Rapid identification of Candida glabrata using a new commercial kit

Candida glabrata is an emergent pathogen with diminished susceptibility to azoles, thus a rapid identification of this yeast could be of help to choose the appropriate treatment. GLABRATA RTT (Fumouze Diagnostics, France) is a new C. glabrata identification test. To evaluate its utility in the clinical laboratory daily routine, we prospectively tested 168 yeasts isolated in our hospital. GLABRATA RTT results had a sensitivity of 98.4% and a specificity of 100%. The combination of CHROMagar Candida isolation medium and GLABRATA RTT test allowed the identification of the four most common species in the clinical practice (Candida albicans, Candida tropicalis, Candida krusei and C. glabrata).     

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.     

Molecular typing of clinical Candida strains using random amplified polymorphic DNA and contour‐clamped homogenous electric fields electrophoresis

Aims: This report describes an investigation into the genetic profiles of 38 Candida albicans and 19 Candida glabrata strains collected from a dental hospital of Monastir (Tunisia) and the Laboratory of Parasitology, Farhat Hached Hospital of Sousse (Tunisia), using two typing methods: random amplified polymorphic DNA (RAPD) and contour‐clamped homogenous electric fields (CHEF). Methods and Results: The two methods (RAPD and CHEF electrophoresis) were able to identify clonal‐related isolates from different patients. RAPD method using two primers (CA1 and CA2) exhibited the highest discriminatory power by discriminating 22 genotypes for C. albicans with CA1 oligonucleotides and 19 genotypes with CA2 primer. For C. glabrata, 17 genotypes were obtained when both primers CA1 and CA2 were combined. The CHEF karyotyping of C. albicans has discriminated only 17 different karyotypes. Conclusion: The genotype of each isolate and genotypic difference among C. albicans and C. glabrata isolates were patient specific and not associated with the site of infection, geographic origin or date of isolation. Significance and Impact of the Study: Identification of relatedness between Candida species using molecular approaches with high discriminatory power is important in determining adequate measures for interruption of transmission of this yeast.     

Prevalence of yeast-like fungi and evaluation of several virulence factors from feral pigeons in Seoul, Korea

Aims: Studies of pigeon‐borne yeasts have tended to focus on species, such as Cryptococcus neoformans and Candida albicans, with scant attention to feral pigeons in Korea. We studied the prevalence of yeasts from faecal samples of feral pigeons obtained in various public places in Seoul, Korea, and assessed their potential capacity as human pathogens. Methods and Results: Three hundred and six pigeon faeces samples were collected at city squares and parks in 21 localities in Seoul and Seoul Grand Park and analysed for yeast with conventional methods. Of the 306 samples, 126 (41·2%) were positive for yeast. Seventeen species of yeast were identified. The most frequent species were Candida glabrata (34·1%), Candida famata (12·7%), Cryptococcus albidus (14·3%) and Cryptococcus laurentii (7·9%). The yeast isolates were tested for virulence. Of the 116 isolates (ten isolates missing), 70·7% (n = 82) grew at 37°C. All the Cryptococcus spp. isolates possessed a capsule, 16·4% (n = 19) produced melanin, and 33·6% (n = 39) produced proteinase. Two Ca. glabrata, a Ca. famata and Ca. albicans as well as three C. neoformans, a C. laurentii and Ca. albicans isolates had three virulence factors. Accordingly, 29·3% (n = 34) isolates possessed more than two virulence factors except capsule formation. Conclusions: These results of this study indicate that feral pigeons harbour a variety of yeasts and are a reservoir of human pathogenic fungi. Significance and Impact of the Study: This study is the first time about the microflora (fungi) presents in faecal samples collected from a variety of public areas throughout Seoul, Korea.     

An Evaluation of Risk Factors in Pregnant Women with <Emphasis Type="Italic">Candida</Emphasis> Vaginitis and the Diagnostic Value of Simultaneous Vaginal and Rectal Sampling

In this study, we investigated the epidemiological characteristics of VVC among pregnant women. We conducted a prospective survey among 372 pregnant women to investigate the prevalence, clinical forms, etiological agents, and predisposing factors of VVC. In addition, we determined the relationship between vaginal and rectal flora by simultaneously obtaining one high vaginal swab and one rectal swab from each patient using sterile cotton-tipped swabs. Furthermore, we compared the recovery and identification performances of chromID Candida agar to Sabouraud dextrose agar with gentamicin and chloramphenicol. Clinically and mycologically confirmed cases of VVC were detected in 139 (37.4%) and vaginal colonization described in 42 (11.3%) of 372 pregnant women. Rectal cultures were also positive in 98 of the 139 (70.5%) VVC cases. Candida albicans and C. glabrata were identified in vaginal samples in 58.0 versus 19.0% and from rectal samples in 49.0 versus 13.5%, respectively. Increases in gestational week and gravidae were identified to be statistically significant in patients with acute VVC (AVVC) and symptomatic recurrent VVC (RVVC), and asymptomatic RVVC (P = 0.04 and P = 0.03, respectively). In the laboratory diagnosis of VVC, specifically tailored chromogenic media are reliable tools for both the recovery and rapid identification of common Candida spp., particularly C. albicans, as well as for the detection of polyfungal populations in vaginal samples (P > 0.05). In addition, rectal colonization is a common finding in cases of AVVC and symptomatic-RVVC cases and corresponds well with the presence of the same yeast species in the vagina.     

