scholarly journals Effect of Heat Inactivation for the Detection of Severe Acute Respiratory Syndrome-Corona Virus-2 (SARS-CoV-2) with Reverse Transcription Real Time Polymerase Chain Reaction (rRT-PCR): Evidence from Ethiopian Study

2021 ◽  
Author(s):  
Belete Woldesemayat Hailemariam ◽  
Gebremedihin Gebremicael ◽  
Kidist Zealias ◽  
Amelework Yilma ◽  
Sisay Adane ◽  
...  
Keyword(s):  
Pearson Correlation ◽  
False Negative ◽  
Rna Extraction ◽  
Pcr Detection ◽  
N Gene ◽  
Heat Inactivation ◽  
P Value ◽  
Rt Pcr ◽  
Specimen Handling ◽  
Ct Value

Abstract Background: Coronavirus disease 2019 (COVID-19) specimen handling needs a major concern due to the virus has a potential of easily transmittable to health care workers and laboratory personnel. Heat inactivation before nucleic acid isolation might permit safe testing, even though, the effect of heat inactivation on SARS-CoV-2 RT-PCR detection results needs to be determined. Methods: An experimental study was conducted in Ethiopian Public Health Institute (EPHI) from September 25 to October 15, 2020. A total of 188 Oro-pharyngeal swabs were collected from COVID-19 suspected cases, referred to EPHI for SARS COV-2 testing during the study period. One group of the sample was inactivated at 56 °C heat for 30 min, and the other group was stored at 4°C for a similar period of time. RNA extraction and detection were done by DAAN Gene extraction and detection kit. Abbott m2000 RT-PCR was used for amplification and detection. Data analysis was done by using SPSS version 23.0; Chi-square and Pearson correlation test for qualitative and semi-quantitative analysis were used. P-value < 0.05 was considered as statistically significant.Results: Out of 188 total samples, 117 (62.2 %) and 118 (62.8%) were positive for ORF1a/b and N gene respectively before inactivation. Whereas after inactivation, 111 (59 %) was ORF1a/b and 116 (61.7 %) was N gene positive. Rate of positivity between groups was not statistically significant (p>0.05). The mean CT value difference between the two groups of ORF1a/b gene and N gene was 0.042 (95 % CI, -0.247- 0.331; t= 0.28; p = 0.774) and 0.38 (95% CI, 0.097 - 0.682; t =2.638; p = 0.010) respectively.Conclusion: Heat inactivation at 56 ℃ for 30 min has not statistically significant effect for the qualitative rRT-PCR detection of SARS-CoV-2. However, the finding also showed that there was statistically significant CT value increment after heat inactivation compared to untreated samples. Therefore, false-negative results with high CT value results (CT > 35) were found to be the challenge of this protocol. Hence alternative inactivation methods should be investigated and further studies should be considered.

scholarly journals Overhauling a faulty control in the CDC-recommended SARS-CoV-2 RT-PCR test panel

2020 ◽  
Author(s):  
Rob J. Dekker ◽  
Wim A. Ensink ◽  
Selina van Leeuwen ◽  
Han Rauwerda ◽  
Timo M. Breit
Keyword(s):  
False Positive ◽  
False Negative ◽  
Rna Extraction ◽  
N Gene ◽  
Rt Pcr ◽  
P Gene ◽  
Reverse Primer ◽  
Probe Set ◽  
Pcr Test ◽  
Test Control

ABSTRACTTo battle the COVID-19 pandemic, widespread testing for the presence of the SARS-CoV-2 virus is worldwide being employed by specific real-time RT-PCR (rRT-PCR) of viral RNA. The CDC has issued a recommended panel of PCR-based test sets that entail several primer/probe sets that target the SARS-CoV-2 N-gene, but also one that targets the human RNase P gene (h-RP) as a positive control for RNA extraction and/or reverse-transcription (RT) efficacy.We discovered that the CDC-recommended h-RP primer/probe set has a faulty design, because both PCR primers are located in the same exon, which allows for unwanted PCR-amplification of background genomic DNA (gDNA). By removing RNA from nose-swab samples by an RNase treatment, we showed that the presence of gDNA in samples resulted in false-positive signals for the h-RP test control. This is rather serious, because it could lead to false-negative test outcomes, since the CDC interpretation of an absent SARS-CoV-2 rRT-PCR signal plus a positive h-RP rRT-PCR signal is interpreted as “2019-nCoV not detected”, whereas a false-positive h-RP rRT-PCR signal resulting from amplification of gDNA should be interpreted as “Invalid Result” and the procedure should be repeated.In order to overhaul the faulty h-RP rRT-PCR primer/probe set with minimal modification, we designed and tested several new h-RP reverse primers. Replacement of the CDC-recommended PCR reverse primer with our selected exon-exon junction reverse primer corrected the problem of false-positive results with this important SARS-CoV-2 RT-PCR test control and thus eliminated the problem of potential false-negative COVID-19 diagnoses.

