Yiel Hea Seo

Evaluation of Platelet Indices for Differential Diagnosis of Thrombocytosis by ADVIA 120

BACKGROUNDnFor the diagnosis of essential thrombocythemia (ET), no single clinical or laboratory finding of diagnostic value is available and a differential diagnosis of other myeloproliferative neoplasms or reactive thrombocytosis (RT) is needed. Following recent developments in automated blood cell analyzers, various platelet indices can now be measured. In this study, we analyzed whether platelet counts and 6 platelet indices can be used for the differentiation of ET from RT in patients with a platelet count of 600x10(3)/microL or more.nnnMETHODSnThe subjects studied were 31 patients with ET and 224 patients with RT. The platelet counts, mean platelet volume (MPV), plateletcrit (PCT), platelet distribution width (PDW), mean platelet mass (MPM), mean platelet component concentration (MPC) and large platelets (LPLT) were measured by ADVIA 120 (Bayer Diagnostics, USA). The mean values of each item were compared between the two patient groups and the sensitivity and specificity of each item in the diagnosis of ET were determined by ROC curve analysis.nnnRESULTSnIn essential thrombocythemia, all parameters except MPC were significantly higher than in reactive thrombocytosis. For the diagnosis of ET, the sensitivity and specificity were: 74.2% and 84.4%, when the platelet count was > or = 820 x 10(3)/microL; 80.6% and 80.0%, when the plateletcrit was > or = 0.63%; and 64.5% and 99.1%, respectively, when LPLT was > or = 23 x 10(3)/microL.nnnCONCLUSIONSnThe platelet counts and platelet indices are useful for the differential diagnosis of thrombocytosis. The plateletcrit and LPLT are particularly useful for the diagnosis of ET when the platelet count is markedly increased.

Comparison of sputum and nasopharyngeal swabs for detection of respiratory viruses

Diagnostic tests for respiratory viral infections use traditionally either nasopharyngeal washes or swabs. Sputum is representative of the lower respiratory tract but is used rarely for viral testing. The aim of this study was to compare the detection rates of respiratory viruses from nasopharyngeal swabs and sputum using a multiplex real‐time reverse transcription‐polymerase chain reaction (RT‐PCR). Adults who were admitted or presented to the clinics of Gil Medical Center with acute respiratory symptoms were recruited from 1 November 2012 to 31 March 2013. Paired specimens of nasopharyngeal swabs and sputum were obtained from 154 subjects, and RNA was extracted and tested for 16 different respiratory viruses using the Anyplex II RV16 Detection kit (Seegene, Seoul, Korea). The positive rate was 53% (81/154) for nasopharyngeal swabs and 68% (105/154) for sputum (Pu2009<u20090.001). One hundred thirty‐four viruses were identified for 107 illnesses. Influenza A virus, RSV A, HRV, coronavirus OC43, and adenovirus were detected more frequently in sputum samples than in nasopharyngeal swabs (Pu2009<u20090.001). Importantly, 12 of 44 (27%) influenza A infections and 11 of 27 (41%) RSV infections were positive in only sputum samples. The detection rates of respiratory viruses from sputum samples were significantly higher than those from nasopharyngeal swabs in adults using real‐time multiplex RT‐PCR. These findings suggest that sputum would benefit for the detection of respiratory viruses by nucleic acid amplification tests (NAATs) in patients who produce sputum. Further studies are needed to establish standardized RNA extraction methods from sputum samples. J. Med. Virol. 86:2122–2127, 2014.

A Case of B-cell Lymphoma, Unclassifiable, with Features Intermediate between Diffuse Large B-cell Lymphoma and Burkitt Lymphoma in a Korean Child

