Rapid Antifungal Susceptibility Determination for Yeast Isolates By

Rapid Antifungal Susceptibility Determination for Yeast Isolates by Use of Etest Performed Directly on Blood Samples from Patients with Fungemiaᰔ Jesu´s Guinea,1,2,3* Sandra Recio,1 Pilar Escribano,1 Marta Torres-Narbona,1 Teresa Pelaéz,1,2,3 Carlos Sańchez-Carrillo,1,2 Marta Rodríguez-Creíxems,1,2,3 and Emilio Bouza1,2,3 Clinical Microbiology and Infectious Diseases Department, Hospital General Universitario Gregorio Maranõń, Universidad Complutense de Madrid, Madrid, Spain1; CIBER de Enfermedades Respiratorias (CIBER RES CD06/06/0058), Palma de Mallorca, Spain2; and Microbiology Department, School of Medicine, Universidad Complutense de Madrid, Madrid, Spain3

Received 25 November 2009/Returned for modiﬁcation 20 January 2010/Accepted 10 March 2010

We prospectively determined the antifungal susceptibility of yeast isolates causing fungemia using the Etest on direct blood samples (195 prospectively collected and 133 laboratory prepared). We compared the Etest direct (24 h of incubation) with CLSI M27-A3 and the standard Etest methodologies for fluconazole, voriconazole, posaconazole, isavuconazole, caspofungin, and amphotericin B. Strains were classified as susceptible, resistant, or nonsusceptible using CLSI breakpoints (voriconazole breakpoints were used for posaconazole and isavuconazole). Cate- gorical errors between Etest direct and CLSI M27-A3 for azoles were mostly minor. No errors were detected for caspofungin, and high percentages of major errors were detected for amphotericin B. For the azoles, false susceptibility (very major errors) was found in only two (0.6%) isolates (Candida tropicalis and C. glabrata). False resistance (major errors) was detected in 46 (14%) isolates for the three azoles (in 23 [7%] after excluding posaconazole). Etest direct of posaconazole yielded a higher number of major errors than the remaining azoles, especially for C. glabrata, Candida spp., and other yeasts. Excluding C. glabrata, Candida spp., and other yeasts, the remaining species did not yield major errors. Etest direct for fluconazole, voriconazole, isavuconazole, and caspofungin shows potential as an alternative to the CLSI M27-A3 procedure for performing rapid antifungal susceptibility tests on yeast isolates from patients with fungemia. Etest direct is a useful tool to screen for the presence of azole-resistant and caspofungin- nonsusceptible strains.

The incidence of fungemia continues to rise in many insti- to considerable increases in the number of adverse events and tutions throughout the world, and Candida is one of the lead- in the cost of treatment (2). ing pathogens isolated from blood (15, 28). Amphotericin B The combination of an increasing number of antifungal- and fluconazole have been widely used for the treatment of resistant isolates and the cost of the new antifungal agents fungemia. However, newly licensed antifungal agents (voricon- makes antifungal susceptibility testing a necessity. The refer- azole, posaconazole, and the echinocandins) and other azoles ence antifungal susceptibility testing method for yeasts is the currently under investigation (isavuconazole) have expanded Clinical and Laboratory Standards Institute (CLSI; formerly the antifungal armamentarium. NCCLS) testing standard M27-A3. However, this method re- The mortality rate of fungemia remains high (30%) and is quires pure-culture isolates, and results of antifungal suscep- clearly correlated with delayed initiation of effective antifungal tibility testing are not available until 48 to 72 h after the therapy (11, 17). Antifungal therapy is considered inappropri- isolation of fungi in blood. ate when it is omitted, when the agent administered has no The Etest performed directly on blood samples may expedite antifungal activity against the infecting organism, or when its antifungal testing and provide results in 24 h. Our group has serum concentrations are subtherapeutic. previously demonstrated that the Etest performed directly on A growing proportion of Candida isolates obtained from samples from the lower respiratory tract is a rapid and accurate blood samples have reduced antifungal susceptibility to flucon- procedure for antimicrobial susceptibility testing of bacteria in azole and other antifungal agents (14, 25). Patients with can- patients with ventilator-associated pneumonia (4, 5). We com- didemia caused by Candida strains with high MICs for flucon- pared the results of the Etest performed directly on positive blood azole or voriconazole and echinocandins can have a worse cultures with yeasts grown in Bactec blood bottles with the results prognosis (22–24). Consequently, systematic use of empirical of CLSI M27-A3 in isolates from patients with fungemia and antifungal agents with broad-spectrum in vitro activity has led blood samples generated from previously characterized isolates. (This study was partially presented at the 20th Conference of the European Congress of Clinical Microbiology and Infec- tious Diseases [ECCMID] in Vienna, Austria, 2010 [abstract * Corresponding author. Mailing address: Servicio de Microbiología no. P-838] [13a].) Clínica y Enfermedades Infecciosas-VIH, Hospital General Universi- tario Gregorio Maranõń C/Dr. Esquerdo 46, 28007 Madrid, Spain. Phone: 34 915867163. Fax: 34 915044906. E-mail: jguineaortega MATERIALS AND METHODS @yahoo.es. Setting, study period, and episodes of fungemia. The study was carried out ᰔ Published ahead of print on 14 April 2010. prospectively from February 2007 to July 2009 in a 1,750-bed tertiary hospital

2205 2206 GUINEA ET AL. J. CLIN.MICROBIOL.

