CURRENT STATE AND FUTURE PROSPECTS IN VIRAL DIAGNOSIS

Transcription

1 CURRENT STATE AND FUTURE PROSPECTS IN VIRAL DIAGNOSIS Richard L. Hodinka, Ph.D. University of South Carolina School of Medicine Greenville Health System

2 Isolation of Viruses in Culture Science 1949; 109:85-87

3 Diagnostic Virology Over Time Rotavirus by Electron Microscopy Suckling Mice Inoculation for Enterovirus Detection Histopathology for CMV Egg Inoculation for Influenza Complement Fixation and Hemagglutination Inhibition Serologic Assays Plaque Reduction Neutralization Antibody Test

4 Diagnostic Virology Over Time Conventional Tube and Rapid Shell Vial/Plate Culture Rapid Antigen Detection Immunoassays for Antibody Detection Direct Immunofluorescence Assays

5 PCR Through the Ages Year 1953 Discovery of DNA double helix structure 1967 Thomas Brock reports on isolation of extremophilic bacterium Thermus aquaticus 1970 Enzymatic assay used to replicate short piece of DNA using a single primer 1976 Taq DNA polymerase, one of best known thermostable enzymes, isolated from Thermus aquaticus 1983 PCR technique created by Kary B. Mullis 1985 First publication of PCR by Cetus Corporation appears in Science 1986 Purified Taq polymerase first used in PCR 1988 PerkinElmer introduces first automated thermal cycler 1989 Science declares Taq polymerase molecule of year 1993 Kary Mullis awarded Nobel Prize in Chemistry 1994 Real-time PCR introduced

6 Transformational Technologies Nucleic acid amplification tests Broad syndrome-specific molecular multiplex screening tests Point-of-care molecular testing Digital PCR Genomic Sequencing MALDI-TOF MS Host Response Diagnostics

7 Molecular Diagnostics Has rapidly evolved over the years Numerous technological advances Now the accepted standard for the diagnosis and monitoring of many microbial pathogens Significant clinical benefit being shown Bacteria Viruses Fungi Parasites

8 Value of Molecular Assays QUALITATIVE TESTS Early detection of infections ID new microorganisms Detect microbes that are: Uncultivable Fastidious or slow-growing Too dangerous to be amplified in culture Nonviable or present in extremely low numbers or in small specimen volumes Molecular epidemiology QUANTITATIVE TESTS Associate infection with disease Monitor efficacy of therapy Predict treatment failure; emergence of drug resistance Assess progression of disease Facilitate understanding of natural history and pathogenesis of organisms

9 Clinical Benefits of Rapid and Accurate Diagnosis Informing timely and effective antimicrobial therapy Preventing secondary spread of infection Shortening hospital stays Reducing costs of unnecessary tests Provide a specific diagnosis; early informed decision making Help manage high-risk patients (e.g., cancer, transplant, HIV, those in ICU, those with underlying co-morbidity) Education and increased clinical awareness Rapid outbreak ID at local, regional, national, and global levels

10 Real-Time PCR Technology Introduced in mid-1990s Has reached the greatest maturity Now the new gold-standard More sensitive and specific than any combination of culture/radts Can detect, quantify, and genotype Multiplex capabilities; co-infections identified Displacing more traditional methods Excellent sensitivity and specificity Major impact on patient care

11 Fluorescence With real-time PCR, the more copy numbers of nucleic acid present, the sooner an increase in fluorescence is detected Cycle Threshold (C T ) 1 Cycle Number 40

12 Nanotechnology The Newest Wave Has been applied to molecular testing Assay miniaturization; compact platforms; speed and simplicity for use by all labs and beyond Sample-to-result automation High multiplex capability; syndrome-specific testing Point of care testing to accurately detect multiple organisms, type them when appropriate, screen for drug resistance if desirable, and even quantify at some level

13 Selected Molecular Platforms Specimen In-Result Out Cepheid GeneXpert Roche cobas Liat System Alere i System Luminex ARIES Atlas Genetics Enigma Diagnostics Micronics Cirrus Dx T-COR 8 BioFire FilmArray Nanosphere Verigene SP QuantuMDx Janssen Diagnostics Rheonix Encompass Optimum GenMark Dx esensor Veredus VereChip Great Basin Portrait Focus Dx Simplexa/3M Cycler Quidel Savanna & Solana Meridian Illumigene BD Max System ELITe InGenius Systems Biomeme Fluoresentric, Inc. GeneWEAVE VivoDx

14 Syndrome-Specific Testing Highly multiplexed PCR platforms One sample-multiple results Comprehensive panels of probable pathogens causing a particular syndrome Designed to directly probe specimens (respiratory, stool, CSF, blood, urogenital) and positive blood culture bottles for an array of microorganisms

15 BioFire FilmArray System Closed system for sample preparation, nested multiplex PCR, and analysis Chemical circuits in a pouch; sample to result in ~65-70 min Fully automated instrument; integrated electropneumatic systems

16 The FilmArray Reaction Pouch 16 Reagent Storage (freeze dried, RT) Silica bead beating to release nucleic acids Chemical Circuit Board 1 st stage multiplex PCR 2 nd stage nested PCR Magnetic bead NA extraction RT for RNA Targets N2 H3 Influenza A High density array with >100 individual 2 nd stage PCR wells; each well contains one reaction and results are generated from analysis of melt curves Bocavirus NP Matrix

