Parapsilosis Sensu Stricto Research Articles

The identification and differentiation of the Candida parapsilosis complex species by polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region of the rDNA.

Currently, it is accepted that there are three species that were formerly grouped under Candida parapsilosis: C. para- psilosis sensu stricto, Candida orthopsilosis, andCandida metapsilosis. In fact, the antifungal susceptibility profiles and distinct virulence attributes demonstrate the differences in these nosocomial pathogens. An accurate, fast, and economical identification of fungal species has been the main goal in mycology. In the present study, we searched sequences that were available in the GenBank database in order to identify the complete sequence for the internal transcribed spacer (ITS)1-5.8S-ITS2 region, which is comprised of the forward and reverse primers ITS1 and ITS4. Subsequently, an in silico polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to differentiate the C. parapsilosis complex species. Ninety-eight clinical isolates from patients with fungaemia were submitted for analysis, where 59 isolates were identified as C. parapsilosis sensu stricto, 37 were identified as C. orthopsilosis, and two were identified as C. metapsilosis. PCR-RFLP quickly and accurately identified C. parapsilosis complex species, making this method an alternative and routine identification system for use in clinical mycology laboratories.

The Black Yeast Exophiala dermatitidis and Other Selected Opportunistic Human Fungal Pathogens Spread from Dishwashers to Kitchens.

We investigated the diversity and distribution of fungi in nine different sites inside 30 residential dishwashers. In total, 503 fungal strains were isolated, which belong to 10 genera and 84 species. Irrespective of the sampled site, 83% of the dishwashers were positive for fungi. The most frequent opportunistic pathogenic species were Exophiala dermatitidis, Candida parapsilosis sensu stricto, Exophiala phaeomuriformis, Fusarium dimerum, and the Saprochaete/Magnusiomyces clade. The black yeast E. dermatitidis was detected in 47% of the dishwashers, primarily at the dishwasher rubber seals, at up to 106 CFU/cm2; the other fungi detected were in the range of 102 to 105 CFU/cm2. The other most heavily contaminated dishwasher sites were side nozzles, doors and drains. Only F. dimerum was isolated from washed dishes, while dishwasher waste water contained E. dermatitidis, Exophiala oligosperma and Sarocladium killiense. Plumbing systems supplying water to household appliances represent the most probable route for contamination of dishwashers, as the fungi that represented the core dishwasher mycobiota were also detected in the tap water. Hot aerosols from dishwashers contained the human opportunistic yeast C. parapsilosis, Rhodotorula mucilaginosa and E. dermatitidis (as well as common air-borne genera such as Aspergillus, Penicillium, Trichoderma and Cladosporium). Comparison of fungal contamination of kitchens without and with dishwashers revealed that virtually all were contaminated with fungi. In both cases, the most contaminated sites were the kitchen drain and the dish drying rack. The most important difference was higher prevalence of black yeasts (E. dermatitidis in particular) in kitchens with dishwashers. In kitchens without dishwashers, C. parapsilosis strongly prevailed with negligible occurrence of E. dermatitidis. F. dimerum was isolated only from kitchens with dishwashers, while Saprochaete/Magnusiomyces isolates were only found within dishwashers. We conclude that dishwashers represent a reservoir of enriched opportunistic pathogenic species that can spread from the dishwasher into the indoor biome.

Members of the Candida parapsilosis Complex and Candida albicans are Differentially Recognized by Human Peripheral Blood Mononuclear Cells

