Comparative Analysis of Clinical Samples Showing Weak Serum Reaction on AutoVue System Causing ABO Blood Typing Discrepancies.

BACKGROUND: ABO blood typing in pre-transfusion testing is a major component of the high workload in blood banks that therefore requires automation. We often experienced discrepant results from an automated system, especially weak serum reactions. We evaluated the discrepant results by the reference manual method to confirm ABO blood typing. METHODS: In total, 13,113 blood samples were tested with the AutoVue system; all samples were run in parallel with the reference manual method according to the laboratory protocol. RESULTS: The AutoVue system confirmed ABO blood typing of 12,816 samples (97.7%), and these results were concordant with those of the manual method. The remaining 297 samples (2.3%) showed discrepant results in the AutoVue system and were confirmed by the manual method. The discrepant results involved weak serum reactions (<2+ reaction grade), extra serum reactions, samples from patients who had received stem cell transplants, ABO subgroups, and specific system error messages. Among the 98 samples showing ≤1+ reaction grade in the AutoVue system, 70 samples (71.4%) showed a normal serum reaction (≥2+ reaction grade) with the manual method, and 28 samples (28.6%) showed weak serum reaction in both methods. CONCLUSIONS: ABO blood tying of 97.7% samples could be confirmed by the AutoVue system and a small proportion (2.3%) needed to be re-evaluated by the manual method. Samples with a 2+ reaction grade in serum typing do not need to be evaluated manually, while those with ≤1+ reaction grade do.

Identification of Mycobacterial Antigens in Human Urine by Use of Immunoglobulin G Isolated from Sera of Patients with Active Pulmonary Tuberculosis.

Point-of-care (POC) diagnostic testing of tuberculosis (TB) is a tremendous unmet need. In this study, four urinary mycobacterial antigens were identified through two independent approaches using IgG capture and immunodepletion methods. Among these, ModC was validated by a multiple reaction monitoring (MRM) method. As expected, the biomarkers elevated the clinical validity of TB diagnosis when combined with preexisting markers.

Incidence, clinical features, and prognostic impact of CALR exon 9 mutations in essential thrombocythemia and primary myelofibrosis: an experience of a single tertiary hospital in Korea.

We evaluated the incidence, clinical characteristics, and prognostic impact of calreticulin (CALR) mutations in essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients. In all, 48 ET and 14 PMF patients were enrolled, and the presence of CALR mutations was analyzed by direct sequencing. Patients were classified into three subgroups according to Janus kinase 2 (JAK2) V617F and CALR mutation status, and their clinical features and prognosis were compared. CALR mutations were detected in 15 (24.2%) patients, and the incidence increased to 50.0% in 30 JAK2 V617F mutation-negative cases. These included 11 patients with three known mutations (c.1092_1143del [seven cases], c.1154_1155insTTGTC [three cases], and c.1102_1135del [one case]) and 4 patients with novel mutations. ET patients carrying CALR mutation were younger, had lower white blood cell counts, and experienced less thrombosis during follow-up than those carrying JAK2 V617F mutation, while both patient groups showed similar clinical features and prognosis. In ET patients without JAK2 V617F mutation, CALR mutation did not significantly affect clinical manifestation and prognosis. In conclusion, CALR mutation analysis could be a useful diagnostic tool for ET and PMF in 50% of the cases without JAK2 V617F mutations. The prognostic impact of CALR mutations needs further investigation.

Incidence and prognostic impact of DNMT3A mutations in Korean normal karyotype acute myeloid leukemia patients.

BACKGROUND: DNA methyltransferase 3A (DNMT3A) mutation was recently introduced as a prognostic indicator in normal karyotype (NK) AML and we evaluated the incidence and prognostic impact of DNMT3A mutations in Korean NK AML patients. METHODS: Total 67 NK AML patients diagnosed during the recent 10 years were enrolled. DNMT3A mutations were analyzed by direct sequencing and categorized into nonsynonymous variations (NSV), deleterious mutations (DM), and R882 mutation based on in silico analysis results. Clinical features and prognosis were compared with respect to DNMT3A mutation status. RESULTS: Three novel (I158M, K219V, and E177V) and two known (R736H and R882H) NSVs were identified and the latter three were predicted as DMs. DNMT3A NSVs, DMs, and R882 mutation were identified in 14.9%-17.9%, 10.3%-10.4%, and 7.5% of patients, respectively. DNMT3A mutations were frequently detected in FLT3 ITD mutated patients (P=0.054, 0.071, and 0.071 in NSV, DMs, and R882 mutation, resp.) but did not affect clinical features and prognosis significantly. CONCLUSIONS: Incidences of DNMT3A NSVs, DMs, and R882 mutation are 14.9%-17.9%, 10.3%-10.4%, and 7.5%, respectively, in Korean NK AML patients. DNMT3A mutations are associated with FLT3 ITD mutations but do not affect clinical outcome significantly in Korean NK AML patients.

