Efficacy of two PBO long lasting insecticidal nets against natural populations of Anopheles gambiae s.l. in experimental huts, Kolokopé, Togo
Efficacy of two PBO long lasting insecticidal nets against natural populations of Anopheles gambiae s.l. in experimental huts, KolokopeÂ, Togo
Guillaume K. Ketoh 2 3
Koffi M. Ahadji-Dabla 2 3
Joseph Chabi 1 3
Adjovi D. Amoudji 2 3
Georges Y. Apetogbo 2 3
Fantchè Awokou 0 3
Isabelle A. Glitho 2 3
0 Programme National de Lutte contre le Paludisme (PNLP) , Ministère de la Sant eÂ, LomeÂ , Togo
1 Noguchi Memorial Institute for Medical Research (NMIMR), University of Ghana , Legon, Accra , Ghana
2 Insect Pest and Insect Vector Management/Ecotoxicology, UniteÂ de Recherche en Ecotoxicologie (URET), Laboratoire d'Entomologie Appliqu eÂe (LEA), FaculteÂ des Sciences, Universit eÂ de Lom eÂ , LomeÂ , Togo
3 Editor: Basil Brooke, National Institute for Communicable Diseases , SOUTH AFRICA
LLINs containing an insecticide plus the synergist, piperonyl butoxide (PBO) have been designed for increased efficacy against pyrethroid-resistant malaria vectors. In this study, two LLINs with PBO, PermaNet® 3.0 and Olyset® Plus, and a pyrethroid-only LLIN, Yorkool®, were evaluated in experimental huts against a free-flying, wild population of Anopheles gambiae s.l. in KolokopeÂ , a cotton cultivated area of Togo. WHO susceptibility tube tests and subsequent molecular assays determine the An. gambiae s.l. populations to be resistant to pyrethroids and DDT with both target site kdr and metabolic resistance mechanisms involved in the resistance observed. Anopheles gambiae s.s. and An. coluzzi were present in sympatry though the kdr (L1014F) mutation was observed at a higher frequency in An. gambiae s.s. The experimental hut results showed that both PermaNet® 3.0 and Olyset® Plus nets induced similar levels of deterrence, exophily, and reduced blood feeding rate against wild An. gambiae s.l. in contrast to the pyrethroid only LLIN, Yorkool®. The proportion of wild An. gambiae s.l. killed by unwashed PermaNet® 3.0 was significantly higher than unwashed Olyset® Plus (corrected mortality 80.5% compared to 66.6%). Similar blood feeding inhibition rates were observed for unwashed PermaNet® 3.0 and Olyset® Plus; however, PermaNet® 3.0 washed 20 times demonstrated significantly higher blood feeding inhibition rate than Olyset® Plus washed 20 times (91.1% compared with 85.6% respectively). Yorkool® performed the worst for all the parameters evaluated. In an area of pyrethroid resistance of An. gambiae s.l involving kdr target site and metabolic resistance mechanisms, LLINs with PBO can provide additional protection in terms of reduction in blood feeding and increase in mosquito mortality compared to a pyrethroid-only net, and should be considered in malaria vector control strategies.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: The authors received no specific funding
for this work.
Competing interests: The authors have declared
that no competing interests exist.
Long lasting insecticidal nets (LLINs) continue to be one of the primary interventions against
malaria vectors. Currently, a LLIN is expected to retain its biological activity for at least 20
standard washes under laboratory conditions and three years of recommended use under field
conditions, as defined in WHO guidelines . WHO Pesticide Evaluation Scheme (WHOPES)
Phase II experimental hut studies are conducted on nets that pass Phase I laboratory
investigations to determine comparative efficacy against free-flying, wild mosquito populations.
In Togo, insecticide susceptibility status of Anopheles populations was effectively reported
for the first time in 2005 [
]. Pyrethroid resistance with the presence of kdr (L1014F) mutation
was recently detected in malaria vectors in the south of the country [
]. It was also known that
carboxylesterases (COEs), glutathione-S-transferases (GSTs) and cytochrome P450-dependent
monoxygenases (P450s) are the three main groups of enzymes involved in the metabolic
resistance to pyrethroids used for malaria vector control [
LLINs containing an insecticide plus the synergist piperonyl butoxide (PBO) have been
designed for increased efficacy against pyrethroid-resistant malaria vectors. PBO is an
inhibitor of mixed function oxidases (MFO) implicated in pyrethroid resistance, and also increases
the rate of insecticide uptake through the mosquito cuticle [
]. In 2014, at the time of the
evaluation, two LLINs with PBO were available: PermaNet1 3.0 and Olyset1 Plus 
PermaNet1 3.0 is a LLIN with PBO and deltamethrin incorporated on polyester side panels and a
mixture of deltamethrin and PBO incorporated in the polyethylene top panel. Olyset1 Plus
LLIN is made of polyethylene netting incorporating permethrin and PBO. A third LLIN with
PBO, Veeralin1 LN received WHOPES interim recommendation in 2016 . In contrast to
LLINs with PBO, the pyrethroid-only LLIN included in this study, Yorkool1 is a
multifilament polyester net coated with deltamethrin.
