ArticlePDF Available

Pilot study on the combination of an organophosphate-based insecticide paint and pyrethroid-treated long lasting nets against pyrethroid resistant malaria vectors in Burkina Faso

Authors:
  • Research Institute of Health Sciences/ Centre Muraz
  • UPB/IRSS/Centre Muraz, Bobo-Dioulasso
  • Institut de recherche en Sciences de la Santé (IRSS)/Centre Muraz, Bobo-Dioulasso, Burkina Faso

Figures

Content may be subject to copyright.
Acta
Tropica
148
(2015)
162–169
Contents
lists
available
at
ScienceDirect
Acta
Tropica
jo
u
r
n
al
homep
age:
www.elsevier.com/locate/actatropica
Pilot
study
on
the
combination
of
an
organophosphate-based
insecticide
paint
and
pyrethroid-treated
long
lasting
nets
against
pyrethroid
resistant
malaria
vectors
in
Burkina
Faso
Beatriz
Mosqueiraa,,
Dieudonné
D.
Somab,
Moussa
Namountougoub,
Serge
Podab,
Abdoulaye
Diabatéb,
Ouari
Alib,
Florence
Fournetc,
Thierry
Baldetd,
Pierre
Carnevalee,
Roch
K.
Dabiréb,
Santiago
Mas-Comaa
aDepartamento
de
Parasitologia,
Facultad
de
Farmacia,
Universidad
de
Valencia,
Av
Vicent
Andrés
Estellés
s/n,
Burjassot,
46100
Valencia,
Spain
bInstitut
de
Recherche
en
Sciences
de
la
Santé
(IRSS)/Centre
Muraz,
Bobo-Dioulasso
01
BP
545,
Burkina
Faso
cInstitut
de
Recherche
pour
le
Développement
(IRD),
BP
64501,
34394
Montpellier
Cedex
5,
France
dCirad,
UMR15
CMAEE;
INRA,
UMR1309
CMAEE,
Montpellier,
France
eImmeuble
Le
Majoral,
Avenue
de
la
Tramontane,
34420
Portiragnes
Plage,
France
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
15
January
2015
Received
in
revised
form
18
March
2015
Accepted
11
April
2015
Available
online
7
May
2015
Keywords:
Inesfly
5A
IGRTM
LLINs
Pyrethroid
resistance
Anopheles
coluzzii
Malaria
control
a
b
s
t
r
a
c
t
A
pilot
study
to
test
the
efficacy
of
combining
an
organophosphate-based
insecticide
paint
and
pyrethroid-treated
Long
Lasting
Insecticide
Treated
Nets
(LLINs)
against
pyrethroid-resistant
malaria
vector
mosquitoes
was
performed
in
a
real
village
setting
in
Burkina
Faso.
Paint
Inesfly
5A
IGRTM,
com-
prised
of
two
organophosphates
(OPs)
and
an
Insect
Growth
Regulator
(IGR),
was
tested
in
combination
with
pyrethroid-treated
LLINs.
Efficacy
was
assessed
in
terms
of
mortality
for
12
months
using
Early
Morning
Collections
of
malaria
vectors
and
30-minute
WHO
bioassays.
Resistance
to
pyrethroids
and
OPs
was
assessed
by
detecting
the
frequency
of
L1014F
and
L1014S
kdr
mutations
and
Ace-1RG119S
muta-
tion,
respectively.
Blood
meal
origin
was
identified
using
a
direct
enzyme-linked
immunosorbent
assay
(ELISA).
The
combination
of
Inesfly
5A
IGRTM and
LLINs
was
effective
in
killing
99.9–100%
of
malaria
vec-
tor
populations
for
6
months
regardless
of
the
dose
and
volume
treated.
After
12
months,
mortality
rates
decreased
to
69.5–82.2%.
The
highest
mortality
rates
observed
in
houses
treated
with
2
layers
of
insec-
ticide
paint
and
a
larger
volume.
WHO
bioassays
supported
these
results:
mortalities
were
98.8–100%
for
6
months
and
decreased
after
12
months
to
81.7–97.0%.
Mortality
rates
in
control
houses
with
LLINs
were
low.
Collected
malaria
vectors
consisted
exclusively
of
Anopheles
coluzzii
and
were
resistant
to
pyrethroids,
with
a
L1014
kdr
mutation
frequency
ranging
from
60
to
98%
through
the
study.
About
58%
of
An.
coluzzii
collected
inside
houses
had
bloodfed
on
non-human
animals.
Combining
Inesfly
5A
IGRTM
and
LLINs
yielded
a
one
year
killing
efficacy
against
An.
coluzzii
highly
resistant
to
pyrethroids
but
sus-
ceptible
to
OPs
that
exhibited
an
anthropo-zoophilic
behaviour
in
the
study
area.
The
results
obtained
in
a
real
setting
supported
previous
work
performed
in
experimental
huts
and
underscore
the
need
to
study
the
impact
that
this
novel
strategy
may
have
on
clinical
malaria
and
malaria
exposure
in
children
in
a
similar
area
of
high
pyrethroid
resistance
in
South-Western
Burkina
Faso.
©
2015
Elsevier
B.V.
All
rights
reserved.
1.
Background
Malaria
transmission
occurs
in
97
countries,
putting
about
3.4
billion
people
at
risk
(WHO,
2013).
In
Africa,
it
is
estimated
that
Corresponding
author.
Tel.:
+34
96
354
4298.
E-mail
addresses:
bmosqueira@yahoo.com
(B.
