Automating the Illumina DNA library preparation kit for whole genome sequencing applications on the flowbot ONE liquid handler robot.

Whole-genome sequencing (WGS) is currently making its transition from research tool into routine (clinical) diagnostic practice. The workflow for WGS includes the highly labor-intensive library preparations (LP), one of the most critical steps in the WGS procedure. Here, we describe the automation of the LP on the flowbot ONE robot to minimize the risk of human error and reduce hands-on time (HOT). For this, the robot was equipped, programmed, and optimized to perform the Illumina DNA Prep automatically. Results obtained from 16 LP that were performed both manually and automatically showed comparable library DNA yields (median of 1.5-fold difference), similar assembly quality values, and 100% concordance on the final core genome multilocus sequence typing results. In addition, reproducibility of results was confirmed by re-processing eight of the 16 LPs using the automated workflow. With the automated workflow, the HOT was reduced to 25 min compared to the 125 min needed when performing eight LPs using the manual workflow. The turn-around time was 170 and 200 min for the automated and manual workflow, respectively. In summary, the automated workflow on the flowbot ONE generates consistent results in terms of reliability and reproducibility, while significantly reducing HOT as compared to manual LP.

Prospective and systematic screening for invasive aspergillosis in the ICU during the COVID-19 pandemic, a proof of principle for future pandemics.

The diagnostic performance of a prospective, systematic screening strategy for COVID-19 associated pulmonary aspergillosis (CAPA) during the COVID-19 pandemic was investigated. Patients with COVID-19 admitted to the ICU were screened for CAPA twice weekly by collection of tracheal aspirate (TA) for Aspergillus culture and PCR. Subsequently, bronchoalveolar lavage (BAL) sampling was performed in patients with positive screening results and clinical suspicion of infection. Patient data were collected from April 2020-February 2022. Patients were classified according to 2020 ECMM/ISHAM consensus criteria. In total, 126/370 (34%) patients were positive in screening and CAPA frequency was 52/370 (14%) (including 13 patients negative in screening). CAPA was confirmed in 32/43 (74%) screening positive patients who underwent BAL sampling. ICU mortality was 62% in patients with positive screening and confirmed CAPA, and 31% in CAPA cases who were screening negative. The sensitivity, specificity, positive and negative predictive value (PPV & NPV) of screening for CAPA were 0.71, 0.73, 0.27 and 0.95 respectively. The PPV was higher if screening was culture positive compared to PCR positive only, 0.42 and 0.12 respectively. CAPA was confirmed in 74% of screening positive patients, and culture of TA had a better diagnostic performance than PCR. Positive screening along with clinical manifestations appeared to be a good indication for BAL sampling since diagnosis of CAPA was confirmed in most of these patients. Prospective, systematic screening allowed to quickly gain insight into the epidemiology of fungal superinfections during the pandemic and could be applicable for future pandemics.

During the COVID-19 pandemic we screened for COVID-19 associated pulmonary aspergillosis (CAPA) in patients with COVID-19 admitted to the intensive care unit. Screening enabled us to quickly gain insight into the epidemiology of patients with a superinfection with Aspergillus.

A multicenter evaluation of the QIAstat-Dx meningitis-encephalitis syndromic test kit as compared to the conventional diagnostic microbiology workflow.

PURPOSE: Rapid diagnosis and treatment of infectious meningitis and encephalitis (ME) is critical to minimize morbidity and mortality. Recently, Qiagen introduced the CE-IVD QIAstat-Dx ME panel (QS-ME) for syndromic diagnostic testing of meningitis and encephalitis. Some data on the performance of the QS-ME in comparison to the BioFire FilmArray ME panel are available. In this study, the performance of the QS-ME is compared to the current diagnostic workflow in two academic medical centers in the Netherlands. METHODS: A total of 110 cerebrospinal fluid samples were retrospectively tested with the QS-ME. The results obtained were compared to the results of laboratory-developed real-time PCR assays (LDTs), IS-pro, bacterial culture, and cryptococcal antigen (CrAg) testing. In addition, the accuracy of the QS-ME was also investigated using an external quality assessment (EQA) panel consisting of ten samples. RESULTS: Four of the 110 samples tested failed to produce a valid QS-ME result. In the remaining 106 samples, the QS-ME detected 53/53 viral targets, 38/40 bacterial targets, and 7/13 Cryptococcus neoformans targets. The discrepant bacterial results consisted of two samples that were previously tested positive for Listeria monocytogenes (CT 35.8) and Streptococcus pneumoniae (CT 40), respectively. The QS-ME detected one additional result, consisting of a varicella-zoster virus signal (CT 35.9), in a sample in which both techniques detected Streptococcus pyogenes. Finally, 100% concordance was achieved in testing a blinded bacterial ME EQA panel. CONCLUSION: The QS-ME is a relevant addition to the syndromic testing landscape to assist in diagnosing infectious ME.

