Antifungal Resistance in Cryptococcal Infections.

Antifungal therapy, especially with the azoles, could promote the incidence of less susceptible isolates of Cryptococcus neoformans and C. gattii species complexes (SC), mostly in developing countries. Given that these species affect mostly the immunocompromised host, the infections are severe and difficult to treat. This review encompasses the following topics: 1. infecting species and their virulence, 2. treatment, 3. antifungal susceptibility methods and available categorical endpoints, 4. genetic mechanisms of resistance, 5. clinical resistance, 6. fluconazole minimal inhibitory concentrations (MICs), clinical outcome, 7. environmental influences, and 8. the relevance of host factors, including pharmacokinetic/pharmacodynamic (PK/PD) parameters, in predicting the clinical outcome to therapy. As of now, epidemiologic cutoff endpoints (ECVs/ECOFFs) are the most reliable antifungal resistance detectors for these species, as only one clinical breakpoint (amphotericin B and C. neoformans VNI) is available.

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.

Evaluation of the Sensititre YeastOne and Etest in Comparison with CLSI M38-A2 for Antifungal Susceptibility Testing of Three Azoles, Amphotericin B, Caspofungin, and Anidulafungin, against Aspergillusfumigatus and Other Species, Using New Clinical Breakpoints and Epidemiological Cutoff Values.

Aspergillosis is an invasive fungal disease associated with high mortality. Antifungal susceptibility testing (AFST) is receiving increasing consideration for managing patients, as well as for surveilling emerging drug resistance, despite having time-consuming and technically complex reference methodologies. The Sensititre YeastOne (SYO) and Etest methods are widely utilized for yeasts but have not been extensively evaluated for Aspergillus isolates. We obtained Posaconazole (POS), Voriconazole (VCZ), Itraconazole (ITC), Amphotericin B (AMB), Caspofungin (CAS), and Anidulafungin (AND) minimum inhibitory concentrations (MICs) for both the Etest (n = 330) and SYO (n = 339) methods for 106 sequenced clinical strains. For 84 A. fumigatus, we analyzed the performance of both commercial methods in comparison with the CLSI-AFST, using available cutoff values. An excellent correlation could be demonstrated for Etest-AMB and Etest-VCZ (p < 0.01). SYO-MICs of AMB, VCZ, and POS resulted in excellent essential agreement (>93%), and >80% for AMB, VCZ, and ITC Etest-MICs. High categoric agreement was found for AMB, ITC, and CAS Etest-MICs (>85%) and AMB SYO-MICs (>90%). The considerable number of major/very major errors found using Etest and SYO, possibly related to the proposed cutoffs and associated with the less time-consuming processes, support the need for the improvement of commercial methods for Aspergillus strains.

Early clinical and microbiological predictors of outcome in hospitalized patients with cryptococcal meningitis.

BACKGROUND: Cryptococcal meningitis causes high mortality in immunocompromised and immunocompetent patients. The objective of this study was to identify early predictors of clinical outcome, available at the first days of hospitalization, in patients with cryptococcal meningitis in a tertiary center in Brazil. METHODS: Ninety-six cases of cryptococcal meningitis with clinical, epidemiological and laboratory data, and identification and antifungal susceptibility of the strains were analyzed. Quantitative CSF yeast counts were performed by direct microscopic exam with a Fuchs-Rosenthal cell counting chamber using an institutional protocol. Univariable and multiple analyses using logistic regression were performed to identify predictors, available at the beginning of hospitalization, of in-hospital mortality. Moreover, we performed a secondary analysis for a composite outcome defined by hospital mortality and intensive care unit transfer. RESULTS: The species and the antifungal susceptibility were not associated with the outcomes evaluated. The variables significantly associated with the mortality were age (OR = 1.08, 95% CI 1.02-1.15), the cerebrospinal fluid (CSF) yeasts count (OR = 1.65, 95% CI 1.20-2.27), systemic arterial hypertension (OR = 22.63, 95% CI 1.64-312.91) and neurological impairment identified by computed tomography (OR = 41.73, 95% CI 3.10-561.65). At the secondary analysis, CSF yeast count was also associated with the composite outcome, in addition to the culture of Cryptococcus spp. from bloodstream and cerebral toxoplasmosis. The associations were consistent with survival models evaluated. CONCLUSIONS: Age and CSF yeast count were independently associated with in-hospital mortality of patients with cryptococcal meningitis but Cryptococcus species identification and antifungal susceptibility were not associated with the outcomes. Quantitative CSF yeast counts used in this study can be evaluated and implemented in other low and middle-income settings.

