Gye Cheol Kwon

Distribution of Pseudomonas-Derived Cephalosporinase and Metallo-β-Lactamases in Carbapenem-Resistant Pseudomonas aeruginosa Isolates from Korea.

The emergence of carbapenem resistance among Pseudomonas aeruginosa is an increasing problem in many parts of the world. In particular, metallo-β-lactamases (MBLs) and AmpC β- lactamases are responsible for high-level resistance to carbapenem and cephalosporin. We studied the diversity and frequency of β-lactamases and characterized chromosomal AmpC β- lactamase from carbapenem-resistant P. aeruginosa isolates. Sixty-one carbapenem-resistant P. aeruginosa isolates were collected from patients in a tertiary hospital in Daejeon, Korea, from January 2011 to June 2014. Minimum inhibitory concentrations (MICs) of four antimicrobial agents were determined using the agar-dilution method. Polymerase chain reaction and sequencing were used to identify the various β-lactamase genes, class 1 integrons, and chromosomally encoded and plasmid-mediated ampC genes. In addition, the epidemiological relationship was investigated by multilocus sequence typing. Among 61 carbapenem-resistant P. aeruginosa isolates, 25 isolates (41.0%) were MBL producers. Additionally, 30 isolates producing PDC (Pseudomonas-derived cephalosporinase)-2 were highly resistant to ceftazidime (MIC50 = 256 μg/ml) and cefepime (MIC50 = 256 μg/ml). Of all the PDC variants, 25 isolates harboring MBL genes showed high levels of cephalosporin and carbapenem resistance, whereas 36 isolates that did not harbor MBL genes revealed relatively low-level resistance (ceftazidime, p < 0.001; cefepime, p < 0.001; imipenem, p = 0.003; meropenem, p < 0.001). The coexistence of MBLs and AmpC β-lactamases suggests that these may be important contributing factors for cephalosporin and carbapenem resistance. Therefore, efficient detection and intervention to control drug resistance are necessary to prevent the emergence of P. aeruginosa possessing this combination of β-lactamases.

Emergence of Acinetobacter pittii harboring New Delhi metallo-β-lactamase genes in Daejeon, Korea.

Carbapenemase production has been reported worldwide in gram-negative bacteria, including Acinetobacter species. We detected carbapenemase-producing Acinetobacter pittii in clinical isolates in Daejeon, Korea. Twenty-one ertapenem-resistant A. pittii isolates screened with a disk diffusion method were characterized by using the Epsilon test, four multiplex PCR assays, and a multilocus sequence typing (MLST) scheme. A total of 21 A. pittii isolates harbored the metallo-β-lactamase (MBL) gene blaIMP-1 or blaNDM-1. Nineteen isolates containing blaIMP-1 were resistant to imipenem and meropenem, but two isolates harboring blaNDM-1 were susceptible to them. The sequence types (STs) of the two New Delhi MBL (NDM-1)-producing A. pittii isolates were ST70 and ST207, which differed from the STs (ST63, ST119, ST396, and a novel ST) of the IMP-1-producing A. pittii. This is the first report on NDM-1-producing A. pittii isolates in Korea. Our results emphasize that the study of NDM-1-producing gram-negative bacteria should involve carbapenem-susceptible as well as carbapenem-resistant isolates.

