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 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 A Novel Multiplex qRT-PCR Assay to Detect SARS-CoV-2 Infection: High Sensitivity and Increased Testing Capacity

2020 ◽  
Vol 8 (7) ◽  
pp. 1064 ◽  
Author(s):  
Sara Petrillo ◽  
Giovanna Carrà ◽  
Paolo Bottino ◽  
Elisa Zanotto ◽  
Maria Chiara De Santis ◽  
...  
Keyword(s):  
Internal Control ◽  
False Negative ◽  
Rna Extraction ◽  
Simultaneous Detection ◽  
High Sensitivity ◽  
N Gene ◽  
Qrt Pcr ◽  
P Gene ◽  
Global Pandemic ◽  
Pcr Method

Rapid and sensitive screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to limit the spread of the global pandemic we are facing. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is currently used for the clinical diagnosis of SARS-CoV-2 infection using nasopharyngeal swabs, tracheal aspirates, or bronchoalveolar lavage (BAL) samples. Despite the high sensitivity of the qRT-PCR method, false negative outcomes might occur, especially in patients with a low viral load. Here, we developed a multiplex qRT-PCR methodology for the simultaneous detection of SARS-CoV-2 genome (N gene) and of the human RNAse P gene as internal control. We found that multiplex qRT-PCR was effective in detecting SARS-Cov-2 infection in human specimens with 100% sensitivity. Notably, patients with few copies of SARS-CoV-2 RNA (<5 copies/reaction) were successfully detected by the novel multiplex qRT-PCR method. Finally, we assessed the efficacy of multiplex qRT-PCR on human nasopharyngeal swabs without RNA extraction. Collectively, our results provide evidence of a novel and reliable tool for SARS-CoV-2 RNA detection in human specimens, which allows the testing capacity to be expanded and the RNA extraction step to be bypassed.

scholarly journals Superiority of MALDI-TOF Mass Spectrometry over Real-Time PCR for SARS-CoV-2 RNA Detection

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 730
Author(s):  
Magda Rybicka ◽  
Ewa Miłosz ◽  
Krzysztof Piotr Bielawski
Keyword(s):  
Mass Spectrometry ◽  
Gold Standard ◽  
Viral Genome ◽  
False Negative ◽  
Rt Pcr ◽  
Rna Detection ◽  
Maldi Tof ◽  
Negative Results ◽  
False Negative Results ◽  
Pcr Test

At present, the RT-PCR test remains the gold standard for early diagnosis of SARS-CoV-2. Nevertheless, there is growing evidence demonstrating that this technique may generate false-negative results. Here, we aimed to compare the new mass spectrometry-based assay MassARRAY® SARS-CoV-2 Panel with the RT-PCR diagnostic test approved for clinical use. The study group consisted of 168 suspected patients with symptoms of a respiratory infection. After simultaneous analysis by RT-PCR and mass spectrometry methods, we obtained discordant results for 17 samples (10.12%). Within fifteen samples officially reported as presumptive positive, 13 were positive according to the MS-based assay. Moreover, four samples reported by the officially approved RT-PCR as negative were positive in at least one MS assay. We have successfully demonstrated superior sensitivity of the MS-based assay in SARS-CoV-2 detection, showing that MALDI-TOF MS seems to be ideal for the detection as well as discrimination of mutations within the viral genome.

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 Perspective of Arguments in Public Health

2021 ◽  
Vol 9 (1) ◽  
pp. 44-45
Author(s):  
Dinesh Kumar
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Respiratory Symptoms ◽  
False Negative ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Negative Report ◽  
Polymerase Chain ◽  
Time Of Admission ◽  
Pcr Test

