molecular methods: the future in clinical practice · molecular methods: the future in clinical...

2015 Annual Scientific Meeting of College of Pathologists, Academy of Medicine Malaysia & 40th Anniversary

Celebration of the Pathology Advocates

Molecular methods: the future in clinical practice

13th to 14th June 2015, Berjaya Times Square Hotel, Kuala Lumpur, Malaysia

Professor Dr Ng Kee Peng (BSc, PhD, MBBS, DTM&H, FRCPath)

1

Comprehensive Role of Molecular Diagnostics

Description Diagnostic tests to complement traditional risk factors

Applied to high-risk patients to identify disease early

Used for definitive diagnosis and general cancer typing

Assess severity and/or risk of recurrence

Inform adjuvant chemo decision

Used to predict efficacy or safety response to specific treatments

Recurrence monitoring

Monitoring for treatment efficacy

Clinical Implications

Implement wellness programs proactively

Nip disease in the bud with early treatment

Refer to the appropriate

specialist

Determine whether treatment is necessary

Do not waste unproductive therapy

Control disease progression with changes in treatment

Monitoring Therapy Selection

Staging and Prognosis

Diagnosis Screening Risk Assessment

Source: DxInsights White Paper January 2012

Diagnostics can help clinicians optimally manage patients through the continuum of

care.

HIV: A Progression in Diagnosis and Treatment

1990 2000 2010 1980

2011

The first over the counter (OTC) HIV

test being reviewed by the

FDA

1981

Official beginning of the HIV/AIDS epidemic with the MMWR report of 5 cases of PCP

1983-84

Isolation of virus

1985

First HIV antibody test licensed by FDA detects antibodies to HIV and aids in diagnosis

1992

FDA licenses first rapid HIV test for diagnosis of HIV

1994

FDA approves first oral test for diagnosis of HIV

1996

FDA approves first viral load testing that measures HIV in the blood, specifically the number of copies of viral RNA per one mL of blood

2007

Trofile launched, a companion diagnostic to

the drug Selzentry , indicated for patients

infected with CCR5-tropic HIV

AMA recommends resistance testing to help determine a patient's initial antiretroviral regimen only if there are factors that indicate an increased risk for resistance

HIV diagnostics now span entire spectrum of disease management, from screening to diagnosis to treatment selection to monitoring

Source: DxInsights White Paper January 2012

The evolution of diagnostics for HIV identification and subsequent care is a good example of the

diagnostic industry progress over time.

Molecular Diagnostics

Conventional PCR

Real Time PCR Probe-based real-time PCR or TaqMan PCR: requires a pair of PCR primers and an

additional fluorogenic probe which is an oligonucleotide with both a reporter fluorescent dye and

a quencher dye .

Intercalator-based method or SYBR Green method: requires a double-stranded DNA dye in

the PCR reaction which binds to newly synthesized double-stranded DNA and gives

fluorescence.

Digital PCR

4

Conventional Polymerase Chain Reaction

Denaturing-Annealing-Extension process

DNA polymerase: Add nucleotide at 3’ end 5

Conventional PCR

6

Laboratory Methods To Identify Mycobacterial Species following

Growth Detection with the BACTEC MGIT 960 System

Biochemical Tests

GenProbe Technology (AccuProbe)

M. tuberculosis complex, M. avium-intracellulare complex, M.

avium, M. intracellulare, M. kansasii, and M. gordonae.

BD ProbeTec ET system

High Performance Liquid Chromatography (HPLC)

GenoType test systems HAIN LIFESCIENCE

7

Molecular Methods To Identify Mycobacterial Species following

Growth Detection with the BACTEC MGIT 960 System UMMC

GenoType Mycobacterium CM: cultivated samples

GenoType Mycobacterium AS

GenoType MTBC

GenoType MTBDR plus

GenoType Mycobacteria Direct : pulmonary and extrapulmonary

direct patient materials OR culture

GenoType test systems HAIN LIFESCIENCE

8

2014 2013

TOTAL SAMPLES 14341 12112 TOTAL PATIENTS 7410 5880 MTBC 880 758

BCG 3 1

M.TUBERCULOSIS 7 5

TOTAL NTM 453 456

M.FORTUITUM 157 189

M.ABSCESSUS 110 107

M. species 35 27

M.INTRACELLULARE 32 43

M.CHELONAE 18 9

M.MUCOGENICUM 18 1

M.KANSAII 16 17

M.GORDONAE 15 19

M.LENTIFLAVUM 14 13

M.AVIUM 13 12

M.INTERJECTUM 7

M.CELATUM 6 5

M.SCROFULACEUM 6 8

M.SZULGAI 2 3

M.GASTRI 1

M.GENAVENSE 1 1

M.SIMIAE 1

M.SMEGMATIS 1 1

M.HAEMOPHILUM 1

Molecular Diagnostics: Mycobacteriology

9

A combination of conventional PCR, amplifies and simultaneously detects or

quantifies targeted DNA molecule in real time within one reaction vessel.

