Identification of the novel HLA-A*24:589 allele in a Korean deceased donor.

The HLA-A*24:589 allele differs from A*24:02:01:01 by one nucleotide substitution in codon 145 (CGC > GGC).

Novel Pathogenic Variant (c.1171A>T) in PHF21A in a Female with Intellectual Disability and Craniofacial Anomalies.

Background: PHF21A, along with EXT2 and ALX4, is one of the causative genes of Potocki-Shaffer syndrome (PSS), a rare contiguous disorder involving chromosome region11p11.2. PHF21A has been associated with intellectual developmental disorders and craniofacial anomalies and suggested as a candidate for more extended phenotypes. However, variants in PHF21A and its associated phenotypes are yet to be fully explored, since reports on cases with variants affecting this gene are few worldwide. We present a novel heterogeneous variant in PHF21A in a 26-year-old Korean female. Methods: The patient's clinical manifestations were recorded and physical examination, cognitive assessment, brain imaging, metabolic screening, and cytogenetic testing including whole exome sequencing were pursued. Results: Whole exome sequencing identified a de novo nonsense variant c.1171A>T (p.Lys391Ter), affecting the AT-hook domain. The patient showed an extended phenotypic spectrum along with intellectual developmental disorders and craniofacial anomalies, such as attention-deficit hyperactivity disorder, epilepsy, overgrowth, and hypotonia. Variants affecting the AT-hook domain are few in PSS, however, the phenotypic spectrum of the patient was in line with previously reported cases. Conclusion: This case further reinforced and adds to the extended data on the phenotypes associated with PHF21A haploinsufficiency.

Development and evaluation of a multiplex loop-mediated isothermal amplification (LAMP) assay for differentiation of Mycobacterium tuberculosis and non-tuberculosis mycobacterium in clinical samples.

INTRODUCTION: The rapid and accurate diagnosis of tuberculosis (TB) is important to reduce morbidity and mortality rates and risk of transmission. Therefore, molecular detection methods such as a real-time PCR-based assay for Mycobacterium tuberculosis (MTB) have been commonly used for diagnosis of TB. Loop-mediated isothermal amplification (LAMP) assay was believed to be a simple, quick, and cost-effective isothermal nucleic acid amplification diagnostic test for infectious diseases. In this study, we designed an in-house multiplex LAMP assay for the differential detection of MTB and non-tuberculosis mycobacterium (NTM), and evaluated the assay using clinical samples. MATERIAL AND METHODS: For the multiplex LAMP assay, two sets of specific primers were designed: the first one was specific for IS6110 genes of MTB, and the second one was universal for rpoB genes of mycobacterium species including NTM. MTB was confirmed with a positive reaction with both primer sets, and NTM was identified with a positive reaction by only the second primer set without a MTB-specific reaction. Total 333 clinical samples were analyzed to evaluate the multiplex LAMP assay. Clinical samples were composed of 195 positive samples (72 MTB and 123NTM) and 138 negative samples. All samples were confirmed positivity or negativity by real-time PCR for MTB and NTM. Analytical sensitivity and specificity were evaluated for the multiplex LAMP assay in comparison with acid fast bacilli staining and the culture method. RESULTS: Of 123 NTM samples, 121 were identified as NTM and 72/72 MTB were identified as MTB by the multiplex LAMP assay. False negative reactions were seen only in two NTM positive samples with co-infection of Candida spp. All 138 negative samples were identified as negative for MTB and NTM. Analytical sensitivity of the multiplex LAMP assay was 100% (72/72) for MTB, and 98.4% (121/123) for NTM. And the specificity of assay was 100% (138/138) for all. CONCLUSIONS: Our newly designed multiplex LAMP assay for MTB and NTM showed relatively good sensitivity in comparison with previously published data to detect isolated MTB. This multiplex LAMP assay is expected to become a useful tool for detecting and differentiating MTB from NTM rapidly at an acceptable sensitivity.

Sheathless Shape-Based Separation of Candida Albicans Using a Viscoelastic Non-Newtonian Fluid.

