No systematic difference was observed between rabbit-50 and rabbit-100 in the agreement by the Bland and Altman method (B&A). The mean difference was 4.54% (-22.54-31.62) in BFS and 1.71% (-28.71-32.12) in M72 for rabbit-50 versus rabbit-100. Similar M24, M72 and lower BFS was measured by membrane-50 compared to rabbit-100. No systematic difference was observed in the agreement between membrane-50 and rabbit-100, by B&A. The mean difference was 9.06% (-11.42-29.64) for BSF and -5.44% (-50.3-39.45) for M72. Both membrane-50, rabbit-50 and rabbit-100 predicted the superiority of Interceptor® G2 over Interceptor® ITN for the resistant strain on M72. Conclusion: These results demonstrate that WHO tunnel tests using rabbit bait may be run with 50 mosquitoes to increase sample sizes needed for bio-efficacy durability monitoring of ITNs in "free-flying" bioassays. Using a membrane feeder with 50 mosquitoes is a potential replacement for the WHO tunnel bioassay with animal bait if control blood feeding rates can be improved to 50% because blood feeding impacts mosquito survival after exposure to insecticides.

Comparison of cone bioassay estimates at two laboratories with different Anopheles mosquitoes for quality assurance of pyrethroid insecticide-treated nets.

BACKGROUND: Quality assurance (QA) of insecticide-treated nets (ITNs) delivered to malaria-endemic countries is conducted by measuring physiochemical parameters, but not bioefficacy against malaria mosquitoes. This study explored utility of cone bioassays for pre-delivery QA of pyrethroid ITNs to test the assumption that cone bioassays are consistent across locations, mosquito strains, and laboratories. METHODS: Double-blinded bioassays were conducted on twenty unused pyrethroid ITNs of 4 brands (100 nets, 5 subsamples per net) that had been delivered for mass distribution in Papua New Guinea (PNG) having passed predelivery inspections. Cone bioassays were performed on the same net pieces following World Health Organization (WHO) guidelines at the PNG Institute of Medical Research (PNGIMR) using pyrethroid susceptible Anopheles farauti sensu stricto (s.s.) and at Ifakara Health Institute (IHI), Tanzania using pyrethroid susceptible Anopheles gambiae s.s. Additionally, WHO tunnel tests were conducted at IHI on ITNs that did not meet cone bioefficacy thresholds. Results from IHI and PNGIMR were compared using Spearman's Rank correlation, Bland-Altman (BA) analysis and analysis of agreement. Literature review on the use of cone bioassays for unused pyrethroid ITNs testing was conducted. RESULTS: In cone bioassays, 13/20 nets (65%) at IHI and 8/20 (40%) at PNGIMR met WHO bioefficacy criteria. All nets met WHO bioefficacy criteria on combined cone/tunnel tests at IHI. Results from IHI and PNGIMR correlated on 60-min knockdown (KD60) (rs = 0.6,p = 0.002,n = 20) and 24-h mortality (M24) (rs = 0.9,p < 0.0001,n = 20) but BA showed systematic bias between the results. Of the 5 nets with discrepant result between IHI and PNGIMR, three had confidence intervals overlapping the 80% mortality threshold, with averages within 1-3% of the threshold. Including these as a pass, the agreement between the results to predict ITN failure was good with kappa = 0.79 (0.53-1.00) and 90% accuracy. CONCLUSIONS: Based on these study findings, the WHO cone bioassay is a reproducible bioassay for ITNs with > 80% M24, and for all ITNs provided inherent stochastic variation and systematic bias are accounted for. The literature review confirms that WHO cone bioassay bioefficacy criteria have been previously achieved by all pyrethroid ITNs (unwashed), without the need for additional tunnel tests. The 80% M24 threshold remains the most reliable indicator of pyrethroid ITN quality using pyrethroid susceptible mosquitoes. In the absence of alternative tests, cone bioassays could be used as part of pre-delivery QA.

The Centres for Disease Control light trap (CDC-LT) and the human decoy trap (HDT) compared to the human landing catch (HLC) for measuring Anopheles biting in rural Tanzania.

