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Syndicat des Jeunes
Biologistes Médicaux
Marseille, 14 décembre 2013

ESI-TOF PLEX ID
UNE NOUVELLE TECHNIQUE DE PRISE
EN C...
DIRECT SEQUENCING : A COST-EFFECTIVE AND
CONVENIENT APPROACH IN MICROBIOLOGY ?

  could identify the isolates, gene resi...
A NOVEL STRATEGY IN MICROBIOLOGY

I
(direct patient
specimens)

NA extraction

II
Multiplex
PCR

III
ESI-TOF MS

(single-p...
From Sept 2001, 11 to June 2009, 29
The assay was able to detect
and characterize the new flu
pandemic stain H1N1 in 2009
...
5
MASS SPECTROMETER ANALYSIS
lower mass amplicon travel faster

Spectral Signal

Mass Spectrometer
(ESI)

(TOF)

Ion Source
...
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

...
During the deconvolution m/z-data are converted
in molecule mass (Kda) From molecule mass the
basecount/- composition is c...
PLEX-ID : IDENTIFICATION

10
PATHOGEN DETECTIONS, MIXTURE, QUANTIFICATION

ie 3 BACTERIA IN THE SAMPLE

Semi quantification
Clincial research study res...
ASSAYS FOR RUO IN 2013 :

- BAC ASSAY : identifies more than 6000 species of
bacteria, 40 species of Candida and 4 antibio...
ASSAYS FOR RUO IN 2012 : BAC AND FUNGAL ASSAYS
Broad Fungal

BAC SPECTRUM

6 samples /plate
Assay Kit Configuration
Amplification Kit:
• 10 Assay plates (10 plates, 60 assays)
– Primers
– Calibrant
– DNA Polymerase...
KIT BAC SPECTRUM > 6000 species.
Larges Primers
Couvrant Bactéries
Primers Couvrant
Protéobactéries
Primers Couvrant
Gamma...
Kit Broad Fungal > 2000 species

16
PLEX ID Viral IC Spectrum
Exemples d’espèces identifiées

12 échantillons (plasma et autres) par plaque
Temps par cycle de...
BAC Spectrum BC Plate
(Exemple)
Assay Reporting Format
UNIVERSITY PARIS DIDEROT
HOPITAL SAINT LOUIS
PCR-ESI TOF MS
(IBIS/ABBOTT PLEX ID)
MICROBIOLOGICAL AND CLINICAL RESULTS
ADENOVIRUS IN HEMATOPOETIC STEM CELLS RECIPIENTS

 - 7 species A  G -

 > 60 serotypes
 Various diseases in IC
 Enter...
ADENOVIRUS INFECTIONS IN HSCT PATIENTS
VIRAL IC IN ESI-TOF (PLEX ID) VS REAL TIME PCR
 Objectives
 PLEX ID Sensitivity a...
SPECIES / SEROTYPE AGREEMENT :
SEQUECING vs ESI-TOF (PLEX ID)
Species/Serotype

n

PlexID

A31

16

A31

A12

4

A12

F41
...
SIMULTANEOUS DETECTION OF OTHER VIRUSES
IN ADV-INFECTED PATIENTS
PCR ESI-TOF MS with VIRAL IC assay is allowing a large an...
WORKFLOW STUDY
IN
MOLECULAR VIROLOGY
July 2012

25
Molecular viral testing for IC patients (St Louis
Hospital)

CMV

EBV

Integrated extraction
amplification system

Adv, Pa...
ORGANISATION DU TRAVAIL
45mn pour 96 pts
1h30

2h

6à8h

27
SET-UP OF THE STUDY
• Tested on ESI-TOF PLEX ID in parallel to the Routine process

Starting point for

Final point for

t...
ROUTINE

ESI MS
PLEXID CMV Whole
blood

1,79

0

1621801

CMV = 13 + / 79

Sample ID

CV CMV Abbott
realTime
whole

124840...
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,...
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

12...
ROUTINE PCR VS PCR – ESI - TOF – MS WORKFLOW
FINAL POINT FOR TIME MEASUREMENT

PLEX ID = 28 Hours WITH 1
TECHNICIANS
VS

R...
BACTERIOLOGY
MYCOLOGY IN IC PATIENTS

PLEX ID & SEPSIS
PLEX ID & CASE REPORTS

33
1-C ALBICANS INFECTION EARLY DETECTION IN PLEX ID

 hemoculture negative the patient being onto
capsofungine
 C albicans...
2-PULMONARY ACTINOMYCOSIS DETECTED
BY PLEX ID

