Internal Quality Control (IQC) Antimicrobial Susceptibility Tests Using Disk Diffusion
National AMR Surveillance Network, NCDC
National Programme on Containment of Antimicrobial Resistance National Centre for Disease Control, New Delhi April 2019
CONTENTS I. Scope ...... 2
II. Selection of Strains for Quality Control ...... 2
III. Maintenance and Testing of QC Strains...... 3
Figure 1: Flow Chart: Maintenance of QC strains in a bacteriology lab...... 4
IV. Quality Control (QC) Results—Documentation - Zone Diameter ...... 5
V. QC Conversion Plan ...... 5
1. The 20- or 30-Day Plan ...... 5
2. The 15-Replicate (3× 5 Day) Plan ...... 5
3. Implementing Weekly Quality Control Testing ...... 6
4. Out-of-Range Results with Quality Control Strains and Corrective Action ...... 6
Figure 2: Flow Chart: Quality Control Plan in bacteriology lab ...... 7
5. Weekly Quality Control Testing – Out-of-Range Result Due to Identifiable Error ...... 8
6. Weekly Quality Control Testing – Out-of-Range Result Not Due to Identifiable Error ...... 9
7. Additional Corrective Action- Checklist ...... 11
8. Reporting Patient Results When Out-of-Range Quality Control Results Are Observed ...... 12
9. Accuracy Checklist before Reporting the Patient AST Results...... 12
10. End-Point Interpretation Control ...... 13
VI. Appendix ...... 14
Table 1: Disk Diffusion QC Ranges for QC Strains ...... 14
Table 2: Disk Diffusion QC Ranges for Non-fastidious QC Strains and β-Lactam Combination Agents. . 15
Table 3: Characteristics of Routine QC Strains for Antimicrobial Susceptibility Tests ...... 16
Table 4: Conditions for Disk diffusion Antimicrobial Susceptibility Test for Non-fastidious Organisms . 17
Table 5: Plan 15-Replicate (3×5 Day): Acceptance Criteria and Recommended Action* ...... 18
Table 6: Internal Quality Control for monitoring AST by disk diffusion log sheet ...... 19
VII. Annexures ...... 20
a. Mueller Hinton Agar (MHA) ...... 20
a.1 Media QC recording format ...... 20 b. Antimicrobial Disks & Quality Specifications ...... 21
c. Storage of Antimicrobial Disks...... 21
d. Batch or Lot Quality Control ...... 22
e. Inoculum Preparation for Disk Diffusion Tests ...... 22
VIII. References ...... 23
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Internal Quality Control (IQC) for Antimicrobial Susceptibility Tests Using Disk Diffusion
Quality control (QC) are procedures used to assure that a test run is valid and results are accurate and reliable.All participating laboratories in the NCDC AMR surveillance network should have internal quality control procedures in place. This SOP is intended to provide guidance on performing internal QC for disk diffusion antimicrobial susceptibility tests (AST) in the department of Microbiology/Clinical Microbiology laboratory of a hospital.
I. Scope This SOP gives an overview of the purpose of IQC for Disc Diffusion Antimicrobial Susceptibility Tests: i. To ensure all testing materials including media and antibiotic disks are maintained properly by the laboratory. ii. Description of the selection, maintenance and testing of QC strains iii. The recommended frequency for performing QC testing including the amount of testing required to reduce QC frequency from daily to weekly. iv. Suggestions for troubleshooting out-of-range results with QC strains v. Factors to consider before reporting patient results when out-of-range QC results are observed vi. Guidance for confirming noteworthy or uncommon results encountered when testing patient isolates
II. Selection of Strains for Quality Control i. Procure strains from a recognized source (e.g., ATCC, NCTC) for QC of antimicrobial susceptibility tests (disk diffusion) to ensure the test system is working properly ii. Select and include routine QC strains as recommended by CLSI document1, 2, iii. CLSI-recommended QC strains should produce results within the expected range listed in table 1 to 2 of Appendix. iv. The characteristics of QC strains provided under the national AMR surveillance programme are described in table 3 Appendix (routine QC strains). v. Use the recommended testing conditions for the respective organism group as in table 4 Appendix. vi. Select QC strains that most closely resemble the organism isolated from the clinical specimen. vii. For commercial test systems, follow manufacturer’s recommendations for all QC procedures including the QC strains recommended by the manufacturer
