Nontraditional approaches to quality control of antimicrobial susceptibility tests

Clinical Microbiology Newsletter Vol. 12, No. 9

May 1, 1990

Nontraditional Approaches to Quality Control of Antimicrobial Susceptibility Tests Janet A. Hindler, MCLS MT(ASCP) Technical Specialist Department of Pathology, Clinical Microbiology Section UCLA Medical Center Los Angeles, CA 90024-1713 Quality control (QC) is an essential part of any clinical laboratory procedure and is defined as the series of steps taken to make certain that test resuits are accurate and reproducible. Antimicrobial susceptibility tests are somewhat unique in that they depend on growth (or no growth) of bacteria, which is often unpredictable and sensitive to changes in the environment. Therefore, it is essential that technical variables in the test system are well standardized and controlled and that personnel performing the tests thoroughly understand the components of the test system. This must all be considered when developing a program for QC of antimicrobial susceptibility tests.

Traditional Approaches Accrediting and licensing agencies require that laboratories regularly test reference strains of known antimicrobial susceptibility profiles as part of a QC program. A familiar and practical approach for testing such QC reference strains is described in detail by the National Committee for Clinical Laboratory Standards (NCCLS) (1, 2). The QC strains currently recommended for routine disk diffusion and dilution tests include: Escherichia coli ATCC 25922, CMNEEJ12(9)65-72,1990

Staphylococcus aureus ATCC 25923 (for disk diffusion), S. aureus ATCC 29213 (for dilution tests), Pseudomonas aeruginosa ATCC 27853, E. coli ATCC 35218, and Enterococcus faecalis ATCC 29212. When results with these reference strains agree with predefined criteria, the test system is considered to be in control. Multiple parameters of the test system are controlled (some of which are controlled to a greater degree than others) by testing the QC strains. Some of these include: • Antimicrobial potency • Test medium (e.g., pH; cation [Ca + +, Mg ÷ +] concentration; thymidine content; medium composition, growth factors; agar depth [disk diffusion]) • Incubation conditions (temperature, atmosphere time) • Standardization of inoculum and inoculation technique • Batch contamination • Instrument performance • Measurement of endpoints As would be expected, tests of the standard QC reference strains are limited in controlling test results of patient isolates. Some of the parameters not readily controlled are: • Individual antimicrobial/organism test problem (disk, well, tube) • Sporadic contamination of broth systems (a purity plate helps detect problems) • Sporadic instrument malfunction Elsevier

• NaC1 content for oxacillin broth tests with staphylococci • Subjective reading of "difficult" endpoints (i.e., "trailing") • Interpretation of results, use of appropriate interpretive criteria • Transcription errors • Individual technical errors Additionally, patient isolates may differ considerably from the recommended QC reference strains and their testing is only modestly controlled by performance testing of the suggested QC reference strains. Some examples of these include:

• Streptococcus pneumoniae and other nonenterococcal streptococci • Corynebacterium spp. • Other fastidious organisms • Organisms with "unusual" growth characteristics

In T h i s Issue Nontraditional Approaches to Quality Control of Antimicrobial Susceptibility Tests . . . . . . . . . . . . 65 Some practical suggestions for assuring the quality of your test results Son of Anaerobic Susceptibility Testing--Revisited . . . . . . . . . . . . . A reiteration of the problems with and clinical relevance of existing methods, along with examples of promising changes

69

0196-4399/90/$0.00 + 02.20

• mucoid P. aeruginosa • dwarf E. coli • Organisms with unusual resistance characteristics • oxacillin-resistant staphylococci • organisms with inducible resistance (Forthcoming NCCLS protocols will include QC reference strains for Haemophilus influenzae and Neisseria gonorrhoeae tests.) In addition to regular testing of QC reference strains, participation in proficiency testing programs may be considered as another "traditional" QC measure for antimicrobial susceptibility tests. Assuming that proficiency survey samples are handled in a manner similar to patient samples, satisfactory performance when testing these isolates provides further assurance that your laboratory is generating accurate results. Let's ask ourselves the following question: "Was our most recent discomfort about reporting antimicrobial results on a patient isolate due to (i) a QC reference strain demonstrating outof-control results or (ii) our observation of "unusual" results on the specific isolate? The answer for most technologists would probably be the latter. It is obvious that satisfactory performance testing of the recommended QC reference strains is extremely valuable; however, this alone does not ensure consistently accurate and reproducible results upon testing patient isolates. Thus, additional measures must be included in an antimicrobial susceptibility testing QC program.

