Direct RT-qPCR Assay for the Detection of SARS-CoV-2 in Saliva Samples

Since mid-2020 there have been complexities and difficulties in the standardisation and administration of nasopharyngeal swabs. Coupled with the variable and/or poor accuracy of lateral flow devices, this has led to increased societal ‘testing fatigue’ and reduced confidence in test results. Consequently, asymptomatic individuals have developed reluctance towards repeat testing, which remains the best way to monitor COVID-19 cases in the wider population. On the other hand, saliva-based PCR, a non-invasive, highly sensitive, and accurate test suitable for everyone, is gaining momentum as a straightforward and reliable means of detecting SARS-CoV-2 in symptomatic and asymptomatic individuals. Here, we provide an itemised list of the equipment and reagents involved in the process of sample submission, inactivation and analysis, as well as a detailed description of how each of these steps is performed

Performance evaluation of a non-invasive one-step multiplex RT-qPCR assay for detection of SARS-CoV-2 direct from saliva

Polymerase chain reaction (PCR) has proven to be the gold-standard for SARS-CoV-2 detection in clinical settings. The most common approaches rely on nasopharyngeal specimens obtained from swabs, followed by RNA extraction, reverse transcription and quantitative PCR. Although swab-based PCR is sensitive, swabbing is invasive and unpleasant to administer, reducing patient compliance for regular testing and resulting in an increased risk of improper sampling. To overcome these obstacles, we developed a non-invasive one-step RT-qPCR assay performed directly on saliva specimens. The University of Nottingham Asymptomatic Testing Service protocol simplifies sample collection and bypasses the need for RNA extraction, or additives, thus helping to encourage more regular testing and reducing processing time and costs. We have evaluated the assay against the performance criteria specified by the UK regulatory bodies and attained accreditation (BS EN ISO/IEC 17,025:2017) for SARS-CoV-2 diagnostic testing by the United Kingdom Accreditation Service. We observed a sensitivity of 1 viral copy per microlitre of saliva, and demonstrated a concordance of > 99.4% between our results and those of other accredited testing facilities. We concluded that saliva is a stable medium that allows for a highly precise, repeatable, and robust testing method

Characterisation of a Novel Producer, the Antimicrobial Properties and the Molecular Target of Zelkovamycin

The rise of antimicrobial resistance poses a threat to the treatment of infections caused by pathogenic and opportunistic bacteria, including Staphylococcus aureus and Pseudomonas aeruginosa. In a time of relentless search for new antibiotics, the recent isolation of a novel producer of zelkovamycin offered the opportunity to investigate the unidentified biosynthetic gene cluster of the antibiotic and encouraged the elucidation of its antimicrobial properties and uncertain target. In the first part of this work, the polyphasic characterisation the novel actinomycete strain 32-07 is presented. Based on its genome and 16S rRNA sequence, the microorganism was found to belong to the genus Actinomadura. Its phenotypic and genotypic characteristics, however, distinguished the isolate from its closest relative and its classification as Actinomadura graeca was therefore proposed. The new species was also confirmed as a producer of the antibiotic zelkovamycin, for which the putative biosynthetic cluster was identified. The antimicrobial properties of zelkovamycin against P. aeruginosa PAO1-L and S. aureus SH1000 are elucidated in the second part of the study. The antibiotic was confirmed effective against S. aureus and its previously unreported activity against P. aeruginosa was uncovered and characterised. In both species, zelkovamycin appeared to easily permeate cell membranes, although a marked increase in its activity was recorded against an efflux-deficient P. aeruginosa mutant. It is therefore postulated that zelkovamycin may be subject to active efflux in Gram-negative bacteria. The last part of this manuscript focuses on the investigation and identification of the molecular target of zelkovamycin. Each of several single amino acid substitutions in a single protein were found to confer S. aureus high resistance to the antibiotic, confirming the role of the protein as the target for zelkovamycin

Direct RT-qPCR Assay for the Detection of SARS-CoV-2 in Saliva Samples

Since mid-2020 there have been complexities and difficulties in the standardisation and administration of nasopharyngeal swabs. Coupled with the variable and/or poor accuracy of lateral flow devices, this has led to increased societal ‘testing fatigue’ and reduced confidence in test results. Consequently, asymptomatic individuals have developed reluctance towards repeat testing, which remains the best way to monitor COVID-19 cases in the wider population. On the other hand, saliva-based PCR, a non-invasive, highly sensitive, and accurate test suitable for everyone, is gaining momentum as a straightforward and reliable means of detecting SARS-CoV-2 in symptomatic and asymptomatic individuals. Here, we provide an itemised list of the equipment and reagents involved in the process of sample submission, inactivation and analysis, as well as a detailed description of how each of these steps is performed

Characterisation of a Novel Producer, the Antimicrobial Properties and the Molecular Target of Zelkovamycin

