Evaluation of extraction-free RT-PCR methods for faster and cheaper detection of SARS-CoV-2 using two commercial systems.

OBJECTIVE: When using high-throughput batched diagnostic platforms based on RT-PCR for SARS-CoV-2 detection, avoidance of the conventional nucleic acid extraction step can help to reduce the turnaround time and increase processivity. This approach can also spare reagents and plasticware, which have experienced a shortage during the initial waves of the pandemic, reducing the overall testing costs. METHODS: This study evaluated the performance of extraction-free protocols based on simple dilution of the specimen in sterile RNAse free water (with or without a heating step) in comparison to standard RNA extraction protocols, using two commercial kits for molecular detection of SARS-CoV-2 (Allplex™ SARS-CoV-2 assay and Allplex™ SARS-CoV-2/FluA/FluB/RSV assay) in nasopharyngeal swabs (NPS). RESULTS: Compared with conventional protocols, extraction-free protocols based on sample dilution without a heating step exhibited a lower analytical sensitivity: 74.0% and 82.1% with the Allplex™ SARS-CoV-2 assay (tested with 139 NPS samples) and the Allplex™ SARS-CoV-2/FluA/FluB/RSV assay (tested with 69 NPS samples), with a mean increase of Ct values of +2.04 and +1.32, respectively. Most false negative results were observed with sampled low viral load. Including a step of heat exposure did not improve but actually decreased the analytical sensitivity of the assay. CONCLUSIONS: Results confirmed that extraction-free protocols could be a faster and cheaper approach to SARS-CoV-2 detection in NPS samples, which could improve processivity of diagnostic platforms.

Effect of consumption of ancient grain bread leavened with sourdough or with baker's yeast on cardio-metabolic risk parameters: a dietary intervention trial.

The aim of this study was to compare the effect of consumption of ancient grain "Verna" bread obtained by two different leavening agents, sourdough (SD) and baker's yeast (BY), on inflammatory parameters and cardiometabolic risk factors. Seventeen clinically healthy subjects were included to consume SD or BY bread for 4 weeks each, and blood analyses were carried out. The consumption of "Verna" bread obtained with both leavening agents led to a significant improvement of LDL cholesterol. A reduction of -10.6% and -8.53% was observed after replacement with SD and BY bread, respectively. A significant increase in fasting blood glucose (+6%) was observed only after the intervention with BY bread. A 10.7% decrease of vascular endothelial growth factor was found after the SD bread replacement period. The consumption of "Verna" bread resulted significantly associated with an improvement in the cardiometabolic and inflammatory profile. However, only consumption of BY bread determined a significant increase in blood glucose levels.

Effects of an Olive By-Product Called Pâté on Cardiovascular Risk Factors.

BACKGROUND: The nutraceutical effects of Olea europaea L. products are mainly due to phenolic compounds. During olive milling, most of the total phenols remain in the process by-products. AIM: We aimed to evaluate the effects of a specific by-product of olive oil called "pâté" (OlP) administered as tablets, on cardiovascular and metabolic risk factors. METHODS: The study was a crossover trial with 2 intervention periods. Nineteen participants (mean age: 38 years) took 4 tablets/day of either olive pâté (corresponding to 30 mg/day of hydroxytyrosol) or placebo for 2 months followed by a 2-month washout and another 2 months of crossed over treatment. RESULTS: After the intervention with pâté, participants showed a statistically significant reduction in plasma levels of total cholesterol (-10.8 mg/dL), LDL cholesterol (-10.8 mg/dL) and urea (-4.1 mg/dL), and a significant increase in calcium levels (+0.3 mg/dL). Leukocyte response to exogenous oxidative stress was significantly reduced (-12.8%) and levels of the antioxidant transcription factor Nrf-2 increased by 88.9%. Plasma levels of the pro-inflammatory protein MCP-1 were significantly reduced (-9.0 pg/mL). CONCLUSION: In conclusion, the intake of OlP showed positive effects on several cardiovascular risk factors, demonstrating the nutraceutical potential of a widely available but, to date, underestimated olive oil by-product.

