Biologically meaningful coverage indicators for eliminating malaria transmission.

Mosquitoes, which evade contact with long-lasting insecticidal nets and indoor residual sprays, by feeding outdoors or upon animals, are primary malaria vectors in many tropical countries. They can also dominate residual transmission where high coverage of these front-line vector control measures is achieved. Complementary strategies, which extend insecticide coverage beyond houses and humans, are required to eliminate malaria transmission in most settings. The overwhelming diversity of the world's malaria transmission systems and optimal strategies for controlling them can be simply conceptualized and mapped across two-dimensional scenario space defined by the proportion of blood meals that vectors obtain from humans and the proportion of human exposure to them which occurs indoors

Using adult mosquitoes to transfer insecticides to Aedes aegypti larval habitats.

Vector control is a key means of combating mosquito-borne diseases and the only tool available for tackling the transmission of dengue, a disease for which no vaccine, prophylaxis, or therapeutant currently exists. The most effective mosquito control methods include a variety of insecticidal tools that target adults or juveniles. Their successful implementation depends on impacting the largest proportion of the vector population possible. We demonstrate a control strategy that dramatically improves the efficiency with which high coverage of aquatic mosquito habitats can be achieved. The method exploits adult mosquitoes as vehicles of insecticide transfer by harnessing their fundamental behaviors to disseminate a juvenile hormone analogue (JHA) between resting and oviposition sites. A series of field trials undertaken in an Amazon city (Iquitos, Peru) showed that the placement of JHA dissemination stations in just 3-5% of the available resting area resulted in almost complete coverage of sentinel aquatic habitats. More than control mortality occurred in 95-100% of the larval cohorts of Aedes aegypti developing at those sites. Overall reductions in adult emergence of 42-98% were achieved during the trials. A deterministic simulation model predicts amplifications in coverage consistent with our observations and highlights the importance of the residual activity of the insecticide for this technique

Challenges for Malaria Elimination in Zanzibar: Pyrethroid Resistance in Malaria Vectors and Poor Performance of Long-Lasting Insecticide Nets.

Long-lasting insecticide treated nets (LLINs) and indoor residual house spraying (IRS) are the main interventions for the control of malaria vectors in Zanzibar. The aim of the present study was to assess the susceptibility status of malaria vectors against the insecticides used for LLINs and IRS and to determine the durability and efficacy of LLINs on the island. Mosquitoes were sampled from Pemba and Unguja islands in 2010--2011 for use in WHO susceptibility tests. One hundred and fifty LLINs were collected from households on Unguja, their physical state was recorded and then tested for efficacy as well as total insecticide content. Species identification revealed that over 90% of the Anopheles gambiae complex was An. arabiensis with a small number of An. gambiae s.s. and An. merus being present. Susceptibility tests showed that An. arabiensis on Pemba was resistant to the pyrethroids used for LLINs and IRS. Mosquitoes from Unguja Island, however, were fully susceptible to all pyrethroids tested. A physical examination of 150 LLINs showed that two thirds were damaged after only three years in use. All used nets had a significantly lower (p < 0.001) mean permethrin concentration of 791.6 mg/m2 compared with 944.2 mg/m2 for new ones. Their efficacy decreased significantly against both susceptible An. gambiae s.s. colony mosquitoes and wild-type mosquitoes from Pemba after just six washes (p < 0.001). The sustainability of the gains achieved in malaria control in Zanzibar is seriously threatened by the resistance of malaria vectors to pyrethroids and the short-lived efficacy of LLINs. This study has revealed that even in relatively well-resourced and logistically manageable places like Zanzibar, malaria elimination is going to be difficult to achieve with the current control measures

Effective autodissemination of pyriproxyfen to breeding sites by the exophilic malaria vector Anopheles arabiensis in semi-field settings in Tanzania

