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UR-9825 is a new broad-spectrum triazole antifungal agent with a good pharmacokinetic profile and excellent bioavailability. It shows high in-vitro activity and efficacy in models of systemic candidosis in rats and rabbits, comparing favourably with fluconazole. The purpose of this study was to evaluate the in-vitro activity of UR-9825 and to compare it with that of fluconazole and itraconazole against 283 clinical isolates of Candida spp. UR-9825 was more potent against Candida spp. than both fluconazole and itraconazole, even against some Candida albicans and Candida krusei isolates with decreased susceptibility to fluconazole (MIC 16 mg/L). [ABSTRACT FROM AUTHOR]

We report the in-vitro susceptibility of 27 clinical isolates of Scedosporium apiospermum and 43 of Scedosporium prolificans. S. apiospermum was resistant to fluconazole and flucytosine, with variable susceptibility to amphotericin B, itraconazole, ketoconazole and susceptible to miconazole. Voriconazole was much more active than fluconazole and flucytosine, more active than amphotericin B, fluaconazole and ketoconazole and was as active as miconazole against S. apiospermum isolates. Voriconazole and the other six antifungal agents showed low activity against S. prolificans isolates. [ABSTRACT FROM PUBLISHER]

Objectives: Cryptococcosis remains a severe global health concern, underscoring the urgent need for rapid and reliable diagnostic solutions. Point-of-care tests (POCTs), such as the cryptococcal antigen semi-quantitative (CrAgSQ) lateral flow assay (LFA), offer promise in addressing this challenge. However, their subjective interpretation poses a limitation. Our objectives encompass the development and validation of a digital platform based on Artificial Intelligence (AI), assessing its semi-quantitative LFA interpretation performance, and exploring its potential to quantify CrAg concentrations directly from LFA images., Methods: We tested 53 cryptococcal antigen (CrAg) concentrations spanning from 0 to 5000 ng/ml. A total of 318 CrAgSQ LFAs were inoculated and systematically photographed twice, employing two distinct smartphones, resulting in a dataset of 1272 images. We developed an AI algorithm designed for the automated interpretation of CrAgSQ LFAs. Concurrently, we explored the relationship between quantified test line intensities and CrAg concentrations., Results: Our algorithm surpasses visual reading in sensitivity, and shows fewer discrepancies (p < 0.0001). The system exhibited capability of predicting CrAg concentrations exclusively based on a photograph of the LFA (Pearson correlation coefficient of 0.85)., Conclusions: This technology's adaptability for various LFAs suggests broader applications. AI-driven interpretations have transformative potential, revolutionizing cryptococcosis diagnosis, offering standardized, reliable, and efficient POCT results., (© 2024. The Author(s).)

Background: A Diagnostic Laboratory Hub (DLH) was set up in Guatemala to provide opportunistic infection (OI) diagnosis for people with HIV (PWH)., Methods: Patients newly presenting for HIV, PWH not receiving antiretrovirals (ARVs) for >90 days but returned to care (Return/Restart), and PWH on ARVs with symptoms of OIs (ARV treatment) were prospectively included. Screening for tuberculosis, nontuberculous mycobacteria (NTM), histoplasmosis, and cryptococcosis was done. Samples were couriered to the DLH, and results were transmitted electronically. Demographic, diagnostic results, disease burden, treatment, and follow-up to 180 days were analyzed., Results: In 2017, 1953 patients were included, 923 new HIV infections (an estimated 44% of all new HIV infections in Guatemala), 701 on ARV treatment, and 315 Return/Restart. Three hundred seventeen (16.2%) had an OI: 35.9% tuberculosis, 31.2% histoplasmosis, 18.6% cryptococcosis, 4.4% NTM, and 9.8% coinfections. Histoplasmosis was the most frequent AIDS-defining illness; 51.2% of new patients had <200 CD4 cells/mm 3 with a 29.4% OI incidence; 14.3% of OIs in new HIV infections occurred with CD4 counts of 200-350 cells/mm 3 . OIs were the main risk factor for premature death for new HIV infections. At 180 days, patients with OIs and advanced HIV had 73-fold greater risk of death than those without advanced disease who were OI-free., Conclusions: The DLH OI screening approach provides adequate diagnostic services and obtains relevant data. We propose a CD4 screening threshold of <350 cells/mm 3 . Mortality remains high, and improved interventions are required, including expansion of the DLH and access to antifungal drugs, especially liposomal amphotericin B and flucytosine., (© The Author(s) 2019. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

The epidemiology of Candida parapsilosis and the closely related species C. orthopsilosis and C. metapsilosis has changed in recent years, justify the need to identify this complex at the species level. In this study we investigate the intergenic spacer 1 (IGS1) of the ribosomal DNA (rDNA) to evaluate the utility of this gene region as a phylogenetic molecular marker and the suitability of a high-resolution melting (HRM) strategy based on this region for identification of members of the C. parapsilosis spp. complex. We sequenced the IGS1 and the internal transcribed spacer (ITS) regions of the rDNA from 33 C. parapsilosis sensu lato strains. Although both regions are useful in identifying species, comparative sequence analysis showed that the diversity in the IGS1 region was higher than in the ITS sequences. We also developed an HRM analysis that reliably identifies C. parapsilosis spp. complex based on the amplification of 70 bp in the IGS1 region. All isolates were correctly identified with a confidence interval >98%. Our results demonstrate that HRM analysis based on the IGS1 region is a powerful tool for distinguishing C. parapsilosis from cryptic species., (© The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The aim of this study was the development and validation of a fast and simple high performance liquid chromatography method for measuring voriconazole in human serum using ravuconazole as an external standard. The experience of the reference laboratory in therapeutic drug monitoring of voriconazole is also reported. This method is based on the precipitation of proteins in human serum and detection by HPLC/UV. Chromatographic separation is achieved using an isocratic solvent delivery with detection at 255 nm and a run time of 7 min. The assay was validated according to international guidelines and was also applied to the analysis of 141 trough serum samples from patients treated with voriconazole. All validation parameters met the criteria set out in FDA guidelines for bioanalytical methods. A high interpatient and intrapatient variability was observed in clinical samples. This method is accurate enough to perform therapeutic drug monitoring in patients receiving voriconazole treatment., (Copyright © 2012 Elsevier España, S.L. All rights reserved.)

