Detection of <Emphasis Type="Italic">Candida</Emphasis><Emphasis Type="Italic">dubliniensis</Emphasis> in Venezuela

Over the past decades there has been a significant increase in fungal infections caused by Candida species, and continues to be common in immunocompromised individuals infected with the human immunodeficiency virus (HIV). Although Candida albicans remains the fungal species most frequently isolated as an opportunistic oral pathogen, other non-albicans are often identified in this cohort of patients, including C. dubliniensis. This yeast is closely related to and shares many phenotypic characteristics with C. albicans. Colonies of these two species appear morphologically identical when not grown on special media. The shared phenotypic characteristics of C. dubliniensis and C. albicans suggest that many C. dubliniensis isolates may have been misidentified as C. albicans in the past. The present studies aim is to recover and identify C. dubliniensis, and presumptive clinical C. albicans, from the oral cavities of HIV-seropositive individuals, comparing conventional media to obtain a simple, low-cost and reliable identification system for C. dubliniensis. A total of 16 isolates (3,98%) had been obtained from 402 HIV infected individuals with recurrent oropharyngitis and were identified as C. dubliniensis. Out of these C. dubliniensis isolates 19% were resistant, with MICs above 64 μg/ml to fluconazole. This constitutes, to the authors knowledge the first recovery of this organism in Venezuela.     

Isolation of <Emphasis Type="Italic">Candida dubliniensis</Emphasis> for the First Time in Cali,Colombia, and its Identification with Phenotyping Methods

Summary   Candida dubliniensis is an emerging pathogenic yeast isolated mainly from the oral cavity of HIV-infected patients. The close phenotypic and genotypic relationship between C. albicans and C. dubliniensis has led to incorrectly identifying isolates of C. dubliniensis as C. albicans. The oral cavities of 107 diabetic patients were studied in Cali, Colombia, and 72 colonies of Candida, with shades of green on CHROMagar Candida culture media, were obtained. Various phenotypic tests were carried out, which included germ tube formation and production of chlamydospores on corn meal Agar. Additionally, growth studies were carried out at 42°C and 45°C and on Sabouraud agar with 6.5%, sodium chloride. Identification of C. dubliniensis with these tests was confirmed with API 20C Aux. We identified 65 and 7 colonies of C. albicans and C. dubliniensis, respectively. This is the first time that C. dubliniensis is identified with phenotypic methods in Colombia.     

DRBC agar: a new tool for <Emphasis Type="Italic">Candida dubliniensis</Emphasis> identification

Candida dubliniensis pathogenic species, which shares many phenotypic features with C. albicans, may be misidentified in the microbiology laboratory. The growth on DRBC agar at 25 °C was shown to be a new tool for differentiation between C. dubliniensis and C. albicans. All 27 isolates of C. dubliniensis showed in this medium rough colonies (peripheral hyphal fringes) and abundant chlamydospore production, while all 103 isolates of C. albicans showed smooth colonies without fringes or chlamydospores. DRBC agar allowed the differentiation of C. albicans from C. dubliniensis with 100 % sensitivity and specificity.     

Lack of Candida africana and Candida dubliniensis in vaginal Candida albicans isolates in Turkey using HWP1 gene polymorphisms

Candida africana differs from the common strains of C. albicans and C. dubliniensis morphologically, physiologically, genetically, and, in particular, clinically. This fungal pathogen is primarily recovered from genital specimens, especially in vaginal specimens. In this investigation, we reexamined 195 vaginal C. albicans isolates for the presence of C. africana and C. dubliniensis by using hyphal wall protein 1 (HWP1) gene polymorphisms. All study isolates were confirmed to be C. albicans, and none were verified as either C. africana or C. dubliniensis. In conclusion, the HWP1 gene polymorphisms offer a useful tool in the discrimination of C. africana, C. albicans, and C. dubliniensis. Further studies may highlight the pathogenesis and importance of this yeast in vulvovaginal candidiasis.     

Response to Oxidative Stress in Eight Pathogenic Yeast Species of the Genus <Emphasis Type="Italic">Candida</Emphasis>

In the course of an infection, the formation of reactive oxygen species by phagocytes and the antioxidant defense mechanisms of microorganisms play a crucial role in pathogenesis. In this study, isolates representing 8 pathogenic Candida species—Candida albicans, Candida dubliniensis, Candida famata, Candida glabrata, Candida guilliermondii, Candida krusei, Candida parapsilosis and Candida tropicalis—were compared with regard to their resistance to oxidative stress in vitro. We evaluated degree of resistance, induction of oxidative damage, capacity to adapt, and induction of antioxidant enzymes. The species showed variable sensitivity to oxidative attack. C. albicans, C. glabrata, and C. krusei were more resistant to oxidative stress under the conditions tested; C. parapsilosis and C. tropicalis presented medium resistance; and C. dubliniensis, C. famata, and C. guilliermondii were more sensitive. The overall greater resistance to oxidative stress of C. albicans and C. glabrata may provide an advantage to these species, which are the major causative agents of candidiasis.     