In this experimental hut trials, the primary objective was to determine the comparative
efficacy between LLINs with PBO and a pyrethroid-only net in an area of pyrethroid resistance
with the involvement of metabolic resistance mechanisms. As per standard outcomes
measures for experimental hut trials, efficacy was measured in terms of blood-feeding inhibition,
deterrence, induced exophily and mortality. Characterisation of the wild mosquito population
including species composition, susceptibility to the pyrethroid active ingredient in the LLINs
(deltamethrin and permethrin), frequency of the target site mutation (kdr L1014F) and
upregulated metabolic enzymes were also determined. The trial was conducted from June to
December 2013 in a West African experimental hut design at KolokopeÂ, Togo.
This evaluation was conducted in experimental huts located at KolokopeÂ, Togo (07Ê47'59'N,
01Ê18'00E) from June to December 2013. The village is situated in the plateau region of the
country and at 200 km from LomeÂ. The area is a cotton cultivation site covering approximately
236 hectares and produces an estimated 1000 tons of cotton per year. To protect farms,
pyrethroid based insecticides are commonly used to spray fields [
]. The region is characterised by
a long rainy season from March to October and a dry season from November to February. The
annual rainfall is estimated to 1300-1500mm per year.
WHO susceptibility Test
To characterise the wild An. gambiae s.l. mosquito population in KolokopeÂ, WHO
susceptibility tests were conducted according to WHO standard protocols . Mosquitoes were assayed
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using WHO discriminating dosages of nine insecticides belonging to four chemical classes: (1)
pyrethroids (0.05% deltamethrin, 0.75% permethrin and 0.05% lambdacyhalothrin), (2)
organochlorine (4% DDT), (3) organophosphates (1% fenitrothion; 5% malathion and 0.4%
chlorpyrifos methyl), and (4) carbamates (0.1% propoxur and 0.1% bendiocarb). In addition,
synergist assays with 5% PBO impregnated papers were conducted to determine the presence
of metabolic mechanisms such as P450 enzymes. Anopheles gambiae s.l. mosquito larvae were
collected in the surroundings of the village and reared to adults at the field site laboratory.
Twenty to twenty-five non-blood fed female An. gambiae s.l., aged 3±5 days were exposed for
one hour to the different insecticides and two hours specifically for fenitrothion. For the
synergist assay, mosquitoes were pre-exposed to PBO for one hour before exposure to the
insecticide for an additional hour. The number of mosquitoes knocked down was recorded at 60
minutes and mortality recorded after 24 hours . Tests with silicone and olive oil
impregnated papers were run in parallel and served as controls. Following the susceptibility tests, all
mosquitoes (including controls) were kept at −20ÊC for further identification of An. gambiae
species complex and characterization of the kdr mutation.
Species identification and kdr L1014F detection
Anopheles specimens were randomly selected from the susceptibility testing and analyzed
using SINE-PCR for species identification [
]. The detection of kdr L1014F was conducted
following the methods of Martinez-Torres et al.[
] with additional confirmation using real
time-PCR following the protocol of Bass et al. [
Experimental hut design
The experimental huts are made of concrete bricks with a corrugated iron roof, a ceiling of
thick polyethylene sheeting, and a concrete base surrounded by a water-filled channel to
prevent entry of ants [
]. Mosquito access is via four window slits constructed from pieces of
metal, fixed at an angle to create a funnel with a 1 cm wide gap. Mosquitoes fly upward to
enter through the gap and downwards to exit; this precludes or greatly limits exit though the
aperture enables the majority of entering mosquitoes to be accounted for. A single verandah
trap made of polyethylene sheeting and screening mesh measuring 2 m long, 1.5 m wide and
1.5 m high, projects from the back wall of each hut. Movement of mosquitoes between hut and
verandah is unimpeded during the night.
