The Basics: Using CLSI Antimicrobial Susceptibility Testing Standards

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1 The Basics: Using CLSI Antimicrobial Susceptibility Testing Standards Janet A. Hindler, MCLS, MT(ASCP) UCLA Health System Los Angeles, California, USA 1

2 Learning Objectives Describe information contained in M02 and M07 and identify how these documents differ from the M100 tables. List ways to identify new information contained in M100. Discuss how M02, M07, and M100 can aid development of a quality program for antimicrobial susceptibility testing (AST) and reporting in clinical and public health laboratories. Discuss several procedural details of disk diffusion testing that could lead to errors if not performed as recommended. 2

3 The CLSI Subcommittee on Antimicrobial Susceptibility Testing develops AST standards. The subcommittee includes experts in infectious diseases, pharmaceuticals, and clinical laboratory practice. 3

4 Six Important Points to Ensure Quality AST Results 1. Only test bacteria that are likely to be causing an infection. (Not addressed in CLSI AST standards) 2. Use a standardized testing method. 3. Test and report antimicrobial agents appropriate for the organism and infection site. 4. Perform quality control (QC) routinely to ensure the AST is working properly. 5. Report results in a clear and concise manner so the physician will understand them. 6. Communicate highly significant findings (eg, multidrug resistant organisms) to appropriate resources (eg, infection control) in a timely manner. 4

5 CLSI AST Standards for Routine AST The Tables 2013 Disk Diffusion 2012 MIC MIC

6 CLSI AST Standards Standards that describe how to perform tests Disk diffusion: M02-A11 (2012)* Minimal inhibitory concentrations (MICs) (reference broth and agar dilution): M07-A9 (2012)* M100-S23 tables (2013) Suggested drugs to test/report Interpretive criteria (breakpoints) QC ranges * M02, M07 updated every three years M100 updated at least annually 6

7 US Clinical Laboratories and CLSI AST Standards CLSI publishes standard consensus reference methods. Clinical laboratories can use: CLSI test method as written Method that performs comparably to CLSI reference method (eg, US Food and Drug Administration [FDA] cleared diagnostic AST device) Each laboratory must customize testing and reporting to meet the needs of their facilities. Must interact with stakeholders (Infectious Diseases, Pharmacy, Infection Control, etc.) 7

8 Clinical Laboratory Can Use a CLSI Reference Method Disk diffusion (Kirby-Bauer) Broth microdilution (or agar dilution) MIC 8

9 or a Commercial System 9

10 If using a commercial AST system, follow the manufacturer s instructions precisely! 10

11 M02 and M07 Describe Steps for Performing AST Select fresh isolated colonies from agar plate. Prepare inoculum suspension. Standardize turbidity of inoculum suspension. Inoculate plate/tray/panel. Incubate. Read results. Interpret results. Confirm results. Report results. Follow timing rules! 11

12 M02-A11 Partial Table of Contents (similar for M07-A9) 12

13 Two Most Critical Steps in Performing Disk Diffusion Test Inoculum preparation M02-A11, Section 8 Examining zones of inhibition M02-A11, Section

14 Prepare inoculum Suspension. Select fresh ( 24-hour) colonies. Example: Direct colony suspension method 0.5 McFarland standard 14

15 Mix well. 0.5 McFarland standard Standardize inoculum suspension (manually or spectrophotometrically). 15

16 Swab plate. Remove sample. 16

17 Incubate overnight. Add disks. 17

18 Measuring Zones (1) Transmitted Light Reflected Light 18

19 Measuring Zones (2) Measure zone diameter using reflected light except for: Staphylococcus spp. linezolid Enterococcus spp. vancomycin Use transmitted light for these only and do not ignore ANY growth! 19

20 Measuring Zones (3) When using reflected light: Ignore miniscule colonies at zone s edge Proteus spp. ignore swarm within zone Trimethoprim, sulfonamides disregard slight growth (look for 80% reduction of the lawn of growth). 20

21 Measuring Zones (4) Proteus spp. ignore swarm 21

22 Measuring Zones (5) Measure zone of complete inhibition, except trimethoprimsulfamethoxazole and sulfonamides (ignore fine growth within obvious zone). 22

23 Measuring Zones (6) P. aeruginosa resistant subpopulation E. coli contaminated with enterococci repeat test Do not ignore colonies within zone. 23

