Increased tolerance of Anopheles gambiae s.s. to chemical insecticides after exposure to agrochemical mixture

Resistance of mosquitoes to insecticides is mainly attributed to their adaptation to insecticide-based vector control interventions. Although pesticides used in agriculture have been frequently mentioned as an additional force driving the selection of resistance, only a few studies were dedicated to validate this hypothesis. The objective of this study was to investigate the effect of exposure of the malaria mosquito, Anopheles gambiae s.s. larvae for 72h to sub-lethal concentrations of the agrochemical mixture (pesticides, herbicides and fungicides). Their subsequent tolerances were measured to deltamethrin (pyrethroid), DDT (organochlorine) and bendiocarb (carbamate) currently used for vector control. The mean LC50 was determined and tolerance ratios for larvae exposed to agrochemical comparatively with unexposed larvae were calculated and expressed as fold increased tolerance. Bioassays revealed a significant increase in larval tolerance to detamethrin (1.83-2.86 fold), DDT (1.31-1.53 fold) and bendiocarb (1.14-1.19 fold) following exposure to 0.1 µM and 1µM agrochemical mixture. The observed increased tolerance in this study is likely to be based on metabolic resistance mechanisms. Overall, this study reveals the potential of agrochemicals to increase the tolerance of mosquito larvae to chemical insecticides

Insecticide susceptibility status of human biting mosquitoes in Muheza, Tanzania

Background: There has been a rapid emergence in insecticide resistance among mosquito population to commonly used public health insecticides. This situation presents a challenge to chemicals that are currently used to control mosquitoes in sub-Saharan African. Furthermore, there is limited information on insecticide susceptibility status of human-biting mosquitoes in some areas of Tanzania. This study aimed to determine insecticide susceptibility status of human biting mosquitoes in a rural area of north-eastern Tanzania.Methods: The study was conducted in two villages in Muheza district, Tanzania. Insecticide susceptibility bioassays were performed according to the World Health Organization standard operating procedures on two to five-day old human biting mosquitoes. The mosquitoes of each species were exposed to four classes of insecticides commonly used for malaria vector control. Mosquito mortality rates (%) were determined after 24 hours post insecticide exposure.Results: Mosquito species tested were Anopheles gambiae s.l., An. funestus, Aedes aegypti, and Culex quinquefasciatus species. Real-time PCR have showed that the main sibling species of An. gambiae complex and An. funestus group were An. gambiae s. s. (58.2%) and An. funestus s. s. (91.1%), respectively. All mosquitoes, except Ae. aegypti formosus were susceptible to pirimiphos-methyl (0.25%). An. gambiae s. l. was found to be resistant to permethrin (0.75%) but showed possibility of resistance to DDT (4%) and bendiocarb (0.1%). Our findings have shown that, An. funestus was fully susceptible to all insecticide tested.Conclusion: The present study has revealed different levels of insecticide susceptibility status to four classes of commonly used insecticides in the most common mosquito vectors of human diseases in north-eastern Tanzania. The findings of the present study call for integrated vector control interventions.

\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

Combination of Insecticide Treated Nets and Indoor Residual Spraying in Northern Tanzania Provides Additional Reduction in Vector Population Density and Malaria Transmission Rates Compared to Insecticide Treated Nets Alone: A Randomised Control Trial.

Indoor residual spraying (IRS) combined with insecticide treated nets (ITN) has been implemented together in several sub-Saharan countries with inconclusive evidence that the combined intervention provides added benefit. The impact on malaria transmission was evaluated in a cluster randomised trial comparing two rounds of IRS with bendiocarb plus universal coverage ITNs, with ITNs alone in northern Tanzania. From April 2011 to December 2012, eight houses in 20 clusters per study arm were sampled monthly for one night with CDC light trap collections. Anopheles gambiae s.l. were identified to species using real time PCR Taq Man and tested for the presence of Plasmodium falciparum circumsporozoite protein. ITN and IRS coverage was estimated from household surveys. IRS coverage was more than 85% in two rounds of spraying in January and April 2012. Household coverage with at least one ITN per house was 94.7% after the universal coverage net campaign in the baseline year and the proportion of household with all sleeping places covered by LLIN was 50.1% decreasing to 39.1% by the end of the intervention year. An.gambiae s.s. comprised 80% and An.arabiensis 18.3% of the anopheline collection in the baseline year. Mean An.gambiae s.l. density in the ITN+IRS arm was reduced by 84% (95%CI: 56%-94%, p = 0.001) relative to the ITN arm. In the stratum of clusters categorised as high anopheline density at baseline EIR was lower in the ITN+IRS arm compared to the ITN arm (0.5 versus 5.4 per house per month, Incidence Rate Ratio: 0.10, 95%CI: 0.01-0.66, p-value for interaction <0.001). This trial provides conclusive evidence that combining carbamate IRS and ITNs produces major reduction in Anopheles density and entomological inoculation rate compared to ITN alone in an area of moderate coverage of LLIN and high pyrethroid resistance in An.gambiae s.s

