Toenail onychomycosis in a Portuguese geriatric population

Onychomycosis is a common fungal infection of the nail but few data of mycological features in geriatric Portuguese population are yet available. The aim of this study was to perform a mycological examination and characterization of fungal nail pattern of a geriatric population from the north of Portugal clinically suspected of onychomycosis. A total of 108 patients attending the Podology Service in the Centro Hospitalar do Alto Ave (Portugal) from October 2007 to January 2009 were enrolled. All were suspected of having onychomycosis by the abnormal appearance of their nails. From these, 59.3% were diabetic. Distal and lateral subungual onychomycosis was the more common clinical pattern followed by total dystrophic onychomycosis. In 21.3% cases, every nail in both feet had an abnormal appearance. In 86%, the hallux was involved in at least one foot. Fifty samples were culture positive, and fifty-four isolates were reported regardless of the questionable pathogenicity of the infectious agent. In three cases, clinical feature of the nail, direct microscopy, and culture were consistent with Scopulariopsis infection. Fusarium spp. were identified in three cases; however, only one isolate was preceded by the observation of branching septate filaments by direct microscopy. No mixed infections with dermatophytes were reported. Trichophyton rubrum was the dermatophyte most frequently isolated (83.3%) followed by Trichophyton interdigitale. In Portugal, onychomycosis is still viewed by general population as a cosmetic condition. Health risk is enhanced in geriatrics that only perceived the severity of their condition when experiencing further foot complications that include bacterial infection and pain

Silver nanoparticles as an antimicrobial agent of Tricophyton rubrum

Resistance to antimicrobial agents, such as amphotericin B, fluconazole, itraconazole, and voriconazole, by pathogenic bacteria and fungi has been increasing at an alarming rate and has become a serious problem. Microorganisms, such as bacteria, moulds, yeasts, and viruses are often pathogenic to humans. There is a pressing need to search for new antimicrobial agents. Amongst inorganic antimicrobial agents, silver has been employed most widely since ancient times to fight infections. The antimicrobial activities of silver, silver ions, and silver compounds are well known Fungi can be employed for biosynthesis of nanoparticles hence avoiding the use of hazardous chemicals for synthesis. However, the effects of mycological synthesized silver nanoparticles against fungal dermatophytes are not well understod. In this work were used inorganic and biogenic routes for the synthesis of silver nanoparticles. In the biogenic route, extracts from Aspergillus oryzae and Penicillium chrysogenum were used. In the inorganic route glucose was used as the reducing agent and polyvinylpyrrolidone as the stabilizer. The nanoparticles were characterized by various techniques. Biogenic nanoparticles from A. ozyzae and P. chrysogenum showed an average size from 19-51 nm to 51-85 nm, respectively. Nanoparticles synthesized by inorganic route had a mean size of 74 nm as determined by Dynamic Light Scattering. The antimicrobial potential activity was tested against strains of Tricophyton rubrum and the silver nanoparticles from P. chrysogenum had antimicrobial effects against T. rubrum strains. The synthesis parameters in future studies should be studied to take full advantage of the potentail for filamentous fungi to synthesise silver nanoparticles

MALDI-TOF ICMS: capability, potentiality and limits in the fast identification of Trichophyton rubrum from clinical cases occurrence in Portuguese health centres

Objective: Trichophyton rubrum is presently the most common worldwide pathogen causing dermatophytoses such as tinea corporis, tinea capitis, tinea pedis, and onychomycosis [1]. The main aim of the present work was assess MALDI-TOF ICMS as a fast and reliable technique in the identification of T. rubrum from clinical cases occurrence in the Portuguese health centres, and evaluates the potentialities and limits of this new microbial identification technique on the taxonomy of these infectious dermatophytes. Methods: Fungi were grown for 10 days in solid medium (SDA, Sabouraud Dextrose Agar) and then the mycelia were direct transferred from the SDA plate to the MALDI stainless steel template and mixed with 1 ml MALDI matrix solution (75 mg/ml 2,5-dihydroxybenzoic acid in ethanol/water/acetonitrile [1:1:1] with 0.03% trifluoroacetic acid). The sample mixtures were air dried at room temperature. The analyses were performed in our laboratory on an Axima LNR system (Kratos Analytical, Shimadzu, Manchester, UK) equipped with a nitrogen laser (337 nm). The mass range from m/z = 2,000 to 20,000 Da was recorded. Escherichia coli strain DH5a with known mass values of ribosomal proteins was used for external calibration. The fungi classification was performed on the SARAMIS software (AnagnosTec mbH, Potsdam- Golm, Germany). Molecular biology was used when appropriated with PCR based-technology. The presence of a 203-bp PCR product confirmed T. rubrum identification. Results: All strains were accurately and consistently identified as T. rubrum by MALDI-TOF ICMS combined to SARAMIS database analysis. Spectral mass analysis proven to be a rapid method since the analysis took only a few minutes to perform with the benefit of any laborious sample preparation procedures or any expensive chemical reagent was needed. Conclusions: The fungal spectral analysis by MALDI-TOF ICMS was as good as molecular biology in order to identify T. rubrum but much faster and cheaper

