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Summary Introduction: The SARS-CoV-2 coronavirus, that causes the COVID-19 disease, has become a global public health problem that requires the implementation of rapid and sensitive diagnostic tests. Aim(s): To evaluate and compare the sensitivity of LAMP assay to a standard method and use RT-LAMP for the diagnosis of SARS-CoV-2 in clinical samples from Colombian patients. Method(s): A descriptive and cross-sectional study was conducted. A total of 25 nasopharyngeal swab samples including negative and positive samples for SARS-CoV-2 were analyzed, through the RT-LAMP method compared to the RT-qPCR assay. Result(s): LAMP method detected ~18 copies of the N gene, in 30 min, evidenced a detection limit similar to the standard method, in a shorter time and a concordance in RT-LAMP of 100% with the results. Conclusion(s): RT-LAMP is a sensitive, specific, and rapid method that can be used as a diagnostic aid of COVID-19 disease.Copyright © 2021. All Rights Reserved.
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The additional impact of emission-reduction measures in North China (NC) during autumn and winter on the air quality of downwind regions is an interesting but less addressed topic. The mass concentrations of routine air pollutants, the chemical compositions, and sources of fine particles (PM2.5) for January 2018, 2019, and 2020 at a megacity of Central China were identified, and meteorology-isolated by a machine-learning technique. Their variations were classified according to air mass direction. An unexpectedly sharp increase in emission-related PM2.5 by 22.7% (18.0 mug m-3) and 25.7% (19.4 mug m-3) for air masses from local and NC in 2019 was observed compared to those of 2018. Organic materials exhibited the highest increase in PM2.5 compositions by 6.90 mug m-3 and 6.23 mug m-3 for the air masses from local and NC. PM2.5 source contributions related to emission showed an upsurge from 1.39 mug m-3 (biomass burning) to 24.9 mug m-3 (secondary inorganic aerosol) in 2019 except for industrial processes, while all reduced in 2020. From 2018 to 2020, the emission-related contribution of coal combustion to PM2.5 increased from 10.0% to 19.0% for air masses from the local area. To support the priority natural gas quotas in northern China, additional coal in cities of southern China was consumed, raising related emissions from transportation activities and road dust in urban regions, as well as additional biofuel consumption in suburban or rural regions. All these activities could explain the increased primary PM2.5 and related precursor NO2. This study gave substantial evidence of air pollution control measures impacting the downwind regions and promote the necessity of air pollution joint control across the administration.Copyright © 2023 Elsevier Ltd
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Sensitive and rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a vital goal in the ongoing COVID-19 pandemic. We present in this comprehensive work, for the first time, detailed fabrication and clinical validation of a point of care (PoC) device for rapid, onsite detection of SARS-CoV-2 using a real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP) reaction on a polymer cartridge. The PoC system, namely PATHPOD, consisting of a standalone device (weight less than 1.2 kg) and a cartridge, can perform the detection of 10 different samples and two controls in less than 50 min, which is much more rapid than the golden standard real-time reverse-transcription Polymerase Chain Reaction (RT-PCR), typically taking 16-48 h. The novel total internal reflection (TIR) scheme and the reactions inside the cartridge in the PoC device allow monitoring of the diagnostic results in real-time and onsite. The analytical sensitivity and specificity of the PoC test are comparable with the current RT-PCR, with a limit of detection (LOD) down to 30-50 viral genome copies. The robustness of the PATHPOD PoC system has been confirmed by analyzing 398 clinical samples initially examined in two hospitals in Denmark. The clinical sensitivity and specificity of these tests are discussed.
