scholarly journals A Prospective Diagnostic Study to Measure the Accuracy of Detection of SARS-CoV-2 Variants Of Concern (VOC) Utilising a Novel RT-PCR GENotyping Algorithm in an in silico Evaluation (VOC-GENIE)

2021 ◽  
Author(s):  
Daryl Borley ◽  
R.A. Trevor ◽  
Alex G. Richter ◽  
Stephen Kidd ◽  
Nick Cortes ◽  
...  
Keyword(s):  
In Silico ◽  
Diagnostic Study ◽  
Rt Pcr ◽  
Pcr Genotyping

scholarly journals A prospective diagnostic study to measure the accuracy of detection of SARS-CoV-2 Variants Of Concern (VOC) utilising a novel RT-PCR GENotyping algorithm in an In silico Evaluation (VOC-GENIE)

2021 ◽  
Author(s):  
Daryl Borley ◽  
R A Trevor ◽  
Alex Richter ◽  
Stephen Kidd ◽  
Nick Cortes ◽  
...  
Keyword(s):  
Immune Evasion ◽  
In Silico ◽  
Diagnosis And Treatment ◽  
Diagnostic Study ◽  
Data Set ◽  
In Situ Pcr ◽  
Treatment Patient ◽  
Patient Pathway ◽  
Pcr Genotyping

Background SARS-CoV-2 variants of concern (VOCs) have been associated with higher rate of transmission, and evasion of immunisation and antibody therapeutics. Variant sequencing is widely utilized in the UK. However, only 0.5% (~650k) of the 133 million cumulative positive cases worldwide were sequenced (in GISAID) on 08 April 2021 with 97% from Europe and North America and only ~0.25% (~320k) were variant sequences. This may be due to the lack of availability, high cost, infrastructure and expert staff required for sequencing. Public health decisions based on a non-randomised sample of 0.5% of the population may be insufficiently powered, and subject to sampling bias and systematic error. In addition, sequencing is rarely available in situ in a clinically relevant timeframe and thus, is not currently compatible with diagnosis and treatment patient care pathways. Therefore, we investigated an alternative approach using polymerase chain reaction (PCR) genotyping to detect the key single nucleotide polymorphisms (SNPs) associated with increased transmission and immune evasion in SARS-CoV-2 variants. Methods We investigated the utility of SARS-CoV-2 SNP detection with a panel of PCR-genotyping assays in a large data set of 640,482 SARS-CoV-2 high quality, full length sequences using a prospective in silico trial design and explored the potential impact of rapid in situ variant testing on the COVID-19 diagnosis and treatment patient pathway. Results Five SNPs were selected by screening the published literature for a reported association with increased transmission and / or immune evasion. 344881 sequences contained one or more of the five SNPs. This algorithm of SNPs was found to be able to identify the four variants of concern (VOCs) and sequences containing the E484K and L452R escape mutations. Interpretation The in silico analysis suggest that the key mutations and variants of SARS-CoV-2 may be reliably detected using a focused algorithm of biologically relevant SNPs. This highlights the potential for rapid in situ PCR genotyping to compliment or replace sequencing or to be utilized instead of sequences in settings where sequencing is not feasible, accessible or affordable. Rapid detection of variants with in situ PCR genotyping may facilitate a more effective COVID-19 diagnosis and treatment patient pathway.

scholarly journals Triage of Patients Suspected of COVID-19 in Chronic Hemodialysis: Eosinophil Count Differentiates Low and High Suspicion of COVID-19

2020 ◽  
Vol 10 (1) ◽  
pp. 4
Author(s):  
Romain Vial ◽  
Marion Gully ◽  
Mickael Bobot ◽  
Violaine Scarfoglière ◽  
Philippe Brunet ◽  
...  
Keyword(s):  
Predictive Value ◽  
Eosinophil Count ◽  
Characteristic Curve ◽  
Final Analysis ◽  
Hemodialysis Patients ◽  
Chronic Hemodialysis ◽  
Screening Methods ◽  
Dialysis Session ◽  
Diagnostic Study ◽  
Rt Pcr

