Maria Helena Galdino Figueiredo-Carvalho

Phenotypical properties associated with virulence from clinical isolates belonging to the Candida parapsilosis complex

The production of virulence attributes in three reference strains and 11 clinical isolates primarily identified as Candida parapsilosis was evaluated. Morphological and phenotypical tests were not able to discriminate among the three species of the C. parapsilosis complex; consequently, molecular methods were applied to solve this task. After employing polymerase chain reaction-based methods, nine clinical strains were identified as C. parapsilosis sensu stricto and two as C. orthopsilosis. Protease, catalase, and hemolysin were produced by all 14 strains, while 92.9% and 78.6% of strains secreted, respectively, esterase and phytase. No phospholipase producers were detected. Mannose/glucose, N-acetylglucosamine, and sialic acid residues were detected at the surface of all strains, respectively, in high, medium, and low levels. All strains presented elevated surface hydrophobicity and similar ability to form biofilm. However, the adhesion to inert substrates and mammalian cells was extremely diverse, showing typical intrastrain variations. Overall, the strains showed (1) predilection to adhere to plastic over glass and the number of pseudohyphae was more prominent than yeasts and (2) the interaction process was slightly enhanced in macrophages than fibroblasts, with the majority of fungal cells detected inside them. Positive/negative correlations were demonstrated among the production of these virulence traits in C. parapsilosis complex.

Candida haemulonii complex: species identification and antifungal susceptibility profiles of clinical isolates from Brazil

OBJECTIVES The emerging fungal pathogens comprising the Candida haemulonii complex (Candida haemulonii, Candida haemulonii var. vulnera and Candida duobushaemulonii) are notable for their antifungal resistance. Twelve isolates with phenotypic similarity to C. haemulonii were recovered from patients in Brazilian hospitals. Here we aimed to identify these isolates by a molecular approach, using the current classification of this fungal complex, and to evaluate their antifungal susceptibility profiles. METHODS The fungal isolates were rechecked to certify their authentication by mycology methodologies and then characterized by ITS1-5.8S-ITS2 gene sequencing. A susceptibility assay was performed using the broth microdilution method published by CLSI (M27-A3/M27-S3). RESULTS Based on biochemical tests, all Brazilian isolates were identified as C. haemulonii. After employing ITS sequencing, five isolates were identified as C. haemulonii, four as C. duobushaemulonii and three as C. haemulonii var. vulnera. All 12 clinical isolates were resistant to amphotericin B (MICs ranged from 2 to >16 mg/L) and fluconazole (MICs ≥ 64 mg/L). One isolate of C. haemulonii var. vulnera and two isolates of C. duobushaemulonii were susceptible-dose dependent to itraconazole, while the remaining isolates (75%) were resistant to this antifungal. Eight out of 12 isolates (66.7%) were resistant to voriconazole (MICs ≥ 16 mg/L), while all isolates were susceptible to caspofungin (MICs ≤ 0.5 mg/L). CONCLUSIONS Our results reinforce the importance of molecular identification in differentiating species of the C. haemulonii complex. Moreover, the antifungal multiresistant profile of clinical isolates of the C. haemulonii complex represents a challenge to the treatment of such infections.

Melanins Protect Sporothrix brasiliensis and Sporothrix schenckii from the Antifungal Effects of Terbinafine.

Terbinafine is a recommended therapeutic alternative for patients with sporotrichosis who cannot use itraconazole due to drug interactions or side effects. Melanins are involved in resistance to antifungal drugs and Sporothrix species produce three different types of melanin. Therefore, in this study we evaluated whether Sporothrix melanins impact the efficacy of antifungal drugs. Minimal inhibitory concentrations (MIC) and minimal fungicidal concentrations (MFC) of two Sporothrix brasiliensis and four Sporothrix schenckii strains grown in the presence of the melanin precursors L-DOPA and L-tyrosine were similar to the MIC determined by the CLSI standard protocol for S. schenckii susceptibility to amphotericin B, ketoconazole, itraconazole or terbinafine. When MICs were determined in the presence of inhibitors to three pathways of melanin synthesis, we observed, in four strains, an increase in terbinafine susceptibility in the presence of tricyclazole, a DHN-melanin inhibitor. In addition, one S. schenckii strain grown in the presence of L-DOPA had a higher MFC value when compared to the control. Growth curves in presence of 2×MIC concentrations of terbinafine showed that pyomelanin and, to a lesser extent, eumelanin were able to protect the fungi against the fungicidal effect of this antifungal drug. Our results suggest that melanin protects the major pathogenic species of the Sporothrix complex from the effects of terbinafine and that the development of new antifungal drugs targeting melanin synthesis may improve sporotrichosis therapies.

