Sarah S. Gonçalves

Prevalence rates and antifungal susceptibility profiles of the Candida parapsilosis species complex: results from a nationwide surveillance of candidaemia in Brazil

The genetically heterogeneous taxon Candida parapsilosis was recently reclassified into three species: Candida parapsilosis, Candida orthopsilosis and Candida metapsilosis. The prevalences of these species among 141 bloodstream isolates tested in Brazil were 88% for C. parapsilosis, 9% for C. orthopsilosis, and 3% for C. metapsilosis. Except for three C. orthopsilosis isolates that were considered resistant to 5-flucytosine, all isolates representing the different species of this complex were susceptible to polyenes, triazoles and caspofungin.

Epidemiology and molecular mechanisms of antifungal resistance in Candida and Aspergillus.

The significant increase in the use of antifungal agents, both for the treatment of candidiasis and invasive aspergillosis and as azole fungicides in agricultural crop protection has resulted in the emergence of resistant clinical isolates, particularly to triazoles and echinocandins. Notably, among isolates that were primarily sensitive to fluconazole such as Candida parapsilosis and Candida tropicalis have witnessed an emerging resistance development. Also for echinocandins, the occurrence of Candida isolates with lower susceptibility to these drugs has been reported, which is possibly due to its broad clinical use. Triazole resistance among Aspergillus fumigatus and other Aspergillus species is commonly found in European and Asian countries. Specific mutations are associated with azole resistance in A. fumigatus and these mutations are now reported globally from six continents. Therefore, we highlight the need to conduct antifungal resistance surveillance studies using clinical isolates of Candida and Aspergillus in different geographical regions and monitoring of the infection rates in distinct population groups for early detection of resistance to these drugs and implementation of efficient policies for infection control and treatment.

Aspergillus novoparasiticus: a new clinical species of the section Flavi

During a survey on the incidence of Aspergillus in clinical environments, we found some interesting isolates that were morphologically similar to Aspergillus parasiticus, but differed in the color of the colonies and in the pattern of their conidial ornamentation. In the present study, those isolates were characterized using a polyphasic approach. A phylogenetic analysis was carried out, based on partial fragments of the acetamidase (amdS) and O-methyltransferase (omtS) genes and the internal transcribed spacer (ITS) region of rDNA. This information was combined with a detailed morphological and physiological study that included aflatoxin production and assimilation profiles of different carbon and nitrogen sources. The phenotypic and genotypic results support the proposal of a new species, Aspergillus novoparasiticus, phylogenetically placed in a distinct sister clade to that of A. parasiticus. The former has lobate-reticulate conidia and does not produce aspergillic acid on AFPA or organic acids on CREA, while A. parasiticus has echinulate conidia and produces aspergillic and organic acids. In addition, this new species, as well as A. parasiticus, produces aflatoxins B1, B2, G1 and G2.

Cryptic and Rare Aspergillus Species in Brazil: Prevalence in Clinical Samples and In Vitro Susceptibility to Triazoles

ABSTRACT Aspergillus spp. are among the most common causes of opportunistic invasive fungal infections in tertiary care hospitals. Little is known about the prevalence and in vitro susceptibility of Aspergillus species in Latin America, because there are few medical centers able to perform accurate identification at the species level. The purpose of this study was to analyze the distribution of cryptic and rare Aspergillus species among clinical samples from 133 patients with suspected aspergillosis admitted in 12 medical centers in Brazil and to analyze the in vitro activity of different antifungal drugs. The identification of Aspergillus species was performed based on a polyphasic approach, as well as sequencing analysis of the internal transcribed spacer (ITS) region, calmodulin, and β-tubulin genes and phylogenetic analysis when necessary. The in vitro susceptibility tests with voriconazole, posaconazole, and itraconazole were performed according to the CLSI M38-A2 document (2008). We demonstrated a high prevalence of cryptic species causing human infection. Only three isolates, representing the species Aspergillus thermomutatus, A. ochraceus, and A. calidoustus, showed less in vitro susceptibility to at least one of the triazoles tested. Accurate identifications of Aspergillus at the species level and with in vitro susceptibility tests are important because some species may present unique resistance patterns against specific antifungal drugs.

