Molecular Diagnosis of Infectious Diseases

Gregory J. Tsongalis, Ph.D.

Professor of Pathology

Director, Molecular Pathology

Dartmouth Medical School

Dartmouth Hitchcock Medical Center

Norris Cotton Cancer Center

Lebanon, NH

*

DHMC, Lebanon, NH

*

Boston, MA

CANADA

California

Flo

rida

Texas

The Human Genome Project

February 2001

A major impact on microbial genomics.

• 1995, first complete genome sequence of a free-living organism, Haemophilus influenzae

• Since then 1,554 complete bacterial genomes

• Species pangenome contains set of core genes that are common and set of dispensable genes that are absent in at least one strain.

• Approximately 90% of bacterial genome codes for protein whereas <2% of human genome codes for protein

Relman DA. N Engl J Med 2011;365:347-357

Microbial Genomics

Why Molecular Pathology? • Genetics

• previously unavailable tests

• carrier detection

• risk assessment

• Infectious diseases

• turn-around time

• inability to culture

• microscopic interpretation and competency

• quantitative analysis

• genotyping

• Heme/Oncology

• confirmation

• minimal residual disease

• therapeutics

• Identity testing

• most polymorphic molecule

• Therapeutics/PGx

Applications of Molecular Analyses to

Infectious Diseases

• Qualitative detection

• Quantitative detection

• Identification/speciation

• Microbial “Identity” Testing

• Genotyping/Drug Resistance

Molecular Infectious Disease (Molecular Testing Methods)

• Nucleic acid (DNA, RNA) specimens:

• Blood

• CSF

• Tissue (fresh, frozen FFPE)

• Urine

• Stool

• Amniotic fluid

• Other body fluids

• Microbial isolate

Nucleic Acid Extraction Technologies

1980- Manual Extractions:

1. Make your own buffers

2. pH your own buffers

3. Lab math

4. TAT = 1.5 days

5. Low throughput

2000- Automated Extractions:

1. Reagent contracts

2. Consumables

3. Maintenance contracts

4. TAT = 20-45 minutes

5. Higher throughput

Automation of NA Extraction

Extracton Type Instrument

Magnetic silica particles COBAS AmpliPrep, EZI,

MagnaPure Compact and

LC, Maxwell 16, NucliSens

EasyMag

Magnetic charge switch particles iPrep Purification Instrument

Silica vacuum manifold plate 6100/6700 Automated

Nucleic Acid Workstation,

QIAxtractor

Iron oxide particles m2000sp

Silica spin columns QIAcube

Persing, et.al. 2011. Molecular Microbiology – Diagnostic Principles and Practice (2nd Ed.) C. Hill, p123.

Extraction Issues to be Considered

• Specimen type

• Extraction Method

• Inhibitors

• Target sequences (DNA vs RNA)

• Controls (type and number)

Nucleic acid probes for culture confirmation and direct

detection (DNA and RNA probes)

Nucleic acid amplification (“NAT”) technologies

– Sequence amplification

• Polymerase chain reaction (PCR) and real time PCR

• Transcription-mediated amplification (TMA and NASBA)

• Strand displacement amplification (SDA)

• Ligase chain reaction (LCR)

• Loop-Mediated Isothermal Amplification (LAMP)

– Signal amplification

• Branched-chain DNA (bDNA)

• Hybrid capture

• Invader (Cleavase)

Sequencing – Sanger or Next Generation

Microarrays and Mass Spectrometry

Molecular Infectious Disease (Molecular Testing Methods)

Sample

Preparation Amplification Detection

Controls for Qualitative Molecular Diagnostic Testing

1. 2. 3. Extraction control

(pre- or post-)

Blank control

Negative control

Positive control

Internal amplification control (IAC)

(competitive vs non-competitive) efficiency inhibitors

?

