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Mosquitoes and bednets: testing the spatial positioning ofinsecticide on nets and the rationale behind combinationinsecticide treatments
R. M. OXBOROUGH*,{, F. W. MOSHA*, J. MATOWO*, R. MNDEME*, E. FESTON*,
J. HEMINGWAY{ and M. ROWLAND{
*Kilimanjaro Christian Medical Centre, P.O. Box 3010, Moshi, Tanzania{London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, U.K.{Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, U.K
Received 23 April 2008, Revised 3 July 2008,
Accepted 7 July 2008
The recent development of pyrethroid resistance of operational significance in Anopheles gambiae s.l. is a major
threat to the control of malaria in West Africa. The so-called ‘2-in-1’ bednet, in which the top of the net is treated
with a non-pyrethroid insecticide and the sides with pyrethroid, has been proposed as a way of maintaining efficacy
in the wake of such resistance. A host-seeking female Anopheles mosquito must contact both the top and sides of a
‘2-in-1’ net, however, for such nets to be useful in resistance management.
In the present study, the interaction between mosquitoes and insecticide-treated bednets (ITN) was explored by
restricting the insecticide to particular surfaces of the nets (top only or sides only) and then testing these nets,
untreated nets and nets treated on all their surfaces in experimental huts, under simulated field conditions. Over the
6-week trial, there was no significant difference in An. arabiensis mortality between nets treated with pyrethroid on
the top only (39.2%), sides only (39.6%) and all surfaces (39.7%), thus indicating that a female An. arabiensis
usually contacts both the top and sides of a bednet during its host-seeking behaviour. The data on blood-feeding
indicated, however, that the insecticide used on the sides of the net may be more important in preventing mosquito
biting than that on the top.
These results support the rationale behind the ‘2-in-1’ nets. Such nets may have advantages over the use of nets
treated on all surfaces with a mixture of insecticides that includes a non-pyrethroid component. With the ‘2-in-1’,
the more toxic component can be deployed on the top of the net, away from human contact, while the more
repellent pyrethroid can be restricted to the sides, to prevent blood-feeding.
With the scaling-up of ITN coverage and the need to preserve pyrethroid efficacy, more consideration should be
given to switching from pyrethroid-only nets to ‘combination’ nets that have been treated with a pyrethroid and
another insecticide. Since the mosquitoes that act as malarial vectors may contact all surfaces of a bednet during
their host-seeking, spatial heterogeneity in insecticide levels over the surface of a net may not reduce that net’s
overall efficacy. Nets with a rather uneven distribution of insecticide (such as those that might be produced using
home-treatment insecticide kits) may therefore be no less effective, prior to washing, than nets with a more even
distribution of insecticide (such as long-lasting insecticidal nets produced under factory conditions).
A key target set out in the global strategic plan
of the Roll Back Malaria Partnership is ‘for
80% of people at risk from malaria to be
protected by 2010 through locally appropriate
vector control such as insecticide-treated nets
(ITN) and indoor residual spraying (IRS)’
(WHO, 2005). To achieve this goal, the
insecticides used to treat bednets or house
surfaces must be efficacious in reducing
mosquito feeding on human blood, by
personally protecting those who sleep under
the ITN or by the community-wide mass
killing of mosquitoes. The main biological
Reprint requests to: R. M. Oxborough, London School ofHygiene and Tropical Medicine, Keppel Street, LondonWC1E 7HT, U.K.E-mail: [email protected]; fax:z44(0)207299 4720.
Annals of Tropical Medicine & Parasitology, Vol. 102, No. 8, 717–727 (2008)
# 2008 The Liverpool School of Tropical Medicine
DOI: 10.1179/136485908X337553
threat to sustaining malaria control through
use of ITN and IRS is the development of
insecticide resistance in the mosquitoes that
act as malarial vectors.
The gene that confers knockdown
resistance to pyrethroids (kdr) is already
widespread in Anopheles gambiae s.s. in
many areas of western Africa (Chandre
et al., 1999; Etang et al., 2006; Pinto et al.,
2006) and has also been reported in parts of
eastern Africa (Vulule et al., 1994).