Recently, an argument was put forth because a symptomatic and positive patient for CoVID-19 turned tested negative after 7 days, so discharged from the hospital. Both at the time of admission and discharge real-time reverse transcriptase Polymerase Chain Reaction (RT-PCR) was done for testing of CoVID-19. Immediately, patient again developed respiratory symptoms and was admitted to hospital again. Amidst of current CoVID-19 pandemic, a question was asked “What is the specificity of the Real Time-Polymerase Chain Reaction (RT-PCR) test for COVID-19?” with an assumption that what if at the time of discharge the disease is present in patient but test turned out to be negative? In response to that a counter statement was posed that “It is the sensitivity that should be asked rather than specificity”. It was based on the implication of primary question that was implying false negative report of the RT-PCR. It means, since patient was discharged with negative result that could be false negative.

scholarly journals Reducing False Negative PCR Test for COVID-19

2020 ◽  
Vol 9 (3) ◽  
pp. 408-410
Author(s):  
Fatemeh Bahreini ◽  
Rezvan Najafi ◽  
Razieh Amini ◽  
Salman Khazaei ◽  
Saeid Bashirian
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
False Negative ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Creative Commons ◽  
Negative Results ◽  
False Negative Results ◽  
Polymerase Chain ◽  
Pcr Test

As the SARS-CoV-2 (COVID-19) pandemic spreads rapidly, there is need for a diagnostic test with high accuracy to detect infected individuals especially those without symptoms. Real-time polymerase chain reaction (RT-PCR) is a common molecular test for diagnosing SARS-CoV-2. If some factors are not taken into consideration when performing this test, it can have a relatively large number of false negative results. In this article, we discuss important considerations that could lead to false negative test reduction. Key words: • SARS-CoV-2 • COVID-19 • Real time polymerase chain reaction • RT-PCR test • Diagnosis • False negatives • Genetics • Emerging disease   Copyright © 2020 Bahreini et al. Published by Global Health and Education Projects, Inc. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0)which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in this journal, is properly cited.

scholarly journals Estimating the false-negative test probability of SARS-CoV-2 by RT-PCR

2020 ◽  
Vol 25 (50) ◽  
Author(s):  
Paul S Wikramaratna ◽  
Robert S Paton ◽  
Mahan Ghafari ◽  
José Lourenço
Keyword(s):  
Symptom Onset ◽  
False Positive ◽  
False Negative ◽  
Positive Test ◽  
Rt Pcr ◽  
Negative Test ◽  
False Positive Test ◽  
True Number ◽  
Test Probability ◽  
False Negative Test

Background Reverse-transcription PCR (RT-PCR) assays are used to test for infection with the SARS-CoV-2 virus. RT-PCR tests are highly specific and the probability of false positives is low, but false negatives are possible depending on swab type and time since symptom onset. Aim To determine how the probability of obtaining a false-negative test in infected patients is affected by time since symptom onset and swab type. Methods We used generalised additive mixed models to analyse publicly available data from patients who received multiple RT-PCR tests and were identified as SARS-CoV-2 positive at least once. Results The probability of a positive test decreased with time since symptom onset, with oropharyngeal (OP) samples less likely to yield a positive result than nasopharyngeal (NP) samples. The probability of incorrectly identifying an uninfected individual due to a false-negative test was considerably reduced if negative tests were repeated 24 hours later. For a small false-positive test probability (<0.5%), the true number of infected individuals was larger than the number of positive tests. For a higher false-positive test probability, the true number of infected individuals was smaller than the number of positive tests. Conclusion NP samples are more sensitive than OP samples. The later an infected individual is tested after symptom onset, the less likely they are to test positive. This has implications for identifying infected patients, contact tracing and discharging convalescing patients who are potentially still infectious.

scholarly journals Detection and Species Determination of Malaria Parasites by PCR: Comparison with Microscopy and with ParaSight-F and ICT Malaria Pf Tests in a Clinical Environment