scholarly journals Viral Dynamics and Real-Time RT-PCR Ct Values Correlation with Disease Severity in COVID-19

Diagnostics ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1091
Author(s):  
Ali A. Rabaan ◽  
Raghavendra Tirupathi ◽  
Anupam A Sule ◽  
Jehad Aldali ◽  
Abbas Al Mutair ◽  
...  
Keyword(s):  
Viral Load ◽  
Real Time ◽  
Disease Severity ◽  
False Negative ◽  
Influential Factors ◽  
Confirmatory Test ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Viral Kinetics ◽  
Ct Value

Real-time RT-PCR is considered the gold standard confirmatory test for coronavirus disease 2019 (COVID-19). However, many scientists disagree, and it is essential to understand that several factors and variables can cause a false-negative test. In this context, cycle threshold (Ct) values are being utilized to diagnose or predict SARS-CoV-2 infection. This practice has a significant clinical utility as Ct values can be correlated with the viral load. In addition, Ct values have a strong correlation with multiple haematological and biochemical markers. However, it is essential to consider that Ct values might be affected by pre-analytic, analytic, and post-analytical variables such as collection technique, specimen type, sampling time, viral kinetics, transport and storage conditions, nucleic acid extraction, viral RNA load, primer designing, real-time PCR efficiency, and Ct value determination method. Therefore, understanding the interpretation of Ct values and other influential factors could play a crucial role in interpreting viral load and disease severity. In several clinical studies consisting of small or large sample sizes, several discrepancies exist regarding a significant positive correlation between the Ct value and disease severity in COVID-19. In this context, a revised review of the literature has been conducted to fill the knowledge gaps regarding the correlations between Ct values and severity/fatality rates of patients with COVID-19. Various databases such as PubMed, Science Direct, Medline, Scopus, and Google Scholar were searched up to April 2021 by using keywords including “RT-PCR or viral load”, “SARS-CoV-2 and RT-PCR”, “Ct value and viral load”, “Ct value or COVID-19”. Research articles were extracted and selected independently by the authors and included in the present review based on their relevance to the study. The current narrative review explores the correlation of Ct values with mortality, disease progression, severity, and infectivity. We also discuss the factors that can affect these values, such as collection technique, type of swab, sampling method, etc.

scholarly journals Performance of Unobserved Self-Collected Nasal Swabs for Detection of SARS-CoV-2 by RT-PCR Utilizing a Remote Specimen Collection Strategy

2021 ◽  
Author(s):  
Ron M Kagan ◽  
Amy A Rogers ◽  
Gwynngelle A Borillo ◽  
Nigel J Clarke ◽  
Elizabeth M Marlowe
Keyword(s):  
Return To Work ◽  
Screening Program ◽  
False Negative ◽  
N Gene ◽  
Rt Pcr ◽  
Pooled Testing ◽  
Specimen Collection ◽  
Asymptomatic Patients ◽  
Nasal Swabs ◽  
The Impact

Abstract Background The use of a remote specimen collection strategy employing a kit designed for unobserved self-collection for SARS-CoV-2 RT-PCR can decrease the use of PPE and exposure risk. To assess the impact of unobserved specimen self-collection on test performance, we examined results from a SARS-CoV-2 qualitative RT-PCR test for self-collected specimens from participants in a return-to-work screening program and assessed the impact of a pooled testing strategy in this cohort. Methods Self-collected anterior nasal swabs from employee return to work programs were tested using the Quest Diagnostics SARS-CoV-2 RT-PCR EUA. The Ct values for the N1 and N3 N-gene targets and a human RNase P (RP) gene control target were tabulated. For comparison, we utilized Ct values from a cohort of HCP-collected specimens from patients with and without COVID-19 symptoms. Results Among 47,923 participants, 1.8% were positive. RP failed to amplify for 13/115,435 (0.011%) specimens. The median (IQR) Cts were 32.7 (25.0-35.7) for N1 and 31.3 (23.8-34.2) for N3. Median Ct values in the self-collected cohort were significantly higher than those of symptomatic, but not asymptomatic patients. Based on Ct values, pooled testing with 4 specimens would have yielded inconclusive results in 67/1,268 (5.2%) specimens but only a single false-negative result. Conclusions Unobserved self-collection of nasal swabs provides adequate sampling for SARS-CoV-2 RT-PCR testing. These findings alleviate concerns of increased false negatives in this context. Specimen pooling could be used for this population as the likelihood of false negative results is very low due when using a sensitive, dual-target methodology.

