Automated Antibiotic Susceptibility Testing: Comparative Evaluation of Four Commercial Systems and Present State

Antimicrobial Susceptibility Testing

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Automated Antibiotic Susceptibility Testing: Comparative Evaluation of Four Commercial Systems and Present State Julia Kiehlbauch, PhD," Joanne M. Kendle, MS,f Larry G. Carlson, MS,f Fritz D. Schoenknecht, MD,; and James J . Plorde, MDtt

Antibiotic susceptibility testing (AST) of all the technical procedures in clinical microbiology laboratories has received the earliest attention for possible automation. Numerous attempts at automation or at least mechanization resulted, since the details of this procedure-diluting and dispensing microorganisms and antimicrobics, reading endpoints, and interpreting results-are all very suitable for the replacement of manual processes by automated ones. It will b e convenient to keep simple mechanization of an existing procedure separate from automation of parts or all of it and to recognize entirely new technical principles. How fast results can b e generated and how accurate and reproducible they are constitute other important considerations. This article will, after a brief recall of past attempts, report on an in-use evaluation of four commercially available systems, two semiautomatic adaptations of the microtitration principle, and two fully automated instruments in the laboratories of one of the authors. Throughout this discussion, principles will b e stressed rather than extensive technical details. Questions of quality control, early reading of results, flexibility as to drug and microorganisms, and cost will b e addressed. Some conclusions will b e drawn.

" Enteric Bacteriology Section, Centers for Disease Control, Atlanta, Georgia f Seattle Veterans Administration Medical Center, Seattle, Washington j University of Washington School of Medicine, Seattle, Washington Clinics in Laboratory Medicine-Vol.

9, No. 2, June 1989

320

J. KIEHLBAUCH, J. KENDLE,L. CARLSON, F. SCHOENKNECHT AND J. PLORDE

EARLY ATTEMPTS AT MECHANIZATION

One of the earliest practical and successful devices still in use .~~ devices have been in is the Steers inoculum r e p l i ~ a t o r Similar use for phage typing procedures as well. They allow simultaneous inoculation of some 30 strains of bacteria onto the surface of regular agar plates containing the antibiotic to be tested in a suitable serial dilution. A large number of minimum inhibitory concentration (MIC) determinations can be accomplished economically. This simple technique is still in use in larger institutions and hospitals. Details for the use of agar dilution systems are in the fourth edition of the American Society of Microbiology (ASM) Manual of Clinical Microbiology. l7 The adaptation of this principle to broth dilution susceptibility testing goes back probably to the Takatsy microtitration technique, originally designed for viral and serological Subsequently, many manual, mechanized, and semiautomatic systems for AST have been commercially developed, based on miniaturized broth dilution techniques. Plastic trays with up to 96 wells are inoculated with wire loops or other devices, delivering 0.05 ml. Initial problems with static electricity and evaporation have been solved and the technique is at least ten times faster, and of course much more economical in materials than conventional or macro tube dilution procedures. Standardization and improved quality control have made microtitration techniques a major means of AST in many laboratories throughout the world. Again, details of use can b e found in the ASM Manual of Clinical Microbiology17 and in the documents of the National Committee for Clinical Laboratory Standards (NCCLS).I9>20 The agar diffusion test has not been overlooked by fertile minds bent on increasing efficiency. Motor-driven spiral inoculating devices, disk applicators for 100-mm and 150-mm plates, and various readout systems have been developed and are widely used, the latter mainly for industrial assay purposes.

EARLY ATTEMPTS AT AUTOMATION

Mechanization of AST really did not introduce any new principles, unless one wants to call miniaturization new. Automation, on the other hand, even in the earlier stages, introduced some new technology, which is worth reviewing briefly. Even devices that did not become production models are enlightening, and the principles involved might well b e revived and used in the future. Several instruments based on particle counting, electrical impedance technology, and light scattering concepts are particularly

interesting and should not be forgotten, even though three of the described devices never reached the market, at least not for the purpose of detecting bacterial resistance to antimicrobics. A fourth system, the Autobac, went into production and was the first commercially available and FDA approved machine. Particle Counting

Technicon's automated antibiotic susceptibility system (TAAS) is probably the first fully automated device for AST developed in this country." Except for loading the machine with a suspension of the organisms to be tested, the process is fully automatic. After incubation of the organisms for 150 minutes in the presence and absence of antimicrobic and after the addition of formalin, the number of bacteria in the wells with the drug is compared to the number in control wells without it. Electronic analysis and recording of results in the form of a ratio on a strip chart recorder complete the process. At the heart of this system is a well designed and functioning reverse dark field optical system as a particle counter. Although comparative evaluations yielded good correlation with conventional standardized methods, the machine never reached production stage. The expense and the sheer bulk of the equipment-equivalent to more than two household refrigerators-might have played a role; in addition formalin fumes were generated at the end of the incubation time. Another interesting point emerging from final cost efficiency considerations was the realization that AST is not likely to ever be a volumegenerating screening test, since it is a secondary procedure that will b e requested only in a limited number of positive clinical isolates. Electrical Impedance

Since electrical impedance is a measure of the resistance to flow of an alternating electrical current and since it varies with the change occurring in culture media as the result of microbial growth, its indirect correlation with bacterial growth can be utilized. This principle was first applied to blood culturing and then was shown to b e promising in 6-hour impedance MIC tests using inocula that were higher by a factor of 10 than the ones employed in the standard overnight MIC test.5 For reasons that were never quite clear, this interesting technique that had good initial results was never commercially developed. Light Scattering Methods

The first approach used differential light scattering, and its potential for susceptibility testing was based on the observation of changes in differential light scattered from bacterial suspen-

