Detection and quantification of fluconazole within Candida glabrata biofilms

Candida infections are often associated with biofilms and consequent high resistance to most common drugs (e.g. azoles). These resistance mechanisms are not only associated with the biofilm yeast physiology, but also with the presence of a diffusional barrier imposed by the biofilm matrix; however, the real biochemical role of the biofilm components remains very unclear. So, in order to further clarify this issue, we intend to determine, for the first time, fluconazole in biofilms within both supernatants and matrices. Candida biofilms were formed in the presence of fluconazole, and it was recovered from both supernatant and matrix cell-free fractions. Then, high-pressure liquid chromatography was used to identify and quantify the amount of drug that was present in the two fractions. Moreover, this study also showed that the presence of fluconazole in both fractions indicated that the drug administrated did not completely reach the cells, so this phenomena can easily be associated with lower biofilm susceptibility, since the drug administered did not completely reach the cells.This work was supported by the Programa Operacional, Fatores de competitividade-COMPETE and by national funds through FCT-Fundacao para a Ciencia e a Tecnologia on the scope of the projects FCT PTDC/SAU-MIC/119069/2010, RECI/EBB-EBI/0179/2012, PEst-OE/EQB/LA0023/2013 and Celia F. Rodrigue's SFRH/BD/93078/2013 PhD grant. The authors thank the Project "BioHealth-Biotechnology and Bioengineering approaches to improve health quality,'' Ref. NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON. 2-O Novo Norte), QREN, FEDER. We also would like to acknowledge Pfizer (R), S.A., for the kindly donation of fluconazole

A Cross-Sectional Study of People with Epilepsy and Neurocysticercosis in Tanzania: Clinical Characteristics and Diagnostic Approaches.

Neurocysticercosis (NCC) is a major cause of epilepsy in regions where pigs are free-ranging and hygiene is poor. Pork production is expected to increase in the next decade in sub-Saharan Africa, hence NCC will likely become more prevalent. In this study, people with epilepsy (PWE, n=212) were followed up 28.6 months after diagnosis of epilepsy. CT scans were performed, and serum and cerebrospinal fluid (CSF) of selected PWE were analysed. We compared the demographic data, clinical characteristics, and associated risk factors of PWE with and without NCC. PWE with NCC (n=35) were more likely to be older at first seizure (24.3 vs. 16.3 years, p=0.097), consumed more pork (97.1% vs. 73.6%, p=0.001), and were more often a member of the Iraqw tribe (94.3% vs. 67.8%, p=0.005) than PWE without NCC (n=177). PWE and NCC who were compliant with anti-epileptic medications had a significantly higher reduction of seizures (98.6% vs. 89.2%, p=0.046). Other characteristics such as gender, seizure frequency, compliance, past medical history, close contact with pigs, use of latrines and family history of seizures did not differ significantly between the two groups. The number of NCC lesions and active NCC lesions were significantly associated with a positive antibody result. The electroimmunotransfer blot, developed by the Centers for Disease Control and Prevention, was more sensitive than a commercial western blot, especially in PWE and cerebral calcifications. This is the first study to systematically compare the clinical characteristics of PWE due to NCC or other causes and to explore the utility of two different antibody tests for diagnosis of NCC in sub-Saharan Africa

Hsp90 governs dispersion and drug resistance of fungal biofilms

Fungal biofilms are a major cause of human mortality and are recalcitrant to most treatments due to intrinsic drug resistance. These complex communities of multiple cell types form on indwelling medical devices and their eradication often requires surgical removal of infected devices. Here we implicate the molecular chaperone Hsp90 as a key regulator of biofilm dispersion and drug resistance. We previously established that in the leading human fungal pathogen, Candida albicans, Hsp90 enables the emergence and maintenance of drug resistance in planktonic conditions by stabilizing the protein phosphatase calcineurin and MAPK Mkc1. Hsp90 also regulates temperature-dependent C. albicans morphogenesis through repression of cAMP-PKA signalling. Here we demonstrate that genetic depletion of Hsp90 reduced C. albicans biofilm growth and maturation in vitro and impaired dispersal of biofilm cells. Further, compromising Hsp90 function in vitro abrogated resistance of C. albicans biofilms to the most widely deployed class of antifungal drugs, the azoles. Depletion of Hsp90 led to reduction of calcineurin and Mkc1 in planktonic but not biofilm conditions, suggesting that Hsp90 regulates drug resistance through different mechanisms in these distinct cellular states. Reduction of Hsp90 levels led to a marked decrease in matrix glucan levels, providing a compelling mechanism through which Hsp90 might regulate biofilm azole resistance. Impairment of Hsp90 function genetically or pharmacologically transformed fluconazole from ineffectual to highly effective in eradicating biofilms in a rat venous catheter infection model. Finally, inhibition of Hsp90 reduced resistance of biofilms of the most lethal mould, Aspergillus fumigatus, to the newest class of antifungals to reach the clinic, the echinocandins. Thus, we establish a novel mechanism regulating biofilm drug resistance and dispersion and that targeting Hsp90 provides a much-needed strategy for improving clinical outcome in the treatment of biofilm infections

