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BACKGROUND: PCSK9 (proprotein convertase subtilisin-kexin type 9) chaperones the hepatic LDLR (low-density lipoprotein receptor) for lysosomal degradation, elevating serum LDL (low-density lipoprotein) cholesterol and promoting atherosclerotic heart disease. Though the major effect on the hepatic LDLR comes from secreted PCSK9, the details of PCSK9 reuptake into the hepatocyte remain unclear. In both tissue culture and animal models, HSPGs (heparan sulfate proteoglycans) on hepatocytes act as co-receptors to promote PCSK9 reuptake. We hypothesized that if this PCSK9:HSPG interaction is important in humans, disrupting it with unfractionated heparin (UFH) would acutely displace PCSK9 from the liver and increase plasma PCSK9. METHODS: We obtained remnant plasma samples from 160 subjects undergoing cardiac catheterization before and after administration of intravenous UFH. PCSK9 levels were determined using a commercial enzyme-linked immunosorbent assay. RESULTS: Median plasma PCSK9 was 113 ng/mL prior to UFH and 119 ng/mL afterward. This difference was not significant (P=0.83 [95% CI, -6.23 to 6.31 ng/mL]). Equivalence testing provided 95% confidence that UFH would not raise plasma PCSK9 by > 4.7%. Among all subgroups, only subjects with the lowest baseline PCSK9 concentrations exhibited a response to UFH (8.8% increase, adj. P=0.044). A modest correlation was observed between baseline plasma PCSK9 and the change in plasma PCSK9 due to UFH (RS=-0.3634; P<0.0001). CONCLUSIONS: Administration of UFH does not result in a clinically meaningful effect on circulating PCSK9 among an unselected population of humans. The results cast doubt on the clinical utility of disrupting the PCSK9:HSPG interaction as a general therapeutic strategy for PCSK9 inhibition. However, the observations suggest that in selected populations, disrupting the PCSK9:HSPG interaction could still affect PCSK9 reuptake and offer a therapeutic benefit.
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Heparina , Pró-Proteína Convertase 9 , Animais , Humanos , Pró-Proteína Convertase 9/metabolismo , Serina Endopeptidases , Pró-Proteína Convertases/metabolismo , Proteoglicanas de Heparan Sulfato , Receptores de LDL/metabolismo , LDL-Colesterol , SubtilisinasRESUMO
BACKGROUND: As global confirmed cases and deaths from coronavirus disease 2019 (COVID-19) surpass 100 and 2.2 million, respectively, quantifying the effects of the widespread treatment of remdesivir (GS-5734, Veklury) and the steroid dexamethasone is becoming increasingly important. Limited pharmacokinetic studies indicate that remdesivir concentrations in serum decrease quickly after dosing, so its primary serum metabolite GS-441524 may have more analytical utility. OBJECTIVES: We developed and validated a method to quantify remdesivir, its metabolite GS-441524 and dexamethasone in human serum. METHODS: We used LC-MS/MS and applied the method to 23 serum samples from seven patients with severe COVID-19. RESULTS: The method has limits of detection of 0.0375 ng/mL for remdesivir, 0.375 ng/mL for GS-441524 and 3.75 ng/mL for dexamethasone. We found low intra-patient variability, but significant inter-patient variability, in remdesivir, GS-441524 and dexamethasone levels. CONCLUSIONS: The significant inter-patient variability highlights the importance of therapeutic drug monitoring of COVID-19 patients and possible dose adjustment to achieve efficacy.