The rise of antimicrobial resistance poses a threat to the treatment of infections caused by pathogenic and opportunistic bacteria, including Staphylococcus aureus and Pseudomonas aeruginosa. In a time of relentless search for new antibiotics, the recent isolation of a novel producer of zelkovamycin offered the opportunity to investigate the unidentified biosynthetic gene cluster of the antibiotic and encouraged the elucidation of its antimicrobial properties and uncertain target. In the first part of this work, the polyphasic characterisation the novel actinomycete strain 32-07 is presented. Based on its genome and 16S rRNA sequence, the microorganism was found to belong to the genus Actinomadura. Its phenotypic and genotypic characteristics, however, distinguished the isolate from its closest relative and its classification as Actinomadura graeca was therefore proposed. The new species was also confirmed as a producer of the antibiotic zelkovamycin, for which the putative biosynthetic cluster was identified. The antimicrobial properties of zelkovamycin against P. aeruginosa PAO1-L and S. aureus SH1000 are elucidated in the second part of the study. The antibiotic was confirmed effective against S. aureus and its previously unreported activity against P. aeruginosa was uncovered and characterised. In both species, zelkovamycin appeared to easily permeate cell membranes, although a marked increase in its activity was recorded against an efflux-deficient P. aeruginosa mutant. It is therefore postulated that zelkovamycin may be subject to active efflux in Gram-negative bacteria. The last part of this manuscript focuses on the investigation and identification of the molecular target of zelkovamycin. Each of several single amino acid substitutions in a single protein were found to confer S. aureus high resistance to the antibiotic, confirming the role of the protein as the target for zelkovamycin

Updated definition of pulmonary hypertension and outcome after transcatheter aortic valve implantation

Objective: The European Society of Cardiology guidelines have recently defined new cut-offs for pulmonary hypertension (PH) and pulmonary vasculature resistance (PVR; median pulmonary artery pressure (mPAP) >20 instead of 25 mm Hg and PVR >2 instead of 3 Wood unit). The prognostic value of this updated classification after transcatheter aortic valve implantation (TAVI) is unknown. Methods: 579 consecutive patients treated by TAVI with preprocedural right heart catheterisation evaluation were included. Patients were grouped as: (1) no PH, (2) isolated precapillary/combined (I-PreC/Co) PH and (3) isolated postcapillary PH (I-PoC). All-cause death, cardiovascular death and hospitalisations for heart failure (HF) were evaluated at follow-up. We also analysed the prognostic role of residual postprocedural PH. Results: Out of 579 patients, 299 (52%) had PH according to the new criteria compared with 185 (32%) according to the previous ones. Overall median age was 82 years, while 55.3% patients were male. Patients with PH were more frequently diagnosed with chronic obstructive pulmonary disease and atrial fibrillation and were characterised by higher surgical risk as compared with patients without PH.At a median follow-up of 2.9 years, the presence of PH according to previous definition was associated with worse survival (p<0.001) and HF hospitalisation (p=0.002) rates, irrespective of PVR values. With newer cut-offs, PH was associated with worse outcomes only in patients with increased PVR, while no differences were found between patients with PH and normal PVR values and those without PH. Postprocedural mPAP normalisation was observed in 45% of the cases, but it was associated with improved long-term survival only in the I-PoC PH group. Conclusions: New ESC PH cut-offs increased the number of PH diagnoses. The presence of PH, particularly in the setting of increased PVR, identify patients at higher risk for postprocedural mortality and rehospitalisation. Normalisation of PH was associated with better survival only in I-PoC group

Coronary Access After Transcatheter Aortic Valve Replacement With Commissural Alignment: The ALIGN-ACCESS Study

Background: Coronary access (CA) after transcatheter aortic valve replacement (TAVR) with supra-annular transcatheter heart valves (THV) can be challenging. Specific Evolut R/Pro and Acurate Neo THVs orientations are associated with reduced neo-commissure overlap with coronary ostia, while SAPIEN 3 THV cannot be oriented. With the ALIGN-ACCESS study (TAVR With Commissural Alignment Followed by Coronary Access), we investigated the impact of commissural alignment on the feasibility of CA after TAVR. Methods: We performed coronary angiography after TAVR with intra-annular SAPIEN 3, supra-annular Evolut R/Pro, and Acurate Neo THVs in 206 patients. Evolut THVs were implanted aiming for commissure alignment. Alignment of Acurate Neo was retrospectively assessed in 36, intentionally attempted in 26 cases. The primary end point was the rate of unfeasible and nonselective CA after TAVR. Results: Thirty-eight percent of patients received SAPIEN 3, 31.1% Evolut Pro/R, 30.1% Acurate Neo THV. Final valve orientation was favorable to commissural alignment in 85.9% of Evolut and 69.4% of Acurate Neo cases (with intentional alignment successful in 88.5%). Selective CA was higher for SAPIEN 3 than for aligned and misaligned supra-annular THVs (95% versus 71% versus 46%, P<0.001). Cannulation of at least one coronary was unfeasible with 11% misaligned supra-annular, 3% aligned supra-annular, and 0% SAPIEN 3 THVs. Independent predictors of unfeasible/nonselective CA were implantation of a misaligned supra-annular THV (odds ratio, 4.59 [95% CI, 1.81-11.61]; P<0.01), sinus of Valsalva height (odds ratio, 0.83 [95% CI, 0.7-0.98]; P=0.03), and THV-sinus of Valsalva relation (odds ratio, 1.06 [95% CI, 1.02-1.1]; P<0.01). Conclusions: Commissural alignment improves the rate of selective CA after TAVR with supra-annular THVs. Nevertheless, aligned supra-annular THVs carry higher risk of unfeasible/nonselective CA than SAPIEN 3. Patients with a misaligned supra-annular THV, low sinus of Valsalva, and higher THV-sinus of Valsalva relation are at highest risk of impaired CA after TAVR

