Internal Quality Assurance. Jenny Andrews Secretary of the BSAC Working Party on Susceptibility Testing

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1 Internal Quality Assurance Jenny Andrews Secretary of the BSAC Working Party on Susceptibility Testing

2 Data provided by the BSAC Acceptable MIC ranges for control strains Acceptable zone diameter ranges for susceptible control strains Acceptable zone diameter ranges for control strains with a mechanism of resistance

3 To be remembered Acceptable ranges are for use with the BSAC Standardized Methods ONLY Do not assume that MIC values are always the same on Iso-Sensitest agar & Mueller Hinton agar (MHA) e.g. azithromycin MICs for Salmonella typhi are higher on Mueller Hinton agar than on Iso-Sensitest agar. In this case there is an impact on the MIC BP used to interpret susceptibility and isolates may appear resistant when tested on MHA (Przybylo, Burns & Qamruddin JAC 2012). Acceptable zone diameter ranges for control strains are affected by test conditions e.g. size of inoculum (BSAC = semi-confluent growth, EUCAST = confluent growth), medium etc. Acceptable ranges for control strains (and zone diameter BPs for tests) cannot be used with other standardized methods

4 Table 2a: Susceptible control strains or control strains with low-level resistance that have been chosen to monitor test performance of antimicrobial susceptibility testing Organism Either Or Characteristics Escherichia coli NCTC (ATCC 25922) Staphylococcus aureus NCTC (ATCC 25923) Pseudomonas aeruginosa NCTC (ATCC 27853) Enterococcus faecalis NCTC (ATCC 29212) NCTC NCTC 6571 NCTC Susceptible Susceptible Susceptible Susceptible Haemophilus influenzae NCTC Susceptible Streptococcus pneumoniae NCTC (ATCC 49619) Neisseria gonorrhoeae NCTC (ATCC 49226) Low-level resistant to penicillin Low-level resistant to penicillin Pasteurella multocida NCTC 8489 Susceptible Bacteroides fragilis NCTC 9343 (ATCC 25285) Susceptible Bacteroides thetaiotaomicron ATCC Susceptible Clostridium perfringens NCTC 8359 (ATCC 12915) Susceptible

5 Table 2b: Control strains with a resistance mechanism that can be used to confirm that the method will detect resistance Organism Strain Characteristics Escherichia coli NCTC TEM-1 ß- lactamaseproducer Staphylococcus aureus NCTC MecA positive, methicillin resistant Haemophilus influenzae NCTC (ATCC 49247) Resistant to ß- lactams (ßlactamasenegative)

6 BSAC Acceptable MIC Ranges for Control Strains Control of MIC Determinations Tables 7-10 provide target MIC (mg/l) values for recommended control strains by BSAC methodology. MICs should be within one two-fold dilution of the target values i.e. target MIC 1 mg/l acceptable range mg/l.

7 Source of Zone Diameter Data BSAC Field Study (5 members of the Working Party & 14 diagnostic laboratories) Rolling programmes (25 diagnostic laboratories) e.g. 10 laboratories acceptable ranges for 12 agents v N. gonorrhoeae Diagnostic Laboratories e.g. New Cross Hospital in Wolverhampton, Withington Hospital in Manchester NB. The BSAC welcome any zone data for control strains

8 Calculation of 2.5 and 97.5 percentile points, to obtain a range encompassing 95% of observations Obtain at least 100 observations (zone diameters mm) for the control strain tested by BSAC methodology. Zone diameter (mm) No. Observations Cumulative No. Observations Cumulative % Statistical advice Dr. T. Marshall Birmingham University

9 Example of plot of zone diameter v cumulative percentage Cumulative % Zone diameter (mm) Acceptable range mm

10 Rolling programme: Confirmation of acceptable range E. coli ATCC 25922: ampicillin 25 ug disc CT25 (Oxoid) CT1 (Oxoid) CT24 (In-house) CT6 (Oxoid) CT2 (Oxoid) Centre Each centre undertook 50 tests on different occasions

