Présentation de la technologie Plex-ID par le Pr François Simon lors du Congrès 2013 du Syndicat des Jeunes Biologistes Médicaux (SJBM) à Marseille

1. Syndicat des Jeunes
Biologistes Médicaux
Marseille, 14 décembre 2013
ESI-TOF PLEX ID
UNE NOUVELLE TECHNIQUE DE PRISE
EN CHARGE MICROBIOLOGIQUE
CHU
Saint
Louis
Paris
GH Saint Louis – Lariboisière-F Widal
Prof François SIMON

2. 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
2
Nature Reviews Genetics 13, 601-612 (September 2012) Xavier Didelot et al

3. A NOVEL STRATEGY IN MICROBIOLOGY
I
(direct patient
specimens)
NA extraction
II
Multiplex
PCR
III
ESI-TOF MS
(single-plex)
6 - 8 hours
IV
Convert
mass to base
composition

4. 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 identified
at 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

5. 5

7. MASS SPECTROMETER ANALYSIS
lower mass amplicon travel faster
Spectral Signal
Mass Spectrometer
(ESI)
(TOF)
Ion Source
Ion Source
Generation of of Ions
Generation Ions
Mass Analysis
Mass Analysis
Seperation of of Ions
Seperation Ions
Signal Processing
Masses to Base Compositions
#
Mass
Base Count
Quantity
1 35875.03 A25G35C30T26
4260
2 35297.70 A29G33C27T25
1948
3 35619.87 A26G36C29T24
1555
4 36196.21 A23G37C31T26
1306
5 35297.70 A29G33C27T26
1949
6 33734.22 A19G21C17T27
1000
Detector
Detector
Detection of of Ions
Detection Ions
Base Compositions Map
to Microbes
7

8. PCR PRODUCTS IDENTIFICATION
PRINCIPLE OF MASS SPECTROMETRY
different A, C, T, G in
your PCR
8.5 g
Poids: 36,2 g
7.5 g
7.8 g
different coins in your
pocket
.
5.74 g
?
1 x 1 Euro
5 x 20 Cent
8

9. During the deconvolution m/z-data are converted
in molecule mass (Kda) From molecule mass the
basecount/- composition is calculated
9

10. PLEX-ID : IDENTIFICATION
10

11. PATHOGEN DETECTIONS, MIXTURE, QUANTIFICATION
ie 3 BACTERIA IN THE SAMPLE
Semi quantification
Clincial research study results on blood
culture samples obtained at LABCONOWL, Germany
S.
aureus
(A30G29
C30T29)
Amplitu
de
Detection of mixture
3
0
0
0
K.
pneumoniae
1
5
0
0
(A26G32C
28T30)
E. coli
(A27G33
C27T29)
0
3
5.
6
3
6.
molecular
1
mass (kDa)
3
6.
6
11

12. ASSAYS FOR RUO IN 2013 :
- 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…

13. ASSAYS FOR RUO IN 2012 : BAC AND FUNGAL ASSAYS
Broad Fungal
BAC SPECTRUM
6 samples /plate

14. 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
• Kit Component
Part Number
– Beat-beating tubes (zirconium/yttrium)
MG-00361
– Ultra-Clean SDS, 20%
MG-00349
– Microparticles
MT-00416
– Wash 1
MT-00371
– Wash 2
MG-00351
– Elution Buffer
MG-00350
Quantitiy
60
10
10
10
10
10
BAC Spectrum Control Kit: 5 x 12
– Negative Control (2 kits required per sample prep kit)
MG-00354
5
Pro K Kit
– Proteinase K
MT-00600
10
For Training Purposes Only – Not for Publication or Distribution Outside Customer Training

15. KIT BAC SPECTRUM > 6000 species.
Larges Primers
Couvrant Bactéries
Primers Couvrant
Protéobactéries
Primers Couvrant
Gamma Protéobactéries
Primers Couvrant
Fusobactéries
Primers Couvrant
Staphlocoques

16. Kit Broad Fungal > 2000 species
16

17. PLEX ID Viral IC Spectrum
Exemples d’espèces identifiées
12 échantillons (plasma et autres) par plaque
Temps par cycle de PCR: 2 heures
Temps total< 8 h
17

18. BAC Spectrum BC Plate
(Exemple)

19. Assay Reporting Format

20. UNIVERSITY PARIS DIDEROT
HOPITAL SAINT LOUIS
PCR-ESI TOF MS
(IBIS/ABBOTT PLEX ID)
MICROBIOLOGICAL AND CLINICAL RESULTS

