Antifungal susceptibility testing

ANNLDO 3(3) 17-26, 1986

ISSN 0738-1751

VOLUME 3, NUMBER 3, MARCH 1986

EDITORIAL BOARD Editor

Associate Editors

DANIEL AMSTERDAM, PhD,

RONALD N. JONES, MD,

State University of New York at Buffalo and Erie County Laboratory

Kaiser-Permanente Medical Care Program

HAROLD C. NEU, MD, College of Physicians and Surgeons, Columbia University

CLYDETHORNSBERRY, PhD, Center for Infectious Diseases, Centers for Disease Control

LOWELLS. YOUNG, MD, UCLA School of Medicine

ANTIFUNGAL SUSCEPTIBILITY TESTING* Recent Findings and Experience EDITOR'S NOTE D. AMSTERDAM Antifungal Susceptibility

Test'ing: Recent Findings and Experience

17

University of Arizona College of Medicine and Section of Infectious Diseases, Veterans Administration Medical Center, Tucson, Arizona

17

]. N. GALGIANI

Managing the Use and Cost of Antimicrobial Agents

JOHN N. GALGIANI

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T. R. BEAM, Jr

It was not that long ago, at least as recently as 1977, that the need to test for antifungal agents did not seem pressing. The reasons were several. First, standard methods for testing fungi did not exist.

Even had the methods existed, there were no options for using several or selecting among various agents because there was only one drug available, namely amphotericin B. It was used to treat systemic infection, and resistance to this compound had not yet surfaced. Also, there was some contention about in vitro reliability and

*Portions of this paper were presented at a symposium, "Clinical implications of antimicrobial resistance: Mechanisms, testing problems, and epidemiology," held November 21 and 22, 1985, in Philadelphia, PA, and sponsored by the Eastern Pennsylvania Branch of theAmerican Society for Microbiology. The symposium proceedings, edited by P. Actor, E. Hinks, and J. Schockman, will be published later this year by Plenum Press, NY.

Editor's Note In this issue, Dr. John Galgiani summarizes recent interlaboratory data and experience with antifungal susceptibility testing. Dr. Galgiani first commented on these matters in a previous issue of The A M N (1(5):40, May 1984)i Although considerable progress has been made since that time, there are as yet no definitive guidelines or approved National Committee for Clinical Laboratory Standards (NCCLS) procedures for evaluating these compounds.

ELSEVIER

In another section, Dr. Thomas Beam reviews some current measures that are being undertaken to educate and direct health care providers to become cognizant o f and involved in efforts that will ensure appropriate use of antimicrobial agents and in turn (hopefully) reduce costs. In the several applications and approaches he discusses, users have employed IBM-PC compatabile dedicated software to assist in the implentation of their programs. As a consequence o t t h e s e endeavors, these programs have resulted in more appropriate application of

antimicrobics in several different settings. The issues that Dr. Beam touches upon' have been raised in previous A M N newsletters and readers may wish to review some of these. (See Levy S: Antibiotics in the environment: Threat versus benefit AMN 1(2), February 1984; and Gardner DA, Lorian V: Bacterial susceptibility to antibiotics remains virtually stable. A M N 2(2), February 1985. The Editor's notes and comments in these issues provide additional references.)

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THE ANTIMICROBICNEWSLETTER,VOLUME3, NUMBER3, MARCH1986

TABLE 1. Currently Available Agents for Treating Systemic Fungal Infections Antifungal drug

Amphotericin B Flucytosine Miconazole Ketoconazole

Year drug first available

1957 1971 1978 1981

clinical relevance of in vitro susceptibility results. Finally, systemic mycoses were considered a relatively small problem, especially when compared with bacterial infections, and the imperative to develop testing methods enjoyed a lower priority. Although that position may have been reasonable in the past, it needs to be reconsidered in light of more recent developments. Table I lists the currently available agents used in the treatment of systemic fungal infections. Obviously, many fewer drugs are listed than are available to treat bacterial infections. However, note that the interval between each successive agent has become progressively shorter. Fourteen years had elapsed after the introduction of amphotericin B before the next agent, flucytosine (5-FC), was introduced. Seven years later, miconazole was released, and only three years after that, ketoconazole became available. Continued proliferation of antifungal agents is likely to continue because the pharmaceutical industry now perceives the field of antifungal therapy to be of much greater importance than it has in the past. This increased interest is due in part to a growing appreciation of the problem of preventing or treating systemic candidiasis and other opportunistic fungal infections. Also, there is interest in treating

