El futuro de la Biología Molecular para el Diagnóstico Clínicopatologiaclinicamexicana.org.mx/minisite/merida/... · 2013-12-17 · Suzane Silbert, Ph.D. Clinical Scientist Esoteric

Suzane Silbert, Ph.D. Clinical Scientist

Esoteric Testing Lab/Pathology Department Tampa General Hospital

Tampa, FL. USA.

El futuro de la Biología Molecular para el Diagnóstico Clínico

PCR:

�  Most common method used at the Molecular Lab

�  Faster turnaround time than culture based methods

�  A lot of advances were noticed since first described

Three Different Steps:

1. Manual Extraction

2. Amplification

3. Detection

•  Whole Procedure: 1 – 3 days •  Quantitative results very difficult/detection not precise

Two Different Steps:

1. Manual Extraction

2. Amplification/ Detection

•  Whole Procedure: 1 – 2 days •  Quantitative results: standard curves

Two Different Steps:

1. Automated Extraction


•  Results: same day •  Quantitative results: standard curves

Isn’t that GREAT?

1. Automated Extraction


•  Results: same day •  Quantitative results: standard curves

It can always get better!!!

1. Extraction

2. Amplification

3. Detection

3 Instruments/ Steps

or or or

Molecular Diagnostic:

Why?

One Instrument, One Step:

Why One Instrument, One Step?

Faster Results Anticipate Treatment

Reduce Length of Hospital Stay

Reduce Overall Costs


Faster Results Anticipate Treatment

Reduce Length of Hospital Stay

Reduce Overall Costs

Save Lives


Save Lives

Especially your Boss Life… Always under Saving Costs Pressure!!!

“Sample in – Results out”

Molecular Biology: 1 instrument, 1 step

GeneXpert:

GX-1

GX-16

GeneXpert Infinity-48

GeneXpert® Module GX-2 GX-4

Cartridge:

All Testing Done Within Cartridge

�  Sample Prep

�  Amplification

�  Detection

Vortex and Dispense Sample into Port S

2

Insert Cartridge and Start Assay

3

Insert Swab into Elution Reagent Vial and

Break at Score

1

Total Hands-On time <1 Minute. Results in ~ 1h.

