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Viable virus shedding during SARS-CoV-2 reinfection

2021; Elsevier BV; Volume: 9; Issue: 7 Linguagem: Inglês

10.1016/s2213-2600(21)00219-8

2213-2619

Andrew G. Letizia, Darci R Smith, Yongchao Ge, Irene Ramos, Rachel Sealfon, Carl Goforth, Ana S. González-Reiche, Sindhu Vangeti, Dawn L. Weir, Hala Alshammary, Hua‐Wei Chen, Mary-Catherine George, Alessandra Soares‐Schanoski, Rhonda A. Lizewski, Stephen E. Lizewski, Jan Marayag, Clare M. Miller, Edgar Nunez, Chad K. Porter, Ernesto Santa Ana, Megan A. Schilling, Victor A. Sugiharto, Peifang Sun, Michael Termini, Adriana van de Guchte, Olga G. Troyanskaya, Harm van Bakel, Stuart C. Sealfon,

Respiratory viral infections research

The reinfection risk in individuals previously infected with SARS-CoV-2 is about a fifth of those never infected.1Hansen CH Michlmayr D Gubbels SM Mølbak K Ethelberg S Assessment of protection against reinfection with SARS-CoV-2 among 4 million PCR-tested individuals in Denmark in 2020: a population-level observational study.Lancet. 2021; 397: 1204-1212Summary Full Text Full Text PDF PubMed Scopus (438) Google Scholar, 2Letizia AG Ge Y Vangeti S et al.SARS-CoV-2 seropositivity and subsequent infection risk in healthy young adults: a prospective cohort study.Lancet Respir Med. 2021; (published online April 15.)https://doi.org/10.1016/S2213-2600(21)00158-2Summary Full Text Full Text PDF PubMed Scopus (110) Google Scholar Whether reinfected individuals shed viable virus has been identified as an important question relevant to pandemic control.3Ledford H Coronavirus reinfections: three questions scientists are asking.Nature. 2020; 585: 168-169Crossref PubMed Scopus (44) Google Scholar In a prospective cohort study in The Lancet Respiratory Medicine,2Letizia AG Ge Y Vangeti S et al.SARS-CoV-2 seropositivity and subsequent infection risk in healthy young adults: a prospective cohort study.Lancet Respir Med. 2021; (published online April 15.)https://doi.org/10.1016/S2213-2600(21)00158-2Summary Full Text Full Text PDF PubMed Scopus (110) Google Scholar we identified 19 cases of reinfection in people who at study entry were seropositive for both SARS-CoV-2 receptor binding domain and full-length spike protein, tested negative on three nasal swab PCR tests over a 2-week quarantine period, and subsequently developed a positive PCR test at least 2 weeks after leaving quarantine.2Letizia AG Ge Y Vangeti S et al.SARS-CoV-2 seropositivity and subsequent infection risk in healthy young adults: a prospective cohort study.Lancet Respir Med. 2021; (published online April 15.)https://doi.org/10.1016/S2213-2600(21)00158-2Summary Full Text Full Text PDF PubMed Scopus (110) Google Scholar We have now investigated whether these SARS-CoV-2-reinfected individuals shed viable virus. Viral transport media was available from the first and some subsequent PCR-positive tests from 16 (84%) of 19 reinfected participants. Samples were cultured for SARS-CoV-2 in Vero E6 cells expressing TMPRSS24Matsuyama S Nao N Shirato K et al.Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells.Proc Natl Acad Sci USA. 2020; 117: 7001-7003Crossref PubMed Scopus (947) Google Scholar in T25 cm2 flasks and monitored for cytopathic effect for 4 days followed by a second passage onto fresh cells to allow additional time for virus amplification. Samples were simultaneously titred by plaque assay in Vero E6/TMPRSS2 cells to quantify the level of detectable infectious virus. Viable virus was detected in only four (25%) of 16 participants, and only once in each, with titres ranging from 1·7 to 5·5 log10 plaque-forming units per mL. Serology and PCR cycle threshold (Ct) values using the US Food and Drug Administration-authorised Thermo Fisher TaqPath COVID-19 Combo Kit (Thermo Fisher Scientific, Waltham, MA, USA) were compared in those with viable virus detected (shedders) and those without viable virus detected (non-shedders; table; appendix p 2). At the time of positive viral culture, samples from shedders had lower Ct values. Although all participants had detectable SARS-CoV-2 spike IgG at study enrolment, none of the four shedders and only four (33%) of the 12 non-shedders had detectable serum neutralisation activity (limit of detection was 50% inhibitory dilution of 20) in serum at the time of enrolment into the study (methods as described2Letizia AG Ge Y Vangeti S et al.SARS-CoV-2 seropositivity and subsequent infection risk in healthy young adults: a prospective cohort study.Lancet Respir Med. 