Candida species causing fungal keratitis: molecular identification, antifungal susceptibility, biofilm formation, and clinical aspects.

The study aimed to evaluate the clinical aspects, molecular identification, biofilm formation, and antifungal susceptibility profile of Candida species isolated from fungal keratitis. Thirteen Candida isolates from 13 patients diagnosed with Candida keratitis were retrieved and grown in pure culture. Species identification was performed by micromorphology analysis and ITS-rDNA sequencing. The broth microdilution method tested the minimum inhibitory concentration (MIC) of four antifungal drugs (fluconazole, amphotericin B, voriconazole, and anidulafungin). The biofilms were cultured and incubated with antifungal drugs for 24 h. The XTT reduction assay measured the biofilm activity. Biofilm MICs were calculated based on a 50% reduction in metabolic activity compared with the activity of the drug-free control. Among isolates, two were C. albicans, 10 were C. parapsilosis (sensu stricto), and one was C. orthopsilosis. All isolates were classified as susceptible or intermediate to all four antifungal drugs. Four isolates were very low biofilm producers (30%). Nine isolates were biofilm producers, and all biofilm samples were unsusceptible to all drugs tested. Previous ocular surgery was the most common underlying condition for fungal keratitis (84.6%), and C. parapsilosis was the most frequent Candida species (76.9%). Four patients (30.7%) needed keratoplasty, whereas two (15.3%) required evisceration. The biofilm formation ability of Candida isolates decreased antifungal susceptibility compared with planktonic cells. Despite in vitro antifungal susceptibility, almost half of the patients were unresponsive to clinical treatment and needed surgery.

Trends towards lower azole susceptibility among 200 Candida tropicalis bloodstream isolates from Brazilian medical centres.

OBJECTIVES: Candida tropicalis is one of the three most frequent species causing candidaemia in Latin America. Despite the high prevalence of C. tropicalis in candidaemia cases in Brazil, little is known about the trends in fluconazole susceptibility over time. The objective of this study was to evaluate temporal trends in azole resistance rates among C. tropicalis bloodstream isolates from patients treated in six Brazilian medical centres over a 12-year period. METHODS: We selected 200 C. tropicalis bloodstream isolates from six medical centres in Brazil collected between 2007 and 2018. Species identification was confirmed by MALDI-TOF/MS. Antifungal susceptibility testing for four antifungal agents was performed by the Clinical and Laboratory Standards Institute (CLSI) microbroth method. RESULTS: Overall, rates of non-susceptibility were 4% and 3.5% to fluconazole and voriconazole, respectively. All isolates were susceptible to amphotericin B and only one isolate was resistant to echinocandins. CONCLUSION: Although we failed to demonstrate statistical differences in the rates of azole resistance documented during the period of analysis, trends towards lower susceptibility to fluconazole and voriconazole were shown.

Species diversity, antifungal susceptibility and phenotypic and genotypic characterisation of Exophiala spp. infecting patients in different medical centres in Brazil.

The Exophiala genus is responsible for many superficial and invasive infections resulting from black fungi. Identification of Exophiala at the species level is based on morphological observations complemented by molecular tests. The aim of this study was to identify 23 clinical isolates of Exophiala spp. and evaluate the antifungal susceptibility to seven different agents. Molecular identification was based on an analysis of ITS region of rDNA using genomic databases. The micromorphology was evaluated by microculture and scanning electron microscopy. The susceptibility tests were performed using the antifungal agents 5-fluorocytosine (5-FC), amphotericin B (AMB), itraconazole (ITC), voriconazole (VRC), posaconazole (PSC), caspofungin (CFG) and terbinafine (TRB). The ITS analysis identified 100% of the following isolates as: E. dermatitidis (8), E. xenobiotica (6), E. bergeri (4), E. oligosperma (3), E. spinifera (1) and E. mesophila (1). The antifungal susceptibility tests showed that the triazoles compounds were in vitro the most active agents against Exophiala. ITS sequencing enabled the accurate identification of the 23 tested isolates. The triazoles, particularly itraconazole and posaconazole, exhibited MIC values lower than AMB, CAS and 5-FC. Although the guidelines do not indicate AMB for treatment against Exophiala spp., this study showed activity for all of the tested species, except E. mesophila.

