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Chromoblastomycosis and leprosy are chronic diseases with high prevalence in tropical and subtropical regions. Brazil is one of the countries with the highest incidence and prevalence for both diseases, however, reports of co-infections are scarce. The aim of this study was to describe three cases of chromoblastomycosis-leprosy co-infection in patients from Mato Grosso state, Brazil. A review of chromoblastomycosis-leprosy co-infection was performed of English, Portuguese and Spanish publications in LILACS, SciELO, PubMed and Web of Science databases using the descriptors (chromoblastomycosis OR cromoblastomicose OR cromoblastomicosis) AND (leprosy OR hanseníase OR lepra), without time period delimitation. Nineteen cases were included, 16 cases were published in 11 articles, plus the three cases reported in the current study. Most reported coninfection cases came from Brazil. Majority of the patients were male with a mean age of 52.2 years. Farmer was the main occupational activity reported. In 12 patients, the clinical signs and symptoms of leprosy started first. No contacts with patients affected by leprosy, armadillos or history of injuries at the anatomical site of chromoblastomycosis lesions were reported. Five leprosy patients who received steroid treatment for leprosy reactions or neuropathies, were diagnosed with chromoblastomycosis during immunosuppressive therapy. Four cases (21.1%) were reported among the elderly patients. Co-infections in patients with chromoblastomycosis or leprosy are uncommon, but the possibility should always be considered, especially if the patient is undergoing immunosuppressive treatment or is elder.

Relatório de Estágio de Natureza Profissional no âmbito do Mestrado Enfermagem Comunitária apresentada na Escola Superior de Saúde do Instituto Politécnico de Viana do Castelo O envelhecimento demográfico é uma realidade inquietante. Portugal, à semelhança de outros países, tem vindo a registar um aumento da longevidade e da população idosa. Há, portanto, maior ênfase na qualidade de vida (QdV) da pessoa idosa, transformando-a num foco importante para políticas de saúde. Torna-se relevante analisar este conceito e de que forma é percecionado e influenciado para obter um conhecimento profundo da realidade do envelhecimento e elencar um conjunto de ações numa lógica de co-construção que permita repensar o envelhecimento. Desta forma, elaborou-se um trabalho de investigação com o objetivo principal de conhecer a perceção de QdV dos idosos institucionalizados. Para tal, realizou-se um estudo de abordagem quantitativa, observacional, transversal, analítico-correlacional, tendo sido utilizado como instrumento de avaliação de percepção de QdV a WHOQOL-Bref. Da amostra de 181 idosos com idades compreendidas entre os 65 e os 96 anos resultou numa perceção de QdV mediana (51.8 %), mas com valores preocupantes no âmbito do domínio físico (48.1%). Nos estudos de comparação constatou-se a existência de várias variáveis implicadas na perceção de QdV. Notou-se uma melhor QdV nas mulheres no domínio das relações sociais (p=0.032), níveis de perceção de QdV melhores no domínio físico (p=0.009) em idosos mais novos, a escolha da institucionalização é um fator preditor de QdV no total (p=0.025), idosos que recebem visitas apresentam melhor QdV no domínio das relações sociais (p=0.015), a participação dos idosos em atividades proporcionadas pelas instituições aumenta a perceção de QdV no domínio psicológico (p=0.001), o consumo de bebidas alcoólicas sobe a QdV no domínio perceção de QdV e saúde (p=0.007), os hábitos tabágicos melhoram a perceção da QdV no domínio psicológico (p=0.007), ter um sono reparador é positivo para a QdV no domínio físico (p=0.043), a utilização de um equipamento adaptativo diminui a perceção de QdV no domínio físico (p=0.036) e a toma de medicação ansiolítica está associada a piores níveis de perceção de QdV no domínio físico (p=0.005). Verificou-se a uma boa mobilidade e a uma boa condição de saúde está associada a uma melhor perceção de QdV (p

INTRODUCTION: Chromoblastomycosis is a disease caused by melanized fungi, primarily belonging to the genera Fonsecaea and Cladophialophora, mainly affecting individuals who are occupationally exposed to soil and plant products. This research aimed to determine the clinical, epidemiological and laboratory characteristics of chromoblastomycosis in the state of Mato Grosso, Brazil. MATERIALS AND METHODS: Patients diagnosed with chromoblastomycosis treated at the Júlio Müller University Hospital, Cuiabá, Brazil, from January 2015 to December 2020, whose isolates were preserved in the Research Laboratory of the Faculty of Medicine of the Federal University of Mato Grosso. Isolates were identified by partly sequencing the Internal Transcribed Spacer (ITS) and β-tubulin (BT2) loci. AFLP fingerprinting was used to explore the genetic diversity. Susceptibility to itraconazole, voriconazole, 5-fluorocytosine, terbinafine and amphotericin B was determined by the broth microdilution technique. RESULTS: Ten patients were included, nine were male (mean age=64.1 years). Mean disease duration was 68 years. Lesions were mainly observed in the lower limbs. Predominant clinical forms were verrucous and scarring. Systemic arterial hypertension and type II diabetes mellitus were the predominant comorbidities. Leprosy was the main concomitant infectious disease. Fonsecaea pedrosoi was the unique etiological agent identified with moderate genetic diversity (H=0.3934-0.4527; PIC=0.3160-0.3502). Antifungal agents with the highest activity were terbinafine, voriconazole and itraconazole. CONCLUSION: Chromoblastomycosis is affecting the poor population in rural and urban areas, mainly related to agricultural activities, with F. pedrosoi being the dominant etiologic agent. All isolates had low MICs for itraconazole, voriconazole and terbinafine, confirming their importance as therapeutic alternatives for chromoblastomycosis.

