ARTICLE | doi:10.20944/preprints201610.0022.v1
Subject: Biology And Life Sciences, Animal Science, Veterinary Science And Zoology Keywords: P. pseudoannulata; Cadmium; Transcriptome; RT-qPCR
Online: 8 October 2016 (11:07:25 CEST)
Pardosa pseudoannulata is one of the most common wandering spiders in agricultural fields and a potentially good bioindicator for heavy metal contamination. However, little is known about the mechanism by which spiders respond to heavy metals at the molecular level. In this study, high-throughput transcriptome sequencing was employed to characterize the de novo transcriptome of the spiders and to identify differentially expressed genes (DEGs) after cadmium exposure. We obtained 60,489 assembled unigenes, 18,773 of which were annotated in the public databases. Ultimately, 3450 cDNA simple sequence repeats were identified and validated as potential molecular markers in the unigenes. A total of 2939, 2491 and 3759 DEGs were detected among the three libraries of two Cd-treated groups and the control. Functional enrichment analysis revealed that metabolism processes and digestive system function were predominately enriched in response to Cd stress. At the cellular and molecular levels, significantly enriched pathways in lysosomes and phagosomes as well as replication, recombination and repair demonstrated that oxidative damage resulted from Cd exposure. Based on the selected DEGs, certain critical genes involved in defence and detoxification were analysed. These results may elucidate the molecular mechanism underlying spiders' responses to heavy metal stress.
ARTICLE | doi:10.20944/preprints202306.1920.v1
Subject: Biology And Life Sciences, Biology And Biotechnology Keywords: Amorphophallus; reference genes; RT-qPCR; gene expression
Online: 27 June 2023 (13:43:45 CEST)
Real-time fluorescent quantitative PCR (RT-qPCR) is the most classic and widely used technology for evaluating the expression level of target gene. In order to select the proper internal reference genes for RT-qPCR analysis in Amorphophallus konjac (Araceae), eight candidate internal reference genes, including 25S ribosomal RNA gene (25S rRNA), 18S ribosomal RNA gene (18S rRNA), actin gene (ACT), glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH), Ubiquitin gene (UBQ), β-tubulin gene (β-TUB), eukaryotic elongation factor 1-αgene(eEF-1α), and eukaryotic translation initiation factor 4α-1 gene (eIF-4α) were selected and tested the corresponding expression level in different tissues at different growing stages. The results showed that 25S rRNA, 18S rRNA, and ACT at the reproductive periods, eEF-1α and eIF-4α at the nutritional periods, and eEF-1α, UBQ, and ACT at different leaf developmental periods had a stable level of gene expression, respectively. These results might be useful for the study of gene function in A. konjac.
COMMUNICATION | doi:10.20944/preprints202004.0206.v2
Subject: Biology And Life Sciences, Immunology And Microbiology Keywords: COVID-19; RT-qPCR; liquid phase immunoassay
Online: 30 April 2020 (05:10:43 CEST)
The World Health Organisation declared Covid-19 as a pandemic on 11th March 2020. The main approach to tackle Covid-19 worldwide is to screen and provide supportive care to patients. For screening purpose RT-qPCR based test are used as an initial detections assay. The test is expensive, time consuming and not suitable for mass scale screening/ confirmation requirement. A recent advancement is development of Immunoassay procedures (liquid Phase tests or bed side 10-20minute strip test). In order to help and accelerate bringing life to normal after lock down, Pakistan is in dire need to develop and adopt the immunoassay procedures for mass scale screening and confirmation of COVID-19 infection. It is cheap and easy to perform without a lab requirement.
ARTICLE | doi:10.20944/preprints202310.0104.v1
Subject: Biology And Life Sciences, Virology Keywords: Hepatitis A; Hepatitis E; Norovirus; Real-time RT-qPCR
Online: 3 October 2023 (04:25:03 CEST)
Enteric viruses are the major cause of gastroenteritis and enteric hepatitis worldwide, but in some areas like Saudi Arabia little data is known about their presence in water sources. The available information from clinical samples is not enough to figure their actual prevalence. The aim of this study was to gather information for the first time in Saudi Arabia on the presence of Norovirus (NoV) genogroup GI and GII, hepatitis A virus (HAV) and hepatitis E virus (HEV) in water. For this purpose, thirteen monthly samples were collected in lake Wadi Hanifa and surrounding wells from December 2014 to November 2015. Viruses were detected and quantified by Real-time RT-qPCR. Despite HEV findings were anecdotic, our results highlight interesting behaviors of the other viruses. There was a higher prevalence of noroviruses in Wadi Hanifa samples than in well water samples (46.43% vs12.5% of NoV GI; 66.67% vs8.33% of NoV GII). On the contrary, similar levels of HAV positivity were observed (40.48% in surface water vs 43.06% in well water). Also, a strong influence of flooding events on HAV and NoV GI occurrence was observed in both surface and well water samples, being NoV GII apparently not affected.
COMMUNICATION | doi:10.20944/preprints202306.2007.v1
Subject: Biology And Life Sciences, Virology Keywords: SARS-CoV-2; RNA genome assembly; RT-qPCR; SPAdes
Online: 29 June 2023 (02:12:07 CEST)
During the recent pandemics of COVID-19, sequencing technics became powerful tool for gaining information about the SARS-CoV-2 virus and using this knowledge in our advantage. Thanks to this advantage, scientists over the world were able to search for emerging variations, watching the virus evolving in real time. Assembly of the virus genomes is crucial part of getting this kind of useful information. In our study, we sequenced 79 samples from nasopharyngeal swabs of COVID-19 patients. Positivity on disease was evaluated by qRT-PCR. By studying assembly success rate, we noticed, that Ct-value of qRT-PCR can predict success of genome assembly and SARS-CoV-2 lineage assignment. In this work we described the relationship between Ct-value and further steps of genome construction and its assignment to specific viral strain.
COMMUNICATION | doi:10.20944/preprints202203.0010.v1
Subject: Biology And Life Sciences, Virology Keywords: SARS-CoV-2; variant; Omicron; Delta; antigen; RT-qPCR
Online: 1 March 2022 (09:01:19 CET)
Rapid antigen detection tests (RAD) are commonly used for the diagnosis of SARS-CoV-2 infections. However, with the continuous emergence of new variants of concern (VOC) presenting various mutations potentially affecting the nucleocapsid protein, the analytical performances of these assays should be frequently reevaluated. One-hundred and twenty samples were selected and tested with both RT-qPCR and five commercial RAD commonly sold in Belgian pharmacies. Of these, direct whole genome sequencing identified the strains present in 116 samples, of which 70 were Delta and 46 were Omicron. Sensitivity across a wide range of Ct values (13.5 to 35.7; median = 21.3) were comparable and ranged from 70.0% to 77.1% for Delta strains and from 69.6% to 78.3% for Omicron strains. When taking swabs with a low viral load (Ct > 25), poor performances were observed for the Delta strains (20.0 to 40.0%) and, even more so, for Omicron strains (0.0 to 23.1%). Two devices failed to detect all samples (n = 13) containing Omicron strains with a low viral load. The poor performance observed with low viral loads is an important limitation of RAD, which is not sufficiently highlighted in the instruction for use of these devices.
COMMUNICATION | doi:10.20944/preprints202202.0158.v1
Subject: Biology And Life Sciences, Virology Keywords: SARS-CoV-2; wastewater; passive sampler; autosampler; RT-qPCR
Online: 11 February 2022 (08:38:07 CET)
Wastewater-based surveillance is emerging as an important tool for COVID-19 pandemic trending. Current methods of wastewater collection, such as grab and auto-composite sampling, have drawbacks that impede effective surveillance, especially from small catchments with limited accessibility. Passive samplers, which are more cost-effective and require fewer resources to process, are promising candidates for monitoring wastewater for SARS-CoV-2. Here, we compared traditional auto sampling with passive sampling for SARS-CoV-2 detection in wastewater. Torpedo-style 3D printed passive sampler device containing both cotton swabs and electronegative filter membranes was used. Between April and June 2021, fifteen passive samplers were placed at a local hospital wastewater outflow alongside an auto sampler. Reverse transcription and quantitative polymerase chain reaction (RT-qPCR) was used to detect SARS-CoV-2 in the samples after processing and RNA extraction. The swab and membrane of the passive sampler showed similar detection rates and Ct values for SARS-CoV-2 RNA for the N1 and N2 gene targets. The passive method performed as well as the grab/auto sampling, with no significant differences between N1 and N2 Ct values. There were discrepant results on two days with negative grab/auto samples and positive passive samples, which might be related to the longer duration of passive sampling in the study. Overall, the passive sampler was rapid, reliable and cost-effective, and could be used as an alternative sampling method for the detection of SARS-CoV-2 in wastewater.
ARTICLE | doi:10.20944/preprints202009.0324.v1
Subject: Biology And Life Sciences, Virology Keywords: MERS-CoV; camel; seroprevalence; transmission; ELISA; RT-qPCR; slaughterhouse
Online: 15 September 2020 (03:54:34 CEST)
Background: MERS-CoV is a zoonotic virus that have emerged in humans in 2012 and caused severe respiratory illness with mortality rate of 34.4%. Since its appearance, MERS-CoV have been reported in 27 countries and most of these cases were in Saudi Arabia. So far, dromedaries are considered to be the intermediate host and the only known source of human infection. Method: This study was designed to determine the seroprevalence and the infection rate of MERS-CoV in slaughtered food-camels in Riyadh, Saudi Arabia. A total of 171 nasal swabs along with 161 serum samples were collected during the winter; from January to April 2019. Nasal swabs were examined by Rapid test and RT-qPCR to detect MERS-CoV RNA, while serum samples were tested primarily using S1-based ELISA Kit to detect MERS-CoV (IgG) antibodies and subsequently by MERS pseudotyped viral particles (MERSpp) neutralization assay for confirmation. Genetic diversity of the positive isolates was determined based on the amplification and sequencing of the spike gene. Results: Our results showed high prevalence (38%) of MERS-CoV infection in slaughtered camels and high seropositivity (70.81%) during the time of the study. These data indicate previous and ongoing MERS-CoV infection in camels. Phylogenic analysis revealed relatively low genetic variability among our isolated samples. When these isolates were aligned against published spike sequences of MERS-CoV, deposited in global databases, there was sequence similarity of 94%. Conclusion: High seroprevalence and high genetic stability of MERS-CoV in camels indicating that camels pose a public health threat. The widespread of MERS-CoV infections in camels increases the risk of future zoonotic transmission into people with direct contact with these infected camels. This study confirms re-infections in camels, highlighting a challenge for vaccine development when it comes to protective immunity.
ARTICLE | doi:10.20944/preprints202006.0280.v1
Subject: Medicine And Pharmacology, Obstetrics And Gynaecology Keywords: antenatal stress; hair cortisol; term-placentae; RT-qPCR; human
Online: 21 June 2020 (16:30:08 CEST)
Anxiety, chronical stress and depression during pregnancy are considered to affect the offspring, presumably through placental dysregulation. We have studied the term placentae of pregnancies clinically monitored with the Beck’s Anxiety Inventory (BAI) and Edinburgh Postnatal Depression Scale (EPDS). A cutoff threshold for BAI/EPDS of 10 classed patients into an Index group (>10, n=23) and a Control group (<10, n=23). Cortisol concentrations in hair (HCC) were periodically monitored throughout pregnancy and delivery. Expression differences of main glucocorticoid pathway genes: i.e. corticotropin-releasing hormone (CRH), 11β-hydroxysteroid dehydrogenase (HSD11B2), glucocorticoid receptor (NR3C1), as well as other key stress biomarkers (Arginine Vasopressin, AVP and O-GlcNAc transferase, OGT) were explored in medial placentae using real-time qPCR and western blotting. Moreover, gene expression changes were considered for their association with HCC, offspring, gender and birthweight. A significant dysregulation of gene expression for CRH, AVP and HSD11B2 genes was seen in the Index group, compared to controls, while OGT and NR3C1 expression remained similar between groups. Placental gene expression of the stress-modulating enzyme 11β-hydroxysteroid dehydrogenase (HSD11B2) was related to both hair cortisol levels (Rho= 0.54; p<0.01) and the sex of the newborn in pregnancies perceived as stressful (Index, p<0.05). Gene expression of CRH correlated with both AVP (Rho= 0.79; p<0.001) and HSD11B2 (Rho= 0.45; p<0.03), and also between AVP with both HSD11B2 (Rho= 0.6; p<0.005) and NR3C1 (Rho= 0.56; p<0.03) in the Control group but not in the Index group; suggesting a possible loss of interaction in the mechanisms of action of these genes under stress circumstances during pregnancy.
ARTICLE | doi:10.20944/preprints202305.0001.v1
Subject: Medicine And Pharmacology, Epidemiology And Infectious Diseases Keywords: SARS-CoV-2; COVID-19; Children; Seroprevalence; RT-qPCR; Iran
Online: 1 May 2023 (00:16:12 CEST)
A population-based seroepidemiological and molecular survey for detection of earlier and re-cent SARS-CoV-2 infection was done in children aged 14 years or less in Tehran between 19 September 2020 and 21 June 2021. Demographic data, COVID-19 symptoms and infection status were recorded and IgG antibodies and RNA of SARS-CoV-2 were detected in sera and nasopha-ryngeal swab samples, respectively. Out of 1517 participants, cardinal symptoms of COVID-19 (fever >38 oC and/or cough and/or diarrhea) were detected in 18% and serological history of SARS-CoV-2 infection and PCR positivity were confirmed in 33.2% and 10.7% of the weighted-population, respectively. SARS-CoV-2 infection was significantly higher among 10–14-year-old children. Active infection was significantly higher in symptomatic children and during autumn 2020 and spring 2021. The RT-qPCR positivity was related to contact with in-fected persons. RT-qPCR positivity was significantly higher among families with a lower socio-economic status, while no association between RT-qPCR- or seropositivity was determined with household size, underlying diseases, or gender. In conclusion, high SARS-CoV-2 infection prev-alence and seroprevalence was detected in children in Tehran in different seasons. The infection was significantly higher in older age children, and those with a positive-history of close contact with infected cases and/or lower socioeconomic status.
ARTICLE | doi:10.20944/preprints202303.0330.v1
Subject: Arts And Humanities, Film, Radio And Television Keywords: infecção experimental; Aedes albopictus; febre amarela; reemergência; RT‒qPCR; isolamento viral
Online: 20 March 2023 (02:01:29 CET)
The risk of the emergence and reemergence of zoonoses is high in regions that are under anthropogenic actions, as they contribute to the risk of vector disease transmission. Yellow fever (YF) is among the main pathogenic arboviral disease in the world, and the Culicidae Aedes albopictus has been proposed to have the potential to transmit yellow fever virus (YFV). This mosquito inhabits both urban and wild environments, and under experimental conditions, it has been shown to be susceptible to infection by YFV. In this study, the vector competence of the mosquito Ae. albopictus for the YFV was investigated. Female Ae. albopictus were exposed to non-human primates (NHP) of the genus Callithrix infected with YFV for blood meal. Subsequently, on the 14th and 21st days post infection, the legs, heads, thorax/abdomen and saliva of the arthropods were collected and analyzed by viral isolation and molecular analysis techniques to verify the infection, dissemination and transmission. The presence of YFV was detected in saliva samples through viral isolation and in the head, thorax/abdomen and legs both by viral isolation and by molecular detection. The susceptibility of Ae. Albopictus to YFV confers a potential risk of reemergence of urban YF in Brazil.
ARTICLE | doi:10.20944/preprints202308.1098.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: Transcriptional meta-analysis; molecular docking; RT-qPCR; Bioinformatics; Structural-based virtual screening
Online: 15 August 2023 (11:47:28 CEST)
Gastric cancer (GC) is a highly heterogeneous, complex disease and the fifth most common cancer worldwide (about one million cases and 784 000 deaths worldwide in 2018). CG is lately diagnosed and guarantees a poor prognosis for GC (the 5-year survival rate is less than 20%, but in early detection can reach 90%). This study evaluated the transcriptional profile in tumor gastric samples to find genes highly expressed during tumor establishment and use the related proteins as targets to find new anticancer molecules. Data was collected at Gene Expression Omnibus (GEO) bank to obtain 3 dataset matrices that analyze gastric tumor tissue versus normal gastric tissue, performed microarray using GPL570 platform, and from different sources. The genes found in silico analysis were confirmed in several lines of GC cells by RT-qPCR. The protein data bank was used to find the correspondent proteins. Then a structural-based virtual screening was done to find molecules, and docking analysis was done using the DockThor server. Our results of transcriptomic analysis, together with RT-qPCR, confirm the high expression of the genes AJUBA, FBXL13, CCDC69, CD80 and NOLC1 in GC lines. Based on that, the correspondent proteins were used in SBVS analysis. Five molecules, one each target, MCULE-2386589557-0-6, MCULE-7343047040-0-1, MCULE-5230409338-0-3, MCULE-9178344200-0-1 and MCULE -5881513100-0-29. All molecules have favorable pharmacokinetic, pharmacodynamic and toxicological properties. Molecular docking analysis revealed that the molecules interact with proteins in critical sites for their activity. Using a virtual screening approach, a molecular docking study was performed for proteins encoded by genes that play important roles in cellular functions for carcinogenesis. Combining a systematic collection of public microarray data with a comparative meta-profiling, RT-qPCR, SBVS, and molecular docking analysis provided a suitable approach to find genes involved in GC and work the correspondent protein to strive for new molecules with anticancer properties.
