Method-Dependent Epidemiological Cutoff Values for Detection of Triazole Resistance in Candida and Aspergillus Species for the Sensititre YeastOne Colorimetric Broth and Etest Agar Diffusion Methods.

Although the Sensititre Yeast-One (SYO) and Etest methods are widely utilized, interpretive criteria are not available for triazole susceptibility testing of Candida or Aspergillus species. We collected fluconazole, itraconazole, posaconazole, and voriconazole SYO and Etest MICs from 39 laboratories representing all continents for (method/agent-dependent) 11,171 Candida albicans, 215 C. dubliniensis, 4,418 C. glabrata species complex, 157 C.guilliermondii (Meyerozyma guilliermondii), 676 C. krusei (Pichia kudriavzevii), 298 C.lusitaniae (Clavispora lusitaniae), 911 C.parapsilosissensu stricto, 3,691 C.parapsilosis species complex, 36 C.metapsilosis, 110 C.orthopsilosis, 1,854 C.tropicalis, 244 Saccharomyces cerevisiae, 1,409 Aspergillus fumigatus, 389 A.flavus, 130 A.nidulans, 233 A.niger, and 302 A.terreus complex isolates. SYO/Etest MICs for 282 confirmed non-wild-type (non-WT) isolates were included: ERG11 (C. albicans), ERG11 and MRR1 (C. parapsilosis), cyp51A (A. fumigatus), and CDR2 and CDR1 overexpression (C. albicans and C. glabrata, respectively). Interlaboratory modal agreement was superior by SYO for yeast species and by the Etest for Aspergillus spp. Distributions fulfilling CLSI criteria for epidemiological cutoff value (ECV) definition were pooled, and we proposed SYO ECVs for S. cerevisiae and 9 yeast and 3 Aspergillus species and Etest ECVs for 5 yeast and 4 Aspergillus species. The posaconazole SYO ECV of 0.06 µg/ml for C. albicans and the Etest itraconazole ECV of 2 µg/ml for A. fumigatus were the best predictors of non-WT isolates. These findings support the need for method-dependent ECVs, as, overall, the SYO appears to perform better for susceptibility testing of yeast species and the Etest appears to perform better for susceptibility testing of Aspergillus spp. Further evaluations should be conducted with more Candida mutants.

Posaconazole MIC Distributions for Aspergillus fumigatus Species Complex by Four Methods: Impact of cyp51A Mutations on Estimation of Epidemiological Cutoff Values.

Estimating epidemiological cutoff endpoints (ECVs/ECOFFS) may be hindered by the overlap of MICs for mutant and nonmutant strains (strains harboring or not harboring mutations, respectively). Posaconazole MIC distributions for the Aspergillus fumigatus species complex were collected from 26 laboratories (in Australia, Canada, Europe, India, South and North America, and Taiwan) and published studies. Distributions that fulfilled CLSI criteria were pooled and ECVs were estimated. The sensitivity of three ECV analytical techniques (the ECOFFinder, normalized resistance interpretation [NRI], derivatization methods) to the inclusion of MICs for mutants was examined for three susceptibility testing methods (the CLSI, EUCAST, and Etest methods). The totals of posaconazole MICs for nonmutant isolates (isolates with no known cyp51A mutations) and mutant A. fumigatus isolates were as follows: by the CLSI method, 2,223 and 274, respectively; by the EUCAST method, 556 and 52, respectively; and by Etest, 1,365 and 29, respectively. MICs for 381 isolates with unknown mutational status were also evaluated with the Sensititre YeastOne system (SYO). We observed an overlap in posaconazole MICs among nonmutants and cyp51A mutants. At the commonly chosen percentage of the modeled wild-type population (97.5%), almost all ECVs remained the same when the MICs for nonmutant and mutant distributions were merged: ECOFFinder ECVs, 0.5 µg/ml for the CLSI method and 0.25 µg/ml for the EUCAST method and Etest; NRI ECVs, 0.5 µg/ml for all three methods. However, the ECOFFinder ECV for 95% of the nonmutant population by the CLSI method was 0.25 µg/ml. The tentative ECOFFinder ECV with SYO was 0.06 µg/ml (data from 3/8 laboratories). Derivatization ECVs with or without mutant inclusion were either 0.25 µg/ml (CLSI, EUCAST, Etest) or 0.06 µg/ml (SYO). It appears that ECV analytical techniques may not be vulnerable to overlap between presumptive wild-type isolates and cyp51A mutants when up to 11.6% of the estimated wild-type population includes mutants.