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III. Maintenance and Testing of QC Strains i. For long-term storage, maintain stock cultures at −20°C or below in 10% to 15% glycerol in Tryptic soy broth (TSB), alternatively strains can be lyophilized. Temperature lower than −60°C or liquid nitrogen is preferred for long term storage as some QC strains, particularly those with plasmid-mediated resistance (e.g., E. coli ATCC 35218), have been shown to lose the plasmid when stored at temperatures higher than −60°C. ii. Revive the original frozen or freeze-dried ATCC strain as per instructions in the kit insert, culture on Tryptic soy agar plate and incubate. Label this as F1 culture plate. FollOw steps as mentioned in Figure 1 iii. From the F1 culture plate, make at least 12/52 F1 (either 12 monthly or 52 weekly) Tryptic soy broth (TSB) glycerol stock vials which will last for one year for each QC strain. Label these as F1/month or F1/week (e.g. F1/Jan; F1/Feb…..F1/Dec; F1/week1; F1/week2……….F1/week52) QC strain glycerol stock and store them at −20°C or below. iv. For each month/week use particular F1-month/week stock. For eg. for the month of January use F1/Jan and for February use F1/Feb and so on; in case weekly stock vials use F1/week1 for week1 and F1/week52 for last week of the calendar year. v. Subsequently from one F1/month or F1/week QC strain glycerol stock subculture on TSA plate/slant to get F2 weekly working QC strain for 1 week. This step is to be repeated every week. After one month next F1/ month vial or after a week next F1/week vial is to be revived. vi. Store F2 weekly working QC strain plate/slant at 2 to 8°C or as appropriate for the organism type vii. F2 weekly working QC strain will be sub cultured for the preparation of F3 QC strain to obtain isolated colonies. Always use fresh subcultures (e.g., overnight incubation) for inoculum suspension preparation for AST. viii. Discard F2 weekly working QC strain after one week. Repeat step (e), starting with F1/month or F1/week QC strain glycerol stock for getting next F2 weekly working QC strain for week 2, and so on. ix. After a month, discard F1/month or after a week, discard F1/week QC strain glycerol stock.
Note: If QC test appears contaminated or if QC results are questionable, it may be necessary to revive a new F1 QC strain glycerol stock. Test QC strains using the same materials and methods that are used to test clinical isolates.
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Figure 1: Flow Chart: Maintenance of QC strains in a bacteriology lab
Original ATCC QC Strain
Revive on Tryptic Soy Agar (TSA) plate
Prepare at least 12 / 52 TSB glycerol vials to get F1 12 monthly/ 52 weekly QC strain glycerol stock (Store at−20°C or below)
Revive and Inoculate F1 for the month/week on TSA plate/slant for every week
After 1 month/1week end,
Week 1 Week 2 Week 3 Week 4 discard F1 and pull another F1 Glycerol stock from freezer
F2 Working Weekly QC Strain
F3 Day 1 F3 Day 2 F3 Day 3 F3 Day 4 F3 Day 5 F3 Day 6 2 2 2 F2 Working weekly Strain will be discarded after 1 week and fresh plate will be taken for next week
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IV. Quality Control (QC) Results—Documentation - Zone Diameter Document results from all disk diffusion QC test on a QC log sheet (Sample of QC log sheet at Annexure Table 6.). Acceptable zone diameter QC ranges for a single QC test (single-drug/single-organism combination) for select antibiotics are listed in table 1 and 2 in Appendix. Results for QC strains must fall within CLSI specified ranges for testing to be acceptable.
Note: Refer CLSI M100-29th Ed, tables 4 A1- A 2 Page 150-157 for other antibiotics and β-lactam combinations.
Results for QC strains must fall within CLSI specified ranges for testing to be acceptable.
Frequency of Quality Control Testing Two plans are available to demonstrate satisfactory performance with daily QC testing before going to weekly QC testing. 1. 20- or 30-day plan 2. 15-replicate (3× 5 day) plan. Either plan allows a laboratory to perform weekly QC testing once satisfactory performance with daily testing of QC strains is documented.
V. QC Conversion Plan (Performance Criteria for Reducing Quality Control Frequency to Weekly) 1. The 20- or 30-Day Plan i. Test all applicable QC strains for 20 or 30 consecutive test days, and document results. ii. When no more than 1 out of 20 or 3 out of 30 consecutive tests turn out to be outside acceptable limit, performance will be considered satisfactory to proceed to weekly testing. If not, take corrective action. 2. The 15-Replicate (3× 5 Day) Plan iii. Test three replicates of each applicable QC strain using individual inoculum preparations for five consecutive test days and document results. iv. Upon successful completion of the 15-replicate (3 × 5 day) plan, lab can proceed to go to weekly QC testing. v. If completion of the 15-replicate (3 × 5 day) plan is unsuccessful, take corrective action as appropriate, and continue daily QC testing.(Refer Table 5 Appendix) 5 | P a g e
Note: testing on “consecutive test days” means testing of QC strains each day tests are performed on isolates. It does not refer to calendar day.
