Joowon Park

Evaluation of a Multiplex Real-time PCR Assay for the Detection of Respiratory Viruses in Clinical Specimens

Background In this study, we evaluated the analytical performance and clinical potential of a one-step multiplex real-time PCR assay for the simultaneous detection of 14 types of respiratory viruses using the AdvanSure RV real-time PCR Kit (LG Life Sciences, Korea). Methods Three hundred and twenty clinical specimens were tested with the AdvanSure RV real-time PCR Kit and conventional multiplex reverse transcription (RT)-PCR assay. The assay results were analyzed and the one-step AdvanSure RV real-time PCR Kit was compared with the conventional multiplex RT-PCR assay with respect to the sensitivity and specificity of the detection of respiratory viruses. Results The limit of detection (LOD) was 1.31 plaque-forming units (PFU)/mL for human rhinoviruses (hRVs), 4.93 PFU/mL for human coronavirus HCoV-229E/NL63, 2.67 PFU/mL for human coronavirus HCoV-OC43, 18.20 PFU/mL for parainfluenza virus 1 (PIV)-1, 24.57 PFU/mL for PIV-2, 1.73 PFU/mL for PIV-3, 1.79 PFU/mL for influenza virus group (Flu) A, 59.51 PFU/mL for FluB, 5.46 PFU/mL for human respiratory syncytial virus (hRSV)-A, 17.23 PFU/mL for hRSV-B, 9.99 PFU/mL for human adenovirus (ADVs). The cross-reactivity test for this assay against 23 types of non-respiratory viruses showed negative results for all viruses tested. The agreement between the one-step AdvanSure multiplex real-time PCR assay and the conventional multiplex RT-PCR assay was 98%. Conclusions The one-step AdvanSure RV multiplex real-time PCR assay is a simple assay with high potential for specific, rapid and sensitive laboratory diagnosis of respiratory viruses compared to conventional multiplex RT-PCR.

Human telomerase reverse transcriptase (hTERT): a target molecule for the treatment of cisplatin-resistant tumors.

BACKGROUND Human telomerase reverse transcriptase (hTERT) is a catalytic enzyme that is required for telomerase activity (TA) and cancer progression. Telomerase inhibition or inactivation increases cellular sensitivity to UV irradiation, DNA-damaging agents, the tyrosine kinase inhibitor, imatinib, and pharmacological inhibitors, such as BIBR1532. hTERT is associated with apoptosis. Some patients show drug-resistance during anti-cancer drug treatment and the cancer cell acquire anti-apoptotic mechanism. Therefore, we attempted to study correlation between hTERT and drug-resistance. METHODS To study the correlation between protein level and activity of hTERT and drug-resistance, Western blotting and telomerase repeat amplification protocol (TRAP) assays were performed. To investigate whether hTERT contributes to drug resistance in tumor cells, we transiently decreased hTERT levels using small interfering RNA (siRNA) in T24/R2 cells. RESULTS hTERT knockdown increased Bax translocation into the mitochondria and cytochrome C release into the cytosol. Caspase inhibitors, especially Z-VAD-FMK, rescued this phenomenon, suggesting that the stability or expression of hTERT might be regulated by caspase activity. CONCLUSIONS These data suggest that hTERT might be a target molecule for drug-resistant tumor therapy.

Congenital Zinc Deficiency from Mutations of the SLC39A4 Gene as the Genetic Background of Acrodermatitis Enteropathica

Acrodermatitis enteropathica (AE) is an autosomal recessive disorder with the clinical triad of acral dermatitis, diarrhea and alopecia. AE is known to be caused by mutations of the SLC39A4 gene on the chromosome band 8q24.3, encoding the zinc transporter in human. An 8-month-old Korean boy presented with eczematous changes on the inguinal area and knees and was diagnosed with AE. Blood tests revealed a markedly decreased level of plasma zinc, and his symptoms improved on oral zinc replacement. To confirm the diagnosis of AE from congenital zinc deficiency, direct sequencing analysis of SLC39A4 was performed and revealed that he was compound heterozygous for a known missense mutation (Arg95Cys) and a novel splicing mutation in the donor site of intron 7 (c.1287+2T>C). Family study showed that his parents were heterozygous carriers of the mutations. To the best of our knowledge, this is the first report of genetically confirmed AE in Korea.

