The First Identification of Cryptosporidium parvum Virus-1 (CSpV1) in Hanwoo (Bos taurus coreanae) Calves in Korea.

Cryptosporidium is an obligate coccidian parasite that causes enteric diseases in bovine species. A double-stranded RNA virus associated with C. parvum oocysts, Cryptosporidium parvum virus-1 (CSpV1), has been characterized. However, the relationship between the abovementioned coccidian parasite and the virus has not been studied in the context of the known clinical outcomes. This study aimed to characterize the prevalence and molecular traits of CSpV1 in diarrheal feces of Hanwoo (Korean indigenous cattle) calves. Of the 140 fecal samples previously tested for C. parvum, which were obtained from Hanwoo calves aged 60 days, 70 tested positive and 70 tested negative. These samples were included in this study. By using the polymerase chain reaction (PCR) analysis targeting the RdRp gene of CSpV1, we detected CSpV1 in 28 samples (20.0%), with infection rates of 31.4% (22/70) in C. parvum-positive and 8.6% (6/70) in C. parvum-negative samples. CSpV1 samples detected in the same farm were clustered together. To the best of our knowledge, this is the first study to report the prevalence and molecular characteristics of CSpV1 in Hanwoo calves in the Republic of Korea, providing important insights into the relationship between C. parvum and CSpV1 in bovine hosts.

Establishment of Korean Pediatric Reference Intervals for Estradiol using Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: Ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) is a reliable and accurate method for measuring steroid hormone levels. There is an increasing need for sensitive and precise methods to measure estradiol in pediatric patients. Here, we established reference intervals for estradiol in healthy children using a UHPLC-MS/MS-based method for the first time in South Korea. METHODS: Serum estradiol was measured using a Sciex Triple QuadTM 6500 + UHPLC-MS/MS (Sciex, Framingham, MA, USA). Reference intervals for estradiol were established according to the CLSI document EP28-A3c:2008. The reference intervals were validated using serum samples from 634 pediatric patients, including neonates, children, and adolescents. Among them, 389 specimens were used in analysis of the specimen acceptance time. Statistical analysis was performed using MedCalc (MedCalc, Ostend, Belgium) and Analyse-it (Analyse-it Software Ltd., Leeds, United Kingdom) software. RESULTS: Reference intervals for boys (n = 297) were <16.6, <7.3, <19.0, <30.5, 7.6-96.5, and 10.6-134.4 pmol/L among those aged <1, 1-5, 6-9, 10-11, 12-14, and 15-17 years, respectively. Reference intervals for girls (n = 337) were <114.7, <24.2, <34.8, 8.0-177.0, 10.4-480.5, and 9.1-486.7 pmol/L among those aged <1, 1-5, 6-9, 10-11, 12-14, and 15-17 years, respectively. Overall, there was no effect of specimen acceptance time on estradiol measurements in boys or girls, except for that in the group aged 10-11 years. CONCLUSIONS: The reference intervals for healthy children were validated using a UHPLC-MS/MS-based method. The highly analytical sensitive UHPLC-MS/MS method may be useful for estradiol determination in pediatric patients.

Complete chloroplast genome of Exochorda serratifolia (Rosaceae).

Exochorda serratifolia (pearlbush) is a rosid shrub found in northeast Asia, including the Korean peninsula. This ornamental plant has white inflorescences and strong insect resistance; however, its genetic diversity is poorly understood and a complete plastid genome is unavailable. Here, we determined the complete chloroplast genome of E. serratifolia through de novo assembly using next-generation sequencing. The E. serratifolia chloroplast genome was 160,558 bp, comprising a large single-copy (LSC) region of 88,514 bp, a small single-copy (SSC) region of 19,308 bp, and two inverted repeat regions (IRs) of 26,368 bp each. We annotated 112 genes: 78 protein-coding genes, 30 tRNA genes, and four rRNA genes. This reference plastid genome increases our understanding of the phylogenetic relationships of E. serratifolia among Rosaceae plants.

Pharmacokinetics and Genetic Factors of Atorvastatin in Healthy Korean Subjects.