BACKGROUND: Vector mosquito biting intensity is an important measure to understand malaria transmission. Human landing catch (HLC) is an effective but labour-intensive, expensive, and potentially hazardous entomological surveillance tool. The Centres for Disease Control light trap (CDC-LT) and the human decoy trap (HDT) are exposure-free alternatives. This study compared the CDC-LT and HDT against HLC for measuring Anopheles biting in rural Tanzania and assessed their suitability as HLC proxies. METHODS: Indoor mosquito surveys using HLC and CDC-LT and outdoor surveys using HLC and HDT were conducted in 2017 and in 2019 in Ulanga, Tanzania in 19 villages, with one trap/house/night. Species composition, sporozoite rates and density/trap/night were compared. Aggregating the data by village and month, the Bland-Altman approach was used to assess agreement between trap types. RESULTS: Overall, 66,807 Anopheles funestus and 14,606 Anopheles arabiensis adult females were caught with 6,013 CDC-LT, 339 indoor-HLC, 136 HDT and 195 outdoor-HLC collections. Indoors, CDC-LT caught fewer An. arabiensis (Adjusted rate ratio [Adj.RR] = 0.35, 95% confidence interval [CI]: 0.27-0.46, p < 0.001) and An. funestus (Adj.RR = 0.63, 95%CI: 0.51-0.79, p < 0.001) than HLC per trap/night. Outdoors, HDT caught fewer An. arabiensis (Adj.RR = 0.04, 95%CI: 0.01-0.14, p < 0.001) and An. funestus (Adj.RR = 0.10, 95%CI: 0.07-0.15, p < 0.001) than HLC. The bias and variability in number of mosquitoes caught by the different traps were dependent on mosquito densities. The relative efficacies of both CDC-LT and HDT in comparison to HLC declined with increased mosquito abundance. The variability in the ratios was substantial for low HLC counts and decreased as mosquito abundance increased. The numbers of sporozoite positive mosquitoes were low for all traps. CONCLUSIONS: CDC-LT can be suitable for comparing mosquito populations between study arms or over time if accuracy in the absolute biting rate, compared to HLC, is not required. CDC-LT is useful for estimating sporozoite rates because large numbers of traps can be deployed to collect adequate mosquito samples. The present design of the HDT is not amenable for use in large-scale entomological surveys. Use of HLC remains important for estimating human exposure to mosquitoes as part of estimating the entomological inoculation rate (EIR).

Influence of testing modality on bioefficacy for the evaluation of Interceptor® G2 mosquito nets to combat malaria mosquitoes in Tanzania.

BACKGROUND: Insecticide-treated net (ITN) durability is evaluated using longitudinal bioefficacy and fabric integrity sampling post-distribution. Interceptor® G2 was developed for resistance management and contains two adulticides: alpha-cypermethrin and chlorfenapyr; it is a pro-insecticide that is metabolized into its active form by mosquito-detoxifying enzymes and may be enhanced when the mosquito is physiologically active. To elucidate the impact of bioassay modality, mosquito exposures of the alphacypermethrin ITN Interceptor® and dual adulticide Interceptor® G2 were investigated. METHODS: This study evaluated the performance of Interceptor® G2 compared to Interceptor® against local strains of mosquitoes in Tanzania. Unwashed and 20× times washed nets were tested. Efficacy of ITNs was measured by four bioassay types: (1) World Health Organisation (WHO) cone test (cone), (2) WHO tunnel test (tunnel), (3) Ifakara ambient chamber test (I-ACT) and (4) the WHO gold standard experimental hut test (hut). Hut tests were conducted against free-flying wild pyrethroid metabolically resistant Anopheles arabiensis and Culex quinquefasciatus. Cone, tunnel and I-ACT bioassays used laboratory-reared metabolically resistant An. arabiensis and Cx. quinquefasciatus and pyrethroid susceptible Anopheles gambiae sensu stricto and Aedes aegypti. RESULTS: Against resistant strains, superiority of Interceptor® G2 over Interceptor® was observed in all "free-flying bioassays". In cone tests (which restrict mosquito flight), superiority of Interceptor® over Interceptor® G2 was recorded. Mortality of unwashed Interceptor® G2 among An. arabiensis was lowest in hut tests at 42.9% (95% CI: 37.3-48.5), although this increased to 66.7% (95% CI: 47.1-86.3) by blocking hut exit traps so mosquitoes presumably increased frequencies of contact with ITNs. Higher odds of mortality were consistently observed in Interceptor® G2 compared to Interceptor® in "free-flying" bioassays using An. arabiensis: tunnel (OR = 1.42 [95% CI:1.19-1.70], p < 0.001), I-ACT (OR = 1.61 [95% CI: 1.05-2.49], p = 0.031) and hut (OR = 2.53 [95% CI: 1.96-3.26], p < 0.001). Interceptor® and Interceptor® G2 showed high blood-feeding inhibition against all strains. CONCLUSION: Both free-flying laboratory bioassays (WHO Tunnel and I-ACT) consistently measured similarly, and both predicted the results of the experimental hut test. For bioefficacy monitoring and upstream product evaluation of ITNs in situ, the I-ACT may provide an alternative bioassay modality with improved statistical power. Interceptor G2® outperformed Interceptor ® against pyrethroid-resistant strains, demonstrating the usefulness of chlorfenapyr in mitigation of malaria.

"In starvation, a bone can also be meat": a mixed methods evaluation of factors associated with discarding of long-lasting insecticidal nets in Bagamoyo, Tanzania.

