AIM: To determine intestinal permeability, the serum tumor necrosis factor (TNF)-α level and urine nitric oxide (NO) metabolites are altered in liver cirrhosis (LC) with or without ascites.
METHODS: Fifty-three patients with LC and 26 healthy control subjects were enrolled in the study. The intestinal permeability value is expressed as the percentage of polyethylene glycol (PEG) 400 and 3350 retrieval in 8-h urine samples as determined by high performance liquid chromatography. Serum TNF-α concentrations and urine NO metabolites were determined using an enzyme-linked immunosorbent assay (ELISA) and Greiss reaction method, respectively.
RESULTS: The intestinal permeability index was significantly higher in patients with LC with ascites than in healthy control subjects or patients with LC without ascites (0.88 ± 0.12 vs 0.52 ± 0.05 or 0.53 ± 0.03, P < 0.05) and correlated with urine nitrite excretion (r = 0.98). Interestingly, the serum TNF-α concentra-tion was significantly higher in LC without ascites than in control subjects or in LC with ascites (198.9 ± 55.8 pg/mL vs 40.9 ± 12.3 pg/mL or 32.1 ± 13.3 pg/mL, P < 0.05). Urine nitrite excretion was significantly higher in LC with ascites than in the control subjects or in LC without ascites (1170.9 ± 28.7 μmol/L vs 903.1 ± 55.1 μmol/L or 956.7 ± 47.7 μmol/L, P < 0.05).
CONCLUSION: Increased intestinal macromolecular permeability and NO is probably of importance in the pathophysiology and progression of LC with ascites, but the serum TNF-α concentration was not related to LC with ascites.
Intestinal permeability; Tumor necrosis factor-α; Nitric oxide; Liver cirrhosis; Ascites
We evaluated three commercial colistin susceptibility testing methods using 213 bloodstream Acinetobacter isolates identified by gene sequencing. Compared to the agar dilution reference method, excellent categorical agreements (both 99.1%) were observed using Vitek 2 and Etest, compared to 87.3% (95.7% for Acinetobacter baumannii and 80.7% for non-baumannii Acinetobacter isolates) using MicroScan.
Canine NK cells still are not well-characterized due to the lack of information concerning specific NK cell markers and the fact that NK cells are not an abundant cell population. In this study, we selectively expanded the canine cytotoxic large granular lymphocytes (CLGLs) that exhibit morphologic, genetic, and functional characteristics of NK cells from normal donor PBMCs. The cultured CLGLs were characterized by a high proportion of CD5(dim) expressing cells, of which the majority of cells co-expressed CD3 and CD8, but did not express TCRαβ and TCRγδ. The phenotype of the majority of the CLGLs was CD5(dim)CD3+CD8+ TCRαβ−TCRγδ−CD4−CD21−CD11c+/−CD11d+/−CD44+. The expression of mRNAs for NK cell-associated receptors (NKG2D, NKp30, NKp44, Ly49, perforin, and granzyme B) were highly upregulated in cultured CLGLs. Specifically, NKp46 was remarkably upregulated in the cultured CLGLs compared to PBMCs. The mRNAs for the NKT-associated iTCRα gene in CLGLs was present at a basal level. The cytotoxic activity of the CLGLs against canine NK cell-sensitive CTAC cells was remarkably elevated in a dose-dependent manner, and the CLGLs produced large amounts of IFN-γ. The antitumor activity of CLGLs extended to different types of canine tumor cells (CF41.Mg and K9TCC-pu-AXC) without specific antigen recognition. These results are consistent with prior reports, and strongly suggest that the selectively expanded CLGLs represent a population of canine NK cells. The results of this study will contribute to future research on canine NK cells as well as NK cell-based immunotherapy.
Canine; Cytotoxic large granular lymphocytes; NK cells; Expansion; Cytotoxicity
The aim of this study was to investigate the frequency of autoantibodies with mimicking specificity by using the dilution technique, to assess the usefulness of the combination of the dilution technique and red blood cell (RBC) phenotyping, and to establish a pre-transfusion testing algorithm in patients with warm autoantibodies.
