Sejong Chun

Species-Specific Difference in Antimicrobial Susceptibility Among Viridans Group Streptococci

Background Viridans group streptococci (VGS) are both commensal microbes and potential pathogens. Increasing resistance to penicillin in VGS is an ongoing issue in the clinical environment. We investigated the difference in susceptibility and resistance to penicillin among various VGS species. Methods In total 1,448 VGS isolated from various clinical specimens were analyzed over a two-yr period. Identification and antimicrobial susceptibility test was performed by the automated VITEK 2 system (bioMerieux, France) or the MicroScan MICroSTREP system (Siemens, Germany). Results Among the 1,448 isolates, 412 were isolated from blood (28.4%). Streptococcus mitis group was the most frequently isolated (589 isolates, 40.7%), followed by the S. anginosus group (290 isolates, 20.0%), S. sanguinis group (179 isolates, 12.4%) and S. salivarius group (57 isolates, 3.9%). In total, 314 isolates could not be identified up to the species level. The overall non-susceptibility to penicillin was observed to be 40.0% (resistant, 11.2% and intermediately resistant, 28.8%) with uneven distribution among groups; 40.2% in S. sanguinis group (resistant, 5.0% and intermediately resistant, 35.2%), 60.3% in S. mitis group (resistant, 20.9% and intermediately resistant, 39.4%), 78.9% in S. salivarius group (resistant, 8.8% and intermediately resistant, 70.1%), and 6.2% in S. anginosus group (resistant, 1.7% and intermediately resistant, 4.5%). Conclusions Antimicrobial resistance patterns towards penicillin show differences among various VGS; this should be considered while devising an effective antimicrobial treatment against VGS.

Natural killer cell subsets and receptor expression in peripheral blood mononuclear cells of a healthy Korean population: Reference range, influence of age and sex, and correlation between NK cell receptors and cytotoxicity

BACKGROUND The purpose of this study was to identify CD56bright and CD56dim natural killer (NK) cell subsets and analyze their receptors expression in a healthy Korean population, and to determine whether receptor expression correlates with age, sex, and cytotoxicity. MATERIALS AND METHODS We performed multicolor flow cytometry assays to analyze the expression of various NK cell receptors (CD16, NKG2A, NKG2C, NKG2D, CD57, DNAM-1, CD8a, CD62L, NKp30, and NKp46) on both CD3-/CD56dim and CD3-/CD56bright NK cells in whole-blood samples from 122 healthy donors. The expression of these receptors was compared according to age (<30years, n=22, 30-60years, n=73 and >60years, n=27) and gender (male, n=61, female, n=61). NK cell cytotoxicity assays were performed with peripheral blood mononuclear cells (PBMCs) from 18 individuals. The results were compared to the expression levels of NKp30 and NKp46 receptors. RESULTS A normal reference range for NK cell receptor expression in two NK cell subsets was established. NKp46 and NKG2D expression gradually decreased with age (p<0.01 and p<0.05, respectively) whereas NK cell proportion and numbers, frequencies of CD56dim cells, and CD57 expression increased with age (p<0.01 in all cases). Men showed greater NK cell proportion and numbers, frequencies of CD56dim cells, and CD57 expression compared to those of women (p<0.05 and p<0.001; p<0.01 and p<0.01, respectively). Notably, the expression of NKp46 was negatively correlated with NK cell frequency (r=-0.42, p<0.001). Furthermore, NK cell cytotoxicity was found to positively correlate with NCR expression (p=0.02), but not NK cell proportion (p=0.80). CONCLUSION We have established a profile of NK cell surface receptors for a Korean population, and revealed that age and gender have an effect on the expression of NK cell receptors in the population. Our data might explain why neither NK cell numbers nor proportions correlate with NK cell cytotoxicity.

Evaluation of alpha-fetoprotein as a screening marker for hepatocellular carcinoma in hepatitis prevalent areas.

