AST Flashcards
Define AST
Antimicrobial susceptibility testing is a lab procedure designed to assess the effectiveness of specific antimicrobial agents
What are the three main purposes of AST
Guiding Clinical Therapy
Antibiotic Resistance Awareness
Identify and Monitor Resistance Profiles
Talk about the role of AST in guiding clinical therapy
Guides appropriate antimicrobial therapy based on the identiffied pathogens and their resistance patterns i.e. what they are susceptible too
Means targeted therapy rather than relying on broad-spectrum antimicrobials
Improve treatment outcomes, reduce side effects and shorten recovery times
Oftten comes too late to actually impact treatment decisions i.e. by the time we have the results the critical phase of illness has already passed
Talk about the role of AST in awareness for antibiotic resistance
AST provides information about the resistance profiles of bacteria within a specific population or geographical area
This knowledge is essential for clinicians to understand the local resistance trends and make informed decisions
e.g. penicillin resistant S. pneumonia where there can be 50% resistance in some regions but only about 5-10% in Ireland thus penicillin is the recommended treatment option in Ireland
Talk about the role of AST in identifying and monitoring resistance profiles
Detect pathogens that exhibit resistance to specific antimicrobials, including multi-drug-resistant organisms (MDROs)
Preventing the spread of resistant infections in hospitals and the broader community (Infection Control) - become increasingly more difficult (1.2 days on average in hospital)
Reveal trends in resistance patterns, showing which antimicrobials are becoming less effective over time
Monitor the emergence of resistant strains, adjust treatmnet guidelines and develop new policies for infection control
What are the four different ways AST can be carried out?
Phenotypic AST
Genotypic AST
Mechanistic AST
Expert Rules
What is phenotypic ASt?
Measurement of the minimum inhibitory concentration (MIC) and the interpretation of breakpoints
EUCAST breakpoints
What is genotypic AST
Detection of specific resistance genes such as mecA, vanA/B, Big 5 CPE that confer resistace
What is mechanistic AST
Detection of resistance mechanism such as Beta-lactamase detection
Can infer success of antibiotics from this
What are expert rules?
Conditional rules that link specific resistance mechanisms to treatment decisions
e.g. rules that guide the VITEK
Involves looking at a resistance profile and making assumptions from this
Pros and cons to this as the landscape of resistace is constantly changing hence the need to mannually check VITEK sens
What are the two different ways phenotypic AST can be carried out?
Qualitative:
- Disc diffusion
Quantitative:
- Macro broth
- Micro broth
- Micro well e.g. VITEK automated microwells or microtitres for fungi
- Etest
Talk about the disc diffusion method of AST, principle and development
The diameter of the inhibition zone is inversely proportional to the minimum inhibitory concentration (MIC) i.e. the larger the zone the less resistant
Based on the standardised Kirby-Bauer disc diffusion approach developed in 1966 and adapted for EUCAST mIC
What does MIC stand for?
minimum inhibitory concentration
Talk about how you would carry out disc diffusion AST
Identify colony of interest -> since AST is species specific its important to ID correctly
Select agar: Mueler Hinton Agar for non-fastidious, MG + 5% defibrinated horse blood + 20 mg/L B-NAD for fastidious
Ensure agar is batch accepted
Ensure agar is at room temperature and dry
Inoculate with a heavy 0.5 McFarland standard
Maintain 15 minute rule
Apply disks within 15 mins
Incubate within 15 mins
What agar is used for fastidous disc diffusion
mueller hinton agar + 5% mechanically defibrinated horse blood + 20 mg/L B-NAD
Talk about batch acceptance of MH agar, give two examples of where there can be issues with agar
Each new batch must be tested with ATCC strains to ensure they fall within EUCAST QC ranges
Variations in Ca2+ and Mg2+ can affect aminoglycoside inhibition zones for P. aeruginosa
Excess thymine/thymidine can reduce trimethoprim-sulfamethoxazole zones for E. faecalis
Talk about drying and storage of agar plates for disc diffusion
Agar must be at room temperature prior to inoculation
Surfaces must be dry i.e. not straight from fridge
Excess moisture can cause fuzzy zone edges and/or haze
How should Disc Diffusion agar be inoculated?
