Frederick L. Kiechle, MD, PhDMedical Director, Clinical Pathology

Memorial Healthcare SystemPathology Consultants of South Broward, LLP

Hollywood, FL 33021

Point-of-Care Molecular Diagnostics: The Future

Outline Molecular Diagnostics 3 steps

Miniaturization for POCT application

Proof of Concept Cepheid GeneXpert Enigma Diagnostics DxNA LLC: GeneSTAT IQUUM: Liat analyzer

DNA Sequencing Genia and Oxford Nanopore

Conclusions

Molecular Diagnostics

AACC CPOCT 9/18/20143

The use of DNA/RNA to test for specific states of disease or health

Infectious DiseasesOncologyPharmacogenomicsGenetic Disease ScreeningPersonalized MedicineCoagulation

Three Basic Steps

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Extraction & Purification of nucleic acid

Amplification (making copies)

Detection of amplifiedproduct

Real-Time PCR

End-Product Detection

Microarrays

Luminex (similar to flow analysis)

Sequencing

Features of POCT Molecular Device

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Hand held Portable Cost/test < $25.00 Total time to result < 30 minutes All 3 molecular steps in unit use device Results = clear with no interpretation Accuracy and precision Full process controls (aka internal controls) are extracted, amplified

and detected along with target

Kiechle & Holland. Clin Lab Med 2009; 29:555-560.

Software Features

1. Input user / Patient ID

2. Control assay & System performance

3. Perform data analyses and test interpretation

4. Wireless connectivity

5. Multiplexing and Melting curve analysis

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Miniaturization Using Microfluidics

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Behavior, precise control and manipulation of fluids constrained to small (submillimeter) scale

Micro features

• Small volume (nL = 10-9 L/ pl = 10-12 L)• Small size• Low energy consumption• Effects of the micro domain

Used in development of

• Inject printheads• DNA chips• Lab-on-a-chip technology

• Sia S, Kricka L. Lab Chip 2008; 8:1982-1983.

POCT Molecular Diagnosis in Canadian Urban Mobile Health Clinics

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Interviewed 9 mobile clinic directors and supervisors from 6 clinics

All supported further development of rapid molecular testing using: microfluidics microchip-based platform with integrated sample prep, genetic

amplification, and detection on one chip

Device ~ $1,000; disposable microchip $1 each

Infectious disease detection: CT, NG, HPV, HIV

Navid EL, et al. Point of Care 2011; 10:40-44.

POCT: Benefit to Clients

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Reduced waiting times

Fewer or no follow up visits

Immediate discussion of

results and Rx

Improve healthcare

accessibilityNavid EL et al. Point of Care 2011; 10:40-44

February 2012

Cepheid GeneXpert Features Single step assay

Place premeasured reagents into labeled parts in the cartridge

70 minutes for most assays

Includes sample processing control to monitor reaction steps and inhibitors

Enigma Diagnostics

www.enigmadiagnostics.com

Founded in 2004

Porton Down, Salisbury UK

POC infectious disease CE mark flu A/B + RSV

1/14/14 Development: MDR-TB

February 2012

Assembled Assay Cartridge

ECP Capillary

RSC Special Report. 2009;(No.317):238-244.

DxNA LLC St. George, Utah

POC Infectious Disease

GeneSTAT

+ 2009 A /H1N1 influenza test, not FDA approved, but authorized by FDA for Emergency Use. Used Roche High Pure RNA Isolation Kit

+ Also Avian flu H5N1 test evaluated in Vietnam in 2010

1/2/14 acquired PathoGene LLC: MSSA/MRSA; Flu A/B; Coccidioidomycosis in development

GeneSTATScreen shot and results

Device LoadedCraig Mosman of DxNA in Hanoi 4/25/2010

GeneSTAT Features Portable, battery powered One button operation after sample is loaded Built to be CLIA waved Disposable, pocket-sized, single use plastic unit Patented close system and photometric cell Sonication with high frequency sound waves to lysis cells PCR chemistry is freeze dried; shelf life 6 months at room

temperature CE marked in 2011 Cartridge test ID with RFID embedded

IQUUMLiat™ Analyzer Advantages

Rapid sample-to-result automation with quantitative analysis

Completely closed system eliminates contamination and allows nucleic acid testing in any setting

Flexible platform is adaptable to various assays and analytes

Flexible tube divided into sealed segments

IQUUM Update Purchased by Roche Molecular Diagnostics April 7, 2014

Liat Influenza A/B Assay CE marked and FDA cleared

Cost/Sample:$400K ‐ >$1M $25K‐$50K $1K‐$5K Genia <$100

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Genia Technologies, Inc.

Mountain View, California

6/2/14 acquired by Roche Diagnostics

Single molecule nanopore-based sequencing by synthesis

Distinguishes 4 bases by detecting 4 different sized tags released from 5´-phosphate modified nucleotides

Sci Rep 2, 682: DOI:10.1038/step00684 (2012).

Genia Technology

Figure 1. Schematic of a single molecule DNA sequencing by a nanopore with phosphate tagged nucleotides. Genia’s NanoTag sequencing approach identifies DNA sequences not by detecting the nucleotides themselves with the nanopore, but by measuring the current changes caused by the passage of each of four different tags that are released from the incorporated nucleotide during the polymerase reaction.

Time (ms)

DNA Sequencing To Go

Oxford Nanopore's MinION device. Credit: Oxford Nanopore

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Oxford Nanopore

MinION – Disposable DNA Sequencing Size of USB memory stick < $900.00 Currently 2,000 nanopores

2013: 8,000 nanopores – 20 node installation – complete human genome sequence in 15 minutes Specimen – whole blood, no extraction or amplification required

2014: New membrane to support nanopore 10,000 base sequenced at one time

Science 2014;343:829-830

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Nanopore Sensing

This diagram shows a protein nanopore set in an electrically resistant membrane bilayer. An ionic current is passed through the nanopore by setting a voltage across this membrane. If an analyte passes through the pore or near its aperture, this event creates a characteristic disruption in current. Measurement of that current makes it possible to identify the molecule in question. For example, this system can be used to distinguish between the four standard DNA bases G, A, T and C, and also modified bases. It can be used to identify target proteins and small molecules, or to gain rich molecular information, for example to distinguish between the enantiomers of ibuprofen or study molecular binding dynamics.

Conclusion

1. The interest in the miniaturization of the three distinct steps in PCR-based molecular methods is currently under intense investigation.

2. Soon, portable, hand-held, inexpensive POCT devices, equivalent to currently offered full-size systems will become available to aid in the detection of mutations or in the identification of infectious agents.

References1. Holland CA, Kiechle FL. Point-of-care molecular

systems – past, present and future. Current Opinion Microbiol 2005; 8:504-509.

2. Kiechle FL, Holland CA. Point-of-care testing and molecular diagnostics: Miniaturization required. Clin Lab Med 2009; 29:555-560.

3. Agrawal N, Ugaz VM. A buoyancy-driven compact thermocycler for rapid PCR. Clin Lab Med 2007; 27:215-223.

The End

FINITO

FINThank You