Treatment arms. Washed and unwashed LLINs were evaluated using experimental huts
for their effects on free-flying, wild mosquitoes and for their ability to deter entry, repel or
drive mosquitoes out of houses (i.e. induced exophily), induce mortality, and inhibit
bloodfeeding. Yorkool1 LLIN was used as a positive control and untreated polyester net was used as
a negative control.
The following treatment arms were tested using seven nets per arm for the study:
1. Untreated net
2. PermaNet1 3.0 unwashed
3. PermaNet1 3.0 washed 20 times
4. Olyset1 Plus unwashed
5. Olyset1 Plus washed 20 times
6. Yorkool1 unwashed
7. Yorkool1 washed 20 times
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Washing of the nets. The nets were washed according to standard WHO Phase II
washing procedure. Nets were washed in aluminium bowls containing 10 litres of clean water and
containing 2g/litre of soap ("Savon de Marseille") using manual agitation. Nets were rinsed
twice and dried horizontally in the shade then stored at ambient temperature between daily
washes. One day regeneration time was considered between each washing of all the nets
following previous study results of the same treatment arms [
Before testing in the experimental huts, the nets (including control) were deliberately
holed. Six holes were made in each net: two holes in each of the long sides and one hole at each
short side. Each hole measured 4cm x 4cm.
Each week, the treatment arms were rotated among the huts according to a Latin square
scheme. Seven nets were used per treatment arm and each of the seven nets was tested one
night during the week. At the end of the week, the huts were carefully cleaned and aired to
remove potential contamination. The treatment was then rotated to a different hut.
Study design. Adult volunteers slept under each individual net per night. They were
recruited among the inhabitants of the villages close to the site. Nets were evaluated from 23
June to 22 August 2013 for the first Latin square and from 20 October to 19 December 2013
for a second Latin square, corresponding to 98-night collections per hut to obtain sufficient
number of mosquitoes for adequate statistical analysis.
Sleepers were rotated randomly among huts each night of the study. They entered a hut at
dusk and remain inside until dawn. In the morning, dead and alive mosquitoes were collected
from the floor of the hut as well as from the veranda traps and inside the nets; resting
mosquitoes were collected using aspirators from inside the net, from the walls and roof of the hut, and
veranda traps. Mosquitoes were scored by location as dead or alive and as fed or unfed. Alive
mosquitoes were placed in disposable cups and provided with access to 10% sugar solution for
24 hours to assess delayed mortality.
The primary outcomes measured in experimental huts were:
· deterrence (reduction in hut entry relative to the control hut fitted with untreated nets);
· induced exophily (the proportion of mosquitoes that exited early and were found in exit
· blood-feeding inhibition (the reduction in blood feeding compared with that in the control
· Immediate and delayed mortality (the proportion of mosquitoes that were killed).
Outcome measures were calculated as per standard procedures. The primary analysis was a
test of the non-inferiority of the candidate LLINs with PBO (PermaNet1 3.0 and Olyset1
Plus) washed 20 times relative to the standard LLIN (Yorkool1) washed 20 times. According
to WHOPES, a candidate LLIN is considered to meet the Phase II efficacy criteria if, after 20
washes, it performs as well as or better than the reference LN when washed 20 times in terms
of blood feeding inhibition and mortality.
The percentage personal protection was calculated as follows :
% personal protection 100
BFC = total number of blood fed females in the control hut
BFT = total number of blood-fed female mosquitoes in the treated hut
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The insecticidal effect or overall killing effect of a treatment was calculated using the
following formula :
Overall insecticidal effect
DT = total number of dead mosquitoes in the treated hut
DC = total number of dead mosquitoes in the control hut
TC = total number of mosquitoes collected in the control hut
WHO cone bioassays. Cone bioassays were conducted according to the WHO procedures
 on one net of each treatment arm before the first wash on the 27 May 2013, for a 2nd time
when all washings were completed on 17 June 2013, and for a 3rd time at the end of the field
experiment on the nets used in huts. For each net, 5 cones each were placed on the 5 sections
of the net (roof and 4 sides). Ten females of An. gambiae s.s. Kisumu, the susceptible reference
strain were introduced per cone and exposed for 3 min to the net giving an average of 50
mosquitoes. Knockdown was recorded 60 minutes after exposure and mortality was checked 24
hours after exposure. Bioassays were also conducted against wild An. gambiae s.l. from
KolokopeÂ. Mosquitoes were collected at larval stage from the site, brought to the insectary, and
reared until adults. WHO cone bioassays were conducted on non-blood fed adults 3 to 4 days
Chemical content analysis of nets. Chemical analysis was conducted on LLIN samples
pre-washing, post-washing, and post hut trial. Each net sample (10cm x 10cm) was
homogenized and an analytical portion of 300mg was taken for determination of permethrin,
deltamethrin, and/or PBO. Following CIPAC (Collaborative International Pesticide Analytical
Council) methods deltamethrin, deltamethrin R-isomer, and PBO were extracted by heating
under reflux for 60 min with xylene and were determined by gas chromatography with flame
ionization detection (GC-FID) using the internal standard calibration. Permethrin and PBO
was extracted in a water bath with heptane for 45 minutes and similarly determines by
The analysis of each mosquito species that entered the huts was compared among the different
treatment arms by a non-parametric Kruskal-Wallis test. The proportion of mosquitoes that
exited early, the proportion that were killed within the hut and the proportion that successfully
blood fed was compared by species and then analyzed using a logistic regression or generalized
linear mixed models, which provide a framework for regression modeling of non-normal
outcome data using XLSTAT software (version 2011).