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Intro: Deoxyribozymes (Dz) are short synthetic DNA oligonucleotides that catalyze the cleavage of a phosphodiester bond between nucleotides in the presence of divalent metal ions. The use of DNAzymes in the in vitro diagnostics increases the specificity and versatility of the analysis. Method(s): We took the well-studied Dz 10-23 with high catalytic activity as the basis of our system. The biosensor is divided into two fragments according to the binary probe principle (Dz1 and Dz2), which consist of target RNA binding sites, a fluorescent substrate (Fsub), and half of the Dz 10-23 catalytic center sequence. Assembly of the Dz 10-23 active center with subsequent Fsub cleavage and registration of a fluorescent signal is possible only if the target RNA is present in the sample. Finding(s): To assess the diagnostic potential of the biosensor, we measured FAM fluorescence in a solution containing synthetic RNA 35 nucleotides long (nip35) corresponding to the NiV target sequence, Fsub labeled with the FAM-BHQ1 and Dz_NiV pair. A mixture of Dz_NiV and Fsub was used as a control. The detection limit of the target RNA reached 5 nM, the signal development time was 30 minutes at a temperature of 37 C . Discussion(s): The specificity of Dz_NiV was evaluated in the presence of synthetic RNAs from six other RNA viruses of similar length: Hendra, Machupo, Sabia, Junin, Guanarito, and SARS-CoV. A fluorescent signal was recorded only in the presence of nip35 in the reaction mixture. The efficiency of Dz_NiV on a long fragment was tested using a plasmid with a cloned target sequence. The site is about 700 b.p. was amplified by PCR, followed by transcription. Conclusion(s): It was developed the highly specific biosensor Dz_NiV for the detection of Nipah virus RNA with a sensitivity limit of 5 nM at 37 C .Copyright © 2023
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The deadliness associated with the COVID-19 disease caused by the SARS-CoV-2 virus plunged the entire global community into the worst times of this century. It was globally realized that a timely diagnosis, effective treatment, and prevention were the key factors in its management. Responding to the emergent scenario, sequencing of the genome of this virus was performed and shared with the scientific community in the nick of time. Thereafter, diverse sets of test kits to detect the SARS-CoV-2 and to detect the antibodies in the patients of the COVID-19 were developed at the war scale. It was indeed a war but with a microscopic spiky package of 50–200 nanometres in diameter having a genome of about 29.9 kb encoding deadly tools in its arsenal. For the reason, patients of the COVID-19 exhibit diverse symptoms from mild influenza-like to potentially fatal ones that overlap with other respiratory diseases, only efficient testing was essential during the early stages of infection to identify COVID-19 patients among others. The diverse test kits designed exclusively for rapid and accurate outcomes proved instrumental in identifying individuals among asymptomatics, presymptomatics, and symptomatics. The test kits have also been playing an appreciable role in identifying communities with hot spots to facilitate proper management. To meet the demand of higher throughput and simplification of the testing process, novel ways were devised that did not otherwise allow the testing spree to get hit with pandemic supply bottlenecks. Mechanistic models have played an essential role in shaping public health policy. The regulatory agencies, both at the world health and the regional public health levels, shared the knowledge and experience on the test kits that helped in the development and improvement in the testing capability and efficiency of the testing infrastructure. The information about the emergence of variants of the SARS-CoV-2 happening due to intrinsic behavior of the viral genomes drew attention of the test kit developers, regulatory agencies, and end-users to be vigilant over the test outcomes. Offering a mechanistic approach, in this chapter, testing strategies for the detection of SARS-CoV-2 virus and COVID-19 disease are delineated. © The Author(s), under exclusive licence to Springer Nature Singapore Pte Ltd. 2021.
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Background: Remdesivir (RDV) is a broad-spectrum nucleotide analog antiviral approved for the treatment of COVID-19 in patients who are hospitalized or non-hospitalized and at risk of progressing to severe disease. Here we present SARS-CoV-2 resistance analyses from the Phase 3 PINETREE trial. Method(s): PINETREE was a double-blind, placebo-controlled trial of nonhospitalized participants (N=562) with COVID-19 and >=1 risk factor for disease progression, randomized to receive RDV or placebo once-daily for 3 days. The whole genome of SARS-CoV-2 was sequenced from nasopharyngeal swabs collected at days 1 (baseline), 2, 3, 7, and 14 using next-generation sequencing. Emergent amino acid substitutions in SARS-CoV-2 from participants treated with RDV were tested in a replicon system to determine if they alter sensitivity to RDV. Result(s): Resistance analysis criteria included all participants in the RDV group and 50% in the placebo group with viral load above the lower limit of detection for the viral load assay. Of 281 participants who met these criteria, baseline and postbaseline sequencing data were available for 115/130 (88.5%) participants in the RDV group and 129/151 (85.4%) participants in the placebo group (Table 1). Among these, emergent substitutions in Nsp12 were observed in 8/115 (7.0%) in the RDV group and 7/129 (5.4%) in the placebo group. A total of 7 emergent amino acid substitutions in Nsp12 were observed in the RDV group, but not in the placebo group. Among these, only one substitution from one participant (A376V;first detected at day 14), showed reduced in vitro susceptibility to RDV, with a half-maximal effective concentration (EC50) fold-change of 12.6 compared with a wildtype reference. The participant achieved clinical recovery by day 14. None of the other substitutions impacted RDV susceptibility (EC50 fold-change <=1.4). Emergent substitutions in Nsp8, Nsp10, Nsp13, or Nsp14 were detected in 10/115 (8.7%) of participants in the RDV group and 10/129 (7.8%) in the placebo group, with substitutions in the RDV group showing similar susceptibility to RDV as the wildtype reference (EC50 fold-change <=2.3). Conclusion(s): Overall, emergent substitutions in the SARS-CoV-2 replication complex including Nsp12 were observed with similar frequency in the RDV and placebo groups, with only one participant developing a substitution associated with reduced in vitro RDV susceptibility, indicating a high barrier to the development of RDV resistance in COVID-19 patients.
