No Remdesivir Resistance Observed in the Phase 3 Severe and Moderate COVID-19 SIMPLE Trials.

Remdesivir (RDV) is a broad-spectrum nucleotide analog prodrug approved for the treatment of COVID-19 in hospitalized and non-hospitalized patients with clinical benefit demonstrated in multiple Phase 3 trials. Here we present SARS-CoV-2 resistance analyses from the Phase 3 SIMPLE clinical studies evaluating RDV in hospitalized participants with severe or moderate COVID-19 disease. The severe and moderate studies enrolled participants with radiologic evidence of pneumonia and a room-air oxygen saturation of ≤94% or >94%, respectively. Virology sample collection was optional in the study protocols. Sequencing and related viral load data were obtained retrospectively from participants at a subset of study sites with local sequencing capabilities (10 of 183 sites) at timepoints with detectable viral load. Among participants with both baseline and post-baseline sequencing data treated with RDV, emergent Nsp12 substitutions were observed in 4 of 19 (21%) participants in the severe study and none of the 2 participants in the moderate study. The following 5 substitutions emerged: T76I, A526V, A554V, E665K, and C697F. The substitutions T76I, A526V, A554V, and C697F had an EC50 fold change of ≤1.5 relative to the wildtype reference using a SARS-CoV-2 subgenomic replicon system, indicating no significant change in the susceptibility to RDV. The phenotyping of E665K could not be determined due to a lack of replication. These data reveal no evidence of relevant resistance emergence and further confirm the established efficacy profile of RDV with a high resistance barrier in COVID-19 patients.

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.

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.

Improved diagnosis of viral encephalitis in adult and pediatric hematological patients using viral metagenomics.

Metagenomic sequencing is a powerful technique that enables detection of the full spectrum of pathogens present in any specimen in a single test. Hence, metagenomics is increasingly being applied for detection of viruses in clinical cases with suspected infections of unknown etiology and a large number of relevant potential causes. This is typically the case in patients presenting with encephalitis, in particular when immunity is impaired by underlying disorders. In this study, viral metagenomics has been applied to a cohort of hematological patients with encephalitis of unknown origin. Because viral loads in cerebrospinal fluid of patients with encephalitis are generally low, the technical performance of a metagenomic sequencing protocol with viral enrichment by capture probes targeting all known vertebrate viral sequences was studied. Subsequently, the optimized viral metagenomics protocol was applied to a cohort of hematological patients with encephalitis of unknown origin. Viral enrichment by capture probes increased the viral sequence read count of metagenomics on cerebrospinal fluid samples 100 - 10.000 fold, compared to unenriched metagenomic sequencing. In five out of 41 (12%) hematological patients with encephalitis, a virus was detected by viral metagenomics which had not been detected by current routine diagnostics. BK polyomavirus, hepatitis E virus, human herpes virus-6 and Epstein Barr virus were identified by this unbiased metagenomic approach. This study demonstrated that hematological patients with encephalitis of unknown origin may benefit from early viral metagenomics testing as a single step approach.

Retrospective Validation of a Metagenomic Sequencing Protocol for Combined Detection of RNA and DNA Viruses Using Respiratory Samples from Pediatric Patients.

Viruses are the main cause of respiratory tract infections. Metagenomic next-generation sequencing (mNGS) enables unbiased detection of all potential pathogens. To apply mNGS in viral diagnostics, sensitive and simultaneous detection of RNA and DNA viruses is needed. Herein, were studied the performance of an in-house mNGS protocol for routine diagnostics of viral respiratory infections with potential for automated pan-pathogen detection. The sequencing protocol and bioinformatics analysis were designed and optimized, including exogenous internal controls. Subsequently, the protocol was retrospectively validated using 25 clinical respiratory samples. The developed protocol using Illumina NextSeq 500 sequencing showed high repeatability. Use of the National Center for Biotechnology Information's RefSeq database as opposed to the National Center for Biotechnology Information's nucleotide database led to enhanced specificity of classification of viral pathogens. A correlation was established between read counts and PCR cycle threshold value. Sensitivity of mNGS, compared with PCR, varied up to 83%, with specificity of 94%, dependent on the cutoff for defining positive mNGS results. Viral pathogens only detected by mNGS, not present in the routine diagnostic workflow, were influenza C, KI polyomavirus, cytomegalovirus, and enterovirus. Sensitivity and analytical specificity of this mNGS protocol were comparable to PCR and higher when considering off-PCR target viral pathogens. One single test detected all potential viral pathogens and simultaneously obtained detailed information on detected viruses.

Gene Mosaicism Screening Using Single-Molecule Molecular Inversion Probes in Routine Diagnostics for Systemic Autoinflammatory Diseases.

Diagnosis of systemic autoinflammatory diseases (SAIDs) is often difficult to achieve and can delay the start of proper treatments and result in irreversible organ damage. In several patients with dominantly inherited SAID, postzygotic mutations have been detected as the disease-causing gene defects. Mutations with allele frequencies <5% have been detected, even in patients with severe phenotypes. Next-generation sequencing techniques are currently used to detect mutations in SAID-associated genes. However, even if the genomic region is highly covered, this approach is usually not able to distinguish low-grade postzygotic variants from background noise. We, therefore, developed a sensitive deep sequencing assay for mosaicism detection in SAID-associated genes using single-molecule molecular inversion probes. Our results show the accurate detection of postzygotic variants with allele frequencies as low as 1%. The probability of calling mutations with allele frequencies ≥3% exceeds 99.9%. To date, we have detected three patients with mosaicism, two carrying likely pathogenic NLRP3 variants and one carrying a likely pathogenic TNFRSF1A variant with an allele frequency of 1.3%, confirming the relevance of the technology. The assay shown herein is a flexible, robust, fast, cost-effective, and highly reliable method for mosaicism detection; therefore, it is well suited for routine diagnostics.