Human RNase Research Articles

Development of an efficient one-step real-time reverse transcription polymerase chain reaction method for severe acute respiratory syndrome-coronavirus-2 detection.

The general methods to detect the RNA of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) in clinical diagnostic testing involve reverse transcriptases and thermostable DNA polymerases. In this study, we compared the detection of SARS-CoV-2 by a one-step real-time RT-PCR method using a heat-resistant reverse transcriptase variant MM4 from Moloney murine leukemia virus, two thermostable DNA polymerase variants with reverse transcriptase activity from Thermotoga petrophila K4 and Thermococcus kodakarensis KOD1, or a wild-type DNA polymerase from Thermus thermophilus M1. The highest performance was achieved by combining MM4 with the thermostable DNA polymerase from T. thermophilus M1. These enzymes efficiently amplified specific RNA using uracil-DNA glycosylase (UNG) to remove contamination and human RNase P RNA amplification as an internal control. The standard curve was obtained from 5 to 105 copies of synthetic RNA. The one-step real-time RT-PCR method’s sensitivity and specificity were 99.44% and 100%, respectively (n = 213), compared to those of a commercially available diagnostic kit. Therefore, our method will be useful for the accurate detection and quantification of SARS-CoV-2.

Human ribonuclease 1 serves as a secretory ligand of ephrin A4 receptor and induces breast tumor initiation

Human ribonuclease 1 (hRNase 1) is critical to extracellular RNA clearance and innate immunity to achieve homeostasis and host defense; however, whether it plays a role in cancer remains elusive. Here, we demonstrate that hRNase 1, independently of its ribonucleolytic activity, enriches the stem-like cell population and enhances the tumor-initiating ability of breast cancer cells. Specifically, secretory hRNase 1 binds to and activates the tyrosine kinase receptor ephrin A4 (EphA4) signaling to promote breast tumor initiation in an autocrine/paracrine manner, which is distinct from the classical EphA4-ephrin juxtacrine signaling through contact-dependent cell-cell communication. In addition, analysis of human breast tumor tissue microarrays reveals a positive correlation between hRNase 1, EphA4 activation, and stem cell marker CD133. Notably, high hRNase 1 level in plasma samples is positively associated with EphA4 activation in tumor tissues from breast cancer patients, highlighting the pathological relevance of the hRNase 1-EphA4 axis in breast cancer. The discovery of hRNase 1 as a secretory ligand of EphA4 that enhances breast cancer stemness suggests a potential treatment strategy by inactivating the hRNase 1-EphA4 axis.

Buccal swabs as non-invasive specimens for detection of severe acute respiratory syndrome coronavirus-2.

IntroductionThe current gold standard for detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA involves subjecting nasopharyngeal or oropharyngeal swabs to reverse transcription quantitative PCR (RT-qPCR). However, both sample types need to be collected by trained professionals. Using self-collected buccal swabs as an alternative could simplify and accelerate diagnosis of coronavirus disease 2019 (COVID-19).ObjectiveTo assess self-collected buccal swab samples as an alternative method for SARS-CoV-2 detection in patients with COVID-19.MethodsBuccal swab samples were self-collected by 73 patients with COVID-19. Total RNA was extracted using Qiagen kits. RNA encoding the SARS-CoV-2 Env protein and human RNase P as an internal control was amplified using the TRUPCR® SARS-CoV-2 RT-qPCR kit version 2.1 and a Bio-Rad CFX96 Real-Time Detection System.ResultThe sensitivity of RT-qPCR from buccal swabs was 58.9% (43/73; 95% confidence interval [CI] 46.77%–70.27%) and that of RT-qPCR from saliva was 62.90% (39/62; 95% CI 49.69%–74.84%) taking positive SARS-CoV-2 RT-qPCR from nasopharyngeal swabs as the gold standard.ConclusionSelf-collected buccal swabs are promising alternatives to nasopharyngeal or oropharyngeal swabs for SARS CoV-2 detection.

Expression and function of human ribonuclease 4 in the kidney and urinary tract.

