Catheter-related blood stream infection caused by Millerozyma farinosa in an immunocompetent patient: a case report and a brief review of the literature.

Millerozyma farinose is a halotolerant yeast that has recently been described as an emerging human pathogen, especially in immunocompromised patients. Both the diagnostic process and treatment options are still unclear. Here, we report a case of an immunocompetent oncological patient who developed a catheter-related bloodstream infection (CRBSI) with a concomitant respiratory tract infection caused by M. farinosa. In this report, we discuss how prompt microbiological identification and attentive evaluation of the patient's clinical status can play a significant role in the appropriate management of infections caused by uncommon fungi. MALDI-TOF technology has also substantially improved the timely diagnosis of rare fungi. Furthermore, our diagnosis was subsequently confirmed by 5.8S rRNA sequencing. In our patient, the rapid diagnosis of fungaemia was crucial, together with catheter removal and the initiation of antifungal treatment, for the patient's clinical improvement.

Rifaximin use favoured micafungin-resistant Candida spp. infections in recipients of allogeneic hematopoietic cell transplantation.

Damage to gut mucosa following conditioning regimens may favour bacterial infections that can trigger graft versus host disease (GvHD) in patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT). Rifaximin, an oral and non-absorbable antibiotic, has been recently proposed as effective prophylaxis to reduce bacterial infections in the gut and consequently acute GvHD in this setting. The present study evaluated safety and outcomes of HSCT patients that were treated with rifaximin prophylaxis at Perugia University Hospital. Rifaximin prophylaxis was introduced as standard of care in HSCT patients in May 2018. We retrieved data from 118 consecutive transplants, and we compared the outcomes of rifaximin-treated patients with historical controls that did not receive antibiotic prophylaxis. While incidences of neutropenic fever, documented bacterial infections, and aGvHD were similar in the two groups, we found an increased frequency of invasive candidiasis and clinically relevant Candida spp. infections in rifaximin-treated patients (5 patients vs 1 patient, 25% [± 0.99%] vs 1% [± 0.01%], p < .0001). Three rifaximin-treated patients experienced life-threating candidemia (2 C. krusei, 1 C. orthopsilosis). Rifaximin was the only factor that increased the risk of Candida spp. infections. Rifaximin could have contributed to microbiome disruption which favoured an outbreak of life-threatening Candida infections. This important complication forced us to halt its use. Larger, prospective studies are needed to assess the impact of rifaximin prophylaxis on incidence of bacterial infections, aGvHD, and survival of HSCT patients.

Azole resistance in Aspergillus isolates by different types of patients and correlation with environment - An Italian prospective multicentre study (ARiA study).

BACKGROUND: A wide range of frequency of azole-resistance in A fumigatus in different patient populations worldwide was observed threatening to reduce therapeutic options. OBJECTIVES: Estimate the prevalence of azole-resistance, investigate the molecular mechanisms of resistance, compare the genotypes of resistant clinical isolates with those from the surrounding environment. METHODS: Aspergillus isolates were collected by seven Italian hospital microbiology laboratories. Strains were isolated from different clinical samples from unselected patients. The azole-resistance was evaluated using screening test and microdilution EUCAST method. The molecular mechanism of resistance was performed sequencing the cyp51A gene. Resistant isolates were genotyped by microsatellite analysis and their profiles compared with those of azole-resistant isolates from previous Italian studies. RESULTS: 425 Aspergillus isolates from 367 patients were analysed. The azole-resistance rates were 4.9% and 6.6% considering all Aspergillus spp. isolates and the A fumigatus sensu stricto, respectively. All resistant isolates except one were from a single hospital. Two rare azole-resistant species were identified: A thermomutatus and A lentulus. The predominant resistance mechanism was TR34 /L98H. No correlation between the clinical resistant strains and environmental isolates from patients' home/work/ward was observed. The analysis of the molecular correlation between the resistant clinical strains collected in the present study and those of environmental and clinical origin collected in previous Italian studies reveals a progressive diversification of azole-resistant genotypes starting from a founder azole-resistant genotype. CONCLUSIONS: This study confirms the trend of azole-resistance rate in Italy, showing a geographical difference. Data reinforce the importance of surveillance programmes to monitor the local epidemiological situation.

Yeast-like filamentous fungi: Molecular identification and in vitro susceptibility study.

