# Presentation: 454427

## Using the CLSI / NCCLS AST Standards Most Effectively!

**Janet Fick Hindler, MCLS MT(ASCP)***
**UCLA Medical Center**
**Los Angeles, CA**
**jhindler@ucla.edu**
*** ***

****also working as a consultant with the Association of Public Health Laboratories with support from CDC ***

## At the conclusion of this talk, you will be able to......

**Describe the overall purpose and scope of the ****CLSI/NCCLS antimicrobial susceptibility testing (AST) standards.**
**Review the ****contents of the standards.**
**Explain where and how to find ****changes in the most recent standards**** including M100-S15 which was published in January 2005. **

## CDC AST CD-ROM

**Modes of action / mechanisms of resistance**
**Methods**
**Gram-positive bacteria**
**Gram-negative bacteria**

*ordering information at...*
**www.phppo.cdc.gov/dls/master/default.asp**

## Askit

**ASM Division C Homepage**
**Join “DivC Listserver”**
**- Discuss issues with peers**
**- Search archives**

**http://www.asm.org/division/c/index.htm**

**Askit**

## Askit

**“****Askit” Main Screen**
**- Type keywords**
**- Ask question**
**- Get answer from “expert”**
**- Search archives**

**Askit**
**archives**

## Routine Susceptibility Tests

**CLSI disk diffusion reference method (Kirby Bauer)**
**Automated zone reader****   **_**[www.biomic.com](http://www.biomic.com/)**_
**Broth microdilution MIC**
**CLSI reference method**
**Commercial systems**
**Etest  **_**[www.abbiodisk.com](http://www.abbiodisk.com/)**_
**Automated / semi-automated systems**
**MicroScan (Dade Behring)   ****www.dadebehring.com**
**Vitek (bioMerieux)    ****www.biomerieux.com**
**Phoenix – (Becton Dickinson)  ****www.bd.com**
**Sensititre (Trek)    ****www.****trek****ds.com**

## If using an FDA-cleared commercial AST system, always follow manufacturer’s instructions in the package insert...

**“****Note 4:**** ****Acceptable MIC QC limits**** for FDA-cleared antimicrobial susceptibility tests may differ slightly from acceptable CLSI QC limits.  Users of each device should ****utilize**** ****manufacturer’s procedures and QC limits**** as indicated in the instructions for use.”**

**CLSI M100-S15 Table 3C (M7)**

## The CLSI / NCCLS AST Standards

- CLSI / NCCLS

## CLSI Standards - 2005

**M100-S15 Tables ****(2005)***
**	****...****..to be used with text documents explaining how to perform the tests....**
**	****	****M2-A8   Disk Diffusion**** (2003)****
**	****	****M7-A6   MIC**** (2003)****

- New!

*** M100 updated yearly**
****M2, M7 updated every 3 years**
- _[www.clsi.org](http://www.clsi.org/)__ _
**(610) 688-0100**

## The “Tables” - M100-S15

**“****Updated information in this edition”**
**“****Introduction to tables”**** - how to use tables**
**Drugs to ****test/report**
**Tables 1 – 1B**** **
**Interpretive**** criteria **
**Tables 2A – 2L****            **
**Quality control**** ranges **
**Table 3 – 3C**
**Other tables**
**           **

***Check Table of Contents ***
***in M100-S15 pages 7-9***

## Updated information in M100-S15

**Updated information in M100-S15**

## Antimicrobial AgentGlossary I and II

**Class, subclass, generic name**
**Abbreviations / PO, IM, IV**
**“****Identical” abbreviations for same drug in U.S.**

## CLSI M100-S15 Glossary I  Part 1, Example 

| Antimicrobial Class | Antimicrobial Subclass | Agents Included; Generic Names |
| --- | --- | --- |
| penicillins | penicillin | penicillin |
|  | penicillinase- stable penicillins | cloxacillin
dicloxacillin
methicillin
nafcillin
oxacillin |

## CLSI M100-S15 Glossary II Example 

| Antimicrobial Agent | Agent Abbrev. | Routes of Administration |  |  | Drug Class |
| --- | --- | --- | --- | --- | --- |
|  |  | PO | IV | IM |  |
| Amikacin | AN, AK, Ak, AMI, AMK |  | X | X | aminoglycoside |
| Ampicillin | AM, Am, AMP | X | X | X | penicillin |

## Table 1

**Table 1**

## Table 1Select Drugs to Test/Report

**Recommendations by ****organism group**
**Lists ****primary, secondary agents**** of choice **
**Use results from one drug to ****predict**** another**
**Obtain input from ****Medical Staff**** when developing your test/report protocol**

## Test/Report Groups

**A****  Primary Test/Report**
**B  ****Primary Test/Report Selectively**
**C****  Supplemental, Report Selectively**
**U****  Supplemental, Urine Only**

