Candida Haemulonii Research Articles

Invasive Candida auris infections in Kuwait hospitals: epidemiology, antifungal treatment and outcome.

Candida auris is a recently recognized yeast pathogen, which has attracted worldwide attention due to its multidrug-resistant nature and associated high mortality rates. Its persistence in hospital environment and propensity of nosocomial transmission underscores the need of continuous monitoring to prevent outbreaks. Since the first case of C. auris candidemia in May, 2014, we have identified 17 additional invasive cases, which are described here. Identity of 17 isolates originating from proven or possible cases of invasive C. auris infection and identified as Candida haemulonii by Vitek 2 yeast identification system was confirmed by PCR-sequencing of rDNA. Information about risk factors, treatment and outcomes were retrospectively retrieved from case files. Antifungal susceptibility testing was performed by Etest. Thirteen cases of candidemia and 4 cases of other invasive infections were detected in 6 hospitals across Kuwait. Major risk factors included adult patients with cancer, diabetes, gastrointestinal/liver diseases and extended (> 25 days) hospital stay. All isolates were resistant to fluconazole. Additionally, 5 and 4 isolates were also resistant to voriconazole and amphotericin B, respectively. Despite antifungal treatment, 9 of 15 patients died. Most patients (n = 12) were hospitalized in 2 hospitals that are in close proximity, whereas 5 other patients were from 3 hospitals that are situated > 10km apart. Occurrence of successive cases of invasive C. auris infections with resulting mortality in nine patients suggests persistence of this multidrug-resistant yeast in major hospitals in Kuwait. Early detection by continuous surveillance and enforcement of infection control measures are recommended.

Genome Sequence of a Multidrug-Resistant Candida haemulonii Isolate from a Patient with Chronic Leg Ulcers in Israel.

ABSTRACTCandida haemulonii is an emerging multidrug-resistant yeast that can cause invasive candidiasis. Here, we report the first genome sequence of C. haemulonii (isolate B11899) generated using PacBio sequencing technology. The estimated genome size was 13.3 Mb, with a GC content of 45.19%.

Increasing prevalence, molecular characterization and antifungal drug susceptibility of serial Candida auris isolates in Kuwait.

Candida auris is an emerging yeast pathogen of global significance. Its multidrug-resistant nature and inadequacies of conventional identification systems pose diagnostic and therapeutic challenges. This study investigated occurrence of C. auris in clinical specimens in Kuwait and its susceptibility to antifungal agents. Clinical yeast strains isolated during 3.5-year period and forming pink-colored colonies on CHROMagar Candida were studied by wet mount examination for microscopic morphology and Vitek 2 yeast identification system. A simple species-specific PCR assay was developed for molecular identification and results were confirmed by PCR-sequencing of rDNA. Antifungal susceptibility testing of one isolate from each patient was determined by Etest. The 280 isolates forming pink-colored colonies on CHROMagar Candida, were identified by Vitek 2 as Candida haemulonii (n = 166), Candida utilis (n = 49), Candida kefyr (n = 45), Candida guilliermondii (n = 9), Candida famata (n = 6) and Candida conglobata (n = 5). Species-specific PCR and PCR-sequencing of rDNA identified 166 C. haemulonii isolates as C. auris (n = 158), C. haemulonii (n = 6) and Candida duobushaemulonii (n = 2). C. auris isolates originated from diverse clinical specimens from 56 patients. Of 56 C. auris isolates tested, all were resistant to fluconazole, 41/56 (73%) and 13/56 (23%) were additionally resistant to voriconazole and amphotericin B, respectively. Eleven (20%) isolates were resistant to fluconazole, voriconazole and amphotericin B. One isolate was resistant to caspofungin and micafungin. Increasing isolation of C. auris in recent years from diverse clinical specimens including bloodstream shows that C. auris is an emerging non-albicans Candida species in Kuwait causing a variety of infections. Inability of conventional identification methods to accurately identify this pathogen and multidrug-resistant nature of many strains calls for a greater understanding of its epidemiology, risk factors for acquiring C. auris infection and management strategies in high-risk patients. This is the first comprehensive study on the emergence of this multidrug-resistant yeast from Kuwait and the Middle East.