REVIEW | doi:10.20944/preprints202106.0320.v1
Subject: Environmental And Earth Sciences, Water Science And Technology Keywords: RT-qPCR; assay validity; standard curve; quality assurance; quality control; wastewater surveillance
Online: 11 June 2021 (14:10:51 CEST)
The coronavirus disease 2019 (COVID-19) pandemic has led to wastewater surveillance becoming an important tool for monitoring the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within communities. As a result, molecular methods, in particular reverse transcription-quantitative PCR (RT-qPCR), have been employed to generate large data sets aimed at the detection and quantification of SARS-CoV-2 in wastewater. Although RT-qPCR is rapid and sensitive, there is no standard method that fits all use cases, there are no certified quantification standards and experiments are carried out using numerous different assays, reagents, instruments, and data analysis protocols. These variations can lead to the reporting of erroneous quantitative data resulting in potentially misleading interpretations and conclusions. We have reviewed the SARS-CoV-2 wastewater surveillance literature focusing on the variability of RT-qPCR data as revealed by inconsistent standard curves and associated parameters. We find that variation in these parameters and deviations from best practices as described in The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines suggest a lack of reproducibility and reliability in quantitative measurements of SARS-CoV-2 RNA in wastewater.
ARTICLE | doi:10.20944/preprints202307.1362.v1
Subject: Public Health And Healthcare, Health Policy And Services Keywords: SARS-CoV-2; Omicron; Wastewater-based epidemiology; RT-qPCR; SIR model; SIRS model
Online: 20 July 2023 (11:44:24 CEST)
The COVID-19 pandemic caused by the SARS-CoV-2 virus has inflicted significant mortality and morbidity worldwide. Continuous virus mutations have led to the emergence of new variants. The Omicron BA.1 sub-lineage prevailed as the dominant variant globally at the beginning of 2022 but was subsequently replaced by BA.2 in numerous countries. Wastewater-based epidemiology (WBE) offers an efficient tool for capturing viral shedding from infected individuals, enabling early detection of potential pandemic outbreaks without relying solely on community cooperation and clinical testing resources. This study integrated RT-qPCR assays for detecting general SARS-CoV-2 and its variants levels in wastewater into a modified triple susceptible-infected-recovered-susceptible (SIRS) model. The emergence of the Omicron-BA.1 variant was observed, replacing the presence of its predecessor, the Delta variant. Comparative analysis between the wastewater data and the modified SIRS model effectively described the BA.1 and subsequent BA.2 waves, with the decline of the Delta variant aligning with its diminished presence below the detection threshold in wastewater. This study demonstrates the potential of WBE as a valuable tool for future pandemics. Furthermore, by analyzing the sensitivity of different variants to model parameters, we are able to deduce real-life values of cross-variant immunity probabilities, emphasizing the asymmetry in their strength.
ARTICLE | doi:10.20944/preprints201912.0093.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: mixed linear model; genotyping-by-sequencing; functional validation; RT-qPCR; resistance genes; GWAS
Online: 7 December 2019 (12:41:39 CET)
Meloidogyne javanica causing root-knot nematode in soybean is an important problem in soybean areas, leading to several yield losses. Some accessions have been identified carrying resistance loci to this nematode specie. In this study, a set of 317 soybean accessions were characterized for resistance to M. javanica. Genome-wide association study (GWAS) was performed using SNPs from genotyping-by-sequencing (GBS), and a region of 29.2 Kbp on chromosome 13 was identified. The haplotype analysis showed that SNPs were able to discriminate susceptible and resistant accessions, leading to 25 accessions sharing the resistance locus. Furthermore, 5 accessions may be new M. javanica resistance sources. The screening of the SNPs in the USDA soybean germplasm showed that several accessions previous reported as resistance to other nematodes also showed the resistance haplotype on chromosome 13. High levels of concordance among the phenotypes of Brazilian cultivars and the SNPs in chromosome 13 were observed. A in silico analysis of the mapped region on soybean genome revealed a presence of 5 genes with structural similarity with major resistance genes. The expression levels of the candidate genes in the interval demonstrated a potential pseudogene, and other two model genes up-regulated in the resistance source after pathogen infection. The SNPs associated to the region conferring resistance is a important tool for introgression of the resistance by marker-assisted selection in soybean breeding programs.
ARTICLE | doi:10.20944/preprints202311.1062.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: small RNA; Pseudomonas; nitrogen; NtrC; gene expression; RT-qPCR; nitrogen metabolism; carbon/nitrogen homeostasis
Online: 16 November 2023 (10:34:33 CET)
The residual content of the only carbon source increases at the end of the exponential growth phase when P. putida BS3701 cells are cultured under nitrogen deficiency. Moreover, the mRNA quantity of some genes involved in carbon catabolism decreases. There are no available data on regulators capable of stimulating the target mRNA degradation in pseudomonads under nitrogen deficiency in the literature. We have applied both comparative genomics methods and a number of biomolecular ones to identify new small P. putida BS3701 RNAs. This is due to the fact that the formation of ncRNA-mRNA duplexes, recognized by RNases, is one of the main mechanisms for reducing the number of transcripts in a cell. We have used the following selection criteria: 1) the presence of the interest gene of the NtrC binding site and a sigma54-dependent promoter in the upstream area, 2) the conservative location of the interest gene in the P. putida and P. aeruginosa genomes, 3) increase in the product quantity of the interest gene in a cell under nitrogen deficiency on two different carbon sources (succinate and naphthalene), 4) the product detection of the interest gene in a fraction enriched with small RNAs. The expression of two new small RNAs has increased twice or more times under nitrogen deficiency on both succinate and naphthalene: expression of ng171 and ng379. ng379 contains narK RNA motif.
ARTICLE | doi:10.20944/preprints202004.0216.v1
Subject: Biology And Life Sciences, Immunology And Microbiology Keywords: COVID-19; SARS-CoV-2; field work; community; diagnosis; rapid detection; inactivation; RT-qPCR
Online: 14 April 2020 (08:41:04 CEST)
Outbreaks of coronavirus disease 2019 (COVID-19) have been recorded in different countries across the globe. The virus is highly contagious, hence early detection, isolation, and quarantine of infected patients will play an important role in containing the viral spread. Diagnosis in a mobile lab can aid to find infected patients in time. Here, we develop a field-deployable diagnostic workflow that can reliably detect COVID-19. Instruments used in this workflow could easily fit in a mobile cabin hospital and also be installed in the community. Different steps from sample inactivation to detection were optimized to find the fastest steps and portable instruments in detection of COVID-19. Each step was compared to that of the normal laboratory diagnosis set-up. From the results, our proposed workflow (80 min) was two times faster compared to that of the normal laboratory workflow (183 min) and a maximum of 32 samples could be detected at each run. Additionally, we showed that using 1% Rewocid WK-30 could inactivate the novel coronavirus directly without affecting the overall detection results. Comparison of our workflow using an in-house assay to that of a commercially acquired assay produced highly reliable results. From the 250 hospital samples tested, there was a high concordance 247/250 (98.8%) between the two assays. The in-house assay sensitivity and specificity were 116/116 (100%) and 131/134 (97.8%) compared to that of the commercial assay. Based on these results, we believe that our workflow is fast, reliable, adaptable and most importantly, field deployable.
ARTICLE | doi:10.20944/preprints202304.0574.v1
Subject: Biology And Life Sciences, Plant Sciences Keywords: ABA biosynthesis; ABA perception; Fragaria chiloensis; fruit ripening; NCED; PYR/PYL receptors; phylogenic analysis; RT-qPCR
Online: 19 April 2023 (08:43:03 CEST)
Hormones act as master ripening regulators. In non-climacteric fruit ABA plays a key role in ripening. Recently we confirmed in Fragaria chiloensis fruit that in response to ABA treatment the fruit induces ripening associated changes such as softening and color development. In consequence with these phenotypic changes, transcriptional variations associated with cell wall disassembly and anthocyanins biosynthesis were reported. As ABA stimulates ripening of F. chiloensis fruit, the molecular network involved in ABA metabolism was analyzed. Therefore, the expression level of genes involved in ABA biosynthesis and ABA perception were quantified during development of the fruit. Four NCED/CCDs and 6 PYR/PYLs family members were identified in F. chiloensis. Bioinformatics analyses confirmed the existence of key domains related to functional properties. Through RT-qPCR analyses the level of transcripts were quantified. FcNCED1 codifies a protein that displays crucial functional domains and the level of transcripts increased as the fruit develops and ripens, in parallel with the increment in ABA. In addition, FcPYL4 codifies for a functional ABA receptor and its expression follows an incremental pattern during ripening. The study concludes that FcNCED1 is involved in ABA biosynthesis, meanwhile FcPYL4 participates in ABA perception during ripening of F. chiloensis fruit.
ARTICLE | doi:10.20944/preprints202204.0091.v1
Subject: Medicine And Pharmacology, Epidemiology And Infectious Diseases Keywords: SARS-CoV-2; SARS-CoV; RT-PCR; Sanger sequencing; RT-qPCR; receptor-binding domain (RBD); N-terminal domain (NTD); Omicron; multi-allelic SNPs; false-positive
Online: 12 April 2022 (03:57:29 CEST)
Both SARS-CoV-2 and SARS-CoV initially appeared in China and spread to other parts of the world. SARS-CoV-2 has generated a COVID-19 pandemic causing more than 6 million human deaths worldwide while the SARS outbreak quickly ended in six months with a global total of 774 reported deaths. One of the factors contributing to this stunning difference in the outcome between these two outbreaks is the inaccuracy of the RT-qPCR tests for SARS-CoV-2, which generated a large number of false-negative and false-positive test results that have misled patient management and public health policy-makers. This article presented Sanger sequencing evidence to show that the RT-PCR diagnostic protocol established in 2003 for SARS-CoV can in fact detect SARS-CoV-2 accurately due to the well-known nonspecific PCR amplification of DNAs with similar sequences. Using nested RT-PCR followed by Sanger sequencing to retest 50 patient samples collected in January, 2022 and sold as RT-qPCR positive reference confirmed 21 (42%) were false-positive. Although the other 29 positive isolates were categorized as Omicron variant by partial sequencing of the N gene, and the RBD and the NTD of the S gene, 9 (31%) showed focal to complete sequencing failure in the S gene segments due to multi-allelic SNPs. During the course of the study, an Omicron variant isolate containing a BA.1 NTD and a BA.2 RBD in its S gene was also detected. Routine partial S gene sequencing of all PCR-positive samples can timely discover multi-allelic SNPs and viral recombination in the circulating variants for investigation of their impacts on vaccine efficacies, therapeutics and diagnostics.
REVIEW | doi:10.20944/preprints202009.0526.v1
Subject: Biology And Life Sciences, Virology Keywords: COVID-19 testing; molecular diagnostics; immunological testing; RT-qPCR; ELISA; pool PCR; lateral flow assay; rapid assay
Online: 23 September 2020 (03:33:12 CEST)
Accurate diagnosis at an early stage of infection is essential for the successful management of any contagious disease. The COVID-19, caused by the SARS-CoV-2 virus is a pandemic that has affected 214 countries affecting more than 30.8 million people causing 0.957 million deaths as of third week of September, 2020. The primary diagnosis of the infection is done either by the molecular technique of RT-qPCR by detecting portions of the RNA of the viral genome or through immunodiagnostic tests by detecting the viral proteins or the antibodies produced by the host. As the demand for the test increased rapidly many naive manufacturers entered the market with novel kits and more and more laboratories also entered the diagnostic arena making the test result more error-prone. There are serious debates globally and regionally on the sensitivity and specificity of these tests and about the overall accuracy and reliability of the tests for decision making on control strategies. The significance of the test is also complexed by the presence of asymptomatic carriers, re-occurrence of infection in cured patients as well as by the varied incubation periods of the infection and shifting of the viral location in the host tissues. In this paper, we review the techniques available for SARS-CoV-2 diagnosis and probable factors that can reduce the sensitivity and specificity of the different test methods currently in vogue. We also provide a check-list of factors to be taken care to avoid fallacious practices to reduce false positive and false negative results by the clinical laboratories
ARTICLE | doi:10.20944/preprints202208.0392.v1
Subject: Biology And Life Sciences, Virology Keywords: Severe Acute Respiratory Syndrome-CoV-2 (SARS-CoV2), COVID-19, molecular diagnostics, real-time polymerase chain reaction (RT-qPCR)
Online: 23 August 2022 (03:55:44 CEST)
Background: Coronavirus disease (COVID-19) is an infectious disease caused by the SARS-CoV-2. In Colombia, many commercial methods are now available to perform the RT-qPCR assays, and the laboratories must evaluate its diagnostic accuracy to ensure reliable results to suspected COVID-19 patients. The purpose of the study was to compare four commercial RT-qPCR assays for detection of SARS-CoV2 virus, from nasopharyngeal swab samples referred to Laboratorio Carvajal IPS, SAS of Tunja, Boyacá - Colombia. Methods: This prospective study was conducted on 152 samples of respiratory tract samples (Nasopharyngeal Swab) from patients with suspected SARS-CoV-2 infection. Diagnostic accuracy of GeneFinderTM COVID-19 Plus RealAmp (In Vitro diagnostic), One-Step Real-Time RT-PCR (Vitro Master diagnostica), Berlin modified protocol and gold standard Berlin protocol (Berlin Charite Probe One-Step RT-qPCR Kit, New England Biolabs) as reference was assessed. Operational characteristics were estimated in terms of sensitivity, specificity, agreement, and predictive values. Results: Using Berlin Charite Probe One-Step RT-qPCR Kit as reference, the sensitivity/specificity for the diagnostic tests were found to be GeneFinderTM COVID-19 Plus RealAmp Kit 100%/92.7%, One-Step Real-Time RT-PCR, One-Step Real-Time RT-PCR 92.75%/67.47%, and Berlin modified protocol 100%/96.39%. The results of four commercially available methods were found to be consistent with those obtained from Berlin Modified protocol analysis for % of the samples and showed good agreement (κ= 0.96). Concordant SARS-CoV2 negative and positive RT-qPCR results were reported for xxx and xxx samples, respectively. Summarize something about the Ct. Conclusion: Our data demonstrate that all commercially available methods are rapid and reliable for the identification of SARS-CoV-2 virus associated with COVID-19. One-Step RT-qPCR Kit and GeneFinderTM COVID-19 Plus RealAmp assay show optimal sensitivity compared with Belin modified protocol. In addition, there is no significant correlation between xxxxx
ARTICLE | doi:10.20944/preprints202310.0519.v1
Subject: Biology And Life Sciences, Virology Keywords: Grapevine leafroll disease; Closteroviridae; GLRaV-3cDNA clones; RT-qPCR; Western blot; vacuum agro-infiltration; agro-pricking; agro-drenching; agro-injection; Koch’s postulates
Online: 9 October 2023 (15:17:00 CEST)
Grapevine leafroll disease (GLRD) is the most globally prevalent and destructive disease complex responsible for significant reductions in grape yield and quality as well as wine production. GLRD is associated with several positive-strand RNA viruses of the family Closteroviridae, designated grapevine leafroll-associated viruses (GLRaVs). However, the specific etiological role of any of these GLRaVs in GLRD has not been demonstrated. Even though GLRaV-3 is considered the chief GLRD agent, little is known about the molecular, cellular and pathological properties of this virus. Such knowledge gap is due to multiple factors, including unavailability of biologically active virus cDNA clones and the lack of reliable experimental systems for launching grapevine infection using such clones. In this work, we have tested four methods for inoculating tissue cultured grapevine plantlets with cDNA clones of GLRaV-3: i) vacuum agro-infiltration; ii) agro-pricking; iii) agro-drenching and iv) agro-injection. We have shown that vacuum agro-infiltration was the most effective among these methods. Further, we examined the impacts of different experimental conditions on the survival and infectivity rate of grapevines after infiltration. To verify the infectivity rate for different treatments, we used RT-PCR, RT-qPCR, and Western blotting. We have found that humidity plays a critical role in the survival of plantlets after agro-infiltration and that the use of RNA silencing suppressor, and dormancy treatment both had strong effects on the infection rates. To our best knowledge, the experimental protocol reported here is the most effective system for launching infection of grapevine using cDNA clones of grapevine viruses featuring up to 70% infection rate. This system has strong potential to facilitate grapevine virology research including fulfillment of Koch’s postulates for GLRD and other major virus diseases as well as the molecular, cellular, and pathological properties of GLRaVs and potentially other important grapevine viruses.
