automated methods for bacterial identification

1. GENERAL BACTERIOLOGY:
LABORATORY DIAGNOSIS OF
BACTERIAL INFECTIONS

2. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
LABORATORY DIAGNOSIS OF BACTERIAL
INFECTIONS
Useful for the following purposes:
▰ Identification
▰ Treatment
▰ Surveillance purpose
▰ For outbreak investigation
▰ To start PEP (post-exposure prophylaxis)
▰ To initiate appropriate infection control measures
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3. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
LABORATORY DIAGNOSIS OF BACTERIAL
INFECTIONS (Cont..)
Comprises of several steps
▰ Specimen collection
▰ Direct detection
▰ Culture
▰ Identification and Antimicrobial susceptibility test
▰ Serology
▰ Molecular methods
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4. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
General Principles
Following principles should be followed while collecting the specimen:
▰ Standard precautions
▰ Before antibiotics start
▰ Contamination with indigenous flora should be avoided
▰ Swabs - convenient but considered inferior to tissue, aspirate and body fluids
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5. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
NEED FOR AUTOMATED METHODS
▰ Hospital Acquired Infection a threat to Medical Profession
▰ The Clinicians need Quicker Results
▰ Automation enters into several areas in Microbiology
▰ Automation reduces errors and innovative
▰ Lesser Manpower More volume of work
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6. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
Desired Objectives in Automation
▰ Reexamination of laboratory functional steps
▰ Phlebotomy
▰ Sample labeling
▰ Transportation
▰ Pre-, peri-, and post-analytical processing
▰ Laboratory automation for improved efficiency and error reduction
▰ Create an informatics continuum
▰ Process Control vs. LIS function
▰ Auto-verified and auto-interpreted data
▰ Predictive genomics and the passive home monitoring paradigm
▰ TOT Accuracy, Precision,
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7. CULTURE, IDENTIFICATION
AND AST
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8. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
Automated Blood Culture Techniques
▰ Continuous automated monitoring: Blood culture bottles - periodically
monitored for the microbial growth - every 10 minutes by the instrument.
▰ Composition: Tryptic soy broth and/or brain heart infusion broth + polymeric
resin beads - adsorb and neutralize the antimicrobials present in blood
specimen.
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9. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
Automated Blood Culture Techniques (Cont..)
▰ Specimens: Used for culture of blood, bone marrow and sterile body fluids.
▰ More sensitive: Higher yield of positive cultures from clinical specimens
▰ Rapid: Less time than conventional methods
▰ Less labor intensive: fully-automated
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10. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
Automated Systems
BacT/ALERT 3D:
▰ Principle - colorimetric detection of growth.
▰ When bacteria multiply - produce CO2 -
increases the pH - changes the color of a
blue-green sensor present at the bottom of
the bottle to yellow
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11. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
BacT/AlerT 3D culture system
▰ This is the first automated non-radiometric and non-invasive culture system
that continuously monitors system for culture of bacteria (both aerobic and
anaerobic), fungi and mycobacteria.
▰ All these bacteria can be cultured using different media as prescribed.
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12. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
▰ This is a closed system and works on the colorimetric principle of detection of
CO2 produced by the organisms.
▰ The CO2 causes a lowering of the pH of the medium, which in turn produces a
colour change in a sensor attached to the CO2-sensitive base of each bottle.
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13. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
Automated Systems (Cont..)
BacT/ALERT VIRTUO (bioMerieux): Advanced
form of BacT/ALERT – has several
advantages, such as:
▰ Automatic loading and unloading of
bottles,
▰ Faster detection of growth,
▰ Determine volume of blood present in
bottle
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14. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
The bioMérieux BacT/ALERT® 3D
▰ It provides an optimal environment for the recovery of a wide
range of pathological organisms, including bacteria, yeasts and
mycobacteria;
▰ utilizing proprietary plastic culture bottles ensuring added safety to
the user.
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15. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
Automated Systems (Cont..)
BACTEC (BD Diagnostics)
▰ Principle - fluorometric detection of growth.
▰ Uses an oxygen-sensitive fluorescent dye present in the medium.
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16. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
Principles of functioning of BacT alert Monitors
▰ Microorganisms multiply in the media, generating CO2.
▰ As CO2 increases, the sensor in the bottle turns a lighter colour.
▰ Measuring reflected light, the BacT/ALERT 3D monitors and detects color
changes in the sensor.
▰ Algorithms analyze the data to determine positivity, and the laboratory is
notified immediately with visual and audible alarms
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17. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
Advantages of automation
▰ Automation becomes need of the Hour
▰ Automation improves quality of services
▰ Industry flourishes too
▰ Automation increases efficacy and eliminates individual variations.
▰ elimination of subjective variability
▰ Savings in media and reagents
▰ Automation combined with Laboratory Information management
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18. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
Barcoding
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▰ Bar coding replacing the Manual reading – Reduces errors
▰ The key to this real-time automation was real time barcode labelling of
all sample carriers (such as bags, tubes, dishes, bottles) provided by
Kiestra's Barcode system.
▰ Kiestra's BarcodA automatically places an optical barcode on all tubes,
bottles and petri-dishes that contains important information such as
composition, sell-by date etc

19. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
Quality replacing Quantitity
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▰ Quality issues are becoming increasingly important in diagnostic
laboratories. The fact of quality is no longer sufficient and we must now
develop mechanisms to assure consumers, the public and, most
importantly, ourselves of the continuing quality of our service.
▰ Moving towards a quality-assured system is not easy, requiring a
meticulous attention to detail in all areas of a laboratory's working and
organization

20. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
Automation gained the Universal acceptance
▰ A further plus for lab automation is that it promotes consistency and
quality.
▰ Without automation, lab tasks that are necessarily repetitive can lead to
inconsistent or inappropriate ways of work and, from there, to improper
treatment, longer patient stays, medication errors and unwanted drug
side-effects
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21. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
Disadvantages of automated systems
▰ High cost of the instrument and culture bottles,
▰ Inability to observe the colony morphology as liquid medium is used.
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22. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
Evacuation and Replacement (Cont..)
▰ Automated system (Anoxomat): Automatically
evacuates air - replaces by hydrogen gas from
a cylinder.
▰ Catalyst - sachet containing aluminum pellets
coated with palladium.
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23. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
Absorption of Oxygen by Chemical Methods (Cont..)
GENbag (bioMérieux):
▰ Consists of an airtight transparent bag with a generator sachet - rapidly
produces carbon dioxide and creates anaerobic environment.
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24. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
Automated Systems for Bacterial Identification
(Cont..)
▰ MALDI–TOF (Matrix-assisted laser desorption/ionization time-of-flight)
▰ VITEK 2 (bioMérieux) - automated identification and antimicrobial
susceptibility test.
▰ Phoenix (BD Diagnostics) - automated identification and antimicrobial
susceptibility test
▰ MicroScanWalkAway system (Beckman Coulter) - automated identification
and antimicrobial susceptibility test.
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25. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
MALDI-TOF
▰ Can identify bacteria, fungi, and mycobacteria with a turnaround time of few
minutes and with absolute accuracy.
▰ Two systems are commercially available: VITEK MS (bioMérieux) and Biotyper
system (Bruker).
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26. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
MALDI-TOF (Cont..)
Principle
▰ Examines the pattern of ribosomal proteins present in the organism.
▰ Sample preparation: The colony of an organism - smeared onto a well of the
slide and one drop of matrix solution (composed of cyano-hydroxy-cinnamic
acid) - added to the same well and mixed - slide is loaded in the system.
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27. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
MALDI-TOF (Cont..)
▰ Steps after loading: three steps occurring in three chambers of the system.
 1. Ionization chamber
 2. Analyzer
 3. Detector
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28. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
MALDI-TOF (Cont..)
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29. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
VITEK 2 automated system
▰ Perform both identification and antimicrobial susceptibility testing (AST) of
bacteria and yeast.
▰ Principle of VITEK for identification: Uses colorimetric reagent card containing
64 wells; each well contains an individual test substrate.
▰ Result of identification - usually available within 4–6 hours.
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30. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
VITEK 2 automated system (Cont..)
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31. ANTIMICROBIAL
SUSCEPTIBILITY TEST
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32. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
Automated Antimicrobial Susceptibility Tests
▰ VITEK 2 identification and antimicrobial sensitivity system
▰ Phoenix System (Becton Dickinson)
▰ Micro Scan Walk Away system.
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33. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
VITEK 2 Automated System for AST
▰ Principle - microbroth dilution.
▰ Uses reagent card containing 64 wells - contain doubling dilution of
antimicrobial agents. The
▰ Organism suspension - added to the wells (Figure 3.3.23 and Table 3.3.7)
▰ Cards are incubated in the system at 35.5 ±1°C.
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34. Essentials of Medical Microbiology by Apurba S Sastry © Jaypee Brothers Medical Publishers
VITEK 2 Automated System for AST (Cont..)
▰ Reading - taken once in every 15 minutes.
▰ Results - available within 8-10 hours for
gram negative bacilli and 16-18 hours for
gram-positive cocci.
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