scholarly journals SARS-CoV-2 detection using digital PCR for COVID-19 diagnosis, treatment monitoring and criteria for discharge

2020 ◽  
Author(s):  
Renfei Lu ◽  
Jian Wang ◽  
Min Li ◽  
Yaqi Wang ◽  
Jia Dong ◽  
...  
Keyword(s):  
Viral Load ◽  
Limit Of Detection ◽  
False Negative ◽  
Digital Pcr ◽  
Detection Sensitivity ◽  
Rt Pcr ◽  
Pharyngeal Swab ◽  
Clinical Specimens ◽  
Positive Results ◽  
Negative Detection

SummaryBackgroundSARS-CoV-2 nucleic acid detection by RT-PCR is one of the criteria approved by China FDA for diagnosis of COVID-19. However, inaccurate test results (for example, high false negative rate and some false positive rate) were reported in both China and US CDC using RT-PCR method. Inaccurate results are caused by inadequate detection sensitivity of RT-PCR, low viral load in some patients, difficulty to collect samples from COVID-19 patients, insufficient sample loading during RT-PCR tests, and RNA degradation during sample handling process. False negative detection could subject patients to multiple tests before diagnosis can be made, which burdens health care system. Delayed diagnosis could cause infected patients to miss the best treatment time window. False negative detection could also lead to prematurely releasing infected patients who still carry residual SARS-CoV-2 virus. In this case, these patients could infect many others. A high sensitivity RNA detection method to resolve the existing issues of RT-PCR is in need for more accurate COVID-19 diagnosis.MethodsDigital PCR (dPCR) instrument DropX-2000 and assay kits were used to detect SARS-CoV-2 from 108 clinical specimens from 36 patients including pharyngeal swab, stool and blood from different days during hospitalization. Double-blinded experiment data of 108 clinical specimens by dPCR methods were compared with results from officially approved RT-PCR assay. A total of 109 samples including 108 clinical specimens and 1 negative control sample were tested in this study. All of 109 samples, 26 were from 21patients reported as positive by officially approved clinical RT-PCR detection in local CDC and then hospitalized in Nantong Third Hospital. Among the 109 samples, dPCR detected 30 positive samples on ORFA1ab gene, 47 samples with N gene positive, and 30 samples with double positive on ORFA1ab and N genes.ResultsThe lower limit of detection of the optimize dPCR is at least 10-fold lower than that of RT-PCR. The overall accuracy of dPCR for clinical detection is 96.3%. 4 out 4 of (100 %) negative pharyngeal swab samples checked by RT-PCR were positive judged by dPCR based on the follow-up investigation. 2 of 2 samples in the RT-PCR grey area (Ct value > 37) were confirmed by dPCR with positive results. 1 patient being tested positive by RT-PCR was confirmed to be negative by dPCR. The dPCR results show clear viral loading decrease in 12 patients as treatment proceed, which can be a useful tool for monitoring COVID-19 treatment.ConclusionsDigital PCR shows improved lower limit of detection, sensitivity and accuracy, enabling COVID-19 detection with less false negative and false positive results comparing with RT-PCR, especially for the tests with low viral load specimens. We showed evidences that dPCR is powerful in detecting asymptomatic patients and suspected patients. Digital PCR is capable of checking the negative results caused by insufficient sample loading by quantifying internal reference gene from human RNA in the PCR reactions. Multi-channel fluorescence dPCR system (FAM/HEX/CY5/ROX) is able to detect more target genes in a single multiplex assay, providing quantitative count of viral load in specimens, which is a powerful tool for monitoring COVID-19 treatment.

scholarly journals ddPCR: a more sensitive and accurate tool for SARS-CoV-2 detection in low viral load specimens

2020 ◽  
Author(s):  
Tao Suo ◽  
Xinjin Liu ◽  
Jiangpeng Feng ◽  
Ming Guo ◽  
Wenjia Hu ◽  
...  
Keyword(s):  
Viral Load ◽  
Limit Of Detection ◽  
False Negative ◽  
Digital Pcr ◽  
Clinical Samples ◽  
Double Blind Study ◽  
Rt Pcr ◽  
Current Standard ◽  
Double Blind ◽  
Sensitivity Specificity

