Publications by authors named "Keith R Jerome"

250 Publications

Evaluation of cell-based and surrogate SARS-CoV-2 neutralization assays.

J Clin Microbiol 2021 Jul 21:JCM0052721. Epub 2021 Jul 21.

Vaccine and Infectious Diseases Division, Fred Hutch Cancer Research Center, Seattle WA.

Determinants of protective immunity against SARS-CoV-2 infection require the development of well-standardized, reproducible antibody assays. This need has led to the emergence of a variety of neutralization assays. Head-to-head evaluation of different SARS-CoV-2 neutralization platforms could facilitate comparisons across studies and laboratories. Five neutralization assays were compared using forty plasma samples from convalescent individuals with mild-to-moderate COVID-19: four cell-based systems using either live recombinant SARS-CoV-2 or pseudotyped viral particles created with lentivirus (LV) or vesicular stomatitis virus (VSV) packaging and one surrogate ELISA-based test that measures inhibition of the spike protein receptor binding domain (RBD) binding its receptor, human angiotensin converting enzyme 2 (hACE2). Vero, Vero E6, HEK293T expressing hACE2, and TZM-bl cells expressing hACE2 and transmembrane serine protease 2 were tested. All cell-based assays showed 50% neutralizing dilution (ND50) geometric mean titers (GMTs) that were highly correlated (Pearson = 0.81-0.89) and ranged within 3.4-fold. The live-virus assay and LV-pseudovirus assays with HEK293T/hACE2 cells showed very similar mean titers: 141 and 178, respectively. ND50 titers positively correlated with plasma IgG targeting SARS-CoV-2 spike and RBD ( = 0.63-0.89), but moderately correlated with nucleoprotein IgG ( = 0.46-0.73). ND80 GMTs mirrored ND50 data and showed similar correlation between assays and with IgG concentrations. The VSV-pseudovirus assay and LV-pseudovirus assay with HEK293T/hACE2 cells in low and high-throughput versions were calibrated against the WHO SARS-CoV-2 IgG standard. High concordance between the outcomes of cell-based assays with live and pseudotyped virions enables valid cross-study comparison using these platforms. 249.
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http://dx.doi.org/10.1128/JCM.00527-21DOI Listing
July 2021

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

J Med Virol 2021 Jun 25. Epub 2021 Jun 25.

Department of Laboratory Medicine and Pathology, Virology Division, University of Washington School of Medicine, Seattle, Washington, USA.

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. Genotyping by RT-ddPCR offers an alternative to rapidly detect VOCs through discrimination of specific alleles such as N501Y, which is associated with increased transmissibility and virulence. Here we describe the first cases of the B.1.1.7 lineage of SARS-CoV-2 detected in Washington State by using a combination of reverse-transcription polymerase chain reaction (RT-PCR), RT-ddPCR, and next-generation sequencing. We initially screened 1035 samples positive for SARS-CoV-2 by our CDC-based laboratory-developed assay using ThermoFisher's multiplex RT-PCR COVID-19 assay over four weeks from late December 2020 to early January 2021. S gene target failures (SGTF) were subsequently assayed by RT-ddPCR to confirm four mutations within the S gene associated with the B.1.1.7 lineage: a deletion at amino acid (AA) 69-70 (ACATGT), deletion at AA 145, (TTA), N501Y mutation (TAT), and S982A mutation (GCA). All four targets were detected in two specimens; follow-up sequencing revealed a total of 9 mutations in the S gene and phylogenetic clustering within the B.1.1.7 lineage. Next, we continued screening samples for SGTF detecting 23 additional B.1.1.7 variants by RT-ddPCR and confirmed by sequencing. As VOCs become increasingly prevalent, molecular diagnostic tools like RT-ddPCR can be utilized to quickly, accurately, and sensitively distinguish more contagious lineages of SARS-CoV-2.
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http://dx.doi.org/10.1002/jmv.27155DOI Listing
June 2021

Variants of concern are overrepresented among post-vaccination breakthrough infections of SARS-CoV-2 in Washington State.

Clin Infect Dis 2021 Jun 24. Epub 2021 Jun 24.

Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA.

Across 20 vaccine breakthrough cases detected at our institution, all 20 (100%) infections were due to variants of concern (VOC) and had a median Ct of 20.2 (IQR=17.1-23.3). When compared to 5174 contemporaneous samples sequenced in our laboratory, VOC were significantly enriched among breakthrough infections (p < .05).
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http://dx.doi.org/10.1093/cid/ciab581DOI Listing
June 2021

Anti-SARS-CoV-2 antibody levels measured by the Advise Dx SARS-CoV-2 assay are concordant with previously available serologic assays but are not fully predictive of sterilizing immunity.

J Clin Microbiol 2021 Jun 24:JCM0098921. Epub 2021 Jun 24.

Department of Laboratory Medicine and Pathology, University of Washington Medical Center, Seattle, WA.

With the availability of widespread SARS-CoV-2 vaccination, high-throughput quantitative anti-spike serological testing will likely become increasingly important. Here, we investigated the performance characteristics of the recently FDA authorized semi-quantitative anti-spike AdviseDx SARS-CoV-2 IgG II assay compared to the FDA authorized anti-nucleocapsid Abbott Architect SARS-CoV-2 IgG, Roche elecsys Anti-SARS-CoV-2-S, EuroImmun Anti-SARS-CoV-2 ELISA, and GenScript surrogate virus neutralization assays and examined the humoral response associated with vaccination, natural protection, and breakthrough infection. The AdviseDx assay had a clinical sensitivity at 14 days post-symptom onset or 10 days post PCR detection of 95.6% (65/68, 95% CI: 87.8-98.8%) with two discrepant individuals seroconverting shortly thereafter. The AdviseDx assay demonstrated 100% positive percent agreement with the four other assays examined using the same symptom onset or PCR detection cutoffs. Using a recently available WHO International Standard for anti-SARS-CoV-2 antibody, we provide assay unit conversion factors to international units for each of the assays examined. We performed a longitudinal survey of healthy vaccinated individuals, finding median AdviseDx immunoglobulin levels peaked seven weeks post-first vaccine dose at approximately 4,000 IU/mL. Intriguingly, among the five assays examined, there was no significant difference in antigen binding level or neutralizing activity between two seropositive patients protected against SARS-CoV-2 infection in a previously described fishing vessel outbreak and five healthcare workers who experienced vaccine breakthrough of SARS-CoV-2 infection - all with variants of concern. These findings suggest that protection against SARS-CoV-2 infection cannot currently be predicted exclusively using antibody assays against wildtype SARS-CoV-2 spike. Further work is required to establish protective correlates for SARS-CoV-2 infection.
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http://dx.doi.org/10.1128/JCM.00989-21DOI Listing
June 2021

Tracking SARS-CoV-2 Spike Protein Mutations in the United States (2020/01 - 2021/03) Using a Statistical Learning Strategy.

bioRxiv 2021 Jun 15. Epub 2021 Jun 15.