Voclosporin and the Antiviral Effect Against SARS-CoV-2 in Immunocompromised Kidney Patients.

Introduction: Immunocompromised kidney patients are at increased risk of prolonged SARS-CoV-2 infection and related complications. Preclinical evidence demonstrates a more potent inhibitory effect of voclosporin on SARS-CoV-2 replication than tacrolimus in vitro. We investigated the potential antiviral effects of voclosporin on SARS-CoV-2 in immunocompromised patients. Methods: First, we conducted a prospective, randomized, open-label, proof-of-concept study in 20 kidney transplant recipients (KTRs) on tacrolimus-based immunosuppression who contracted mild to moderate SARS-CoV-2 infection. Patients were randomized to continue tacrolimus or switch to voclosporin. Second, we performed a post hoc analysis on SARS-CoV-2 infections in 216 patients with lupus nephritis (LN) on standard immunosuppression who were randomly exposed to voclosporin or placebo as part of a clinical trial that was conducted during the worldwide COVID-19 pandemic. Results: The primary end point was clearance of SARS-CoV-2 viral load and that did not differ between voclosporin-treated KTRs (median 12 days, interquartile range [IQR] 8-28) and tacrolimus-treated KTRs (median 12 days, IQR 4-16) nor was there a difference in clinical recovery. Pharmacokinetic analyses demonstrated that, when voclosporin trough levels were on-target, SARS-CoV-2 viral load dropped significantly more (ΔCt 7.7 [3.4-10.7]) compared to tacrolimus-treated KTRs (ΔCt 2.7 [2.0-4.3]; P = 0.035). In voclosporin-exposed patients with LN, SARS-CoV-2 infection was detected in 6% (7/116) compared to 12% (12/100) in placebo-exposed patients (relative risk [RR] 1.4 [0.97-2.06]). Notably, no voclosporin-exposed patients with LN died from severe SARS-CoV-2 infection compared to 3% (3/100) in placebo-exposed patients (RR 2.2 [1.90-2.54]). Conclusion: This proof-of-concept study shows a potential positive risk-benefit profile for voclosporin in immunocompromised patients with SARS-CoV-2 infection. These results warrant further investigations on voclosporin to establish an equipoise between infection and maintenance immunosuppression.

A core-genome multilocus sequence typing scheme for the detection of genetically related Streptococcus pyogenes clusters.

The recently observed increase in invasive Streptococcus pyogenes infections causes concern in Europe. However, conventional molecular typing methods lack discriminatory power to aid investigations of outbreaks caused by S. pyogenes. Therefore, there is an urgent need for high-resolution molecular typing methods to assess genetic relatedness between S. pyogenes isolates. In the current study, we aimed to develop a novel high-resolution core-genome multilocus sequence typing (cgMLST) scheme for S. pyogenes and compared its discriminatory power to conventional molecular typing methods. The cgMLST scheme was designed with the commercial Ridom SeqSphere+ software package. To define a cluster threshold, the scheme was evaluated using publicly available data from nine defined S. pyogenes outbreaks in the United Kingdom. The cgMLST scheme was then applied to 23 isolates from a suspected S. pyogenes outbreak and 117 S. pyogenes surveillance isolates both from the Netherlands. MLST and emm-typing results were used for comparison to cgMLST results. The allelic differences between isolates from defined outbreaks ranged between 6 and 31 for isolates with the same emm-type, resulting in a proposed cluster threshold of <5 allelic differences out of 1,095 target loci. Seven out of twenty-three (30%) isolates from the suspected outbreak had an allelic difference of <2, thereby identifying a potential cluster that could not be linked to other isolates. The proposed cgMLST scheme shows a higher discriminatory ability when compared to conventional typing methods. The rapid and simple analysis workflow allows for extended detection of clusters of potential outbreak isolates and surveillance and may facilitate the sharing of sequencing results between (inter)national laboratories.