Supplemental Strategies Inclusion of Supplemental QC Strains In addition to using the NCCLS-recommended QC reference strains, a laboratory may elect periodically to augment QC by testing selected in-

house organisms. Since detection of oxacillin-resistant S. aureus (ORSA) is subject to minor alterations in test conditions, including an ORSA isolate would seem reasonable. Similarly, there is significant concern that "rapid systems" do not detect beta-lactam resistance in Enterobacteriaceae, and laboratories that use a rapid system might want to test an isolate of Enterobacter cloacae resistant to ampicillin and several cephalosporins. One source for supplemental QC strains is proficiency surveys because program coordinators often select organisms that provide significant challenges for clinical laboratories (3). The attractive features about using proficiency survey organisms are that (i) all accredited clinical microbiology laboratories obtain these, and (ii) substantial information is often provided with the scores and the data reflecting the performance of many laboratories using a variety of test systems. Alternatively, supplemental QC organisms could be obtained from other microbiologists or the American Type Culture Collection. With these sources, laboratories are often faced with starting from scratch to establish any QC limits. Some manufacturers of commercial systems are recommending that user laboratories test additional strains that they have selected specifically for QC of their test system. Companies should provide these organisms as well as any other organisms with specific antimicrobial susceptibility characteristics that you might wish to examine. The resistance characteristics of some of these strains may not be stable and must be considered when a discrepancy is noted. A practical strategy for periodic testing of supplemental QC strains would be to use them to: • Make certain the test system can reliably detect a specific type of resistance (e.g., ORSA) • Train students and new technologists

• Periodically check proficiency of technologists performing testing • Trouble-shoot specific problems • Establish confidence in a new test system used in your laboratory

Technologist Proficiency Because significant judgment is required when performing antimicrobial susceptibility tests, a primary focus should be on making certain technologists are proficient in all aspects of the testing; this includes knowledge about how to verify the accuracy of individual patient results. Checklists customized to your laboratory's needs can be periodically distributed to technologists for self-assessment to make certain the most critical aspects of testing are understood. An example of the items that might be included on a proficiency checklist for the disk diffusion test would include: indications for using this method and its limitations; storage, handling, and characteristics of test materials and equipment; methods for preparation of inocula; specific modifications for testing H. influenzae, S. pneumoniae, N. gonorrhoeae, and Staphylococcus spp., etc. Technologists should be encouraged to seek assistance when they recognize that their knowledge in specific areas is lacking.

Antibiograms An antibiogram, or the antimicrobial susceptibility profile of a bacterial isolate to a battery of antimicrobial agents, is an extremely useful tool in helping to verify the accuracy of antimicrobial test results. Its use in verifying an organism's identification cannot be overemphasized (4). Certain bacteria often have predictable or "typical" susceptibility or drug-resistance patterns and the identity of a particular organism can be checked against its expected antibiogram. To optimize use of antibiograms, technologists must become familiar with typical antibiograms and develop an understanding of the rela-

NOTE. No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. No suggested test or procedure should be carried out unless, in the reader's judgment, its risk is justified. Because of rapid advances in the medical sciences, we recommend that the independent verification of diagnoses and drug dosages should he made. Discussions, views and recommendations as to medical procedures, choice of drugs and drug dosages are the responsibility of the authors.