TABLE 1. Number of yeast cells per ml of the 0.5 McFarland Etest direct on blood samples (ETdir). ETdir was prepared by pouring 10 to suspensions of 10 different species of yeastsa 20 drops from Bactec bottles onto 150-mm RPMI 1640 agar plates supplemented with 2% glucose (Tec-Laim, Madrid, Spain). The sample was further streaked Species CFU/mlb Cells/fieldc across the surface of the agar plates, which were allowed to dry for 15 min. Etest T. mucoides 4.5 ϫ 106 Ͻ1 strips (fluconazole, voriconazole, posaconazole, isavuconazole, amphotericin B, Rhodotorula spp. 1.9 ϫ 106 4–5 and caspofungin) were placed onto the surface and incubated at 35°C for 24 h. C. tropicalis 3.3 ϫ 106 5–6 ETdir MICs could be interpreted in 85% of isolates after 24 h of incubation. All S. cerevisiae 1.2 ϫ 106 Ͻ1 Cryptococcus isolates, one C. albicans isolate, four C. glabrata isolates, one C. C. parapsilosis 1.3 ϫ 106 4–5 parapsilosis isolate, two R. mucilaginosa isolates, and four S. cerevisiae isolates C. glabrata 4.3 ϫ 106 Ͼ20 required 48-h incubation due to poor growth. A further eight S. cerevisiae isolates C. albicans 1.4 ϫ 106 Ͻ1 had to be incubated for 6 days. C. kefyr 2.1 ϫ 106 Ͼ10 Fifteen (7.5%) of the 200 Bactec blood culture bottles contained the following C. krusei 3.1 ϫ 106 2–3 bacterial copathogens: Enterococcus faecalis (n ϭ 1), Klebsiella pneumoniae (n ϭ 6 T. inkin 1.4 ϫ 10 Ͻ1 1), Lactobacillus acidophilus (n ϭ 1), Staphylococcus aureus (n ϭ 1), and coag- ϭ a ulase-negative Staphylococcus spp. (n 11). In four of these bottles (2%), One isolate each of the species listed was used to prepare a 0.5 McFarland ϭ suspension. bacterial growth (coagulase-negative Staphylococcus spp. [n 3] and E. faecalis b CFU present in the 0.5 McFarland suspension that was further incubated in [n ϭ 1]) interfered with interpretation of the ETdir MICs, and they were ex- the Bactec bottles. Counts were determined using a Neubauer chamber and cluded from the analysis. A strain from another blood culture was not available microscopy (ϫ40). and was also excluded. A total of 328 bottles (195 prospectively collected and 133 c A 0.5-ml sample of each suspension was inoculated in the Bactec bottle and laboratory prepared) were further analyzed. further incubated. When the result was positive, we prepared a Gram stain of Reference procedures: CLSI M27-A3 microdilution and Etest standard each bottle. Using microscopy (ϫ100), we counted the number of cells in 10 different fields and divided it by 10. (ETsd) (7). The antifungal drugs obtained as reagent-grade powders were isavuconazole (Basilea Pharmaceutica International Ltd., Basel, Switzerland), voriconazole and fluconazole (Pfizer Pharmaceutical Group, New York, NY), posaconazole (Schering-Plough, Kenilworth, NJ), amphotericin B (Sigma, Ma- (Hospital General Universitario Gregorio Maranõń, Madrid, Spain) serving a drid, Spain), and caspofungin (Merck Research Laboratories, Rahway, NJ). population of 715,000 inhabitants. Blood samples from patients with fungemia Stock solutions of caspofungin and fluconazole were prepared in sterile distilled were inoculated in Bactec blood culture bottles (Becton Dickinson Microbiology water; the remaining agents were prepared in dimethyl sulfoxide (Sigma, Madrid, Systems, Cockeysville, MD) and incubated in the automated Bactec NR9240 Spain). The final concentrations of drugs in the trays were 0.015 to 16 ␮g/ml instrument for no fewer than 5 days. (posaconazole, isavuconazole, voriconazole, and amphotericin B), 0.125 to 128 An episode of fungemia was defined as the isolation of yeasts in Bactec blood ␮g/ml (fluconazole), and 0.008 to 8 ␮g/ml (caspofungin). The inoculated trays culture bottles. In patients with multiple blood culture sets containing yeast were incubated at 35°C and read at 24 h (caspofungin and fluconazole) and 48 h isolates, episodes were defined as follows: (i) a different yeast species isolated in (azoles and amphotericin B). Incubation was prolonged for Cryptococcus spp. subsequent blood culture sets or (ii) the same species isolated 1 month after the and R. mucilaginosa (72 h) or S. cerevisiae (3 to 6 days) until fungal growth made last blood sample was drawn. According to this definition, a total of 200 episodes it possible to interpret the MIC endpoint, defined as the lowest concentration at of fungemia occurred in 194 patients. which growth is inhibited by 50% (7). Quality control was ensured by testing C. Samples and fungal isolates. Bactec bottles with a Gram stain revealing krusei ATCC 6258 and C. parapsilosis ATCC 22019 isolates. All results were elements compatible with yeasts were selected. Only one bottle from the first within the recommended CLSI limits. blood culture set of each episode (n ϭ 200) was selected. The distribution of The ETsd was performed using yeast suspensions adjusted to a 0.5 McFarland microorganisms isolated was as follows: Candida albicans (n ϭ 82), Candida standard. A swab was dipped into the suspension and streaked across the surface parapsilosis (n ϭ 64), Candida glabrata (n ϭ 20), Candida tropicalis (n ϭ 15), Pichia guilliermondii (formerly Candida guilliermondii)(n ϭ 3), C. albicans plus of the agar plates, which were allowed to dry for 15 min. Etest strips of the six C. parapsilosis (n ϭ 3), C. albicans plus C. glabrata (n ϭ 2), Rhodotorula muci- antifungal agents were placed onto the surface and further incubated at 35°C for laginosa (n ϭ 2), Candida dubliniensis (n ϭ 2), Candida krusei (n ϭ 1), Arxula 48 h. The MICs were read according to the manufacturer’s instructions. adeninivorans (n ϭ 1), Cryptococcus neoformans var. neoformans (n ϭ 1), Statistical and data analysis. As the Etest strips contain a continuous gra- Kluyveromyces marxianus (formerly Candida kefyr)(n ϭ 1), Saccharomyces cer- dient of antifungal drug, the MICs obtained by the ETdir and ETsd were evisiae (n ϭ 1), Trichosporon inkin (n ϭ 1), and Trichosporon mucoides (n ϭ 1). increased to the concentration of the next 2-fold dilution matching the drug All isolates were subcultured and stored at Ϫ70°C for antifungal susceptibility dilution scale used for the CLSI procedure. All the MICs obtained were testing (CLSI and Etest standard procedures). further converted to log2 MICs. Due to the low number of episodes caused by species of Candida with reduced The mean MIC of each antifungal (obtained by the three different methods) was susceptibility to azoles or by non-Candida yeasts, we inoculated Bactec bottles calculated. We used the t test to compare the antifungal activity determined by each with blood from nonfungemic/bacteremic patients with the following strains: C. method. The alpha value was set at 0.05, and all P values were two-tailed. glabrata (n ϭ 47), C. krusei (n ϭ 30), C. neoformans var. grubii (n ϭ 15), C. ETdir was compared with CLSI M27-A3 and ETsd procedures, which are neoformans var. neoformans (n ϭ 13), S. cerevisiae (n ϭ 14), Dipodascus capitatus considered the gold standards. ETsd was also compared with CLSI M27-A3. ϭ ϭ (formerly Blastoschizomyces capitatus)(n 9), T. mucoides (n 2), C. tropicalis Agreement between the methods was considered to be essential when the log ϭ 2 (n 2) resistant to multiple azoles (fluconazole, voriconazole, posaconazole, and MIC measured by each method was within Ϯ2 or fewer than 2-fold dilutions of ϭ isavuconazole), and Cryptococcus gattii (n 1). the next one (8, 13, 21). In each case, a 0.5-ml volume of a suspension (0.5 McFarland standard) of In addition, strains and antifungal agents were compared to calculate categor- each strain was inoculated into the bottles, which were reincubated until growth ical agreement using the CLSI M27-A3 breakpoints, as follows: voriconazole (Յ1 of yeast was detected. To estimate the number of yeast cells per ml of the 0.5 ␮g/ml, susceptible; 2 ␮g/ml, susceptible-dose dependent; Ն4 ␮g/ml, resistant); McFarland suspensions, we chose 10 strains of different species and counted the fluconazole (Յ8 ␮g/ml, susceptible; 16 to 32 ␮g/ml, susceptible-dose dependent; number of cells using a Neubauer chamber (Table 1). If the Bactec bottle gave Ն64 ␮g/ml, resistant); caspofungin (Յ2 ␮g/ml, susceptible; Ն4 ␮g/ml, nonsus- a positive result, we also counted the number of yeast cells using microscopy. A previous study showed that variations in the yeast inoculum in the Bactec bottle ceptible). In the absence of breakpoints for posaconazole and isavuconazole, the Ͼ ␮ had no impact on the MIC (6). breakpoints for voriconazole were used. Strains with MICs of 1 g/ml for All isolates were identified using culture characteristics and biochemical re- amphotericin B were considered resistant (7). The procedures were considered actions by means of CHROMAgar (Tec-Laim, Madrid, Spain) and ID 32C to be in categorical agreement when they resulted in the same susceptibility (bioMe´rieux, Marcy-l’Etoile, France). Strains of S. cerevisiae, D. capitatus, Tricho- category (e.g., susceptible or resistant). Errors were categorized as very major sporon spp., P. guilliermondii, A. adeninivorans, and K. marxianus were identified (ETdir indicated susceptible and gold standard indicated resistant), major by sequencing the ITS1–5.8S–ITS2 region, as described by White et al. (27). C. (ETdir indicated resistant and gold standard indicated susceptible), or minor neoformans and C. gattii strains were identified by means of amplified fragment (there was a single categorical shift between two results, e.g., susceptible to length polymorphism (AFLP) fingerprint analysis. susceptible-dose dependent) (8). VOL. 48, 2010 ANTIFUNGAL DIRECT Etest ON BLOOD SAMPLES WITH YEASTS 2207

