R O M N E Y H U M P H R I E S

S E P T E M B E R 2 0 1 4

QUALITY CONTROL FOR AST

ENSURING QUALITY OF AST RESULTS

AST QC

Good media / reagent quality

Equipment properly serviced

Staff are competent

Review patient results

RESPONSIBILITIES FOR AST QC

AST Manufacturer

• Antimicrobial is stable

• Antimicrobial labeled

properly

• Antimicrobial potency

is correct

• Complies with GMP

Laboratory

• Reagents stored

appropriately

• Staff are trained &

competent

• Protocols are followed

STANDARD QC STRAINS

WHICH QC STRAIN DO I TEST?

IF performing CLSI reference testing

(MIC or DD).

IF using a diagnostic manufacturer –

follow their QC recommendations!

M100 S24 APPENDIX C

Use for βlactam/βlactamase inhibitor combinations

Use for all antimicrobials, unless otherwise stated

WHY DO I HAVE TO TEST PSA 27853 VS CARBAPENEMS?

Agent E. coli

ATCC 25922

P. aeruginosa ATCC 27853

Doripenem 0.015–0.06 0.12–0.5

Ertapenem 0.004–0.015 2–8

Imipenem 0.06–0.25 1–4

Meropenem

0.008–0.06 0.25–1

Goal is to get “on scale” endpoints for carbapenems 25922 MICs

are too low (will always test “≤”

OTHER RECENT CHANGES TO “ROUTINE” QC

Table Organism Group Routine QC Change For use ONLY to QC:

2A Enterobacteriaceae Added P. aeruginosa ATCC 27853

carbapenems

2B-1 Pseudomonas

aeruginosa

Deleted E. coli ATCC 25922 NA

2B-2 Acinetobacter spp. E. coli ATCC 25922 tetracyclines trimethoprim-sulfa

2B-3 Burkholderia cepacia E. coli ATCC 25922

chloramphenicol minocycline

trimethoprim-sulfa

2B-4 Stenotrophomonas maltophilia

E. coli ATCC 25922 chloramphenicol minocycline trimethoprim-sulfa

2B-5 Other Non-

Enterobacteriaceae

E. coli ATCC 25922

chloramphenicol

tetracyclines sulfonamides trimethoprim-sulfa

WHAT ARE THOSE “SUPPLEMENTAL” QC STRAINS FOR?

• “Routine” QC Strain

• Test regularly (daily or weekly)

• “Supplemental” QC strain

• May have “S” or “R” characteristic specific for one or more

“special” AST tests

• EXAMPLE: ATCC BAA-977 has includible clindamycin resistance

• Use to assess new test

• Use for training purposes

• Use for competency

• DO NOT need to perform regularly (weekly or daily)

HOW OFTEN SHOULD AST QC BE DONE?

• Daily OR

• On days when patient isolates are

tested OR

• Weekly (after alternate QC plan

implemented)

• 20 – or 30 – day plan

• New, alternative 3 x 5 plan

REMINDER: 20- 30- DAY PLAN

• Perform testing for 20 or 30 consecutive test days,

and document results

• Convert to weekly QC if:

• No more than 1 out of 20 OR

• No more than 3 out of 30 MICs for each antimicrobial

agent/organism combination are outside acceptable MIC

limits

• THEN:

• Perform weekly QC testing AND

• Whenever any reagent component of the test is changed

(new lot / new shipment)

3 X 5 PLAN

• Perform 3 tests / day for 5 days

• 3 separate inocula for each replicate

• If results unacceptable, test another 5 days

• Advantages:

• Possible to identify issues sooner

• Takes less time

• Takes fewer resources

• Statistically comparable to 20-30 dya plan

• Accepted by CAP

• Pending CLIA

Test 3 replicates of each QC strain for 5

days (different inoculum)

0-1 of 15 out of

range? 4 or more of 15 out

of range?

Test another 3 x 5

days

2-3 of 30

out of

range?

PASS. Convert

to weekly QC

2-3 of 15 out of

range?

FAIL.

Continue daily QC

M100-S24.

Yes Yes

Yes No

SOURCES OF IDENTIFIABLE ERROR

QC strain

• Wrong QC strain used

• Improper storage

• Inadequate maintenance

(same F2 for >1 month)

• Contamination

• Nonviable

• Changes (loss of plasmid)

Supplies

• Improper storage

• Contamination

• Damages plates / panels

• expired

Process: not following SOP, transcription error.