Coumaric acid analogues inhibit growth and melanin biosynthesis in Cryptococcus neoformans and potentialize amphotericin B antifungal activity.

Fungal infections are on the rise, since the imunocompromised population is increasing due to AIDS/HIV, organ transplant and chemotherapy. Many environmental and pathogenic fungi are able to accomplish melanin biosynthesis as a virulence factor to promote host invasion. Melanized cells are more resistant to radiation, oxidative and osmotic stresses; also melanin confers an advantage in vivo, since melanized cells are more resistant to phagocytic engulfment and oxidative stress caused by the host defense cells and by some antifungal drugs, such as fluconazole (FCZ) and amphotericin B (AmB). Brown, red or black melanin pigments can be produced by the polyketide pathway (DHN-melanin) or from dihydroxyphenols, such as L-DOPA (L-3,4-dihydroxyphenylalanine) and L-tyrosine by polyphenoloxidases. Among several pathogenic fungi, Cryptococcus neoformans is a melanized yeast that causes pneumonia and meningoencephalitis in immunocompromised patients. The knockout of the laccase genes or other interruptions on melanin biosynthetic pathway generates cryptococcal strains with attenuated virulence in an animal model. In this study 16 analogues of coumaric and cinnamic acid were evaluated as possible tyrosinase inhibitors. We have identified some valuable inhibitors of C. neoformans growth and melanin biosynthesis disruption agents. The results showed that coumaric acid derivatives (1a-c), the ketones (3a-b) and 2-allylphenol (7c) are significant inhibitors of tyrosinase and melanization of the fungus. Two analogues (1b and 3b) were selected as promising antimelanogenic agents to be combined with AmB, showing to promote 16-fold reduction in the AmB fungicidal concentration with no appreciable cytotoxicity to mammalian cells. The data suggest that inhibition of the melanin biosynthesis by these compounds may increase the susceptibility of the cells to the oxidative stress generated by AmB. In summary, our data show that C. neoformans can be a suitable model system to test novel inhibitors that target melanin biosynthesis, and novel compounds for adjunct therapy against C. neoformans were identified.

Usefulness of yeast cell counting and lack of clinical correlation of the antifungal susceptibility testing results in management of AIDS-associated cryptococcal meningitis

Purpose of Review Cryptococcal meningitis is one of the most seriously opportunistic infections in people living with HIV. We evaluated clinical and laboratorial features (minimum inhibitory concentrations for fluconazole, initial fungal burden in cerebrospinal fluid) and risk factors associated with in-hospital mortality. Recent Findings There is no good evidence for the use ofminimum inhibitory concentrations for fluconazole in routine practice for the management of cryptococcosis patients. Counting yeast cells at cerebrospinal fluid can predict positive culture by not death. Summary Data from 46 cryptococcal meningitis patients were reviewed, retrospectively. Patients who presented yeast cell count greater than 400 yeast cells/µ in their initial cerebrospinal fluid sample were associated with higher mortality (p = 0.014); moreover, the yeast cell count is an easy and cheap assay, with high values possibly associated to poor prognosis. Additionally, we verified no significant differences between fluconazole susceptibility profile, molecular type, clinical presentation, cytological analyses, time to sterilize the cerebrospinal fluid, agent recovering out of central nervous system, previous diagnosis of cryptococcal meningitis or usage of fluconazole, and overall mortality

Autofix: uma tecnologia para fixação segura de tubo orotraqueal / Pre-hospital nursing interventions: narrative review

Objetivo: construir e validar de uma tecnologia diferenciada para fixação de tubo orotraqual em pacientes críticos. Metodologia: trata-se de um estudo de desenvolvimento metodológico, para construção e validação de um protótipo para fixação segura denominado "autofix". Participaram da pesquisa 15 juízes especialistas profissionais da saúde, equipe multiprofissional, médicos intensivistas, enfermeiros especialistas e fisioterapeutas intensivistas. A validação de conteúdo evidenciou todos os itens como excelentes IVC (≥ 0,78), teve excelente IVC global de 0,97, evidenciando que o instrumento poderá favorecer o paciente e equipe para uma assistência humanizada e qualificada. A tecnologia construída e validada foi desenvolvida para atuar como estratégia de segurança e humanização do paciente crítico em uso da ventilação mecânica. (AU)

