key: cord-0979802-vt2sqolu
authors: Lesho, Emil; Reno, Lisa; Newhart, Donna; Clifford, Robert; Vasylyeva, Olga; Hanna, John; Yu, Stephanie; Bress, Jonathan; Walsh, Edward
title: Temporal, Spatial, and Epidemiologic Relationships of SARS-CoV-2 Gene Cycle Thresholds: A Pragmatic Ambi-directional Observation
date: 2020-08-25
journal: Clin Infect Dis
DOI: 10.1093/cid/ciaa1248
sha: 622ae918ee2586bea49ebad82c4d7e504fddc00a
doc_id: 979802
cord_uid: vt2sqolu

Prospective serial sampling of 70 patients revealed clinically relevant cycle thresholds (Ct) occurred 9, 26, and 36 days after symptom onset. Race, gender, or corticosteroids did not appear to influence RNA-positivity. Retrospective analysis of 180 patients revealed that initial Ct did not correlate with requirement for admission or intensive care

The optimal timing of public health decisions, including when to discontinue isolation precautions (IP) for patients hospitalized with SARS-CoV-2 infection remains unclear. Federal, state, and professional guidelines are not consistently harmonized. In many locations, the availability of testing remains limited, and repeated testing after symptom resolution for patient disposition further stresses scarce resources.

Despite several reports of viral RNA kinetics [1-6], U.S. data, especially those correlating viral kinetics to infection control practice, are scarce. Also unclear is whether dexamethasone, now widely used, prolongs RNA-positivity. Against this backdrop, we sought to evaluate temporal and spatial associations between demographic factors, disease severity, corticosteroid use and SARS-CoV-2 real-time PCR cycle threshold (RT-PCR Ct) to inform the application of IP.

From 03/14/2020 to 06/01/2020, nasopharyngeal (NP) swabs from SARS-CoV-2 infected hospitalized patients were prospectively collected weekly from admission. Samples were collected in a standardized protocol using flocked or polyester swabs applied to each middle turbinate and rotated for 5-10 seconds, placed in 3 ml viral transport medium, and immediately analyzed using an emergency-use authorized laboratory-developed-EUA assay or commercial platforms. Each platform targeted the RNA-dependent RNA polymerase, N, ORF1ab, S, and/or PanSARS-E genes. The These were excluded from both groups.) To be included in the steroid group, patients had to receive at least one full day of therapy, defined as >/=4 mg of methylprednisolone or >/=7.5 mg prednisone or prednisolone per 24 hrs.

Separately, a regional registry of SARS-CoV-2 positive ambulatory and hospitalized patients was retrospectively queried. The registry had been created on 03/21/20 to capture detailed clinical data from every patient with a positive SARS-CoV-2 RT-PCR in a five-hospital healthcare system in upstate NY. It currently contains data from 399 RT-PCR positive patients.

Symptom onset (SO) was determined from the history and defined as how many days the patient felt ill before presenting for care and having a positive test. For the retrospective sample, author JH manually reviewed every record.

ANOVA, Mann-Whitney U, and the 2-sample percent defective tests were performed in R or Minitab.

Prospective sample 235 NP samples from 70 hospitalized patients were prospectively evaluated. The demographic, co-morbidities, outcomes, and isolation status are presented in Table 1 

Regarding the prospectively studied cohort, exposure to corticosteroids did not appear to prolong RNA positivity; however, our sample size might be too small to detect a difference.

Nonetheless, to our knowledge it is the first study of a diverse cohort U.S. patients to include steroid exposure in analysis of Ct kinetics for IP application [7] . It was previously reported that the RT-A c c e p t e d M a n u s c r i p t 6 PCR Ct breakpoint above which virus was uncultivable varies from > 24 to  34, and perhaps higher for severely immunocompromised patients [1, 2, 8] . In our study, linear regression derived from a diverse cohort of 70 patients with various disease severities revealed that a Ct of 24, 34, and negative (>40) occurred 9, 26, and 36 days after SO (Supplemental Figure 2 ). This timing is consistent with those from a cohort of nine patients with relatively mild disease that included viral culture [3] , study that included cultures, but did not provide number of patients [2] and, a smaller study evaluating viral kinetics in saliva [4] . Together, these data offer reassurance that discontinuation of enhanced isolation/ PPE after day 10 is probably safe for immunocompetent patients who are improving and afebrile. Waiting for a completely negative Ct is probably unnecessary and contributes to avoidable consumption of personal protective equipment.