AbstractReal time fluorescent quantitative PCR (RT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. However, due to the low viral load in patient throats and the limitations of RT-PCR, significant numbers of false negative reports are inevitable, which results in failure to timely diagnose, early treat, cut off transmission, and assess discharge criteria. To improve this situation, an optimized droplet digital PCR (ddPCR) was used for detection of SARS-CoV-2, which showed that the limit of detection of ddPCR is significantly lower than that of RT-PCR. We further explored the feasibility of ddPCR to detect SARS-CoV-2 nucleic acid from 77 clinical throat swab samples, including 63 suspected outpatients with fever and 14 supposed convalescents who were about to discharge after treatment, and compared with RT-PCR in terms of the diagnostic accuracy. In this double-blind study, we tested, surveyed subsequently and statistically analyzed 77 clinical samples. According to our study, 26 samples from COVID-19 patients with RT-PCR negative were detected as positive by ddPCR. No FPRs of RT-PCR and ddPCR were observed. The sensitivity, specificity, PPV, NPV, NLR and accuracy were improved from 40% (95% CI: 27–55%), 100% (95% CI: 54–100%), 100%, 16% (95% CI: 13–19%), 0.6 (95% CI: 0.48–0.75) and 47% (95% CI: 33–60%) for RT-PCR to 94% (95% CI: 83–99%), 100% (95% CI: 48–100%), 100%, 63% (95% CI: 36–83%), 0.06 (95% CI: 0.02–0.18) and 95% (95% CI: 84–99%) for ddPCR, respectively. Moreover, 14 (42.9 %) convalescents still carry detectable SARS-CoV-2 after discharge. Overall, ddPCR shows superiority for clinical diagnosis of SARS-CoV-2 to reduce the false negative reports, which could be a powerful complement to the current standard RT-PCR. It also suggests that the current clinical practice that the convalescent after discharge continues to be quarantined for at least 2 weeks is completely necessary which can prevent potential viral transmission.

scholarly journals Distribution of SARS-CoV-2 PCR Cycle Threshold Values Provide Practical Insight Into Overall and Target-Specific Sensitivity Among Symptomatic Patients

2020 ◽  
Vol 154 (4) ◽  
pp. 479-485 ◽  
Author(s):  
Blake W Buchan ◽  
Jessica S Hoff ◽  
Cameron G Gmehlin ◽  
Adriana Perez ◽  
Matthew L Faron ◽  
...  
Keyword(s):  
Viral Load ◽  
Limit Of Detection ◽  
False Negative ◽  
Age Group ◽  
Rt Pcr ◽  
Patient Age ◽  
Cycle Threshold ◽  
Negative Results ◽  
Patient Âgé ◽  
False Negative Results

Abstract Objectives We examined the distribution of reverse transcription polymerase chain reaction (RT-PCR) cycle threshold (CT) values obtained from symptomatic patients being evaluated for coronavirus disease 2019 (COVID-19) to determine the proportion of specimens containing a viral load near the assay limit of detection (LoD) to gain practical insight to the risk of false-negative results. We also examined the relationship between CT value and patient age to determine any age-dependent difference in viral load or test sensitivity. Methods We collected CT values obtained from the cobas severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) assay corresponding to 1,213 combined nasopharyngeal-oropharyngeal specimens obtained from symptomatic individuals that were reported as positive or presumptive positive for SARS-CoV-2. CT values were stratified by SARS-CoV target and patient age group. Results In total, 93.3% to 98.4% of specimens demonstrated CT values greater than 3× the assay LoD, at which point false-negative results would not be expected. The mean of CT values between age groups was statistically equivalent with the exception of patients in age group 80 to 89 years, which demonstrated slightly lower CTs. Conclusions Based on the distribution of observed CT values, including the small proportion of specimens with values near the assay LoD, there is a low risk of false-negative RT-PCR results in combined nasopharyngeal-oropharyngeal specimens obtained from symptomatic individuals.

scholarly journals Direct Quantitation of SARS-CoV-2 Using Droplet Digital PCR in Suspected Samples With Very Low Viral Load

2021 ◽  
Author(s):  
Michela Deiana ◽  
Chiara Piubelli ◽  
Antonio Mori ◽  
Gian Paolo Chiecchi ◽  
Giulia La Marca ◽  
...  
Keyword(s):  
Viral Load ◽  
Real Time ◽  
False Negative ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Hospitalized Patients ◽  
Lower Sensitivity ◽  
Rt Pcr ◽  
Viral Genes ◽  
Viral Target

