Amphotericin B, antifungal susceptibility, bloodstream infections, Candida spp., posaconazole, sus-

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1 ORIGINAL ARTICLE /j x Epidemiology of candidaemia and antifungal susceptibility patterns in an Italian tertiary-care hospital A. Bedini 1, C. Venturelli 2, C. Mussini 1, G. Guaraldi 1, M. Codeluppi 1, V. Borghi 1, F. Rumpianesi 2, F. Barchiesi 3 and R. Esposito 1 1 Clinic of Infectious and Tropical Diseases, University of Modena, 2 Microbiology Department, Azienda Policlinico of Modena, Modena and 3 Clinic of Infectious Diseases, University of Ancona, Ancona, Italy ABSTRACT The epidemiological and antifungal susceptibility data for 94 episodes of candidaemia in an Italian tertiary-care hospital between January 2000 and August 2003 were evaluated by prospective laboratorybased surveillance. The incidence of fungaemia was 0.90 episodes patient-days, and the most common species isolated were Candida albicans (40.4%), Candida parapsilosis (22.3%), Candida tropicalis (16.0%) and Candida glabrata (12.8%). Among 24 patients who received antifungal prophylaxis, nonalbicans Candida spp. were more prevalent than C. albicans (p 0.012). The 30-day mortality rate was high (38.2%), particularly for haematological (71.4%) and solid-organ transplant patients (50.0%), and in individuals with C. tropicalis and C. glabrata bloodstream infections (60.0% and 50.0%, respectively). In-vitro susceptibility tests demonstrated that 95% of the isolates were susceptible to amphotericin B (MIC < 2 mg L), 98.1% to posaconazole (MIC < 1 mg L), 95.8% to flucytosine (MIC < 32 mg L) and fluconazole (MIC < 64 mg L), and 94.7% to itraconazole (MIC < 1 mg L). Posaconazole was active (MIC 0.5 mg L) against all three isolates of Candida krusei, which had reduced susceptibility to both fluconazole and itraconazole. Overall, non-albicans Candida spp. accounted for 60% of the episodes of candidaemia, which could be related to the use of antifungal prophylaxis. Resistance is still uncommon in Candida spp. recovered from blood cultures. The in-vitro activity of posaconazole is encouraging, and this agent could play an important role in the management of invasive candidiasis, including episodes caused by inherently less susceptible species such as C. krusei. Amphotericin B, antifungal susceptibility, bloodstream infections, Candida spp., posaconazole, sus- Keywords ceptibility Original Submission: 23 September 2004; Revised Submission: 13 May 2005; Accepted: 29 June 2005 Clin Microbiol Infect 2006; 12: INTRODUCTION During the past two decades, the frequency of invasive fungal infections has increased dramatically in hospitalised patients throughout the world, and Candida has now emerged as one of the leading causes of bloodstream infections (BSIs) [1,2]. Data from North American and European surveillance programmes of hospitalacquired bacteraemia have revealed that isolates of Candida spp. are the fourth most common cause [3,4], accounting for 8 10% of nosocomial BSIs Corresponding author and reprint requests: A. Bedini, Clinic of Infectious and Tropical Diseases, University of Modena, Via del pozzo 71, Modena, Italy andreabedini@yahoo.com [1]. Risk-factors for invasive candidiasis include improvements in intensive care strategies (i.e., central venous catheters, mechanical ventilation, hyper-alimentation), prolonged stays in intensive care units (ICUs), the development of more aggressive surgical techniques, and the prolongation of survival of critically-ill patients [5]. Two other important factors, observed mainly in cancer patients, are colonisation of mucous membranes by yeasts, and neutropenia, resulting from increased use of antibiotics and anti-neoplastic agents, respectively [6]. The crude mortality rate of candidaemia is high (38 75%) [3,4,7], and the attributable mortality has been estimated at 25 38% [8,9]. During the past 15 years, the prevalence of infections caused by non-albicans Candida spp. has increased Ó 2006 Copyright by the European Society of Clinical Microbiology and Infectious Diseases

