Long-term SARS-CoV-2 shedding was observed from the upper respiratory tract of a female immunocompromised patient with chronic lymphocytic leukemia and acquired hypogammaglobulinemia. Shedding of infectious SARS-CoV-2 was observed up to 70 days, and genomic and subgenomic RNA up to 105 days past initial diagnosis. The infection was not cleared after a first treatment with convalescent plasma, suggesting limited impact on SARS-CoV-2 in the upper respiratory tract within this patient. Several weeks after a second convalescent plasma transfusion, SARS-CoV-2 RNA was no longer detected. We observed marked within-host genomic evolution of SARS-CoV-2, with continuous turnover of dominant viral variants. However, replication kinetics in Vero E6 cells and primary human alveolar epithelial tissues were not affected. Our data indicate that certain immunocompromised patients may shed infectious virus for longer durations than previously recognized. Detection of subgenomic RNA is recommended in persistently SARS-CoV-2 positive individuals as a proxy for shedding of infectious virus.
In this report, we describe long-term SARS-CoV-2 shedding in an immunocompromised patient with chronic lymphocytic leukemia (CLL) and acquired hypogammaglobulinemia out to 105 days past the initial positive test. Although the exact time-point when the patient acquired the SARS CoV-2 is unknown, it is likely that the exposure occurred in the long-term care facility where she 251 resided between February 19-25, 2020, just shortly before a large COVID-19 outbreak was identified in that facility on February 28, 2020. The patient remained asymptomatic throughout the course of infection despite the isolation of infectious SARS-CoV-2 49 and 70 days past the initial diagnosis, much longer than shedding of infectious virus up to day 20 as reported previously (van 255 Kampen et al., 2020). The information available to date on SARS-CoV-2 infection in immunocompromised patients, including those with cancers such as CLL, is limited, and mostly focuses on disease severity and outcome (He et al., 2020a, Paneesha et al., 2020, Baumann et al., 258 2020, Furstenau et al., 2020, Jin et al., 2020, Soresina et al., 2020, Zhu et al., 2020, Fill et al., 259 2020). Although it is difficult to extrapolate from a single patient, our data suggest that long-term shedding of infectious virus may be a concern in certain immunocompromised patients. Given that immunocompromised patients could have prolonged shedding and may not have typical symptoms of COVID-19, symptom-based strategies for testing patients and discontinuing transmission-based precautions, as recommended by the CDC (CDC, 2020b), may fail to detect whether certain patients are shedding infectious virus. The patient eventually cleared the SARS-CoV-2 infection from the upper respiratory tract, after the development of low neutralizing antibody titers. How the virus was cleared and the effect of convalescent plasma on clearance of virus is unknown. The initial administration of convalescent plasma was followed by a decreased viral load in nasal swabs, but viral loads subsequently increased, despite administration of a second dose of convalescent plasma comprising higher antibody titers. Therapeutic administration of convalescent plasma is focused at treatment of severe or life-threatening COVID-19. Several clinical trials are investigating the efficacy of convalescent plasma, but currently the efficacy of convalescent plasma therapy on COVID-19 outcome remains equivocal (Mira et al., 2020, Salazar et al., 2020). The limited impact of the convalescent plasma treatment on clearance of SARS-CoV-2 could be due to the fact that IV administered antibodies do not distribute well to the nasal epithelium (Ikegami et al., 2020) compared to the lower respiratory tract (Mira et al., 2020). Throughout the course of infection there was marked within-host genomic evolution of SARS279 CoV-2. Deep sequencing revealed a continuously changing virus population structure with turnover in relative frequency of the observed genotypes over the course of infection. Within SARS-CoV-2 there is generally relatively limited within-host variation reported, and over the course of infection the major SARS-CoV-2 population remains identical (Jary et al., 2020, Shen et al., 2020, Capobianchi et al., 2020). Potential factors contributing to the observed within-host evolution is the prolonged infection and the compromised immune status of the host, possibly resulting in a different set of selective pressures compared to an immune-competent host. These differential selective pressures may have allowed a larger genetic diversity with a continuous turnover of dominant viral species throughout the course of the infection. While some sequence variants remain consistent throughout the duration of infection, we also observed variants unique to individual time points, such as the spike deletions observed at day 49 and day 70. Previously reported spike deletions, distinct from those reported herein, were observed at relatively low frequency in clinical samples, but were enriched upon virus isolation (Andres