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The destruction of red cells during blood collection or with the processing of the sample continues to occur at a high rate, especially among emergency department (ED) patients. This can produce pre-analytical laboratory errors, particularly for potassium. We determined the incidence of hemolyzed samples and discuss the potential medical impact for hypokalemic patients who potassium level is artificially normal (pseudoeukalemia).Potassium results were obtained for a 6-month period. Using a measured hemolysis index (HI), hemolysis was present in 3.1 % for all potassium ordered (n=94,783) and 7.5 % for ED orders (n=22,770). Most of these samples were reported as having high normal result or were hyperkalemic. There were 22 hemolytic samples with a potassium of

This study aimed to evaluate and compare the performance of three anti-S and one anti-N assays that were available to the project in detecting antibody levels after three commonly used SARS-CoV-2 vaccines (Pfizer, Moderna, and Johnson & Johnson). It also aimed to assess the association of age, sex, race, ethnicity, vaccine timing, and vaccine side effects on antibody levels in a cohort of 827 individuals. In September 2021, 698 vaccinated individuals donated blood samples as part of the Association for Diagnostics & Laboratory Medicine (ADLM) COVID-19 Immunity Study. These individuals also participated in a comprehensive survey covering demographic information, vaccination status, and associated side effects. Additionally, 305 age- and gender-matched samples were obtained from the ADLM 2015 sample bank as pre-COVID-19-negative samples. All these samples underwent antibody level analysis using three anti-S assays, namely Beckman Access SARS-CoV-2 IgG (Beckman assay), Ortho Clinical Diagnostics VITROS Anti-SARS-CoV-2 IgG (Ortho assay), Siemens ADVIA Centaur SARS-CoV-2 IgG (Siemens assay), and one anti-N antibody assay: Bio-Rad Platelia SARS-CoV-2 Total Ab assay (BioRad assay). A total of 827 samples (580 COVID-19 samples and 247 pre-COVID-19 samples) received results for all four assays and underwent further analysis. Beckman, Ortho, and Siemens anti-S assays showed an overall sensitivity of 99.5%, 97.6%, and 96.9%, and specificity of 90%, 100%, and 99.6%, respectively. All three assays indicated 100% sensitivity for individuals who received the Moderna vaccine and boosters, and over 99% sensitivity for the Pfizer vaccine. Sensitivities varied from 70.4% (Siemens), 81.5% (Ortho), and 96.3% (Beckman) for individuals who received the Johnson & Johnson vaccine. BioRad anti-N assays demonstrated 46.2% sensitivity and 99.25% specificity based on results from individuals with self-reported infection. The highest median anti-S antibody levels were measured in individuals who received the Moderna vaccine, followed by Pfizer and then Johnson & Johnson vaccines. Higher anti-S antibody levels were significantly associated with younger age and closer proximity to the last vaccine dose but were not associated with gender, race, or ethnicity. Participants with higher anti-S levels experienced significantly more side effects as well as more severe side effects (e.g., muscle pain, chills, fever, and moderate limitations) (p < 0.05). Anti-N antibody levels only indicated a significant correlation with headache. This study indicated performance variations among different anti-S assays, both among themselves and when analyzing individuals with different SARS-CoV-2 vaccines. Caution should be exercised when conducting large-scale studies to ensure that the same platform and/or assays are used for the most effective interpretation of the data.

BACKGROUND: Liquid chromatography-high-resolution mass spectrometry (LC-HR-MS) has emerged as a powerful analytical technology for compound screening in clinical toxicology. To evaluate the potential of LC-HR-MS3 in detecting toxic natural products, a spectral library of 85 natural products (79 alkaloids) that contains both MS2 and MS3 mass spectra was constructed and used to identify the natural products. Samples were analyzed using an LC-HR-MS3 method and the generated data were matched to the spectral library to identify the natural products. METHODS: To test the performance of the LC-HR-MS3 method in different sample matrices, the 85 natural product standards were divided into three groups to separate structural isomers and avoid ion suppression effects caused by co-elution of multiple analytes. The grouped analytes were spiked into drug-free serum and drug-free urine to produce contrived clinical samples. RESULTS: The compound identification results of the 85 natural products in urine and serum samples were obtained. The match scores using both MS2 and MS3 mass spectra and those using only MS2 mass spectra were compared at 10 different analyte concentrations. The two types of data analysis provided identical identification results for the majority of the analytes (96% in serum, 92% in urine), whereas, for the remaining analytes, the MS2-MS3 tree data analysis had better performance in identifying them at lower concentrations. CONCLUSION: This study shows that in comparison to LC-HR-MS (MS2), LC-HR-MS3 can increase the performance in identification of a small group of the toxic natural products tested in serum and urine specimens.

