Today's Hottest Molecular Technologies in Infectious Disease Testing with a Review of What's Best in Next Generation Tests. Joel A. Lefferts, Ph.D.

Transcription

1 Today's Hottest Molecular Technologies in Infectious Disease Testing with a Review of What's Best in Next Generation Tests Joel A. Lefferts, Ph.D. Dartmouth-Hitchcock Medical Center Dartmouth Medical School Norris Cotton Cancer Center Overview Basics of Molecular Testing Regulatory issues Reimbursement Current Technologies New Technologies for the Future

2 Why Consider Molecular Testing? Slow-growing/difficult-to-culture organisms Shorter TATs (MRSA screening) Quantitative Testing (HIV, HCV) Genotyping Often more expensive Molecular Testing for Infectious Diseases: The Basics

3 Molecular Testing for Infectious Diseases Target: Microbial, Viral, Fungal (DNA/RNA) Purpose: Qualitative (yes/no) Quantitative (how much) Genotyping (Prognosis or drug selection/prediction) Molecular Testing for Infectious Diseases (Assay Chemistries) Traditional PCR (DNA) or RT-PCR (RNA) Post-PCR analysis (potential for contamination) Gel electrophoresis Capillary Electrophoresis Hybridization to beads, array, etc. Sequencing

4 Post-PCR Analysis Agarose Gel Electrophoresis Post-PCR Analysis Capillary Electrophoresis _ Dye Signal Size (nt)

5 Post-PCR Analysis Luminex Bead Arrays Real-Time PCR Target sequence is amplified in the presence of a reporter (probe) Instrument excites & detects reporter throughout PCR Signal intensity is directly proportional to the amount of amplified DNA Threshold cycle (Ct) determination; the cycle at which target is first detected

6 Real-time PCR Reporter dye Quencher Real-time PCR Threshold Cycle Threshold Value Threshold penetration Threshold Line

7 Molecular Testing for Infectious Diseases: Regulatory Issues Categories of Assays IVD (in vitro diagnostic device) Commercial Kit Complete Protocol (Specimen Results) Commercial bias: IVDs more common for high volume testing List of FDA approved assays

8 List of FDA approved assays Categories of Assays -- LDT (Laboratory Developed Test) Not FDA regulated Developed and validated within lab More rigorous in-house validation needed Lab is completely responsible for assay performance With or without ASRs RUO Assays?? Unknown components; reimbursement?

9 More on ASRs (2007 Guidance) Analyte-Specific Reagents: active ingredient Primer pair Probe (real-time PCR) Manufacturer may not: Provide instructions/protocol Bundle multiple ASRs to form a kit Designate a specific platform/instrument Kits sold as ASRs in the past have been discontinued or submitted to FDA for IVD status Molecular Testing for Infectious Diseases: Reimbursement Issues

10 Reimbursement Issues 1. CPT coding: test/procedural codes 2. Testing in-house/reference labs CPT Coding Test coding Chlamydia trachomatis, amplified probe technique Procedural coding Molecular isolation Amplification Enzymatic digestion Separation

11 CPT Coding Test coding ($48.50) Chlamydia trachomatis, amplified probe technique $48.50 Procedural coding ($40.22) Molecular isolation $ Amplification $ Enzymatic digestion $ Separation $5.60 Reimbursement Issues Comparing cost to perform testing in-house to reimbursement It may be worth introducing testing even if it isn t profitable sending testing to reference lab may result in greater loss.

12 Molecular Testing for Infectious Diseases: Current Technologies Real-time PCR Instruments Applied Biosystems 7500 (96 well plate) Roche LightCycler (32 capillaries) Cepheid SmartCycler (16 Samples; Random Access)

13 Sample Preparation Amplification Detection/Analysis Goal: To combine and automate these steps Roche Ampliprep

14 Roche Ampliprep Quantitative Real-time RT-PCR Assays HIV, HCV, HBV Up to 24 or 48 specimens per batch Can be fully automated Set-up and walk away Results in ~5 hours Large footprint (not benchtop) GeneXpert System

15 I-CORE Module Components Swab GeneXpert Dx System Reagents S Elution Reagent Cartridge

16 GeneXpert System Multiple Modules (1,4, 16, Infinity) Smaller Footprint Random Access Automated Sample Preparation Growing Test Menu CE-IVD -Flu A -MTB/RIF -C. difficile -vana/vanb -MRSA/SA Nasal, SSTI & BC -MRSA -GBS -EV -BCR-ABL -FII/FV