3. Implementing Weekly Quality Control Testing i. Implement Weekly QC testing once satisfactory performance with daily QC testing has been documented. ii. Along with weekly testing, QC will be performed whenever reagent component of the test is changed. iii. If any weekly test result is out of range, take corrective action. 4. Out-of-Range Results with Quality Control Strains and Corrective Action
i. Out-of-range QC results can be categorized into 1) random, 2) identifiable, or 3) system related. ii. Out-of-range results with QC strains due to random or identifiable errors can usually be resolved by a single repeat of the QC test. iii. Out-of-range QC tests are often the result of contamination or the use of an incorrect QC strain; corrective action should first include repeating the test with a pure culture of a freshly sub cultured QC strain. iv. Out-of-range QC results that are due to a problem with the test system usually do not correct when the QC test is repeated and may indicate a serious problem that can adversely affect patient results. Every out-of-range QC result must be investigated.
Note: See disk diffusion Troubleshooting Guide in M100, 29th edition Table 4D for trouble shooting and corrective action for out-of –range results with QC strains.
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Figure 2: Flow Chart: Quality Control Plan in bacteriology lab
Quality Control using ATCC
Reconstitute the ATCC Strain
Make 52 aliquots and freeze
Weekly use one aliquot to prepare
working culture
Use 30days/15 days conversion plan every time new discs/media obtained Use fresh culture Trouble shoot in case of every time while errors putting QC procedure Switch to weekly plan Once conversion achieved
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5. Weekly Quality Control Testing – Out-of-Range Result Due to Identifiable Error i. Identify the reason for an out-of-range result, correct the problem, document the reason, and retest the QC strain on the day the error is observed. If the repeated result is within range, no further corrective action is required. ii. Identifiable reasons for the out-of-control results may include, but are not limited to: a. QC strain i. Use of the wrong QC strain ii. Improper storage iii. Inadequate maintenance (e.g., use of the same F2 subculture for > 1 month) Contamination iv. Non-viability v. Changes in the organism (eg, mutation, loss of plasmid) b. Testing supplies i. Improper storage or shipping conditions ii. Contamination iii. Use of a defective agar plate (eg, too thick or too thin) iv. Use of damaged (e.g., cracked) plates / expired materials (disks) c. Testing process i. Inoculum suspensions incorrectly prepared or adjusted ii. Inoculum prepared from a plate incubated for the incorrect length of time Inoculum prepared from differential or selective media containing antimicrobial agents or other growth-inhibiting compounds iii. Use of the wrong incubation temperature or conditions iv. Use of wrong disk, ancillary supplies v. Improper disk placement (e.g., inadequate contact with the agar) or disk falls off agar vi. Incorrect reading or interpretation of test results vii. Transcription error d. Equipment o Not functioning properly or out of calibration (e.g., pipettes)
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6. Weekly Quality Control Testing – Out-of-Range Result Not Due to Identifiable Error If the reason for out-of-range result with the QC strain cannot be identified, perform corrective action, as follows, to determine if the error is random. Test out-of-range antimicrobial agent/organism combination on the day error is observed or as soon as F2 or F3 subculture of the QC strain is available. If the repeat results are in range, evaluate all QC results available for the antimicrobial agent/organism combination when using the same lot numbers of materials that were used when the out-of-range QC result was observed. If five acceptable QC results are available, no additional days of QC testing are needed. Different scenarios of inconsistent QC results and action to be taken is given below for better guidance a. Scenario #1:
Ampicillin E. coli ATCC® Day Lot Lot Result Action 25922; acceptable range: Number Number 15 to 22 mm Week (Disks) (MHA)
1 1 3564 16481 18
2 1 3564 16481 19
3 1 3564 16481 18
4 1 3564 16481 19
5 1 3564 16481 14 Out of range. Repeat QC same day.
5 2 3564 16481 17 In range. Five acceptable in-range QC tests for E. coli ATCC® 25922 with ampicillin disks lot 3564 and MHA lot 16481. Resume weekly QC testing.
For more trouble shooting / suggestive action refer to CLSI guideline M100-29th Ed. Table 4D, Pg.164-166.
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1 1 9661 16785 18
2 1 9661 16785 19
3 1 9661 16785 14 Out of range. Repeat QC same day.
3 2 9661 16785 18 In range. Three acceptable in-range QC tests for E. coli ATCC® 25922 with ampicillin disks lot 9661 and MHA lot 16785. Repeat QC 2 more consecutive days.
3 3 9661 16785 18 In range.
3 4 9661 16785 17 In range. Five acceptable in-range QC tests for E. coli ATCC® 25922 with ampicillin disks lot 9661 and MHA lot 16785. Resume weekly QC testing. c. Scenario#3: Ampicillin E. coli Day Lot Lot Result Action ATCC® 25922; Number Number acceptable range: 15 (Disks) (MHA) to 22 mm Week
1 1 8541 16922 17
2 1 8541 16922 21
3 1 8541 16922 28 Out of range. Repeat QC same day.
