A full-term pregnant patient was admitted in March 2020 for a scheduled Cesarean delivery, before being tested according to a universal inpatient screening protocol for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). During surgery, the patient developed a fever and required oxygen supplementation. The facility implemented contact and droplet isolation precautions due to suspicion for COVID-19. The patient was transferred to a negative pressure room, the baby was isolated in the neonatal intensive care unit (NICU), and the patient’s husband was instructed to self-isolate at home until the SARS-CoV-2 test results were available.
A specimen obtained via nasopharyngeal swab was sent to a commercial laboratory for reverse transcriptase polymerase chain reaction (RT-PCR) testing. However, due to delays in receiving those results, another sample was tested two days later with a newly developed in-house test and a third sample was sent to the state public health laboratory. The in-house test returned as positive for SARS-CoV-2. Blood and urine cultures showed no growth, and a chest radiograph was unremarkable.
When the patient left the hospital on her fourth postpartum day, she was in stable clinical condition. She reported feeling well, without cough, fever, or shortness of breath. Because she was assumed to be positive for SARS-CoV-2, but clinically healthy, she was advised to isolate herself at home for 14 days, and the hospital initiated contact tracing of all exposed persons, in consultation with the local public health department. In addition, because she had a newborn and a toddler at home, she was encouraged to be vigilant about wearing a mask, to stay in one room of the house if possible, and to focus on hand hygiene and surface cleaning.
Two days after the mother’s discharge, the commercial and state lab tests were both reported as negative. The patient was concerned about SARS-CoV-2 transmission, so she chose to bottle-feed rather than breastfeed, despite her pediatrician’s encouragement to breastfeed. An infant hearing test was deferred as the tester did not want to risk exposure to SARS-CoV-2. Ultimately, the patient and her baby did well clinically, although the false positive result caused substantial anxiety and alterations in home care (personal and parental) and health care during the critical transitional period of caring for a newborn child. The patient and her family were extremely forgiving of the error, appreciating the uncertainties related to the pandemic and the care and communication from health care staff.
A thorough root-cause analysis subsequently determined that the positive test run on the in-house platform was due to cross-contamination from a neighboring positive sample. A follow-up test conducted on a remnant sample from the in-house assay, one week later, confirmed that the patient was negative. When it was determined that the in-house test was falsely positive, testing was shifted to other commercial platforms and to a second-generation in-house test. Since that time, more than 50,000 tests have been run, and there have been no other known cases of false positive tests due to cross-contamination
By Stephen A. Martin, MD, EdM, Gordon D. Schiff, MD, Sanjat Kanjilal, MD, MPH
False positive test results can lead to considerable anxiety on the part of patients and clinicians. In this case, there was additional complexity due to the patient’s post-partum status and the potential for transmission of a newly discovered pandemic virus to susceptible hosts, including other patients, family, and particularly a newborn infant. We have come to learn a great deal about COVID-19 in the ensuing months, but the first months of the pandemic were full of unknowns. The United States also had scarce testing capacity, leading to rationing and delays in receiving test results.1
Ironically, false positive results also contributed to early delays in introducing SARS-CoV-2 RT-PCR testing in the United States. After initial SARS-CoV-2 RT-PCR test kits from the US Centers for Disease Control and Prevention (CDC) were distributed in early February 2020, state public health laboratories performing basic quality checks encountered positive results when testing their negative control specimens of purified water.2 The kits were suspended by CDC for diagnostic use because one of three primer/probe sets was generating these incorrect results, potentially due to contamination before shipment. On February 26, 2020, states were instructed to remove this set and begin diagnostic testing; no patients received false positive test results.3
The patient in this case was affected by limited national testing capacity in these early weeks of the pandemic, leading to delays in commercial laboratory testing. As a result, there was understandable urgency to use an in-house test with faster turnaround. What was not known to the patient, her clinicians, or the hospital lab was that the patient’s particular specimen was contaminated due to carry-over of RNA from neighboring true positive specimens during the nucleic acid extraction and amplification steps. Extremely sensitive molecular assays are susceptible to analytic errors such as this one;4 preventing such errors depends upon fastidious care in conducting the tests and multiple quality control checks. However, in the early days of this pandemic, the stresses and sense of panic might have undermined standard operating procedures when evaluating novel tests for a novel microbe.
