INTRODUCTION

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Institutions across the United States have reported an increase in their rate of nosocomial fungal infections (3). In the 1980s Candida species were responsible for approximately three-quarters of these fungal infections (3, 24), with Candida albicans being the most commonly isolated (59.7%) species (3). The greatest increase has been noted in bloodstream infections (2, 3), focused primarily in critical care units (3, 35). The rise in fungemia has been striking, ranging from 75% in small (200 beds) nonteaching hospitals to 487% in large (>500 beds) teaching hospitals (2). High rates of morbidity and mortality are associated with candidal infections in immunocompromised patients (3, 15, 23, 24, 35, 37, 38), and early diagnosis can be difficult. With Candida bloodstream infections accounting for the highest rates of mortality, Pittet and colleagues found that even single positive cultures could not be ignored (26). Mindful of these factors, the use of antifungal agents for prophylaxis and therapy has grown (1, 28). Recent reports have also suggested an increasing prevalence of yeast isolates resistant to the newer azole class of antifungal agents (27, 29), implying that future antifungal treatment and prophylaxis may be more difficult.

We undertook the current study to (i) determine the prevalence of resistance in yeast isolates colonizing non-AIDS, immunocompromised patients at Northwestern Memorial Hospital (NMH) and (ii) to assess any association of increased use of imidazoles, particularly fluconazole, to the increasing rate of resistance of yeasts to these agents.

	MATERIALS AND METHODS

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Prevalence of imidazole resistance in yeast isolates from non-AIDS, immunocompromised patients. Rectal swabs were obtained from patients hospitalized for therapy for leukemia or lymphoma and from patients undergoing bone marrow or solid-organ transplantation between June 1995 and January 1996 as part of our routine screening program for the detection of vancomycin-resistant enterococci. Swabs were inoculated onto in-house-prepared 5% sheep's blood agar plates containing 10 µg of vancomycin and 20 µg of gentamicin/ml of agar. Colonies from all plates with growth of more than five yeast colonies were collected for yeast identification and susceptibility testing. One hundred one yeast strains were recovered from 97 non-AIDS, immunocompromised patients for susceptibility testing. A review of the medication histories was performed for all but four of the patients, whose charts encompassing their hospitalization period when the swab for culture was obtained were unavailable. An assessment of any antifungal agent administration for a period of 6 months prior to obtaining the specific swab for culture was performed. The susceptibility test results for these isolates were then compared to the susceptibility patterns for the fresh clinical isolates obtained during 1994 and 1995, the times just before and during our surveillance study. Susceptibility testing was also performed with fresh clinical isolates obtained from 1996 and 1997 to assess any ongoing trends in susceptibility patterns.

Determining prevalence of azole resistance in archived yeast isolates. Archived C. albicans isolates that had been recovered from blood and other sterile body fluids between 1984 and 1993, along with all fresh clinical isolates obtained from 1994 through 1997, were tested. From 1994 through 1997, isolates from blood and other sterile body fluids, excluding urine, were routinely tested. Yeast isolates from other body sites were tested only by specific physician request. The yeasts were identified to the species level by the NMH Clinical Microbiology Laboratory by standard methods. These conventional biochemical methods included tests for carbohydrate assimilation, fermentation of maltose, cornmeal agar morphology, and utilization of urea. The carbohydrate assimilation test used glucose, maltose, sucrose, lactose, raffinose, trehalose, and cellobiose discs on yeast nitrogen agar plates.

