We describe a large series of patients with chronic obstructive pulmonary disease (COPD) and probable invasive pulmonary aspergillosis (IPA), and the risk factors and incidence of the disease in patients with isolation of Aspergillus from lower respiratory tract samples. From 2000 to 2007, we retrospectively studied all patients admitted with COPD and isolation of Aspergillus (239; 16.3/1000 admissions). Multivariate logistic regression and survival curves were used. Fifty-three patients had probable IPA (3.6 cases of IPA per 1000 COPD admissions). IPA affects at least 22.1% of patients with COPD and isolation of Aspergillus in culture. In 33 of the 53 patients with probable IPA, serum galactomannan was determined; in 14 (42.4%) of these, the result was positive. Five variables were independent predictors of IPA with statistical significance: admission to the intensive-care unit, chronic heart failure, antibiotic treatment received in the 3 months prior to admission, the accumulated dosage of corticosteroids equivalent to >700 mg prednisone received in the 3 months prior to admission, and the similar accumulated dosage of corticosteroids received from admission to the first clinical isolation of Aspergillus. Multivariate analysis gave an area under the curve of 0.925 (95% CI 0.888-0.962; p <0.001). The overall mean survival of the cohort was 64.1% (28.3% for IPA patients and 75.2% for non-IPA patients). The median number of days of survival was 48 (95% CI 33.07-62.92). However, we found statistically significant differences between patients with IPA (29 days; 95% CI 20.59-37.40) and patients without IPA (86 days; 95% CI 61.13-110.86) (log rank, p <0.001).

Use of S. cerevisiae probiotics should be carefully reassessed, particularly in immunosuppressed or critically ill patients.

Among the filamentous (mold) fungal infections, those caused by Aspergillus fumigatus and other Aspergillus spp. are the most common; these infections are associated with high morbidity and mortality, especially in the immunocompromised host (3,7,15,33). The triazoles itraconazole, voriconazole, and posaconazole have a broad spectrum of in vitro activity against molds and are important therapeutic agents for the systemic treatment and prevention of severe mold infections, including aspergillosis (33). Although acquired azole resistance in Aspergillus spp. has been documented since the late 1990s (8), it was uncommon. However, these reports have increased in the last few years, especially in Europe (4, 13, 24, 25, 31). The azoles act by blocking the pathway of ergosterol biosynthesis, specifically the enzymes 14-␣-sterol demethylases A and B. These cytochrome enzymes are encoded by cyp51 (A and B) genes in A. fumigatus and other Aspergillus spp. (19). More importantly, multiazole resistance or cross-resistance has been associated with point mutations in the cyp51A gene in A. fumigatus by substitution of the glycine at position 54 and methionine at position 220 by different amino acids (4, 9, 12), but substitution of L98H and 2 copies of 34 bp in the cyp51A promoter and other mutations also have been identified (13,16,20,25,31). The TR/L98H point mutation has been responsible for an increased level of cyp51A expression (25,29,31), and several mutations have been associated with patient failure on triazole treatment (13, 29, 31); however, other host and drug factors cannot be ignored (e.g., azole bioavailability).The Clinical and Laboratory Standards Institute (CLSI) has developed a reference broth microdilution method for antifungal susceptibility testing of molds (CLSI M38-A2 document) (5). The availability of reference methodologies has enabled the recognition of the complexity of cross-resistance among triazoles (9,18,20,22,24,25) and the proposal for epidemiologic cutoff values (ECVs) for A. fumigatus and itraconazole, posaconazole, and voriconazole by both CLSI (22) and the European Committee of Antibiotic Susceptibility Testing (AFST-EUCAST) (24) methodologies. Clinical breakpoints are not available for mold testing by the CLSI methodology versus any antifungal agent. However, breakpoints based on MIC distributions, pharmacokinetic and pharmacodynamic (PK/PD) parameters, animal data, and clinical experience have been proposed for the EUCAST reference method for A. fumigatus and the three triazoles (30). In the absence of clinical breakpoints, ECVs could help to characterize the susceptibility of Aspergillus isolates to itraconazole, posaconazole, and voriconazole and to monitor the emergence of strains with mutations in the cyp51A gene and/or reduced antifungal triazole

MICs measured using CLSI yeast nitrogen base (YNB) medium instead of CLSI RPMI medium for C. neoformans were evaluated. CLSI RPMI medium ECVs for distributions originating from at least three laboratories, which included >95% of the modeled WT population, were as follows: fluconazole, 8 g/ml (VNI, C. gattii nontyped, VGI, VGIIa, and VGIII), 16 g/ml (C. neoformans nontyped, VNIII, and VGIV), and 32 g/ml (VGII); itraconazole, 0.25 g/ml (VNI), 0.5 g/ml (C. neoformans and C. gattii nontyped and VGI to VGIII), and 1 g/ml (VGIV); posaconazole, 0.25 g/ml (C. neoformans nontyped and VNI) and 0.5 g/ml (C. gattii nontyped and VGI); and voriconazole, 0.12 g/ml (VNIV), 0.25 g/ml (C. neoformans and C. gattii nontyped, VNI, VNIII, VGII, and VGIIa,), and 0.5 g/ml (VGI). The number of laboratories contributing data for other molecular types was too low to ascertain that the differences were due to factors other than assay variation. In the absence of clinical breakpoints, our ECVs may aid in the detection of isolates with acquired resistance mechanisms and should be listed in the revised CLSI M27-A3 and CLSI M27-S3 documents.