GeneXpert: Procedure

Women’s Health

GeneXpert Test Portfolio: By the end of 2014

Healthcare Associated Infections

Critical Infectious Disease

CLIA-Waived Testing

Oncology & Genetics

HemosIL FII & FV

Virology

Between Now & 2014 •  Targeting 10 New Tests Commercially Available,

Bringing Total to 25 Tests

MRSA/SA BC

SA Nasal Complete

vanA for VRE

MRSA/SA SSTI

MRSA C. difficile

C. diff/Epi

EV Flu GBS CT/NG SA Flu A/B Panel

HIV Quant

BCR/ABL Monitor

V2

HPV BCR/ABL Monitor

V1 CT/NG

Norovirus

MRSA/SA SSTI

MTB/RIF Breast CA Rx Strat

HCV Quant

HBV Quant

Vaginitis

CE-IVD

CE-IVD CE-IVD CE-IVD CE-IVD

MDRO

CE-IVD

Bladder Cancer Monitor

CE-IVD

CT

Vaginitis Vaginosis

CE-IVD

CE-IVD

EV Flu

MTB/RIF

GeneXpert Test Portfolio: By the end of 2016

Healthcare Associated Infections

Critical Infectious Disease

CLIA-Waived Testing

Oncology & Genetics

Virology

Microbial ID

Candidemia

HIV Quant HCV Quant MRSA/

SA BC

SA Nasal Complete

vanA for VRE

MRSA/SA SSTI

MRSA C. difficile

C. diff/Epi Norovirus

Gastro B Panel

BCR/ABL Monitor

V2 HemosIL FII & FV

BCR/ABL Monitor V1

Breast CA RX Strat

Breast Cancer Metastasis

Risk

SA

CT/NG

Flu A/B Panel

MRSA/SA SSTI EBV Quant

BKV

HBV Quant

HCV Genotype

Group A Strep

Vaginitis Group A Strep

MDRO VAP MRSA/SA

VAP Gram Negative

RSV

CMV Quant

VZV

CE-IVD

CE-IVD CE-IVD

CE-IVD CE-IVD CE-IVD

Vaginitis/Vaginosis

CE-IVD

CE-IVD

46 Tests Available Globally

Bladder Cancer Monitor

CE-IVD

Bladder Sympt

GBS CT/NG

HPV

CE-IVD

CT

Vaginitis Vaginosis

CE-IVD

HSV Typing Lesion

Women’s Health

CE-IVD

FilmArray – BioFire (Idaho Technologies)

Sample in Results out

Hands-on-Time: 2 Minutes

Add Sample to Pouch

Add Buffer to Pouch

FilmArray: Procedure

The FilmArray Pouch/Cartridge:

Reagent Storage

Chemical Circuit Board

Sample Extraction & Purification

1st Stage Multiplex

PCR

2nd Stage PCR

Results = 1h

FilmArray:

1.  Adenovirus

2.  Coronavirus HKU1

3.  Coronavirus NL63

4.  Human Metapneumovirus

5.  Influenza A

6.  Influenza A H1

7.  Influenza A H1 2009

8.  Influenza A H3

9.  Influenza B

10. Parainfluenza 1




14. Respiratory Syncytial Virus

15. Rhinovirus /Enterovirus

Respiratory Virus Panel:

FilmArray:

1.  S. aureus

2.  S. coagulase neg

3.  L. monocytogenes

4.  Enterococcus sp

5.  Streptococcus sp

6.  S. pneumoniae

7.  S. agalactiae

8.  S. pyogenes

Positive Blood Cultures: 9. Enterobacteriaceae

10. E. cloacae

11. E. coli

12. K. pneumoniae

13. S. marcescens

14. Acinetobacter sp

15. A. baumanii

16. H. influenzae

17. H. flu non typeable

18. N. meningitidis

19. P. aeruginosa

20. C. albicans

21. C. glabrata

22. C. krusei

23. C. parapsolosis

24. C. Tropicalis

25. mecA

26. vanA

27. vanB

28. KPC

Focus: “Direct Amplification Disc” - DAD

Sample in Results out 8 samples/disc

Load Sample

Load Reaction Mix

Place disc on instrument; Results available in ≤ 60

minutes

Swab in Transport Media

Transfer Pipette

Required Materials

Reaction Mix

Focus: Procedure

PCR Setup

0.5 hours

PCR Run

1.5 hours

Total Time

4 hours

Extraction Room PCR Setup Amplification Room

“Traditional” Assay

Assay Setup 2 min

PCR

30-60 min Total Time

≈1 hour

Performed in Single Room/Area

“Sample to Answer” Assay

Sample Prep DNA Extraction

2 hours

Tests Format Status

Flu A/B & RSV Direct Amplification Disc FDA cleared, 2012

HSV-1 & 2 Direct Amplification Disc FDA Submission, 2013

MRSA Direct Amplification Disc FDA Submission, 2013

Group A Streptococcus

Direct Amplification Disc

FDA Submission 2013

Group B Streptococcus

Direct Amplification Disc In Development

M. tuberculosis Direct Amplification Disc In Development

HIV Direct Amplification Disc In Development

Tests:

All of these 3 instruments are GREAT:

�  Excellent Sensitivity and Specificity �  Fast TAT �  Reliable �  Not Labor Intensive + Walkway

Number of tests available

Cost

Open System

GeneXpert 10 > 20 Expensive No

Filmarray 2 Panels Expensive (?) No

Focus < 5 Less Expensive No

LIMITATION

BD MAX™:

Only Open System: Sample in Results out

BD MAX™:

Reagents:

Open System

Extraction

Master Mix IVD

PCR: Microfluidic Cartridge

•  24 lanes per cartridge •  Each lane capable of performing an independent PCR reaction •  Supports multiple PCR technologies:

•  TaqMan® •  Scorpions®/Molecular Beacon •  SYBR® Green •  Melt Curve analysis •  Quantitative Molecular Analysis