B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL) (intermediate DLBCL/BL), is a heterogeneous group with some features resembling DLBCL and others resembling BL. Here, we report a case of intermediate DLBCL/BL in a Korean child. A 2-yr-old male was admitted for evaluation and management of left hip pain. Immunohistochemistry of a biopsy of the femur neck revealed tumor cells positive for CD20, CD10, BCL2, BCL6, and Ki67. A bone marrow (BM) aspirate smear revealed that 49.3% of all nucleated cells were abnormal lymphoid cells, composed of large- and medium-sized cells. Immunophenotyping of the neoplastic cells revealed positivity for CD19, CD10, CD20, and sIg lambda and negativity for CD34, Tdt, and myeloperoxidase (MPO). Cytogenetic and FISH analyses showed a complex karyotype, including t(8;14)(q24.1;q32) and IGH-MYC fusion. Intensive chemotherapy was initiated, including prednisone, vincristine, L-asparaginase, daunorubicin, and central nervous system prophylaxis with intrathecal methotrexate (MTX) and cytarabine. One month after the initial diagnosis, BM examination revealed the persistent of abnormal lymphoid cells; cerebrospinal fluid cytology, including cytospin, showed atypical lymphoid cells. The patient was treated again with cyclophosphamide, vincristine, prednisone, adriamycin, MTX, and intrathecal MTX and cytarabine. The patient died of sepsis 5 months after the second round of chemotherapy.

Nontuberculous Mycobacteria Isolated from Respiratory Specimens during Recent Two Years: Distribution and Clinical Significance

Background: The isolation of nontuberculous mycobacteria (NTM) has been increasing worldwide as well as its clinical importance. The aim of this study was to investigate the distribution and clinical significance of NTM that has been isolated from respiratory specimens during a recent two-year period at a tertiary hospital. Methods: We analyzed respiratory samples that were obtained between January 2009 and December 2010 for AFB culture. We retrospectively reviewed the electronic medical records of these patients to obtain both clinical and radiologic information. NTM pulmonary disease was defined by using the guidelines provided by the America Thoracic Society/Infectious Diseases Society of America. Results: Among the 1,601 specimens that resulted in a positive AFB culture, 310 (19.4%) were NTM. In 189 patients, the most common isolate was M. aviumintracellulare complex (MAC) (127, 67.2%), which was then followed by M. abscessus (31, 16.4%), M. fortuitum (10, 5.3%), M. kansasii (9, 4.8%), and other NTM species. Of these, 93 (49.2%) patients were diagnosed with NTM pulmonary disease. MAC, M. abscessus, and M. kansasii were more virulent than the other species. None of the cases of NTM pulmonary disease were caused by M. fortuitum, M. chelonae, M. peregrinum, M. terrae complex, or M. gordonae. Conclusion: In Korea, the prevalence of NTM isolates is increasing, as are the cases of pulmonary disease. The pathogenic potential of NTM differs enormously by species and as a result the treatment of NTM lung disease depends on which species has caused the infection. The isolation and identification of NTM isolated from respiratory specimens are mandatory in order for clinical microbiology laboratories to make an accurate diagnosis and suggest the proper treatment of the NTM disease. (Korean J Clin Microbiol 2012;15:98-103)

Impact of Revised Penicillin Breakpoints for Streptococcus pneumoniae (CLSI M100-S18) on the Penicillin Susceptibility Rate

Background: In January 2008, the Clinical and Laboratory Standards Institute (CLSI) published revised penicillin breakpoints for Streptococcus pneumoniae according to clinical presentation and the route of penicillin administration. The aim of this study was to evaluate the impacts of the new penicillin breakpoints on the susceptibility rates of S. pneumoniae isolated from blood. Methods: A total of 156 non-duplicated S. pneumoniae strains recovered from blood of hospitalized patients were collected between January 2003 and December 2008. Penicillin and cefotaxime susceptibility tests were performed using an E-test (AB Biodisk, Solna, Sweden). Results of the penicillin susceptibility tests were analyzed using the former and new CLSI guidelines. Results: Of the 156 S. pneumoniae strains isolated from blood, penicillin susceptibility under the former CLSI guidelines resulted in 42.3% susceptible, 42.3% intermediate, and 15.4% resistant states. According to the new CLSI guidelines (nonmeningitis, parenteral), 87.8% of isolates were susceptible, 9.6% were intermediate, and 2.6% were resistant to penicillin. Conclusion: When the new CLSI guidelines are applied, the penicillin susceptibility rate of S. pneumoniae strains isolated from blood is considerably increased. This suggests that penicillin should still be useful for the treatment of nonmeningeal pneumococcal infections and that the use of broad-spectrum antimicrobials should not replace this treatment. (Korean J Clin Microbiol 2010;13:68-72)