Mosqueira),
dieusoma@yahoo.fr
(D.D.
Soma),
namountougou
d@yahoo.fr
(M.
Namountougou),
sergepoda71@yahoo.fr
(S.
Poda),
a
diabate@hotmail.com
(A.
Diabaté),
ouari
ali@yahoo.fr
(O.
Ali),
florence.fournet@ird.fr
(F.
Fournet),
thierry.baldet@cirad.fr
(T.
Baldet),
pjcarnevale2001@yahoo.fr
(P.
Carnevale),
dabire
roch@hotmail.com
(R.K.
Dabiré),
s.mas.coma@uv.es
(S.
Mas-Coma).
in
2012
alone,
about
165
million
people
suffered
from
malaria
and
about
562,000
people
died
from
causes
attributed
to
malaria.
About
86%
of
those
deaths
were
among
children
under
5
years
of
age
(WHO,
2013).
In
addition
to
the
devastating
impact
on
human
health,
malaria
also
imposes
an
enormous
economic
bur-
den,
estimated
at
1.3%
of
economic
growth
per
year
in
sub
Saharan
Africa
(WHO,
2013).
Primary
prevention
of
malaria
on
a
large
scale
is
essentially
achieved
through
vector
control
using
mostly
Long
Lasting
Insecticide
Treated
Nets
(LLINs)
and,
to
a
lesser
extent,
Indoor
Residual
Spraying
(IRS)
(WHO,
2013).
Between
2008
and
2010,
254
million
LLINs
were
supplied
to
countries
in
sub-Saharan
Africa
(WHO,
2013).
All
currently
recommended
LLINs
are
treated
http://dx.doi.org/10.1016/j.actatropica.2015.04.010
0001-706X/©
2015
Elsevier
B.V.
All
rights
reserved.
B.
Mosqueira
et
al.
/
Acta
Tropica
148
(2015)
162–169
163
with
pyrethroids.
Protection
using
IRS
reached
58
million
people
in
Africa
representing
8%
of
the
global
population
at
risk
in
2012
as
reported
by
National
Malaria
Control
Programmes
(WHO,
2013).
In
2009,
pyrethroids
were
estimated
to
account
for
about
75%
of
IRS
coverage,
while
DDT
was
the
second
most
widely
used
insecticide;
carbamates
and
organophosphates
(OPs)
represented
only
small
percentages
of
global
usage
(WHO,
2012).
LLINS
and
IRS
remain
efficient
and
cost-effective
tools
for
malaria
control
across
a
large
number
of
settings
(Lengeler
and
Sharp,
2003).
The
raised
levels
of
pyrethroid
resistance
among
malaria
mosquito
vectors
(Chandre
et
al.,
1999;
Diabaté
et
al.,
2004;
Dabiré
et
al.,
2012)
and
subsequent
reports
on
reduced
efficacy
of
pyrethroid-based
vector
control
tools
(Toé
et
al.,
2014)
are
a
source
of
concern.
However,
there
is
yet
no
final
consensus
on
whether
kdr
based
modifica-
tions
reduce
significantly
the
efficacy
of
insecticides
operationally
speaking
(Briët
et
al.,
2013;
Hemingway,
2014).
Furthermore,
the
use
of
LLINs
is
advocated
because,
when
well
used
and
intact,
it
will
help
reduce
bloodfeeding
thus
increasing
individual
protection
(Trape
et
al.,
2014).
Apart
from
the
issue
of
pyrethroid
resistance,
there
are
operational
obstacles
surrounding
LLINs
(Toé
et
al.,
2009;
MCHIP/USAID/PNLP,
2013)
and
IRS
(Najera
and
Zaim,
2001)
poten-
tially
rendering
these
tools
less
operationally
effective
in
protecting
againt
malaria.
To
summarize,
LLINs
and
IRS
remain
the
corner-
stone
in
malaria
vector
control
but
there
is
a
growing
need
to
find
alternative
malaria
vector
control
strategies
that
can
be
added
to
the
list
of
tools
to
choose
from
(Beier
et
al.,
2008).
The
National
Malaria
Control
Programme
(“Programme
National
de
Lutte
contre
le
Paludisme”—PNLP)
of
the
Ministry
of
Health
in
Burkina
Faso,
dis-
tributed
more
than
8
million
nets
were
to
a
population
of
around
16
million
targeted
to
the
population
at
risk,
children
under
5
years
old
and
pregnant
women
(MCHIP/USAID/PNLP,
2013).
Thus,
rather
than
departing
from
LLINs,
the
strategy
implemented
in
this
study
enforced
their
use,
in
line
with
the
PLNP
efforts.
The
LLINs
in
this
study
were
PermaNet®2.0
that
had
been
dis-
tributed
in
the
area
by
the
PNLP
in
2013
(MCHIP/USAID/PNLP,
2013)
and
were
confirmed
by
the
team
to
be
well
used
by
the
population
and
intact.
Insecticide
paint
Inesfly
5A
IGRTM is
a
“cocktail”
consist-
ing
of
two
OPs,
chlorpyriphos
and
diazinon,
and
an
insect
growth
regulator
(IGR),
pyriproxyfen.
The
paint
was
applied
on
plastic
sheetings
with
no
need
of
special
equipment
and
placed
in
real
houses,
in
a
village
in
the
Kou
Valley,
South-Western
Burkina
Faso,
where
there
is
high
pyrethroid
resistance
among
malaria
mosquito
vectors
per
the
high
frequency
of
the
L1014F
kdr
mutation
(Dabiré
et
al.,
2008,
2009).