The systemic infections caused by members of the Candida parapsilosis complex are currently associated to high morbility and mortality rates, and are considered as relevant as those caused by Candida albicans. Since the fungal cell wall is the first point of contact with the host cells, here we performed a comparison of this organelle in members of the C. parapsilosis complex, and its relevance during interaction with human peripheral blood mononuclear cells (PBMCs). We found that the wall of the C. parapsilosis complex members is similar in composition, but differs to that from C. albicans, with less mannan content and more β-glucan and porosity levels. Furthermore, lectin-based analysis showed increased chitin and β1,3-glucan exposure at the surface of C. parapsilosis sensu lato when compared to C. albicans. Yeast cells of members of the C. parapsilosis complex stimulated more cytokine production by human PBMCs than C. albicans cells; and this significantly changed upon removal of O-linked mannans, indicating this wall component plays a significant role in cytokine stimulation by C. parapsilosis sensu lato. When inner wall components were exposed on the wall surface, C. parapsilosis sensu stricto and C. metapsilosis, but not C. orthopsilosis, stimulated higher cytokine production. Moreover, we found a strong dependency on β1,3-glucan recognition for the members of the C. parapsilosis complex, but not for live C. albicans cells; whereas TLR4 was required for TNFα production by the three members of the complex, and stimulation of IL-6 by C. orthopsilosis. Mannose receptor had a significant role during TNFα and IL-1β stimulation by members of the complex. Finally, we demonstrated that purified N- and O-mannans from either C. parapsilosis sensu lato or C. albicans are capable to block the recognition of these pathogens by human PBMCs. Together; our results suggest that the innate immune recognition of the members of the C. parapsilosis complex is differential of that reported for C. albicans. In addition, we propose that purified cell wall mannans can be used as antagonist to block specific receptors on innate immune cells.

Identification to Species Level of Candida parapsilosis Complex into Sites Oral Cavity in a Cohort of Argentinos Patients

Candida parapsilosis is a complex of three species (Cp. sensu stricto, Candida orthopsilosis and Candida methapsilosis) due to genetic heterogeneity. Currently, it is the second most isolated in yeast bloodstream infections in Latin America, Asia and Europe. In Argentina and globally is no data on the distribution and behavior of the species that make up this complex in oral niches. Knowledge becomes important in the event that the mouth can be a potential source of candidemia and / or invasive infections by this yeast in patients with other risk factors source. Objective: To identify to species C. parapsilosis complex in clinical isolates obtained from various oral niches, of a cohort of Argentinos patients with different bucodentarias clinical situations. Methods: Retrospective, transversal and descriptive study using 31 clinical isolates of oral cavity, cryopreserved and obtained from immunocompetent patients with and without periodontal disease, and presence or absence of intraoral appliances; previously recognized by conventional methods such as C. parapsilosis, and recovered for molecular characterization endpoint PCR using specific primers. Results: 100% (31/31) of the isolates were positive for Candida parapsilosis sensu stricto. 77.5% of these strains were recovered in oral inflammatory conditions, and 22.5% in terms of oral health. Being statistically significant difference (p=0). Conclusions: C. parapsilosis sensu stricto is a common colonizing the oral mucosa, especially in pathological conditions.

Candida parapsilosis Sensu Stricto: A Common Colonizing in Oral Mucosa of Argentine Subjects

Candida parapsilosis is a complex made up of three species (Candida parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis) which differ genetically. In Argentina and worldwide, no data is available on the distribution and behavior of the complex in oral cavity niches, and there is little information on its epidemiology. Aim: To characterize Argentine isolates of the C. parapsilosis complex from different oral cavity sites and other ecological niches. Methodology: Retrospective, cross-sectional, descriptive study on a collection of isolates which were previously identified by conventional methods as parapsilosis complex, in order to distinguish the species by using end-point PCR with specific primers derived from a single sequence in the ITS1-5.8SrRNA-ITS2 region. Results: 95% of the isolates were identified as Cp. sensu stricto, which was recovered with higher probability from oral mucosa sites, under pathological conditions, and in presence of intraoral appliances. Seventy-four percent of the strains were recovered under conditions of immunocompetence, and 100% of the isolates had resistant phenotype to flucytosine. Conclusions: C. parapsilosis sensu stricto is a common species in different ecological niches. It is more likely to be recovered under conditions of immunocompetence. Dysbiosis of the mouth favors the growth of Cp. sensu stricto, which under these conditions may become a source for cross-transmission of more or less virulent strains by direct person-to-person contact, and a potential source of candidemia or invasive infections through hematogenous dissemination of strains with increased pathogenicity.

