Realising the potential of simple isothermal molecular tools for field diagnosis of FMD

Transcription

1 Realising the potential of simple isothermal molecular tools for field diagnosis of FMD Emma Howson Research Scientist, Vesicular Disease Reference Laboratory Group, The Pirbright Institute 1

2 The Problem: Field deployment of RT-LAMP Requirements for in situ diagnostics: Three elements of a molecular test (sample preparation, amplification, detection) Ready-to-use kit Reagents compatible with field deployment 2

3 The Solution: Lyophilised reagents Thermostable reagents Experimental or field use rrt-lamp Field use 3

4 Molecular LFDs: Singleplex format Biotin Anti-biotin antibody linked to latex beads Flc TEST LINE Anti-Flc antibody CONTROL LINE* Anti-Ig antibody *control represents a read line indicating LFD has run correctly 4

5 Test --- Control --- Lyophilised singleplex RT-LAMP: Analytical sensitivity comparable to rrt-pcr Laboratory concordance (rrt-pcr/wet/dry) Dilution series of RNA standard Comparison with the gold-standard Callahan rrt-pcr Copy number of RNA standard RT-LAMP (RNA standard)* Negative rrt-pcr /- - Wet reagents Lyophilised reagents rrt-lamp rrt-lamp /- - *RNA standard supplied by Graham Freimanis 5

6 Expansion of simple sample preparation: From the laboratory to the field Cattle samples from experimental infection Serum samples OP* fluid samples 0dpc 1dpc 2dpc 3dpc 0dpc 1dpc 2dpc 3dpc rrt-pcr Extracted RNA rrt-lamp Neat NS NS NS + 1 in 5 dilution NS From Jose Gonzales 1 in 10 dilution *oesophageal-pharyngeal (probang) **Non-specific amplification 6

7 Test --- Control --- Detection of FMDV in clinical animals: Two days after the appearance of clinical signs Animal ear tag Negative** Mobile rrt-pcr* Foot Epithelium rrt-lamp Serum OP fluid rrt-lamp rrt-lamp Epithelial samples positive by rrt-pcr were also positive by Ag-LFD King et al., 2012 (data from Alexandersen et al. 2003) *mobile rrt-pcr is ~one log less sensitive than the OIE laboratory gold standard **Negative water control 7

8 Test --- Control --- Detection of FMDV in late infection: Ten days after the appearance of clinical signs Animal ear tag Negative Mobile rrt-pcr* Mouth Epithelium rrt-lamp Serum OP fluid rrt-lamp rrt-lamp Epithelial samples were all negative on Ag-LFD *mobile rrt-pcr is ~one log less sensitive than the OIE laboratory gold standard King et al., 2012 (data from Alexandersen et al. 2003) 8

9 Test --- Control --- Detection of FMDV in clinically normal cattle: One month after the appearance of clinical signs (Serengeti) Animal ear tag Serum OP fluid rrt-lamp rrt-lamp previously displayed clinical symptoms never displayed clinical symptoms 9

10 Test --- Control --- Detection of FMDV in clinically normal cattle: ~Six weeks after the report of clinical signs (Morogoro) Animal number Serum OP fluid rrt-lamp N/A N/A N/A N/A N/A N/A N/A N/A rrt-lamp * * Farm 1 Farm 2 *confirmed by mobile rrt-pcr 10

11 Design of field protocols - separation of animal sampling and pre / post LAMP procedures - disposable on site - use of closed systems Use in different FMD situations - outbreaks in non-endemic vs endemic regions - species, serotypes, strains, environments Operator characteristics and sample quality - samples affecting results Field observations: looking to the future vs Source The Pirbright Institute Source EuFMD 11

12 Summary RT-LAMP and - simple sample preparation protocols - simple amplification and detection methods - performed in situ on OP fluid, serum and epithelium - lyophilised reagents suitable for field deployment Sample collection to result calling in < 30 minutes 12

13 Co-authors Veronica Fowler (Pirbright) Sarah Cleaveland (University of Glasgow) Bryony Armson (Pirbright) Donald King (Pirbright) Valerie Mioulet (Pirbright) Miki Madi (Pirbright) Christopher Kasanga (Sokoine University) Sengiyumva Kandusi (Sokoine University) Acknowledgements Jose Gonzales (Pirbright) Graham Freimanis (Pirbright) WRLFMD staff Raphael Sallu (TVLA) Raphael Mahemba Shabani Magumu field staff Optigene Ltd. 13