~ions.~O A monochromatic, vertically polarized helium-neon laser beam (632.8 nm) served as the light source in the photometer. The recorded light scattering pattern reflects the size, shape, structure, and size distribution of the bacteria suspended in the liquid growth medium. Changes in the morphology of susceptible organisms as the result of added antimicrobial agents will, in turn, alter the light scattering pattern from the one recorded initially as a baseline and thus theoretically allow detection of resistance within minutes. The exact concentration of the bacterial suspension, however, proved to be so critical that it became technically too difficult and ultimately impractical to obtain reproducibly the required initial light scattering pattern against which the bacterial suspension after addition of antimicrobics had to be compared. A carefully conducted study2' in a national reference center using a prototype instrument (Differential One) was unable to confirm the claims by the original investigator^,^ at least not insofar as they were extended to application of this interesting principle to rapid AST. Autobac System

The second approach involving light scattering led to the first commercially produced and successful instrument for automated AST. The Autobac, originally developed by Pfizer, operates on the principle of forward light scattering at a fixed angle of 35". The light scattering photometer, which also standardizes the inoculum, reads the amount of growth in each chamber of the cuvette and automatically compares the amount of growth in the presence of an antimicrobial agent with the amount of growth in the control. The instrument then prints out a numerical ratio between 0 and 1 called a light scattering index. This index figure is translated to reports of susceptible, intermediate, and resistant. In addition to the photometer, the components of the system include a transparent plastic multichambered cuvette within which the inoculated broth is automatically distributed to 12 test chambers and one control chamber and into which the antimicrobial agents are delivered on impregnated paper disks. These so-called elution disks contain precise amounts of antimicrobic, usually considerably less than on the disks used for a standardized disk diffusion test. A disk dispenser loads the cuvettes with the panel of elution disks. An incubator-shaker operating at 35°C agitates the cuvettes for 3 hours or longer. If growth is insufficient as indicated by a light scattering index of 0.90 or less in the control chamber, the cuvette in question is simply transferred manually back into the shaker-incubator. The system performed well in a large multicenter trial2' and was subsequently modified to yield results expressed in MIC ranges.24

EVALUATION OF FOUR RECENTLY DEVELOPED COMMERCIAL INSTRUMENTS

As an increasing number of instruments for the performance of AST have been developed and marketed, the task of evaluating their suitability for the clinical laboratory has become increasingly complex. Factors to be considered prior to implementing such systems in the laboratory include their accuracy and reproducibility vis-a-vis standard methods, flexibility in the choice of antimicrobial agents, frequency of technical and quality control problems, the technologist and turn around times required, and the capital and recurring costs of the individual systems. At the Seattle Veterans Administration Medical Center (SVAMC), we had a unique opportunity to evaluate four instrumented methods in relationship to the issues outlined previously. Two fully automated, photometry-based systems, the Automicrobic System (AMS) and Avantage (AVAN) provide "walk-away7' convenience and rapid turn around times, but utilize an unconventional testing procedure. The remaining two systems, the MicroScan (MSCAN) and Sceptor (SCEPT), are instrumented adaptations of the microtiter methodology, the former utilizing a machine and the latter visual determination of endpoints. The organisms chosen for inclusion in this study were selected from diverse sources. They included fresh, clinical isolates; organisms known to present special challenges to automated systems assembled by the Centers for Disease Control (CDC) in Atlanta, Georgia; and previously isolated and stocked multiresistant grampositive and gram-negative bacteria.

Test Organisms

A total of 414 gram-negative bacilli and 181 gram-positive cocci were tested (Table 1).The fresh clinical isolates were all recovered from specimens submitted to the SVAMC Microbiology Laboratory as were the frozen multiresistant organisms, excepting 23 strains of methicillin-resistant Staphylococcus aureus recovered in the laboratories of the University of Washington Hospital and Harborview Medical Center. The group of organisms specifically designed to challenge automated susceptibility testing systems was provided by Dr. Clyde Thornsberry at the CDC. Susceptibility Test Procedures

In the Automicrobic System (Vitek Systems, Hazelwood, MO) dried antibiotics are contained in the microwells of plastic cards

Table 1. Test Organisms ISOLATE CATEGORY ORGANISM

Gram-negative Acinetobacter Citrobacter amalonaticus diversus freundii Enterobacter agglomerans aerogenes cloacae Escherichia coli Hafnia alvei Klebsiella oxytoca pneumoniae Morganella morganii Proteus vulgaris mirabilis Providencia rettgeri stuurtii Pseudomonas aeruginosa maltophilia putida Sematia liquefaciens marcescens Shgella s p p . Gram-positive Staphylococcus aureus epidermidis Streptococcus enterococcus faecalis faecium durans boois equinus Totals

Clinical

CDC

Multiresistant

Total

10

1

0

11

3 13 15

0 0 0

0 4 4

3 17 19

1 10 20 38 0

1 10 7 19 1

0 2 4 5 0

2 22 31 62 1

11 29 9

2 8 2

3 4 5

16 41 16

5

3 10

0 0

8 32

22

5

5

19

2

4 11

14 32

18 8 2

29 0 0

0 0 0

47 8 2

0 14 0

3 6 1

0 6 0

3 26 1

28 19

19 21

28 0

40

19 19 2 1 1 1

14

342

173

75 33 21 4 2 4 2

2 2 1 3 1

80

595

roughly the size and shape of a bank credit card; test organisms are emulsified in 0.45 per cent saline to a 1.0 McFarland standard, diluted 1:20, and placed in a filling unit. Here the card is charged, sealed, and placed in the incubator-reader module. Bacterial growth is monitored turbidimetrically in the presence and absence of antibiotic. Results are provided in the form of a category call along with an MIC calculated on the basis of regression analysis. Reporting time is 5 to 7 hours for Enterobacteriaceae and