Toxicology
studies
performed
so
far
support
the
paint’s
safety
(Spanish
Ministry
of
Health
and
Consumer
Affairs
(SMHCA),
1996;
International
Center
of
Training
and
Medical
Investigations
(ICTM),
2003;
National
Center
of
Tropical
Diseases,
2004).
Inesfly
5A
IGRTM has
been
evaluated
previously
under
exper-
imental
conditions
in
South
America
against
the
Chagas
disease
vector
Triatoma
infestans
(Dias
and
Jemmio,
2008;
Amelotti
et
al.,
2009;
Maloney
et
al.,
2013).
Tests
were
also
performed
following
the
WHO
Pesticide
Eval-
uation
Scheme
(WHOPES)
procedures
(WHO,
1996)
on
Inesfly
5A
IGRTM in
the
laboratory
(Phase
I),
against
100%
OP-resistant
Culex
quinquefasciatus
(Mosqueira
et
al.,
2010a),
and
in
experimental
houses
in
the
field
(Phase
II),
against
wild
pyrethroid-resistant
populations
of
the
main
malaria
vector,
Anopheles
gambiae,
and
pest
mosquito,
Cx.
quinquefasciatus
in
Benin
(Mosqueira
et
al.,
2010b).
In
the
laboratory,
one
year
after
treatment
delayed
mortality
was
93–100%
even
against
OP-resistant
females
on
non-porous
surfaces
like
hard
plastic
or
softwood
(Mosqueira
et
al.,
2010a).
Pyriprox-
ifen
was
added
to
the
paint
to
confer
and
additional
angle
of
attack
against
mosquito
females
once
the
OP
effect
diminishes
over
time.
The
effect
of
pyriproxyfen
has
been
studied
in
the
lab,
where
it
was
shown
that
pyriproxyfen
had
an
effect
on
the
fecundity,
fertility
and
adult
emergence
of
exposed
adult
females
once
the
lethal
effect
of
OPs
diminished
over
time
even
against
OP-resistant
mosquitoes
(Mosqueira
et
al.,
2010a).
In
the
field,
on
porous
surfaces
made
of
cement,
mortality
rates
were
90–100%
against
pyrethroid-resistant
mosquito
populations
six
months
after
treatment.
Nine
months
after
treatment,
mortality
rates
in
huts
treated
with
two
layers
was
still
about
90–93%
against
An.
gambiae
and
55%
against
Cx.
quinquefasciatus,
both
resistant
to
pyrethroids
(Mosqueira
et
al.,
2010b).
In
addition,
a
high
spatial
long
term
mortality
(96–100%)
was
obtained
for
12
months
in
the
field
on
mosquitoes
that
were
kept
at
distances
of
one
meter
overnight,
never
entering
in
direct
contact
with
treated
surfaces
(Mosqueira
et
al.,
2010b,
2013).
The
objective
of
the
present
study
was
to
assess
the
efficacy
of
Paint
Inesfly
5A
IGRTM in
combination
with
pyrethroid-treated
LLINs
in
real-life
houses
in
a
village
setting.
This
pilot
study
sup-
ported
the
previous
Phase
II
studies
performed
in
experimental
huts
(Mosqueira
et
al.,
2010b)
and
provided
useful
information
on
the
method
to
apply
the
paint,
perform
the
mosquito
collections
and
mosquito
populations,
in
preparation
for
the
forthcoming
large
scale
Phase
III
cluster
randomized
controlled
evaluation
to
assess
the
impact
of
this
combination
strategy
on
the
incidence
of
clini-
cal
malaria
and
malaria
exposure
in
children
aged
from
6
months
to
14
years
old
in
a
similar
area
of
high
pyrethroid
resistance
in
South-Western
Burkina
Faso.
2.
Methods
2.1.
Study
site
and
mosquitoes
The
study
was
conducted
in
the
Kou
Valley,
a
rice
growing
area
in
South-Western
Burkina
Faso,
West
Africa.
It
is
located
at
30
km
in
the
North
of
Bobo-Dioulasso
(lat.
112314N
and
long.
42442W)
and
is
composed
of
7
villages
with
a
total
of
4470
habitants
in
2013.
The
study
was
conducted
specifically
at
the
VK1
village
(Fig.
1).
Irrigation
has
existed
in
this
area
since
1972,
and
is
now
semi-permanent
with
two
crops
grown
per
year:
from
February
to
June
during
the
dry
season
and
from
July
to
November
during
the
rainy
season.
The
study
area
was
chosen
because
of
its
high
malaria
transmission,
its
high
frequency
of
the
L1014F
kdr
muta-
tion,
rendering
local
malaria
vector
populations
highly
resistant
to
pyrethroids
and
DDT
(Dabiré
et
al.,
2008,
2009).
Both
An.
gambiae
(former
An.
gambiae
S
form)
and
Anopheles
coluzzii
(former
An.
gam-
biae
M
form)
coexist
in
sympatry
in
the
study
area,
but
An.
coluzzii
is
preponderant
within
the
rice
field
habitats.
As
part
of
the
neces-
sary
background
information,
the
exact
species
were
determined
molecularly
(Santolamazza
et
al.,
2008).
The
study
was
performed
continuously
for
six
months,
from
June
to
December
2013,
and
then
again
in
June
2014,
12
months
after
treatment.
2.2.
Insecticide
paint
and
LLINs
Inesfly
5A
IGRTM contains
two
organophosphates,
chlorpyriphos
(1.5%)
and
diazinon
(1.5%),
and
an
insect
growth
regulator
(IGR),
pyriproxyfen
(0.063%),
as
active
ingredients.
