Risks associated with pathogenic fungi isolated from surgical centers, intensive care units, and materials sterilization center in hospitals. Risks associated with pathogenic fungi isolated from critical hospital areas.

The hospital environment requires special attention to air quality, since it needs to be healthy for the protection of patients and health professionals in order to prevent them against hospital infections. The objective of this study was to isolate, identify and evaluate the susceptibility profile of isolated fungi from two hospitals. For air sampling the impaction (Spin Air, IUL®) and passive sedimentation methods were used. For the isolation of fungi from surfaces, contact plates (RODAC®) were used. The identification of the fungi was performed by observing the macroscopic and microscopic aspects of the colonies, whereas for better visualization of fruiting structures, the microculture technique was performed on slides. To evaluate the susceptibility profile, the broth microdilution test recommended by CLSI was performed. Thirty-five isolates were identified: Aspergillus flavus (12), Aspergillus fumigatus (11), Aspergillus niger (1), Aspergillus terreus (2), Penicillium spp. (7), and Fusarium spp. (2) in the hospitals evaluated. All isolates had a minimum inhibitory concentration (MIC) more than 128 µg/ml for fluconazole; 0.5 to 4.0 µg/ml for amphotericin B (hospital 1), and all isolates from haospital 2 had MIC ≥2.0 µg/ml. In hospital 1, MIC for posaconazole ranged from 0.25 µg/ml to ≥32 µg/ml, and hospital 2 ranged from 0.5 to 1.0 µg/ml. The monitoring and evaluation of air quality and surfaces are essential measures for prevention and control of hospital infections, as these microorganisms are becoming increasingly resistant to antimicrobial agents, thus making treatment difficult, especially in immunocompromised individuals.

Antifungal and cytotoxicity activities of the fresh xylem sap of Hymenaea courbaril L. and its major constituent fisetin.

BACKGROUND: The great potential of plants as Hymenaea courbaril L (jatoba) has not yet been throughly explored scientifically and therefore it is very important to investigate their pharmacological and toxicological activities to establish their real efficacy and safety. This study investigated the cytotoxicity of xylem sap of Hymenaea courbaril L and its bioactivity against the fungi Cryptococcus neoformans species complex and dermatophytes. METHODS: The fresh xylem sap of H. courbaril was filtered resulting in an insoluble brown color precipitate and was identified as fisetin. In the filtrate was identified the mixture of fisetinediol, fustin, 3-O-methyl-2,3-trans-fustin and taxifolin, which were evaluated by broth microdilution antifungal susceptibility testing against C. neoformans species complex and dermatophytes. The fresh xylem sap and fisetin were screened for cytotoxicity against the 3T3-A31 cells of Balb/c using neutral red uptake (NRU) assay. RESULTS: The fresh xylem sap and the fisetin showed higher in vitro activity than the filtrate. The xylem sap of H. courbaril inhibited the growth of dermatophytes and of C. neoformans with minimal inhibition concentration (MIC) < 256 µg/mL, while the fisetin showed MIC < 128 µg/mL for these fungi. Fisetin showed lower toxicity (IC50 = 158 µg/mL) than the fresh xylem sap (IC50 = 109 µg/mL). CONCLUSION: Naturally occurring fisetin can provide excellent starting points for clinical application and can certainly represent a therapeutic potential against fungal infections, because it showed in vitro antifungal activity and low toxicity on animal cells.

Lodderomyces elongisporus as causative organism of oropharyngeal infections - Two case reports.

We report two cases of patients with oropharyngeal infection by Lodderomyces elongisporus. The identification of the two isolates was confirmed after sequencing the ITS1 and ITS4 regions. The antifungal susceptibility test revealed low MIC values for the different antifungals tested. This is the first reported case of L. elongisporus present during an oropharyngeal infection and describes the laboratory methodology employed in the diagnosis.

Acremonium kiliense: case report and review of published studies.

