Molecular Phylogenetic Analysis of Paracoccidioides Species Complex Present in Paracoccidioidomycosis Patient Tissue Samples.

Paracoccidioidomycosis (PCM) is the main and most prevalent systemic mycosis in Latin America, that until recently, it was believed to be caused only by Paracoccidioides brasiliensis (P. brasiliensis). In 2006, researchers described three cryptic species: S1, PS2, PS3, and later, another one, PS4. In 2009, Paracoccidioides lutzii (Pb01-like) was described, and in 2017, a new nomenclature was proposed for the different agents: P. brasiliensis (S1), P. americana (PS2), P. restrepiensis (PS3), and P. venezuelensis (PS4). These species are not uniformly distributed throughout Latin America and, knowing that more than one cryptic species could coexist in some regions, we aimed to identify those species in patients' biopsy samples for a better understanding of the distribution and occurrence of these recently described species in Botucatu region. The Hospital of Medical School of Botucatu-UNESP, which is a PCM study pole, is located in São Paulo State mid-west region and is classified as a PCM endemic area. Genotyping analyses of clinical specimens from these patients that have been diagnosed and treated in our Hospital could favor a possible correlation between genetic groups and mycological and clinical characteristics. For this, molecular techniques to differentiate Paracoccidioides species in these biopsies, such as DNA extraction, PCR, and sequencing of three target genes (ITS, CHS2, and ARF) were conducted. All the sequences were analyzed at BLAST to testify the presence of P. brasiliensis. The phylogenetic trees were constructed using Mega 7.0 software and showed that 100% of our positive samples were from S1 cryptic species, therefore P. brasiliensis. This is important data, demonstrating the predominance of this species in the São Paulo State region.

Aspects related to biofilm production and antifungal susceptibility of clinically relevant yeasts of the genus Trichosporon.

Trichosporonosis corresponds to a systemic fungal disease that leads to high mortality rates and is frequently associated with medical devices. It affects immunosuppressed patients in particular and is strongly linked to acquired human immunodeficiency, organ and tissue transplants, and malignant hematologic diseases such as leukemia and lymphomas. Trichosporon infections have been increasingly reported worldwide; however, little information is available either about their characteristics or the causative microorganism. Thus, the aims of the present study were: to investigate 59 yeasts of the genus Trichosporon by verifying the biofilm formation capacity of isolates; to analyze the susceptibility patterns of planktonic cells against the antifungals fluconazole, itraconazole, amphotericin-B, voriconazole, and caspofungin by comparing European Committee for Antimicrobial Susceptibility Testing (EUCAST) broth microdilution technique with the commercial method Etest; and to assess the susceptibility patterns of biofilm cells (sessile) against the same antifungals through broth microdilution. The ability to form biofilm on the surface of polystyrene plates was noted for all isolates, and 54.3% of samples were considered strong producers. Comparison between the antifungal susceptibility techniques evidenced that Etest showed higher and discordant minimum inhibitory concentrations (MICs) from those obtained by the microdilution method, especially for fluconazole, itraconazole, and caspofungin. Considering the susceptibility of biofilms, most species had high MIC50 and MIC90 against the tested antifungals, showing 4-to-66-fold higher concentrations for amphotericin B and 2-to-33-fold greater concentrations for caspofungin. These results highlight the importance of further studies with Trichosporon spp. for comparison between laboratory findings and in vivo response, considering both the susceptibility tests and the behavior of biofilm cells against drugs.

This study investigated 59 isolates of the medically important yeast Trichosporon in relation to their ability to form biofilms and the susceptibility of biofilms to antifungal agents. All isolates were able to produce biofilms and biofilms showed lower antifungal susceptibility.

Outbreak of equine pythiosis in a southeastern region of Brazil: Environmental isolation and phylogeny.

