Saeam Shin

Mortality risk factors of Acinetobacter baumannii bacteraemia.

Abstract

Evaluation of the Xpert Clostridium difficile Assay for the Diagnosis of Clostridium difficile Infection

Infection with Clostridium difficile is a growing concern because of the increasing prevalence and spread of nosocomial infections. Emergence of the hypervirulent 027/NAP1/BI strain is also notable. Existing diagnostic methods have low sensitivity or are time-consuming. Therefore, establishing a rapid and accurate microbiological diagnostic assay is needed. We evaluated the Xpert C. difficile assay (Xpert CD assay; Cepheid, USA) to detect toxigenic C. difficile. This assay is a real-time multiplex PCR assay that can be used to detect toxigenic C. difficile strains and differentiate the C. difficile presumptive 027/NAP1/BI strain. A total of 253 loose stool specimens were collected and toxigenic cultures, VIDAS C. difficile A & B assays (VIDAS CDAB assay; bioMérieux, France), and the Xpert CD assay were performed. In comparison to toxigenic cultures, the sensitivity, specificity, and positive and negative predictive values were 100%, 94.6%, 83.1%, and 100%, respectively, for the Xpert CD assay and 40.8%, 98.0%, 100%, and 88.9%, respectively, for VIDAS CDAB assay. Because of the low prevalence of the PCR ribotype 027 in Korea, the evaluation of the usefulness of the Xpert CD assay for screening for the 027 strain was limited. The Xpert CD assay provides great sensitivity in diagnosing toxigenic C. difficile infection. In addition, this method has excellent usability because it is simple and fast.

Comparison Study of the Rates of Manual Peripheral Blood Smear Review From 3 Automated Hematology Analyzers, Unicel DxH 800, ADVIA 2120i, and XE 2100, Using International Consensus Group Guidelines

CONTEXT In the clinical laboratory, it is important both to reduce the number of peripheral blood slide reviews to save time and money and to avoid reporting false results. OBJECTIVE To determine differences in the slide review rates of 3 widely used automated hematologic analyzers, the Unicel DxH 800 (Beckman Coulter Inc, Fullerton, California), ADVIA 2120i (Siemens Diagnostics, Tarrytown, New York), and XE 2100 (Sysmex, Kobe, Japan), using International Consensus Group for Hematology Review guidelines. DESIGN A total of 1485 samples were tested, and 300 were manually reviewed. Slide review rates, sensitivity, specificity, and false-positive and false-negative rates were estimated using consensus group rules and compared using χ(2) tests, Fisher exact tests, or generalized estimating equations. RESULTS Unicel DxH 800, ADVIA 2120i, and XE 2100 showed 22.8%, 20.2%, and 28.6% slide review rates; 14.3%, 14.3%, and 9.7% false-negative rates; and 13.7, 11.3%, and 17.3% false-positive rates, respectively. All analyzers showed significantly higher false-negative rates than that of the consensus group (2.9%). CONCLUSIONS False-negative rates were higher than the recommended levels. Among 3 automated hematologic analyzers, XE 2100 showed the highest rate of slide review. Because the present study clearly shows that the slide review rates have distinct characteristics among the studied analyzers, each individual laboratory should consider selecting the most appropriate analyzer according to clinical characteristics. Analyzers with high sensitivity may be advantageous in outpatient settings for screening patients, whereas analyzers with high specificity may be beneficial in inpatient settings for efficient patient care.

Routine Chromosomal Microarray Analysis is Necessary in Korean Patients With Unexplained Developmental Delay/Mental Retardation/Autism Spectrum Disorder

Background All over the world, chromosomal microarray (CMA) is now the first tier diagnostic assay for genetic testing to evaluate developmental delay (DD), mental retardation (MR), and autism spectrum disorder (ASD) with unknown etiology. The average diagnostic yield of the CMA test is known to be about 12.2%, while that of conventional G-banding karyotype is below 3%. This study aimed to assess the usefulness of CMA for the purpose of clinical diagnostic testing in the Korean population. Methods We performed CMA and multiplex ligation-dependent probe amplification (MLPA) tests in 96 patients with normal karyotype and unexplained DD, MR, or ASD. The CMA was conducted with CytoScan 750K array (Affymetrix, USA) with an average resolution of 100 kb. Results Pathogenic copy number variations (CNVs) were detected in 15 patients by CMA and in two patients by MLPA for four known microdeletion syndromes (Prader-Willi/Angelman syndrome, DiGeorge syndrome, Miller-Dieker syndrome and Williams syndrome) designated by National Health Insurance system in Korea. The diagnostic yield was 15.6% and 2.1%, respectively. Thirteen (13.5%) patients (excluding cases with pathogenic CNVs) had variants of uncertain clinical significance. There was one patient with a 17.1-megabase (Mb) region of homozygosity on chromosome 4q. Conclusions Our findings suggest the necessity of CMA as a routine diagnostic test for unexplained DD, MR, and ASD in Korea.