It allows measurement during amplification for quantitative or qualitative

results:

Qualitative detection (Yes/No answer)

Absolute Quantification (result: e.g., copies/ml)

Relative Quantification (result: e.g., relative ratio)

It allows measurements at the endpoint of PCR:

Endpoint Genotyping (allelic discrimination)

It allows melting curve analysis subsequent to PCR amplification used for:

Product identification with SYBR Green I

Detection of known mutations using e.g., HybProbes

Detection of unknown mutations with Gene scanning

Real-Time Polymerase Chain Reaction (Real-Time PCR)

10

Types of Fluoresent Probe: Oligonucleotide hybridization

• DNA binding protein probe

• Binds to all double-stranded DNA in PCR reaction

• Example: SYBR Green, cyanine dye

• Advantage: Flexibility, not sequence specific

• Drawbacks: Bind to double stranded DNA—specificity issue

SYBR Green

11

For Internal Use Only

Commonly used fluorescent Probes

A lot of diagnostic tools available

Costs

Time-consuming

Labor-intensive

Technically

complex/requires specific

expertise

Lack sensitivity and

specificity

Limited coverage

Overlapping

symptomology

Need to order multiple

tests specific for

suspected organisms

Unavailability of tests

for many organisms

Confounded by:

Low yield

?

Core Molecular diagnostics services

COBAS Ampliprep /COBAS TaqMan 48

High volume

Multiple assay platform

Automated

14

Sample preparation MGP Technology Principle COBAS Ampliprep

Principle of Roche PCR

15


TaqMan Analyzer: Roche Patented technology

Use TaqMan probe : dual fluorescent dye-labeled probe attached covalently:

5’-end known as the reporter dye ; and 3’-end known as the quencher dye

16

Assay uses an Internal Control/Quantitation Standards (IC/QS)


AmpErase : recombinant Uracil-N-Glucosylase (UNG), cuts out any carryover amplicon, proven contamination control

Phase matched thermal cycling for mismatch tolerance

Primer/probe modification for mismatch tolerance

Optimized enzyme : thermostable recombinant enzyme Thermus specie

Z05 DNA Polymerase (Z05)

Global surveillance Program and Bioinformatics Tools: Tools for identify

potential target sequences for next generation primers and

probes to further improve genotype coverage of the viral tests.

17

2013 2014

HIV 1999 2095

HBV 529 775

CMV 396 380

HCV 302 240

3226 3490

Molecular diagnostics in UMMC: management

HPV Test Integrated genotyping assay

CT/NG

18

Transcription Mediated Amplification

HIV-1,2

HCV

HBV

HIV-1,2

HCV HBV

IC

Magnetic

microparticle

Amplified

RNA

HIV-1,2 HCV HBV

IC

Dual

light

emission

IC

•Multiple targets can be amplified •Can be qualitative or quantitative •No transfers, no wash steps

Procedure occurs in a single tube

Sample preparation and extraction:

• Serum, plasma including heparinized

• Viral lysis and target capture

• Hybridization of viral nucleic acids and

Internal Control by specific capture probes

• Binding to magnetic particles

• Wash removes unbound substances

Amplification process to enzymatically replicate

billions of copies of RNA

• Reverse transcriptase

• T7 RNA polymerase

Isothermal reaction

Amplicon is primarily RNA

Simultaneous detection of analyte and internal

control following HPA and DKA

19

Nucleic Acid Technologies

20

Virus Growth After Infection (HIV-1)

• Sensitivity of detection differs with test used

Doubling time : 0.85 days

106

103

ID-NAT

MP-NAT

p24Ag

0 5 7 15 19

Viral load

copies/mL

Days

Busch MP et al. Transfusion 2005;45:254-264, Assal A et al. Transfusion 2009;49:289-300, Weusten J et al, Transfusion 2011;51:203-15

Anti-HIV Ab

21


106

103

ID-NAT

MP-NAT

0 3 5 65

Viral load

copies/mL


Anti-HCV Ab

Virus Growth After Infection (HCV) • Sensitivity of detection differs with test used

22

Virus Growth After Infection (HBV)

• Sensitivity of detection differs with test used


106

103

MP-NAT

HBsAg

0 (15) 21 28 36

Days

Viral load

copies/mL

ID-NAT


23

SYNDROMIC APPROACH

Syndromic approach is a new infectious disease testing using a

single reagent with more and more disease incorporated into the

diagnostic algorithm.