2 76 Clinical Microbiology and Infection, Volume 12 Number 1, January 2006 exponentially, so that these organisms now account for > 50% of episodes of fungaemia in various surveys. The widespread use of azoles has been suggested as the main factor responsible for this changing epidemiology [10 15]. Candida albicans is generally susceptible to all the antifungal agents available currently. Among the non-albicans Candida spp., Candida tropicalis and Candida parapsilosis are both generally susceptible to azoles, but C. tropicalis is less susceptible to fluconazole than is C. albicans. Candida glabrata is intrinsically more resistant to antifungal agents, particularly to fluconazole. Candida krusei is intrinsically resistant to fluconazole, and infections caused by this species are associated strongly with previous fluconazole prophylaxis and neutropenia. Candida lusitaniae, accounting for 1 2% of candidaemias, is susceptible to azoles, but has higher intrinsic resistance to amphotericin B [16]. The increase in invasive fungal infections, the associated high mortality rate, and the emergence of antifungal resistance, have all driven the search for more potent antifungal drugs. Posaconazole is a novel antifungal triazole, with potent activity against the yeasts and filamentous fungi that cause systemic infections [17 19]. The aims of the present study were to investigate the epidemiology of candidaemia and to determine the antifungal susceptibility patterns of isolates of Candida spp. from a tertiary-care hospital in Italy. MATERIALS AND METHODS Surveillance Prospective surveillance of nosocomial candidaemia was conducted at the University Hospital of Modena, Italy, between January 2000 and August The hospital has 990 beds, with two adult ICUs, one neonatal ICU, 28 medical wards and 11 surgical wards, including two centres for solidorgan transplantations (kidney and liver multivisceral transplants). Surveillance was based on data from the microbiology laboratory. Episodes of candidaemia were defined by at least two positive blood cultures yielding Candida spp. during a single hospitalisation period. Demographic data, reasons for hospitalisation, antifungal prophylaxis and outcome were collected from medical records. Organism identification Blood specimens were processed by an automated blood culture system (BACTEC 9240; Becton Dickinson, Sparks, MD, USA), and viable yeasts were subcultured on Sabouraud dextrose agar (Labobasi, Vallauris, France). Species identification was based on germ tube production, distinctive colour, and morphology on Candida ID2 Agar (biomérieux, Marcy l Etoile, France), together with sugar assimilation profiles obtained using the API ID 32C System (biomérieux). All isolates were stored at ) 80 C in glycerol 10% v v. Susceptibility testing The antifungal agents tested were fluconazole (Pfizer Inc., New York, NY, USA), posaconazole (Schering-Plough Research Institute, Kenilworth, NJ, USA), itraconazole (Janssen Research Foundation, Beerse, Belgium), and amphotericin B and flucytosine (Sigma Chemical, Milano, Italy). MICs were determined according to NCCLS guidelines [20]. Final concentrations ranged from to 4 mg L for itraconazole and posaconazole, mg L for fluconazole and flucytosine, and mg L for amphotericin B. MICs were read visually; for posaconazole, itraconazole, fluconazole and flucytosine, the MIC was the lowest drug concentration that resulted in an obvious inhibition of growth (c. 50%) compared with that of the growth control; for amphotericin B, the MIC was the concentration in the first well that was optically clear. C. krusei ATCC 6258 and C. albicans ATCC were used as control strains in all tests. The interpretative breakpoints for fluconazole, itraconazole and flucytosine were those suggested previously [20,21]. Since in-vitro susceptibility breakpoints for amphotericin B and posaconazole have not yet been established, susceptible breakpoints of < 2 mg L for amphotericin B and < 1 mg L for posaconazole were used, based on pharmacokinetic data [22]. Statistical analyses The chi-square test was used to evaluate the differences in prevalence between C. albicans and non-albicans Candida spp. in relation to antifungal prophylaxis and the underlying diseases of patients, to evaluate the 30-day outcome of patients in relation to different wards of admission, causes of hospitalisation and the species of Candida responsible for the fungaemia, and to evaluate the correlation between in-vitro susceptibility to fluconazole, itraconazole and posaconazole and the use of azole prophylaxis. To investigate the degree of crossresistance between azoles, the correlation coefficients between the MICs of fluconazole, itraconazole and posaconazole were determined by the Spearman rho non-parametric method. p < 0.05 was considered significant for both analyses. RESULTS AND DISCUSSION In total, 94 isolates of Candida spp. were collected from blood cultures taken from 86 hospitalised patients. In eight (9.3%) patients, there was simultaneous or subsequent isolation of two different Candida spp. Forty-eight (56%) patients were male, and the median age was 61 years (range 0 89 years). The overall incidence of candidaemia was 0.90 episodes patient-days. The most common species isolated was C. albicans (38 episodes, 40.4%), followed by C. parapsilosis

