Nicolina Dias

Trends in the use of protozoa in the assessment of wastewater treatment

Increasing environmental pollution and the continuous development of new chemicals and drugs has led to ever growing concern about the potential effects of these compounds directly or indirectly on human health. As concerns water pollution, protozoa seem to be an excellent tool to assess both toxicity and pollution: they are regarded as biological indicators of pollution when their presence or absence can be related to particular environmental conditions, and they are considered test organisms when a species or population is used to evaluate the toxicity of relevant toxic compounds. Thus, an integrated approach is being developed to assess how toxic compounds affect the different biological levels of organisation--from the community level to the species level--of ciliated protozoa. The present paper reports and discusses the current state of the art of this approach.

Morphological and physiological changes in Tetrahymena pyriformis for the in vitro cytotoxicity assessment of Triton X-100

Non-ionic surfactants such as Triton X-100 have been widely used in industrial processing and in cleaning products for almost 50 years, being effective and economic emulsifying, wetting agents, dispersants and solubilizers. Cleaning products containing these surfactants are disposed of mainly by discharge into wastewater, which receives biological treatment in wastewater treatment systems. However, surface-active agents interact with eukaryotic cell membranes leading to biological damage at high concentrations. Tetrahymena pyriformis was used here as model organism to assess the effects of Triton X-100 through a series of in vitro cytotoxicity tests. Growth rates and morphological changes were, by their simplicity and reproducibility, the simplest toxicological assays. Cytoskeleton analysis seemed to be related with phagocytosis rate. Viability was evaluated by two different tests. Calcein AM/EthD-1 was used to assess T. pyriformis membrane damage during the 48-h experiment. The colorimetric MTT assay proved to be highly sensitive even at very short periods of Triton X-100 exposure. Tests performed in this study included simple and fast bioassays that provide overall information on the morphological and physiological state of cells exposed to different non-lytic and lytic concentrations of Triton X-100.