3 2 8541 16922 21 In range. Three acceptable in-range QC tests for E. coli ATCC® 25922 with ampicillin disks lot 8541 and MHA lot 16922. Repeat QC 2 more consecutive days 3 3 8541 16922 30 Out of range. Repeat QC same day.
3 4 8541 16922 28 Out of range. Perform corrective action if results for a QC strain/antimicrobial agent combination are out of range and the error is not identifiable on two consecutive days of testing/if > 3 results for a QC strain/antimicrobial agent combination are out of range during 30 consecutive days of testing. Daily QC to be continued until final resolution is achieved. See section 4.11
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7. Additional Corrective Action- Checklist Additional corrective action is required if repeat results with QC strains are still out of range. It is possible that the problem is due to a system error rather than a random error. If repeat QC errors are observed then iii. Continue Daily QC tests until final resolution of the problem is achieved. iv. If necessary, obtain a new QC strain (either from stock cultures or a reliable source) and new lots of materials (including new turbidity standards), possibly from different manufacturers. v. If the problem appears to be related to a manufacturer, contact and provide the manufacturer with the test results and lot numbers of materials used. vi. It may be helpful to exchange QC strains and materials with another laboratory (may be Reference Lab) in order to determine the root cause of out-of-range QC results where the reason is not identifiable
As part of the corrective actions, make certain that you check all of the following:
1. Zone diameters were measured correctly. ☐ Yes ☐ No 2. Turbidity standard is homogeneous and free of clumps. ☐ Yes ☐ No
3. Inoculum suspension was prepared properly. ☐ Yes ☐ No
4. Test materials were stored properly. ☐ Yes ☐ No
5. Test materials have not reached or passed their expiration dates ☐ Yes ☐ No 6. Incubator temperature and atmosphere are within the specified range and ☐ Yes ☐ No other equipment is properly maintained. 7. QC strain used for testing is acceptable. ☐ Yes ☐ No 8. QC strain maintained as per recommendations ☐ Yes ☐ No 9. The person performing testing has been certified as competent to perform ☐ Yes ☐ No the test 10. The pH of the medium is in the established range ☐ Yes ☐ No 11. The depth of the agar in the plates is 4 mm. ☐ Yes ☐ No
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8. Reporting Patient Results When Out-of-Range Quality Control Results Are Observed
When an out-of-range result occurs when testing QC strains or when corrective action is necessary, each patient test result must be carefully examined to determine if it can be reliably reported. Factors to consider may include, but are not limited to: Size and direction of QC strain error (e.g., slightly increased OR significantly increased OR decreased zone size) Actual patient result and its proximity to the interpretive breakpoint Results with other QC organisms Results with other antimicrobial agents Usefulness of the particular QC strain/antimicrobial agent as an indicator for a procedural or storage issue (e.g., inoculum dependent, heat labile)
Options to consider for patient results include: Suppress the results for an individual antimicrobial agent Review individual patient or cumulative data for unusual patterns Use an alternative test method or a reference laboratory until the problem is resolved
9. Accuracy Checklist before Reporting the Patient AST Results.
To ensure that antimicrobial susceptibility test results on patients’ isolates are accurate review all results, make certain: 1. Results with QC strains are within the acceptable range. ☐ Yes ☐ No 2. Growth is satisfactory. ☐ Yes ☐ No
3. Test is not contaminated (mixed) ☐ Yes ☐ No
4. The overall susceptibility profile or antibiograms are consistent with expected results for agents tested and the identification of the isolate; check results for ☐ Yes ☐ No all antimicrobial agents tested and not just those that will be reported. 5. The results from individual antimicrobial agents within a specific drug class follow the established hierarchy of activity rules (eg, third-generation cephalosporins are more active than first- or second-generation cephalosporins ☐ Yes ☐ No against Enterobacteriaceae).
6. The isolate is susceptible to those antimicrobial agents for which resistance has not been documented (e.g. vancomycin and Streptococcus spp.) and for which ☐ Yes ☐ No only “susceptible” interpretive criteria exist in CLSI M100 S 29, 2019 7. Atypical resistance, if present, is confirmed. ☐ Yes ☐ No
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While Testing Patient Isolates confirm the above checklist before Reporting Results
NB: Multiple test parameters are monitored by following the QC recommendations. However, acceptable results derived from testing QC strains do not guarantee accurate results when testing patient isolates.
Further confirm unusual or inconsistent results by checking for Previous results on the patient (e.g. did the patient previously have the same isolate with an unusual antibiogram?) Previous QC performance (e.g., is there a similar trend or observation with recent QC testing?) Problems with the testing supplies, process, or equipment If a reason for the unusual or inconsistent result for the patient’s isolate cannot be ascertained, a repeat of the susceptibility test or the identification, or both, may be needed. Use an alternative test method or a reference laboratory until the problem is resolved
Note: Refer CLSI M100 29th edition page 212-217, Appendix A, page 204. Suggestions for Confirming Resistant, Intermediate, or Non-susceptible Antimicrobial Susceptibility Test Results and Organism Identification
Each laboratory must develop its own policy for confirmation of unusual or inconsistent AST results. This policy should emphasize those results that may significantly impact patient care.
