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Today's Hottest Molecular Today's Hottest Molecular Technologies in Infectious Disease Technologies in Infectious Disease
Testing Testing with a Review of What's Best in Next Generation Testswith a Review of What's Best in Next Generation Tests
Joel A. Lefferts, Ph.D.
Dartmouth-Hitchcock Medical CenterDartmouth Medical School
Norris Cotton Cancer Center
Overview
• Basics of Molecular Testing
• Regulatory issues
• Reimbursement
• Current Technologies
• New Technologies for the Future
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…
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
Post-PCR Analysis
Agarose Gel Electrophoresis
Post-PCR Analysis
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
125 130 135 140 145 150 155 160 165 170 175 180 185
_
Size (nt)
Dye
Sig
nal
138.35
139.44
140.46
151.03152.12
153.14
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
125 130 135 140 145 150 155 160 165 170 175 180 185
_
Size (nt)
Dye
Sig
nal
138.35
139.44
140.46
151.03152.12
153.14
Capillary Electrophoresis
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
Real-time PCR
Reporter dyeQuencher
Real-time PCR
Threshold Line
Thresholdpenetration
Threshold Cycle
Threshold Value
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 assayshttp://www.amptwo.org/FDATable/FDATable.doc
List of FDA approved assayshttp://www.amptwo.org/FDATable/FDATable.doc
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?
More on ASRs (2007 Guidance)http://www.fda.gov/medicaldevices/deviceregulationandguidance/guidancedocuments/ucm078423.htm
• 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
Reimbursement Issues
• 1. CPT coding: test/procedural codes
• 2. Testing in-house/reference labs
CPT Coding
Test codingTest coding
8749187491 Chlamydia Chlamydia trachomatis, amplified trachomatis, amplified probe techniqueprobe technique
Procedural codingProcedural coding
83890 Molecular isolation83890 Molecular isolation
83898 Amplification83898 Amplification
83892 Enzymatic digestion83892 Enzymatic digestion
83894 Separation83894 Separation
CPT Coding
Test coding ($48.50)Test coding ($48.50)
87491 Chlamydia 87491 Chlamydia trachomatis, amplified probe trachomatis, amplified probe techniquetechnique
$48.50$48.50
Procedural coding ($40.22)Procedural coding ($40.22)
83890 Molecular isolation83890 Molecular isolation
$5.60$5.60
83898 Amplification83898 Amplification
$23.42$23.42
83892 Enzymatic digestion83892 Enzymatic digestion
$5.60$5.60
83894 Separation83894 Separation
$5.60$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.
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)
Sample Preparation Amplification Detection/Analysis
Goal: To combine and automate these steps
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)
Roche Ampliprep
GeneXpert®
System
I-CORE™ Module
S
Swab
Reagents
Elution Reagent
Cartridge
GeneXpert® Dx System
Components
•Multiple Modules (1,4, 16, Infinity)•Smaller Footprint•Random Access•Automated Sample Preparation•Growing Test Menu
GeneXpert® System
CE-IVD-Flu A-MTB/RIF-C. difficile-vanA/vanB-MRSA/SA Nasal, SSTI & BC-MRSA-GBS-EV-BCR-ABL -FII/FV
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
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
•Rapid
•Ease of Use
•Small Footprint
•Cost Effective
•Flexible
•Reliable Results
Raw Specimen to Result in 45 - 90 min!
Fully Automated Nucleic Acid Testing System
BD MAX System (previously HandyLab Jaguar)
Specimen Transfer
Sample
HandyLab Sample
Preparation Tube
Reagent & Specimen Loading
2-D Custom Bar Code
Cartridge
Rack Loading
Load Microfluidic Cartridge
Cartridge carrier
Ready to Close Door
Reagent Strip
Patient Sample
PCR Cartridge
Close the Door To Start RunCurrently in
Development• Chlamydia
trachomatis (CT)*
• Neisseria
gonorrhoeae (GC)*
• Group B Strep*
•HSV
•HSV Typing
• CMV
• Influenza A & B
•MRSA
• RSV
• TB
• Trichomonas
• Adenovirus
• Bordatella
• BK
• JC
•HHV6
• EBV
• Enterovirus
•M. pneumoniae
* Entering Clinical Trail
Targeted Assays
The Verigene System Nanosphere, Inc.
http://www.nanosphere.us/
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.
The Verigene System Nanosphere, Inc.
• Respiratory Virus Assay (RVNATSP)
• Flu A, Flu B, RSV
• FDA cleared
• Moderately Complex designation (CLIA)
The Verigene® RVNATSP• Workflow Detail for RVNATSP
– 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 Use09-0028-A
48
The Verigene® RVNATSP• Workflow Detail for RVNATSP
– 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 Use09-0028-A
49
The Verigene System Nanosphere, Inc.
• Respiratory Virus Assay (RVNATSP)
TIGRIS and Panther GenProbe, Inc.
• Fully Automated
• Laboratory Information System (LIS) interface
• Utilizes RNA amplification technology (TMA)
TIGRIS GenProbe, Inc.
Panther (the next generation) Gen-Probe, Inc.
3M Integrated Cycler
Simplexa Assays: Focus Diagnostics (Quest) and 3M
Conclusions• Current molecular testing can difficult to set-
up
• 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.
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.
Staffing Technologies
Test Menu
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
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