ARTICLE | doi:10.20944/preprints202109.0421.v1
Subject: Biology And Life Sciences, Immunology And Microbiology Keywords: laboratory practical class; undergraduate teaching; qPCR
Online: 24 September 2021 (09:04:57 CEST)
From gene expression studies to identifying microbes quantitative polymerase chain reaction (qPCR) is widely used in research and medical diagnostics. In transmittable diseases like the Ebola outbreak in West Africa (2014-2016), or the present SARS-CoV2 pandemic qPCR plays a key role in the detection of infected patients. Although the technique itself is decades old with reliable approaches (eg. TaqMan essay) in the diagnosis of pathogens many people showed distrust in it during the SARS-CoV2 outbreak. This came mainly from not understanding or misunderstanding the principles of qPCR. This situation motivated us to design a simple laboratory practical class, in which students have opportunities to understand the underlying principles of qPCR and its advantages in microbiological diagnosis. Moreover, during the exercise, students can develop skills such as handling experimental assays, and the ability to solve problems, discuss their observations. Finally, this activity brings them closer to the clinical practice and they can see the impact of the science on real life. The class is addressed to undergraduate students of biological sciences.
ARTICLE | doi:10.20944/preprints202212.0009.v1
Subject: Chemistry And Materials Science, Analytical Chemistry Keywords: Glyphosate; Aptamer; qPCR; SYBR Green I; sensor
Online: 1 December 2022 (04:00:13 CET)
Glyphosate (GLYP) is a broad-spectrum, non-selective, organic phosphine post emergence herbicide registered for use on many food and non-food field. Herein, we developed a biosensor (Mbs@dsDNA) based on carboxylated modified magnetic beads incubated with NH2-polyA and then hybridized with polyT-glyphosate aptamer and complementary DNA. Afterward, a quantitative detection method based on qPCR was established. When the glyphosate aptamer on Mbs@dsDNA specifically recognized glyphosate, a complementary DNA is released and then enters the qPCR signal amplification process. The linear range of the method was 0.1-5 μg/mL, and the detection limit was set at 0.1 μg/mL. The recoveries in tap water were ranged from 103.4 ~ 104.9%, and the relative standard deviations (RSDs) were < 1%. The aptamer proposed in this study has a good potential for recognizing glyphosate. The detection method combined with qPCR might have a good application prospect in detecting and supervising other pesticide residues.
REVIEW | doi:10.20944/preprints202308.1678.v1
Subject: Public Health And Healthcare, Physical Therapy, Sports Therapy And Rehabilitation Keywords: CONV-RT; FLASH-RT; ultra-high-dose-rate; beam characteristics; pulsed beams; normal tissue sparing
Online: 24 August 2023 (04:10:58 CEST)
FLASH-RT represents a novel therapeutic radiation modality that holds remarkable potential for mitigating radiation therapy’s adverse side effects. This cutting-edge technology allows for the sparing of healthy tissue while precisely targeting cancerous cells by administering an ultra-high dose-rates of typically between 10 and 30 Gy in less than a few hundred milliseconds. FLASH-RT has demonstrated impressive results in small-animal models, prompting scientists to adapt and advance existing technologies to make it a viable treatment option for humans. However, producing the ultra-high-dose-rate radiation required for the therapy remains a significant challenge. Several radiation sources, such as very high energy electrons (VHEEs), low energy electrons, x-rays, and protons, have been studied for their ability to deliver the necessary dose. Among them, FLASH-x-ray has gained the most attention owing to its capacity to penetrate deeply seated tumours. Despite the complexity of the process, the potential advantages of FLASH-RT make it an exciting area of research. To achieve the FLASH effect, high-frequency, pulsed irradiated accelerator technology can be employed. Sparing healthy tissue, it may allow for more aggressive and effective cancer treatments, leading to a better quality of life for patients. Ongoing research and development will be necessary to refine and optimize this approach to radiation therapy.
ARTICLE | doi:10.20944/preprints202308.2102.v1
Subject: Biology And Life Sciences, Neuroscience And Neurology Keywords: BCI, rt-fMRI, MI, DWGC, svm
Online: 31 August 2023 (09:43:14 CEST)
This article presents a method for extracting neural signal features to identify the imagination of left and right hand grasping movements. A functional magnetic resonance imaging (fMRI) experiment is employed to identify four brain regions with significant activations during motor imagery(MI) and the effective connections between these regions of interest (ROIs) were calculated using Dynamic Window-level Granger Causality (DWGC). Then, a real-time fMRI(rt-fMRI) classification system for left and right hand MI is developed using the Open-NFT platform. The experimental results show that incorporating effective connections can enhance the average accuracy of real-time three-class classification (rest, left hand and right hand) by 3% in comparison to traditional multivoxel pattern classification analysis(MVPA). Moreover, it significantly improves classification accuracy during the initial stage of MI tasks while reducing the latency effects in real-time decoding. The study suggests that the effective connections obtained through the DWGC method serve as valuable features for real-time decoding of MI using fMRI. Moreover, they exhibit higher sensitivity to changes in brain states. This research offers theoretical support and technical guidance for extracting neural signal features in the context of fMRI-based studies.
ARTICLE | doi:10.20944/preprints202308.1650.v1
Online: 23 August 2023 (10:08:12 CEST)
Peste des Petits Ruminants (PPR), a highly contagious viral disease, causes significant economic losses in sheep and goats. Laboratory diagnosis is crucial to disease control and eradication. Since PPRVs have recently adopted new hosts, genetic diversity within isolates of the same lineage is more likely than if they were host-specific, sheep and goats most often. ELISA detects antibodies and antigens in serology. However, mishandling, environmental conditions (temperature and humidity), storage, and sensitivity issues of commercially available ELISA kits prevent rapid PPR virus detection in third-world countries like Pakistan. In the 2020-21 outbreaks in Pakistan, 325 blood samples, 19 swabs, and 6 virus tissue samples of sheep and goats were collected from Gilgit, Islamabad, and Fateh Jang. These virus isolates were submitted to NCBI for partial N gene sequencing. Antigen was prepared from indigenous virus Using semi-purified antigen from PPRV, an indirect ELISA for the detection of PPR antibodies in goat and sheep serum was developed using MW600922 grown in Vero cells. Dilutions of 1:200 serums and 1:32 antigens improve antibody detection. The N-gene-based analysis revealed a better understanding of PPRV genetic characterization. This study develops an RT-PCR assay to detect PPRV by targeting the N-protein gene. This assay offers an accurate and affordable diagnostic tool for PPRV and partial genome sequencing. Results described that PPRV isolates from the current study have 99.73% and 99.4% similarity to previously published Lahore and Faisalabad isolates from Pakistan, respectively. Compared to previous isolates from NCBI data, the virus showed high divergence rates with the Turkish strain. The comparative analysis between Commercial kit (c-ELISA) and I-ELISA revealed that the former had a high sensitivity of 90.60% and specificity of 85.23% compared with the cELISA kit. By comparing I-ELISA and VNT, we find that the current assay is 100% specific and 82.14 % sensitive. Based on these results, serological surveys for PPR antibodies, on a larger scale, in small ruminants can be conducted with indirect ELISA rather than competitive ELISA. Furthermore, the cost and storage efficiency was highly significant because the currently developed method has a low production cost, which is much lesser than the commercially available kit. Our findings demonstrated a significant breakthrough in terms of cost-effectiveness and storage efficiency, and they are highly recommended for developing countries.
REVIEW | doi:10.20944/preprints201707.0003.v1
Subject: Medicine And Pharmacology, Veterinary Medicine Keywords: prion, cervids, PMCA, RT-QuIC, diagnosis
Online: 3 July 2017 (17:34:35 CEST)
Since chronic wasting disease (CWD) was first identified nearly 50 years ago in a captive mule deer herd in the Rocky Mountains of the United States, it has slowly spread across North America through the natural and anthropogenic movement of cervids and their carcasses. As the endemic areas have expanded, so has the need for rapid, sensitive, and cost effective diagnostic tests – especially those which take advantage of samples collected antemortem. Over the past two decades, strategies have evolved from the recognition of microscopic spongiform pathology and associated immunohistochemical staining of the misfolded prion protein to enzyme-linked immunoassays capable of detecting the abnormal prion conformer in postmortem samples. In a history that parallels the diagnosis of more conventional infectious agents, both qualitative and real-time amplification assays have recently been developed to detect minute quantities of misfolded prions in a range of biological and environmental samples. With these more sensitive and semi-quantitative approaches has come a greater understanding of the pathogenesis and epidemiology of this disease in the native host. Because the molecular pathogenesis of prion protein misfolding is broadly analogous to the misfolding of other pathogenic proteins, including Aβ and α-synuclein, efforts are currently underway to apply these in vitro amplification techniques towards the diagnosis of Alzheimer’s disease, Parkinson’s disease, and other proteinopathies. Chronic wasting disease – once a rare disease of Colorado mule deer – now represents one of the few naturally occurring protein misfolding disorders which might allow continued development and implementation of novel diagnostic strategies in an animal model.
REVIEW | doi:10.20944/preprints202210.0291.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: eDNA; eRNA; fish disease; surveillance; hydrolysis; degradation; qPCR
Online: 19 October 2022 (14:45:21 CEST)
Organisms release their nucleic acid in the environment including the DNA and RNA which can be used to detect their presence. eDNA/eRNA techniques are being used in different sectors to identify organisms from soil, water, air, and ice since long. The advancement in technology led to easier detection of different organisms without impacting the environment and the organism itself. These methods are being employed in different areas including surveillance, history, and conservation. eDNA and eRNA methods are being extensively used in aquaculture and fisheries setting to understand the presence of different fish species and pathogens in water. However, there are some challenges associated with the reliability of the results because of the degradation of nucleic acid by several factors. In aquaculture there are several diseases and parasites detected with these methods. In this review we discuss different aquaculture diseases and parasites detected with eDNA/eRNA approach and the fate of these nucleic acids when subjected to different water quality and environmental parameters. This review intends to help the researcher about the potential of eDNA/eRNA based detection of pathogens in aquaculture; this will be useful to predict the potential outbreak before it occurs. Along with that this paper intends to make people understand several factors that degrade and can hamper the detection of these nucleic acids.
ARTICLE | doi:10.20944/preprints201809.0413.v1
Subject: Biology And Life Sciences, Immunology And Microbiology Keywords: Paenibacillus larvae; optimized qPCR; quantification; honey; hive debris
Online: 20 September 2018 (14:13:42 CEST)
The application of quantitative PCR (qPCR) as a routine method to detect and enumerate Paenibacillus larvae in honey and hive debris could greatly speed up the estimation of prevalence and outbreak risk of the American foulbrood (AFB) disease of Apis mellifera. However, none of the qPCR tests described so far has been officially proposed as a standard procedure for P. larvae detection and enumeration for surveillance purposes. Therefore, in this study inclusivity, exclusivity and sensitivity in detection of P. larvae spores directly in samples of honey and hive debris were re-evaluated for the previously published qPCR methods. To this aim recently acquired P. larvae sequence data were considered to assess inclusivity in silico and more appropriate non-target species were used to verify exclusivity experimentally. This led to the modification of one of the previously described methods resulting in a new test capable to allow the detection of P. larvae spores in honey and hive debris down to 1 CFU/g. The application of the qPCR test optimized in this study can allow to reliably detect and quantify P. larvae in honey and hive debris, thus circumventing the disadvantages of late AFB diagnosis based on clinical symptoms and possible underestimation of spore numbers that is the main drawback of culture-dependent procedures.
ARTICLE | doi:10.20944/preprints202308.1692.v1
Subject: Biology And Life Sciences, Parasitology Keywords: HRM; Leishmania; Americas; leishmaniasis; qPCR; parasite typing; HSP70; diagnosis.
Online: 24 August 2023 (09:48:48 CEST)
High Resolution Melting Analysis (HRM) has been pointed as a suitable alternative method to detect and identify Leishmania species. Herein, we aimed to evaluate the sensitivity, specificity, accuracy, and limitations of a HSP70-HRM protocol both as a diagnostic scheme applied in clinical samples and as a species typing tool for laboratory research and reference services. Our data reveal the pronounced species-typing potential of the HSP70-HRM in DNA from cultured parasites. For clinical samples, however, we advise caution due to the parasite load-dependent accuracy. In the light of these findings and considering the importance of parasite load determination for clinical and research purposes we recommend the integration of the presented typing scheme and the previously published Leishmania quantifying approach as combined tools for clinicians, surveillance, and research.
ARTICLE | doi:10.20944/preprints202209.0267.v1
Subject: Biology And Life Sciences, Agricultural Science And Agronomy Keywords: spore trap; qPCR; Moniliasis; Theobroma cacao; Frosty pod rot
Online: 19 September 2022 (07:33:31 CEST)
Frosty pod rot, caused by Moniliophthora roreri, is the most damaging disease of cacao in Latin America. However, to better comprehend its epidemiology, we must understand its dissemination and proliferation. Still, we ignore how loads of M. roreri spores fluctuate across growing seasons since we lack a reliable technique to quantify M. roreri spores in the fields. Therefore, we developed a method that uses a spore trap to capture M. roreri spores and qPCR to quantify them. This study demonstrated that this technique could quantify 3.9 x104 M. roreri spores with a 95 % confidence level. However, it could not differentiate between M. roreri and its close relative, M. perniciosa. Despite this limitation, we could detect and quantify Moniliophthora spores from environmental samples taken from a cacao field. This technique can help the phytopathologist address studies more accurately in disseminating cacao pathogens.
ARTICLE | doi:10.20944/preprints202011.0213.v1
Subject: Biology And Life Sciences, Anatomy And Physiology Keywords: Gekkota; reptiles; DNA-seq; sex chromosomes; sex determination; qPCR
Online: 5 November 2020 (14:14:53 CET)
Geckos demonstrate a remarkable variability in sex determination systems, but our limited knowledge prohibits accurate conclusions on the evolution of sex determination in this group. Eyelid geckos (Eublepharidae) are of particular interest, as they encompass species with both environmental and genotypic sex determination. We identified for the first time the X-specific gene content in the Yucatán banded gecko, Coleonyx elegans, possessing X1X1X2X2/X1X2Y multiple sex chromosomes by comparative genome coverage analysis between sexes. The X-specific gene content of Coleonyx elegans was revealed to be partially homologous to genomic regions linked to the chicken autosomes 1, 6 and 11. A qPCR-based test was applied to validate a subset of X-specific genes by comparing the difference in gene copy numbers between sexes, and to explore the homology of sex chromosomes across 11 eublepharid, two phyllodactylid and one sphaerodactylid species. Homologous sex chromosomes are shared between Coleonyx elegans and Coleonyx mitratus, two species diverged approximately 34 million years ago, but not with other tested species. As far as we know, the X-specific gene content of Coleonyx elegans / Coleonyx mitratus was never involved in the sex chromosomes of other gecko lineages, indicating that the sex chromosomes in this clade of eublepharid geckos evolved independently.
ARTICLE | doi:10.20944/preprints202309.0559.v1
Subject: Medicine And Pharmacology, Veterinary Medicine Keywords: RT-QuIC; Chronic Wasting Disease; Diagnostics; Optimization
Online: 7 September 2023 (15:45:02 CEST)
Real-time quaking-induced conversion (RT-QuIC) assays have become common in the detection of chronic wasting disease (CWD) and are very sensitive provided the assay duration is sufficient. However, a prolonged assay duration may lead to non-specific signal amplification. The wide range of pre-defined assay durations in current RT-QuIC applications presents a need for optimization of the RT-QuIC assay duration. In this study, receiver operating characteristic (ROC) analysis was applied to optimize assay duration for detection of CWD in obex and retropharyngeal lymph node (RLN) tissue specimens. Two different fluorescence thresholds were used: a fixed threshold based on background fluorescence (Tstdev) and a max-point ratio (maximum/background fluorescence) threshold (TMPR) to determine CWD positivity. The optimal assay duration was 27 h for both obex and RLN based on Tstdev, and 27 and 28 h for obex and RLN, respectively, based on TMPR. The optimized assay durations were then evaluated for screening CWD in white-tailed deer from an affected farm. Results by RT-QuIC using optimized duration based on Tstdev and TMPR were in 100% and 92.3 % or higher agreement with those by the widely used screening assay, ELISA. In comparison, when using a 40 h assay duration, the agreement between RT-QuIC and ELISA reduced to 89.2% or higher, and the RT-QuIC results were significantly (p < 0.05) different from those using optimum durations. These findings demonstrated that the application of ROC analysis for the optimization of assay duration could improve the RT-QuIC assay for screening CWD in white-tailed deer.