Improved DNA‐FISH for cytometric detection of Candida spp

Aims: We developed improved methods for DNA‐based fluorescence in situ hybridization (FISH) for rapid detection of Candida spp. and Candida albicans via flow cytometry. Methods and Results: Two previously reported C. albicans‐targeted DNA probes were evaluated against whole cells of C. albicans and related Candida species using a rapid, high‐temperature hybridization protocol. One probe (CalB2208) was shown for the first time to be suitable as a FISH probe. Although cell labelling for both probes was relatively bright, we were able to substantially improve our results by altering fixation and hybridization conditions. For fixation, a 60 : 40 mixture of 10% buffered formalin and ethanol was most effective. Probe intensity was improved as much as ten‐fold through the use of unlabelled helper probes, and buffer containing 0·9 mol l^?1 NaCl plus 10% formamide yielded the best hybridizations for both probe/helper cocktails. Although optimal labelling occurred with longer hybridizations, we found that C. albicans could be completely differentiated from the nontarget yeast Rhodotorula glutinis after only 15 min using the brightest probe (Calb‐1249) and that a formal washing step was not required. Specificities of probe/helper cocktails under optimal conditions were determined using a panel of target and nontarget cell types, including four strains of Candida dubliniensis. Calb‐1249 cross‐reacted slightly with Candida parapsilosis and strongly with both Candida tropicalis and C. dubliniensis. In contrast, we found that CalB2208 was exclusive for C. albicans. The molecular basis of this specificity was confirmed by DNA sequencing. Conclusions: We describe DNA probe‐based approaches for rapid and bright labelling of Candida spp. and for specific labelling of C. albicans without cross‐reaction with C. dubliniensis. Our work improves upon previously described methods. Significance and Impact of the Study: The methods described here for rapid FISH‐based detection of Candida spp. may have applications in both clinical and food microbiology.     

<Emphasis Type="Italic">Candida dubliniensis</Emphasis>: Epidemiology and Phenotypic Methods for Identification

Candida dubliniensis is an emerging pathogen first described in 1995, which shares many phenotypic features with Candida albicans and therefore may be misidentified in microbial laboratories. Despite various phenotypic techniques described in the literature to differentiate the two species, the correct identification of C. dubliniensis remains problematic due to phenotypic similarities between these species. Thus, as the differences between both are best characterized at genetic levels, several molecular methods have been proposed to provide a specific and rapid identification of this species. Epidemiological studies have shown that C. dubliniensis is prevalent throughout the world and it is primarily associated with oral carriage and oropharyngeal infections in patients infected with human immunodeficiency virus (HIV). However, data acquired from its isolation from other healthy and immunocompromised patients are variable, and there is still no real consensus on the epidemiological relevance of this species. In this article, we review the various phenotypic methods used in the identification of C. dubliniensis and the epidemiological impact of this new species.     

First Isolation of <Emphasis Type="Italic">Candida dubliniensis</Emphasis> from Oral Cavities of Dermatological Patients in Nanjing,China

Candida dubliniensis is an emerging pathogen capable of causing both superficial and systemic infections. Although C. dubliniensis and C. albicans are phenotypically similar, the two species differ in terms of epidemiology and the ability to rapidly develop resistance to fluconazole. C. dubliniensis is primarily associated with oral candidiasis of human immunodeficiency virus (HIV)-infected individuals. In this study, we describe the first recovery of C. dubliniensis from oral cavities of non-HIV-infected patients with dermatological diseases in Nanjing, China. The isolates were phenotypically characterized as C. dubliniensis by their production of brown rough colonies and chlamydospores on tobacco agar and their inability to grow on hypertonic Sabouraud dextrose agar or to assimilate xylose or α-methyl-d-glycoside. The species identification was subsequently confirmed by amplification and sequencing of the internal transcribed spacer region (ITS). Three C. dubliniensis isolates out of 128 (2.3%) presumptive C. albicans/C. dubliniensis ones were finally identified. Further sequence analysis separated the three isolates into two of the four reported ITS genotypes. Antifungal susceptibility testing showed that they were susceptible to fluconazole, itraconazole, voriconazole, micafungin, and amphotericin B. This study adds to the accumulating evidence that C. dubliniensis is widely distributed in non-HIV-infected populations worldwide.     

