A high throughput immuno-affinity mass spectrometry method for detection and quantitation of SARS-CoV-2 nucleoprotein in human saliva and its comparison with RT-PCR, RT-LAMP, and lateral flow rapid antigen test.

OBJECTIVES: Many reverse transcription polymerase chain reaction (RT-PCR) methods exist that can detect SARS-CoV-2 RNA in different matrices. RT-PCR is highly sensitive, although viral RNA may be detected long after active infection has taken place. SARS-CoV-2 proteins have shorter detection windows hence their detection might be more meaningful. Given salivary droplets represent a main source of transmission, we explored the detection of viral RNA and protein using four different detection platforms including SISCAPA peptide immunoaffinity liquid chromatography-mass spectrometry (SISCAPA-LC-MS) using polyclonal capture antibodies. METHODS: The SISCAPA-LC MS method was compared to RT-PCR, RT-loop-mediated isothermal amplification (RT-LAMP), and a lateral flow rapid antigen test (RAT) for the detection of virus material in the drool saliva of 102 patients hospitalised after infection with SARS-CoV-2. Cycle thresholds (Ct) of RT-PCR (E gene) were compared to RT-LAMP time-to-positive (TTP) (NE and Orf1a genes), RAT optical densitometry measurements (test line/control line ratio) and to SISCAPA-LC-MS for measurements of viral protein. RESULTS: SISCAPA-LC-MS showed low sensitivity (37.7 %) but high specificity (89.8 %). RAT showed lower sensitivity (24.5 %) and high specificity (100 %). RT-LAMP had high sensitivity (83.0 %) and specificity (100.0 %). At high initial viral RNA loads (<20 Ct), results obtained using SISCAPA-LC-MS correlated with RT-PCR (R2 0.57, p-value 0.002). CONCLUSIONS: Detection of SARS-CoV-2 nucleoprotein in saliva was less frequent than the detection of viral RNA. The SISCAPA-LC-MS method allowed processing of multiple samples in <150 min and was scalable, enabling high throughput.

Operation Moonshot: rapid translation of a SARS-CoV-2 targeted peptide immunoaffinity liquid chromatography-tandem mass spectrometry test from research into routine clinical use.

OBJECTIVES: During 2020, the UK's Department of Health and Social Care (DHSC) established the Moonshot programme to fund various diagnostic approaches for the detection of SARS-CoV-2, the pathogen behind the COVID-19 pandemic. Mass spectrometry was one of the technologies proposed to increase testing capacity. METHODS: Moonshot funded a multi-phase development programme, bringing together experts from academia, industry and the NHS to develop a state-of-the-art targeted protein assay utilising enrichment and liquid chromatography tandem mass spectrometry (LC-MS/MS) to capture and detect low levels of tryptic peptides derived from SARS-CoV-2 virus. The assay relies on detection of target peptides, ADETQALPQRK (ADE) and AYNVTQAFGR (AYN), derived from the nucleocapsid protein of SARS-CoV-2, measurement of which allowed the specific, sensitive, and robust detection of the virus from nasopharyngeal (NP) swabs. The diagnostic sensitivity and specificity of LC-MS/MS was compared with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) via a prospective study. RESULTS: Analysis of NP swabs (n=361) with a median RT-qPCR quantification cycle (Cq) of 27 (range 16.7-39.1) demonstrated diagnostic sensitivity of 92.4% (87.4-95.5), specificity of 97.4% (94.0-98.9) and near total concordance with RT-qPCR (Cohen's Kappa 0.90). Excluding Cq>32 samples, sensitivity was 97.9% (94.1-99.3), specificity 97.4% (94.0-98.9) and Cohen's Kappa 0.95. CONCLUSIONS: This unique collaboration between academia, industry and the NHS enabled development, translation, and validation of a SARS-CoV-2 method in NP swabs to be achieved in 5 months. This pilot provides a model and pipeline for future accelerated development and implementation of LC-MS/MS protein/peptide assays into the routine clinical laboratory.

High non-adherence rates to secondary prevention by chemical adherence testing in patients with TIA.