Diagnostic Usefulness of the Janus Kinase 2 Mutation in non-BCR/ABL Myeloproliferative Disorders

Background We investigated the Janus kinase 2 (JAK2) mutation and its diagnostic value in patients suffering with non BCR/ABL myeloproliferative diseases (nMPD) or other reactive conditions. Methods We reviewed the clinical records of 83 patients who underwent bone marrow (BM) examinations with suspect of nMPD. The diagnoses of nMPD were made based on the WHO criteria since 2001 and the PVSG criteria before 2001. The JAK2 mutation was examined by PCR in 54 patients whose BM samples were available. Results The JAK2 mutation was detected in 25 patients (46%); 12 of 26 patients with essential thrombocythemia (ET), 9 of 12 patients with polycyhtemia vera (PV), one of 7 patients with chronic idiopathic myelofibrosis (CIM) and one patient with unclassifiable MPD. Additionally, JAK2 mutation was detected in each one patient with secondary polycythemia and reactive thrombocytosis. These two patients and two other patients among the JAK2 mutated ET did not meet the WHO PV criteria due to their initial low hemoglobin levels. These patients had liver cirrhosis and hypersplenism due to Budd-Chiari syndrome (1), gastrointestinal bleeding (1) or the initial hemoglobin level was slightly below the level as provided by the criteria, but the level showed a rising pattern despite cytoreductive therapy (2). With the results of the JAK2 mutation available, 4 patients disease could be re-diagnosed as PV. Finally, the positive rate of the JAK2 mutation was 81% in PV, 48% in ET and 14% in CIM. The presence of JAK2 mutation closely correlated with PV (p=0.001), leukocytosis (p=0.001) and an increased cellularity of BM (p=0.024). Conclusions The JAK2 mutation may help differentiate nMPD from secondary cytosis. Therefore, it should be incorporated into the guidelines for the nMPD work-up for making a more accurate diagnosis and administering proper treatment.

The Prognostic Value of Serum Levels of Heart-Type Fatty Acid Binding Protein and High Sensitivity C-Reactive Protein in Patients With Increased Levels of Amino-Terminal Pro-B Type Natriuretic Peptide

Background Amino-terminal pro-B type natriuretic peptide (NT-proBNP) is a well-established prognostic factor in heart failure (HF). However, numerous causes may lead to elevations in NT-proBNP, and thus, an increased NT-proBNP level alone is not sufficient to predict outcome. The aim of this study was to evaluate the utility of two acute response markers, high sensitivity C-reactive protein (hsCRP) and heart-type fatty acid binding protein (H-FABP), in patients with an increased NT-proBNP level. Methods The 278 patients were classified into three groups by etiology: 1) acute coronary syndrome (ACS) (n=62), 2) non-ACS cardiac disease (n=156), and 3) infectious disease (n=60). Survival was determined on day 1, 7, 14, 21, 28, 60, 90, 120, and 150 after enrollment. Results H-FABP (P<0.001), NT-proBNP (P=0.006), hsCRP (P<0.001) levels, and survival (P<0.001) were significantly different in the three disease groups. Patients were divided into three classes by using receiver operating characteristic curves for NT-proBNP, H-FABP, and hsCRP. Patients with elevated NT-proBNP (≥3,856 pg/mL) and H-FABP (≥8.8 ng/mL) levels were associated with higher hazard ratio for mortality (5.15 in NT-proBNP and 3.25 in H-FABP). Area under the receiver operating characteristic curve analysis showed H-FABP was a better predictor of 60-day mortality than NT-proBNP. Conclusions The combined measurement of H-FABP with NT-proBNP provides a highly reliable means of short-term mortality prediction for patients hospitalized for ACS, non-ACS cardiac disease, or infectious disease.

