Automations in

antimicrobial
susceptibility

Dr.Sridevi D
Senior Resident,
PIMS.
VITEK 2 ID/AST SYSTEM

• Most widely used automated system in India

Available in 2 formats:
• VITEK 2 system-fully automated system
• VITEK 2 Compact system-near automated system
VITEK 2 system

• Fully automated
• Consists of one processing
chamber
2 formats
• VITEK2 60→accommodate
60 cards
• VITEK2 XL
→accommodate 120 cards
VITEK2 COMPACT SYSTEMS

• Near automated
• Two processing chambers
2 formats:
• 30 cards
• 60 cards
VITEK2 for Identification
Cards for Identification
 Polystyrene cards with microwells
 Each well has a mini-biochemical reaction chamber containing
lyophilised reagents
 Phenotypic tests based on carbon source utilization, enzymatic
activities etc
Available Identification cards

Neisseria- Anaerobic and

Gram-negative Gram-positive
Haemophilus Corynebacteria
ID (GN) card ID (GP) card
ID (NH) card ID (ANC) card

BCL card (for CBC card (For

Yeast ID (YST)
Bacillus Corynebacteriu
card
species) m species)
Procedure

Matched to desired
turbidity
Pure isolated colonies 3 mL of 0.45% sterile Colony suspended in 3 mL of
18-24 hours old 0.45% sterile saline
saline

Loaded into the instrument

 Principle
• Advanced colorimetry technology
• Utilizes ‘Analytical profile index
(API)’
• Has a database on various
phenotypic tests from known
strains-determines unique reaction
pattern
• Software compares phenotypic test
result of test isolate =Expected
unique reaction pattern
GN card Identification
 Results
Confidence level % Probability

Excellent 96-99

Very good 93-95

Good 89 - 92
Can be reported after
matching with colony
characteristics
Acceptable 85 - 88

Low discrimination 2 to 3 organism with Carefully evaluated + matched with colony

split ID
characteristics+ perform biochemical tests
Inconclusive >3 organism
Or 0

Unidentified Organism Repeat test

2 types of analysis

• End point analysis

• Kinetic analysis
2 types of analysis
• Reading taken every 15
• End point analysis minutes ANC,NH,Y
• Identification obtained ST CARDS
only after completion

• Kinetic analysis
• Reading taken every 15
minutes GP & GN
• But analysis stops-When CARDS
profile matches minimum
of one organism with
good discrimination
VITEK2 automated
AST system
 VITEK2 AST CARD

• Comprises 64 wells - one growth control well and remaining drug wells
• Drug well→ pre-coated with increased concentration of various
antimicrobial agent
• Growth control well → contains only growth medium –no antimicrobial
agent
Commonly used cards
N405 panel N406 panel
Amikacin Ertapenem Amikacin Gentamicin

Amoxicillin/clavulanic Fosfomycin Aztreonam Imipenem

acid
Cefepime Gentamicin Cefepime Meropenem

Ceftriaxone Imipenem Ceftazidime Levofloxacin

Cefoperazone/sulbactam Meropenem Cefoperazone/ Minocycline

sulbactam
Cefuroxime Piperacillin/ Colistin Piperacillin/tazobactam
tazobactam
Ciprofloxacin Tigecycline Ciprofloxacin Tigecycline
Colistin Trimethoprim- Fosfomycin Trimethoprim-
sulfamethoxazole sulfamethoxazole
 N407 panel N235 panel

Ampicillin-sulbactam Doxycycline Amikacin Ertapenem

Amoxicillin/ Fosfomycin
Cefepime ESBL Clavulanate
cefoxitin Levofloxacin Ampicillin Gentamicin
Cefalotin Nalidixic acid
Ceftazidime-avibactam Meropenem
Cefixime Nitrofurantoin
Ceftizoxime Netilmicin
Ceftazidime Norfloxacin
Ceftolozane-tazobactam Polymyxin B
Ceftriaxone Ofloxacin
Chloramphenicol Tetracycline Ciprofloxacin Piperacillin/
Tazobactam
Colistin Tobramycin Ticarcillin Trimethoprim/
Sulfamethoxazole
 P628 panel ST-03 panel
Benzyl penicillin Linezolid Ampicillin Levofloxacin

Cefoxitin screen Nitrofurantoin Benzyl penicillin Linezolid

Ciprofloxacin Oxacillin
Clindamycin Rifampicin Cefotaxime Moxifloxacin

Daptomycin Teicoplanin Ceftriaxone Rifampicin

Erythromycin Tetracycline Chloramphenicol Teicoplanin
Gentamicin Tigecycline
Clindamycin Tetracycline
High level Trimethoprim-
gentamicin sulfamethoxazole Erythromycin Tigecycline
Inducible Vancomycin Gentamicin Trimethoprim-
clindamycin sulfamethoxazole
resistance
Inducible clindamycin Vancomycin
Levofloxacin resistance
PROCEDURE for AST
• AST suspension tubes are prepared manually
• Organism inoculated in 3 mL of 0.45% saline-matched to 0.5-0.63
McF(First tube)
• 3 mL of 0.45% saline is taken in second tube
• 145 µL/280 µL transferred to 2nd tube
ID AST AST
CARD CARD CARD

