Determining Genotypic Drug Resistance by Ion Semiconductor Sequencing With the Ion AmpliSeq™ TB Panel in Multidrug-Resistant Mycobacterium tuberculosis Isolates

BACKGROUND: We examined the feasibility of a full-length gene analysis for the drug resistance-related genes inhA, katG, rpoB, pncA, rpsL, embB, eis, and gyrA using ion semiconductor next-generation sequencing (NGS) and compared the results with those obtained from conventional phenotypic drug susceptibility testing (DST) in multidrug-resistant Mycobacterium tuberculosis (MDR-TB) isolates. METHODS: We extracted genomic DNA from 30 pure MDR-TB isolates with antibiotic susceptibility profiles confirmed by phenotypic DST for isoniazid (INH), rifampin (RIF), ethambutol (EMB), pyrazinamide (PZA), amikacin (AMK), kanamycin (KM), streptomycin (SM), and fluoroquinolones (FQs) including ofloxacin, moxifloxacin, and levofloxacin. Enriched ion spheres were loaded onto Ion PI Chip v3, with 30 samples on a chip per sequencing run, and Ion Torrent sequencing was conducted using the Ion AmpliSeq TB panel (Life Technologies, USA). RESULTS: The genotypic DST results revealed good agreement with the phenotypic DST results for EMB (Kappa 0.8), PZA (0.734), SM (0.769), and FQ (0.783). Agreements for INH, RIF, and AMK+KM were not estimated because all isolates were phenotypically resistant to INH and RIF, and all isolates were phenotypically and genotypically susceptible to AMK+KM. Moreover, 17 novel variants were identified: six (p.Gly169Ser, p.Ala256Thr, p.Ser383Pro, p.Gln439Arg, p.Tyr597Cys, p.Thr625Ala) in katG, one (p.Tyr113Phe) in inhA, five (p.Val170Phe, p.Thr400Ala, p.Met434Val, p.Glu812Gly, p.Phe971Leu) in rpoB, two (p.Tyr319Asp and p.His1002Arg) in embB, and three (p.Cys14Gly, p.Asp63Ala, p.Gly162Ser) in pncA. CONCLUSIONS: Ion semiconductor NGS could detect reported and novel amino acid changes in full coding regions of eight drug resistance-related genes. However, genotypic DST should be complemented and validated by phenotypic DSTs.

Sigma-Metrics of Electrolyte Tests From a Recently Launched New-Generation Proficiency Testing Program of the Korean Association of Quality Assurance for Clinical Laboratory

No abstract available.

A Case of Thrombotic Thrombocytopenic Purpura with Exacerbation after Incomplete Remission of Therapeutic Plasma Exchange / 대한수혈학회지

Thrombotic thrombocytopenic purpura (TTP) is a rare blood disorder with a mortality rate of over 90% without prompt treatment. It is caused by congenital, idiopathic, or secondary diseases; idiopathic TTP is mainly associated with deficiency of ADAMTS13, a von Willebrand factor cleaving protease or ADAMTS13 inhibitors. The long-term survival rate of TTP has improved since the introduction of therapeutic plasma exchange (TPE), and the therapeutic aims have also been established. However, deciding on the end-point and appropriate treatment method requires careful assessment of clinical conditions of patients. The present study reports a case of a 33-year-old male patient with reduced ADAMTS13 activity and ADAMTS13 inhibitor, who developed symptoms after an early termination of TPE with improved symptoms, which finally improved with retreatment and additionally corticosteroid. We report our case with relevant literature review on TPE in TTP with this case.

Quality Management of Clinical Chemistry Tests in Blood Center / 대한수혈학회지

The main goal of transfusion medicine is safe and appropriate blood transfusion in all situations. To accomplish this, it is essential to have a high level quality management system for the entire process from blood donation to transfusion. Regulations regarding blood management have been adopted and strictly managed in Korea since 2007. Blood center's blood management tasks should establish appropriate quality management systems to ensure the safe supply of blood, as well as the basic resources of personnel, facilities and equipment in accordance with laws and regulations governed by the Ministry of Health and Welfare in Korea. The purpose of this review is to examine the contents and processes for quality control of clinical chemistry tests in Korean blood centers.

