Multicenter Evaluation of the QIAstat-Dx and the BioFire Multiplex Panel Tests for the Detection of Respiratory Pathogens.

Syndromic multiplex panel testing enables simultaneous detection of multiple respiratory pathogens, but limited data is available on the comparative diagnostic performance of different testing systems. In this multicenter prospective study, we aimed to compare the QIAstat-Dx Respiratory Panel 2.0 (QIAstat-Dx-RP2.

Revealing the unseen: next generation sequencing for early detection of drug-resistant cytomegalovirus variants upon letermovir prophylaxis failure.

In allogeneic hematopoietic cell transplant (HCT)-recipients, prophylactic management strategies are essential for preventing CMV-reactivation and associated disease. We report on a 63-year-old male patient with a D-/R+ CMV-serostatus, who showed ongoing low-level CMV-replication post-HCT despite receiving letermovir prophylaxis. Sanger-sequencing failed to detect drug resistance mutations (DRM) until CMV-pneumonitis developed, revealing a UL56-C325R-DRM linked to high-level letermovir resistance.

Impact of nucleic acid extraction procedures on human papillomavirus (HPV) detection and genotyping.

Human papillomavirus (HPV) infections are often asymptomatic, but some of the >200 HPV genotypes confer a high risk for precancerous cervical lesions and cervical cancer. Current clinical management of HPV infections relies on reliable nucleic acid testing detection and genotyping. We prospectively compared nucleic acid extraction without and with prior centrifugation enrichment for detecting and genotyping HPV in cervical swabs with atypical squamous or glandular cells.

Usefulness of the GenMark ePlex RPP assay for the detection of respiratory viruses compared to the FTD21 multiplex RT-PCR.

Cartridge-based multiplex panels covering numerous pathogens offer an advantage of minimal hands-on-time and short time to result to commercial RT-PCR assays. In this study, we evaluated the performance of the ePlex respiratory pathogen panel (RPP) compared to the Fast Track Diagnostics Respiratory pathogens 21 multiplex RT-PCR assay (FTD21) using 400 clinical respiratory samples. Discrepant results were further analysed by a reference nucleic acid amplification testing (NAT) and a composite reference approach was used for final interpretation.

Epidemiology and precision of SARS-CoV-2 detection following lockdown and relaxation measures.

Objectives: Detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is key to the clinical and epidemiological assessment of CoVID-19. We cross-validated manual and automated high-throughput testing for SARS-CoV-2-RNA, evaluated SARS-CoV-2 loads in nasopharyngeal-oropharyngeal swabs (NOPS), lower respiratory fluids, and plasma, and analyzed detection rates after lockdown and relaxation measures.

Methods: Basel-S-gene, Roche-E-gene, and Roche-cobas®6800-Target1 and Target2 were prospectively validated in 1344 NOPS submitted during the first pandemic peak (Week 13).

Comparing cytomegalovirus diagnostics by cell culture and quantitative nucleic acid testing in broncho-alveolar lavage fluids.

Many clinical laboratories have replaced virus isolation in cell-culture (VIC) for cytomegalovirus (CMV) by quantitative-nucleic-acid testing (QNAT), rendering clinically relevant CMV-replication difficult to distinguish from CMV-shedding or latent infection. We compared direct VIC in 1109 consecutive bronchoalveolar lavage fluids (BALFs) and a well-validated CMV-QNAT (Basel-CMV-UL111a-77bp). In the retrospective Group 1 (N = 694) and Group 2 (N = 303), CMV-QNAT was performed within 48 h from 2-fold and 10-fold concentrated total nucleic acid (TNA) eluates, respectively.

Hunting coronavirus by transmission electron microscopy - a guide to SARS-CoV-2-associated ultrastructural pathology in COVID-19 tissues.

Transmission electron microscopy has become a valuable tool to investigate tissues of COVID-19 patients because it allows visualisation of SARS-CoV-2, but the 'virus-like particles' described in several organs have been highly contested. Because most electron microscopists in pathology are not accustomed to analysing viral particles and subcellular structures, our review aims to discuss the ultrastructural changes associated with SARS-CoV-2 infection and COVID-19 with respect to pathology, virology and electron microscopy. Using micrographs from infected cell cultures and autopsy tissues, we show how coronavirus replication affects ultrastructure and put the morphological findings in the context of viral replication, which induces extensive remodelling of the intracellular membrane systems.

Epidemiology of Severe Acute Respiratory Syndrome Coronavirus 2 Emergence Amidst Community-Acquired Respiratory Viruses.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in China as the cause of coronavirus disease 2019 in December 2019 and reached Europe by late January 2020, when community-acquired respiratory viruses (CARVs) are at their annual peak. We validated the World Health Organization (WHO)-recommended SARS-CoV-2 assay and analyzed the epidemiology of SARS-CoV-2 and CARVs.