2nd TUBE
Contd…

AST- N405, AST- N406, AST- 145µL

N235
AST-P628,AST-ST03,AST-YS08 280µL
PRINCIPLE
• Modified broth microdilution method coupled with growth curve
algorithm Modified
Reference
Broth micro Broth micro dilution
method
dilution
Only few selected intermittent
All serial doubling dilution of drug concentration are tested
agents tested to obtain true/actual
MIC
Growth pattern obtained

Projected MIC is estimated

Interpretation: VITEK software is equipped

with clinical breakpoints(CLSI/EUCAST)
Preparing an AST panel

• VITEK card has 64 wells with one N405 panel

growth control well antibiotic Calling range Testing dilutions
≤1 to ≥ 64
• Calling range(reporting range) of the Amikacin 2,4,16,48

drug is wide Amoxicillin/ ≤2/1 to ≥ 32/16 4/2,16/8, 32/16

clavulanic acid
• But, only few intermittent drug Cefepime ≤0.12 to ≥ 32 0.25, 1, 4, 16, 32
concentrations are included in Ceftriaxone ≤0.25 to ≥ 64 0.12, 0.25, 1, 4,
VITEK panel 16 32

Cefoperazone/ ≤8 to ≥ 64 8,16,32
sulbactam
Cefuroxime ≤1 to ≥ 64 2,8,32
Ciprofloxacin ≤0.06 to ≥ 4 0.06,0.12,0.5, 1
 Only intermittent drug
Example: Amikacin/E.coli concentration are tested

Broth micro dilution Modified BMD

Testing dilutions 4
dilutions dilutions
64 µg/mL 64 µg/mL
48 µg/mL 48 µg/mL Growth pattern of these 4
wells
16 µg/mL Calling range 16 µg/mL
8 µg/mL dilutions : 7 8 µg/mL
4 µg/mL 4 µg/mL
PROJECTED MIC
2 µg/mL 2 µg/mL
1 µg/mL 1 µg/mL

reference MIC
Growth curve of a VITEK well
MIC is determined by comparing
growth pattern of test isolate to
growth pattern of large no. of
reference isolate with known
MICs(present in VITEK database)
Reference isolates and reference MIC

• Manufacturer used >1800 GN isolates and 1200 GP isolates with

various resistance mechanism
• These isolates were tested by BMD to determine actual MIC (known
as reference MIC/rMIC)
• All these isolates were tested by VITEK over large range of drug
concentrations
Advanced Expert System(AES)
Advanced Expert System(AES)

• Automated tool-to verify and validate AST results

• It also modifies the interpretation of AST results in case of
discrepancy
• Results are provided with various coloured confidence indicators
for which specific action needs to be taken
Result Interpretation Action
Green colour MIC value of ‘all’ antimicrobials completely • Results are reliable
fit to an expected pattern of phenotype • Can be reported
CONSISTENT

Yellow colour MIC value of ‘one’ antimicrobial does not fit Can be reported only after review
CONSISTENT WITH the expected pattern for a known phenotype by the clinical microbiologist.
CORRECTION
Red colour MIC pattern of >1 antimicrobial not matched • Cannot be reported
to that of a known phenotype. • Must be re-confirmed by re-
INCONSISTENT
testing

Purple colour Do not have predefined phenotypes database, • Preferable to perform alternate
but AST results are available AST method.
ANALYSIS NOT • If not feasible, MIC interpreted
PERFORMED using clinical breakpoints

Black colour No clinical breakpoints have been defined for Cannot be reported for that
this organism/antimicrobials combination organism
VITEK SCREEN
 Important step -matching the phenotypes in
the database

MIC DISTRIBUTION

N.V.TYPICAL TYPICAL V.TYPICAL TYPICAL N.V.TYPICAL

Ranking system to show confidence of phenotype match
Best
phenotype
INTERPRETATION

• S/I/R based on pre-stored database of clinical breakpoints from

standard source –CLSI , EUCAST, FDA, literature
 Calibration standards-daily
Cleaning done monthly
0.5% hypochlorite,/3-25%
H2O2/ Quaternary ammonium
compound

Saline sterility
TROUBLE check
SHOOTING

Pure isolated
colonies
No contamination
16-18 hours old

Appropriate storage of
cards as per instructions
QUALITY CONTROL

• To check the quality of cards, instruments and preparation techniques

• ATCC strains are used for Identification cards and AST cards
• Frequency as per local guidelines
 QUALITY CONTROL
STRAINS
Strain for AST N405 E.coli ATCC 25922, P.aeruginosa ATCC27853

Strain for AST N406 P.aeruginosa ATCC27853, E.coli ATCC 35218

Strain for AST N235 E.coli ATCC 25922, P.aeruginosa ATCC27853

E. faecalis ATCC 29212,Staph.aureus ATCC

Strain for AST P628 29213

Strain for AST ST03 Streptococcus pneumoniae ATCC 49619

Strain for BL-BLI E.coli ATCC 35218

Candida parapsilosis ATCC 22019, C.krusei

Strain for AST YS08 ATCC 6258
REFERENCES
“Technology should not aim to replace
humans,rather amplify human capabilities”
-Doug Engelhard
THANK YOU