Multicenter performance evaluation of the Alinity m CMV assay for quantifying cytomegalovirus DNA in plasma samples.

Monitoring of cytomegalovirus (CMV) viral load is critical for informing treatment decisions in order to prevent the severe health consequences of CMV infection or reactivation of latent CMV in immunocompromised individuals. This first field evaluation examined the analytical and clinical performance of the Alinity m CMV assay. Analytical performance was assessed with a commercially available six-member panel, while the clinical performance evaluation compared the Alinity m CMV assay to the RealTime CMV assay and a laboratory-developed test (LDT) as the test of record at three large hospital-based clinical laboratories. Precision of the Alinity m CMV assay was demonstrated with total standard deviation (SD) between 0.08 and 0.28 Log IU/mL. A total of 457 plasma specimens were tested on the Alinity m CMV assay and compared to the test of record at each site (n = 304 with RealTime CMV and n = 153 with LDT CMV). The Alinity m CMV assay had excellent correlation (correlation coefficient r ≥0.942) in comparison to the RealTime CMV or LDT CMV assays. The mean observed bias ranged from -0.03 to 0.34 Log IU/mL. Median onboard turnaround time of Alinity m CMV was less than 3 h. When the CMV assay is run on the Alinity m system, it has the capacity to shorten time to result and, therefore, to therapy.

Multi-site performance evaluation of the Alinity m Molecular assay for quantifying Epstein-Barr virus DNA in plasma samples.

Detection and monitoring of acute infection or reactivation of Epstein-Barr virus (EBV) are critical for treatment decision-making and to reduce the risk of EBV-related malignancies and other associated diseases in immunocompromised individuals. The analytical and clinical performance of the Alinity m EBV assay was evaluated at two independent study sites; analytical performance was assessed by evaluating precision with a commercially available 5-member EBV verification panel, while the clinical performance of the Alinity m EBV assay was compared to the RealTime EBV assay and a laboratory-developed test (LDT) as the routine test of record (TOR). Analytical analysis demonstrated standard deviation (SD) between 0.08 and 0.13 Log IU/mL. A total of 300 remnant plasma specimens were retested with the Alinity m EBV assay, and results were compared to those of the TOR at the respective study sites (n = 148 with the RealTime EBV assay and n = 152 with the LDT EBV assay). Agreement between Alinity m EBV and RealTime EBV or LDT EBV assays had kappa values of 0.88 and 0.84, respectively, with correlation coefficients r of 0.956 and 0.912, while the corresponding observed mean bias was -0.02 and -0.19 Log IU/mL. The Alinity m EBV assay had a short median onboard turnaround time of 2:40 h. Thus, the Alinity m system can shorten the time to results and, therefore, to therapy.

Panton-Valentine Leukocidin-Positive CC398 MRSA in Urban Clinical Settings, the Netherlands.

We report detection of Panton-Valentine leukocidin-positive clonal complex 398 human-origin methicillin-resistant Staphylococcus aureus L2 in the Netherlands. This hypervirulent lineage originated in the Asia-Pacific Region and could become community-acquired in Europe after recurrent travel-related introductions. Genomic surveillance enables early detection to guide control measures and help limit spread of pathogens in urban settings.

Performance of Five Metagenomic Classifiers for Virus Pathogen Detection Using Respiratory Samples from a Clinical Cohort.

