Variation in False-Negative Rate of Reverse Transcriptase Polymerase Chain Reaction–Based SARS-CoV-2 Tests by Time Since ExposureFREE
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Abstract
Background:
Objective:
Design:
Setting:
Patients:
Measurements:
Results:
Limitation:
Conclusion:
Primary Funding Source:
Methods
Source Data
How Cases Were Defined
Statistical Analysis
Model for Estimating False-Negative Rate and False Omission Rate by Time Since Exposure
Sensitivity Analyses
Code and Data Availability
Role of the Funding Source
Results
Probability of a False-Negative Result Among SARS-CoV-2–Positive Patients, by Day Since Exposure
Posttest Probability of Infection if RT-PCR Result is Negative (1 Minus Negative Predictive Value)
Variation in Posttest Probability of Infection if RT-PCR Result is Negative, by Pretest Probability
Sensitivity Analyses
Discussion
Supplemental Material
References
Information & Authors
Information
Published In
History
Keywords
- Allergy and immunology
- COVID-19
- Diagnostic medicine
- Diagnostic techniques
- Health care
- Health care providers
- Hospital medicine
- Infectious diseases
- Molecular biology
- Patients
- Polymerase chain reaction
- Prevention, policy, and public health
- Pulmonary diseases
- Pulmonary medicine
- Research and reporting methods
- Respiratory infections
- Respiratory system
- Safety
- Safety equipment
- Upper respiratory tract infections
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Variation in False-Negative Rate of Reverse Transcriptase Polymerase Chain Reaction–Based SARS-CoV-2 Tests by Time Since Exposure. Ann Intern Med.2020;173:262-267. [Epub 13 May 2020]. doi:10.7326/M20-1495
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Influence of viral concentration on false negatives
Limits of Detetction
Delays in testing as a source of COVID-19 false negative results.
The article by Kucirka and colleagues [1] highlights a major weakness in our reliance for diagnosis and public health surveillance on reverse transcriptase polymerase chain reaction (RT-PCR) based tests using nasopharyngeal (NP) or oropharyngeal (OP) sampling to determine community spread of “severe acute respiratory syndrome 2” (SARS-CoV-2) virus infection, which leads to “coronavirus disease 2019” (COVID-19). For mild to moderately ill patients, the window of reliable detection seems to be 7-10 days from onset of symptoms based on studies referenced by Kucirka [1], in addition to findings from Pan [2] and He [3].
Government-run COVID-19 assessment centers (at least in Canada) have been fraught with booking delays from the time that symptomatic patients call 811 for an appointment to the time of actual testing. In my experience, patients have reported waiting 5 days on average (ranging from 3 days to 2 weeks), while already feeling unwell for several days. Systemic delays in testing among probable community cases raises the risk of preventable false negative results. The following example illustrates this problem in practice.
On 22 April 2020, three patients called our office requesting COVID-19 testing, since they were still waiting to receive a centralized booking. All three were brought into the clinic immediately and tested using NP viral swab kits left over from the annual influenza surveillance program. Two of three patients were reported positive for SARS-CoV-2 infection the following day. One of the confirmed cases was a parent with a spouse and two children at home with similar symptoms. The older child started to become sick at the same time as the case, while the spouse became ill a few days later followed by the younger child. As the clinic did not have sufficient kits for testing the rest of the family, they contacted the local assessment center. When tested on 1 May 2020 (7 days later), the older child was “negative”, the spouse was initially “indeterminate” (later classified as “positive”) and the younger child was “positive”. Detection of SARS-CoV-2 virus in the upper airway appears to be short-lived in most moderately ill cases similar to that of influenza viruses [3].
Decentralizing testing back into community-based clinics as part of routine medical care and reducing backlogs in centralized assessment centers by not testing persons with symptoms beyond 7-10 days would reduce the risk of false negative tests. As we open up the economy, we require smarter surveillance that better tracks community spread.
[1] Kucirka LM, Lauer SA, Laeyendecker O, Boon D, Lessler J. Variation in False-Negative Rate of Reverse Transcriptase Polymerase Chain Reaction-Based SARS-CoV-2 Tests by Time Since Exposure. Ann Intern Med. 2020 May 13. doi: 10.7326/M20-1495. Available: https://www.acpjournals.org/doi/full/10.7326/M20-1495 (accessed 2020 May 25).
[2] Pan Y, Zhang D, Yang P, Poon LLM, Wang Q. Viral load of SARS-CoV-2 in clinical samples. Lancet Infect Dis. 2020;20(4):411‐412. doi:10.1016/S1473-3099(20)30113-4. Available: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7128099/ (accessed 2020 May 25).