First description of Candida nivariensis in Brazil: antifungal susceptibility profile and potential virulence attributes

This study evaluated the antifungal susceptibility profile and the production of potential virulence attributes in a clinical strain of Candida nivariensis for the first time in Brazil, as identified by sequencing the internal transcribed spacer (ITS)1-5.8S-ITS2 region and D1/D2 domains of the 28S of the rDNA. For comparative purposes, tests were also performed with reference strains. All strains presented low planktonic minimal inhibitory concentrations (PMICs) to amphotericin B (AMB), caspofungin (CAS), and voriconazole. However, our strain showed elevated planktonic MICs to posaconazole (POS) and itraconazole, in addition to fluconazole resistance. Adherence to inert surfaces was conducted onto glass and polystyrene. The biofilm formation and antifungal susceptibility on biofilm-growing cells were evaluated by crystal violet staining and a XTT reduction assay. All fungal strains were able to bind both tested surfaces and form biofilm, with a binding preference to polystyrene (p < 0.001). AMB promoted significant reductions (≈50%) in biofilm production by our C. nivariensis strain using both methodologies. This reduction was also observed for CAS and POS, but only in the XTT assay. All strains were excellent protease producers and moderate phytase producers, but lipases were not detected. This study reinforces the pathogenic potential of C. nivariensis and its possible resistance profile to the azolic drugs generally used for candidiasis management.

Different scenarios for Candida parapsilosis fungaemia reveal high numbers of mixed C. parapsilosis and Candida orthopsilosis infections.

Nosocomial fungal bloodstream infections (BSI) are increasing significantly in hospitalized patients and Candida parapsilosis has emerged as an important pathogen responsible for numerous outbreaks. The objective of this study was to evaluate C. parapsilosis sensu lato infection scenarios, regarding species distribution and strain relatedness. One hundred isolates of C. parapsilosis sensu lato derived from blood cultures and catheter tips were analysed by multiplex microsatellite typing and by sequencing D1/D2 regions of the ribosomal DNA. Our results indicate that 9.5 % of patients presented infections due to C. parapsilosis and Candida orthopsilosis, 57.1 % due to C. parapsilosis, 28.3 % due to C. orthopsilosis and 4.8 % due to Candida metapsilosis. Eighty per cent of the C. parapsilosis BSIs were due to a single strain that was also identified in the catheter, but in 10 % of the cases C. parasilosis was identified in the catheter but the BSI was due to C. orthopsilosis. There is a significant probability that C. parapsilosis isolates collected from the same patient at more than 3 months interval are of different strains (P = 0.0179). Moreover, several isolates were identified persistently in the same hospital, infecting six different patients. The incidence of polyfungal BSI infections with C. parapsilosis and C. orthopsilosis is reported herein for the first time, emphasizing the fact that the species identified in the catheter is not always responsible for the BSI, thus impacting the treatment strategy. The observation that strains can remain in the hospital environment for years highlights the possible existence of reservoirs and reinforces the need for accurate genotyping tools, such as the markers used for elucidating epidemiological associations and detecting outbreaks.

Minimal inhibitory concentration distributions and epidemiological cutoff values of five antifungal agents against Sporothrix brasiliensis.

BACKGROUND Sporothrix brasiliensis is the most virulent sporotrichosis agent. This species usually responds to antifungal drugs, but therapeutic failure can occur in some patients. Antifungal susceptibility tests have been performed on this species, but no clinical breakpoints (CBPs) are available. In this situation, minimal inhibitory concentration (MIC) distributions and epidemiological cutoff values (ECVs) support the detection of identification of resistant strains. OBJECTIVES To study the MIC distributions of five antifungal drugs against S. brasiliensis and to propose tentative ECVs. METHODS MICs of amphotericin B (AMB), itraconazole (ITR), ketoconazole (KET), posaconazole (POS), and terbinafine (TRB) against 335 S. brasiliensis strains were determined by the Clinical and Laboratory Standards Institute broth microdilution method. FINDINGS The proposed ECV, in µg/mL, for AMB, ITR, KET, POS, and TRB were 4.0, 2.0, 1.0, 2.0, and 0.25, respectively. Percentages of wild-type strains in our population for the above antifungal drugs were 98.48, 95.22, 95.33, 100, and 97.67%, respectively. MAIN CONCLUSIONS These ECVs will be useful to detect strains with resistance, to define CBPs, and to elaborate specific therapeutic guidelines for S. brasiliensis. Rational use of antifungals is strongly recommended to avoid the emergence of resistant strains and ensure the therapeutic effectiveness of sporotrichosis.

