Efficacy of a Mosaic Long-Lasting Insecticide Net , PermaNet 3 . 0 , againstWild Populations of Culex quinquefasciatus in Experimental Huts in Togo

1 Kintampo Health Research Centre, Ghana Health Service, Ministry of Health, P.O. Box 200, Kintampo, Ghana 2 Laboratoire d’Entomologie Appliquée (LEA), Faculté des Sciences, Université de Lome, P.O. Box 1515, Lomé, Togo 3 Centre de Recherche Entomologique de Cotonou (CREC), Institut de Recherche Pour le Développement (IRD), 01 P.O. Box 4414 RP, Cotonou, Benin Anopheles Biology and Control (ABC) Network, Institut de Recherche Pour le Développement (IRD), P.O. Box 1386, Dakar, Senegal


Background
Insecticide treated nets are recommended by the World Health Organisation (WHO) for malaria control in malaria endemic countries [1][2][3] and for control of other diseases of medical importance [4].ey offer individual and community protection against malaria [3,5], sometimes reducing morbidity by as much as 50% and global mortality by 20-30% [6].New techniques for long-lasting insecticide treatment of nets provide solution for the need to regularly re-treat nets [7].For acceptability at community level, nets should as well offer protection to individuals against nuisance vectors such as Culex and Mansonia mosquito species [5,8,9].
Many infectious diseases in Africa are caused by Culex mosquitoes [10] such as West Nile Virus (WVN), Ri Valley Fever (RVF) and Bancroian �lariasis [4].Most organized efforts to control populations of C. quinquefasciatus (C.fatigans, C. pipiens fatigans, and C. pipiens quinquefasciatus) in Asia and elsewhere have been directed at the immature stages [11,12].Most Culex species rest outdoors, except for C. quinquefasciatus, which is a domestic mosquito, over 50% resting on nonsprayable surfaces in houses, such as mosquito nets, clothes, hangings and furniture [13].
Directly and indirectly, the use of pesticides has resulted in selection of broad spectrum organophosphate and carbamate resistance against C. Quinquefasciatus [14], and pyrethroid resistance with the most commonly selected resistance mechanism observed as increased activity of Esta 2 and Estb 2 carboxylesterases; A2 and B2 esterases on an earlier classi�cation [15,16].One mechanism of resistance to pyrethroids is increased reduction in intrinsic insensitivity of the insect nervous system [17], and a second mechanism involves increased insecticide metabolism [18].
Currently, pyrethroid insecticides are the recommended compounds for the treatment of mosquito nets for malaria control [19], because they have low mammalian toxicity and fast acting properties against mosquitoes [20].Resistance in many disease vectors to pyrethroids threatens this recommendation and jeopardises the success of spraying efforts and bed net impregnation [21].Several strategies have been proposed for resistance management.Notably, to use a pyrethroid and a nonpyrethroid insecticide in combination on the same mosquito net, either separately or as a mixture [22].A few studies have shown that exposure to the P450 monooxygenase inhibitor, piperonyl butoxide (PBO) caused a partial reversal of Permethrin resistance [23].Piperonyl butoxide (PBO) is a pesticide synergist.It does not have pesticidal properties.However, when added to insecticide mixtures, typically pyrethrin, pyrethroid, and carbamate insecticides, their potency is increased considerably.It is a potent cytochrome P450 inhibitor and this family of enzymes acts as the principal detoxi�cation pathway for many pesticides.Inhibiting the detoxi�cation pathway allows higher unmetabolised systemic concentrations of the active insecticide to remain within the target insect for a longer period.
Urbanization continues to create favourable grounds for breeding of nuisance mosquitoes such as Culex, with strong levels of pyrethroid resistance [24,25].Developing alternative chemicals and/or vector control strategies using long-lasting insecticide nets (LLIN) to maintain an effective control of resistant mosquito populations has, therefore, become a priority.New methods and strategies are urgently needed [20], and regular resistance surveillance or monitoring research activities timely are welcomed.
Due to recent developments in bioactive �ber technology, other long-lasting insecticide-treated materials, such as curtains, tents, clothing, blankets, and plastic sheeting have been tested as potential alternatives to mosquito nets [21,26].However, they proved to be effective only in addition to mosquito nets or in limited epidemiological or cultural settings [1,19].
Similar to a multicentre WHO commissioned phase II trial [27], this research evaluated the new technology net (PermaNet 3.0) in verandah-trap huts in Togo against free-�ying wild Culex quinquefasciatus.Efficacy was expressed in terms of blood feeding inhibition, deterrence, induced exophily, and mortality.In parallel, susceptibility of Culex quinquefasciatus to four groups of insecticides (DDT, permethrin, carbosulfan, deltamethrin, bendiocarb, and chlorpyrifos methyl) was evaluated with and without a synergist (PBO).