BRIEF REPORT | doi:10.20944/preprints202005.0193.v1
Subject: Medicine And Pharmacology, Epidemiology And Infectious Diseases Keywords: COVID-19; diagnostic accuracy; CT; RT-PCR
Online: 11 May 2020 (12:35:08 CEST)
Introduction: Clinicians have been struggling with the optimal diagnostic approach of patients with suspected COVID-19. We evaluated the added value of chest CT over RT-PCR alone. Methods: Consecutive adult patients with suspected COVID-19 presenting to the emergency department (Academic Medical Center, Amsterdam University Medical Centers, the Netherlands) from March 16th to April 16th were retrospectively included if they required hospital admission and underwent chest CT and RT-PCR testing for SARS-CoV-2 infection. The CO-RADS classification was used to assess the radiological probability of COVID-19, where a score of 1-2 was considered as negative, 3 as indeterminate, and 4-5 as positive. CT results were stratified by initial RT-PCR results. For patients with a negative RT-PCR but a positive CT, serology or multidisciplinary discussion after clinical follow-up constituted the final diagnosis. Results: 258 patients with suspected COVID-19 were admitted, of which 239 were included because they had both CT and RT-PCR testing upon admission. Overall, 112 patients (46.9%) had a positive initial RT-PCR, and 14 (5.9%) had a positive repeat RT-PCR. Of 127 patients with a negative or indeterminate initial RT-PCR, 38 (29.9% [95%CI 21.3-39.3%]) had a positive CT. Of these, 13 had a positive RT-PCR upon repeat testing, and 5 had positive serology. The remaining 20 patients were assessed in a multidisciplinary consensus meeting, and for 13 it was concluded that COVID-19 was ‘very likely’. Of 112 patients with a positive initial RT-PCR result, CT was positive in 104 (92.9% [95%CI 89.3-97.5%]). Conclusion: In a high-prevalence emergency department setting, chest CT showed high probability of COVID-19 (CO-RADS 4-5) in 29.9% of patients with a negative or indeterminate initial RT-PCR result. As the majority of these patients had proven or ‘very likely’ COVID-19 after follow-up, we believe that CT helps in the identification of patients who should be admitted in isolation.
REVIEW | doi:10.20944/preprints202005.0142.v1
Online: 8 May 2020 (12:26:46 CEST)
COVID-19 was identified in Wuhan, China in in December 2019, and rapidly spread worldwide, being declared global pandemic one month later on 30 January 2020. Since its emergence, COVID-19 has raised global concerns associated with drastic measures that were never adopted in any previous outbreak, to contain the situation as early as possible. The 2019 novel corona virus (2019-nCoV) or SARS-CoV-2 is the causative agent of COVID-19. 2019-nCoV genetic sequence was rapidly identified within few days since the first reported cases and RT-PCR kits became available for COVID-19 diagnosis. However, RT-PCR diagnosis carries a risk of false-negative results, therefore additional serologic test are needed. The most important approach in the battle against COVID-19 is rapid diagnosis of suspicious cases, timely therapeutic intervention and isolation to avoid community spread. In this review, we summarize the clinical scenario that raises suspicion of COVID-19 and available laboratory diagnostics.
ARTICLE | doi:10.20944/preprints202311.0196.v1
Subject: Biology And Life Sciences, Immunology And Microbiology Keywords: Vulvovaginal Candidiasis; qPCR; Candida species, antifungal susceptibility, resistance-related mutations
Online: 3 November 2023 (04:02:59 CET)
Vulvovaginal candidiasis (VVC) is a prevalent condition affecting women worldwide. This study aimed to develop a rapid qPCR assay for accurate identification of VVC etiological agents and reduced azole susceptibility. One hundred and twenty nine vaginal samples from an outpatient clinic (Bilbao-Spain) were analyzed using culture-based methods and multiplex qPCR targeting fungal species, Candida albicans being the predominant species. Antifungal susceptibility tests revealed reduced azole susceptibility in three (3,48%) isolates. Molecular analysis identified several mutations in genes associated with azole resistance and novel mutations in TAC1 and MRR1 genes were also identified, which could contribute to drug resistance.
ARTICLE | doi:10.20944/preprints202306.1718.v1
Subject: Biology And Life Sciences, Virology Keywords: Pacific oysters; OsHV-1; latent; persistent; qPCR; nested PCR; hemocytes
Online: 25 June 2023 (05:24:35 CEST)
Ostreid herpesvirus 1 (OsHV-1) is one of the most economically important pathogens of Pacific oysters. Understanding the pathogenesis of this virus is critical to developing tools to control outbreaks on shellfish farms. OsHV-1 is genetically related to vertebrate herpesviruses, which have a lytic and a latent stage, with the latent stage capable of being reactivated to the lytic stage. Here, OsHV-1 latency in Pacific oysters was investigated in experimentally and naturally infected oysters. Lytic infection in one-year-old oysters injected with the Tomales Bay strain of OsHV-1 was detectable between 1 and 4 days post-infection (dpi) but was not detectable after 5 dpi. The infected oysters shed 1102 to 1104 DNA copies/ml during the 4-day acute phase. At 21 dpi, the recovered oysters were temperature and chemically stressed and were found to shed 1104 to 1105 DNA copies/ml over a period of 48 h. Lytic shedding was not detectable in two-year-old oysters injected similarly with the same strain of OsHV-1; however, the OsHV-1 genome was detectable by qPCR in the adductor muscle, gill, mantle, and hemocytes within the first 3 dpi, after which it became undetectable. No OsHV-1 was detectable in the adductor muscle, gill, or mantle from experimentally infected oysters on days 15 and 21 post-infection or from oysters sampled 9 months after surviving an OsHV-1 mortality event; however, OsHV-1 DNA could be detected in hemocytes of both experimentally infected oysters at 21 dpi and naturally infected oysters. In addition, lytic viral gene transcription was detectable in hemocytes of experimentally infected oysters between 1 and 21 dpi and in hemocytes of naturally infected oysters.
ARTICLE | doi:10.20944/preprints202107.0604.v2
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: COVID-19; reverse transcription; qPCR; SARS-CoV-2; molecular diagnosis
Online: 3 August 2021 (15:34:05 CEST)
The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an established tool for the diagnosis of RNA pathogens. Its potential for automation has caused it to be used as a presence/absence diagnostic tool even when RNA quantification is not required. This technology has been pushed to the forefront of public awareness by the COVID-19 pandemic, as its global application has enabled rapid and analytically sensitive mass testing, with the first test targeting three viral genes published within days of the publication of the SARS-CoV-2 genomic sequence. One of those, targeting the RNA-dependent RNA polymerase gene, has been heavily criticised for supposed scientific flaws at the molecular and methodological level and this criticism has been extrapolated to doubts about the validity of RT-qPCR for COVID-19 testing in general. We have analysed this assay in detail and our findings reveal some limitations, but also highlight the robustness of the RT-qPCR methodology for SARS-CoV-2 detection. Whilst our data show that some errors can be tolerated, it is always prudent to confirm that primer and probe sequences complement their intended target, since when errors do occur, they may result in a reduction in the analytical sensitivity. However, in this case it is unlikely that a mismatch will result in poor specificity or significant number of false positive SARS-CoV-2 diagnoses, especially as this is routinely checked by diagnostic laboratories as part of their quality assurance.
ARTICLE | doi:10.20944/preprints201708.0007.v1
Subject: Biology And Life Sciences, Immunology And Microbiology Keywords: natural mineral water; free living protozoa; Nontuberculous mycobacteria; Legionella; qPCR
Online: 3 August 2017 (09:11:03 CEST)
Italian Directives recommends the good quality of natural mineral waters but literature data assert a potential risk from several microorganisms colonizing wellsprings and mineral water bottling plants. Aim of study is the identification of microorganisms from spring waters (SW) and bottled mineral waters (BMW) samples. Methods: Routine microbiological indicators, Legionella spp., Nontuberculous mycobacteria (NTM), protozoa (FLA) and physical-chemical parameters were assessed in 24 SW and 10 BMW samples performing culture methods and molecular tests as PCR and qPCR. Results: In 33 out of 34 samples no cultivable bacteria were isolated with the exception of 83 CFU/L of Mycobacterium gilvum, detected in one warm rich-mineralized SW. qPCR showed the presence of Legionella genomic units in 24% of samples (mean 2,9x102±1,7x102 GU/L) and NTM genomic units in 18% of samples (mean 5,7x103±4,1x103 GU/L). Vermamoeba vermiformis and Acanthamoeba polyphaga were recovered respectively in 70% of BMW samples (counts from 1,3x103 to 1,2x105) and 42% of SW samples (counts from 1,1x103 to 1,3x104). Vahlkampfia spp. was detected in 42% of SW and 70% of BMW samples (mean 1,3x104 ±2,9x103 GU/L). Conclusion: The study highlights a low rate of microbial risk and the importance of risk assessment in natural mineral waters.
BRIEF REPORT | doi:10.20944/preprints202306.1122.v1
Subject: Biology And Life Sciences, Animal Science, Veterinary Science And Zoology Keywords: alphavirus; Costa Rica; Venezuelan equine encephalitis; RT-PCRs
Online: 15 June 2023 (10:19:06 CEST)
Alphavirus species are globally distributed zoonoses, primarily transmitted by arthropods. In Costa Rica, Venezuelan equine encephalitis virus (VEEV) and Eastern equine encephalitis virus (EEEV) are endemic. The objective of this study is to detect these viruses in brain samples from equines displaying nervous signs. For this purpose, four published universal RT-PCR methods were compared. The most sensitive and specific RT-PCR method was used to test a total of 70 brain samples, including 40 from bovines and 30 from equines, all exhibiting nervous signs. In the positive cases, eight different brain regions were extracted and tested using this RT-PCR. Positive cases were confirmed through sequencing. Among the four universal RT-PCR assays, Torii RT-PCR demonstrated the highest sensitivity and specificity for diagnosing VEEV and EEEV. Not all assessed brain regions showed DNA amplification. None of the bovine brains was positive and out of the 30 equine brain samples, only four tested positive, and sequencing confirmed two of these samples as VEEV subtype IE. Torii RT-PCR successfully detected VEEV in pools of the hip-pocampus, spinal cord, and basal nuclei, making these brain regions suitable for diagnosing this virus. None of the samples were positive for EEEV.
ARTICLE | doi:10.20944/preprints202306.1956.v1
Subject: Biology And Life Sciences, Aquatic Science Keywords: COI; teleostei; nucleotide diversity; haplotypic diversity; qPCR; demographic history; environmental DNA
Online: 28 June 2023 (07:37:41 CEST)
Fish tissue samples from 203 adult individuals were collected in the main ports and markets of the Pacific coast of Panama. Molecular identification based on cytochrome oxidase I gene segment of all species was verified GENBANK reference sequences. 34 species from 14 families (Ariidae, Lutjanidae, Caranjidae, Scianidae, Centropomidae, Serranidae, Scombridae, Sphyraenidae, Haemulidae, Gerreidae, Stromotidae, Lobotidae, Malacanthidae, Mugilidae) were identified at species molecular level from 164 sequences. Also, three Caribbean species were molecularly identified among the analyzed samples (Mycteroperca xenarcha, Paralonchurus brasilensis and Lobotes surinamensis). Species diversity was slightly higher in the Gulf of Panama than in the Gulf of Chiriquí. For species with 5 or more individual sequences, genetic diversity and genetic connectivity parameters such as: total number of haplotypes (H), haplotype diversity (Hd), and nucleotide diversity (π) were calculated. Overall, pelagic-migratory species showed higher values of genetic diversity than coastal and estuarine species with some exceptions. Connectivity between Gulf areas was compared using values of genetic distances and genetic differentiation (Fst). High level of connectivity observed between Gulf of Chiriqui and Gulf of Montijo indicates the existence of a single stock in that area for the following species: Scomberomorus sierra, Caranx caninus and Lutjanus guttatus. Demographic history of the most common species was examined using Tajima’s D values suggesting population expansion for two snapper species, L. peru and L. argentiventris, having significant and higher values. Another important contribution from this research was the production of primers and dual labeled probes for environmental DNA detection using qPCR for the five most abundant species (spotted rose snapper, yellow snapper, green jack, Pacific crevalle jack and the Pacific sierra fish). These markers represent a new set of tools for environmental DNA (eDNA) detection and molecular traceability of three commercially important fish species along the supply chain including landing sites and markets of the main fishery areas.
ARTICLE | doi:10.20944/preprints202202.0286.v1
Subject: Biology And Life Sciences, Immunology And Microbiology Keywords: cholera; Vibrio cholerae; Vibrio cholerae O1; endemic; toxigenic; abundance and qPCR
Online: 23 February 2022 (06:45:35 CET)
Cholera is a severe diarrheal disease caused by Vibrio cholerae, a natural inhabitant of brackish water. Effective control of cholera outbreaks depends on prompt detection of the pathogen from clinical specimens and tracking its source in the environment. Although the epidemiology of chol-era is well studied, rapid detection of V. cholerae remains a challenge, and data on its abundance in environmental sources are limited. Here, we describe a sensitive molecular quantification as-say by qPCR, which can be used on-site in low resource settings directly on water without the need for DNA extraction. This newly optimized method exhibited 100% specificity for total V. cholerae as well as V. cholerae O1 and allowed detection of as few as three target genome copies per reaction. The limit of detection is as low as 5 × 10E3 genome copies/L of water after concentrat-ing biomass from the sample. The ability to perform qPCR directly on water samples, portable features of the equipment, stability of the reagents at 4°C and user-friendly online software facili-tate fast quantitative analysis of V. cholerae. These characteristics make this assay extremely use-ful for field research in resource-poor settings and could support continuous monitoring in cholera endemic areas.
ARTICLE | doi:10.20944/preprints201908.0124.v1
Subject: Chemistry And Materials Science, Medicinal Chemistry Keywords: plasmonics; nanomedicine; theranostics; copper; VEGF; glioblastoma; differentiated neuroblastoma; peptidomimetics; qPCR; actin.
Online: 11 August 2019 (07:13:00 CEST)
Angiogenin (ANG), an endogenous protein that plays a key role in cell growth and survival, has been scrutinised here as promising nanomedicine tool for the modulation of pro-/ anti-angiogenic processes in brain cancer therapy. Specifically, peptide fragments from the putative cell membrane binding domain (residues 60-68) of the protein were used in this study to obtain peptide-functionalised spherical gold nanoparticles (AuNPs) of about 10 nm and 30 nm in optical and hydrodynamic size, respectively. Different hybrid biointerfaces were fabricated by peptide physical adsorption (Ang60-68) or chemisorption (the cysteine analogous Ang60-68Cys) at the metal nanoparticle surface, and the cellular assays were performed in the comparison with ANG-functionalised AuNPs. Cellular treatments were performed both in basal and in copper-supplemented cell culture medium, to scrutinise the synergic effect of the metal, which is another known angiogenic factor. Two brain cell lines were investigated in parallel, namely tumour glioblastoma (A172) and neuron-like differentiated neuroblastoma (d-SH-SY5Y). Results on cell viability/proliferation, cytoskeleton actin, angiogenin translocation and VEGF release pointed to the promising potentialities of the developed systems as anti-angiogenic tunable nanoplaftforms in cancer cells treatment.
