CLINICAL ARTICLES. Clinical Infectious Diseases 1997; 24: by The University of Chicago. All rights reserved /97/ $02.

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1 124 CLINICAL ARTICLES Patterns of Fluconazole Susceptibility in Isolates from Human Immunodeficiency Virus Infected Patients with Oropharyngeal Candidiasis Due to Candida albicans Fernando Laguna, Juan L. Rodriguez-Tudela, Joaquin V. Martinez-Suarez, Rosa Polo, Eulalia Valencia, Teresa M. Diaz-Guerra, Fernando Dronda, and Federico Pulido From the Servicio de Enfermedades Infecciosas, Centro de InvestigaciOn Clinica; the Unidad de Micologia, Centro Nacional de Microbiologia, Instituto de Salud Carlos III; Unidad de Enfermedades Infecciosas y Microbiologia, Hospital General Penitenciario; and Unidad de VIH, Hospital 12 de Octubre, Madrid We evaluated 119 episodes of oropharyngeal candidiasis due to C. albicans to study the patterns of fluconazole susceptibility of the isolates and the characteristics of the patients and to confirm the correlation between fluconazole susceptibility of isolates and therapeutic outcome. Sixty-one isolates were considered susceptible to fluconazole (MICs, 0.5 Ag/mL), 33 were intermediate (MICs, ,ug/mL), and 25 were resistant (MICs, 16.0 aug/ml). Patients infected with resistant strains had significantly lower CD4+ cell counts and a less recent AIDS diagnosis than patients infected with intermediate or susceptible strains. Previous fluconazole therapy and prophylaxis were significantly more frequent for patients infected with resistant and intermediate strains (P <.001). Decreased susceptibility to ketoconazole and itraconazole was observed in resistant and intermediate strains. Fluconazole treatment was ineffective for patients infected with resistant isolates; however, high doses of ketoconazole or itraconazole were successful for nine (81%) of them. Different patterns of fluconazole susceptibility among C. albicans strains are correlated with patients' characteristics and with therapeutic outcomes. Oropharyngeal candidiasis is the commonest opportunistic infection in patients infected with HIV, occurring in up to 90% of them. Candida albicans is the species most frequently isolated from patients with AIDS who have oral thrush [1], and antifungal therapy without microbiological confirmation is appropriate. Treatment with azole derivatives is effective for both oral thrush and esophageal candidiasis [2-4]. Fluconazole is an orally active, triazole antifungal agent that is less toxic, is better tolerated, and has greater in vivo activity against C. albicans than ketoconazole, and it is absorbed more reliably [5]. Fluconazole is associated with significantly greater rates of endoscopic and clinical cure than is ketoconazole for HIV-infected patients with oral or esophageal candidiasis [2, 3]. However, oropharyngeal or esophageal, candidiasis recurs Received 24 January 1996; revised 2 October Financial support: This work was supported in part by Fondo de Investigaciones Sanitarias (grant no. 96/0598) and the ComisiOn Interministerial de Ciencia y Tecnologia, both of Spain. T.M.D.G. is a fellow of the Instituto de Salud Carlos III. Reprints or correspondence: Dr. F. Laguna Cuesta, Servicio de Enfermedades Infecciosas, Centro de InvestigaciOn Clinica, c/sinesio Delgado 12, Madrid, Spain. Clinical Infectious Diseases 1997; 24: by The University of Chicago. All rights reserved /97/ $02.00 within 3 months of successful treatment in up to 80% of patients treated with azole therapy [4, 6]. In such settings some studies have shown the efficacy of daily or weekly fluconazole therapy for preventing recurrence of oropharyngeal [7-9] or esophageal candidiasis [10]. In recent years, an increased number of patients have had oral thrush caused by C. albicans strains that are clinically and microbiologically resistant to fluconazole, most of them were being chronically treated with fluconazole [11-21]. However, these reports must be evaluated with caution, because antifungal susceptibility testing remains unstandardized, and appropriate studies to correlate in vitro results with in vivo response to antifungal drugs have not been extensively accomplished [22]. The National Committee for Clinical Laboratory Standards (NCCLS) has recommended a broth macrodilution method for antifungal susceptibility testing [23]. However, several studies have demonstrated good agreement between macrodilution and microdilution antifungal susceptibility testing [24-26]. Recently, two of us [27] developed a microdilution method using RPMI supplemented with glucose (RPMI-2% glucose), which facilitates the reading of growth inhibition of azole drugs without changing the MICs determined with standard RPMI. This method shows an excellent correlation between in vitro yeast susceptibility to azoles and clinical outcome after antifungal treatment in HIV-infected patients with oropharyngeal candidiasis [28].

