New Molecular Approaches to Identify 21st Century Microbes - Dr Melissa Miller - November 2010...

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New Molecular Approaches to New Molecular Approaches to New Molecular Approaches to New Molecular Approaches to Identify 21st Century Microbes Identify 21st Century Microbes Directly from Patient Specimens Directly from Patient Specimens Directly from Patient Specimens Directly 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 Laboratory A it Di t Cli i l Mi bi l I l Associate Director, Clinical Microbiology-Immunology Laboratory N b 18 2010 November 18, 2010

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Presented by Dr. Miller at the 40th Annual Symposium "Diagnostic and Clinical Challenges of 20th Century Microbes", held on Nov 18, 2010 in Philadelphia.

Transcript of New Molecular Approaches to Identify 21st Century Microbes - Dr Melissa Miller - November 2010...

Page 1: New Molecular Approaches to Identify 21st Century Microbes - Dr Melissa Miller - November 2010 Symposium

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

Page 2: New Molecular Approaches to Identify 21st Century Microbes - Dr Melissa Miller - November 2010 Symposium

OutlineOutline

• Where are we now?

• Where are we going?» Terminal RFLP» Next generation sequencing» Mass spectrometry

• Challenges

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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

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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

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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)

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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

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The problem of mixed infectionsThe problem of mixed infectionsThe problem of mixed infectionsThe problem of mixed infections

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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

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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

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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

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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)

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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

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NGS: Tools for pathogen discoveryNGS: Tools for pathogen discoveryNGS: Tools for pathogen discoveryNGS: Tools for pathogen discovery

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Next Gen SequencersNext Gen SequencersNext Gen SequencersNext Gen Sequencers

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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

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NGS: 454NGS: 454NGS: 454NGS: 454

Nature Biotechnology 26, 1117 - 1124 (2008)

Video: http://www.youtube.com/watch?v=bFNjxKHP8Jc

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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/

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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

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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

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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

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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

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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

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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

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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.

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MALDIMALDI TOF PublicationsTOF PublicationsMALDIMALDI--TOF PublicationsTOF Publications

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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

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MassTagMassTag PCRPCRMassTagMassTag PCRPCR

Briese et al., Emerg Infect Dis. 2005 Feb;11(2):310-3

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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

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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

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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

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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

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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

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Step 4: Step 4: DeconvolutionDeconvolution with Reverse with Reverse ComplimentarityComplimentaritypp p yp yYields an Unambiguous Base CountYields an Unambiguous Base Count

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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

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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 ( )

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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!

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So you’re still skeptical... So you’re still skeptical...

Thank you to Dr. Donna Wolk (U Arizona) for sharing her MS slides/images.