In this study we present the results of a therapeutic drug monitoring retrospective analysis involving 14 patients with several underlying diseases who were receiving voriconazole for the treatment of fungal infections. A simple high performance liquid chromatography assay with ultraviolet detection was used in the drug monitoring. We report here that serum concentrations were highly variable and unpredictable in most patients. We also found that lack of response was more frequent in patients with levels persistently lower than 1 mg/l. The number of samples with voriconazole concentrations below 1 mg/l was significantly higher in patients who exhibited therapeutic failures (88% versus 27%; P < 0.001). In addition, the period of time in which voriconazole concentrations were maintained below 1 mg/l was slightly higher in patients in the failure group. We suggest that serum concentration should be individually quantified for patients receiving voriconazole therapy. Further prospective studies are needed to clarify the potential benefit of the individualization of treatment.

A duplex Real Time PCR (RT-PCR) assay for detecting DNA of members of the genus Fusarium has been developed and validated by using two mouse models of invasive infection. The duplex RT-PCR technique employed two specific molecular beacon probes targeting a highly conserved region of the fungal rDNA gene. This technique showed a detection limit of 10 fg DNA per μl of sample and a specificity of 100%. The sensitivity in a total of 48 samples from a murine model of Fusarium solani infection was 93.9% for lung tissues and 86.7% for serum samples. In comparison, the sensitivity in a total of 45 samples of a F. oxysporum murine model infection was 87% for lung tissues and 42.8% for serum samples. This molecular technique could be a reliable method for the quantification and the evaluation of the disease in animal models and for the clinical diagnosis of fusariosis.

The performance of a real-time PCR-based assay was retrospectively analyzed (according to European Organization for Research and Treatment of Cancer/Mycosis Study Group criteria) in the samples of patients with invasive aspergillosis. A total of 711 serial samples (356 whole-blood and 355 serum samples) from 38 adult patients were analyzed. The Aspergillus fumigatus PCR assay results were positive for 89 of 356 (25%) whole-blood samples and 90 of 355 (25.35%) serum samples. Positive PCR results were seen in 29 of 31 (93.5%) patients for which serum was analyzed and in 31 of 33 (93.9%) cases with whole-blood specimens. Both blood and serum samples were available in 26 cases, and significant differences were not observed in this subgroup of cases. The average number of threshold cycles (C(T)) for positive blood samples was 37.6, and the average C(T) for serum was 37.4. The DNA concentration ranged between 2 and 50 fg per μl of sample, with average DNA concentrations of 10.2 and 11.7 fg in positive blood and serum samples, respectively (P > 0.01). The performance of this PCR-based quantitative assay was similar for both serum and blood samples. We recommend serum samples as the most convenient hematological sample to use for Aspergillus DNA quantification when serial determinations are done.

We have developed a two-step method based on high-resolution melting (HRM) that reliably identifies species from the Cryptococcus species complex (Cryptococcus neoformans var. grubii, Cryptococcus neoformans var. neoformans, and Cryptococcus gattii). Our results indicate that HRM can provide a fast protocol to identify and distinguish among the main Cryptococcus species.

Candida krusei is a fungal pathogen of interest for the scientific community for its intrinsic resistance to fluconazole. Little is known about the interaction of this yeast with host immune cells. In this work, we have characterized the outcome of the interaction between C. krusei and murine macrophages. Once C. krusei was internalized, we observed different phenomena. In a macrophage-like cell line, C. krusei survived in a significant number of macrophages and induced filamentation and macrophage explosion. Phagocytosis of C. krusei led to actin polymerization around the yeast cells at the site of entry. Fluorescent specific staining with anti-Lamp1 and LysoTracker indicated that after fungal internalization, there was a phagolysosome maturation defect, a phenomenon that was more efficient when the macrophages phagocytosed killed yeast cells. Using cell line macrophages, we also observed macrophage fusion after cell division. When we used primary resident peritoneal macrophages in addition to macrophage explosion, we also observed a strong chemotaxis of uninfected macrophages to regions where C. krusei-infected macrophages were present. We also noticed yeast transfer phenomena between infected macrophages. Primary macrophages inhibited pseudohypha elongation more efficiently than the macrophage-like cell line, suggesting that C. krusei infection was better controlled by the former macrophages. Primary macrophages induced more tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) than the macrophage-like cell line. Our results demonstrate that C. krusei can exploit the macrophages for replication, although other different outcomes are also possible, indicating that the interaction of this pathogen with phagocytic cells is very complex and regulated by multiple factors.

We studied the effects of Amphotericin B (AmB) on Cryptococcus neoformans using different viability methods (CFUs enumeration, XTT assay and propidium iodide permeability). After 1h of incubation, there were no viable colonies when the cells were exposed to AmB concentrations ≥ 1 mg/L. In the same conditions, the cells did not become permeable to propidium iodide, a phenomenon that was not observed until 3h of incubation. When viability was measured in parallel using XTT assay, a result consistent with the CFUs was obtained, although we also observed a paradoxical effect in which at high AmB concentrations, a higher XTT reduction was measured than at intermediate AmB concentrations. This paradoxical effect was not observed after 3h of incubation with AmB, and lack of XTT reduction was observed at AmB concentrations higher than 1mg/L. When stained with dihydrofluorescein, AmB induced a strong intracellular oxidative burst. Consistent with oxidative damage, AmB induced protein carbonylation. Our results indicate that in C. neoformans, Amphotericin B causes intracellular damage mediated through the production of free radicals before damage on the cell membrane, measured by propidium iodide uptake., (Copyright © 2011 Institut Pasteur. Published by Elsevier SAS. All rights reserved.)

