NCCLS Standards for Antimicrobial Susceptibility Tests

Transcription

1 CE Update Microbiology III NCCLS Standards for Antimicrobial Susceptibility Tests Clyde Thornsberry, PhD T he most requested test of the clinical microbiology laboratory today is probably t h e antimicrobial susceptibility test, and c e r t a i n l y a major role of the clinical microbiology laboratory is to aid the clinician in determining the best therapeutic regimen for the patient with an infection. In spite of this obvious demand, it was a p p r o x i m a t e l y four decades after Fleming discovered penicillin in 1929 that a "standardized method" was published 1 and a p p r o x i m a t e l y five decades before a "standard" for doing an antimicrobial susceptibility test was written. 2 In the 1940s Rammelkamp and Maxoh' reported on a broth dilution test for penicillin and Staphylococcus aureus, and o t h e r s ' '' reported on agar diffusion tests using filter paper disks. Many methods using one or more disks were recommended during the next several years, but the most significant research on a diffusion method was done by Kirby and his colleagues at the University of Washington during the 1950s and 1960s. Their work culminated in the publication of their classic paper in 1966.' One of the several important contributions of this group was that they offered a standardized method. They used the same culture medium in all tests, adjusted the inoculum, maintained the same conditions of incubation every time, used disks with various but predeter- From the Antimicrobics and Infection Mechanisms Branch, Hospital Infections Program, Center for Infectious Diseases, Centers for Disease Control. Atlanta. GA mined concentrations of antimicrobial, and measured zone diameters. A predetermined set of interpretive criteria was used to categorize the susceptibility as susceptible, intermediate, or resistant. Another factor contributing to the development of standardized susceptibility tests in the United States was the 1968 ruling by the Supreme Court, which approved the right of the Food and Drug Administration (FDA) to certify commercial antimicrobial disks before their sale. 6 This ruling had farreaching effects, and one by-product was the adoption of the Bauer-Kirby method for inclusion in the inserts for the commercial disks. 7 This action meant that the Federal government through FDA had adopted and recommended this method. The Centers for Disease Control (CDC) also recommended this method and has been active in further standardization of the method. fi Another organization that has been concerned about standard methods in all areas of the clinical laboratory is the National Committee for Clinical Laboratory Standards (NCCLS). Begun in 1966 and incorporated in 1968, NCCLS is a nonprofit organization whose members represent the clinical science professions, government, and industry. Its purpose is to develop standards on a national and international level and to promote voluntary acceptance and use of t h e s e standards. NCCLS develops standards through subcommittees composed of experts in the field. For example, the Subcommittee for Disk Susceptibility Tests is composed of microbiologists and physicians (including infectious disease experts and pathologists) from hospitals, universities, government, and industry. The subcommittee is also advised by several other persons from these professions and institutions. The subcommittee is led by a chairman and is under the general administration of the chairman of the Area Committee of Microbiology. When a standard is developed, it is submitted through the area committee chairman to the Board of Directors, who give final approval. Thus, each standard receives consensus approval. An NCCLS standard is developed in three stages. The first stage is the proposed standard, written and approved by the subcommittee itself and then submitted to the Area Chairman and the Board of Directors. If approved by them, it is printed and made available for purchase by anyone interested in the subject. Comments are solicited from the users and readers. These comments are collated for response by the subcommittee chairman and for use in revision of the proposed standard. After being available for at least one year, the proposed standard is ready for revision to a tentative standard, the second stage of development. The tentative standard goes through the same consensus approval process. If approved, after at least another year the tentative standard can be revised to the final, approved standard stage, the third stage of development. If approved this time, it then goes into a three-year revision cycle, ie, it is reviewed at the end of three years and LABORATORY MEDICINE VOL. 14, NO. 9, SEPTEMBER