gram-positive cocci and up to 1 5 hours for nonfermenting gramnegative rods. In this study the AMS GNS-B (gram-negative organisms) and GPS (gram-positive organisms) cards were inoculated according to manufacturer's directions and read using the R2.04 version of their software. The Avantage system (Abbott Diagnostics, North Chicago, IL) also measures bacterial growth turbidimetrically and calculates an MIC based on the organism's growth curve in the presence of the antibiotic as compared to a control well without antibiotics. The system consists of a plastic cuvette divided into 1 0 sections; one section serves as a control and antibiotic elution disks are placed into the other nine sections. The cuvette was filled with 1 0 ml of Avantage Culture Media (Abbott) prior to adding 100 mcl of the same 1.0 McFarland suspension as that used to inoculate the AMS system. For all tests involving gram-positive organisms, 0.1 ml erythromycin enzyme inducer (Abbott) was added to each cuvette. During the course of the study, the 1.03 version of the Abbott software was utilized. The MicroScan system (American MicroScan, Rahway, NJ) was used in conjunction with the AutoScan 4 (American MicroScan) and the 13.00 version of the manufacturer's software. For each test, four to five colonies were used to inoculate a 4.0-ml brain heart infusion broth (BHIB; BBL Microbiology Systems, Cockeysville, Md). The resulting suspension was then incubated at 35"C for 4 to 6 hours. This growth was adjusted in another BHIB tube to a 0.5 McFarland standard. An inoculum of 0.5 ml of the standardized suspension was added to 20 ml of sterile distilled water containing 0.02 per cent TWEEN 80 (Fisher Chemical Company, Fair Lawn, NJ). The inoculated TWEEN suspension was poured into a seed tray. The lid, with replicator prongs designed to deliver 5 mcl of suspension, was placed on the tray and then used to inoculate the panel. Panels were incubated at 35°C for 1 6 to 20 hours (48 hours for oxacillin wells) prior to being read with the Autoscan 4. Gram negative and gram positive MIC panels (American MicroScan) were used in this study. The Sceptor system (Johnston Laboratories, Towson, MD) was used in conjunction with gram negative and gram positive MIC panels. Four to five colonies were selected from a blood agar plate (BAP) and emulsified in 3 ml of Trypticase soy broth (TSB; BBL Microbiology Systems, Cockeysville, MD). The inoculated TSB was incubated at 35°C for 2 to 6 hours prior to standardizing to a density equivalent to a 0.5 McFarland in a second TSB tube. A dilution loop (Johnston Laboratories) was used to transfer a 20 mcl drop to a tube of Sceptor GN broth (Johnston Laboratories). The GN broth was poured into a sterile inoculating cup (Johnston Laboratories) which had been placed into the Sceptor prep station

(Johnston Laboratories), a semiautomated pipetting device for inoculation of the appropriate panels. Panels were incubated at 35 "C for 16 to 20 hours (48 hours for oxacillin wells) prior to being read visually on a Sceptor reading station linked to a Digital Profession computer equipped with Data Management System version 2.00 (Johnston Laboratories). NCCLS breakpoints were utilized. The NCCLS (Kirby-Bauer) reference disk diffusion procedure, used as previously described,lg served as the reference method. The following BBL disks (BBL Microbiology Systems, Cockeysville, Md) were used: amikacin, 30 pg; ampicillin, 10 pg; carbenicillin, 100 pg; cefoxitin, 30 pg; cefotaxime, 30 pg; cephalothin, 30 pg; chloramphenicol, 30 pg; clindamycin, 2 pg; erythromycin, 15 pg; gentamicin, 10 pg; kanamycin, 30 pg; moxalactam, 30 pg; nitrofurantoin, 300 pg; novobiocin, 5 pg; oxacillin, 1pg; penicillin, 10 U; piperacillin, 100 pg; polymyxin By300 pg; trimethoprim-sulfamethoxazole, 1.25/23.75 pg; tetracycline, 30 pg; tobramycin, 10 pg; and vancomycin, 30 pg. Data Analysis

All data accumulated in each of the five systems were entered into a dBase I11 Plus data base (Ashton-Tate, Torrance, CA). Results from the eight antibiotics used in all systems for the gram-negative organisms (ampicillin, amikacin, cephalothin, chloramphenicol, cefotaxime, gentamicin, tobramycin, and trimethoprim-sulfamethoxazole) and from the nine antibiotics used for gram-positive organisms (ampicillin, cephalothin, chloramphenicol, clindamycin, erythromycin, gentamicin, oxacillin, penicillin, and vancomycin) were analyzed. Carbenicillin was tested against gram-negative bacilli with the Kirby-Bauer method and in all test systems except the AMS. Discrepancies were defined as follows: a very major error (VME) was one in which the test method result was susceptible and the reference method resistant, a major error (MAE) was one in which the reference method indicated the organism was susceptible and the test method indicated resistant, and a minor error (MIE) was one in which either method called the organism intermediate, while the other method called it susceptible or resistant; both very major and major errors were considered essential errors (ESE). When three or more of the test systems demonstrated major or very major discrepancies with the reference method for an individual antibiotic-microbe combination, the data for that combination were excluded from analysis. Complete agreement (CA) was calculated using the formula [n - (VME MAE MIE)]/n X 100. Essential agreement (EA) was defined by the formula [n (VME MAE)]/n X 100. Analysis to determine signi,icance of differences was done using the cross-tabulation procedure of the

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AUTOMATED ANTIBIOTIC SUSCEPTIBILITY TESTING

SPSS-PC package (SPSS Inc., Chicago, IL). Alevel o f p considered significant.

