Candida isolates causing candidemia show different degrees of virulence in Galleria mellonella.

We aim to assess intra- and interspecies differences in the virulence of Candida spp. strains causing candidemia using the invertebrate Galleria mellonella model. We studied 739 Candida spp. isolates (C. albicans [n = 373], C. parapsilosis [n = 203], C. glabrata [n = 92], C. tropicalis [n = 53], and C. krusei [n = 18]) collected from patients with candidemia admitted to Gregorio Marañon Hospital (Madrid, Spain). Species-specific infecting inocula (yeast cells/larva) were adjusted (5 × 105 [C. albicans, and C. tropicalis], 2 × 106-5 × 106 [C. parapsilosis, C. glabrata, and C. krusei]) and used to infect 10 larvae per isolate; percentage of survival and median survival per isolate were calculated. According to the interquartile range of the median survival, isolates with a median survival under P25 were classified as of high-virulence and isolates with a median survival over P75 as of low virulence. The median survival of larvae infected with different species was variable: C. albicans (n = 2 days, IQR <1-3 days), C. tropicalis (n = 2 days, IQR 1.5-4 days), C. parapsilosis (n = 2 days, IQR 2-3.5 days), C. glabrata (n = 3 days, IQR 2-3 days), and C. krusei (n = 7 days, 6.5->8 days) (P < .001). Differences in virulence among species were validated by histological examination (day +1 post-infection) in the larvae infected by the isolates of each virulence category and species. Virulence-related gene expression in C. albicans isolates did not reach statistical significance. We report species-specific virulence patterns of Candida spp. and show that isolates within a given species have different degrees of virulence in the animal model.

Persistent Candidemia in adults: underlying causes and clinical significance in the antifungal stewardship era.

To investigate the causes and the clinical significance of persistent candidemia (PC) in adults diagnosed in a tertiary hospital with an active antifungal stewardship program. Retrospective cohort including all adults with candidemia from 2010 to 2018. PC was defined as any positive follow-up blood culture (BC) obtained ≥ 5 days from the first BCs yielding the same Candida species. PC was detected in 35/255 (13.7%) patients. There were no differences regarding antifungal adequacy in PC vs. non-PC (94.3% vs. 82.3%, p = 0.084) and primary source control (63.3% vs. 76.4%, p = 0.172) at the time of the follow-up BCs. The average time until source control (2 [0-37] vs. 2 days [0-44], p = 0.311) or adequate antifungal treatment (2 [0-26] vs. 2 days [- 2-10], p = 0.748) was similar. Patients with PC had more non-ocular complications (31.4% vs. 10.5%, p = 0.002). No impact on 30-day mortality was observed (31.4% vs. 22.3%, p = 0.238). The only independent factor associated with PC was to have a previously undetected site of infection [OR 4.28, 95%CI (1.77-10.34), p = 0.001]. Persistent candidemia was not associated with inadequate or delayed therapeutic management, nor higher 30-day mortality rates. Timely screening and control of unexpected infection sources are encouraged to shorten hospitalization and improve patient care.

Does the composition of polystyrene trays affect Candida spp. biofilm formation?

The biofilm formation ability of Candida species seems to have a role in the prognosis of patients with candidemia. Biofilm formation is usually tested using 96 well flat bottom polystyrene microtiter plates, although the type of plastic used is not commonly reported. This study compares biofilm formation by Candida spp. on six types of plates from three brands (three non-tissue-treated and three tissue-treated). Thirty isolates of each of the following species were selected: C. albicans, C. parapsilosis, C. glabrata, C. tropicalis, as well as 15 isolates of C. krusei (n = 135 isolates) from patients with candidemia. Biofilm production was evaluated by measuring biomass production and metabolic activity. Our results show higher biomass production and metabolic activity of biofilms formed on non-tissue-treated plates in comparison to those formed on tissue-treated plates (P < .001). We only found significant differences in metabolic activity of biofilms formed on non-tissue-treated plates (P < .003). All comparisons including biofilm formation and metabolic activity among plates of the same brand yielded higher biofilm formation on non-treated plates compared to treated plates (P < .001). Significant difference in biomass production by C. parapsilosis was only seen when comparing between the various tissue-treated plastics (P < .03). In contrast, comparisons of different non-tissue-treated tray brands yielded significant metabolic activity differences for all species except for C. parapsilosis (P < .05). Biofilm formation and metabolic activity is significantly affected by the plastic composition of non-tissue-treated trays leading to increased biofilm formation.

