scholarly journals Pooling of Nasopharyngeal Swab Specimens for SARS-CoV-2 detection by RT-PCR

2020 ◽  
Author(s):  
Ignacio Torres ◽  
Eliseo Albert ◽  
David Navarro
Keyword(s):  
Large Population ◽  
Rdrp Gene ◽  
Contact Tracing ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Case Identification ◽  
E Gene ◽  
Sample Pooling ◽  
Community Transmission ◽  
Pcr Targeting

ABSTRACTSystematic testing of large population groups by RT-PCR is mandatory to Covid-19 case identification and contact tracing in order to minimize the likelyhood of resurgence in contagion. Sample pooling for RT-PCR has been effectively used to detect community transmission of SARS CoV-2. Nevertheless, this procedure may decrease the sensitivity of RT-PCR assays due to specimen dilution. We evaluated the efficacy of this strategy for diagnosis of Covid-19 using a sensitive commercially-available RT-PCR targeting SARS CoV-2 E and RdRp genes in a single reaction. A total of 20 mini-pools containing either 5 (n=10) or 10 (n=10) nasopharyngeal exudates collected in universal transport medium were made, each of which including a unique positive NP specimen. Positive specimens yielding CT <32 for the E gene (6 out of 10) or <35.2 for the RdRP gene (7 out of 10) were detected in mini-pools of both sizes. In contrast, most NP samples displaying CTs > 35.8 for the E gene or 35.7 for the RdRP gene remained undetected in mini-pools of 5 specimens (3/4 and 2/3, respectively) or in mini-pools of 10 samples (4/4 and 3/3, respectively.

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 Assessment of Successful qRT-PCR of SARS-CoV-2 Assay in Pool Screening Using Isopropyl Alcohol Purification Step in RNA Extraction

2021 ◽  
Vol 2021 ◽  
pp. 1-6
Author(s):  
Mayank Gangwar ◽  
Alka Shukla ◽  
Virendra Kumar Patel ◽  
Pradyot Prakash ◽  
Gopal Nath
Keyword(s):  
Isopropyl Alcohol ◽  
Rna Extraction ◽  
Group Testing ◽  
Research Work ◽  
Rt Pcr ◽  
Purification Step ◽  
E Gene ◽  
Qrt Pcr ◽  
Pcr Targeting ◽  
Pooled Samples

The study is aimed at establishing the optimal parameters for RNA purification of pooled specimens, in SARS-CoV-2 assay. This research work evaluates the difference of extracted RNA purity of pooled samples with and without treatment with isopropyl alcohol and its effect on real-time RT-PCR. As per the protocol of the Indian Council of Medical Research (ICMR), 5 sample pools were analysed using qRT-PCR. A total of 100 pooled samples were selected for the study by mixing 50 μL of one COVID-19 positive nasopharyngeal/oropharyngeal (NP/OP) specimen and 50 μL each of 4 known negative specimens. Pool RNA was extracted using the column-based method, and 1 set of pooled extracted RNA was tested as such, while RNA of the second set was treated additionally with chilled isopropyl alcohol (modified protocol). Further, the purity of extracted RNA in both the groups was checked using Microvolume Spectrophotometers (Nanodrop) followed by RT-PCR targeting E-gene and RNaseP target. The results showed that the purity index of extracted RNA of untreated pooled specimens was inferior to isopropyl alcohol-treated templates, which was observed to be 85% sensitivity and 100% specificity. The average Cq (E gene) in the unpurified and purified pool RNA group was 34.66 and 31.48, respectively. The nanodrop data suggested that purified RNA concentration was significantly increased with an average value of 24.73 ± 1.49   ng / uL , which might be the reason for high sensitivity and specificity. Thus, this group testing of SARS-CoV-2 cases using pools of 5 individual samples would be the best alternative for saving molecular reagents, personnel time, and can increase the overall testing capacity. However, purity of RNA is one of the important determinants to procure unfailing results, thus, this additional purification step must be included in the protocol after RNA has been extracted using commercially available kit before performing qRT-PCR.

