WHO/CDS/WHOPES/GCDPP/2005.

11

GUIDELINES FOR
LABORATORY AND FIELD TESTING
OF LONG-LASTING INSECTICIDAL
MOSQUITO NETS

COMMUNICABLE DISEASE CONTROL,

PREVENTION AND ERADICATION
WHO PESTICIDE EVALUATION SCHEME (WHOPES)
© World Health Organization 2005

All rights reserved.

The designations employed and the presentation of the material in this

publication do not imply the expression of any opinion whatsoever on the
part of the World Health Organization concerning the legal status of any
country, territory, city or area or of its authorities, or concerning the
delimitation of its frontiers or boundaries.

The mention of specific companies or of certain manufacturers’ products

does not imply that they are endorsed or recommended by the World
Health Organization in preference to others of a similar nature that are not
mentioned. Errors and omissions excepted, the names of proprietary
products are distinguished by initial capital letters.

All reasonable precautions have been taken by the World Health

Organization to verify the information contained in this publication.
However, the published material is being distributed without warranty of
any kind, either express or implied. The responsibility for the
interpretation and use of the material lies with the reader. In no event shall
the World Health Organization be liable for damages arising from its use.

Preparation of this document has been funded by the Global Collaboration

for Development of Pesticides for Public Health (GCDPP).
CONTENTS

Page

1. INTRODUCTION ................................................................. 1

2. LABORATORY TESTING (PHASE I)................................ 3

2.1 Regeneration and wash resistance................................ 3

2.1.1 Regeneration time .......................................... 3

2.1.2 Wash resistance.............................................. 4
2.1.3 WHO washing procedure............................... 4

2.2 Efficacy ........................................................................ 5

2.2.1 Bioassays........................................................ 5
2.2.2 Tunnel tests..................................................... 6
2.2.3 Supplementary tests........................................ 7

3. SMALL-SCALE FIELD TRIALS (PHASE II)..................... 9

3.1 Efficacy and impact on mosquito behaviour................ 9

3.2 Perceived side-effects................................................. 13

4. LARGE-SCALE FIELD TRIALS (PHASE III) ................. 15

1. INTRODUCTION
The purpose of this document is to provide specific and
standardized procedures and guidelines for testing long-lasting
insecticidal mosquito nets (LNs) for personal protection and malaria
control. It is intended to harmonize the testing procedures carried
out to generate data for registration and labelling of such products
by national authorities.

An LN is a factory-treated mosquito net expected to retain its

biological activity for a minimum number of standard World Health
Organization (WHO) washes and a minimum period of time under
field conditions. Currently, an LN would be expected to retain
biological activity for at least 20 standard WHO washes under
laboratory conditions and 3 years of recommended use under field
conditions, as defined in these guidelines. The guidelines do not
include the testing/evaluation of products for long-lasting post-
factory treatment of mosquito nets, which will be subject to separate
WHO guidelines, or of the LNs that may use insecticides not
currently recommended by WHO for such application.1 Rather, they
reflect the current state of knowledge on LN technology and will be
subject to revision as more information becomes available.

The guidelines were reviewed and recommended by the WHO

Pesticide Evaluation Scheme (WHOPES) Informal Consultation on
the development of guidelines for testing/evaluation of long-lasting
insecticidal mosquito nets, held at WHO headquarters in Geneva,
Switzerland, on 4–7 April 2005.2

1
http://whqlibdoc.who.int/hq/2003/WHO_CDS_WHOPES_2002.5_Rev.1.pdf
(accessed 20 April 2005).
2
The report will be available at:
http://www.who.int/whopes/gcdpp/publications/en/

1
The document includes laboratory, small- and large-scale field
studies to determine the efficacy and operational acceptability of an
LN, as summarized below. Although some observations on the
safety of such nets will be carried out in the field, a preliminary
safety assessment has to be undertaken, following the generic risk
assessment model developed by WHO for this purpose,3 before any
field study can be done. In addition, the physical properties of the
fabric and factors relating to its structural integrity should conform
to WHO specifications for netting materials.4

