Seung Jung Kee

Biofilm Production by Isolates of Candida Species Recovered from Nonneutropenic Patients: Comparison of Bloodstream Isolates with Isolates from Other Sources

ABSTRACT Biofilm production has been implicated as a potential virulence factor of some Candida species responsible for catheter-related fungemia in patients receiving parenteral nutrition. We therefore compared clinical bloodstream isolates representing seven different Candida species to each other and to those from other anatomical sites for the capacity to form biofilms in glucose-containing medium. Potential associations between the capacity to form biofilms and the clinical characteristics of fungemia were also analyzed. Isolates included the following from nonneutropenic patients: 101 bloodstream isolates (35 C. parapsilosis, 30 C. albicans, 18 C. tropicalis, 8 C. glabrata, and 10 other Candida species isolates) and 259 clinical isolates from other body sites (116 C. albicans, 53 C. glabrata, 43 C. tropicalis, 17 C. parapsilosis, and 30 other Candida species isolates). Organisms were grown in Sabouraud dextrose broth (SDB) containing a final concentration of 8% glucose to induce biofilm formation, as published previously. Biofilm production was determined by both visual and spectrophotometric methods. In this medium, biofilm production by C. albicans isolates was significantly less frequent (8%) than that by non-C. albicans Candida species (61%; P < 0.0001). The overall proportion of non-C. albicans Candida species isolates from the blood that produced biofilms was significantly higher than that of non-C. albicans Candida isolates obtained from other sites (79% versus 52%; P = 0.0001). Bloodstream isolates of C. parapsilosis alone were significantly more likely to be biofilm positive than were C. parapsilosis isolates from other sites (86% versus 47%; P = 0.0032). Non-C. albicans Candida species, including C. parapsilosis, were more likely to be biofilm positive if isolates were derived from patients whose candidemia was central venous catheter (CVC) related (95%; P < 0.0001) and was associated with the use of total parenteral nutrition (TPN) (94%; P < 0.005). These data suggest that the capacity of Candida species isolates to produce biofilms in vitro in glucose-containing SDB may be a reflection of the pathogenic potential of these isolates to cause CVC-related fungemia in patients receiving TPN.

Candida haemulonii and Closely Related Species at 5 University Hospitals in Korea: Identification, Antifungal Susceptibility, and Clinical Features

Background. Candida haemulonii, a yeast species that often exhibits antifungal resistance, rarely causes human infection. During 2004-2006, unusual yeast isolates with phenotypic similarity to C. haemulonii were recovered from 23 patients (8 patients with fungemia and 15 patients with chronic otitis media) in 5 hospitals in Korea. Methods. Isolates were characterized using D1/D2 domain and ITS gene sequencing, and the susceptibility of the isolates to 6 antifungal agents was tested in vitro. Results. Gene sequencing of the blood isolates confirmed C. haemulonii group I (in 1 patient) and Candida pseudohaemulonii (in 7 patients), whereas all isolates recovered from the ear were a novel species of which C. haemulonii is its closest relative. The minimum inhibitory concentration (MIC) ranges of amphotericin B, fluconazole, itraconazole, and voriconazole for all isolates were 0.5-32 microg/mL (MIC(50), 1 microg/mL), 2-128 microg/mL (MIC(50), 4 microg/mL), 0.125-4 microg/mL (MIC(50), 0.25 microg/mL), and 0.03-2 microg/mL (MIC(50), 0.06 microg/mL), respectively. All isolates were susceptible to caspofungin (MIC, 0.125-0.25 microg/mL) and micafungin (MIC, 0.03-0.06 microg/mL). All cases of fungemia occurred in patients with severe underlying diseases who had central venous catheters. Three patients developed breakthrough fungemia while receiving antifungal therapy, and amphotericin B therapeutic failure, which was associated with a high MIC of amphotericin B (32 microg/mL), was observed in 2 patients. Conclusions. Candida species that are closely related to C. haemulonii are emerging sources of infection in Korea. These species show variable patterns of susceptibility to amphotericin B and azole antifungal agents.

Species-Specific Differences in the Susceptibilities of Biofilms Formed by Candida Bloodstream Isolates to Echinocandin Antifungals

ABSTRACT The echinocandin susceptibilities of bloodstream Candida isolates growing in a biofilm was investigated. Within the therapeutic range of concentrations of each drug, caspofungin and micafungin were active against biofilms formed by Candida albicans or C. glabrata but not those formed by C. tropicalis or C. parapsilosis.