scholarly journals Prospective Study Comparing Deep Throat Saliva With Other Respiratory Tract Specimens in the Diagnosis of Novel Coronavirus Disease 2019

2020 ◽  
Vol 222 (10) ◽  
pp. 1612-1619 ◽  
Author(s):  
Christopher K C Lai ◽  
Zigui Chen ◽  
Grace Lui ◽  
Lowell Ling ◽  
Timothy Li ◽  
...  
Keyword(s):  
Prospective Study ◽  
Pearson Correlation ◽  
False Negative ◽  
False Negative Rate ◽  
Productive Cough ◽  
Rt Pcr ◽  
Virus Shedding ◽  
Positive Rate ◽  
Novel Coronavirus ◽  
Rna Concentration

Abstract Background Self-collected specimens have been advocated to avoid infectious exposure to healthcare workers. Self-induced sputum in those with a productive cough and saliva in those without a productive cough have been proposed, but sensitivity remains uncertain. Methods We performed a prospective study in 2 regional hospitals in Hong Kong. Results We prospectively examined 563 serial samples collected during the virus shedding periods of 50 patients: 150 deep throat saliva (DTS), 309 pooled-nasopharyngeal (NP) and throat swabs, and 104 sputum. Deep throat saliva had the lowest overall reverse-transcriptase polymerase chain reaction (RT-PCR)-positive rate (68.7% vs 89.4% [sputum] and 80.9% [pooled NP and throat swabs]) and the lowest viral ribonucleic acid (RNA) concentration (mean log copy/mL 3.54 vs 5.03 [sputum] and 4.63 [pooled NP and throat swabs]). Analyses with respect to time from symptom onset and severity also revealed similar results. Virus yields of DTS correlated with that of sputum (Pearson correlation index 0.76; 95% confidence interval, 0.62–0.86). We estimated that the overall false-negative rate of DTS could be as high as 31.3% and increased 2.7 times among patients without sputum. Conclusions Deep throat saliva produced the lowest viral RNA concentration and RT-PCR-positive rate compared with conventional respiratory specimens in all phases of illness. Self-collected sputum should be the choice for patients with sputum.

scholarly journals Association of COVID-19 RT-qPCR test false-negative rate with patient age, sex and time since diagnosis

2020 ◽  
Author(s):  
Matan Levine-Tiefenbrun ◽  
Idan Yelin ◽  
Hedva Uriel ◽  
Jacob Kuint ◽  
Licita Schreiber ◽  
...  
Keyword(s):  
False Negative ◽  
False Negative Rate ◽  
Disease Spread ◽  
Positive Test ◽  
N Gene ◽  
P Value ◽  
True Positive ◽  
Younger Patients ◽  
Viral Loads ◽  
Negative Rate

AbstractBackgroundRoutine testing for SARS-CoV-2 in the community is essential for guiding key epidemiological decisions from the quarantine of individual patients to enrolling regional and national preventive measures. Yet, the primary testing tool, the RT-qPCR based testing, is notoriously known for its low sensitivity, i.e. high risk of missed detection of carriers. Quantifying the false-negative rate (FNR) of the RT-qPCR test at the community settings and its dependence on patient demographic and disease progression is therefore key in designing and refining strategies for disease spread prevention.MethodsAnalyzing 843,917 test results of 521,696 patients, we identified false-negative (FN) and true-positive (TP) results as negative and positive results preceded by a COVID-19 diagnosis and followed by a later positive test. Regression analyses were used to determine associations of false-negative results with time of sampling after diagnosis, patient demographics and viral loads based on RT-qPCR Ct values of the next positive tests.FindingsThe overall FNR was 22.8%, which is consistent with previous studies. Yet, this rate was much lower at the first 5 days following diagnosis (10.7%) and only increased in later dates. Furthermore, the FNR was strongly associated with demographics, with odds ratio of 1.74 (95% CI: 1.58-1.90) for women over men and 1.36 (95% CI: 1.34-1.39) for 10 years younger patients. Finally, FNR was associated with viral loads (p-value 0.0005), with a difference of 1.50 (95% CI: 0.70-2.30) between the average Ct of the N gene in a positive test following a false-negative compared to a positive test following a true-positive.InterpretationOur results show that in the first few days following diagnosis, when results are critical for quarantine decisions, RT-qPCR testing is more reliable than previously reported. Yet the reliability of the test result is reduced in later days as well as for women and younger patients, where the viral loads are typically lower.FundingThis research was supported by the ISRAEL SCIENCE FOUNDATION (grant No. 3633/19) within the KillCorona – Curbing Coronavirus Research Program.