The objective of this study was to establish modified cutoff values of serum alpha-fetoprotein (AFP) according to hepatitis status. While AFP is used as a serum marker in the diagnosis or monitoring of hepatocellular carcinoma (HCC), its use as a screening method to the general population is controversial. We evaluated its screening performance in a hepatitis prevalent East Asian population, and suggest different cutoff values according to the individuals hepatitis status. We evaluated the performance of AFP as a screening test in 48,123 consecutive Koreans during the period from March, 2012 to August, 2013 who underwent routine health checks at a single institution. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated with fixed cutoff and with modified cutoffs according the individuals hepatitis status. A total of 24 out of 48,123 subject (0.05%) were newly diagnosed with HCC after screening. Among the 1,874 subject with positive hepatitis B virus surface antigen (HBsAg), 17 (0.91%) developed HCC, compared with two out of 393 (0.51%) individuals with hepatitis C virus antibody (anti-HCV). Five out of 45,855 (0.01%) subject with neither HBsAg nor anti-HCV developed HCC. Compared to the performance of a fixed cutoff, specificity, PPV, and NPV improved without sacrificing sensitivity when applying modified cutoff. In conclusion, our findings suggest that AFP with modified cutoffs according to the individuals hepatitis status might be a useful screening marker for HCC in hepatitis prevalent areas.

ABO Mistyping of cis -AB Blood Group by the Automated Microplate Technique

Background: The cis-AB phenotype, although rare, is the relatively most frequent of ABO subgroups in Koreans. To prevent ABO mistyping of cis-AB samples, our hospital has applied a combination of the manual tile method with automated devices. Herein, we report cases of ABO mistyping detected by the combination testing system. Methods: Cases that showed discrepant results by automated devices and the manual tile method were evaluated. These samples were also tested by the standard tube method. The automated devices used in this study were a QWALYS-3 and Galileo NEO. Exons 6 and 7 of the ABO gene were sequenced. Results: 13 cases that had the cis-AB allele showed results suggestive of the cis-AB subgroup by manual methods, but were interpreted as AB by either automated device. This happened in 87.5% of these cases by QWALYS-3 and 70.0% by Galileo NEO. Genotyping results showed that 12 cases were ABO*cis-AB01/ABO*O01 or ABO*cis-AB01/ABO*O02, and one case was ABO*cis-AB01/ ABO*A102. Conclusion: Cis-AB samples were mistyped as AB by the automated microplate technique in some cases. We suggest that the manual tile method can be a simple supplemental test for the detection of the cis-AB phenotype, especially in countries with relatively high cis-AB prevalence.

Possible Transfusion-Related Acute Lung Injury Following Convalescent Plasma Transfusion in a Patient With Middle East Respiratory Syndrome