Serum samples from 71 patients with warm autoantibodies were tested using the dilution technique. Among them, 25 samples were adsorbed with allogeneic ZZAP (a combination of dithiothreitol and enzyme) or polyethylene glycol (PEG) and their RBC phenotypes were determined. Thirty-nine patients were transfused with our pre-transfusion testing algorithm using a combination of dilution technique and RBC phenotyping.
Autoantibodies with mimicking specificity were detected by the dilution technique in 26.8% (19/71) of the patients and most of them were directed against Rh system antigens. The agreement of the results obtained with the dilution technique in combination with RBC phenotyping and those from ZZAP or PEG adsorption was 100% (18/18) in patients who have autoantibodies with mimicking specificity and/or alloantibodies. No clinical symptoms indicating severe acute or delayed hemolytic transfusion reactions were reported in the 39 patients transfused with our pre-transfusion testing algorithm.
Autoantibodies with mimicking specificity detected by the dilution technique in patients with warm autoantibodies are relatively frequent, can be discriminated from alloantibodies by employing a combination of dilution technique and RBC phenotyping, and might not appear to cause severe acute or delayed hemolytic transfusion reactions.
Autoantibody; Dilution technique; Mimicking specificity
Natural killer T (NKT) cells are known to play a protective role in the immune responses of mice against a variety of infectious pathogens. However, little is known about the detailed information of NKT cells in patients with Mycobacterium tuberculosis infection. The aims of this study were to examine NKT cell levels and functions in patients with active M. tuberculosis infection, to investigate relationships between NKT cell levels and clinical parameters, and to determine the mechanism responsible for the poor response to α-galactosylceramide (α-GalCer). NKT cell levels were significantly lower in the peripheral blood of pulmonary tuberculosis and extrapulmonary tuberculosis patients, and the proliferative responses of NKT cells to α-GalCer were also lower in patients, whereas NKT cell levels and responses were comparable in latent tuberculosis infection subjects and healthy controls. Furthermore, this NKT cell deficiency was found to be correlated with serum C-reactive protein levels. In addition, the poor response to α-GalCer in M. tuberculosis-infected patients was found to be due to increased NKT cell apoptosis, reduced CD1d expression, and a defect in NKT cells. Notably, M. tuberculosis infection was associated with an elevated expression of the inhibitory programmed death-1 (PD-1) receptor on NKT cells, and blockade of PD-1 signaling enhanced the response to α-GalCer. This study shows that NKT cell levels and functions are reduced in M. tuberculosis-infected patients and these deficiencies were found to reflect the presence of active tuberculosis.
The emerging fungal pathogens Candida haemulonii and Candida pseudohaemulonii often show high-level resistance to amphotericin B (AMB). We compared the utilities of five antifungal susceptibility testing methods, i.e., the Etest using Mueller-Hinton agar supplemented with glucose and methylene blue (Etest-MH), the Etest using RPMI agar supplemented with glucose (Etest-RPG), the Vitek-2 yeast susceptibility system, and the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution methods, for the detection of AMB-resistant isolates of C. haemulonii and closely related species. Thirty-eight clinical isolates (8 C. haemulonii, 10 C. pseudohaemulonii, and 20 Candida auris isolates) were analyzed. Of the 18 C. haemulonii and C. pseudohaemulonii isolates, 18, 15, 18, 10, and 9 exhibited AMB MICs of >1 μg/ml by the Etest-MH, Etest-RPG, Vitek-2, CLSI, and EUCAST methods, respectively. All 20 C. auris isolates showed AMB MICs of ≤1 μg/ml by all five methods. Of the methods, the Etest-MH generated the broadest distribution of AMB MICs for all 38 isolates and showed the best discrimination between the C. haemulonii and C. pseudohaemulonii isolates (4 to 32 μg/ml) and those of C. auris (0.125 to 0.5 μg/ml). Taking the Etest-MH as the reference method, the essential agreements (within two dilutions) for the Etest-RPG, Vitek-2, CLSI, and EUCAST methods were 84, 92, 55, and 55%, respectively; the categorical agreements were 92, 92, 79, and 76%, respectively. This study provides the first data on the efficacy of the Etest-MH and its excellent agreement with Vitek-2 for discriminating AMB-resistant from AMB-susceptible isolates of these Candida species.
