key: cord-0778310-0kyf7ikr authors: Bullis, Sean S.M.; Crothers, Jessica W.; Wayne, Shawn; Hale, Andrew J. title: A Cautionary Tale of False-Negative Nasopharyngeal COVID-19 Testing date: 2020-05-05 journal: IDCases DOI: 10.1016/j.idcr.2020.e00791 sha: c27d323cc9527571b8066018cbf7aa84b79c1cc6 doc_id: 778310 cord_uid: 0kyf7ikr Abstract There remains diagnostic uncertainty regarding the sensitivity of reverse transcription polymerase chain reaction in detection of SARS-CoV-2 from nasopharyngeal specimens. We present a case where two nasopharyngeal specimens were negative, followed by a positive sputum sample. Serial testing for COVID-19 is indicated in patients with high pretest probability of disease. We present the case of a patient that, based on known exposure to a COVID-19 positive family member, typical symptoms, suggestive labs, and consistent imaging, had a high pre-test probability of having COVID-19, yet tested negative on two successive NP RT-PCRs. Only on the third COVID-19 sample, taken from sputum, was the patient ultimately correctly diagnosed. As appropriate precautions were stopped after the second negative NP swab, several medical personnel were potentially exposed to SARS-CoV-2 in the process. In the setting of a high pretest probability for COVID-19, a negative NP RT-PCR result (and in the case presented, multiple negative results) may represent a false negative. Given a growing appreciation in the literature for both heterogeneity of presentation and disease severity, it is critical to have a clear sense of COVID-19 testing performance (2) . It is unclear why our patient's first two nasopharyngeal swabs for SARS-CoV-2 were negative. Possible explanations include improper collection or handling technique, viral load below the detectable limit of the assay, or diminished upper airway viral shedding. The latter possibly reflects the natural history of the disease wherein duration of viral shedding (which may precede symptom onset by several days) was observed to be as few as eight days to as many as 37 (1); additionally, it is conceivable that the patient's immunocompromised state may have contributed. The precise test characteristics of a single NP RT-PCR for detection of SARS-CoV-2 are unknown. Available data suggest a range of sensitivities that likely increase with repetition. This may relate to varying assays based on the country of origin, as well as the reference standard used for a positive or presumptive positive test (e.g., viral culture, radiographic findings). Table 1 outlines the observed sensitivities from several recent publications (2) (3) (4) (5) . At the early stages of a novel disease when the clinical sensitivity of a given assay is poorly understood, its analytic J o u r n a l P r e -p r o o f sensitivity, or limit of detection (LoD), can offer a useful point of reference and comparability. The LoDs reported across the two assays employed in this patient's case however were derived from varying methodologies resulting in entirely different units of measurement (RNA copies/L vs. TCID50/mL), making early comparisons difficult. The genes targeted in these two assays also differ significantly. While the N1 and N2 genes are included in the CDC assay, Roche® targets the nonstructural ORF1a gene of SARS-CoV-2 in combination with the E gene (envelope protein) of the broader Sarbecovirus group. The relative clinical sensitivity and specificity of these targets are unknown. In a recent study (2) involvement. Based on these observations, the authors theorized a de-isolation protocol that not only considers date from symptom onset as is suggested by CDC (7), but also viral load. In a similar study, To and colleagues sought to ascertain viral load dynamics of SARS-CoV-2 in the posterior oropharynx and tracheal aspirates, as well as antibody kinetics and the viral genome, by collecting serial swabs from 23 patients (8) . They found that viral loads correlated positively with age and peaked shortly after symptom onset and subsequently declined. Of note, they found that in one third of the cohort viral RNA could still be detected from the posterior oropharynx after 20 days. This observation questions the feasibility of incorporating viral load into deisolation protocols, as it remains unknown whether these patients are continuing to shed live virus versus inactive virions coated in neutralizing antibody. Finally, further complicating test interpretation is the notion that RT-PCR positivity may be intermittent as highlighted by Lan and colleagues, where four patients who had recovered from COVID-19 and tested negative by two oropharyngeal swabs separated by 24 hours, subsequently tested positive again (9) . The presented case serves as a cautionary tale on the limitations of the current state of diagnostic testing for COVID-19. In patients with a high pre-test probability of COVID-19, a single negative NP RT-PCR may be insufficient to rule-out disease. Additional studies ascertaining precise test characteristics will be essential in correctly identifying cases and avoiding the consequences that accompany improper de-isolation of patients who receive a false-negative result. Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study Detection of SARS-CoV-2 in different types of clinical specimens Sensitivity of chest CT for COVID-19: comparison to RT-PCR Correlation of chest CT and RT-PCR testing in coronavirus disease 2019 (COVID-19) in China: A Report of 1014 Cases Profiling early humoral response to diagnose novel coronavirus disease (COVID-19). Clin Infect Dis 2020. Epub ahead of print Virological assessment of hospitalized patients with COVID-2019. Nature 2020. Epub ahead of print Interim guidelines for collecting, handling, and testing clinical specimens from persons for Coronavirus Disease Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study Positive RT-PCR test results in patients recovered from COVID-19. JAMA 2020. Epub ahead of print