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U.S. Department of Health and Human Services

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XVI. Appendix to Class II Special Controls Guidance Document: Antimicrobial Susceptibility Test (AST) Systems

  TABLE 1: Recommendations for Antimicrobial Susceptibility Devicesa 

 

MIC/BP
Formats

  Fastidiousb

Additional methods of
Inoculation/Readingc

Number of Sites (including 1 in-house)

3

3

3

OrganismsFreshd Clinical/stocke

100/site

75/site

0

CDC Challengef

75/one site

50/one site

75 or 50/one site

Reproducibilityg

25/site or 10x3x3/site

25/site or 10x3x3/site

25/site or 10x3x3/site

Interpretive Standards

FDA

FDA

FDA

Stability (3 lots)

Real time (on file)

Real time (on file)

Real time (on file)

QC Reference and
Test Device Results
CLSI Strains

20 results/site

20 results/site

20 results/site

(Other Mfg. Recommended)

Optional

Optional

Optional

On-scale

At least 1

At least 1

At least 1

Inoculum density checkh

QC, reproducibility, fresh

QC, reproducibility, fresh

QC, reproducibility

CLSI Reference MethodMIC

MIC

MIC

  1. See Tables 5, 8, and 9 for statistical calculation examples and evaluable results.
  2. For Fastidious organisms such as Streptococcus, Haemophilus, anaerobes, etc. that have an CLSI approved standard methodology, FDA interpretive criteria and quality control recommendations, refer to CLSI approved standard M100 4 Table 1A. The routine testing of rare isolates is not recommended.
  3. Minimal data to establish performance should be presented for each procedural option of the method of inoculation (growth, direct colony suspension etc.), reading of results (visual vs. automated), or combinations of options.
  4. Fresh clinical - an organism isolated from a clinical specimen which has been on an agar plate for less than 7 days and not frozen.
  5. Stock organisms - any organism from a clinical specimen which has been isolated greater than 7 days prior to testing. Should not include organisms for which the antimicrobial agent is not intended. Selection should be supplemental based on the listing in the FDA approved pharmaceutical antimicrobial agent package insert and should not comprise more than 50% of the clinical isolates.
  6. Challenge - CDC or reference laboratory source with known results (preferably on-scale) to be tested on the test system. Organisms that are intended for the testing with the antimicrobial agent as stated in the FDA approved pharmaceutical antimicrobial agent package insert (microbiology section), should be selected for testing on the test device.
  7. All on-scale results.
  8. Inoculum density check should be performed daily on the QC isolates, on reproducibility isolates, and 10% of fresh isolates. Alternate approaches may be substituted if the inoculum method uses a spectrophotometric device.

  TABLE 2: Recommendations for the Removal of Limitations from Antimicrobial Susceptibility Devicesa

Items

Performance

Insufficient Resistant
Strains

Quality Controlb

Reproducibility

Number of Sitesc

3

1

3

3

Organisms

Fresh or
Stock
Clinical

100/site

75d

0

NA

Challenge

75/one site

as needed

0

 

Reproducibilityae

NA

NA

NA

25/site or 10x3x3/site

Quality Control

Daily

Daily

20

Daily

  1. For Statistically evaluable numbers see Tables 5, 8 and 9
  2. To be used to demonstrate that the QC ranges are now in the same ranges as the FDA/CLSI
  3. One may be in-house
  4. A minimum of 75 organisms either resistant or clustered near the susceptible threshold.
  5. On-scale

Note:

  1. If changes have been made to the device to alter the overall performance, the testing should include all organisms previously tested.
    Refer to Table 1.
  2. Perform testing for all procedural options.