BackgroundSubstance use increases risk of cardiovascular events, particularly among women with additional risk factors like housing instability. While multiple substance use is common among unstably housed individuals, relationships between multiple substance use and cardiovascular risk factors like blood pressure are not well characterized.MethodsWe conducted a cohort study between 2016 and 2019 to examine associations between multiple substance use and blood pressure in women experiencing homelessness and unstable housing. Participants completed six monthly visits including vital sign assessment, interview, and blood draw to assess toxicology-confirmed substance use (e.g., cocaine, alcohol, opioids) and cardiovascular health. We used linear mixed models to evaluate the outcomes of systolic and diastolic blood pressure (SBP; DBP).ResultsMean age was 51.6 years; 74 % were women of color. Prevalence of any substance use was 85 %; 63 % of participants used at least two substances at baseline. Adjusting for race, body mass index and cholesterol, cocaine was the only substance significantly associated with SBP (4.71 mmHg higher; 95 % CI 1.68, 7.74) and DBP (2.83 mmHg higher; 95 % CI 0.72, 4.94). Further analysis found no differences in SBP or DBP between those with concurrent use of other stimulants, depressants, or both with cocaine, compared to those who used cocaine only.ConclusionsCocaine was the only substance associated with higher SBP and DBP, even after accounting for simultaneous use of other substances. Along with interventions to address cocaine use, stimulant use screening during cardiovascular risk assessment and intensive blood pressure management may improve cardiovascular outcomes among women experiencing housing instability.

BackgroundPCSK9 (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.MethodsWe 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.ResultsMedian 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

ObjectivesThe objective of this prospective study was to investigate the role of adaptive immunity in response to SARS-CoV-2 vaccines.Design and methodsA cohort of 677 vaccinated individuals participated in a comprehensive survey of their vaccination status and associated side effects, and donated blood to evaluate their adaptive immune responses by neutralizing antibody (NAb) and T cell responses. The cohort then completed a follow-up survey to investigate the occurrence of breakthrough infections.ResultsNAb levels were the highest in participants vaccinated with Moderna, followed by Pfizer and Johnson & Johnson. NAb levels decreased with time after vaccination with Pfizer and Johnson & Johnson. T cell responses showed no significant difference among the different vaccines and remained stable up to 10 months after the study period for all vaccine types. In multivariate analyses, NAb responses (

Loss-of-function CYP2C19 variants are associated with increased cumulative ischemic outcomes warranting CYP2C19 genotyping prior to clopidogrel administration. TAILOR-PCI was an international, multicenter (40 sites), prospective, randomized trial comparing rapid point of care (POC) genotype-guided vs. conventional anti-platelet therapy. The performance of buccal-based rapid CYP2C19 genotyping performed by non-laboratory-trained staff in TAILOR-PCI was assessed. Pre-trial training and evaluation involved rapid genotyping of 373 oral samples, with 99.5% (371/373) concordance with Sanger sequencing. During TAILOR-PCI, 5302 patients undergoing PCI were randomized to POC rapid CYP2C19 *2, *3, and *17 genotyping versus no genotyping. At 12 months post-PCI, TaqMan genotyping determined 99.1% (2,364/2,385) concordance with the POC results, with 90.7-98.8% sensitivity and 99.2-99.6% specificity. In conclusion, non-laboratory personnel can be successfully trained for on-site instrument operation and POC rapid genotyping with analytical accuracy and precision across multiple international centers, thereby supporting POC genotyping in patient-care settings, such as the cardiac catheterization laboratory.Clinical Trial Registration: https://www.clinicalTrials.gov (Identifier: NCT01742117).

ContextCardiovascular disease (CVD) and heart failure (HF) are major causes of mortality in low-income populations and differ by sex. Risk assessment that incorporates cardiac biomarkers is common. However, research evaluating the utility of biomarkers rarely includes controlled substances, which may influence biomarker levels and thus influence CVD risk assessment.Materials and methodsWe identified the effects of multiple substances on soluble "suppression of tumorigenicity 2" (sST2), a biomarker of adverse cardiac remodelling, in 245 low-income women. Adjusting for CVD risk factors, we examined associations between substance use and sST2 over six monthly visits.ResultsMedian age was 53 years and 74% of participants were ethnic minority women. An sST2 level > 35 ng/mL (suggesting cardiac remodelling) during ≥1 study visit was observed in 44% of participants. In adjusted analysis, higher sST2 levels were significantly and positively associated with the presence of cocaine (Adjusted Linear Effect [ALE]:1.10; 95% CI:1.03-1.19), alcohol (ALE:1.10; 95% CI:1.04-1.17), heroin (ALE:1.25; 95% CI:1.10-1.43), and the interaction between heroin and fentanyl use.ConclusionResults suggest that the use of multiple substances influences the level of sST2, a biomarker often used to evaluate cardiovascular risk. Incorporating substance use alongside cardiac biomarkers may improve CVD risk assessment in vulnerable women.