Objective: To construct pre-hospital nursing intervention algorithms for trauma victims. Methodology: Literature Narrative Review, between 2008 and 2019, in the main databases. Two independent reviewers carried out the critical evaluation, extraction and synthesis of data. The construction of the algorithms resulted from the interpretive process of the narrative review by three experts. The theoretical model of Virginia Henderson was used. Results: Seventeen documents were obtained, six were included in the development of the metaparadigm Health, Person and Environment and 16 were included in the development and construction of assessment, diagnosis and nursing interventions algorithms for trauma victims. Conclusions: The review enabled the operationalization of Henderson's theoretical model for pre-hospital care, allowing the creation of algorithms to guide nursing practice. (AU)

Objetivo: Construir algoritmos de intervención de enfermería prehospitalaria para víctimas de traumatismos. Metodología: Revisión narrativa de literatura, entre 2008 y 2019, en las principales bases de datos. Dos revisores independientes realizaron la evaluación crítica, extracción y síntesis de los datos. La construcción de los algoritmos fue el resultado del proceso interpretativo de la revisión por parte de tres expertos. Se utilizó el modelo de Virginia Henderson. Resultados: Se obtuvieron diecisiete documentos, seis se incluyeron en el desarrollo de los metaparadigmas y 16 se incluyeron en el desarrollo y construcción de algoritmos de evaluación, diagnóstico e intervenciones de enfermería. Conclusiones: La revisión permitió la operacionalización del modelo de Henderson para la atención prehospitalaria, permitiendo la creación de algoritmos. (AU)

Fatores microbiológicos da criptococose e sua importância na carga da doença em pacientes atendidos em um hospital de referência do Estado de São Paulo / Not available

Introdução: A meningite criptocócica causa elevada mortalidade, sobretudo em pacientes acometidos de alguma condição imunossupressora. O objetivo deste estudo foi identificar fenômenos de baixa suscetibilidade a antifúngicos e outros preditores clínicos que possam explicar falha terapêutica e recidiva da neurocriptococose Metodologia: Foram avaliados 96 casos com coleta de dados clínicos epidemiológicos e laboratoriais. Os isolados foram identificados quanto a genótipo molecular, suscetibilidade de anfotericina B (AMB) e fluconazol (FCZ) pela determinação da concentração inibitória mínima (Minimal Inhibitory Concentration, MIC), nível de heteroresistência ao FCZ (NHF) e determinação do tempo de morte frente AMB (Time-Kill, TK). Foram selecionados isolados heterorresistentes para análise quantitativa de DNA por PCR em tempo Real, expressão de bombas de efluxo por citometria de fluxo e isolados tolerantes a AMB para estudo de resistência ao estresse oxidativo. Foi realizada análise univariável e múltipla usando regressão logística para identificar preditores de óbito hospitalar e de um desfecho composto definido pelo óbito, encaminhamento para unidade de terapia intensiva ou recidiva 6 meses após alta hospitalar. Resultados: A maioria dos pacientes eram imunodeprimidos, com CD4 de 2 a 722 cel./mm3 e 96,7% eram portadores do HIV. Foram identificados 93 isolados de C. neoformans, sendo 76 do genótipo VNI e 17 VNII e 3 C. gattii, todos VGII. MIC de AMB variou de 0,012 a 0,94 mg/L e MIC de FCZ estiverem entre 0,12 e 64 mg/L. Resistência a FCZ (MIC>16mg/L) foi maior em VNI do que em VNII (p=0,03). Dentre os isolados VNI, 64,5% sofreu atividade fungicida até as 24h (TK24) de exposição à AMB e 6 cepas VNI não sofreram ação fungicida (TK>72). A maioria dos isolados VNII (64,7%) apresentou TK24. Os 3 isolados VGII sofreram atividade fungicida a partir de TK24. A maioria dos isolados VNI, VNII e todos os isolados VGII apresentaram alto NHF (>32mg/L). Diferença no NHF de acordo com os genótipos foi observada (p=0,005). No modelo múltiplo, as variáveis associadas significativamente ao óbito foram: idade em anos (OR=1,08;IC95%=1,02-1,15), contagem de leveduras no líquido cefalorraquidiano em logaritmo (LCR) (OR=1,66;IC95%=1,21-2,28) e uma variável composta por hipertensão arterial sistêmica ou diagnóstico de edema cerebral ou dilatação ventricular por tomografia (OR=35,68;IC95%=4,97-256,31). Para o desfecho composto, as variáveis associadas foram: contagem de leveduras do 1D em logaritmo (OR=1,50; IC95%=;1,20-1,86; p=<0,001), cultura de sangue positiva para Cryptococcus spp. (OR=3,30; IC95%=0,86-12,59; p=0,08) e descrição de neurotoxoplasmose (OR=18,62; IC95%=1,85-187,5; p=0,01). As associações foram consistentes em modelos de sobrevida. Conclusão: Foi possível descrever genótipos mais frequentes e identificar fatores genéticos, como aumento da expressão de genes e bombas de efluxo, relacionados à resistência aos fármacos. Nenhum dos testes de suscetibilidade esteve associado com os desfechos. Variáveis obtidas nos primeiros dias de internação mostraram utilidade para predizer o prognóstico em pacientes com meningite criptocóccica. Estes preditores podem ajudar a identificar os casos com maior potencial de óbito e que necessitam da otimização dos recursos terapêuticos.

