Clinical Microbiology Laboratory Workflow PDF

Clinical Microbiology Laboratory Workflow Antimicrobial Susceptibility Testing (AST) & Interpretation Amelia Sofjan, PharmD, BCPS [email protected] Fall 2023 1 References • Werth BJ et al. Ch e122: Laboratory Tests to Direct Antimicrobial Chemotherapy. Pharmacotherapy: A Pathophysiologic Approach, 1...

Clinical Microbiology Laboratory Workflow Antimicrobial Susceptibility Testing (AST) & Interpretation Amelia Sofjan, PharmD, BCPS [email protected] Fall 2023 1 References • Werth BJ et al. Ch e122: Laboratory Tests to Direct Antimicrobial Chemotherapy. Pharmacotherapy: A Pathophysiologic Approach, 11 ed. 2019. McGraw‐ Hill Education. • Bottom of slides 2 Objectives 1. Recognize the clinical microbiology lab workflow 2. Identify the utility and impact of rapid diagnostic tests (RDTs) on patient care 3. Compare and contrast methods for antimicrobial susceptibility testing (AST) 4. Accurately interpret MIC, breakpoints, and antibiotic susceptibility results 3 Patient case JS • 75 year‐old male JS presents to the ER – Fever (101.3oF) – Chills – Back pain • Obtain cultures – Urine culture – Blood cultures (2 sets) • Each set = 1 aerobic bottle, 1 anaerobic bottle 4 What the pharmacist sees days later 5 Systematic Approach to Select Antimicrobials 1.Confirm infection 2.Obtain specimen to identify pathogen (sometimes) 3.Select antimicrobial therapy 4.Monitor response and optimize therapy as needed 6 Objective 1: Recognize the clinical microbiology laboratory workflow 7 How to identify pathogen • • Direct examination of tissues/stains  Gram stain, special stain/ink Culture  Specimen: various body fluids  Most definitive method for identification  Usually takes days 8 Clinical microbiology lab workflow (blood culture example) Blood draw 5-72 hours Identification and susceptibility Blood culture incubation Subculture and incubation Blood culture signals (+) Gram stain 12-24 hours 24 hours 30 minutes 9 Providers receive information piece‐by‐ piece (not all at once) Clue #1 Gram stain Clue #2 Bug ID Mystery solved! AST report Sometimes bug ID and AST report are released simultaneously 10 CULTURES ARE GREAT BUT WE WANT FASTER RESULTS! 11 Objective 2: Identify the utility and impact of rapid diagnostic tests (RDTs) on patient care 12 How to identify pathogen • Rapid diagnostic tests (RDTs)  Fluorescence in situ hybridization (FISH)  Nucleic acid amplification (NAAT)  Mass spectrometry • Serology  Antigen or antibody tests 13 Rapid diagnostic tests (RDTs) cut one or more intermediate steps 5-72 hours Blood draw Identification and susceptibility Blood culture incubation Subculture and incubation 24 hours Blood culture signals (+) Gram stain 30 minutes 12-24 hours 14 Fluorescence in situ hybridization (FISH) • Use fluorescent probes to detect the presence or absence of specific DNA/RNA sequences Probe is specific for a particular bug 15 https://www.youtube.com/watch?v=b81DcJC1jAs Nucleic acid amplification (NAAT) • Examples – Polymerase chain reaction (PCR) – Reverse-transcriptase PCR (RT-PCR) 16 https://www.youtube.com/watch?v=gubLAtn2o4s Example of multiplex PCR technology 17 https://www.biofiredx.com/ 18 Mass spectrometry • Matrix‐assisted laser desorption/ionization time‐ of‐flight mass spectrometry (MALDI‐TOF) 19 MALDI‐TOF machine 20 Utility of RDTs • RDTs provide rapid bug identification (only 1 RDT also provides rapid AST results) • Some RDTs detect presence of resistance genes – Examples: mecA, vanA/B • DOES NOT replace culture. Why? – Some RDTs do not detect ALL pathogens – We still have to grow the bug to test it against antibiotics! 21 Impact of RDTs plus real‐time antimicrobial stewardship • Optimize anti‐infective use – Faster time to stopping unnecessary therapy – Faster time to initiating active therapy – Faster time to de-escalating therapy • Improve patient outcomes – Decreased hospital length of stay – Decreased mortality • Reduce healthcare cost 22 Timbrook TT et al. Clin Infect Dis 2017;64:15-23. Example of how RDTs “change the game” 23 Patient case JS cont. Scenario 1: RDT NOT available • Patient JS was initiated on empiric therapy with vancomycin + cefepime. • On hospital day 2, his culture results in the electronic medical record are updated: – Urine culture: negative to date – Blood cultures: pending. • Aerobic bottle #1 Gram stain: gram positive cocci in clusters • Aerobic bottle #2 Gram stain: gram positive cocci in clusters • JS is