10. End-Point Interpretation Control Monitor end-point interpretation periodically to minimize variation in the interpretation of zone sizes among observers. All laboratory personnel who perform these tests should independently read a selected set of tests. Record the results and compare to the results obtained by an experienced reader; or, when using QC strains, compare to the expected results from M-100 Tables 4 A1-2. Generally, zone measurement readings from several individuals should not vary more than ± 2 mm.
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VI. Appendix Table 1: Disk Diffusion QC Ranges for QC Strains Disk Diffusion QC Ranges ( mm)
Antimicrobial Disk Content E. coli Staphylococcus aureus ATCC Pseudomonas aeruginosa ATCC Agent ATCC 25922 25923 27853 Amikacin 30 µg 19–26 20–26 18–26 Ampicillin 10 µg 15–22 27–35 – Azithromycin 15 µg - 21-26 - Aztreonam 30 µg 28-36 - 23-29 Cefazolin 30 µg 21-27 29-35 - Cefepime 30 µg 31-37 23-29 25-31 Cefotaxime 30 µg 29-35 25-31 18-22 Cefoxitin 30 µg 23-29 23-29 - Ceftaroline 30 µg 26-34 26-35 - Ceftazidime 30 µg 25-32 16-20 22-29 Ceftriaxone 30 µg 29-35 22-28 17-23 Cefuroxime 30 µg 20-26 27-35 - Chloramphenicol 30 µg 21-27 19-26 - Ciprofloxacin 5 µg 29-37 22-30 25-33 Clindamycin 2µg - 24-30 - Doxycycline 30 µg 18-24 23-29 - Ertapenem 10 µg 29-36 24-31 13-21 Erythromycin 15 µg - 22-30 - Fosfomycin b 200 µg 22-30 25-33 - Gentamicin c 10 µg 19-26 19-27 17-23 Imipenem 10 µg 26-32 - 20-28 Linezolid 30 µg - 25-32 - Meropenem 10 µg 28-35 29-37 27-33 Minocycline 30 µg 19-25 25-30 - Nalidixic acid 30 µg 22-28 - - Nitrofurantoin 300 µg 20-25 18-22 - Penicillin 10 units - 26-37 - Teicoplanin 30 µg - 15-21 - Tetracycline 30 µg 18-25 24-30 - Tobramycin 10 µg 18-26 19-29 20-26 TMP/SXT 1.25/23.75 µg 23-29 24-32 - Vancomycin 30 µg - 17-21 - a. Refer M100-S29 Tables 4A1-2 Pgs 150-157 for complete list. When a Commercial test system is used for AST refer to manufacturer’s instructions for QC test recommendations and QC ranges b. The 200-µg fosfomycin disk should contain 50 µg of glucose-6-phosphate. c. For control ranges of gentamicin 120-µg and streptomycin 300-µg disk use E.faecalis ATCC 29212(gentamicin:16–23 mm; streptomycin: 14–20 mm).
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Table 2: Disk Diffusion QC Ranges for Non-fastidious QC Strains and β-Lactam Combination Agents. (Un supplemented Mueller-Hinton medium)
E. coli P. ATCC aeruginosa S. aureus E. coli ATCC 25922 ATCC ATCC25923 35218 a,b
Antimicrobial Agent 27853 Disk β- β-lactamase Content Inducible lactamase negative, mecA TEM-1 AmpC negative negative MIC QC ranges, mm Amoxicillin- 20/10 µg 18–24 – 28–36 17–22 clavulanate (2:1) Piperacillin 100 µg 24–30 25–33 – 12–18 Piperacillin- 100/10 24–30 25–33 27–36 24–30 tazobactam µg
QC strain is recommended for routine QC of these combination antibiotic discs
a. Careful attention to organism maintenance (e.g. minimal subcultures) and storage (e.g. −60°C or below) is especially important for this QC strain because spontaneous loss of the plasmid encoding the β‐lactamase has been documented. If stored at temperatures above −60°C or if repeatedly sub cultured, the strain may lose its resistance characteristics and QC results may be outside the acceptable ranges. b. To confirm the integrity of the QC strain (E.coli ATCC 35218), test the β‐lactam agent highlighted in orange by disk diffusion when the strain is first sub cultured from a frozen stock culture. In-range results for the single agent indicate the QC strain is reliable for QC of β‐lactam combination agents. It is not necessary to check the QC strain again with a single agent until a new frozen stock culture is put into use, providing recommendations for handling QC strain are followed.