Rapid and accurate identification of Candida albicans from among other candida species is critical for cost-effective treatment and antifungal drug assays. Shape is a critical biomarker indicating cell type, cell cycle, and environmental conditions; however, most microfluidic techniques have been focused only on size-based particle/cell manipulation. This study demonstrates a sheathless shape-based separation of particles/cells using a viscoelastic non-Newtonian fluid. The size of C. albicans was measured at 37 °C depending on the incubation time (0 h, 1 h, and 2 h). The effects of flow rates on the flow patterns of candida cells with different shapes were examined. Finally, 2-h-incubated candida cells with germ tube formations (≥26 µm) were separated from spherical candida cells and shorter candida cells with a separation efficiency of 80.9% and a purity of 91.2% at 50 µL/min.

A single-center, single-blind study to evaluate the clinical sensitivity, specificity, and agreement between Elecsys Anti-HBc II and Elecsys Anti-HBc in a Korean population.

BACKGROUND: Anti-HBc IgG is almost always detected in hepatitis B virus (HBV)-infected individuals and persists in resolved infections. In certain cases, anti-HBc IgG is the only serological marker and anti-HBc-positive result generally means anti-HBc total positivity. OBJECTIVES: To evaluate the clinical sensitivity and specificity of an investigational medical device, Elecsys Anti-HBc II, using samples from the Korean population. Agreement between Elecsys Anti-HBc II and its widely utilized predecessor Elecsys Anti-HBc was also evaluated. STUDY DESIGN: Residual serum or plasma samples stored at below -20 °C without individual identifiers were used in this study. This study had 106 randomly selected HBV deoxyribonucleic acid (DNA)-positive samples used for evaluating clinical sensitivity. For clinical specificity, a total of 239 both HBV DNA and hepatitis B surface antigen-negative samples, which were anti-HBc-negative by Elecsys Anti-HBc, were used. Agreement between Elecsys Anti-HBc and Elecsys Anti-HBc II was evaluated in total 345 samples. The Architect Anti-HBc II was used as a confirmatory test regarding discrepancies between Elecsys Anti-HBc and Elecsys Anti-HBc II results. RESULTS: The clinical sensitivity and specificity of Elecsys Anti-HBc II were found to be 99.06% and 100%, respectively. In total, 345 samples showed 100% agreement. Both positive and negative agreements were also 100%. CONCLUSIONS: The clinical performance of Elecsys Anti-HBc II was confirmed as sufficient in Korean samples. Elecsys Anti-HBc II demonstrated an exceptional performance, exceeding the requirements of the Ministry of Food and Drug Safety and confirming its reliability as an in vitro diagnostic device for HBV diagnosis in Korea.

Diagnostic Performance and Comparative Evaluation of the Architect, Liaison, and Platelia Epstein-Barr Virus Antibody Assays.

BACKGROUND: Epstein-Barr Virus (EBV) is one of the most prevalent causes of viral infection in humans. EBV infection stage (acute, past, or absent infection) is typically determined using a combination of assays that detect EBV-specific markers, such as IgG and IgM antibodies against the EBV viral capsid antigen (VCA) and IgG antibodies against the EBV nuclear antigen (EBNA). We compared the diagnostic performance and agreement of results between three commercial EBV antibody assays using an EBV performance panel (SeraCare Life Science, Milford, MA, USA) as a reference. METHODS: EBV antibody tests of EBV VCA IgM, VCA IgG, and EBNA IgG antibodies were performed by the Architect (Abbott Diagnostics, Wiesbaden, Germany), Liaison (DiaSorin, Saluggia, Italy), and Platelia (Bio-Rad, Marnes-la-Coquette, France) assays. Agreement between the three assays was evaluated using 279 clinical samples, and EBV DNA and antibody test results were compared. RESULTS: The three EBV antibody assays showed good diagnostic performance with good and excellent agreement with the performance panel (kappa coefficient, >0.6). The overall VCA IgM positivity rate was higher in EBV DNA-positive samples than in EBV DNA-negative samples for all three EBV antibody assays (P=0.02). The three EBV antibody assays exhibited good agreement in results for the clinical samples. CONCLUSIONS: The diagnostic performance of the three EBV antibody assays was acceptable, and they showed comparable agreement in results for the clinical samples.

