Practical approach to Antimicrobial susceptibility testing (AST) and quality

control

A/Professor John Ferguson, Microbiologist &

Infectious Diseases Physician, Pathology North, University of Newcastle, NSW,

Australia jferguson@hnehealth.nsw.gov.au

Increasing trust in and relevance of your laboratory service

• Implement quality management approach

• Prioritise the most important testing – if resources limited, don’t neglect most important tests

• Critical result notification to clinician and document this

• Improve result availability – networked computerised database (OpenMRS or WHONET)

• Maintain close communication with clinicians :

– regular surveys- find out what they need and what is not working well

– seek out the clinician leaders and speak with them

– promptly notify test changes or unavailability of specific tests

• Provide an annual report to clinicians and management including cumulative antibiograms

Remember – there is a patient depending on you at the end of every result

Laboratory quality management

World Health Organization. Laboratory Quality Management System 2009

A central resource

ISO9001 (15189) -based.

Testing : always consider all elements

Importance of reliable AST

• Critical results for clinicians for individual patient management

• Cumulative antibiogram results can help with guideline decisions – which antibiotic will reliably cover certain organisms?

• New or emerging resistance constant threat- importation or locally arising

WRONG OR DELAYED CORRECT ANTIBIOTIC => INCREASED MORTALITY

Antimicrobial resistance testing

• What testing standard to use? – CLSI vs EUCAST • Media production and blood agar type • Quality control – internal and EQA essential • What antibiotics to test against what species? • How to report the results? • When should results be withheld?

[what antibiotics are available to clinicians? What antibiotics are recommended in standard treatment guidelines?

Priority bacteria for local AST testing and reporting

• Enterobacteriaceae (E. coli, Klebsiella etc) • Salmonella, Shigella + • Haemophilus influenzae • Staphylococcus aureus • Beta-haemolytic streptococcal species • [Enterococci

Reference level testing- MIC by E-test -CSF or blood isolates of S. pneumoniae and H. influenzae -Blood isolates of alphahaemolytic streptococci or enterococci from patients with known endocarditis

Advantages of EUCAST

Strive to be transparent:

–Open, public consultation as part of their decision making process with published comments and rebuttals on website.

–National breakpoint committees

–EMA, ECDC

–Industry

–Public

–Rationale documents on website

–Decision on zone diameter breakpoints and how they relate to MIC values available on website

http://Eucast.org

Methods for susceptibility testing

1) Phenotypic test methods:

– Reference Method: MIC values determined by the ISO reference method broth microdilution method and clinical breakpoints defined by Standards organisations

– Proxy MIC methods:

●agar dilution,

●gradient tests (e-test),

●automated broth microdilution systems (e.g. Vitek2, Phoenix, Microscan).

– Predict susceptibility and resistance

– Quantifiable

– Disc AST is a carefully designed ‘proxy’ or substitute for direct MIC determination methods

Methods for susceptibility testing

2) Genotypic test methods:

•Detection of a specific resistance gene (PCR) or its product

MecA, vanA, vanB, betalactamase, PBP2a etc.

•Whole genome sequencing

Bioinformatics approach to document presence of known resistance genes or mutational sequences associated with resistance

•Rapid

•Useful for epidemiological purposes

•Predict resistance, not sensitivity (yet)

•Not quantifiable

Methods for susceptibility testing

3) By deduction – extrapolation rules

•If MRSA (cefoxitin resistant) then report all betalactam antibiotics R

•If erythromycin resistant, then report all macrolides as R

Some rules predict susceptibility, others resistance

NB. Species based intrinsic resistance characters- no testing – just assume resistance based on the species ID – e.g. Proteus species and nitrofurantoin; Pseudomonas and ampicillin or ceftriaxone

See eucast.org for useful uptodate intrinsic resistance tables

MIC ‘wild type distributions’ and ECOFFs: definitions

WILD TYPE– a microorganism is DEFINED as wild-type for a species by the absence of acquired and mutational resistance mechanisms, and is CATEGORISED as wild type by applying the appropriate cut-off value in a defined phenotypic test system.

ECOFF (epidemiological cut-off) – the value that discriminates the wild-type strains from strains with acquired resistance to an agent. Ie is the highest MIC value of the isolate devoid of any phenotypically expressed resistance.

CLINICAL BREAKPOINT - The clinical breakpoint is the MIC concentration defined by man to predict clinical success and failure: S ≤ mg/L R > mg/L

MIC wild type distributions and ECOFFs

MIC wild type distributions and ECOFFs

CLINICAL BREAKPOINTS

Are determined by:

–MIC distributions of the target organisms

–Pharmacokinetics of the agent in target patients

–Pharmacodynamics of the agent in relation to the target organism

–Dose and mode of administration

–Clinical targets (indications)

–Target organisms (indications)

–Clinical outcome data for target infections

EUCAST or CLSI do all of this for us!

CLINICAL BREAKPOINTS

SENSITIVE – a microorganism is defined as susceptible by a level of antimicrobial activity associated with a high likelihood of therapeutic success

INTERMEDIATE – a microorganism is defined as intermediate by a level of antimicrobial activity associated with uncertain therapeutic effect. The implication is that an infection due to the organism may be successfully treated in sites where the agent is physically concentrated or when a high dosage of drug can be used.