TABLE 2. Essential agreement between ETdir, CLSI, and ETsd for the MICs of six antifungal agents

Ϯ b Essential agreement (% of MICs within 2 log2) No. of Species a Procedure Fluconazole strains Voriconazole Posaconazole Isavuconazole Amphotericin B Caspofungin (24 h/48 h) C. albicans 81 ETdir vs CLSIc 44.4/71.6 76.6 61.7 79.1 39.5 83.9 ETdir vs ETsdd 88.9 80.2 59.3 79 83.9 86.4 ETsd vs CLSIc 72.8/69 87.7 87.7 92.6 69.1 90.1

C. glabrata 66 ETdir vs CLSIc 84.8/60.6 86.4 25.7 45.5 77.3 97 ETdir vs ETsdd 81.8 84.8 63.6 71.2 50 98.5 ETsd vs CLSIc 51.5/72.7 89.5 44.5 56.1 77.3 95.5

C. parapsilosis 62 ETdir vs CLSIc 75.8/90.3 91.9 74.2 93.5 32.3 87.1 ETdir vs ETsdd 92 93.5 87.2 92 70.9 100 ETsd vs CLSIc 46.6/85.5 96.8 77.4 95.2 64.5 88.7

C. krusei 31 ETdir vs CLSIc 10/83.9 96.8 90.3 77.4 64.5 93.5 ETdir vs ETsdd 96.8 93.6 96.8 100 96.8 100 ETsd vs CLSIc 10/71 96.8 93.5 100 67.7 87.1

C. tropicalis 17 ETdir vs CLSIc 64.8/58.8 52.9 58.8 29.4 47.1 82.4 ETdir vs ETsdd 88.2 64.7 58.8 52.9 88.2 94.1 ETsd vs CLSIc 64.7/82.3 76.5 76.5 70.6 94.1 88.2

Candida spp.e 6 ETdir vs CLSIc 66.6/66.6 66.6 50 66.7 16.7 83.3 ETdir vs ETsdd 66.6 83.3 83.3 83.3 66.6 83.3 ETsd vs CLSIc 83.3/66.6 83.3 50 66.7 66.7 100

Mixedf 4 ETdir vs CLSIc 75/75 100 100 100 25 50 ETdir vs ETsdd 50 75 50 75 100 75 ETsd vs CLSIc 75/100 100 50 50 75 100

Cryptococcus spp. 30 ETdir vs CLSIc 96.7g 100 16.7 90 50 100 ETdir vs ETsdd 96.3 90 93.3 90 36.7 96.7 ETsd vs CLSIc 90 93.3 43.3 86.7 0 96.7