Clinical Microbiology Newsletter (ISSN 0196-4399) is issued twice monthly in one indexed volume by Elsevier Science Publishing Co., 655 Avenue of the Americas, New York, NY 10010. Subscription prices per year: $98.00 including postage and handling in the United States, Canada, and Mexico. Add $43.00 for postage in the rest of the world. Second-class postage paid at New York, NY, and at additional mailing offices. Postmaster: send address changes to Clinical Microbiology Newsletter, Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010.

66

0196-4399/90/$0.00 + 02.20

© 1990 Elsevier Science Publishing Co., Inc.

Clinical Microbiology Newsletter 12:9,1990

tionship among the drugs that are tested. It is important to differentiate typical from atypical antibiograms. Because of numerous factors, not the least of which is the increasing use of antimicrobial agents, organisms with atypical antibiograms are becoming more common. However, atypical antibiograms, where results cannot be duplicated, may occur as a result of technical or clerical errors. These can often be recognized if laboratories help technologists to recognize atypical antibiograms consistently and provide a strategy that will minimize the reporting of erroneous results when testing patient isolates. Computer reporting systems often have a "built-in" capability for basic antibiogram checks. However, these do not preclude the need for technologists to check results that are generated. An example that illustrates typical and atypical antibiograms is shown in Table 1. In developing an antibiogram check program the following should be provided: • Descriptions of the relatedness of drugs tested (e.g., activity hierarchy). For example, the activity hierarchy of the three generations of cephalosporins against the Enterobacteriaceae is: 3rd > 2nd > 1st. • Descriptions of typical antibiograms for given species (e.g., the typical S. aureus is resistant to penicillin and susceptible to clindamycin, erythromycin, oxacillin, and vancomycin). • Informal updates to advise staff of the prevalence of a particular "atypical" antibiogram at a given time (e.g., increased incidence of nosocomial infections due to gentamicin-resistant Providencia rettgeri). Additionally, descriptions of the types of antimicrobial resistance (or susceptibility) that when reported (or missed) are likely to impact on patient care should be repeatedly emphasized. Examples would include P. aeruginosa resistant to amikacin, gentamicin, and tobramycin, particularly when recovered from systemic infections, ORSA from any source, and S. pneumoniae from CSF relatively resistant to peni-

Clinical Microbiology Newsletter 12:9,1990

TABLE I. Antibiograms---E. coli Result Patterna Antimicrobial

1

2

3

4

5

Amikacin Ampicillin Cephalothin Cefotaxime Cefuroxime Gentamicin Ticarcillin Tobramycin Trimethoprim/sulfamethoxazole

S S S S S S S S S

S R S S S S R S S

R R R S S R R R R

R S S S S S S S S

S R S R S S R S S

a1 2 3 4

Typical and most common antibiogram. Typical but less common antibiogram. Atypical, but possible, antibiogram (results of related drugs are reasonable). Atypical antibiogram; very unusual to encounter an isolate resistant to amikacin and susceptible to gentamicin and tobramycin. 5 Atypical antibiogram; unusual to encounter resistance to a third-generation cephalosporin if an isolate is susceptible to first- (cephalothin) and second- (cefuroxime) generation agents. S, susceptible; R, resistant.

cillin. Once an atypical antibiogram is identified, one must determine what, if anything, should be done to verify these results. A common misconception is that verification of atypical antibiograms always necessitates repeat testing. An example of a more cost-effective approach to verify atypical antibiograms would involve the following steps: (1) Check for transcriptional errors. (2) Reexamine agar disk diffusion plate, MIC tray, purity plate, etc., to make certain that the test appears satisfactory. Subtle problems may not always be detected upon initial examination of the test. (3) Check previous reports on the patient to determine whether this atypical antibiogram represents a repeat occurrence. (4) Repeat antimicrobial tests, identification tests, or both. In some situations it may be helpful to use an alternative test method to verify unusual results. We may elect to perform an MIC or chloramphenicol acetyltransferase test on a CSF isolate of H. influenzae that has a chloramphenicol zone diameter 1 mm below the breakpoint for susceptibility. We should always perform an MIC test on an S. pneumoniae isolate that demonstrates zones of <20 mm around a 1 I~g oxacillin disk to determine if the isolate is resistant (MIC >1.0 ~g/ml) or rela-