Dear Editor, Korea suffered from an outbreak of the Middle East Respiratory Syndrome coronavirus (MERS-CoV) in May 2015 [1]. This endemic was the largest to have occurred outside of Saudi Arabia. Currently, a curative treatment for MERS is unavailable. Passive immunotherapy using convalescent plasma from recovering patients is suggested for positive clinical effects [2,3]. However, the use of human plasma has potential risks including anaphylactic shock, transfusion-associated circulatory overload (TACO), and transfusion-related acute lung injury (TRALI) [4]. We describe our experience with administering a convalescent plasma infusion to a MERS patient that resulted in possible TRALI, which might have further accelerated the pulmonary manifestation of MERS in the patient. A previously healthy, 32-yr-old male subject had contact with a MERS patient on May 28, 2015. He developed symptoms of productive cough and fever on June 8 and was admitted for evaluation. After confirmation of MERS-CoV by detection of the upE and ORF1a genes of MERS-CoV with a real-time polymerase chain reaction (qPCR) assay (Kogene Biotech, Seoul, Korea), he was treated with oral administration of ribavirin and lopinavir/ritonavir with a single dose of interferon α-2a. However, the patients clinical manifestation showed a stagnant course. Therefore, convalescent plasma therapy was planned. The convalescent plasma donor was a cured 22-yr-old female patient. She had no previous history of gestation, and no record of transfusion was found. The donors blood was screened for hemoglobin (>12.0 g/dL), hepatitis B virus, HIV, and hepatitis C virus by both serologic and nucleic acid testing (negative), for syphilis (negative) by serologic test, alanine aminotransferase (<65 IU), and MERS-CoV RNA (negative), and was without any other contraindication for plasmapheresis donation other than a seven-day interval between donation and termination of treatment. The ABO/RhD blood type of the donor was identical to that of the patient. In addition, the donor was retrospectively tested for the presence of anti-HLA class I and II antibodies and anti-human neutrophil antigen (HNA) antibodies, which were all negative. On June 16, apheresis was performed, and 500 mL of plasma was collected without any adverse effects on the donor. The patient received 250 mL of the product immediately following plasma collection, and the remaining 250 mL was preserved, which was later discarded after the subsequent adverse reaction was observed in the recipient. The collected plasma was not pathogen-inactivated. The patient developed respiratory distress within two hours after transfusion. Clinical and laboratory features of the patient before and after TRALI are described in Fig. 1. The virological aspect of the patient differed from the patients respiratory symptoms. While the threshold cycle (Ct) value of MERS-CoV qPCR performed on the patients lower respiratory tract specimen showed little change from initial diagnosis to the time of convalescent plasma infusion, it increased afterwards, which may be suggestive of decreased viral load. Fig. 1 Clinical and laboratory features of the patient before and after transfusion-related acute lung injury. (A) PaO2/FiO2 ratio, (B) oxygen saturation (pulse oximetry) and partial pressure of oxygen (PaO2) and carbon dioxide (PaCO2) of arterial blood, (C) ... TRALI is defined as a new onset of acute lung injury (ALI) within six hours of transfusion, with evidence of hypoxia (PaO2/FiO2 ≤300 mmHg or SpO2 <90% of room air) and radiological evidence. Additionally, it does not require evidence of left atrial hypertension, preexisting ALI, or temporal relationship to an alternative risk factor for ALI. In our case, as the onset of hypoxia happened two hours after convalescent plasma infusion and both SpO2 (Oxygen saturation as measured by pulse oximetry) and PaO2/FiO2 (Fraction of inspired oxygen) levels met the criteria for TRALI with no auscultative findings of circulatory overload, TRALI was suspected. Since MERS can also result in ALI, we recognized that a temporal risk factor existed; thus, our patient met the criteria for possible TRALI. As both antibodies for HLA and HNA were negative, the underlying mechanism is thought to be non-antibody mediated. The finding that the Ct value in the qPCR increased after transfusion suggested that passive immunotherapy could decrease the viral burden of MERS-CoV. However, further investigation with a controlled study and a larger number of subjects is required to determine the clinical benefits of this therapy. As in any other blood component donation, precautions are needed to prevent adverse transfusion effects. To specifically prevent antibody-mediated TRALI, it is recommended that plasma be processed from male donors only [5]. However, the majority of potential donors for convalescent plasma were female nurses. Thus, the male-only protocol was waived during the MERS outbreak. A case of non-HLA antibody-mediated TRALI after convalescent plasma use in an Ebola virus disease patient was recently reported [6]. Our results that a convalescent plasma infusion to a MERS patient led to possible TRALI suggest that convalescent plasma therapy should be cautiously approached, especially regarding the possibility of TRALI.

Performance Evaluation of the Serum Thyroglobulin Assays With Immunochemiluminometric Assay and Immunoradiometric Assay for Differentiated Thyroid Cancer

