# Presentation: 608281

## Antimicrobial Susceptibility Testing (AST): Current Issues & Implications for Public Health

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

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

**Discuss ****current issues**** in antimicrobial susceptibility testing and antimicrobial resistance.**
**List current ****sources for information ****on antimicrobial susceptibility testing.**
**Describe the ****effects**** of these issues on ****public health.**

## Current Issues in AST  

**Identify ****methods**** to detect and report “emerging resistance”**
**Reliable**
**Practical for clinical laboratories**
**Communicate**** AST and reporting issues to clinical labs **
**Assess performance**** of clinical labs in detecting “emerging resistance”**

## Communicate AST & Reporting Issues to Clinical Labs  

**Primary resources**** for continuing education**
**Workshops / seminars**
**Internet **
**Publications**
**CDC / NLTN**** efforts**
**MASTER website (****www.phppo.cdc.gov/dls/master/default.asp)**
**AST CD ROM (available late 2002)**
**NLTN workshops, teleconferences, other**

## Detect / Report 

**Detect / Report **
**Emerging Resistance**

**Clinical Lab**

**Public Health Lab**
**(Define role)**

## Public Health Lab as A Resource for Clinical Labs in AST & Reporting

**Performs ****AST**** (confirms unusual results) **
**Answers ****questions**
**Identifies other resources, when needed**
**Provides ****news**** “bulletins”**
**Provides ****CE**

**Much Variability Among PH Labs!**

## Clinical Lab Need

**  ****Knowledge of “resources” for AST & reporting in their community ....particularly when a ****“real problem”**** arises.**** **

## Assess Performance of Clinical Labs in Detecting Emerging Resistance

**Inspections**
**Proficiency surveys**
**Post CE monitoring**
**(Competency assessment)**

## Contemporary Resistance Concerns

***Staphylococcus aureus***
**MRSA – new tests**
**VISA**
**VRSA**
***Streptococcus pneumoniae***
**Reporting using new breakpoints**

## Contemporary Resistance Concerns (con’t)

***Streptococcus *****spp*****.***
**Beta *****Streptococcus***** Group B (in pregnancy) and clindamycin / erythromycin**
***Enterobacteriaceae  ***
**Extended-spectrum beta-lactamases (ESBLs)**

## NCCLS Standards - 2002

**M2-A7     Disk Diffusion**
**M7-A5     MIC**
**M100-S12 Tables*******

***Updated annually**
- _[www.nccls.org](http://www.nccls.org/)__ (610) 688-0100_

## Staphylococcus spp. 

**P’ase stable     ****	****penicillin (e.g., oxacillin)**
**vancomycin +/- gentamicin +/- rifampin**

**a cephalosporin, vancomycin, ß-lac/ß-lac inhibitor combo, imipenem or meropenem, clindamycin, a fluoroquinolone**
**linezolid, quin-dalfo, a fluoroquinolone, a tetracycline, trim-sulfa**

**The Medical Letter; 2001; 43:69.**

**MSS**

**MRS**

**Alternative Drugs**

**1st Choice Drugs**

**Organism**

## MRSA	 (ORSA)

**Genetic determinant – *****mecA***
***MecA***** codes for ****PBP2a**

**MRSA**

## MRSA Test Methods

**NCCLS disk diffusion or MIC**
**Commercial method **
**Agar screen**
***mecA***** assay **
**PBP2a assay**

## MRSA

**“****Isolates of staphylococci that are shown to carry the *****mecA***** gene, or that produce ****PBP2a****, the gene product, should be reported as oxacillin resistant.”**

**NCCLS M100-S12**

## PBP2a

## Can mecA or PBP2a Assays be Used Alone?

**MRSA?**
**MSSA?**
**Are these tests consistently reliable “on the bench”?**
**Are results for other drugs routinely needed? **

## Staphylococcus - ß-Lactams

**Pen****	****Ox****	****               Comments**
**  ****S****	**** ****S****	****S**** to penicillins, cephems, ****	****	****	****	****carbapenems**
**  ****R****	**** ****S****	****R**** to ß-lactamase labile pens; ****  ****	****	****	****S**** to ß-lactamase stable pens; ****	****	****	****S****  to ß-lac / ß-lac inhibitor ****	****	****	****	****combos, cephems, carbapenems**
**  ****R****	**** ****R****	****R**** to all ß-lactams **