Risk of reoperation in bioprosthetic valve patients with indication for long-term anticoagulation. Results from the observational retrospective multicentre PLECTRUM study.

Objective: Several factors should be considered when a prosthetic heart valve, bioprosthetic valve (BV) or mechanical valve is to be implanted: thrombogenicity, life expectancy and the risk of reoperation. Methods: We conducted an observational retrospective multicentre study among Italian Thrombosis Centers on patients with BV on long-term vitamin K antagonist (VKA) treatment to evaluate the risk of reoperation and the rate of bleeding and thrombotic events. Results: We analysed 612 patients (median age 71.8 years) with BV on long-term VKA treatment for the presence of atrial fibrillation (AF) (78.4%) or other indications (21.6%). Thirty-four major bleeding events (rate 1.1×100 patient-years) and 29 thromboembolic events (rate 0.9×100 patient-years) were recorded, and 46 patients (rate 1.5×100 patient-years) underwent reoperation. The rate of reoperation was higher among younger patients: 32.9% in patients <60 years and 3.9% in patients ≥60 years (relative risk (RR) 3.8, 95% CI 2.1 to 7.2; p=0.0001). When patients were analysed according to age <65 or ≥65 years and <75 or ≥70 years, younger patients still were at higher risk for reoperation (RR 3.1, 95% CI 1.7 to 6.0 and 3.7, 95% CI 1.7 to 8.6, respectively). Conclusions: Our findings suggest that the threshold of 65 years for implanting a BV should be carefully evaluated, considering the high risk for reoperation and the high risk of AF occurrence with persisting need for long-term anticoagulation. The high risk for reoperation of young patients implanted with BV and the availability of a safer and easier way to conduct VKA treatment, such as the use of point-of-care devices, should be considered when the type of valve must be chosen.

Cardiovascular benefits from ancient grain bread consumption: findings from a double-blinded randomized crossover intervention trial.

Ancient grain varieties have been shown to have some beneficial effects on health. Forty-five clinically healthy subjects were included in a randomized, double-blinded crossover trial aimed at evaluating the effect of a replacement diet with bread derived from ancient grain varieties versus modern grain variety on cardiovascular risk profile. After 8 weeks of intervention, consumption of bread obtained by the ancient varieties showed a significant amelioration of various cardiovascular parameters. Indeed, the ancient varieties were shown to result in a significant reduction of total cholesterol, low-density lipoprotein (LDL)-cholesterol and blood glucose, whereas no significant differences during the phase with the modern variety were reported. Moreover, a significant increase in circulating endothelial progenitor cells were reported after the consumption of products made from the ancient "Verna" variety. The present results suggest that a dietary consumption of bread obtained from ancient grain varieties was effective in reducing cardiovascular risk factors.

Light transmittance aggregometry induced by different concentrations of adenosine diphosphate to monitor clopidogrel therapy: a methodological study.