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< 0.05 was

Quality Control

All panels were quality controlled weekly and with each new lot number during the course of the study. Gram-negative panels were quality controlled at least five times during the course of the study. Gram-positive panels were quality controlled at least 1 2 times. The Kirby-Bauer test was quality controlled twice weekly in accordance with the National Committee for Clinical Laboratory Standards (NCCLS) recommendation^.^^ Additional Studies

Throughout the course of the study, records were kept of any problems encountered in performing susceptibility tests using each of the systems, including technologist errors, hardware and software failures, manipulations other than those prescribed in the manufacturer's standard operating instructions, skip wells, test failures, and failure to achieve susceptibility results after three consecutive attempts. Time motion studies were performed on five consecutive days during the second month of the study to determine the "handson" personnel time required to complete each test. These included observing and recording the times required to ready the test panels for use, prepare the inocula, charge panels and place them under incubation, record pertinent identifying data as well as the reading and recording of results. Nonpersonnel preparation times were recorded separately and added to the personnel times to obtain total preparation times. The elapsed time from the initiation to the completion of susceptibility testing was recorded as turn around time. The purchase costs to the SVAMC of the appropriate hardware and software and the cost of their implementation were obtained from hospital records; warranty costs were excluded. Direct costs for antibiotic panels and other expendables were similarly determined. Personnel costs were calculated from technologists' salaries. Because of the difficulties in accounting indirect and overhead costs in the Veterans Administration, these were excluded from our calculations.

Impact of Isolate Source on Accuracy

The ESE rates for the fresh clinical isolates, multiresistant stock organisms, and the CDC challenge organisms are presented

328

J. KIEHLBAUCH, J. KENDLE,L. CARLSON,F. SCHOENKNECHT AND J. PLORDE Table 2. Essential Errors by Organism Group and System PER CENT ORGANISM GROUP

Clinical

SYSTEM

CDC

Multiresistant

Gram-negative AMS AVAN MSCAN SCEPT Gram-positive AMS AVAN MSCAN SCEPT

in Table 2. Interestingly, the ESE for the CDC organisms was not substantially higher than those of the fresh, clinical isolates and multiresistant stock organisms. For this reason, the accuracy rates for organisms from all three sources were consolidated. Accuracy by System and Gram Stain Category of Organisms

Gram-NegativeOrganisms. The consolidated results for the organisms from all sources for each testing system are presented in Table 3. The rates of EA of each were quite similar (AMS, 95 per cent; AVAN, 94 per cent; MSCAN, 97.5 per cent; SCEPT, 96.9 per cent) but VME were significantly higher in the AMS (1.6 per cent) and Avantage (3.2 per cent) than in the two microtiter procedures (0.2 per cent, 0.3 per cent). The Microscan gave the best overall results with the lowest percentage of MIE, MAE, andVME. The Sceptor had similar rates of ESE, but had the highest percentage of MIE (13 per cent), indicating that ESE were minimized by assigning a larger proportion of organisms to intermediate categories. Table 3. Summary of Errors by Organism Group and System ERROR CATEGORY

SYSTEM

Gram-negative AMS AVAN MSCAN SCEPT Gram-positive AMS AVAN MSCAN SCEPT

None (Per Cent)

Minor (Per Cent)

Major (Per Cent)

Very Major (Per Cent)

BLANKS

TOTAL

Gram-Positive Organisms. Oxacillin and gentamicin data were analyzed only for staphylococci; cephalothin results were analyzed only for oxacillin-susceptible staphylococci. Ampicillin results were analyzed for enterococci. Like the gram-negative test organisms, the EA of the AMS (97.6 per cent), MicroScan (96.5 per cent), and Sceptor (98.1 per cent) for the gram-positive battery were extremely close. Although VME with the AMS (1.4 per cent) were still higher than those seen in the microtiter systems (0.6 per cent, 0.7 per cent), the differences were substantially less than those seen with the gram-negative organisms. The Avantage, in contrast, demonstrated high MAE (7.3 per cent) and VME (2.9 per cent) rates, resulting in an ESE rate of over 1 0 per cent. Accuracy by System and Antimicrobial Agent Gram-Negatiue Organisms. The ESE rates with chloramphenicol were more than twice the mean system ESE for the AMS (11.8 per cent vs. 5.1 per cent), MicroScan (6.3 per cent vs. 2.4 per cent), and Sceptor (6.3 per cent vs. 2.9 per cent) systems (Table 4). The Sceptor, in addition, demonstrated relatively high ESE rates when testing susceptibility to ampicillin (4.6 per cent vs. 2.9 per cent) and trimethoprim-sulfamethoxazole (4.9 per cent vs. 2.9 per cent). The Avantage had high ESE rates for trimethoprimsulfamethoxazole and all of the /3-lactam antimicrobic, except ceTable 4. Essential Errors by Antimicrobial Agent and System AMS DRUG

Gram-negative organisms Amikacin Ampicillin Carbenicillin Cephalothin Chloramphenicol Cefotaxime Gentamicin Tobramycin TrimethoprimSulfamethoxazole Total Gram-positive organisms Ampicillin Cephalothin Chloramphenicol Clindamycin Erythromycin Gentamicin Oxacillin Penicillin G Vancomycin Total

AVAN

MSCAN

(PERCENT)

8 ( 1.9) 26 ( 6.3) 17 ( 4.1) 49 (11.8) 3 ( 0.7) 21 ( 5.1) 23 ( 5.6) 21 ( 5.1)

10 ( 2.4) 45 (11.1) 34 ( 8.4) 44 (10.9) 8 ( 2.0) 14 ( 3.4) 16 ( 3.9) 23 ( 5.7) 34 ( 8.4)