 June 2012 Miss Cham…42 y o
Pulmonary mass without diagnosis
Transthoraxic...
 3 PULMONARY FUSOBACTERIOSIS DETECTION
BY PLEX ID
Pulmonary mass without diagnosis
Transthoraxic biopsies
ESI TOF MS : F ...
IC PATIENT H CAPSULATUM :

positive detection in BAL by ESI TOF (PLEX ID)
19 days before the 1st positive culture

 Liver...
Success stories - cont
 – Testis mass from unknowm origin in HIV –infected
Pt : Plex ID identified a T pallidum onto the ...
PRELIMINARY CONCLUSIONS ONTO PCR-ESI-TOF MS

A highly valuable system that will change the workflow
and the organization o...
PERSPECTIVES 2014
A new system « Next Plex »
- lighter, higher throughput with unitary
cartridge for Bac, Fungal and virus...
Next PLEX 2013-2014
Room 1
nBB

nSP

Assay Strip & Carrier
Private LAN

nAC

nTC

nDS

nMS
NEXTPLEX ASSAY LAUNCH MENU

Pneumonia
• BAC
Pneumonia
• Fungal

BSI/Sepsis
• BAC Blood
Stream
Infections

Severe
Infection...
 Séverine Mercier – Delarue
 Linda Feghoul

VIROLOGY

 Jérôme Le Goff
 Jean Louis Pons

BACTERIOLOGY

 Jean Luc Donay...
Plex ID Congrès SJBM Marseille 2013
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Plex ID Congrès SJBM Marseille 2013

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Publié le

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

Publié dans : Santé & Médecine
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Plex ID Congrès SJBM Marseille 2013

  1. 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. 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. 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. 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. 5
  6. 6. 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
  7. 7. 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
  8. 8. During the deconvolution m/z-data are converted in molecule mass (Kda) From molecule mass the basecount/- composition is calculated 9
  9. 9. PLEX-ID : IDENTIFICATION 10
  10. 10. 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
  11. 11. 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…
  12. 12. ASSAYS FOR RUO IN 2012 : BAC AND FUNGAL ASSAYS Broad Fungal BAC SPECTRUM 6 samples /plate
  13. 13. 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
  14. 14. KIT BAC SPECTRUM > 6000 species. Larges Primers Couvrant Bactéries Primers Couvrant Protéobactéries Primers Couvrant Gamma Protéobactéries Primers Couvrant Fusobactéries Primers Couvrant Staphlocoques
  15. 15. Kit Broad Fungal > 2000 species 16
  16. 16. 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
  17. 17. BAC Spectrum BC Plate (Exemple)
  18. 18. Assay Reporting Format
  19. 19. UNIVERSITY PARIS DIDEROT HOPITAL SAINT LOUIS PCR-ESI TOF MS (IBIS/ABBOTT PLEX ID) MICROBIOLOGICAL AND CLINICAL RESULTS
  20. 20. 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
  21. 21. 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
  22. 22. 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
  23. 23. 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
  24. 24. WORKFLOW STUDY IN MOLECULAR VIROLOGY July 2012 25
  25. 25. 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
  26. 26. ORGANISATION DU TRAVAIL 45mn pour 96 pts 1h30 2h 6à8h 27
  27. 27. 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
  28. 28. 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
  29. 29. 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
  30. 30. 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
  31. 31. 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
  32. 32. BACTERIOLOGY MYCOLOGY IN IC PATIENTS PLEX ID & SEPSIS PLEX ID & CASE REPORTS 33
  33. 33. 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
  34. 34. 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
  35. 35.  3 PULMONARY FUSOBACTERIOSIS DETECTION BY PLEX ID Pulmonary mass without diagnosis Transthoraxic biopsies ESI TOF MS : F nucleatum in accordance with the scanner
  36. 36. 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.
  37. 37. 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
  38. 38. 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
  39. 39. 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
  40. 40. Next PLEX 2013-2014 Room 1 nBB nSP Assay Strip & Carrier Private LAN nAC nTC nDS nMS
  41. 41. NEXTPLEX ASSAY LAUNCH MENU Pneumonia • BAC Pneumonia • Fungal BSI/Sepsis • BAC Blood Stream Infections Severe Infections • BAC Sterile Fluids • Fungal • Viral IC
  42. 42.  Séverine Mercier – Delarue  Linda Feghoul VIROLOGY  Jérôme Le Goff  Jean Louis Pons BACTERIOLOGY  Jean Luc Donay  Stéphane Bretagne  Jean Menotti –Alexandre Alanio MYCOLOGY

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