key: cord-0966550-5n2qrmj1
authors: Tarhini, Hassan; Recoing, Amélie; Bridier-Nahmias, Antoine; Rahi, Mayda; Lambert, Céleste; Martres, Pascale; Lucet, Jean-Christophe; Rioux, Christophe; Bouzid, Donia; Lebourgeois, Samuel; Descamps, Diane; Yazdanpanah, Yazdan; Le Hingrat, Quentin; Lescure, François-Xavier; Visseaux, Benoit
title: Long term SARS-CoV-2 infectiousness among three immunocompromised patients: from prolonged viral shedding to SARS-CoV-2 superinfection
date: 2021-02-08
journal: J Infect Dis
DOI: 10.1093/infdis/jiab075
sha: 117c296a8c1812eefb4fba60c5fd02c24ec102a7
doc_id: 966550
cord_uid: 5n2qrmj1

Guidelines for stopping COVID-19 patient isolation are mainly symptom-based, with isolation for 10 to 20 days depending on their condition. Here, we describe three deeply immunocompromised patients, each with different clinical evolutions. Asymptomatic carriage, symptom resolution, or superinfection with a second SARS-CoV-2 strain were observed, all leading to prolonged infectious viral shedding several months. We followed the patients epidemiological, clinical, serological data, infectiousness using viral culture and viral mutations accumulated over time. Understanding underlying mechanisms and frequency of prolonged infectiousness is crucial to adapt current guidelines and strengthen the use of systematic PCR testing before stopping isolation in immunocompromised populations.

The COVID-19 pandemic has severely disrupted healthcare systems and socioeconomic activities. The SARS CoronaVirus 2 (SARS-CoV-2), has caused large outbreaks in, bars, workplaces, households, and healthcare institutions. In the latter, patient's management and isolation is critical.

Several important questions, important to determine prevention policies, remain unanswered as the duration of infectiousness and, consequently the duration of isolation in healthcare institutions. The CDC recommends a 10-day isolation period for afebrile COVID-19 patients with mild/moderate clinical presentation and improvement of other symptoms since at least 24h. This period is extended for up to 20 days for patients with severe infection and/or severe immunosuppression. A negative SARS-CoV2 RT-PCR control is not mandatory but is encouraged in immunocompromised patients before stopping isolation [1] . For hospitalized patients in France, the isolation period should be at least 14 days after symptom onset and 48h after their resolution. This period is extended up to 24 days for severe infections or immunocompromised patients [2] . Several studies described prolonged positive RT-PCR above 15 days post symptom onset for less than 5% of hospitalized patients, but without viral culture testing [3] . However, three cases of long-term infectious shedding with high viral loads were recently reported in the literature with viral shedding for 35, 70 and 119 days [4] [5] [6] .

Here, we describe three deeply immunocompromised patients, each presenting a different clinical evolution. All three lead to prolonged viral shedding with high viral load for several months. We explored their epidemiological, clinical, serological data and infectiousness using viral culture and viral mutations accumulated over time.

Respiratory samples and sera were collected from the patients as a part of their routine clinical care. The research was approved by the local ethics committee, N° CER-2020-6.

A c c e p t e d M a n u s c r i p t

The respiratory samples were tested using either the Cobas® SARS-CoV-2 (Roche, Switzerland) [7] or the NeumoDX® (QIAgen, Germany) using the IP2 Institute Pasteur and the WHO E gene primers [8] . E gene cycle threshold (Ct) value was used as a proxy for viral load.

Anti-SARS-CoV-2 nucleocapsid (N) and spike (S) IgG were detected using a chemiluminescent microparticle immunoassay (Architect, Abbott, USA) and an ELISA assay (EuroImmun, Lubeck, Germany), respectively.

Vero E6 cells (ATCC, reference R CRL-1586) were cultured in Dulbecco's modified Eagle's medium (DMEM, GibcoTM) with 10% of heat-inactivated fetal bovine serum (FBS, GibcoTM) at 37°C and 5% of CO 2 . Briefly, 200µL of respiratory samples viral transport media mixed with 800µL of DMEM were filtered and inoculated in 12-wells plates containing 1.10 5 cells for 1 hour before adding of 500µL of DMEM with 4% of FBS. After a 6-day incubation, cytopathogenic effect assessment and RT-PCR quantification in cells supernatant were performed to assess the production of new virions. A previously cultured SARS-CoV-2 strain was systematically added as a positive control to ensure cell sensitivity. A c c e p t e d M a n u s c r i p t

Reads were filtered using the Nanofilt and Nanostat python scripts [9] . They were mapped on the reference genome Wuhan Hu-1 (Genbank ID NC_045512.2) using minimap2.

Alignment coverage, depth, and general quality were assessed using in-house R scripts.

Variant calling was performed with bcftools suite, and the proportions of each variant in each sample were retrieved with an in-house R script. Finally, for patient 3, mutations located on the same amplicon tiles were recovered using R to assess their linkage.

