Different Within-Host Viral Evolution Dynamics in Severely Immunosuppressed Cases with Persistent SARS-CoV-2

Abstract

A successful Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variant, B.1.1.7, has recently been reported in the UK, causing global alarm. Most likely, the new variant emerged in a persistently infected patient, justifying a special focus on these cases. Our aim in this study was to explore certain clinical profiles involving severe immunosuppression that may help explain the prolonged persistence of viable viruses. We present three severely immunosuppressed cases (A, B, and C) with a history of lymphoma and prolonged SARS-CoV-2 shedding (2, 4, and 6 months), two of whom finally died. Whole-genome sequencing of 9 and 10 specimens from Cases A and B revealed extensive within-patient acquisition of diversity, 12 and 28 new single nucleotide polymorphisms, respectively, which suggests ongoing SARS-CoV-2 replication. This diversity was not observed for Case C after analysing 5 sequential nasopharyngeal specimens and one plasma specimen, and was only observed in one bronchoaspirate specimen, although viral viability was still considered based on constant low Ct values throughout the disease and recovery of the virus in cell cultures. The acquired viral diversity in Cases A and B followed different dynamics. For Case A, new single nucleotide polymorphisms were quickly fixed (13-15 days) after emerging as minority variants, while for Case B, higher diversity was observed at a slower emergence: fixation pace (1-2 months). Slower SARS-CoV-2 evolutionary pace was observed for Case A following the administration of hyperimmune plasma. This work adds knowledge on SARS-CoV-2 prolonged shedding in severely immunocompromised patients and demonstrates viral viability, noteworthy acquired intra-patient diversity, and different SARS-CoV-2 evolutionary dynamics in persistent cases.

Keywords: COVID-19; SARS-CoV-2; diversity; genomics; immunosuppressed; persistence; viral viability.

Figure 1

Treatment history for the study cases. Day 0 corresponds to diagnosis. For Case A, only the therapies administered during the period he was managed in our institution are indicated. Anti-inflammatory antibodies refer to either tozilizumab or anakinra. The graphs at the lower part of each case´s panel represent the Ct values for the positive RT-PCRs along the infection.

Figure 2

Chronological acquisition of diversity for the three study cases (days are indicated at the upper file; days coloured in pink correspond to plasma, in blue to bronchoaspirate, and without colour to nasopharyngeal specimens). On the left side, the SARS-CoV-2 genetic content is indicated. Single nucleotide polymorphisms are indicated in orange, when identified as fixed, and in yellow when they are found as minority/intermediate variants. In the right panel, the coordinates and specific substitutions are shown (nonsynonymous changes are highlighted in bold). At the bottom panel, Ct values for the specimens used in the sequencing analysis are shown. Dots out of the lines correspond to specimens other than nasopharyngeal (plasma and bronchoaspirate). All sequencing data covered >96% of the reference genome, with a 100X depth. The positions with an “X” correspond to the only 2 positions involved in the diversity analysis, which were not sufficiently covered in two specimens among those in the study.