Levels of Angiotensin and Kinin Metabolite Peptides Related to COVID-19 Severity.

In addition to crucial roles in normal human biology, peptide metabolites of the renin-angiotensin (RAS) and kallikrein-kinin systems (KKS) have been reported to be altered in COVID-19 patients. Here, we evaluate new data on RAS and KKS peptides in COVID-19 patient serum obtained from a recently developed, fully validated, and optimized stable isotope labeling LC-MS peptide assay. We found that the RAS peptides angiotensin (ANG) 1, 2, 1-5, and 1-7 were downregulated compared to COVID-free surrogate controls, while the KKS peptides Brad, Brad 1-8, and Brad 1-7 were upregulated. This paper focuses on uncovering the possible diagnostic value of these peptides using receiver operating characteristic (ROC) analyses of these data. ROC plots confirmed that all of the analyte peptides in 80 serum samples from COVID-19 patients were significantly altered from "normal" values of the control samples. The best diagnostic sensitivities and selectivities for COVID vs no COVID were found in ROC plots for Brad and Brad 1-7 (both 99% sensitivity, 100% selectivity). We then analyzed levels of all the peptides grouped according to preassigned values of the World Health Organization (WHO) COVID-19 Severity Index. ROC plots differentiated patients with a high WHO severity index from those with a low WHO severity index with moderate success, with BRAD (73% sensitivity, 79% selectivity) and Ang 1-7 (75% sensitivity, 65% selectivity) giving the best diagnostic performance. Results suggest the possible diagnostic value of these peptides as biomarkers to help identify moderate and serious COVID-19 cases at relatively early stages.

Microfluidic Immunoarray for Point-of-Care Detection of Cytokines in COVID-19 Patients.

The "cytokine storm" often induced in COVID-19 patients contributes to the onset of "acute respiratory distress syndrome" (ARDS) accompanied by lung infection and damage, multiorgan failure, and even death. This large increase in pro-inflammatory cytokines in blood may be related to severity. Rapid, on-demand cytokine analyses can thus be critical to inform treatment plans and improve survival rates. Here, we report a sensitive, low-cost, semiautomated 3D-printed microfluidic immunoarray to detect 2 cytokines and CRP simultaneously in a single 10 µL serum sample in 25 min. Accuracy was validated by analyzing 80 COVID-19 patient serum samples, with results well correlated to a commercial Meso Scale protein immunoassay. Capture antibodies immobilized in detection microwells in a flat well plate-type flow chamber facilitate the immunoassay, with a programmable syringe pump automatically delivering reagents. Chemiluminescence signals were captured in a dark box with a CCD camera integrated for 30 s. This system was optimized to detect inflammation biomarkers IL-6, IFN-γ, and CRP simultaneously in blood serum. Ultralow limits of detection (LODs) of 0.79 fg/mL for IL-6, 4.2 fg/mL for CRP, and 2.7 fg/mL for IFN-γ with dynamic ranges of up to 100 pg/mL were achieved. ROC statistical analyses showed a relatively good diagnostic value related to the samples assigned WHO COVID-19 scores for disease severity, with the best results for IL-6 and CRP. Monitoring these biomarkers for coronavirus severity may allow prediction of disease severity as a basis for critical treatment decisions and better survival rates.

Decreased progenitor TCF1 + T-cells correlate with COVID-19 disease severity.

COVID-19, caused by SARS-CoV-2, can lead to a severe inflammatory disease characterized by significant lymphopenia. However, the underlying cause for the depletion of T-cells in COVID-19 patients remains incompletely understood. In this study, we assessed the presence of different T-cell subsets in the progression of COVID-19 from mild to severe disease, with a focus on TCF1 expressing progenitor T-cells that are needed to replenish peripheral T-cells during infection. Our results showed a preferential decline in TCF1+ progenitor CD4 and CD8+ T-cells with disease severity. This decline was seen in various TCF1+ subsets including naive, memory and effector-memory cells, and surprisingly, was accompanied by a loss in cell division as seen by a marked decline in Ki67 expression. In addition, TCF1+ T-cells showed a reduction in pro-survival regulator, BcL2, and the appearance of a new population of TCF1 negative caspase-3 expressing cells in peripheral blood from patients with severe disease. The decline in TCF1+ T-cells was also seen in a subgroup of severe patients with vitamin D deficiency. Lastly, we found that sera from severe patients inhibited TCF1 transcription ex vivo which was attenuated by a blocking antibody against the cytokine, interleukin-12 (IL12). Collectively, our findings underscore the potential significance of TCF1+ progenitor T-cells in accounting for the loss of immunity in severe COVID-19 and outline an array of markers that could be used to identify disease progression.