IntroductionThe use of illicitly manufactured synthetic opioids, specifically fentanyl and its analogs, has escalated exponentially in the United States over the last decade. Due to the targeted nature of drug detection methods in clinical laboratories and the ever-evolving list of synthetic opioids of concern, alternative analytical approaches are needed.MethodsUsing the fentanyl analog screening (FAS) kit produced by the Centers for Disease Control and Prevention (CDC), we developed a liquid chromatography-high resolution mass spectrometry (LC-HRMS) synthetic opioid spectral library and data acquisition method using information dependent acquisition of product ion spectra. Chromatographic retention times, limits of detection and matrix effects, in urine and serum, for the synthetic opioids in the FAS kit (n = 150) were established. All urine and serum specimens sent to a clinical toxicology laboratory for comprehensive drug testing in 2019 (n = 856) and 2021 (n = 878) were analyzed with the FAS LC-HRMS library to determine the prevalence of fentanyl analogs and other synthetic opioids, retrospectively (2019) and prospectively (2021).ResultsThe limit of detection (LOD) of each opioid ranged from 1 to 10 ng/mL (median, 2.5 ng/mL) in urine and 0.25-2.5 ng/mL (median, 0.5 ng/mL) in serum. Matrix effects ranged from -79 % to 86 % (median, -37 %) for urine, following dilution and direct analysis, and -80 % to 400 % (median, 0 %) for serum, following protein precipitation. The prevalence of fentanyl/fentanyl analogs in serum samples increased slightly from 2019 to 2021 while it remained the same in urine. There were only 2 samples identified that contained a fentanyl analog without the co-occurrence of fentanyl or fentanyl metabolites. Analysis of the established MS/MS spectral library revealed characteristic fragmentation patterns in most fentanyl analogs, which can be used for structure elucidation and drug identification of future analogs.ConclusionsThe LC-HRMS method was capable of detecting fentanyl analogs in routine samples sent for comprehensive drug testing. The method can be adapted to accommodate testing needs for the evolving opioid epidemic.

ContextSARS-CoV-2 infects cells via the angiotensin converting enzyme 2 (ACE2) receptor, whose downstream effects "counterbalance" the classical renin angiotensin aldosterone system (RAAS).ObjectiveWe aimed to determine to what extent circulating RAAS biomarker levels differ in persons with and without COVID-19 throughout the disease course.MethodsWe measured classical (renin, aldosterone, aldosterone/renin ratio [ARR], Ang2, ACE activity) and nonclassical (ACE2, Ang1,7) RAAS biomarkers in hospitalized COVID-19 patients vs SARS-CoV-2 negative controls. We compared biomarker levels in cases with contemporaneous samples from control patients with upper respiratory symptoms and a negative SARS-CoV-2 PCR test. To assess RAAS biomarker changes during the course of COVID-19 hospitalization, we studied cases at 2 different times points ∼ 12 days apart. We employed age- and sex-adjusted generalized linear models and paired/unpaired t tests.ResultsMean age was 51 years for both cases (31% women) and controls (50% women). ARR was higher in the first sample among hospitalized COVID-19 patients vs controls (P = 0.02). ACE activity was lower among cases at their first sample vs controls (P =

BackgroundCardiovascular disease (CVD) and heart failure (HF) are disproportionately high in people living with HIV and differ by sex. Few CVD-related studies focus on drug use, yet it is common in low-income women living with HIV (WLWH) and increases cardiac dysfunction.SettingWe recruited unsheltered and unstably housed WLWH from San Francisco community venues to participate in a six-month cohort study investigating linkages between drug use, inflammation, and cardiac dysfunction.MethodsAdjusting for CVD risk factors, co-infections, medications, and menopause, we examined the effects of toxicology-confirmed drug use and inflammation (C-reactive protein, sCD14, sCD163 and sTNFR2) on levels of NT-proBNP, a biomarker of cardiac stretch and HF.ResultsAmong 74 WLWH, the median age was 53 years and 45 % were Black. At baseline, 72 % of participants had hypertension. Substances used included tobacco (65 %), cannabis (53 %), cocaine (49 %), methamphetamine (31 %), alcohol (28 %), and opioids (20 %). Factors significantly associated with NT-proBNP included cannabis use (Adjusted Relative Effect [ARE]: -39.6 %) and sTNFR2 (ARE: 65.5 %). Adjusting for heart failure and restricting analyses to virally suppressed persons did not diminish effects appreciably. Cannabis use was not significantly associated with sTNFR2 and did not change the association between sTNFR2 and NT-proBNP.ConclusionsAmong polysubstance-using WLWH, NT-proBNP levels signaling cardiac stretch were positively associated with sTNFR2, but 40 % lower in people who used cannabis. Whether results suggest that cardiovascular pathways associated with cannabis use mitigate cardiac stress and dysfunction independent of inflammation in WLWH who use multiple substances merits further investigation.

