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2.2 Microbiology > Celine - Automation > Flashcards

Celine - Automation Flashcards

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1
Q

Why is micro slow to change over to automation?
(5)

A

Micro too complex to automate -> there is a large variety of specimens, specimen collection systems, specimen processing and culture requirements

Automation can’t replace subjective decision, making skills and interpretative expertise required

High cost - complexity of specimens poor return on instrument investment

Scale of economy lost in micro - large scale tracking automation systems under-utilised

Microbiologists don’t like change

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2
Q

What are the four main drivers for automation in micro

A

Increased demands on service increase by 10% per year

More challenges - mutli-resistant organisms, new pathogens

Reduced funding - reduced budget and staff number

Accreditation - need for standardisation and traceability

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3
Q

Explain why automation is being introduced to the micro lab
(3)

A

Lab under immense pressure to produce faster, better, standardised results in face of increasing work volume

Microbiologist recognise that automation need not replace cognitive decision-making but rather replace tedious, repetitive steps

Automation offers the opportunity to reduce TAT, optimise workflow and reduce costs

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4
Q

What happens on Day 1
(4)

A

Specimen in
- Microscopy
- Processing
- Culture
- Isolate

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5
Q

What happens on day 2?

A

Identify
Susceptibility

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6
Q

Comment on automation for day 1 specimen processing
(2)

A

Automated systems for specimen preparation and culture are complex

Front end processing only recently developed and only evident in large diagnostic micro labs

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7
Q

Comment on automation for day 2, pathogen

A

Austomated systems for ID and AST first developed widespread use in all diagnostic micro labs

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8
Q

Where is the most manual work done in micro

A

Plate and broth medium inoculation
Medium and plate reading

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9
Q

Write about the role of automation in Day 1: Microscopic investigation of clinical specimen
(6)

A

Microscopic investigation consists of direct gram stain and/or white cell count (CSF and Urine)

Direct gram stain can be automated but microscopy is still manual

Microscopic cell count increasingly automated

Urine is the most common sample in the lab

Automation of urine microscopy using fluorescence flow cytometry

Negative screening method for urine -> 60-80% negative, if WCC count normal no need for culture

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10
Q

What machine can be used for urine microscopy?

A

Sysmex UF-5000 - urine microscopy

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11
Q

How does the Sysmex UF-5000 work?
(3)

A

Urine flow cytometer

Enumerates WBC, RBC, bacteria and epithelial cells by cell size and staining with fluorescent dyes

105 samples/hour

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12
Q

Comment on the role of automation in Day 1 processing and culture
(6)

A

Remains largely manual

Front end processing evolving at a rapid pace

Liquid samples readily compatible with automation but swabs required manual handling

Liquid-based ESwab facilitated change

The Eswab sits in liquid media - vortex so flocked head releases organism into liquid media -> this allows for automated smear preparation for gram stain and for plate inoculation

New generation of liquid-based specimen processors now in large diagnostic labs with high specimen throughput

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13
Q

Explain the role of Eswabs in automation

A

The Eswab sits in liquid media - vortex so flocked head releases organism into liquid media -> this allows for automated smear preparation for gram stain and for plate inoculation

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14
Q

Give an example of a specimen processor used in the labs, capable of plate culture

A

WASP Processor (Copan Diagnostics)

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15
Q

What is the WASP processor and how does it work
(8)

A

2 robot arms which move specimens and plates

Can work with all specimen types

Has 9 media silos and a capacity of 370 plates

Uses reusable chrome loops

Can inoculate 180 plates per hour

Can gram stain prep

Capable of auto decapping and recapping

Only one installed in Ireland

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16
Q

Write about the role of automation in blood culture processing
(4)

A

Automation is important for the early detection of BSIs -> rapid diagnosis, prognosis and appropriate therapy

Automation for Blood cultures is well established

BacT/Alert SD is the most common BC system available

BacT/Alert SD allows for the continuous monitoring for detection of bacteria, fungi and mycobacteria

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17
Q

What is a BC bottle
(3)

A

A blood culture bottle is used to investigate BSI

Blood is injected into bottles with culture media at bedside

The BC is then sent to the lab

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18
Q

What is the most common BC automation system

A

BacT/Alert 3D

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19
Q

What is the principle behind the BacT detecion system
(5)