Anti-D in Rh(D)-negative pregnant women: are at-risk pregnancies and deliveries receiving appropriate prophylaxis?

OBJECTIVE: Although anti-D prophylaxis has greatly reduced the rate of Rh-immunization, there remain women who sensitize during or after pregnancy because of inadequate prophylaxis. The purpose of this study was to compare adherence to prophylaxis recommendations for antenatal and postnatal anti-D immunoglobulin administration. METHODS: We conducted a retrospective cohort study of all pregnancies recorded at the Royal Victoria Hospital between 2001 and 2006 to determine the rates of antenatal and postnatal prophylaxis in Rh(D)-negative women. We compared adherence to anti-D prophylaxis recommendations between our institution's physician-dependent antenatal approach and the protocol-based postpartum approach. Logistic regression analysis was used to estimate the odds ratio and 95% confidence intervals of determinants of non-adherence to current recommendations for anti-D prophylaxis. RESULTS: Antenatal administration was analyzed in 1868 pregnancies in eligible Rh-negative women. Among these women, 85.7% received appropriate antenatal prophylaxis and 98.5% of eligible women received appropriate postnatal prophylaxis. Factors independently associated with non-adherence to antepartum prophylaxis included first visit in the third trimester (P < 0.001), transfer from an outside hospital (P = 0.03), and physician licensing before 1980 (P = 0.04). CONCLUSION: Unlike hospital-based protocol-dependent systems, physician-dependent systems for antenatal anti-D prophylaxis remain subject to errors of omission. A more standardized system is needed to ensure effective antenatal prophylaxis.

Parathyroid hormone-related protein (PTHrP) and malignancy.

PTHrP (parathyroid hormone related protein) is an important mediator of malignancy-related tumor progression and hypercalcemia that shares considerable homology and functionality with parathyroid hormone. In this chapter, we review what has been elucidated to date regarding PTHrP's role in malignancies. Starting with a review of calcium metabolism and regulation, we then summarize the discovery and structure of PTHrP and development of sensitive immunoassays for specific measurement. Subsequently, we explore its role in tumor progression, with emphasis on the primary tumor as well as skeletal and non-osseus metastases. We then consider the clinical implications of PTHrP in cancer before concluding with a discussion of both established and potential treatments for malignancy associated hypercalcemia and bone metastases.

Geriatric Syndromes in Older Adults Hospitalized with COVID-19 in Montreal, Canada.

Background: Older adults are more vulnerable to severe infection and mortality due to COVID-19. They have atypical presentations of the disease without respiratory symptoms, making early diagnosis clinically challenging. We aimed to compare the baseline characteristics, presentation, and disease course of older (≥70 yrs & ≥90 yrs) and younger (<70 yrs) patients hospitalized with COVID-19. Methods: Retrospective review of 429 consecutive patients hospitalized at two tertiary care hospitals in Montreal, Canada, with PCR-confirmed COVID-19. Baseline characteristics, presentation, in-hospital complications, and outcomes were recorded. Desegregation by age was performed to compare older versus younger individuals. Additional subgroup analyses were performed amongst patients ≥70 stratifying by sex, living situation, and those presenting with geriatric syndromes compared to those without. Results: Patients ≥70 (n=260) presented less frequently with respiratory symptoms compared to patients <70 (n=169) (52% vs. 32%). 11% of patients ≥70 and 24% of patients ≥90 presented with a geriatric syndrome as their sole symptom compared to 3% of those <70. Older adults were more likely to develop disease complications (including delirium, acute kidney injury, and hypernatremia) and had higher in-hospital mortality (32% vs. 13%). Subgroup analyses revealed heightened vulnerability to complications in older men, those from long-term care, and those with at least one geriatric syndrome upon presentation. Conclusions: Older adults presenting to hospital with COVID-19 often have no respiratory symptoms and can present with only a geriatric syndrome. New geriatric syndromes in older individuals should trigger evaluation for COVID-19 and consideration for early initiation of multidisciplinary care to prevent complications.

Safety and effectiveness concerns of lopinavir/ritonavir in COVID-19 affected patients: a retrospective series.