17 BioFire FilmArray Panels Available (US-IVD, Health Canada IVD, CE- IVD Europe) Respiratory Panel 20 targets; viruses, bacteria) Blood Culture ID Panel 27 targets; Gram +/Gram- bacteria, yeast (Candida spp.), antibiotic resistance genes GI Panel 22 targets; bacteria, protozoa, viruses Meningitis/Encephalitis Panal 14 targets; bacteria, viruses, yeast

18 Multiplex PCR Assays Redefining the diagnosis of infectious disease Can have significant impact on patient care and management Particularly important to help narrow down causative pathogen(s) when more than one pathogen can cause same clinical presentation

19 Point-of-Care Molecular Technology Over the years, molecular testing has become increasingly automated and efficient Progressive advances in amplification chemistries, microfluidics and miniaturized detectors Paved way for introduction and use of compact sample in-results out diagnostic devices

20 Outcomes of New Molecular Revolution U.S. $18 billion dollar market by 2016 for POC Portability Lower Cost Less Sample Assay Miniaturization Less Reagents Decentralize Testing Faster Turnaround Desire is to have self-contained, fully integrated sample-to-report devices that accept raw, untreated specimens, perform all of the molecular steps, and provide interpreted test results in < 1 h Improved Healthcare Open/Expand Market

21 Desire to Use for POC Service Doctor s offices Drugstore clinics In the field At home Not confined to regulated laboratory environments Accelerated turnaround times for results

22 Compact Molecular Systems Roche Cobas Liat Alere i System Cepheid GeneXpert Omni

23 Fluoresentric Handheld Device Uses smartphone for optics, data collection, telemetry XCR extreme chain reaction; blazing fast amplification (5 min) Integrated fluorescence detection Extremely low power requirement; no moving parts, no fluidic movement Low cost portable instrument Low cost reagents Compatible with existing PCR

24 Point-of-Care Molecular Testing Beginning to enter clinical practice throughout the world Paradigm shift towards decentralized testing Especially suited for applications where fast turnaround is desirable where centralized laboratory services face limitations in rural areas and places that are hard to reach In resource-limited countries Poses diverse technological, economic and organizational challenges

25 Digital PCR New approach to nucleic acid detection and quantification Unlike real-time quantitative PCR, quantifies DNA in a sample without the need for a standard curve Provides precise absolute quantification of nucleic acids

26 Life Technologies QuantStudio 12K QuantStudio 3D RainDance RainDrop Digital PCR Bio-Rad QX100 ddpcr Fluidigm BioMark HD

27 Sequencing Technology Amplify specific sequences Perform sequencing and computer-assisted analysis, and query genetic database Traditional Sanger Sequencing Next Generation/Whole Genome Sequencing (NGS/WGS)

28 Clinical Value of NGS: Agnostic Testing Has potential to dramatically revolutionize clinical virology/microbiology Ultimate pathogen multiplex assay Identify any expected or unexpected pathogens from single specimen or as isolates Identify rare pathogens not frequently on differential Identify novel, highly divergent pathogens from a sample (metagenomics) Detect virulence determinants and genetic markers/variants of drug resistance Track outbreaks of infection

29 Next-Generation Desktop Sequencers NextSeg miseg Ion PGM miniseq Ion Proton Ion S5 MiniION

30 Mainstreaming of Sequencing Routine clinical testing will be a reality with time Need for development of simplified solutions for all phases of testing Sample preparation Sequencing Data analysis Result interpretation Need to address clinical relevance of finding a fragment of nucleic acid that may not correlate with patient s disease Need access to well-vetted databases

31 MALDI-TOF MS in Clinical Virology Matrix-assisted laser desorption/ionization timeof-flight mass spectrometry Based on the detection of the mass of molecules Until now, main use has been to identify and type bacteria from a positive culture; used some for fungi, mycobacteria, and parasites Rapid and inexpensive; accurate results with simple sample preparation and minimal technical time Has applications in Clinical Virology

32 MALDI-TOF MS Clark EA, Kaleta EJ, Arora A, Wolk DM Clin Microbiol Rev 26:

33 MALDI-TOF MS Systems Bruker MALDI Biotyper CA System BioMerieux Vitek MS

34 MALDI-TOF MS Applications in Virology Identification of viruses from clinical samples Detection of mutations and strain variation Screening of viral subtypes Identification of antiviral resistance Epidemiology of viral infections

35 Host Response Diagnostics Analysis of transcriptome of host Transcriptome is full range of mrnas produced in a particular cell or tissue or expressed by an organism Can measure the expression of thousands of genes at the same time Generation of gene expression profiles can describe changes in the host transcriptome in response to a particular condition or treatment Can be coupled with metabolomics and/or proteomics

36 PNAS : PLoS One :e52198 Nature Scientific Reports :33752

37 Ribosome Profiling Innovative technique Uses deep sequencing to monitor gene expression at the level of translation rather than transcription Providing novel insights into the identities and amounts of proteins being produced in cells infected with viruses J Virol

38 Conclusions Significant advances have been made in field of clinical virology over the years Continuous introduction of newer technologies Will need to be adequately trained on these methods and well informed about the availability and utility of such tests for optimum integration into clinical care Continuous education and regular interactions between clinical virologists and healthcare providers will be vital