Background: Statins are the most popular agents for the primary and secondary prevention of cardiovascular disease; however, the pharmacokinetic parameters and associated genetic factors in the Korean population have not been fully elucidated. This study explored the pharmacokinetic properties of atorvastatin and the association between genetic variations and atorvastatin pharmacokinetics in healthy Korean subjects. Methods: Atorvastatin (80 mg) was administered to 35 healthy Korean volunteers. Plasma levels of atorvastatin and its metabolites were measured sequentially using liquid chromatography-tandem mass spectrometry from 0 to 24 h after atorvastatin administration. Customized next-generation sequencing analysis was performed covering all coding exons of 15 genes, as well as 46 single-nucleotide variants in 29 genes related to statin pharmacokinetics. Results: The mean area under the concentration-time (AUC) and Cmax (maximum peak concentration) were 269.0 ng/ml∙h and 84.3 ng/ml, respectively, which were approximately two times higher than those reported in Caucasians. Genetic analysis revealed that eight genetic variants in ABCB1, ABCG2, APOA5, CETP, and CYP7A1 contributed to the AUC of atorvastatin. The atorvastatin AUC0-24 h prediction model was developed based on age and eight genetic variants using multivariate linear regression (adjusted R 2 = 0.878, p < 0.0001). Conclusion: This study shows that the pharmacokinetic properties of atorvastatin in Koreans are different from those in Caucasians and that atorvastatin AUC0-24 h could be predicted based on age and eight genetic variants of ABCB1, ABCG2, APOA5, CETP, and CYP7A1.

Immunosuppressive Drug Measurement by Liquid Chromatography Coupled to Tandem Mass Spectrometry: Interlaboratory Comparison in the Korean Clinical Laboratories.

BACKGROUND: Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is increasingly used for immunosuppressive drug tests. However, most LC-MS/MS tests are laboratory-developed and their agreement is unknown in different Korean laboratories. This interlaboratory comparison study evaluated test reproducibility and identified potential error sources. METHODS: Test samples containing three concentrations of tacrolimus, sirolimus, everolimus, cyclosporine, and mycophenolic acid were prepared by pooling surplus samples from patients undergoing routine therapeutic drug monitoring and tested in duplicate in the participating 10 clinical laboratories. Reconstitution and storage experiments were conducted for the commonly used commercial calibrator set. The robust estimators of reproducibility parameters were calculated. Spearman's rank correlation coefficient (rho, ρ) was used to evaluate the correlation between drugs. Multiple linear regression was used to determine whether the experimental conditions alter the calibration curves. RESULTS: The reproducibility coefficient of variation exceeded 10% only for sirolimus concentrations 1 and 2 (10.8% and 12.5%, respectively) and everolimus concentrations 1 and 2 (12.3% and 11.4%, respectively). The percent difference values showed weak correlations between sirolimus and everolimus (ρ=0.334, P =0.175). The everolimus calibration curve slope was significantly altered after reconstitution following prolonged 5°C storage (P =0.015 for 14 days; P =0.025 for 28 days); the expected differences at 6 ng/mL were 0.598% for 14 days and 0.384% for 28 days. CONCLUSIONS: LC-MS/MS test reproducibility for immunosuppressive drugs seems to be good in the Korean clinical laboratories. Continuous efforts are required to achieve test standardization and harmonization, especially for sirolimus and everolimus.

Test utilization for the diagnosis of vitamin B12 and folate deficiency in local clinics in Korea.

BACKGROUND: Current guidelines pertaining to diagnosing macrocytic anemia in association with vitamin B12 and folate deficiency recommend that vitamin B12, folate, homocysteine, and methylmalonic acid assays should be assessed concurrently due to their close relationship in metabolism. We aimed to investigate the completion of these assays in local clinics and hospitals without in-house clinical laboratories in Korea. METHODS: We retrospectively reviewed data from the laboratory information system between September 25, 2017, and June 30, 2019, to investigate usage rates of vitamin B12, folate, homocysteine, and methylmalonic acid assays in patients with macrocytic anemia. RESULTS: During the study period, 14 894 Korean adults among 109 524 (13.6%) total hemoglobin-tested subjects underwent concurrent erythrocyte mean corpuscular volume (MCV) tests. Among these 14,894 adults, 265 (1.2%) from 94 local clinics or hospitals without in-house clinical laboratories in Korea had macrocytic anemia. Furthermore, among these 265 adults, only one woman underwent serum vitamin B12 and folate assay and one man underwent serum homocysteine testing during the study period. No patients among the 265 individuals with macrocytic anemia received erythrocyte folate or methylmalonic acid testing (with either serum, plasma, random urine, or 24-hour collected urine). CONCLUSIONS: The results of this study provide basic information regarding utilization rates of assays in association with vitamin B12 and folate deficiency. Making more data available is expected to improve rates of testing in patients with macrocytic anemia in local clinics and hospitals without in-house clinical laboratories in Korea.

Exploring the role of copper and selenium in the maintenance of normal thyroid function among healthy Koreans.

