newton handout

8/6/2019 Newton Handout

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Application of Molecular TechniquesFor Smaller Clinical Microbiology

Laboratories

Duane W. Newton, Ph.D., D(ABMM)Director, Clinical Microbiology Laboratories

Disclosures

Member, Roche MolecularDiagnostics Advisory Board

Research support: BD-GeneOhm,bioMrieux, HandyLabs, RocheDiagnostics


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Overview Many options for molecular testing

Focus on FDA-cleared/approvedassays and systems

Not all-inclusive

Provide framework for makingdecisions based on institutional or

customer needs

Molecular diagnostics

Extraction of nucleic acid fromsample

Detection of target-specificsequences

Hybridization or amplification

Real-time detection


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Detection of nucleic acid targets fordiagnosis

organism-specific genes or virulence factors

Quantitation of nucleic acid targets formanagement

viral load testing

Detection of gene mutations:

resistance testing

Molecular diagnostics

Why Use Molecular Diagnostic Tests?

When conventional methods are:

1. Too slow (e.g., Mycobacterium, Legionella)

2. Too insensitive (asymptomatic HIV, viralinfection of central nervous system, etc.)

3. Too cumbersome (e.g., virus isolation)

4. Not available (unculturable agents: HPV, HCV)

5. Too dangerous (HIV, BT agents)


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Higher sensitivity and specificity

Shorter turn-around-time

Overall reduction in patient care costs

The Promises of Molecular Diagnostics

Some Organisms Detected byMolecular Methods

M. tuberculosis

Legionellae

B. burgdorferii

H. influenzae

B. pertussis

N. meningitidis

T. pallidum

H. pylori

F. tularensis

C. difficileE. coli

T. whipelii

van, mec

HIV

HTLV

CMV

HSV

HHV

VZV

EBV

Hepatides

HPVRubella

Influenza

Rhino

Entero

Adeno

Rabies

Parvo B19

Arbo

Yellow Fever

Lassa

JC/BK

CandidaeCryptococcus

Trypanosoma

Toxoplasma

Naegleria.


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Levels of Service All labs performing molecular diagnostic

tests should be qualified to perform high-complexity testing as described in CLIA1988

Level 1 labs

Little experience with NAATs

Lack of resources to conduct extensive verificationof test performance characteristics

Use FDA cleared tests

Levels of Service

Level 2

Greater experience with NAATs

Greater resources available to documenttest performance characteristics

FDA cleared kits, RUOs, ASRs

Level 3

Greatest experience with NAATs

Design, develop and verify in-house tests


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Staffing All levels

Individual skills For low volume laboratories, mostly

manual procedures (potentially)

Good organizational skills

Detail oriented

Manual dexterity

Good eyesight

Physical Considerations

All levels?

Laboratory Space

Four steps

Reagent prep

Sample Prep

Amplification Detection


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Physical Considerations In a perfect world, each step carried

out in a separate room

Real world, limited space, configureas individual areas within one or tworooms

Physical Considerations

Label each area

Each step should provideunidirectional workflow from preampto detection.

Separation and airflow/traffic control

protect against carryover contamination


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Physical Considerations Other control measures include:

Environmental monitoring

Decontamination with bleach

Enzymatic inactivation of amplifiedtarget with UNG

Surface irradiation

Equipment Considerations

All levels?

Inventory could include

Dedicated pipettors for each area

Barrier tips

Biological safety cabinet or dead air box

Incubators

Heat blocks

Water baths

Vortex

Microcentrifuge


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Equipment Considerations

Label items appropriately so that if theybecome mobile, it will be noticed andappropriate decontamination can takeplace

New Developments

Extraction procedures simplified

Automated systems

FDA-approved/cleared products


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Targets to Consider CT/NG

MRSA

GBS

C. difficile

Enterovirus

CT/NG Molecular Testing

STD diagnosis/screening

Improves detection ~30%

Potential large outpatient volumes


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MRSA Surveillance Rate of MRSA is increasing

MRSA infections significant

Higher mortality

Longer hospitalizations

Increased costs

Colonization increases risk ofinfection

MRSA Surveillance

There is no consensus

SHEA and HICPAC differ in theirrecommendations on the scope ofactive surveillance cultures

Decision needs to be made on aninstitutional basis

Culture vs. Molecular?


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Group B Streptococcus Prenatal screening for colonization

Week 35-37

Culture vs. PCR Rapid detection for emergent deliveries

Need for susceptibility results

Potential large outpatient volumes

C. difficile

C diff associated diarrhea (CDAD)

Limitations with EIA tests

Labor intensive

Poor performance

Diagnostic and infection controlimplications


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Enterovirus Self-limiting viral meningitis

Difficult to distinguish from bacterialmeningitis

Benign clinical course

Culture is slow and labor-intensive

Need results in


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Gen-Probe Aptima CT/NG

Manual orautomatedextraction anddetection

DTS 400, 800, 1600

FDA-approved

Roche COBAS Amplicor

CT/NG

Manual orautomatedextraction,automateddetection

24-48 tube format

FDA-approved


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Abbott Real-time CT/NG m2000 platform

Automatedextraction anddetection

96 well format

FDA-approved

BD Viper

CT/NG


FDA-approved


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Gen-Probe Tigris CT/NG


180 tube carousel

FDA-approved

BD GeneOhm

C. difficile, MRSA,StaphSR, GBS

Manual extraction,real-time PCRdetection

16-sample block

(CepheidSmartCycler)

FDA-approved


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Cepheid GeneXpert MRSA (screening,

SSTI, bloodcultures), GBS,Enterovirus


FDA-approved

GeneXpert Infinity


>2000 tests/24hr


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Factors to Consider

Speed

Performance

Cost

Financial aspects

Costs vs. billing potential

Excluding CT/NG, costs generally $25-50/test

More competitive pricing for CT/NG

Equipment + reagents + tech time = costs

Billing:

CPT 87491 Amplified CT

CPT 87591 Amplified NG CPT 87621 Amplified HPV

Expected reimbursement $49.04 each

Reimbursement : ?


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Coming Soon? Expanded menu on existing platforms

New systems in development: Gen-Probe Panther

CT/NG

HandyLabs Jaguar GBS, CT/NG

Roche cobas 4800 CT/NG, HPV

ThirdWave/Hologic HPV

Dont forget

Approved specimen types

Approved collection/transport systems

Space requirements

Appropriate use/interpretation


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