scholarly journals Validation of a Methodology for the Detection of Severe Acute Respiratory Syndrome Coronavirus 2 in Saliva by Real-Time Reverse Transcriptase-PCR

2021 ◽  
Vol 9 ◽  
Author(s):  
Daniel F. Escobar ◽  
Pablo Díaz ◽  
Diego Díaz-Dinamarca ◽  
Rodrigo Puentes ◽  
Pedro Alarcón ◽  
...  

In January 2021, the Chilean city of Concepción experienced a second wave of coronavirus 2019 (COVID-19) while in early April 2021, the entire country faced the same situation. This outbreak generated the need to modify and validate a method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in saliva, thereby expanding the capacity and versatility of testing for COVID-19. This study was conducted in February 2021 in the Chilean city of Concepción during which time, the town was under total quarantine. The study participants were mostly symptomatic (87.4%), not hospitalized, and attended care centers because of their health status rather than being asked by the researchers. People coming to the health center in Concepción to be tested for COVID-19 (via reverse transcriptase polymerase chain reaction [RT-PCR]) from a specimen of nasopharyngeal swab (NPS) were then invited to participate in this study. A total of 131 participants agreed to sign an informed consent and to provide saliva and NPS specimens to validate a method in terms of sensitivity, specificity, and statistical analysis of the cycle threshold (Ct) values from the RT-PCR. Calculations pertaining to the 127 participants who were ultimately included in the analysis showed sensitivity and specificity at 94.34% (95% CI: 84.34–98.82%) and 98.65% (95% CI: 92.70–99.97%), respectively. The saliva specimen showed a performance comparable to NPS as demonstrated by the diagnostic parameters. This RT-PCR method from the saliva specimen is a highly sensitive and specific alternative compared to the reference methodology, which uses the NPS specimen. This modified and validated method is intended for use in the in vitro diagnosis of SARS-CoV-2, which provides health authorities in Chile and local laboratories with a real testing alternative to RT-PCR from NPS.

Gut Pathogens ◽  
2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Andreas Papoutsis ◽  
Thomas Borody ◽  
Siba Dolai ◽  
Jordan Daniels ◽  
Skylar Steinberg ◽  
...  

Abstract Background SARS-CoV-2 has been detected not only in respiratory secretions, but also in stool collections. Here were sought to identify SARS-CoV-2 by enrichment next-generation sequencing (NGS) from fecal samples, and to utilize whole genome analysis to characterize SARS-CoV-2 mutational variations in COVID-19 patients. Results Study participants underwent testing for SARS-CoV-2 from fecal samples by whole genome enrichment NGS (n = 14), and RT-PCR nasopharyngeal swab analysis (n = 12). The concordance of SARS-CoV-2 detection by enrichment NGS from stools with RT-PCR nasopharyngeal analysis was 100%. Unique variants were identified in four patients, with a total of 33 different mutations among those in which SARS-CoV-2 was detected by whole genome enrichment NGS. Conclusion These results highlight the potential viability of SARS-CoV-2 in feces, its ongoing mutational accumulation, and its possible role in fecal–oral transmission. This study also elucidates the advantages of SARS-CoV-2 enrichment NGS, which may be a key methodology to document complete viral eradication. Trial registration ClinicalTrials.gov, NCT04359836, Registered 24 April 2020, https://clinicaltrials.gov/ct2/show/NCT04359836?term=NCT04359836&draw=2&rank=1).


Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2388
Author(s):  
Brendan Russ ◽  
Friedhelm Pfeiffer ◽  
Mike Dyall-Smith

Halovirus HF2 was the first member of the Haloferacalesvirus genus to have its genome fully sequenced, which revealed two classes of intergenic repeat (IR) sequences: class I repeats of 58 bp in length, and class II repeats of 29 bp in length. Both classes of repeat contain AT-rich motifs that were conjectured to represent promoters. In the present study, nine IRs were cloned upstream of the bgaH reporter gene, and all displayed promoter activity, providing experimental evidence for the previous conjecture. Comparative genomics showed that IR sequences and their relative genomic positions were strongly conserved among other members of the same virus genus. The transcription of HF2 was also examined by the reverse-transcriptase-PCR (RT-PCR) method, which demonstrated very long transcripts were produced that together covered most of the genome, and from both strands. The presence of long counter transcripts suggests a regulatory role or possibly unrecognized coding potential.


2006 ◽  
Vol 69 (9) ◽  
pp. 2210-2216 ◽  
Author(s):  
MARÍA BELÉN MAYORAL ◽  
ROSARIO MARTÍN ◽  
PABLO E. HERNÁNDEZ ◽  
ISABEL GONZÁLEZ ◽  
TERESA GARCÍA

A fast and sensitive reverse transcriptase PCR (RT-PCR) method was developed for the detection of viable Kluyveromyces marxianus in yoghurt. Yeast-specific primers were used with the RT-PCR to evaluate the suitability of 18S rRNA as a target for the detection of viable yeasts in pure culture and yoghurt. The RT-PCR assay was able to detect down to 102 CFU ml−1 in yoghurt samples contaminated with viable yeast cells. Application of the RT-PCR method to commercial yoghurt samples demonstrated the utility of this technique for detection of low concentrations of viable yeast cells in naturally contaminated dairy products. The 18S rRNA molecule is an appropriate target for cell viability assessment because of its limited persistence after cell death and the resultant high level of sensitivity of the assay.


