a new era in access to high performance diagnostics?...• david paton • eleanor cottam • jemma...

Molecular Characterisation & Diagnostics Group

Institute for Animal Health

A new era in access to high performance diagnostics?

Donald King

Molecular Characterisation and Diagnostics Group, IAH


1. FMDV-specific assays have been developed to test a wide range of samples – address different clinical circumstances

2. Likely to be increased demand for testing in future

3. Devolved and POC formats offer potential to change the way that testing is organised and implemented

Summary and Prospects:


Virus isolation

(CTY or IBRS2)

Ag ELISA

Time to report result (hrs)

1-4 days

~4 hours

Current assays for FMDV detection

AutomatedTaqMan®RT-PCR

~5 hours

1 10 100


Automated real-time RT-PCR• First Case: 2 pan-serotype assays in routine use

– 5’UTR and 3D

• 3D assay selected in early stages of outbreaks

• Automated extraction equipment used to

prepare nucleic acid

– Roche MagNA PURE LC

– Qiagen BioRobot Universal

• Qiagen: interim protocol using “off-deck” lysis

• Up to 84 samples could be processed in

~5hours

• Highest demand: 311 samples/day


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FMD 2007 - daily submissions:

August September

•Outbreaks in southern England occurred in 2 distinct

phases

Date

Da

ily s

ubm

issio

ns


Sample type Number

submitted rRT-PCR

Ag-ELISA VI

Epithelial suspension

50 50 49 50

Tissue suspension 1 1 1 1

Vesicular fluid 2 1 1 1

Fluid 3 3 - -

Serum 3115 3086 - 585

EDTA-blood 32 32 - 32

Probang 36 36 - 36

Swab 6 6 1 3

Faecal suspension 1 1 - 1

Total samples tested 3246 3216 52 709

% of samples tested 99.1 1.6 21.8

•rRT-PCR was widely used to test submitted material

FMD 2007 – laboratory test selection:


DAYS

ME

AS

UR

EM

EN

T

14654321

1 Rapid confirmation of clinical signs

2 Active surveillance for infected animals(including pre-clinical cases)

87

Diagnostic windows

● ● ● ●

FMD virus

in blood

Clinical

lesionsantibody

response

3 sero-surveillencefor FMDV exposed animals

Representative “in contact” cattle data from Alexandersen et al., 2003 and unpublished data from IAH

What are we trying to do?


IP2c: pre-clinical diagnosis

• Additional holding from an IP

• All animals closely checked for clinical signs

• with negative results

• 19 of 58 animals rRT-PCR positive blood

samples

• Subsequent testing by VI supported these findings

• Indicates near simultaneous infection of multiple

animals

• First use of real-time preclinical diagnosis for

FMD in field


Active surveillance: use of rRT-PCR for preclinical detection

• Intensively Patrolled Areas (IPA) established

within protection zone (PZ) around Egham

• Visited every-other day

• Clinical examination

• Blood samples collected and tested for FMDV

by rRT-PCR

• 14 “high risk” cattle herds

• ~ 500 animals

• Reduced un-necessary slaughter

• Is this realistic for a larger outbreak????


Infected animal

LOCAL

CLINICAL

OBSERVATION

LABORATORY

DIAGNOSIS

(Local or NRL)

INTERNATIONAL

REFERENCE

LABS

Samples for:•FMDV detection•Serotype-characterisation•Serology•In-vitro vaccine matching•VP1 sequencing•Full-genome sequencing•Cross-protection studies

New technologies/new opportunities


AssaySpecificity

AssaySensitivity

New assay formats: considerations

• Reliability– Simple-to-use

– Non-specialist

• Performance– Limit of detection

– Ability to correctly identify infected animals with diverse FMDV strains

• Speed

• Scalability

• Cost ?


