Comparative performances, under laboratory ? Comparative performances, under laboratory conditions,

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Comparative performances, under laboratory conditions,of seven pyrethroid insecticides used for impregnationof mosquito nets*Jean-Marc Hougard,1 Stephane Duchon,2 Frederic Darriet,2 Morteza Zaim,3 Christophe Rogier,4 & Pierre Guillet3Objective To compare the efficacy of seven pyrethroid insecticides for impregnation of mosquito nets, six currently recommended byWHO and one candidate (bifenthrin), under laboratory conditions.Methods Tests were conducted using pyrethroid-susceptible and pyrethroid-resistant strains of Anopheles gambiae and Culexquinquefasciatus. Knock-down effect, irritancy and mortality were measured using standard WHO cone tests. Mortality and blood-feeding inhibition were also measured using a baited tunnel device.Findings For susceptible A. gambiae, alpha-cypermethrin had the fastest knock-down effect. For resistant A. gambiae, the knock-down effect was slightly slower with alpha-cypermethrin and much reduced following exposure to the other insecticides, particularlybifenthrin and permethrin. For susceptible C. quinquefasciatus, the knock-down effect was significantly slower than in A. gambiae,particularly with bifenthrin, and no knock-down effect was observed with any of the pyrethroids against the resistant strain. Bifenthrinwas significantly less irritant than the other pyrethroids to susceptible and resistant A. gambiae but there was no clear ranking ofpyrethroid irritancy against C. quinquefasciatus. In tunnels, all insecticides were less toxic against C. quinquefasciatus than againstA. gambiae for susceptible strains. For resistant strains, mortality was significant with all the pyrethroids with A. gambiae but not withC. quinquefasciatus. Inhibition of blood-feeding was also high for susceptible strains of both species and for resistant A. gambiae butlower for resistant C. quinquefasciatus; bifenthrin had the greatest impact.Conclusions Efficacy for impregnation of mosquito nets against A. gambiae was greatest with alpha-cypermethrin. Bifenthrin is likelyto have a significant comparative advantage over other pyrethroids in areas with pyrethroid resistance because of its much strongerimpact on the nuisance mosquito, C. quinquefasciatus, despite its slower knock-down effect and irritancy. Selection of pyrethroids formosquito vector control and personal protection should take into account the different effects of these insecticides, the status ofpyrethroid resistance in the target area, and the importance of nuisance mosquitoes, such as C. quinquefasciatus.Keywords Pyrethrins; Insecticides, Botanical/toxicity; Anopheles; Culex; Bedding and linens; Insecticide resistance; Comparativestudy (source: MeSH, NLM ).Mots cles Pyrethrine; Insecticides phytogenes/toxicite; Anopheles; Culex; Literie et linge; Resistance aux insecticides; Etudecomparative (source: MeSH, INSERM ).Palabras clave Piretrinas; Insecticidas botanicos/toxicidad; Anopheles; Culex; Ropa de cama y ropa blanca; Resistencia a insecticida;Estudio comparativo (fuente: DeCS, BIREME ).Bulletin of the World Health Organization 2003;81:324-333.Voir page 331 le resume en francais. En la pagina 331 figura un resumen en espanol.IntroductionThe widespread distribution of insecticide-impregnated mos-quito nets is a major component of the WHO global strategyfor malaria control, especially in sub-Saharan Africa, wheremore than 90% of malaria cases are reported annually (1). Todate, six pyrethroid insecticides the only group ofinsecticides currently considered suitable for impregnation ofmosquito nets have been evaluated by the WHO PesticideEvaluation Scheme (WHOPES) and recommended for thispurpose: alpha-cypermethrin, cyfluthrin, deltamethrin andlambdacyhalothrin (alpha-cyano pyrethroids), and etofenproxand permethrin (non-cyano pyrethroids) (24).Pyrethroid resistance of malaria vectors has alreadydeveloped in several malarious countries (57), and the absenceof a suitable alternative insecticide class for impregnation ofmosquito nets may undermine the gains in malaria control andpersonal protection being made through improved coverage* This investigation was carried out by the Institut de Recherche pour le Developpement, Montpellier, France, as part of the WHO Pesticide Evaluation Scheme (WHOPES). Themention of specific companies and/or products does not in any way imply that they are recommended or endorsed by WHO in preference over others that are not mentioned.1 Head, Vector Control Research Unit (RU016), Institut Francais pour le Developpement, 911 Av. Agropolis, BP 64501, 34394 Montpellier Cedex 5, France(email: hougard@mpl.ird.fr). Correspondence should be addressed to this author.2 Engineer, Institut de Recherche pour le Developpement, Montpellier, France.3 Communicable Diseases Control, Prevention and Eradication, World Health Organization, Geneva, Switzerland.4 Institut de Medecine Tropicale du Service de Sante des Armees, Marseille, France.Ref. No. 01-1552Research324 Bulletin of the World Health Organization 2003, 81 (5)with treated nets. Recent field studies in pyrethroid-resistantareas of Cote dIvoire, in experimental huts (8, 9) and on a largerscale (10, 11), indicated that pyrethroid-impregnated mosquitonets reduce malaria transmission despite a high frequency of theknock-down resistance (kdr) gene. A WHO consultationrecommended that this should be confirmed in other studies,especially where pyrethroid-resistance mechanisms other thanthe kdr gene may be involved (12).Protection against nuisance insects, especially Culexquinquefasciatus, which keep people awake at night, is the mainmotivation for the use of mosquito nets. However, pyrethroidresistance in this species is already widespread in the tropicalworld, including Africa (13).Bifenthrin, a non-alpha-cyano pyrethroid, is used againsta broad range of agricultural pests and has emerged as apromising candidate for malaria vector control in WHOPES-supervised trials in India, Mexico, Thailand, and UnitedRepublic of Tanzania (14), and in field studies in Cote dIvoire(15). Bifenthrin has been suggested for treatment of mosquitonets in view of its high efficacy against Anopheles gambiae, themajor malaria vector in Africa, and C. quinquefasciatus (16).However, further testing and evaluation of the compound forsuch applications is needed, given its particular attributes, i.e.slower knock-down effect, lower irritability and higher toxicityto C. quinquefasciatus