Biofilm formation and adhesive/invasive properties of <Emphasis Type="Italic">Candida dubliniensis</Emphasis> in comparison with <Emphasis Type="Italic">Candida albicans</Emphasis>

Candida dubliniensis and Candida albicans are closely related spp. exhibiting differences in their virulence potency. This study compared clinical isolates of C. dubliniensis with C. albicans from HIV patients with oropharyngeal candidiasis (OPC) and standard strains in power to form biofilm and their adhesive and invasive properties. Members of both spp. were able to form strong biofilms. However, SEM microscopy confirmed that C. albicans undergoes the more effective yeast-to-hyphae transition than C. dubliniensis with prevalent yeast form and limited ability to form filaments. Kinetic patterns indicated that while the first 30 min are critical for sufficient attachment to a polystyrene surface, adhesion to human carcinoma cell lines (Caco-2 and TR 146) needs additional time with maximal saturation observed at 240 min for both spp. The invasion process was tested on 3D RHE (reconstituted human epithelium) with Caco-2 or TR 146 on the collagen surface. C. albicans rapidly produced hyphae that penetrated the tissue layer, demonstrating substantive invasion within 21 h. In contrast, C. dubliniensis attached to the tissue surface and proliferated, suggesting the formation of a biofilm-like structure. After 21 h, C. dubliniensis was able to penetrate the RHE layer and invade unusually, with a cluster of the yeast cells.     

Heteroduplex mobility assay of the 26S rDNA D1/D2 region for differentiation of clinically relevant Candida species

The Heteroduplex Mobility Assay (HMA) method using the PCR amplified D1/D2 region of the 26S rDNA was tested for the differentiation of clinically relevant Candida species. Strains belonging to the same species are not expected to form heteroduplexes in this assay when their PCR products are mixed. D1/D2 HMA experiments between all Candida type strains tested showed heteroduplex formation, including Candida albicans and Candida dubliniensis. There was no heteroduplex formation when most clinical and non-type strains were tested against the type strain of their presumptive species, except when C. albicans WVE and C.␣dubliniensis TAI were analysed. Additional HMA experiments, phenotypic characterisation, and D1/D2 sequencing identified these isolates as Candida tropicalis and Candida parapsilosis, respectively. HMA provides a rapid and relatively simple molecular tool for the differentiation of potentially pathogenic Candida species.     

Hypertonic Sabouraud Dextrose Agar as a Substrate for Differentiation of Candida dubliniensis

In this study, we aimed to detect the proportion of Candida dubliniensis among yeast strains previously identified as C. albicans by using several phenotypic methods and PCR. For this purpose, we screened 300 strains by using phenotypic tests suggested for the identification of C. dubliniensis in the literature, but we detected high proportion of false-positive reactions. Only two strains (0.6%) were detected as true C. dubliniensis by PCR and API ID 32C methods. Moreover, these two strains gave the expected results with all the phenotypic tests, including modified salt tolerance test for C. dubliniensis. In conclusion, none of the phenotypic methods, except for the modified salt tolerance test, revealed 100% successful results in discrimination of C. albicans and C. dubliniensis species. However, in the tobacco agar test, the rate of false positivity was as low as 0.6%. We suggest that in the case of absence of PCR and other automatized identification systems, these two phenotypic tests can be used in routine laboratories to obtain a presumptive result.     

Four novel <Emphasis Type="Italic">Candida</Emphasis> species in the <Emphasis Type="Italic">Candida albicans</Emphasis>/<Emphasis Type="Italic">Lodderomyces elongisporus</Emphasis> clade isolated from the gut of flower beetles

Flower-visiting beetles belonging to three species of Cetoniidae were collected on three mountains near Beijing, China, and yeasts were isolated from the gut of the insects collected. Based on the 26S rDNA D1/D2 domain and internal transcribed spacer (ITS) region sequence analysis and phenotypic characterization, four novel anamorphic yeast species located in the Candida albicans/Lodderomyces elongisporus clade were identified from 18 of the strains isolated. The new species and type strains are designated as Candida blackwellae AS 2.3639^T (=CBS 10843^T), Candida jiufengensis AS 2.3688^T (=CBS 10846^T), Candida oxycetoniae AS 2.3656^T (=CBS 10844^T), and Candida pseudojiufengensis AS 2.3693^T (=CBS 10847^T). C. blackwellae sp. nov. was basal to the branch formed by C. albicans and C. dubliniensis with moderately strong bootstrap support. The closest relative of C. oxycetoniae was L. elongisporus. C. jiufengensis sp. nov. and C. pseudojiufengensis sp. nov. were closely related with each other and formed a branch in a subclade represented by C. parapsilosis and L. elongisporus.     