Background: Cryptococcal meningitis causes high mortality in immunocompromised patients. The objective of this study was to identify the phenomena of low susceptibility to antifungal and other clinical predictors that may explain therapeutic failure and relapse of neurocryptococcosis Methodology: It was analyzed 96 cases with clinical and epidemiological data. The respective isolates were identified for genotype, susceptibility profile by Minimal Inhibitory Concentration (MIC), FCZ heteroresistance level (NHF), and time to death determination against 1 mg / L BMA (Time-Kill, TK). We isolated heteroresistant DNA expression analysis by real-time PCR, expression of efflux pumps by flow cytometry and, some isolates tolerant to AMB were selected to study resistance to oxidative stress. Univariable and multiple analyses using logistic regression were performed to identify predictors of in-hospital mortality and a composed outcome defined by death, referral to the intensive care unit and relapse 6 months after hospital discharge. Results: Most of the patients were immunocompromised, with CD4 range from 2 to 722 cells/mm3 and 96.7% patients HIV-positive. It was analyzed 93 strains of Cryptococcus neoformans of which 76 were genotype VNI and 17 were VNII and 3 were C. gattii, all were VGII. AMB MIC ranged from 0.012 to 0.94 mg/L and FCZ MIC were between 0.12 and 64 mg/L. Resistance to FCZ (MIC>16mg/L) was higher to VNI than VNII (p=0.03). Among the VNI strains, 64.5% had fungicidal activity up to 24h (TK24) of exposure to AMB and 6 VNI did not present this activity until 72h (TK> 72). Most VNII strains (64.7%) had TK24. The 3 VGII strains presented fungicidal activity from TK24. According to the MIC, all strains were susceptible to AMB. The majority of VNI strains (93.4%) and VNII (76.5%) and 3 VGII strains showed high NHF (>32mg/L) and it was observed statistical difference according to the genotypes VNI and VNII (p=0.005). At the multiple analysis, the variables significantly associated with the death were the age in years (OR=1.08,95%CI=1.02-1.15), the cerebrospinal fluid (CSF) yeasts count-log (OR=1.66,95%CI=1.21-2.28), and a variable composed of systemic arterial hypertension or diagnosis of cerebral edema or ventricular dilatation by tomography (OR=35.68,95%CI=4.97-256.31). At the composed outcome, the variables associated were: CSF yeasts count-log (OR=1,50; IC95%=;1,20-1,86; p=<0,001), positive blood culture for Cryptococcus spp. (OR=3,30; IC95%=0,86-12,59; p=0,08) and neurotoxoplasmosis (OR=18,62; IC95%=1,85-187,5; p=0,01). The associations were consistent at survival models. Conclusion: It was possible to describe more frequent genotypes and to identify genetic factors, such as increased gene expression and efflux pumps, related to drug resistance. The antifungal susceptibilities were not associated with the outcomes. were not associated with outcomes. Variables available in the first days of hospitalization showed utility to predict the prognosis in patients with cryptococcal meningitis. These predictors can help to identify the cases with higher potential of death and that require the optimization of the therapeutic resources.

Caryocar brasiliense oil improves cardiac function by increasing Serca2a/PLB ratio despite no significant changes in cardiovascular risk factors in rats.

