The Molecular Identification and Antifungal Susceptibility of Clinical Isolates of Aspergillus Section Flavi from Three French Hospitals.

(1) Background: Aspergillus flavus is a cosmopolitan mold with medical, veterinary, and agronomic concerns. Its morphological similarity to other cryptic species of the Flavi section requires molecular identification techniques that are not routinely performed. For clinical isolates of Aspergillus section Flavi, we present the molecular identification, susceptibility to six antifungal agents, and clinical context of source patients. (2) Methods: One hundred forty fungal clinical isolates were included in the study. These isolates, recovered over a 15-year period (2001-2015), were identified based on their morphological characteristics as belonging to section Flavi. After the subculture, sequencing of a part of the ß-tubulin and calmodulin genes was performed, and resistance to azole antifungals was screened on agar plates containing itraconazole and voriconazole. Minimum inhibitory concentrations were determined for 120 isolates by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution method. (3) Results: Partial ß-tubulin and calmodulin sequences analysis showed that 138/140 isolates were A. flavus sensu stricto, 1 isolate was A. parasiticus/sojae, and 1 was A. nomiae. Many of the isolates came from samples collected in the context of respiratory tract colonization. Among probable or proven aspergillosis, respiratory infections were the most frequent, followed by ENT infections. Antifungal susceptibility testing was available for isolates (n = 120, all A. flavus ss) from one hospital. The MIC range (geometric mean MIC) in mg/L was 0.5-8 (0.77), 0.5-8 (1.03), 0.125-2 (0.25), 0.03-2 (0.22), 0.25-8 (1.91), and 0.03-0.125 (0.061) for voriconazole, isavuconazole, itraconazole, posaconazole, amphotericin B, and caspofungin, respectively. Two (1.67%) isolates showed resistance to isavuconazole according to current EUCAST breakpoints with MICs at 8 mg/L for isavuconazole and voriconazole. One of these two isolates was also resistant to itraconazole with MIC at 2 mg/L. (4) Conclusions: The present characterization of a large collection of Aspergillus belonging to the Flavi section confirmed that A. flavus ss is the predominant species. It is mainly implicated in respiratory and ENT infections. The emergence of resistance highlights the need to perform susceptibility tests on section Flavi isolates.

Occurrence and species diversity of human-pathogenic Mucorales in commercial food-stuffs purchased in Paris area.

Mucormycoses are life-threatening fungal diseases that affect a variety of patients including those with diabetes mellitus or hematological malignancies. The responsible agents, the Mucorales, are opportunistic pathogens originating from the environment such as soil or decaying organic matter. The aim of the present study was to assess the prevalence and diversity of human-pathogenic species of Mucorales in commercially available foodstuffs in France. All food samples were purchased from January 2014 to May 2015 in France. A total of 159 dried food samples including spices and herbs (n = 68), herbal tea (n = 19), cereals (n = 19), vegetables (n = 14), and other foodstuffs (n = 39) were analyzed. Each strain of Mucorales was identified phenotypically, and molecular identification was performed by ITS sequencing. From the 28 (17.6%) samples that were culture-positive for Mucorales, 30 isolates were recovered. Among the isolates, 13 were identified as Rhizopus arrhizus var. arrhizus, 10 R. arrhizus var. delemar, two Rhizopus microsporus, one Lichtheimia corymbifera, three Lichtheimia ramosa, and one Syncephalastrum racemosum. Culture-positive samples originated from different countries (Europe, Asia) and brands. The samples most frequently contaminated by Mucorales were spices and herbs (19/68, 27.9%), followed by herbal tea (2/19, 10.5%), cereals (2/19, 10.5%), other food products (5/39, 12.8%). The present study showed that human-pathogenic Mucorales were frequently recovered from commercially available foodstuffs in France with a large diversity of species. The potential danger represented by Mucorales present in food for immunocompromised patients should be further analyzed.

In vitro combination of voriconazole with micafungin against azole-resistant clinical isolates of Aspergillus fumigatus from different geographical regions.

In vitro interaction of voriconazole with micafungin was evaluated against 33 clinical Aspergillus fumigatus isolates, including azole-resistant (n=31) and -susceptible (n=2) isolates. Interaction was synergistic for only 1 resistant isolate carrying the TR34/L98H mutation. No antagonistic effects were observed for 96.8% of azole-resistant isolates.

