key: cord-0908408-op0s9eiu authors: Monrad, Ida; Sahlertz, Signe Risgaard; Nielsen, Stine Sofie Frank; Pedersen, Louise Ørnskov; Petersen, Mikkel Steen; Kobel, Carl Mathias; Tarpgaard, Irene Harder; Storgaard, Merete; Mortensen, Klaus Leth; Schleimann, Mariane H; Tolstrup, Martin; Vibholm, Line K title: Persistent SARS-CoV-2 Infection in Immunocompromised Host Displaying Treatment Induced Viral Evolution date: 2021-06-04 journal: Open Forum Infect Dis DOI: 10.1093/ofid/ofab295 sha: 8365ae9453d937a922b760a0173396770ede125c doc_id: 908408 cord_uid: op0s9eiu We report a COVID-19 case with SARS-CoV-2 persisting beyond 333 days in an immunocompromised patient with chronic lymphocytic leukemia, asymptomatically carrying infectious SARS-CoV-2 at day 197 post diagnosis. Additionally, viral sequencing indicates major changes in the spike protein over time, temporally associated with convalescent plasma treatment. SARS-CoV-2 infection is associated with a robust antibody response and development of T cell immunity [1] [2] [3] . Control of the pandemic has been based on testing, isolation, and contact tracing. Accordingly, health authorities advice self-isolation until 24-48 hours after recovery 4 . However, viable virus may persist for prolonged periods in certain severely immunocompromised individuals [5] [6] [7] . The immunological prerequisites for complete clearance of SARS-CoV-2 infection are thus unclear. Additionally, concern is growing regarding the emergence of new SARS-CoV-2 variants, including B.1.1.7 and B.1.351, and their implications for future control of the COVID-19 pandemic. Increasing numbers of mutational changes in the spike protein genome pose a risk of reinfection, viral escape from present treatment modalities and lowering vaccine efficacy [7] [8] [9] . Here, we report a case of persistent SARS-CoV-2 infection in an immunocompromised patient with chronic lymphocytic leukemia (CLL), asymptomatically carrying infectious SARS-CoV-2 at day 197 post diagnosis. Viral sequencing showed accumulation of mutations over time and indicated major changes in the spike protein, temporally associated with convalescent plasma treatment. In May 2020, an asymptomatic 75-year-old Caucasian male with a history of CLL since 2011 tested SARS-CoV-2 positive by reverse transcriptase polymerase chain reaction (RT-PCR) analysis of a pharyngeal swab specimen (day 0, Fig. 1A ) as part of a routine screening. A timeline of the clinical history is depicted in fig. 1A , indicating timepoints for sampling of pharyngeal swabs for viral outgrowth (black arrows) and blood samples for immunological analysis (orange arrows). The patient previously received rituximab (5 doses administered in 2015, 3 doses administered in 2017), fludarabine and cyclophosphamide (3 doses administered in 2017), bendamustine (4 doses administered in 2015) and most recently Ibrutinib (administered daily from January 2018 to June 2020). He developed fever and shortness of breath and was admitted to hospital on day 12 with chest X-ray showing bilateral infiltrates, but discharged on day 17 following clinical and biochemical improvement. Recurrence of fever and respiratory symptoms resulted in readmission on day 22. Chest-CT scan showed progression of ground-glass opacities and the patient was placed on ventilation in the intensive care unit (ICU). A 10-day remdesivir treatment was initiated on day 24 while ibrutinib was paused, after which he was discharged in recovery on day 33. Following recurrence of respiratory symptoms, the patient was readmitted on day 54 and subsequently transferred to the ICU on day 60 for high-flow oxygen, dexamethasone and a 5-day remdesivir treatment. Despite initial improvement in clinical parameters and inflammatory biomarkers, the patient received convalescent plasma (CP) (2 x 300 ml, both with an IgG ratio of 4.46 measured by semi-quantitative ELISA from Euroimmun 10 ) on day 70, due to relapse in fever. After recovery to an asymptomatic state the patient was discharged for home-isolation on day 101, despite being continuously RT-PCR positive. In an attempt to eradicate this asymptomatic viral carrier-state the patient was readmitted for a double dose of CP on days 127-128 (Day 127: 2 x 