**4. Antifungal susceptibility**

In vitro susceptibility testing of the 984 BSI isolates of Candida species against amphotericin B, fluconazole, voriconazole, caspofungin and anidulafungin revealed that when globally analyzed *Candida* strains causing BSI are rarely resistant to a wide number of antifungal agents. However, the resistance rates among the different species vary a lot as it can be shown on Table 4.


a All the isolates except one exhibit decreased susceptibility (SDD) to fluconazole. b All the isolates are SDD to voriconazole.

Table 4. Antifungal susceptibility test results for selected species of *Candida* isolated during prospective, sentinel surveillance in Spain from June 2008 to June 2009

There were no statistically significant differences when the risk mentioned above were

In vitro susceptibility testing of the 984 BSI isolates of Candida species against amphotericin B, fluconazole, voriconazole, caspofungin and anidulafungin revealed that when globally analyzed *Candida* strains causing BSI are rarely resistant to a wide number of antifungal agents. However, the resistance rates among the different species vary a lot as it can be

> MIC (ug/ml)

1.0 0.5 1.0 0 (0.00)

1.0 0.5 1.0 0 (0.00)

2.0 0.25 0.75 0 (0.00)

1 2 2 (1.89)

Range 50% 90%

 Fluconazole 0.125–64 0.5 2.0 10 (2.07) Voriconazole < 0.03–4 0.03 0.03 5 (1.04) Caspofungin <0.03–4 0.25 2.0 3 (0.62) Anidulafungin <0.03–2 0.03 0.03 0 (0.00)

*(204)* Amphotericin B 0.25–1.0 1.0 1.0 0 (0.00) Fluconazole 0.125–64 0.5 4.0 11 (5.39) <sup>a</sup> Voriconazole 0.03–2 0.03 0.125 3 (1.47) <sup>b</sup> Caspofungin 0.125–64 1.0 2.0 12 (5.88) Anidulafungin 0.03–64 1.0 2.0 7 (3.43) *C. glabrata (134)* Amphotericin B 0.25–1.0 0.5 1.0 0 (0.00)

 Fluconazole 1.0–64 4.0 32 25 (18.67) Voriconazole 0.03–4.0 0.125 0.5 3 (2.24) Caspofungin 0.03–0.5 0.06 0.125 0 (0.00) Anidulafungin 0.06–0.5 0.06 0.125 0 (0.00)

 Voriconazole 0.03–8 0.06 0.125 2 (1.89) Caspofungin 0.03–0.5 0.06 0.125 0 (0.00) Anidulafungin 0.03–0.5 0.03 0.125 0 (0.00)

 Fluconazole 16–128 4.0 8.0 21 (100.00) Voriconazole 0.03–4.0 0.25 1.0 2 (9.52) Caspofungin 0.125–8 0.125 1.0 1 (4.76) Anidulafungin 0.03–8 0.06 0.25 1 (4.76) a All the isolates except one exhibit decreased susceptibility (SDD) to fluconazole. b All the isolates are

Table 4. Antifungal susceptibility test results for selected species of *Candida* isolated during

prospective, sentinel surveillance in Spain from June 2008 to June 2009

128

No. of resistant or SDD isolates

analyzed for the pediatric population of patients.

**4. Antifungal susceptibility** 

Species Antifungal

*C. albicans (483)* Amphotericin B 0.125–

*C. tropicalis (106)* Amphotericin B 0.125–

*C. krusei (21)* Amphotericin B 0.125–

Fluconazole 0.25–

agent

shown on Table 4.

*C. parapsilosis*

SDD to voriconazole.

When we considered the *C. glabrata* isolates obtained during the study only the 81.33% of them were were susceptible to fluconazole and 97.76% were susceptible to voriconazole, but on the contrary, 97.93% and 98.96% of the isolates of *C. albicans* were susceptible to fluconazole and voriconazole respectively. The proportion of isolates that was resistant to the studied azole drugs was comparable with that observed in the other recently published studies. (Messer et al., 2009, Pemán et al., 2011).

The antifungal activities of voriconazole, fluconazole, amphotericin B, caspofungin and anidulafungin against the 984 *Candida spp* isolated during the study period are summarized in Table 4. Among the azole compounds, voriconazole was the most active drug overall with an MIC90 of 0.25 µg/ml. Against *C. albicans*, *C. parapsilosis* and *C. tropicalis* isolates, voriconazole (MIC90 range 0.03-0.25 µg/ml) was much more active than fluconazole (MIC90, 2-4). Although these differences in the drug activity, both azole compounds showed lower MICs for fluconazole and voriconazole for the species mentioned before when compared to *C. glabrata* and *C. krusei* isolates. Despite this good susceptibility profile, we found five *Candida albicans* isolates with a MIC greater than 4 µg/ml to voriconazole and two *C. krusei* isolates that had a voriconazole MIC of 2 ug/ ml. All these isolates were also resistant to fluconazole.