ObjectiveThree 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.MethodsRemnant 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.ResultsOnly 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.ConclusionSolid organ transplant patients should consider protecting themselves against future SARS-CoV-2 infection.

Pregnancy confers unique immune responses to infection and vaccination across gestation. To date, there are limited data comparing vaccine- and infection-induced neutralizing Abs (nAbs) against COVID-19 variants in mothers during pregnancy. We analyzed paired maternal and cord plasma samples from 60 pregnant individuals. Thirty women vaccinated with mRNA vaccines (from December 2020 through August 2021) were matched with 30 naturally infected women (from March 2020 through January 2021) by gestational age of exposure. Neutralization activity against the 5 SARS-CoV-2 spike sequences was measured by a SARS-CoV-2-pseudotyped spike virion assay. Effective nAbs against SARS-CoV-2 were present in maternal and cord plasma after both infection and vaccination. Compared with WT spike protein, these nAbs were less effective against the Delta and Mu spike variants. Vaccination during the third trimester induced higher cord-nAb levels at delivery than did infection during the third trimester. In contrast, vaccine-induced nAb levels were lower at the time of delivery compared with infection during the first trimester. The transfer ratio (cord nAb level divided by maternal nAb level) was greatest in mothers vaccinated in the second trimester. SARS-CoV-2 vaccination or infection in pregnancy elicits effective nAbs with differing neutralization kinetics that are influenced by gestational time of exposure.

BackgroundThe SARS-CoV-2 virus has mutated and evolved since the inception of the COVID-19 pandemic bringing into question the future effectiveness of current vaccines and antibody therapeutics. With evolution of the virus updated methods for the evaluation of the immune response in infected and vaccinated individuals are required to determine the durability of the immune response to SARS-CoV-2 variants.MethodsWe developed a multiplexed surrogate virus neutralization test (plex-sVNT) that simultaneously measures the ability of antibodies in serum to inhibit binding between angiotensin converting enzyme-2 (ACE2) and 7 SARS-CoV-2 trimeric spike protein variants, including wild type, B.1.1.7(α), B.1.351(β), P.1(γ), B.1.617.2(δ), B.1.617.1(κ), and B.1.429(ε). The assay was validated against a plaque reduction neutralization test (PRNT).We evaluated 170 samples from 97 COVID-19 patients and 281 samples from 188 individuals that received the Pfizer-BioNTech or Moderna mRNA vaccines.ResultsThe plex-sVNT demonstrated >96% concordance with PRNT. Antibody neutralization activity was significantly reduced for all SARS-CoV-2 variants compared to wild type in both the infected and vaccinated cohorts. There was a decline in overall antibody neutralization activity, within both cohorts, out to 5 months post infection or vaccination, with the rate of decline being more significant for the vaccinated.ConclusionsThe plex-sVNT provides a correlative measure to PRNT and a convenient approach for evaluating antibody neutralization against SARS-CoV-2 variants. Neutralization of SARS-CoV-2 variants is reduced compared to wild type and declines over the ensuing months after exposure or vaccination within each cohort, however it is still unknown what degree of neutralizing capacity is protective.

ObjectiveDetermine the COVID-19 seroconversion rate for patients with multiple myeloma receiving a COVID-19 vaccine.Materials and methodsAfter 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.ResultsThree 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.ConclusionAfter receiving a COVID-19 vaccine, most patients with MG produce anti-SARS-CoV-2 antibodies comparable to levels in uninfected vaccinated healthy control patients.

IntroductionViral infections have been implicated in the initiation of the autoimmune diseases. Recent reports suggest that a proportion of patients with COVID-19 develop severe disease with multiple organ injuries. We evaluated the relationship between COVID-19 severity, prevalence and persistence of antinuclear and other systemic and organ specific autoantibodies as well as SARS-CoV-2 infection specific anti-nucleocapsid (N) IgG antibodies and protective neutralizing antibody (Nab) levels.MethodsSamples from 119 COVID-19 patients categorized based on their level of care and 284 healthy subjects were tested for the presence and persistence of antinuclear and other systemic and organ specific autoantibodies as well as SARS-CoV-2 and neutralizing antibody levels.ResultsThe data shows significantly increased levels of anti RNP-A, anti-nucleocapsid and neutralizing antibody among patients receiving ICU care compared to non-ICU care. Furthermore, subjects receiving ICU care demonstrated significantly higher nucleocapsid IgG levels among the RNP-A positive cohort compared to RNP-A negative cohort. Notably, the expression of anti RNP-A antibodies is transient that reverts to non-reactive status between 20 and 60 days post symptom onset.ConclusionsCOVID-19 patients in ICU care exhibit significantly higher levels of transient RNP-A autoantibodies, anti-nucleocapsid, and SARS-CoV-2 neutralizing antibodies compared to patients in non-ICU care.