the less severe but much more prevalent problems of vaginal candidiasis and fungal infections of the skin and nails. A second change relates to the earlier belief that one could assume the susceptibility of a specific fungus by knowing its species. This is now known not always to be the case. Resistance to amphotericin B in sterol-deficient Candida and the identification of a few imidazoleresistant yeasts have both been reportedY 3 With the increased use of these antifungal agents, it would not be too surprising if resistance became more prevalent. If that occurs, sensitivity will not be predictable without test results obtained on a patient's specific isolate. Ultimately, this may be the most pressing reason to develop standardized antifungal tests. Development of standardized antifungal tests would also benefit the pharmaceutical industry as it looks for new agents. There is no question that in vitro screening is much easier and faster than in vivo studies when in vitro methods are reliable. Further discussion of this point is beyond the scope of this paper, but preclinical applications of in vitro testing constitute another area where improved susceptibility testing of fungi would be of benefit. To put the situation with antifungal agents into a larger historical perspective, it may be useful to consider the evolution of susceptibility testing of bacteria. Recall that 1937 was the approximate year in which sulfonamides were introduced. However, it was some 30 years later that the work of Kirby, Bauer, Sherris, and many others provided meaningful standards of this and other antimicrobial agents. In this context, although we certainly would like to have

fungal testing standardized now, the field is not really out of keeping with the precedents in other groups of antimicrobials. If it is agreed that the availability to the clinical laboratory of standardized methods for testing antifungal agents is a desirable goal, then how should such a goal be achieved? In my view, it is simplest to first examine the in vitro testing methods in themselves. Once there is an understanding of the factors that influence such tests, it will be easier to select a testing method for developing the essential correlations with both laboratory animal infections and clinical results of treatment. What follows is an overview of recent studies that bear on both the methodology of in vitro testing and the beginnings at estimating their predictive value. Also, the recent activities of the National Committee for Clinical Laboratory Standards (NCCLS) will be reviewed.

TEST CONDITIONS THAT INFLUENCE TEST RESULTS Several test conditions have been identified as having significant effects on the results from antifungal drug testing. The most important of these are listed in Table 2. This list shows remarkable

TABLE 2. Test Conditions That Influence Broth Dilution Susceptibility of Fungi" Variable

Inoculum size Medium composition pH Incubation temperature Time of reading

Literature references

5-9 7, 10-16 12, 13, 16-18 8, 12, 17, 19 5-9, 11, 18, 20-23

a Partial list.

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THE ANTIMICROBICNEWSLETTER,VOLUME3, NUMBER3, MARCH1986

TABLE 3. In Vitro Activity of Ketoconazole at Various pH Values Against 23 Strains of C. albicans in Sabouraud Glucose Agar

pH of medium buffered SGA

MIC range ( #g/ml)

3 4 5 6 7 SGA"

2.5-40 1.25-10 0.16-0.63 0.02-0.08 _< 0.01-0.04 5-80

MICs0 14.5 2.4 0.32 0.03 0.01 26.0

MICgo 19.3 4.65 0.58 0.04 0.02 38.0

a Unbuffered medium. Initial pH was 5.3. Data from Minagawa et al. 18 SGA= Sabouraud glucose agar; MIG = minimal inhibitory concentration.

similarities to the variables that influence the testing of antibacterial agents. Although all of these variables for standardizing in vitro testing require close attention, by way of example I shall focus on one of them, medium composition, in the light of some recent work. Minagawa and co-workers is reported a striking effect of pH on the activity of ketoconazole. A summary of some of those findings is shown in Table 3. It seems quite clear that ketoconazole's activity is inhibited in unbuffered media that has a pH of 4 to 5. By buffering the pH towards the physiologic range, ketoconazole's activity increases. Table 3 illustrates another important point as well. In the acidic media the range of minimal inhibitory concentrations (MICs) is very broad, ranging from 5 to 80 #g/mL. However, as the pH is raised, the range narrows so that at pH of 7.0 all isolates agree within two-fold of each other. This suggests that all