than other pyrethroids recommended forbednet impregnation.Other studies on the efficacy, under laboratory condi-tions, of pyrethroid insecticides for impregnation of mosquitonets have been conducted in situations that did not permitdirect comparison in terms of impact on mosquito mortality,knock-down effect, irritancy, and blood-feeding inhibition(1719). The present study was undertaken, among otherobjectives, to gain a better understanding of how differences inthese effects may translate in terms of efficacy of impregnatedbednets, and to determine whether bifenthrin has anyadvantage over other pyrethroids in this regard.MethodsTestsThe efficacy of a pyrethroid used for impregnation ofmosquito nets is the result of the insecticides intrinsic activityand the behaviour of the target mosquito in response to it. Thisis of particular relevance for fast-acting insecticides, such aspyrethroids and DDT, with knock-down and irritant proper-ties. The intrinsic activity can be tested with adult mosquitoesusing WHO cones (20), a device which forces tarsal contactwith the impregnated netting material. This test does notindicate overall insecticide efficacy under field conditions,however, because the forced contact does not permit naturalavoidance behaviour. The tunnel test provides a bettersimulation of field conditions. It has given results comparablewith those obtained in the field in experimental huts (21),particularly for mortality and blood-feeding inhibition. In thisstudy both these tests were used.InsecticidesFormulations and concentrations for alpha-cypermethrin,cyfluthrin, deltamethrin, lambdacyhalothrin, etofenprox andpermethrin were selected in accordance with WHO recom-mendations (Table 1 (available at: www.who.int/bulletin)).Bifenthrin was added because of encouraging results obtainedpreviously. Tests were carried out at the WHO-recommendedconcentrations for impregnation of bednets against malariavectors and at one-quarter of the recommended concentra-tions, as it was thought that the lower concentration might be amore sensitive indicator for detecting differences amongproducts.MosquitoesTwo laboratory strains of A. gambiae and two of C. quinque-fasciatus were used. The reference susceptible strains ofA. gambiae (Kisumu), originating from Kenya, and C. quinque-fasciatus (S-Lab), originating from California (22), have beencolonized for many years and are free from any detectableinsecticide-resistance mechanism. The resistant strain ofA. gambiae (VKPR), originating from Burkina Faso, wasalready strongly resistant to permethrin when collected in thefield and has been maintained under constant permethrinselection at each generation (23). The resistant strain ofC. quinquefasciatus (BKPER) was collected in Cote dIvoire andhas also been maintained under continuous selection withpermethrin (24). Both are homozygous for the kdr gene (25,26) with a 40-fold resistance factor (by topical application) (20).The C. quinquefasciatus-resistant strain also has a monooxygen-ase-resistance mechanism (13). Resistant and susceptiblestrains were checked every 3 months for resistance statusand R-genotype.Substrates and treatmentTarsal contact tests were conducted using netting material(warp-knitted multifilament polyester 100 denier, mesh156 (Siamdutch, Thailand)) treated with formulated productas recommended by WHO (27). Pieces of netting(25 cm 6 25 cm) were treated with insecticide at theWHO-recommended concentration and at one-quarter of thisdose, using the formulated product diluted with deionizedwater. The pieces were folded into three equal parts one way,then into three equal parts the other way to give nine layers andeach piece placed in a disposable Petri dish. A quantity offormulation corresponding to the specific absorbency of thenetting and prepared immediately prior to the treatment wasdropped evenly onto the surface of each piece. The pieces werethen carefully squeezed by hand (hands protected by plasticgloves) to ensure an even distribution of the solution and thatno solution remained, and left in the dishes to dry. Tests weremade 510 days after impregnation to ensure that depositswere of similar ages.Tarsal contact with treated netting materialKnock-down effect and mortality resulting from tarsal contactwith netting material were measured using standard WHOplastic cones and a 3-minute exposure time (20). Duringexposure, mosquitoes did not stay long on the cone wall andcones were closed with a polyethylene plug, which does notprovide an attractive resting site. Five non-blood-fed femalesaged 25 days were introduced per cone. On each piece ofnetting 24 cones were attached. Tests were conducted at25 + 2 oC under subdued lighting. After exposure, theinsects were grouped in batches of 20 in 150-ml plastic cupsand held for 24 hours at 27 + 2 oC and 80 + 10% relativehumidity, with honey solution provided. Each piece of nettingwas tested using a total of 50 mosquitoes consisting of tenreplicates of 5 mosquitoes each to allow for inter-batch325Bulletin of the World Health Organization 2003, 81 (5)Comparative efficacy of pyrethroids for impregnation of mosquito netsvariability. The number of knocked-down mosquitoes wasrecorded at fixed intervals (every 210 minutes depending onknock-down rates) for 60 minutes. The observed times to50% knock-down (median knock-down time, KDT50), and2575% knockdown (KDT2575) of mosquitoes were re-corded. Separate KaplanMeier estimates of the proportion ofknocked-down mosquitoes were plotted for each insecticide.The equality of risk of knock-down between insecticides wastested using Coxs proportional hazards models (28). Tiedknock-down times were treated by an exact partial methodconsidering time as discrete. The proportional hazardassumption was assessed graphically by plotting survivalcurves for each insecticide and using a test based onSchoenfeld residuals (29). These tests were conducted inparallel with a control with no insecticide. Mortality ratesobserved after 24 hours were corrected using the Abbottformula (30) and binomial exact 95% confidence intervals(CIs) were calculated for the corrected values. The insecticideswere compared using Fishers exact test.Irritancy testsFemale, non-blood-fed mosquitoes, aged 25 days, wereintroduced individually into plastic cones applied to treatednetting material. After an adaptation time of exactly 60 sec, thetime elapsed between the first landing and the following take-off of themosquito was recorded as the time to first take-off(FT). The observation was not continued