300 ml, with an IgG ratio of 8.24. Day 128: 1 x 300 ml with an IgG ratio of 4.72 and 1 x 300 ml with an IgG ratio of 5.44). Virus eradication is thus far unsuccessful, as weekly performed RT-PCR tests remain positive for SARS-CoV-2, with the last sample acquired on day 333 just prior to submission of this report. Additional antibiotic treatment is depicted in supplementary figure S1. Data was obtained from electronic patient records. Pharyngeal swab samples were taken on day 126 and 197, during asymptomatic home isolation and collected in transport media optimized for viral outgrowth. RNA was extracted and viral load was quantified using droplet digital PCR (ddPCR). Furthermore, Vero E6/TMPRSS2 cells were inoculated with swab transport media, and subsequently assessed for viral outgrowth. Input swab material was 1D ) representing a broad disease severity spectrum 11 . The patient's SARS-CoV-2 specific antibody response was more equivalent to that of 10 healthy pre-pandemic controls collected in 2015, compared to convalescent COVID-19 patients 12 . Presence of SARS-CoV-2 neutralizing antibodies in patient plasma taken on day 126 was further assessed through SARS-CoV-2 spike pseudovirus neutralization. Five-fold dilutions of patient plasma were incubated with SARS-CoV-2 Spike expressing pseudovirus, carrying an eGFP reporter gene. Infection of Vero E6/TMPRSS2 cells was determined by flow cytometry as eGFP expression. The patient's plasma-neutralizing capacity was within the 95% confidence interval of 10 healthy prepandemic controls, and thus unable to neutralize SARS-CoV-2 spike (fig. 1E ). Flow cytometry analyses of lymphocyte populations were performed on day 126, as described in supplementary methods and results. The patient had a total CD3 + lymphocyte count within the standard reference range ( fig. S2) , and an essentially normal T-cell subset distribution (table S1) , albeit with few naïve and slightly elevated numbers of memory T cells. Total NK cell counts were also within the standard reference range. The absolute CD19 + B lymphocyte count was approximately 10fold above upper normal limit, dominated by B-cells of a marginal-zone-like phenotype (IgD + CD27 + CD38 -) consistent with his known B cell malignancy. 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Science (80-) Escape from neutralizing antibodies 1 by SARS-CoV-2 spike protein variants Convalescent plasma therapy for B-cell-depleted patients with protracted COVID-19 Pharyngeal swabs for viral outgrowth was sampled on day 126 and 197 post diagnosis (black arrows) while blood samples for immunologic and serological assessments were sampled on day 126 (orange arrow). B) Pharyngeal swab samples taken on day 126 and 197 post diagnosis, was used for RNA extraction and subsequent droplet digital PCR (ddPCR) for quantification of viral load. C) Vero E6/TMPRSS2 cells were inoculated with swab transport media, and subsequently assessed for viral outgrowth. Input swab material was washed away after 24 hours of inoculation, after which cell supernatant containing viral outgrowth was collected and used for RNA extraction and subsequent ddPCR, with viral copies increasing over a 5-day time period. D) Meso Scale analysis of SARS-CoV-2 specific IgG towards full-length spike, the spike receptor-binding domain (RBD), N-terminal domain (NTD), and nucleocapsid Case patient plasma (red) is shown in the context of the 95% confidence interval (grey shaded area) of 10 pre-pandemic controls (Controls). F) Illumina sequencing of SARS-CoV-2 RNA from clinical samples taken at day 24 (tracheal secretion), day 59 (bronchoalveolar lavage), day 80 (pharyngeal swab), day 126 (pharyngeal swab), day 142 (pharyngeal swab), day 155 (pharyngeal swab) and day 197 (pharyngeal swab) We thank the case for kindly donating sample material and participating in this study. The VSV-eGFP pseudovirus reporter system and the Vero E6/TMPRSS2 cells was kindly obtained through Dr Stefan Pöhlmann. All authors declare no conflicts of interest. Verbal and written consent was obtained from the patient allowing analyses of samples and publication of the report. This study has been approved by The Central Denmark Region Committees on Health Research Ethics.