Viral metagenomics is increasingly applied in clinical diagnostic settings for detection of pathogenic viruses. While several benchmarking studies have been published on the use of metagenomic classifiers for abundance and diversity profiling of bacterial populations, studies on the comparative performance of the classifiers for virus pathogen detection are scarce. In this study, metagenomic data sets (n = 88) from a clinical cohort of patients with respiratory complaints were used for comparison of the performance of five taxonomic classifiers: Centrifuge, Clark, Kaiju, Kraken2, and Genome Detective. A total of 1144 positive and negative PCR results for a total of 13 respiratory viruses were used as gold standard. Sensitivity and specificity of these classifiers ranged from 83 to 100% and 90 to 99%, respectively, and was dependent on the classification level and data pre-processing. Exclusion of human reads generally resulted in increased specificity. Normalization of read counts for genome length resulted in a minor effect on overall performance, however it negatively affected the detection of targets with read counts around detection level. Correlation of sequence read counts with PCR Ct-values varied per classifier, data pre-processing (R2 range 15.1-63.4%), and per virus, with outliers up to 3 log10 reads magnitude beyond the predicted read count for viruses with high sequence diversity. In this benchmarking study, sensitivity and specificity were within the ranges of use for diagnostic practice when the cut-off for defining a positive result was considered per classifier.

Longitudinal Monitoring of DNA Viral Loads in Transplant Patients Using Quantitative Metagenomic Next-Generation Sequencing.

INTRODUCTION: Immunocompromised patients are prone to reactivations and (re-)infections of multiple DNA viruses. Viral load monitoring by single-target quantitative PCRs (qPCR) is the current cornerstone for virus quantification. In this study, a metagenomic next-generation sequencing (mNGS) approach was used for the identification and load monitoring of transplantation-related DNA viruses. METHODS: Longitudinal plasma samples from six patients that were qPCR-positive for cytomegalovirus (CMV), Epstein-Barr virus (EBV), BK polyomavirus (BKV), adenovirus (ADV), parvovirus B19 (B19V), and torque teno-virus (TTV) were sequenced using the quantitative metagenomic Galileo Viral Panel Solution (Arc Bio, LLC, Cambridge, MA, USA) reagents and bioinformatics pipeline combination. Qualitative and quantitative performance was analysed with a focus on viral load ranges relevant for clinical decision making. RESULTS: All pathogens identified by qPCR were also identified by mNGS. BKV, CMV, and HHV6B were additionally detected by mNGS, and could be confirmed by qPCR or auxiliary bioinformatic analysis. Viral loads determined by mNGS correlated with the qPCR results, with inter-method differences in viral load per virus ranging from 0.19 log10 IU/mL for EBV to 0.90 log10 copies/mL for ADV. TTV, analysed by mNGS in a semi-quantitative way, demonstrated a mean difference of 3.0 log10 copies/mL. Trends over time in viral load determined by mNGS and qPCR were comparable, and clinical thresholds for initiation of treatment were equally identified by mNGS. CONCLUSIONS: The Galileo Viral Panel for quantitative mNGS performed comparably to qPCR concerning detection and viral load determination, within clinically relevant ranges of patient management algorithms.

Livestock-associated methicillin-resistant Staphylococcus aureus epidemiology, genetic diversity, and clinical characteristics in an urban region.

Objectives: While Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA), defined as CC398, is a well-known pathogen among those working with livestock, there are indications that LA-MRSA prevalence among the general population is increasing. However, the clinical impact in urban areas remains unknown. The aim of this study was to assess the genetic epidemiology and clinical characteristics of LA-MRSA in an urban area with a limited livestock population. Methods: In this retrospective study, we evaluated LA-MRSA strains that were collected between 2014 and 2018 from patients who received clinical care in a single urban area in Netherlands. Patient files were assessed for livestock exposure data, clinical findings, and contact tracing information. Next-generation sequencing (NGS) analysis in combination with wgMLST was conducted to assess genetic diversity and relatedness and to detect virulence and resistance genes. Results: LA-MRSA strains were cultured from 81 patients, comprising 12% of all the MRSA strains found in seven study laboratories between 2014 and 2018. No livestock link was found in 76% of patients (n = 61), and 28% of patients (n = 23) had an infection, mostly of the skin or soft tissue. Contact tracing had been initiated in 14 cases, leading to the identification of two hospital transmissions: a cluster of 9 cases and one of 2 cases. NGS data were available for 91% (n = 75) of the patients. wgMLST confirmed the clusters detected via contact tracing (n = 2) and identified 5 additional clusters without a known epidemiological link. Relevant resistance and virulence findings included the PVL virulence gene (3 isolates) and tetracycline resistance (79 isolates). Conclusion: LA-MRSA may cause a relevant burden of disease in urban areas. Surprisingly, most infections in the present study occurred in the absence of a livestock link, suggesting inter-human transmission. These findings and the presence of PVL and other immune evasive complex virulence genes warrant future surveillance and preventative measures.