Other yeastsh 31 ETdir vs CLSIc 53.9/64.5 67.7 58 74.1 67.7 87.1 ETdir vs ETsdd 70.9 64.5 62.7 64.5 58.1 90.3 ETsd vs CLSIc 64.5/83.8 74.1 64.5 74.2 64.5 87.1

Total 328 ETdir vs CLSIc 59.2/75 83.5 55.2 72.9 55.5 89.3 ETdir vs ETsdd 86.9 84.1 72.9 80.2 69.5 94.2 ETsd vs CLSIc 63/79.8 89.3 70.9 80 64.3 91.5

a ETdir MICs for Cryptococcus isolates, 1 C. albicans isolate, 4 C. glabrata isolates, 1 C. parapsilosis isolate, 2 R. mucilaginosa isolates, and 13 S. cerevisiae isolates were read after Ն48 h of incubation due to poor fungal growth on RPMI agar plates. b Ϯ Percentages of MICs that fell within 2 log2 of the MICs obtained by the gold standard procedure. c CLSI considered the gold standard. d ETsd considered the gold standard. e Including C. dubliniensis (n ϭ 2), K. marxianus (n ϭ 1), and P. guilliermondii (n ϭ 3). f Mixed fungemia corresponded to four episodes caused by two different species: C. albicans plus C. parapsilosis (n ϭ 2) and C. albicans plus C. glabrata (n ϭ 2). A bottle containing C. albicans and C. parapsilosis was excluded due to bacterial overgrowth (coagulase-negative Staphylococcus). g Due to the absence of growth of Cryptococcus strains, MICs of ﬂuconazole by CLSI M27-A3 after 24 h of incubation were not obtained. h Including S. cerevisiae (n ϭ 15), D. capitatus (n ϭ 9), T. mucoides (n ϭ 3), R. mucilaginosa (n ϭ 2), T. inkin (n ϭ 1), and A. adeninivorans (n ϭ 1).

RESULTS that obtained by CLSI (P Ͻ 0.05). For amphotericin B, the mean MIC obtained by CLSI was higher than that obtained by ETsd Antifungal activity of the agents obtained by the three pro- and ETdir and the ETdir mean MIC was higher than that ob- cedures. The overall antifungal activity (MIC ) of each antifun- 90 tained by ETsd. For the remaining comparisons, including caspo- gal agent determined by the ETdir, ETsd, and CLSI procedures fungin, the differences did not reach statistical significance. was as follows: posaconazole (ETdir/ETsd/CLSI), Ͼ32/8/0.5; isavuconazole, 2/1/0.25; fluconazole, Ͼ256/Ͼ256/32; voriconazole, Table 2 summarizes the essential agreement found between 1/0.5/0.5; amphotericin B, 4/1/4; and caspofungin, Ͼ32/Ͼ32/16. the three procedures. In general, the highest agreement was For posaconazole, isavuconazole, and fluconazole (trays contain- found between ETdir and ETsd. Table 3 shows the percentage ing this drug were interpreted after 24 h and 48 h of incubation), of strains included in each susceptibility category (susceptible, the mean MICs obtained by ETdir or ETsd were significantly susceptible-dose dependent, resistant, or nonsusceptible) for higher than those obtained by CLSI (P Ͻ 0.05). For voriconazole, each antifungal agent. Table 4 summarizes the categorical the mean MIC obtained by ETdir was significantly higher than agreement found between the three procedures compared. 2208 GUINEA ET AL. J. CLIN.MICROBIOL.

TABLE 3. Percentage of isolates included in susceptibility categories by ETdir, CLSI, and ETsd for the MICs of six antifungal agents

% MICa No. of Amphotericin Species Procedure Fluconazole (24 h/48 h) Voriconazole Posaconazole Isavuconazole Caspofungin strains B S SDD R S SDD R S SDD R S SDD R S R S Non S

C. albicans 81 ETdir 100 0 0 100 0 0 100 0 0 100 0 0 83.9 16.1 100 0 CLSI 100/98.8 0/1.2 0/0 100 0 0 100 0 0 100 0 0 33.3 66.7 100 0 ETsd 100 0 0 100 0 0 100 0 0 100 0 0 84.6 15.4 100 0

C. glabrata 66 ETdir 34.8 33.3 31.8 89.4 7.6 3 28.8 16.7 54.5 66.7 21.2 12.1 34.8 65.2 100 0 CLSI 92.4/71.2 4.5/21.2 3/7.6 95.5 3 1.5 93.9 4.6 1.5 95.5 4.5 0 9.1 90.9 100 0 ETsd 31.8 43.9 24.2 97 0 3 31.8 18.2 50 86.4 7.6 6.1 92.4 7.6 100 0

C. parapsilosis 62 ETdir 100 0 0 98.4 1.6 0 100 0 0 100 0 0 90.3 9.7 100 0 CLSI 98.4/100 1.6/0 0/0 100 0 0 100 0 0 100 0 0 4.8 95.2 100 0 ETsd 98.4 1.6 0 100 0 0 100 0 0 100 0 0 93.5 6.5 100 0

C. krusei 31 ETdir 3.2 6.5 90.3 96.8 3.2 0 96.8 3.2 0 83.9 16.1 0 74.2 25.8 100 0 CLSI 13.3/6.5 86.7/32.3 0/61.3 100 0 0 100 0 0 96.8 3.2 0 0 100 100 0 ETsd 3.2 6.5 90.3 87.1 9.7 3.2 93.5 6.5 0 83.9 12.9 3.2 87.1 12.9 100 0

C. tropicalis 17 ETdir 88.2 0 11.8 88.2 0 11.8 88.2 0 11.8 76.4 11.8 11.8 82.4 17.6 100 0 CLSI 82.4/82.4 0/0 17.6/17.6 82.4 0 17.6 82.4 0 17.6 88.2 0 11.8 0 100 100 0 ETsd 88.2 0 11.8 88.2 0 11.8 88.2 0 11.8 88.2 0 11.8 76.5 23.5 100 0