Changes in the spectrum of clinically important fungal infection have been observed in recent years. Acremonium species has been responsible for eumycotic mycetomas but has also been increasingly implicated in systemic fungal diseases. A case of Acremonium kiliense fungemia with proven involvement of the lungs in an allogeneic hematopoietic stem cell patient is reported. A high-resolution computed tomography scan of the lungs showed nodules in both lungs. Multiple cultures of blood demonstrated narrow septate hyphae, cylindrical conidia, and solitary tapering phialides and microconidia that remained grouped in slimy heads. The isolate was identified as A. kiliense based on its morphological characteristics and DNA sequence analysis. Susceptibility testing of the clinical isolate was performed to four antifungal agents. Amphotericin B, fluconazole and itraconazole were found to be inactive in vitro against the isolate; however, it was found to be sensitive to voriconazole. This last drug was indicated, and a high-resolution computed tomography scan of the lungs was normal after 10 days. One year later, the patient was free of symptoms and her blood culture was negative for fungi. Thus, voriconazole was effective in treatment for life-threatening A. kiliense infections. In this work, we performed an overview of worldwide clinical infections caused by A. kiliense.

Distribution and antifungal susceptibility profiles of Candida species isolated from people living with HIV/AIDS in a public hospital in Goiânia, GO, Brazil.

Oropharyngeal candidiasis (OPC) is the most common opportunistic fungal infection of the oral cavity and is a significant clinical problem, particularly in immunocompromised individuals, such as people living with HIV/AIDS (PLWHA). Although Candida albicans is the most frequent pathogen, at least 30 species capable of causing infection have been described. Identifying the infecting organism is necessary because the species respond differently to therapy, and antifungal susceptibility testing is important to determine the appropriate treatment. This study aimed to determine the epidemiological, clinical, and mycological profiles of OPC in hospitalized PLWHA. Clinical samples were collected from 103 PLWHA with suspected candidiasis admitted to the Hospital Estadual of Doenças Tropicais/Hospital Anuar Auad of Goiania, Goias, Brazil, for 14 months. Candida species were identified using phenotypic microbiological techniques and molecular analysis performed by PCR using species-specific primers. The antifungal susceptibility pattern of the isolates against the six antifungal agents was determined using the broth microdilution method. Here, female individuals were the most affected by OPC, presenting a higher risk of oral colonization by Candida spp. The main clinical manifestation was pseudomembranous candidiasis. The number of cases of candidiasis was 87.3% (90/103), with C. albicans being the most common species, followed by C. tropicalis and C. glabrata. In the susceptibility pattern, non-albicans Candida showed higher resistance to than C. albicans. The fast and accurate identification of Candida spp. is very important to identify therapeutic agents for the treatment of oral candidiasis in PLWHA.

In silico-chemogenomic repurposing of new chemical scaffolds for histoplasmosis treatment.

BACKGROUND: Histoplasmosis is a systemic form of endemic mycosis to the American continent and may be lethal to people living with HIV/AIDS. The drugs available for treating histoplasmosis are limited, costly, and highly toxic. New drug development is time-consuming and costly; hence, drug repositioning is an advantageous strategy for discovering new therapeutic options. OBJECTIVE: This study was conducted to identify drugs that can be repositioned for treating histoplasmosis in immunocompromised patients. METHODS: Homologous proteins among Histoplasma capsulatum strains were selected and used to search for homologous targets in the DrugBank and Therapeutic Target Database. Essential genes were selected using Saccharomyces cerevisiae as a model, and functional regions of the therapeutic targets were analyzed. The antifungal activity of the selected drugs was verified, and homology modeling and molecular docking were performed to verify the interactions between the drugs with low inhibitory concentration values and their corresponding targets. RESULTS: We selected 149 approved drugs with potential activity against histoplasmosis, among which eight were selected for evaluating their in vitro activity. For drugs with low minimum inhibitory concentration values, such as mebendazole, everolimus, butenafine, and bifonazole, molecular docking studies were performed. A chemogenomic framework revealed lanosterol 14-α-demethylase, squalene monooxygenase, serine/threonine-protein kinase mTOR, and the ß-4B tubulin chain of H. capsulatum, respectively, as the protein targets of the drugs. CONCLUSIONS: Our strategy can be used to identify promising antifungal targets, and drugs with repositioning potential for treating H. capsulatum.

Phytochemical Analysis and Antimicrobial, Antinociceptive, and Anti-Inflammatory Activities of Two Chemotypes of Pimenta pseudocaryophyllus (Myrtaceae).