The emergence and establishment of SARS-CoV-2 variants of interest (VOI) and variants of concern (VOC) highlight the importance of genomic surveillance. We propose a statistical learning strategy (SLS) for identifying and spatiotemporally tracking potentially relevant Spike protein mutations. We analyzed 167,893 Spike protein sequences from US COVID-19 cases (excluding 21,391 sequences from VOI/VOC strains) deposited at GISAID from January 19, 2020 to March 15, 2021. Alignment against the reference Spike protein sequence led to the identification of viral residue variants (VRVs), i.e., residues harboring a substitution compared to the reference strain. Next, generalized additive models were applied to model VRV temporal dynamics, to identify VRVs with significant and substantial dynamics (false discovery rate q-value <0.01; maximum VRV proportion > 10% on at least one day). Unsupervised learning was then applied to hierarchically organize VRVs by spatiotemporal patterns and identify VRV-haplotypes. Finally, homology modelling was performed to gain insight into potential impact of VRVs on Spike protein structure. We identified 90 VRVs, 71 of which have not previously been observed in a VOI/VOC, and 35 of which have emerged recently and are durably present. Our analysis identifies 17 VRVs ∼91 days earlier than their first corresponding VOI/VOC publication. Unsupervised learning revealed eight VRV-haplotypes of 4 VRVs or more, suggesting two emerging strains (B1.1.222 and B.1.234). Structural modeling supported potential functional impact of the D1118H and L452R mutations. The SLS approach equally monitors all Spike residues over time, independently of existing phylogenic classifications, and is complementary to existing genomic surveillance methods.
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http://dx.doi.org/10.1101/2021.06.15.448495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8219100PMC
June 2021

Association of Inherited Chromosomally Integrated Human Herpesvirus 6 with Neurologic Symptoms and Management after Allogeneic Hematopoietic Cell Transplantation.

Transplant Cell Ther 2021 Jun 7. Epub 2021 Jun 7.

Fred Hutchinson Cancer Research Center, Vaccine and Infectious Disease Division, Seattle, Washington; Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, Seattle, Washington. Electronic address:

Reactivation of human herpesvirus 6 (HHV-6) after allogeneic hematopoietic cell transplantation (HCT) is associated with neurologic complications, but the impact of donor and/or recipient inherited chromosomally integrated HHV-6 (iciHHV-6) on post-HCT central nervous system (CNS) symptoms and diagnostic and therapeutic interventions is not well understood. The aims of the present study were (1) to compare the cumulative incidence of CNS symptoms in the first 100 days following allogeneic HCT among patients with donor and/or recipient iciHHV-6 (iciHHV-6)with that of patients with neither donor nor recipient iciHHV-6 (iciHHV-6) and (2) to assess the role of HHV-6 detection in driving potentially unnecessary interventions in iciHHV-6 patients. We performed a retrospective matched cohort study of 87 iciHHV-6 and 174 iciHHV-6 allogeneic HCT recipients. HHV-6 testing was performed at the discretion of healthcare providers, who were unaware of iciHHV-6 status. The cumulative incidence of CNS symptoms was similar in iciHHV-6 (n = 37; 43%) and iciHHV-6 HCT recipients (n = 81; 47%; P = .63). HHV-6 plasma testing was performed in similar proportions of iciHHV-6 (n = 6; 7%) and iciHHV-6 (9%) patients and was detected in all tested iciHHV-6 HCTs and 2 (13%) iciHHV-6 HCTs. This resulted in more frequent HHV-6-targeted antiviral therapy after iciHHV-6 HCT (odds ratio, 12.8; 95% confidence interval, 1.5 to 108.2) with associated side effects. HHV-6 plasma detection in 2 iciHHV-6 patients without active CNS symptoms prompted unnecessary lumbar punctures. The cumulative incidence of CNS symptoms was similar after allogeneic HCT involving recipients or donors with and without iciHHV-6. Misattribution of HHV-6 detection as infection after iciHHV-6 HCT may lead to unnecessary interventions. Testing for iciHHV-6 may improve patient management.
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http://dx.doi.org/10.1016/j.jtct.2021.05.029DOI Listing
June 2021

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

J Clin Virol 2021 07 14;140:104869. Epub 2021 May 14.

Department of Laboratory Medicine and Pathology, Virology Division, University of Washington, Seattle, WA, United States; Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, United States. Electronic address:

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. Here, we validate the performance characteristics of the Alinity m SARS-CoV-2 assay in comparison with the Roche cobas 6800 and Hologic Panther Fusion platforms. Across 178 positive and 195 negative nasopharyngeal swab specimens (C range 14.30-38.84), the Alinity m detected one additional positive specimen that was found to be negative on the Roche cobas 6800 (PPA 100%, NPA 99.5%). Across a separate set of 30 positive and 174 negative nasopharyngeal swab specimens (C range 14.1-38.5), the Alinity m had 100% positive and negative agreement with the Hologic Panther Fusion. Using SeraCare SARS-CoV-2 RNA standards, the assay limit of detection was verified to be two-fold more sensitive than the parameters stated by the SARS-CoV-2 AMP kit package insert, at 50 virus copies/mL. Assay specificity was 100% over 20 specimens positive for other respiratory viruses and intraday precision was 100% concordant with <2% CV. These data illst u illustrate the Abbott Alinity m system's high concordance with reference assays and analyti high analytical for SARS-CoV-2 molecular detection.
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http://dx.doi.org/10.1016/j.jcv.2021.104869DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8118701PMC
July 2021

A highly multiplexed droplet digital PCR assay to measure the intact HIV-1 proviral reservoir.

Cell Rep Med 2021 Apr 12;2(4):100243. Epub 2021 Apr 12.

Department of Obstetrics & Gynecology, University of Washington, Seattle, WA, USA.

Quantifying the replication-competent HIV reservoir is essential for evaluating curative strategies. Viral outgrowth assays (VOAs) underestimate the reservoir because they fail to induce all replication-competent proviruses. Single- or double-region HIV DNA assays overestimate it because they fail to exclude many defective proviruses. We designed two triplex droplet digital PCR assays, each with 2 unique targets and 1 in common, and normalize the results to PCR-based T cell counts. Both HIV assays are specific, sensitive, and reproducible. Together, they estimate the number of proviruses containing all five primer-probe regions. Our 5-target results are on average 12.1-fold higher than and correlate with paired quantitative VOA (Spearman's ρ = 0.48) but estimate a markedly smaller reservoir than previous DNA assays. In patients on antiretroviral therapy, decay rates in blood CD4 T cells are faster for intact than for defective proviruses, and intact provirus frequencies are similar in mucosal and circulating T cells.
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http://dx.doi.org/10.1016/j.xcrm.2021.100243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8080125PMC
April 2021

Viral genomes reveal patterns of the SARS-CoV-2 outbreak in Washington State.

