Prolonged SARS-CoV-2 Positivity in Immunocompetent Patients: Virus Isolation, Genomic Integrity, and Transmission Risk

Abstract

Current guidelines for patient isolation in COVID-19 cases recommend a symptom-based approach, averting the use of control real-time reverse transcription PCR (rRT-PCR) testing. However, we hypothesized that patients with persistently positive results by RT-PCR for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could be potentially infectious for a prolonged time, even if immunocompetent and asymptomatic, which would demand a longer social isolation period than presently recommended. To test this hypothesis, 72 samples from 51 mildly symptomatic immunocompetent patients with long-lasting positive rRT-PCR results for SARS-CoV-2 were tested for their infectiousness in cell culture. The serological response of samples from those patients and virus genomic integrity were also analyzed. Infectious viruses were successfully isolated from 34.38% (22/64) of nasopharynx samples obtained 14 days or longer after symptom onset. Indeed, we observed successful virus isolation up to 128 days. Complete SARS-COV-2 genome integrity was demonstrated, suggesting the presence of replication-competent viruses. No correlation was found between the isolation of infectious viruses and rRT-PCR cycle threshold values or the humoral immune response. These findings call attention to the need to review current isolation guidelines, particularly in scenarios involving high-risk individuals. IMPORTANCE In this study, we evaluated mildly symptomatic immunocompetent patients with long-lasting positive rRT-PCR results for SARS-CoV-2. Infectious viruses were successfully isolated in cell cultures from nasopharynx samples obtained 14 days or longer after symptom onset. Indeed, we observed successful virus isolation for up to 128 days. Moreover, SARS-CoV-2 genome integrity was demonstrated by sequencing, suggesting the presence of replication-competent viruses. These data point out the risk of continuous SARS-CoV-2 transmission from patients with prolonged detection of SARS-CoV-2 in the upper respiratory tract, which has important implications for current precaution guidelines, particularly in settings where vulnerable individuals may be exposed (e.g., nursing homes and hospitals).

Keywords: COVID-19; COVID-19 nucleic acid testing; SARS-CoV-2; immunocompetent; persistence; virus shedding.

FIG 1

Patient enrollment and sample selection. The figure demonstrates the number of patients that had samples tested by rRT-PCR for SARS-CoV-2 and remained positive after 14 days and the percentage from these samples that were selected for virus isolation in cell culture.

FIG 2

Percentage of patients with persistently positive SARS-CoV-2 rRT-PCR result by days after symptom onset (DASO). Results are the percentage of COVID-19 patients (n = 1,064) followed until a negative rRT-PCR result was achieved. Red area represents the percentage of patients with persistently positive rRT-PCR until 14 DASO (71.33%). The longest documented positive rRT-PCR persistence was at 144 days after symptom onset (one patient).

FIG 3

Frequency of viral isolation and humoral response in SARS-CoV-2 PCR+ persistent samples. (A) Number of nasopharyngeal swab samples with positive and negative viral isolation in culture from persistent SARS-CoV-2 PCR+ patients collected at 14 days after symptom onset or longer. Samples (n = 61) were inoculated in susceptible Vero E6 and 293/ACE2 cells, and viral production was tested by virus plaque-forming assay. Black bars represent samples with positive viral isolation, and white bars represent samples negative for virus isolation. (B) Distribution of positive (full circle) and negative (hollow circle) viral isolation in culture among NP swab samples from patients with SARS-CoV-2 PCR+ over time (days after symptom onset). Only one sample was available from patients P05, P06, and P10-12. (C) Cycle threshold (C_T) values of rRT-PCR for SARS-CoV-2 N1 and N2 target genes from NP swab samples with positive and negative virus isolation in culture. Lines represent mean ± SEM values. (D) Serum samples time matched with nasopharyngeal swab samples were tested for the presence of IgG, IgA, and IgM against the SARS-CoV-2 spike protein. Dot plots represent optical density (OD)/cutoff values of samples with positive and negative virus isolation. Lines represent mean ± SEM values.

FIG 4

Genomic description and phylogenetic analyses of SARS-CoV-2 from persistently infected immunocompetent patients. (A) Panel exhibiting all nonsynonymous mutations identified in each sequence by Nextclade v1.7.1 (“x” marks presence), indicating the high degree of similarity of the characterized genomes. Noticeably, most pairs of sequences characterized from the same patients did not present any exclusively shared mutation (synapomorphy). (B) Sequences were aligned with a comprehensive set of B.1.1.33 sequences available on the GISAID EpiCoV database (n = 1,538) and a maximum likelihood tree was inferred with IQ-Tree v2.0.3, under the GTR+F+I+G4 model. The tree indicates the novel genome group with diverse branches along the tree, although often with limited support values, likely reflecting the high nucleotide identity in the data set. Only two pairs of samples from patients 20 (5912 and 8255) and 28 (6936 and 8222) cluster together, likely due to the presence of an exclusive synonymous mutation for each, G24040T and A3409G, respectively. Branch colors indicate SH-aLRT support values, as indicated in the color gradient bar on the left. The scale bar marks substitutions per site. Irrelevant branches have been collapsed, and the tree was rooted in the oldest sequence available in the data set for visualization purposes only.