\ud Detection and Monitoring of Insecticide Resistance in Malaria Vectors in Tanzania Mainland\ud

\ud Vector control is a major component of the global strategy for malaria control which aims to prevent parasite transmission mainly through interventions targeting adult Anopheline vectors. Insecticide treated nets (ITNs) and indoor residual spraying (IRS) are the cornerstone of malaria vector control programmes. These major interventions in most cases use pyrethroid insecticides which are also used for agricultural purposes. With widespread development of resistance to pyrethroid insecticides in malaria vectors raises concern over the sustainability of insecticide-based interventions for malaria control. Therefore, close monitoring of performance of the insecticides against malaria vectors is essential for early detection and\ud management of resistance. To measure pyrethroid susceptibility in populations of malaria vectors in Tanzania and to test the efficacy of LLINs/ITNs and insecticide residues on sprayed wall substrates in the IRS operation areas. In 2011 the National Institute for Medical Research (NIMR) in collaboration with National Malaria Control Programme (NMCP) conducted large scale surveillance to determine the countrywide susceptibility levels of malaria vectors to insecticides used for both public health and agricultural purposes. Anopheles gambiae Giles s.l. were collected during national surveys and samples of LLINs/ITNs in the 14 sentinel sites and houses from the IRS areas were randomly selected for bioassays to test the efficacy and insecticide residual effects on sprayed wall substrates respectively. Wild adult mosquitoes for susceptibility testing were collected by resting catches indoors. Net traps (outdoors and indoors) were set up to enhance catches. WHO Susceptibility kits were used to test for resistance status using test papers: Lambdacyhalothrin 0.05%, Deltamethrin 0.05%, Permethrin 0.75%, DDT 4%, Propoxur 0.1% and Fenitrothion 1%. The quality of the test paper was checked against a laboratory susceptible An. gambiae Kisumu strain. Knockdown effect and mortality were measured in standard WHO susceptibility tests and cone bio-efficacy tests. Whereas, con bioassays on treated walls and ITNs were conducted using the laboratory susceptible An. gambiae Kisumu strain. The results from the surveillance recorded continued susceptibility of malaria vectors to commonly used insecticides. However, there were some isolated cases of resistance and/or reduced susceptibility to pyrethroid insecticides which may not compromise the current vector control interventions in the country. Anopheles gambiae s.l. showed resistance (15-28%) to each of the pyrethroids and to DDT but not to Organophosphates (Propoxur 0.1%), and Carbamates (Fenitrothion 1%). The information obtained from this surveillance is expected to be used to guide the National Malaria Control Programme on the rational selection of insecticides for malaria vector control and for the national mitigation plans for management and containment of malaria vector resistance in the country. The current observation warrants more vigilant monitoring of the susceptibility of malaria mosquitoes to commonly used insecticides in areas found with resistance and/or reduced levels of susceptibility of malaria vectors to insecticides, particularly in areas with heavy agricultural and/or public health use of insecticides where resistance is likely to develop. The current survey covered malaria vectors only and not the non malaria vectors (nuisance) mosquitoes such as Culex. Similar monitoring of insecticide susceptibility of this non malaria vectors may be needed to ensure public motivation for sustained use of ITNs/LLINs in the country. The surveillance leading to these results received funding from PMI/USAID through RTI International with Sub Agreement Number 33300212555.\u

Novel use of stir bar sorptive extraction (SBSE) as a tool for isolation of oviposition site attractants for gravid Culex quinquefasciatus

Mosquitoes such as Culex quinquefasciatus Say (Diptera: Culicidae) are important vectors of organisms that cause disease in humans. Research into the development of effective standardized odour baits for blood-fed females (oviposition attractants), to enable entomological monitoring of vector populations, is hampered by complex protocols for extraction of physiologically active volatile chemicals from natural breeding site water samples, which have produced inconsistent results. Air entrainment and solvent extraction are technically demanding methods and are impractical for use in resource poor environments where mosquito-borne disease is most prevalent. This study reports the first use of a simple, robust extraction technique, stir bar sorptive extraction (SBSE), to extract behaviourally active small lipophilic molecules (SLMs) present in water samples collected from Cx. quinquefasciatus breeding sites in Tanzania. Extracts from a pit latrine and from a cess pool breeding site attracted more gravid Cx. quinquefasciatus in pair choice bioassays than control extracts, and coupled gas chromatography-electroantennography (GC-EAG) allowed tentative identification of 15 electrophysiologically active chemicals, including the known oviposition attractant, skatole (3-methylindole). Here, we have demonstrated, using simple pair choice bioassays in controlled laboratory conditions, that SBSE is effective for the extraction of behaviourally and electrophysiologically active semiochemicals from mosquito breeding site waters. Further research is required to confirm that SBSE is an appropriate technique for use in field surveys in the search for oviposition cues for Cx. quinquefasciatus

Using a New Odour-Baited Device to Explore Options for Luring and Killing Outdoor-Biting Malaria Vectors: A Report on Design and Field Evaluation of the Mosquito Landing Box.