Clinical relevance of high-resolution single nucleotide polymorphism array in patients with relapsed acute lymphoblastic leukemia with normal karyotype: a report of three cases.

We report three patients with normal karyotype (NK) ALL, who showed genetic aberrations as determined by high-resolution single nucleotide polymorphism array (SNP-A) analysis at both diagnosis and relapse. We evaluated the clinical relevance of the SNP-A assay for the detection of subtle changes in the size of affected genetic lesions at relapse as well as the prognostic value of the assay. In our patients, application of the SNP-A assay enabled sensitive detection of cryptic changes affecting clinically important genes in NK ALL. Therefore, this assay seems to be more advantageous compared to other conventional methods such as FISH assay, HemaVision (DNA Technology, Denmark), and conventional karyotyping for the detection of an "unstable genotype" at relapse, which may be associated with microscopic clonal evolution and poor prognosis. Further comprehensive studies are required to confirm the issues presented by our case patients in this report.

Semi-quantitative measurement of a specific glycoform using a DNA-tagged antibody and lectin affinity chromatography for glyco-biomarker development.

Aberrant glycosylation-targeted disease biomarker development is based on cumulative evidence that certain glycoforms are mass-produced in a disease-specific manner. However, the development process has been hampered by the absence of an efficient validation method based on a sensitive and multiplexed platform. In particular, ELISA-based analytical tools are not adequate for this purpose, mainly because of the presence of a pair of N-glycans of IgG-type antibodies. To overcome the associated hurdles in this study, antibodies were tagged with oligonucleotides with T7 promoter and then allowed to form a complex with corresponding antigens. An antibody-bound specific glycoform was isolated by lectin chromatography and quantitatively measured on a DNA microarray chip following production of fluorescent RNA by T7-trascription. This tool ensured measurement of targeted glycoforms of multiple biomarkers with high sensitivity and multiplexity. This analytical method was applied to an in vitro diagnostic multivariate index assay where a panel of hepatocellular carcinoma (HCC) biomarkers comprising alpha-fetoprotein, hemopexin, and alpha-2-macroglobulin (A2M) was examined in terms of the serum level and their fuco-fractions. The results indicated that the tests using the multiplexed fuco-biomarkers provided improved discriminatory power between non- hepatocellular carcinoma and hepatocellular carcinoma subjects compared with the alpha-fetoprotein level or fuco-alpha-fetoprotein test alone. The developed method is expected to facilitate the validation of disease-specific glycan biomarker candidates.

Evaluation of propidium monoazide real-time PCR for early detection of viable Mycobacterium tuberculosis in clinical respiratory specimens.

BACKGROUND: Conventional acid-fast bacilli (AFB) staining cannot differentiate viable from dead cells. Propidium monoazide (PMA) is a photoreactive DNA-binding dye that inhibits PCR amplification by DNA modification. We evaluated whether PMA real-time PCR is suitable for the early detection of viable Mycobacterium tuberculosis (MTB) in clinical respiratory specimens. METHODS: A total of 15 diluted suspensions from 5 clinical MTB isolates were quadruplicated and subjected to PMA treatment and/or heat inactivation. Eighty-three AFB-positive sputum samples were also tested to compare the ΔCT values (CT value in PMA-treated sputum samples-CT value in non-PMA-treated sputum samples) between culture-positive and culture-negative specimens. Real-time PCR was performed using Anyplex MTB/NTM Real-Time Detection (Seegene, Korea), and the CT value changes after PMA treatment were compared between culture-positive and culture-negative groups. RESULTS: In MTB suspensions, the increase in the CT value after PMA treatment was significant in dead cells (P=0.0001) but not in live cells (P=0.1070). In 14 culture-negative sputum samples, the median ΔCT value was 5.3 (95% confidence interval [CI], 4.1-8.2; P<0.0001), whereas that in 69 culture-positive sputum samples was 1.1 (95% CI, 0.7-2.0). In the ROC curve analysis, the cutoff ΔCT value for maximum sensitivity (89.9%) and specificity (85.7%) for differentiating dead from live cells was 3.4. CONCLUSIONS: PMA real-time PCR is a useful approach for differentiating dead from live bacilli in AFB smear-positive sputum samples.