Resistance to Echinocandins in Candida Can Be Detected by Performing the Etest Directly on Blood Culture Samples.

We examined the rapid evaluation of susceptibility to echinocandins in Candida spp. using the Etest performed directly on positive blood cultures and anidulafungin-containing agar plates. We prospectively collected 80 positive blood cultures (Bactec-FX system, Becton-Dickinson, Cockeysville, MD, USA) with echinocandin-susceptible Candida spp. (n = 60) and echinocandin-intermediate Candida parapsilosis (n = 20) from patients with candidemia. Additionally, blood culture bottles of nonfungemic/bacteremic patients were spiked with 35 echinocandin-resistant Candida species isolates. A total of 2 to 4 drops of medium from each bottle were stroked directly onto both RPMI 1640 agar plates with micafungin and anidulafungin Etest strips (ETDIR) and Sabouraud agar plates containing 2 mg/liter of anidulafungin. The isolates were tested according to the EUCAST method and Etest standard (ETSD). Essential and categorical agreement between the methods was calculated. The essential agreement and categorical agreement between the EUCAST method and ETDIR and ETSD were both >97.4%. The essential agreement between ETDIR and the EUCAST method for both echinocandins was >97%. The categorical agreement between the FKS sequence and ETDIR was 97.4%. The ETDIR MICs of anidulafungin and micafungin (≥0.19 mg/liter and ≥0.064 mg/liter, respectively) effectively separated all susceptible FKS wild-type isolates from the resistant FKS mutant isolates. The categorical agreement (62.6%) between the EUCAST method and growth on anidulafungin-containing plates was poor, with the best agreement observed for Candida glabrata (94.2%). When performed directly on positive blood cultures from patients with candidemia, the Etest with micafungin and anidulafungin is a reliable procedure for the rapid testing of susceptibility to echinocandins in Candida species isolates.

Mutant Prevention Concentration and Mutant Selection Window of Micafungin and Anidulafungin in Clinical Candida glabrata Isolates.

We report the mutant prevention concentration (MPC) and mutant selection window (MSW) for micafungin and anidulafungin administered to treat Candida glabrata We also determine the mutation frequency. We studied 20 echinocandin-susceptible, fluconazole-intermediate, and FKS wild-type C. glabrata isolates. Adjusted inocula were stroked directly onto Sabouraud agar plates containing different concentrations of micafungin or anidulafungin and visually inspected daily for up to 5 days of incubation. Individual colonies growing on the plates containing echinocandins at 1 mg/liter were selected for antifungal susceptibility testing. The FKS genes of the resulting individual phenotypically resistant colonies were sequenced, and the MPC, MSW, and mutation frequency were determined. Biofilm was quantified, and the growth kinetics and virulence (Galleria mellonella model) of the resulting individual FKS mutant colonies were studied. For micafungin and anidulafungin, we found similar results for the MPC (0.06 to 2 mg/liter and 0.25 to 2 mg/liter, respectively), MSW (0.015 to 2 mg/liter for both echinocandins), and mutation frequency (3.7 × 10-8 and 2.8 × 10-8, respectively). A total of 12 isolates were able to grow at 1 mg/liter on echinocandin-containing plates, yielding a total of 32 phenotypically resistant colonies; however, FKS2 mutations (ΔF658, S663P, W715L, and E655A) were observed only in 21 colonies. We did not find differences in biofilm formation, the kinetic parameters studied, or the median survival of larvae infected by wild-type isolates and the resulting individual FKS2 mutant colonies. Echinocandin concentrations lower than 2 mg/liter can lead to selection of resistance mutations in C. glabrata isolates in vitro.

