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Abstract Background Bio-efficacy and residual activity of insecticides used for indoor residual spraying (IRS) and long-lasting insecticide nets (LLINs) were assessed against laboratory-reared and wild populations of the malaria vector, Anopheles arabiensis in south eastern Tanzania. Implications of the findings are examined in the context of potential synergies and redundancies where IRS and LLINs are combined. Methods Bioassays were conducted monthly for six months on three LLIN types (Olyset® PermaNet 2.0®,and Icon Life®) and three IRS treatments (2 g/m2 pirimiphos-methyl, 2 g/m2 DDT and 0.03 g/m2 lambda-cyhalothrin, sprayed on mud walls and palm ceilings of experimental huts). Tests used susceptible laboratory-reared An. arabiensis exposed in cones (nets and IRS) or wire balls (nets only). Susceptibility of wild populations was assessed using WHO diagnostic concentrations and PCR for knock-down resistance (kdr) genes. Results IRS treatments killed ≥ 85% of mosquitoes exposed on palm ceilings and ≥ 90% of those exposed on mud walls, but up to 50% of this toxicity decayed within 1–3 months, except for DDT. By 6th month, only 7.5%, 42.5% and 30.0% of mosquitoes died when exposed to ceilings sprayed with pirimiphos-methyl, DDT or lambda-cyhalothrin respectively, while 12.5%, 36.0% and 27.5% died after exposure to mud walls sprayed with the same insecticides. In wire-ball assays, mortality decreased from 98.1% in 1st month to 92.6% in 6th month in tests on PermaNet 2.0®, from 100% to 61.1% on Icon Life® and from 93.2% to 33.3% on Olyset® nets. In cone bioassays, mortality reduced from 92.8% in 1st month to 83.3% in 6th month on PermaNet 2.0®, from 96.9% to 43.80% on Icon Life® and from 85.6% to 14.6% on Olyset®. Wild An. arabiensis were 100% susceptible to DDT, 95.8% to deltamethrin, 90.2% to lambda cyhalothrin and 95.2% susceptible to permethrin. No kdr gene mutations were detected. Conclusions In bioassays where sufficient contact with treated surfaces is assured, LLINs and IRS kill high proportions of susceptible An. arabiensis mosquitoes, though these efficacies decay gradually for LLINs and rapidly for IRS. It is, therefore, important to always add intact nets in sprayed houses, guaranteeing protection even after the IRS decays, and to ensure accurate timing, quality control and regular re-spraying in IRS programmes. By contrast, adding IRS in houses with intact LLINs is unlikely to improve protection relative to LLINs alone, since there is no guarantee that unfed vectors would rest long enough on the sprayed surfaces, and because of the rapid IRS decay. However, there is need to clarify these effects using data from observations of free flying mosquitoes in huts. Physiological susceptibility of An. arabiensis in the area remains 100% against DDT, but is slightly reduced against pyrethroids, necessitating caution over possible spread of resistance. The loss of LLIN toxicity, particularly Olyset® nets suggests that protection offered by these nets against An. arabiensis may be primarily due to physical bite prevention rather than insecticidal efficacy.

Abstract Background Gold-standard tests of mosquito repellents involve exposing human volunteers to host-seeking mosquitoes, to assess the protective efficacy of the repellents. These techniques are not exposure-free and cannot be performed prior to toxicological evaluation. It is postulated that synthetic lures could provide a useful assay that mimics in-vivo conditions for use in high-throughput screening for mosquito repellents. Methods This paper reports on a semi-field evaluation of repellents using a synthetic blend of human derived attractants for the malaria vector, Anopheles gambiae sensu stricto Different concentrations of known repellents, N, N diethyl-3-methylbenzamide (deet) and Para-methane-3, 8, diol (PMD) were added into traps baited with the synthetic blend, and resulting changes in mosquito catches were measured. Results All test concentrations of deet (0.001% to 100%) reduced the attractiveness of the synthetic blend. However, PMD was repellent only at 0.25%. Above this concentration, it significantly increased the attractiveness of the blend. There was no relationship between the repellent concentrations and the change in mosquito catches when either deet (r2 = 0.033, P = 0.302) or PMD (r2 = 0.020, P = 0.578) was used. Conclusion It is concluded that while some repellents may reduce the attractiveness of synthetic human odours, others may instead increase their attractiveness. Such inconsistencies indicate that even though the synthetic attractants may provide exposure-free and consistent test media for repellents, careful selection and multiple-repellent tests are necessary to ascertain their suitability for use in repellent screening. The synthetic odour blend tested here is not yet sufficiently refined to serve as replacement for humans in repellent testing, but may be developed further and evaluated in different formats for exposure free repellent testing purposes.

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.