scholarly journals Real-time RT-PCR detection of Citrus bark cracking viroid (CBCVd) in hops including an mRNA-based internal positive control

2020 ◽  
Vol 127 (6) ◽  
pp. 763-767
Author(s):  
Luitgardis Seigner ◽  
Marion Liebrecht ◽  
Linda Keckel ◽  
Katharina Einberger ◽  
Carolin Absmeier
Keyword(s):  
Real Time ◽  
Detection Method ◽  
Plant Protection ◽  
False Negative ◽  
Positive Control ◽  
Pcr Detection ◽  
Economic Losses ◽  
Rt Pcr ◽  
Validation Data ◽  
Internal Positive Control

Abstract Citrus bark cracking viroid (CBCVd), formerly known as pathogen in the genus Citrus and first detected in Slovenian hops in 2014, threatens hop production as it leads to important economic losses. Reduction in yield and quality and even death of the infected plants within a few years are typical observations due to CBCVd infections of hops. The viroid is easily transmitted and spreads rapidly. As it cannot be controlled by plant protection measures, avoiding its introduction into hop gardens and eradicating first centres of infection are of utmost importance. An indispensable prerequisite is a reliable detection method suitable for large-scale routine testing. In this study, the development of primers and probe for real-time RT-PCR for sensitive CBCVd detection is described. To exclude “false negative” results, a nad5 mRNA-based internal positive control was included. To our knowledge, this is the first time such a duplex real-time RT-PCR detection method for CBCVd at least in hops is described. In addition, first method validation data are presented.

scholarly journals Efficient SARS-CoV-2 detection in unextracted oro-nasopharyngeal specimens by rRT-PCR with the Seegene Allplex™ 2019-nCoV assay

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Wesley Freppel ◽  
Natacha Merindol ◽  
Fabien Rallu ◽  
Marco Bergevin
Keyword(s):  
Detection Rate ◽  
High Efficiency ◽  
False Negative ◽  
Rna Extraction ◽  
Free Water ◽  
Cost Savings ◽  
Global Scale ◽  
Proteinase K ◽  
N Gene ◽  
Infected People

Abstract Background The fight against the COVID-19 pandemic has created an urgent need to rapidly detect infected people. The challenge for clinical laboratories has been finding a high throughput, cost-efficient, and accurate testing method in the context of extraction reagents shortage on a global scale. To answer this need, we studied SARS-CoV-2 detection in oro-nasopharyngeal (ONP) swabs stored in Universal Transport Media (UTM) or in RNase-free water by rRT-PCR with Seegene Allplex™ 2019-nCoV assay without RNA extraction. Results Optimal results were obtained when swabs stored in UTM were diluted 1/5 and 1/2 in RNase-free water. Thermal lysis before rRT-PCR testing slightly improved detection rate. In addition, proteinase K (PK) treatment allowed for a significant reduction of invalid results and increased sensitivity for detection of low viral load specimens. In a panel of positive samples with all 3 viral genes amplified and N gene Cycle threshold values (Ct values) from 15 to 40, our detection rate was 98.9% with PK and 94.4% without. In a challenging panel of low positive samples with only the N gene being detectable at Ct values > 30, detection rate was increased from 53.3 to 76.7% with the addition of PK, and invalid rate fell off from 18.3 to 0%. Furthermore, we demonstrated that our method reliably detects specimens with Ct values up to 35, whereas false negative samples become frequent above this range. Finally, we show that swabs should be stored at − 70 °C rather than 4 °C when testing cannot be performed within 72 h of collection. Conclusion We successfully optimized the unextracted rRT-PCR process using the Seegene Allplex™ 2019-nCoV assay to detect SARS-CoV-2 RNAs in nasopharyngeal swabs. This improved method offers cost savings and turnaround time advantages compared to automated extraction, with high efficiency of detection that could play an important role in the surveillance of Covid-19.

