a. 379 laboratories provided quantitative results, e.g (DD method) to 35.4% (MIC method) of all participants; see Table 2.

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1 AND QUANTITATIVE PRECISION (SAMPLE UR-01, 2017) Background and Plan of Analysis Sample UR-01 (2017) was sent to API participants as a simulated urine culture for recognition of a significant pathogen colony count, to perform the identification of the organism, and to test the organism by routinely used antimicrobial susceptibility testing (AST) method/product. The sample contained an Escherichia coli strain (ATCC 25922), a quality control (QC) organism commonly applied by AST quality assurance procedures for disk diffusion (DD) or dilution MIC methods. 1-4 The E. coli is pan-susceptible to all frequently utilized Gram-negative-active antimicrobial agents. The use of this QC organism in an AST proficiency challenge provides the opportunity to analyze survey results for recognizing significant activity of antimicrobials against a susceptible strain. Furthermore, participant laboratories were requested to forward an additional response form with quantitative results (inhibitory zone diameters for DD; MICs for dilution tests), as well as the AST interpretive category criteria used to define susceptibility or resistance. 3-5 The overall response statistics included 16,610 potentially gradable category results; a total of 99.3% (16,496) were reported as susceptible. Among these over 1,100 laboratories, 379 participants forwarded quantitative AST results including 35.4 and 41.1% for MIC and DD test users, respectively (Tables 1-6). The interpretive category criteria utilized by each type of test/device applied are tabulated in Table 1. The breakpoint category criteria of the Clinical and Laboratory Standards Institute (CLSI) were most often used at 66.2% of laboratories overall (range, %); and USA-FDA breakpoints were applied by 19.0% (range, %) of participants. 3 Non-USA or unlisted/unknown criteria were cited by 14.8% of API Survey respondents (Table 1). 4,5 Table 1. API Survey subscriber use of antimicrobial susceptibility breakpoint organization criteria 3-5 for sample UR-01 (2017), by listed AST method or commercial product applied (379 responses). a AST breakpoint organizations AST method used by API participant (%): Disk Diffusion (DD) BD Phoenix MicroScan Vitek 2 CLSI FDA Other Not listed Listed a. 379 laboratories provided quantitative results, e.g (DD method) to 35.4% (MIC method) of all participants; see Table 2.

2 Categorical and Quantitative Accuracy Analyses Overall Categorical Accuracy (CA) for DD and MIC methods Table 2 lists the CA of both major AST testing groups (DD and commercial MIC devices) for the 25 mosttested antimicrobial agents. These tested drugs had 100 reported categorical results with the greatest number of responses for ampicillin (1,044), trimethoprim-sulfamethoxazole (TMP-SMX; 1,039), nitrofurantoin (1,027), gentamicin (1,023), ciprofloxacin (984) and cefazolin (953). Laboratory categorical responses for DD users (90 TMP-SMX values) were greatly outnumbered by commercial MIC test users (949 TMP-SMX values; greatest number for gentamicin at 967 results). Approximately 91.5% of participating laboratories utilized a MIC-based system with only 8.5% using DD; 2 see Table 2. Table 2. Categorical accuracy (CA) for DD and MIC methods 1, 2 when testing UR-01 (2017), an E. coli quality control (QC) organism (ATCC 25922). a Antimicrobial agent Expected No. tests (% CA) for: b category a DD method MIC method Amikacin Susceptible 10 (100.0) 440 (99.8) Amoxicillin-Clavulanate Susceptible 43 (97.7) 489 (99.4) Ampicillin Susceptible 81 (95.1) 963 (98.7) Ampicillin-Sulbactam Susceptible 10 (100.0) 780 (99.5) Aztreonam Susceptible 2 (100.0) 382 (99.2) Cefazolin Susceptible 38 (86.8) 915 (99.2) Cefepime Susceptible 5 (100.0) 705 (99.3) Cefotaxime Susceptible 3 (100.0) 319 (99.1) Cefoxitin Susceptible 5 (80.0) 350 (99.1) Ceftazidime Susceptible 12 (100.0) 629 (99.7) Ceftriaxone Susceptible 41 (100.0) 839 (99.8) Cefuroxime Susceptible 11 (81.8) 419 (98.8) Ciprofloxacin Susceptible 84 (100.0) 900 (99.4) Ertapenem Susceptible 4 (100.0) 586 (99.3) Gentamicin Susceptible 56 (100.0) 967 (99.8) Imipenem Susceptible 13 (100.0) 577 (99.1) Levofloxacin Susceptible 30 (100.0) 896 (99.8) Meropenem Susceptible 1 (100.0) 406 (99.3) Nitrofurantoin Susceptible 89 (98.9) 938 (99.7) Piperacillin-Tazobactam Susceptible 10 (100.0) 798 (99.4) Tetracycline Susceptible 35 (100.0) 418 (99.5) Ticarcillin-Clavulanate Susceptible 3 (100.0) 121 (100.0) TMP-SMX Susceptible 90 (98.9) 949 (99.9) Tobramycin Susceptible 11 (100.0) 747 (99.9) Trimethoprim Susceptible 7 (100.0) 105 (99.0) a. E. coli ATCC is a pan-susceptible QC strain. 3 b. CA determined from approximately 90 and 967 participants using DD and MIC methods, respectively; see gentamicin and trimethoprim-sulfamethoxazole (TMP-SMX) data.

