Virulence potential of Candida albicans isolated from oral cavity of patients with chronic renal failure on hemodialysis

Objective In patients with chronic renal failure (PCRF), the frequency of colonization of the oral cavity by yeasts of genus Candida spp. is high compared with healthy individuals. These yeasts have virulence factors that may contribute to the persistence of colonization and the development of these infections. The aim of this study was evaluate aspects of virulence from Candida albicans isolated from oral cavity of PCRF on dialysis. Methods This study was initially conducted with 49 clinical samples of C. albicans. The virulence factors assayed were produce of biofilm, germ tube, determination of adherence in oral epithelial cells and evaluation of resistance to the antimicrobial action of neutrophils and mononuclear cells. Results All isolates were highly efficient in forming biofilms on poly- styrene microplates, where 94% of the samples formed 4 + biofilm. Used as a screening test, of which three isolates were selected with different degrees of ability to form biofilm to assess other indicators of virulence. Overall, the isolates exhibited different characteristics regarding the virulence factors analyzed. It was also observed that the hypophosphorous acid (HOCl), production, one of leading inflammatory mediators with fungicidal action, also varied especially when the neutrophils, and not mononuclear cells, were stimulated with different samples. (Figure 1). Conclusion Therefore, our results indicate that C. albicans, is not only the most common species in the oral cavity of CRFP on dialysis, but also it presents the main virulence attributes, which reinforces the importance of monitoring of these patients towards the prevention of fungal infections

Evaluation of propolis and its subproduct as an inhibitor of growth and biofilm formation in vaginal yeast from pregnant women

Objectives The treatment of vulvovaginal candidiasis (VVC) is still unsatisfactory, especially in pregnant women, being promising to the utilization of alternative therapies. Propolis extract solution (PES) has demonstrated antifungal efficacy and low toxicity. In addition, the subproduct of propolis extract solution (SPES) is produced during the process of preparing PES and is usually discarded, but can still sub- mit substances responsible for biological effects, such as the polyphenols, responsible for the therapeutic activity of propolis. SPES have not been investigated or used as an antimicrobial agent. Thus, the objective of the present study was to investigate the effect of PES and SPES on Candida spp. isolated from the vaginal material of pregnant women. Methods Vaginal samples from 291 pregnant women were collected and cultivated for yeasts, which were identified by the classical method and performing susceptibility tests against PES, SPES and conventional antifungal agents. The anti-biofilm effect and cytotoxicity tests of the PES and SPES were evaluated. Results In 38.48% (112/291) of culture was positive for Candida species. There were patients with two different species, being a total of 115 yeasts (82.61% C. albicans; 6.08% C. glabrata; 5.22% C. tropi- calis; 5.22% C. parapsilosis and 0.87% C. krusei). PES and SPES were effective, even against isolates resistant to conventional antifungal (Table 1) and reduced about 25% C. tropicalis biofilm, besides presenting its low toxicity in the concentrations of fungicides. Conclusion Thus, in addition to the PES, SPES can also be a promising alternative treatment, especially in this population

Genotypic variability and antifungal susceptibility of Candida spp. isolated from hospital surfaces and hands of healthcare professionals