3 Bedini et al. Candidaemia and antifungal susceptibility patterns 77 (21 episodes, 22.3%), C. tropicalis (15 episodes, 16.0%), C. glabrata (12 episodes, 12.8%), C. krusei (three episodes, 3.2%), C. lusitaniae (two episodes, 2.1%), Candida guilliermondii (two episodes, 2.1%) and Candida pelliculosa (one episode, 1.1%). The reasons for hospitalisation are shown in Table 1. Overall, 36% of patients with candidaemia were from medical wards, 35% from surgical wards and 29% from ICUs. No significant differences were observed with respect to the prevalence of C. albicans and non-albicans Candida spp. between the different wards and the reasons for admission. However, for the nine patients with neutropenia (defined as < 500 neutrophils mm 3 ), all the episodes were caused by non-albicans Candida spp. (p < 0.001; three C. tropicalis, two C. glabrata, two C. krusei, one C. guilliermondii, one C. lusitaniae). The overall incidence of candidaemia observed in the hospital was in agreement with the results of another Italian study (1.14 episodes patient days) [23], but was higher than that observed in a large European survey (0.44 episodes patient-days) [24]. In Modena, C. albicans accounted for only 40.4% of cases and, among the nonalbicans spp., C. parapsilosis and C. tropicalis were most prevalent. C. albicans accounted for > 50% of BSIs and C. tropicalis for only 7% of the episodes in these two surveys. A possible explanation for these differences could be the absence of haematological and neutropenic patients, who are at higher risk of C. tropicalis BSI [25,26], in the study of Luzzati et al. [23], while a larger sample size could account for the different species distribution described by Tortorano et al. [24]. Before diagnosis, 24 patients had received antifungal prophylaxis: 14 received fluconazole, four itraconazole, and six nystatin. In this subset of patients, non-albicans and non-parapsilosis Candida spp. were significantly more prevalent Table 1. Underlying diseases that caused hospitalisation of patients suffering from candidaemia Underlying disease Solid neoplasia 30 (32.0) Abdominal surgery 17 (18.1) Trauma 12 (12.7) Solid-organ transplantation a 10 (10.6) Haematological neoplasia 7 (7.4) Other illness b 18 (19.2) No. (%) of episodes a Includes liver transplantation (nine episodes) and multivisceral transplantation (one episode). b Includes neurological diseases (six episodes), fetal immaturity (six episodes), respiratory diseases (five episodes) and AIDS (one episode). Table 2. Prevalence of Candida spp. in relation to antifungal prophylaxis Species No antifungal prophylaxis (n = 70) Antifungal prophylaxis a (n = 24) Candida albicans 34 (48.6%) 4 (16.7%) Candida parapsilosis 18 (25.7%) 3 (12.5%) Candida tropicalis 9 (12.9%) 6 (25.0%) Candida glabrata 6(8.6%) b 6 (25.0%) Others 3 (4.2%) 5 (20.8%) c a Antifungal agents: nystatin (6), fluconazole (14) and itraconazole (4). b Includes Candida krusei (1), Candida guilliermondii (1) and Candida pelliculosa (1). c Includes C. krusei (2), Candida lusitaniae (2), and C. guilliermondii (1). (p 0.002; Table 2). These data agree with previous reports describing the increasing rate of colonisation and infection caused by natively resistant Candida spp. (C. glabrata and C. krusei) in patients receiving azoles as prophylaxis [10,15]. Overall, the crude mortality rate at 1 month after the diagnosis of fungaemia was 38.2%, in agreement with previous results [6,23,24]. No significant differences in the 30-day mortality rate were observed with respect to the ward at the onset of candidaemia, or the reasons for hospitalisation and neutropenia (data not shown). Haematological (71.4%) and solid-organ transplant (50.0%) patients showed the highest 30-day mortality rate. Table 3 compares the 30-day mortality rate of patients with C. albicans (21.1%) and nonalbicans Candida infections (42.8%; p 0.012). Patients