improving clinically 24 25 Patient case JS cont. Scenario 2: RDT available • Patient JS was initiated on empiric therapy with vancomycin + cefepime. • On hospital day 2, his culture results in the electronic medical record are updated: – Urine culture: negative to date – Blood cultures: pending. • Aerobic bottle #1 Gram stain: gram positive cocci in clusters • Aerobic bottle #2 Gram stain: gram positive cocci in clusters • RDT result on blood cultures: positive for MSSA • JS is improving clinically 26 27 Take home points 1. The clinical microbiology lab workflow involves a series of sequential steps; therefore, data are released to providers in a step‐wise fashion. 2. RDTs in combination with real time antimicrobial stewardship has revolutionized the care of patients with infectious diseases by: – – – Optimizing anti-infective use Improving patient outcomes Reducing healthcare cost 28 Objective 3. Compare and contrast methods for antimicrobial susceptibility testing (AST) 29 AST is the last step of the clinical microbiology lab workflow Blood draw 5-72 hours Identification and susceptibility Blood culture incubation Subculture and incubation Blood culture signals (+) Gram stain 12-24 hours 24 hours 30 minutes 30 Why do we perform antimicrobial susceptibility testing (AST)? Other diseases Infectious diseases Drug Drug We need to measure the affinity (potency) of a drug to a bug (receptor) in each patient. Host Bug 31 Two common ways to measure antibiotic potency or affinity to the bug 1. Minimum inhibitory concentration (MIC) = lowest antimicrobial concentration that inhibits the visible growth of a standard inoculum of organism after ~24 hrs of incubation under standard growth conditions 2. Zone of inhibition (ZOI) ZOI 32 Common Methods for AST • Liquid media – Broth macrodilution  MIC – Broth microdilution  MIC • Solid media ― Epsilometer strip or “E test”  MIC ― Disk diffusion  zone of inhibition (ZOI) • CONVENTIONAL automated ID/AST systems  MIC • RAPID automated ID/AST systems  MIC 33 Liquid media 34 Broth mAcrodilution 35 MIC determination Inoculate each test tube with 105-6 CFU/mL bacteria, then incubate 0.25 µg/mL 0.5 µg/mL 1.0 µg/mL 2.0 µg/mL 4.0 µg/mL 8.0 µg/mL 16 µg/mL Increasing antibiotic concentration 36 Broth mAcrodilution: use, pros, and cons • Use – Research labs • Pros – Accurate MIC • Cons – Tedious – Labor-intensive – Error in preparing antibiotic solutions – Lots of reagents and space 37 Broth mIcrodilution • CLSI reference method = gold standard for MIC determination • Non‐CLSI reference method 38 CLSI reference method – panel prep with antibiotics and bacteria Control Prepare 2‐fold serial dilution of antibiotic stocks in growth media Increasing antibiotic concentration (µg/mL) Inoculate each well with 5 x10^5 CFU/ml bacteria, then incubate. Dispense 0.05 to 0.1 ml of antibiotic from each test tube into each well in microtiter tray (except control wells) PCN AMX CFZ CTX CFP CIP GTX IMI Increasing antibiotic concentration (µg/mL) 39 PCN AMX CFZ CTX CFP CIP GTX IMI 128 64 32 8 16 4 2 0.5 1 0.25 0.12 Control CLSI reference method – MIC determination MIC (µg/mL) 0.5 0.5 0.5 1.0 0.25 0.25 0.25 0.25 Increasing antibiotic concentration (µg/mL) Inspect for growth in wells with naked eye 40 Broth mIcrodilution CLSI reference method: use, pros, and cons • Use – Research labs • Pros – Gold standard for MIC determination – Cheap – Less reagents and space • Cons – Tedious – Labor-intensive – Error in preparing antibiotic solutions 41 Broth microdilution NON‐CLSI reference method: use, pros, cons • What’s different? • Pros – Premade or premanufactured microtiter trays – Automated panel reader to inspect growth • Use – Clinical micro labs • Cons – Inflexibility of drug selections 42 Solid media 43 E test 44 45 MIC determination via E test 46 E test use, pros, and cons • Use – Clinical micro labs to test new antibiotics • Pros – Flexibility of drug selections • Cons – Expensive – Generally correlate well with MICs via broth dilution BUT… systemic biases toward higher or lower MICs for certain bug-drug combinations 47 Disk diffusion (Kirby‐Bauer) 48 Disk diffusion (Kirby‐Bauer) Place commercially-prepared, fixed concentration, paper antibiotic disk onto agar, then incubate Bacteria on agar plate 49 Zone of inhibition determination 50 Disk diffusion use, pros, and cons • Use • Cons – Clinical micro labs as a workhorse – NO MIC • Pros – – – – – Cheap Reliable Easily interpreted Flexibility of drug selections Automated reader? 