Performance Evaluation of New Automated Hepatitis B Viral Markers in the Clinical Laboratory Two Quantitative Hepatitis B Surface Antigen Assays and an HBV Core-Related Antigen Assay

We evaluated quantitative hepatitis B surface antigen (qHBsAg) assays and a hepatitis B virus (HBV) core-related antigen (HBcrAg) assay. A total of 529 serum samples from patients with hepatitis B were tested. HBsAg levels were determined by using the Elecsys (Roche Diagnostics, Indianapolis, IN) and Architect (Abbott Laboratories, Abbott Park, IL) qHBsAg assays. HBcrAg was measured by using Lumipulse HBcrAg assay (Fujirebio, Tokyo, Japan). Serum aminotransferases and HBV DNA were respectively quantified by using the Hitachi 7600 analyzer (Hitachi High-Technologies, Tokyo, Japan) and the Cobas AmpliPrep/Cobas TaqMan test (Roche). Precision of the qHBsAg and HBcrAg assays was assessed, and linearity of the qHBsAg assays was verified. All assays showed good precision performance with coefficients of variation between 4.5% and 5.3% except for some levels. Both qHBsAg assays showed linearity from 0.1 to 12,000.0 IU/mL and correlated well (r = 0.9934). HBsAg levels correlated with HBV DNA (r = 0.3373) and with HBcrAg (r = 0.5164), and HBcrAg also correlated with HBV DNA (r = 0.5198; P < .0001). This observation could provide impetus for further research to elucidate the clinical usefulness of the qHBsAg and HBcrAg assays.

Assessment of real-time PCR method for detection of EGFR mutation using both supernatant and cell pellet of malignant pleural effusion samples from non-small-cell lung cancer patients

Abstract Background: EGFR mutation is an emerging biomarker for treatment selection in non-small-cell lung cancer (NSCLC) patients. However, optimal mutation detection is hindered by complications associated with the biopsy procedure, tumor heterogeneity and limited sensitivity of test methodology. In this study, we evaluated the diagnostic utility of real-time PCR using malignant pleural effusion samples. Methods: A total of 77 pleural fluid samples from 77 NSCLC patients were tested using the cobas EGFR mutation test (Roche Molecular Systems). Pleural fluid was centrifuged, and separated cell pellets and supernatants were tested in parallel. Results were compared with Sanger sequencing and/or peptide nucleic acid (PNA)-mediated PCR clamping of matched tumor tissue or pleural fluid samples. Results: All samples showed valid real-time PCR results in one or more DNA samples extracted from cell pellets and supernatants. Compared with other molecular methods, the sensitivity of real-time PCR method was 100%. Concordance rate of real-time PCR and Sanger sequencing plus PNA-mediated PCR clamping was 98.7%. Conclusions: We have confirmed that real-time PCR using pleural fluid had a high concordance rate compared to conventional methods, with no failed samples. Our data demonstrated that the parallel real-time PCR testing using supernatant and cell pellet could offer reliable and robust surrogate strategy when tissue is not available.