Low and constant micafungin concentrations may be sufficient to lead to resistance mutations in FKS2 gene of Candida glabrata.

We studied the ability of five echinocandin-susceptible C. glabrata isolates to acquire in vitro resistance to anidulafungin and micafungin. All isolates became phenotypically resistant after 2-4 days of exposure to low and constant micafungin concentrations (P < .05). Mutations in the HS1 region of the FKS2 gene were found in all isolates. The acquisition of resistance was not related to the previous use of antifungal treatment in the patients or the presence of mutations at MSH2 gene. We found differences (P < .0001) in the median survival of Galleria mellonella larvae infected with FKS2 mutant isolates (5 days) and wild-type isolates (3 days).

Frequency of the Paradoxical Effect Measured Using the EUCAST Procedure with Micafungin, Anidulafungin, and Caspofungin against Candida Species Isolates Causing Candidemia.

We report data on the frequency of the paradoxical effect of echinocandins against Candida spp. (n = 602 incident isolates) using the EUCAST definitive document EDef 7.2 procedure. The paradoxical effect for one or more echinocandins was observed in 16% of the isolates. However, differences between species were found, and the paradoxical effect was more common in Candida tropicalis (P < 0.001). Caspofungin was the drug in which the paradoxical effect was most common, followed by anidulafungin and micafungin (P < 0.001).

The Etest Performed Directly on Blood Culture Bottles Is a Reliable Tool for Detection of Fluconazole-Resistant Candida albicans Isolates.

We assessed the ability of the Etest performed directly on positive blood cultures (ETDIR) to detect fluconazole susceptibility in 6 fluconazole-resistant and 12 fluconazole-susceptible Candida albicans isolates, according to CLSI M27-A3 and EUCAST EDef 7.2 procedures. Categorical agreement between ETDIR and broth microdilution was 100% when the trays were incubated at 25°C and trailing effect was ruled out. ETDIR is a reliable procedure when screening for the presence of fluconazole resistance in C. albicans.

In Vitro Exposure to Increasing Micafungin Concentrations Easily Promotes Echinocandin Resistance in Candida glabrata Isolates.

We assessed the in vitro susceptibility of five echinocandin-susceptible Candida glabrata isolates after exposure to micafungin. The direct exposure to plates at different micafungin concentrations resulted in the inhibition of growth at 0.062 µg/ml. The progressive exposure was performed on plates using 0.031 µg/ml of micafungin and sequential propagation on plates containing the next 2-fold concentration; the MICs of micafungin and anidulafungin increased sequentially, and all the isolates became echinocandin resistant, showing fks2 mutations.

Candida guilliermondii Complex Is Characterized by High Antifungal Resistance but Low Mortality in 22 Cases of Candidemia.

The objectives of our study were to describe the characteristics of patients with Candida guilliermondii candidemia and to perform an in-depth microbiological characterization of isolates and compare them with those of patients with C. albicans candidemia. We described the risk factors and outcomes of 22 patients with candidemia caused by the C. guilliermondii complex. Incident isolates were identified using molecular techniques, and susceptibility to fluconazole, anidulafungin, and micafungin was studied. Biofilm formation was measured using the crystal violet assay (biomass production) and the XTT reduction assay (metabolic activity), and virulence was studied using the Galleria mellonella model. Biofilm formation was compared with that observed for C. albicans The main conditions predisposing to infection were malignancy (68%), immunosuppressive therapy (59%), and neutropenia (18%). Clinical presentation of candidemia was less severe in patients infected by the C. guilliermondii complex than in patients infected by C. albicans, and 30-day mortality was lower in C. guilliermondii patients (13.6% versus 33.9%, respectively; P = 0.049). Isolates were identified as C. guilliermondiisensu stricto (n = 17) and Candida fermentati (n = 5). The isolates produced biofilms with low metabolic activity and moderate biomass. The G. mellonella model showed that C. guilliermondii was less virulent than C. albicans (mean of 6 days versus 1 day of survival, respectively; P < 0.001). Patients with candidemia caused by the C. guilliermondii complex had severe and debilitating underlying conditions. Overall, the isolates showed diminished susceptibility to fluconazole and echinocandins, although poor biofilm formation and the low virulence were associated with a favorable outcome.

