A Retrospective Study of Factors Contributing to the Performance of an Interferon-Gamma Release Assay Blood Test for Tuberculosis Infection.

BACKGROUND: Tuberculosis (TB) remains a significant global health concern. Accurate detection of latent TB infection is crucial for effective control and prevention. We aimed to assess the performance of an interferon-gamma release assay blood test (QuantiFERON-TB Gold Plus [QFT-Plus]) in various clinical contexts and identify conditions that affect its results. METHODS: We conducted a retrospective analysis of 31 000 QFT-Plus samples collected from 26 000 subjects at a tertiary hospital in South Korea over a 4-year period and compared the rates of positivity and indeterminate results across diverse clinical situations. We also analysed the contribution of the QuantiFERON TB2 tube to the test's sensitivity and determined optimal cutoff values for 3 hematologic parameters to distinguish false-negative results. These cutoff values were validated in a separate cohort of subjects with microbiologically confirmed subclinical TB. RESULTS: Rates of QFT-Plus positivity and indeterminate results were disparate across diagnoses. The TB2 tube increased QFT-Plus sensitivity by 4.1% (95% CI, 1.1%-7.0%) in patients with subclinical TB. Absolute lymphocyte count ≤1.19 × 109/L, absolute neutrophil count ≥5.88 × 109/L, and neutrophil-to-lymphocyte ratio ≥4.33 were effective criteria to discriminate false-negative QFT-Plus results. Application of the hematologic criteria, individually or combined with mitogen response <10 IU/mL, substantially improved performance in the main study cohort and the validation cohort. CONCLUSIONS: These findings highlight the influence of clinical context and patient hematologic profiles on QFT-Plus results. To minimise neglected latent TB infections due to false-negative QFT-Plus results, serial retesting is advisable in patients with severe lymphopenia or neutrophilia, particularly when the mitogen response is <10 IU/mL.

Applicability of the cobas 6800 System for Epstein-Barr viral load quantitation using whole-blood specimens.

OBJECTIVES: This study aimed to evaluate the analytical performance of the cobas 6800 System (Roche Diagnostics) and assess the feasibility of using whole-blood specimens instead of plasma. METHODS: The analytical performance of the cobas EBV test (Roche Diagnostics) was evaluated. Thereafter, 120 clinical samples were collected to compare the cobas EBV test and the artus EBV RG PCR Kit (Qiagen). The results of the cobas EBV test conducted using paired plasma as well as 5× and 10× diluted whole-blood specimens were compared with those of the artus EBV RG PCR Kit performed using whole blood. RESULTS: The precision of the cobas EBV test was acceptable, and its linearity was confirmed to be within the range of 2.85 to 6.89 log IU/mL. Cross-reactivity was not observed. The best qualitative agreement (Cohen κ = 0.733) was observed using 5× diluted whole blood; the best quantitative correlation (Spearman correlation coefficient = 0.6865) was observed using 10× diluted whole blood. CONCLUSIONS: A significant discrepancy was observed in the results obtained from the 2 assays because of the different specimens used. We observed, however, that diluting whole blood before conducting the cobas EBV test effectively resolved polymerase chain reaction inhibition and viscosity issues, leading to an acceptable correlation with the results from the artus EBV RG PCR Kit conducted using whole blood.

Cell aggregation activates small GTPase Rac1 and induces CD44 cleavage by maintaining lipid raft integrity.