INTRODUCTION: Transient ischaemic attack (TIA) clinics are important for secondary prevention of fatal or disabling stroke. Non-adherence to prescribed medications is an important reason for treatment failure but difficult to diagnose. This study ascertained the utility of a novel biochemical tool in the objective biochemical diagnosis of non-adherence. METHODS: One-hundred consecutive urine samples collected from patients attending the TIA clinic, at a tertiary centre, were analysed for presence or absence of prescribed cardiovascular medications using liquid chromatography-mass spectrometry (LC-MS/MS). Patients were classified as adherent or non-adherent, respectively. Demographic and clinical characteristics were compared between the two cohorts. Univariate regression analyses were performed for individual variables and model fitting was undertaken for significant variables. RESULTS: The mean duration of follow-up from the index event was 31 days [standard deviation (SD): 18.9]. The overall rate of non-adherence for at least one medication was 24%. In univariate analysis, the number of comorbidities [3.4 (SD: 1.9) vs. 2.5 (1.9), P = 0.032] and total number of all prescribed medications [6.0 (3.3) vs 4.4 (2.1), P = 0.032] were higher in the non-adherent group. On multivariate analysis, the total number of medications prescribed correlated with increased non-adherence (odds ratio: 1.27, 95% Confidence Intervals: 1.1-1.5, P = 0.01). CONCLUSIONS: LC-MS/MS is a clinically useful tool for the diagnosis of non-adherence. Nearly a quarter of TIA patients were non-adherent to their cardiovascular medications Addressing non-adherence early may reduce the risk of future disabling cardiovascular events.

Nonadherence in Hypertension: How to Develop and Implement Chemical Adherence Testing.

Nonadherence to antihypertensive medication is common, especially in those with apparent treatment-resistant hypertension (true treatment-resistant hypertension requires exclusion of nonadherence), and its routine detection is supported by clinical guidelines. Chemical adherence testing is a reliable and valid method to detect adherence, yet methods are unstandardized and are not ubiquitous. This article describes the principles of chemical adherence testing for hypertensive patients and provides a set of recommendations for centers wishing to develop the test. We recommend testing should be done in either of two instances: (1) in those who have resistant hypertension or (2) in those on 2 antihypertensives who have a less than 10 mm Hg drop in systolic blood pressure on addition of the second antihypertensive medication. Furthermore, we recommend that verbal consent is secured before undertaking the test, and the results should be discussed with the patient. Based on medications prescribed in United Kingdom, European Union, and United States, we list top 20 to 24 drugs that cover >95% of hypertension prescriptions which may be included in the testing panel. Information required to identify these medications on mass spectrometry platforms is likewise provided. We discuss issues related to ethics, sample collection, transport, stability, urine versus blood samples, qualitative versus quantitative testing, pharmacokinetics, instrumentation, validation, quality assurance, and gaps in knowledge. We consider how to best present, interpret, and discuss chemical adherence test results with the patient. In summary, this guidance should help clinicians and their laboratories in the development of chemical adherence testing of prescribed antihypertensive drugs.

Cov-MS: A Community-Based Template Assay for Mass-Spectrometry-Based Protein Detection in SARS-CoV-2 Patients.

Rising population density and global mobility are among the reasons why pathogens such as SARS-CoV-2, the virus that causes COVID-19, spread so rapidly across the globe. The policy response to such pandemics will always have to include accurate monitoring of the spread, as this provides one of the few alternatives to total lockdown. However, COVID-19 diagnosis is currently performed almost exclusively by reverse transcription polymerase chain reaction (RT-PCR). Although this is efficient, automatable, and acceptably cheap, reliance on one type of technology comes with serious caveats, as illustrated by recurring reagent and test shortages. We therefore developed an alternative diagnostic test that detects proteolytically digested SARS-CoV-2 proteins using mass spectrometry (MS). We established the Cov-MS consortium, consisting of 15 academic laboratories and several industrial partners to increase applicability, accessibility, sensitivity, and robustness of this kind of SARS-CoV-2 detection. This, in turn, gave rise to the Cov-MS Digital Incubator that allows other laboratories to join the effort, navigate, and share their optimizations and translate the assay into their clinic. As this test relies on viral proteins instead of RNA, it provides an orthogonal and complementary approach to RT-PCR using other reagents that are relatively inexpensive and widely available, as well as orthogonally skilled personnel and different instruments. Data are available via ProteomeXchange with identifier PXD022550.

Non-adherence to heart failure medications predicts clinical outcomes: assessment in a single spot urine sample by liquid chromatography-tandem mass spectrometry (results of a prospective multicentre study).