First report of sporadic cases of Candida auris in Colombia

BackgroundCandida auris is a recently reported Candida species that is phenotypically similar to Candida haemulonii and related to hospital outbreaks. This organism can be misidentified as Candida haemulonii, Candida famata, Candida catenulata, or Rhodotorula glutinis by phenotypic approaches. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and DNA sequence analysis using internal transcribed spacer rDNA bar-coding provide an accurate identification. Case reportsThree cases of C. auris infection in patients with risk factors for fungal infection (one admitted to the intensive care unit, one with lymphoma, and one with HIV; all three with previous antibiotic use) are reported; these infections were not epidemiologically related. Yeast isolates were recovered from blood, ocular secretion, and bronchoalveolar lavage and were misidentified as C. catenulata and Candida albicans by the phenotypic MicroScan method. The isolates were confirmed to be C. auris by means of MALDI-TOF MS and DNA sequence analysis. Antifungal susceptibility testing was performed on these C. auris isolates, which exhibited high minimum inhibitory concentrations to triazoles and amphotericin B. One patient survived and the other two died. Only one of these deaths was related to fungemia. ConclusionsC. auris is an emerging and opportunistic multidrug-resistant human pathogen. It is necessary to strengthen measures to achieve an accurate and quick identification and also to avoid its dissemination. This will require improvements in health and infection control measures, as well as the promotion of antifungal stewardship in healthcare facilities.

Comparative evaluation of sensititre yeast one panel against broth microdilution method for testing Candida auris and Candida haemulonii susceptibility: A pilot study

BACKGROUND: Candidal infection due to non-Candida albicans is on the rise and implicated to cause significant morbidity and mortality. Although empirical treatment can be provided, the sensitivity pattern varies drastically in different species of Candida. The conventional broth microdilution method for antifungal susceptibility is labour intensive. Data on antifungal susceptibility testing (AFST) by colorimetric yeast one sensititre method are available for many Candida species but not for Candida auris and Candida haemulonii. AIM: The utility of colorimetric yeast one plate was compared to conventional broth microdilution method (CLSI M27-A3 guidelines) for five isolates each of C. haemulonii and C. auris along with CLSI quality control strain Candida krusei ATCC 6258. MATERIALS AND METHODS: A total of 10 isolates of C. auris/haemulonii, confirmed by sugar assimilation and DNA sequencing were chosen. AFST was performed by conventional broth microdilution and colorimetric yeast one sensititre plates. STATISTICAL ANALYSIS: The analysis was performed by Carl Pearson's correlation coefficient. RESULTS: All isolates showed high MIC (>64 μg/mL) for Fluconazole by both methods. Strains of C. auris were found to have high MIC (>64 μg/mL) for Amphotericin B by conventional method. The degree of agreement between the two methods was 100%, 96.83% and 90.03% for Fluconazole, Posaconazole and Itraconazole, respectively, for the 10 isolates. The degree of agreement was 100%, 95.70% and 93.83% and 100%, 97.97% and 86.24% for Fluconazole, Posaconazole and Itraconazole for C. auris and C. haemulonii, respectively. CONCLUSION: AFST by colorimetric yeast one plate is less time-consuming, easy to perform and interpret, however further studies with a large number of isolates are required to test Echinocandins and Amphotericin B.

Emerging multidrug-resistant Candida species.

To describe the epidemiology, strategies for early detection, and clinical management of infections caused by the most commonly found multidrug-resistant (MDR) Candida spp. Increasing numbers of reports describing invasive infections by MDR Candida auris and Candida glabrata has been reported in medical centers worldwide. We checked all papers published along the last 10 years describing epidemiological, diagnostic, and clinical aspects of infections by MDR Candida spp., with emphasis on C. auris and C. glabrata spp. C. auris has been reported in 15 countries and multidrug resistance rates is usually above 30%. Horizontal transmission is a great concern regarding C. auris. C. glabrata ranks the second most reported Candida spp. in deep-seated infections from United States and some European Centers, although multidrug resistance rates above 10% are restricted to some US centers. Candida haemulonii complex isolates with poor susceptibility to azoles and amphotericin B have been isolated in superficial and deep-seated infections, whereas Candida guilliiermondii complex isolates with poor susceptibility to azoles and echinocandins have been recovered from catheter-related bloodstream infections. Other potential MDR Candida species are Candida krusei, Candida lusitaniae, Candida kefyr, Yarrowia (Candida) lypolitica, and Candida rugosa.

A new approach by optical coherence tomography for elucidating biofilm formation by emergent Candida species.

