Identification of Candida species isolated from patients in intensive care unit and in vitro susceptibility to fluconazole for a 3-year period

Species level identification of Candida and antifungal susceptibility testing is not generally performed in routine laboratory practice. There is limited information about the distribution of Candida species and antifungal susceptibility in Turkey. In this study, we aimed at identifying Candida isolates to species level from various samples obtained from patients treated in an intensive care unit between 2002 and 2005 and to evaluate fluconazole susceptibilities of the isolates. A total of 320 Candida isolates obtained from 270 patients were identified by conventional methods and using API (Candida and/or 20C AUX) system. Antifungal susceptibility testing was performed by broth microdilution method. Candida albicans was isolated with the highest frequency (65.6%) followed by C. parapsilosis (11.3%), C. glabrata (8.8%) and C. tropicalis (7.8%). Of all the isolates, 92.9% revealed susceptibility to fluconazole. Susceptibility to fluconazole was highest for C. albicans followed by C. parapsilosis and C. glabrata. The MIC(90) values for C. albicans, C. parapsilosis, C. glabrata and C. tropicalis were 1, 2, 8 and 4 mug ml(-1) respectively. Fluconazole remains effective against both C. albicans and the majority of non-albicans Candida species. In this study, we determine the distribution of Candida species and evaluate the susceptibilities of the isolates, particularly for the azoles.     

Potent anti-microbial activity of traditional Chinese medicine herbs against Candida species

Anti-candidial activities of eight traditional Chinese medicinal (TCM) herbs were evaluated against six different Candida species. TCM preparations were screened for antifungal activity using a standard agar diffusion assay. Following identification of potential candidate herbs, their minimum inhibitory concentration (MIC) values were determined using the standardised NCCLS M-27A broth microdilution assay. Among TCM herbs, Rhizoma Coptidis had potent antifungal activity against Candida glabrata, Candida krusei and Candida tropicalis, but not against Candida albicans, Candida dubliniensis and Candida parapsolosis. The MIC values of the Rhizoma Coptidis against C. glabrata, C. krusei and C. tropicalis were 50, 50 and 100 microg ml(-1) respectively. We report here, for the first time, the potent antifungal activity of Rhizoma Coptidis and Cortex phellodendri Chinesis on three different non-albicans Candida species, C. glabrata, C. krusei and C. tropicalis and hence their possible use as therapeutic agents.     

In vitro susceptibility of 545 isolates of Candida spp. to four antifungal agents

Summary. The in vitro susceptibility to amphotericin B, fluconazole, itraconazole and ketoconazole of 545 Candida strains from patients treated at the University Hospital of the Canaries was determined by means of a microdilution test. The distribution of the species was as follows: Candida albicans (342), Candida tropicalis (70), Candida glabrata (68), Candida parapsilosis (65). Of Candida albicans isolates, 8.5% and 7.6% showed resistance to itraconazole and fluconazole respectively. Of C. tropicalis isolates 34.3%, 27.1% and 2.9% were resistant to itraconazole, fluconazole and ketaconazole respectively. For C. glabrata , 10.3% and 4.4% of the isolates under study demonstrated resistance to fluconazole and itraconazole respectively. Only 4.6% and 1.5% of C. parapsilosis isolates demonstrated resistance to fluconazole and itraconazole respectively. C. tropicalis was the most resistant strain and C. parapsilosis the most sensitive. The greatest percentages of resistance in vitro were seen with the triazoles.
Zusammenfassung. Es wurde die Empfindlichkeit von 545 Candida -Stämmen für Amphotericin B, Fluconazol, Itraconazol und Ketoconazol in vitro in einem Mikrodilutionstest bestimmt. Bei den Stämmen handelte es sich um Isolate von Patienten, die in der Universitäts-Klinik der Kanarischen Inseln behandelt worden waren. Das Untersuchungsgut war wie folgt verteilt: 342 Candida albicans , 70 C. tropicalis , 68 C. glabrata , 65 C. parapsilosis. Bei C. albicans waren 8.5% gegen Itraconazol und 7.6% gegen Fluconazol resistent. Bei C. tropicalis wurden 34.3% resistent gegenüber Itraconazol befundet, 27.1% gegen Fluconazol und 2.9% gegen Ketoconazol. Bei C. glabrata waren 10.3% resistent gegen Fluconazol und 4.4% gegen Itraconazol. Candida parapsilosis wurde zu 4.6% gegen Fluconazol und zu 1.5% gegen Itraconazol als resistent befundet. Somit erwies sich C. tropicalis als die resistenteste und C. parapsilosis als die sensibelste Art.     

