A Case of Early Re-infection with SARS-CoV-2 LCDR Derek Larson1, LTC Sterling L. Brodniak2, Logan J. Voegtly3,4, Regina Z. Cer3,4, Lindsay A. Glang3,4, Francisco J. Malagon3,4, Kyle A. Long3,4, Ronald Potocki5, Darci R. Smith6, LTC Charlotte Lanteri7, CAPT Timothy Burgess7, Kimberly A Bishop-Lilly4 us cr ip t Division of Infectious Disease, Fort Belvoir Community Hospital, Fort Belvoir, VA, United States of America 2 Department of Family Medicine, Fort Belvoir Community Hospital, Fort Belvoir, VA, United States of America Leidos, VA, United States of America 4 Genomics & Bioinformatics Department, Biological Defense Research Directorate, Naval Medical an 3 5 M Research Center, MD, United States of America Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD, United Immunodiagnostics Department, Biological Defense Research Directorate, Naval Medical Research pt e 6 d States of America Center, MD, United States of America Infectious Disease Clinical Research Program, Department of Preventive Medicine & Biostatistics, ce 7 Ac Uniformed Services University, Bethesda, MD, United States of America Published by Oxford University Press for the Infectious Diseases Society of America 2020. This work is written by (a) US Government employee(s) and is in the public domain in the US. Downloaded from https://academic.oup.com/cid/advance-article/doi/10.1093/cid/ciaa1436/5908892 by guest on 14 October 2020 1 Corresponding Author LCDR Derek Larson, D.O. Mailing Fort Belvoir Community Hospital 9300 DeWitt Loop Downloaded from https://academic.oup.com/cid/advance-article/doi/10.1093/cid/ciaa1436/5908892 by guest on 14 October 2020 Fort Belvoir, VA 22060 us cr ip t Email: Derek.T.Larson2.mil@mail.mil Secondary email: LarsonDT@gmail.com Phone: 571-231-1588 Ac ce pt e d M an Fax: 571-231-6641 2 Dear Editor, It is with great interest that we read the first report of re-infection from SARS-CoV-2, which represented an important data point in the ongoing COVID-19 pandemic [1-3]. Questions have arisen regarding the timing and severity of re-infections, for which we offer a case report of t us cr ip 42-year-old healthy male military healthcare provider presented with cough, subjective fever, and myalgias on 21 March following a workplace COVID-19 exposure and tested positive by SARS-CoV-2 RT-PCR (Figure 1). Physical examination was unrevealing and supportive outpatient management M excellent health for the following 51 days. an was pursued [4]. Clinical resolution of illness occurred by day 10, and he returned to baseline On 24 May he presented with fevers, cough, shortness of breath and gastrointestinal symptoms, d following a confirmed new household exposure to COVID-19. The physical examination revealed a pt e temperature of 100.2 Fahrenheit, pulse of 119 beats per minute with a blood pressure of 124/87 mmHg, respirations of 24 breaths per minute with oxygen saturation of 92-94% on ambient air. A ce chest X-ray demonstrated a pulmonary infiltrate. Multiplex respiratory RT-PCR testing was negative but SARS-CoV-2 RT-PCR was again positive. Notably, symptoms were significantly worse when Ac compared with the initial syndrome. Serum collected on 1 June demonstrated presence of SARSCoV-2 spike IgG antibodies. Samples were collected as part of the IRB-approved protocol IDCRP-085. Viral culture was attempted but was unsuccessful. RNA sequencing was performed via the ARTIC nCoV-2019 Sequencing protocol [12], the YouSeq SARS-CoV-2 Coronavirus NGS Library prep kit, and SuperScript IV (ThermoFisher Scientific). The consensus genome was generated and Single Nucleotide Variants 3 Downloaded from https://academic.oup.com/cid/advance-article/doi/10.1093/cid/ciaa1436/5908892 by guest on 14 October 2020 symptomatic re-infection within 90 days. (SNVs) were determined [5]. Global lineage was determined using a subset of SARS- CoV-2 genomes available from the Global Initiative on Sharing All Influenza Data repository (GISAID accessed, June 24, 2020). Alignments were performed [6] and a Maximum Likelihood tree was generated [7]. The SARS-CoV-2 genome from the re-infection sample was deposited in NCBI GenBank under Accession MT840184. t us cr ip fragments totaling 4,126 bp and distributed across the genome. Sequencing of the sample from the patient’s second illness yielded a nearly coding complete genome of 27,268 bp. One discrete 50 bp region of zero coverage was observed. Phylogenetic