Overview of BD MAX Operation:

Pipet the Sample

Load reagents and specimens

Place racks and cartridges on

BD MAXTM

Create worklist

Close the door to initiate run

Tampa General Hospital x BD MAX:

October, 2011

May, 2012

March, 2013


1° Validation: Streptococcus β-hemolitico do grupo B – GBS

•  Total= 75 clinical samples tested

•  Vaginal and rectal swabs collected and sent to the lab for GBS PCR testing

•  In the lab, swabs were inoculated in LIM broth and incubated for >18h at 37°C

•  Two tests were performed and the results were compared:

Cepheid SmartGBS® x BD MAXTM GBS



Positive Negative

Positive 48

0

Negative 3 24

BD MAX™ GBS Sm

artG

BS®



•  S. pyogenes •  E. faecalis •  S. pneumoniae •  S. aureus •  S. epidermidis •  E. coli •  K. pneumoniae •  E. cloacae •  S. marcescens •  P. aeruginosa •  C. albicans •  C. tropicalis •  N. gonorrheae •  A. baumannii •  HSV-1 e HSV-2 Total = 16 diferent species tested. All of them negative for GBS

Specificity Test

Dilution BD MAXTM SmartGBS®

1/100,000 Positive Positive

1/1,000,000 Positive Positive

1/10,000,000 Positive Negative

1/100,000,000 Negative Negative

1/1,000,000,000 Negative Negative

Limit of Detection



•  The BD MAX™ GBS assay has excellent sensitivity and specificity

•  The BD MAX™ GBS assay also demonstrated greater analytic

sensitivity based on the LOD and appeared to be more sensitive in detecting GBS positive clinical samples than the Cepheid SmartGBS® assay

•  Finally, the BD MAX™ GBS assay offer a “sample in - results

out” format of test, allowing faster results and a less labor intensive procedure

Conclusions:


2° Validation: Staphylococcus aureus resistant to Methicilina - MRSA

•  Total= 247 clinical samples tested

•  Nare swabs collected and sent to the lab for MRSA PCR testing

•  2 swabs/patient were collected – 1 for each test: GeneXpert MRSA® x BD MAXTM MRSA



Positive Negative

Positive 30 1

Negative 12(1) 204

BD MAX™ MRSA

Gen

eXpe

rt M

RSA®

1) 5 samples tested by an “in-house” PCR had positive results.



•  CoNS •  S. pneumoniae •  A. viridans •  M. luteus •  Group A Streptococcus •  Group B Streptococcus •  H. influenzae •  C. albicans •  E. faecalis •  Influenzae virus •  RSV •  Human metapneumovirus •  Rhinovirus •  Coronavirus •  Enterovirus

Total = 15 different species. All of them negative for MRSA

Specificity Test

Dilution BD MAXTM GeneXpert®






Limit of Detection



•  The BD MAX ™ MRSA assay was demonstrated to have an excellent sensitivity, specificity and LOD for MRSA isolates

•  Out of 247 samples tested, 13 had different results when both assays were compared.

•  One sample was positive by the GeneXpert and negative by the the BD MAXTM and 12 samples were negative by the GeneXpert and positive by the BD MAXTM.

•  Five out of the 12 samples positive just by the BD MAXTM were also tested by an in-house PCR test. All of them were positive for MRSA.

•  The BD MAX™ MRSA assay appeared to be at least as sensitive in detecting MRSA positive samples than the GeneXpert MRSA® assay.

Conclusions:


3° Validation: Clostridium difficile

•  Total= 137 clinical samples testes

•  1 loose/liquid stool container was received for C. difficile PCR testing

•  Sample were divided for 2 tests: GeneXpert C. difficile® x BD MAXTM C. difficile


