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In the Swiss HIV Cohort Study, 61/222 (27%) HIV-suppressed persons with chronic hepatitis B virus (HBV) infection had HBV replication after two years on tenofovir, of whom 77% were suppressed thereafter. Self-reported adherence to therapy and HBV viral load at tenofovir initiation were predictors of persistent replication.

Background Human adenovirus (HAdV) is an important pathogen seen in clinical practice. Long-term studies may help better understand epidemiological trends and changes in circulating genotypes over time. Purpose Using a large biobank of samples from hospitalized, adenovirus-positive patients over a 20-year period, we aimed to analyze long-term epidemiological trends and genotypic relatedness among circulating HAdV strains. Methods Based on samples from hospitalized patients confirmed to be HAdV positive in Bern, Switzerland, from 1998 to 2017, and on their associated demographic and clinical data, we identified epidemiological trends and risk factors associated with HAdV infection. HAdV genotyping was performed by PCR amplification and sequencing of the hypervariable hexon gene. The obtained sequences were phylogenetically compared with sequences from international HAdV strains. Results HAdV was identified in 1302 samples tested. Cases of HAdV infection were reported throughout the years with no clear seasonality. Upper respiratory tract samples, conjunctivitis swabs, and stool had the highest positivity rate (56.2%, 18.7%, and 14.2% of the cases, respectively). HAdV infection was highest among children ≤4 years old. Increased number of HAdV cases were observed in years 2009 (n = 110) and 2010 (n =112). HAdV8 was the predominant genotype among patients older than 20 years, and was mostly associated with ophthalmic infection. Predominant genotypes among children ≤4 years old were HAdV1, HAdV2, and HAdV3, which were mostly associated with respiratory tract infections. Recurring peaks of increased HAdV cases were evidenced every 4 years among children ≤4 years old. Conclusion Our study gives novel insights on long-term epidemiological trends and phylogenetic relatedness among circulating HAdV strains in Switzerland, country in which little data on HAdV prevalence and diversity was so far available.

BackgroundMorbidity and mortality of COVID-19 is increased in patients with B-cell depleting therapies, the majority of which also show compromised vaccination-induced immune responses. Herein, we report on the trajectories of anti-SARS-CoV-2 antibodies in patients from the original RituxiVac study compared to healthy volunteers and investigate the immunogenicity of a third vaccination in previously non-responding patients.MethodsA follow-up evaluation was performed in volunteers and patients from the RituxiVac Study (NCT04877496), which investigated the humoral and cell-mediated immune response after SARS-CoV-2 mRNA vaccination in patients with a history with anti-CD20 depleting therapies (rituximab or ocrelizumab). The current population included 33 patients and 26 healthy volunteers with initial humoral vaccine response and 32 non-responding patients. Primary outcome was anti-SARS-CoV-2 antibody trajectories in vaccine responders 4.3 months (median; interquartile range [IQR]: 3.6 – 4.8 months) after first evaluation and humoral responses after a third vaccine dose in previous non-responders. Antibody decay rates were compared using analysis of covariance in linear regression.ResultsIn patients with detectable anti-spike IgG antibodies after two-dose vaccination, circulating anti-spike IgG persisted in 88% (29/33) of patients 5.6 months after the second vaccination (median; IQR: 5.1-6.7) compared to 92% (24/26) of healthy volunteers 6.8 months after the second dose (IQR: 6.0-7.1) (p=0.7). Antibody decay rates were comparable between patients and controls with −0.54 signal/cut-off (s/c) units per month (IQR −0.72 to −0.45) and −0.60 s/c units per month (IQR: −0.88 to −0.44), p=0.70. Two-dose responders with loss of circulating antibodies at follow-up (n=4/33, 12%) had lower initial antibody concentrations (pConclusionThe present study reveals comparable antibody reduction rates between patients with CD20-depleting treatment history and healthy volunteers, but inefficient humoral responses to a third dose of SARS-CoV-2 mRNA vaccines in the majority of two-dose non-responders. There is a need for individually tailored vaccination strategies in immunocompromised patients that could be stratified by B cell counts and initial level of antibody titers. (Funded by Bern University Hospital, ClinicalTrials.gov number, NCT04877496)