  TABLE 3: Presentation of Summary Data for Both Challenge and Clinical Data

Clinical Data

Organism Group Total Tested # EA %EA Total Evaluable # EA of Evaluable %EA of Evaluable # CA %CA # R # vmj # maj # min
K. pneumoniae 797493.7646292.27088.620018
P. aeruginosa 969093.8918593.48891.740017
C. freundii 262180.8181472.22388.510003
E. aerogenes 222195.5212195.22195.52001
E. cloacae 5753935048925189.513114
M. morganii 151493.3121291.7151007000
P. mirabilis 342676.5342976.53410033000
E. coli 928592.4625988.78794.643212
S. marcescens 5049984848100479432003
Acinetobacter spp 414097.6292896.63482.914007
TOTAL 51247392.442940694.647091.82143435

Challenge

Organism Group Total Tested # EA %EA Total Evaluable # EA of Evaluable %EA of Evaluable # CA %CA # R # vmj # maj # min
K. pneumoniae 10101001010100101006002
P. aeruginosa 201890171588.2199515100
C. freundii 1010100991009902010
E. aerogenes 5480437551000011
E. cloacae 551002210051004001
Aeromonas sp. 54804410051000000
P. mirabilis 1099086759904002
S. marcescens 101080991009902001
Acinetobacter spp 10880551008802102
TOTAL 857891.8686392.67992.935229

Clinical and Challenge Combined

Organism Group Total Tested # EA %EA Total Evaluable # EA of Evaluable %EA of Evaluable # CA %CA # R # vmj # maj # min
  All organisms 59755192.349746994.454992.02495644

TABLE 4: Example of Reporting Format for Quality Control Data

Antimicrobial agent: ___________________________

QC OrganismExpected Result

 

Reference Result Frequency

New Device Frequency

E. coli ATCC 259220.25 - 1.0 mg/mL

 

Site 1

Site 2

Site 3

Site 1

Site 2

Site 3

<.25

 

 

 

1

1

 

.25

14

18

14

4

14

14

.5

6

2

6

4

5

6

1.0

 

 

 

11

 

 

>1.0

 

 

 

 

 

 

E. cloacae Ref 1611

2 - 8 mg/mL

 

<2

 

 

 

 

 

 

2

 

 

 

12

14

4

4

14

15

12

2

6

5

8

6

5

8

8

 

11

>8

 

>

 

>

 

>

 

>

 

>

 

Pseudomonas aeruginosa ATCC 27853

0.5 - 2 mg/mL

 

<.25

>

 

 

2

 

 

2

.5

10

10

18

 

2

 

1

5

8

2

20

18

18

2

5

2

 

 

 

4

4

 

 

 

 

 

 

Enterococcus faecalis ATCC 29212

4 - 16 mg/mL

 

2

 

 

 

 

 

 

4

18

2

18

20

12

6

8

2

18

2

 

8

14

16

 

>

 

>

 

>

 

>

 

>

 

  • Performed daily with a minimum of 20 per site.
  • List all reference and test results including out of range results that required repeat testing.
  • To be used for all procedural options.

TABLE 5: Sample Table Format for Device Performance

Antimicrobic agent: Oxacillin
 Test Results  Reference Results
 ≤ 0.25a0.512 S4  R 8≥ 16
≤ 0.256aa1   a
0.5102100212  a
1a1081  a
2 Sa6111  a
4 Ra     a
≥ 8a   aa31a
Evaluable Results 116414   
 Organism: Staphylococcus aureus
Evaluation
Overall EAb 
108/11693.1%
EA based on evaluable resultsab 
3/1127.3%
CA based on interpretationc 
 100%
Antimicrobic agent: Oxacillin
 Test Results  Reference Results
 ≤ 0.25 S0.5 R1248≥ 16
≤ 0.25 S29aa    a
0.5 Ra     a
1a     1a
2a 11 1a
4a   1 a
≥ 8a  712a12a52a
Evaluable Results  1811 
 Organism: Staphylococcus epidermidis
Evaluation
Overall EAb 
108/11693.1%
EA based on evaluable resultsab 
3/1127.3%
CA based on interpretationc 
 100%
Antimicrobic agent: Oxacillin
 Test Results  Reference Results
 ≤ 0.25 S0.5 R1248≥ 16
≤ 0.25 S25a1a vmj    1a
0.5 Ra83   a
1a     1a
2a     a
4a     a
≥ 81a (maj)   2a3a26a
Evaluable Results 83    
see footnotes on Table 5A
 Organism: other CNS
Evaluation
Overall EAb 
67/7095.7%
EA based on evaluable resultsab 
11/11100%
CA based on interpretationc 
67/70100%