International Evaluation of MIC Distributions and Epidemiological Cutoff Value (ECV) Definitions for Fusarium Species Identified by Molecular Methods for the CLSI Broth Microdilution Method.

The CLSI epidemiological cutoff values (ECVs) of antifungal agents are available for various Candida spp., Aspergillus spp., and the Mucorales. However, those categorical endpoints have not been established for Fusarium spp., mostly due to the difficulties associated with collecting sufficient CLSI MICs for clinical isolates identified according to the currently recommended molecular DNA-PCR-based identification methodologies. CLSI MIC distributions were established for 53 Fusarium dimerum species complex (SC), 10 F. fujikuroi, 82 F. proliferatum, 20 F. incarnatum-F. equiseti SC, 226 F. oxysporum SC, 608 F. solani SC, and 151 F. verticillioides isolates originating in 17 laboratories (in Argentina, Australia, Brazil, Canada, Europe, Mexico, and the United States). According to the CLSI guidelines for ECV setting, ECVs encompassing ≥97.5% of pooled statistically modeled MIC distributions were as follows: for amphotericin B, 4 µg/ml (F. verticillioides) and 8 µg/ml (F. oxysporum SC and F. solani SC); for posaconazole, 2 µg/ml (F. verticillioides), 8 µg/ml (F. oxysporum SC), and 32 µg/ml (F. solani SC); for voriconazole, 4 µg/ml (F. verticillioides), 16 µg/ml (F. oxysporum SC), and 32 µg/ml (F. solani SC); and for itraconazole, 32 µg/ml (F. oxysporum SC and F. solani SC). Insufficient data precluded ECV definition for the other species. Although these ECVs could aid in detecting non-wild-type isolates with reduced susceptibility to the agents evaluated, the relationship between molecular mechanisms of resistance (gene mutations) and MICs still needs to be investigated for Fusarium spp.

Prospective multicenter international surveillance of azole resistance in Aspergillus fumigatus.

To investigate azole resistance in clinical Aspergillus isolates, we conducted prospective multicenter international surveillance. A total of 3,788 Aspergillus isolates were screened in 22 centers from 19 countries. Azole-resistant A. fumigatus was more frequently found (3.2% prevalence) than previously acknowledged, causing resistant invasive and noninvasive aspergillosis and severely compromising clinical use of azoles.

Multicenter evaluation of MIC distributions for epidemiologic cutoff value definition to detect amphotericin B, posaconazole, and itraconazole resistance among the most clinically relevant species of Mucorales.

Clinical breakpoints (CBPs) have not been established for the Mucorales and any antifungal agent. In lieu of CBPs, epidemiologic cutoff values (ECVs) are proposed for amphotericin B, posaconazole, and itraconazole and four Mucorales species. Wild-type (WT) MIC distributions (organisms in a species-drug combination with no detectable acquired resistance mechanisms) were defined with available pooled CLSI MICs from 14 laboratories (Argentina, Australia, Canada, Europe, India, Mexico, and the United States) as follows: 10 Apophysomyces variabilis, 32 Cunninghamella bertholletiae, 136 Lichtheimia corymbifera, 10 Mucor indicus, 123 M. circinelloides, 19 M. ramosissimus, 349 Rhizopus arrhizus, 146 R. microsporus, 33 Rhizomucor pusillus, and 36 Syncephalastrum racemosum isolates. CLSI broth microdilution MICs were aggregated for the analyses. ECVs comprising ≥95% and ≥97.5% of the modeled populations were as follows: amphotericin B ECVs for L. corymbifera were 1 and 2 µg/ml, those for M. circinelloides were 1 and 2 µg/ml, those for R. arrhizus were 2 and 4 µg/ml, and those for R. microsporus were 2 and 2 µg/ml, respectively; posaconazole ECVs for L. corymbifera were 1 and 2, those for M. circinelloides were 4 and 4, those for R. arrhizus were 1 and 2, and those for R. microsporus were 1 and 2, respectively; both itraconazole ECVs for R. arrhizus were 2 µg/ml. ECVs may aid in detecting emerging resistance or isolates with reduced susceptibility (non-WT MICs) to the agents evaluated.

