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BACKGROUND: Detailed understanding of the immune response to severe acute respiratory syndrome coronavirus (SARS-CoV)-2, the cause of coronavirus disease 2019 (CO-VID-19) has been hampered by a lack of quantitative antibody assays. OBJECTIVE: The objective was to develop a quantitative assay for IgG to SARS-CoV-2 proteins that could be implemented in clinical and research laboratories. METHODS: The biotin-streptavidin technique was used to conjugate SARS-CoV-2 spike receptor-binding domain (RBD) or nucleocapsid protein to the solid phase of the ImmunoCAP. Plasma and serum samples from patients hospitalized with COVID-19 (n = 60) and samples from donors banked before the emergence of COVID-19 (n = 109) were used in the assay. SARS-CoV-2 IgG levels were followed longitudinally in a subset of samples and were related to total IgG and IgG to reference antigens using an ImmunoCAP 250 platform. RESULTS: At a cutoff of 2.5 µg/mL, the assay demonstrated sensitivity and specificity exceeding 95% for IgG to both SARS-CoV-2 proteins. Among 36 patients evaluated in a post-hospital follow-up clinic, median levels of IgG to spike-RBD and nucleocapsid were 34.7 µg/mL (IQR 18-52) and 24.5 µg/mL (IQR 9-59), respectively. Among 17 patients with longitudinal samples, there was a wide variation in the magnitude of IgG responses, but generally the response to spike-RBD and to nucleocapsid occurred in parallel, with peak levels approaching 100 µg/mL, or 1% of total IgG. CONCLUSIONS: We have described a quantitative assay to measure IgG to SARS-CoV-2 that could be used in clinical and research laboratories and implemented at scale. The assay can easily be adapted to measure IgG to mutated COVID-19 proteins, has good performance characteristics, and has a readout in standardized units.
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Anticorpos Antivirais/sangue , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , COVID-19/imunologia , Imunoglobulina G/sangue , SARS-CoV-2/imunologia , Biomarcadores/sangue , COVID-19/virologia , Humanos , Estudos Longitudinais , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: One-third of all healthcare dollars are wasted, primarily in the form of clinician-ordered unnecessary diagnostic tests and treatments. Medical education has likely played a central role in the creation and perpetuation of this problem. We aimed to create a curriculum for medical students to promote their contribution to high-value care conversations in the clinical environment. METHODS: At a large university medical center between March 2017 and February 2018, we implemented a 3-phase curriculum combining multimodal educational initiatives with individual and group reflection for third-year medical students during their 12-week long Internal Medicine clerkship rotation. Students were asked to identify examples of clinical decision making that lacked attention to high-value care, propose solutions to the identified situation, and pinpoint barriers to the implementation of effective solutions using a structured reflection framework and then participate in a debrief debate with fellow students. To assess the curriculum, reflective narratives were coded by frequency and codes were compared with one another and with relevant high-value care literature to identify patterns and themes. RESULTS: In total, 151 medical students participated in phase 1 and 119 in phase 3. For phase 2, 126 reflective narratives (94.7% participation rate) comprised 226 problems, 280 solutions, and 179 barriers. CONCLUSIONS: When provided appropriate resources, medical students are able to identify relevant examples of low-value care, downstream solutions, and barriers to implementation through a structured reflection curriculum comprising written narratives and in-person debate.
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Currículo/tendências , Processos Grupais , Meditação/psicologia , Educação de Graduação em Medicina/métodos , Humanos , Meditação/métodosAssuntos
Testes Genéticos , Triagem Neonatal , Humanos , Recém-Nascido , Genômica , Promoção da SaúdeRESUMO
BACKGROUND: Cystic fibrosis (CF) is a complex autosomal recessive disease that continues to present unique diagnostic challenges. Because CF was first described in 1938, there has been a substantial growth of genetic and phenotypic information about the disorder. During the past few years, as more evidence has become available, a consortium of international experts determined that the 2008 guidelines from the CF Foundation needed to be reviewed and updated. CONTENT: The goal of this review is to highlight the latest advances in CF multidisciplinary care, together with the recent updates to the 2017 CF Foundation diagnostic guidelines. SUMMARY: Data from newborn screening programs, patient registries, clinical databases, and functional research have led to a better understanding of the CF transmembrane conductance regulator (CFTR) gene. Recent consensus guidelines have provided recommendations for clinicians and laboratorians to better assist with interpretation of disease status and related CF mutations. The highly recommended Clinical and Functional Translation of CFTR project should be the first resource in the evaluation of disease severity for CF mutations. Screen-positive newborns and patients with high clinical suspicion for CF are always recommended to undergo confirmatory sweat chloride testing with interpretations based on updated reference intervals. Every patient diagnosed with CF should receive genotyping, as novel molecular therapies are becoming standard of practice. The future of CF management must consider healthcare system disparities as CF transitions from a historically childhood disease to a predominantly adult epidemic.
