Wellcome Open Research 2018, 3:71 Last updated: 06 JUL 2018
RESEARCH ARTICLE
Experimental huts trial of the efficacy of pyrethroids/piperonyl
butoxide (PBO) net treatments for controlling multi-resistant
populations of Anopheles funestus s.s. in Kpomè, Southern
Benin [version 1; referees: awaiting peer review]
Romaric Akoton
Akadiri Yessoufou
Francis Zeukeng
Charles S. Wondji
1,2, Genevieve M. Tchigossou1,2, Innocent Djègbè2,3,
1, Michael Seun Atoyebi
2,4, Eric Tossou1,2,
5, Pelagie Boko6, Helen Irving7, Razack Adéoti2, Jacob Riveron7,
7, Kabirou Moutairou1, Rousseau Djouaka
2
1University of Abomey, Calavi, Abomey-Calavi, 526, Benin
2AgroEcoHealth Platform, International Institute of Tropical Agriculture, Cotonou, 0932, Benin
3National University of Sciences, Technologies, Engineering and Mathematics of Abomey, Abomey, 123, Benin
4Cell Biology and Genetics Unit, Department of Zoology, University of Ibadan, Ibadan, Nigeria
5Faculty of Sciences, Department of Biochemistry, University of Yaounde I, Yaounde, 812, Cameroon
6National malaria and Neglected diseases control program, Ministry of Health, Cotonou, Benin
7Liverpool School of Tropical Medicine, Liverpool, L3 5QA , UK
v1
First published: 13 Jun 2018, 3:71 (doi: 10.12688/wellcomeopenres.14589.1)
Latest published: 13 Jun 2018, 3:71 (doi: 10.12688/wellcomeopenres.14589.1)
Abstract
Background: Insecticides resistance in Anopheles mosquitoes limits
Long-Lasting Insecticidal Nets (LLIN) used for malaria control in Africa,
especially Benin. This study aimed to evaluate the bio-efficacy of current LLINs
in an area where An. funestus s.l. and An. gambiae have developed
multi-resistance to insecticides, and to assess in experimental huts the
performance of a mixed combination of pyrethroids and piperonyl butoxide
(PBO) treated nets on these resistant mosquitoes.
Methods: The study was conducted at Kpomè, Southern Benin. The
bio-efficacy of LLINs against An. funestus and An. gambiae was assessed
using the World Health Organization (WHO) cone and tunnel tests. A
released/recapture experiment following WHO procedures was conducted to
compare the efficacy of conventional LLINs treated with pyrethroids only and
LLINs with combinations of pyrethroids and PBO. Prior to huts trials, we
confirmed the level of insecticide and PBO residues in tested nets using high
performance liquid chromatography (HPLC).
Results: Conventional LLINs (Type 2 and Type 4) have the lowest effect
against local multi-resistant An. funestus s.s. and An. coluzzii populations from
Kpomè. Conversely, when LLINs containing mixtures of pyrethroids and PBO
(Type 1 and Type 3) were introduced in trial huts, we recorded a greater effect
against the two mosquito populations (P < 0.0001). Tunnel test with An.
funestus s.s. revealed mortalities of over 80% with this new generation of LLINs
(Type 1 and Type 3),while conventional LLINs produced 65.53 ± 8.33%
mortalities for Type 2 and 71.25 ±7.92% mortalities for Type 4. Similarly,
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mortalities ranging from 77 to 87% were recorded with the local populations of
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Wellcome Open Research 2018, 3:71 Last updated: 06 JUL 2018
mortalities ranging from 77 to 87% were recorded with the local populations of
An. coluzzii.
Conclusion: This study suggests the reduced efficacy of conventional LLINs
(Pyrethroids alone) currently distributed in Benin communities where
Anopheles populations have developed multi-insecticide resistance. The new
generation nets (pyrethroids+PBO) proved to be more effective on
multi-resistant populations of mosquitoes.
Keywords
An. funestus s.s., An. coluzzii, Pyrethroids, PBO, LLINs, Multi-resistance
controlling
Corresponding author: Romaric Akoton (romuluscoulys01@yahoo.fr)
Author roles: Akoton R: Data Curation, Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation; Tchigossou GM: Data
Curation, Visualization, Writing – Review & Editing; Djègbè I: Data Curation, Supervision; Yessoufou A: Validation, Visualization; Atoyebi MS:
Data Curation, Investigation, Writing – Review & Editing; Tossou E: Data Curation, Investigation; Zeukeng F: Data Curation, Formal Analysis,
Investigation; Boko P: Conceptualization, Data Curation, Investigation, Methodology, Supervision; Irving H: Data Curation, Visualization; Adéoti
R: Data Curation, Visualization; Riveron J: Data Curation, Methodology, Visualization; Wondji CS: Conceptualization, Supervision, Validation,
Writing – Review & Editing; Moutairou K: Methodology, Supervision, Validation; Djouaka R: Conceptualization, Funding Acquisition, Project
Administration, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing
Competing interests: No competing interests were disclosed.
How to cite this article: Akoton R, Tchigossou GM, Djègbè I et al. Experimental huts trial of the efficacy of pyrethroids/piperonyl butoxide
(PBO) net treatments for controlling multi-resistant populations of Anopheles funestus s.s. in Kpomè, Southern Benin [version 1;
referees: awaiting peer review] Wellcome Open Research 2018, 3:71 (doi: 10.12688/wellcomeopenres.14589.1)
Copyright: © 2018 Akoton R et al. This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Grant information: The study was supported by Wellcome Trust [099864] to RD.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
First published: 13 Jun 2018, 3:71 (doi: 10.12688/wellcomeopenres.14589.1)
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Wellcome Open Research 2018, 3:71 Last updated: 06 JUL 2018
Introduction
Malaria is responsible for about 438,000 deaths with an
estimated 214 million disease cases annually1. Malaria vector
control tools have been encouraging worldwide, resulting in a
decreased morbidity and mortality as of 2016 compared to the
20002. Unfortunately, as this disease has reduced globally, it has
been a different case in Africa, where malaria is still a serious
challenge2. Long lasting insecticide-treated nets (LLINs) are
major components of malaria control tools, and they have helped
to combat malaria disease when in good conditions and properly
used2. LLINs are effective, simple to use, easy to deliver to
rural communities, and cost-effective when used in highly
endemic malaria areas3.