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Tratamento Farmacológico da COVID-19 , Espectrometria de Massas em Tandem , Adenosina/análogos & derivados , Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/uso terapêutico , Cromatografia Líquida , Dexametasona , Furanos , Humanos , Pirróis , SARS-CoV-2 , TriazinasRESUMO
BACKGROUND: Biomarkers have been widely explored for coronavirus disease 2019 diagnosis. Both viral RNA or antigens (Ag) in the respiratory system and antibodies (Ab) in blood are used to identify active infection, transmission risk, and immune response but have limitations. This study investigated the diagnostic utility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (N-Ag) in serum. METHODS: We retrospectively studied 208 randomly selected cases with SARS-CoV-2 infection confirmed by viral RNA test in swabs. N-Ag concentrations were measured in remnant serum samples, compared to viral RNA or Ab results, and correlated to electronic health records for clinical value evaluation. RESULTS: Serum N-Ag was detected during active infection as early as day 2 from symptom onset with a diagnostic sensitivity of 81.5%. Within 1 week of symptom onset, the diagnostic sensitivity and specificity reached 90.9% (95% CI, 85.1%-94.6%) and 98.3% (95% CI, 91.1%-99.9%), respectively. Moreover, serum N-Ag concentration closely correlated to disease severity, reflected by highest level of care, medical interventions, chest imaging, and the length of hospital stays. Longitudinal analysis revealed the simultaneous increase of Abs and decline of N-Ag. CONCLUSIONS: Serum N-Ag is a biomarker for SARS-CoV-2 acute infection with high diagnostic sensitivity and specificity compared to viral RNA in the respiratory system. There is a correlation between serum N-Ag concentrations and disease severity and an inverse relationship of N-Ag and Abs. The diagnostic value of serum N-Ag, as well as technical and practical advantages it could offer, may meet unsatisfied diagnostic and prognostic needs during the pandemic.
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Teste para COVID-19/métodos , COVID-19 , Proteínas do Nucleocapsídeo de Coronavírus/sangue , Anticorpos Antivirais/sangue , COVID-19/diagnóstico , Humanos , Proteínas do Nucleocapsídeo , Fosfoproteínas/sangue , RNA Viral , Estudos Retrospectivos , SARS-CoV-2 , Sensibilidade e EspecificidadeRESUMO
Laboratory tests for Clostridioides difficile infection (CDI) rely on the detection of free toxin or molecular detection of toxin genes. The Singulex Clarity C. diff toxins A/B assay is a rapid, automated, and ultrasensitive assay that detects C. difficile toxins A and B in stool. We compared CDI assays across two prospective multicenter studies to set a cutoff for the Clarity assay and to independently validate the performance compared with that of a cell culture cytotoxicity neutralization assay (CCCNA). The cutoff was set by two sites testing fresh samples from 897 subjects with suspected CDI and then validated at four sites testing fresh samples from 1,005 subjects with suspected CDI. CCCNA testing was performed at a centralized laboratory. Samples with discrepant results between the Clarity assay and CCCNA were retested with CCCNA when the Clarity result agreed with that of at least one comparator method; toxin enzyme immunoassays (EIA), glutamate dehydrogenase (GDH) detection, and PCR were performed on all samples. The cutoff for the Clarity assay was set at 12.0 pg/ml. Compared to results with CCCNA, the Clarity assay initially had 85.2% positive agreement and 92.4% negative agreement. However, when samples with discrepant results between the Clarity assay and CCCNA in the validation study were retested by CCCNA, 13/17 (76.5%) Clarity-negative but CCCNA-positive samples (Clarity+/CCCNA-) became CCCNA-, and 5/26 (19.2%) Clarity+/CCCNA- samples became CCCNA+, resulting in a 96.3% positive agreement and 93.0% negative agreement between Clarity and CCCNA results. The toxin EIA had 59.8% positive agreement with CCCNA. The Clarity assay was the most sensitive free-toxin immunoassay, capable of providing CDI diagnosis in a single-step solution. A different CCCNA result was reported for 42% of retested samples, increasing the positive agreement between Clarity and CCCNA from 85.2% to 96.3% and indicating the challenges of comparing free-toxin results to CCCNA results as a reference standard.
