Activities of fluconazole and voriconazole against bloodstream isolates of Candida glabrata and Candida krusei: a 14-year study in a Spanish tertiary medical centre

International Journal of Antimicrobial Agents 31 (2008) 266–271

Short communication

Activities of fluconazole and voriconazole against bloodstream isolates of Candida glabrata and Candida krusei: a 14-year study in a Spanish tertiary medical centre Guillermo Quind´os a,∗ , Luis Octavio S´anchez-Vargas a,b , Mar´ıa Villar-Vidal a , Elena Eraso a , Miriam Alkorta c , Jos´e Luis Hern´andez-Almaraz c a

b

Departamento de Inmunolog´ıa, Microbiolog´ıa y Parasitolog´ıa, Facultad de Medicina y Odontolog´ıa, Universidad del Pa´ıs Vasco, Aptdo. 699, E-48080 Bilbao, Spain Departamento de Estomatolog´ıa, Instituto de Ciencias Biom´edicas, Universidad Aut´onoma de Ciudad Ju´arez, Ciudad Ju´arez, Chihuahua, Mexico c Servicio de Microbiolog´ıa, Hospital Universitario de Cruces, Barakaldo, Spain Received 3 September 2007; accepted 19 September 2007

Abstract The aim of this study was to evaluate the in vitro activities of voriconazole and fluconazole against Candida glabrata and Candida krusei isolated from blood during a 14-year period (1990–2003) at the tertiary care hospital of Cruces (Barakaldo, Spain). The in vitro activities of fluconazole and voriconazole against 28 isolates of C. glabrata and 15 isolates of C. krusei were determined by the Clinical and Laboratory Standards Institute disk diffusion method. Of the 28 C. glabrata isolates tested, 24 (85.7%) were susceptible (S) to fluconazole, 2 (7.1%) were susceptible dose-dependent (S-DD) and 2 (7.1%) were resistant (R). All C. krusei isolates were classified as R to fluconazole. Resistance to voriconazole was observed in one isolate each of C. glabrata (3.6%) and C. krusei (6.7%), and one isolate of each species was S-DD. These results were confirmed by the Sensititre® YeastOne and Etest methods, with good comparative results. Voriconazole was very active in vitro against C. glabrata and C. krusei blood isolates and the resistance observed was not related to the introduction of voriconazole in the therapeutic schedule of the hospital. These facts support the usefulness of voriconazole as a therapeutic tool for candidaemia caused by these species. © 2007 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Fluconazole; Voriconazole; Disk; Candidaemia; Candida glabrata; Candida krusei

1. Introduction The incidence of fungal infections has increased significantly, particularly among immunodeficient and intensive care patients. Candida albicans is the most common cause of mycoses in hospitalised patients. However, the rate of candidaemia caused by non-albicans Candida species is increasing [1]. Among these candidiases, Candida glabrata and Candida krusei infections are common (15–25% of bloodstream Candida isolates worldwide) and are difficult to treat owing to their reduced susceptibility to common antifungal agents,

∗

Corresponding author. Tel.: +34 94 601 2854; fax: +34 94 601 3495. E-mail address: [email protected] (G. Quind´os).

resulting in a high mortality rate [2]. Candida glabrata may be more common in older persons and in patients with haematological malignancies [3], and C. krusei has been associated with the highest mortality among candidaemias [4]. Moreover, decreased susceptibility to new triazole agents, such as voriconazole and posaconazole, has been described in some clinical isolates [2]. For these reasons, appropriate treatment of invasive candidiasis requires rapid identification of the aetiological agent, followed by reliable and accurate in vitro antifungal susceptibility testing. The Clinical and Laboratory Standards Institute (CLSI) Subcommittee for Antifungal Testing has developed standardised broth microdilution [5] and disk diffusion [6] methods for in vitro susceptibility testing of Candida spp. These methods are reproducible and accurate and provide clinically useful information. The disk

0924-8579/$ – see front matter © 2007 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2007.09.010

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diffusion method, including fluconazole and voriconazole, is a simpler alternative and could guide treatment. The aim of this study was to evaluate the in vitro susceptibility to voriconazole and fluconazole of C. glabrata and C. krusei isolated over a 14-year period (1990–2003) at the tertiary care hospital of Cruces (Barakaldo, Spain).