2 revised if necessary. It can, however, be reviewed and revised at any time during the three-year period if there is reason to do so. It became obvious soon after the disk diffusion standard was developed that a dire need existed for a mechanism to r e v i s e or add new i n f o r m a t i o n without going through the regular consensus process. For example, when new antimicrobials are approved, information on disk mass, interpretive breakpoints, exceptions, etc, must be added to the standard. But to wait for the third-year revision would be unacceptable unless it was already late ih the third year. This problem was solved through the issuance of supplements, which contain for the most part updated tables that can be inserted into the standard document. The present disk diffusion document has had two informational supplements and would be ready for a third one, except that the standard document itself is soon to be revised. The standard sets forth specific criteria for performing a test. The intention is that the standard method is not to be modified. It may or may not be particularly suitable for routine use in a laboratory. For example, most microbiologists do not find the standard for susceptibility testing of anaerobic bacteria 9 to be a method that they would use routinely, but it is a standard to which they can compare the results of another test that they might use routinely. Most standards are, however, adaptable to routine use, as with the disk diffusion and broth microdilution methods. NCCLS has recognized that there is a need for methods that are developed through the consensus process but are meant to be modifiable if needed or desired for routine use and, thus, are not standards. Such documents are called NCCLS voluntary guidelines. For example, a guideline is now being developed for microdilution susceptibility tests on anaerobic bacteria. Disk Diffusion Standard The disk diffusion standard, "Performance Standards for Antimicrobic Disk Susceptibility Tests," was first proposed in 1971 and tentatively approved in The first edition was approved in 1976 and the second edition in The present designation of this standard is M2-A2-S2, meaning that standard M2 is approved, in the second revision, and has had two supplements. 10 In the development of this stand a r d, t h e m e t h o d of B a u e r a n d colleagues' as published by the FDA 7 was used as the platform on which the NCCLS standard would be written. During the development, many questions arose concerning some details of the methods. These were generally resolved by small collaborative studies involving the members of the subcommittee. The report by Ericsson and S h e r r i s " on an International Collaborative Study (ICS) was also used extensively. Although the standard method is e s s e n t i a l l y t h a t of B a u e r and colleagues, the subcommittee also recognized that most of the organisms that could be tested by the BauerKirby method could also be reliably tested by the agar overlay method. 12 Since t h i s m e t h o d h a d been adequately studied to confirm its accur a c y a n d r e p r o d u c i b i l i t y, it w a s included as an alternative. The disk diffusion standard is divided into five sections: (1) an introd u c t o r y s e c t i o n ; (2) a s e c t i o n on indications for susceptibility testing, the selection of antimicrobial disks to be tested, interpretation (ie, categorization of results based on zone diameters), and the limitations of the methods; (3) a section describing the materials and reagents to be used; (4) a section detailing the procedures to be used for the Bauer-Kirby method, the agar overlay method, for quality control, and for special disk diffusion tests on certain special or fastidious bacteria; and (5) a section citing a few of the appropriate references. Finally, there are the tables that show suggested basic sets of drugs to be tested, interpretive breakpoints for categorization of results, and control limits for use in quality control. The general methods for doing these diffusion tests do not change much from year to year, but a study of the differences between the first proposed standard (1971) and the second edition with the second supplement (1982) reveals the great changes that have occurred in the number of antimicro LABORATORY MEDICINE VOL. 14, NO. 9, SEPTEMBER 1983 bial agents available to test and the changes in susceptibility of certain "universally susceptible" pathogens to drugs of choice. For example, in the first proposed standard only one cephalosporin (cephalothin) was recommended for testing, but in the latest supplement (M2-A2-S2) there are, in addition to cephalothin, two secondgeneration cephalosporins (cefamandole and cefoxitin) and three thirdgeneration cephalosporins (cefotaxime, moxalactam, and cefoperazone) listed in t h e t a b l e of i n t e r p r e t i v e standards (breakpoints were not given for cefoperazone because they were not approved by the FDA at the time of publication). Several other newer drugs are also listed, and many more will have to be considered for the next revision. In 1971, at the time of the first proposed standard, strains of methicillinresistant S aureus were seldom isolated, nor were they a problem in the United States, and Hemophilus influenzae, Neisseria gonorrhoeae, and Streptococcus pneumoniae were all considered to be universally susceptible to the appropriate drugs of choice. In the latest document (M2-A2-S2), however, there is a special note on testing methicillin-resistant staphylococci, and special sections on testing H influenzae, N gonorrhoeae, and S pneumoniae. Application of the Standard How should this standard be used in the clinical microbiology laboratory? (1) It is essential that the tests be done as outlined in the standard. To do this, the standard must be up-todate. For example, at the time of this writing, the second supplement must be in use. Only with the most updated tables can correct interpretations be made. (2) Use Table 1 as a guide in selecting the antimicrobials to be tested. There was no intention to imply that all of these drugs need be tested, but rather that only those be selected that fulfill the needs of the patients and clinicians served by the laboratory. In some cases drugs are grouped, and one can be selected on the basis of individual needs, eg, selected methicillin or oxacillin or nafcillin.

