scholarly journals An accurate model for SARS-CoV-2 pooled RT-PCR test errors

2021 ◽  
Vol 8 (11) ◽  
Author(s):  
Yair Daon ◽  
Amit Huppert ◽  
Uri Obolski
Keyword(s):  
Infected Individual ◽  
False Negative ◽  
False Negative Rate ◽  
Positive Outcome ◽  
Error Rates ◽  
Infection Prevalence ◽  
Rt Pcr ◽  
Multiple Samples ◽  
Infected Sample ◽  
Pcr Test

Pooling is a method of simultaneously testing multiple samples for the presence of pathogens. Pooling of SARS-CoV-2 tests is increasing in popularity, due to its high testing throughput. A popular pooling scheme is Dorfman pooling: test N individuals simultaneously, if the test is positive, each individual is then tested separately; otherwise, all are declared negative. Most analyses of the error rates of pooling schemes assume that including more than a single infected sample in a pooled test does not increase the probability of a positive outcome. We challenge this assumption with experimental data and suggest a novel and parsimonious probabilistic model for the outcomes of pooled tests. As an application, we analyse the false-negative rate (i.e. the probability of a negative result for an infected individual) of Dorfman pooling. We show that the false-negative rates under Dorfman pooling increase when the prevalence of infection decreases. However, low infection prevalence is exactly the condition when Dorfman pooling achieves highest throughput efficiency. We therefore urge the cautious use of pooling and development of pooling schemes that consider correctly accounting for tests’ error rates.

scholarly journals Variation in False Negative Rate of RT-PCR Based SARS-CoV-2 Tests by Time Since Exposure

2020 ◽  
Author(s):  
Lauren M Kucirka ◽  
Stephen A Lauer ◽  
Oliver Laeyendecker ◽  
Denali Boon ◽  
Justin Lessler
Keyword(s):  
Symptom Onset ◽  
Infected Individual ◽  
False Negative ◽  
False Negative Rate ◽  
Published Data ◽  
Rt Pcr ◽  
Considerable Uncertainty ◽  
Negative Rate ◽  
High Clinical Suspicion ◽  
Cubic Polynomial

ABSTRACTSARS-CoV-2 RT-PCR based tests are being used to “rule out” infection among high-risk individuals such as exposed inpatients and healthcare workers. It is critical to understand how the predictive value of the test varies with time from exposure and symptom onset in order to avoid being falsely reassured by negative tests. As such, the goal of our study was to estimate the false negative rate by day since infection. We used previously published data on RT-PCR sensitivity on samples derived from nasal swabs by day since symptom onset (n=633) and fit a cubic polynomial spline to calculate the false negative rate by day since exposure and symptom onset. Over the four days of infection prior to the typical time of symptom onset (day 5) the probability of a false negative test in an infected individual falls from 100% on day one (95% CI 69-100%) to 61% on day four (95% CI 18-98%), though there is considerable uncertainty in these numbers. On the day of symptom onset, the median false negative rate was 39% (95% CI 16-77%). This decreased to 26% (95% CI 18-34%) on day 8 (3 days after symptom onset), then began to rise again, from 27% (95% CI 20-34%) on day 9 to 61% (95% CI 54-67%) on day 21. Care must be taken when interpreting RT-PCR tests for SARS-CoV-2 infection, particularly if performed early in the course of infection, when using these results as a basis for removing precautions intended to prevent onward transmission. If there is high clinical suspicion, patients should not be ruled out on the basis of RT-PCR alone, and the clinical and epidemiologic situation should be carefully considered.

scholarly journals Inflated false-negative rates in pooled RT-PCR tests of SARS-CoV-2

2020 ◽  
Author(s):  
Yair Daon ◽  
Amit Huppert ◽  
Uri Obolski
Keyword(s):  
Infected Individual ◽  
False Negative ◽  
False Negative Rate ◽  
Infection Prevalence ◽  
Policy Makers ◽  
Trade Off ◽  
Dilution Effects ◽  
Parameter Values ◽  
Positive Results ◽  
Sample Dilution

AbstractBackgroundPooling is a popular strategy for increasing SARS-CoV-2 testing throughput. A common pooling scheme is Dorfman pooling: test N individuals simultaneously. If the first test is positive — retest each individual.MethodsUsing a probabilistic model, we analyze the false-negative rate (i.e., the probability of a negative result for an infected individual) of Dorfman pooling. Our model is conservative in that it ignores sample dilution effects, which can only worsen pooling performance.ResultsWe show that one can expect a 60-80% increase in false-negative rates under Dorfman pooling, for reasonable parameter values. Moreover, we show that the false-negative rates under Dorfman pooling increase when the prevalence of infection decreases.DiscussionIn most pooling schemes, identifying an infected individual requires positive results in multiple tests and hence substantially deteriorates false-negative rates. Furthermore, this phenomenon is more pronounced when infection prevalence is low — exactly when pooling is most efficient. Thus, pooling presents an inherent trade-off: it is most efficient when it is least accurate. The deterioration of false-negative rates and the aforementioned trade-off are inherent problems of pooling schemes and should be kept in mind by practitioners and policy makers.

