Evaluation of Antifungal Susceptibility Testing with Microdilution and Etest Methods of <Emphasis Type="Italic">Candida</Emphasis> Blood Isolates

Candida species that show an increasing number of clinical and/or microbiological resistance to several antifungals and are the most common agents of invasive fungal infections. The aim of this study was to investigate the in vitro susceptibility of Candida blood isolates to antifungal agents (amphotericin B, fluconazole, itraconazole, and voriconazole) by comparative use of the CLSI reference microdilution method and Etest. Four hundred Candida blood isolates (215 Candida albicans, 185 non-albicans Candida strains) were included in the study. The broth microdilution test was performed according to the CLSI M27 A2 document. Etest was carried out according to the manufacturer’s instructions. The MIC results obtained with reference microdilution were compared with those obtained with the Etest by using percent and categorical agreements. According to MIK₉₀ values, voriconazole was the most active and itraconazole was the least active drug in vitro against all Candida species. Other than voriconazole, statistically significant differences were found when the susceptibility of Candida albicans and non-albicans Candida spp. to amphotericin B, fluconazole, and itraconazole were compared. These antifungal agents were found to be more active to C. albicans. Among the non-albicans Candida species, the lowest MIC values were obtained for Candida parapsilosis isolates. When the standard method was compared with Etest, the total agreement was higher for C. albicans than for non-albicans species, especially for fluconazole and voriconazole. In view of the findings, it was concluded that itraconazole showed the lowest activity against all Candida species. Etest could be an alternative method in assessing the in vitro antifungal susceptibility of Candida spp., but it is more convenient to use the microdilution method for studying in vitro susceptibility of non-albicans species, in particular for those possessing high MIC values against azoles.     

The carriage of Candida species on the dorsal surface of the tongue: the correlation with the dental, periodontal and prosthetic status in elderly subjects

Objectives: To screen the carriage status of Candida species, especially Candida albicans and its genotype in an epidemiological survey and to investigate its correlation with the dental, periodontal and prosthetic status of healthy elderly subjects. Materials and methods: Microbiological samples were collected from the dorsum of the tongue of 366 subjects, aged 75, and cultured on CHROMagar medium. The carriage status of Candida spp. and the distribution of C. albicans genotypes by a polymerase chain reaction (PCR) method were analysed and compared with the dental, periodontal and prosthetic status of the subjects. Results: A high carriage rate (68.6%) of Candida spp. and the predominant species of C. albicans (72.1%) were found in this study. The prevalence, density and multi‐species of Candida spp. were significantly related to the presence of a dental prosthesis. In C. albicans, genotype A predominated (56.4%) and genotype D showed a higher prevalence (12.5%) than previous reports. When comparing Candida spp. carriage with the oral status, significant positive correlations were found with the presence of any dental prosthesis, missing teeth, the number of retained roots and the percentage of sites showing bleeding on probing (BOP), while significant negative correlations were found with the number of teeth present, sound and filled teeth. Conclusions: Candida carriage on the dorsum of the tongue in healthy elderly is significantly associated with the dental, periodontal and prosthetic status, especially the presence of a dental prosthesis. As the complexity of the prosthesis being worn increased, the relative risk of Candida carriage and the numbers and multi‐species of Candida increased accordingly.     

Effect of growth of Candida spp. in the presence of various glucocorticoids on the adherence to human buccal epithelial cells

In vitro culturing of three different yeast species with a number of glucocorticoids altered their adherence ability in two ways: Incubation with dexamethasone and triamcinolone acetonide promoted the adherence in general (the increase in adherence ranged between 17% and 44%), whilst growth in the presence of cortisone acetate or hydrocortisone blocked the adherence (inhibition ranged from 16% to 32%). No statistical difference in the adherence capabilities of different growth phases of C. albicans was noted, and the effects of glucocorticoids persisted irrespective of the phase of growth used. An attempt to explain the differences in adherence of the Candida spp. investigated, in the presence of various steroids, on the basis of variation in their structural configurations and/or steroid-receptor interaction is given.     