In Benin, malaria control is hugely dependent on LLINs and
indoor residual spraying (IRS)4,5. In October 2014, there was a
country-wide distribution campaign of mosquito nets to ensure
universal coverage, with the free distribution of 6,077,272 LLINs
to 2,199,522 households surveyed6. After this exercise, LLINs
utilization by children under five rose from 70% in 2012, to
73% in 20146. However, the emergence and spread of resistant
malaria vectors to insecticidal components used for treating these
nets have threatened the earlier progress made with this malaria
vector control tool7–9. Resistance to insecticides ofone of the
main malaria vectors, An. funestus against control tools has since
become a serious challenge facing the quest for malaria elimination in Africa. Reported cases of resistance are available
in countries such as Cameroon10, Uganda11, Mozambique12,
Malawi13,14, Ghana15, Nigeria16 and Benin17,18. There are also
multiple mechanisms that are driving observed resistance in this
mosquito population, although over-expression of detoxification
genes remains the main driving force of insecticide resistance in
this Anopheles species15,19. Another observation is that resistance
mechanisms are known to differ from one mosquito population to
another suggesting the contribution of geographical differences
in resistance profiling15,20. This revelation is of serious concern
because it is becoming a significant threat to existing malaria
control tools. Recently, a study by Agossa et al.9 in the northern
part of Benin, showed that the efficacy of existing malaria vector
control tools treated with pyrethroid have decreased in wild
An. gambiae s.l. populations. Considering the fact that An. funestus
and An. gambiae have developed resistance to almost all classes
of insecticides across Benin17,21,22, it might follow a similar trend
as the above study. Indeed, there is a serious quest for alternative
insecticides since pyrethroids are becoming less effective with
recorded reports of resistance in malaria vectors23. Pyrethroids are
very safe, acceptable and suitable for LLINs, but degrade very
fast, especially when exposed to sunlight, which can be avoided
if nets are well preserved24. A different insecticide resistance
management approach combining a chemical synergist,
piperonyl butoxide (PBO), with pyrethroids on net fibres could
be a promising way to fight insecticide resistance. PBO, a synergist capable of inhibiting the action of oxidase enzymes, has
potential to combat the growing problem of oxidase based pyrethroid resistance in mosquito vectors species. Two types of long
lasting nets treated with permethrin+PBO and deltamethrin+
PBO are the new generation of LLINs for improved resistance
management25,26. These new generation nets have shown their
efficacy on some resistant populations of Anopheles in experimental hut trials25,27. In hut trials, the new generation of LLINs
increases mortality and inhibits blood feeding against
pyrethroid-resistant An. gambiae in some Africa regions23,25,26,28.
In Nigeria, the efficacy of LLINs treated with deltamethrin
+PBO was highly effective on resistant An. gambiae compared
with standard treated nets with no PBO29. Also, in Southern Africa
(Mozambique), this combination proved to be more effective
against resistant An. funestus and An. gambiae12,20.
Due to the widespread of insecticide resistance in most
populations of An. funestus from South to the North of
Benin17,18,21, it is important to assess the efficacy of currently
used LLINs (conventional LLINs) and also conduct in experimental huts a comparative assessment of the performance of the
new generation of treated LLINs (Pyrethroids+PBO) against
conventional LLINs currently used by communities in areas of
Benin where the main malaria vectors An. gambiae and
An. funestus have developed multiple resistance to insecticides.
Methods
Study area
The assessment was conducted in the rural locality of Kpomè
(6° 23' N, 2° 13'E) located in South Benin, approximately
81 km from Cotonou. The study area has a sub-tropical climate,
receives 1,100 mm of mean rainfall annually and has a mean
monthly temperature between 27 and 31°C. The rainfall pattern
in this area is similar to other southern localities of Benin, with
two rainy seasons and two dry seasons. The constant presence of water bodies in this locality favors the development of
An. funestus and other mosquito species18. Previous studies
carried out in Kpomè showed that An. funestus s.s. is mainly
predominant during the dry season and transitional periods, and
exhibited high resistance to permethrin and deltamethrin with
mortalities rates (World Health Organization (WHO) susceptibility tests) of 13% and 46.5% respectively17. P450s are the main
family of detoxification enzymes involved in observed pyrethroid resistance in the An. funestus population in this locality17.
An. gambiae populations from this same locality have also
developed multi-resistance to several insecticides families30.
This set of available environmental and entomological data has
prompted building of seven experimental huts at Kpomè for trials
to identify best LLINs types for improved control of insecticide
resistant populations of malaria vectors.
Collection of mosquitoes for planed experiments
Early morning collections of blood-fed, semi-gravid and fully
gravid females of resistant An. funestus resting inside houses were
collected using electric aspirators between 06h00 and 10h00 in
June 2017 (Consent from head of household was obtained prior
to collection). These mosquitoes were identified morphologically
using Gillies and De Meillon31 and Gillies and Coetzee32 key as
An. funestus were kept in small cups and immediately transported
to the laboratory (Relative humidity of 70–80% and a temperature
of 25–30 °C) until fully gravid (for blood-fed and semi-gravid
females). Eggs were obtained from F0generation (Collected
females from the field) using the forced egg laying method33
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and were allowed to hatch to obtain F1 generation to be used for
different experiments.
Still in the same locality, An. gambiae breeding sites were
surveyed and their larvae collected using dipping methods34 and
reared in the insectary to adult stage for different experiments.
Species characterization for An. funestus and An. gambiae
A subset (100 An. funestus and 100 An. gambiae) of mosquitoes to be used for various assays were subjected to molecular speciation prior to assays. DNA of An. funestus s.l. used for
forced-laying was extracted using the Livak method35. Speciesspecific targets were amplified for DNA using method described
by Koekemoer et al.36, before distinct separations on a 1.5%
agarose gel electrophoresis. For An. gambiae, we used also the
protocol of Livak35, for DNA extraction followed by an amplification based on the protocol described by Fanello et al.37 then
after, amplified products were migrated on agarose gel for
describing banding patterns.
Insecticide susceptibility pattern of mosquito populations
used in this study
Prior to assays, a subset of An. funestus and An. gambiae were
subjected to insecticide susceptibility tests to confirm resistance
levels of collected Anopheles species from Kpomè. Similarly,
An. gambiae Kisumu were also tested to confirm their susceptibility. Unfed F1 Anopheles female mosquitoes (2–5 days old) were
therefore tested with 2 insecticides: 0.75% permethrin (type I
pyrethroid) and 0.05% deltamethrin (type II pyrethroid) by using
WHO susceptibility tests38. Approximately 100 mosquitoes
(4 replicates of 25 mosquitoes) were used per test. The knockdown rate of mosquito exposed to the insecticides was recorded
each 5 min, during 1 h exposure-period. A 10 % of sugar solution was made available to survivors. This test was made under
observation at 25°C and 80% relative humidity laboratory
condition. Mortality was recorded 24h after exposure to each
insecticide. According to WHO criteria38, vectors were considered as being susceptible to a given insecticide if mortality rate
was ≥ 98 %, resistant if mortality was <90 % or possibly resistant
if mortality was between 90 and 98 %.
Piperonyl butoxide (PBO) synergist tests
According to the level of observed resistance against permethrin and deltamethrin, and because of pyrethroids resistant,
An. funestus population has been shown to express P450s genes
more than in previous studies19,39,40, 2–5 days old F1 mosquitoes
were pre-exposed to 4% PBO paper for 1 h and immediately
exposed to 0.75% permethrin and 0.05% deltamethrin for 1 h.
Two controls were used during this experiment. The first control
was the mosquitoes exposed to untreated papers without PBO,
and the second comprised of mosquitoes exposed to paper treated
with PBO only. Mortalities were recorded 24h post exposure
and were later compared to the un-synergized group in order to
evaluate the potential role of cytochromes P450 genes in the
observed resistance.
Characteristics of long-lasting insecticidal nets used during
the various assessments
Five types of LLINs were used for the phase I (Cone and tunnel
tests) and phase II (experimental hut) evaluations.
The Type 1 LLINs made of monofilament polyethylene (100
mesh size) fabric treated with deltamethrin at 4 g/kg±25% and
piperonyl butoxide (PBO) at 25g/kg±25%, side panels made
of multifilament polyester fabric with a strengthened border
treated with deltamethrin at 2.1 g/kg±25%.
The Type 2 LLINs was made of multifilament polyester fabric
(100 mesh size), treated with a deltamethrin only (no PBO added)
at 1.4 g/kg±25%.