Antimicrobial peptides are essential host defense mechanisms that prevent urinary tract infections. Recent studies have demonstrated that peptides in the ribonuclease A superfamily have antimicrobial activity against uropathogens and protect the urinary tract from uropathogenic Escherichia coli (UPEC). Little is known about the antibacterial function or expression of ribonuclease 4 (RNase 4) in the human urinary tract. Here, we show that full-length recombinant RNase 4 peptide and synthetic amino-terminal RNase 4 peptide fragment have antibacterial activity against UPEC and multidrug-resistant (MDR)-UPEC. RNASE4 transcript expression was detected in human kidney and bladder tissue using quantitative real-time PCR. Immunostaining or in situ hybridization localized RNase 4 expression to proximal tubules, principal and intercalated cells in the kidney's collecting duct, and the bladder urothelium. Urinary RNase 4 concentrations were quantified in healthy controls and females with a history of urinary tract infection. Compared with controls, urinary RNase 4 concentrations were significantly lower in females with a history of urinary tract infection. When RNase 4 was neutralized in human urine or silenced in vitro using siRNA, urinary UPEC replication or attachment to and invasion of urothelial and kidney medullary cells increased. These data show that RNase 4 has antibacterial activity against UPEC, is expressed in the human urinary tract, and can contribute to host defense against urinary tract infections.NEW & NOTEWORTHY Ribonuclease 4 (RNase 4) is a newly identified host defense peptide in the human kidney and bladder. RNase 4 kills uropathogenic Escherichia coli (UPEC) and multidrug-resistant UPEC. RNase 4 prevents invasive UPEC infection and suppressed RNase 4 expression may be a risk factor for more severe or recurrent urinary tract infection.

Enhanced throughput of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) real-time RT-PCR panel by assay multiplexing and specimen pooling

A multiplex real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assay for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was developed based on the same primer and probe sequences of an existing U.S. CDC Emergency Use authorized test panel, targeting SARS-CoV-2 N1, N2 and human RNase P genes in singleplex. Both singleplex and multiplex assays demonstrated linear dynamic ranges of 8 orders of magnitude and analytical limits of detection of 5 RNA transcript copies/reaction. Both assays showed 100 % agreement with 364 previously characterized clinical specimens (146 positive and 218 negative) for detection of SARS-CoV-2 RNA. To further increase testing throughput, 40 positive and 20 negative four-specimen pools were tested by the multiplex assay and showed 97.75 % and 100 % congruence with individual specimen tests, respectively. rRT-PCR assay multiplexing and sample pooling, individually or in combination, can substantially increase throughput of SARS-CoV-2 testing.

Development and performance evaluation of the first in-house multiplex rRT-PCR assay in Bangladesh for highly sensitive detection of SARS-CoV-2

BackgroundThe emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic is posing a great threat to global health and economy. Due to the lack of broad diagnostic setup, consistent reagent supply lines, and access to laboratory instruments and equipment, it is undoubtedly an enormous burden for developing countries to face the crisis. ObjectivesTo develop a cost-effective, reliable and sensitive multiplex assay for SARS-CoV-2 screening which would expand the testing capacities of a developing and low-income country like Bangladesh. Study designInitially a singleplex and then a multiplex real-time reverse-transcriptase PCR assays were developed targeting 2 nucleocapsid genes of SARS-CoV-2, and the human RNase P gene as an internal control using laboratory-made mastermixes. Three sets of primer- probes were designed for each of the target genes and one set was optimized for the final reaction set-up. Limit of detection, cross-reactivity and reproducibility were checked in order to assess the sensitivity and specificity of the assays, and validation was done using clinical specimens. ResultsClinical evaluation of the new assays using 240 nasopharyngeal swabs showed 100 % sensitivity, specificity, and accuracy in detecting SARS-CoV-2 infection in human. Equal efficiency and concordant results were observed between the singleplex and multiplex approaches. Notably, the kit was able to detect SARS-CoV-2 RNA at very low concentration upto 5 copies/reaction. ConclusionThis is the first locally developed multiplex rRT-PCR kit in Bangladesh providing rapid and low-cost screening of COVID-19 which would be valuable for infection prevention and clinical management in the perspective of Bangladesh.

