Molecular Identification and In Vitro Antifungal Susceptibility of Aspergillus Isolates Recovered from Otomycosis Patients in Western China

Mycopathologia - Aspergillus species are the most common causative agents involved in otomycosis. In this study, 45 Aspergillus isolates were obtained from patients with otomycosis in western China...     

Significance of Molecular Identification and Antifungal Susceptibility of Clinically Significant Yeasts and Moulds in a Global Antifungal Surveillance Programme

The increasing diversity of opportunistic fungi causing serious invasive fungal infections (IFI) has been documented. Accurate identification (ID) is important in guiding therapy, determining prognosis for IFIs and in epidemiological surveys. We assessed the utility of PCR-based methods for the ID of yeasts and moulds that either were uncommon, failed conventional ID, or represented unusual biochemical or phenotypic profiles of common species. Among 1,790 viable fungal clinical isolates received during the SENTRY Program in 2010, 322 strains from 40 study sites had ID confirmed by molecular methods. Isolates were previously identified in participant institutions. Yeasts that were not confirmed by morphology on CHROMagar, growth at 45?°C (Candida albicans/dubliniensis), or assimilation of trehalose (C. glabrata) as well as non-Candida yeasts and all moulds were amplified and sequenced using primers amplifying one or more of the following genes: ITS, 28S, β-tubulin (Aspergillus spp.), TEF (Fusarium spp.), IGS (Trichosporon spp.). The isolates selected for molecular ID included 149 isolates of Candida species, 77 of Aspergillus species, 73 non-Candida yeasts, and 23 other moulds (a total of 41 different species). Overall, the ID determined by the submitting site was confirmed for 189 isolates (58.7?%): Aspergillus spp. (64.1?% correct); Candida spp. (60.1?% correct); non-Candida yeasts (58.9?% correct); non-Aspergillus moulds (30.4?% correct). Species with high levels of concordance between conventional and molecular ID included A. fumigatus (95.0 %), C. lusitaniae (100?%), C. dubliniensis (92.3?%), C. kefyr (100?%), and C. neoformans (90.2?%). Only 50.0?% of isolates of C. albicans and 59.1?% of C. glabrata selected due to unusual phenotypic or biochemical features were found to be correctly identified by the submitting site. Molecular methods for the identification of fungal pathogens are an important adjunct to the conventional identification of many less common clinically relevant yeasts and moulds including species of Candida with unusual or erroneous phenotypic or biochemical profiles. Molecular confirmation of fungal identification is essential in epidemiological surveys such as SENTRY.     

Molecular Identification and Antifungal Susceptibilities of Black Aspergillus Isolates from Otomycosis Cases in Hungary

Otomycosis, also known as fungal otitis externa, has been used to describe a fungal infection of the external auditory canal, but sometimes involving the middle ear. Many fungal species have been identified as infectious agents in otomycosis, with Aspergillus and Candida species being the most common. Among aspergilli, Aspergillus niger is the most commonly described species in the literature. In this study, 14 black Aspergillus strains were analyzed, which were isolated from otomycosis cases in Hungary between 2010 and 2011. These strains were identified as A. niger according to conventional morphological methods. Species identification was based on sequencing of part of the calmodulin gene. Our results indicate that instead of A. niger, A. awamori and A. tubingensis are the predominant species that cause ear infections in Southern Hungary. Antifungal susceptibility tests were carried out against four antifungal drugs: amphotericin B, itraconazole, ketoconazole and terbinafine. All isolates were found to exhibit low in vitro MIC values to amphotericin B, terbinafine and itraconazole. However, the examined isolates exhibited high in vitro MIC values to ketoconazole.     

Genotyping and Antifungal Drug Susceptibility of Trichosporon asahii Isolated from Chinese Patients

As a major pathogenic agent of trichosporonosis, Trichosporon asahii can cause life-threatening infection in immunocompromised patients. In this study, we analyzed the genotypes of the intergenic spacer (IGS) 1 region of the rRNA gene and the antifungal drug susceptibility of eight T. asahii isolates obtained from Chinese patients. Five genotypes were identified from the eight isolates, including three novel genotypes, three genotype 1, and two genotype 4. The eight T. asahii isolates were resistant to amphotericin B, 5-flucytosine, and terbinafine, but were highly sensitive to fluconazole (FLC), itraconazole (ITC), and voriconazole (VRC). The mean minimum inhibitory concentrations (MICs) of FLC and VRC were significantly lower than those reported in most other countries, while that of ITC was slightly higher. Our results suggest that genotypes of the T. asahii isolated from China are different from those of other countries, and azole drugs appeared to be more effective on the Chinese isolates. These results provide new insights into the epidemiology and antifungal treatment for T. asahii.     