Molecular epidemiology of Candida species isolated from urine at an intensive care unit

Candida spp. has been the leading microorganism isolated from the urine specimens of patients hospitalized at the Anesthesiology and Reanimation intensive care unit (ICU) of Dokuz Eylul University Hospital, Izmir, since 1998. This study was undertaken to investigate the clonal relationship of Candida urine isolates in order to find the mode of spread among the patients. Epidemiological surveillance of 38 Candida albicans, 15 Candida tropicalis and 12 Candida glabrata recovered from the urine specimens of patients who were hospitalized in the ICU between June 11, 2000 and October 15, 2001 was carried out by antifungal susceptibility testing and randomly amplified polymorphic DNA (RAPD) analysis. Two short primers [Cnd3 (5'-CCAGATGCAC-3') and Cnd4 (5'-ACGGTACACT-3')] were used for RAPD. None of the isolates had high minimal inhibitory concentration (MIC) values (>1 microg ml(-1)) against amphotericin B with MIC50 values of 0.5 microg ml(-1), 0.5 microg ml(-1) and 0.125 microg ml(-1) for C. albicans, C. tropicalis and C. glabrata isolates, respectively. However, three C. glabrata isolates were resistant and one C. albicans and five C. glabrata isolates were dose-dependent susceptible (D-DS) to fluconazole. Among C. albicans isolates 19 and 20 patterns were detected with primers Cnd3 and Cnd4, respectively. When primers Cnd3 and Cnd4 were evaluated together, three and four genotypes were identified for C. tropicalis and C. glabrata isolates, respectively. Our results suggest that the source of C. albicans isolates was mostly endogenous. It is difficult to interpret the mode of spread of C. tropicalis and C. glabrata urine isolates as we obtained insufficient banding patterns for these species.     

Detection of Candida dubliniensis in oropharyngeal samples from human immunodeficiency virus infected and non-infected patients and in a yeast culture collection

The incidence of Candida dubliniensis in oropharyngeal swabs of 132 human immunodeficiency virus (HIV)-positive and 89 HIV-negative patients was determined. The samples were plated onto CHROMagar Candida medium and 82 strains, presumptively identified as C. albicans or C. dubliniensis, were further investigated (temperature test, chlamydoconidia production, specific primer PCR). In addition, 487 collection strains (isolated from clinical samples and previously identified as C. albicans on the basis of a positive germ tube test) were screened in order to identify C. dubliniensis isolates. Two C. dubliniensis strains were isolated from two HIV-positive patients without oral candidiasis. Candida dubliniensis was not isolated from 89 HIV-negative patients nor was it identified among the collection strains.     

Oropharyngeal carriage of Candida species in HIV-infected patients in India

The present investigation represents the first study of oropharyngeal carriage of Candida and other yeasts in HIV-infected patients in India. One hundred and fifty HIV-positive patients were investigated by culturing their swish samples on plates of CHROMagar Candida. Ninety-eight patients (65.3%) were positive for Candida and four (2.7%) were positive for other yeasts. Among them, the first Indian C. dubliniensis isolate has been recovered. Molecular typing of selected C. albicans isolates by AP-PCR revealed two major genotypes based on the banding patterns. The susceptibilities of 30 Candida isolates to five antifungal agents including the new triazole voriconazole were determined in a micro-dilution test, according to the NCCLS protocol M 27. All the 22 C. albicans isolates were susceptible to five antimycotic agents (flucytosine, amphotericin B, fluconazole, voriconazole and itraconazole) except one isolate (VPCI-122), which was resistant to flucytosine (MIC > or = 64 mg l-1). The azole-resistant isolates reported here endorse the role of antifungal susceptibility testing whenever antifungal treatment with azoles is planned.     