Multiple Brain Abscesses Caused by Nocardia asiatica in a Patient With Systemic Lupus Erythematosus: The First Case Report and Literature Review

Dear Editor, Nocardia species are uncommon pathogens that affect immunosuppressed patients; although cerebral nocardiosis is a rare condition, it is associated with significant morbidity and mortality [1]. Because Nocardia species exhibit different antibiotic susceptibilities, accurate species identification is important for prognoses. To the best of our knowledge, this is the first case of Nocardia asiatica brain abscesses reported in a systemic lupus erythematosus (SLE) patient. A 51-yr old man visited our emergency department on May 2016 complaining of left leg weakness, dysarthria, dizziness, nausea, vomiting, and uncontrolled fever lasting three days. His past medical history consisted of SLE (diagnosed in August 2002) treated intermittently with steroid and platelet transfusion because of severe thrombocytopenia. In addition, in April 2015, he was diagnosed as having diabetes; however, no medical treatment had been undertaken. His last admission to hospital, due to severe thrombocytopenia (6×10/L), was two months prior to this presentation. He was subsequently treated with danazol (400 mg twice daily), hydroxychloroquine (200 mg twice daily), methotrexate (15 mg/week), and prednisolone (15 mg/day). At presentation, the patient’s temperature was 39.1°C, and blood tests indicated a white blood cell count of 11.03×10/L with a differential count of 76.2% neutrophils. Serum C-reactive protein (71.9 mg/L) and erythrocyte sedimentation rate (28 mm/ hr) were elevated. Brain magnetic resonance imaging (MRI) revealed multiple contrast-enhanced lesions in both cerebral and cerebellar hemispheres (Fig. 1A). A subsequent brain abscess aspiration removed 5 mL of a yellowish aspirate; Gram staining of the aspirate revealed gram-positive filamentous branched bacilli, and specimen culturing on blood agar plates for 48 hr at 37°C under aerobic conditions yielded white, rough, and dry colonies, which also presented gram-positive filamentous branched bacilli and were modified acid fast bacilli stain-positive (Fig. 1BE). 16S rRNA gene sequencing was performed for isolate identification according to the CLSI guidelines with primer pair forward 4F and reverse 801R [2]. The isolate 16S rRNA sequence (671 bp; GenBank accession number KY417120) showed 100% homology with N. asiatica (KC333452.1) and N. abscessus (GU471235.1). Alternative gene targets, such as the secA1 gene, are necessary for accurate species discrimination in the Nocardia asteroides group, because several N. asiatica, N. abscessus, N. asteroides, and N. arthritidis strains share ≥99.6% identity [2]. Thus, gene amplification and additional sequencing of secA1 were performed with primer pair forward F47 and reverse ConR. The results (497; KY417121) showed 99.4% (494/497) and

Screening PCR Versus Sanger Sequencing: Detection of CALR Mutations in Patients With Thrombocytosis.

Background Mutations in calreticulin (CALR) have been reported to be key markers in the molecular diagnosis of myeloid proliferative neoplasms. In most previous reports, CALR mutations were analyzed by using Sanger sequencing. Here, we report a new, rapid, and convenient system for screening CALR mutations without sequencing. Methods Eighty-three bone marrow samples were obtained from 81 patients with thrombocytosis. PCR primers were designed to detect wild-type CALR (product: 357 bp) and CALR with type 1 (product: 302 bp) and type 2 mutations (product: 272 bp) in one reaction. The results were confirmed by Sanger sequencing and compared with results from fragment analysis. Results The minimum detection limit of the screening PCR was 10 ng for type 1, 1 ng for type 2, and 0.1 ng for cases with both mutations. CALR type 1 and type 2 mutants were detected with screening PCR with a maximal analytical sensitivity of 3.2% and <0.8%, respectively. The screening PCR detected 94.1% (16/17) of mutation cases and showed concordant results with sequencing in the cases of type 1 and type 2 mutations. Sanger sequencing identified one novel mutation (c.1123_1132delinsTGC). Compared with sequencing, the screening PCR showed 94.1% sensitivity, 100.0% specificity, 100.0% positive predictive value, and 98.5% negative predictive value. Compared with fragment analysis, the screening PCR presented 88.9% sensitivity and 100.0% specificity. Conclusions This screening PCR is a rapid, sensitive, and cost-effective method for the detection of major CALR mutations.