Invasive fungal infections (IFIs) caused by filamentous fungi still have high rates of mortality, associated with difficulties in early detection of the infection and therapeutic limitations. Consequently, a useful approach is to prevent patients at risk of fungal infection from coming into contact with conidia of Aspergillus and other mould species. This document describes the recommendations for preventing IFI caused by filamentous fungi worked out by Spanish experts from different medical and professional fields. The article reviews the incidence of IFI in different risk populations, and questions related to environmental measures for prevention, control of hospital infections, additional procedures for prevention, prevention of IFI outside of hospital facilities and antifungal prophylaxis are also analysed., (© 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.)

We report the case of a 72-year-old female renal transplant recipient with a nodular lesion in the distal phalange of the third left finger produced by a dematiaceous fungus that was identified as Phomopsis longicolla. She was treated with itraconazole and terbinafine and later with voriconazole, without response. The patient underwent a surgical resection with lesion-free edge and continued on voriconazole. One year later she was asymptomatic and had not developed new lesions., (© 2010 John Wiley & Sons A/S.)

Nonfermentative yeasts, such as Cryptococcus spp., have emerged as fungal pathogens during the last few years. However, standard methods to measure their antifungal susceptibility (antifungal susceptibility testing [AST]) are not completely reliable due to the impaired growth of these yeasts in standard media. In this work, we have compared the growth kinetics and the antifungal susceptibilities of representative species of nonfermentative yeasts such as Cryptococcus neoformans, Cryptococcus gattii, Cryptococcus albidus, Rhodotorula spp., Yarrowia lipolytica, Geotrichum spp., and Trichosporon spp. The effect of the growth medium (RPMI medium versus yeast nitrogen base [YNB]), glucose concentration (0.2% versus 2%), nitrogen source (ammonium sulfate), temperature (30°C versus 35°C), shaking, and inoculum size (10(3), 10(4), and 10(5) cells) were analyzed. The growth rate, lag phase, and maximum optical density were obtained from each growth experiment, and after multivariate analysis, YNB-based media demonstrated a significant improvement in the growth of yeasts. Shaking, an inoculum size of 10(5) CFU/ml, and incubation at 30°C also improved the growth kinetics of organisms. Supplementation with ammonium sulfate and with 2% glucose did not have any effect on growth. We also tested the antifungal susceptibilities of all the isolates by the reference methods of the CLSI and EUCAST, the EUCAST method with shaking, YNB under static conditions, and YNB with shaking. MIC values obtained under different conditions showed high percentages of agreement and significant correlation coefficient values between them. MIC value determinations according to CLSI and EUCAST standards were rather complicated, since more than half of isolates tested showed a limited growth index, hampering endpoint determinations. We conclude that AST conditions including YNB as an assay medium, agitation of the plates, reading after 48 h of incubation, an inoculum size of 10(5) CFU/ml, and incubation at 30°C made MIC determinations easier without an overestimation of MIC values.

The present article is an update of the literature on fungemia in onco-hematologic patients. A multidisciplinary group of Spanish physicians with an interest in this field selected the most important papers published lately. Papers from the fields of epidemiology, risk factors, pathogenesis, diagnosis, outcome, prevention and treatment are discussed. Important aspects of these studies include the assessment of different strategies in the management of fever in neutropenic patients. Moreover, early identification of patients at risk of fungal infections, as well as identification of patients at risk for fluconazole-resistant strains are topics of increasing interest., (Copyright © 2011 Elsevier España S.L. All rights reserved.)

We have studied infection of Cryptococcus neoformans in the non-vertebrate host Galleria mellonella with particular interest in the morphological response of the yeast. Inoculation of C. neoformans in caterpillars induced a capsule-independent increase in haemocyte density 2 h after infection. C. neoformans manifested a significant increase in capsule size after inoculation into the caterpillar. The magnitude of capsule increase depended on the temperature, being more pronounced at 37°C than at 30°C, which correlated with an increased virulence of the fungus and reduced phagocytosis at 37°C. Capsule enlargement impaired phagocytosis by haemocytes. Incubation of the yeast in G. mellonella extracts also resulted in capsule enlargement, with the polar lipidic fraction having a prominent role in this effect. During infection, the capsule decreased in permeability. A low proportion of the cells (<5%) recovered from caterpillars measured more than 30 µm and were considered giant cells. Giant cells recovered from mice were able to kill the caterpillars in a manner similar to regular cells obtained from in vivo or grown in vitro, establishing their capacity to cause disease. Our results indicate that the morphological transitions exhibited by C. neoformans in mammals also occur in a non-vertebrate host system. The similarities in morphological transitions observed in different animal hosts and in their triggers are consistent with the hypothesis that the cell body and capsular responses represent an adaptation of environmental survival strategies to pathogenesis.

Background: Histoplasmosis and paracoccidioidomycosis (PCM) have increased in Spain in recent years, due firstly to the migration from endemic regions and secondly to travelers returning from these regions. In non-endemic areas, diagnosis of both diseases is hampered by the lack of experience, long silent periods, and the resemblance to other diseases such as tuberculosis and sarcoidosis., Methods: A total of 39 cases of imported histoplasmosis and 6 cases of PCM diagnosed in the Spanish Mycology Reference Laboratory since 2006 were analyzed. Microbiological diagnosis was performed using classical methods and also a specific real-time polymerase chain reaction (RT-PCR) assay for each microorganism., Results: We had 9 cases of probable histoplasmosis in travelers and 30 cases in immigrants, 29 of whom were defined as proven. Paracoccidioidomycosis (PCM) cases were either immigrants or people who had lived for a long period of time in endemic regions, all of whom were classified as proven cases. Cultures showed a good sensitivity in detecting Histoplasma capsulatum in immigrants with proven histoplasmosis (73%); however, growth was very slow. The fungus was never recovered in traveler patients. Paracoccidioides brasiliensis was isolated in a culture only in one case of the proven PCM. Serological methods were not very reliable in immunocompromised patients with histoplasmosis (40%). A PCR-based technique for histoplasmosis detected 55.5% of the cases in travelers (probable cases) and 89% of the cases in immigrants (proven). The PCR method for PCM detected 100% of the cases., Conclusions: These kinds of mycoses are increasingly frequent in non-endemic areas, and newer and faster techniques should be used to reach an early diagnosis. The RT-PCR techniques developed appear to be sensitive, specific, and fast and could be helpful to detect those mycoses. However, it is also essential that physicians perform differential diagnosis in individuals coming from endemic areas., (© 2010 International Society of Travel Medicine.)

