Antimicrobial Susceptibility Testing: General Principles and Contemporary Practices

Transcription

1 973 Antimicrobial Susceptibility Testing: General Principles and Contemporary Practices James H. Jorgensen and Mary Jane Ferraro From the Department of Pathology, The University of Texas Health Science Center, San Antonio, Texas; and the Microbiology Laboratory, Massachusetts General Hospital, Boston, Massachusetts One of the most important tasks in the clinical microbiology tration of antibiotic that prevents visible growth represents the laboratory is the performance of antimicrobial susceptibility endpoint of the test, the MIC. tests on significant bacterial isolates. The goal of susceptibility The precision of this method is considered to be plus or testing is to predict the likely outcome of treating a patient s minus one two-fold concentration, in large part because of the infection with a particular antimicrobial agent. Empirical ther- practice of manually preparing serial two-fold dilutions of the apy continues to be effective for some bacterial pathogens antibiotics [5]. The advantages of this technique include the because resistance mechanisms have not yet been acquired or generation of a quantitative result (the MIC) and the examination are still rare; examples include penicillin therapy for group A of a substantial number of bacterial cells because of the streptococcal infections, erythromycin for legionellosis, and large initial inoculum provided by the relatively large volume penicillin for N. meningitidis infections in the United States. of broth in each of the tubes. The principal disadvantages of Susceptibility testing is useful and important for the common the macrodilution method include the tedious manual task of bacterial species that are not predictably susceptible to drugs preparing the antibiotic solutions for each individual test, the of choice because of acquired resistance mechanisms (e.g., relatively large amount of reagents and space required for such members of the Enterobacteriaceae, Pseudomonas species, tests, and the possibility of making errors in the preparation of Staphylococcus species, Enterococcus species, Streptococcus antibiotic concentrations. pneumoniae, Haemophilus influenzae, and Neisseria gonor- The modification of the tube-dilution test that has made broth rhoeae). The two important and related functions of susceptibil- dilution testing very popular is the miniaturization and mechaity testing are the detection of frank resistance and the quantita- nization afforded by use of small, disposable plastic microdilutive measurement of susceptibility to antimicrobial agents with tion trays for performance of susceptibility tests (figure 1). some species that may have direct therapeutic relevance, e.g., Such trays ordinarily contain 96 wells, each holding a volume the magnitude of the penicillin and cephalosporin MICs for of 100 ml which allows Ç12 antibiotics to be tested in a Streptococcus pneumoniae [1 3]. range of eight two-fold dilutions in a single tray. Although the antibiotic dilutions were prepared directly in the microtiter trays in the early phase of the development of this technique [5], Overview of Currently Available Susceptibility Testing contemporary microdilution trays are typically prepared in pro- Methods duction-line fashion with use of dispensing instruments that Broth Macrodilution and Microdilution Tests aliquot precise volumes of antibiotics into the individual wells of trays from large-volume tubes containing the diluted antibi- One of the earliest methods of antimicrobial susceptibility otics in broth. In this manner, hundreds of identical microdilutesting was the macrobroth or tube-dilution method. This procetion trays can be prepared from a single master set of dilutions dure involves preparing two-fold dilutions of antibiotics (e.g., in a relatively brief period. Pre-prepared microdilution antibi- 1 mg/ml, 2 mg/ml, 4 mg/ml, 8 mg/ml, or 16 mg/ml) in a otic panels are purchased in most clinical microbiology laboraliquid bacterial growth medium [4]. Typically, eight or more tories. Some prepared antibiotic panels can be stored frozen concentrations of a drug are prepared in a final medium volume until needed for performance of individual tests; other products of 1 2 ml per tube. The antibiotic-containing tubes are then contain freeze-dried antibiotics that allow them to be stored at inoculated with a standardized bacterial suspension of Ç5 room temperature. The cost for the pre-prepared panels ranges cfu/ml in each tube. After overnight incubation at 35 C, from approximately $6 to $9 each. the tubes are examined for macroscopically visible evidence Microdilution panels are inoculated by using the same cell of bacterial growth in the form of turbidity. The lowest concendensity as that used in the tube-dilution test, i.e., cfu/ml [4]. Inoculation is most often accomplished by using a mechanized instrument or a disposable plastic template device Received 17 November 1997; revised 9 December that transfers 1 5 ml of a standardized bacterial suspension Reprints or correspondence: Dr. James H. Jorgensen, Department of Pathology, simultaneously into each well of the microdilution tray. After University of Texas Health Science Center, 7703 Floyd Curl Drive, San hours of incubation, MICs are determined in exactly Antonio, Texas the same manner as in the tube-dilution test, except that some Clinical Infectious Diseases 1998;26: form of viewing device is needed to facilitate inspection of 1998 by The University of Chicago. All rights reserved /98/ $03.00 each of the wells for evidence of growth. Such devices include

