Anna Prigitano

Cross-Reactivity of Fusarium spp. in the Aspergillus Galactomannan Enzyme-Linked Immunosorbent Assay

ABSTRACT Nine of 11 hematological patients with disseminated/deep-seated Fusarium infection tested at least twice for Aspergillus galactomannan (GM) had repeated positive results in the absence of Aspergillus isolation in culture. The centrifuged supernatants of 12 Fusarium isolates were tested by a GM enzyme-linked immunosorbent assay (EIA). All the isolates produced positive reactions when tested undiluted. These results show cross-reactivity of Fusarium spp. with Aspergillus GM that may constitute a drawback with respect to the specificity of the Platelia EIA.

INVASIVE FUNGAL INFECTIONS IN THE INTENSIVE CARE UNIT: A MULTICENTRE, PROSPECTIVE, OBSERVATIONAL STUDY IN ITALY (2006-2008)

Critically ill patients admitted to intensive care units (ICU) are highly susceptible to healthcare‐associated infections caused by fungi. A prospective sequential survey of invasive fungal infections was conducted from May 2006 to April 2008 in 38 ICUs of 27 Italian hospitals. A total of 384 fungal infections (318 invasive Candida infections, three cryptococcosis and 63 mould infections) were notified. The median rate of candidaemia was 10.08 per 1000 admissions. In 15% of cases, the infection was already present at the time of admission to ICU. Seventy‐seven percent of Candida infections were diagnosed in surgical patients. Candida albicans was isolated in 60% of cases, Candida glabrata and Candida parapsilosis in 13%, each. Candida glabrata had the highest crude mortality rate (60%). Aspergillus infection was diagnosed in 32 medical and 25 surgical patients. The median rate was 6.31 per 1000 admissions. Corticosteroid treatment was the major host factor. Aspergillosis was demonstrated to be more severe than candidiasis as the crude mortality rate was significantly higher (63% vs. 46%), given an equal index of severity, Simplified Acute Physiology Score (SAPS‐II). The present large nationwide survey points out the considerable morbidity and mortality of invasive fungal infections in surgical as well as medical patients in ICU.

Species Distribution and In Vitro Antifungal Susceptibility Patterns of 75 Clinical Isolates of Fusarium spp. from Northern Italy

ABSTRACT Fusarium isolates from 75 Italian patients were identified by molecular methods, and their susceptibilities to antifungals were tested in vitro. Fusarium verticillioides was the species most frequently isolated from deep-seated infections, and F. solani was the species most frequently isolated from superficial infections. F. solani isolates showed high azole MICs, while F. verticillioides isolates showed low posaconazole MICs.

In vitro activity of conventional antifungal drugs and natural essences against the yeast-like alga Prototheca

BACKGROUND Two outbreaks of mastitis due to the yeast-like alga Prototheca zopfii recently occurred in dairy herds in Lombardia (Italy) involving 180 and 150 lactating cows, respectively. OBJECTIVES To determine the in vitro susceptibility of Prototheca isolates to conventional antifungal agents and to essential oils. METHODS Twenty P. zopfii isolated from milk during these outbreaks, six P. zopfii isolated from fresh water and two Prototheca sp. reference strains were submitted to antifungal susceptibility testing by broth microdilution assay following the CLSI guidelines for yeasts. RESULTS The tested isolates were shown to be resistant to fluconazole and caspofungin. A wide range of voriconazole MICs was observed. In contrast, amphotericin B, itraconazole and posaconazole appeared active with MICs < or = 1 mg/L. Bergamot and tea tree oils seemed to exert an interesting activity against this yeast-like alga. CONCLUSIONS Difficulties in treating animals with conventional drugs and the potent in vitro activity of essential oils demonstrated here raise the interest in further investigations on the therapeutic use of these non-conventional natural products.

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.

Four-Year Persistence of a Single Candida albicans Genotype Causing Bloodstream Infections in a Surgical Ward Proven by Multilocus Sequence Typing

ABSTRACT The present study represents the first application of multilocus sequence typing to retrospectively investigate a suspected outbreak of Candida albicans bloodstream infection cases that occurred in the same hospital ward between July 1987 and October 1991. Results demonstrated that eight bloodstream infections were caused by the same strain, endemic in the ward, over a 4-year period.

Azole resistance in Aspergillus fumigatus clinical isolates from an Italian culture collection

ABSTRACT The aims of the study were to investigate the prevalence of azole resistance among Aspergillus fumigatus clinical isolates. A total of 533 clinical isolates that had been collected between 1995 and 2006, from 441 patients, were screened. No resistance was detected in isolates collected between 1995 and 1997. Starting in 1998, the resistance rate was 6.9%; a total of 24 patients (6.25%) harbored a resistant isolate. The TR34/L98H substitution was found in 21 of 30 tested isolates.