Background Measurement of postoperative serum thyroglobulin (Tg) is important for detecting persistent or recurrent differentiated thyroid cancer. We evaluated the analytic performance of the DxI 800 assay (Beckman Coulter, USA) for serum Tg and anti-thyroglobulin antibodies (TgAbs) in comparison with that of the GAMMA-10 assay (Shinjin Medics Inc., Korea) for serum Tg and RIA-MAT 280 assay (Stratec, Germany) for TgAb. Methods We prospectively collected blood samples from 99 patients thyroidectomized for thyroid cancer. The functional sensitivity was investigated in standards and human serum. Precision and linearity were evaluated according to the guidelines of the Clinical and Laboratory Standards Institute. The correlation between the two assays was assessed in samples with different Tg ranges. Results The functional sensitivity of the DxI 800 assay for serum Tg was between 0.0313 and 0.0625 ng/mL. The total CV was 3.9–5.6% for serum Tg and 5.3–6.9% for serum TgAb. The coefficient of determination (R2) was 1.0 and 0.99 for serum Tg and TgAb, respectively. The cut-offs for serum TgAb were 4.0 IU/mL (DxI 800) and 60.0 IU/mL (RIA-MAT 280), and the overall agreement was 68.7%. The correlation between the two assays was excellent; the correlation coefficient was 0.99 and 0.88 for serum Tg and TgAb, respectively. Conclusions The DxI 800 is a sensitive assay for serum Tg and TgAb, and the results correlated well with those from the immunoradiometric assays (IRMA). This assay has several advantages over the IRMA and could be considered an alternative test for Tg measurement.

Comparison of BacT/Alert FAN and FAN Plus Bottles with Conventional Medium for Culturing Cerebrospinal Fluid

ABSTRACT We compared the BacT/Alert system FAN and FAN Plus media to conventional media for culturing cerebrospinal fluid (CSF) with 2,545 samples. FAN/FAN Plus bottles showed better performance for isolating microorganisms in CSF than conventional media (positive rate, 7.2% [182/2,545] versus 3.1% [80/2,545]). The incremental recovery rate of Cryptococcus neoformans from FAN Plus bottles was higher than that from FAN bottles.

The synonymous nucleotide substitution RHD 1056C>G alters mRNA splicing associated with serologically weak D phenotype

D antigen is one of the most clinically significant blood group antigens. Variation of the RHD gene can cause weak D or partial D phenotypes. While most variations are missense substitutions with amino acid changes, those without are called “silent” or “synonymous” substitutions. Synonymous substitutions often have little effect on the protein, not altering the phenotype. However, effect on splicing can affect end‐product protein. We report a new synonymous variation, RHD 1056C>G, that resulted in weak D phenotype, and predicted its effect with various in silico methods.

X-ray as Irradiation Alternative for K562 Feeder Cell Inactivation in Human Natural Killer Cell Expansion

Background/Aim: γ-Irradiation has been proven to be the most effective method to inactivate K562 cells, but γ-irradiators are not available in some institutes. This study was designed to compare the effects of X-ray and γ-irradiation on K562 cells in natural killer (NK) cell expansion. Materials and Methods: To expand NK cells, isolated peripheral blood mononuclear cells (PBMCs) were co-cultured with γ-irradiated or X-ray-treated K562 cells plus IL-2 and IL-15. Characteristics of expanded NK cells were identified by flow cytometry. Results: NK cell expansion rate tended be to lower in the X-ray-treated group (68.9±32.6) than the γ-irradiated group (78±28.7), but the difference was not significant (p=0.39). Furthermore, NK cell functions or receptor expression were similar in the two groups. Conclusion: Our results suggest that X-ray treatment can be used as an alternative to γ-irradiation for K562 cells inactivation in human NK cell expansion.

Application of a portable microscopic cell counter for the counting of residual leukocytes in leukoreduced apheresis platelet concentrates in a hospital blood bank

While a portable microscopic cell counter has been evaluated to enumerate residual white blood cells (WBCs) in red blood cells and platelet concentrates at blood centers, it has not yet been assessed in a hospital blood bank. We investigated the performance of this device and evaluated its accuracy, along with its benefits in time management. Residual WBCs from each of 100 apheresis platelet specimens were measured manually using a Nageotte chamber, along with flow cytometry methods and an ADAM-rWBC automated instrument (NanoEnTek, Seoul, South Korea). The efficiency was calculated by measuring the time required for the analysis of one specimen ten times consecutively. Flow cytometry and the ADAM-rWBC were able to detect four sporadic cases that had residual WBCs exceeding 1/μL that were not detected by the manual method. Analysis time was the shortest with the ADAM-rWBC, followed by flow cytometry and the manual method. Our data suggest that hospital blood banks require quality control of residual WBCs; among the methods evaluated in this study, the portable microscopic cell counter offers the best time efficiency.