## Table 1

**Test/report groups (selective reporting)**
**Drugs in ****“boxes”**
**“****ors” ****between some drugs in boxes**
**Footnotes**
***“******Rx”****** *****comments**
***“******Warning”****** *****comments**

## Reporting Second Line Drugs – Selective Reporting

**Resistance to primary agents**
**Specific body site**
**Patient intolerance of primary agents**
**Patient not responsive**
**Polymicrobial infection**
**Infection at multiple sites**

## Pseudomonas aeruginosa

**	****	****ceftazidime ****	****	****S**
**	****	****ciprofloxacin****	****	****S**
**	****	****gentamicin ****	****	****S**
**	****	****piper-tazobactam ****	****S**
** **

- Fresno 11/00

## Pseudomonas aeruginosa

**amikacin****	****S****	****	**
**ceftazidime ****	****R**
**ciprofloxacin****	****S**
**gentamicin ****	****R**
**imipenem****	****S**
**piper-tazobactam ****	****R**
**tobramycin****	****R**
** **

- Fresno 11/00

## Should We Use a Selective (Cascade) Reporting Protocol?

**Yes,**** with approval of medical staff**
**Yes,**** because it helps MDs focus on prescribing the drug(s) that is:**
**Narrowest spectrum**
**Lest toxic**
**Least costly**
**Maybe not****....if there are other ways to encourage ****“prudent prescribing”**** in our institution**

## From CLSI Table 1Enterobacteriaceae

**Cefmetazole**
**Cefoperazone**
**Cefotetan**
**Cefoxitin**

## From CLSI Table 1  Enterobacteriaceae

**Cefotaxime ****or**
**Ceftizoxime ****or**
**Ceftriaxone**

## Enterobacter cloacae 

**	****	****ampicillin****	****	****	****R**
**	****	****cefazolin****	****	****	****R**
**	****	****cefotaxime****	****	****R**
**	****	****gentamicin****	****	****S**
**	****	****trimeth/sulfa****	****	****R**
**  “****Cefotaxime-R *****E. cloacae***** are ceftriaxone-R****”**

## Enterobacter cloacae 

**  ****	****	****ampicillin****	****	****	****R**
**  ****	****	****cefazolin****	****	****	****R**
**  ****	****	****cefotaxime****	****	****S**
**  ****	****	****gentamicin****	****	****S**
**  ****	****	****trimeth/sulfa****	****	****R**
**  ****“****Cefotaxime-S *****E. cloacae***** are usually ceftriaxone-S”**

## CLSI “Warning” Comments 

**Some drug/bug combinations show activity in vitro but are clinically ineffective**
** **

## Warning Comment Table“Dangerously Misleading” Bug/Drug Combinations

**Salmonella, Shigella****	**
**Listeria**
**ESBL producers**

**1st, 2nd generation    cephalosporins**
**aminoglycosides**
**cephalosporins**
**aztreonam**
**cephalosporins**
**penicillins**

## Warning Comment Table“Dangerously Misleading” Bug/Drug Combinations

**MRSA, MRCNS**
**Enterococci**

**   ****all beta-lactams**
**   ****cephalosporins**
**   ****clindamycin**
**   ****trimeth-sulfa**
**   ****aminoglycosides   ****	****(ex. high conc)****	**

## “Warning” Comments Example 

**“*****Warning*****:**** For oxacillin-resistant *****S. aureus***** and coagulase-negative staphylococci (MRS), other ********-lactam agents, i. e., penicillins, ********-lactam/********-lactamase inhibitor combinations,  cephems and carbapenems, may appear active in vitro but are not effective clinically.  Results for these drugs should be reported as resistant or should not be reported. This is because most cases of documented MRS infections have responded poorly to ********-lactam therapy, or because convincing clinical data have yet to be presented that document clinical efficacy for these agents****.**

**CLSI M100-S15 Table 2C (M2, M7)**

## Staphylococcus aureus 

**	****	****cefazolin****	****	****	**** S  R* ****	****	**
**	****	****clindamycin     ****	**** R****	****	**
** ****	**** ****	****erythromycin****	****	**** R**
** ****	**** ****	****oxacillin****	****	****	**** R**
** ****	****	****penicillin****	****	****	**** R**
** ****	****	****vancomycin****	****	**** S**

## Reporting for CSF Isolates 

**	****“****Warning:**** Routine reporting of the following on any species of bacteria included in this document isolated from CSF could be dangerously misleading for patient care (i.e., the bacteria included in Tables 2A-2J)”**

**CLSI M100-S15 Table 1, 1A (M2, M7)**

## DO NOT Report on CSF

**agents administered by oral route only**
**1****st**** and 2****nd**** generation cephalosporins (ex. cefuroxime sodium)**
**clindamycin**
**macrolides**
**tetracyclines**
**fluoroquinolones**

