EQAS 2008 Salmonella and Campylobacter

CRL workshop, April 23-24, 2009

Susanne Karlsmose (suska@food.dtu.dk)

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Outline of Salm/Camp EQAS 2008• AST of eight Salmonella and eight Campylobacter• New participants were supplied with original reference

strains ATCC 25922 and ATCC 33560 for QC testing• Participants’ results and comments submitted through a

secured web-based data entry program, using individual logins and passwords

• Instant individual evaluation report • Report comparing and evaluating all results• If only 75% of the results were correct (strain/antimicrobial

combination), results should be further analysed, possibly omitted

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Participation in the Salm/Camp EQAS, 2008

No contact to Luxembourg

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Issues important for differences in performanceGoal for this EQAS: that all NRL’s perform AST with less than

7% incorrect interpretations

Further investigation of a laboratory’s deviations includes looking into:

• Method• Media, disk concentrations• Reading of results• Interpretation guidelines used• Use of a QA-system (eg. QC strain)• Routine• …

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Methods and guidelines• MIC methods are recommended (For Campylobacter,

reference to M45-A: an appearance of any zone of inhibition when performing DD would require MIC determination for accurate categorisation of susceptibility)

• Interpretation guidelines for MIC results are given in the protocol

• For interpretation of zone diameters (Salmonella), the laboratory’s routine should be followed (however ‘sensitive’ and ‘resistant’ only, is accepted)

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Salmonella Campylobacter

Perc

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MIC

DD or E-test

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Comparison to former EQASs

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Salmonella Campylobacter

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EQAS 2006EQAS 2007EQAS 2008

Internal control strain

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Salmonella results – pr. lab

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Deviation % (all tested antimicrobials)

Deviation % (antimicrobials recommended by EFSA)

Acceptance limit

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0-1 >1-3 >3-5 >5-7 >7-9 >9-11 >11-13 >13-15 >15-17

Total deviation % (Salmonella )

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Salmonella results – intervals

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Salmonella – deviations on antimicrobials% correct

Ampicillin, AMP 99.5Cefotaxime, CTX 99.5Ceftazidime, CAZ 94.7

Ceftiofur, XNL 100.0Chloramphenicol, CHL 99.5

Ciprofloxacin, CIP 90.5Gentamicin, GEN 98.6Nalidixic acid, NAL 99.5Streptomycin, STR 96.8

Sulphonamides, SMX 99.5Tetracycline, TET 99.1

Trimethoprim, TMP 100.0

No CLSI guidelines for MIC-values, ARBAO-value is used

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CAZ – towards Salmonella• Results from the test strain/antimicrobial combination S3.3 and

ceftazidime were only 60% correct – Strain was ESBL-producing:

• Expected MIC for cefotaxime was >4

RESISTANT• Expected MIC for ceftazidime was <0.5

should be

interpreted as RESISTANT due to resistance towards other cephalosporins

REMEMBER to regard all cephalosporins resistant if one cephalosporin shows resistance!

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CIP – towards Salmonella

• Ciprofloxacin (9.5% deviation)– Low cut-off value is used (epidemiological cut off value is

lower than the CLSI clinical breakpoint) => when performing DD the resistance is not seen

– However, Salmonella resistant to nalidixic acid should also be interpreted as resistant to ciprofloxacin (protocol)

• Qnr-gene in S3.1 (75% correct for the S3.1/ciprofloxacin combination)

– Nalidixic acid – sensitive– Ciprofloxacin – low level resistant

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CIP – if performing DD

If you see reduced susceptibility towards ciprofloxacin– Check the nalidixic acid result

If this is resistant, ciprofloxacin should also be interpreted resistant (due to low epidemiological cut off value lower than the CLSI clinical breakpoint)

If this is sensitive, you may want to check for a qnr- gene (e.g. by PCR)

• Percentage that reported the test strain ESBL-positive

• All used combinations of CTX, CAZ and XNL were successful

=> however, some failed to regard all cephalosporins resistant if one cephalosporin showed resistance

Strain S3.1(CTX M-15 / SHV 12)

Strain S3.3(CTX M-9)

Strain S3.5(CTX M-15 like)

CTX, CAZ, XNL 6/6 (100%) 4/5 (80%) 5/6 (83%)

CTX, CAZ 11/12 (92%) 12/13 (92%) 13/13 (100%)

CTX, XNL 2/2 (100%) 2/2 (100%) 2/2 (100%)

CTX 2/2 (100%) 2/2 (100%) 2/2 (100%)

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ESBL-producing test strains – detection

• Especially the result from the confirmatory test CAZ/Cl:CAZ deviated from the expected (however, only confirmed/not confirmed is evaluated)

Strain S3.1(CTX M-15 / SHV 12)

Strain S3.3(CTX M-9)

Strain S3.5(CTX M-15 like)

CTX/Cl:CTX 22/23 (96%) 20/22 (91%) 21/21 (100%)

CAZ/Cl:CAZ 22/23 (96%) 10/18 (56%) 22/23 (96%)

Confirmed ESBL 23/24 (96%) 22/24 (92%) 23/24 (96%)

FOXS 24/24 (100%) 24/24 (100%) 24/24 (100%)

AmpC not confirmed 23/24 (96%) 24/24 (100%) 23/24 (96%)

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ESBL-producing test strains – confirmation

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S3.3 – CAZ/Cl:CAZ, confirmatory test

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Confirmation of ESBL-production – methods

6065707580859095

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Increase in zone diameter MIC ratio