Schizophyllum commune, a basidiomycetous fungus, rarely causes disease in humans. We report a rare case of allergic fungal sinusitis caused by S. commune in a 14-yr-old girl. The patient presented with nasal obstruction and a purulent nasal discharge. Materials obtained during endoscopic surgery of the frontal recess revealed allergic mucin and a few fungal hyphae. A potato dextrose agar (PDA) culture from the allergic mucin yielded a rapidly growing white woolly mold. Although no distinctive features including hyphae bearing spicules or a clamp connection were present, the case isolate disclosed compatible mycological features including growth at 37℃, susceptibility to cycloheximide, and production of a tart and disagreeable smell. S. commune was confirmed by sequence analysis of the internal transcribed spacer region and D1/D2 regions of the 26S ribosomal DNA. We believe this is the first report of allergic fungal sinusitis caused by S. commune in Korea. Moreover, this report highlights the value of gene sequencing as an identification tool for non-sporulating isolates of S. commune.
Schizophyllum commune; Sinusitis; Sequencing
Multilocus sequence typing (MLST) has been successfully applied to the epidemiology of Candida albicans isolates not only within the hospital setting but also in multiple locations nationwide. We performed MLST to investigate the genetic relatedness among bloodstream infection (BSI) isolates of C. albicans recovered from 10 Korean hospitals over a 12-month period. The 156 isolates yielded 112 unique diploid sequence types (DSTs). While 95 DSTs were each derived from a single isolate, 17 DSTs were shared by 61 isolates (39.1%). Interestingly, 111 (71.1%) isolates clustered within previously known clades, and 29 (18.6%) clustered within a new clade that includes strains of Asian origin previously typed as singletons. This MLST study was complemented by restriction endonuclease analysis of genomic DNA using BssHII (REAG-B) in order to evaluate whether strains with identical DSTs and originating from the same hospital corresponded to nosocomial clusters. Importantly, only those isolates with a strong epidemiological relationship showed ≥95% identical REAG-B types. Our results indicate that REAG-B typing can be complementary to MLST but should be limited to the investigation of isolates of identical DSTs and when interhuman transmission is suspected.
This study investigated the spectrum of chromosomal abnormalities in 325 leukemia patients and developed optimal profiles of leukemic fusion genes for multiplex RT-PCR. We prospectively analyzed blood and bone marrow specimens of patients with acute leukemia. Twenty types of chromosomal abnormalities were detected in 42% from all patients by commercially available multiplex RT-PCR for detecting 28 fusion genes and in 35% by cytogenetic analysis including FISH analysis. The most common cytogenetic aberrations in acute myeloid leukemia patients was PML/PARA, followed by AML1/MGT8 and MLL1, and in acute lymphoid leukemia patients was BCR/ABL, followed by TEL/AML1 and MLL1 gene rearrangement. Among the negative results for multiplex RT-PCR, clinically significant t(3;3)(q21;q26.2), t(8;14)(q24;q32) and i(17)(q10) were detected by conventional cytogenetics. The spectrum and frequency of chromosomal abnormalities in our leukemia patients are differed from previous studies, and may offer optimal profiles of leukemic fusion genes for the development of new molecular detection systems.
Leukemia; Chromosomal Abnormalities; Molecular Detection System
To date, most clinical data on pro-gastrin-releasing peptide (proGRP) have been based on serum concentrations. This study evaluated the agreement between proGRP levels in fresh serum and plasma in patients with various lung diseases. Pairs of serum and EDTA plasma were collected from 49 healthy individuals. At the same time, EDTA plasma of 118 lung cancer patients and 23 patients with benign pulmonary diseases were prospectively collected. Compared to serum, plasma proGRP concentrations were higher by an average of 103.3%. Plasma proGRP was higher in malignancy (336.4 ± 925.4 pg/mL) than in benign conditions (40.1 ± 11.5 pg/mL). Small cell lung cancer (SCLC) patients showed higher levels of proGRP (1,256.3 ± 1,605.6 pg/mL) compared to other types of lung cancer. Based on the ROC curve analyses at a specificity of 95%, the diagnostic sensitivity of plasma proGRP was estimated to be 83.8% in distinguishing SCLC from all the other conditions, and 86.5% for discriminating SCLC from the nonmalignant cases. Among the SCLC cases, limited stage disease had lower levels of plasma proGRP than extensive disease. When measuring circulating levels of proGRP, the use of plasma is preferred over serum. Plasma proGRP has a potential marker for discriminating SCLC from nonmalignant conditions or non-small cell lung cancer.
pro-gastrin-releasing peptide (31-98); Serum; Plasma; Small Cell Lung Carcinoma
We evaluated the efficacy of multilocus sequence typing (MLST) for assessing the genetic relationship among Candida albicans isolates from patients with candidemia in a hospital setting.