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Infecções por Clostridium/diagnóstico , Enterotoxinas/isolamento & purificação , Fezes/química , Imagem Individual de Molécula/métodos , Adolescente , Adulto , Idoso , Técnicas Bacteriológicas , Criança , Pré-Escolar , Clostridioides difficile , Testes Imunológicos de Citotoxicidade/métodos , Feminino , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Sensibilidade e Especificidade , Adulto JovemRESUMO
Background Biological variation studies have shown that the complete blood count (CBC) has narrow within-individual variation and wide group variation, indicating that the use of reference intervals (RIs) is challenging. The aim of this study was to examine differences in CBC RIs according to race/ethnicity in a multiethnic population at a hospital in San Francisco in hopes of improving the medical utility of CBC testing. Methods Subject data were obtained by screening CBC results from the medical records of outpatients meeting certain criteria who visited Zuckerberg San Francisco General Hospital from April 2017 to January 2018. From these records, sex- and race/ethnicity-specific CBC RIs were calculated as the 2.5th to 97.5th percentiles. Results From a total of 552 subjects, 47.9% were male (65 White, 50 Black, 71 Hispanic and 54 Asian) and 52.1% were female (51 White, 39 Black, 122 Hispanic and 72 Asian). The RIs of neutrophil, lymphocyte and eosinophil counts; and hemoglobin, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) showed significant differences (p<0.05) among the four racial/ethnic groups: neutrophil, lymphocyte and eosinophil counts; and MCHC in males, and hemoglobin, MCV, MCH and MCHC in females. Conclusions Race/ethnicity-specific CBC RIs should be taken into consideration in a multiethnic population to better interpret patient status and make progress toward precision medicine.
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Contagem de Células Sanguíneas/estatística & dados numéricos , Contagem de Células Sanguíneas/normas , Grupos Raciais/classificação , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valores de Referência , Estudos RetrospectivosRESUMO
BACKGROUND: Serum level of microRNA-122 (miR-122) has been reported as a sensitive diagnostic biomarker for detecting liver injury, comparable to the aminotransferases. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities are increased in other conditions, such as acute skeletal muscle injury (ASMI). We determined whether miR-122 is nonspecifically increased in patients suffering from ASMI. METHODS: We measured ALT, AST, creatine kinase (CK), and miR-122 in 3 groups: healthy controls (n = 24), patients with ASMI (total n = 29, 11 with recreational drug use and 18 without recreational drug use), and patients with acute liver injury (ALI; n = 14). RESULTS: Levels of ALT, AST, and CK increased 83%, 97%, and 100% for patients with ASMI and 100% for all 3 enzymes in ALI patients. In contrast, miR-122 increased in 34% of patients with ASMI (44.4% with recreational drug use and 18.2% without recreational drug use) and 100% of ALI patients. In 2 drug-induced liver injury cases, miR-122 increased about 12-24 hours before ALT and AST. CONCLUSION: Recreational drug misuse is associated with both rhabdomyolysis and drug-induced liver injury (DILI). The traditional liver function markers AST and ALT were nonspecifically increased in the majority of patients with ASMI. miR-122 is only increased in patients at risk for DILI and demonstrates superior specificity for liver injury.
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OBJECTIVE: Determine the COVID-19 seroconversion rate for patients with multiple myeloma receiving a COVID-19 vaccine. MATERIALS AND METHODS: After 45 patients received their second COVID-19 vaccine dose, their serum IgG antibodies were measured: 22 with monoclonal gammopathy (MG) of unknown significance, 3 with smoldering myeloma, 2 with light chain amyloidosis, and 18 with MG (9 in remission, 6 out of remission, and 3 with free light-chain gammopathy alone). A second serum specimen was retained for 16 patients with MG. Their antibody levels were compared to those of 78 uninfected healthy vaccinated control patients. RESULTS: Three patients with MG had low antibody levels on blood collected 98, 100, and 113 days after the initial vaccine dose (2 with MG of unknown significance and 1 with hypogammaglobulemia). The other 40 patients with MG (seroconversion rate 93%) and both patients with amyloidosis produced antibodies. Relative to days after vaccination, patients with MG had lower antibody levels than control patients. CONCLUSION: After receiving a COVID-19 vaccine, most patients with MG produce anti-SARS-CoV-2 antibodies comparable to levels in uninfected vaccinated healthy control patients.
