...recently, there existed minimal guidance on the use of LC-MS for clinical diagnostics. An LDT is defined as an in vitro diagnostic test that is designed, manufactured, validated, and used within a ...single laboratory. ...no 2 LC-MS methods for the same analyte are alike.
Deaths from opioid use are increasing in the US, with a growing proportion due to synthetic opioids. Until 2013, sporadic outbreaks of fentanyl and fentanyl analogs contaminating the heroin supply ...caused some deaths in heroin users. Since then, fentanyl has caused deaths in every state and fentanyl and its analogs have completely infiltrated the North American heroin supply. In 2014, the first illicit pills containing fentanyl, fentanyl analogs, and other novel synthetic opioids such as U-47700 were detected. These pills, which look like known opioids or benzodiazepines, have introduced synthetic opioids to more unsuspecting customers. As soon as these drugs are regulated by various countries, new compounds quickly appear on the market, making detection difficult and the number of cases likely underreported. Standard targeted analytical techniques such as GC-MS (gas chromatography mass spectrometry) and LC-MS/MS (liquid chromatography tandem mass spectrometry) can detect these drugs, but novel compound identification is aided by nontargeted testing with LC-HRMS (liquid chromatography high resolution mass spectrometry). Fentanyl, fentanyl analogs and other novel synthetic opioids are all full agonists of varying potencies at the μ-opioid receptor, leading to typical clinical effects of miosis and respiratory and central nervous system depression. Due to their high affinity for μ-opioid receptors, larger doses of naloxone are required to reverse the effects than are commonly used. Synthetic opioids are an increasingly major public health threat requiring vigilance from multiple fields including law enforcement, government agencies, clinical chemists, pharmacists, and physicians, to name a few, in order to stem its tide.
This article is part of the Special Issue entitled ‘Designer Drugs and Legal Highs.’
•Fentanyl use is increasing due to illicit pills, ease of purchase, and contamination of the North American heroin supply.•As soon as they are regulated, new synthetic opioids appear, making detection difficult and number of cases underreported.•Synthetic opioids are μ-opioid agonists of varying potencies, leading to respiratory and central nervous system depression.•Due to high μ-opioid affinity, larger naloxone doses are required to reverse drug effects than are commonly used.
For mass spectrometry (MS) testing in the clinical laboratory, postimplementation monitoring for quality is just as important as method development and validation but often receives less attention. ...Quality-assurance monitoring for liquid chromatography-tandem MS (LC-MS/MS) testing should be proactive rather than reactive and should monitor the entire testing process. An LC-MS/MS quality-assurance plan should cover overall batch review parameters, individual peak review parameters, system and reagent changes, and assessment of long-term accuracy. This article discusses Clinical Laboratory Improvement Amendments' regulations as they apply to LC-MS/MS-based testing and reviews available guidelines for LC-MS/MS quality assurance and postimplementation monitoring.
Abstract
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.
Abstract
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.
•Demonstration of the utility of real-time comprehensive drug testing for acute care pediatric cases.•Results, when negative, rule-out toxic exposure, allowing for investigation into alternative ...causes of symptoms.•Results, when positive, lead to appropriate medical and social interventions.•Results inconsistent with medical history were determined to be due to accidental ingestion of prescribed medication or an illicit substance.
Drug testing typically follows a one-size-fits-all approach that is inadequate in some clinical scenarios, such as child maltreatment, neglect, and unintentional drug exposure. Results from immunoassay-based testing, which are non-specific, insensitive, and far from comprehensive, can lead to unintended consequences for children and their families.
The objective of this retrospective case series study is to evaluate the utility of real-time (0–1 day) comprehensive drug testing as an alternative to immunoassay-based testing in the pediatric acute care setting.
Comprehensive drug testing results obtained by mass spectrometry testing and associated medical data for all pediatric cases (0–12 years) at one institution from 2019 to 2022 were included in the analysis. The final case series (n = 7) included all cases from patients <3 years with comprehensive drug testing results that were inconsistent with medication history and/or toxicology results by immunoassay.
Comprehensive drug testing by mass spectrometry was ordered for 174 urine and blood samples representing 97 patients (0–12 years) from 2019 to 2022. Of these, 76 cases were from patients <3 years old; results were consistent with medication history and confirmatory for immunoassay results (n = 34), consistent with medication history (n = 14), confirmatory for immunoassay results (n = 10), negative (n = 9), or medical history was incomplete (n = 2). The remaining 7 cases were included in the final case series.