24 Measuring Zones (7) Measure zone of growth NOT zone of hemolysis when using bloodsupplemented media. 24

25 Poor Growth (need confluent lawn) 25

26 M100-S23 Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Third Informational Supplement 26

27 Summary of Changes 27

28 The M100 Tables (1) Instructions for use of tables Drugs to test/report Table 1A nonfastidious bacteria Table 1B fastidious bacteria Table 1C anaerobes Interpretive criteria (breakpoints), examples Table 2A Enterobacteriaceae Table 2G Streptococcus pneumoniae Table 2J Anaerobes Supplemental tables, example Table 2D Supplemental Table 1. Screening Tests for High-Level Aminoglycoside Resistance (HLAR) and Vancomycin MIC 8 μg/ml in Enterococcus spp. 28

29 The M100 Tables (2) QC ranges Tables 3A 3B: disk diffusion Tables 4A 4E: MIC Other Tables antimicrobial solution/media preparation Appendixes, examples Patient results that should be confirmed Intrinsic resistance profiles of common species Anaerobes cumulative antibiograms Antimicrobial agent glossary 29

30 Instructions for Use of Tables 30

31 Instructions for us of Tables Strategy for selecting drugs to test/report (and selective reporting) Definitions of susceptible (S), intermediate (I), resistant (R), nonsusceptible (NS) Definitions of Warning and Therapy comments List of screening tests More 31

32 M100 Tables 1A 1C Select drugs to test/report. Determine if one drug can predict results for another. Identify drugs that are dangerous to report. Add comments to report. 32

33 Group A Group B Table 1A. Drugs to Test/Report (1) 33

34 Group C Group U Do NOT report on isolates from sources other than urine! Table 1A. Drugs to Test/Report (2) 34

35 Selective (or Cascade) Reporting Report (first line) drugs with narrowest spectrum, lowest toxicity, lowest cost first. Report others if: Resistant to first line (primary) From specific body site 35

36 Selective Reporting Example Pseudomonas aeruginosa Cefepime Ciprofloxacin Gentamicin Piperacillin-tazobactam S S S S 36

37 Selective Reporting Example Pseudomonas aeruginosa Amikacin S Cefepime R Ciprofloxacin S Gentamicin R Meropenem S Piper-tazobactam R Tobramycin R Selectively reported 2. Amikacin selectively reported when gentamicin-r and tobramycin-r 3. Meropenem selectively reported when cefepime- R and piperacillintazobactam -R 1. Tobramycin selectively reported when gentamicin-r 37

38 Should We Use Selective Reporting? Yes, with approval of physicians Maybe not, if there are other ways to encourage prudent prescribing in our institution Each laboratory must decide if selective reporting is right for them and which drugs to selectively report on individual species or organism groups. 38

39 Table 1A Enterobacteriaceae Acinetobacter spp. OR No OR Drugs in boxes have similar spectrum of activity against organism group. If OR, you can extrapolate results for two drugs. If no OR, you cannot extrapolate. 39

40 Serratia marcescens OR Example Ampicillin Cefazolin Cefotaxime Gentamicin Trimethoprim-sulfa... R R R S S Cefotaxime-R S. marcescens are ceftriaxone-r. 40

41 No OR Example Imipenem-S Meropenem-R Laboratory reported imipenem S Patient treated with meropenem No OR between imipenem and meropenem for Acinetobacter spp Cannot extrapolate results Patient died Lesho E, Wortmann G, Moran K, Craft D. Fatal Acinetobacter baumannii infection with discordant carbapenem susceptibility. Clin Infest Dis. 2005;41(5):

42 * some drugs can be reliably tested by MIC method only Table 1A. Drugs to Test/Report 42

43 CLSI Warning Comments Some organism/antimicrobial combinations show activity in vitro (ie, they test S ) but are clinically ineffective. DO NOT report as Susceptible. 43

44 Drug/Bug Combinations That Do Not Work in Patient Even if S in Test Organisms Salmonella spp., Shigella spp. Oxacillin-R staphylococci Enterococcus spp. Do Not Report as S First- and second- generation cephalosporins cephamycins aminoglycosides any beta-lactam (except ceftaroline for S. aureus) aminoglycosides (except high conc) cephalosporins clindamycin trimethoprim-sulfamethoxazole 44

