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

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

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

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

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

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.

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.

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.

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

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

4. Insecticide susceptibility

4. Insecticide susceptibility

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

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.

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.

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.

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.

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.

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.

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.

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

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.

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

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

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.

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

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)

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.

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

Questions ?