Objectives Candida spp. are responsible for 9095% of hematogenous fungal infections. In Brazil and Latin America, C. albicans is the most common specie, followed by C. parapsilosis and C. tropicalis. Infections caused by Candida spp. may have their origin in exogenous sources, transmitted to patients via contaminated infusions, biomedical devices or even by the hands of the hospital staff members. Molecular biology techniques such as Randomly Amplified Polymorphic DNA (RAPD) can show that the strains found in anatomical sites or abi- otic surfaces have the same pattern genome.Moreover, in the last decades it has been observed increasing the number of yeasts isolated from hospital environment resistant to antifungals. Thus, the aim of this study was to determine the susceptibility to antifungals and intraspecies similarity among isolates of different hospital surfaces and hands of healthcare professionals. Methods The study was conducted with 25 isolates of Candida spp.: 5 strains of C. albicans and 5 strains of C. parapsilosis isolated from hospital surfaces. 5 strains of C. albicans, 5 strains of C. parapsilosis and 5 strains of C. tropicalis isolated from hands of healthcare professionals. Professionals and surfaces belonged to intensive care units. The minimal inhibitory concentration (MIC) was determined to voriconazole (VOR), fluconazole (FLZ), amphotericin B (AMB) and micafungin (MFG) according to M27-A3 of the Clinical and Labora- tory Standards Institute (CLSI). To determine the intra-species similarity, 3 primers were used: P4 (50 -AAGAGCCCGT-30 ), OPA-18 (50AGCTGACCGT30) and OPE-18 (50GGACTGCAGA 30). RAPD pro- files were analyzed using BioNumerics software version 4.6. The study was approved by the Ethics in research involving human subjects, CAAE 0448.0.093.000-11 protocol. Results In relation to susceptibility testing (Table 1), it is important to highlight that C. parapsilosis showed 80% of MFG resistance. C. albicans and C. tropicalis showed reduced susceptibility to VOR, and resistence of the AMB was observed for C. albicans (20%). All amplifi- cations revealed distinct polymorphic bands. Genetic distances between each of the isolates were calculated and cluster analysis was used to generate a dendrogram showing relationships between them. The analysis of all primers showed similarity greater than 80% between strains of hands and hospital surfaces for intraspecies. Conclusion Our work shows that, healthy people and hospital surfaces may be colonized by different species yeast. Furthermore, the strains studied had relative resistance to antifungal drugs most frequently used in clinical practice. Finally, there was a high similarity between samples from hands (hospital staff members) and surfaces, providing an infection risk to susceptible individuals. Healthy people working in hospitals can carry yeasts on their hands with the same potential virulence, and which therefore offer the same risk of infection. This information should be considered when preventive measures are established. Attention to the colonization of hands and surfaces should not be restricted to high-risk units such as NICUs, but should also include other sections of hospitals

Assessment of in vitro biofilm formation and antifungal susceptibility of Candida albicans isolates from vulvovaginal candidiasis

Objectives Vulvovaginal candidiasis (VVC) is an inflammation of the genital mucosa, which mainly affects the vulva and vagina. Candida spp. are considered commensal fungus, however, when there is imbalance in the microbiota or the host immune system is compromised, these can become pathogenic. C. albicans is responsible for most cases of VVC and is able of expressing mechanisms which allow the colonization or infection in the host. These factors related yeasts, including the growth of strains resistant to antifungal agents and virulence attributes (such as biofilm formation) are important in the development of VVC. In this sense, the objective of this study was to evaluate the in vitro biofilm formation and susceptibility to antifungal of C. albicans isolates from patients with vulvovaginal candidiasis. Methods For the study were analyzed 30 clinical isolates of Candida albicans. The clinical isolates were separated in groups of 10 samples of the according to symptoms presented by the patients: asymptomatic (AS), vulvovaginal candidiasis (VVC) and recurrent vulvo- vaginal candidiasis (RVVC). For all isolates were analyzed biofilm formation and minimal inhibitory concentration (MIC) for fluconazole and nystatin. The MIC was performed according to M27-A3 protocol of the Clinical Laboratory Standards Institute. Biofilm forming ability was assessed through quantification of total biomass by crystal violet (CV) staining, performed on 96-well microplates containing a cellular suspension of 1 9 107 cells ml1 and incubated for 24 h at 37°C. Results Antifungal susceptibility testing is showed in table 1. The isolates were tested to the two antifungals. The MIC raging from 0.125 to 2 lg ml1 for fluconazole and 1 to 4 lg ml1 to nystatin. The figure 1 show the quantification of the total biomass. It was evident that all the C. albicans isolates were able to form biofilm, although differences occurred depending on the isolated and consequently the group. Importantly it was noted that, in general, VVC and RVVC groups had similar capacity biofilm formation. On the other hand, these groups had less total biomass (average Abs = 1,091 ` 0.88) compared with AS group (average Abs = 1,521 ` 1.32). Conclusion Although all the samples analyzed are sensitive to anti- fungals tested research of resistant strains is relevant, since recurrences are related to cases of VVC. Nystatin and fluconazole were effective in small concentrations for the isolates analysed. All samples were able to form biofilm and the average of the group of asymptomatic patients greater than the others. Thus, the capacity to form- ing biofilm is an important virulence factor in the persistence of microorganisms in infectious processes and represent an increase in resistance to antifungal and host defense