t(12;17)(p13;q12)/TAF15-ZNF384 Rearrangement in Acute Lymphoblastic Leukemia

Dear Editor, In ALL, cytogenetic subgroups according to recurrent genetic abnormalities are used to classify patients for risk stratification and to introduce them to the proper therapeutic strategies—such as the use of tyrosine kinase inhibitors in the case of t(9;22)(q34;q11.2)/BCR-ABL1 fusion [1]. Therefore, the identification of genetic aberrations is clinically significant and crucial in understanding the leukemogenesis mechanisms. t(12;17)(p13;q11.2)/TAF15-ZNF384 rearrangement is a rare chromosomal abnormality [2]. It has been reported in lymphoid as well as myeloid leukemia and is thought to be involved in the early differentiation of common progenitors [3]. Although some cases of the disease have reported a poor outcome [4,5], the clinical characteristics and prognostic impact of TAF15-ZNF384 are not well characterized, since not all the previous studies investigated the precise molecular consequences of the translocation. We report a B-ALL case with t(12;17)(p13;q11.2) detected by cytogenetic analysis and confirmed by direct sequencing of the TAF15-ZNF384 transcripts, which substantiated genes involved in the rearrangement. A 32-month-old girl was admitted following four days of fever and erythema of both legs. Initial complete blood counts demonstrated pancytopenia, with hemoglobin level of 5.5 g/dL, white blood cell counts of 2.63×109/L, and platelet counts of 123×109/L. Leukemic blasts up to 5% were observed in peripheral blood and 84.9% in bone marrow. The leukemic blasts varied in size, with scanty and occasionally granulated cytoplasms. Blasts were positive for CD34, CD19, CD13, CD33, cytoplasmic CD79a, and terminal deoxynucleotidyl transferase (TdT), and negative for CD2, CD7, CD10, CD14, myeloperoxidase, and cytoplasmic CD3, indicating pro-B cell stage ALL. The reverse transcription-polymerase chain reaction using the HemaVision kit (DNA technology, Aarhus, Denmark) and fluorescence in situ hybridization using Vysis IGH/MYC/CEP8 tri-color dual fusion, CDKN2A (p16)/CEP9 dual spot, and MLL Break Apart probes (Abbott Molecular, Abbott Park, IL, USA) showed negative results. G(Giemsa)-T(Trypsin)-G-banding analysis using the bone marrow sample revealed a karyotype of 46,XX,t(12;17)(p13;q11.2)[8]/46,XX[11] (Fig. 1A). To confirm the TAF15-ZNF384 fusion transcript, complementary DNA was synthesized from total RNA, amplified, and sequenced by using primers specific for TAF15 and ZNF384 [6]. The fusion transcript, amplified by using specific primers, was approximately 800 bp in length (Fig. 1B). Sequence alignment of the amplified product revealed breakpoints between exon 9 of TAF15 and exon 3 of ZNF384 (Fig. 1C). Fig. 1 (A) G(Giemsa)-T(Trypsin)-G-banding analysis using the bone marrow sample revealed a translocation involving the breakpoint on chromosome 12p13 and 17q11.2. (B) Agarose gel electrophoresis of the TAF15-ZNF384 fusion transcript obtained from patient (approximately ... Diagnostic lumbar puncture and computed tomography ruled out the central nervous system (CNS) involvement. She achieved complete remission by day 35 following a single course of standard risk induction chemotherapy, including cytarabine, methotrexate, vincristine, hydrocortisone, and I-asparaginase. Thereafter, she received high-dose cyclophosphamide consolidation and intrathecal methotrexate CNS prophylaxis, followed by high-risk vincristine and methotrexate maintenance, and has been in remission for eight months after the initial diagnosis. The t(12;17)(p13;q11), t(12;17)(p13;q12), or t(12;17)(p11-12;q11-12), for which the breakpoint assignment differs slightly, was first described in 1982 by Kaneko et al. [7]. Its molecular fusion gene, TAF15-ZNF384, was shown to be involved in tumorigenesis in 2002 [2]. To the best of our knowledge, there have been 10 cases of TAF15-ZNF384 fusion confirmed using molecular studies (Table 1). In addition to the early B-cell morphology, coexpression of myeloid markers and a lack of expression of CD10 are common immunophenotypic features of this entity [8]. There are conflicting reports regarding the prognosis of cases with t(12;17) [3,5,6]. Owing to its low incidence, the treatment protocol differs between institutions, and the statistical significance of t(12;17) in terms of clinical outcome has not been analyzed to date [3]. Studies with a larger ALL patient group displaying such changes are required in order to determine the prognostic impact of TAF15-ZNF384 fusion. Table 1 Summary of acute leukemia cases with the TAF15-ZNF384 fusion transcript confirmed by molecular studies Primers and probes for TAF15-ZNF384 are not usually included in commercial kits for screening leukemia translocations, so this abnormality may be missed in routine clinical settings. We suggest that special attention be paid when a translocation between 12p13 and 17q11 is suspected and that additional studies for TAF15-ZNF384 may be useful in ALL diagnoses. Including this fusion transcript in the initial screening panel can assist in identifying underdiagnosed cases and distinguishing ambiguity of t(12;17), therefore establishing their incidence and clinical significance.