A population-based survey was conducted to investigate the epidemiology of and antifungal resistance in Spanish clinical strains of filamentous fungi isolated from deep tissue samples, blood cultures, and respiratory samples. The study was conducted in two different periods (October 2010 and May 2011) to analyze seasonal variations. A total of 325 strains were isolated in 29 different hospitals. The average prevalence was 0.0016/1,000 inhabitants. Strains were identified by sequencing of DNA targets and susceptibility testing by the European Committee for Antimicrobial Susceptibility Testing reference procedure. The most frequently isolated genus was Aspergillus, accounting for 86.3% of the isolates, followed by Scedosporium at 4.7%; the order Mucorales at 2.5%; Penicillium at 2.2%, and Fusarium at 1.2%. The most frequent species was Aspergillus fumigatus (48.5%), followed by A. flavus (8.4%), A. terreus (8.1%), A. tubingensis (6.8%), and A. niger (6.5%). Cryptic/sibling Aspergillus species accounted for 12% of the cases. Resistance to amphotericin B was found in 10.8% of the isolates tested, while extended-spectrum triazole resistance ranged from 10 to 12.7%, depending on the azole tested. Antifungal resistance was more common among emerging species such as those of Scedosporium and Mucorales and also among cryptic species of Aspergillus, with 40% of these isolates showing resistance to all of the antifungal compounds tested. Cryptic Aspergillus species seem to be underestimated, and their correct classification could be clinically relevant. The performance of antifungal susceptibility testing of the strains implicated in deep infections and multicentric studies is recommended to evaluate the incidence of these cryptic species in other geographic areas.

x Although Clinical and Laboratory Standards Institute (CLSI) clinical breakpoints (CBPs) are available for interpreting echinocandin MICs for Candida spp., epidemiologic cutoff values (ECVs) based on collective MIC data from multiple laboratories have not been defined. While collating CLSI caspofungin MICs for 145 to 11,550 Candida isolates from 17 laboratories (Brazil, Canada, Europe, Mexico, Peru, and the United States), we observed an extraordinary amount of modal variability (wide ranges) among laboratories as well as truncated and bimodal MIC distributions. The species-specific modes across different laboratories ranged from 0.016 to 0.5 g/ml for C. albicans and C. tropicalis, 0.031 to 0.5 g/ml for C. glabrata, and 0.063 to 1 g/ml for C. krusei. Variability was also similar among MIC distributions for C. dubliniensis and C. lusitaniae. The exceptions were C. parapsilosis and C. guilliermondii MIC distributions, where most modes were within one 2-fold dilution of each other. These findings were consistent with available data from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (403 to 2,556 MICs) for C. albicans, C. glabrata, C. krusei, and C. tropicalis. Although many factors (caspofungin powder source, stock solution solvent, powder storage time length and temperature, and MIC determination testing parameters) were examined as a potential cause of such unprecedented variability, a single specific cause was not identified. Therefore, it seems highly likely that the use of the CLSI species-specific caspofungin CBPs could lead to reporting an excessive number of wild-type (WT) isolates (e.g., C. glabrata and C. krusei) as either non-WT or resistant isolates. Until this problem is resolved, routine testing or reporting of CLSI caspofungin MICs for Candida is not recommended; micafungin or anidulafungin data could be used instead.

Clostridium difficile is the most frequently identified enteric pathogen in patients with nosocomially acquired, antibiotic-associated diarrhea. The drugs most commonly used to treat diseases associated with C. difficile are metronidazole and vancomycin. Most clinical laboratories assume that all C. difficile isolates are susceptible to metronidazole and vancomycin. We report on the antimicrobial susceptibilities of 415 C. difficile isolates to metronidazole and vancomycin over an 8-year period (1993 to 2000). The overall rate of resistance to metronidazole at the critical breakpoint (16 g/ml) was 6.3%. Although full resistance to vancomycin was not observed, the overall rate of intermediate resistance was 3.1%. One isolate had a combination of resistance to metronidazole and intermediate resistance to vancomycin. Rates of resistance to metronidazole and vancomycin were higher among isolates from human immunodeficiency virus-infected patients. Molecular typing methods proved the absence of clonality among the isolates with decreased susceptibilities to the antimicrobials tested.Clostridium difficile-associated diarrhea (CDAD) is the most common nosocomial diarrhea in adults, occurring mainly in patients with prior antimicrobial therapy. The disease has variable incidences and severities in different hospital populations (2,6,22,25,27,38) The most serious cases of CDAD require antimicrobial therapy with agents which are active against C. difficile. Metronidazole is the drug of choice due to its in vitro activity, its efficacy by either the oral or the intravenous route of administration, its presumed lower potential for selection of vancomycin-resistant Enterococcus (VRE), and the low cost of treatment with the drug. Nowadays, vancomycin is considered a second-line drug, mainly due to the potential for the selection of VRE and its high cost. Both drugs are considered equivalent in efficacy, but infection recurrence rates of 15 to 35% have been reported for both drugs (3, 7).In vitro determination of the susceptibility of C. difficile to these antibiotics is not routinely performed, as it is broadly accepted that C. difficile is regularly and predictably susceptible to metronidazole and vancomycin. The assay method is timeconsuming, and the use of susceptibility breakpoints is based on the therapeutic levels of drugs in serum and not on the levels in the intraluminal area, where higher drug concentrations can be achieved (1,19). At present, metronidazole resistance in C. difficile is considered anecdotal and vancomycin resistance has been, to the best of our knowledge, reported only once (16), although on that occasion the method used was not the present standard.In 1994, we sounded the alarm when we reported on the first series of metronidazole-resistant C. difficile isolates (T. Peláez, R. Sánchez, R. Blázquez, P. Catalán, P. Muñoz, and E. Bouza, Abstr. 34th. Intersci. Conf. Antimicrob. Agents Chemother., abstr. E-34, p. 50, 1994). The aim of the present study is to report on the decrease in the rates of suscept...

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