New Developments in Molecular DiagnosticsApril 8, 2011Montreal, QC Astrid Petrich, PhD SickKids, Toronto, ON
Objectives • To provide an overview of the current state of molecular microbiology diagnostics in Canadian laboratories. • To review some of the factors involved with laboratories offering new molecular assays in microbiology. • To discuss some of the new developments in technology and instrumentation in molecular microbiology diagnostics.
Disclosures • I have tried many of the instruments and kits over the years • No financial arrangements • No contracts • Some on-going evaluations for which I have received free kits (artus, Seegene)
Outline • Where are we with molecular diagnostics? • Extraction • Real-time • Multiplexing • Systems that do it all • Sequencing • The new and wonderful? • Or I wish we could have one of those.
What do most of us have? • A thermal cycler (old school, conventional, block PCR) • Molecular method for CT/GC detection • Real-time PCR instrument • Older versions • Newer versions • Automated or semi-automated extraction • Access to sequencing (send-out), if not doing on-site
Assays many of us are using • CT/GC • MRSA • VRE • HSV, Enterovirus in CSF • FluA (other respiratory)
Assays some of us are doing • Transplant screening and monitoring • CMV, EBV, BK, Adenovirus • HPV • B. pertussis • WNV • HepC, HepB • HIV • Norovirus • 16s PCR plus sequencing • C. difficile
Assays we would like to do • Everything • More multiplexes for “disease states” • Respiratory • Gastro • Encephalitis • Transplant • Resistance beyond
Some of the reasons we don’t • Cost of the test • Cost of the instrumentation • Trained staff • Commercially available assays • Health Canada approved • Sensitivity • Add on test, still need to do culture • What do we do with the results?
Pre-Analytical • Quality of sample • Sample type • Few changes in the last 20 years • One exception-Flocked swab for respiratory and other specimens • Should we be spending more time on this?
Extraction • Most important part of analysis? • Quantity, Quality • Lack of inhibitors • Complete lysis of cells • RNA, DNA, both • Throughput • Different methods for different sample types • Hands-on time • Expertise
Nucleic Acid Extraction • Semi-automated • Automated • Instruments with kits • EZmag (bioMérieux) • M48, MDX, (Qiagen) • MagNA Pure LC (Roche) • Kits with generic instruments
Thermal cyclers • Faster • More compact • More features (gradients) • Cheaper • Other options for detection
Real-time • significant improvements in the diagnosis of infectious disease. • Increased sensitivity and specifity • Increased speed • Allows multiplexing • Greatest impact is probably its use for the quantitation of target organisms in samples. • six orders of magnitude • closed-tube format removes need for post-amplification manipulation reduces risk of contamination • Costs more (reagents and instrumentation)
Real-time NAAT instruments • LightCycler 1.5, 2.0 • ABI 7700 • Smartcycler (Cepheid) Newer • Corbett Rotorgene 6000 • LightCycler 480, LightCycler 1536 • Stratagene: Mx4000, Mx3000P, Mx3005P • Applied Biosystems: ABI 7300, ABI 7500, ABI 7500 Fast, ABI 7900 Fast HT with automation accessory, ABI StepOne • Eppendorf: Mastercyclereprealplex • BioRad: MiniOpticon, MyiQ, Opticon2, Chromo4, iQ5 Good comparison of features of Real-time instruments http://www.horizonpress.com/pcr/qPCR-machines.html
Quantitation • Monitoring of transplant patients • CMV, EBV, BK, Adenovirus? • Efficacy of treatment • Prognosis • Resistance • Standardization? • QA • Other agents? • FluA
Self-collected influenza H1N1 viral loads: Smieja, Petrich, Mahony et al. [Late-breaker] International Congress of Chemotherapy and Infections, Toronto June 2009
High Resolution Melt • SNP analysis • Identification • Resistance
Systems that do it all for you • From sample extraction • to amplification • to detection
Gen-Probe • Tigris DTS • Soon to lauch-Panther for low to medium throughput laboratories
Tigris DTS • Target capture magnetic bead • No opening of tubes • Aptima kits • CT/GC • T. vaginalis • HPV • HIV-1 • HepC • 3.5 hrs for first results • 100 samples every 3 hrs following first results
Roche Diagnostics AmpliPrep • Automates the sample extraction process • Bar coded reagents packaged in ready-to-use spill-proof cassettes with self-sealing caps for increased stability • Up to 72 samples and 4 different assays can be loaded onto the system at the same time • Works in combination with the COBAS® TaqMan® 48 Analyzer, COBAS® TaqMan® Analyzer or Amplicor Analyzer to automate sample preparation, amplification, and quantitation of RNA or DNA
Roche (COBAS®) TaqMan® Analyzer • Automated and fully docked systems available with COBAS® AmpliPrep/COBAS® TaqMan® HIV-1, HCV, and HBV tests • 96-test capacity • 4 independently controlled thermal cyclers • Amplifies and detects RNA and DNA within hours • Quantitation standard and closed-tube format • Reduces the risk of sample contamination, optimizes workflow • Assays-HIV-1 (quant and qual), HCV, HBV AMPLICOR Analyzer • Bar-coded data entry • 48 sample capacity per run • Test-specific reagents • Multiplexing- up to 6 detections per amplified sample • Assays-HIV-1, CMV, CT/GC, HCV
Roche (COBAS®) TaqMan® 48 Analyzer • Two thermal cyclers can run independent PCR profiles simultaneously • One platform separately configurable to run IVD assays and up to 10 user-defined applications • Amplifies and detects RNA and DNA within hours • Requires no user interaction after sample loading completed • Optimizes workflow, minimizes contamination • Small, compact footprint • 48 samples at a time
Qiagen • QIAsymphony RGQ®, an automated system for molecular testing, • process a range of sample types • composed of modules that can be used as stand-alone systems or combined into a fully integrated system. • QIAsymphony SP for sample preparation • QIAsymphony AS for assay setup • Rotor-Gene® Q real-time PCR thermocycler.