Effect of antineoplastic agents and X-irradiation on the adherence of Candida spp. to human buccal epithelial cells in vitro

The role of chemotherapy, X-irradiation and a combination of both on the phenomenon of adherence of yeast to buccal epithelial cells (BEC) was investigated in vitro. Growth of three Candida spp. in the presence of eight of eleven antineoplastic agents led to reduction of adherence of the isolates tested (reduction between 30% and 61% of the control value), and this effect was observed whether exponential or stationary phase Candida cells were used. Exposure of C. albicans to various doses of radiation also led to a reduction in adherence of this yeast to BEC between 31% and 53% of the control value. This reduction was shown to be dose related. Similar results were obtained when BEC were exposed to radiation, and the effect of radiation treatment was accentuated when both yeast and BEC were irradiated simultaneously. Furthermore, treating C albicans with a combination of chemotherapy and radiation led to the greatest reduction in adherence of yeast to BEC compared to when the yeast was treated with either chemotherapy or radiation alone (reduction between 63% to 74% as compared with control). The possible mechanism/s involved in reduction of adherence of yeast to BEC are discussed.     

Action mechanisms of probiotics on Candida spp. and candidiasis prevention: an update

Due to the high incidence of fungal infections caused by Candida species and their increasing resistance to antimicrobial treatments, alternative therapies such as probiotics have been studied. It has been show that several species of the genus Lactobacillus have anti-Candida activity, probably by direct inhibition, through competition for adhesion sites or production of secondary metabolites, and by indirect inhibition, through stimulation of the immune system of their host. However, the mechanisms of inhibition of these probiotics on Candida species have not yet been fully elucidated since this effect is related to more than one inhibition pathway. In the literature, several in vitro and in vivo studies have been developed seeking to elucidate the probiotics mechanisms of action. These studies have been focused on C. albicans inhibition assays, including analysis of antimicrobial activity, adherence capacity, biofilms formation, filamentation and interference on virulence genes, as well as assays of experimental candidiasis in invertebrate and vertebrate models. In this context, the purpose of this review was to gather different studies focused on the action mechanism of probiotic strains on Candida sp. and to discuss their impact on the candidiasis prevention.     

Systemic Candida infection in University Hospital 1997–1999: the distribution of Candida biotypes and antifungal susceptibility patterns

A total of 102 Candida species were isolated from blood cultures from January 1997 to October 1999. Using assimilation of carbohydrate test, 52 (51.0%) of the Candida sp. were identified as C. parapsilosis, 25.5% (26) were C. tropicalis. C. albicans made up 11.8% (12), 6.9% (7) were C. rugosa, 3.8% (4) C. glabrata and 1% (1) C. guilliermondii. No C. dubliniensis was found in the study. In vitro antifungal susceptibility tests showed that all Candida species were sensitive to nystatin, amphotericin B and ketoconazole. Although all isolates remained sensitive to fluconazole, intermediate susceptibility was found in 3 C. rugosa isolates. Antifungal agents with high frequency of resistance were econazole, clotrimazole, miconazole and 5-fluorocytosine. Candida species found to have resistance to these antifungal agents were non-C. albicans. This revised version was published online in June 2006 with corrections to the Cover Date.     

Candida-associated denture stomatitis

Candida-associated denture stomatitis was demonstrated by its cultivation in 171 out of 240 patients examined with partial or total dentures. After taking smears from lesions of the oral mucosa (tongue, cheeks, palate) and the contiguous denture surface by cotton wool swabs and inoculating them onto Sabouraud glucose agar and CHROMagarCandida, individual yeast species were identified by a germ tube. filamentous, and assimilation tests employing the commercial kit AuxaColor. SevenCandida species were identified in smears from the oral mucosa lesions and the contiguous denture surface:C. albicans (95 patients),C. tropicalis (26).C. parapsilosis (20),C. krusei (14),C. guilliermondii (12),C. lusitaniae (1) andC. freyschusii (1). Diabetes mellitus, neoplastic diseases, chemotherapy, radiotherapy, and broad-spectrum antibiotic therapy were identified as some of the large number of factors predisposing patients to stomatitis prothetica.     