**NCCLS M100-S12**

## Staphylococcus aureus 

**  **
** ****	****	**** ****clindamycin       ****	**** S****	****	**
** ****	****	**** ****erythromycin****	****   ****	**** S**
**  ****	****	**** ****oxacillin****	****	****   ****	**** S**
** ****	**** ****	**** ****penicillin****	****	****   ****	**** R**
** ****	****	**** ****vancomycin****	****   ****	**** S**
**“****Cefazolin and other beta-lactams (except amoxicillin, ampicillin, and penicillins) are active against oxacillin-S and penicillin-R staphylococci.”**

## VISA* (GISA**)

**Mostly MRSA **
**Vanco MIC=8 **μ**g/ml**
**Japan, USA (MI, NY, NJ, CA), Europe**
**Patients previously Rx with vanco**

*** vancomycin-intermediate *****S. aureus***
****glycopeptide-intermediate *****S. aureus***
** ****(vancomycin is a glycopeptide)**

## Detection of VISA

**MicroScan ON****	****   8**
**Etest****	**** 6-8**
**Sensititre****	**** 4,8**
**Vitek****	****   4**
**BHI-Van (6 **μ**g/ml)             growth**
**MicroScan rapid              ********2, ******** 16**
**Disk diffusion               inadequate**
** **

**Tenover, et al. 1998. JCM. 36:1020**

**MIC ****(μg/ml)**

## Case Study VISA - Pt. JB (UCLA)

**27 y.o. referral patient **
**liver transplant candidate**
**Bile drainage (liver abscesses)**
**2 strains VISA**

**Marlowe et al. 2001. JCM. 39:2637**

##    Case Study - VISA  Pt. JB

**                               ****MIC (********g/ml) ****	**** **
**Strain  ****   *****mecA*****     OX      ****Van ****    **
**     ****1****	****         ****- ****           0.5      ****8****           **
**     ****2****	****         ****+****          >16      ****8****           ****         **
** **

## 48h BAP

**48h BAP**
**VISA - Pt. JB**

**VISA**

**MRSA**

##  Confirmation of VISA

**If ****VISA**** suspected:**
**Repeat ID and susceptibility tests  **
**Contact your institution’s infection control department**
**Contact CDC at **_**[SEARCH@cdc.gov](mailto:SEARCH@cdc.gov)**_
**Contact your local health department**
**Save isolate**

## VRSA – 1st Case 

**Michigan **
**40 y.o., diabetic on dialysis**
**Previous catheter site with MRSA**
**Previous vancomycin Rx (6.5 wks)**

**MMWR 51:565-567, July 5, 2002**

## VRSA – 1st Case (con’t) 

**VRSA with***** mecA***** and *****vanA***** genes**
**Patient also had VRE (*****vanA*****)**
**Vancomycin MIC =1024 ****μ****g/ml**
**S to chloramphenicol, linezolid, minocycline, quin-dalfo, tetracycline, TMP-SMZ**

**MMWR 51:565-567, July 5, 2002**

## VRSA – 2nd Case 

**Pennsylvania **
**Chronic foot ulcer**
**VRSA with***** mecA***** and *****vanA***** genes**
**Vancomycin MIC = 32 ********g/ml**
**DD zone = 12 mm**
**Van (6 ********g/ml) BHI screen = growth**
**S to chloramphenicol, linezolid, minocycline, quin-dalfo, rifampin, TMP-SMZ **

**MMWR  51:902. Oct. 11, 2002**

## Streptococcus pneumoniae

**penicillin G or V;**
**amoxicillin  **

**If pen **** 0.06 ****μg/ml**** ****(S)...**

**a cephalosporin; erythromycin; clarithromycin; azithromycin; levofloxacin, gatifloxacin, or moxifloxacin; meropenem; imipenem; trim-sulfa; clindamycin; a tetracycline**

