Exploring viral aetiology in upper respiratory tract infections: insights from metagenomic next-generation sequencing in Swiss outpatients before and during the SARS-CoV-2 pandemic.

AIMS OF THE STUDY: Upper respiratory tract infections are among the most common reasons for primary care consultations. They are diagnosed predominantly based on clinical assessment. Here, we investigated the benefit of viral metagenomic next-generation sequencing (mNGS) in an outpatient setting. METHODS: This prospective cross-sectional study included immunocompetent patients with acute upper respiratory tract infections. General practitioners collected pharyngeal swabs and demographic and clinical data. Specimens were analysed using viral mNGS and conventional tests. RESULTS: Two hundred seventy-seven patients were recruited by 21 general practitioners between 10/2019 and 12/2020, of which 91% had a suspected viral aetiology. For 138 patients (49.8%), mNGS identified one or more respiratory viruses. The mNGS showed a high overall agreement with conventional routine diagnostic tests. Rhinoviruses were the most frequently detected respiratory viruses (20.2% of patients). Viral mNGS reflected the influenza wave in early 2020 and the SARS-CoV-2 pandemic outbreak in Switzerland in March 2020. Notably, rhinoviruses continued to circulate despite non-pharmaceutical hygiene measures. CONCLUSIONS: Viral mNGS allowed the initial diagnosis to be retrospectively re-evaluated. Assuming reduced turnaround times, mNGS has the potential to directly guide the treatment of upper respiratory tract infections. On an epidemiological level, our study highlights the utility of mNGS in respiratory infection surveillance, allowing early detection of epidemics and providing information crucial for prevention.

Risk Factors for Nontuberculous Mycobacteria Infections in Solid Organ Transplant Recipients: A Multinational Case-Control Study.

BACKGROUND: Risk factors for nontuberculous mycobacteria (NTM) infections after solid organ transplant (SOT) are not well characterized. Here we aimed to describe these factors. METHODS: Retrospective, multinational, 1:2 matched case-control study that included SOT recipients ≥12 years old diagnosed with NTM infection from 1 January 2008 to 31 December 2018. Controls were matched on transplanted organ, NTM treatment center, and post-transplant survival greater than or equal to the time to NTM diagnosis. Logistic regression on matched pairs was used to assess associations between risk factors and NTM infections. RESULTS: Analyses included 85 cases and 169 controls (59% male, 88% White, median age at time of SOT of 54 years [interquartile range {IQR} 40-62]). NTM infection occurred in kidney (42%), lung (35%), heart and liver (11% each), and pancreas transplant recipients (1%). Median time from transplant to infection was 21.6 months (IQR 5.3-55.2). Most underlying comorbidities were evenly distributed between groups; however, cases were older at the time of NTM diagnosis, more frequently on systemic corticosteroids and had a lower lymphocyte count (all P < .05). In the multivariable model, older age at transplant (adjusted odds ratio [aOR] 1.04; 95 confidence interval [CI], 1.01-1.07), hospital admission within 90 days (aOR, 3.14; 95% CI, 1.41-6.98), receipt of antifungals (aOR, 5.35; 95% CI, 1.7-16.91), and lymphocyte-specific antibodies (aOR, 7.73; 95% CI, 1.07-56.14), were associated with NTM infection. CONCLUSIONS: Risk of NTM infection in SOT recipients was associated with older age at SOT, prior hospital admission, receipt of antifungals or lymphocyte-specific antibodies. NTM infection should be considered in SOT patients with these risk factors.

Epidemiology and outcomes of bone and joint infections in solid organ transplant recipients.

Bone and joint infection (BJI) epidemiology and outcomes in solid organ transplant recipients (SOTr) remain largely unknown. We aim to describe BJI in a multi-center cohort of SOTr (Swiss Transplant Cohort Study). All consecutive SOTr with BJI (01.05.2008-31.12.2019) were included. A nested case-control study to identify risk factors for BJI was performed. Among 4482 patients, 61 SOTr with 82 BJI were included, at an incidence of 1.4% (95% CI 1.1-1.7), higher in heart and kidney-pancreas SOTr (Gray's test p < .01). Although BJI were predominately late events (median of 18.5 months post-SOT), most infections occurred during the first year post-transplant in thoracic SOTr. Diabetic foot osteomyelitis was the most frequent infection (38/82, 46.3%), followed by non-vertebral osteomyelitis (26/82, 31.7%). Pathogens included Gram-positive cocci (70/131, 53.4%), Gram-negative bacilli (34/131, 26.0%), and fungi (9/131, 6.9%). BJI predictors included male gender (OR 2.94, 95% CI 1.26-6.89) and diabetes (OR 2.97, 95% CI 1.34-6.56). Treatment failure was observed in 25.9% (21/81) patients and 1-year mortality post-BJI diagnosis was 14.8% (9/61). BJI remain a rare event in SOTr, associated with subtle clinical presentations, high morbidity and relapses, requiring additional studies in the future.