Phase Type of study Activities

Phase I Laboratory • Regeneration of insecticide and wash
resistance
• Efficacy

Phase II Small-scale • Wash resistance

field trials • Efficacy and impact on vector
behaviour
• Safety observations

Phase III Large-scale • Long-lasting efficacy

field trials • Community acceptance
• Safety observations

3
http://whqlibdoc.who.int/hq/2004/WHO_PCS_04.1.pdf (accessed 20 April
2005).
4
http://www.who.int/malaria/vectorcontrol.html

2
2. LABORATORY TESTING (PHASE I)

The objectives of the laboratory testing are to determine the

efficacy and wash resistance of an LN and to study the dynamics of
the insecticide on the fibre. The aim of these experiments is not to
simulate washing that would be experienced under field conditions
but rather to provide a consistent, repeatable protocol that would
allow for comparisons between different laboratories and different
LN products.

The test includes:

o determination of the period of time required for full

regeneration of the LN after washing;
o determination of the efficacy and wash resistance of the LN
against susceptible vector species.

A certificate of chemical analysis must be provided by the

manufacturer to ensure that the concentration of the active
ingredient is within +25% of the declared content.

2.1 Regeneration and wash resistance

2.1.1 Regeneration time

In order to determine the time period required for regeneration of an

LN after standard washing and holding at 30 °C, bioassays (as
outlined below) are carried out at 24-hour intervals on net samples
washed and dried once and three times consecutively until initial
biological activity is restored; nets washed three times are expected
to deplete surface insecticide on the net, whereas nets washed once

3
may not. Insecticide bioavailability (efficacy) curves will be
established and compared for nets washed once and three times
consecutively. The time required (in days) to reach the plateau is
the period required for regeneration of the net. If the two curves are
different, the longer period will be adopted as the washing interval
in Phase I and Phase II studies to ensure that wash resistance is not
overestimated. Details of standard washing (see 2.1.3) and
bioassays (see 2.2.1) are provided below.

2.1.2 Wash resistance

The resistance of an LN to washing will be determined through

standard bioassays carried out on nets washed at intervals required
for regeneration (as determined above), using the standard WHO
wash, and dried and held at 30 °C. Bioassays will be done after 0, 1,
5, 10, 15 and 20 washes or more as necessary. Each bioassay should
be done just before the next wash. Regression curves should be
drawn using respectively percentage mortality and knock down
(KD) versus number of washes. The number of washes providing
mortality and/or KD above the cut-off point (more than 80%
mortality after 24 hours and/or above 95% KD after 60 minutes
post-exposure) is reported. If an LN falls below the cut-off point,
the study should continue until 20 washes are reached; a tunnel test
(see 2.2.2) should then be conducted.

2.1.3 WHO washing procedure

Net samples (25 cm x 25 cm) will be individually introduced into

1-l beakers containing 0.5 l deionized water, with 2 g/l soap5
(pH 10–11) added just before and fully dissolved. Beakers will be

5
Currently, “Savon de Marseille” is recommended as the standard soap. Further
standardization, including the use of products recommended by the International
Organization for Standardization, or other standard products, is necessary.

4
immediately introduced into a water bath at 30 °C and shaken for
10 minutes at 155 movements per minute. The samples are then
removed and rinsed twice for 10 minutes in clean, deionized water
in the same shaking conditions as stated above. Nets are dried at
room temperature and stored at 30 °C in the dark between washes.