A total of 45 C. albicans isolates from 21 patients with candidemia were analyzed. The MLST results were compared with results obtained by Southern blot hybridization (C1 fingerprinting) and pulsed-field gel electrophoresis (PFGE). PFGE analysis included karyotyping and restriction endonuclease analysis of genomic DNAs using BssHII (REAG-B) and SfiI (REAG-S).
The 45 isolates yielded 20 unique diploid sequence types (DSTs) by MLST, as well as 12 karyotypes, 15 REAG-B patterns, 13 REAG-S patterns, and 14 C1 fingerprinting types. Microevolution among intra-individual isolates was detected in 6, 5, 3, 5, and 7 sets of isolates by MLST (1 or 2 allelic differences), REAG-B, REAG-S, C1 fingerprinting, and a combination of all methods, respectively. Among 20 DSTs, 17 were unique, and 3 were found in more than 1 patient. The results of 2 DSTs obtained from 9 patient isolates were in agreement with REAG and C1 fingerprinting patterns. However, the remaining DST, which was shared by 2 patient isolates, showed 2 different PFGE and C1 fingerprinting patterns. In addition, 3 sets of isolates from different patients, which differed in only 1 or 2 alleles by MLST, also exhibited different PFGE or C1 fingerprinting patterns.
MLST is highly discriminating among C. albicans isolates, but it may have some limitations in typing isolates from different patients, which may necessitate additional analysis using other techniques.
Candia albicans; Multilocus sequence typing; Pulsed-field gel electrophoresis; Southern hybridization; Genotyping
All-trans retinoic acid (ATRA)/anthracycline chemotherapy is beneficial in newly diagnosed acute promyelocytic leukemia (APL); however, it is important to identify patients with high-risk disease to increase the cure rate. We investigated the outcome of ATRA/anthracycline chemotherapy and clinicobiological correlations of FLT3/ITD and NPM1 mutations in APL patients.
Induction therapy included oral ATRA (45 mg/m2/day) and idarubicin (12 mg/m2/day, intravenous, on days 2, 4, and 6). Patients achieving complete remission (CR) received 3 courses of ATRA combined with reinforced consolidation therapy. Mutations were analyzed using GeneScan and polymerasae chain reaction assays of bone marrow samples obtained from patients at diagnosis.
Forty-five (84.9%) of 53 eligible patients achieved CR. The overall relapse rate was 8.9%, and the 3-year overall survival (OS) and leukemia-free survival (LFS) were 84.9±4.9% and 77.5±6.0%, respectively. The NPM1 mutation was not found in any patient, while the FLT3/ITD mutation was found in 10 (20.0%) patients. Of the FLT3/ITD+ patients, 80% belonged to the high-risk group, defined according to the presenting WBC and platelet counts. Among the patients who achieved CR, those who were FLT3/ITD+ had a higher relapse rate than those FLT3/ITD-. FLT3/ITD+ patients also had a significantly lower 3-year LFS than FLT3/ITD- patients. Multivariate analysis of the LFS showed that the FLT3/ITD mutation was independently associated with a shorter overall LFS, after adjusting for pretreatment risk stratification.
This study investigated the clinical outcome of newly diagnosed APL patients treated with ATRA/anthracycline chemotherapy. Patients carrying the FLT3/ITD mutation had more aggressive clinical features and a poorer clinical outcome.
Acute promyelocytic leukemia; FLT3; Prognosis
chronic atrial fibrillation; atrial tissue; mitochondrial DNA; mutation
Nucleophosmin (NPM1) gene and fms-like tyrosine kinase 3 gene-internal tandem duplication (FLT3-ITD) mutations are the most frequent mutations in patients with cytogenetically normal (CN)-AML. We analyzed the prognostic impact of these mutations and their interactions in adults with CN-AML.