The
formulation
is
vinyl
white-coloured
paint
with
an
aqueous
base,
with
the
active
ingredients
residing
within
CaCO3
and
resin
microcapsules,
allow-
ing
a
gradual
release
of
active
ingredients.
Microcapsules
range
from
one
to
several
hundred
micrometers
in
size.
The
paint
was
applied
on
plastic
sheetings
with
no
need
of
special
equipment,
just
a
regular
brush
and
gloves.
Polypropylene
plastic
sheeting
was
bought
at
the
local
market
and
consisted
of
big
plastic
rolls
cut
and
fit
into
the
study
houses.
The
plastic
sheeting
was
used
to
homog-
enize
test
surfaces
as
some
houses
were
made
of
adobe
and
some
of
cement.
The
plastic
sheeting
was
then
placed
on
the
superior
two
thirds
of
interior
house
walls
and
ceilings.
The
lower
part
of
all
walls
was
left
untreated
for
up
to
1
m
for
all
houses
to
reduce
direct
164
B.
Mosqueira
et
al.
/
Acta
Tropica
148
(2015)
162–169
Fig.
1.
Location
of
VK1
at
Kou
Valley
in
South-Western
Burkina
Faso.
exposure
to
both,
babies
and
young
toddlers.
The
LLINs
in
this
study
were
PermaNet®
2.0,
made
of
multifilament
polyester
netting
(100
denier)
factory
impregnated
with
deltamethrin
at
55
mg/m2
in
a
wash-resistant
binder
system
that
had
been
distributed
locally
by
the
PNLP
in
2013.
All
nets
were
checked
prior
to
the
study
and
were
found
to
be
intact
and
correctly
used
by
the
owners.
2.3.
Early
morning
collections
(EMCs)
Inesfly
5A
IGRTM was
evaluated
in
14
real-life
village
houses
at
VK1.
The
14
houses
at
VK1
were
chosen
based
on
owners’
wish
to
participate
and
equivalence
in
dimensions.
The
control
houses
consisted
on
plastic
sheetings
with
no
paint,
but
with
intact
LLINs.
For
the
treated
houses,
paint
was
applied
on
plastic
sheetings
with
one
or
two
layers
of
insecticide
paint
at
1
kg
commercial
prod-
uct/6
m2,
that
is
0.51
g
a.i.
per
m2.
Huts
treated
with
two
layers
had
the
first
layer
diluted
in
20%
water
following
recommendations
of
the
manufacturer.
The
ceilings
of
certain
houses
were
also
covered
with
painted
plastic
sheeting
per
the
configuration
below.
The
dif-
ferent
configurations
were
treated
in
duplicate,
in
two
houses
each.
Experimental
hut
studies
commonly
use
one
single
hut
per
config-
uration
(WHO,
2006).
However,
because
this
study
was
done
using
real
houses
that
were
similar
but
not
identical
to
each
other,
we
used
two
houses
per
configuration,
collected
for
several
nights
in
a
row
and
performed
the
week
of
blank
collections
prior
to
treat-
ment
and
rotated
the
volunteers.
Configurations
were
designed
to
allow
the
evaluation
of
a
potential
volume
effect
and
dose
effect.
(1)
2
houses
=
control
sheeting
with
no
paint
+
LLIN.
(2)
2
houses
=
regular
paint
1
layer
+
insecticide
paint
1
layer
on
walls
only
+
LLIN.
(3)
2
houses
=
regular
paint
1
layer
+
insecticide
paint
1
layer
on
walls
and
ceiling
+
LLIN.
(4)
2
houses
=
insecticide
paint:
1
layer
on
walls
only
+
LLIN.
(5)
2
houses
=
insecticide
paint:
1
layer
on
walls
and
ceiling
+
LLIN.
(6)
2
houses
=
insecticide
paint:
2
layers
on
walls
only
+
LLIN.
(7)
2
houses
=
insecticide
paint:
2
layers
on
walls
and
ceiling
+
LLIN.
Mortality
was
the
entomological
indicator
evaluated
during
this
pilot
study
under
real
conditions.
As
there
was
no
verandah,
the
exito-repellent
effect
generally
assessed
in
Phase
II
WHOPES
protocols,
could
not
be
implemented.
Similarly,
although
house
dimensions
were
similar,
the
number
and
size
of
openings
(win-
dows
and
doors)
were
too
different
to
reliably
evaluate
the
deterrent
effect
and
bloodfeeding
inhibition.
Before
any
treated
sheetings
were
applied,
mosquito
collections
took
place
for
1
full
week
just
with
LLINs
to
ensure
there
that
there
was
no
difference
between
houses
in
attractiveness
to
mosquitoes.
Between
June
and
December
2013
and
again
in
June
2014,
mosquito
collections
were
performed
nightly
at
VK1.
The
study
was
approved
by
the
Ethics
Committee
of
“Institut
de
Recherche
en
Sciences
de
la
Santé”
(IRSS)
at
Centre
Muraz.
Sixteen
volunteers
18
years
old
or
older
were
recruited
from
the
population
at
VK1–2
volunteers
served
as
back
ups
in
case
it
was
needed.
After
being
informed
about
the
study
and
discussing
it,
these
volunteers
provided
an
informed
consent
in
writing
or
with
a
finger
print
if
illiterate.
The
volunteers
received
training
on
mosquito
collection
procedures.