For the retrospectively analyzed cohort, initial Ct could not be used to differentiate those needing admission from those released after emergency department evaluation. Nor could initial Ct values be used to distinguish between those patients needing intensive care versus ward level care.

Our study has several limitations. First, the cultivation of the virus was not available due to safety concerns. Second, prospective data come from a single center. Third, a single assay could not be used for the entire duration of the observation period due to the initial limitation of test systems or reagents. As mentioned, most of the tests were performed on the laboratory-developed-EUA assay, and the variation was negligible as indicated by the CVs. The volume of sample required per assay, along with the ongoing shortage of kits and reagents, precludes re-testing samples on all platforms simultaneously. Nonetheless, it has recently been shown that commercially available and laboratorydeveloped platforms are quite comparable [9-11, S1-4] .

Despite these limitations, our findings are notable for several reasons. We provide actionable RT-PCR Ct values, other than negative, that can complement infection control decisions based on illness duration and symptom resolution. Furthermore, reports from non-university-based locations are underrepresented in research, and studies are carried out in large academic institutions. However, the majority of health care in the U.S. is provided at non-university-based facilities. Our data help A c c e p t e d M a n u s c r i p t 7 close this gap. 20% of the prospectively evaluated patients reverted to RT-PCR positivity after symptom improvement and defervescence for > 72 hours. Table 1 .In contrast, a previously reported study that found only 3% reverted to positivity [12] . Finally, although race and gender are known to affect SARS-CoV-2-related morbidity and mortality, they did not appear to influence RNA-positivity and therefore provide reassurance that infection control measures do not require adjustment for race or gender.

In conclusion, regression analysis of 235 patient samples from 70 patients was consistent with and supported by culture-derived data from other, albeit smaller or more homogenous studies [1-3].

RT-PCR Ct obtained at day nine can inform IP decisions. 43% of patients re-tested 4 weeks after SO had detectable RNA. Retrospective analysis of a 180-patient registry revealed that duration of RNApositivity did not appear to be affected by race or gender. Initial Ct did not correlate with requirement for admission or intensive care. These data provide a foundation for hypothesis generation and more in-depth and well controlled follow up studies. A c c e p t e d M a n u s c r i p t M a n u s c r i p t 

Viral RNA load as determined by cell culture as a management tool for discharge of SARS-CoV-2 patients from infectious diseases wards

Predicting infectious SARS-CoV-2 from diagnostic samples

Virological assessment of hospitalized cases of coronavirus disease 2019

Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study

Viral load dynamics and disease severity in patents infected with SARS-CoV-2 in Zhejiang province, China

Profile of RT-PCR for SARS-CoV-2: a preliminary study from 56 COVID-19 patients

Low-dose corticosteroid therapy does not delay viral clearance in patients with COVID-19

Disposition of patients with COVID-19 infection whose respiratory specimens remain SARS-CoV-2 PCR-positive

Comparison of commercially available and laboratory developed assays for in vitro detection of SARS-CoV-2 in clinical laboratories

Comparison of SARS-CoV-2 detection from nasopharyngeal swab samples by the Roche cobas 6800 SARS-CoV-2 test and a laboratory-developed rea-time RT-PCR test

Comparison of two high-throughput reverse transcription-polymerase chain reaction systems for the detection of severe acute respiratory syndrome coronavirus 2

Assessment of patients who tested positive for COVID-19 after recovery

A c c e p t e d M a n u s c r i p t 11 A c c e p t e d M a n u s c r i p t