Lipid rafts are highly ordered membrane domains that are enriched in cholesterol and glycosphingolipids and serve as major platforms for signal transduction. Cell detachment from the extracellular matrix (ECM) triggers lipid raft disruption and anoikis, which is a barrier for cancer cells to metastasize. Compared to single circulating tumor cells (CTCs), our recent studies have demonstrated that CD44-mediatd cell aggregation enhances the stemness, survival and metastatic ability of aggregated cells. Here, we investigated whether and how lipid rafts are involved in CD44-mediated cell aggregation. We found that cell detachment, which mimics the condition when tumor cells detach from the ECM to metastasize, induced lipid raft disruption in single cells, but lipid raft integrity was maintained in aggregated cells. We further found that lipid raft integrity in aggregated cells was required for Rac1 activation to prevent anoikis. In addition, CD44 and γ-secretase coexisted at lipid rafts in aggregated cells, which promoted CD44 cleavage and generated CD44 intracellular domain (CD44 ICD) to enhance stemness of aggregated cells. Consequently, lipid raft disruption inhibited Rac1 activation, CD44 ICD generation, and metastasis. Our findings reveal two new pathways regulated by CD44-mediated cell aggregation via maintaining lipid raft integrity. These findings also suggest that targeting cell aggregation-mediated pathways could be a novel therapeutic strategy to prevent CTC cluster-initiated metastasis.

Routine breast milk monitoring using automated molecular assay system reduced postnatal CMV infection in preterm infants.

Human cytomegalovirus (CMV) transmitted through breast milk poses fatal risks to preterm infants. However, current molecular assay systems often do not accommodate breast milk samples. In this study, we evaluated the analytical and clinical performance of the measurement procedure of CMV load in breast milk utilizing the Cobas CMV test on the Cobas 6,800 system. This was enabled by incorporating a simple independent sample preparation procedure before the application of samples on the automated assay system. Clinical data from electronic medical records were retrospectively analyzed. Breast milk samples from mothers of preterm infants born before 33 weeks of gestation were screened for CMV using the automated assay system. CMV positivity rates in breast milk and neonatal samples and the CMV transmission rate were calculated. Furthermore, to validate the analytical accuracy of the overall measurement procedure with newly obtained residual breast milk samples, the linearity of the measurement procedure was assessed, and a simplified sample preparation method was validated against a conventional method. The CMV positivity rates in maternal breast milk and neonatal samples were 57.8 and 5.2%, respectively. The CMV transmission rate through breast milk was 7.7%. No significant differences in gestational age or birth weight were found between the CMV-negative and CMV-positive neonates. The linearity of the procedure was observed within a range of 1.87-4.73 log IU/mL. The simplified sample preparation method had an equivalent or even improved CMV detection sensitivity than the conventional method. Incorporating a simple independent sample preparation procedure effectively resolved any potential issues regarding the application of breast milk on the automated assay system. Our approach contributed to reduced vertical transmission of CMV by providing a convenient and reliable method for the monitoring of breast milk CMV positivity for clinicians.

Evaluating the Efficiency of the Cobas 6800 System for BK Virus Detection in Plasma and Urine Samples.

We evaluated the overall performance of the Cobas 6800 BKV test in detecting BK virus (BKV). We examined the imprecision of the Cobas 6800 BKV test and compared the qualitative and quantitative results obtained from the Cobas 6800 BKV test and the Real-Q BKV quantification assay. We assessed 88 plasma and 26 urine samples collected between September and November 2022 from patients with BKV infection using the Real-Q BKV quantitative assay. The lognormal coefficient of variation indicated that the inter-assay precision of the Cobas 6800 BKV test ranged from 13.86 to 33.83%. A strong correlation was observed between the quantitative results obtained using the Cobas 6800 BKV test and the Real-Q BKV quantification assay for plasma samples. The Spearman's rank correlation coefficients (ρ) for plasma, polymerase chain reaction (PCR) media-stabilized urine, and raw urine samples were 0.939, 0.874, and 0.888, respectively. Our analyses suggest that the Cobas 6800 BKV test is suitable for clinical applications owing to the strong correlation between the results obtained using this test and the Real-Q BKV quantification assay in plasma and urine samples. Furthermore, utilizing fresh raw urine samples can be a viable approach for the Cobas 6800 BKV test as it is less labor- and time-intensive.

Identification of the novel HLA-A*24:585:02 allele using next-generation sequencing.