51 52 CONVENTIONAL automated ID/AST systems These are NOT rapid diagnostic tests (RDT) previously mentioned 53 CONVENTIONAL automated ID/AST system “automates” the last step Blood draw 5-72 hours Identification and susceptibility Blood culture incubation Subculture and incubation Blood culture signals (+) Gram stain 12-24 hours 24 hours 30 minutes 54 Function • Bug identification (ID) through biochemical tests • Automated AST (3‐16 hrs) through “automated broth microdilution” • Examples – Microscan WalkAway – premade microtiter trays – BD Phoenix – premade microtiter trays – Vitek 2 – small plastic reagent “cards” 55 Use • Clinical micro labs as a workhorse 56 Pros • MIC • Faster AST results (vs manual liquid/solid media) • Cost savings due to shorter length of hospital stay • Data storage – Epidemiologic reports, antibiogram • Run multiple samples in machine at once 57 Cons • Inflexibility of drug selections • Expensive (initial & maintenance) • Unreliable for fastidious or anaerobic bacteria 58 RAPID automated ID/AST system: Accelerate PhenoTest BC Kit This is considered a rapid diagnostic test (RDT) 59 Accelerate PhenoTest BC Kit “automates” the last step AND eliminates subculture/incubation Blood draw 5-72 hours Identification and susceptibility Blood culture incubation Subculture and incubation Blood culture signals (+) Gram stain 12-24 hours 24 hours 30 minutes 60 Accelerate PhenoTest BC Kit: cool technology but NOT a magic bullet! • Use – Clinical micro lab as an ADD ON machine • Pros: – Only RDT that provides rapid ID AND AST report • Cons: – – – – $$ Processes only blood culture samples Processes only 1 sample per machine Only able to detect 14 bacteria and 2 fungi 61 62 So many methods…who determines the “standard methodology?” USA: Clinical Laboratory Standards Institute (CLSI) & FDA Europe: European Committee on Antimicrobial Susceptibility testing (EUCAST) 63 Which antimicrobials to test? 64 Which antimicrobials to test? • CLSI lists agents that are appropriate for testing for each organism • Depends on hospital’s AST method and formulary • Bottom line: cannot test every drug! CLSI list agents that can be used as alternatives – Example of CLSI comment on Staphylococci: 65 Homework 3. Objective: Compare and contrast methods for antimicrobial susceptibility testing (AST) • • • • • • • Which one is solid vs liquid media? Which one(s) provides MIC? Use: research lab vs clinical micro lab? Flexible vs inflexible drug selections? Which one(s) are workhorses in clinical micro labs? Automated vs non-automated? Conventional vs rapid automated ID/AST? 66 Objective 4. Accurately interpret MIC, breakpoints, and antibiotic susceptibility results 67 Limitations of MICs • Limitations lie within its definition • MIC = lowest antimicrobial concentration that inhibits the visible growth of a standard inoculum of organism after ~24 hrs of incubation under standard growth conditions • Bottomline: MIC is an imperfect measure of antibiotic potency 68 Why are MICs still the standard potency test for antibiotics? 1. 2. 3. 4. Simple Relatively reproducible Can be easily related to Pk data Has withstood the test of time 69 Whichever antibiotic susceptibility testing method you use, you either get a MIC or a zone of inhibition…. how do you interpret these numbers? 70 How do these MICs become S or R? 71 Breakpoints translate MIC or zone of inhibition • Breakpoint = – a chosen drug concentration (mcg/mL) which categorizes MICs into interpretive categories – a zone diameter (mm) which categorizes zone of inhibitions into interpretive categories • Set by CLSI & FDA in USA and EUCAST in Europe • Unique for each bug‐drug combination based on: – Bug’s MIC distribution – Drug’s Pk/Pd – Clinical trial data 72 Interpretive categories • Susceptible (S) – Isolate inhibited by usually achievable concentrations of an agent used at recommended doses • Susceptible Dose‐Dependent (S‐DD) – Susceptibility dependent on dosing regimen • Intermediate (I) – Clinical response is likely less than that of a susceptible isolate 73 Interpretive categories • Resistant (R) – Isolate NOT inhibited by usually achievable concentrations of an agent used at recommended doses and/or resistance mechanisms are likely and – Clinical efficacy has not been reliably shown • Nonsusceptible (NS) – Used if there are rare occurrences of resistant strains  (ie. combines the “I” and “R” categories into 1 