scholarly journals Prospective comparison of saliva and nasopharyngeal swab sampling for mass screening for COVID-19

2020 ◽  
Author(s):  
◽  
mathieu nacher ◽  
magalie demar
Keyword(s):  
Mass Screening ◽  
Saliva Sample ◽  
Field Testing ◽  
Contact Tracing ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Prospective Comparison ◽  
Asymptomatic Patients ◽  
Patient Groups ◽  
Trained Staff

Current testing for COVID-19 relies on quantitative reverse-transcriptase polymerase chain reaction from a nasopharyngeal swab specimen. Saliva samples have advantages regarding ease and painlessness of collection, which does not require trained staff and may allow self-sampling. We enrolled 776 persons at various field-testing sites and collected nasopharyngeal and pooled saliva samples. 162 had a positive COVID-19 RT-PCR, 61% were mildly symptomatic and 39% asymptomatic. The sensitivity of RT-PCR on saliva samples versus nasopharygeal swabs varied depending on the patient groups considered or on Ct thresholds. There were 10 (6.2%) patients with a positive saliva sample and a negative nasopharyngeal swab, all of whom had Ct values<25. For symptomatic patients for whom the interval between symptoms onset and sampling was <10 days sensitivity was 77% but when excluding persons with isolated Ngen positivity (54/162), sensitivity was 90%. In asymptomatic patients, the sensitivity was only 24%. When we looked at patients with Cts <30, sensitivity was 83% or 88.9% when considering 2 genes. The relatively good performance for patients with low Cts suggests that Saliva testing could be a useful and acceptable tool to identify infectious persons in mass screening contexts, a strategically important task for contact tracing and isolation in the community.

scholarly journals Clinical Performance of the Luminex NxTAG CoV Extended Panel for SARS-CoV-2 Detection in Nasopharyngeal Specimens from COVID-19 Patients in Hong Kong

2020 ◽  
Vol 58 (8) ◽  
Author(s):  
Jonathan Hon-Kwan Chen ◽  
Cyril Chik-Yan Yip ◽  
Jasper Fuk-Woo Chan ◽  
Rosana Wing-Shan Poon ◽  
Kelvin Kai-Wang To ◽  
...  
Keyword(s):  
Hong Kong ◽  
High Throughput ◽  
Clinical Performance ◽  
Diagnostic System ◽  
Molecular Diagnostic ◽  
N Gene ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Gene Target ◽  
E Gene

ABSTRACT In December 2019, the coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was first reported in the Hubei province of China and later spread all over the world. There was an urgent need of a high-throughput molecular test for screening the COVID-19 patients in the community. The Luminex NxTAG CoV extended panel is a high-throughput FDA emergency use-authorized molecular diagnostic assay for SARS-CoV-2 detection. This system targets three genes (ORF1ab, N, and E genes) of SARS-CoV-2, the ORF1ab region of SARS-CoV, and the ORF5 region of MERS-CoV. In this study, we evaluated the diagnostic performance of this system with nasopharyngeal swab specimens of 214 suspected COVID-19 patients in Hong Kong. The results were compared with our routine COVID-19 reverse transcription-PCR (RT-PCR) protocol with a LightMix SarbecoV E-gene kit and an in-house RdRp/Hel RT-PCR assay. The NxTAG CoV extended panel demonstrated 97.8% sensitivity and 100% specificity to SARS-CoV-2 in nasopharyngeal specimens. On low-viral load specimens, the sensitivity of the NxTAG panel could still maintain at 85.71%. Strong agreement was observed between the NxTAG panel and the routine COVID-19 RT-PCR protocol (kappa value = 0.98). Overall, the E gene target of the NxTAG panel demonstrated the highest sensitivity among the three SARS-CoV-2 targets, while the N gene targets demonstrated the least. In conclusion, the NxTAG CoV extended panel is simple to use, and it has high diagnostic sensitivity and specificity to SARS-CoV-2 in nasopharyngeal specimens. We recommend this diagnostic system for high-throughput COVID-19 screening in the community.

scholarly journals Evaluation of Commercial qPCR Kits for Detection of SARS-CoV-2 in Pooled Samples