BD Diagnostics • BD Max The new BD MAX™ • fully-automated open molecular biology platform allowing • multiple specimen types and assays simultaneously. • The Jaguar™ platform, initially developed by HandyLab, commercialised by BD as the BD MAX™ system
BD • Reagent strips for 1 to 24 samples per run • Microfluidic real-time PCR • Up to 144 tests per day (38 000 tests a year) • Extraction from multiple specimen types (blood, urine, swabs, etc.) • Ability to run multiple tests simultaneously (e.g. testing for S. aureus and Group B Streptotoccus at the same time) • 45 minutes to 2 hours, depending on the number of samples analysed
Abbott Diagnostics M2000 • Real-time PCR Amplification and Detection • 5 excitation, 5 emission filters • maxRatio Data Analysis • M2000sp • M2000rt
Abbott • Barcoded Laboratory Tubes • Open Mode • Flexible protocol for various • sample types and volumes • 24 to 96 samples • RealTime HIV-1 • RealTime HCV • RealTime HCV Genotype II • RealTime HBV • RealTime High Risk HPV • RealTime CT/NG • RealTime CT • RealTime CMV • RealTime HIV-1 Qualitative • Parvo B19 PCR Kit • HSV 1/2 PCR Kit • VZV PCR Kit • EBV PCR Kit • M. tuberculosis • SARS RT-PCR Kit
Multiplexing • New technologies have allowed for multiplexing with minimal loss of sensitivity • Seegene (Alere) • xTag (Luminex) • Multicode (Eragen) • ResPlex (Qiagen)
XtagLumionex • RVP respiratory panel • In development • Gastrointestinal panel • Fungal panel Eragen • MulticodeRTx HSV 1 and 2 • MultiCode-RTx System Neisseria gonorrhoeae • Others in development
ResPlex(Qiagen) • ResPlex 1 bacterial pathogens and adenovirus • ResPlex 2 viral pathogens • Others in development Seegene (Alere) • Respiratory panels • Gastrointestinal panels • Meningitis panels • STI panels • HPV • Mycobacteria panels
Multiplexing and Quantitation PrimeraDx • Multiplex PCR with continuous sampling and separation by capillary electrophoresis • Herpesviruses • C. difficile • Respiratory viruses • Others in development • Cost!
MALDI-TOF Mass Spectrometry Matrix-assisted laser desorption/ionization (MALDI) • Soft ionization technique • Allows analysis of biomolecules which fragment when ionized i • Matrix solution is mixed with the analyte (e.g. whole cells) on a MALDI analysis plate • Solvent is added to the sample resulting into a recrystallized matrix with analyte molecules spread throughout the crystals. • A current is generated resulting in movement of the fragmented biomolecules towards a detector based on size smaller molecules move faster) • A pattern is generated based on the size and number of molecules
MALDI/TOF spectra are used for the identification of microorganisms such as bacteria or fungi. • A colony of the microbe in question is smeared directly onto a dry plate (96 wells), overlayed with matrix • Mass spectra generated are analyzed by dedicated software and compared with stored profiles. • 96 wells in 40 mins. • Species diagnosis by this procedure is faster, more accurate and cheaper than immunological or biochemical tests.
Representative mass spectral outputs H. N. Shah, As published in CLI November 2005
Representative mass spectral outputs H. N. Shah, As published in CLI November 2005
Concordance between Conventional Routine Identification (Vitek; bioMérieux) and Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry Identification (Brucker Mass Spectrometer and Database Complemented with Local Database). Seng P et al. Clin Infect Dis. 2009;49:543-551 © 2009 by the Infectious Diseases Society of America
Sequencing • Applications • ID of known organisms • Cultured isolates • Sterile body fluids • Typing • Resistance detection
Sanger Sequencing • Expensive • Hands on time • 500-700 bases per run • Overnight?
Sequencing for ID • Does my ID give me the same ID as your ID? • Cultured isolate vs. clinical specimen? • What are you sequencing? -16S rDNA -HSP genes? -whole genome? • What will the changes be to taxonomy • What does it mean if it is not a recognized pathogen • What about contaminating nucleic acid?
Pyromark Q24 • PCR product of interest • Up to 24 samples can be prepared in parallel using Vacuum Workstation (~15 mins) to clean up PCR product. • Two formats-pre-programed sequence of nucleotide presented vs. sequential addition (G-A-T-C)n • High Volume instrument also available Pyromark Q96 • Real time addition and viewing of results • 50-100 bases added
High through-put • Genome sequencing? • Sequence of all data and pulling out important?