CONTEXT: Originally developed for treatment of human immunodeficiency virus (HIV), the antiviral combination lopinavir/ritonavir (LPV/r) is being repurposed for treating the novel coronavirus disease (COVID-19) despite minimal experience in this markedly different population and an in-vitro derived EC50 against SARS-CoV-2 several hundred-fold greater than for HIV. We present a case series including a case of severe hyponatremia and a 32-fold overdose raising safety and effectiveness concerns in COVID-19 patients. METHODS: We measured LPV trough concentrations in 12 patients and reviewed their clinical charts for side effects known to occur in HIV patients. FINDINGS: Compared to established LPV trough concentrations in HIV patients, concentrations in COVID-19 patients were 3-fold greater (19.37 ± 10.12 mcg/mL versus 6.25 mcg/mL). In addition, cholestasis and dyslipidemia toxicity thresholds were exceeded in 12/12 and 11/12 patients respectively. No patients achieved the presumed therapeutic concentration. Side effects included gastrointestinal symptoms (5/12), electrolyte imbalances (4/12), liver enzyme disturbances (5/12) and triglyceride elevations (2/12). CONCLUSION: No patients reached presumed therapeutic LPV concentrations despite experiencing side effects and exceeding cholestasis and dyslipidemia toxicity thresholds. This raises concerns for the safety and effectiveness of LPV/r. Clinicians should consider closely monitoring for side effects and not necessarily attribute them to COVID-19.

Massive acetaminophen overdose: effect of hemodialysis on acetaminophen and acetylcysteine kinetics.

CONTEXT: Early onset acidosis from mitochondrial toxicity can be observed in massive acetaminophen poisoning prior to the development of hepatotoxicity. In this context, the efficacy of acetylcysteine to reverse mitochondrial toxicity remains unclear and hemodialysis may offer prompt correction of acidosis. Unfortunately, toxicokinetics of acetaminophen and acetylcysteine during extracorporeal treatments hemodialysis have seldom been described. CASE DETAILS: An 18-year-old woman presented to the emergency department 60 minutes after ingestion of 100 g of acetaminophen, and unknown amounts of ibuprofen and ethanol. Initial assessment revealed an agitated patient. Her mental status worsened and she required intubation for airway protection. Investigations showed metabolic acidosis with lactate peaking at 8.6 mmol/L. Liver and coagulation profiles remained normal. Acetaminophen concentration peaked at 981 µg/ml (6496 µmol/L). Pending hemodialysis, the patient received 100 g of activated charcoal and an acetylcysteine infusion at 150 mg/kg over 1 hour, followed by 12.5 mg/kg/h for 4 hours. During hemodialysis, the infusion was maintained at 12.5 mg/kg/h to compensate for expected removal before it was decreased to 6.25 mg/kg for 20 hours after hemodialysis. The patient rapidly improved during hemodialysis and was discharged 48 hours post-admission. TOXICOKINETICS: The acetaminophen elimination half-life was 5.2 hours prior to hemodialysis, 1.9-hours during hemodialysis and 3.6 hours post hemodialysis. The acetaminophen and acetylcysteine clearances by A-V gradient during hemodialysis were 160.4 ml/min and 190.3 ml/min, respectively. Hemodialysis removed a total of 20.6 g of acetaminophen and 17.9 g of acetylcysteine. CONCLUSION: This study confirms the high dialyzability of both acetaminophen and acetylcysteine. Hemodialysis appears to be a beneficial therapeutic option in cases of massive acetaminophen ingestion with coma and lactic acidosis. Additionally, these results suggest that the infusion rate of acetylcysteine must be more than double during hemodialysis to compensate for its ongoing removal and provide similar plasma concentrations to the usual acetylcysteine regimen.

Review of the effect of intravenous lipid emulsion on laboratory analyses.

