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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
Outline
• Where are we now?

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

• Challenges
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
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
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)
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
The problem of mixed infections
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
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
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
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)
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
NGS: Tools for pathogen discovery
Next Gen Sequencers
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
NGS: 454




          Nature Biotechnology 26, 1117 - 1124 (2008)


Video: http://www.youtube.com/watch?v=bFNjxKHP8Jc
NGS: 454
• General principle of pyrosequencing: detection of
  pyrophosphate release upon nucleotide i
       h    h t     l             l tid incorporation
                                                    ti




                         http://454.com/
Pyrogram of Raw Data




               Video: http://www.pyrosequencing.com/DynPage.aspx?id=7454

Ronaghi M Genome Res. 2001;11:3-11
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
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
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
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
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
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.
MALDI-
MALDI-TOF Publications
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
MassTag PCR




Briese et al., Emerg Infect Dis. 2005 Feb;11(2):310-3
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
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
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
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
Step 3: Collect Spectral Output of ESI-MS
                                   ESI-
Electrospray
 Ionization




          3




                                    Courtesy E. Johnson
Step 4: Deconvolution with Reverse Complimentarity
   p                                  p          y
         Yields an Unambiguous Base Count
Step 5: “Multi-primer Triangulation” compares base
        “Multi-
compositions to a curated database to define genus
                    and species
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)   )
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!
So you’re still skeptical...




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

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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
  • 7. The problem of mixed infections
  • 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
  • 13. NGS: Tools for pathogen discovery
  • 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
  • 16. NGS: 454 Nature Biotechnology 26, 1117 - 1124 (2008) Video: http://www.youtube.com/watch?v=bFNjxKHP8Jc
  • 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
  • 27. MassTag PCR Briese et al., Emerg Infect Dis. 2005 Feb;11(2):310-3
  • 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.