Accurate Identification of Candida parapsilosis (Sensu Lato) by Use of Mitochondrial DNA and Real-Time PCR

ABSTRACT Candida parapsilosis is the Candida species isolated the second most frequently from blood cultures in South America and some European countries, such as Spain. Since 2005, this species has been considered a complex of 3 closely related species: C. parapsilosis, Candida metapsilosis, and Candida orthopsilosis. Here, we describe a real-time TaqMan-MGB PCR assay, using mitochondrial DNA (mtDNA) as the target, which readily distinguishes these 3 species. We first used comparative genomics to locate syntenic regions between these 3 mitochondrial genomes and then selected NADH5 as the target for the real-time PCR assay. Probes were designed to include a combination of different single-nucleotide polymorphisms that are able to differentiate each species within the C. parapsilosis complex. This new methodology was first tested using mtDNA and then genomic DNA from 4 reference and 5 clinical strains. For assay validation, a total of 96 clinical isolates and 4 American Type Culture Collection (ATCC) isolates previously identified by internal transcribed spacer (ITS) ribosomal DNA (rDNA) sequencing were tested. Real-time PCR using genomic DNA was able to differentiate the 3 species with 100% accuracy. No amplification was observed when DNA from other species was used as the template. We observed 100% congruence with ITS rDNA sequencing identification, including for 30 strains used in blind testing. This novel method allows a quick and accurate intracomplex identification of C. parapsilosis and saves time compared with sequencing, which so far has been considered the “gold standard” for Candida yeast identification. In addition, this assay provides a useful tool for epidemiological and clinical studies of these emergent species.

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).

In Vitro Antifungal Susceptibility of Clinically Relevant Species Belonging to Aspergillus Section Flavi

ABSTRACT The in vitro antifungal susceptibility of 77 isolates belonging to different clinically relevant species of Aspergillus section Flavi, including those of different phylogenetic clades of A. flavus, was tested for nine antifungal agents using a microdilution reference method (CLSI, M38-A2). Terbinafine and the echinocandins demonstrated lower MICs/MECs for all species evaluated, followed by posaconazole. Amphotericin B showed MICs ≥ 2 μg/ml for 38 (49.4%) of the 77 isolates tested.

Molecular phylogeny and phenotypic variability of clinical and environmental strains of Aspergillus flavus

Aspergillus flavus is the second most common cause of aspergillosis infection in immunocompromised patients and is responsible for the production of aflatoxins. Little is known about the population structure of A. flavus, although recent molecular and phenotypic data seem to demonstrate that different genetic lineages exist within this species. The aim of this study was to carry out a morphological, physiological, and molecular analysis of a set of clinical and environmental isolates to determine whether this variability is due to species divergence or intraspecific diversity, and to assess whether the clinical isolates form a separate group. The amdS and omtA genes were more phylogenetically informative than the other tested genes and their combined analysis inferred three main clades, with no clear distinction between clinical and environmental isolates. No important morphological and physiological differences were found between the members of the different clades, with the exception of the assimilation of d-glucosamine, which differentiates the members of the clade II from the others.

Natural occurrence of tenuazonic acid and Phoma sorghina in Brazilian sorghum grains at different maturity stages

A survey of 100 samples of sorghum grains was carried out to determine Phoma spp. and tenuazonic acid (TA) contamination using molecular tools and LC-MS/MS. Sorghum samples were obtained at the following four grain maturity stages: milk (S1), soft dough (S2), hard dough (S3), and physiological maturity (S4). The results revealed a good correlation between Phoma and TA occurrence during grain development. The samples showed Phoma contamination with frequencies ranging from 2.4% (S1) to 87.4% (S4), and the molecular identification revealed P. sorghina as the only Phoma specie isolated. Tenuazonic acid was found in sorghum grains at all maturity stages. In S2, S3 and S4, 100% of the samples showed TA contamination with levels ranging from 20 to 1234µg/kg. Low levels of TA were detected in 36% of the samples collected at S1 stage. This is the first report of tenuazonic acid in Brazilian sorghum grains.

Melanized fungal infections in kidney transplant recipients: contributions to optimize clinical management

OBJECTIVES This is a retrospective and observational study addressing clinical and therapeutic aspects of melanized fungal infections in kidney transplant recipients. METHODS We retrospectively reviewed medical records of all patients admitted between January 1996 and December 2013 in a single institution who developed infections by melanized fungi. RESULTS We reported on 56 patients aged between 30 and 74 years with phaeohyphomycosis or chromoblastomycosis (0.54 cases per 100 kidney transplants). The median time to diagnosis post-transplant was 31.2 months. Thirty-four (60.8%) infections were reported in deceased donor recipients. Fifty-one cases of phaeohyphomycosis were restricted to subcutaneous tissues, followed by two cases with pneumonia and one with brain involvement. Most dermatological lesions were represented by cysts (23/51; 45.1%) or nodules (9/51; 17.9%). Exophiala spp. (34.2%) followed by Alternaria spp. (7.9%) were the most frequent pathogens. Graft loss and death occurred in two patients and one patient, respectively. Regarding episodes of subcutaneous phaeohyphomycosis, a complete surgical excision without antifungal therapy was possible in 21 of 51 (41.2%) patients. Long periods of itraconazole were required to treat the other 30 (58.8%) episodes of subcutaneous disease. All four cases of chromoblastomycosis were treated only with antifungal therapy. CONCLUSIONS Melanized fungal infections should be considered in the differential diagnosis of all chronic skin lesions in transplant recipients. It is suggested that the impact of these infections on graft function and mortality is low. The reduction in immunosuppression should be limited to severely ill patients.