New Developments in Molecular
Diagnostics
April 8, 2011
Montreal, QC
Astrid Petrich, PhD
SickKids, Toronto, ON
Objectives
• To provide an overview of the current state
of molecular microbiology diagnostics in
Canadian laboratories.
• To review some of the factors involved with
laboratories offering new molecular assays
in microbiology.
• To discuss some of the new developments in
technology and instrumentation in molecular
microbiology diagnostics.
Disclosures
• I have tried many of the instruments and kits
over the years
• No financial arrangements
• No contracts
• Some on-going evaluations for which I have
received free kits (artus, Seegene)
Outline
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Where are we with molecular diagnostics?
Extraction
Real-time
Multiplexing
Systems that do it all
Sequencing
The new and wonderful?
– Or I wish we could have one of those.
What do most of us have?
• A thermal cycler (old school, conventional, block
PCR)
• Molecular method for CT/GC detection
• Real-time PCR instrument
– Older versions
– Newer versions
• Automated or semi-automated extraction
• Access to sequencing (send-out), if not doing onsite
Assays many of us are using
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CT/GC
MRSA
VRE
HSV, Enterovirus in CSF
FluA (other respiratory)
Assays some of us are doing
• Transplant screening and monitoring
– CMV, EBV, BK, Adenovirus
• HPV
• B. pertussis
• WNV
• HepC, HepB
• HIV
• Norovirus
• 16s PCR plus sequencing
• C. difficile
Assays we would like to do
• Everything
• More multiplexes for “disease states”
– Respiratory
– Gastro
– Encephalitis
– Transplant
• Resistance beyond
Some of the reasons we don’t
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Cost of the test
Cost of the instrumentation
Trained staff
Commercially available assays
– Health Canada approved
• Sensitivity
– Add on test, still need to do culture
• What do we do with the results?
Pre-Analytical
• Quality of sample
• Sample type
• Few changes in the
last 20 years
• One exception-Flocked
swab for respiratory
and other specimens
• Should we be
spending more time on
this?
Extraction
• Most important part of analysis?
• Quantity, Quality
• Lack of inhibitors
• Complete lysis of cells
• RNA, DNA, both
• Throughput
• Different methods for different sample
types
• Hands-on time
• Expertise
Extraction
Nucleic Acid Extraction
• Semi-automated
• Automated
• Instruments with kits
– EZmag (bioMérieux)
– M48, MDX, (Qiagen)
– MagNA Pure LC (Roche)
• Kits with generic instruments
Extraction
Thermal cyclers
• Faster
• More compact
• More features
(gradients)
• Cheaper
• Other options for
detection
Real-time
• significant improvements in the diagnosis of
infectious disease.
• Increased sensitivity and specifity
• Increased speed
• Allows multiplexing
• Greatest impact is probably its use for the
quantitation of target organisms in samples.
• six orders of magnitude
• closed-tube format removes need for postamplification manipulation reduces risk of
contamination
• Costs more (reagents and instrumentation)
Real-time NAAT instruments
• LightCycler 1.5, 2.0
• ABI 7700
• Smartcycler (Cepheid)
Newer
– Corbett Rotorgene 6000
– LightCycler 480, LightCycler 1536
– Stratagene: Mx4000, Mx3000P, Mx3005P
– Applied Biosystems: ABI 7300, ABI 7500, ABI 7500 Fast,
ABI 7900 Fast HT with automation accessory, ABI
StepOne
– Eppendorf: Mastercycler ep realplex
– BioRad: MiniOpticon, MyiQ, Opticon2, Chromo4, iQ5
Good comparison of features of Real-time instruments
http://www.horizonpress.com/pcr/qPCR-machines.html
Quantitation
• Monitoring of transplant patients
– CMV, EBV, BK, Adenovirus?
• Efficacy of treatment
• Prognosis
• Resistance
• Standardization?
• QA
• Other agents?