10 (2.4) 11 (2.7) 7 (1.7) 10 (2.4) 26 (6.3) 2 (0.5) 8 (1.9) 12 (2.9) 5 (1.2)

0 (0.0) 19 (4.6) 7 (1.7) 12 (2.9) 26 (6.3) 2 (0.5) 14 (3.4) 9 (2.2) 20 (4.9)

168 ( 5.1)

228 ( 6.2)

89 (2.4)

109 (2.9)

1 ( 1.8) 1 ( 1.6) 3 ( 1.8) 21 (12.7) 12 ( 7.2) 6 ( 5.5) 1 3 (11.9) 61 (36.7) 1 ( 0.6) 119 (10.2)

0 (0.0) 6 (8.2) 4 (2.2) 3 (1.7) 5 (2.8) 10 (8.0) 8 (7.0) 3 (7.3) 0 (0.0) 39 (3.4)

-

0( 0( 2( 9( 7( 4( 5( 4( 0( 31 (

0.0) 0.0) 1.1) 5.0) 3.9) 3.5) 4.3) 2.2) 0.0) 2.4)

(PERCENT)

SCEPT

(PERCENT)

(PERCENT)

0 3 2 3 2 2 5 7 0 34

(0.0) (4.1) (1.1) (1.7) (1.1) (1.7) (4.3) (3.9) (0.0) (1.9)

fotaxime. In general, amikacin and cefotaxime gave low ESE rates in all systems. Gram-Positive Organisms. ESE rates with oxacillin significantly exceeded the mean system errors for the AMS (4.3 per cent vs. 2.4 per cent), Avantage (11.9 per cent vs. 10.2 per cent), MicroScan (7.0 per cent vs. 3.4 per cent), and Sceptor (4.3 per cent vs. 1.9 per cent) systems; excepting the Avantage, the same was true for penicillin. Excessive clindamycin errors were demonstrated in the two growth curve-based methods, as were excessive cephalothin errors in the microtiter systems. Ampicillin, chloramphenicol, and vancomycin gave low ESE rates in all systems. Antibiotic-Organism Combination Errors

The high ESE rates demonstrated by individual antibiotics were usually concentrated within certain organism groups. Some antibiotic-bacteria ("drug-bug") combinations demonstrated ESE rates of greater than 15 per cent for the gram-negative panels. Chloramphenicol versus Serratia spp. and miscellaneous nonfermenters (Acinetobacterspp. and pseudomonads other than P. aeruginosa) seemed to be problematic for every system except Avantage. The p-lactam errors demonstrated by the latter system were most pronounced with Enterobacter spp. and Proteae. Among the gram-positive organisms, oxacillin-resistant staphylococci were largely responsible for the high ESE rate seen with that antibiotic. ESE rates of more than 15 per cent were seen in the Avantage system for clindamycin, gentamicin, and oxacillin; in the MicroScan for penicillin G and gentamicin; and in the Sceptor for oxacillin. The MicroScan did not produce ESE rates in excess of 15 per cent for any antimicrobic-staphylococcal combination. TechnicaI Problems

The extra manipulations and repeat testing required, the test failures, and, for microtiter systems, the number of panels that contained "skip wells" are recorded in Table 5. The need for extra manipulations was encountered almost exclusively with the Avantage and MicroScan systems. Most of the problems with the former Table 5 . Problems Encountered During Study SYSTEM PROBLEM

AMS

Microscan

Sceptor

Avantage

Kirby-Bauer

Skip wells Manipulations Repeat testing Failures

N/A 0 48 2

16 28 11 0

40 1 18 0

N/A 76 173 22

N1-4 0 18 0

Table 6. Quality Control SYSTEM

CATEGORY

Organisms' Gram negative

Gram positive

AMS

Microscan

Sceptor

Avantage

Kirby-Bauer

ECOL 25922 PSAR 27833

ECOL 25922 PSAR 27833 KLOX AMSlOl

ECOL 25922 PSAR 27833

ECOL 25922 PSAR 27833

SAUR 29213 STFC 29212

SAUR 29213 STFC 29212

ECOL 25922 PSAR 27833 ENTC 35030 SERR 33670 SAUR 29213 STFC 29212 BCER 11778 PSAR 27853

SAUR 25913

SAUR 25913

No. performed Gram negative Gram positive Failures Gram negative Gram positive

5 12

5 12

5 12

5 12

5 12

0 0

13t 264

0 5

0 0

0 11

" Organisms: Ecol, E. coli; Psar, P. aeruginosa; Klox, K. oxytoca; Entc, Enterococci; Serr, S. marcescens; Saur, S. aureus; Stfc, S. faecalis; Bcer, Bacillus cereus; Psar, P. aeruginosa. t Microscan: 1 Ecol-Amik, 2 Ecol-Gent, 3 Ecol-Tobr, 1 Ecol-Tris, 1 Klox-Gent, 1 Klox-Tobr, 1 Klox-Tetr, 1 Klox-Tobr. t Microscan: 8 Saur-Ampi, 10 Saur-Peng, 2 Saur-Tetr, 1 Saur-Gent, 1 Stfc-Eryt, 1 Stfc-Gent, 1 Stfc-Tetr, 1 Stfc-0xac.Sceptor: 1 Bcer-Peng, 1 Saur-Amik, 1 Saur-Tris, 1 Stfc-Amik, 1 Stfc-Gent. K-B: 2 Saur-Ampi, 1 Saur-Ceph, 2 Saur-Chlo, 3 Saur-Clin, 1 Saur-Oxac, 1 Saur-Peng, 1 Saur-Vanc.

