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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.

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.

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.