At
the
first
suspicion
of
malaria,
volunteers
were
provided
with
the
curative
treatment
recommended
by
the
PNLP
in
Burkina
Faso.
Fur-
thermore,
all
houses
were
checked
and
had
intact
well
used
LLINs.
Volunteers
rotated
houses
each
night
to
avoid
bias
while
avoiding
contamination
between
houses.
The
lower
part
of
doors
were
cov-
ered
with
cloth
to
reduce
the
number
of
scavengers
from
entering
houses.
Houses
were
broomed
every
morning
and
every
evening
to
remove
scavengers
that
made
it
in
through
other
openings.
There
was
one
volunteer
sleeping
per
house.
Mosquito
collections
were
performed
to
assess
mortality
rates.
Volunteers
would
enter
their
houses
at
18:00
h,
one
volunteer
per
house,
and
sleep
under
LLINs
until
5:30
h,
when
they
would
be
awaken
to
close
the
windows
(that
had
been
left
open
during
the
night
as
it
is
commonly
done
in
the
area).
Once
windows
were
closed
at
5:30
h,
the
volunteer
B.
Mosqueira
et
al.
/
Acta
Tropica
148
(2015)
162–169
165
proceeded
to
collect
mosquitoes
within
the
house.
After
classifying
mosquito
females
as
dead
or
alive,
they
were
put
in
observation
for
delayed
mortality
assessments
after
24
h.
All
mosquitoes
were
then
conserved
in
silica
gel
at
20 C
to
identify
the
species,
resistance
status
and
source
of
blood
meal.
2.4.
Residual
efficacy
tests
Thirty-minute
standard
WHO
cone
bioassays
(WHO,
1998)
were
carried
out
using
2–4
days
old
unfed
females
of
An.
gambiae
Kisumu,
a
reference
strain
susceptible
to
all
insecticides
reared
at
the
IRSS/Centre
Muraz
insectarium.
The
local
population
identified
molecularly
as
An.
coluzzii
and
resistant
to
pyrethroids
was
reared
at
the
insectarium
from
field
caught
larvae
to
the
adult
stage
and
was
also
tested
in
parallel
to
An.
gambiae
Kisumu.
For
each
house,
10
females
were
introduced
in
5
cones
placed
on
five
sides
of
the
house
(4
walls
and
ceiling)
for
30
min.
Cones
were
not
placed
on
LLINs.
Delayed
mortality
was
observed
24
h
later.
Tests
were
performed
monthly
at
T0,
T1,
T3,
T6
and
T12
after
treatment.
2.5.
Molecular
analysis
on
resistance
The
detection
of
kdr
resistance
genes
was
performed
following
protocols
developed
for
the
L1014F
kdr
mutation
(Martinez-Torres
et
al.,
1998),
for
the
L1014S
kdr
mutation
(Ranson
et
al.,
2000),
as
well
as
the
detection
of
the
Ace-1RG119S
mutation
(Weill
et
al.,
2004).
Testing
took
place
each
month
for
5
months
after
treat-
ment
on
An.
coluzzii
females
collected
in
control
houses
and
houses
treated
with
1
or
2
layers
of
insecticide
paint
on
walls
and
ceiling.
2.6.
Determination
of
blood
meal
source
Blood
meal
identification
was
performed
using
a
direct
enzyme-
linked
immunosorbent
assay
(ELISA)
(Beier
et
al.,
1988).
The
choice
of
antibodies
tested
was
based
on
the
animals
that
are
more
fre-
quent
in
the
study
area.
Six
antibodies
were
tested:
human,
dog,
sheep,
donkey,
cattle
and
pig.
These
antibodies,
marked
with
per-
oxidase,
were
kept
at
+4 C.
Bloodfed
Anopheles
females
collected
during
EMCs
from
June
to
December
2013
in
control
houses
and
houses
treated
with
1
or
2
layers
of
insecticide
paint
on
walls
and
ceiling
were
tested.
A
total
of
425
females
identified
molecularly
as
An.
coluzzii
were
tested
from
each
of
those
3
configurations
(>140
per
configuration)
to
determine
the
source
of
the
blood
meal.
2.7.
Statistical
analysis
Results
on
mortality
were
compiled
and
analyzed
using
Epi-
Info
Version
6
to
test
for
any
significant
difference
in
mortality
rates
between
the
different
configurations
via
Chi
square
tests.
A
95%
confidence
interval
was
applied.
When
mortality
rates
in
con-
trol
huts
were
between
5
and
20%
Abbott’s
mortality
correction
formula
was
applied.
Because
bioassay
tests
are
subject
to
varia-
tions,
a
99%
confidence
interval
was
applied.
The
allelic
frequency
of
each
mutation
(kdr
and
ace-1R)
was
calculated
using
the
formula
F(R)
=
(2RR
+
RS)/2n
where
n
is
the
total
sample
size,
using
GenePop
version
4.
3.
Results
3.1.
Early
morning
collections
(EMC)
No
difference
in
house
attractiveness
was
found
prior
to
treat-
ment.
An.
coluzzii
(former
An.
gambiae
form
M)
was
the
only
An.
gambiae
s.l.
species
present
in
the
study
area
as
established
from
the
molecular
analysis
performed
during
the
study.
Between
June
Table
1
Mortality
rates
on
wild
populations
of
Anopheles
coluzzii
at
VK1
using
EMCs.
Aver-
ages
taken
for
each
configuration,
2
houses
per
configuration.
C
=
control
with
LLINs
only;
RP
=
regular
Paint;
IP
=
insecticide
paint;
T
=
time
in
months
since
treatment.