2 974 Jorgensen and Ferraro CID 1998;26 (April) Figure 1. A 96-well microbroth dilution antimicrobial susceptibility testing tray (right) with a disposable tray inoculator (left). The Etest (AB BIODISK; Solna, Sweden) is a commercial product based on the principle of establishment of an antimicro- bial gradient in an agar medium as a means of determining susceptibility [4]. The Etest consists of very thin plastic strips that are impregnated on the underside with a dried antibiotic concentration gradient and are marked on the upper surface with a concentration scale for visual interpretation of results. Up to five or six strips may be placed in a radial fashion on the surface of an appropriate 150-mm round agar plate that has been inoculated in a manner similar to that for a disk diffusion test (figure 2). After overnight incubation, the tests are read by viewing the strips from the top with the plate lids removed. The antibiotic gradient, which forms in the agar adjacent to the Etest strips, gives rise to an elliptical-shaped inhibitory area around each strip. The MIC is determined by the intersection of the bacterial growth ellipse with the Etest strip (figure 2). The Etest shares with the disk diffusion test the intrinsic flexibility of drug selection and testing because of the method of applying selected strips to the surface of various agar test plates. However, the cost of Etest strips is much greater than that of disks (approximately $2 to $3 per strip vs. $.20 per disk) and represents one of the main limitations of this approach. Numerous published studies have indicated that MICs determined by using the Etest generally compare within one twofold dilution interval with those determined by conventional broth or agar dilution methods [8 11]. The Etest is especially a parabolic magnifying mirror with a support stand that allows visualization of the wells of the tray from its underside, a light box that allows visualization from the top, or more complex viewers that may incorporate microcomputer-assisted data recording systems or automated photometer/tray readers. The latter devices provide a computer-printed report and also facilitate data storage and retrieval for periodic generation of cumulative antibiograms for organisms that have been tested. The advantages of the microdilution susceptibility procedure include the economy of reagents and space due to the miniaturization of the test, the reproducibility of results due to preparation of a large number of trays from the same antibiotic dilution series, the convenience of having pre-prepared trays available when needed, the generation of a quantitative result (the MIC), and the convenience of the computerized reporting systems that are available from the commercial microdilution tray manufacturers. It is also possible to identify common bacteria by incorporating biochemical substrates in some of the wells of a microdilution tray, thus allowing simultaneous identification and antimicrobial susceptibility to be determined in a single tray [6]. For these reasons, the commercially prepared broth microdilution method has become the most popular susceptibility testing method available to clinical microbiology laboratories in the United States, as indicated in a survey by the College of American Pathologists [7]. Indeed, that survey showed that the broth microdilution procedure was used routinely in 46% of responding laboratories, disk diffusion in 32%, and rapid automated test systems in 20%. The main disadvantage of the microdilution method is the inflexibility of drug selections available in the standard commercially prepared antibiotic panels. Agar Dilution Tests The agar dilution susceptibility test represents one of the earliest methods used for determining bacterial susceptibility to antibiotics. The test is performed by incorporating two-fold concentrations of antibiotics in an agar growth medium dispensed into individual petri plates [4]. Each plate represents a single concentration of one antibiotic; most often, multiple plates are used to allow testing of multiple drugs in relevant concentrations. Plates are inoculated by applying a small, controlled-volume droplet of a standardized bacterial suspension onto the surface of the agar. A 1 3 ml drop containing Ç bacterial cells is usually used. Multiprong inoculating devices allow different isolates to be tested on each plate. However, six to twelve plates may be required for testing of a full concentration range of each drug. Inoculated plates are incubated for hours at 35 C. The lowest concentration of antibiotic that will prevent macroscopically visible growth of the bacteria is the endpoint of the test, the MIC. Advantages of the agar dilution test include the ability to test a large number of isolates simultaneously at a relatively low cost, the provision of a quantitative (i.e., MIC) result, and the ability to test some fastidious organisms that do not grow well in broth media (e.g., N. gonorrhoeae and anaerobic bacteria). Disadvantages of this procedure include the fact that it is very time consuming if only a few isolates are to be tested, the requirement that antibiotic plates be prepared fresh or stored for only limited periods before use, and the fact that pre-prepared plates are not available from commercial sources. Antimicrobial Gradient Method