Chromoblastomycosis (CBM) is a chronic cutaneous and subcutaneous mycosis caused by black, dimorphic, and filamentous fungi of the Herpothrichiellaceae family, such as species of the genus Fonsecaea. These fungi can switch between the saprophytic forms (conidia and hyphae) and the pathogenic form, the muriform cells (MCs), which is considered an essential mechanism for fungal virulence. Nearly all types of cells can produce membranous structures formed by a lipid bilayer that communicate extracellularly with other cells, known as “extracellular vesicles” (EVs), which may act as virulence factors, as observed for several species of pathogenic fungi. Our findings demonstrated for the first time that F. pedrosoi, F. nubica, and F. erecta produce EVs in response to nutritional conditions. The EVs varied in sterol and protein contents, size, and morphology. Moreover, the EVs induced different cytokine and nitric oxide release patterns by bone marrow-derived macrophages (BMDMs). The EVs activated IL-1β production, possibly acting as the first signal in inflammasome activation. Unlike the pathogenic species, the EVs isolated from F. erecta did not significantly stimulate TNF and IL-10 production in general. Overall, these results demonstrated that different species of Fonsecaea produce EVs capable of modulating pro- and anti-inflammatory cytokine and nitric oxide production by BMDMs and that growth conditions affected the immunomodulatory capacities of the EVs as well as their size, content, and morphology.

The black yeast-like fungus Arthrocladium fulminans is known from strains that cause severe and eventually fatal disseminated infections in immunocompromised patients. Given the dramatic outcome of this clinical case, it is essential to understand the virulence potential of this species. The fungus is a member of the family Trichomeriaceae, at some phylogenetic distance from the Herpotrichiellaceae where most infectious fungi in the order Chaetothyriales are located. Main ecological preferences among Trichomeriaceae include colonization of exposed inert surfaces. Currently, black yeasts genomes that are available in public databases cover members of the families Herpotrichiellaceae and Cyphellophoraceae. In the present report, we sequenced the genome of the first member and only clinical representative of the family Trichomeriaceae.

Made available in DSpace on 2020-12-12T01:57:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-03-01 National Council for Scientific Research Federal Agency for Scientific Organizations Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Chromoblastomycosis is a neglected disease characterized by cutaneous, subcutaneous or disseminated lesions. It is considered an occupational infectious disease that affects mostly rural workers exposed to contaminated soil and vegetal matter. Lesions mostly arise after a traumatic inoculation of herpotrichiellaceous fungi from the Chaetothyriales order. However, the environmental niche of the agents of the disease remains obscure. Its association with insects has been predicted in a few studies. Therefore, the present work aimed to analyze if social insects, specifically ants, bees, and termites, provide a suitable habitat for the fungi concerned. The mineral oil flotation method was used to isolate the microorganisms. Nine isolates were recovered and phylogenetic analysis identified two strains as potential agents of chromoblastomycosis, i.e., Fonsecaea pedrosoi CMRP 3076, obtained from a termite nest (n = 1) and Rhinocladiella similis CMRP 3079 from an ant exoskeleton (n = 1). In addition, we also identified Fonsecaea brasiliensis CMRP 3445 from termites (n = 1), Exophiala xenobiotica CMRP 3077 from ant exoskeleton (n = 1), Cyphellophoraceae CMRP 3103 from bees (n = 1), Cladosporium sp. CMRP 3119 from bees (n = 1), Hawksworthiomyces sp. CMRP 3102 from termites (n = 1), and Cryptendoxyla sp. from termites (n = 2). The environmental isolate of F. pedrosoi CMRP 3076 was tested in two animal models, Tenebrio molitor and Wistar rat, for its pathogenic potential with fungal retention in T. molitor tissue. In the Wistar rat, the cells resembling muriform cells were observed 30 d after inoculation. Graduate Program in Microbiology Parasitology and Pathology Federal University of Paraná University Center Campo Real Federal University of Paraná Graduate Program in Pharmacology UFPR Medical Department Federal University of Maranhão Embrapa Amazônia Oriental CPATU Westerdijk Fungal Biodiversity Institute Department of Biochemistry and Microbiology Institute of Biological Sciences UNESP-São Paulo State University Division of Microbial Resources CPQBA University of Campinas Center of Expertise in Mycology of Radboud University Medical Center Canisius Wilhelmina Hospital Department of Biochemistry and Microbiology Institute of Biological Sciences UNESP-São Paulo State University CNPq: 312811/2018–7 CAPES: Brazil CAPES: Finance Code 001, Brasilia

With the COVID-19 pandemic, many diagnostic tests (molecular or immunological) were rapidly standardised, given the urgency of the situation, many are still in the process of being validated. The main objective of this study was to review the aspects of the diagnostic kits approved in Brazil and their application in the different federative units to gather epidemiological information. In order to achieve these objectives, a survey was carried out on the data available at the regulatory agency (ANVISA) and in the literature. The main countries that have registered products in Brazil are China (51.4%), Brazil (16.6%), South Korea (9.2%), USA (8.8%) and Germany (3.6%). The methodologies of these products are based on the detection of nucleic-acid (15.8%), antigen (13%) and antibody (71.2%). In the immunological tests, it was verified that the sensitivity ranged from 55 to 100% and the specificity from 80 to 100%. The percentage of cases in the samples tested in Brazil is elevated in almost all federative units since eight states showed 40% of positive cases in tested samples, while 18 states displayed between 20 and 40%. In conclusion, this review showed that Brazil is dependent on external technology to respond to pandemics, epidemics and endemics disease and needs to improve its biotechnological scheme to solve further diseases outbreaks.

The antimicrobial potential of cranberry hydro alcoholic extracts (CrE) was evaluated against Escherichia coli isolated from women with urinary tract infection (UTI). CrE was diluted based on the percentage of proanthocyanidins (PACs) in extract powder for final concentrations: 1.26%; 2.52%; 3.35%, 5.03% and 10.06%. CrE antimicrobial potential was evaluated by disk and well diffusion assays, and by in vitro direct action against E. coli. Antibacterial action was observed for all performed tests: minimal inhibitory concentration (MIC) was 1.26% PACs per disk diffusion assay and 2.52% of PACs by well diffusion assay. The in vitro antimicrobial direct action against E. coli resulted 3.8 Log10 cycles reduction for a concentration of 5.03% of PACs. One of the isolates showed multi resistance to antibiotics. But it was also inhibited more than any of the antibiotic tested in well diffusion assay. Only for concentrations 1.26%, 2.52% and 3.45% the inhibition of Escherichia coli by cranberry extract was dose-dependent, i.e directly proportional to the concentration of PACs. The results indicate a inhibitory action high potential of CrE. However, more in vitro and in vivo analysis can be performed to fix which the best concentration of CrE capable of causing a real beneficial effect on UTI´s.