of the isolates in this panel are quite similarly inhibited by ketoconazole and that much of the variability in the unbuffered medium could be related to the rates of change in pH during incubation. For this reason, media for fungal testing might need to be buffered, or at least have the initial pH adjusted to the physiologic range at the beginning of the test procedure. That pH could similarly effect 5-FC results was investigated by David Calhoun in my laboratory. He studied two different synthetic media, yeast nitrogen broth (YNB), 19 a medium that is normally unbuffered and acidic, and "synthetic amino acid media, fungal" (SAAMF), 20 a medium that is normally buffered to 7.4 with Mops/ Tris. Initial results supported this possibility, as is shown in Table 4. The mean results from a panel of 12 isolates of Candida albicans, when tested in YNB, gave lower end-

TABLE 4. Geometric mean susceptibility of 12 strains of C. albicans to flucytosine by broth dilution testing in differently formulated media Media

pH

Buffer

MIC

ICI/2

YNB SAAMF YNB SAAMF

5.6 7.4 7.4 5.6

None Mops/Tris Mops/Tris None

0.03 2.6 1.0 0.03

0.02 0.47 0.17 0.04

YNB = yeast nitrogen broth; SAAMF = synthetic amino acid media, fungal. Data from Calhoun and Galgiani. ~6

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points than did the same isolates in SAAMF. When the pH conditions were reversed by switching the buffer between media, the results also were reversed. However, with additional experiments, it was found that the Mops/Tris buffer itself rather than pH was actually the source of these differences. As the Mops/Tris concentration was decreased or when Mops/Tris was replaced with other buffers such as Hepes or phosphate, the susceptibility increased despite maintaining a physiologic pH. These results indicate that detailed analysis of media effects probably should be carried out separately for each new agent.

CORRELATIONS BETWEEN LABORATORY TESTS AND TREATMENT RESULTS The correlation of susceptibility test results with those of treatment in experimental or clinical infections is essential for determmining the practical value of the in vitro information. Such correlations are starting to be developed for antifungal agents. An important work was published by Stiller and his many collaborators, z4 In that report, four groups of ten isolates, each of C. albicans, were studied. Each group has been found to have different in vitro sensitivities for 5-FC. Each isolate then was used separately to infect mice intravenously and the effect of 5FC treatment in this murine model was determined. The in vitro and in vivo results were then compared by several statistical methods. From these studies, Stiller et a124 were able to demonstrate a significant relationship between the activity of 5-FC as determined by susceptibility testing and the efficacy of 5-FC treatment. They also found that the relationship was not very strong, indicating that other factors might be involved in determining outcome.

~) 1986 by ELSEVIERSCIENCEPUBLISHINGCO., INC.

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We have retested 36 of the 40 strains used in those studies, and have measured their susceptibility by slightly different methods. Specifically, SAAMF buffered with Hepes to pH 7.4 was substituted for YNB, and drug dilutions were prepared so that turbidimetric (IC~) endpoints were obtained for all susceptible isolates. These modifications resulted in IC~ values that demonstrated as significant an association and with virtually identical Spearman rank'correlation coefficients as had been reported by Stiller et al. A very different type of approach was taken by Ryley et al. 2 For those studies, isolates were used from two patients with chronic mucocutaneous candidiasis. These patients initially had responded to treatment with ketoconazole, but had subsequently relapsed many months later while still receiving therapy. Ryley et al z demonstrated that with certain methods of in vitro testing, the isolates appeared significantly different from a panel of Candida isolates from many other sources. Ryley et al also used these patients' isolates and several others for treatment studies in mice, demonstrating that ketoconazole was much less effective in treating disease caused by these isolates than disease caused by several other strains of Candida. In this study, a similar correlation between in vitro inhibition and in vivo efficacy was seen with another imidazole (ICI-153,066). Other laboratories have extended these findings to Fluconazole, a drug which Pfizer, Inc. (New York, NY) is now developing, zS,z6 These various studies indicate that resistance to ketoconazole may result in resistance to other members of that class of drug. Clearly, the above-mentioned studies leave many questions unanswered and much more work needs to be done in this area. However, I find it very encouraging that such studies are now being published.