Real-Time PCR

Permits rapid target Id; <30 min

Eliminate post-PCR processing

Highly specific; Hybridization probes

Allows multiplexing

Permits quantification

Signal intensity is directly proportional to the amount of

amplified DNA

Threshold cycle (Ct) determination; the cycle at which

target is first detected

Detection Chemistries

Intercalating/binding dyes

–SYBR® green I

Dual label probes/ Quenched probes

-TaqMan® (5’ nuclease assay)

-Molecular beacons, Scorpions

-FRET probes

Increase in Reporter Signal “Reports”

Amplification of Target

Ct: Primary Signal Analysis

Threshold Line

Threshold

penetration

Threshold Cycle

Threshold Value

Melt Curve Analysis

• Following amplification temperature is slowly increased (ie. 60oc to 95oc, 0.2oc/sec)

• Strands denature

• Dye released, fluorescence decreases

• Melting curve; Temperature (x) vs. Fluorescence (y)

• Tm function of %GC and length

Melt Curve Analysis

Phase Transition

Target

Primer dimer

Melt Curve Analysis

BKV JCV

Sample

Preparation Amplification Detection/Analysis

PCR Testing Steps

Automated extraction + Real Time PCR = STAT DNA Analysis

• Positive or negative

• Increased sensitivity

• Decreased TAT

• Low to high throughput

• Becoming more automated

• Applications are limitless but questionable

clinical utility

• Major impact with real time PCR

Molecular Infectious Disease (Qualitative)

• Misperception of prevalence

• Asymptomatic

• Non-specific symptoms

• Coinfections

• Unreliable diagnostic tests

• Asymptomatic individuals serve as a

reservoir of infection

Issues: high volume, STD (social), high throughput

“THE SILENT EPIDEMIC”

Qualitative Molecular Infectious Disease Testing

(Chlamydia trachomatis)

Abbott LCx® System

Sample Prep Amplification Detection

LCx®

Analyzer LCx®

Thermocycler

LCR Amplification

(LCx® MEIA System)

MEIA particle linked to enzymatic

conversion

of fluorescent dyes

BD Viper (ProbeTec System)

GenProbe TIGRIS DTS

Assay Gene Target NAAT Amp. Control

M2000 v2

(Abbott)

Plasmid (2 targets) Real-time

PCR

Yes

ProbeTec

(BD)

Plasmid SDA Yes

Hybrid Capture II

(Digene)

Plasmid and

genome

Hybridization No

PACE 2 CT

(Gen-Probe)

23S rRNA Hybridization No

Aptima Combo 2 23S rRNA TMA No

Aptima CT 16S rRNA TMA No

TaqMan48 v2 Plasmid and omp1 PCR Yes

Molecular Infectious Disease Molecular Diagnostic Methods for C. trachomatis

• Home brew (Laboratory Developed Tests)

• Analyte Specific Reagents

• Primers > PCR > Gel

• Real time PCR

• Near patient testing???

Qualitative Molecular Infectious Disease Testing (Non-Kit Based Assays)

1.

2.

Bam HI-W repetitive region (296 bp)

Conserved Among EBV Strains

Qualitative Molecular Infectious Disease Testing (EBV)

NE

GA

TIV

E

PO

SIT

IVE

SP

EC

IME

N 1

SP

EC

IME

N 1

SP

EC

IME

N 2

SP

EC

IME

N 2

DN

A M

AR

KE

R

EBV BETA-ACTIN

Real Time PCR Detection of EBV

EBV DNA,

5000, 500, 50 and 5

copies/ml

Target gene = BNT p143ORF38

• PCR is both sensitive and specific

• Viability (post treatment still detectable)

• Use of multiple primer sets

• Specimen types for PCR testing

• Nasopharyngeal swab (Dacron not Ca alginate

or use of a mucolytic agent to avoid PCR

inhibition)

• Other specimens may serve as alternatives

• Other laboratory testing (Culture or DFA)

• Bordet and Gengou (Starch Blood Agar)