Author(s):  

SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2) is a novel coronavirus identified for the first time in Wuhan (China) in 2019, responsible of the current pandemic infection known as Coronavirus-19 disease (COVID-19). Wide range of clinical presentation of COVD -19 has been observed, from asymptomatic carriers to ARDS. The common signs and symptoms of SARS-CoV-2 infection include fever, fatigue, dry cough, and dyspnoea; the severity of the disease is due to the impairment of the respiratory function. The radiological findings include a large variety of lesions; bilateral interstitial pneumonia is the most concerning presentation of COVID-19. Pleural involvement has been described in a minority of cases: pleural thickening had been observed in 32% of cases whereas pleural effusion is uncommon being described in only 5%. Furthermore, pleural involvement has been significantly associated with a worse prognosis. Coronavirus 2 (SARS-CoV-2), beyond the nasopharyngeal swab, has been detected in other samples; up to now, data about RT-PCR specific results in the pleural fluid of patients suffering from coronavirus disease 2019 5 (COVID-19) are very limited. The current gold standard for diagnosis is nucleic acid detection by real time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) in nasopharyngeal swab. In this report, a case of a positive RT-PCR for Sars-Cov-2 in the pleura fluid and in the naso- pharyngeal swab of a patient affected by bilateral interstitial pneumonia and severe respiratory failure is described. As the presence of SARS-Cov-2 in the pleural fluid seems to be associated to a poor prognosis, physicians should carry out the specific RT-PCR assay both in the nasopharyngeal swab and in the pleural sample also when the fluid amount is very scarce and not recognizable in the chest X ray. Furthermore, the analysis of multiple samples allows to increase the test reliability.


2020 ◽  
Author(s):  
Maulin Patel ◽  
Junad Chowdhury ◽  
Matthew Zheng ◽  
Osheen Abramian ◽  
Steven Verga ◽  
...  

AbstractIntroductionCurrently the main diagnostic modality for COVID-19 (Coronavirus disease-2019) is reverse transcriptase polymerase chain reaction (RT-PCR) via nasopharyngeal swab which has high false negative rates. We evaluated the performance of high-resolution computed tomography (HRCT) imaging in the diagnosis of suspected COVID-19 infection compared to RT-PCR nasopharyngeal swab alone in patients hospitalized for suspected COVID-19 infection.MethodsThis was a retrospective analysis of 324 consecutive patients admitted to Temple University Hospital. All hospitalized patients who had RT-PCR testing and HRCT were included in the study. HRCTs were classified as Category 1, 2 or 3. Patients were then divided into four groups based on HRCT category and RT-PCR swab results for analysis.ResultsThe average age of patients was 59.4 (±15.2) years and 123 (38.9%) were female. Predominant ethnicity was African American 148 (46.11%). 161 patients tested positive by RT-PCR, while 41 tested positive by HRCT. 167 (52.02%) had category 1 scan, 63 (19.63%) had category 2 scan and 91 (28.35%) had category 3 HRCT scans. There was substantial agreement between our radiologists for HRCT classification (κ = 0.64). Sensitivity and specificity of HRCT classification system was 77.6 and 73.7 respectively. Ferritin, LDH, AST and ALT were higher in Group 1 and D-dimers levels was higher in Group 3; differences however were not statistically significant.ConclusionDue to its high infectivity and asymptomatic transmission, until a highly sensitive and specific COVID-19 test is developed, HRCT should be incorporated into the assessment of patients who are hospitalized with suspected COVID-19.Key PointsKey QuestionCan High Resolution CT chest (HRCT) improve diagnostic accuracy of current Nasopharyngeal swab in suspected COVID-19 patients?Bottom LineIn this retrospective analysis, our novel HRCT classification identified 20% of all COVID-19 patients who had negative nasopharyngeal reverse transcriptase polymerase chain reaction (RT-PCR) tests but had HRCT findings consistent with COVID-19 pneumonia. These patients were ruled out for other infections and laboratory markers were similar to other RT-PCR positive patientsWhy Read onOur new HRCT classification when combined with RT-PCR can improve diagnostic accuracy while promptly improving triaging in COVID-19 patients.