ARTICLE | doi:10.20944/preprints201710.0011.v1
Subject: Biology And Life Sciences, Forestry Keywords: Pine pitch canker; Galicia; spore trap; air sampling; qPCR; seasonal dynamics
Online: 2 October 2017 (16:00:11 CEST)
The airborne inoculum of Fusarium circinatum, the fungal pathogen causing Pine Pitch Canker (PPC), is one of the main means of spread of the disease in forest stands and forest nurseries. Since this world-wide known pathogen was introduced in Europe, its biology in this newly infected area still remains scarcely known. To shed more light on this topic, we set an experiment on a naturally PPC infected forest of Monterey pine in Galicia (NW Spain) with the following two goals: (i) to describe the seasonal spore dispersal pattern during one year of regular sampling and (ii) to assess the spatial spore dispersal pattern around the infested plot. Portable rotating arm spore traps were used and complemented with meteorological measurements. The abundance of F. circinatum spores in the samples was evaluated by quantitative PCR (qPCR) with hydrolysis probe. The results showed almost permanent occurrence of the air inoculum throughout the whole year, being detected in 27 of the 30 samplings. No clear temporal trends were observed, but higher air inoculum was favoured by previous lower air temperatures and lower leaf wetness. Conversely, neither rainfall nor air humidity seemed to have any significant importance. The spatial spread of the inoculum was noted to be successful up to a distance of 1000 m in the wind direction, even with winds of just 5 m s-1. Our study shows that rotating arm spore traps combined with qPCR may be an efficient tool for F. circinatum detection.
ARTICLE | doi:10.20944/preprints202307.1387.v1
Subject: Biology And Life Sciences, Virology Keywords: seed transmission; tobamovirus; diagnostic method; natural hosts; RT-PCR
Online: 20 July 2023 (07:26:38 CEST)
Tomato brown rugose fruit virus (ToBRFV) is a causal agent of severe emergent diseases in Solanaceae hosts with agronomic relevance, such as tomato and pepper. Herein, we analyzed the seed transmission rate and efficacy of different seed disinfection treatments and performed a comparison of molecular biology techniques seeking rapid and sensitive detection in seed lots. We evaluated several total RNA extraction and RT-PCR protocols using a distinct combination of PCR primers to test for the presence of the ToBRFV Mexican strain in tobacco host. Our results showed that the percentage of seed and seedlings transmission was less than 1% and that a 3% sodium hypochlorite solution was effective as a seed disinfection treatment. Finally, the most sensitive molecular method identified for virus detection consisted of a CTAB-Trizol RNA extraction followed by nested PCR using primers reported by Dovas et al. (2004). Additionally, we tested potential natural hosts in selected Cucurbitaceae and Solanaceae species. Our results showed that the ToBRFV Mexican strain is capable of experimentally infecting eggplant (Solanum melongena), tomatillo (Physalis ixocarpa), and tobacco (Nicotiana rustica).
ARTICLE | doi:10.20944/preprints202304.0512.v1
Subject: Biology And Life Sciences, Biology And Biotechnology Keywords: grape skins; seeds; polyphenolic composition; antioxidant potential; RT-HPLC
Online: 18 April 2023 (10:22:09 CEST)
Abstract: The aim of this study was to identify and quantify polyphenolic compounds in skin extracts from four Bulgarian grape varieties and compare to those of seed extracts. The values of total phenolic contents (TPC), flavonoids, anthocyanins, procyanidins, ascorbic acid in grape skin extracts were determined. The antioxidant capacities of skin extracts were evaluated by DPPH, ABTS, FRAP and CUPRAC methods. The TPC values of skin extracts were 2-3 times lower than those of seed extracts. The significant difference between total parameters values of individual grape varieties were also found. According to the TPC and antioxidant capacity of skin extracts the different grape varieties were in the following order: Marselan ≥ Pinot Noir ˃ Cabernet Sauvignon ˃ Tamyanka. The individual compounds in the grape skin extracts were determined by RP-HPLC and compared with those of seed extracts. The determined composition of skin extracts was significant different from seed extracts composition. Qualitative and quantitative evaluation of the procyanidins and catechins in the skins were carried out. A correlation between phenolic contents, individual composition and antioxidant capacity of different extracts was found. The studied grape extracts have a potential to be applied as natural antioxidants in the pharmaceutical and food industries.
ARTICLE | doi:10.20944/preprints202207.0449.v1
Subject: Medicine And Pharmacology, Dermatology Keywords: CKD-aP; ion channels; action potential; hyperpolarization; RT-PCR
Online: 29 July 2022 (04:50:39 CEST)
Expression levels of Cav3.2, BKCa, and anoctamin 1 were previously found to be significantly elevated in patients with chronic kidney disease–associated pruritus (CKD-aP). On the other hand, the expression of TRPV1 was significantly reduced. We further compared CKD patients with and without CKD-aP in terms of the expression levels of several ion channels in the skin, including in peripheral nerve endings。Based on CKD-aP severity, subjects were divided into two groups: non-CKD-aP (no or slight pruritus; n = 16) and CKD-aP (mild, moderate, or severe pruritus; n = 16). Skin samples were obtained from the forearm or elbow during arteriovenous fistula surgery. We used quantitative real-time polymerase chain reaction to measure the skin expression levels of the following ion channels in the skin: Nav1.7, Kv7.2, TREK1, HCN2, TrkA, and Piezo2. RT-PCR analyses showed that CKD-aP patients had significantly higher levels of TREK1 and Piezo2 transcripts and significantly lower levels of HCN2 transcripts than non–CKD-aP patients. No significant differences were noted between groups in the expression of Nav1.7 or TrkA. Moreover, Kv7.2 transcripts were not detected in either group. In skin samples collected from CKD-aP patients, ion channel expression patterns were altered to enhance hyperpolarization of pruriceptive neurons.
ARTICLE | doi:10.20944/preprints202102.0525.v1
Subject: Chemistry And Materials Science, Analytical Chemistry Keywords: HIV-RT; ribonuclease H; dual inhibitors; docking; putative binding
Online: 23 February 2021 (15:51:40 CET)
Current therapeutic protocols for the treatment of HIV infection consist of the combination of diverse anti-retroviral drugs in order to reduce the selection of resistant mutants and to allow for the use of lower doses of each single agent to reduce toxicity. However, avoiding drugs interactions and patient compliance are issues not fully accomplished so far. In this respect the identification of single agents with multitarget potential might represent the ideal solution. Accordingly, a small library of biphenylhydrazo 4-arylthiazoles derivatives has been synthesised and evaluated to investigate the ability of the new derivatives to simultaneously inhibit both associated functions of HIV reverse transcriptase. All compounds were active towards the two functions, although at different concentrations. The substitution pattern on the biphenyl moiety appears relevant to determine the activity.
REVIEW | doi:10.20944/preprints202005.0316.v1
Subject: Biology And Life Sciences, Virology Keywords: RT-PCR; seroconversion; serum biomarkers; SARS-CoV2; neutralizing antibodies
Online: 20 May 2020 (04:10:24 CEST)
The progression of the recent COVID-19 pandemic surprised political authorities as well as scientists. The possibility to design powerful strategies for health care and preserving economic and social activities strongly relies on the capacity to monitor correctly the virus spreading and the immune response in the symptomatic and asymptomatic population. The available data relative to the first pandemic months indicate that the test reliability was progressively improved but also that the extremely variable methodologies used in the diagnostic studies generated data that are often not comparable. This condition prevents a simple metadata analysis for the identification of reliable diagnostics guidelines. Nevertheless, there are converging evidences that combinations of complementary approaches may enable more precise identification of virus infection. Furthermore, it appears that the similarities between SARS-CoV2 and the related types SARS-CoV1 and MERS that caused outbreaks in the last 20 years can be exploited to infer some information for which no direct evidence is still available
ARTICLE | doi:10.20944/preprints201905.0037.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: Bos taurus; skin; bovine dermcidin; RT-PCR; antimicrobial activity
Online: 6 May 2019 (08:35:59 CEST)
Description of a novel bovine antimicrobial peptide and its antimicrobial spectrum. RNA isolation and RT-PCR were done from various tissues. DCD peptide was synthesized, and antimicrobial activity was analyzed. Bovine dermcidin gene contains five exons intermittent by 4 introns. Bovine DCD-mRNA was 398 bp with ORF 336 bp. Bovine DCD was expressed in skin and blood. Analysis of the amino acid compositions revealed that cysteine was repeated 6 times indicating the presence of 3 disulfide bonds that play role in the peptide stability. Staphylococcus epidermidis, Streptococcus bovis, and Enterococcus faecalis were affected by Bovine DCD peptide. Highest antimicrobial effect was at 50 and 100 µg/ml. The effect on Escherichia coli and Candida albicans was slightly low. In all bacteria, Bovine DCD peptide activity did not affect by varying pH values, but in Staphylococcus aureus, the activity was affected greatly at pH 4.5 and 5.5. The optimum salt concentrations were 100 and 50 mM NaCl with all bacterial strains and E. coli, respectively. In case of C. albicans, the antimicrobial activity of Bovine DCD peptide decreased with increasing the pH value regardless the NaCl concentration. The pH 6.5 of the sweat buffer was the optimum for the Bovine DCD peptide activity.
ARTICLE | doi:10.20944/preprints202305.0869.v1
Subject: Biology And Life Sciences, Virology Keywords: Jingmenvirus group; Alongshan virus; qPCR; Flavivirus; Yanggou tick virus; tick-borne viruses
Online: 11 May 2023 (14:25:01 CEST)
The recently discovered Jingmenvirus group includes viruses with a segmented genome, RNA of a positive polarity, and several proteins with distant homology to the proteins of the members of the genus Flavivirus. Some Jingmenvirus group members, namely Alongshan virus (ALSV) and Jingmen tick virus, are reported to be tick-borne human pathogens, causing a wide variety of symptoms. ALSV is widely distributed in Eurasia, yet there is no reliable assay for its detection. Here, we describe a qPCR system for the detection of ALSV. Our data show that this system can detect as low as 104 copies of ALSV in the probe. It shows no amplification with common tick-borne viruses circulating in Eurasia, Yanggou tick virus—another member of the Jingmenvirus group—or some known members of the genus Flavivirus. The qPCR system was tested have no non-specific signal for Ixodes ricinus, I. persulcatus, Dermacentor reticulatus, D. marginatus, Haemaphysalis concinna, and H. japonica ticks. Overall, the qPCR system described here can be used for reliable and quantitative ALSV detection.
ARTICLE | doi:10.20944/preprints202311.0999.v1
Subject: Medicine And Pharmacology, Emergency Medicine Keywords: emergency department; ED time targets; acute ischemic stroke; rt-PA
Online: 16 November 2023 (02:32:20 CET)
Background and objectives: Although intravenous tissue plasminogen activator (rt-PA) has been shown to be effective in the treatment of acute ischemic stroke (AIS) only a small proportion of stroke patients receive this drug. Low administration rate is mainly due to delayed presentation of patients to the emergency department (ED) or lack of a stroke team/unit in most of the hospitals. So, the aim of this study is to analyze ED time targets and the rate of rt-PA intravenous administration after the initial admission of patients with AIS in an ED from a traditional healthcare center (without a neurologist or stroke team/unit). Methods: To analyze which factors, influence administration of rt-PA we split the general sample (n=202) in two groups: group No rt-PA (n=137) and group rt-PA (n=65) based on performing or no intravenous thrombolysis. Results: Analyzing ED time targets for all sample we found that the median onset-to-ED door time was 180 minutes (IQR, 120-217.5 minutes), door-to-physician time was 4 minutes (IQR, 3-7 minutes), door-to-CT time was 52 minutes (IQR, 48-55 minutes), and door-in-door-out time was 61 minutes (IQR, 59-65 minutes). ED time targets such as door-to physician time (p=0.245), door-to-CT time (p=0.219), door-to-door-out time (p=0.24), or NIHSS at admission to Neurology department (p=0.405), or NIHSS after 24 h (p=0.9) did not have a statistically significant effect on administration or no rt-PA treatment in patients included in our study. Only highest Door-to-CT time was statistically significantly correlated with death outcome. Conclusion: In our study, rt-PA administration rate was higher than expected. Statistically significant correlation between ED time targets was found between lower Onset-to-ED door time with administration of rt-PA treatment (p<0.001) and between highest Door-to-CT time and death outcome.
ARTICLE | doi:10.20944/preprints202210.0255.v1
Subject: Engineering, Electrical And Electronic Engineering Keywords: controller; centralized; distributed; CHIL; fault-tolerant; MMC; prototype; Opal-RT
Online: 18 October 2022 (07:30:28 CEST)
Centralized control algorithm limits the hardware flexibility of a Modular Multilevel Converter (MMC). Therefore, industrial MMC applications have started adopting distributed control structures. Even though distributed controller reduces a single point of failure risk compared to the centralized controller, the failure risk of the entire control systems increases due to the number of local controllers. However, the distributed controller can be programmed in such a way as to bypass the faulty local controller and take care of the power modules with the adjacent local controllers. This paper implements a modular distributed fault-tolerant controller for a scaled laboratory MMC prototype. Experimental results show that an MMC can operate without interruption, even under two local controller failures. Besides, the experimental results are verified with the Opal-RT, a real-time simulator with the same controller in a Control Hardware in Loop (CHIL) environment.
ARTICLE | doi:10.20944/preprints202208.0509.v1
Subject: Biology And Life Sciences, Virology Keywords: FTA cards; foot-and-mouth disease; RT-PCR; field; sequence
Online: 30 August 2022 (05:45:36 CEST)
Foot and Mouth Disease (FMD) is a viral disease, widespread and highly contagious affecting mainly cloven-hoofed domestic and wild animals. FMD can lead to high economic losses due to reduction in animals’ production such as drop of milk production, loss of body weight and high mortality rate in young ruminants. Sixteen nasal swabs and epithelial tissues were collected from animals showing typical clinical signs of FMD during the last FMD outbreak in Libya in 2018-2019. Blood samples, swabs and epithelial tissues impressions on Flinders Technology Associates (FTA) cards were shipped to the FMD Reference laboratory in Brescia, Italy, and tested for the detection of FMD viruses. Nucleic acids were extracted from the FTA cards and molecular testing of the examined FTA cards confirmed that the FMD virus circulating in Libya was serotype O. Sequencing analysis of the FMD virus VP1 gene confirmed that FMD virus strain was serotype O/East Africa 3 (O/EA-3) topotype. The phylogenetic of the VP-1 gene of the FMD virus showed very high nucleotide identity (99.8%) between the virus circulating in Libya and the Algerian FMD virus strains isolated in Algeria on 2018 and 2019.
ARTICLE | doi:10.20944/preprints202103.0689.v1
Subject: Engineering, Civil Engineering Keywords: COVID-19; mobility patterns; Rt; changepoint; modeling; Portugal; Longitudinal Study
Online: 29 March 2021 (12:31:54 CEST)
This study analyzes the relationship between the spread of the SARS-CoV-2 virus (COVID-19) and the mobility patterns of the Portuguese population. By reducing mobility, the idea is that contacts are reduced, countering the spread of the virus in the community. As an indicator of the spread of the virus, the reproduction number (Rt) was used. Data from Google's Community Mobility Reports was used to evaluate changes in mobility patterns. This report uses location data from Android mobile phone users. The locations are divided into retail and recreation, grocery and pharmacy, parks, transit stations, workplaces and residential. In this year of the COVID-19 crisis in Portugal, population mobility patterns have changed over the various phases of the pandemic. At first, all mobility was affected uniformly, with the population avoiding much of the activity outside the home. In a second phase, there was some adaptation, and the areas considered to be of lower risk had less impact, emphasizing the changes in the relationship between daily life and the workplace.
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: COVID-19; SARS-CoV-2; RT-PCR; control strategy; Morocco
Online: 5 November 2020 (10:40:41 CET)
Historically, 2020 will be remembered as the year of COVID-19 pandemic, that limited people move and their social habits. This virus, becomes the first daily tracked public health burden that leads to more than 1,2 million deaths in the World by the end of October 2020, and until now no countries' best control strategies are cited as the example of long term success.Since March 2nd, 2020, date of the first SARS-COV-2 detected case in Morocco; many decisions were adopted as COVID-19 control strategies. If the first period of COVID-19 noticed a few numbers of cases and deaths, the second half from July is marked with an exponential increase of the number of cases and a spread in almost all provinces with more intensive care needs and more deaths. The policy analysis approach was followed as a method to define the pitfalls themes and to compare with the updated available international information any significant similarities or divergences. Thus, this report has the aim to present an overview of how the COVID-19 pandemic was dealt in Morocco during these 200 days, by highlighting some discrepancies with corrective advice provided in the first version of the manuscript to be confronted by a Moroccan health policymaker’s community, then to notice the decisions adopted during another one month by the MoH to get better future control results against COVID-19.Unfortunately, despite the MoH decision to increase the diagnostic facilities for molecular tests, the overall number of laboratory tests do not go further than 21000 tests per day. The actual insidious transmission is a dangerous blind threat that dissipates the collaborating efforts, due to this limited number of confirmation and follows up tests.Accurate, standardised and up to ten times per day tests of molecular biology represented by RT-qPCR, to interpret results following the time progress of cycle quantification values is the primary action to be done to enhance the overall control strategy.