Effectiveness of photoelectrocatalysis treatment for the inactivation of Candida parapsilosis sensu stricto in planktonic cultures and biofilms

Photoelectrocatalysis (PEC) has become an attractive technology to disinfect microorganisms from aqueous environments because of its ability to degrade microorganisms without producing any harmful by-products. PEC methodology was applied to inactivate Candida parapsilosis sensu stricto in planktonic and biofilm cells, and its efficiency was compared to that of photolytic and photocatalytic treatment. In the case of planktonic (free-floating) cells, PEC promoted the inactivation of 106 CFUmL−1 after 3min of treatment, and the addition of silver nanoparticles to the Ti/TiO2 electrodes had virtually no effect on the disinfection period, although it caused a decrease by a factor of 1.5 in the mineralization efficiency. After 60, 10 and 60min of treatment, PEC methodology reduced 106 CFUmL−1 on fungal biofilms and provided 71, 91 and 92% mineralization for biofilm cells performed on polyvinyl chloride (PVC), silicone, and polytetrafluoroethylene (PTFE), respectively. These results indicated that PEC is a promising process for disinfection of water contaminated with C. parapsilosis sensu stricto in both planktonic and biofilm cells.

Simple, Low-Cost Detection of Candida parapsilosis Complex Isolates and Molecular Fingerprinting of Candida orthopsilosis Strains in Kuwait by ITS Region Sequencing and Amplified Fragment Length Polymorphism Analysis.

Candida parapsilosis has now emerged as the second or third most important cause of healthcare-associated Candida infections. Molecular studies have shown that phenotypically identified C. parapsilosis isolates represent a complex of three species, namely, C. parapsilosis, C. orthopsilosis and C. metapsilosis. Lodderomyces elongisporus is another species phenotypically closely related to the C. parapsilosis-complex. The aim of this study was to develop a simple, low cost multiplex (m) PCR assay for species-specific identification of C. parapsilosis complex isolates and to study genetic relatedness of C. orthopsilosis isolates in Kuwait. Species-specific amplicons from C. parapsilosis (171 bp), C. orthopsilosis (109 bp), C. metapsilosis (217 bp) and L. elongisporus (258 bp) were obtained in mPCR. Clinical isolates identified as C. parapsilosis (n = 380) by Vitek2 in Kuwait and an international collection of 27 C. parapsilosis complex and L. elongisporus isolates previously characterized by rDNA sequencing were analyzed to evaluate mPCR. Species-specific PCR and DNA sequencing of internal transcribed spacer (ITS) region of rDNA were performed to validate the results of mPCR. Fingerprinting of 19 clinical C. orthopsilosis isolates (including 4 isolates from a previous study) was performed by amplified fragment length polymorphism (AFLP) analysis. Phenotypically identified C. parapsilosis isolates (n = 380) were identified as C. parapsilosis sensu stricto (n = 361), C. orthopsilosis (n = 15), C. metapsilosis (n = 1) and L. elongisporus (n = 3) by mPCR. The mPCR also accurately detected all epidemiologically unrelated C. parapsilosis complex and L. elongisporus isolates. The 19 C. orthopsilosis isolates obtained from 16 patients were divided into 3 haplotypes based on ITS region sequence data. Seven distinct genotypes were identified among the 19 C. orthopsilosis isolates by AFLP including a dominant genotype (AFLP1) comprising 11 isolates recovered from 10 patients. A rapid, low-cost mPCR assay for detection and differentiation of C. parapsilosis, C. orthopsilosis, C. metapsilosis and L. elongisporus has been developed.