Background: Malaria elimination is unlikely to occur if vector control efforts focus entirely on transmission occurring indoors without addressing vectors that bite outdoors and outside sleeping hours. Additional control tools such as spatial repellents may provide the personal protection required to fill this gap. However, since repellents do not kill mosquitoes it is unclear if vectors will be diverted from households that use spatial repellents to those that do not., Methods: A crossover study was performed over 24 weeks in Kilombero, Tanzania. The density of resting and blood-engorged mosquitoes and human blood index (HBI) of malaria vector species per household was measured among 90 households using or not using 0.03 % transfluthrin coils burned outdoors under three coverage scenarios: (i) no coverage (blank coils); (ii) complete coverage of repellent coils; and (iii) incomplete coverage of repellent and blank coils. Mosquitoes were collected three days a week for 24 weeks from the inside and outside of all participating households using mosquito aspirators. Paired indoor and outdoor human landing collections were performed in three random households for six consecutive nights to confirm repellent efficacy of the coils and local vector biting times., Results: The main vectors were Anopheles arabiensis and Anopheles funestus (sensu stricto), which fed outdoors, outside sleeping hours, on humans as well as animals. Anopheles arabiensis landings were reduced by 80 % by the spatial repellent although household densities were not reduced. The HBI for An. arabiensis was significantly higher among households without repellents in the incomplete coverage scenario compared to houses in the no coverage scenario (Odds ratio 1.71; 95 % CI: 1.04-2.83; P = 0.03). This indicated that An. arabiensis mosquitoes seeking a human blood meal were diverted from repellent users to non-users. The repellent coils did not affect An. funestus densities or HBI., Conclusions: Substantial malaria vector activity is occurring outside sleeping hours in the Kilombero valley. Repellent coils provided some protection against local An. arabiensis but did not protect against local (and potentially pyrethroid-resistant) An. funestus. Pyrethroid-based spatial repellents may offer a degree of personal protection, however the overall public health benefit is doubtful and potentially iniquitous as their use may divert malaria vectors to those who do not use them.

Background: Before topical repellents can be employed as interventions against arthropod bites, their efficacy must be established. Currently, laboratory or field tests, using human volunteers, are the main methods used for assessing the efficacy of topical repellents. However, laboratory tests are not representative of real life conditions under which repellents are used and field-testing potentially exposes human volunteers to disease. There is, therefore, a need to develop methods to test efficacy of repellents under real life conditions while minimizing volunteer exposure to disease., Methods: A lotion-based, 15% N, N-Diethyl-3-methylbenzamide (DEET) repellent and 15% DEET in ethanol were compared to a placebo lotion in a 200 sq m (10 m × 20 m) semi-field system (SFS) against laboratory-reared Anopheles arabiensis mosquitoes and in full field settings against wild malaria vectors and nuisance-biting mosquitoes. The average percentage protection against biting mosquitoes over four hours in the SFS and field setting was determined. A Poisson regression model was then used to determine relative risk of being bitten when wearing either of these repellents compared to the placebo., Results: Average percentage protection of the lotion-based 15% DEET repellent after four hours of mosquito collection was 82.13% (95% CI 75.94-88.82) in the semi-field experiments and 85.10% (95% CI 78.97-91.70) in the field experiments. Average percentage protection of 15% DEET in ethanol after four hours was 71.29% (CI 61.77-82.28) in the semi-field system and 88.24% (84.45-92.20) in the field., Conclusions: Semi-field evaluation results were comparable to full-field evaluations, indicating that such systems could be satisfactorily used in measuring efficacy of topically applied mosquito repellents, thereby avoiding risks of exposure to mosquito-borne pathogens, associated with field testing.

Background: Current malaria vector control programmes rely on insecticides with rapid contact toxicity. However, spatial repellents can also be applied to reduce man-vector contact, which might ultimately impact malaria transmission. The aim of this study was to quantify effects of airborne pyrethroids from coils and DDT used an indoor residual spray (IRS) on entomological parameters that influence malaria transmission., Methods: The effect of Transfluthrin and Metofluthrin coils compared to DDT on house entry, exit and indoor feeding behaviour of Anopheles gambiae sensu lato were measured in experimental huts in the field and in the semi-field. Outcomes were deterrence--reduction in house entry of mosquitoes; irritancy or excito-repellency--induced premature exit of mosquitoes; blood feeding inhibition and effect on mosquito fecundity., Results: Transfluthrin coils, Metofluthrin coils and DDT reduced human vector contact through deterrence by 38%, 30% and 8%, respectively and induced half of the mosquitoes to leave huts before feeding (56%, 55% and 48%, respectively). Almost all mosquitoes inside huts with Metofluthrin and Transfluthrin coils and more than three quarters of mosquitoes in the DDT hut did not feed, almost none laid eggs and 67%, 72% and 70% of all mosquitoes collected from Transfluthrin, Metofluthrin and DDT huts, respectively had died after 24 hours., Conclusion: This study highlights that airborne pyrethroids and DDT affect a range of anopheline mosquito behaviours that are important parameters in malaria transmission, namely deterrence, irritancy/excito-repellency and blood-feeding inhibition. These effects are in addition to significant toxicity and reduced mosquito fecundity that affect mosquito densities and, therefore, provide community protection against diseases for both users and non-users. Airborne insecticides and freshly applied DDT had similar effects on deterrence, irritancy and feeding inhibition. Therefore, it is suggested that airborne pyrethroids, if delivered in suitable formats, may complement existing mainstream vector control tools.

