Positive selection underlies repeated knockout of ORF8 in SARS-CoV-2 evolution.

Knockout of the ORF8 protein has repeatedly spread through the global viral population during SARS-CoV-2 evolution. Here we use both regional and global pathogen sequencing to explore the selection pressures underlying its loss. In Washington State, we identified transmission clusters with ORF8 knockout throughout SARS-CoV-2 evolution, not just on novel, high fitness viral backbones.

Cytomegalovirus breakthrough and resistance during letermovir prophylaxis.

Letermovir is a relatively new antiviral for prophylaxis against cytomegalovirus (CMV) after allogeneic hematopoietic cell transplantation (HCT). CMV-seropositive HCT recipients who received letermovir prophylaxis from 2018 to 2020 at our center were evaluated for letermovir resistance and breakthrough CMV reactivation. Two-hundred twenty-six letermovir recipients were identified and 7/15 (47%) with CMV DNAemia ≥200 IU/mL were successfully genotyped for UL56 resistance.

Rapid and accurate identification of SARS-CoV-2 Omicron variants using droplet digital PCR (RT-ddPCR).

Background: Some mutations in the receptor binding domain of the SARS-CoV-2 Spike protein are associated with increased transmission or substantial reductions in vaccine efficacy, including in recently described Omicron subvariants. The changing frequencies of these mutations combined with their differing susceptibility to available therapies have posed significant problems for clinicians and public health professionals.

Objective: To develop an assay capable of rapidly and accurately identifying variants including Omicron in clinical specimens to enable case tracking and/or selection of appropriate clinical treatment.

Predicting infectivity: comparing four PCR-based assays to detect culturable SARS-CoV-2 in clinical samples.

With the COVID-19 pandemic caused by SARS-CoV-2 now in its second year, there remains an urgent need for diagnostic testing that can identify infected individuals, particularly those who harbor infectious virus. Various RT-PCR strategies have been proposed to identify specific viral RNA species that may predict the presence of infectious virus, including detection of transcriptional intermediates (e.g.

A SARS-CoV-2 Nucleocapsid Variant that Affects Antigen Test Performance.

More than one year into a global pandemic, SARS-CoV-2 is now defined by a variety of rapidly evolving variant lineages. Several FDA authorized molecular diagnostic tests have been impacted by viral variation, while no reports of viral variation affecting antigen test performance have occurred to date. While determining the analytical sensitivity of the Quidel Sofia SARS Antigen FIA test (Sofia 2), we uncovered a high viral load specimen that repeatedly tested negative by this antigen test.

Specific allelic discrimination of N501Y and other SARS-CoV-2 mutations by ddPCR detects B.1.1.7 lineage in Washington State.

Real-time epidemiological tracking of variants of concern (VOCs) can help limit the spread of more contagious forms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), such as those containing the N501Y mutation. Typically, genetic sequencing is required to be able to track VOCs in real-time. However, sequencing can take time and may not be accessible in all laboratories.

Performance characteristics of the Abbott Alinity m SARS-CoV-2 assay.

Mass molecular diagnostic testing for the SARS-CoV-2 pandemic has drawn on laboratory developed tests, commercial assays, and fully-automated platforms to accommodate widespread demand. The Alinity m instrument by Abbott is capable of detecting several clinically relevant pathogens and has recently received FDA emergency use authorization for SARS-CoV-2 molecular testing. The Alinity m performs automatic sample preparation, RT-PCR assembly, amplification, detection, and result calculation in under two hours.

Analytical Sensitivity of the Abbott BinaxNOW COVID-19 Ag Card.

Multiple rapid antigen (Ag) tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have recently received emergency-use authorization (EUA) from the U.S. Food and Drug Administration (FDA).

Human Metapneumovirus Infection and Genotyping of Infants in Rural Nepal.

Background: Acute respiratory tract infections are a serious clinical burden in infants; human metapneumovirus (HMPV) is an important etiological agent. We investigated genotypic variation and molecular epidemiological patterns among infants infected with HMPV in Sarlahi, Nepal, to better characterize infection in a rural, low-resource setting.

Methods: Between May 2011 and April 2014, mid-nasal swabs were collected from 3528 infants who developed respiratory symptoms during a longitudinal maternal influenza vaccine study.

Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step.

The ongoing COVID-19 pandemic has created an unprecedented need for rapid diagnostic testing. The World Health Organization (WHO) recommends a standard assay that includes an RNA extraction step from a nasopharyngeal (NP) swab followed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect the purified SARS-CoV-2 RNA. The current global shortage of RNA extraction kits has caused a severe bottleneck to COVID-19 testing.