Candida parapsilosis (sensu lato) isolated from hospitals located in the Southeast of Brazil: Species distribution, antifungal susceptibility and virulence attributes

Candida parapsilosis (sensu lato), which represents a fungal complex composed of three genetically related species – Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis, has emerged as an important yeast causing fungemia worldwide. The goal of the present work was to assess the prevalence, antifungal susceptibility and production of virulence traits in 53 clinical isolates previously identified as C. parapsilosis (sensu lato) obtained from hospitals located in the Southeast of Brazil. Species forming this fungal complex are physiologically/morphologically indistinguishable; however, polymerase chain reaction followed by restriction fragment length polymorphism of FKS1 gene has solved the identification inaccuracy, revealing that 43 (81.1%) isolates were identified as C. parapsilosis sensu stricto and 10 (18.9%) as C. orthopsilosis. No C. metapsilosis was found. The geographic distribution of these Candida species was uniform among the studied Brazilian States (São Paulo, Rio de Janeiro and Espírito Santo). All C. orthopsilosis and almost all C. parapsilosis sensu stricto (95.3%) isolates were susceptible to amphotericin B, fluconazole, itraconazole, voriconazole and caspofungin. Nevertheless, one C. parapsilosis sensu stricto isolate was resistant to fluconazole and another one was resistant to caspofungin. C. parapsilosis sensu stricto isolates exhibited higher MIC mean values to amphotericin B, fluconazole and caspofungin than those of C. orthopsilosis, while C. orthopsilosis isolates displayed higher MIC mean to itraconazole compared to C. parapsilosis sensu stricto. Identical MIC mean values to voriconazole were measured for these Candida species. All the isolates of both species were able to form biofilm on polystyrene surface. Impressively, biofilm-growing cells of C. parapsilosis sensu stricto and C. orthopsilosis exhibited a considerable resistance to all antifungal agents tested. Pseudohyphae were observed in 67.4% and 80% of C. parapsilosis sensu stricto and C. orthopsilosis isolates, respectively. The secretion of phytase (93% versus 100%), aspartic protease (88.4% versus 90%), esterase (20.9% versus 50%) and hemolytic factors (25.6% versus 40%) was detected in C. parapsilosis sensu stricto and C. orthopsilosis isolates, respectively; however, no phospholipase activity was identified. An interesting fact was observed concerning the caseinolytic activity, for which all the producers (53.5%) belonged to C. parapsilosis sensu stricto. Collectively, our results add new data on the epidemiology, antifungal susceptibility and production of potential virulence attributes in clinical isolates of C. parapsilosis complex.

Candida parapsilosis Resistance to Fluconazole: Molecular Mechanisms and In Vivo Impact in Infected Galleria mellonella Larvae.

Candida parapsilosis is the main non-albicans Candida species isolated from patients in Latin America. Mutations in the ERG11 gene and overexpression of membrane transporter proteins have been linked to fluconazole resistance. The aim of this study was to evaluate the molecular mechanisms in fluconazole-resistant strains of C. parapsilosis isolated from critically ill patients. The identities of the nine collected C. parapsilosis isolates at the species level were confirmed through molecular identification with a TaqMan qPCR assay. The clonal origin of the strains was checked by microsatellite typing. The Galleria mellonella infection model was used to confirm in vitro resistance. We assessed the presence of ERG11 mutations, as well as the expression of ERG11 and two additional genes that contribute to antifungal resistance (CDR1 and MDR1), by using real-time quantitative PCR. All of the C. parapsilosis (sensu stricto) isolates tested exhibited fluconazole MICs between 8 and 16 μg/ml. The in vitro data were confirmed by the failure of fluconazole in the treatment of G. mellonella infected with fluconazole-resistant strains of C. parapsilosis. Sequencing of the ERG11 gene revealed a common mutation leading to a Y132F amino acid substitution in all of the isolates, a finding consistent with their clonal origin. After fluconazole exposure, overexpression was noted for ERG11, CDR1, and MDR1 in 9/9, 9/9, and 2/9 strains, respectively. We demonstrated that a combination of molecular mechanisms, including the presence of point mutations in the ERG11 gene, overexpression of ERG11, and genes encoding efflux pumps, are involved in fluconazole resistance in C. parapsilosis.