ImportanceMost healthcare institutions require employees to be vaccinated against SARS-CoV-2 and many also require at least one booster.ObjectiveWe determine the impact of vaccine type, demographics, and health conditions on COVID-19 vaccine side effects in healthcare professionals.DesignA COVID-19 immunity study was performed at the 2021 American Association for Clinical Chemistry Annual Scientific meeting. As part of this study, a REDCap survey with cascading questions was administered from September 9, 2021 to October 20, 2021. General questions included participant demographics, past and present health conditions, smoking, exercise, and medications. COVID-19 specific questions asked about SARS-CoV-2 vaccine status and type, vaccine-associated side effects after each dose including any boosters, previous infection with COVID-19, diagnostic testing performed, and type and severity symptoms of COVID-19.ResultsThere were 975 participants (47.1% male, median age of 50 years) who completed the survey. Pfizer was the most commonly administered vaccine (56.4%) followed by Moderna (32.0%) and Johnson & Johnson (7.1%). There were no significant differences in vaccine type received by age, health conditions, smoking, exercise, or type or number of prescription medications. Side effects were reported more frequently after second dose (e.g., Moderna or Pfizer) (54.1%) or single/only dose of Johnson & Johnson (47.8%). Males were significantly more likely to report no side effects (p < 0.001), while females were significantly more likely to report injection site reactions (p < 0.001), fatigue (p < 0.001), headache (p < 0.001), muscle pain (p < 0.001), chills (p = 0.001), fever (p = 0.007), and nausea (p < 0.001). There was a significant upward trend in participants reporting no side effects with increasing age (p < 0.001). There were no significant trends in side effects among different races, ethnicities, health conditions, medications, smoking status or exercise. In multivariate logistic regressions analyses, the second dose of Moderna was associated with a significantly higher risk of side effects than both the second dose of Pfizer and the single dose of Johnson & Johnson.Conclusions and relevanceYounger people, females, and those receiving the second dose of Moderna had more COVID-19 vaccine side effects that per self-report led to moderate to severe limitations. As reported in other studies, the increase in side effects from Moderna may be explained by higher viral mRNA concentrations but be associated with additional protective immunity.

BackgroundThe levels of circulating troponin are principally required in addition to electrocardiograms for the effective diagnosis of acute coronary syndrome. Current standard-of-care troponin assays provide a snapshot or momentary view of the levels due to the requirement of a blood draw. This modality further restricts the number of measurements given the clinical context of the patient. In this communication, we present the development and early validation of non-invasive transdermal monitoring of cardiac troponin-I to detect its elevated state.MethodsOur device relies on infrared spectroscopic detection of troponin-I through the dermis and is tested in stepwise laboratory, benchtop, and clinical studies. Patients were recruited with suspected acute coronary syndrome.ResultsWe demonstrate a significant correlation (r = 0.7774, P

Studies are needed to evaluate the safety and effectiveness of mRNA SARS-CoV-2 vaccination during pregnancy, and the levels of protection provided to their newborns through placental transfer of antibodies. Here, we evaluate the transplacental transfer of mRNA vaccine products and functional anti-SARS-CoV-2 antibodies during pregnancy and early infancy in a cohort of 20 individuals vaccinated during late pregnancy. We find no evidence of mRNA vaccine products in maternal blood, placenta tissue, or cord blood at delivery. However, we find time-dependent efficient transfer of IgG and neutralizing antibodies to the neonate that persists during early infancy. Additionally, using phage immunoprecipitation sequencing, we find a vaccine-specific signature of SARS-CoV-2 Spike protein epitope binding that is transplacentally transferred during pregnancy. Timing of vaccination during pregnancy is critical to ensure transplacental transfer of protective antibodies during early infancy.

BackgroundBiomarkers 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.MethodsWe 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.ResultsSerum 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.ConclusionsSerum 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.

Background Individuals infected with HIV have an increased risk of developing cardiovascular disease; yet, the underlying mechanisms remain unknown. Recent evidence has implicated the Tie-2 tyrosine kinase receptor system and its associated ligands ANG1 (angiopoietin 1) and ANG2 (angiopoietin 2) in maintaining vascular homeostasis. In the general population, lower ANG1 levels and higher ANG2 levels are strongly correlated with the development of cardiovascular disease. In this study, we aim to investigate the associations of HIV infection with angiopoietin levels and endothelial dysfunction. Methods and Results In this cross-sectional study, we compared measures of ANG1, ANG2, and endothelial dysfunction using flow-mediated vasodilation of the brachial artery in 39 untreated subjects infected with HIV, 47 treated subjects infected with HIV, and 46 uninfected subjects from the SCOPE (Observational Study of the Consequences of the Protease Inhibitor Era) cohort. Compared with uninfected controls, treated individuals infected with HIV had 53.1% lower mean ANG1 levels (P

The kinetics of IgG avidity maturation during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection was studied. The IgG avidity assay, using a novel label-free immunoassay technology, revealed a strong correlation between IgG avidity and days since symptom onset. Peak readings were significantly higher in severe than mild disease cases.