More and more syndromic approach is highly associated with the

use of multiplex PCR to detect simultaneously the most important

pathogens involved in a syndrome.

Laboratory Standpoint:

Improve efficiency and decrease testing costs

Expand testing capabilities

Improved services

Improve time to results (no reflex testing)

Reduce technical time (single assay)

Reduce ancillary testing

Reduce send out tests ($$$)

Provide additional revenue

24

TOCE (Tagging oligonucleotide cleavage and extension)

Tagging portion

5’ 3’

Catcher: Artificial template

Extension

5’

5’ 3’

3’

Real-time detection

Duplex Catcher is independent of target amplicon, leading to the consistent Tm.

Pre-determined (or Controlled) Tm allows for multiple target detection in a single channel

61.5 66.5

71.0 76.0 81.0

Temperature (°C)

-d(R

FU

)/dT

140

120

100

80

60

40

20

0

50 60 70 80 90

26

74, 31,168, 18.5% 27

28 25, 168, 14.9%

MagPlex®-TAG™ Microspheres •superparamagnetic microspheres

•No sequence cross-hybridization

•No hybridization to genomic DNA

•24 nucleotides long

•Contains three bases

Luminex xTAG® Technology

30

xTAG® Assay Workflow

Total hands-on time ~ 1 hrs

Total elapsed time ~ 4 hrs

0.5 hr 2.5 hr < 1.0 hr

32

MAGPIX

• Multiplex 50 test in single reaction

• Compact, Portable and Affordable

• Analyze both nucleic acid and proteins

• User-developed assays

LED/Image-Based Analysis - MAGPIX Interrogate label with

green LED (525 nm)

(Quantification)

Interrogate bead with red

LED (635 nm)

(Identification)

Identify and quantify with

CCD imager

Beads in Chamber Magnetic

Capture

33

xTAG® Respiratory Virus Panel Fast v2

Pathogen Targets for xTAG RVP Fast v2

Influenza A 4-plex detection for

Non-specific influenza A

H1 Subtype

H3 Subtype

H1N1 (2009) subtype

Influenza B

Adenovirus

Parainfluenza Virus 4-plex detection for Parainfluenza Virus 1, 2, 3 and 4

Respiratory Synctial Virus

Bocavirus

Metapneumovirus

Coronvirus 4-plex detection for

Coronavirus 229E

Coronavirus NL63

Coronavirus OC43

Coronavirus HKU1

Entero-Rhinovirus

• Detects 18 respiratory viral targets • TAT of 5 hours (including extraction) • Detects the 2009 H1N1 pandemic influenza subtype • Contain Primers for Non-specific Influenza A detection

34

0

50

100

RVP IF VI

Pe

rce

nta

ge

Positive rate of RVP, IF and viral isolation

Number of viral culture positive specimens 15 (7.5%)

Number of direct IF positive specimens 45 (22.6%)

Number of xTAG RVP Fast v2 assay positive specimens 156 (78.4%)

35 199

Respiratory viruses

isolated

by viral culture (n)

Single virus detected by

xTAG RVP Fast v2 assay

(n)a

Multiple viruses detected by

xTAG RVP Fast v2 assay (n)a

Total respiratory

viruses detected by

xTAG RVP Fast v2

assay (%) Consistent (57, 28.6%) RSV (7) RSV (5) RSV/HBoV (1)

RSV/AdV (1)

7 (3.5)

AdV (3) AdV (2) AdV/HRV/HEV (1) 3 (1.5)

Influenza A (2) Influenza A H3 subtype (1) - 2 (1.0)

Influenza A H1N1 subtype (1)

Influenza B (1) Influenza B (1) - 2 (1.0)

PIV-2 (1) - PIV-2/HBoV (1) 1 (0.5)

Negative (43) Negative (43) 43 (21.6)

Inconsistent (142, 71.4%) Influenza B (1) HRV/HEV (1) - 1 (0.5)

Negative (141) HRV/HEV (58)

RSV (27)

HMPV (9)

PIV-1 (3)

HBoV (2)

PIV-3 (2)

PIV-4 (1)

Influenza A H3 subtype (1)