THE ANTIMICROBIC NEWSLETTER, VOLUME 3, NUMBER 3, MARCH 1986

CURRENT ACTIVITIES OF THE NATIONAL COMMUNITY FOR CLINICAL LABORATORY STANDARDS From these examples it should be clear that fungal testing, like susceptibility testing of bacteria, is influenced by numerous test conditions, and that fungal testing has, in the hands of some investigators, predictive value for treatment efficacy. However, very little had been done to standardize the field or to coordinate the efforts of those interested in improving clinical testing of fungi. For these advances, credit should be given to the efforts of several persons. They include Dr. Arthur Barry, who proposed to the NCCLS that fungal testing be standardized by the activities of a subcommittee under its auspices, Dr. John Sherris who provided a great deal of encouragement, especially in the early stages of the subcommittee's formation, and the many members, advisors, and observers of the subcommittee who have volunteered their time. As a result of these

efforts, the NCCLS established in 1983 a Subcommittee on Antifungal Tests. Since then the subcommittee has been involved in three projects. First, it has carried out a questionnaire survey of antifungal testing practices among NCCLS members. Second, it supported an independent study by several investigators to test the reproducibility of a commonly used procedure for ,5-FC and amphotericin B testing. Finally, the subcommittee has completed a report that reviews the current status of antifungal testing and proposes some recommendations for future work. Turning to the survey results first, the questionnaire was sent to 350 hospitals with 300 or more beds. Nearly two-thirds of the questionnaires were completed. Of respondents, 47 (19%) indicated that they tested fungi for their susceptibility to antifungal agents. However, most tested fewer than 20 isolates in the previous three years. Candida albicans was the species and amphotericin B and 5-FC were the agents most frequently

Figure 1.

Relative susceptibility of five strains of C. albicans and one strain of S. cerevisiae to 5-FC as determined by seven different laboratories using the

same broth dilution method. Each symbol represents individual strains. Columns A-G represent results within different laboratories. Symbols within shaded boxes indicate that those strains had identical MICs as determined by that laboratory.

I

NCCLS Antifungal Susceptibility Study: 5-FC Rank Order of Isolates

M )st esi ~tant i

]st ita nt

RLs~

© 1986 by ELSEVIER SCIENCE PUBLISHING CO., INC.

A

B

C

D

E

F

G

Laboratory

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THE ANTIMICROBICNEWSLETTER,VOLUME3, NUMBER3, MARCH1986

tested. A large majority of laboratories used a broth dilution method of some sort, usually that of Shado m y and Espinel-Engroff. 27 Furthermore, over 90% of laboratories doing tests reported that the primary use of their results was for patient management. The homogeneity of the survey results made the job of designing an initial interlaboratory study considerably easier. In keeping with the survey, five isolates of C. albicans were selected: four were from the different groups used by Stiller et al,24 and had varying sensitivities to 5-FC; the fifth isolate was supplied by Dr. William Merz and had previously been characterized as sterol-deficient. In addition, an isolate of Saccharomyces cerevisiae also was included in the study. The broth dilution method as published in the fourth edition of the American Society for Microbiology Manual of Clinical Microbiology was used to study the susceptibility of these isolates to 5-FC and amphotericin B. Seven separate laboratories participated, each performing replicate tests both on the same day and on three separate days. The complete details of that study have been published elsewhere. 28 To summarize the results, agreement of replicates within each laboratory was very good: nearly 95% of replicates performed on the same day gave identical results, and 95% of replicates performed on different days agreed within one 2-fold dilution of each other. However, when results for different isolates tested in different laboratories were compared, the agreement was not nearly as close. For various combinations of drugs and yeasts, the variability among laboratories ranged from 8- to 512-fold. Despite these major discrepancies, the results also showed that generally all laboratories reported the same rank order for the panel of isolates as is shown for 5-FC in Figure 1. Since all laboratories agreed on the relative sensitivities of the isolates,

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it seems the difficulty was in agreement on the endpoint. SUMMARY The current status of antifungal susceptibility tests is very much in flux. There is ample evidence that current testing of fungi is at this point poorly standardized, and therefore the results from such tests are difficult to apply to patient management. On the other hand, there is now considerable interest in collaborative efforts to change this situation and all evidence to date indicates that standardization is a reasonable and clearly achievable goal. In the NCCLS Subcommittee Report, 20 several steps were proposed to reach that objective. Among others, they include: 1) Establishing a panel of fungal isolates to be used in further studies; 2) Establish performance standards for a single media that might be used to test all known classes of antifungal agents; and 3) Determine and correct the sources of error that result in variability of the broth dilution method. Now that the problem has been more sharply defined, work to eliminate interlaboratory variability and to determine test correlation with treatment results should proceed. Hopefully work during the next few years will result in antifungal tests that are just as useful as today's tests for antibacterial agents.