Etiology of fungaemia and catheter colonisation in Argentinean paediatric patients

Yeast strains obtained from blood cultures and catheters from intensive care units (ICU) and hospitalised oncology paediatrics were studied. Yeast were the first cause of catheter colonisation (51/627), and the third cause of bloodstream infection (44/6065). In catheter, the most frequent species were Candida albicans (34%), C. parapsilosis (27.7%) and C. tropicalis (15%). In blood, C. albicans (40.8%), C. parapsilosis (26.6%), C. tropicalis (15%). Malassezia furfur and Malassezia sympodialis were isolated from catheters from ICU patients. All isolates were susceptible to amphotericin B, 88.8% to itraconazole and 91.9% to fluconazole. Candida albicans and C. tropicalis strains resistant to fluconazole and itraconazol were detected. These results reveal a change in the predominant role of C. albicans as cause of candidemia in hospitalised children and the emergence of antifungal resistant species. These variations emphasise the importance of performing a permanent surveillance to observe and assess them.     

Fluconazole and itraconazole susceptibilities of Candida spp. isolated from oropharyngeal specimens and blood cultures of paediatric haematology/oncology patients

This study examined the in vitro susceptibilities to fluconazole and itraconazole of isolates of Candida spp. from surveillance oropharyngeal specimens and blood cultures from paediatric patients with malignancy. The species distribution of 100 isolates from oropharyngeal specimens was C. albicans 86%, C. glabrata 7%, C. lusitaniae 4%, C. parapsilosis 2% and C. tropicalis 1%. From a total of nine isolates from blood cultures the species distribution was C. albicans 33.3%, C. parapsilosis 33.3 % and C. guilliermondii 33.3%. Only three of the oropharyngeal isolates were resistant to fluconazole (MIC > or = 64 mg l(-1)) and only two were resistant to itraconazole (MIC > or = 1 mg l(-1)). None of the blood culture isolates was resistant to either agent. At this centre, C. albicans is the predominant species from oropharyngeal specimens, but non-albicans Candida species predominate in blood cultures. Although resistance to fluconazole and itraconazole is rare at present, continued surveillance is warranted to monitor trends in species distribution and antifungal susceptibility.     

Vergleich von drei Antimykotika-Empfindlichkeitsprüfungs-Methoden für die In-vitro-Testung von Candida-Isolaten von Patienten mit HIV-Infektion

Zusammenfassung. Es wurden die MHK-Werte für Fluconazol von 90 Candida -Isolaten (56 C. albicans , 15 C. glabrata , 9 C. krusei und 10 C. tropicalis ) untersucht. Zur Antimykotika-Resistenztestung wurden die Mikrodilutionsmethoden nach dem Protokoll M27-P des NCCLS (M27-Pmicro) mit RPMI 1640-Medium sowie der Methode nach Troke & Pye mit HR-Medium (HRmicro) als auch die Agardiffusionsmethode mit dem Etest (YNB-Agar) verwendet. Die Ergebnisse mit diesen Methoden wurden mit den klinischen Befunden verglichen. Sämtliche C. albicans -Isolate von AIDS-Patienten mit Fluconazol-refraktärer Candidose hatten MHK-Werte entweder ≥ 6,25 μg/ml (M27-Pmicro) oder ≥ 25 μg/ml (HRmicro und Etest). Andererseits lagen die MHK-Werte von C. albicans -Isolaten bei AIDS-Patienten mit Fluconazol-empfindlicher Candidose teil-weise ebenfalls über diesen Endpunkten. Ein mögliches Ansprechen auf eine Fluconazol-Therapie kann somit nicht zwangsläufig von dem MHK-Wert abgeleitet werden.
Summary. The MIC values of fluconazole were determined for 96 Candida isolates (56 C. albicans , 15 C. glabrata , 9 C. krusei and 10 C. tropicalis ). The methods employed for antifungal susceptibility testing were: microdilution according to the protocol M27-P of the NCCLS (M 27-Pmicro) using RPMI 1640 medium or HR medium following Troke & Pye (HRmicro) as well as the agar diffusion method by means of the Etest (YNB agar). The in vitro results were compared with the clinical outcome of patients. All C. albicans isolates received from AIDS patients with fluconazole-refractory candidosis showed MICs of ≥ 6,25 mg/ml (M27-Pmicro) or ≥ 25 mg/ml (MRmicro) and Etest). On the other hand some MICs of C. albicans isolates from AIDS patients with fluconazole-sensitive candidosis were also beyond these breakpoints. Therefore a possible success of a fluconazole therapy cannot unequivocally be predicted from the MIC value determined in vitro.     