Preparations from Pimenta pseudocaryophyllus (Gomes) L.R. Landrum (Myrtaceae) have been widely used in Brazilian folk medicine. This study aims to evaluate the antimicrobial activity of the crude ethanol extracts, fractions, semipurified substances, and essential oils obtained from leaves of two chemotypes of P. pseudocaryophyllus and to perform the antinociceptive and anti-inflammatory screening. The ethanol extracts were purified by column chromatography and main compounds were spectrally characterised (1D and 2D (1)H and (13)C NMR). The essential oils constituents were identified by GC/MS. The broth microdilution method was used for testing the antimicrobial activity. The abdominal contortions induced by acetic acid and the ear oedema induced by croton oil were used for screening of antinociceptive and anti-inflammatory activities, respectively. The phytochemical analysis resulted in the isolation of pentacyclic triterpenes, flavonoids, and phenol acids. The oleanolic acid showed the best profile of antibacterial activity for Gram-positive bacteria (31.2-125 µg mL(-1)), followed by the essential oil of the citral chemotype (62.5-250 µg mL(-1)). Among the semipurified substances, Ppm5, which contained gallic acid, was the most active for Candida spp. (31.2 µg mL(-1)) and Cryptococcus spp. (3.9-15.6 µg mL(-1)). The crude ethanol extract and fractions from citral chemotype showed antinociceptive and anti-inflammatory effects.

Antimicrobial activity of Hymenaea martiana towards dermatophytes and Cryptococcus neoformans.

The biological activity of crude extract and fractions of Hymenaea martiana was evaluated against a panel of human pathogenic fungi. The crude extracts and hydroalcoholic fractions (E) showed a high activity against Cryptococcus neoformans species complex isolates with MICs between 2 and 64 µg ml(-1). The methanolic (C) and butanolic (D) fractions were the most active against Trichopyton rubrum, Trichopyton mentagrophytes and Microsporum canis with MICs between 8 and 256 µg ml(-1). None of the extracts was active against the yeast Malassezia furfur, Malassezia obtusa and Malassezia sympodialis.

Punicalagin triggers ergosterol biosynthesis disruption and cell cycle arrest in Cryptococcus gattii and Candida albicans : Action mechanisms of punicalagin against yeasts.

Punicalagin is a phenolic compound extracted from Lafoensia pacari A. St.-Hil (Lythraceae) leaves. It has demonstrated interesting activity against pathogenic fungi, e.g., Cryptococcus gattii and Candida albicans, by inhibiting fungi growth in a minimum inhibitory concentration (MIC) at 4 µg/mL. However, the mechanisms behind its antifungal action are not well understood. In this study, certain parameters were investigated, by transmission electron microscopy, ergosterol synthesis inhibition, and flow cytometry analyses, to gain insight into the possible biological targets of punicalagin (4 or 16 µg/mL) against yeast cells. Data showed that, in contrast to untreated cells, punicalagin triggered severe ultrastructural changes in C. gattii and C. albicans, such as disorganization of cytoplasmic content and/or thickened cell walls. In addition, it caused a decrease in yeast plasma membrane ergosterol content in a concentration-dependent manner. However, it was unable to bring about significant fungal cell membrane rupture. On the other hand, punicalagin (16 µg/mL) significantly arrested C. albicans and C. gattii cells at the G0/G1 phase, with a consequent reduction in cells at the G2/M phase in both fungi isolates, and thereby prevented progression of the normal yeast cell cycle. However, these alterations showed no involvement of reactive oxygen species overproduction in C. albicans and C. gattii cells, although punicalagin triggered a significant loss of mitochondrial membrane potential in C. albicans. These findings suggest that punicalagin is a promising plant-derived compound for use in developing new antifungal therapies.

Susceptibility testing of Candida albicans isolated from oropharyngeal mucosa of HIV(+) patients to fluconazole, amphotericin B and Caspofungin. killing kinetics of caspofungin and amphotericin B against fluconazole resistant and susceptible isolates.