Long-Term Stability of Selected Serum Biomarkers in Leftover Samples / 임상검사와정도관리

BACKGROUND: The purpose of this study was to assess the quality of long-term-stored leftover blood samples, and to evaluate the long-term stability of selected serum biomarkers such as proteins, enzymes, electrolytes, and tumour markers. METHODS: Stored blood samples were transferred to our biobank after being used to conduct tests for routine medical examinations in one health care institution, and were preserved at or below -70degrees C for 4 years. We analysed 24 biomarkers whose levels had been reported 4 years ago and tested them using the same analyser, reagents, and methods by utilizing an ADVIA Centaur Immunoassay System (Siemens Healthcare Diagnostics, USA) or an ADVIA 2400 Chemistry System (Siemens, USA). RESULTS: A total of 15 out of the 24 tested biomarkers showed significant differences in paired Student t-tests (P0.975). Two biomarkers, creatinine and rheumatoid arthritis factor, showed no significant differences but were poorly correlated with previously analysed data. Aspartate aminotransferase, alanine aminotransferase, hepatitis B virus (HBV) surface antigen, and insulin levels were discordant according to their reference ranges. A total of 3 biomarkers, C-reactive protein, cancer antigen 125, and HBV surface antibody, showed no significant differences and good correlations without discordant data. CONCLUSIONS: Our findings showed that long-term storage for more than 4 years can result in a considerable bias for variable biomarkers. Only 3 of the 24 biomarkers evaluated were found to be stable biomarkers. Long-term storage of leftover samples is not recommended for most chemical analyses.

Serum Pro-hepcidin as an Predictor of Iron Deficiency in Anemic Patients

BACKGROUND: Hepcidin has recently been known as a negative regulatory hormone of iron. Hepcidin precursor, pro-hepcidin has been used as a surrogate and reported to be related to iron deficiency. We investigated serum pro-hepcidin levels in patients with iron deficiency anemia (IDA), anemia of chronic disorder (ACD) and ACD concomitant iron deficiency (ACD/ID) to assess its usefulness as a marker of iron deficiency and examined whether its level is associated with anemia, iron status or inflammation profiles involved in the synthesis of hepcidin. METHODS: We enrolled 50 patients with IDA, 46 with ACD, 12 with ACD/ID and 60 healthy controls. Complete blood cell count, iron parameters (iron, TIBC, trasferrin saturation, ferritin), C-reactive protein (CRP) and serum pro-hepcidin were measured. RESULTS: Patients with iron deficiency, the IDA group and ACD/ID group had lower serum pro-hepcidin levels than healthy controls and the ACD group. The cutoff value of pro-hepcidin for detecting iron deficiency was 230 ng/mL (sensitivity 88.1%, specificity 51.2%). Patients with increased CRP showed higher mean pro-hepcidin level than those with normal CRP and the difference was significant in the IDA group (P=0.02). And serum pro-hepcidin level was positively correlated with CRP level (r=0.30, P=0.04) in the IDA group but not with hemoglobin. CONCLUSIONS: In patients with anemia, pro-hepcidin measurement may be useful for differentiating anemia patients with iron deficiency, IDA and ACD/ID from those with ACD. Serum pro-hepcidin levels may be more affected by inflammation than by the degree of anemia.