2.2 Efficacy

2.2.1 Bioassays

Five susceptible,6 non-blood fed, 2–5-day old Anopheles7 (species

to be stated in the test report) mosquitoes will be exposed to netting
materials (25 cm x 25 cm) for 3 minutes, under standard WHO
cones8 (Figure 1), after which they are held for 24 hours with
access to sugar solution. KD is measured after 60 minutes post-
exposure and mortality after 24 hours. At least 50 mosquitoes on
each net (10 replicates) and samples from four different nets should
be tested. Results should be reported for each net tested along with
the pooled results (5 x 10 x 4 = 200 mosquitoes). Mosquitoes
exposed to untreated nets are used as controls. Bioassays will be
carried out at 25 + 2 °C and 75 + 10% RH.

Nets washed at least 20 times that cause >80% mortality and/or

>95% KD meet the criteria to undergo Phase II testing.

6
Susceptibility should be confirmed at least twice per year using standard
WHO susceptibility test kits.
7
Caution should be exercised in comparing results obtained using different
Anopheles species.
8
WHO tube tests may be used pending determination of appropriate and
equivalent cut-off values.

5
If coloured LNs are marketed, their bioavailability curves should be
compared with those of white nets and, if significantly different,
should be considered a separate product requiring full testing and
evaluation.

2.2.2 Tunnel tests

The efficacy of treated nets can be underestimated if judged only

based on standard cone bioassays. This is specially the case with
insecticides that have a high excito-repellent effect, such as
permethrin and etofenprox. Therefore, the efficacy (mortality and
blood feeding inhibition) of LNs washed 20 times or more that no
longer meet the criteria of standard cone bioassays will be studied
in the laboratory, by releasing non-blood fed female anopheline
mosquitoes, aged 5–8 days, in a tunnel (square section 25 cm x
25 cm) made of glass, 60 cm length (Figure 2). At each end of the
tunnel, a 25-cm square cage is fitted (extension) and covered with
polyester netting. At one third of the length, a disposable cardboard
frame is placed with the treated netting sample. The surface of
netting “available” to mosquitoes is 400 cm2 (20 cm x 20 cm), with
nine holes each 1 cm in diameter: one hole is located at the centre
of the square; the other eight are equidistant and located at 5 cm
from the border.

In the shorter section of the tunnel, a bait (e.g. guinea-pig for

An. gambiae) is placed, unable to move. In the cage at the end of
the longer section of the tunnel, 100 females are introduced at
18:00. Females are free to fly in the tunnel but have to make contact
with the piece of netting and locate the holes in it before passing
through to reach the bait.

6
The following morning, at 09:00, the mosquitoes are removed and
counted separately from each section of the tunnel and the
immediate mortality is recorded. Live females are placed in plastic
cups with honey solution; delayed mortality is recorded after
24 hours. During tests, cages are maintained at 27 °C + 2 °C and
80% + 10% relative humidity under subdued light.

Several tunnels will be used simultaneously, one tunnel with

untreated netting always being used as a negative control. Blood-
feeding inhibition is assessed by comparing the proportion of blood-
fed females (alive or dead) in treated and control tunnels. Overall
mortality is measured by pooling the immediate and delayed
(24-hour) mortalities of mosquitoes from the two sections of the
tunnel.

Nets washed at least 20 times that cause mortality >80% and or

blood-feeding inhibition >90% in tunnel tests meet the criteria to
undergo Phase II testing.

2.2.3 Supplementary tests

Chemical assays. Chemical assays of the total insecticide content of

the netting (following the methodology recommended by the
manufacturer) before and after wash resistance studies will support
better interpretation of the results.

Other assays, such as the median time to knock-down test, may

provide useful supplementary information on the bioavailability of
the insecticide on the LN after washing. However, caution should
be exercised in comparing different insecticide products.

7
Figure 1. Cone bioassay on mosquito nets (courtesy of Dr Vincent
Corbel, Institut de Recherche pour le Développement (IRD),
Montpellier, France).

Figure 2. A tunnel made of glass for the study of the efficacy of

insecticide-treated mosquito nets (courtesy of Dr Vincent Corbel,
Institut de Recherche pour le Développement (IRD), Montpellier,
France).