Background: In coronavirus disease-2019 (COVID-19) patients, there is increasing evidence of neuronal injury by the means of elevated serum neurofilament light chain (sNfL) levels. However, the role of systemic inflammation and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–specific immune response with regard to neuronal injury has not yet been investigated. Methods: In a prospective cohort study, we recruited patients with mild–moderate ( n = 39) and severe ( n = 14) COVID-19 and measured sNfL levels, cytokine concentrations, SARS-CoV-2-specific antibodies including neutralizing antibody titers, and cell-mediated immune responses at enrollment and at 28(±7) days. We explored the association of neuro-axonal injury as by the means of sNfL measurements with disease severity, cytokine levels, and virus-specific immune responses. Results: sNfL levels, as an indicator for neuronal injury, were higher at enrollment and increased during follow-up in severely ill patients, whereas during mild–moderate COVID-19, sNfL levels remained unchanged. Severe COVID-19 was associated with increased concentrations of cytokines assessed [interleukin (IL)-6, IL-8, interleukin-1 beta (IL-1β), and tumor necrosis factor-alpha (TNF-α)], higher anti-spike IgG and anti-nucleocapsid IgG concentrations, and increased neutralizing antibody titers compared with mild–moderate disease. Patients with more severe disease had higher counts of defined SARS-CoV-2-specific T cells. Increases in sNfL concentrations from baseline to day 28(±7) positively correlated with anti-spike protein IgG antibody levels and with titers of neutralizing antibodies. Conclusion: Severe COVID-19 is associated with increased serum concentration of cytokines and subsequent neuronal injury as reflected by increased levels of sNfL. Patients with more severe disease developed higher neutralizing antibody titers and higher counts of SARS-CoV-2-specific T cells during the course of COVID-19 disease. Mounting a pronounced virus-specific humoral and cell-mediated immune response upon SARS-CoV-2 infection did not protect from neuro-axonal damage as by the means of sNfL levels.

We are reporting on the observation of a large, under-sequenced region of the S gene of the SARS-CoV2 Delta variant genomes, identified in sequences originating from various sequencing centres worldwide (e.g. USA, India, England, Switzerland, France, Germany). This poorly sequenced region was identified from the early phases of the Delta variant spread and the phenomenon is still ongoing. As many commonly-used protocols rely on amplicon-based sequencing procedures, we investigated the likely origin of the issue. We established its biological origin as resulting from mutations in the viral genomes at primer binding sites. We designed and evaluated new PCR primers to circumvent this issue in order to complement the ARTIC v3 set, and validated their performance for the sequencing of circulating Delta variants.

Background B-cell-depleting therapies increase the risk of morbidity and mortality due to COVID-19. Evidence-based SARS-CoV-2 vaccination strategies for patients on B-cell-depleting therapies are scarce. We aimed to investigate humoral and cell-mediated immune responses to SARS-CoV-2 mRNA-based vaccines in patients receiving CD20-targeted B-cell-depleting agents for autoimmune disease, malignancy, or transplantation. Methods The RituxiVac study was an investigator-initiated, single-centre, open-label study done at the Bern University Hospital (Bern, Switzerland). Patients with a treatment history of anti-CD20-depleting agents (rituximab or ocrelizumab) and with no previous history of SARS-CoV-2 infection were enrolled between April 26 and June 30, 2021, for analysis of humoral and cell-mediated immune responses (by interferon-γ [IFNγ] release assay) at least 4 weeks after completing vaccination against SARS-CoV-2. Healthy controls without a history of SARS-CoV-2 infection were also enrolled at least 4 weeks after completing vaccination against SARS-CoV-2. All study participants received two doses of either the Pfizer-BioNTech BNT162b2 vaccine or the Moderna mRNA-1273 vaccine. The primary outcome was the proportion of patients with a history of anti-CD20 treatment who showed a humoral immune response against the SARS-CoV-2 spike protein in comparison with immunocompetent controls. Prespecified secondary endpoints were the effect of anti-CD20 therapy (including time since last treatment and cumulative dose) on humoral or cell-mediated immune responses to SARS-CoV-2 vaccination, and biomarkers of immunocompetence. This study is registered with ClinicalTrials.gov, NCT04877496. Findings The final study population comprised 96 patients and 29 immunocompetent controls. The median age of patients was 67 years (IQR 57-72) and of controls was 54 years (45-62), and 51 (53%) of 96 patients and 19 (66%) of 29 controls were female. The median time since last anti-CD20 treatment was 1·07 years (IQR 0·48-2·55) and the median cumulative dose of an anti-CD20 depleting agent was 2·80 g (1·50-5·00). Anti-spike IgG antibodies were detected in 47 (49%) of 96 patients 1·79 months (IQR 1·16-2·48) after the second vaccine dose compared to 29 (100%) of 29 controls 1·81 months (1·17-2·48) after the second vaccine dose (p7·6 months; positive predictive value 0·78), peripheral CD19+ cell count (>27 cells per μL; positive predictive value 0·70), and CD4+ lymphocyte count (>653 cells per μL; positive predictive value 0·71) were predictive of humoral vaccine response (area under the curve [AUC] 67% [95% CI 56-78] for time since last anti-CD20 therapy, 67% [55-80] for peripheral CD19+ count, and 66% [54-79] for CD4+ count). Interpretation This study provides further evidence of blunted humoral and cell-mediated immune responses elicited by SARS-CoV-2 mRNA vaccines in patients with a history of CD20 B-cell-depleting treatment. Lymphocyte subpopulation counts were associated with vaccine response in this highly vulnerable population. On validation, these results could help guide both the administration of SARS-CoV-2 vaccines and B-cell-depleting agents in this population. Funding Bern University Hospital.