A pork sausage was produced with low sodium content (1.64%) to which Lactobacillus sakei was added with the aim of developing a meat pork sausage for cooking and having technological, organoleptic, and hygienic advantages. The lactic acid bacteria (LAB) L. sakei, Lactococcus sp., and Pediococcus pentosaceus were submitted to extreme pH, temperature, and NaCl conditions. Lactobacillus sakei was used in pork sausage because of its resistance to different culture conditions and its antimicrobial potential. The food-borne pathogens Listeria monocytogenes Scott A, Enterococcus faecalis, and Staphylococcus aureus were used as indicator microorganisms to evaluate the antimicrobial activity of selected LAB strains. Salmonella enterica serotype Choleraesuis is a common pathogen of pigs. To the raw sausage product containing L. sakei and nonpathogenic endogenous microbiota, we added about >104 and 5 CFU/g of S. enterica serotype Choleraesuis to evaluate the inhibitory potential of L. sakei towards this pathogen. Salmonella Choleraesuis was inhibited in the presence of L. sakei over 7 days of storage of the meat product (about 3.0 log cycles reduction). Lactobacillus sakei significantly increased inhibition when compared with the nonfermented sausage. Thus, L. sakei BAS0117 played an important role as an additional hurdle in the fermented meat pork sausage during storage.

Fungal peritonitis is frequent on peritoneal dialysis, with rare cases by Exophiala dermatitidis. A 25-month-old female admitted to the pediatric ICU with acute renal failure was submitted to peritoneal dialysis. After 10 days patient presented fever. Peritoneal fluid culture showed yeast colonies molecularly identified as E. dermatitidis. Patient was treated with voriconazole and hemodialysis. The literature was reviewed. Disseminated infections are frequently fatal, but appropriate diagnose and therapeutic led to cure in this case. Keywords: Phaeohyphomycosis, Fungal infection, Dialysis, Therapeutic management, Molecular diagnosis

Made available in DSpace on 2021-06-25T10:59:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-05-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Environmental pollution with alkylbenzene hydrocarbons such as toluene is a recurring phenomenon. Their toxicity and harmful effect on people and the environment drive the search for sustainable removal techniques such as bioremediation, which is based on the microbial metabolism of xenobiotic compounds. Melanized fungi present extremophilic characteristics, which allow their survival in inhospitable habitats such as those contaminated with hydrocarbons. Screening methodologies for testing the microbial assimilation of volatile organic compounds (VOC) are scarce despite their importance for the bioremediation of hydrocarbon associated areas. In this study, 200 strains of melanized fungi were isolated from four different hydrocarbon-related environments by using selective methods, and their biodiversity was assessed by molecular and ecological analyses. Seventeen genera and 27 species from three main orders, namely Chaetothyriales, Cladosporiales, and Pleosporales, were identified. The ecological analysis showed a particular species distribution according to their original substrate. The isolated strains were also screened for their toluene assimilation potential using a simple and inexpensive methodology based on miniaturized incubations under controlled atmospheres. The biomass produced by the 200 strains with toluene as the sole carbon source was compared against positive and negative controls, with glucose and with only mineral medium, respectively. Nineteen strains were selected as the most promising for further investigation on the biodegradation of alkylbenzenes. Center for the Study of Social Insects São Paulo State University (UNESP) GIRO Institute of Agrifood Research and Technology (IRTA), Torre Marimon Basic Pathology Department Federal University of Paraná (UFPR) Division of Microbial Resources CPQBA University of Campinas (Unicamp) Center for the Study of Social Insects São Paulo State University (UNESP) CAPES: 2013/01556-2 CNPq: 2013/01556-2 FAPESP: 2013/01556-2 CNPq: 312811/2018-7.

Não há dúvidas de que a pandemia de Covid-19 é um choque dramático na saúde e na vida da humanidade. Aqueles que descartam ou menosprezam este efeito estão a ignorar algo decisivo. O impacto económico que a pandemia gerou, é muito superior a qualquer outra coisa que o mundo tenha visto desde 1945. O surto de coronavírus constitui um grande choque económico para a União Europeia, tendo sido determinado regularmente, medidas económicas coordenadas. Relativamente à questão propriamente produtiva, o problema dominante vem do lado da procura. Por tanto a função do Estado tem de ser sustentar o rendimento dos cidadãos, em particular os mais afetados. Tendo como principal foco as empresas. Ou seja, salvar as empresas da crise é também proteger as fontes de receita fiscal. Com regularidade, têm sido aplicadas medidas fiscais destinadas às empresas das regiões e setores mais afetados (por exemplo, diferimento do pagamento dos impostos sobre o rendimento das sociedades, das contribuições para a segurança social e do Imposto sobre o Valor Acrescentado (IVA); adiantamento dos pagamentos pelo Estado e saneamento dos pagamentos em atraso; abatimentos fiscais; apoio financeiro direto). A grande incógnita está no impacto final que a crise terá na solidez económica do país, o que depois determina a sua capacidade de liquidar as dívidas contraídas ao longo destes meses. Esta pandemia será marcada como uma das mais desafiantes e perturbadoras da história do mundo.

Chromoblastomycosis is a chronic, cutaneous or subcutaneous mycosis characterized by the presence of muriform cells in host tissue. Implantation disease is caused by melanized fungi related to black yeasts, which, in humid tropical climates, are mainly members of the genus Fonsecaea. In endemic areas of Brazil, F. pedrosoi and F. monophora are the prevalent species. The current hypothesis of infection is traumatic introduction via plant materials, especially by plant thorns. However, isolation studies have demonstrated a low frequency of the agents in environmental substrates. The present study aimed to detect F. pedrosoi and F. monophora in shells of babassu coconuts, soil, plant debris, and thorns from endemic areas of chromoblastomycosis in Maranhão state, northern Brazil, using Rolling Circle Amplification (RCA) with padlock probes as a new environmental screening tool for agents of chromoblastomycosis. In addition to molecular screening, the environmental samples were analyzed by fungal isolation using mineral oil flotation. The limit of detection of the RCA method was 2.88 × 107 copies of DNA per sample for the used padlock probes, indicating that this represents an efficient and sensitive molecular tool for the environmental screening of Fonsecaea agents. In contrast, with isolation from the same samples using several selective methods, no agents of chromoblastomycosis were recovered.