Although kdr initially appeared to be no
obstacle to malaria control in many areas
(Henry et al., 2005), the recent emergence
and spread of pyrethroid resistance and/or
kdr in the M form of An. gambiae may
severely limit the effectiveness of ITN and
IRS (N’Guessan et al., 2007; Sharp et al.,
2007). In southern Africa, the emergence of
An. funestus with metabolic pyrethroid resis-
tance is believed to be the main reason why
the malaria burden in Kwazulu Natal rose
seven-fold between 1995 and 1999
(Hargreaves et al., 2000).
It has been suggested that, for vector
control, existing organophosphates and car-
bamates might be suitable alternatives to
pyrethroids (Kolaczinski et al., 2000;
Hougard et al., 2003; Asidi et al., 2004,
2005). As mosquitoes find such insecticides
less irritant and excito-repellent than pyre-
throids, they make longer contact with nets
treated with these insecticides than with
pyrethroid-treated nets (Hougard et al.,
2003). Nets treated with organophosphates
or carbamates therefore tend to cause
greater mosquito mortality than pyre-
throid-treated nets but give less personal
protection (Hougard et al., 2003). In theory
at least, the use of nets that have been
treated not only with a pyrethroid but also
with one of these alternative insecticides
should give high levels of mosquito mortal-
ity and personal protection while reducing
the selection pressure for resistance. If such
‘combination’ nets are to be effective in the
management of resistance, however, each
insecticide component has to kill most of
those mosquitoes that are resistant to the
other component (Mani, 1985; Tabashnik,
1990). The only mosquitoes that should
survive exposure to the nets are the very rare
double mutants that carry resistance to both
insecticides. Theoretical models indicate
that, provided a minority of other mosqui-
toes evades contact with either insecticide
and is free to mate with the rare double
mutants, selection of resistance should still
be slow to evolve (Taylor and Georghiou,
1979). In practice, mixtures of insecticides
work in more subtle ways than can be
predicted using deterministic population
genetics. For example, at high coverages,
an excito-repellent component may stimu-
late pick up of the other insecticide and
enhance mortality (Denholm and Rowland,
1992).
Compared with the use of a mixture of
insecticides on the whole net, the treating of
the roof of a bednet with one insecticide and
the sides with another (to give a so-called ‘2-
in-1’ net) has potential benefits. For exam-
ple, deployment of the more toxic compo-
nent on the roof of the net may reduce any
health risks to those who sleep under the
net. It is suggested that the close proximity
of the two insecticides on the net effectively
means that the two act like a mixture, with
similar resistance-management benefits
(Guillet et al., 2001). As the warm air and
carbon dioxide that emanate from the
sleeper move upwards thermally (Guillet et
al., 2001; Mathenge et al., 2004), the
assumption is that host-seeking mosquitoes
usually explore an occupied bednet from the
top downwards. With a net that has a non-
pyrethroid insecticide on its top and a
pyrethroid on its sides, it might therefore
be expected that a host-seeking mosquito
would pick up a lethal dose of the non-
pyrethroid before being driven away from
the sleeper by the excito-repellent pyre-
throid on the sides. For such ‘2-in-1’ nets
to be useful as a tool for resistance manage-
ment, it is important that the host-seeking
mosquito contacts both the top and sides of
the net, so that a mosquito that is resistant
to one component will still contact the other
718 OXBOROUGH ET AL.
component and be killed by it. The aim of
the present study was to determine whether
host-seeking An. arabiensis do contact both
the top and sides of an occupied bednet, by
comparing mortality and blood-feeding in
experimental huts containing occupied bed-
nets that had been treated on the top only,
on the sides only, or on all surfaces with the
same concentration of a pyrethroid insecti-
cide (lambdacyhalothrin).