Prognostic Impact of Lymphocyte Subpopulations in Peripheral Blood after Hematopoietic Stem Cell Transplantation for Hematologic Malignancies.

BACKGROUND: We prospectively evaluated prognostic value of lymphocyte subpopulations in peripheral blood of allogeneic hematopoietic stem cell transplant (HSCT) recipients. METHODS: 113 allogeneic HSCT (47 sibling matched, 37 unrelated matched, 29 haploidentical)-performed patients diagnosed as AML (n = 66), ALL (n = 28), and MDS (n = 19) were prospectively enrolled. 14 lymphocyte subpopulations were quantified by flow cytometry of PB at specific time-points after HSCT, and their prognostic impacts were analyzed. RESULTS: At 1, 2, and 3 months post-HSCT, significant adverse impact on overall survival (OS) and/or event free survival (EFS) was exhibited by low levels of natural killer (NK) cells (≤32 and ≤90/µL at 1 and 2 months on OS and EFS); regulatory T cells (≤1/µL) on EFS at 2 months; and B cells (≤19 and ≤92/µL for OS and EFS at 3 months). At 12 months, low levels of T cells (≤1180/µL), helper/inducer (H/I) T cells (≤250/µL), cytotoxic/suppressor (C/S) T cells (≤541/µL), and NK cells (≤138/µL) were associated with significantly higher risk of relapse. Low levels of T cells (≤879/µL) and C/S T cells (≤541/µL), and high level of naïve thymic T cells (>115/µL) showed a significant association with poor OS; low levels of C/S T cells (≤541/µL) and NK cells (≤138/µL) showed a significant adverse impact on EFS. CONCLUSIONS: Low levels of NK cells, regulatory T cells, and B cells at early stage post-HSCT are adverse prognostic indicators. At late stage, low levels of T cells and their subpopulations, NK cells, and high level of naïve thymic T cells are adverse prognostic indicators. © 2017 International Clinical Cytometry Society.

Successful kidney transplantation across a positive complement-dependent cytotoxicity crossmatch by using C1q assay-directed, bortezomib-assisted desensitization: A case report.

RATIONALE: Human leukocyte antigen (HLA) is the major immunologic barrier in kidney transplantation (KT). Various desensitization protocols to overcome the HLA barrier have increased the opportunity for transplantation in sensitized patients. In addition, technological advances in solid-phase assays have permitted more comprehensive assessment of donor-specific antibodies. Although various desensitization therapies and immunologic techniques have been developed, the final transplantation decision is still based on the classic complement-dependent cytotoxicity (CDC) crossmatch (XM) technique. Some patients who fail to achieve negative XM have lost their transplant opportunities, even after receiving sufficient desensitization therapies. PATIENT CONCERNS: A 57-year-old male with end-stage renal disease secondary to chronic glomerulonephritis was scheduled to have a second transplant from his son, but CDC XM was positive. DIAGNOSES: Initial CDC XM (Initial T-AHG 1:32) and flow-cytometry XM were positive. Anti-HLA-B59 donor specific antibody was detected by Luminex single antigen assay. INTERVENTIONS: Herein, we report a successful case of KT across a positive CDC XM (T-AHG 1:8 at the time of transplantation) by using C1q assay-directed, bortezomib-assisted desensitization. After confirming a negative conversion in the C1q donor-specific antibody, we decided to perform KT accepting a positive AHG-CDC XM of 1:8 at the time of transplantation. OUTCOMES: The posttransplant course was uneventful and a protocol biopsy at 3 months showed no evidence of rejection. The patient had excellent graft function at 12 months posttransplant. LESSONS: The results of XM test and solid-phase assay should be interpreted in the context of the individual patient.

The clinicopathological relevance of pretransplant anti-angiotensin II type 1 receptor antibodies in renal transplantation.

