The Efficacy of Vectron 20 WP, Etofenprox, for Indoor Residual Spraying in Areas of High Vector Resistance to Pyrethroids and Organochlorines in Zambia

The selection of insecticide resistance in malaria vectors has the potential to compromise any insecticide-based vector control programme. To ensure that the insecticides used for indoor residual spraying and insecticide-treated nets in Zambia remain effective and their choice is evidence based, insecticide resistance surveillance and monitoring are essential. This study assessed and compared the residual efficacy of etofenprox (Vectron 20 WP), an ether pyrethroid, at 0.1 g/m2 with pyrethroids: bifenthrin (Bistar 10 WP) and lambda-cyhalothrin (Icon 10 CS) at 25 mg/m2 for indoor residual spraying. We also assessed the resistance status of etofenprox to local malaria vectors, An. funestus s.s and An. gambiae s.s, using World Health Organization standard protocols. The residual efficacy of Vectron 20 WP on cement, rendered walls of houses lasted for four months with 100% mortality. By the eighth month, the killing effect had reduced to 73.8% compared to 63.3% for bifenthrin and 77.0% for lambda-cyhalothrin. Susceptibility tests using standard World Health Organization assays on An. gambiae s.s showed susceptibility to etofenprox (0.1%) but some resistance was detected to Anopheles funestus s.s. The product is recommended as an ideal insecticide for indoor residual spraying for malaria control in Zambia as part of a resistance management programme in selected areas of the country.


Introduction
Globally, about 515 million malaria cases with over one million deaths occur annually in tropical and subtropical regions [1]. e greatest toll is exacted in sub-Saharan Africa, particularly in children and pregnant women [2,3]. is huge burden of disease is as a consequence of the excellent vectorial competence of the three major vectors of malaria: Anopheles gambiae s.s, Anopheles arabiensis, and Anopheles funestus [4,5]. Malaria remains a major public health challenge and continues to severely undermine the socioeconomic growth in sub-Saharan Africa [6]. In the absence of a vaccine, much of the malaria control efforts rely primarily on effective treatment with antimalaria drugs and prevention through transmission-blocking vector control interventions [7,8]. Nevertheless, effectiveness of malaria control is threatened by the increasing levels of both drug resistance in Plasmodium parasites [9] and insecticide resistance in Anopheles vectors [10].
e main thrust contemporary vector control interventions, that is, indoor residual spraying (IRS) and insecticide treated nets (ITNs), are insecticide based [11]. However, the arsenal of insecticides is so limited with only four classes being available for control. Presently there are only 12 registered insecticides for IRS and 6 for ITNs [12,13]. Moreover, indiscriminate and persistent utilization of insecticides invariably exacerbate resistance development in the vectors that they are intended to control [14][15][16]. Insecticide resistance in malaria vectors to pyrethroids and dichlorodimethyl-trichloroethane (DDT) has been reported across sub-Saharan Africa [16]. In Zambia, resistance has been detected in both An. gambiae s.s and An. funestus s.s to pyrethroids and DDT [17]. ere is an urgent need for the identi�cation of alternative effective insecticides for vector control.
In this light, Vectron 20 WP was evaluated for potential use for IRS in the national malaria control programme in Zambia. Etofenprox, a pyrethroid-like insecticide devoid of an ester bond, has very low mammalian toxicity and the highest safety factor [18,19]. A few laboratory and �eld trials have been carried out with this insecticide in different countries [18][19][20]. e product is recommended for indoor residual spraying by World Health Organization Pesticide Evaluation Scheme (WHOPES) at 0.1-0.3 g/m 2 with an effective duration for 3-6 months [18].
is study evaluated the persistence of the biological efficacy of Vectron 20 WP IRS against Anopheles gambiae s. s and An. funestus s.s (Diptera: Culicidae) in areas of high insecticide resistance in �eld tropical conditions of Zambia.

