Clinical performance of the STANDARD M10 SARS-CoV-2 rapid RT-PCR assay in patients visiting an emergency department.

In emergency departments, rapid screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was important for arranging limited isolation resources and patient care during the coronavirus disease 2019 (COVID-19) pandemic. STANDARD M10 SARS-CoV-2 (SD Biosensor) is a recently developed cartridge-based RT-PCR that provides a turnaround time of 1 h, which is shorter than that for conventional RT-PCR. This study evaluated the clinical performance of STANDARD M10 in patients visiting an emergency department. From March to June 2022, two specimens were collected from patients visiting an emergency department. Each specimen comprised one nasopharyngeal and one oropharyngeal swab. Respective specimens underwent rapid RT-PCR using STANDARD M10 and conventional RT-PCR using Allplex SARS-CoV-2 (Seegene). When discordant results occurred, specimens undergoing the STANDARD M10 were retested with the Allplex to exclude specimen variations. Retest results replaced initial results of the Allplex. Clinical performance of STANDARD M10 was compared with Allplex. The study enrolled 1971 patients. COVID-19 prevalence was 6.2% based on the Allplex. Compared with the Allplex, overall agreement, positive percent agreement, and negative percent agreement of STANDARD M10 were 99.5% (95% CI: 99.1%-99.8%), 95.9% (95% CI: 90.8%-98.3%), and 99.8% (95% CI: 99.4%-99.9%), respectively. Nine discordant results were all positive on droplet digital PCR, except for one specimen that was positive with STANDARD M10. The STANDARD M10 showed reliable diagnostic performance for detecting SARS-CoV-2 from patients visiting in emergency departments and is a useful tool in emergency healthcare systems because of its easy-to-use cartridge-based assay and short resulting time for detecting SARS-CoV-2.

Comparison of Throat and Nasopharyngeal Swabs for the Molecular Detection of Enterovirus in Pediatric Patients.

BACKGROUND: Enterovirus infections frequently occur in children worldwide. Molecular assays are widely used to detect enterovirus. Nasopharyngeal swabs (NPS) and throat swabs (TS) are common specimen types used in clinical practice. Here, the reliability of TS for detecting enterovirus in pediatric patients was compared with that of NPS using real-time reverse transcription polymerase chain reaction (RT-rPCR). METHODS: Results obtained using the Allplex Respiratory Panel 2 (Seegene, Korea) for NPS (NPS-RP) and Accu-Power EV Real-time RT-PCR (Bioneer, Korea) for TS (TS-EV), which were performed simultaneously between September 2017 to March 2020, were initially compared. Cross examination (Allplex Respiratory Panel 2 assay using TS and AccuPower EV assay with NPS) was performed for specimens collected between July 2019 to March 2020 to evaluate the performance of the enterovirus assays based on each specimen type. RESULTS: Among the 742 case results of initial tests, 597 cases (80.5%) tested negative in both assays, and 91 cases (12.6%) tested positive in both assays. Fifty-four discrepant results were observed: 39 cases (5.3%) tested positive in TS-EV and negative in NPS-RP, and 15 cases (2.0%) tested positive in NPS-RP and negative in TS-EV. The overall percent agreement was 92.7%. In the 99 cases cross examined, overall percent agreements were 98.0%, 94.9%, 92.9%, and 89.9% for TS-EV vs. TS-RP, NPS-RP vs. NPS-EV, TS-EV vs. NPS-EV, and NPS-RP vs. TS-RP, respectively. CONCLUSIONS: TS yields a high agreement rate with NPS in detecting enterovirus, regardless of single-plex or multiplex RT-rPCR assays. Thus, TS could be a good alternative specimen in pediatric patients who are reluctant to NPS sampling.

A Case of Marrow Fibrosis with Megakaryocytic Hyperplasia after Short-Term use of Granulocyte Colony-Stimulating Factor.