A clear understanding of the pharmacodynamic properties of antifungal agents is important for the adequate treatment of fungal infections like candidiasis. For certain antifungal agents, the determination of Minimal Fungicidal Concentration (MFC) and time kill curve could be clinically more relevant than the determination of the Minimal Inhibitory Concentration (MIC). In this study, MIC and MFC to fluconazole, amphotericin B and caspofungin against C. albicans isolates and the killing patterns obtained with caspofungin and amphotericin B against susceptible and resistant strains to fluconazole were determined. The results of MICs showed that all C. albicans isolates were highly susceptible to amphotericin B, but two isolates were fluconazole resistant. The comparative analysis between MIC and MFC showed that MFC of fluconazole was fourfold higher than MIC in 41.9% of the C. albicans isolates. Same values of MFC and MIC of amphotericin B and caspofungin were found for 71% of the isolates. Correlation between time kill curves and MFC of amphotericin B and caspofungin against all 4 isolates tested was observed. The caspofungin killing effect was more evident at MFC in 6 hours of incubation than at MIC in this time, suggesting dependence of concentration. The similarity of results of time-kill curve and MFC values indicate that determination of MFC is an alternative for the detection of the fungicidal activity of these drugs.

Antifungal potential of punicalagin against Cryptococcus neoformans species complex.

This study evaluated the antifungal activity and cytotoxicity profile of the ellagitannin punicalagin, a compound extracted from the L. pacari A. St.-Hil (Lythraceae) leaf, against Cryptococcus neoformans species complex. Minimum inhibitory concentrations (MIC) were checked using the broth microdilution method. Minimum fungicidal concentrations (MFC) and time of death were used to confirm the antifungal activity of the compound. The in vitro cytotoxicity of punicalagin was tested in BALB/c3T3 fibroblasts and A549 human lung cancer cell line, while the hemolytic potential was tested on sheep erythrocytes. The morphological changes induced in yeast strains by the presence of punicalagin were also analyzed. Tested on eight isolates of the C. neoformans complex punicalagin showed MIC of 0.5 to 4.0 µg/mL and MFC> 256 µg/mL. Punicalagin also demonstrated a good growth inhibitory activity in time-kill curves, but it was not able to achieve a statistically significant reduction of fungal growth suggesting a fungistatic effect of the compound. In vitro cytotoxicity studies using the two cell lines showed that punicalagin has low activity on these cells and no activity on sheep erythrocytes. Morphological changes were seen in the yeasts strains studied when treated with punicalagin. Therefore, punicalagin is a potential antifungal for important pathogenic yeasts and presents a low cytotoxicity profile associated with no hemolytic effects.

[Phenotyping of bacteria isolated in blood cultures from critical patients]. / Fenotipagem de bactérias isoladas em hemoculturas de pacientes críticos.

Bloodstream infections are a serious complication among critical patients. The detection of microbial pathogens in blood cultures is an important diagnostic tool. This study aimed to isolate and characterize bacteria from the blood of patients admitted to the clinical intensive care unit of a teaching hospital, between April 2003 and April 2004. The bacteria isolated were identified by biochemical/enzymatic tests and detection of the antimicrobial susceptibility profile using the disk diffusion method. A total of 304 blood cultures from 195 patients were evaluated. Forty-nine (16.1%) of the blood cultures presented microbe development. The predominant species was Pseudomonas stutzeri (18.2%). Gram-positive cocci and enterobacteria showed greater resistance to ampicillin; vancomycin and linezolid were the most active agents for Gram-positive cocci and carbapenems for Gram-negative rods. Because of the impact of bloodstream infection within the hospital context, additional studies are needed in order to give backing for prevention and control measures.

Invasive fungal infection in patients with hematologic disorders in a Brazilian tertiary care hospital.