Prevalence and phenotypic evaluation of Candida dubliniensis in pregnant women with vulvovaginal candidosis in a university hospital in Ankara

Candida dubliniensis is very similar to Candida albicans in terms of genotypic and phenotypic characteristics. As the hormonal milieu of the vagina during pregnancy, characterised by a lack of maternal cell-mediated immunity, enhances Candida colonisation and serves as a risk factor for symptomatic expression, investigation into the isolation of C. dubliniensis in vaginal discharges of pregnant women with vulvovaginal candidosis was made. A total of 77 Candida isolates obtained from 60 patients positive for vulvovaginal candidosis collected from 218 pregnant women were investigated for C. dubliniensis subsistence. In total 41 Candida species phenotypically identified as C. albicans on the basis of a positive germ tube test and carbohydrate assimilation tests were screened for the presence of C. dubliniensis. Phenotypic tests for differentiation of C. dubliniensis from C. albicans, such as growth at 42 and 45 degrees C on Sabouraud dextrose agar, appearance on CHROMagar and colony morphology on Cornmeal-Tween-80 agar and Staib agar were carried out. Only one strain (2.43%) was phenotypically identified as C. dubliniensis. According to our study, a combination of at least five phenotypic methods is necessary for an exact diagnosis of C. dubliniensis. Large-scale studies of pregnant women are required to discover the aetiological importance of this yeast.     

Yeasts species distribution in Neonatal Intensive Care Units in northeast Argentina

The aim of this study was to determine the distribution and antifungal susceptibility profile of yeast species isolated from neonates in Neonatal Intensive Care Units (NICU) in northeast of Argentina. With this purpose 92 strains isolated from 25 blood stream cultures, 20 venous catheters, 23 suprapubic aspirations and 24 rectal swabs were studied. Candida albicans and C. parapsilosis appeared with similar frequencies (36%) in blood stream isolates. Candida parapsilosis (50%) was the most frequent catheters colonizer and C. tropicalis (54.2%) was the most frequent yeast associated with gastrointestinal tract colonization. Candida krusei, C. glabrata and Trichosporon cutaneum appeared with a very low frequency. A high rate of susceptibility to amphotericin B, fluconazole, and itraconazole was observed.     

Genotypic relatedness of yeast isolates from women infected with human immunodeficiency virus and their children.