21. ADENOVIRUS IN HEMATOPOETIC STEM CELLS RECIPIENTS
 - 7 species A  G -
 > 60 serotypes
 Various diseases in IC
 Enteritis – Cystitis
 Hepatitis – Pneumonia - Encephalitis
 Disseminated disease  Mortality ≈ 50%
 Persistent infection in children  Adv C
 Nosocomial infections

22. ADENOVIRUS INFECTIONS IN HSCT PATIENTS
VIRAL IC IN ESI-TOF (PLEX ID) VS REAL TIME PCR
 Objectives
 PLEX ID Sensitivity and specificity in ADV detection in HSCT
patients on plasma
 Plex ID ADV genotyping versus traditional sequencing
93 positive plasma from
60 ADV-infected patients
22

23. SPECIES / SEROTYPE AGREEMENT :
SEQUECING vs ESI-TOF (PLEX ID)
Species/Serotype
n
PlexID
A31
16
A31
A12
4
A12
F41
5
F41
B (B3, B7, B11, B undet)
13
B
C
101
20
58
17
1
5
C
C2 n=10
C5 n=8
D56
1
D
D undet
5
D
C1
C2
C5
C6
C undet
Undetermined
A31
B
C (n-4)
serotype identification
Species identification
The base composition
matched with several
serotypes

24. 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 +
BKV +
EBV +
CMV +
JCV +
HSV-1 +
n
39
32
31
10
9
24

25. WORKFLOW STUDY
IN
MOLECULAR VIROLOGY
July 2012
25

26. Molecular viral testing for IC patients (St Louis
Hospital)
CMV
EBV
Integrated extraction
amplification system
Adv, Parvo,
HSV1/2, VZV,
BK virus
HHV6
HHV8
Extraction
Extraction
Enterovirus
Integrated extraction
amplification system
Commercial & In house
Real time assay quantification
In house
Real time quantification
(Slide from F Simon)
WORKFLOW STUDY
October 1st, 2012
26
© 2012 Abbott
26

27. ORGANISATION DU TRAVAIL
45mn pour 96 pts
1h30
2h
6à8h
27

28. SET-UP OF THE STUDY
• Tested on ESI-TOF PLEX ID in parallel to the Routine process
Starting point for
Final point for
time measurement
time measurement
Registration of tube in LIS
Result available for biologist
28

29. ROUTINE
ESI MS
PLEXID CMV Whole
blood
1,79
0
1621801
CMV = 13 + / 79
Sample ID
CV CMV Abbott
realTime
whole
1248401
PLEX ID
CMV DETECTION
ONTO WHOLE BLOOD
Viral IC 2
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

30. VIRAL IC : BK VIRUS DETECTION IN PLASMA
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

31. VIRAL IC-2 : HHV-6 &HHV-7 detection in whole blood
ROUTINE LOG HHV6
whole blood
HHV-7 = 13 + / 29
2,41
87
2,45
50
120701988701
10 + /29
120701892001
120701725901
HHV-6 =
VIRAL IC 2
whole blood
BAC SP
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

32. 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

33. BACTERIOLOGY
MYCOLOGY IN IC PATIENTS
PLEX ID & SEPSIS
PLEX ID & CASE REPORTS
33

34. 1-C ALBICANS INFECTION EARLY DETECTION IN PLEX ID
 hemoculture negative the patient being onto
capsofungine
 C albicans DNA detected in PLEX ID
- Liver biopsy culture +
- Clinical and therapeutical
confirmation of nodular candidiasis

35. 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

36.  3 PULMONARY FUSOBACTERIOSIS DETECTION
BY PLEX ID
Pulmonary mass without diagnosis
Transthoraxic biopsies
ESI TOF MS : F nucleatum in accordance with the
scanner

37. 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.

38. 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

39. PRELIMINARY CONCLUSIONS ONTO PCR-ESI-TOF MS
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

40. 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
40

41. Next PLEX 2013-2014
Room 1
nBB
nSP
Assay Strip & Carrier
Private LAN
nAC
nTC
nDS
nMS

42. NEXTPLEX ASSAY LAUNCH MENU
Pneumonia
• BAC
Pneumonia
• Fungal
BSI/Sepsis
• BAC Blood
Stream
Infections
Severe
Infections
• BAC Sterile
Fluids
• Fungal
• Viral IC

43.  Séverine Mercier – Delarue
 Linda Feghoul
VIROLOGY
 Jérôme Le Goff
 Jean Louis Pons
BACTERIOLOGY
 Jean Luc Donay
 Stéphane Bretagne
 Jean Menotti –Alexandre Alanio
MYCOLOGY