Molecular Identification of <Emphasis Type="Italic">Candida orthopsilosis</Emphasis> Isolated from Blood Culture

The incidence of candidemia and invasive candidiasis have increased markedly due to the increasing number of immunocompromised patients. There are five major medically important species of Candida with their frequency of isolation in the diminishing order namely Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata and Candida krusei. In addition, there are numerous other species of Candida which differ in their genetic makeup, virulence properties, drug susceptibilities and sugar assimilation capabilities. In this report, an unusual Candida species was isolated from the blood of two leukaemic patients. Conventional culture and biochemical tests identified the Candida species as C. parapsilosis. Using fungal-specific oligonucleotide primers ITS1 and ITS4, we managed to amplify the ribosomal RNA gene and its internal transcribed spacer region from the genomic DNA of these isolates. The PCR products were then purified and subjected to automated DNA sequencing using BLAST and CLUSTAL sequence analysis identified these isolates to be Candida orthopsilosis. Candida orthopsilosis is a new species recently identified in 2005, being morphologically indistinguishable from C. parapsilosis and was previously classified as a subspecies of C. parapsilosis. This report highlights the importance of complementing traditional culture and biochemical-based identification methods with DNA-based molecular assays such as PCR as the latter is more superior in terms of its discriminatory power and speed.     

Pigmentation and autofluorescence of Cryptococcus species after growth on tryptophan and anthranilic acid media

Cryptococcus neoformans and Candida albicans produced a pink pigment from media containing tryptophan. Approximately 30% of the C. neoformans strains produced large amounts of the pink (purple after 6 days) pigment in the absence of light whereas 70% of the Cryptococcus neoformans strains, as well as C. laurentii, C. albidus, C. diffluens, and C. albicans also produced the pink pigment with light being required for significant early production (2–6 days). Significant production did occur for Cryptococcus but not Candida species in the dark after extended incubation (10–25 days). C. terreus produced brown pigments from tryptophan and C. luteolus produced a trace of a buff pigment. Most Candida species produced either pink or brown pigments but not both. In contrast, many Cryptococcus species producing the pink pigment simultaneously produced brown pigments. C. terreus, C. albidus, and C. diffluens produced brown pigments from anthranilic acid whereas C. neoformans, C. laurentii, C. luteolus, and the medically important Candida species did not produce significant amounts of pigments from anthranilic acid. Cryptococcus and Candida species were autofluorescent when tryptophan was a major nitrogen source whereas yeast cell autofluorescence was not observed when anthranilic acid was used. Pigmentation of some Cryptococcus species also the substrate.Operated for the U.S. Department of Energy under contract number EY-76-C-05-0033This article is based on work supported by the Division of Biomedical and Environmental Research.     

Interactions ofCandida albicans with bacteria and salivary molecules in oral biofilms

The yeastCandida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells.C. albicans andCandida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind toStreptococcus gordonii NCTC 7869 while two otherCandida species (Candida krusei andCandida kefyr) do not. Adherence ofC. albicans was greatest when the yeast had been grown at 30° C to mid-exponential growth phase. For 21 strains ofC. albicans there was a positive correlation between the ability to adhere toS. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence ofC. albicans toS. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins ofC. albicans are co-expressed.     

