Laura Judith Marcos-Zambrano

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.

Antifungal Resistance to Fluconazole and Echinocandins Is Not Emerging in Yeast Isolates Causing Fungemia in a Spanish Tertiary Care Center

ABSTRACT 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).

Micafungin at Physiological Serum Concentrations Shows Antifungal Activity against Candida albicans and Candida parapsilosis Biofilms

ABSTRACT We assessed the in vitro activity of micafungin against preformed Candida biofilms by measuring the concentration of drug causing the most fungal damage and inhibition of regrowth. We studied 37 biofilm-producing Candida spp. strains from blood cultures. We showed that micafungin was active against planktonic and sessile forms of Candida albicans strains and moderately active against Candida parapsilosis sessile cells. Concentrations of micafungin above 2 μg/ml were sufficiently high to inactivate regrowth of Candida sessile cells.

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.

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 β-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.

Is Biofilm Production a Predictor of Catheter-Related Candidemia?

Catheter-related candidemia (CRC) is typically a biofilm related disease, but it is mostly unknown if the production of biofilm is a feature exclusively shown by Candida spp. isolates causing CRC. We performed an in vitro biofilm assay using Candida isolates obtained from the blood of patients with candidemia. We demonstrated that biofilm production was not a good predictor of catheter-related candidemia. Also, we demonstrated that there was no difference in the mortality of candidemia patients infected by biofilm-forming isolates and those in which the infection is caused by nonbiofilm-forming species.

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.

Comparison between the EUCAST Procedure and the Etest for Determination of the Susceptibility of Candida Species Isolates to Micafungin

ABSTRACT We compared the ability of the EUCAST EDef 7.2 and the Etest to detect the susceptibility to micafungin of 160 Candida and non-Candida clinical isolates. Agreement was higher when Etest MICs were obtained after 24 h of incubation; essential agreement was 90%, and categorical agreement was >90%. False susceptibility was seen only for Candida krusei (10%), and false resistance was observed in 6% of the isolates, ranging from 2.6% (C. glabrata) to 13% (C. albicans).

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.