Primary Cutaneous Coccidioidomycosis in an Italian Nun Working in South America and Review of Published Literature.

Coccidioidomycosis is a systemic disease caused by the dimorphic fungus Coccidioides, endemic in parts of the Southwestern USA and Central and South America. Two species, Coccidioides immitis and Coccidioides posadasii, were differentiated. Primary cutaneous coccidioidomycosis (PCC) has been reported rarely. An unusual case of PCC characterized by a persistent solitary lesion diagnosed in Italy in an immunocompetent Italian nun living in Argentina is described. The isolate was identified by sequence analysis as C. posadasii. Antibody screening was negative. A total of 39 cases of PCC have been reported in the literature. Infections occurred as a consequence of traumatic implantation in a natural setting in endemic areas or of accidental inoculation in laboratory workers. Importance of accurate investigation of travel history and of occupational hazards to laboratory workers is outlined.

European Confederation of Medical Mycology (ECMM) epidemiological survey on invasive infections due to Fusarium species in Europe.

In order to better understand the epidemiology of fusariosis in Europe, a survey collecting information on the clinical characteristics of the patients infected by Fusarium as well as on the infecting isolates was launched. A total of 76 cases of invasive fusariosis occurring from January 2007 to June 2012 were collected and Fusarium isolates were identified by sequencing the translation elongation factor 1α (TEF) gene. Also, antifungal susceptibility was tested by broth microdilution according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Etest. Disseminated disease was considered proven in 46 cases and probable in 17 cases. Localised infection was seen in 13 cases. Gibberella fujikuroi species complex (SC), including Fusarium verticillioides and F. proliferatum, and F. solani SC were the most frequent aetiology of disseminated and localised infections, respectively. The crude mortality rate was 46 %, the highest associated with F. solani SC (67 %) and F. proliferatum (62.5 %). A wide range of antifungal susceptibilities was observed. Amphotericin B was the most potent antifungal in vitro, and itraconazole the least effective. The azoles exhibited lower minimum inhibitory concentrations (MICs) against F. verticillioides strains, with posaconazole having a slightly better performance, while F. solani SC isolates were resistant to all three azoles tested. The essential agreement between the Etest and the EUCAST method was 100 % for itraconazole and voriconazole, and 96 % for amphotericin B and posaconazole. In conclusion, we confirm that fusariosis is a rare but severe event in Europe, that G. fujikuroi SC is the predominant cause of deep infections and that different species have different antifungal in vitro susceptibility patterns.

Azole-resistant Aspergillus fumigatus in the environment of northern Italy, May 2011 to June 2012.

In recent years acquired azole resistance in Aspergillus fumigatus has been increasingly reported and a dominant mechanism of resistance (TR34/L98H) was found in clinical and environmental isolates. The aim of the present study was to investigate the prevalence of azole resistance in environmental A. fumigatus isolates collected in northern Italy. A. fumigatus grew from 29 of 47 soil samples analysed. Azole-resistant isolates were detected in 13% (6/47) of the soil samples and in 21% (6/29) of the soil samples containing A. fumigatus. High minimal inhibitory concentrations (MIC) of itraconazole (≥16 mg/L) and posaconazole (≥0.5 mg/L) were displayed by nine isolates from six different soil samples, namely apple orchard (1 sample), rose pot compost (2 samples), and cucurbit yields (3 samples). Seven isolates had a MIC=2 mg/L of voriconazole. Seven of nine itraconazole and posaconazole resistant isolates harboured the same TR34/L98H mutation of cyp51A. These findings, together with the occurrence of resistant clinical isolates, suggest that azole resistance should be considered in primary patient care.

A 1-year prospective survey of candidemia in Italy and changing epidemiology over one decade.