External Quality Assessment of SARS-CoV-2 Sequencing: an ESGMD-SSM Pilot Trial across 15 European Laboratories.

This first pilot trial on external quality assessment (EQA) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) whole-genome sequencing, initiated by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Study Group for Genomic and Molecular Diagnostics (ESGMD) and the Swiss Society for Microbiology (SSM), aims to build a framework between laboratories in order to improve pathogen surveillance sequencing. Ten samples with various viral loads were sent out to 15 clinical laboratories that had free choice of sequencing methods and bioinformatic analyses. The key aspects on which the individual centers were compared were the identification of (i) single nucleotide polymorphisms (SNPs) and indels, (ii) Pango lineages, and (iii) clusters between samples. The participating laboratories used a wide array of methods and analysis pipelines. Most were able to generate whole genomes for all samples. Genomes were sequenced to various depths (up to a 100-fold difference across centers). There was a very good consensus regarding the majority of reporting criteria, but there were a few discrepancies in lineage and cluster assignments. Additionally, there were inconsistencies in variant calling. The main reasons for discrepancies were missing data, bioinformatic choices, and interpretation of data. The pilot EQA was overall a success. It was able to show the high quality of participating laboratories and provide valuable feedback in cases where problems occurred, thereby improving the sequencing setup of laboratories. A larger follow-up EQA should, however, improve on defining the variables and format of the report. Additionally, contamination and/or minority variants should be a further aspect of assessment.

Viral metagenomic sequencing in the diagnosis of meningoencephalitis: a review of technical advances and diagnostic yield.

INTRODUCTION: Meningoencephalitis patients are often severely impaired and benefit from early etiological diagnosis, though many cases remain without identified cause. Metagenomics as pathogen agnostic approach can result in additional etiological findings; however, the exact diagnostic yield when used as a secondary test remains unknown. AREAS COVERED: This review aims to highlight recent advances with regard to wet and dry lab methodologies of metagenomic testing and technical milestones that have been achieved. A selection of procedures currently applied in accredited diagnostic laboratories is described in more detail to illustrate best practices. Furthermore, a meta-analysis was performed to assess the additional diagnostic yield utilizing metagenomic sequencing in meningoencephalitis patients. Finally, the remaining challenges for successful widespread implementation of metagenomic sequencing for the diagnosis of meningoencephalitis are addressed in a future perspective. EXPERT OPINION: The last decade has shown major advances in technical possibilities for using mNGS in diagnostic settings including cloud-based analysis. An additional advance may be the current established infrastructure of platforms for bioinformatic analysis of SARS-CoV-2, which may assist to pave the way for global use of clinical metagenomics.

Increasing diagnostic possibilities using the geneLEAD VIII platform for detection of SARS-CoV-2.

At the time SARS-CoV-2 was identified as the cause of coronavirus disease 2019 (COVID-19) no in vitro diagnostic (IVD) tests were available since it was a new virus. Very shortly after the release of the genomic sequence of SARS-CoV-2, laboratory-developed tests (LDTs) were developed, made available and implemented in several laboratories in the Netherlands and globally. In this study, the performance of an E-gene Sarbeco specific real-time reverse-transcriptase PCR (RT-PCR) was verified on the open modus of the geneLEAD VIII sample-to-answer platform. The results obtained from 134 clinical samples, of which 63 had been tested positive, showed almost complete concordance compared to the same PCR on the routine diagnostic systems and that was validated according to the national reference standard. The only discordant sample tested positive using the routine diagnostic workflow with a cycle threshold (CT) value of 37.7, while the sample tested negative using the geneLEAD VIII workflow. In addition, good performance was achieved in analyzing a blinded SARS-CoV-2 external quality assurance (EQA) panel. Implementation of the geneLEAD VIII platform as routine diagnostic tool resulted in testing 871 clinical samples with 115 positive results. In conclusion, the geneLEAD VIII SARS-CoV-2 workflow presented in this study showed excellent diagnostic performance and with a rapid turnaround time of approximately two hours it proved a valuable option for STAT SARS-CoV-2 testing in the absence of (rapid, CE-IVD) point-of-care testing platforms.