RESISTANT – a microorganism is defined as resistant by a level of antimicrobial activity associated with a high likelihood of therapeutic failure.

CLINICAL BREAKPOINTS

EUCAST disc test- Technical Aspects

● Disc diffusion method is based a well –known technique (Kirby-Bauer)

● The 15-15-15 minute rule:

– Use the inoculum within 15 minutes of preparation (and ALWAYS within 60 minutes)

– Apply discs within 15 minutes of inoculating plates

– Start incubation within 15 minutes of application of discs

EUCAST - Technical Aspects (Media)

Mueller-Hinton II (MH) *

● Enterobacteriaceae

● Pseudomonas

● Staphylococci

● Enterococci

Mueller-Hinton with 5% horse blood and 20mg/L β-NAD (MH-F) for fastidious organisms**:

● S.pneumoniae and other streptococci

● H.influenzae

● others

* Lower concentration of thymidine than MHA

** NB- Not feasible in peripheral laboratories in PNG – probable referral function

EUCAST - Technical Aspects (Media)

Mueller-Hinton II agar

Lower concentration of thymidine

– enables reliable trimethoprim-sulfamethoxazole testing

Controlled concentration of magnesium and calcium ions

– Important for aminoglycoside testing (gentamicin, tobramycin, amikacin)

Inoculation

EUCAST - Technical Aspects (Inoculation)

EUCAST - Technical Aspects (Discs)

● Apply the discs firmly to the surface of the inoculated AND DRIED plate ; Do not move the discs once applied

● Loss of potency of antimicrobial agents in discs results in reduced zone diameters - a common source of error

– Discs (including those in dispensers) should be stored in sealed containers and protected from light.

– Store disc stocks at -20°C unless otherwise indicated by the supplier

– Store working supplies of discs at <8°C

– Allow discs to warm to room temperature before opening containers.

● 6 discs are the maximum possible number on a 90mm plate

EUCAST - Technical Aspects (Incubation)

● Invert plates and incubate within 15 minutes of disc application.

● Stacking plates – uneven heating of plates.

● Incubation conditions

EUCAST - Technical Aspects (Incubation)

Avoid short incubation periods – must be 18 hrs at least

EUCAST - Technical Aspects (Endpoint reading)

EUCAST - Technical Aspects (Endpoint reading)

Read zones where no obvious growth is detected by the unaided eye with the plate held at about 30cm from the eye.

EUCAST - Technical Aspects (Endpoint reading)

● S.aureus and benzylpenicillin

– Examine with transmitted light

● Disc diffusion is a reliable method for detection of penicillinase producers if zone diameter is measured correctly AND the zone edge is closely inspected

Deviations from the method - Common sources of error

● Materials used

– Mueller-Hinton agar

● Cations, thymidine, pH etc

● Manufacturing and storage

– Discs

● Compliance

● Storage

● Expiry date

Deviations from the method - Common sources of error

● Not adhering to the EUCAST methodology

– 0.5 McFarland standard – too heavy or too light inoculum

– Compliance with 15-15-15 minute rule

● Reading of zones – Follow the instructions!

Internal QC

QC: required ATCC strains

Quality control organisms (ATCC)

QC- control ranges are defined by antibiotic

Internal QC

Internal QC- plate reading exercises for staff

– Suggested strains:

● E. coli ATCC 25922, S. aureus ATCC 29213 (MH

agar)

– All staff read zones from the same plate

● Repeat reading for the same organism twice

● Compare results and discuss with staff

● Repeat exercises with same organisms until everyone gets the same results (mean±1mm)

External quality assurance

• Crucial addition to internal QC

• Unknown organisms for characterisation

• Track the methods and the results (e.g. MIC or zone size

• Track how each lab reports the results

• Feedback regularly with expert commentary

• Facilitate standardised approaches

WHO Global Action Plan 2015 Adopted May 2015 by World Health Assembly

All nations will have to draft their own 2 year action plans- principles-

– Whole of society engagement including a ‘One health’ approach

– Prevention first- emphasis on infection control

– Strengthen surveillance and research

– Preserve equitable access to antimicrobials

– Sustainability

Control of antibiotic resistance: microbiology expertise and testing underpins many key elements

www.react.org

Role of pathology services in antimicrobial stewardship

• Microbiological diagnosis – enables directed antimicrobial treatment – more effective patient cure

– Adequate clinical specimens

– High quality culture methods and AST

• Communication of critical results

– Liaison with clinicians to give therapeutic advice

– Commenting on reports

– Cascade reporting

• Cumulative antibiograms- essential for guideline development

• Education of prescribers Reference- AMS guide for Hospitals Edition 2 2017, Australia to come!

Excerpt from John Hunter Hospital (Aust) non-urine antibiogram (part )

• S= extrapolated result

• R= intrinsic resistance

• N/a – not available/tested

www.aimed.net.au/antibiograms

Thank you! More reading

http://Idmicnepal.net : infectious diseases and microbiology; guides to AST

http://Hicsiganz.org : lab quality management sections

http://aimed.net.au : practical antibiotic stewardship

jferguson@hnehealth.nsw.gov.au