BACKGROUND: Administration of granulocyte colony-stimulating factors G-SCFs can cause diagnostic challenges because of morphologic alteration of hematopoietic cells. METHODS: We experienced a patient who showed distinctive clinical and morphologic findings after short time use of G-CSF. The clinical information and examination results of the morphology of bone marrow (BM) specimen and karyotype were analyzed by reviewing relevant literature. RESULTS: White blood cell (WBC) counts of the patient were unresponsive to G-CSF and marrow fibrosis and megakaryocytic hyperplasia were accompanied with increase of blasts in BM. Presence of malignant clones was confirmed by cytogenetic aberrations of monosomy 7. CONCLUSIONS: We concluded, BM study including cytogenetic analysis should be performed when such clinical findings are encountered and the possibility of hematologic malignancy should be considered.

Clinical Utility of SARS-CoV-2 Rapid Antigen Test in the Emergency Department.

BACKGROUND: Rapid screening for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was important in the emergency department during the coronavirus disease 2019 (COVID-19) pandemic. Real-time polymerase chain reaction (RT-PCR) is the standard method for detecting SARS-CoV-2, but it requires several hours to provide results. Instead, the rapid antigen test (RAT) has a short turnaround time and can be used at the bedside but shows low sensitivity. To overcome these shortcomings, the clinical utility of stepwise testing of RAT with RT-PCR in the emergency department was analyzed. METHODS: Patients who underwent SARS-CoV-2 RAT (SD Biosensor or Abbott) and RT-PCR (Seegene Allplex or GeneXpert) testing simultaneously at the emergency department in South Korea from January 2021 to March 2022 were enrolled. We compared the performance status of RAT with that of RT-PCR and evaluated the clinical utility of RAT as a screening tool for patients visiting the emergency department. RESULTS: A total of 7,574 patients were included. The overall prevalence of COVID-19 was 1.9% (146/7,574). The sensitivity and specificity of the RAT were 69.2% and 99.9%, respectively, and the positive and negative predictive values were 96.2% and 99.4%, respectively. Based on the cycle threshold (Ct) of the E gene, the sensitivity was 86.0% in patients with Ct < 26, but the sensitivity was 9.3% in patients with Ct ≥ 26. CONCLUSIONS: In the COVID-19 pandemic, RAT can be used as supplement test for the screening strategy using RT-PCR in the emergency department because it is rapid, highly specific, and relatively sensitive in patients with high viral load.

Genomic Mutation Profiles of Patients with Acute Myeloid Leukemia in Korea: a Single-Center Experience.

BACKGROUND: The emergence of next-generation sequencing (NGS) is currently leading the diagnosis of acute myeloid leukemia (AML) and its treatment using a more genetic-level approach. The study aimed to find clinical and prognostic correlations with genomic mutation profiles in Korean patients with AML using NGS. METHODS: This retrospective study enrolled a total of 30 patients who were newly diagnosed with AML from February 2021 to October 2022 in Korea. NGS was used to identify the genetic profiles of 40 genes relevant to AML. The clinical and laboratory data of the patients were analyzed with their genomic mutation profiles. RESULTS: NGS revealed at least one mutation in all patients, with a range of one to seven mutations (median of three mutations). Mutations were commonly associated with TET2, CEBPA, RUNX1, FLT3, IDH2, NPM1, and SRSF2 genes. The TET2 mutation correlated with older (77 vs. 72) patients, and the FLT3 mutation was associated with a higher WBC count (33.4 x 109/L vs. 6.4 x 109/L). The RUNX1 mutation correlated with a lower (44.0 x 109/L vs. 65.5 x 109/L) platelet count, and the NPM1 mutation showed a higher number of blasts in peripheral blood (56.5% vs. 13.0%). Among 16 patients who received induction chemotherapy, mutations in SRSF2, ASXL1, PHF6, SF3B1, and PTPN11 were detected only in patients who failed to achieve complete remission (CR). Meanwhile, mutations in NRAS, TP53, IKZF1, DNMT3A, SH2B3, U2AF1, and WT1 were detected in patients who achieved CR. CONCLUSIONS: Clinical and prognostic correlations were observed according to genomic mutation profiles detected by NGS in Korean patients with AML. An NGS study with a larger cohort of patients would be beneficial to establish the significant prognostic impact on patients with AML.