NPM1 mutation (NPM1mut) and FLT3-ITD mutation (FLT3-ITD+) were analyzed by GeneScan and PCR assays of bone marrow samples obtained from 121 adult patients with CN-AML (age≤60 years at diagnosis).
The incidence of FLT3-ITD+ was higher in the NPM1mut group than in the wild-type NPM1 gene (NPM1wt) group. The patients were divided according to their mutation status into the NPM1mut/FLT3-ITD (isolated NPM1mut), NPM1mut/FLT3-ITD+ or NPM1wt/FLT3-ITD-, and NPM1wt/FLT3-ITD+ (isolated FLT3-ITD+) groups. The isolated NPM1mut group showed significantly better clinical outcomes in terms of relapse rate, 5-year relapse-free survival (RFS), and overall survival (OS) than the other groups. In contrast, the isolated FLT3-ITD+ group had a higher relapse rate and shorter RFS and OS than the other groups. The 5-year RFS rate was much higher among the patients who underwent allogeneic stem cell transplantation (alloSCT) than among those treated with high-dose cytarabine chemotherapy (HDAC) only as consolidation therapy in the isolated NPM1mut group and the NPM1mut/FLT3-ITD+ or NPM1wt/FLT3-ITD- group.
Adult patients with CN-AML carrying isolated NPM1mut and isolated FLT3-ITD+ exhibit different clinical outcomes than those with NPM1mut/FLT3-ITD+ or NPM1wt/FLT3-ITD-. Although isolated NPM1mut leads to favorable clinical outcomes of CN-AML, the role of alloSCT in such patients remains to be considered.
NPM1; FLT3-ITD; Acute myeloid leukemia; Normal karyotype
This study was undertaken primarily to test the hypothesis that mitochondrial DNA (mtDNA) mutations may be associated with aplastic anemia. Complete mtDNA nucleotide sequence was analyzed in nine and eight bone marrow specimens from Korean patients with aplastic anemia and healthy individuals, respectively. We found a large number of polymorphisms as well as apparent new mutations in both patients and controls throughout the entire mtDNA genome; 12 mutations harbored amino acid changes in patients and none of the mutations in controls produced amino acid changes. There were heteroplasmic mutations and more nonsynonymous mtDNA changes observed in patients, so the mean number of mtDNA aberrations of bone marrow cells showed statistically significant difference overall between patients (mean=25.6) and controls (mean=12.8) (p=0.019). Our data may support an association of mtDNA aberrations with aplastic anemia.
Anemia, Aplastic; DNA, Mitochondrial; Mutation
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.
The incidence of Candida bloodstream infections (BSI) has increased over the past two decades. The rank order of occurrence and the susceptibility to antifungals of the various Candida species causing BSI are important factors driving the establishment of empirical treatment protocols; however, very limited multi-institutional data are available on Candida bloodstream isolates in Korea.
Materials and Methods
We investigated the susceptibility to azole antifungals and species distribution of 143 Candida bloodstream isolates recovered from eight university hospitals over a six-month period. Minimal inhibitory concentrations (MICs) of fluconazole, itraconazole, and voriconazole for each isolate were determined by the broth microdilution method of the Clinical and Laboratory Standards Institute (CLSI).
species recovered most frequently from the blood cultures was C. albicans (49%), followed by C. parapsilosis (22%), C. tropicalis (14%), and C. glabrata (11%). The MIC ranges for the Candida isolates were 0.125 to 64 µg/mL for fluconazole, 0.03 to 2 µg/mL for itraconazole, and 0.03 to 1 µg/mL for voriconazole. Overall, resistance to fluconazole was found in only 2% of the Candida isolates (3/143), while the dose-dependent susceptibility was found in 6% (8/143). The resistance and dose-dependent susceptibility of itraconazole were found in 4% (6/143) and 14% (20/143) of the isolates, respectively. All bloodstream isolates were susceptible to voriconazole (MIC, ≤ 1 µg/mL).
