Presented by Dr. Miller at the 40th Annual Symposium "Diagnostic and Clinical Challenges of 20th Century Microbes", held on Nov 18, 2010 in Philadelphia.
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New Molecular Approaches to Identify 21st Century Microbes - Dr Melissa Miller - November 2010 Symposium
1. New Molecular Approaches to
Identify 21st Century Microbes
Directly from Patient Specimens
Melissa B. Miller, Ph.D., D(ABMM)
Associate Professor, Pathology and Laboratory Medicine
Director, Clinical Molecular Microbiology Laboratory
Associate Di
A i t Director, Clinical Microbiology-Immunology
t Cli i l Mi bi l I l
Laboratory
November 18, 2010
N b 18
2. Outline
• Where are we now?
• Where are we going?
» Terminal RFLP
» Next generation sequencing
» Mass spectrometry
• Challenges
3. Progression of Molecular Detection in
g
the last 10 years
• Uniplex real-time PCR
• Targeted multiplex detection
• Real-time PCR
• Suspension bead arrays
• PNA-FISH
• Direct sequencing from patient samples
4. Direct sequencing from patient
q g p
samples
• Most common target 16S rRNA g
g gene, or other ribosomal
genes
• Limited to “sterile” sites (i.e., no endogenous flora) and to the
identification of one organism unless amplicons are cloned
5. Direct sequencing from patient
samples
• Endocarditis
» Goldenberger et al., 1997 (N=18)
• Compared to valve and blood cultures
• DNA detected in 16/18, species-level N=4,
ge us e e
genus-level N=7
» Breitkopf et al., 2005 (N=51)
• Sens/Spec: direct seq 41%/100% vs. culture
7.8%/94%
7 8%/94%
» Marin et al., 2007 (N=35)
• Sens/Spec: direct seq 96%/95% (compared to
Duke criteria and blood cultures)
6. Direct sequencing from patient
samples
• Bone/joint infections
» Fenollar et al., J Clin Microbiol., 2006
• N=525, positive N=139
• 90.5% concordance with culture
• 16 false-negative culture results
g
• 7 mixed infections
» Fihman et al., J Infect., 2007
• 51 patient with suspected infections; 18 controls
• PCR/seq sensitivity: 73%, culture: 97%
• PCR/seq specificity: 95%, culture: 86%
» Vandercam et al J Mol Diagn 2008
al., Diagn.,
• N=41 (prosthetic), N=28 controls
• 65% culture-positive, 91% PCR/seq positive
• 82% concordance
d
• 7/9 patients culture-negative received antibiotics
8. Detection of Microbial
Populations
• Terminal Restriction Fragment Length
Polymorphism (T-RFLP) Profiling
• N t generation sequencing
Next ti i
• Mass spectrometry
» MALDI-TOF
» PCR/MS
9. T-RFLP Profiling
• 16S rRNA gene is amplified using fluorescently labeled
primer(s).
primer(s)
• The mixture of amplicons is then subjected to a restriction
enzyme digestion (four-cutter).
• The mixture of fragments is separated by capillary
electrophoresis and the sizes of the different terminal
fragments are determined.
determined
www.appliedbiosystems.com
10. T-RFLP Profiling
• Has been used to analyze environmental samples, oral
flora i l di evaluation of th efficacy of periodontal
fl including l ti f the ffi f i d t l
disease treatments (Sakamoto et al., 2004), and CF lungs
(Stressmann et al., 2010)
Combined primers
Bacterial
Fungal
Archaeal
11. T-RFLP Profiling
• Advantages
» No a priori knowledge needed of sample
contents
» Identifies “non-cultureable” bacteria
» Inexpensive
» Easy to perform
• Disadvantages
g
» Accuracy/validation of database
» Cannot retrieve sequences so one peak could
represent multiple species
» Very complex communities are over-simplified
(20-50 peaks)
12. Next Generation Sequencing
(NGS)
• Also called: deep sequencing, high-
throughput sequencing
• General characteristics
» Amplification of genetic material by PCR
» Li ti of amplified material t a solid surface
Ligation f lifi d t i l to lid f
» Sequence of the target genetic material
• Sequence by synthesis (
q y y (labelled nucleotides or
pyrosequencing)
• Sequence by ligation
» Sequencing done in a massively parallel
fashion and sequence information is captured
by software
15. Next Gen Sequencers
Roche
R h (454) Illumina Genome
Ill i G
Sequencing platform ABI SOLiD HeliScope
FLX Analyzer
Sequencing-by-
Sequencing-by-
Sequencing Pyrosequencing synthesis with Sequencing by
synthesis with
chemistry on solid support reversible ligation
virtual terminators
terminators
Template
None (single
amplification Emulsion PCR Bridge PCR Emulsion PCR
molecule)
method
Read length ~400 bp 36-175 bp ~50 bp 30–35 bp
Sequencing
S i
400 Mb/run/8h >17Gb/run/3-6d 10-15 Gb/run/6d 21-28 Gb/run/8d
throughput
17. NGS: 454
• General principle of pyrosequencing: detection of
pyrophosphate release upon nucleotide i
h h t l l tid incorporation
ti
http://454.com/
18. Pyrogram of Raw Data
Video: http://www.pyrosequencing.com/DynPage.aspx?id=7454
Ronaghi M Genome Res. 2001;11:3-11
19. NGS
• Advantages