Evidence from comparative genomics for a complete sexual cycle in the 'asexual' pathogenic yeast <Emphasis Type="Italic">Candida glabrata</Emphasis>

Background  

Candida glabrata is a pathogenic yeast of increasing medical concern. It has been regarded as asexual since it was first described in 1917, yet phylogenetic analyses have revealed that it is more closely related to sexual yeasts than other Candida species. We show here that the C. glabrata genome contains many genes apparently involved in sexual reproduction.     

Identification of <Emphasis Type="Italic">Candida</Emphasis> Species Isolated from Bovine Mastitic Milk and Their In Vitro Hemolytic Activity in Western Turkey

In this study, identification of 207 Candida isolates, previously isolated from mastitic bovine quarter milk samples at the level of genus, was made using API 20 C AUX system. The most frequently isolated species were Candida krusei (34.8%), followed by Candida rugosa (16.4%), Candida kefyr (12.6%), Candida albicans (10.1%), and Candida tropicalis (9.2%). Less common isolates were Candida zeylanoides (5.8%), Candida parapsilosis (4.3%), Candida guilliermondii (3.4%), Candida famata (1.9%), and Candida glabrata (1.5%). Additionally, in vitro hemolytic activity of all Candida strains were also examined in the present study. C. krusei (72 isolates), C. kefyr (26), C. albicans (21), C. tropicalis (19), C. zeylanoides (12), and C. glabrata (3) demonstrated both alpha and beta hemolysis at 48-h postinoculation. Only alpha hemolysis was detected in C. rugosa (34), C. guilliermondii (7), and C. famata (4), while C. parapsilosis (9) did not show any hemolytic activity after incubation for 72 h. Statistically significant difference (P < 0.001) was determined between the beta-hemolytic activities of Candida strains. The hemolytic activities of C. zeylanoides, C. albicans and C. kefyr were higher than other strains. This is the first study to describe variable hemolysis types exhibited by different Candida strains isolated from bovine mastitic milk in Turkey.     

Strain differentiation of pathogenic yeasts by the killer system

High sensitivity rates to the activity of killer toxins produced by 25 species of yeasts belonging to the genera Candida, Hansenula, Pichia, Rhodotorula, Saccharomyces and Trichosporon have been observed among 112 yeast isolates (25 Cryptococcus neoformans, 29 C. glabrata, 16 C. parapsilosis, 20 C. pseudotropicalis and 22 C. tropicalis). The highest sensitivity has been observed among the C. parapsilosis isolates, the lowest in C. glabrata strains. Genera Pichia and Hansenula proved to have the greatest killer activity. A killer system, formerly used for differentiating C. albicans isolates within the species, proved to be valid as epidemiological marker when applied to 112 strains of pathogenic yeasts.     

Effect of the incubation conditions on the production of patulin by Penicillium griseofulvum isolated from wheat

A total of 290 Candida isolates from patients were investigated for in vitro proteinase production. Overall, sixty percent of these strains were found to be proteinase producers. Of the C. albicans strains, 81.4% of the significant isolates in contrast to 19.7% of nonsignificant isolates were proteinase producers, the difference being statistically significant (P<0.001). Amongst the different Candida species, the proteinase production was found not only in Candida albicans, but also in C. tropicalis, C. parapsilosis and C. glabrata. Thus this in vitro method of demonstration of proteinase may be a good adjunct to smear and culture examination in identifying pathogenic Candida species from anatomical sites where they can also be present as commensals.     