Subject: Medicine And Pharmacology, Epidemiology And Infectious Diseases Keywords: COVID-19; RT-PCR test; pre-test probability; positive rate
Online: 5 May 2020 (06:04:41 CEST)
Several articles have reported that the low number of COVID-19 cases in Japan is attributed to the small number of diagnostic RT-PCR tests performed. The criticism is based on the low number of the tests performed, and they suspected there would be more potential cases in Japan. The use of pre-test probability among tested subjects is proposed in order to evaluate of the sufficiency of test availability instead of the number of the tests. The pre-test probability means the average probability, i.e., ‘suspicion level’, of having coronavirus among the tested subject in a country. The higher pre-test probability is regarded as less sufficient opportunity of the tests, and the test availability could be evaluated by the pre-test probability. Thus, potential problems of underestimation of COVID-19 cases by insufficient amount of the test would be clear by using pre-test probability. The comparison of the pre-test probability could be replaced with that of the positive rate of the test because of the linear relationship between them under the assumption of common sensitivity and specificity. Japan shows the third lowest rate (8.6%), and is considered that the considerably sufficient number of the tests have been performed. In conclusion, the positive rate of the test as a surrogate index of the pre-test probability is useful to evaluate the sufficiency of test amount instead of the number of the tests performed. In present, the potential problem of underestimation by insufficient test availability would be less serious in Japan.
ARTICLE | doi:10.20944/preprints201911.0042.v1
Subject: Biology And Life Sciences, Endocrinology And Metabolism Keywords: olivo-ponto-cerebellar atrophy (OPCA); amyotrophic lateral sclerosis (ALS); tauopathy; leukodystrophy; mass spectrometry; RT-qPCR; Ceramide Synthase (CERS2/CERS1); Serine Palmitoyltransferase 2 (Sptlc2); neutral Sphingomyelinase (Smpd3); neutral Ceramidase (Asah2); Fatty Acid Elongase (Elovl1/4/5); SCA34; SCA38; acid Sphingomyelinase (ASMase, Smpd1)
Online: 5 November 2019 (03:04:02 CET)
Ataxin-2 (ATXN2) acts during stress-responses, modulating mRNA translation and nutrient metabolism. Atxn2 knockout mice exhibit progressive obesity, dyslipidemia and insulin resistance. Conversely, the progressive ATXN2 gain-of-function due to polyGlutamine (polyQ) expansions leads to a dominantly inherited neurodegenerative process named spinocerebellar ataxia type 2 (SCA2), with early adipose tissue loss and late muscle atrophy. We tried to understand lipid dysregulation in a SCA2 patient brain and in an authentic mouse model. Thin layer chromatography of a patient cerebellum was compared to the lipid metabolome of Atxn2-CAG100-KnockIn (KIN) mouse spinocerebellar tissue. The human pathology caused deficits of sulfatide, galactosylceramide, cholesterol, C22/24-sphingomyelin and gangliosides GM1a/GD1b, despite quite normal levels of C18-sphingomyelin. Cerebellum and spinal cord from the KIN mouse showed a consistent decrease of various ceramides, with a significant elevation of sphingosine in the more severely affected spinal cord. Deficiency of C24/26-sphingomyelins contrasted with excess C18/20-sphingomyelin. Spinocerebellar expression profiling revealed consistent reductions of CERS protein isoforms, of Sptlc2 and Smpd3, but upregulation of Cers2 mRNA, as prominent anomalies in the ceramide-sphingosine metabolism. Reduction of Asah2 mRNA correlated to deficient S1P levels. In addition, downregulations for the elongase Elovl1, Elovl4, Elovl5 mRNAs and ELOVL4 protein explain the deficit of very-long-chain sphingomyelin. Reduced ASMase protein levels correlated to the accumulation of long-chain sphingomyelin. Overall, a deficit of myelin lipids was prominent in SCA2 nervous tissue at prefinal stage, not compensated by transcriptional adaptation of several metabolic enzymes. Myelination is controlled by mTORC1 signals, so our human and murine observations are in agreement with the known role of ATXN2 yeast, nematode and mouse orthologs as mTORC1 inhibitors and autophagy promoters.
ARTICLE | doi:10.20944/preprints202306.1385.v2
Subject: Engineering, Other Keywords: BCS superconductivity; RT superconductivity; B-doped Q-carbon; B-doped diamond
Online: 30 October 2023 (09:01:40 CET)
We present atomic structures and nonequilbrium synthesis of new class of materials, where the basic structural unit is a diamond tetrahedron. When units of one, two, and three tetrahedra are randomly packed, we create distinct phases of amorphous Q-carbon. Four tetrahedra in two adjacent layers lead to crystalline diamond lattice, which has four missing tetrahedra alternately. When these four missing tetrahedra are filled, we create subunit cell of crystalline Q-diamond. Theoretical calculations show that superconducting transition temperature (Tc) in 50 atomic % B-doped Q-diamond can reach near room temperature at ambient pressures. This is consistent with our earlier results using low-loss EELS measurements in 50 atomic % B-doped Q-carbon, which had mostly amorphous QB3 phase mixed with some crystalline Q-diamond phase. These EELS results showed that the Tc for these samples was in between 90K and 300K. Theoretical calculations of density of states, Eliashberg function, electron-phonon interaction parameter, and root-mean-square and logarithmic average of frequency in crystalline Q-diamond show Tc in the range of 268K to 300K, which is in a complete agreement with our EELS results in QB3.
BRIEF REPORT | doi:10.20944/preprints202105.0342.v1
Subject: Medicine And Pharmacology, Immunology And Allergy Keywords: COVID-19; SARS-CoV-2; nasopharyngeal swab; RNA extraction; RT-PCR
Online: 14 May 2021 (14:46:42 CEST)
Background: the devastating outbreak of COVID-19 poses serious challenges for the diagnostics laboratories, which are often facing global shortage of reagents and equipment. With the aim of increasing the diagnostic throughput for SARS-CoV-2 molecular test, the purpose of this study was to validate an additional RNA extraction method respect to those already recommended by WHO and the US Centers for Disease Control and Prevention (CDC). Methods: a new protocol for RNA extraction from nasopharyngeal swab was set up, adapting the Qiagen RNeasy 96 plate and validated on a set of 100 clinical samples analyzed in parallel by Roche-Magnapure method (already recommended by CDC guidelines). Results: the internal control and target genes analysis showed a good agreement between the two extraction methods indicating that the two methods can be considered equivalent and that the RNeasy-adapted method can be applied for the SARS-CoV-2 diagnostics. The addition of this new extraction method resulted in a throughput increase for SARS-CoV-2 molecular test of about 2000 samples/month during the initial months of the pandemic emergency in which the lack of reagents for the extraction led to an insufficient sample processing throughput of the analysis of the swabs.
ARTICLE | doi:10.20944/preprints201901.0212.v1
Subject: Biology And Life Sciences, Agricultural Science And Agronomy Keywords: DHAV-1; DHAV-3; Phylogenetic analysis; One-tube RT-PCR; Simultaneously
Online: 22 January 2019 (11:08:19 CET)
The co-circulation of duck hepatitis A virus subtypes 1 (DHAV-1) and 3 (DHAV-3) in ducklings has resulted in significant economic losses. Because ducklings infected with DHAV-1 or DHAV-3 show similar clinical signs and gross lesions, it is important to discriminate these subtypes as early as possible for better clinical management. On the basis of multiple alignments of the 5′-noncoding region sequences of strains DHAV-1 and DHAV-3, universal and type-specific primers were designed and synthesized. Using the primers in a one-tube reverse transcription-PCR (RT-PCR) assay, reference strains of DHAV-1 and DHAV-3 (isolated over a span of 60 years and covering many different countries) were successfully amplified, indicating that the primer sequences were completely conserved. The amplicon sequences results and the sizes of amplicons from reference DHAV-1 and DHAV-3 isolates correlated completely with their genotypes. Moreover, with this one-tube RT-PCR system, the amplicon sizes of liver samples of reference DHAV-1- or DHAV-3-infected birds matched perfectly with their respective genotypes, as determined by virus isolation and neutralization tests. No other RNA viruses of duck origin were detected with the synthesized primers. The sensitivity of viral RNA detection was 10 pg. With this system, 20% genotype 1, 45% genotype 3, and 9% co-infection of the two genotypes were detected in 55 clinical samples. This novel approach could be used for the rapid genotyping DHAV-1 and/or DHAV-3 infection in routine clinical surveillance or epidemiologic screening.
ARTICLE | doi:10.20944/preprints201705.0173.v1
Subject: Biology And Life Sciences, Animal Science, Veterinary Science And Zoology Keywords: Echinococcus granulosus; Calmodulin; Ca2+-binding protein; Immunohistochemical localization; Quantitative RT-PCR
Online: 24 May 2017 (08:07:35 CEST)
Echinococcus granulosus is a harmful cestode parasite which could cause Cystic Echinococcosis in humans, various livestock species and wild animals. Calmodulin (CaM), a Ca2+ sensor protein, is widely expressed in eukaryotes and mediates a variety of cellular signaling activities. In our study, the CaM in Echinococcus granulosus (rEgCaM) was successfully cloned and the molecular and biochemical characterizations of rEgCaM were identified. The results showed that rEgCaM was a highly conserved calcium-binding protein, consisting of 149 amino acids. Immunoblot analysis revealed that rEgCaM could be identified using E. granulosus infected sheep serum. The usage of rEgCaM as antigen was evaluated by indirect ELISA which exhibited a high sensitivity of 90.3% but low specificity (47.1%). rEgCaM was mainly located in the tegument tissues and parenchymal region of protoscoleces, the tegument and inner body of adult worm and predominantly expressed in the germinal layer. The mRNA expression of rEgCaM in PSCs were gradually decreased with the increase death of PSCs. In electrophoretic mobility tests and ANS assays, rEgCaM showed a typical calcium-binding protein characteristics. This is the first report on CaM from E. granulosus and rEgCaM is considered to be involved in some important biological function of E. granulosus as a calcium-binding protein.
SHORT NOTE | doi:10.20944/preprints202306.1658.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: qPCR assay; probe-based detection; SYBR green; human KRAS gene; human gastric CloTest samples
Online: 23 June 2023 (10:50:59 CEST)
Quantitative polymerase chain reaction (qPCR) is the gold standard approach for detecting a variety of pathogens either via probe-based or SYBR green detection chemistry. Generally, probe-based detection is more specific than SYBR green chemistry. This report describes work done in using a FAM probe targeting a specific segment of the human KRAS gene for detecting presence and relative abundance of the gene in human gastric CloTest samples. Results indicate ineffective detection of the human KRAS gene in samples that are shown to harbor the gene through a similar qPCR assay using SYBR green detection chemistry. Such observations point to inability of the designed FAM probe to bind to target segment of the human KRAS gene that suggests a region of rapid genomic evolution that require close surveillance in the public health community. Given its rapid genomic evolution, this probe region may be an important region in the human KRAS gene playing critical functional roles in either molecular recognition or other structural functions. Overall, human KRAS gene may be undergoing rapid genomic evolution, and qPCR probes targeting specific segment of the gene may be rendered ineffective in short time clinically, thereby, making SYBR green qPCR assay a more reliable choice.
ARTICLE | doi:10.20944/preprints202107.0548.v1
Subject: Engineering, Automotive Engineering Keywords: ANN; COVID-19; CT; mRNA; MRI; RT-PCR; SARS-CoV-2; XCR
Online: 23 July 2021 (15:02:40 CEST)
Accurate early diagnosis of COVID-19 viral pneumonia, primarily in asymptomatic people is essential to reduce the spread of the disease, the burden on healthcare capacity, and the overall death rate. It is essential to design affordable and accessible solutions to distinguish pneumonia caused by COVID-19 from other types of pneumonia. In this work, we propose a reliable approach based on deep transfer learning that requires few computations and converges faster. Experimental results demonstrate that our proposed framework for transfer learning is a potential and effective approach to detect and diagnose types of pneumonia from chest X-ray images with a test accuracy of 94.0%.
ARTICLE | doi:10.20944/preprints202003.0413.v1
Subject: Medicine And Pharmacology, Pharmacology And Toxicology Keywords: coronavirus; drug; COVID-19; SARS; MERS; ontology; ChEBI; NDF-RT; DrON; bioinformatics
Online: 29 March 2020 (01:58:40 CET)
Coronavirus-infected diseases have posed great threats to human health. In past years, highly infectious coronavirus-induced diseases, including COVID-19, SARS, and MERS, have resulted in world-wide severe infections. Our literature annotations identified 110 chemical drugs and 26 antibodies effective against at least one human coronavirus infection in vitro or in vivo. Many of these drugs inhibit viral entry to cells and viral replication inside cells or modulate host immune responses. Many antimicrobial drugs, including antimalarial (e.g., chloroquine and mefloquine) and antifungal (e.g., terconazole and rapamycin) drugs as well as antibiotics (e.g., teicoplanin and azithromycin) were associated with anti-coronavirus activity. A few drugs, including remdesivir, chloroquine, favipiravir, and tocilizumab, have already been reported to be effective against SARS-CoV-2 infection in vitro or in vivo. After mapping our identified drugs to three ontologies ChEBI, NDF-RT, and DrON, many features such as roles and mechanisms of action (MoAs) of these drugs were identified and categorized. For example, out of 57 drugs with MoA annotations in NDF-RT, 47 have MoAs of different types of inhibitors and antagonists. A total of 29 anticoronaviral drugs are anticancer drugs with the antineoplastic role. Two clustering analyses, one based on ChEBI-based semantic similarity, the other based on drug chemical similarity, were performed to cluster 110 drugs to new categories. Moreover, differences in physicochemical properties among the drugs were found between those inhibiting viral entry and viral replication. A total of 163 host genes were identified as the known targets of 68 anti-coronavirus drugs, resulting in a network of 428 interactions among these drugs and targets. Chlorpromazine, dasatinib, and anisomycin are the hubs of the drug-target network with the highest number of connected target proteins. Many enriched pathways such as calcium signaling and neuroactive ligand-receptor interaction pathways were identified. These findings may be used to facilitate drug repurposing against COVID-19.
ARTICLE | doi:10.20944/preprints201909.0142.v1
Subject: Medicine And Pharmacology, Urology And Nephrology Keywords: uremic pruritus; ion channels; cell signaling; Cav3.2 calcium channel; RT-PCR; skin
Online: 14 September 2019 (19:00:34 CEST)
Background: We investigated ion channels at the skin, including peripheral nerve endings, which serve as output machines and molecular integrators of many pruritic inputs mainly received by multiple G protein-coupled receptors (GPCRs). Methods: Based on the level of chronic kidney disease–associated pruritus (CKD-aP), subjects were divided into two groups: non-CKD-aP (no or slight pruritus; n=12) and CKD-aP (mild, moderate, or severe pruritus; n=11). Skin samples were obtained from the forearm or elbow during operations on arteriovenous fistulas. We measured ion channels expressed at the skin, including peripheral nerve endings by RT-PCR: Nav1.8, Kv1.4, Cav2.2, Cav3.2, BKCa, Anoctamin1, TRPV1, TRPA1, and ASIC. Results: Expression of Cav3.2, BKCa, and anoctamin1 was significantly elevated in patients with CKD-aP. On the other hand, expression of TRPV1 was significantly reduced in these patients. We observed no significant difference in the levels of Cav2.2 or ASIC between subjects with and without CKD-aP. TRPA1, Nav1.8，and Kv1.4 were not expressed. Conclusions: It was concluded that this greater difference in expression of ion channels at the skin tissue including specific for cutaneous peripheral nerve endings in CKD patients with CKD-aP may increase generator potential related to itching.
REVIEW | doi:10.20944/preprints202104.0481.v1
Subject: Environmental And Earth Sciences, Waste Management And Disposal Keywords: COVID-19; SARS-CoV-2; Wastewater; Surveillance; False-positive; False-negative; RT-PCR
Online: 19 April 2021 (13:08:13 CEST)
Wastewater surveillance for pathogens using the reverse transcription-polymerase chain reaction (RT-PCR) is an effective, resource-efficient tool for gathering additional community-level public health information, including the incidence and/or prevalence and trends of coronavirus disease-19 (COVID-19). Surveillance of SARS-CoV-2 in wastewater may provide an early-warning signal of COVID-19 infections in a community. The capacity of the world’s environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is rapidly increasing. However, there are no standardized protocols nor harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can lead to false-positive and -negative errors in the surveillance of SARS-CoV-2, culminating in recommendations and strategies that can be implemented to identify and mitigate these errors. Recommendations include, stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, amplification inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly during a low incidence of SARS-CoV-2 in wastewater. Corrective and confirmatory actions must be in place for inconclusive and/or potentially significant results (e.g., initial onset or reemergence of COVID-19 in a community). It will also be prudent to perform inter-laboratory comparisons to ensure results are reliable and interpretable for ongoing and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance was demonstrated during this global crisis. In the future, wastewater will play an important role in the surveillance of a range of other communicable diseases.
