Plex ID Congrès SJBM Marseille 2013

CHU Saint Louis Paris

ESI-TOF PLEX ID

UNE NOUVELLE TECHNIQUE DE PRISE EN CHARGE MICROBIOLOGIQUE

GH Saint Louis – Lariboisière-F Widal

Prof François SIMON

Syndicat des Jeunes Biologistes Médicaux

Marseille, 14 décembre 2013

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DIRECT SEQUENCING : A COST-EFFECTIVE AND CONVENIENT APPROACH IN MICROBIOLOGY ?

could identify the isolates, gene resistance to antibiotics of bacterial/fongi pathogens

next-generation sequencing will soon be sufficiently fast, accurate and cheap to be used in routine clinical microbiology practice

Nature Reviews Genetics 13, 601-612 (September 2012) Xavier Didelot et al

A NOVEL STRATEGY IN MICROBIOLOGY

I

NA extraction

(direct patient specimens) Multiplex

PCR (single-plex)

ESI-TOF MS

II III IVConvert mass to base composition

6 - 8 hours

From Sept 2001, 11 to June 2009, 29

The assay was able to detect and characterize the new flu

pandemic stain H1N1 in 2009

…An untypable influenza A strain was identifiedat a surveillance site of the Naval Health Research Center by a new diagnostic device.

…Further characterization by PCR assay and electrospray ionization mass spectrometry indicated a swine-origin virus,

– D Faix, et al. New England Journal of Medicine, June 29, 2009

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MASS SPECTROMETER ANALYSIS

Mass Spectrometer(ESI) (TOF)(ESI) (TOF)

Ion Source Mass Analysis Detector

Generation of Ions Seperation of Ions Detection of Ions

Ion Source Mass Analysis Detector

Generation of Ions Seperation of Ions Detection of Ions

Ion Source Mass Analysis Detector

Generation of Ions Seperation of Ions Detection of Ions

Ion Source Mass Analysis Detector

Generation of Ions Seperation of Ions Detection of Ions

Spectral Signal

Signal ProcessingMasses to Base Compositions

# Mass Base Count Quantity1 35875.03 A25G35C30T26 42602 35297.70 A29G33C27T25 19483 35619.87 A26G36C29T24 15554 36196.21 A23G37C31T26 13065 35297.70 A29G33C27T26 19496 33734.22 A19G21C17T27 1000

Base Compositions Mapto Microbes

lower mass amplicon travel faster

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800 900 1000 1100 1200 1300m/z

800 900 1000 1100 1200 1300m/z

MW = 32,588.90 amuA28 G29 C25 T24

Penny = 2.500 gNickel = 3.950 gDime = 2.268 gQuarter = 5.670 g

A = 313.0576 amuG = 329.0526 amuC = 289.0464 amuT = 304.0461 amu

Weight = 4.6 grams 2 dimes

??

.. ... .

Mass spectrum

Scale

.. ... .

.

800 900 1000 1100 1200 1300m/z

800 900 1000 1100 1200 1300m/z

MW = 32,588.90 amuA28 G29 C25 T24

Penny = 2.500 gNickel = 3.950 gDime = 2.268 gQuarter = 5.670 g

A = 313.0576 amuG = 329.0526 amuC = 289.0464 amuT = 304.0461 amu

Weight = 4.6 grams 2 dimes

??

.. ... .

Mass spectrum

Scale

.. ... .

7.5 g

7.8 g

5.74 g

8.5 g

7.5 g

7.8 g

5.74 g

8.5 g Poids: 36,2 g

1 x 1 Euro5 x 20 Cent

?

different coins in your pocket

different A, C, T, G in your PCR

PCR PRODUCTS IDENTIFICATIONPRINCIPLE OF MASS SPECTROMETRY

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During the deconvolution m/z-data are converted in molecule mass (Kda) From molecule mass the

basecount/- composition is calculated

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PLEX-ID : IDENTIFICATION

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PATHOGEN DETECTIONS, MIXTURE, QUANTIFICATION

35.6

36.1

36.6

3000

1500

0

molecular mass (kDa)

S. aureus(A30G29C30T29)

K. pneumoniae(A26G32C

28T30)

E. coli(A27G33C27T29)

Am

pli

tud

eClincial research study results on blood culture samples obtained at LABCON-OWL, Germany

Detection of mixture ie 3 BACTERIA IN THE SAMPLE

Semi quantification

- BAC ASSAY : identifies more than 6000 species of bacteria, 40 species of Candida and 4 antibiotic resistance markers (kpc, mec A, van A, van B) direct for sample

- Broad Fungal ASSAY : identifies more than 350 families and over 2,000 species of fungi including Aspergillus, Fusarium, Penicillium, Clasvipora and Cryptococcus direct from sample