Green synthesis of silver nanoparticles using Artemisia turcomanica leaf extract and the study of anti-cancer effect and apoptosis induction on gastric cancer cell line (AGS).

The current study was aimed (1) to study the green synthesis of silver nanoparticles using Artemisia turcomanica leaf extract, (2) to investigate the induction of apoptosis by biologically synthesized silver nanoparticles in gastric cancer cell line (AGS) and (3) to compare their anti-cancer potential with commercial silver nanoparticles. The specification and morphology of the phytosynthesized AgNPs were evaluated using transmission electron microscopy (TEM), scanning electron microscopy (SEM), UV-visible spectroscopy, X-ray diffraction and Fourier transform infrared spectroscopy (FTIR). The nanoparticles synthesized were of an average size of 22 nm. The cytotoxicity of biological and commercial nanoparticles was investigated in gastric cancer cells (AGS) as well as normal fibroblast cells (L-929) by MTT assay. By increasing the concentration of phytosynthesized and commercial silver nanoparticles, a decrease was observed in the cell viability. Increased apoptosis was observed in the cells treated with biological silver nanoparticles compared to untreated cells (p < .001). Based on these findings, it was inferred that biologically synthesized silver nanoparticles induced apoptosis, and showed a cytotoxic and anti-cancer effect against gastric cancer cell lines in a dose- and time-dependent manner. Biologically synthesized nanoparticles may possess higher anti-cancer properties than commercial silver nanoparticles.

cyp51A gene silencing using RNA interference in azole-resistant Aspergillus fumigatus.

An increasing number of reports have described the emergence of acquired resistance of Aspergillus fumigatus to azole compounds. The primary mechanism of resistance in clinical isolates is the mutation of the azole drug target enzyme, which is encoded by the cyp51A gene. The aim of this study was to evaluate the impact of silencing the cyp51A gene in azole-resistant A. fumigatus isolates. A 21-nucleotide small-interfering RNA (siRNA) was designed based on the cDNA sequence of the A. fumigatus cyp51A gene. After silencing the cyp51A gene in germinated conidia (15, 20, 25 and 50 nM), azole-resistant A. fumigatus was cultured on broth media and gene expression was analysed by measuring the cyp51A mRNA level using RT-PCR assay. Hyphae were successfully transfected by siRNA and expression of the cyp51A gene was significantly reduced by siRNA at the concentration of 50 nM (P ≤ 0.05). In addition, at this siRNA concentration, the minimum inhibitory concentration of itraconazole for the treated cells was decreased, compared with that for untreated control cells, from 16 to 4 µg/ml.

Endocarditis due to a co-infection of Candida albicans and Candida tropicalis in a drug abuser.

In recent decades the incidence of Candida endocarditis has increased dramatically. Despite the application of surgery and antifungal therapy, Candida endocarditis remains a life-threatening infection with significant morbidity and mortality. We report a 37-year-old male drug abuser presenting with high fever, chest pain, loss of appetite and cardiac failure. His echocardiography revealed mobile large tricuspid valve vegetations. Fungal endocarditis was confirmed by culturing of the resected vegetation showing mixed growth of Candida albicans and Candida tropicalis, although three consecutive blood cultures were negative for Candida species. Phenotypic identification was reconfirmed by sequencing of the internal transcribed spacer (ITS rDNA) region. The patient was initially treated with intravenous fluconazole (6 mg kg(-1) per day), followed by 2 weeks of intravenous amphotericin B deoxycholate (1 mg kg(-1) per day). Although MICs were low for both drugs, the patient's antifungal therapy combined with valve replacement failed, and he died due to respiratory failure.

Environmental study of azole-resistant Aspergillus fumigatus with TR34/L98H mutations in the cyp51A gene in Iran.

Azole resistance in Aspergillus is emerging in European and Asian countries. As azoles are mainstay of therapy in the management of aspergillosis, azole resistance has serious implications in patient management. We report the emergence of resistance to triazoles in environmental Aspergillus fumigatus isolates in Iran. The TR34/L98H mutation was the only resistance mechanism. Overall 3.3% of the A. fumigatus isolates from hospital surroundings in Sari and Tehran had the same TR34/L98H STRAf genotype and were related to some resistant clinical and environmental TR34/L98H isolates from the Netherlands and India. It is emphasised that routine resistance surveillance studies focusing on environmental and clinical samples are warranted to yield the true prevalence of azole resistance in A. fumigatus in Iran.