Background: Serological tests are a powerful tool in the monitoring of infectious diseases and the detection of host immunity. However, manufacturers often provide diagnostic accuracy data generated through biased studies and the performance in clinical practice is essentially unclear. Objectives: We aimed to determine the diagnostic accuracy of various serological testing strategies for (a) identification of patients with previous coronavirus disease-2019 (COVID-19) and (b) prediction of neutralizing antibodies against SARS-CoV-2 in real-life clinical settings. Methods: We prospectively included 2’573 consecutive health-care workers and 1’085 inpatients with suspected or possible previous COVID-19 at a Swiss University Hospital. Various serological immunoassays based on different analytical techniques (enzyme-linked immunosorbent assays, ELISA; chemiluminescence immunoassay, CLIA; electrochemiluminescence immunoassay, ECLIA; lateral-flow immunoassay, LFI), epitopes of SARS-CoV-2 (nucleocapsid, N; receptor-binding domain, RBD; extended RBD, RBD+; S1 or S2 domain of the spike [S] protein, S1/S2), and antibody subtypes (IgG, pan-Ig) were conducted. A positive real-time PCR test from a nasopharyngeal swab was defined as previous COVID-19. Neutralization assays with live SARS-CoV-2 were performed in a subgroup of patients to assess neutralization activity (n=201). Results: The sensitivity to detect patients with previous COVID-19 was ≥85% in anti-N ECLIA (86.8%) and anti-S1 ELISA (86.2%). Sensitivity was 84.7% in anti-S1/S2 CLIA, 84.0% in anti-RBD+ LFI, 81.0% in anti-N CLIA, 79.2% in anti-RBD ELISA, and 65.6% in anti-N ELISA. The specificity was 98.4% in anti-N ECLIA, 98.3% in anti-N CLIA, 98.2% in anti-S1 ELISA, 97.7% in anti-N ELISA, 97.6% in anti-S1/S2 CLIA, 97.2% in anti-RBD ELISA, and 96.1% in anti-RBD+ LFI. The sensitivity to detect neutralizing antibodies was ≥85% in anti-S1 ELISA (92.7%), anti-N ECLIA (91.7%), anti-S1/S2 CLIA (90.3%), anti-RBD+ LFI (87.9%), and anti-RBD ELISA (85.8%). Sensitivity was 84.1% in anti-N CLIA, and 66.2% in anti-N ELISA. The specificity was ≥97% in anti-N CLIA (100%), anti-S1/S2 CLIA (97.7%), and anti-RBD+ LFI (97.9%). Specificity was 95.9% in anti-RBD ELISA, 93.0% in anti-N ECLIA, 92% in anti-S1 ELISA, and 65.3% in anti-N ELISA. Diagnostic accuracy measures were consistent among subgroups. Conclusions: The diagnostic accuracy of serological tests for SARS-CoV-2 antibodies varied remarkably in clinical practice, and the sensitivity to identify patients with previous COVID-19 deviated substantially from the manufacturer’s specifications. The data presented here should be considered when using such tests to estimate the infection burden within a specific population and determine the likelihood of protection against re-infection.

