1. Point-of-Care PCR Testing for Respiratory pathogens:- Multiplexed Viral Panels David Squirrell, KTN Meeting September 2013

2. 1)Enigma Diagnostics: the company 2)The ML instrument platform for P-o-C molecular diagnostics 3)Assay cartridge design and function 4)Respiratory virus panel assay development 2

3. 3 1)Founded in 2004 as a VC-funded spinout from the MoD 2)Currently around 40 staff 3)Mainly based at Porton Down in Wiltshire with an administrative office in London and a cartridge “fill-and-pack” facility in San Diego 4)Assay development and R&D at Porton 5)ISO 13485 accredited 6)Our focus is on respiratory diseases Enigma Diagnostics

4. 4 Enigma ® ML System Manufacturing Instruments Supply Chain & Assembly by Tecan (Switzerland/Austria) Cartridge Manufacture by Hi-P (Singapore) Assays Developed and Packaged into Cartridges (US and USA)

5. 5 1)Initially Enigma followed two strands of development: a fieldable instrument designed for biological agent detection and veterinary pathogen detection in the field (MoD funded) a clinical instrument for point of care use (DoH funded) 2)The system developed for clinical use proved too expensive to manufacture for both the instrument and the consumable 3)The system for field use was technically successful, but from a commercial view, the market is too fragmented (both temporally and spatially): so the focus of the business became clinical diagnostics 4)The ML instrument has been produced by adapting the field system’s technical approach to clinical targets 2)The ML instrument platform for P-o-C molecular diagnostics

6. 6 Development of ML System

7. 7 ML system ● The ML system:  Designed for ease of use (US CLIA waiver)  All reagents are room temperature stable  No reagent additions or manipulations of the consumable are required other than adding the sample tube  The system, with a built-in user interface and barcode readers, makes data entry and sample handling simple

8. CARTRIDGE LOADING Barcode Reader Graphical user interface with touchscreen Between 1 and 6 bolt-on processing modules Tray opened from touchscreen for operator to insert cartridge Control module with printer

9. 3) Assay cartridge design and function 2. Transfer to Tube3. Place Tube in Cartridge 4. Enter Data 5. Load Instrument 1. Take Sample 6. READ RESULTS 9 9

10. 10 ML Cartridge Tube (+ Swab) Elution Buffer Wet Area Foil Stopper Sample Well FD Reagent Pots and Foil (up to 3) Cutter Wand Diaphragm (x 2) Pipette ECP Pipette Cartridge Base ECP Main Cartridge Body Lysis Buffer Wash Wells (x 3) Barcode Label Spare Well

11. x-drive Internal barcode reader Sample sensor Tool sensor z-drive Gripper assembly The Enigma® ML: Processing Module Thermal cycling contacts Completely independent modules capable of running a full assay test, Reports result to CM upon completion Low maintenance: no fluidics- all sample interfaces via the consumable Thermal module and optics block with independent calibration in each PM

12. 12 Magnetic beads ● Fast collection of magnetic beads without forming clumps ● Simple mixing of beads for washing and extraction ● Heated tip to speed up extraction (can also be used to incubate enzyme reactions) ● Rare earth magnet enables high temperature extraction or enzyme denaturation 0 seconds 5 seconds10 seconds No magnet No Bead Clumping

13. ROBOTICS AND PROCESSING “waist”portion operates grabber for picking up tools from cartridge; integral heater (for sample processing: tip operates pipettor tools; magnet on tip for transferring magnetic particles. Grabber on X-axis gantry: (Z-1axis) with Plunger on co-axial Z-2 axis:

14. Enigma ML Storyboard for Automated Boom-based Extraction

15. Process control PCR / RT-PCR enzyme mix Sample W1 W2 W3 W4 Piercer Wand Sample pipettor PCR pipettor Stopper ECP Elution buffer Lysis buffer Sample tube Wash wells Magnetic beads Spare pot Enigma ML Cartridge Layout KEY to functional components  Mechanical  Liquid transfer  Bead transfer  Heating       

16. 1. PREPARING THE CARTRIDGE The Piercer is used to open foils in the order indicated.  1 8 2 7 3 4 5 6 9

17. 2. RELEASE THE SAMPLE The Sample tube is pushed forward into the tube holder to rupture the foil on its cap allowing sample to flow into the Sample well which is assisted by shaking the tray.