Hb variants in Korea: effect on HbA1c using five routine methods

Abstract Background: Quantification of glycated hemoglobin (HbA1c) is a challenge in patients with hemoglobin (Hb) variants. We evaluated the impact of various Hb variants on five routine HbA1c assays by comparing with the IFCC reference measurement procedure (RMP). Methods: Whole blood samples showing warning flags or no results on routine HPLC HbA1c assays were confirmed for Hb variants and were submitted to HbA1c quantification using Sebia Capillarys 2 Flex Piercing, Roche Tina-quant HbA1c Gen. 2, Bio-Rad Variant II Turbo 2.0, ADAMS HA-8180, Tosoh G8 standard mode, and IFCC RMP using LC-MS. Results: Among 114 samples, the most common variants were Hb G-Coushatta (n=47), Queens (n=41), Ube-4 (n=11), Chad (n=4), Yamagata (n=4), G-His-Tsou (n=2), G-Taipei (n=1), Fort de France (n=1), Hoshida (n=1), and two novel variants (Hb α-globin, HBA 52 Gly>Cys and Hb β-globin, HBB 146 His>Asn). In terms of control samples, all the result of HbA1c were “acceptable”, within the criteria of ±7% compared to IFCC RMP target values. However, percentage of “unacceptable” results of samples with Hb variants were 16% for Capillarys 2, 7% for Tina-quant, 51% for Variant II Turbo 2.0, 95% for G8 standard mode, and 89% for HA-8180. The Capillarys 2 and HA-8180 assay did not provide the results in 5 and 40 samples with Hb variants, respectively. Conclusions: HbA1c results from five routine assays in patients with relatively common Hb variants in Korea showed various degrees of bias compared to those of IFCC RMP. Therefore, laboratories should be aware of the limitation of their methods with respect to interference from Hb variants found commonly in their local population and suggest an alternative HbA1c quantification method.

Persistence of Multidrug-Resistant Acinetobacter baumannii Isolates Harboring blaOXA-23 and bap for 5 Years.

The emergence and dissemination of carbapenemase-producing Acinetobacter baumannii isolates have been reported worldwide, and A. baumannii isolates harboring blaOXA-23 are often resistant to various antimicrobial agents. Antimicrobial resistance can be particularly strong for biofilm-forming A. baumannii isolates. We investigated the genetic basis for carbapenem resistance and biofilm-forming ability of multidrug-resistant (MDR) clinical isolates. Ninety-two MDR A. baumannii isolates were collected from one university hospital located in the Chungcheong area of Korea over a 5-year period. Multiplex PCR and DNA sequencing were performed to characterize carbapenemase and bap genes. Clonal characteristics were analyzed using REP-PCR. In addition, imaging and quantification of biofilms were performed using a crystal violet assay. All 92 MDR A. baumannii isolates involved in our study contained the blaOXA-23 and bap genes. The average absorbance of biomass in Bap-producing strains was much greater than that in non-Bap-producing strains. In our study, only three REP-PCR types were found, and the isolates showing type A or type B were found more than 60 times among unique patients during the 5 years of surveillance. These results suggest that the isolates have persisted and colonized for 5 years, and biofilm formation ability has been responsible for their persistence and colonization.

A case of acute promyelocytic leukemia concomitant with plasma cell myeloma.