Background: The reference test for SARS-CoV-2 detection is the reverse transcriptase real time PCR (real time RT-PCR). However, evidences reported that real time RT-PCR has a lower sensitivity compared with the droplet digital PCR (ddPCR) leading to possible false negative in low viral load cases. Methods: We used ddPCR for viral genes N1 and N2 on 20 negative (no detection) samples from symptomatic hospitalized COVID-patients presenting fluctuating real time RT-PCR results and 10 suspected samples (Ct value>35) from asymptomatic not hospitalized subjects. Results: ddPCR performed on RNA revealed 65% of positivity for at least one viral target in the hospitalized patients group of samples (35% for N1 and N2, 10% only for N1 and 20% only for N2) and 50% in the suspected cases (30% for N1 and N2, while 20% only for N2). On hospitalized patients’ samples, we applied also a direct ddPCR approach on the swab material, achieving an overall positivity of 83%. Conclusion: ddPCR, in particular the direct quantitation on swabs, shows a sensitivity advantage for the SARS-CoV-2 identification and may be useful to reduce the false negative diagnosis, especially for low viral load suspected samples.

scholarly journals Multi-site Evaluation of SARS-CoV-2 Spike Mutation Detection Using a Multiplex Real-time RT-PCR Assay

2021 ◽  
Author(s):  
Carolin Bier ◽  
Anke Edelmann ◽  
Kathrin Theil ◽  
Rolf Schwarzer ◽  
Maria Deichner ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
False Negative ◽  
Analytical Sensitivity ◽  
Test Results ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Clinical Specimens ◽  
Negative Results ◽  
Global Pandemic

Background. SARS-CoV-2 causes COVID-19, which can be fatal and is responsible for a global pandemic. Variants with increased transmissibility or the potential to evade immunity have emerged and represent a threat to global pandemic control. Variants of concern (VOC) can be identified by sequencing of viral RNA, or by more rapid methods for detection of subsets of signature mutations. Methods. We developed a multiplex, real-time RT-PCR assay (cobas SARS-CoV-2 Variant Set 1) for the qualitative detection and differentiation of three key SARS-CoV-2 mutations in the viral spike protein: del 69-70, E484K and N501Y. Analytical sensitivity and accuracy were evaluated at three testing sites using clinical specimens from patients infected with SARS-CoV-2 variants belonging to several different lineages, including B.1.1.7, B.1.351, and P.1. Results. The limit of detection for E484K was between 180 and 620 IU/mL for the three different isolates tested. For N501Y, the LOD was between 270 and 720 IU/mL (five isolates), while for del 69-70, it was 80 - 92 IU/mL (two isolates). Valid test results were obtained with all clinical specimens that were positive using routine diagnostic tests. Compared to sequencing (Sanger and next-generation), test results were 100% concordant at all three loci; no false positive or false negative results were observed. Conclusions. Data collected at three independent laboratories indicates excellent performance and concordance of cobas SARS-CoV-2 Variant Set 1 with sequencing. New sets of primers and probes that target additional loci can be rapidly deployed in response to the identification of other emerging variants.

scholarly journals Comparison of real-time PCR and droplet digital PCR for the detection of Xylella fastidiosa in plants

2019 ◽  
Author(s):  
Enora Dupas ◽  
Bruno Legendre ◽  
Valérie Olivier ◽  
Françoise Poliakoff ◽  
Charles Manceau ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Xylella Fastidiosa ◽  
False Negative ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Detection Sensitivity ◽  
Plant Sample ◽  
The European Union