Pythiosis is a disease caused by the oomycete Pythium insidiosum, mainly reported in equines, dogs and humans and directly transmitted through contaminant zoospores in aquatic environments. We report the first outbreak of equine pythiosis in five equines. Wound samples were submitted for diagnostic testing including mycological culture and nested PCR. Treatment approaches consisted of conventional and alternative therapies. Microbiological analyses were performed using water samples from the riverbanks close to where the animals had grazed. All animals were positive for P. insidiosum cultures, and two animals responded successfully to alternative therapy (ozone therapy). After culture and molecular analysis of environmental samples, the presence of P. insidiosum in one section of the Tietê River was confirmed through a 99% sequence identity. Phylogenetic analyses using the cytochrome oxidase II gene showed that the animal isolates clustered in clade I and the environmental isolates clustered in clade III. Although the environmental and wound isolates belonged to different genetic clades, we concluded that the Tietê River is an important source of infection by P. insidiosum and that research concerning environmental isolation of P. insidiosum from rivers and lakes should be strongly facilitated in Brazil.

Comparing the phenotypic, genotypic, and proteomic identification of Trichosporon species: A globally emerging yeast of medical importance.

Trichosporon spp. are widely distributed in the nature, comprising species that inhabit different ecological niches and can be found in the water, soil, and body surface of animals and humans. Such microorganisms have been classically associated with superficial infections; however, in the last decades, they have also been related to disseminated infections in immunocompromised patients, behaving as opportunistic agents, which demands rapid and accurate species identification for efficient therapy. Concordance level between the traditional phenotypic method and the molecular technique (gold standard) in the identification of all 59 Trichosporon samples was 59.3%. Identification concordance between MALDI-TOF spectrometry and the molecular technique was 71.2%. No isolate of environmental origin was identifiable by MALDI-TOF mass spectrometry (MS), and 100% of such environmental isolates were discordant for IGS region sequencing and phenotypic characterization. Both comparisons evidenced greatest concordance in the identification of T. asahii. The species T. debeurmannianum, T. dermatis, T. venhuisii and T. insectorum were not properly identified by both MALDI-TOF MS and the phenotypic technique. MALDI-TOF MS, in particular, seems to be appropriate to investigate yeasts of the genus Trichosporon; however, database updates are still necessary, especially for species that are not common in the clinical routine. With the aim of helping understand the aspects involved in early and accurate diagnosis of infections caused by this opportunistic agent, the present study compared the phenotypic, molecular (IGS region) and mass-spectrometry (MALDI-TOF) identification of 59 yeasts of the genus Trichosporon which had clinical and environmental origin and were kept in a mycology collection.

The present study compared the phenotypic, genotypic, and mass-spectrometry (MALDI-TOF) identification of 59 yeasts of the genus Trichosporon. MALDI-TOF MS, in particular, seems to be appropriate to investigate this yeasts when compared to a molecular technique (gold standard).

Paracoccidioides brasiliensis Isolated from Nine-Banded Armadillos (Dasypus novemcinctus) Reveal Population Structure and Admixture in the Amazon Basin.

Paracoccidioidomycosis is an endemic fungal disease to Latin America caused by at least five species-level genotypes of Paracoccidioides, named P. lutzii, P. brasiliensis (S1a and S1b populations), P. americana, P. restrepiensis, and P. venezuelensis. In this manuscript, we report on Paracoccidioides sp. sampling efforts in armadillos from two different areas in Brazil. We sequenced the genomes of seven Paracoccidioides isolates and used phylogenomics and populations genetics for genotyping. We found that P. brasiliensis and P. lutzii are both present in the Amazon region. Additionally, we identified two Paracoccidioides isolates that seem to be the result of admixture between divergent populations within P. brasiliensis sensu stricto. Both of these isolates were recovered from armadillos in a P. lutzii endemic area in Midwestern Brazil. Additionally, two isolates from human patients also show evidence of resulting from admixture. Our results suggest that the populations of P. brasiliensis sensu stricto exchange genes in nature. More generally, they suggest that population structure and admixture within species is an important source of variation for pathogenic fungi.

Antifungal activity of liriodenine on agents of systemic mycoses, with emphasis on the genus Paracoccidioides.