Our findings show that C. albicans is the most common cause of Candida-related BSI, followed by C. parapsilosis, and that the rates of resistance to azole antifungals are still low among bloodstream isolates in Korea.
Candida species; antifungal susceptibility; bloodstream infections; triazoles
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.
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.
Chimerism in humans is a rare phenomenon often initially identified in the resolution of an ABO blood type discrepancy. We report a dispermic chimera who presented with mixed field in his B antigen typing that might have been mistaken for the B3 subtype. The propositus is a healthy Korean male blood donor. Neither his clinical history nor initial molecular investigation of his ABO gene explained his mixed field agglutination with murine anti-B. Chimerism was suspected, and 9 short tandem repeat (STR) loci were analyzed on DNA extracted from blood, buccal swabs, and hair from this donor and on DNA isolated from peripheral blood lymphocytes from his parents. The propositus' red blood cells demonstrated mixed field agglutination with anti-B. Exon 6 and 7 and flanking intronic regions of his ABO gene were sequenced and revealed an O01/O02 genotype. B allele haplotype-specific PCR, along with exon 6 and 7 cloning and sequencing demonstrated a third ABO allele, B101. Four STR loci demonstrated a pattern consistent with a double paternal chromosome contribution in the propositus, thus confirming chimerism. His karyotype revealed a mosaic pattern: 32/50 metaphases were 46,XY and 18/50 metaphases demonstrated 47,XYY.
Chimerism; ABO Blood Type; Mosaicism; XYY Karyotype
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.
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.
At present, the clinical breakpoints (CBPs) of both fluconazole and voriconazole are available only for 3 common Candida species in the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) methods. Epidemiological cutoff values (ECVs) were recently applied to both methods to detect the emergence of acquired resistance (i.e., non-wild-type isolates) among 5 common Candida species.
We performed a nationwide study to determine the fluconazole and voriconazole susceptibility of Candida bloodstream isolates (BSIs) using both the CLSI and EUCAST methods. A total of 423 BSIs of 5 Candida species were collected from 8 hospitals. The azole susceptibilities were assessed on the basis of the species-specific CBPs and ECVs.
Of the 341 BSIs of 3 common Candida species (i.e., C. albicans, C. tropicalis, and C. parapsilosis), 0.3% and 0.9%, 0.0% and 1.5% of isolates were categorized as fluconazole and voriconazole resistant according to the CLSI and EUCAST CBPs, respectively. Of 423 total BSIs, 1.4% and 2.6% had fluconazole minimum inhibitory concentrations (MICs) exceeding the ECVs according to the CLSI and EUCAST, respectively; 1.0% and 2.1% had voriconazole MICs exceeding the ECVs according to the CLSI and EUCAST, respectively. Categorical agreement between the methods using ECVs was 98.3% for fluconazole and 98.3% for voriconazole.
The EUCAST and CLSI methods using ECVs provide highly concordant results. Moreover, non-wild-type isolates with possibly acquired azole resistance were rare among the BSIs of 5 common Candida species in Korea.
Candida; Fluconazole; Voriconazole; EUCAST; CLSI; epidemiological cutoff value
The discovery of a single point mutation in the JAK2 gene in patients with BCR/ABL-negative myeloproliferative neoplasms (MPNs) has not only brought new insights and pathogenesis, but also has made the diagnosis of MPNs much easier. Although, to date, several mechanisms for the contribution of single JAK2V617F point mutation to phenotypic diversity of MPNs have been suggested in multiple studies, but it is not clear how a unique mutation can cause the phenotypic diversity of MPNs. In this study, our results show that allelic expression imbalance of JAK2 V617F mutant frequently occurs and contributes to phenotypic diversity of BCR-ABL-negative MPNs. The proportion of JAK2 V617F mutant allele was significantly augmented in RNA levels as compared with genomic DNA differently by distinct MPNs subtypes. In detail, preferential expression of JAK2 mutant allele showed threefold increase from the cDNA compared with the genomic DNA from patients with essential thrombocythemia and twofold increase in polycythemia vera. In conclusion, allelic expression imbalance of JAK2 V617F mutant proposes another plausible mechanism for the contribution of single JAK2 point mutation to phenotypic diversity of MPNs.