PURPOSE: The aim of this study was to assess the epidemiology of candidemia and antifungal susceptibility profiles of Candida isolates in Italy through a prospective surveillance study and to evaluate changes compared to a previous survey performed in one Italian region (Lombardy) in 1997-1999. METHODS: A prospective laboratory-based surveillance of candidemia was performed in Italy from January to December 2009. For each case a questionnaire was filled in, and the first isolate was collected and tested for in vitro antifungal susceptibility. RESULTS: During our 12-month survey, 467 episodes of candidemia were reported from 34 centres (30 located in Lombardy) and 464 isolates collected. Candida albicans was the predominant species (overall incidence 50.4 %), but the proportion varied considerably from 52.1 % in Lombardy hospitals to 45.2 % hospitals located outside this region. The second most frequent species was C. glabrata in Lombardy and C. parapsilosis in other regions. Comparison of the 1997-1999 and 2009 data on episodes of candidemia in Lombardy revealed a threefold increase in incidence (from 0.38 to 1.19 per 1,000 admissions), aging of infected patients, decline in crude mortality (from 35 to 27.1 %) and an increased proportion of C. glabrata etiology (from 12.8 to 20.3 %). Susceptibility testing confirmed the broad activity of amphotericin B and echinocandins. Decreased susceptibility to fluconazole was found in 24.9 % of the tested isolates. CONCLUSIONS: The results of this latest survey confirm the high rate of candidemia in Italy and show changes in some of the epidemiological tracts, such as aging of infected patients, increased proportion of C. glabrata infections, increased diagnosis in medical wards, and improvement in patients' survival.

Looking for Candida nivariensis and C. bracarensis among a large Italian collection of C. glabrata isolates: results of the FIMUA working group.

Two recently described pathogenic Candida species, C. nivariensis and C. bracarensis, share many phenotypic characteristics with C. glabrata and are easily misidentified as such. The aim of this study was to determine the occurrence of these cryptic species in Italy. One thousand yeast isolates collected in 14 Italian regions and identified as C. glabrata by phenotypic and biochemical methods were included in this study: 928 were screened on CHROMagar and 72 were analysed by a multiplex PCR. None of these cryptic species was identified despite the nationwide distribution and the variety of biological origin of the isolates.

Increased mortality in young candidemia patients associated with presence of a Candida albicans general-purpose genotype.

The yeast Candida albicans causes life-threatening candidemia. A general-purpose genotype (GPG; corresponds to clade 1) causes more infections than other C. albicans genotypes. To investigate if GPG strains also cause higher mortality, we developed a duplex PCR assay which was 98% accurate in identifying GPG strains in an international collection of strains typed with probe Ca3. We applied the assay to 635 European C. albicans candidemia isolates. Of these, 18% conformed to the GPG genotype, 4% were of a borderline genotype, and 78% were of a non-GPG genotype, broadly consistent with genotype distributions in earlier studies. The prevalence of GPG strains was increased in females and in younger patients, exceeding 40% in infants aged ≤1 year. Logistic regression confirmed sex and age as significant determinants of GPG prevalence. Across the entire patient cohort, there was no difference in mortality for patients infected with GPG strains or other strains (36% versus 37%). However, mortality in patients aged ≤48 years was 33% for infection with GPG strains but only 15% for infection with other strains (z test; P < 0.01). Mortality rates associated with GPG and non-GPG strains were comparable in older patients (39% versus 46%). A logistic regression analysis confirmed the age-dependent impact of genotype on mortality. Thus, GPG strains may be more virulent than other strains in younger patients. Because candidemia is usually caused by endogenous strains, our PCR assay could potentially be used as a risk assessment tool for identifying younger patients most at risk of death from candidemia.

Mycosands: Fungal diversity and abundance in beach sand and recreational waters - Relevance to human health.

