Collaborative Evaluation of the UniScept Quantitative Antimicrobial ...

JOURNAL OF CLINICAL MICROBIOLOGY, June 1984, p. 733-735

Vol. 19, No. 6

0095-1137/84/060733-03$02.00/0 Copyright © 1984, American Society for Microbiology

Collaborative Evaluation of the UniScept Quantitative Antimicrobial Susceptibility Test HENRY D.

ISENBERG,'* RICHARD F. D'AMATO,2 GEOFFREY A. McKINLEY,3

LISA HOCHSTEIN,2

AND

JANE

SAMPSON-SCHERER1

Long Island Jewish-Hillside Medical Center, New Hyde Park, New York 110421; The Catholic Medical Center of Queens and Brooklyn, Jamaica, New York 114322, and Analytab Products, Plainview, New York J18033 Received 22 December 1983/Accepted 22 February 1984

UniScept (Analytab Products, Plainview, N.Y.) is a commercially prepared microdilution antimicrobial susceptibility system for the determination, in a single tray, of antimicrobial MICs for gram-negative and gram-positive bacteria and those isolated from urinary tract infections. The system showed excellent correlation with the reference microdilution approach for organisms from clinical specimens and with stock and reference cultures. Intra- and interlaboratory reproducibility was high. The rapid increase of new antimicrobial agents, the requirements of clinicians to select a suitable drug in the face of increasing and changing bacterial resistance patterns, and the need of clinical microbiologists to meet these challenges in a cost-effective fashion have encouraged the appearance of several commercial microdilution antimicrobial susceptibility systems. They are available as frozen trays or in an airdried form and are directed specifically against either grampositive or gram-negative bacteria and in specific configurations for organisms isolated from urinary tract cultures. Revisions of these antimicrobial panels are required frequently with the availability of new drugs. UniScept (Analytab Products, Plainview, N.Y.) is a lyophilized microdilution system that contains several concentrations of 22 antimicrobial agents in a single tray suitable for the determination of the susceptibility of both gram-positive and gram-negative bacteria. This report summarizes a comparison of the UniScept system with a standard microdilution method.

isolates encountered in practice. Table 1 summarizes the institutional distribution of these bacteria. The bacteria were isolated and identified in accordance with the approaches outlined in the Manual of Clinical Microbiology (2), and their antimicrobial susceptibility was determined with both systems (see below). Inoculum preparation. All inocula were prepared from organisms cultivated for 18 to 20 h on Trypticase soy agar with 5% defibrinated sheep blood (BBL Microbiological Systems, Cockeysville, Md.) at 35°C. Several colonies were suspended in 5.0 ml of 0.85% NaCl to a density that matched a 0.5 McFarland turbidity standard. A 1:100 dilution of the standardized suspension in 0.85% NaCl served as the inoculum for the experimental UniScept system. The same standardized suspension diluted 1:100 in Mueller-Hinton broth (BBL) served as inoculum for the standard microdilution system. Both systems were inoculated with a mechanical, 10-tip, hand-operated device that delivered 50 ,ul per well. Antimicrobial susceptibility test. (i) Reference method. All microdilution trays were prepared in one laboratory for use in all studies by the three participating laboratories as directed in the proposed standard by the National Committee on Clinical Laboratory Standards, (3). All three laboratories performed the dilution susceptibility test in accordance with this proposal. (ii) UniScept. The MIC component of the UniScept system consists of a 120-well tray with the antimicrobial concentration ranges shown in Table 2. The doubling dilutions of the antimicrobial agents in Mueller-Hinton broth are lyophilized, and a well for growth control is present. The trays can be used for either gram-positive or gram-negative bacteria. After 18 to 20 h of incubation at 35°C, results are determined by visual inspection with a convex-mirror-enlarged image. The same antimicrobial agents were used in the preparation of the reference microdilution trays. The MIC was that concentration of each antimicrobial agent that prevented discernible growth of the test organism. Quality control. E. coli ATCC 25922, P. aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213, and Streptococcus faecalis ATCC 29212 were used daily to control the performance of both systems. RESULTS Each UniScept result was compared with the matching reference microdilution MIC determined with the same