In this case, the family was significantly inconvenienced by this testing error as they avoided breastfeeding, were isolated at home for a 14-day quarantine period, and experienced “substantial anxiety.” It is important to note that the American Academy of Pediatrics, the CDC, and the World Health Organization encourage women with suspected or confirmed COVID-19 to initiate or continue breastfeeding, if desired, with mask use and hand hygiene. However, these protective measures and reduced skin-to-skin contact time may lead to decreased breast milk production. False positive results may harm the tested person and their contacts in other ways, during a pandemic.5 These impacts may include: 1) unnecessary isolation, including unnecessarily missed work or school days; 2) new outbreaks when uninfected people given false positive test results are subsequently housed with truly infected people in residential settings; 3) missed or inappropriate treatment as COVID-19 therapies (e.g., monoclonal antibodies) are given and the actual diagnosis is not treated; 4) a false sense of security and vaccine refusal as people may think they have already had COVID-19 and are thus immune; 5) undue stress; and 6) loss of trust in testing.5,6
Despite the false-positive error the patient experienced in this case, the true positive rate for RT-PCR testing is exceedingly high. FDA submissions for both RT-PCR and antigen testing for SARS-CoV-2 often list specificities of 98-100%. More than 200 tests related to SARS-CoV-2 have received Emergency Use Authorization (EUA) from FDA.7 To accelerate medical product availability during an emergency, the EUA standard is less rigorous than other FDA approval/clearance standards. FDA considers both the analytic performance of COVID-19 tests—or, accuracy measured in a laboratory—and the clinical performance, for tests to receive authorization.8 Clinical performance, however, may be lower in community practice than in the manufacturer’s testing due to viral dynamics, sampling technique, and—in the case of antigen testing—antigen-antibody interaction error.9 An additional type of false positive RT-PCR result occurs when nonviable SARS-CoV-2 particles are detected weeks to months after a person’s recovery from COVID-19. Current routine testing cannot distinguish viral components (RNA or proteins) from the contagious whole virus. Recognizing the potential for this error, CDC recommends avoiding follow-up testing for patients with a positive SARS-CoV-2 result for three months after the onset of symptoms.10
While false positive results from RT-PCR are rare, a policy suggestion has been made for frequent, rapid, mass population-based antigen testing, which has a higher false positive rate than RT-PCR tests.11,12 Even if antigen test specificity is as high as 98% in practice, many (even most) positive results will be false in a population with low prevalence of SARS-CoV-2 (e.g., ≤2%).13,14,15 False positive antigen results led the FDA to issue an urgent national warning in early November 2020 cautioning about this error.13 The World Health Organization (WHO) recommends against use of antigen testing in such low prevalence circumstances because of the low positive predictive value.14 The alternative is to immediately confirm all positive antigen results with RT-PCR in low prevalence16 or asymptomatic populations,15 a strategy subject to further potential error and delay. Of course, errors with SARS-CoV-2 testing are not limited to analytical and clinical performance. Challenges in communication from clinicians to patients affect all testing, both in terms of actually conveying results and also having the implications of those results understood.17,18
For a test to be considered accurate, an accurate reference is required. Since the start of the pandemic, defining a reference (“gold”) standard for the detection of SARS-CoV-2 and the related diagnosis of COVID-19 has been difficult because of RT-PCR’s limited sensitivity.19-29 Pooled inpatient studies using regression modeling have found test sensitivity ranging from 0% on the day after exposure to SARS-CoV-2 to 80-89% in the first four days after onset of symptoms.30-32 Limited outpatient studies have found similarly variable RT-PCR sensitivity.33,34 Recently updated guidance from the Infectious Disease Society of America (IDSA) reports the sensitivity of RT-PCR from the upper airway to still be only 71%.35 Although the ddPCR (droplet digital) technique may improve RT-PCR sensitivity,36,37 it is not currently in routine use.
It is also important for clinicians to recognize that antigen testing has even more significant limitations than RT-PCR in current clinical practice. WHO14 reports that antigen testing has a percent positive agreement ranging from 0-94% when compared with RT-PCR, depending on the specific study design and the test brand that was evaluated. Lower-range clinical performance continues to be found in recent studies, especially among asymptomatic individuals,29,31-41 and the operating characteristics of antigen tests may be inflated by the choice of comparator(s).42 CDC therefore recommends all negative antigen tests in symptomatic people be confirmed by molecular testing.15 Clinicians should appreciate the risk and consequences of providing mistaken reassurance from false negative SARS-CoV-2 results.42,43
Thus, as we enter the next phases of the COVID-19 pandemic, it will be important to learn from the errors and limitations of our practices during its initial months. How to strategically deploy and interpret testing for SARS-CoV-2 will remain central in overcoming this highly contagious, potentially lethal, and—as seen in this case—anxiety-producing virus.