Morphogenesis control in Candida albicans and Candida dubliniensis through signaling molecules produced by planktonic and biofilm cells

Morphogenesis control by chemical signaling molecules is beginning to be highlighted in Candida biology. The present study focuses on morphogenic compounds produced in situ by Candida albicans and Candida dubliniensis during planktonic and biofilm growth that may at least partially substantiate the effect promoted by supernatants in morphogenesis. For both species, planktonic versus biofilm supernatants were analyzed by headspace-solid-phase microextraction and gas chromatography-mass spectrometry. Both planktonic cells and biofilm supernatants of C. albicans and C. dubliniensis contained isoamyl alcohol, 2-phenylethanol, 1-dodecanol, E-nerolidol, and E,E-farnesol. Alcohol secretion profiles were species, culture mode, and growth time specific. The addition of exogenous alcohols to the cultures of both species inhibited the morphological transition from the yeast to the filamentous form by up to 50%. The physiological role of these alcohols was put to evidence by comparing the effects of a 96-h cultured supernatant with synthetic mixtures containing isoamyl alcohol, 2-phenylethanol, E-nerolidol, and E,E-farnesol at concentrations determined herein. All synthetic mixtures elicited a morphological effect similar to that observed for the corresponding supernatants when used to treat C. albicans and C. dubliniensis cultures, except for the effect of the 96-h C. dubliniensis planktonic supernatant culture on C. albicans. Overall, these results reveal a group of alcohol extracellular signaling molecules that are biologically active with C. albicans and C. dubliniensis morphogenesis.     

Prevalence of <Emphasis Type="Italic">Candida dubliniensis</Emphasis> Fungemia in Argentina: Identification by a Novel Multiplex PCR and Comparison of Different Phenotypic Methods

Candida dubliniensis is an emerging pathogen that can cause invasive disease in patients who have a variety of clinical conditions. C. dubliniensis is often misidentified as Candida albicans by clinical laboratories. In Argentina, incidence data are still scarce, and only one systemic infection has been reported. This study aims to determine the prevalence of C. dubliniensis in blood samples in Argentina, to evaluate a novel PCR multiplex as well as several phenotypic methods for the identification of this yeast, and to know the susceptibility profile of isolates against seven antifungal drugs. We have found that prevalence in Argentina appears to be lower than that reported in other countries, occurring only in 0.96% of the Candidemia cases recovered in 47 hospitals during a 1-year period. All C. dubliniensis clinical isolates included in this study were genetically identical when comparing ITS genes sequences. This is in agreement with the previous studies suggesting little genetic variation within this species. The novel multiplex PCR proved to be 100% sensitive and specific for the identification of C. dubliniensis. Therefore, we propose its use as a rapid and inexpensive method for laboratories having access to molecular techniques. Although no single phenotypic test has proved to be infallible, both colony morphology on tobacco agar, as well as abundant chlamydospore formation on both tobacco agar and on sunflower seed agar, may be used as a presumptive differentiation method in routine mycology laboratories. It has been suggested that C. dubliniensis may have higher propensity to develop azole antifungal drug resistance than C. albicans. In this study, one of the five clinical isolates of C. dubliniensis was resistant to fluconazole.     

<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> inhibits <Emphasis Type="Italic">in-vitro Candida</Emphasis> biofilm development

Background  

Elucidation of the communal behavior of microbes in mixed species biofilms may have a major impact on understanding infectious diseases and for the therapeutics. Although, the structure and the properties of monospecies biofilms and their role in disease have been extensively studied during the last decade, the interactions within mixed biofilms consisting of bacteria and fungi such as Candida spp. have not been illustrated in depth. Hence, the aim of this study was to evaluate the interspecies interactions of Pseudomonas aeruginosa and six different species of Candida comprising C. albicans, C. glabrata, C. krusei, C. tropicalis, C. parapsilosis, and C. dubliniensis in dual species biofilm development.     

