Post on 24-May-2018
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CURRENT STATE AND FUTURE PROSPECTS IN VIRAL DIAGNOSIS
Richard L. Hodinka, Ph.D.
University of South Carolina School of Medicine
Greenville Health System
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 NeutralizationAntibody Test
Diagnostic Virology Over Time
Conventional Tube and Rapid Shell Vial/Plate CultureRapid Antigen Detection
Direct Immunofluorescence AssaysImmunoassays for Antibody Detection
PCR Through the AgesYear
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
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
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
Value of Molecular Assays
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
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
QUALITATIVE TESTS QUANTITATIVE TESTS
Clinical Benefits of Rapid and Accurate Diagnosis
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
Informing timely and
effective antimicrobial
therapy
Preventing secondary spread
of infection
Shortening hospital stays
Reducing costs of
unnecessary tests
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
Cycle Number
Flu
ore
scence
Cycle Threshold (CT)
With real-time PCR,
the more copy numbers of nucleic acid present,
the sooner an increase in fluorescence is detected
1 40
NanotechnologyThe 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
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
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
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
The FilmArray Reaction Pouch
Matrix
H3
N2
Bocavirus
NP
Influenza A
High density array with >100
individual 2nd stage PCR wells;
each well contains one reaction
and results are generated from
analysis of melt curves
Chemical
Circuit Board
Reagent Storage
(freeze dried, stable @ RT)
Silica bead beating to
release nucleic acids
Magnetic bead
NA extraction
RT for
RNA Targets
1st stage multiplex PCR
2nd stage nested PCR
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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
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
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
Outcomes of New Molecular Revolution
Assay Miniaturization
Portability
Lower Cost
Less Sample
Less Reagents
Decentralize Testing
Faster Turnaround
Improved Healthcare
Open/Expand Market
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
U.S. $18 billion dollar market by 2016 for POC
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
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
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
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
RainDance RainDrop Digital PCR
Bio-Rad QX100 ddPCR
Fluidigm BioMark HD
Life Technologies
QuantStudio 3D
QuantStudio 12K
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)
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
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
MALDI-TOF MS in Clinical Virology
Matrix-assisted laser desorption/ionization time-of-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
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
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
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 2015. 896164-6166
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