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Objectives: The objective is to develop a low-cost, practical, portable aptasensor platform for the diagnosis of COVID-19. Materials -Methods: Amino-terminated aptamers to be used for the design of an aptasensor were synthesized by SELEX method, and interaction of aptamers with SARS-CoV-2 S1 protein was investigated by isothermal titration calorimetry (ITC). Gold electrodes were used to design the biosensor platform. After the electrode surface was functionalized with cysteamine, the amino-terminated aptamer was conjugated to the surface via glutaraldehyde crosslinker. Then, the surface characterization and analytical parameters of the designed sensing platform were determined by adding commercial S1 proteins on the surface using differential pulse voltammetry (DPV), cyclic voltammetry (CV) and impedance spectroscopy (EIS). To evaluate the working performance of the system, S1 proteins were added to the synthetic serum samples using the standard addition method and the measurements were repeated. Result(s): Surface characterization of the platform designed with EIS and CV measurements was performed and it was found that the modification was successfully performed. In addition, DPV results and analytical parameters of the platform (calibration plot, limit of detection(LOD) , repeatability, coefficient of variation) were determined and the working performance of system was evaluated. Moreover, working performance of the biosensor in real samples and its specificity for COVID -19 were determined by experiments with synthetic serum and influenza A and B proteins. Conclusion(s): According the results, the system has potential to be used for the detection of COVID -19, and also it can be rapidly adapted in different pandemic situations that may occur in the future.
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Background: Omicron subvariants questioned the efficacy of the approved therapies for the early COVID-19. In vitro data show that remdesivir (RDV), molnupiravir (MLN), and nirmatrelvir/ritonavir (NMV/r) all retained activity against all sub-lineages, while poor neutralizing activity was observed for Sotrovimab (SOT) and Tixagevimab/cilgavimab (TIX/CIL). No data about the risk of clinical failure or even in vivo antiviral activity are available. Method(s): Single-center observational comparison study enrolling all consecutive patients (pts) seen for care with a confirmed SARS-CoV-2 Omicron diagnosis and who met the AIFA criteria for eligibility for treatment with RDV, MLN, NMV/r, TIX/CIL, or SOT. Treatment allocation was subject to drug availability, time from symptoms onset, and comorbidities. Nasopharyngeal swab (NPS) VL was measured on day 1 (D1) and D7 and was expressed by log2 cycle threshold (CT) scale. Comparisons between treatment groups were made by Chi-square, and Wilcoxon paired tests. Primary endpoint was D1-D7 VL variation. Potential decrease in VL and average treatment effect (ATE) were calculated from fitting marginal linear regression models weighted for calendar month of drug initiation, duration of symptoms, and immunodeficiency using NMV/r as the comparator trial arm. Result(s): A total of 971 pts received treatments (SOT 321, MLN 231, NMV/r 211, TIX/CIL 70, and RDV 138): female 457 (47%), median age 67 yrs (IQR 56-78), 93% vaccinated;12% with negative baseline serology. At D1, median time from symptoms onset was 3 days (IQR 2,4). 