scholarly journals Influence of Different Inactivation Methods on Severe Acute Respiratory Syndrome Coronavirus 2 RNA Copy Number

2020 ◽  
Vol 58 (8) ◽  
Author(s):  
Hailong Chen ◽  
Rui Wu ◽  
Yuan Xing ◽  
Quanli Du ◽  
Zerun Xue ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Copy Number ◽  
False Negative ◽  
Viral Rna ◽  
Medical Laboratory ◽  
High Temperature Treatment ◽  
N Gene ◽  
Clinical Samples ◽  
Heat Inactivation ◽  
Nasopharyngeal Swab

ABSTRACT The outbreak of coronavirus disease 2019 (COVID-19) has spread across the world and was characterized as a pandemic. To protect medical laboratory personnel from infection, most laboratories inactivate the virus causing COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in clinical samples before testing. However, the effect of inactivation on the detection results remains unknown. Here, we used a digital PCR assay to determine the absolute SARS-CoV-2 RNA copy number in 63 nasopharyngeal swab samples and assess the effect of inactivation methods on viral RNA copy number. Viral inactivation was performed by three different methods: (i) incubation with the TRIzol LS reagent for 10 min at room temperature, (ii) heating in a water bath at 56°C for 30 min, and (iii) high-temperature treatment, including autoclaving at 121°C for 20 min, boiling at 100°C for 20 min, and heating at 80°C for 20 min. Compared to the amount of RNA in the original sample, TRIzol treatment destroyed 47.54% of the nucleocapsid protein (N) gene and 39.85% of open reading frame (ORF) 1ab. For samples treated at 56°C for 30 min, the copy number of the N gene and ORF 1ab was reduced by 48.55% and 56.40%, respectively. The viral RNA copy number dropped by 50 to 66% after heating at 80°C for 20 min. Nearly no viral RNA was detected after autoclaving at 121°C or boiling at 100°C for 20 min. These results indicate that inactivation reduced the quantity of detectable viral RNA and may cause false-negative results, especially in weakly positive cases. Thus, use of the TRIzol reagent rather than heat inactivation is recommended for sample inactivation, as the TRIzol reagent had the least effect on the RNA copy number among the tested methods.

scholarly journals Asymptomatic screening of SARS-CoV-2 (COVID-19) virus RNA using reverse transcriptase loop-mediated isothermal amplification (RT-LAMP)

2021 ◽  
Author(s):  
Rebecca Allsopp ◽  
Caroline Cowley ◽  
Ruth Barber ◽  
Carolyn Jones ◽  
Christopher Holmes ◽  
...  
Keyword(s):  
Diagnostic Performance ◽  
Screening Program ◽  
False Negative ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Molecular Diagnostic ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Clinical Patient ◽  
The University

Abstract This study demonstrates the diagnostic performance of SARS-CoV-2 RT-LAMP assays, comparing the performance of genomic versus sub-genomic sequence target with subsequent application in an asymptomatic screening population. An RT-LAMP workflow was developed using synthetic positive control RNA and the diagnostic sensitivity and specificity was then determined using clinical patient samples processed through the diagnostic RT-PCR service within the University Hospitals of Leicester NHS Trust. 92 RT-PCR clinically positive and 88 RT-PCR clinically negative swab samples along with 78 clinically positive and 63 clinically negative saliva samples were equally detected at 100% DSe and 100% DSp for all samples reporting a Ct < 20. DSe for all samples reporting a Ct < 30 reduced slightly to around 95% (100% DSp) for both the single genomic (large open reading frame; orf1a) and dual sub-genomic (nucleocapsid plus envelope) targeting RT-LAMP assays. Lastly, the diagnostic performance of a saliva direct workflow was only about 50% that of the saliva RNA extraction workflow. Subsequently, a swab based RNA -RT-LAMP assay was implemented to ISO 15189:2012 standards supporting an advisory COVID-19 screening program for staff and students at the University of Leicester between October and December 2020. Within a 24-hour period, total nucleic acid extraction was followed by genomic target RT-LAMP plus an internal total RNA control to mitigate the possibility of false negative reporting. SARS-CoV-2 RT-LAMP positive samples were confirmed by an RT-PCR test in an NHS diagnostic laboratory and results were included within national statistics. Nine confirmed positive samples were detected in 1680 symptom free individuals (equivalent to 540 cases per 100,000) thus demonstrating the utility of RT-LAMP molecular diagnostic tool for the detection of SARS-CoV-2 in an asymptomatic population.

Sign in / Sign up

Export Citation Format

Share Document