The novel oral glucan synthase inhibitor SCY-078 shows in vitro activity against sessile and planktonic Candida spp.

Objectives: We studied the antifungal activity of SCY-078 (an orally bioavailable 1,3-ß -d- glucan synthesis inhibitor), micafungin and fluconazole against the planktonic and sessile forms of 178 Candida and non- Candida isolates causing fungaemia in patients recently admitted to a large European hospital. Methods: The in vitro activity of SCY-078, micafungin and fluconazole against the planktonic form of the isolates was assessed using EUCAST EDef 7.3 and CLSI M27-A3. Antibiofilm activity was assessed using the XTT reduction assay. Results: SCY-078 and micafungin showed potent in vitro activity against Candida and non- Candida isolates. The in vitro activity of both drugs was similar, but SYC-078 displayed significantly lower MIC values than micafungin against Candida parapsilosis and non- Candida isolates, whereas micafungin displayed significantly lower MIC values for the remaining species ( P <0.001). In contrast, SCY-078 and micafungin showed essentially the same activity against the biofilms with the exception of Candida glabrata , in which the micafungin sessile MIC values were significantly lower ( P <0.001). These observations were confirmed by assessing biofilm structure by scanning electron microscopy after antifungal treatment. Conclusions: Our study showed that the high in vitro activity of SCY-078 against invasive Candida isolates in both sessile and planktonic forms is comparable to that of micafungin.

Biofilm Production and Antibiofilm Activity of Echinocandins and Liposomal Amphotericin B in Echinocandin-Resistant Yeast Species.

The echinocandins and liposomal amphotericin B are active against biofilm produced by echinocandin-susceptible Candida strains. However, few data have been reported on the production of biofilm by echinocandin-resistant isolates and their antifungal susceptibility. We studied the production of biofilm by fks mutant Candida strains and intrinsically echinocandin-resistant non-Candida isolates and the susceptibility of both entities to liposomal amphotericin B and echinocandins. We analyzed the production of biofilm by isolates from patients with fungemia (fks mutant Candida, n = 5; intrinsically echinocandin-resistant non-Candida, n = 12; and Candida wild type, n = 10). Biofilm formation was measured to classify strains according to biomass (crystal violet assay) and metabolic activity (XTT reduction assay). Preformed biofilms were tested against liposomal amphotericin B, caspofungin, micafungin, and anidulafungin. The sessile MIC was defined as the antifungal concentration yielding a 50% or 80% reduction in the metabolic activity of the biofilm compared to that of the growth control (SMIC50 and SMIC80, respectively). fks mutant Candida isolates formed biofilms in a fashion similar to that of Candida wild-type strains. The echinocandins had the highest activity against biofilms formed by wild-type Candida isolates, followed by fks mutant Candida isolates and non-Candida isolates. Liposomal amphotericin B had the highest activity against fks mutant Candida biofilms. The formation of biofilm by echinocandin-resistant strains was similar to that of wild-type strains, although resistance to echinocandins remained high.

Comparison of the antifungal activity of micafungin and amphotericin B against Candida tropicalis biofilms.