with C. parapsilosis infections had a poorer outcome than those with C. albicans fungaemia (mortality rate, 33.3% vs. 21.1%), but this difference was not statistically significant. C. tropicalis and C. glabrata were associated with a poorer outcome (mortality rates of 60.0% and 50.0%, respectively), but only C. tropicalis infection correlated significantly with a worse prognosis than C. albicans (p 0.006). These findings are consistent with those described by Viscoli et al. [6] in cancer patients. The antifungal susceptibility patterns of the Candida isolates are summarised in Table 4. The Table 3. Thirty-day mortality rate of patients with fungaemia: comparison between episodes caused by Candida albicans and non-albicans Candida spp. Candidaemia episodes in 86 patients, n (mortality rate %) C. albicans vs. non-albicans spp. 38 (21.1) vs. 56 (42.8) C. albicans vs. C. parapsilosis 38 (21.1) vs. 21 (33.3) C. albicans vs. C. tropicalis 38 (21.1) vs. 15 (60.0) C. albicans vs. C. glabrata 38 (21.1) vs. 12 (50.0) C. albicans vs. other species a 38 (21.1) vs. 8 (25.0) a C. krusei (n =3),C. lusitaniae (n =2),C. guilliermondii (n =2),C. pelliculosa (n =1). p p

4 78 Clinical Microbiology and Infection, Volume 12 Number 1, January 2006 Table 4. In-vitro activities of five antifungal agents against 94 bloodstream isolates of Candida spp. from 86 hospitalised patients Organisms (no. of isolates) Compound Range MIC50 MIC90 C. albicans (38) C. parapsilosis (21) C. tropicalis (15) C. glabrata (12) No. of No of isolates isolates R a (%) S-DD (%) Amphotericin B (0) Flucytosine (0) 1 (2.6) Fluconazole (0) 2 (5.2) Itraconazole (0) 4 (10.5) Posaconazole (0) Amphotericin B (0) Flucytosine (0) 0 (0) Fluconazole (0) 1 (4.7) Itraconazole (0) 8 (38.0) Posaconazole (0) Amphotericin B (0) Flucytosine to > > 64 3 (20) 0 (0) Fluconazole 0.25 to > (6.6) 0 (0) Itraconazole to > (6.6) 10 (66.6) Posaconazole 0.03 to > (6.6) Amphotericin B Flucytosine to > (8.3) 0 (0) Fluconazole (8.3) 0 (0) Itraconazole (8.3) 9 (75.0) Posaconazole (8.3) Other species Amphotericin B (12.5) (8) b Flucytosine (25.0) Fluconazole (25) 2 (25.0) Itraconazole (37.5) 4 (50.0) Posaconazole All Amphotericin B (1.0) organisms (94) Flucytosine to > (4.2) 3 (31.9) Fluconazole to > (4.2) 5 (5.3) Itraconazole to > (5.3) 35 (37.2) Posaconazole to > (2.1) R, resistant; S-DD, susceptible dose dependent; S, susceptible. a Susceptibility breakpoints (mg L): amphotericin B 2 (R); fluconazole 8(S), (S-DD), 64 (R); itraconazole (S), (S-DD), 1 (R); posaconazole 1 (R); flucytosine 8 (S), (S-DD), 64 (R). b C. krusei (n =3),C. lusitaniae (n =2),C. guilliermondii (n =2),C. pelliculosa (n =1). The resistant isolates were: one C. lusitaniae (resistant to amphotericin B), one C. krusei (resistant to itraconazole) and two C. krusei (resistant to itraconazole and fluconazole). MICs for the two control strains were within the expected range (data not shown). Amphotericin B was the most active agent in vitro, with MIC 50 and MIC 90 values of 0.25 and 1 mg L, respectively. Only one C. lusitaniae isolate, recovered from a patient who had not received antifungal treatment, was resistant to amphotericin B (MIC 2 mg L). However, these results may not be conclusive, since the RPMI 1640 medium may not be optimal for testing susceptibility to this drug [27]. The susceptibility of the isolates to flucytosine was 95.8%. Flucytosine was active against all C. albicans (MIC mg L) and C. parapsilosis (MIC mg L) isolates. MIC 90 values for the eight less common Candida spp. (including three isolates of C. krusei) were 16 mg L. Interestingly, C. tropicalis was the least susceptible species, with an MIC 90 of > 64 mg L (three (20%) of 15 isolates resistant). Although a low percentage (8.0%) of resistance to flucytosine has been reported among C. tropicalis isolates [28], a resistance rate of 30.0% has also been reported [29]. The MIC 90 for C. glabrata was 0.25 mg L; only one of the 12 C. glabrata isolates was resistant to flucytosine (MIC > 64 mg L; resistance rate 8.3%). None of the patients had received flucytosine before the diagnosis of candidaemia. Among the azoles, posaconazole was the most potent agent in vitro (MIC mg L), followed by itraconazole (MIC mg L) and fluconazole (MIC 90 8mg L). Overall, the frequency of resistance to fluconazole was as low (4.2%) as the dosedependent susceptibility (5.3%). All C. albicans and C. parapsilosis isolates were susceptible to