First report of Candida auris in America: Clinical and microbiological aspects of 18 episodes of candidemia.

OBJECTIVES: Characterization of a hospital outbreak of Candida auris candidemia that involved 18 critically ill patients in Venezuela. METHOD: Bloodstream isolates of C. auris obtained from 18 patients admitted at a medical center in Maracaibo, between March, 2012 and July, 2013 were included. Species identification was confirmed by ITS rDNA sequencing. Isolates were subsequently typed by amplified fragment length polymorphism fingerprinting (AFLP). Susceptibility testing was performed according to CLSI. Clinical data were collected from all cases by using a standard clinical form. RESULTS: A total of 13 critically ill pediatric and 5 adult patients, with a median age of 26 days, were included. All were previously exposed to antibiotics and multiple invasive medical procedures. Clinical management included prompt catheter removal and antifungal therapy. Thirteen patients (72%) survived up to 30 days after onset of candidemia. AFLP fingerprinting of all C. auris isolates suggested a clonal outbreak. The isolates were considered resistant to azoles, but susceptible to anidulafungin and 50% of isolates exhibited amphotericin B MIC values of >1 µg/ml. CONCLUSIONS: The study demonstrated that C. auris is a multiresistant yeast pathogen that can be a source of health-care associated infections in tertiary care hospitals with a high potential for nosocomial horizontal transmission.

Neonatal Candidemia Caused by Candida haemulonii: Case Report and Review of Literature.

Candidemia is a frequent condition in Neonatal Intensive Care Units (NICU) and usually complicates the newborns clinical course. Several factors are responsible for candidiasis, such as prematurity and use of broad-spectrum antibiotics, and in these cases, there are the involvement of various Candida species, as C. albicans, and C. parapsilosis. However, other species as C. haemulonii has been rarely described in candidemia cases, being considered an emergent pathogen. Thus, we report a case of neonatal candidemia by C. haemulonii and a review of literature of fungemia by this yeast. The patient was a neonate with gestational age of 26 weeks and birth weight of 660 g hospitalized in a NICU from a Brazilian hospital. The identification of the etiological agent was performed by phenotypic methods, scanning electron microscopy, sequencing of the ITS region of rDNA, and mass spectrometry. Antifungal susceptibility testing was carried out according to the Clinical Laboratories and Standards Institute guidelines. The newborn was diagnosed with candidemia by C. haemulonii resistant to amphotericin B with minimal inhibitory concentration (MIC) of 8 µg/mL, sensitive to fluconazole (MIC: 8 µg/mL) and voriconazole (MIC: 0.12 µg/mL). The treatment with fluconazole (12 mg/kg/day) was established with good outcome. Candidemia by C. haemulonii is still being limited to a few sporadic cases in adults with endemic and restricted occurrences in neonates. Usually, the therapy with amphotericin B is ineffective against this species. Our results showed the importance of the mycological diagnosis associated to antifungigrama for the successful clinical management followed by important epidemiological data.

A quick and low-cost PCR-based assay for Candida spp. identification in positive blood culture bottles.