3 The graded CA for the DD method was 97.8% (2.2% false-resistant or intermediate errors). The lowest CA among drugs tested by DD was for three cephalosporins (cefazolin [86.8%], cefoxitin [80.0%], cefuroxime [81.8%]), each having a smaller sample size of 5 to 38 results. In contrast, the MIC method CA was 99.5% overall, ranging from 98.7% (ampicillin) to 100.0% (ticarcillin-clavulanate); falsenonsusceptible results were quite rare for TMP-SMX (0.1%), nitrofurantoin (0.3%), aminoglycosides ( %) and the fluoroquinolones ( %). Several antimicrobials of potential clinical utility for UTI therapy were reported by a small number (14-78) of laboratories. Cefixime (17 responses), cefotetan (78), cefpodoxime (25), doripenem (44), nalidixic acid (14) and norfloxacin (32) were tested with excellent CA rates. The reporting of inappropriate drugs for this pathogen and/or infection site is considered unacceptable. These occurrences included: azithromycin (three results), chloramphenicol (one), clarithromycin (one), colistin (one), erythromycin (one), moxifloxacin (12), penicillin (three) and vancomycin (two). Some recently FDA-approved antimicrobials having expanded activity versus the Enterobacteriaceae and an UTI clinical indication should be considered for AST reporting, such as ceftazidime-avibactam and ceftolozane-tazobactam. Only three participants among the more than 1,100 laboratories reported ceftolozane-tazobactam MIC results. CA and QA Analyses of the DD method 2 Table 3 lists the DD zones of inhibition for the 18 drugs most reported on the supplemental quantitative result form. CA among these tallied antimicrobials was 97.3% (97.8% overall; Table 2) and the median zone diameter in mm for each drugs was within the published QC range published in CLSI 100-S27. 3 However, the QA results (% of zones within the published QC guidances) were compromised at only 89.3%. The antimicrobials having the highest rates of "out-of control" results were: amoxicillinclavulanate (28.6%), imipenem (20.0%), cefazolin and tetracycline (18.7%) and ampicillin-sulbactam (16.7%). A total of 660 zones were forwarded by DD users for these analyses.

4 Table 3. Categorical (CA) and quantitative accuracy (QA) of the Kirby-Bauer disk diffusion (DD) method 2 when testing UR-01, a simulated UTI sample containing E. coli ATCC 25922, the commonly used quality control (QC) organism. Antimicrobial Breakpoint Reference QC Median Acceptable DD results (%) tested zone (mm) zone range (mm) zone (mm) CA a QA b Amikacin Amoxicillin-Clavulanate Ampicillin Ampicillin-Sulbactam Cefazolin Cefepime Ceftazidime Ceftriaxone Ciprofloxacin Gentamicin Imipenem Levofloxacin Meropenem Nitrofurantoin Piperacillin-Tazobactam Tetracycline Tobramycin TMP-SMX c a. Graded categorical DD response (susceptible) for listed agents that ranged from only 1 test (meropenem) to 90 results (TMP-SMX). b. Acceptable quantitative (inhibiting zone diameter) results within published QC ranges. 3 c. TMP-SMX = Trimethoprim-Sulfamethoxazole. CA and QA Analyses of the BD Phoenix MIC Device Three commercial MIC devices were tabulated separately (Tables 4-6), each having enough results to calculate both CA and QA (compared to published MIC QC ranges) rates. 3 Table 4 lists the BD Phoenix results (2.8% of MIC reporting sites) for the 18 antimicrobial agents that allowed comparisons of the most reported MIC to the breakpoint criteria and reference MIC QC range, and to calculate the CA and QA.