A Case of Neonatal Alloimmune Thrombocytopenia due to Anti-HLA-B35

Neonatal alloimmune thrombocytopenia (NAIT) is induced by maternal antibodies to fetal platelet alloantigens. Because the main cause of NAIT is incompatibility to platelet specific antibodies, NAIT due to HLA antibodies are relatively rare. We managed a case of NAIT induced by maternal anti-HLA-B35 antibodies. The patient was a second born male. He had no petechiae or purpura at birth. He was admitted to the hospital due to fever for 5 days and a platelet count of 106x10(9)/L. The fever subsided after admission but on the 2nd day of admission, petechiae developed on the chest wall and the platelet count decreased to 25x10(9)/L. Other laboratory findings included C-reactive protein, prothrombin time, and partial thromboplastin time were normal. His mother's platelet count was normal and she had no history of bleeding. Anti-HLA-B35, B52, B56, C3, and C14 were identified in the mother's serum by a panel reactive antibody test and HLA-B35 antigen was identified in the father's and patient's sera. These finding suggested that maternal Anti-HLA-B35 antibody was a response to neonatal HLA-B35 antigen inherited from the father. The patient received concentrated platelet and intravenous immunoglobulin. The platelet count rose to 248x10(9)/L and was maintained thereafter.

Usefulness of Bile Cultures and Predictive Factors for Bacteriobilia in Percutaneous Cholecystostomy in Patients with Acute Cholecystitis / 대한진단검사의학회지

BACKGROUND: Bile cultures have been used to diagnose and predict the prognosis of acute cholecystitis (AC). As the standard treatment for AC has changed, the appropriate timing and clinical usefulness of bile cultures should be reevaluated. We analyzed the incidence of positive bile cultures in cholecystostomy and cholecystectomy, and attempted to see if a positive bile culture is related to the laboratory and imaging parameters and postoperative infections. METHODS: Included in the study were 86 patients with AC who underwent percutaneous cholecystostomy (PC) and then laparoscopic cholecystectomy (LC). We performed hematologic, biochemical, and radiological analyses at admission and bile cultures with each surgical procedure. The patients were followed for two months for postoperative infections. RESULTS: Bile cultures were positive in 40.7% of the patients at PC, significantly higher than at LC (12.8%). The group with positive cultures showed a higher median age and elevated levels of alkaline phosphatase (ALP) and total bilirubin (TB) than the group with negative cultures. Univariate analysis identified three preoperative factors as predictors of positive bile cultures: age (>55 yr), ALP (>100 IU/L) and TB (>1.2 mg/dL). Infectious complications after LC were mild and the incidence of postoperative infections was not different between the groups. CONCLUSIONS: The sensitivity of bile cultures is low for diagnosing AC, and the adequate timing of bile cultures is at PC, rather than LC. An old age and factors (ALP & TB) manifesting an advanced stage of bile stasis are associated with positive bile cultures. No correlation was found between positive bile cultures and postoperative infections.

Usefulness of Bile Cultures and Predictive Factors for Bacteriobilia in Percutaneous Cholecystostomy in Patients with Acute Cholecystitis / 대한진단검사의학회지

BACKGROUND: Bile cultures have been used to diagnose and predict the prognosis of acute cholecystitis (AC). As the standard treatment for AC has changed, the appropriate timing and clinical usefulness of bile cultures should be reevaluated. We analyzed the incidence of positive bile cultures in cholecystostomy and cholecystectomy, and attempted to see if a positive bile culture is related to the laboratory and imaging parameters and postoperative infections. METHODS: Included in the study were 86 patients with AC who underwent percutaneous cholecystostomy (PC) and then laparoscopic cholecystectomy (LC). We performed hematologic, biochemical, and radiological analyses at admission and bile cultures with each surgical procedure. The patients were followed for two months for postoperative infections. RESULTS: Bile cultures were positive in 40.7% of the patients at PC, significantly higher than at LC (12.8%). The group with positive cultures showed a higher median age and elevated levels of alkaline phosphatase (ALP) and total bilirubin (TB) than the group with negative cultures. Univariate analysis identified three preoperative factors as predictors of positive bile cultures: age (>55 yr), ALP (>100 IU/L) and TB (>1.2 mg/dL). Infectious complications after LC were mild and the incidence of postoperative infections was not different between the groups. CONCLUSIONS: The sensitivity of bile cultures is low for diagnosing AC, and the adequate timing of bile cultures is at PC, rather than LC. An old age and factors (ALP & TB) manifesting an advanced stage of bile stasis are associated with positive bile cultures. No correlation was found between positive bile cultures and postoperative infections.