17 Molecular Testing for Infectious Diseases: What s Next? Clinical Molecular Demands on Future Technologies/Platforms Automation (Specimen to Results) Less Expensive Less complex Multiplexing? (Respiratory Viral Panels) More Compact

18 Clinical Molecular Demands on Future Technologies/Platforms Random Access for some applications Large batches for other applications Point-of-care? More IVD assays ASRs and LDTs more suited for larger/academic/reference laboratories BD MAX System (previously HandyLab Jaguar) Fully Automated Nucleic Acid Testing System Rapid Ease of Use Small Footprint Cost Effective Flexible Reliable Results Raw Specimen to Result in min!

19 Specimen Transfer Sample HandyLab Sample Preparation Tube Reagent & Specimen Loading 2-D Custom Bar Code

20 Cartridge Rack Loading

21 Load Microfluidic Cartridge Cartridge carrier Ready to Close Door Patient Sample PCR Cartridge Reagent Strip

22 Close the Door To Start Run Currently in Development Chlamydia trachomatis (CT)* Neisseria gonorrhoeae (GC)* Group B Strep* HSV HSV Typing CMV Targeted Assays Influenza A & B MRSA RSV TB Trichomonas Adenovirus Bordatella BK JC HHV6 EBV Enterovirus M. pneumoniae * Entering Clinical Trail The Verigene System Nanosphere, Inc.

23 The Verigene System Nanosphere, Inc. Random Access Expandable platform Highly sensitive detection by gold nanoparticles Infectious Disease, Genetic, and Pharmacogenomic Testing Protein-based assays in development (Troponin I) The Verigene System Nanosphere, Inc.

24 The Verigene System Nanosphere, Inc. Respiratory Virus Assay (RVNAT SP ) Flu A, Flu B, RSV FDA cleared Moderately Complex designation (CLIA) The Verigene RVNAT SP Workflow Detail for RVNAT SP Collect specimen Use a Nylon- or Rayon-tipped nasopharyngeal swab for specimen collection. After collection, place swab in 3 ml Universal Transport Medium (UTM). Break swab shaft and cap the tube. Prepare Verigene SP* Scan Test Cartridge ID with barcode scanner. Open drawer on Verigene SP and load Extraction Tray, Amplification Tray, Tip Holder Assembly, and Test Cartridge. The Verigene SP automatically verifies that the correct consumables have been loaded. Scan or manually input patient ID using the barcode scanner or Reader. Approved For Customer Use A 48

25 The Verigene RVNAT SP Workflow Detail for RVNAT SP Begin Test Aliquot 200 μl of UTM into Sample Well of Extraction Tray. Latch the drawer clamp and close the drawer. Testing begins automatically. Inside the Verigene SP (automated processes) Lysis buffer and MMPs are added to the sample to extract nucleic acids. Purified nucleic acids are transferred to the Amplification Tray for RT-PCR and UDG decontamination. RT-PCR product is pipetted into the Test Cartridge for test processing. Primary and secondary hybridization occur on the array (slide). 200 μl Approved For Customer Use A 49 The Verigene System Nanosphere, Inc. Respiratory Virus Assay (RVNAT SP )

26 TIGRIS and Panther GenProbe, Inc. Fully Automated Laboratory Information System (LIS) interface Utilizes RNA amplification technology (TMA) TIGRIS GenProbe, Inc.

27 Panther (the next generation) Gen-Probe, Inc. 3M Integrated Cycler

28 Simplexa Assays: Focus Diagnostics (Quest) and 3M Conclusions Current molecular testing can difficult to setup Introduction of automation is making molecular applications more accessible Molecular diagnostics is a small community, ask around. Trendy science does not always equal good science.

29 Predictions: The Next Five Years. More molecular testing being performed in smaller labs and as point of care testing. Automated and/or random access platforms will predominate the market. Track systems will be introduced for larger molecular labs. Test Menu Staffing Technologies

30 Dartmouth-Hitchcock Medical Center and Dartmouth Medical School Molecular Pathology Gregory Tsongalis, PhD (director) Claudine Bartels, PhD (supervisor) Samantha Allen Heather Bentley Betty Dokus Susan Gallagher Arnie Hawk Rebecca O Meara Elizabeth Reader Brian Ward Translational Research Lab Gregory Tsongalis, PhD (co-director) Wendy Wells, MD (co-director) Mary Claire Schwab Rebecca O Meara Carol Hart