Background: In recent years, evidence of Rift Valley fever (RVF) transmission during inter-epidemic periods in parts of Africa has increasingly been reported. The inter-epidemic transmissions generally pass undetected where there is no surveillance in the livestock or human populations. We studied the presence of and the determinants for inter-epidemic RVF transmission in an area experiencing annual flooding in southern Tanzania., Methodology: A cross-sectional sero-survey was conducted in randomly selected cattle, sheep and goats in the Kilombero river valley from May to August 2011, approximately four years after the 2006/07 RVF outbreak in Tanzania. The exposure status to RVF virus (RVFV) was determined using two commercial ELISA kits, detecting IgM and IgG antibodies in serum. Information about determinants was obtained through structured interviews with herd owners., Findings: An overall seroprevalence of 11.3% (n = 1680) was recorded; 5.5% in animals born after the 2006/07 RVF outbreak and 22.7% in animals present during the outbreak. There was a linear increase in prevalence in the post-epidemic annual cohorts. Nine inhibition-ELISA positive samples were also positive for RVFV IgM antibodies indicating a recent infection. The spatial distribution of seroprevalence exhibited a few hotspots. The sex difference in seroprevalence in animals born after the previous epidemic was not significant (6.1% vs. 4.6% for females and males respectively, p = 0.158) whereas it was significant in animals present during the outbreak (26.0% vs. 7.8% for females and males respectively, p<0.001). Animals living >15 km from the flood plain were more likely to have antibodies than those living <5 km (OR 1.92; 95% CI 1.04-3.56). Species, breed, herd composition, grazing practices and altitude were not associated with seropositivity., Conclusion: These findings indicate post-epidemic transmission of RVFV in the study area. The linear increase in seroprevalence in the post-epidemic annual cohorts implies a constant exposure and presence of active foci transmission preceding the survey.

Background: Long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) are commonly used together in the same households to improve malaria control despite inconsistent evidence on whether such combinations actually offer better protection than nets alone or IRS alone., Methods: Comparative tests were conducted using experimental huts fitted with LLINs, untreated nets, IRS plus untreated nets, or combinations of LLINs and IRS, in an area where Anopheles arabiensis is the predominant malaria vector species. Three LLIN types, Olyset®, PermaNet 2.0® and Icon Life® nets and three IRS treatments, pirimiphos-methyl, DDT, and lambda cyhalothrin, were used singly or in combinations. We compared, number of mosquitoes entering huts, proportion and number killed, proportions prevented from blood-feeding, time when mosquitoes exited the huts, and proportions caught exiting. The tests were done for four months in dry season and another six months in wet season, each time using new intact nets., Results: All the net types, used with or without IRS, prevented >99% of indoor mosquito bites. Adding PermaNet 2.0® and Icon Life®, but not Olyset® nets into huts with any IRS increased mortality of malaria vectors relative to IRS alone. However, of all IRS treatments, only pirimiphos-methyl significantly increased vector mortality relative to LLINs alone, though this increase was modest. Overall, median mortality of An. arabiensis caught in huts with any of the treatments did not exceed 29%. No treatment reduced entry of the vectors into huts, except for marginal reductions due to PermaNet 2.0® nets and DDT. More than 95% of all mosquitoes were caught in exit traps rather than inside huts., Conclusions: Where the main malaria vector is An. arabiensis, adding IRS into houses with intact pyrethroid LLINs does not enhance house-hold level protection except where the IRS employs non-pyrethroid insecticides such as pirimiphos-methyl, which can confer modest enhancements. In contrast, adding intact bednets onto IRS enhances protection by preventing mosquito blood-feeding (even if the nets are non-insecticidal) and by slightly increasing mosquito mortality (in case of LLINs). The primary mode of action of intact LLINs against An. arabiensis is clearly bite prevention rather than insecticidal activity. Therefore, where resources are limited, priority should be to ensure that everyone at risk consistently uses LLINs and that the nets are regularly replaced before being excessively torn. Measures that maximize bite prevention (e.g. proper net sizes to effectively cover sleeping spaces, stronger net fibres that resist tears and burns and net use practices that preserve net longevity), should be emphasized.