Methods of <Emphasis Type="BoldItalic">Candida dubliniensis</Emphasis> identification and its occurrence in human clinical material

Candida dubliniensis was reported as a new species in 1995. This species is often misidentified as Candida albicans. The aims of this work were to determine the occurrence of C. dubliniensis in various clinical materials, to evaluate several ways to identify it and to examine the genetic variability of isolates. Among 7706 isolates originally identified as C. albicans, 237 were identified as C. dubliniensis (3.1%). Most of the C. dubliniensis isolates were obtained from the upper and lower respiratory tract (61.4 and 22.9%). Five phenotypic methods including latex agglutination were used (cultivation on CHROMagar Candida, on Staib agar, at 42 °C and in medium with 6.5% NaCl), but only cultivation on the medium with an increased concentration of NaCl and latex agglutination gave reliable results. Species-specific polymerase chain reaction was used as the confirmation method. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry provided less reliable results. In fact, 78.9% of C. dubliniensis isolates had scores above 1.7. However, the rest of them (21.1%) were also identified as C. dubliniensis even when the scores were lower than 1.7. Divergences among C. dubliniensis strains were evaluated by means of pulsed-field gel electrophoresis. Eighty-six selected C. dubliniensis isolates showed a 69.6% level of similarity. The results of this study expand the knowledge of the incidence, means of identification and genotypic divergence of C. dubliniensis isolates.     

Differential Phytate Utilization in <Emphasis Type="Italic">Candida</Emphasis> species

The present study was undertaken to evaluate and characterize the phytase activity in different Candida species. A total of 113 Candida isolates representing eight species were examined for phytase activity by an agar plate assay using the calcium salt of phytic acid as the sole phosphorus source. A phytase-positive phenotype was identified by the formation of a clear halo around a fungal colony. Cell-bound differential phytase activity was observed in Candida isolates at inter- and intra-species levels. Although phytase activity was not affected by the supplementation of external phosphate in C. albicans, C. dubliniensis, C. glabrata, and C. kefyr, elevated phytase activity was evident in C. guilliermondii, C. krusei, C. parapsilosis, and C. tropicalis in phosphate-free medium. Further characterization showed that, in general, relatively higher phytase activity was observed at more acidic pHs, and the phytase activity increased with incubation temperature, reaching a maximum at 55 or 65°C. Taken together, the findings demonstrated, for the first time, differential phytase activities in different Candida species. Phytase activity may be a contributing factor to fungal survival and proliferation within the human gastrointestinal tract, where nutrients are usually scarce.     

Usefulness of the Non-conventional <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> Model to Assess <Emphasis Type="Italic">Candida</Emphasis> Virulence

Invasive candidiasis is caused mainly by Candida albicans, but other Candida species have increasing etiologies. These species show different virulence and susceptibility levels to antifungal drugs. The aims of this study were to evaluate the usefulness of the non-conventional model Caenorhabditis elegans to assess the in vivo virulence of seven different Candida species and to compare the virulence in vivo with the in vitro production of proteinases and phospholipases, hemolytic activity and biofilm development capacity. One culture collection strain of each of seven Candida species (C. albicans, Candida dubliniensis, Candida glabrata, Candida krusei, Candida metapsilosis, Candida orthopsilosis and Candida parapsilosis) was studied. A double mutant C. elegans AU37 strain (glp-4;sek-1) was infected with Candida by ingestion, and the analysis of nematode survival was performed in liquid medium every 24 h until 120 h. Candida establishes a persistent lethal infection in the C. elegans intestinal tract. C. albicans and C. krusei were the most pathogenic species, whereas C. dubliniensis infection showed the lowest mortality. C. albicans was the only species with phospholipase activity, was the greatest producer of aspartyl proteinase and had a higher hemolytic activity. C. albicans and C. krusei caused higher mortality than the rest of the Candida species studied in the C. elegans model of candidiasis.     

Candida albicans and Candida tropicalis in oral candidosis: quantitative analysis, exoenzyme activity, and antifungal drug sensitivity

Candida albicans and C. tropicalis obtained from whole saliva of patients presenting signs of oral candidosis were assayed for quantification of colony forming units, exoenzyme activity (phospholipase and proteinase) and antifungal drug sensitivity (amphotericin B, fluconazole and itraconazole) by the reference method of the Clinical and Laboratory Standards Institute. The number of colony forming units per milliliter varied according to the Candida species involved and whether a single or mixed infection was present. Proteinase activity was observed in both C. albicans and C. tropicalis, but phospholipase activity was noted only in C. albicans. In vitro resistance to antifungals was verified in both species, but C. tropicalis appears to be more resistant to the tested antifungals than C. albicans.