Effect of voriconazole on Candida tropicalis biofilms: Relation with ERG genes expression

Candida tropicalis has emerged as the third most prevalent fungal pathogens and its ability to form biofilms has been considered one of the most important virulence factors, since biofilms represent high tolerance to antifungal agents. However, the mechanisms of C. tropicalis biofilm resistance to antifungals remain poorly understood. Thus, the main aim of this work was to infer about the effect of voriconazole on the formation and control of C. tropicalis biofilms and disclose its relationship with ERG genes' expression. Planktonic cells tolerance of several C. tropicalis clinical isolates to voriconazole was determined through of antifungal susceptibility test, and the effect of this azole against C. tropicalis biofilm formation and pre-formed biofilms was evaluated by cultivable cells determination and total biomass quantification. ERG genes expression was analyzed by quantitative real-time polymerase chain reaction. This work showed that C. tropicalis resistance to voriconazole is strain dependent and that voriconazole was able to partially control biofilm formation, but was unable to eradicate C. tropicalis pre-formed biofilms. Moreover, C. tropicalis biofilms resistance to voriconazole seems to be associated with alterations of sterol content in the cell membrane, resulting in ERG genes overexpression. Voriconazole is unable to control C. tropicalis biofilms, and the overexpression of ERG genes is a possible mechanism of biofilm resistance.TheauthorsthanktheFCTfortheStrategic Project of the UID/BIO/04469/2013 unit, FCT and European Union funds (FEDER/COMPETE) for the project RECI/BBBEBI/0179/2012 (FCOMP-01-0124-FEDER-027462). We also would like to acknowledge Pfizer , S.A. for the kindly donation of voriconazole

Genetics of Mechanosensation in the Heart

Mechanosensation (the ultimate conversion of a mechanical stimulus into a biochemical signal) as well as mechanotransduction (transmission of mechanically induced signals) belong to the most fundamental processes in biology. These effects, because of their dynamic nature, are particularly important for the cardiovascular system. Therefore, it is not surprising that defects in cardiac mechanosensation, are associated with various types of cardiomyopathy and heart failure. However, our current knowledge regarding the genetic basis of impaired mechanosensation in the cardiovascular system is beginning to shed light on this subject and is at the centre of this brief review

Biofilms of non-Candida albicans Candida species : quantification, structure and matrix composition

Most cases of candidiasis have been attributed to C. albicans, but recently, non- Candida albicans Candida (NCAC) species have been identified as common pathogens. The ability of Candida species to form biofilms has important clinical repercussions due to their increased resistance to antifungal therapy and the ability of yeast cells within the biofilms to withstand host immune defenses. Given this clinical importance of the biofilm growth form, the aim of this study was to characterize biofilms produced by three NCAC species, namely C. parapsilosis, C. tropicalis and C. glabrata. The biofilm forming ability of clinical isolates of C. parapsilosis, C. tropicalis and C. glabrata recovered from different sources, was evaluated by crystal violet staining. The structure and morphological characteristics of the biofilms were also assessed by scanning electron microscopy and the biofilm matrix composition analyzed for protein and carbohydrate content. All NCAC species were able to form biofilms although these were less extensive for C. glabrata compared with C. parapsilosis and C. tropicalis. It was evident that C. parapsilosis biofilm production was highly strain dependent, a feature not evident with C. glabrata and C. tropicalis. Scanning electron microscopy revealed structural differences for biofilms with respect to cell morphology and spatial arrangement. Candida parapsilosis biofilm matrices had large amounts of carbohydrate with less protein. Conversely, matrices extracted from C. tropicalis biofilms had low amounts of carbohydrate and protein. Interestingly, C. glabrata biofilm matrix was high in both protein and carbohydrate content. The present work demonstrates that biofilm forming ability, structure and matrix composition are highly species dependent with additional strain variability occurring with C. parapsilosis.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/28341/2006, PDTC/BIO/61112/200

Development of a High-Throughput Candida albicans Biofilm Chip

We have developed a high-density microarray platform consisting of nano-biofilms of Candida albicans. A robotic microarrayer was used to print yeast cells of C. albicans encapsulated in a collagen matrix at a volume as low as 50 nL onto surface-modified microscope slides. Upon incubation, the cells grow into fully formed “nano-biofilms”. The morphological and architectural complexity of these biofilms were evaluated by scanning electron and confocal scanning laser microscopy. The extent of biofilm formation was determined using a microarray scanner from changes in fluorescence intensities due to FUN 1 metabolic processing. This staining technique was also adapted for antifungal susceptibility testing, which demonstrated that, similar to regular biofilms, cells within the on-chip biofilms displayed elevated levels of resistance against antifungal agents (fluconazole and amphotericin B). Thus, results from structural analyses and antifungal susceptibility testing indicated that despite miniaturization, these biofilms display the typical phenotypic properties associated with the biofilm mode of growth. In its final format, the C. albicans biofilm chip (CaBChip) is composed of 768 equivalent and spatially distinct nano-biofilms on a single slide; multiple chips can be printed and processed simultaneously. Compared to current methods for the formation of microbial biofilms, namely the 96-well microtiter plate model, this fungal biofilm chip has advantages in terms of miniaturization and automation, which combine to cut reagent use and analysis time, minimize labor intensive steps, and dramatically reduce assay costs. Such a chip should accelerate the antifungal drug discovery process by enabling rapid, convenient and inexpensive screening of hundreds-to-thousands of compounds simultaneously