- The performance of RT-PCR assays for SARS-CoV-2 in clinical practice varies widely due to temporal changes in the dynamics of viral shedding, techniques of specimen collection, and a small but real potential for laboratory errors such as cross-contamination.
- Interpretation of the results of any test with imperfect specificity and sensitivity must take into account the prior probability of contracting the disease, which, in this case, means (when used for symptomatic or exposed patients) both a patient's clinical probability and the population-based prevalence of active COVID-19 infection. The fact that so many people with SARS-CoV-2 infection are both asymptomatic (or presymptomatic) and contagious further complicates such interpretations.
- The unprecedented stresses and uncertainties of the early weeks of the pandemic placed demands on everyone in the health system, leading to improvised actions and testing approaches. More recently, clinical practices and laboratory procedures related to SARS-CoV-2 have improved, leading to more reliable testing and better outcomes. However, as SARS-CoV-2 testing still lacks a reference gold standard and the policies regarding SARS-CoV-2 tests remain adjusted in response to the public health emergency,44 we should be cautious in our test interpretation while we pursue evidence on the best strategies for test utilization and interpretation.
- The post-partum patient in this case who experienced a false positive testing error illustrates the consequences of such errors for both new mothers and their newborn infants (e.g., needless isolation, anxiety, interruption of breastfeeding, and deferral of screening tests), as well as the importance and power of timely disclosure, apology, and learning and improvement from errors.
Stephen A. Martin, MD, EdM
Barre Family Health Center, UMassMemorial Health Care
Department of Family Medicine and Community Health
University of Massachusetts Medical School
Gordon D. Schiff, MD
Brigham and Women’s Hospital
Harvard Medical School
Sanjat Kanjilal, MD, MPH
Department of Population Medicine, Harvard Medical School & Harvard Pilgrim Healthcare Institute
Associate Medical Director of Clinical Microbiology
Brigham and Women’s Hospital
Harvard Medical School
The long-standing process for submitting PSNet WebM&M case submissions is anonymous. Users may contribute by submitting a case at the following link: https://psnet.ahrq.gov/webmm/submit-case.
Periodically, the Primary-Care Research in Diagnosis Errors (PRIDE) Learning Network, a collaborative project convened by the Brigham and Women’s Hospital Center for Patient Safety Research and Practice and the State of Massachusetts Betsy Lehman Center for Patient Safety, contributes cases and commentaries from their monthly discussions of diagnosis error cases to PSNet. PRIDE is funded by a grant from the Gordon and Betty Moore Foundation. This case was produced in cooperation with the PRIDE Learning Network. We acknowledge the assistance of the PRIDE project director Maria Mirica, PhD, in preparing this Case and Commentary.
- Shear MD, Goodnough A, Kaplan S, Fink S, Thomas K, Weiland N. The Lost Month: How a Failure to Test Blinded the U.S. to Covid-19. New York Times. 2020. Available at: https://www.nytimes.com/2020/03/28/us/testing-coronavirus-pandemic.html. Accessed January 20, 2021.
- Willman D. Contamination at CDC lab delayed rollout of coronavirus tests. Washington Post. 2020. Available at: https://www.washingtonpost.com/investigations/contamination-at-cdc-lab-…. Accessed November 12, 2020.
- Jernigan D, Centers for Disease Control and Prevention (CDC). Revision to Test Instructions: CDC 2019 Novel Coronavirus (nCoV) Real-Time RT-PCR Diagnostic Panel (EUA200001). 2020. Available at: https://www.aphl.org/Materials/Signed_CDC_Letter_to_PHLs-N3_Removal_Ins…. Accessed February 1, 2021.
- Tang YW, Schmitz JE, Persing DH, Stratton CW. Laboratory diagnosis of COVID-19: Current issues and challenges. J Clin Microbiol. 2020;58(6). doi:10.1128/JCM.00512-20.
- Surkova E, Nikolayevskyy V, Drobniewski F. False-positive COVID-19 results: hidden problems and costs. Lancet Respir Med. 2020;8(12):1167–1168. doi:10.1016/S2213-2600(20)30453-7.
- Healy B, Khan A, Metezai H, Blyth I, Asad H. The impact of false positive COVID-19 results in an area of low prevalence. Clin Med (Northfield Il). 2020;21(1):clinmed.2020-0839. doi:10.7861/clinmed.2020-0839.
- US Food and Drug Administration. In Vitro Diagnostics EUAs. Available at: https://www.fda.gov/medical-devices/coronavirus-disease-2019-covid-19-e…. Accessed February 1, 2021.