EMCs
=
early
morning
collections.
Numbers
in
the
same
column
sharing
a
letter
superscript
do
not
differ
significantly
(p
>
0.05).
%
Mortality
in
Anopheles
coluzzii
collected
via
EMCs
T1
T3
T6
T12
C
(LLINs)
9.5a5.2a8.9a7.6a
RP/1
layer
+
IP/1
layer
walls
+
LLINs
100b100b100b78.6b
RP/1
layer
+
IP/1
layer
walls
+
ceiling
+
LLINs
100b100b100b69.5b
IP/1
layer
walls
+
LLINs 100b100b100b78.9b
IP/1
layer
walls
+
ceiling
+
LLINs
100b100b100b79.9b
IP/2
layers
walls
+
LLINs
100b99.9b100b78.5b
IP/2
layers
walls
+
ceiling
+
LLINs
100b100b100b82.2b
and
December
2013
and
June
2014,
a
total
of
3903
females
belong-
ing
to
the
An.
gambiae
complex
identified
molecularly
as
An.
coluzzii,
were
collected
in
all
houses
combined.
Full
collections
started
one
month
after
treatment
(Table
1).
For
the
first
6
months,
the
mortal-
ity
rates
observed
in
houses
treated
with
the
insecticide
paint
were
97–100%.
Globally,
6
months
after
treatment,
all
houses
treated
with
the
insecticide
paint,
with
1
or
2
layers,
on
walls
or
on
walls
and
ceiling,
presented
100%
mortality
rates
against
wild
popula-
tions
of
An.
coluzzii
whether
they
were
bloodfed
or
not
and
were
statistically
significantly
different
from
control
(p
<
0.001).
By
T12,
mortalities
were
still
high
and
significantly
different
from
control
(p
<
0.001),
but
rates
had
slightly
decreased
to
69.5–82.2%.
The
high-
est
mortality
rates
12
months
after
treatment
were
observed
in
houses
treated
with
2
layers
of
insecticide
paint
and
a
larger
volume
(82.2%).
No
statistically
significant
differences
were
found
between
treated
houses
at
T12.
Mortality
rates
observed
in
control
houses
with
no
insecticide
paint
but
with
LLINs
ranged
from
5.2
to
9.5%,
throughout
the
study
(Table
1).
3.2.
Residual
efficacy
tests
Thirty-minute
standard
WHO
cone
bioassays
on
An.
gambiae
“Kisumu”
and
local
populations
of
An.
coluzzii
from
VK1,
yielded
mortality
rates
of
98–100%
in
all
houses
treated
with
insecticide
paint
(Table
2)
regardless
of
the
configuration.
Mortality
in
control
houses
was
lower
and
significantly
different
from
treated
houses,
but
because
mortality
was
over
5%
(but
always
less
than
20%),
the
Abbott
formula
was
applied.
Mortality
rates
were
100%
at
T6
against
both
An.
gambiae
“Kisumu”
and
local
populations
of
An.
coluzzii
from
VK1,
in
all
treated
houses.
Mortality
rates
at
T12
were
still
98–100%
in
all
houses
against
An.
gambiae
“Kisumu”.
In
the
case
of
the
local
An.
coluzzii
from
VK1,
12
months
after
treatment
mortality
rates
were
97%
in
houses
treated
with
2
layers
of
insecticide
paint
on
walls
and
ceiling,
but
slightly
lower
mortalities
were
observed
in
the
other
configurations.
These
differences
were
not
statistically
significant
(p
>
0.05).
Mortality
rates
observed
in
control
houses
with
LLINs
only
ranged
from
1.7%
to
10.9%
(Table
2).
Again,
cones
were
only
placed
on
walls
and
ceiling,
not
on
LLINs.
3.3.
Molecular
Analysis
on
resistance
3.3.1.
Allelic
frequency
of
the
L1014F
and
L1014S
kdr
mutations
All
houses
contained
pyrethroid
treated
LLINs.
Also,
because
the
Anopheles
females
collected
in
treated
houses
were
dead
and
around
89%
to
94%
of
the
females
were
alive
in
control
houses,
no
comparisons
could
be
done
between
dead
and
alive
mosquitos
within
each
given
configuration.
Thus,
comparisons
were
done
overtime
between
control
houses
with
LLINs
and
treated
houses
with
LLINs
and
1
or
2
layers
of
insecticide
paint.
Overall,
An.
coluzzii
females
at
VK1
were
pyrethroid
resistant:
the
allelic
frequency
of
the
L1014F
kdr
mutation
was
high,
ranging
from
60
to
98%
(Table
3)
166
B.
Mosqueira
et
al.
/
Acta
Tropica
148
(2015)
162–169
Table
2
Residual
efficacy
tests
on
(A)
Anopheles
gambiae
“Kisumu”
and
(B)
Anopheles
coluzzii
VK1
using
WHO
test
cones.
Averages
taken
for
each
configuration,
2
houses
per
configuration.
C
=
control
with
LLINs
only;
RP
=
regular
paint;
IP
=
insecticide
paint;
T
=
time
in
months
since
treatment.
Numbers
in
the
same
column
sharing
a
letter
superscript
do
not
differ
significantly
(p
>
0.05).
Molecular
analysis
on
resistance
Allelic
frequency
of
the
L1014F
and
L1014S
kdr
mutations
Anopheles
coluzzii
VK1
(B).