Virulence Factors and Antifungal Susceptibility in Candida Species Isolated from Dermatomycosis Patients

Mycopathologia - Dermatomycoses caused by Candida spp. are increasingly common, however there are few reports in the literature regarding their epidemiology, pathogenesis and antifungal...     

Antifungal Activity of Phenyllactic Acid against Molds Isolated from Bakery Products

Phenyllactic acid (PLA) has recently been found in cultures of Lactobacillus plantarum that show antifungal activity in sourdough breads. The fungicidal activity of PLA and growth inhibition by PLA were evaluated by using a microdilution test and 23 fungal strains belonging to 14 species of Aspergillus, Penicillium, and Fusarium that were isolated from bakery products, flours, or cereals. Less than 7.5 mg of PLA ml⁻¹ was required to obtain 90% growth inhibition for all strains, while fungicidal activity against 19 strains was shown by PLA at levels of ≤10 mg ml⁻¹. Levels of growth inhibition of 50 to 92.4% were observed for all fungal strains after incubation for 3 days in the presence of 7.5 mg of PLA ml⁻¹ in buffered medium at pH 4, which is a condition more similar to those in real food systems. Under these experimental conditions PLA caused an unpredictable delaying effect that was more than 2 days long for 12 strains, including some mycotoxigenic strains of Penicillium verrucosum and Penicillium citrinum and a strain of Penicillium roqueforti (the most widespread contaminant of bakery products); a growth delay of about 2 days was observed for seven other strains. The effect of pH on the inhibitory activity of PLA and the combined effects of the major organic acids produced by lactic acid bacteria isolated from sourdough bread (PLA, lactic acid, and acetic acid) were also investigated. The ability of PLA to act as a fungicide and delay the growth of a variety of fungal contaminants provides new perspectives for possibly using this natural antimicrobial compound to control fungal contaminants and extend the shelf lives of foods and/or feedstuffs.     

The Molecular Identification and Antifungal Susceptibilities of Aspergillus Species Causing Otomycosis in Tochigi,Japan

Aspergillus species are the most common pathogenic fungi involved in otomycosis, an infection of the outer ear canal. In this study, we examined the incidence of Aspergillus infections and the antifungal susceptibilities of 30 Aspergillus species isolates from patients with otomycosis who visited Saiseikai Utsunomiya Hospital between August 2013 and July 2016. Based on the morphological test results, the strains were identified as Aspergillus niger sensu lato (20 strains), A. terreus sensu lato (7 strains), and A. fumigatus sensu lato (3 strains). In contrast, the molecular identifications based on analyzing the isolates’ partial β-tubulin gene sequences revealed them to be A. niger sensu stricto (12 strains), A. tubingensis (8 strains), A. terreus sensu stricto (7 strains), and A. fumigatus sensu stricto (3 strains). The antifungal susceptibility test results indicated that strains of A. tubingensis and A. niger sensu stricto displayed lower susceptibilities to ravuconazole, compared with the other isolates. The Aspergillus strains from this study showed low minimum inhibitory concentrations toward the azole-based drugs efinaconazole, lanoconazole, and luliconazole. Therefore, these topical therapeutic agents may be effective for the treatment of otomycosis.

     

Prevalence and Antifungal Susceptibility of Yeasts Obtained from the Oral Cavity of Elderly Individuals

Infections caused by Candida yeasts are common in elderly individuals. Seventy-five isolates of Candida spp. were obtained from saliva samples of 136 institutionalized elderly individuals resident in six retirement homes of Belo Horizonte, Brazil. Forty-seven isolates (62.66%) were identified as Candida albicans, 15 (20%) as C. tropicalis, 7 (9.33%) as C. glabrata, 4 (5.33) as C. parapsilosis, and 2 (2.67%) as C. guilliermondii. Among the 136 elderly individuals studied, 49 (36%) were male and 87 (64%) were female. Ages ranged from 60 to 90 years old. Sixty-three (46.3%) of the institutionalized individuals were denture wearers and, among them, 53 (84.1%) carried Candida yeasts in the oral cavity. Forty-four subjects presented lesions in the oral mucosa and among these, 36 (82%), had positive culture for Candida spp. The samples were tested for the in vitro susceptibility to amphotericin B, itraconazole, fluconazole and 5-flucytosin, and great variations were observed in the minimum inhibitory concentrations (MIC) of these drugs according to the species.     