ARTICLE | doi:10.20944/preprints202311.0621.v1
Subject: Biology And Life Sciences, Animal Science, Veterinary Science And Zoology Keywords: enteric virus; picobirnavirus; brazilian animals; page, rt-pcr; rdrp gene; phylogenetic analysis; genogroup I
Online: 9 November 2023 (11:24:00 CET)
This study aimed to detect picobirnavirus (PBV) in the fecal samples of wild and domestic animals from 2014 to 2016 in the Amazon biome. Fecal samples from different animals, including birds (n=41) and mammals (n=217) were used. The PAGE test showed negativity for PBV. However, 32 samples (12.4%, 32/258) showed positive results in RT-PCR analyses. Among the positive sam-ples, pigs and cats, both with 28.12% (9/32), registered the highest frequencies. In phylogenetic analysis, eight sequences from positive samples grouped in the Genogroup 1 of PBV (PBV GI). PBV occurrence was significantly related to cats and pigs, but not other mammals or birds, in-dependently of their geographical origin. Nucleotide analysis demonstrated similarity among the feline group, but the absence of a defined structure between the clades. PBVs are highly wide-spread viruses that can affect the most diverse types of hosts in the Amazon biome, including humans.
ARTICLE | doi:10.20944/preprints202103.0292.v1
Subject: Medicine And Pharmacology, Immunology And Allergy Keywords: cancer; COVID-19; symptoms; healthcare workers; anosmia; dysgeusia; ageusia; France; serological test; RT-PCR
Online: 10 March 2021 (16:17:48 CET)
Background: Cancer patients may fail to distinguish COVID-19 symptoms such as anosmia, dysgeusia/ageusia, anorexia, headache, and fatigue, which are frequent after cancer treatments. We aimed to identify symptoms associated with COVID-19 and to assess the strength of their association in cancer and cancer-free populations. Methods: The prospective multicenter cohort study PAPESCO-19 included 878 cancer patients and 940 healthcare workers (HCWs) systematically tested for SARS-CoV-2-specific antibodies. Participants reported the results of routine screening RT-PCR and thirteen COVID-19 symptoms. Backward logistic regression identified the symptom combinations significantly associated with COVID-19. Results: COVID+ proportions were similar in patients (8%) and HCWs (9.5%, p=0.26), whereas symptomatic proportions were lower in patients (32%) than HCWs (52%, p<0.001). Anosmia, anorexia, fever, headache, and rhinorrhea together accurately discriminated (c-statistic=0.7027) COVID-19 cases in patients. Anosmia, dysgeusia/ageusia, muscle pain, intense fatigue, headache, and chest pain better discriminated (c-statistic=0.8830) COVID-19 cases in HCWs. Anosmia had the strongest association in patients (OR=7.48, 95% CI: 2.96–18.89) and HCWs (OR=5.71, 95% CI: 2.21–14.75). Conclusions: COVID-19 symptoms and their diagnostic performance differ in cancer patients and HCWs. Anosmia is associated with COVID-19 for patients, while dysgeusia/ageusia are not. Cancer patients deserve tailored preventive measures due to their particular COVID-19 symptom pattern.
REVIEW | doi:10.20944/preprints202006.0284.v1
Subject: Biology And Life Sciences, Virology Keywords: SARS-CoV-2; RT-PCR; antibody; zoonotic; animal transmission; genomics; asymptomatic fraction; herd immunity
Online: 23 June 2020 (13:30:11 CEST)
Since December 2019, a rapid increase in the number of SARS-CoV-2 (COVID-19) cases was reported worldwide, despite strict infection control and lock down measures. Current paper investigated the actual facts behind this rapid increase in the number of cases. Study of genomic sequence reveals that domestic and wild animals were likely ancestors and zoonotic source for SARS-CoVs, MERS-CoVs, and SARS-CoV-2. Strong evidence suggest that these viruses already existed and replicated in animals and humans during past several decades, exhibiting diverse mutations, evolutions and self-limiting diseases, except during outbreaks. Serious zoonotic reservoir investigations are required to investigate animal transmission of SARS-CoVs and SARS-CoV-2 to limit current pandemic. This might be the reason of increasing number of cases via animals. SARS-CoV-2 has been retrospectively isolated in different studies in August 2019, several months before Wuhan announced. Hence, there is a possibility that viruses existed, went undetected, infecting subclinically, in past several years, and SARS-CoV-2 antigens and neutralizing antibodies may have been present in humans since long time. This might be another reason of increasing number of cases by screening as mass screening and antigen or antibody testing was not carried out in the past years. Randomized controlled trials are required to investigate human to human transmission by touch, as the current evidence is limited with conflicting results. As all SARS-CoVs are basically respiratory viruses, droplet precautions and infection control measures are essential, especially for hospital staff. Increased number of SARS-CoV-2 asymptomatic, or subclinical cases are detected worldwide. This silent phase of transmission can be beneficial for humans. Lack of symptoms eventually lessen virus transmission and reduce the pathogen's long-term survival and provide humoral herd immunity up to several years. Hence, seropositivity with diverse antibodies develops against mutating SARS-CoVs which will confer strong immunity during epidemics. Strategies such as identification, contact tracing and quarantine are costly and practically difficult. Hence, asymptomatic persons can continue their work with droplet precautions and standard infection control procedures, while symptomatic or sick persons can isolate themselves in their homes without the need for strict quarantine until clinical recovery, with reduced hospital visits and minimizing chances of hospital acquired infections. RT-PCR has low sensitivity and specificity, carries a high risk of handling live virus antigens, and requires difficult protocols. As viral load also sharply declines after few days of onset of infection, this technique might overlook infection. Furthermore, SARS-CoV-2 infection may be present in blood when oropharyngeal swabs are negative by RT-PCR. Additionally, RT-PCR usually gives false negative and false positive results and must be interpreted cautiously. This might be again a reason of increasing number of cases by false positive RT-PCR reporting. Moreover, antibodies against SARS-CoVs develop robustly in serum even by reduced amount of antigens. In contrast to RT-PCR, ELISA for diagnosing antibodies against SARS-CoV-2 demonstrates 100% specificity and 100% sensitivity, even in clinically asymptomatic individuals. These antibodies can be used for serologic surveys, monitoring and screening. However, screening tests for SARS-COV-2 should be avoided in unhygienic public places by nasopharyngeal swabs, which carry a high risk of further transmission, co-infection or superinfection. Such highly infectious virus must be isolated and tested in highly sterilized laboratory. Further strict international laws and policies are required to stop the possible spread of experimental viruses, biological warfare and bioterrorism.
ARTICLE | doi:10.20944/preprints202308.0372.v3
Subject: Social Sciences, Tourism, Leisure, Sport And Hospitality Keywords: complex indicator; isolated action; weighting system; simultaneous action; machine learning(ML); decision tree(C-RT)
Online: 16 October 2023 (08:35:18 CEST)
Analytics have become increasingly popular among sports in recent years, providing valuable information regarding physical and mental health. Many athletes have found that analytics during training can help to improve their performance, reduce the risk of injury, and enhance their overall well-being. This paper aims to improve the results of handball players by applying a method for measuring the influence of different trials on aggregate performance calculated per each athlete in order to qualify for the Olympic Games. By separating the isolated action of each trial, the result is an additional influence caused by the interaction of factors or the simultaneous action. That might explain why the neuromuscular feedback loop is utterly necessary to perform handball motor actions. Further on, the ML analysis can help identify areas for improvement, optimize training programs, and enhance overall team performance.
ARTICLE | doi:10.20944/preprints202307.1532.v1
Subject: Biology And Life Sciences, Virology Keywords: SARS-CoV-2; COVID-19; pandemic; variants; RT-PCR; sequencing; surveillance; vaccination; vaccine breakthrough; comorbidity.
Online: 21 July 2023 (13:55:29 CEST)
New Jersey was among the first states impacted by the COVID-19 pandemic, with one of the highest overall death rates in the nation. Nevertheless, relatively few reports have been published focusing specifically on New Jersey. Here we report on molecular, clinical, and epidemiologic observations from the largest healthcare network in the state, in a cohort of vaccinated and unvaccinated individuals with laboratory-confirmed SARS-CoV-2 infection. We conducted molecular surveillance of SARS-CoV-2-positive nasopharyngeal swabs collected in nine hospitals from December 2020 through June 2022, using both whole genome sequencing (WGS) and a real-time RT-PCR screening assay targeting spike protein mutations found in variants of concern (VOC) within our region. De-identified clinical data were obtained retrospectively, including demographics, COVID-19 vaccination status, ICU admission, ventilator support, mortality, and medical history. Statistical analyses were performed to identify associations between SARS-CoV-2 variants, vaccination status, clinical outcomes, and medical risk factors. A total of 5,007 SARS-CoV-2-positive nasopharyngeal swabs were successfully screened and/or sequenced. Variant screening identified three predominant VOC, including Alpha (n =714), Delta (n =1,877), and Omicron (n =1,802). Omicron isolates were further sub-typed as BA.1 (n =899), BA.2 (n =853), and BA.4/BA.5 (n =50); the remaining 614 isolates were classified as “Other”. Approximately 31.5% (1,577/5,007) of the samples were associated with vaccine breakthrough infections, which increased in frequency following the emergence of Delta and Omicron. Severe clinical outcomes included ICU admission (336/5007 = 6.7%), ventilator support (236/5007 = 4.7%), and mortality (430/5007 = 8.6%), with increasing age being the most significant contributor to each (p <0.001). Unvaccinated individuals accounted for 79.7% (268/336) of ICU admissions, 78.3% (185/236) of ventilator cases, and 74.4% (320/430) of deaths. Highly significant (p <0.001) increases in mortality were observed in individuals with cardiovascular disease, hypertension, cancer, diabetes, and hyperlipidemia, but not with obesity, thyroid disease, or respiratory disease. Significant differences (p <0.001) in clinical outcomes were also noted between SARS-CoV-2 variants, including Delta, Omicron BA.1, and Omicron BA.2. Vaccination was associated with significantly improved clinical outcomes in our study, despite an increase in breakthrough infections associated with waning immunity, greater antigenic variability, or both. Underlying comorbidities contributed significantly to mortality in both vaccinated and unvaccinated individuals, with increasing risk based on the total number of comorbidities. Real-time RT-PCR-based screening facilitated timely identification of predominant variants using a minimal number of spike protein mutations, with faster turnaround time and reduced cost compared to WGS. Continued evolution of SARS-CoV-2 variants will likely require ongoing surveillance for new VOCs, with real-time assessment of clinical impact.
ARTICLE | doi:10.20944/preprints202203.0040.v1
Subject: Biology And Life Sciences, Virology Keywords: SARS-CoV-2; rapid antigen test; RT-PCR test; COVID-19; image processing; Raspberry Pi
Online: 2 March 2022 (08:06:49 CET)
At-home rapid antigen test (RAT) kits for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are valuable public health tools during the present coronavirus disease (COVID-19) pandemic. They provide fast identification of coronavirus infection, which can help to reduce the transmission rates and burden on the healthcare system. However, they have lower sensitivity when compared with the reverse transcription polymerase chain reaction (RT-PCR) tests. One of the reasons for the lower sensitivity is due to the RAT color indicators being indistinct or invisible to the naked eye after the measurements. For this reason, we propose a systematic approach, through which we investigated anonymously provided at-home RAT kit results by using our in-house open source image processing scripts developed for affordable Raspberry Pi computer and Raspberry Pi HQ camera systems (available at https://github.com/kmiikki/ratcv). Therefore, we aimed at minimizing the human-related analysis errors for such kits. We believe that our framework can contribute to reduced the delayed quarantines of infected individuals and spreading of the current infectious disease.
ARTICLE | doi:10.20944/preprints202105.0381.v1
Subject: Environmental And Earth Sciences, Water Science And Technology Keywords: SARS-CoV-2; wastewater monitoring; environmental surveillance; RT-LAMP; building-level; near-source; passive sampling
Online: 17 May 2021 (10:07:04 CEST)
Community-level wastewater monitoring for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA has demonstrated useful correlation with both coronavirus disease 2019 (COVID-19) case numbers and clinical testing positivity. Wastewater monitoring on college campuses has demonstrated promising predictive capacity for the presence and absence of COVID-19 cases. However, to date, such monitoring has largely relied upon composite or grab samples and reverse transcription quantitative PCR (RT-qPCR) techniques, which limits the accessibility and scalability of wastewater monitoring. In this study, we piloted a workflow that uses tampons as passive swabs for collection and reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect SARS-CoV-2 RNA in wastewater. Results for the developed workflow were available same day, with a time to result following tampon swab collection of approximately three hours. The RT-LAMP 95% limit of detection (76 gene copies reaction-1) was greater than RT-droplet digital PCR (ddPCR; 3.3 gene copies reaction-1). Nonetheless, during a building-level wastewater monitoring campaign conducted in the midst of weekly clinical testing of all students, the workflow demonstrated a same-day positive predictive value (PPV) of 33% and negative predictive value (NPV) of 80% for incident COVID-19 cases. The NPV is comparable to that reported by wastewater monitoring using RT-qPCR. These observations suggest that even with lower analytical sensitivity the tampon swab and RT-LAMP workflow offers a cost-effective and rapid approach that could be leveraged for scalable same-day building-level wastewater monitoring for COVID-19.
ARTICLE | doi:10.20944/preprints202104.0688.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: droplet digital PCR; real time RT-PCR; SARS-CoV-2; false negative; viral load; diagnosis
Online: 26 April 2021 (20:09:26 CEST)
Background: The reference test for SARS-CoV-2 detection is the reverse transcriptase real time PCR (real time RT-PCR). However, evidences reported that real time RT-PCR has a lower sensitivity compared with the droplet digital PCR (ddPCR) leading to possible false negative in low viral load cases. Methods: We used ddPCR for viral genes N1 and N2 on 20 negative (no detection) samples from symptomatic hospitalized COVID-patients presenting fluctuating real time RT-PCR results and 10 suspected samples (Ct value>35) from asymptomatic not hospitalized subjects. Results: ddPCR performed on RNA revealed 65% of positivity for at least one viral target in the hospitalized patients group of samples (35% for N1 and N2, 10% only for N1 and 20% only for N2) and 50% in the suspected cases (30% for N1 and N2, while 20% only for N2). On hospitalized patients’ samples, we applied also a direct ddPCR approach on the swab material, achieving an overall positivity of 83%. Conclusion: ddPCR, in particular the direct quantitation on swabs, shows a sensitivity advantage for the SARS-CoV-2 identification and may be useful to reduce the false negative diagnosis, especially for low viral load suspected samples.
REVIEW | doi:10.20944/preprints202004.0201.v1
Subject: Biology And Life Sciences, Virology Keywords: diagnosis; detection kits; RT-PCR; immunoassay; false negative; false positive; sensitivity; point-of-care (POC)
Online: 12 April 2020 (16:50:05 CEST)
The COVID-19 pandemic has created huge damage to society and brought panics around the world. Such panics can be ascribed to the seemingly deceptive features of the COVID-19: compared to other deadly viral outspreads, it has medium transmission and mortality rates. As a result, the severity of this virus was deeply underestimated by the society at the beginning of the outbreak. Based on this, in this review, we define the viruses with features similar to those of COVID-19 as the Panic Zone viruses. To contain those viruses, accurate and fast diagnosis followed by effective isolation and treatment of patients are pivotal at the early stage of virus breakouts. This is especially true when there is no cure or vaccine available for a transmissible disease, which is the case for current COVID-19 pandemic. As of April 2020, more than one hundred kits for the COVID-19 diagnosis on the market are surveyed in this review. It is of critical importance to rationally use these kits for the efficient management and control of the Panic Zone viruses. Therefore, we discuss guidelines to select diagnostic kits at different outbreak stages of the Panic Zone viruses, COVID-19 in particular. While it is of utmost importance to use detection kits with low false negativity at the early stage of an outbreak, the low false positivity gains its importance at later stages of the outbreak. Finally, since a massive attack from a viral pandemic requires a massive defense from the whole society, we urge both government and private sectors to research and develop affordable point-of-care (POC) detection kits, which can be used massively by the general public (and therefore called as massive POC) to contain Panic Zone viruses in future.