The species belonging to the genus Fonsecaea are the main causative agents of chromoblastomycosis. The invasive potential of Fonsecaea differs significantly among its various sibling species. Moreover, the lack of clarity on the virulence and availability of precise markers to distinguish and detect Fonsecaea species is attributed to the different ways of dissemination and pathogenicity. Therefore, the present study aimed to propose new molecular tools to differentiate between sibling species causing chromoblastomycosis. We used an infection model of chromoblastomycosis in BALB/c to study species-specific molecular markers for the in vivo detection of Fonsecaea species in biological samples. Specific primers based on the CBF5 gene were developed for Fonsecaea pedrosoi, Fonsecaea monophora, Fonsecaea nubica, and Fonsecaea pugnacius. In addition, a padlock probe was designed for F. pugnacius based on ITS sequences. We also assessed the specificity of Fonsecaea species using in silico, in vitro, and in vivo assays. The results showed that markers and probes could effectively discriminate the species in both clinical and environmental samples, enabling bioprospecting of agents of chromoblastomycosis, thereby elucidating the infection route of the disease.

Eucalyptus is the main species for the forestry industry in Brazil. Biotechnology and, more recently, gene editing offer significant opportunities for rapid improvements in Eucalyptus breeding programs. However, the recalcitrance of Eucalyptus species to in vitro culture is also a major limitation for commercial deployment of biotechnology techniques in Eucalyptus improvement. We evaluated various clones of Eucalyptus urophylla for their in vitro regeneration potential identified a clone, BRS07-01, with considerably higher regeneration rate (85%) in organogenesis, and significantly higher than most works described in literature. Endophytic bacteria are widely reported to improve in vitro plant growth and development. Hence, we believe that inclusion of endophytic plant growth promoting bacteria enhanced was responsible for the improved plantlets growth and development of this clone under in vitro culture. Metagenomic analysis was performed to isolate and characterize the prominent endophytic bacteria on BRS07-01 leaf tissue in vitro micro-cultures, and evaluate their impact on plant growth promotion. The analysis revealed the presence of the phyla Firmicutes (35%), Proteobacteria (30%) and much smaller quantities of Actinobacteria, Bacteroidetes, Gemmatimonadetes, Crenarchaeota, Euryarchaeota and Acidobacteria. Of the thirty endophytic bacterial strains isolated, eleven produced indole-3-acetic acid. Two of the isolates were identified as Enterobacter sp. and Paenibacillus polymyxa, which are nitrogen-fixing and capable of phosphate and produce ammonium. These isolates also showed similar positive effects on the germination of common beans (Phaseolus spp.). The isolates will now be tested as a growth promoter in Eucalyptus in vitro cultures. Graphical abstract for the methodology using cultivation independent and dependent methodologies to investigate the endophytic bacteria community from in vitro Eucalyptus urophylla BRS07-01.

Most human epidemiological and clinical studies use visual inspection of the hair and scalp to diagnose Pediculus humanus capitis , however this method has low sensitivity to diagnose active infestations (presence of nymphs and adult lice). Vacuuming the hair and scalp has been used as a diagnostic method, but there are no previous data comparing its effectiveness with visual inspection. The aim of this study was to determine the prevalence of overall infestation (nits and trophic stages), of active infestation by Pediculus humanus capitis , and to evaluate the effectiveness of vacuuming in comparison with the visual inspection. Visual inspection was performed by three examiners and vacuuming of the scalp by one investigator, with an adapted vacuum cleaner. A total of 166 children aged 4 to 10 years old were randomly selected from public schools in Southern Brazil. Considering the positive results obtained by both methods, the prevalence of overall infestation was 63.3%, whereas active infestation was 18.7%. The visual inspection was more effective on diagnosing overall infestation, however, its effectiveness to detect active infestation was lower, ranging from 0.6% (RR=3%, p

Chromoblastomycosis is one of the most prevalent implantation fungal infections caused by melanized fungi, affecting individuals with certain risk factors with high morbidity due to its recalcitrant nature. It is difficult to identify the etiological agents and thus a suitable reproductive molecular identification method applicable in developing countries has been investigated. We report the identification of four different fungal causative agents of chromoblastomycosis by reverse line blotting hybridization (RLB) based on biotin-labeled PCR products and amine labeled probes to hybridize. Sixty five reference strains, including type strains, i.e. Fonsecaea pedrosoi, F. monophora, F. nubica, and Phialophora verrucosa, obtained from the CBS-KNAW were included in this study. Internal transcribed spacer 1 (ITS1) regions of relevant species were aligned and adjusted using BIONUMERICS v. 4.61 in order to design four specific probes to identify informative nucleotide polymorphisms. The final identification of these species by RLB assay was concordant with ITS sequencing and showed 100% specificity with no cross hybridization, able to identify all tested strains. The time and cost were less compare to other routine identification methods such as sequencing. This assay allows sensitive and specific simultaneous detection and identification of a different fungal species.

The black yeast genus Exophiala includes numerous potential opportunistic species that potentially cause systematic and disseminated infections in immunocompetent individuals. Species causing systemic disease have ability to grow at 37–40 °C, while others consistently lack thermotolerance and are involved in diseases of cold-blooded, waterborne vertebrates and occasionally invertebrates. We explain a fast and sensitive assay for recognition and identification of waterborne Exophiala species without sequencing. The ITS rDNA region of seven Exophiala species (E. equina, E. salmonis, E. opportunistica, E. pisciphila, E. aquamarina, E. angulospora and E. castellanii) along with the close relative Veronaea botryosa was sequenced and aligned for the design of specific padlock probes for the detection of characteristic single-nucleotide polymorphisms. The assay demonstrated to successfully amplify DNA of target fungi, allowing detection at the species level. Amplification products were visualized on 1% agarose gels to confirm specificity of probe–template binding. Amounts of reagents were reduced to prevent the generation of false positive results. The simplicity, tenderness, robustness and low expenses provide padlock probe assay (RCA) a definite place as a very practical method among isothermal approaches for DNA diagnostics.

