Viral primary infections and reactivations are common complications in patients after solid organ transplantation (SOT) and hematopoietic stem cell transplantation (HSCT) and are associated with high morbidity and mortality. Among these patients, viral infections are frequently associated with viremia. Beyond the usual well-known viruses that are part of the routine clinical management of transplant recipients, numerous other viral signatures or genomes can be identified in the blood of these patients.

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 at Geneva University Hospitals. We performed metagenomics next-generation sequencing (mNGS) analysis using a validated pipeline and de novo analysis on pooled 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 patients with fatal outcomes, 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: Clinically unrecognized viral infections were identified in 28% of highly immunocompromised allo-HSCT recipients with steroid-refractory/dependent GvHD in consecutive samples. 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, or re-infection.

Meningitis, encephalitis, and myelitis are various forms of acute central nervous system (CNS) inflammation, which can coexist and lead to serious sequelae. Known aetiologies include infections and immune-mediated processes. Despite advances in clinical microbiology over the past decades, the cause of acute CNS inflammation remains unknown in approximately 50% of cases. High-throughput sequencing was performed to search for viral sequences in cerebrospinal fluid (CSF) samples collected from 26 patients considered to have acute CNS inflammation of unknown origin, and 10 patients with defined causes of CNS diseases. In order to better grasp the clinical significance of viral sequence data obtained in CSF, 30 patients without CNS disease who had a lumbar puncture performed during elective spinal anaesthesia were also analysed. One case of human astrovirus (HAstV)-MLB2-related meningitis and disseminated infection was identified. No other viral sequences that can easily be linked to CNS inflammation were detected. Viral sequences obtained in all patient groups are discussed. While some of them reflect harmless viral infections, others result from reagent or sample contamination, as well as index hopping. Altogether, this study highlights the potential of high-throughput sequencing in identifying previously unknown viral neuropathogens, as well as the interpretation issues related to its application in clinical microbiology.

False-positive results in the diagnostic of meningitis and encephalitis pose important challenges. This study aimed to determine false-positive rates for Haemophilus influenzae in cerebrospinal fluids evaluated by the BioFire FilmArray^® Meningitis/Encephalitis Panel. We conducted a retrospective study of all H. influenzae-positive FilmArray^®. Meningitis/Encephalitis Panel results from June 2016 to October 2019 in two Swiss university hospitals. Cases were classified as true positive, likely true-positive, and likely false-positive results according to cerebrospinal fluid culture, H. influenzae-specific quantitative real-time PCR (qPCR), and Gram staining, as well as culture of other materials. We performed 3,082 panels corresponding to 2,895 patients: 0.6% of the samples (18/3,082) were positive for H. influenzae. Culture and H. influenzae-specific qPCR were performed on 17/18 (94.4%) and 3/18 (16.7%) cerebrospinal fluid samples, respectively; qPCR was negative in all cases. Among 17 samples sent for culture, 10 concerned patients were not treated with antibiotics prior to lumbar puncture. Only 1/17 revealed growth of H. influenzae and was classified as a true positive. We further classified 3/18 (16.7%) cases with the identification of Gram-negative rods in the cerebrospinal fluid or positive blood cultures for H. influenzae as likely true-positive and 14/18 (77.8%) cases as likely false-positive. Diagnostic results should always be interpreted together with the clinical presentation, cerebrospinal fluid analysis, and other available microbiological results. All H. influenzae-positive results should be viewed with special caution and a H. influenzae-specific qPCR should be systematically considered.