key: cord-0785230-edvyp1fs
authors: Leung, Wayne F.; Chorlton, Samuel; Tyson, John; Al-Rawahi, Ghada N.; Jassem, Agatha N.; Prystajecky, Natalie; Masud, Shazia; Deans, Gregory D.; Chapman, Michael G.; Mirzanejad, Yazdan; Murray, Melanie C.M.; Wong, Patrick H.P.
title: COVID-19 in an Immunocompromised Host: Persistent Shedding of Viable SARS-CoV-2 and Emergence of Multiple Mutations, a Case Report.
date: 2021-10-29
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2021.10.045
sha: 6cc8baf6a416728e50691de001b6e20742e93576
doc_id: 785230
cord_uid: edvyp1fs

We report a case of a 21-year-old woman with refractory B-cell acute lymphocytic leukemia presenting with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), who remained positive for SARS-CoV-2 by viral culture for 78 days and by polymerase chain reaction (PCR) for 97 days. Sequencing of repeat samples over time demonstrated an increasing and dynamic repertoire of mutations.

Keywords: SARS-CoV-2, immunocompromised, sequencing, mutation, viral culture, case report Introduction

Throughout the severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) pandemic, clinicians have faced the challenge of interpreting the results of SARS-CoV-2 to distinguish infectious versus non-infectious virus particles. Previously, immunocompromised patients have remained SARS-CoV-2 polymerase chain reaction (PCR) positive up to 151 days (Choi et al., 2020) . Although detection of viral genomic material does not confirm presence of infectious SARS-CoV-2, continued viral shedding raises questions regarding potential for disease transmission and viral mutation, particularly among the severely immunocompromised, where SARS-CoV-2 is detected weeks after symptom onset. In this setting viral culture may be used to assess whether the virus retains infective potential. A study of 129 immunocompetent and immunocompromised SARS-CoV-2 patients demonstrated that virus could be cultured for a median of eight days and a maximum of 20 days after symptom onset (van Kampen et al., 2021) .

Recently, concern is growing around accumulation of SARS-CoV-2 genome mutations that may confer increased infectivity, pathogenicity, or immune escape. In particular, variants of concern such as P. 1, B.1.351, B.1.617.2 and B.1.1.7 (O'Toole et al., 2021) , have mutations identified as Gamma, Beta, Delta, and Alpha in the spike protein receptor-binding domain (Rambaut et al., 2020) , a site critical for initial binding of virus to the angiotensin converting enzyme 2 (ACE2) receptor in the upper respiratory tract. Estimates suggest that SARS-CoV-2 lineages acquire 1-2 mutations per month as they transit through successive hosts (Duchene et al., 2020) . However, case reports have documented accelerated viral evolution in immunocompromised hosts who are susceptible to prolonged SARS-CoV-2 infection and viral replication (Avanzato et al., 2020; Choi et al., 2020; Kemp et al., 2021) , which is thought to have led to emergence of the Alpha variant. In one such case, a patient with chronic lymphocytic leukemia and acquired hypogammaglobulinemia had SARS-CoV-2 cultured at 70 days, while viral RNA was detected at 105 days after infection (Avanzato et al., 2020) . Continuous viral variant turnover demonstrated new, previously undocumented variants (Avanzato et al., 2020) .

Another individual with marginal B-cell lymphoma and previous chemotherapy received convalescent plasma treatment, and over 101 days, genomic analysis demonstrated emergence of spike protein mutations (Kemp et al., 2021) . Finally, in a case by Choi et al. (2020) October 21 st , 2020, five days after her third inotuzumab dose (day 1 of her symptoms), she experienced chills, sore throat, and myalgia after community exposure to SARS-CoV-2. On day 7, an outpatient nasopharyngeal swab was collected, and SARS-CoV-2 RNA was detected by our laboratory-developed RT-qPCR (LeBlanc et al., 2020) (cycle threshold [Ct] value 16.60).

Her initial mild symptoms resolved within two weeks and public health deemed her not contagious. On day 30, she received a fourth dose of inotuzumab and two days later developed fever and breathlessness. On day 33 she was admitted to hospital with fever of 38.7°C and progressive dyspnea. Oxygen saturation was 70-74% on room air requiring 7 L/min of oxygen by face mask. SARS-CoV-2 RNA was detected by nasopharyngeal swab (Ct 16.35). Initial chest computed tomography (CT) scan showed extensive bilateral ground glass opacities consistent with SARS-CoV-2 pneumonia, with no evidence of pulmonary embolism. She received dexamethasone 6 mg intravenously daily for 10 days, and piperacillin-tazobactam for possible bacterial pneumonia until day 35. Remdesivir was not initiated due to mild liver enzyme elevation and time since symptom onset. She progressed to require high-flow oxygen with 70% fraction of inspired oxygen (FiO 2 ). Given marked hypoxemia and LDH of 568 U/L (reference < 220 U/L), pneumocystis pneumonia was treated empirically with trimethoprim-sulfamethoxazole (from day 42 to day 63) and dexamethasone continued, tapering to 3 mg daily from day 43 onwards. She was also treated with voriconazole (day 43-58) for possible coronavirus disease associated pulmonary aspergillosis (CAPA). Lower respiratory specimens to assess for pneumocystis or aspergillosis were never obtained given lack of sputum production and instability for bronchoscopy with ongoing hypoxemia without intubation.