Candida spp. 6 ETdir 100 0 0 100 0 0 83.3 0 16.7 83.3 0 16.7 100 0 100 0 CLSI 83.3/83.3 16.7/16.7 0/0 100 0 0 100 0 0 100 0 0 66.7 33.3 100 0 ETsd 83.3 0 16.7 100 0 0 83.3 16.7 0 66.7 16.7 16.7 100 0 100 0

Mixed 4 ETdir 100 0 0 100 0 0 100 0 0 100 0 0 75 25 100 0 CLSI 100/100 0/0 0/0 100 0 0 100 0 0 100 0 0 25 75 100 0 ETsd 50 50 0 100 0 0 50 0 50 75 25 0 75 25 100 0

Cryptococcus 30 ETdir 96.7 3.3 0 100 0 0 100 0 0 100 0 0 100 0 0 100 CLSI 93.3b 6.7b 0b 100 0 0 100 0 0 100 0 0 96.7 3.3 0 100 ETsd 86.7 13.3 0 100 0 0 100 0 0 100 0 0 100 0 3.3 96.7

Other yeasts 31 ETdir 54.8 9.7 35.5 83.9 0 16.1 51.6 9.7 38.7 87.1 3.2 9.7 53.3 46.7 54.8 45.2 CLSI 92.3/71 0/19.4 7.7/9.6 96.8 3.2 0 80.6 19.4 0 100 0 0 13.3 86.7 54.8 45.2 ETsd 77.4 3.2 19.4 93.5 0 6.5 61.3 9.7 29 96.8 3.2 0 90 10 51.6 48.4

Total 328 ETdir 72.6 8.5 18.9 95.2 2.1 2.7 79.9 4.7 15.5 89 6.7 4.3 73.1 26.9 86.6 13.4 CLSI 87.3/80.5 10.3/10.4 2.4/9.1 97.9 0.9 1.2 96 2.8 1.2 98.2 1.2 0.6 22.6 77.4 86.6 13.4 ETsd 72 11.9 16.2 97 0.9 2.1 80.5 5.5 14 93.9 3.7 2.4 93 7 86.6 13.4

a Percentages of ETdir, CLSI, and ETsd MICs that were within the breakpoints chosen for ﬂuconazole (Յ8, susceptible ͓S͔; 16–32, susceptible-dose dependent ͓SDD͔; Ն64, resistant ͓R͔), voriconazole (Յ1, S; 2, SDD; Ն4, R), posaconazole and isavuconazole (same breakpoints as those used for voriconazole), amphotericin B(Յ1, S; Ն2, R), and caspofungin (Յ2, S; Ͼ2, nonsusceptible ͓non S͔). b Due to the absence of growth of Cryptococcus strains, MICs of ﬂuconazole by CLSI M27-A3 after 24 h of incubation were not obtained.

Agreement between ETdir and CLSI for the six agents stud- isolate of C. albicans (ETdir indicated susceptible and CLSI ied. For fluconazole, with few exceptions, essential agreement indicated susceptible-dose dependent). Very major errors for between the ETdir and CLSI was higher when MICs were fluconazole, voriconazole, and posaconazole were detected in determined after 48 h of incubation. When C. krusei was ex- one isolate of C. tropicalis. Excluding a very major error found cluded from the analysis, the overall essential agreement found in C. krusei (ETdir MIC ϭ 16 ␮g/ml; CLSI MIC ϭ 64 ␮g/ml), was as follows: ETdir versus CLSI, 66.3% and 72.6% (for MICs the remaining major errors occurred only in C. glabrata or obtained after 24 h and 48 h of incubation, respectively); ETdir other yeasts. Taking into account the intrinsic fluconazole re- versus ETsd, 86.5%; and ETsd versus CLSI, 69.1% and 79.3% sistance of C. krusei, ETdir and ETsd detected fluconazole (for MICs obtained after 24 h and 48 h of incubation, respec- resistance in 90.3% of the isolates. tively). Agreement was near 100% for yeast species without Among the azoles, ETdir and CLSI showed the highest fluconazole breakpoints proposed (e.g., Cryptococcus). Over- essential agreement for voriconazole. This was particularly all, CLSI tended to classify a higher number of isolates as high for species showing reduced or intrinsic fluconazole re- susceptible. However, the percentages observed with the three sistance (C. glabrata and C. krusei). A categorical agreement of methods were comparable for species showing low azole resis- 94.3% was found between ETdir and CLSI. Errors for C. tance (C. albicans, C. parapsilosis, and C. tropicalis). For flu- glabrata were mostly minor. Only two very major errors were conazole, comparisons of ETdir/ETsd with CLSI yielded a detected in a fluconazole-resistant C. glabrata isolate (correctly lower number of errors when MICs were read at 48 h. An classified by ETdir as resistant) and in another isolate of C. 83.5% categorical agreement was found between ETdir and tropicalis. For C. parapsilosis, errors were minor in only one CLSI, and most errors were minor, especially for C. glabrata. isolate (ETdir indicated susceptible-dose dependent and CLSI We found only a minor error between ETdir and CLSI in one indicated susceptible). TABLE 4. Very major, major, and minor errors found between ETdir and CLSI, ETdir and ETsd, and CLSI and ETsd for the MICs of six antifungal agents

% of errors by typea No. of Fluconazole (24 h/48 h) Voriconazole Posaconazole Isavuconazole Amphotericin B Caspofungin Species Procedure strains Very Very Very Very Very Very Minor Major Minor Major Minor Major Minor Major Major Major major major major major major major

C. albicans 81 ETdir and CLSIb 0/1.2 0/0 0/0 0 0 0 0 0 0 0 0 0 53.1 2.5 0 0 ETdir and ETsdc 0 0 0 0 0 0 0 0 0 0 0 0 13.6 0 0 0 ETsd and CLSIb 0/1.2 0/0 0/0 0 0 0 0 0 0 0 0 0 0 64.2 0 0