[3] He X, Lau EHY, Wu P, et al. Temporal dynamics in viral shedding and transmissibility of COVID-19. Nat Med. 2020;26(5):672‐675. doi:10.1038/s41591-020-0869-5. Available: https://www.nature.com/articles/s41591-020-0869-5 (accessed 2020 May 25).
Refrain from modeling
We agree with the spirit of article regarding a cautious approach in removing isolation precautions and using clinical suspicion along with RT-PCR for suspected SARS-CoV-2 infection. Kucirka et al(1) conclusion that deeming a patient COVID-free on the basis of a single negative RT-PCR test is fraught with peril, especially in those early in the course of the infection. We also appreciate that these authors posted their data and code and by so doing gave readers like us the opportunity to offer specific critique; this is the way science advances.
We have two concerns. First, the authors did not include Young et al (2) which offered serial RT-PCR data on 18 patients in Singapore, leading us to wonder what other sources of data were missed. Second, and perhaps more important, we are concerned that data are so sparse that the results are based more on model assumptions than data. The authors identified 7 studies providing results of serial RT-PCR and time of symptom onset. They assume that exposure occurred 5 days prior to onset of symptoms. The authors then fit a bayesian hierarchical logistic model with a smoothing function in order to draw inference on the false-negative rate by day since exposure. Only one study, Danis et al (3) , had RT-PCR results for specimens obtained before the patients were symptomatic, however careful examination of that paper reveals that this consists of 4 sequential negative tests in a single patient (case 13) on days 2 to 5 before symptom onset. That is their entire data for the presymptomatic period.
Given this, it is impossible to make any inferences for the presymptomatic days and it would have been wiser to refrain from modeling and simply say that these data cannot tell us when any positive results can be expected but that it is clear that there will be negative RT-PCR tests in both in the early symptomatic days and the pre-symptomatic days.
1. Kucirka LM, Lauer SA, Laeyendecker O, Boon D, Lessler J. Variation in False-Negative Rate of Reverse Transcriptase Polymerase Chain Reaction–Based SARS-CoV-2 Tests by Time Since Exposure. Annals of Internal Medicine. 2020 May 13.
2. Young BE, Ong SW, Kalimuddin S, Low JG, Tan SY, Loh J, Ng OT, Marimuthu K, Ang LW, Mak TM, Lau SK. Epidemiologic features and clinical course of patients infected with SARS-CoV-2 in Singapore. Jama. 2020 Apr 21;323(15):1488-94.
3. Danis K, Epaulard O, Bénet T, Gaymard A, Campoy S, Bothelo-Nevers E, Bouscambert-Duchamp M, Spaccaferri G, Ader F, Mailles A, Boudalaa Z. Cluster of coronavirus disease 2019 (Covid-19) in the French Alps, 2020. Clinical Infectious Diseases. 2020 Apr 11.
What is the Value of Reverse Transcriptase Polymerase Chain Reaction–Based SARS-CoV-2 Tests for Screening Asymptomatic Individuals?
Kucirka and colleagues recently published an important analysis that accounts for time from exposure and symptom onset to obtain insights into the diagnostic performance of RT-PCR testing for infection with SARS-CoV-2 (1). Their analysis contributes to a better understanding of the sensitivity (1- false negative rate) of the RT-PCR test in the pre-symptomatic phase. The authors assume that test sensitivity is low (~0%) shortly after exposure and only increases to a reasonable value of ~60% at symptom onset (5 days later). The post-test probability of infection after a negative test remains substantial among pre-symptomatic individuals, especially when the population prevalence is high.
There is growing support among governments, public health agencies, and healthcare systems worldwide to adopt universal screening strategies despite direct evidence of benefit from screening asymptomatic individuals. Guidelines for universal screening from the Infectious Disease Society of America (IDSA) are based on the assumption that the sensitivity of the RT-PCR test found in symptomatic individuals can be applied to asymptomatic individuals (2). The authors’ findings however cast doubt on the validity of IDSA’s assumption and raise questions that are critical to address before developing and implementing an effective strategy for screening asymptomatic individuals.
First, the test sensitivity data from Kucirka and colleagues’ analysis only apply to the subgroup of pre-symptomatic individuals, i.e. those who develop symptoms later on, while widespread screening policies must be applicable to all asymptomatic individuals including up to 80% of infected individuals who never develop symptoms (3). Second, due to lack of data, the modeled sensitivity of the RT-PCR test in the pre-symptomatic phase depended on the assumed length of the incubation period. When assuming a 5-day period, the test sensitivity gradually declined to zero toward time of exposure (top panel in Figure 2). However, it is also plausible that shortly after exposure, sensitivity would increase more steeply and then level off (4). Lastly, the wide credible intervals limit the ability to assess diagnostic accuracy with confidence.