In addition to approval from the traditional head of district, ethics approval was obtained from
national ethics committee and the Ministry of Health of Togo. Sleeper volunteers were
informed of the objective of this study and informed consent was obtained from each
volunteer. A medical doctor was on hand during the trial to respond to any side effects of the treated
nets or to treat any cases of fever. Any confirmed case of P. falciparum parasitaemia was treated
with Coartem (artemether 20mg/lumefantrine 120 mg). Perceived adverse or beneficial side
effects of the washed and unwashed nets were also noted by the seven volunteers during the
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WHO susceptibility tests
The results of the susceptibility testing in Table 1 showed that An. gambiae s.l. population of
KolokopeÂ is resistant to both pyrethroids and DDT, but susceptible to organophosphates and
carbamates. Among the pyrethroids, particularly low mortality (1.2%) was recorded for
lambdacyhalothrin. Resistance to deltamethrin and permethrin were 14.8% and 7.5% mortalitiy
Alongside WHO susceptibility tests, synergist assays conducted with pre-exposure to PBO
enhanced the mortality of permethrin from 7.5% to 92.8% and deltamethrin from 14.8% to
Species identification and determination of resistant mechanisms
The results of the species identification and the kdr genotype are shown in the Table 2. Out of
the 270 An. gambiae s.l. analyzed, 133 (49.3%) were An. coluzzii and 137 (50.7%) were An.
gambiae s.s. The frequency of the kdr mutation (L1014F) was 0.62 within the population of An.
coluzzii and 0.96 for An. gambiae s.s.
WHO cone bioassays
Full bioefficacy (meeting WHO cut-offs of >80% mortality or >95% knockdown) was
observed in all unwashed and washed nets against susceptible An. gambiae Kisumu (Table 3).
Bioassays against the wild resistant populations of An. gambiae s.l. from KolokopeÂ
demonstrated that PermaNet1 3.0 unwashed (roof portions containing deltamethrin and PBO)
retained full bioefficacy before and after the hut trial (Table 4). PermaNet1 3.0 washed 20
times and Olyset1 Plus (unwashed and washed) reported low bioefficacy against wild resistant
populations. Yorkool1 a deltamethrin-only net delivered nearly no mortality against wild
resistant An. gambiae s.l populations irrespective to the wash status.
Experimental hut trial
In total, 4,716 mosquitoes were collected in the experimental huts during the evaluation: 2,591
(54.9%) were An. gambiae s.l., 1,037 (22.0%) were Culex species, and 1,088 (23.1%) were other
species predominantly Mansonia. The trial results are outlined in Table 5 for An. gambiae s.l.
An. gambiae s.l.
Control hut. During the 98-night collections, 835 culicidae were collected in the control
hut. Among them 465 An. gambiae s.l. were recorded. A mean number of 5 Anopheles females
were caught per night and 84.1% of them were blood fed. This corresponded to an average of
3.9 bites per man per night in the control. Natural exophily (13.1%) and natural mortality
remained low throughout the experiment (1.9%).
Treated huts. A significant reduction in entry rates (deterrence) was noted with both
unwashed PermaNet1 3.0 and Olyset1 Plus yielding a deterrence of 33.3% and 34.6%,
respectively compared with the negative control (p<0.0001). The same trend was observed for
washed PermaNet1 3.0 and Olyset1 Plus with a deterrence of 25.2% and 23.4%, respectively
(p<0.0001). Both unwashed and washed LLINs with PBO deterred more An. gambiae s.l.
compared with unwashed and washed pyrethroid-only Yorkool1 LLIN. Notably, the deterrence of
unwashed and washed Yorkool1 did not differ significantly compared to the untreated net
(p = 0.181 and p = 0.143, respectively).