The majority of microorganisms present a community lifestyle, establishing biofilm ecosystems. However, little is known about its formation in emergent Candida species involved in catheter-related infections. Thus, various techniques may be used in the biofilm detection to elucidate structure and clinical impact. In this context, we report the ability of emergent Candida species (Candida haemulonii, C. lusitaniae, C. pelliculosa, C.guilliermondii, C. famata and C. ciferrii) on developing well structured biofilms with cell viability and architecture, using optical coherence tomography (OCT). This new approach was compared with XTT analyses and Scanning Electron Microscopy (SEM). A positive correlation between oxidative activity (XTT) and OCT results (r = 0.8752, p < 0.0001) was observed. SEM images demonstrated cells attachment, multilayer and morphologic characteristics of the biofilm structure. C. lusitaniae was the emergent species which revealed the highest scattering extension length and oxidative metabolism when evaluated by OCT and XTT methods, respectively. Herein, information on C. ciferri biofilm structure were presented for the first time. The OCT results are independently among Candida strains and no species-specific pattern was observed. Our findings strongly contribute for clinical management based on the knowledge of pathogenicity mechanisms involving emergent yeasts.

Candida haemulonii sensu lato: Update of the Determination of Susceptibility Profile in Argentina and Literature Review

This review summarizes the information available of Candida haemulonii sensu lato, and updates the in vitro susceptibility profile of the isolates of these species from Argentina. C. haemulonii sensu lato causes fungemia in low frequency in adults and children; however, the knowledge about this emerging yeast is relevant since it is considered as a multi-resistant pathogen. The discovery of new antifungal molecules, the determination of epidemiological cutoff value and clinical breakpoints for some yeasts of clinical impact, and the update of techniques to determine the in vitro susceptibility profile to yeast have generated some available information, although, for C. haemulonii sensu lato, this information is not always useful for clinical application. We determined the susceptibility profile of C. haemulonii sensu lato strains isolated in Argentina, as a first step to establish the epidemiological cutoff values in our region. We also review the current situation in other countries.

Antifungal Potential of Copper(II), Manganese(II) and Silver(I) 1,10-Phenanthroline Chelates Against Multidrug-Resistant Fungal Species Forming the Candida haemulonii Complex: Impact on the Planktonic and Biofilm Lifestyles.

Candida haemulonii, Candida haemulonii var. vulnera and Candida duobushaemulonii, which form the C. haemulonii complex, are emerging etiologic agents of fungal infections known to be resistant to the most commonly used antifungals. The well-established anti-Candida potential of metal complexes containing 1,10-phenanthroline (phen) ligands encouraged us to evaluate different copper(II), manganese(II), and silver(I) phen chelates for their ability to inhibit planktonic growth and biofilm of C. haemulonii species complex. Two novel coordination complexes, {[Cu(3,6,9-tdda)(phen)2].3H2O.EtOH}n and [Ag2(3,6,9-tdda)(phen)4].EtOH (3,6,9-tddaH2 = 3,6,9-trioxaundecanedioic acid), were synthesized in a similar fashion to the other, previously documented, sixteen copper(II), manganese(II), and silver(I) chelates employed herein. Three isolates of each C. haemulonii species complex were used and the effect of the metal chelates on viability was determined utilizing the CLSI standard protocol and on biofilm-growing cells using the XTT assay. Cytotoxicity of the chelates was evaluated by the MTT assay, employing lung epithelial cells. The majority of the metal chelates were capable of interfering with the viability of planktonic-growing cells of all the fungal isolates. The silver complexes were the most effective drugs (overall geometric mean of the minimum inhibitory concentration (GM-MIC) ranged from 0.26 to 2.16 μM), followed by the manganese (overall GM-MIC ranged from 0.87 to 10.71 μM) and copper (overall GM-MIC ranged from 3.37 to >72 μM) chelates. The manganese chelates (CC50 values ranged from 234.51 to >512 μM) were the least toxic to the mammalian cells, followed by the silver (CC50 values ranged from 2.07 to 13.63 μM) and copper (CC50 values ranged from 0.53 to 3.86 μM) compounds. When tested against mature biofilms, the chelates were less active, with MICs ranging from 2- to 33-fold higher levels when compared to the planktonic MIC counterparts. Importantly, manganese(II), copper(II), and silver(I) phen chelates are relatively cheap and easy to synthesize and they offer significant antifungal chemotherapeutic potential for the treatment of highly resistant pathogens.