Efficacy of interceptor® G2, a long-lasting insecticide mixture net treated with chlorfenapyr and alpha-cypermethrin against Anopheles funestus: experimental hut trials in north-eastern Tanzania.

BACKGROUND: The effectiveness of long-lasting insecticidal nets (LLIN), the primary method for preventing malaria in Africa, is compromised by evolution and spread of pyrethroid resistance. Further gains require new insecticides with novel modes of action. Chlorfenapyr is a pyrrole insecticide that disrupts mitochrondrial function and confers no cross-resistance to neurotoxic insecticides. Interceptor® G2 LN (IG2) is an insecticide-mixture LLIN, which combines wash-resistant formulations of chlorfenapyr and the pyrethroid alpha-cypermethrin. The objective was to determine IG2 efficacy under controlled household-like conditions for personal protection and control of wild, pyrethroid-resistant Anopheles funestus mosquitoes. METHODS: Experimental hut trials tested IG2 efficacy against two positive controls-a chlorfenapyr-treated net and a standard alpha-cypermethrin LLIN, Interceptor LN (IG1)-consistent with World Health Organization (WHO) evaluation guidelines. Mosquito mortality, blood-feeding inhibition, personal protection, repellency and insecticide-induced exiting were recorded after zero and 20 washing cycles. The trial was repeated and analysed using multivariate and meta-analysis. RESULTS: In the two trials held in NE Tanzania, An. funestus mortality was 2.27 (risk ratio 95% CI 1.13-4.56) times greater with unwashed Interceptor G2 than with unwashed Interceptor LN (p = 0.012). There was no significant loss in mortality with IG2 between 0 and 20 washes (1.04, 95% CI 0.83-1.30, p = 0.73). Comparison with chlorfenapyr treated net indicated that most mortality was induced by the chlorfenapyr component of IG2 (0.96, CI 0.74-1.23), while comparison with Interceptor LN indicated blood-feeding was inhibited by the pyrethroid component of IG2 (IG2: 0.70, CI 0.44-1.11 vs IG1: 0.61, CI 0.39-0.97). Both insecticide components contributed to exiting from the huts but the contributions were heterogeneous between trials (heterogeneity Q = 36, P = 0.02). WHO susceptibility tests with pyrethroid papers recorded 44% survival in An. funestus. CONCLUSIONS: The high mortality recorded by IG2 against pyrethroid-resistant An. funestus provides first field evidence of high efficacy against this primary, anthropophilic, malaria vector

Experimental hut evaluation of a novel long-lasting non-pyrethroid durable wall lining for control of pyrethroid-resistant Anopheles gambiae and Anopheles funestus in Tanzania.