A

If organisms are present in blood they will multiply in nutritious liquid media in BC bottle to generate CO2

CO2 production lowers the pH of the medium and produces a colour change in a sensor at the base of each bottle

Resins in the media neutralise antimicrobials in patient blood enhancing early recovery of pathogens

The bottles are constantly agitated and read every 10 minutes

When the bottle flags positive the bottle is removed from the BacTAlert -> withdraw a sample and perform a gram stain and culture on appropriate culture plates

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20
Q

What is present in BC bottles that neutralise antimicrobials?

A

Resins

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21
Q

What two aspects of confirming identification on Day 2 is compatible with automation

A

Detection of biochemical properties via Vitek/Manual

Determine protein profile via MALDI

22
Q

In general what is the Vitek 2 capable of doing

A

Identifying biochemical properties

Antimicrobial Susceptibility Testing

23
Q

In general what is the MALDI TOFF capable of doing

A

Id only based on protein profile

24
Q

In general what is the pheonix capable of?

A

Biochemical properties and AST

25
Q

Why is automation so important in pathogen identification
(2)

A

These systems (Vitek and MALDI) are reproducible, reduce hands on time and quicker turn around time

Systems are also interfaced with hospital computer systems - automate result entry

26
Q

What is the Vitek comprised of and how does it work?

A

Filler-sealer module -> draw bacterial suspension in 64 microwell card

Incubator and photometer to detect optical density for bacterial growth and biochemical reaction colour

27
Q

What are the five ID cards for the Vitek 2?
(5)

A

Gram negative
Gram positive
YST(Yeast)
Neisseria/Haemophilus
ANC (anaerobe)

28
Q

Explain in your own words how the vitek is used
(6)

A

Select the correct ID card

The Vitek will draw the bacterial suspension into the 64 microwells of the ID cardd

These wells contain test substrates which are used to measure a variety of metabolic activities, enzyme hydrolysis and growth in presence of inhibitory substrate

The growth/colour change is determined every 15 minutes

The series of 64 biochemical reactions is then compared to a database of known organisms

Gives results in 8 to 16 hours

29
Q

What is measured by the Vitek?
(2)

A

Metabolic activities

Enzyme hydrolysis and growth in presence of inhibitory substrate

30
Q

What does MALDI TOF stand for

A

Matrix-Assisted Laser Desorption/Ionisation - Time of Flight

31
Q

What is the MALDI-TOF MS used for

A

MALDI-TOF mass spectrometry used for species ID

32
Q

What are some of the benefits of MALDI?
(6)

A

Widely used for ID of bacteria and fungi

Its reproducible and sensitive - powerful tool

Analysis is very short -> TAT only takes 6 minutes/bacteria

Simple sample preparation and result acquisition

Cost is 5 times cheaper than conventional ID

MALDI analyses the protein composition of the organism -> there is no need to wait for growth -> can test directly on colony

33
Q

How is the MALDI used/How does it work?
(8)

A

Prepared single colonies from Day 2 plate used (directly or inactivated)

Spot colony on biotarget teflon plate

Add matrix (crystallized molecules), air dry, place on MALDI

Sample ionised with laser burst, releasing ‘cloud’ of proteins

Proteins accelerated with an electric charge through a flight tube to mass spectrometer

Time of flight recorder, lighter proteins travel faster than slower heavier ones

Protein profile determined by mass/charge ratio

Protein profile of test organism compared with profile database of well-characterized strains

34
Q

How do you prepare your colonies for MALDI

A

Single colonies from Day 2 plate used directly or inactivated with alcohol/formic acid which allows for cell wall disruption and protein extraction

35
Q

Where do you add your colony for the MALDI?

A

On biotarget teflon plate

36
Q

What do you add to your colony on the teflon plate for MALDI

A

Add matrix (crystallized molecules), air dry then place on MALDI

37
Q

What is the point of the matrix for MALDI

A

Acts as a buffer between bacteria and laser and helps ionise the sample, carrying it along the flight tube

38
Q

What is the most common MALDI-TOF instrument

A

Bruker Biotyper system

39
Q

Write about the Burker Biotyper system, how accurate is it?