These guidelines are an update of recommendations for the diagnosis of invasive fungal infections by the Spanish Society of Clinical Microbiology and Infectious Diseases (SEIMC) published in 2004 (Enferm Infecc Microbiol Clin. 2004, 22:32-9). In this updated version of the guidelines, a comprehensive review of the most recent diagnostic innovations and levels of evidence to recommend those diagnostic procedures are included. We first analyse conventional diagnostic methods, microscopic examination and culture, underlining their limitations which have led to the development of alternative methods, such as fungal antigen and DNA quantification. Those alternative methods of diagnosis are analysed by fungal infection. We also briefly review the methods for molecular identification of fungal species and recommendations for carrying out susceptibility tests for antifungal drugs, including reference procedures, commercial techniques and their indications., (Copyright © 2010 Elsevier España, S.L. All rights reserved.)

Although Candida albicans (CA) is the most common cause of Candida bloodstream infections (BSIs), recent studies have observed an increasing percentage of candidaemias caused by non-albicans Candida species (NAC). In the present study, we attempted to identify the predictors of candidaemia due to NAC compared to CA. We analyzed data from an active population-based surveillance in Barcelona (Spain) from January 2002 to December 2003. Factors associated with NAC fungaemia were determined by multivariate analysis. A total of 339 episodes of Candida BSI, in 336 patients (median age 63 years, interquartile range: 41-72 years), were included. CA was the most commonly isolated (52%), followed by Candida parapsilosis (23%), Candida tropicalis (10%), Candida glabrata (8.6%), Candida krusei (3.4%) and other NAC spp. (3%).Overall, 48% of cases were due to NAC spp. Multivariate logistic regression analysis identified factors associated with a risk of BSI due to NAC spp.: having received a haematologic transplant (OR 10.8; 95% CI 1.31-90.01; p 0.027), previous fluconazole exposure (OR 4.47; 95% CI 2.12-9.43; p <0.001) and neonatal age (OR 4.42; 95% CI 1.63-12.04; p 0.004). Conversely, previous CA colonization (OR 0.33; 95% CI 0.19-0.57; p 0.001) and previous antibiotic use (OR 0.42; 95% CI 0.21-0.85; p 0.017) were associated with CA fungaemia compared to NAC. In conclusion, NAC candidaemia comprised 48% of cases in our series. Predictors of NAC include having received a haematologic transplant, neonatal age and previous fluconazole use., (© 2010 The Authors. Journal Compilation © 2010 European Society of Clinical Microbiology and Infectious Diseases.)

The interaction between fungal pathogens with the host frequently results in morphological changes, such as hyphae formation. The encapsulated pathogenic fungus Cryptococcus neoformans is not considered a dimorphic fungus, and is predominantly found in host tissues as round yeast cells. However, there is a specific morphological change associated with cryptococcal infection that involves an increase in capsule volume. We now report another morphological change whereby gigantic cells are formed in tissue. The paper reports the phenotypic characterization of giant cells isolated from infected mice and the cellular changes associated with giant cell formation. C. neoformans infection in mice resulted in the appearance of giant cells with cell bodies up to 30 microm in diameter and capsules resistant to stripping with gamma-radiation and organic solvents. The proportion of giant cells ranged from 10 to 80% of the total lung fungal burden, depending on infection time, individual mice, and correlated with the type of immune response. When placed on agar, giant cells budded to produce small daughter cells that traversed the capsule of the mother cell at the speed of 20-50 m/h. Giant cells with dimensions that approximated those in vivo were observed in vitro after prolonged culture in minimal media, and were the oldest in the culture, suggesting that giant cell formation is an aging-dependent phenomenon. Giant cells recovered from mice displayed polyploidy, suggesting a mechanism by which gigantism results from cell cycle progression without cell fission. Giant cell formation was dependent on cAMP, but not on Ras1. Real-time imaging showed that giant cells were engaged, but not engulfed by phagocytic cells. We describe a remarkable new strategy for C. neoformans to evade the immune response by enlarging cell size, and suggest that gigantism results from replication without fission, a phenomenon that may also occur with other fungal pathogens.

The EUCAST and the CLSI have established different breakpoints for fluconazole and Candida spp. However, the reference methodologies employed to obtain the MICs provide similar results. The aim of this work was to apply supervised classification algorithms to analyze the clinical data used by the CLSI to establish fluconazole breakpoints for Candida infections and to compare these data with the results obtained with the data set used to set up EUCAST fluconazole breakpoints, where the MIC for detecting failures was >4 mg/liter, with a sensitivity of 87%, a false-positive rate of 8%, and an area under the receiver operating characteristic (ROC) curve of 0.89. Five supervised classifiers (J48 and CART decision trees, the OneR decision rule, the naïve Bayes classifier, and simple logistic regression) were used to analyze the original cohort of patients (Rex's data set), which was used to establish CLSI breakpoints, and a later cohort of candidemia (Clancy's data set), with which CLSI breakpoints were validated. The target variable was the outcome of the infections, and the predictor variable was the MIC or dose/MIC ratio. For Rex's data set, the MIC detecting failures was >8 mg/liter, and for Clancy's data set, the MIC detecting failures was >4 mg/liter, in close agreement with the EUCAST breakpoint (MIC > 4 mg/liter). The sensitivities, false-positive rates, and areas under the ROC curve obtained by means of CART, the algorithm with the best statistical results, were 52%, 18%, and 0.7, respectively, for Rex's data set and 65%, 6%, and 0.72, respectively, for Clancy's data set. In addition, the correlation between outcome and dose/MIC ratio was analyzed for Clancy's data set, where a dose/MIC ratio of >75 was associated with successes, with a sensitivity of 93%, a false-positive rate of 29%, and an area under the ROC curve of 0.83. This dose/MIC ratio of >75 was identical to that found for the cohorts used by EUCAST to establish their breakpoints (a dose/MIC ratio of >75, with a sensitivity of 91%, a false-positive rate of 10%, and an area under the ROC curve of 0.90).

