New Molecular Approaches to Identify 21st Century Microbes - Dr Melissa Miller - November 2010...
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Transcript of New Molecular Approaches to Identify 21st Century Microbes - Dr Melissa Miller - November 2010...
New Molecular Approaches toNew Molecular Approaches toNew Molecular Approaches to New Molecular Approaches to Identify 21st Century Microbes Identify 21st Century Microbes
Directly from Patient SpecimensDirectly from Patient SpecimensDirectly from Patient SpecimensDirectly from Patient Specimens
Melissa B. Miller, Ph.D., D(ABMM)Melissa B. Miller, Ph.D., D(ABMM)Associate Professor, Pathology and Laboratory Medicine
Director, Clinical Molecular Microbiology LaboratoryA i t Di t Cli i l Mi bi l I lAssociate Director, Clinical Microbiology-Immunology
Laboratory
N b 18 2010November 18, 2010
OutlineOutline
• Where are we now?
• Where are we going?» Terminal RFLP» Next generation sequencing» Mass spectrometry
• Challenges
Progression of Molecular Detection in Progression of Molecular Detection in ggthe last 10 yearsthe last 10 years
• Uniplex real-time PCR
• Targeted multiplex detection• Real-time PCR• Suspension bead arrays• PNA-FISH
• Direct sequencing from patient samples
Direct sequencing from patient Direct sequencing from patient q g pq g psamplessamples
• Most common target 16S rRNA gene, or other ribosomal g ggenes
• Limited to “sterile” sites (i.e., no endogenous flora) and to the identification of one organism unless amplicons are clonedidentification of one organism unless amplicons are cloned
Direct sequencing from patient Direct sequencing from patient samplessamplessamplessamples
• Endocarditis» Goldenberger et al., 1997 (N=18)
• Compared to valve and blood cultures• DNA detected in 16/18, species-level N=4,
genus-level N=7ge us e e» 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)
Direct sequencing from patient Direct sequencing from patient samplessamplessamplessamples
• Bone/joint infections» Fenollar et al., J Clin Microbiol., 2006
• N=525, positive N=139• 90.5% concordance with culture• 16 false-negative culture resultsg• 7 mixed infections
» Fihman et al., J Infect., 2007• 51 patient with suspected infections; 18 controls51 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» Vandercam et al., J Mol Diagn., 2008• N=41 (prosthetic), N=28 controls• 65% culture-positive, 91% PCR/seq positive
82% d• 82% concordance• 7/9 patients culture-negative received antibiotics
The problem of mixed infectionsThe problem of mixed infectionsThe problem of mixed infectionsThe problem of mixed infections
Detection of Microbial Detection of Microbial PopulationsPopulationsPopulationsPopulations
• Terminal Restriction Fragment Length Polymorphism (T-RFLP) Profiling
N t ti i• Next generation sequencing
• Mass spectrometry• Mass spectrometry» MALDI-TOF» PCR/MS
TT RFLP ProfilingRFLP ProfilingTT--RFLP ProfilingRFLP 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 determinedfragments are determined.
