Explosive nosocomial outbreak of SARS-CoV-2 in a rehabilitation clinic: the limits of genomics for outbreak reconstruction.

BACKGROUND: Nosocomial outbreaks of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are frequent despite implementation of conventional infection control measures. An outbreak investigation was undertaken using advanced genomic and statistical techniques to reconstruct likely transmission chains and assess the role of healthcare workers (HCWs) in SARS-CoV-2 transmission. METHODS: A nosocomial SARS-CoV-2 outbreak in a university-affiliated rehabilitation clinic was investigated, involving patients and HCWs, with high coverage of pathogen whole-genome sequences (WGS). The time-varying reproduction number from epidemiological data (Rt) was estimated, and maximum likelihood phylogeny was used to assess genetic diversity of the pathogen. Genomic and epidemiological data were combined into a Bayesian framework to model the directionality of transmission, and a case-control study was performed to investigate risk factors for nosocomial SARS-CoV-2 acquisition in patients. FINDINGS: The outbreak lasted from 14th March to 12th April 2020, and involved 37 patients (31 with WGS) and 39 employees (31 with WGS), 37 of whom were HCWs. Peak Rt was estimated to be between 2.2 and 3.6. The phylogenetic tree showed very limited genetic diversity, with 60 of 62 (96.7%) isolates forming one large cluster of identical genomes. Despite the resulting uncertainty in reconstructed transmission events, the analyses suggest that HCWs (one of whom was the index case) played an essential role in cross-transmission, with a significantly greater fraction of infections (P<2.2e-16) attributable to HCWs (70.7%) than expected given the number of HCW cases (46.7%). The excess of transmission from HCWs was higher when considering infection of patients [79.0%; 95% confidence interval (CI) 78.5-79.5%] and frail patients (Clinical Frailty Scale score >5; 82.3%; 95% CI 81.8-83.4%). Furthermore, frail patients were found to be at greater risk for nosocomial COVID-19 than other patients (adjusted odds ratio 6.94, 95% CI 2.13-22.57). INTERPRETATION: This outbreak report highlights the essential role of HCWs in SARS-CoV-2 transmission dynamics in healthcare settings. Limited genetic diversity in pathogen genomes hampered the reconstruction of individual transmission events, resulting in substantial uncertainty in who infected whom. However, this study shows that despite such uncertainty, significant transmission patterns can be observed.

Unmasking viral sequences by metagenomic next-generation sequencing in adult human blood samples during steroid-refractory/dependent graft-versus-host disease.

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. Video abstract.

High-throughput sequencing for the aetiologic identification of viral encephalitis, meningoencephalitis, and meningitis. A narrative review and clinical appraisal.

BACKGROUND: Viral aetiologies are the most common cause of central nervous system (CNS) infections. Approximately one-half of CNS infections remain of undetermined origin. High-throughput sequencing (HTS) brought new perspectives to CNS infection investigations, allowing investigation of viral aetiologies with an unbiased approach. HTS use is still limited to specific clinical situations. OBJECTIVES: The aim of this review was to evaluate the contribution and pitfalls of HTS for the aetiologic identification of viral encephalitis, meningoencephalitis, and meningitis in CNS patient samples. SOURCES: PubMed was searched from 1 January 2008 to 2 August 2018 to retrieve available studies on the topic. Additional publications were included from a review of full-text sources. CONTENT: Among 366 studies retrieved, 29 used HTS as a diagnostic technique. HTS was performed in cerebrospinal fluid and brain biopsy samples of 307 patients, including immunocompromised, immunocompetent paediatric, and adult cases. HTS was performed retrospectively in 18 studies and prospectively in 11. HTS led to the identification of a potential causal virus in 41 patients, with 11 viruses known and ten not expected to cause CNS infections. Various HTS protocols were used. IMPLICATIONS: The additional value of HTS is difficult to quantify because of various biases. Nevertheless, HTS led to the identification of a viral cause in 13% of encephalitis, meningoencephalitis, and meningitis cases in which various assays failed to identify the cause. HTS should be considered early in clinical management as a complement to routine assays. Standardized strategies and systematic studies are needed for the integration of HTS in clinical management.

Human pegivirus persistence in human blood virome after allogeneic haematopoietic stem-cell transplantation.

OBJECTIVES: Because commensal viruses are defined by the immunologic tolerance afforded to them, any immunomodulation, such as is received during haematopoietic stem-cell transplantation, may shift the demarcation between innocuous viral resident and disease-causing pathogen. METHODS: We analysed by deep-sequencing the plasma virome of 40 allogeneic haematopoietic stem-cell transplantation patients 1 month after transplantation. Because human pegivirus (HPgV) was highly prevalent, we performed a 1-year screening of 122 plasma samples by specific real-time reverse transcription PCR assay. We used the log-rank test and the Gray test to assess association with outcomes, and the Mann-Whitney test and multivariable linear regression model to assess association with T-cell reconstitution. RESULTS: Polyomaviruses (PyV) (20/40 patients), anelloviruses (16/40), pegiviruses (14/40) and herpesviruses (14/40) were most frequently identified, including ten cytomegalovirus; three Epstein-Barr virus; two herpes simplex virus type 1; one human herpesvirus 6b and one human herpesvirus 7; 18 Merkel cell-PyV; two BK-PyV; three PyV-6; and one JC-PyV. Papillomavirus and adenovirus were identified in 11 and two patients, respectively. The HPgV specific real-time reverse transcription PCR screening identified 51 of 122 positive samples, high virus loads and persistent infections up to 1 year after transplantation. Comparison between patients with or without HPgV infection at time of transplantation did not reveal a significant difference in infections, engraftment, survival, graft vs. host disease, relapse or immune reconstitution. CONCLUSIONS: The blood virome after allogeneic haematopoietic stem-cell transplantation includes several DNA viruses, notably herpesviruses and PyV. Among RNA viruses, HPgV is highly prevalent and persists for several months, and it thus may deserve special attention in further research on immune reconstitution.