Planktonic growth and biofilm formation profiles in Candida haemulonii species complex.

Candida haemulonii species complex have emerged as multidrug-resistant yeasts able to cause fungemia worldwide. However, very little is known regarding their physiology and virulence factors. In this context, planktonic growth and biofilm formation of Brazilian clinical isolates of Candida haemulonii (n = 5), Candida duobushaemulonii (n = 4), and Candida haemulonii var. vulnera (n = 3) were reported. Overall, the fungal planktonic growth curves in Sabouraud dextrose broth reached the exponential phase in 48h at 37°C. All the clinical isolates formed biofilm on polystyrene in a time-dependent event, as judged by the parameters evaluated: biomass (crystal violet staining), metabolic activity (XTT reduction), and extracellular matrix (safranin incorporation). No statistically significant differences were observed when the average measurements among the three Candida species were compared regarding both planktonic and biofilm lifestyles; however, typical isolate-specific differences were clearly noticed in fungal growth kinetics.

BRIGHT ASTEROIDS IN THE RED SEA - AN EMERGING PATHOGEN CANDIDA HAEMOLUNII: ARE AZOLES THE TREATMENT OPTION?

This is a case study of a 15-year-old female who on chemotherapy and central line catheter-related bloodstream candidemia presented with recurrentepisodes of candidemia, was febrile and breathless. She was diagnosed of Candida haemulonii fungemia. Echinocandins was started as this species ofCandida is usually resistant to the commonly used antifungal agents. On treatment, the patient was afebrile and discharged.Keywords: Candida haemulonii, Candidemia, Case report, Emerging pathogen

First Nine Cases of Candida auris Infection Reported in Central America: Importance of Acurate Diagnosis and Susceptibility Testing

Background Candida auris is an emerging multidrug-resistant pathogen associated with invasive infections and high mortality. This report describes the first 9 cases of C. auris in Central America in a hospital in Panama City, Panama, and highlights the challenges of accurate identification and methods for susceptibility testing.MethodsIsolates initially identified at a Panama City acute care hospital during July–October 2016 as Candida haemulonii (a common misidentification for C. auris) or Candida species by Vitek® 2 automated system (bioMérieux) were further characterized by molecular methods. Antifungal susceptibility testing was performed and results were compared between standard and reference methodologies. Patient demographic, clinical, and laboratory data were collected from the medical record.ResultsA total of 14 isolates from 9 hospitalized patients were confirmed as C. auris. Isolates were from urine (11), blood (1), catheter tip (1) and pleural fluid (1). Results of susceptibility testing were highly discrepant between automated and reference techniques for fluconazole (92% resistant vs. 77%, respectively) and amphotericin B (100% vs. 8%). Six (67%) patients were male, and the mean age was 53 years (range 42–78). All patients were admitted to the intensive care unit and were mechanically ventilated. Seven (78%) patients died.Conclusion C. auris is present in Central America. Healthcare facilities in the region should be vigilant for this concerning pathogen, particularly given challenges in its identification and need for infection control precautions. Although automated testing overestimated amphotericin B resistance, most initial isolates were susceptible by reference testing.Disclosures All authors: No reported disclosures.

Nosocomial fungemia by Candida auris: First four reported cases in continental Europe

BackgroundCandida auris is an emerging multidrug-resistant yeast that can cause invasive infections and is associated with high mortality. It is typically resistant to fluconazole and voriconazole and, some cases, also to echinocandins and amphotericin B. This species, phylogenetically related to Candida haemulonii, is frequently misidentified by commercial identification techniques in clinical laboratories; therefore, the real prevalence of C. auris infections may be underestimated. AimsTo describe the clinical and microbiological features of the first four cases of C. auris fungemia episodes observed in the European continent. MethodsThe four patients were hospitalized in the adult surgical intensive care unit. A total of 8 isolates (two per patient) from blood and catheter tip were analyzed. ResultsAll isolates were misidentified as Saccharomyces cerevisiae by AuxaColor 2, and as Candida sake by API ID20C. VITEK MS technology misidentified one isolate as Candida lusitaniae, another as C. haemulonii and could not identify the other six. C. auris identification was confirmed by ITS rDNA sequencing. All isolates were fluconazole (MIC >256mg/l) and voriconazole (MIC 2mg/l) resistant and susceptible to posaconazole, itraconazole, echinocandins and amphotericin B. ConclusionsC. auris should be regarded as an emerging pathogen, which requires molecular methods for definitive identification. Our isolates were highly resistant to fluconazole and resistant to voriconazole, but susceptible to the other antifungals tested, which emphasizes the importance of accurately identifying this species to avoid therapeutic failures.