Sporotrichosis caused by sporothrix mexicana, Portugal

To the Editor: Sporotrichosis is a subcutaneous fungal infection present worldwide that is caused by traumatic inoculation or inhalation of spores of the dimorphic fungus Sporothrix schenckii complex (1–3). However, molecular studies have shown that the S. schenckii complex constitutes several cryptic infectious species (i.e., S. albicans, S. brasiliensis, S. globosa, S. luriei, S. mexicana, and S. schenckii). Marimon et al. (4) demonstrated 3 major clades grouped into 6 putative phylogenetic species. The natural habitats of these species are soil and plants. The species showed distinct pathologic behavior, antifungal responses, and phenotypes, which suggests that optimal clinical treatment may depend on the taxon involved in the sporotrichosis (1). Human infections have been reported primarily from the Americas, including Latin America (3,5). Asia (e.g., Malaysia, India, Japan), Africa, and Australia are also regions where infections are endemic (6). Although infections are rare in Europe, a case of human infection (7) and a case of an animal infection (8) have been described in southern Italy. We report a case of human sporotrichosis in which S. mexicana was isolated from a patient in Portugal. n nA 34-year-old man sought care at a podiatry clinic in Vila Nova de Famalicao, Portugal, in 2009 for multiple polymorphous eruptions and ulcers on both feet. There was no obvious cause of the disease. Although the patient had traveled to Malaysia in 2003 and had worn open footwear every day, he did not recall receiving a skin wound. In 2004 in Portugal, subcutaneous nodules appeared in both feet, became ulcerated, and spontaneously healed. By 2005, more severe lesions had appeared and became a chronic infection in both feet and lower limbs. The symptoms were diagnosed erroneously as dyshidrotic eczema, and treatment with topical corticosteroids was unsuccessful. n nSeveral skin fragments of the lesions were submitted for mycological assessment. Fungi were not found on potassium hydroxide slides of all samples. Filamentous fungal colonies were observed after 7 days of culture on Sabouraud dextrose agar slopes at 25°C. The fungus had hyaline septate hyphae, with hyaline and dematiaceous conidia compatible with Sporothrix spp. The isolate was accessed and preserved in the Micoteca da Universidade do Minho (MUM, Braga, Portugal) fungal culture collection and given the accession code MUM 11.02. n nThe macroscopic features and sporulation were analyzed by using cornmeal and potato dextrose agars. Clusters of intercalary or terminal conidia were formed by sympodial growth from differentiated conidiophores on both media. Sympodial conidia were hyaline or slightly pigmented. Sessile conidia were predominantly subglobose, obovoidal or ellipsoidal, and 3.35 ± 0.41 µm long by 2.30 ± 0.32 µm wide (Figure, panel A). A teleomorph was not observed. The colony diameter on potato dextrose agar after 21 days of incubation attained 40 mm at 30°C and 5 mm at 37°C. The yeast form was achieved by incubating the isolate on brain heart infusion agar on slants at 35°C ± 2°C for 7 days in a single subculture. n n n nFigure n nA) Photomicrograph of sympodial and sessile conidia of Sporothrix mexicana obtained by using a transmitted differential interference contrast microscope. The isolate was obtained from a patient in Portugal in 2009 and archived in the Micoteca da Universidade ... n n n nDextrose, sucrose, and raffinose assimilation tests were performed in triplicate by using yeast nitrogen base medium. The strain assimilated dextrose, sucrose, and raffinose, showing phenotypic characteristics typical of S. mexicana and S. schenckii (2). In contrast, type reference strain S. brasiliensis CBS 120339 was included in the test, and it was able to assimilate only dextrose. n nA presumptive identification based on phenotypic characteristics allowed us to classify this fungus as S. mexicana, although this species has an atypical morphologic profile. The diameter of colonies grown at 30°C and 37°C are smaller than those proposed by Marimon and collaborators but much closer to those of S. schenckii (2). These differences could be attributable to the intraspecific variation of this single isolate. n nGenomic DNA was obtained from the yeast phase of S. mexicana MUM 11.02, and the partial sequencing of the nuclear calmodulin gene was based on the amplicon generated by PCR reaction by using CL1 and CL2A primers (2,3). Sequencing was performed at Fundacao Oswaldo Cruz, Rio de Janeiro, Brazil. A BLAST analysis (www.ncbi.nlm.nih.gov/BLAST) comparing the sequence of the calmodulin gene with sequences {type:entrez-nucleotide,attrs:{text:AM398382,term_id:157954195}}AM398382, {type:entrez-nucleotide,attrs:{text:AM398393,term_id:157954199}}AM398393, {type:entrez-nucleotide,attrs:{text:AM117444,term_id:114145258}}AM117444, {type:entrez-nucleotide,attrs:{text:AM116899,term_id:114145044}}AM116899, and {type:entrez-nucleotide,attrs:{text:AM116908,term_id:114145062}}AM116908 in the GenBank database confirmed the identity of this isolate as S. mexicana. The MUM 11.02 isolate showed 99% similarity with the sequences of S. mexicana (i.e., GenBank accession no. {type:entrez-nucleotide,attrs:{text:AM398393,term_id:157954199}}AM398393) with high bootstrap support values (Figure, panel B). The calmodulin sequence of MUM 11.02 was deposited in GenBank as {type:entrez-nucleotide,attrs:{text:JF970258,term_id:349592773}}JF970258. n nIn vitro susceptibility tests with fluconazole, itraconazole, and terbinafine were performed by the microdilution method (9) and revealed MICs of 128 µg/mL, 32 µg/mL, and 0.5–1.0 µg/mL, respectively, which corresponds to the findings of Marimon et al. (1) for S. mexicana. Thus, S. mexicana is an emerging cause of human sporotrichosis.