for the very fewmosquitoes that did not take off at least once after 256 sec. Foreach test, 50 mosquitoes were used. A simple program usingthe internal clock of a laptop computer was developed to runthis test and to analyse the results by grouping mosquitoes byclasses of first take-off time. The times taken for 50% and 2575%ofmosquitoes to leave the treated surface (median time tofirst take-off, FT50, and FT2575, respectively) were alsorecorded. Separate KaplanMeier estimates of the proportionof mosquitoes that had left the treated surface were plotted foreach insecticide. The equality of risk of take-off betweeninsecticides was tested using Coxs proportional hazardsmodels (28). Tied FT times were analysed using Breslowsmethod (31). The proportional hazard assumption wasassessed graphically by plotting survival curves for eachinsecticide and by using a test based on Schoenfeld residuals(29). When the proportional hazard assumption was rejected,the equality of risk of take-off between insecticides was testedusing the PetoPetoPrentice test (32). Fairly constantconditions of lighting (subdued) and air temperature(25 + 2 oC) were maintained during the test. The numberof take-offs has also been proposed as a measure of irritancy,but this is not a reliable indicator, especially for fast-actinginsecticides (21).Tunnel testsThe basic equipment consisted of a section of square glasstunnel (25 cm 6 25 cm), 60 cm in length, similar to that usedby Elissa & Curtis (33) and described in detail by Chandre et al.(21). A disposable cardboard frame mounted with a treatednetting sample was placed across the tunnel 25 cm from oneend. The surface area of netting accessible to mosquitoes was400 cm2 (20 cm 6 20 cm) with nine holes, each 1 cm indiameter: one hole was located at the centre of the square, theeight others were equidistant and located at 5 cm from theborder. In the shorter section of the tunnel, a bait (guinea pigforA. gambiae, quail forC. quinquefasciatus) was placed, unable tomove. Animals used as baits were selected at random. At eachend of the tunnel, a 30-cm square cage was fitted and coveredwith polyester netting. In the cage at the end of the longersection of the tunnel, 100 female, non-blood-fed mosquitoes,aged 58 days, were introduced at 18:00 hours. Females werefree to fly in the tunnel but had tomake contact with the treatedpiece of netting and locate the holes in it before passingthrough to reach the bait. After a blood meal, they usually flewto the cage at the end of the short section of the tunnel andrested. The following morning, at 09:00 hours, the mosquitoeswere removed and counted separately from each section of thetunnel and the immediate mortality was recorded. Live femaleswere placed in plastic cups with honey solution provided;delayed mortality was recorded after 24 hours. During tests,cages weremaintained in a climatic chamber at 27 + 2 oC and80 + 10% relative humidity under subdued light. Five tunnelswere used simultaneously in the same climatic chamber, onetunnel, with untreated netting always being used as a control.Each net sample was used no more than twice within the sameweek and was then discarded. Blood-feeding inhibition wasassessed by comparing the proportion of blood-fed females(alive or dead) in treated and control tunnels. For eachexperiment with insecticide-treated net, the expected numberof blood-fed females was calculated by multiplying the totalnumber of females tested by the proportion of blood-fedfemales observed among the total of tested females in thecontrol tunnel. Percentage blood-feeding inhibition (BFI) wascalculated by dividing the number of non-fed females by theexpected number of blood-fed females; 95% CIs wereestimated according to the binomial distribution, andinsecticides were compared using Fishers exact test. Overallmortality was measured by pooling the immediate and delayed(24-hour) mortalities of mosquitoes from the two sections ofthe tunnel. Mortality rates in treated conditions were correctedusing the Abbott formula (30) and binomial exact 95% CIswere calculated for the corrected values. Insecticides werecompared using Fishers exact test.Statistical analysisThe differences in outcome variables (mortality, blood-feedinginhibition and irritancy) between the insecticides were analysedseparately for the two insecticide concentrations with Stata7.0 statistical software (34), using the Bonferroni correction totake into account the multiplicity of tests (comparisons of21 pairs of insecticides). The effects of two insecticides wereconsidered to be significantly different when the P-value wasless than 0.05/21 = 0.00238.ResultsKnock-down effectAnopheles gambiaFor susceptible A. gambiae, all tested pyrethroids were fast-acting at theWHO-recommended concentration, with KDT50values of 412 min (Fig.1, Table 2 (available at: www.who.int/bulletin)). For four pyrethroids, including bifenthrin, the valuesof the KDT2575 were in the range 410 min; for three theywere outside this range, one (alpha-cypermethrin) with lowerKDT2575 values and the two others (deltamethrin andlambdacyhalothrin) with higher KDT2575 values. At one-quarter of the WHO-recommended concentration, differ-326 Bulletin of the World Health Organization 2003, 81 (5)Researchences were greater; alpha-cypermethrin and deltamethrin hadthe shortest and longest KDT50, respectively; the value forbifenthrin was similar to those for the other five insecticides.With the resistant strain, the KDT50 values at the higherconcentration were six-to-eight times longer for cyfluthrin,deltamethrin and etofenprox, and two-to-three times longerfor alpha-cypermethrin and lambdacyhalothrin, but more than10 times longer for bifenthrin and permethrin. At the lowerconcentration, alpha-cypermethrin still had the shortestKDT50; almost no knock-down was observed with etofen-prox, lambdacyhalothrin and permethrin.Culex quinquefasciatusAt the WHO-recommended concentration, the KDT50 valuesfor susceptible C. quinquefasciatus (Fig. 2, Table 2) were morethan twice those recorded with susceptible A. gambiae, exceptfor etofenprox, lambdacyhalothrin and permethrin. TheKDT50 for permethrin was the shortest, and not significantlydifferent from the value observed with susceptible A. gambiae.The KDT50 for bifenthrin was three-to-15 times longer thanthe values observed with the other insecticides; this differencewas also observed with the lower concentration. With theresistant strain, only alpha-cypermethrin retained some knock-down effect at the WHO recommended concentration. Forthe