involved correction of mechanical jams of the unit responsible for dispensing antimicrobic disks into the individual sections of the test cuvette. In the MicroScan system, the effort was devoted to popping bubbles that had formed in the microtiter wells following inoculation. At t h e time of the study, the manufacturer felt this was necessary to maintain the accuracy of the automatic reader. Repeat was required most frequently with the AMS and Avantage, two fully automated systems. In the Avantage, 19 per cent of all tests on gram-negative organisms and 29 per cent of tests on grampositive bacteria had to be repeated. For the AMS, the retesting rates for gram-negative and gram-positive organisms were 6 per cent and 11 per cent, respectively. Susceptibility tests on organisms that failed to yield a result after three attempts (that is, failures) were a problem almost unique to the Avantage system; the only exceptions involved two strains of Streptococcus equinus that did not grow in the AMS test cards. Quality Control and Reproducibility Studies

Quality control procedures and results are listed in Table 6. Although a few problems were encountered in the quality control of the gram-positive Sceptor panels, only the MicroScan results failed to fall within the manufacturer's recommended control limits. Specifically, aminoglycoside MICs for the gram-negative quality control organisms were frequently one dilution higher than the control limits allowed. In spite of this, results were always

332

J. KIEHLBAUCH, J. KENDLE,L. CARLSON, F. SCHOENKNECHT AND J. PLORDE

Table 7. Time Motion Study SYSTEM CATEGORY

AMS

MicroScan

Sceptor

Auantage

Kirby-Bauer

Technologist time (min.) Total preparation time (min.) Turn around time (hrs.)

10.7

6.2

11.8

5.2

8.9

13.0

66.2

11.8

5.2

68.9

6.1

16.6

16.1

6.5

17.7

in the susceptible category as recommended. For the grampositive quality control organisms, problems were primarily encountered in the testing of S. aureus against ampicillin and penicillin. Time Management and Cost Analysis

The "hands-on" technologist time required was least with the Avantage and greatest for AMS and Sceptor (Table 7). However, since the "hands-on" time for all three procedures was concentrated in a single time period, these systems were preferred by the majority of technologists. The MicroScan, which requires preincubation steps, was more difficult to incorporate smoothly into the workflow of the laboratory. The abbreviated turn around time for the AMS and Avantage generally resulted in the availability of the test results prior to the end of the day shift. The cost supply and personnel costs of performing a single test by the four test procedures and the Kirby-Bauer reference method are listed in Table 8. For gram-negative organisms, the costs relative to the Kirby-Bauer were: Avantage, 1.6;AMS, 1.8; MicroScan, 2.0; and Sceptor, 2.6. Ratios for the gram-positive organisms were similar. The lower operating costs of the fully automated systems (AMS, AVAN) were offset by a corresponding cost of the initial capital outlay for these systems. The Kirby-Bauer was the least expensive and the most flexible system overall. Table 8. Cost Analusis bu Sustem in Dollars SYSTEM CATEGORY

AMS

Microscan

Sceptor

Avantage

Kirby-Bauer

Instrument Personnel Supplies Gram negative Gram positive Recurring costs Gram negative Gram positive

69,500 1.52

39,500 0.88

23,400 1.68

41,300 0.75

1.28

3.49 3.49

4.78 5.87

5.49 5.26

3.58 4.10

1.50 2.12

5.01 5.01

5.66 6.75

7.17 6.94

4.33 4.85

2.78 3.40

DISCUSSION

The Antimicrobic System from the company of the same name, Avantage from Abbott Diagnostics, MicroScan System from American MicroScan, and Sceptor from Johnston Laboratories are four of the most actively marketed AST instruments in this country. Although there have been a number of published evaluations of each of these semiautomated and automated instruments," to our knowledge none have compared all four for accuracy, convenience, and cost in a single, controlled, prospective study. To ensure a fair and reliable evaluation of accuracy, the four systems were tested with a large (n = 595), diverse group of gram-positive and gram-negative bacteria, including fresh clinical isolates (n = 342), multiresistant stock organisms (n = 80), and a CDCprovided collection of bacteria (n = 173) specifically designed to challenge antimicrobial susceptibility testing procedures. To evaluate convenience of use, careful records were kept of the technical and quality control problems encountered with each system, and time motion studies were performed to determine how well each system fit into the flow of laboratory work. Costs for equipment acquisition, personnel work time, and expendable supplies were also tabulated. Overall Accuracy

Gradus, Baker, and Thornsberrys have tried to define the acceptable levels of accuracy for new AST procedures by setting arbitrary limits for total (51 0 per cent), essential ( 5 5 per cent), and very major errors (51per cent). Total and essential errors of 5 1 0 and 1 5 per cent, respectively, obviously imply that there should b e CA for 9 0 per cent or more of the antimicrobic-organism combinations and EA for 95 per cent or more. In the present study, t h e CA rates reached 90 per cent only for the MicroScan system and, then, only for gram-negative bacteria. These rates are lower than those reported by other authors for these same test method^.^, There is no ready explanation for these differences. Although it is possible that the large percentage of multiresistant and CDC challenge organisms included in our study contributed to higher error rates,21the similarity in ESE rates demonstrated for our fresh clinical isolates, multiresistant stock organisms, and CDC challenge organisms make this unlikely. The performance of the susceptibility tests by clinical technologists within the daily work flow of the clinical laboratory undoubtedly induced errors that could have been avoided in a research laboratory. Finally, the use of the Kirby-Bauer disk diffusion test as our reference method "See references 1-3, 6 , 8 - 1 0 , 12-14, 18, 21-24, 27-29.