Invasive fungal infections (IFI) due to filamentous fungi still have high rates of mortality associated with the difficulties of early detection of the infection and their therapeutic limitations. Consequently, a useful approach is to prevent patients at risk of fungal infection from getting in contact with conidia of Aspergillus and other mould species. This document describes the recommendations to prevent IFI due to filamentous fungi, prepared by Spanish experts from different medical and professional fields. The paper reviews the incidence of the IFI in different risk populations and the questions related to environmental measures of prevention, control of hospital infections, additional procedures for prevention, prevention of IFI outside hospitals, as well as antifungal prophylaxis., (Copyright 2009 Elsevier España, S.L. All rights reserved.)

Susceptibility testing of fungi and development of interpretative breakpoints has become increasingly important due to the growing incidence of invasive fungal infections, the number and classes of antifungals, and the emerging reports of acquired resistance. The subcommittee on antifungal susceptibility testing of the European Committee on Antibiotic Susceptibility Testing (EUCAST) has developed standards for susceptibility testing of fermentative yeasts and molds as well as proposing breakpoints for fluconazole and voriconazole against Candida. The aim of this work is to describe the EUCAST process of setting breakpoints for antifungals. Five aspects are evaluated during the process of developing breakpoints: 1) the most common dosage used in each European country, 2) the definition of the wild-type population for each target microorganism at the species level and the determination of epidemiological cutoffs, 3) the drug's pharmacokinetics and 4) pharmacodynamics, including Monte Carlo simulations, and 5) the correlation of MICs with clinical outcome of patients treated with the compound. When insufficient data are available (e.g., due to lack of information on the clinical outcome of infections caused by isolates with an elevated MIC), epidemiological cutoff values, rather than breakpoints, are recommended until the necessary information becomes available., (Copyright 2010 Prous Science, S.A.U. or its licensors. All rights reserved.)

Abstract Infections caused by rarely encountered fungal pathogens have increased in recent decades. The present study describes a disseminated infection caused by Phialemonium curvatum, and reviews the literature in an effort to summarize prior experiences with this unusual pathogen. The clinical and microbiological characteristics of a new case due to Phialemonium are presented. The case is analysed with 19 other which have appeared in the literature since 1986. Ten cases were sporadic infections, while the others were associated with three small outbreaks. In all but our patient the skin's natural barrier was compromised (15/20 [75%]) and immunosuppression was a factor in the majority of cases (14/20 [70%]). Dissemination was noted in 83% (5/6) of the immunocompetent patients and in 57% (8/14) of immunocompromised patients. Endocarditis was the most frequent form of infection (8/20 [40%]). Blood cultures were positive in 92% (12/13) of those with disseminated disease. The mortality rate was 54% (7/13) among those with disseminated infections, but fatal outcomes were not observed in patients receiving treatment with itraconazole, voriconazole or posaconazole. The in vitro susceptibility of Phialemonium indicated a more consistent level of activity for voriconazole and posaconazole. Although infections usually occur when there is a breakdown in the skin the skin barrier or host defences are weakened, our case points out that infections due to Phialemonium species may occur in patients without these risk factors. The most frequent form of Phialemonium infections is endovascular, often with endocarditis and positive blood cultures, associated with high mortality rates. Treatment with the new triazoles is associated with improved survival.

To date, no reference standard for therapy for zygomycosis has been established because there are insufficient clinical data with which to make such a judgement. Knowledge of the species responsible for the infection and its antifungal susceptibility profile has become increasingly important in the management of patients. Amphotericin B is the most active drug against all the species involved, followed by posaconazole, whereas voriconazole has no activity. Echinocandins are completely inactive in vitro, but may be an interesting option when used in combination with other drugs.

European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints classify Candida strains with a fluconazole MIC < or = 2 mg/liter as susceptible, those with a fluconazole MIC of 4 mg/liter as representing intermediate susceptibility, and those with a fluconazole MIC > 4 mg/liter as resistant. Machine learning models are supported by complex statistical analyses assessing whether the results have statistical relevance. The aim of this work was to use supervised classification algorithms to analyze the clinical data used to produce EUCAST fluconazole breakpoints. Five supervised classifiers (J48, Correlation and Regression Trees [CART], OneR, Naïve Bayes, and Simple Logistic) were used to analyze two cohorts of patients with oropharyngeal candidosis and candidemia. The target variable was the outcome of the infections, and the predictor variables consisted of values for the MIC or the proportion between the dose administered and the MIC of the isolate (dose/MIC). Statistical power was assessed by determining values for sensitivity and specificity, the false-positive rate, the area under the receiver operating characteristic (ROC) curve, and the Matthews correlation coefficient (MCC). CART obtained the best statistical power for a MIC > 4 mg/liter for detecting failures (sensitivity, 87%; false-positive rate, 8%; area under the ROC curve, 0.89; MCC index, 0.80). For dose/MIC determinations, the target was >75, with a sensitivity of 91%, a false-positive rate of 10%, an area under the ROC curve of 0.90, and an MCC index of 0.80. Other classifiers gave similar breakpoints with lower statistical power. EUCAST fluconazole breakpoints have been validated by means of machine learning methods. These computer tools must be incorporated in the process for developing breakpoints to avoid researcher bias, thus enhancing the statistical power of the model.