BACKGROUND: Between 2000 and 2019, more than 1.8 billion long-lasting insecticidal nets (LLINs) were distributed in Africa. While the insecticidal durability of LLINs is around 3 years, nets are commonly discarded 2 years post distribution. This study investigated the factors associated with the decision of users to discard LLINs. METHODS: A mixed-method sequential explanatory approach using a structured questionnaire followed by focus group discussions (FGDs) to collect information on experiences, views, reasons, how and when LLINs are discarded. Out of 6,526 households that responded to the questionnaire of LLINs durability trial, 160 households were randomly selected from the households in four villages in Bagamoyo Tanzania for FGDs but only 155 households participated in the FGDs. Five of the household representatives couldn't participate due to unexpected circumstances. A total of sixteen FGDs each comprising of 8-10 adults were conducted; older women (40-60 years), older men (40-60 years), younger women (18-39 years), younger men (18-39 years). During the FGDs, participants visually inspected seven samples of LLINs that were "too-torn" based on Proportionate Hole Index recommended by the World Health Organization (WHO) guidelines on LLIN testing, the nets were brought to the discussion and participants had to determine if such LLINs were to be kept or discarded. The study assessed responses from the same participants that attended FGD and also responded to the structured questionnaire, 117 participants fulfilled the criteria, thus data from only 117 participants are analysed in this study. RESULTS: In FGDs, integrity of LLIN influenced the decision to discard or keep a net. Those of older age, women, and householders with lower income were more likely to classify a WHO "too-torn" net as "good". The common methods used to discard LLINs were burning and burying. The findings were seen in the quantitative analysis. For every additional hole, the odds of discarding a WHO "too-torn" LLIN increased [OR = 1.05 (95%CI (1.04-1.07)), p < 0.001]. Younger age group [OR = 4.97 (95%CI (3.25-7.32)), p < 0.001], male-headed households [OR = 6.85 (95%CI (4.44 -10.59)), p < 0.001], and wealthy households [OR = 3.88 (95%CI (2.33-6.46)), p < 0.001] were more likely to discard LLINs. CONCLUSION: Integrity of LLIN was the main determinant for discarding or keeping LLINs and the decision to discard the net is associated with socioeconomic status of the household, and the age and gender of respondents. WHO "too torn" nets are encouraged to be used instead of none until replacement, and disposal of nets should be based on recommendation.

Single blinded semi-field evaluation of MAÏA® topical repellent ointment compared to unformulated 20% DEET against Anopheles gambiae, Anopheles arabiensis and Aedes aegypti in Tanzania.

BACKGROUND: N,N-Diethyl-3-methylbenzamide (DEET) topical mosquito repellents are effective personal protection tools. However, DEET-based repellents tend to have low consumer acceptability because they are cosmetically unappealing. More attractive formulations are needed to encourage regular user compliance. This study evaluated the protective efficacy and protection duration of a new topical repellent ointment containing 15% DEET, MAÏA® compared to 20% DEET in ethanol using malaria and dengue mosquito vectors in Bagamoyo Tanzania. METHODS: Fully balanced 3 × 3 Latin square design studies were conducted in large semi-field chambers using laboratory strains of Anopheles gambiae sensu stricto, Anopheles arabiensis and Aedes aegypti. Human volunteers applied either MAÏA® ointment, 20% DEET or ethanol to their lower limbs 6 h before the start of tests. Approximately 100 mosquitoes per strain per replicate were released inside each chamber, with 25 mosquitoes released at regular intervals during the collection period to maintain adequate biting pressure throughout the test. Volunteers recaptured mosquitoes landing on their lower limbs for 6 h over a period of 6 to 12-h post-application of repellents. Data analysis was conducted using mixed-effects logistic regression. RESULTS: The protective efficacy of MAÏA® and 20% DEET was not statistically different for each of the mosquito strains: 95.9% vs. 97.4% against An. gambiae (OR = 1.53 [95% CI 0.93-2.51] p = 0.091); 96.8% vs 97.2% against An. arabiensis (OR = 1.08 [95% CI 0.66-1.77] p = 0.757); 93.1% vs 94.6% against Ae. aegypti (OR = 0.76 [95% CI 0.20-2.80] p = 0.675). Average complete protection time (CPT) in minutes of MAÏA® and that of DEET was similar for each of the mosquito strains: 571.6 min (95% CI 558.3-584.8) vs 575.0 min (95% CI 562.1-587.9) against An. gambiae; 585.6 min (95% CI 571.4-599.8) vs 580.9 min (95% CI 571.1-590.7) against An. arabiensis; 444.1 min (95% CI 401.8-486.5) vs 436.9 min (95% CI 405.2-468.5) against Ae. aegypti. CONCLUSIONS: MAÏA® repellent ointment provides complete protection for 9 h against both An. gambiae and An. arabiensis, and 7 h against Ae. aegypti similar to 20% DEET (in ethanol). MAÏA® repellent ointment can be recommended as a tool for prevention against outdoor biting mosquitoes in tropical locations where the majority of the people spend an ample time outdoor before going to bed.