Subject: Medicine And Pharmacology, Immunology And Allergy Keywords: SARS-CoV-2; COVID-19; RT-PCR; nucleic acids; direct testing; loop-mediated isothermal amplification; LAMP
Online: 5 April 2021 (12:21:19 CEST)
The availability of simple SARS-CoV-2 detection methods is crucial to contain the COVID-19 pandemic. This study examined whether a commercial LAMP assay can reliably detect SARS-CoV-2 genomes directly in respiratory samples without having to extract nucleic acids (NA) beforehand. Nasopharyngeal swabs (NPS, n = 220) were tested by real-time reverse transcription (RT)-PCR and with the LAMP assay. For RT-PCR, NA were investigated. For LAMP, NA from 26 NPS in viral transport medium (VTM) were tested. The other 194 NPS were analyzed directly without prior NA extraction [140 samples in VTM; 54 dry swab samples stirred in phosphate buffered saline]. Ten NPS were tested directly by LAMP using a sous-vide cooking unit. The isothermal assay demonstrated excellent specificity (100%) but moderate sensitivity (68.8%), with a positive predictive value of 1 and a negative predictive value of 0.65 for direct testing of NPS in VTM. The use of dry swabs, even without NA extraction, improved the analytical sensitivity; up to 6% of samples showed signs of inhibition. The LAMP could be performed successfully with a sous-vide cooking unit. This technique is very fast, requires little laboratory resources and can replace rapid antigen tests or verify reactive rapid tests on site.
BRIEF REPORT | doi:10.20944/preprints202105.0526.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: SARS-CoV-2 virus; complete genome sequencing; COVID-19 RT-PCR testing; Spike protein; vi-ral variants
Online: 21 May 2021 (15:12:17 CEST)
A growing number of emerging SARS-CoV-2 variants is being identified worldwide, potentially impacting the effectiveness of current vaccines. We report the data obtained in several Italian regions involved in the SARS-CoV-2 variant monitoring from the beginning of the epidemic and spanning the period from October 2020 to March 2021.
ARTICLE | doi:10.20944/preprints202006.0275.v1
Subject: Medicine And Pharmacology, Hematology Keywords: SARS-CoV-2; COVID-19; real-time RT-PCR; COVID-19 symptoms; COVID-19 hematological findings; Bangladesh
Online: 21 June 2020 (14:47:03 CEST)
Objective: SARS-Cov-2 infection or COVID-19 is a global pandemic. From the time of identification to till, multiple clinical symptoms and parameters have been identified by the researchers of various countries and regions regarding the diagnosis and presentations of COVID-19 disease. In this manuscript, we investigated the primary symptoms and basic hematological presentations of SARS-CoV-2 infection among the Bangladeshi patients. Methodology: We have collected the disease history of mild to moderate degree of COVID-19 patients; hematological and biochemical on admission reports of moderate degree COVID-19 patients. All of them were tested positive for SARS-CoV-2 by RT-PCR in different institutes in Bangladesh. Results: According to this study though COVID-19 patients in Bangladesh commonly presented with fever, cough, fatigue, shortness of breath, and sore throat, but symptoms like myalgia, diarrhea, skin rash, headache, Abdominal pain/cramp, nausea, vomiting, restlessness, and a higher temperature of >1000F have a greater presentation rate and more frequent than other published studies. CRP and Prothrombin time was found to increase in all the patients. Serum ferritin, ESR, SGPT, and D-Dimer were found increased among 53.85%, 80.43, 44%, and 25% patients respectively. 17.39% of the patients had leukocytosis and neutrophilia. 28.26% of patients presented with lymphocytopenia. 62.52% of patients had mild erythrocytopenia. Conclusion: Despite some similarities, our study has evaluated a different expression in presenting symptoms in the case of COVID-19 patients in Bangladesh. CRP, Prothrombin time, serum ferritin, ESR, SGPT, D-Dimer, erythrocytopenia, and lymphocytopenia can be initial diagnostic hematological findings and assessment for prognosis COVID-19 disease. Also, gender variations have a different scenario of clinical and laboratory appearance in this region.
ARTICLE | doi:10.20944/preprints202304.0971.v1
Subject: Medicine And Pharmacology, Pulmonary And Respiratory Medicine Keywords: 2019-nCoV; COVID-19; RT-PCR; SARS-CoV-2; Community-acquired pneumonia; Chest CT; microwave radiometry; temperature measurement
Online: 26 April 2023 (08:02:32 CEST)
Background. Chest CT is widely regarded as a dependable imaging technique for detecting pneumonia in COVID-19 patients, but there is growing interest in microwave radiometry (MWR) of the lungs as a possible substitute for diagnosing lung involvement. Aim. The aim of the study is to examine the utility of the MWR approach as a screening tool for diagnosing pneumonia with complications in patients with COVID-19. Methods. Our study involved two groups of participants. The control group consisted of 50 individuals (24 male and 26 female) between the ages of 20 to 70 years who underwent clinical evaluations and had no known medical conditions. The main group included 142 participants (67 men and 75 women) between the ages of 20 to 87 years who were diagnosed with COVID-19 complicated by pneumonia and were admitted to the emergency department between June 2020 to June 2021. Skin and lung temperatures were measured at 14 points, including 2 additional reference points, using a previously established method. Lung temperature data were obtained with the MWR2020 (RTM-01-RES) (MMWR LTD, Edinburgh, UK), a CE Class I device. All participants underwent clinical evaluations, laboratory tests, chest CT scans, MWR of the lungs, and reverse transcriptase polymerase chain reaction (RT-PCR) testing for SARS-CoV-2. Results. The MWR exhibits a high predictive capacity as demonstrated by its sensitivity of 98.6% and specificity of 84.0%. Conclusions. MWR of the lungs can be a valuable substitute for chest CT in diagnosing pneumonia in patients with COVID-19, especially in situations where chest CT is unavailable or impractical.
ARTICLE | doi:10.20944/preprints202309.1546.v1
Subject: Physical Sciences, Chemical Physics Keywords: RT-TAO-DFT; TAO-DFT; multi-reference character; real-time electron dynamics; time-dependent properties; high-order harmonic generation
Online: 22 September 2023 (11:40:32 CEST)
Thermally-assisted-occupation density functional theory (TAO-DFT) has been an efficient electronic structure method for studying the ground-state properties of large electronic systems with multi-reference character over the past few years. To explore the time-dependent (TD) properties of electronic systems (e.g., subject to an intense laser pulse), in this work, we propose a real-time (RT) extension of TAO-DFT, denoted as RT-TAO-DFT. Besides, we employ RT-TAO-DFT to study the high-order harmonic generation (HHG) spectra and related TD properties of molecular hydrogen H2 at the equilibrium and stretched geometries, aligned along the polarization of an intense linearly polarized laser pulse. The TD properties obtained with RT-TAO-DFT are compared with those obtained with the widely used time-dependent Kohn-Sham (TDKS) method. In addition, issues related to the possible spin-symmetry breaking effects in the TD properties are discussed.
ARTICLE | doi:10.20944/preprints202309.1037.v1
Subject: Medicine And Pharmacology, Clinical Medicine Keywords: respiratory syncytial virus; influenza A virus; influenza B virus; SARS-CoV-2; rapid RT-PCR; diagnostic accuracy; point-of-care testing
Online: 15 September 2023 (05:38:13 CEST)
SARS-CoV-2, influenza A/B virus (IAV/IBV) and respiratory syncytial virus (RSV) are among the common viruses causing acute respiratory infections. Clinical diagnosis to differentiate these viruses is challenging due to similar clinical presentations, thus laboratory-based real-time RT PCR is the gold standard for diagnosis. The study aimed to evaluate the diagnostic performance of STANDARD M10 Flu/RSV/SARS-CoV-2 (SD Biosensor Inc, Seoul, Korea). This retrospective study was conducted with archived respiratory samples positive and negative for SARS-CoV-2, IAV, IBV or RSV. The evaluation study involved a total of 322 respiratory samples comprising 215 positive samples (49 SARS-CoV-2, 48 IAV, 53 IBV, 65 RSV) and 107 negative samples. All samples were tested with both STANDARD M10 and either Xpert Xpress SARS-CoV-2 or Xpert Xpress Flu/RSV (Cepheid, USA). The sensitivity, specificity, positive predictive value and negative predictive value rates of STANDARD M10 were very similar to Xpert Xpress SARS-CoV-2 or Xpert Xpress Flu/RSV ranges for each virus (98-100%). The overall agreement was 99.4% with 99.1% agreement for positive samples and 100% agreement for negative samples. In conclusion, the STANDARD M10 point-of-care test is suitable for rapid simultaneous detection of SARS-CoV-2, IAV, IBV and RSV.
ARTICLE | doi:10.20944/preprints202308.1465.v1
Subject: Public Health And Healthcare, Public Health And Health Services Keywords: Adolescent; children; 12-17 years COVID-19 vaccine; fully vaccinated; partially vaccinated; vaccination status; RT-PCR-positive; Pfizer; BNT162b2; vaccine effectiveness
Online: 22 August 2023 (03:02:19 CEST)
Qatar experienced five SARS-CoV-2 waves dominated sequentially by the original virus, Alpha, Beta, Omicron BA.1 and BA.2, and Omicron BA.4 and BA.5, in addition to a prolonged low-incidence phase dominated by the Delta variant. The two-dose primary series of BNT162b2 (Pfizer-BioNTech) COVID-19 vaccine against SARS-CoV-2 infection has been approved for use in 10µg formulations among children and adolescents aged 12-17 years as of May 16, 2021. This study aimed at estimating the effectiveness of the 30µg BNT162b2 Pfizer-BioNTech mRNA COVID-19 vaccine against the pre-Omicron variants of SARS-CoV-2 infection in children and adolescents aged 12-17 years residing in Qatar. A test-negative matched case-control study was conducted where any child or adolescent aged 12-17 years who had been tested for SARS-CoV-2, RT-PCR tests performed on nasopharyngeal or oropharyngeal swabs, as part of contact tracing, between June and November 2021 and eligible to receive the BNT162b2 vaccine as per the national guidelines. Data regarding 14,161 children/adolescents meeting inclusion-exclusion criteria were retrieved from the national Surveillance and Vaccine Electronic System (SAVES). Of the total, 3.1% (444) were positive for SARSCoV-2. More than half (55.96%) were vaccinated with two doses of Pfizer-BioNTech-mRNA COVID-19 vaccine. Amongst those immunized with two doses, 1.2% tested positive for SARS CoV2, while 5.6% amongst the unvaccinated tested positive. The vaccine effectiveness was calculated to be 79%. Pfizer-BioNTech mRNA COVID-19 vaccine provides protection from COVID-19 infection for children/adolescents, hence it is crucial to ensure they receive the recommended vaccines.
ARTICLE | doi:10.20944/preprints201809.0054.v1
Subject: Engineering, Electrical And Electronic Engineering Keywords: Active Front-End converter; back-to-back converter; PMSG; THD; Type-4 wind turbine; wind energy system; Opal-RT Technologies®
Online: 4 September 2018 (05:02:15 CEST)
In this paper, the active front-end (AFE) converter topology for the total harmonic distortion (THD) reduction in a wind energy system (WES) is used. A higher THD results in serious pulsations in the wind turbine (WT) output power and in several power losses at the WES. The AFE converter topology improves capability, efficiency and reliability in the energy conversion devices; by modifying a conventional back-to-back converter, from using a single voltage source converter (VSC) to use pVSC connected in parallel the AFE converter is generated. The THD reduction is done by applying a different phase shift angle at the carrier of digital sinusoidal pulse width modulation (DSPWM) switching signals of each VSC. To verify the functionality of the proposed methodology, the WES simulation in Matlab-Simulink® is analyzed, and the experimental laboratory tests using the concept of rapid control prototyping and the real-time simulator Opal-RT® Technologies is achieved. The obtained results show a type-4 WT with total output power of 6MVA, generating a THD reduction up to 5.5 times at the WES.
ARTICLE | doi:10.20944/preprints202109.0415.v2
Subject: Biology And Life Sciences, Virology Keywords: Delta variant; Variants of concern; Variants of interest; SARS-CoV-2; Spike protein; Nested RT-PCR; Sanger sequencing; Amino acid mutations; ACE2 RBD; N-terminal domain (NTD)
Online: 2 November 2021 (10:40:46 CET)
As SARS-CoV-2 continues to spread among human populations, genetic changes occur and accumulate in the circulating virus. Some of these genetic changes have caused amino acid mutations, including deletions, which may have potential impact on critical SARS-CoV-2 countermeasures, including vaccines, therapeutics, and diagnostics. Considerable efforts have been made to categorize the amino acid mutations of the angiotensin-converting enzyme 2 (ACE2) receptor binding domain (RBD) of the spike (S) protein along with certain mutations in other regions within the S protein as specific variants in an attempt to study the relationship between these mutations and the biological behavior of the virus. However, the currently used whole genome sequencing surveillance technologies can test only a small fraction of the positive specimens with high viral loads and often generate uncertainties in nucleic acid sequencing that needs additional verification for precision determination of mutations. This article introduces a generic protocol to routinely sequence a 437-bp nested RT-PCR cDNA amplicon of the ACE2 RBD and a 490-bp nested RT-PCR cDNA amplicon of the N-terminal domain (NTD) of the S gene for detection of the amino acid mutations needed for accurate determination of all variants of concern and variants of interest according to the definitions published by the U.S. Centers for Disease Control and Prevention. This protocol was able to amplify both nucleic acid targets into cDNA amplicons to be used as templates for Sanger sequencing on all 16 clinical specimens that were positive for SARS-CoV-2.
ARTICLE | doi:10.20944/preprints202307.1155.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: miRNA; piRNA; mRNA; CA125; progesterone receptor (PGR); new-generation sequencing (NGS); quantitative RT-PCR; serous ovarian carcinoma; borderline cystadenoma; benign cystadenoma; formalin-fixed paraffin-embedded (FFPE) blocks; blood plasma; cytoreduction
Online: 18 July 2023 (09:58:12 CEST)
Progesterone receptor (PGR) expression level determines biological characteristics of the serous ovarian carcinoma, and low PGR expression appears to be associated with chemoresistance and worse outcome. In this study, we aimed to find relationships between tumor progesterone receptor level and RNA-profile (miRNAs, piwiRNAs, and mRNAs), determining its biological characteristics and behavior. For this purpose, we applied next generation sequencing of small noncoding RNAs, quantitative RT-PCR, and immunohistochemistry to analyze FFPE and frozen tumor samples as well as blood plasma from patients with benign cystadenoma (BSC), serous borderline tumour (SBT), low-grade and high-grade serous ovarian carcinoma (LGSOC and HGSOC, respectively). We found significant upregulation of MMP7 and MUC16 and downregulation of PGR in LGSOC and HGSOC in comparison with BSC. The tissue content of miR-199a-5p, miR-214-3p, miR-424-3p, miR-424-5p, miR-125b-5p significantly inversely correlated with the expression level of MUC16 and blood serum CA125 concentration, and significantly directly correlated with the PGR expression level in tumor tissue. On the contrary, the tissue content of miR-16-5p, miR-17-5p, miR-20a-5p, miR-93-5p, responsible for epithelial-mesenchymal transition (EMT) of the cell, significantly directly correlated with the blood serum CA125 concentration and significantly inversely correlated with the PGR expression level in the tumor tissue. Levels of the EMT-associated miRNAs significantly directly correlated with the content of hsa_piR_022437, hsa_piR_009295, hsa_piR_020813, hsa_piR_004307, hsa_piR_019914 in tumor tissues. Among them, expression level of hsa_piR_004307 significantly inversly correlated with the PGR expression level in the tumor. Two optimal logistic regression models were developed based on the quantitation of hsa_piR_020813, miR-16-5p, hsa_piR_022437 or hsa_piR_004307, hsa_piR_019914, miR-93-5p in the tumor tissue, both of which significantly diagnose PGR-negative tumor phenotype with 93% sensitivity. According to FunRich3.1.3 functional enrichment analysis tool, 72 gene-targets of miRNA and piRNA identified here as markers of PGR-negative ovarian tumor phenotype were proven to be mutated in different cancers such as ovarian, breast, colorectal, liver, stomach, lung, endometrial, thyroid cancer. Among them, the blood plasma levels of miR-16-5p and hsa_piR_022437 can be used to diagnose PGR-negative tumor phenotype with 86% sensitivity before surgery and chemotherapy to choose the treatment strategy for this most aggressive type of ovarian cancer (for instance, neoadjuvant chemotherapy followed by cytoreduction in combination with hyperthermic intraperitoneal chemotherapy) to increase the effectiveness of treatment and longevity of the patient.