3 CID 1998;26 (April) Antimicrobial Susceptibility Testing 975 Figure 2. A Staphylococcus aureus isolate tested by the antimicrobial gradient method. Etest strips include (clockwise), ampicillin/ sulbactam (AB), trimethoprim-sulfamethoxazole (TS), cefotaxime (CT), vancomycin (VA), and cefuroxime (XM). The MIC of each agent is determined by the intersection of the lawn of organism growth with the strip. An MIC scale is inscribed on the upper surface of each strip. useful for testing fastidious organisms such as H. influenzae, S. pneumoniae, and anaerobic bacteria [8, 12, 13]. This is in part because the strips can be placed on special enriched media or in a special incubation atmosphere (e.g., an anaerobic or CO 2 -enriched atmosphere) and because fewer antibiotics may need to be tested against fastidious organisms, allowing accommodation of the relatively high cost of the Etest strips. A potential drawback of the Etest method, however, is the application of MIC interpretive criteria derived from broth dilution testing (which does not require incubation with increased levels of CO 2 for capnophilic bacteria) to MICs obtained with Etest strips on agar media (which requires incubation in a CO 2 - enriched atmosphere). Differences in MICs have been noted with antibiotics that are particularly ph sensitive, such as the macrolide and lincosamide antibiotics, under these different incubation conditions [14]. Users can consult the Etest package insert to determine the specific organism antimicrobial agent combinations that have been cleared for use by the U.S. Food and Drug Administration (FDA). Disk Diffusion Test (Bauer-Kirby Procedure) One of the simplest and most reliable susceptibility testing methods is the disk diffusion or Bauer-Kirby procedure [4, 15]. This method has been widely studied and well standardized over a number of years. The test is performed by applying a standardized inoculum of Ç cfu/ml to the surface of a large (150-mm diameter) Mueller-Hinton agar plate. Up to 12 commercially prepared, fixed-concentration filter-paper antibiotic disks are placed on the inoculated agar surface. Plates are incubated for hours in ambient air at 35 C before the results are determined. The diameters of the zones of growth inhibition around each of the antibiotic disks are measured to the nearest millimeter by viewing the plate with reflected light when it is held a few inches above a black, nonreflecting background. The diameter of the zone of inhibition is related to the sus- ceptibility of the isolate and to the rate of diffusion of the drug through the agar medium. The zone diameter correlates inversely with the approximate MIC for that antibiotic, i.e., linear regression analysis of zone diameters plotted against log 2 MIC values demonstrate a consistent relationship. However, in practice, the results of a disk diffusion test are interpreted by comparing the measured zone diameter with the interpretive criteria published by the National Committee on Clinical Laboratory Standards (NCCLS) [16] and included in the disk s FDA-approved product insert. The results of the disk diffusion test are qualitative in that a category of susceptibility (susceptible, intermediate, or resistant) is derived from the test rather than an MIC. However, one commercially available computer software system makes it possible to calculate an approximate MIC with some organisms and antibiotics by entering zone-diameter values that are programatically compared with standard curves for a species and drug with use of the linear regression formula [17]. The disk diffusion procedure, in addition to being well standardized and reproducible, is a simple test that does not require any special equipment, provides categorical results easily interpreted by all clinicians, and is the most flexible of the available tests because of the selection of individual disks in user-defined test batteries. It is also the least costly of all susceptibility methods (cost, approximately $1.50 to $4 per test for materials). The disadvantages of the disk test are the fact that it is not mechanized or automated and the fact that some fastidious or slow-growing bacteria may not be accurately tested by this approach. However, the disk test may be modified for testing

4 976 Jorgensen and Ferraro CID 1998;26 (April) some fastidious bacteria (e.g., H. influenzae, N. gonorrhoeae, and unusual resistance phenotypes [19]. The Expert System S. pneumoniae, and other streptococci) through use of specialized contains three levels of analysis that may be selected or deactipretive media, incubation conditions, and different zone size inter- vated independently by laboratory personnel. Level one rules criteria [16]. However, because zone sizes are affected are aimed at detection of technical errors (atypical results be- by the media used and by the different growth rates of organ- cause of mixed cultures, improper inoculum density, or organism isms being tested, MIC tests are more accurate with anaerobic identification errors). Second-level rules indicate when the bacteria, and perhaps, with fastidious bacteria other than those normal intrinsic resistance of a species is not detected or when described above. unlikely resistance patterns occur or