BACKGROUND: A novel, insecticide-treated, durable wall lining (ITWL), which mimics indoor residual spraying (IRS), has been developed to provide prolonged vector control when fixed to the inner walls of houses. PermaNet® ITWL is a polypropylene material containing non-pyrethroids (abamectin and fenpyroximate) which migrate gradually to the surface. METHODS: An experimental hut trial was conducted in an area of pyrethroid-resistant Anopheles gambiae s.l. and Anopheles funestus s.s. to compare the efficacy of non-pyrethroid ITWL, long-lasting insecticidal nets (LLIN) (Interceptor®), pyrethroid ITWL (ZeroVector®), and non-pyrethroid ITWL + LLIN. RESULTS: The non-pyrethroid ITWL produced relatively low levels of mortality, between 40-50% for An. funestus and An. gambiae, across all treatments. Against An. funestus, the non-pyrethroid ITWL when used without LLIN produced 47% mortality but this level of mortality was not significantly different to that of the LLIN alone (29%, P = 0.306) or ITWL + LLIN (35%, P = 0.385). Mortality levels for An. gambiae were similar to An. funestus with non-pyrethroid ITWL, producing 43% mortality compared with 26% for the LLIN. Exiting rates from ITWL huts were similar to the control and highest when the LLIN was present. An attempt to restrict mosquito access by covering the eave gap with ITWL (one eave open vs four open) had no effect on numbers entering. The LLIN provided personal protection when added to the ITWL with only 30% blood-fed compared with 69 and 56% (P = 0.001) for ITWL alone. Cone bioassays on ITWL with 30 min exposure after the trial produced mortality of >90% using field An. gambiae. CONCLUSIONS: Despite high mortality in bioassays, the hut trial produced only limited mortality which was attributed to pyrethroid resistance against the pyrethroid ITWL and low efficacy in the non-pyrethroid ITWL. Hut ceilings were left uncovered and may have served as a potential untreated refuge. By analogy to IRS campaigns, which also do not routinely treat ceilings, high community coverage with ITWL may still reduce malaria transmission. Restriction of eave gaps by 75% proved an inadequate barrier to mosquito entry. The findings represent the first 2 months after installation and do not necessarily predict long-term efficacy

Malaria entomological profile in Tanzania from 1950 to 2010: a review of mosquito distribution, vectorial capacity and insecticide resistance

In Sub Saharan Africa where most of the malaria cases and deaths occur, members of the Anopheles gambiae species complex and Anopheles funestus species group are the important malaria vectors. Control efforts against these vectors in Tanzania like in most other Sub Saharan countries have failed to achieve the set objectives of eliminating transmission due to scarcity of information about the enormous diversity of Anopheles mosquito species and their susceptibility status to insecticides used for malaria vector control. Understanding the diversity and insecticide susceptibility status of these vectors and other factors relating to their importance as vectors (such as malaria transmission dynamics, vector biology, ecology, behaviour and population genetics) is crucial to developing a better and sound intervention strategies that will reduce man-vector contact and also manage the emergency of insecticide resistance early and hence a success in malaria control. The objective of this review was therefore to obtain the information from published and unpublished documents on spatial distribution and composition of malaria vectors, key features of their behaviour, transmission indices and susceptibility status to insecticides in Tanzania. All data available were collated into a database. Details recorded for each data source were the locality, latitude/longitude, time/period of study, species, abundance, sampling/collection methods, species identification methods, insecticide resistance status, including evidence of the kdr allele, and Plasmodium &nbsp;falciparum sporozoite rate. This collation resulted in a total of 368 publications, encompassing 806,273 Anopheles mosquitoes from 157 geo-referenced locations being collected and identified across Tanzania from 1950s to 2010. Overall, the vector species most often reported included An. gambiae complex (66.8%), An. funestus complex (21.8%), An. gambiae s.s. (2.1%) and An. arabiensis (9%). A variety of sampling/collection and species identification methods were used with an increase in molecular techniques in recent decades. Only 32.2% and 8.4% of the data sets reported on sporozoite analysis and entomological inoculation rate (EIR), respectively which highlights the paucity of such important information in the country. Studies demonstrated efficacy of all four major classes of insecticides against malaria vectors in Tanzania with focal points showing phenotypic resistance. About 95% of malaria entomological data was obtained from north- eastern Tanzania. This shows the disproportionate nature of the available information with the western part of the country having none. Therefore it is important for the country to establish entomological surveillance system with state of the art to capture all vitally important entomological indices including vector bionomics in areas of Tanzania where very few or no studies have been done. This is vital in planning and implementing evidence based malaria vector control programmes as well as in monitoring the current malaria control interventions

Effectiveness of a long-lasting piperonyl butoxide-treated insecticidal net and indoor residual spray interventions, separately and together, against malaria transmitted by pyrethroid-resistant mosquitoes: a cluster, randomised controlled, two-by-two factorial design trial.