BackgroundHuman Adenoviruses (HAdVs) are highly contagious pathogens of clinical importance, especially among the pediatric population. Studies on comparative viral genomic analysis of cases associated with severe and mild infections due to HAdV are limited. Using whole-genome sequencing (WGS), we investigated whether there were any differences between circulating HAdV strains associated with severe infections (meningitis, sepsis, convulsion, sudden infant death syndrome, death, and hospitalization) and mild clinical presentations in pediatric patients hospitalized between the years 1998 and 2017 in a tertiary care hospital group in Bern, Switzerland covering a population base of approx. 2 million inhabitants. The HAdV species implicated in causing severe infections in this study included HAdV species C genotypes (HAdV1, HAdV2, and HAdV5). Clustering of the HAdV whole-genome sequences of the severe and mild cases did not show any differences except for one sample (isolated from a patient presenting with sepsis, meningitis, and hospitalization) that formed its own cluster with HAdV species C genotypes. This isolate showed intertypic recombination events involving four genotypes, had the highest homology to HAdV89 at complete genome level, but possessed the fiber gene of HAdV1, thereby representing a novel genotype of HAdV species C. The incidence of potential recombination events was higher in severe cases than in mild cases. Our findings confirm that recombination among HAdVs is important for molecular evolution and emergence of new strains. Therefore, further research on HAdVs, particularly among susceptible groups, is needed and continuous surveillance is required for public health preparedness including outbreak investigations.

BackgroundB-cell depleting therapies increase COVID19 morbidity and mortality. For this specific population, evidence-based vaccination strategies are lacking. Here, we investigated humoral and cell mediated immune responses to SARS-CoV2 mRNA-based vaccines in patients receiving CD20-B-cell depleting agents for autoimmune disease, malignancy, or transplantation.MethodsPatients at the Bern University Hospital with a treatment history of anti-CD20 depleting agents (rituximab or ocrelizumab) were enrolled for analysis of humoral and cell-mediated immune responses (by interferon-γ release assay) after completing vaccination against SARS-CoV2. Primary outcome was the the anti-spike antibody response in anti-CD20-treated patients (n=96) in comparison to immunocompetent controls (n=29).ResultsAnti-spike IgG antibodies were detected in 49% of patients 1.79 months after the second vaccine dose (interquartile range, IQR: 1.16-2.48) compared to 100% of controls (p27/µl), and CD4+ lymphocyte count (>653/µl) predicted humoral vaccine response (area under the curve [AUC]: 67% [CI 56-78], 67% [CI 55-80] and 66% [CI 54-79], (positive predictive value [PPV]: 0.78, 0.7 and 0.71).ConclusionThis study provides evidence for blunted humoral and cell-mediated immune responses elicited by SARS-CoV2 mRNA vaccines in patients with CD20-depleting treatment history. Lymphocyte subpopulation counts are associated with vaccine response in this highly vulnerable population. (Funded by Bern University Hospital, ClinicalTrials.gov number, NCT04877496)

Background The most sensitive method to detect SARS-CoV-2 relies on rRT-PCR; however, viral RNA can be detected weeks/months after clinical resolution. Since rRT-PCR cannot discern between non- and infectious virus, it is unclear whether the presence of viral RNA after recovery reflects infectious SARS-CoV-2. However, recent studies suggest a positive correlation between antigen rapid tests (Ag-RDT) and virus isolation that is more suited to assess contagiousness. Objectives To assess the utility of SARS-CoV-2 diagnostic tests in different settings we evaluated the performance of Ag-RDT-based and a cell culture-based SARS-CoV-2 assay in comparison to rRT-PCR. Study design A total of 61 Nasopharyngeal-Swabs tested positive by cobas��� SARS-CoV-2 rRT-PCR were in parallel evaluated with the Roche Ag-RDT and a cell culture-based assay to detect SARS-CoV-2. Results SARS-CoV-2 was successfully isolated in 51/61 samples corresponding to 83.6%, which was 97.3% or 96.2% when considering samples with E-gene Ct-value