A heavy inoculum of 0.5 McFarland should be made of -> corresponds to 102 x10^8 CFU/ml of an E. coli
According to EUCAST you should select well -isolated colonies from overnight growth on non-selective medium
4-6 hour incubation is also suitable
Why should you not pick from selective agar?
Selective agar contains antimicrobials already - hene the need for a purity plate prior to AST
What is the 15-15-15 rule
Inoculate agar within 15 mins of making suspension
Apply discs within 15 mins of inoculating
Incubate agar within 15 mins of applying discs
How does disc diffusion for gram negs compare to that of gram positives?
For gram-negative bacteria, remove excess fluid by pressing and turning the swab against the inside of the tube to avoid over-inoculation
For gram-positive theres no need to do this
What is the only exception to the 0.5 MacFarland for disc diffusion?
S. pneumoniae is suspended to a 0.5 McFarland from blood agar but to 1.0 if from a chocolate agar plate
(google this)
How should discs by applied for disc diffusion?
Apply disks within 15 minutes of inoculation
Disks must be in close and even contact with the agar surface
The number of disks on a plate should be limited to avoid overlapping of zones and interference between agents
It is important that zone diameters can be reliably measured
5 to 6 disks max per plate
How should you incubate disk diffusion plates?
Invert plates making sure disks stay attached to surface
Incubate at 35 degrees in air with 4-6% CO2 for 18+_2 hours
AST must be read within 18 hours of incubation hence why E. Colis were put up after lunch and read first thing in the morning
How should you read disk diffusion
First check for confluent lawn, dont want any gaps in lan
For MH agar hold plate over a black, non-reflective surface and read from the back with reflected light
For MH-F agar read from the front with the lid removed illuminated with reflected light
Measure diameter of zone of inhibition to the nearest mm
What should you do if you have distinct colonies within your zone?
Check for purity -> is it the same organism -> repeat if not
If cultures are pure then take colonies into account when reading result
Should subculture any colonies within the zone and repeat AST
How should you read proteus AST
Ignore swarming of colonies
Howshould you read haemolysis on disk diffusion
Read the inhibition of growth and not the inhibition of haemolysis
B-haemolysins diffuse in agar -> they are usually free from growth
a-haemolysins do not diffuse -> there is often growth within areas of a-haemolysis
Zone edges accompanied with a-haemolysis is most common with S. pneumoniae and B-lactam antibiotics
How are zone sizes interpretted?
Check ATCC controls - zone diameters of ATCC control organisms must fall within accepted range
Criteria for test organism
- zone diamters of test organisms compared to interpretive clinical breakpoints for relevant species
Categorised as S, I or R
What are some methods of zone reading?
Callipers
Ruler
Stencils
Semi-auromated systems
- reduces operator variability in reading plates
- provides auomatic interpretation of zone diameters
- zone readers linked to LIS
What is the MIC, how are they used
The lowest concentration of an antimicrobial agent that prevents visible growth of a microorganism after a specified period
Clinical breakpoints determine if organism is suseptible, susceptible at increased exposure or resistant
What is meant by susceptible?
Organism is susceptible at standard dosing regimen
i.e. there is a high likelihood of therapeutic success using a a standard dosing regimen of the agent
What is susceptible, increased eposure?
There is a high likelihood of therapeutic success because exposure to the agent is increased by adjusting the dosing regimen or by itc concentration at the site of infection
It can be used but must increase conentration -> prevents heavy reliance on newer drugs in an attempt to reduce resistance against these
What is defined as resistant
A miroorganism is categorised as Resistant when there is a high likelihood of therapeutic failure even when there is increased exposure
What does a EUCAST NA result mean?
It means EUCAST wont stand over any result that falls within these breakpoints
Its usually seen with drugs used for screenining moreso than treatment e.g. cefoxitin
What is an area of technical uncertainty (ATU)?
A term used by EUCAST to indicate potential confusion in interpreting antimicrobial susceptibiliy testing (AST) results, whether through MIC determination or disk diffusion
It is defined by specific MIC values or inhibition zone diameters -> it is not a susceptibility category like susceptible (S) etc
What should you do with an area of technical uncertainty in the lab?