BACKGROUND: Selenium and iodine are trace elements well known to have important roles in the synthesis and metabolism of thyroid hormones. However, the effects of other trace elements on thyroid hormones are still inconclusive. We investigated the association between several trace elements and thyroid hormones. METHODS: The data of 448 subjects who were measured for both, trace elements and TSH/free T4, at the Heath Checkup Center were retrospectively reviewed. The presence of thyroiditis (from thyroid echogenicity) and thyroid nodules were reviewed in the subjects who underwent thyroid ultrasonography. RESULTS: Blood concentrations of manganese, copper, selenium, and molybdenum were associated with TSH or free T4. After adjusting for age, sex, BMI, smoking, and alcohol consumption, blood copper levels were positively associated with free T4 in both sexes and selenium levels were positively associated with free T4 in women. There was no association between trace elements and thyroiditis. Blood copper concentration had a weak non-linear association with the presence of thyroid nodules. CONCLUSIONS: This study demonstrated that blood concentrations of copper and selenium were significantly associated with free T4 in healthy Korean subjects with sufficient iodine intake suggesting their role in maintaining normal thyroid function.

Assessment of 7 trace elements in serum of patients with nontuberculous mycobacterial lung disease.

Nontuberculous mycobacterial (NTM) lung diseases are an emerging cause of pulmonary infection, becoming more common in the clinical setting as incidence of NTM lung diseases steadily increases worldwide. Trace elements are essential micronutrients and are known to play many important roles in infectious diseases. We investigated the concentrations of trace elements in patients with NTM lung disease and compared these values to patients with pulmonary tuberculosis and healthy controls. A case-control study was conducted to evaluate the serum trace element concentrations in 95 patients with NTM lung disease, 97 patients with pulmonary tuberculosis, and 99 healthy control subjects. The serum concentrations of 7 trace elements (cobalt, copper, chromium, manganese, molybdenum, selenium, and zinc) were measured using inductively coupled plasma-mass spectrometry. We also analyzed demographic data, clinical outcomes, and other biochemical parameters. The median serum concentrations of copper and molybdenum were higher in patients with NTM lung disease (109 vs. 91 µg/dL, p < 0.001 and 1.70 vs. 0.96 µg/L, p < 0.001). In contrast, the median serum concentrations of selenium and zinc were significantly lower in patients with NTM lung disease than in healthy controls (105 vs. 115 µg/L, p < 0.001 and 94 vs. 102 µg/dL, p < 0.001). Compared to patients with pulmonary tuberculosis, the serum concentrations of molybdenum and zinc were higher in patients with NTM lung disease, while cobalt and copper concentrations were lower (p < 0.001). Correlations among trace element concentrations were observed (copper and zinc, r = -0.367; cobalt and molybdenum, r = -0.360; selenium and zinc, r = 0.335; and manganese and zinc, r = 0.327, respectively). None of the 7 trace elements were associated with treatment outcomes. Patients with NTM lung disease showed different serum trace element concentrations. Our study indicates that altered trace element status is associated with mycobacterial disease. Further study investigating the clinical significance of individual trace elements and their association with nutritional status in patients with NTM lung disease would be required.

Serum Trace Elements and Their Associations with Breast Cancer Subgroups in Korean Breast Cancer Patients.

The relationships between serum levels of trace elements and breast cancer remain relatively unknown. In this study, we investigate serum levels of seven trace elements in Korean breast cancer patients compared to controls without breast cancer. Serum trace element levels were determined using inductively coupled plasma mass spectrometry in Korean breast cancer patients before initiation of breast cancer treatment. Korean females without breast cancer served as a control group. Trace element levels were measured in the discovery cohort (n = 287) and were validated in an independent cohort (n = 142). We further investigated possible associations between trace element levels and the presence of lymph node metastasis, distant metastasis, or triple-negative breast cancer among breast cancer patients in subgroup analyses. Serum manganese and molybdenum levels were significantly higher (p < 0.05) in breast cancer patients than in controls. Serum copper levels were significantly higher in breast cancer patients with distant metastasis, while selenium levels were significantly lower. Other trace elements were neither significantly different between breast cancer patients and controls nor between subgroups of breast cancer patients. Our study provides insights about the potential roles and impacts of trace elements through an assessment of the associations between trace elements and breast cancer.

Neuroprotective Effects of the Methanol Extract of Kimchi, a Korean Fermented Vegetable Food, Mediated Via Suppression of Endoplasmic Reticulum Stress and Caspase Cascade Pathways in High-Cholesterol Diet-Fed Mice.

