Session III Accurate and rapid diagnostic tests are an essential component of initiatives to control and eradicate transboundary viral diseases such as foot-and-mouth disease (FMD). Tests are deployed to confirm initial cases of diseases, monitor the spread of the virus, characterise the causative viral strain, and to provide evidence of disease freedom after outbreaks. This presentation reviews progress to develop new diagnostic tools that can be used to detect and characterise viruses that cause important livestock diseases, including a “molecular toolbox” of new lineage-specific real-time RT-PCR assays that have been developed to quickly identify which FMDV lineage is present in samples. Away from laboratory testing, recent data demonstrates that simple sample preparation protocols and new equipment that utilize lyophilized reagents allow real-time RT-PCR or isothermal RT-LAMP assays to be performed in simple laboratories or in field settings in under 60 minutes.

1. An introduction to novel diagnostic
technologies:
Innovation in molecular diagnosis, next generation sequencing and
molecular epidemiology for FMD and FAST diseases
Donald King
Co-authors: Natasha Edwards, Andrew Shaw, Britta Wood,
Valerie Mioulet, Jemma Wadworth, Nick Knowles

2. Academia/Government
• IZSLER, Italy - Santina Grazioli, Emi Brocchi and Colleagues
• NCFAD, Canada - Charles Nfon, Min Yang and Colleagues
• SUA, Tanzania - Chris Kasanga and colleagues
• KVS, Kenya - Eunice Chepkwony and colleagues
• TVLA, Tanzania – Deogratius Mshanga and colleagues
• NAHDIC, Ethiopia – Daniel Gizaw and colleagues
• University of Glasgow - Tiziana Lembo, Sarah Cleaveland,
Richard Reave, Richard Orton, Julien Reboud, Jonathan Cooper
• University of Miyazaki - Wataru Yamazaki
• EuFMD
Commercial companies
• Tetracore (USA) - Rolf Rauh and Bill Nelson
• Optigene (UK) - Duncan Clark and colleagues
• Global Access Diagnostics (UK) – Alison Wakeham and colleagues
… and many past members of the WRLFMD
… and many livestock owners
David Paton
Partnerships are essential – acknowledgements

3. Suspect case
of disease
LOCAL
CLINICAL
OBSERVATION
LABORATORY
DIAGNOSIS
New Technologies
Two key drivers:
• Point-of-decision tests
• Earliest recognition of exotic diseases
• Particularly for disease-free countries
• Improved global surveillance
• More dynamic disease surveillance
• Clear line-of-sight between field and epi teams
• Ensure best samples are collected
Changing the rules for diagnostics?
>40% of samples
received to the FMD
Network are NVD

4. • Largely driven by advances in
human “personal” medicine
• Increased awareness of these
testing methods due to COVID-19
• >2 billion LFDs used in the US
(Oct 21 – May 22)
1. Immunoassays
o Lateral-flow devices
2. Molecular tests
oMobile PCR assays
o Isothermal tests
3. New “third generation”
sequencing approaches
Detection platforms and technologies
Tricorder has three
functions; Sensing,
Computing and Recording

5. Bridging the “Valley of Death”
Basic and
applied research
Marketing and
Commercialization
Resources
• Many tremendous ideas do not proceed beyond the first peer-
reviewed paper!
• Is a test really needed? – what is the demand?
• Should we engage with the commercial sector at an earlier stage?

6. • Quick and simple to perform
o Rapid confirmation of FMD in the field (<10
mins)
o Also useful in the Lab for triage of samples
• Similar assay performance to lab-based
Ag-ELISA
• Pan-serotype and “typing” versions are
available
o For FMD, poster by Foglia (Session III)
o For LSD, poster by Baselli (Session III)
• Relatively limited uptake for routine use
• Difficulties to access LFDs from Svanova
• Do costs inhibit the use of LFDs for
routine/wide-scale testing?
FMDV
Pos
FMDV
Neg
Lateral-flow devices
for antigen detection

7. Paulo-Fupi Raphael in Tanzania
• FMDV can be detected by real-
time RT-PCR using nucleic acid
recovered from positive LFDs
• RNA is stable > 1 month
• Possible to generate larger RT-
PCR amplicons
o Strain characterisation
o “live” virus recovery following
transfection
• Use of LFDs to ship FMDV
positive material to Ref. Labs?
o Posters by Bernelin-Cottet and
Romey (Session III)
Not only local testing
What else can we use LFDs for?

8. Isothermal amplification (LAMP and RT-LAMP)
• Lab and mobile PCR
o Portable instrumentation
o Pan-serotypic or lineage-specific assays
• Nucleic acid amplification at a single
temperature (isothermal)
o Such as LAMP – Loop-mediated
isothermal amplification
o No need for fragile precision
instrumentation
o More suitable for use in the field?
• Many assays have been developed
for livestock pathogens
• Does access to hardware inhibit the
uptake of these assays?
NA amplification technologies

9. Howson et al., (2017) Transboundary and Emerging Diseases
“Ready-to-use”: lyophilised reagents
• Different formats evaluated for RT-PCR and RT-LAMP:
 Fluorescence-based detection
 LFD – for dual-labeled LAMP products
• Lyophilization of reagents did not adversely affect the
performance of the assays
• Simple-to-use sample prep. pipelines can be employed
• Use in East Africa to detect FMDV in acutely infected as well as
convalescent cattle

10. Progress favours simple solutions
• Simplicity is often delivered through “hidden” complexity
Heater
LFD slot
Battery Pack
Space for smartphone
• Smartphone-based heater with sealed chip for LAMP

11. Sequencing is becoming easier and cheaper
European Pharmaceutical Review
• Technology is no longer the limiting factor
• 2019-22: >10 million submissions generated for SARS-CoV-2
• Data deluge: analyses of data is now a significant challenge!

12. MinION sequencing – for regional/national labs?
• Rapid and portable sequencing
method for genome sequencing
• Sample-to-sequence <24 hrs
• Equipment requirements: PCR
machine, Qubit, MinION,
• Preliminary data indicates that the
protocols can be applied for FMDV
lineages
• (talk – Shaw, this session)
• Is this platform a step towards
democratising viral sequencing?
… or are there other factors that
need consideration?
Brown et al., 2021 – capsid sequencing protocol

13. Imagining a “new” diagnostic pipeline
Sample
Testing
in
situ
Testing
at
Lab
Shipment of
inactivated material
• Is FMDV present?
• What serotype is it?
• Immediate feedback
Simple-to-use
sampling protocols
• Sequence analyses
• Characterization of
field isolates
Satya Parida and Francois Maree

14. • Selecting the “winning” technology
• Bridging the gap between assay
development and marketing of tests
o Engagement of end-users – does the test
have a practical utility?
o Demand: Is the market big enough to
warrant investment?
o Freedom to exploit IP
o Partnerships between commercial and
academic sectors
• Acceptance of tests (and their results)
o How are the results used?
o Validation: involvement of WOAH?
o “Data” need context to become
“information”
Conclusions – some key challenges and considerations