BACKGROUND: Progress in malaria control is under threat by wide-scale insecticide resistance in malaria vectors. Two recent vector control products have been developed: a long-lasting insecticidal net that incorporates a synergist piperonyl butoxide (PBO) and a long-lasting indoor residual spraying formulation of the insecticide pirimiphos-methyl. We evaluated the effectiveness of PBO long-lasting insecticidal nets versus standard long-lasting insecticidal nets as single interventions and in combination with the indoor residual spraying of pirimiphos-methyl. METHODS: We did a four-group cluster randomised controlled trial using a two-by-two factorial design of 48 clusters derived from 40 villages in Muleba (Kagera, Tanzania). We randomly assigned these clusters using restricted randomisation to four groups: standard long-lasting insecticidal nets, PBO long-lasting insecticidal nets, standard long-lasting insecticidal nets plus indoor residual spraying, or PBO long-lasting insecticidal nets plus indoor residual spraying. Both standard and PBO nets were distributed in 2015. Indoor residual spraying was applied only once in 2015. We masked the inhabitants of each cluster to the type of nets received, as well as field staff who took blood samples. Neither the investigators nor the participants were masked to indoor residual spraying. The primary outcome was the prevalence of malaria infection in children aged 6 months to 14 years assessed by cross-sectional surveys at 4, 9, 16, and 21 months after intervention. The endpoint for assessment of indoor residual spraying was 9 months and PBO long-lasting insecticidal nets was 21 months. This trial is registered with ClinicalTrials.gov, number NCT02288637. FINDINGS: 7184 (68·0%) of 10 560 households were selected for post-intervention survey, and 15 469 (89·0%) of 17 377 eligible children from the four surveys were included in the intention-to-treat analysis. Of the 878 households visited in the two indoor residual spraying groups, 827 (94%) had been sprayed. Reported use of long-lasting insecticidal nets, across all groups, was 15 341 (77·3%) of 19 852 residents after 1 year, decreasing to 12 503 (59·2%) of 21 105 in the second year. Malaria infection prevalence after 9 months was lower in the two groups that received PBO long-lasting insecticidal nets than in the two groups that received standard long-lasting insecticidal nets (531 [29%] of 1852 children vs 767 [42%] of 1809; odds ratio [OR] 0·37, 95% CI 0·21-0·65; p=0·0011). At the same timepoint, malaria prevalence in the two groups that received indoor residual spraying was lower than in groups that did not receive indoor residual spraying (508 [28%] of 1846 children vs 790 [44%] of 1815; OR 0·33, 95% CI 0·19-0·55; p<0·0001) and there was evidence of an interaction between PBO long-lasting insecticidal nets and indoor residual spraying (OR 2·43, 95% CI 1·19-4·97; p=0·0158), indicating redundancy when combined. The PBO long-lasting insecticidal net effect was sustained after 21 months with a lower malaria prevalence than the standard long-lasting insecticidal net (865 [45%] of 1930 children vs 1255 [62%] of 2034; OR 0·40, 0·20-0·81; p=0·0122). INTERPRETATION: The PBO long-lasting insecticidal net and non-pyrethroid indoor residual spraying interventions showed improved control of malaria transmission compared with standard long-lasting insecticidal nets where pyrethroid resistance is prevalent and either intervention could be deployed to good effect. As a result, WHO has since recommended to increase coverage of PBO long-lasting insecticidal nets. Combining indoor residual spraying with pirimiphos-methyl and PBO long-lasting insecticidal nets provided no additional benefit compared with PBO long-lasting insecticidal nets alone or standard long-lasting insecticidal nets plus indoor residual spraying. FUNDING: UK Department for International Development, Medical Research Council, and Wellcome Trust

Evaluation of ICON Maxx, a long-lasting treatment kit for mosquito nets: experimental hut trials against anopheline mosquitoes in Tanzania.