Methods: Nasopharyngeal/oropharyngeal swabs (NOPS) from 7663 patients were prospectively tested by the Basel S-gene and WHO-based E-gene (Roche) assays in parallel using the Basel N-gene assay for confirmation.

Brief validation of the novel GeneXpert Xpress SARS-CoV-2 PCR assay.

The clinical and epidemiologic management of the SARS-CoV-2 pandemic is critically dependent on molecular assays with short turn-around time. We validated the novel Xpert Xpress SARS-CoV-2 assay using a commercial nucleic acid testing (Roche Cobas 6800). We found an excellent concordance over a range of SARS-CoV-2 loads and across established human coronaviruses.

Comparing the Cobas Liat Influenza A/B and respiratory syncytial virus assay with multiplex nucleic acid testing.

Influenza virus and respiratory syncytial virus (RSV) detection with short turn-around-time (TAT) is pivotal for rapid decisions regarding treatment and infection control. However, negative rapid testing results may come from poor assay sensitivity or from influenza-like illnesses caused by other community-acquired respiratory viruses (CARVs). We prospectively compared the performance of Cobas Liat Influenza A/B and RSV assay (LIAT) with our routine multiplexNAT-1 (xTAG Respiratory Pathogen Panel; Luminex) and multiplexNAT-2 (ePlex-RPP; GenMark Diagnostics) using 194 consecutive nasopharyngeal swabs from patients with influenza-like illness during winter 2017/2018.

Cytomegalovirus sequence variability, amplicon length, and DNase-sensitive non-encapsidated genomes are obstacles to standardization and commutability of plasma viral load results.

Background: Cytomegalovirus (CMV) management post-transplantation relies on quantification in blood, but inter-laboratory and inter-assay variability impairs commutability. An international multicenter study demonstrated that variability is mitigated by standardizing plasma volumes, automating DNA extraction and amplification, and calibration to the 1st-CMV-WHO-International-Standard as in the FDA-approved Roche-CAP/CTM-CMV. However, Roche-CAP/CTM-CMV showed under-quantification and false-negative results in a quality assurance program (UK-NEQAS-2014).

Novel Human Polyomavirus Noncoding Control Regions Differ in Bidirectional Gene Expression according to Host Cell, Large T-Antigen Expression, and Clinically Occurring Rearrangements.

Human polyomavirus (HPyV) DNA genomes contain three regions denoted the early viral gene region (EVGR), encoding the regulatory T-antigens and one microRNA, the late viral gene region (LVGR), encoding the structural Vp capsid proteins, and the noncoding control region (NCCR). The NCCR harbors the origin of viral genome replication and bidirectional promoter/enhancer functions governing EVGR and LVGR expression on opposite DNA strands. Despite principal similarities, HPyV NCCRs differ in length, sequence, and architecture.

Enterovirus detection in patients with acute gastroenteritis in Switzerland.

Acute gastroenteritis (GE) has a major impact on morbidity and mortality worldwide, yet comprehensive data regarding infectious agents including enteroviruses are scarce. We hypothesized that enteroviruses constitute a significant cause of acute GE. We analyzed 677 stool samples from 504 patients, which had been submitted for suspected infectious GE.

Donor-derived, metastatic urothelial cancer after kidney transplantation associated with a potentially oncogenic BK polyomavirus.

BK polyomavirus has been linked to urothelial carcinoma in immunosuppressed patients. Here, we performed comprehensive genomic analysis of a BK polyomavirus-associated, metachronous, multifocal and metastatic micropapillary urothelial cancer in a kidney transplant recipient. Dissecting cancer heterogeneity by sorting technologies prior to array-comparative genomic hybridization followed by short tandem repeat analysis revealed that the metastatic urothelial cancer was of donor origin (4-year-old male).

Human BK Polyomavirus Plasmid pBKV (34-2) (Dunlop) Contains Mutations Not Found in the Originally Published Sequences.

The plasmid pBKV (34-2) (ATCC 45025) contains the entire BK polyomavirus Dunlop genome. Sequencing revealed 12 point mutations compared to the GenBank sequence, but only 4 point mutations compared to the published sequence. The origin of these differences is unknown, but may impact virological as well as diagnostic research and development.

Sp1 sites in the noncoding control region of BK polyomavirus are key regulators of bidirectional viral early and late gene expression.

Unlabelled: In kidney transplant patients with BK polyomavirus (BKPyV) nephropathy, viral variants arise bearing rearranged noncoding control regions (rr-NCCRs) that increase viral early gene expression, replicative fitness, and cytopathology. rr-NCCRs result from various deletions and duplications of archetype NCCR (ww-NCCR) sequences, which alter transcription factor binding sites (TFBS). However, the role of specific TFBS is unclear.