The HLA-A*24:585:02 allele differs from HLA-A*24:02:01:01 allele by three nucleotides within codons -16 and -15.

ICAM-1-suPAR-CD11b Axis Is a Novel Therapeutic Target for Metastatic Triple-Negative Breast Cancer.

Accumulating evidence demonstrates that circulating tumor cell (CTC) clusters have higher metastatic ability than single CTCs and negatively correlate with cancer patient outcomes. Along with homotypic CTC clusters, heterotypic CTC clusters (such as neutrophil-CTC clusters), which have been identified in both cancer mouse models and cancer patients, lead to more efficient metastasis formation and worse patient outcomes. However, the mechanism by which neutrophils bind to CTCs remains elusive. In this study, we found that intercellular adhesion molecule-1 (ICAM-1) on triple-negative breast cancer (TNBC) cells and CD11b on neutrophils mediate tumor cell-neutrophil binding. Consequently, CD11b deficiency inhibited tumor cell-neutrophil binding and TNBC metastasis. Furthermore, CD11b mediated hydrogen peroxide (H2O2) production from neutrophils. Moreover, we found that ICAM-1 in TNBC cells promotes tumor cells to secrete suPAR, which functions as a chemoattractant for neutrophils. Knockdown of uPAR in ICAM-1+ TNBC cells reduced lung-infiltrating neutrophils and lung metastasis. Bioinformatics analysis confirmed that uPAR is highly expressed in TNBCs, which positively correlates with higher neutrophil infiltration and negatively correlates with breast cancer patient survival. Collectively, our findings provide new insight into how neutrophils bind to CTC to facilitate metastasis and discover a novel potential therapeutic strategy by blocking the ICAM-1-suPAR-CD11b axis to inhibit TNBC metastasis.

Identification of the novel HLA-C*01:242 allele using next-generation sequencing.

The HLA-C*01:242 allele differs from HLA-C*01:02:01:01 allele by a single nucleotide in codon 281.

Cell aggregation prevents anoikis and induces CD44 cleavage by maintaining lipid raft integrity to promote triple negative breast cancer metastasis.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, and metastasis is the major cause of cancer morbidity and mortality. Therefore, it is urgent to discover novel therapeutic targets and develop effective treatments for this lethal disease. Circulating tumor cells (CTCs) are considered "seeds of metastasis". Compared to single CTCs, our previous studies have demonstrated that CD44 homophilic interaction mediates CTC aggregation to enhance the stemness, survival and metastatic ability of aggregated cells. Importantly, the presence of CD44+ CTC clusters correlates with a poor prognosis in breast cancer patients. Here, we further investigated the underlying mechanism of how CD44-mediated cell aggregation promotes TNBC metastasis. We found that cell detachment, which mimics the condition when tumor cells detach from the extracellular matrix (ECM) to metastasize, induces lipid raft disruption in single cells, but lipid rafts integrity is maintained in aggregated cells. We further found that lipid rafts integrity in aggregated cells is required for Rac1 activation to prevent anoikis. In addition, CD44 and γ-secretase coexisted at lipid rafts in aggregated cells, which promotes CD44 cleavage and generates CD44 intracellular domain (CD44 ICD) to enhance stemness. Consequently, lipid rafts disruption inhibited Rac1 activation, CD44 ICD generation and metastasis. These data reveal a new mechanism of cell aggregation-mediated TNBC metastasis via maintaining lipid raft integrity after cell detachment. The finding provides a potential therapeutic strategy to prevent CTC cluster-initiated metastasis by disrupting lipid raft integrity and its-mediated downstream pathways.

Comparison of humoral immunogenicity in solid organ transplant recipients after third-dose mRNA vaccine with homologous or heterologous schedules: An observational study.