The kinetics of IgG avidity maturation during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection was studied. The IgG avidity assay, using a novel label-free immunoassay technology, revealed a strong correlation between IgG avidity and days since symptom onset. Peak readings were significantly higher in severe than mild disease cases.

ObjectiveTo investigate maternal immunoglobulins' (IgM, IgG) response to SARS-CoV-2 infection during pregnancy and IgG transplacental transfer, to characterise neonatal antibody response to SARS-CoV-2 infection, and to longitudinally follow actively and passively acquired antibodies in infants.DesignA prospective observational study.SettingPublic healthcare system in Santa Clara County (California, USA).ParticipantsWomen with symptomatic or asymptomatic SARS-CoV-2 infection during pregnancy and their infants were enrolled between 15 April 2020 and 31 March 2021.OutcomesSARS-CoV-2 serology analyses in the cord and maternal blood at delivery and longitudinally in infant blood between birth and 28 weeks of life.ResultsOf 145 mothers who tested positive for SARS-CoV-2 during pregnancy, 86 had symptomatic infections: 78 with mild-moderate symptoms, and 8 with severe-critical symptoms. The seropositivity rates of the mothers at delivery was 65% (95% CI 0.56% to 0.73%) and the cord blood was 58% (95% CI 0.49% to 0.66%). IgG levels significantly correlated between the maternal and cord blood (Rs=0.93, p

Methods designed to measure severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) humoral response include virus neutralization tests to determine antibody neutralization activity. For ease of use and universal applicability, surrogate virus neutralization tests (sVNTs) based on antibody-mediated blockage of molecular interactions have been proposed. A surrogate virus neutralization test was established on a label-free immunoassay platform (LF-sVNT). The LF-sVNT analyzes the binding ability of SARS-CoV-2 spike protein receptor-binding domain (RBD) to angiotensin-converting enzyme 2 (ACE2) after neutralizing RBD with antibodies in serum. The LF-sVNT neutralizing antibody titers (50% inhibitory concentration [IC50]) were determined from serum samples (n = 246) from coronavirus disease 2019 (COVID-19) patients (n = 113), as well as the IgG concentrations and the IgG avidity indices. Although there was variability in the kinetics of the IgG concentrations and neutralizing antibody titers between individuals, there was an initial rise, plateau, and then in some cases a gradual decline at later time points after 40 days after symptom onset. The IgG avidity indices, in the same cases, plateaued after an initial rise and did not show a decline. The LF-sVNT can be a valuable tool in research and clinical laboratories for the assessment of the presence of neutralizing antibodies to COVID-19. This study is the first to provide longitudinal neutralizing antibody titers beyond 200 days post-symptom onset. Despite the decline of IgG concentration and neutralizing antibody titer, IgG avidity index increases, reaches a plateau, and then remains constant up to 8 months postinfection. The decline of antibody neutralization activity can be attributed to the reduction in antibody quantity rather than the deterioration of antibody quality, as measured by antibody avidity.

BackgroundAs 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.ObjectivesWe developed and validated a method to quantify remdesivir, its metabolite GS-441524 and dexamethasone in human serum.MethodsWe used LC-MS/MS and applied the method to 23 serum samples from seven patients with severe COVID-19.ResultsThe 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.ConclusionsThe significant inter-patient variability highlights the importance of therapeutic drug monitoring of COVID-19 patients and possible dose adjustment to achieve efficacy.

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.