Background: Daily management to shield chronic dialysis patients from SARS-CoV-2 contamination makes patient care cumbersome. There are no screening methods to date and a molecular biology platform is essential to perform RT-PCR for SARS-CoV-2; however, accessibility remains poor. Our goal was to assess whether the tools routinely used to monitor our hemodialysis patients could represent reliable and quickly accessible diagnostic indicators to improve the management of our hemodialysis patients in this pandemic environment. Methods: In this prospective observational diagnostic study, we recruited patients from La Conception hospital. Patients were eligible for inclusion if suspected of SARS-CoV-2 infection when arriving at our center for a dialysis session between March 12th and April 24th 2020. They were included if both RT-PCR result for SARS-CoV-2 and cell blood count on the day that infection was suspected were available. We calculated the area under the curve (AUC) of the receiver operating characteristic curve. Results: 37 patients were included in the final analysis, of which 16 (43.2%) were COVID-19 positive. For the day of suspected COVID-19, total leukocytes were significantly lower in the COVID-19 positive group (4.1 vs. 7.4 G/L, p = 0.0072) and were characterized by lower neutrophils (2.7 vs. 5.1 G/L, p = 0.021) and eosinophils (0.01 vs. 0.15 G/L, p = 0.0003). Eosinophil count below 0.045 G/L identified SARS-CoV-2 infection with AUC of 0.9 [95% CI 0.81—1] (p < 0.0001), sensitivity of 82%, specificity of 86%, a positive predictive value of 82%, a negative predictive value of 86% and a likelihood ratio of 6.04. Conclusions: Eosinophil count enables rapid routine screening of symptomatic chronic hemodialysis patients suspected of being COVID-19 within a range of low or high probability.

scholarly journals Best Molecular Tools to Investigate Coronavirus Diversity in Mammals: A Comparison

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1975
Author(s):  
Petra Drzewnioková ◽  
Francesca Festa ◽  
Valentina Panzarin ◽  
Davide Lelli ◽  
Ana Moreno ◽  
...  
Keyword(s):  
Literature Review ◽  
In Silico ◽  
Host Shift ◽  
Molecular Tools ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Field Samples ◽  
Low Sensitivity

Coronaviruses (CoVs) are widespread and highly diversified in wildlife and domestic mammals and can emerge as zoonotic or epizootic pathogens and consequently host shift from these reservoirs, highlighting the importance of veterinary surveillance. All genera can be found in mammals, with α and β showing the highest frequency and diversification. The aims of this study were to review the literature for features of CoV surveillance in animals, to test widely used molecular protocols, and to identify the most effective one in terms of spectrum and sensitivity. We combined a literature review with analyses in silico and in vitro using viral strains and archive field samples. We found that most protocols defined as pan-coronavirus are strongly biased towards α- and β-CoVs and show medium-low sensitivity. The best results were observed using our new protocol, showing LoD 100 PFU/mL for SARS-CoV-2, 50 TCID50/mL for CaCoV, 0.39 TCID50/mL for BoCoV, and 9 ± 1 log2 ×10−5 HA for IBV. The protocol successfully confirmed the positivity for a broad range of CoVs in 30/30 field samples. Our study points out that pan-CoV surveillance in mammals could be strongly improved in sensitivity and spectrum and propose the application of a new RT-PCR assay, which is able to detect CoVs from all four genera, with an optimal sensitivity for α-, β-, and γ-.

scholarly journals Evaluation of 2-Thioxoimadazolidin-4-One Derivatives as Potent Anti-Cancer Agents through Apoptosis Induction and Antioxidant Activation: In Vitro and In Vivo Approaches

Molecules ◽  
2021 ◽  
Vol 27 (1) ◽  
pp. 83
Author(s):  
Mohamed S. Nafie ◽  
Ahmed I. Khodair ◽  
Hebat Allah Y. Hassan ◽  
Noha M. Abd El-Fadeal ◽  
Hanin A. Bogari ◽  
...  
Keyword(s):  
Cell Cycle ◽  
In Silico ◽  
Hepg2 Cells ◽  
Flow Cytometric Analysis ◽  
Apoptosis Induction ◽  
Rt Pcr ◽  
Anti Cancer ◽  
Ic50 Values

