New tools for detecting insecticide New tools for detecting insecticide ...

New tools for detecting insecticide resistance: future perspective Mark Paine on behalf b h lf off the th VPMT/PQK Groups G

Resistance Monitoring

Usually too late

R i t Resistance d detection t ti methods th d Test

Pros

Cons

Bioassay

Phenotype Standard methodologies

Insectary Resistance high frequency Too late for pre-emptive action Mechanism?

Biochemical

Mechanism Some prediction of cross resistance

Infrastructure Specimens stored frozen Resistance high frequency

Molecular ((PCR,, HOLA,, SSOP)

Dried specimens Exact mechanism Mode of action (CROSS RESISTANCE) q ((EARLY Detect at low freq DETECTION)

Infrastructure Few specific metabolic markers

© The Liverpool School of Tropical Medicine

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Tools for the Future Reduce transmission of mosquito mosquito-borne borne pathogens New Insecticides Decision Support Vector Population Monitoring Tools Insecticide Quantification Kits

Vector Population Monitoring Tool (VPMT) iimproved d resistance i t monitoring it i ttools l th thatt are applicable li bl iin disease endemic countries

Courtesy Brian Sharp

Diagnostics 9 9 9 X

Vector species ID Parasite infection Target-site insensitivity Metabolic resistance

RR R+ 20 Cycle


25

30

35

40

++

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Current Toolkit Anopheles gambiae

• Knockdown-resistance Bass ett all Malaria B M l i Journal J l 2007; 2007 Mzilahowa M il h ett all 2008 Med Vet Entomo; Ridl et al Malaria Journal 2008

• Acetylcholinesterase Bass et al 2010 Pesticide Biochemisty and Physiology

• GABA receptors Nikou et al (in prep)

• Species identification Bass et al Malaria Journal 2007 Bass et al Acta Tropica 2008

• Plasmodium infection Bass et al Malaria Journal 2008


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VPMT pipeline for metabolic resistance candidates Phenotype populations for insecticide resistance

Phenotype Candidate Validate

Identify candidate loci associated with insecticide resistance Gene expression group- microarrays, qPCR Genotyping group- association mapping

Validate candidates are the likely cause of resistance Enzyme characterization group

Technology Field test

Develop DEC appropriate technology for detecting the validated resistance locus

Field test the kits in DEC setting Africa Africa, Latin America and South East Asia © The Liverpool School of Tropical Medicine

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Current Status: Detox g genes overexpressed p in permethrin resistant Anopheles gambiae Gh Ghana

P450

Location

CYP6P3

Ghana (Dodowa); Benin (Akron and Gbedjromede); Nigeria (Orogun)

CYP6M2

Ghana (Odumasy and d Dodowa); d ) Benin B (Akron and Gbedjromede); Ni Nigeria i (Orogun (O )

CYP325A3

Kenya y (Kisumu (RSP)); Nigeria (Ipokia)

CYP6Z1

Kenya (Kisumu (RSP))

CYP6Z2

Ghana (Odumasy)

CYP6AK1

Ghana (Dodowa);

Muller et al. al (2008) PLoS Genetics

Benin

Djouaka et al. al (2008) BMC Genomics. © The Liverpool School of Tropical Medicine

Muller, 2008 PLoS Gen

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Functionally validated insecticide resistance genes H3C C

C Br

9Recomb. expression –Characterization: 9Recomb Characterization: Detailed Cross Resistance spectrum

CH3

Br

C H

C H

CH

O C

H C

O

C N

O

Deltamethrin

Enzyme Activity 0 min

90 min


P450

Perm (min-1)

Delta (min-1)

DDT (min-1)

CYP6P3 3.1 31

26 2.6

0

CYP6M2 6.0

1.2

0

CYP6Z2

0

0

0

Muller et al. (2008) PLoS Genetics

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9R i t 9Resistance genes id identified tifi d A battery of P450s are associated with pyrethroid resistance in An. gambiae • CYP6M2 and CYP6P3 are most consistently over expressed 9 They metabolise permethrin and d l deltamethrin h with h roughly hl similar l kinetics k •

These are important markers of resistance and targets for insecticide development .


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Designing insecticides that are resistant to detoxification i.e. Stick around and attack the organism Predictive model to stymie metabolism

Experimental validation

C1 is NOT metabolised by CYP6P3 – a major resistance gene Resistance cured © The Liverpool School of Tropical Medicine

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Insecticide Quality Assurance: vital element in planning successful - sustainable interventions

Is it the right dose? Available tools for measuring insecticides on ITM

HPLC‐GS

Antibody (ELISA)

Bioassays

Expensive (>$20) ‐ technically demanding; skilled staff,

Demand for user friendly, cost-effective tests © The Liverpool School of Tropical Medicine

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New Technology gy

Guilbault G ilba lt (1966); (1966) Kaur Ka r et al (2009), Green et al (2009)


Morou et al 2008, Dowd et al 2010)

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A GSTe2-dehydrochlorinase biosensor assay for DDT Good correlation with HPLC in preliminary field trials (India, Uganda)

H+ and Cl‐ ions released by GSTE2 are easily detected (Cl strip test or pH) 9 Highly Hi hl specific ifi for f th insecticidal the i ti id l Patent G01N 33/573 ppDDT. © The Liverpool School of Tropical Medicine

NEXT: Roll out DDT IRS Kit via WHO programs (i.e., India) 14

Malaria Decision Support pp System y

Information can feed back to improve decision making in the management of malaria


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Future: An integrated set of tools for speciation, infection status and resistance mechanisms

Arrays

Regulatory sequences –

Species ID markers

Target site Resistance

Pathogen/ parasite

Metabolic Resistance

gDNA (ETOH/dry)

Biofilm


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Man vs Mosquito

LSTM

Rothamsted Research

Prof Lin Field Dr Martin Donnelly Dr Martin Williamson Dr Hilary Ranson Prof Janet Hemingway Dr Chris Bass SNPing/Illumina Dr David Weetman Dr Craig Wilding Keith Steen

y of Crete University Dr John Vontas Dr Dimitra Nikou Evangelia g Morou

Gene expression data Colorado State University Dr Pie Muller Prof Bill Black Dr Emma Warr Dr Karla Saavedra-Rodriguez Enzyme characterization Dr Mark Paine Dr Bradley Stevenson Patricia Pignatelli Dr Andrew Dowd A d St Andy Steven Hanafy Ismail

Sponsors

Amanda Ball John Morgan