Methods
2.1.Study Area.Togo lies 6.17 ∘ north, 1.35 ∘ .It is bordered by Burkina Faso to the north, Benin to the east, and Ghana to the west.e study area, Akodésséwa district, is situated in the urban district in the eastern part of Lomé with vibrant economic activities.e area is characterized by scattered water bodies, inland water impoundments, and water run-offs from the Bè lagoon.Vegetable crop cultivation is predominant along the boundaries of the Lagoon at the periphery closer to the Atlantic sea.ere are also scattered settlements along the boundaries of the lagoon and various activities such as cars, trucks, and stone washing bays designated at intervals primarily due to the available water source from the lagoon.

Speci�cations of Nets �sed for
Evaluation.PermaNet 3.0 is manufactured by Vestergaard Frandsen SA.All the nets in the trial were of the same size: 120 cm × 190 cm × 150 cm.PermaNet 2.0 had speci�cation as follows: deltamethrin (25 mg/m 2 ) coated on polyethylene mono�lament net fabric.PermaNet 3.0 has the following speci�cation: 85 mg/m 2 deltamethrin in net and 115 mg/m 2 in 70 cm boarder (i.e., 2.8 g/kg deltamethrin load in 75 denier net fabric).e roof of the net (upper part) had deltamethrin and piperonyl butoxide (PBO) incorporated into the mono�lament (220 denier with 4 g/kg deltamethrin; 15 g/kg PBO).

Initial Treatment of Nets prior to Trial.
For the 20 minimum WHO resistance to washing, nets were washed with "savon de Marseille" 2 g/litre (soap : H 2 O) with approximately 2 degree hardness tap water.Washing was done in a total of 10 litres of water (H 2 O) for 3 minutes le for 4 minutes and washed again for 3 minutes using a wooden ladle (each wash agitation in different direction of rotation).ey were rinsed twice in 10 litres of clean water and air dried in shade.Subsequent washes were repeated on each day for 20 days (20 times), 18th January-6th February, 2008.Before the start of the trial, nets were deliberately holed; six holes per net 2 cm × 2 cm.Two holes located on the longer sides and one hole each on the shorter sides.

Efficacy and Effect of Nets on Mosquito Behaviour.
Washed and unwashed LLIN were evaluated in experimental huts for their effects on free-�ying, wild Cx.quinquefasciatus mosquitoes and for their ability to deter entry, repel, or drive mosquitoes out of houses, induce mortality, and inhibit blood-feeding.An untreated net was used as a negative control.A PermaNet 2.0 (55 mg/m 2 deltamethrin) was used as a reference net.A net conventionally treated with deltamethrin and washed just before exhaustion was also used as another reference net.e point of exhaustion was determined by washing the conventionally treated net following Phase II protocol.Aer each washing, bioassays using WHO cones were performed.e last wash for which the net still causes >80% mortality or >95% KD was considered to be the number of washes required before exhaustion.Finally, a PermaNet 2.0 washed 20 times was tested for comparison with Per-maNet 3.0.Treatment arms were as follows: (1) Untreated net (same fabric-polyester on the side/ polyethylene on top-and same design as of Per-maNet 3.0, that is, with a border in the �rst 70 cm);

Chemical
Assay.An additional net per treatment arm was not actually placed in huts but kept for chemical assays.Pieces of nettings (4×50 cm 2 ) were taken from each of the �ve locations of these six nets according to WHO speci�cations before any washing to be subjected to chemical analysis.Aer washes were completed, pieces again were taken from each of the �ve locations of these six nets (including the unwashed nets) and shipped for chemical analysis.At the end of the experimental hut trial, new pieces were taken from each of the �ve locations of the six nets tested in the �eld (one per treatment arm).All samples for chemical analysis were sent to �estergaard Frandsen SA aer the �eld trial. .e �ve treatments (including the untreated control net) were rotated through each of the six huts using a Latin square rotation design.A treatment was allocated to a hut for six consecutive nights before being rotated to another hut.A different net was used on each of the six nights of rotation.Sleepers were rotated between huts on successive nights in order to reduce the effect of variation in individual attractiveness to mosquitoes.e primary outcomes measured in experimental huts were (i) deterrence (reduction in hut entry relative to the control huts);

Larval and Pupal Mosquito
(ii) induced exophily (proportion of mosquitoes trying to exit early found in verandah-trap); (iii) blood-feeding inhibition (reduction in blood feeding compared to control huts); (iv) immediate and delayed mortality (proportion of dead mosquitoes).