Background: Hepatocellular carcinoma (HCC) is one of the most widespread malignancies and is reported as the fourth most prevalent cause of cancer deaths worldwide. Therefore, we aimed to investigate the probable mechanistic cytotoxic effect of the promising 2-thioxoimidazolidin-4-one derivative on liver cancer cells using in vitro and in vivo approaches. The compounds were tested for the in vitro cytotoxic activity using MTT assay, and the promising compound was tested in colony forming unit assay, flow cytometric analysis, RT-PCR, Western blotting, in vivo using SEC-carcinoma and in silico to highlight the virtual mechanism of action. Both compounds 4 and 2 performed cytotoxic effects against HepG2 cells with IC50 values of 0.017 and 0.18 μM, respectively, compared to Staurosporine and 5-Fu as reference drugs with IC50 values of 5.07 and 5.18 µM, respectively. Compound 4 treatment revealed apoptosis induction by 19.35-fold (11.42% compared to 0.59% in control), arresting the cell cycle at G2/M phase. Moreover, studying gene expression that plays critical roles in cell cycle and apoptosis by RT-PCR demonstrated that compound 4 enhances the expression of the pro-apoptotic genes p53, PUMA, and Caspase 3, 8, and 9, and impedes the anti-apoptotic Bcl-2 gene in the HepG2 cells. It can also inhibit the PI3K/AKT pathway at both gene and protein levels, which was reinforced by the in silico predictions of the molecular docking simulations towards the PI3K/AKT proteins. Finally, in vivo study verified that compound 4 has a promising anti-cancer activity through activating antioxidant levels (CAT, SOD and GSH) and ameliorating hematological, biochemical, and histopathological findings.

scholarly journals Diagnostic accuracy of SARS-CoV-2 rapid antigen detection testing in symptomatic and asymptomatic children in the clinical setting

2021 ◽  
Author(s):  
Arnaud L'Huillier ◽  
Matthieu Lacour ◽  
Debora Sadiku ◽  
Mehdi A Gadiri ◽  
Loraine De Siebenthal ◽  
...  
Keyword(s):  
Infectious Virus ◽  
Rapid Test ◽  
World Health ◽  
Diagnostic Study ◽  
Rt Pcr ◽  
Test Device ◽  
Asymptomatic Children ◽  
Polymerase Chain ◽  
Health Organization ◽  
Post Onset

ABSTRACT Importance. Antigen-based rapid diagnostic tests (RDTs) have shown good sensitivity for SARS-CoV-2 detection in adults and are used in children despite the lack data from children. Objective. We evaluated the diagnostic performance of the PanbioTM-COVID-19 Ag Rapid Test Device (P-RDT) in symptomatic and asymptomatic children against reverse-transcription polymerase chain reaction (RT-PCR) on nasopharyngeal swabs (NPS). Design. Prospective diagnostic study from 11.2020 to 03.2021 Setting. Single-center Participants. Consecutive symptomatic and asymptomatic participants 0-16yo Intervention. Two NPS for both RT-PCR and P-RDT Main outcome. P-RDT sensitivity and specificity Results. Eight-hundred and twenty-two participants completed the study, of which 533 (64.9%) were symptomatic. Among the 119 (14.5%) RT-PCR positive patients, the overall P-RDT sensitivity was 0.66 (95%CI 0.57-0.74). Mean viral load (VL) was higher among P-RDT positive than negative ones (p<0.001). Sensitivity was 0.87 in specimens with VL>1.0E6 copies/mL (95%CI 0.87-1.00), which is the accepted cut-off for the presence of infectious virus, and decreased to 0.67 (95%CI 0.59-0.76) for specimens >1.0E3 copies/mL. Among symptomatic participants, the P-RDT displayed a sensitivity of 0.73 (95%CI 0.64-0.82), which peaked at 1.00 at 2 days post onset of symptoms (DPOS; 95%CI 1.00-1.00), then decreased to 0.56 (95%CI 0.23-0.88) at 5 DPOS. There was a trend towards lower P-RDT sensitivity in symptomatic children <12 years (0.62 [95%CI 0.45-0.78]) versus > 12 years (0.80 [95%CI 0.69-0.91]; p=0.09). VL which was significantly lower in asymptomatic participants than in symptomatic ones (p<0.001). The P-RDT displayed a sensitivity of 0.43 (95%CI 0.26-0.61). Specificity was 1.00 in symptomatic and asymptomatic children (95%CI 0.99-1.00). Conclusion and relevance. The overall respective 73% and 43% sensitivities of P-RDT in symptomatic and asymptomatic children was below the 80% cut-off recommended by the World Health Organization. These findings are likely explained by lower VLs in children at the time of diagnosis. As expected, we observed a direct correlation between VL and P-RDT sensitivity as well as variation of sensitivity according to DPOS, a major determinant of VL. These data highlight the limitations of RDTs both in symptomatic and asymptomatic children, with the potential exception in early symptomatic children >12yrs where sensitivity reached 80%.