5 Table 4. Categorical (CA) and quantitative accuracy (QA) of the BD Phoenix MIC device when testing UR-01, a simulated UTI sample containing E. coli ATCC 25922, the commonly used quality control (QC) organism. 3 Antimicrobial tested Breakpoint MIC (µg/ml) Reference MIC QC range (µg/ml) Most reported MIC (%) Acceptable MIC result (%) CA a QA b Amikacin (100) Amoxicillin-Clavulanate 8/4 2/1-8/4 4/2 (75) Ampicillin (100) Ampicillin-Sulbactam 8/4 2/1-8/4 4/2 (100) Cefazolin (75) Cefepime (100) Ceftazidime (88) Ceftriaxone (80) Ciprofloxacin (100) Gentamicin (50) Imipenem (100) Levofloxacin (100) Meropenem (60) Nitrofurantoin (100) Piperacillin-Tazobactam 16/4 1/4-4/4 2/4 (70) Tetracycline (50) Tobramycin (100) TMP-SMX c 2/38 0.5/ /9.5 (100) a. Graded categorical MIC response (susceptible) for listed agents across all tabulated MIC testing devices. b. Acceptable quantitative results (MIC in µg/ml) within published QC range for this brand of commercial MIC device. 3 c. TMP-SMX = Trimethoprim-Sulfamethoxazole. The CA rate across these listed agents was 99.5%, the same as for all reported antimicrobials (Table 2). When the reported BD Phoenix MIC results were compared to the QC range of the E. coli ATCC range 3 for each drug, two concerns were observed: 1) the lowest reported MIC was not within the published QC range 3 or 2) the most reported MIC value was at the upper limit of the QC range. Such results do not allow this commonly used QC strain to be routinely useful for one-half of the 18 drugs and with four additional drugs, only the highest MIC in the QC range was tested; thus requiring the testing of additional QC organisms to assure QA of this test device. The QA for BD Phoenix using this QC strain could only be calculated at 56.0% (range, %) due to the limited number of MIC dilutions reported by this device against the monitored, commonly tested agents (Table 4). CA and QA Analyses of the MicroScan MIC System Table 5 lists the MicroScan device results (used by 43.6% of MIC reporting sites) for the 18 tabulated antimicrobials. When comparing the most reported MIC results for each drug, the dominant MIC was a

6 concentration less than or equal to the published susceptible breakpoint (example, ampicillin at 8 µg/ml). This occurred at 90% for 11 of 18 drugs. For ceftriaxone, the most reported MIC was 8 µg/ml, three log2 dilution steps greater than the current breakpoint published by several organizations 3-5 and the USA- FDA. CA (99.5%) for MicroScan was acceptable, but the QA analysis was nearly impossible to calculate because the reported MIC results were generally well above the published QC range for each drug, e.g. 15 of 18 drugs assessed. 3 The remaining three antimicrobials (amoxicillin-clavulanate, ampicillin, ampicillin-sulbactam) had the reported MIC at the upper limit of the MIC QC range (Table 5). The overall QA for the 18 drugs was only 19.1%. Table 5. Categorical (CA) and quantitative accuracy (QA) of the MicroScan MIC device when testing UR-01, a simulated UTI sample containing E. coli ATCC 25922, the commonly used quality control (QC) organism. 3 Antimicrobial tested Breakpoint MIC (µg/ml) Reference MIC QC range (µg/ml) Most reported MIC (%) Acceptable MIC result (%) CA a QA b Amikacin (98) Amoxicillin-Clavulanate 8/4 2/1-8/4 8/4 (94) Ampicillin (96) Ampicillin-Sulbactam 8/4 2/1-8/4 8/4 (92) Cefazolin (69) Cefepime (52) Ceftazidime (90) Ceftriaxone (60) Ciprofloxacin (97) Gentamicin (82) Imipenem (68) Levofloxacin (95) Meropenem (66) Nitrofurantoin (99) Piperacillin-Tazobactam 16/4 1/4-4/4 16/4 (95) Tetracycline (98) Tobramycin (93) TMP-SMX c 2/38 0.5/9.5 2/38 (95) a. Graded categorical MIC response (susceptible) for listed agents across all tabulated MIC testing devices. b. Acceptable quantitative results (MIC in µg/ml) within published QC range for this brand of commercial MIC device. 3 c. TMP-SMX = Trimethoprim-Sulfamethoxazole. Of additional concern, two cefazolin MIC results were reported as either 2 or 8 µg/ml for this UTI sample. This implies use of differing device products with acceptable and unacceptable QA for this QC strain. Application of these products by survey participants should follow manufacturer QC instructions as