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Table 3: Characteristics of Routine QC Strains for Antimicrobial Susceptibility Tests
Organism Disk Diffusion QC Strains MIC Tests Other Tests Comments Characteristics Tests
E. faecalis Nonfastidious • Vancomycin Assess suitability of
ATCC 29212 gram- positive agar medium for bacteria sulfonamide or • HLAR tests trimethoprim disk • High-level diffusion tests a mupirocin resistance MIC test Escherichia coli β-lactamase Nonfastidious Nonfastidious ATCC 25922 negative gram-negative gram- negative bacteria bacteria
E. coli TEM-1 β -lactam β -lactam ATCC 35218 combination combination agents agents
Pseudomonas Inducible AmpC Nonfastidious Nonfastidious Assess suitability aeruginosa β-lactamase gram-negative gram- negative of cation content ATCC 27853b bacteria bacteria in each batch/lot of CAMHB.
Staphylococcus β-lactamase Nonfastidious High-level Little value in aureus negative gram-positive mupirocin MIC testing due ATCC 25923 mecA negative bacteria resistance disk to its extreme mupA negative diffusion test susceptibility to Inducible most drugs clindamycin resistance disk diffusion test (D- zone test)
S. aureus Weak β - Nonfastidious Oxacillin salt Assess suitability ATCC 29213 lactamase– gram- positive agar of cation content producing strain bacteria High-level in each batch/lot mecA negative mupirocin of MHB for mupA negative resistance MIC Daptomycin broth test microdilution
a. Disk diffusion and MIC end points should be easy to read as 80% or greater reduction in growth if the medium Inducible has acceptable levels of thymidine. b. May develop resistance to β -lactam antimicrobial agents after repeated subcultures. clindamycin c. Minimize this risk by sub culturing from a frozen stock culture at least monthly or whenever the strainresistance demonstrates MIC results outside the acceptable range. test Penicillin zone- edge test
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Table 4: Conditions for Disk diffusion Antimicrobial Susceptibility Test for Non- fastidious Organisms (Including routine QC Strains)
Organism/ CLSI Medi 0.5 McFarland Incubation Incubation Minimal QC Required Organism Group M100 um Inoculum Time Table Direct colony Escherichia coli ATCC suspension in 25922 broth or saline, or growth P. aeruginosa ATCC Enterobacteriacea method 35°C± 2°C; ambient 27853 2A MHA 16–18 hours e air for carbapenems
E. coli ATCC 35218 (for β-lactam/β-lactamase inhibitor combinations) Direct colony P. aeruginosa ATCC suspension in 27853 broth or saline, Pseudomonas 35°C± 2°C; ambient 2B-1 MHA or growth 16–18 hours E. coli ATCC 35218 (for β aeruginosa method air -lactam/ β -lactamase inhibitor combinations)
Direct colony P. aeruginosa ATCC suspension in 27853 broth or saline, or growth E. coli ATCC 25922 for Acinetobacter method 35°C± 2°C; ambient tetracyclines and 2B-2 MHA 20–24 hours spp. air trimethoprim- sulfamethoxazole E. coli ATCC® 35218 (for β -lactam/ β -lactamase inhibitor combinations) Direct colony 35°C ± 2°C; ambient 16–18 hours *Staphylococcus suspension in air (Testing at Staphylococcus aureus 24 hours for spp. 2C MHA broth or saline temperatures above ATCC 25923 cefoxitin with 35°C may not detect CoNS MRS) Direct colony Staphylococcus aureus suspension in 16–18 hours ATCC 25923 **Enterococcus 35°C± 2°C; ambient 2D MHA broth or saline, 24 hours for spp air or growth vancomycin method * Examine linezolid zones carefully with transmitted light; Vancomycin disk diffusion test is not recommended for S.aureus and CoNS ** Examine vancomycin zones carefully with transmitted light for small colonies or haze inside the zones of inhibition; any growth =resistance
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Table 5: Plan 15-Replicate (3×5 Day): Acceptance Criteria and Recommended Action* Number Out of Range Conclusion From Initial Number Out of Range Conclusion After Repeat With Initial Testing Testing (Based on 15 After Repeat Testing Testing (Based on 15 Replicates) Replicates) (Based on All 30 Replicates) 0–1 Plan is successful. N/A N/A Convert to weekly QC testing 2–3 Test another 3 2–3 Plan is successful. replicates for 5 days Convert to weekly QC testing ≥ 4 Plan fails. Investigate ≥ 4 Plan fails. Investigate and and take corrective take corrective action as action as appropriate. appropriate. Continue QC Continue QC each each test day test day *Assess each QC strain/antimicrobial agent combination separately.