Up-regulation of reactive oxygen species (ROS) and resistance to Fas-mediated apoptosis in the C33A cervical cancer cell line transfected with IL-18 receptor

Abstract Cervical cancer cells were transfected with a newly discovered interleukin (IL)-18 receptor to investigate the effect of endogenous IL-18 on the regulation of immune-related factors such as Fas (CD95/Apo-1)/Fas ligand and intercellular adhesion molecules. Transfection of the IL-18 receptor selectively induced a slight enhancement of the Fas via the up-regulation of intracellular reactive oxygen species and IL-18 in cervical carcinoma C33A cells, whereas there were no effects on the expression of p53, intercellular adhesion molecules-1 and Fas ligand. Neither IL-18 receptor transfection nor recombinant IL-18 enhanced interferon-γ production in C33A cells. Thus, IL-18 receptor transfection induced IL-18 expression and enhanced intracellular reactive oxygen species and Fas expression in C33A cells in an interferon-γ-independent pathway. However, treatment with agonistic anti-Fas antibody did not induce the apoptosis of C33A/IL-18 receptor transfectants, suggesting that either reactive oxygen species play a key role in resisting the Fas-induced apoptosis of C33A cells, or Fas was not functional. These results show that C33A/IL-18 receptor cells are resistant to the apoptosis and thus can survive against the immune surveillance and activated immune cells. Our results thus suggest that IL-18 and IL-18 receptor, together, may play a role in immunoregulation or in inflammation by augmenting the levels of IL-18 and reactive oxygen species in C33A cells.

Evaluation of iQ200 Automated Urine Microscopy Analyzer

BACKGROUND Microscopic examination of urine sediment is one of the most commonly performed tests in the clinical laboratory. However, manual microscopic sediment examination is labor-intensive, time-consuming and imprecise. In this study, we evaluated the analytical performance and clinical usefulness of a recently introduced image-based automated urinalysis system, Iris iQ200 (Iris Diagnostics, USA). METHODS We assessed the iQ200 for linearity, precision and carryover rate using patients samples and quality control materials. On 337 urine samples, urine sediment analyses performed by the iQ200 were compared with manual microscopy results. RESULTS The iQ200 showed a good linearity (r2>0.99) for all cellular components analyzed. Within-run and total CVs on urine specimens and quality control samples were less than 10% except for within-run CV for the samples with low concentration of the squamous epithelial cells. The carryover rates were 0.21% for RBCs and 1.92% for WBCs. The agreement rates within one grade between the iQ200 and manual microscopy for RBCs, WBCs, and squamous epithelial cells were 93.8%, 94.2% and 96.9%, respectively. CONCLUSIONS Since the iQ200 showed a reliable analytical performance and good concordance with manual microscopy, it could be useful in the clinical practice as a screening procedure.

Evaluation of SeeplexTM pneumobacter multiplex PCR kit for the detection of respiratory bacterial pathogens in pediatric patients.

BACKGROUND Rapid identification of the causative agent among potential bacterial and viral pathogens is important for the management of acute respiratory disease. In this study, we evaluated the analytical performance and clinical usefulness of a recently-introduced multiplex PCR assay, Seeplex Pneumobacter detection kit (Seegene Inc., Korea) for the identification of respiratory bacterial pathogens. METHODS One hundred and eighty one nasopharyngeal aspirates were collected from pediatric patients with respiratory symptoms and analysed by multiplex PCR for the detection of Streptococcus pneumoniae (S.P), Haemophilus influenzae (H.I), Mycoplasma pneumoniae (M.P), Chlamydophila pneumoniae (C.P), Bordetella pertussis (B.P) and Legionella pneumophila (L.P). A comparison of multiplex PCR with conventional culture for the isolation of S.P and H.I was performed on 112 specimens. The cross reactivity of multiplex PCR was also evaluated. RESULTS Of 181 cases, 81 cases were positive by multiplex PCR (44.8%): 52 cases for S.P (28.7%), 47 cases for H.I (26.0%), 9 cases for M.P (5.0%), 3 cases for B.P (1.7%) and 1 case for C.P (0.6%) including multiple infection cases. The agreement rates between multiplex PCR and culture for S.P and H.I were 92.9% (kappa index=0.84, P<0.001) and 91.1% (kappa index=0.75, P<0.001), respectively. There was no cross reactivity with common bacterial and viral pathogens. CONCLUSIONS Seeplex Pneumobacter detection kit could be a useful screening tool for the rapid detection of respiratory bacterial pathogens. Further studies with lower respiratory tract specimens would be needed for the clinical evaluation of S. pneumoniae and H. influenzae detected by multiplex PCR.

Extramedullary blast crisis of secondary CML accompanying marrow fibrosis.

which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. A 54-year-old man was referred to our hospital with right flank pain. Three years ago, he was diagnosed with gastric mucosa-associated lymphoid tissue (MALT) lymphoma and successfully treated with radiotherapy. CBC showed a WBC count of 24. Bone marrow (BM) examination showed granulocytic and megakaryocytic proliferation with moderate dysplastic megakaryopoiesis (A; H&E stain, ×200), and diffuse reticulin fibrosis (B; reticulin stain, ×400). Primary myelofibrosis was the first diagnostic consideration after BM study. Chromosomal analysis, however, showed t(9;22)(q34;q11.2), indicating CML. Concurrent abdomen computerized tomography revealed enlarged inguinal lymph nodes. Inguinal lymph node biopsy showed diffuse infiltration of immature cells (C; H&E stain, ×400), which were positive for myeloperoxidase (D). BCR/ABL1 rearrangement was demonstrated by fluorescence in-situ hybridization analysis, and a diagnosis of granulocytic sarcoma (GS) was made. Accompanying extramedullary myeloid tumor, CML was classified as blastic phase. Secondary CML with a simultaneous manifestation of GS is rare. Combining morphological and molecular-cytogenetic approaches can help detect the coexistence of both neoplasms, especially in CML cases with fewer typical morphologic features.