Present all fresh and stock results for organisms in a genus/species in a separate diagonal. For example all Staphylococcus aureus results from all sites would appear in the first diagonal.

Antimicrobic agent: Oxacillin
 Test Results  Reference Results
 ≤ 0.120.250.51 S2 I4 R≥ 8
≤ 0.12259aa    a
0.25a42   a
0.56a42   a
1 Sa 101051a
2 Ia 291011a
4 Ra   810a
≥ 8a    1a53a
Evaluable Results 816192322 
 Organism: All
Evaluation
Overall EAb 
398/40797.8%
EA based on evaluable resultsab 
85/8896.6%
CA based on interpretationc 
371/407100%
CA Minord 
35/4078.6%
CA Majore 
00%
CA Very Minorf 
1/761.3%

a Results are not considered evaluable if they fall in this box

b Essential Agreement (EA) = (# exact agreement or within plus or minus one two-fold dilution of the reference method / total # organisms tested) X 100

c Category Agreement (CA) = (# with interpretive agreement to the reference method / total # organisms tested) X 100

d Minor Discrepancy (min) = (# min discrepancies based on interpretation / total # organisms tested) X 100

e Major Discrepancy (maj) = (# maj discrepancies based on interpretation / total # susceptible organisms by reference method) X 100

f Very Major Discrepancy (vmj) = (# vmj discrepancies based on interpretation / totla # resistant organisms by reference method) X 100

To be used for:

  • All fresh and stock organisms for each site presented in one 2A diagonal.
  • All fresh and stock organisms for all sites combined presented in one 2A diagonal.
  • All challenge organisms presented in one 2A diagonal chart.
  • All challenge organisms presented in one 2A diagonal chart for each method variation.
  • All organisms (challenge, fresh, stock) combined and presented in one 2A diagonal chart. Do not combine different method variations.
     

TABLE 6: Presentation of Reproducibility Results a by Organism

  Difference in the number of wells
between test result and test mode
  
  Off-
scale
   -2    -1    0    +1    +2 Off-
scale
Site 1
result
Site 2
result
Site 3
result
Test
Modeb
P. aeruginosa   3   0.50.50.50.5
P. aeruginosa   21  4222
P. aeruginosa  12   16323232
P. aeruginosa   21  81688
E. coli   3   2222
E. coli  12   8161616
E. coli   21  4222
E. coli   3   1111
E. coli  12   2444
E. coli   3   32323232
M. morganii  12   1616816
C. diversus   21  0.50.510.5
C. freundii   21  1211
C. freundii   3   16161616
E. cloacae   21  16321616
E. cloacae   2 1 0.50.520.5
E. cloacae   3   4444
P. mirabilis   3   8888
P. mirabilis   3   2222
S. marcescens   21  2111
S. marcescens   21  32161616
S. marcescens   21  4222
K. pneumoniae   2  13232>3232
K. pneumoniae   21  0.250.50.250.25
P. stuartii 1 2   1616416
            
Total 14581011    
Between-site reproducibilityc

73/75 = 97.3%d
72/75 = 96%e

a Results in the table are occurrences of the difference in the number of wells between the test result and the test mode. The study is based on 25 on-scale organisms, tested at 3 sites.
b Most frequent new test result. If there is no mode, the median should be used.
c Total number of results that fall within 1 well (+/-1) of the mode result divided by total number of results. This should be calculated for the best and worst case if some of the values are off-scale while some are on-scale. If all three results are off-scale they should still be included in the calculation as part of the best-worst case calculations. For this study the denominator would always be 75 whether the results are off or on-scale.
d Best case calculation for reproducibility assuming the off-scale result is within one well from the mode.
e Worst case calculation for reproducibility assuming the off-scale result is greater than one well from the mode.