Urban Pigeons (<Emphasis Type="Italic">Columba livia</Emphasis>) as a Potential Source of Pathogenic Yeasts: A Focus on Antifungal Susceptibility of <Emphasis Type="Italic">Cryptococcus</Emphasis> Strains in Northeast Brazil

To investigate pigeons as a potential source of pathogenic yeast species, 47 samples of pigeon droppings and 322 samples from pigeon cloacae were evaluated. The samples were also collected from trees located near the pigeon habitats, in the city of Fortaleza, Ceará, Northeast Brazil. In addition, we evaluated the in vitro antifungal susceptibility of these environmental Cryptococcus strains to amphotericin B, azoles and caspofungin. C. neoformans var. neoformans (n = 10), C. laurentii (n = 3), Candida spp. (n = 14), Rhodotorula mucilaginosa (n = 6) and Trichosporon sp. (n = 3) were isolated from pigeon droppings. In contrast, only Candida spp. (n = 4), Trichosporon sp. (n = 3) and R. mucilaginosa (n = 2) were recovered from cloacae specimens. Only Candida glabrata (n = 1) was recovered from plant samples. Azole resistance was detected in only one environmental strain of Cryptococcus, which was resistant to itraconazole (MIC = 1 μg/ml). As expected, all Cryptococcus strains were resistant to caspofungin. In summary, the present study confirms that urban pigeons are a potential source of Cryptococcus spp. and other pathogenic yeasts. Additionally, antifungal resistance was observed in one environmental strain of Cryptococcus neoformans var. neoformans in Northeast Brazil.     

First Environmental Isolations of <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> and <Emphasis Type="Italic">Cryptococcus gattii</Emphasis> in Tunisia and Review of Published Studies on Environmental Isolations in Africa

Cryptococcus neoformans and Cryptococcus gattii are pathogenic yeasts that cause cryptococcosis. These fungi were commonly associated with pigeon droppings and plant materials. The habitat of these pathogens has not been yet studied in Tunisia, although the ecology of these yeasts must be elucidated in order to establish surveillance programs and to prevent infections. The aim of this survey was to recover C. neoformans and C. gattii environmental isolates from pigeon droppings and plant materials in different areas of Sfax region, Tunisia. Nine hundred and fifty samples from leaves, wood, flowers, fruits and soil around trunk bases of 40 almond (Prunus dulcis) and 60 eucalyptus trees were collected as well as 250 pigeon droppings samples from different sites: buildings (n = 150), houses (n = 50) and zoo (n = 50). The identification of Cryptococcus neoformans complex was confirmed using the ID32C auxanogram panel (BioMérieux, Marcy l’Etoile, France); species were determined by multiplex PCR using the CN70 and CN49 primers, and mating type was determined by PCR. C. neoformans was recovered from 26 specimens of pigeon droppings (10.4%). This yeast was obtained more frequently from dry droppings (9.2%) than from moist droppings (1.2%). The mating type was determined. All the 31 environmental strains of C. neoformans and C. gattii were MATα. Out of 700 samples tested from 100 trees, only 5 isolates of Cryptococcus neoformans species complex were recovered (0.6%), two isolates of C. gattii and one isolate of C. neoformans were recovered from the wood of E. camaldulensis trees, and only two isolates of C. gattii were recovered from the wood of almond trees (Prunus dulcis Mill. var. zaaf and var. achek). These two Tunisian almond tree varieties were recorded for the first time in Africa as hosts for C. gattii. These results add new information to the ecology and epidemiology of C. neoformans species complex in Tunisia.     

Frequency of <Emphasis Type="Italic">Candida</Emphasis> spp. in the Oral Cavity of Brazilian HIV-Positive Patients and Correlation with CD4 Cell Counts and Viral Load

The aim of this study was to evaluate the prevalence of Candida spp., and particularly C. dubliniensis, among oral isolates from Brazilian HIV-positive patients correlating these results with CD4 cell counts and viral load. Forty-five individuals (23 female and 22 male) diagnosed as HIV-positive by ELISA and Western-blot, under anti-retroviral therapy for at least 1 year and without oral candidosis signals were included in the study. The control group was constituted by 45 healthy individuals, matched to the test group in relation to age, gender, and oral conditions. Oral rinses were collected and the identification was performed by phenotypic tests. The existence of C. dubliniensis among the isolates was analyzed using a validated multiplex PCR assay. Candida spp. were detected at significantly higher number in the oral cavity of HIV-positive patients in relation to the controls (P = 0.0008). C. albicans was the most frequently isolated species in both groups. In the HIV group, C. glabrata, C. lipolytica, C. krusei, C. guilliermondii, and C. parapsilosis were also identified. In the control group, we additionally identified C. tropicalis and C. dubliniensis. Two isolates (1.9%, 2/108) from control individuals were identified as C. dubliniensis and this species was not verified in the HIV group. Candida spp. counts were statistically lower (P = 0.0230) in the oral cavity of patients with low viral load (<400 copies/mm3). Candida spp. counts did not differ statistically among groups with different levels of CD4 cells counts (P = 0.1068).     