Endoplasmic reticulum (ER) stress-related unfolded peptide accumulation is closely associated with the development of neurodegenerative diseases known as protein misfolding disorders. The antioxidative properties of kimchi, a traditional Korean fermented vegetable dish, have been well established. In this study, the neuroprotective effects of the kimchi methanol extract (KME) were examined in high-cholesterol diet (HCD)-fed mice. The animals were fed a HCD, with oral administration of either KME (KME group, 200 mg·kg bw-1·day-1, n = 10) or distilled water (Control group, n = 10) for 8 weeks. Compared with the levels in the control group, the reactive oxygen species, peroxynitrite, and lipid peroxidation levels in the brain were significantly decreased in the KME group (P < .05), whereas the glutathione level was increased (P < .05). In addition, the ER stress biomarkers, phospho-eukaryotic initiation factor 2 subunit α, glucose-regulated protein 78, X-box binding protein 1, inositol-requiring enzyme 1, and C/EBP homologous protein and the nuclear factor-kappaB-mediated inflammation were significantly reduced in the KME group (P < .05). In contrast, the expression levels of antioxidative enzymes regulated by nuclear factor erythroid 2-related factor-2 were elevated (P < .05). The amyloid-beta expression levels of the KME group were lower than that of the control group (P < .05). Moreover, the expression levels of Bcl-2-associated X, and caspases-3 and -9 were downregulated, with a concomitant upregulation of B cell lymphoma 2 (P < .05). Accordingly, KME provide neuronal cell protection via suppressing ER stress and caspase cascade signaling.

Development of Genome-Wide SSR Markers from Angelica gigas Nakai Using Next Generation Sequencing.

Angelica gigas Nakai is an important medicinal herb, widely utilized in Asian countries especially in Korea, Japan, and China. Although it is a vital medicinal herb, the lack of sequencing data and efficient molecular markers has limited the application of a genetic approach for horticultural improvements. Simple sequence repeats (SSRs) are universally accepted molecular markers for population structure study. In this study, we found over 130,000 SSRs, ranging from di- to deca-nucleotide motifs, using the genome sequence of Manchu variety (MV) of A. gigas, derived from next generation sequencing (NGS). From the putative SSR regions identified, a total of 16,496 primer sets were successfully designed. Among them, we selected 848 SSR markers that showed polymorphism from in silico analysis and contained tri- to hexa-nucleotide motifs. We tested 36 SSR primer sets for polymorphism in 16 A. gigas accessions. The average polymorphism information content (PIC) was 0.69; the average observed heterozygosity (HO) values, and the expected heterozygosity (HE) values were 0.53 and 0.73, respectively. These newly developed SSR markers would be useful tools for molecular genetics, genotype identification, genetic mapping, molecular breeding, and studying species relationships of the Angelica genus.

The effects of anti-insulin antibodies and cross-reactivity with human recombinant insulin analogues in the E170 insulin immunometric assay.

BACKGROUND: Insulin assays are affected by varying degrees of interference from anti-insulin antibodies (IAs) and by cross-reactivity with recombinant insulin analogues. We evaluated the usefulness of the E170 insulin assay by assessing IA effects and cross-reactivity with 2 analogues. METHODS: Sera were obtained from 59 type 2 diabetes patients receiving continuous subcutaneous insulin infusion and 18 healthy controls. Insulin levels were determined using an E170 analyzer. To investigate the effects of IAs, we performed IA radioimmunoassays, and analyzed the differences between directly measured insulin (direct insulin) and polyethylene glycol (PEG)-treated insulins (free, IA-unbound; total, IA-bound and unbound insulin). We performed in-vitro cross-reactivity tests with insulin aspart and insulin glulisine. RESULTS: In IA-positive patients, E170 free insulin levels measured using the E170 analyzer were significantly lower than the direct insulin levels. The mean value of the direct/free insulin ratio and IA-bound insulin, which were calculated as the difference between total and free insulin, increased significantly as endogenous IA levels increased. The E170 insulin assay showed low cross-reactivities with both analogues (< 0.7%). CONCLUSIONS: IAs interfered with E170 insulin assay, and the extent of interference correlated with the IA levels, which may be attributable to the increase in IA-bound insulin, and not to an error in the assay. The E170 insulin assay may measure only endogenous insulin since cross-reactivity is low. Our results suggest that the measurement of free insulin after PEG pre-treatment could be useful for beta cell function assessment in diabetic patients undergoing insulin therapy.

Determination of carbohydrate-deficient transferrin levels by using capillary electrophoresis in a Korean population.