Definition of trAnscatheter heart Valve orIeNtation in biCuspId aortic valve: The DA VINCI pilot study

ObjectivesTo assess the impact of conventional transcatheter heart valve (THV) commissural alignment techniques on THV/coronary overlap and coronary access (CA) after transcatheter aortic valve replacement (TAVR) in bicuspid aortic valve (BAV). BackgroundSpecific Evolut Pro/Pro + and Acurate Neo2 THV orientations are associated with reduced neo-commissural overlap with coronary ostia in tricuspid aortic anatomy. Whether standard orientation techniques are effective also in the setting of BAV anatomy has not been studied. MethodsThe DA VINCI (Definition of trAnscatheter aortic Valve orIeNtation in biCuspId aortic valve) pilot study is a prospective registry enrolling consecutive patients with severe BAV stenosis undergoing TAVR with last generation supra-annular tall-frame THVs implanted with a cusp overlap view-based commissural alignment. Patients underwent pre- and post-TAVR computed tomography (CT) and coronary angiography. The study endpoint was the rate of favorable THV/coronary overlap, defined as an angle > 40 degrees between the THV commissural post and coronary ostia. Other endpoints were the rates of successful THV alignment with respect to the raphe and of selective CA after TAVR. Moreover, different virtual THV alignment models were tested to identify which one would produce the lower degree of THV/coronary overlap. ResultsThirty-four patients with type 1 BAV with right-left raphe undergoing TAVR (23 with Evolut Pro/Pro + and 11 with Acurate Neo2) were included. At pre-TAVR CT, moderate/severe cusp asymmetry was found in 50% of patients, severe coronary ostia eccentricity was observed in 47.1% for the RCA vs. 8.8% for the LCA (P < 0.007). Correct TVH orientation was achieved in 29 cases. At post-TAVR CT, optimal THV alignment/mild misalignment to the raphe was observed in 86.2%, but a moderate/severe overlap with the coronaries was seen in 13.7% for the RCA and 44.8% for the LCA (P = 0.019). After TAVR, selective RCA cannulation was possible in 82.8% vs. 75.9% for the LCA (P = 0.74), while combined selective CA of both coronaries was possible in less than two-thirds of the patients. Virtual THV alignment in the coronary ostia overlap view assuming a hypothetical circular THV expansion would produce an optimal THV/coronary overlap in almost 90% of cases. ConclusionGiven cusp asymmetry and coronary ostia eccentricity of BAV combined with potential THV asymmetrical expansion, conventional commissural alignment techniques are associated with higher rates of THV misalignment and of moderate/severe neo-commissure overlap with the coronary ostia as compared to tricuspid aortic stenosis, resulting in lower rates of selective CA after TAVR. A modified THV orientation technique based on the coronary ostia overlap view might be preferable in BAV patients

Real-World Experience With a Large Bore Vascular Closure Device During TAVI Procedure: Features and Predictors of Access-Site Vascular Complications

Backgrounds: Among vascular closure devices (VCDs), the novel collagen plug-based MANTA VCD is the first designed for large bore percutaneous access. We aimed to assess the features and predictors of access-site vascular complications in an unselected trans-femoral transcatheter aortic valve replacement (TF-TAVR) population. Methods: Patients undergoing large bore arteriotomy closure with 18F MANTA VCD following TF-TAVR at a large tertiary care center from September 2019 to January 2021 were prospectively analyzed. Primary Outcome was the MANTA VCD access-site-related complications according to Valve Academic Research Consortium-3 (VARC) definitions. Its incidence and predictors were evaluated. Results: Eighty-eight patients (median age 82 years, 48% male, 3.3 median Society of Thoracic Surgeons score) undergoing TF-TAVR were included, mostly (63%) treated with a self-expandable device and with outer diameter sizes varied from 18F to 24-F. MANTA VCD technical success rate was 98%, while 10 patients (11%) experienced MANTA VCD access-site vascular complications which included 8% of minor complications and only to 2% of major events resulting in VARC type ≥2 bleeding. Vessel occlusion/stenosis (60%), perforation (20%), and pseudoaneurysm/dissection/hematoma (20%) occurred, but all were managed without surgical treatment. Independent predictors of failure were age (p = 0.04), minimum common femoral artery diameter (CFA) (p < 0.01), sheath-to-femoral-artery ratio (SFAR) (p < 0.01), and a lower puncture height (p = 0.03). A CFA diameter <7.1 mm with a SFAR threshold of 1.01 were associated with VCD failure. Conclusions: In a more comers TF-TAVR population, MANTA VCD was associated with reassuring rates of technical success and major access-site vascular complications. Avoiding lower vessel size and less puncture site distance to CFA bifurcation might further improve outcomes