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Table 6: Internal Quality Control for monitoring AST by disk diffusion log sheet Disk diffusion QC ranges (in mm) Antimicrobial Disk Lot no. E. coli Staph aureus Pseud E. coli agent content ATCC 25922 ATCC 25923 aeruginosa ATCC 35218 ATCC 27853
Amikacin 30 µg 19–26 20–26 18–26 Ampicillin 10 µg 15–22 27–35
Azithromycin 15 µg 21-26 Aztreonam 30 µg 28-36 23-29 Cefazolin 30 µg 21-27 29-35 Cefepime 30 µg 31-37 23-29 25-31 Cefotaxime 30 µg 29-35 25-31 18-22 Cefoxitin 30 µg 23-29 23-29 Ceftazidime 30 µg 25-32 16-20 22-29 Ceftriaxone 30 µg 29-35 22-28 17-23 Cefuroxime 30 µg 20-26 27-35 Chloramphenicol 30 µg 21-27 19-26 Ciprofloxacin 5 µg 29-37 22-30 25-33 Clindamycin 2 µg 24-30 Coamoxyclav 20/10 µg 18–24 28–36 17–22 Cotrimoxazole 1.25/23.75 µg 23-29 24-32 Doxycycline 30 µg 18-24 23-29 Ertapenem 10 µg 29-36 24-31 13-21 Erythromycin 15 µg 22-30 Fosfomycin 1 200 µg 22-30 25-33 Gentamicin 2 10 µg 19-26 19-27 17-23 Imipenem 10 µg 26-32 20-28 Linezolid 30 µg 25-32 Meropenem 10 µg 28-35 29-37 27-33 Nitrofurantoin 300 µg 20-25 18-22 Norfloxacin 10 µg 28-35 17-28 22-29 Penicillin 10 units 26-37 Piperacillin 100 µg 24–30 25–33 12–18 Piperacillin- 100/10 µg 24–30 25–33 27–36 24–30 Tazobactam Teicoplanin 30 µg 15-21 Tetracycline 30 µg 18-25 24-30 Tobramycin 10 µg 18-26 19-29 20-26 Vancomycin 30 µg 17-21
1 Fosfomycin 200-µg disk should contain 50 µg of glucose-6-phosphate 2 For Gentamicin HLAR use 120-µg disk (& to monitor MH agar quality) use E. faecalis ATCC 29212; QC range: 16–23 mm
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VII. Annexures
Guidance on MHA and Disks a. Mueller Hinton Agar (MHA) a. Prepare MHA from a commercially available dehydrated base according to the manufacturer’s instructions. b. Immediately after autoclaving, allow the agar to cool in a 45 to 50°C water bath. c. Pour freshly prepared and cooled medium into glass or plastic flat-bottomed Petri on a level, horizontal surface to give a uniform depth of approximately 4 mm (60 to 70 mL of medium for plates with a diameter of 150 mm and 25 to 30 mL for plates with a diameter of 100 mm). d. Fresh plates should be used the same day or stored in a refrigerator (2- 8ºC). e. Examine a representative sample of each batch of plates for sterility by incubating at 35°C ± 2°C for 24 hours or longer f. Plates are stable for seven days, but could have a longer shelf life if precautions are taken to prevent drying (wrapped in plastic to minimize evaporation) and QC is in range at the time of testing. g. Just before use, if excess moisture is visible on the surface, plates should be placed in an incubator (35ºC) or, with lids ajar, in a laminar-flow hood at room temperature until the moisture evaporates (usually 10 to 30 minutes). h. Check the pH of each batch of MHA as reference in the a.1 QC reporting format below. i. The pH of MHA should be between 7.2 and 7.4 at room temperature (refer to CLSI guidelines M02 and manufacturer instructions for further details). j. If the pH is less than 7.2 certain drugs will appear to lose potency (eg, aminoglycosides, macrolides), whereas other antimicrobial agents may appear to have excessive activity (e.g., tetracycline). If the pH is greater than 7.4, the opposite effects can be expected.