**The Medical Letter; 2001; 43:69.**

**1st Choice Drugs**

**Alternative Drugs**

## Streptococcus pneumoniae

**penicillin G IV or **
**   ****ceftriaxone or cefotaxime****	****  **

**If pen 0.12 - 1 ****μg/ml**** ****(I)...**

**levofloxacin, gatifloxacin, or moxifloxacin; vancomycin**
**clindamycin**

**The Medical Letter; 2001; 43:69.**

**1st Choice Drugs**

**Alternative Drugs**

## Streptococcus pneumoniae

**Meningitis:**
**  ****vancomycin + **
**    ****cefotaxime or ceftriaxone +/- rifampin**
**Other:**
**  ****as above or levofloxacin, gatifloxacin, or moxifloxacin  **

**If pen ******** 2 ****μg/ml**** ****(R)...**

**meropenem; imipenem         **
**quinupristin/dalfopristin         ****	****linezolid**

**The Medical Letter; 2001; 43:69.**

**1st Choice Drugs**

**Alternative Drugs**

## Streptococcus pneumoniaeBreakpoints

**Ceftriaxone and Cefotaxime**
**Originally based on treatment of ****meningitis****  **
**NCCLS saw need for breakpoints for treatment of respiratory infections**
**New non-meningitis breakpoints**
**Penicillin**
**Originally based on treatment of ****meningitis**
**No change in breakpoints**

## Streptococcus pneumoniae

**	****	****	****	****	****	**
**	****	****	****	****	****             ****S****	****     Int         R**
**Ceftriaxone or cefotaxime**
**	****(meningitis)****	****	****          ******** 0.5****	****        1         ******** 2**
**Ceftriaxone or cefotaxime**
**	****(non-meningitis) ****	****	****  ******** 1****	****        2         ******** 4**
**Penicillin                              ******** 0.06    0.12-1     ******** 2****	****	****	**
**	****	****	****	****	****	**

**MIC (μg/ml)**

**NCCLS M100-S12**

## Streptococcus pneumoniae Penicillin

** “****High doses of IV penicillin (e.g. at least 2 million units every 4 hours in adults with normal renal function ) or similarly ampicillin (e.g. 2 g at every 6 hours) are effective in treating pneumococcal pneumonia due to strains in the ****intermediate**** category.”**

**NCCLS M100-S12**

## S. pneumoniae (CSF)

**	****	**
**	****	**
**	****	****ceftriaxone ****	****	**
**	****	****    ****(meningitis)****	**** ********0.25 S **
**	****	****penicillin****	****	**** ********0.03 S**
**	****	****vancomycin ****	****	****    0.5 S**
**Patients with meningitis require therapy with maximum doses of ceftriaxone.**

_**MIC (μg/ml)**_** **

## S. pneumoniae (blood)

**	****	**
**	****	****ceftriaxone ****	****	**
**	****	****  ****(meningitis)****	****	****     1 I**
**	****	****ceftriaxone****	****  **
**	****	****  ****(non-meningitis)           1 S**
**	****	****erythro, trim-sulfa****	****      R****	**** **
**	****	****levofloxacin ****	****	****  0.5 S**
**	****	****penicillin****	****	****	****   1.0 I**
**	****	****vancomycin ****	****	****  0.5 S**
**Pts. with meningitis require therapy with max doses of ceftriax; High dose IV pens (e.g. at least 2 mil U every 4 h in adults with normal renal function) or amp (e.g. 2 g at every 6 h) are effective in treating pneumococcal pneumonia due to strains in the penicillin “int” category.**

_**MIC (μg/ml)**_** **

## Beta Streptococcus spp. 

**Beta Group       penicillin G or V     clindamycin; erythromycin; **
** ****A, C, G****	****	****         a cephalosporin; vancomycin; ****	****	****         clarithromycin; azithromycin**
**Group B            penicillin G or         a cephalosporin; vancomycin;  ****	****        ampicillin                 erythromycin**
**                       **

**The Medical Letter; 2001; 43:69.**

**1st Choice Drugs**

**Alternative Drugs**

## Throat Culture 

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

## Beta Streptococcus spp.Erythromycin/Clindamycin

**Erythromycin and/or clindamycin are alternatives in penicillin-allergic patients**
**Group A**** - 5-10% erythromycin-R**
**Group B**** - up to 25% erythromycin-R and 15% clindamycin-R (prophylaxis in pregnant women issue)**