The rapid spread of the SARS-CoV-2 lineages B.1.1.7 (N501Y.V1) throughout the UK, B.1.351 (N501Y.V2) in South Africa, and P.1 (B.1.1.28.1; N501Y.V3) in Brazil has led to the definition of variants of concern (VoCs) and recommendations for lineage specific surveillance. In Switzerland, during the last weeks of December 2020, we established a nationwide screening protocol across multiple laboratories, focusing first on epidemiological and microbiological definitions. In January 2021, we validated and implemented an N501Y-specific PCR to rapidly screen for VoCs, which are then confirmed using amplicon sequencing or whole genome sequencing (WGS). A total of 13,387 VoCs have been identified since the detection of the first Swiss case in October 2020, with 4194 being B.1.1.7, 172 B.1.351, and 7 P.1. The remaining 9014 cases of VoCs have been described without further lineage specification. Overall, all diagnostic centers reported a rapid increase of the percentage of detected VOCs, with a range of 6 to 46% between 25 to 31 of January 2021 increasing towards 41 to 82% between 22 to 28 of February. A total of 739 N501Y positive genomes were analysed and show a broad range of introduction events to Switzerland. In this paper, we describe the nationwide coordination and implementation process across laboratories, public health institutions, and researchers, the first results of our N501Y-specific variant screening, and the phylogenetic analysis of all available WGS data in Switzerland, that together identified the early introduction events and subsequent community spreading of the VoCs., Microorganisms, 9 (4)

The rapid spread of the SARS-CoV-2 lineages B.1.1.7 (N501Y.V1) throughout the UK, B.1.351 (N501Y.V2) in South Africa, and P.1 (B.1.1.28.1; N501Y.V3) in Brazil has led to the definition of variants of concern (VoCs) and recommendations by the European Center for Disease Prevention and Control (ECDC) and World Health Organization (WHO) for lineage specific surveillance. In Switzerland, during the last weeks of December 2020, we established a nationwide screening protocol across multiple laboratories, focusing first on epidemiological definitions based on travel history and the S gene dropout in certain diagnostic systems. In January 2021, we validated and implemented an N501Y-specific PCR to rapidly screen for VoCs, which are then confirmed using amplicon sequencing or whole genome sequencing (WGS). A total of 3492 VoCs have been identified since the detection of the first Swiss case in October 2020, with 1370 being B1.1.7, 61 B.1.351, and none P.1. The remaining 2061 cases of VoCs have been described without further lineage specification. In this paper, we describe the nationwide coordination and implementation process across laboratories, public health institutions, and researchers, the first results of our N501Y-specific variant screening, and the phylogenetic analysis of all available WGS data in Switzerland, that together identified the early introduction events and subsequent community spreading of the VoCs.

BACKGROUND Healthcare workers are more frequently exposed to SARS-CoV-2 than the general population. Little is known about healthcare settings outside of hospitals. We studied the seroprevalence of SARS-CoV-2 among healthcare workers in outpatient facilities and retirement or nursing homes in the Canton of Solothurn, Switzerland in the first wave of the COVID-19 pandemic. METHODS Longitudinal seroprevalence study among healthcare workers with examinations at baseline and 2 months between June and September 2020. The Abbott SARS-CoV-2 IgG and Liaison/Diasorin SARS-CoV-2 S1/S2 IgG assay were used to detect antibodies against SARS-CoV-2. All participants provided demographic information. We report descriptive statistics and calculated the seroprevalence with 95% confidence intervals. RESULTS We included 357 healthcare workers; their median age was 43 years (interquartile range 29-54), and 315 (88.2%) were female. Forty-nine (13.7%) were physicians, 87 (24.4%) practice assistants and 221 (61.9%) nurses. Overall seroprevalence among healthcare workers in outpatient facilities and retirement or nursing homes was 3.4% (12/357). The 12 seropositive healthcare workers were all nurses (12/221, 5.5%); 11 worked at retirement or nursing homes and one at the hospital's outpatient clinic. Symptoms such as loss of smell or taste, shortness of breath, and fever were more prevalent among seropositive healthcare workers than seronegative healthcare workers. No close contact had detectable antibodies against SARS-CoV-2. CONCLUSIONS Seroprevalence among healthcare workers was low, but higher among nursing staff of retirement or nursing homes. Healthcare workers at private practices were able to protect themselves well during the first wave of the COVID-19 pandemic.