Susceptibility testing. Susceptibility testing was performed by a broth microdilution method that we have described previously (8) and that is based on the approved National Committee for Clinical Laboratory Standards guideline for a broth macrodilution reference method (16). Sterile, plastic microtiter trays (MIC 2000; Dynatech Laboratories, Chantilly, Va.) with round-bottom wells were used to prepare panels for susceptibility testing with an automated tray-dispensing system (Quick Spense II; Dynatech Laboratories). The microtiter plates contained two separate media: Eagle's minimal essential medium and RPMI 1640 (Bio-Whittaker, Walkersville, Md.). The trays for this study assessed five drugs, each at four different concentrations: amphotericin B (Bristol-Myers Squibb, Princeton, N.J.) at 0.5, 1, 2, and 4 µg/ml; flucytosine (Roche Laboratories, Nutley, N.J.) at 2.5, 5, 10, and 20 µg/ml; fluconazole (Pfizer Pharmaceuticals Group, New York, N.Y.) at 2.5, 5, 10, and 20 µg/ml; ketoconazole (Janssen Pharmaceutica, Titusville, N.J.) at 1.25, 2.5, 5, and 10 µg/ml; and miconazole (Janssen Pharmaceutica) at 1.25, 2.5, 5, and 10 µg/ml. Additionally, routine testing of itraconazole (Janssen Pharmaceutica) at 1.25, 2.5, 5, and 10 µg/ml against fresh clinical isolates was begun in 1996. Trays were stored at 85°C until they were ready for use.

Antifungal agent use determination. The NMH Pharmacy Department purchase data for the years of 1989 through 1996 were reviewed, and the number of doses of fluconazole administered per year was calculated. Fluconazole was approved for use at our hospital in late 1989, with use first beginning in 1990.

	RESULTS

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The 101 colonizing yeast isolates that were recovered from 97 non-AIDS, immunocompromised patients included the following: 70 C. albicans, 15 Candida (Torulopsis) glabrata, 8 Candida krusei, 7 Candida tropicalis, and 1 Candida parapsilosis isolates. Among the C. albicans strains, two were resistant to all three azoles, two strains were resistant to two azoles, and four isolates were resistant to a single azole. The only other yeast in which multidrug resistance against the azoles occurred was C. krusei, among which seven isolates were resistant to two azole drugs and one isolate was resistant to only a single azole. As expected, all of the eight C. krusei strains were resistant to fluconazole.

Review of the records available for 93 patients with 97 fungal infections revealed that 31 yeast isolates were retrieved from patients who had received at least one antifungal agent prior to obtaining a swab for surveillance culture, and 8 (26%) of these isolates were resistant to the agent that the patient had received. Ten of the patients had received nystatin (n = 7) or clotrimazole (n = 3). These were not considered therapy with the potential for selecting strains resistant to the agents tested, leaving 21 yeast isolates from patients who had received an antifungal agent relevant to our investigation (Table 1). Table 1 indicates the specific agent to which the yeast demonstrated in vitro resistance, the number of days prior to organism isolation that therapy was begun, and whether treatment with the indicated agent was discontinued prior to isolation of the resistant yeast. For 66 isolates from 64 patients who were not given an antifungal prior to obtaining a sample for culture, the susceptibility test results were similar, with 13 (20%) isolates being resistant to at least one imidazole agent. The susceptibility patterns for individual species from among the entire group of isolates tested are tabulated in Table 2. The susceptibility patterns of these yeasts colonizing the gastrointestinal tracts of non-AIDS, immunocompromised patients were also compared to the susceptibility patterns of the clinical fungal isolates whose susceptibilities to the antifungal agents were tested. These clinical isolates were obtained just preceding (1994) and during (1995) our study. These results are compared in Table 3.

The 70 C. albicans isolates whose results are compared in Table 3 were recovered from 68 patients. Nine (12.9%) of the patients from whom these isolates were retrieved had been on antifungal agents before the rectal swab for culture was obtained (Table 1). The systemically active medications included fluconazole and amphotericin B. The length of time that the patients were on an antifungal agent before the rectal swab for culture was obtained ranged from 3 to 25 days, and the total duration of treatment (before and after culture) ranged from 5 to 38 days. Only one isolate was resistant to the drug that the patient had received before the rectal swab for culture was obtained. This patient had received fluconazole daily for 4 days before the rectal swab for culture was obtained.

Eight C. krusei isolates were recovered from eight patients. Interestingly, seven patients had received an antifungal medication before the rectal swab for culture was obtained. (Table 1). The medications included fluconazole, itraconazole, and amphotericin B. Therapy was started from 7 to 39 days before the rectal swab for culture was obtained, and the total length of treatment ranged from 1 to 34 days. Six of the patients infected with C. krusei had been started on fluconazole therapy from 7 to 39 days before the rectal swab for culture was obtained, with the total length of treatment ranging from 3 to 34 days. All six patients had received this azole within at least 5 days before the rectal swab for culture, which was positive, was obtained. Another patient colonized with an amphotericin B-resistant organism had received 1 day of amphotericin B therapy 8 days prior to recovery of the isolate (Table 1).