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High exophily rates were induced by PermaNet1 3.0 and Olyset1 Plus. In contrast,
exophily rates were low for the Yorkool1 washed and unwashed arms (28.5% and 22.9%
A decrease of the number of blood fed mosquitoes was observed with all six treatments and
especially with unwashed PermaNet1 3.0 and Olyset1 Plus, recording 93.9% and 96.1% blood
feeding inhibition, respectively. However, the bloodfeeding inhibition of both unwashed
LLINs with PBO were not significantly different (p = 0.263), the blood feeding inhibition of
PermaNet1 3.0 washed 20 times was significantly higher (91.1%) than Olyset1 Plus washed
20 times (85.6%) (p = 0.043).
A significantly higher corrected mortality of 80.5% was observed with PermaNet1 3.0
unwashed arm compared to other treatments (p<0.05). The corrected mortality of Olyset1
Plus unwashed was 66.6%. As with other parameters washed Yorkool1 LLIN performed the
worst with a corrected mortality of 40.7%.
Personal protection and insecticidal effect. The personal protection rates measured for
PermaNet1 3.0 unwashed and washed were 95.9% and 93.4% respectively; Olyset1 Plus
unwashed and washed, 97.4% and 89.0% respectively and Yorkool1 unwashed and washed,
80.3 and 65/0% respectively.
The insecticidal effect showed a similar trend: PermaNet1 3.0 unwashed and washed,
55.3% and 46.7% respectively; Olyset1 Plus unwashed and washed, 44.3% and 42.4%
respectively and Yorkool1 unwashed and washed, 46.2% and 37.2% respectively.
Other species. Similar outcomes like those of An. gambiae s.l. were noted for all the other
species including Culex and Mansonia species for induced exophily, blood feeding inhibition
and mortality parameters. Data on Culex species are available from S1 Table.
kdr mutation (L1014F)
Values in the same column sharing the same letter superscript do not differ significantly (P> 0.05)
n/a = not applicable
The mean concentration of deltamethrin, permethin, and PBO against the target
concentration indicated by the manufacturer is outlined in Table 6. At the start of the trial, prior to any
washing, all LLINs reported active ingredient concentrations within the target range provided
by the manufacturer. Substantial active ingredient loss was noted on the side portions
(deltamethrin only) of PermaNet1 3.0. A similar loss of deltamethrin was also observed with
Yorkool1 net. Also, the loss of PBO active ingredient after 20 washes was 44.0% and 56.6% lower
than the initial concertation for Olyset1 Plus and PermaNet1 3.0, respectively.
An. gambiae s.lis the main malaria vector in KolokopeÂ. PCR testing for species identification
found both An. coluzzii and An. gambiae s.s living in sympatry at similar proportions. WHO
susceptibility testing conducted from larval collections determined resistance to pyrethroid
insecticides and DDT. Higher kdr frequency was found in An. gambiae s.s. (96%) compared to
An. coluzzi (62%). This data is in line with previous works done in Burkina Faso
demonstrating a similar predominance of the kdr allele frequency of An. gambiae s.s. [17±19].
Synergist assays conducted using pre-exposure to PBO restored the susceptibility against
permethrin and deltamethrin, thus indicating resistance mediated by elevated P450
monoValues in the same column sharing the same letter superscript do not differ significantly (P> 0.05)
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Letters in the same row sharing a letter superscript do not differ significantly (P> 0.05)
Olyset1 Plus 0
oxygenase mechanisms. Similar trends were observed in CoÃte d'Ivoire and Benin where
synergist assays using PBO also indicated the involvement of P450s [
]. Similarly, previous
studies have reported kdr L1014F mutation  and other resistance mechanisms [
] in Togo.
The DDT and pyrethroid resistance observed at the experimental hut site in KolokopeÂ is likely
conferred by both kdr L1014F and metabolic mechanisms. However, microplate enzyme
activity experiments should be conducted to further explore the level and role metabolic
mechanisms play in insecticide resistance in Togo. The confirmation of elevated P450 based
mechanisms as indicated by the synergist assays also demonstrates the increased efficacy
expected from LLINs with PBO (PermaNet13.0 and Olyset1 Plus) against natural resistant
An. gambiae s.l. populations.