379 (39%) pts were infected with BA.1, 215 (22%) with BA.2, 372 with BA.4/5 (38%), and 5 with BQ.1 (0,5%). D1 mean viral load was 4.02 log2. Adjusted analysis (ATE) showed that NMV/r significantly reduced VL compared to all the other drugs in pts infected with all sublineages, (Fig.1A-B) while less evidence for a difference vs. TIX/CIL was seen in those infected with BA.2 (p=0.05) (Fig.1 C-D). Conclusion(s): In this analysis of in vivo early VL reductions, NMV/r appears to be the drug showing the greatest antiviral activity, regardless of the underlying subvariant, perhaps with the exception of TIX/CIL in people infected with BA.2 for which there was less evidence for a difference. In the Omicron era, due to the high prevalence of vaccinated people and in absence of clinical events, VL is one of the possible alternative endpoints which guarantees adequate statistical power. Fig 1 SARS-CoV-2 RNA levels at D1 and D7 in patients treated with Nirmatrelvir/ ritonavir, Sotrovimab, Molnupiravir, Remdesivir, and Tixagevimab/cilgavimab. Dot-plots showing the comparison of viral loads detected at D1 and D7 and the variation of RNA levels observed between the two time-points by intervention in (A) all patients treated with Nirmatrelvir/ritonavir (n=211), Sotrovimab (n=321), or Molnupiravir (n=231), or Remdesivir (n=138), or Tixagevimab/ cilgavimab (n=136);(C) patients with Omicron BA.2 infection treated with Nirmatrelvir/ritonavir (n=58), Sotrovimab (n=81), or Molnupiravir (n=21), or Remdesivir (n=37), or Tixagevimab/cilgavimab (n=18);(D) patients with Omicron BA.4/5 infection treated with Nirmatrelvir/ritonavir (n=102), Sotrovimab (n=92), or Molnupiravir (n=110), or Remdesivir (n=16), or Tixagevimab/cilgavimab (n=52). Viral RNA levels are expressed as log2 CT values. The horizontal dashed line represents the limit of detection (CT: 40.0), values >=40 are considered negative. Mean of log2 CT values, and SD are shown in the graph. Statistical analysis of the differences in viral loads by intervention as compared to Nirmatrelvir/ritonavir was performed by Mann-Whitney test. Potential decrease in VL and average treatment effect (ATE) were calculated from fitting marginal linear regression models weighted for calendar month of drug initiation, duration of symptoms, and immunodeficiency using NMV/r as the comparator trial arm. Results are shown (B) for patients infected with all Omicron sublineages and (D) for those infected with Omicron BA.2 sublineage.
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The use of saliva directly in RT-PCR reactions may be interesting, particularly in screenings for the detection of DNA/RNA of pathogens, due to the advantages of time and costs. However, its use faces some difficulties that essentially result from the saliva being a matrix with a variable composition and which contains inhibitors of the polymerization reaction. Saliva samples obtained from healthy donors were spiked with SARS-CoV-2 RNA and 3 viral targets were detected by RT-PCR. This work was considered exempt from review by an institutional ethical review board, because it comprises use of completely anonymized specimens obtained voluntarily and informed. The efficiency of RT-PCR reactions was calculated in the presence of variable amounts of saliva and the effect of saliva pre-treatment on the performance of each reaction was also analysed. It was found that saliva has no significant influence on the mean efficiency of each individual reaction, affecting mainly the beginning of the exponential phase of amplification, resulting in a significant increase in Ct values. This effect which is target dependent, presents a highly between-person variability (DELTACt = 0.93 to 11.36) and affects RNA/DNA detection and the limit-of-detection, but can be partly reversed by a treatment of saliva prior to the reaction. The work presented contributes to a better understanding of the advantages and limitations of using saliva directly in RT-PCR. This work was supported by ProjectNORTE-01-0145- FEDER-072545- SAICTCOVID/72545/2020 and was the result of the projects: UIDB/05937/2020 and UIDP/05937/2020- Centre for Research and Development in Agrifood Systems and Sustainability-funded by national funds, through FCT-Fundacao para a Ciencia e a Tecnologia.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.