genetically linked or re- lated resistances are not detected. Third-level algorithms attempt Short-Incubation Automated Instrument Systems to explain resistance phenotypes on the basis of the most likely resistance mechanisms [20] and may offer comments on Use of instruments can allow performance of susceptibility drug selection or contraindications because of likely crosstests in a shorter period than manual methods because the resistance to related compounds. Vitek also offers an additional sensitive optical detection systems of current instruments allow information management system for sorting and storing of test measurement of subtle changes in bacterial growth. There are data and generation of periodic statistical reports (antibiograms). only two instruments presently cleared by the FDA for use in The Vitek data management system may also include the United States that are capable of generating rapid (3 10 programming (called IMPACT) to assist pharmacy personnel hours) susceptibility test results [18]. The MicroScan Walk- in identifying patients who may be receiving antibiotics to Away (Dade Microscan, West Sacramento, CA) is a large, which their isolates are resistant, patients who have bacterial self-contained incubator/reader device that can simultaneously isolates recovered and tested but who are not receiving antimi- incubate and analyze from 40 to 96 microdilution trays [6, 18]. crobial therapy, or patients who are receiving antimicrobial The WalkAway utilizes standard size microdilution trays that therapy in the absence of susceptibility data. Pharmacy reports are hydrated and inoculated manually and then placed in one may be obtained on-line through a remote Vitek terminal in of the incubator slots in the instrument. The type of test to be the pharmacy, or the report may be generated as a hard copy performed is indicated on an instrument-readable bar code label for later review by pharmacy personnel. placed on the tray. The instrument incubates the trays for the MicroScan has also introduced software to provide antimi- appropriate period, examining them periodically with a pho- crobial susceptibility data to hospital pharmacies. The Pharmtometer or fluorometer to determine growth development. LINK option for the WalkAway system uses Windows software Overnight incubation can be done with the WalkAway by using (Microsoft; Redmond, WA) and the ability to transfer information turbidimetric readings of conventional panels, or rapid testing from the instrument to a pharmacy computer or to print a is available by using fluorogenic substrate-containing panels. PharmALERT report to help pharmacy personnel recognize The Vitek System (biomérieux Vitek, Hazelwood, MO) was patients receiving inappropriate or excessive treatment [19]. originally designed for use in the space exploration efforts of The Vitek IMPACT and Microscan PharmLINK programs are the 1970s as an onboard test system for the spacecraft of the functionally quite similar approaches to linking pharmacies National Aeronautics and Space Administration. Because of with microbiology laboratories to facilitate appropriate antibiotic the Vitek System s design objectives, it is highly automated use and to control antibiotic costs [19]. and uses very compact plastic reagent cards (similar in size to The potential clinical benefit of performing antimicrobial a credit card) that contain microliter quantities of antibiotics susceptibility tests in 3 10 hours rather than after overnight and test media in 30 or 45 well formats. The Vitek System incubation has been difficult to measure. Three studies [21 allows for repetitive turbidimetric monitoring of bacterial 23] have shown that providing rapid susceptibility testing is growth during an abbreviated incubation period. The instru- likely to result in a more timely change to appropriate antimicrobial ment can be configured to accommodate 30, 60, 120, or 240 therapy than is conventional testing after overnight inment simultaneous tests. The susceptibility cards allow testing of cubation. One of these studies [23] documented substantial common, rapidly growing gram-positive and gram-negative direct cost savings attributable to earlier correct diagnosis, aerobic bacteria in a period of 4 10 hours [18]. which resulted in fewer additional laboratory tests, fewer invasive The WalkAway and Vitek instruments can also perform simultaneous procedures, and shortened stays in intensive care units. identifications of gram-negative or gram-positive That study also surprisingly demonstrated lower mortality bacteria and merge and print identification and antimicrobial among patients whose results were obtained by using the rapid susceptibility results in a single computer report [18]. The man- instrument method. However, these benefits require aggressive ufacturers of both instruments have focused recently on enhancements laboratory reporting strategies to bring the rapid results to the to the computer software used to interpret and attention of physicians for appropriate action [21, 22]. It is not report susceptibility results. The manufacturer of the Vitek likely that rapid reporting of susceptibility test results will lead System has developed software called the Expert System, to measurable improvements in patient care unless more aggressive which analyzes susceptibility test results for atypical patterns laboratory reporting programs are initiated.