In Vitro Comparison of the Internal Ribosomal Entry Site Activity from Rodent Hepacivirus and Pegivirus and Construction of Pseudoparticles.

The 5' untranslated region (5' UTR) of rodent hepacivirus (RHV) and pegivirus (RPgV) contains sequence homology to the HCV type III internal ribosome entry sites (IRES). Utilizing a monocistronic expression vector with an RNA polymerase I promoter to drive transcription, we show cell-specific IRES translation and regions within the IRES required for full functionality. Focusing on RHV, we further pseudotyped lentivirus with RHV and showed cell surface expression of the envelope proteins and transduction of murine hepatocytes and we then constructed full-length RHV and RPgV replicons with reporter genes. Using the replicon system, we show that the RHV NS3-4A protease cleaves a mitochondrial antiviral signaling protein reporter. However, liver-derived cells did not readily support the complete viral life cycle.

Tissue microenvironment dictates the fate and tumor-suppressive function of type 3 ILCs.

Innate lymphoid cells (ILCs) have been classified into "functional subsets" according to their transcription factor and cytokine profiles. Although cytokines, such as IL-12 and IL-23, have been shown to shape plasticity of ILCs, little is known about how the tissue microenvironment influences the plasticity, phenotype, and function of these cells. Here, we show clearly demarcated tissue specifications of Rorc-dependent ILCs across lymphoid and nonlymphoid organs. Although intestinal Rorc fate map-positive (Rorcfm+) ILCs show a clear ILC3 phenotype, lymphoid tissue-derived Rorcfm+ ILCs acquire an natural killer (NK) cell/ILC1-like phenotype. By adoptively transferring Rorcfm+ ILCs into recipient mice, we show that ILCs distribute among various organs and phenotypically adapt to the tissue environment they invade. When investigating their functional properties, we found that only lymphoid-tissue resident Rorcfm+ ILCs can suppress tumor growth, whereas intestinal Rorcfm- ILC1s or NK cells fail to inhibit tumor progression. We thus propose that the tissue microenvironment, combined with ontogeny, provides the specific function, whereas the phenotype is insufficient to predict the functional properties of ILCs.

IL17A-Mediated Endothelial Breach Promotes Metastasis Formation.

The role of the IL23/IL17A axis in tumor-immune interactions is a matter of controversy. Although some suggest that IL17A-producing T cells (TH17) can suppress tumor growth, others report that IL17A and IL23 accelerate tumor growth. Here, we systematically assessed the impact of IL17A-secreting lymphocytes in several murine models of tumor lung metastasis. Genetic fate mapping revealed that IL17A was secreted within lung metastases predominantly by γδ T cells, whereas TH17 cells were virtually absent. Using different tumor models, we found Il17a(-/-) mice to consistently develop fewer pulmonary tumor colonies. IL17A specifically increased blood vessel permeability and the expression of E-selectin and VCAM-1 by lung endothelial cells in vivo. In transgenic mice, specific targeting of IL17A to the endothelium increased the number of tumor foci. Moreover, the direct impact of IL17A on lung endothelial cells resulted in impaired endothelial barrier integrity, showing that IL17A promotes the formation of lung metastases through tumor-endothelial transmigration.

T cell contamination in flow cytometry gating approaches for analysis of innate lymphoid cells.

Innate lymphoid cells (ILCs) differ from T and B cells as they do not express genetically rearranged antigen receptors. The most prominent member of this group, NK cells, can be identified by numerous surface receptors such as natural cytotoxicity receptors (NCRs). However, novel groups of ILCs have recently been described and classified based on fate-determining transcription factors and cytokines being produced, similarly to T helper cells. Due to the lack of exclusive markers, ILCs are primarily defined by the paucity of lineage markers. Using RORc-fate-mapping mice, we found that the common lineage exclusion using CD3 yields an ILC population containing a large proportion of T cells with recombined TCR loci and low expression of CD3. Thus, we suggest adding CD5 as a marker for thorough elimination of T cells to avoid erroneous interpretations of ILC function in immunity.