Insecticide Susceptibility WHO Tube test.

Insecticide Resistance in Mosquitoes

Introduction
• Vector-borne diseases - major causes of sickness,
disability and death worldwide
• More than 80% of the world’s population lives in areas
with transmission of one or more of these diseases
• Mosquito-borne diseases - the largest contributor to human vector-borne disease
burden
• Malaria, dengue, chikungunya, lymphatic filariasis, Zika virus disease, yellow fever,
Japanese encephalitis

Introduction
• Malaria - 219 million cases globally &
400,000 deaths every year
• Dengue - Most prevalent viral infection
transmitted by Aedes mosquitoes
- More than 3.9 billion people in over 129
countries are at risk
- 96 million symptomatic cases & 40,000
deaths every year
Source: Fact Sheet, Vector-borne Diseases, WHO (2020)

Introduction
• Vector control - a key role in reducing the burden of these diseases.
• Prevention of diseases for which vaccines or treatments (either prophylactic or curative)
are not available relies heavily on vector control.
• For diseases for which treatments are available, such as malaria, vector control remains
the most widely used prevention strategy and has historically led to the greatest
reductions in disease burden.

Introduction
• Integrated Vector Management

Introduction
• Insecticides - most widely used and effective vector control interventions.
- Insecticide-treated nets (ITNs)
- Indoor residual spraying (IRS)
- Larvicide
- Space spraying

Introduction
• Classification of insecticides

Introduction
• ITNs – pyrethroids
• IRS - pyrethroids, carbamates, organophosphates and organochlorines
• Space spraying - pyrethroids and organophosphates

Introduction
• As a result of the wide use of insecticides for disease vector control and in agriculture,
mosquito resistance to insecticides in four classes traditionally widely used for vector
control – organophosphates, carbamates, pyrethroids and the organochlorine
dichlorodiphenyltrichloroethane (DDT) – has emerged in major disease vectors and
spread across all regions of the world
• Mosquito-borne diseases are now resurgent, largely because of the insecticide resistance

What is Insecticide Resistance?

Development of Insecticide Resistance

Mechanism of Insecticide Resistance
• Target site resistance - modifications of the molecular target site of the insecticide in the insect
body (i.e. mutations in the voltage-sensitive sodium channel gene, known as “kdr” mutations)
• Metabolic resistance - an increase in the activity of insecticide-detoxifying enzymes within the
insect body (i.e. cytochrome P450 monooxygenases, esterases, carboxylesterases and glutathione
S-transferases).
• Cuticular resistance
• Behavioural resistance

Rational for Entomological Surveillance

Evaluation of Insecticide Resistance
• Test procedures are available to characterize the following aspects of vector resistance to
insecticides:
- presence of phenotypic insecticide resistance in a vector population
- intensity of phenotypic resistance
- effect of a synergist in restoring susceptibility to an insecticide in a resistant
vector population
- mechanisms responsible for phenotypic resistance

• Three standard procedures for evaluation of insecticide resistance:
1. WHO tube test
2. WHO bottle bioassay
3. CDC bottle bioassay.

• Bioassay - direct response-to-exposure test,
• Mosquito mortality 24 hours after exposure to a known standard concentration of an
insecticide (e.g. the discriminating concentration) for a period of 1 hour.
• Testing mosquito susceptibility to insecticides that can impregnate filter papers.
• For insecticides that are unstable on filter papers or that cannot be impregnated on filter
papers - the WHO bottle bioassay
Insecticide discriminating concentration - The concentration of an insecticide that, when a
sample of mosquitoes is exposed to a surface treated with it for a standard period of time,
reliably kills susceptible mosquitoes so that any survivors may be assumed to be resistant.

Equipment, Reagents & Consumables
Equipment - used multiple times except impregnated papers (max 6 times)
6 green-dotted holding tubes 2 yellow-dotted control exposure tubes 4 red-dotted exposure tubes

• 6 steel clips (rings) to hold white papers
in the holding tubes
• 6 copper clips (rings) to hold insecticide-
or oil-impregnated (control) papers in
the exposure tubes

• 6 slide units
• 6 sheets of 15 cm x 12 cm white paper for lining the holding tubes
• 2 aspirators with glass tubes, straight
• 2 pieces of rubber tubing, 60 cm long
• 1 roll of adhesive tape
• 1 label

• Impregnated papers:
each box contains 8 sheets of insecticide-impregnated papers
• Control papers:
each box contains 8 sheets of carrier oil-impregnated papers to be used as the
control

Insecticide DCs for WHO tube tests with Anopheles spp. mosquitoes

Insecticide DCs for WHO tube tests with Aedes spp. mosquitoes

Insecticide DCs for WHO tube tests with Culex spp. mosquitoes

Health, Safety & Environmental Protection
• Before using any chemical compound, laboratory staff should read and
understand the risk assessment, material safety data sheets and the control of
substances hazardous to health assessment for each chemical used.
• Appropriate personal protective equipment must be worn at all times when
handling insecticides, including laboratory coat, gloves, safety glasses and a face
mask when weighing out chemicals.
• Ensure all working areas are clear of other materials and cleaned prior to
performing the test.
• All staff working in the laboratory must have received laboratory induction
training and the training must be documented in the individual’s training file.
• When working with mosquitoes, minimize mosquito escape by keeping all doors
and windows shut. If any mosquitoes escape, immediately use an electric bat to
electrocute them.