1999 ◽  
Vol 37 (5) ◽  
pp. 1269-1273 ◽  
Author(s):  
Jill M. Tham ◽  
Szu Hee Lee ◽  
Theresa M. C. Tan ◽  
Robert C. Y. Ting ◽  
Ursula A. K. Kara
Keyword(s):  
False Positive ◽  
False Negative ◽  
Dried Blood Spots ◽  
Species Differentiation ◽  
Clinical Environment ◽  
Species Determination ◽  
Easy Method ◽  
Two Samples ◽  
Positive Results ◽  
Pcr Test

A rapid procedure for the diagnosis of malaria infections directly from dried blood spots by PCR amplification was evaluated with samples from 52 patients. Plasmodium infections were identified with a genus-specific primer set, and species differentiation betweenPlasmodium falciparum and Plasmodium vivax was analyzed by multiplex PCR. The PCR test with any of the three primer sets was able to detect as few as four parasites per microliter by gel electrophoresis or by nonisotopic paper hybridization chromatography. The diagnoses obtained by PCR correlated closely with those obtained by Giemsa staining except for two samples observed to have mixed P. falciparum-P. vivax infections. These were initially missed by microscopic analysis. In comparison with antigen-capture assays forP. falciparum, the PCR assays were able to detect three infections that were missed by the ParaSight-F test. The PCR test was negative for nine ParaSight-F-positive samples and one ICT Malaria Pf-positive sample, and these were confirmed to be false-positive results. The PCR thus gave no false-negative or false-positive results. Patients undergoing antimalarial therapy were also monitored by the PCR assay. Four of seven patients who were PCR positive forP. vivax at the time of discharge were later readmitted to the hospital with a recurrence of P. vivax infection. We would like to propose that PCR is a sensitive and easy method that can serve as a useful addition to microscopy for the diagnosis and the clinical monitoring of treatment of malaria.

scholarly journals Coronavirus Disease 2019 (COVID-19) in Italy: Double reading of chest CT examination

2020 ◽  
Author(s):  
Reginelli Alfonso ◽  
Grassi Roberto ◽  
Feragalli Beatrice ◽  
Belfiore Maria Paola ◽  
Montanelli Alessandro ◽  
...  
Keyword(s):  
Predictive Value ◽  
False Negative ◽  
Viral Pneumonia ◽  
Ct Diagnosis ◽  
Rt Pcr ◽  
Ct Examination ◽  
Polymerase Chain ◽  
Sensitivity Specificity ◽  
Age Range ◽  
Pcr Test

Abstract OBJECTIVETo assess the performance of the second reading of chest Compute Tomography (CT) examinations by expert radiologists in patients with discordance between the reverse transcription real-time fluorescence polymerase chain reaction (RT-PCR) test for COVID-19 viral pneumonia and the first CT report.MATERIALS AND METHODS.Three hundred seventy-heigth patients were included in this retrospective study (121 women and 257 men; 71 years of median age - range, 29–93 years) subjected to RT-PCR test for suspicious COVID-19 infection. All patients were subjected to CT examination in order to evaluate the pulmonary disease involvement by COVID-19. CT images were reviewed first by two radiologists who identified COVID-19 typical CT patterns and then reanalyzed by anoter two radiologists using a CT structured report for COVID-19 diagnosis.RESULTS.The median temporal window between RT-PCRs execution and CT scan was 0 days with a range of [-9, 11] days. RT-PCR test was resulted positive in 328/378 (86.8%). Discordance between RT-PCR and CT findings for viral pneumonia was revealed in 60 cases. The second reading changed the CT diagnosis in 16/60 (26.7%) cases contributing to increase the concordance with the RT-PCR. Among these 60 cases, 8 were false negative with positive RT-PCR, and 36 were false positive with negative RT-PCR. Sensitivity, specificity, positive predictive value and negative predictive value of CT were respectively of 97.3%, 53.8%, 89.0%, and 88.4%.CONCLUSION.Double reading of CT could increase the diagnostic confidence of radiological interpretation in COVID-19 patients. Using expert second readers could reduce the rate of discrepant cases between RT-PCR results and CT diagnosis for COVID-19 viral pneumonia.

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