Conversational analysis of medical discourse in rehabilitation: a study in Korea.

OBJECTIVE: The purpose of this study is to conduct an analysis of actual communicative behaviors, including nonverbal ones, between physicians and patients in rehabilitation. DESIGN: Sixteen videotaped physician-patient interactions in a rehabilitation center in Korea were transcribed. And three coders placed utterances in transcripts into categories while watching the videotapes. RESULTS: Consistent with results of previous studies, patient active communicative behaviors varied considerably depending on individual patients. The findings revealed that, on average, patients' active communicative behaviors were less than 9% of patients' total utterances. In particular, almost half of the patients (N = 7) did not even ask one single question. The results also showed that physicians' empathic communicative acts averaged less than 3% of physicians' total utterances. In addition, among physicians' nonverbal behaviors, eye-contact showed significant correlation with physician empathic listening and supportive talks. CONCLUSIONS: The findings of this study indicated a considerable lack of empathic communicative behaviors of physicians in Korea. This might have led to infrequent use of active communicative behaviors by patients. In addition, the importance of physician's partnership for enhancement of active communicative behaviors of patients was demonstrated. To better serve patients, physicians who wish to improve their communication should first identify components of their empathic communicative behaviors that need improvement and then attempt to refine their skills.

Prevalence of plasmid-mediated quinolone resistance and its association with extended-spectrum beta-lactamase and AmpC beta-lactamase in Enterobacteriaceae.

BACKGROUND: We investigated the prevalence of plasmid-mediated quinolone resistance and its association with extended-spectrum beta-lactamase (ESBL) and AmpC beta-lactamase in Enterobacteriaceae. METHODS: A total of 347 non-duplicated isolates of Enterobacteriaceae were collected between August and October 2006 from 2 hospitals. Qnr determinant screening was conducted using PCR amplification, and all positive results were confirmed by direct sequencing. Qnr-positive strains were determined on the basis of the presence of ESBL and AmpC beta-lactamase genes. RESULTS: The qnr gene was detected in 47 of 347 clinical Enterobacteriaceae isolates. Among the 47 qnr-positive strains, Klebsiella pneumoniae (N=29) was the most common, followed by Escherichia coli (N=6), Enterobacter cloacae (N=6), Citrobacter freundii (N=5), and Enterobacter aerogenes (N=1). These isolates were identified as qnrA1 (N=6), 8 qnrB subtypes (N=40), and qnrS1 (N=1). At least 1 ESBL was detected in 38 of the 47 qnr-positive strains. Qnr-positive strains also showed high positive rates of ESBL or AmpC beta-lactamase, such as TEM, SHV, CTX-M, and DHA. DHA-1 was detected in 23 of 47 qnr-positive strains, and this was co-produced with 1 qnrA1 and 22 qnrB4. Strains harboring MIR-1T and CMY were also detected among the qnr-positive strains. Antimicrobial-resistance rates of qnr-positive strains to ciprofloxacin, levofloxacin, norfloxacin, nalidixic acid, and moxifloxacin were 51.1%, 46.8%, 46.8%, 74.5%, and 53.2%, respectively. CONCLUSIONS: The qnr genes were highly prevalent in Enterobacteriaceae, primarily the qnrB subtypes. They were closely associated with EBSL and AmpC beta-lactamase.

High rates of plasmid-mediated quinolone resistance QnrB variants among ciprofloxacin-resistant Escherichia coli and Klebsiella pneumoniae from urinary tract infections in Korea.

