Duration of SARS-CoV-2 viral culture positivity among different specimen types.

To the Editor,

We read with great interest the article Prolonged SARS-CoV-2 cell culture replication in respiratory samples from patients with severe COVID-19 by Folgueira et al., which reported that patients with severe coronavirus disease 2019 (COVID-19) might shed culturable virus [1]. Current guidelines recommend that individuals with COVID-19 can discontinue isolation without retesting 10 days after either symptom onset or a positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) test among asymptomatic persons [2]. That recommendation was made because previous studies did not isolate SARS-CoV-2 from individuals beyond 9 days after symptom onset [3]. Additionally, positive viral culture has been rarely detected in clinical specimens with a PCR cycle threshold (Ct) value greater than 34 cycles of amplification. With increasing use of oral fluid specimens for the detection of SARS-CoV-2 [4], we sought to determine the performance of oral fluid versus nasopharyngeal specimens for the duration and detection of culturable virus.

Outpatient SARS-CoV-2 healthcare-worker-observed, self-collected oral mucosal fluid specimen testing was conducted as part of routine public health activities at drive-through testing venues in Los Angeles County (Curative, Inc., San Dimas, CA, USA) [5]. Eligible adults tested positive for SARS-CoV-2 RNA with Ct values lower than 34.0 on a Food and Drug Administration Emergency Use Authorized test system. A study physician contacted and enrolled participants after obtaining informed consent. In participant homes, the physician collected oral mucosal fluid and nasopharyngeal swab specimens in viral transport media for viral culture within 7 days of original screening and specimen collection [5]. Repeat RT-PCR testing was performed on oral mucosal fluid specimens [5]. Viral culture was performed at the biosafety laboratory level 3 containment facility at the George Washington University. The institutional review board was reviewed by Advarra, IRB number: Pro00045881.

Over a 10-day period from 21 to 31 August 2020, 390 individuals were screened for eligibility. Of those, 30 participants were enrolled. Participants had a median age of 28 years (interquartile range (IQR) 23–33 years) and 13 of 30 (43.3%) participants were women (Table 1 ). At the time of the initial testing, 29 of 30 (96.7%) participants were symptomatic; all participants reported symptoms at the time of study specimen collection. The median number of days from initial screening to study participation was 3 days (IQR 3–4 days). Among participants, the median duration of symptoms at the time of testing was 5 days (IQR 4– 6 days).

Table 1

Age, PCR cycle threshold values, days since symptom onset, and symptoms among persons that tested positive and negative using culture for SARS-CoV-2

Variable	Culture-positive (n = 24)	Culture-negative (n = 4)
Age (years), median (IQR)	29.7 (25.7–33.2)	26.6 (23.3–30.5)
Ct value, median (IQR)	29.7 (25.3–33.5)	26.6 (22.2–33)
Days since symptom onset, median (IQR)	5 (4–6)	6 (5–7)
Symptoms, n/N (%)
Headache	23/24 (95.8)	3/4 (75)
Tiredness	18/24 (75)	4/4 (100)
Muscle pain	18/24 (75)	4/4 (100)
Sore throat	17/24 (70.8)	3/4 (75)
Chills	14/24 (58.3)	2/4 (50)
Joint pain	14/24 (58.3)	1/4 (25)
Diarrhea, stomach discomfort	14/24 (58.3)	4/4 (100)
Fever	13/24 (54.2)	2/4 (50)
Loss of taste or smell	11/24 (45.8)	3/4 (75)
Shortness of breath	10/24 (41.6)	3/4 (75)
Chest discomfort	9/24 (37.5)	3/4 (75)
Difficulty breathing	8/24 (33.3)	2/4 (50)
Required hospitalization or emergency department visit	2/24 (8.3)	0/4 (0)

Of the 30 participants, 28 (93.3%) had a positive SARS-CoV-2 RT-PCR test. Of those 28 participants with a positive SARS-CoV-2 test, the median Ct value was 29.07 (IQR 23.81–33.03). Among the 28 specimens, 24 (85.7%) viral culture tests were positive. Among nasopharyngeal swab specimens, 23 of 28 (82.1%) specimens were culture positive. Among oral mucosal fluid specimens, 9 of 28 (32.1%) specimens were culture positive. Among those with any positive viral culture, the median duration from symptom onset to time of specimen collection was 5 days (range 1–14 days). Among those with any positive viral culture, 3 of 28 (10.7%) participants had a positive viral culture (one oral mucosal fluid and three nasopharyngeal) more than 9 days from symptom onset. Among those with any positive viral culture, 4 of 28 (14.2%) participants had a positive viral culture in oral mucosal fluid specimens with Ct value greater than 34.0 cycles (upper range 37.73 cycles).

We tested participants for SARS-CoV-2 with viral culture in an outpatient setting. We found that culturable SARS-CoV-2 can be detected for most individuals up to 6 days after symptom onset; however, we detected SARS-CoV-2 by culture up to 14 days from the time of symptom onset in a few individuals. We also found that SARS-CoV-2 could be cultured from oral mucosal fluid specimens, including specimens with Ct values as high as 37.73 cycles.

Our study is not without limitations. Specimens were not sequenced in this study and were assumed to be wild-type virus. It is possible that higher Ct values were due to primer mismatches from mutations in the virus. Negative controls remained negative and there were no signs of cross-contamination. To reduce the risk of PCR inhibition, imperfect controls and improper culture interpretation, PCR specimens were run on different plates with different batches of controls, and cultures were verified with PCR testing.

Although our study sample was small and not representative of all people with COVID-19, because of the selection of those with Ct values less than 34 cycles, we found that some individuals may have a positive viral culture for longer than current CDC guidelines for the duration of isolation. Other studies have described differences in Ct values in infected individuals by the anatomical site of specimen collection [5]. We also observed differences in SARS-CoV-2 culture results based on anatomical sites. More studies are needed to understand the risk of virus transmission based on virus culture results and Ct values through the testing of exposed contacts.

Transparency declaration

NK and CM are consultants for Curative. NF is employed by Curative. JDK is the Medical Director of Curative.

Funding

None received.

Authors' contributions

NK contributed to conceptualization, methodology, analysis, sample collecting, supervision, investigation and writing the original draft and review. CM contributed to methodology, analysis, investigation, validation, review and editing. NF contributed to conceptualization, methodology, analysis, investigation, review, and editing. JDK contributed to supervision, interpretation, review, and editing.