scholarly journals Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Mutational Pattern in the Fourth Pandemic Phase in Greece

2022 ◽  
Vol 44 (1) ◽  
pp. 329-335
Author(s):  
Panagiotis Halvatsiotis ◽  
Sofia Vassiliu ◽  
Panagiotis Koulouvaris ◽  
Kalliopi Chatzantonaki ◽  
Konstantinos Asonitis ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Rural Areas ◽  
Gene Mutations ◽  
Threshold Value ◽  
Rapid Review ◽  
Nasopharyngeal Swab ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Spike Gene ◽  
Copy Numbers

The aim of this study is to investigate the circulating variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from Athens and from rural areas in Greece during July and August 2021. We also present a rapid review of literature regarding significant SARS-CoV-2 mutations and their impact on public health. A total of 2500 nasopharyngeal swab specimens were collected from suspected COVID-19 cases (definition by WHO 2021b). Viral nucleic acid extraction was implemented using an automatic extractor and the RNA recovered underwent qRT-PCR in order to characterize the specimens as positive or negative for SARS-CoV-2. The positive specimens were then used to identify specific Spike gene mutations and characterize the emerging SARS-CoV-2 variants. For this step, various kits were utilized. From the 2500 clinical specimens, 220 were tested positive for SARS-CoV-2 indicating a prevalence of 8.8% among suspected cases. The RT-PCR Ct (Cycle threshold) Value ranged from 19 to 25 which corresponds to medium to high copy numbers of the virus in the positive samples. From the 220 positive specimens 148 (67.3%) were from Athens and 72 (32.7%) from Greek rural areas. As far as the Spike mutations investigated: N501Y appeared in all the samples, D614G mutation appeared in 212 (96.4%) samples with a prevalence of 87.2% in Athens and 98.6% in the countryside, E484K had a prevalence of 10.8% and 12.5% in Athens and the rural areas, respectively. K417N was found in 18 (12.2%) samples from Athens and four (5.6%) from the countryside, P681H was present in 51 (34.5%) Athenian specimens and 14 (19.4%) specimens from rural areas, HV69-70 was carried in 32.4% and 19.4% of the samples from Athens and the countryside, respectively. P681R had a prevalence of 87.2% in Athens and 98.6% in rural areas, and none of the specimens carried the L452R mutation. 62 (28.2%) samples carried the N501Y, P681H, D614G and HV69-70 mutations simultaneously and the corresponding variant was characterized as the Alpha (UK) variant (B 1.1.7). Only six (2.7%) samples from the center of Athens had the N501Y, E484K, K417N and D614G mutations simultaneously and the virus responsible was characterized as the Beta (South African) variant (B 1.351). Our study explored the SARS-CoV-2 variants using RT-PCR in a representative cohort of samples collected from Greece in July and August 2021. The prevalent mutations identified were N501Y (100%), D614G (96.4%), P681R (90.1%) and the variants identified were the Delta (90.1%), Alpha (28.2%) and Beta (2.7%).

scholarly journals Evaluation of an Automated High-Throughput Liquid-Based RNA Extraction Platform on Pooled Nasopharyngeal or Saliva Specimens for SARS-CoV-2 RT-PCR

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 615
Author(s):  
Allen Wing-Ho Chu ◽  
Cyril Chik-Yan Yip ◽  
Wan-Mui Chan ◽  
Anthony Chin-Ki Ng ◽  
Dream Lok-Sze Chan ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
High Throughput ◽  
Rna Extraction ◽  
Nasopharyngeal Swab ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Nucleic Acid Extraction ◽  
Suitable Alternative ◽  
Pcr Inhibitors ◽  
Pcr Targeting

SARS-CoV-2 RT-PCR with pooled specimens has been implemented during the COVID-19 pandemic as a cost- and manpower-saving strategy for large-scale testing. However, there is a paucity of data on the efficiency of different nucleic acid extraction platforms on pooled specimens. This study compared a novel automated high-throughput liquid-based RNA extraction (LRE) platform (PHASIFYTM) with a widely used magnetic bead-based total nucleic acid extraction (MBTE) platform (NucliSENS® easyMAG®). A total of 60 pools of nasopharyngeal swab and 60 pools of posterior oropharyngeal saliva specimens, each consisting of 1 SARS-CoV-2 positive and 9 SARS-CoV-2 negative specimens, were included for the comparison. Real-time RT-PCR targeting the SARS-CoV-2 RdRp/Hel gene was performed, and GAPDH RT-PCR was used to detect RT-PCR inhibitors. No significant differences were observed in the Ct values and overall RT-PCR positive rates between LRE and MBTE platforms (92.5% (111/120] vs 90% (108/120]), but there was a slightly higher positive rate for LRE (88.3% (53/60]) than MBTE (81.7% (49/60]) among pooled saliva. The automated LRE method is comparable to a standard MBTE method for the detection of SAR-CoV-2 in pooled specimens, providing a suitable alternative automated extraction platform. Furthermore, LRE may be better suited for pooled saliva specimens due to more efficient removal of RT-PCR inhibitors.