» Massive parallel sequencing- high throughput
» Use universal primer on adaptors (no need for prior
sequence knowledge)
q g )
» No bacterial cloning
» Faster, less labor = more cost-effective
» Hi h sensitivity than array-based d
Higher ii i h b d detection
i
» Suitable for pathogen discovery
• Disadvantages
» Cost of equipment
» Core equipment not in CLIA space
» Bioinformatics/analysis is complex
20. Protein Mass Spectrometry
• Three functional units (under high vacuum allows
unhindered movement of i
hi d d t f ions)
)
» Ionization source: Ionized samples easier to manipulate
» Analyzer: Ions separate according to mass-to-charge ratios (m/z)
» Detector: Detects separated ions and identifies their relative
abundance
• Data System
» Data system control: Signals sent to data system and formatted in
a m/z spectrum
21. MALDI-
MALDI-TOF
• Matrix Assisted Laser Desorption Ionization (MALDI)-
Time of Fli ht (TOF)
Ti f Flight
» Bruker Daltonics MALDI BioTyper (TM)
» BD and b o e eu a so have MALDI in t e p pe e
a d bioMerieux also a e the pipeline
• Sample mixed with UV-
absorbing acid matrix
and spotted on a MALDI
plate
• L
Laser I di ti f
Irradiation forms
an excited plume
• Proton transfer from the
matrix forms ions
22. MALDI-
MALDI-TOF
• Ions accelerated by applying high voltage
• Kinetic energy is inversely related to the mass to charge
ratio (m/z)
» Heavier ions travel slower than lighter ions
» Ion arrival is measured as a current to create spectrum
D
or
Detecto
m/z
V
23. Bruker Biotyper system
• Measures high-abundance proteins, including ribosomal
proteins
t i
» IVD-CE Mark 2009, RUO in US
• Identification/classification based on characteristic protein
expression patterns
» Gram positive and negative bacteria
» Yeasts and multicellular fungi
• http://www.bdal.com/solutions/clinical/microorganism-id/details.html
24. Bruker MALDI BioTyper Workflow
1. Select a Colony 2. Smear a thin-
Unknown layer onto Target
Microorganism Plate or perform
rapid organic
extraction &
spot supernatant
6. Match patterns
to database to
identify 3. Add MALDI
species Matrix
5. Data Interpretation 4. Generate
MALDI-TOF
MALDI TOF
Profile Spectrum
* For research use only in the U.S.
26. PCR-
PCR-MS
• PCR plus atmospheric p
p p pressure chemical
ionization (APCI) = MassTag PCR
• PCR plus MALDI-TOF = Sequenom
MassARRAY® System with iSEQ™
• PCR plus Electrospray Ionization Time of
Flight (ESI-TOF) = Abbott/Ibis PLEX-ID
28. Sequenom MassARRAY® System
MassARRAY®
• M
Mass CLEAVE™ - M
MassARRAY Li id H dl
ARRAY Liquid Handler
Mutation Research
Volume 573, 2005, Pages 83-95
For research use only
29. Abbott/Ibis T5000 Plex-ID
Plex-
• Couples amplification of targets (PCR) with mass
spectrometry to obtain sequence-based id tifi ti
t t t bt i b d identification
without sequencing
• Simultaneously detects and identifies broad groups of
organisms
» KNOWN and UNKNOWN t
d targets
t
» Speed: 4 – 8 hours, batch
» High analytical sensitivity
» Automation
For research use only
30. Step 1: Sample Prep and Broad Range PCR (Multiple
p p p g ( p
primers amplify rDNA & specific genes)
16 wells per
sample
Hofstadler, S.A. et al. 2005, IJMS, 242, 23-41
31. Step 2: Sample Cleanup and ESI-TOF
ESI-
• Amplicons are dissolved in a volatile solvent and pushed
through a tiny, charged, capillary
th h ti h d ill
• Negative charges repel & liquid is aerosolized
• Analyte is moved to mass spectrometer
» Mass is analyzed with time of flight
32. Step 3: Collect Spectral Output of ESI-MS
ESI-
Electrospray
Ionization
3
Courtesy E. Johnson
33. Step 4: Deconvolution with Reverse Complimentarity
p p y
Yields an Unambiguous Base Count
34. Step 5: “Multi-primer Triangulation” compares base
“Multi-
compositions to a curated database to define genus
and species
35. Examples
• Palacios et al., N Engl J Med, 2008; 358:991-8
» A new arenavirus in a cluster of fatal transplant-associated
disease (NGS)
• Palacios et a , PLoS O e, 2009; 4:e8540
a ac os al., oS One, 009; e85 0
» Streptococcus pneumoniae coinfection is correlated with the
severity of H1N1 pandemic influenza (MassTag)
• G t Kl i et al., M l C ll P b
Grant-Klein t l Mol Cell Probes, 2010 24 219 28
24:219-28
» Rapid identification of vector-borne flaviviruses by mass
spectrometry (
p y (PCR/MS) )
36. Challenges
• From research to clinical diagnostics
» FDA-cleared platforms/assays
» Standards, validation, QC, QA
» Cost-effectiveness
• Proof f
P f of causation
ti
• Presence vs. absence of microbiota
• What is the gold standard?
• How to craft a clinically relevant report?
• Resistance data
Molecular technologies are rapidly evolving
Ready or not– Change is coming!
37. So you’re still skeptical...
Thank you to Dr. Donna Wolk (U Arizona)
for sharing her MS slides/images.