BACKGROUND: Endemic systemic mycoses remain a health challenge, since these opportunistic diseases are increasingly infecting immunosuppressed patients. The simultaneous use of antifungal compounds and other drugs to treat infectious or non-infectious diseases has led to several interactions and undesirable effects. Thus, new antifungal compounds should be investigated. The present study aimed to evaluate the activity of liriodenine extracted from Annona macroprophyllata on agents of systemic mycoses, with emphasis on the genus Paracoccidioides. METHODS: The minimum inhibitory concentration (MIC) and minimum fungicide concentration (MFC) were determined by the microdilution method. The cellular alterations caused by liriodenine on a standard P. brasiliensis (Pb18) strain were evaluated by transmission and scanning electron microscopy. RESULTS: Liriodenine was effective only in 3 of the 8 strains of the genus Paracoccidioides and in the Histoplasma capsulatum strain, in a very low concentration (MIC of 1.95 µg.mL-1); on yeasts of Candida spp. (MIC of 125 to 250 µg.mL-1), including C. krusei (250 µg.mL-1), which has intrinsic resistance to fluconazole; and in Cryptococcus neoformans and Cryptococcus gattii (MIC of 62.5 µg.mL-1). However, liriodenine was not effective against Aspergillus fumigatus at the studied concentrations. Liriodenine exhibited fungicidal activity against all standard strains and clinical isolates that showed to be susceptible by in vitro tests. Electron microscopy revealed cytoplasmic alterations and damage to the cell wall of P. brasiliensis (Pb18). CONCLUSION: Our results indicate that liriodenine is a promising fungicidal compound that should undergo further investigation with some chemical modifications.

Rabies virus and Histoplasma suramericanum coinfection in a bat from southeastern Brazil.

Bats are essential to the global ecosystem, but their ability to harbour a range of pathogens has been widely discussed, as well as their role in the emergence and re-emergence of infectious diseases. This paper describes the first report of coinfection by two zoonotic agents, rabies virus (RABV) and the fungus Histoplasma suramericanum in a bat. The bat was from the Molossus molossus species, and it was found during the daytime in the hallway of a public psychiatric hospital in a municipality in São Paulo State, southeastern Brazil. RABV infection was diagnosed by the direct fluorescent antibody test and mouse inoculation test. The fungus was isolated by in vitro culture. Both diagnoses were confirmed by molecular techniques. Phylogenetic analysis showed that the fungus isolate had proximity to H. suramericanum in the Lam B clade, while the RABV isolate was characterized in the Lasiurus cinereus lineage. Since the M. molossus bat was found in a peri-urban transition area (urban/peri-urban), the possibility of cross-species transmission of this RABV lineage becomes more plausible, considering that this scenario may provide shelter for both M. molossus and L. cinereus. These are relevant findings since there has been an increase in bat populations in urban and peri-urban areas, particularly due to environmental modifications and anthropogenic impacts on their habitat. Thus, the detection of two zoonotic agents in a bat found in a public hospital should raise awareness regarding the importance of systematic surveillance actions directed towards bats in urban areas.

Antifungal activity of liriodenine on agents of systemic mycoses, with emphasis on the genus Paracoccidioides

Endemic systemic mycoses remain a health challenge, since these opportunistic diseases are increasingly infecting immunosuppressed patients. The simultaneous use of antifungal compounds and other drugs to treat infectious or non-infectious diseases has led to several interactions and undesirable effects. Thus, new antifungal compounds should be investigated. The present study aimed to evaluate the activity of liriodenine extracted from Annona macroprophyllata on agents of systemic mycoses, with emphasis on the genus Paracoccidioides. Methods: The minimum inhibitory concentration (MIC) and minimum fungicide concentration (MFC) were determined by the microdilution method. The cellular alterations caused by liriodenine on a standard P. brasiliensis (Pb18) strain were evaluated by transmission and scanning electron microscopy. Results: Liriodenine was effective only in 3 of the 8 strains of the genus Paracoccidioides and in the Histoplasma capsulatum strain, in a very low concentration (MIC of 1.95 µg.mL-1); on yeasts of Candida spp. (MIC of 125 to 250 µg.mL-1), including C. krusei (250 µg.mL-1), which has intrinsic resistance to fluconazole; and in Cryptococcus neoformans and Cryptococcus gattii (MIC of 62.5 µg.mL-1). However, liriodenine was not effective against Aspergillus fumigatus at the studied concentrations. Liriodenine exhibited fungicidal activity against all standard strains and clinical isolates that showed to be susceptible by in vitro tests. Electron microscopy revealed cytoplasmic alterations and damage to the cell wall of P. brasiliensis (Pb18). Conclusion: Our results indicate that liriodenine is a promising fungicidal compound that should undergo further investigation with some chemical modifications.(AU)