Ganciclovir (GCV)-resistant cytomegalovirus (CMV) infection is a common problem among solid organ transplant (SOT) recipients without prior CMV immunity (CMV D+/R-). GCV-resistant CMV represents a particular challenge for CMV management. Letermovir is a recently licensed antiviral agent for primary CMV prophylaxis in allogenic hematopoietic stem cell transplant (HSCT) recipients. Given the favorable safety profile and its oral bioavailability letermovir may be considered a valuable off-label option for secondary prophylaxis of GCV-resistant CMV in SOT recipients. Here, we describe our experience with letermovir as secondary prophylaxis for GCV-resistant CMV in two renal transplant recipients and review the literature in regard of previously published cases. Letermovir resistance emerged after a few months of secondary prophylaxis in the two renal transplant recipients. In both cases, the previously described UL56 C325Y letermovir resistance mutation was detected. In vitro studies of letermovir suggest a relatively low genetic barrier to resistance. Therefore, caution is warranted when using letermovir as secondary prophylaxis for GCV-resistant CMV infection.

Enteroviruses are small RNA viruses that affect millions of people each year by causing an important burden of disease with a broad spectrum of symptoms. In routine diagnostic laboratories, enteroviruses are identified by PCR-based methods, often combined with partial sequencing for genotyping. In this proof-of-principle study, we assessed direct RNA sequencing (DRS) using nanopore sequencing technology for fast whole-genome sequencing of viruses directly from clinical samples. The approach was complemented by sequencing the corresponding viral cDNA via Illumina MiSeq sequencing. DRS of total RNA extracted from three different enterovirus-positive stool samples produced long RNA fragments, covering between 59% and 99.6% of the most similar reference genome sequences. The identification of the enterovirus sequences in the samples was confirmed by short-read cDNA sequencing. Sequence identity between DRS and Illumina MiSeq enterovirus consensus sequences ranged between 94% and 97%. Here, we show that nanopore DRS can be used to correctly identify enterovirus genotypes from patient stool samples with high viral load and that the approach also provides rich metatranscriptomic information on sample composition for all life domains.

Enteroviruses are small RNA viruses that affect millions of people each year by causing an important burden of disease with a broad spectrum of symptoms. In routine diagnostic laboratories, those viruses are identified by PCR based methods, often combined with partial sequencing for genotyping. In this proof-of-principle study, we assessed direct RNA sequencing (DRS) using nanopore sequencing technology for fast whole-genome sequencing of viruses directly from clinical samples. Results of the approach were complemented with those obtained by sequencing the corresponding viral cDNA via Illumina MiSeq sequencing. DRS of total RNA extracted from three different enterovirus-positive stool samples produced long RNA fragments, covering between 59% to 99.6 % of the best reference genomes. The identification of the enterovirus sequences in the sample was confirmed by the short-read cDNA sequencing. Sequence identity between DRS and Illumina MiSeq enterovirus consensus sequences ranged between 94-97%. Here we show that nanopore DRS can be used to correctly identify the genotypes of enteroviruses from patient stool samples with high viral load.

The clinical course of SARS-CoV-2 infection (COVID-19) in children with hematologic malignancies is unclear. We describe the diagnosis, treatment and outcome of a 4-year-old boy with high-risk acute lymphoblastic leukemia and COVID-19. Regardless of immunosuppressive induction chemotherapy his symptoms remained moderate. He received only supportive treatment. Seroconversion occurred in a similar period as in immunocompetent adults. Despite prolonged myelosuppression he did neither acquire secondary infections nor did the treatment delay caused by the infection have a measurable negative impact on the residual disease of acute lymphoblastic leukemia. Intriguingly, residual leukemia even decreased even though he did not receive any antileukemic therapy.

Amplicon sequencing of the 16S rRNA gene is commonly used for the identification of bacterial isolates in diagnostic laboratories and mostly relies on the Sanger sequencing method. The latter, however, suffers from a number of limitations, with the most significant being the inability to resolve mixed amplicons when closely related species are coamplified from a mixed culture. This often leads to either increased turnaround time or absence of usable sequence data. Short-read next-generation sequencing (NGS) technologies could solve the mixed amplicon issue but would lack both cost efficiency at low throughput and fast turnaround times., Amplicon sequencing of the 16S rRNA gene is commonly used for the identification of bacterial isolates in diagnostic laboratories and mostly relies on the Sanger sequencing method. The latter, however, suffers from a number of limitations, with the most significant being the inability to resolve mixed amplicons when closely related species are coamplified from a mixed culture. This often leads to either increased turnaround time or absence of usable sequence data. Short-read next-generation sequencing (NGS) technologies could solve the mixed amplicon issue but would lack both cost efficiency at low throughput and fast turnaround times. Nanopore sequencing developed by Oxford Nanopore Technologies (ONT) could solve those issues by enabling a flexible number of samples per run and an adjustable sequencing time. Here, we report on the development of a standardized laboratory workflow combined with a fully automated analysis pipeline LORCAN (long read consensus analysis), which together provide a sample-to-report solution for amplicon sequencing and taxonomic identification of the resulting consensus sequences. Validation of the approach was conducted on a panel of reference strains and on clinical samples consisting of single or mixed rRNA amplicons associated with various bacterial genera by direct comparison to the corresponding Sanger sequences. Additionally, simulated read and amplicon mixtures were used to assess LORCAN’s behavior when dealing with samples with known cross-contamination levels. We demonstrate that by combining ONT amplicon sequencing results with LORCAN, the accuracy of Sanger sequencing can be closely matched (>99.6% sequence identity) and that mixed samples can be resolved at the single-base resolution level. The presented approach has the potential to significantly improve the flexibility, reliability, and availability of amplicon sequencing in diagnostic settings.