2. Materials and methods 2.1. Study design A total of 35 blood isolates of C. glabrata and 21 isolates of C. krusei recovered from 555 episodes of candidaemia treated at the tertiary care hospital of Cruces were collected between 1990 and 2003. However, only 28 blood isolates of C. glabrata and 15 isolates of C. krusei were recovered and included in this study.

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ceptible dose-dependent (S-DD), zone diameter 15–18 mm; and resistant (R), zone diameter ≤14 mm. The corresponding disk test breakpoints for voriconazole were as follows: S, ≥17 mm; S-DD, 14–16 mm; and R, ≤13 mm [2]. 2.4. Etest method Etest strips (AB BIODISK, Solna, Sweden) containing concentrations ranging from 0.002 ␮g/mL to 32 ␮g/mL for fluconazole, itraconazole and voriconazole were used for comparative testing. The Etest method was performed as described previously [7] on RPMI 1640 agar plates. Minimum inhibitory concentrations (MICs) were read where the edge of the inhibition ellipse intersected the MIC scale on the Etest strip after 24 h of incubation in air at 37 ◦ C. The interpretive susceptibility criteria were based on the MIC breakpoints recommended for fluconazole in the CLSI M27A2 reference method [5] and for voriconazole by Pfaller et al. [2].

2.2. Identification of microorganisms 2.5. Broth microdilution test The isolates were identified by standard procedures such as morphology on cornmeal agar plates, germ tube production in serum and biochemical patterns determined with ID 32 C panels (bioM´erieux, Madrid, Spain). The isolates were subcultured onto Candida ID2 agar (bioM´erieux) for 24 h at 37 ◦ C to ensure purity. Three methods were used for in vitro susceptibility testing of fluconazole and voriconazole: antifungal drug diffusion as described in CLSI document M44-A [6]; Etest; and broth microdilution by Sensititre® YeastOne 3. Additionally, the in vitro activity of itraconazole was studied by the latter two methods, and 5-fluorocytosine, amphotericin B and ketoconazole activity by Sensititre YeastOne 3. Quality control was performed in accordance with CLSI document M44-A using C. albicans ATCC 90028, Candida parapsilosis ATCC 22019 and C. krusei ATCC 6258. 2.3. Disk diffusion test Disk diffusion testing of fluconazole and voriconazole was performed as described in CLSI document M44-A [6]. Agar plates (90 mm diameter) containing Mueller–Hinton agar (Difco, Becton Dickinson, Sparks, MD) supplemented with 2% glucose and 0.5 ␮g of methylene blue per mL at a depth of 4 mm were used. The agar surface was inoculated using a swab dipped in a cell suspension adjusted to the turbidity of a 0.5 McFarland standard. Fluconazole (25 ␮g) and voriconazole (1 ␮g) disks (supplied by Pfizer Espa˜na S.A., Madrid, Spain and manufactured by Becton Dickinson, Sparks, MD) were placed onto the surfaces of the plates and the plates were incubated in air at 37 ◦ C and read at 18–24 h. Zone diameter endpoints were read manually with a caliper (Mitutoyo, Tokyo, Japan). The interpretive criteria for fluconazole disk diffusion testing were those published by the CLSI [6] and were as follows: susceptible (S), zone diameter ≥19 mm; sus-