The goal of most studies published on sand contaminants is to gather and discuss knowledge to avoid faecal contamination of water by run-offs and tide-retractions. Other life forms in the sand, however, are seldom studied but always pointed out as relevant. The Mycosands initiative was created to generate data on fungi in beach sands and waters, of both coastal and freshwater inland bathing sites. A team of medical mycologists and water quality specialists explored the sand culturable mycobiota of 91 bathing sites, and water of 67 of these, spanning from the Atlantic to the Eastern Mediterranean coasts, including the Italian lakes and the Adriatic, Baltic, and Black Seas. Sydney (Australia) was also included in the study. Thirteen countries took part in the initiative. The present study considered several fungal parameters (all fungi, several species of the genus Aspergillus and Candida and the genera themselves, plus other yeasts, allergenic fungi, dematiaceous fungi and dermatophytes). The study considered four variables that the team expected would influence the results of the analytical parameters, such as coast or inland location, urban and non-urban sites, period of the year, geographical proximity and type of sediment. The genera most frequently found were Aspergillus spp., Candida spp., Fusarium spp. and Cryptococcus spp. both in sand and in water. A site-blind median was found to be 89 Colony-Forming Units (CFU) of fungi per gram of sand in coastal and inland freshwaters, with variability between 0 and 6400 CFU/g. For freshwater sites, that number was 201.7 CFU/g (0, 6400 CFU/g (p = 0.01)) and for coastal sites was 76.7 CFU/g (0, 3497.5 CFU/g). For coastal waters and all waters, the median was 0 CFU/ml (0, 1592 CFU/ml) and for freshwaters 6.7 (0, 310.0) CFU/ml (p < 0.001). The results advocate that beaches should be monitored for fungi for safer use and better management.

Evolution of fungemia in an Italian region.

BACKGROUND: Fungemia represents a public health concern. Knowing aetiology and activity of the antifungals is critical for the management of bloodstream infections. Therefore, surveillance on local/international levels is desirable for a prompt administration of appropriate therapy. METHODS: Data on fungi responsible for fungemia and antifungal susceptibility profiles were collected from a laboratory-based surveillance over 2016-2017 in 12 hospitals located in Lombardia, Italy. The trend of this infection in twenty years was analysed. RESULTS: A total of 1024 episodes were evaluated. Rate of candiaemia progressively increased up to 1.46/1000 admissions. C.albicans was the most common species (52%), followed by C. parapsilosis (15%) and C glabrata (13%). As in the previous surveys the antifungal resistance is rare (echinocandins<2%, fluconazole 6%, amphotericin B 0.6%). Fungi other than Candida were responsible for 18 episodes: Cryptococcus neoformans (5 cases), Fusarium spp. (4), Magnusiomyces clavatus (3), Saccharomyces cerevisiae (3), Rhodotorula spp. (2), Exophiala dermatitidis (1). All fungi, except S.cerevisiae, were intrinsically resistant to echinocandins. Some isolates showed also elevated azole MIC. CONCLUSIONS: No particular changes in terms of species distribution and antifungal susceptibility patterns was noted. However, surveillance programs are needed to monitor trends in antifungal resistance, steer stewardship activities, orient empirical treatment.

Prospective multicentre study on azole resistance in Aspergillus isolates from surveillance cultures in haematological patients in Italy.

OBJECTIVES: This study was conducted to assess the prevalence of azole resistance in Aspergillus isolates from patients with haematological malignancies or who were undergoing haematopoietic stem cell transplantation and to identify the molecular mechanism of resistance. METHODS: In this 28-month prospective study involving 18 Italian centres, Aspergillus isolates from surveillance cultures were collected and screened for azole resistance, and mutations in the cyp51A gene were identified. Resistant isolates were genotyped by microsatellite analysis, and the allelic profiles were compared with those of resistant environmental and clinical isolates from the same geographical area that had been previously genotyped. RESULTS: There were 292 Aspergillus isolates collected from 228 patients. The isolates belonged mainly to the section Fumigati (45.9%), Nigri (20.9%), Flavi (16.8%) and Terrei (4.8%). Three isolates showed itraconazole resistance: Aspergillus fumigatus sensu stricto, Aspergillus lentulus (section Fumigati) and Aspergillus awamori (section Nigri). The itraconazole resistance rates were 1% and 1.48% considering all Aspergillus spp. isolates and the Aspergillus section Fumigati, respectively. The prevalence of azole resistance among all the patients was 1.3%. Among patients harbouring A. fumigatus sensu stricto isolates, the resistance rate was 0.79%. The A. fumigatus isolate, with the TR34/L98H mutation, was genotypically distant from the environmental and clinical strains previously genotyped. CONCLUSIONS: In this study, the Aspergillus azole resistance rate was 1% (3/292). In addition to A. fumigatus sensu stricto, A. lentulus and A. awamori azole-resistant isolates were identified. Therefore, it is important have a correct identification at the species level to address a rapid therapy better, quickly understand the shift towards cryptic species and have an updated knowledge of the local epidemiology.