7 well as to assure QA with QC organisms having MIC values within published ranges. 3, 4 Furthermore, refer to current breakpoint criteria tables, to interpret the cefazolin MIC results when testing UTI isolates ( 16 µg/ml as susceptible and as a surrogate predictor for selected oral cephalosporins). 3-5 The oral cephalosporins predicted as active by the susceptibility to cefazolin are: cefaclor, cefdinir, cefpodoxime, cefprozil, cefuroxime, cephalexin and loracarbef. 3 CA and QA Analyses of the Vitek 2 MIC System Table 6 presents the Vitek 2 device results (used by 53.6% of MIC reporting sites) for the 18 tabulated antimicrobial agents. MIC results from this product are calculated from testing several drug concentrations and reported for a validated MIC range for each agent. The most reported calculated MIC values are found in Table 6, and are usually at the lowest extreme of the calculated MIC range but generally extends below the published breakpoints. 3-5 Table 6. Categorical (CA) and quantitative accuracy (QA) of the Vitek 2 MIC device when testing UR-01, a simulated UTI sample containing E. coli ATCC 25922, the commonly used quality control (QC) organism. 3 Antimicrobial Breakpoint Reference MIC Most reported Acceptable MIC result (%) tested MIC (µg/ml) QC range (µg/ml) MIC (%) CA a QA b Amikacin (97) Amoxicillin-Clavulanate 8/4 2/1-8/4 4/2 (75) Ampicillin (84) Ampicillin-Sulbactam 8/4 2/1-8/4 2/1 (64) Cefazolin (100) Cefepime (99) Ceftazidime (100) Ceftriaxone (100) Ciprofloxacin (99) Gentamicin (99) Imipenem (99) Levofloxacin (99) Meropenem (100) Nitrofurantoin (99) Piperacillin-Tazobactam 16/4 1/4-4/4 4/4 (99) Tetracycline (100) Tobramycin (99) TMP-SMX c 2/38 0.5/9.5 1/19 (98) a. Graded categorical MIC response (susceptible) for listed agents across all tabulated MIC testing devices. b. Acceptable quantitative results (MIC in µg/ml) within published QC range for this brand of commercial MIC device. 3 c. TMP-SMX = Trimethoprim-Sulfamethoxazole.

8 This device produced more MIC results that could be assessed for QA versus the E. coli ATCC QC strain (Table 6). 3 Where MIC results could be evaluated for QA, % of MIC values were within the QC range (11 of 18 drugs). However, and like other MIC devices, Vitek 2 often does not extend the calculated MIC dilution range significantly below the published breakpoint concentration. 3 The overall QA for Vitek 2 versus the 18 drugs was 60.9% (MicroScan at 19.1%; BD Phoenix at 56.0%), and the DD method was 89.3%. Major Findings and Points of Concern from Analysis of this Sample (UR-01, 2017) Major Findings for E. coli ATCC UTI Sample Over 1,100 laboratories participated in this survey, approximately 40% forwarding quantitative AST results (MICs and zone diameters) Commercial MIC-based devices (Vitek 2 > MicroScan > BD Phoenix) dominated AST use at >90.0% among 1,610 potentially gradable responses. Interpretive breakpoint criteria applied to AST results were: CLSI (66.2%) > USA-FDA (19%) > others. CA results for 25 drugs were: DD (97.8% correct) and all commercial MIC devices (99.5%). QA Findings (% of results within published CLSI QC ranges) For DD method the QA was at 89.3%, highest "out-of-control" rates for amoxicillin-clavulanate and some other β-lactams. For BD Phoenix device the QA was at 56.0% For MicroScan products the QA was only 19.1% For Vitek 2 system the QA was 60.9% Points of Concern DD QA was below 90% overall Commercial MIC method products rarely or do not directly test/calculate MIC endpoints in a dilution range were the E. coli ATCC can be used as an effective QC strain. This fact limits precision use of MIC results to guide clinical therapy (dosing and TDM) via evolving "Antibiotic Stewardship" programs. Some results forwarded by participating laboratories imply that category breakpoint interpretations are not current with regulatory or other breakpoint publications. 3-5

9 Recommendations Correct/re-evaluate any test procedure where unacceptable categorical grades were received. Re-evaluate AST QC procedures to assure that QC strains are routinely tested to provide measures of QA for each antimicrobial, not just a CA metric. Assure that current category interpretive breakpoints are being applied locally and/or via utilized commercial AST product software. 3-5 References 1. Clinical and Laboratory Standards Institute M07-A10. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard- tenth edition. Wayne, PA. CLSI, Clinical and Laboratory Standards Institute M02-A12. Performance standards for antimicrobial disk susceptibility tests; Twelfth Edition. Wayne, PA. CLSI, Clinical and Laboratory Standards Institute M100-S27. Performance standards for antimicrobial susceptibility testing: 27th informational supplement. Wayne, PA. CLSI, EUCAST. Breakpoint tables for interpretation of MICs and zone diameters. Version 7.0, January Available at Accessed January USCAST. Breakpoint tables for interpretations of MICs and Zone Diameters, Version 3.0, January Available at Accessed March 2017.

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