The broth microdilution test was performed using the Sensititre YeastOne 3 (Trek Diagnostic Systems, East Grinstead, UK). This test consists of a 96-well microtitre plate containing dried amphotericin B, fluconazole, itraconazole, ketoconazole, 5-fluorocytosine and voriconazole with incorporated Alamar BlueTM , which changes from blue to pink in the presence of microbial growth. The drug concentrations ranged from 0.008 ␮g/mL to 16 ␮g/mL for amphotericin B, itraconazole, ketoconazole and voriconazole, from 0.125 ␮g/mL to 256 ␮g/mL for fluconazole and from 0.03 ␮g/mL to 64 ␮g/mL for 5-fluorocytosine. Sensititre YeastOne 3 testing was carried out in accordance with the manufacturer’s instructions, as follows: 20 ␮L of the inoculum suspension was added to 11 mL of RPMI 1640 broth to obtain a working suspension (ca. 1.5–5 × 103 cells/mL), of which 100 ␮L was added to each well. The panels were sealed and incubated in air at 37 ◦ C and read at 24 h by comparing the colour of the medium in each well with those of the reading mask provided by the manufacturer. If no growth was noted in the control well at 24 h, a second reading was made after an additional 24 h of incubation. Classification of isolates in terms of their susceptibilities to fluconazole, itraconazole and 5-fluorocytosine was based on the MIC breakpoints recommended for these drugs in the CLSI M27-A2 reference method [5]. MIC breakpoints for voriconazole were those described by Pfaller et al. [2]: S, ≤1 ␮g/mL; S-DD, 2 ␮g/mL; and R, ≥4 ␮g/mL.

3. Results and discussion Several investigators have recently highlighted an increase in non-albicans Candida species among hospitalised

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patients, and C. glabrata and C. krusei have been frequently reported as common aetiological agents of candidaemia. The reported decrease in the proportion of Candida bloodstream infections due to C. albicans and the increase in the proportion of C. glabrata candidaemias might have been mediated by one or more risk factors in addition to selection for species less susceptible to azoles [1]. During the last 6-year period (1999–2004), the average incidence of fungaemia at Cruces Hospital was 1.3 new episodes/10 000 patient-days of stay in hospital (range 1.1–1.4 episodes); the average incidence was significantly higher in children (incidence 1.97 episodes). These incidence rates are higher than those reported in Europe by the European Confederation of Medical Mycology Societies survey (0.31–0.44 episodes) [1] but lower than those reported in the USA (1.5 episodes) [8]. It has been described [1] that the rate of candidaemia increases with the hospital bed size. In our hospital, non-albicans Candida species were more frequently isolated than C. albicans (57.7% vs. 42.3% of isolates). Overall, 42.3% (105/248) of fungaemia episodes were due to C. albicans, followed by C. parapsilosis (36.3%), C. glabrata (9.7%), Candida tropicalis (4.4%), C. krusei (4%) and other Candida spp. (4.3%). The rates of isolation from blood of C. krusei (4%) and C. glabrata (9.7%) at our institution are also similar to the 2–4% and 9–12% rates, respectively, reported in other studies in Europe [1] and lower than those reported in the USA (12–24%) for C. glabrata. Of the total 35 C. glabrata and 21 C. krusei isolates archived at the Hospital de Cruces, 7 C. glabrata and 6 C. krusei failed to grow or were missed. Tables 1–3 present the in vitro susceptibilities of isolates to fluconazole and voriconazole. The results reported for the three quality control strains included on each day of testing were within the accepted limits for all the antifungal agents and methods. Table 1 summarises the in vitro susceptibilities of 28 isolates of C. glabrata and 15 isolates of C. krusei to fluconazole and voriconazole as determined by CLSI disk diffusion testing. The percentages of isolates of C. glabrata in each category (S, S-DD, and R) were, respectively, 85.7%, 7.1% and 7.1% for fluconazole and 92.9%, 3.6% and 3.6% for voriconazole. For C. krusei, the percentages of isolates in each category (S, S-DD and R) were, respectively, 0.0%,