This work was supported in part by the Veterans Administration, Eli Lilly and Company, Janssen Pharmaceutica, Bristol Meyers Company, Burroughs Wellcome Company, Miles Laboratories, Inc., Syntex Laboratories, Inc., and E.R. Squibb & Sons, Inc. I also would like to thank John E. Bennett for making available the isolates and detailed results from reported 24 studies. Laurinda Crawford contributed valuable technical assist-

21

ance to the conduct of some of our

work.

REFERENCES

1. Dick JD, Merz WG, Saral R: Incidence of polyene-resistant yeasts recovered from clinical specimens. Antimicrob Agents Chemother 218:158-163, 1980. 2. Ryley JF, Wilson RG, Barrett-Bee KJ: Azole resistance in Candida albicans. Sabouraudia 22:53-63, 1984. 3. Stiller RL, Bennett JE, Scholer HJ, Wall M, Polak A, Stevens DA: Susceptibility to 5-fluorocytosine and prevalence of serotypes in 402 Candida albicans isolates from the United States. Antimicrob Agents Chemother 22:482-487, 1982. 4. Ericsson HM, Sherris JC: Antibiotic sensitivity study. Report of an international collaborative study. Acta Pathol Microbiol Scand Sect B 217:1-90(S), 1971. 5. Brass CB, Shainhouse JZ, Stevens DA: Variability of agar dilutionreplicator method of yeast susceptibility testing. Antimicrob Agents Chemother 15:763-768, 1979. 6. Galgiani JN, Stevens DA: Turbidimetric studies of growth inhibition of yeasts with three drugs: Inquiry into inoculum-dependent susceptibility testing, time of onset of drug effect and implications for current and newer methods. Antimicrob Agents Chemother 15:763-768, 1978. 7. Odds FC: Interactions among amphotericin B, 5-fluorocytosine, ketoconazole and miconazole against pathogenic fungi in vitro. Antimicrob Agents Chemother 22:763-770, 1982. 8. Block ER, Jennings AE, Bennett JE: Variables influencing susceptibility testing of Cryptococcus neoformans to 5-fluorocytosine. Antimicrob Agents Chemother 4:392-395, 1973. 9. Galgiani JN, Stevens DA: Antimicrobial susceptibility testing of yeasts: A turbidimetric technique independent of inoculum size. Antimicrob Agents Chemother 10:721-726, 1976. 10. Utz C, Shadomy S: New medium for in vitro susceptibility studies with amphotericin B. Antimicrob Agents Chemother 10:776-777, 1976.

© 1986 by ELSEVIERSCIENCEPUBLISHINGCO., INC.

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11. Hoeprich PD, Saubolle MA, Houston AC: Susceptibility testing of fungi. In A Bondi, JT Bartola, JE Prier (eds), The clinical laboratory as an aid in chemotherapy of infectious disease. Baltimore, University Park Press, 1977, pp 101-106. 12. Holt RJ, Newman RL: Laboratory assessment of the antimycotic drug clotrimazole. J Clin Pathol 25:1089-1097, 1972. 13. Bannatyne RM, Cheung R: Susceptibility of Candida albicans to miconazole. Antimicrob Agents Chemother 13:1040-1041, 1978. 14. Polak A, Grenson M: Interference between the uptake of pyrimadines and purines in yeast. Pathol Microbiol 39:37-38, 1973. 15. Polack A, Scholer HJ: Mode of action of 5-fluorocytosine and mechanisms of resistance. Chemotherapy 21:113-130, 1973. 16. Calhoun DA, Galgiani JN: Analysis of pH and buffer effect of flucytosine activity in broth dilution susceptibility testing of Candida albicans in two synthetic media. Antimicrob Agents Chemother 26:364367, 1984. 17. Johnson B, White RJ, Williamson GM: Factors influencing the susceptibility of Candida albicans to the polyenoic antibiotics nystatin and amphotericin B. J Gen Microbiol 104:325-333, 1978. 18. Minagawa H, Kitawra K, Nakamizo N: Effects of pH on the activ-