CONTEXT: Although the clinical use of intravenous lipid emulsion therapy for the treatment of lipophilic drug toxicity is increasing, the focus of most publications is on outcome in laboratory animals or in patients. An unintended consequence of intravenous lipid emulsion is the creation of extremely lipemic blood, which may interfere with the laboratory analysis or interpretation of common analytes. OBJECTIVE: The American Academy of Clinical Toxicology has established a lipid emulsion workgroup to review the evidence and produce recommendations on the use of this novel therapy for drug toxicity. The aim of this subgroup is to review the available evidence regarding the effect of intravenous lipid emulsion on common laboratory testing, which often forms the basis of the appraisal of the balance between benefits and potential adverse events. METHODS: We performed a comprehensive review of the literature. Relevant articles were determined based upon a predefined methodology. Package inserts of manufacturers' assays were collected. Article inclusion required that the article met predefined inclusion criteria with the agreement of at least two members of the subgroup. RESULTS: We included thirty-six articles in the final analysis. Evaluation of the reviewed analytes revealed heterogeneity with regards to the assessment of the effect of intravenous lipid emulsion in terms of consistency and magnitude of effect across the different analytic platforms. CONCLUSIONS: The measurements of a number of common analytes can be markedly affected by the lipemia produced by lipid emulsions such that they cannot always be interpreted in the way that most physicians use this information in typical clinical situations. In fact, a lack of appreciation of this effect may lead to unintentional treatment errors. Because the effect of the lipemia produced is dependent on the reagents and laboratory platform used, it would be useful for all future reports to clearly document sample handling, reagents and laboratory platform used, as well as any procedures employed to reduce the lipid content.

Remission of hormone-refractory prostate cancer attributed to Essiac.

Essiac is a popular complementary and alternative medicine (CAM) that is utilized by many cancer patients in North America. Much anecdotal reporting exists about its cancer-fighting qualities, but so far no clinical trials have been preformed to validate those claims. We describe here the case of a 64-year-old man whose hormone-refractory prostate cancer responded well to Essiac tea.

Methodology for AACT evidence-based recommendations on the use of intravenous lipid emulsion therapy in poisoning.

Intravenous lipid emulsion (ILE) therapy is a novel treatment that was discovered in the last decade. Despite unclear understanding of its mechanisms of action, numerous and diverse publications attested to its clinical use. However, current evidence supporting its use is unclear and recommendations are inconsistent. To assist clinicians in decision-making, the American Academy of Clinical Toxicology created a workgroup composed of international experts from various clinical specialties, which includes representatives of major clinical toxicology associations. Rigorous methodology using the Appraisal of Guidelines for Research and Evaluation or AGREE II instrument was developed to provide a framework for the systematic reviews for this project and to formulate evidence-based recommendations on the use of ILE in poisoning. Systematic reviews on the efficacy of ILE in local anesthetic toxicity and non-local anesthetic poisonings as well as adverse effects of ILE are planned. A comprehensive review of lipid analytical interferences and a survey of ILE costs will be developed. The evidence will be appraised using the GRADE system. A thorough and transparent process for consensus statements will be performed to provide recommendations, using a modified Delphi method with two rounds of voting. This process will allow for the production of useful practice recommendations for this therapy.

Analytical interferences resulting from intravenous lipid emulsion.

CONTEXT: Lipid resuscitation therapy using intravenous lipid emulsion (IVLE) for drug overdoses has gained widespread use. However, there is little information regarding its adverse effects. OBJECTIVES: We performed lipemic interference studies on typical automated platforms to investigate the potential of lipid resuscitation therapy to interfere with the reliability and turnaround time of analytes that would be of interest in acute intoxications. We also tested methods to minimize interferences. MATERIALS AND METHODS: Serum pools were supplemented with increasing concentrations of Intralipid-20%(®) (0-30%). Analyses were performed on Beckman-Coulter DXC800 and DXI and Roche Modular-P. Analytes demonstrating significant interference were re-measured after centrifugation (14 000 × g for 10 minutes). RESULTS: Triglyceride and glycerol-blanked triglyceride concentrations were similar in IVLE-free samples. However, with addition of IVLE, concentrations were markedly different (139 vs. 76 mmol/L). There was no appreciable interference on the troponin-I, sodium, potassium, chloride, calcium, bicarbonate or urea assays. Albumin and magnesium assays demonstrated significant interference. Amylase, lipase, phosphate, creatinine, total protein, ALT, CK and bilirubin became unmeasurable in IVLE-supplemented samples. Whereas glucose measurement by potentiometry was free of interference, colorimetric methodology was error prone. Centrifugation removed > 90% of glycerol-blanked triglyceride (max = 5.8 mmol/L), dramatically reducing lipid interferences. DISCUSSION: IVLE results in appreciable analytical interferences at concentrations demonstrated in lipid resuscitation therapy. Of particular concern is the marked interference on glucose and magnesium, which may result in unsuccessful and potentially harmful interventions. Major implications for patient care include reporting of incorrect results and delays in the reporting of time-sensitive results. Whenever possible, blood samples should be collected prior to initiating lipid therapy. Interferences can be minimized by brief centrifugation at relatively low speeds on equipment readily available in most core labs.