2 CID 1997;24 (February) Fluconazole Susceptibility of C. albicans from HIV-Positive Patients 125 Using this method, therefore, we studied HIV-infected patients with oral thrush due to C. albicans to learn the patterns of susceptibility of the isolates to antifungal drugs, to confirm the correlation between the in vitro activity of antifungal drugs and the in vivo response to therapy (especially for patients with increased MICs of fluconazole), and to compare the epidemiological and clinical characteristics between the different groups of patients. Patients and Methods Patient population. We included only patients with HIV infection and oropharyngeal candidiasis due to C. albicans who were evaluated at the Service of Infectious Diseases of the Centro de InvestigaciOn Clinica and at the Unit of Microbiology and Infectious Diseases of the Hospital General Penitenciario in Madrid. Clinical information regarding previous oral thrush episodes, azole therapy, length of secondary antifungal prophylaxis, CD4 + cell counts, response to antifungal therapy, and HIV status was obtained from patients and clinical charts; we had no previous knowledge of the microbiological results. Clinical resolution of oral thrush was defined as the absence of oral candidal lesions following 10 days of antifungal therapy. Mycological cure was not evaluated. For evaluating clinical outcome of antifungal therapy, we included all episodes treated with different therapeutic schemes and of which the outcomes were directly assessed. Secondary antifungal prophylaxis was usually started after monthly recurrence of oral thrush; fluconazole (200 mg weekly) or ketoconazole (400 mg three times weekly) was administered, according to the decision of the physician in charge of each patient. On the basis of a preliminary study [28], C. albicans strains were classified as fluconazole-resistant if the MICs were _-16.0 pg/ml. On the basis of our expanding experience, oral isolates for which MICs of fluconazole ranged between 1.0 ii,g/ml and 8.0 ug/ml were considered intermediate, and those for which MICs were -0.5 pg/ml were considered susceptible. Data from 40 patients have been published in part elsewhere [28]. Patients with mixed oral thrush due to C. albicans and nonalbicans Candida species were not included in the study. AIDS was diagnosed according to the 1987 criteria of the Centers for Disease Control and Prevention. Culture procedure. Samples were taken (with sterile swabs) from oral lesions of the patients. The specimens were inoculated onto Sabouraud dextrose agar (Oxoid, Basingstoke, UK) and were incubated for 1 week at 28 C. Strain identification was made by the germ tube test, carbohydrate assimilation and fermentation, and morphology on corn meal agar. Susceptibility testing. MICs were determined by a microbroth dilution method (described previously [29]) with use of RPMI-2% glucose medium [27]. The concentrations of ketoconazole and fluconazole ranged from p,g/ml to p,g/ml, while itraconazole because of its poor solubility and amphotericin B were tested at concentrations between 0.03 yg/ml and 16.0 ag/ml. Inocula were prepared according to the method described by Espinel-Ingroff et al. [24], and the microtiter trays were inoculated with an automatic pipette programmed to deliver 10 to each well (yielding suspensions that initially contained 10 7 cfu/l). Following incubation at 35 C for 24 hours, the trays were shaken for 5 minutes and turbidity was read spectrophotometrically at 405 nm (630 nm for amphotericin B) with a Mios Merck automatic plate reader (Merck Igoda S.A., Madrid). For the azoles, the MIC endpoint (IC 1/2) was defined as the lowest drug concentration that met the following criterion: %T %Tk (100 %Tk), where T is transmission and k is a drug-free well [30]. The MIC of amphotericin B was taken as the lowest drug concentration that allowed no visible growth. The following control strains were included in the study: Candida krusei ATCC 6258 and Candida parapsilosis ATCC They have well-defined reference ranges for MICs of amphotericin B, flucytosine, fluconazole, itraconazole, and ketoconazole. They were tested five times, on different days, with the spectrophotometric method and use of RPMI-2% glucose. The results were compared with the values established by Pfaller et