AIMS: Liquid chromatography-mass spectrometry (LC-MS/MS) is an objective new technique to assess non-adherence to medications. We used this method to study the prevalence, predictors and outcomes of non-adherence in patients with heart failure with reduced left ventricular ejection fraction (HFrEF). METHODS AND RESULTS: This study included 1296 patients with HFrEF from BIOSTAT-CHF, a study that aimed to optimise guideline-recommended therapies. Angiotensin-converting enzyme inhibitors (ACEi), angiotensin receptor blockers (ARBs), mineralocorticoid receptor antagonists, ß-blockers and loop diuretics were measured in a single spot urine sample at 9 months using LC-MS/MS. The relationship between medication non-adherence and the composite endpoint of all-cause death or heart failure hospitalisation, over a median follow-up of 21 months, was evaluated. Non-adherence to at least one prescribed medication was observed in 45.9% of patients. The strongest predictor of non-adherence was non-adherence to any of the other medication classes (P < 0.0005). Regional differences within Europe were observed. On multivariable analyses, non-adherence to ACEi/ARBs and ß-blockers was associated with an increased risk of the composite endpoint [hazard ratio (HR) 1.38, 95% confidence interval (CI) 1.09-1.95, P = 0.008 and HR 1.48, 95% CI 1.12-1.96, P = 0.006, respectively). Non-adherence to ß-blockers was also associated with an increased risk of death (HR 2.48, 95% CI 1.67-3.68, P < 0.0005). Patients who were non-adherent to loop diuretics were healthier and had a decreased risk of the composite endpoint (HR 0.69, 95% CI 0.51-0.93, P = 0.014). Non-adherence to mineralocorticoid receptor antagonists was not related to any clinical outcome. CONCLUSION: Non-adherence to medications, assessed by a single urine test, is common and predicts clinical outcomes in patients with HFrEF.

Objective measures of non-adherence in cardiometabolic diseases: a review focused on urine biochemical screening.

Cardiometabolic diseases are among the most prevalent and harmful conditions worldwide. They are complex, comorbid conditions that require polypharmacy - a known contributor to non-adherence in cardiovascular disease (CVD) and diabetes mellitus (DM). Suboptimal adherence is associated with poor disease control, which increases the risk of hospitalizations, mortality, and preventable financial implications. However, until recently, the lack of a gold standard for non-adherence testing in cardiometabolic diseases has been the major barrier for understanding true prevalence and mortality consequences. Recent European guidelines have endorsed biochemical testing as the preferred measure for non-adherence in CVD, with urinary screening methods being the most clinically widespread. The diagnostic and therapeutic benefits incurred to health service resources by use of biochemical non-adherence testing are vast, as hospitalizations and associated economic burdens are reduced, and tailored therapies are increased. However, biochemical testing can only signify a snap shot of adherence behavior, and true adherence may be skewed by pharmacokinetic factors. This review summarizes current literature regarding the prevalence, impact, and reasons of non-adherence in cardiometabolic disease. The benefits of current adherence diagnostic tools have been appraised, where urine in biochemical testing has been focused upon and evaluated against other matrices.

Biochemical Urine Testing of Adherence to Cardiovascular Medications Reveals High Rates of Nonadherence in People Attending Their Annual Review for Type 2 Diabetes.

OBJECTIVE: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a new method to objectively and robustly detect nonadherence. We applied this technique to study nonadherence to cardiovascular medications in people with type 2 diabetes (T2DM). RESEARCH DESIGN AND METHODS: Routine urine samples, received at the time of the annual diabetes review from 228 people with T2DM in primary care, were assessed for adherence by LC-MS/MS. RESULTS: A total of 28.1% patients (N = 64) were nonadherent to antidiabetic, antihypertensive, and/or lipid-lowering medications. Nonadherence to statins was the highest at 23.7%, and nonadherence to oral hypoglycemic agents was 9.3%. HbA1c, albumin-to-creatinine ratio, and lipid profiles were significantly higher in the patients who were nonadherent compared with those who were adherent to treatment. CONCLUSIONS: This unique study shows that routine urine samples can be used for adherence testing screening by LC-MS/MS and has demonstrated high nonadherence rates especially to statins in people with T2DM. Future intervention studies using LC-MS/MS as a diagnostic/therapeutic tool may help to improve clinical outcomes.