Sci Transl Med 2021 05 3;13(595). Epub 2021 May 3.

Seattle Children's Research Institute, Seattle, WA 98101, USA.

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has gravely affected societies around the world. Outbreaks in different parts of the globe have been shaped by repeated introductions of new viral lineages and subsequent local transmission of those lineages. Here, we sequenced 3940 SARS-CoV-2 viral genomes from Washington State (USA) to characterize how the spread of SARS-CoV-2 in Washington State in early 2020 was shaped by differences in timing of mitigation strategies across counties and by repeated introductions of viral lineages into the state. In addition, we show that the increase in frequency of a potentially more transmissible viral variant (614G) over time can potentially be explained by regional mobility differences and multiple introductions of 614G but not the other variant (614D) into the state. At an individual level, we observed evidence of higher viral loads in patients infected with the 614G variant. However, using clinical records data, we did not find any evidence that the 614G variant affects clinical severity or patient outcomes. Overall, this suggests that with regard to D614G, the behavior of individuals has been more important in shaping the course of the pandemic in Washington State than this variant of the virus.
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http://dx.doi.org/10.1126/scitranslmed.abf0202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8158963PMC
May 2021

Optimization of AAV vectors to target persistent viral reservoirs.

Virol J 2021 Apr 23;18(1):85. Epub 2021 Apr 23.

Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, Seattle, WA, USA.

Gene delivery of antiviral therapeutics to anatomical sites where viruses accumulate and persist is a promising approach for the next generation of antiviral therapies. Recombinant adeno-associated viruses (AAV) are one of the leading vectors for gene therapy applications that deliver gene-editing enzymes, antibodies, and RNA interference molecules to eliminate viral reservoirs that fuel persistent infections. As long-lived viral DNA within specific cellular reservoirs is responsible for persistent hepatitis B virus, Herpes simplex virus, and human immunodeficiency virus infections, the discovery of AAV vectors with strong tropism for hepatocytes, sensory neurons and T cells, respectively, is of particular interest. Identification of natural isolates from various tissues in humans and non-human primates has generated an extensive catalog of AAV vectors with diverse tropisms and transduction efficiencies, which has been further expanded through molecular genetic approaches. The AAV capsid protein, which forms the virions' outer shell, is the primary determinant of tissue tropism, transduction efficiency, and immunogenicity. Thus, over the past few decades, extensive efforts to optimize AAV vectors for gene therapy applications have focused on capsid engineering with approaches such as directed evolution and rational design. These approaches are being used to identify variants with improved transduction efficiencies, alternate tropisms, reduced sequestration in non-target organs, and reduced immunogenicity, and have produced AAV capsids that are currently under evaluation in pre-clinical and clinical trials. This review will summarize the most recent strategies to identify AAV vectors with enhanced tropism and transduction in cell types that harbor viral reservoirs.
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http://dx.doi.org/10.1186/s12985-021-01555-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8067653PMC
April 2021

An international, interlaboratory ring trial confirms the feasibility of an open-source, extraction-less "direct" RT-qPCR method for reliable detection of SARS-CoV-2 RNA in clinical samples.

medRxiv 2021 Apr 16. Epub 2021 Apr 16.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used worldwide to test and trace the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). "Extraction-less" or "direct" real time-reverse transcription polymerase chain reaction (RT-PCR) is an open-access qualitative method for SARS-CoV-2 detection from nasopharyngeal or oral pharyngeal samples with the potential to generate actionable data more quickly, at a lower cost, and with fewer experimental resources than full RT-qPCR. This study engaged 10 global testing sites, including laboratories currently experiencing testing limitations due to reagent or equipment shortages, in an international interlaboratory ring trial. Participating laboratories were provided a common protocol, common reagents, aliquots of identical pooled clinical samples, and purified nucleic acids and used their existing in-house equipment. We observed 100% concordance across laboratories in the correct identification of all positive and negative samples, with highly similar cycle threshold values. The test also performed well when applied to locally collected patient nasopharyngeal samples, provided the viral transport media did not contain charcoal or guanidine, both of which appeared to potently inhibit the RT-PCR reaction. Our results suggest that open-access, direct RT-PCR assays are a feasible option for more efficient COVID-19 coronavirus disease testing as demanded by the continuing pandemic.
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http://dx.doi.org/10.1101/2021.04.10.21254091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8057246PMC
April 2021

Phylogenetic estimates of SARS-CoV-2 introductions into Washington State.

medRxiv 2021 Apr 7. Epub 2021 Apr 7.

Background: The first confirmed case of SARS-CoV-2 in North America was identified in Washington state on January 21, 2020. We aimed to quantify the number and temporal trends of out-of-state introductions of SARS-CoV-2 into Washington.

Methods: We conducted a phylogenetic analysis of 11,422 publicly available whole genome SARS-CoV-2 sequences from GISAID sampled between December 2019 and September 2020. We used maximum parsimony ancestral state reconstruction methods on time-calibrated phylogenies to enumerate introductions/exports, their likely geographic source (e.g. US, non-US, and between eastern and western Washington), and estimated date of introduction. To incorporate phylogenetic uncertainty into our estimates, we conducted 5,000 replicate analyses by generating 25 random time-stratified samples of non-Washington reference sequences, 20 random polytomy resolutions, and 10 random resolutions of the reconstructed ancestral state.

Results: We estimated a minimum 287 separate introductions (median, range 244-320) into Washington and 204 exported lineages (range 188-227) of SARS-CoV-2 out of Washington. Introductions began in mid-January and peaked on March 29, 2020. Lineages with the Spike D614G variant accounted for the majority (88%) of introductions. Overall, 61% (range 55-65%) of introductions into Washington likely originated from a source elsewhere within the US, while the remaining 39% (range 35-45%) likely originated from outside of the US. Intra-state transmission accounted for 65% and 28% of introductions into eastern and western Washington, respectively.

Conclusions: There is phylogenetic evidence that the SARS-CoV-2 epidemic in Washington is continually seeded by a large number of introductions, and that there was significant inter- and intra-state transmission. Due to incomplete sampling our data underestimate the true number of introductions.
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http://dx.doi.org/10.1101/2021.04.05.21254924DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8045029PMC
April 2021

SARS-CoV-2 ORF6 Disrupts Bidirectional Nucleocytoplasmic Transport through Interactions with Rae1 and Nup98.

mBio 2021 04 13;12(2). Epub 2021 Apr 13.

Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA

RNA viruses that replicate in the cytoplasm often disrupt nucleocytoplasmic transport to preferentially translate their own transcripts and prevent host antiviral responses. The accessory protein ORF6 has previously been shown to be a major inhibitor of interferon production in both severe acute respiratory syndrome coronavirus (SARS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we show SARS-CoV-2-infected cells display an elevated level of nuclear mRNA accumulation compared to mock-infected cells. We demonstrate that ORF6 is responsible for this nuclear imprisonment of host mRNA, and using a cotransfected reporter assay, we show this nuclear retention of mRNA blocks expression of newly transcribed mRNAs. ORF6's nuclear entrapment of host mRNA is associated with its ability to copurify with the mRNA export factors, Rae1 and Nup98. These protein-protein interactions map to the C terminus of ORF6 and can be abolished by a single amino acid mutation in Met58. Overexpression of Rae1 restores reporter expression in the presence of SARS-CoV-2 ORF6. SARS-CoV ORF6 also interacts with Rae1 and Nup98. However, SARS-CoV-2 ORF6 more strongly copurifies with Rae1 and Nup98 and results in significantly reduced expression of reporter proteins compared to SARS-CoV ORF6, a potential mechanism for the delayed symptom onset and presymptomatic transmission uniquely associated with the SARS-CoV-2 pandemic. We also show that both SARS-CoV and SARS-CoV-2 ORF6 block nuclear import of a broad range of host proteins. Together, these data support a model in which ORF6 clogs the nuclear pore through its interactions with Rae1 and Nup98 to prevent both nuclear import and export, rendering host cells incapable of responding to SARS-CoV-2 infection. SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), is an RNA virus with a large genome that encodes multiple accessory proteins. While these accessory proteins are not required for growth , they can contribute to the pathogenicity of the virus. We demonstrate that SARS-CoV-2-infected cells accumulate poly(A) mRNA in the nucleus, which is attributed to the accessory protein ORF6. Nuclear entrapment of mRNA and reduced expression of newly transcribed reporter proteins are associated with ORF6's interactions with the mRNA export proteins Rae1 and Nup98. SARS-CoV ORF6 also shows the same interactions with Rae1 and Nup98. However, SARS-CoV-2 ORF6 more strongly represses reporter expression and copurifies with Rae1 and Nup98 compared to SARS-CoV ORF6. Both SARS-CoV ORF6 and SARS-CoV-2 ORF6 block nuclear import of a wide range of host factors through interactions with Rae1 and Nup98. Together, our results suggest ORF6's disruption of nucleocytoplasmic transport prevents infected cells from responding to the invading virus.
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http://dx.doi.org/10.1128/mBio.00065-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092196PMC
April 2021

Endogenously Produced SARS-CoV-2 Specific IgG Antibodies May Have a Limited Impact on Clearing Nasal Shedding of Virus during Primary Infection in Humans.

Viruses 2021 03 20;13(3). Epub 2021 Mar 20.

Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

While SARS-CoV-2 specific neutralizing antibodies have been developed for therapeutic purposes, the specific viral triggers that drive the generation of SARS-CoV-2 specific IgG and IgM antibodies remain only partially characterized. Moreover, it is unknown whether endogenously derived antibodies drive viral clearance that might result in mitigation of clinical severity during natural infection. We developed a series of non-linear mathematical models to investigate whether SARS-CoV-2 viral and antibody kinetics are coupled or governed by separate processes. Patients with severe disease had a higher production rate of IgG but not IgM antibodies. Maximal levels of both isotypes were governed by their production rate rather than different saturation levels between people. Our results suggest that an exponential surge in IgG levels occurs approximately 5-10 days after symptom onset with no requirement for continual antigenic stimulation. SARS-CoV-2 specific IgG antibodies appear to have limited to no effect on viral dynamics but may enhance viral clearance late during primary infection resulting from the binding effect of antibody to virus, rather than neutralization. In conclusion, SARS-CoV-2 specific IgG antibodies may play only a limited role in clearing infection from the nasal passages despite providing long-term immunity against infection following vaccination or prior infection.
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http://dx.doi.org/10.3390/v13030516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8003723PMC
March 2021

Risk factors for seasonal human coronavirus lower respiratory tract infection after hematopoietic cell transplantation.

Blood Adv 2021 04;5(7):1903-1914

Vaccine and Infectious Disease Division and.

Data are limited regarding risk factors for lower respiratory tract infection (LRTI) caused by seasonal human coronaviruses (HCoVs) and the significance of virologic documentation by bronchoalveolar lavage (BAL) on outcomes in hematopoietic cell transplant (HCT) recipients. We retrospectively analyzed patients undergoing allogeneic HCT (4/2008-9/2018) with HCoV (OC43/NL63/HKU1/229E) detected by polymerase chain reaction during conditioning or post-HCT. Risk factors for all manifestations of LRTI and progression to LRTI among those presenting with HCoV upper respiratory tract infection (URTI) were analyzed by logistic regression and Cox proportional hazard models, respectively. Mortality rates following HCoV LRTI were compared according to virologic documentation by BAL. A total of 297 patients (61 children and 236 adults) developed HCoV infection as follows: 254 had URTI alone, 18 presented with LRTI, and 25 progressed from URTI to LRTI (median, 16 days; range, 2-62 days). Multivariable logistic regression analyses showed that male sex, higher immunodeficiency scoring index, albumin <3 g/dL, glucose >150 mg/dL, and presence of respiratory copathogens were associated with occurrence of LRTI. Hyperglycemia with steroid use was associated with progression to LRTI (P < .01) in Cox models. LRTI with HCoV detected in BAL was associated with higher mortality than LRTI without documented detection in BAL (P < .01). In conclusion, we identified factors associated with HCoV LRTI, some of which are less commonly appreciated to be risk factors for LRTI with other respiratory viruses in HCT recipients. The association of hyperglycemia with LRTI might provide an intervention opportunity to reduce the risk of LRTI.
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http://dx.doi.org/10.1182/bloodadvances.2020003865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8015796PMC
April 2021

Hydroxychloroquine with or without azithromycin for treatment of early SARS-CoV-2 infection among high-risk outpatient adults: A randomized clinical trial.

EClinicalMedicine 2021 Mar 27;33:100773. Epub 2021 Feb 27.

Division of Allergy and Infectious Diseases, University of Washington, United States.

Background: Treatment options for outpatients with COVID-19 could reduce morbidity and prevent SARS-CoV-2 transmission.