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Objectives: The Panbio COVID-19 Ag Rapid Test Device (Panbio COVID-19 Ag, Abbott Rapid Diagnostics) is a lateral flow immunochromatographic assay targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleoprotein in nasopharyngeal specimens for the diagnosis of coronavirus disease 2019 (COVID-19). This study aimed to verify the performance of the Panbio COVID-19 Ag for implementation in clinical laboratories. Method(s): Sixty nasopharyngeal swab specimens (30 positive and 30 negative) dipped in transport medium, and COVID-19 was confirmed using real-time RT-PCR using Allplex SARS-CoV-2 assay (Seegene), were tested using the Panbio COVID-19 Ag. Reproducibility was evaluated using positive and negative control materials. Sensitivity and specificity were calculated based on the results of realtime RT-PCR as the standard test method. Result(s): Reproducibility was confirmed by the consistent results of repeated tests of the quality control materials. The overall sensitivity and specificity of Panbio COVID-19 Ag were 50.0% and 100.0%, respectively. Panbio COVID-19 Ag demonstrated high sensitivity (88.2%) in analyzing the detection limit cycle threshold (Ct) value of 26.67 provided by the manufacturer as a positive criterion, and the sensitivity was 100.0% for the positive criterion of Ct values <25, although it was less sensitive for Ct > 25. Conclusion(s): Considering the high sensitivity for positive samples with Ct values <25 and the rapid turnaround of results, Panbio COVID-19 Ag can be used in clinical laboratories to diagnose COVID-19 in limited settings. Copyright © 2023 Ewha Womans University College of Medicine and Ewha Medical Research Institute.
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Background: Metformin has in vitro activity against SARS-CoV-2. In a published phase 3, quadruple-blinded, placebo-controlled randomized trial of outpatient COVID-19 therapy, metformin resulted in a 42% reduction in ER visits/hospitalizations/deaths by day 14, 58% reduction in hospitalizations/ death by day 28, and 42% reduction in Long Covid through 10 months. This analysis presents the results of viral load sampling performed during that clinical trial. Method(s): Covid-Out trial (NCT04510194) enrolled adults aged 30 to 85 within 3 days of a documented SARS-CoV-2 infection and < 7 days after symptom onset. The trial randomized 1323 participants to metformin (1000mg/day days 2-5;1500mg/day days 6 to 14), ivermectin, fluvoxamine, and/or exact-matching placebo in a 2x3 factorial trial design. Nasal swabs for viral load were an optional component, self-collected from the anterior nares on day 1, 5, and 10. Viral loads were measured via RT-qPCR using N1 and N2 targets in the SARSCoV- 2 nucleocapsid protein, with relative Ct values converted to absolute copy number via calibration to droplet digital PCR. A linear Tobit regression model was used to assess change over time while accounting for left censoring due to the viral load limit of detection. Results were adjusted for other treatment allocations within the factorial design, vaccination status, and baseline viral load. Repeated measures were accounted for using clustered standard errors within participants. Result(s): Samples were available from n = 945, 871, and 775 participants on days 1, 5, and 10, respectively. The mean change from baseline to followup was -0.64 log10 copies/mL (95%CI, -1.16 to -0.13) for metformin versus placebo, which equates to a 4.4-fold greater decrease. The mean change in SARS-CoV-2 from baseline to day 5 was -0.48 log10 copies/mL, and was -0.81 log10 copies/mL from baseline to day 10. The anti-viral effect increased with increased metformin dosing days 6-14. The antiviral effect was larger in those unvaccinated (mean -0.95 log copies/mL) than vaccinated (mean -0.39 log copies/mL). There was no change in viral load vs. placebo for ivermectin or fluvoxamine. Conclusion(s): Metformin lowered SARS-CoV-2 viral load in this quadrupleblinded, randomized clinical trial. The temporal relationship to dose titration suggests a dose-dependent effect. The magnitude of antiviral effect was similar to nirmatrelvir at day 5, greater than nirmatrelvir at day 10. Metformin is safe, widely available, and has few contraindications.
