Susceptibility testing and antifungal agents

C I l I l l"e a 1M"l e r o b"lO 1o g Newsletter April 151 1980

Copyright © 1980 by (3. K. Hall & Co. ISSN0196-4399 i

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Susceptibility Testing and Antifungal Agents William G. Merz Johns Hopkins Hospital Baltimore, Marylar~ 21205 The microbiology laboratory plays a key role in establishing the diagnosis of systemic fungal infections, by recovering fungi in culture and/or detecting fungal constituents or metabolic products in body fluids. The laboratory's role in proriding pertinent information to help manage patients with documented systemic fungal infections is, however, less clear. The value in providing susceptibility data on fungal isolates will be the subject of this report. The choice of antifungal agents that are available for treatment of systemic mycoses is still limited and represented only by amphotericin B, 5-fluorocytosine, and miconazole.

PolyeneAntibiotics Amphotericin B (AMP-B) is a heptaene, polyene antibiotic that was isolated in 1956 from a strain of Streptomyces nodosus (9). It is only one of several polyene antibiotics that have been described since Hazen and Brown (10) first characterized nystatin, a substance isolated from Streptomyces noursei. AMP-B is the only polyene antibiotic that is available for parenteral treatment of systemic mycoses. The usefulness of most other polyenes is limited by their low solubility, poor stability, and their high toxicity. AMP-B's spectrum of antifungal activity has been established and confh'med by many investigators (27). The majority of the pathogenic and opportunistic fungi are susceptible to AMP-B at concentrations of less than 2 /~g/ml. Certain isolates of Sporothrix schenckii, Aspergillus spp., and Petriellidium boydii may require AMP-B concentrations of 6/~g to 100/~g/ml for inhibition. AMP-B and other polyene anm

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tibiotics alter fungal membranes by binding to sterols. The most common sterol, ergosterol, binds the antibiotic, resulting in the structural rearrangement of the cell membrane. This rearrangement leads to an altered membrane permeability with resulting leakage of potassium ions, phosphate ions, carboxylic acids, and eventually larger molecules, leading to reduced uptake of amino acids and glucose. In the laboratory, AMP-B resistant fungi have been induced by either serial transfer of an isolate in media containing increasing concentrations of AMP-B or by use of mutagenic agents (1, 18). The cell membranes of some resistant isolates have increased total sterol concentration, but they have decreased amounts of ergosterol or other delta 5-7 sterols. The development of resistance in vivo has not been observed frequently. Six strains of AMP-B resistant yeasts are reported in the literature, and we have seen an additional resistant isolate of Candida tropicalis (7, 22, 23, 25). Five of these seven strains have been recovered from patients receiving therapy for hematologic malignancies, and six patients had received systemic polyene antibiotics. Polyene-resistant isolates are suspected to be less pathogenic than susceptible isolates, although, recently, AMP-B resistant strains of C. lusitaniae from blood (23) and C. tropicalis from autopsy material (unpublished) have been reported. Since AMP-B resistant fungi have been recovered from specimens obtained from patients who are simultaneously treated with systemic AMP-B therapy and cytotoxic drugs, there is some speculation that the two together may play a .role in the development of AMP-B resistance. Techniques employed for the susceptibility testing of fungi to AMP-B have included: broth-dilution (27), microdilu-

tion (20), agar-dilution (5, 22), and disk-diffusion methods (26). Since AMP-B is insoluble in water, it must be dissolved in an organic solvent, usually dimethyl sulfoxide (DMSO). In addition, AMP-B is both light- and temperature-labile. Reagents must be prepared frequently and incubation temperature employed for susceptibility testing may have to be lowered when testing slower growing, f'damentous fungi. Attempts have been made to alter the structure of AMP-B and the other polyene antibiotics in order to produce a more soluble, less toxic derivative that will retain antifungal activity. The most promising derivative is amphotericin B methyl ester (AME) prepared by Mechlinski and Schaffner (21). It is water soluble, and animal studies have shown it to be less nephrotoxic and hepatotoxic than AMP-B (16). The in vitro antifungal activity of the AME determined is equal to, or slightly less active than, that of AMP-B, depending on the organism. Howarth (12) found AME to be slightly less active than AMP-B against the yeast phase of the dimorphic fungi. Only a few organisms displayed higher minimal inhibitory concentrations (MIC's) with AME. Filamentous forms of the dimorphic fungi, members of the phycomycetes, species of Aspergillus, dermatophytes, and agents of chromomycosis were less susceptible to AME than to AMP-B. The slightly lower activity of AME compared to AMP-B should not limit its value, however, since considerably more drug can be given and higher peak blood levels attained. Animal and clinical trials are pending. 5-Fiuorocytosine Another antifungal drug available for treatment of certain systemic mycoses is 5-fluorocytosine (5-FC). It is used