Veronaea botryosa: molecular identification with amplified fragment length polymorphism (AFLP) and in vitro antifungal susceptibility.

Inter- and intraspecific genomic variability of 18 isolates of Veronaea botryosa originating from clinical and environmental sources was studied using amplified fragment length polymorphism (AFLP). The species was originally described from the environment, but several severe cases of disseminated infection in apparently healthy individuals have been reported worldwide. All tested strains of V. botryosa, identified on the basis of sequencing and phenotypic and physiological criteria prior to our study, were confirmed by AFLP analysis, yielding a clear separation of V. botryosa as a rather homogeneous group from related species. In vitro antifungal susceptibility testing resulted in MIC90s across all strains in increasing order posaconazole (0.25 µg/ml), itraconazole (1 µg/ml), voriconazole (4 µg/ml), terbinafine (4 µg/ml), caspofungin (8 µg/ml), anidulafungin (8 µg/ml), isavuconazole (16 µg/ml), amphotericin B (16 µg/ml), and fluconazole (32 µg/ml). Overall, the isolates showed a uniform pattern of low MICs of itraconazole and posaconazole, but high MICs for remaining agents. The echinocandins (caspofungin and anidulafungin) had no activity against V. botryosa. There was no statistically significant difference between susceptibilities of environmental (n = 11) and clinical (n = 7) isolates of V. botryosa (P > 0.05).

Chromoblastomycosis due to Fonsecaea pedrosoi and F. monophora in Cuba.

We report two cases of chromoblastomycosis due to Fonsecaea pedrosoi and F. monophora in otherwise healthy Cuban males. Direct microscopic examination of biopsies revealed muriform cells, the hallmark of chromoblastomycosis. The suspected agents were recovered in culture, identified on the basis of morphological criteria and confirmed by sequencing of the internal transcribed spacer regions of rDNA. Final treatment consisted of surgical excision. The patients were successfully cured since there was no relapse after a follow-up of more than a year. In vitro antifungal susceptibility testing of both isolates showed that itraconazole and posaconazole had potent activity. High MICs of amphotericin B (2 µg/ml), fluconazole (>64 µg/ml), anidulafungin (8 µg/ml) and caspofungin (8 µg/ml) were found.

A case of onychomycosis caused by Aspergillus candidus.

Based on epidemiological studies, Aspergillus candidus has been demonstrated as an emerging fungal agent of toenail onychomycosis. Here we report a case of a toenail infection caused by A. candidus in a healthy 60-year-old woman. Based on macroscopic and microscopic characteristics of the culture as well as nucleotide sequencing of 28S region, the causative agent was identified as A. candidus.

Volumetric assessment of airborne indoor and outdoor fungi at poultry and cattle houses in the Mazandaran Province, Iran.

The aim of this study was to assess the volume of airborne fungi in the indoor and outdoor environment of poultry and cattle houses in the Mazandaran Province in Iran. Indoor and outdoor air of twenty cattle houses and twenty-five poultry houses were sampled using a single-stage impactor, which draws air at 20 L min-1 and impacts sampled material onto Petri plates containing malt extract agar. The plates were incubated at 30 °C for seven days, after which the resulting colonies were counted. The fungi were identified and counted microscopically and macroscopically. A total of 4,662 fungal colonies were isolated from 90 plates collected from indoor and outdoor air of cattle and poultry houses. Cladosporium (55.3 %), yeast (10.0 %), and Aspergillus (9.4 %) were the most common findings. The concentration of airborne fungi in cattle and poultry houses ranged from 10 CFU m-3 to 1700 CFU m-3 in indoor and 10 CFU m-3 to 2170 CFU m-3 in outdoor environments. Cladosporium had the highest mean indoor (424.5 CFU m-3) and outdoor (449.7 CFU m-3) air concentration in the cattle houses. In the poultry houses, the highest mean concentrations were measured for Cladosporium (551.0 CFU m-3) outdoors and yeast (440.7 CFU m-3) indoors. These levels might present an occupational risk, but threshold levels for these environments have yet to be established worldwide.