MATERIALS AND METHODS
Study Area and Insecticide Treatments
Evaluation of the lambdacyhalothrin-treated
nets was carried out under both field
conditions (in experimental huts at
Mabogini field station, in Lower Moshi,
northern Tanzania, in an area of rice
irrigation) and in a laboratory setting (con-
tact bio-assays were conducted at the
Kilimanjaro Christian Medical Centre, in
Moshi, northern Tanzania). The only sig-
nificant human-biting mosquitoes in the
Lower Moshi area are An. arabiensis and
Culex quinquefasciatus (Ijumba et al., 2002).
Test materials were rectangular polyester
bednets. There were four types of net, with
three nets/type. Three nets were left whole
and not impregnated, as controls. Each of
the rest was cut to separate the top from the
four sides and then the top, the four sides or
the whole net was impregnated, with 18 mg
lambdacyhalothrin/m2, before the net was
sewn back together. Care was taken to
ensure there was no gap in the area of
stitching between the top and sides of each
reconstructed net. The top had an area of
2.9 m2 whereas, together, the four sides
covered 17.1 m2. Three replicate nets were
made for each of the three ‘treatments’ and
the untreated control.
Contact Bio-assay
Each of the 12 mosquito nets was subjected
to contact (cone) bio-assays (WHO, 2006)
on two occasions: after the net was sewn
together but immediately before it was used
in the experimental-hut trials and, again,
several weeks after the field trials had
finished. At each time-point, sugar-fed, 2-
to 5-day-old, laboratory-reared An. arabien-
sis (Dondotha) were tested on each net —
three replicates of five mosquitoes each on
the top of each net and three replicates of
five mosquitoes each on two sides of each
net, giving 45 test mosquitoes/net. The
mosquitoes were exposed to the net surface
for 3 min and then transferred to paper cups
for mortality assessment 24 h later.
Experimental-hut Evaluation
The four experimental huts used in the 6-
week field trial were based on the design
described by Smith (1964) and Smith and
Webley (1969), with some slight modifica-
tions (reduction of eave space, addition of
hardboard ceilings lined with hessian cloth,
replacement of supporting pillars with a
concrete floor surrounded by a water filled
moat, and improved screening of the ver-
andah). The total verandah catch was
doubled to adjust for the loss of mosquitoes
through the open verandahs.
During the trial, the nine treated nets
(treated 2–3 days before) and the three
untreated nets were rotated through each of
the four huts. The volunteers who slept
under the bednets were rotated between
huts on successive nights, in order to reduce
potential bias caused by inter-individual
differences in attractiveness to the local
mosquitoes. The direction of the two open
verandahs in each hut was routinely chan-
ged with each treatment rotation, to mini-
mise the potential confounding effect of a
preferential escape route. None of the nets
was holed during the trial.
Each morning during the trial (at
07.00 hours), mosquitoes were collected
from inside the net, the window (exit) traps,
and the ceiling, walls and floors of the
verandahs and room. They were kept for
species identification, determination of
gonotrophic stage, and mortality counts.
MOSQUITOES AND BEDNETS 719
All members of the An. gambiae complex
identified by morphological characteristics
were assumed to be An. arabiensis, based on
the results of previous cytotaxonomic and
PCR-based identifications of the mosqui-
toes in the study area (Ijumba et al., 2002;
Kulkarni et al., 2006). Mosquitoes were
held in paper cups and provided with 10%
glucose solution for 24 h before their
mortality was scored.
All the data collected were double-
entered and analysed to show the effect of
each type of impregnation (top only, sides
only, or all net surfaces) on the exiting ‘rate’
(proportion, of all of the mosquitoes col-
lected, that came from the verandahs and
exit traps), blood-feeding ‘rate’ (proportion,
of all of the mosquitoes collected, that had
blood-fed) and mortality ‘rates’ [propor-
tions, of all the mosquitoes collected, that
were found dead in the morning (immediate
mortality) and after a further 24 h]. Each of
these outcome variables for each type of
impregnation was compared with the corre-
sponding values for the controls, and also
with each of the other types of impregna-
tion, using logistic regression and version
8.0 of the Stata software package
(StataCorp, College Station, TX). A P-
value of ,0.05 was considered indicative
of a statistically significant difference.