BACKGROUND: Insecticide-treated nets are the primary method of preventing malaria. To remain effective, the pyrethroid insecticide must withstand multiple washes over the lifetime of the net. ICON(®) Maxx is a 'dip-it-yourself' kit for long-lasting treatment of polyester nets. The twin-sachet kit contains a slow-release capsule suspension of lambda-cyhalothrin plus binding agent. To determine whether ICON Maxx meets the standards required by the World Health Organization Pesticide Evaluation Scheme (WHOPES), the efficacy and wash fastness of ICON Maxx was evaluated against wild, free-flying anopheline mosquitoes. METHODS: ICON Maxx was subjected to bioassay evaluation and experimental hut trial against pyrethroid-susceptible Anopheles gambiae, Anopheles arabiensis and Anopheles funestus. Mosquito mortality, blood feeding inhibition and personal protection were compared between untreated nets, conventional lambda-cyhalothrin treated nets (CTN) washed either four times (cut-off threshold) or 20 times, and ICON Maxx-treated nets either unwashed or washed 20 times. RESULTS: In bioassay, ICON Maxx demonstrated superior wash resistance to the CTN. In the experimental hut trial, ICON Maxx killed 75 % of An. funestus, 71 % of An. gambiae and 47 % of An. arabiensis when unwashed and 58, 66 and 42 %, respectively, when 20 times washed. The CTN killed 52 % of An. funestus, 33 % of An. gambiae and 30 % of An. arabiensis when washed to the cut-off threshold of four washes and 40, 40 and 36 %, respectively, when 20 times washed. Percentage mortality with ICON Maxx 20 times washed was similar (An. funestus) or significantly higher (An. gambiae, An. arabiensis) than with CTN washed to the WHOPES cut-off threshold. Blood-feeding inhibition with ICON Maxx 20 times washed was similar to the CTN washed to cut-off for all three species. Personal protection was significantly higher with ICON Maxx 20 times washed (66-79 %) than with CTN washed to cut-off (48-60 %). CONCLUSIONS: Nets treated with ICON Maxx and washed 20 times met the approval criteria set by WHOPES for Phase II trials in terms of mortality and blood-feeding inhibition. This finding raises the prospect of conventional polyester nets and other materials being made long-lastingly insecticidal through simple dipping in community or home, and thus represents a major advance over conventional pyrethroid treatments

Risk factors for malaria infection prevalence and household vector density between mass distribution campaigns of long-lasting insecticidal nets in North-western Tanzania.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are the most widely deployed vector control intervention in sub-Saharan Africa to prevent malaria. Recent reports indicate selection of pyrethroid insecticide resistance is widespread in mosquito vectors. This paper explores risk factors associated with malaria infection prevalence and vector density between mass distribution campaigns, changes in net coverage, and loss of protection in an area of high pyrethroid resistance in Northwest Tanzania. METHODS: A cross sectional malaria survey of 3456 children was undertaken in 2014 in Muleba district, Kagera region west of Lake Victoria. Vector density was assessed using indoor light traps and outdoor tent traps. Anophelines were identified to species using PCR and tested for Plasmodium falciparum circumsporozoite protein. Logistic regression was used to identify household and environmental factors associated with malaria infection and regression binomial negative for vector density. RESULTS: LLIN use was 27.7%. Only 16.9% of households had sufficient nets to cover all sleeping places. Malaria infection was independently associated with access to LLINs (OR: 0.57; 95% CI 0.34-0.98). LLINs less than 2 years old were slightly more protective than older LLINs (53 vs 65% prevalence of infection); however, there was no evidence that LLINs in good condition (hole index < 65) were more protective than LLINs, which were more holed. Other risk factors for malaria infection were age, group, altitude and house construction quality. Independent risk factors for vector density were consistent with malaria outcomes and included altitude, wind, livestock, house quality, open eaves and LLIN usage. Indoor collections comprised 4.6% Anopheles funestus and 95.4% Anopheles gambiae of which 4.5% were Anopheles arabiensis and 93.5% were Anopheles gambiae sensu stricto. CONCLUSION: Three years after the mass distribution campaign and despite top-ups, LLIN usage had declined considerably. While children living in households with access to LLINs were at lower risk of malaria, infection prevalence remained high even among users of LLINs in good condition. While effort should be made to maintain high coverage between campaigns, distribution of standard pyrethroid-only LLINs appears insufficient to prevent malaria transmission in this area of intense pyrethroid resistance