scholarly journals Analysis of factors associated early diagnosis in coronavirus disease 2019 (COVID-19)

2020 ◽  
Author(s):  
Jinwei Ai ◽  
Junyan Gong ◽  
Limin Xing ◽  
Renjiao He ◽  
Fangtao Tian ◽  
...  
Keyword(s):  
Early Diagnosis ◽  
Ct Scan ◽  
Lymphocyte Count ◽  
False Negative ◽  
False Negative Rate ◽  
Diagnostic Value ◽  
Rt Pcr ◽  
Likelihood Ratios ◽  
High False Negative Rate ◽  
Pcr Test

AbstractBackgroundThe pandemic of coronavirus disease 2019 (COVID-19) has become the first concern in international affairs as the novel coronavirus (SARS-CoV-2) is spreading all over the world at a terrific speed. The accuracy of early diagnosis is critical in the control of the spread of the virus. Although the real-time RT-PCR detection of the virus nucleic acid is the current golden diagnostic standard, it has high false negative rate when only apply single test.ObjectiveSummarize the baseline characteristics and laboratory examination results of hospitalized COVID-19 patients. Analyze the factors that could interfere with the early diagnosis quantitatively to support the timely confirmation of the disease.MethodsAll suspected patients with COVID-19 were included in our study until Feb 9th, 2020. The last day of follow-up was Mar 20th, 2020. Throat swab real-time RT-PCR test was used to confirm SARS-CoV-2 infection. The difference between the epidemiological profile and first laboratory examination results of COVID-19 patients and non-COVID-19 patients were compared and analyzed by multiple logistic regression. Receiver operating characteristic (ROC) curve and area under curve (AUC) were used to assess the potential diagnostic value in factors, which had statistical differences in regression analysis.ResultsIn total, 315 hospitalized patients were included. Among them, 108 were confirmed as COVID-19 patients and 207 were non-COVID-19 patients. Two groups of patients have significance in comparing age, contact history, leukocyte count, lymphocyte count, C-reactive protein, erythrocyte sedimentation rate (p<0.10). Multiple logistic regression analysis showed age, contact history and decreasing lymphocyte count could be used as individual factor that has diagnostic value (p<0.05). The AUC of first RT-PCR test was 0.84 (95% CI 0.73-0.89), AUC of cumulative two times of RT-PCR tests was 0.92 (95% CI 0.88-0.96) and 0.96 (95% CI 0.93-0.99) for cumulative three times of RT-PCR tests. Ninety-six patients showed typical pneumonia radiological features in first CT scan, AUC was 0.74 (95% CI 0.60-0.73). The AUC of patients’ age, contact history with confirmed people and the decreased lymphocytes were 0.66 (95% CI 0.60-0.73), 0.67 (95% CI 0.61-0.73), 0.62 (95% CI 0.56-0.69), respectively. Taking chest CT scan diagnosis together with patients age and decreasing lymphocytes, AUC would be 0.86 (95% CI 0.82-0.90). The age threshold to predict COVID-19 was 41.5 years, with a diagnostic sensitivity of 0.70 (95% CI 0.61-0.79) and a specificity of 0.59 (95% CI 0.52-0.66). Positive and negative likelihood ratios were 1.71 and 0.50, respectively. Threshold of lymphocyte count to diagnose COVID-19 was 1.53×109/L, with a diagnostic sensitivity of 0.82 (95% CI 0.73-0.88) and a specificity of 0.50 (95% CI 0.43-0.57). Positive and negative likelihood ratios were 1.64 and 0.37, respectively.ConclusionSingle RT-PCR test has relatively high false negative rate. When first RT-PCR test show negative result in suspected patients, the chest CT scan, contact history, age and lymphocyte count should be used combinedly to assess the possibility of SARS-CoV-2 infection.

scholarly journals FAST: a Flexible, Accurate and Speedy Test for COVID-19

2020 ◽  
Author(s):  
Linjiajie Fang ◽  
Bing-Yi Jing ◽  
Shen Ling ◽  
Qing Yang
Keyword(s):  
Close Contact ◽  
False Negative ◽  
Group Testing ◽  
False Negative Rate ◽  
Rt Pcr ◽  
False Negatives ◽  
Improve Efficiency ◽  
Negative Rate ◽  
Pcr Test

AbstractAs the COVID-19 pandemic continues worldwide, there is an urgent need to detect infected patients as quickly and accurately as possible. Group testing proposed by Technion [1][2] could improve efficiency greatly. However, the false negative rate (FNR) would be doubled. Using USA as an example, group testing would have over 70,000 false negatives, compared to 35,000 false negatives by individual testing.In this paper, we propose a Flexible, Accurate and Speedy Test (FAST), which is faster and more accurate than any existing tests. FAST first forms small close contact subgroups, e.g. families and friends. It then pools subgroups to form larger groups before RT-PCR test is done. FAST needs a similar number of tests to Technion’s method, but sharply reduces the FNR to a negligible level. For example, FAST brings down the number of false negatives in USA to just 2000, and it is seven times faster than individual testing.