other insecticides there was no knock-down or a very longKDT50. At the lower concentration, none of the insecticideshad any knock-down effect.MortalityThe percentage mortalities observed in WHO cones andtunnels are summarized in Table 3 and Table 4, respectively(available at: www.who.int/bulletin). As expected, there was astrong difference in efficacy between the susceptible andresistant strains, particularly for C. quinquefasciatus, whereresistance almost or completely prevented mortality with allcompounds, except permethrin under WHO cones. WithresistantA. gambiae, permethrin showed remarkable efficacy inthe tunnel test, which was greater at the lower than the higherconcentration, as earlier reported by Hodjati & Curtis (35). Atthe WHO-recommended concentration under WHO cones,alpha-cypermethrin was as effective as deltamethrin againstsusceptibleA. gambiae, slightly more effective than etofenprox,and significantly more effective than the other insecticides.The results were similar at the lower concentration. Alpha-cypermethrinwas clearly themost effective insecticide in termsof mortality.IrritancyWith susceptible A. gambiae (Fig. 3, Table 5 (available at:www.who.int/bulletin)), the FT50 values for alpha-cyperme-thrin, cyfluthrin, deltamethrin, and lambdacyhalothrin werecomparable at both concentrations. The most irritatingtreatments were the two etofenprox concentrations and thelower concentration of permethrin. Bifenthrin was by far theleast irritant. With resistant A. gambiae, the FT50 values for327Bulletin of the World Health Organization 2003, 81 (5)Comparative efficacy of pyrethroids for impregnation of mosquito netsalpha-cypermethrin, cyfluthrin, lambdacyhalothrin, and per-methrin at the higher concentration were similar to those withthe susceptible strain; for etofenprox, deltamethrin andbifenthrin, they were 1.83.7 times higher. At the lowerconcentration, the irritant effect remained almost unchanged,except for bifenthrin, for which a 1.5-fold increase was noted.For susceptible C. quinquefasciatus (Fig. 4, Table 5), theirritant effects did not greatly differ among the insecticides,including bifenthrin, except that a significantly longer FT50was observed for deltamethrin at the higher concentration.With the resistant strain, the FT50 values were higherespecially for bifenthrin and deltamethrin at the higherconcentration, and bifenthrin, etofenprox and permethrin atthe lower concentration.Blood-feeding inhibitionBlood-feeding inhibition values are summarized in Table 6(available at: www.who.int/bulletin). With susceptible A. gam-biae andC. quinquefasciatus, there were no significant differencesbetween insecticides at either concentration. With C. quinque-fasciatus, significant differences in blood-feeding inhibitionbetween the susceptible and resistant strains were observed atboth concentrations. The differences were less pronounced forsusceptible and resistant A. gambiae. With resistant C. quinque-fasciatus, bifenthrin performed best at both concentrations. Atthe higher concentration, cyfluthrin, etofenprox, lambdacyha-lothrin and permethrin had similar activities; deltamethrin wassignificantly less effective.Overall insecticidal activityThe results on knock-down, mortality, irritancy and blood-feeding inhibition were graded into six categories (05; Table 7and Annexes 14 (available at www.who.int/bulletin)).Rankings were established for each concentration on the basisof cumulative scores derived by combining the scores for thetwo mosquito species, susceptible or resistant, as indicated inTable 8 and Table 9 (available at: www.who.int/bulletin). Theresults for knock-down effect, irritancy and mortality obtainedin the cone test were grouped as shown in Table 8. Alpha-cypermethrin performed significantly better than the otherproducts regardless of mosquito species and strain; bifenthrinwas significantly less efficient. The results for mortality andblood-feeding inhibition obtained in the tunnel test weregrouped as shown in Table 9. Bifenthrin and, to a lesser extent,alpha-cypermethrin performed best regardless of mosquitospecies and resistance status.DiscussionTo our knowledge, this laboratory evaluation is the mostcomprehensive comparative study yet undertaken on theefficacy of pyrethroids for impregnation of mosquito nets. Itinvolved the use of susceptible and resistant strains of two328 Bulletin of the World Health Organization 2003, 81 (5)Researchmosquito species of public health importance and permittedthe assessment of three aspects of the insecticide activity(knock-down effect, irritancy, and mortality under WHOcones) andmortality and blood-feeding inhibition in free-flyingmosquitoes in tunnels, where the result depends on variousinteracting factors, including irritancy and knock-down effect.Insecticide activity under WHO conesThe activity under WHO cones varied significantly frominsecticide to insecticide and between mosquito species andstrains. The knock-down effect noted with the two susceptiblemosquito species was rapid and related to concentration,whatever the insecticide tested (KDT50 was longer at the lowerconcentration). KDT50 increased dramatically with resistantA. gambiae and the knock-down effect almost completelydisappeared with resistant C. quinquefasciatus. Mortality ratesobserved following forced tarsal contact in WHO cones ortunnels clearly showed the overall good performances of alpha-cypermethrin. Its efficacy at 20 mg/m2 has been confirmed inCote dIvoire by Koffi et al. (36) with laboratory and wildsusceptible populations of A. gambiae but not with wild kdr-resistant populations. Mortality under cones was sometimeslower than expected, e.g. for cyfluthrin and lambdacyhalothrin.In other studies (37, 38), these insecticides have commonlyshown 100% mortality when tested at the WHO-recom-mended concentration. However, mortality should not beconsidered alone, since a high irritant effect can considerablyreduce tarsal contact with treated netting material, even withforced contact under WHO cones. For example, in the case ofpermethrin, which has a high irritant effect, mortality of lessthan 80% has commonly been observed with susceptibleA. gambiae under WHO cones at the recommended concen-tration (62% in the present study).Irritant effect of insecticidesThe irritant effect was not closely related to insecticideconcentration, as observed previously for susceptible A. gam-biae with DDT (39). Irritancy differed from insecticide toinsecticide: bifenthrin was much less irritant toA. gambiae thanthe other