**1Schrag et al. 2002. ****MMWR Recomm Rep. 51:1-22.**** **

## Extended-Spectrum ß-lactamases (ESBLs)

**Result from mutation of common ****ß****-lactamase genes (e.g., *****bla*****TEM-1****, *****bla*****SHV-1**** )**
**Inactivate extended-spectrum ****ß****-lactam agents**
**Genes located on plasmids (often with other ****R**** genes)**
**Approx. 100 TEM and 30 SHV types**

**Bradford, P. 2001. CMR. 14:933.**

## Significance of ESBLs

**Cause nosocomial infections**
**Serious, infections. ****Treatment failures**** (esp. bacteremia) when patient treated with 3rd gen. cephalosporins.**

## ESBLs – Testing 	

**Organisms:*****	******	******	******	******	******	******	******        E. coli, Klebsiella***** spp.**
**Screen test:****	****	****	****	****	****	**** Decreased susceptibility to extended-spectrum ß-lactams**
**Phenotypic confirmatory test:****	****	****	****       ß-lactam activity restored by ß-lactamase inhibitor (e.g. clavulanic acid)**
**Other clues:**** ****	****	****	****	****	****                       “S” to cephamycins; multi-R**

**NCCLS M100-S12**

## Klebsiella pneumoniae   ?ESBL

**cefoxitin****	****        1 S **
**ciprofloxacin  ****	****     0.5 S**
**imipenem              ******** 0.25 S**
**pip-tazo    ****	****	****   8 S**
**am, cfaz, gm, T-S           R**
**    *******hold cefepime, cefotaxime, if ****“S”**

_**MIC (μg/ml)**_

**This *****K. pneumoniae***** is suspicious for extended-spectrum beta-lactamase (ESBL) production; confirmatory tests pending.**

**PRELIM:**

- Fresno 11/00

## ESBL Phenotypic Confirmatory Test 

**Test:****	**
**cefotaxime**
**cefotaxime/clavulanic acid**
**ceftazidime**
**ceftazidime/clavulanic acid  **
**Results:**
**clavulanic acid restores activity of cefotaxime or ceftazidime or both**

**NCCLS M100-S12**

## ESBL Confirmatory Test

**ESBL Confirmatory Test**

**Ceftaz/CA**

**Cefotax/CA**

**Ceftaz**

**Cefotax**

## ESBL Reporting 

**Confirmed ESBLs**** - report “R” for:**** **
**cephalosporins (**_**not**_** cephamycins)**
**penicillins (**_**not**_** ß-lac inhibitor combos)**
**aztreonam****  **

**NCCLS M100-S12**

## Klebsiella pneumoniae   ESBL

**cefepime****	****	****  2 S R**
**cefoxitin****	****           1 S**
**cefotaxime****	****	****  1 S R **
**ciprofloxacin****	****         0.5 S**
**imipenem****	****       0.25 S**
**pip-tazo****	****	****      8 S****	**
**am, cefaz, gent, T-S                R   **
** **

_**MIC (μg/ml)**_

**Confirmatory tests for this *****K. pneumoniae***** indicate unusual resistance [extended-spectrum beta-lactamase (ESBL) production]; ID consult suggested.**

**FINAL:**

## Issues - ESBLs

**Reporting ESBLs from urine isolates (e.g. acute cystitis)**
**Changing results for ****other ********-lactams**** (e.g. pip-tazobactam)**
**Detection of.....**
**all types of ESBLs (********100 TEM and 30 SHV)**
**ESBLs present with other “R” mechanisms**
**ESBLs beyond *****E. coli***** and *****Klebsiella***** spp.**
**other ********-lactamases (e.g. plasmid-mediated ampC)**

## NCCLS M39-A Guideline

**“****Analysis and Presentation of Cumulative Antimicrobial Susceptibility Test Data”**
**Focus - clinical laboratories**
**Guide MDs on ****empiric**** therapy**