AbstractXylella fastidiosa (Xf) is a quarantine plant pathogen bacterium originating from the Americas and that has emerged in Europe in 2013. Xf can be detected directly on plant macerate using molecular methods such as real-time PCR, which is a sensitive technique. However, some plants may contain components that can act as PCR reaction inhibitors, which can lead to false negative results or an underestimation of the bacterial concentration present in the analyzed plant sample. Droplet digital PCR (ddPCR) is an innovative tool based on the partitioning of the PCR reagents and the DNA sample into thousands of droplets, allowing the quantification of the absolute number of target DNA molecules present in a reaction mixture, or an increase of the detection sensitivity. In this study, a real-time PCR protocol, already used for Xf detection in the framework of official surveys in the European Union, was transferred and optimized for Xf detection using ddPCR. This new assay was evaluated and compared to the initial real-time PCR on five plant matrices artificially inoculated and on naturally infected plants. In our conditions, this new ddPCR enabled the detection of Xf on all artificially inoculated plant macerates with a similar limit of detection, or a slight benefit for Quercus ilex. Moreover, ddPCR improved diagnostic sensitivity as it enabled detection of Xf in samples of Polygala myrtifolia or Q. ilex that were categorized as negative or close to the limit of detection using the real-time PCR. Here, we report for the first time a ddPCR assay for the detection of the bacterium Xf.

scholarly journals Highly accurate and sensitive diagnostic detection of SARS-CoV-2 by digital PCR

2020 ◽  
Author(s):  
Lianhua Dong ◽  
Junbo Zhou ◽  
Chunyan Niu ◽  
Quanyi Wang ◽  
Yang Pan ◽  
...  
Keyword(s):  
Viral Load ◽  
Reverse Transcription ◽  
Clinical Symptoms ◽  
Limit Of Detection ◽  
Digital Pcr ◽  
Pharyngeal Swab ◽  
Positive Rate ◽  
Novel Coronavirus ◽  
Sensitivity Specificity ◽  
Close Contacts

BACKGROUNDThe outbreak of COVID-19 caused by a novel Coronavirus (termed SARS-CoV-2) has spread to over 140 countries around the world. Currently, reverse transcription quantitative qPCR (RT-qPCR) is used as the gold standard for diagnostics of SARS-CoV-2. However, the positive rate of RT-qPCR assay of pharyngeal swab samples are reported to vary from 30∼60%. More accurate and sensitive methods are urgently needed to support the quality assurance of the RT-qPCR or as an alternative diagnostic approach.METHODSWe established a reverse transcription digital PCR (RT-dPCR) protocol to detect SARS-CoV-2 on 194 clinical pharyngeal swab samples, including 103 suspected patients, 75 close contacts and 16 supposed convalescents.RESULTSThe limit of blanks (LoBs) of the RT-dPCR assays were ∼1.6, ∼1.6 and ∼0.8 copies/reaction for ORF 1ab, N and E genes, respectively. The limit of detection (LoD) was 2 copies/reaction. For the 103 fever suspected patients, the sensitivity of SARS-CoV-2 detection was significantly improved from 28.2% by RT-qPCR to 87.4% by RT-dPCR. For close contacts, the suspect rate was greatly decreased from 21% down to 1%. The overall sensitivity, specificity and diagnostic accuracy of RT-dPCR were 90%, 100% and 93 %, respectively. In addition, quantification of the viral load for convalescents by RT-dPCR showed that a longer observation period was needed in the hospital for elderly patients.CONCLUSIONRT-dPCR could be a confirmatory method for suspected patients diagnosed by RT-qPCR. Furthermore, RT-dPCR was more sensitive and suitable for low viral load specimens from the both patients under isolation and those under observation who may not be exhibiting clinical symptoms.

scholarly journals Sensitivity evaluation of 2019 novel coronavirus (SARS-CoV-2) RT-PCR detection kits and strategy to reduce false negative

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0241469
Author(s):  
Yunying Zhou ◽  
Fengyan Pei ◽  
Mingyu Ji ◽  
Li Wang ◽  
Huailong Zhao ◽  
...  
Keyword(s):  
Viral Load ◽  
Nucleic Acid ◽  
False Negative ◽  
Detection Sensitivity ◽  
Nucleic Acid Detection ◽  
Rt Pcr ◽  
Current Standard ◽  
Mixed Sample ◽  
Asymptomatic Patients ◽  
Selection Of

The early detection and differential diagnosis of respiratory infections increase the chances for successful control of COVID-19 disease. The nucleic acid RT-PCR test is regarded as the current standard for molecular diagnosis. However, the maximal specificity confirmation target ORF1ab gene is considered to be less sensitive than other targets in clinical application. In addition, recent evidence indicated that the initial missed diagnosis of asymptomatic patients with SARS-CoV-2 and discharged patients with “re-examination positive” might be due to low viral load, and the ability of rapid mutation of SARS-CoV-2 also increases the rate of false-negative results. Moreover, the mixed sample nucleic acid detection is helpful in seeking out the early community transmission of SARS-CoV-2 rapidly, but the detection kit needs ultra-high detection sensitivity. Herein, the lowest detection concentration of different nucleic acid detection kits was evaluated and compared to provide direct evidence for the selection of kits for mixed sample detection or make recommendations for the selection of validation kit, which is of great significance for the prevention and control of the current epidemic and the discharge criteria of low viral load patients.