In vitro activity of amphotericin B against Aspergillus terreus isolates from different countries and regions

Invasive aspergillosis is the most common infection caused by filamentous fungi which is observed in immunocompromised patients. Amphotericin B deoxycholate was considered the “gold standard” for therapy of invasive aspergillosis for more than three decades. However, the recently published American and European guidelines recommend intravenous or oral voriconazole as primary treatment and lipid-based amphotericin B as an alternative 1, 2. Aspergillus fumigatus is the Aspergillus species most commonly associated with invasive aspergillosis, A. flavus is second most common, and A. terreus accounts for a minority of cases. However, an increasing frequency of this last species has been reported in some institutions, where it was isolated from up to 12.5% of the cases 3,4. In addition, the A. terreus resistance to amphotericin B observed in vitro 5,6 and in vivo 7,8 represents a serious concern. The aim of the present study was to investigate the in vitro susceptibility to amphotericin B of A. terreus patient isolates from the USA and Europe. A total of 145 A. terreus clinical isolates, mainly from respiratory tract and nails, were analysed. The isolates were identified on the basis of morphologic characters. Ninety-seven were collected in Europe, that is 61 from Italy (Igiene Università Milano, IUM collection) and 37 from Belgium and France (IHEM collection at Scientific Institute of Public Health, Bruxelles, Belgium). A total of 47 were collected in USA, and among these 24 were from the West Coast and 23 from the northeastern section of the country. The in vitro susceptibility to amphotericin B was investigated by the broth microdilution method, according to the Clinical Laboratory Standards Institute (formerly the National Committee Clinical Laboratory Standards) reference method for filamentous fungi 9. Tests were performed in the Department of Public Health-Microbiology-Virology, University of Milan, Italy. The A. terreus conidia were harvested after the isolates were subcultured on potato dextrose agar at 35°C for 3-5 days and suspended in normal saline containing 0.025% Tween 20. The conidial suspensions were counted with a hemocytometer and the inocula adjusted to a final concentration of 4 x 103 to 5 x 104 conidia/ml. Amphotericin B (Sigma, Milano, Italy) was dissolved in dimethyl sulfoxide and then diluted twofold to final concentrations ranging from 0.03 to 16 mg/L 9. The dilutions in the microtiter plates were prepared and used for testing within 24 hours. Testing was performed in RPMI 1640 without sodium bicarbonate (Sigma), buffered to pH 7.0 with 0.165 M MOPS (Sigma) supplemented with 0.03% Lglutamine (Sigma). Reference strains Candida parapsilosis ATCC 22019 and C. krusei ATCC 6258 were used as quality control isolates for susceptibility testing. Petromyces alliaceus IUM 81-713 was included in each testing session as an amphotericin B-resistant control. Tests were performed in duplicate. Isolates showing different results in the two runs were tested again and the MIC most frequently obtained was utilized. The minimal inhibitory concentration (MIC, mg/L) endpoint was the lowest drug concentration showing no visible growth after 48 hours of incubation at 35°C. Bartlett’s test was used to test the homogeneity of variances across geographic groups and Kruskal-Wallis test to compare the groups showing statistically significant differences. Table 1 shows the MIC profiles of the isolates by geographic origin. Amphotericin B at a concentration ≤1 mg/L was able to inhibit the growth of 97% (59 out of 61) of the isolates from Italy, and 84% of the isolates from Belgium and France. The American isolates, 96% of which from the West Coast but only 61% from the Northeast, were inhibited by this amphotericin B concentration. In this last group four isolates were clearly resistant, being inhibited only by a concentration of 4 and 16 mg/L. These results show a different pattern of susceptibility to amphotericin B of A. terreus isolates from difREPRINT

Antifungal susceptibility profiles of Candida isolates from a prospective survey of invasive fungal infections in Italian intensive care units

The antifungal susceptibility pattern of 302 Candida isolates collected during an Italian survey on invasive fungal infections in an intensive care setting was investigated. The results were correlated with some epidemiological data and compared with the antifungal profiles obtained in a previous survey. No resistance to echinocandins was detected. The overall resistance levels to fluconazole, posaconazole and voriconazole were 12.6, 6.0 and 7.1 %, respectively. Candida tropicalis and Candida parapsilosis accounted for more than half of all the fluconazole resistant isolates. Reduced susceptibility to fluconazole is not uncommon among isolates (12.3 %) and appears to be increasing, particularly among C. parapsilosis isolates, which showed an increase in resistant isolates from 2 % in the 1990s to 25.8 % in the present study. Routine antifungal susceptibility testing of this species is therefore recommended.

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.