A new era in access to high performance diagnostics?

Transcription

1 A new era in access to high performance diagnostics? Donald King Molecular Characterisation and Diagnostics Group, IAH Institute for Animal Health

2 Summary and Prospects: 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

3 Current assays for FMDV detection Virus isolation (CTY or IBRS2) 1-4 days Ag ELISA ~4 hours Automated TaqMan RT-PCR ~5 hours Time to report result (hrs)

4 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

5 FMD daily submissions: Outbreaks in southern England occurred in 2 distinct phases August September 0 03/08 08/08 13/08 18/08 23/08 28/08 02/09 07/09 12/09 17/09 22/09 27/09 02/10 07/10 12/10 17/10 Daily submissions Date

6 FMD 2007 laboratory test selection: rrt-pcr was widely used to test submitted material Sample type Number AgrRT-PCR submitted ELISA VI Epithelial suspension Tissue suspension Vesicular fluid Fluid Serum EDTA-blood Probang Swab Faecal suspension Total samples tested % of samples tested

7 Diagnostic windows What are we trying to do? 2 Active surveillance for infected animals (including pre-clinical cases) 1 Rapid confirmation of clinical signs Clinical lesions 3 sero-surveillence for FMDV exposed animals antibody response MEASUREMENT FMD virus in blood DAYS Representative in contact cattle data from Alexandersen et al., 2003 and unpublished data from IAH

8 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

9 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????

10 New technologies/new opportunities 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

11 New assay formats: considerations Assay Sensitivity Assay Specificity Reliability Simple-to-use Non-specialist Performance Limit of detection Ability to correctly identify infected animals with diverse FMDV strains Speed Scalability Cost?

12 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

13 Rapid detection of FMDV in the field: Portable PCR platform Smiths Bio-Seeq Vet TM Talks by Carmelo Volpe and 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

14 Non-invasive sampling Air-samplers (MesoSystems) Hand held Simple-to-use BioCapture 650 Cattle 3 dpi BioCapture BioBadge Log10 FMDV copies detected by rrt-pcr after 5 minute collection near animals infected with FMDV (serotype Asia-1) (Ryan et al., 2007) BioBadge 100 Integrated with FMDV detector? Static located in high-risk areas?

15 Alternative detector technologies Isothermal amplification (RT-LAMP) 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 1 Fluorescence (d RN) Time in minutes Dukes et al., 2006

16 Detection and characterisation? Viral MicroArray Potential Benefits: Thousands of experiments in parallel Differential diagnosis Viral Typing of FMDV isolates Clarification of mixed infections Portable formats emerging O UKG 12/2001 A DEFRA funded Biochip project (April 06-09)

17 Field testing vs centralised testing Roles 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 1 st case (in a FMD-free country)? Strain characterisation Reduced requirement for live virus? Inexpensive methods to ship samples to Reference Laboratories?

18 Future organisation of tests? Infected animal LOCAL CLINICAL OBSERVATION supporting LABORATORY DIAGNOSIS (Local or NRL) supporting INTERNATIONAL REFERENCE LABS Use for: Rapid confirmation and negation (?) of clinical signs for secondary cases First cases in FMD-free countries? Surveillance screening Strain typing Molecular epidemiology (VP1 & CG) Future prospects: Serotyping LFDs Serotyping RT-PCRs LFDs for serology Isothermal assays Microarrays? Others?

19 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 notifiable diseases

20 Summary and Prospects: 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

21 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