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(IQC) Antimicrobial Susceptibility Tests Using Disk Diffusion 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 1 | P a g e 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 2 | P a g e 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. 3 | P a g e 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
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