%
Mortality
in
Anopheles
coluzzii
using
WHO
test
cones
Anopheles
gambiae
Kisumu
(A)
Anopheles
coluzzii
VK1
(B)
T0
T1
T3
T6
T12
T0
T1
T3
T6
T12
C
(LLINs)
10.9a7.9a6.1a5.6a6.9a1.7a2.6a2.9a2.1a2.1a
RP
+
IP/1
layer
walls
+
LLINs
100b100b100b100b99.0b100b100b100b98.9b90.9b
RP
+
IP/1
layer
walls
+
ceiling
+
LLINs
100b100b98.1b100b99.0b100b100b100b99.0b91.3b
IP/1
layer
walls
+
LLINs
100b100b98.0b100b99.0b100b100b100b100b85.0b
IP/1
layer
walls
+
ceiling
+
LLINs 100b100b100b100b98.1b100b100b100b100b81.8b
IP/2
layers
walls
+
LLINs 100b100b100b100b100
100b100b100b98.8b88.9b
IP/2
layers
walls
+
ceiling
+
LLINs
100b100b100b100b100
100b100b100b100b97.0b
Table
3
Distribution
of
the
frequency
of
L1014F
and
L1014S
kdr
mutations
in
Anopheles
coluzzii
in
VK1.
C
=
control
with
LLINs
only;
IP
=
insecticide
paint;
n
=
number
of
mosquitoes
tested;
T
=
time
in
months
since
treatment;
F(kdr)
=
frequency
of
the
mutation
kdr;
p
(HW)
=
value
for
Hardy–Weinberg
equilibrium
hypothesis;
“–”
=
non
determinable.
Treatments
Month
n
SS
RS
RR
F(L1014F
kdr)
p
(HW)
SS
RS
RR
F(L1014S
kdr)
p
(HW)
C
(LLINs) T0
30
7
3
20
0.717
0.0001
30
0
0
0
T1
30
0
0
30
0.98
30
0
0
0
T2
30
11
2
17
0.6
0
30
0
0
0
T3
31
8
3
20
0.694
0
31
0
0
0
T4
30
7
0
23
0.767
0
30
0
0
0
T5
25
5
0
20
0.8
0
25
0
0
0
IP/1
layer
walls
+
ceiling
+
LLINs T0
30
3
0
27
0.9
0.0001
30
0
0
0
T1
28
1
0
27
0.964
30
0
0
0
T2
30
4
3
23
0.817
0.002
27
3
0
0.05
1
T3
31
6
1
24
0.79
0
30
1
0
0.016
T4
29
3
6
20
0.793
0.066
24
5
0
0.086
1
T5
30
4
7
19
0.75
0.048
23
7
0
0.117
1
IP/2
layers
walls
+
ceiling
+
LLINs
T0
30
4
0
26
0.867
0
30
0
0
0
T1
31
0
0
31
0.98
30
0
0
0.001
T2
30
4
0
26
0.867
0
30
0
0
0
T3
31
9
0
22
0.71
0
31
0
0
0
T4
29
3
0
26
0.897
0.0001
29
0
0
0
T5
30
4
10
16
0.7
0.378
20
10
0
0.167
0.563
with
no
significant
difference
between
alive
specimens
collected
from
the
control
and
dead
specimens
collected
from
the
treated
houses
during
the
period
tested,
up
to
5
months
after
treatment.
Similarly,
no
increasing
or
decreasing
trends
were
identified
on
the
allelic
frequency
overtime.
The
L1014S
kdr
was
not
found
in
the
samples
collected
in
control
houses
with
LLINs
and
was
weakly
detected
in
the
heterozygous
form
in
houses
treated
with
1
layer
starting
at
T2,
T4
and
T5,
and
in
houses
treated
with
2
layers,
at
T5,
though
only
in
the
heterozygote
form
(Table
3).
3.3.2.
Allelic
frequency
of
the
mutation
Ace-1R
The
Ace1Rmutation
was
detected
at
low
allelic
frequencies
and
was
heterozygous.
It
was
only
randomly
found
at
T0
and
T5
in
the
control
houses
at
frequencies
of
8.3
and
4.0%,
respectively
(Table
4)
and
at
no
point
in
the
treated
houses.
3.3.3.
Determination
of
the
bloodfeeding
origin
There
were
no
statistical
differences
between
control
houses,
houses
treated
with
1
insecticide
paint
layer,
and
houses
with
2
insectide
paint
layers
(Table
5).
The
averages
of
all
houses
combined
from
T0
to
T6,
showed
about
27%
of
females
had
fed
on
humans,
about
58%
on
other
animals
and
about
16%
on
both.
All
in
all,
the
rate
of
zoophily
was
high
(58%).
Of
the
females
having
bloodfed
on
other
animals
(non
human),
about
45%
of
them
had
not
blood
fed
on
any
of
the
domestic
animals
chosen
as
the
most
typical
blood
meal
sources
in
the
area.
Of
the
identified
domestic
animals,
cattle
remained
the
most
common
blood
meal
source
(Table
5).
4.
Discussion
The
study
area
was
chosen
based
on
parameters
such
as
insecti-
cide
resistance
and
malaria
transmission
levels
(Dabiré
et
al.,
2008,
2009).
In
addition,
the
team
was
drawn
by
the
population’s
inter-
est
on
the
paint
and
the
efforts
that
home
owners
had
previously
undergone
to
try
to
paint
the
interior
of
their
homes
and
the
edges
of
windows
and
doors
when
their
economic
level
allowed
it.
From
that
standpoint,
the
study
area
presented
an
optimal
profile
to
per-
form
a
pilot
study
on
the
efficacy
of
combining
an
OP-based
paint
and
LLINs.