## % Susceptible 2001 – Exmp.

**	****	****	****   ****n****     ****Am   Cf  Ctx   Cip  Gm   Pp  T-S**
** *****E. coli*****          *****729*****    66    81    98****	**** 97****	****97    66****	****76**
** *****E. cloacae*****   *****144*****      -      -      66****	**** 95****	****88****	****65****	****88**
** *****P. aerug*****       *****221*****      -      -****	**** 10****	**** 79****	****81****	****76****	**** -****	**

## CAP Checklist MIC.21950 (2001) 

**Does the procedure manual address unusual or ****inconsistent antimicrobial susceptibility testing results****?**
**Testing QC strains doesn’t guarantee accurate patient results**
**Unusual or inconsistent results should be investigated**

## Verification Rules - Exmps. Biologically implausible or infrequent  

**Repeat test**
**amik-R + gent-R + tob-R Enterobacteriaceae**
**amp-R *****E. faecalis***
**imipenem-R Enterobacteriaceae**
**Reexamine or repeat test**
**amp-S *****Klebsiella***** spp.**

**NCCLS M39-A*****      ***

## Enterobacter aerogenes 

**	****	****ampicillin****	****R**
**	****	****cefazolin****	****R**
**	****	****ceftizoxime****	****S**
**	****	****gentamicin****	****S**
**	****	****imipenem****	****R***
**	****	****trimeth-sulfa****	****S**
**  **

*** ”R” likely due to drug deterioration**

## Conclusions

**Clinical laboratory detection of emerging resistance**
**Labs must have access to current testing / reporting guidelines**
**Lab personnel must be competent in testing**
**Resources for “problems” in emerging resistance must be available to labs**
***Staphylococcus aureus***
***mecA***** or PBP2a tests can be used to identify MRSA **
**VISA are uncommon and may be difficult to detect**
**VRSA are rare and can be detected readily; however the resistance may be unstable**

## Conclusions (con’t)

***Streptococcus pneumoniae***
**The new NCCLS breakpoints for ceftriaxone and cefotaxime should be reported**
**MICs should be performed on CSF isolates ASAP**
**A comment should be added to explain the meaning of a penicillin “Int” result on non-CSF isolates**
***Streptococcus *****spp*****.***
**Group B streptococci from penicillin-allergic pregnant women should be tested with clindamycin and/or erythromycin**

## Conclusions (con’t)

***Enterobacteriaceae ***
***E. coli***** and *****Klebsiella***** spp. should be tested for ESBL production **
**Urine isolates associated with acute uncomplicated cystitis may not require ESBL testing **
**There are currently no standard NCCLS methods for detecting ESBLs in isolates other than *****E. coli***** and *****Klebsiella***** spp.**
**Local resistance patterns**
**When possible, NCCLS M39-A should be followed for preparing a cumulative antibiogram report**

## Conclusions (con’t)

**Verifying results on patient isolates**
**Each laboratory should develop a written protocol for verification of unusual or atypical patient results prior to reporting them**
**Resources**
**a listing of**** ****antimicrobial susceptibility testing resources can be found on MASTER website**

## Additional References

**Livermore, D. M., T. G. Winstanley, and K. P. Shannon. 2001. Interpretative reading: recognizing the unusual and inferring resistance mechanisms from resistance phenotypes. J. Antimicrob. Chemother. 48:87-102.**
**Websites:**
**http://www.cdc.gov/drugresistance/factsheets/index.htm**
**CDC antimicrobial susceptibility testing fact sheets**
** **
- _[http://](http://www.cdc.gov/drugresistance/)__[www.cdc.gov/drugresistance](http://www.cdc.gov/drugresistance/)__[/](http://www.cdc.gov/drugresistance/)_
**CDC drug resistance**
** **
**http://www.cdc.gov/drugresistance/community/**
**CDC antibiotic resistance **
** **
- _[http://](http://www.asmusa.org/division)__[www.asmusa.org/division/c/index.htm](http://www.asmusa.org/division)_
**ASM Division C (clinical microbiology) website includes “Askit” feature**

## Thank you!

**www.phppo.cdc.gov/dls/master/default.asp**