A 66-years-old African male was admitted on June 4, 2020 for loss of autonomy, and M a n u s c r i p t

On April 15, patient 2, a heart-transplanted 71-year-old European male patient receiving an immunosuppressive treatment (prednisone, mycophenolic acid, belatacept), was hospitalized for asthenia, dry cough, myalgia, and low-grade fever for 1-week. He also presents diabetes mellitus and chronic kidney disease (GFR 35 mL/min). He had neither dyspnea nor oxygen requirements throughout his hospital stay. NP swabs tested positive for SARS-CoV-2 at admission and day 14 with minimal COVID-19 involvement on CT scan (<10%). At day 39, he was discharged after clinical improvement, despite persistent positive PCR at day 32 (21 Ct).

On June 23, 76 days after initial symptoms' onset, the patient presented with dry cough, dyspnea, and oxygen requirement. He was admitted to the intensive care unit for cardiac decompensation due to underlying respiratory infection. CT scan showed worsened COVID-19 compatible lesions (40%). He had lymphopenia with CD4 <200/mm3 and CD19 <20/mm3. SARS-CoV-2 whole-genome was obtained from a NP swab at day 2, a lower respiratory tract sample at day 73, and from a NP swab after symptom's relapse at day 84. Sequences from days 2 and 73 were similar, aside from a C5147T mutation detected in at 68% frequency at day 2 (figure 1). On day 84, we detected the appearance of seven mutations at frequencies close to 70%. Two pairs of close mutations were present in the same PCR tiles. The mutations in these pairs were strongly linked, i.e., about 99% of tiles amplicons contained either none or both mutations (supp. Table 1 Those seven new mutations were never present at a 100% frequency. Moreover, we observed a strong linkage for two pairs of these mutations. This, along with the symptom relapse, reinforces the hypothesis of a superinfection with a probable cohabitation of two viral strains. The patient homeless condition also allowed multiple re-exposure until day 52, however, the symptom relapse on day 73 suggest a nosocomial infection. No sequence data from the other patients or healthcare workers of the ward could be explored to explore the infection source. Isolation precaution were maintained and observed during the whole hospitalization.

Two cases of prolonged viral shedding for more than 100 days were previously described in two patients presenting B-cell immunodeficiency [4, 10] . Interestingly, all our patients also presented deep CD19 depletion. Convalescent plasma to reduce viral shedding in such population could be evaluated. The T-cell immunity, which could not be explored in our patients, may also play a role in prolonged viral shedding. Several observational studies identified patients with positive SARS-CoV-2 RT-PCR 100 days after their initial detection [11, 12] . However, differentiation between re-infections and prolonged viral shedding was not established. Moreover, these studies did not follow patient's infectiousness using viral culture, as we did here. Although several case reports published in different countries showed SARS-CoV-2 reinfection [13] [14] [15] a superinfection with a second SARS-CoV-2 strain has not been described to date.

In conclusion, immunodeficiency plays a major role in prolonged viral shedding that can be observed in immunocompromised patients without any respiratory symptom (patient 1), late symptom relapse (patient 2), or with SARS-CoV-2 superinfection (patient 3). Further studies are needed to better understand the frequency and dynamic of long-term viral infectiousness. In the meantime, guidelines should recommend virological assessment of infectiousness, using viral culture and/or Ct value measure (low CT value), prior to stopping isolation in immunocompromised patients.

Duration of Isolation and Precautions for Adults with COVID-19

Covid-19 : délai de transfert dans un service de soins de suite ou un Ehpad

Shedding of infectious virus in hospitalized patients with coronavirus disease-2019 (COVID-19): duration and key determinants. medRxiv

Prolonged SARS-CoV-2 replication in an immunocompromised patient

Case Study: Prolonged infectious SARS-CoV-2 shedding from an asymptomatic immunocompromised cancer patient

Prolonged SARS-CoV-2 shedding and mild course of COVID-19 in a patient after recent heart transplantation

Multicenter comparison of the Cobas 6800 system with the RealStar RT-PCR kit for the detection of SARS-CoV-2

NanoPack: visualizing and processing long-read sequencing data

Persistence and Evolution of SARS-CoV-2 in an Immunocompromised Host

Prevalence of Asymptomatic SARS-CoV-2 Infection : A Narrative Review

Prevalence of SARS-CoV-2 in Spain (ENE-COVID): a nationwide, population-based seroepidemiological study

Asymptomatic Reinfection in 2 Healthcare Workers From India With Genetically Distinct Severe Acute Respiratory Syndrome Coronavirus 2

COVID-19) Re-infection by a Phylogenetically Distinct Severe Acute Respiratory Syndrome Coronavirus 2 Strain Confirmed by Whole Genome Sequencing

Genomic evidence for reinfection with SARS-CoV-2: a case study. The Lancet Infectious Diseases

M a n u s c r i p t FUNDING This study was supported in part by the ANRS (Agence Nationale de la Recherche sur le SIDA et les hépatites virales), the PhyloCoV study, funded by the FRM (Fondation pour la Recherche Médicale) and the TheraCoV study, funded by the ANR (Agence Nationale pour la Recherche). The authors did not present conflict of interests with the current work. A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t Figure 1