RESULTS
Contact Bio-assays
In the contact bio-assays, which were con-
ducted before the hut trials, similarly high
mortalities (.65%) were observed on all the
lambdacyhalothrin-treated samples, and
relatively low mortalities (,12%) were
observed on all the untreated surfaces
(Fig. 1). This indicates that no cross-con-
tamination had occurred during the treat-
ments and the sewing of the net pieces back
together. Contact bio-assays conducted sev-
eral weeks after the conclusion of the hut
trial still showed high mortality for the net
parts that had been treated with insecticide
(ranging from 87% to 100%), confirming
FIG. 1. The results of the cone bio-assays conducted immediately prior to the experimental-hut trial, showing the
mortality obtained with the tops (%) and sides (&) of the treated and untreated nets. The vertical lines indicate
95% confidence intervals.
720 OXBOROUGH ET AL.
insecticide integrity during the trial period.
By this time, the untreated tops of the nets
that had been treated only on their sides
had, unfortunately, become contaminated
with the insecticide, presumably during
storage after the trial (giving mortality up
to 78%).
Experimental-hut Trials
Anopheles arabiensis
NUMBERS CAUGHT/NIGHT. In general, the
number of An. arabiensis caught each night
in each experimental hut did not differ
significantly between the three types of
treatment or between each type of treatment
and the untreated control. Exceptionally, on
the third night of the trial, five times as
many An. arabiensis were caught in the hut
with one of the fully treated nets (155 in
total) as in any of the other huts. Although
this result did not cause undue bias to the
analysis (non-parametric statistics), it did
skew the total for that particular treatment
(Table 1).
EXITING. In huts with the untreated nets, a
total of 90.8% of the An. arabiensis exited to
the verandahs. In the huts with any of the
treated nets, however, the corresponding pro-
portions were significantly higher (Table 1).
BLOOD-FEEDING. Blood-feeding was signifi-
cantly rarer in the huts with any type of
treated net than in the huts with the
untreated nets (Table 1). Although similar
levels of blood-feeding were seen with the
nets that only had their sides treated (16.2%
blood-fed) as with the nets treated on all
surfaces (12.7%), the latter nets gave a
significantly lower level of blood-feeding
than seen with the nets that only had their
tops treated (18.1%).
MORTALITY. The three types of net treat-
ment induced similar levels of An. arabiensis
mortality 24 h post-exposure, and these
levels were all significantly higher than the
corresponding mortalities seen with the
untreated nets (Table 1). There were no
significant differences in mortality between
the three types of treatment. The temporal
trend seen in mortality over the 6 weeks of the
trial was consistent within each treatment and
did not differ between treatments (Fig. 2).
Culex quinquefasciatus
NUMBERS CAUGHT/NIGHT. The mean num-
bers of Cx. quinquefasciatus seen in the hut
collections ranged from 2.6 to 4.6/night.
Significantly fewer mosquitoes were caught
in the huts with the nets that had been
treated only on their sides than in those with
the nets that had been treated on all of their
surfaces (Table 2). As there is no obvious
cause for this difference and there was no
similar trend in the An. arabiensis collections
TABLE 1. The results of trials of pyrethroid (lambdacyhalothrin) treatments on bednets, against Anopheles ara-
biensis in experimental huts*
Untreated net
Part of net treated, at 18 mg/m2:
Top only Sides only All surfaces
Total no. of females caught 422a 497a 551a 769a
Females caught/night 11.7 13.8 15.3 21.4
OUTCOME VARIABLE (%) AND (95% CONFIDENCE INTERVAL)
Exiting 90.8 (87.6–93.2)a 95.8 (93.6–97.2)b 96.4 (94.4–97.6)b 97.4 (96.0–98.3)b
Blood-feeding 24.6 (20.8–29.0)a 18.1 (15.0–21.7)b 16.2 (13.3–19.5)bc 13.1 (10.9–15.7)c
Blood-feeding inhibition – 26.4 34.1 46.7
Mortality 24 h post-exposure
Observed 9.7 (7.2–12.9)a 39.2 (35.0–43.6)b 39.6 (35.6–43.7)b 39.7 (36.3–43.2)b
Corrected for control mortality – 32.7 33.1 33.2
*Within each row, values sharing the same superscript letter do not differ significantly (P.0.05).