BACKGROUND: Differences in the susceptibility of Candida species to antifungal drugs make identification to the species level important for clinical management of candidemia. Molecular tests are not yet standardized or available in most clinical laboratories, although such tests can reduce the time required for species identification, as compared to the conventional culture-based methods. To decrease laboratory costs and improve diagnostic accuracy, different molecular methods have been proposed, including DNA extraction protocols to produce pure DNA free of PCR inhibitors. The objective of this study was to validate a new format of molecular method, based on the internal transcribed spacer (ITS) of the rDNA gene amplification followed by sequencing, to identify common and cryptic Candida species causing candidemia by analyzing DNA in blood culture bottles positive for yeasts. METHODS: For DNA extraction, an "in-house" protocol based on organic solvent extraction was tested. Additional steps of liquid nitrogen incubation followed by mechanical disruption ensured complete cell lysis, and highly pure DNA. One hundred sixty blood culture bottles positive for yeasts were processed. PCR assays amplified the ITS region. The DNA fragments of 152 samples were sequenced and these sequences were identified using the GenBank database (NCBI). Molecular yeast identification was compared to results attained by conventional method. RESULTS: The organic solvent extraction protocol showed high reproducibility in regards to DNA quantity, as well as high PCR sensitivity (10 pg of C. albicans DNA and 95% amplification on PCR). The identification of species at the molecular level showed 97% concordance with the conventional culturing method. The molecular method tested in the present study also allowed identification of species not commonly implicated in human infections. CONCLUSIONS: This study demonstrated that our molecular method presents significant advantages over the conventional yeast culture identification method by providing accurate results within 24 hours, in contrast to at least 72 hours required by the automated conventional culture method. Additionally, our molecular method allowed the identification of mixed infections, as well as infections due to emergent fungal pathogens. This economical DNA extraction method developed in our laboratory provided high-quality DNA and 60% cost savings compared to commercial methods.

Molecular identification of melanised non-sporulating moulds: a useful tool for studying the epidemiology of phaeohyphomycosis.

Subcutaneous infections caused by melanised fungi have been increasingly reported among transplant patients, and these infections have the potential for blood and visceral dissemination. Some moulds, such as Mycelia sterilia, cannot grow and sporulate on different media, making their identification impossible by conventional methods. The fast and accurate identification of melanised fungi at the species level is important because species may have tropism to different organs and different susceptibilities to antifungal agents. Molecular tools have been reported to be helpful for the species identification of non-sporulating moulds. Our goal was to identify the species of M. sterilia isolates obtained from clinical samples of transplant patients using sequences of ITS and the D1/D2 regions of rDNA. Clinical samples were obtained from eight kidney transplant recipients who developed subcutaneous fungal infections. The diagnosis was confirmed by histopathology and conventional culture. Histopathology showed septated, melanised hyphae, and the cultures identified non-sporulating fungi. Therefore, the DNA from the M. sterilia isolates was subjected to PCR amplification and sequencing of the ITS and D1/D2 regions. Genus/species identification was obtained by comparison with gene banks. We obtained the following identifications: Alternaria sp. (2), Cochliobolus lunatus/Curvularia lunata (2), Cochliobolus hawaiiensis/Bipolaris hawaiiensis (1), Ochroconis sp. (1), Medicocopsis romeroi/Pyrenochaeta romeroi (1) and Nigrograna mackinnonii/Pyrenochaeta mackinnonii (1).

Candida mesorugosa sp. nov., a novel yeast species similar to Candida rugosa, isolated from a tertiary hospital in Brazil.

Candida rugosa is a yeast species that is emerging as a causative agent of invasive infection, particularly in Latin America. Recently, C. pseudorugosa was proposed as a new species closely related to C. rugosa. We evaluated in this investigation the genetic heterogeneity within the C. rugosa species complex. All clinical isolates used in this study were identified phenotypically as C. rugosa but were genotypically different from the C. rugosa type, ATCC 10571. RAPD marker analysis revealed less than 83% similarity between our clinical isolates and the C. rugosa type strain. The D1/D2 region sequences of our clinical isolates showed 98% identity with C. rugosa but only 94-95% identity with C. pseudorugosa. The ITS rDNA sequences of the Brazilian isolates showed 91% identity with the C. rugosa ATCC 10571 ITS sequence. Network and Bayesian analyses of ITS and housekeeping gene sequences separated our clinical isolates into different branches from C. rugosa type strain. These differences are sufficient to reassign our isolates to a distinct species, named C. mesorugosa.

Changes in cell wall synthesis and ultrastructure during paradoxical growth effect of caspofungin on four different Candida species.

