Duration of Infectious Virus Shedding by SARS-CoV-2 Omicron Variant–Infected Vaccinees

Kenichiro Takahashi; Masahiro Ishikane; Mugen Ujiie; Noriko Iwamoto; Nobumasa Okumura; Testsuro Sato; Maki Nagashima; Ataru Moriya; Michiyo Suzuki; Masayuki Hojo; Takayuki Kanno; Shinji Saito; Sho Miyamoto; Akira Ainai; Minoru Tobiume; Takeshi Arashiro; Tsuguto Fujimoto; Tomoya Saito; Masaya Yamato; Tadaki Suzuki; Norio Ohmagari

Disclosures

Emerging Infectious Diseases. 2022;28(5):998-1001. 

In This Article

The Study

We conducted our retrospective study on leftover clinical samples collected from Omicron-infected patients in Japan during November 29–December 18, 2021. We sequenced the Omicron variant by using whole-genome sequencing as described[2] and uploaded the consensus sequences to GISAID (https://www.gisaid.org) (Table).

For cases detected by SARS-CoV-2 testing at airport quarantines, samples collected for diagnosis (saliva or nasopharyngeal) were transported to the NIID to confirm Omicron. We used the residual samples for this study. The date of sample collection of the first Omicron-positive sample for each patient was defined as the diagnosis date (day 0). Nasopharyngeal samples were collected serially during hospitalization, stored at −80°C, and transported to NIID.

We quantified SARS-CoV-2 RNA by using quantitative reverse transcription PCR (qRT-PCR) and virus isolation testing. We performed qRT-PCR as described previously.[6] We measured Cq values (i.e., viral RNA levels) by using qRT-PCR targeting the SARS-CoV-2 nucleocapsid gene (Appendix Figure 1, https://wwwnc.cdc.gov/EID/article/28/5/22-0197-App1.pdf). We analyzed samples with Cq values that were reported as negative after 40 cycles by substituting a value of 45. We performed the virus isolation assay according to described procedure.[7] All laboratory analyses were performed at the NIID.

To examine infectious virus shedding, we classified samples according to date of diagnosis, date of symptom onset, and date of symptom resolution. For cases in which multiple samples were collected in each time segment, we used the sample with the highest amount of viral RNA (i.e., lowest Cq values) in each time segment for each case for comparison. For data analysis and visualization, we used GraphPad Prism version 8.4.3 (https://www.graphpad.com). To compare the Cq values, we used Mann-Whitney t and Friedman tests with Dunn multiple comparisons. Statistical significance was set at p<0.05.

All 18 case-patients had been vaccinated >14 days before coronavirus disease (COVID-19) diagnosis (Table). The median (interquartile range [IQR]) duration between vaccination and diagnosis was 117 (71–131) days. Of the 18 case-patients, 15 were symptomatic and 3 were asymptomatic.

Among the 101 serially collected samples analyzed (85 nasopharyngeal and 16 saliva), we detected infectious virus in 10 (9.9%) from 10 patients (8 symptomatic and 2 asymptomatic) (Figure 1, panel A; Appendix Tables 1, 2,). The viral RNA levels analyzed by using qRT-PCR were significantly higher in samples with the infectious virus than without (p<0.0001) (Figure 1, panel A). Infectious virus was detected up to 9 days after diagnosis; the highest proportion of virus isolates (41.7%) was found in samples collected 2–5 days after diagnosis, and no isolates were detected 10 days after diagnosis (Figure 1, panel B; Appendix Figure 3, panel A).

Figure 1.

SARS-CoV-2 RNA level and infectious virus shedding in upper respiratory samples from symptomatic patients infected with the SARS-CoV-2 Omicron variant, Japan, November 29–December 18, 2021. A) SARS-CoV-2 RNA levels and presence of the infectious virus, by date of symptom onset. Each closed circle indicates case-patients from whom virus was isolated. Numbers above each plot indicate the proportion of case-patients from whom virus was isolated in each period. Black lines indicate median Cq values and error bars interquartile ranges; dotted lines indicate negative cutoff values. *Before symptom onset. B) SARS-CoV-2 RNA levels and presence of infectious virus, by date of symptom resolution. Closed circles indicate patients from whom virus was isolated. Numbers above each plot indicate the proportion of persons from whom virus was isolated in each period. Black lines indicate median Cq values and error bars interquartile ranges; dotted lines indicate cutoff values. †Before symptom resolution. Cq, quantification cycle; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

We detected infectious virus in the samples of 20%–30% symptomatic patients, ranging from before they were symptomatic to 9 days after symptom onset, but we detected no infectious virus beyond 10 days after symptom onset (Figure 2, panel A; Appendix Table 3, Figure 2, panel B, Figure 3, panel B). For ≈30% of case-patients, infectious virus shedding was detected up to 2 days after symptom resolution, but no virus was detected beyond 3 days after symptom resolution (Figure 2, panel B; Appendix Table 4, Figure 3, panel C). Many of the first samples collected were saliva samples. Of note, the results of only nasopharyngeal samples did not differ from samples including saliva after 2 days of diagnosis (Appendix Figure 4, panels A, B).

Figure 2.

SARS-CoV-2 RNA level and infectious virus shedding in all upper respiratory samples from patients infected with the SARS-CoV-2 Omicron variant, Japan, November 29–December 18, 2021. A) SARS-CoV-2 RNA levels in NP swab samples (open circles) and saliva (closed circles) with or without infectious virus. Red lines indicate median Cq values and error bars interquartile ranges; dotted lines indicate negative cutoff values. The Cq values between samples from which infectious virus was isolated and samples from which infectious virus was not isolated were compared by using the Mann-Whitney test. B) SARS-CoV-2 RNA levels and presence of infectious virus organized by the days after diagnosis. Red circles indicate symptomatic case-patients; blue circles indicate asymptomatic case-patients; each closed circle indicates case-patients from whom virus was isolated. Numbers above each plot indicate the proportion of case-patients from whom virus was isolated in each period. Black lines indicate median Cq values and error bars interquartile ranges; dotted lines indicate negative cutoff values. Cq, quantification cycle; NP, nasopharyngeal; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; +, with infectious virus; –, without infectious virus.

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