Development and Translation of Mass Spectrometry in the Fields of Non-Adherence and Covid-19

Non-adherence is the failure to follow prescribed treatment regimens. In cardiometabolic diseases (e.g., hypertension, type 2 diabetes mellitus etc), non-adherence is directly linked with increased risk of hospitalisation and early all-cause mortality. These endpoints are completely avoidable. However, the incidence of non-adherence may occur in >25% of patients. New chemical adherence tests (CAT) have recently been adopted for clinical use in the UK (circa. 2014), and these represent the gold-standard measure for determining adherence. Using liquid-chromatography tandem mass spectrometry (LC-MS/MS), biomatrices (e.g., urine/plasma) are tested qualitatively for the presence of medications to indicate ingestion. Clinically, CAT is not widespread (only available in specialist clinics), it is slow (>60 minutes per sample), and it poses a risk of false results (quantitative data may identify sporadic dosing). This thesis developed a set of guidelines regarding the use of CAT, which was published and endorsed by the European Society of Hypertension Adherence Working Group. Then, Fast ( The initial aims of the thesis then shifted due to the COVID-19 pandemic. Here, the Department of Health and Social Care (DHSC) set a UK-wide directive to rapidly develop and implement novel SARS-CoV-2 testing devices for mass screening (“Operation Moonshot”). The latter half of this work follows the role of this thesis in Operation Moonshot. A proteomic LC-MS/MS assay was developed. Using a novel viral peptide-capture approach, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid peptides were successfully resolved from human saliva for the determination of active infection. This assay was then tested against the gold-standard, reverse transcriptase polymerase chain reaction (RT-PCR), to assess its clinical utility.</p

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

<div><p>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 <i>emotional responses</i> and <i>donation behavior</i> have generally found that help appeals focusing on a single victim elicit more prosocial responses, while studies examining <i>policy support</i> have found the opposite. The present studies investigate these effects while addressing potential confounds that may have arisen in previous research. Study 1 (<i>N</i> = 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 (<i>N</i> = 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.</p></div

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

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.</p

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.</p