scholarly journals Direct detection of SARS-CoV-2 antisense and sense genomic RNA in human saliva by semi-autonomous fluorescence in situ hybridization: A proxy for contagiousness?

PLoS ONE ◽  
2021 ◽  
Vol 16 (8) ◽  
pp. e0256378
Author(s):  
Gijsbert J. Jansen ◽  
Marit Wiersma ◽  
Willem J. B. van Wamel ◽  
Inge D. Wijnberg

Saliva is a matrix which may act as a vector for pathogen transmission and may serve as a possible proxy for SARS-CoV-2 contagiousness. Therefore, the possibility of detection of intracellular SARS-CoV-2 in saliva by means of fluorescence in situ hybridization is tested, utilizing probes targeting the antisense or sense genomic RNA of SARS-CoV-2. This method was applied in a pilot study with saliva samples collected from healthy persons and those presenting with mild or moderate COVID-19 symptoms. In all participants, saliva appeared a suitable matrix for the detection of SARS-CoV-2. Among the healthy, mild COVID-19-symptomatic and moderate COVID-19-symptomatic persons, 0%, 90% and 100% tested positive for SARS-CoV-2, respectively. Moreover, the procedure allows for simultaneous measurement of viral load (‘presence’, sense genomic SARS-CoV-2 RNA) and viral replication (‘activity’, antisense genomic SARS-CoV-2 RNA) and may yield qualitative results. In addition, the visualization of DNA in the cells in saliva provides an additional cytological context to the validity and interpretability of the test results. The method described in this pilot study may be a valuable diagnostic tool for detection of SARS-CoV-2, distinguishing between ‘presence’ (viral load) and ‘activity’ (viral replication) of the virus. Moreover, the method potentially gives more information about possible contagiousness.

1998 ◽  
Vol 72 (9) ◽  
pp. 7263-7269 ◽  
Author(s):  
J. Lindsay Oaks ◽  
Travis C. McGuire ◽  
Catherine Ulibarri ◽  
Timothy B. Crawford

ABSTRACT The equine infectious anemia virus (EIAV) often results in lifelong subclinical infection following early episodes of clinical disease. To identify the cellular reservoirs of EIAV during subclinical infection, horses were infected with EIAV and allowed to develop subclinical infections. Horses with acute disease served as a basis for comparison. The tissue distribution, replication status, location of infected cells, and viral load were characterized by PCR for proviral DNA and reverse transcriptase PCR for viral RNA, in situ hybridization, and in situ PCR. Proviral DNA was widespread in tissues regardless of disease status. Viral gag and env RNAs were also detected in tissues of all horses regardless of disease status. Plasma viral RNA (viremia) could be detected in some, but not all, horses with subclinical infections. In situ assays determined that a primary cellular reservoir and site of viral replication during subclinical infection is the macrophage. During subclinical infection, viral load was decreased 4- to 733-fold and there was decreased viral RNA expression within infected cells. These data indicate that viral replication continues at all times, even in horses that are clinically quiescent. Moreover, restricted viral replication at the cellular level is associated with clinical remission.


2019 ◽  
Author(s):  
Nikki Theofanopoulou ◽  
Katherine Isbister ◽  
Julian Edbrooke-Childs ◽  
Petr Slovák

BACKGROUND A common challenge within psychiatry and prevention science more broadly is the lack of effective, engaging, and scale-able mechanisms to deliver psycho-social interventions for children, especially beyond in-person therapeutic or school-based contexts. Although digital technology has the potential to address these issues, existing research on technology-enabled interventions for families remains limited. OBJECTIVE The aim of this pilot study was to examine the feasibility of in-situ deployments of a low-cost, bespoke prototype, which has been designed to support children’s in-the-moment emotion regulation efforts. This prototype instantiates a novel intervention model that aims to address the existing limitations by delivering the intervention through an interactive object (a ‘smart toy’) sent home with the child, without any prior training necessary for either the child or their carer. This pilot study examined (i) engagement and acceptability of the device in the homes during 1 week deployments; and (ii) qualitative indicators of emotion regulation effects, as reported by parents and children. METHODS In this qualitative study, ten families (altogether 11 children aged 6-10 years) were recruited from three under-privileged communities in the UK. The RA visited participants in their homes to give children the ‘smart toy’ and conduct a semi-structured interview with at least one parent from each family. Children were given the prototype, a discovery book, and a simple digital camera to keep at home for 7-8 days, after which we interviewed each child and their parent about their experience. Thematic analysis guided the identification and organisation of common themes and patterns across the dataset. In addition, the prototypes automatically logged every interaction with the toy throughout the week-long deployments. RESULTS Across all 10 families, parents and children reported that the ‘smart toy’ was incorporated into children’s emotion regulation practices and engaged with naturally in moments children wanted to relax or calm down. Data suggests that children interacted with the toy throughout the duration of the deployment, found the experience enjoyable, and all requested to keep the toy longer. Child emotional connection to the toy—caring for its ‘well-being’—appears to have driven this strong engagement. Parents reported satisfaction with and acceptability of the toy. CONCLUSIONS This is the first known study investigation of the use of object-enabled intervention delivery to support emotion regulation in-situ. The strong engagement and qualitative indications of effects are promising – children were able to use the prototype without any training and incorporated it into their emotion regulation practices during daily challenges. Future work is needed to extend this indicative data with efficacy studies examining the psychological efficacy of the proposed intervention. More broadly, our findings suggest the potential of a technology-enabled shift in how prevention interventions are designed and delivered: empowering children and parents through ‘child-led, situated interventions’, where participants learn through actionable support directly within family life, as opposed to didactic in-person workshops and a subsequent skills application.