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OBJECTIVES: Antigen rapid diagnostic tests (Ag-RDTs) play an important role in the diagnosis of SARS-CoV-2. They are easier, quicker, and less expensive than the 'reference standard' RT-PCR and therefore widely in use. Reliable clinical data with respect to Ag-RDT performance in SARS-CoV-2 Omicron variants of concern (VOCs) are limited. Consequently, the objective of this study was to determine the impact different VOCs-especially Omicron-have on the clinical performance of an Ag-RDT. METHODS: We compared the clinical performance of the Sofia SARS-CoV-2 Ag-RDT to RT-PCR in a real-world, single-centre study in a clinical point-of-care setting in patients admitted to a large hospital via the emergency department from 2 November 2020 to 4 September 2022. RESULTS: Among 38 434 Ag-RDT/RT-PCR tandems taken, 1528 yielded a SARS-CoV-2 positive RT-PCR test result, with a prevalence of 4.0% (95% CI, 3.8-4.2). Overall sensitivity of the Ag-RDT was 63.7% (95% CI, 61.3-66.1) and overall specificity was 99.6% (95% CI, 99.5-99.6). Ag-RDT sensitivity was dependent on viral load (VL), because the sensitivity increased to 93.2% (95% CI, 91.5-94.6) in samples with a VL > 106 SARS-CoV-2 copies/mL. Furthermore, the Ag-RDT was more sensitive in men, and older patients. Variant-dependent sensitivity assessment showed that the sensitivity was significantly lower in Omicron-VOC (64.1%; 95% CI, 60.5-67.6) compared with SARS-CoV-2 wild-type samples (70.0%; 95% CI, 59,8-78,6) (binomial test; p value < 0.001). Analysing the limits of detection showed a 27 times higher 95% limit of detection for the Omicron-VOC BA.5 compared with the SARS-CoV-2 wild-type. DISCUSSION: Ag-RDT sensitivity for detection of patients with lower VLs and with Omicron-VOC is reduced, limiting the effectiveness of Ag-RDTs. However, Ag-RDTs are still an unreplaceable tool for widely available, quick, and inexpensive point-of-care SARS-CoV-2 diagnostics.
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Molnupiravir, a broad-spectrum antiviral is an isopropyl ester prodrug of beta-D-N4-hydroxycytidine. Molnupiravir targets RNA-dependent RNA-polymerase enzyme of the viruses. A new stability-indicating HPLC-method was developed to determine related substances and assay of molnupiravir. Separation was achieved by using Shim-pack GWS C18 column. The method was validated according to current ICH requirements. The calibration plot gave a linear relationship for all known analytes over the concentration range from LOQ to 200%. LOD and LOQ for all known analytes were found in 0.05-0.08 microg mL-1 and 0.12-0.20 microg mL-1, respectively, the mean recovery was found to be 97.79-102.44 %. Study showed that the method, results of robustness, solution stability studies are precise and within the acceptable limits. Molnupiravir was found to degrade in acid, alkali, and oxidative conditions, and was stable in thermal, moisture, and photolytic degradation condition. The method is simple, accurate, precise, and reproducible for routine purity analysis of drug-samples.Copyright © 2022 Indian Drug Manufacturers' Association. All rights reserved.
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Kabasura Kudineer is a polyherbal decoction of the Siddha medical system (an Indian system of medicine), traditionally used to cure fever, colds, coughs, and respiratory ailments. The government of India had recommended Kabasura Kudineer as one of many preventive/treatment measures for COVID-19. Kabasura Kudineer Choornam is an admixed coarse powder of 15 herbs and its decoction is Kabasura Kudineer. The chemical constituents in the 15 herbs used for the preparation of the Choornam are known but the constituents present in the Kabasura Kudineer (decoction) are unidentified. Piperine, vasicine and eugenol are known for their potent activity against respiratory tract infections;hence, they were selected as marker compounds. The present work was planned to simultaneously quantify piperine, vasicine and eugenol in Kabasura Kudineer by the HPTLC method. The optimised mobile phase was toluene: ethyl acetate: methanol: ammonia (5:9:3:0.5, v/v/v/v), and the scanning was carried out at 287 nm. The Rf values of piperine, vasicine and eugenol were found to be 0.70, 0.32 and 0.82, respectively. The linearity range of piperine and vasicine was 500-3000 ng spot-1 and it was 10-60 ng spot-1 for eugenol. The quantities of piperine, vasicine and eugenol in Kabasura Kudineer (100 mL) were 0.03, 0.056 and 0.035 % w/v, respectively. This developed method can be used to simultaneously quantify piperine, vasicine and eugenol in any polyherbal formulation.Copyright © 2023, Informatics Publishing Limited and Society for Biocontrol Advancement. All rights reserved.