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 A verified genomic reference sample for assessing performance of cancer panels detecting small variants of low allele frequency

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Wendell Jones ◽  
Binsheng Gong ◽  
Natalia Novoradovskaya ◽  
Dan Li ◽  
Rebecca Kusko ◽  
...  
Keyword(s):  
Quality Control ◽  
Allele Frequency ◽  
Liquid Biopsy ◽  
Limit Of Detection ◽  
Reference Sample ◽  
Digital Pcr ◽  
Detection Sensitivity ◽  
Analytical Accuracy ◽  
And Performance ◽  
Reference Samples

Abstract Background Oncopanel genomic testing, which identifies important somatic variants, is increasingly common in medical practice and especially in clinical trials. Currently, there is a paucity of reliable genomic reference samples having a suitably large number of pre-identified variants for properly assessing oncopanel assay analytical quality and performance. The FDA-led Sequencing and Quality Control Phase 2 (SEQC2) consortium analyze ten diverse cancer cell lines individually and their pool, termed Sample A, to develop a reference sample with suitably large numbers of coding positions with known (variant) positives and negatives for properly evaluating oncopanel analytical performance. Results In reference Sample A, we identify more than 40,000 variants down to 1% allele frequency with more than 25,000 variants having less than 20% allele frequency with 1653 variants in COSMIC-related genes. This is 5–100× more than existing commercially available samples. We also identify an unprecedented number of negative positions in coding regions, allowing statistical rigor in assessing limit-of-detection, sensitivity, and precision. Over 300 loci are randomly selected and independently verified via droplet digital PCR with 100% concordance. Agilent normal reference Sample B can be admixed with Sample A to create new samples with a similar number of known variants at much lower allele frequency than what exists in Sample A natively, including known variants having allele frequency of 0.02%, a range suitable for assessing liquid biopsy panels. Conclusion These new reference samples and their admixtures provide superior capability for performing oncopanel quality control, analytical accuracy, and validation for small to large oncopanels and liquid biopsy assays.

scholarly journals False Negative Mitigation in Group Testing for COVID-19 Screening

2021 ◽  
Vol 8 ◽  
Author(s):  
Amir Reza Alizad Rahvar ◽  
Safar Vafadar ◽  
Mehdi Totonchi ◽  
Mehdi Sadeghi
Keyword(s):  
Viral Load ◽  
False Negative ◽  
Group Testing ◽  
False Negative Rate ◽  
Screening Strategy ◽  
Web Interface ◽  
Rt Pcr ◽  
Promising Candidate ◽  
Testing Method ◽  
High False Negative Rate

After lifting the COVID-19 lockdown restrictions and opening businesses, screening is essential to prevent the spread of the virus. Group testing could be a promising candidate for screening to save time and resources. However, due to the high false-negative rate (FNR) of the RT-PCR diagnostic test, we should be cautious about using group testing because a group's false-negative result identifies all the individuals in a group as uninfected. Repeating the test is the best solution to reduce the FNR, and repeats should be integrated with the group-testing method to increase the sensitivity of the test. The simplest way is to replicate the test twice for each group (the 2Rgt method). In this paper, we present a new method for group testing (the groupMix method), which integrates two repeats in the test. Then we introduce the 2-stage sequential version of both the groupMix and the 2Rgt methods. We compare these methods analytically regarding the sensitivity and the average number of tests. The tradeoff between the sensitivity and the average number of tests should be considered when choosing the best method for the screening strategy. We applied the groupMix method to screening 263 people and identified 2 infected individuals by performing 98 tests. This method achieved a 63% saving in the number of tests compared to individual testing. Our experimental results show that in COVID-19 screening, the viral load can be low, and the group size should not be more than 6; otherwise, the FNR increases significantly. A web interface of the groupMix method is publicly available for laboratories to implement this method.

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