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 Pooling of Upper Respiratory Specimens Using a SARS-CoV-2 Real-time RT-PCR Assay Authorized for Emergency Use in Low-Prevalence Populations for High-Throughput Testing

2020 ◽  
Vol 7 (11) ◽  
Author(s):  
Gwynngelle A Borillo ◽  
Ron M Kagan ◽  
Russell E Baumann ◽  
Boris M Fainstein ◽  
Lamela Umaru ◽  
...  
Keyword(s):  
Real Time ◽  
False Negative ◽  
False Negative Rate ◽  
Nucleic Acid Amplification ◽  
Rt Pcr ◽  
Pooled Testing ◽  
Negative Results ◽  
Single Positive ◽  
Upper Respiratory ◽  
Respiratory Specimens

Abstract Background Nucleic acid amplification testing is a critical tool for addressing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Specimen pooling can increase throughput and conserve testing resources but requires validation to ensure that reduced sensitivity does not increase the false-negative rate. We evaluated the performance of a real-time reverse transcription polymerase chain reaction (RT-PCR) test authorized by the US Food and Drug Administration (FDA) for emergency use for pooled testing of upper respiratory specimens. Methods Positive specimens were selected from 3 prevalence groups, 1%–3%, &gt;3%–6%, and &gt;6%–10%. Positive percent agreement (PPA) was assessed by pooling single-positive specimens with 3 negative specimens; performance was assessed using Passing-Bablok regression. Additionally, we assessed the distributions of RT-PCR cycle threshold (Ct) values for 3091 positive specimens. Results PPA was 100% for the 101 pooled specimens. There was a linear relationship between Ct values for pooled and single-tested specimens (r = 0.96–0.99; slope ≈ 1). The mean pooled Ct shifts at 40 cycles were 2.38 and 1.90, respectively, for the N1 and N3 targets. The median Cts for 3091 positive specimens were 25.9 (N1) and 24.7 (N3). The percentage of positive specimens with Cts between 40 and the shifted Ct was 1.42% (N1) and 0.0% (N3). Conclusions Pooled and individual testing of specimens positive for SARS-CoV-2 demonstrated 100% agreement, which demonstrates the viability of pooled specimens for SARS-COV-2 testing using a dual-target RT-PCR system. Pooled specimen testing can help increase testing capacity for SARS-CoV-2 with a low risk of false-negative results.

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.

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 Microfluidic Nano-Scale qPCR Enables Ultra-Sensitive and Quantitative Detection of SARS-CoV-2

Processes ◽  
2020 ◽  
Vol 8 (11) ◽  
pp. 1425
Author(s):  
Xin Xie ◽  
Tamara Gjorgjieva ◽  
Zaynoun Attieh ◽  
Mame Massar Dieng ◽  
Marc Arnoux ◽  
...  
Keyword(s):  
Viral Infections ◽  
Limit Of Detection ◽  
False Negative ◽  
False Negative Rate ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
Viral Loads ◽  
Nano Scale ◽  
Negative Rate

A major challenge in controlling the COVID-19 pandemic is the high false-negative rate of the commonly used RT-PCR methods for SARS-CoV-2 detection in clinical samples. Accurate detection is particularly challenging in samples with low viral loads that are below the limit of detection (LoD) of standard one- or two-step RT-PCR methods. In this study, we implemented a three-step approach for SARS-CoV-2 detection and quantification that employs reverse transcription, targeted cDNA preamplification, and nano-scale qPCR based on a commercially available microfluidic chip. Using SARS-CoV-2 synthetic RNA and plasmid controls, we demonstrate that the addition of a preamplification step enhances the LoD of this microfluidic RT-qPCR by 1000-fold, enabling detection below 1 copy/µL. We applied this method to analyze 182 clinical NP swab samples previously diagnosed using a standard RT-qPCR protocol (91 positive, 91 negative) and demonstrate reproducible and quantitative detection of SARS-CoV-2 over five orders of magnitude (<1 to 106 viral copies/µL). Crucially, we detect SARS-CoV-2 with relatively low viral load estimates (<1 to 40 viral copies/µL) in 17 samples with negative clinical diagnosis, indicating a potential false-negative rate of 18.7% by clinical diagnostic procedures. In summary, this three-step nano-scale RT-qPCR method can robustly detect SARS-CoV-2 in samples with relatively low viral loads (<1 viral copy/µL) and has the potential to reduce the false-negative rate of standard RT-PCR-based diagnostic tests for SARS-CoV-2 and other viral infections.

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.

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