Dear Editor AML is a myeloid clonal malignancy characterized by the accumulation of abnormal immature myeloid cells in the bone marrow. The occurrence of therapy-related AML as a late complication of cytotoxic therapy is well documented [1], and several regimens for the treatment of plasma cell myeloma (PCM) have been associated with the development of myeloid neoplasms, such as AML, myelodysplastic syndrome, and myeloproliferative neoplasm [2]. The simultaneous occurrence of AML with various plasma cell dyscrasias without prior exposure to chemotherapy or radiotherapy is extremely rare, and most reported cases are of the myelomonocytic or monocytic subtype [3-6]. Herein, we report the first case of simultaneous acute promyelocytic leukemia (APL) and PCM presentation without previous exposure to chemotherapy or radiotherapy. A 60-yr-old man with a history of diabetic nephropathy, hypertension, and acute myocardial infarction with subsequent congestive heart failure presented to the emergency department with a 1-week history of general weakness, poor oral intake, and nausea. Neither hepatomegaly nor splenomegaly was detected on physical examination. The initial complete blood counts were the following: hemoglobin (11.5 g/dL, reference range: 13.5-17.4 g/dL), white blood cell count (2.7×109/L, reference range: 3.8-10.0×109/L), platelet count (378×109/L, reference range: 130-400×109/L), and white blood cell differential indicated 1% band neutrophils; 29% neutrophils; 62% lymphocytes; 2% monocytes; 4% eosinophils; and 2% basophils. Initial biochemical tests determined levels of total protein, (7.1 g/dL, reference range: 6.5-8.0 g/dL), albumin (2.9 g/dL, reference range: 4.0-5.0 g/dL), creatinine (5.1 mg/dL, reference range: 0.8-1.2 mg/dL), total calcium (6.6 mg/dL, reference range: 8.7-10.6 mg/dL), phosphorus (4.8 mg/dL, reference range: 2.5-4.7 mg/dL), β2 microglobulin (9.29 mg/L, reference range: 0-3.0 mg/L), and serum IgG (3,018 mg/dL, reference range: 680-1,620 mg/dL). Capillary electrophoresis (Sebia, Norcross, GA, USA) of serum and urine proteins showed a monoclonal peak (1.41 g/dL, 21.1% in serum; 0.22 g/dL, 8.3% in urine) in the gamma-globulin region, and immunofixation electrophoresis confirmed monoclonal gammopathy of IgG, kappa type. The ratios of serum and urine kappa/lambda light chains were 1.69 (reference range: 0.26-1.65) and 5.33 (reference range: 0.26-1.65), respectively. Radiologically, no osteolytic lesions were detected, but spinal magnetic resonance imaging (MRI) showed abnormal bone marrow signal intensity related to PCM. A bone marrow aspirate was composed of 10% myeloblasts, 23% abnormal hypergranular promyelocytes containing Auer rods, and 16% plasma cells. Myeloblasts and abnormal promyelocytes stained strongly positive for myeloperoxidase (MPO). The cellularity of biopsy was 70% and diffuse infiltration of two distinct populations consisting of leukemic myeloid cells (myeloblasts and abnormal promyelocytes) and plasma cells was observed. Immunohistochemical staining of the marrow biopsy showed significant positivity for CD138 and kappa light chain (Fig. 1). Flow cytometric analysis demonstrated blastic cells expressing CD13, CD33, and cytoplasmic MPO, but not HLA-DR, CD34, or CD15, consistent with the characteristics of APL. Cytogenetic analysis revealed a karyotype of 46,XY,t(15;17)(q22;q21)[4]/46,XY[16], and FISH confirmed nuc ish (PML,RARA)x3(PML con RARAx2)[64/200]. Nested reverse transcriptase-polymerase chain reaction (Seegene, Seoul, Korea) determined rearrangement of the PML/RARA gene. On the basis of these findings, we diagnosed the patient with APL concomitant with PCM, and the patient was initially treated with all-trans retinoic acid (ATRA) for 1 week. The patient refused additional chemotherapy because of his deteriorating physical condition after administration of ATRA and was discharged after 4 months of supportive care. Fig. 1 (A) Bone marrow aspiration showing abnormal promyelocytes (yellow arrow), Faggot cells (black arrow), and plasma cells (red arrow) (Wright stain, ×1,000) and (B) Immunohistochemical stain with CD138, (left, ×100), kappa light chain (middle, ... AML and PCM are different disease entities, and most concomitant cases result from leukemia that develops because of chemotherapy for preceding myeloma. Undiagnosed or untreated PCM co-presenting with AML is rare, with only 13 reports in the literature to date [7-9]. To our knowledge, this is the first reported case of APL concomitant with PCM worldwide. In this case, the diagnosis of APL was evident because of the presence of abnormal promyelocytes, Faggot cells, and the PML/RARA gene rearrangement. Before concluding that plasmacytosis is associated with clonal hematologic malignancy or PCM, reactive plasmacytosis should be ruled out because plasmacytosis can co-occur with AML [10], in which interleukin-6 production by leukemic blasts may stimulate plasma cell growth [11]. The patients various chronic conditions confounded the assessment of CRAB (hypercalcemia, renal insufficiency, anemia, and bony lesion) to prove end-organ damage by PCM. In this case, anemia could be attributed to the underlying chronic diseases, and the decrease of albumin and elevation of creatinine levels usually accompanied by PCM were masked by diabetic nephropathy. Elevation of total protein levels was not prominent, and hypercalcemia was also not observed because of the patients nephropathy. As MRI is more sensitive than a skeletal survey, the increased signal intensity detected by MRI determined bony lesions in PCM [12]. The creatinine level in this patient was stable (3 mg/dL), but was aggravated on presentation possibly because of PCM, and urgent hemodialysis was needed. In addition, bone marrow with 16% plasma cells was noted, which is not usually seen in reactive plasmacytosis, where plasma cells typically do not exceed 10% [13]. The presence of >10% plasma cells, monoclonal gammopathy by electrophoresis, kappa restriction by immunohistochemical stain, aggravation of kidney function, and bony lesions in MRI lead to the conclusion of clonal plasmacytosis, and a final diagnosis of APL concomitant with PCM was made. The proposed reasons for concurrent presentation of 2 different hematologic malignancies are abnormal multipotent stem cells, environmental risk factors, repeated infections that result in the development of a leukemic clone, and decreased immune surveillance, which may result in failure to eliminate leukemic clones [6, 11, 14]. In cases of APL followed by chemotherapy for PCM, a regimen of ATRA or allogeneic stem cell transplantation can be considered treatment options [1, 15], but treatment regimens for concomitant malignancies are not fully established because the co-occurrence of APL and PCM is rare. In this case, treatment with ATRA was begun with the intent to add idarubicine; however, additional chemotherapy was discontinued because the patient developed sepsis with aggravated general weakness. Aside from 1 patient who received stem cell transplantation [9], there have been no reports on survival of patients with concomitant AML and PCM who received chemotherapy, and the time interval from diagnosis to death in most cases was <5 months. The prognosis of this patient was poor because of his deteriorating condition despite appropriate supportive care. In conclusion, we described the first case of concomitant APL and PCM, suggesting that 2 distinct diseases can present coin cidentally without previous chemotherapy. Although rare, plasmacytosis can occur in AML, and clonality should be ruled out because of the simultaneous presentation of these 2 different hematological malignancies.