may have exaggerated the minor error rates. A study by Thornsberry et a129showed that comparing results from MIC systems to those obtained by disk diffusion testing increased minor errors while decreasing the percentage of very major errors. This is compatible with the moderately high minor error rates and low CA seen in our study. Accordingly, despite CA rates generally less than 90 per cent, EA for three of the four methods equalled or exceeded the suggested 95 per cent level for both gram-negative (AMS, 95 per cent; AVAN, 94 per cent; MSCAN, 97.5 per cent; SCEPT, 96.9 per cent) and gram-positive organisms (AMS, 97.6 per cent; AVAN, 89.8 per cent; MSCAN, 96.5 per cent; SCEPT 98.1 per cent). However, only MicroScan and Sceptor, the two microtiter procedures, achieved VME rates of 1per cent or less. Although the results of the two are close, MicroScan displayed slightly better accuracy, demonstrating higher CA rates and lower MIE and VME rates; Sceptor appeared to achieve its low ESE rate at the expense of minor errors, indicating that a larger proportion of its test results fell into the intermediate category than did those of the MicroScan. The CA and EA rates of the AMS were equivalent to those of the two microtiter systems, as were its corresponding MIE and MAE rates. In the vital category of VME, however, it exceeded both the rates of the microtiter systems and the suggested 1per cent limit (1.6 and 1.4 per cent for gram-negative and gram-positive organisms, respectively); the differences in VME between AMS and the microtiter systems were relatively smaller for gram-positive organisms. Our AMS data coincide with that from other published studies which have demonstrated this system to be slightly less accurate than the Mi~roScan.~, 21 The poorest performer was the Avantage. It produced EA rates less than 95 per cent, CA rates less than 90 per cent, and VME rates that were double those demonstrated by the AMS system, dramatically exceeding the suggested 1per cent level for both gram-negative (3.2 per cent) and gram-positive (2.9 per cent) organisms. Moreover, its ESE rate for the gram-positive organisms (10.2 per cent) was disturbingly high; ironically, its MIE rate was very low, providing it with the best CA rate of the four systems for this group of organisms. Two other studies have demonstrated the Avantage to perform less well than the AMS.lO.l2 Accuracy by Antimicrobic Agent and Microorganism

As noted in previous studies, errors were not randomly distributed across all antimicrobic-bacteria combinations, but were concentrated in relatively few. For gram-negative organisms, ESE for chloramphenicol were a problem for the AMS, MicroScan, and Sceptor systems, whereas thep-lactam antibiotics proved the most difficult challenge for the Avantage. The chloramphenicol errors

were seen primarily among Serratia species and nonfermentative organisms other than P. aeruginosa. Although these particular combinations, generally, have not been reported as problematic, It is both chloramphenicol and Serratia individually have.9.12,18.22 also of interest that Baker et a12reported that chloramphenicol was among the most difficult antimicrobial agents for the AutoScan 4 to read. In contrast, the testing of chloramphenicol in the Avantage produced low error rates; however, this system had difficulty with ampicillin, carbenicillin, and cephalothin. The errors were seen primarily in the testing of Enterobacter spp. and the Proteae. Inducible p-lactam resistance by strains of Enterobacter had been previously investigated by Sherris and c o - ~ o r k e r sand ~~~~ ascribed to high level mutational events. This phenomenon was thought to be responsible for the false susceptible results with Enterobacter strains and the p-lactam antibiotics encountered during the collaborative evaluation of the Abbott MS-2 instrument, an earlier version of the A ~ a n t a g eDuring . ~ ~ the normal 4- to 6-hour test period, complete suppression of the organisms had occurred, but 8 to 14 hours after antibiotic exposure, resistant mutants emerged. Barnes et a13noted similar problems when testing the MS-2. The AMS also experienced difficulty in the testing of ampicillin, but not cephalothin, against these same organism g r o ~ p s . l , lIt~ ,is~ interesting ~ to speculate that the somewhat longer test period utilized by the AMS vis-a-vis the Avantage system, may have allowed better detection of inducible resistance to the p-lactam antibiotics by Enterobacters and other genera of Enterobacteriaceae, thus explaining its higher level of concordance with the Kirby-Bauer technique during the testing of these "drugbug" combinations. One of the most notable findings was the low level of error displayed in the testing of Escherichia coli against all antimicrobics and P. aeruginosa against the aminoglycosides. Since these two species accounted for over one quarter of the organisms tested, it is unlikely that the failure to detect errors was due to an inadequate sample. Amikacin and cefotaxime produced extremely low error levels against all test organisms. For gram-positive cocci, excessive ESE rates were seen among the methicillin-resistant staphylococci. The excessive rates involved three antimicrobics in the Avantage, two in the MicroScan, one in the Sceptor, and none in the AMS. A literature review by Gradus and coworkers published in 1985' revealed that the most common antimicrobic - gram-positive bacterial combinations producing excessive error rates were methicillin-resistant S. aureus versus methicillin and enterococci versus gentamicin. Other high error rate combinations cited in the literature have included enterococci versus clindamycin and cephalothin3 and .~~ et staphylococci versus cefamandole and g e n t a m i ~ i n Henry