MOSQUITOES AND BEDNETS 721
made at the same time, this difference is
considered to be a ‘type-II’ error (showing a
statistical difference when, in truth, there is
none).
EXITING. Exiting of Cx. quinquefasciatus ran-
ged from 81.5% to 89.1%, with no significant
differences between the three types of net
treatment or between each type of treatment
and the untreated control (Table 2).
BLOOD-FEEDING. Relative to the untreated
control, the nets treated only on their tops
produced the smallest reduction in the
blood-feeding of Cx. quinquefasciatus, and
the nets treated only on their sides produced
the greatest reduction (73.4%). There were,
however, no significant differences between
the three types of net treatment (Table 2).
MORTALITY. Although the 24-h mortality of
Cx. quinquefasciatus with the treated nets
ranged from 19.5% to 27.2% (each type of
treated net giving significantly higher mor-
tality than the untreated control), there were
no significant differences in such mortality
FIG. 2. Changes in mortality of Anopheles arabiensis entering experimental huts over the 6-week trial period,
showing the values recorded in the first 2 weeks (%), third and fourth weeks (&) and last 2 weeks (&). The vertical
lines indicate 95% confidence intervals.
TABLE 2. The results of trials of pyrethroid (lambdacyhalothrin) treatments on bednets, against Culex quinquefas-
ciatus in experimental huts*
Untreated net
Part of net treated, at 18 mg/m2:
Top only Sides only All surfaces
Total no. of females caught 119ab 128ab 92a 166b
Females caught/night 3.3 3.6 2.6 4.6
OUTCOME VARIABLE (%) AND (95% CONFIDENCE INTERVAL)
Exiting 81.5 (73.5–87.5)a 89.1 (82.4–93.4)a 88.0 (79.7–93.3)a 88.6 (82.8–92.6)a
Blood-feeding 24.4 (17.5–32.9)a 13.3 (8.4–20.3)b 6.5 (3.0–13.8)b 12.0 (7.9–17.9)b
Blood-feeding inhibition – 45.5 73.4 50.8
Mortality 24 h post-exposure
Observed 6.7 (3.4–12.9)a 19.5 (13.6–27.3)b 27.2 (19.1–37.1)b 27.1 (20.9–34.4)b
Corrected for control mortality – 13.7 22.0 21.9
*Within each row, values sharing the same superscript letter do not differ significantly (P.0.05).
722 OXBOROUGH ET AL.
between the three types of net treatment
(Table 2).
DISCUSSION
In the present hut trials, the nets treated
only on their tops gave similar 24-h mortal-
ity of An. arabiensis as the nets treated only
on their sides, even though a net treated on
all of its surfaces had a six-fold greater area
of treated material than a net treated on the
top only. There appear to be three possible
reasons for this observation.
The first possibility is that each An.
arabiensis in the trials contacted the sides
or the top of the net (but not both the top
and sides), with an equal chance of contact-
ing each. If this were the case, however, An.
arabiensis mortality with the nets treated on
all of their surfaces should have been
approximately equal to the sum of the
mortality on the nets treated on their sides
only (39.6%) and the mortality on the nets
treated on their tops only (39.2%) — that is,
about 80% (whereas the observed value was
39.7%).
A second possibility is that, at some stage
during the 6-week trial, the tops of the nets
that had been treated only on their sides
became contaminated with insecticide from
the sides of the nets, so that, in effect, the
nets resembled the nets treated on all of
their surfaces. Although some support for
this possibility comes from the results of the
post-trial bio-assays (in which it did appear
that the tops of the nets that had been
treated only on their sides had become
contaminated with insecticide), it seems
likely that most, if not all, of the contamina-
tion occurred after the trial, when the nets
were left folded, in storage, for several
weeks. When mortality in the 6-week trial
was broken down into three fortnightly
periods, mortality with the nets treated only
on their sides showed the same temporal
trend as the mortalities recorded on the
other types of treated net (Fig. 2), with no
indication of the gradual contamination of
the tops of the nets during the course of
the trial.