BACKGROUND Malaria vector control strategies that target adult female mosquitoes are challenged by the emergence of insecticide resistance and behavioural resilience. Conventional larviciding is restricted by high operational costs and inadequate knowledge of mosquito-breeding habitats in rural settings that might be overcome by the juvenile hormone analogue, Pyriproxyfen (PPF). This study assessed the potential for Anopheles arabiensis to pick up and transfer lethal doses of PPF from contamination sites to their breeding habitats (i.e. autodissemination of PPF). METHODS A semi-field system (SFS) with four identical separate chambers was used to evaluate PPF-treated clay pots for delivering PPF to resting adult female mosquitoes for subsequent autodissemination to artificial breeding habitats within the chambers. In each chamber, a tethered cow provided blood meals to laboratory-reared, unfed female An. arabiensis released in the SFS. In PPF-treated chambers, clay pot linings were dusted with 0.2 - 0.3 g AI PPF per pot. Pupae were removed from the artificial habitats daily, and emergence rates calculated. Impact of PPF on emergence was determined by comparing treatment with an appropriate control group. RESULTS Mean (95%CI) adult emergence rates were (0.21 +/- 0.299) and (0.95 +/- 0.39) from PPF-treated and controls respectively (p < 0.0001). Laboratory bioassay of water samples from artificial habitats in these experiments resulted in significantly lower emergence rates in treated chambers (0.16 +/- 0.23) compared to controls 0.97 +/- 0.05) (p < 0.0001). In experiments where no mosquitoes introduced, there were no significant differences between control and treatment, indicating that transfer of PPF to breeding sites only occurred when mosquitoes were present; i.e. that autodissemination had occurred. Treatment of a single clay pot reduced adult emergence in six habitats to (0.34 +/- 0.13) compared to (0.98 +/- 0.02) in the controls (p < 0.0001), showing a high level of habitats coverage amplification of the autodissemination event. CONCLUSION The study provides proof of principle for the autodissemination of PPF to breeding habitats by malaria vectors. These findings highlight the potential for this technique for outdoor control of malaria vectors and call for the testing of this technique in field trials

Adaptation and evaluation of the bottle assay for monitoring insecticide resistance in disease vector mosquitoes in the Peruvian Amazon

<p>Abstract</p> <p>Background</p> <p>The purpose of this study was to establish whether the "bottle assay", a tool for monitoring insecticide resistance in mosquitoes, can complement and augment the capabilities of the established WHO assay, particularly in resource-poor, logistically challenging environments.</p> <p>Methods</p> <p>Laboratory reared <it>Aedes aegypti </it>and field collected <it>Anopheles darlingi </it>and <it>Anopheles albimanus </it>were used to assess the suitability of locally sourced solvents and formulated insecticides for use with the bottle assay. Using these adapted protocols, the ability of the bottle assay and the WHO assay to discriminate between deltamethrin-resistant <it>Anopheles albimanus </it>populations was compared. The diagnostic dose of deltamethrin that would identify resistance in currently susceptible populations of <it>An. darlingi </it>and <it>Ae. aegypti </it>was defined. The robustness of the bottle assay during a surveillance exercise in the Amazon was assessed.</p> <p>Results</p> <p>The bottle assay (using technical or formulated material) and the WHO assay were equally able to differentiate deltamethrin-resistant and susceptible <it>An. albimanus </it>populations. A diagnostic dose of 10 μg a.i./bottle was identified as the most sensitive discriminating dose for characterizing resistance in <it>An. darlingi </it>and <it>Ae. aegypti</it>. Treated bottles, prepared using locally sourced solvents and insecticide formulations, can be stored for > 14 days and used three times. Bottles can be stored and transported under local conditions and field-assays can be completed in a single evening.</p> <p>Conclusion</p> <p>The flexible and portable nature of the bottle assay and the ready availability of its components make it a potentially robust and useful tool for monitoring insecticide resistance and efficacy in remote areas that require minimal cost tools.</p

Simplified Models of Vector Control Impact upon Malaria Transmission by Zoophagic Mosquitoes

