Slide 1: 3base: The Science Behind EasyScreen Molecular Diagnostic Assays
3base™ The Science Behind EasyScreen™ Molecular Diagnostic Assays Nucleic Acid Conversion Technology 1
Slide 2: Topics
Topics • 3base™ Technology Overview • Technical Advantages of 3base™ • Validation of 3base™ Specificity • Validation of EasyScreen Assays – C. difficile, screening panels • Validation of GS1 automation • Future developments 2
Slide 3: Overview of 3base
Overview of 3base™ Basic technical functionality: Moving from 4base to 3base™ Regular Cell Lysis and Preparation Steps Native Sequence Original (Native) 4-base 3base™ Microbial genome proprietary Sodium Bisulfite Treatment Converted 3base™ Microbial Genome 3base™ Sequence Standard Real-Time PCR Inst
Slide 4: Developed to Improve Subtype Similarity
Developed to Improve Subtype Similarity e.g. Non-Converted Influenza Sequences Influenza A virus H5N1 TGTGTGTGCA GGGATAATTG Influenza A virus H7N3 TGTATATGTA GGGACAATTG Influenza A virus H5N8 TGTGTTTGTA GAGACAACTG Influenza A virus H5N3 TGTATATGTA GGGACAATTG Influenza A virus H5N2 TGTGTTTGCA GAGATAA
Slide 5: Conversion to 3base Improves Subtype Similarity and Reduces Variation
Conversion to 3base™ Improves Subtype Similarity and Reduces Variation – Enabling Analysis via PCR e.g. Non-Converted Influenza Sequences 3base™ Converted Influenza Sequences Influenza A virus H5N1 TGTGTGTGCA GGGATAATTG Influenza A virus H5N1 TGTGTGTGTA GGGATAATTG Influenza A virus H7N3 TGTATATGTA G
Slide 6: 3base Technology
3base™ Technology The cornerstone of EasyScreen™ assays • Universally applicable to all specimen types - converts and detects both DNA and RNA – Works with multiple specimen types simultaneously – Stool is first commercially available • Allows for simple multiplexed assays – More targets detected pe
Slide 7: Technical Advantages of 3base Technology
Technical Advantages of 3base™ Technology 7
Slide 8: Technical Advantages of 3base Assays
Technical Advantages of 3base™ Assays One primer set detects multiple species Universal primer F1 Gi|4566774|G 2007 TAACTACTGA TTTGAAAAAA TACTGTCTCA ACTGGAGATT TGAAAGTACA Parainfluenza-1 Gi|67906100| 10794 TAACTACTGA TCTTGCTAAA TACTGTCTTC AATGGAGATA TCAGACCATA Parainfluenza-2 Gi|332716|Gb 5805 TAACA
Slide 9: Validation of 3base Specificity
Validation of 3base™ Specificity 9
Slide 10: Validation of Specificity
Validation of Specificity • Single copy genes may be amplified from a 3base™ converted human genome • High-Risk HPV identified as hardest challenge 10
Slide 11: HPV Validation Study Summary
HPV Validation Study Summary • The HGS tests had a statistically significant higher PPV than the HC2 (Digene) test (P < 0.001) • The HGS test has a lower rate of false positives at the same level of sensitivity as the HC2 (Digene) test (P< 0.001) • The HGS test and the HC2 test showed no statistical
Slide 12: Validation of EasyScreen Clinical Performance
Validation of EasyScreen™ Clinical Performance 12
Slide 13: EasyScreen C. difficile Detection and Reflex Kits
EasyScreen™ C. difficile Detection and Reflex Kits • Rapid real-time PCR kit for detection of C.difficile • Reflex kit to identify hypervirulent subtypes Tom Riley/Kerry Carson to present at ECCMID 2014: sensitivity 93.7% PPVs 96.8% 13
Slide 14: Hypervirulent Strains of C. difficile
Hypervirulent Strains of C. difficile Identified in Australia 2010 14
Slide 15: EasyScreen Gastrointestinal Screening Panels
EasyScreen™ Gastrointestinal Screening Panels Detection Kit Targets EasyScreen™ Enteric Salmonella spp., Shigella spp., Campylobacter spp., Yersinia Bacterial Detection Kit entercolitica, Listeria monocytogenes, C. difficile, Aeromonas (REF: EB003) hydrophila, Vibrio cholera/parahaemolyticus, Shiga
Slide 16: EasyScreen Protozoan Panel
EasyScreen™ Protozoan Panel High performance vs microscopy and RT-PCR Overall Overall Method No. Samples No of Positive samples and sensitivity Sensitivitya Specificityb Blastocystis Cryptosporidium D. fragilis E. complex Giardia 92-100% 100% EasyScreen 358 96% (51/53) 100% (9/9) 95% (41/43) 92% (22
Slide 17: Clinical Sensitivity and Specificity
Clinical Sensitivity and Specificity As presented by Lee Thomas, Westmead Hospital, Sydney, at ASM 2013 Pathogen detected EasyScreen™ Sensitivity Specificity Additional % % pathogens • EasyScreen™ assays identified 79 Viruses (Noro, Rota, Adeno, 69 100 97.1% 25 Astro) infections that were missed usi
Slide 18: Validation of GS1 automation
Validation of GS1 automation 18
Slide 19: The New GS1
The New GS1 Optimised and easy to use for improved workflow • Single platform to perform all sample processing steps (nucleic acid extraction) & set-up of PCR plates (96 or 384 well format) • Optimised for all EasyScreen™ Assays • Improves sample throughput • Eliminates set up errors • Open system f
Slide 20: Clinical Validation of GS1
Clinical Validation of GS1 Ongoing evaluation by Damien Stark, SydPath, Sydney, 221 Specimens Pathogen Conventional Methods* EasyScreen™ • EasyScreen™ Campylobacter 7 9 assays identified 44 Salmonella 8 9 infections that Shigella 5 6 were missed using C. difficile 3 7 traditional methods Yersinia -
Slide 21: Future Developments
Future Developments • Exciting new screening assays in development: – MRSA – Mycobacterium tuberculosis/avium – Meningitis – STIs – Pneumonia – Influenza Detection and Typing – Respiratory Tract Viral Infections 21
Slide 22: Team and Acknowledgements
Team and Acknowledgements • Genetic Signatures Scientific Staff – Dr Douglas Millar – Dr Shoo Peng Siah – Ms Jiny Nair – Dr Kiran Kaur – Mr Christopher French • Tom Olma and Lee Thomas ICPMR • Thomas Karagiannis and Peter Huntington PaLMS • Damien Stark and Tamalee Roberts SydPath 22
Slide 23: Team and Acknowledgements
Team and Acknowledgements • Independent confirmatory PCR/OCP/Tissue Culture – Diane Grote,Virology Department, The Children's Hospital, Westmead – Steven Siarakas, Department of Microbiology and Infectious Diseases, Concord Hospital, Hospital Rd, Concord – Rogan Lee, Parasitology, CIDMLS, Westmead H
Slide 24: Genetic Signatures
al ] Genetic ¢ 9 Signatures