Virological Aspects of COVID-19 in Patients with Hematological Malignancies: Duration of Viral Shedding and Genetic Analysis

Abstract

Coronavirus disease 2019 (COVID-19) has been associated with a significant fatality rate and persistent evolution in immunocompromised patients. In this prospective study, we aimed to determine the duration of excretion of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 37 Tunisian patients with hematological malignancies (40.5% with lymphoma and 37.8% with leukemia). In order to investigate the accumulation of viral mutations, we carried out genetic investigation on longitudinal nasopharyngeal samples using RT-PCR and whole-genome sequencing. Patients' samples were collected until the RT-PCR results became negative. SARS-CoV-2 infection was symptomatic in 48.6% of cases with fever, and cough was symptomatic in 61% of cases; the mortality rate was estimated to be 13.5%. The duration of viral RNA shedding ranged from 7 to 92 days after onset; it exceeded 18 days in 79.4% of cases. An intermittent PCR positivity was observed in two symptomatic patients. Persistent PCR positivity, defined as the presence of viral RNA for more than 30 days, was found in 51.4% of cases. No significant differences were observed for age, sex, type of hematological malignancy, or COVID-19 evolution between this group and a second one characterized by non-persistent PCR positivity. Lymphopenia was an independent predictor of prolonged SARS-CoV-2 RNA detection (p = 0.04). Three types of variants were detected; the most frequent was the Omicron. Globally, the mean intra-host variability in the SARS-CoV-2 genome was 1.31 × 10^-3 mutations per site per year; it was 1.44 × 10^-3 in the persistent group and 1.3 × 10^-3 in the non-persistent group. Three types of mutations were detected; the most frequent were nucleotide substitutions in the spike (S) gene. No statistically significant difference was observed between the two groups as to the type and mean number of observed mutations in the whole genome and the S region (p = 0.650). Sequence analysis revealed the inclusion of one to eight amino acid-changing events in seventeen cases; it was characterized by genetic stability from the third to the twentieth day of evolution in six cases. For the two patients with intermittent PCR positivity, sequences obtained from samples before and after negative PCR were identical in the whole genome, confirming an intra-host evolution of the same viral strain. This study confirms the risk of persistent viral shedding in patients with hematological malignancies. However, persistence of PCR positivity seems to be correlated only with a continuous elimination of viral RNA debris. Additional studies based on cell culture analysis are needed to confirm these findings.

Keywords: SARS-CoV-2 infection; hematological malignancies; viral shedding; whole-genome sequencing.

Figure 1

Viral shedding of Sars-cov-2 in hematological malignancy patients (n = 37) and lineage assignment, Tunisia.

Figure 2

Intermittent PCR positivity in two symptomatic patients.

Figure 3

Phylogenetic tree built using the Nextclade online software (https://clades.nextstrain.org/tree, accessed on 2 March 2023) and based on n = 75 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases detected in Tunisian patients with hematological malignancy [23]. The circles represent the Tunisian sequences in comparison with published sequences from all over the world. Five different clades were detected at the Aziza Othmana Hospital in this type of population: the 20I (Alpha) (n = 1; 2.7%), the 21J (Delta) (n = 2; 5.4%), the 21K (Omicron) (n = 15; 40.5%), the 21L (Omicron) (n = 6; 16.2%), and the 22B (Omicron) (n = 13; 35.1%).The most observed variant was Omicron (n = 34; 91.8%).

Figure 4

Values of entropy for the changes in AA after analyses of 75 global SARS-COV-2 genomes in Tunisian patients with hematological malignancies.