[Learning how to face emergencies. A project dealing with first aid aimed at primary and secondary school students]. / Le emergenze. Un progetto di primo soccorso rivolto agli studenti delle scuole primarie e secondarie superiori.

The authors describe the planning, making and evaluation of a project aimed at primary school students attending their 4th year and at secondary school students attending their 5th year designed to promote first aid procedures in emergency situations. An educational video has been filmed to support these projects.

[Quality assessment of health promotion web sites]. / Analisi della qualità dei siti web riguardanti la promozione della salute.

The continuous expansion of the use of Internet has determined an increase in the number of web sites, including those related to health promotion. Even if this new tool provides a faster way to find the desired information, the high number of different sites can confuse the user especially when they present inconsistent data. For this reason, according to the principles contained in the Minerva Manual, we have built a template to evaluate the quality of health care related web sites, and we have verified its validity applying it to ten different sites. The results of this analysis show that the attention to the requirements varies according to the target users, the site focus and the sponsoring agency. Finally we have proposed an ideal web site, choosing, for each requirement in the evaluation grid, the approach that we considered best among the ones adopted by the ten examined sites.

A prospective international Aspergillus terreus survey: an EFISG, ISHAM and ECMM joint study.

OBJECTIVES: A prospective international multicentre surveillance study was conducted to investigate the prevalence and amphotericin B susceptibility of Aspergillus terreus species complex infections. METHODS: A total of 370 cases from 21 countries were evaluated. RESULTS: The overall prevalence of A. terreus species complex among the investigated patients with mould-positive cultures was 5.2% (370/7116). Amphotericin B MICs ranged from 0.125 to 32 mg/L, (median 8 mg/L). CONCLUSIONS: Aspergillus terreus species complex infections cause a wide spectrum of aspergillosis and the majority of cryptic species display high amphotericin B MICs.

[Tobacco smoking among medical students of the University of Milano, Italy]. / Indagine conoscitiva sull'abitudine al fumo tra gli studenti di medicina e chirurgia dell'Università degli studi di Milano.

In order to evaluate smoking habits among the future medical class an anonymous questionnaire about tobacco smoking was submitted to 822 students of the University of Milano in six academic years, from 1998/99 to 2003/04. A total of 184 students (22.4%) were smoker 463 (56.3%) had never smoked and 161 (19.6%) had previously smoked. More than half of these last had quit smoking during the university years. Smoking habits resulted influenced by the habits in the family (parents and brothers) and among the friends. Anti-smoking campaigns among medical students are needed taking into account their future role in health promotion and in prevention of tobacco smoking and its consequences.

Fusarium musae as cause of superficial and deep-seated human infections.

BLAST analysis in GenBank of 60 Fusarium verticillioides clinical isolates using the sequence of translation elongation factor 1-alpha allowed the identification of four F. musae confirming that this species is not a rare etiology of superficial and deep infections and that its habitat is not restricted to banana fruits.

Genotypic variation and antifungal susceptibilities of Candida pelliculosa clinical isolates.

At the Istituto Ricovero Cura Carattere Scientifico, Ospedale Maggiore di Milano, Italy, Candida pelliculosa accounted for 3.3 and 4.4 % of all Candida species other than Candida albicans collected during 1996 and 1998, respectively. Genetic variability was investigated by electrophoretic karyotyping and inter-repeat PCR, and the susceptibility to five antifungal agents of 46 strains isolated from 37 patients during these 2 years was determined. Combination of the two typing methods yielded 14 different DNA types. Although the majority of DNA types were randomly distributed among different units, one DNA type was significantly more common in patients hospitalized in a given unit compared with those from other wards (P=0.034), whereas another DNA type was more frequently isolated in patients hospitalized during 1996 than in those hospitalized during 1998 (P=0.002). Fluconazole, itraconazole and posaconazole MIC90 values were 16, 1 and 4 microg ml-1, respectively. All isolates but three were susceptible in vitro to flucytosine. All isolates were susceptible in vitro to amphotericin B. These data suggest that there are possible relationships among strains of C. pelliculosa, wards and time of isolation. Amphotericin B seems to be the optimal drug therapy in infections due to this yeast species.