We found that there was a statistically significant moderate linear correlation between fluconazole and voriconazole MICs (*r* = 0.574; *P* ≤ 0.01). having higher voriconazole MICs those isolates from patients who received fluconazole before the candidemia episode when compared to those without previous exposure to fluconazole (MIC90s of 0.25 µg/ml and 0.06 µg/ml, respectively; *P* ≤ 0.05

Table 5 summarize the risk factors we identified during the study with a candidemia episode due to an isolate with decreased susceptibility (SDD or resistant) to fluconazole using univariate statistical techniques.


a Only statistically significant variables are summarized in the table

Table 5. Summary of univariate statistical analysis between fluconazole susceptible isolates vs. resistant or SDD ones

We found that this condition was associated with neoplasia (9% versus 3%; *P* ≤ 0.01), current neutropenia (16% versus 4%; *P* ≤ 0.01), and prior fluconazole use (14% versus 3%; *P* ≤ 0.001). These independent factors identified using the univariate statistical approach, were analyzed more deeply using a repeated measures logistic regression model. We obtained significant results for neoplasia (odds ratio, 2.9; 95% confidence interval, 1.4 to 5.9; *P* ≤ 0.05) and prior use of fluconazole (odds ratio, 3.8; 95% confidence interval, 1.7 to 8.2; *P* ≤ 0.01). (Table 6).

Epidemiology of Bloodstream *Candida* spp. Infections

Total number of isolates

(Adapted and modified from Pemán et al., 2001 and Pfaller & Diekema, 2007).

Despite this fact, it seems that probably a combination of factors may have affected the overall rates of fungemia cases in Spain. The differences appeared in the average age of our patients when compared to other surveillance series from the United States (Ostrosky-Zeichner et al., 2003, Pappas et al., 2003), are probably due to the high proportion of children in our study, especially in the Spanish hospitals located in the Southern part of the country (32% of children in the Spanish hospitals from the Southern part of the country compared to 9% described in the study from the United States) (data not shown). Therefore, this condition reflects that there are great differences among patients of different geographical locations as it was mentioned in the introduction and the demographical composition and lastly the risk factors, could be very different from one population to another. (Table 7). In fact, the number of cases of invasive candidemia was not homogeneous across the country., the distribution of the clinical isolates obtained during the study period along four different

Diekema, 2007) (see Table 7).

Country and period

Observed During a Surveillance Study Conducted in Spain 23

combination of multiple factors, including differences in medical care resources, transplantation programs, implementation of infection control measures in hospitals, empirical antifungal therapy and prophylaxis for high-risk patients. Another possibility is that our series may not reflect the current trends, but the data from a prospective study held in Spain and recently published by Peman *et al.* support our data (Pemán et al., 2011, Pfaller &

> *C. albicans*

USA 1992-1993 837 52 21 10 12 4 USA 1993-1995 79 56 15 10 15 - USA 1995-1997 1593 46 14 12 20 2 USA 1995-1998 934 53 10 12 20 3 USA 1998-2000 935 45 13 12 24 2 USA 2001-2004 2773 51 14 7 22 2 USA 2008-2009 1354 48 17 10 18 2 Canada 1992-94 415 69 10 7 8 1 Latin America 1995-1996 145 37 25 24 4 1 Latin America 2001-2004 1565 50 16 20 7 2 Asia-Pacific 2001-2004 1344 56 16 14 10 2 Taiwan 1994-2000 1095 50 14 21 12 <1 Europe 1992-94 249 49 11 11 10 9 Europe 1997-99 2089 56 13 7 14 2 Europe 2001-2004 2515 60 12 9 10 5 Norway 1991-2003 1415 70 6 7 13 2 Denmark 2003-2004 307 63 4 4 20 3 Spain 2002-2003 351 51 23 10 9 4 Spain 2001-2006 1997 47 19 10 12 5 Spain 2008-2009 984 49 21 13 11 2 Spain 2009-2010 1377 45 29 12 8 2 Table 7. Summary of geographical differences in species distribution in Candida BSI isolates.

% of total by most representative species

*C. tropicalis*

*C. glabrata*

*C. krusei* 

*C. parapsilosis*


a Only statistically significant variables are summarized in the table.

Table 6. Summary of multivariate statistical analysis of risk factors for candidemia caused by fluconazole susceptible isolates vs. resistant or SDD ones a

Caspofungin and anidulafungin resistance was low (16 cases for caspofungin and 8 cases for anidulafungin) (Table 4). Despite this low rate of in vitro resistance to echinocandins of the isolates studied MICs from *C. parapsilosis* and *C. guilliermondi* were higher compared to the MIC obtained from other *Candida spp*. as it has been described in others studies.