Manual versus automated streaking system in clinical microbiology laboratory: Performance evaluation of Previ Isola for blood culture and body fluid samples

The process of plate streaking has been automated to improve routine workflow of clinical microbiology laboratories. Although there were many evaluation reports about the inoculation of various body fluid samples, few evaluations have been reported for blood. In this study, we evaluated the performance of automated inoculating system, Previ Isola for various routine clinical samples including blood.

Discriminating myelodysplastic syndrome and other myeloid malignancies from non-clonal disorders by multiparametric analysis of automated cell data

BACKGROUND We investigated the usefulness of novel complete blood count (CBC) data for discriminating myeloid malignancies from non-clonal CBC abnormalities. METHODS Data were obtained during routine CBC tests of 119 samples from 37 myelodysplastic syndrome (MDS) patients, 92 samples from 45 myeloproliferative neoplasm (MPN) patients, and 15 samples from 11 chronic myelogenous leukemia (CML) patients using a DxH800 (Beckman Coulter). Data obtained from patients with hypocellular bone marrow and from those with other non-clonal diseases with CBC abnormalities were included in the comparisons. RESULTS For cell population data of neutrophils, the means of median, upper median, lower median, and low angle light scatters were significantly lower in MDS patients than in patients without hematological malignancies. Low hemoglobin density (LHD) did not significantly differ between the MDS and non-clonal cytopenia patients, but it was significantly higher in the MPN and CML patients. We selected 13 parameters and scored the MDS diagnosis using cut-off values obtained from receiver operating characteristic (ROC) curve analysis. Using a score > 9, MDS was distinguished from non-clonal cytopenia with a sensitivity of 92.4% and a specificity of 85.4%. CONCLUSIONS Multiparametric analyses of new automated parameters are useful for discriminating MDS from non-clonal cytopenia.