category  “NS”) 74 Breakpoints example Enterobacterales (e.g., E. coli) Abx Disk Interpretive categories content and zone diameter (mcg) breakpoints (nearest whole mm) Interpretive categories and MIC breakpoints (mcg/mL) S I R S I R Cipro 5 ≥21 16-20 ≤15 ≤1 2 ≥4 Levo 5 ≥17 14-16 ≤13 ≤2 4 ≥8 75 Where can I find breakpoints? • CLSI website • http://em100.edaptivedocs.net/dashboard.aspx • M100 document – free & updated yearly 76 The levofloxacin MIC for an E. coli isolate is 0.5 mcg/mL. How do you interpret this MIC? Enterobacteriaceae (e.g., E. coli) Abx Disk content (mcg) Zone diameter interpretive criteria (nearest whole mm) MIC interpretive criteria (mcg/mL) S I R S I R Cipro 5 ≥21 16-20 ≤15 ≤1 2 ≥4 Levo 5 ≥17 14-16 ≤13 ≤2 4 ≥8 A. B. C. D. Susceptible Intermediate Resistant Indeterminate 77 The ciprofloxacin zone of inhibition diameter for an E. coli isolate is 13 mm. How do you interpret this zone of inhibition diameter? Enterobacteriaceae (e.g., E. coli) Abx Disk content (mcg) Zone diameter interpretive criteria (nearest whole mm) MIC interpretive criteria (mcg/mL) S I R S I R Cipro 5 ≥21 16-20 ≤15 ≤1 2 ≥4 Levo 5 ≥17 14-16 ≤13 ≤2 4 ≥8 A. B. C. D. Susceptible Intermediate Resistant Non-susceptible 78 Breakpoints: caution • Breakpoints usually consider drug concentration in serum (not at site of infection) • “S” DOES NOT guarantee success • “R” DOES NOT guarantee failure 79 Example – Scenario A • S. aureus is susceptible (S) to drug A per AST report – This is assuming drug A achieves 100 mcg/mL (typical serum concentration) • If the site of infection is the bone and drug A only achieves a concentration of 10 mcg/mL in the bone, is drug A guaranteed to work against S. aureus in this case? A. Yes B. No • Bottomline: “S” DOES NOT guarantee success 80 Example – Scenario B • E. coli is resistant (R) to drug B per AST report – This is assuming drug B achieves 10 mcg/mL (typical serum concentration) • If the site of infection is the bladder and drug B achieves a concentration of 100 mcg/mL in the bladder, is drug B guaranteed to fail against E. coli in this case? A. Yes B. No • Bottomline: “R” DOES NOT guarantee failure 81 Breakpoints may change • Breakpoints should/can change as bugs evolve & new Pk/Pd and clinical data emerge • Example: – Initially the “susceptible” MIC breakpoint for drug A and bug X is set at 2 mcg/mL (ie, MIC ≤2 mcg/mL = susceptible) – New clinical studies show that patients who are infected with bug X with MICs of 2 mcg/mL are experiencing clinical failure – Should the susceptible MIC breakpoint remain at 2 mcg/mL? 82 AST summary (example: testing Morganella Morganii against various antibiotics) Breakpoints translate MIC or ZOI to S, SDD, I, R, NS AST method Liquid media MIC Solid media MIC or ZOI Conventional automated ID/AST system MIC Rapid automated ID/AST system MIC 83 Systematic Approach to Select Antimicrobials 1.Confirm infection 2.Obtain specimen to identify pathogen (sometimes) 3.Select antimicrobial therapy 4.Monitor response and optimize therapy as needed Providers use AST report to select 84 DEFINITIVE therapy How should providers interpret the AST report? 85 Patient A is infected with P. aeruginosa. Given the following susceptibility report, which is the most optimal antibiotic? Drug MIC (mcg/mL) Interpretive category Piperacillin/tazobactam 4 S Meropenem 2 S Levofloxacin 1 R A. B. C. D. Pip/tazo Meropenem Levofloxacin Not enough information 86 AST interpretation: pearls and caution • CANNOT compare MIC values of different antibiotics to each other. Why? – Each drug achieves different concentration in the body – Each drug has a unique breakpoint for each bug • Many BUG, HOST, and DRUG factors to consider • AST report is only ONE piece of the puzzle! 87 Summary: put the following steps in chronological order Gram stain the organism AST yields either an MIC or zone of inhibition for each bug-drug combination Confirm infection Release gram stain result to clinicians (CLUE #1) Perform antimicrobial susceptibility testing (AST) on the organism Start empiric therapy Assess for growth of organism in culture Obtain culture Release organism identification to clinicians (CLUE #2) Release AST report to clinicians (CLUE #3) Identify the organism Clinician uses AST report and bug, drug, host factors to choose an optimal definitive therapy MICs or zone of inhibitions are translated by breakpoints to interpretive categories Subculture organism onto agar plate and incubate 88