New Molecular Approaches to Identify 21st Century MicrobesDirectly from Patient Specimens      Melissa B. Miller, Ph.D., D...
Outline• Where are we now?• Where are we going?  » Terminal RFLP  » Next generation sequencing  » Mass spectrometry• Chall...
Progression of Molecular Detection in   g          the last 10 years       • Uniplex real-time PCR       • Targeted multip...
Direct sequencing from patient                q      g      p                  samples• Most common target 16S rRNA g     ...
Direct sequencing from patient           samples• Endocarditis   » Goldenberger et al., 1997 (N=18)      • Compared to val...
Direct sequencing from patient           samples• Bone/joint infections   » Fenollar et al., J Clin Microbiol., 2006      ...
The problem of mixed infections
Detection of Microbial          Populations• Terminal Restriction Fragment Length  Polymorphism (T-RFLP) Profiling• N t ge...
T-RFLP Profiling• 16S rRNA gene is amplified using fluorescently labeled  primer(s).  primer(s)• The mixture of amplicons ...
T-RFLP Profiling• Has been used to analyze environmental samples, oral  flora i l di evaluation of th efficacy of periodon...
T-RFLP Profiling• Advantages  » No a priori knowledge needed of sample    contents  » Identifies “non-cultureable” bacteri...
Next Generation Sequencing           (NGS)• Also called: deep sequencing, high-  throughput sequencing• General characteri...
NGS: Tools for pathogen discovery
Next Gen Sequencers
Next Gen Sequencers                       Roche                       R h (454)         Illumina Genome                   ...
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 ...
Pyrogram of Raw Data               Video: http://www.pyrosequencing.com/DynPage.aspx?id=7454Ronaghi M Genome Res. 2001;11:...
NGS• Advantages  » Massive parallel sequencing- high throughput  » Use universal primer on adaptors (no need for prior    ...
Protein Mass Spectrometry• Three functional units (under high vacuum allows  unhindered movement of i    hi d d           ...
MALDI-                     MALDI-TOF• Matrix Assisted Laser Desorption Ionization (MALDI)-  Time of Fli ht (TOF)  Ti     f...
MALDI-                        MALDI-TOF• Ions accelerated by applying high voltage• Kinetic energy is inversely related to...
Bruker Biotyper system• Measures high-abundance proteins, including ribosomal  proteins     t i   » IVD-CE Mark 2009, RUO ...
Bruker MALDI BioTyper Workflow1. Select a Colony                                        2. Smear a thin-    Unknown       ...
MALDI-MALDI-TOF Publications
PCR-                PCR-MS• PCR plus atmospheric p        p         p     pressure chemical  ionization (APCI) = MassTag P...
MassTag PCRBriese et al., Emerg Infect Dis. 2005 Feb;11(2):310-3
Sequenom MassARRAY® System            MassARRAY®• M  Mass CLEAVE™ - M                 MassARRAY Li id H dl                ...
Abbott/Ibis T5000 Plex-ID                               Plex-• Couples amplification of targets (PCR) with mass  spectrome...
Step 1: Sample Prep and Broad Range PCR (Multiple   p       p      p              g        (   p      primers amplify rDNA...
Step 2: Sample Cleanup and ESI-TOF                                  ESI-• Amplicons are dissolved in a volatile solvent an...
Step 3: Collect Spectral Output of ESI-MS                                   ESI-Electrospray Ionization          3        ...
Step 4: Deconvolution with Reverse Complimentarity   p                                  p          y         Yields an Una...
Step 5: “Multi-primer Triangulation” compares base        “Multi-compositions to a curated database to define genus       ...
Examples• Palacios et al., N Engl J Med, 2008; 358:991-8   » A new arenavirus in a cluster of fatal transplant-associated ...
Challenges• From research to clinical diagnostics    » FDA-cleared platforms/assays    » Standards, validation, QC, QA    ...
So you’re still skeptical...Thank you to Dr. Donna Wolk (U Arizona)   for sharing her MS slides/images.
Upcoming SlideShare
Loading in …5
×

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

3,234 views
2,977 views

Published on

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

Published in: Health & Medicine
2 Comments
0 Likes
Statistics
Notes
  • Good!!!!!!!!!!!
       Reply 
    Are you sure you want to  Yes  No
    Your message goes here
  • A clear, comprehensive and really useful summary
       Reply 
    Are you sure you want to  Yes  No
    Your message goes here
  • Be the first to like this

No Downloads
Views
Total views
3,234
On SlideShare
0
From Embeds
0
Number of Embeds
11
Actions
Shares
0
Downloads
93
Comments
2
Likes
0
Embeds 0
No embeds

No notes for slide

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

  1. 1. New Molecular Approaches to Identify 21st Century MicrobesDirectly 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. 2. Outline• Where are we now?• Where are we going? » Terminal RFLP » Next generation sequencing » Mass spectrometry• Challenges
  3. 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. 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. 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. 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. 7. The problem of mixed infections
  8. 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. 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. 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. 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. 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. 13. NGS: Tools for pathogen discovery
  14. 14. Next Gen Sequencers
  15. 15. Next Gen Sequencers Roche R h (454) Illumina Genome Ill i GSequencing 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. 16. NGS: 454 Nature Biotechnology 26, 1117 - 1124 (2008)Video: http://www.youtube.com/watch?v=bFNjxKHP8Jc
  17. 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. 18. Pyrogram of Raw Data Video: http://www.pyrosequencing.com/DynPage.aspx?id=7454Ronaghi M Genome Res. 2001;11:3-11
  19. 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. 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. 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. 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. 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. 24. Bruker MALDI BioTyper Workflow1. 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.
  25. 25. MALDI-MALDI-TOF Publications
  26. 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. 27. MassTag PCRBriese et al., Emerg Infect Dis. 2005 Feb;11(2):310-3
  28. 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. 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. 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. 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. 32. Step 3: Collect Spectral Output of ESI-MS ESI-Electrospray Ionization 3 Courtesy E. Johnson
  33. 33. Step 4: Deconvolution with Reverse Complimentarity p p y Yields an Unambiguous Base Count
  34. 34. Step 5: “Multi-primer Triangulation” compares base “Multi-compositions to a curated database to define genus and species
  35. 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. 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. 37. So you’re still skeptical...Thank you to Dr. Donna Wolk (U Arizona) for sharing her MS slides/images.

×