THE ANTIMICROBIC NEWSLETTER, VOLUME 3, NUMBER 3, MARCH 1986

ity of ketoconazole against Candida albicans. Antimicrob Agents Chemother 23:105-107, 1983. 19. Mazens MF, Andrews GP, Bartlett RC: Comparison of microdilution and broth dilution techniques for the susceptibility testing of yeasts to 5-fluorocytosine and amphotericin B. Antimicrob Agents Chemother 15:475-477, 1979. 20. Hoeprich PD, Finn PD: Obfuscation of the activity of antifungal antimicrobials by culture media. J Infect Dis 126:353-361, 1972. 21. Shadomy S, Paxton L, EspinelIngroff A, Shadomy J: In vitro studies with miconazole and miconazole nitrate. J Antimicrob Chemother 3:147-152, 1977. 22. Utz C, Shadomy S: Antifungal activity of 5-fluorocytosine as measured by disk diffusion susceptibility testing. J Infect Dis 135:970-974, 1977. 23. Aerts F, DeBrabander M, van den Bossche H, van Cutsem J, Borgers M: The activity of ketoconazole in mixed cultures of fungi and human fibroblasts. Mykosen 23:53-67, 1980. 24. Stiller RL, Bennett JE, Scholer HJ, Wall HJ, Polak A, Stevens DA: Correlation of in vitro susceptibility test results with in vivo response: Flucytosine therapy in a systemic candidiasis model. J Infect Dis 147:1070-1076, 1983.

fungal activity of the triazole UK49,858 in the treatment of experimental Candida pyelonephritis and Cryptococcal meningitis. In International Society for Human and Animal Mycology Ninth International Congress, Program and Abstracts. Abstr R2-6, 1985. 26. Rogers TE, Galgiani JN: Antifungal activity of UK-49, 858 against Candida albicans in vitro and in rats after intravenous infection. In press. Antibiot Agents Chemother. 27. Shadomy S, Espinel-Ingroff A: Susceptibility testing with antifungal drugs. In E. Lennette (ed), Manual of Clinical Microbiology, 3rd edition. Washington, DC, 1980, pp 647-653. 28. Calhoun DL, Roberts GD, Galgiani JN, Bennett JE, Feingold DS, Jorgensen J, Kobayashi GS, Shadomy S: Results of a survey of antifungal susceptibility testing in the United States and interlaboratory comparison of broth dilution testing of flucytosine and amphotericin B. J Clin Microbiol 23:298301, 1986. 29. National Committee for Clinical Standards: Antifungal susceptibility testing; Committee report. NCCLS publication M20-CR. Villanova, PA, NCCLS, 1985.

25. Perfect JR, Durack DT: The anti-

MANAGING THE USE AND COST OF ANTIMICROBIAL AGENTS Thomas R. Beam, Jr Infectious Diseases, Buffalo Veterans Administration Medical Center, Department Of Medicine, State University of New York at Buffalo, Buffalo, New York

T h e p r o b l e m s of overuse, misuse, and abuse of antibiotics are worldwide in scope. P r o p e r application of these d r u g s can be life-saving. I n a p p r o p r i a t e use p r o m o t e s e m e r -

gence of resistant bacteria, develo p m e n t of suprainfection, needless toxicity, and w a s t e of health care resources. Several investigators in the United States have developed prog r a m s to define the m a g n i t u d e of the problem, i m p r o v e prescribing habits, and reduce expenditures. T h e p u r p o s e s of this article are to

~) 1986 by ELSEVIERSCIENCE PUBLISHING CO., INC.

s u m m a r i z e the approaches being t a k e n and e n c o u r a g e health care professionals to b e c o m e involved in this endeavor.

S C O P E OF T H E P R O B L E M T h e r e are several w a y s to analyze the a m o u n t of antibiotics used in the United States. Bulk p r o d u c t i o n

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