Reactivation and dynamics of cytomegalovirus and Epstein-Barr virus after rabbit antithymocyte globulin and cyclosporine for aplastic anemia.

OBJECTIVES: This study aimed to identify the natural course of cytomegalovirus (CMV)/Epstein-Barr virus (EBV) after rabbit antithymocyte globulin and cyclosporine (rATG-CsA) for aplastic anemia (AA). METHODS: In 113 prospectively observed AA patients treated with rATG-CsA, the CMV/EBV cohort was classified into two groups by baseline viremic status: no viremia (CMV-G1, n = 112; EBV-G1, n = 98) and the presence of viremia (CMV-G2, n = 1; EBV-G2, n = 13). RESULTS: In CMV-G1, the mean CMV load increased up to 3 months but was completely resolved from 6 months. The mean EBV load of EBV-G1 showed a peak at 1 month and then gradually decreased over time but remained detectable throughout the observation period. EBV-G2 showed fluctuating EBV dynamics. With reactivation rates of 38.4% in CMV-G1 and 62.2% in EBV-G1, a longer time to rATG-CsA from diagnosis and a lower absolute lymphocyte count at 1 month from rATG-CsA were significantly associated with CMV and EBV reactivation, respectively. The mean peak CMV and EBV loads of patients with CMV-related (3.5%) and EBV-related (0.9%) diseases were evidently higher than those of the remaining patients without CMV and EBV diseases in the respective cohort. CONCLUSION: Considering frequent reactivation and distinct courses of CMV/EBV, virologic surveillance is recommended after rATG-CsA for AA.

Clinical usefulness of iQ200/iChem Velocity workstation for screening of urine culture.

BACKGROUND: Clinical microbiology laboratories are asked to process large numbers of urine specimens for culture, but only 20-40% of them are positive. Therefore, a rapid, reliable screening method is necessary to speed up the reporting of a negative result. In this study, we evaluated the iQ200/iChem workstation, which is a combination of digital imaging software and a strip reader to predict negative urine culture. METHOD: A total of 1942 urine specimens were processed through both culture and iQ200/ iChem workstation. We analyzed the performance using two definition of positive urine culture; one or two potential uropathogens at a concentration of ≥105 CFU/ml and ≥ 104 CFU/ml. We assessed combinations of parameters (ASP; all small particles, WBC; leukocyte, BACT; bcteria, LE; leukocyte esterase) applying various cut-offs which can achieve the negative predictive value (NPV) ≥97% and culture reduction rate ≥ 50%. RESULTS: The culture positive rate was 12.8 and 18.4% applying the criteria of ≥105 CFU/ml and ≥ 104 CFU/ml, respectively. The area under the curve (AUC) of each parameter for ≥105 CFU/ml / ≥104 CFU/ml bacteriuria was 795 /0.719 for WBC, 0.722 / 0.701 for ASP and 0.740 /0.704 for bacteria. Therefore, we investigated the combination of the parameters. With the fixed parameter of BACT≥1/HPF and positive LE, the combinations of WBC ≥ 4/HPF and ASP ≥8500/µl or WBC ≥ 6/HPF and ASP≥5500/µl showed good performance for detecting ≥105 CFU/ml uropathogen. The ranges of sensitivity, specificity, negative predictive value and culture reduction rate were 91.5-92.3%, 49.8-52.6%, 97.7-97.9% and 50.4-53.0%, respectively. However, none of the combined setting yielded acceptable range of NPV for detecting ≥104 CFU/ml uropathogen (NPV 92.9-94.9%). Enterococcus spp. was the most common uropathogen causing the false negative results (55.7%), and also the main pathogen among the positive culture of 104-5 CFU/ml bacteriuria (45%). CONCLUSIONS: iQ200/iChem workstation was excellent in detection of ≥105 CFU/ml uropathogen, but unsatisfactory in detection of 104-5 CFU/ml uropathogen and Enterococcus spp. It can be useful for screening of urine specimens to reduce bacterial culture. However, notice from clinician will be necessary for specimens from the patients with high risk for UTI, such as pregnant woman, infant, elderly or immune compromised patients.