– FluA
Self-collected influenza H1N1 viral loads:
Influenza viral load (copies/mL)
100000000
10000000
1000000
100000
Patient 1
10000
Patient 2
Patient 3
1000
100
10
1
1
2
3
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8
Days from symptom onset
Smieja, Petrich, Mahony et al. [Late-breaker] International
Congress of Chemotherapy and Infections, Toronto June 2009
High Resolution Melt
• SNP analysis
• Identification
• Resistance
Systems that do it all for you
• From sample extraction
• to amplification
• to detection
Gen-Probe
• Tigris DTS
• Soon to lauch-Panther for low to
medium throughput laboratories
Tigris DTS
• Target capture magnetic bead
• No opening of tubes
• Aptima kits
– CT/GC
– T. vaginalis
– HPV
– HIV-1
– HepC
• 3.5 hrs for first results
• 100 samples every 3 hrs following first results
Roche Diagnostics
AmpliPrep
• Automates the sample extraction process
• Bar coded reagents packaged in ready-to-use spillproof cassettes with self-sealing caps for increased
stability
• Up to 72 samples and 4 different assays can be
loaded onto the system at the same time
• Works in combination with the COBAS® TaqMan®
48 Analyzer, COBAS® TaqMan® Analyzer or
Amplicor Analyzer to automate sample
preparation, amplification, and quantitation of RNA
or DNA
Roche (COBAS®)
TaqMan® Analyzer
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Automated and fully docked systems available with COBAS®
AmpliPrep/COBAS® TaqMan® HIV-1, HCV, and HBV tests
96-test capacity
4 independently controlled thermal cyclers
Amplifies and detects RNA and DNA within hours
Quantitation standard and closed-tube format
Reduces the risk of sample contamination, optimizes workflow
Assays-HIV-1 (quant and qual), HCV, HBV
AMPLICOR Analyzer
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Bar-coded data entry
48 sample capacity per run
Test-specific reagents
Multiplexing- up to 6 detections per amplified sample
• Assays-HIV-1, CMV, CT/GC, HCV
Roche (COBAS®)
TaqMan® 48 Analyzer
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Two thermal cyclers can run independent PCR profiles
simultaneously
One platform separately configurable to run IVD assays and up to 10
user-defined applications
Amplifies and detects RNA and DNA within hours
Requires no user interaction after sample loading completed
Optimizes workflow, minimizes contamination
Small, compact footprint
48 samples at a time
Qiagen
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QIAsymphony RGQ®, an automated
system for molecular testing,
process a range of sample types
composed of modules that can be used
as stand-alone systems or combined
into a fully integrated system.
QIAsymphony SP for sample
preparation
QIAsymphony AS for assay setup
Rotor-Gene® Q real-time PCR
thermocycler.
BD Diagnostics
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BD Max The new BD MAX™
fully-automated open molecular biology platform allowing
multiple specimen types and assays simultaneously.
The Jaguar™ platform, initially developed by HandyLab,
commercialised by BD as the BD MAX™ system
BD
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Reagent strips for 1 to 24 samples per run
Microfluidic real-time PCR
Up to 144 tests per day (38 000 tests a year)
Extraction from multiple specimen types (blood, urine, swabs,
etc.)
• Ability to run multiple tests simultaneously (e.g. testing for S.
aureus and Group B Streptotoccus at the same time)
• 45 minutes to 2 hours, depending on the number of samples
analysed
Abbott Diagnostics
M2000
• Real-time PCR Amplification and Detection
• 5 excitation, 5 emission filters
• maxRatio Data Analysis
• M2000sp
• M2000rt
Abbott
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RealTime HIV-1
RealTime HCV
RealTime HCV Genotype II
RealTime HBV
RealTime High Risk HPV
RealTime CT/NG
RealTime CT
RealTime CMV
RealTime HIV-1 Qualitative
Parvo B19 PCR Kit
HSV 1/2 PCR Kit
VZV PCR Kit
EBV PCR Kit
M. tuberculosis
SARS RT-PCR Kit
• Barcoded Laboratory Tubes
• Open Mode
• Flexible protocol for various
sample types and volumes
• 24 to 96 samples
Multiplexing
• New technologies have allowed for
multiplexing with minimal loss of
sensitivity
• Seegene (Alere)
• xTag (Luminex)
• Multicode (Eragen)
• ResPlex (Qiagen)
Xtag Lumionex
• RVP respiratory panel
• In development
– Gastrointestinal panel
– Fungal panel
Eragen
• Multicode RTx HSV 1 and 2
• MultiCode-RTx System Neisseria
gonorrhoeae
• Others in development
ResPlex(Qiagen)
• ResPlex 1 bacterial pathogens and
adenovirus
• ResPlex 2 viral pathogens
• Others in development
Seegene (Alere)
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Respiratory panels
Gastrointestinal panels
Meningitis panels
STI panels
HPV
Mycobacteria panels
Multiplexing and Quantitation
PrimeraDx
• Multiplex PCR with continuous sampling and
separation by capillary electrophoresis
• Herpesviruses
• C. difficile
• Respiratory viruses
• Others in development
• Cost!