Changes in Karyotype and Azole Susceptibility of Sequential Bloodstream Isolates from Patients with Candida glabrata Candidemia

ABSTRACT We examined the changes in genotypes and azole susceptibilities among sequential bloodstream isolates of Candida glabrata during the course of fungemia and the relationship of these changes to antifungal therapy. Forty-one isolates were obtained from 15 patients (9 patients who received antifungal therapy and 6 patients who did not) over periods of up to 36 days. The isolates were analyzed using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) and tested for antifungal susceptibility to fluconazole, itraconazole, and voriconazole. PFGE typing consisted of electrophoretic karyotyping and restriction endonuclease analysis of genomic DNA by use of NotI (REAG-N). The 41 isolates yielded 23 different karyotypes and 11 different REAG-N patterns but only 3 MLST types. The sequential strains from each patient had identical or similar REAG-N patterns. However, they had two or three different karyotypes in 6 (40%) of 15 patients. The isolates from these six patients exhibited the same or similar azole susceptibilities, and five patients did not receive antifungal therapy. Development of acquired azole resistance in sequential isolates was detected for only one patient. For this patient, an isolate of the same genotype obtained after azole therapy showed three- or fourfold increases in the MICs of all three azole antifungals and exhibited increased expression of the CgCDR1 efflux pump. This study shows that karyotypic changes can develop rapidly among sequential bloodstream strains of C. glabrata from the same patient without antifungal therapy. In addition, we confirmed that C. glabrata could acquire azole resistance during the course of fungemia in association with azole therapy.

Microevolution of Candida albicans Strains during Catheter-Related Candidemia

ABSTRACT We examined microevolution in a series of Candida albicans strains isolated from patients with catheter-related candidemia. Sixty-one isolates (29 from blood, 18 from catheters, 10 from urine, and 4 from other sites) were obtained from 15 patients who were admitted to the same hospital over a 3-year period. Isolates were analyzed by using Southern hybridization with the C1 fragment of Ca3 as a probe (C1 fingerprinting) and pulsed-field gel electrophoresis (PFGE). PFGE typing consisted of electrophoretic karyotyping (EK) and restriction endonuclease analysis of genomic DNA (REAG) by using SfiI (REAG-S) and BssHII (REAG-B). When catheter isolates were compared with blood isolates from the same patient, catheter isolates from 5 of 14 patients (36%) exhibited minor band differences (microevolution) relative to blood isolates in either C1 fingerprinting (n = 4), REAG-S (n = 3), or REAG-B (n = 5) profiles, although they had identical EK patterns. However, the other sequential isolates from each patient, which had identical EK patterns, showed the same REAG and C1 fingerprinting patterns. Both fingerprinting methods revealed that two distinct genotypes were shared by isolates from seven patients in a neonatal intensive care unit, suggesting two nosocomial clusters. Except for two catheter isolates from the index patients of each cluster, no consecutive isolates collected from each of the two clusters showed any microevolution during the 2- or 7-month cluster periods. The findings suggest that in catheter-related candidemia, some C. albicans strains undergo microevolution during catheter colonization.

Kodamaea ohmeri Isolates from Patients in a University Hospital: Identification, Antifungal Susceptibility, and Pulsed-Field Gel Electrophoresis Analysis

ABSTRACT Data on clinical isolates of Kodamaea (Pichia) ohmeri, an emerging fungal pathogen, are scarce. Over the past 5 years, we identified yeast isolates from nine patients with fungemia as K. ohmeri by using the API 20C system. Here, we reanalyzed these isolates first by sequencing the internal transcribed spacer 2 (ITS2) regions and then by growing the isolates on CHROMagar Candida medium and subjecting them to pulsed-field gel electrophoresis (PFGE). Based on their ITS2 sequences, six of the nine isolates were confirmed as K. ohmeri, while the others were identified as Candida haemulonii (n = 2) and Candida parapsilosis (n = 1). PFGE karyotyping of the K. ohmeri isolates revealed similar major bands, and their colonies showed a characteristic color change from pink to blue when grown on CHROMagar Candida medium for more than 48 h. For K. ohmeri, the ranges of MICs of fluconazole, voriconazole, caspofungin, and micafungin were 2 to 32 μg/ml, 0.03 to 0.5 μg/ml, 0.125 to 0.25 μg/ml, and 0.03 to 0.06 μg/ml, respectively. Restriction endonuclease analysis of genomic NotI-digested DNA (REAG-N) from isolates from different patients produced unique patterns, suggesting that the fungemia had occurred sporadically. This study determined that ITS2 sequence data, PFGE karyotypes, and CHROMagar Candida chromogenic culture medium are reliable diagnostic tools for identifying K. ohmeri while REAG-N is useful for genotyping the clinical isolates of K. ohmeri.