Pooling of SARS-CoV-2 samples to increase molecular testing throughput.

Background: SARS-CoV-2 testing demand has outpaced its supply. Pooling samples for lower risk populations has the potential to accommodate increased demand for SARS-CoV-2 molecular testing.

Objective: To evaluate the sensitivity, specificity, and reproducibility of 4-way pooling of SARS-CoV-2 specimens for high-throughput RT-PCR.

Multiplexing primer/probe sets for detection of SARS-CoV-2 by qRT-PCR.

Background: The novel respiratory virus SARS-CoV-2, responsible for over 380,000 COVID-19 related deaths, has caused significant strain on healthcare infrastructure and clinical laboratories globally. The pandemic's initial challenges include broad diagnostic testing, consistent reagent supply lines, and access to laboratory instruments and equipment. In early 2020, primer/probe sets distributed by the CDC utilized the same fluorophore for molecular detection - requiring multiple assays to be run in parallel - consuming valuable and limited resources.

Comparative Performance of SARS-CoV-2 Detection Assays using Seven Different Primer/Probe Sets and One Assay Kit.

More than 100,000 people worldwide are known to have been infected with SARS-CoV-2 beginning in December 2019. The virus has now spread to over 93 countries including the United States, with the largest cluster of US cases to date in the Seattle metropolitan area in Washington. Given the rapid increase in the number of local cases, the availability of accurate, high-throughput SARS-CoV-2 testing is vital to efforts to manage the current public health crisis.

DIRECT RT-qPCR DETECTION OF SARS-CoV-2 RNA FROM PATIENT NASOPHARYNGEAL SWABS WITHOUT AN RNA EXTRACTION STEP.

The ongoing COVID-19 pandemic has caused an unprecedented need for rapid diagnostic testing. The Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) recommend a standard assay that includes an RNA extraction step from a nasopharyngeal (NP) swab followed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect the purified SARS-CoV-2 RNA. The current global shortage of RNA extraction kits has caused a severe bottleneck to COVID-19 testing.

Validation of SARS-CoV-2 detection across multiple specimen types.

Background: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused considerable disruption across the world, resulting in more than 235,000 deaths since December 2019. SARS-CoV-2 has a wide tropism and detection of the virus has been described in multiple specimen types, including various respiratory secretions, cerebrospinal fluid, and stool.

Objective: To evaluate the accuracy and sensitivity of a laboratory modified CDCbased SARS-CoV-2 N1 and N2 assay across a range of sample types.

Metagenomic Analysis Reveals Clinical SARS-CoV-2 Infection and Bacterial or Viral Superinfection and Colonization.

Background: More than 2 months separated the initial description of SARS-CoV-2 and discovery of its widespread dissemination in the United States. Despite this lengthy interval, implementation of specific quantitative reverse transcription (qRT)-PCR-based SARS-CoV-2 tests in the US has been slow, and testing is still not widely available. Metagenomic sequencing offers the promise of unbiased detection of emerging pathogens, without requiring prior knowledge of the identity of the responsible agent or its genomic sequence.

Comparative Performance of SARS-CoV-2 Detection Assays Using Seven Different Primer-Probe Sets and One Assay Kit.

Nearly 400,000 people worldwide are known to have been infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) beginning in December 2019. The virus has now spread to over 168 countries including the United States, where the first cluster of cases was observed in the Seattle metropolitan area in Washington. Given the rapid increase in the number of cases in many localities, the availability of accurate, high-throughput SARS-CoV-2 testing is vital to efforts to manage the current public health crisis.

Phylogenetic characterization of rhinoviruses from infants in Sarlahi, Nepal.

Problem: Rhinoviruses (RVs), the most common causes of acute respiratory infections in young children and infants, are highly diverse genetically.

Objective: To characterize the RV types detected with respiratory illness episodes in infants in Nepal.

Study Methods: Infants born to women enrolled in a randomized trial of maternal influenza immunization in rural, southern Nepal were followed with household-based weekly surveillance until 180 days of age.

Large, Stable, Contemporary Interspecies Recombination Events in Circulating Human Herpes Simplex Viruses.

Background: The ubiquitous human pathogens, herpes simplex virus (HSV)-1 and HSV-2, are distinct viral species that diverged approximately 6 million years ago. At least 4 small, ancient HSV-1 × HSV-2 interspecies recombination events have affected the HSV-2 genome, with recombinants and nonrecombinants at each locus circulating today. However, it is unknown whether interspecies recombination can affect other loci and whether new recombinants continue to be generated.