Measurement of Teicoplanin Concentration With Liquid Chromatography-Tandem Mass Spectrometry Method Demonstrates the Usefulness of Therapeutic Drug Monitoring in Hematologic Patient Populations.

BACKGROUND: Teicoplanin is a glycopeptide antibiotic that has become increasingly popular with the spread of methicillin-resistant Staphylococcus aureus. The aim of the study was to develop and validate an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for teicoplanin, and analyze trough teicoplanin concentrations achieved in patients with hematological diseases. METHODS: The UHPLC-MS/MS method for teicoplanin was developed, validated, and applied in a retrospective analysis of trough plasma teicoplanin concentrations from 305 patients receiving standard dose, and 17 patients receiving therapeutic drug monitoring (TDM)-guided individualized dose. RESULTS: The linear range was 3.9-52.9 mg/L. The imprecision was less than 12%, the limits of detection and quantification were less than 0.13 and 0.72 mg/L, respectively. The sample carry-over and ion suppression were insignificant. In the standard dose group, the median teicoplanin concentrations were 7.5 mg/L (days 3-5) and 8.9 mg/L (on days 6-8); and the proportion of trough levels achieving ≥10 mg/L was 20% (days 3-5) and 38% (days 6-8), respectively. In the TDM-guided individualized dose group, median teicoplanin concentration was higher (16.9 mg/L), and the proportion of trough levels ≥10 mg/L was also higher (77%) when compared with the standard dose group. CONCLUSIONS: Based on these results, the present UHPLC-MS/MS method can be considered suitable for routine TDM of teicoplanin. Also, based on the insufficient trough teicoplanin concentrations achieved with standard dose regimen, and the higher trough teicoplanin concentrations achieved with TDM-guided individualized dose regimen, this study highlights the importance of TDM of teicoplanin, especially in high-risk patient groups.

Systemic Epstein-Barr Virus-positive T-Cell Lymphoproliferative Disease of Childhood With Good Response to Steroid Therapy.

Systemic Epstein-Barr virus (EBV)-positive T-cell lymphoproliferative disease of childhood is a rare disease and has a very fulminant clinical course with high mortality. A 21-month-old female patient was referred to our hospital with a 1 week history of fever and was subsequently diagnosed with systemic Epstein-Barr virus-positive T-cell lymphoproliferative disease of childhood. After starting treatment with dexamethasone, she showed early defervescence and improvement of laboratory parameters, and has remained disease-free after stopping steroid treatment, although longer follow-up is necessary. Our report underscores the possibility that this disease entity may be heterogenous in terms of prognosis.

FLT3 expression and IL10 promoter polymorphism in acute myeloid leukemia with RUNX1-RUNX1T1.

We investigated the correlation between FLT3 expression and IL10 gene promoter polymorphism in acute myeloid leukemia with RUNX1-RUNX1T1 and their clinical significance. FLT3 mRNA expression was measured by real-time quantitative PCR (qPCR) and immunohistochemical staining (IHC) on bone marrow (BM) leukemic cells. IL10 gene promoter polymorphisms including rs1800896 (G-1082A), rs1800871 (C-819T), and rs1800872 (C-592T) were genotyped by direct sequencing. Among 45 enrolled patients, 32 (71.1 %) exhibited FLT3 overexpression, whose FLT3 mRNA level was higher than normal cut-off value (0.02). The IHC results also consisted with FLT3 mRNA expression data achieved by qPCR. The FLT3 mRNA level was significantly different among 3 IHC staining groups (P < 0.0001); 0.031 ± 0.041, 0.106 ± 0.097 and 0.588 ± 0.573 in IHC negative, intermediate and positive group, respectively. Interestingly, the FLT3 expression level was correlated with the percentage of BM CD34 positive cells (R = 0.360, P = 0.016). The elevated FLT3 expression at initial BM were decreased after remission and maintained lower than the cut-off level. FLT3 expression was not dependent on IL10 gene promoter polymorphisms. FLT3 overexpression itself did not demonstrate significant effects on overall survival (OS). However, it is notable that IL10 rs1800896 GA genotype tended to have a lower estimated mean OS (20.1 months) compared to GG genotype (54.6 months), but the statistical significance was not derived because of limited number of patients in this study (P = 0.072). Further studies including more type of leukemia and patients may be helpful to understand the relations between cytokine genotype and FLT3 expression and their prognostic impact.