TABLE 6A: Presentation of Reproducibility Resultsa by Organism and Site

   Difference in the number of wells between new test result and test mode   
     Off-scale    -2    -1    0    +1    +2    Off-scale    Test Modeb

 

 

 

 

Site 1

 

 

 

 

E. coli 1  63   0.5
E. coli 2   9   2
E. coli 3  63   2
Pseudomonas 4   36  16
Pseudomonas 5  35  132
Klebsiella 6   8 1 8
Enterobacter 7   81  8
Serratia 8   45  4
Serratia 9   54  16
Proteus 10  54   1
         
Total  20521611 
Within-Site Reproducibilityc

89/90 = 98.9%d
88/90 = 97.8%e

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Site 2

 

 

 

 

 

 

 

  

 

 

 

 

E. coli 1

 

 

3

6

 

 

 

0.5

E. coli 2

 

 

 

9

 

 

 

2

etc…

 

 

 

 

 

 

 

 

a Results in the table are occurrences of the difference in the number of wells between the test result and the test mode. The study is based on 10 on-scale organisms, tested in triplicate on 3 separate days at 3 sites.
b Most frequent test result.
c Total number of results that fall within 1 well (+/-1) of the mode result divided by total number of results. This should be calculated for the best and worst case if some of the values are off-scale while some are on-scale. If all results are off-scale they should still be included in the calculation. For this study the denominator would be ninety whether they are all on scale or not.
d Best case calculation for reproducibility assuming the off-scale result is within one well from the mode.
e Worst case calculation for reproducibility assuming the off-scale result is greater than one well from the mode.

TABLE 6B: Presentation of Reproducibility Resultsa by Organism, Pooled Across Sites 

   Difference in the number of wells between new test result and test mode   
     Off-scale    -2    -1    0    +1    +2    Off-scale    Test Modeb
All Sites

 

 

 

 

 

 

 

 

 

E. coli 1  918   0.5
E. coli 2   252  2
E. coli 3 39123  2
Pseudomonas 4  6129  16
Pseudomonas 5  217  832
Klebsiella 6  71451 8
Enterobacter 7  5148  8
Serratia 8  4158  4
Serratia 9  3204  16
Proteus 10  31410  1
Total 3481614918 
 Between-site reproducibilityc

266/270 = 98.5%d
258/270 = 95.6%e

a Results in the table are occurrences of the difference in the number of wells between the test result and the test mode. The study is based on 10 on-scale organisms, tested in triplicate on 3 separate days at 3 sites.
b Most frequent test result.
c Total number of results that fall within 1 well (+/-1) of the mode result divided by Total number of results. This should be calculated for the best and worst case if some of the values are off-scale while some are on-scale. If all results are off-scale they should be included in the calculation with a denominator of 270 for all calculations.
d Best case calculation for reproducibility assuming the off-scale result is within one well from the mode.
e Worst case calculation for reproducibility assuming the off-scale result is greater than one well from the mode