The elusive power of the individual victim: Failure to find a difference in the effectiveness of charitable appeals focused on one compared to many victims.

Previous research has offered conflicting findings regarding the influence of help appeals that feature an individual victim compared to a group of victims. Studies examining emotional responses and donation behavior have generally found that help appeals focusing on a single victim elicit more prosocial responses, while studies examining policy support have found the opposite. The present studies investigate these effects while addressing potential confounds that may have arisen in previous research. Study 1 (N = 924) compares the effects of help appeals that focus on either a) an identified individual, b) an identified group, c) statistics describing many individuals, or d) statistics paired with an individual. Study 1 also examines how the location of the individual(s) in need moderates observed effects. Study 2 (N = 1,085) compares the effects of help appeals that describe either an identified individual or statistics about many individuals, while fully crossing the text manipulation with either a) no imagery, b) an image of an individual, or c) a map indicating areas of poverty. In both Study 1 and Study 2 no significant differences were found between the individual and the group conditions.

Time required to initiate outbreak and pandemic observational research.

PURPOSE: Observational research focused upon emerging infectious diseases such as Ebola virus, Middle East respiratory syndrome, and Zika virus has been challenging to quickly initiate. We aimed to determine the duration of start-up procedures and barriers encountered for an observational study focused upon such infectious outbreaks. MATERIALS AND METHODS: At 1 pediatric and 5 adult intensive care units, we measured durations from protocol receipt to a variety of outbreak research milestones, including research ethics board (REB) approval, data sharing agreement (DSA) execution, and patient study screening initiation. RESULTS: The median (interquartile range) time from site receipt of the protocol to REB submission was 73 (30-126) days; to REB approval, 158 (42-188) days; to DSA completion, 276 (186-312) days; and to study screening initiation, 293 (269-391) days. The median time from REB submission to REB approval was 43 (13-85) days. The median time for all start-up procedures was 335 (188-335) days. CONCLUSIONS: There is a lengthy start-up period required for outbreak-focused research. Completing DSAs was the most time-consuming step. A reactive approach to newly emerging threats such as Ebola virus, Middle East respiratory syndrome, and Zika virus will likely not allow sufficient time to initiate research before most outbreaks are advanced.

Support Needs for Canadian Health Providers Responding to Disaster: New Insights from a Grounded Theory Approach.

INTRODUCTION: An earlier descriptive study exploring the various supports available to Canadian health and social service providers who deployed to the 2010 earthquake disaster in Haiti, indicated that when systems are compromised, professionals are at physical, emotional and mental risk during overseas deployment. While these risks are generally well-identified, there is little literature that explores the effectiveness of the supports in place to mitigate this risk. This study provides evidence to inform policy development regarding future disaster relief, and the effectiveness of supports available to responders assisting with international disaster response. METHODS: This study follows Strauss and Corbin's 1990 structured approach to grounded theory to develop a framework for effective disaster support systems. N=21 interviews with Canadian health and social service providers, who deployed to Haiti in response to the 2010 earthquake, were conducted and analyzed. Resulting data were transcribed, coded and analysed for emergent themes. RESULTS AND DISCUSSION: Three themes were identified in the data and were used to develop the evolving theory. The interview data indicate that the experiences of responders are determined based on an interaction between the individual's 'lens' or personal expectations, as well as the supports that an organization is able to provide. Therefore, organizations should consider the following factors: experience, expectations, and supports, to tailor a successful support initiative that caters to the needs of the volunteer workforce.

A scoping review of patient discharge from intensive care: opportunities and tools to improve care.

BACKGROUND: We conducted a scoping review to systematically review the literature reporting patient discharge from ICUs, identify facilitators and barriers to high-quality care, and describe tools developed to improve care. METHODS: We searched Medline, Embase, CINAHL, and the Cochrane Central Register of Controlled Trials. Data were extracted on the article type, study details for research articles, patient population, phase of care during discharge, and dimensions of health-care quality. RESULTS: From 8,154 unique publications we included 224 articles. Of these, 131 articles (58%) were original research, predominantly case series (23%) and cohort (16%) studies; 12% were narrative reviews; and 11% were guidelines/policies. Common themes included patient and family needs/experiences (29% of articles) and the importance of complete and accurate information (26%). Facilitators of high-quality care included provider-patient communication (30%), provider-provider communication (25%), and the use of guidelines/policies (29%). Patient and family anxiety (21%) and limited availability of ICU and ward resources (26%) were reported barriers to high-quality care. A total of 47 tools to facilitate patient discharge from the ICU were identified and focused on patient evaluation for discharge (29%), discharge planning and teaching (47%), and optimized discharge summaries (23%). CONCLUSIONS: Common themes, facilitators and barriers related to patient and family needs/experiences, communication, and the use of guidelines/policies to standardize patient discharge from ICU transcend the literature. Candidate tools to improve care are available; comparative evaluation is needed prior to broad implementation and could be tested through local quality-improvement programs.