al. [31] and Rex et al. [32]. Statistical analysis. The characteristics of the patients in the three groups were compared by the x2 test and Mann- Whitney U-test as appropriate. Logistic regression was used to estimate the adjusted odds ratio for lack of response to antifungal therapy. Odds were adjusted for confounding effects of CD4 + lymphocyte counts. P values of <.05 were considered statistically significant. Statistical analyses were done with RSIGMA software (Horns Hardware S.A., Madrid, 1990). Results Patients' Characteristics From January 1992 to December 1994, a total of 110 HIVinfected patients and 303 oropharyngeal isolates were evaluated for the study. For microbiological purposes we included only one strain per patient, except when the isolated strains were associated with different MICs of fluconazole. We studied all patients with oral thrush due to C. albicans for which the MIC of fluconazole was p,g/ml and a control group of patients with oral thrush due to C. albicans for which the MIC was 0.5 //g/ml. During the follow-up, three patients infected with fluconazole-susceptible C. albicans acquired oral thrush, from which the isolates had intermediate susceptibility. In addition, four patients whose isolates had been intermediately susceptible became infected with fluconazole-resistant strains. Furthermore, one patient had different episodes caused by C. albicans strains that were susceptible, intermediate, and resistant, respectively,

3 126 Laguna et al. CID 1997;24 (February) to fluconazole during the follow-up. In summary, we included 110 HIV-infected patients and 119 C. albicans strains in our analyses. Twenty-five patients infected with resistant strains, 33 with intermediate strains, and 61 with susceptible isolates were identified. The demographic characteristics are summarized in table 1. The three groups were similar in terms of age and risk factor for HIV infection. AIDS had been previously diagnosed for more patients whose isolates were resistant (100%) or intermediate (97%) than patients whose strains were susceptible (62%) (P <.001). In addition, patients with resistant isolates had a less recent AIDS diagnosis than did patients with intermediate and susceptible strains (P <.05). The median CD4 + cell count was 12/mm3 (range, 5-56/mm3) in patients with resistant isolates, 20/mm3 (range, 0-140/mm 3) (P =.025) in patients with intermediate isolates, and 35/mm3 (range, 3 297/mm 3) (P <.001) in patients with susceptible strains. Antifungal Prophylaxis and Susceptibility to Azoles All patients in the resistant and intermediate groups and 34 of 61 (53%) in the susceptible group had recurrent oral thrush (P <.001). The median duration of recurrent oropharyngeal candidiasis in the resistant and intermediate groups was 24 and 20 months, respectively (ranges, 2-56 months and months) and 8 months in the susceptible group (range, 1-94 months) (P <.001). Twenty-three (92%) of 25 patients in the resistant group and 29 (88%) of 33 in the intermediate group had been treated with fluconazole before detection of fluconazole failure. By contrast, only 26 (43%) of 61 patients in the susceptible group had previously received this drug (P <.001). Twenty-one (84%) of 25 patients infected with resistant strains, 31 (94%) of 33 patients with intermediate isolates, and 11 (18%) of 61 patients with susceptible strains had received antifungal prophylaxis (P <.001). Secondary antifungal prophylaxis with fluconazole (200 mg weekly) or with ketoconazole (400 mg three times weekly) was respectively given to 18 and 2 resistant-group patients, to 25 and 5 intermediate-group patients, and to 5 and 6 susceptible-group patients (P <.001 for fluconazole prophylaxis). Daily fluconazole prophylaxis was given to one patient each in the resistant and intermediate groups following meningeal cryptococcosis. These prophylactic treatments were begun a median of 45 weeks (range, weeks) before the detection of resistant strains, 43 weeks (range, weeks) before detection of intermediate strains, and 17 weeks (range, weeks) before detection of susceptible strains (P <.05) (table 2). MICs of antifungal drugs for 50% of strains (MIC 50) and for 90% of strains (MIC 90) are summarized in table 3. For C. albicans strains with decreased susceptibility to fluconazole, the MIC 50 and MIC 90 