Influenza A H1N1 subtype (1)

Influenza B (1)

HRV/HEV/HBoV (12)

HRV/HEV/AdV (7)

HRV/HEV/RSV (4)

HRV/HEV/HMPV (3)

HRV/HEV/PIV-1 (3)

HRV/HEV/PIV-2 (1)

HRV/HEV/PIV-3 (1)

HBoV/RSV (1)

RSV/PIV-4 (1)

RSV/AdV (1)

RSV/HCoV NL63 subtype (1)

HRV/HEV/RSV/AdV (1)

141 (70.9)

Respiratory viruses isolated by viral isolation or detection by xTAG RVP Fast v2 assay

36

14,199, 7.0%

Respiratory viruses

detected

by direct IF staining

of clinical

specimens(n)

Single virus detected

by xTAG RVP Fast v2

assay (n)

Multiple viruses

detected

by xTAG RVP Fast

v2 assay (n)

Total respiratory

virus detected by

xTAG RVP Fast

v2 assay (%)

Consistent

(88, 44.2%)

RSV (35) RSV (27) RSV/HRV/HEV (3)

RSV/HBoV (2)

RSV/AdV (2)

RSV/PIV 4 (1)

35 (17.6)

AdV (1) AdV (1) - 1 (0.5)

Influenza A (2) Influenza A H3 subtype (1)

Influenza A H1N1 subtype

(1)

- 2 (1.0)

HMPV (5) HMPV (4) HMPV/HRV/HEV (1) 5 (2.5)

PIV-1 (1) PIV-1 (1) - 1 (0.5)

PIV-3 (1) PIV-3 (1) - 1 (0.5)

Negative (43) Negative (43) 43 (21.6)

Inconsistent

(111, 55.8%)

Negative (111) HRV/HEV (59)

RSV (5)

HMPV (5)

PIV-1 (2)

HBoV (2)

Influenza B (2)

PIV-3 (1)

PIV-4 (1)

Influenza A H3 subtype (1)

Influenza A H1N1 subtype

(1)

AdV (1)

HRV/HEV/HBoV (12)

HRV/HEV/AdV (8)

HRV/HEV/RSV (1)

HRV/HEV/HMPV (2)

HRV/HEV/PIV-1 (3)

HRV/HEV/PIV-2 (1)

HRV/HEV/PIV-3 (1)

PIV-2/HBoV (1)

RSV/HCoV NL63

subtype (1)

HRV/HEV/RSV/AdV

(1)

111 (55.8)

Respiratory viruses detected by direct IF or xTAG RVP Fast v2 assay

37 45,199,22.6%

• Viruses

– Adenovirus 40/41

– Rotavirus A

– Norovirus GI/GII

xTAG® GPP Detected Pathogens

• Bacteria and bacterial toxins

– Clostridium difficile toxin A/B

– Salmonella

– Shigella

– Campylobacter

– Escherichia coli O157

– Enterotoxigenic E. coli (ETEC) LT/ST

– Yersinia enterocolitica

– Vibrio cholerae

– Shiga-like Toxin producing E. coli (STEC) stx 1/stx 2

• Parasites

– Giardia lamblia

– Cryptosporidium

– Entamoeba hystolytica

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GPP assay

9. 98 5.1% 39

One system. Fully integrated.

BIOFIRE Diagnostics: FilmArray:

40

• Up to 27

different

targets in one

test

Comprehensive

• 2 minutes of

hands-on time

• 60 minutes run

time

Fast

• No precise

pipetting

required

• Simple sample

preparation

Easy

FilmArray: The Fastest Way to Better Results

41

5. The lid of the sample injection vial is closed and the vial is inverted 3 times to mix the sample

6. The sample/buffer mixture is injected into the pouch through the red inlet port

2:00 1:59 1:58

2. Hydration solution is injected into the pouch through the blue inlet port

3. Sample buffer is added to the sample injection vial 1. The pouch is inserted into the loading block The FilmArray instrument is now ready to set-up