Different classes of hydrolytic enzymes produced by multidrug-resistant yeasts comprising the Candida haemulonii complex.

The production of enzymes in clinical isolates of Candida haemulonii (Ch, n = 5), Candida duobushaemulonii (Cd, n = 4) and Candida haemulonii var. vulnera (Chv, n = 3) was identified by agar plate. Aspartic protease, phytase, caseinolytic and hemolytic activities were detected in all the isolates. A distinct scenario was evidenced regarding the production of lipases. In this way, 80%, 50% and 100% of Ch, Cd and Chv, respectively, were phospholipase producers. Regarding esterase activity, 100%, 50% and 66.7% of Ch, Cd and Chv, respectively, were positive isolates. Esterase activity was significantly higher in isolates recovered from cutaneous candidiasis compared with those recovered from body fluids.

Identification and Antifungal Susceptibility Profiles of Candida haemulonii Species Complex Clinical Isolates from a Multicenter Study in China

Candida haemulonii complex (Candida haemulonii, Candida haemulonii var. vulnera, and Candida duobushaemulonii) consists of emerging pathogens. Thirty-one isolates from 14 hospitals in China were studied for their species classification and antifungal susceptibilities. Performances of molecular (i.e., ribosomal DNA [rDNA] internal transcribed spacer [ITS] sequencing, D1/D2 sequencing, and ITS sequencer-based capillary gel electrophoresis [SCGE]) and phenotypic identification methods in species identification were compared. Twenty-six (83.9%) of 31 isolates were identified as C. haemulonii and 5 isolates were identified as C. duobushaemulonii by ITS sequencing as the reference method; results obtained by D1/D2 sequencing and ITS SCGE were concordant with those obtained by ITS sequencing for all (100%) of the isolates. All 31 isolates were identified as C. haemulonii by the Vitek matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system (bioMérieux, France), whereas the Bruker MS system (Bruker Daltoniks, Germany) correctly provided species identification for 77.4% and 100% of isolates using cutoff scores for species of ≥2.0 and ≥1.70, respectively. The Vitek 2 compact (bioMérieux) only identified 9 (29%) of 31 isolates. All isolates showed high MICs for amphotericin B (range, 2 to >8 μg/ml) and fluconazole (≥128 μg/ml) but low MICs (≤0.5 μg/ml) for the echinocandins. Our results reinforce the need for MALDI-TOF MS and/or molecular differentiation of species within the C. haemulonii complex. The multiresistant antifungal susceptibility profile of these isolates represents a challenge to therapy.

Aspectos epidemiológicos de la sepsis, en unidades de cuidados intensivos Santa Marta, Colombia

Determinar los aspectos epidemiológicos relacionados con sepsis en los pacientes atendidos en las unidades de cuidados intensivos de adultos de Santa Marta. Se realizó un estudio descriptivo transversal en el año 2014; muestreo aleatorio simple, en pacientes atendidos en unidades de cuidado intensivo adulto (UCIA) en Santa Marta. Se utilizó una muestra de 100 pacientes mayores de 18 años con diagnóstico de sepsis, las variables estudiadas fueron: edad, sexo, agente causal, principales focos de infección entre otros. Como resultado se encontró que la edad promedio de los casos fue de 63, los principales microorganismos causales fueron las bacterias gram negativas con un 76%, siendo la Echerichia coli, Klebsiella pneumoniae, Pseudomona aureuginosa y Serratia marcensens las más aisladas, en segundo lugar, las bacterias gram positivas con un 20%, principalmente el Staphylococcus aureus, y por último los hongos con un 4%, siendo Candida albicans y Candida haemulonii las levaduras causantes. Los focos infecciosos más frecuentes son; el abdominal (30%), seguido del tracto urinario (22%) y la piel y tejidos blandos (18%). Se concluye que los principales agentes causales de sepsis son las bacterias gram negativas y el principal foco primario infeccioso es la sepsis de origen abdominal.