BACKGROUND: Solid organ transplant recipients (SOTRs) are susceptible to severe coronavirus disease 2019 (COVID-19); however, immunogenicity studies of the Omicron variants per vaccination schedules are still lacking. We examined humoral immunogenicity following third-dose mRNA vaccine administration in Korean SOTRs who received primary COVID-19 vaccine series on homologous or heterologous schedules. METHODS: We recruited SOTRs at Severance Hospital from October 27, 2021, to March 31, 2022. Blood samples were collected between 14 days and 5 months after the second and third mRNA vaccine (BNT162b2 or mRNA-1273) doses. SARS-CoV-2 anti-spike IgG titer was analyzed. The neutralization inhibition rate was analyzed using the surrogate neutralization assay for the wild-type, Delta, and Omicron variants. RESULTS: No significant differences existed in the SARS-CoV-2 anti-spike IgG positivity rate between the homologous BNT162b2/BNT162b2/BNT162b2 (85%) and other heterologous groups (83% of ChAdOx1/ChAdOx1/BNT162b2, 90% of ChAdOx1/ChAdOx1/mRNA-1273, and 78% of ChAdOx1/BNT162b2/BNT162b2). No significant difference existed in the neutralization inhibition rates between the four groups for wild-type, Delta, and Omicron variants. Median neutralization inhibition rates against the Omicron variant (2-5%) were significantly lower than those against the wild-type (87-97%) and Delta (55-89%) variants (P < 0.001). CONCLUSIONS: Regardless of the schedule, the neutralization inhibition rate against the Omicron variant was poor; therefore, additional preventive measures are required in such high-risk populations.

Identification of the novel HLA-DQA1*01:03:07 allele using next-generation sequencing.

The HLA-DQA1*01:03:07 allele differs from HLA-DQA1*01:03:01:01 allele by a single nucleotide in codon -8.

Identification of the novel HLA-DPA1*02:89 allele using next-generation sequencing.

The HLA-DPA1*02:89 allele differs from HLA-DPA1*02:02:02:01 allele by a single nucleotide in codon 224.

Guidelines for the Laboratory Diagnosis of Monkeypox in Korea.

While the coronavirus disease 2019 pandemic is ongoing, monkeypox has been rapidly spreading in non-endemic countries since May 2022. Accurate and rapid laboratory tests are essential for identifying and controlling monkeypox. Korean Society for Laboratory Medicine and the Korea Disease Prevention and Control Agency have proposed guidelines for diagnosing monkeypox in clinical laboratories in Korea. These guidelines cover the type of tests, selection of specimens, collection of specimens, diagnostic methods, interpretation of test results, and biosafety. Molecular tests are recommended as confirmatory tests. Skin lesion specimens are recommended for testing in the symptomatic stage, and the collection of both blood and oropharyngeal swabs is recommended in the presymptomatic or prodromal stage.

Assessment of disease activity in patients with rheumatoid arthritis using plasma tumour M2-pyruvate kinase test.