Experimental Sites. Zambia is situated in the Southern
African region with a population of approximately 13 million, 45% of whom are below the age of �een [21]. e climatic conditions in the country include three distinct seasons; warm and rainy season (November-April), cool and dry season (April-August), and a hot and dry season (August-November). In winter the temperatures fall to as low as 4 ∘ C and during summer the temperature rises to as high as 38 ∘ C and varies as a function of altitude. Residual efficacy studies were carried out at Bauleni in Lusaka district where formal structures are predominant. Mosquitoes for susceptibility assays were collected from IRS operational areas of high insecticide resistance, particularly, Chipulukusu in Ndola district and Malata in Katete districts.
Vectron 20 WP was applied at 0.1 g active ingredient (etofenprox) per meter square of spray surface, Bistar 10 WP and Icon 10 CS at 25 mg active ingredient (bifenthrin and lambda-cyhalothrin)/m 2 , respectively. All applications were done with the Hudson X-Pert compression spray pumps (H.D Hudson Manufacturing Company, USA) aer calibration of 8001E nozzle according to standard procedure. Insecticides were mixed in 10 liters of clean water in the spray can and pressurized to 55 Psi. e spraying coincided with national malaria control programme spraying campaign and was conducted by competent spray operators trained in accordance with the standard training guidelines [22].

Cone Bioassay
Test. e residual efficacy of etofenprox (Vectron 20 WP), an ether pyrethroid, at 0.1 g/m 2 was compared with pyrethroids: bifenthrin (Bistar 10 WP) and lambda-cyhalothrin (Icon 10 CS) at 25 mg/m 2 and control. Residual efficacy of all insecticides was determined on randomly selected sprayed cement surfaces of uniform texture. e controls (no insecticide) were conducted on untreated card boxes �xed on the wall. Contact bioassays of three replicates (15 mosquitoes per dose/surface) using WHOsupplied bioassay cones were performed 1 day aer spraying, and subsequently on the same day of each month for 7 months.
World Health Organisation standard bioassays were conducted [23,24] using sugar-fed, 48 to 72 h old female An. gambiae s.s Kisumu strain from a colony maintained at the National Malaria Control Centre in Lusaka, Zambia. ree replicates of the WHO cones were adhered to the top, middle, and bottom of the surface sprayed with respective insecticides. Knockdown of mosquitoes was recorded 60 min aer releasing them in cones. Aer exposure, mosquitoes were transferred to cups and held for 24 hrs with 10% glucose solution-soaked cotton wool before percentage mortality was calculated. e WHOPES criterion for persistence is, at least 70% mortality at 8-week period [24]. Indoor temperature and relative humidity were recorded using digital thermometer and hygrometer, respectively, each time contact bioassays were carried out.

Vector Susceptibility.
Wild Blood-fed adult female An. gambiae s.l and An. funestus s.l were collected from houses using torches and backpack aspirators in operational areas of vector control interventions. Collected mosquitoes were transported to the laboratory and transferred to individual oviposition tubes and females allowed to lay eggs that were reared to F1 adults at 26 ± 2 ∘ C and 70-80% relative humidity. Susceptibility of sugar-fed 2-5-day-old F1 progeny was determined for DDT (4%), malathion (5%), lambdacyhalothrin (0.05%), bendiocarb 0.1%, deltamethrin (0.05%), and etofenprox (0.1%) using the standard WHO tube method [23]. All papers were supplied by WHO.
2.6. Statistics. e residual efficacy data on Vectron 20 WP was compared with that of conventional pyrethroids, Bistar 10 WP and Icon 10 CS and control. e signi�cance of the differences in the mosquito mortalities was analyzed by chisquare test and odds ratio to assess the efficacy of the given formulation of insecticide.  Figure 1). e residual effect of Vectron 20 WP and Bistar 10 WP (>80% mortality in cone bioassays) lasted for �ve months. Comparing the persistence of etofenprox (0.1 mg/m 2 ) with that of bifenthrin (25 mg/m 2 ) and lambdacyhalothrin (25 mg/m 2 ) on the cement wall surfaces using An. gambiae Kisumu strain showed no signi�cant difference ( > ). At 8 months the mortality reduced to 73.8% for etofenprox, 63.3% for bifenthrin, and 77.0% for lambdacyhalothrin. e controls for the three insecticides registered no mortality throughout the study period. Readings for indoor temperature and relative humidity ranged from 22 to 26 ∘ C and 38 to 62%, respectively, in the houses and from 24 to 26.7 ∘ C and 76 to 90% in the insectary and showed signi�cant changes at 24 hourly readings ( Table 2).