primarily for treatment of cryptococcosis, cand!d:"sis, and chromomycosis. 5-FC is a synt6etic drug that is absorbed by the gastrointestinal tract. The mechanism of action has been studied most recently with isolates ofC. albicans (24). 5-FC is taken up by fungal cells (uptake can be inhibited by cytosine and adenine) and is deaminated to 5-fluorouracil (5-FU). Intracellular 5-FU is incorporated into r-RNA and t-RNA, altering their structure, thereby affecting normal protein synthesis. Resistance may develop because of enzyme deficiencies, competition with normal metabolites, or both. Because resistance may develop with some frequency, susceptibility tests must be performed on all yeast isolates. 5-FC resistance in organisms not exposed previously to 5-FC (primary resistance) and the development of resistance during therapy are common. The MIC used to define resistance varies from 12 to 32pg/ml. Primary resistance can be demonstrated to be present in isolates of Candida albicans and otherCandida sp. Shadomy (27) has reported that 42% ofC. albicans isolates are resistant to 5-FC, whereas Speller and Davies (29) found only 4% of strains to be resistant. Primary 5-FC resistance in strains of C. Neoformans is much less common. The susceptibility of yeasts isolated at the Johns Hopkins Hospital, as determined by the agar dilution procedure, is presented in Table 1. These data, obtained with strains recovered from patients who did not receive 5-FC, indicate that primary resistance among yeasts is not uncommon. Resistance of C. albicans has been correlated (6) with specific serotypes; resistance being more common in serotype B. Variation in the frequency of serotypes between geographic areas may explain these differences reported in the literature. The development of secondary resistance during therapy with 5-FC occurs more frequently with Cryptococcus neoformans than with other yeasts, and stresses the need to test isolates recovered during the course of therapy for their susceptibility to 5-FC. Susceptibility tests with 5-FC have been performed using broth-dilution (27), agar-dilution (22), disk-diffusion (26), and microdilution (20) methods. Results are significantly influenced by the media used, the length of incubation,

and the inoculum size (3). Media containing cytosine and pyrimidine bases inhibit the activity of 5-FC. Yeast nitrogen-base medium is most frequently used because of the low concentration of these substances. Inoculum size is also critical since the yeast cell population may be heterogeneous in relation to its susceptibility to 5-FC. To avoid selecting the few resistant cells, the inoculum size should be kept at under 104 organisms/ml. The use of a combination of AMP-B and a second drug has been shown to have great advantages in vitro. Sub-MIC concentrations of AMP-B alter the membrane of the fungi and result in the increased uptake of a second anti-fungal agent. Combinations of 5-FC, rifampin, and tetracycline with AMP-B have been shown to be effective against different species of fungi in vitro and have been reviewed by Kobayashi and Medoff (17). Animal investigations are now confirming the value of these combinations. Clinically, the combination of AMP-B and 5-FC has been used effectively in treating cases of cryptococcal meningitis and systemic candidiasis. Imidazoles Among the imidazole group of antifungal agents, clotrimazole, econazole, miconazole, and ketoconazole all have significant antifungal activity. They are all structurally related compounds that are insoluble in water. At present, only miconazole is available for parenteral

administration. Miconazole has been shown (4) to cause decreased oxidase, peroxidase, and catalase activities in fungi with the probable accumulation of hydrogen peroxide and the inhibition of ergosterol biosynthesis, by the absence of C14 demethylation (11, 31). Miconazole has been demonstrated to have a broad spectrum of activity against fungi. Most dimorphic fungi, Petriellidium boydii, and the dematiaceous fungi (5, 8, 28, 32) are susceptible in vitro to from 1-101a g of miconazole/ml. There have been reports of successful treatment of coccidioidomycosis, petriellidiosis, and opportunistic yeasts infection (14, 15, 19, 30). Treatment failures have also been reported (8), and in these cases there was no correlation between in vitro susceptibility data and the clinical response. Therefore, the value of susceptibility tests for miconazole is unclear at this time. Ketoconazole is another imidazole with promising in vitro antifungal activity. Clinical trials and animal investigations are continuing in the evaluation of this drug. In conclusion, susceptibility testing of fungi has not, to date, played an important role in the microbiology laboratory. Susceptibility testing need not be routinely done for AMP-B, since susceptibility is predictable and resistance is rare. In addition, when organisms may be inhibited by higher than attainable AMP-B drug levels, or in diseases like invasive aspergillosis where response may be