Diagnostics ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 472 ◽  
Author(s):  
Vlad Petrovan ◽  
Virgil Vrajmasu ◽  
Ana Cristina Bucur ◽  
Dan Sebastian Soare ◽  
Eugen Radu ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rdrp Gene ◽  
Rna Dependent Rna Polymerase ◽  
Easy Method ◽  
E Gene ◽  
Suitable Target ◽  
Sample Pooling ◽  
Preliminary Study ◽  
Commercial Kits ◽  
Pooled Samples

Due to the current pandemic, a global shortage of reagents has drawn interest in developing alternatives to increase the number of coronavirus tests. One such alternative is sample pooling. We compared commercial kits that are used in COVID-19 diagnostics in terms of their sensitivity and feasibility for use in pooling. In this preliminary study, we showed that pooling of up to 80 samples did not affect the efficacy of the kits. Additionally, the RNA-dependent RNA polymerase (RdRp) gene is a more suitable target in pooled samples than the envelope (E) gene. This approach could provide an easy method of screening a large number of samples and help adjust different governmental regulations.

CORRELATION OF PROGNOSIS OF COVID POSITIVE PATIENTS BETWEEN REAL TIME RT-PCR AND CT-VALUES OF E. GENE, N.GENE, RDRP GENE

2021 ◽  
pp. 1-3
Author(s):  
Sreyoshi Som ◽  
Indraneel Dasgupta
Keyword(s):  
Real Time ◽  
Virus Disease ◽  
The Other ◽  
Rdrp Gene ◽  
N Gene ◽  
Absolute Amount ◽  
Target Sequence ◽  
Rt Pcr ◽  
E Gene ◽  
Corona Virus

INTRODUCTION: COVID-19 – Corona virus disease-19 – A respiratory illness caused by severe acute respiratory syndrome corona virus 2 (SARS-CoV-2), colloquially referred to as coronavirus. Real-time PCR is conducted to quantify the absolute amount of target sequence or to compare relative amount of a target sequence among samples. This technique monitors amplication of the target in real time via a target specic uorescent signal emitted during amplication. During most real times PCR, a considerable amount of background uorescence occurs. Inspite of this fact PCR uorescent dyes and probes should be sequence specic. This CT value or cycle threshold value in a real time RT- PCR is dened as the number of cycles required for the uorescent signal to cross the threshold i.e., exceeds background level). This is inversely proportional to the amount of target nuclei in the sample. i.e., lower the CTlevel the greater the amount of target nuclei in the sample. AIM: To explore the association of the mutation pattern of covid-19 genetic variation with the severity and fatality of patients with covid-19 viral illness. Factors considered are Age, Sex, and Duration of hospital stay and outcome of patient. MATERIALS AND METHODS: The study was a comparative, correlation, retrospective, lab values based study. All covid+ve patients tested at Peerless hospital, Kolkata by RT-PCR on admission. It is imperative to choose reference genes whose expression levels are not expected to change during our experiment. Common housekeeping genes include actin, alpha-tubuliun, gapdh and ubiquitin. it is wise to use at least two reference gene for one study may not be suitable for another. Total 370 participants were present in this study. RESULT: In our study, 114(30.8%) COVID positive patients were in E GENE GR 11-20, 147(39.7%) COVID positive patients were in E GENE GR 21-30 and 109(29.5%) COVID positive patients were in E GENE GR 31-40. 163(44.1%) COVID positive patients were in N GENE GR 11-20, 132(35.7%) COVID positive patients were in N GENE GR 21-30 and 75(20.3%) COVID positive patients were in N GENE GR 31-40. 228(61.6%) COVID positive patients were in RdRp GENE GR 11-20, 112(30.3%) COVID positive patients were in RdRp GENE GR 21-30 and 30(8.1%) COVID positive patients were in RdRp GENE GR 31-40. We found that, 30(8.1%) COVID positive patients died and 340 (91.9%) patients have survived. CONCLUSION: The patients who died had signicantly lower RT-PCR CT-values OF E. GENE, N.GENE, RdRp GENE than patients who survived. We concluded that the time of onset of symptoms were earlier for patients with lower CTvalue, than the other group who survived. So we concluded after correlating CT-values of real time RT-PCR and E. GENE, N.GENE, RdRp GENE, that the patients with lower CTvalues had more severity and had poor prognosis than the other group with higher RT-PCR CT-values of E.GENE, N, GENE, RdRp GENE

scholarly journals Clinical evaluation of the Roche/SD Biosensor rapid antigen test with symptomatic, non-hospitalized patients in a municipal health service drive-through testing site