Background: Pyruvate kinase M2 (PKM2) is an enzyme that regulates the final process of glycolysis and exists in tetrameric and dimeric forms. The dimeric form of PKM2, also known as tumour M2-PK, increases when aerobic glycolysis is augmented, a feature observed in rheumatoid arthritis (RA). We investigated whether plasma tumour M2-PK is elevated in patients with RA and whether its levels correlate with disease activity. Methods: Plasma levels of tumour M2-PK were measured for patients with RA (n=151), those with osteoarthritis (OA) (n=37), and controls (n=37). We evaluated the association between plasma tumour M2-PK and continuous variables using Pearson's correlation analysis, and multivariate logistic regression analysis to determine the association between plasma tumour M2-PK and disease activity status. Knee synovial tissue blocks from patients with RA and OA were subjected to real-time quantitative PCR (qPCR) using two different primers for PKM2 and tumour M2-PK immunohistochemical (IHC) staining. Results: The tumour M2-PK level significantly correlated with the disease activity score in 28 joints (DAS28)-erythrocyte sedimentation rate (ESR) (r=0.546, p<0.001) and DAS28-C-reactive protein (CRP) (r=0.589, p<0.001). Moreover, repeat testing of tumour M2-PK levels in 20 patients revealed a significant decline in tumour M2-PK levels after reduction in inflammation (p<0.001). Area under the receiver operating characteristic curve (AUROC) analysis demonstrated that upon incorporation of tumour M2-PK, ESR, and CRP, the area under the curve was 0.962 for distinguishing moderate/high from remission/low disease activity. Adjusted logistic regression also revealed that a tumour M2-PK >43.9 U/mL (OR 3.672, p=0.042) independently predicted moderate/high disease activity status. Furthermore, tumour M2-PK levels in patients with RA were significantly higher than in those with OA and controls (all p<0.001). However, no differences were found in PKM2 expression in RA and OA synovial tissues as assessed by qPCR, and IHC analysis revealed negligible tumour M2-PK expression in the synovial tissues. Conclusion: Circulating plasma tumour M2-PK levels may be a clinically useful indicator for evaluating disease activity and RA diagnosis.

Identification of the novel HLA-DRB4*01:162N allele using next-generation sequencing.

The HLA-DRB4*01:162N allele differs from HLA-DRB4*01:03:01:01 allele by a single nucleotide in codon 131.

Matched Versus Mixed COVID-19 Vaccinations in Korean Solid Organ Transplant Recipients: An Observational Study.

BACKGROUND: Solid organ transplant recipients (SOTRs) are vulnerable to severe coronavirus disease 2019 (COVID-19) and exhibit poor antibody responses to COVID-19 vaccines. Herein, we compared the humoral immunogenicity of a mixed vaccine (ChAdOx1 nCoV-19 [ChAd]/BNT162b2 [BNT]) with that of conventional matched vaccines (mRNA, adenoviral vector [AdV-Vec]) in SOTRs. METHODS: Serum samples were collected at Severance Hospital (Seoul, Korea) between September and October 2021 (14 d-5 mo after COVID-19 vaccination; V2). The severe acute respiratory syndrome coronavirus 2 antispike IgG titer (BAU/mL; ELISA) and neutralization inhibition (percentage; neutralization assay) were compared between vaccination groups overall and stratified by V2 (poststudy vaccination visit) timing. RESULTS: Of the 464 participants, 143 (31%) received mRNA vaccines, 170 (37%) received AdV-Vec vaccines, and 151 (33%) received mixed vaccines (all ChAd/BNT). The geometric mean titer for the ChAd/BNT group was 3.2-fold higher than that of the AdV-Vec group (geometric mean ratio, 3.2; confidence interval, 1.9-5.4) but lower than that of the mRNA group (geometric mean ratio, 0.4; confidence interval, 0.2-0.7). Neutralization inhibition in the ChAd/BNT group was 32%, which was higher than that in the AdV-Vec group (21%; P < 0.001) but lower than that in the mRNA group (55%; P = 0.02). There was no difference in geometric mean titer by V2 timing (ChAd/BNT, 45 versus 31, days 14-60; mRNA, 28 versus 15, days 61-150). CONCLUSIONS: The ChAd/BNT group showed higher humoral immunogenicity than the AdV-Vec group, with similar immunogenicity to the mRNA vaccine. Nevertheless, immunogenicity following the primary vaccination series was poor in all vaccine groups, supporting the justification for booster vaccination in SOTRs.

Utility of Epstein-Barr Viral Load in Blood for Diagnosing and Predicting Prognosis of Lymphoma: A Comparison with Epstein-Barr Virus-Encoded RNA in Situ Hybridization.

