The QuantuMDx Q-POC SARS-CoV-2 RT-PCR assay for rapid detection of COVID-19 at point-of-care: preliminary evaluation of a novel technology.

Accurate and rapid point-of-care (PoC) diagnostics are critical to the control of the COVID-19 pandemic. The current standard for accurate diagnosis of SARS-CoV-2 is laboratory-based reverse transcription polymerase chain reaction (RT-PCR) assays. Here, a preliminary prospective performance evaluation of the QuantuMDx Q-POC SARS-CoV-2 RT-PCR assay is reported. Between November 2020 and March 2021, 49 longitudinal combined nose/throat (NT) swabs from 29 individuals hospitalised with RT-PCR confirmed COVID-19 were obtained at St George's Hospital, London. In addition, 101 mid-nasal (MN) swabs were obtained from healthy volunteers in June 2021. These samples were used to evaluate the Q-POC SARS-CoV-2 RT-PCR assay. The primary analysis was to compare the sensitivity and specificity of the Q-POC test against a reference laboratory-based RT-PCR assay. The overall sensitivity of the Q-POC test compared with the reference test was 96.88% (83.78- 99.92% CI) for a cycle threshold (Ct) cut-off value for the reference test of 35 and 80.00% (64.35-90.95% CI) without altering the reference test's Ct cut-off value of 40. The Q-POC test is a sensitive, specific and rapid PoC test for SARS-CoV-2 at a reference Ct cut-off value of 35. The Q-POC test provides an accurate option for RT-PCR at PoC without the need for sample pre-processing and laboratory handling, enabling rapid diagnosis and clinical triage in acute care and other settings.

Genetic obesity increases pancreatic expression of mitochondrial proteins which regulate cholesterol efflux in BRIN-BD11 insulinoma cells.

Pancreatic ß-cells are sensitive to fluctuations in cholesterol content, which can damage the insulin secretion pathway, contributing to the aetiology of type 2 diabetes mellitus. Cholesterol efflux to (apo)lipoproteins, via ATP-binding cassette (ABC) transporter A1 (ABCA1), can prevent intracellular cholesterol accumulation; in some peripheral cells, ABCA1-dependent efflux is enhanced by promotion of cholesterol trafficking to, and generation of Liver X receptor (LXR) ligands by, mitochondrial sterol 27-hydroxylase (Cyp27A1 (cytochrome P450 27 A1/sterol 27-hydroxylase)) and its redox partners, adrenodoxin (ADX) and ADX reductase (ADXR). Despite this, the roles of mitochondrial cholesterol trafficking (steroidogenic acute regulatory protein [StAR] and 18-kDa translocator protein [TSPO]) and metabolising proteins in insulin-secreting cells remain wholly uncharacterised. Here, we demonstrate an increase in pancreatic expression of Cyp27A1, ADXR, TSPO and LXRα, but not ADX or StAR, in obese (fa/fa) rodents compared with lean (Fa/?) controls. Overexpression of Cyp27A1 alone in BRIN-BD11 cells increased INS2 expression, without affecting lipid metabolism; however, after exposure to low-density lipoprotein (LDL), cholesterol efflux to (apo)lipoprotein acceptors was enhanced in Cyp27A1-overexpressing cells. Co-transfection of Cyp27A1, ADX and ADXR, at a ratio approximating that in pancreatic tissue, stimulated cholesterol efflux to apolipoprotein A-I (apoA-I) in both basal and cholesterol-loaded cells; insulin release was stimulated equally by all acceptors in cholesterol-loaded cells. Thus, genetic obesity increases pancreatic expression of Cyp27A1, ADXR, TSPO and LXRα, while modulation of Cyp27A1 and its redox partners promotes cholesterol efflux from insulin-secreting cells to acceptor (apo)lipoproteins; this response may help guard against loss of insulin secretion caused by accumulation of excess intracellular cholesterol.

Intracellular cholesterol transporters and modulation of hepatic lipid metabolism: Implications for diabetic dyslipidaemia and steatosis.

AIMS/HYPOTHESES: To examine hepatic expression of cholesterol-trafficking proteins, mitochondrial StarD1 and endosomal StarD3, and their relationship with dyslipidaemia and steatosis in Zucker (fa/fa) genetically obese rats, and to explore their functional role in lipid metabolism in rat McArdle RH-7777 hepatoma cells. METHODS: Expression of StarD1 and StarD3 in rat liver and hepatoma samples were determined by Q-PCR and/or immunoblotting; lipid mass by colorimetric assays; radiolabelled precursors were utilised to measure lipid synthesis and secretion, and lipidation of exogenous apolipoprotein A-I. RESULTS: Hepatic expression of StarD3 protein was repressed by genetic obesity in (fa/fa) Zucker rats, compared with lean (Fa/?) controls, suggesting a link with storage or export of lipids from the liver. Overexpression of StarD1 and StarD3, and knockdown of StarD3, in rat hepatoma cells, revealed differential effects on lipid metabolism. Overexpression of StarD1 increased utilisation of exogenous (preformed) fatty acids for triacylglycerol synthesis and secretion, but impacted minimally on cholesterol homeostasis. By contrast, overexpression of StarD3 increased lipidation of exogenous apoA-I, and facilitated de novo biosynthetic pathways for neutral lipids, potentiating triacylglycerol accumulation but possibly offering protection against lipotoxicity. Finally, StarD3 overexpression altered expression of genes which impact variously on hepatic insulin resistance, inducing Ppargcla, Cyp2e1, Nr1h4, G6pc and Irs1, and repressing expression of Scl2a1, Igfbp1, Casp3 and Serpine 1. CONCLUSIONS/INTERPRETATION: Targeting StarD3 may increase circulating levels of HDL and protect the liver against lipotoxicity; loss of hepatic expression of this protein, induced by genetic obesity, may contribute to the pathogenesis of dyslipidaemia and steatosis.