Purification and partial characterization of a fibrinolytic enzyme from the fruiting body of the medicinal and edible mushroom Pleurotus ferulae

A fibrinolytic metalloprotease with in vitro fibrinolytic effects was purified from the edible mushroom Pleurotus ferulae using several chromatography steps including anion and ion exchange, gel filtration, and fast protein liquid chromatography columns. The molecular mass of the enzyme was estimated to be 20.0?kDa, as determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fibrin zymography. The protease was active at 50°C, and pH 4.0, 5.0, and 8.0. The fibrinolytic activity of the enzyme was inhibited by ethyleneglycol-bis-(2-aminoethyl)-N,N,N′,N′ tetraacetic acid and strongly inhibited by two metal ions, Cu and Mg. In vitro assays evaluating fibrinolytic activity on a fibrin plate, fibrin turbidity, and thrombolytic activity on fibrin clots using human fibrinogen and human thrombin revealed that the enzyme could hydrolyze fibrin polymers directly and inhibit the formation of fibrin clots. In activated partial thromboplastin time (APTT) and prothrombin time assays, the enzyme strongly prolonged the APTT, which detects an activity of intrinsic and common pathways. The enzyme showed strong in vivo protective effect against mortality/paralysis from epinephrine plus collagen-induced acute thromboembolism in in vivo model. Our findings suggest that the enzyme may have a potential for treatment and prevention of thrombosis-relative diseases.     

Fecal fungal flora of pediatric healthy volunteers and immunosuppressed patients

Most hematogenous candidiasis originates from endogeneous host flora. Fungal flora of gastrointestinal system are important source of infection especially in immunosupressed patients. The purpose of this study was to investigate the fecal fungal flora of pediatric patients with hematologic malignancy or disorders and to compare the results with healthy volunteers. For this purpose, fungal etiological agents were investigated retrospectively in stool samples of 80 patients followed in Bone marrow transplantation and Hematology–Oncology units. The diagnosis of patients were as follows: 26 acute myelogeneous leukemia, 19 acute lymphocytic leukemia, 5 lymphoma, 3 chronic myelogeneous leukemia, 2 solid tumor, 4 neuroblastoma and 21 hematologic disorders. In patients, totally 102 fungal growth was detected and 42 (41.2%) C. albicans and 51 (50%) non-albicans Candida species and 9 (8.8%) yeast other than Candida and mould was isolated. The results were compared prospectively with growth in stool samples of 61 healthy children. C. albicans was detected in 16 (43.2%) and non-albicans Candida species in 15 (40.5%) and yeasts other than Candida and mould in 6 (16.2%) of 37 fungal growth in controls. Non-albicans Candida species growth was found significantly higher and C. glabrata was more prevelant in patients than in controls (p < 0.001).     

Comparison of PCR-RFLP with 21-plex PCR and rDNA Sequencing for Identification of Clinical Yeast Isolates

Non-albicans Candida species and other rare yeasts have emerged as major opportunistic pathogens in fungal infections. Identification of opportunistic yeasts in developing countries is mainly performed by phenotypic assay, which are time-consuming and prone to errors. The aim of the present study was to evaluate PCR-RFLP as a routinely used identification technique for the most clinically important Candida species in Iran and make a comparison with a novel multiplex PCR, called 21-plex PCR. One hundred and seventy-three yeast isolates from clinical sources were selected and identified with sequence analysis of the D1/D2 domains of rDNA (LSU rDNA) sequencing as the gold standard method. The results were compared with those obtained by PCR-RFLP using MspI restriction enzyme and the 21-plex PCR. PCR-RFLP correctly identified 93.4% of common pathogenic Candida species (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and P. kudriavsevii (=?C. krusei)) and was able to identify 45.5% of isolates of the uncommon yeast species compared to the D1/D2 rDNA sequencing. Compared with PCR-RFLP, all common Candida species and 72.7% of uncommon yeast species were correctly identified by the 21-plex PCR. The application of the 21-plex PCR assay as a non-sequence-based molecular method for the identification of common and rare yeasts can reduce turnaround time and costs for the identification of clinically important yeasts and can be applied in resource-limited settings.

