Download as PDF, PPTX
Uploaded byEastern Pennsylvania Branch ASM
New Molecular Approaches to Identify 21st Century Microbes - Dr Melissa Miller - November 2010 Symposium
The document discusses advancements in molecular techniques for identifying microbes directly from patient specimens, focusing on methods such as terminal restriction fragment length polymorphism (T-RFLP), next-generation sequencing (NGS), and mass spectrometry. It outlines the progression and challenges faced in molecular detection over the last decade, highlighting various studies demonstrating the efficacy of these methods in clinical microbiology. Furthermore, it emphasizes the need for standardized protocols and cost-effectiveness in transitioning from research to clinical diagnostics.
More Related Content
Similar to New Molecular Approaches to Identify 21st Century Microbes - Dr Melissa Miller - November 2010 Symposium
New Molecular Approaches to Identify 21st Century Microbes - Dr Melissa Miller - November 2010 Symposium
- 1. New Molecular Approachesto 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 arewe now? • Where are we going? » Terminal RFLP » Next generation sequencing » Mass spectrometry • Challenges
- 3. Progression of MolecularDetection 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 frompatient 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 frompatient 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 frompatient 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 ofmixed 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 • 16SrRNA 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 • Hasbeen 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 forpathogen discovery
- 14.
- 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 • Generalprinciple of pyrosequencing: detection of pyrophosphate release upon nucleotide i h h t l l tid incorporation ti http://454.com/
- 18. Pyrogram of RawData 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 BioTyperWorkflow 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.
- 25.
- 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 etal., 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: SamplePrep 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: SampleCleanup 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: CollectSpectral Output of ESI-MS ESI- Electrospray Ionization 3 Courtesy E. Johnson
- 33. Step 4: Deconvolutionwith Reverse Complimentarity p p y Yields an Unambiguous Base Count
- 34. Step 5: “Multi-primerTriangulation” compares base “Multi- compositions to a curated database to define genus and species
- 35. Examples • Palacios etal., 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 researchto 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 stillskeptical... Thank you to Dr. Donna Wolk (U Arizona) for sharing her MS slides/images.