0.0% and 100.0% for fluconazole and 86.7%, 6.7% and 6.7% for voriconazole. Table 2 summarises the in vitro antifungal susceptibilities of 28 isolates of C. glabrata and 15 isolates of C. krusei determined by Etest and by Sensititre YeastOne 3. The geometric mean MICs (␮g/mL) for the tested agents against C. glabrata were: 5-fluorocytosine, 0.05; amphotericin B, 0.04; fluconazole, 28.98 and 4.83 (by Sensititre YeastOne 3 and Etest, respectively); itraconazole, 1.05 and 1.43 (by Sensititre YeastOne 3 and Etest, respectively); ketoconazole, 0.66; and voriconazole, 0.39 and 0.06 (by Sensititre YeastOne 3 and Etest, respectively). The geometric mean MICs (␮g/mL) for the tested agents against C. krusei were: 5-fluorocytosine, 7.64; amphotericin B, 0.09; fluconazole, 55.71 and 56.60 (by Sensititre YeastOne 3 and Etest, respectively); itraconazole, 0.38 and 1.57 (by Sensititre YeastOne 3 and Etest, respectively); ketoconazole, 0.60; and voriconazole, 0.31 and 0.29 (by Sensititre YeastOne 3 and Etest, respectively). Commercial methods, such as Etest or Sensititre YeastOne, have shown an excellent correlation for in vitro antifungal testing between both as well as with the American and European broth microdilution methods [7,9,10]. This correlation has been better for voriconazole but there have been some discrepancies for fluconazole and C. glabrata. The results of the present study agree with those previously described [7,9,10]. Voriconazole was highly active against isolates of both species tested (MIC for 90% of the organisms (MIC90 ) = 0.25 ␮g/mL for C. glabrata and MIC90 = 1 ␮g/mL for C. krusei), including those for which the MICs of itraconazole (MIC90 = 2 ␮g/mL for C. glabrata and MIC90 = 1 ␮g/mL for C. krusei) and fluconazole (MIC90 = 2 ␮g/mL for C. glabrata and MIC90 = 1 ␮g/mL for C. krusei) were high. On the basis of the chosen breakpoints, 100% of the C. krusei isolates and 92.8% of the C. glabrata isolates were classified as susceptible to voriconazole. 5-Fluorocytosine displayed a moderate activity against C. krusei (MIC90 = 8 ␮g/mL) but was highly active against C. glabrata (MIC90 = 0.03 ␮g/mL). The MIC90 value of ketoconazole was 2 ␮g/mL for both C. glabrata and C. krusei. Amphotericin B displayed similar high activity against both species (MIC90 = 0.06 ␮g/mL and 0.125 ␮g/mL, respectively).

Table 1 In vitro susceptibilities of Candida glabrata and Candida krusei blood isolates to fluconazole and voriconazole as determined by Clinical and Laboratory Standards Institute disk diffusion testing Antifungal agent

No. of isolates (%) C. glabrata (n = 28)

Fluconazolea Voriconazoleb

C. krusei (n = 15)

S

S-DD

R

S

S-DD

R

24 (85.7) 26 (92.9)

2 (7.1) 1 (3.6)

2 (7.1) 1 (3.6)

0 13 (86.7)

0 1 (6.7)

15 (100) 1 (6.7)

S, susceptible; S-DD, susceptible dose-dependent; R, resistant. a Interpretive criteria for fluconazole: S, zone diameter ≥19 mm; S-DD, zone diameter 15–18 mm; R, zone diameter ≤14 mm. b Interpretive criteria for voriconazole: S, zone diameter ≥17 mm; S-DD, zone diameter 14–16 mm; and resistant, zone diameter ≤13 mm.