Use of CHROMagar in the Differentiation of Common Species of <Emphasis Type="Italic">Candida</Emphasis>

CHROMagar has been reported to be useful for the rapid and accurate identification of Candida species. We tested 135 isolates of Candida species isolated from oropharyngeal candidiasis in HIV patients and found that it was useful in the presumptive identification of Candida albicans and Candida krusei. Occasional strains of C. tropicalis produced colonies with a greenish tinge making it difficult to differentiate from C. albicans.     

<Emphasis Type="Italic">Candida neustonensis</Emphasis> sp. nov., a novel ascomycetous yeast isolated from the sea surface microlayer in Taiwan

A new ascomycetous yeast species, Candida neustonensis is proposed in this study based on four strains (SN92^T, SN47, SJ22, SJ25) isolated from sea surface microlayer in Taiwan. These four yeast strains were morphologically, physiologically and phylogenetically identical to each other. No sexual reproduction was observed on 5% malt extract agar, corn meal agar, V8 agar, McClary’s acetate agar and potato-dextrose agar. Phylogenetic analysis of the sequences of the D1/D2 domain of the large subunit (LSU) rRNA gene places C. neustonensis as a member of the Pichia guilliermondii clade, it also reveals that the phylogenetically closest relatives of C. neustonensis are C. fukuyamaensis (4.4% divergence), C. xestobii (4.4% divergence) and P. guilliermondii (4.5% divergence). C. neustonensis also is clearly distinguished from other known species in the P. guilliermondii clade based on the results of physiology tests. From these comparison analyses, the following novel yeast species is proposed: Candida neustonensis sp. nov., with strain SN92^T (= BCRC 23108^T = JCM 14892^T = CBS 11061^T) as the type strain.     

Isolation of yeasts from bovine milk in Belgium

A total of 436 milk samples from non-infected and 80 from infected quarters were investigated: 24.5% of the samples collected from non-infected and 55% of those collected from infected quarters were positive. Normal milk yielded not less than 16 different species and among them many potentially pathogenic yeast species such as C. parapsilosis, C. guilliermondii, C. tropicalis, C. glabrata and T. asahii, all five able to grow at 40 ° C. In contrast, the yeasts isolated from infected quarters were from 3 species: C. kefyr, C. catenulata and C. lambica, which were also among the yeasts species recovered from normal milk. Among the three species, only one i.e. Candida kefyr is able to grow above 40 ° C and from there can be considered as potentially pathogenic, even if bacterial association is necessary to cause mastitis.     

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.     

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.

     

Comparison of genomes of eight species of sections <Emphasis Type="Italic">Linum</Emphasis> and <Emphasis Type="Italic">Adenolinum</Emphasis> from the genus<Emphasis Type="Italic"> Linum</Emphasis> based on chromosome banding,molecular markers and RAPD analysis

Karyotypes of species sects. Linum and Adenolinum have been studied using C/DAPI-banding, Ag-NOR staining, FISH with 5S and 26S rDNA and RAPD analysis. C/DAPI-banding patterns enabled identification of all homologous chromosome pairs in the studied karyotypes. The revealed high similarity between species L. grandiflorum (2n = 16) and L. decumbens by chromosome and molecular markers proved their close genome relationship and identified the chromosome number in L. decumbens as 2n = 16. The similarity found for C/DAPI-banding patterns between species with the same chromosome numbers corresponds with the results obtained by RAPD-analysis, showing clusterization of 16-, 18- and 30-chromosome species into three separate groups. 5S rDNA and 26S rDNA were co-localized in NOR-chromosome 1 in the genomes of all species investigated. In 30-chromosome species, there were three separate 5S rDNA sites in chromosomes 3, 8 and 13. In 16-chromosome species, a separate 5S rDNA site was also located in chromosome 3, whereas in 18-chromosome species it was found in the long arm of NOR-chromosome 1. Thus, the difference in localization of rDNA sites in species with 2n = 16, 2n = 30 and 2n = 18 confirms taxonomists opinion, who attributed these species to different sects. Linum and Adenolinum, respectively. The obtained results suggest that species with 2n = 16, 2n = 18 and 2n = 30 originated from a 16-chromosome ancestor.     

The first clinical isolates of <Emphasis Type="Italic">Candida dubliniensis</Emphasis> in Slovakia

Candida dubliniensis, yeast closely related to Candida albicans, is a new pathogen associated mainly with infections of immunocompromised hosts. In this study, we report the first isolation of three isolates of C. dubliniensis in Slovakia. The first selection of both C. albicans and C. dubliniensis from the other Candida species was done on the basis of specific green color of primoculture grown on CHROMagar Candida. The presumptive identification was completed by supplemental tests: germ-tube formation, production of chlamydospores, ability or inability to grow at 42 and 45,°C and by commercial set API 20C AUX. Parallely, the discrimination between both species was performed by PCR assay using primers specific for Candida dubliniensis