Identification of clinical isolates Trichophyton rubrum using a rapid and accurate mass spectral analysis (MALDI‐TOF ICMS)

condition called “ringworm” in man. The affinity for keratinized tissues by dermatophytes, implies in most of the cases, that the infection remains restricted to the nonliving cornified layers of the skin, nails, and hair [1]. Among dermatophytes, the species Trichophyton rubrum is of particular clinical interest for man because is the most common agent of human dermatophytoses. Macro and micro‐morphological examination combined to physiological analysis of primary isolates grown in selective culture media are still the most used methods in routine laboratory. Besides their low specificity, an accurate diagnosis may take 3 to 4 weeks to be achieved Modern identification methods involve molecular biology by using PCR technology based on differential sequence elements. It is gradually becoming clearer that microbial identification and authentication requires a polyphasic approach to generate quality data which are accurate and useful [2]. Microbial mass spectral analysis has been progressively more incorporated to the polyphasic approach to improve the accuracy of the microbial identification issue. Matrix Assisted Laser Desorption Ionization Time of Flight Intact Cell Mass Spectroscopy (MALDI‐TOF ICMS) is becoming an alternative to DNA‐dependent methods so it has been already successfully applied to the rapid identification and classification of microorganisms [3].The aim of this work was to test the applicability of MALDI‐TOF ICMS for identifying clinical isolates of T. rubrum. In this study twenty clinical isolates of T. rubrum were grown on Sabouraud culture medium. Plates were incubated for 7 days at 25 ºC. All the isolates were identified both macroscopically and microscopically. From the same plate, a tiny sample (about 50 mg) was transferred to stainless steel templates. A 0.5 ml of dihydroxy‐benzoic acid (DHB) matrix solution was added to the sample and air dried. Peak lists of individual samples were compared with the superspectra database generating a ranked list of matching spectra from SARAMIS software. All strains were accurately and consistently identified as T. rubrum by MALDI‐TOF ICMS combined to SARAMIS database analysis. Spectral mass analysis proven to be a rapid method, as the analysis took only a few minutes to perform with the benefit of any laborious sample preparation procedures or any expensive chemical reagent was needed

Oxygenated monoterpenes-rich volatile oils as potential antifungal agents for dermatophytes

Essential oils (EOs) extracted from Lavandula luisieri and Cymbopogon citratus were tested for their antifungal activity against ten clinical isolates of dermatophytes isolated from cases of tinea pedis. Inhibition of conidial germination and antifungal drug/EO combination assay were tested on two ATCC reference strains of Trichophyton rubrum and Trichophyton mentagrophytes. EOs were characterised by high amount of oxygenated monoterpenes in their composition. Strong antifungal activity was observed for the majority of clinical strains, and fungicidal activity was demonstrated. Positive interaction between L. luisieri EO combined with terbinafine was observed against terbinafine-resistant strain (Tr ATCC MYA-4438). Significative reduction of the germination was observed above 100 g mL1. Both oils were safe to macrophage mammalian cells at tested concentration. This study describes the antifungal activity of L. luisieri and C. citratus EOs against dermatophytes, which could be useful in designing new formulations for topical treatments.This work was supported by Portuguese Foundation for Science and Technology (FCT) [grant number PEST-C/FIS/UI607/2013], [grant number UID/BIO/04469/2013], [grant number PEst-OE/EQB/LA0023/2013] and European Community fund FEDER [grant number FCOMP-01-0124-FEDER-007025], [grant number RECI project: FCOMP-01-0124-FEDER-027462].The authors thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013. Nicolina Dias acknowledges the project “Consolidating Research Expertise and Resources on Cellular and Molecular Biotechnology at CEB/IBB”, Ref. FCOMP-01-0124-FEDER-027462

Micoteca da Universidade do Minho (MUM): implementation of a quality management system based on ISO 9001:2008

[Excerpt] Micoteca da Universidade do Minho (MUM) is a filamentous fungi culture collection established in May 1996, hosted by the Biological Engineering Centre, which is a centre of excellence integrated in the Institute for Biotechnology and Bioengineering (IBB).The mission of MUM is to provide the highest quality services to our customers, collecting, maintaining and supplying fungal strains and their associated information for teaching and research in biotechnology and life sciences. MUM intends to be a centre of knowledge, information and training in mycology, operating at a global level and under national and international regulations. In order to keep its high standards MUM has implemented a Quality Management System (QMS) based on the normative reference ISO 9001:2008.MUM has defined three processes, those being: Material Reception Process, Material Preservation Process and Material Supply Process; and has also developed a documental base for all the QMS including proceedings, standard operating procedures, forms, as well as quality objectives and goals for a continuous improvement