Fonsecaea pedrosoi, a melanized fungal pathogen that causes Chromoblastomycosis, a human disease with a worldwide distribution. Biolistic is a widely used technique for direct delivery of genetic material into intact cells by particles bombardment. Another well-established transformation method is Agrobacterium-mediated transformation (ATMT), which involves the transfer of a T-DNA from the bacterium to the target cells. In F. pedrosoi there are no reports of established protocols for genetic transformation, which require optimization of physical and biological parameters. In this work, intact conidia of F. pedrosoi were particle bombarded and subjected to ATMT. In addition, we proposed hygromycin B, nourseothricin and neomycin as dominant selective markers for F. pedrosoi and vectors were constructed. We tested two parameters for biolistic: the distance of the particles to the target cells and time of cells recovery in nonselective medium. The biolistic efficiency was 37 transformants/μg of pFpHYG, and 45 transformants/μg of pAN7.1. Transformants expressing GFP were successfully obtained by biolistic. A co-culture ratio of 10: 1 (bacterium: conidia) and co-incubation time of 72 h yielded the largest number of transformants after ATMT. Southern blot analysis showed the number of foreign DNA insertion into the genome is dependent upon the plasmid used to generate the mutants. This work describes for the first time two efficient methods for genetic modification of Fonsecaea and these results open new avenues to better understand the biology and pathogenicity of the main causal agent of this neglected disease.

Chromoblastomycosis is a chronic, cutaneous or subcutaneous mycosis characterized by the presence of muriform cells in host tissue. Implantation disease is caused by melanized fungi related to black yeasts, which, in humid tropical climates, are mainly members of the genus Fonsecaea. In endemic areas of Brazil, F. pedrosoi and F. monophora are the prevalent species. The current hypothesis of infection is traumatic introduction via plant materials, especially by plant thorns. However, isolation studies have demonstrated a low frequency of the agents in environmental substrates. The present study aimed to detect F. pedrosoi and F. monophora in shells of babassu coconuts, soil, plant debris, and thorns from endemic areas of chromoblastomycosis in Maranhão state, northern Brazil, using Rolling Circle Amplification (RCA) with padlock probes as a new environmental screening tool for agents of chromoblastomycosis. In addition to molecular screening, the environmental samples were analyzed by fungal isolation using mineral oil flotation. The limit of detection of the RCA method was 2.88 × 107 copies of DNA per sample for the used padlock probes, indicating that this represents an efficient and sensitive molecular tool for the environmental screening of Fonsecaea agents. In contrast, with isolation from the same samples using several selective methods, no agents of chromoblastomycosis were recovered.

A pork sausage was produced with low sodium content (1.64%) to which

BackgroundTraditionally known as a common contaminant, Rhodotorula mucilaginosa is among the leading causes of invasive fungal infections by non-candida yeasts. They affect mainly immunocompromised individuals, often mimicking the cryptococcosis infection, despite invasive infections by Rhodotorula are still not well explained. Thus, here we aimed to characterize microbiologically clinical isolates of R. mucilaginosa isolated from colonization of a patient with chronic renal disease (CKD), as well as to evaluate their phylogeny, antifungal susceptibility, virulence, and pathogenicity in order to infer the potential to become a possible infection.Methodology/Principal FindingsFor this study, two isolates of R. mucilaginosa from oral colonization of a CKD patient were isolated, identified and characterized by classical (genotypic and phenotypic) methods. Susceptibility to conventional antifungals was evaluated, followed by biofilm production, measured by different techniques (total biomass, metabolic activity, colony forming units and extracellular matrix quantification). Finally, the pathogenicity of yeast was evaluated by infection of Tenebrio molitor larvae.All isolates were resistant to azole and sensitive to polyenes and they were able to adhere and form biofilm on the abiotic surface of polystyrene. In general, similar profiles among isolates were observed over the observed periods (2, 24, 48 and 72 hours). Regarding extracellular matrix components of biofilms at different maturation ages, R. mucilaginosa was able to produce eDNA, eRNA, proteins, and polysaccharides that varied according to time and the strain. The death curve in vivo model showed a large reduction in the survival percentage of the larvae was observed in the first 24 hours, with only 40% survival at the end of the evaluation.Conclusions/SignificanceWe infer that colonization of chronic renal patients by R. mucilaginosa offers a high risk of serious infection. And also emphasize that the correct identification of yeast is the main means for an efficient treatment.Author SummaryThe genus Rhodotorula is known to be a common contaminant, however, it has been increasing in the last years, reports of different forms infections by this yeast, reaching mainly individuals with secondary diseases or with low immunity. However, very little is known about the mechanism that triggers the disease. Thus, this study aims to characterize microbiologically clinical isolates of R. mucilaginosa isolated from a patient with chronic renal disease, as well as to evaluate their phylogeny, antifungal susceptibility, virulence, and pathogenicity in order to infer the potential to become a possible infection. It was possible to characterize in general the clinical isolates, to determine that they are resistant to an important class of the antifungal agents which are the azoles. In addition, they are able to adhere and to form biofilm on abiotic surfaces, this skill represents an important factor of virulence, which would guarantee their presence in medical devices, such as catheters, surfaces. These biofilm works as true reservoirs of these fungi disseminate and cause serious infections. This pathogenic potential was reinforced by a great reduction of survival in the larvae infected with this yeast. Therefore our results infer a high risk of infection to patients who are colonized by R. mucilaginosa.

The orderChaetothyriales(Pezizomycotina,Ascomycetes) harbours obligatorily melanised fungi and includes numerous etiologic agents of chromoblastomycosis, phaeohyphomycosis and other diseases of vertebrate hosts. Diseases range from mild cutaneous to fatal cerebral or disseminated infections and affect humans and cold-blooded animals globally. In addition,Chaetothyrialescomprise species with aquatic, rock-inhabiting, ant-associated, and mycoparasitic life-styles, as well as species that tolerate toxic compounds, suggesting a high degree of versatile extremotolerance. To understand their biology and divergent niche occupation, we sequenced and annotated a set of 23 genomes of main the human opportunists within theChaetothyrialesas well as related environmental species. Our analyses included fungi with diverse life-styles, namely opportunistic pathogens and closely related saprobes, to identify genomic adaptations related to pathogenesis. Furthermore, ecological preferences ofChaetothyrialeswere analysed, in conjuncture with the order-level phylogeny based on conserved ribosomal genes. General characteristics, phylogenomic relationships, transposable elements, sex-related genes, protein family evolution, genes related to protein degradation (MEROPS), carbohydrate-active enzymes (CAZymes), melanin synthesis and secondary metabolism were investigated and compared between species. Genome assemblies varied from 25.81 Mb (Capronia coronata) to 43.03 Mb (Cladophialophora immunda). The bantiana-clade contained the highest number of predicted genes (12 817 on average) as well as larger genomes. We found a low content of mobile elements, with DNA transposons from Tc1/Mariner superfamily being the most abundant across analysed species. Additionally, we identified a reduction of carbohydrate degrading enzymes, specifically many of the Glycosyl Hydrolase (GH) class, while most of the Pectin Lyase (PL) genes were lost in etiological agents of chromoblastomycosis and phaeohyphomycosis. An expansion was found in protein degrading peptidase enzyme families S12 (serine-type D-Ala-D-Ala carboxypeptidases) and M38 (isoaspartyl dipeptidases). Based on genomic information, a wide range of abilities of melanin biosynthesis was revealed; genes related to metabolically distinct DHN, DOPA and pyomelanin pathways were identified. TheMAT(MAtingType) locus and other sex-related genes were recognized in all 23 black fungi. Members of the asexual generaFonsecaeaandCladophialophoraappear to be heterothallic with a single copy of eitherMAT-1-1orMAT-1-2in each individual. AllCaproniaspecies are homothallic as bothMAT1-1andMAT1-2genes were found in each single genome. The genomic synteny of theMAT-locus flanking genes (SLA2-APN2-COX13) is not conserved in black fungi as is commonly observed inEurotiomycetes, indicating a unique genomic context forMATin those species. The heterokaryon (het) genes expansion associated with the low selective pressure at theMAT-locus suggests that a parasexual cycle may play an important role in generating diversity among those fungi.