of both ketoconazole and itraconazole were higher than in the control group. It is noteworthy that we did not observe an increase in the MICs of amphotericin B against oral isolates with decreased susceptibility to fluconazole The results obtained with our antifungal susceptibility testing method with use of RPMI-2% glucose [27] are compared in table 4 with those obtained by the NCCLS M27-P broth macrodilution method [23]. As can be seen in the table, all MICs are in agreement except those of ketoconazole against C. parapsilosis ATCC 22019, which are lower with the alternative method than with the NCCLS method. Table 1. Demographic characteristics of patients with oropharyngeal candidiasis, as related to susceptibility of their isolates to fluconazole. Data for patients whose isolates were: Characteristic Susceptible (n = 61) Intermediate (n = 33) Resistant (n = 25) P value Age (y) Median NS Range Risk factor for HIV infection, no. (%) of patients Intravenous drug use 44 (72) 25 (76) 18 (72) NS Homosexuality (male) 8 (13) 5 (14) 3 (12) Others 9 (15) 3 (10) 4(16) Previous AIDS diagnosis, no. (%) of patients 38 (62) 32 (97)* 25 (100)* <.001 Time since diagnosis of AIDS (mo) Median *t <.05 Range No. of CD4 + cells per mm 3 Median 35 20* 12*t <.05 Range * Significantly different from the susceptible group. t Significantly different from the intermediate group.

4 CID 1997;24 (February) Fluconazole Susceptibility of C. albicans from HIV-Positive Patients 127 Table 2. Antifungal prophylaxis and fluconazole therapy among patients with oropharyngeal candidiasis, as related to susceptibility of their isolates to fluconazole. No. (%) of patients or other data, per susceptibility group Characteristic Susceptible (n = 61) Intermediate (n = 33) Resistant (n = 25) P value Previous fluconazole therapy 26 (43) 29 (88)* 23 (92)* <.001 Antifungal prophylaxis 11 (18) 31 (94)* 21 (84)* <.001 Fluconazole prophylaxis 5 (8) 25 (79)* 19 (76)* <.001 Length of prophylaxis (w) Median 17 43* 45* <.05 Range * Significantly different from susceptible group. Response to Antifungal Therapy Clinical response to antifungal treatment is shown in table 5. After adjustment for CD4+ lymphocyte counts, infection with fluconazole-resistant isolates was found to be an independent risk factor for lack of response. Overall, clinical cure was achieved in all patients treated with fluconazole, and for 39 (95%) of 41 ketoconazole-treated patients' C. albicans strains the MIC of fluconazole was - 2 p,g/ml. However, our experience in the treatment of oral thrush due to C. albicans strains for which the MICs of fluconazole range from 1 tg/ml to 2 p,g/ml is still limited. Among 6 patients infected with C. albicans strains for which the MICs of fluconazole were 4.0,u,g/mL, 9 courses of therapy with fluconazole were evaluated: 4 of 5 patients (80%) were Table 3. MICs of antifungal drugs against C. albicans strains, as related to susceptibility of the isolates of fluconazole. Antifungal drug MICs (p,g/ml) to indicated drug, per fluconazole susceptibility group Susceptible (n = 61) Intermediate (n = 33) Resistant (n = 25) P value Fluconazole Median Range Ketoconazole Median * 0.12t 1 <.001 Range Itraconazole Median * 1 tt <.00 1 Range Amphotericin B Median NS Range * Significantly different from the susceptible group (P <.05 for MIC of ketoconazole and P <.001 for MIC of itraconazole). t Significantly different from the susceptible group. 1 Significantly different from the intermediate group (P <.001). not cured with a fluconazole dose of 100 mg/d, although these subjects were clinically cured with oral fluconazole (200 mg/d; 3 patients) or intravenous fluconazole (400 mg/d). Among 18 patients whose oral isolates were associated with fluconazole MICs of 8.0 ii,g/ml, none of 15 were cured with 100 mg/d of fluconazole but 7 (50%) of 14 responded to 200 mg daily. On the other hand, six (60%) of 10 patients were cured with ketoconazole or itraconazole treatment (400 mg/d). All patients infected with resistant strains were treated with oral fluconazole (u200 mg/d) without resolution of their infection. Moreover, therapy with intravenous fluconazole (400 mg/d) was ineffective in six patients. Another two patients were treated with intravenous fluconazole (1,200 mg/d), and one was cured. However, ketoconazole therapy (400 mg/d) was effective in five of 14 patients (35.7%); the MICs of ketoconazole for responders (median, 0.06 ii,g/ml; range, p,g/ml) were lower than for nonresponders (median, 0.25 p,g/ml, range, p,g/ml). Furthermore, six (75%) of eight patients were successfully treated with ketoconazole at a dosage of 800 mg/d. Itraconazole therapy was effective in 1 of 6 patients at a dosage of 400 mg/d and in each of 3 patients treated at a dosage of 600 mg/d. Seventeen patients were treated also with intravenous amphotericin B (0.5 mg/[kg d]) over 7-14 days, and 15 of them (89%) were cured. In all cases that were cured with treatment, antifungal therapy was only transiently effective, and fast recurrence of oral thrush was universal after the therapy ended. Discussion The results of this study extend those of previous reports regarding the positive correlation of in vitro susceptibility antifungal testing and in vivo response to therapy in HIV-infected patients with oropharyngeal candidiasis due to C. albicans [1, 14, 19, 33, 34]. In this study we observed two very different clinical and microbiological patterns relating to resistance and susceptibility of C. albicans strains to fluconazole. As has been

5 128 Laguna et al. CID 1997;24 (February) Table 4. Comparison of MICs determined by the NCCLS broth macrodilution reference method (M27-P) and an alternative microdilution test with RPMI-2% glucose, against quality control strains of Candida. Quality control strain, antifungal agent Range of MIC values (µg/ml) per indicated method Reference method* RPMI-2% glucose microdilutiont MIC80 IC1/2 C. krusei ATCC 6258 Fluconazole Itraconazole Ketoconazole C. parapsilosis ATCC Fluconazole Itraconazole Ketoconazole * NCCLS broth macrodilution method (M27-P) [23]. t See [27]. Five repetitive determinations (on different days) were made for the three antifungals. See section in text on susceptibility testing for definition of IC 1/2. previously stated, patients with fluconazole-resistant oral thrush had a more severe HIV infection, as indicated by the very low CD4 + cell counts and a higher prevalence of AIDS [20, 33]. They also had a more lengthy history of oral thrush and more frequent use of fluconazole treatments and fluconazole prophylaxis than did patients in the fluconazole-susceptible group [17, 34]. However, for some patients' oral strains the MICs of fluconazole ranged from 1.0 pig/ml to 8.0 pg/ml; these patients had epidemiological and clinical similarities with resistant-group patients, and some eventually had oral thrush with high fluconazole resistance. These isolates were classified as intermediates because we did not have enough clinical data to classify them as susceptible or resistant. The study and objective validation of fluconazole-resistant oropharyngeal candidiasis have been limited because antifungal susceptibility tests remain unstandardized and the different methods can produce discordant results in different laboratories [22]. With the exception of MICs of ketoconazole against C. parapsilosis ATCC 22019, the MICs obtained with our method are in total agreement with those determined by the NCCLS for quality control strains. However, Rex et al. [32] noted on five occasions an MIC of ketoconazole of 0.03 big/ ml for C. parapsilosis ATCC Furthermore, with our method the endpoint is estimated at 24 hours, and this could be an explanation for the lower MIC. With our antifungal susceptibility testing method we noted excellent correlation between MICs and clinical outcome after fluconazole treatment in patients with oral thrush, in cases of both susceptible and intermediate/resistant strains. In fact, we observed a gradated relationship between MIC and response to fluconazole therapy. It is necessary to achieve a general consensus as to the definition of clinical resistance to fluconazole in the treatment of oropharyngeal candidiasis and the correlation with MICs of fluconazole [20, 331 In our experience, clinical resistance to fluconazole (100 mg/d for 10 days) appeared in all but one patient with C. albicans isolates for which the MIC of fluconazole was n4.0 µg/ml. At a fluconazole dosage of 200 mg/d over 10 days, 50% of patients whose oral isolates had fluconazole MICs of 8.0 big/ml were in the clinically fluconazole-resistant group, as were 100% of patients whose isolates had fluconazole MICs of 16.0 pg/ml. At this point we cannot suggest a fluconazole MIC of 8.0 µg/ml as a definitive Table 5. Response of patients to antifungal therapy, as related to susceptibility of their isolates to fluconazole. No. of patients cured/no. treated, per MIC (µg/ml) of fluconazole against isolates Antifungal drug, dosage (mg/d) (n = 61) 1 (n = 6) 2 (n = 3) 4 (n = 6) 8 (n = 18) (n = 25) Fluconazole /21 4/4 1/1 1/5 0/15 0/ /5 3/3 7/14 0/ /3 0/ iv 1/1 0/6 Ketoconazole /37 2/2 2/2 4/7 5/ /2 1/2 6/8 Itraconazole 400 2/3 1/ /1 3/3 Amphotericin B 0.5 mg/(kg d) iv 3/3 15/17