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Amiloidose , COVID-19 , Mieloma Múltiplo , Paraproteinemias , Formação de Anticorpos , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Humanos , SARS-CoV-2RESUMO
OBJECTIVE: Three SARS-CoV-2 vaccinations and boosters are available. We determined whether solid organ transplant patients mounted an immune response to the vaccinations and whether the antibodies had neutralizing activity compared to healthcare worker controls and monoclonal gammopathy patients. METHODS: Remnant plasma was obtained from vaccinated solid organ transplant, allogeneic stem cell transplant, monoclonal gammopathy patients, and healthcare worker controls. Samples positive on a SARS-CoV-2 IgG assay (detects spike protein and nucleocapsid) were run on a SARS-CoV-2 in vitro neutralizing antibody assay and a nucleocapsid-specific SARS-CoV-2 IgG assay. RESULTS: Only 25% of solid organ transplant patients produced antibodies to SARS-CoV-2 vaccination. Of these, 90% had neutralizing activity against wild type virus, but reduced activity to the variants compared to monoclonal gammopathy patients and healthcare worker controls, particularly the delta variant, for which only 50% had neutralizing antibody activity. CONCLUSION: Solid organ transplant patients should consider protecting themselves against future SARS-CoV-2 infection.
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COVID-19 , Transplante de Órgãos , Paraproteinemias , Anticorpos Neutralizantes , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Humanos , Imunidade , Imunoglobulina G , SARS-CoV-2 , VacinaçãoRESUMO
Serosurveillance provides a unique opportunity to quantify the proportion of the population that has been exposed to pathogens. Here, we developed and piloted Serosurveillance for Continuous, ActionabLe Epidemiologic Intelligence of Transmission (SCALE-IT), a platform through which we systematically tested remnant samples from routine blood draws in two major hospital networks in San Francisco for SARS-CoV-2 antibodies during the early months of the pandemic. Importantly, SCALE-IT allows for algorithmic sample selection and rich data on covariates by leveraging electronic health record data. We estimated overall seroprevalence at 4.2%, corresponding to a case ascertainment rate of only 4.9%, and identified important heterogeneities by neighborhood, homelessness status, and race/ethnicity. Neighborhood seroprevalence estimates from SCALE-IT were comparable to local community-based surveys, while providing results encompassing the entire city that have been previously unavailable. Leveraging this hybrid serosurveillance approach has strong potential for application beyond this local context and for diseases other than SARS-CoV-2.
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COVID-19/epidemiologia , Registros Eletrônicos de Saúde/estatística & dados numéricos , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Pandemias , SARS-CoV-2/isolamento & purificação , São Francisco/epidemiologia , Estudos Soroepidemiológicos , Adulto JovemRESUMO
Serosurveillance provides a unique opportunity to quantify the proportion of the population that has been exposed to pathogens. Here, we developed and piloted Serosurveillance for Continuous, ActionabLe Epidemiologic Intelligence of Transmission (SCALE-IT), a platform through which we systematically tested remnant samples from routine blood draws in two major hospital networks in San Francisco for SARS-CoV-2 antibodies during the early months of the pandemic. Importantly, SCALE-IT allows for algorithmic sample selection and rich data on covariates by leveraging electronic medical record data. We estimated overall seroprevalence at 4.2%, corresponding to a case ascertainment rate of only 4.9%, and identified important heterogeneities by neighborhood, homelessness status, and race/ethnicity. Neighborhood seroprevalence estimates from SCALE-IT were comparable to local community-based surveys, while providing results encompassing the entire city that have been previously unavailable. Leveraging this hybrid serosurveillance approach has strong potential for application beyond this local context and for diseases other than SARS-CoV-2.