Search

Contains fulltext : 238214.pdf (Publisher’s version ) (Open Access) Rapid diagnostics for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are paramount for reducing the spread of the current pandemic. During additional seasonal epidemics with influenza A/B and respiratory syncytial virus (RSV), the clinical signs and symptoms cannot be distinguished easily from SARS-CoV-2. Therefore, a new assay combining four targets in the form of the new Xpert Xpress SARS-CoV-2/Flu/RSV assay was evaluated. The assay was compared to the Xpert Xpress SARS-CoV-2, Xpert Xpress Flu/RSV, Seegene Flu/RSV, influenza A/B r-gene® and RSV/hMPV r-gene®. A total of 295 nasopharyngeal and throat swabs were tested at four institutes throughout Europe including 72 samples positive for SARS-CoV-2, 65 for influenza A, 47 for influenza B, and 77 for RSV. The sensitivity of the new assay was above 95% for all targets, with the highest for SARS-CoV-2 (97.2%). The overall correlation of SARS-CoV-2 Ct values between Xpert Xpress SARS-CoV-2 assay and Xpert Xpress SARS-CoV-2/Flu/RSV assay was high. The agreement between Ct values above 30 showed the multiplex giving higher Ct values for SARS-CoV-2 on average than the singleplex assay. In conclusion, the new assay is a rapid and reliable alternative with less hands-on time for the detection of not one, but four upper respiratory tract pathogens that may circulate at the same time.

During the course of the SARS-CoV-2 pandemic reports of mutations with effects on spreading and vaccine effectiveness emerged. Large scale mutation analysis using rapid SARS-CoV-2 Whole Genome Sequencing (WGS) is often unavailable but could support public health organizations and hospitals in monitoring transmission and rising levels of mutant strains. Here we report a novel WGS technique for SARS-CoV-2, the EasySeq™ RC-PCR SARS-CoV-2 WGS kit. By applying a reverse complement polymerase chain reaction (RC-PCR), an Illumina library preparation is obtained in a single PCR, thereby saving time, resources and facilitating high-throughput screening. Using this WGS technique, we evaluated SARS-CoV-2 diversity and possible transmission within a group of 173 patients and healthcare workers (HCW) of the Radboud university medical center during 2020. Due to the emergence of variants of concern, we screened SARS-CoV-2 positive samples in 2021 for identification of mutations and lineages. With use of EasySeq™ RC-PCR SARS-CoV-2 WGS kit we were able to obtain reliable results to confirm outbreak clusters and additionally identify new previously unassociated links in a considerably easier workaround compared to current methods. Furthermore, various SARS-CoV-2 variants of interest were detected among samples and validated against an Oxford Nanopore sequencing amplicon strategy which illustrates this technique is suitable for surveillance and monitoring current circulating variants.

Background SARS-CoV-2 is taking a huge toll on society while influenza and RSV detection are also becoming more important. These viruses pose a high burden on health care. Rapid and accurate diagnostics for these pathogens are important for swift triage in the hospital. Fast molecular point of care test (mPOCT) assays for these pathogens can prove an alternative. Here a multi-center evaluation of the Xpert® Xpress SARS-CoV-2/Flu/RSV assay is reported. Study design The Xpert® Xpress SARS-CoV-2/Flu/RSV assay was compared to three reference assays at three Dutch medical microbiology laboratories. An external quality assessment panel consisting of 16 specimens containing SARS-CoV-2, influenza viruses, RSV or human seasonal coronaviruses, or a combination thereof were used. Clinical specimens containing SARS-CoV-2 (n = 57), influenza viruses (n = 21) or RSV (n = 12), at a wide range of relevant concentrations were used. One laboratory also tested zoonotic avian and swine influenza viruses, and eight relevant SARS-CoV-2 variants. Results The Xpert® Xpress SARS-CoV-2/Flu/RSV assay showed equal performance compared to the reference assays. All SARS-CoV-2 variants of interest and variants of concern were accurately detected. Human seasonal coronaviruses were not detected. All four circulating seasonal influenza virus subtypes/lineages and both RSV types were accurately detected as well as a set of recent zoonotic avian and swine influenza viruses. The clinical specimens showed 98.2% concordance using this assay. Conclusion The Xpert® Xpress SARS-CoV-2/Flu/RSV assay is a good alternative for accurate detection for SARS-CoV-2, influenza type A virus, influenza type B virus and RSV types A and B detection in a short timeframe.