BACKGROUND: Caryocar brasiliense (pequi) oil is high in monounsaturated fat acids (MUFA), especially oleic, and in carotenoids, which have been associated with protection against cardiovascular disease. However, this food is poorly studied in this context, especially in the cardiac function. Therefore, we investigated the effects of a long-term intake of pequi oil in systemic cardiovascular risk factors and in the ex vivo cardiac function of rats. METHODS: Previously, we determined fatty acids and carotenoids in pequi oil. Next, male rats were divided in C - control group feed a standard diet, and PO - pequi oil group fed the same diet added pequi oil (+2.25 g.100 g-1). After 15 weeks, plasma lipids, glucose, insulin, blood pressure, heart rate, hepatic lipids were accessed and visceral fat pads were harvested. Hearts were used for the ex vivo cardiac function, histologic assays, SERCA2a and phospholanban (PLB) determinations. RESULTS: In agreement with scientific data, pequi oil had expressive amounts MUFA, especially oleic acid, and carotenoids. Hepatic triglycerides (TG) were reduced by pequi oil intake (p < 0.05). All others cardiovascular risk factors were not changed. The intrinsic heart rate was lower in PO group (p < 0.05). SERCA2a content was higher in this group (p < 0.05), without affecting PLB. Also, SERCA2a/PLB ratio increased in PO group (p < 0.05). CONCLUSION: Pequi oil intake improved cardiac function ex vivo, despite no significant changes in systemic cardiovascular risk factors. The higher lipid offer in pequi oil diet, its composition in oleic acid and carotenoids could be related to those effects.

Pain in onco-hematologic patients and its association with analgesia / Caracterização da dor em pacientes onco-hematológicos e sua associação com a analgesia

ABSTRACT BACKGROUND AND OBJECTIVES: It is understood that in light of the increasing number of cancer diagnoses, one should intensify the quality of assistance offered to those patients, aiming at improving quality of life and minimizing suffering of patients and their relatives. Nursing assistance should be enhanced and focused on cancer patients' pain evaluation, to help pain characterization and, as a consequence, to improve multiprofessional assistance. This study aimed at characterizing onco-hematologic patients' pain and at associating it to offered analgesia. METHODS: This is a descriptive, prospective and longitudinal study developed with 20 patients with onco-hematologic disease admitted to the hematology unit, Hospital Universitário Walter Cantídeo. Visual analog scale and pain numerical scale were applied, in addition to multidimensional pain evaluation scale and McGil questionnaire. RESULTS: Primary cause of pain was intestinal infection. Acute pain lasting the whole day has prevailed. According to multidimensional pain evaluation scale, most commonly reported acute descriptor was deep, and of chronic persistent. Most commonly mentioned word of McGill questionnaire was unbearable, being the evaluative category the most commonly used. When evaluating analgesia, it was noted that 12 out of 20 medical prescriptions were incompatible with World Health Organization recommendations. CONCLUSION: Scales are extremely important to measure and characterize pain. The nursing team should be at the front for its implementation and use in hospital units, especially in cancer units due to the high incidence of this symptom.

RESUMO JUSTIFICATIVA E OBJETIVOS: Entende-se que diante da crescente incidência no diagnóstico de câncer, deve-se intensificar a qualidade da assistência oferecida a esses pacientes, com o intuito de melhorar a qualidade de vida e amenizar o sofrimento dos pacientes e dos familiares. A assistência de enfermagem deve ser aprimorada e focada na avaliação da dor do paciente oncológico, para facilitar a caracterização da dor e, consequentemente, melhorar o tratamento multiprofissional. O objetivo deste estudo foi caracterizar a dor dos pacientes onco-hematológicos e associar com a analgesia ofertada. MÉTODOS: Estudo descritivo, prospectivo, longitudinal, desenvolvido com 20 pacientes que apresentaram diagnóstico de doença onco-hematológica e que estivesse internado na unidade de hematologia do Hospital Universitário Walter Cantídeo. Foram aplicadas as escalas analógicas visual e numérica da dor, multidimensional de avaliação de dor e o questionário de McGill. RESULTADOS: A principal causa de dor foi por infecção intestinal. Prevaleceu a dor aguda e que durava o dia todo. Pela escala multidimensional de avaliação de dor o descritor agudo mais relatado foi profundo e do crônico persistente. A palavra mais citada no McGill foi insuportável, com a categoria avaliativa sendo a mais utilizada. Na avaliação da analgesia, percebeu-se que 13 das 20 prescrições médicas estavam incompatíveis com as recomendações da Organização Mundial da Saúde. CONCLUSÃO: As escalas são de extrema importância na mensuração e caracterização da dor. A enfermagem deve estar à frente na sua implantação e utilização nas unidades hospitalares, de maneira especial nas oncológicas pela alta incidência desse sintoma.