5 CID 1998;26 (April) Antimicrobial Susceptibility Testing 977 One of the previously cited shortcomings of rapid susceptibility testing methods has been lessened ability to detect some forms of inducible antimicrobial resistance [24 27]. In some instances, a short incubation period of 3 6 hours has not been sufficient for induction of an enzyme (such as a b-lactamase) that would destroy the antibiotic being tested [28]. Thus, the rapid test might indicate susceptibility to an antibiotic, whereas a conventional overnight test would allow time for enzyme induction resulting in obvious resistance. However, significant improvements in these FDA-approved instruments have been made, in large part through modifications of the instruments computer software so that either prolonged incubation for problematic organism-drug combinations is provided or susceptibility results are edited by using expert software to prevent unlikely results from being reported [19, 29 31]. In spite of these enhancements, surveillance for testing errors still ultimately rests with knowledgeable, well-trained laboratory personnel. The possibility of labor savings through increased efficiency of automation has not been realized in microbiology laboratories to the degree that it has been achieved in clinical chemistry and hematology laboratories. Some labor savings may result in large clinical microbiology laboratories where many automated susceptibility and identification tests are performed daily. However, in the era of cost containment, it is important to note that the most economical susceptibility test method currently available is the disk diffusion test [32]. Although the microdilution and rapid automated instrument methods offer several direct or ancillary benefits, with rare exception, they are more costly than the disk diffusion test. Selection of Drugs for Routine Susceptibility Testing Laboratory personnel must report the antimicrobial agents that are most appropriate for the organism isolated, for the site of the infection, and for the clinical practice setting served by the laboratory [33]. The battery of antimicrobial agents tested and reported routinely should relate directly to the institution s formulary. The NCCLS provides a table that lists the antimicrobial agents appropriate for testing the Enterobacteriaceae, Pseudomonas, and other gram-negative nonfermenters of glucose; staphylococci; enterococci; streptococci; Haemophilus species; and N. gonorrhoeae [16, 34]. The listing includes recommendations regarding the agents that are the most important to test routinely and those that may be tested selectively on the basis of an institution s formulary or prescribing policies or only by special request or under special circumstances (e.g., for epidemiological reasons). It is important to next determine the availability of antimicrobial agents used for a laboratory s routine testing methodology. Because of the inherent ability to select individual disks for testing, the Bauer-Kirby disk diffusion procedure offers the greatest flexibility of current methods. However, for practical purposes, only 12 agents can be tested on a single agar plate. Similarly, most broth microdilution trays contain 96 wells, effectively limiting the number of agents tested or the range of dilutions of each drug that can be included. This may also mean that no more than 12 agents can be tested in a microdilution tray if full concentration ranges of each agent are to be included for routine determination of MICs. Similar limits on the total number of drugs that can be tested simultaneously exist with the Vitek cards. Therefore, with any of these methods, there are practical limits to how many antimicrobial agents can be tested routinely. Manufacturers of commercially prepared antibiotic panels have attempted to deal with this problem by offering a number of different standard drug panel configurations or by including fewer dilutions of each drug in a single panel (thus allowing more drugs to be tested per tray or card) [33]. Despite these options, the increasing number of new drugs makes it unlikely that every laboratory can exactly represent its formulary among a manufacturer s standard antibiotic panels. Alternative possibilities include purchasing custom commercial panels that match precisely the institution s formulary, or perhaps, testing other antimicrobial agents that have activities that are essentially the same as the desired formulary drugs. The NCCLS susceptibility testing documents [16, 34] list groups of some antimicrobial agents with nearly identical activities that can provide practical alternatives for testing. Since periodic changes are likely in any institution s formulary because of changing clinical and economic needs, updating a laboratory s testing batteries represents an ongoing task. Interpretation of Susceptibility Test Results The results of a susceptibility test must be interpreted by laboratory personnel before a report is communicated