pyrethroids, while etofenprox provided the highestirritancy, whatever the resistance status. The irritant effect wassignificantly reduced in resistant strains, as observed previouslywith permethrin versus resistantA. gambiae (40). The extent ofthis reduction differed between insecticides and mosquitospecies, however, being generally greater with C. quinquefascia-tus than with A. gambiae.Tunnel testIn the tunnel test, all the insecticides performed well againstsusceptible A. gambiae in terms of mortality and blood-feedinginhibition, even at the lower concentration. This last observa-tion is of great importance, since the first wash of a treatedmosquito net is expected to remove up to 50% of theinsecticide, and every subsequent wash 2530%, leaving about329Bulletin of the World Health Organization 2003, 81 (5)Comparative efficacy of pyrethroids for impregnation of mosquito netsone-quarter of the original content after three-to-four washes(41). Our results were in line with unpublished field data thatshowed that mosquito nets were still effective after three-to-four washes. These results encouraged WHO to review theexisting guidelines (42) in recommending in the next versionthe systematic re-treatment of nets after three washes or at leastonce a year. Pyrethroid resistance significantly decreasedmortality of A. gambiae but did not dramatically interfere withblood-feeding inhibition. These results confirmed thatmosquito nets treated with pyrethroids are still effective inreducing humanvector contact (8, 9) and malaria morbidity(10, 11) in resistant strains.ConclusionsFor susceptible C. quinquefasciatus in tunnels, only bifenthrincaused high mortality at the lower concentration and mortalitywith the resistant strain was extremely low or nil with all theinsecticides. Contrary to Miller & Curtis, who observed alower but not significant feeding rate with bifenthrin thanwith other treatments (43), we observed a significantinhibition of blood-feeding with this compound, even at thelower concentration. These results are of great importancesince C. quinquefasciatus is responsible for most mosquitonuisance worldwide and is increasing in Africa and Asiabecause of the expansion of favourable habitats that usuallyaccompanies urbanization. To gain better acceptance andcompliance in the use of insecticide-impregnated mosquitonets, the nets should have a noticeable impact in reducing thispest nuisance. Alpha-cypermethrin performed best in boththe cone and the tunnel test, whatever the mosquito speciesand resistance status. Bifenthrin also performed well in thetunnel test but showed the least effective performances interms of knock-down effect, irritancy and mortality aftershort-term exposure. This demonstrates the fact that theimpact of impregnated mosquito nets results from a complexinteraction of factors, which cannot easily be dissociated fromeach other. Mortality and blood-feeding inhibition undertunnels should be considered as among the most importantattributes of insecticides for use in impregnation of mosquitonets since these tests provide results comparable with thoseobtained in experimental hut studies (21).Our results with bifenthrin and those obtained by othersunder laboratory (16) and field (15) conditions suggest that thisinsecticide is a promising pyrethroid for impregnation ofmosquito nets because of its much stronger impact onC. quinquefasciatus.When selecting pyrethroids for mosquito vector controland personal protection, specific attention should be given tothe various properties of these insecticides, the behaviouralresponse of the target mosquito species, the pyrethroidresistance status in the area and the importance of nuisance,especially that due to C. quinquefasciatus. n330 Bulletin of the World Health Organization 2003, 81 (5)ResearchAcknowledgementsThis study was supported financially by the WHOPesticide Evaluation Scheme and the Institut deRecherche pour le Developpement, Paris, France. Weare grateful to the following companies for providingpyrethroid insecticide formulations: Aventis (deltame-thrin and permethrin), Bayer AG (cyfluthrin), BASF(alpha-cypermethrin), FMC Corporation (bifenthrin),Mitsui Chemical Inc. (etofenprox), and Syngenta (lamb-dacyhalothrin).Conflicts of interest: none declared.ResumePerformances comparees, dans des conditions de laboratoire, de sept insecticides de la classedes pyrethrinodes utilises pour limpregnation des moustiquairesObjectif Comparer lefficacite de sept insecticides de la classe despyrethrinodes pour limpregnation des moustiquaires, six recom-mandes par lOMS et un produit candidat, la bifenthrine, dans desconditions de laboratoire.Methodes Les tests ont ete realises sur des souches dAnophelesgambiae et de Culex quinquefasciatus sensibles et resistantes auxpyrethrinodes. Leffet knock-down , leffet irritant et lamortalite ont ete mesures au moyen de tests standard de lOMSavec pieges coniques. La mortalite et linhibition de la prise derepas de sang ont egalement ete mesurees au moyen de tunnelscontenant un appat.Resultats Chez A. gambiae sensible aux pyrethrinodes, lalpha-cypermethrine avait leffet knock-down le plus rapide. Chez lessouches resistantes, cet effet etait legerement plus lent aveclalpha-cypermethrine et beaucoup plus reduit apres exposition auxautres insecticides, notamment la bifenthrine et la permethrine.Chez C. quinquefasciatus sensible, leffet knock-down etaitsignificativement plus lent que chez A. gambiae, en particulier avecla bifenthrine, et chez les souches resistantes il etait nul quel quesoit le pyrethrinode utilise. La bifenthrine etait significativementmoins irritante que les autres pyrethrinodes pour les A. gambiaesensibles et resistants, mais il ny avait pas de difference marqueeau niveau de leffet irritant contre C. quinquefasciatus. Dans lestunnels, les insecticides etaient tous moins toxiques chezC. quinquefasciatus que chez A. gambiae en ce qui concerne lessouches sensibles. Pour les souches resistantes, on a observe unemortalite importante avec tous les pyrethrinodes chez A. gambiaemais non chez C. quinquefasciatus. Linhibition de la prise de repasde sang etait forte chez les souches sensibles des deux especes etchez les souches resistantes de A. gambiae mais plus faible chez lessouches resistantes de C. quinquefasciatus ; la bifenthrine avaitlimpact le plus marque.Conclusion Lefficacite pour limpregnation des moustiquairescontre A. gambiae etait maximale avec lalpha-cypermethrine. Labifenthrine possede probablement un avantage relatif importantsur les autres