2019 ◽  
Vol 7 (9) ◽  
pp. 1422-1424
Author(s):  
Aminah Dalimunthe ◽  
Poppy Anjelisa Zaitun Hasibuan ◽  
Denny Satria

AIM: To investigated the activities of chloroform fractions at pH 7 of Litsea cubeba Lour. Fruits and heartwoods (CF-7F and CF-7H) in decrease expression of PI3KCA, Akt-1 and Akt-2 genes towards cervical cancer cell culture (HeLa) experiments in vitro. MATERIAL AND METHODS: CF-7F and CF-7H (12.5 and 25 µg/mL) were tested for its potential inhibition on gene expression of PI3KCA, Akt-1 and Akt-2 genes by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) method. RESULT: CF-7F and CF-7H were showed the activity to reduce the expression of PI3KCA, Akt-1 and Akt-2 genes. CONCLUSION: Our results suggest that CF-7F and CF-7H significantly inhibit the expression of PI3KCA, Akt-1 and Akt-2 genes.


2020 ◽  
Author(s):  
Andreas Papoutsis ◽  
Thomas Borody ◽  
Siba Dolai ◽  
Jordan Daniels ◽  
Skylar Steinberg ◽  
...  

Abstract Background SARS-CoV-2 has been detected not only in respiratory secretions, but also in stool collections. Here were sought to identify SARS-CoV-2 by enrichment NGS from fecal samples, and to utilize whole genome analysis to characterize SARS-CoV-2 mutational variations in COVID-19 patients. Results Study participants underwent testing for SARS-CoV-2 from fecal samples by whole genome enrichment NGS (n = 14), and RT-PCR nasopharyngeal swab analysis (n = 12). The concordance of SARS-CoV-2 detection by enrichment NGS from stools with RT-PCR nasopharyngeal analysis was 100%. Unique variants were identified in four patients, with a total of 33 different mutations among those in which SARS-CoV-2 was detected by whole genome enrichment NGS. Conclusion These results highlight the potential viability of SARS-CoV-2 in feces, its ongoing mutational accumulation, and its possible role in fecal-oral transmission. This study also elucidates the advantages of SARS-CoV-2 enrichment NGS, which may be a key methodology to document complete viral eradication.


Author(s):  
Alainna J Jamal ◽  
Mohammad Mozafarihashjin ◽  
Eric Coomes ◽  
Jeff Powis ◽  
Angel X Li ◽  
...  

Abstract We enrolled 91 consecutive inpatients with COVID-19 at 6 hospitals in Toronto, Canada, and tested 1 nasopharyngeal swab/saliva sample pair from each patient using real-time RT-PCR for severe acute respiratory syndrome coronavirus 2. Sensitivity was 89% for nasopharyngeal swabs and 72% for saliva (P = .02). Difference in sensitivity was greatest for sample pairs collected later in illness.


Zygote ◽  
2003 ◽  
Vol 11 (3) ◽  
pp. 245-252 ◽  
Author(s):  
Dong-Wook Han ◽  
Sang-Jin Song ◽  
Sang Jun Uhum ◽  
Jeong-Tae Do ◽  
Nam-Hyung Kim ◽  
...  

Incomplete reprogramming of the donor cell nucleus after nuclear transfer (NT) probably leads to the abnormal expression of developmentally important genes. This may be responsible for the low efficiency of cloned animal production. Insulin-like growth factor 2 (IGF2) and IGF2 receptor (IGF2R) are imprinted genes that play important roles in preimplantation development. To obtain an insight into abnormal gene expression after nuclear transfer, we assessed the transcription patterns of IGF2-IGF2R in single in vitro fertilised and cloned embryos by reverse-transcription polymerase chain reaction (RT-PCR). IGF2R expression did not differ significantly but IGF2 was more highly expressed in cloned embryos than in IVF embryos (p < 0.05). This was confirmed by a quantitative RT-PCR method. Thus, incomplete reprogramming may induce abnormal transcription of IGF2 in cloned embryos.


2020 ◽  
Vol 37 (10) ◽  
pp. 1055-1060 ◽  
Author(s):  
Ziyi Yang ◽  
Yi Liu

Objective The aim of this study is to summarize currently available evidence on vertical transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Study Design A systematic review was conducted following the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-analysis Statement. Results A total of 22 studies comprising 83 neonates born to mothers diagnosed with coronavirus disease 2019 were included in the present systematic review. Among these neonates, three were confirmed with SARS-CoV-2 infection at 16, 36, and 72 hours after birth, respectively, by nasopharyngeal swab real-time polymerase chain reaction (RT-PCR) tests; another six had elevated virus-specific antibody levels in serum samples collected after birth, but negative RT-PCR test results. However, without positive RT-PCR tests of amniotic fluid, placenta, or cord blood, there is a lack of virologic evidence for intrauterine vertical transmission. Conclusion There is currently no direct evidence to support intrauterine vertical transmission of SARS-CoV-2. Additional RT-PCR tests on amniotic fluid, placenta, and cord blood are needed to ascertain the possibility of intrauterine vertical transmission. For pregnant women infected during their first and second trimesters, further studies focusing on long-term outcomes are needed. Key Points


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