BACKGROUND\ud \ud High coverage of personal protection measures that kill mosquitoes dramatically reduce malaria transmission where vector populations depend upon human blood. However, most primary malaria vectors outside of sub-Saharan Africa can be classified as "very zoophagic," meaning they feed occasionally (<10% of blood meals) upon humans, so personal protection interventions have negligible impact upon their survival.\ud \ud METHODS AND FINDINGS\ud \ud We extended a published malaria transmission model to examine the relationship between transmission, control, and the baseline proportion of bloodmeals obtained from humans (human blood index). The lower limit of the human blood index enables derivation of simplified models for zoophagic vectors that (1) Rely on only three field-measurable parameters. (2) Predict immediate and delayed (with and without assuming reduced human infectivity, respectively) impacts of personal protection measures upon transmission. (3) Illustrate how appreciable indirect communal-level protection for non-users can be accrued through direct personal protection of users. (4) Suggest the coverage and efficacy thresholds required to attain epidemiological impact. The findings suggest that immediate, indirect, community-wide protection of users and non-users alike may linearly relate to the efficacy of a user's direct personal protection, regardless of whether that is achieved by killing or repelling mosquitoes. High protective coverage and efficacy (≥80%) are important to achieve epidemiologically meaningful impact. Non-users are indirectly protected because the two most common species of human malaria are strict anthroponoses. Therefore, the small proportion of mosquitoes that are killed or diverted while attacking humans can represent a large proportion of those actually transmitting malaria.\ud \ud CONCLUSIONS\ud \ud Simplified models of malaria transmission by very zoophagic vectors may be used by control practitioners to predict intervention impact interventions using three field-measurable parameters; the proportion of human exposure to mosquitoes occurring when an intervention can be practically used, its protective efficacy when used, and the proportion of people using it

Reducing biting rates of Aedes aegypti with metofluthrin: investigations in time and space

Background: Indoor residual spraying is key to dengue control in Cairns and other parts of northern Queensland, Australia, where Aedes aegypti is prevalent, but the strategy faces challenges with regards to slow application time and, therefore, community coverage. A faster potential improvement might be the use of polyethylene netting impregnated with the volatile pyrethroid metofluthrin (SumiOne™). This formulation was assessed in rooms in three houses in Cairns, Australia. One emanator was placed in each room and cages of 10 female Aedes aegypti were exposed at distances of 1 and 3 m. Knockdown and landings on a human hand were counted before metofluthrin exposure and at 10, 30, 60, 90 and 120 min during exposure. In addition, two trials continued over 48 h of exposure to assess the long-term sublethal effects of metofluthrin on caged mosquitoes. Results: Percentage landing rates fell to 0-2.5% in the first 10 min of exposure. Knockdown was most evident between 10 and 30 min (54% at 1 m and 33% at 3 m). Distance from the emanator strongly affected the results: mosquitoes at 3 m exhibited less knockdown and more landings than those at 1 m. As room volume increased, knockdown decreased and the number of landing increased. There is a cumulative mortality and landing inhibition and, for mosquitoes exposed to metofluthrin for > 48 h, mortality was 100% at 1 m and 90% at 3 m. Of those still alive, a small number continued to land and bite. After being removed from metofluthrin-treated rooms, exposed insect cages were found to reducing landing rates for up to 2 h. Conclusions: Despite only moderate levels of knockdown during the initial hours of exposure, metofluthrin emanators were effective in reducing mosquito landing rates, especially within 1 m, even when exposed on an open veranda. The evaluation methods and results described in this paper will help inform the optimal conditions of deployment of metofluthrin emanators. These devices have the potential to reduce contact between humans and urban disease vectors faster than indoor residual spraying so supplement our current arsenal of dengue control tools

Effect of Serotype and Strain Diversity on Dengue Virus Replication in Australian Mosquito Vectors

Dengue virus (DENV) is the most important mosquito-borne viral pathogen of humans, comprising four serotypes (DENV-1 to -4) with a myriad of genotypes and strains. The kinetics of DENV replication within the mosquito following ingestion of a blood meal influence the pathogen’s ability to reach the salivary glands and thus the transmission potential. The influence of DENV serotype and strain diversity on virus kinetics in the two main vector species, Aedes aegypti and Ae. albopictus, has been poorly characterized. We tested whether DENV replication kinetics vary systematically among serotypes and strains, using Australian strains of the two vectors. Mosquitoes were blood fed with two strains per serotype, and sampled at 3, 6, 10 and 14-days post-exposure. Virus infection in mosquito bodies, and dissemination of virus to legs and wings, was detected using qRT-PCR. For both vectors, we found significant differences among serotypes in proportions of mosquitoes infected, with higher numbers for DENV-1 and -2 versus other serotypes. Consistent with this, we observed that DENV-1 and -2 generally replicated to higher RNA levels than other serotypes, particularly at earlier time points. There were no significant differences in either speed of infection or dissemination between the mosquito species. Our results suggest that DENV diversity may have important epidemiological consequences by influencing virus kinetics in mosquito vectors

The Dynamics of Pyrethroid Nesistance in Anopheles Arabiensis from Zanzibar and an Assessment of the Underlying Genetic Basis.