2021; (published online April 15.)https://doi.org/10.1016/S2213-2600(21)00158-2Summary Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Only one (25%) of the four who shed virus was symptomatic when viable virus was detected.TableBaseline serology, clinical features, and SARS-CoV-2 Ct levels in reinfected participants with samples cultured for viable virusVirus culture titreDifference*Difference in mean for the continuous variables and difference in percentage for binary variables.Positive (n=4)Negative (n=12)Baseline IgG S titre (log10)2·8 (0·2)2·9 (0·7)−0·08 (−0·88 to 0·72), p=0·834Baseline ID50 detected (>20)04 (33%)−33% (−87 to 21), p=0·207PCR positive >7 days3 (75%)4 (33%)42% (−20 to 100), p=0·166Symptomatic1 (25%)3 (25%)−0% (−57 to 57), p=1·000N gene Ct16·2 (4·0)31·9 (6·1)−15·64 (−22·68 to −8·60), p=0·0003S gene Ct17·8 (4·1)32·3 (6·2)−14·52 (−21·76 to −7·28), p=0·0007ORF1ab gene Ct16·8 (3·9)31·6 (6·1)−14·83 (−21·88 to −7·78), p=0·0005Data are mean (SD) and n (%). Ranges are 95% CIs. When a participant has multiple PCR-positive samples from more than one visit day, only the data from the visit day with the lowest mean Ct values of the three viral genes were included in the data summary. Ct=PCR cycle threshold. ID50=50% inhibitory dilution.* Difference in mean for the continuous variables and difference in percentage for binary variables. Open table in a new tab Data are mean (SD) and n (%). Ranges are 95% CIs. When a participant has multiple PCR-positive samples from more than one visit day, only the data from the visit day with the lowest mean Ct values of the three viral genes were included in the data summary. Ct=PCR cycle threshold. ID50=50% inhibitory dilution. All reinfections occurred before December, 2020, when the B.1.1.7 variant was first reported in the USA. Full-length viral genome was recovered from seven (44%) of 16 participants studied by viral transport medium culturing, including all four culture-positive individuals (methods previously described5Letizia AG Ramos I Obla A et al.SARS-CoV-2 transmission among marine recruits during quarantine.N Engl J Med. 2020; 383: 2407-2416Crossref PubMed Scopus (72) Google Scholar). Although the strains isolated all had the Asp614Gly spike protein mutation and two of the isolates had the Leu18Phe spike protein mutation, they did not have mutations associated with the B.1.1.7, P.1, B.1.351, or other variants of concern as currently defined by the US Centers for Disease Control and Prevention (appendix p 3). Limitations of our study are the use of virus culture as a proxy for capacity for transmission without direct study of transmission, the small sample size, the absence of specimens available for culturing from all reinfected participants, and the inability to compare potential for transmissibility between those who are infected for the first time and those who are reinfected. Overall, our findings suggest that about a quarter of young healthy individuals with subsequent SARS-CoV-2 reinfection shed viable virus. Some of these individuals were asymptomatic and could unknowingly transmit SARS-CoV-2 to others. All authors declare no competing interests. This study was supported by the Defense Health Agency and Defense Advanced Research Projects Agency. AGL, DRS, CG, DLW, HWC, RAL, SEL, JM, EN, CKP, ESA, MS, VAS, PS, and MT are military service members or government service employees. This work was prepared as part of their official duties. Title 17, US Code §105 provides that copyright protection under this title is not available for any work of the US Government. Title 17, US code §101 defines a US Government work as a work prepared by a military service member or employee of the US Government as part of that person's official duties. The views expressed in the article are those of the authors and do not necessarily express the official policy and position of the US Navy, the Department of Defense, the US Government, or the institutions affiliated with the authors. Download .pdf (.23 MB) Help with pdf files Supplementary appendix SARS-CoV-2 seropositivity and subsequent infection risk in healthy young adults: a prospective cohort studySeropositive young adults had about one-fifth the risk of subsequent infection compared with seronegative individuals. Although antibodies induced by initial infection are largely protective, they do not guarantee effective SARS-CoV-2 neutralisation activity or immunity against subsequent infection. These findings might be relevant for optimisation of mass vaccination strategies. Full-Text PDF