BackgroundSARS-CoV-2 infection can be detected indirectly by measuring the host immune response. For some viruses, antibody concentrations correlate with host protection and viral neutralization, but in rare cases, anti-viral antibodies can promote disease progression. Elucidation of the kinetics and magnitude of the SARS-CoV-2 antibody response is essential to understand the pathogenesis of COVID-19 and identify potential therapeutic targets.MethodsSera (n=533) from patients with RT-PCR confirmed COVID-19 (n=94 with acute infections and n=59 convalescent patients) were tested using a high-throughput quantitative IgM and IgG assay that detects antibodies to the spike protein receptor binding domain and nucleocapsid protein. Individual and serial samples covered the time of initial diagnosis, during the disease course, and following recovery. We evaluated antibody kinetics and correlation between magnitude of the response and disease severity.ResultsPatterns of SARS-CoV-2 antibody production varied considerably. Among 52 patients with 3 or more serial specimens, 44 (84.6%) and 42 (80.8%) had observed IgM and IgG seroconversion at a median of 8 and 10 days, respectively. Compared to those with milder disease, peak measurements were significantly higher for patients admitted to the intensive care unit for all time intervals between 6 and 20 days for IgM, and all intervals after 5 days for IgG.ConclusionsHigh sensitivity assays with a robust dynamic range provide a comprehensive picture of host antibody response to SARS-CoV-2. IgM and IgG responses were significantly higher in patients with severe than mild disease. These differences may affect strategies for seroprevalence studies, therapeutics and vaccine development.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can be detected indirectly by measuring the host immune response. For some viruses, antibody concentrations correlate with host protection and viral neutralization, but in rare cases, antiviral antibodies can promote disease progression. Elucidation of the kinetics and magnitude of the SARS-CoV-2 antibody response is essential to understand the pathogenesis of coronavirus disease 2019 (COVID-19) and identify potential therapeutic targets. METHODS: Sera (n = 533) from patients with real-time polymerase chain reaction-confirmed COVID-19 (n = 94 with acute infections and n = 59 convalescent patients) were tested using a high-throughput quantitative immunoglobulin M (IgM) and immunoglobulin G (IgG) assay that detects antibodies to the spike protein receptor binding domain and nucleocapsid protein. Individual and serial samples covered the time of initial diagnosis, during the disease course, and following recovery. We evaluated antibody kinetics and correlation between magnitude of the response and disease severity. RESULTS: Patterns of SARS-CoV-2 antibody production varied considerably. Among 52 patients with 3 or more serial specimens, 44 (84.6%) and 42 (80.8%) had observed IgM and IgG seroconversion at a median of 8 and 10 days, respectively. Compared to those with milder disease, peak measurements were significantly higher for patients admitted to the intensive care unit for all time intervals between 6 and 20 days for IgM, and all intervals after 5 days for IgG. CONCLUSIONS: High-sensitivity assays with a robust dynamic range provide a comprehensive picture of host antibody response to SARS-CoV-2. IgM and IgG responses were significantly higher in patients with severe than mild disease. These differences may affect strategies for seroprevalence studies, therapeutics, and vaccine development.

BackgroundData regarding symptoms in the lactating mother-infant dyad and their immune response to COVID-19 mRNA vaccination during lactation are needed to inform vaccination guidelines.MethodsFrom a prospective cohort of 50 lactating individuals who received mRNA-based vaccines for COVID-19 (mRNA-1273 and BNT162b2), blood and milk samples were collected prior to first vaccination dose, immediately prior to 2nd dose, and 4-10 weeks after 2nd dose. Symptoms in mother and infant were assessed by detailed questionnaires. Anti-SARS-CoV-2 antibody levels in blood and milk were measured by Pylon 3D automated immunoassay and ELISA. In addition, vaccine-related PEGylated proteins in milk were measured by ELISA. Blood samples were collected from a subset of infants whose mothers received the vaccine during lactation (4-15 weeks after mothers' 2nd dose).ResultsNo severe maternal or infant adverse events were reported in this cohort. Two mothers and two infants were diagnosed with COVID-19 during the study period before achieving full immune response. PEGylated proteins were not found at significant levels in milk after vaccination. After vaccination, levels of anti-SARS-CoV-2 IgG and IgM significantly increased in maternal plasma and there was significant transfer of anti-SARS-CoV-2-Receptor Binding Domain (anti-RBD) IgA and IgG antibodies to milk. Milk IgA levels after the 2nd dose were negatively associated with infant age. Anti-SARS-CoV-2 IgG antibodies were not detected in the plasma of infants whose mothers were vaccinated during lactation.ConclusionsCOVID-19 mRNA vaccines generate robust immune responses in plasma and milk of lactating individuals without severe adverse events reported.

IntroductionThe use of controlled substances like cocaine increases the risk of cardiovascular disease (CVD) and myocardial infarction (MI). However, outside of alcohol and tobacco, substance use is not included in CVD risk assessment tools. We identified the effects of using multiple substances (nicotine/cotinine, cannabis, alcohol, cocaine, methamphetamine, heroin and other opioids) on cardiac injury measured by high-sensitivity troponin (hsTnI) in homeless and unstably housed women.MethodsWe recruited 245 homeless and unstably housed women from shelters, free meal programs and street encampments. Participants completed six monthly study visits. Adjusting for traditional CVD risk factors, we examined longitudinal associations between substance use and hsTnI.ResultsMedian participant age was 53 years and 74 % were ethnic minority women. At baseline, 76 % of participants had hypertension, 31 % were HIV-positive, 8% had a history of a prior MI and 12 % of prior stroke. The most commonly used substances were cotinine/nicotine (80 %), cannabis (68 %) and cocaine (66 %). HsTnI exceeding the 99th percentile (14.7 ng/L) - a level high enough to signal possible MI - was observed in 14 participants during >1 study visit (6%). In adjusted analysis, cocaethylene and fentanyl were significantly associated with higher hsTnI levels.ConclusionsFentanyl use and the co-use of cocaine and alcohol are associated with myocardial injury, suggesting that the use of these substances may act as long-term cardiac insults. Whether risk counseling on these specific substances and/or including their use in CVD risk stratification would improve CVD outcomes in populations where substance use is high merits further investigation.

