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 threatimportation 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 * Lower concentration of thymidine than MHA Mueller-Hinton with 5% horse blood and 20mg/L β-nad (MH-F) for fastidious organisms**: S.pneumoniae and other streptococci H.influenzae others ** 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