The
fact
that
classical
WHOPES
Phase
II
experimental
huts
were
not
used
posed
some
liminations
on
the
measurement
of
certain
entomological
parameters
(discussed
throughout
the
text),
but
allowed
the
assessment
of
how
the
Phase
III
trial
may
be
implemented.
Furthermore,
the
results
obtained
in
this
pilot
study
supported
previous
findings
observed
during
the
WHOPES
Phase
I
in
the
laboratory
and
Phase
II
study
in
experimental
huts
in
the
South
of
Benin
using
the
same
paint,
Inesfly
5A
IGRTM,
in
terms
of
entomological
mortality
rates,
the
porosity
of
materials
and
the
notion
of
volume
effect
discussed
below.
In
this
pilot
study,
the
combination
of
the
insecticide
paint
Inesfly
5A
IGRTM consisting
of
two
different
OPs
with
an
IGR,
and
pyrethroid-treated
LLINs
was
able
to
control
An.
coluzzii
(former
An.
gambiae
form
M)
popula-
tions
yielding
mortality
rates
of
100%
for
6
months
after
treatment
regardless
of
the
treatment
configuration
in
terms
of
volume
(walls
or
walls
+
ceiling),
dose
of
insecticide
paint
and
number
of
layers.
With
time,
however,
houses
with
two
layers
of
insecticide
paint
and
a
larger
volume
benefited
with
a
higher
long
term
efficacy.
The
mortality
rates
observed
during
mosquito
collections
on
the
pilot
B.
Mosqueira
et
al.
/
Acta
Tropica
148
(2015)
162–169
167
Table
4
Allelic
frequency
and
genotype
of
the
Ace-1Rmutation
in
Anopheles
coluzzii
at
VK1.
C
=
control
with
LLINs
only;
IP
=
insecticide
paint;
n
=
number
of
mosquitoes
tested;
T
=
time
in
months
since
treatment;
f(119S)
=
allelic
frequency
of
the
mutation
ace-1
119S;
p
(HW)
=
value
for
Hardy–Weinberg
equilibrium
hypothesis;
“–”
=
non
determinable.
Treatment
Month
n
Genotypes
f(119S)
[95%CI]
p
(HW)
119G
119G
119S
119G
119S
119S
C
(LLINs) T0
30
25
5
0
0.083
[0.00–0.18]
1
T1
30
30
0
0
0
T2
30
30
0
0
0
T3
30
30
0
0
0
T4
30
30
0
0
0
T5
23
21
2
0
0.04
[0.00–0.12]
1
IP/1
layer
walls
+
ceiling
+
LLINs T0
30
30
0
0
0
T1
30
30
0
0
0
T2
30
30
0
0
0
T3
30
30
0
0
0
T4
30
30
0
0
0
T5
30
30
0
0
0
IP/2
layers
walls
+
ceiling
+
LLINs
T0
30
30
0
0
0
T1
30
30
0
0
0
T2
30
30
0
0
0
T3
30
30
0
0
0
T4
30
30
0
0
0
T5
24
24
0
0
0
Table
5
Analysis
of
the
blood
source
of
bloodfed
Anopheles
coluzzii
collected
using
EMCs
at
VK1.
C
=
control
with
LLINs
only;
IP
=
insecticide
paint;
n
=
numbers
of
mosquitoes
tested;
T0–T6
=
period
from
June
to
December
2013
when
collected
Anopheles
coluzzii
were
pooled
and
randomly
tested
for
bloodfeeding
source.
Numbers
in
the
same
column
sharing
a
letter
superscript
do
not
differ
significantly
(p
>
0.05).
Treatment
Anopheles
coluzzii
females
tested
(T0–T6)
Humans
Other
animals
Mixed
n
%
Cattle
Sheep
Donkey
Pig
Dog
Other
n
%
n
%
C
(LLINs)
141
35
24.8a16
8
18
7
5
39
93
66.0 a13
9.2a
IP/1
layer
walls
+
ceiling
+
LLINs 143
51
35.7a21
4
3
5
4
33
70
49.0a22
15.4a
IP/2
layers
walls
+
ceiling
+
LLINs
141
28
19.9 a30
3
9
0
2
38
82
58.2a31
22.0a
Total
425
114
26.8
67
15
30
12
11
110
245
57.6
66
15.5
study
in
VK1,
in
real
houses,
were
also
supported
by
the
long-term
residual
tests
using
WHO
cone
tests.
Mortality
rates
in
all
treated
houses
remained
98.9–100%
for
6
months
against
both
An.
gambiae
“Kisumu”
(the
insecticide-susceptible
laboratory
reference
strain)
and
the
pyrethroid-resistant
An.
coluzzii
populations
in
VK1.
Results
obtained
12
months
after
treatment
using
WHO
cones
confirm
that,
in
the
long
term,
houses
with
two
layers
and
a
larger
volume
per-
formed
best.
The
results
obtained
using
EMCs
and
WHO
cones
are
in
consistence
with
previous
studies
performed
in
an
experimen-
tal
field
setting
in
Benin
with
the
same
paint
(Mosqueira
et
al.,
2010b),
where
huts
treated
with
two
layers
of
insecticide
paint
and,
particularly,
a
larger
volume
had
a
longer
lasting
efficacy.
The
observed
volume
effect
was
in
line
with
previous
observations
during
the
Phase
II
trial
in
the
South
of
Benin
(Mosqueira
et
al.,
2010b)
and
a
study