3 (3) Test the standard reference organisms regularly. Although daily testing has been advocated in the past and is still preferred by many, most experts have now agreed that weekly testing will be adequate (providing the user has demonstrated an acceptable level of accuracy and reproducibility), and will be much more cost-effective. These strains should also be tested each time there is a change in materials used in the test or when new personnel do the tests. It is not enough, however, to merely test these strains; the results must be compared with the expected results given in Table 3 of the standard. For example, an individual test with E coli ATCC and amikacin should yield a zone diameter that falls between 19 and 26 mm. If it does not (95% of the time), then corrective action should be taken. Dilution Standard The NCCLS dilution standard, "Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically," was published as a proposed standard in 1980 but has recently been approved as a tentative standard (January, 1983, published March, 1983). The present NCCLS designation is.' 3 The dilution s t a n d a r d describes three methods for doing minimum inhibitory concentration (MIC) tests on bacteria that grow aerobically: the agar dilution method and the broth macrodilution and microdilution methods. With these three methods, achievable results are the same or within one log2 dilution. For routine use, most microbiologists prefer the microdilution method, and most commercial systems are of this type. These commercial microdilution systems have proved to be accurate and reproducible and are recommended for routine use, but users should prepare their own plates when the microdilution test is to be used as a standard. As with the disk methods, many facets of these methods were studied in collaboration with members of the s u b c o m m i t t e e and others. One example was the selection and charact e r i z a t i o n of s t a n d a r d r e f e r e n c e strains. It became obvious that the strains used to control the disk dif- fusion test were not adequate for the dilution tests. The ideal set of control strains for dilution tests would yield midpoint MICs for all drug-organism combinations. The strains currently recommended are E coli ATCC 25922, P aeruginosa ATCC (also recommended in the disk diffusion standard), S aureus ATCC 29213, and Streptococcus faecalis ATCC These four strains do not fill the needs for all combinations, but do present a usable set. The expected MICs for these strains of bacteria are given in Table 5 of the standard. Two parts of the standard should be singled out because each is, to some degree, controversial. First, the recommendation is made that the Mueller-Hinton broth be supplemented with calcium and magnesium. Although there is no clinical data to support this decision, the subcommittee members believed that data obtained in this medium more accurately reflected the in vivo situation. The second point of controversy is Table 1, in which MICs for all drugs are put into categories of susceptibility. These categories are patterned after those described in the ICS report. 11 In the proposed standard we called these categories very susceptible, moderately susceptible, moderately resistant, and resistant. The moderately susceptible category was used for those cases in which maximal safe dosage should be used. The moderately resistant category was used for those cases in which a drug was concentrated, principally in the urinary tract, and if the infection occurred in these areas, then the patient might respond to treatment with the drug. The resistant category was for those cases in which the organism would be resistant under any circumstances. After some experience with this system, we believed that our main problem lay with the use of the moderately resistant category, particularly in the possibility of toxic doses of some drugs if the meaning of the category was not thoroughly understood. As a result, we made some changes in the tentative standard. There are still four categories, but they differ as follows. The first three categories are susceptible, moderately susceptible, and resistant. These are intended for use with systemic infections, with m o d e r a t e l y susceptible meaning that the maximal safe dosage should be used. The fourth category is c a l l e d "conditionally susceptible" and is meant for those cases, eg, the urinary tract, in which the patient might respond because the drug is concentrated. Application of the Dilution Standard How should the dilution standard be used in the clinical microbiology laboratory? (1) These methods may be used as standards to which other methods can be compared, or they can be used as a "routine test." Decide which method to use and whether to use it routinely. According to present usage patterns, in most cases the choice would be broth microdilution; if so, decide whether to prepare plates in the laboratory or to use a commercial system. In the latter case results should compare favorably with those obtained with the standard method. (2) Use t h e s t a n d a r d reference strains at predetermined intervals of