to a patient s physician. In general, drugs with the lowest MICs for a given bacterial isolate are the best for treatment of an infection due to that isolate. However, optimal interpretation of MICs requires knowledge of the pharmacokinetics of a particular drug in humans as well as accumulated information on the success of that drug in eradicating bacteria at various body sites [35]. In practice, this is best accomplished by referring to an expert source such as the NCCLS, which publishes interpretive criteria for MICs of all relevant antibiotics for most bacteria [34]. Indeed, both MICs and disk diffusion zone diameters must be interpreted by using a table of values that relate to the proven clinical and microbiological efficacy of each antibiotic [16, 34]. The NCCLS zone size and MIC interpretive criteria are established by careful analysis of three kinds of data: microbiological data (e.g., a comparison of MICs vs. zone sizes against a large number of bacterial strains), pharmacokinetic data (e.g., levels of an antibi- otic in serum, CSF, urine, other secretions, and tissues), and clinical study results obtained during the phase before FDA approval and marketing of an antibiotic [36]. Thus, MIC interpretive criteria are not based simply on a comparison of serum levels of an antibiotic and MIC values. However, zone size

6 978 Jorgensen and Ferraro CID 1998;26 (April) may be required daily, or it may only be necessary once a week [16, 34]. A susceptibility test should never be performed with an unstandardized inoculum or a mixed culture [16, 34]. For that reason, direct susceptibility tests that incorporate the use of a patient s infected body fluid cannot be recommended, even for obtaining a presumptive result. What Is Acceptable Test Accuracy? A number of criteria have been proposed for defining accept- able accuracy of susceptibility testing methods. The terms very major error and major error have been used to describe false- susceptible or false-resistant results, respectively [28]. A false- susceptible result could lead to a patient being treated with a drug to which an organism is resistant. In contrast, a report of false resistance could result in administration of an unnecessar- ily expensive or toxic antibiotic instead of a less costly agent. In evaluations of new susceptibility testing methods it is im- portant to examine a representative number of strains that are resistant to various drugs to verify the ability of the new test to detect resistance [32]. It is also important to determine a new test s performance by examining a number of susceptible strains to determine the extent of major errors that might be expected in a typical clinical laboratory setting. The FDA criterion is that very major errors attributable to a new test procedure or instrument method should be õ1.5% for all individual species tested [38]. Furthermore, the rate of major errors cannot exceed 3%. Other slightly less stringent criteria for acceptable results, which take into account the inherent reproducibility of standard susceptibility testing methods and the difficulties inherent in recognizing some subtle resistance mechanisms, have been proposed [32]. Strengths and Weaknesses of Current Test Methods interpretive criteria are based in large part on direct correlations of MICs and zone sizes [36]. Susceptibility to an antibiotic indicates that an infecting organism should be eradicated by therapy with that antibiotic at the dosage normally recommended for that type of infection and species [35]. Conversely, when an MIC or zone size indicates resistance, an organism should not be inhibited by the concentrations of the antibiotic achieved with the dosages normally used [16, 34]. An intermediate result indicates that a microorganism falls into a range of susceptibility for which the MIC approaches or exceeds the concentration of antibiotic that can ordinarily be achieved and for which clinical response is likely to be less than with a susceptible strain. Exceptions can occur if an antibiotic is highly concentrated in a body fluid such as urine or if higher than normal dosages of an antibiotic can be administered safely (as is the case with some penicillins and cephalosporins). At times, an intermediate result can also mean that certain variables in the susceptibility test may not have been properly controlled and that the values have fallen into a buffer zone separating susceptible from resistant strains [16, 34]. It is important to be aware that the tables used for susceptibil- ity test interpretations represent the most current criteria that have been reviewed and accepted by the NCCLS. Indeed, the NCCLS documents are updated frequently, usually once a year. Use of old or outdated NCCLS tables could result in a serious shortcoming in the reporting of patients results. There is no objective evidence that reporting MICs is any more relevant clinically than reporting a categorical result of susceptible, intermediate, or