Yeast-like filamentous fungi, collected in Italy from 1985 to 2018, were submitted to molecular identification and antifungal susceptibility testings. Clinical isolates were identified as Magnusiomyces capitatus (28), M. clavatus (18), and Geotrichum candidum (2). M. clavatus was prevalent among blood isolates (18/24), M. capitatus among isolates from other biological materials. The intrinsic echinocandin resistance was confirmed. Both species had low minimum inhibitory concentrations (MICs) of itraconazole, posaconazole, and voriconazole, while M. clavatus had lower MIC of flucytosine and higher MIC of isavuconazole than M. capitatus. The intrinsic resistance of these species to echinocandins could be the reason of the recent increase of M. clavatus bloodstream infections.

Epidemiological trends of cryptococcosis in Italy: Molecular typing and susceptibility pattern of Cryptococcus neoformans isolates collected during a 20-year period.

In the present study clinical data and isolates from cases of cryptococcosis recorded during clinical surveys carried out in Italy from 1997 to 2016, were investigated. Molecular typing and antifungal susceptibility testing were performed in order to delineate the epidemiological trend of cryptococcosis in Italy and to define wild-type population for four different antifungal compounds. During the studied period, a total of 302 cases collected from 32 centers of 11 Italian regions were recorded. Analysis of clinical data showed a significant increase of frequency (from 7% to 38%) of cryptococcosis in human immunodeficiency virus (HIV)-negative patients primarily with hematologic malignancies and solid organ transplantations. The prevalence of the molecular types has significantly changed during the study period, showing an increase of VNIII isolates from 11% to 41% in HIV-negative patients, and a decrease of VNIV isolates from 36% to 16%. Antifungal susceptibility testing allowed us to calculate the epidemiological cut-off for flucytosine (1 mg/l), fluconazole (8 mg/l), itraconazole (0.5 mg/l), and voriconazole (0.25 mg/l). Most of the isolates were wild-type strains. Comparison of the MIC distributions according to molecular types showed that VNIV isolates had lower MICs for fluconazole and itraconazole than the VNI and VIII isolates. The current study emphasizes that the epidemiology of cryptococcosis in Italy has significantly changed over the last decades.

Evaluation of Real Time PCR Aspergillus spp. in bronchoalveolar lavage samples.

The present study investigated the improvement in the diagnosis of invasive pulmonary aspergillosis (IPA) adding a molecular test on bronchoalveolar lavage (BAL) to the routine diagnostic approach including microscopy, culture and galactomannan (GM) immunoassay. A total of 133 BAL samples were retrospectively tested for the Aspergillus DNA: 112 samples were from immunocompromised patients at risk of invasive fungal infection and 21 were from patients not at risk and without clinical evidence of IPA. The latter samples were used to identify the cut-off of positivity for the molecular test. Applying the cut-off quantity of 50 copies/reaction, the PCR test had 90% sensitivity and 97% specificity and resulted the most sensitive, specific and accurate among those evaluated. The statistical analysis showed that the probability that a patient is not affected by IPA is 99% when the three tests (PCR, GM and culture) are concordantly negative.

Azole Resistance in Aspergillus fumigatus Clinical Isolates from an Italian Culture Collection.

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.

CAND-LO 2014-15 study: changing epidemiology of candidemia in Lombardy (Italy).

PURPOSE: The aim of this study was to monitor recent changes in the epidemiology of candidemia and in the antifungal susceptibility profiles of Candida isolates in one Italian region (Lombardy) in 2014-2015 in comparison with two other studies performed in the same area in 1997-1999 and in 2009. METHODS: A laboratory-based surveillance was conducted in 11 microbiology laboratories. Identification of Candida isolates from 868 episodes and antifungal susceptibility testing (YeastOne) was performed locally. RESULTS: A progressive increase in the rate of candidemia up to 1.27/1000 admissions and 1.59/10,000 patient days was documented. In all the three surveys, Candida albicans remains the most frequently isolated species, ranging from 52 to 59 % of the etiology of BSIs. The epidemiological shift to the more resistant C. glabrata, observed between 1997-1999 and 2009 surveys, was not confirmed by our more recent data. The pattern of etiology of BSIs occurred in 2014-2015 overlaps that of the 90s. Acquired antifungal resistance is a rare event. No isolate had an amphoterin B minimal inhibitory concentration (MIC, mg/L) value higher than the epidemiological cutoff. All the echinocandin MIC distributions are typical for wild-type organisms except for those of two C. glabrata isolates. Fluconazole resistance declined from 24.9 % in the 2009 survey to 5.4 % in the recent one. CONCLUSIONS: Data from regional surveys may highlight the influence of therapeutic practices on the epidemiology of Candida BSIs and may optimize empirical therapies.