Methods: In this randomized, double-blind, three-arm (1:1:1) placebo-equivalent controlled trial conducted remotely throughout the United States, adult outpatients with laboratory-confirmed SARS-CoV-2 infection were recruited. Participants were randomly assigned to receive hydroxychloroquine (HCQ) (400 mg BID x1day, followed by 200 mg BID x9days) with or without azithromycin (AZ) (500 mg, then 250 mg daily x4days) or placebo-equivalent (ascorbic acid (HCQ) and folic acid (AZ)), stratified by risk for progression to severe COVID-19 (high-risk vs. low-risk). Self-collected nasal swabs for SARS-CoV-2 PCR, FLUPro symptom surveys, EKGs and vital signs were collected daily. Primary endpoints were: (a) 14-day progression to lower respiratory tract infection (LRTI), 28-day COVID-19 related hospitalization, or death; (b) 14-day time to viral clearance; secondary endpoints included time to symptom resolution (ClinicalTrials.gov: NCT04354428). Due to the low rate of clinical outcomes, the study was terminated for operational futility.

Findings: Between 15th April and 27th July 2020, 231 participants were enrolled and 219 initiated medication a median of 5.9 days after symptom onset. Among 129 high-risk participants, incident LRTI occurred in six (4.7%) participants (two control, four HCQ/AZ) and COVID-19 related hospitalization in seven (5.4%) (four control, one HCQ, two HCQ/AZ); no LRTI and two (2%) hospitalizations occurred in the 102 low-risk participants (one HCQ, one HCQ/AZ). There were no deaths. Among 152 participants with viral shedding at enrollment, median time to clearance was 5 days (95% CI=4-6) in HCQ, 6 days (95% CI=4-8) in HCQ/AZ, and 8 days (95% CI=6-10) in control. Viral clearance was faster in HCQ (HR=1.62, 95% CI=1.01-2.60,  = 0.047) but not HCQ/AZ (HR=1.25,  = 0.39) compared to control. Among 197 participants who met the COVID-19 definition at enrollment, time to symptom resolution did not differ by group (HCQ: HR=1.02, 95% CI-0.63-1.64,  = 0.95, HCQ/AZ: HR=0.91, 95% CI=0.57-1.45,  = 0.70).

Interpretation: Neither HCQ nor HCQ/AZ shortened the clinical course of outpatients with COVID-19, and HCQ, but not HCQ/AZ, had only a modest effect on SARS-CoV-2 viral shedding. HCQ and HCQ/AZ are not effective therapies for outpatient treatment of SARV-CoV-2 infection.

Funding: The COVID-19 Early Treatment Study was funded by the Bill & Melinda Gates Foundation (INV-017062) through the COVID-19 Therapeutics Accelerator. University of Washington Institute of Translational Health Science (ITHS) grant support (UL1 TR002319), KL2 TR002317, and TL1 TR002318 from NCATS/NIH funded REDCap. The content is solely the responsibility of the authors and does not necessarily represent the views, decisions, or policies of the institutions with which they are affiliated. PAN and MJA were supported by the Mayo Clinic Windland Smith Rice Comprehensive Sudden Cardiac Death Program. ClinicalTrials.gov number NCT04354428.
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http://dx.doi.org/10.1016/j.eclinm.2021.100773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7912360PMC
March 2021

Cytomegalovirus-specific T-cell reconstitution following letermovir prophylaxis after hematopoietic cell transplantation.

Blood 2021 07;138(1):34-43

Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA.

Decreased cytomegalovirus (CMV)-specific immunity after hematopoietic cell transplantation (HCT) is associated with late CMV reactivation and increased mortality. Whether letermovir prophylaxis-associated reduction in viral exposure influences CMV-specific immune reconstitution is unknown. In a prospective cohort of allogeneic HCT recipients who received letermovir, we compared polyfunctional CMV-specific T-cell responses to those of controls who received PCR-guided preemptive therapy before the introduction of letermovir. Thirteen-color flow cytometry was used to assess T-cell responses at 3 months after HCT following stimulation with CMV immediate early-1 (IE-1) antigen and phosphoprotein 65 (pp65) antigens. Polyfunctionality was characterized by combinatorial polyfunctionality analysis of antigen-specific T-cell subsets. Use of letermovir and reduction of viral exposure were assessed for their association with CMV-specific T-cell immunity. Polyfunctional T-cell responses to IE-1 and pp65 were decreased in letermovir recipients and remained diminished after adjustment for donor CMV serostatus, absolute lymphocyte count, and steroid use. Among letermovir recipients, greater peak CMV DNAemia and increased viral shedding were associated with stronger CD8+ responses to pp65, whereas the CMV shedding rate was associated with greater CD4+ responses to IE-1. In summary, our study provided initial evidence that letermovir may delay CMV-specific cellular reconstitution, possibly related to decreased CMV antigen exposure. Evaluating T-cell polyfunctionality may identify patients at risk for late CMV infection after HCT.
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http://dx.doi.org/10.1182/blood.2020009396DOI Listing
July 2021

Hospitalization and mortality associated with SARS-CoV-2 viral clades in COVID-19.

Sci Rep 2021 02 26;11(1):4802. Epub 2021 Feb 26.

Department of Ophthalmology, University of Washington School of Medicine, 325 9th Avenue, Campus Box 359608, Seattle, WA, 98104, USA.

The COVID-19 epidemic of 2019-20 is due to the novel coronavirus SARS-CoV-2. Following first case description in December, 2019 this virus has infected over 10 million individuals and resulted in at least 500,000 deaths world-wide. The virus is undergoing rapid mutation, with two major clades of sequence variants emerging. This study sought to determine whether SARS-CoV-2 sequence variants are associated with differing outcomes among COVID-19 patients in a single medical system. Whole genome SARS-CoV-2 RNA sequence was obtained from isolates collected from patients registered in the University of Washington Medicine health system between March 1 and April 15, 2020. Demographic and baseline clinical characteristics of patients and their outcome data including their hospitalization and death were collected. Statistical and machine learning models were applied to determine if viral genetic variants were associated with specific outcomes of hospitalization or death. Full length SARS-CoV-2 sequence was obtained 190 subjects with clinical outcome data. 35 (18.4%) were hospitalized and 14 (7.4%) died from complications of infection. A total of 289 single nucleotide variants were identified. Clustering methods demonstrated two major viral clades, which could be readily distinguished by 12 polymorphisms in 5 genes. A trend toward higher rates of hospitalization of patients with Clade 2 infections was observed (p = 0.06, Fisher's exact). Machine learning models utilizing patient demographics and co-morbidities achieved area-under-the-curve (AUC) values of 0.93 for predicting hospitalization. Addition of viral clade or sequence information did not significantly improve models for outcome prediction. In summary, SARS-CoV-2 shows substantial sequence diversity in a community-based sample. Two dominant clades of virus are in circulation. Among patients sufficiently ill to warrant testing for virus, no significant difference in outcomes of hospitalization or death could be discerned between clades in this sample. Major risk factors for hospitalization and death for either major clade of virus include patient age and comorbid conditions.
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http://dx.doi.org/10.1038/s41598-021-82850-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7910290PMC
February 2021

In silico detection of SARS-CoV-2 specific B-cell epitopes and validation in ELISA for serological diagnosis of COVID-19.