Analytical performance of the Abbott ID NOW 2.0 assay for SARS-CoV-2 detection in clinical samples from symptomatic patients.

We evaluated the analytical performance of ID NOW™ COVID-19 2.0 assay versus conventional real-time reverse transcription-polymerase chain reaction (RT-PCR) using a total of 792 clinical samples from nasopharyngeal and oropharyngeal swabs, stored in frozen universal transport medium samples. Positive percent agreement (PPA) and negative percent agreement of ID NOW were 97.6 % and 100 %, respectively. The overall percent agreement between ID NOW and RT-PCR was 99.5 %. The PPA of ID NOW in detecting SARS-CoV-2 in 164 RT-PCR positive patients, all of whom had symptoms related COVID-19, was 97.1 % within 8 days since symptom onset, 97.9 % from 8 to 14 days since symptom onset, and 97.6 % after 14 days since symptom onset, with no significant difference between the days since symptom onset. The ID NOW assay demonstrated good performance, providing a rapid and randomly accessible alternative to conventional RT-PCR for timely SARS-CoV-2 detection, particularly in situations requiring rapid results for patient care.

A comprehensive Exdia TRF-LFIA for simultaneous quantification of GFAP and NT-proBNP in distinguishing ischemic and hemorrhagic stroke.

The goal of this study is to create a highly sensitive time-resolved fluorescence lateral flow immunoassay (TRF-LFIA) capable of concurrently measuring glial fibrillary acidic protein (GFAP) and the N-terminal fragment of B-type natriuretic peptide precursor (NT-proBNP). This assay is designed as a diagnostic tool and aims to provide an algorithm for stroke management, specifically for distinguishing between Ischemic stroke (IS) and Hemorrhagic stroke (HS). However, LFIA to quantify simultaneous serum NT-proBNP and GFAP are not yet available. We have developed and validated a novel TRF-LFIA for the simultaneous quantitative detection of NT-proBNP and GFAP. The sensitivity and reproducibility of the immunoassay were significantly improved by employing specific monoclonal antibodies linked to europium nanoparticles (EuNPs) that specifically target NT-proBNP and GFAP. The detection area on the nitrocellulose membrane featured sandwich-style complexes containing two test lines for NT-proBNP and GFAP, and one Control line. The fluorescence intensity of these test lines and control line was measured using an in-house developed Exdia TRF-Plus analyzer. As proof-of-concept, we enrolled patients suspected of having a stroke who were admitted within a specific time frame (6 h). A small amount of clinical specimen (serum) was used. To optimize the LFIA, an EuNPs conjugated antibodies were investigated to improve the detection sensitivity and decrease the background signal as well shorten the detection time. The Exdia TRF-LFIA cartridge offers a wide linear dynamic detection range, rapid detection, high sensitivity, and specificity. The limit of detection was determined to be 98 pg/mL for NT-proBNP and 68 pg/mL for GFAP, with minimal cross-reactivity. There were 200 clinical human serum samples that were used to evaluate this platform with high correlation. By combining the results of NT-proBNP and GFAP, we formulated an algorithm for the clinical assessment of Ischemic Stroke (IS) and Hemorrhagic Stroke (HS). According to our proposed algorithm, the combination of GFAP and NT-proBNP emerged as the most effective biomarker combination for distinguishing between IS and HS. Exdia TRF-LFIA shows great potential as a supplemental method for in vitro diagnostics in the laboratory or in other point-of-care testing (POCT) applications. Its development substantially decreases the diagnosis time for IS and HS. The proposed algorithm not only minimizes treatment delays but also lowers medical costs for patients.