Paradoxical growth (PG) has been described for echinocandins and is characterized by cell growth at drug concentrations above the MIC. In this study, two isolates each of Candida albicans, C. tropicalis, C. orthopsilosis, and C. parapsilosis, all of which displaying PG in response to caspofungin, were subjected to MIC, minimal fungicidal concentration (MFC), and time-kill curve assays to evaluate the levels of PG. Cell wall components and ultrastructural modifications of the PG cells were also investigated. The results showed that when cell growth and survival were evaluated by MFC or time-kill curve assays, high concentrations of caspofungin did not show fungicidal activity against PG cells. Furthermore, for C. parapsilosis and C. orthopsilosis, time-kill curves were more discriminatory than MFCs in detecting the PG effect. The four different Candida species studied demonstrated similar alterations in cell wall components and ultrastructure associated with PG. In PG cells, ß-1,3-glucan content decreased from 2.7- to 7.8-fold, whereas chitin content increased from 4.0- to 6.6-fold. An electron microscopy study of the PG cells revealed morphological alterations, clumping of cells, enlarged cells, the absence of filamentation, abnormal septa, and accumulation of chitin in the cell wall. Also, PG cells basically exhibited a single dark high-density layer in the cell wall, indicating the loss of the ß-1,3-glucan layer. Our results present novel details about the ultrastructural alterations that occur in C. albicans, C. parapsilosis, C. orthopsilosis, and C. tropicalis during PG and show that chitin is the major component of the cell walls of PG cells. Stimulation of chitin synthesis may represent a rescue mechanism against caspofungin activity.

Bloodstream infections due to Trichosporon spp.: species distribution, Trichosporon asahii genotypes determined on the basis of ribosomal DNA intergenic spacer 1 sequencing, and antifungal susceptibility testing.

The reevaluation of the genus Trichosporon has led to the replacement of the old taxon Trichosporon beigelii by six new species. Sequencing of the ribosomal DNA (rDNA) intergenic spacer 1 (IGS1) is currently mandatory for accurate Trichosporon identification, but it is not usually performed in routine laboratories. Here we describe Trichosporon species distribution and prevalence of Trichosporon asahii genotypes based on rDNA IGS1 sequencing as well as antifungal susceptibility profiles of 22 isolates recovered from blood cultures. The clinical isolates were identified as follows: 15 T. asahii isolates, five Trichosporon asteroides isolates, one Trichosporon coremiiforme isolate, and one Trichosporon dermatis isolate. We found a great diversity of different species causing trichosporonemia, including a high frequency of isolation of T. asteroides from blood cultures that is lower than that of T. asahii only. Regarding T. asahii genotyping, we found that the majority of our isolates belonged to genotype 1 (86.7%). We report the first T. asahii isolate belonging to genotype 4 in South America. Almost 50% of all T. asahii isolates exhibited amphotericin B MICs of >or=2 microg/ml. Caspofungin MICs obtained for all the Trichosporon sp. isolates tested were consistently high (MICs >or= 2 microg/ml). Most isolates (87%) had high MICs for 5-flucytosine, but all of them were susceptible to triazoles, markedly to voriconazole (all MICs

The generation and utilization of a cancer-oriented representation of the human transcriptome by using expressed sequence tags.

Whereas genome sequencing defines the genetic potential of an organism, transcript sequencing defines the utilization of this potential and links the genome with most areas of biology. To exploit the information within the human genome in the fight against cancer, we have deposited some two million expressed sequence tags (ESTs) from human tumors and their corresponding normal tissues in the public databases. The data currently define approximately 23,500 genes, of which only approximately 1,250 are still represented only by ESTs. Examination of the EST coverage of known cancer-related (CR) genes reveals that <1% do not have corresponding ESTs, indicating that the representation of genes associated with commonly studied tumors is high. The careful recording of the origin of all ESTs we have produced has enabled detailed definition of where the genes they represent are expressed in the human body. More than 100,000 ESTs are available for seven tissues, indicating a surprising variability of gene usage that has led to the discovery of a significant number of genes with restricted expression, and that may thus be therapeutically useful. The ESTs also reveal novel nonsynonymous germline variants (although the one-pass nature of the data necessitates careful validation) and many alternatively spliced transcripts. Although widely exploited by the scientific community, vindicating our totally open source policy, the EST data generated still provide extensive information that remains to be systematically explored, and that may further facilitate progress toward both the understanding and treatment of human cancers.