Validation and Optimization of the Ion Torrent S5 XL Sequencer and Oncomine Workflow for BRCA1 and BRCA2 Genetic Testing

In this study, we validated the analytical performance of BRCA1/2 sequencing using Ion Torrents new bench-top sequencer with amplicon panel with optimized bioinformatics pipelines. Using 43 samples that were previously validated by Illuminas MiSeq platform and/or by Sanger sequencing/multiplex ligation-dependent probe amplification, we amplified the target with the Oncomine™ BRCA Research Assay and sequenced on Ion Torrent S5 XL (Thermo Fisher Scientific, Waltham, MA, USA). We compared two bioinformatics pipelines for optimal processing of S5 XL sequence data: the Torrent Suite with a plug-in Torrent Variant Caller (Thermo Fisher Scientific), and commercial NextGENe software (Softgenetics, State College, PA, USA). All expected 681 single nucleotide variants, 15 small indels, and three copy number variants were correctly called, except one common variant adjacent to a rare variant on the primer-binding site. The sensitivity, specificity, false positive rate, and accuracy for detection of single nucleotide variant and small indels of S5 XL sequencing were 99.85%, 100%, 0%, and 99.99% for the Torrent Variant Caller and 99.85%, 99.99%, 0.14%, and 99.99% for NextGENe, respectively. The reproducibility of variant calling was 100%, and the precision of variant frequency also showed good performance with coefficients of variation between 0.32 and 5.29%. We obtained highly accurate data through uniform and sufficient coverage depth over all target regions and through optimization of the bioinformatics pipeline. We confirmed that our platform is accurate and practical for diagnostic BRCA1/2 testing in a clinical laboratory.

Establishing Quality Control Ranges for Antimicrobial Susceptibility Testing of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus: A Cornerstone to Develop Reference Strains for Korean Clinical Microbiology Laboratories

Quality control (QC) processes are being performed in the majority of clinical microbiology laboratories to ensure the performance of microbial identification and antimicrobial susceptibility testing by using ATCC strains. To obtain these ATCC strains, some inconveniences are encountered concerning the purchase cost of the strains and the shipping time required. This study was focused on constructing a database of reference strains for QC processes using domestic bacterial strains, concentrating primarily on antimicrobial susceptibility testing. Three strains (Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus) that showed legible results in preliminary testing were selected. The minimal inhibitory concentrations (MICs) and zone diameters (ZDs) of eight antimicrobials for each strain were determined according to the CLSI M23. All resulting MIC and ZD ranges included at least 95% of the data. The ZD QC ranges obtained by using the CLSI method were less than 12 mm, and the MIC QC ranges extended no more than five dilutions. This study is a preliminary attempt to construct a bank of Korean QC strains. With further studies, a positive outcome toward cost and time reduction can be anticipated.

Detection of Immunoglobulin Heavy Chain Gene Clonality by Next-Generation Sequencing for Minimal Residual Disease Monitoring in B-Lymphoblastic Leukemia

Minimal residual disease (MRD) following B-lymphoblastic leukemia (B-ALL) treatment has gained prognostic importance. Clonal immunoglobulin heavy chain (IGH) gene rearrangement is a useful follow-up marker in B-ALL owing to its high positivity rate. We evaluated the performance and clinical applicability of a next-generation sequencing (NGS) assay for IGH rearrangement in B-ALL MRD monitoring. IGH rearrangement was tested by using fluorescence PCR-fragment analysis and the NGS assay in eight B-ALL patients. The NGS assay was run on two platforms: the Ion Torrent PGM (Thermo Fisher Scientific, USA) (18 samples from 1st to 7th patients) and the MiSeq system (Illumina, USA) (four samples from 8th patient). All initial diagnostic samples and four follow-up samples were positive for clonal IGH rearrangement with fluorescence PCR-fragment analysis and the NGS assay, and six follow-up samples were positive only with NGS. In one case with BCR-ABL1 translocation, BCR-ABL1 quantitative PCR was negative but the NGS IGH assay was positive just prior to full-blown relapse, suggesting the high sensitivity and clinical utility of the NGS assay. The NGS assay is proposed for MRD monitoring in B-ALL Additional studies are needed to confirm the clinical implications of cases showing positive results only in NGS.