Supports for health and social service providers from Canada responding to the disaster in haiti.

UNLABELLED: In January 12, 2010, a 7.0 magnitude earthquake shook Port-au-Prince, Haiti. The massive disaster made it difficult for local Haitian community officials to respond immediately, leaving the country reliant on foreign aid and international and non-governmental relief organizations. This study explores the effectiveness of various supports that were made available to health and social service providers in Haiti, by focusing on their lived experiences pre-deployment, on-site and post-deployment. The paper provides a qualitative exploration of participant perceptions with respect to the success of their performance in response, and relevant literature describing the various supports provided to health and social service providers responding to disasters. METHODS: A single, semi-structured interview was conducted with Canadian health professionals (n=21) who deployed to Haiti during the time of, or after, the 2010 earthquake. The study uses Strauss and Corbin's structured approach to grounded theory to identify main themes and relationships in the interviews. RESULTS: The interviews indicate that training, and psychological and emotional supports for health and social service providers require improvement to enhance the experience and effectiveness of their work. CONCLUSIONS: Findings indicate that supports are most effective when they are tailored to the volunteers. The paper highlights future research stemming from the grounded theory findings.

Is oestrogen neuroprotective?

Neuro-protection in this context is an important concept in the treatment of patients in the early, prodromal phase of psychosis, otherwise known as the 'At Ultra High Risk Mental State'. Neuro-protection as described here refers to the use of agents to control the process of apoptosis, which occurs more rapidly in the earliest phases of schizophrenia. There is a need to identify medications with fewer side effects than anti-psychotics in order to treat at risk mental states, or prodromal psychosis. Studies have shown that schizophrenia occurs in males at an earlier age than females. Later, at about the time of the menopause, there is a second peak in the incidence of psychosis (schizophrenia) in women. Hence it has been suggested that Oestrogen may be neuroprotective. Studies have shown that the addition of oestradiol to anti-psychotics in the treatment of schizophrenia in females increased the efficacy of the treatment, which suggests that oestrogen does indeed have a neuroprotective action. However oestrogen has never been used in 'at ultra high risk mental states', perhaps because of concern regarding side effects.

Use of antibiotics for sore throat and incidence of quinsy.

BACKGROUND: Quinsy is the most common major suppurative complication of sore throat. Evidence on the effectiveness of antibiotics in preventing quinsy is sparse. AIM: To assess the incidence of quinsy and the pattern of presentation, and to identify variables that predict the development of quinsy. DESIGN OF STUDY: Case-control study. SETTING: UK-wide primary care. METHOD: Retrospective analysis of data from the General Practice Research Database (GPRD) for the years 1995-1997. RESULTS: There were 606 recorded cases of patients with quinsy, but only 192 (31%) of these patients presented following an initially uncomplicated sore throat. Patients with quinsy were more likely to be aged 21-40 years (odds ratio [OR] = 2.5, 95% confidence interval [CI] = 1.7 to 3.6, compared with other ages), smokers (OR = 2.5, 95% CI = 1.8 to 3.5), and male (OR = 1.6, 95% CI = 1.1 to 2.2). Quinsy developed very quickly for most patients (median of 2 days after tonsillitis and 3 days after a sore throat). For cases initially labelled as tonsillitis, there was a nonsignificant trend of antibiotics preventing quinsy (OR = 0.6, 95% CI = 0.3 to 1.3), but no evidence that antibiotics prevent quinsy for cases labelled as sore throat or pharyngitis (OR = 1.2, 95% CI = 0.7 to 2.2). CONCLUSION: Most patients with quinsy develop the condition rapidly, and many do not present with a respiratory tract infection to their GP first. The current low doses of antibiotics used in modern community settings may be less likely to protect against quinsy than the trial evidence suggests.