scholarly journals A Rapid and Consistent Method To Identify SARS-CoV-2 Variants By RT-PCR

2021 ◽  
Author(s):  
Marco Fabiani ◽  
Katia Margiotti ◽  
Manuela Sabatino ◽  
Antonella Viola ◽  
Alvaro Mesoraca ◽  
...  
Keyword(s):  
Defense Mechanisms ◽  
Immune Escape ◽  
Low Cost ◽  
Gene Mutations ◽  
Nucleotide Polymorphisms ◽  
Rt Pcr ◽  
Spike Gene ◽  
Natural Defense ◽  
The Uk ◽  
Next Generation Sequencing Ngs

Abstract Background Since 2020, the COVID-19 pandemic spread worldwide causing health, economic, and social distresses. Containment strategy relay on rapid and consistent methodology for molecular detection and characterization. The emerging variants of concern (VOCs) are currently associated with increased infectivity, and immune escape (natural defense mechanisms as well as a vaccine). Several VOCs has been detected and include lineage B.1.1.7 first identified in the UK, linage B.1.351 in South Africa, and lineage P.1 (B.1.1.28.1) in Brazil. Here we validated a rapid and low-cost technique to distinguish B.1.1.7, B.1.351 and P.1 SARS-CoV-2 variants by detecting Spike gene mutations using RT-PCR methodology. Results We recruited 77 positive patients affected by Coronavirus Disease-19 (COVID-19). Specific Real-time reverse transcription-polymerase chain reaction (RT-PCR) was employed targeting single nucleotide polymorphisms (SNPs) to screen Spike protein mutations. All data were validated by next generation sequencing (NGS) methodology and using sequence from a public database.Among 77 COVID-19 positive samples we could discriminate with 100% of concordance all the investigated SARS-CoV-2 variants when comparing with NGS method. Conclusions PCR-based assays for identification of circulating VOCs of SARS-CoV-2 resulted in a rapid method to identify the specific SARS-CoV-2 variants allowing a better survey of the spread of the virus and its transmissibility in the pandemic phase.

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 ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Reverse Transcriptase ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Health Authorities ◽  
Specific Alternative ◽  
Pcr Method ◽  
Sensitivity Specificity ◽  
Study Participants

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.

scholarly journals Sensitivity of Nasopharyngeal Swabs and Saliva for the Detection of Severe Acute Respiratory Syndrome Coronavirus 2

2020 ◽  
Author(s):  
Alainna J Jamal ◽  
Mohammad Mozafarihashjin ◽  
Eric Coomes ◽  
Jeff Powis ◽  
Angel X Li ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Saliva Sample ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Sample Pair

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.

AN EPIDEMIOLOGY-CLINICAL CORRELATION OF VIRAL LOAD AS MEASURED BY CYCLE THRESHOLD VALUE BY RT-PCR IN COVID-1

2021 ◽  
pp. 1-2
Author(s):  
Atrikumar P. Patel ◽  
Palak Shah ◽  
Pavan Acharya ◽  
Monila N. Patel
Keyword(s):  
Polymerase Chain Reaction ◽  
Viral Load ◽  
Severe Acute Respiratory Syndrome ◽  
Threshold Value ◽  
Clinical Correlation ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Oropharyngeal Swab ◽  
Polymerase Chain ◽  
Novel Coronavirus

The 2019 novel coronavirus [severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)] was rst documented in December 2019 in Wuhan, China, and spread across the globe resulting in [1]. signicant global morbidity and mortality Diagnosis of COVID-19 is mainly done by nasopharyngeal and oropharyngeal swab RT-PCR (Reverse transcriptase - polymerase chain reaction). Real time RT-PCR is of great interest today for detection of SARS- CoV-2 due to its benets as a specic assay.

scholarly journals Vertical Transmission of Severe Acute Respiratory Syndrome Coronavirus 2: A Systematic Review

2020 ◽  
Vol 37 (10) ◽  
pp. 1055-1060 ◽  
Author(s):  
Ziyi Yang ◽  
Yi Liu
Keyword(s):  
Systematic Review ◽  
Severe Acute Respiratory Syndrome ◽  
Amniotic Fluid ◽  
Cord Blood ◽  
Vertical Transmission ◽  
Meta Analysis ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Serum Samples ◽  
Virus Specific Antibody