Identification of yeasts isolated from raffia wine (Raphia hookeri) produced in Côte d'Ivoire and genotyping of Saccharomyces cerevisiae strains by PCR inter-delta.

Raffia wine is a traditional alcoholic beverage produced in several African countries where it plays a significant role in traditional customs and population diet. Alcoholic fermentation of this beverage is ensured by a complex natural yeast flora which plays a decisive role in the quality of the final product. This present study aims to evaluate the distribution and the diversity of the yeast strains isolated in raffia wine from four sampling areas (Abengourou, Alépé, Grand-Lahou and Adzopé) in Côte d'Ivoire. Based on the D1/D2 domain of the LSU rDNA sequence analysis, nine species belonging to six genera were distinguished. With a percentage of 69.5% out of 171 yeast isolates, Saccharomyces cerevisiae was the predominant species in the raffia wine, followed by Kodamaea ohmeri (20.4%). The other species isolated were Candida haemulonii (4.1%), Candida phangngensis (1.8%), Pichia kudriavzevii (1.2%), Hanseniaspora jakobsenii (1.2%), Candida silvae (0.6%), Hanseniaspora guilliermondii (0.6%) and Meyerozyma caribbica (0.6%). The molecular characterization of S. cerevisiae isolates at the strain level using the PCR-interdelta method revealed the presence of 21 profiles (named I to XXI) within 115 isolates. Only four profiles (I, III, V and XI) were shared by the four areas under study. Phenotypic characterization of K. ohmeri strains showed two subgroups for sugar fermentation and no diversity for the nitrogen compound assimilations and the growth at different temperatures.

Identification of Candida haemulonii Complex Species: Use of ClinProTools(TM) to Overcome Limitations of the Bruker Biotyper(TM), VITEK MS(TM) IVD, and VITEK MS(TM) RUO Databases.

Candida haemulonii is now considered a complex of two species and one variety: C. haemulonii sensu stricto, Candida duobushaemulonii and the variety C. haemulonii var. vulnera. Identification (ID) of these species is relevant for epidemiological purposes and for therapeutic management, but the different phenotypic commercial systems are unable to provide correct species ID for these emergent pathogens. Hence, we evaluated the MALDI-TOF MS performance for the ID of C. haemulonii species, analyzing isolates/strains of C. haemulonii complex species, Candida pseudohaemulonii and Candida auris by two commercial platforms, their databases and softwares. To differentiate C. haemulonii sensu sctricto from the variety vulnera, we used the ClinProToolsTM models and a single-peak analysis with the software FlexAnalysisTM. The BiotyperTM database gave 100% correct species ID for C. haemulonii sensu stricto, C. pseudohaemulonii and C. auris, with 69% of correct species ID for C. duobushaemulonii. Vitek MSTM IVD database gave 100% correct species ID for C. haemulonii sensu stricto, misidentifying all C. duobushaemulonii and C. pseudohaemulonii as C. haemulonii, being unable to identify C. auris. The Vitek MSTM RUO database needed to be upgraded with in-house SuperSpectra to discriminate C. haemulonii sensu stricto, C. duobushaemulonii, C. pseudohaemulonii, and C. auris strains/isolates. The generic algorithm model from ClinProToolsTM software showed recognition capability of 100% and cross validation of 98.02% for the discrimination of C. haemulonii sensu stricto from the variety vulnera. Single-peak analysis showed that the peaks 5670, 6878, or 13750 m/z can distinguish C. haemulonii sensu stricto from the variety vulnera.

Susceptibility to antifungal agents and enzymatic activity of Candida haemulonii and Cutaneotrichosporon dermatis isolated from soft corals on the Brazilian reefs.