  TABLE 7: Report Format for Inoculum Density

ORGANISMaNUMBER TESTEDSOURCEMETHODbMEANMINUMUMMAXIMUMSTD. DEV.
S. aureus ATCC #20QC ATCCReference6 X 10 52 x 10 59 x 10 5 
S. aureusATCC #20QC ATCCDirect inoculum5 x 10 52 x 10 58 x 10 5 
S. aureusATCC #20QC ATCCGrowth inoculum5 x 10 52 x 10 56 x 10 5 
MRSA13Reproducibility, clinicalDirect inoculum7 x 10 54 x 10 56 x 10 6 
MRSA13Reproducibility, clinicalGrowth inoculum6 X 10 52 x 10 58 x 10 6 
MSSE3ReproducibilityDirect inoculum8 x 10 55 x 10 57 x 10 5 
MSSE3ReproducibilityGrowth inoculum7 x 10 54 x 10 512 x 10 5 
MRSE19ReproducibilityDirect inoculum6 X 10 52 x 10 58 x 10 6 
MRSE19ReproducibilityGrowth inoculum7 x 10 55 x 10 57 x 10 5 
Enterococcus4ClinicalDirect inoculum  9 x 10 5 
Enterococcus4ClinicalGrowth inoculum    
MSSA15ClinicalDirect inoculum    
MSSA15ClinicalGrowth inoculum    
   Direct inoculum    
   Growth inoculum    
  1. Data should be available upon request for “by site” evaluation, by organism, etc.
  2. Inoculum density should be performed on all methods of inoculation unless a standardized method (photometric device) is used.

  TABLE 8: Number of VMJ Discrepancies as a Function of the Number of Resistant Organisms Tested

Number of
Resistant
Organisms
Acceptable
Number of
Discrepancies
Estimated
Ratea
95% Confidence
Intervalb for True
VMJ Rate
4800.00(0.00, 7.40)
5000.00(0.00, 7.11)
6000.00(0.00, 5.96)
7000.00(0.00, 5.13)
7211.39(0.04, 7.50)
8011.25(0.03, 6.77)
9011.11(0.03, 6.04)
9422.13(0.26, 7.48)
10022.00(0.24, 7.04)
11021.82(0.22, 6.41)
12032.50(0.52, 7.13)
13032.31(0.48, 6.60)
14042.86(0.78, 7.15)
15042.67(0.73, 6.69)
16053.13(1.00, 7.20)
17052.94(0.94, 6.78)
18063.33(1.21, 7.16)
19073.68(1.48, 7.48)
20073.50(1.40, 7.12)
25083.20(1.38, 6.24)
30093.00(1.37, 5.64)
400112.75(1.37, 4.88)
500132.60(1.39, 4.41)
600152.50(1.40,4.09)
700192.43(1.42,3.86)
  1. Est. Rate = estimated vmj rate = number of vmj discrepancies divided by number of resistant organisms.
  2. Exact confidence intervals based on the binomial distribution.

  TABLE 9: Essential Agreement as Function of the Number of Evaluable Organisms Tested

Number of
Evaluablea
Organisms
Acceptable
Number of
Disagreements
Estimated
Essential
Agreement (EA)b
95% Confidence
Intervalc for
True EA
350100.00 %(90.00, 100.00)
54198.15(90.11, 99.95)
55198.18(90.28, 99.95)
60198.33(91.06, 99.96)
65198.46(91.72, 99.96)
70297.14(90.06, 99.65)
75297.33(90.70, 99.68)
80297.50(91.26, 99.70)
85396.47(90.03, 99.27)
90396.67(90.57, 99.31)
95396.84(91.05, 99.34)
100496.00(90.07, 98.90)
110496.36(90.95, 99.00)
120595.83(90.54, 98.63)
130695.38(90.22, 98.29)
140695.71(90.91, 98.41)
150795.33(90.62, 98.10)
160895.00(90.39, 97.82)
170994.71(90.19, 97.55)
1801094.44(90.02, 97.30)
1901094.74(90.53, 97.45)
2001194.50(90.37, 97.22)
  1. Evaluable (e.g. on-scale) organisms are those that fall within the test range of the reference and have the opportunity for a result on the test method that could also be on-scale. Any reference result that falls in the < or > category is considered not evaluable.
  2. Estimated Essential Agreement = percent agreement = number of evaluable test results that are equal to or with in one dilution of the expected result divided by number of organisms that are evaluable.
  3. Exact confidence intervals based on the binomial distribution.