BACKGROUND: Carbohydrate-deficient transferrin (CDT) levels have rarely been determined in an Asian population. We evaluated the analytical performance of a test for measuring CDT levels by using capillary electrophoresis (EP). METHODS: We determined the precision of CDT measurement by using capillary EP and nephelometry and compared the CDT values obtained using both the methods. We included healthy control subjects, abstinent patients with liver disease, and individuals consuming varying amounts of alcohol. RESULTS: The CDT measurement by using capillary EP were correlated well with those CDT measurement by using nephelometry, N Latex CDT assay, Y=0.5706X+1.581, R=0.930. The results obtained from both methods showed good qualitative agreement with each other (κ coefficient=0.61). Genetic variants of transferrin isoforms were detected in 4.1% of the tested population. Both the CDT and γ-glutamyl transpeptidase (GGT) levels in the abstinent patients with liver disease were significantly higher than those in healthy abstinent individuals (0.9% vs. 0.5%, 109.5 mg/dL vs. 28.5 mg/dL, respectively), but the difference in CDT values in the 2 groups was less pronounced for the CDT values. Individuals who had a mean daily alcohol intake of more than 60 g/day showed significantly higher CDT levels than those who had a mean daily alcohol intake of less than 60 g/day (1.9% vs. 0.7%, P=0.03). CONCLUSIONS: The CDT test using capillary EP showed good performance, and this method has several advantages such as automation and detection of variant forms. Thus, CDT can be a more useful marker than GGT for monitoring alcohol abstinence, especially in patients with liver disease.

Differences in circulating dendritic cell subtypes in pregnant women, cord blood and healthy adult women.

Different subtypes of dendritic cells (DC) influence the differentiation of naíve T lymphocytes into T helper type 1 (Th1) and Th2 effector cells. We evaluated the percentages of DC subtypes in peripheral blood from pregnant women (maternal blood) and their cord blood compared to the peripheral blood of healthy non pregnant women (control). Circulating DC were identified by flow cytometry as lineage (CD3, CD14, CD16, CD19, CD20, and CD56)-negative and HLA-DR-positive cells. Subtypes of DC were further characterized as myeloid DC (CD11c(+)/CD123(+/-)), lymphoid DC (CD11c(-)/CD123(+++)) and less differentiated DC (CD11c(-)/CD123(+/-)). The frequency of DC out of all nucleated cells was significantly lower in maternal blood than in control (P<0.001). The ratio of myeloid DC/lymphoid DC was significantly higher in maternal blood than in control (P<0.01). HLA-DR expressions of myeloid DC as mean fluorescence intensity (MFI) were significantly less in maternal blood and in cord blood than in control (P<0.001, respectively). The DC differentiation factors, TNF-alpha and GM-CSF, released from mononuclear cells after lipopolysaccharide stimulation were significantly lower in maternal blood than in control (P<0.01). The distribution of DC subtypes was different in maternal and cord blood from those of non-pregnant women. Their role during pregnancy remains to be determined.

[Current status of external quality assessment of syphilis test in Korea].

BACKGROUND: Current status of external quality assessment (EQA) of laboratory tests for syphilis in Korea was analyzed to find out the problems that should be improved in the future. METHODS: Based on the data from the external quality assessment program performed twice a year by the Immunoserology Subcommittee of the Korean Association of Quality Assurance for Clinical Laboratory from the year 2004 to 2006, discordance rates were analyzed according to the test method and commercial kit used. RESULTS: Among the laboratories participating in the EQA program for syphilis test, about 90% of them used non-treponemal tests and about 55% treponemal tests. The non-treponemal tests included RPR (rapid plasma reagin) and VDRL tests used in 88% (363/412) and 11% (45/412), respectively, of the laboratories. The discordance rates were 2.2% for RPR test and 3.6% for VDRL. For the treponemal tests, Treponema pallidum hemagglutination assay (TPHA) was used in 60-76% and Immunochromatography assay (ICA) in about 30% of the laboratories in 2006. A high discordance rate of over 10% was reported in both TPHA and in ICA methods, possibly due to a low titer (1:1 in VDRL) of EQA samples in 2005. Analysis of the accumulated data from year 2004 to 2006 showed that the discordance rates of TPHA, ICA, and FTA-ABS were 4.6%, 3.7%, and 2.7%, respectively. CONCLUSIONS: For syphilis tests, RPR test, TPHA, and ICA are mainly used in Korea. A high discordance rate is still reported in TPHA and ICA, especially when testing samples with a low titer. Further analysis of data and education of laboratory personnel are needed for the improvement of the EQA program.