3° Validation: Clostridium difficile toxin B

Positive Negative

Positive 37 4

Negative 1 95

BD MAX™ C. difficile

Gen

eXpe

rt C

. diff

icile

®



E. coli, E. faecium, E. cloacae

E. faecalis, S. marcescens,

K. pneumoniae, C. albicans,

P. aeruginosa, B. fragilis,

S. aureus, S. epidermidis,

S. sonnei, S. enteritica,

V. parahemolyticus

C. jejuni, Adenovirus 41

Coxsackie A15, Echovirus 27

L. monocytogenes, C. perfringens,

C. sordelii, C. sporogenes

Total = 22 different species. All of them negative for C.difficile

Specificity Test

Dilution BD MAXTM GeneXpert®

1/10 Positive Positive

1/100 Positive Positive



1/100,000 Negative Negative


Limit of Detection



•  Both, Cepheid GeneXpert toxin B PCR and Clostridium difficile toxin B had similar performance in detecting C. difficile toxin B genes in stool samples.

•  There were 4 samples positive with the GeneXpert and negative with the BD MAXTM.

•  These 4 samples had been frozen and thawed and all had cycle numbers on the GeneXpert at 35 or higher.

•  The sample that was positive on the BD MAXTM was a fresh sample and was positive at 37 cycles.

•  The BD MAXTM represents an effective option for detection of C. difficile with good sensitivity and specificity. The system allows for simple sample in – answer out processing of up to 24 samples in about 2 hours.

Conclusions:

Collaboration Study:

BD MAX™

Develop and Validate a Test to Detect the Presence of VRE in Rectal Swab Samples


BD MAX


•  Sent primer sequences to BioGX

•  Have BD MAX™ and clinical samples

for future validation


•  Worked on TGH primers and on dual

GeneTAG probe sequences targeting different

regions within the flanking primers

•  Custom manufacturing of the test using the

BioGX technology


New LDT vanA Assay:

•  TGH LDT primers and probes were custom mixed

and lyophilized with buffer, dNTPs, MgCl2 and

enzyme, in a pellet sufficient for 8 reactions

•  This BioGX Sample-Ready™ (SR) reagents include

all necessary components for amplification on the

BD Max platform

•  The lyophilized pellet is rehydrated with

molecular grade water

New LDT vanA Assay:

•  A Sample Prep Control (SPC)/internal

amplification control (IAC) reagent is also included.

•  LDT primers and probes are included in the

lyophilized PCR reagents for detection of this SPC

DNA sequence as well

•  The SPC cake has to be rehydrated in 900µL of

molecular grade water.

•  It includes a blue dye for visual verification of its

addition

Extraction Tube"PCR Tube"

1"2"3"

Tube position"

Procedure on the BD MAX:

12.5µL of mastermix was dispensed into microtubes that

fit into the BD MAX strip

3"

Extraction Tube

Results Analysis:

Color of Detection = 5; Reading Channels= 6 (1 for HRM)

Color Dye Wavelength – Reading Channels

Green FAM 475/520

Yellow TET, VIC 530/565

Orange Texas Red, ROX 585/630

Red Cy5 630/665

Crimson Cy5.5 680/715

Results – PCR Analysis:

Results – LDT Test:

Examples of vanA target results, displayed in the 530/565 channel (vanA P1)

Examples of vanA target results, displayed in the 585/630 channel (vanA P2)

Results – LDT Test:

Examples of SPC/IAC results, displayed in the 680/715 channel (SPC)


1"2"3"

Tube position"

Validation:

•  182 clinical rectal swab samples were tested

•  Specimens were collected either by ESwabs (Copan) or Traditional

Swabs (LQ Stuart, BD BBL).

•  In the lab, both types of swabs were vortexed in 1mL of liquid Amies

(ESwab medium)

•  After vortexing, 250µL of the liquid Amies was added to the Expert

vanA buffer and 500µL of the solution was added to the BD EXK3

extraction buffer tube, to provide approximately the same volume of

sample processed in extraction on both systems

•  The GeneXpert vanA assay was processed using the procedure

specified in the package insert

•  LDT vanA on the BD MAXTM was processed as described


1"2"3"

Tube position"