et al., 2020, Liu et 292 al., 2020d). Similar to these reports, the spike deletion in the isolate at day 49 was observed as a minor variant in the patient sample but was also selected for during passage upon virus isolation. In contrast to the previously reported deletions at the cleavage sites, both spike deletions observed at day 49 and 70 in the patient are located in the NTD of S1, a region distal from the receptor binding site. These deleted residues are not modelled in a number of spike structures suggesting that this region is conformationally labile. Although the NTD has been identified as an antigenic target (Brouwer et al., 2020, Chi et al., 2020, Liu et al., 2020a), no clear difference in virus neutralization was observed between the two patient isolates and the prototype USA/WA1/2020 SARS-CoV-2 isolate.Despite genetic changes in the SARS-CoV-2 isolated from the patient, replication kinetics did not significantly change when compared to the USA/WA1/2020 virus in Vero E6 cells and primary human alveolar epithelial tissues. This indicates that, most likely, the infectious virus shed by the patient would still be able to establish a productive infection in contacts upon transmission, assuming that viral growth kinetics in vitro are a suitable surrogate for virus fitness in vivo. Moreover, despite prolonged replication exclusively in the upper respiratory tract, the virus was still able to replicate in epithelial cells derived from the lower respiratory tract, suggestive that it could still cause pneumonia. Many current infection control guidelines assume that persistently PCR positive patients are shedding residual RNA and not infectious virus, with immunocompromised patients thought to remain infectious for no longer than 20 days after symptom onset (CDC, 2020a). Here, we show that certain patients may shed infectious, replication-competent virus for much longer durations than previously recognized (van Kampen et al., 2020). Whereas infectious virus could be detected up to day 70, sgRNA, a molecular marker for active SARS-CoV-2 replication (Speranza et al., 2020), could be detected up until day 105. An immunocompromised state has been identified as a risk factor for the development of severe disease and complications from COVID-19 (CDC, 320 2020b). A wide variety of conditions and treatments can alter the immune system and cause immunodeficiency, creating opportunities for prolonged viral replication and shedding of infectious SARS-CoV-2. While this reports focuses on the long-term shedding of one immunocompromised patient, an estimated 3 million people in the US have some form of immunocompromising condition, including HIV infection, solid-organ transplant recipients, haemopoietic stem-cell transplants, patients receiving chemotherapy and patients receiving corticosteroids (Kunisaki and Janoff, 2009). This transient or chronic immunocompromised patient population is at higher risk of respiratory disease complications with respiratory infections such as influenza A virus and SARS-CoV-2 (Kunisaki and Janoff, 2009). Prolonged shedding of pH1N1 shedding was observed in immunocompromised patients with a variety of immunocompromising conditions during the previous pandemic in 2009, such as cancer patients on chemotherapy and solid-organ transplant recipients (van der Vries et al., 2013). For the SARS-CoV-2 related Middle East respiratory syndrome coronavirus (MERS-CoV), prolonged shedding up to 38 days was observed in patients with myelodysplastic syndrome, autologous peripheral blood stem cell transplantation for treatment of large B-cell lymphoma and a patient with a peripheral T-cell lymphoma (Kim et al., 2017). MERS-CoV shedding was higher and longer in experimentally infected non-human primates immunosuppressed with cyclophosphamide and dexamethasone providing experimental support for the impact of immunosuppression on virus-host dynamics observed here (Prescott et al., 2018).
Limitations of the Study
A limitation of the present study is that it comprises only a single case, making it difficult to draw general conclusions on the use of convalescent plasma in clearance of the virus, potential alternative mechanisms involved in virus clearance and the frequency of persistent SARS-CoV-2 infection and shedding in patients with other immunocompromising conditions. Identification of additional cases of persistent infection and long-term shedding of infectious virus are needed so the infection dynamics can be studied in this diverse population in more detail. Understanding the mechanism of virus persistence and eventual clearance will be essential to providing appropriate treatment and preventing transmission of SARS-CoV-2, as persistent infection and prolonged shedding of infectious SARS-CoV-2 might occur more frequently. Because immunocompromised patients are often cohorted in hospital settings, a more nuanced approach to testing these individuals is warranted, and the presence of persistently positive patients by performing SARS CoV-2 gRNA and sgRNA analyses on clinical samples should be investigated.
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