The impact of enhanced screening for carbapenemase-producing Enterobacterales in an acute care hospital in South Korea.

BACKGROUND: Carbapenemase-producing Enterobacterales (CPE) poses a significant challenge to infection control in healthcare settings. Active screening is recommended to prevent intra-hospital CPE transmission. METHODS: CPE screening was initiated at a 660-bed hospital in South Korea in September 2018, targeting patients previously colonized/infected or admitted to outside healthcare facilities (HCFs) within 1 month. Universal intensive care unit (ICU) screening was performed at the time of admission. After a hospital-wide CPE outbreak in July-September 2019, the screening program was enhanced by extending the indications (admission to any HCF within 6 months, receipt of hemodialysis) with weekly screening of ICU patients. The initial screening method was changed from screening cultures to the Xpert Carba-R assay. The impact was assessed by comparing the CPE incidence per 1000 admissions before (phase 1, September 2018-August 2019) and after instituting the enhanced screening program (phase 2, September 2019-December 2020). RESULTS: A total of 13,962 (2,149 and 11,813 in each phase) were screened as indicated, among 49,490 inpatients, and monthly screening compliance increased from 18.3 to 93.5%. Compared to phase 1, the incidence of screening positive patients increased from 1.2 to 2.3 per 1,000 admissions (P = 0.005) during phase 2. The incidence of newly detected CPE patients was similar (3.1 vs. 3.4, P = 0.613) between two phases, but the incidence of hospital-onset CPE patients decreased (1.9 vs. 1.1, P = 0.018). A significant decrease was observed (0.5 to 0.1, P = 0.014) in the incidence of patients who first confirmed CPE positive through clinical cultures without a preceding positive screening. Compared to phase 1, the median exposure duration and number of CPE contacts were also markedly reduced in phase 2: 10.8 days vs. 1 day (P < 0.001) and 11 contacts vs. 1 contact (P < 0.001), respectively. During phase 2, 42 additional patients were identified by extending the admission screening indications (n = 30) and weekly in-ICU screening (n = 12). CONCLUSIONS: The enhanced screening program enabled us to identify previously unrecognized CPE patients in a rapid manner and curtailed a hospital-wide CPE outbreak. As CPE prevalence increases, risk factors for CPE colonization can broaden, and hospital prevention strategies should be tailored to the changing local CPE epidemiology.

Plasma Glial Fibrillary Acidic Protein and N-Terminal Pro B-Type Natriuretic Peptide: Potential Biomarkers to Differentiate Ischemic and Hemorrhagic Stroke.

Acute stroke management is critically time-sensitive and challenging. Blood-based biomarkers that can differentiate acute ischemic stroke (IS) from hemorrhagic stroke (HS) can greatly facilitate triage and early management. Admission blood samples obtained within 6 h of stroke symptom onset were analyzed in a derivation/validation design. GFAP, N-FL, NT-proBNP, copeptin, neutrophils (%), NLR, and platelet counts were assessed in the derivation cohort. The informative markers and the derived cutoff values were evaluated in the validation cohort. GFAP > 703 pg/mL showed a PPV of 76.9% and NPV of 95.8% for differentiating HS from IS. Multiple logistic regression analysis showed that GFAP and NT-proBNP were independent variables associated with IS and HS differentiation. Furthermore, applying a combined cutoff (GFAP > 703 pg/mL and NT-proBNP ≤ 125 pg/mL) for HS detection increased the PPV in both the derivation and validation cohorts (93.3% and 100%, respectively). GFAP and NT-proBNP levels were validated as informative blood biomarkers in the differentiation of IS and HS and using a combination of GFAP and NT-proBNP is suggested as a feasible strategy to differentiate stroke subtypes in the hyperacute phase of stroke.