On day 65, she acutely deteriorated with fever, neutrophil count of 0.8 x 10 9 /L (reference 2.0-8.0 x 10 9 /L), and oxygen requirement increase to 95% FiO 2 . Given the palliative nature of her ALL and in keeping with her wishes, she was not transferred to intensive care. Chest CT scan demonstrated new left apical consolidation with cavitation on background of persistent disease. Dexamethasone was increased to 6 mg daily, and voriconazole restarted in case deterioration was due to CAPA. Day 67 serum galactomannan was negative, but voriconazole continued. Oxygen requirements remained at 95% FiO2.

Repeat nasopharyngeal swab (day 78) was again positive (Ct 23.84). Viral culture demonstrated in vitro infective viability in T25 cell cultures, as detectable cytopathic effect with confirmation by SARS-CoV-2 qPCR (E and RdRP gene, laboratory-developed). Serology assessing for total antibodies to SARS-CoV-2 spike 1 receptor binding domain (Siemens ADVIA Centaur XP) from day 87 was non-reactive. Rubella anti-IgG testing (Siemens ADVIA Centaur XP) on same day was equivocal, confirming a general lack of humoral immunity in this patient with up-to-date vaccination status. She received a trial of remdesivir (day 91-98). Other therapies including interleukin-6 inhibitors, neutralizing antibodies, and convalescent plasma were not considered as they were either not in use in Canada or there was lack of adequate evidence at that time, or she did not meet criteria for enrolment in clinical trials. SARS-CoV-2 RNA was again detected in nasopharyngeal swabs from day 91 (Ct 26.67) and 97 (Ct 28.00), though viral culture was negative in both day 91 and 97. She required progressively higher oxygen requirements, and after discussion with family, she transitioned to end-of-life care and passed away on day 98.

Sequencing of this patient's virus was conducted five times over three months (Table 1) , using previously published methods (Hogan et al., 2021) . All sequencing attempts produced high-quality data spanning the near complete genome (up to 99.4%) (Figure 2A are discussed below where information is available ( Figure 2C ).

Herein we present a case of prolonged shedding and mutation of SARS-CoV-2 in an immunocompromised host. Positive viral culture at 78 days after symptom onset was consistent with case reports of SARS-CoV-2 in other immunocompromised hosts (Avanzato et al., 2020; Choi et al., 2020; Kemp et al., 2021) , and longer than reported early in the pandemic, where infectious virus shedding occurred to day 20 (van Kampen et al., 2020).

Our report demonstrates a prolonged SARS-CoV-2 infection in a patient who received inotuzumab. While there have been no case reports specifically documenting associations between anti-CD22 monoclonal antibodies such as inotuzumab and prolonged SARS-CoV-2 infections, this has been previously reported in patients receiving rituximab, eculizumab (Choi et al., 2020) and vincristine (Kemp et al., 2021) . The dynamic viral population with accelerated evolution over time as seen in our case resulted in development of twelve mutations (eleven with predicted coding alterations) in three months; however, in the final sample there were seven mutations including six protein coding changes, which matched the overall expected mutation rate (Duchene et al., 2020) . Interestingly, though twelve mutations were observed in total, only seven were present at day 97, emphasizing the ongoing viral evolution and selection within a host ( Figure 2C ).

Of the persistent mutations, several are previously described. The S494P surface mutation on the S protein was shown in silico to increase complementarity between the receptorbinding domain of SARS-CoV-2 and ACE2 (Chakraborty, 2021) , possibly increasing viral virulence. This mutation also acts as an escape mutation, selected for with administration of camelid nanobodies (engineered heavy-chain-only antibodies) for SARS-CoV-2 treatment (Koenig et al., 2021) . The Y144del surface mutation is found in B.1.1.7 and B.1.351 variants, and may convey immune escape based on decreased antibody binding (Wang et al., 2021) . Prevalence of 7/12 viral mutations increased over time, while five mutations were seen only once ( Figure 2C ). While fixation of several mutations did occur, others were observed to arise and then not detected, suggestive of a combination of selective pressures and a diverse viral population. Our patient developed eleven non-synonymous and one synonymous mutations; the development of more non-synonymous than synonymous mutations was consistent with other reports (Choi et al., 2020; Khatamzas et al., 2021) .

The mutation profile shifted markedly from day 91 to 97 ( Figure 2C ) which coincided with remdesivir treatment (day 91-98). In addition to three mutations that were present in high frequencies by day 78, six new protein coding mutations were observed, four of which occurred in the S and N genes ( Figure 2C ). This dynamic change may warrant further investigation in relation to possible remdesivir association. None of these mutations were found in literature to be remdesivir associated mutations.

In this case of prolonged SARS-CoV-2 viral evolution in an immunocompromised host, we demonstrated viral viability and variable acquisition of 12 mutations over three months.

Ongoing infection and viral evolution of SARS-CoV-2 in immunocompromised individuals may result in variants, which may in turn have increased virulence and potential for immune escape.

In addition, many current infection control guidelines assume that persistently PCR-positive individuals are shedding residual RNA and not infectious virus, and this case report highlights the potential of using viral culture to confirm the presence of infectious SARS-CoV-2 in select patients.

The authors have no conflicts of interest to disclose.

Not applicable.

Consent was obtained from the patient's mother. Ethics approval not applicable. The following mutations are identified differences (>25% read fraction) between the sequenced viral samples and the Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1, NCBI Reference Sequence: NC_045512.2 (National Centre for Biotechnology Information, 2020). Grey text indicates synonymous amino acid changes, (ref) reference Wuhan-Hu-1 sequence, (alt) identified sequence mutation and (aa) amino acid alteration due to mutation.

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