- VIRAL IC II ASSAY for IC patients : Herpes 1-8, ADV, polyomaV, HEV ParvoV…

ASSAYS FOR RUO IN 2013 :

ASSAYS FOR RUO IN 2012 : BAC AND FUNGAL ASSAYS

BAC SPECTRUM

6 samples /plate

Broad Fungal

Assay Kit Configuration

Amplification Kit: • 10 Assay plates (10 plates, 60 assays)

– Primers– Calibrant– DNA Polymerase

• 10 Extraction Control vials• 10 % BSA, vials• Protocol for processing PLEX-ID BAC Spectrum SF Assay Plates (1x)

Ultra-Clean Sample Preparation Kit (Promega): 10 x 6

BAC Spectrum Control Kit: 5 x 12

Pro K Kit

• Kit Component Part Number Quantitiy– Beat-beating tubes (zirconium/yttrium) MG-00361 60– Ultra-Clean SDS, 20% MG-00349 10– Microparticles MT-00416 10– Wash 1 MT-00371 10– Wash 2 MG-00351 10– Elution Buffer MG-00350 10

– Negative Control (2 kits required per sample prep kit) MG-00354 5

– Proteinase K MT-00600 10

For Training Purposes Only – Not for Publication or Distribution Outside Customer Training

Larges Primers Couvrant Bactéries

Primers Couvrant Protéobactéries

Primers Couvrant Gamma Protéobactéries

Primers Couvrant Fusobactéries

Primers Couvrant Staphlocoques

KIT BAC SPECTRUM > 6000 species.

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Kit Broad Fungal > 2000 species

1717

PLEX ID Viral IC SpectrumExemples d’espèces identifiées

Temps par cycle de PCR: 2 heures

Temps total< 8 h

12 échantillons (plasma et autres) par plaque

BAC Spectrum BC Plate (Exemple)

Assay Reporting Format

UNIVERSITY PARIS DIDEROTHOPITAL SAINT LOUIS

PCR-ESI TOF MS (IBIS/ABBOTT PLEX ID)

MICROBIOLOGICAL AND CLINICAL RESULTS

- 7 species A G -

> 60 serotypes

Various diseases in IC Enteritis – CystitisHepatitis – Pneumonia - EncephalitisDisseminated disease Mortality ≈ 50%

Persistent infection in children Adv C

Nosocomial infections

ADENOVIRUS IN HEMATOPOETIC STEM CELLS RECIPIENTS

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ADENOVIRUS INFECTIONS IN HSCT PATIENTSVIRAL IC IN ESI-TOF (PLEX ID) VS REAL TIME PCR

ObjectivesPLEX ID Sensitivity and specificity in ADV detection

in HSCT patients on plasmaPlex ID ADV genotyping versus traditional

sequencing

93 positive plasma from

60 ADV-infected patients

Species/Serotype n PlexID

A31 16 A31

A12 4 A12

F41 5 F41

B (B3, B7, B11, B undet) 13 B

C C1 C2 C5 C6 C undet

10120581715

C

C2 n=10C5 n=8

D56 1 D

D undet 5 D

Undetermined A31 B

C (n-4)

Species identificationThe base composition matched with several serotypes

serotype identification

SPECIES / SEROTYPE AGREEMENT :SEQUECING vs ESI-TOF (PLEX ID)

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nBKV + 39EBV + 32CMV + 31JCV + 10HSV-1 + 9

SIMULTANEOUS DETECTION OF OTHER VIRUSES IN ADV-INFECTED PATIENTS

PCR ESI-TOF MS with VIRAL IC assay is allowing a large and combined detection of others viruses in the sample during the same run

93 plasma ADV +

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WORKFLOW STUDY IN

MOLECULAR VIROLOGY July 2012

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Molecular viral testing for IC patients (St Louis Hospital)

WORKFLOW STUDYOctober 1st, 2012

26© 2012 Abbott

CMV EBVHHV6HHV8

Integrated extraction amplification system

Extraction

In house Real time

quantification

Extraction

Adv, Parvo, HSV1/2, VZV,

BK virusEnterovirus

Commercial & In house Real time assay quantification

Integrated extraction amplification system

(Slide from F Simon)