© 1990 Elsevier Science Publishing Co., Inc.

tively resistant (MIC 0.12-1.0 i~g/ml) to penicillin. Additionally, it may be appropriate to solicit the assistance of a reference laboratory that specializes in antimicrobial testing to verify virtually "unheard of" atypical results (e.g., vancomycin-resistant S. aureus). Some situations may warrant assistance from your local Public Health Department (e.g., the isolation of an S. pneumoniae strain that is resistant to penicillin, with a penicillin MIC of 8 ~g/ml). Technologists, often with supervisorial assistance, must determine when to report a result that requires verification by repeat testing. Obviously, the report should not be delayed; however, an erroneous result may have a greater adverse effect on patient care than a delayed report. Each situation must be evaluated individually. In our laboratory, we have developed a list of conditions that always require verification (Table 2). Our rationale for including some of these is based on the impact that erroneous results might have on patient care. Additionally, some of these represent situations where we have had significant problems in the past or represent situations that are very unlikely to occur. The clinical impact of an erroneous report can be illustrated by ORSA. Patients infected with oxacillin-susceptible S. aureus are usually treated with a penicillinase-resistant penicillin such as oxacillin. In contrast, the more

0196-4399/90/$0.00 + 02.20

67

T A B L E 2. Conditions requiring verification of antimicrobial susceptibility

test results 1. Oxacillin-resistant S. aureus ~'b 2. Gentamicin- + tobramicin- + amikacin-resistant gram-negative bacilli a,b 3. Penicillin-resistant or relatively-resistant S. pneumoniae (CDC addresses penicillinresistant isolates only)a,b 4. Ampicillin- + chloramphenicol-resistant H. influenzae ~'b 5. Chloramphenicol-resistant H. influenzae b 6. Cephalosporin-resistant H. influenzae b 7. Penicillin-resistant or moderately susceptible Streptococcus spp. b 8. Vancomycin-resistant or moderately susceptible gram-positive organisms (except Lactobacillus spp., Leuconostoc spp. or Pediococcus spp.) b 9. Enterococcus spp. with high-level resistance to streptomycin or gentamicin or bothb 10. Imipenem-resistant or moderately susceptible gram-negative bacilli (except Pseudomonas maltophilia, Pseudomonas cepacia, or Proteus/Providencia spp.) 11. Ciprofloxacin-resistant gram-negative bacilli (except P. maltophilia or P. cepacia) or S. aureus

12. 13. 14. 15.

Trimethoprirn/sulfamethoxazole-resistant P. maltophilia b Isolate where antibiogram is "atypical" for the species Isolate resistant to all relevant drugs Isolate where results of related drugs do not correlate

Patients with these organisms require contact isolation per CDC InfectionControl Guidelines (5). b Requires repeat testing unless patient had same isolate from another recent culture. a

costly and toxic vancomycin is the drug of choice for ORSA infections (6). Additionally, the hospitalized patient with ORSA requires isolation. Thus the added cost as well as clinical and psychosocial consequences for the patient of an ORSA report are significant, and maximum precautions must be taken when reporting this type of result. Reporting vancomycin resistance for coagulase-negative staphylococci isolated from one bottle of a blood culture set may not be regarded as significant by the general practitioner who suspects this to be a contaminant. However, this same information compiled into the laboratory's cumulative antimicrobial statistics may create minor chaos among the infectious disease specialists. Many of the examples stated thus far suggest specific types of resistance that should be verified. The more difficult check would be to verify "susceptible" results. Most of us would agree that it would be necessary to verify results when an ampicillin- and cephalothinsusceptible E. cloacae is encountered. However, in many situations there is no " c l u e " to suggest that a susceptible result may be erroneous. Reporting erroneously susceptible results can obviously have a significant clinical impact. For example, penicillin is the