Mosquitoes that bite people outdoors can sustain malaria transmission even where effective indoor interventions such as bednets or indoor residual spraying are already widely used. Outdoor tools may therefore complement current indoor measures and improve control. We developed and evaluated a prototype mosquito control device, the 'Mosquito Landing Box' (MLB), which is baited with human odours and treated with mosquitocidal agents. The findings are used to explore technical options and challenges relevant to luring and killing outdoor-biting malaria vectors in endemic settings. Field experiments were conducted in Tanzania to assess if wild host-seeking mosquitoes 1) visited the MLBs, 2) stayed long or left shortly after arrival at the device, 3) visited the devices at times when humans were also outdoors, and 4) could be killed by contaminants applied on the devices. Odours suctioned from volunteer-occupied tents were also evaluated as a potential low-cost bait, by comparing baited and unbaited MLBs. There were significantly more Anopheles arabiensis, An. funestus, Culex and Mansonia mosquitoes visiting baited MLB than unbaited controls (P<=0.028). Increasing sampling frequency from every 120 min to 60 and 30 min led to an increase in vector catches of up to 3.6 fold (P<=0.002), indicating that many mosquitoes visited the device but left shortly afterwards. Outdoor host-seeking activity of malaria vectors peaked between 7:30 and 10:30pm, and between 4:30 and 6:00am, matching durations when locals were also outdoors. Maximum mortality of mosquitoes visiting MLBs sprayed or painted with formulations of candidate mosquitocidal agent (pirimiphos-methyl) was 51%. Odours from volunteer occupied tents attracted significantly more mosquitoes to MLBs than controls (P<0.001). While odour-baited devices such as the MLBs clearly have potential against outdoor-biting mosquitoes in communities where LLINs are used, candidate contaminants must be those that are effective at ultra-low doses even after short contact periods, since important vector species such as An. arabiensis make only brief visits to such devices. Natural human odours suctioned from occupied dwellings could constitute affordable sources of attractants to supplement odour baits for the devices. The killing agents used should be environmentally safe, long lasting, and have different modes of action (other than pyrethroids as used on LLINs), to curb the risk of physiological insecticide resistance

Indoor application of attractive toxic sugar bait (ATSB) in combination with mosquito nets for control of pyrethroid-resistant mosquitoes.

BACKGROUND: Attractive toxic sugar bait (ATSB) sprayed onto vegetation has been successful in controlling Anopheles mosquitoes outdoors. Indoor application of ATSB has yet to be explored. The purpose of this study was to determine whether ATSB stations positioned indoors have the potential to kill host-seeking mosquitoes and constitute a new approach to control of mosquito-borne diseases. METHODS: Insecticides were mixed with dyed sugar solution and tested as toxic baits against Anopheles arabiensis, An. Gambiae s.s. and Culex quinquefasciatus in feeding bioassay tests to identify suitable attractant-insecticide combinations. The most promising ATSB candidates were then trialed in experimental huts in Moshi, Tanzania. ATSB stations were hung in huts next to untreated mosquito nets occupied by human volunteers. The proportions of mosquitoes killed in huts with ATSB treatments relative to huts with non-insecticide control treatments huts were recorded, noting evidence of dye in mosquito abdomens. RESULTS: In feeding bioassays, chlorfenapyr 0.5% v/v, boric acid 2% w/v, and tolfenpyrad 1% v/v, mixed in a guava juice-based bait, each killed more than 90% of pyrethroid-susceptible An. Gambiae s.s. and pyrethroid-resistant An. arabiensis and Cx. quinquefasciatus. In the hut trial, mortality rates of the three ATSB treatments ranged from 41-48% against An. arabiensis and 36-43% against Cx. quinquefasciatus and all were significantly greater than the control mortalities: 18% for An. arabiensis, 7% for Cx. quinquefasciatus (p<0.05). Mortality rates with ATSB were comparable to those with long lasting insecticidal nets previously tested against the same species in this area. CONCLUSIONS: Indoor ATSB shows promise as a supplement to mosquito nets for controlling mosquitoes. Indoor ATSB constitute a novel application method for insecticide classes that act as stomach poisons and have not hitherto been exploited for mosquito control. Combined with LLIN, indoor use of ATSB has the potential to serve as a strategy for managing insecticide resistance

