Plex ID Congrès SJBM Marseille 2013
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Transcript of 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 +
25
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