MATERIALS AND METHODS Bacteria. (i) Stock organisms. Stock organisms consisted of American Type Culture Collection organisms and bacteria selected from laboratory stocks that represented a spectrum of response to various antimicrobial agents. These organisms were established as stock cultures for the consistency of their susceptibility profiles with established reference methods. The bacteria were stored at -70°C until used. These organisms were tested in each laboratory on 3 successive days to ascertain the reproducibility of the systems. The following bacteria were used to this end: Staphylococcus aureus, seven isolates, five of which were methicillin resistant; Staphylococcus epidermidis, two isolates; Streptococcus faecalis, four isolates; Streptococcus faecium, two isolates; Pseudomonas aeruginosa, five isolates, three of which were resistant to all aminocyclitols included in the panel; Escherichia coli, four isolates; Klebsiella pneumoniae, three isolates; Serratia marcescens, one isolate; Enterobacter cloacae, one isolate; and Shigella flexneri, one isolate. (ii) Clinical specimens. Approximately 100 bacterial isolates were selected from clinical specimens in each participating laboratory to represent the normal distribution of *

Corresponding author. 733

734

ISENBERG ET AL.

J. CLIN. MICROBIOL.

TABLE 1. Distribution of bacteria by institution No. of isolates testeda Organism LIJ-HMC CMC API Citrobacter freundii ...........0.......... 0 2 Enterobacter cloacae .................... 6 7 5 Escherichia coli ........................ 13 14 12 Klebsiella pneumoniae ......... 8 7 ......... 5 Proteus mirabilis ....................... 3 3 5 Providencia spp. ....................... 1 0 2 Pseudomonas aeruginosa ................ 8 7 5 Salmonella spp ......................... 3 3 4 Serratia marcescens .......... .......... 5 5 5 Shigella spp. .......................... 3 3 3 Enterococcus ........................... 16 13 16 Staphylococcus aureus ......... ......... 27 24 25 7 9 8 Coagulase-negative staphylococci ......... a LIJ-HMC, Long Island Jewish-Hillside Medical Center; CMC, Catholic Medical Center; API, Analytab Products, Inc.

standard inoculum and incubated and read at the same time. The comparison is expressed as an MIC ratio (UniScept MIC/reference MIC), similar to that used by Gavan et al. (1). When the two MICs were identical, the ratio was 1; when the UniScept MIC was greater than the reference method, the ratios were 2, 4, etc.; and when the UniScept MIC was less than the reference method, the ratios were 0.5, 0.25, etc. The two systems were considered in agreement when the MIC ratios ranged from 0.5 to 2.0 (±1 doubling dilution). Table 3 demonstrates the results with staphylococci. The agreement ranged from 87 to 100% for the panel of antimicrobial agents. Methicillin-resistant staphylococci were detected accurately by both approaches. The methicillin-resistant bacteria had been screened and confirmed as resistant to the drug before inclusion in the study. The UniScept formulation for methicillin contains NaCl to insure detection of methicillin resistance. TABLE 2. Antimicrobial agents and their dilutions

Agent'

Agent" Tetracycline (GN) ................................