A new culture medium for recovering the agents of Cryptococcosis from environmental sources.

The isolation of Cryptococcosis agents from environmental samples may be difficult due to the presence of groups of fast-growing fungi. We propose a new culture medium based on a modification of Dichloran Rose-Bengal Chloramphenicol Medium (DRBCm) to detect colonies of Cryptococcus neoformans. Our results indicate that DRBCm is superior to the classical Bird Seed Agar in its ability to detect colonies of C. neoformans.

A new culture medium for recovering the agents of Cryptococcosis from environmental sources

The isolation of Cryptococcosis agents from environmental samples may be difficult due to the presence of groups of fast-growing fungi. We propose a new culture medium based on a modification of Dichloran Rose-Bengal Chloramphenicol Medium (DRBCm) to detect colonies of Cryptococcus neoformans. Our results indicate that DRBCm is superior to the classical Bird Seed Agar in its ability to detect colonies of C. neoformans.

Report of filamentous forms in a mating type VNI clinical sequential isolates of Cryptococcus neoformans from an HIV virus-infected patient

We reported a cryptococcal meningitis Aids-patient infected with a mating type VNI isolate showing filamentous cells in direct examination of cerebrospinal fluid. Clinical data, outcome, treatment features and microbiological findings were discussed

Effects of probiotic fermented milk on biofilms, oral microbiota, and enamel.

The aim of this study was to evaluate in vitro and in vivo the effects of 2 brands of probiotic fermented milk on biofilms, oral microbiota, and enamel. For the in situ experiment, ten volunteers wore palatine devices containing four blocks of bovine dental enamel over 3 phases, during which 20% sucrose solution, Yakult® (Treatment A), and Batavito® (Treatment B) were dropped on the enamel blocks. Salivary microbial counts were obtained and biofilm samples were analyzed after each phase. For the in vivo experiment, the same ten volunteers drunk Yakult® (Treatment C) and Batavito® (Treatment D) in two phases. Saliva samples were collected for microbial analysis after each phase. The in situ study showed that in comparison with Treatment A, Treatment B resulted in fewer total cultivable anaerobes and facultative microorganisms in biofilms, higher final microhardness, lower percentage change in surface hardness, and smaller integrated subsurface enamel hardness. In the in vivo study, Treatment D resulted in a reduction in the counts of all microorganisms. The results suggested that the probiotic fermented milk Batavito®, but not Yakult®, reduced the amount of oral microorganisms and mineral loss in bovine enamel.

Report of filamentous forms in a mating type VNI clinical sequential isolates of Cryptococcus neoformans from an HIV virus-infected patient.

We reported a cryptococcal meningitis Aids-patient infected with a mating type VNI isolate showing filamentous cells in direct examination of cerebrospinal fluid. Clinical data, outcome, treatment features and microbiological findings were discussed.

Estudo de características dos agentes de criptococose: genótipos, polimorfismo, melanização e suscetibilidade a antifúngicos / Study of characteristics of cryptococcosis agents; genotypes, polymorphism, melanization and antifungal susceptibility

Os objetivos deste trabalho foram determinar frequência e características de polimorfismo celular em isolados clínicos de Cryptococcus spp. e utilizar novo método de inóculo individualizado para teste de suscetibilidade a antifúngicos (TSA) utilizando isolados de pacientes hospitalizados. O estudo de polimorfismo analisou 112 isolados de C. neoformans (Cn) e 65 de C. gattii (Cg), empregandomicroscopia ótica e microscopia eletrônica de transmissão (MET), análise de melanização, TSA a fluconazol (FCZ) e anfotericina B (AmB) por E-test® e curvas de morte (Time Kill Curves, TK) para AmB, tipagem molecular e mating-type (MAT). O estudo do novo método empregou a carga fúngica de 6 pacientes em suas amostras de líquido cefalorraquidiano (LCR) no momento do diagnóstico,7°dia e 14°dia de terapia antifúngica para CSNC. As cepas foram classificadas segundo tipo molecular e MAT, TSA com FCZ e AMB e teste de combinação por tabuleiro do xadrez. A frequência de polimorfismo celular foi baixa (4%;7/177), mas nunca antes relatada em isolados de micoteca. Foi inédito o encontro de polimorfismo em isolados de sangue, indicando a facilidade de disseminação decélulas atípicas pela corrente sanguínea, assim como em isolado de LCR. Todos os fenótipos polimórficos apresentavam células titãs (100%; 7/7) e, em menor porcentagem, pseudohifas (43%; 3/7). O fenômeno de polimorfismo parece ser exclusivo de Cn, desde que não foi observado em Cg. A espessura da parede das células titãs apresentou a mesma variação (350 nm a 900 nm) em relação àscélulas...