18. 3. ADD THE SAMPLE PROCESSING CONTROL The Sample pipettor is used to transfer sample into the freeze dried Process control (MS2) and to mix it in.  MIX

19. 4. ADD SAMPLE TO THE LYSIS REAGENT The Sample pipettor then transfers the sample plus Process control into the Lysis well containing guanidium salts. 

20. 5. ADD BEADS TO LYSED SAMPLE The Wand is used to transfer the magnetic beads into the lysis well. 

21. 6. BIND RELEASED NUCLEIC ACID The Wand is used to mix the sample with lysis buffer and to keep the magnetic beads suspended. Back and forward tray vibration may be used to assist the process. Extracted nucleic acid binds to the beads.  MIX

22. 7. TRANSFER BEADS FOR 1 st WASH The Wand is used to transfer the magnetic beads into the first Wash well containing ethanol & guanidum. 

23. 8. 1 st WASH The Wand is used to mix the magnetic beads in the wash solution. Back and forward tray vibration may be used to assist the process by helping to keep the beads suspended.  MIX

24. 9. TRANSFER BEADS FOR 2 nd WASH The Wand is used to transfer the magnetic beads into the second Wash well containing ethanol & guanidium. 

25. 10. 2 nd WASH The Wand is used to mix the magnetic beads in the wash solution. Back and forward tray vibration may be used to assist the process by helping to keep the beads suspended.  MIX

26. 11. TRANSFER BEADS FOR 3 rd WASH The Wand is used to transfer the magnetic beads into the third Wash well containing ethanol. 

27. 12. 3 rd WASH The Wand is used to mix the magnetic beads in the wash solution. Back and forward tray vibration may be used to assist the process by helping to keep the beads suspended.  MIX

28. 13. TRANSFER BEADS FOR 4 th AND FINAL WASH The Wand is used to transfer the magnetic beads into the fourth Wash well containing ethanol. 

29. 14. 4 th WASH The Wand is used to mix the magnetic beads in the wash solution. Back and forward tray vibration may be used to assist the process by helping to keep the beads suspended.  MIX

30. 15. TRANSFER BEADS FOR THE ELUTION STEP The Wand is used to transfer the magnetic beads into the Elution well (this has a smaller volume than the Wash wells to allow concentration of the extracted nucleic acid), but there is an additional step before the beads are released. 

31. 16. ELUTION BUFFER HEATING The Wand is used to heat the elution buffer (at this point the beads are attached to the tip of the Wand). At the end of the heating step the Wand is lifted out of the buffer, the magnet is withdrawn and then the Wand is lowered back into the buffer to release the beads. HEAT 

32. 17. NUCLEIC ACID ELUTION The Wand is used to mix the magnetic beads in the elution solution. Back and forward tray vibration may be used to assist the process by helping to keep the beads suspended.  MIX

33. 18. BEAD REMOVAL The Wand is used to dump the beads in the nearest Wash well. No mixing as such is required. 

34.  19. REHYDRATION OF PCR MIX WITH ELUTED NUCLEIC ACID The PCR pipettor is used to add eluted nucleic acid to the freeze dried PCR mix.

35.  20. FILL ECP CAPILLARY The PCR pipettor is used to transfer the final mix to fill the ECP capillary.

36.  21. SEAL WITH OIL The PCR pipettor is then used to add mineral oil to overlay the PCR reaction mix.

37.  22. STOPPER ECP CAPILLARY The Stopper is used to seal the ECP capillary.

38.  23. CAP LYSIS The Piercer is used to cap the Lysis well.