MOLECULAR DETECTION OF COVID -19 BY TRUENAT RT-PCR IN A TERTIARY CARE HOSPITAL IN DELHI NCR REGION

Introduction: COVID-19 is a severe infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) affected lives of millions of people , responsible for millions of death affecting health care systems worldwide seriously. To diagnose Aims and Objectives: COVID -19 infection in a tertiary care hospital using TrueNat RTPCR and to categorise and co - relate various Ct values with viral load. Material and Methods: Oropharyngeal and nasopharyngeal swab specimens were collected from the patients following standard protocols and were inserted into the viral lysis medium tube. Specimen is transferred from viral lysis medium to automatic extracted device for extraction of RNA and then into RT-PCR analyser for reaction to start automatically. Test detects the screening E gene and conrmatory RdRp /Orf1a gene and human RNase P. Of the 1025 patients subjected to COVID - 19- RTPCR 630 (61%) were male Results : s and 395 (39%) were females. 26% (269/1025) of patients were conrmed COVID positive and 72% (747/1025) were negative. Age group 21-30 showed maximum positive cases followed by age group 51-60 years. High viral load was seen in 41% cases whereas maximum no. of conrmed positive had low viral loads. Rapid and Conclusion: accurate diagnostic methods are required for early detection along with precautionary measures for timely therapeutic interventions and prophylaxis to control and prevent the Spread of highly contagious COVID-19.

The Selectivity for Tumor Cells of Nuclear-Directed Cytotoxic RNases Is Mediated by the Nuclear/Cytoplasmic Distribution of p27KIP1.

Although single targeted anti-cancer drugs are envisaged as safer treatments because they do not affect normal cells, cancer is a very complex disease to be eradicated with a single targeted drug. Alternatively, multi-targeted drugs may be more effective and the tumor cells may be less prone to develop drug resistance although these drugs may be less specific for cancer cells. We have previously developed a new strategy to endow human pancreatic ribonuclease with antitumor action by introducing in its sequence a non-classical nuclear localization signal. These engineered proteins cleave multiple species of nuclear RNA promoting apoptosis of tumor cells. Interestingly, these enzymes, on ovarian cancer cells, affect the expression of multiple genes implicated in metabolic and signaling pathways that are critic for the development of cancer. Since most of these targeted pathways are not highly relevant for non-proliferating cells, we envisioned the possibility that nuclear directed-ribonucleases were specific for tumor cells. Here, we show that these enzymes are much more cytotoxic for tumor cells in vitro. Although the mechanism of selectivity of NLSPE5 is not fully understood, herein we show that p27KIP1 displays an important role on the higher resistance of non-tumor cells to these ribonucleases.

Crystal structure of human RPP20-RPP25 proteins in complex with the P3 domain of lncRNA RMRP

Human RNase MRP ribonucleoprotein complex is an essential endoribonuclease involved in the processing of ribosomal RNAs, mitochondrial RNAs and certain messenger RNAs. Its RNA subunit RMRP catalyzes the cleavage of substrate RNAs, and the protein components of RNase MRP are required for activity. RMRP mutations are associated with several types of inherited developmental disorders, but the pathogenic mechanism is largely unknown. Recent structural studies shed lights on the catalytic mechanism of yeast RNase MRP and the closely related RNase P; however, the structural and catalytic mechanism of RMRP in human RNase MRP complex remains unclear. Here we report the crystal structure of the P3 domain of RMRP in complex with the RPP20 and RPP25 proteins of human RNase MRP, which shows that the P3 RNA binds to a conserved positively-charged surface of the RPP20-RPP25 heterodimer through its distal stem and internal loop regions. The disease-related mutations of RMRPP3 are mostly located at the protein-RNA interface and are likely to weaken the binding of P3 to RPP20-RPP25. Moreover, the structure reveals a homodimeric organization of the entire RPP20-RPP25-RMRPP3 complex, which might mediate the dimerization of human RNase MRP complex in cells. These findings provide structural clues to the assembly and pathogenesis of human RNase MRP complex and also reveal a tetrameric feature of RPP20-RPP25 evolutionarily conserved with that of the archaeal Alba proteins.