• Antibiotic selection plate (Cephalexin)

• Synthetic media

Molecular Infectious Disease (Bordetella pertussis)

Bordetella Target Genes

IS481 500-1,000 present not present present

IS1001 10-20 not present present present

PTxs1 1 present present not present

pertussis parapertussis holmesii Copy#

• Specimen types

• Extraction efficiencies

• Automation vs manual

• Importance of controls

• Assessing performance characteristics

– Sensitivity, specificity, precision, accuracy

– Limit of blank, limit of detection

• Armbruster and Pry. Clin Biochem Rev 2008;29 (Si):S49-

S52

• Regulatory: FDA vs ASR vs RUO

Qualitative Molecular Infectious Disease Testing

(What did we learn?)

• How much is present

• Limit of quantification (LoQ)

• Automation

• Low to high throughput

• Which applications are clinically relevant

• Is the LLoQ the same as LoD?

• Why quant vs qual?

Molecular Infectious Disease (Quantitative)

Qualitative Genotyping Quantitative

Molecular Infectious Disease (HIV-1)

HIV-1 Subtypes

• Group M (Major)

– Subtypes A - H

• Group O (Outlier)

• Group N (New)

HIV-1 Viral Load Testing

• Not diagnostic (?)

– Designed to monitor infection

– Sensitivity may be higher than proviral DNA PCR

• Indications

– Disease progression

– Prognosis

– Response to antiviral agents

• 0.5 log10 units is considered clinically significant

Molecular Assays for the Qualitative Detection of HIV-1 RNA

Assay Method Target Application

COBAS® AmpliScreen

HIV-1 Test, v1.5 (IVD)

RT-PCR HIV-1 gag gene Qualitative detection of

viral RNA from plasma,

organs and tissues

COBAS®

AmpliPrep/COBAS®

TaqMan® HIV-1

Qualitative Test (RUO)

PCR HIV-1 gag gene Qualitative detection of

HIV-1 RNA and proviral

DNA in plasma,

anticoagulated fresh

whole blood and dried

blood spots

COBAS® TaqScreen

MPX Test (IVD)

PCR Multiple Simultaneous testing

for multiple viruses in a

single sample: HIV-1

group M, HIV-1 group

O, HIV-2, HCV and

HBV

Molecular Assays for Quantification of HIV-1 RNA Assay Method Target Dynamic Range

(copies/mL)

Roche Amplicor HIV

Monitor v1.5 test,

Standard (IVD)

RT-PCR HIV-1 gag gene 400 – 750,000

Roche Amplicor HIV

Monitor v1.5 test,

Ultrasensitive (IVD)

RT-PCR HIV-1 gag gene 50 – 100,000

Roche Cobas

AmpliPrep/Cobas TaqMan

HIV Test v.1.0 (IVD)

Real-time PCR - TaqMan HIV-1 gag gene

48 – 10,000,000

Roche Cobas

AmpliPrep/Cobas TaqMan

HIV Test v.2.0

(IVD)

Real-time PCR - TaqMan

HIV-1 gag gene, LTR 20 – 10,000,000

Siemens Versant HIV RNA

3.0 Assay (IVD)

bDNA HIV-1 pol gene 75 – 500,000

Biomerieux NucliSENS

EasyQ® HIV-1 V2.0 (RUO)

NASBA HIV-1 gag gene 25 – 10,000,000

Abbott RealTime HIV-1

Assay (IVD)