Adult Mosquito Collection in Experimental
Huts. Adult volunteers (sleepers) were consented and slept in the huts during the six-week-trial period (one complete Latin square rotation).Sleepers enter huts at dusk and remained till dawn.
Mosquitoes were collected as per location and scored as dead or alive in the laboratory aer initial species identi�cation.
Live mosquitoes were subsequently put into paper cups, provided with honey solution, and delayed mortality observed 24 hrs aer collection.Preliminary collection of mosquitoes was done in huts prior to start of trial to eliminate bias in attractiveness of sleepers to mosquitoes.In doing so, sleepers were rotated in 6 × 6 hut-night collection scheme to adjust for any bias in results interpretation.

Statistical
Analysis.e number of mosquitoes of each species entering the huts, the proportion of mosquitoes that exited early, the proportion that was killed within the hut, and the proportion that successfully blood fed was compared by species and analysed using Mann Whitney rank test for numeric data and logistic regression for proportional data (e.g., XL-Stat soware).Chi-square statistics were done using Fisher exact test.e clustering of observations made in one night, and controlling for any variation between huts and sleepers was controlled for.Comparisons between treatments were made by successively dropping treatments from the overall comparison, and this process allowed each treatment to be compared with every other one.

Ethical Considerations and Perceived Side Effects.
Adult male participants were eligible for the trial.ey were consented and objectives and procedures of the trial explained to them both in English and French.ey were given an information consent sheet in English and in French to be taken away for further or future explanations of trial objectives and procedures to them by an independent person.ey were then �nally consented and made to sign a consent form before commencement of the trial.ey were provided malaria prophylaxis during the entire period of the trial and two weeks aer the completion of the trial.e trial received ethical clearance (endorsement) from the Ministry of Health of Togo and the University of Togo (Faculty of Science).Sleepers in huts were questioned at the end of the experiment about perceived adverse or bene�cial side effects of the nets they had slept under.

Results
Susceptibility of Culex quinquefasciatus to tested insecticides is low (Figure 1).is ranged from 6% for deltamethrin to 50% for the organophosphate chlorpyrifos methyl.e picture did not differ signi�cantly when PBO was applied for 1 hr contact before each insecticide.Mortalities increased for all insecticides except for the organophosphate CM (a decrease from 50% mortality to 37% in tested insect populations).
Based on trial endpoints (Table 5), PermaNet 3.0 in the unwashed state deterred 16.84% of Culex mosquitoes.Aer 20 washes, the net deterred 5.79% of mosquitoes from entering huts which is slightly lower than deterrence (6.84%) offered by the unwashed PermaNet 2.0.PermaNet 3.0 was able to inhibit blood feeding by 70.97% and induce mosquitoes to exit huts by 50.48% at zero wash.Aer 20 washes, the net performed equally well by inducing 42.91% mosquitoes to exit huts and inhibited 67.06% mosquitoes from blood feeding.e polyester net conventionally treated and washed just before exhaustion did not have any sig-ni�cant effect on blood feeding inhibition nor mortality.Exophily was signi�cantly induced by all treatments, except the net washed to exhaustion.Induced exophily was particularly signi�cant with PermaNet 3.0 (64% and 69% for the washed and unwashed nets, resp.).Similarly, an important decrease of blood feeding was observed for PermaNet 3.0 (67% and 71% for the washed and unwashed, resp.).To a lesser extent, PermaNet 2.0 inhibited 38% and 46% (washed and unwashed, resp.) of mosquitoes from successful blood feeding.Overall efficacy was good for Perma Net 3.0 aer 20 washes (69%) and when not washed (76%).1) con�rms the multiresistance status of Cx. quinquefasciatus worldwide and particularly in Africa [28] and therefore calls for better and comprehensive investigation of its resistance mechanisms locally.Some of the females have been kept by CREC to perform PCR diagnostic tests and biochemical assays for a better knowledge of resistant mechanisms.2), the fact that all treated nets are fully effective against the susceptible reference strain of An. gambiae is an indication that deltamethrin is fully bioavailable regardless of the treatment, even when  a Values (or data) in the same column sharing a letter superscript do not differ signi�cantly (  ).