11 Rolling programme: Confirmation of acceptable range E. coli NCTC 10418: 75 ug piperacillin disc. Zone diameter (mm) CT4 (in-house) CT8 (Oxoid) CT20 (biomerieux) CT14 (Oxoid) CT12 (in-house) Centre Each centre undertook 50 tests on different occasions

12 Use of control data to monitor the performance of disc diffusion tests Testing is acceptable if no more than1 in every 20 results is outside the limits of acceptability. If 2 or more results fall out of the acceptable range this requires immediate investigation. Look for trends within the limits of acceptability e.g. tendency for zones to be at the limits of acceptability; tendency for zones to be consistently above or below the mean; gradual drift in zone diameters. Quality Assurance will often pick up trends before the controls go out of range. Prepared by Trevor Winstanley for the BSAC recommendations

13 Potential sources of error in disc diffusion antimicrobial susceptibility testing: Test conditions Excessive pre-incubation before discs applied Excessive pre-diffusion before plates incubated Incorrect incubation temperature (35 0 C for cefoxitin v staphylococci) Incorrect incubation atmosphere (false metronidazole R ) Incorrect incubation time (VanB resistance in enterococci) Inadequate illumination of plates when reading Incorrect reading of zone edges (cefoxitin for the detection of MRSAs) Incorrect template (wrong agent or wrong version of the guidelines) Prepared by Trevor Winstanley for the BSAC recommendations

14 Potential sources of error in disc diffusion antimicrobial susceptibility testing: medium Required susceptibility testing agar not used Not prepared as required by the manufacturer s instructions Batch to batch variation Antagonists present (e.g. with sulphonamides and trimethoprim) Incorrect ph Incorrect divalent cation concentration (can be an issue when testing daptomycin by gradient methods need 50 mg/l calcium in the medium) Incorrect depth of agar plates (4mm ± 0.5 mm) Agar plates not level Expiry date exceeded Prepared by Trevor Winstanley for the BSAC recommendations

15 Potential sources of error in disc diffusion antimicrobial susceptibility testing: antimicrobial discs Wrong agent or content used Labile agent possibly deteriorated Light sensitive agent left in light Incorrect storage leading to deterioration Disc containers opened before reaching room temperature Incorrect labelling of disc dispensers Expiry date exceeded Prepared by Trevor Winstanley for the BSAC recommendations

16 Potential sources of error in disc diffusion antimicrobial susceptibility testing: control strains Contamination Mutation Incorrect inoculum density Uneven inoculation Old culture used (maintenance of control strains available on the BSAC web site) Prepared by Trevor Winstanley for the BSAC recommendations

17 Reporting Susceptibility Results when Controls Indicate Problems Microbiologists must use a pragmatic approach, as results from repeat testing are not available on the same day. If results with control strains are out of range the implications for test results need to be assessed. Control results out of range If control zones are below range but test results are susceptible, or control zones are above range but test results are resistant, investigate possible sources of error but report the test results. Otherwise it may be necessary to suppress reports on affected agents, investigate and retest. Atypical results If results are atypical with clinical isolates, the purity of the isolate and identification should be confirmed and the susceptibility repeated. Suppress the results for individual agents and retest. Prepared by Trevor Winstanley for the BSAC recommendations

18 What to do if the problem is not resolved Contact the BSAC (HQ or the Secretary of the Working party) BSAC will forward the information to the appropriate manufacturer for their comment

19 Conclusion: BSAC Provides A list of susceptible control strains for monitoring performance A list of control strains with a resistance mechanism that can be used to confirm that the method will detect resistance Acceptable MIC ranges for control strains Acceptable zone diameter ranges for control strains

20 Conclusion: BSAC Provides Advice on how to monitor the performance of disc testing Advice on the investigations that are necessary if the control data indicates there is a problem Advice on reporting susceptibility results when controls indicate a problem Support for laboratories that cannot resolve problems with control strain performance

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