Identification and Aflatoxin Production of Molds Isolated from Country Cured Hams

Of 562 molds isolated from country cured hams, 403 isolates were of the genus Penicillium, 121 were Aspergillus, and 36 were Cladosporium, Alternaria, and other genera.     

Electrophoretic Karyotypes of Candida Yeasts Recurrently Isolated from Single Patients

Candida strains were isolated repeatedly from single patients during recurrent episodes of Candida infection in a hospital, and their electrophoretic karyotypes were analyzed by pulsed-field gel electrophoresis using CHEF system. When only C. albicans (in 6 patients) or C. glabrata (in 1 patient) were recurrently isolated, their karyotypes from each patient were almost identical to one another, suggesting that they carried single type of the yeast. When multiple species were recovered from single patients (6 cases), the karyotypes of the most frequently recovered yeast species were almost identical with respect to each patient. The electrophoretic karyotype analysis has been proved to be useful for epidemiological studies because the method can tell not only the species identification but also the differences among the strains of the same species.     

Antifungal Susceptibility and Pathogenic Potential of Environmental Isolated Filamentous Fungi Compared with Colonizing Agents in Immunocompromised Patients

Infection is a major cause of morbidity and mortality in bone marrow transplant recipients and in patients with hematological malignancies. The source of infection is almost always endogenous flora or the hospital environment. The present study evaluated bone marrow transplant recipients and patients with hematological malignancies colonized and/or infected with filamentous fungi. During 1 year, environmental air samples were also taken from the bone marrow transplant unit by a modification of gravity air-setting plate (GASP) methodology. Fusarium spp. were the most prevalent genus in the fall and Cladosporium spp. in the winter. Clinically isolated strains grew better at 37 °C than environmental strains. According to NCCLS M-38P methods, environmental Aspergillus strains showed higher MICs to miconazol and itraconazol, and clinical Fusarium strains were less susceptible to fluconazole.     

In Vitro Antifungal Susceptibility of Malassezia pachydermatis Strains Isolated from Dogs with Chronic and Acute Otitis Externa

Malassezia pachydermatis is a yeast that is frequently involved as a secondary/perpetuating factor in canine otitis externa. Topical therapies with different antifungal agents, mainly azole compounds, are generally successful in controlling the yeast overgrowth, but treatment failure and rapid recurrences are common. This study compared the in vitro antifungal susceptibility of M. pachydermatis isolates obtained from chronic and acute cases of otitis externa. The aim was to assess the possible onset of resistance mechanisms in isolates involved in long-lasting episodes with poor response to treatment. We evaluated the in vitro susceptibility to miconazole (MCZ) and clotrimazole (CTZ) of 42 isolates of M. pachydermatis obtained from dogs with chronic (group A, n = 25) and acute otitis (group B, n = 17), using a modified CLSI M27-A3 microdilution method. All isolates were inhibited by the antifungal agents employed, but Malassezia isolates from group A were significantly associated with higher minimum inhibitory concentration (MIC) values for both agents (Median MIC values: MCZ group A 2 µg/ml, group B 1 µg/ml; CTZ group A 8 µg/ml, group B 4 µg/ml). These findings prove that these isolates had a reduced in vitro susceptibility to the antifungal agents tested. However, it is unlikely that this could have any influence on the outcome of a topical treatment. Indeed, marketed products include concentrations of the tested agents that largely exceed even the highest MICs found in this study (in most cases at least 1,000 × the MIC, or greater). In conclusion, this study suggests that isolates of M. pachydermatis involved in chronic cases of canine external otitis and exposed to repeated antifungal treatments are unlikely to develop mechanisms of resistance of clinical relevance.     

Development of Reference Procedures for Broth Microdilution Antifungal Susceptibility Testing of Yeasts with Standardized Endpoint Determination

Standard guidelines for the broth microdilution antifungal susceptibility testing of amphotericin B, flucytosine, fluconazole, miconazole and itraconazole are reported. These are a modification of the method developed by the National Committee for Clinical Laboratory Standards (NCCLS) on the following two points: standardization of the means of endpoint determination and the inclusion of miconazole and itraconazole in the testing. MIC was determined to be when the positive control had a turbidity of 0.2 at the 630 nm wavelength. The endpoint was 80% inhibition for azoles and 100% inhibition for other drugs. The method provided good reproducibility, and a wide range of MIC distribution was observed in all antifungal agents except amphotericin B.     

Molecular Identification and Antifungal Susceptibility of Clinical Isolates of Sporothrix schenckii Complex in Medellin,Colombia

Background

Sporotrichosis is a subcutaneous mycosis that affects humans and other animals. Infection prevails in tropical and subtropical countries. Until a few years ago, it was considered that two varieties of Sporothrix schenckii caused this mycosis, but by applying molecular taxonomic markers, it has been demonstrated that there are several cryptic species within S. schenckii complex which varies in susceptibility, virulence, and geographic distribution.