Pediculosis is a disease caused by the insect Pediculus humanus capitis that mainly occurs in childhood. A comparative study was carried out evaluating groups of schoolchildren with (group A) and without pediculosis (group B) to analyse the characteristics of the scalp microbiota. Samples were collected by swab using Stuart transport medium and incubate in Sabouraud dextrose agar with tetracycline to analyse the fungal microbiota and in blood agar to assess the bacterial microbiota. The isolates identity was confirmed by sequencing of the 16S and 18S regions of the ribosomal DNA gene for bacteria and fungi, respectively. The analysis of the 186 isolates led to the identification of 35 bacteria and 40 fungi in group A and 47 bacteria and 64 fungi in group B. The results indicate differences in bacterial and fungal species in the groups analysed. In the observed bacterial microbiota, Staphylococcus capitis occurred more frequently than Staphylococcus epidermidis in group A vs B. Among fungal isolates, Debaryomyces sp. was more frequent in group B vs A. Our findings showed scalp microbiota alterations in children with pediculosis, meriting future studies to analyse the relationship between these agents and their impact on human health.

Following a fatal case of Cryptococcus neoformans meningitis in a child with X-linked hyper-immunoglobulin M syndrome (XHIGM), we evaluated the fungal isolate in an experimental infection in a mouse model with respect to microbiology, epidemiology, virulence and response to therapy. The minimum inhibitory concentrations for antifungals in the susceptibility test were 0.5 mg/L for amphotericin B, 4.0 mg/L for fluconazole and 0.12 mg/L for voriconazole. Evaluation of pathogenicity by means of an experimental infection in BALB/c mice showed that fungus isolated from the blood and cerebrospinal fluid of the child was able to disseminate, reaching the spleen, lungs and brain, where it caused significant macroscopic alterations in the size and texture of each organ. Treatment of infected mice with amphotericin B reduced the fungal load in the spleen and lungs, but not in the brain.

Aim: To evaluate and characterize the etiopathogenesis of the fusarial onychomycosis in an ex vivo study through fragments of sterile human nail, without the addition of any nutritional source. Materials & methods: The infection and invasion of Fusarium oxysporum in the nail were evaluated by scanning electron microscopy (SEM), CFU, matrix, histopathology and Fourier Transform Infrared Spectrometer coupled to an equipment with diamond accessory (FTIR-ATR). Results: F. oxysporum infected and invaded across the nail, regardless of application face. However, the dorsal nail surface was the strongest barrier, while the ventral was more vulnerable to infection and invasion process. The fungal–nail interaction resulted in the formation of a dense biofilm. Conclusion: F. oxysporum infect and invade the healthy human nail, resulting in biofilm formation. Therefore, F. oxysporum is likely a primary onychomycosis agent.

A aposta em produzir produtos e serviços diferenciadores na área turístico-cultural tem vindo a adolescer significativamente, criando cada vez mais uma dependência do uso de determinados serviços no dia-a-dia de cada individuo. Neste sentido, o principal objetivo deste projeto é articular e relacionar a Programação Cultural e a Hotelaria, com base em contributos metodológicos capazes de fundamentar não só a importância e o interesse dos recursos turístico-culturais em si, mas sobretudo na sua aglomeração em áreas territoriais que apresentem um potencial de interesse cultural capaz de suportar intervenções estratégicas e financeiras futuras. O conhecimento empírico do território permite identificar claramente essas áreas de interesse no âmbito da oferta cultural, nomeadamente pelo nível de atração turística que geram. A orientação seguida na concetualização deste projeto foi no sentido de se criar uma interação cultural entre a Comunidade e a Hotelaria, tendo como principal foco propor uma ideia de projeto que permita desenvolver Programação Cultural em Hotelaria, articulando os recursos turístico-culturais existentes e um conjunto de atividades culturais que permita dinamizar os espaços hoteleiros, criando assim desta forma um novo conceito cultural. A Região Centro de Portugal será o caso de estudo, por ser uma unidade administrativa onde existe grande diversidade de recursos turístico-culturais, sendo considerados como uma alavanca potenciadora da identidade cultural, onde se identificará uma potencial localização geográfica para o projeto PULSAR Centro, com recurso à modelação em Sistemas de Informação Geográfica. Com este estudo pretende-se conceber um empreendimento turístico-cultural, que proporcione uma motivação de cariz cultural que também justifique a sua escolha e permanência na Região, baseada numa oferta de atividades culturais que de forma integrada e articulada se espelhem numa Programação Cultural a oferecer ao turista e à comunidade em geral.