The aims of this study were to investigate the prevalence of qnrA, qnrB, and qnrS determinants and their molecular characteristics in ciprofloxacin-resistant isolates of Escherichia coli and Klebsiella pneumoniae from urinary tract infections (UTI) in Korea. A total of 202 nonduplicated clinical isolates of ciprofloxacin-resistant E. coli (n = 143) and K. pneumoniae (n = 59) were collected between July 2005 and August 2006. The qnr determinant screening was carried out by PCR amplification of qnrA, qnrB, and qnrS, and all positive results were confirmed by direct sequencing of the PCR products. For qnr-positive strains and their conjugants, antimicrobial susceptibility tests and pulsed-field gel electrophoresis (PFGE) were performed. The qnrB gene was detected in 41 of the 202 isolates. Among 33 of 59 (55.9%) K. pneumoniae isolates showing qnrB, 29 isolates contained the qnrB4 gene, 3 isolates had the qnrB2 gene, and 1 isolate had the qnrB6 gene. All 8 (5.6%) of the qnrB-positive isolates among the 143 E. coli strains possessed the qnrB4 gene. The minimum inhibitory concentrations (MICs) of ciprofloxacin for the transconjugants were 0.03-2 mug/ml, representing an increase of 4- to 256-fold relative to the recipient, E. coli J53Az(r). Resistances to various other antimicrobial agents also were transferred with the plasmid. The PFGE analysis revealed indistinguishable or closely related patterns in several strains and highly diverse patterns in general. QnrB variants, especially the qnrB4 subtype, are highly prevalent in ciprofloxacin-resistant E. coli and K. pneumoniae from UTI in Korea. The emergence of plasmid-mediated quinolone resistance may contribute by several means to the rapid increase in bacterial resistance to these drugs.

Prevalence, characteristics, and molecular epidemiology of macrolide and fluoroquinolone resistance in clinical isolates of Streptococcus pneumoniae at five tertiary-care hospitals in Korea.

The genes erm(B), mef(A), and both erm(B) and mef(A) were identified in 42.6, 10.1, and 47.3%, respectively, of the erythromycin-resistant Streptococcus pneumoniae isolates. Of the strains, 3.8% were nonsusceptible to levofloxacin and had 1 to 6 amino acid changes in the quinolone resistance-determining region, including a new mutation, Asn94Ser, in the product of parC. Levofloxacin with reserpine was highly specific for efflux screening.

Evaluation of a colorimetric antifungal susceptibility test by using 2,3-diphenyl-5-thienyl-(2)-tetrazolium chloride.

A colorimetric antifungal susceptibility test was performed using 2,3-diphenyl-5-thienyl-(2)-tetrazolium chloride. Among 24 strains of Candida species, no trailing growth was found. In 22 and 20 strains, the MICs obtained in the colorimetric assay were within two dilutions of those obtained by the National Committee for Clinical Laboratory Standards method for ketoconazole and itraconazole, respectively.

Reduction of contamination of mycobacterial growth indicator tubes with a modified antimicrobial combination.

Culture in the fluorimetric Mycobacteria Growth Indicator Tube (MGIT) treated with a combination of vancomycin, amphotericin B, and nalidixic acid (VAN) showed growth of most strains of 31 mycobacterial species with a less-than-1-day delay. The results were similar to those in the MGIT with polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin, but with respiratory specimens, the MGIT with VAN showed a lower contamination rate with no change in the detection rate or time.

Early Screening of oxacillin-resistant Staphylococcus aureus and Staphylococcus epidermidis from blood culture.

The timely detection of blood-borne pathogens is one of the most important functions of the microbiology laboratory. Recently, methicillin-resistant staphylococci have become the most important pathogens seen by the laboratory. The purpose of this study was to evaluate Staphy agar, a novel screening medium, for the detection methicillin-resistant Staphylococcus aureus, S. epidermidis, or other coagulase-negative staphylococci (CNS) from positive blood cultures showing Gram-positive cocci in clusters. Eighty-six blood cultures that yielded Gram-positive cocci in clusters were included in this study. The organisms were finally identified by the Vitek system, and oxacillin resistance was confirmed by polymerase chain reaction (PCR)-based mecA gene detection. The identification and oxacillin resistance of all S. aureus strains showed complete agreement with the Vitek and PCR results. The presumptive detection of S. epidermidis and other CNS were consistent with the Vitek system in 94.7%, and the screening of oxacillin resistance was consistent with the result of PCR in 92.1% of 38 strains. The Staphy agar method is reliable and rapid for differentiating Gram-positive cocci in clusters in blood and for determining their methicillin resistance.