The objective of this study was to compare polymerase chain reaction (PCR) fingerprinting with other molecular typing methods as an epidemiologic tool to investigate the transmission of Candida strains between HIV-positive mothers and their children. Forty-nine yeast strains (including Candida albicans, Candida glabrata, Rhodotorula rubra, Candida tropicalis, Candida famata, Candida dubliniensis, Saccharomyces cerevisiae) from 30 individuals (15 children and 15 HIV-infected mothers or accompanying person) were isolated. Colonization/infection with yeast was observed in 80% of all individuals in the oral cavity, and in 33% from hand cultures, respectively. Thirteen out of 15 children (86%) and 12 out of 15 adults (80%) were colonized/infected with yeasts. Candida dubliniensis strains were found in four HIV-infected women but not in children. The results with an arbitrarily primed (AP)-PCR mediated genotyping assay using phage M13 core sequence were compared with the hybridization patterns using the species-specific DNA probe CARE-2 for the C. albicans isolates. Typing of non-C. albicans strains was done using AP-PCR in comparison with pulsed-field gel electrophoresis (PFGE). Twenty-six C. albicans strains gave two different genotypes by AP-PCR but 16 genotypes by CARE-2 hybridization. The CARE-2 probe appeared to have a higher discriminatory power compared with the primer 'M13'-mediated AP-PCR in typing C. albicans isolates.     

Detection of Candida dubliniensis in oropharyngeal samples of Turkish HIV-positive patients

The incidence of Candida dubliniensis in immunocomprimised patients in Turkey has not yet been determined. In this study the presence of C. dubliniensis in oral rinse samples of human immunodeficiency virus (HIV)-positive patients and healthy controls were investigated. Phenotypic tests like inability of growth at 45 degrees C, colony formation on Staib agar, intracellular beta-D-glucosidase activity, carbohydrate assimilation profiles and polymerase chain reaction with species-specific primers (DUBF and DUBR) were carried out for differentiation of C. dubliniensis. Of the 35 patients, four (11.4%) had C.dubliniensis in their oral cavity. Antifungal susceptibility testing of these C. dubliniensis isolates showed fluconazole MICs ranging from <0.06 to 32 microg ml(-1) and amphotericin B from <0.06 to 0.25 microg ml(-1). One isolate was dose-dependently susceptible to fluconazole (32 microg ml(-1)). This study demonstrates C. dubliniensis in HIV-positive patients from Turkey.     

Candidaemia in a London teaching hospital: analysis of 128 cases over a 7-year period

In a retrospective analysis of 128 cases of Candida bloodstream infections in a London teaching hospital between 1995 and 2001, the incidence of candidaemia increased from 0.2/1000 admissions in 1995 to 0.5 and 0.4/1000 admissions in 2000 and 2001, respectively. Risk factors for candidaemia included the presence of intravascular (IV) lines (88%), admission to intensive care (51%), parenteral nutrition (35%), multiple antibiotics (74%), corticosteroid therapy (12%), cancer chemotherapy (11%), renal transplantation (5%) and neutropenia (3%). The sources of infection were IV lines (77%), the urinary tract (7%) and the gastrointestinal tract (7%). Serious infective complications (endocarditis, endophthalmitis or brain abscess) were noted in 6% of cases. The most frequently isolated species were Candida albicans (64%), C. glabrata (20%), C. tropicalis (9%) and C. parapsilosis (5%). The overall fluconazole-resistance rate of Candida spp. was 7% (MIC > or = 64 mg l-1). All the C. albicans isolates were sensitive to fluconazole (MIC < or = 8 mg l-1) whereas 20% of non-C. albicans isolates (27% of C. glabrata and 14%C. tropicalis) were resistant. The mortality rate (35%) was lower than in other reports and may be due to the early recognition of candidaemia and the prompt removal of IV lines together with the initiation of appropriate antifungal therapy. Regular surveillance of local Candida species, resistance profiles and risk factors is important in order to identify patients at risk and to develop empirical treatment protocols to reduce the incidence and mortality of candidaemia.     

Emergence of Candida tropicalis as the major cause of fungaemia in India.

The fungal isolates (n = 191) from 1970 consecutive blood cultures over a 9-year period were identified. Candida tropicalis was the predominant isolate accounting for 66% of all isolates whereas Candida albicans ranked only a distant second with 21%. The difference was highly significant (P < 0.00001) The other isolates included C. parapsilosis (5.8%), C. glabrata (2.1%), C. pelliculosa (1.2%), C. lipolytica (1.0%) C. krusei (1.0%) and Trichosporon beigelii (2.6%). The isolates were obtained predominantly from postoperative patients on therapy with more than one antibiotic.     