A sensitive in-house RT-PCR genotyping system for combined detection of plasma HIV-1 and assessment of drug resistance

2006 ◽  
Vol 133 (2) ◽  
pp. 137-145 ◽  
Author(s):  
Kim Steegen ◽  
Els Demecheleer ◽  
Nancy De Cabooter ◽  
Dieudonné Nges ◽  
Marleen Temmerman ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Rt Pcr ◽  
Combined Detection ◽  
Hiv 1 ◽  
Pcr Genotyping

scholarly journals Analyzing Thiol-Dependent Redox Networks in the Presence of Methylene Blue and Other Antimalarial Agents with RT-PCR-Supported in silico Modeling

2012 ◽  
Vol 6 ◽  
pp. BBI.S10193 ◽  
Author(s):  
J. Zirkel ◽  
A. Cecil ◽  
F. Schäfer ◽  
S. Rahlfs ◽  
A. Ouedraogo ◽  
...  
Keyword(s):  
Experimental Data ◽  
Methylene Blue ◽  
Combination Therapy ◽  
In Silico ◽  
Network Effects ◽  
Metabolic Flux ◽  
Biological Effects ◽  
Chloroquine Resistance ◽  
Rt Pcr ◽  
The Face

Background In the face of growing resistance in malaria parasites to drugs, pharmacological combination therapies are important. There is accumulating evidence that methylene blue (MB) is an effective drug against malaria. Here we explore the biological effects of both MB alone and in combination therapy using modeling and experimental data. Results We built a model of the central metabolic pathways in P. falciparum. Metabolic flux modes and their changes under MB were calculated by integrating experimental data (RT-PCR data on mRNAs for redox enzymes) as constraints and results from the YANA software package for metabolic pathway calculations. Several different lines of MB attack on Plasmodium redox defense were identified by analysis of the network effects. Next, chloroquine resistance based on pfmdr/ and pfcrt transporters, as well as pyrimethamine/sulfadoxine resistance (by mutations in DHF/DHPS), were modeled in silico. Further modeling shows that MB has a favorable synergism on antimalarial network effects with these commonly used antimalarial drugs. Conclusions Theoretical and experimental results support that methylene blue should, because of its resistance-breaking potential, be further tested as a key component in drug combination therapy efforts in holoendemic areas.

scholarly journals Anti-dengue activity of super critical extract and isolated oleanolic acid of Leucas cephalotes using in vitro and in silico approach

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Sulochana Kaushik ◽  
Lalit Dar ◽  
Samander Kaushik ◽  
Jaya Parkash Yadav
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Oleanolic Acid ◽  
Plant Extract ◽  
In Silico ◽  
Rt Pcr ◽  
Ft Ir ◽  
Isolated Compound ◽  
Layer Chromatography