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Fibrose Cística/diagnóstico , Fibrose Cística/terapia , Genoma Humano , Cloretos/metabolismo , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Bases de Dados Genéticas , Edição de Genes , Humanos , Recém-Nascido , Mutação , Triagem Neonatal , Guias de Prática Clínica como Assunto , Suor/metabolismoRESUMO
Prothrombin (FII) is activated to α-thrombin (IIa) by prothrombinase. Prothrombinase is composed of a catalytic subunit, factor Xa (fXa), and a regulatory subunit, factor Va (fVa), assembled on a membrane surface in the presence of divalent metal ions. We constructed, expressed, and purified several mutated recombinant FII (rFII) molecules within the previously determined fVa-dependent binding site for fXa (amino acid region 473-487 of FII). rFII molecules bearing overlapping deletions within this significant region first established the minimal stretch of amino acids required for the fVa-dependent recognition exosite for fXa in prothrombinase within the amino acid sequence Ser(478)-Val(479)-Leu(480)-Gln(481)-Val(482). Single, double, and triple point mutations within this stretch of rFII allowed for the identification of Leu(480) and Gln(481) as the two essential amino acids responsible for the enhanced activation of FII by prothrombinase. Unanticipated results demonstrated that although recombinant wild type α-thrombin and rIIa(S478A) were able to induce clotting and activate factor V and factor VIII with rates similar to the plasma-derived molecule, rIIa(SLQâAAA) with mutations S478A/L480A/Q481A was deficient in clotting activity and unable to efficiently activate the pro-cofactors. This molecule was also impaired in protein C activation. Similar results were obtained with rIIa(ΔSLQ) (where rIIa(ΔSLQ) is recombinant human α-thrombin with amino acids Ser(478)/Leu(480)/Gln(481) deleted). These data provide new evidence demonstrating that amino acid sequence Leu(480)-Gln(481): 1) is crucial for proper recognition of the fVa-dependent site(s) for fXa within prothrombinase on FII, required for efficient initial cleavage of FII at Arg(320); and 2) is compulsory for appropriate tethering of fV, fVIII, and protein C required for their timely activation by IIa.
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Glutamina/metabolismo , Leucina/metabolismo , Protrombina/química , Protrombina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Fator Va/genética , Fator Va/metabolismo , Fator Xa/genética , Fator Xa/metabolismo , Glutamina/genética , Humanos , Leucina/genética , Dados de Sequência Molecular , Proteína C/genética , Proteína C/metabolismo , Processamento de Proteína Pós-Traducional , Protrombina/genética , Tromboplastina/genética , Tromboplastina/metabolismoRESUMO
Single chain factor V (fV) circulates as an Mr 330,000 quiescent pro-cofactor. Removal of the B domain and generation of factor Va (fVa) are vital for procoagulant activity. We investigated the role of the basic amino acid region 1000-1008 within the B domain of fV by constructing a recombinant mutant fV molecule with all activation cleavage sites (Arg(709)/Arg(1018)/Arg(1545)) mutated to glutamine (fV(Q3)), a mutant fV molecule with region 1000-1008 deleted (fV(ΔB9)), and a mutant fV molecule containing the same deletion with activation cleavage sites changed to glutamine (fV(ΔB9/Q3)). The recombinant molecules along with wild type fV (fV(WT)) were transiently expressed in COS-7L cells, purified, and assessed for their ability to bind factor Xa (fXa) prior to and following incubation with thrombin. The data showed that fV(Q3) was severely impaired in its interaction with fXa before and after incubation with thrombin. In contrast, KD(app) values for fV(ΔB9) (0.9 nM), fVa(ΔB9) (0.4 nM), and fV(ΔB9/Q3) (0.7 nM) were similar to the affinity of fVa(WT) for fXa (0.3 nM). Two-stage clotting assays revealed that although fV(Q3) was deficient in its clotting activity, fV(ΔB9/Q3) had clotting activity comparable with fVa(WT). The kcat value of prothrombinase assembled with fV(ΔB9/Q3) was minimally affected, whereas the Km value of the reaction was increased 57-fold compared with the Km value obtained with prothrombinase assembled with fVa(WT). These findings strongly suggest that amino acid region 1000-1008 of fV is a regulatory sequence protecting the organisms from spontaneous binding to fXa and unnecessary prothrombinase complex formation, which in turn results in catastrophic physiological consequences.