Dilution range

~~~~~~~~(p.g/ml)

1-16 4-8 1-16 Methicillin (S) .................................... ..................... 0.5-8 Erythromycin 0.5-8 ...................... Clindamycin ...................... 0.5-8 Vancomycin Amikacin ....................................... 2-32 Gentamicin ...................................... 0.5-8 ...................... 0.5-8 Tobramycin Trimethoprim-sulfamethoxazole .............. 1-19-4-76 Nitrofurantoin 16-64 ..................... ................... 1-16 Chloramphenicol Cefotaxime 2-32 ...................... Cefamandole ..................... 2-32 Cefoxitin ........................................ 2-32 2-32 Cephalothin (GN, S) .............................. 4-64 ..................... Cefoperazone Moxalactam ...................................... 4-64 Mezlocillin 16-256 ...................... 8-512 Piperacillin ....................................... Carbenicillin 8-512 ..................... ....................... 0.25-16 Ampicillin Penicillin ........................................ 0.06-16 a GN, Gram-negative organisms; GP, gram-positive organisms; 5,

Tetracycline (GP)

staphylococci.

TABLE 3. Staphylococcal MIC ratiosa No. of tests with the follow-0.25 0.5

Amikacin ...................

Ampicillin .................. Cephalothin ................. Chloramphenicol ............ Clindamycin ................ Erythromycin ............... Gentamicin ................. Methicillin .................. Nitrofurantoin ............... Penicillin ...................

Tetracycline ................ Tobramycin ................. Trimethoprimsulfamethoxazole ..........

ratio':

ing MIC

Antimicrobial agent

1 1 1 3 0 0 1 3 0 0 1 1

3 9 7 50 0 2 4 23 3 3 0 5

1

2

%

Agree-4

44 41 11 66 17 7 89 2 1 46 1 0 98 2 0 97 1 0 84 9 2 66 7 1 97 0 0 66 18 13 99 0 0 89 3 2

mentC 88.0 92.0 98.0 97.0 100.0 100.0 97.0 96.0 100.0 87.0 99.0 97.0

0 1

6 91 3 0 100.0 2 68 28 1 98.0 a A total of 100 staphylococcal isolates were used, including 76 S. aureus and 24 coagulase-negative staphylococci. b UniScept MIC/reference MIC. C Overall percent agreement was 96.4%.

Vancomycin ................

Table 4 summarizes the results with 45 enterococcal isolates and underlines the degree of agreement; only gentamicin results agreed less than 93%. All MIC ratios for the gram-negative rods showed no appreciable differences between the two test modalities (Table 5). Table 6, summarizing the 4,439 bacterium-antimicrobial agent combinations, supports the conclusion that the UniScept MIC system performs at the same level as the reference method. This comparison between the UniScept system and the reference method was analyzed with emphasis on the interpretive standards susceptible, moderately susceptible, and resistant (3); the conditionally susceptible category, addressing the susceptibility of organisms isolated from lower urinary tract infections in which achievable antimicrobial levels greatly exceed those possible in plasma, was not considered in the interpretation of our results. Analyses of interpretive standards were applied to the findings. For the

...................

TABLE 4. Enterococcal MIC ratios' No. of tests with the follow-

ing MIC

Antimicrobial agent

ratio': 2

>4

Amikacin ................... Ampicillin ..................

0 0 3 0

1 32 10 8 36 1 21 21 0 5 32 8

2 0 0 0

Gentamicin ................. Nitrofurantoin ............... Penicillin ...................

0 0 0 0 3

4 4 3 8 1 11

s0.25 0.5

Chloramphenicol ............ Clindamycin ................ Erythromycin ............... 3 Tetracycline ................ Tobramycin ................. Trimethoprimsulfamethoxazole ..........

1

32 23 42 28 44 25

ment'

95.6

6 0 7 11 0 0 9 0 0 0 6 0

100.0 93.3 100.0 93.3 75.6 100.0 100.0 100.0 93.3

0 0

100.0 97.7

0 0 45 0 1 7 33 4 a A total of 45 enterococcal isolates were used. b UniScept MIC/reference MIC. c Overall percent agreement was 95.7%.

Vancomycin ................

% Agree-

VOL. 19, 1984

UNISCEPT MIC EVALUATION

TABLE 5. MIC ratios of gram-negative rodsa No. of tests with the follow-

%

ing MIC ratio': Agrees0.25 0.5 1 2 -4 ment' 6 96 40 3 96.6 Amikacin ................... 2 95.9 Ampicillin .................. 0 12 104 25 6 90.5 Carbenicillin ................ 12 35 98 0 2 90.5 4 122 7 12 Cefamandole ................ 2 93.2 8 122 7 4 Cefoperazone ............... 6 9 122 8 3 94.6 Cefotaxime ................. 5 94.6 Cefoxitin ................... 5 23 105 11 3 95.2 Cephalothin ................. 6 24 110 6 1 Chloramphenicol ............ 0 16 115 16 0 100.0 97.3 Gentamicin ................. 3 14 111 18 1 93.2 , 9 125 3 4 Mezlocillin ........6 97.3 Moxalactam ................. 1 12 127 4 3 98.6 2 25 115 5 0 Nitrofurantoin. 91.2 Piperacillin ................. 5 6 124 4 8 98.6 Tetracycline ................ 1 4 118 23 1 98.0 Tobramycin ................. 3 32 104 8 0 Trimethoprim4 140 1 0 98.6 sulfamethoxazole .......... 2 a A total of 147 isolates were used, including 20 P. aeruginosa and 127 enteric bacteria. b UniScept MIC/reference MIC. C Overall perceht agreement was 95.5%. Antimicrobial Agentt

.......

........