2020 ◽  
Author(s):  
Zsὁfia Iglὁi ◽  
Jans Velzing ◽  
Janko van Beek ◽  
David van de Vijver ◽  
Georgina Aron ◽  
...  
Keyword(s):  
Viral Load ◽  
Rapid Test ◽  
Contact Tracing ◽  
Gene Copy ◽  
Nasopharyngeal Swab ◽  
List Type ◽  
Antigen Test ◽  
Rt Pcr ◽  
Outbreak Management ◽  
Testing Site

AbstractBackgroundRapid detection of infectious individuals is essential in stopping the further spread of SARS-CoV-2. Although rapid antigen test is not as sensitive as the gold standard RT-PCR, the time to result is decreased by day(s), strengthening the effectiveness of contact tracing.MethodsThe Roche/SD Biosensor lateral flow antigen rapid test was evaluated in a mild symptomatic population at a large drive through testing site. A second nasopharyngeal swab was directly tested with the rapid test on site and results were compared to RT-PCR and virus culture. Date of onset and symptoms were analysed using data from a clinical questionnaire.ResultsWe included 970 persons with complete data. Overall sensitivity and specificity were 84.9% (CI95% 79.1-89.4) and 99.5% (CI95% 98.7-99.8) which translated into a positive predictive value of 97.5% (CI95% 94.0-99.5) under the current regional PCR positivity of 19.2%. Sensitivity for people with high loads of viral RNA (ct <30, 2.17E+05 E gene copy/ml) and who presented within 7 days since symptom onset increased to 95.8% (CI95% 90.5-98.2). Band intensity and time to result correlated strongly with viral load thus strong positive bands could be read before the recommended time. Around 98% of all viable specimen with ct <30 were detected successfully indicating that the large majority of infectious people can be captured with this test.ConclusionAntigen rapid tests can detect mildly symptomatic cases in the early phase of disease thereby identifying the most infectious individuals. Using this assay can have a significant value in the speed and effectiveness of SARS-CoV-2 outbreak management.SummaryPeople with early onset and high viral load were detected with 98.2% sensitivity.97% of individuals in which virus could be cultured were detected by the rapid test.This test is suitable to detect mild symptomatic cases.

scholarly journals COVID-19 infection in the palatine tonsil tissue and detritus: the detection of the virus compartment with RT-PCR

2021 ◽  
Vol 14 (2) ◽  
pp. e239108
Author(s):  
Hamsu Kadriyan ◽  
Bayu Tirta Dirja ◽  
Dewi Suryani ◽  
Didit Yudhanto
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Palatine Tonsil ◽  
Rdrp Gene ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Recurrent Tonsillitis ◽  
Igg Antibody ◽  
Tonsil Tissue

Two patients suffering from chronic recurrent tonsillitis were reported. The first patient was confirmed infected with COVID-19, 3 weeks prior to tonsillectomy. The detritus and tonsil specimen were further analysed through real-time PCR (RT-PCR) and revealed amplification of the fragment N and ORF1ab genes of SARS-CoV-2. The second patient had a negative IgM and positive IgG antibody for COVID-19; however, the nasopharyngeal swab indicated negative for SARS-CoV-2. Tonsillectomy was performed 2 weeks after the swab; the tonsil specimen was analysed through RT-PCR and revealed amplification of the N2 and RdRp gene of SARS-CoV-2. According to both results, the presence of the SARS-CoV-2 gene remains to be detected in tonsil and/or detritus after 2–3 weeks after recovery. Hence, it is suggested that it is necessary to use adequate protection when performing tonsillectomy on early recovered patients with COVID-19. Furthermore, tonsillectomy would be more advisable to be performed after the fourth week after recovery from COVID-19.