Resting behaviour of Anopheles gambiae s.l. and its implication on malaria transmission in Uyui District, western Tanzania

An entomological survey to determine resting behaviour and species composition of malaria vectors was carried out in Uyui District in western Tanzania in May 2009. Mosquitoes were collected using indoor resting catch, window exit trap and outdoor &ldquo;bed-net&rdquo; techniques. They mosquitoes were identified using morphological key and polymerase chain reaction (PCR). A total of 672 Anopheles gambiae sensu lato were collected. Of these, 661 (98.4%) were collected outdoor whereas few (1.6%) were collected indoor. The exit trap catch: mechanical aspirator catch ratio was 1:1.75. The overall indoor resting density of An. gambiae s.l. as determined by mechanical aspirator and exit trap was 0.7 and 0.5 mosquitoes per room, respectively. The overall density of the host-seeking as determined by bed net trap outdoor was 44.1 mosquitoes per person. A sample of 44 specimens taken randomly from morphologically identified An.gambiae s.l. population was further analyzed to species level using PCR techniques. Of these 44 specimens 26 (59%) and 18 (41%) were Anopheles arabiensis and Anopheles gambiae sensu stricto respectively. This study contributes to the understanding of the distribution of malaria vectors with respect to species composition and their resting behaviour that could contribute to vector control operations in western Tanzania. A longitudinal study considering dry and wet seasons is recommended to provide more information on the seasonal distribution, abundance and biting behaviour of malaria vectors in the study area

The Impact of Pyrethroid Resistance on the Efficacy of Insecticide-Treated Bed Nets against African Anopheline Mosquitoes: Systematic Review and Meta-Analysis

Background Pyrethroid insecticide-treated bed nets (ITNs) help contribute to reducing malaria deaths in Africa, but their efficacy is threatened by insecticide resistance in some malaria mosquito vectors. We therefore assessed the evidence that resistance is attenuating the effect of ITNs on entomological outcomes. Methods and Findings We included laboratory and field studies of African malaria vectors that measured resistance at the time of the study and used World Health Organization–recommended impregnation regimens. We reported mosquito mortality, blood feeding, induced exophily (premature exit of mosquitoes from the hut), deterrence, time to 50% or 95% knock-down, and percentage knock-down at 60 min. Publications were searched from 1 January 1980 to 31 December 2013 using MEDLINE, Cochrane Central Register of Controlled Trials, Science Citation Index Expanded, Social Sciences Citation Index, African Index Medicus, and CAB Abstracts. We stratified studies into three levels of insecticide resistance, and ITNs were compared with untreated bed nets (UTNs) using the risk difference (RD). Heterogeneity was explored visually and statistically. Included were 36 laboratory and 24 field studies, reported in 25 records. Studies tested and reported resistance inconsistently. Based on the meta-analytic results, the difference in mosquito mortality risk for ITNs compared to UTNs was lower in higher resistance categories. However, mortality risk was significantly higher for ITNs compared to UTNs regardless of resistance. For cone tests: low resistance, risk difference (RD) 0.86 (95% CI 0.72 to 1.01); moderate resistance, RD 0.71 (95% CI 0.53 to 0.88); high resistance, RD 0.56 (95% CI 0.17 to 0.95). For tunnel tests: low resistance, RD 0.74 (95% CI 0.61 to 0.87); moderate resistance, RD 0.50 (95% CI 0.40 to 0.60); high resistance, RD 0.39 (95% CI 0.24 to 0.54). For hut studies: low resistance, RD 0.56 (95% CI 0.43 to 0.68); moderate resistance, RD 0.39 (95% CI 0.16 to 0.61); high resistance, RD 0.35 (95% CI 0.27 to 0.43). However, with the exception of the moderate resistance category for tunnel tests, there was extremely high heterogeneity across studies in each resistance category (chi-squared test, p<0.00001, I2 varied from 95% to 100%). Conclusions This meta-analysis found that ITNs are more effective than UTNs regardless of resistance. There appears to be a relationship between resistance and the RD for mosquito mortality in laboratory and field studies. However, the substantive heterogeneity in the studies' results and design may mask the true relationship between resistance and the RD, and the results need to be interpreted with caution. Our analysis suggests the potential for cumulative meta-analysis in entomological trials, but further field research in this area will require specialists in the field to work together to improve the quality of trials, and to standardise designs, assessment, and reporting of both resistance and entomological outcomes