Management and outcome of CSF-JC virus PCR-negative PML in a natalizumab-treated patient with MS.

Objective: To describe the diagnosis and management of a 49-year-old woman with multiple sclerosis (MS) developing a progressive hemiparesis and expanding MRI lesion suspicious of progressive multifocal leukoencephalopathy (PML) 19 months after starting natalizumab.

Results: Polyomavirus JC (JCV)-specific qPCR in CSF was repeatedly negative, but JCV-specific antibodies indicated intrathecal production. Brain biopsy tissue taken 17 weeks after natalizumab discontinuation and plasmapheresis was positive for JCV DNA with characteristic rearrangements of the noncoding control region, but histology and immunohistochemistry were not informative except for pathologic features compatible with immune reconstitution inflammatory syndrome.

CMX001 (1-O-hexadecyloxypropyl-cidofovir) inhibits polyomavirus JC replication in human brain progenitor-derived astrocytes.

Polyomavirus JC (JCV) replication causes progressive multifocal leukoencephalopathy (PML), a frequently fatal brain disease in immunodeficient patients, yet antiviral drugs are lacking. We characterized the lipid conjugate 1-O-hexadecyloxypropyl-cidofovir (CMX001) regarding JCV (Mad-4) replication in human brain progenitor-derived astrocytes (PDA) and the simian virus 40 (SV40) large-T-antigen-expressing COS-7 cells up to 7 days postinfection (dpi). We examined JCV loads by PCR, the infection rate by immunofluorescence, and host cell toxicity by WST-1 and BrdU incorporation assays.

1-O-hexadecyloxypropyl cidofovir (CMX001) effectively inhibits polyomavirus BK replication in primary human renal tubular epithelial cells.

Antiviral drugs for treating polyomavirus BK (BKV) replication in polyomavirus-associated nephropathy or hemorrhagic cystitis are of considerable clinical interest. Unlike cidofovir, the lipid conjugate 1-O-hexadecyloxypropyl cidofovir (CMX001) is orally available and has not caused detectable nephrotoxicity in rodent models or human studies to date. Primary human renal proximal tubular epithelial cells were infected with BKV-Dunlop, and CMX001 was added 2 h postinfection (hpi).

Rearranged JC virus noncoding control regions found in progressive multifocal leukoencephalopathy patient samples increase virus early gene expression and replication rate.

Polyomavirus JC (JCV) infects ∼ 60% of the general population, followed by asymptomatic urinary shedding in ∼ 20%. In patients with pronounced immunodeficiency, including HIV/AIDS, JCV can cause progressive multifocal leukoencephalopathy (PML), a devastating brain disease of high mortality. While JCV in the urine of healthy people has a linear noncoding control region called the archetype NCCR (at-NCCR), JCV in brain and cerebrospinal fluid (CSF) of PML patients bear rearranged NCCRs (rr-NCCRs).

The polyomavirus BK agnoprotein co-localizes with lipid droplets.

Agnoprotein encoded by human polyomavirus BK (BKV) is a late cytoplasmic protein of 66 amino acids (aa) of unknown function. Immunofluorescence microscopy revealed a fine granular and a vesicular distribution in donut-like structures. Using BKV(Dunlop)-infected or agnoprotein-transfected cells, we investigated agnoprotein co-localization with subcellular structures.

Clinical polyomavirus BK variants with agnogene deletion are non-functional but rescued by trans-complementation.

High-level replication of polyomavirus BK (BKV) in kidney transplant recipients is associated with the emergence of BKV variants with rearranged (rr) non-coding control region (NCCR) increasing viral early gene expression and cytopathology. Cloning and sequencing revealed the presence of a BKV quasispecies which included non-functional variants when assayed in a recombinant virus assay. Here we report that the rr-NCCR of BKV variants RH-3 and RH-12, both bearing a NCCR deletion including the 5' end of the agnoprotein coding sequence, mediated early and late viral reporter gene expression in kidney cells.

Antibody responses to recombinant polyomavirus BK large T and VP1 proteins in young kidney transplant patients.

BK virus (BKV)-specific immunity is critical for polyomavirus-associated nephropathy, but antibody responses are incompletely defined. We compared the hemagglutination inhibition assay (HIA) with immunoglobulin G enzyme immunoassays (EIA) to BKV proteins expressed in baculovirus-infected insect cells. N-terminal, internal, and C-terminal domains of the BKV large T antigen (BKLT) were fused to glutathione S-transferase (GST), yielding GST-BKLTD1, GST-BKLTD2, and GST-BKLTD3, respectively.