45 DO NOT Report on CSF Isolates Little or No Active Drug Gets into CSF Agents administered by oral route only First- and second- generation cephalosporins (except cefuroxime parenteral) and cephamycins Clindamycin Macrolides Tetracyclines Fluoroquinolones Abbreviation: CSF, cerebrospinal fluid. 45

46 Urine Isolates DO NOT report routinely on urine isolates. Azithromycin Chloramphenicol Clarithromycin Clindamycin Erythromycin Report ONLY on urine isolates. Drugs in Group U in M100-S23 Table 1A For example, nitrofurantoin. 46

47 How should we interpret results? Table 2A. Interpretive Criteria (Breakpoints) 47

48 48 Why might an antimicrobial agent be listed in Table 2 but not in Table 1? Drug may have a clinical indication for the organism group but is generally not appropriate for routine testing and reporting in United States. Listed as test/report group O (other) Drug may not be FDA-approved Listed as test/report group Inv (investigational) Table 2A. Interpretive Criteria (Breakpoints)

49 Some Organism-Drug Combinations Have Susceptible-Only Breakpoints S. pneumoniae Vancomycin MIC (µg/ml) (Note: to date, vancomycin NS has not been reported in S. pneumoniae) Susc Int Res Vancomycin Investigate any nonsusceptible (NS) result. Repeat identification and AST. Save isolate. Send to reference laboratory (test by CLSI MIC method). 49

50 Table 2C Supplemental Table 1. Screening Tests for S. aureus Group 50

51 How can we ensure accurate results? Table 3A. QC Organism Ranges 51

52 Appendix A. Confirm Patient s Results (all results should be confirmed but some require additional attention) 52

53 Additional Quality Control Tables QC testing frequency Tables 3C, 4F Troubleshooting guide Tables 3D, 4G Summary of QC strains and their use Appendix C 53

54 Appendix B. Intrinsic Resistance Enterobacteriaceae 54

55 Glossaries I and II Class, subclass, generic name Abbreviations: PO = per OS (oral); IM = intramuscular; IV = intravenous. 55

56 Glossaries of Antimicrobial Agents 56

57 Artificial Intelligence (eg, expert software) Helps to: Flag atypical/inconsistent results. Suggest confirmatory tests. Report appropriate drugs. Edit S or I results to R. Add comments to report. AST but still need informed clinical laboratory scientists! 57

58 CLSI Website Find information from AST meetings. Order CLSI AST products. subcommittee-on-ast/ 58

59 An online interactive searchable version of M100-S23 enables you to easily Access M100-S23 all you need is a Web connection. Dynamically filter by tables, organisms, and agents. View only the information you want to see. Customize for your institution s formulary. Find additional CLSI AST standards and guidelines within the em100 Resource Center. Available at 59

60 Clinical Microbiology Portal Ask questions. Retrieve protocols. Join LISTSERVs. Also from ASM: Clinical Microbiology Procedures Handbook Some Additional Resources 60

61 The following summary slides will not be discussed and are presented for participant s review. 61

62 Summary (1) CLSI develops standards and guidelines for many laboratory procedures including AST. CLSI M100 tables for AST are updated each year; standards describing how to perform disk diffusion tests (M02) and how to perform reference MIC tests (M07) are updated every three years. When using a commercial AST, the manufacturer s recommendations must be followed precisely. 62

63 Summary (2) Selective reporting of antimicrobial agents may encourage prescribing of narrowerspectrum agents and may be appropriate for some facilities. It is dangerous to extrapolate results from one drug to another unless there is an or connecting the two in CLSI tables. Some organism/antimicrobial combinations cannot be reliably tested by the disk diffusion method. 63

64 Summary (3) Some organism/antimicrobial combinations do not work in the patient even though they may appear susceptible in vitro. Some antimicrobial agents are ineffective in treating infections at specific body sites. Some antimicrobial agents are only effective in treating urinary tract infections ( U category in M100). Nonsusceptible results should be confirmed before reporting. 64

65 Summary (4) Interpretive criteria may require revision as new information about organisms or antimicrobial agents becomes available. All patients results must be examined before reporting. Uncommon results should be confirmed or verified. Artificial intelligence or expert software can be extremely helpful to highlight potential problem results. Antimicrobial agent glossaries in M100 list classes and routes of administration for antimicrobial agents. 65

66 66

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