resistant in the majority of cases [35, 37]. Reporting of MICs could aid a physician in selecting from among a group of similar drugs for treatment of infective endocarditis or osteomyelitis, for which therapy is likely to be protracted. For virtually all other infections, categorical results provide clinicians with the information necessary to select appropriate therapy. The trend has been toward the use of either broth microdilution Quality Control Procedures or automated instrument methods over the disk diffusion test. However, the Bauer-Kirby test may be regaining some It is essential that any susceptibility test method provide popularity because of its inherent flexibility and cost-effectivereproducible results within a given laboratory from day to day. ness. One of the problems most often mentioned with the commercial It is also necessary for patient care purposes that results generated microdilution or automated systems is the inflexibility in one laboratory are comparable to those generated in of the standard antibiotic batteries or test panels. Thus, the other laboratories. One of the best ways to assure reproducibility inherent flexibility of the disk diffusion test allows the testing and comparability is to test known control strains with well- of any 12 antibiotics that are deemed appropriate. Other assets defined susceptibility to a wide array of drugs. Such strains of the disk diffusion procedure include the fact that the interpre- are recommended by the NCCLS for routine monitoring in tive category results susceptible, intermediate, and resistant individual clinical laboratories [16, 34]. These strains include should be readily understood by all clinicians. The latter has both gram-positive and gram-negative bacteria similar to those not always been the case with MIC results. In fact, a survey likely to be encountered in clinical laboratories. The control conducted by the Infectious Diseases Society of America in strains should be tested whenever new reagents are introduced the 1980s indicated that MIC results may be misinterpreted and on a regular basis along with patients bacterial isolates. by some physicians and that categorical results may be prefera- Depending on a laboratory s demonstrated proficiency, this ble [37].

7 CID 1998;26 (April) Antimicrobial Susceptibility Testing 979 As indicated earlier, the broth microdilution susceptibility the isolation of colonies on an agar surface, should be developed. test has become the most popular method in American clinical Moreover, efforts should be invested toward developing laboratories. Its advantages include the quantitative nature of new automated equipment that could provide faster results and MICs, rather than a strict categorical result; the fact that it may also save money by virtue of lower reagent costs and reduced be performed with some organisms for which the disk test may labor requirements. To accomplish this it will likely be necessary not be standardized; and the computerized systems that are to devise a different methodologic approach than exists available from the commercial suppliers. However, an MIC presently. Improved optical detection methods or novel approaches method should not be chosen on the grounds that MICs per to the detection of bacterial proliferation are urgently se are more valuable to physicians. As stated above, many needed. One possibility might be the use of genetic probes or physicians who are not infectious diseases specialists are not gene amplification techniques [42], which could allow rapid familiar with the MICs of most contemporary antibiotics. detection of certain well-defined resistance mechanisms (e.g., An acknowledged shortcoming of the rapid, automated susceptibility the genes responsible for methicillin resistance in staphylomechanical testing instruments is that the possibility of a serious cocci, vancomycin resistance in enterococci, and quinolone failure mandates that a backup testing procedure resistance in gram-negative bacilli). The extent of the applica- such as disk diffusion or manual, overnight microdilution test- tion of this strategy to routine susceptibility testing is not clear ing is maintained, since fluorogenic or kinetic turbidimetric at this moment. The number of different b-lactamases and tests cannot be interpreted manually. In addition, the concept aminoglycoside-inactivating enzymes currently recognized that automated susceptibility instruments reduce labor requirements likely precludes the routine application of our current molecular through increased efficiency has been overstated. Labor genetic methods. Thus, it seems likely that phenotypic mea- savings are often negligible when these instruments are compared sures of the susceptibility of bacterial isolates to antimicrobial with efficient manual test methods, including the disk agents will continue to be clinically relevant for years to come. diffusion procedure [39]. 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