Current routine practice and clinico-pathological characteristics associated with acute promyelocytic leukemia in Korea

Background Acute promyelocytic leukemia (APL) can be life threatening, necessitating emergency therapy with prompt diagnosis by morphologic findings, immunophenotyping, cytogenetic analysis, or molecular studies. This study aimed to assess the current routine practices in APL and the clinico-pathologic features of APL. Methods We reviewed the medical records of 48 Korean patients (25 men, 23 women; median age, 51 (20-80) years) diagnosed with APL in 5 university hospitals between March 2007 and February 2012. Results The WBC count at diagnosis and platelet count varied from 0.4 to 81.0 (median 2.0)×109/L and 2.7 to 124.0 (median 54.5)×109/L, respectively. The median values for prothrombin time and activated partial thromboplastin time were 14.7 (11.3-44.1) s and 29 (24-62) s, respectively. All but 2 patients (96%) showed a fibrin/fibrinogen degradation product value of >20 µg/mL. The D-dimer median value was 5,000 (686-55,630) ng/mL. The t(15;17)(q22;q12 and PML-RARA fusion was found in all patients by chromosome analysis and/or multiplex reverse transcriptase-polymerase chain reaction (RT-PCR), with turnaround times of 8 (2-19) d and 7 (2-13) d, respectively. All patients received induction chemotherapy: all-trans retinoic acid (ATRA) alone (N=11, 26%), ATRA+idarubicin (N=25, 58%), ATRA+cytarabine (N=3, 7%), ATRA+idarubicin+cytarabine (N=4, 9%). Conclusion Since APL is a medical emergency and an accurate diagnosis is a prerequisite for prompt treatment, laboratory support to implement faster diagnostic tools to confirm the presence of PML-RARA is required.

Large cell lymphoma as initial presentation of undetected chronic lymphocytic leukemia.

which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. A 46-year-old man having back pain for 3 weeks showed multiple lymphadenopathy and epidural mass (T7-T10) on physical examination, computed tomography, and magnetic resonance imaging. Initial complete blood cell counts mature-appearing lymphocytes (62%) (A; Wright stain, ×1,000). Spinal bone biopsy showed diffuse infiltration of large mononuclear cells with prominent nucleoli (B; hematoxylin-eosin stain, ×400), and partial juxtaposition of large cells and small lymphoid cells (C). Large cells were CD10-, CD20-, and CD79a-positive and CD3-, CD5-, and cyclin D1-negative. The patient was diagnosed with diffuse large B-cell lymphoma (DLBL). Further, bone marrow (BM) aspirate showed several small lymphoid cells (37%), and large immature cells with occasional cytoplasmic vacuolations (D; Wright stain, ×1,000). Flow cytometric analysis of BM aspirate demonstrated that small lymphoid cells (CD45+/low side scatter) were CD5-, CD19-, CD20-, and CD23-positive and TdT-, CD10-, and FMC7-negative, consistent with chronic lymphocytic leukemia (CLL). Transformation of CLL to DLBL occurs in 1-10% of CLL cases and has a poor prognosis. The clonality of both neoplasms was not determined in this case. Despite several chemotherapy cycles, the patient died 3 months after diagnosing DLBL.

Microangiopathic hemolytic anemia as initial presentation of recurrent colon cancer

Fig. 1. Schistocytes, polychromasia and nucleated RBC (arrow) in peripheral blood (Wright stain, ×1,000). Patients with thrombocytopenia are often recommended to undergo a variety of tests, much of which may not be indicated and/or diagnostically helpful. In this series, we found that although a large percentage of patients underwent multiple laboratory tests and pathologic evaluation, these tests often did not offer significant insight into the etiology of the thrombocytopenia. This is congruent with results from other studies specifically focusing on bone marrow biopsies in the workup of thrombocytopenia [1, 2] and further adds to the literature with regard to autoimmune and infectious workup. Additionally, nearly half of the patients in this series had a platelet count >100×10/L, which may in some cases be considered a normal variant [3]. In summary, evaluation of thrombocytopenia should vary based on individual patient history and risk factors rather than a one-size-fits-all testing panel. Our study was based on a small cohort of patients but is likely representative of clinical practice at many large tertiary care centers. Large-scale prospective studies may be helpful to define the optimal workup for thrombocytopenia and formulate a protocol for cost-effective workup by stratification of clinical risk factors.