Bioefficacy. Before any washing (Table
using the long lasting technology.e 3 washes required just before exhaustion (Table 1) are a little bit higher than the 2 washes usually observed with other Phase II evaluations (Dawa net, K-O Tables 1, 2, and 3 and NetProtect).is slight difference can be considered as an acceptable "biological variability" between bioassays from one test to another.Resistance to washing before or aer �eld testing (Tables 3 and 4) is remarkably important on both PermaNet 3.0 (including the one with roof treated with PBO) and PermaNet 2.0 (despite the fact that a slight but signi�cant decrease in mortality had been observed on PermaNet 2.0 washed 20 times even before �eld testing).

Field Efficacy.
Results obtained with Cx. quinquefasciatus in the control arm (untreated) do not differ radically from those obtained with An. gambiae in other experimental hut studies.Natural exophily is comparable (30-40%) as well as natural mortality (5-10%).Blood feeding is, however, less important with this species (48%) compared to the rates obtained with An. gambiae (i.e., 76% in September in Kou Valley, Burkina Faso).Additional studies against Culex species are needed to know whether this difference in blood feeding is interspeci�c (difference in vector behaviour) and not due to intertrial variability.In a nutshell, Cx. quinquefasciatus is less affected by PermaNet 3.0 than An.gambiae: deterrence is practically T 3: Resistance to washing before �eld testing.Aer all washes were completed (February 10, 2008), nets were tested before being installed in experimental huts (Table 3).All treated nets were fully effective in terms of knockdown effect and mortality except PermaNet 2.0 washed 20 times (  ).Knockdown and mortality were recorded at 10 minutes interval using WHO cones, aer washing and before �eld testing, aer 3 min exposure of susceptible reference strain of An. gambiae (Kisumu strain).zero, mortality is very low (only 7% overall insecticidal effect with PermaNet 3.0 unwashed), and induced exophily is moderate.However, blood feeding inhibition remains as elevated as for pyrethroid resistant An. gambiae in Burkina Faso, leading to maintain a good personal protection despite the high level of pyrethroid resistance of local populations of Cx. quinquefasciatus.Moreover, this personal protection is much higher with PermaNet 3.0, washed and unwashed (69 and 76%, resp.)than with PermaNet 2.0 (49 and 22%, resp.).e fact that mortality is low and blood feeding inhibition is high is probably the result of the behavioural response of Cx. quinquefasciatus to pyrethroids: contact time with the treated fabrics is not long enough to kill mosquitoes but enough to prevent blood feeding.e efficacy of the new mosaic net is better explained by the overall personal protection it offers to an individual.Comparatively, it performed better in protection of sleepers from mosquito bites which was nearly two times that of the conventionally used net.is protective effect is higher than the conventional net, aer the recommended 20 minimum washes.Aer trials, contact bioassays revealed that all nets were effective in killing susceptible Anopheles gambiae (kisumu strain) except the manually treated net �ust washed to exhaustion.is con�rms the rapid loss of efficacy of nets manually treated.Also, these results support multicountry trials [11] that demonstrated the ability of retention of insecticide properties by long lasting insecticide nets even aer 20 minimum washes.

Conclusions
Resistance in Culex quinquefasciatus is high in Lomé, particularly in Akodésséwa.Further toxicology studies of water samples from breeding sites of this species are recommended to give concrete and better understanding of the high resistance observed in this study.Innovative vector control measures are seriously encouraged and constant research on vector species dynamics is welcomed, as it demonstrates at any given time vector resistance and susceptibility status.
e fact that all treated nets (before any washing) were fully effective against the susceptible reference strain of An. gambiae is an indication that deltamethrin is fully bioavailable regardless of the treatment, even when using the long lasting technology.e new technology incorporated in the mosaic net evaluated is good.However, Cx. quinquefasciatus is less affected by PermaNet 3.0.Deterrence is practically negligible and mortality very low.However, blood feeding inhibition remains high which resultantly gives a good personal protection despite the high level of pyrethroid resistance of local populations of Cx. quinquefasciatus.Moreover, this personal protection is much higher with PermaNet 3.0, washed and unwashed (69% and 76%, resp.)than with PermaNet 2.0 (22% and 49%) for washed and unwashed, respectively.
Results for PermaNet 3.0 are rather encouraging.Per-maNet 3.0 succeeded in reducing blood feeding and thus nuisance.It is well known that in order for LLINS to be used adequately against malaria vectors, they also need to be effective against nuisance pest mosquitoes, particularly Cx. quinquefasciatus in tropical urban environment, as they will be widely accepted by the local populations.