Comparative analysis of viral shedding in pediatric and adult subjects with central nervous system-associated enterovirus infections from 2013 to 2015 in Switzerland.

BACKGROUND: Several enterovirus (EV) genotypes can result in aseptic meningitis, but their routes of access to the central nervous system remain to be elucidated and may differ between the pediatric and adult populations. OBJECTIVE: To assess the pattern of viral shedding in pediatric and adult subjects with acute EV meningitis and to generate EV surveillance data for Switzerland. STUDY DESIGN: All pediatric and adult subjects admitted to the University Hospitals of Geneva with a diagnosis of EV meningitis between 2013 and 2015 were enrolled. A quantitative EV real-time reverse transcriptase (rRT)-PCR was performed on the cerebrospinal fluid (CSF), blood, stool, urine and respiratory specimens to assess viral shedding and provide a comparative analysis of pediatric and adult populations. EV genotyping was systematically performed. RESULTS: EV positivity rates differed significantly between pediatric and adult subjects; 62.5% of pediatric cases (no adult case) were EV-positive in stool and blood for subjects for whom these samples were all collected. Similarly, the EV viral load in blood was significantly higher in pediatric subjects. Blood C-reactive protein levels were lower and the number of leucocytes/mm3 in the CSF were higher in non-viremic than in viremic pediatric subjects, respectively. A greater diversity of EV genotypes was observed in pediatric cases, with a predominance of echovirus 30 in children ≥3 years old and adults. CONCLUSION: In contrast to adults, EV-disseminated infections are predominant in pediatric subjects and show different patterns of EV viral shedding. This observation may be useful for clinicians and contribute to modify current practices of patient care.

Enterovirus and Parechovirus viraemia in young children presenting to the emergency room: Unrecognised and frequent.

BACKGROUND: Human Enterovirus (EV) and Parechovirus (HPeV) are well recognised as agents causing disease in neonates, but their importance is poorly described in the general paediatric population consulting with a suspicion of infection. OBJECTIVE: We investigated the prevalence of EV- or HPeV-associated infections in children presenting to a paediatric emergency department with a suspicion of infection. STUDY DESIGN: Plasma specimens collected in our paediatric emergency room for clinical reasons were screened by specific real-time RT-PCR for the presence of EV and HPeV. RESULTS: Based on an analyses of 233 plasma specimens, up to 6.9% and 2.6% were positive for EV and HPeV, respectively. Amongst the population <3y.o, prevalence of EV and HPeV viraemia was 11% and 3.7%, respectively. Importantly, 56.3% of positive EV specimens were detected in infants >3 months of age. CONCLUSION: The prevalence of EV and HPeV viraemia in children <3 years old is largely underestimated. Our results confirm that EV should be suspected and included in the work-up in children >3 months of age and not restricted to neonates.

Toscana virus meningitis case in Switzerland: an example of the ezVIR bioinformatics pipeline utility for the identification of emerging viruses.

Toscana virus (TOSV) represents a frequent cause of viral meningitis in the Mediterranean Basin that remains neglected in neighbouring countries. We report a documented TOSV meningitis case in a traveller returning from Tuscany to Switzerland. While routine serological and PCR assays could not discriminate between TOSV and Sandfly fever Naples virus infection, a high-throughput sequencing performed directly on the cerebrospinal fluid specimen and analysed with the ezVIR pipeline provided an unequivocal viral diagnostic. TOSV could be unequivocally considered as the aetiological agent, proving the potential of ezVIR to improve standard diagnostics in cases of infection with uncommon or emerging viruses.

Appearance of a novel measles G3 strain in multiple European countries within a two month period, 2010.

During late 2010, a previously unrecognised strain of measles genotype G3 virus was identified in five different European countries by the World Health Organization Measles and Rubella Laboratory Network.Apart from one, none had a travel history to south-east Asia, the usual source of G3 viruses, although epidemiological links could be established between some of the cases. This case series illustrates the value of genotyping and sequencing in tracking measles infections, and identifying otherwise unrecognised chains of transmission.

[Clinical and genetic diversity of rhinoviruses]. / Rhinovirus : diversité clinique et génomique.

Human rhinoviruses are the most common cause of viral respiratory infections across all age groups, from the neonate to the elderly patient. The benign nature of most of these infections as well as the difficulty to isolate the causative agent limits our perception of its real clinical impact. Molecular diagnostic tools have allowed to better characterize the variety of clinical presentations which are not limited to the common cold alone. It is now clearly established that rhinoviruses infect both the upper and lower tracheobronchial tree which may also be the site of viral replication. Moreover, the virus is the cause of significant complications in patients at risk such as those with asthma or highly immunocompromised hosts. The use of molecular screening techniques shows the very high diversity of circulating strains which are not only limited to known serotypes and has allowed the identification of new subgroups previously unknown. Detailed analysis of the genomic organization shows a common phylogeny between certain subgroups of rhinoviruses and enteroviruses and sheds light on the constraints modelling the evolution of human Picornaviridae. Furthermore, detailed analysis of the CRE structures shows that this structure is not only conserved for each species, but is also located on a specific region for each of these species.