MALDI-TOF Mass Spectrometry
Matrix-assisted laser desorption/ionization (MALDI)
• Soft ionization technique
• Allows analysis of biomolecules which fragment
when ionized i
• Matrix solution is mixed with the analyte (e.g. whole
cells) on a MALDI analysis plate
• Solvent is added to the sample resulting into a
recrystallized matrix with analyte molecules spread
throughout the crystals.
• A current is generated resulting in movement of the
fragmented biomolecules towards a detector based
on size smaller molecules move faster)
• A pattern is generated based on the size and
number of molecules
• MALDI/TOF spectra are used for the identification of
microorganisms such as bacteria or fungi.
• A colony of the microbe in question is smeared
directly onto a dry plate (96 wells), overlayed with
matrix
• Mass spectra generated are analyzed by dedicated
software and compared with stored profiles.
• 96 wells in 40 mins.
• Species diagnosis by this procedure is faster, more
accurate and cheaper than immunological or
biochemical tests.
Representative mass spectral outputs
H. N. Shah, As published in CLI November 2005
Representative mass spectral outputs
H. N. Shah, As published in CLI November 2005
Concordance between Conventional Routine Identification (Vitek; bioMérieux) and
Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Mass
Spectrometry Identification (Brucker Mass Spectrometer and Database
Complemented with Local Database).
Seng P et al. Clin Infect Dis. 2009;49:543-551
© 2009 by the Infectious Diseases Society of America
Sequencing
• Applications
– ID of known organisms
• Cultured isolates
• Sterile body fluids
– Typing
– Resistance detection
Sanger sequencing
Sanger Sequencing
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Expensive
Hands on time
500-700 bases per run
Overnight?
Sequencing for ID
• Does my ID give me the same ID as your ID?
• Cultured isolate vs. clinical specimen?
• What are you sequencing?
-16S rDNA
-HSP genes?
-whole genome?
• What will the changes be to taxonomy
• What does it mean if it is not a recognized pathogen
• What about contaminating nucleic acid?
Pyrosequencing
Pyromark Q24
• PCR product of interest
• Up to 24 samples can
be prepared in parallel
using Vacuum Workstation
(~15 mins) to clean up
PCR product.
• Two formats-preprogramed sequence of
nucleotide presented vs.
sequential addition (G-AT-C)n
• High Volume instrument
also available Pyromark
Q96
• Real time addition and
viewing of results
• 50-100 bases added
High through-put
• Genome sequencing?
• Sequence of all data and pulling out
important?
Sequencing Workflow
 Sample input: Genomic DNA,
BACs, amplicons, cDNAGeneration of
small
 DNA fragments via Nebulization
 Ligation of A/B-Adaptors flanking
singlestranded DNA fragments
 Emulsification of beads and
fragments in water-in-oil microreactors
 Clonal amplification of fragments
bound to beads in microreactors
 Sequencing and base calling
(400,000 reads per run)
High-throughput Sequencing
Plex-ID (Abbott Diagnostics)
IBIS
• The coupling of broad PCR amplification with
electrospray Time-of-flight Mass Spectrometry (ESI
– TOF – MS)
• Post PCR amplicons are measured with accurate
mass to determine their unique base compositions
and compared against a database of known targets
• Rapid and sensitive identification of known,
unknown, and emerging microorganisms without
culture.
• Broad identification
Bacteria, Viruses, Fungi, Protozoa
• High resolution genotyping and strain
identification
• Detects complex mixtures of microbes
Expected and unexpected microbes in a
single test
• Rapid detection – no culture required
Detection and identification in < 1 shift
(6-8 hrs)
• Determine drug resistance and virulence
IBIS workflow
• Extract nucleic acid from sample
• Amplify nucleic acids
to measure
• Use broad-range, unbiased PCR primers
(conserved universal areas with variable
regions in between)
• Analyze nucleic acid using mass spec
• Identify the organism using base recognition
fingerprints
• Eight hours start to finish
Analysis
Really neat stuff with huge
potential
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Macro and microarrays
Microfluidics
Sequencing
New identification methods
What does the future hold?
• More automation
• More clinical interpretation
– What does it mean to have that
virus/bacteria/fungi in that specimen?
– What does that mutation mean for
resistance/pathogenicity?
• More research into correlating what we find
with clinical information
• More bioinformatics