Abstract Backgrounds Leucas cephalotes is a common ethnomedicinal plant widely used by traditional healers for the treatment of Malaria and other types of fever. Oleanolic acid and its derivatives have been reported for various types of pharmacological activities, such as anti-inflammatory, antioxidant, anticancer, hepatoprotective, anti-HIV and anti-HCV activity. Methods L.cephalotes plant extracts were prepared by supercritical fluid extraction (SFE) method and oleanolic acid was isolated by preparatory thin-layer chromatography. The compound was identified and characterize by using ultraviolet-visible spectroscopy (UV-VIS), Fourier transform infra-Red spectroscopy (FT-IR) and high-performance thin-layer chromatography (HPTLC). The structure of the compound was elucidated by proton nuclear magnetic resonance (1HNMR) and carbon nuclear magnetic resonance (1CNMR) and the purity checked by differential scanning calorimetry (DSC). The MTT assay was used to determine the toxicity of plant extract and oleanolic acid using a microplate reader at 595 nm. The anti-dengue activity of plant extract and oleanolic acid was tested in vitro and in silico using real-time RT-PCR. Results The optimum yield of the extract was obtained at 40 °C temperature and 15Mpa pressure. The maximum non-toxic dose (MNTD) of plant extract and oleanolic acid were found as 46.87 μg/ml and 93.75 μg/ml, respectively in C6/36 cell lines. UV spectrophotometer curve of the isolated compound was overlapped with standard oleanolic acid at 232 nm. Superimposed FT-IR structure of the isolated compound was indicated the same spectra at 3433, 2939, 2871, 1690, 1500,1463, 1387, 1250, 1209, 1137 and 656 position as per marker compound. HPTLC analysis showed the retention factor of L. cephalotes extract was 0.19 + 0.06 as similar to the standard oleanolic acid chromatogram. The NMR structure of the isolated compound was identified as similar to the marker oleanolic acid structure. DSC analysis revealed the purity of isolated oleanolic acid was 98.27% with a melting point of 311.16 °C. Real-time RT PCR results revealed that L. cephalotes supercritical extract and isolated oleanolic acid showed 100 and 99.17% inhibition against the dengue − 2 virus when treated with MNTD value of plant extract (46.87 μg/ml) and the test compound (93.75 μg/ml), respectively. The molecular study demonstrated the binding energy of oleanolic acid with NS1and NS5 (non-structural protein) were − 9.42 & -8.32Kcal/mol, respectively. Conclusions The SFE extract L. cephalotes and its active compound, oleanolic acid inhibiting the activity of dengue-2 serotype in the in vitro and in silico assays. Thus, the L.cephalotes plant could be an excellent source for drug design for the treatment of dengue infection.

scholarly journals Triage of Patients Suspected of COVID-19 in Chronic Hemodialysis: Eosinophil Count Differentiates Low and High Suspicion of COVID-19

2020 ◽  
Author(s):  
Romain Vial ◽  
Marion Gully ◽  
Mickael Bobot ◽  
Violaine Scarfoglière ◽  
Philippe Brunet ◽  
...  
Keyword(s):  
Predictive Value ◽  
Eosinophil Count ◽  
Characteristic Curve ◽  
Final Analysis ◽  
Hemodialysis Patients ◽  
Chronic Hemodialysis ◽  
Screening Methods ◽  
Dialysis Session ◽  
Diagnostic Study ◽  
Rt Pcr

Abstract Background: Daily management to shield chronic dialysis patients from SARS-CoV-2 contamination makes patient care cumbersome. There are no screening methods to date and a molecular biology platform is essential to perform RT-PCR for SARS-CoV-2; however, accessibility remains poor. Our goal was to assess whether the tools routinely used to monitor our hemodialysis patients could represent reliable and quickly accessible diagnostic indicators to improve the management of our hemodialysis patients in this pandemic environment.Methods: In this prospective observational diagnostic study, we recruited patients from La Conception hospital. Patients were eligible for inclusion if suspected of SARS-CoV-2 infection when arriving at our center for a dialysis session between March 12th and April 24th 2020. They were included if both RT-PCR result for SARS-CoV-2 and cell blood count on the day that infection was suspected were available. We calculated the area under the curve (AUC) of the receiver operating characteristic curve.Results: 37 patients were included in the final analysis, of which 16 (43.2%) were COVID-19 positive. For the day of suspected COVID-19, total leukocytes were significantly lower in the COVID-19 positive group (4.1 vs 7.4 G/L, p=0.0072) and were characterized by lower neutrophils (2.7 vs 5.1 G/L, p=0.021) and eosinophils (0.01 vs 0.15 G/L, p=0.0003). Eosinophil count below 0.045 G/L identified SARS-CoV-2 infection with AUC of 0.9 [95% CI 0.81-1] (p<0.0001), sensitivity of 82%, specificity of 86%, a positive predictive value of 82%, a negative predictive value of 86% and a likelihood ratio of 6.04.Conclusions :Eosinophil count enables rapid routine screening of chronic hemodialysis patients suspected of being COVID-19 within a range of low or high probability.

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