Setting Up the FilmArray Is Easy

2 minutes of

hands-on time

1:57 1:56 1:55 1:54 1:53 1:52 1:51 1:50 1:49 1:48 1:47 1:46 1:45 1:44 1:43 1:42 1:41 1:40 1:39 1:38 1:37 1:36 1:35 1:34 1:33 1:32 1:31 1:30 1:29 1:28 1:27 1:26 1:25 1:24 1:23 1:22 1:21 1:20 1:19 1:18 1:17 1:16 1:15 1:14 1:13 1:12 1:11 1:10 1:09 1:08 1:07 1:06 1:05 1:04 1:03 1:02 1:01 1:00 0:59 0:58 0:57 0:56 0:55 0:54 0:53 0:52 0:51 0:50 0:49 0:48 0:47 0:46 0:45 0:44 0:43 0:42 0:41 0:40 0:39 0:38 0:37 0:36 0:35 0:34 0:33 0:32 0:31 0:30 0:29 0:28 0:27 0:26 0:25 0:24 0:23 0:22 0:21 0:20 0:19 0:18 0:17 0:16 0:15 0:14 0:13 0:12 0:11 0:10 0:09 0:08 0:07 0:06 0:05 0:04 0:03 0:02 0:01 0:00

4. The sample is added to the sample injection vial using the transfer pipette

2:00

42

Sample Extraction, Amplification, and Detection: It’s All in the Pouch

The FilmArray pouch is loaded into the FilmArray instrument 43

8. The FilmArray performs a melt to confirm the presence or absence of assay-specific temperature signatures of the second stage PCR product for each well in the array

2. Nucleic acids bound by magnetic beads move from the lysis chamber to the purification chamber. A wash buffer removes cellular and pathogen debris

3. An elution buffer removes purified nucleic acids from the magnetic beads

4. Nucleic acids move to the first-stage PCR chamber. Reverse transcriptase converts target RNA to DNA, followed by a high-order multiplex PCR

5. Products from the first-stage PCR are diluted to remove any remaining PCR primers

6. First-stage PCR products are added to fresh master mix and are aliquoted into each well of the array

7. Each well is pre-spotted with a single pair of second-stage PCR primers, resulting in specific amplification of target DNA only. A fluorescent double-stranded DNA binding dye monitors each reaction

1. Sample moves into lysis chamber. Cells and pathogens are lysed by bead beating, releasing nucleic acids

65:00

Sample extraction, amplification, and detection: It’s all in the pouch

65 minutes

run-time

64:00 63:00 62:00 61:00 60:00 59:00 58:00 57:00 56:00 55:00 54:00 53:00 52:00 51:00 50:00 49:00 48:00 47:00 46:00 45:00 44:00 43:00 42:00 41:00 40:00 39:00 38:00 37:00 36:00 35:00 34:00 33:00 32:00 31:00 30:00 29:00 28:00 27:00 26:00 25:00 24:00 23:00 22:00 21:00 20:00 19:00 18:00 17:00 16:00 15:00 14:00 13:00 12:00 11:00 10:00 09:00 08:00 07:00 06:00 05:00 04:00 03:00 02:00 01:00 00:00 65:00

44

20 targets

Respiratory

Panel

27 targets

Blood Culture

Identification

Panel

Gastrointestinal

Panel

22 targets

• 3 bacteria

• 17 viruses

• 19 bacteria

• 5 yeast

• 3 antibiotic

resistance genes

• 13 bacteria

• 5 viruses

• 4 parasites

FilmArray : One system, many applications.

Meningitis

Encephalitis

Panel

15 targets

• 6 bacteria

• 8 viruses

• 1 fungus

Sept. 2015

45

Cepheid GeneXpert System

• on-demand - full walkaway automation

• just load a biological sample and the system does the rest

• simultaneously detects TB and rifampicin drug resistance which is a reliable indicator for MDR-TB

• provides accurate results in less than two hours so that patients can be offered proper treatment on the same day

• has minimal bio-safety requirements, training, and can be housed in non-conventional laboratories

RTmPCR

46

I. “There was a lot of concern among patients and some of the panelists that pathologists were not

doing everything we could to support the concept of active surveillance” . “Patients perceived

pathologists as not being engaged in the treatment dilemmas they faced”. NIH State-of-the-Science conference , CAP TODAY April 9, 2015

II. Shifting the paradigm

Conventional microbiological / culture methods

Molecular methods with supports of conventional microbiology laboratory facilities.

Budget.

Integration of molecular laboratory facilities and platforms.

Point of care provision.

Teaching and training of postgraduates .

III. Laboratory development tests.

Fungal genomic initiative

UM Fungal db

IV. Emergence of new technologies .

Third generation PCR

V. Next-generation sequencing-based genome diagnostics/ clinical

genomic

Major focus is cancer diagnosis and therapy

The Future of Molecular Diagnostics in Clinical Practice

Fluidigm Technology

Droplet Digital PCR 47

Thank you

48

molecular methods: the future in clinical practice · molecular methods: the future in clinical...

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