1. BSAC rolling programme for devising acceptable limits for control strains Jenny Andrews The BSAC Standardized Method Development Centre

2. Acceptable limits for control strains Used by diagnostic laboratories to monitor the performance of testing. Used by diagnostic laboratories to monitor the performance of testing. There are gaps in the tables that need filling. There are gaps in the tables that need filling. At present S. aureus is used to control testing of fastidious organisms and this may not be ideal because the growth requirements of fastidious organisms are different to that of staphylococci. At present S. aureus is used to control testing of fastidious organisms and this may not be ideal because the growth requirements of fastidious organisms are different to that of staphylococci.

3. Findings from a previous BSAC study Confirmed that agar poured `in-house’ to a depth between 3.5-4.5 mm, and pre-poured plates from Oxoid and bioMerieux gave acceptable results by BSAC methodology. Confirmed that agar poured `in-house’ to a depth between 3.5-4.5 mm, and pre-poured plates from Oxoid and bioMerieux gave acceptable results by BSAC methodology. When combined data from these plates was used to calculate the acceptable limits for all of the antibiotic/strain combinations tested, the ranges were similar to those found in previous `field studies’. So it was proposed that this procedure could be used to fill the gaps in the tables. When combined data from these plates was used to calculate the acceptable limits for all of the antibiotic/strain combinations tested, the ranges were similar to those found in previous `field studies’. So it was proposed that this procedure could be used to fill the gaps in the tables.

4. Programme design After discussion the Working Party decided that it would be prudent if acceptable limits were calculated using data from several laboratories rather than one centre. After discussion the Working Party decided that it would be prudent if acceptable limits were calculated using data from several laboratories rather than one centre. However, SMDC acceptable ranges could be used as a guide for comparison. However, SMDC acceptable ranges could be used as a guide for comparison.

5. Request for help Needed the help of laboratories using the BSAC method, but unsure if laboratories would take part. Needed the help of laboratories using the BSAC method, but unsure if laboratories would take part. Letter sent to known users of the BSAC method Letter sent to known users of the BSAC method Asking if they would take part and select from a list of antibiotic/strain combinations those they were prepared to test Asking if they would take part and select from a list of antibiotic/strain combinations those they were prepared to test 20 laboratories agreed to take part 20 laboratories agreed to take part

6. QC Study SMDC calculated acceptable limits by testing discs 40 times each on ISA poured to a depth of 3.5, 4 & 4.5mm and pre- poured plates from Oxoid & bioMerieux. 200 observations SMDC calculated acceptable limits by testing discs 40 times each on ISA poured to a depth of 3.5, 4 & 4.5mm and pre- poured plates from Oxoid & bioMerieux. 200 observations The participating laboratories were asked to test discs (supplied by SMDC) 5 times on 10 separate occasions. 250 observations. The participating laboratories were asked to test discs (supplied by SMDC) 5 times on 10 separate occasions. 250 observations.

7. How are the acceptable limits for the control strains calculated? Obtain at least 200 zone diameters for the strain/antibiotic disc combination. Obtain at least 200 zone diameters for the strain/antibiotic disc combination. Determine the number of observations at each zone diameter. Determine the number of observations at each zone diameter. Calculate the cumulative % at each zone diameter. Calculate the cumulative % at each zone diameter. Construct a graph of cumulative % versus zone diameter. Construct a graph of cumulative % versus zone diameter. Read off the zones equivalent to 2.5% & 97.5% of observations to give the acceptable range. Read off the zones equivalent to 2.5% & 97.5% of observations to give the acceptable range.

8. Plot of cumulative % v zone diameter

9. E. coli ATCC 25922 CT2 (Chester MS) CT2 (Chester MS) CT6 (Oxoid) CT6 (Oxoid) CT1 (Oxoid) CT1 (Oxoid) CT25 (Oxoid) CT25 (Oxoid) CT24 (In-house) CT24 (In-house) Pip/tazo Piperacillin Nalidixic acid Levofloxacin Ampicillin

10. E. coli NCTC 10418 CT4 (Taunton PHL) CT4 (Taunton PHL) CT8 (In-house) CT8 (In-house) CT14 (bioMerieux) CT14 (bioMerieux) CT12 (Chester PHL) CT12 (Chester PHL) CT 20 (Oxoid) CT 20 (Oxoid) Pip/tazo Piperacillin Chloramphenicol Colistin

11. E. faecalis ATCC 29212 CT17 (Oxoid) CT17 (Oxoid) CT15 (E&O) CT15 (E&O) CT16 (Taunton MS) CT16 (Taunton MS) CT29 (bioMerieux) CT29 (bioMerieux) CT10 (Leeds PHL) CT10 (Leeds PHL) Pip/tazo Meropenem Imipenem Linezolid Azithromycin

12. E. coli ATCC 25922: Ampicillin 25 ug Participant zones compared with SMDC acceptable limits Observations

13. E. coli NCTC 10418 : Chloramphenicol 10 ug Participant zones compared with SMDC acceptable limits Observations

14. E. faecalis ATCC 29212: Azithromycin 15 ug Participant zones compared with SMDC acceptable limits Observations

15. E. faecalis ATCC 29212: Linezolid 10 ug Participant zones compared with SMDC acceptable limits CT10 (Leeds PHL) Observations