Molecular identification and antifungal susceptibility profiles of Candida parapsilosis complex species isolated from culture collection of clinical samples.

Candida parapsilosis is a common yeast species found in cases of onychomycosis and candidemia associated with infected intravascular devices. In this study, we differentiated Candida parapsilosis sensu stricto, Candida orthopsilosis , and Candida metapsilosis from a culture collection containing blood and subungual scraping samples. Furthermore, we assessed the in vitro antifungal susceptibility of these species to fluconazole, itraconazole, voriconazole, posaconazole, amphotericin B, and caspofungin. Differentiation of C. parapsilosis complex species was performed by amplification of the secondary alcohol dehydrogenase (SADH) gene and digestion by the restriction enzyme Ban I. All isolates were evaluated for the determination of minimal inhibitory concentrations using Etest, a method for antifungal susceptibility testing. Among the 87 isolates, 78 (89.7%) were identified as C. parapsilosis sensu stricto , five (5.7%) were identified as C. orthopsilosis , and four (4.6%) were identified as C. metapsilosis . Analysis of antifungal susceptibility showed that C. parapsilosis sensu strictoisolates were less susceptible to amphotericin B and itraconazole. One C. parapsilosis sensu stricto isolate was resistant to amphotericin B and itraconazole. Moreover, 10.2% of C. parapsilosis sensu stricto isolates were resistant to caspofungin. Two C. parapsilosis sensu strictoisolates and one C. metapsilosis isolate were susceptible to fluconazole in a dose-dependent manner. We reported the first molecular identification of C. parapsilosiscomplex species in State of Goiás, Brazil. Additionally, we showed that although the three species exhibited differences in antifungal susceptibility profiles, the primary susceptibility of this species was to caspofungin.

Yeast microbiota of natural cavities of manatees (Trichechus inunguis and Trichechus manatus) in Brazil and its relevance for animal health and management in captivity.

The aim of this study was to characterize the yeast microbiota of natural cavities of manatees kept in captivity in Brazil. Sterile swabs from the oral cavity, nostrils, genital opening, and rectum of 50 Trichechus inunguis and 26 Trichechus manatus were collected. The samples were plated on Sabouraud agar with chloramphenicol and incubated at 25 °C for 5 days. The yeasts isolated were phenotypically identified by biochemical and micromorphological tests. Overall, 141 strains were isolated, of which 112 were from T. inunguis (Candida albicans, Candida parapsilosis sensu stricto, Candida orthopsilosis, Candida metapsilosis, Candida guilliermondii, Candida pelliculosa, Candida tropicalis, Candida glabrata, Candida famata, Candida krusei, Candida norvegensis, Candida ciferri, Trichosporon sp., Rhodotorula sp., Cryptococcus laurentii) and 29 were from T. manatus (C. albicans, C. tropicalis, C. famata, C. guilliermondii, C. krusei, Rhodotorula sp., Rhodotorula mucilaginosa, Rhodotorula minuta, Trichosporon sp.). This was the first systematic study to investigate the importance of yeasts as components of the microbiota of sirenians, demonstrating the presence of potentially pathogenic species, which highlights the importance of maintaining adequate artificial conditions for the health of captive manatees.

Species Distribution, Virulence Factors, and Antifungal Susceptibility Among Candida parapsilosis Complex Isolates Recovered from Clinical Specimens.