BACKGROUND: Anti-angiotensin II type 1 receptor antibodies (AT1R-Abs) have been suggested as a risk factor for graft failure and acute rejection (AR). However, the prevalence and clinical significance of pretransplant AT1R-Abs have seldom been evaluated in Asia. METHODS: In this multicenter, observational cohort study, we tested the AT1R-Abs in pretransplant serum samples obtained from 166 kidney transplant recipients. Statistical analysis was used to set a threshold AT1R-Abs level at 9.05 U/mL. RESULTS: Pretransplant AT1R-Abs were detected in 98/166 (59.0%) of the analyzed recipients. No graft loss or patient death was reported during the study period. AT1R-Abs (+) patients had a significantly higher incidence of biopsy-proven AR than AT1R-Abs (-) patients (27.6 versus 10.3%, P = 0.007). Recipients with pretransplant AT1R-Abs had a 3.2-fold higher risk of AR within a year of transplantation (P = 0.006). Five study subjects developed microcirculation inflammation (score ≥2). Four of them were presensitized to AT1R-Abs. In particular, three patients had a high titer of anti-AT1R-Abs (>22.7 U/mL). CONCLUSIONS: Pretransplant AT1R-Abs is an independent risk factor for AR, especially acute cellular rejection, and is possibly associated with the risk of antibody-mediated injury. Pretransplant assessment of AT1R-Abs may be useful for stratifying immunologic risks.

Successful kidney transplantation after desensitization in a patient with positive flow crossmatching and donor-specific anti-HLA-DP antibody: A Case report.

BACKGROUND: Traditionally, the presence of antibodies against human leukocyte antigen (HLA)-C and DP was considered to be associated with only a low risk of antibody-mediated rejection (ABMR) in kidney transplantation (KT), because the antigenicities of these proteins are weak. However, the clinical effects of HLA-C and -DP donor-specific HLA antibodies (DSHAs) have recently been reevaluated. METHODS: Here, we report the case of a retransplant patient with positive flow cytometry crossmatch (FCXM) and high level of HLA-DP DSHA who was desensitized using rituximab, plasmapheresis, and intravenous immunoglobulin. RESULTS: The epitope-based antibody reactivity was identified that the positive B-cell FCXM in our patient was attributable to the specific epitope. The patient underwent a successful retransplantation and has continued to do well for 10 month after KT. CONCLUSION: If an HLA-DP DSHA is present, it is important to detect any mismatched HLA-DP epitope pretransplantation and to monitor HLA-DP levels carefully. According to previous reports, anti-HLA-DP DSHA can induce ABMR soon after transplantation, but such ABMR can be prevented by pretransplantation desensitization and careful monitoring of DSHA levels.

Detecting primary drug-resistant mutations in Korean HIV patients using ultradeep pyrosequencing.

HIV primary resistance, drug resistance in treatment-naïve patients, is an emerging public health issue. The prevalence of HIV primary resistance mutations down to the level of 1% minor variants was investigated using ultradeep pyrosequencing (UDPS) in HIV-positive Korean blood donors and in treatment naïve chronic patients for the comparison. The entire pol region was sequenced from 25 HIV-positive blood donors, and 18 treatment-naïve chronic HIV patients. UDPS was successful in 19 blood donors and 18 chronic patients. In total, 1,011,338 sequence reads were aligned, and 28,093 sequence reads were aligned on average per sample. The prevalence of HIV primary resistance mutations in the HIV-positive blood donors and chronic HIV patients were 63.2% and 44.4% according to UDPS, respectively. Protease inhibitor (PI) drugs demonstrated different patterns in HIV-positive blood donors and chronic HIV patients, whereas non-nucleoside reverse transcriptase inhibitors (NNRTI), nucleoside reverse transcriptase inhibitors (NRTI), and integrase inhibitor (INI) drugs showed similar patterns between the two groups. Higher level of primary resistance prevalence was observed mainly because UDPS method could detect mutations in minor variants with 1-10% frequency. The higher resistance prevalence was observed in HIV-positive blood donors than in chronic patients. Considering that treatments for HIV-infected patients were recently amended to start at an earlier stage, information about degree of drug resistance to each drug between the two groups would help to establish future policies, design additional clinical trials, assess HIV patient care in Korea.

Comparison of Six Automated Treponema-Specific Antibody Assays.