The Type 3 LLINs was treated with 20 g/kg of permethrin and 10 g/kg
of PBO in the whole polyethylene net fibres (150 mesh size).
The Type 4 LLINs made of polyethylene fibers treated with
permethrin only (no PBO) at 20 g/kg, incorporated during fibers
extrusion (150 mesh size).
The Type 5 was an untreated net, a multifilament polyester (100
mesh size) fabric with neither insecticide nor PBO treatments.
All nets used had sizes of 160 cm wide, 180 cm long and 150 cm
high. All types of nets treated and control nets were procured by
the Liverpool School of Tropical Medicine (LSTM), UK, properly
wrapped and shipped to us at IITA for our various trials.
WHO cone tests with of 5 types of nets under tests
Four cones were fixed in contact to 25 × 25 cm pieces of nets
taken from the sides and top panels of LLINs (Methods in
Anopheles Research, 2010). 2 to 5 days old individuals from
the 3 colonies of Anopheles mosquitoes (resistant-An. funestus
Kpomè, Resistant-An. gambiae Kpomè and SusceptibleAnopheles Kisumu) were exposed to nets for 3 min, after which
they were transferred into recovery paper cups and provided with
cotton wool soaked in a 10 % honey solution. A minimum of
50mosquitoes was tested for each net. At least three pieces per
net was used for this test. Mosquito knock-down rate (kd) was
recorded at 1h post-exposure period and the mortality rate was
determined 24 h post-exposure. The mortality rate was corrected
using Abbott’s formula if needed. These tests were conducted
at a room temperature and relative humidity of 25–30°C and
70–80% respectively.
WHO tunnel tests with fragments of the 5 types of nets
under tests
Tunnel test was carried out with the same samples of LLINs
used for cone test. Adults An. funestus, An. gambiae and
Kisumu strain were also used for this test41. Adult mosquitoes
aged between 5 to 8 days were released at 6.00 PM in the first
compartment C1 of a 60-cm long tunnel made of glass divided
by a transverse netting (25cm x 25 cm) insert, fitted onto a frame
that slots across the tunnel. The LLINs fragments used had been
pierced (1-cm diameter holes) to allow mosquitoes to pass
through it into the tunnel to the compartment C2 where a guinea
pig was placed for mosquitoes feeding. Each guinea pig was used
only once for this study. Guinea pigs were sourced from local
markets where they are sold for food consumption. At 8.00 AM
of the following morning, mosquitoes were collected from both
compartments and transferred into plastic cups. The mortality
and feeding status (blood-fed or unfed) of each mosquitoes
collected from the tunnel were recorded. Blood-feeding rate and
penetration rate across the tunnel were also assessed.
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Wellcome Open Research 2018, 3:71 Last updated: 06 JUL 2018
Experimental hut trials of the efficacy of the 5 Net Types on
insecticide resistant mosquitoes
Experimental huts newly built in Kpomè are specially designed
to test the efficacy of different vector control products against
freely entering mosquitoes under natural but controlled conditions. This facility was used for our release and recaptures tests.
Huts were typical of the West African model as recommended by
WHO41. The 3.5 × 2 × 2 m huts were made from concrete bricks,
with a corrugated iron top and a ceiling of thick polyethylene
sheeting lined, and each was built on a concrete base surrounded
by a water-filled moat to exclude ants. Mosquito access was
through 4 window slits, constructed from pieces of iron fixed at
an angle to create a funnel with a 1-cm gap, present on 3 sides
of the huts. Mosquitoes had to fly upward to enter through the
gaps and downwards to exit; this precluded or limited exodus
through the aperture and enabled us to account for most entering
mosquitoes. A veranda trap made of concrete bricks and mesh
screening (2 m long × 1.5 m wide × 1.5 m high) projected from
the back wall of each hut. Movement of mosquitoes between a
room and the veranda was unimpeded.
Study design
The described 5 types of mosquito nets were assessed against
pyrethroid resistant An. funestus and An. gambiae. The control
mosquito population used was only An. gambiae Kisumu as we
had neither laboratory/field susceptible An. funestus, nor field
susceptible An. gambiae.
The following five comparison arms were tested in separate huts:
1- LLINs Type 1 (deltamethrin + PBO)
2-LLINs Type 2 (deltamethrin only)
3-LLINs Type 3 (permethrin + PBO)
4-LLNIs Type 4 (permethrin only)
5-Type 5 Untreated Polyester net (control)
Blank assessment of hut attractiveness
Prior to introducing nets into huts, we conducted preliminary
experiments which showed the huts to be evenly attractive to
mosquitoes. Briefly, assessment for freely-entered mosquitoes
in the hut was conducted during 2 weeks and the attractiveness
effects of each hut were evaluated. Adult male volunteers
slept under the untreated net in the huts from 20:00 hours to
05:00 hours each night after cleaning the hut to remove any
spiders and ants. To minimize biases in individual attractiveness, sleepers were rotated between huts on successive nights
throughout the 2 weeks.
Blank assessment of huts lethality
Still prior to assessments, an initial series of bioassays was
conducted to determine the mortality of susceptible mosquitoes
exposed to various surfaces in the huts to know the lethal effect
of the huts. Bioassays were performed with WHO cones tests
attached to the surfaces with masking tape. In each hut, surfaces
tested included doors, walls, screening-mesh of veranda, ceiling and floor. Ten females of An. gambiae Kisumu strain of
2 to 5 days old were put into each cone for 30 min. After this
exposure time, they were removed from the cone and put into
plastic cups covered with untreated mosquito net and given
access to 10% honey solution and mortalities recorded after 24h.
Release and recapture experiment
A release/recapture experiment was conducted in experimental
huts with resistant populations of An. funestus and An. gambiae
both from Kpomè and a susceptible An. gambiae Kisumu.
These 3 populations of mosquitoes were released different days
into huts where the 5 described types of nets were erected. The
experiment was conducted as described by WHO protocols41. The
main trial was conducted in August 2017. The treatments were
allocated randomly to five experimental huts in study site. Each
net was deliberately holed with six 4cm×4cm holes to simulate
a worn net. Before experimental hut evaluation, adult volunteers
had been recruited among the inhabitants of the villages where
experimental huts were implemented and informed consent to
participate in the study was given beforehand and, chemoprophylaxis was provided during the trial. Female mosquitoes aged
5 days were released in each hut at 20:00 h in the night and
monitored till morning. Early in the morning, released
mosquitoes were recaptured from the hut, veranda and inside the
nets and were scored as dead or alive and as fed or unfed. Live
mosquitoes were kept in small cups containing sugar solution for
24 hours to assess the delayed mortality. Entomological effects of
treatments were compared in-between nets and with the untreated
net (control Net Type 5). Target entomological parameters
monitored included: induced exiting, blood-feeding inhibition and
mortality.
(i) insecticide-induced exiting, i.e. the proportion of mosquitoes
found in hut verandahs relative to control huts; (ii) blood-feeding
inhibition, i.e. the proportional reduction in blood-feeding relative
to untreated nets; and (iii) mortality, the proportion of mosquitoes
killed (immediate plus delayed).