The 15 C. glabrata isolates were recovered from 15 patients. Four patients (26.6%) had received either amphotericin B or an azole antifungal agent (fluconazole) prior to obtaining the rectal swab (Table 1). Treatment was started 5 to 22 days before the rectal swab for culture was obtained. However, all the isolates were susceptible to the antifungal agents tested. Seven C. tropicalis isolates were recovered from seven patients. One patient had received amphotericin B and fluconazole, and the organism from that patient was not resistant to either agent. The patient colonized with C. parapsilosis had not received an antifungal agent prior to obtaining the rectal swab for culture.

The archived C. albicans isolates were from blood and sterile body fluids other than urine, and the fresh C. albicans isolates obtained and tested during 1994 through 1997 were recovered from normally sterile body sites including blood, bone, pleural and peritoneal fluids, tissue such as liver and lung, cerebrospinal fluid, and urinary tract and respiratory tract secretions. During the first 2 years (1994 and 1995), 36 (1994) and 37 (1995) of these C. albicans isolates were from blood and sterile body sites other than urine (Table 4). A similar number of isolates (n = 36) was found in 1996, with 34 isolates recovered in 1997. 

Fluconazole was placed on our hospital formulary in late 1989 and was first used in 1990 (Table 4). Since then, of the imidazoles used in our hospital annually, fluconazole is used more than 90% of the time. Resistance to these agents did not subsequently appear until 1993, after 3 years of continuous fluconazole use. Regression analysis of these data indicates a correlation between fluconazole use and in vitro susceptibility results (P = 0.0147; R² = 0.432). Statistical assessment by a two-tailed paired t test gave a similar association (P = 0.0041). Since 1993, the percentage of C. albicans isolates remaining susceptible to the three azole antifungal agents fluconazole, ketoconazole, and miconazole has remained relatively stable: 77 to 90%, 92 to 100%, and 73 to 95%, respectively. The 1997 data suggest that, overall, azole activity is not worsening. The susceptibilities of the invasive strains were comparable to or better than those found when all isolates were considered together. Itraconazole appears to have the least in vitro activity against C. albicans strains at our institution, with resistance observed among 20% of all isolates tested in 1996 and resistance observed among 12% of all isolates tested in 1997 (Table 4).

	DISCUSSION

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While C. albicans remains the most frequently isolated yeast causing fungemia (3, 24), there has been a rise in the prevalence of infections with other Candida species (19, 24, 25). Data from the National Nosocomial Infections Surveillance system indicate that Candida species were the fourth most common cause of bloodstream infections and the sixth most common pathogen causing nosocomial infections from October 1986 to December 1990 (10). Between October 1986 and April 1996 Candida species were the fifth most common cause of nosocomial bloodstream infections and the seventh most common cause of nosocomial infections (17, 34). During 1996 at NMH, Candida species ranked fourth in prevalence, along with enterococci, as a cause of bloodstream infection, with each being responsible for 5.1% of infections.

Fungal infections are particularly problematic in immunocompromised patients. In cancer patients, risk factors include the use of increased doses of chemotherapeutic agents that cause greater damage to mucosal surfaces, prolonged myelosuppression, the underlying neoplastic process itself, the type of immunosuppression present, and the level of fungal colonization (12). Other risk factors include the presence of indwelling catheters (3, 12, 21, 23, 24, 37), the presence of flora- altering broad-spectrum antimicrobial agents (12, 23, 33, 37), and prior hemodialysis or azotemia (23). With bone marrow transplant patients, an additional risk factor can be the presence of graft-versus-host disease (12). Solid-organ transplant recipients have many risk factors that are specific to the type of transplant as well as the underlying disease, and risk factors for these patients also include the type and dosage of immunosuppressive medications (21). Fungal infections occur in 43 to 93% of human immunodeficiency virus-infected patients, with the rate of infection rising as the CD4 lymphocyte count falls below 200 cells/mm³ (18).