**CLSI M100-S15 Table 1, 1A (M2, M7)**

## Not routinely reported on organisms isolated from the urinary tract...

**Azithromycin**
**Chloramphenicol**
**Clarithromycin**
**Clindamycin**
**Erythromycin **

**CLSI M100-S15 Table 1, 1A (M2, M7)**

## CLSI “Rx” Comments 

**Suggest that CLSI comment (or a modification of CLSI comment) be added to report to enhance MD understanding of appropriate therapy.**
**  **

## Rx: CommentsExample

***Rx:***** Combination therapy of penicillin or ampicillin, plus an aminoglycoside, is usually indicated for serious enterococcal infections, such as endocarditis.   **

**CLSI M100-S15 Table 1 (M2, M7)**

## E. faecalis (blood)

**	****	****ampicillin ****	****       S**
**	****	****vancomycin          S**
**	****	****gent synergy   ****	****     R**
**	****	****strep synergy ****	****     S**
**“****Serious enterococcal infections need combination therapy with ampicillin or penicillin plus an aminoglycoside.  Synergy occurs only when both drugs in the combination are “S””.**

## “S” Only Breakpoint

**	****“****For some organism/antimicrobial combinations, the absence of resistant strains precludes defining any results categories other than ****“susceptible”.****  For strains yielding results suggestive of a ****“non-susceptible”**** category, ****organism identification and antimicrobial susceptibility test results should be confirmed****.  Subsequently, the isolates should be saved and submitted to a reference laboratory that will confirm results using an CLSI reference dilution method.”**

**CLSI M100-S15 Introduction (M2, M7)**

## S. pneumoniae - Vancomycin MIC (μg/ml)

**    ****	****	****    ****	****	**_**Susc**_**     **_**Int**_**      **_**Res**_
**vancomycin****	****  ****	******** 1.0****	**** -****	****     -**
** **

*****_**investigate any non- “S” isolate**_
**..Repeat ID and AST**
**..Save isolate**
**..Send to reference lab**

## Table 2Interpretive Criteria (Breakpoints)

**Separate Table 2 for ****each organism group**** – use appropriate Table 2**
**Do not “borrow”**** breakpoints from one Table for a different organism group**
**Interpretive criteria:**
**Susceptible**
**Intermediate**
**Resistant**

## Table 2B

**Table 2B**

## Incubation Temperature Range

***Staphylococcus***** spp.**
**33-35********C (do not exceed 35********C) **
**Potential problems detecting MRSA and MR-coagulase-negative staphylococci (MRCoNS) at >35********C **
**All other organism / antimicrobials **
**35********C +/- 2********C **

**CLSI M100-S15 Table 2 (M2, M7)**

## Drugs not on T-2 List for a specific organism group....

**Do not test without questioning**
**Exmp.  *****Pseudomonas aeruginosa***
**Cefoxitin**
**Cefoxitin does not have activity against *****Pseudomonas aeruginosa***** and is not indicated for this species.**

## Developing Protocols for Testing and Reporting

- Test/Report

## When Test?

****** ****2 potential pathogens**
**Unpredictable antibiograms**

## Throat Culture 

**Many Group A Streptococcus**
**“****Group A Streptococcus remains universally susceptible to penicillin”**

## Sputum Culture 

**GS:**
**	****Many oral flora**
**	****Many WBCs**
**	****Many GNR**
**Culture:**
**	****Many Normal Flora**
**	****Many *****Klebsiella pneumoniae***

**1 potential pathogen**
**Perform AST**

## Leg Wound Culture 

**GS:**
**	****Many GPC clusters**
**	****Many pleomorphic GPR**
**Culture:**
**	****Many coag-neg staphylococci**
**	****Many diphtheroids**
**	****Few *****E. coli-like *****colonies**
**	****Few***** Proteus spp.***

## Leg Wound Culture(if MD insists!) 

**GS:**
**	****Many GPC clusters**
**	****Many pleomorphic GPR**
**Culture:**
**	****Many coag-neg staphylococci*******
**	****Many diphtheroids**
**	****Few *****E. coli**********
**	****Few***** Proteus mirabilis**********

***”Susceptibility results reported per Dr. Jones’ request.”**

## Blood Culture

** “****Coagulase-negative staphylococcus isolated from one culture only.  Susceptibility testing is not routinely done as this organism frequently represents skin contamination in blood cultures.  If testing is indicated, please contact lab within 24 h.” **

## How Often Should I Test?

**Test duplicate isolates from similar body sites after 3-4 days**
**CLSI M2-A8, M7-A6:**
**	****	****“****Resistance**** may develop in 3-4 days for: **
***	******	******	******Enterobacter, Citrobacter, Serratia***** - 3rd gen****	****	****	****  cephalosporins**
***	******	******	******P. aeruginosa***** - all drugs**
***	******	******	******Staphylococcus***** – quinolones****”**
**Sterile sites**** – daily  (JH suggestion)**

