Background: Viral infections are common complications following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Allo-HSCT recipients with steroid-refractory/dependent graft-versus-host disease (GvHD) are highly immunosuppressed and are more vulnerable to infections with weakly pathogenic or commensal viruses. Here, twenty-five adult allo-HSCT recipients from 2016 to 2019 with acute or chronic steroid-refractory/dependent GvHD were enrolled in a prospective cohort of patients at Geneva University Hospitals. We performed metagenomics next-generation sequencing (mNGS) analysis using a validated viral pipeline and de novo analysis on pooled stored routine plasma samples collected throughout the period of intensive steroid treatment or second-line GvHD therapy to identify weakly pathogenic, commensal and unexpected viruses.

Results: Median duration of intensive immunosuppression was 5.1 months (IQR 5.5). GvHD-related mortality rate was 36%. mNGS analysis detected viral nucleotide sequences in 24/25 patients. Sequences of ≥3 distinct viruses were detected in 16/25 patients, Anelloviridae (24/25) and human pegivirus-1 (9/25) were the most prevalent. In 7/25 patients with fatal outcomes, unexpected viral sequences, not assessed by routine investigations, were identified with mNGS and confirmed by RT-PCR. These cases included usutu virus (1), rubella virus (1 vaccine-strain and 1 wild-type), novel human astrovirus (HAstV) MLB2 (1), classic HAstV (1), human polyomavirus 6 and 7 (2), cutavirus (1), and bufavirus (1).

Conclusions: Unexpected, opportunistic and protracted viral infections were identified in 28% of highly immunocompromised allo-HSCT recipients with steroid refractory/dependent GvHD. These identified viruses have all been previously described in humans, but have poorly understood clinical significance. Rubella virus identification raises the possibility of re-emergence from past infections or vaccinations.