68

0196-4399/90/$0.00 + 02.20

drug of choice for the patient with endocarditis due to a penicillin-susceptible viridans Streptococcus. However, the addition of an aminoglycoside would be warranted when treating this same patient, were he or she to have a penicillin-resistant or moderately susceptible isolate (7). To reiterate, the antibiogram check is only one component of the antimicrobial susceptibility testing QC program and probably allows us to identify potentially false resistant results better than those that are falsely susceptible. One check to minimize reporting of false-susceptible results would be to make certain that growth of the organism in the test system is sufficient. It is sometimes

tempting to read results even when growth on the disk diffusion plate or MIC tray is "light." Reliable results can be obtained with a disk diffusion test only when there is a lawn of confluent growth surrounding the zones of inhibition. Similarly, 3 + or 4 + turbidity or a large button of growth in the growth control well is needed in microdilution MIC tests. The dangers in reading results from tests that do not demonstrate adequate growth are obvious. All of the results generated by the laboratory are important. However, it is obvious that the consequences of certain reports will have a much greater impact than others. Therefore it is critically important for us to obtain insight into the results that are likely to have the greatest clinical impact and act on these appropriately.

Other Components of an Antimicrobial Susceptibility Testing QC Program In addition to the above, other measures can be taken to ensure reporting of accurate and reproducible antimicrobial susceptibility results on patient isolates. As mandated by accrediting agencies, supervisory review of laboratory reports will undoubtedly identify some errors that might have gone undetected. Periodic review of cumulative susceptibility statistics may uncover subtle inconsistencies. For example, if 5% of Enterobacter, Serratia, or Citrobacter are susceptible to ampicillin, an insufficient inoculum is probably being used (8). Boyce and colleagues observed an increase in the

TABLE 3. Components of a QC program for antimicrobial susceptibility tests 1. Satisfactorily perform required antimicrobial susceptibility test QC as mandated by accrediting agencies. This includes daily or weekly testing of QC reference strains and participation in a proficiency testing program. 2. Become knowledgeable in all aspects of the tests used and understand their limitations. Theoretical as well as technical knowledge is essential. 3. Become proficient in performing the tests you use. 4. Use antibiograms to check results on patient isolates; verify atypical antibiograms when necessary. 5. Incorporate a functional supervisory review process to review all reported results. 6. Develop an understanding of the most clinically significant results and strive to prevent errors that are likely to have a clinical impact.

© 1990ElsevierSciencePublishingCo., Inc.

Clinical MicrobiologyNewsletter12:9,1990

number of ORSA in their patient population and discovered defective oxacillin disks, a problem that went virtually undetected with routine testing of the QC reference strains (9). As summarized in Table 3, there are multiple components to an effective program for QC of antimicrobial susceptibility tests that will ensure good results when testing patient isolates. In addition to testing the QC reference strains as recommended by NCCLS, we must maintain a continual awareness of the critical parameters involved in test performance, the typical results that we would expect to encounter for a given organism, and the impact that specific results might have on patient care. Undoubtedly, it is critical that the entire microbiology laboratory staff participate in this ongoing program.

References 1. National Committee for Clinical Laboratory Standards. 1988. Performance standards for antimicrobial disk susceptibility tests--4th ed. Tentative Standard: M2-T4. NCCLS, Villanova, PA. 2. National Committee for Clinical Laboratory Standards. 1988. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically-2nd ed. Tentative Standard: M7-T2. NCCLS, Villanova, PA.

microorganisms, p. 810-824. In V. Lorian (ed.), Antibiotics in laboratory medicine. Williams & Wilkins, Baltimore. 5. CDC guidelines for isolation precautions in hospitals: nosocomial infections. 1983. Infect. Control 4:245-249.