Sci Rep 2021 02 22;11(1):4290. Epub 2021 Feb 22.

Seattle Structural Genomics Center for Infectious Disease (SSGCID), Seattle, WA, USA.

Rapid generation of diagnostics is paramount to understand epidemiology and to control the spread of emerging infectious diseases such as COVID-19. Computational methods to predict serodiagnostic epitopes that are specific for the pathogen could help accelerate the development of new diagnostics. A systematic survey of 27 SARS-CoV-2 proteins was conducted to assess whether existing B-cell epitope prediction methods, combined with comprehensive mining of sequence databases and structural data, could predict whether a particular protein would be suitable for serodiagnosis. Nine of the predictions were validated with recombinant SARS-CoV-2 proteins in the ELISA format using plasma and sera from patients with SARS-CoV-2 infection, and a further 11 predictions were compared to the recent literature. Results appeared to be in agreement with 12 of the predictions, in disagreement with 3, while a further 5 were deemed inconclusive. We showed that two of our top five candidates, the N-terminal fragment of the nucleoprotein and the receptor-binding domain of the spike protein, have the highest sensitivity and specificity and signal-to-noise ratio for detecting COVID-19 sera/plasma by ELISA. Mixing the two antigens together for coating ELISA plates led to a sensitivity of 94% (N = 80 samples from persons with RT-PCR confirmed SARS-CoV-2 infection), and a specificity of 97.2% (N = 106 control samples).
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http://dx.doi.org/10.1038/s41598-021-83730-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900118PMC
February 2021

Association of HHV-6 With Outcomes in CMV-Seronegative Liver Transplant Recipients With CMV-Seropositive Donors Receiving Preemptive Antiviral Therapy.

Transplantation 2020 Dec 29. Epub 2020 Dec 29.

University of Pittsburgh, and VA Pittsburgh Healthcare System, Pittsburgh, PA University of California Los Angeles Medical Center, Los Angeles, CA Mayo Clinic, Rochester, MN Emory University, Atlanta, GA University of Pittsburgh and University of Pittsburgh Medical Center, Pittsburgh PA Fred Hutchinson Cancer Research Center, Seattle, WA University of Washington, Seattle, WA.

Background: Risk factors, virologic parameters and outcomes associated with HHV-6 viremia in high-risk donor CMV-seropositive and recipient CMV-seronegative (D+R-) liver transplant recipients in the current era are incompletely defined.

Methods: The study population consisted of patients in the preemptive therapy (PET) arm of a randomized, controlled trial of PET versus valganciclovir prophylaxis for CMV prevention in D+R- liver transplant recipients. Weekly blood samples through 100 days in the PET group were tested for HHV-6 viremia using a real-time quantitative PCR. Assessments included virologic characteristics and relationship with CMV, risk factors and impact of HHV-6 viremia with outcomes through 12 months post-transplant.

Results: HHV-6 viremia at any level developed in 42% (40/96). Older patient age (p=0.03), longer hospitalization (p=0.015), and ICU stay at transplantation (p=0.029) were significantly associated with high-grade viremia. Concurrent HHV-6 and CMV viremia was associated with earlier onset of HHV-6 viremia (p=0.004), higher HHV-6 AUC (p=0.043), and higher peak HHV-6 viral load (p=0.006) vs HHV-6 viremia alone. High-grade viremia was independently associated with biopsy-proven rejection within 12 months (p=0.045) post-transplant.

Conclusions: Among D+R- liver transplant recipients receiving valganciclovir as PET, high-grade HHV-6 viremia was associated with increased age and critical-illness in ICU at time of transplant and was independently associated with allograft rejection.
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http://dx.doi.org/10.1097/TP.0000000000003604DOI Listing
December 2020

Occurrence and Timing of Subsequent Severe Acute Respiratory Syndrome Coronavirus 2 Reverse-transcription Polymerase Chain Reaction Positivity Among Initially Negative Patients.

Clin Infect Dis 2021 01;72(2):323-326

Department of Anesthesiology and Pain Medicine, University of Washington School of Medicine, Seattle, Washington, USA.

Using data for 20 912 patients from 2 large academic health systems, we analyzed the frequency of severe acute respiratory syndrome coronavirus 2 reverse-transcription polymerase chain reaction test discordance among individuals initially testing negative by nasopharyngeal swab who were retested on clinical grounds within 7 days. The frequency of subsequent positivity within this window was 3.5% and was similar across institutions.
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http://dx.doi.org/10.1093/cid/ciaa722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7314163PMC
January 2021

CRISPR-Cas9 gene editing of hepatitis B virus in chronically infected humanized mice.

Mol Ther Methods Clin Dev 2021 Mar 26;20:258-275. Epub 2020 Nov 26.

Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

Chronic hepatitis B virus (HBV) infection is a major public health problem. New treatment approaches are needed because current treatments do not target covalently closed circular DNA (cccDNA), the template for HBV replication, and rarely clear the virus. We harnessed adeno-associated virus (AAV) vectors and CRISPR- ()Cas9 to edit the HBV genome in liver-humanized FRG mice chronically infected with HBV and receiving entecavir. Gene editing was detected in livers of five of eight HBV-specific AAV-Cas9-treated mice, but not control mice, and mice with detectable HBV gene editing showed higher levels of Cas9 delivery to HBV human hepatocytes than those without gene editing. HBV-specific AAV-Cas9 therapy significantly improved survival of human hepatocytes, showed a trend toward decreasing total liver HBV DNA and cccDNA, and was well tolerated. This work provides evidence for the feasibility and safety of gene editing for chronic HBV infections, and it suggests that with further optimization, this approach may offer a plausible way to treat or even cure chronic HBV infections.
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http://dx.doi.org/10.1016/j.omtm.2020.11.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7803634PMC
March 2021

SARS-CoV-2 Viral Load on Admission Is Associated With 30-Day Mortality.

Open Forum Infect Dis 2020 Dec 3;7(12):ofaa535. Epub 2020 Nov 3.