Equipment: not functioning properly

WHAT IF MY QC ISN’T DUE TO AN IDENTIFIABLE ERROR?

• QC ranges are established to include >=95% of

results from routine testing

• Some errors are random!

• Perform “corrective Action”:

• Perform QC as soon as possible

• **new** If 5 acceptable QC results are available, no

additional days of QC testing are needed.

• Old: had to do 5 days of QC

Weekly QC

Out-of-range (error not identifiable)

Retest (same day)

Result in range?

≥5 recent results for

same lot in range?

Retest result out-of-

range?

Yes, test daily until

5 results available

Any result

out of

range?

PASS. Resume

weekly QC

testing

Result in range?

<5 recent results for

same lot in range?

FAIL.

Corrective Action

M100-S24.

See Q&A section

Yes Yes

No Yes

EXAMPLES

Week Day Result Action

1 1 4

2 1 8

3 1 8

4 1 4

5 1 16 Out of QC, repeat

next day

5 2 8 In range. 5

acceptable QC tests

Week Day Result Action

1 1 4

2 1 8

3 1 16 Out of QC, repeat

next day

3 2 4 In range: 3

acceptable results

(need 2 more)

3 3 8 In range

3 4 8 In range. 5

acceptable QC tests

E. Coli ATCC 25922 and ampicillin

Acceptable Range: 2-8 μg/ml

Lot: 9661

Scenario 1 Scenario 2

M100 S24 (page 216 Q&A)

UH-OH, MORE RESULTS OUT OF QC!

• Probable system error

• Daily QC must be continued until final resolution of

problem!

• Ideas on how to fix this:

• New QC strain

• New lots of materials (incl. new turbidity standards)

• If problem appears to be related to manufacturer, contact them!

• Might need to use another test method

WHAT ABOUT MY PATIENT RESULTS?

• Don’t forget to carefully review each patient result

• Approaches will differ, based on degree and

direction of QC errors

• Things to consider:

• Suppress result for antimicrobial out of QC

• Review individual patient / cumulative data for unusual

patterns (should be doing this anyway)

• Use an alternative method if necessary

EXAMPLE 1: DISK DIFFUSION QC WEEKLY RESULTS

QC Strain Drug Acceptable

Range Result Comment

P. aeruginosa

ATCC 27853

gentamicin 16-21 14 Reason for error; disk not flat on agar surface

tobramycin 19-25 23

E. coli

ATCC 25922

gentamicin 19-26 22

tobramycin 18-26 22

Identifiable error = disk not placed correctly Corrective action: Repeat QC test; OK to report patient results

EXAMPLE 2: DISK DIFFUSION QC

WEEKLY RESULTS

QC Strain Drug Acceptable

Range Result Comment

P. aeruginosa

ATCC 27853

gentamicin 16-21 12 Reason for error not identifiable but used new lot of media (lot A)

tobramycin 19-25 15 Reason for error not identifiable but used new lot of media (lot A)

E. coli

ATCC 25922

gentamicin 19-26 22

tobramycin 18-26 22

Error not Identifiable (possibly media??)

Note: lab should QC new lot of media before using for

patient isolates

SUGGESTED CORRECTIVE ACTION

EXAMPLE 2

• Test new lot of media (lot B) • Suspected problem is lot A media

• Probably contains too high concentration of cations (calcium, magnesium)

• Decreased activity for aminoglycosides with P. aeruginosa

• Elevated cations do not affect aminoglycosides with Enterobacteriaceae

• E. coli ATCC 25922 QC in control

CORRECTIVE ACTION DOCUMENTATION FOR

EXAMPLE 2

P. AERUGINOSA ATCC 27853

GENTAMICIN ZONE = 12 MM

ACCEPTABLE RANGE = 16-21 MM

Date/ Initial

Antimicrobial

Agent Result

Describe problem

Corrective Action taken to resolve

problem

Patient Results affected (yes/no)

Comments

8/8/08

RH

Gentamicin

12 mm

Mueller Hinton agar (lot A) showing small zone with gentamicin and P. aeruginosa; E. coli QC with gentamicin OK.

Obtain new lot of media (Lot B). Repeat QC with Lot A and Lot B. Lot A 12 mm

Lot B 18 mm

No Lot A media probably too high in calcium or magnesium content. Discard Lot A.