The emergence of pyrethroid resistance in the malaria vector, Anopheles arabiensis, threatens to undermine the considerable gains made towards eliminating malaria on Zanzibar. Previously, resistance was restricted to the island of Pemba while mosquitoes from Unguja, the larger of the two islands of Zanzibar, were susceptible. Here, we characterised the mechanism(s) responsible for resistance on Zanzibar using a combination of gene expression and target-site mutation assays. WHO resistance bioassays were conducted using 1-5d old adult Anopheles gambiae s.l. collected between 2011 and 2013 across the archipelago. Synergist assays with the P450 inhibitor piperonyl-butoxide were performed in 2013. Members of the An. gambiae complex were PCR-identified and screened for target-site mutations (kdr and Ace-1). Gene expression in pyrethroid resistant An. arabiensis from Pemba was analysed using whole-genome microarrays. Pyrethroid resistance is now present across the entire Zanzibar archipelago. Survival to the pyrethroid lambda-cyhalothrin in bioassays conducted in 2013 was 23.5-54.3% on Unguja and 32.9-81.7% on Pemba. We present evidence that resistance is mediated, in part at least, by elevated P450 monoxygenases. Whole-genome microarray scans showed that the most enriched gene terms in resistant An. arabiensis from Pemba were associated with P450 activity and synergist assays with PBO completely restored susceptibility to pyrethroids in both islands. CYP4G16 was the most consistently over-expressed gene in resistant mosquitoes compared with two susceptible strains from Unguja and Dar es Salaam. Expression of this P450 is enriched in the abdomen and it is thought to play a role in hydrocarbon synthesis. Microarray and qPCR detected several additional genes putatively involved in this pathway enriched in the Pemba pyrethroid resistant population and we hypothesise that resistance may be, in part, related to alterations in the structure of the mosquito cuticle. None of the kdr target-site mutations, associated with pyrethroid/DDT resistance in An. gambiae elsewhere in Africa, were found on the islands. The consequences of this resistance phenotype are discussed in relation to future vector control strategies on Zanzibar to support the ongoing malaria elimination efforts on the islands

El Niño Southern Oscillation, overseas arrivals and imported chikungunya cases in Australia: A time series analysis.

BACKGROUND: Chikungunya virus (CHIKV) is an emerging mosquito-borne pathogen circulating in tropical and sub-tropical regions. Although autochthonous transmission has not been reported in Australia, there is a potential risk of local CHIKV outbreaks due to the presence of suitable vectors, global trade, frequent international travel and human adaptation to changes in climate. METHODOLOGY/PRINCIPAL FINDINGS: A time series seasonal decomposition method was used to investigate the seasonality and trend of monthly imported CHIKV cases. This pattern was compared with the seasonality and trend of monthly overseas arrivals. A wavelet coherence analysis was applied to examine the transient relationships between monthly imported CHIKV cases and southern oscillation index (SOI) in time-frequency space. We found that the number and geographical distribution of countries of acquisition for CHIKV in travellers to Australia has increased in recent years. The number of monthly imported CHIKV cases displayed an unstable increased trend compared with a stable linear increased trend in monthly overseas arrivals. Both imported CHIKV cases and overseas arrivals showed substantial seasonality, with the strongest seasonal effects in each January, followed by each October and July. The wavelet coherence analysis identified four significant transient relationships between monthly imported CHIKV cases and 6-month lagged moving average SOI, in the years 2009-2010, 2012, 2014 and 2015-2016. CONCLUSION/SIGNIFICANCE: High seasonal peaks of imported CHIKV cases were consistent with the high seasonal peaks of overseas arrivals into Australia. Our analysis also indicates that El Niño Southern Oscillation (ENSO) variation may impact CHIKV epidemics in endemic regions, in turn influencing the pattern of imported cases