To conclude, there remains considerable uncertainty around the sensitivity of the RT-PCR test in asymptomatic individuals. We concur with IDSA’s recommendations that as cities, countries and healthcare systems move towards reopening, well-designed diagnostic accuracy studies should become a high-priority research focus to better understand the variation in test sensitivity over the course of infection (5). The value of acquiring additional information from such studies could be used to inform decision models to more effectively guide screening policies.
References
Methodological Limitations
Comment: I read with great interests Kucirka et al.(1) that estimated the false-negative rate by day since infection based on seven published studies and estimated the probability of a false-negative result in an infected person on day 1 is 100% (95% CI, 100% to 100%). I identified two major limitations in the paper that could reject their conclusion.
Firstly, although, they estimated the probability of a false-negative before onset, their data include only one before-onset case that reported in Danis et al.(2). Estimation based on a small sample could be unreliable and robustness of the results should be examined. In the Sensitivity analysis section, they reported unchanged false-positive curves for repeated analysis excluding one out of seven studies in the Supplement Figure 3. However, the credible interval of estimates that bring important information on robustness of the results is not reported and curves for false-negative is not displayed. I replicated analysis using their code and data (3), then obtained the probability of a false-negative result in an infected person on day 1 is 100% with widened 95% CI of (39.6% to 100%) excluding Danis et al.. Moreover, I examined the estimates of their model that determines the shape of daily false-negative and false-positive curves. As showed below, sign of coefficient revers for log(t), magnitude of coefficient are drastically changed for log(t)^2 and log(t)^3, and statistical significance of coefficient changed for log (t) and log(t)^2. All data logit(pj,t) = -0.12 4.25 log(t) - 31.76 log(t)^2 14.12 log(t)^3 (95%CI= -0.63, 0.40) (-6.32, 15.48) (-48.72, -15.54) (4.82, 23.93) Excluding Danis et al logit(pj,t) = 0.13 - 11.69 log(t) -2.03 log(t)^2 2.57 log (t)^3 (-0.46, 0.70) (-14.30, -9.13) (-4.35, 0.29) (0.17, 4.91)
Secondly, the paper neglects the nature of data that leads to model misspecification and biased estimates. According to Supplement Table 2 that summarize source data, original papers reported results of daily testing or repeated measure of subjects. In spite of that, results are tabulated into study by days from onset, then hierarchical regression model that incorporate study heterogeneity is estimated that assumes test results from the same subject are independent. Enough size of pre-onset cases, proper data handling and modeling that reflect nature of data are necessary to obtain reliable results. Careful model diagnosis must be conducted to reach a proper conclusion.
Acknowledgement I appreciate Dr. Lauer SA. for publish data and script.
Reference
Authors' Response to Comment Posted by Takhar et al
In response to online comments raised by Takhar et al. (May 29, 2020), We understand that there may have been studies that we missed, although the paper by Young et al was published after our search period of Arpil 15, 2020. This is why, as the commenter’s state, we made all code and data publicly available so that the analysis can be updated as more studies are published. We have re-run the analysis with the data from Young et al. and have found no qualitative differences in results.
We recognize that there are legitimate potential criticisms of extrapolating from trends occurring after symptom-onset to make estimates about the pre-symptomatic period; however, we felt that these extrapolations carried a very important clinical message in terms of the likely high false negative rate and high level of uncertainty about the performance of the test in this period. As more studies begin to report data on testing in pre-symptomatic individuals, it will be important to update these results.
We recognize that, in retrospect, there are some modeling assumptions we could have made to further highlight the uncertainty of estimates in this period; for instance, treating the time of symptom onset as a random variable. These changes would have increased represented uncertainty intervals in certain periods, but not changed the core results.
We respect the commenters’ difference in opinion about the utility of this modeling but continue to believe it provides important insights that we hope will be further informed by later work.
Good review … on COVID-19 false negative report
Thanks for such an elaborate review of a subject which has many areas of unseen universe. It also was helpful in knowing this topic at large.
We at Indian areas go for HRCT Thorax if we were in doubt of result.
Many time it is seen that inadequate sample collection or collected with improper techniques create such reports dilemma.
Thank you again.
Nicely shown stastitical data on CoVID.
Thanks for posting such elaborate statistics on SARS-COV-2. We have noticed such results in last year or so. And have gone with HRCT reports for same.
It is indeed needed to know such values and must be clinically judged on diseased state.