INTRODUCTION:: Invasive fungal infections (IFIs) are an important complication in immunocompromised individuals, particularly neutropenic patients with hematological malignancies. In this study, we aimed to verify the epidemiology and diagnosis of IFIs in patients with hematologic problems at a tertiary hospital in Goiânia-GO, Brazil. METHODS:: Data from 117 patients, involving 19 cases of IFIs, were collected. The collected data included diagnosis methods, demographics, clinical characteristics, and in vitro susceptibility to different antifungal agents. Among the 19 cases, 12 were classified as proven IFI and 7 as probable invasive aspergillosis with detection of galactomannan in blood and presence of lung infiltrates in radiographic images. Logistic regression analysis showed that the proven and probable IFIs were associated with increased risk of death. Statistical analysis demonstrated that age, sex, and underlying disease were not independently associated with risk of death in IFI patients. RESULTS:: Most bloodstream isolates of Candida spp. exhibited low minimum inhibitory concentrations (MICs) to all antifungal agents tested. Voriconazole and amphotericin had the lowest MICs for Aspergillus spp. and Fusarium spp., but Fusarium spp. showed the least susceptibility to all antifungals tested. Amphotericin B, fluconazole, and itraconazole were found to be inactive in vitro against Acremonium kiliense; but this fungus was sensitive to voriconazole. CONCLUSIONS:: Considering the high number of IFI cases, with crude mortality rate of 6%, we could conclude that IFIs remain a common infection in patients with hematological malignancies and underdiagnosed ante mortem. Thus, IFIs should be monitored closely.

Antifungal susceptibilities of clinical and environmental isolates of Cryptococcus neoformans in Goiânia city, Goiás, Brazil.

We evaluated the antifungal activities of amphotericin B, fluconazole, itraconazole and voriconazole in 70 Cryptococcus neoformans strains obtained from cerebrospinal fluid from AIDS patients and 40 C. neoformans strains isolated from the environment. Four clinical isolates were identified as C. neoformans var. gattii. The susceptibility test was done using a broth microdilution method according to NCCLS M27-A2. Range minimal inhibitory concentrations (MICs) for C. neoformans clinical isolates were 0.06-1.0 microg/mL for amphotericin B, 0.125-8 microg/mL for fluconazole, 0.03-0.5 microg/mL for itraconazole and 0.03-0.25 microg/mL for voriconazole. C. neoformans environmental isolates showed range MICs 0.015-0.125 microg/mL, 0.25-2.0 microg/mL, 0.007-0.125 microg/mL and 0.03-0.25 microg/mL for amphotericin B, fluconazole, itraconazole and voriconazole respectively. The MICs results obtained from clinical and environmental isolates showed similar pattern of susceptibility and no resistance has been found in our isolates.

Characterization of Cryptococcus neoformans isolated from urban environmental sources in Goiânia, Goiás State, Brazil.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis as the most frequent clinical presentation in immunocompromised patients, mainly in people infected by HIV. This fungus is an environmental encapsulated yeast, commonly found in soil enriched with avian droppings and plant material. A total of 290 samples of pigeon and the other avian droppings, soil, ornamental trees and vegetable material associated with Eucalyptus trees were collected to study environmental sources of Cryptococcus species in Goiânia, Goiás State. The determination of varieties, serotypes and the susceptibility in vitro to fluconazole, itraconazole and amphotericin B of C. neoformans isolates were performed. C. neoformans var. grubii (serotype A) was found in 20.3% (36/177) of pigeon dropping samples and in 14.3% (5/35) of samples of Eucalyptus. None of the environmental isolates of C. neoformans showed in vitro resistance to three antifungal agents. The knowledge of major route for human cryptococcal infection (inhalation of infectious particles from saprophytic sources) and a total of 60 C. neoformans isolates obtained from AIDS patients with cryptococcal meningitis between October 2001 and April 2002 justify the study of the habitats of these yeasts as probable sources of cryptococcosis in this city.

Molecular identification and antifungal susceptibility profiles of Candida parapsilosis complex species isolated from culture collection of clinical samples.