Chemical analysis of net used in the experimental hut trial
Prior to the trial, chemical analysis were conducted on pieces
of nets (pieces from holes made on nets) from the five Net
Types erected in each hut. This experiment was to confirming the
presence, or absence, and the concentration of pyrethroids and
PBO in each net to be used in this trial. For the LLINs Type 1, the
side panels and top panel were tested separately. Chemical analysis was conducted using high performance liquid chromatography
(HPLC) machine (Agilent technology1260 infinity, Germany).
Deltamethrin, permethrin and PBO was extracted using acetonitrile
as solvent and the mixture was sonicated for 15min. Afterwards,
the solution without the net was transferred into a new flask
and filtered through a 0.45µm PTFE syringe filter into an HPLC
vial for analysis. For HPLC analysis, standard solutions of each
insecticide (Permethrin cat no. 45614, Deltamethrin cat no.45423
and PBO cat no.45626) purchased from Sigma Aldrich were
prepared from stock solution in acetonitrile. Standard curve of
each insecticide were drawn. The HPLC system condition was as
follow: mobile phase: Acetonitrile /H2O (90:10), C18 Column,
flow rate: 1ml/min, injection volume: 50µl and UV detector
wavelength: 226nm.The quantities of insecticides were calculated
based on the peak area and expressed in g/kg of net.
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Data analysis
Data from bioassays were compared between each net using
MedCalc easy-to-use online statistical software, version 18.2.142,
while the Fisher’s exact test was used to test for significant
difference of mortality rates. Significance between treatments
was set at 5% level. The proportion of mosquitoes that exited
early (induced exophily), the proportion that was killed within
the hut (mortality) and the proportion that successfully blood-fed
(blood feeding rate) were compared and analyzed using the
logistic regression with treatments as fixed effects and huts,
sleepers as random effects (STATA 9 Software).
mortality rates were also recorded for An. coluzzii population
from Kpomè against permethrin (19.27±3.52%) and deltamethrin (60.11±7.19%). No mortality was recorded when subsets
of these mosquitoes species were exposed to papers with no
insecticides (Control).
Synergist assay with PBO
When permethrin and deltamethrin were combined with PBO
(Figure 2), mortalities in An. funestus s.s. rose from 14.84% to
96.51% (permethrin) and from 44.15% to 100% (deltamethrin). However with An. coluzzii population, mortalities
reached 95% with deltamethrin + PBO whereas, the combination permethrin + PBO lifted up the mortality from 19.27% to
69.67%.
Results
Molecular speciation of An. funestus and An. gambiae
All 100 samples of An. funestus and 100 samples of An. gambiae
from Kpomè analyzed for molecular speciation were An. funestus
s.s. and An. coluzzii respectively (Table 1).
Chemical analysis of insecticide contains of nets used in
the experimental hut trial
HPLC analysis conducted on net fiber showed that deltamethrin
concentration in the side of the Net Type 1(1.570±0.024 g/kg)
and in the roof (3.762±0.019g/kg) were all within the standard dose (2.1g/kg±25% on sides and 4g/kg ±25% in the roof)
(Table 2).A mean dose of insecticide from the five sides of
treated Net Type 2(0.994±0.013g/kg) and treated net Type 3
(16.065±0.244 g/kg) analyzed using HPLC were closer to the
standard doses (1.4g/kg ±25% and 20g/kg ±25% respectively)
as recommended by manufacturers. Similarly for Type 4, the
Insecticide susceptibility profiles of Anopheles populations
used in this study
An. gambiae Kisumu was used in this test as a control for checking the quality of impregnated papers. A mortality rate of 100%
was recorded with permethrin and deltamethrin treated papers.
With local populations of An. funestus s.s. we recorded 24 h
post-exposure, mortalities of 14.84±2.32% and 44.15±2.23%
for permethrin and deltamethrin respectively (Figure 1). Low
Table 1. Distribution of members of Anopheles groups collected in June
2017 in Kpomè, Southern Benin.
Mosquito
species
Anopheles subjected
to molecular
speciation
An. funestus s.s. An. coluzzii
An. funestus s.l.
100
100
/
An. gambiae s.l.
100
/
100
0 (n=50)
100 (n=50)
An. gambiae Kisumu
100 (n=50)
0 (n=50)
60.11 (n=90)
An. coluzzii
Control
19.27 (n=90)
deltamethrin
permethrin
0 (n=50)
An. funestus s.s.
44.15 (95)
14.88 (n=88)
0
20
40
60
80
100
% Mortality rates
Figure 1. Insecticide susceptibility profiles of Anopheles to permethrin and deltamethrin. Error bars represent standard error of the
mean.
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Wellcome Open Research 2018, 3:71 Last updated: 06 JUL 2018
0 (n=50)
0 (n=50)
95 (n=100)
An. coluzzii
60.11 (n=90)
Control
PBO
69.67 (n=70)
deltamethrin+ PBO
19.27 (n=97)
deltamethrin
permethrin+PBO
0 (n=50)
permethrin
0 (n=50)
100 (n=70)
An. funestus s.s.
44.15 (n=95)
96.51 (n=62)
14.88 (n=88)
0
20
40
60
80
100
% Mortality rates
Figure 2. Insecticide susceptibility profiles of Anopheles to pyrethroids when combined with piperonyl butoxide (PBO). Error bars
represent standard error of the mean.
Table 2. Concentrations of insecticides and synergist in the net fragments
when analyzed by HPLC techniques.
Net
Net
Types sections
Chemicals
Units Standard
Recorded
concentrations concentrations
1
Sides
deltamethrin
g/Kg
2.1 ± 25%
1.570 ± 0.024
Roof
deltamethrin
g/Kg
4 ± 25%
3.762 ± 0.019
PBO
g/Kg
25 ± 25%
26.210 ± 0.057
2
Sides/Roof
deltamethrin
g/Kg
1.4 ± 25%
0.994 ± 0.013
3
Sides/Roof
permethrin
g/Kg
20 ± 25%
16.065 ± 0.244
Sides/Roof
PBO
g/Kg
10 ± 25%
11.016 ± 0.003
4
Sides/Roof
permethrin
g/Kg
20 ± 25%
23.702 ± 0.003
5
Sides/Roof
deltamethrin
g/Kg
0
0
Sides/Roof
permethrin
g/Kg
0
0
Sides/Roof
PBO
g/Kg
0
0
PBO - piperonyl butoxide, HPLC - high performance liquid chromatography
permethrin concentration for sides/top panels (23.702±0.003 g/kg)
was within the standard dose. No presence of insecticides traces on
the untreated net (Type 5) was revealed by this HPLC analysis.
WHO cone tests with the 5 types of nets
A total of 275, 461 and 462 females of An. funestus s.s., An. coluzzii
and Kisumu strain respectively aged 2-5 days were exposed to the
five sides of nets following WHO standard cones protocol. No
mortality was recorded with mosquitoes exposed to untreated Net
Type 5 after 24 hrs. An. gambiae Kisumu showed full susceptibility to all treated net. The Net Type 2 had the lowest lethal effect
on both resistant An. funestus s.s. (56.67±6.38%) and An. coluzzii
(34.67±4.11%) from Kpomè (Figure 3). However, when they
were exposed to Net Type 1 containing PBO + deltamethrin, the
mortality rate rose from 56.67 % to 95.77% for An. funestus s.s and
a mean mortality of 69.54 ± 2.66% was recorded for An. coluzzii
population showing that Net Type 1 is more effective against these
Anopheles species compared to Net Type 2 (P < 0.0001).