pyrethrinodes dans les zones de resistance a cetteclasse dinsecticides en raison de son impact beaucoup plusmarque sur le moustique nuisant, C. quinquefasciatus, malgre uneffet knock-down plus lent et un effet irritant plus faible. Lechoix des pyrethrinodes destines a la lutte contre les moustiquesvecteurs et a la protection individuelle devra tenir compte desdifferents effets de ces insecticides, de letat de la resistance auxpyrethrinodes dans la zone concernee et de limportance desespeces nuisantes telles que C. quinquefasciatus.ResumenResultados comparativos, en condiciones de laboratorio, de siete insecticidas piretroides utilizadospara impregnar los mosquiterosObjetivo Comparar la eficacia como tratamiento de impregna-cion de mosquiteros, en condiciones de laboratorio, de sieteinsecticidas piretroides: seis recomendados actualmente por laOMS, y un producto experimental, la bifentrina.Metodos Se hicieron pruebas con cepas de Anopheles gambiae yCulex quinquefasciatus sensibles y resistentes a los piretroides. Losefectos de cada, irritacion y mortalidad se midieron utilizando laspruebas ordinarias con conos de la OMS. Tambien se midieron lamortalidad y la inhibicion de la hemoingestion, empleando paraello un dispositivo tuneliforme con cebo.Resultados En los ejemplares de A. gambiae sensibles, la alfa-cipermetrina tuvo el efecto de cada mas rapido, mientras que en losresistentes dicho efecto fue ligeramente mas lento con la alfa-cipermetrina y mucho menor tras la exposicion a los otrosinsecticidas, particularmente la bifentrina y la permetrina. EnC. quinquefasciatus sensible, el efecto de cada fue significativa-mente mas lento que en A. gambiae, en particular con la bifentrina, yen el caso de la cepa resistente no se observo tal efecto con ningunode los piretroides. La bifentrina fue significativamente menos irritanteque los otros piretroides para A. gambiae, sensible y resistente, perono se observaron diferencias claras en el poder de irritacion entre lospiretroides en el caso de C. quinquefasciatus. En los dispositivostuneliformes, todos los insecticidas fueron menos toxicos contraC. quinquefasciatus que contra A. gambiae para las cepas sensibles.Considerando las cepas resistentes, la mortalidad fue importante contodos los piretroides en el caso de A. gambiae, pero no en el deC. quinquefasciatus. La inhibicion de la hemoingestion fue altatambien en las cepas sensibles de ambas especies y en A. gambiaeresistente, pero inferior en C. quinquefasciatus resistente; el maximoefecto fue el conseguido con la bifentrina.Conclusion La alfa-cipermetrina fue el producto mas eficaz comotratamiento de impregnacion de los mosquiteros contra A. gam-biae. La bifentrina puede presentar ventajas comparativasimportantes frente a otros piretroides en las areas con resistenciaa estos productos, debido a su mucho mayor efecto en el mosquitocausante de molestias C. quinquefasciatus, pese a la mayor lentitudde su efecto de cada y de irritacion. A la hora de seleccionar lospiretroides para combatir los mosquitos vectores y asegurar laproteccion personal, deberan tenerse en cuenta los diferentesefectos de estos insecticidas, la situacion de resistencia a lospiretroides en la zona en cuestion y la importancia de los mosquitoscausantes de molestias, como C. quinquefasciatus.331Bulletin of the World Health Organization 2003, 81 (5)Comparative efficacy of pyrethroids for impregnation of mosquito netsReferences1. Lines J. Mosquito nets and insecticides for net treatment: a discussion ofexisting and potential distribution systems in Africa. Tropical Medicine andInternational Health 1996;1:616-32.2. Lines JD, Zaim M. Insecticide products: treatment of mosquito nets at home.Parasitology Today 2000;16:91-2.3. Zaim M, Aitio A, Nakashima N. Safety of pyrethroid-treated nets. Medical andVeterinary Entomology 2000;14:1-5.4. 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Geneva; World Health Organization, 1997.WHO document WHO/CTD/AFRO/97.4. Available from: URL: http://whqlib-doc.who.ch/hq/1997/CTD_MAL_AFRO_97.4.pdf43. Miller JE, Curtis CF. Testing net impregnating agents with mosquitoes releasedto fly freely in a room. Transactions of the Royal Society of Tropical Medicineand Hygiene 1995;89:241-344.333Bulletin of the World Health Organization 2003, 81 (5)Comparative efficacy of pyrethroids for impregnation of mosquito netsTable 1. List and characteristics of the seven test pyrethroid insecticidesInsecticide Formulationa Trade name Sample ref. Tested concentration(mg/m2)Alpha-cypermethrin SC 10% Fendona R1811-187 40 and 10Bifenthrin ME 0.3% Talstar PL99-0189 25 and 6.25Cyfluthrin EW 5% Solfac 233-926-708 50 and 12.5Deltamethrin SC 1% K-Othrin LELH10169700 25 and 6.25Etofenprox EW 10% Vectron MN-106 200 and 50Lambdacyhalothrin CS 2.5% Icon BSNIC-1614 20 and 5Permethrin EC 10% Peripel LEEHI0189800 500 and 125a CS = Capsule suspension; EC = emulsifiate concentrate; EW = emulsion, oil in water; ME = micro-emulsion; SC = suspension concentrate.Table 2. Median knock-down times (KDT50) and times to knock-down of 2575% (KDT2575) of mosquitoes in susceptibleand resistant strains of Anopheles gambiae and Culex quinquefasciatus for seven pyrethroid insecticides used to impregnatemosquito netsTime (min)Insecticide Concentration A. gambiae C. quinquefasciatus(mg/m2)Susceptible Resistant Susceptible ResistantKDT501 KDT2575 KDT501 KDT2575 KDT501 KDT2575 KDT501 KDT2575WHO-recommended concentrationAlpha-cypermethrin 40 4a 26 (51)2 10a 1015 (49) 10a 812 (50) > 60a 20 to > 60 (54)Bifenthrin 25 8b 610 (53) > 60b > 60 to > 60 (53) 60b 50> 60 (51) > 60a, b > 60 to > 60 (51)Cyfluthrin 50 8b 610 (50) 60c d 40 to > 60 (50) 20c 1030 (51) > 60a, b > 60 to > 60 (52)Deltamethrin 25 10c 812 (50) 60c 40 to > 60 (50) 20c 2025 (51) > 60b > 60 to > 60 (51)Etofenprox 200 6b 46 (50) 40c, d 20 to > 60 (51) 8d 58 (50) > 60a, b > 60 to > 60 (52)Lambdacyhalothrin 20 12c 820 (51) 40d 3050 (50) 12e 1015 (54) > 60b > 60 to > 60 (49)Permethrin 500 6b 410 (50) > 60b > 60 to > 60 (50) 4f 26 (50) > 60a, b > 60 to > 60 (51)25% WHO-recommended concentrationAlpha-cypermethrin 10 6a 48 (51) 15a 1520 (53) 10a 812 (49) > 60a > 60 to > 60 (51)Bifenthrin 6.25 10b, d 612 (51) > 60b 60 to > 60 (54) > 60b 60> 60 (50) > 60a > 60 to > 60 (50)Cyfluthrin 12.5 10b, d 815 (51) > 60b 60 to > 60 (51) 20c 2020 (49) > 60a > 60 to > 60 (50)Deltamethrin 6.25 15c 1225 (50) > 60b 50 to > 60 (49) 40d 2540 (51) > 60a > 60 to > 60 (50)Etofenprox 50 10b, c 815 (60) > 60b > 60 to > 60 (49) 8e 510 (51) > 60a > 60 to > 60 (51)Lambdacyhalothrin 