Los microorganismos endófitos viven dentro de plantas sanas y su aislamiento e identificación pueden favorecer las aplicaciones biotecnológicas. Los microorganismos endofíticos fueron encontrados en callos de Ilex paraguariensis cultivados in vitro durante cinco meses. Se usó la secuenciación de la región 16S rRNA para identificar dos aislados como Bacillus megaterium. La presencia de células bacterianas en los espacios intercelulares de los cultivos de callos se detectó mediante análisis ultraestructurales. Los aislamientos también se evaluaron para la producción de ácido indol acético (AIA) y su potencial en la promoción del crecimiento de las plántulas de Phaseolus vulgaris L. La síntesis de ácido AIA en presencia y ausencia de L-triptófano se detectó por análisis colorimétrico para ambos aislamientos. En presencia de extractos de las cepas IPC06 e IPC07, las plántulas de P. vulgaris crecieron más rápido en comparación con las plantas control sin el extracto en pruebas in vitro. Palabras clave: yerba mate, 16S rRNA, secuenciación, cultivo de tejidos

Onychomycosis is a chronic fungal infection of nails, commonly caused by dermatophyte fungi, primarily species of Trichophyton. Because of the limited drug arsenal available to treat general fungal infections and the frequent failure of onychomycosis treatment, the search for new therapeutic sources is essential, and topical treatment with natural products for onychomycosis has been encouraged. Propolis, an adhesive resinous compound produced by honeybees (Apis mellifera), has shown multiple biological properties including significant antifungal and anti-biofilm activities in vitro. In spite of promising in vitro results, in vivo results have not been reported so far. This study assessed an ethanol propolis extract (PE) as a topical therapeutic option for onychomycosis, including its characterization in vitro and its applicability as a treatment for onychomycosis (from bench to clinic). The in vitro evaluation included analysis of the cytotoxicity and the antifungal activity against the planktonic cells and biofilm formed by Trichophyton spp. We also evaluated the capacity of PE to penetrate human nails. Patients with onychomycosis received topical PE treatments, with a 6-month follow-up period. The results of the in vitro assays showed that PE was non-toxic to the cell lines tested, and efficient against both the planktonic cells and the biofilm formed by Trichophyton spp. The results also showed that PE is able to penetrate the human nail. The results for PE applied topically to treat onychomycosis were promising, with complete mycological and clinical cure of onychomycosis in 56.25% of the patients. PE is an inexpensive commercially available option, easy to obtain and monitor. Our results indicated that PE is a promising natural compound for onychomycosis treatment, due to its ability to penetrate the nail without cytotoxicity, and its good antifungal performance against species such as Trichophyton spp. that are resistant to conventional antifungals, both in vitro and in patients.

In this study, we analyzed the antimicrobial activity of extracts harvested from 17 endophytic fungi isolated from the medicinal plant Schinus terebinthifolius. Morphological and molecular analyses indicated that these fungal species belonged to the genera Alternaria, Bjerkandera, Colletotrichum, Diaporthe, Penicillium, and Xylaria. Of the endophytes analyzed, 64.7 % produced antimicrobial compounds under at least one of the fermentation conditions tested. Nine isolates produced compounds that inhibited growth of Staphylococcus aureus, four produced compounds that inhibited Candida albicans, and two that inhibited Pseudomonas aeruginosa. The fermentation conditions of the following endophytes were optimized: Alternaria sp. Sect. Alternata—LGMF626, Xylaria sp.—LGMF673, and Bjerkandera sp.—LGMF713. Specifically, the carbon and nitrogen sources, initial pH, temperature, and length of incubation were varied. In general, production of antimicrobial compounds was greatest when galactose was used as a carbon source, and acidification of the growth medium enhanced the production of compounds that inhibited C. albicans. Upon large-scale fermentation, Alternaria sp. Sect. Alternata—LGMF626 produced an extract containing two fractions that were active against methicillin-resistant S. aureus. One of the extracts exhibited high activity (minimum inhibitory concentration of 18.52 µg/mL), and the other exhibited moderate activity (minimum inhibitory concentration of 55.55 µg/mL). The compounds E-2-hexyl-cinnamaldehyde and two compounds of the pyrrolopyrazine alkaloids class were identified in the active fractions by gas chromatography-mass spectrometry.

Information about microbial functionality in agricultural soils is still scarce, and in this study we used a shotgun metagenomic approach to compare different soil [conventional tillage (CT) with plowing and disking, and no-tillage (NT) with direct sowing into the residues of previous crops], and crop [crop succession (CS, soybean-summer/wheat-winter) or rotation (CR, soybean/maize-summer)/wheat/lupine/oat-winter)] managements in a 13-year-old field experiment in southern Brazil. Differences were detected between NT and CT in some functional subsystems, e.g., NT had more sequences associated with the metabolism of aromatic compounds, which might be related to higher capacity to degrade pesticides, more sequences of the adenylate cyclase (cAMP) pathway, which might confer stability to the microbial community, among others. On the other hand, CT showed more sequences related to carbohydrate metabolism, what could be related with a lower content of organic matter and need to metabolize a broader range of carbon sources. Also, we detected differences related to crop management, e.g., crop rotation showed more sequences in the metabolism of amino acids and derivatives and carbohydrate subsystems, what might result from higher diversity of crop-residues added to the soil. However, it was notable that the differences in the diversity of taxa previously shown in the same experiment was far greater than the functional diversity reported now, emphasizing a high level of microbial functional redundancy.

The aim of this study was to develop and evaluate a padlock probe based on the Rolling Circle Amplific ation (RCA), which targeted to 16S-23S rDNA region of S. mutans . The specificity of developed padlock probe was te sted for DNA within a panel strains, including S. mutans isolated from the saliva and reference strains of the genus Streptococcus , as well as total DNA samples of biofilm and saliv a. The results were positive either for DNA samples of S. mutans or DNA samples recovered from the biofilm and sali va revealing the specificity of designed padlock probe. The padlock probe based on the RCA was prove d to be an effective, reproducible method for S. mutans detection and demonstrated the possibility of a ra pid detection and accurate identification of S. mutans infection.