Influence of Laminarin in colonisation process of Candida albicans

Objective Candida albicans is responsible for the majority of cases of vulvovaginal candidiasis (VVC), one of the most important candidal virulence factors is the ability to adhere to host surfaces. Chemotherapies that seek to improve the host immune response are an alterna- tive to control fungal infections. b-glucans are polymeric carbohydrates that have been reported to modulate human inflammatory responses in vitro and in vivo. The aim of this study was to determine the influence of Laminarin (LAM) a b-glucan on C. albicans virulence, namely colonisation of HeLa cells. Methods To assess the role of LAM in the cell colonization process, HeLa cells were previously treated or not with 3 mg mL1 of LAM (b-glucan extracted from Laminarina digitata) for 30 min at 37 °C, 5% CO2. Three clinical isolates (5V, 7V and 9V) obtained from female vaginal secretions and one reference strain (ATCC 90028) were used in the study. These strains were separated according to symptoms presented by the patients. Colonization assays were assessed for 2 h incubation at 37 °C, 5% CO2, with 2x105 HeLa cells mL1 treated or not with LAM and 1x107 yeast mL1 of different clinical isolates of C. albicans. After colonization assays, adherent C. albicans cells were harvested by detaching the cervical cells mono- layer to evaluation of viable cells (colony forming units). Results In this study, LAM significantly decreased the interaction of VVC clinical isolates with Hela cells (Figure 1). For ASS and VVC iso- lates, there was a similar reduction in the number of viable cells during colonization process, approximately one log (P < 0.05). Moreover, RVVC isolate showed a reduction more expressive, approximately two log (P < 0.05). Conclusion The pathogenesis of VVC involves the initial adherence of the yeast to the vaginal mucosa, followed by asymptomatic colonisation, ultimately leading to infection (symptomatic vaginitis). This study was able to show that LAM a b-glucan can negatively modulate the process of interaction between HeLa cells and Candida albicans. These results show that this carbohydrate might be a promising agent for preventing the first contact between yeast and vaginal epithelium, and consequently the development of VVC

Propolis potential activity against Candida tropicalis adhered cells and its biofilms

Objectives: Invasive fungal infections, such as candidiasis, represent a public health problem of major importance, and Candida tropicalis has been highlighted among the main agents of candidiasis. One of the major contributions to C. tropicalis virulence is its versatility in adapting to a variety of different habitats and the formation of surface attached microbial communities known as biofilms. Moreover, from the clinical perspective, the most important feature of Candida biofilms is its role in increasing tolerance to conventional antifungal therapy. This scenario encourages the search for alternative therapies. Natural matrixes, such as propolis, compromise a multitude of bioactive properties, in particular phenolic extracts have evidenced significant antimicrobial properties against a multiple of opportunist invaders, including Candida species. Thus, the main objective of the present work was to evaluate the potential antifungal effect of propolis against Candida tropicalis biofilms. Methods: This study was conducted with four strains of C. tropicalis and one reference strain, from the American Type Culture Collection (ATCC 40042). Biofilm formation were carried out on 96-well microplates containing a cellular suspension of 1x105 cells/mL and incubated for 24 h at 37°C. Pre-formed C. tropicalis biofilms were treated with propolis (ranging from 0.47 to 1.42 mg/ml), during 24 h at 37°C and its effect assessed through quantification of the number of colony forming unit (CFU). Results: It was evident that all C. tropicalis strains tested were able to form biofilm and that propolis was able to reduce around 40% and 50% of the pre-formed biofilm. Moreover, in general the propolis effect was similar among all the C. tropicalis clinical isolates strains Conclusions: These data are promising, since they open important perspectives regarding new antifungal agents, much more effective and safer than the currently available to treat and to prevent C. tropicalis infections

