Emocolture o Biologia Molecolare?

Dr Andrea Rocchetti

• Affermazione 1

• Per emocoltura si intende la coltura di campioni di sangue prelevati in condizioni di asepsi.

• Argomentazione

• Si tratta di un metodo diagnostico fondamentale per la diagnosi microbiologica di batteriemia in quanto consente di confermare il sospetto clinico di sepsi, di accertarne l’agente eziologico e di studiarne la sensibilità in vitro agli antibiotici.

• Bibliografia essenziale

• CLSI – Principles and Procedures for Blood Cultures; Approved Guideline. – CLSI document M47-A. Wayne, PA; Clinical and Laboratory Standards Institute; 2007.

•

Focalizzare velocemente la terapia antibiotica può portare a:

Usare responsabilmente l’antibiotico, evitando il rischio di sviluppo diceppi microbici multiresistenti

Aumentare la sopravvivenza del paziente settico:

MIGLIOR OUTCOME CLINICO

Abbassare i costi ospedalieri, riducendo la spesa associata ad un lungotrattamento antibiotico e alla lunga degenza del paziente

Blood

Draw

Gram

StainPositiveApprox. 1 Hr.

2Hrs.

Standard Testing

Blood

Culture

32 Hrs. 1,40 min. 65,32Hrs.

35 Hrs

ORA DI CAMBIO DELLA TERAPIA

?

Anno 2013

Avvio incubatore emocolture decentrato all’ Ospedale Infantile e Urgenze Laboratorio Analisi o area ritenuta strategica.

Il tempo al gram rappresenta il momento cardine del passaggio dalla terapia antibiotica empirica alla terapia mirata.

Inoltre questo tempo rappresenta il punto nevralgico delle analisi di “tecnology improvement” e di valorizzazione delle risorse organizzative.

COLLOCAZIONE IN

PRONTO SOCCORSO DI

UN MODULO PER

EMOCOLTURA

COLLEGATO IN REMOTO

CON IL LABORATORIO

© 2016 BD. BD and the BD Logo are trademarks of Becton, Dickinson and Company.11

Blood Culture Satellite Solutions

MALDI TOF

< 20-30 minuti

BatteriFunghiMicobatteri

< 20-30 minuti< 20-30 minuti

15 minuti

Verigene

8 batteri G-CTX-MKPCNDMVIMIMPOXA12 atteri G+Mec AVanAVanB

< 5 minuti

< 2,5 – 3 ore

8 batteri G-CTX-MKPCNDMVIMIMPOXA12 batteri G+Mec AVanAVanB

E PLEX

5 < minuti

< 2 ore

33 batteri G-CTX-MKPCNDMVIMIMPOXA24 Batteri G+Mec AMec CVanAVanB

BioFire FilmArray

13batteri G-KPC8 batteri G+Mec AVanA5 Candide spp

< 5 minuti

< 65 minuti

Sample Extraction, Amplification, and Detection: It’s All in the Pouch

The FilmArray pouch is loaded into the FilmArray instrument

8. The FilmArray performs a melt to confirm the presence or absence of assay-specific temperature signatures of the second stage PCR product for each well in the array

2. Nucleic acids bound by magnetic beads move from the lysis chamber to the purification chamber. A wash buffer removes cellular and pathogen debris

3. An elution buffer removes purified nucleic acids from the magnetic beads

4. Nucleic acids move to the first-stage PCR chamber. Reverse transcriptase converts target RNA to DNA, followed by a high-order multiplex PCR

5. Products from the first-stage PCR are diluted to remove any remaining PCR primers

6. First-stage PCR products are added to fresh master mix and are aliquotedinto each well of the array

7. Each well is pre-spotted with a single pair of second-stage PCR primers, resulting in specific amplification of target DNA only. A fluorescent double-stranded DNA binding dye monitors each reaction

1. Sample moves into lysis chamber. Cells and pathogens are lysed by bead beating, releasing nucleic acids

65:00

Sample extraction, amplification, and detection: It’s all in the pouch

65 minutes

run-time

64:0063:0062:0061:0060:0059:0058:0057:0056:0055:0054:0053:0052:0051:0050:0049:0048:0047:0046:0045:0044:0043:0042:0041:0040:0039:0038:0037:0036:0035:0034:0033:0032:0031:0030:0029:0028:0027:0026:0025:0024:0023:0022:0021:0020:0019:0018:0017:0016:0015:0014:0013:0012:0011:0010:0009:0008:0007:0006:0005:0004:0003:0002:0001:0000:0065:00

Automated Results Analysis

102 individual 2nd stage PCR wells

Each well contains one reaction

All targets tested in triplicate

Melt curves generated for each well

ACCELERATE

< 2 minuti

• 90 minuti per una identificazione

• 5 ore per un antibiogramma con MIC

• 7 ore il tempo totale

FISH IDENTIFICATION

FUNGALC. albicans

C. glabrata

AO - Acridine Orange

S. lugdunensis

S. aureusCoNS spp.

E. faecalis

E. faeciumS. pneumoniae

S. agalactiaeStrep spp.

GRAM POSITIVEEnterobacter spp. E. coli

Klebsiella spp. Proteus spp.

Citrobacter spp S. marcescens

P. aeruginosa A. baumannii

GRAM NEGATIVE

Fluorescence In-Situ Hybridization

Universal Bacterial Probes in all ID channels Note: Visual demonstrates proportional usage of flow channels Actual channel usage is alternate/balanced

Automated Sample Preparation Identification& Quantitation

Sample Filtration

(GEF)

Cell Immobilization

(EKC)

Target ID, Universal and

Polymicrobial Detection

Quantitation of

individual cells

Susceptibility Analysisand Reporting

Image analysis via

proprietary algorithmsMIC determination and

SIR interpretation

TECHNOLOGY OVERVIEW

Proprietary Supercomputing

MORPHOKINETIC CELLULAR ANALYSIS

Rapid Direct From Specimen MIC Reporting

Identification results guide selection of antimicrobials for susceptibility testing. Bacteria are grown in the presence of a single concentration of each antimicrobial. Growth response is analyzed using time-lapse imaging of individual bacteria.

Microscopy Images Computed into Many Individual Growth Curves

Founder cells Clonal growth Growth

Resistant

Susceptible

Time

Log(

mas

s)

Founder cells Clonal growth Lysis

TRACK GROWTH/LYSIS OF LIVE INDIVIDUAL BACTERIAL CELLS

Time

Log(

mas

s)

DID T2

< 10 minuti

Candida albicansCandida glabrataCandida tropicalisCandida KruseiCandida parapsilosis

Average Time to Identification

-

Molecular

Direct from patient blood sample

4.3 hours*Dependent on Blood Culture Positivity

49 hrs.†

51 hrs.†

72 hrs.+

129 hrs.+

Time Savings: 2-7 Days

19

Automated ID/AST

MALDI-TOF

Nanosphere

BioFire

T2

Susceptibility

4-48 hours

Species ID

1 hour – 2 days

Blood Culture

1-5 Days

BC+ andGram Stain

~1 hour

n = 617

n = 207

n = 198

n = 212

Pathogens were:

Gram+ 54.8%

Gram- 32.6%

Candida sp. 2%

Polymicrobial 10.5%

Among subjects with PCR testing, 81% of organisms isolated were detectable by FA BCID panel

* For all groups, MALDI-TOF MS or pathogen identification of colonies

isolated from positive BCBs and rapid testing for MRSA colonies were used

Median time from Gram stain result to organism identification was

shorter in both intervention groups (both 1.3 hours) versus the

control group (22 hours) (P < .0001)

INT1

INT2

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