     

Correlation between factors associated with the removable partial dentures use and Candida spp. in saliva

doi: 10.1111/j.1741‐2358.2010.00390.x
Correlation between factors associated with the removable partial dentures use and Candida spp. in saliva Objectives: To correlate the presence and number of Candida spp. in the saliva of wearers of removable partial dentures retained with precision attachments with the proportion of metal/acrylic resin present in the dentures. Methods: Saliva samples from 40 removable partial denture wearers (test) and one paired sample of individuals, non‐wearers of any type of removable denture (control) were collected, seeded, and the colony forming units of Candida counted and identified. The metal/acrylic resin proportion of each denture was quantified, using silicone plates pressed over each denture. Results: Candida spp. was found in the saliva of 80% of the individuals in the test group and 65% of the control, with C. albicans being the most prevalent species. The test group presented with the highest number of colony forming units of Candida per ml of saliva, and there was weak correlation between this number and the metal and resin area of the denture (Pearson’s coefficient of correlation). Greater prevalence and a higher number of colony forming units of Candida per ml of saliva occurred in removable partial denture wearers (p = 0.04) with a weak positive correlation between the metal and resin area and the number of colony forming units of Candida per ml of saliva. However, this correlation was more significant for the area of resin.     

Species Distribution and Antifungal Susceptibility Profile of Oral Candida Isolates from HIV-infected Patients in the Antiretroviral Therapy Era

In this study, we investigated the yeasts colonization of genus Candida, including C. dubliniensis, isolated of HIV-infected patients oral cavities and we accessed in vitro susceptibility pattern of the Candida isolates to four antifungal agents. Out of 99 patients investigated, 62 (62.6%) were colonized with yeasts. C. albicans was the prevailing species (50%). C. dubliniensis isolates were not recovered in our study. We verified that 8.1% of the yeasts isolated were resistant to fluconazole, 8.1% to itraconazole and 3.2% to voriconazole. The isolates demonstrated very low voriconazole MICs, in which 79% (49/62) presented values of 0.015 μg/ml. All Candida isolates were susceptible to amphotericin B. The results reported here showed that although C. albicans continues to be present in one-half of oral Candida carriage of HIV-infected patients, Candida non-albicans species are increasing among these patients. Besides, the findings of resistant isolates endorse the role of antifungal susceptibility testing whenever antifungal treatment with azoles is planned.     

Experiments on in vivo biofilm formation and in vitro adhesion of Candida species on polysiloxane liners

doi:10.1111/j.1741‐2358.2009.00329.x
Experiments on in vivo biofilm formation and in vitro adhesion of Candida species on polysiloxane liners Objectives: Microorganisms may colonise polysiloxane soft liners leading to bio‐deterioration. The aim of this study was to investigate in vitro adhesion and in vivo biofilm formation of Candida species on polysiloxane surfaces. Methods: The materials used in this study were Molloplast B, GC Reline soft, Mollosil Plus, Silagum Comfort and Palapress Vario. The in vitro retention of clinical isolates of Candida albicans to the relining and denture‐base materials by microscopic (scanning electron microscopy, SEM), conventional culturing methods and antimicrobial properties of these materials were studied. Candida found on materials and mucosa following long‐term use were identified and quantified, and biofilms covering the surfaces were investigated by SEM. Results: There was a significant decrease in the number of cells attached in vitro to saliva‐coated surfaces compared with non‐treated surfaces. An oral Candida carriage of 78% was found. Candida albicans, C. glabrata, C. intermedia and C. tropicalis were identified. In vivo biofilm formation on the liners appeared as massive colonisation by microorganisms. Conclusions: The results of the in vitro experiments suggest that salivary film influences early colonisation of different C. albicans strains. The film layer also minimises the differences among different strains. The Candida carriage of these patients was similar to denture‐wearing patients without soft liners.     