Our objectives were to describe the frequency and characteristics of polymorphismamong clinical isolates of Cryptococcus spp. and use new method of individualizedinoculum for the test of susceptibility to antifungal agents (TSA) against theetiological agent of cryptococcosis of the central nervous system (CSNC) inhospitalized patients. The study of polymorphism analyzed 112 isolates of C.neoformans (Cn) and 65 of C. gattii (Cg) using optical microscopy andtransmission electron microscopy (TEM), analysis of melanization, TSA forfluconazole (FCZ) by E-Test® and time kill curves method (TK) for amphotericin B(AmB), molecular typing and mating-type (MAT). The study of new methodemployed the fungal load of 6 patients in their cerebrospinal fluid (CSF) samples atdiagnosis, 7th and 14th day of therapy for CSNC. The strains were classifiedaccording to molecular type and MAT, TSA using FCZ or AmB and drugcombination using AMB and 5-flucytosine (5FC) by chequerboard, and TK. Thefrequency of cell polymorphism was low (4%; 7/177), but never before reported inisolates of collection. The occurrence of polymorphism in blood isolates wasunprecedented, indicating the ease of dissemination of atypical cells into thebloodstream, as well as in CSF. All polymorphic phenotypes showed titan cells(100%; 7/7), and, at a lower percentage, formed pseudohyphae (43%; 3/7). Thephenomenon of polymorphism seems to be unique in Cn, since it was notobserved in Cg. The wall thickness of the titans cells presented the same variation(350 nm to 900 nm) compared to regular cells. Low melanization was observed in71.4% (5/7) of polymorphic isolates, two isolates showed both patterns ofmelanization. All isolates with regular cells showed high level of melanization. Lowsusceptibility (MIC, >8 mg/L) was observed in almost half of the isolates with titancells, indicating that polymorphism may be related to resistance to FCZ. The newmethod of TSA with individualized...

Effects of probiotic fermented milk on biofilms, oral microbiota, and enamel

The aim of this study was to evaluate in vitro and in vivo the effects of 2 brands of probiotic fermented milk on biofilms, oral microbiota, and enamel. For the in situ experiment, ten volunteers wore palatine devices containing four blocks of bovine dental enamel over 3 phases, during which 20% sucrose solution, Yakult® (Treatment A), and Batavito® (Treatment B) were dropped on the enamel blocks. Salivary microbial counts were obtained and biofilm samples were analyzed after each phase. For the in vivo experiment, the same ten volunteers drunk Yakult® (Treatment C) and Batavito® (Treatment D) in two phases. Saliva samples were collected for microbial analysis after each phase. The in situ study showed that in comparison with Treatment A, Treatment B resulted in fewer total cultivable anaerobes and facultative microorganisms in biofilms, higher final microhardness, lower percentage change in surface hardness, and smaller integrated subsurface enamel hardness. In the in vivo study, Treatment D resulted in a reduction in the counts of all microorganisms. The results suggested that the probiotic fermented milk Batavito®, but not Yakult®, reduced the amount of oral microorganisms and mineral loss in bovine enamel.