We analyzed changes in hepatitis B virus and hepatitis delta virus (HDV) viral loads (VL) during tenofovir-containing antiretroviral therapy among patients with a replicating HDV infection in the Swiss HIV Cohort Study. Only 28.6% experienced a ≥2.0 log reduction in HDV RNA, and 14.3% had undetectable HDV VL within 5 years.

BACKGROUND Acyclovir (ACV) is the most commonly used drug for herpes simplex virus (HSV) infection therapy. Prolonged antiviral therapy or prophylaxis in immunocompromised patients may promote the development of drug-resistant strains. Due to the high polymorphism in genes involved in drug resistance, phenotypic methods, although work-intensive, are still required to test drug susceptibility. Real-time cell analysis (RTCA) based methods could offer a rapid and less labor-intensive alternative for phenotypic testing of ACV resistance. OBJECTIVE To investigate the utility of a new RTCA based assay (RTCAA) to test acyclovir susceptibility of HSV clinical isolates. STUDY DESIGN Four reference strains and 93 clinical isolates (60 HSV-1 and 33 HSV-2) were tested by RTCAA. In the presence of ACV concentrations from 2.2 to 140.8 μM, Vero cells were infected with different virus dilutions. IC50 values were calculated by dose-response curve (DRC) with area-under-curve (AUC) method. The reference strains and 22 clinical isolates were additionally tested by dye-uptake assay, and IC50 values of both methods were compared. RESULTS IC50 values from RTCAA and dye-uptake assays were positively correlated (Spearman's rho = 0.897, p

Enteroviruses affect millions of people worldwide and are of significant clinical importance. The standard method for enterovirus identification and genotyping still relies on Sanger sequencing of short diagnostic amplicons. In this study, we assessed the feasibility of nanopore sequencing using the new flow cell &ldquo, Flongle&rdquo, for fast, cost-effective, and accurate genotyping of human enteroviruses from clinical samples. PCR amplification of partial VP1 gene was performed from multiple patient samples, which were multiplexed together after barcoding PCR and sequenced multiple times on Flongle flow cells. The nanopore consensus sequences obtained from mapping reads to a reference database were compared to their Sanger sequence counterparts. Using clinical specimens sampled over different years, we were able to correctly identify enterovirus species and genotypes for all tested samples, even when doubling the number of barcoded samples on one flow cell. Average sequence identity across sequencing runs was &gt, 99.7%. Phylogenetic analysis showed that the consensus sequences achieved with Flongle delivered accurate genotyping. We conclude that the new Flongle-based assay with its fast turnover time, low cost investment, and low cost per sample represents an accurate, reproducible, and cost-effective platform for enterovirus identification and genotyping.