Virus-Like Particles as Positive Controls for COVID-19 RT-LAMP Diagnostic Assays.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a rapid and inexpensive isothermal alternative to the current gold standard reverse transcription quantitative polymerase chain reaction (RT-qPCR) for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, unlike RT-qPCR, there are no consensus detection regions or optimal RT-LAMP methods, and most protocols do not include internal controls to ensure reliability. Naked RNAs, plasmids, or even RNA from infectious COVID-19 patients have been used as external positive controls for RT-LAMP assays, but such reagents lack the stability required for full-process control. To overcome the lack of proper internal and external positive controls and the instability of the detection RNA, we developed virus-like particles (VLPs) using bacteriophage Qβ and plant virus cowpea chlorotic mottle virus (CCMV) for the encapsidation of target RNA, namely a so-called SARS-CoV-2 LAMP detection module (SLDM). The target RNA is a truncated segment of the SARS-CoV-2 nucleocapsid (N) gene and human RNase P gene (internal control) as positive controls for RT-qPCR and RT-LAMP. Target RNAs stably encapsidated in Qβ and CCMV VLPs were previously shown to function as full-process controls in RT-qPCR assays, and here we show that SLDMs can fulfill the same function for RT-LAMP and swab-to-test (direct RT-LAMP with heat lysis) assays. The SLDM was validated in a clinical setting, highlighting the promise of VLPs as positive controls for molecular assays.

Understanding false positives and the detection of SARS-CoV-2 using the Cepheid Xpert Xpress SARS-CoV-2 and BD MAX SARS-CoV-2 assays

Several real-time RT-PCR assays have received Emergency Use Authorization from the United States Food and Drug Administration. The BD MAX™ SARS-CoV-2 assay, run by the BD MAX™ system, is a qualitative test that detects the SARS-CoV-2 specific nucleocapsid phosphoprotein gene regions, N1 and N2. The human RNase P gene is used as the endogenous nucleic acid extraction control. The Cepheid Xpert® Xpress SARS-CoV-2 assay, run by the GeneXpert system, detects the pan-sarbecovirus E gene and the N2 region of the N gene. We evaluated the performance characteristics of the BD and Cepheid assays using matched patient samples. We also analyzed comparative Ct values for both assays using 183 positive samples tested at this facility. In addition, we mitigated reporting false positive results without relying on interpretive software. We found that both systems showed comparable sensitivity. We found an approximately 3.5% false positive rate from the BD MAX™ system results.

Performance of Unobserved Self-Collected Nasal Swabs for Detection of SARS-CoV-2 by RT-PCR Utilizing a Remote Specimen Collection Strategy.

BackgroundThe use of a remote specimen collection strategy employing a kit designed for unobserved self-collection for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse transcription polymerase chain reaction (RT-PCR) can decrease the use of personal protective equipment (PPE) and exposure risk. To assess the impact of unobserved specimen self-collection on test performance, we examined results from a SARS-CoV-2 qualitative RT-PCR test for self-collected specimens from participants in a return-to-work screening program and assessed the impact of a pooled testing strategy in this cohort.MethodsSelf-collected anterior nasal swabs from employee return-to-work programs were tested using the Quest Diagnostics Emergency Use Authorization SARS-CoV-2 RT-PCR. The cycle threshold (Ct) values for the N1 and N3 N-gene targets and a human RNase P (RP) gene control target were tabulated. For comparison, we utilized Ct values from a cohort of health care provider–collected specimens from patients with and without coronavirus disease 2019 symptoms.ResultsAmong 47 923 participants, 1.8% were positive. RP failed to amplify for 13/115 435 (0.011%) specimens. The median (interquartile range) Cts were 32.7 (25.0–35.7) for N1 and 31.3 (23.8–34.2) for N3. Median Ct values in the self-collected cohort were significantly higher than those of symptomatic but not asymptomatic patients. Based on Ct values, pooled testing with 4 specimens would have yielded inconclusive results in 67/1268 (5.2%) specimens but only a single false-negative result.ConclusionsUnobserved self-collection of nasal swabs provides adequate sampling for SARS-CoV-2 RT-PCR testing. These findings alleviate concerns of increased false negatives in this context. Specimen pooling could be used for this population, as the likelihood of false-negative results is very low when using a sensitive, dual-target methodology.

Bioaerosol Sampling for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a Referral Center with Critically Ill Coronavirus Disease 2019 (COVID-19) Patients March-May 2020.