BackgroundCurrent transmission rates of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are still increasing and many countries are facing second waves of infections. Rapid SARS-CoV-2 whole-genome sequencing (WGS) is often unavailable but could support public health organizations and hospitals in monitoring and determining transmission links. Here we report the use of reverse complement polymerase chain reaction (RC-PCR), a novel technology for WGS of SARS-CoV-2 enabling library preparation in a single PCR saving time, resources and enables high throughput screening. Additionally, we show SARS-CoV-2 diversity and possible transmission within the Radboud university medical center (Radboudumc) during September 2020 using RC-PCR WGS.MethodsA total of 173 samples tested positive for SARS-CoV-2 between March and September 2020 were selected for whole-genome sequencing. Ct values of the samples ranged from 16 to 42. They were collected from 83 healthcare workers and three patients at the Radboudumc, in addition to 64 people living in the area around the hospital and tested by the local health services. For validation purposes, nineteen of the included samples were previously sequenced using Oxford Nanopore Technologies and compared to RC-PCR WGS results. The applicability of RC-PCR WGS in outbreak analysis for public health service and hospitals was tested on six suspected clusters containing samples of healthcare workers and patients with an epidemiological link.FindingsRC-PCR resulted in sequencing data for 146 samples. It showed a genome coverage of up to 98,2% for samples with a maximum Ct value of 32. Comparison to Oxford Nanopore technologies gives a near-perfect agreement on 95% of the samples (18 out of 19). Three out of six clusters with a suspected epidemiological link were fully confirmed, in the others, four healthcare workers were not associated. In the public health service samples, a previously unknown chain of transmission was confirmed.Significance statementSAR-CoV-2 whole-genome sequencing using RC-PCR is a reliable technique and applicable for use in outbreak analysis and surveillance. Its ease of use, high-trough screening capacity and wide applicability makes it a valuable addition or replacement during this ongoing SARS-CoV-2 pandemic.FundingNoneResearch in contextEvidence before this studyAt present whole genome sequencing techniques for SARS-CoV-2 have a large turnover time and are not widely available. Only a few laboratories are currently able to perform large scale SARS-CoV-2 sequencing. This restricts the use of sequencing to aid hospital and community infection prevention.Added value of this studyHere we present clinical and technical data on a novel Whole Genome Sequencing technology, implementing reverse-complement PCR. It is able to obtain high genome coverage of SARS-CoV-2 and confirm and exclude epidemiological links in 173 healthcare workers and patients. The RC-PCR technology simplifies the workflow thereby reducing hands on time. It combines targeted PCR and sequence library construction in a single PCR, which normally takes several steps. Additionally, this technology can be used in concordance with the widely available range of Illumina sequencers.Implications of all the available evidenceRC-PCR whole genome sequencing technology enables rapid and targeted surveillance and response to an ongoing outbreak that has great impact on public health and society. Increased use of sequencing technologies in local laboratories can help prevent increase of SARS-CoV-2 spreading by better understanding modes of transmission.