Six different Treponema (TP)-specific immunoassays were compared to the fluorescent treponemal antibody absorption (FTA-ABS) test. A total of 615 samples were tested. The overall percent agreement, analytical sensitivity, and analytical specificity of each assay compared to the FTA-ABS test were as follows: Architect Syphilis TP, 99.2%, 96.8%, and 100%; Cobas Syphilis, 99.8%, 99.4%, and 100%; ADVIA Centaur Syphilis, 99.8%, 99.4%, and 100%; HISCL Anti-TP assay kit, 99.7%, 98.7%, and 100%; Immunoticles Auto3 TP, 99.0%, 97.5%, and 99.6%; Mediace TPLA, 98.0%, 98.1%, and 98.0%. All results that were discrepant between the TP-specific assays were associated with samples from noninfectious cases (11 immunoassay false positives and 7 from previous syphilis cases). Our study demonstrated that TP-specific immunoassays generally showed high sensitivities, specificities, and percentages of agreement compared to FTA-ABS, with rare cases of false-positive or false-negative results. Therefore, most TP-specific immunoassays are acceptable for use in screening for syphilis. However, it is important to perform a thorough review of a patient's clinical and treatment history for interpreting the results of syphilis serology.

Comparison and clinical utility evaluation of four multiple allergen simultaneous tests including two newly introduced fully automated analyzers.

BACKGROUND: We compared the diagnostic performances of two newly introduced fully automated multiple allergen simultaneous tests (MAST) analyzers with two conventional MAST assays. METHODS: The serum samples from a total of 53 and 104 patients were tested for food panels and inhalant panels, respectively, in four analyzers including AdvanSure AlloScreen (LG Life Science, Korea), AdvanSure Allostation Smart II (LG Life Science), PROTIA Allergy-Q (ProteomeTech, Korea), and RIDA Allergy Screen (R-Biopharm, Germany). We compared not only the total agreement percentages but also positive propensities among four analyzers. RESULTS: Evaluation of AdvanSure Allostation Smart II as upgraded version of AdvanSure AlloScreen revealed good concordance with total agreement percentages of 93.0% and 92.2% in food and inhalant panel, respectively. Comparisons of AdvanSure Allostation Smart II or PROTIA Allergy-Q with RIDA Allergy Screen also showed good concordance performance with positive propensities of two new analyzers for common allergens (Dermatophagoides farina and Dermatophagoides pteronyssinus). The changes of cut-off level resulted in various total agreement percentage fluctuations among allergens by different analyzers, although current cut-off level of class 2 appeared to be generally suitable. CONCLUSIONS: AdvanSure Allostation Smart II and PROTIA Allergy-Q presented favorable agreement performances with RIDA Allergy Screen, although positive propensities were noticed in common allergens.

Reconstitution of lymphocyte subpopulations after hematopoietic stem cell transplantation: comparison of hematologic malignancies and donor types in event-free patients.

The reconstitution of different immunocyte subsets after hematopoietic stem cell transplantation (HSCT), follows different timelines. We prospectively investigated changes in lymphocyte subsets after HSCT and their associations with primary diagnosis, conditioning regimen, and HSCT type in event-free patients. A total of 95 patients (48 with acute myeloid leukemia, 22 with acute lymphoid leukemia, and 25 with myelodysplastic syndrome) who underwent allogeneic HSCT (34 sibling matched, 37 unrelated matched, and 24 haploidentical HSCT) but did not experience any events such as relapse or death were enrolled in this study. Lymphocyte subpopulations (T cells, helper/inducer T cells, cytotoxic/suppressor T cells, memory T cells, regulatory T cells, natural killer (NK) cells, NK-T cells, and B cells) were quantified by flow cytometry of peripheral blood from recipients 7 days before and 1, 2, 3, 6, and 12 months after HSCT. Leukocyte counts recovered within 1 month after HSCT. However, the number of T and B lymphocytes recovered at 2 months after HSCT. NK cell counts recovered shortly after haploidentical HSCT. However, T lymphocytes and their subpopulations showed delayed recovery after haploidentical HSCT. Lymphocyte subsets showed different sequential patterns according to HSCT type but no differences were seen according to primary diagnosis or conditioning regimen.