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Fator V/química , Sequência de Aminoácidos , Animais , Células COS , Bovinos , Chlorocebus aethiops , Fator V/genética , Fator V/metabolismo , Fator Xa/química , Fator Xa/genética , Fator Xa/metabolismo , Humanos , Camundongos , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Trombina/química , Trombina/genética , Trombina/metabolismoRESUMO
BACKGROUND: Biannual instrument-correlation studies are required for nonwaived assays performed on multiple instruments. OBJECTIVE: To determine the feasibility of using College of American Pathologists (CAP) Quality Cross Check-Chemistry and Therapeutic Drug Monitoring (CZQ) to assess instrument correlations among multiple analyzers, analyzer models, and Clinical Laboratory Improvement Amendments (CLIA) licenses for 55 unique analytes. METHODS: Instrument correlation studies were performed on 9 Abbott ARCHITECT instruments (c4000 [n = 4], c8000 [n = 2], and c16000 [n = 3]) over 3 CLIA licenses using CZQ materials. The mean (SD) values, concentration difference, percent bias, and peer data for each individual level of CZQ were determined for each individual analyzer. Acceptable concentration and percentage for each analyte were set using criteria from CAP or other reputable sources such as the American Association of Bioanalysts or the Royal College of Pathologists of Australasia. Peer data were provided by CAP with the CZQ kit. RESULTS: Correlations using CZQ materials showed that 94.5% of assays studied were within the acceptability criteria by percent bias only and 98.2% were within acceptability criteria by concentration difference. CONCLUSIONS: The use of CZQ provides support to standardized correlation studies among instruments within and across separate CLIA licenses. However, widespread adoption of CZQ may be limited due to concerns regarding matrix effects, analyte ranges, and ease of data analysis.
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Monitoramento de Medicamentos , Humanos , Monitoramento de Medicamentos/métodos , Monitoramento de Medicamentos/instrumentação , Monitoramento de Medicamentos/normas , Controle de Qualidade , Estados Unidos , PatologistasRESUMO
OBJECTIVES: The goal of this study was to assess hospital compliance with federal price transparency mandates and barriers to pricing information in Tennessee. METHODS: All hospitals websites were queried for gross, cash, and BlueCross BlueShield of Tennessee prices for 8 high-frequency laboratory tests in 2 Centers for Medicare & Medicaid Services-mandated pricing sources: (1) a machine-readable file of all available services and (2) a consumer-friendly display of 300 shoppable services. Barriers, including click counts, data availability, and intrahospital price discrepancies, were noted. RESULTS: Of the 145 Tennessee hospitals assessed, 97.2% were noncompliant with the Centers for Medicare & Medicaid Services final rule. Subanalysis of available machine-readable files, price estimators, and shoppable services files demonstrated 49.6%, 95.1%, and 78.6% noncompliance, respectively. Barriers to pricing information included requiring protected health information (55.9%), missing at least 1 pricing source (7.6%), having no pricing sources available (6.2%), and involving more than 3 clicks to access the cash price in machine-readable files (54.1%) and price estimators (68.6%.) Average intrahospital discrepancy for basic metabolic panel cash prices across pricing sources was $101.30 (range, $0-1012.40). CONCLUSIONS: Our study showed high levels of noncompliance with price transparency laws, inconsistent and inaccessible pricing, and continued challenges facing patients in Tennessee.
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BACKGROUND: Immunoassays are important for routine clinical testing and medical diagnosis. However, they are limited by cross-reactivity especially at low analyte concentrations. There is a critical need to investigate compounds that can interfere with immunoassays. Herein, we describe the identification of canrenone, a spironolactone metabolite that falsely increases progesterone concentrations on the Abbott Architect i2000 Immunoassay. METHODS: Serum samples and assay diluents were spiked with spironolactone or canrenone and progesterone concentrations were measured on the Architect i2000 and Immulite XPi immunoassay platforms. Blood samples from patients taking spironolactone were analyzed with liquid chromatography-tandem mass spectrometry to evaluate the intrinsic response of progesterone concentrations to the presence of canrenone. RESULTS: We measured approximately 10-fold higher progesterone concentrations on the Abbott Architect i2000 compared to reference immunoassay analyzers (Siemens Immulite XPi and Roche Cobas e601/602), suggesting an analytical error which is unique to the Architect i2000 antibody and/or assay conditions. By measuring serum progesterone after addition of spironolactone or canrenone to serum samples, we found that canrenone falsely increased progesterone on the Architect i2000 immunoassay. However, this interference was more pronounced at low serum progesterone concentrations. Moreover, a strong positive correlation was seen between canrenone and measured serum progesterone concentrations. CONCLUSIONS: Our investigations are important for individuals who require progesterone measurements using the Architect i2000 immunoassay, especially because it is unlikely for clinicians to order canrenone measurements alongside progesterone measurements for individuals taking spironolactone. Further research is needed to determine whether canrenone can influence progesterone measurements on other immunoassay systems.