Risk Factors and Outcomes of Candidemia Caused by Biofilm-Forming Isolates in a Tertiary Care Hospital

Very few data exist on risk factors for developing biofilm-forming Candida bloodstream infection (CBSI) or on variables associated with the outcome of patients treated for this infection. METHODS AND FINDINGS: We identified 207 patients with CBSI, from whom 84 biofilm-forming and 123 non biofilm-forming Candida isolates were recovered. A case-case-control study to identify risk factors and a cohort study to analyze outcomes were conducted. In addition, two sub-groups of case patients were analyzed after matching for age, sex, APACHE III score, and receipt of adequate antifungal therapy. Independent predictors of biofilm-forming CBSI were presence of central venous catheter (odds ratio [OR], 6.44; 95% confidence interval [95% CI], 3.21-12.92) or urinary catheter (OR, 2.40; 95% CI, 1.18-4.91), use of total parenteral nutrition (OR, 5.21; 95% CI, 2.59-10.48), and diabetes mellitus (OR, 4.47; 95% CI, 2.03-9.83). Hospital mortality, post-CBSI hospital length of stay (LOS) (calculated only among survivors), and costs of antifungal therapy were significantly greater among patients infected by biofilm-forming isolates than those infected by non-biofilm-forming isolates. Among biofilm-forming CBSI patients receiving adequate antifungal therapy, those treated with highly active anti-biofilm (HAAB) agents (e.g., caspofungin) had significantly shorter post-CBSI hospital LOS than those treated with non-HAAB antifungal agents (e.g., fluconazole); this difference was confirmed when this analysis was conducted only among survivors. After matching, all the outcomes were still favorable for patients with non-biofilm-forming CBSI. Furthermore, the biofilm-forming CBSI was significantly associated with a matched excess risk for hospital death of 1.77 compared to non-biofilm-forming CBSI. CONCLUSIONS: Our data show that biofilm growth by Candida has an adverse impact on clinical and economic outcomes of CBSI. Of note, better outcomes were seen for those CBSI patients who received HAAB antifungal therapy

Prevalence of anogenital HPV infection, related disease and risk factors among HIV-infected men in inner-city Johannesburg, South Africa: baseline findings from a cohort study.

BACKGROUND: Persistent high-risk human papillomavirus (HR-HPV) infection is associated with the development of anogenital cancers, particularly in men living with HIV (MLWH). We describe the prevalence of anogenital HPV infection, abnormal anal cytology and anogenital warts (AGWs) in MLWH in Johannesburg, and explore whether HPV infection and receipt of antiretroviral treatment is associated with detection of abnormal anal cytology and AGWs. METHODS: We enrolled a cohort of 304 sexually-active MLWH ≥18 years, who completed a questionnaire and physical examination. Genital swabs were collected from all men and intra-anal swabs from 250 (82%). Swabs were tested for HPV DNA and genotypes, and anal smears graded using the Bethesda classification. Factors associated with anogenital disease were assessed by logistic regression models. RESULTS: Two thirds were receiving antiretroviral treatment, for a median 33 months (IQR = 15-58) and 54% were HIV-virologically suppressed. Only 5% reported ever having sex with men. Among 283 genital swabs with valid results, 79% had any HPV, 52% had HR-HPV and 27% had >1 HR-HPV infection. By comparison, 39% of the 227 valid intra-anal swabs had detectable HPV, 25% had any HR-HPV and 7% >1 HR infection. While most anal smears were normal (51%), 20% had ASCUS and 29% were LSIL. No cases had HSIL or cancer. Infection with >1 HR type (adjusted OR [aOR] = 2.39; 95%CI = 1.02-5.58) and alpha-9 types (aOR = 3.98; 95%CI = 1.42-11.16) were associated with having abnormal cytology. Prevalence of AGWs was 12%. Infection with any LR type (aOR = 41.28; 95%CI = 13.57-125.62), >1 LR type (aOR = 4.14; 95%CI = 1.60-10.69), being <6 months on antiretroviral treatment (aOR = 6.90; 95%CI = 1.63-29.20) and having a CD4+ count <200 cells/μL (aOR = 5.48; 95%CI: 1.60-18.78) were associated with having AGWs. CONCLUSIONS: In this population, anogenital HR-HPV infection and associated low-grade disease is common, but severe anal dysplasia was not detected. Findings reinforce the need for HPV vaccination in men for preventing both AGWs and HR-HPV infection. Given the absence of anal HSILs, however, the findings do not support the use of anal screening programmes in this population