8
3. SMALL-SCALE FIELD TRIALS
(PHASE II)

The efficacy of LNs in terms of blood-feeding inhibition,

deterrence, induced exophily and mortality will be studied in
experimental huts (Figure 3) using susceptible, free-flying, wild
mosquitoes.

The small-scale field studies include:

o determination of the efficacy of washed and unwashed LNs

and their impact on the behaviour of susceptible wild
mosquitoes (anophelines and, where possible, culicines);
o recording the perceived side-effects of the LN among users.

3.1 Efficacy and impact on mosquito behaviour

The impact of washed and unwashed LNs on the blood-feeding

inhibition of susceptible (confirmed by WHO susceptibility tests),
free-flying, wild mosquitoes (anophelines and, where possible,
culicines), their tendency to be repelled or driven out of houses and
their mortality will be assessed using experimental huts (fitted with
entry slots to prevent escape of mosquitoes), exit traps and/or
screened veranda; and with mechanisms for excluding ants and
other scavengers that might carry off dead mosquitoes from the huts
during the night.

Untreated nets will be used as a negative control. A net

conventionally treated with the same insecticide using the
WHO-recommended concentration and formulation(s) that is
washed to just before exhaustion should also be used as a positive

9
control. The point of exhaustion should be determined at the field
site by washing the conventionally treated net using the Phase II
protocol. WHO cone bioassays are performed after each wash. The
last wash for which the net still causes >80% mortality or >95% KD
is considered to be the number of washes required before
exhaustion.

The following treatment arms should be tested:

o Unwashed LN
o LN washed 20 times
o LN washed according to manufacturer’s claim (or
maximum washes determined in Phase I)
o Polyester, conventionally treated net washed under
Phase II conditions until just before exhaustion (see
previous paragraph)
o Polyester, conventionally treated net washed 20 times
o Untreated net (use of the same fabric and mesh size as
the test LN is preferred).

The nets are washed according to a protocol adapted from the

standard WHO washing procedure used in Phase I, and on cycles
equal to the number of days necessary for the full regeneration of
the insecticide (bioavailability) established in Phase I. Nets are
washed in 10 l of water (preferably well-water or de-chlorinated
water with a maximum hardness of 5 dh) and manual agitation for
10 minutes at approximately 20 rotations per minute. Nets will be
thoroughly rinsed twice in fresh water and dried horizontally in the
shade. The nets will be stored at ambient temperature between
washes. Washing should be carefully planned to ensure that all
material is ready at the same time.

10
Each week, the treatment arms are rotated among the huts according
to a Latin square scheme (Annex 1). Five nets are used per treatment
arm and each net is tested one night during the week. At the end of
the week, the huts are carefully cleaned and aired to remove
potential contamination. The trial should continue for a multiple of
6 weeks to ensure complete rotation through the huts. In most cases,
12 weeks should be long enough to obtain sufficient numbers of
mosquitoes for adequate statistical analysis. Before use in the study,
each net (including control) should have a total of six holes (4 cm x
4 cm) cut in the sides using sharp scissors to simulate the conditions
of a torn net (two holes on each large side and one on each small
side).

Two additional LNs from the same production batch should be

obtained from industry so that after random allocation to
experimental huts, pieces of the netting from the two LNs can be
sent for chemical residue analysis. Furthermore, two additional nets
are treated conventionally, so that samples of such nets can also be
sent for chemical analysis, in order to confirm the initial
concentration of the insecticide on mosquito nets. For this purpose,
four samples (30 cm2 x 30 cm2) are cut along a diagonal across the
roof and three samples along a diagonal across each side, using
sharp scissors. The samples are rolled up and placed in labelled,
new, clean aluminium foil prior to analysis. The samples from each
net will be combined to provide the average target concentration of
the insecticide on each net.

Ethical clearance should be obtained from the appropriate

institutions and authorities before starting the study.