6 CID 1997; 24 (February) Fluconazole Susceptibility of C. albicans from HIV-Positive Patients 129 breakpoint to establish microbiological resistance when a dosage of 200 mg/d is used for evaluating clinical outcome. Treatment success with this dosage in some patients and failure in others make it necessary to ascertain if other factors, such as fluconazole serum levels, play a role in the outcome for these patients. However, all patients with oral thrush due to oral isolates for which the MICs of fluconazole were.-16.0,ug/ml showed signs of clinical resistance after fluconazole treatment at a dosage of a200 mg/d. On the basis of these data and with use of our antifungal susceptibility method, we could establish new microbiological breakpoints for fluconazole in oral thrush due to C. albicans in HIV-positive patients: resistant, MICs of,ug/ml; intermediate, MICs of ,u,g/mL, and susceptible, MICs of p,g/ml. Fluconazole-resistant oropharyngeal candidiasis has been described in patients who have received (1) treatment with a single fluconazole dose for each episode [20], (2) repeated conventional therapy [16], (3) treatment with daily fluconazole prophylaxis [19], and (4) as shown in most of our cases, weekly fluconazole prophylaxis [33]. However, five of our patients' C. albicans strains had decreased susceptibility to fluconazole despite the fact that no previous fluconazole treatment had been administered. One of the patients had only two oral thrush episodes, which were treated with ketoconazole, in the month prior to having fluconazole-resistant oropharyngeal candidiasis. Unfortunately, we did not take oral samples during the first episode and we cannot be sure if this isolate had primary resistance to fluconazole that had not previously been observed or if the resistant strain was acquired by nosocomial spread [18]. We have shown, as have other investigators, decreased susceptibility in vitro to ketoconazole and itraconazole; the MIC 50 and MIC90 values were several times higher for fluconazoleresistant or intermediate C. albicans strains than for fluconazole-susceptible strains [34]. This decreased susceptibility to azoles could be due to the development of diverse mechanisms of resistance such as reduced cell permeability or target site alteration [35], which could affect the susceptibility of yeasts to ketoconazole or itraconazole. However, correlation between ketoconazole MICs or itraconazole MICs and outcome after treatment with these drugs is probably more difficult to establish because of the pharmacokinetic" characteristics of both drugs: intestinal absorption in patients with severe immunodeficiency is low [36], and several interactions with some drugs frequently used for patients with AIDS have been noted [37]. In our experience, after the development of fluconazoleresistant oropharyngeal candidiasis with use of increasing fluconazole dosages to >400 mg/d, the drug is no longer useful, and it is expensive. At present, there is no consensus for a therapeutic approach to this increasing problem [35]. Intravenous amphotericin B is highly effective in these patients [20, 33, 38], but its use is limited by toxicity and patients' compliance. Our findings show that ketoconazole or itraconazole treatment is useful for fluconazole-resistant oropharyngeal candidiasis. Among patients unresponsive to fluconazole, high doses of these drugs were effective in 76.9% of those who experienced no significant toxic effects. Some previous reports have described a low rate of success with itraconazole for fluconazole-resistant oral thrush, although in most cases itraconazole was given at a low dosage [11, 16, 39]. 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