fluconazole (MIC 90 1mg L). The rates of fluconazole resistance among C. glabrata (one (8.3%) of 12) and C. tropicalis (one (6.6%) of 15) were consistent with those found in other surveillance studies [30]. Fluconazole was active against only six of the eight less common Candida spp.; two of the three C. krusei isolates in this subgroup had fluconazole MICs of 64 mg L. In three of the four episodes of candidaemia caused by fluconazoleresistant Candida spp. (one C. glabrata and two C. krusei), the patients had received fluconazole prophylaxis before diagnosis. The dose-dependent susceptibility to itraconazole was high (37.2%), particularly for C. glabrata (75%) and C. tropicalis (66.6%). However, the resistance profile for itraconazole (5.3%) was similar to that for fluconazole. The five itraconazole-resistant isolates comprised one C. glabrata (MIC 4 mg L), one C. tropicalis (MIC > 4 mg L) and three C. krusei isolates (MIC 1 mg L). In two cases (one C. krusei and one C. tropicalis), the patients had not received any prophylaxis, while the other three patients had received fluconazole before the diagnosis of fungaemia, suggesting the possible emergence of cross-resistance between the two azoles [29]. Posaconazole was two-fold more active than itraconazole against all the species of Candida tested; the MIC 90 values for C. albicans, C. parapsilosis, C. tropicalis, C. glabrata and the rarer Candida spp. were 0.125, 0.125, 0.25, 0.25 and 0.5 mg L, respectively. Furthermore, posaconazole was active (MIC 0.5 mg L) against the three isolates of C. krusei, all of which exhibited reduced susceptibility to both fluconazole and itraconazole. One isolate each of C. glabrata and C. tropicalis, both resistant to fluconazole and itraconazole, were resistant to posaconazole (MIC 2 and

5 Bedini et al. Candidaemia and antifungal susceptibility patterns 79 >4 mg L, respectively). It is of note that the C. glabrata isolate was recovered from a patient who had received fluconazole prophylaxis; thus, it is possible that it had acquired resistance to fluconazole, and had become cross-resistant to posaconazole [29]. For the C. tropicalis isolate, the most likely explanation is rare primary resistance to all three azoles. Table 5 shows the relationship between in-vitro antifungal susceptibility and exposure to azole prophylaxis. Candida spp. isolated from patients who had received azoles before the onset of fungaemia were significantly less susceptible to fluconazole, itraconazole and posaconazole (p 0.006, p and p 0.026, respectively). These findings suggest that azole prophylaxis could be responsible for the emergence of cross-resistance between fluconazole, itraconazole and posaconazole, or that it could account for the selection of Candida spp. that are less susceptible to azoles. No significant correlations were observed between the MICs of fluconazole, itraconazole and posaconazole (data not shown). In conclusion, the incidence of candidaemia at this institution was consistent with the data obtained from other studies in the USA and Europe [31,32,33]. C. albicans remains the most prevalent species, but non-albicans Candida spp. accounted for 60% of episodes. One factor that may contribute to the emergence of non-albicans Candida spp. is the use of antifungal prophylaxis [10 15]. The relatively high proportion of C. parapsilosis isolates, typically related to the use of central venous catheters and parenteral nutrition [32 34], raises the possibility of inadequate Table 5. Relationship between in-vitro antifungal susceptibility and exposure to azole prophylaxis in 94 episodes of candidaemia in 86 hospitalised patients Susceptibility No azole prophylaxis (76 episodes) Azole prophylaxis a (18 episodes) p Fluconazole S S-DD 3 2 R 1 3 Itraconazole S S-DD 26 9 R 2 3 Posaconazole b S* S** 11 7 R 1 1 R, resistant; S, susceptible. a Antifungal agents: fluconazole (n = 14) and itraconazole (n =4). b For comparison purposes, susceptibility to posaconazole was divided into S* ( mg L) and S** ( mg L). infection control practices. The severity of Candida BSI was confirmed by the 30-day mortality rate of 38.2%. Although significant differences were not observed when considering the causes of hospitalisation, haematological and solid-organ transplant patients had poorer outcomes. C. tropicalis and C. glabrata were related to the highest mortality rates. 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