ObjectivesTo develop: (1) two validated risk prediction models for coronavirus disease-2019 (COVID-19) positivity using readily available parameters in a general hospital setting; (2) nomograms and probabilities to allow clinical utilisation.MethodsPatients with and without COVID-19 were included from 4 Hong Kong hospitals. The database was randomly split into 2:1: for model development database (n = 895) and validation database (n = 435). Multivariable logistic regression was utilised for model creation and validated with the Hosmer-Lemeshow (H-L) test and calibration plot. Nomograms and probabilities set at 0.1, 0.2, 0.4 and 0.6 were calculated to determine sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV).ResultsA total of 1330 patients (mean age 58.2 ± 24.5 years; 50.7% males; 296 COVID-19 positive) were recruited. The first prediction model developed had age, total white blood cell count, chest x-ray appearances and contact history as significant predictors (AUC = 0.911 [CI = 0.880-0.941]). The second model developed has the same variables except contact history (AUC = 0.880 [CI = 0.844-0.916]). Both were externally validated on the H-L test (p = 0.781 and 0.155, respectively) and calibration plot. Models were converted to nomograms. Lower probabilities give higher sensitivity and NPV; higher probabilities give higher specificity and PPV.ConclusionTwo simple-to-use validated nomograms were developed with excellent AUCs based on readily available parameters and can be considered for clinical utilisation.

Appropriate use and interpretation of serological tests for assessments of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure, infection and potential immunity require accurate data on assay performance. We conducted a head-to-head evaluation of ten point-of-care-style lateral flow assays (LFAs) and two laboratory-based enzyme-linked immunosorbent assays to detect anti-SARS-CoV-2 IgM and IgG antibodies in 5-d time intervals from symptom onset and studied the specificity of each assay in pre-coronavirus disease 2019 specimens. The percent of seropositive individuals increased with time, peaking in the latest time interval tested (>20 d after symptom onset). Test specificity ranged from 84.3% to 100.0% and was predominantly affected by variability in IgM results. LFA specificity could be increased by considering weak bands as negative, but this decreased detection of antibodies (sensitivity) in a subset of SARS-CoV-2 real-time PCR-positive cases. Our results underline the importance of seropositivity threshold determination and reader training for reliable LFA deployment. Although there was no standout serological assay, four tests achieved more than 80% positivity at later time points tested and more than 95% specificity.

BackgroundThe accuracy and speed by which acute myocardial infarction (AMI) is excluded are an important determinant of emergency department (ED) length of stay and resource utilization. While high-sensitivity troponin I (hsTnI) >99th percentile (upper reference level [URL]) represents a "rule-in" cutpoint, our purpose was to evaluate the ability of the Beckman Coulter hsTnI assay, using various level-of-quantification (LoQ) cutpoints, to rule out AMI within 3 hours of ED presentation in suspected acute coronary syndrome (ACS) patients.MethodsThis multicenter evaluation enrolled adults with >5 minutes of ACS symptoms and an electrocardiogram obtained per standard care. Exclusions were ST-segment elevation or chronic hemodialysis. After informed consent was obtained, blood samples were collected in heparin at ED admission (baseline), ≥1 to 3, ≥3 to 6, and ≥6 to 9 hours postadmission. Samples were processed and stored at -20°C within 1 hour and were tested at three independent clinical laboratories on an immunoassay system (DxI 800, Beckman Coulter). Analytic cutpoints were the URL of 17.9 ng/L and two LoQ cutpoints, defined as the 10 and 20% coefficient of variation (5.6 and 2.3 ng/L, respectively). A criterion standard MI diagnosis was adjudicated by an independent endpoint committee, blinded to hsTnI, and using the universal definition of MI.ResultsOf 1,049 patients meeting the entry criteria, and with baseline and 1- to 3-hour hsTnI results, 117 (11.2%) had an adjudicated final diagnosis of AMI. AMI patients were typically older, with more cardiovascular risk factors. Median (IQR) presentation time was 4 (1.6-16.0) hours after symptom onset, although AMI patients presented ~0.5 hour earlier than non-AMI. Enrollment and first blood draw occurred at a mean of ~1 hour after arrival. To evaluate the assay's rule-out performance, patients with any hsTnI > URL were considered high risk and were excluded. The remaining population (n = 829) was divided into four LoQ relative categories: both hsTnI  LoQ (Lo-Hi cohort); first > LoQ and second  LoQ (Hi-Hi cohort). In patients with any hsTnI result 3 hours after the onset of suspected ACS symptoms, with at least two Beckman Coulter Access hsTnI LoQ had inadequate sensitivity and NPV.