Pyrosequencing-based quantitative measurement of CALR mutation allele burdens and their clinical implications in patients with myeloproliferative neoplasms

BACKGROUND We developed a pyrosequencing-based method for the quantification of CALR mutations and compared the results using Sanger sequencing, fragment length analysis (FLA), digital-droplet PCR (ddPCR), and next-generation sequencing (NGS). METHODS Method validation studies were performed using cloned plasmid controls. Samples from 24 patients with myeloproliferative neoplasms were evaluated. RESULTS Among the 24 patients, 15 had CALR mutations (7 type 1, 2 type 2, and 6 other mutations). The type 1 or type 2 mutation-positive results from pyrosequencing exhibited 100% concordance with the Sanger sequencing results. One novel CALR mutation was not detected by pyrosequencing. The CALR mutation allele burdens measured by pyrosequencing were slightly lower than those measured by FLA but slightly higher than the results obtained using ddPCR. Pyrosequencing exhibited high correlations with both methods. The mutation allele burdens estimated by NGS were significantly lower than those measured by pyrosequencing. An increased CALR mutation allele burden was associated with overt primary myelofibrosis. Patients with >70% mutation allele burdens in myeloid cells had a significantly longer time from diagnosis (P = 0.007), more bone marrow fibrosis (P = 0.010), and lower hemoglobin (P = 0.007). CONCLUSIONS Pyrosequencing was a useful rapid sequencing method to determine the burden of CALR mutations.

Evaluation of the LABGEO PT10 Point-Of-Care Testing Device: Comparison of Analyte Measurements in Capillary Whole Blood and Lithium Heparin Whole Blood Samples With Those in Central Laboratory

Point‐of‐care (POC) testing device has been widely used because of its rapid availability of results making diagnosis and management as early as possible. Capillary blood can reduce the difficulty of obtaining samples compared to venous blood and allows the prompt testing results. In this study, we evaluated the usefulness of capillary blood in Samsung LABGEO PT10.

Fully international system of units-traceable glycated hemoglobin quantification using two stages of isotope-dilution high-performance liquid chromatography–tandem mass spectrometry

Glycated hemoglobin (HbA1c), defined as hemoglobin (Hb) molecules having a stable adduct of glucose on the N-terminal of the β-chains, has been endorsed as a diagnostic tool for diabetes mellitus and a prediction indicator for the development of diabetes complications. Here we describe an accurate procedure using two stages of isotope dilution-ultra performance liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) for the quantification of HbA1c that provides full traceability to International System of Units (SI). First, synthetic peptides representing specific markers of HbA1c (G-hexa) and hemoglobin A0 (Hexa) were certified by amino acid analysis via acid hydrolysis as reference materials (RMs) for the next step. For this peptide certification, three amino acids (proline, valine, and leucine) were determined by hydrolysis with 10M hydrochloric acid at 130°C for 48h followed by ID-LC-MS/MS. Then, HbA1c content in blood was quantified with the ratio of specific proteolytic peptides from HbA1c and HbA0 via enzyme digestion using ID-LC-MS/MS with the certified peptides as RMs and isotope-labeled peptides as internal standards. Results demonstrate complete traceability to SI-units throughout this procedure. Reliability was confirmed through comparative studies with commercially available RMs for HbA1c, and other routine HbA1c diagnostic methods as well. Following full method validation, we applied this procedure to the certification of candidate hemolysate-certified RMs for HbA1c content, as well as 52 real clinical samples. All of the results showed the suitability of this method to act as a primary reference measurement procedure for HbA1c in complex biological samples.