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CAZ/Cl:CAZ

CTX/Cl:CTX

Confirmation ofESBL

Results uploaded to the database (proportion in accordance with the expected):

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ESBL-producing test strains – conclusionsESBL-producing microorganisms is an emerging problem, and it

should be high-priority for all NRL-AR’s to be able to detect these problem strains, therefore:

• The NRLs decided on the WS 2008 that testing for ESBL- production is be mandatory in the EQASs

Remember:• Strains resistant to one cephalosporin should be interpreted

resistant to all cephalosporins – regardless of the value obtained

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Campylobacter – deviations on antimicrobialsEQAS 2008 % correct

Chloramphenicol, CHL 100.0Ciprofloxacin, CIP 97.5Erythromycin, ERY 97.7*Gentamicin, GEN 99.0Nalidixic acid, NAL 96.4Streptomycin, STR 98.4Tetracycline, TET 96.4

Originally 92.7%, however, some results were omitted

Majority of deviations are from disk diffusion tests

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Omitted results, Campylobacter• C3.6/erythromycin (72% correct)

– 6/25 labs obtained an MIC of 1 µg/mL or below => S– 1/25 labs obtained an interpretation as S by DD– Expected result was >32 µg/mL => R

Results for this strain/antimicrobial combination omitted

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Campylobacter results – pr. lab

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Deviation % (antimicrobials recommended by EFSA)

Deviation % (all tested antimicrobials)

Acceptance limit

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0-1

>1-3

>3-5

>5-7

>7-9

>9-11

>11-13

>13-15

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>17-19

>19-21

>21-23

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>25-27

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Total deviation % (Campylobacter )

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Campylobacter results – intervals

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Methods, Campylobacter

• Results obtained by disk diffusion on Campylobacter will not be included in future EQASs

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QC strains – Salmonella, MICEQAS 2008 MIC determination E. coli ATCC 25922

Proportion of labs outside QC range

Obtained values in MIC steps (min/max)Antimicrobial Below lower QC limit Above upper QC limit

Ampicillin, AMP 0/20 (0%) - -Cefotaxime, CTX 0/21 (0%) - -Cefoxitin, FOX 0/1 (0%) - -Ceftazidime, CAZ 0/16 (0%) - -Ceftiofur, XNL 0/3 (0%) - -Chloramphenicol, CHL 0/20 (0%) - -Ciprofloxacin, CIP 3/21 (14%) - 1 stepGentamicin, GEN 0/21 (0%) - -Nalidixic acid, NAL 0/21 (0%) - -Streptomycin, STR 0/21 (0%) - -Sulphonamides, SMX 0/14 (0%) - -Tetracycline, TET 0/21 (0%) - -Trimethoprim, TMP 0/21 (0%) - -

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QC strains – Salmonella, disc diffusionEQAS 2008 Disk diffusion E. coli ATCC 25922

Proportion of labs outside QC range

Obtained values in mm zones (min/max)Antimicrobial Below lower QC limit Above upper QC limit

Ampicillin, AMP 0/6 (0%) - -Cefotaxime, CTX 0/4 (0%) - -Cefoxitin, FOX 0/5 (0%) - -Ceftazidime, CAZ 0/4 (0%) - -Ceftiofur, XNL 0/3 (0%) - -Chloramphenicol, CHL 0/6 (0%) - -Ciprofloxacin, CIP 0/6 (0%) - -Gentamicin, GEN 0/6 (0%) - -Imipenem, IMI 0/4 (0%) - -Nalidixic acid, NAL 0/6 (0%) - -Streptomycin, STR 0/6 (0%) - -Sulphonamides, SMX 1/3 (33%) 1 -Tetracycline, TET 0/6 (0%) - -Trimethoprim, TMP 0/5 (0%) - -

• All labs performing MIC on Campy, uploaded QC-strain data

EQAS 2008 MIC determination C. jejuni ATCC 33560

Proportion of labs outside QC range

Obtained values in MIC steps (min/max)

Antimicrobial Below lower QC limit Above upper QC limit

Chloramphenicol, CHL 1/16 (6%) 1 step -

Ciprofloxacin, CIP 1/22 (4%) - 1 step

Erythromycin, ERY 4/22 (18%) 2 steps 1 step

Gentamicin, GEN 1/20 (5%) 1 step -

Nalidixic acid, NAL 3/20 (15%) 3 steps -

Tetracycline, TET 3/20 (15%) 1 step 2 steps

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QC strains – Campylobacter, MIC

• The proportion within the QC intervals in 2007: 83.8%• The proportion within the QC intervals in 2008: 89.2%• Two laboratories each had four of the 13 deviations

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Summing up• 27 labs participated in the Salmonella trial – 24 labs

performed with deviation levels below the acceptance level, of which 17 labs had no deviations

• 26 labs participated in the Campylobacter trial – 25 labs performed with deviation levels below the acceptance level, of which 16 labs had no deviations

The lab with a high deviation percent (Campy)• Has received training (March 2009)

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Salmonella, new goal?

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Acceptance limit

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Deviation % (all tested antimicrobials)

Deviation % (antimicrobials recommended by EFSA)

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Salmonella – cip and ceph deviations omitted

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Campylobacter, new goal?

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Deviation % (antimicrobials recommended by EFSA)

Deviation % (all tested antimicrobials)

Acceptance limit

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Deviation % (antimicrobials recommended by EFSA)

Deviation % (all tested antimicrobials)

Acceptance limit

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Campylobacter – DD-results omitted

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Thanks for your attention!

(Additional) questions and/or items for discussion?