Report of terbinafine resistant Trichophyton spp. in Italy: Clinical presentations, molecular identification, antifungal susceptibility testing and mutations in the squalene epoxidase gene.

BACKGROUND: Numerous reports of resistance to terbinafine in Trichophyton spp. from all over the world are arousing justified attention and concern. Point mutations in the gene that encodes the squalene epoxidase (SQLE) enzyme are responsible for these therapeutic resistances. OBJECTIVES: Primary objective of the study was to describe first isolates of Trichophyton spp. resistant to terbinafine among the patients treated between September 2019 and June 2022 at the Dermatology Units of Ospedale Maggiore Policlinico and San Bortolo Hospital. Secondary objective was to study the resistance mechanism. METHODS: Patients with confirmed Trichophyton spp. infection has been treated with systemic and topical terbinafine. Patients were then re-evaluated 12 weeks after the therapy. Patients with incomplete or absent response to terbinafine underwent a new skin scraping for direct mycological examination, new identification of dermatophyte species from culture and MALDI-TOF, molecular species identification, antifungal susceptibility testing and molecular analysis of SQLE gene. RESULTS: We identified five patients without clinical response to treatment with terbinafine. The DNA sequencing of the ITS region identified one Trichophyton rubrum and four Trichophyton indotineae. The T. rubrum strain showed minimum inhibitory concentration (MIC) (90% growth inhibition) of 4 mg/L for terbinafine. The four T. indotineae strains showed a MICs range of 0.25-4 mg/L for terbinafine. The analysis of the SQLE gene in the T. rubrum strain showed a nucleotide substitution generating a missense mutation (L393F). The SQLE gene sequencing in the T. indotineae strains showed a nucleotide substitution generating a missense mutation (F397L) in two strains, a nucleotide substitution L393S in one strain and a nucleotide substitution F415C in another strain. CONCLUSIONS: We report the first cases of terbinafine-resistant Trichophyton isolates in the Italian population. Solid antifungal management programs will be needed to promote more responsible use of antimycotics and preserve their therapeutic efficacy to control antifungal resistance.

Environmental and bioclimatic factors influencing yeasts and molds distribution along European shores.

The present study employed data collected during the Mycosands survey to investigate the environmental factors influencing yeasts and molds distribution along European shores applying a species distribution modelling approach. Occurrence data were compared to climatic datasets (temperature, precipitation, and solar radiation), soil datasets (chemical and physical properties), and water datasets (temperature, salinity, and chlorophyll-a concentration) downloaded from web databases. Analyses were performed by MaxEnt software. Results suggested a different probability of distribution of yeasts and molds along European shores. Yeasts seem to tolerate low temperatures better during winter than molds and this reflects a higher suitability for the Northern European coasts. This difference is more evident considering suitability in waters. Both distributions of molds and yeasts are influenced by basic soil pH, probably because acidic soils are more favorable to bacterial growth. Soils with high nitrogen concentrations are not suitable for fungal growth, which, in contrast, are optimal for plant growth, favored by this environment. Finally, molds show affinity with soil rich in nickel and yeasts with soils rich in cadmium resulting in a distribution mainly at the mouths of European rivers or lagoons, where these metals accumulate in river sediments.

ICU environmental surfaces are a reservoir of fungi: species distribution in northern Italy.

BACKGROUND: Preventing and reducing nosocomial infections is a public health goal. Concern about healthcare-associated fungal infections has increased in recent years due to the emergence and spread of new pathogens, increasing antifungal resistance and outbreaks in hospital settings. AIM: To investigate the presence of medically relevant fungal species on environmental surfaces in 12 intensive care units of eight hospitals in Milan, Italy. METHODS: Environmental samplings, using contact plates on surfaces near bed stations and medical workstations, were conducted between November 2019 and January 2020. Fungi isolated were identified, and some were tested in vitro for antifungal susceptibility. FINDINGS: In total, 401 environmental samples were collected from 61 bed stations and 17 medical workstations. Positive samples were found in all hospitals except one, with positivity rates ranging from 4% to 24.2%. Filamentous fungi were found mainly on infusion pumps (23.2%) and patient tables (21.2%), whereas yeasts were found mainly on computers (25%) and floors (10.9%). Fungi were isolated from 12% of total samples. Filamentous fungi, mainly Aspergillus fumigatus, grew in 70.8% of positive samples, and yeasts grew in 27.1%, mainly Candida parapsilosis (42.8%) and Candida glabrata (28.6%). Fungi were detected near patients' beds and on surfaces at workstations, indicating potential for environment-to-patient, patient-to-patient and healthcare worker-to-patient transmission. CONCLUSIONS: Surveillance in hospital settings through environmental sampling may be an important component of fungal infection prevention.

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.

Tobacco and e-cigarette smoking habits among Italian healthcare students.

BACKGROUND: Healthcare professionals might play a significant role in tobacco control. The aims of this study were to investigate tobacco cigarette and electronic cigarette (e-cigarette) consumption among university students enrolled in courses of the healthcare area, and to understand whether training in healthcare could induce to smoking cessation. STUDY DESIGN: Cross-sectional survey using a self-administered, structured questionnaire. METHODS: Tobacco smoking habits of 560 students of four different medical area courses at the University of Milan, enrolled both in the first and in the last course year during the 2017-2018 academic year, were collected. RESULTS: The prevalence of smokers was 34.8%, almost the same for males and females, and higher in Italian students compared to foreigners. Smoking prevalence was higher among employed (46.9%) than unemployed (35.1%) students, without differences between healthcare and non-healthcare workers. The influence of family, and particularly friends, is confirmed to be relevant. About 25% of respondents tried e-cigarettes, mainly smokers (44.6%) and former smokers (38.6%) with the goal of quitting smoking. More than 44% were dual users of both tobacco cigarettes and e-cigarettes. Comparing smoking habits between first-year and final-year students, only students of healthcare assistance course showed a significant drop in smoking during the university studies. CONCLUSIONS: The implementation of specific educational curricula on smoking dangers and on smoking cessation techniques might have the double effect of supporting students in quitting smoking and of properly preparing them for their future task of helping people to quit smoking.

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.

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.

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.

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.

Trichophyton concentricum in skin lesions in children from the Salomon Islands.

AIM: Aim of the paper was to report cases of Tinea imbricata, a mycosis caused by the anthropophilic dermatophyte Trichophyton concentricum, observed in 2012 in Guadalcanal, the largest of the Salomon islands. METHODS: During 2012, several cases of Tinea imbricata, called bakwa by local people, were observed in the Little Samaritan Hospital in Guadalcanal. Skin scrapings collected from three young patients were examined in Italy to confirm the clinical diagnosis. The fungus grown on culture was morphologically identified and submitted to sequencing of the ITS1-ITS2 region. RESULTS: The diagnosis obtained by visual inspection of the skin lesions, characterised by concentric and lamellar plaques of scale often involving large part of the body, was confirmed mycological investigations. A prevalence of 15% of Tinea imbricata in this population was hypothesized. The fungus grown on culture was morphologically identified as Trichophyton concentricum and identification was confirmed sequencing the ITS1-ITS2 region. Patients were treated with potassium permanganate solution soaked gauze followed by colloidal sulfur and salicylic acid cream application. However, the efficacy of the antifungal treatment was difficult to evaluate due to the poor compliance of the patients and the remoteness of the villages. CONCLUSION: Italian clinicians and mycologists should be aware of this fungal infection because the increased number of international travels and of migration rise the spread of infections previously restricted to limited geographical areas.

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.