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Here, we report the development of an indirect ELISA antibodies detection method for African swine fever virus (ASFV). Two purified monoclonal antibodies (mAbs) against ASFV p30 and p54 protein were used as targets and a phage-displayed 12-mer peptide library was used to conduct four rounds of biopanning to screen peptide epitopes, then amino acids GGG was used as a linker to synthesize tandem-epitope peptide of ASFV p30 and p54 protein which was used as coating antigen. The optimum reaction conditions of indirect ELISA were determined by chessboard titration, and clinical serum samples were used to evaluate the specificity, sensitivity, stability and conformity of this method. The biopanning experiment indicated that 146PAEPYTT152 was a core domain of the B cell linear epitope of p54 protein. The optimization results of ELISA reaction conditions showed that the tandem-epitope peptide coupled with ovalbumin (OVA) at N-terminal had low background of non-specific serum reaction. And the optimum reaction effect was obtained when the polypeptide antigen was coated with carbonate buffer in 2 mug.mL-1, the serum was diluted 100-fold with blocking solution (1% gelatin solution), and the HRP-antibody was diluted 5 000 times with 0.05% PBST solution. The cut-off value was determined to be 0.339. Furthermore, the results of specificity, sensitivity and stability tests showed that there is no cross-reaction in positive serum samples of classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), porcine parvovirus (PPV) and porcine pseudorabies virus (PRV), the detection limit of ASFV positive sera is 1:1 600, and the method had high repeatability. Finally, Total 320 swine serum samples were detected simultaneously by the present established method and commercial ASFV antibody detection kit. The results showed that the relative specificity and sensitivity of the two methods were 97.6% and 97.3%, respectively. And the coincidence rate was 97.5%. In conclusion, this method showed good specificity, sensitivity, repeatability and coincidence rate, that had the potential value of developing clinical diagnostic kit.Copyright © 2022 Editorial Board, Institute of Animal Science of the Chinese Academy of Agricultural Sciences. All rights reserved.
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Herein, we establish a novel isothermal digital amplification system termed digital nicking and extension chain reaction system-based amplification (dNESBA) by utilizing the isothermal NESBA technique and the newly developed miniaturized fluorescence monitoring system (mFMS). dNESBA enables parallel isothermal NESBA reactions in more than 10,000 localized droplet microreactors and read the fluorescence signals rapidly in 150 s by mFMS. This system could identify the genomic RNA (gRNA) extracted from target respiratory syncytial virus A (RSV A) as low as 10 copies with remarkable specificity. The practical applicability of dNESBA was also successfully verified by reliably detecting the gRNA in the artificial sputum samples with excellent reproducibility and accuracy. Due to the intrinsic advantages of isothermal amplifying technique including the elimination of the requirement of thermocycling device and the enhanced portability of the miniaturized read-out equipment, the dNESBA technique equipped with mFMS could serve as a promising platform system to achieve point-of-care (POC) digital molecular diagnostics, enabling absolute and ultra-sensitive quantification of various infectious pathogens even in an early stage.Copyright © 2023
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This study aimed to investigate the usefulness of cerebral regional oxygen saturation (rSO2) during the initial 5 and 10 minutes of cardiopulmonary resuscitation (CPR) compared with an initial rSO2 and mean rSO2 during entire CPR to predict the futility of resuscitation for patients without of-hospital-cardiac arrest (OHCA). This was a prospective study involving 52 adult patients presenting in OHCA and whose cerebral rSO2 values were measured until either CPR was terminated or sustained return of spontaneous circulation (ROSC) was achieved. Receiver operating characteristics analyses were used to evaluate which time and type of measurement is better to predict non-ROSC. The area under the curve (AUC) of each rSO2 value according to measurement time (overall, initial 5 minutes and 10 minutes) were the highest value of 0.743, 0.724, and 0.739, mean values of 0.724, 0.677 and 0.701 and rSO2 (Changes in values of regional cerebral oxygen) value of 0.722, 0.734 and 0.724, respectively, while all of the initial values had a poor AUC (<0.7) and also were not statistically significant. The optimal cut-off value of each rSO2 values during overall, initial 5 minutes and 10 minutes were the highest value of 26% (sensitivity, 53.9% specificity, 92.3%), 24% (sensitivity, 56.4% specificity, 92.3%), and 30% (sensitivity, 61.5% specificity, 84.6%), mean value of 15.2%, 15.3% and 16%, respectively. None of the patients with a persistent rSO2 <=18% during the overall period achieved ROSC. Initial 5 minutes and 10 minutes cerebral rSO2 values an out-of-hospital-cardiac arrest (OHCA) are a better predictor in deciding the futility of CPR, compared to initial and overall measurements.Copyright © 2023 The Author(s). Published by MRE Press.