C. glabrata 66 ETdir and CLSIb 37.9/48.5 0/0 24.2/9.1 10.7 1.5 3 21.2 0 48.5 21.2 0 10.6 4.5 30.3 0 0 ETdir and ETsdc 34.8 0 1.5 9.1 1.5 1.5 31.8 9.1 10.6 28.8 0 7.6 59.1 1.5 0 0 ETsd and CLSIb 48.5/57.6 0/0 18.2/7.6 3 0 1.5 22.7 0 43.9 9.1 0 3 1.5 84.8 0 0

C. parapsilosis 62 ETdir and CLSIb 0/0 0/0 0/0 1.5 0 0 0 0 0 0 0 0 1.6 87.1 0 0 ETdir and ETsdc 1.6 0 0 1.6 0 0 0 0 0 0 0 0 9.7 6.5 0 0 ETsd and CLSIb 0/0 0/0 0/0 0 0 0 0 0 0 0 0 0 0 88.7 0 0

C. krusei 31 ETdir and CLSIb 90/32.2 0/0 6.7/3.2 3.2 0 0 3.2 0 0 19.3 0 0 74.2 25.8 0 0 ETdir and ETsdc 6.5 0 0 12.9 3.2 0 9.7 0 0 22.6 3.2 0 3.2 16.2 0 0 ETsd and CLSIb 90.3/3.2 0/0 3.2/0 9.7 0 3.2 6.5 0 0 16.1 3.2 0 0 87.1 0 0

C. tropicalis 17 ETdir and CLSIb 0/0 5.9/5.9 0/0 0 5.9 0 0 5.9 0 11.8 0 0 0 82.3 0 0 ETdir and ETsdc 0 0 0 0 0 0 0 0 0 11.8 0 0 11.8 5.9 0 0 ETsd and CLSIb 0/0 5.9/5.9 0/0 0 5.9 0 0 5.9 0 0 0 0 0 76.5 0 0

Candida spp.d 6 ETdir and CLSIb 16.7/16.7 0/0 0/0 0 0 0 0 0 16.7 0 0 16.7 0 33.3 0 0 ETdir and ETsdc 0 16.7 0 0 0 0 0 0 0 16.7 0 0 0 0 0 0 ETsd and CLSIb 0/0 0/0 0/0 0 0 0 16.7 0 0 16.7 0 16.7 0 33.3 0 0

Mixede 4 ETdir and CLSIb 0/0 0/0 0/0 0 0 0 0 0 0 0 0 0 0 50 0 0 ETdir and ETsdc 50 0 0 0 0 0 0 50 0 25 0 0 0 0 0 0 ETsd and CLSIb 50/50 0/0 0/0 0 0 0 0 0 50 25 0 0 0 50 0 0

Cryptococcus 30 ETdir and CLSIb 10f 0f 0f 00 0 0 0 0 00 0 0 3.300 ETdir and ETsdc 16.7 0 0 0 0 0 0 0 0 0 0 0 0 0 3.3 0 ETsd and CLSIb 19.4f 0f 0f 0 0 0 0 0 0 0 0 0 0 3.3 0 3.3

Other yeastsg 31 ETdir and CLSIb 3.8/9.7 0/0 26.9/22.6 3.2 0 16.1 16.1 0 29 3.2 0 9.7 6.6 46.7 0 0 ETdir and ETsdc 12.9 0 22.6 0 0 9.8 12.9 3.2 12.9 6.5 0 9.7 40 3.3 0 3.2 ETsd and CLSIb 3.8/22.6 0/0 7.7/0 3.2 0 6.5 29 0 9.7 3.2 0 0 0 74.4 3.2 0

Total 328 ETdir and CLSIb 18.2/15.2 0.3/0.3 8.6/4.3 3 0.6 2.1 6.1 0.3 12.8 7.3 0 3.4 24.5 52.9 0 0 ETdir and ETsdc 11.3 0.6 2.4 3 0.6 1.3 8.8 2.7 3.4 9.8 0.3 2.4 22.6 2.8 0.4 0.4 ETsd and CLSIb 19.2/18.6 0.3/0.3 4.5/1.5 3 0.6 2.1 8.2 0.3 10.4 4.7 0 12.2 0.3 70.7 0.3 0.3

a Errors were categorized as very major (ETdir indicated susceptible and gold standard indicated resistant), major (ETdir indicated resistant and gold standard indicated susceptible), or minor (there was a single categorical shift between two results, e.g., susceptible to susceptible-dose dependent). b CLSI considered the gold standard. c ETsd considered the gold standard. d Including C. dubliniensis (n ϭ 2), K. marxianus (n ϭ 1), and P. guilliermondii (n ϭ 3). e Mixed fungemia corresponded to four episodes caused by two different species: C. albicans plus C. parapsilosis (n ϭ 2) and C. albicans plus C. glabrata (n ϭ 2). A bottle containing C. albicans and C. parapsilosis was excluded due to bacterial overgrowth (coagulase-negative Staphylococcus). f Due to the absence of growth of Cryptococcus strains, MICs of ﬂuconazole by CLSI M27-A3 after 24 h of incubation were not obtained. g Including S. cerevisiae (n ϭ 15), D. capitatus (n ϭ 9), T. mucoides (n ϭ 3), R. mucilaginosa (n ϭ 2), T. inkin (n ϭ 1), and A. adeninivorans (n ϭ 1). . 48, 2010 ANTIFUNGAL DIRECT Etest ON BLOOD SAMPLES WITH YEASTS 2209 OL V 2210 GUINEA ET AL. J. CLIN.MICROBIOL.