OBJECTIVES: Candida tropicalis is the fourth most common cause of candidaemia in hospitalized patients and associated mortality is high. C. tropicalis frequently causes biofilm-related infections. Echinocandins and amphotericin B show potent in vitro activity against C. albicans biofilms, but their activity against C. tropicalis biofilms has received little attention. METHODS: We studied production of biofilm by 54 C. tropicalis isolates from blood and the antifungal susceptibility of these isolates to micafungin, amphotericin B and liposomal amphotericin B. Biofilm production was measured using the crystal violet assay to determine biomass and the XTT reduction assay to determine metabolic activity. The antifungal susceptibility of planktonic and sessile cells was measured using the EUCAST EDef 7.2 procedure and XTT reduction assay, respectively. The sessile MIC endpoint of SMIC80 was defined as an 80% reduction in the metabolic activity of the biofilm treated with the antifungal compared with the control well. RESULTS: The three drugs were very active against the isolates in planktonic form, with micafungin showing the highest activity (P < 0.001). Micafungin was the most active agent against C. tropicalis biofilms (P < 0.001). In contrast, liposomal amphotericin B showed poor antifungal activity. CONCLUSIONS: Micafungin was the most active drug against C. tropicalis biofilm. Although the echinocandins and liposomal amphotericin B are considered very active against Candida spp. biofilms, this is not true for C. tropicalis, as liposomal amphotericin B showed poor antifungal activity against biofilms.

Susceptibility of Candida albicans biofilms to caspofungin and anidulafungin is not affected by metabolic activity or biomass production.

Micafungin is more active against biofilms with high metabolic activity; however, it is unknown whether this observation applies to caspofungin and anidulafungin and whether it is also dependent on the biomass production. We compare the antifungal activity of anidulafungin, caspofungin, and micafungin against preformed Candida albicans biofilms with different degrees of metabolic activity and biomass production from 301 isolates causing fungemia in patients admitted to Gregorio Marañon Hospital (January 2007 to September 2014). Biofilms were classified as having low, moderate, or high metabolic activity according XTT reduction assay or having low, moderate, or high biomass according to crystal violet assay. Echinocandin MICs for planktonic and sessile cells were measured using the EUCAST E.Def 7.2 procedure and XTT reduction assay, respectively. Micafungin showed the highest activity against biofilms classified according to the metabolic activity and biomass production (P < .001). The activity of caspofungin and anidulafungin was not dependent on the metabolic activity of the biofilm or the biomass production. These observations were confirmed by scanning electron microscopy. None of the echinocandins produced major changes in the structure of biofilms with low metabolic activity and biomass production when compared with the untreated biofilms. However, biofilm with high metabolic activity or high biomass production was considerably more susceptible to micafungin; this effect was not shown by caspofungin or anidulafungin.

Scope and frequency of fluconazole trailing assessed using EUCAST in invasive Candida spp. isolates.

Trailing is a well-known phenomenon that is defined as reduced but persistent visible growth of Candida spp. at fluconazole concentrations above the MIC. Trailing is commonly detected using the CLSI M27-A3 method, although little is known about its frequency when investigated with EUCAST. We assessed the frequency and scope of fluconazole trailing after using EUCAST EDef 7.2. against a large number of Candida spp. isolates from patients with candidemia. We studied 639 fluconazole-susceptible non-krusei Candida spp. isolates from 570 patients admitted to Gregorio Marañón Hospital. Isolates were tested in vitro for fluconazole susceptibility according to the EUCAST EDef 7.2 procedure; trailing was defined as the presence of any residual growth in wells containing fluconazole concentrations above the MIC. According to the mean percentage of trailing observed, isolates were classified as residual trailers (0.1-5%), slight trailers (6%-10%), moderate trailers (11%-15%), and heavy trailers (>15%). The relationship between trailing and genotyping was assessed. The mean overall percentage of trailing was 6.8%, with C. albicans and C. tropicalis showing the highest percentages (9.75% and 9.29%, respectively; P < .001). C. albicans and C. tropicalis had the highest percentage of heavy trailers (>15%). Trailing was not genotype-specific. Fluconazole trailing was observed frequently when EUCAST was used for antifungal susceptibility testing, particularly in isolates of C. albicans and C. tropicalis The cut-off proposed enabled us to classify the isolates according to the degree of trailing and can be used as the basis for future studies to evaluate the clinical impact of this phenomenon.