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a.1 Media QC recording format
Media QC record Name of the medium ATCC strains used Expected reactions Strain 1 Strain 2 Strain 3 Acceptable Expected reactions obtained Unacceptable Expected reactions not obtained
Date of preparation Lot number Expiry Autoclave PH Physical Sterility Growth Reviewed Date examination check check with by ATCC strains Hold time Temperature
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For each batch of media prepared, in addition to fertility check, perform following quality control
Positive controls QC Strain Tested for Expected results
Escherichia coli ATCC® Nonfastidious gram-negative bacteria Good growth 25922 Nonfastidious gram-negative bacteria Pseudomonas Assess suitability of cation content in Good growth aeruginosa ATCC® 27853 each batch/lot of CAMHB Staphylococcus Nonfastidious gram-positive bacteria Good growth aureus ATCC® 25923 E. faecalis ATCC 29212 Assess suitability of medium for sulfonamide or trimethoprim disk diffusion tests Negative control Un-Inoculated medium No growth b. Antimicrobial Disks & Quality Specifications Purchase Disks from a reliable commercial vendor. a) Check for following at the time of receiving: The disks should be accompanied, at minimum, by a certificate of analysis stating the content of the disks. Lot number, expiration date, and assurance that they were tested and performed according to established QC specifications. c. Storage of Antimicrobial Disks a. Refrigerate the cartridges at 8°C or below or freeze at −14°C or below in a non-frost-free freezer until needed. b. Sealed packages of disks that contain drugs from the ß-lactam class should be stored frozen (-20ºC), except for a small working supply, which may be refrigerated for at most one week. Some labile agents (e.g, imipenem, cefaclor and clavulanic acid combinations) may retain greater stability if stored frozen until the day of use. c. Remove the disk cartridges from the refrigerator or freezer 1–2 hours before use to equilibrate to room temperature before use. d. Once a cartridge of disks has been removed from its sealed package, place it in a tightly sealed, desiccated container for storage. e. Refrigerate the container with the disk –dispensing apparatus when not in use. f. Use only those disks that have not reached the expiry date stated on the label. Discard disks when they reach the expiry date.
21 | P a g e d. Batch or Lot Quality Control a. Check each batch or new lot of MHA /antibiotic discs with the appropriate QC strains before or concurrent with their first use for testing patient isolates. Performance tests with each lot of MHA must conform to the control limits of disk QC ranges for organisms and antibiotics. b. If zone diameter measurements obtained do not fall within acceptable range (Table 2-3 Appendix B) proceed with corrective action. c. From each batch of prepared media, keep MHA plates and other agar media not containing antimicrobials overnight at 37ºC for sterility checking prior to inoculation. d. Maintain records to include, at a minimum, the lot numbers, expiry dates, and date of use of all materials and reagents used in performing susceptibility tests. e. Inoculum Preparation for Disk Diffusion Tests
i. Turbidity Standard for Inoculum Preparation a. Use a BaSO4 turbidity standard equivalent to a 0.5 McFarland standard to standardize the inoculum for susceptibility testing. b. Verify the correct density (0.08 to 0.13) of the standard using a spectrophotometer at absorbance 625nm. c. The density of BaSO4 standard must be verified monthly, otherwise should be made fresh. For further details see Annexure
ii. Direct colony suspension method for Inoculum Preparation Prepare a saline suspension of the isolate from an overnight incubated agar plate (use a nonselective medium, such as blood agar) to obtain 0.5 McFarland turbidity (1.5 x 108 cfu/ ml of E. coli ATCC®25922). iii. Growth method for Inoculum Preparation a. With a sterile straight wire touch the top of each of four to five colonies of the same morphological type, and inoculate tryptic soya or any suitable broth. b. Incubate tube at 35ºC till turbidity of 0.5 McFarland tube or more is achieved (usually 2 to 6 hrs). Then with sterile normal saline adjust turbidity to exactly 0.5 McFarland. c. Use of the spectrophotometric method is preferable over visual determination. d. For visual comparison with the 0.5 McFarland standard, look through the broths in transmitted light against a white background with contrasting black lines.
iv. Inoculation of Test Plates a. Inoculate MHA plate within 15 minutes of adjusting the inoculum turbidity to 0.5 McFarland standards. b. Dip a sterile cotton swab into the suspension, rotate several times, and gently press onto the inside wall of the tube above the fluid level to remove excess inoculum from the swab.
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c. Inoculate the dried surface of an MHA plate by streaking three times over the entire surface of the agar, with the plate rotated approximately 60o each time to ensure even distribution of the inoculum. d. Make a final sweep of the swab around the agar rim. The lid may be left ajar for 3 to 5 minutes but no longer than 15 minutes to allow any excess surface moisture to be absorbed before the drug-impregnated discs are applied.
v. Application of Disks to Inoculated Agar Plates e. Selected antimicrobial discs should be dispensed evenly onto the agar plate individually or with dispending apparatus. f. They discs must be distributed evenly so they are no closer than 24mm from centre to centre. g. No more than 12 discs are applied on a 150 mm plate or 6 discs on a 100 mm plate or 5 discs on a 90mm plate h. Each disc must be pressed down to ensure complete contact with the agar surface. i. Dispensing too near to the edge of the plate should be avoided. j. Because some of the drugs diffuse instantaneously, a disc should not be relocated once it has come in contact with the agar surface. k. Incubation– Invert the plates and place in an incubator set at 35º ± 2ºC within 15 minutes after discs are applied.
VIII. References 1. CLSI. Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard— Twelfth Edition. CLSI document M02-A12. Wayne, PA: Clinical and Laboratory Standards Institute; 2015. 2. CLSI. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-eighth edition. CLSI document M100-A28 edition. Wayne, PA: Clinical and Laboratory Standards Institute; 2018.
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