Contains fulltext : 220114.pdf (Publisher’s version ) (Closed access) BACKGROUND: With the outbreak of SARS-CoV-2, rapid diagnostics are paramount to contain the current pandemic. The routinely used realtime RT-PCR is sensitive, specific and able to process large batches of samples. However, turnaround time is long and in cases where fast obtained results are critical, molecular point of care tests (POCT) can be an alternative. Here we report on a multicenter evaluation of the Cepheid Xpert Xpress SARS-CoV-2 point-of-care test. STUDY DESIGN: The Xpert Xpress SARS-CoV-2 assay was evaluated against the routine in-house real-time RT-PCR assays in three medical microbiology laboratories in The Netherlands. A sensitivity and specificity panel was tested consisting of a dilution series of SARS-CoV-2 and ten samples containing SARS-CoV-2 and a range of other seasonal respiratory viruses. Additionally, 58 samples of patients positive for SARS-CoV-2 with different viral loads and 30 tested negative samples in all three Dutch laboratories using an in-house RT-PCR, were evaluated using Cepheids Xpert Xpress SARS-CoV-2 cartridges. RESULTS: Xpert Xpress SARS-CoV-2 point of care test showed equal performance compared to routine in-house testing with a limit of detection (LOD) of 8.26 copies/mL. Other seasonal respiratory viruses were not detected. In clinical samples Xpert Xpress SARS-CoV-2 reaches an agreement of 100 % compared to all in-house RT-PCRs CONCLUSION: Cepheids GeneXpert Xpert Xpress SARS-CoV-2 is a valuable addition for laboratories in situations where rapid and accurate diagnostics are of the essence.

OBJECTIVE: Plasmacytoid dendritic cells (PDCs) are a critical source of type I interferons (IFNs) that can contribute to the onset and maintenance of autoimmunity. Molecular mechanisms leading to PDC dysregulation and a persistent type I IFN signature are largely unexplored, especially in patients with systemic sclerosis (SSc), a disease in which PDCs infiltrate fibrotic skin lesions and produce higher levels of IFNalpha than those in healthy controls. This study was undertaken to investigate potential microRNA (miRNA)-mediated epigenetic mechanisms underlying PDC dysregulation and type I IFN production in SSc. METHODS: We performed miRNA expression profiling and validation in highly purified PDCs obtained from the peripheral blood of 3 independent cohorts of healthy controls and SSc patients. Possible functions of miRNA-618 (miR-618) on PDC biology were identified by overexpression in healthy PDCs. RESULTS: Expression of miR-618 was up-regulated in PDCs from SSc patients, including those with early disease who did not present with skin fibrosis. IFN regulatory factor 8, a crucial transcription factor for PDC development and activation, was identified as a target of miR-618. Overexpression of miR-618 reduced the development of PDCs from CD34+ cells in vitro and enhanced their ability to secrete IFNalpha, mimicking the PDC phenotype observed in SSc patients. CONCLUSION: Up-regulation of miR-618 suppresses the development of PDCs and increases their ability to secrete IFNalpha, potentially contributing to the type I IFN signature observed in SSc patients. Considering the importance of PDCs in the pathogenesis of SSc and other diseases characterized by a type I IFN signature, miR-618 potentially represents an important epigenetic target to regulate immune system homeostasis in these conditions.

Objective. Plasmacytoid dendritic cells (PDCs) are a critical source of type I interferons (IFNs) that can contribute to the onset and maintenance of autoimmunity. Molecular mechanisms leading to PDC dysregulation and a persistent type I IFN signature are largely unexplored, especially in patients with systemic sclerosis (SSc), a disease in which PDCs infiltrate fibrotic skin lesions and produce higher levels of IFN alpha than those in healthy controls. This study was undertaken to investigate potential microRNA (miRNA)-mediated epigenetic mechanisms underlying PDC dysregulation and type I IFN production in SSc. Methods. We performed miRNA expression profiling and validation in highly purified PDCs obtained from the peripheral blood of 3 independent cohorts of healthy controls and SSc patients. Possible functions of miRNA-618 (miR-618) on PDC biology were identified by overexpression in healthy PDCs. Results. Expression of miR-618 was up-regulated in PDCs from SSc patients, including those with early disease who did not present with skin fibrosis. IFN regulatory factor 8, a crucial transcription factor for PDC development and activation, was identified as a target of miR-618. Overexpression of miR-618 reduced the development of PDCs from CD34+ cells in vitro and enhanced their ability to secrete IFN alpha, mimicking the PDC phenotype observed in SSc patients. Conclusion. Up-regulation of miR-618 suppresses the development of PDCs and increases their ability to secrete IFN alpha, potentially contributing to the type I IFN signature observed in SSc patients. Considering the importance of PDCs in the pathogenesis of SSc and other diseases characterized by a type I IFN signature, miR-618 potentially represents an important epigenetic target to regulate immune system homeostasis in these conditions., Supported by Reumafonds (project 13-2-304). Dr. Rossato's work was supported by the European Commission (IEF Marie Curie Actions fellowship 622811; project MicroSCAP). Dr. Affandi's work was supported by Reumafonds (grant NR-10-1-301) and the Netherlands Organization for Scientific Research (NWO; Mosaic grant 017.008.014). Dr. Broen's work was supported by a personal Veni grant from the NWO (project 91614041). Dr. Radstake's work was supported the European Research Council (starting grant ERC-2011-StG; project Circumvent).