Adult volunteers sleep under the nets, and mosquitoes are collected
the next morning. Informed consent should be obtained from all
volunteers participating in the study. Effective chemoprophylaxis

11
should be provided where appropriate, and volunteers should be
medically supervised. Sleepers are rotated randomly among huts
each night of the study. They shall enter the hut at dusk and remain
inside until dawn. In the morning, dead mosquitoes are collected
from the floor of the hut as well as from the exit traps and inside the
nets; resting mosquitoes are collected using aspirators from inside
the net and from the walls and roof of the hut and exit traps.
Mosquitoes are scored by location as dead or alive and as fed or
unfed. Live mosquitoes should be placed in small cups and
provided with access to sugar solution for 24 hours to assess
delayed mortality.

The primary outcomes measured in experimental huts are: the

deterrence (reduction in hut entry relative to the control huts fitted
with untreated nets); induced exophily (the proportion of
mosquitoes that exit early and are found in exit traps); blood-
feeding inhibition (the reduction in blood feeding compared with
that in the control huts); and immediate and delayed mortality (the
proportion of mosquitoes that are killed). The first three of these
outcomes are indicators of personal protection, and benefit
individual users. The fact that blood-seeking females are killed is
also important because community-wide use of treated nets can, in
some circumstances, produce a “mass effect”, i.e. a reduction in the
density of infective mosquitoes in the area and, consequently,
protection of the whole community, including those not using
treated nets.

For statistical analysis, the number of mosquitoes entering the huts,

the proportion of mosquitoes that exit early, the proportion that are
killed within the hut and the proportion that successfully blood feed
should be compared by species with the hut as the repeat unit.

12
The primary criteria in evaluating an LN in experimental huts
should be blood-feeding inhibition and mortality in the prominent
malaria vectors at the study site. An LN washed 20 times or more
should perform equal to or better than a conventionally treated net
washed until just before exhaustion. The maximum number of
washes to be claimed by a manufacturer will be the one the LN can
withstand under Phase II (experimental huts).

3.2 Perceived side-effects

The sleepers in the huts should be questioned about perceived

adverse or beneficial side-effects of the LNs and of the
conventionally treated and untreated nets.

Note: In view of the long-term studies that may be required to fully

test or evaluate an LN product, interim recommendations on its use
for malaria prevention and control may be given subject to the
following: use of WHO-recommended insecticides in making the
LN; satisfactory completion of laboratory and small-scale field
testing; and confirmation that after at least 20 standard WHO
washes the LN performs equal to or better than a conventionally
treated net washed until just before exhaustion. It is assumed that in
such circumstances the available information on the performance of
the conventionally treated nets will assist in anticipating the
performance of the LN product in operational settings.

13
Figure 3. Design of two experimental huts commonly used in west
and east Africa (top: United Republic of Tanzania, courtesy of
Professor C.F. Curtis, London School of Hygiene and Tropical
Medicine; bottom: west Africa, courtesy of Dr J.M. Hougard,
Institut de Recherche pour le Développement (IRD), Benin).

14
4. LARGE-SCALE FIELD TRIALS
(PHASE III)
The efficacy, longevity and fabric integrity as well as the
community acceptance of an LN will be studied in household
randomized trials lasting at least 3 years and in comparison with
conventionally treated nets, using the same insecticide, at the WHO
recommended dose (highest dose of the range). Conventionally
treated nets will be followed for up to 1 year or until biological
activity, as measured in bioassays, declines to a level that is
significantly lower than that observed in the LN.9 The efficacy will
be determined through regular bioassays and chemical residue
analysis at the start and completion of the study.

Ethical clearance should be obtained from the appropriate

institutions and authorities before starting the study.

Statistical power to detect changes over time in the performance of

the LN should be calculated separately for the outcome variables;
the maximum number of samples required shall be used for the
calculation of the number of households to be included in each arm
of the study (LNs versus conventionally treated nets). In most
studies, 30 nets sampled every 6 months is adequate. In calculating
the total number of nets required for the study, consideration should
be given to possible losses and to nets that need replacing during
both the sampling programmes and the study period.