Department of Laboratory Medicine and Pathology, University of Washington School of Medicine, Seattle, Washington, USA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load on admission was associated with a significantly increased 30-day mortality (odds ratio [OR], 4.20; 95% CI, 1.62-10.86), and anti-SARS-CoV-2 nucleocapisid IgG seropositivity on admission trended toward a reduced 30-day mortality (OR, 0.43; 95% CI, 0.15-1.26). Reporting of quantitative SARS-CoV-2 viral load and serologic assays may offer prognostic clinical information.
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http://dx.doi.org/10.1093/ofid/ofaa535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7665729PMC
December 2020

Evaluation of SARS-CoV-2 neutralization assays for antibody monitoring in natural infection and vaccine trials.

medRxiv 2020 Dec 8. Epub 2020 Dec 8.

Determinants of protective immunity against SARS-CoV-2 infection require the development of well-standardized, reproducible antibody assays to be utilized in concert with clinical trials to establish correlates of risk and protection. This need has led to the appearance of a variety of neutralization assays used by different laboratories and companies. Using plasma samples from COVID-19 convalescent individuals with mild-to-moderate disease from a localized outbreak in a single region of the western US, we compared three platforms for SARS-CoV-2 neutralization: assay with live SARS-CoV-2, pseudovirus assay utilizing lentiviral (LV) and vesicular stomatitis virus (VSV) packaging, and a surrogate ELISA test. Vero, Vero E6, HEK293T cells expressing human angiotensin converting enzyme 2 (hACE2), and TZM-bl cells expressing hACE2 and transmembrane serine protease 2 (TMPRSS2) were evaluated. Live-virus and LV-pseudovirus assay with HEK293T cells showed similar geometric mean titers (GMTs) ranging 141-178, but VSV-pseudovirus assay yielded significantly higher GMT (310 95%CI 211-454; p < 0.001). Fifty percent neutralizing dilution (ND50) titers from live-virus and all pseudovirus assay readouts were highly correlated (Pearson = 0.81-0.89). ND50 titers positively correlated with plasma concentration of IgG against SARS-CoV-2 spike and receptor binding domain (RBD) ( = 0.63-0.89), but moderately correlated with nucleoprotein IgG ( = 0.46-0.73). There was a moderate positive correlation between age and spike (Spearman's rho=0.37, p=0.02), RBD (rho=0.39, p=0.013) and nucleoprotein IgG (rho=0.45, p=0.003). ND80 showed stronger correlation with age than ND50 (ND80 rho=0.51 (p=0.001), ND50 rho=0.28 (p=0.075)). Our data demonstrate high concordance between cell-based assays with live and pseudotyped virions.
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http://dx.doi.org/10.1101/2020.12.07.20245431DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7743084PMC
December 2020

Clinical, laboratory, and temporal predictors of neutralizing antibodies against SARS-CoV-2 among COVID-19 convalescent plasma donor candidates.

J Clin Invest 2021 02;131(3)

Department of Medicine, University of Washington, Seattle, Washington, USA.

BACKGROUNDSARS-CoV-2-specific antibodies may protect from reinfection and disease, providing rationale for administration of plasma containing SARS-CoV-2-neutralizing antibodies (nAbs) as a treatment for COVID-19. Clinical factors and laboratory assays to streamline plasma donor selection, and the durability of nAb responses, are incompletely understood.METHODSPotential convalescent plasma donors with virologically documented SARS-CoV-2 infection were tested for serum IgG against SARS-CoV-2 spike protein S1 domain and against nucleoprotein (NP), and for nAb.RESULTSAmong 250 consecutive persons, including 27 (11%) requiring hospitalization, who were studied a median of 67 days since symptom onset, 97% were seropositive on 1 or more assays. Sixty percent of donors had nAb titers ≥1:80. Correlates of higher nAb titers included older age (adjusted OR [AOR] 1.03 per year of age, 95% CI 1.00-1.06), male sex (AOR 2.08, 95% CI 1.13-3.82), fever during illness (AOR 2.73, 95% CI 1.25-5.97), and disease severity represented by hospitalization (AOR 6.59, 95% CI 1.32-32.96). Receiver operating characteristic analyses of anti-S1 and anti-NP antibody results yielded cutoffs that corresponded well with nAb titers, with the anti-S1 assay being slightly more predictive. nAb titers declined in 37 of 41 paired specimens collected a median of 98 days (range 77-120) apart (P < 0.001). Seven individuals (2.8%) were persistently seronegative and lacked T cell responses.CONCLUSIONnAb titers correlated with COVID-19 severity, age, and sex. SARS-CoV-2 IgG results can serve as useful surrogates for nAb testing. Functional nAb levels declined, and a small proportion of convalescent individuals lacked adaptive immune responses.FUNDINGThe project was supported by the Frederick National Laboratory for Cancer Research with support from the NIAID under contract number 75N91019D00024, and was supported by the Fred Hutchinson Joel Meyers Endowment, Fast-Grants, a New Investigator award from the American Society for Transplantation and Cellular Therapy, and NIH contracts 75N93019C0063, 75N91019D00024, and HHSN272201800013C, and NIH grants T32-AI118690, T32-AI007044, K08-AI119142, and K23-AI140918.
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http://dx.doi.org/10.1172/JCI144930DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7843229PMC
February 2021

Detection of parvovirus B19 and human herpesvirus 6 in pediatric dilated cardiomyopathy: Impact after heart transplantation.

Ann Pediatr Cardiol 2020 Oct-Dec;13(4):301-308. Epub 2020 Sep 1.

Department of Laboratory Medicine, Division of Virology, University of Washington, Seattle, WA, USA.

Objectives: The aim of this study is to evaluate HHV-6 and PVB19 infection using polymerase chain reaction (PCR) and immunofluorescent assay (IFA) in the myocardium of pediatric patients with dilated cardiomyopathy (DCM) and the impact of viral persistence in the cardiac allograft after heart transplantation (HT).

Methods: Multiplex droplet digital PCR was used to analyze the prevalence of viral sequences in myocardial samples from 48 pediatric DCM patients and 10 control subjects. Of the 48 DCM patients, 44 underwent HT. After HT, consecutive endomyocardial biopsy (EMB) samples were analyzed for the presence of PVB19 and HHV-6 antigens using IFA and the patients were evaluated for rejections, coronary vasculopathy, and graft loss.

Results: Of the 48 DCM patients, 14 had positive viral PCR results in explanted/autopsy hearts. Among them, PVB19 was found in 8/48, HHV6 in 4/48, both PVB19 and HHV6 in 1/48, and enterovirus in one, but no adenovirus was found. The EMB samples obtained after HT were positive for PVB19 and HHV-6 in 7/44 and 3/44 cases, respectively. Viral presence in both the explanted heart and the cardiac allograft was demonstrated in 4 patients, 3 of whom were positive for PVB19, and one of whom was positive for HHV-6 pretransplant. Coronary vasculopathy and graft loss were more common in patients with PVB19-positive myocardial tissues versus those who were PVB19-negative.