INTRODUCTION: Candida parapsilosis is a common yeast species found in cases of onychomycosis and candidemia associated with infected intravascular devices. In this study, we differentiated Candida parapsilosis sensu stricto, Candida orthopsilosis , and Candida metapsilosis from a culture collection containing blood and subungual scraping samples. Furthermore, we assessed the in vitro antifungal susceptibility of these species to fluconazole, itraconazole, voriconazole, posaconazole, amphotericin B, and caspofungin. METHODS: Differentiation of C. parapsilosis complex species was performed by amplification of the secondary alcohol dehydrogenase (SADH) gene and digestion by the restriction enzyme Ban I. All isolates were evaluated for the determination of minimal inhibitory concentrations using Etest, a method for antifungal susceptibility testing. RESULTS: Among the 87 isolates, 78 (89.7%) were identified as C. parapsilosis sensu stricto , five (5.7%) were identified as C. orthopsilosis , and four (4.6%) were identified as C. metapsilosis . Analysis of antifungal susceptibility showed that C. parapsilosis sensu strictoisolates were less susceptible to amphotericin B and itraconazole. One C. parapsilosis sensu stricto isolate was resistant to amphotericin B and itraconazole. Moreover, 10.2% of C. parapsilosis sensu stricto isolates were resistant to caspofungin. Two C. parapsilosis sensu strictoisolates and one C. metapsilosis isolate were susceptible to fluconazole in a dose-dependent manner. CONCLUSIONS: We reported the first molecular identification of C. parapsilosiscomplex species in State of Goiás, Brazil. Additionally, we showed that although the three species exhibited differences in antifungal susceptibility profiles, the primary susceptibility of this species was to caspofungin.

Accuracy in clinical examinations for the diagnosis of vulvovaginitis by CANDIDA SPP. and IN VITRO susceptibility to the main antifungals

Vulvovaginal candidiasis (VVC) is a common infection. This work aims to determine the positive predictive value (PPV) of the clinical diagnosis of VVC and to characterize Candida species isolated from the vaginal mucosa. This cross-sectional study was conducted from February 2016 to February 2017 at the Gynecology and Obstetrics Outpatient Clinic of the Hospital das Clínicas, in Goiânia, Goiás State, Brazil. The study included samples of vaginal secretion from 55 women who complained of vaginal discharge and itching as their main symptoms. The PPV of the clinical diagnosis of VVC was estimated in comparison to the laboratory culture method. The phenotypic methods and molecular tests were performed to identify Candida spp. In vitro susceptibility of Candida spp. isolates to fluconazole, itraconazole, clotrimazole, nystatin, and amphotericin B was determined using the broth microdilution assay. Yeast growth using the enzymes protease, phospholipase, and hemolysin was carried out in media containing respectively bovine albumin, egg yolk, and sheep erythrocytes. A PPV of 61.8% (34/55) was determined. Among the 55 vulvovaginal samples collected, we identified 36 isolates in which C. albicans was the most common species. High resistance to fluconazole and low minimal inhibitory concentration (MIC) values for clotrimazole, nystatin and amphotericin B were observed. All isolates were proteinase and hemolysin producers, while seven strains were phospholipase negative. The clinical diagnosis of VVC presented a moderate PPV, which meant that cultures had to be conducted in the laboratory to confirm infection. The high resistance to fluconazole and itraconazole indicated the importance of the in vitro susceptibility test.

[Tinea capitis in children from Goiânia, Brazil]. / Tinha do couro cabeludo em crianças de Goiânia, Brasil.

During the period January 1999 to July 2002 a total de 164 cases of Tinea capitis were diagnosed by mycological examination in Instituto de Patologia Tropical e Saúde Pública of the Universidade Federal de Goiás. Of the 164 patients 94 (57.3%) were males, with an age of 3 months to 13 years. Laboratory studies were performed by direct examination with 20% KOH and cultivated on Mycobiotic agar medium and Sabouraud dextrose agar with chloramphenicol. The following species were identified: Microsporum canis (71.3%), Trichophyton tonsurans (11%), Trichophyton mentagrophytes (7.9%), Trichophyton rubrum (6.7%), and Microsporum gypseum (3%). Our study showed that the most frequent rise of scalp infection was a zoophylic fungi, called Microsporum canis.

[Antifungal activity of Caryocar brasiliensis (Caryocaraceae) against Cryptococcus neoformans]. / Atividade antifúngica de Caryocar brasiliensis (Caryocaraceae) sobre Cryptococcus neoformans.

The widespread occurrence of cryptococcosis mainly in immunocompromised patients and the side effects of available drugs which are effective against this mycosis have led investigators to search for new antimycotic agents. Caryocar brasiliensis derived compounds were investigated against Cryptococcus neoformans using the agar dilution method. Based on MIC values, the best results were obtained with a concentration of < 250 g/mL of cuticular waxes of the Caryocar brasiliensis leaf collected during the dry period (170.8mm of precipitation) which inhibited the growth of 91.3% (21/23) Cryptococcus neoformans isolates.