The Candida parapsilosis complex has emerged as an important fungal pathogen. In spite of this, relatively little is known about its characteristics. Thus, the purposes of this study were (1) to determine by BanI-RFLP-assay the occurrence of C. parapsilosis complex species among 81 clinical isolates primarily identified as C. parapsilosis; (2) to evaluate their in vitro production of virulence factors; and (3) to compare their susceptibility profiles, grown as planktonic cells and biofilms, against amphotericin B, fluconazole, voriconazole, and caspofungin by following the Clinical and Laboratory Standards Institute (CLSI) guidelines. Seventy-seven isolates (95%) were identified as C. parapsilosis sensu stricto, 2 (2.5%) as C. orthopsilosis, and 2 (2.5%) as C. metapsilosis. Protease activity was detected in 29 (37.7%) isolates of C. parapsilosis sensu stricto, whereas only 7 (9.1%) exhibited phospholipase activity. None of the C. metapsilosis or C. orthopsilosis was able to produce protease or phospholipase. Biofilm production was detected in 35 (43.2%) isolates, among which 33 were C. parapsilosis sensu stricto and 2 were C. orthopsilosis. Antifungal resistance was uncommon; only one C. metapsilosis was fluconazole resistant. However, biofilm-producing isolates showed a marked resistance to all antifungal agents tested, particularly to voriconazole. This knowledge could be of clinical relevance for guiding therapeutic decisions.

Candida parapsilosis complex induces local inflammatory cytokines in immunocompetent mice.

Despite the increasing incidence of the Candida parapsilosis complex in the clinical setting and high mortality rates associated with disseminated infection, the host-fungus interactions regarding Candida parapsilosis sensu stricto and the closely related species C. orthopsilosis and C. metapsilosis remains blurred. In this study, we analyzed inflammatory cytokines levels and histopathology as well as fungal burden in spleen, kidney and lung of mice infected with six strains of the "psilosis" group with different enzymatic profiles. Strong interleukin 22 (IL-22) and tumor necrosis factor α (TNF-α) responses were observed in analyzed organs from infected mice (P<.0001) regardless of the species and enzymatic profile. TNF-α and IL-22 levels were related with spleen inflammation and fungal load. Fungal cells were detected only in spleen and kidney of infected mice, especially by day 2 post-challenge. The kidney showed glomerular retraction and partial destruction of renal tubules. Our data suggest that a strong inflammatory response, mainly of IL-22 and TNF-α, could be involved in Candida parapsilosis complex infection control.

Biofilm formation and genetic variability of BCR1 gene in the Candida parapsilosis complex

BackgroundCandida parapsilosis sensu stricto, Candida orthopsilosis, and Candida metapsilosis are cryptic species that belong to the C. parapsilosis complex, which has been increasingly associated to fungemia in various geographic regions, principally due to the capability of these yeasts to form biofilms on indwelling medical devices. BCR1 is one of the most studied genes related to Candida spp. biofilms. AimsTo evaluate the biofilm forming capability of a subset of 65 clinical isolates of the C. parapsilosis complex using two conventional approaches, and to look for an association between the biofilm forming phenotype and genetic variants of a fragment of BCR1. MethodsThe biofilm determination was carried out by crystal violet staining and tetrazolium reduction assay. On the other hand, a segment of BCR1 gene was sequenced by Sanger methodology. ResultsC. parapsilosis sensu stricto was statistically associated with a low biofilm production phenotype, while C. orthopsilosis was significantly associated with both phenotypes (high and low biofilm producers). According to the BCR1 sequence analysis, genetic variability was detected in C. orthopsilosis and C. metapsilosis without a particular biofilm formation phenotype association. ConclusionsUnder the adopted experimental design, C. parapsilosis sensu stricto was associated with the low biofilm phenotype and C. orthopsilosis with both phenotypes (high and low biofilm producers). On the other hand, an association between a biofilm forming phenotype and a particular genetic variant of the analyzed BCR1 fragment was not found.

Clinical characteristics and treatment outcomes of patients with candidaemia due to Candida parapsilosis sensu lato species at a medical centre in Taiwan, 2000-12.