Household factors associated with access to insecticide-treated nets and house modification in Bagamoyo and Ulanga districts, Tanzania.

BACKGROUND: Insecticide-treated nets (ITNs) and house modifications are proven vector control tools, yet in most regions, full coverage has not been achieved. This study investigates household factors associated with access to ITNs and house modification in Tanzania. METHODS: Baseline cross-sectional survey data from previous studies on spatial repellants and indoor residual spray evaluation was analysed from 6757 households in Bagamoyo (60 km north of Dar es Salaam) and 1241 households in Ulanga (a remote rural area in southeast Tanzania), respectively. Regression models were used to estimate the associations between the outcomes: population access to ITNs, access to ITN per sleeping spaces, window screens and closed eaves, and the covariates household size, age, gender, pregnancy, education, house size, house modification (window screens and closed eaves) and wealth. RESULTS: Population access to ITNs (households with one ITN per two people that stayed in the house the previous night of the survey) was 69% (n = 4663) and access to ITNs per sleeping spaces (households with enough ITNs to cover all sleeping spaces used the previous night of the survey) was 45% (n = 3010) in Bagamoyo, 3 years after the last mass campaign. These findings are both lower than the least 80% coverage target of the Tanzania National Malaria Strategic Plan (Tanzania NMSP). In Ulanga, population access to ITNs was 92% (n = 1143) and ITNs per sleeping spaces was 88% (n = 1093), 1 year after the last Universal Coverage Campaign (UCC). Increased household size was significantly associated with lower access to ITNs even shortly after UCC. House modification was common in both areas but influenced by wealth. In Bagamoyo, screened windows were more common than closed eaves (65% vs 13%), whereas in Ulanga more houses had closed eaves than window screens (55% vs 12%). CONCLUSION: Population access to ITNs was substantially lower than the targets of the Tanzania NMSP after 3 years and lower among larger households after 1 year following ITN campaign. House modification was common in both areas, associated with wealth. Improved access to ITNs and window screens through subsidies and Behaviour Change Communication (BCC) strategies, especially among large and poor households and those headed by people with a low level of education, could maximize the uptake of a combination of these two interventions.

Long-lasting insecticidal nets retain bio-efficacy after 5 years of storage: implications for malaria control programmes.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are the most sustainable and effective malaria control tool currently available. Global targets are for 80% of the population living in malaria endemic areas to have access to (own) and use a LLIN. However, current access to LLINs in endemic areas is 56% due to system inefficiencies and budget limitations. Thus, cost-effective approaches to maximize access to effective LLINs in endemic areas are required. This study evaluated whether LLINs that had been stored for 5 years under manufacturer's recommended conditions may be optimally effective against Anopheles mosquitoes, to inform malaria control programmes and governments on the periods over which LLINs may be stored between distributions, in an effort to maximize use of available LLINs. METHODS: Standard World Health Organization (WHO) bioassays (cone and tunnel test) were used to evaluate the bio-efficacy and wash resistance of Olyset® and DawaPlus® 2.0 (rebranded Tsara® Soft) LLINs after 5 years of storage at 25 °C to 33.4 °C and 40% to 100% relative humidity. In addition, a small scale Ifakara Ambient Chamber test (I-ACT) was conducted to compare the bio-efficacy of one long stored LLINs to one new LLIN of the same brand, washed or unwashed. LLINs were evaluated using laboratory reared fully susceptible Anopheles gambiae sensu stricto (s.s.) (Ifakara strain) and pyrethroid resistant Anopheles arabiensis (Kingani strain). RESULTS: After 5 years of storage, both unwashed and washed, Olyset® and DawaPlus® 2.0 (Tsara® Soft) LLINs passed WHO bio-efficacy criteria on knockdown (KD60) ≥ 95%, 24-h mortality ≥ 80% and ≥ 90% blood-feeding inhibition in WHO assays against susceptible An. gambiae s.s. DawaPlus® 2.0 LLINs also passed combined WHO bioassay criteria against resistant An. arabiensis. Confirmatory I-ACT tests using whole nets demonstrated that long-stored LLINs showed higher efficacy than new LLINs on both feeding inhibition and mortality endpoints against resistant strains. CONCLUSIONS: Even after long-term storage of around 5 years, both Olyset® and DawaPlus® 2.0 LLINs remain efficacious against susceptible Anopheles mosquitoes at optimal storage range of 25 °C to 33.4 °C for temperature and 40% to 100% relative humidity measured by standard WHO methods. DawaPlus® 2.0 (Tsara® Soft) remained efficacious against resistant strain.

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.