Discussion
Insecticide resistance has developed in Zambia and now threatening the successful malaria control programme [17]. Although only a few operational �eld studies have been conducted with Vectron 20 WP, various laboratory and simulated �eld efficacy studies have been conducted [18][19][20] and the results of the present study corroborate these studies.
is efficacy study demonstrates that Vectron 20 WP, an ether pyrethroid, is as effective and persistent as the conventional pyrethroid insecticide, Bistar 10 WP and Icon 10 CS in contact bioassay �eld trials. Vectron 20 WP has been shown to have limiting factors in the �eld such as its short duration of activity and its tendency of leaving white stains on the sprayed surfaces [19]. Additionally, it is worth mentioning that the residual efficacy of this product may be compromised if it is applied on highly porous surfaces such as mud walls. However, results from this study demonstrate an improved residual efficacy of over �ve months on cement rendered walls. In Zambia, the active malaria transmission runs from November to April [29]. As such, the product would be appropriate in reducing transmission during this period.
Although Vectron 20 WP has very minimal mammalian toxicity and has the highest safety factor [18], studies on the susceptibility status of local vectors to this ether pyrethroid and DDT are essential before it can be used as a safer alternative for indoor residual spray against malaria vectors. Earlier susceptibility tests conducted in Zambia using standard WHO diagnostic doses of insecticide impregnated papers on An. gambiae s.s revealed full susceptibility to DDT (4%), lambda-cyhalothrin (0.05%), and deltamethrin (0.05%) [11]. However, following intensive vector control efforts, variable levels of insecticide resistance have been detected in both An. gambiae s.s and An. funestus to pyrethroids and organochlorine (DDT). Signi�cantly high levels of resistance have been detected in indoor residual spraying operational areas compared to insecticide treated nets localities. Crossresistance between pyrethroids and DDT mediated by the knock down resistance (kdr) mutation has been detected in some areas of the country [17]. Plans are underway to deploy Vectron 20 WP operationally in areas with detectable susceptibility to this insecticide in Zambia.
Malaria has a country-wide endemicity in Zambia [30]. Successful implementation of effective malaria control has resulted in a shi in the epidemiology of the disease culminating in three distinct strata across the country [30]. Pesticide utilization both in agriculture on the high-value insecticide intensive crops and in public health for IRS and ITNs is on the increase in Zambia. Selection pressure in An. gambiae s.s in Ndola on the Copperbelt province could in large part be ascribed to the extensive IRS and ITNs programmes as well as the mining activities in the area. On the other hand, the high levels of resistance in An. funestus in Katete district in Eastern province could be as a result of agricultural use of insecticides on cotton and ITNs distribution, and to a lesser extent on IRS. In this study, An. gambiae s.s from Ndola, an area with high kdrmediated-cross resistance between DDT and pyrethroids was fully susceptibile to etofenprox, malathion, and bendiocarb. Complete resistance to deltamethrin, lambda-cyhalothrin, and DDT was detected (Table 1). However, recent resistance surveillance results have indicated very high levels of etofenprox resistance in An. gambiae s.s in Kitwe on the Copperbelt and An. funestus s.s in Mbinga in eastern province. is could be a function of the focal nature of the insecticide resistance phenomenon. e suspected resistance of An. funestus s.s to etofenprox, bendiocarb, lambda-cyhalothrin, and DDT detected in Katete district indicates that products could still be used operationally but with rigorous insecticide resistance surveillance in place.
Vectron 20 WP is as effective and persistent on cement walls as the competitive Bistar 10 WP and Icon 10 CS. e detected susceptibility of An. gambiae s.s and An. funestus to etofenprox, malathion, and bendiocarb provides an opportunity for establishing a rational insecticide resistance management strategy once the spatial heterogeneity and underlying mechanisms are determined. Vectron 20 WP is environmentally friendlier and more effective against the major malaria vectors An. gambiae s.s and An. funestus and could be rotated with organophosphates or carbamates. erefore, it is recommended as an ideal insecticide for IRS for malaria control in Zambia as part of the insecticide resistance management strategy in selected areas of the country following expansive and rigorous surveillance.  Authors' Contribution E. Chanda designed and coordinated the study, participated in data collection, analyzed the data, and draed the paper. A. Kandyata and j. chanda assisted with data collection, analysis, and interpretation. F. N. Phiri and L. Muzia participated in �eld data collection. M. Kamuliwo managed the project and reviewed the paper.

Con�ict o� �nterests
e author of the paper does not have a direct �nancial relation with the commercial identity mentioned in the paper that might lead to a con�ict of interest for any of the authors. e authors declare that they have no competing interests.