Acquired echinocandin resistance in a Candida krusei blood isolate confirmed by mutations in the fks1 gene.

We describe a case of bloodstream infection caused by a Candida krusei strain that developed echinocandin resistance during caspofungin therapy. Three mutations were found in the HS1 region of the fks1 gene, two of them have never been reported either in C. krusei nor in C. albicans.

Yeast Bloodstream Infections in the COVID-19 Patient: A Multicenter Italian Study (FiCoV Study).

Fungemia is a co-infection contributing to the worsening of the critically ill COVID-19 patient. The multicenter Italian observational study FiCoV aims to estimate the frequency of yeast bloodstream infections (BSIs), to describe the factors associated with yeast BSIs in COVID-19 patients hospitalized in 10 hospitals, and to analyze the antifungal susceptibility profiles of the yeasts isolated from blood cultures. The study included all hospitalized adult COVID-19 patients with a yeast BSI; anonymous data was collected from each patient and data about antifungal susceptibility was collected. Yeast BSI occurred in 1.06% of patients, from 0.14% to 3.39% among the 10 participating centers. Patients were mainly admitted to intensive or sub-intensive care units (68.6%), over 60 years of age (73%), with a mean and median time from the hospitalization to fungemia of 29 and 22 days, respectively. Regarding risk factors for fungemia, most patients received corticosteroid therapy during hospitalization (61.8%) and had a comorbidity (25.3% diabetes, 11.5% chronic respiratory disorder, 9.5% cancer, 6% haematological malignancies, 1.4% organ transplantation). Antifungal therapy was administered to 75.6% of patients, mostly echinocandins (64.5%). The fatality rate observed in COVID-19 patients with yeast BSI was significantly higher than that of COVID-19 patients without yeast BSI (45.5% versus 30.5%). Candida parapsilosis (49.8%) and C. albicans (35.2%) were the most fungal species isolated; 72% of C. parapsilosis strains were fluconazole-resistant (range 0-93.2% among the centers). The FiCoV study highlights a high prevalence of Candida BSIs in critically ill COVID-19 patients, especially hospitalized in an intensive care unit, a high fatality rate associated with the fungal co-infection, and the worrying spread of azole-resistant C. parapsilosis.

Fungal diversity in lake and sea beaches of Italy: Relevance to human health.

Fungal diseases correlated to beach sand or water have not yet been demonstrated due to the lack of epidemiological studies. This study aims to illustrate the fungal population in beach sands of the two largest Italian lakes and in sands and waters of Mediterranean coasts of Southern Italy to contribute to the identification and assessment of causes of microbiological pollution that might impair bathers health. A great difference was observed between the two lakes, where the total of colony-forming units (CFU) ranged from 33.3 to 1049.9 CFU/g. For coastal sands, the total CFU ranged from 216.7 to 538.8 CFU/g, and for coastal waters the total ranged from 185 to 368.7 CFU/ml. The survey revealed the prevalence of opportunistic pathogenic moulds, mainly Aspergillus spp. (A. niger and A. fumigatus) and Penicillium spp., both in freshwater and costal bathing sites. Dermatophytes and yeasts were not detected in the freshwater sands while they were found at low load in coastal waters (3.3 CFU/ml) and sands (1.7 CFU/g). Differences were observed between urban and non-urban coastal beaches with regard to isolation of dermatophytes only from one urban beach. The present study reports a great diversity of fungi in sand and water of bathing beaches confirming that the Mediterranean region has a greater variety of fungal species.

Cross-reactivity of Fusarium spp. in the Aspergillus Galactomannan enzyme-linked immunosorbent assay.

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.

Exploring Mitogenomes Diversity of Fusarium musae from Banana Fruits and Human Patients.