Therapeutic drug monitoring of voriconazole helps to decrease the percentage of patients with off-target trough serum levels.

We monitored trough voriconazole serum concentrations from 107 patients (n = 258 samples) at 6 hospitals in Madrid. Most of the patients were male (67%) and had the following underlying conditions: hematological cancer (42%), solid organ transplantation (15%), chronic obstructive pulmonary disease (14%), human immunodeficiency virus infection (8.4%), solid cancer (5.6%), and other (29%). The indication for voriconazole administration was aspergillosis treatment (74.6%) and prophylaxis (14%). The main reasons for voriconazole trough drug monitoring were initiation of treatment/prophylaxis (33%), patient monitoring (47%), and suspected toxicity (3.5%). Levels (µg/ml) were subtherapeutic (<1; 18.2%), on-target (1-5.5; 71.3%), and high (>5.5; 10.5%). The samples percentage with on-target levels was significantly lower for the first sample than for subsequent samples (62.6% vs. 77.5%). "Subsequent samples," "admission in nonpediatric wards," "voriconazole used for treatment of invasive aspergillosis," and "use of proton pump inhibitors" were predictors of voriconazole therapeutic levels (≥1 µg/ml).

Sputum and bronchial secretion samples are equally useful as bronchoalveolar lavage samples for the diagnosis of invasive pulmonary aspergillosis in selected patients.

In the absence of histopathology studies of lung biopsies, the bronchoalveolar lavage (BAL) sample is preferred for the diagnosis of invasive pulmonary aspergillosis. Isolation of Aspergillus fumigatus from sputum and bronchial secretion samples are commonly interpreted as colonization or laboratory contamination, particularly in nonneutropenic patients. We studied if sputum/bronchial secretions and BAL samples are equally useful for the diagnosis of invasive pulmonary aspergillosis. We retrospectively selected 14 patients with proven (n = 1) or probable (n = 13) invasive pulmonary aspergillosis from whose samples A. fumigatus had been simultaneously isolated in BAL and sputum/bronchial secretions between 2006 and 2012. The isolates were identified by sequencing the ß-tubulin gene and genotyped using the STRAf assay. Matches between BAL and sputum/bronchial secretions were observed in patients with identical genotypes in BAL and sputum/bronchial secretions. All patients had clinically suspected pneumonia, before the diagnosis of invasive pulmonary aspergillosis. The sample from which A. fumigatus was initially isolated was collected as a result of the presence of fever (50%), abnormal radiological findings (100%), and/or pneumonia that did not respond to antibiotics (36%). The underlying conditions varied, although the most common predisposing factors were hematological malignancies (21.5%) and COPD (43%). In 13 of the 14 patients (93%), we found matching genotypes in the BAL and the sputum/bronchial secretion samples. Genotyping showed that samples of sputum or bronchial secretions were equally useful as samples of BAL for the diagnosis of invasive pulmonary aspergillosis.

Antifungal resistance to fluconazole and echinocandins is not emerging in yeast isolates causing fungemia in a Spanish tertiary care center.