Conclusions: There is an association between PVB19 and HHV-6 infection and DCM in children. The study suggests the persistence of PVB19 and HHV-6 in the host can lead to subsequent viral reactivation in the transplanted heart, even in those recipients who do not have active myocarditis. PVB19 in the cardiac allograft tended toward higher adverse post-HT events.
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http://dx.doi.org/10.4103/apc.APC_124_19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7727911PMC
September 2020

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

J Clin Microbiol 2021 02 18;59(3). Epub 2021 Feb 18.

Department of Laboratory Medicine and Pathology, Virology Division, University of Washington, Seattle, Washington, USA

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). Although less sensitive than molecular detection methods, rapid antigen testing offers the potential for inexpensive, quick, decentralized testing. Robust analytical sensitivity data in comparison to reverse transcription-quantitative PCR (qRT-PCR) are currently lacking for many rapid antigen tests. Here, we evaluated the analytical sensitivity of the Abbott BinaxNOW COVID-19 Ag card using SARS-CoV-2-positive clinical specimens quantified by reverse transcription-droplet digital PCR (RT-ddPCR) and multiple FDA EUA qRT-PCR platforms using RNA standards. Initial and confirmatory limits of detection for the BinaxNOW COVID-19 Ag card were determined to be equivalent to 4.04 × 10 to 8.06 × 10 copies/swab. We further confirmed this limit of detection with 72 additional clinical samples positive for SARS-CoV-2 in either phosphate-buffered saline or viral transport medium. One hundred percent of samples with viral loads of >40,000 copies/swab were detected by rapid antigen testing. These data indicate that the BinaxNOW COVID-19 Ag card has an analytical sensitivity approximately equivalent to a generic qRT-PCR cycle threshold ( ) value of 29 to 30.
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http://dx.doi.org/10.1128/JCM.02880-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8106729PMC
February 2021

Hydroxychloroquine as Postexposure Prophylaxis to Prevent Severe Acute Respiratory Syndrome Coronavirus 2 Infection : A Randomized Trial.

Ann Intern Med 2021 03 8;174(3):344-352. Epub 2020 Dec 8.

University of Washington, Seattle, Washington (H.C.S., T.T.S., M.L.K., K.K.T., S.M., H.S.H., L.K., M.W., C.C., H.Y.C., J.M.B.).

Background: Effective prevention against coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently limited to nonpharmaceutical strategies. Laboratory and observational data suggested that hydroxychloroquine had biological activity against SARS-CoV-2, potentially permitting its use for prevention.

Objective: To test hydroxychloroquine as postexposure prophylaxis for SARS-CoV-2 infection.

Design: Household-randomized, double-blind, controlled trial of hydroxychloroquine postexposure prophylaxis. (ClinicalTrials.gov: NCT04328961).

Setting: National U.S. multicenter study.

Participants: Close contacts recently exposed (<96 hours) to persons with diagnosed SARS-CoV-2 infection.

Intervention: Hydroxychloroquine (400 mg/d for 3 days followed by 200 mg/d for 11 days) or ascorbic acid (500 mg/d followed by 250 mg/d) as a placebo-equivalent control.

Measurements: Participants self-collected mid-turbinate swabs daily (days 1 to 14) for SARS-CoV-2 polymerase chain reaction (PCR) testing. The primary outcome was PCR-confirmed incident SARS-CoV-2 infection among persons who were SARS-CoV-2 negative at enrollment.

Results: Between March and August 2020, 671 households were randomly assigned: 337 (407 participants) to the hydroxychloroquine group and 334 (422 participants) to the control group. Retention at day 14 was 91%, and 10 724 of 11 606 (92%) expected swabs were tested. Among the 689 (89%) participants who were SARS-CoV-2 negative at baseline, there was no difference between the hydroxychloroquine and control groups in SARS-CoV-2 acquisition by day 14 (53 versus 45 events; adjusted hazard ratio, 1.10 [95% CI, 0.73 to 1.66];  > 0.20). The frequency of participants experiencing adverse events was higher in the hydroxychloroquine group than the control group (66 [16.2%] versus 46 [10.9%], respectively;  = 0.026).

Limitation: The delay between exposure, and then baseline testing and the first dose of hydroxychloroquine or ascorbic acid, was a median of 2 days.

Conclusion: This rigorous randomized controlled trial among persons with recent exposure excluded a clinically meaningful effect of hydroxychloroquine as postexposure prophylaxis to prevent SARS-CoV-2 infection.

Primary Funding Source: Bill & Melinda Gates Foundation.
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http://dx.doi.org/10.7326/M20-6519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7732017PMC
March 2021

Human Metapneumovirus Infection and Genotyping of Infants in Rural Nepal.

J Pediatric Infect Dis Soc 2021 Apr;10(4):408-416

Department of Laboratory Medicine & Pathology, University of Washington, Seattle, Washington, USA.

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. Sequencing glycoprotein genes permitted genotyping and analyses among subtypes.

Results: HMPV was detected by reverse-transcriptase polymerase chain reaction (RT-PCR) in 187 (5%) infants, with seasonality observed during fall and winter months. Phylogenetic investigation of complete and partial coding sequences for the F and G genes, respectively, revealed that 3 genotypes were circulating: A2, B1, and B2. HMPV-B was most frequently detected with a single type predominating each season. Both HMPV genotypes exhibited comparable median viral loads. Clinically significant differences between genotypes were limited to increased cough duration and general respiratory symptoms for type B.

Conclusions: In rural Nepal, multiple HMPV genotypes circulate simultaneously with an alternating predominance of a single genotype and definitive seasonality. No difference in viral load was detected by genotype and symptom severity was not correlated with RT-PCR cycle threshold or genotype.
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http://dx.doi.org/10.1093/jpids/piaa118DOI Listing
April 2021

Incidence of Health Care-Associated COVID-19 During Universal Testing of Medical and Surgical Admissions in a Large US Health System.

Open Forum Infect Dis 2020 Oct 14;7(10):ofaa435. Epub 2020 Sep 14.

University of Washington School of Medicine, Department of Anesthesiology & Pain Medicine, Seattle, Washington, USA.

Concerns about severe acute respiratory syndrome coronavirus 2 exposure in health care settings may cause patients to delay care. Among 2992 patients testing negative on admission to an academic, 3-hospital system, 8 tested positive during hospitalization or within 14 days postdischarge. Following adjudication of each instance, health care-associated infection incidence ranged from 0.8 to 5.0 cases per 10 000 patient-days.
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http://dx.doi.org/10.1093/ofid/ofaa435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7543563PMC
October 2020
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