The cases highlight the value of real-time comprehensive drug testing in acute pediatric cases. Testing results can rule out toxic exposure from the diagnostic differential when negative, and lead to appropriate medical and social interventions when positive.
Untargeted data acquisition on high-resolution mass spectrometers (HRMSs) has been used in clinical toxicology for screening and identifying unknown compounds in patient samples. A common modality ...for untargeted HRMS data acquisition is information-dependent acquisition (IDA), which analyzes the most abundant small molecules within an acquisition cycle. This process can potentially lead to false negatives of clinically relevant compounds at low concentrations. Sequential window acquisition of all theoretical fragment ion spectra (SWATH) has emerged as a method of unbiased, untargeted HRMS data acquisition in which no spectral data are lost. SWATH has yet to be optimized and assessed for use in clinical toxicology.
We developed a variable-window SWATH method (vSWATH) and compared it to IDA by limit of detection studies in drug-supplemented urine (81 compounds) and against a retrospective cohort of 50 clinical urine samples characterized by LC-MS/MS.
vSWATH had a lower limit of detection than IDA for 33 (41%) drugs and metabolites added into urine samples. Both IDA and vSWATH were equivalent in discovering compounds from clinical urine samples and confirmed 26 additional compounds not previously discovered by targeted LC-MS/MS. Lastly, the unbiased acquisition of spectra in vSWATH allowed for identification of 5 low-abundance compounds missed by IDA.
This vSWATH method for clinical toxicology demonstrated equivalent analytical sensitivity and specificity for untargeted drug screening and identification in urine samples. vSWATH provided the additional benefit of collecting all tandem mass spectrometry spectra in a sample, which could be useful in discovering low-abundance compounds not discovered by IDA.
Abstract
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.
In sera (n = 533) from patients with COVID-19 (n = 153), immunoglobulin M and immunoglobulin G responses, as measured by a quantitative immunoassay, were significantly higher in patients with severe than mild disease. These differences may affect strategies for seroprevalence studies, therapeutics, and vaccine development.
Although T cells are likely players in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunity, little is known about the phenotypic features of SARS-CoV-2-specific T cells associated ...with recovery from severe coronavirus disease 2019 (COVID-19). We analyze T cells from 34 individuals with COVID-19 with severity ranging from mild (outpatient) to critical, culminating in death. Relative to individuals who succumbed, individuals who recovered from severe COVID-19 harbor elevated and increasing numbers of SARS-CoV-2-specific T cells capable of homeostatic proliferation. In contrast, fatal COVID-19 cases display elevated numbers of SARS-CoV-2-specific regulatory T cells and a time-dependent escalation in activated bystander CXCR4+ T cells, as assessed by longitudinal sampling. Together with the demonstration of increased proportions of inflammatory CXCR4+ T cells in the lungs of individuals with severe COVID-19, these results support a model where lung-homing T cells activated through bystander effects contribute to immunopathology, whereas a robust, non-suppressive SARS-CoV-2-specific T cell response limits pathogenesis and promotes recovery from severe COVID-19.
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•Dysfunctional spike-specific T cells are characteristic of severe COVID-19•Spike-specific CD127+ Th1 cells are increased in survivors of severe COVID-19•Spike-specific Treg cells and IL6+ CD8+ T cells are increased in fatal COVID-19•Escalation of activated lung-homing CXCR4+ T cells is associated with fatal COVID-19
Conducting CyTOF on cells from affected individuals, Neidleman et al. identify features of SARS-CoV-2-specific T cells predicting survival of severe COVID-19. Fatal COVID-19 is also characterized by escalating activation of bystander lung-homing CXCR4+ T cells. Boosting SARS-CoV-2-specific T effector responses while diminishing CXCR4-mediated homing may help recovery from severe disease.
The worldwide diversity of HIV-1 presents an unprecedented challenge for vaccine development. Antigens derived from natural HIV-1 sequences have elicited only a limited breadth of cellular immune ...responses in nonhuman primate studies and clinical trials to date. Polyvalent 'mosaic' antigens, in contrast, are designed to optimize cellular immunologic coverage of global HIV-1 sequence diversity. Here we show that mosaic HIV-1 Gag, Pol and Env antigens expressed by recombinant, replication-incompetent adenovirus serotype 26 vectors markedly augmented both the breadth and depth without compromising the magnitude of antigen-specific T lymphocyte responses as compared with consensus or natural sequence HIV-1 antigens in rhesus monkeys. Polyvalent mosaic antigens therefore represent a promising strategy to expand cellular immunologic vaccine coverage for genetically diverse pathogens such as HIV-1.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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