Differences between individual human houses can confound results of studies aimed at evaluating indoor vector control interventions such as insecticide treated nets (ITNs) and indoor residual insecticide spraying (IRS). Specially designed and standardised experimental huts have historically provided a solution to this challenge, with an added advantage that they can be fitted with special interception traps to sample entering or exiting mosquitoes. However, many of these experimental hut designs have a number of limitations, for example: 1) inability to sample mosquitoes on all sides of huts, 2) increased likelihood of live mosquitoes flying out of the huts, leaving mainly dead ones, 3) difficulties of cleaning the huts when a new insecticide is to be tested, and 4) the generally small size of the experimental huts, which can misrepresent actual local house sizes or airflow dynamics in the local houses. Here, we describe a modified experimental hut design - The Ifakara Experimental Huts- and explain how these huts can be used to more realistically monitor behavioural and physiological responses of wild, free-flying disease-transmitting mosquitoes, including the African malaria vectors of the species complexes Anopheles gambiae and Anopheles funestus, to indoor vector control-technologies including ITNs and IRS. Important characteristics of the Ifakara experimental huts include: 1) interception traps fitted onto eave spaces and windows, 2) use of eave baffles (panels that direct mosquito movement) to control exit of live mosquitoes through the eave spaces, 3) use of replaceable wall panels and ceilings, which allow safe insecticide disposal and reuse of the huts to test different insecticides in successive periods, 4) the kit format of the huts allowing portability and 5) an improved suite of entomological procedures to maximise data quality.

Background: Disease transmitting mosquitoes locate humans and other blood hosts by identifying their characteristic odor profiles. Using their olfactory organs, the mosquitoes detect compounds present in human breath, sweat and skins, and use these as cues to locate and obtain blood from the humans. These odor compounds can be synthesized in vitro, then formulated to mimic humans. While some synthetic mosquito lures already exist, evidence supporting their utility is limited to laboratory settings, where long-range stimuli cannot be investigated., Methodology and Principal Findings: Here we report the development and field evaluation of an odor blend consisting of known mosquito attractants namely carbon dioxide, ammonia and carboxylic acids, which was optimized at distances comparable with attractive ranges of humans to mosquitoes. Binary choice assays were conducted inside a large-cage semi-field enclosure using attractant-baited traps placed 20 m apart. This enabled high-throughput optimization of concentrations at which the individual candidate attractants needed to be added so as to obtain a blend maximally attractive to laboratory-reared An. gambiae. To determine whether wild mosquitoes would also be attracted to this synthetic odor blend and to compare it with whole humans under epidemiologically relevant conditions, field experiments were conducted inside experimental huts, where the blend was compared with 10 different adult male volunteers (20-34 years old). The blend attracted 3 to 5 times more mosquitoes than humans when the two baits were in different experimental huts (10-100 metres apart), but was equally or less attractive than humans when compared side by side within same huts., Conclusion and Significance: This highly attractive substitute for human baits might enable development of technologies for trapping mosquitoes in numbers sufficient to prevent rather than merely monitor transmission of mosquito-borne diseases.

Background: Gold-standard tests of mosquito repellents involve exposing human volunteers to host-seeking mosquitoes, to assess the protective efficacy of the repellents. These techniques are not exposure-free and cannot be performed prior to toxicological evaluation. It is postulated that synthetic lures could provide a useful assay that mimics in-vivo conditions for use in high-throughput screening for mosquito repellents., Methods: This paper reports on a semi-field evaluation of repellents using a synthetic blend of human derived attractants for the malaria vector, Anopheles gambiae sensu stricto Different concentrations of known repellents, N, N diethyl-3-methylbenzamide (deet) and Para-methane-3, 8, diol (PMD) were added into traps baited with the synthetic blend, and resulting changes in mosquito catches were measured., Results: All test concentrations of deet (0.001% to 100%) reduced the attractiveness of the synthetic blend. However, PMD was repellent only at 0.25%. Above this concentration, it significantly increased the attractiveness of the blend. There was no relationship between the repellent concentrations and the change in mosquito catches when either deet (r2 = 0.033, P = 0.302) or PMD (r2 = 0.020, P = 0.578) was used., Conclusion: It is concluded that while some repellents may reduce the attractiveness of synthetic human odours, others may instead increase their attractiveness. Such inconsistencies indicate that even though the synthetic attractants may provide exposure-free and consistent test media for repellents, careful selection and multiple-repellent tests are necessary to ascertain their suitability for use in repellent screening. The synthetic odour blend tested here is not yet sufficiently refined to serve as replacement for humans in repellent testing, but may be developed further and evaluated in different formats for exposure free repellent testing purposes.