Page 7 of 18
Wellcome Open Research 2018, 3:71 Last updated: 06 JUL 2018
100
100
95.77
100
100
100
92.69
Kisumu (n≥78)
An. coluzzii (n≥80)
% Mortality rates
80
69.54
An. funestus s.s. (n≥50)
56.67
60
34.67
40
25.83
23.33
20
6
0
0
1
2
3
Treated Net Types
4
0
0
5
Untreated Net
Type
Figure 3. Cone tests performed on different net Types with using pyrethroids resistant Anopheles funestuss.s. and An. coluzzii. Error
bars represent standard error of the mean.
A similar trend was observed when An. funestus s.s. were
exposed to Net Type 4. Recorded mortality rate with Net Type 4
was 23.3 ± 3.59 %. When PBO was added into the net fibers for
Net Type 3 (PBO + permethrin), a significant increase of the
mortality rate was recorded (mortality rate with Net Type 3 = 92.69
± 3.59%; χ2 = 52.352, P < 0.0001).In contrast, when An. coluzzii
mosquitoes from Kpomè were introduced into the various cones,
mortalities slightly rose from 6% to 25.83% with Net Type 3.
WHO tunnel tests with fragments of the 5 types of nets
When the three mosquito populations (An. gambiae Kisumu,
An. funestus s.s. and An. coluzzii) were separately released in
tunnels with the untreated Net Type 5, we globally recorded
over 45% penetration rates for all 3 mosquito species. When the
untreated net was replaced by Net Type 2 and Net Type 1, the
penetration rate decreased respectively from 85.66% to 3.4%
and 0% for An. gambiae Kisumu (Table 3). For the resistant
An. funestus s.s., we recorded an inhibition of the penetration in Net Type 2 (9.32%) and with side sections of Net Type 1
(1.79%) when compare to the untreated Net Type 5 (66.85%). No
An. funestus s.s. was able to pass through the top section of the
Net Type 1. When the pyrethroid resistant An. coluzzii population was released in tunnels containing fragments of treated Net
Type 2 (deltamethrin only), side and top sections of Net Type
1, we recorded respectively penetration rates of 9.66±2.84%,
7.84±0.86% and 3.66 ±1.22% in the second compartment (C2)
of tunnels. With fragments of Net Type 4 (bigger mesh size and
treated with permethrin only), we recorded a similar penetration
rate as with the control untreated Net Type 5 in resistant population
of An. funestus s.s. However remarkable decreased penetration of
this mosquito species through the Net Type 3 (PBO+permethrin)
was observed. With Net Type 4 containing only permethrin and
Net Type 3 containing permethrin+PBO, we recorded limited
entry of An. coluzzii into compartment C2; 16.07±4.44% and
55.52±1.24% penetration rates respectively.
A high blood feeding rate was observed in the insecticide
resistant An. funestus s.s. population (76.54±3.46%), the resistant
An. coluzzii (33.87±10.96%) and Kisumu strains (91.92±0.81%)
released in tunnel containing untreated Net Type 5 (Table 3).
However, no Kisumu was able to blood feed in the presence of
Net Type 1, Net Type 2 and Net Type 3. Only a single mosquito
(Kisumu) was able to blood feed in the presence of Net Type 4
containing permethrin and having large mesh sizes. Generally,
all treated nets provided more blood feeding inhibition with
An. funestus s.s. compare to An. coluzzii. Indeed, blood feeding
was inhibited at respectively 91.98%, 97.66% and 100% with Net
Type 2, Net Type 1(Side) and Net Type 1 (Top) in An. funestus
s.s. In An. coluzzii population, Net Type 2, Net Type 1 (Side) and
Net Type 1 (Top) provided respectively 71.48%, 80.30% and
100% blood feeding inhibition rates. Against resistant An. funestus s.s., blood feeding inhibition rates with Net Type 4 (41.75%)
was significantly lower than Net Type 4 (100%). Respectively
9.71% and 63.92% blood feeding inhibition rates were obtained
with Net Type 4 and Type 3 in An. coluzzii.
Furthermore, lethal effect of all LLINs ranged from 99% to 100%
against susceptible An. gambiae Kisumu strain. However, the
mortality rate recorded in resistant An. coluzzii population with
Net Type 2 increased when we exposed this mosquito species to
Net Type 1. The same observation was noted with Net Type 2 and
Type 1 (side and top sections) against resistant An. funestus s.s.
where mortality rate rose from 65.53% to 92.47% and to 98%
respectively. Less than 80% mortality was recorded in presence of
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Wellcome Open Research 2018, 3:71 Last updated: 06 JUL 2018
Table 3. Bio -efficacy of Long-Lasting Insecticidal Nets (LLIN) against resistant Anopheles funestus s.s. and
An. coluzzii and laboratory susceptible ‘Kisumu’ strain in tunnel test Standard errors are 95% confidence
interval.
Species
Mosquito net
Types
Susceptible
An. gambiae
(Kisumu)
1 (Top panel)
92
0
0
100
100
1 (Side panel)
97
0
0
100
100
2
99
3.40 (±1.73)
0
100
99.17 (±0.83)
3
93
1.22 (±1.22)
0
100
100
4
100
11.10 (±5.22)
1.02 (±1.02)
98.9
100
5
100
85.66 (±3.43)
91.92 (±0.81)
/
0
1 (Top panel)
82
3.66 (±1.22)
0
100
87.80 (±2.44)
1 (Side panel)
89
7.84 (±0.86)
6.67 (±2.02)
80.30
63 (±2.12)
2
92
9.66 (±2.84)
9.66 (±2.84)
71.48
56.82 (±6.82)
3
82
16.07 (±4.44)
12.22 (±0.60)
63.92
77.88 (±3.52)
4
72
55.52 (±1.24)
30.58 (±0.85)
9.71
45.91 (±2.66)
5
77
45.65 (±6.07) 33.87 (±10.96)
/
0
Resistant
An. coluzzii
1 (Top panel)
Resistant
An. funestus s.s.
1 (Side panel)
Released % Penetrating
number
% Blood % Blood feeding
feeding
inhibition
% Overall
mortality
50
0
0
100
98 (±2)
51
1.79 (±1.79)
1.79 (±1.79)
97.66
92.47 (±3.18)
2
51
9.32 (±4.97)
6.13 (±2.56)
91.98
65.53 (±8.33)
3
53
5.57 (±1.57)
0
100
96.21 (±0.21)
4
54
66.67
44.58 (±1.25)
41.75
71.25 (±7.92)
5
51
66.85 (±9.15)
76.54 (±3.46)
/
0
Net Type 4 against An. funestus s.s., while 96% of mortality rate
with Net Type 3. High lethal action was provided by Net Type 3
(77.88%) against An. coluzzii (Table 3) compared to the Net Type
4(45.91%). Zero mortality was recorded with untreated Net Type 5
against the three Anopheles mosquito populations.
Table 4. Mean number of mosquitoes collected per
night in the seven experimental huts over 12 nights
prior to phase II evaluation. Standard errors are 95%
confidence interval.
Hut number
Blank assessment of experimental huts attractiveness
A total of 603 mosquitoes were allowed to freely enter the seven
experimental huts during the 12 trial nights. The mean number
of mosquitoes collected in huts was high in hut N°7 (18.41)
followed by hut N°2 (8.41). The mean number of mosquito per
night was almost similar in huts N° 6, 5, 4 and 3. The hut N°1
showed a relatively low attractiveness (Table 4). However,
similar attractiveness in terms of Anopheles mosquitoes was
observed between the hut N° 1, 2 and 5. The recorded mean
numbers of Anopheles mosquito in hut N° 3, 4, 6 and 7, which are
similar, were higher than the others.