Electrophoretic Karyotype Analysis of Sequential Candida parapsilosis Isolates from Patients with Persistent or Recurrent Fungemia

ABSTRACT We assessed the genetic relatedness of sequential isolates ofCandida parapsilosis during persistent or recurrent fungemia and the effect of central venous catheter (CVC) removal. Serial isolates of C. parapsilosis were obtained from 17 patients with persistent or recurrent fungemia over periods of up to 5 months. Forty-eight C. parapsilosis isolates from the blood of 17 patients were analyzed by electrophoretic karyotyping (EK) with pulsed-field gel electrophoresis (PFGE), revealing 25 different karyotypes. The strains sequentially isolated from each of seven patients whose fungemia resolved following CVC removal had the same karyotype. Two patients with fungemia that cleared without CVC removal each had two sequential isolates with different karyotypes. In six (75%) of the eight patients whose fungemia was recurrent even after CVC removal, the karyotypes of the pre- and post-CVC removal isolates were different, implying the emergence of a new strain. Overall, the sequential strains from each patient had identical karyotypes in 53% (9 of 17) of the patients and two different karyotypes in 47% (8 of 17). This study shows that EK with PFGE is useful for investigating persistent or recurrent fungemia due to C. parapsilosis and that recurrent fungemia due to C. parapsilosis is more likely caused by reinfection with a second strain.

Differences in biofilm production by three genotypes of Candida parapsilosis from clinical sources

Three distinct genotypes of Candida parapsilosis (group I, II, and III) have been identified among clinical isolates but their clinical significance remains unclear. We investigated the distribution of C. parapsilosis genotypes in isolates from blood, all other sites from patients, and the hands of health care workers (HCWs), and we examined the relationship between genotype and biofilm positivity. The 53 bloodstream isolates and 38 of 39 HCW isolates were categorized as group I, whereas the 67 non-blood isolates taken from patients were distributed in groups I (n=43), II (n=13), and III (n=11). Biofilm positivity was observed in 77% (103 of 134) of group I isolates versus 0% (0 of 25) of non-group I (groups II and III) isolates (P < 0.01). There was no difference in biofilm production among group I isolates from blood (81%), other clinical specimens (72%), and the hands of HCWs (73%). This study has shown that biofilm production differs among three genotypes of C. parapsilosis isolates and that a majority of C. parapsilosis isolates from the bloodstream (100%), the hands of HCWs (97%), and all other sites from patients (64%) belong to group I, which has the ability to produce biofilm.

Expression of CgCDR1, CgCDR2, and CgERG11 in Candida glabrata biofilms formed by bloodstream isolates.

Many studies have shown that Candida biofilms are highly resistant to fluconazole. The mechanisms of acquired fluconazole resistance of Candida glabrata in non-biofilms have been elucidated, but the relevance of them relative to C. glabrata in biofilms are unknown. We examined by real-time PCR the expression of CgCDR1, CgCDR2, and CgERG11 of four bloodstream isolates of C. glabrata during the early (6 h), intermediate (15 h), and mature (48 h) phases of biofilm development. We found high levels of biofilm formation in vitro. At 6 and 15 h, the biofilms exhibited, in comparison to planktonic cells, approximately 1.5- and 3.3-fold upregulation of CgCDR1 and 0.5- and 3.1-fold upregulation of CgCDR2, respectively. However, at 48 h, neither gene was upregulated. In comparison, the expression of CgERG11 did not significantly increase during any of the three phases. Our results indicate a temporary increase in the expression of both CgCDR1 and CgCDR2 during the intermediate phase of C. glabrata biofilm development. In addition, the data collected at 48 h suggest that CgCDR1, CgCDR2, and CgERG11 may not play a role in the azole resistance of C. glabrata in mature biofilms.

Gonadotropin-releasing hormone stimulation test for precocious puberty.

Background Gonadotropin-releasing hormone (GnRH) stimulation test is the gold standard to identify central precocious puberty (CPP). This test requires multiple blood samples at different time points to measure gonadotropin levels, and is therefore expensive, time-consuming, and uncomfortable for patients. We aimed to simplify the GnRH stimulation test to require fewer blood samples. Methods A study of 166 girls with precocious puberty was undertaken. Blood samples were obtained at 0, 15, 30, 45, 60, 90, and 120 min after GnRH administration, and the levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured. For each parameter, the sensitivities and specificities were estimated and ROC curves were constructed. Results One hundred and twenty-eight patients (77.1%) were diagnosed for CPP. Peak LH levels were achieved 30 min after GnRH stimulation in patients with CPP. Further, 98.4% of the 45-min samples were diagnostic for CPP, and the cumulative frequency of LH values of ≥5 IU/L was 100% at 45 min. Using this cut-off value for LH, the ROC curve for LH at 45 min showed the highest sensitivity (98.4%) and specificity (100%) in the diagnosis of CPP. Conclusions Values of LH measured from a single blood sample obtained at 45 min in the GnRH stimulation test may be adequate for the diagnosis of CPP. Two samples, taken at 30 and 45 min after stimulation, were able to accurately diagnose CPP in 100% of the patients in this study.