Table 1. 5-FC Susceptibility Tests of Yeasts Isolated at JHH No. Resistant* Isolates

No. Tested

% Resistant

472

1217

39

78

263

30

C. parapsilosis

6

43

14

C. guilliermondii

0

13

0

C. krusei

2

12

17

C. pseudotropicalis

0

12

0

Torulopsis glabrata

3

173

2

Cryptococcus neoformans

1

l0

10

Candida albicans C. tropicalis

*Resistancedefinedas yeast with MIC of greater than 12 ~g/ml.

poor, alternative drugs are not available. 5-FC is an alternative agent in only a limited number o f infections and is used infrequently. When 5-FC is used, however, susceptibility tests should be performed. Presently, susceptibility testing o f fungi to miconazole is unnecessary. Further evaluation is needed before the value o f susceptibility testing can be assessed. When susceptibility testing o f fungi is performed, the tests employed are not currently standardized. This lack o f standardization may thus lead to variation in test results. Before the role o f the microbiology laboratory, in providing pertinent information relative to the management o f documented infections, can be established, new antifungal agents must be developed and susceptibility tests must be standardized. References 1. Athar, M. A., and H. I. Winner. 1971. The development of resistance by Candida species to polyene antibiotics in vitro. J. Med. Microbiol. 4:505. 2. Bannatyne, R. M., and R. Cheung. 1977. Comparative susceptibility of Candida albicans to amphotericin B and amphotericin B methyl ester. Antimicrob. Agents Chemother. 12:449. 3. Block, E. R., A. E. Jennings, and J. E. Bennett. 1973. Variables influencing susceptibility testing of Cryptococcus neoformans to 5fluorocytosine. Antimicrob. Agents Chemother. 4:392. 4. de Nollin, H. Van Belle, F. Goossens, F. Thone, and M. Borgers. 1977. Cytochemical and biochemical studies of yeasts after in vitro exposure to miconazole. Antimicrob. Agents Chemother. 11:500. 5. Dixon, D. M., G. E. Wagner, S. Shadomy, and H. J. Shadomy. 1978. I n vitro comparison of the antifungal activities of R34,000, miconazole and amphotericin B. Chemotherapy 24:364. 6. Drouhet, E., L. Mercier-Soucy, and S. Montplaisir. 1974. Nouvelles observations sur la sensibilite et la resistance des Candida aux 5-fluoropyrimidines Relations avec l'ecologie et le serotype du C. albicans. Bull. Soc. Fr. Mycol. Med. 3:9. 7. Drutz, D. J., and R. I. Lehrer. 1978. Development of amphotericin Bresistant Candida tropicalis in a patient with defective leukocyte function. Am. J. Med. Sci. 276:77. 8. Fisher, J. F., R. J. Duma, S. M.