5 12b, c 820 (51) > 60b > 60 to > 60 (52) 15f 1220 (50) > 60a > 60 to > 60 (49)Permethrin 125 8d 612 (50) > 60a, b > 60 to > 60 (51) 6g 48 (51) > 60a > 60 to > 60 (50)1 KDT50 values with different superscripts (a g) within the same group of concentrations and in the same column differ significantly (Coxs model).2 Figures in parentheses are number of insects tested.ResearchA Bulletin of the World Health Organization 2003, 81 (5)Table 3. Mortality rates in WHO cone tests for susceptible and resistant strains of Anopheles gambiae and Culex quinquefasciatusfor seven pyrethroid insecticides used to impregnate mosquito netsMortality rateInsecticide Concentration A. gambiae C. quinquefasciatus(mg/m2)Susceptible Resistant Susceptible Resistantn1 %2 (95% CI)3 n %2 (95% CI) n %2 (95% CI) n %2 (95% CI)WHO-recommended concentrationAlpha-cypermethrin 40 51 100a (93100) 49 94a (8399) 50 100a (93100) 54 4a, b (013)Bifenthrin 25 51 61b (4674) 53 8b (218) 51 18b (831) 51 2a, b (010)Cyfluthrin 50 50 74b, c (6085) 50 0b (07) 51 33b, c (2148) 52 4a, b (013)Deltamethrin 25 50 100a (93100) 50 14b (627) 51 98a (90100) 51 2a, b (010)Etofenprox 200 50 94a, c (8399) 51 6b (116) 50 18b (931) 52 0a (07)Lambdacyhalothrin 20 51 43b (2958) 50 0b (07) 54 50c (3664) 49 0a (07)Permethrin 500 50 60b (4574) 50 2b (011) 50 38b, c (2553) 51 22b (1135)25% WHO-recommended concentrationAlpha-cypermethrin 10 51 100a (93100) 53 70a (5682) 49 100a (93100) 51 0a (07)Bifenthrin 6.25 51 24b (1337) 54 7b (218) 50 2b (011) 50 0a (07)Cyfluthrin 12.5 51 47b, c (3362) 51 8b (219) 49 16b, c (730) 50 0a (07)Deltamethrin 6.25 50 92a (8198) 49 4b (014) 51 86a (7494) 50 2a, b (011)Etofenprox 50 50 56c (4170) 49 4b (014) 51 8b, c (219) 51 0a (07)Lambdacyhalothrin 5 51 43b, c (2958) 52 2b (010) 50 18b, c (931) 49 0a (07)Permethrin 125 50 44b, c (3059) 51 2b (010) 51 25c (1440) 50 20b (1034)1 n = No. of insects tested.2 % in the same column with different superscripts (a c) within the same group of concentrations, differ significantly (Fishers exact test).3 95% CI = 95% confidence interval.Table 4. Mortality rates in tunnel tests with susceptible and resistant strains of Anopheles gambiae and Culex quinquefasciatusfor seven pyrethroid insecticides used to impregnate mosquito netsMortality rateInsecticide Concentration A. gambiae C. quinquefasciatus(mg/m2)Susceptible Resistant Susceptible Resistantn1 %2 (95% CI)3 n %2 (95% CI) n %2 (95% CI) n %2 (95% CI)WHO-recommeded concentrationAlpha-cypermethrin 40 92 100a (96100) 91 59a (4970) 89 87a (7893) 101 3a (18)Bifenthrin 25 94 100a (96100) 87 33b, d (2444) 97 91a (8396) 97 4a (110)Cyfluthrin 50 97 95a (8898) 98 21b, c (1431) 97 87a (7893) 83 0a (04)Deltamethrin 25 93 97a (9199) 99 38a, d (2949) 94 43b (3253) 99 0a (04)Etofenprox 200 88 98a (92100) 93 9c (416) 106 21c (1330) 100 1a (05)Lambdacyhalothrin 20 92 99a (94100) 100 17b, c (1026) 97 78a (6986) 96 1a (06)Permethrin 500 95 95a (8898) 96 10c (518) 96 77a (6785) 99 0a (04)25% WHO-recommended concentrationAlpha-cypermethrin 10 92 95a, b (8898) 97 24a, e (1633) 96 65a (5474) 98 1a (06)Bifenthrin 6.25 84 99a, b (94100) 84 49b, c, e (3860) 96 99b (94100) 79 0a (05)Cyfluthrin 12.5 99 90b (8295) 99 5d (211) 111 36c (2746) 100 0a (04)Deltamethrin 6.25 94 94a, b (8798) 92 39e (2950) 94 33c (2443) 111 0a (03)Etofenprox 50 81 100a (96100) 94 9a, d (416) 93 28c (1938) 99 0a (04)Lambdacyhalothrin 5 92 99a, b (94100) 93 0d (04) 91 79a (6987) 102 0a (04)Permethrin 125 97 96a, b (9099) 95 66c (5676) 95 35c (2545) 95 2a (07)1 n = No. of insects tested.2 % in the same column with different superscripts (ae) within the same group of concentrations, differ significantly (Fishers exact test).3 95% CI = 95% confidence interval.Comparative efficacy of pyrethroids for impregnation of mosquito netsBBulletin of the World Health Organization 2003, 81 (5)Table 5. Time to first take-off (FT50) and time to take-off of 2575% (FT25-75) of mosquitoes in WHO cone tests with susceptibleand resistant strains of Anopheles gambiae and Culex quinquefasciatus for seven pyrethroid insecticides used to impregnatemosquito netsTime (sec)Insecticide Concentration A. gambiae C. quinquefasciatus(mg/m2)Susceptible Resistant Susceptible ResistantFT501 FT2575 FT501 FT2575 FT501 FT2575 FT501 FT2575WHO-recommended concentrationAlpha-cypermethrin 40 11a, c 518 (50)2 16a, d 647 (50) 11a 514 (50) 16a, c, d* 1092 (50)Bifenthrin 25 39b 1079 (51) 145b 24 to > 256 (50) 12a 627 (50) > 256 b* 23 to > 256 (50)Cyfluthrin 50 13a 825 (50) 19a, d 745 (50) 31b 1569 (51) 51b, c* 21131 (51)Deltamethrin 25 12a 722 (50) 29d 12125 (51) 97c 31256 (26) 159a, b, c* 15 to > 256 (25)Etofenprox 200 7c 413 (50) 13a 546 (50) 29b 678 (50) 45a, b, c, d* 13 to > 256 (50)Lambdacyhalothrin 20 11a 817 (50) 15a, d 641 (50) 9a 618 (50) 20d* 932 (50)Permethrin 500 11a 720 (50) 13a 433 (50) 38b 1564 (51) 32a, c, d* 1247 (51)25% WHO-recommended concentrationAlpha-cypermethrin 10 9a, c 519 (50) 24a*, d* 1638 (50) 18a 1128 (50) 37a, d* 21139 (50)Bifenthrin 6.25 38b 14140 (50) 219b* 27 to > 256 (50) 31b 1268 (50) > 256b* > 256 to > 256 (31)Cyfluthrin 12.5 11a 522 (50) 11c* 626 (50) 35b 15106 (51) 10c* 734 (50)Deltamethrin 6.25 19a 828 (50) 31a* 1675 (50) 22a, b 1350 (51) 54a, c, d* 6 to > 256 (50)Etofenprox 50 6c, d 411 (50) 19a, c* 7232 (50) 34b 1671 (50) 131d* 44 to > 256 (50)Lambdacyhalothrin 5 15a 828 (50) 20a, c* 856 (50) 12a 823 (50) 22a, c* 960 (50)Permethrin 125 6d 39 (50) 19a, c* 940 (50) 20a, b 1036 (51) 89d* 32 to > 256 (50)1 FT50 values with different superscripts (ad) within the same group of concentrations and in the same column differ significantly (Coxs model).2 Figures in parentheses are the numbers of insects tested.