Disk Diffusion QC

Troubleshooting

Guide

WHEN DOES 1 VS. 5 VS. NEW AQP REQUIRED?

M100

S24,

Table 5F

CLSI M02-A11.

CLSI M07-A9.

MAINTAINING QUALITY CONTROL STRAINS

• Rehydrate new stock culture or obtain strain from

frozen

• Subculture to appropriate media (F1) store at 2-

8C (as appropriate)

• Subculture to approprirate media (F2) store at 2-

8C (as appropriate)

• Use this for QC on day 1 and to make F3

• Every 7 days, prepare a new F2 from F1

• Every 4 weeks, discard F1 and start again

MAINTAINING QUALITY CONTROL STRAINS (2)

E. coli ATCC 35218; K. pneumoniae ATCC 700603

• Beta-lactamase producers

• Beta-lactamase is plasmid-mediated

• Store at -60C or lower to prevent loss of plasmid; if plasmid is lost, strain would appear susceptible to beta-lactams

DON’T FORGET, TESTING QC

STRAINS IS JUST PART OF QC!

• Reviewing results of patient’s isolates:

• Testing routine QC strains doesn’t ensure every result on a patient’s isolate is accurate

• Patient results may be erroneous due to:

• mixed culture, misidentification

• individual drug/bug problem

• other technical error

EXAMPLE

• S. agalactiae isolated from blood and wound

cultures from an 82 year old woman with diabetes

and right ankle pain / swelling

Penicillin 0.06 S

Cefotaxime 0.12 S

Clindamycin >32 R

Erythromycin >32 R

Vancomycin 4 NS

What should we review?

Do ID and AST results correlate?

Were appropriate drugs reported?

Are results from similar drugs (class) OK?

Were appropriate comments added?

APPENDIX A M100 S24

SUGGESTED STEPS TO CONFIRM

ID AND AST RESULTS:

1.Check for transcription errors, contamination, or defective panel, plate, or card.

2.Check previous reports on the patient to determine if the isolate was encountered and confirmed earlier.

3.Repeat organism ID and AST with initial method to ensure they reproduce.

4.Confirm organism ID with second method performed in-house or at a referral laboratory.

5.Confirm AST results with second method (eg, in-house or referral laboratory). The second method might be:

A CLSI reference method (eg, broth microdilution, agar dilution, or disk diffusion) or

An FDA-cleared commercial test.

EXAMPLE: CONFIRM VANCOMYCIN- RESISTANT S. AGALACTIAE

ID AST Results

Initial tests Automated method

Automated method

Vancomycin 4 µg/ml

ID: Streptococcus agalactiae

Repeat tests Automated method

Etest

w or w/o

Automated method

Etest:

Vancomycin 4 µg/ml

Automated:

Vancomycin 4 µg/ml

ID: Streptococcus agalactiae

Repeat tests Conventional biochemicals and/or 16S sequencing

None S. agalactiae

Forward to local or state public health laboratory CDC

ADDITIONAL RESOURCES…

SHOULD WE EDIT ANY “S” OR “I” TO “R” BASED ON INTRINSIC RESISTANCE PROFILE FOR AN ISOLATE?

Any editing rule is based on ID and AST results being accurate!!! So, we must consider….

• Competency of personnel doing testing

• Testing pure cultures – use purity plates for broth MIC tests

• Reliability of organism ID

• Reliability of AST results

• Impact to patient care

• Situations where it would be wise to confirm results

CLSI “S” TO “R” EDITING RULES

Organisms Criteria Edit any “S” to

”R”

Staphylococcus

spp.

Oxacillin “R” All ß-lactams

except ceftaroline

Staphylococcus

spp.

Beta streptococci

Inducible

clindamycin

resistance

Clindamycin

UCLA ADDITIONAL “S” TO “R”

EDITING RULES (BASED ON INTRINSIC R)

Organisms Edit “S” to ”R”

Citrobacter braakii/ freundii

Enterobacter aerogenes

Enterobacter cloacae

Hafnia alvei

Morganella morganii

Proteus vulgaris

Providencia rettgeri / stuartii

Serratia marcescens

ampicillin

ampicillin-sulbactam

cefazolin

Klebsiella oxytoca

Klebsiella pneumoniae

ampicillin