Pre-Pandemic Distribution of Bacterial Species in Nasopharyngeal Swab Specimens from Pediatric and Adult Patients Detected via RT-PCR Using the Allplex Respiratory Panel.

Background: Recently, panel-based molecular diagnostics for the simultaneous detection of respiratory viruses and bacteria in nasopharyngeal swab (NPS) specimens have been highlighted. We identified the distribution of bacterial species in NPS specimens collected from pediatric and adult patients by employing RT-PCR (Allplex respiratory panel 4, RP4, Seegene) to estimate its applicability in a panel-based assay for detecting respiratory viruses. Methods: We used 271 and 173 NPS specimens from pediatric and adult patients, respectively. The results of the Allplex RP4 panel using NPS (NPS-RP4) from adult patients were compared with those of the Seeplex PneumoBacter ACE Detection assay (Seegene), which used sputum for testing (sputum-Seeplex). Results: A total of 147 specimens (54.2%) were positive for the NPS-RP4 panel in pediatric patients. There were 94, 77, 10, 3, 3, and 2 specimens that were positive for Haemophilus influenzae (HI), Streptococcus pneumoniae (SP), Mycoplasma pneumoniae (MP), Chlamydia pneumoniae (CP), Bordetella pertussis (BP), and B. parapertussis (BPP), respectively. Among 173 adult patients, 39 specimens (22.5%) were positive in the NPS-RP4. Thirty specimens were positive for HI, and 13 were positive for SP. One specimen tested positive for both MP and Legionella pneumophila (LP). CP, BP, and BPP results were all negative. However, 126 specimens (72.8%) had positive results with sputum-Seeplex (99 SP, 59 HI, three LP, and two MP), and the overall percentage of agreement between the two assays was 39.3% in the adult patients. Conclusions: Bacterial species in NPS from more than half of pediatric patients were detected. Performing the Allplex RP4 assay with NPS revealed additional respiratory bacteria that are not detected in current clinical practices, which do not include bacterial testing, demanding the use of sputum specimens. However, the use of NPS showed low agreement with standard assays using sputum in adult patients. Thus, more research is needed to develop a reliable RT-PCR method using NPS specimens in adult patients.

Contribution of proteoglycan osmotic swelling pressure to the compressive properties of articular cartilage.

The negatively charged proteoglycans (PG) provide compressive resistance to articular cartilage by means of their fixed charge density (FCD) and high osmotic pressure (π(PG)), and the collagen network (CN) provides the restraining forces to counterbalance π(PG). Our objectives in this work were to: 1), account for collagen intrafibrillar water when transforming biochemical measurements into a FCD-π(PG) relationship; 2), compute π(PG) and CN contributions to the compressive behavior of full-thickness cartilage during bovine growth (fetal, calf, and adult) and human adult aging (young and old); and 3), predict the effect of depth from the articular surface on π(PG) in human aging. Extrafibrillar FCD (FCD(EF)) and π(PG) increased with bovine growth due to an increase in CN concentration, whereas PG concentration was steady. This maturation-related increase was amplified by compression. With normal human aging, FCD(EF) and π(PG) decreased. The π(PG)-values were close to equilibrium stress (σ(EQ)) in all bovine and young human cartilage, but were only approximately half of σ(EQ) in old human cartilage. Depth-related variations in the strain, FCD(EF), π(PG), and CN stress profiles in human cartilage suggested a functional deterioration of the superficial layer with aging. These results suggest the utility of the FCD-π(PG) relationship for elucidating the contribution of matrix macromolecules to the biomechanical properties of cartilage.

One-step, aid-mediated method for modification of glass surfaces with N-hydroxysuccinimide esters and its application to the construction of microarrays for studies of biomolecular interactions.