39.  24. CAP SAMPLE The Sample pipettor is used to cap the Sample well.

40.  25. RT-qPCR

41. 26. RESULTS ANALYSIS The fluorescence data are analysed in the Processing module by the embedded algorithm and then the results are sent to the Control module for display and printout.

42. 27. CARTRIDGE EJECTION

43. 43 1)In Europe, 50% of GP visits are for respiratory infections 2)The majority of these are for viral infections, especially rhinoviruses which cause the common cold 3)The principle respiratory viral diseases of concern are influenza in old and young age groups and respiratory syncytial virus in infants 4)Antivirals rather than antibacterial antibiotics are required for treatment 5)Molecular diagnostics have become the standard for these infections 4) Respiratory virus panel assay development

44. 44 Three Peak Resolution on Single Dye Channel H5N1 H274Y mutant H1N1 WT H1N1 H274Y mutant or H5N1 WT Multiplexing is achieved by end-point melt analysis

45. Influenza A/B & RSV Influenza A (channel 1) Influenza B (Channel 2) RSV (Channel 1) Identifies Utilises trans (one primer acts as acceptor in dual hyb format) hybridisation signalling to multiplex 5 primer pairs & 7 probes in a single closed tube assay  Influenza A  Influenza B  Respiratory Syncytial Virus (RSV)  Sample Extraction control (MS2) Partnered with GSTT and Edinburgh Royal Infirmary for clinical data Detects Influenza Respiratory Syncytial Virus (RSV) an example of multiplexed dual hyb probes and melt analysis using just two fluorescent channels

46. 46 A DLS_05736 (true +VE) DLS_05746 (true +VE) DLS_05751 (true +VE) DLS_05754 (false –ve) Influenza A/B & RSV

47. No of Strains analysed Time PeriodConservation of G residue All human strains33502008-201395% H1N1 only21922008-201398% H1N1 only7052010-201399% H3N2 only11142008-201392% H3N2 only6282011-201288% H3N2 only2132012-201372% Conservation of the G residue in H3N2 lower than in H1N1 and dropping over time Sequence data suggested the undetected Inf A was an H3N2 strain H1N1 strains not variant Only 5% variable across H1N1 strains

48. Highly Multiplexed Respiratory Viral Assays 48 Inf A/B, RSV AssayRVP 1 AssayRVP 2 Assay TargetsCallTargetsCallTargetsCall Influenza AInf A +/-Influenza AInf A +/-Influenza AInf A +/- Influenza BInf B +/-Influenza BInf B +/-Influenza BInf B +/- RSV ARSV +/-RSV ARSV +/-RSV ARSV +/- RSV B Control+/-Parainfluenza 1Para +/-Parainfluenza 1Para +/- Parainfluenza 2 Parainfluenza 3 Rhino ARhino +/-Rhino ARhino +/- Rhino B Rhino C Control+/-Adenovirus AAdeno +/- Adenovirus B Adenovirus C Control+/-

49. 49 RVP-1 Utilises trans (one primer acts as acceptor in dual hyb format) hybridisation signalling to multiplex 9 primer pairs & 11 probes in a single closed tube assay  Influenza A  Influenza B  Respiratory Syncytial Virus (RSV)  Rhinovirus A/B/C  Parainfluenza virus 1/2/3  Sample Extraction control (MS2) Partnered with GSTT and Edinburgh Royal Infirmary for clinical data PIV 1/2/3 Rhino A/B/C INFB RSVA INFA RSVB MS2 705nm 640nm 670nm640nm Influenza A (Channel 3) Influenza B (Channel 2 or 4 if crossover cannot be controlled) RSV (Channel 3) Parainfluenza virus (Channel 1) Rhinovirus (Channel 1) Identifies Detects Influenza Respiratory Syncytial Virus (RSV) Parainfluenza virus Rhinovirus an example of multiplexed dual hyb probes and melt analysis using three fluorescent channels