CURRENT STATE AND FUTURE PROSPECTS IN VIRAL DIAGNOSIS

Richard L. Hodinka, Ph.D.

University of South Carolina School of Medicine

Greenville Health System

Isolation of Viruses in Culture

Science 1949; 109:85-87

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

16

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

Compact Molecular Systems

Alere i System

Roche Cobas Liat

Cepheid GeneXpert Omni

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

Next-Generation Desktop Sequencers

Ion PGM

Ion Proton

Ion S5

NextSegmiSeg

miniSeq

MiniION

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

Clark EA, Kaleta EJ, Arora A, Wolk DM 2013. Clin Microbiol Rev 26:547-603

MALDI-TOF MS Systems

Bruker MALDI Biotyper CA System

BioMerieux Vitek MS

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

PNAS 2013. 110:12792-12797

PLoS One 2013. 8:e52198

Nature Scientific Reports 2016. 6:33752

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