analysis placed this virus in lineage B.1.26 and an the genome encoded the D614G variation in the spike protein [8]. Comparison of the partial sequence obtained from the initial infection with the nearly complete sequence obtained from the M re-infection identified several potential variations, including one high confidence variation. d The clinical, epidemiological, and sequencing data of this case suggest early re-infection with SARS- pt e CoV-2, only 51 days after resolution of initial infection. Importantly, this was observed in a young immunocompetent patient. In contrast to the case reported by To et al., this second infection was ce more severe, potentially due to immune enhancement, acquisition of a more pathogenic strain, or Ac perhaps a greater inoculum of infection as the second exposure was from within the household. 4 Downloaded from https://academic.oup.com/cid/advance-article/doi/10.1093/cid/ciaa1436/5908892 by guest on 14 October 2020 A partial genome sequence was obtained from the initial clinical infection, consisting of sequence NOTES Disclaimer The views expressed in this article are those of the author and do not necessarily reflect the official policy or position of Fort Belvoir Community Hospital, the Defense Health Agency, Department of Defense, U.S. Navy, or U.S. Government. Several of the authors are U.S. Government employees. us cr ip t protection under this title is not available for any work of the United States Government.’ Title 17 U.S.C. §101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person’s official duties. The identification of specific products, scientific instrumentation, or organization is considered an an integral part of the scientific endeavor and does not constitute endorsement or implied M endorsement on the part of the author, DoD, or any component agency. Funding d This work was supported by [WUN A1417] and [GEIS P0013_20_AH_01.01] to KBL for virus isolation, pt e sequencing, and bioinformatic analyses; and IDCRP-085 is funded by the Defense Health Program. ce Conflicts of Interest Ac The authors of this study report no financial conflicts of interest. 5 Downloaded from https://academic.oup.com/cid/advance-article/doi/10.1093/cid/ciaa1436/5908892 by guest on 14 October 2020 This work was prepared as part of their official duties. Title 17 U.S.C. § 105 provides that ‘Copyright References: 1. To KKW, Hung IFN, Ip JD, et al. COVID-19 re-infection by a phylogenetically distinct SARScoronavirus-2 strain confirmed by whole genome sequencing. Clin Infect Dis. [published online ahead of print, 2020 Aug 25] doi: 10.1093/cid/ciaa1275 2. Mathew D, Giles JR, Baxter AE, et al. Deep immune profiling of COVID-19 patients reveals distinct us cr ip t doi: 10.1126/science.abc8511 3. Cento V, Colagrossi L, Nava A, et al. Persistent positivity and fluctuations of SARS-CoV-2 RNA in clinically-recovered COVID-19 patients. J Infect. 2020;S0163-4453(20)30405-9. doi: 10.1016/j.jinf.2020.06.024 an 4. Larson DT, Sherner J, Gallagher K et al. Clinical outcomes of COVID-19 with evidence-based supportive care. Clin Infect Dis. [published online ahead of print, 2020 May 30] M doi:10.1093/cid/ciaa678 5. Grubaugh, N.D., Gangavarapu, K., Quick, J. et al. An amplicon-based sequencing framework for d accurately measuring intrahost virus diversity using PrimalSeq and iVar. Genome Biol20, 8 (2019). pt e doi: 10.1186/s13059-018-1618-7 14 6. Katoh K, Misawa K, Kuma K, Miyata T. MAFFT: a novel method for rapid multiple sequence alignment ce based on fast Fourier transform. Nucleic Acids Res. 2002;30(14):3059-3066. doi:10.1093/nar/gkf436 7. Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, von Haeseler A, Lanfear R: IQ- Ac TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. MolBiolEvol 2020;37(5):1530-1534 8. Korber B, Fischer WM, Gnanakaran S, et al. Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus. Cell. [published online ahead of print, 2020 Jul 3] 2020;S0092-8674(20)30820-5. doi:10.1016/j.cell.2020.06.043 19 6 Downloaded from https://academic.oup.com/cid/advance-article/doi/10.1093/cid/ciaa1436/5908892 by guest on 14 October 2020 immunotypes with therapeutic implications. Science. [published online ahead of print, 2020 Jul 15] d pt e ce us cr ip an M t Downloaded from https://academic.oup.com/cid/advance-article/doi/10.1093/cid/ciaa1436/5908892 by guest on 14 October 2020 Ac Figure 1. Timeline of symptoms and testing. 7