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

scholarly journals Performance of Oropharyngeal Swab Testing Compared With Nasopharyngeal Swab Testing for Diagnosis of Coronavirus Disease 2019—United States, January 2020–February 2020

2020 ◽  
Author(s):  
Monita R Patel ◽  
Darin Carroll ◽  
Emily Ussery ◽  
Hilary Whitham ◽  
Christopher A Elkins ◽  
...  
Keyword(s):  
United States ◽  
Polymerase Chain Reaction ◽  
Severe Acute Respiratory Syndrome ◽  
Nasopharyngeal Swab ◽  
Threshold Values ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Illness Onset ◽  
Oropharyngeal Swab ◽  
Polymerase Chain

Abstract Among 146 nasopharyngeal (NP) and oropharyngeal (OP) swab pairs collected ≤7 days after illness onset, Real-Time Reverse Transcriptase Polymerase Chain Reaction assay for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 RT-PCR) diagnostic results were 95.2% concordant. However, NP swab cycle threshold values were lower (indicating more virus) in 66.7% of concordant-positive pairs, suggesting NP swabs may more accurately detect the amount of SARS-CoV-2.

scholarly journals Detection of SARS Coronavirus in Patients with Severe Acute Respiratory Syndrome by Conventional and Real-Time Quantitative Reverse Transcription-PCR Assays

2004 ◽  
Vol 50 (1) ◽  
pp. 67-72 ◽  
Author(s):  
Leo L M Poon ◽  
Kwok Hung Chan ◽  
On Kei Wong ◽  
Timothy K W Cheung ◽  
Iris Ng ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
N Gene ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
Copy Numbers ◽  
Stool Samples ◽  
Pcr Assays

Abstract Background: A novel coronavirus (CoV) was recently identified as the agent for severe acute respiratory syndrome (SARS). We compared the abilities of conventional and real-time reverse transcription-PCR (RT-PCR) assays to detect SARS CoV in clinical specimens. Methods: RNA samples isolated from nasopharyngeal aspirate (NPA; n = 170) and stool (n = 44) were reverse-transcribed and tested by our in-house conventional RT-PCR assay. We selected 98 NPA and 37 stool samples collected at different times after the onset of disease and tested them in a real-time quantitative RT-PCR specific for the open reading frame (ORF) 1b region of SARS CoV. Detection rates for the conventional and real-time quantitative RT-PCR assays were compared. To investigate the nature of viral RNA molecules in these clinical samples, we determined copy numbers of ORF 1b and nucleocapsid (N) gene sequences of SARS CoV. Results: The quantitative real-time RT-PCR assay was more sensitive than the conventional RT-PCR assay for detecting SARS CoV in samples collected early in the course of the disease. Real-time assays targeted at the ORF 1b region and the N gene revealed that copy numbers of ORF 1b and N gene sequences in clinical samples were similar. Conclusions: NPA and stool samples can be used for early diagnosis of SARS. The real-time quantitative RT-PCR assay for SARS CoV is potentially useful for early detection of SARS CoV. Our results suggest that genomic RNA is the predominant viral RNA species in clinical samples.

scholarly journals Implications in the quantitation of SARS-CoV2 copies in concurrent nasopharyngeal swabs, whole mouth fluid and respiratory droplets

2021 ◽  
Author(s):  
Priya Kannian ◽  
Bagavad Gita Jayaraman ◽  
Swarna Alamelu ◽  
Chandra Lavanya ◽  
Nagalingeswaran Kumarasamy ◽  
...  
Keyword(s):  
Disease Severity ◽  
Severe Disease ◽  
Cross Sectional Study ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Cross Sectional ◽  
Virus Shedding ◽  
Mild Disease ◽  
Copy Numbers ◽  
Respiratory Droplets