Evaluation of the Genmark ePlex® and QIAstat-Dx® respiratory pathogen panels in detecting bacterial targets in lower respiratory tract specimens.

BACKGROUND: The ePlex® and QIAstat-Dx® respiratory pathogen panels detect multiple respiratory pathogens, mainly viruses but also Legionella pneumophila, Mycoplasma pneumoniae and Bordetella pertussis. The assays have been marketed for use in nasopharyngeal swab specimens. For diagnosing bacterial pneumonia, lower respiratory tract (LRT) specimens are indicated. Aim of this study was to evaluate the performance of these syndromic panels for these three bacterial targets in samples from the LRT. Fifty-six specimens were collected from our repositories, five negative samples and fifty-one samples which had been previously tested positive with the routine diagnostic real-time PCR assays for Legionella spp. (N = 20), Bordetella spp. (N = 16) or M. pneumoniae (N = 15). RESULTS: The QIAstat-Dx Respiratory Panel V2 (RP) assay detected all of the L. pneumophila and B. pertussis positive samples but only 11/15 (73.3 %) of the M. pneumoniae targets. The ePlex Respiratory Pathogen Panel (RPP) assay detected 10/14 (71.4 %) of the L. pneumophila targets, 8/12 (66.7 %) of the B. pertussis positive samples and 13/15 (86.7 %) of the M. pneumoniae targets. CONCLUSIONS: No false-positive results were reported for all three bacterial pathogens by both assays. The clinical performance of both assays depended highly on the bacterial load in the sample and the type of specimen under investigation.

Viral metagenomic sequencing in a cohort of international travellers returning with febrile illness.

BACKGROUND: Diagnosis of infections in returning international travellers can be challenging because of the broad spectrum of potential infectious etiologies potentially involved. Viral metagenomic next-generation sequencing (mNGS) has the potential to detect any virus present in a patient sample and is increasingly being used for difficult to diagnose cases. The aim of this study was to analyze the performance of mNGS for viral pathogen detection in the clinical setting of international travellers returning with febrile illness. METHODS: Thirty-eight serum samples from international travellers returning with febrile illness and presenting at the outpatient clinic of the Leiden University Medical Center in the Netherlands in the time period 2015-2016 were selected retrospectively. Samples were processed for viral metagenomic sequencing using a probe panel capturing all known vertebrate viruses. Bioinformatic analysis was performed using Genome Detective software for metagenomic virus detection. Metagenomic virus findings were compared with viral pathogen detection using conventional methods. RESULTS: In 8 out of the 38 patients (21%), a pathogenic virus was detected by mNGS. All viral pathogens detected by conventional assays were also detected by mNGS: dengue virus (n=4 patients), Epstein-Barr virus (n=2), hepatitis B virus (n=1). In addition, mNGS resulted in additional pathogenic findings in 2 patients (5%): dengue virus (n=1), and hepatitis C virus (n=1). Non-pathogenic viruses detected were: GB virus C (n=1) and torque teno viruses (n=3). High genome coverage and depth using capture probes enabled typing of the dengue viruses detected. CONCLUSIONS: Viral metagenomics has the potential to assist the detection of viral pathogens and co-infections in one step in international travellers with a febrile syndrome. Furthermore, viral enrichment by probes resulted in high genome coverage and depth which enabled dengue virus typing.

Benchmark of thirteen bioinformatic pipelines for metagenomic virus diagnostics using datasets from clinical samples.