Fungal diseases of the respiratory tract

The proportion ofCandida and non-Candida species in the clinical material from patients. with respiratory-tract diseases was determined.C. albicans was isolated in 102 cases. An additional 89 strains of yeasts, isolated in association with respiratory diseases, belonged to 10 non-albicans Candida spp. andCryptococcus spp. The prevailing species, which occurred in 47 cases, wasC. parapsilosis. C. tropicalis, C. glabrata, andC. guilliermondii were isolated in 12, 10, and 9 cases, respectively. Four strains ofC. krusei and three strains ofC. lusitaniae and one strain each ofC. freyschussii, C. robusta, C. zeylanoides, andCryptococcus neoformans were also isolated.     

Phenotypic variability and therapeutic implications of Candida species in patients with oral lichen planus

We investigated the prevalence and phenotypic variation of Candida species in oral lichen planus (OLP) and the therapeutic implications of our findings. Eighty patients with clinically and histopathologically confirmed cases of OLP (64 non-erosive, 16 erosive) and a control group of 80 healthy individuals with no predisposing factors for oral candidiasis were examined for evidence of Candida infection. Oral swabs and smears were obtained for cytology and culture. Identification, speciation and antifungal susceptibility tests of Candida isolates were performed using an automated microbial identification system. Fifty percent of erosive OLP cases, 28% of non-erosive cases and none of the controls showed evidence of Candida. Candida albicans was found predominantly in non-erosive OLP, while other Candida species were predominate in erosive OLP. Non-Candida albicans isolates (C. glabrata, C. krusei) were resistant to the commonly used antifungals, clotrimazole and fluconazole. Candida infection is common in cases of OLP. We recommend antifungal sensitivity testing prior to antifungal therapy for the erosive form of OLP.     

Oxidation-induced destabilization of the fibrinogen αC-domain dimer investigated by molecular dynamics simulations

Upon activation, fibrinogen is converted to insoluble fibrin, which assembles into long strings called protofibrils. These aggregate laterally to form a fibrin matrix that stabilizes a blood clot. Lateral aggregation of protofibrils is mediated by the αC domain, a partially structured fragment located in a disordered region of fibrinogen. Polymerization of αC domains links multiple fibrin molecules with each other enabling the formation of thick fibrin fibers and a fibrin matrix that is stable but can also be digested by enzymes. However, oxidizing agents produced during the inflammatory response have been shown to cause thinner fibrin fibers resulting in denser clots, which are harder to proteolyze and pose the risk of deep vein thrombosis and lung embolism. Oxidation of Met⁴⁷⁶ located within the αC domain is thought to hinder its ability to polymerize disrupting the lateral aggregation of protofibrils and leading to the observed thinner fibers. How αC domains assemble into polymers is still unclear and yet this knowledge would shed light on the mechanism through which oxidation weakens the lateral aggregation of protofibrils. This study used temperature replica exchange molecular dynamics simulations to investigate the αC-domain dimer and how this is affected by oxidation of Met⁴⁷⁶. Analysis of the trajectories revealed that multiple stable binding modes were sampled between two αC domains while oxidation decreased the likelihood of dimer formation. Furthermore, the side chain of Met⁴⁷⁶ was observed to act as a docking spot for the binding and this function was impaired by its conversion to methionine sulfoxide.     

Synergy of human neutrophils with fluconazole in killing Candida species

The killing of Candida species by human neutrophils in a long-term 24-h assay and possible synergy with fluconazole (FCZ) for killing was investigated. The test medium (TM) consisted of RPMI-1640, penicillin and streptomycin (P/S), and 10% fresh autologous serum. TM alone was highly fungistatic for Candida species compared to TM without serum. When neutrophils were cocultured in TM with Candida species for 24 h the inoculum colony-forming units (CFU) were always significantly reduced (killing) by 58 to 99%. FCZ was tested over a range of 1–500 g/ml, and though almost always fungistatic itself, it synergized with neutrophils for significantly increased killing of C. albicans (isolate Sh27) (P<0.01) and C. albicans (isolate 94-20) (P<0.05). Killing of non-albicans Candida species was so efficient in the absence of FCZ that demonstration of synergy with FCZ was difficult.