AbstractImportanceThe nasopharyngeal swab (NPS) is considered the ideal diagnostic specimen for Covid-19, while WMF is recently promoted due to collection simplicity and importance in disease transmission. There is limited knowledge on the relative viral load in these samples – NPS, whole mouth fluid (WMF) and respiratory droplets (RD; another important source in transmission), on how the loads vary with disease severity and on how much virus is shed.ObjectiveTo quantify and compare SARS-CoV2 copies in the NPS, WMF and RD samples, and correlate with disease severity.DesignCross sectional study.SettingTertiary care multi-speciality hospital with limited resources in a low-to-middle income country.ParticipantsEighty suspected COVID-19 patients were recruited from the COVID-19 out-patient clinic and hospital isolation wards.InterventionConcurrent NPS, WMF and RD samples were collected from all the recruited patients and tested for SARS-CoV2 copies by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR).Main outcomes and measuresThe main outcome was COVID-19 measured by SARS-CoV2 quantitative RT-PCR in NPS samples. COVID-19 disease severity was determined according to NIH criteria. Virus shedding was defined as the presence of SARS-CoV2 copies in the WMF and RD samples.ResultsSARS-CoV2 was detected in 55/80 (69%) of the NPS samples. Of these 55, WMF and RD samples were positive in 44 (80%) and 17 (31%), respectively. The concordance of WMF with NPS was 84% (p=0.02). SARS-CoV2 copy numbers were comparable in the NPS (median: 8.74×10^5) and WMF (median: 3.07×10^4), but lower in RD samples (median: 3.60×10^2). Patients with mild disease had higher copies in the NPS (median: 3.46×10^6), while patients with severe disease had higher copies in the WMF (median: 1.34×10^6) and RD samples (median: 4.29×10^4). The 25-75% interquartile range of NPS SARS-CoV2 copies was significantly higher in the WMF (p=0.0001) and RD (p=0.01) positive patients.Conclusion and relevanceSARS-CoV2 copies are highest in NPS samples. WMF is a reliable surrogate sample for diagnosis. High copy numbers in the NPS imply initial virological phase and higher risk of virus shedding via WMF and RD.Key pointsQuestionHow the numbers of SARS-CoV2 copies in nasopharyngeal swab (NPS) samples might reflectvirus shedding from the whole upper aerodigestive tract and indicatedisease severity?FindingsIn this cross-sectional study involving 80 suspected COVID-19 patients, the data indicate higher SARS-CoV2 copies in NPS samples of patients with mild disease,and in the whole mouth fluid (WMF) and respiratory droplet (RD) samples of patients with severe disease. Patients with higher SARS-CoV2 copies in the NPS shed the virus in the WMF and RD samples at statistically higher levels.MeaningHigh SARS-CoV2 copies in NPS samples imply initial virological phase withhigh levels of shedding through both WMF and RD.

scholarly journals Highly sensitive and specific detection of the SARS-CoV-2 Delta variant by double-mismatch allele-specific PCR

2021 ◽  
Author(s):  
Jeremy A Garson ◽  
Samuel Badru ◽  
Eleanor L Parker ◽  
Richard S Tedder ◽  
Myra O McClure
Keyword(s):  
Influenza A ◽  
Gene Mutations ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Screening Assay ◽  
Spike Gene ◽  
Specific Pcr ◽  
The World ◽  
Allele Specific ◽  
Syncytial Virus

The highly transmissible Delta variant of SARS-CoV-2 (B.1.617.2), first identified in India, is currently replacing pre-existing variants in Europe, the USA, and many other parts of the world. It is essential to monitor efficiently its spread to help guide public health policies. Genome sequencing is the gold standard for identification of Delta, but is time-consuming, expensive, and unavailable in many regions. We describe here a rapid and relatively inexpensive alternative to sequencing for specific identification of the Delta variant, by application of double-mismatch allele-specific RT-PCR (DMAS-RT-PCR). The technique exploits forward and reverse allele-specific primers, targeting two spike gene mutations, L452R and T478K, within the same amplicon. The discriminatory power of each primer is enhanced by the presence of an additional mismatch located at the fourth nucleotide from the 3' end. Amplicons are detected in real-time by means of a conventional fluorescently-labelled hydrolysis probe. Specificity was assessed by testing a range of well characterised cell culture-derived viral isolates and clinical samples, most of which had previously been fully sequenced. In all cases the results of viral genotyping by DMAS-RT-PCR were entirely concordant with the results of sequencing, and the assay was shown to discriminate reliably between the Delta variant and other variants of concern (Alpha, B.1.1.7 and Beta, B.1.351), and 'wild-type' SARS-CoV-2. Other respiratory viruses, including influenza A and respiratory syncytial virus, were non-reactive in the assay. The sensitivity of DMAS-RT-PCR matched that of the diagnostic SARS-CoV-2 RT-qPCR screening assay, which targets the E gene. Several samples that could not be sequenced due to insufficient virus could successfully be genotyped by DMAS-RT-PCR. The method we describe would be simple to establish in any laboratory that has the ability to conduct PCR assays and should greatly facilitate monitoring of the spread of the Delta variant throughout the world, and its proportional representation in any SARS-CoV-2-infected population.

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