Fluconazole is a water-soluble triazole that is >90% bioavailable after oral administration (4, 5, 29), it is well tolerated, and it has been used extensively for the prophylaxis and treatment of a wide variety of candidal infections (1, 28, 39). Fluconazole and the other azole agents are fungistatic drugs (31); therefore, host defenses play a major role in eradicating infection and effecting a cure. While there still is controversy regarding the optimal use of prophylaxis with antifungal agents to reduce the numbers of systemic fungal infections (12), studies have shown that prophylaxis with fluconazole can reduce the numbers of both systemic and superficial fungal infections (12, 37).

C. krusei is naturally resistant to fluconazole (5, 12, 27), even when high doses are administered to neutropenic mice in an infection model (11). Patients receiving fluconazole prophylaxis while undergoing chemotherapy have been reported to have increased numbers of infections due to C. krusei (22, 38). However, other studies have not found that prophylactic treatment with fluconazole is associated with increased numbers of infections with C. krusei (7, 39). C. krusei has also been isolated from immunocompromised patients never treated with fluconazole (9). It is interesting that six of our eight patients colonized with C. krusei had received recent prior chemotherapy with fluconazole. After completing the current surveillance study, the NMH hematology and oncology unit experienced an outbreak of C. krusei fungemia, but this appeared to be related to the nosocomial transmission of a clonal strain rather than antifungal prophylaxis with fluconazole (20). It is possible that our recovery of C. krusei in the surveillance cultures from this study indicated a nosocomial transmission problem that we only later recognized (20).

Fungal prophylaxis has been associated with the emergence of resistance to fluconazole in Candida species that are usually susceptible (18, 29-31, 36). This seems to be closely associated with advanced AIDS and the total cumulative dose of the azole antifungal agent given (13, 29). One reason for this occurrence may be the fact that fluconazole is commonly given for prolonged periods of time to patients with advanced AIDS and is often prescribed for multiple courses, since patients with advanced AIDS have frequent episodes of candidiasis (14, 27, 32). While this problem is closely linked to AIDS, fluconazole-resistant C. albicans has been isolated from patients with leukemia who were receiving fluconazole prophylaxis (7). However, fluconazole-resistant C. albicans has also been recovered from non-AIDS patients who were not receiving fluconazole prophylaxis (6). In our surveillance study we found only one fluconazole-resistant C. albicans isolate (Table 1) among the isolates from nine patients receiving that agent. This is considerably less than that reported by Maenza and colleagues (14); however, their AIDS patients received treatment for a mean of 231 days and suggests that the risk of the emergence of resistant strains in other types of immunocompromised patients receiving short-term therapy may be lower. Our overall findings support a hypothesis that for our non-AIDS, immunocompromised patients the azole resistance in C. albicans may be more related to the general prevalence of resistant strains in the patient population than to the focused use of fluconazole prophylaxis in selected individuals.

Azole-resistant candidiasis appears to be on the rise, and the reasons for resistance may include incomplete therapy (27), overgrowth of resistant strains, induction of drug resistance in the particular species (27, 29), or colonization and subsequent infection with a resistant organism (29). Our own data suggest that the percentage of organisms resistant to agents like fluconazole may remain stable as long as overall imidazole use is also relatively constant (Table 4). Our data also suggest, on the basis of tests performed in 1996 and 1997, that resistance to itraconazole in C. albicans may be relatively prevalent, even in an environment where itraconazole is not a frequently used imidazole.

In conclusion, while the yeast species colonizing the gastrointestinal tracts of our non-AIDS, immunocompromised patients do not show increased rates of resistance compared to those of our clinical isolates obtained from 1994 through 1997, our data support previous observations that clinical Candida species and related yeast infections are increasing and that the widespread use of imidazoles (such as fluconazole) appears to be associated with emerging resistance to these important antifungal agents in yeasts. As a consequence, in vitro testing of the susceptibility of yeasts to antifungal agents will likely play an ever increasing role in the appropriate selection of antifungal agents for the treatment of fungal infections.