scholarly journals Pooling Nasopharyngeal Swab Specimens to Improve Testing Capacity for SARS-CoV-2 by Real-Time RT-PCR

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Imene Handous ◽  
Naila Hannachi ◽  
Manel Marzouk ◽  
Olfa Hazgui ◽  
Nissaf Bouafif Ep Ben Alaya ◽  
...  
Keyword(s):  
High Viral Load ◽  
Nasopharyngeal Swab ◽  
Individual Sample ◽  
Rt Pcr ◽  
False Negatives ◽  
Pooled Testing ◽  
Sample Pooling ◽  
Sample Testing ◽  
The Mean ◽  
Low Prevalence

Abstract Background The detection of SARS-CoV-2 using qRT-PCR with the pooling of samples can reduce workload and costs especially when the prevalence rate of COVID-19 in a population is low. To analyse the effect of pooling samples on the sensitivity of RT-qPCR for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection, we compared the cycle threshold (Ct) values of pools of 5 and 10 that tested positive with Ct values of individual samples that tested positive in that pool. Twenty positive nasopharyngeal (NP) specimens with low to high viral load were selected and pooled individually with four and nine negative NP. Results In NP specimens, the sensitivity of pools of 5 and 10 were 90 and 85%, compared to individual sample testing, respectively. The RT-qPCR sensitivity of pools of 5 and 10 against individual testing were not significantly different (p > 0.05). Detection of positive samples with low Ct values (< 36) was consistently achieved in pools of 5 and 10. However, there were higher false negatives when samples with high ct values (> 36) were pooled and tested. The mean Ct values obtained with the 5-sample pooled testing exceeded individual sample testing by 1.85 ± 1.09 cycles, while Ct values obtained with the 10-sample pooling exceeded individual sample testing by 3.4 ± 1.65 cycles. Conclusions In a low prevalence setting, testing capacity can be increased by pooling 5 or 10 samples, but the risk of additional false negatives needs to be considered

scholarly journals Prospective Comparison of Saliva and Nasopharyngeal Swab Sampling for Mass Screening for COVID-19

2021 ◽  
Vol 8 ◽  
Author(s):  
Mathieu Nacher ◽  
Mayka Mergeay-Fabre ◽  
Denis Blanchet ◽  
Orelie Benoit ◽  
Tristan Pozl ◽  
...  
Keyword(s):  
Mass Screening ◽  
Saliva Sample ◽  
Field Testing ◽  
Contact Tracing ◽  
N Gene ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Prospective Comparison ◽  
Asymptomatic Patients ◽  
Patient Groups

Current testing for COVID-19 relies on reverse-transcriptase polymerase chain reaction from a nasopharyngeal swab specimen. Saliva samples have advantages regarding ease and painlessness of collection, which does not require trained staff and may allow self-sampling. We enrolled 776 persons at various field-testing sites and collected nasopharyngeal and pooled saliva samples. One hundred sixty two had a positive COVID-19 RT-PCR, 61% were mildly symptomatic and 39% asymptomatic. The sensitivity of RT-PCR on saliva samples vs. nasopharygeal swabs varied depending on the patient groups considered or on Ct thresholds. There were 10 (6.2%) patients with a positive saliva sample and a negative nasopharyngeal swab, all of whom had Ct values &lt;25 for three genes. For symptomatic patients for whom the interval between symptoms onset and sampling was &lt;10 days sensitivity was 77% but when excluding persons with isolated N gene positivity (54/162), sensitivity was 90%. In asymptomatic patients, the sensitivity was only 24%. When we looked at patients with Cts &lt;30, sensitivity was 83 or 88.9% when considering two genes. The relatively good performance for patients with low Cts suggests that Saliva testing could be a useful and acceptable tool to identify infectious persons in mass screening contexts, a strategically important task for contact tracing and isolation in the community.

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