Apiotrichum veenhuisii isolated from a pediatric patient with acute myeloid leukemia: The first case in humans.

This is the first report of the yeast Apiotrichum veenhuisii (formerly Trichosporon veenhuisii) causing disease in humans; its virulence and in vitro behavior against antifungals were also studied. The sample was isolated from biopsy fragments of disseminated lesions on the skin of a pediatric patient with acute myeloid leukemia. The studied virulence factors evidenced that the strain tested negative for secretion of the enzymes proteinase, phospholipase, and hemolysin. The isolate was characterized as low biofilm producer. Except for amphotericin B and voriconazole, the sample presented high minimum inhibitory concentration values against azole and echinocandins.

Infections Caused by Fusarium Species in Pediatric Cancer Patients and Review of Published Literature.

Fusarium species have emerged as responsible for a broad spectrum of infections, including superficial, locally invasive and disseminated ones, especially in the hospital environment. Since there are few reports of invasive and disseminated fusariosis in children, the aim of this study was to report four cases of nosocomial infection caused by this microorganism in children with cancer hospitalized in a public children's hospital located in Brazil. Two of these patients were female and two were male. All patients presented febrile neutropenia, while three patients had acute lymphocytic leukemia and one patient had Wilms' tumor as underlying disease. In two cases, fungi were isolated from blood and identified as Fusarium oxysporum species complex after phenotypic and genotypic studies, while in two other cases fungi were isolated from skin biopsies and identified as Fusarium solani species complex. One patient died 12 days after the onset of cutaneous lesions. All isolates, after susceptibility testing, presented high levels of minimum inhibitory concentration for itraconazole, voriconazole and amphotericin B. Considering the emergence of filamentous fungi as etiologic agents of nosocomial infections, health professionals should be aware of the problems these infections, especially fungal ones, may cause to debilitated patients.

Chemical Composition and Bioactivity of Essential Oil from Blepharocalyx salicifolius.

Natural products represent a source of biologically active molecules that have an important role in drug discovery. The aromatic plant Blepharocalyx salicifolius has a diverse chemical constitution but the biological activities of its essential oils have not been thoroughly investigated. The aims of this paper were to evaluate in vitro cytotoxic, antifungal and antibacterial activities of an essential oil from leaves of B. salicifolius and to identify its main chemical constituents. The essential oil was extracted by steam distillation, chemical composition was determined by gas chromatography/mass spectrometry, and biological activities were performed by a microdilution broth method. The yield of essential oil was 0.86% (w/w), and the main constituents identified were bicyclogermacrene (17.50%), globulol (14.13%), viridiflorol (8.83%), γ-eudesmol (7.89%) and α-eudesmol (6.88%). The essential oil was cytotoxic against the MDA-MB-231 (46.60 µg·mL-1) breast cancer cell line, being more selective for this cell type compared to the normal breast cell line MCF-10A (314.44 µg·mL-1). Flow cytometry and cytotoxicity results showed that this oil does not act by inducing cell death, but rather by impairment of cellular metabolism specifically of the cancer cells. Furthermore, it presented antifungal activity against Paracoccidioides brasiliensis (156.25 µg·mL-1) but was inactive against other fungi and bacteria. Essential oil from B. salicifolius showed promising biological activities and is therefore a source of molecules to be exploited in medicine or by the pharmaceutical industry.