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

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2500

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3500

4000

4500

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5500

125 130 135 140 145 150 155 160 165 170 175 180 185

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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

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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

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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

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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