Candida is a common fungus with the capacity to cause infections in humans. However, most studies have concentrated on clinical isolates and little is known about the identity, ecology and drug resistance of free living species/strains. Here, we isolate eight strains of Candida haemulonii and four strains of Cutaneotrichosporon dermatis from three marine cnidarian zoanthids species (Palythoa caribaeorum, Palythoa variabilis and Zoanthus sociatus) collected from Brazilian coral reefs. Strains were identified by sequencing of the D1/D2 domain LSU rDNA and ITS region. We tested these environmental isolates for their capacity to grow in media with increasing concentration of NaCl, capacity to grow in different temperatures, enzymatic activity and antifungal susceptibility. For C. haemulonii, all strains strongly produced gelatinase, esterase and albuminase and were either able to express lipase, phospholipase and keratinase, but not express urease and DNase. The strains were able to grow at 37°C, but not at 39°C, and except for LMS 40, all of them could grow in a 10% NaCl medium. All isolates were resistant to all antifungals tested, with exception for ketoconazole and tioconazole (MIC=2µg/mL). For C. dermatis, all strains could grow at 39°C and could not express phospholipase, keratinase or gelatinase. However, all were capable of expressing urease, lipase and esterase. Three out of four strains could grow in a 10% NaCl medium, but none grew in a 30% NaCl medium. The strains showed high values of minimal inhibitory concentration. LMPV 90 was resistant to tioconazole, terbinafine, fluconazole and posaconazole, and LMS 38 was resistant to all antifungal agents tested. We discuss the characterization of C. haemulonii and C. dermatis as a possible emerging pathogen due to its animal-related enzymatic arsenal and antifungal resistance.

Detailed MALDI-TOF Mass spectrometry analysis for the discrimination of Candida haemulonii complex species

Introduction Candida haemulonii is now considered to be a complex of two species and one variety : C. haemulonii , Candida duobushaemulonii and the variety Candida haemulonii var. vulnera . The correct identification (ID) of these species is clinically relevant, since resistance to azole derivatives has been reported and amphotericin B has poor in vitro activity against C. duobushaemulonii isolates. Initial evaluation using MALDI-TOF MS for the ID of the C. haemulonii complex species provided promising results, but the discrimination of C. haemulonii from the variety vulnera was problematic, and the performance of the VITEK MS remains unevaluated. Objectives Evaluation of the MALDI-TOF MS performance for the ID of C. haemulonii species ID with two platforms and its’ databases and softwares. Methods 14 C. haemulonii sensu stricto , 9 C. haemulonii var. vulnera and 10 C. duobushaemulonii from hospitals belonging to the Medical school from the University of Sao Paulo were analyzed (ID by ITS1 sequence analysis). A set of reference strains from the CBS-KNAW collection was also included : C. haemulonii CBS5149 T, C. duobushaemulonii CBS7798 T, C. duobushaemulonii CBS7799, and for specificity control, the close related species Candida pseudohaemulonii (CBS10004 and CBS12370). The isolates/strains were cultured on Sabouraud plates and incubated for 48 h at 30 °C before MALDI-TOF MS analysis. Protein extraction protocol with ethanol and formic acid 70 % was carried out according to the Bruker's recommendations. Measurements were performed on a Microflex LT and its standard database (database 3.3.1), and VITEK MS instrument equipped with both IVD and RUO (SARAMIS) databases. To differentiate C. haemulonii (target group) from the variety vulnera (control group), mass spectra analysis models were created with the ClinProTools 3.0 software (Bruker). For each model, the recognition capability (RC) and cross validation (CV) percentage was generated. For single peak analysis, the AUC of each peak for the discrimination of target group from control group was directly obtained from the ClinProTools software. For the peaks with the highest AUC, the detection performances were checked using FlexAnalysis 3.4 (Bruker Daltonics). The signal-to-noise ratios (SN) of the peaks were exported to SPSS 18.0, ROC curves were constructed, and their optimal cut-off values were determined. Results All C. haemulonii sensu stricto isolates/strain had correct species assignment by both Bruker and VITEK MS IVD databases. For the species C. duobushaemulonii, the Bruker database gave correct species ID for 75 % of the isolates/strains. VITEK MS IVD analysis misidentified all C. duobushaemulonii and C. pseudohaemulonii isolates/strains as C. haemulonii (99 % of confidence level). The VITEK MS SARAMIS was unable to give genus/species ID for all isolates/strains. Discrimination of the isolates belonging to the C. haemulonii sensu stricto and to the variety vulnera was not possible with all systems and databases. The ClinProTools models showed values of CV and RC above ≥90 % for the discrimination of target group from control group. The most discriminative peaks were 5107, 6878 and 13,750 m/z (AUC >0,9), with sensibility and specificity of 88,6 %, 80,8 %, 85,8 %, and 96,6 %, 92,9 %, 93,8 %, respectively. The SN cut-off values from the peaks 5107, 6878 and 13,750 m/z for the discrimination of the target group from the control group were 2,5, 3,9 and 6,3, respectively.