Fusarium musae has recently been described as a cross-kingdom pathogen causing post-harvest disease in bananas and systemic and superficial infection in humans. The taxonomic identity of fungal cross-kingdom pathogens is essential for confirming the identification of the species on distant infected hosts. Understanding the level of variability within the species is essential to decipher the population homogeneity infecting human and plant hosts. In order to verify that F. musae strains isolated from fruits and patients are part of a common population and to estimate their overall diversity, we assembled, annotated and explored the diversity of the mitogenomes of 18 F. musae strains obtained from banana fruits and human patients. The mitogenomes showed a high level of similarity among strains with different hosts' origins, with sizes ranging from 56,493 to 59,256 bp. All contained 27 tRNA genes and 14 protein-coding genes, rps3 protein, and small and large ribosomal subunits (rns and rnl). Variations in the number of endonucleases were detected. A comparison of mitochondrial endonucleases distribution with a diverse set of Fusarium mitogenomes allowed us to specifically discriminate F. musae from its sister species F. verticillioides and the other Fusarium species. Despite the diversity in F. musae mitochondria, strains from bananas and strains from human patients group together, indirectly confirming F. musae as a cross-kingdom pathogen.

In Vitro Antifungal Activity of Ibrexafungerp (SCY-078) Against Contemporary Blood Isolates From Medically Relevant Species of Candida: A European Study.

Background: Ibrexafungerp (SCY-078) is the newest oral and intravenous antifungal drug with broad activity, currently undergoing clinical trials for invasive candidiasis. Objective: The aim of this study was to assess the in vitro activity of ibrexafungerp and comparators against a collection of 434 European blood isolates of Candida. Methods: Ibrexafungerp, caspofungin, fluconazole, and micafungin minimum inhibitory concentrations (MICs) were collected from 12 European laboratories for 434 blood isolates, including 163 Candida albicans, 108 Candida parapsilosis, 60 Candida glabrata, 40 Candida tropicalis, 29 Candida krusei, 20 Candida orthopsilosis, 6 Candida guilliermondii, 2 Candida famata, 2 Candida lusitaniae, and 1 isolate each of Candida bracarensis, Candida catenulata, Candida dubliniensis, and Candida kefyr. MICs were determined by the EUCAST broth microdilution method, and isolates were classified according to recommended clinical breakpoints and epidemiological cutoffs. Additionally, 22 Candida auris from different clinical specimens were evaluated. Results: Ibrexafungerp MICs ranged from 0.016 to ≥8 mg/L. The lowest ibrexafungerp MICs were observed for C. albicans (geometric MIC 0.062 mg/L, MIC range 0.016-0.5 mg/L) and the highest ibrexafungerp MICs were observed for C. tropicalis (geometric MIC 0.517 mg/L, MIC range 0.06-≥8 mg/L). Modal MICs/MIC50s (mg/L) against Candida spp. were 0.125/0.06 for C. albicans, 0.5/0.5 for C. parapsilosis, 0.25/0.25 for C. glabrata, 0.5/0.5 for C. tropicalis, 1/1 for C. krusei, 4/2 for C. orthopsilosis, and 0.5/0.5 for C. auris. Ibrexafungerp showed activity against fluconazole- and echinocandin-resistant isolates. If adopting wild-type upper limits, a non-wild-type phenotype for ibrexafungerp was only observed for 16/434 (3.7%) isolates: 11 (4.6%) C. parapsilosis, 4 (5%) C. glabrata, and 1 (2.5%) C. tropicalis. Conclusion: Ibrexafungerp showed a potent in vitro activity against Candida.

Fusarium musae from Diseased Bananas and Human Patients: Susceptibility to Fungicides Used in Clinical and Agricultural Settings.

Fusarium musae belongs to the Fusarium fujikuroi species complex. It causes crown rot disease in banana but also keratitis and skin infections as well as systemic infections in immunocompromised patients. Antifungal treatments in clinical and agricultural settings rely mostly on molecules belonging to the azole class. Given the potential risk of pathogen spread from food to clinical settings, the goal of the work was to define the level of susceptibility to different azoles of a worldwide population of F. musae. Eight fungicides used in agriculture and five antifungals used in clinical settings (4 azoles and amphotericin B) were tested using the CLSI (Clinical and Laboratory Standards Institute) protocol methodology on 19 F. musae strains collected from both infected patients and bananas. The level of susceptibility to the different active molecules was not dependent on the source of isolation with the exception of fenbuconazole and difenoconazole which had a higher efficiency on banana-isolated strains. Minimal inhibitory concentrations (MICs) of the different molecules ranged from 0.12-0.25 mg/L for prochloraz to more than 16 mg/L for tetraconazole and fenbuconazole. Compared to the F. verticillioides, F. musae MICs were higher suggesting the importance of monitoring the potential future spread of this species also in clinical settings.