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Table 2 In vitro susceptibilities of Candida glabrata and Candida krusei blood isolates to antifungal agents as determined by Sensititre® YeastOne 3 and Etest testing Antifungal agent/test method

C. glabrata (n = 28)

C. krusei (n = 15) MIC90 (␮g/mL)

MIC range (␮g/mL)

Susceptible (n (%))a

27 (96.4) ND

8 ND

4–16 ND

2 (13.3) ND

0.01–0.25 ND

ND ND

0.125 ND

0.03–0.25 ND

ND ND

64 8

8 to ≥256 1–96

5 (17.9) 26 (92.9)

128 >256

32–128 24 to >256

0 (0) 0 (0)

Itraconazole Sensititre® YeastOne Etest

2 4

0.25 to ≥16 0.19–8

0 (0) 0 (0)

1 8

0.06–1 0.38 to >32

2 (13.3) 0 (0)

Ketoconazole Sensititre® YeastOne Etest

2 ND

0.125–8 ND

ND ND

2 ND

0.125–2 ND

ND ND

Voriconazole Sensititre® YeastOne Etest

0.25 0.25

0.06–4 0.03–1

26 (92.9) 28 (100)

1 1

0.25–1 0.125–2

15 (100) 14 (93.3)

MIC90 (␮g/mL)

MIC range (␮g/mL)

Susceptible (n

5-Fluorocytosine Sensititre® YeastOne Etest

≤0.03 ND

≤0.03–8 ND

Amphotericin B Sensititre® YeastOne Etest

0.06 ND

Fluconazole Sensititre® YeastOne Etest

(%))a

MIC, minimum inhibitory concentration; MIC90 , MIC for 90% of the organisms; ND, not determined. a Number and percentage of isolates displaying susceptibility, defined as MICs ≤4 ␮g/mL (5-fluorocytosine), ≤8 ␮g/mL (fluconazole), ≤0.125 ␮g/mL (itraconazole) or ≤1 ␮g/mL (voriconazole).

Most fluconazole and/or itraconazole S-DD and R C. glabrata and C. krusei isolates were classified as susceptible to voriconazole (MIC = 0.25 ␮g/mL) by Sensititre YeastOne 3. Only 2 (7.1%) of the 28 C. glabrata iso-

lates showed reduced susceptibility (MIC90 > 1 ␮g/mL) to voriconazole. Both isolates were resistant to fluconazole and itraconazole according to the CLSI breakpoints.

Table 3 Trends in in vitro susceptibilities of Candida glabrata and Candida krusei blood isolates to voriconazole as determined by Clinical and Laboratory Standards Institute disk diffusion testing over a 14-year period Year

No. of candidaemia episodes

C. glabrata n

1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003

32 44 55 33 33 33 36 43 40 45 31 44 40 46

Total

555

1 0 3 2a 0 3 4 1* 3 5* 4*,b 5 1* 3* 35*

C. krusei S-DD (%)

R (%)

0 ND 0 0 ND 0 0 ND 0 0 1 (33.3) 0 ND 0

0 ND 0 1 (50) ND 0 0 ND 0 0 0 0 ND 0

1 (3.6)

1 (3.6)

n 1* 3c,d 1 0 0 3 1 1 2* 1* 0 4 3* 1 21*

S-DD (%)

R (%)

ND 1 (33.3) 0 ND ND 0 0 0 0 ND ND 0 ND 0

ND 1 (33.3) 0 ND ND 0 0 0 0 ND ND 0 ND 0

1 (6.7)

1 (6.7)

S-DD, susceptible dose-dependent; R, resistant; ND, not determined. * Thirteen isolates (7 C. glabrata and 6 C. krusei) from these years were not recovered and were not tested for susceptibility. a The unique clinical C. glabrata isolate resistant to voriconazole was one of the 2 C. glabrata isolated in 1993. This isolate was also resistant to fluconazole. b The unique clinical C. glabrata isolate susceptible dose-dependent to voriconazole was one of the 3 C. glabrata tested in 2000. This isolate was resistant to fluconazole. c The unique clinical C. krusei isolate susceptible dose-dependent to voriconazole was one of the 3 C. krusei isolated in 1991. d The unique clinical C. krusei isolate resistant to voriconazole was one of the 3 C. krusei isolated in 1991.

