**2. Materials and methods**

#### **Study design**

A prospective laboratory-based surveillance was established to monitor the predominant *Candida* species and antifungal resistance patterns of nosocomial and community-acquired invasive *Candida* infections via a network of sentinel hospitals distributed by geographic location across the country.

The participating institutions include 40 medical centers which provide medical care either to adults and children in several medical specialties. Each participant hospital contributed prospectively clinical and epidemiological results (organism identification, date of isolation, hospital location, intrinsic and extrinsic risk factors for candidemia) on clinically significant consecutive blood culture isolates of Candida spp. (one isolate per patient) detected during the 12-month period from June, 2008 through June, 2009. All isolates were saved on agar slants and were sent on a trimestral basis to the Mycology Laboratory at Basurto Hospital for storage, further characterization and reference susceptibility testing.

#### **Clinical definitions**

Clinical and case definitions were according the NHSN (formerly NNISS) methodology. Statements defining a case and other clinical conditions are summarized in Table 1.

#### **Quality control measures of clinical data**

The clinical case report list of each hospital was compared with the isolates received at Basurto Hospital to perform the antifungal susceptibility in order to verify that neither cases nor isolates were missed. Audits of medical records to verify accuracy of data and completeness were performed on 25% of cases.


Table 1. Definitions according to NHSN (formerly NISS) used in this study

#### **In vitro susceptibility testing**

16 Epidemiology Insights

The epidemiology of candidemia has been extensively studied in many countries and there are some large series published in this field (Alonso-Valle et al., 2003, Atunes et al., 2004, Banerjee et al., 1991, Colombo et al., 2006, Diekema et al., 2002, Kao et al., 1999, Messer et al., 2009, San Miguel et al., 2005, Silva et al., 2004, Tortorano et al., 2004, Trick et al., 2002). But, most of the data on candidemia in Spain until recent days are limited to retrospective reviews of medical records or observational studies conducted in a limited geographical area (Almirante et al., 2005, Alonso-Valle et al., 2003, Pemán et al., 2002, Pemán et al., 2011). Regarding the Spanish data available on antifungal resistance is often assessed by occasional surveys or reported in summaries of sporadically occurring cases of treatment failures. The purpose of such investigations is to monitor levels of susceptibility to different agents. However, long-term prospective studies of antifungal susceptibility have the advantage of eliminating a number of variable factors which may affect these assessments. Some of these factors include temporary changes in patterns of Invasive Candida infections (as stated before) and transient alterations in antifungal resistance due to special conditions (e.g. candidemia outbreaks in ICUs). Consequently, the epidemiological data about candidemia and its impact in the healthcare system is unknown, and no reliable nationwide data are available. In order to make a realistic global perspective of invasive Candida BSI, we designed a prospective laboratory-based surveillance study comprising 40 tertiary care hospitals across the country, to assess the incidence, species distribution, frequency of

A prospective laboratory-based surveillance was established to monitor the predominant *Candida* species and antifungal resistance patterns of nosocomial and community-acquired invasive *Candida* infections via a network of sentinel hospitals distributed by geographic

The participating institutions include 40 medical centers which provide medical care either to adults and children in several medical specialties. Each participant hospital contributed prospectively clinical and epidemiological results (organism identification, date of isolation, hospital location, intrinsic and extrinsic risk factors for candidemia) on clinically significant consecutive blood culture isolates of Candida spp. (one isolate per patient) detected during the 12-month period from June, 2008 through June, 2009. All isolates were saved on agar slants and were sent on a trimestral basis to the Mycology Laboratory at Basurto Hospital

Clinical and case definitions were according the NHSN (formerly NNISS) methodology.

The clinical case report list of each hospital was compared with the isolates received at Basurto Hospital to perform the antifungal susceptibility in order to verify that neither cases nor isolates were missed. Audits of medical records to verify accuracy of data and

Statements defining a case and other clinical conditions are summarized in Table 1.

for storage, further characterization and reference susceptibility testing.

antifungal resistance, and risk factors for candidemia.

**2. Materials and methods** 

location across the country.

**Clinical definitions** 

**Quality control measures of clinical data** 

completeness were performed on 25% of cases.

**Study design** 

Antifungal susceptibility tests were performed by using the broth microdilution assay according to the methodology recommended by the CLSI (formerly known as NCCLS), document M27-A2 (NCCLS, 2002) using a microtiter plate. Each isolate was tested against different antifungal drugs at the indicated concentration range suggested in the CLSI document. Quality control (QC) was ensured by testing the CLSI recommended QC strains, *C. krusei* ATCC 6258, and *C. parapsilosis* ATCC 22019.The MIC endpoint for amphotericin B, azoles and echinocandins and interpretative MIC breakpoints for azoles and echinocandins were those suggested by the CLSI document M27-A2, but for the definition of the amphotericin B MIC breakpoints we used the values suggested from a previous study published by Nguyen *et al.* (Nguyen et al., 1998).