* PetoPetoPrentice test was applied to these entries.Table 6. Blood-feeding inhibition in tunnel tests with susceptible and resistant strains of Anopheles gambiae and Culexquinquefasciatus for seven pyrethroid insecticides used to impregnate mosquito netsInsecticide Concentration A. gambiae C. quinquefasciatus(mg/m2)Susceptible Resistant Susceptible Resistantn1 % inhibi- (95% n1 % inhibi- (95% n1 % inhibi- (95% n1 % inhibi- (95%ted2 CI) ted2 CI) ted2 CI) ted2 CI)WHO-recommended concentrationAlpha-cypermethrin 40 23 100a (85100) 83 99a (93100) 66 98a (92100) 93 28a, c, d (1938)Bifenthrin 25 34 97a (85100) 56 79b (6688) 49 100a (93100) 89 93b (8697)Cyfluthrin 50 41 95a (8399) 89 87a, b (7893) 72 100a (95100) 59 49c (3663)Deltamethrin 25 40 98a (87100) 71 94a, b (8698) 31 97a (83100) 70 16d (826)Etofenprox 200 32 100a (89100) 83 88a, b (7994) 80 96a (8999) 93 63c (5373)Lambdacyhalothrin 20 23 100a (85100) 68 93a, b (8498) 73 100a (95100) 89 45a, c (3456)Permethrin 500 41 100a (91100) 69 78b (6787) 49 100a (93100) 85 40a, c (3051)25% WHO-recommended concentrationAlpha-cypermethrin 10 23 91a (7299) 88 76a (6685) 71 100a (95100) 90 39a (2950)Bifenthrin 6.25 30 97a (83100) 54 87a, b (7595) 49 100a (93100) 72 94b (8698)Cyfluthrin 12.5 42 100a (92100) 90 93b (8698) 82 100a (96100) 74 0c (05)Deltamethrin 6.25 41 100a (91100) 66 80a, b (6989) 31 94a (7999) 79 1c, d (07)Etofenprox 50 29 100a (88100) 84 38c (2849) 70 94a (8698) 92 23a, e (1533)Lambdacyhalothrin 5 23 100a (85100) 64 86a, b (7593) 69 100a (95100) 95 13d, e (721)Permethrin 125 42 93a (8199) 69 70a (5780) 48 98a (89100) 82 37a (2648)1 n = No. of insects tested.2 % in the same column with different superscripts (a to e) within the same group of concentrations differ significantly (Fishers exact test).ResearchC Bulletin of the World Health Organization 2003, 81 (5)Table 7. Allocation of an insecticide score on the basis of the results for four main characteristics testedScore KDT50a (min) Mortality (%) FT50b (sec) Blood-feeding inhibition (%)0 > 50 < 20% >50 < 50%1 3049 2039 % 3049 5069 %2 1529 4059 % 2029 7079 %3 1014 6079 % 1519 8089 %4 59 8094 % 1014 9094 %5 < 5 >95 % < 10 > 95 %a Median knock-down time.b Median time to first take-off.Table 8. Ranking of seven pyrethroid insecticides obtained by adding scores from cone test data (knock down, irritancy andmortality) for Anopheles gambiae and Culex quinquefasciatusA. gambiae SSa + A. gambiae SS + A. gambiae RR + A. gambiae RR + AllC. quinque- C. quinque- C. quinque- C. quinque- strainsfasciatus SS fasciatus RRb fasciatus SS fasciatus RRInsecticide Score Rankc Score Rank Score Rank Score Rank Score RankWHO-recommended concentrationAlpha-cypermethrin 26 1 17 1 22 1 13 1 78 1Bifenthrin 12 7 8 7 4 7 0 7 24 7Cyfluthrin 15 6 11 6 7 6 3 5 36 6Deltamethrin 19 2 12 4 9 5 2 6 42 5Etofenprox 19 2 14 2 11 3 6 2 50 2Lambdacyhalothrin 19 2 11 5 14 2 6 2 50 2Permethrin 18 5 13 3 11 3 6 2 48 425% WHO-recommended concentrationAlpha-cypermethrin 25 1 15 1 18 1 8 1 66 1Bifenthrin 6 7 5 7 1 7 0 7 12 7Cyfluthrin 12 6 13 2 7 6 8 1 40 3Deltamethrin 16 3 9 6 8 3 1 6 34 6Etofenprox 15 4 10 4 8 3 3 5 36 4Lambdacyhalothrin 14 5 10 4 8 3 4 3 36 4Permethrin 18 2 12 3 10 2 4 3 44 2a SS = susceptible strain.b RR = resistant strain.c Rank = 1 is the highest, indicating best insecticide performance.Comparative efficacy of pyrethroids for impregnation of mosquito netsDBulletin of the World Health Organization 2003, 81 (5)Table 9. Ranking of seven pyrethroid insecticides obtained by adding scores from tunnel test data (mortality and blood-feedinginhibition) for Anopheles gambiae and Culex quinquefasciatusA. gambiae SSa + A. gambiae SS + A. gambiae RR + A. gambiae RR + AllC. quinque- C. quinque- C. quinque- C. quinque- strainsfasciatus SS fasciatus RRb fasciatus SS fasciatus RRInsecticide Score Rankc Score Rank Score Rank Score Rank Score RankWHO recommended concentrationAlpha-cypermethrin 19 1 10 3 16 1 7 1 52 1Bifenthrin 19 1 14 1 12 3 7 1 52 1Cyfluthrin 19 1 10 3 13 2 4 4 46 3Deltamethrin 17 6 10 3 12 3 5 3 44 4Etofenprox 16 7 11 2 9 7 4 4 40 6Lambdacyhalothrin 18 4 10 3 12 3 4 4 44 4Permethrin 18 4 10 3 10 6 2 7 40 625% WHO-recommended concentrationAlpha-cypermethrin 17 3 9 4 11 2 3 5 40 3Bifenthrin 20 1 14 1 15 1 9 1 58 1Cyfluthrin 15 4 9 4 10 5 4 3 38 5Deltamethrin 14 7 9 4 9 6 4 3 36 6Etofenprox 15 4 10 2 5 7 0 7 30 7Lambdacyhalothrin 18 2 10 2 11 2 3 5 42 2Permethrin 15 4 9 4 11 2 5 2 40 3a SS = susceptible strain.b RR = resistant strain.c Rank = 1 is the highest, indicating best insecticide performance.Annex 1. Insecticide scores for performance against susceptible Anopheles gambiae for seven pyrethroid insecticides in five testsInsecticide Knock-down Irritancy Mortality in Mortality in Blood-feedingeffect WHO cones tunnels inhibitionDosea 25% doseb Dose 25% dose Dose 25% dose Dose 25% dose Dose 25% doseAlpha-cypermethrin 5 5 5 5 5 5 5 4 5 4Bifenthrin 4 4 2 1 3 1 5 5 5 5Cyfluthrin 4 4 4 5 3 2 5 4 4 5Deltamethrin 4 2 5 4 5 4 5 4 5 5Etofenprox 5 4 5 5 4 2 5 5 5 5Lambdacyhalothrin 4 3 4 4 3 2 5 5 5 5Permethrin 4 4 4 5 3 2 4 5 5 4a WHO-recommended concentration for use in impregnating mosquito nets.b One-quarter of WHO-recommended concentration.Annex 2. Insecticide scores for performance against resistant Anopheles gambiae for seven pyrethroid insecticides in five testsInsecticide Knock-down Irritancy Mortality in Mortality in Blood-feedingeffect WHO cones tunnels inhibitionDosea 25% doseb Dose 25% dose Dose 25% dose Dose 25% dose Dose 25% doseAlpha-cypermethrin 4 3 4 3 4 3 4 3 5 3Bifenthrin 0 0 0 0 0 0 0 0 3 4Cyfluthrin 1 0 4 5 0 0 0 0 4 4Deltamethrin 0 0 1 2 1 0 1 0 4 4Etofenprox 1 0 3 2 0 0 0 0 3 4Lambdacyhalothrin 1 0 4 3 0 0 0 0 4 4Permethrin 0 0 4 4 0 0 0 0 3 3a WHO-recommended concentration for use in impregnating mosquito nets.b One-quarter of WHO-recommended concentration.ResearchE Bulletin of the World Health Organization 2003, 81 (5)Annex 3. Insecticide scores for performance against susceptible Culex quinquefasciatus for seven pyrethroid insecticides in five testsInsecticide Knock-down Irritancy Mortality in Mortality in Blood-feedingeffect WHO cones tunnels inhibitionDosea 25% doseb Dose 25% dose Dose 25% dose Dose 25% dose Dose 25% doseAlpha-cypermethrin 4 4 5 4 5 5 4 3 5 5Bifenthrin 0 0 4 2 1 0 4 5 5 5Cyfluthrin 3 3 2 1 1 1 4 1 5 5Deltamethrin 2 2 0 3 5 5 2 1 5 4Etofenprox 4 4 2 2 1 0 1 1 5 4Lambdacyhalothrin 3 3 5 2 2 0 3 3 5 5Permethrin 5 5 2 3 1 1 3 1 5 5a WHO-recommended concentration for use in impregnating mosquito nets.b One-quarter of WHO-recommended concentration.Annex 4. Insecticide scores for performance against resistant Culex quinquefasciatus for seven pyrethroid insecticides in five testsInsecticide Knock-down Irritancy Mortality in Mortality in Blood-feedingeffect WHO cones tunnels inhibitionDosea 25% doseb Dose 25% dose Dose 25% dose Dose 25% dose Dose 25% doseAlpha-cypermethrin 0 0 2 1 0 0 0 0 1 1Bifenthrin 0 0 0 0 0 0 0 0 4 4Cyfluthrin 0 0 1 4 0 0 0 0 2 0Deltamethrin 0 0 0 0 0 0 0 0 0 0Etofenprox 0 0 1 0 0 0 0 0 3 1Lambdacyhalothrin 0 0 3 2 0 0 0 0 2 0Permethrin 0 0 2 0 1 1 0 0 2 1a WHO-recommended concentration for use in impregnating mosquito nets.b One-quarter of WHO-recommended concentration.Comparative efficacy of pyrethroids for impregnation of mosquito netsFBulletin of the World Health Organization 2003, 81 (5)

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