Accurate knowledge of fungemia epidemiology requires identification of strains to the molecular level. Various studies have shown that the rate of resistance to fluconazole ranges from 2.5% to 9% in Candida spp. isolated from blood samples. However, trends in antifungal resistance have received little attention and have been studied only using CLSI M27-A3 methodology. We assessed the fungemia epidemiology in a large tertiary care institution in Madrid, Spain, by identifying isolates to the molecular level and performing antifungal susceptibility testing according to the updated breakpoints of European Committee for Antimicrobial Susceptibility Testing (EUCAST) definitive document (EDef) 7.2. We studied 613 isolates causing 598 episodes of fungemia in 544 patients admitted to our hospital (January 2007 to December 2013). Strains were identified after amplification and sequencing of the ITS1-5.8S-ITS2 region and further tested for in vitro susceptibility to amphotericin B, fluconazole, posaconazole, voriconazole, micafungin, and anidulafungin. Resistance was defined using EUCAST species-specific breakpoints, and epidemiological cutoff values (ECOFFs) were applied as tentative breakpoints. Most episodes were caused by Candida albicans (46%), Candida parapsilosis (28.7%), Candida glabrata (9.8%), and Candida tropicalis (8%). Molecular identification enabled us to better detect cryptic species of Candida guilliermondii and C. parapsilosis complexes and episodes of polyfungal fungemia. The overall percentage of fluconazole-resistant isolates was 5%, although it was higher in C. glabrata (8.6%) and non-Candida yeast isolates (47.4%). The rate of resistance to echinocandins was 4.4% and was mainly due to the presence of intrinsically resistant non-Candida species. Resistance mainly affected non-Candida yeasts. The rate of resistance to fluconazole and echinocandins did not change considerably during the study period.

Micafungin is more active against Candida albicans biofilms with high metabolic activity.

BACKGROUND: The ability to form biofilm enables Candida spp. to cause catheter-related candidaemia. The use of agents with in vitro activity against Candida albicans biofilms, such as micafungin, could obviate catheter removal. The metabolic activity of C. albicans biofilms is strain-dependent, and cell wall formation is thought to be more prominent in biofilms showing high metabolic activity. METHODS: We studied the antifungal activity of micafungin against 265 C. albicans isolates with different degrees of metabolic activity causing fungaemia in 246 patients admitted to Gregorio Marañón Hospital (January 2007 to June 2013). All strains were classified according to the metabolic activity of their biofilm, which was classified as low, moderate and high using XTT. Micafungin MICs for planktonic and sessile cells were assessed using the EUCAST E.Def 7.2 procedure and XTT reduction assay, respectively. The MIC was defined as a 50% and 80% reduction in metabolic activity compared with the control well. RESULTS: Micafungin was uniformly more active against planktonic cells than against sessile cells (MIC50 ≤ 0.015 versus 8 mg/L), although it was not consistently active against all C. albicans biofilms. Isolates with low metabolic activity biofilms showed the lowest susceptibility to micafungin, followed by moderate and high metabolic activity biofilms (P < 0.001). CONCLUSIONS: Our study suggests that the metabolic activity of biofilm may have a role in future evaluations of micafungin for the eradication of C. albicans biofilms (e.g. the lock therapy approach).

Production of biofilm by Candida and non-Candida spp. isolates causing fungemia: comparison of biomass production and metabolic activity and development of cut-off points.

Biofilm production in Candida spp. can be studied by measuring the biomass produced after application of crystal violet stain or by measuring metabolic activity with XTT. Our study is the first in which crystal violet and XTT are compared to analyze the ability of clinically relevant Candida and non-Candida species to produce biofilm. We studied 577 isolates causing fungemia in 512 patients admitted from January 2007 to July 2013. Based on the biomass production measured by crystal violet and the metabolic activity measured by XTT, strains were divided into terciles to establish tentative cut-offs to classify isolates as being low, moderate, or high biofilm-forming and as having low, moderate, or high metabolic activity. Considerable variability in biofilm production and metabolic activity was found both between species and within species. C. tropicalis showed the highest biomass production, whereas C. glabrata showed the highest metabolic activity, and non-Candida species isolates showed the lowest metabolic activity (P<0.0023). The isolates were classified as low metabolic activity, moderate metabolic activity, and high metabolic activity according to their cut-offs by XTT (<0.097, 0.097-0.2, and >0.2) and as low biofilm-forming, moderate biofilm-forming, and high biofilm-forming according to their cut-offs by crystal violet (<0.44, 0.44-1.17, and >1.17). The overall categorical agreement between the procedures was 43.7%, which increased to >50% for C. albicans and C. parapsilosis. XTT and crystal violet are complementary procedures for the study of biofilm production.