Efficacy of PermaNet® 2.0 and PermaNet® 3.0 against insecticide-resistant Anopheles gambiae in experimental huts in Côte d'Ivoire

<p>Abstract</p> <p>Background</p> <p>Pyrethroid resistance in vectors could limit the efficacy of long-lasting insecticidal nets (LLINs) because all LLINs are currently treated with pyrethroids. The goal of this study was to evaluate the efficacy and wash resistance of PermaNet^®3.0 compared to PermaNet^®2.0 in an area of high pyrethroid in Côte d'Ivoire. PermaNet^®3.0 is impregnated with deltamethrin at 85 mg/m²on the sides of the net and with deltamethrin and piperonyl butoxide on the roof. PermaNet^®2.0 is impregnated with deltamethrin at 55 mg/m²across the entire net.</p> <p>Methods</p> <p>The study was conducted in the station of Yaokoffikro, in central Côte d'Ivoire. The efficacy of intact unwashed and washed LLINs was compared over a 12-week period with a conventionally-treated net (CTN) washed to just before exhaustion. WHO cone bioassays were performed on sub-sections of the nets, using wild-resistant <it>An. gambiae </it>and Kisumu strains. Mosquitoes were collected five days per week and were identified to genus and species level and classified as dead or alive, then unfed or blood-fed.</p> <p>Results</p> <p>Mortality rates of over 80% from cone bioassays with wild-caught pyrethroid-resistant <it>An. gambiae </it>s.s were recorded only with unwashed PermaNet^®3.0. Over 12 weeks, a total of 7,291 mosquitoes were collected. There were significantly more <it>An. gambiae </it>s.s. and <it>Culex </it>spp. caught in control huts than with other treatments (P < 0.001). The proportion of mosquitoes exiting the huts was significantly lower with the control than for the treatment arms (P < 0.001). Mortality rates with resistant <it>An. gambiae </it>s.s and <it>Culex </it>spp, were lower for the control than for other treatments (P < 0.001), which did not differ (P > 0.05) except for unwashed PermaNet^®3.0 (P < 0.001), which gave significantly higher mortality (P < 0.001).</p> <p>Conclusions</p> <p>This study showed that unwashed PermaNet^®3.0 caused significantly higher mortality against pyrethroid resistant <it>An. gambiae s.s </it>and <it>Culex </it>spp than PermaNet^®2.0 and the CTN. The increased efficacy with unwashed PermaNet^®3.0 over PermaNet^®2.0 and the CTN was also demonstrated by higher KD and mortality rates (KD > 95% and mortality rate > 80%) in cone bioassays performed with wild pyrethroid-resistant <it>An. gambiae s.s </it>from Yaokoffikro.</p

Field efficacy of a new mosaic long-lasting mosquito net (PermaNet® 3.0) against pyrethroid-resistant malaria vectors: a multi centre study in Western and Central Africa

<p>Abstract</p> <p>Background</p> <p>Due to the spread of pyrethroid-resistance in malaria vectors in Africa, new strategies and tools are urgently needed to better control malaria transmission. The aim of this study was to evaluate the performances of a new mosaic long-lasting insecticidal net (LLIN), i.e. PermaNet^®3.0, against wild pyrethroid-resistant <it>Anopheles gambiae s.l</it>. in West and Central Africa.</p> <p>Methods</p> <p>A multi centre experimental hut trial was conducted in Malanville (Benin), Vallée du Kou (Burkina Faso) and Pitoa (Cameroon) to investigate the exophily, blood feeding inhibition and mortality induced by PermaNet^®3.0 (i.e. a mosaic net containing piperonyl butoxide and deltamethrin on the roof) comparatively to the WHO recommended PermaNet^®2.0 (unwashed and washed 20-times) and a conventionally deltamethrin-treated net (CTN).</p> <p>Results</p> <p>The personal protection and insecticidal activity of PermaNet 3.0 and PermaNet^®2.0 were excellent (>80%) in the "pyrethroid-tolerant" area of Malanville. In the pyrethroid-resistance areas of Pitoa (metabolic resistance) and Vallée du Kou (presence of the L1014F <it>kdr </it>mutation), PermaNet^®3.0 showed equal or better performances than PermaNet^®2.0. It should be noted however that the deltamethrin content on PermaNet^®3.0 was up to twice higher than that of PermaNet^®2.0. Significant reduction of efficacy of both LLIN was noted after 20 washes although PermaNet^®3.0 still fulfilled the WHO requirement for LLIN.</p> <p>Conclusion</p> <p>The use of combination nets for malaria control offers promising prospects. However, further investigations are needed to demonstrate the benefits of using PermaNet^®3.0 for the control of pyrethroid resistant mosquito populations in Africa.</p

Target product profile choices for intra-domiciliary malaria vector control pesticide products: repel or kill?