To investigate the clinical characteristics and treatment outcomes of adult patients with Candida parapsilosis sensu lato candidaemia. We evaluated data in the mycology database of the National Taiwan University Hospital and on patients diagnosed with candidaemia due to C. parapsilosis sensu lato species from 2000 to 2012. Isolates were identified to the species level by conventional identification methods, MALDI-TOF and gene sequencing analysis. A total of 323 adult patients with candidaemia caused by C. parapsilosis sensu lato species were evaluated, including 256 (79.3%) patients with C. parapsilosis sensu stricto, 34 (10.5%) with Candida orthopsilosis and 33 (10.2%) with Candida metapsilosis. There were 222 men and 101 women and the median age was 60 years (range 18-103 years). Among them, 178 (55%) had an underlying diagnosis of cancer. The overall 30 day mortality rate was 25% (n = 80). Multivariate analysis revealed that shock (P < 0.001), antifungal therapy (P = 0.002), central catheter removal (P = 0.02) and abdominal surgery (P = 0.043) were independent prognostic factors for patients with candidaemia due to C. parapsilosis sensu lato species. There were no significant differences in 30 day mortality rate among patients with candidaemia caused by the three different species (P = 0.770). All isolates of C. metapsilosis, C. orthopsilosis and C. parapsilosis sensu stricto were susceptible to voriconazole. WT isolates were susceptible to itraconazole, posaconazole and amphotericin B. There were no significant differences in 30 day mortality among patients with candidaemia caused by C. parapsilosis sensu stricto, C. metapsilosis or C. orthopsilosis. The currently used antifungal agents exhibited good in vitro activities against C. parapsilosis sensu lato species isolates.

Molecular Identification and Antifungal Susceptibility of Candida parapsilosis sensu stricto, Candida orthopsilosis, and Candida metapsilosis in Sousse Region, Tunisia

Candida parapsilosis was recently reclassified into three distinct species, C. parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis. The aim of this study was to assess the prevalence and the antifungal susceptibility of these species among clinical isolates previously identified by phenotypical methods as C. parapsilosis in the laboratory of parasitology of Farhat Hached Hospital (Sousse, Tunisia). Ninety-six clinical isolates were identified by using a multiplex PCR targeting the gene encoding the ribosomal protein SO 40 (RPSO) and a PCR targeting the ITS region. Ninety one (94.8%) isolates were identified as C. parapsilosis sensu stricto, 3 (3.1%) as C. metapsilosis, and 2 (2.1%) as C. orthopsilosis based on PCRITS. These results were concordant with those achieved by Multiplex PCR-RPSO but the prevalence of C. orthopsilosis and C. metapsilosis seems to be very low in the region. Seventeen of the Candida isolates, C. parapsilosis sensu stricto (12), C. metapsilosis (3) and C. orthopsilosis (2) were assayed by ATB Fungus 3® to assess their susceptibility to Flucytosine, Amphotericin B, Fluconazole, Itraconazole and Voriconazole and by E-test® to assess their susceptibility to Caspofungin. All the isolates were susceptible to the antifungals tested.

Candida parapsilosis sensu stricto and the closely related species Candida orthopsilosis and Candida metapsilosis in vulvovaginal candidiasis.

This study aimed to determine the clinical characteristics and in vitro susceptibilities of Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis isolates from patients with vulvovaginal candidiasis (VVC). We analysed 63 vaginal C. parapsilosis specimens. After the molecular analyses, the isolates were characterised as C. parapsilosis sensu stricto (77.8%), C. orthopsilosis (7.9%) and C. metapsilosis (14.3%). The signs and symptoms of VVC caused by C. parapsilosis sensu lato, including itching, erythema and abnormal discharge, were milder than those caused by C. albicans. None of the C. parapsilosis sensu lato isolates were resistant to fluconazole, miconazole or itraconazole. The resistance rates of C. albicans to fluconazole, itraconazole, miconazole and clotrimazole were 2.3, 1.5, 3.1 and 0.8%, respectively. Both C. parapsilosis sensu lato and C. albicans were susceptible to nystatin. The mycological eradication rate at follow-up days 7-14 and 30-35 were 77.8% (49/63) and 76.2% (48/63), respectively, when treated with various antifungal agents and regimens. We conclude that C. parapsilosis sensu stricto and the closely related species C. orthopsilosis and C. metapsilosis were present in the vaginal samples of VVC patients. The symptoms and signs of VVC caused by C. parapsilosis are milder than those caused by C. albicans. The antifungal susceptibility and therapeutic efficacy in patients colonised by C. parapsilosis sensu lato were similar to those observed in C. albicans-colonised patients.