time or whenever different reagents, materials, or personnel are involved. Although I feel less comfortable about weekly testing with the dilution test than with the disk diffusion test, the tentative recommendation for weekly testing is being made. (3) Decide on a method for reporting results. The MICs can be reported without any attempt to categorize the results (ie, susceptible or resistant), both the MIC and category can be reported, or categories alone can be reported. The latter report would be an unusual one. The way in which results are reported is one of the more important parts of the testing. In conjunction with development of a report form, physicians should be advised b e f o r e h a n d if MICs a r e b e i n g reported for the first time. It is also advantageous to provide physicians with booklets containing appropriate data, such as levels of drug t h a t can be achieved in various tissues and body fluids. Standard for Susceptibility Testing of Anaerobic Bacteria The standard for anaerobic bacteria, "Tentative Standard Reference LABORATORY MEDICINE VOL. 14, NO. 9, SEPTEMBER

4 Agar Dilution Procedure for Antimicrobial Susceptibility Testing of Anaerobic Bacteria," was approved as a proposed standard in late 1979 and as a tentative standard in January, Its present NCCLS designation is M l l Work on this standard actually began in 1973 with the informal organization of a working group by NCCLS. They concluded that a dilution reference method would be their goal and that agar dilution was more reproducible than broth dilution for anaerobes. During the development of the s t a n d a r d, an NCCLS collaborative study supported by a contract with the FDA was done to determine variability and reproducibility. As a result of this study, the procedure and control strains given in the standard were selected. The standard is divided into nine sections. The first three sections are introductory, and the next two describe the materials and reagents that can be used in the test. The sixth section describes the procedure, and the seventh has instructions on reading the results. Section 8 deals with quality control and lists eight standard r e f e r e n c e s t r a i n s t h a t a r e recommended for control of the test all eight of these are for control of the media, and three are also for control of the test procedure. The latter three strains are Clostridium perfringens ATCC 13124, Bacteroides fragilis ATCC 25285, and Bacteroides thetaiotaomicron ATCC The expected MICs for these three strains are given in Table 2 of the standard document. The final section lists references. In this procedure, the inoculum is grown in supplemented thioglycollate broth and adjusted to a turbidity equal to a 0.5 McFarland standard. The final "spot" on the plate will contain a p p r o x i m a t e l y 10 5 colony forming units (CFU). The test is done on Wilkins-Chalgren agar. Probably more than the other two standards, this procedure is intended only as a reference standard to which other procedures can be compared, mostly because it is an agar dilution method and is not practical for routine use in most laboratories. The subcommittee is considering and will probably publish alternative methods that yield results comparable to the standard method. These publications will probably be approved guidelines rather than standards and will include broth dilution MIC tests, disk elution and other category tests, and abbreviated versions of the standard method. Use of the Standard How should one use this standard in the clinical microbiology laboratory? (1) Decide whether the agar dilution standard method is suitable for routine use. If not, select an alternative method. (2) Do a series of susceptibility tests using both the standard and alternative methods to confirm that results are similar. (3) If the alternative method is acceptable, control the test with the three reference strains tested at appropriate intervals. If anaerobe tests are done daily, it is probably acceptable to test the control strain weekly after a period of successful use of the test. If tests are done at intervals, test the control strains each time. (4) The results obtained with control strains should be consistent with the expected values given in the standard. Discussion The three NCCLS standards for antimicrobial susceptibility tests have proven to be useful to clinical microbiologists and others who perform antimicrobial susceptibility tests to aid in the selection of therapy, for those who develop new antimicrobials, and for those who develop materials and reagents for use in susceptibility tests. The standards have also contributed significantly to the standardization of susceptibility tests and to the accurate and reproducible performance of t h e s e t e s t s. D a t a from proficiency studies in recent years show that most clinical microbiologists perform these tests extremely well." The role that NCCLS standards have played in this good performance was illustrated in a recent College of American Pathology survey, in which approximately 86% of the participants said that they used the NCCLS standards and package inserts as