Microarray technologies have received considerable attention owing to the fact that they serve as powerful tools for the high-throughput analysis of biomolecular interactions and the identification of bioactive substances that bind to biomolecules. Most of the current methods used to construct microarrays rely on the immobilization of substances on properly derivatized surfaces. Among various functional groups used for this purpose, the N-hydroxysuccinimide (NHS) ester group has been largely employed because it can be readily reacted with amine or hydrazide functionalities in substances of interest. However, the NHS ester group is usually introduced onto the surface of a glass slide by employing inconvenient and time-consuming multistep processes. In recent studies, we have developed an efficient, single step method for derivatization of glass surfaces with NHS ester groups that takes advantage of an acid-mediated reaction of NHS ester functionalized dimethallylsilanes with silanols on the glass surface. Conditions for the surface modification procedure that utilize TfOH rather than Sc(OTf)(3) were found to be superior. Protein and RNA-binding experiments show that glass surfaces modified by employing this method are suitable for efficient immobilization of various substances that are appended by amine, hydrazide, and alcohol functionalities. The microarrays, generated in this way, are applicable to procedures for rapid analysis of protein-protein, protein-glycan, protein-small molecule, and peptide-RNA interactions, as well as for profiling enzyme activities. The newly developed acid-mediated, glass surface modification method should be generally applicable to the preparation of various functional group-modified surfaces.

Detection of BRCA1/2 large genomic rearrangement including BRCA1 promoter-region deletions using next-generation sequencing.

BACKGROUND: Germline mutations in BRCA1 and BRCA2 (BRCA1/2) have been conventionally analyzed by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). Nowadays, next-generation sequencing (NGS) is increasingly being used in clinical genetics. The aim of this study was to evaluate the performance of NGS BRCA1/2 assays by comparing them with the conventional method. MATERIALS AND METHODS: We did BRCA1/2 NGS assays of 108 breast and/or ovarian cancer patients whose BRCA1/2 mutation had been previously analyzed by Sanger sequencing and MLPA using TruSeq Custom Amplicon Design AFP2. Single-nucleotide variations (SNVs) and small insertions or deletions (InDels) were evaluated. In addition, we analyzed large genomic rearrangements (LGRs) using a coverage-based algorithm as well as a revised BRCA1/2 NGS assay (BRCAaccuTest PLUS), which additionally covered a BRCA1 promoter region. RESULTS: The NGS BRCA1/2 assay detected all 20 SNVs and 21 small InDels in 56 patients. Among seven LGRs detected by MLPA, six exonic LGRs were well identified by both NGS BRCA1/2 assays. One pathogenic LGR, located on a BRCA1 promoter region, was successfully identified using revised BRCAaccuTestPLUS. CONCLUSIONS: These results indicated that an NGS BRCA1/2 assay could detect most LGRs including BRCA1 promoter-region deletion as well as SNVs and small InDels. Therefore, it was applicable to clinical BRCA1/2 mutation tests.

Clinical Validity of Next-Generation Sequencing Multi-Gene Panel Testing for Detecting Pathogenic Variants in Patients With Hereditary Breast-Ovarian Cancer Syndrome.

BACKGROUND: Hereditary breast and ovarian cancer syndrome (HBOC) is caused by pathogenic variants in BRCA and other cancer-related genes. We analyzed variants in BRCA gene and other cancer-related genes in HBOC patients to evaluate the clinical validity of next-generation sequencing (NGS) multi-gene panel testing. METHODS: The BRCA1/2 NGS testing was conducted for 262 HBOC patients. Multiplex ligation-dependent probe amplification and direct Sanger sequencing were performed for confirmation. Multi-gene panel testing was conducted for 120 patients who did not possess BRCA1/2 pathogenic variants but met the National Comprehensive Cancer Network criteria. RESULTS: Pathogenic variants in BRCA1/2 were detected in 30 HBOC patients (11.5%). Additionally, four out of the 120 patients possessed pathogenic variants by multi-gene panel testing (3.3%): MSH2 (c.256G>T, p.Glu86*), PMS2 (c.1687C>T, p.Arg563*), CHEK2 (c.546C>A, p.Tyr182*), and PALB2 (c.3351-1G>C). All the four patients had a family history of cancer. CONCLUSIONS: Multi-gene panel testing could be a significant screening tool for HBOC patients, especially for those with a family history of cancer.