The third and most likely possibility to
explain the similar An. arabiensis mortalities
with each type of treated net is that each
host-seeking An. arabiensis in the trial
persistently attempted to penetrate the bed-
net to reach the sleeper and, in doing so,
searched over a large area of the net,
including both the top and the sides. Other
studies delving into the workings of ‘2-in-1’
nets failed to demonstrate that each host-
seeking mosquito generally contacts all
surfaces of a net. The earlier studies used
combinations of insecticides that differed in
toxicity, behavioural effects, position on the
net or surface area covered, however, and
such complexity makes it difficult to provide
adequate controls or allow inferences about
mosquito behaviour on and around the net
to be made (Guillet et al., 2001; Hougard et
al., 2003; Asidi et al., 2005).
Although the bednets used in this study
were un-holed, many of the An. arabiensis
caught in the huts containing untreated nets
had blood-fed (presumably on the volun-
teers under the nets). Encouragingly, how-
ever, treatment of the sides and/or top of a
net with lambdacyhalothrin resulted in
significantly fewer blood-fed An. arabiensis,
confirming the importance of pyrethroids
for personal protection (Miller et al., 1991;
D’Alessandro et al, 1995; Asidi et al., 2005).
Interestingly, treatment of all the surfaces of
a net produced significantly fewer blood-fed
mosquitoes than the treatment of the top
only, lending support to the notion that the
insecticide used on the sides of a net is more
important for personal protection than that
used on the top, because the sleeper is more
likely to be in contact with the sides than the
top (the insecticide chosen for treating the
sides should therefore be repellent). In the
present study, the nets treated only on their
sides should have produced proportionately
fewer blood-fed An. arabiensis than the nets
treated only on their tops; although such a
difference was observed, it did not reach
statistical significance.
MOSQUITOES AND BEDNETS 723
If a difference in contact-repellency was
the key factor in reducing An. arabiensis
blood-feeding with the nets treated on all of
their surfaces, compared with that seen with
the nets treated only on their tops, the nets
treated on all surfaces should have been
associated with a greater degree of exiting.
Although such a difference was observed
(Table 1), it also did not reach statistical
significance. Exiting with the untreated
control was already high, however, since
An. arabiensis is exophilic compared with
An. gambiae s.s. (Mahande et al., 2007).
With Cx. quinquefasciatus, treatment only of
the net top produced the lowest mortality of
all treatments (Table 2). This stands in
contrast with the results for An. arabiensis
and may indicate behavioural differences
between the two species.
The present results support the concept of
the ‘2-in-1’ bednet. To achieve resistance
management, the targeted mosquito must
contact both the treated top of a ‘2-in-1’ net
and the treated sides. Wild, host-seeking
An. arabiensis appear to satisfy this criterion
(present study) and therefore, against this
species at least, ‘2-in-1’ treatment should
have a similar impact, in decreasing the risk
of resistance development, to a mixture of
insecticides applied to all surfaces of a net.
This raises the question of whether a ‘2-in-
1’ bednet has any advantages over a net
treated with a mixture. Published studies on
‘2-in-1’ nets have specifically focused on
organophosphates and carbamates, which
are potent inhibitors of human cholines-
terases (Miller et al., 1991; Kolaczinski et al.,
2000; Asidi et al., 2004). The 2-in-1 method
might be a way of reducing health risks to the
sleeper by deploying such non-pyrethroids
as far from the sleeper as possible (i.e. on the
top of the net).
If two insecticides — one that is repellent
and one that is good at killing mosquitoes —
are to be used on a net, it is more appropriate
to use the non-repellent insecticide on the top
of the net, at a dose sufficient to kill the target
insect, and the repellent insecticide on the
sides, to reduce blood-feeding. Resistance
management with mixtures or ‘2-in-1’ nets
works on the principle of redundant killing:
those insects resistant to one component of
the combination will come into contact and
be killed by the other component (Denholm
and Rowland, 1992). Three assumptions
must be met for optimal use of insecticide
combinations (Tabashnik, 1990): (1) the
insect should not be resistant to both
components; (2) the combination must
maintain its integrity over time, with the
components showing similar decay rates; and
(3) the modes of resistance must be unique.