Oxidative Stress Triggered by Apigenin Induces Apoptosis in a Comprehensive Panel of Human Cervical Cancer-Derived Cell Lines

Recently, the cytotoxic effects of apigenin (4′,5,7-trihydroxyflavone), particularly its marked inhibition of cancer cell viability both in vitro and in vivo, have attracted the attention of the anticancer drug discovery field. Despite this, there are few studies of apigenin in cervical cancer, and these studies have mostly been conducted using HeLa cells. To evaluate the possibility of apigenin as a new therapeutic candidate for cervical cancer, we evaluated its cytotoxic effects in a comprehensive panel of human cervical cancer-derived cell lines including HeLa (human papillomavirus/HPV 18-positive), SiHa (HPV 16-positive), CaSki (HPV 16 and HPV 18-positive), and C33A (HPV-negative) cells in comparison to a nontumorigenic spontaneously immortalized human epithelial cell line (HaCaT). Our results demonstrated that apigenin had a selective cytotoxic effect and could induce apoptosis in all cervical cancer cell lines which were positively marked with Annexin V, but not in HaCaT (control cells). Additionally, apigenin was able to induce mitochondrial redox impairment, once it increased ROS levels and H2O2, decreased the Δψm, and increased LPO. Still, apigenin was able to inhibit migration and invasion of cancer cells. Thus, apigenin appears to be a promising new candidate as an anticancer drug for cervical cancer induced by different HPV genotypes

Propolis: a potential natural product to fight Candida species infections

Aim: To evaluate the effect of propolis against Candida species planktonic cells and its counterpart's biofilms. Materials & methods: The MIC values, time-kill curves and filamentation form inhibition were determined in Candida planktonic cells. The effect of propolis on Candida biofilms was assessed through quantification of CFUs. Results: MIC values, ranging from 220 to 880 µg/ml, demonstrated higher efficiency on C. albicans and C. parapsilosis than on C. tropicalis cells. In addition, propolis was able to prevent Candida species biofilm's formation and eradicate their mature biofilms, coupled with a significant reduction on C. tropicalis and C. albicans filamentation. Conclusion: Propolis is an inhibitor of Candida virulence factors and represents an innovative alternative to fight candidiasis.The authors thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (Cnpq) and Fundação Araucária for the financial support received. Flávia Tobaldini-Valerio acknowledges the financial support of CAPES – Proc. 9469/14-1. The authors also thank FCT for the Strategic 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). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed

COMPARISON BETWEEN FOUR USUAL METHODS OF IDENTIFICATION OF Candida SPECIES

SUMMARY Infection by Candidaspp. is associated with high mortality rates, especially when treatment is not appropriate and/or not immediate. Therefore, it is necessary to correctly identify the genus and species of Candida. The aim of this study was to compare the identification of 89 samples of Candida spp. by the manual methods germ tube test, auxanogram and chromogenic medium in relation to the ID 32C automated method. The concordances between the methods in ascending order, measured by the Kappa index were: ID 32C with CHROMagar Candida(κ = 0.38), ID 32C with auxanogram (κ = 0.59) and ID 32C with germ tube (κ = 0.9). One of the species identified in this study was C. tropicalis,which demonstrated a sensitivity of 46.2%, a specificity of 95.2%, PPV of 80%, NPV of 81.1%, and an accuracy of 80.9% in tests performed with CHROMagar Candida;and a sensitivity of 76.9%, a specificity of 96.8%, PPV of 90.9%, NPV of 91%, and an accuracy of 91% in the auxanogram tests. Therefore, it is necessary to know the advantages and limitations of methods to choose the best combination between them for a fast and correct identification of Candidaspecies