HIV presents one of the highest evolutionary rates ever detected and combination antiretroviral therapy is needed to overcome the plasticity of the virus population and control viral replication. Conventional treatments lack the ability to clear the latent reservoir, which remains the major obstacle towards a cure. Novel strategies, such as CRISPR/Cas9 gRNA-based genome-editing, can permanently disrupt the HIV genome. However, HIV genome-editing may accelerate viral escape, questioning the feasibility of the approach. Here, we demonstrate that CRISPR/Cas9 targeting of single HIV loci, only partially inhibits HIV replication and facilitates rapid viral escape at the target site. A combinatorial approach of two strong gRNAs targeting different regions of the HIV genome can completely abrogate viral replication and prevent viral escape. Our data shows that the accelerating effect of gene-editing on viral escape can be overcome and as such gene-editing may provide a future alternative for control of HIV-infection.

The primary purpose of this study was as follows: (1) to compare bony morphology in subjects with and without ACL injury. The secondary purposes were the following: (2) to compare bony morphology between men and women and (3) to determine whether MRI measurements of the notch correlate with intra-operative measurements. MRI measurements of NW, bicondylar width, medial condyle size and lateral condyle size, and medial-to-lateral condyle ratio (M:L) were taken from 45 subjects with ACL injury and 44 subjects without ACL injury, by two independent observers. In addition, notch width measurements were taken arthroscopically in the 45 injured subjects. Knee morphometrics were compared between men and women. Additionally, values of ACL-injured and non-injured subjects were compared for male and female subjects separately. Correlations between notch measurements taken from MRI and measured arthroscopically were determined. Reliability of the MRI measurements was calculated. Significant differences were found for bicondylar width (P = 0.001), medial condyle width (P = 0.002), and lateral condyle width (P = 0.002) between male and female subjects. When bony morphology was compared between ACL-injured and non-injured subjects, the male subjects showed significant differences for bicondylar width (P = 0.002) and medial condyle width (P = 0.008). For the female subjects, significant differences were found for bicondylar width (P = 0.009) and lateral condyle width (P = 0.002). There was no correlation between notch width measured on the MRI’s and NW measured intra-operatively. The intra- and inter-observer reliability of the MRI measurements was satisfactory. There were differences in bony morphology between men and women and between subjects with and without ACL injury. The bony morphology that was different between ACL-injured and non-injured subjects varied between male and female subjects. Case–control study, Level III.

The primary purpose of the current study is to identify a possible correlation between the femoral intercondylar notch size and the ACL insertion site size. The secondary purpose is to determine if there is a difference between male and female notch widths and insertion site sizes. For this study, 82 patients (41 men and 41 women) with an average age of 24.1 ± 10.0 years (range 13–58 years) undergoing anterior cruciate ligament (ACL) reconstruction were included. Arthroscopic measurements were taken at the base, middle, and top of the notch. Additionally, the notch height was measured at the highest point. The insertion sites of the ACL were identified, marked using electrocautery, and measured. The correlation between notch width and ACL insertion site size was calculated. In addition, differences between men and women with regard to the notch width and ACL insertion site size were determined. Significant positive correlations were found between the notch widths and ACL insertion site measurements and ranged from 0.222 to 0.379 (P