Markowitz, S. Shadomy, A. Espinelo Ingroff, and W. H. Chew. 1978. Therapeutic failures with miconazole. Antimicrob. Agents Chemother. 13:965. 9. Gold, W., H. A. Stout, J. F. Pagano, and R. Donoviek. 1956. P. 579. Amphotericins A and B. Antifungal antibiotics produced by a streptomycete: I. In vitro studies. Antibiotics Annual 19551956. 10. Hazen, E. L., and R. Brown. 1950. Two antifungal agents produced by a soil actinomycete. Science 112:423. 11. Henry, M. J., and H. D. Sisler. 1979. Effects of miconazole and dodecyclimidazole on sterol biosynthesis in Ustilago maydis. Antimicrob. Agents Chemother. 15:603. 12. Howarth, W. R., R. P. Tewari, and M. Solotorovsky. 1975. Comparative in vitro antifungal activity of amphotericin B and amphotericin B methyl ester. Antimicrob. Agents Chemother. 7:58. 13. Hnston, A. C., and P. D. Hoeprich. 1978. Comparative susceptibility of four kinds of pathogenic fungi to amphotericin B and amphotericin B methyl ester. Antimicrob. Agents Chemother. 13:905. 14. Jordan, W. M., G. P. Bodey, V. Rodriguez, S. J. Ketchel, and J. Henney. 1979. Miconazole therapy for treatment of fungal infections in cancer patients. Antimicrob. Agents Chemother. 16:792. 15. Katz, M. E., and P. A. Cassileth. 1977. Disseminated candidiasis in a patient with acute leukemia. Successful treatment with miconazole. J. Am. Med. Assoc. 237:1124. 16. Keim, G. R., P. L. Sibley, Y. H. Yoon, J. S. Kulesza, I. H. Zaidi, M. 5I. Miller, and J. W. Poutsiaka. 1976. Comparative toxicological studies of amphotericin B methyl ester and amphotericin B in mice, rats, and dogs. Antimicrob. Agents Chemother. 10:687. 17. Kobayashi, G. S., and G. Medoff. 1977. Antifungal agents: Recent developments. Ann. Rev. Microbiol. 31:291. 18. Littman, M. L., M. A. Pisano, and R. M. Lancaster. 1958. P. 981. Induced resistance of Candida species to nystatin and amphotericin B. Antibiotics Annual 1957-1958. 19. Lutwick, L. M., W. Rytel, J. P. Ya° Nez, J. N. Galgiani, and D. A. Stevens. 1979. Deep infections from Petriellidium boydii treated with miconazole. J. Am. Med. Assoc. 241:272. 20. Mazens, M. F., G. P. Andrews, and R. C. Bartlett. 1979. Comparison of microdilution and broth dilution techniques for the susceptibility testing

of yeasts to 5-fluorocytosine and amphotericin B. Antimicrob. Agents Chemother. 15:475. 21. Mechlinsid, W., and C. P. Schaffner. 1972. Polyene macrolide derivatives. I N-acylation and esterifieation reactions with amphotericin B. J. Antibiot. 25:256. 22. Merz, W. G., and G. R. Sandford. 1979. Isolation and characterization of a polyene-resistant variant of Candida tropicalis. J. Clin. Microbiol. 9:677. 23. Pappagianis, D., M. S. Collins, R. Hector, and J. Remington. 1979. Development of resistance to amphotericin B in Calutida lusitaniae infecting a human. Antimicrob. Agents Chemother. 16:123. 24. Polak, A., and H. J. Scholer. 1975. Mode of action of 5-fluorocytosine and mechanisms of resistance. Chemotherapy 21:113. 25. Safe, L. M., S. H. Safe, R. E. Subden, and D, J. Morris. 1977. Sterol content and polyene antibiotic resistance in isolates of Candida krusei, Candida parakrusei, and Candida tropicalis. Can. J. Microbiol. 23:398. 26. Saubolle, M. A., and P. D. Hoeprich. 1978. Disk agar diffusion susceptibility testing of yeasts. Antimierob. Agents Chemother. 14:517. 27. Shadomy, S., and A. V. Espinellngroff. 1974. Susceptibility testing of antifungal agents, P. 569. In E. H. Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manual of Clinical Microbiology. American Society for Microbiology, Washington, D.C. 28. Shadomy, S., L. Paxton, A. EsplnelIngroff, and H. J. Shadomy. 1977. In vitro studies with miconazole and miconazole nitrate. J. Antimicrob. Chemother. 3:147-152. 29. Speller, D. C. E., and M. G. Davies. 1973. Sensitivity of yeasts to fluorocytosine. J. Med Microbiol. 6:315. 30. Stevens, D. A., H. B. Levine, and S. C. Deresinski. 1976. Miconazole in coccidioidomycosis. II. Therapeutics and pharmacologic studies in man. Am. J. Med. 60:191. 31. Swamy, K. H. S., M. Sirsi, and G. R. Rao. 1974. Studies of the mechanism of action of miconazole: Effect of miconazole on respiration and cell permeability of Candida albicans. Antimicrob. Agents Chemother. 5:420. 32. Van Cutsem, J. M., and D. Thienpont. 1972. Miconazole, a broad spectrum antimycotic agent with antibacterial activity. Chemotherapy. 17:392.