Several communities should preferably be used, and in each

community the households shall randomly be allocated to each arm
of the study. Baseline information on acceptability, net preferences

9
LNs will be provided to study participants once their conventionally treated nets
have been removed from the study.

15
(e.g. size, colour, shape) and washing habits should be collected in
each community in advance of the study. To distinguish net types,
each net should be given a unique code (using an indelible marker
or sewing a label on each net) and a net master list developed for
later identification and for random selection during follow-ups.

Samples of the consignment of LNs, as well as samples of

conventionally treated nets (for each treatment), should be subject
to chemical assays at the beginning of the trial to ensure that the
target dose of the insecticide has been achieved, as well as at the
completion of the project to facilitate interpretation of the bioassay
data. For this purpose, four samples (30 cm2 x 30 cm2) are cut along
a diagonal across the roof and three samples along a diagonal across
each side of 10 randomly selected nets, using sharp scissors. The
samples are rolled up and placed in labelled, new, clean aluminium
foil prior to assay. The samples from each net will be combined to
provide the average target concentration of the insecticide on each
net. A total of 30 mosquito nets will, however, be samples at the
end of the study, in order to account for the higher variability in
insecticide content expected on nets used under field conditions.

Nets are randomly drawn from the net master list by the principal
investigator and used by the research team for collection of samples
for cone bioassay, at the start of the project and every 6 months
thereafter. Each household sampled for bioassays will also be
interviewed to assess perceived adverse or beneficial side-effects,
net utilization patterns (including early morning observations),
method and number of washes (questionnaire to be developed and
adapted to the local context) and physical integrity of the net (size
and number of holes). The household will be provided with a new
net and removed from the study.

16
To reliably measure washing frequency in the community, another
net within the sampled households or a net in a neighbouring
household will be marked with a water soluble marker. The net will
be revisited 1 month later to check the water soluble mark.

Susceptible, non-blood fed, 2–5-day old laboratory-bred Anopheles

(species to be stated in the report) mosquitoes should preferably be
used for cone bioassay, following the instructions provided in
section 2.1.1. The bioassay will be carried out on a sample (25 cm x
25 cm) that will be cut from the middle of one larger side of the net.
Wild anopheline mosquitoes may be used only if their full
susceptibility to the insecticide has been confirmed. The species and
blood-fed/non-blood fed condition of mosquitoes should be
reported.

If, at the end of 3 years, at least 80% of nets meet the cut-off criteria
for either the WHO cone bioassay test or the tunnel test, then the
product meets the definition for an LN.

17
ANNEX 1. Latin square rotation scheme for testing/evaluation of
six different treatment arms in experimental huts

Treatment rotation
Week Day Hut A Hut B Hut C Hut D Hut E Hut F
1 1 1 2 3 4 5 6
2 1 2 3 4 5 6
3 1 2 3 4 5 6
4 1 2 3 4 5 6
5 1 2 3 4 5 6
2 8 2 3 4 5 6 1
9 2 3 4 5 6 1
10 2 3 4 5 6 1
11 2 3 4 5 6 1
12 2 3 4 5 6 1
3 15 3 4 5 6 1 2
16 3 4 5 6 1 2
17 3 4 5 6 1 2
18 3 4 5 6 1 2
19 3 4 5 6 1 2
4 22 4 5 6 1 2 3
23 4 5 6 1 2 3
24 4 5 6 1 2 3
25 4 5 6 1 2 3
26 4 5 6 1 2 3
5 29 5 6 1 2 3 4
30 5 6 1 2 3 4
31 5 6 1 2 3 4
32 5 6 1 2 3 4
33 5 6 1 2 3 4
6 36 6 1 2 3 4 5
37 6 1 2 3 4 5
38 6 1 2 3 4 5
39 6 1 2 3 4 5
40 6 1 2 3 4 5

18