www.appliedbiosystems.com
TT RFLP ProfilingRFLP ProfilingTT--RFLP ProfilingRFLP Profiling• Has been used to analyze environmental samples, oral
fl i l di l ti f th ffi f i d t lflora including evaluation of the efficacy of periodontal disease treatments (Sakamoto et al., 2004), and CF lungs (Stressmann et al., 2010)
Combined primers
Bacterial
Fungal
Archaeal
TT--RFLP ProfilingRFLP Profiling
• Advantages» No a priori knowledge needed of sample
contents» Identifies “non-cultureable” bacteria» Inexpensive» Inexpensive» Easy to perform
• Disadvantagesg» Accuracy/validation of database » Cannot retrieve sequences so one peak could
represent multiple speciesrepresent multiple species» Very complex communities are over-simplified
(20-50 peaks)
Next Generation Sequencing Next Generation Sequencing (NGS)(NGS)(NGS)(NGS)
• Also called: deep sequencing, high-throughput sequencing
• General characteristics» Amplification of genetic material by PCR
Li ti f lifi d t i l t lid f» Ligation of amplified material to a solid surface» Sequence of the target genetic material
• Sequence by synthesis (labelled nucleotides or q y y (pyrosequencing)
• Sequence by ligation» Sequencing done in a massively parallel» Sequencing done in a massively parallel
fashion and sequence information is captured by software
NGS: Tools for pathogen discoveryNGS: Tools for pathogen discoveryNGS: Tools for pathogen discoveryNGS: Tools for pathogen discovery
Next Gen SequencersNext Gen SequencersNext Gen SequencersNext Gen Sequencers
Next Gen SequencersNext Gen SequencersNext Gen SequencersNext Gen Sequencers
R h (454) Ill i GSequencing platform Roche (454) FLX
Illumina Genome Analyzer ABI SOLiD HeliScope
Sequencing chemistry
Pyrosequencingon solid support
Sequencing-by-synthesis with
reversible terminators
Sequencing by ligation
Sequencing-by-synthesis with
virtual terminators
Template amplification
methodEmulsion PCR Bridge PCR Emulsion PCR None (single
molecule)
Read length ~400 bp 36-175 bp ~50 bp 30–35 bp
S iSequencing throughput 400 Mb/run/8h >17Gb/run/3-6d 10-15 Gb/run/6d 21-28 Gb/run/8d
NGS: 454NGS: 454NGS: 454NGS: 454
Nature Biotechnology 26, 1117 - 1124 (2008)
Video: http://www.youtube.com/watch?v=bFNjxKHP8Jc
NGS: 454NGS: 454NGS: 454NGS: 454• General principle of pyrosequencing: detection of
h h t l l tid i tipyrophosphate release upon nucleotide incorporation
http://454.com/
PyrogramPyrogram of Raw Dataof Raw DataPyrogramPyrogram of Raw Dataof Raw Data
Ronaghi M Genome Res. 2001;11:3-11
Video: http://www.pyrosequencing.com/DynPage.aspx?id=7454
NGSNGSNGSNGS• 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 i i i h b d d i» Higher sensitivity than array-based detection» Suitable for pathogen discovery
• DisadvantagesDisadvantages» Cost of equipment» Core equipment not in CLIA space» Bioinformatics/analysis is complex
Protein Mass SpectrometryProtein Mass SpectrometryProtein Mass SpectrometryProtein Mass Spectrometry• Three functional units (under high vacuum allows
hi d d t f i )unhindered movement of 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 SystemData System» Data system control: Signals sent to data system and formatted in
a m/z spectrum
MALDIMALDI TOFTOFMALDIMALDI--TOFTOF• Matrix Assisted Laser Desorption Ionization (MALDI)-
Ti f Fli ht (TOF)Time of Flight (TOF)» Bruker Daltonics MALDI BioTyper (TM)
» BD and bioMerieux also have MALDI in the pipelinea d b o e eu a so a e t e p pe e
• Sample mixed with UV-absorbing acid matrixabsorbing acid matrix and spotted on a MALDI plateL I di ti f• Laser Irradiation forms an excited plume
• Proton transfer from the matrix forms ions
MALDIMALDI TOFTOFMALDIMALDI--TOFTOF• 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» Heavier ions travel slower than lighter ions» Ion arrival is measured as a current to create spectrum
DD
or
m/z Det
ecto
V
Bruker Biotyper systemBruker Biotyper systemBruker Biotyper systemBruker Biotyper system• Measures high-abundance proteins, including ribosomal
t iproteins» IVD-CE Mark 2009, RUO in US
• Identification/classification based on characteristic proteinIdentification/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
BrukerBruker MALDIMALDI BioTyperBioTyper WorkflowWorkflow
Unknown
1. Select a Colony 2. Smear a thin-layer onto Target Plate or perform
BrukerBruker MALDI MALDI BioTyperBioTyper WorkflowWorkflow
Microorganism Plate or perform rapid organic extraction &
spot supernatant
6. Match patterns to database to
spot supernatant
identify species
3. Add MALDI Matrix
5. Data Interpretation 4. Generate MALDI TOFMALDI-TOF
Profile Spectrum
* For research use only in the U.S.