Candidemia: Evolution of Drug Resistance and Novel Therapeutic Approaches.

Candidemia and invasive candidiasis are the most common healthcare-associated invasive fungal infections, with a crude mortality rate of 25-50%. Candida albicans remains the most frequent etiology, followed by C. glabrata, C. parapsilosis and C. tropicalis. With the exception of a limited number of species (ie: C. krusei, C. glabrata and rare Candida species), resistance to fluconazole and other triazoles are quite uncommon. However, recently fluconazole-resistant C. parapsilosis, echinocandin-resistant C. glabrata and the multidrug resistant C. auris have emerged. Resistance to amphotericin B is even more rare due to the reduced fitness of resistant isolates. The mechanisms of antifungal resistance in Candida (altered drug-target interactions, reduced cellular drug concentrations, and physical barriers associated with biofilms) are analyzed. The choice of the antifungal therapy for candidemia must take into account several factors such as type of patient, presence of devices, severity of illness, recent exposure to antifungals, local epidemiology, organs involvement, and Candida species. The first-line therapy in non-neutropenic critical patient is an echinocandin switching to fluconazole in clinically stable patients with negative blood cultures and azole susceptible isolate. Similarly, an echinocandin is the drug of choice also in neutropenic patients. The treatment duration is 14 days after the first negative blood culture or longer in cases of organ involvement. An early removal of vascular catheter improves the outcome. The promising results of new antifungal molecules, such as the terpenoid derivative ibrexafungerp, the novel echinocandin with an enhanced half-life rezafungin, oteseconazole and fosmanogepix, representative of new classes of antifungals, are discussed.

Cryptococcus neoformans and Cryptococcus gattii Species Complex Isolates on the Slopes of Mount Etna, SICILY, Italy.

This study investigated the presence of Cryptococcus neoformans and Cryptococcus gattii species complex isolates on olive trees growing in the Eastern part of Sicily (Italy) characterized by the presence of the volcano Etna and the ability of these fungal pathogens to sexually reproduce on medium containing volcanic soil. Samples from 124 olive trees were collected from 14 different sites around Mount Etna. Eighteen trees (14.5%) resulted colonized by C. neoformans VNI-αA isolates, one (0.8%) by VNIV-αD isolates, and two (1.6%) by C. gattii VGI-αB isolates. The ability of environmental and reference strains belonging to VNI, VNIV, and VGI molecular types to sexually reproduce on a medium containing volcanic soil was also tested. VNI and VNIV strains were able to produce filaments and basiodiospores more vigorously than on the control medium, whereas VGI strains were not fertile. In conclusion, the present study identified which C. neoformans and C. gattii species complex genotypes are circulating in Eastern Sicily and confirmed the ecological role of olive trees as environmental reservoir of these pathogens. It also showed that Cryptococcus is able to colonize and sexually reproduce in inhospitable environments such as the slopes of a volcano.

Azole-resistant Aspergillus fumigatus in the Italian environment.

OBJECTIVES: Azole resistance in Aspergillus fumigatus environmental and clinical isolates is recognised as an emerging problem worldwide. Development of azole resistance may be environmentally driven because of the massive use of azole fungicides in agriculture. The mechanism of azole resistance is mostly related to mutations in the cyp51A gene. METHODS: A. fumigatus azole resistance in the environment was previously documented in northern Italy. This study extended the research in the agricultural environment also in central and southern Italy and investigated differences in the Italian geographical areas and in the different types of crops. RESULTS: A total of 177 samples (173 soil samples and 4 Dutch bulbs) collected in the period 2014-20117 in 14 Italian regions were analysed. Itraconazole-resistant A. fumigatus isolates grew in 16.9% of the screened samples. Differences were observed in soil samples from the three Italian geographic areas: 12.5% in the north, 15.2% in the centre and 24.1% in the south. Resistant isolates were from different cultivations, treated or officially not treated with azole fungicides. Sequencing of the cyp51A gene confirmed that resistance was mainly associated with the TR34/L98H mutation (29/30 isolates); 1 isolate showed the G54E mutation. CONCLUSIONS: The risk for patients to acquire multi-azole-resistant strains from the environment could have a serious impact on the management of life-threatening invasive infections. The azole resistance rate of 16.9% found in Italy requires suitable monitoring of antifungal susceptibility of clinical isolates.

Species distribution and in vitro antifungal susceptibility patterns of 75 clinical isolates of Fusarium spp. from northern Italy.

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.