- Yohe S, College of American Pathologists. How Good are COVID-19 (SARS-CoV-2). 2020. Available at: https://www.cap.org/member-resources/articles/how-good-are-covid-19-sar…. Accessed November 12, 2020.
- Cohen A, Kessel B, Milgroom M. Diagnosing COVID-19 infection: the danger of over-reliance on positive test results. medRxiv. 2020:2020.04.26.20080911. doi:10.1101/2020.04.26.20080911.
- Duration of Isolation and Precautions for Adults with COVID-19 | CDC. 2021. Available at: https://www.cdc.gov/coronavirus/2019-ncov/hcp/duration-isolation.html. Accessed February 1, 2021.
- Mina MJ, Parker R, Larremore DB. Rethinking Covid-19 Test Sensitivity — A Strategy for Containment. N Engl J Med. 2020. doi:10.1056/nejmp2025631.
- Gill M, Gray M. Mass testing for covid-19 in the UK. BMJ. 2020;371:m4436. doi:10.1136/bmj.m4436.
- US Food and Drug Administration. Potential for False Positive Results with Antigen Tests for Rapid Detection of SARS-CoV-2 - Letter to Clinical Laboratory Staff and Health Care Providers. 2020. Available at: https://www.fda.gov/medical-devices/letters-health-care-providers/poten…. Accessed November 12, 2020.
- World Health Organization. Antigen-detection in the diagnosis of SARS-CoV-2 infection using rapid immunoassays: Interim guidance. 2020. Available at: https://www.who.int/publications/i/item/antigen-detection-in-the-diagno…. Accessed December 4, 2020.
- Centers for Disease Control and Prevention. Interim Guidance for Antigen Testing for SARS-CoV-2. 2020. Available at: https://www.cdc.gov/coronavirus/2019-ncov/lab/resources/antigen-tests-g…. Accessed February 15, 2021.
- European Centre for Disease Prevention and Control. Options for the use of rapid antigen tests for COVID-19 in the EU/EEA and the UK. 2020. Available at: https://www.ecdc.europa.eu/en/publications-data/options-use-rapid-antig…. Accessed December 4, 2020.
- Roy CL, Rothschild JM, Dighe AS, et al. An initiative to improve the management of clinically significant test results in a large health care network. Jt Comm J Qual Patient Saf. 2013;39(11):517–527. doi:10.1016/S1553-7250(13)39068-0.
- Singh H, Vij MS. Eight recommendations for policies for communicating abnormal test results. Jt Comm J Qual patient Saf. 2010;36(5):226–32. doi:10.1016/s1553-7250(10)36037-5.
- Jarrom D, Elston L, Washington J, et al. Effectiveness of tests to detect the presence of SARS-CoV-2 virus, and antibodies to SARS-CoV-2, to inform COVID-19 diagnosis: a rapid systematic review. BMJ Evidence-Based Med. 2020:bmjebm-2020-111511. doi:10.1136/bmjebm-2020-111511.
- Dramé M, Tabue Teguo M, Proye E, et al. Should RT-PCR be considered a gold standard in the diagnosis of COVID-19? J Med Virol. 2020;92(11):2312–2313. doi:10.1002/jmv.25996.
- Bossuyt PM. Testing COVID-19 tests faces methodological challenges. J Clin Epidemiol. 2020;126:172–176. doi:10.1016/j.jclinepi.2020.06.037.
- Watson J, Whiting PF, Brush JE. Interpreting a covid-19 test result. 2020;369:m1808. doi:10.1136/bmj.m1808.
- Wang W, Xu Y, Gao R, et al. Detection of SARS-CoV-2 in Different Types of Clinical Specimens. JAMA. 2020;323(18):1843–1844. doi:10.1001/jama.2020.3786.
- Woloshin S, Patel N, Kesselheim AS. False Negative Tests for SARS-CoV-2 Infection — Challenges and Implications. N Engl J Med. 2020;383(6):e38. doi:10.1056/NEJMp2015897.
- Stites EC, Wilen CB. The Interpretation of SARS-CoV-2 Diagnostic Tests. Med. 2020. doi:10.1016/j.medj.2020.08.001.
- Green DA, Zucker J, Westblade LF, et al. Clinical Performance of SARS-CoV-2 Molecular Tests. McAdam AJ, ed. J Clin Microbiol. 2020;58(8). doi:10.1128/JCM.00995-20.