all0 noted excessive errors in the following combinations: enterococci versus P-lactams, group B streptococci versus gentamicin, coagulase-negative staphylococci versus trimethoprimsulfamethoxazole, and coagulase-negative staphylococci versus vancomycin. Hansen and Freedyg reported that the AMS system had difficulty with the enterococcal-ampicillin, enterococcaltetracycline, and S. aureus-chloramphenicol combinations; Baker and colleagues2 demonstrated high error in the MicroScan system when S. aureus was tested against cephalothin; and Jones et al,13 during the study of the Sceptor, observed a similar phenomenon with staphylococcal-penicillin and S. epidermidb-tetracycline combinations. The failure of the present study to detect most of these same differences may be related, at least in part, to interim changes in the antimicrobic panels provided for these systems. One such change has been the addition of 2 per cent NaCl to the oxacillin wells of the AMS, MicroScan, and Sceptor panels to promote expression of heterologous resistance in methicillin-resistant staphylococci. It is unlikely that a small sample size or specimens from a restricted geographical location could be responsible for the discrepancies, as 181 gram-positive cocci obtained from three different Seattle area hospitals and the CDC were included in the test battery. Technical Problems Overall, repeat testing was required more frequently with gram-positive than gram-negative organisms. Although some of the retesting was necessitated by operator or machine error, the great majority was caused by insufficient growth of the test organism in the initial test. Baker et a12reported more growth failures with gram-positive bacteria tested in the MicroScan. In the present study, two groups of gram-positive organisms accounted for most of the repeated tests. The first was the enterococcal and nonenterococcal Group D streptococci, which posed difficulties for the Kirby-Bauer, AMS, and Avantage. S. epidermidis, particularly those from the CDC collection, were problematic for both Sceptor and Avantage. Likewise, a higher proportion of grampositive than gram-negative organismswere considered as susceptibility testing failures, that is, organisms that failed to produce sensitivity results after three consecutive tests. This was aproblem exclusive to the two fully automated systems, which appear to be more sensitive overall to the growth characteristics of isolates. Failures were highest with the nonenterococcal Group D streptococci (S. equinus > S. bovis), although failures occurred in every organism group and type tested with the Avantage.

"Skipped" antimicrobial wells were frequently encountered with the microtiter based systems. "Skipping" was more prevalent with the Sceptor, in which the inoculating system dispenses one well at a time, than with the MicroScan, which simultaneously inoculates all wells using a replicator apparatus. In the latter system, however, bubbles were frequently formed in the V-bottom wells of the microtiter plates following the inoculation step. According to the manufacturer's instructions, these must b e "popped" prior to inserting them in the AutoScan reader, adding significantly to the number of extra manipulations required in this system. This problem diminished somewhat after panels were moved from storage at - 60°C to - 20°C approximately 1 week prior to use. In apaper published after the initiation of the current study, De Girolami and colleagues6 documented that bubbles have minimal effect on the overall accuracy of the AutoScan reader and suggested that it may be unnecessary to remove them prior to reading. American MicroScan has recently addressed this problem with their release of a product, Inoculum water, which is designed to reduce problems with bubbles. Most of the extra manipulations required of the Avantage were related to "jamming" of the unit that dispenses antibiotic disks into each section of the cuvette. Despite considerable effort, we were unable to correct this problem during the course of the study. Quality Control

Quality control was performed according to manufacturer's recommendations. Not surprisingly, the two microtiter systems, which required the most extensive quality control, also showed the most antimicrobial-organism combinations out of control. MicroScan had the highest number of control failures when testing both gram-negative and gram-positive panels, including major problems when E. coli and Klebsiella oxytoca were tested against amin~gl~cosides. Difficulty in maintaining adequate quality control with the aminoglycosides has been previously reported by Larson et a1,16who attributed variations noted to subtle variations in the systems, such as replicator lids and inoculum size. As the microtiter systems performed with excellent CA when compared t o the Kirby-Bauer method, the quality control failures do not appear to have any adverse effect on overall performance of the two systems. Cost and Time Analysis

Cost analysis revealed, as expected, that instrumented susceptibility testing was more costly than the Kirby-Bauer disk diffusion test. This relatednot only to the capital costs associated with

equipment acquisition, but also to the recurring personnel and supply costs. Not surprisingly, the two fully automated systems (AMS and Avantage) required larger capital outlays than did the microtiter systems. The recurring costs of the Avantage, AMS, Microscan, and Sceptor were all greater than those associated with Kirby-Bauer by factors of 1 . 6 , l .8,2.0, and 2.5, respectively; the Avantage had the lowest personnel and supply costs, whereas the Sceptor exceeded the other three systems in both categories. Unexpectedly, personnel costs for two test systems, the AMS and Sceptor, exceeded those of the disk diffusion test. Interestingly, the technical staff's evaluation of the time required to carry out the tasks associated with susceptibility testing and their general satisfaction with individual testing procedures more closely paralleled the total preparation time rather than the actual "hands-on" subinterval. Moreover, they unanimously expressed the conviction that generating susceptibility results on the same work day as the tests were initiated allowed them to file a final specimen report one day earlier, saving them the time and effort required to reassemble specimens and requisition slips the next day, tasks that were not included in our time-motion studies. SUMMARY

Automation of AST has come quite some way and is here to stay. In particular, fully automated, "hands o f f instruments have great appeal to laboratories with a limited number of well-trained and experienced clinical microbiology personnel. None of the evaluated instruments is perfect, but then neither are the standard or reference techniques. Overnight incubation has been the yardstick since the early days of in vitro AST. Given the usually shorter therapeutic intervals of 4- to 12-hour dosage schedules, it is quite possible that shorter incubation times for in vitro tests will become more of a standard. Until that time, newer, including automatic, techniques need to be evaluated against the more traditional standard methods. Quality control is critical, and since no systematic approach aside from individual manufacturers' suggestions exists, it should be developed by the NCCLS or similar agencies. Quality control might include standards for the evaluation of such equipment and systems because the development of new technology in this area will continue. Overall, reproducibility and accuracy of the instruments and methods evaluated were quite promising and should encourage well-designed studies of clinical correlation and relevance. The AMS equipment has been in use for routine AST in the clinical laboratories of the Seattle Veterans Administration Medical

Center and the University of Washington Hospital. Because of its simplicity and flexibility, the Kirby-Bauer method continues to be an alternate technique for certain important clinical isolates, for instance, blood cultures in both laboratories. Finally, it should be remembered that the most critical function of all such equipment is the reliable detection of resistance.

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