Light transmission aggregation (LTA) is considered the reference method to identify residual platelet reactivity (RPR) in high-risk patients with coronary artery disease on clopidogrel treatment. An international standardization of this technique is still ongoing and different concentrations of adenosine diphosphate (ADP) as the agonist for LTA have been used to evaluate the inhibitory effect of clopidogrel treatment. To evaluate RPR, LTA was performed using different ADP concentrations (2, 5, 10, and 20 µmol/L) in 466 high-risk patients with coronary artery disease on dual antiplatelet therapy who underwent percutaneous coronary intervention and in 46 healthy subjects. A VerifyNow P2Y12 assay was assessed as a point-of care system. Imprecision studies showed higher coefficients of variation for LTA by 2 and 5 µmol/L ADP (healthy subjects: 4.7% and 3.9%; patients: 6.8% and 5.2%, respectively) in comparison with those obtained determining LTA using 10 and 20 µmol/L ADP (healthy subjects: 2.2% and 2.3%; patients: 2.7% and 3.1%, respectively). In patients, a significant difference (P < 0.0001) between mean values of LTA obtained with all ADP concentrations was found, even if LTA data were significantly correlated (at least: rho = 0.88, P < 0.0001). However, data from 10 and 20 µmol/L ADP LTA were very similar and highly concordant (k = 95.9%). All agreements were significant (for all P < 0.0001), in particular the agreement between 10 and 20 µmol/L ADP LTA was very good (k = 0.86, P < 0.0001). A moderate agreement between VerifyNow and both 10 and 20 µmol/L ADP LTA was observed. LTA by 10 and 20 µmol/L ADP gave equivalent percentages of aggregation and highly concordant results in terms of RPR in patients with coronary artery disease on clopidogrel. Significant concordant results were observed between both 10 and 20 µM ADP LTA and VerifyNow. This suggests that a concentration of 10 µmol/L ADP may be considered adequate for the identification of RPR of patients on clopidogrel and should be preferred for standardization LTA.

Comparison of methods for monitoring residual platelet reactivity after clopidogrel by point-of-care tests on whole blood in high-risk patients.

Cardiovascular events are more frequent in high-risk coronary artery disease (CAD) patients on dual antiplatelet therapy with a residual platelet reactivity (RPR) than in those showing inhibition of ADP-inducible platelet activation. It is known that post-interventional RPR is a clinically important entity confirming it as a risk factor for thrombo-ischaemic events. Multiple electrode platelet aggregometry (MEA) on whole blood has been recently proposed as a rapid tool to evaluate RPR in high-risk CAD patients on clopidogrel therapy. It was the aim of this study to detect RPR in 801 high-risk CAD patients on dual antiplatelet therapy comparing MEA with the VerifyNow P2Y12 assay on whole blood and classical light transmission aggregation (LTA) on platelet-rich plasma. ADP (10 microM) was employed as agonist for MEA and LTA. The prevalence of RPR was 20.6% by MEA, 16.1% by LTA and 30.8% by VerifyNow. MEA showed a significant correlation (rho=0.62, p<0.0001) with VerifyNow and a moderate agreement (k=0.52, p<0.001) with 81.5% of concordant values. A significant correlation was found between MEA and LTA (rho=0.71, p<0.001) with a good agreement (k=0.63, p<0.001) and 88.8% of concordant values. MEA in relation to LTA showed a sensitivity of 80% and a specificity of 91%. MEA might represent a reliable method and valid alternative in comparison with other available platelet function assays. It might help to guide antiplatelet therapy and thus improve clinical outcome of high-risk CAD patients.

Assessment of platelet function on whole blood by multiple electrode aggregometry in high-risk patients with coronary artery disease receiving antiplatelet therapy.

This study sought to compare Multiplate impedance platelet aggregometry (IPA) with light transmission aggregometry (LTA) and the PFA-100 for determining the prevalence of residual platelet reactivity (RPR) by the Multiplate IPA in 297 patients with acute coronary syndrome receiving dual antiplatelet therapy. Aggregations were induced by adenosine-5 diphosphate (ADP), arachidonic acid, and collagen. PFA-100 closure times were measured by collagen and ADP and epinephrine (CEPI) cartridges. Significant correlations were observed between Multiplate IPA and LTA after all stimulations (P < .0001) and between Multiplate IPA (arachidonate and collagen) and PFA-100 CEPI closure time (P < .0001 for both). Cutoff values of Multiplate IPA (for all stimulations) were calculated for the identification of RPR. Between the Multiplate IPA and LTA good agreement was found with all 3 agonists (P < .0001 for all). Multiplate IPA might represent a reliable, handy, rapid tool to monitor antiplatelet therapy in clinical practice and for clinical investigations.