Fragmented adipose tissue graft for bone healing: Histological and histometric study in rabbits’ calvaria

Objective The adipose tissue represents an important reservoir of stem cells. There are few studies in the literature with which to histologically evaluate whether or not the adipose tissue graft is really a safe option to achieve bonerepair. This study histologically analyzed the effect of fragmented autogenous adipose tissue grafts on bone healing in surgically created, critical-size defects (CSD) in a rabbit’s calvaria. Study design Forty-two New Zealand rabbits were used in this study. CSD that were 15 mm in diameter were created in the calvarium of each animal. The defects were randomly divided into two groups: in Group C (control),the defect was filled only by a blood clot and, in Group FAT (i.e., fragmented adipose tissue), the defect was filled with fragmented autogenous adipose tissue grafts. The groups were divided into subgroups (n = 7) for euthanasia at 7, 15, and 40 days after the procedure had been conducted. Histologic and histometric analyses were performed. Data were statistically analysed with ANOVA and Tukey’s tests (p < 0.05).Results The amount of bone formation did not show statistically significant differences seven days after the operation, which indicates that the groups had similar amounts of mineral deposition in the earlier period of the repair. Conversely, a significant of amount of bone matrix deposition was identified in the FAT group at 15 and 40 days following the operation, both on the border and in the body of the defect. Such an outcome was not found in the control group. Conclusion In this study, an autologous adipose tissue graft may be considered as likely biomaterial for bone regeneration, since it positively affected the amount of bone formation in surgically created CSD in the rabbits’ calvaria 40 days after the procedure had been performed. Further investigations with a longer time evaluation are warranted to determine the effectiveness of autologous adipose tissue graft in the bone healing (AU)

Fragmented adipose tissue graft for bone healing: histological and histometric study in rabbits' calvaria.

OBJECTIVE: The adipose tissue represents an important reservoir of stem cells. There are few studies in the literature with which to histologically evaluate whether or not the adipose tissue graft is really a safe option to achieve bone repair. This study histologically analyzed the effect of fragmented autogenous adipose tissue grafts on bone healing in surgically created, critical-size defects (CSD) in a rabbit's calvaria. STUDY DESIGN: Forty-two New Zealand rabbits were used in this study. CSD that were 15 mm in diameter were created in the calvarium of each animal. The defects were randomly divided into two groups: in Group C (control), the defect was filled only by a blood clot and, in Group FAT (i.e., fragmented adipose tissue), the defect was filled with fragmented autogenous adipose tissue grafts. The groups were divided into subgroups (n = 7) for euthanasia at 7, 15, and 40 days after the procedure had been conducted. Histologic and histometric analyses were performed. Data were statistically analysed with ANOVA and Tukey's tests (p < 0.05). RESULTS: The amount of bone formation did not show statistically significant differences seven days after the operation, which indicates that the groups had similar amounts of mineral deposition in the earlier period of the repair. Conversely, a significant of amount of bone matrix deposition was identified in the FAT group at 15 and 40 days following the operation, both on the border and in the body of the defect. Such an outcome was not found in the control group. CONCLUSION: In this study, an autologous adipose tissue graft may be considered as likely biomaterial for bone regeneration, since it positively affected the amount of bone formation in surgically created CSD in the rabbits' calvaria 40 days after the procedure had been performed. Further investigations with a longer time evaluation are warranted to determine the effectiveness of autologous adipose tissue graft in the bone healing.

Fragmented adipose tissue transplanted to craniofacial deformities induces bone repair associated with immunoexpression of adiponectin and parathyroid hormone 1-receptor.

Objective : This study analyzed the influence of autogenous white adipose tissue on bone matrix development in critical-size defects created in rabbit calvaria. Materials and methods : A 15-mm-diameter defect was created in the calvaria of 42 rabbits. Twenty-one rabbits were treated with 86 mm(3) of immediate transplant of fragmented white subcutaneous adipose tissue (WSAT); the others constituted the control group (sham). The animals were euthanized at 7, 15, and 40 days postsurgery (n = 7), and the histological data were analyzed by histomorphometry and immunohistochemistry using the anti-adiponectin and parathyroid hormone 1-receptor (PTH1R) antibodies. Results : The calvariae treated with fragmented WSAT demonstrated significant bone formation. These results coincided with the significant presence of immunopositivity to adiponectin and PTH1R in loci, which in turn coincided with the increase in bonelike matrix deposited both in fat tissue stroma and adipocytes' cytoplasm. In contrast, the control group revealed a small amount of bone-matrix deposition and presented scarce PTH1R expression and a lack of immunostain for adiponectin. Conclusion : These results indicate that transplant of fragmented white subcutaneous adipose tissue may be an alternative to treatment of craniofacial bone deformities because adipose tissue suffers from osseous metaplasia and exhibits immunoexpression of the adiponectin and PTH1R, which are proteins associated with bone metabolism.