3. Jones, R. N., D. C. Edson, and the CAP Microbiology Resource Committee. 1983. Special topics in antimicrobial susceptibility testing: test accuracy against methicillin-resistant Staphylococcus aureus, pneumococci, and the sensitivity of 13-1actamase methods. Am. J. Clin. Pathol. 80:609614. 4. Von Graevenitz, A. 1985. The use of antimicrobial agents as tools in epidemiology, identification, and selection of

6. The Medical Letter. 1988. Handbook of Antimicrobial Therapy. The Medical Letter, Inc., New Rochelle, NY. 7. Bisno, A. L., et al. 1981. Treatment of infective endocarditis due to viridans streptococci: AHA committee report. Circulation 63(3):730A-733A. 8. Washington, J. A., II. 1988. Current problems in antimicrobial susceptibility testing. Diagn. Microbiol. Infect. Dis. 9:135-138. 9. Boyce, J., et al. 1988. Spurious oxacillin resistance in Staphylococcus aureus because of defective oxacillin disks. J. Clin. Microbiol. 26:14251427.

tions. Of the six patients who actually received inappropriate therapy (based on culture results only or empirically directed), four died (2). Given these dismal assessments of the impact of susceptibility results on patient care, it is not surprising that anaerobic susceptibility testing is not a well-accepted routine procedure in many clinical laboratories. The knowledge and presumptive identifications of anaerobes in an infected site is likely to be of more practical use to clinicians than are definitive identifications and susceptibility results generated days after the specimen has been received. A cogent overview of technical problems associated with anaerobic susceptibility testing has just been presented in the Newsletter (3). We would like to discuss the issue from a slightly different perspective, highlighting another set of potential problems. The only currently acceptable susceptibility test capable of generating results after overnight incubation is the broth-disk elution procedure (4). The test works well for testing most common antibiotics against clostridia, peptostreptococci, and most non-fragilis Bacteroides. It usually

correlates with agar dilution results for penicillins, chloramphenicol, tetracycline, and metronidazole (5). However, results obtained when the Bacteroidesfragilis group, the most important anaerobes clinically and the most likely to be resistant, is tested against the broader spectrum cephalosporins (cefoxitin, cefotetan, cefoperazone, etc.) and clindamycin, the most commonly used agents, are far from comparable with reference methods (5-8). In most cases, the susceptibilities of the aforementioned anaerobic bacteria to penicillins, chloramphenicol, and metronidazole are actually predictable, reducing the necessity for routine testing. When growth of a gram-negative anaerobic bacillus is seen in the metronidazole tube, it is more likely due to a contaminated disk or a quality control problem than to a resistant organism. Gram-positive bacteria, however, are variably resistant to metronidazole, and resistance among gram-negatives has been reported in France. The broth-disk elution test is unsuitable for testing imipenem because thioglycolate inactivates this antibiotic. Fortunately, imipenem resistance

Editorial Son of Anaerobic Susceptibility Testingm Revisited Ellen Jo Baron, Ph.D. Consultant, Clinical Anaerobic Bacteriology

Research Laboratory Veterans Affairs Wadsworth Medical Center UCLA School of Medicine, Los Angeles, CA

Diane M. Citron, M.S. Associate Director, R. M. Alden Research Laboratory Santa Monica Hospital and Medical Center Santa Monica, CA

Hannah M. Wexler, Ph.D. Director, Microbial Diseases Research

Laboratories Veterans Affairs Wadsworth Medical Center UCLA School of Medicine, Los Angeles, CA

Physician use of antimicrobial susceptibility results is a difficult issue; Cannon et al reported that only 51.7% of physicians were aware of aerobic susceptibility results 72 h after reports were charted (1). A report from the Mayo Clinic found that physicians did not even consider susceptibility results in making therapeutic decisions for 47 of 65 patients with anaerobic infec-

Clinical Microbiology Newsletter 12:9,1990

© 1990 Elsevier Science Publishing Co., Inc.

0196-4399/90/$0.00 + 02.20

69