Anidulafungin is a new echinocandin that acts by inhibiting (1,3)-beta-D-glucan synthesis in the fungal cell wall. This agent is a semisynthetic lipopeptide synthesized from a fermentation product of Aspergillus nidulans. The spectrum of activity of anidulafungin includes Candida and Aspergillus, the two main etiological agents causing invasive fungal infections. This drug is also active against strains of these genera resistant to azoles or amphotericin B. However, anidulafungin is not active against Cryptococcus spp., Trichosporon spp., Fusarium spp. or Mucorales spp. Data on the activity of this drug against other species are limited and do not allow conclusions to be drawn or recommendations to be made. Echinocandin resistance is uncommon and has little clinical relevance.

An increasing number of molecular techniques for the diagnosis of fungal infections have been developed in the last few years, due to the growing prevalence of mycoses and the length of time required for diagnosis when classical microbiological methods are used. These methods are designed to resolve the following aspects of mycological diagnosis: a) Identification of fungi to species level by means of sequencing relevant taxonomic targets; b) early clinical diagnosis of invasive fungal infections; c) detection of molecular mechanisms of resistance to antifungal agents; and d) molecular typing of fungi. Currently, these methods are restricted to highly developed laboratories. However, some of these techniques will probably be available in daily clinical practice in the near future.

The rise of immunocompromised individuals in our society has provoked a significant emergence in the number of patients affected by opportunistic pathogenic yeast. The microorganisms with a major clinical incidence are species from the genera Candida (especially Candida albicans) and Cryptococcus (particularly Cryptococcus neoformans), although there has been a significant increase in other pathogenic yeasts, such as Trichosporon spp. and Rhodotorula spp. In addition, there are an increasing number of patients infected by yeasts that were not previously considered as pathogenic, such as Saccharomyces cerevisiae. The management of these infections is complicated and is highly dependent on the susceptibility profile not only of the species but also of the strain. The available antifungal compounds belong mainly to the polyene, azole and candin families, which show a distinct spectrum of activity. This review summarizes the current knowledge about the use of the main antifungals for treating infections caused by the yeast species with the most significant clinical relevance, including the susceptibility profiles exhibited by these species in vitro.

Cryptococcus neoformans is a facultative intracellular pathogen. The most distinctive feature of C. neoformans is a polysaccharide capsule that enlarges depending on environmental stimuli. The mechanism by which C. neoformans avoids killing during phagocytosis is unknown. We hypothesized that capsule growth conferred resistance to microbicidal molecules produced by the host during infection, particularly during phagocytosis. We observed that capsule enlargement conferred resistance to reactive oxygen species produced by H(2)O(2) that was not associated with a higher catalase activity, suggesting a new function for the capsule as a scavenger of reactive oxidative intermediates. Soluble capsular polysaccharide protected C. neoformans and Saccharomyces cerevisiae from killing by H(2)O(2). Acapsular mutants had higher susceptibility to free radicals. Capsular polysaccharide acted as an antioxidant in the nitroblue tetrazolium (NBT) reduction coupled to beta-nicotinamide adenine dinucleotide (NADH)/phenazine methosulfate (PMS) assay. Capsule enlargement conferred resistance to antimicrobial peptides and the antifungal drug Amphotericin B. Interestingly, the capsule had no effect on susceptibility to azoles and increased susceptibility to fluconazole. Capsule enlargement reduced phagocytosis by environmental predators, although we also noticed that in this system, starvation of C. neoformans cells produced resistance to phagocytosis. Our results suggest that capsular enlargement is a mechanism that enhances C. neoformans survival when ingested by phagocytic cells.

Objectives: To analyse the susceptibility pattern of a collection of Fusarium clinical isolates., Methods: The antifungal susceptibility pattern of 67 isolates of Fusarium was analysed. Strains were identified by morphological and molecular methods by means of sequencing elongation factor alpha., Results and Conclusions: Six different species were identified. Fusarium solani was the most frequently isolated, followed by Fusarium oxysporum, Fusarium proliferatum and Fusarium verticilloides. Amphotericin B was the only drug with in vitro activity (range: 0.015-32 mg/L). The rest of the antifungals tested (itraconazole, voriconazole, ravuconazole, posaconazole and terbinafine) showed very poor activity against Fusarium, confirming the multiresistant nature of this genus.

We report on the correlation of the outcomes for two cohorts of patients who had been treated for candidemia (126 episodes) or oropharyngeal candidiasis (110 episodes) with various doses of fluconazole and the MIC of fluconazole obtained by using the EUCAST standard for fermentative yeasts. Of 145 episodes caused by an isolate with a fluconazole MIC < or =2 mg/liter, 93.7% (136 of 145) responded to fluconazole treatment. The response for those infected with a strain with a MIC of 4 mg/liter was 66% but reached 100% when the dose was greater than 100 mg/day, whereas the response for those infected with strains with MICs > or =8 mg/liter was only 12%. Hence, a MIC of 2 mg/liter or 4 mg/liter was able to predict successful treatment. A cure rate of 93.9% (140 of 149) was achieved when the dose/MIC ratio was > or =100 but fell to 14.6% (16 of 109) when the ratio was less. The dose/MIC required to achieve a response rate of 50% (the 50% effective concentration) was 43.7 for the cohort of patients with oropharyngeal candidiasis. Classification and regression analysis indicated that a dose/MIC of 35.5 was the threshold for the prediction of cure or failure. However, an increase in exposure above this threshold further increased the probability of cure, and all patients were cured when the dose/MIC exceeded 100. Monte Carlo simulations showed a probability of target attainment of 99% at MICs < or =2 mg/liter and a pharmacodynamic target of a dose/MIC ratio of 100, which was equivalent to an unbound fraction of the fluconazole area under the curve versus the MIC of 79.