WHO cone bioassays conducted against susceptible An. gambiae s.s. Kisumu indicated that
all LLINs met WHO cut-offs of greater than 80% mortality or 95% knockdown before and
after the hut trial. Against resistant wild An. gambiae s.l. only PermaNet1 3.0 (roof) unwashed
and washed 20 times were able to exceed 80% mortality performance cut-off. Olyset1 Plus
delivered overall low to minimal bioefficacy against wild pyrethroid resistant Anopheles
populations. Washed and unwashed Yorkool1 nets were found to have no bioefficacy against the
resistant wild An. gambiae s.l. population, even though full bioefficacy was noted against
susceptible An. gambiae s.s. This suggests that pyrethroid-only Yorkool1 nets would not be
effective in this area and would perform like an untreated net.
PermaNet1 3.0 and Olyset1 Plus exhibited high mortality and high blood feeding
inhibition in free flying population of An. gambiae s.l. A similar trend was observed in deterrence
and exophily rates of PermaNet1 3.0 and Olyset1 Plus compared to the pyrethroid-only
Yorkool1. These findings are in accordance with studies of Corbel et al. [
], Tungu et al. [
and Koudou et al. [
] that demonstrated that PermaNet1 3.0 fulfils the WHOPES efficacy
criteria of Phase II studies. An experimental hut study comparing Olyset1 Plus and Olyset1
net also reported similar evidence for the advantage of incorporating PBO with pyrethroid
insecticides in a LLIN for increased efficacy [
Yorkool1 net performed markedly worse than the other LLINs tested on all parameters
measured. In an area with pyrethroid resistant malaria vectors, PermaNet1 3.0 and Olyset1
Plus (LLINs with PBO) can provide additional protection in terms of reduction in blood
feeding and increase in mosquito mortality, compared to a pyrethroid-only net.
The different amount of active ingredient noted for all three LLINs after analysis are similar
to some previous studies [
] and as reported in WHOPES reports [6, 7]. As observed in
the WHOPES report, PermaNet1 3.0 deltamethrin was lost more substantially from the sides
than the roof of the net. It is noted that a study published after this evaluation reported a two
day regeneration time for Olyset1 Plus . While a one day washing interval was used for
this study, the chemical content analysis for both permethrin and PBO were in line with
previous reports [
]. Similarly, the WHOPES report for Yorkool1 reported 21.6% retention
following 20 washes ; this study reported a 62.6% loss of deltamethrin, which corresponds to
a retention rate of 37.4%. The percentage of loss of active ingredient is more considerable for
deltamethrin treated nets than permethrin.
Apart from demonstrating the efficacy of PermaNet1 3.0 and Olyset1 Plus, this is one of
the first experimental hut studies on two available WHOPES approved LLINs with PBO that
emphasises the potential benefit of LLINs with PBO to better control resistant malaria vectors
compared to a pyrethroid-only LLIN. In an area with pyrethroid resistant malaria vectors with
both kdr target site and P450-based metabolic mechanisms, PermaNet1 3.0 and Olyset1 Plus
(LLINs with PBO) can provide additional protection in terms of reduction in blood feeding
and increase in mosquito mortality, compared to a pyrethroid-only net.
To conclude, the present study showed efficacy of LLINs with PBO in experimental huts.
PermaNet1 3.0 and Olyset1 Plus showed significantly better performance against pyrethroid
resistant populations of An. gambiae s.l. than the pyrethroid-only Yorkool1 LN. These results
are encouraging and LLINs with PBO should be taken into consideration in malaria vector
S1 Table. Summary of results obtained for free flying all other species in experimental huts
(98 nights) in KolokopeÂ, Togo.
10 / 12
We are very grateful to the mosquito collectors and the KolokopeÂ villagers for their
commitment and participation. Vestergaard donated PermaNet1 3.0 LLINs. Olyset1 Plus and
Yorkool LLINs were purchased in Kenya and Ghana, respectively. We thank Louis Kouadio N'Dri
and Dora Okyere of Noguchi Memorial Institute for Medical Research for their assistance
with the PCR analyses.
Data curation: Koffi M. Ahadji-Dabla, Joseph Chabi, Adjovi D. Amoudji, Georges Y.
Formal analysis: Koffi M. Ahadji-Dabla, Joseph Chabi.
Supervision: Guillaume K. Ketoh.
Writing ± original draft: Koffi M. Ahadji-Dabla, Joseph Chabi.
Writing ± review & editing: Fantchè Awokou, Isabelle A. Glitho.
WHO. World Health Organization: Report of the fifteenth WHOPES working group meeting: WHO/HQ,
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