Essential agreement between ETdir and CLSI was especially ples can provide results in 24 to 48 h. In 2001, Chang et al. poor for posaconazole, with C. glabrata showing the lowest compared the ETdir with the NCCLS macrodilution proce- values. The essential agreement between ETdir and CLSI for dure in positive blood cultures for five antifungal agents. How- isavuconazole was also low for C. glabrata. For this species, ever, the recent incorporation of the echinocandins and the essential agreement for posaconazole and isavuconazole was new triazoles, together with the development of new break- moderately improved when the ETsd was chosen as the gold points, means that the procedure should be reevaluated (6). standard. The CLSI M27-A3 document has not proposed We studied the ETdir results for posaconazole, isavucon- breakpoints for posaconazole or isavuconazole. Using the azole, fluconazole, voriconazole, amphotericin B, and caspo- same breakpoints as for voriconazole, a high proportion of C. fungin. The highest essential agreement was found for caspo- glabrata strains crept from the category of susceptible (CLSI) fungin and the lowest for posaconazole and amphotericin B. In to resistant (ETdir or ETsd) for posaconazole. Errors for the categorical analysis, the highest correlation was also found posaconazole and isavuconazole were found mostly for C. gla- for caspofungin and the lowest for amphotericin B. Errors brata and several other yeasts. between ETdir and CLSI for azoles were mostly minor. The lowest agreement between ETdir and CLSI was found False susceptibility (very major errors) was detected for for amphotericin B, even when the two Etest procedures were azoles in only two (0.6%) isolates. One C. tropicalis isolate compared. The overall essential agreement was 55.5%, and showed very major errors for fluconazole, posaconazole, and this rose above 80% for only three species. The CLSI M27-A3 voriconazole but not for isavuconazole. C. tropicalis frequently document has not proposed breakpoints for amphotericin B. exhibits azole trailing, and this may interfere with the inter- When susceptibility was defined as an MIC of Յ1 ␮g/ml (7), pretation of MICs (3). In this strain, the 24-hour CLSI MICs of both Etest procedures tended to classify a higher number of the other azoles led to the same susceptibility category. The strains as susceptible than the CLSI. Amphotericin B was the other very major error occurred in a C. glabrata isolate for drug for which the ETdir showed the worst categorical agree- which ETdir indicated susceptibility to voriconazole but resis- ment with the reference procedures (Etest standard or CLSI). tance to fluconazole (CLSI indicated resistance for both The overall categorical agreement was improved when agents), thus revealing the potential of the ETdir to detect the ETsd was chosen as the gold standard (69.5% versus fluconazole-resistant isolates of C. glabrata. Isavuconazole, a 55.5%), with the exception of C. glabrata (39.3%) and other new triazole under phase III evaluations for the treatment of yeasts (58.1%). Agreement was especially low for Cryptococcus candidemia, has a favorable pharmacokinetic profile and an spp. MIC distribution comparable to that of voriconazole (12, 13). Caspofungin was the drug for which ETdir and CLSI showed When the voriconazole breakpoints were used, no very major the highest values of essential and categorical agreement. Both errors were found between the results of ETdir and CLSI for methods presented high degrees of essential agreement isavuconazole. Both essential and categorical agreements be- (Ͼ90%), especially for C. glabrata and C. krusei. The three tween ETdir and CLSI for caspofungin were excellent, indi- procedures were equivalent in terms of classifying isolates as cating the ability of the ETdir to correctly detect isolates that susceptible. No errors were detected between ETdir and CLSI. are susceptible or nonsusceptible to caspofungin, which is widely used for the treatment of candidemia. The MICs ob- DISCUSSION tained by the ETdir for our strains were within the limit of very major errors (Յ1.5%) proposed by the FDA (10), indicating We used the ETdir on blood samples to determine the the high potential of this procedure to screen for the presence antifungal susceptibility of yeasts causing fungemia, and we of azole-resistant or caspofungin-nonsusceptible strains. compared this procedure with the reference microdilution pro- False resistance (major errors) was detected in 46 (14%) cedure CLSI M27-A3 and the Etest standard. Although the isolates for the three azoles (in 23 [7%] after posaconazole was ETdir overestimated azole resistance, it proved to be a rapid, excluded) and in 75 (22.9%) isolates for amphotericin B. The easy, and reliable procedure for the preliminary determination ETdir of posaconazole yielded a higher number of major er- of susceptibility to azoles and caspofungin of yeasts causing rors than those of the remaining azoles, especially in the case fungemia. ETdir is comparable to the CLSI M27-A3 proce- of C. glabrata, Candida spp., and other yeasts. The results dure (7) and results can be obtained in less than 24 h after obtained using ETdir with posaconazole must be interpreted visualization of the yeast in the Gram stain. with caution, as there are no established breakpoints for this Antifungal susceptibility testing of yeast isolates from blood agent and this procedure overestimated antifungal resistance, is a standard of care in many tertiary hospitals. Clinicians can especially in C. glabrata. use this information to optimize the treatment of patients with Excluding C. glabrata, Candida spp., and other yeasts, the fungemia (22, 23). The CLSI M27-A3 procedure is the refer- remaining species did not yield major errors for azoles. How- ence method for antifungal susceptibility testing of yeasts, al- ever, the FDA proposed a limit of Յ3% for major errors. It though it has some limitations, since it is time-consuming and must be emphasized that the ETdir overestimated resistance to labor-intensive and since results are not available until 24 to azoles and that this may lead to the use of broad-spectrum 48 h after pure-culture isolation of the fungal pathogen (7, 18). antifungal agents for empirical treatment (10). However, it is The Etest, an easy-to-perform procedure, can partially com- important to note that the ETdir overestimated the azole re- pensate for these limitations. The ETsd has proven to have a sistance for C. glabrata, although it is still unknown whether good correlation with the CLSI procedure (9, 16, 26). How- azoles should be used for the treatment of episodes caused by ever, the MIC is read after 48 h of incubation and has been this species. Few errors were found for highly prevalent species standardized using pure-culture isolates. ETdir on blood sam- (i.e., C. albicans and C. parapsilosis); ETdir performed partic- VOL. 48, 2010 ANTIFUNGAL DIRECT Etest ON BLOOD SAMPLES WITH YEASTS 2211 ularly well for caspofungin, which is widely used for the treat- 6. Chang, H. C., J. J. Chang, S. H. Chan, A. H. Huang, T. L. Wu, M. C. Lin, and ment of candidemia. Preliminary ETdir results should be con- T. C. Chang. 