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The fluconazole resistance rate was constant in C. krusei during this 14-year period and fluconazole resistance in C. glabrata was sporadic. Four C. glabrata and C. krusei isolates with reduced susceptibility to voriconazole by disk diffusion did not show any relationship to the introduction of voriconazole in the therapy schedule of our institution (Table 3), as all isolates were recovered from blood cultures before 2001 when voriconazole was not used in the treatment of invasive candidiasis. Our susceptibility results for C. glabrata and C. krusei mirror those of other studies in showing a low level of in vitro azole resistance in C. glabrata and the expected high level of resistance in C. krusei [1,9–12]. The 7.1% fluconazole resistance rate observed among C. glabrata isolates in the present study was similar to that found by Pfaller et al. [2] and Hajjeh et al. [8]. Previous surveillance studies demonstrated resistance to fluconazole in C. glabrata varying from 7% to 15%. Candida glabrata fluconazole resistance varies according to the geographic region of origin of blood isolates according to its prevalence as an aetiological agent. In Asia, Europe and South America where C. glabrata is less common, isolates are also less resistant to fluconazole (5–14%). However, the USA shows the highest resistance rate of C. glabrata to fluconazole (>18%) [1,2,9]. Voriconazole susceptibility was very high among C. krusei and C. glabrata isolates. The 3.6% voriconazole resistance rate and the 3.6% of isolates categorised as S-DD observed among C. glabrata isolates in the present study were very similar to those described by Pfaller et al. [2] and other authors [1,10,12,13]. Nguyen and Yu [13] compared the in vitro activity of fluconazole and voriconazole against 67 blood isolates of Candida spp. The MIC for voriconazole ranged from ≤0.015 ␮g/mL to >16 ␮g/mL with a mean of 0.04 ␮g/mL. Higher voriconazole MICs were observed with fluconazole-resistant isolates and the authors suggested that cross-resistance between fluconazole and voriconazole most likely occurs. In their study, voriconazole resistance was only seen in C. albicans and C. tropicalis but not in C. krusei. Drago et al. [10] also demonstrated that voriconazole is very active against C. glabrata isolates (MIC90 = 0.5 ␮g/mL), including those classified as resistant or susceptible dosedependent to fluconazole (7.1% and 69.9%, respectively) or itraconazole (18.7% and 77.3%, respectively). Fluconazole-resistant C. glabrata isolates showed potential cross-resistance with ketoconazole and itraconazole and one isolate was also in vitro resistant to voriconazole. Crossresistance between fluconazole, voriconazole, posaconazole and itraconazole has been described [10,14], most frequently in association with an increased expression of the genes (CgCDR1 and CgCDR2) encoding the CDR efflux pumps [14]. In contrast to C. glabrata, in vitro cross-resistance to voriconazole is distinctly uncommon in C. krusei because voriconazole binds much more effectively to the cytochrome P450 isoenzyme target in C. krusei than does fluconazole [15].

In conclusion, whilst reduced susceptibility to fluconazole and itraconazole is widespread among C. glabrata and C. krusei, most clinical isolates of these species, including those that are resistant or susceptible dose-dependent to fluconazole and/or itraconazole, will display susceptibility to voriconazole in vitro. Our study indicates that resistance to voriconazole is still a fairly restricted and sporadic phenomenon in C. glabrata and C. krusei at our institution, with 95.3% of all yeasts isolated from blood being S or SDD. However, there remains a potential for this phenomenon and in vitro susceptibility testing is thus recommended. In vitro susceptibility testing, when performed correctly using standardised methods, can help clinicians in identifying antifungal drugs with a higher probability of therapeutic efficacy. Funding: The authors were financed in part with grants PI030662, PI050511 and PI061895 from Fondo de Investigaci´on Sanitaria and GIU05/05 from Universidad del Pa´ıs Vasco-Euskal Herriko Unibertsitatea. Competing interests: None declared. Ethical approval: Not required.

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