Blank assessment of experimental huts lethality
The cones bioassay conducted on various surfaces, such as
doors, walls, screening-mesh of veranda, ceiling and floor, of
each hut revealed that all the huts built in Kpomè locality had no
lethal effect on susceptible Anopheles gambiae Kisumu strain. The
mortality rate for all exposed mosquitoes was very low as only
one mosquito died out of the total of 73 exposed in hut N°4 and
N°7 (Table 5).
Overall mosquitoes
Anopheles
1
1.91(1.5 – 2,32)
0.33 (0.19 – 0.47)
2
8.41 (6.93 – 9.89)
0.5 (0.27 – 0.73)
3
4.16 (3.54 – 4.78)
0.83 (0.46 – 1.2)
4
6.83 (6.05 – 7.61)
1.25 (0.82 – 1.68)
5
4.83 (4.41 – 5.25)
0.67 (0.25 – 1.09)
6
5.67 (4.46 – 6.88)
1.17 (0.75 – 1.59)
7
18.41 (15.66 – 21.16)
1.25 (0.8 – 1.7)
Release and recapture experiments
Induced exophily. When Kisumu strain was released in rooms
containing treated nets, we recorded a significant movement
of mosquitoes from the room to the veranda; all treated nets
induced significant exophily rates ranging from 50 to 73%
compared to the untreated net, where the observed exophily was
30% (P< 0.0009). A similar trend was observed with the pyrethroids resistant An. funestus s.s. population from Kpomè with
exophily rates ranging from 30 to 40% with treated nets
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Wellcome Open Research 2018, 3:71 Last updated: 06 JUL 2018
compare to 9.46% with untreated Net Type 5 (Table 6). The
induced exophily rates recorded with An. funestus s.s. was not
significant in between huts containing treated nets (induced
exophily rate ranging from 23 to 34%) (Figure 4). As for the
pyrethroids resistant An. coluzzii, all treated nets induced exophily
rates ranging from 8% to 37% (Figure 5).
Blood feeding. No Anopheles Kisumu bite was recorded when
volunteer sleepers spent nights under all treated nets (100%
blood feeding inhibition). In contrary, when these nets were
replaced with untreated nets (Net Type 5), 71% biting rates
were recorded with Anopheles Kisumu, 52% with pyrethroids
resistant An. funestus s.s. and 42% with resistant An. coluzzii.
Table 5. Lethal effect of experimental huts built at Kpomè,
Southern Benin.
N° of
Dead
Mortality
Tested An.
experimental huts gambiae Kisumu mosquitoes rates (%)
Hut 1
73
0
0
Hut 2
76
0
0
Hut 3
79
0
0
Hut 4
73
1
1.37
Hut 5
73
0
0
Hut 6
74
0
0
Hut 7
73
1
1.37
Table 6. Summary of release-recapture experiment with susceptible strain of Anopheles gambiae (Kisumu) and
resistant An. funestus s.s. and An. coluzzii in experimental huts.
Mosquito nets types Total
recaptured
% Exophily
% Blood feeding
% Personal % Overall Killing
protection
effect
Anopheles gambiae Kisumu
1
138
73.91 (66.59 - 81.24
0
100
97.08
2
139
69.78 (62.15 - 77.42)
0
100
97.08
3
135
51.11 (42.68 - 59.54)
0
100
94.89
4
138
50 (41.66 - 58.34)
0
100
95.62
5
142
30.28 (22.72 - 37.84)
71.83 (64.43 - 79.23)
/
/
Resistant Anopheles coluzzii
1
160
40 (32.41 - 47.59
10 (5.35 - 14.65)
80.24
73.65
2
175
33.71 (26.71 - 40.72)
17.71 (12.06 - 23.37)
61.72
28.49
3
191
34.03 (27.31 - 40.75)
18.32 (12.84 - 23.81)
56.79
72.58
4
190
12.63 (7.91 - 17.36)
23.68 (17.64 - 29.73)
44.44
10.75
5
189
4.76 (1.73 - 7.80)
42.86 (35.80 - 49.91)
/
/
Resistant Anopheles funestus s.s.
1
80
33.75 (23.99 - 44.11)
3.75 (-0.41 - 7.91)
92.30
100
2
96
33.33 (23.90 - 42.76)
21.88 (13.61 - 30.14)
46.15
77.02
3
72
40.28 (28.95 - 51.61)
0
100
87.83
4
109
30.28 (21.65 - 38.90)
29.36 (20.81 - 37.91)
17.94
48.64
5
74
9.46 (2.79 - 16.13)
52.70 (41.33 - 64.08)
/
/
Page 10 of 18
Wellcome Open Research 2018, 3:71 Last updated: 06 JUL 2018
100
92
100
% Percentage
80
58
60
44
34
26
40
Induced exophily
26
23
Blood feeding inhibition
20
0
1
2
3
4
LLINs Types
Figure 4. Induced exophily and blood feeding inhibition rates of resistant population of Anopheles funestus s.s. in experimental huts
with selected treated nets. Error bars represent 95 % confidence interval. LLIN - Long-Lasting Insecticidal Nets.
1FSDFOUBHF
*OEVDFEFYPQIJMZ
#MPPEGFFEJOHJOIJCJUJPO
--*/T5ZQFT
Figure 5. Induced exophily and blood feeding inhibition rates of resistant population of Anopheles coluzzii in experimental huts with
selected treated Nets. Error bars represent 95 % confidence interval. LLIN - Long-Lasting Insecticidal Nets.
More specifically there was blood-feeding inhibition with
An. funestus s.s. populations in presence of Net Type 4 (44%
blood feeding inhibition) and Net Type 2 (58% blood feeding
inhibition) compared to untreated nets. Generally, higher bloodfeeding inhibition rates were provided by Net Type 1 containing deltamethrin + PBO (92% blood feeding inhibition) than
Type 2 containing deltamethrin only (P<0.0001). Similarly, blood
feeding inhibition rates in huts with Net Type 3 containing permethrin + PBO (100% blood feeding inhibition) was higher than
those with Net Type 4 containing permethrin alone (Figure 4)
(Table 6). As for the pyrethroid resistant An. coluzzii, blood
feeding was inhibited more with Net Type 1 which contains
deltamethrin + PBO (76% blood feeding inhibition) than Type 2
which contains deltamethrin only (58% blood feeding inhibition).
Respectively, 44% and 57% blood feeding inhibition rates were
recorded in huts with Net Type 4 and Net Type 3 (Figure 5).
Mortality. All treated nets significantly induced high lethal effect
against susceptible An. gambiae (Kisumu). In huts containing
Net Type 2, 59.38% mortality rate was recorded with resistant
An. funestus s.s. However, Net Type 1 showed a high lethal effect
of 92.5% against this resistant mosquito population. Respectively,
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Wellcome Open Research 2018, 3:71 Last updated: 06 JUL 2018
33.03% and 90.28% mortality rates were recorded in the huts
containing Net Type 4 and Type 3 (Figure 6). Consequently, the
overall killing effect offer by Net Type 1 was significantly higher
than Net Type 2 against resistant An. funestus s.s. (Table 6). Same
thing with Net Type 3 in comparison to Net Type 4. The same
trend was observed against resistant An, coluzzii, where very
low overall killing effect was provided by Net Type 2 (28.49%)
compared to Net Type 1 (73.65%). Mortalities rose from 10.7%
to 71.8%, when resistant An. coluzzii were released in huts
containing respectively Net Type 4 and Type 3. Consequently,
high overall killing effect was provided by Net Type 3 against
this Anopheles specie compared to Net type 4 (Table 6). A combined pyrethroids-PBO Net Type 1 and Type 3 was found to
demonstrate a greater efficacy against these resistant mosquito
populations.