BackgroundPrevious research has shown that rooms of patients with coronavirus disease 2019 (COVID-19) present the potential for healthcare-associated transmission through aerosols containing severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). However, data on the presence of these aerosols outside of patient rooms are limited. We investigated whether virus-containing aerosols were present in nursing stations and patient room hallways in a referral center with critically ill COVID-19 patients.MethodsEight National Institute for Occupational Safety and Health BC 251 2-stage cyclone samplers were set up throughout 6 units, including nursing stations and visitor corridors in intensive care units and general medical units, for 6 h each sampling period. Samplers were placed on tripods which held 2 samplers positioned 102 cm and 152 cm above the floor. Units were sampled for 3 days. Extracted samples underwent reverse transcription polymerase chain reaction for selected gene regions of the SARS-CoV-2 virus nucleocapsid and the housekeeping gene human RNase P as an internal control.ResultsThe units sampled varied in the number of laboratory-confirmed COVID-19 patients present on the days of sampling. Some of the units included patient rooms under negative pressure, while most were maintained at a neutral pressure. Of 528 aerosol samples collected, none were positive for SARS-CoV-2 RNA by the estimated limit of detection of 8 viral copies/m3 of air.ConclusionsAerosolized SARS-CoV-2 outside of patient rooms was undetectable. While healthcare personnel should avoid unmasked close contact with each other, these findings may provide reassurance for the use of alternatives to tight-fitting respirators in areas outside of patient rooms during the current pandemic.

Prevalence of SARS-CoV-2 infection in the workplace: results of a year of investigation in the Marche Nord companies.

Background and aim:The world of work has been profoundly affected by the COVID-19 pandemic since workplace activities involving close contact with coworkers and customers can lead to transmission of SARS-CoV-2 and compromise continuity of operations of workplaces.The Covid-Lab of University of Urbino and the Confindustria Pesaro Urbino association signed an agreement to support the Marche Nord companies in the adoption of anti-contagion safety protocols.Methods:Antibodies detection was performed using a rapid immunochromatographic test. Total RNA from nasopharyngeal swab was subjected to a real-time RT-PCR multiplex for the detection of RdRp specific gene and E gene of the SARS-CoV-2, and the internal control (human RNase P).Results:Between May 2020 and Apr 2021, over 10,000 rapid serological tests had been carried out on workers of 35 companies and in 5% of cases IgG or IgM were found (519). All the 519 swabs gave a valid result (RNAse P Ct≤40) with 105 positive results (20%) for SARS-CoV-2 with a Ct value ≤45. Overall, only 1% of samples resulted positive for viral RNA (105/10000).Conclusions:The University of Urbino set up a rapid-response (within 24 h, generally <6 h) diagnostic centre for SARS-CoV-2 detection (Covid-Lab) allowing the companies to activate the optimal safety path to ensure the health and safety of workers in the workplace. Our observations during this first year of activity, highlight that in the workplace, the infection does not seem to spread if precautionary measures are followed and only 1% (1 worker out of 100) tested positive for the SARS-CoV-2 virus. (www.actabiomedica.it)

Semisynthesis of Human Ribonuclease-S.

Since its conception, the ribonuclease S complex (RNase S) has led to historic discoveries in protein chemistry, enzymology, and related fields. Derived by the proteolytic cleavage of a single peptide bond in bovine pancreatic ribonuclease (RNase A), RNase S serves as a convenient and reliable model system for incorporating unlimited functionality into an enzyme. Applications of the RNase S system in biomedicine and biotechnology have, however, been hindered by two shortcomings: (1) the bovine-derived enzyme could elicit an immune response in humans, and (2) the complex is susceptible to dissociation. Here, we have addressed both limitations in the first semisynthesis of an RNase S conjugate derived from human pancreatic ribonuclease and stabilized by a covalent interfragment cross-link. We anticipate that this strategy will enable unprecedented applications of the "RNase-S" system.

Self-Sampling of Oropharyngeal Swabs Among Healthcare Workers for Molecular Detection of Respiratory Viruses: A Valuable Approach for Epidemiological Studies and Surveillance Programs.