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Favipiravir is a potential repurpose moiety to treat COVID-19 by depletion of virus load in infectious patients. To analyze and separate Favipiravir with remarkable efficiency, X-Bridge C8 column (150 x 4.6 mm, 5 µ) and a solvent phase of 0.1% TEA and acetonitrile (40:60 v/v) with 1-mL/min flow rate were used. The eluted favipiravir and possible degradants were detected at 225 nm. Further, the process was validated by using ICH (Q2R1) guidelines to ensure the method's suitability in the pharmaceutical sector. The RT of Favipiravir was observed at 3.7 min with good linearity of 2 to 30 µg/mL. %RSD of both system and method precision was assessed in the series of 0.32 to 0.98. The mean percentage recovery of Favipiravir was in the range of 99.0–100.4%. The limit of detection (LoD) and limit of quantification (LoQ) were assessed to be 0.024 and 0.084 μg/mL for favipiravir. The outcomes confirmed that the projected approach was economical, insightful, simple and precise with better sensitivity. Investigation of Favipiravir in the incidence of a variety of stressed or forced degradation environments ensures stability indicating quality of the developed approach. © 2023, Dr. Yashwant Research Labs Pvt. Ltd.. All rights reserved.
ABSTRACT
Here we describe a retrospective clinical evaluation of the QIAGEN artus® SARS-CoV-2 Prep&Amp UM RT-PCR assay that detects SARS-CoV-2 RNA without the need for a nucleic acid eluate extraction procedure. Using Roche SARS-CoV-2 RT-PCR on the cobas® 8800 platform as a reference standard, a total of 225 confirmed SARS-CoV-2 positive and 320 negative nasopharyngeal swabs in viral transport media, were used to evaluate the artus® assay. Using the RT-PCR cycle threshold as a semi-quantitative marker of viral load, an assessment of over 370,000 SARS-CoV-2 RT-PCR positive results was used in the design of the reference positive specimen cohort. The viral load of all reference positive specimens used in the evaluation was a unique and accurate representation of the range and levels of SARS-CoV-2 positivity observed over a 13-month period of the COVID-19 pandemic. The artus® RT-PCR detects the presence of SARS-CoV-2 RNA, an internal control, and the human RNase P gene to ensure specimen quality. The diagnostic sensitivity of artus® was 92.89% with a specificity of 100%. To assess the analytical sensitivity, a limit of detection was performed using the 1st WHO NIBSC SARS-CoV-2 international standard, recording a 95% LOD of 1.1 × 103 IU/ml. The total invalid rate of specimens was 7.34% due to a lack of detectable RNase P (Ct >35). The artus® SARS-CoV-2 Prep&Amp UM RT-PCR assay is a new rapid RT-PCR assay, which may be considered to produce acceptable levels of diagnostic sensitivity and specificity whilst potentially halving the laboratory processing time.
ABSTRACT
In the ongoing COVID-19 pandemic, sensitive and rapid on-site detection of the SARS-CoV-2 coronavirus has been one of crucial objectives. A point-of-care (PoC) device called PATHPOD for quick, on-site detection of SARS-CoV-2 employing a real-time reverse-transcription loop-mediated isothermal amplification (RT-rLAMP) reaction on a polymer cartridge. The PATHPOD consists of a standalone device (weighing under 1.2 kg) and a cartridge, and can identify 10 distinct samples and 2 controls in less than 50 minutes. The PATHPOD PoC system is fabricated and clinically validated for the first time in this work © 2023 IEEE.