their authoritative source for quality control and other guidelines (R. N. Jones, MD, personal comm u n i c a t i o n ). O t h e r s t a n d a r d s are b e i n g developed or considered by NCCLS subcommittees (eg, for susceptibility testing of fungi), and it is expected that they will be equally useful. Some interested parties have been concerned that these standards are copyrighted and thus may not be freely available to everyone. This is a legitimate concern but has not proven to be a problem. The standards can be purchased individually from NCCLS, but if an institution is a member of NCCLS it will receive all documents as they are published. The address of NCCLS is: National Committee for Clinical Laboratory Standards, 777 Lancaster Ave, Villanova, PA The status of the standards dealing with antimicrobial susceptibility t e s t i n g is s h o w n in T a b l e I. Ob- Table I: Status of NCCLS Standards for Ant microbial Suscept bility Tests Standard Status M2-A2-S2 Approved 1/76 9/79 Tentative 1/82 Tentative 1/83 Expected Revision Method Organism 5/81 3/ Disk diffusion R. J. Zabransky Agar dilution Grow aerobically not fastidious "Rapid growers" Anaerobes C. Thornsberry Agar dilution Macrodilution Microdilution Chairman R. N. Jones Supplements LABORATORY MEDICINE VOL. 14, NO. 9, SEPTEMBER 1983 Grow aerobically

5 Table II Control Strains for NCCLS Standard Antimicrobial Susceptibility Tests Species ATCC To control Standard E co//' S aureus P aeruginosa 6 fragilis B vulgatus C perfringens P mangnus P variabilis B thetaiotaomicron B thetaiotaomicron P asaccharolyticus S aureus S faecalis Disk diffusion, dilution tests Disk diffusion Disk diffusion, dilution tests and MICs and MICs and MICs Dilution tests Dilution tests M2-A2. M2-A2 M2-A2, viously, these data are applicable at the time of writing (April, 1983) and are subject to change. One of the major advances, partially brought about through the development of these standards, is the recommendations for standard reference strains. These strains are listed in Table II. Note that some are used in more than one standard and for more than one purpose. In conclusion, there are at present three NCCLS standards for antimicrobial susceptibility testing: one for disk diffusion, one for dilution tests, and one for a dilution test with anaerobes. These standards were developed and written by subcommittees of experts in the field and have undergone a consensus approval mechanism. Their main purpose is to serve as standard reference methods, but they may also be used as routine tests. These standards are constantly being updated and are invaluable aids to those who are doing or are interested in antimicrobial susceptibility testing. These standards have been inv a l u a b l e in the s t a n d a r d i z a t i o n of antimicrobial susceptibility tests and are the major source of quality control guidelines for most clinical laboratories. References > Bauer AW. Kirby WMM. Sherris JC. et al: Antibiotic susceptibility testing by standardized single disk method. Am J Clin Pathol 1966:45: Standards. Performance standards for antimicrobic disk susceptibility tests. Proposed Standard ASM-2. Villanova.Pa. NCCLS Rammelkamp CH, Maxon T: Resistance of Staphylococcus aureus to the action of penicillin. Proc Soc Exp Biol Med 1942:51: Vincent JC. Vincent HW: Filter paper disk modification of the Oxford cup penicillin determination. Proc Soc Exp Biol Med 1944:55: Bondi AS. Spaulding EH. Smith DE. et al: Routine method for rapid determination of susceptibility to penicillin and other antibiotics. Am -J Med Sci 1947:213: Wright WW: FDA actions on antibiotic susceptibility disks, in Balows A led): Current Tech- niques for Antibiotic Susceptibility Testing. Springfield. IL. Charles C Thomas pp Antibiotic susceptibilitv disks. Federal Register 1972:37: ' Thornsberry C. Baker CN: The agar diffusion antimicrobial susceptibility test, in Balows A led): Current Techniques for Antibiotic Susceptibility Testing. Charles C Thomas. Springfield. IL pp 3-8. Standards. Tentative standard reference agar dilution procedure for antimicrobial susceptibilitv testing of anaerobic bacteria. Tentative Standard Mll-T. Villanova. Pa. NCCLS National Committee of Clinical Laboratory Standards. Performance standards for antimicrobic disk susceptibility tests, ed 2. Approved Standard M2-A2. Villanova. Pa. NCCLS Ericsson HM. Sherris.JC: Antibiotic sensitivity testing. Report of an International Collaborative Studv. Acta Pathol Microbiol Scand (Suppll 1971:217:1-90. Barry AL. Garcia F. Thrupp LI): Improved single disk method for testing antibiotic susceptibility of rapidlv growing pathogens. Am J Clin Pathol 1970:53: Standards. Standard methods for dilution antimicrobial susceptibility tests for bacteria which grow aerobically. Tentative Standard. Villanova. Pa. NCCLS Jones RN. Edson DC. Marymont JV: Evaluations of antimicrobial susceptibility test proficiency bv the College of American Pathologists Survey Program. ASCP 78: , This is the final article in the current microbiology CE Update series. An examination for CME credit appears on page 582. LABORATORY MEDICINE VOL. 14, NO 9, SEPTEMBER