staphylococci (Table 3), very tnajor errors were 0.3%, mnajor errors were 0.7%, and minor variations were 1.1% (4). The very major errors occurred primarily with amikacin and dti two occasions each with methicillin and chloramphenicol. The interpretation of enterococcal antimicrobial profiles TABLE 6. Summary of MIC ratiosa No. of tests with the follow-

Amikacin ................... Ampicillin ............

Carbenicillin Cefamandole

........

........... ............

Cefotaxime

Cefoxitin

....

...........

Cefoperazone

>4

% Agreement'

172 91 16 206 43 13 98 0 2 122 7 12 122 7 4 122 8 3

93.5 95.2 90.5 90.5 93.2 94.6

ing MIC ratio':

Antimicrobial agent

s0.25 3 1 12 2 6

5 5

0.5

10 29 35 4 8 9

1

2

6 0 3

23 105 11 31 199 8 87 182 17 5 130 10 6 129 7

Gentamicin . ......4........ Methicillin ............ 3 Mezlocillin ............ 6 Moxalactam ........... 1 Nitrofurantoin .......... 2 Penicillin ............. 0 Piperacillin ............ 5 Tetracycline ........... 2 Tobramycin ........... 7

22 218 34 23 66 7 9 125 3 12 127 4 31 254 5 11 94 27 6 124 4 5 261 23 48 218 17

0 0 12 1 4 3 0 13 8 1 2

2 2

10 276 4 9 101 32

0 1

............. ........... ......... ...........

Cephalothin Chloramphenicol

Clindamycin Erythromycin

...........

Trimethoprimsulfamethoxazole

7

3 2 0

94.6

96.4 97.9 100.0 97.9 93.8 97.0

93.2 97.3 99.3 91.0 91.2 99.0 96.9

99.3 97.9 a A total of 292 isolates were used, including 100 staphylococci, 45 enterococci, and 147 gram-negative organisms. b UniScept MIC/reference MIC. C Overall percent agreemerit was 95.8%. ....

Vancomnycin

................

735

revealed very tnajor errors at 0.4%, major errors at 2.2%, and minor errors at 1.1%. The very major and major errors occurred only with aminocyclitols. There were 0.9% very major, 0.9% major, and 1.6% minor interpretive errors for the gram-negative rods (Table 5). The very major and major disparities were distributed randomly among the various beta-lactam antimicrobial agents. For the entire series (Table 6), there were 0.8% very major errors, 0.9% major errors, and 1.4% minor errors. The intralaboratory reproducibility for both methods, including on- and off-scale results, was in excess of 98% for all participants. The interlaboratory reproducibility was 96.7% for the UniScept system and 95.6% for the reference method. For on-scale results only, intralaboratory reproducibility was greater than 951% for the participants and both sy$tems; interlaboratory reproducibility was 92.9% for the UniScept system and 92.4% for the reference method. The quality control organisms, used daily with both systems, demonstrated MICs within the acceptable limits (3). DISCUSSION The results indicate excellent correlation between the standard microdilution and UniScept MIC detertninations. The use of MIC ratios, first advocated by Gavan et al. (1), ensures that differences between the standard and experimental systems are readily discerned. Thus, in a few instances, staphylococci yielded slightly higher MICs with amikacin and penicillin. The discrepancies with methicillin occurred in two instances with S. aureus, once with the standard and once with the UniScept system. Two very major discrepancies with the UniScept system occurred once each with S. epidermidis and Staphylococcus haemolyticus. While the MIC ratio implies two errors by the UniScept system with methicillin-resistant S. aureus in fact it was the standard system that failed to recognize methicillin resistance in one of these instances. Enterococci manifested a trend toward higher MICs with gentamicin, but this drug is advocated in the treatment of enterococcal infections only in combination with beta-lactam agents. This collaborative evaluation indicates that the UniScept system is another excellent alternative for the determination of quantitative antimicrobial susceptibility. It offers the convenience of lyophilized dilution trays containing the drugs and a properly supplemented growth medium for storage without refrigeration. In addition, gram-positive and gram-negative bacteria can be accommodated in one tray. The range of antimicrobial agent concentrations extends to sufficiently high levels to permit the evaluation of organisms isolated from urinary tract infections.

LITERATURIP CITED 1. Gavan, T. L., R. N. Jones, and A. L. Barry. 1980. Evaluation of the sensititre system for quantitative antimicrobial drug susceptibility testing: a collaborative study. Antimicrob. Agents Chemother. 17:464-469. 2. Lennette, E. H., A. Balows, W. J. Hatisler, Jr., and J. P. Truant (ed.). 1980. Manual of clinical microbiology, 3rd ed. American Society for Microbiology, Washington, D.C. 3. National Committee for Clinical Laboratory Standards. 1983. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. M7-T. National Committee for Clinical Laboratory Standards, Villanova, Pa. 4. Thornsberry, C., T. L. Gavan, J. C. Sherris, A. Balows, J. M. Matsen, L. D. Sabath, F. Schoenknecht, L. D. Thrupp, and J. A. Washiigton II. 1975. Laboratory evaluation of rapid, automated susceptibility testing systemn: report of a collaborative study. Antimicrob. Agents Chemother. 7:466-480.