Fourteen Aspergillus fumigatus clinical isolates that exhibited a pattern of reduced susceptibility to triazole drugs were analyzed. The sequences of the cyp51A gene from all isolates showed the presence of a point mutation at t364a, which led to the substitution of leucine 98 for histidine (L98H), together with the presence of two copies of a 34-bp sequence in tandem in the promoter of the cyp51A gene. Quantitative expression analysis (real-time PCR) showed up to an eightfold increase in the level of expression of the cyp51A gene compared to that by the susceptible strain. Three PCR fragments of one azole-resistant strain (strain CM2627) that included the promoter with the tandem repeat and part of cyp51A with the t364a mutation or PCR fragments with only one of the modifications were used to replace the cyp51A gene of an azole drug-susceptible A. fumigatus wild-type strain (strain CM237). Only transformants which had incorporated the tandem repeat in the promoter of the cyp51A gene and the L98H amino acid substitution exhibited similarly reduced patterns of susceptibility to all triazole agents and similarly increased levels of cyp51A expression, confirming that the combination of both alterations was responsible for the azole-resistant phenotype.

Fungal infections are a diagnostic and therapeutic problem of increasing concern due to the frequency and severity of disseminated infection in immunocompromised patients. Culture-based methods are characteristically slow and have poor sensitivity; hence, other methods, based on the detection of fungus-specific genetic, antigenic and metabolic components are being developed to enable early diagnosis and specific treatment. Moreover, reproducible antifungal susceptibility methods that can be adapted for use in clinical laboratories have been standardized to allow in vitro detection of resistance, which correlates with a less favorable clinical outcome. In this paper we review the main microbiological procedures available for the diagnosis of fungal infections and for antifungal susceptibility testing.

Objective: Evaluation of the usefulness of a quantitative real-time polymerase chain reaction-based (RT-PCR) technique for clinical diagnosis of histoplasmosis., Methods: Primers and probes were designed on the basis of sequences from the ITS regions of ribosomal DNA of 20 clinical strains of Histoplasma capsulatum. LightCycler procedures (Roche Applied Science) were used with probes marked by fluorescence resonance energy transfer (FRET). Reproducibility, sensitivity, and specificity were analyzed. In addition, an internal control was designed to identify false negative results by PCR inhibition. The RT-PCR assay was tested in 22 clinical samples from 14 patients with proven histoplasmosis. In addition, 30 samples from patients with febrile neutropenia or mycoses other than histoplasmosis, and from healthy volunteers were analyzed as controls., Results: The limit of detection of the assay was 1 fg of genomic DNA per microl of sample. The PCR-based technique was reproducible and highly specific. Positive results were obtained in 11/14 (78.6%) patients and in 17/22 (77.3%) clinical samples. RT-PCR was positive in 100% of respiratory secretions and bone marrow samples, but only 70% of sera (p < 0.01). Mean fungal DNA value was 23.1 fg/microl in serum and 4.85 x 10(3) fg/microl in respiratory and bone marrow samples. RT-PCR results were positive in serum from three HIV patients for which antibody detection by immunodiffusion was negative. Specificity was 100%, since PCR results were negative for all the control samples., Conclusion: Thes RT-PCR technique is a sensitive, specific method for early diagnosis of histoplasmosis, particularly when respiratory secretions or bone marrow samples are analyzed. The reliability is lower in serum, but it can be used as an additional, complementary technique to culture and serology in HIV patients.

A new real-time PCR-based assay was used for detecting DNA of Histoplasma capsulatum in serum samples collected from four HIV-infected patients with proven histoplasmosis. The assay targeted the ITS1 region of rDNA and its in vitro sensitivity, specificity and reproducibility were evaluated. The technique detected DNA of H. capsulatum in all of the HIV-infected patients with proven histoplasmosis (4/4, 100%). The PCR result was positive for seven of the ten (70%) samples studied. The assay's specificity was determined to be 100%, since the method was negative for 25 other serum samples (10 from patients with proven aspergillosis and 15 from healthy controls). The PCR assay is a new and promising diagnostic alternative and further investigation is warranted.

Azole drug resistance in Aspergillus fumigatus is an uncommon but well-known phenomenon. The analysis of resistance mechanisms at molecular level has identified the bases for A. fumigatus azole resistance. To date, the most prevalent mechanism of azole resistance appears to be the modification of Cyp51, specifically mutations in cyp51A gene. These mutations have been associated with three different antifungal susceptibility profiles: (i) cross-resistance to itraconazole and posaconazole that has been associated with amino acid substitutions at glycine 54 (G54), (ii) elevated MICs to all azole drugs associated with amino acid substitutions at methionine M220, and (iii) cross-resistance to all azole drugs related to the presence of Cyp51A substitutions at leucine 98 for histidine (L98H) linked to a duplication in tandem of a 34 bp repeat in the cyp51A promoter region, which seem to be responsible for increased cyp51A gene expression. Another matter of concern is the increasing reports of isolation of genetic variants of A. fumigatus, originally misidentified as poorly sporulating strains of A. fumigauts, as a causative agents of invasive infection. Many of these isolates belonging to the Aspergillus section Fumigati have been found to be resistant in vitro to multiple antifungal drugs. Current data show that susceptibility profile of these variants could be predictable depending on the species. Resistance among clinical strains of filamentous fungi may become more common in the future associated with the spread of prophylaxis, pre-emptive treatments and specific therapies with antifungal agents.

Objectives: Assessment of a real-time PCR technique for the detection and quantification of fungal DNA in a murine model of pulmonary aspergillosis., Methods: Male ICR specific pathogen-free mice were used in the studies. The animals were divided into groups: immunosuppressed and intranasally inoculated with various inoculum sizes (10(6), 10(5), 10(4), and 10(3) conidia/mL) of a clinical isolate of Aspergillus fumigatus. When symptoms of pulmonary aspergillosis were detected, the mice were killed and the lungs removed for culture and real-time PCR determination. The PCR reactions used primers that amplified a region of Aspergillus spp. ribosomal DNA. Survival time per experimental group was calculated and correlation coefficients with inoculum size, colony counts and PCR results were determined., Results: Average survival time was significantly associated with the size of the inoculum. Pulmonary colony count was positive for 90% of the infected mice, but there was no statistical relationship between count values and either survival time or inoculum size. Real-time PCR was positive in 100% of the animals and was significantly associated with survival time and inoculum size (p < 0.01)., Conclusion: Real-time PCR is a reliable procedure for the quantification and evaluation pulmonary infection due to A. fumigatus in animal models.