Several insecticides new to public health,
such as chlorfenapyr, have shown potential
in initial trials on nets (N’Guessan et al.,
2007; Mosha et al., 2008). Some older
organophosphates that combine low mam-
malian toxicity and low levels of resistance
to insensitive acetylcholinesterase mechan-
isms also show potential (Hemingway et al.,
1984; Kolaczinski et al., 2000). No alter-
native insecticide has the pyrethroids’ twin
attributes of generating excito-repellency
and high mortality in mosquitoes at low
concentration, however, and hence it is
essential that the pyrethroids be preserved
from the threat of resistance if at all possible.
The combining on nets of a non-pyrethroid
with a pyrethroid would have advantages in
all areas of Africa. In areas where most
mosquitoes are susceptibile to pyrethroids,
the non-pyrethroid component (either in
mixture or on the top of the net) would be
expected to kill any pyrethroid-resistant
mosquito that comes into contact with it,
thereby reducing the selection of pyrethroid
resistance, whereas the pyrethroid compo-
nent should continue to kill susceptible
mosquitoes and provide personal protec-
tion against them. In areas where resistance
is already at high frequency, the non-
pyrethroid component would be expected
to kill resistant mosquitoes and, at high
levels of ITN coverage, to reduce malaria
transmission.
In the present study, insecticide combina-
tions were not explored per se. Rather, the
intention was to show, by effects on
724 OXBOROUGH ET AL.
mortality, how host-seeking mosquitoes
contact bednets and pick up insecticide.
This was achieved through tests involving a
single insecticide restricted to given sur-
faces. To have tested a combination at this
stage would have confused the picture (since
no other class of insecticide induces beha-
viour or toxicity in the same way as the
pyrethroids) and would have made inter-
pretation of the data much more difficult.
Other researchers, such as Asidi et al.
(2005), have gone straight to the combined
testing of two insecticides and this has
tended to cloud the picture rather than shed
light on how each component works. By
using just a single insecticide in the present
study, it was possible to show that host-
seeking females of one member of the An.
gambiae complex tend to roam over all
sections of a bednet, including the top
where alternative insecticides might be put.
This sets the scene for further work on
combinations.
With the scaling-up of ITN coverage under
the Global Fund (www.theglobalfund.org)
and the President’s Malaria Initiative
(www.fightingmalaria.gov), there is a grave
risk of accelerating the selection of pyrethroid
resistance. Consideration should be given to
switching from nets treated with a single
insecticide to nets treated with at least two
insecticides, either in the form of a mixture or
as ‘2-in-1’, to help preserve the essential
resource represented by the pyrethroids.
The present data have other important
implications. Aside from ‘2-in-1’ nets and
the problem of resistance, there is concern
that heterogeneity in the pyrethroid content
on the surfaces of individual nets may
reduce effectiveness. The insecticide on nets
treated at home, by dipping, is more uneven
than seen in factory-produced ITN (Yates et
al., 2005; Hill et al., 2006). Even in the era
of long-lasting insecticidal nets, there
remains a significant market for long-lasting
treatment kits in which the insecticide
formulation is mixed in aqueous solution
with a polymer binder that, once dried on
the nets, protects the insecticide from
removal during subsequent washing
(WHO, 2007). The treatments investigated
here indicate that heterogeneity in the
insecticide level over a net may not impact
upon the mortality generated by that net if,
as seen with An. arabiensis, the target
mosquitoes contact multiple surfaces of the
net while host-seeking. Uniform insecticide
impregnation, although desirable from the
perspective of improving the quality of long-
lasting insecticidal nets, may therefore not
be essential for effectiveness.
ACKNOWLEDGEMENTS. The authors thank C.
Masenga, A. Mtui, E. Philip, E. Tillya,
J. Puya, H. Temba, and R. John, for their
field work, and A. Sanga and R. Athuman,
for insectary support. This project was
funded by the Innovative Vector Control
Consortium.
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