Epstein-Barr virus (EBV) is a ubiquitous pathogen that persists in a small portion of B cells after primary infection and is etiologically associated with multiple lymphoma subtypes. We evaluated the clinical utility of EBV real-time quantitative PCR in comparison with the widely used Epstein-Barr virus-encoded RNA (EBER) in situ hybridization (ISH) method in 912 patients with four lymphoma subtypes: diffuse large B-cell lymphoma (DLBCL), extranodal natural killer/T-cell lymphoma (ENKTCL), peripheral T-cell lymphoma (PTCL), and Hodgkin lymphoma. We also assessed the impact of EBV positivity determined from each method or a combination of both methods on mortality using Kaplan-Meier survival analysis and Cox proportional hazard regression. EBV real-time quantitative PCR identified more positive cases than EBER-ISH for all subtypes, except ENKTCL. EBV DNA-positive patients with ENKTCL and PTCL displayed poorer overall survival (OS) than EBV DNA-negative patients (P = 0.0016 and P = 0.0013, respectively). In addition, among those with EBER-positive DLBCL and ENKTL and those with EBER-negative PTCL, OS was significantly worse for EBV DNA-positive patients (P = 0.027, P = 0.0016, and P = 0.0018, respectively). EBER positivity was associated with worse OS for DLBCL (P = 0.037), in reanalyses including only the 862 patients with unambiguous EBER-ISH results. Overall, EBV DNA positivity is a more effective prognostic marker than EBER-ISH status for patients with certain lymphoma subtypes.

A Single-Center Experience on HLA Typing with 11 Loci Next Generation Sequencing in Korean Patients with Hematologic Disease.

The human leukocyte antigen (HLA) system comprises the most polymorphic genes of the human genome and is famous for its potential pathological roles. To accurately type HLA genes and find HLA-matched donors, which are critical for effective hematopoietic transplantation, HLA typing using next-generation sequencing (NGS) was implemented. We aimed to share the experience of HLA typing using NGS in patients with hematologic malignancies and evaluate its association with hematologic diseases. Data from 211 Korean, non-familial patients diagnosed with a hematologic disease were reviewed, and NGS was performed for 11 HLA loci. Three-field HLA typing with G code was successfully achieved for all loci and the known linkage between HLA-DRB3/4/5 and HLA-DRB1 was fully matched. Therefore, NGS-based HLA typing enables a detailed, high-resolution analysis of the HLA system that can help with the selection of suitable donors. Notably, HLA-DRB1*08:02:01G was significantly associated with myelodysplastic syndrome. Although this result confirms the tendency of some alleles to be associated with hematological disorders, this may not be the case in hematologic malignancies. Nonetheless, NGS-based HLA typing data for HLA-DP, HLA-DQ, and HLA-DRB3/4/5 are still warranted for a better understanding of the corresponding locus.

Identification of the novel HLA-A*26:01:75 allele using next-generation sequencing.

The HLA-A*26:01:75 allele differs from HLA-A*26:01:01:01 allele by a single nucleotide in codon -10.3.

Comparison of plaque reduction and focus reduction neutralization tests for the measurement of neutralizing antibody titers against japanese encephalitis virus.

Japanese encephalitis is prevalent throughout the temperate and tropical regions of Asia and is caused by the Japanese encephalitis virus (JEV), a mosquito-borne viral pathogen. The plaque reduction neutralization test (PRNT) is currently recommended as the gold standard test for detecting human antibodies against JEV. The plaque assay is the most widely used method for detecting infectious virions and involves counting discrete plaques in cells. However, it is time-consuming, and results can be subjective (owing to analyst variability during manual plaque counting). The focus reduction neutralization test (FRNT), which is based on an immuno-colorimetric assay, can be used to automatically count foci formed by the JEV. Here, we compared the efficacy of PRNT and FRNT in measuring the neutralizing antibody titers using 102 serum samples from vaccinated and unvaccinated individuals. We observed positive correlations between these neutralization assays against the Nakayama and Beijing strains (R2 = 0.98 and 0.77, respectively). Thus, FRNT may be preferable to PRNT for evaluating the efficacy of JEV vaccines in large-scale serological studies.