Discussion
This study aimed to assess the response of resistant An. funestus
s.s. from Benin to pyrethroid treated nets (current LLINs) and to
combined PBO + pyrethroid nets for improved control of resistant
populations of malaria vectors.
Bio-efficacy of selected LLINs types
Results obtained from the response of susceptible mosquitoes (An. gambiae Kisumu) to treated nets showed that pyrethroid and pyrethroid + PBO treated nets remained effective for
controlling susceptible Anopheles mosquitoes. It was also observed
that the bio-efficacy of nets treated with deltamethrin only (Type
2) was significantly lower when we compared the recorded
mortality rates from the cone test in the resistant populations of
An. funestus s.s. and An. coluzzii and the susceptible strain
Kisumu. These observations further confirm the high pyrethroid
resistance observed in both malaria vectors in Kpomè like in
others localities of Southern Benin17,21. A more recent study
conducted across a South-North transect of Benin, revealed that
100
100
100
99.25
92.5
87.3
90.28
more than 50% of An. gambiae mosquitoes are unaffected by
lethal effects of the current form of Net Type 243. However, in the
Ivory Coast, this net was effective against An. gambiae s.s.44. When
resistant mosquitoes were exposed to the combined deltamethrin-PBO (Net Type 1), the mortality rose from 56.67 to 95.77%
and from 34.67 to 69.54% for respectively An. funestus s.s. and
An. coluzzii. This finding showed the important involvement of
P450s genes in observed pyrethroids resistance in this study and
confirms also the results of synergist bioassays test performed with
these same resistant mosquitoes as almost all individuals were
dead when they were exposed to PBO and immediately after to
deltamethrin.
Similarly, significantly lower mortality of An. funestus s.s.
in the presence of current permethrin treated Net Type 4 was
observed compared to the combined Net Type 3 (permethrin
+ PBO). The loss of bio-efficacy of this current Net Type 4 was
also demonstrated in Malawi, Mozambique and Democratic
Republic of Congo, where recorded mortality rates of An. funestus to Net Type 4 in this study were respectively 3%, 20% and
34%12,13,20. The study conducted in Benin in 2013 demonstrated
the efficacy of combined permethrin-PBO net (Olyset plus)
against resistant An. gambiae25. Surprisingly, only 25.83% of
An. coluzzii was affected by lethal effect of this net in this study.
It could probably due to the presence of other mechanisms
involved in multi-resistance of An. coluzzii from Kpomè like kdr
mutations30. This result supports the relatively low mortality
(69.67%) obtained from the synergist test when we pre-exposed
An. coluzzii to PBO before to permethrin. Therefore, a combined
permethrin - PBO net does not provide a solution to pyrethroid
resistance with An. coluzzii from Kpomè, Southern Benin.
Tunnel test performed on the all net Types used in this study
confirmed the reduced bio-efficacy of only pyrethroids treated
nets, showing a decrease in their effectiveness in areas of high
98.5
Kisumu
An. coluzzii
71.8
80
An. funestus s.s.
% Mortality rates
59.38
60
40
33.03
31
20
10.7
3.52
1.59 0
0
1
2
3
4
5
LLINs Types
Figure 6. Mortality rates of resistant populations of Anopheles funestus s.s. and An. coluzzii and susceptible strain (Kisumu) in
experimental huts. LLIN - Long-Lasting Insecticidal Nets.
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resistance. This observation could be related to the resistance
selection pressure generated by the use and misuse of the same
class of insecticides for malaria vector control in public health
and for pest control in agriculture21,45,46. Indeed, reduced repellent
effect of Net Type 2 against wild resistant Anopheles mosquitoes
compared to high repellent effect against Kisumu strain could
be as a result of their resistance nature. However, crossing of
mosquitoes through Net Type 1was highly inhibited for each
resistant population, even for susceptible strain, penetrating the
compartment C2 of the tunnel containing Net Type 1. Nevertheless, deltamethrin alone used for the treated net (Type 2) continues to have moderate performance against resistant Anopheles
mosquitoes in terms of reducing human and malaria vector
contact and also blood feeding rate.
Crossing rates with Net Type 1 were earlier described by
N’Guessan et al.,27, working with An. gambiae VKPER a strain
originate from Kou valley in Burkina-Faso even after 20 washing times, same elevated crossing rates were still recorded. We
made identical observations for An. coluzzii in this study, with
mosquitoes being no more able to pass through the net section
of Net Type 3 comparing to the Net Type 4.
Similarly, An. funestus s.s. penetration rate recorded in presence
of Net Type 4 (66.67%) was significantly higher than that of
Net Type 3 (5.57%) (χ2 = 42.727; P < 0.0001). These observations suggest the ability of resistant mosquitoes to withstand
the excito-repellency effect of LLINs and penetrate impregnated bed nets thus, feed on humans raising concerns about
the duration of LLIN effective life. This high passage rate
recorded with Net Type 4 could be due to the decreasing repellent effect of permethrin, which was previously pointed out with
An. gambiae9.
But the highest mortality recorded with this Net Type 4
[An. funestus s.s. (71.25%) and An. coluzzii (45.91%)] is in line
with what was reported by Darriet et al.,47 showing that bed nets
treated with pyrethroids (permethrin and deltamethrin) remained
effective even in areas where An. gambiae s.s. are resistant to
these insecticides. This paradox is due to the behavioral changes
in the resistant mosquitoes; they were less repelled by the
insecticide, remained on the only pyrethroid-treated material for
longer periods, and thus received a higher dose of insecticide
leading to the death of the mosquito48.
permethrin resistant An. gambiae s.l. which showed that 100%
blood feeding inhibition with unwashed PermaNet 3.0, a
deltamethrin-PBO combination net27. Blood feeding inhibition
rates of resistant Anopheles mosquitoes with Net Type 4 were
significantly lower than Net Type 3(χ2 = 7.793;P = 0.0052),
suggesting the decreased potency of this standard Net Type 4 in
area where pyrethroids resistance is already spread9,21,22,49,50 but
overall, the effectiveness of LLINs treated only with insecticides
seems to be significantly lower compared to that of nets treated
with insecticides and PBO.
Efficacy against multi-resistant mosquito strains
Experimental huts evaluations conducted in this study showed
that all treated nets induced significant exophily rate ranged from
50 to 73% relatively compared to untreated net at 30% against
susceptible An. gambiae Kisumu. This result indicates that the
current pyrethroid only treated nets continue to exert strong
repulsive action on the Anopheles susceptible strain. There was
no significant difference between the low induced exophily rates
in An. funestus s.s. and An. coluzzii in the hut containing Net
Type 2 and Type 1 (χ2 = 1.837; P = 0.1753). This observation
could be due to the naturally high endophilic behavior of this
Anopheles species51. Contrary, there was a significant difference
between induced exiting rates of Net Type 4 and Type 3 against
resistant An. coluzzii.