- Axell-House DB, Lavingia R, Rafferty M, Clark E, Amirian ES, Chiao EY. The estimation of diagnostic accuracy of tests for COVID-19: A scoping review. J Infect. 2020;81(5):681–697. doi:10.1016/j.jinf.2020.08.043.
- Parikh BA, Bailey TC, Lyons PG, Anderson NW. The brief case: “Not Positive” or “Not Sure”—COVID-19-negative results in a symptomatic patient. J Clin Microbiol. 2020;58(8). doi:10.1128/JCM.01195-20.
- Arnaout R, Lee RA, Lee GR, et al. The Limit of Detection Matters: The Case for Benchmarking Severe Acute Respiratory Syndrome Coronavirus 2 Testing. Clin Infect Dis. 2021. doi:10.1093/cid/ciaa1382.
- 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:M20-1495. doi:10.7326/M20-1495.
- Arevalo-Rodriguez I, Buitrago-Garcia D, Simancas-Racines D, et al. False-negative results of initial RT-PCR assays for COVID-19: A systematic review. PLoS One. 2020;15(12 December). doi:10.1371/journal.pone.0242958.
- Mallett S, Allen AJ, Graziadio S, et al. At what times during infection is SARS-CoV-2 detectable and no longer detectable using RT-PCR-based tests? A systematic review of individual participant data. BMC Med. 2020;18(1). doi:10.1186/s12916-020-01810-8.
- Zhang Z, Bi Q, Fang S, et al. Insight into the practical performance of RT-PCR testing for SARS-CoV-2 using serological data: a cohort study. The Lancet Microbe. 2021. doi:10.1016/s2666-5247(20)30200-7.
- Baron RC, Risch L, Weber M, et al. Frequency of serological non-responders and false-negative RT-PCR results in SARS-CoV-2 testing: A population-based study. Clin Chem Lab Med. 2020;58(12):2131–2140. doi:10.1515/cclm-2020-0978.
- Hanson KE, Caliendo AM, Arias CA, et al. The Infectious Diseases Society of America Guidelines on the Diagnosis of COVID-19: Molecular Diagnostic Testing. Clin Infect Dis. 2021. doi:10.1093/cid/ciab048.
- Alteri C, Cento V, Antonello M, et al. Detection and quantification of SARS-CoV-2 by droplet digital PCR in real-time PCR negative nasopharyngeal swabs from suspected COVID-19 patients. PLoS One. 2020;15(9 September). doi:10.1371/journal.pone.0236311.
- Suo T, Liu X, Feng J, et al. ddPCR: a more accurate tool for SARS-CoV-2 detection in low viral load specimens. Emerg Microbes Infect. 2020;9(1):1259–1268. doi:10.1080/22221751.2020.1772678.
- Pray IW, Ford L, Cole D, et al. Performance of an Antigen-Based Test for Asymptomatic and Symptomatic SARS-CoV-2 Testing at Two University Campuses — Wisconsin, September–October 2020. MMWR Morb Mortal Wkly Rep. 2021;69(5152):1642–1647. doi:10.15585/mmwr.mm695152a3.
- Wise J. Covid-19: Lateral flow tests miss over half of cases, Liverpool pilot data show. BMJ. 2020;371:m4848. doi:10.1136/bmj.m4848.
- Okoye NC, Barker AP, Curtis K, et al. Performance Characteristics of BinaxNOW COVID-19 Antigen Card for Screening Asymptomatic Individuals in a University Setting. J Clin Microbiol. 2021. doi:10.1128/JCM.03282-20.
- Procop GW, Kadkhoda K, Rhoads DD, Gordon SG, Reddy AJ. Home testing for COVID-19: Benefits and limitations. Cleve Clin J Med. 2021. doi:10.3949/ccjm.88a.ccc071.
- Fitzpatrick MC, Pandey A, Wells CR, Sah P, Galvani AP. Buyer beware: inflated claims of sensitivity for rapid COVID-19 tests. Lancet. 2021;397(10268):24–25. doi:10.1016/S0140-6736(20)32635-0.
- Deeks JJ, Raffle AE. Lateral flow tests cannot rule out SARS-CoV-2 infection. BMJ. 2020;371. doi:10.1136/bmj.m4787.
- US Food and Drug Administration. Policy for Coronavirus Disease-2019 Tests During the Public Health Emergency (Revised): Immediately in Effect Guidance for Clinical Laboratories, Commercial Manufacturers, and Food and Drug Administration Staff. May 11, 2020. Available at: https://www.fda.gov/media/135659/download. Accessed March 9, 2021.