Real-time PCR

HIV-1 integrase gene 40 – 10,000,000

Viral Load Testing –

Interpretation of Results

• Results should be presented as Log10 Values

– Prevents clinicians from over interpreting small variations

in viral load

• Changes in viral load must exceed 0.5 log10 (3 fold)

to represent biologically relevant changes in viral

replication

• Due to differences in performance, viral load should

be quantified at follow-up by the same version of the

same assay that was used initially

Viral Load Testing –

Interpretation of Results

• Clinical infections can increase viral load by

as much as 1 log

– HSV infections

– Opportunistic infections

– Vaccinations

• Influenza

• Tetanus

• Pneumococcal

– Viral load testing should not be performed for

1 month following such infections

Patient Monitoring

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

5.50

6.00

1/99 2/99 3/99 5/99 8/99 12/99 4/00 5/00 9/00 2/02 6/02 7/02 8/02

Initiate Rx

Resistance Testing

Vir

al L

oad

(lo

g10

co

pie

s/m

l)

1. Compliance

2. Resistance

HIV-1 Resistance Genotyping

• Determine the sequence RT and protease genes

VGI (FDA approved), ABI, laboratory developed

• Compare sequence to wild type virus Identify mutations

• Associate mutations with resistance Knowledge of genetics of resistance

Data base, rules based system

Different interpretations of same mutations

Viral Genotyping –

Antiretroviral Resistance Testing

FDA Approved Systems Assay Name Manufacturer Methodology

Truegene HIV-1

Genotyping Kit

Siemens Medical

Solutions Diagnostics,

Tarrytown, NY

DNA Sequencing

(polyacrylamide gel

electrophoresis)

ViroSeq HIV-1

Genotyping System

Abbott Molecular, Des

Plaines, IL

DNA Sequencing

(Capillary

electrophoresis)

Viral Genotyping

The BK Virus

• BKV is a member of the polyomavirus family.

• BKV typically presents with respiratory infection and about

80% of adults are seropositive for BKV antibodies.

• After primary infection, the virus enters a latent phase in the

kidneys, brain, and uterus.

• The virus usually remains dormant but may reactivate during

pregancy, HIV infection, diabetes, or after transplant surgery.

Rejected kidney transplant exhibiting BK nephropathy

and chronic rejection.

BKV Reactivation with Immunosupression

• Following Bone Marrow Transplant Hemorrhagic

Cystitis

• Following Renal Transplant polyomavirus-

association nephropathy (PVAN or BKVN)

Typical intranuclear inclusion pre- and post- micro-dissection

a b

Melt Curve Analysis for BKV/JCV From Microdissected Samples

B

0

20

40

60

80

100

120

140

78.1 78.9 79.7 80.4 81.3 82.1 82.9 83.7 84.5 85.2 86.0 86.9

Temperature

-dF

/dT

27412 Glom

27412 Tubules

BK Virus

JC Virus

NTC

A

0

20

40

60

80

100

120

140

78.1 78.9 79.7 80.4 81.3 82.1 82.9 83.7 84.5 85.2 86.0 86.9

Temperature

-dT

/dT

14084 Glom

14084 Tubules

BK virus

JC virus

NTC

Adeyi OA, Belloni DR, Dufresne SD, Schned AR, Tsongalis GJ.

Real-time polymerase chain reaction and laser capture microdissection techniques

in the diagnosis of BK virus infection of renal allografts.

Am J Clin Pathol 124(4):537-542, 2005

Monitoring with PCR

• BK Viruria and Viremia relatively frequent in

immunosuppressed renal transplants

• Positive PCR results alone not very useful

• Quantitative Testing (real-time PCR)

– BKV levels over time

– With viral level cut-offs BKV PCR useful for

determining PVAN (Viscount 2007):

• In Urine: 100% Sensitivity; 78% Specificity

• In Plasma: 100% Sensitivity; 91% Specificity

Requirements for BKV PCR

• Quantitative

• Analytic Specificity: Only detects BKV

• Analytic Sensitivity: Low limit of detection?