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1h30 2 h

45mn pour 96 pts

6 à 8 h

ORGANISATION DU TRAVAIL

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SET-UP OF THE STUDY

Starting point for

time measurement

Registration of tube in LIS Result available for biologist

Final point for

time measurement

• Tested on ESI-TOF PLEX ID in parallel to the Routine process

PLEX ID CMV DETECTION ONTO WHOLE BLOOD

ROUTINE Viral IC 2

Sample IDCV CMV Abbott

realTime whole

ESI MS PLEXID CMV Whole

blood

1248401 1,79 0

1621801 2,75 0

1627101 3,7 0

1248401 3,79 46

1719201 3,89 36

7007549 3,96 176

7034767 4,13 503

1613001 4,17 621

1680701 4,25 63

7059956 4,52 601

7037327 4,71 594

7027809 4,73 494

7045729 4,75 575

CMV = 13 + / 79

PLEXID LEVEL BK

plasma

ROUTINE LOG BK plasma

31 2,5

28 2,81

50 2,83

28 2,83

41 2,88

123 3,92

381 4,35

581 4,44

1005 4,74

1793 5,07

BK viruses = 10 + /79

VIRAL IC : BK VIRUS DETECTION IN PLASMA

VIRAL IC-2 : HHV-6 &HHV-7 detection in whole blood ROUTINE LOG HHV6

whole blood

VIRAL IC 2 whole blood

BAC SP

120701892001 2,41 87

120701725901 2,45 50

120701988701 2,76 0

120701721301 2,87 33

120701853801 2,93 15

120701689601 2,99 121

120701716501 3,01 93

120702225801 3,45 224

120701706201 4,13 258

120701701701 4,44 202

HHV-7 = 13 + / 29

HHV-6 = 10 + /29

ROUTINE PCR VS PCR – ESI - TOF – MS WORKFLOW

FINAL POINT FOR TIME MEASUREMENT

PLEX ID = 28 Hours WITH 1 TECHNICIANS

VS ROUTINE = 48H WITH 3

TECHNICIANS

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PLEX ID & SEPSIS

PLEX ID & CASE REPORTS

BACTERIOLOGYMYCOLOGY IN IC PATIENTS

hemoculture negative the patient being onto capsofungine

C albicans DNA detected in PLEX ID

- Liver biopsy culture + - Clinical and therapeutical confirmation of nodular candidiasis

1-C ALBICANS INFECTION EARLY DETECTION IN PLEX ID

2-PULMONARY ACTINOMYCOSIS DETECTED BY PLEX ID

June 2012 Miss Cham…42 y o Pulmonary mass without diagnosis Transthoraxic biopsy ESI TOF MS identified Actinomyces Naeslundi

3 PULMONARY FUSOBACTERIOSIS DETECTION BY PLEX ID

Pulmonary mass without diagnosis

Transthoraxic biopsies

ESI TOF MS : F nucleatum in accordance with the scanner

IC PATIENT H CAPSULATUM :

positive detection in BAL by ESI TOF (PLEX ID)

19 days before the 1st positive culture

Liver graft 3 years ago , general skin erythrosis following a bath in a swimming pool. Orphan diagnosis. ESI TOF onto skin biopsy : F solani

- Negative samples (65% for BAL) can be considered in < 1-2 days in stead of 3 weeks.

Success stories - cont

– Testis mass from unknowm origin in HIV –infected Pt : Plex ID identified a T pallidum onto the biopsy

- M hominis in a renal liquid-transport before transplantation

– Lariboisière ED : N meningitidis post AB, typed as serotype C by Plex ID

- Positive blood culture at M bovis following bladder instillation PLEX ID : a high predictive negative value

A highly valuable system that will change the workflow and the organization of the microbiology : large panel adapted to medical monitoring

With potential major clinical implications

Ongoing research 2013-2015 :

2013 BAL in Saint Louis 2014 St Louis/Val de Grâce + Londres +

Frankfurt +Brussel +Varsovia + Genève : ICU

PRELIMINARY CONCLUSIONS ONTO PCR-ESI-TOF MS

PERSPECTIVES 2014

A new system « Next Plex » - lighter, higher throughput with unitary cartridge for Bac, Fungal and viruses - mass spectrometer smaller and

easier to use- - more conducive to a clinical

diagnostics setting.- Cheaper - Adapted to bacterio/mycological rapide identifications

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Next PLEX 2013-2014 nBB

nSP

Assay Strip & Carrier

nTC nDS

Private LAN

nAC

nMS

Room 1

NEXTPLEX ASSAY LAUNCH MENU

Pneumonia BSI/SepsisSevere

Infections

• BAC Pneumonia

• Fungal

• BAC Blood Stream Infections

• BAC Sterile Fluids

• Fungal

• Viral IC

Séverine Mercier – Delarue

Linda Feghoul

Jérôme Le Goff

Jean Louis Pons

Jean Luc Donay

Stéphane Bretagne

Jean Menotti –Alexandre Alanio

VIROLOGY

BACTERIOLOGY

MYCOLOGY