1. Entomological monitoring PMI VectorLink
Dereje Dengela
Abt Associates, Inc.
June 12, 2018

2. What does PMI VL entomological Surveillance work entail?
We collect data on six basic and three advanced PMI entomological indicators;
Basic entomological indicators
1. Species composition, abundance and seasonality
2. Vector behavior: feeding location and time
3. Quality assurance and residual efficacy monitoring
4. Insecticide susceptibility
5. Insecticide resistance intensity
6. Mechanism of resistance

3. Ento monitoring continued….
Advanced entomological indicators
Identification of mosquito infectivity
Determine parity rates/ longevity
Determine the host preference of the vectors.

4. Basic PMI entomological indicators
1. Species composition, abundance and seasonality
We determine:
The vectors that exists in particular geographical areas ( morphological identifications/ PCR ID)
Their abundance/ density
Relative proportions
Distributions
Seasonal changes
Impact of interventions on vector behavior or density

5. 1. Species composition, abundance and seasonality
Select sentinel sites. We consider
Representativeness of eco-epidemiological types
vector distribution
vector control interventions
Resources-budget

6. Species composition, abundance and seasonality
Collect baseline data before deploying interventions from both intervention and control areas
Monthly monitoring after interventions
Assess changes pre-post interventions and compare vs between intervention and control sites
Interrupted -time -series design.
Collection methods: HLC, CDC light traps, exit traps, PSC
Morphological identification
Molecular identification – sample mosquitoes collected from different sites and season.

7. 2. Vector behavior: feeding location and time
Determine:
Feeding location: outdoor VS indoor
Feeding time
To understand where and when transmission is occurring
Collection methods: HLC and CDC light traps.

8. 3. Quality assurance and residual efficacy monitoring
To determine
the quality of IRS and ITNs
Bio-efficacy ( residual life)- how long the insecticide last in killing the vectors
Assess airborne/ fumigant effect- Actellic and SumiShield.

9. 3. Quality assurance and residual efficacy monitoring
Method :
cone bioassay attach at 3 different heights ( 0.5m, 1.0 m and 1.5 m)
Use susceptible mosquitoes from insectary or field collected mosquitoes of know susceptibility level.
Test mosquito exposed to insecticide treated surfaces and control to unsprayed surface.
We make sure that different surface types are represented.
Exposure for 30 minutes
Mortality recorded at days 1,2,3,4,5,6,7 of holding period post exposure.
control mortality should no exceed 20% for the result to be valid.
80% mortality is the WHO threshold for efficacy

10. 4. Insecticide susceptibility
To determine vectors susceptibility to a single dose of insecticides
Two methods :WHO tube test and CDC bioassay
The two methods are roughly comparable and not equivalent
Age standardized mosquitoes are exposed to the 2x concentration of an insecticide that kills 100% of the population
WHO method: exposure time 1 hr and mortality recorded at 1, 2 , 3 , 4 , 5, 6, 7 days depending on insecticides

11. 4. Insecticide susceptibility

12. 4. Insecticide susceptibility

13. 4. Insecticide susceptibility
Interpretation of results
> 98% mortality susceptible
90-97% mortality suspected resistance
<90% confirmed resistance

14. INSECTCIDE RESISTANCE
Many vector species of public health importance including mosquitoes have developed resistance to one or more insecticides
What is insecticide resistance?
WHO’s definition
“Is the ability of certain individuals to tolerate doses of toxicants which would prove lethal to the majority of individuals in a normal population of the same species”
Phenotypic resistance is the phenomenon most commonly referred to in the public health. Molecular genotyping of resistance is the identification of the underlying genes that confer the inherited resistance.. Identification of the underlying genes provides evidence of the underlying evolutionary process. It provides understanding of both the degree of resistance expressed in individual insects with the resistance gene and the freq. of such insects in the population.

15. INSECTCIDE RESISTANCE
Insecticide resistance Action Committee (IRAC) defines resistance as
“The selection of heritable characteristic in an insect population that results in the repeated failure of an insecticide product to provide the intended level of control when used as recommended”
based on operational performance of the insecticide
Resistance leading to control failure is the phenomenon most commonly referred to in agriculture. National malaria control programs shouldn’t ,however, wait for control failure to occur before implementing strategies to manage insecticide resistance. There is no acceptable level of control failure in public health, and waiting could result in delaying action until it is too late.

16. INSECTCIDE RESISTANCE
Resistance is a natural phenomenon. Resistance genes appear through random mutations in which individuals are born "resistant”
Individuals who are substantially less susceptible may be present, generally at low frequencies
The emergence of insecticide resistance in a vector population is an evolutionary phenomenon.

