Upload
william66
View
224
Download
0
Embed Size (px)
Citation preview
8/6/2019 Newton Handout
1/20
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
8/6/2019 Newton Handout
2/20
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
8/6/2019 Newton Handout
3/20
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)
8/6/2019 Newton Handout
4/20
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.
8/6/2019 Newton Handout
5/20
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
8/6/2019 Newton Handout
6/20
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
8/6/2019 Newton Handout
7/20
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
8/6/2019 Newton Handout
8/20
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
8/6/2019 Newton Handout
9/20
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
8/6/2019 Newton Handout
10/20
Targets to Consider CT/NG
MRSA
GBS
C. difficile
Enterovirus
CT/NG Molecular Testing
STD diagnosis/screening
Improves detection ~30%
Potential large outpatient volumes
8/6/2019 Newton Handout
11/20
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?
8/6/2019 Newton Handout
12/20
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
8/6/2019 Newton Handout
13/20
Enterovirus Self-limiting viral meningitis
Difficult to distinguish from bacterialmeningitis
Benign clinical course
Culture is slow and labor-intensive
Need results in
8/6/2019 Newton Handout
14/20
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
8/6/2019 Newton Handout
15/20
Abbott Real-time CT/NG m2000 platform
Automatedextraction anddetection
96 well format
FDA-approved
BD Viper
CT/NG
Automatedextraction anddetection
FDA-approved
8/6/2019 Newton Handout
16/20
Gen-Probe Tigris CT/NG
Automatedextraction anddetection
180 tube carousel
FDA-approved
BD GeneOhm
C. difficile, MRSA,StaphSR, GBS
Manual extraction,real-time PCRdetection
16-sample block
(CepheidSmartCycler)
FDA-approved
8/6/2019 Newton Handout
17/20
Cepheid GeneXpert MRSA (screening,
SSTI, bloodcultures), GBS,Enterovirus
Automatedextraction anddetection
FDA-approved
GeneXpert Infinity
Automatedextraction anddetection
>2000 tests/24hr
8/6/2019 Newton Handout
18/20
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 : ?
8/6/2019 Newton Handout
19/20
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
8/6/2019 Newton Handout
20/20