1970 ◽  
Vol 22 (12) ◽  
pp. 1520-1524 ◽  
Author(s):  
E.L. Burwell ◽  
T.E. Sterner ◽  
H.C. Carpenter
Keyword(s):  

BMJ ◽  
2021 ◽  
pp. n1637 ◽  
Author(s):  
Marta García-Fiñana ◽  
David M Hughes ◽  
Christopher P Cheyne ◽  
Girvan Burnside ◽  
Mark Stockbridge ◽  
...  

Abstract Objective To assess the performance of the SARS-CoV-2 antigen rapid lateral flow test (LFT) versus polymerase chain reaction testing in the asymptomatic general population attending testing centres. Design Observational cohort study. Setting Community LFT pilot at covid-19 testing sites in Liverpool, UK. Participants 5869 asymptomatic adults (≥18 years) voluntarily attending one of 48 testing sites during 6-29 November 2020. Interventions Participants were tested using both an Innova LFT and a quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) test based on supervised self-administered swabbing at testing sites. Main outcome measures Sensitivity, specificity, and predictive values of LFT compared with RT-qPCR in an epidemic steady state of covid-19 among adults with no classic symptoms of the disease. Results Of 5869 test results, 22 (0.4%) LFT results and 343 (5.8%) RT-qPCR results were void (that is, when the control line fails to appear within 30 minutes). Excluding the void results, the LFT versus RT-qPCR showed a sensitivity of 40.0% (95% confidence interval 28.5% to 52.4%; 28/70), specificity of 99.9% (99.8% to 99.99%; 5431/5434), positive predictive value of 90.3% (74.2% to 98.0%; 28/31), and negative predictive value of 99.2% (99.0% to 99.4%; 5431/5473). When the void samples were assumed to be negative, a sensitivity was observed for LFT of 37.8% (26.8% to 49.9%; 28/74), specificity of 99.6% (99.4% to 99.8%; 5431/5452), positive predictive value of 84.8% (68.1% to 94.9%; 28/33), and negative predictive value of 93.4% (92.7% to 94.0%; 5431/5814). The sensitivity in participants with an RT-qPCR cycle threshold (Ct) of <18.3 (approximate viral loads >10 6 RNA copies/mL) was 90.9% (58.7% to 99.8%; 10/11), a Ct of <24.4 (>10 4 RNA copies/mL) was 69.4% (51.9% to 83.7%; 25/36), and a Ct of >24.4 (<10 4 RNA copies/mL) was 9.7% (1.9% to 23.7%; 3/34). LFT is likely to detect at least three fifths and at most 998 in every 1000 people with a positive RT-qPCR test result with high viral load. Conclusions The Innova LFT can be useful for identifying infections among adults who report no symptoms of covid-19, particularly those with high viral load who are more likely to infect others. The number of asymptomatic adults with lower Ct (indicating higher viral load) missed by LFT, although small, should be considered when using single LFT in high consequence settings. Clear and accurate communication with the public about how to interpret test results is important, given the chance of missing some cases, even at high viral loads. Further research is needed to understand how infectiousness is reflected in the viral antigen shedding detected by LFT versus the viral loads approximated by RT-qPCR.


1993 ◽  
Vol 104 (4) ◽  
pp. 1187-1197 ◽  
Author(s):  
R.W. Dirks ◽  
F.M. van de Rijke ◽  
S. Fujishita ◽  
M. van der Ploeg ◽  
A.K. Raap