17. INSECTCIDE RESISTANCE
It is assumed that wild –type genes more fit than resistant genes
Fitness cost: Resistant insects- lower productivity, longevity and robustness
In the presence of the insecticide, it is the less susceptible individuals that survive and reproduce
Therefore, the pre-existing mechanisms are selected driving the evolution of resistant
Populations, not species, become resistant (why?)
Resistance is a concept which applies to populations, which are to a degree isolated from the reminder of species concerned. It is also comparative term that relates resistance population to a more susceptible normal population.

19. Resistance Mechanisms(RM)
RM are grouped in to four categories :
Metabolic, target site, reduced penetration and behavioral
Metabolic resistance:
most common resistance mechanism that occurs in insects.
it is based on enzyme systems.
It occurs when increased or modified activity of an enzyme system prevent an insecticide from reaching its intended site of action.
Nearly all of the strains of culex quinquefasciatus which resists a broad range of OP insecticide have been found to posses multiple copies of a gene for esterases, enabling them to overproduce this type of enzyme. In contrast strains of malathion resistant Anopheles have been found with non-elevated levels of an altered form of esterase the specifically metabolizes the OP malathion at a much faster rate than that in susceptible individuals. Metabolic resistance mechanisms have been identified in vector populations for all major classes of insecticides.

20. Resistance Mechanisms(RM)
The three main enzyme systems are: esterases, mono-oxygenases and glutathione S-transferases
(GST).
2. Target-site resistance:
Second most common resistance mechanism.
Insecticides generally act at specific site within the insect( the nervous system)
occurs when the site of action of an insecticide is modified in a resistant strains, such that the insecticide no longer binds effectively
Result: the insect is unaffected or less affected by the insecticide. Kdr=DDT&PY, Ace-1= OP and carbamates.
Reduced susceptibility to Pys conferred by kdr mutations has been confirmed in An. gambiae in West, Central and East Africa.

22. Resistance Mechanisms(RM)
3. Reduced penetration:
Reduces uptake of insecticide due to the modification of the insect cuticle that prevent or slow absorption of insecticides.
Only one study has suggested correlation between cuticle thickness and pyrethroid resistance in An. funestus.
if cuticular resistance emerges, it might have significant impact when combined with other resistance mechanisms.

23. Resistance Mechanisms(RM)
4. Behavioral Resistance
Any modification in insects behavior that helps to avoid the lethal effects of insecticides.
Changes in vectors resting and feeding behavior to minimize contacts with insecticides.
No enough data whether behavioral avoidance is is genetic or adaptive trait.
Eg An. farauti. In Solomon Islands and New Guinea

24. Insecticide Resistance
Cross-resistance: One mechanism confers resistance to more than one insecticide
Example: kdr confers resistance to DDT and pyrethroids
Multiple resistance: occurs when several different resistance mechanisms are present simultaneously in resistant insects.
selection from different insecticide applications
Cross resistance often occurs between insecticide class that have the same mode of action for killing vectors. For example, if a resistance gene creates a change in target site in a vector, it is likely to affect any other insecticides that attack the same target sites, thus conferring the cross resistance. Similarly, an alteration to an enzyme that affects susceptibility to one insecticide may result in cross-resistance to another. Eg, In metabolic resistance, cross- resistance between PY and carbamates associated with mutations in cytochrome P450 enzymes detected. Target site and metabolic resistance can both occur in the same vector population and some times within the same individual. The two types of resistance appear to have different capacities to reduce the effectiveness of insecticide-based vector control interventions, with metabolic resistance being the stronger and more worrying mechanism.

25. Factors that influence resistance development
1)Frequency of application: many applications over a large geographic area quicken the development of resistance
- overuse and misuse
2) Persistence of residues: long persistence of the insecticides for IRS and LLINS have a strong selection effect leading to quicker resistance

26. Factors that influence resistance development
3) Rates of reproduction: short life cycle and high rate of reproduction speeds resistance development
Mosquitoes have short life cycle and high fecundity
4) Population isolation: open population allows migration of susceptible individuals which will have diluting effect

27. 5. Insecticide resistance intensity
To determine the strength of resistance
Initial data showed that more predictive f operational failure
Testing against 2X,5X,10X or 20X the diagnostic concentration
WHO and CDC method could be used.

28. 6. Mechanism of resistance
when resistance is confirmed we need to determine mechanism/s of resistance
Target site insensitivity – Molecular analysis – kdr and ace-1
Metabolic – Synergist assays and bio-chemical

29. B) Advanced Entomology Indicators
1. Mosquito infectivity
To determine whether mosquitoes carry malaria parasites or not
Enzyme-linked immunosorbent assay (ELISA) or
Polymerase chain reaction (PCR)

30. 2. Mosquito longevity Expectation:
Average age of mosquito population will decrease after deployment of IRS and or ITNs
Method; Dissect ovary and determine parity rates.

31. 3. Host preference of mosquitoes.
determine the source of blood meal using ELISA and PCR

32. Questions ?