## How Often Should I Test? (con’t)

**“****In certain circumstances, ****repeat testing**** may be warranted earlier than within 3-4 days...need to know specified situation and severity of the patient’s condition.  Laboratory guidelines on when to perform repeat susceptibility testing should be determined after consultation with the medical staff.”**

**CLSI M2-A8, M7-A6**

## Repeat Schedule – Example

| Specimen obtained on Day: | 1 | 2 | 3 | 4....or 5 |
| --- | --- | --- | --- | --- |
| Complete ID | yes | * | * | yes |
| AST | yes | ** | ** | yes |

**Sputum: *****Klebsiella pneumoniae***

***  Descriptive ID (e.g. lactose+ GNR)**
**** Refer to day 1 result**

## Sputum Culture 

**GS:**
**	****Many oral flora**
**	****Many WBCs**
**	****Many GNR**
**Culture:**
**	****Many Normal Flora**
**	****Many lactose-positive GNR*******
**   ****(see ID/Sensi on culture from 5/22/03)**
***or can report... “Many *****Klebsiella-like*****”, if appropriate**

## Strategy for Handling  “Special” Requests or “Problem” Isolates

**Discuss with MD**
**Explain why not “routine”**
**Explain limited resources in-house**
**Emphasize further testing could be performed, **_**if**__** **__**necessary!**_
**Determine if MD *****really***** needs result for patient management**
**Save isolate for subsequent testing, if necessary**
**Perform supplemental testing**
**In-house (e.g. using disk diffusion, Etest, etc.)**
**Send to “reliable” reference laboratory**

## “For organisms excluded from Tables 2A through 2K (e.g., Campylobacter spp., Corynebacterium spp., Bacillus spp.) studies are not yet adequate to develop reproducible, definitive standards to interpret results. These organisms may require different media, different atmospheres of incubation, or show marked strain-to-strain variation in growth rate. For these microorganisms, consultation with an infectious disease specialist is recommended for guidance in determining the need for susceptibility testing and in the interpretation of results. Published reports in the medical literature and current consensus recommendations for therapy of uncommon microorganisms may obviate the need for testing. If necessary, a dilution method usually will be the most appropriate testing method, and this may require submitting the organism to a reference laboratory.”     Physicians should be informed of the limitations of results and advised to interpret results with caution 

**...****CLSI M100-S15 Introduction**

## “Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently-Isolated or Fastidious Bacteria; Proposed Guideline, CLSI M-45”

**Organism**
**Therapy of choice**
**Testing conditions (MIC method mostly)**
**QC**
**Breakpoints**
**Other**

**Forthcoming 2005!**

## Coryneform bacilli
Moraxella catarrhalis
Aeromonas
Vibrio
Listeria
Erysipelothrix
Campylobacter

**Moraxella catarrhalis**
**Aeromonas**
**Vibrio**
**Listeria**
**Erysipelothrix**
**Campylobacter**

**Lactobacillus, Leuconostoc, Pediococcus **
**Abiotrophia, Granulicatella**
**HACEK**
**Pasteurella**

**“****Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently-Isolated or Fastidious Bacteria; Proposed Guideline, ****CLSI M-45”**

## Corynebacterium spp.

**First Choice Drug**
**     ****penicillin****	****	**

**Alternative Drugs**
**vancomycin**
**erythromycin**
** **
**     **

**Sanford Guide, 2004.**

## Corynebacterium spp.

**Inoculum:****	****direct colony ****	****suspension**
**Incubation:**** ****	****35********C; ambient air; 24h, ****	****48h? **
**Medium:****	****CAMHB + 2.5% LHB **

## Corynebacterium spp.

**erythromycin****	****     >4   R**
**penicillin****	****     >4   R**
**vancomycin ****	****   ********0.5  S**
**	****   **
** **

**Susceptibility results are presumptive; **
**no standard test method available.**

_**MIC (**__****__**g/ml)**_

## Last pages of M2 and M7 sections of M100-S15

**Last pages of M2 and M7 sections of M100-S15**

**Summary of Comments and **
**Subcommittee Responses**

## Summary

**CLSI (formerly NCCLS)**** provides standards for routine AST and reporting in clinical laboratories**
**CLSI AST tables**** are updated annually**
**The ****“Introduction to the Tables”**** and ****footnotes**** contain important information for developing AST and reporting protocols**
**Other resources**** for routine AST include:**
**CDC’s MASTER website**
**ASM’s Askit feature**
**Other websites and publications (please see reference list accompanying this presentation)**

## Thank you!