Since 1997, an emergent fungal disease named lethargic crab disease (LCD) has decimated stocks of the edible mangrove land crab Ucides cordatus (Linnaeus, 1763) (Brachyura: Ocypodidae) along the Brazilian coast, threatening the mangrove ecosystem and causing socioeconomic impacts. Evidence from a variety of sources suggests that the black yeast Exophiala cancerae (Herpotrichiellaceae, Chaetothyriales) has been responsible for such epizootic events. Based on the spatiotemporal patterns of the LCD outbreaks, the well-established surface ocean currents, and the range of ecological traits of Exophiala spp., a marine dispersal hypothesis may be proposed. Using in vitro experiments, we tested the survival and growth of E. cancerae CBS 120420 in a broad combination of salinities, temperatures, and exposure times. While variation in salinity did not significantly affect the growth of colony-forming units (CFUs) (P > 0.05), long exposure times visibly influenced an increase in CFUs growth (P

Citrus black spot disease, caused by the phytopathogen Phyllosticta citricarpa, depreciates the market value of citrus fruits and prevents their exportation to disease-free regions. It may also reduce the productivity of citrus fruit orchards. To identify an alternative to conventional disease control measures, isolates of Diaporthe terebinthifolii, active against P. citricarpa, were selected from an endophytic fungal population of Schinus terebinthifolia leaves. Different culture media were screened to identify the culture medium that afforded the most efficient production of biologically active extracts. A particular fraction (fraction VI) of the extract completely protected orange leaves by inhibiting the germination of P. citricarpa conidia with a minimum inhibitory concentration of 0.003 μg.mL-1. The active constituents in D. terebinthifolii extract fractions were identified by gas chromatography coupled to mass spectrometry as verbanol, phenylethyl alcohol, verbenyl acetate and methyl hexadecanoate. The results obtained strongly suggest the existence of a synergistic effect among the metabolites produced. Thus, these fungal metabolites could be used to control the CBS disease. As the asexual spores of P. citricarpa play an important role in fruit lesion development and disease dispersion, fungal extracts that inhibit the spore germination can be used as an effective alternative for directional disease control.

AIM: To characterize the genetic variability of Streptococcus mutans isolates and to correlate this variability with different colonization profiles observed during dental caries in a sample of children. METHODS: S. mutans samples were isolated from the saliva of 30 children with varying histories of dental caries, and they were characterized according to morphological and biochemical markers and the sequences of their 16S-23S intergenic spacer region. The genetic variability of the isolates was first assessed using Random Amplified Polymorphic DNA (RAPD) markers. Next, the isolates were differentiated by sequencing a specific region of the gene encoding the enzyme glucosyltransferase B (gtfB). RESULTS: Characterization using RAPD markers uncovered significant genetic variability among the samples and indicated the existence of clusters, which allowed us to reconstruct both the origin and clinical history of the disease. By sequencing the 16S-23S intergenic region, it was found that all of the isolates belonged to the species S. mutans. Based on the genetic similarity of the isolates and pattern of amino acid variations identified by partial sequencing of the gtfB gene, base-pair changes were identified and correlated with different virulence patterns among the isolates. CONCLUSIONS: The partial sequencing of the gtfB gene can be a useful tool for elucidating the colonization patterns of S. mutans. As amino acid variations are likely to be correlated with differences in biological risk, molecular characterization, such as that described in this paper, could be the key for assessing the development of dental caries in children.

Cyphellophora is a genus of black yeast-like fungi characterised by having simple phialides with multiseptate, curved conidia. Judging from SSU and LSU data, Cyphellophora was found to be located in a well-supported clade within the Chaetothyriales comprising a number of species occurring on human skin and nail. Cyphellophora is phylogenetically close to Phialophora europaea, P. reptans and P. oxyspora, though morphologically these species produce single-celled phialoconidia rather than multiseptate ones. Pseudomicrodochium suttonii and P. fusarioides have dark colonies and phylogenetically fit in with Cyphellophora; the type species of Pseudomicrodochium, P. aciculare, has similar, septate conidia but has a hyaline thallus. In the present study, multilocus phylogenetic analyses were combined with morphology and physiology. Sequences of the internal transcribed spacer region, the DNA dependent RNA polymerase II largest subunit and the partial beta tubulin gene were analysed for a set of 30 strains. Two novel species, Cyphellophora pauciseptata and Phialophora ambigua were discovered. Cyphellophora eucalypti was reduced to synonymy of C. guyanensis. The role of the studied fungi between colonization and infection of human skin was discussed. Putative virulence factors for these black yeast-like fungi were hypothesized to be the ability to assimilate monoaromatic hydrocarbons, to produce melanin pigments, and to tolerate the temperature of epidermal human skin.

The in vitro activities of eight antifungal drugs against 106 clinical and environmental isolates of waterborne and cutaneous Exophiala species were tested. The MICs and minimum effective concentrations for 90% of the strains tested ( n = 106) were, in increasing order, as follows: posaconazole, 0.063 μg/ml; itraconazole, 0.25 μg/ml; micafungin, 1 μg/ml; voriconazole, 2 μg/ml; isavuconazole, 4 μg/ml; caspofungin, 8 μg/ml; amphotericin B, 16 μg/ml; fluconazole, 64 μg/ml.

The aim of this study was evaluate, two methods for the detection and identification of sulphate reducing bacteria (SRB): ML medium and PCR with specific primers for SRB groups. SRB were detected through the selective medium only on carbon steel, which showed corrosion. Employing specific PCR primer, SBR were detected from all the metallic components assayed, even those that did not present visible corrosion spots, such stainless steel and copper alloys. Despite the presence or absence of corrosion at the later stages effectively by using the selective medium,, the initial stages of the corrosion could only be detected by the amplification of total DNA with SRB specific primers. The early detection of SRB could be employed for preventing the damages on metal surfaces before the installation of corrosion processes. Strategies for reducing the time spent on SRB isolation and identification could be auxiliary tools for controlling the corrosion of materials.

Hemodialysis in patients with chronic renal failure promotes the removal of toxic substances, water, and minerals from the body and often takes place in specialized clinics. Microbial contamination of dialysis fluid is a serious problem in therapy. One of the sources of contamination is the water used to prepare the dialysate. In Brazil, legislation regulating the microbiological quality of water for dialysis does not cover waterborne microbes such as Pseudomonas, mycobacteria, and fungi. The aim of the present study was to quantify, isolate, and identify fungi present in water systems in six hemodialysis units in Curitiba, Parana state, Brazil. Fungi were analyzed by surface plating and membrane filtration. Isolates were identified by morphology, while the dematiaceous fungi were identified by sequencing the rDNA ITS region. It was found that 66 % of the samples presented fungi, while black fungi were present in 46 % of all samples. Twenty-eight isolates from treated water for dialysis and dialysate were identified by sequencing and were found to be Exophiala pisciphila, E. cancerae, E. equina, and Rhinocladiella similis. The presence of dematiaceous fungi may pose a risk for debilitated hospitalized patients.