Antifungal susceptibility and virulence attributes of animal-derived isolates of Candida parapsilosis complex.

This study aimed to identify strains of the Candida parapsilosis complex isolated from animals, as well as to assess their in vitro antifungal susceptibility profile and in vitro production of virulence attributes. We used 28 isolates of C. parapsilosis sensu lato recovered from clinically healthy animals. The strains were characterized phenotypically, followed by molecular identification of the species through PCR-restriction enzyme analysis. The susceptibility of the strains to amphotericin B, itraconazole, voriconazole, fluconazole and caspofungin was assessed through broth microdilution. Additionally, the ability of the strains to produce biofilm, phospholipases and proteases was analysed. Molecular analysis showed 13 C. parapsilosis sensu stricto, 10 Candida orthopsilosis and five Candida metapsilosis strains. In vitro resistance to fluconazole was observed in three strains of C. parapsilosis sensu stricto and two C. metapsilosis. All tested strains were able to form biofilms and 23/28 isolates presented protease production, whilst none was able to produce phospholipases. Our study showed that C. parapsilosis sensu stricto and C. orthopsilosis are the most common species of the C. parapsilosis species complex and that these cryptic species present no significant phenotypical differences.

Nosocomial bloodstream Candida infections in a tertiary-care hospital in South Brazil: a 4-year survey.

The aims of this study were to evaluate the epidemiology of nosocomial candidemia in a tertiary hospital in South Brazil and the in vitro antifungal susceptibility of isolates. Blood strains from 108 patients were identified by PCR-based method. Some 30.5 % of candidemia were caused by Candida tropicalis, 28.7 % were due to Candida albicans, 24.1 % with Candida parapsilosis sensu stricto, 8.3 % with Candida glabrata sensu lato, 1.8 % involved Candida krusei and 6.6 % with other species. Candidemia was more common in intensive care unit settings (66 %). In vitro susceptibility to antifungal drugs was determined by a microdilution method; and new species-specific clinical breakpoints for fluconazole and voriconazole were applied. Overall susceptibility rates were 100 % for itraconazole, 91 % for fluconazole, 98 % for voriconazole and 99 % for amphotericin B. Fluconazole resistance was mostly among C. parapsilosis sensu stricto isolates (26.9 %). Most of the findings reported here agreed with epidemiological features common to other tertiary hospitals in Brazil; but also revealed some peculiarities, such as a high frequency of C. tropicalis associated with candidemia. Besides, high rate of fluconazole resistance among C. parapsilosis stricto sensu isolates was obtained when applying the new species-specific clinical breakpoints.

Killer behavior within the Candida parapsilosis complex

A group of 29 isolates of Candida parapsilosis sensu stricto, 29 of Candida orthopsilosis, and 4 of Candida metapsilosis were assayed for the presence of killer activity using Saccharomyces cerevisiae ATCC 26609 as a sensitive strain. All C. metapsilosis isolates showed killer activity at 25°C while strains of C. parapsilosis sensu stricto or C. orthopsilosis did not exhibit this activity. Sensitivity to killer toxins was evaluated using a set of previously reported killer strains of clinical origin. Only 11 isolates of the C. parapsilosis complex were inhibited by at least one killer isolate without resulting in any clear pattern, except for C. parapsilosis sensu stricto ATCC 22019, which was inhibited by every killer strain with the exception of C. parapsilosis and Candida utilis. The lack of sensitivity to killer activity among isolates of the genus Candida suggests that their toxins belong to the same killer type. Differentiation of species within the C. parapsilosis complex using the killer system may be feasible if a more taxonomically diverse panel of killer strains is employed.