We have determined optimal conditions for the detection of mRNA sequences in cultured cells by nonradioactive in situ hybridization. For this purpose a number of different cell lines have been used: rat 9G cells for the detection of human cytomegalovirus immediate early mRNA, and HeLa as well as 5637 carcinoma cells for the detection of housekeeping gene mRNAs. Extensive optimization of fixation and pretreatment conditions revealed that most intense hybridization signals are obtained when cells are grown on glass microscope slides, fixed with a mixture of formaldehyde and acetic acid, pretreated with pepsin and denatured prior to hybridization. In addition, we also studied the potential of fluorochromized probes for the direct detection of multiple RNA sequences. The optimized in situ hybridization procedure revealed that immediate early mRNA transcripts are, in addition to a cytoplasmic localization, localized within nuclei of rat 9G cells. Double hybridization experiments showed that intron and exon sequences colocalize within the main nuclear signal. In addition, the presence of small, intron-specific, fluorescent spots scattered around the main nuclear signals indicates that intron sequences which are spliced out can be visualized. Additional information about the functioning of cells could be obtained by the detection of mRNA simultaneously with bromodeoxyuridine, incorporated during S-phase, or its cognate protein. The sensitivity of these methods is such that mRNAs of abundantly expressed housekeeping genes can be detected in a variety of cell lines with high signal to noise ratios.


Author(s):  
Miss Payal W. Paratpure

Tracking of public bus location requires a GPS device to be installed, and lots of bus operators in developing countries don't have such an answer in situ to supply an accurate estimation of bus time of arrival (ETA). Without ETA information, it's very difficult for the overall public to plan their journey effectively. In this paper, implementation of an innovative IOT solution to trace the real time location of buses without requiring the deployment of a GPS device is discussed. It uses Bluetooth Low Energy (BLE) proximity beacon to trace the journey of a bus by deploying an Estimate location beacon on the bus. BLE detection devices (Raspberry Pi 4) are installed at selected bus stops along the path to detect the arrival of buses. Once detected, the situation of the bus is submitted to a cloud server to compute the bus ETAs. A field trial is currently being conducted in Johor, Malaysia together with an area bus operator on one single path. Our test results showed that the detection of BLE beacons is extremely accurate and it's feasible to trace the situation of buses without employing a GPS device during a cost-effective way.


2010 ◽  
Vol 134 (5) ◽  
pp. 744-750
Author(s):  
R. Marshall Austin ◽  
Agnieszka Onisko ◽  
Marek J. Druzdzel

Abstract Context.—Evaluation of cervical cancer screening has grown increasingly complex with the introduction of human papillomavirus (HPV) vaccination and newer screening technologies approved by the US Food and Drug Administration. Objective.—To create a unique Pittsburgh Cervical Cancer Screening Model (PCCSM) that quantifies risk for histopathologic cervical precancer (cervical intraepithelial neoplasia [CIN] 2, CIN3, and adenocarcinoma in situ) and cervical cancer in an environment predominantly using newer screening technologies. Design.—The PCCSM is a dynamic Bayesian network consisting of 19 variables available in the laboratory information system, including patient history data (most recent HPV vaccination data), Papanicolaou test results, high-risk HPV results, procedure data, and histopathologic results. The model's graphic structure was based on the published literature. Results from 375 441 patient records from 2005 through 2008 were used to build and train the model. Additional data from 45 930 patients were used to test the model. Results.—The PCCSM compares risk quantitatively over time for histopathologically verifiable CIN2, CIN3, adenocarcinoma in situ, and cervical cancer in screened patients for each current cytology result category and for each HPV result. For each current cytology result, HPV test results affect risk; however, the degree of cytologic abnormality remains the largest positive predictor of risk. Prior history also alters the CIN2, CIN3, adenocarcinoma in situ, and cervical cancer risk for patients with common current cytology and HPV test results. The PCCSM can also generate negative risk projections, estimating the likelihood of the absence of histopathologic CIN2, CIN3, adenocarcinoma in situ, and cervical cancer in screened patients. Conclusions.—The PCCSM is a dynamic Bayesian network that computes quantitative cervical disease risk estimates for patients undergoing cervical screening. Continuously updatable with current system data, the PCCSM provides a new tool to monitor cervical disease risk in the evolving postvaccination era.


2018 ◽  
Vol 69 (1) ◽  
pp. 315-350
Author(s):  
Julia Schirnhofer

Abstract As a phenomenon at the syntax-pragmatics interface, focus marking can cause particular difficulties in adult L2 acquisition and may never be fully acquired, whereas native-like competence can be achieved with formal syntactic properties. The present study examines this so-called Interface Hypothesis by analysing the strategies that monolingual German-speaking learners use to mark information focus in Spanish. Analyses of the test results show that around 97 % of the test subjects prefer to maintain the unmarked constituent order and mark focus in situ, irrespective of their proficiency level. In comparison with Spanish natives (Gabriel 2010, Heidinger 2014), the results show a divergence from the behaviour of native speakers, as the latter use various strategies. This indicates that the German-speaking learners do not make use of the variation of focus marking strategies the Spanish language provides, but rather adhere to in situ focalization, which is also the dominant focus-marking strategy in German. Furthermore, the results of the present study highlight that strategies for marking focus are scarcely taken into account in language teaching classes.


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