Objective

This study aimed to identify the clinical isolates of Sporothrix spp. from patients with sporotrichosis in Medellin, Colombia, using two markers and to evaluate the in vitro susceptibility to itraconazole.

Methods

Thirty-four clinical isolates of Sporothrix spp. from Colombia, three from Mexico, and one from Guatemala were identified through sequencing of the noncoding region ITS-1?+?5.8SDNAr?+?ITS-2 and of the fragment containing exons 3 and 4 of the β-tubulin gene. Clinical isolate sequences were compared with GenBank reference sequences using the BLASTN tool, and then, phylogenetic analysis was performed. Besides, the in vitro susceptibility to itraconazole was evaluated by determining the minimum inhibitory concentrations according to the CLSI M38-A2 method.

Results

Clinical isolates were identified by morphology as Sporothrix spp. Using the molecular markers, ITS and β-tubulin, isolates were identified as S. schenckii sensu stricto (25) and Sporothrix globosa (13). Susceptibility to itraconazole was variable among clinical isolates.

Conclusion

This is the first scientific publication that identifies species that cause sporotrichosis in Colombia, along with the antifungal susceptibility to itraconazole.

     

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).     

Antifungal Susceptibility Profile of Candida Albicans Isolated from Vulvovaginal Candidiasis in Xinjiang Province of China

We investigated the antifungal susceptibility profiles of 207 independent Candida albicans strains isolated from patients with vulvovaginal candidiasis (VVC) in Xinjiang Province of China. Using CLSI M27-A3 and M27-S4 guidelines, anidulafungin and micafungin were the most active drugs against C. albicans showing an MIC₅₀/MIC₉₀ corresponding to 0.016/0.0313 µg/mL, followed by caspofungin (0.25/0.25 µg/mL), posaconazole (0.125/0.5 µg/mL), ravuconazole (0.063/1 µg/mL), itraconazole (0.125/1 µg/mL), amphotericine B (0.5/1 µg/mL), isavuconazole (0.063/2 µg/mL), 5-flucytosine (1/2 µg/mL), voriconazole (0.125/4 µg/mL), and fluconazole (0.5/4 µg/mL). 96.1% (199)–100.0% (207) isolates were sensitive to the three echinocandins tested, amphotericine B and 5-flucytosine. The in vitro activity of triazoles against all isolates tested was variable; itraconazole and voriconazole had reduced the activity to almost half of the isolates (55.1% (114) and 51.2% (106) susceptible, respectively). Fluconazole was active against 76.3% (158) isolates tested. The new triazoles ravuconazole, isavuconazole and posaconazole showed good in vitro potency against 89.9% (186)–95.2% (197) of isolates with the geometric mean MIC (µg/mL) of 0.10, 0.12 and 0.14 µg/mL, respectively. In conclusion, our study indicates that for effective management of systemic candidiasis in Xinjiang Province of China, it is important to determine the susceptibility profiles of isolated C. albicans from patients with VVC.

     

Histopathology,Molecular Identification and Antifungal Susceptibility Testing of Nannizziopsis arthrosporioides from a Captive Cuban Rock Iguana (Cyclura nubila)

Mycopathologia - Chrysosporium-related fungi, the cause of superficial and deep mycoses, are an emerging infectious disease affecting not only reptiles but also immunocompromized humans. However,...     

Characteristics of Yeasts Isolated from Pacific Crab Meat

A total of 202 cultures of yeasts were isolated and characterized from king crab and Dungeness crab meat. A yeastlike organism, resembling Aureobasidium pullulans, and 15 different species distributed among the genera Rhodotorula, Cryptococcus, Torulopsis, Candida, and Trichosporon were represented. Nine of the species grew at 5 C or lower. Although two of the species grew at 37 C, none of the isolates had the characteristics of pathogenic species. Members of the Cryptococcus and Candida failed to grow at 37 C. Furthermore, species of the former genus were not pathogenic to mice. The pigmentation of the Rhodotorula cultures decreased in intensity as the incubation temperature was decreased. Biochemical activities of the different species were studied by use of triglycerides, lecithin, and proteins (casein, gelatin, and crab-meat protein) as substrates. Eight of the species could attack triglycerides; eight, lecithin; five, gelatin; one, casein; and one, crab protein. An organism, tentatively identified as Trichosporon sp., was very active in attacking each of the substrates tested and grew well at 0.5 C.