BACKGROUND\ud \ud The most common pesticide products for controlling malaria-transmitting mosquitoes combine two distinct modes of action: 1) conventional insecticidal activity which kills mosquitoes exposed to the pesticide and 2) deterrence of mosquitoes away from protected humans. While deterrence enhances personal or household protection of long-lasting insecticidal nets and indoor residual sprays, it may also attenuate or even reverse communal protection if it diverts mosquitoes to non-users rather than killing them outright.\ud \ud METHODS\ud \ud A process-explicit model of malaria transmission is described which captures the sequential interaction between deterrent and toxic actions of vector control pesticides and accounts for the distinctive impacts of toxic activities which kill mosquitoes before or after they have fed upon the occupant of a covered house or sleeping space.\ud \ud RESULTS\ud \ud Increasing deterrency increases personal protection but consistently reduces communal protection because deterrent sub-lethal exposure inevitably reduces the proportion subsequently exposed to higher lethal doses. If the high coverage targets of the World Health Organization are achieved, purely toxic products with no deterrence are predicted to generally provide superior protection to non-users and even users, especially where vectors feed exclusively on humans and a substantial amount of transmission occurs outdoors. Remarkably, this is even the case if that product confers no personal protection and only kills mosquitoes after they have fed.\ud \ud CONCLUSIONS\ud \ud Products with purely mosquito-toxic profiles may, therefore, be preferable for programmes with universal coverage targets, rather than those with equivalent toxicity but which also have higher deterrence. However, if purely mosquito-toxic products confer little personal protection because they do not deter mosquitoes and only kill them after they have fed, then they will require aggressive "catch up" campaigns, with behaviour change communication strategies that emphasize the communal nature of protection, to achieve high coverage rapidly

Monitoring mosquitoes in urban Dar es Salaam: Evaluation of resting boxes, window exit traps, CDC light traps, Ifakara tent traps and human landing catches

Ifakara tent traps (ITT) are currently the only sufficiently sensitive, safe, affordable and practical method for routine monitoring host-seeking mosquito densities in Dar es Salaam. However, it is not clear whether ITT catches represent indoors or outdoors biting densities. ITT do not yield samples of resting, fed mosquitoes for blood meal analysis. Outdoors mosquito sampling methods, namely human landing catch (HLC), ITT (Design B) and resting boxes (RB) were conducted in parallel with indoors sampling using HLC, Centers for Disease Control and Prevention miniature light traps (LT) and RB as well as window exit traps (WET) in urban Dar es Salaam, rotating them thirteen times through a 3 × 3 Latin Square experimental design replicated in four blocks of three houses. This study was conducted between 6th May and 2rd July 2008, during the main rainy season when mosquito biting densities reach their annual peak. The mean sensitivities of indoor RB, outdoor RB, WET, LT, ITT (Design B) and HLC placed outdoor relative to HLC placed indoor were 0.01, 0.005, 0.036, 0.052, 0.374, and 1.294 for Anopheles gambiae sensu lato (96% An. gambiae s.s and 4% An. arabiensis), respectively, and 0.017, 0.053, 0.125, 0.423, 0.372 and 1.140 for Culex spp, respectively. The ITT (Design B) catches correlated slightly better to indoor HLC (r(2) = 0.619, P < 0.001, r(2) = 0.231, P = 0.001) than outdoor HLC (r(2) = 0.423, P < 0.001, r(2) = 0.228, P = 0.001) for An. gambiae s.l. and Culex spp respectively but the taxonomic composition of mosquitoes caught by ITT does not match those of the indoor HLC (χ(2) = 607.408, degrees of freedom = 18, P < 0.001). The proportion of An. gambiae caught indoors was unaffected by the use of an LLIN in that house. The RB, WET and LT are poor methods for surveillance of malaria vector densities in urban Dar es Salaam compared to ITT and HLC but there is still uncertainty over whether the ITT best reflects indoor or outdoor biting densities. The particular LLIN evaluated here failed to significantly reduce house entry by An. gambiae s.l. suggesting a negligible repellence effect