A

98% ID at genus level
87% ID at species level
Misidentification most common in S. pneumonia, coagulase negative staph, and shigella Vs E. Coli
The database will expand to resolve many of the current identification problems

40
Q

Comment on the role of automation in AST
(5)

A

Many automated ID systems are adapted to accommodate automated susceptibility testing

Most common automated AST system is the Vitek 2

The AST cards contain 64 micro wells with a range of 8 to 9 antibiotics suitable for a wide range of organisms

Each antibiotic present at concentrations that convers the threshold/breakpoint for susceptible, intermediate and resistant

Employ short incubate with results in 8 to 16 hours

41
Q

AST can be measured using the VITEK for which organisms
(4)

A

Staphylococci
Streptococci
Enterobacterales
Pseudomonas

42
Q

Comment on future total lab automation
(2)

A

There are 2 Total lab automation microbiology systems in development

Th most common TLA system in Micro is the Kiestra

43
Q

What are some common features of the TLA systems
(4)

A

Robotic plate management to automate specimen processing and workup

Conveyor/track systems to move plates to and from incubators

Automated incubators with digital reading stations

Digital cameras capture plate images at timed intervals

44
Q

Is TLA a reality?
(7)

A

Total processing time takes 26 hours and AST appears feasible

They have been said to improve turn around time which could have a positive impact on clinical decisions and patient outcome

But there are few peer-reviewed publications

Benefits are inferred not proven

Need large studies to accurately assess the financial operation and clinical impacts of TLA

There have been 68 installations to date

One study found 2 fold in lab productivity despite 27% increase in workload but there was a decrease in 2.4 staff members

45
Q

What is molecular diagnostics considered the gold standard for?
(5)

A

Non-culturable organisms - viruses, chylamdiae
Fastidious organisms - N. gonorrhoeae
Slow growing orgnaisms - Mycobacteria
Important HCAI pathogens - MRSA, VRE
Multiple potential pathogens implicated

46
Q

Give an example of a piece of automation used for molecular diagnostics

A

COBAS 4800 Ct/Ng assay

47
Q

What is the COBAS 4800 Ct/Ng assay
(5)

A

Multi-plex Realtime PCR-C trachomatis and N. gonorrhoeae

System combines fully automated DNA extraction followed by PCR preparation + Real time PCR detection

97% sensitivity

Specimens include urine samples and genital samples

Processes 94 specimens in 5 hours with only 40 minutes hands-on-time

48
Q

Give the most commonly used automation in molecular diagnostics
(4)

A

GeneXpert Real Time PCR

DNA extraction, amplification and detection in a single cartridge

60 minutes turn around time

Swab samples transferred to cartridges and then inserted into machine

49
Q

Write about the Real-Time Enteric Bio Assay
(6)

A

EntericBio Gastro Panel 1

Real time PCR kit for simultaneous detection Campylobacter spp, Salmonella enterica, Shigella spp and Vero Cytotoxigenic E. Coli (VTEC)

Panel 2 plus Cryptosopidium + Giardia

Multiplex detection of pathogens directly from faeces without pre-enrichment or DNA extraction

PCR performed on Roche 480 Light Cycler Platform

3 hour turn around time

50
Q

Comment on automation in serology
(5)

A

Manual methods utilise ELISA-based micro-titre plate which is very labour intensive

Fully automated systems are widely used for Serology

Main application diagnosis of infection caused by non-culturable/slow growing/fastidious pathogens

Main application in virology for HBV, HCV, HIV, CMV

Main application in syphilis serology - STI agent

51
Q

Give three applications of automation in serology

A

Main application diagnosis of infection caused by non-culturable/slow growing/fastidious pathogens

Main application in virology for HBV, HCV, HIV, CMV

Main application in syphilis serology - STI agent

52
Q

Give an example of an analyser used in the automation of serology
(6)

A

Immunoassay analyser

Assays in Clinical Chemistry and Microbiology

Primary sampling - no sample separation required

Random access - multiple tests performed at the same time

65 sample load

Throughput 50-100 tests/hr

2.2 Microbiology (60 decks)

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