Mosquitoes for the test procedure
• 150 non-blood-fed female mosquitoes
• 3-5 days old
• Starved for 2 hours before using in the test
• During rearing – well nurtured & maintained in uncrowded trays during larval
stage, & uncrowded cages during adult stage
• During exposure period – number per tube be 25 or close to 25 to avoid crowding

Test Procedure
Step 1: Labelling the tubes
• 3 Types of tubes
1. Holding tube with a green dot – clean white paper
2. Control tube with a yellow dot – oil-treated filter paper
3. Exposure tube with a red dot – insecticide-treated filter paper
• Label each tube

Test Procedure
Step 1: Labelling the tubes

Test Procedure
Step 2: Preparing the tubes
Prepare holding tubes (green-dotted)
• Take 6 sheets of clean white papers rolled into a cylinder
shape, and insert one into each of the 6 holding tubes. Fasten
the sheet into position against the wall of the tube using 2
steel rings (clips), 1 at the top end and 1 at the bottom end.
• Place a 16-mesh gauze on each holding tube and screw the
cap into place.
• Attach a slide unit to each of the holding tubes by screwing it
in place at the open end

Test Procedure
Prepare control tubes (yellow-dotted)
• Wearing disposable gloves, roll 2 control papers
into a cylinder shape and insert each one into a
yellow-dotted tube, ensuring that the stamped
label on the paper is on the outer side and is
readable through the transparent tube.
• Fasten the control papers in each tube using 2
copper rings, 1 at the top end and 1 at the
bottom end. Close the tube with a screw cap at
one end (the bottom end).
• Remove the gloves and dispose of them in a
biohazard bag.

Test Procedure
Prepare exposure tubes (red-dotted)
• Wearing disposable gloves, roll 4 insecticide-treated
papers into a cylinder shape and insert each one into
a red-dotted tube, ensuring that the stamped label on
the paper is on the outer side and is readable through
the transparent tube.
• Fasten the insecticide-treated paper in each tube
using 2 copper rings, 1 at the top end and 1 at the
bottom end. Close the tube with a screw cap at one
end (the bottom end).
• Remove the gloves and dispose of them in a
biohazard bag.

Test Procedure
Step 3: Mosquito exposure
Aspirate and insert mosquitoes into the tubes:
• Aspirate 25 adult female mosquitoes from a
mosquito cage into each of the 6 green-dotted
holding tubes through the filling hole on the tube
sliding door.
• A total of 150 mosquitoes is needed to fill the 6
tubes.
• Close the slide unit and set the holding tubes in an
upright position
• Leave the mosquitoes in the holding tubes for 1
hour.

Test Procedure
Transfer the mosquitoes from the holding
tubes to the test tubes (2 control yellow-
dotted and 4 exposure red-dotted tubes): Only
live mosquitoes should be transferred to the
test tubes. Any moribund (i.e. those unable to
fly) or dead mosquitoes should be removed.
• One by one, attach the empty exposure and
control tubes to the vacant position on the
sliding units attached to the holding tubes.
• Carefully slide the units open and gently blow
the mosquitoes from the holding tubes into
the exposure tubes.
• Once all the mosquitoes are in the exposure
tubes, close the slide unit.

Test Procedure
• Record the exact number of live mosquitoes exposed. Some mosquitoes could
have died during the holding or transfer processes.
• Detach the green-dotted holding tubes from the exposure tubes and set them
aside.
• Set the tubes in a vertical position with the mesh screen facing up. Place them in
an area of reduced lighting, to discourage test mosquitoes from resting on the
mesh screen lid.
Leave the mosquitoes in the treatment (red-dotted) and control (yellow-dotted)
tubes for 1 hour.

Test Procedure
Transfer the mosquitoes back to the holding tubes at the end of the 1-hour exposure period.
• Gently blow the mosquitoes back into the green-dotted holding tubes by reversing the procedure
outlined above.
• Detach the exposure tubes from the slide units.
• Place the tubes in an upright position with the mesh screen facing up.
• Place a piece of cotton wool soaked in a 10% sugar solution on the mesh screen of the holding
tubes.
Record the number of knocked down mosquitoes, as per the definition
Hold the mosquitoes in the holding tubes (green-dotted) for 24 hours at 27 °C ± 2 °C and 75% ±
10% relative humidity.

Test Procedure
WHO definitions of knockdown & mortality of mosquitoes post-test

Test Procedure
Mosquito exposure & Post-exposure processes

Test Procedure
Step 4: Record mortality results
• Count and record the number of mosquitoes found dead and alive 24 hours after 1 hour
exposure, as per the definitions
• Enter the data in the recording sheet

Use & Storage of Impregnated Papers
• Do not use the same impregnated paper more than 6 times (equivalent to
exposing 150 mosquitoes in a tube)
• Store at 4–8 °C temperature
• Shelf-life ranges from 2 to 5 years depending on the insecticide

Criteria for Test Rejection
• If the control mortality is >20%, the tests must be discarded and repeated.

Data Recording & Calculation of Test Results

Interpretation of Test Result
7
7

Insecticide Resistance Management Strategies

Rotations
• Rotations of two or more
insecticides with different modes
of action
• If resistance to each insecticide is
rare, any resistance that develops
would be lost in the subsequent
rounds of alternate insecticide
• In vector control, rotation is limited
to when control happens, often
annually.

Insecticide Susceptibility WHO Tube test.

Insecticide Susceptibility WHO Tube test.

More Related Content

Similar to Insecticide Susceptibility WHO Tube test.

More from AnjumFatemaShaikh

Recently uploaded

Insecticide Susceptibility WHO Tube test.