16. E. faecalis ATCC 29212: Pip/tazo 85ug disc Participant zones compared with SMDC acceptable limits Observations

17. E. faecalis ATCC 29212: Pip/tazo 85ug Acceptable limits using data from 5 centres (SMDC 27-32 mm) Observations CT10 (Leeds PHL)

18. E. coli NCTC 10418: Centres where zone diameters outside the SMDC acceptable ranges AntibioticCT4 (Taunton PHL) CT8(Oxoid)CT20 (bioMerieux ) CT14(Oxoid)CT12 (Chester PHL) Pip/tazo85ugX Too large Piperacillin75ugX Chloramphenicol10ugX Colistin25ug

19. Summary of acceptable ranges (mm) for E. coli NCTC 10418 AntibioticSMDC(mm) 5 centres (mm) 4 centres* (mm) SMDC & 4 centres* Chloramphenicol10ug23-2720-2820-2621-27 Piperacillin75ug31-3530-3629-3530-35 Pip/tazo85ug31-3430-3629-3430-35 Colistin25ug15-1916-2016-1915-19 * Excluding CT12

20. E. coli ATCC 25922: Centres where zone diameters outside the SMDC acceptable ranges AntibioticCT25(Oxoid)CT11(Oxoid)CT24(Chester)CT6(Oxoid)CT2(Oxoid) Pip/tazo85ugX Too large X X X X Piperacillin75ugX X X Nalidixic acid 30ugX Too large X X Levofloxacin1ugX X Ampicillin25ugX NB. NCTC E. coli: CT12 (Chester MS) also large zones

21. Summary of acceptable ranges (mm) for E. coli ATCC 25922 AntibioticSMDC(mm) 5 centres (mm) 3 centres* (mm) SMDC & 3 centres* Piperacillin75ug26-3129-3428-3227-32 Pip/tazo85ug26-3028-3228-3226-31 Nalidixic acid 30ug26-3026-3226-3126-32 Levofloxacin1ug29-3328-3628-3428-34 Ampicillin25ug20-2622-2822-2821-28 * Excluding CT24 & CT6

22. E. Faecalis ATCC 29212: Centres where zone diameters outside the SMDC acceptable ranges AntibioticCT17(Oxoid)CT15(E&O)CT16(Taunton)CT28(bioMerieux)CT10(Leeds) Pip/tazo85ugX Too large X Too small Meropenem10ugX Too large X Too small X Imipenem10ugX Linezolid10ugX Too large X Too small Azithromycin15ugX Too large X Too small X Too large

23. Summary of acceptable ranges (mm) for E. faecalis ATCC 29212 AntibioticSMDC(mm) 5 centres (mm) 3 centres* (mm) SMDC & 3 centres* Pip/tazo85ug27-3224-3226-3126-32 Meropenem10ug24-2819-2821-2722-28 Linezolid10ug25-3022-3124-2824-29 Imipenem10ug28-3325-3228-3228-32 Azithromycin15ug15-2011-2114-2115-21 *Excluding CT15 & CT17

24. Conclusions Reasonable to exclude data from the analysis that is outside the acceptable limits generated by SMDC or that is different to the other laboratories Reasonable to exclude data from the analysis that is outside the acceptable limits generated by SMDC or that is different to the other laboratories

25. Conclusions Very worthwhile exercise and the rolling programme is to be continued including looking at fastidious controls. Very worthwhile exercise and the rolling programme is to be continued including looking at fastidious controls. In future studies control strains will be provided. In future studies control strains will be provided. Laboratories asked to test `once daily’ as this reflects `real life’. Laboratories asked to test `once daily’ as this reflects `real life’.

26. Future meeting Intend to have a meeting with the participating laboratories Intend to have a meeting with the participating laboratories Need to look at data from centres where zones are generally different from SMDC & other participants Need to look at data from centres where zones are generally different from SMDC & other participants

27. Acknowledgments QE Hospital, Woolwich, London QE Hospital, Woolwich, London Department of Clinical Medicine, Manchester Department of Clinical Medicine, Manchester Truro PHLS Truro PHLS Rotherham DGH Rotherham DGH Craigavon Area Hospital Craigavon Area Hospital Addenbrookes Hospital, Cambridge Addenbrookes Hospital, Cambridge Singleton PHL, Swansea Singleton PHL, Swansea University Hospital, Birmingham University Hospital, Birmingham Bedford Hospital Bedford Hospital Bristol Royal Infirmary Bristol Royal Infirmary QE2, Hertfordshire QE2, Hertfordshire UCL Hospitals, London UCL Hospitals, London Russells Hall Hospital, West Midlands Russells Hall Hospital, West Midlands Manchester Royal Infirmary Manchester Royal Infirmary New Cross Hospital, Wolverhampton New Cross Hospital, Wolverhampton