INTRODUCTION: Metagenomic sequencing is increasingly being used in clinical settings for difficult to diagnose cases. The performance of viral metagenomic protocols relies to a large extent on the bioinformatic analysis. In this study, the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS) initiated a benchmark of metagenomic pipelines currently used in clinical virological laboratories. METHODS: Metagenomic datasets from 13 clinical samples from patients with encephalitis or viral respiratory infections characterized by PCR were selected. The datasets were analyzed with 13 different pipelines currently used in virological diagnostic laboratories of participating ENNGS members. The pipelines and classification tools were: Centrifuge, DAMIAN, DIAMOND, DNASTAR, FEVIR, Genome Detective, Jovian, MetaMIC, MetaMix, One Codex, RIEMS, VirMet, and Taxonomer. Performance, characteristics, clinical use, and user-friendliness of these pipelines were analyzed. RESULTS: Overall, viral pathogens with high loads were detected by all the evaluated metagenomic pipelines. In contrast, lower abundance pathogens and mixed infections were only detected by 3/13 pipelines, namely DNASTAR, FEVIR, and MetaMix. Overall sensitivity ranged from 80% (10/13) to 100% (13/13 datasets). Overall positive predictive value ranged from 71-100%. The majority of the pipelines classified sequences based on nucleotide similarity (8/13), only a minority used amino acid similarity, and 6 of the 13 pipelines assembled sequences de novo. No clear differences in performance were detected that correlated with these classification approaches. Read counts of target viruses varied between the pipelines over a range of 2-3 log, indicating differences in limit of detection. CONCLUSION: A wide variety of viral metagenomic pipelines is currently used in the participating clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implicating the need for standardization and validation of metagenomic analysis for clinical diagnostic use. Future studies should address the selective effects due to the choice of different reference viral databases.

Hospital transmission of borderline oxacillin-resistant Staphylococcus aureus evaluated by whole-genome sequencing.

Introduction. Staphylococcus aureus is a major cause of hospital infections worldwide. Awareness towards methicillin-resistant S. aureus (MRSA) infections is high but attention towards borderline oxacillin-resistant S. aureus (BORSA) is limited, possibly due to an underestimated clinical relevance, presumption of low incidence and diagnostic limitations.Gap statement. BORSA surveillance has not been routinely implemented, and thus consensus with regard to a definition and infection control measures is lacking.Aim. Our goals were to investigate the occurrence, molecular characteristics and clinical manifestations of BORSA infections in the hospital setting.Methodology. Following an increased incidence in 2016, BORSA cases in 2014/2016 (in our institution) were more specifically evaluated. Medical records were reviewed to investigate epidemiological links, clinical characteristics and outcomes. Resistance and virulence markers were assessed by whole genome sequencing (WGS). Conventional methods: amplified fragment length polymorphism (AFLP) ; multilocus sequence typing (MLST) and multiple locus variable-number tandem repeat analysis (MLVA) were compared with core genome MLST (cgMLST) and whole-genome single nucleotide polymorphism (wgSNP) analysis to confirm genetic clusters.Results. From 2009 to 2013, BORSA comprised 0.1 % of all clinical S. aureus strains. In 2016, the incidence was six-fold higher in comparison to the baseline. Whole-genome SNP and cgMLST confirmed two BORSA clusters among patients with dermatological conditions. Patients with BORSA presented with skin infections, and one case developed a severe invasive infection with a fatal outcome. Infection control measures successfully prevented further transmission in both clusters. WGS findings showed that BORSA strains carried multiple resistance and virulence genes with increased pathogenic potential.Conclusion. WGS and cgMLST effectively characterized and confirmed BORSA clusters among at-risk patients with clinical manifestations ranging from mild skin infections to life-threatening bacteraemia. Clinical awareness and active monitoring are therefore warranted for the timely implementation of infection control measures to prevent BORSA transmission in high-risk patients.

Molecular Epidemiology and Evolutionary Trajectory of Emerging Echovirus 30, Europe.

In 2018, an upsurge in echovirus 30 (E30) infections was reported in Europe. We conducted a large-scale epidemiologic and evolutionary study of 1,329 E30 strains collected in 22 countries in Europe during 2016-2018. Most E30 cases affected persons 0-4 years of age (29%) and 25-34 years of age (27%). Sequences were divided into 6 genetic clades (G1-G6). Most (53%) sequences belonged to G1, followed by G6 (23%), G2 (17%), G4 (4%), G3 (0.3%), and G5 (0.2%). Each clade encompassed unique individual recombinant forms; G1 and G4 displayed >2 unique recombinant forms. Rapid turnover of new clades and recombinant forms occurred over time. Clades G1 and G6 dominated in 2018, suggesting the E30 upsurge was caused by emergence of 2 distinct clades circulating in Europe. Investigation into the mechanisms behind the rapid turnover of E30 is crucial for clarifying the epidemiology and evolution of these enterovirus infections.