MALDIMALDI TOF PublicationsTOF PublicationsMALDIMALDI--TOF PublicationsTOF Publications
PCRPCR--MSMS
• PCR plus atmospheric pressure chemical p p pionization (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
MassTagMassTag PCRPCRMassTagMassTag PCRPCR
Briese et al., Emerg Infect Dis. 2005 Feb;11(2):310-3
SequenomSequenom MassARRAYMassARRAY® System® SystemSequenomSequenom MassARRAYMassARRAY® System® System
M CLEAVE™ M ARRAY Li id H dl• Mass CLEAVE™ - MassARRAY Liquid Handler
Mutation ResearchVolume 573, 2005, Pages 83-95
For research use only
Abbott/Ibis T5000Abbott/Ibis T5000 PlexPlex IDIDAbbott/Ibis T5000 Abbott/Ibis T5000 PlexPlex--IDID• Couples amplification of targets (PCR) with mass
t t t bt i b d id tifi tispectrometry to obtain sequence-based identification without sequencing
• Simultaneously detects and identifies broad groups of organisms
KNOWN d UNKNOWN t t» KNOWN and UNKNOWN targets» Speed: 4 – 8 hours, batch» High analytical sensitivity» High analytical sensitivity» Automation
For research use only
Step 1: Sample Prep and Broad Range PCR (Multiple Step 1: Sample Prep and Broad Range PCR (Multiple p p p g ( pp p p g ( pprimers amplify primers amplify rDNArDNA & specific genes)& specific genes)
Hofstadler, S.A. et al. 2005, IJMS, 242, 23-41
16 wells per sample
Step 2: Sample Cleanup and ESIStep 2: Sample Cleanup and ESI TOFTOFStep 2: Sample Cleanup and ESIStep 2: Sample Cleanup and ESI--TOFTOF• Amplicons are dissolved in a volatile solvent and pushed
th h ti h d illthrough a tiny, charged, capillary• Negative charges repel & liquid is aerosolized • Analyte is moved to mass spectrometerAnalyte is moved to mass spectrometer
» Mass is analyzed with time of flight
Step 3: Collect Spectral Output of ESIStep 3: Collect Spectral Output of ESI MSMSStep 3: Collect Spectral Output of ESIStep 3: Collect Spectral Output of ESI--MSMSElectrospray
Ionization
3
Courtesy E. Johnson
Step 4: Step 4: DeconvolutionDeconvolution with Reverse with Reverse ComplimentarityComplimentaritypp p yp yYields an Unambiguous Base CountYields an Unambiguous Base Count
Step 5: “MultiStep 5: “Multi--primer Triangulation” compares base primer Triangulation” compares base compositions to a curated database to define genus compositions to a curated database to define genus
and speciesand species
ExamplesExamplesExamplesExamples• 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 al., PLoS One, 2009; 4:e8540a ac os et a , oS O e, 009; e85 0» Streptococcus pneumoniae coinfection is correlated with the
severity of H1N1 pandemic influenza (MassTag)G t Kl i t l M l C ll P b 2010 24 219 28• Grant-Klein et al., Mol Cell Probes, 2010 24:219-28» Rapid identification of vector-borne flaviviruses by mass
spectrometry (PCR/MS)p y ( )
ChallengesChallenges
• From research to clinical diagnostics» FDA-cleared platforms/assays» Standards, validation, QC, QA» Cost-effectiveness
P f f ti• Proof of causation• Presence vs. absence of microbiota• What is the gold standard?• What is the gold standard?• How to craft a clinically relevant report?• Resistance data
Molecular technologies are rapidly evolvingReady or not– Change is coming!
So you’re still skeptical... So you’re still skeptical...
Thank you to Dr. Donna Wolk (U Arizona) for sharing her MS slides/images.