Con�ic� o� �n����s�s
e manufacturer of PermaNet 3.0 (Vestergaard Frandsen), played no role in the design or implementation of the research, decision to publish or draing the paper.All authors declare that they have no con�ict of interests.

T 2 :
Initial bioefficacy of the treated nets.
or data) in the same column sharing a letter superscript do not differ signi�cantly (  ).

T 4 :
Resistance to washing aer �eld testing.Aer the end of the �eld trial (March 29, 2008), nets collected from huts were tested.Results yielded knockdown and mortality rates almost similar to those recorded immediately aer washing and before testing.Knockdown and mortality were recorded at 10 minutes interval using WHO cones, aer washing and aer �eld testing, aer 3 min exposure of susceptible reference strain of An. gambiae (Kisumu strainor data) in the same column sharing a letter superscript do not differ signi�cantly (  ).
77. Efficacy Test on Mosquito Nets with Adult Mosquitoes.ree-day-old postemerged female Culex quinquefasciatus from Akodésséwa were subjected to three minutes contact exposure to all sides of each net: PermaNet 3.0 (unwashed and washed 20 times) and PermaNet 2.0 (unwashed and washed 20 times), using WHO cones.Aer exposure, mosquitoes were aspirated back into paper cups and observed for knockdown (KD) at time intervals for one hour and mortality observed aer 24 hours aer exposure.Tests were in replicates of �ve cones per net for the one-technology net (PermaNet 2.0) and �ve cones per side and per roof for the two-technology net (PermaNet 3.0).Mosquitoes were given honey solution and kept at 25 ∘ C, 70 ± 2% relative humidity condition.Culex SLAB strains of mosquitoes were used as susceptible reference strains.Due to high resistance observed in the Culex mosquitoes aer three minutes, a 30 minutes exposure contact time on nets was again tested.
Collections.Immature stages of mosquitoes (larvae and pupae) were prospected in Akodésséwa (N06 ∘ 09 ′ 23.6 ′′ ; E001 ∘ 16 ′ 02.4 ′′ ) for Anopheles species and (N06 ∘ 09 ′ 42.3 ′′ ; E001 ∘ 15 ′ 33.0 ′′ ) for Culex species.Samples were transported to the laboratory (Laboratoire d'Entomologie Appliquée-LEA, University of Lomé) and reared under standard conditions in the insectarium (12 Hr: 12 Hr LD; 72 ± 2% RH).Emerged adults were 4%), DDT (4%), bendiocarb (0.1%), and chlorpyrifos methyl (0.4%).Knockdown (KD) was recorded at time intervals for 1 hr and mortality checked 24 hr aer exposure.Test with untreated paper was performed alongside each test to serve as controls and mortality was corrected (≥5% mortality) with Abbott's formula when necessary.22.8.Evaluation of Mosquito Nets in Experimental Huts.Six (6) experimental huts were constructed with concrete blocks, corrugated iron sheets for the roof, and lined with thick polyethylene sheeting.Huts were situated along a large water swarm area in the Akodésséwa district in Lomé, purposefully envisaged to breed mosquitoes.Each veranda-hut had the following measurements: 1.3 mL, 1.6 mW, 1.8-2 mH (veranda), 1.7 mL, 3.2 mW, 2.1-2.3 mH (hut), typical of the West Africa design hut type for phase II trials.ehuts were spaced at intervals transversely.An exit window trap (30 cm × 60 cm) is located on each side-wall part of the hut and two exit windows on the front-side wall (one on either side of door).A 1 cm (�nger si�e) gap is le on each exit window for mosquito entry into huts.eevaluations run between 11 February and 29 March 2008 T 1: Test for establishing point of exhaustion threshold.
a Values (or data) in the same row sharing a letter superscript do not differ signi�cantly (  ).