It should be a caution to the lab staff that the result falls within a range where interpretation is challenging due to the positioning of breakpoints which can lead to inconsistent result
Importantly the ATU is not due to testing errorss
There is no required action if you get one of these results but if you suspect an error then It is recommended that you repeat the method or try another method
Results should be reported as “uncertain” with a comment
What are expert rules?
Conditional rules that link specific resistance mechanissm to treatment decisions
e.g. mecA in methicillin resistant staph aureus suggests resistance to multiple beta-lactam antibiotics
These rules are constantly changing ang being updated
classes organisms as either expected resistant phenotype (intrinsic resistance) or expected susceptible phenotype
Give an example of how expert rules change over time
ESBLS were historically resistant against cefotaxime but now it depends on MIC
Talk about expected resistant phenotype (Intrinsic Resistance) using expert rules
Refers to species where the vast majority >90% are resistant to specific antibiotics regardless of the isolate’s origin e.g. Bacteroides ID
If a laboratory result shows susceptibility it should be viewed with skepticismm and testing is generally discouraged
Typically results for these antibiotics are reported as resistant without actual testing as resistance is considered intrinsic - clinicians should be advised not to use such antibiotics fot these species
Talk about expected susceptible phenotype (Intrinsic Resistance) using expert rules
Describes species that are almost always (>99%) susceptible to a given antibiotic, as resistance mechanisms of clinical relevance have not been documented
A resistant test result should raise suspicion and prompt a review of the species ID or susceptibility testing method
If a resistant result is believed to be due to an acquired resistance mechanism, it should be confirmed through reference methods, and ideally, genome sequencing
How is AST Quality Controlled?
Control tests should be set up and checked daily, or at least four times per weak, for antibiotics which are part of routine panels
Control tests should always be read and evaluated before reporting ressults for clinical isolates
Each day that tests are set up, examine the results of the last 20 consecutive tests
Examine results for trends and for zones falling consistently above or below the target
If two or more of 20 tests are out of range investigation is required
What is quantitative AST
AST based on MIC - refers to the minimum concentration of an antimicrobial agent required to inhibit the grorwth of a microorganism
Before automated systems such as Vitek, MICs were only done for serious infections, slow-growing organisms or fastidious organisms
How do you establish an MIC, give some exaples of how theyre done
MICs are established by exposing the test organism to a range of twofold dilutions of the antimicrobial agent in an appropriate culture system:
- Macro-broth
- Micro-broth dilution
- Gradient diffusion method: Etest
- Automated-micro-well-dilution-Vitek (2 fold gradiant increase)
What is the reference method for MIC determination?
Agar Dilution MIC is the gold standard
How is agar dilution MIC carried out?
Antimicrobial agents are incorporated into molten agar plates at twofold serial dilutions
Bacteria are inoculated as spots on the surface of he agar
Endpoint interpretation:
- MIC is defined as the lowest concentration of the antimicrobial at which no visible growth is observed
- individual colonies or a fine haze of growth should be disregarded
Nobody actually does this - way too tedious and time consuming -> have to either incorporate antimicorbials in the agar or through spot inoculum
Talk about broth dilution method
Incorporate a range of twofold serial dilutions of the anitmicrobial into the broth
For macrobroth dilution (now obselete) use over 1 ml of broth in tubes
For microbroth dilution only 50-100ul of broth is used in mirotitre plates
Talk about Etests
Test isolate is exposed to a continuous antimicrobial gradient on an inert plastic strip that diffuses into the agar
After incubation an elliptical zone of inhibition forms at the MIC
Follows EUCAST guidelines for disc diffusion
Relies on diffusion yielding optimal results with organisms that grow well overnight
Expensive though at 3euro per strip, control isolates must be tested separately
Talk about Automated AST
VITEK is based on micro-well dilution methods
Carries out both ID and AST
Results in 4-16 hours
Mainly hands-off
Has to be adapted for Eucast guidelines as it will follow american guidelines - hence the need for a scientist to check results
Vitek 2XL in most labs or the phoenix in beaumont