Lateral-flow devices for FMDV Antigen detection

• Talk by Nigel Ferris

• Developed by IAH in collaboration with Brescia and Svanova

• Quick and simple to perform

• Pan-serotypic

• During 2007: used for rapid (<10 mins) confirmation of FMD in the field

• Also useful in the Lab

• LFD marketed by


Rapid detection of FMDV in the field: Portable PCR platform

Smiths Bio-Seeq VetTM

• Talks by Carmelo Volpeand Ken Pierce

• Non-specialist user– Nucleic acid extraction– PCR set-up– Analysis

• 5 independent modules

• Battery operated

• Decontaminate by immersion

• Field trial (Turkey)

• Platform for other livestock diseases


Non-invasive sampling• Air-samplers (MesoSystems)

• Hand held

• Simple-to-use

• Integrated with FMDV detector?

• Static located in high-risk areas?

BioCapture 650

BioBadge 100

BioCapture BioBadgeCattle

3 dpi10.24 11.23

Log10 FMDV copies detected by rRT-PCR after 5 minute collection near animals infected with FMDV (serotype Asia-1) (Ryan et al., 2007)


• Nucleic acid amplification at a single temperature• No need for fragile precision instrumentation

• Basis of disposable device / cost effective• More suitable for use in the field• Very rapid, similar sensitivity to rRT-PCR

Isothermal amplification (RT-LAMP)

0

0.2

0.4

0.6

0.8

1

0 10 20 30 40 50 60

Time in minutes

Flu

ore

sce

nce

(d

RN

)

Dukes et al., 2006

Alternative detector technologies


Detection and characterisation?

Potential Benefits:• Thousands of experiments in parallel• Differential diagnosis• Viral Typing of FMDV isolates• Clarification of mixed infections• Portable formats emerging

DEFRA funded Biochip project (April 06-09)

Viral MicroArray

A

O UKG 12/2001

http://www.bio-chip.co.uk/


Field testing vs centralised testing

• Use of rapid field-based tests to support

diagnosis based on clinical signs• Positives confirmed using LFDs• Support diagnosis (typing) using mobile rRT-PCR• How sample testing during active surveillance

programmes (screening for pre-clinical animals)?

• Role of NRL and International Reference

laboratories• Confirmation of 1st case (in a FMD-free country)?• Strain characterisation• Reduced requirement for “live” virus?• Inexpensive methods to ship samples to Reference

Laboratories?

Roles


Infected animal

LOCALCLINICAL

OBSERVATION

supporting

LABORATORYDIAGNOSIS

(Local or NRL)

supportingINTERNATIONAL

REFERENCE

LABS

Rapid confirmation and negation (?) of

clinical signs for

secondary cases

Serotyping LFDs

Serotyping RT-PCRsLFDs for serology

Isothermal assays

Future organisation of tests?

Use for:

Future

prospects:

First cases in FMD-free countries?Surveillance screening

Strain typing

Molecular epidemiology (VP1 & CG)

Microarrays?

Others?


Future challenges

• Rapid development of technologies

• Key role of commercial partners– Is the market viable?

• Use in FMD-endemic countries

• Availability of technology – Widely available vs

control of local diagnosis/ reporting for notifiablediseases


1. FMDV-specific assays have been developed to test a wide range of samples – address different clinical circumstances

• Role of WRL and NRLs for assay validation

2. Likely to be increased demand for testing in future

• Active surveillance for disease in high risk animals• Pre-clinical testing • High resolution molecular epidemiology

3. Devolved and POC formats offer potential to significantly decrease assay time

• Support of diagnosis based on clinical signs• Integration of different assay formats (virol/sero) • Involvement of end-users and stakeholders is vital

Summary and Prospects:


Acknowledgements:• Scott Reid• Katja Ebert• Nigel Ferris• Geoff Hutchings• Nick Knowles• Juliet Dukes• Yanmin Li • David Paton• Eleanor Cottam• Jemma Wadsworth• Caroline Wright• Zhidong Zhang• Ginette Wilsden• Phil Keel

• Kate Swabey• Pip Hamblin• Sarah Cox• Kasia Bankowska• Andrew Shaw• Annette Saunders• Julie Stirling• John Gloster• Eoin Ryan• Bryan Charleston• Ryan Waters• Bartek Bankowski• Chris Chisholm• Bob Statham

• Carrie Batten

a new era in access to high performance diagnostics?...• david paton • eleanor cottam • jemma...

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