Miniaturization and application of the MTT assay to evaluate metabolic activity of protozoa in the presence of toxicants

This paper describes a critical evaluation of a miniaturised colorimetric assay, using MTT (3‐[4,5‐dimethyl‐thiazol‐2‐yl]‐2,5‐diphenyl‐tetrazolium bromide) reduction, applied to protozoan viability testing. The toxic substances used were copper, zinc, Triton X‐100 (a membrane surfactant) and cycloheximide (an inhibitor of the protein synthesis). The viability assay of the ciliate protozoan Tetrahymena pyriformis was optimised in terms of MTT concentration and incubation time. Since protozoa are non adherent cells the MTT assay was modified in order to maintain the medium in the well. MTT proved to be effective in the measurement of Tetrahymena pyriformis viability. Four hours of MTT incubation followed by 30 minutes of incubation with DMSO were found to be the best incubation times for optical density reading. Furthermore, 10 mg/ml of MTT solution was the concentration that gave higher values of optical densities with minor medium interference.

Synthesis, characterization and antifungal activity of chemically and fungal‐produced silver nanoparticles against Trichophyton rubrum

To characterize and explore the potential in extracellular biosynthesis of silver nanoparticles (AgNPs) by Penicillium chrysogenum and Aspergillus oryzae and to investigate the antifungal effect of chemically vs biologically synthesized AgNPs comparing with conventional antifungal drugs against Trichophyton rubrum.

A comparative study using a fluorescence-based and a direct-count assay to determine cytotoxicity in Tetrahymena pyriformis

A novel cellular cytotoxicity assay using two fluorescent dyes was developed as an alternative method to the standard direct count of viable protozoa under light microscopy. The compound calcein AM is a non-fluorescent substance that diffuses passively across intact cell membranes and is converted by intracellular esterases to the green fluorescent calcein, which is retained in viable cells. The addition of EthD-1 that binds to DNA stained nuclei of dead cells red. The experiments were carried out in order to assess viability in the freshwater ciliate Tetrahymena pyriformis after exposure to eight surfactants, two of each representing one of four ionic class (non-ionic, anionic, cationic and amphoteric), and two heavy metals, copper and zinc, at several concentrations. In earlier time exposure, less than one hour of contact with surfactants at sublethal concentrations, the fluorescent method is more sensitive and provides more accurate results than direct counting under light microscopy. In contrast, with increasing time exposure, the results obtained by the two methods were similar. Calcein was shown to be a poor viability marker in the presence of zinc and copper since the fluorescence intensity was affected by the metal presence. However, the fluorescent method offers new opportunities to use advanced techniques, such as flow cytometry, to assess cytotoxicity in protozoa.

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.

The use of MALDI-TOF ICMS as an alternative tool for Trichophyton rubrum identification and typing

AIMSnIn this study, the potential of matrix-assisted laser desorption/ionization time-of-flight intact cell mass spectrometry (MALDI-TOF ICMS) was investigated for the identification of clinical isolates. The isolates were analyzed at the species and strain level.nnnMETHODSnSpectral identification by MALDI-TOF ICMS was performed for all strains, and compared with the results of sequencing of the internal transcribed spacers (ITS1 and ITS2), and the 5.8S rDNA region. PCR fingerprinting analysis using primers M13, (GACA)4, and (AC)10 was performed in order to assess the intra-specific variability of Trichophyton rubrum strains.nnnRESULTSnThe identification of strains at species level by MALDI-TOF ICMS was in agreement with the previously performed morphological and biochemical analysis. Sequence data confirmed spectral mass identification at species level. Intra-specific variability was assessed. Within the T. rubrum cluster, strains were distributed into smaller highly related sub-groups with a similarity values above 85%.nnnCONCLUSIONSnMALDI-TOF ICMS was shown to be a rapid, low-cost and accurate alternative tool for the identification and strain typing of T. rubrum.

Automated image analysis to improve bead ingestion toxicity test counts in the protozoan Tetrahymena pyriformis

Aims: To improve bead ingestion counts in Tetrahymena pyriformis by automated image analysis as an alternative to direct‐counts.

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

Abstract 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 μg mL−1. 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.