• Wide dynamic range (concentrations above

10 log10 copies/mL)

• Specimen type: Urine and plasma

Plasma MGB vs. Eragen Assays

Urine MGB vs Eragen Assays

BKV PCR Obstacles

• No FDA-approved assays (IVDs): must use

laboratory-developed tests (with ASRs)

• No international standards defining BKV

concentrations

• Limited options for calibrators for standard

curve-Lack of standarization

BKV PCR Obstacles

• Urine specimens can be difficult: PCR

inhibitors

• Assay should be specific for BKV but detect

various strains of BKV equally (robust vs

stringent)

– Gene target

– Primer/probe location

• Performance

• Variants

• Clinical implications

• QC of numerical data

• Automation

Quantitative Molecular Infectious Disease Testing

(What did we learn?)

So what is next in molecular infectious

disease testing?

• Gram positive

• Bacillus (coccobacillus , “diphtheroid”)

• Anaerobe

• Non-spore forming

• Slow grower

• Non-motile

• Many strains are indole & catalase positive

• Frequent contaminant of blood cultures.

Diagnostic challenges of

Propionibacterium acnes

McPherson: Henry's Clinical Diagnosis and Management by

Laboratory Methods, 22nd ed. 2011 Saunders Elsevier

Accessed Online, 8/31/11.

Isabella Martin, M.D.

Habitat

• Three categories:

– Acnes in teenagers & adults.

– Invasive deep-seated infections

• Pacemakers, valves, shunts.

– Surgical wound infections

• Prosthetic joints, spinal hardware.

Infections

Mandell, Douglas, and Bennett's Principles and

Practice of Infectious Diseases, 7th ed. 2009

Churchill Livingstone Accessed online 8/31/2011.

Clinicaladvisor.com Accessed online 8/31/11.

Orthopaedics Quandary

1-2

years

- +

Revision

Arthroplasty

Removal of

hardware

Ultra-sound

guided joint

aspiration

with culture

6 Wks IV

Antibiotics

Implantation

of new

hardware

Oops!

+ -

Good

• Real time PCR Taqman assay.

• Quantitative

– Ct – detect contamination.

• Internal control: beta-actin?

• Multi-plexed?

PCR for P. acnes

The Automation Revolution Continues -2011

BD MAX™ System

(HandyLab Jaguar)

1.fully automates cell lysis,

nucleic acid extraction,

PCR set-up, amplification

and detection

2.24 samples per run

Enigma Diagnostics

Enigma ML

1. portable

2. self-contained

3. ultra-rapid,

laboratory-

standard results

4. point-of-care

testing

Smaller is Better

Idaho Technologies

Film Array

IQuum

Liat Analyzer

PathoGenetix

An automated system for identification and strain typing of pathogens in complex

sample types.

Does not require specific reagents for detection of each pathogen, this approach

uses a single reagent set to create genomic barcodes which are then used to

detect and identify thousands of strains from hundreds of species.

No PCR or other amplification technique is applied.

Genome Sequence Scanning (GSS) technology. GSS enables high throughput,

single molecule DNA analysis to be used for pathogen identification and

characterization in complex biological samples.

Biocartis

Compact platforms whose ease of use will lower the entry barrier to

diagnostic testing.

Developing a platform that integrates sample preparation of nucleic acids,

amplification, detection, and the generation of a result without user

intervention and a detection platform that includes encoded micro carriers, a

micofluidic cartridge, and an instrument for low to high multiplexing

detection of biomarkers.

Lumora

BART (Bioluminescent Assay in Real-Time) is a novel reporter system which is

used with isothermal nucleic acid amplification technologies.

Easy-to-use, affordable, robust hardware.

What if………..

• Miniaturize

• Low cost

• Fast

• Multiplex

• Smart phone

ready

DHMC Molecular Pathology Laboratory and

Translational Research Program

Samantha Allen

Betty Dokus

Susan Gallagher

Carol Hart

Arnold Hawk

Claudine Lefferts, Ph.D.

Joel Lefferts, Ph.D.

Rebecca O’Meara

Elizabeth Reader

Mary Schwab

Heather Steinmetz

Laura Tafe, M.D.

Brian Ward

Eric York