This study aimed at assessing the validity of self-collected (self-sampled) oropharyngeal (OP) swabs among healthcare workers compared to those collected by trained sentinel general practitioners (GP-sampled) from individuals with influenza-like illness (ILI), to be implemented in epidemiological studies and/or surveillance programs of viral pathogens involved in community respiratory infections. In our study, OP swabs were collected from adults (>18 years) with ILI during the 2018–2019 influenza season. Two groups of samples were considered: group 1−131 self-sampled OP swabs collected by healthcare workers after being trained on the sampling procedure; group 2−131 GP-sampled OP swabs collected from outpatients by sentinel GPs operating within the Italian Influenza Surveillance Network. To assess swabbing quality, following RNA extraction, each sample was tested for the presence of the human ribonuclease P gene (RNP) by in-house real-time reverse transcriptase–polymerase chain reaction (RT-PCR). Samples with a cycle threshold (Ct) <35 were considered adequate for further virological analysis. Influenza viruses (IVs), respiratory syncytial virus (RSV), and rhinovirus (RV) genomes were detected by in-house real-time RT-PCR. All samples were positive to RNP detection with Ct <35. The mean Ct value was similar in the two groups (group 1 vs. group 2: 25.93 ± 2.22 vs. 25.46 ± 2.40; p = 0.10). IVs, RSV, and RV positivity rates were 26.7 vs. 52.7% (p < 0.01), 7.6 vs. 9.9% (p = 0.52), and 21.4 vs. 19.9% (p = 0.76), respectively. Self-sampled OP swabs resulted as valid as GP-sampled OP swabs for molecular detection of respiratory viruses. Self-swabbing can thus be a worthwhile strategy for sample collection to implement molecular surveillance of respiratory pathogens and carry out epidemiological studies, easily reaching a larger population size.

Sensitivity of RT-PCR testing of upper respiratory tract samples for SARS-CoV-2 in hospitalised patients: a retrospective cohort study

Background: This study aimed to determine the sensitivity and specificity of reverse transcription PCR (RT-PCR) testing of upper respiratory tract samples from hospitalised patients with coronavirus disease 2019 (COVID-19), compared to the gold standard of a clinical diagnosis. Methods: All RT-PCR testing for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in NHS Lothian, Scotland, United Kingdom between the 7 th of February and 19 th April 2020 (inclusive) was reviewed, and hospitalised patients were identified. All upper respiratory tract RT-PCR tests were analysed for each patient to determine the sequence of negative and positive results. For those who were tested twice or more but never received a positive result, case records were reviewed, and a clinical diagnosis of COVID-19 allocated based on clinical features, discharge diagnosis, and radiology and haematology results. For those who had a negative RT-PCR test but a clinical diagnosis of COVID-19, respiratory samples were retested using a multiplex respiratory panel, a second SARS-CoV-2 RT-PCR assay, and a human RNase P control. Results: Compared to the gold standard of a clinical diagnosis of COVID-19, the sensitivity of a single upper respiratory tract RT-PCR for COVID-19 was 82.2% (95% confidence interval 79.0-85.1%). The sensitivity of two upper respiratory tract RT-PCR tests increased sensitivity to 90.6% (CI 88.0-92.7%). A further 2.2% and 0.9% of patients who received a clinical diagnosis of COVID-19 were positive on a third and fourth test; this may be an underestimate of the value of further testing as the majority of patients 93.0% (2999/3226) only had one or two RT-PCR tests. Conclusions: The sensitivity of a single RT-PCR test of upper respiratory tract samples in hospitalised patients is 82.2%. Sensitivity increases to 90.6% when patients are tested twice. A proportion of cases with clinically defined COVID-19 never test positive on RT-PCR despite repeat testing.

RNase A Treatment Interferes With Leukocyte Recruitment, Neutrophil Extracellular Trap Formation, and Angiogenesis in Ischemic Muscle Tissue.

Background: RNase A (the bovine equivalent to human RNase 1) and RNase 5 (angiogenin) are two closely related ribonucleases. RNase 5 is described as a powerful angiogenic factor. Whether RNase A shares the same angiogenic characteristic, or interferes with vessel growth as demonstrated for arteriogenesis, has never been investigated and is the topic of this present study.Methods and Results: To investigate whether RNase A shows a pro‐ or anti-angiogenic effect, we employed a murine hindlimb model, in which femoral artery ligation (FAL) results in arteriogenesis in the upper leg, and, due to provoked ischemia, in angiogenesis in the lower leg. C57BL/6J male mice underwent unilateral FAL, whereas the contralateral leg was sham operated. Two and seven days after the surgery and intravenous injection of RNase A (50 μg/kg dissolved in saline) or saline (control), the gastrocnemius muscles of mice were isolated from the lower legs for (immuno-) histological analyses. Hematoxylin and Eosin staining evidenced that RNase A treatment resulted in a higher degree of ischemic tissue damage. This was, however, associated with reduced angiogenesis, as evidenced by a reduced capillary/muscle fiber ratio. Moreover, RNase A treatment was associated with a significant reduction in leukocyte infiltration as shown by CD45+ (pan-leukocyte marker), Ly6G+ or MPO+ (neutrophils), MPO+/CitH3+ [neutrophil extracellular traps (NETs)], and CD68+ (macrophages) staining. CD68/MRC1 double staining revealed that RNase A treated mice showed a reduced percentage of M1-like polarized (CD68+/MRC1−) macrophages whereas the percentage of M2-like polarized (CD68+/MRC1+) macrophages was increased.Conclusion: In contrast to RNase 5, RNase A interferes with angiogenesis, which is linked to reduced leukocyte infiltration and NET formation.