Specificity test: •  S. pyogenes

•  vanA negative E. faecalis

•  vanA negative E. faecium

•  P. anaerobius

•  S. aureus

•  S. epidermidis

•  E. coli

•  K. pneumoniae

•  E. cloacae

•  S. marscescens

•  P. aeruginosa

None of the strains tested for specificity was positive using the BD MAX™

vanA LDT

•  P. mirabilis

•  C. albicans

•  B. fragilis

•  A. baumanii

•  L. monocytogenes

•  C. difficile

Results:

GeneXpert LDT Test:

Positive Negative

21 161

Positive Negative

24 158

•  All included samples positive on the GX reported positive on the BD MAX

•  Three samples reported negative on the GX reported positive on the BD

MAX using the LDT


1"2"3"

Costs:

•  TGH: 1,400 VRE PCR tests a month

•  New LDT vanA test on the BD MAX costs $11.39

less than the vanA PCR test used before

•  This represents a saving of:

$15,946.00 /month or $191,352.00 /year


1"2"3"

Tube position"

Conclusions:

•  The new LDT for vanA on the BD MAX™ has excellent sensitivity and specificity

•  It is a user friendly test: “sample in - results out”!

•  Requires minimal hands-on time

•  Provides fast results

•  Represents a significant cost savings

•  It is an excellent alternative method for VRE detection in clinical samples


1"2"3"

Tube position"

Examples LDT in Development:

•  Trichomonas vaginalis

•  Enterovirus + Parechovirus

•  Cystic Fibrosis Panel

•  HBV

•  Atypical pneumonia •  GI Tract

•  And more…

Tampa General Hospital

Tampa General Hospital

TGH: •  1.018 beds •  6.900 employees

•  Afiliated to University of South Florida – USF •  2012: Named by “America’s Best Hospitals” among the best hospitals in 9 specialties

•  Among the leaders of solid transplants in USA

TGH – Esoteric Testing Lab

1.  CMV – quantitative 2.  HSV- detection and typing 3.  HIV Viral Load 4.  HIV qualitative 5.  HCV Viral Load 6.  Chlamydia trachomatis 7.  Neisseria gonorrheae 8.  Mutations Factor V Leiden, Factor II e MTHFR 9.  Molecular Typing of Microrganismos 10.  MRSA 11.  Mycobacterium species, 12.  M. avium/intracellulare 13.  M. tuberculosis 14.  Epstein-Barr Virus Quantitative 15.  B e T-cell rearrangements 16.  BK e JC polyomavirus 17.  Bordetella pertussis 18.  Parvovirus B19 19.  Enterovirus 20.  Influenza A/B 21.  Influenza A Typing 22.  RSV

23.  Human metapneumovirus 24.  Parainfluenza virus 25.  Adenovirus 26.  Rhinovirus 27.  Group B Streptococcus 28.  Mycoplasma pneumoniae 29.  JAK2 mutation 30.  KPC 31.  vanA 32.  C. difficile

33.  Respiratory Painel: FluA/B, RSV, Paraflu 1-4, Coronavirus HKU1 and NL63, Adenovirus, Rhinovirus, Enterovirus, HMPV,

34.  Multiplex for carbapenemases 35.  Multiplex for metalo-betalactamases 36.  Multiplex for ESBL 37.  PCR for qnrs 38.  PCR for ampC 39.  Varicela Zoster 40.  Trichomonas vaginalis

Total mês: ~13.000 exams

Exams Offered/Molecular Diagnostic:

R&D 1.  Suzane Silbert, PhD 2.  Carly Kubasek, MS 3.  Dom Uy, BA 4.  Faris Galambo , BA 5.  Nathan Karakad, BA

Ray Widen, PhD – Director

TGH – Esoteric Testing Lab

1.  Vicki Healer, MT 2.  Alicia Gostnell, MT 3.  Elaine Vendrone, MT 4.  Laura Ferguson, MT 5.  Lisa Wood, MT 6.  Lissa Fenton, MT 7.  Leah Mashburn, MT 8.  Jorge Alves, MT 9.  Josh Kay, MT

Documents

El futuro de la Biología Molecular para el Diagnóstico Clínicopatologiaclinicamexicana.org.mx/minisite/merida/... · 2013-12-17 · Suzane Silbert, Ph.D. Clinical Scientist Esoteric