Bone Marrow Oxalosis in a Patient With Pancytopenia Following Bilateral Nephrectomy

Dear Editor, n nBone marrow (BM) oxalosis is a type of systemic oxalosis wherein oxalate is deposited in BM. It is characterized by cytopenias, leukoerythroblastosis, and hepatosplenomegaly [1] as well as BM findings of calcium oxalate crystals that are birefringent under polarized microscopy and granulomatous structures [2]. Hyperoxaluria (excessive urinary excretion of oxalate) can develop into systemic oxalosis when oxalate is deposited in organs [3]. Hyperoxaluria is classified as primary or secondary. Primary hyperoxaluria is an autosomal recessive disease with defective oxalate metabolism [3] in which the overproduction of oxalate results from an enzyme deficiency in the liver; its clinical presentation involves nephrocalcinosis and renal impairment. Systemic deposition of excess oxalate occurs in the bone and all organs and tissues, except the liver. The retina, arteries, peripheral nervous system, myocardium, thyroid, skin, and BM are the major areas of oxalate deposition. Bone is the most common site, although the bone lesions can mimic clinical renal osteo-dystrophy [4,5]. n nPrimary hyperoxaluria includes three types of enzyme deficiency. The most common form is primary hyperoxaluria type I, with an incidence rate of approximately 1/120,000 live births per year in Europe. It is caused by a mutation in the AGXT gene resulting in a functional defect of the liver enzyme alanine-glyoxylate aminotransferase and represents 80% of primary hyperoxaluria. Primary hyperoxaluria type II is caused by a deficiency of glyoxylate reductase/hydroxypyruvate reductase (GRHPR), and type III is caused by a deficiency of the mitochondrial enzyme HOGA1 [2,3]. Secondary hyperoxaluria occurs when dietary and intestinal absorption of oxalate or intake of oxalate precursors is increased and the intestinal microflora is changed [3]. The clinical presentation of secondary hyperoxaluria is similar to that of primary hyperoxaluria, although systemic oxalosis is less common [3]. We present a case of BM oxalosis with pancytopenia in a patient who had been on hemodialysis after bilateral nephrectomy due to recurrent nephrocalcinosis. n nA 51-yr-old male patient underwent a BM biopsy due to pancytopenia. He had been on hemodialysis via an arterio-venous graft since 2009 when he underwent a bilateral nephrectomy owing to recurrent renal stones and renal failure. One of his siblings had died owing to end-stage renal disease in his or her 30s (the sex is unknown). The patient had no history of a high-oxalate diet or gastrointestinal symptoms. His complete blood count revealed a Hb level of 5.7 g/dL, white blood cell count of 1.2×109/L (absolute neutrophil count 0.81), and platelet count of 98×109/L. Anemia persisted for five years despite treatment with erythropoietin, and leukopenia and thrombocytopenia also developed. Radiography revealed diffuse sclerotic changes and osteolytic lesions in multiple sites that resulted in a spontaneous fracture in his elbow. Endocrinologically, subclinical hypothyroidism was present with an increased thyroid-stimulating hormone level of 5.344 mIU/L (reference range 0.55-4.78 mIU/L), and a near-normal free thyroxine level of 8.1 ng/L (reference range 8.9-17.8 ng/L). Laboratory data showed the following: blood urea nitrogen, 0.36 g/L, creatinine, 43 mg/L, and erythropoietin 33.2 IU/L (reference rage 4.3-29 IU/L). n nTear drop cells and left-shifted neutrophils were observed on a peripheral blood smear. BM aspirates were hemodiluted and showed a few multinucleated giant cells. Extensive oxalate crystals depositions surrounded by macrophages and granulomatous formations were revealed on BM biopsy. The crystals were birefringent under polarized microscopy (Fig. 1). The patient demonstrated systemic oxalosis involving the bone, possibly thyroid, and BM, with no evidence of involvement in the retina, arteries, peripheral nervous system, myocardium, or skin. n n n nFig. 1 n n(A) Pancytopenia showing tear drop cells indicated by the arrow (peripheral blood smear, Wright stain, ×1,000). (B) Bone marrow biopsy shows oxalate crystals surrounded by granuloma (Hematoxylin & Eosin stain, ×200)