Respectively more than 40% and 60% of An. funestus s.s.
and An. coluzzii survived in the hut with Net Type 2. When
resistant Anopheles were released in the hut containing a
combined deltamethrin-PBO net Type 1, less than 15% survived.
However, almost all susceptible mosquito dead to the exposure
with this net. Low lethal effect of Net Type 4 was observed with
the resistant strain of Anopheles compared to Net Type 3. This
result correlates with those reported by Malima et al.52 where the
recorded mortality of An. funestus was 71.6% against nets treated
with permethrin only. Survival rates of these mosquitoes in the
huts suggest that the protective nature of currently used net
Type 4 in Benin is compromised, as previously reported53. These
semi-field controlled experiments confirmed the results from
laboratory phase I evaluations and displays faith in combined
pyrethroids-PBO nets, despite the multiple resistance mechanisms present in these mosquito species4,19,20,21,54–56. However, it is
necessary to further investigate the impact of these multiple
mechanisms on the efficacy of nets treated with pyrethroids only
against An. funestus s.s.
However, the mortality rates in both resistant Anopheles
populations were significantly high in presence of Net Type 3
than Type 4 (χ2 =12.048; P = 0.0005). With Net Type 3 in which
PBO is applied, only about 4% mosquito was able to survive
the exposure. Interestingly, the same observations were found for
Net Type 2 and Type 1 against both resistant mosquito populations. This test gave a slightly synergistic action of PBO on the
roof of Net Type 1 with a mortality of 98% for An. funestus s.s.
compared to the side net (92% mortality).
A combination of the synergist PBO to pyrethroids made
treated nets more efficient as PBO acted both as a metabolic
enzyme inhibitor and as an adjuvant through its effect on
enhanced cuticular penetration of deltamethrin57. The fact that
these new generation nets (Type 1 and Type 3) were able to inhibit
blood feeding more than current nets (Type 2 and Type 4), could
suggest their capability to confer high personal protection
against resistant mosquito biting.
Concerning the control untreated Net Type 5, blood feeding was
inhibited more by both Net Type 2 and Type 1 against resistant An. funestus s.s. This is similar to previous studies with
Studies conducted in Benin and other African countries
showed a loss efficacy of pyrethroids treated net against
An. gambiae9,23,50,58. This research has demonstrated that the
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Wellcome Open Research 2018, 3:71 Last updated: 06 JUL 2018
efficacy of a combined pyrethroids-PBO nets on resistant
malaria vector populations could be a promising strategy against
pyrethroid resistance populations of Anopheles as previously
highlighted20,25,27,28,43,59,60. This study further confirms a role of
oxidases in pyrethroids resistance of An. funestus and the need
to develop nets combining pyrethroid and synergist against
pyrethroid resistant malaria vectors25,33,40,61. Nevertheless, several
other studies have been conducted on the insecticide resistance
management of Anopheles especially An. gambiae, using nonpyrethroid insecticides alone or in mixture of pyrethroids62–67.
These studies also provided relatively good pointers for
management of resistant mosquitoes but the problems with nonpyrethroid ingredients are human toxicity and their irritant
effect62,68. A more recent study conducted by Malima et al.69 in
an area where An. funestus is resistant to pyrethroid, showed that
even when non-pyrethroid insecticide-treated nets with durable
wall lining (ITWL) are used, this cannot guarantee up to 50%
protection against resistant An. funestus.
Conclusion
Pyrethroid resistance in the major malaria vectors An. funestus
s.s. and An. coluzzii in Kpomè is high, and is likely to limit the
impact of currently used LLINs. This study showed that the use of
new generation bed nets could provide additional protection and
reduce malaria burden in endemic environments. This study is of
importance to Malaria control programs for improved control of
pyrethroids resistant malaria vectors in Benin.
Ethical considerations
Approval was obtained from the ethics review boards of the
International Institute of Tropical Agriculture (IITA) ref.PJ/
CC5339. All volunteers recruited to sleep in the experimental
huts gave written and verbal consents. Chemoprophylaxis was
provided to volunteer prior to the hut studies.
Data availability
All data generated and analyzed during this study will be included
in the published article. Raw data are available from Open
Science Framework. Dataset 1: Experimental huts trial of the
efficacy of pyrethroids/piperonyl butoxide (PBO) nets treatments
for controlling multi-resistant populations of Anopheles funestus s.s. in Kpomè, Southern Benin. http://doi.org/10.17605/OSF.
IO/3YRMS.
The data is available under a CC0 1.0 Universal License
Competing interests
No competing interests were disclosed.
Grant information
The study was supported by Wellcome Trust [099864] to RD.
The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Acknowledgements
We are grateful to the Liverpool School of Tropical Medicine for
providing new LLINs used in this study. We are also grateful to
Mr. Claude Gande and Murielle Soglo for their supportive actions
and contributions to this study. Our deep appreciation goes to
Kpomè community, especially our volunteers for their assistance to
successfully conduct the experimental hut activities.
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Open Peer Review
Current Referee Status:
Version 1
Referee Report 06 July 2018
doi:10.21956/wellcomeopenres.15884.r33304
Emmanuel Elanga Ndille
Department of Medical Entomology, Centre for Research on Infectious Diseases (CRID), Yaoundé,
Cameroon
This manuscript describe results of experimental experiences performed to assess the efficacy of of
pyrethroids/piperonyl butoxide (PBO) net treatments for controlling multi-resistant populations of
Anopheles funestus s.s. The study is highly interesting as deals with development of strategies of malaria
control in the context of high resistance to pyrethroid observed in main vectors. The manuscript is well
written and presented, however there are very few minor concerns I would like the authors to take into
consideration so that their work will be more efficient.
Comments:
1. The title could be modified to also take into account An. coluzzi as all the results of the manuscript
are also presenting data from this species
2. 2nd paragraph of the introduction, in the 5th sentence : “resistance to insecticide ofone of ………”
replace “ofone” by “of on”
3. In the section “Methods”, on the part “WHO cone tests with the nets” authors must keep the same
references format. Indeed they should replace “(Methods in Anopheles Research, 2010)” by a
corresponding number to be uniform in all the manuscript.
4. In the text (Release and recapture experiment) the sentence “Target entomological parameters
monitored included: included exiting, blood-feeding inhibition and mortality” could be change by “
target entomological parameters monitored inclided : (i) insecticide-induced exiting, i.e. the
proportion of mosquitoes found in hut verandahs relative to control huts; (ii) blood-feeding
inhibition, i.e. the proportional reduction in blood-feeding relative to untreated nets; and (iii)
mortality, the proportion of mosquitoes killed (immediate plus delayed)”.
Is the work clearly and accurately presented and does it cite the current literature?
Yes
Is the study design appropriate and is the work technically sound?
Yes
Are sufficient details of methods and analysis provided to allow replication by others?
Yes
Page 17 of 18
Wellcome Open Research 2018, 3:71 Last updated: 06 JUL 2018
Yes
If applicable, is the statistical analysis and its interpretation appropriate?
Yes
Are all the source data underlying the results available to ensure full reproducibility?
Yes
Are the conclusions drawn adequately supported by the results?
Yes
Competing Interests: No competing interests were disclosed.
I have read this submission. I believe that I have an appropriate level of expertise to confirm that
it is of an acceptable scientific standard.
Page 18 of 18