Evaluation of CHROMagar Candida for rapid screening of clinical specimens for Candida species

CHROMagar Candida is a new differential culture medium that allows selective isolation of yeasts and simultaneously identifies colonies of Candida albicans, Candida glabrata, Candida tropicalis and Candida krusei. We evaluated this medium and compared it with a reference medium, Sabouraud glucose agar, for the presumptive identification of yeast species isolated directly on the medium from 1150 clinical specimens. A total of 731 specimens showed no growth, 299 isolates (70.2%) showed growth to the same extent on both media. Forty mixed cultures were detected on both media. More than one isolate was detected in 30 of the tested specimens on either CHROMagar (26 specimens) or Sabouraud glucose agar (four specimens). We found a sensitivity of 98.8% and a specificity of 100% for C. albicans, 66.7% and 99.8% for C. tropicalis, 100% and 100% for C. krusei, and 98% and 95.7% for C. glabrata. Regarding these results, CHROMagar Candida is recommended as a useful isolation medium capable of the presumptive identification of yeasts and better detection of mixed cultures in clinical specimens.     

In Vitro Antifungal Activity of Saperconazole (R 66905) Against Candida and Torulopsis Die antimyzetische Aktivität von Saperconazol (R 66905) in vitro gegen Candida und Torulopsis

The susceptibility of several strains of Candida and Torulopsis to saperconazole, a new triazole antifungal compound, was compared to that of ketoconazole. The MICs of the two antifungal agents were determined against 70 strains of Candida albicans, 10 strains of C. guilliermondii, 10 strains of C. krusei, 10 strains of C. parapsilosis, 10 strains of C. pseudotropicalis, 10 strains of C. tropicalis and 15 strains of Torulopsis glabrata. The fungistatic activity was evaluated by the agar dilution method using BHI and casitone media after incubation for 48 hours at 28-30 degrees C. The in vitro activity of saperconazole was similar to that of ketoconazole for most of the Candida spp. tested except for C. krusei in particular. An MIC of less than or equal to 3.12 micrograms/ml for saperconazole was found with 92% of the C. albicans strains tested. In contrast, T. glabrata was more susceptible to ketoconazole.     

Colony Variations in Candida Species

One hundred isolates of seven Candida species and three isolates of Saccharomyces cerevisiae were plated on phloxine B agar and examined for variations in the morphology or colour of colonies that developed. Colony variations were found in 9 of 12 C. parapsilosis isolates, 8 of 13 C. tropicalis isolates, 4 of 9 C. krusei isolates and 12 of 30 C. albicans isolates. None of 23 C. glabrata isolates grew on the test medium. Variant colonies often generated further different colony forms on secondary subculture. The rate of production of fimbriate and rhizoid colonies by two C. albicans isolates varied with agar thickness and the nutrient content of the medium. These results suggest that colony variation is a common property among isolates of many Candida species and that strict control of agar medium thickness and composition is essential for reproducible screening of isolates for colony variations.     

Initial Candida dubliniensis isolate in Candida spp. positive haemocultures in Turkey between 2001 and 2004

Candida dubliniensis which was first recognized in 1995 can be easily misidentified because of its phenotypic similarities with Candida albicans. In this study blood samples of patients from various departments of Ankara University Medical Faculty between January 2001-June 2004 were investigated for the distribution of Candida spp. and the presence of C. dubliniensis. Culture positive 67 fungi were included to the study. Phenotypic tests such as chlamydospore formation, colony morphology on Staib agar, growth at 45 degrees C, carbohydrate assimilation profiles were investigated for identification and differentiation of C. dubliniensis from C. albicans. To confirm the results polymerase chain reaction were used for suspected C. albicans and C. dubliniensis isolates. Among 38 germ tube and chlamydospore forming isolates, 37 of them were found as C. albicans and one as C. dubliniensis. The incidence of C. dubliniensis in our hospital is still low, this is the first C. dubliniensis isolate as an agent of candidaemia reported from Turkey.