2001. Evaluation of Etest for direct antifungal susceptibility testing of yeasts in positive blood cultures. J. Clin. Microbiol. 39:1328–1333. firmed using a standard procedure, such as CLSI M27-A3. 7. Clinical and Laboratory Standards Institute. 2008. Reference method for Antifungal susceptibility testing for amphotericin B is con- broth dilution antifungal susceptibility testing of yeasts. Approved standard CLSI document M27-A3. Clinical and Laboratory Standards Institute, troversial. Microdilution procedures usually yield a narrower Wayne, PA. distribution of MICs than agar-based methods (19). The Etest 8. Espinel-Ingroff, A., E. Cantoń, J. Pemań, M. G. Rinaldi, and A. W. has proven to be more sensitive and reliable than CLSI for Fothergill. 2009. Comparison of 24-hour and 48-hour voriconazole MICs as determined by the Clinical and Laboratory Standards Institute broth mi- detection of resistance to amphotericin B in Candida (20). We crodilution method (M27-A3 document) in three laboratories: results ob- compared the ETdir of amphotericin B with the Etest standard tained with 2,162 clinical isolates of Candida spp. and other yeasts. J. Clin. procedure. Very major errors were detected in 9 (2.7%) iso- Microbiol. 47:2766–2771. 9. Espinel-Ingroff, A., L. Steele-Moore, and J. N. Galgiani. 1994. Evaluation of lates, and this percentage was above 1.5% for C. parapsilosis, 80% inhibition standards for the determination of fluconazole minimum C. krusei, C. tropicalis, and other yeasts. In the absence of inhibitory concentrations in three laboratories. Diagn. Microbiol. Infect. Dis. 20:81–86. consensus for an appropriate method for amphotericin B, 10. Food and Drug Administration. 2003. Class II special controls guidance these results must be interpreted with caution and the use of document: antimicrobial susceptibility testing systems: guidance for industry. ETdir with amphotericin B cannot be recommended. Food and Drug Administration, Washington, DC. 11. Garey, K. W., M. Rege, M. P. Pai, D. E. Mingo, K. J. Suda, R. S. Turpin, and No major or very major errors were detected in C. albicans D. T. Bearden. 2006. Time to initiation of fluconazole therapy impacts or C. parapsilosis, species that could account for 75% of cases mortality in patients with candidemia: a multi-institutional study. Clin. In- of candidemia (1). However, a potential shortcoming of our fect. Dis. 43:25–31. 12. Guinea, J., and E. Bouza. 2008. Isavuconazole: a new and promising anti- study is the low number of azole-resistant or caspofungin- fungal triazole for the treatment of invasive fungal infections. Future Mi- nonsusceptible strains of C. albicans, C. parapsilosis, and C. crobiol. 3:603–615. 13. Guinea, J., T. Pelaéz, S. Recio, M. Torres-Narbona, and E. Bouza. 2008. In tropicalis. vitro antifungal activities of isavuconazole (BAL4815), voriconazole, and We conclude that the ETdir for fluconazole, voriconazole, fluconazole against 1,007 isolates of Zygomycete, Candida, Aspergillus, isavuconazole, and caspofungin has the potential to provide a Fusarium, and Scedosporium species. Antimicrob. Agents Chemother. 52: 1396–1400. rapid preliminary antifungal susceptibility result in yeast iso- 13a.Guinea, J., S. Recio, P. Escribano, T. Pelaéz, M. Torres-Narbona, M. Ro- lates from patients with fungemia in lieu of data obtained using dríguez-Creíxems, C. Sańchez-Carrillo, and E. Bouza. 2010. Antifungal sus- the standard CLSI M27-A3 procedure. ETdir is a useful tool to ceptibility of yeasts isolated from patients with fungaemia: comparison of the E-test on direct blood samples and CLSI M27-A3. Abstr. 20th Conf. Eur. screen for the presence of azoles and caspofungin-nonsuscep- Congr. Clin. Microbiol. Infect. Dis., Vienna, Austria, abstr. P-838. tible strains. Future prospective evaluations of the use of the 14. Krcmery, V., and A. J. Barnes. 2002. Non-albicans Candida spp. causing fungaemia: pathogenicity and antifungal resistance. J. Hosp. Infect. 50:243– ETdir in the clinical setting are warranted to determine the 260. role of this procedure in the optimization of the antifungal 15. Lagrou, K., J. Verhaegen, W. E. Peetermans, T. De Rijdt, J. Maertens, and treatment of patients with fungemia. E. Van Wijngaerden. 2007. Fungemia at a tertiary care hospital: incidence, therapy, and distribution and antifungal susceptibility of causative species. Eur. J. Clin. Microbiol. Infect. Dis. 26:541–547. ACKNOWLEDGMENTS 16. Matar, M. J., L. Ostrosky-Zeichner, V. L. Paetznick, J. R. Rodriguez, E. Chen, and J. H. Rex. 2003. Correlation between E-test, disk diffusion, and We thank Thomas O’Boyle for editing and proofreading the article. microdilution methods for antifungal susceptibility testing of fluconazole and This study does not present any conflicts of interest for its authors. voriconazole. Antimicrob. Agents Chemother. 47:1647–1651. This study was partially financed by grants from Basilea Pharmaceu- 17. Morrell, M., V. J. Fraser, and M. H. Kollef. 2005. Delaying the empiric tica International Ltd., Basel, Switzerland. Etest strips of isavucon- treatment of Candida bloodstream infection until positive blood culture azole were kindly supplied by Basilea Pharmaceutica. Etest strips of results are obtained: a potential risk factor for hospital mortality. Antimi- amphotericin B, fluconazole, voriconazole, posaconazole, and caspo- crob. Agents Chemother. 49:3640–3645. fungin were kindly supplied by AB Biodisk (Solna, Sweden). 18. Ostrosky-Zeichner, L., J. H. Rex, M. A. Pfaller, D. J. Diekema, B. D. Alex- Jesu´s Guinea (CP09/00055) and Marta Torres-Narbona (CM08/ ander, D. Andes, S. D. Brown, V. Chaturvedi, M. A. Ghannoum, C. C. Knapp, D. J. Sheehan, and T. J. Walsh. 2008. Rationale for reading flucon- 00277) are contracted by the Fondo de Investigacioń Sanitaria (FIS). azole MICs at 24 hours rather than 48 hours when testing Candida spp. by the CLSI M27-A2 standard method. Antimicrob. 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