Objectives Highly pathogenic viruses such as EBOV are a threat to routine laboratory workers. Inactivation procedures with Triton X-100 0.1% and/or heat are currently recommended, but have unknown effects on the accuracy of serological testing. Furthermore, virus inactivation by Triton X-100 0.1% was shown to be ineffective in serum. This study aimed to demonstrate virus inactivation in serum by Triton X-100 1% and maintained accuracy of serological testing. Methods A panel of 19 serological tests was run on patient serum samples after treatment with Triton X-100 1%, 0.1%, and 0.1% + heat inactivation at 60°C for 1 h. Mean differences between measurements (bias) were calculated applying the Bland–Altman method. To determine effectiveness of virus inactivation, herpes simplex virus 1 (HSV-1) was spiked into medium containing 90% or 1% serum, and treated with Triton X-100 0.1% or 1%. Infectious titres were then determined on Vero cells. Results Serological measurements showed good agreement between controls and samples treated with Triton X-100 0.1% and 1%, with an estimated bias of 0.6 ± 9.2% (n = 258) and –0.1 ± 18.6% (n = 174), respectively. Discordant qualitative results were rare. Conversely, heat inactivation alone and combined with Triton X-100 0.1% triggered a bias of 17.5 ± 66.4% (n = 200) and 37.9 ± 79.8% (n = 160), respectively. Triton X-100 1% completely inactivated HSV-1 in 1% and 90% serum while Triton X-100 0.1% failed to do so in 90% serum. Conclusions Unlike heat inactivation, Triton X-100 1% enabled accurate serological testing and completely inactivated HSV-1 in serum. This simple method could allow safe routine serological diagnostics in high-risk patients., Graphical abstract Image 1

Background Tablet computers are increasingly being used in hospital patient care and are often colonized with important human pathogens, while the impact of disinfection interventions remains controversial. Method In a prospective hygiene intervention study we consecutively sampled tablet computers exclusively used in a high-resource general internal medicine tertiary care setting with high routine hygiene measures. Our aim was to examine the change in colonizing bacteria on tablet computers before and after the introduction of a mandatory twice daily tablet disinfection intervention. Microbial identification was performed by conventional culture, and the association of bacterial colonization with the intervention was investigated using logistic regression. Results In a total of 168 samples we identified colonizing bacteria in 149 (89%) of samples. While the most commonly identified species were normal skin bacteria, Staphylococcus aureus found in 18 (11%) of samples was the most frequent potential pathogen. We did not detect any Enterococci or Enterobacteriaceae. The disinfection intervention was associated with substantially less overall bacterial colonization (odds ratio 0.16; 95%-CI 0.04–0.56), while specific colonization with Staphylococcus aureus was only slightly decreased (odds ratio 0.46; 95%-CI 0.16–1.29). Conclusion Our results indicate that a twice daily disinfection can still substantially reduce bacterial colonization of in-hospital tablet computers used in a high-resource and high hygiene setting.

Background According to 2016 World Health Organization and United Nations Children’s Fund country estimates, Eritrea has overall high vaccination coverage with immunization rates for three doses of diphtheria/tetanus/pertussis and polio vaccine of 95%, for two doses measles vaccine of 85% and for three doses hepatitis B vaccine of 85%. If confirmed, this could imply that routine basic vaccination of newly arrived Eritreans could be safely omitted. Methods We used stored serum samples from two cross-sectional studies that screened newly arrived Eritrean refugees for infectious diseases. Consenting refugees aged 16 years and older who registered in one of three neighbouring cantons in northwestern Switzerland were enrolled between January 2016 and December 2017. Antibody titers against the following vaccine-preventable diseases were measured (applied thresholds for seroprotection in brackets): diphtheria (>0.1 IU/ml), tetanus (>0.1 IU/ml), measles (>150 mIU/ml), rubella (only for women, >11 IU/ml), varicella (>50 mIU/ml), hepatitis B [hepatitis B surface antigen (HBsAg) Index >0.9, Hepatitis B core antibody (anti-HBc) Index >0.9 and antibodies to HBsAg (anti-HBs) >10 IE/L]. Differences between sex and age groups (≤25 and >25 years) were measured by Fisher’s exact test. Results We analysed samples of 133 study participants (20 women, 15%) with a median age of 25 years (range 16–61). Rates of seropositivity were as follows for women/men, respectively: diphtheria 57.9%/74.8% (difference non-significant), tetanus 94.8%/41.1% (P Conclusion Seroprevalence for vaccine-preventable infections did not meet levels required to confer herd immunity in any of the human-to-human transmissible diseases that were studied. In general, the strategy proposed by the Federal Office of Public Health to offer basic immunization to all newly arrived refugees, including newly arriving Eritrean refugees, is justified.

Eine rasche und zuverlässige Bestimmung von Antibiotikaresistenzen ist für die effiziente und fokussierte Therapie von Infektionskrankheiten unabdingbar. Die Überwachung der Resistenzlage liefert wichtige Informationen zur Festlegung der empirischen Therapie und hilft, Massnahmen gegen die weitere Verbreitung von antibiotikaresistenten Bakterien zu steuern.