Objective: To describe a nosocomial outbreak of gastric mucormycosis caused by Rhizopus microsporus var. rhizopodiformis in five adult patients admitted to an intensive care unit (ICU)., Design: Epidemiological surveillance study., Setting: A 12-bed polyvalent ICU of an acute care teaching hospital in Pamplona, Spain., Patients: Five patients admitted to the ICU requiring artificial ventilation, diagnosis on admission severe pneumonia in four patients and one polytrauma patient, within a 14-week period, were diagnosed with gastric mucormycosis based on microbiological and/or histopathological characteristics. Upper gastrointestinal bleeding was the presenting manifestation in 80% of patients., Interventions: Filamentous fungi isolated at the microbiology laboratory of the hospital were examined at the national Mycology Reference Laboratory in Madrid., Measurements and Results: Rhizopus microsporus var. rhizopodiformis growth was detected in gastric aspiration samples, environmental samples, wooden tongue depressors used to prepare oral medications (and given to patients through a nasogastric catheter), and in some tongue depressors stored in unopened boxes unexposed to the ICU environment. All depressors were purchased from the same supplier. R. microsporus was not isolated from batches purchased at different times from the same supplier and from another supplier. The outbreak terminated when contaminated tongue depressors were withdrawn from use., Conclusions: Wooden tongue depressors contaminated by R. microsporus var. rhizopodiformis used to prepare oral medications caused an outbreak of fungal gastritis with an attributable mortality of 40%. Wooden material should not be used in the hospital setting.

Introduction: A three-month (April-June 2001) cross-sectional study was designed to assess the epidemiological profile of dermatophytosis in Spain., Methods: Sixty-two medical centers belonging to 14 different autonomous regions of Spain took part in the survey. A total of 491 strains were sent the Mycology Unit of the National Microbiology Center together with a form containing information on each patient. Isolates were identified by routine methods., Results: The average age of the patients was 38.7 years and 55.6% were men. The most frequent dermatophytoses were tinea unguium (39.1%), tinea corporis (25.1%), tinea pedis (12.6%), and tinea capitis (11.2%). Trichophyton rubrum (43%) was the most prevalent species. T. mentagrophytes (21.2%) and Microsporum canis (9.8%) were the second and third most common species, respectively. Tinea unguium was significantly associated with urban residence, and tinea corporis with rural residence and contact with animals. T. rubrum was related with urban dermatophytosis and T. mentagrophytes with rural cases. Tinea capitis due to T. tonsurans and T. violaceum was related with patients of North African origin. Tinea unguium was the most prevalent infection by autonomous region, except in Castilla-León, Asturias, Andalucía and Aragón, where tinea corporis was the most common dermatophytosis. Tinea capitis was the most frequent infection in Castilla-La Mancha and Extremadura. T. mentagrophytes was more prevalent than T. rubrum in Valencia, Castilla-La Mancha, Aragón and Murcia., Conclusion: This cross-sectional study shows regional differences in the clinical and microbiological features of dermatophytosis in Spain. Epidemiological surveys are an essential tool for developing strategies for infection control.

Invasive infections by Scedosporium prolificans have increased alarmingly in recent years, mainly in immunosuppressed patients. The epidemiology, pathogenesis and the natural habitat of this pathogen are practically unknown. Isolates of S. prolificans were distinguished from one another by inter-simple-sequence-repeat (ISSR) fingerprinting, a technique based on the high degree of polymorphism of the multisatellite genetic markers used. This technique was found useful for typing 84 isolates of S. prolificans from different countries and sources. The assemblage of S. prolificans isolates tested was extremely diverse, with 35 genotypes present. Several patients were found to have been infected or colonized by more than one strain. Overall, this technique facilitates the epidemiological study of S. prolificans infection.

Objective: To evaluate the intra- and inter-laboratory reproducibility of a new standard for susceptibility testing of fermentative yeasts. This standard is based on the M27-A procedure of the National Committee for Clinical Laboratory Standards (NCCLS), but incorporates several modifications, including spectrophotometric growth-dependent endpoint reading., Methods: Nine laboratories participated in the study. Common material lots were used to test six Candida species (one each of C. albicans, C. tropicalis, C. parapsilosis, C. glabrata, C. krusei, and C. lusitaniae), and two quality control strains (C. krusei ATCC6258 and C. parapsilosis ATCC22019). Triplicate testing on three separate days was performed in microtiter format with RPMI-2% glucose, pH 7.0. Flucytosine, fluconazole and itraconazole were tested. In total, 3888 MIC values were included in the analyses. Reproducibility was calculated by means of agreement (percentage of MICs within one two-fold dilution of the mode) and intraclass correlation coefficient (ICC, maximum value of 1)., Results: The average intra-laboratory agreements were 99% and 96% after 24 h and 48 h of incubation, respectively, with ICCs of 0.98 and 0.97 (P < 0.05). Two strains exhibiting a trailing effect showed intra-laboratory agreement of 92% and ICCs of < 0.91 at 48 h. The inter-laboratory agreement was 94% and 88% after 24 h and 48 h, respectively, with ICCs of 0.93 and 0.91 (P < 0.05). Lower values of agreement and ICCs were obtained for strains exhibiting trailing after 48 h of incubation. Itraconazole yielded the lowest values of reproducibility., Conclusion: The new procedure of EUCAST for antifungal susceptibility testing is a reproducible method within and between laboratories and offers several advantages over the NCCLS approved method.

The combined activity in vitro of amphotericin B/terbinafine and itraconazole/terbinafine was assessed against 17 clinical isolates of Zygomycota using a checkerboard technique. Itraconazole/terbinafine combination exhibited a potent synergistic effect against the most of strains. Amphotericin B/terbinafine combination showed an indifferent interaction for Rhizopus oryzae, and an additive effect for the other species.