The identification and differentiation of the Candida parapsilosis complex species by polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region of the rDNA

Currently, it is accepted that there are three species that were formerly grouped under Candida parapsilosis: C. para- psilosis sensu stricto, Candida orthopsilosis, andCandida metapsilosis. In fact, the antifungal susceptibility profiles and distinct virulence attributes demonstrate the differences in these nosocomial pathogens. An accurate, fast, and economical identification of fungal species has been the main goal in mycology. In the present study, we searched sequences that were available in the GenBank database in order to identify the complete sequence for the internal transcribed spacer (ITS)1-5.8S-ITS2 region, which is comprised of the forward and reverse primers ITS1 and ITS4. Subsequently, an in silico polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to differentiate the C. parapsilosis complex species. Ninety-eight clinical isolates from patients with fungaemia were submitted for analysis, where 59 isolates were identified as C. parapsilosis sensu stricto, 37 were identified as C. orthopsilosis, and two were identified as C. metapsilosis. PCR-RFLP quickly and accurately identified C. parapsilosis complex species, making this method an alternative and routine identification system for use in clinical mycology laboratories.

l‐Dihydroxyphenylalanine induces melanin production by members of the genus Trichosporon

Melanization of members of the genus Trichosporon is poorly described. In this study, six strains, including two clinical isolates, from four different species (Trichosporon asahii, T. asteroides, T. inkin, and T. mucoides) were grown in culture media with or without L-dihydroxyphenylalanine (L-DOPA). Each strain produced a brownish pigment compatible with melanin when cultured in presence of L-DOPA, suggesting that these species are able to produce eumelanin. L-tyrosine was not able to elicit any type of pigment production on cultures. As eumelanin is produced by several fungi during parasitism, this pigment may contribute to Trichosporon virulence.

Evaluation of melanin production by Sporothrix luriei

There is a paucity of studies on the cell biology of Sporothrix luriei, the less common of the pathogenic Sporothrix species worldwide. The production of DHN-melanin, eumelanin, and pyomelanin were evaluated on the mycelial and yeast forms of the S. luriei ATCC 18616 strain. The mycelial form of this species produced only pyomelanin, which protected the fungus against environmental stressors such as ultraviolet light, heat, and cold. The yeast form was unable to produce any of the tested melanin types. The lack of melanin in the parasitic form of S. luriei may be an explanation for its low frequency in human infections.

Comparison of four molecular approaches to identify Candida parapsilosis complex species

Since the description of Candida orthopsilosis and C. metapsilosis in 2005, several methods have been proposed to identify and differentiate these species from C. parapsilosis sensu stricto. Species-specific uniplex polymerase chain reaction (PCR) was performed and compared with sequencing of the D1/D2 region of the LSU 28S rDNA gene, microsatellite typing of C. parapsilosis sensu stricto, and PCR-restriction fragment length polymorphism patterns in the ITS1-5.8S-ITS2 region of the rDNA gene. There was agreement between results of testing of 98 clinical isolates with the four PCR-based methods, with 59 isolates identified as C. parapsilosis sensu stricto, 37 as C. orthopsilosis, and two as C. metapsilosis.Since the description of Candida orthopsilosis and C. metapsilosis in 2005, several methods have been proposed to identify and differentiate these species from C. parapsilosis sensu stricto. Species-specific uniplex polymerase chain reaction (PCR) was performed and compared with sequencing of the D1/D2 region of the LSU 28S rDNA gene, microsatellite typing of C. parapsilosis sensu stricto, and PCR-restriction fragment length polymorphism patterns in the ITS1-5.8S-ITS2 region of the rDNA gene. There was agreement between results of testing of 98 clinical isolates with the four PCR-based methods, with 59 isolates identified as C. parapsilosis sensu stricto, 37 as C. orthopsilosis, and two as C. metapsilosis.