SARS-CoV-2 Detection in Gingival Crevicular Fluid

Understanding the pathophysiology of the coronavirus disease 2019 (COVID-19) infection remains a significant challenge of our times. The gingival crevicular fluid being representative of systemic status and having a proven track record of detecting viruses and biomarkers forms a logical basis for evaluating the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The study aimed to assess gingival crevicular fluid (GCF) for evidence of SARS-CoV-2 in 33 patients who were deemed to be COVID-19 positive upon nasopharyngeal sampling. An attempt was also made to comparatively evaluate it with saliva in terms of its sensitivity, as a diagnostic fluid for SARS-CoV-2. GCF and saliva samples were collected from 33 COVID-19–confirmed patients. Total RNA was extracted using NucliSENS easyMAG (bioMérieux) and eluted in the elution buffer. Envelope gene (E gene) of SARS-CoV-2 and human RNase P gene as internal control were detected in GCF samples by using the TRUPCR SARS-CoV-2 RT qPCR kit V-2.0 (I) in an Applied Biosystems 7500 real-time machine. A significant majority of both asymptomatic and mildly symptomatic patients exhibited the presence of the novel coronavirus in their GCF samples. Considering the presence of SARS-CoV-2 RNA in the nasopharyngeal swab sampling as gold standard, the sensitivity of GCF and saliva, respectively, was 63.64% (confidence interval [CI], 45.1% to 79.60%) and 64.52% (CI, 45.37% to 80.77%). GCF was found to be comparable to saliva in terms of its sensitivity to detect SARS-CoV-2. Saliva samples tested positive in 3 of the 12 patients whose GCF tested negative, and likewise GCF tested positive for 2 of the 11 patients whose saliva tested negative on real-time reverse transcription polymerase chain reaction. The results establish GCF as a possible mode of transmission of SARS-CoV-2, which is the first such report in the literature, and also provide the first quantifiable evidence pointing toward a link between the COVID-19 infection and oral health.

Sensitivity of RT-PCR testing of upper respiratory tract samples for SARS-CoV-2 in hospitalised patients: a retrospective cohort study

Background: This study aimed to determine the sensitivity and specificity of reverse transcription PCR (RT-PCR) testing of upper respiratory tract (URT) samples from hospitalised patients with coronavirus disease 2019 (COVID-19), compared to the gold standard of a clinical diagnosis. Methods: All URT RT-PCR testing for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in NHS Lothian, Scotland, United Kingdom between the 7th of February and 19th April 2020 (inclusive) was reviewed, and hospitalised patients were identified. All URT RT-PCR tests were analysed for each patient to determine the sequence of negative and positive results. For those who were tested twice or more but never received a positive result, case records were reviewed, and a clinical diagnosis of COVID-19 allocated based on clinical features, discharge diagnosis, and radiology and haematology results. For those who had a negative RT-PCR test but a clinical diagnosis of COVID-19, respiratory samples were retested using a multiplex respiratory panel, a second SARS-CoV-2 RT-PCR assay, and a human RNase P control. Results: Compared to the gold standard of a clinical diagnosis of COVID-19, the sensitivity of a single upper respiratory tract RT-PCR for COVID-19 was 82.2% (95% confidence interval 79.0-85.1%). The sensitivity of two upper respiratory tract RT-PCR tests increased sensitivity to 90.6% (CI 88.0-92.7%). A further 2.2% and 0.9% of patients who received a clinical diagnosis of COVID-19 were positive on a third and fourth test; this may be an underestimate of the value of further testing as the majority of patients 93.0% (2999/3226) only had one or two URT RT-PCR tests. Conclusions: The sensitivity of a single RT-PCR test of URT samples in hospitalised patients is 82.2%. Sensitivity increases to 90.6% when patients are tested twice. A proportion of cases with clinically defined COVID-19 never test positive on URT RT-PCR despite repeat testing.