Viral genomic variation and the severity of genital HSV-2 infection as quantified by shedding rate: a viral genome-wide association study.

BACKGROUND: The clinical severity of genital HSV-2 infection varies widely among infected persons with some experiencing frequent genital lesions while others are asymptomatic. The viral genital shedding rate is closely associated with and has been established as a surrogate marker of clinical severity. METHODS: To assess the relationship between viral genetics and shedding, we assembled a set of 145 persons who had the severity of their genital herpes quantified through determination of their HSV genital shedding rate. An HSV-2 sample from each person was sequenced and biallelic variants among these genomes were identified. RESULTS: We found no association between metrics of genome-wide variation in HSV-2 and shedding rate. A viral genome-wide association study (vGWAS) identified the minor alleles of three individual unlinked variants as significantly associated with higher shedding rate (p<8.4x10-5): C44973T (A512T), a non-synonymous variant in UL22 (glycoprotein H); A74534G, a synonymous variant in UL36 (large tegument protein); and T119283C, an intergenic variant. We also found an association between the total number of minor alleles for the significant variants and shedding rate (p=6.6x10-7). CONCLUSIONS: These results add to a growing body of literature for HSV suggesting a connection between viral genetic variation and clinically important phenotypes of infection.

Remote surveillance and detection of SARS-CoV-2 transmission among household members in King County, Washington.

BACKGROUND: Early during the COVID-19 pandemic, it was important to better understand transmission dynamics of SARS-CoV-2, the virus that causes COVID-19. Household contacts of infected individuals are particularly at risk for infection, but delays in contact tracing, delays in testing contacts, and isolation and quarantine posed challenges to accurately capturing secondary household cases. METHODS: In this study, 346 households in the Seattle region were provided with respiratory specimen collection kits and remotely monitored using web-based surveys for respiratory illness symptoms weekly between October 1, 2020, and June 20, 2021. Symptomatic participants collected respiratory specimens at symptom onset and mailed specimens to the central laboratory in Seattle. Specimens were tested for SARS-CoV-2 using RT-PCR with whole genome sequencing attempted when positive. SARS-CoV-2-infected individuals were notified, and their household contacts submitted specimens every 2 days for 14 days. RESULTS: In total, 1371 participants collected 2029 specimens that were tested; 16 individuals (1.2%) within 6 households tested positive for SARS-CoV-2 during the study period. Full genome sequences were generated from 11 individuals within 4 households. Very little genetic variation was found among SARS-CoV-2 viruses sequenced from different individuals in the same household, supporting transmission within the household. CONCLUSIONS: This study indicates web-based surveillance of respiratory symptoms, combined with rapid and longitudinal specimen collection and remote contact tracing, provides a viable strategy to monitor households and detect household transmission of SARS-CoV-2. TRIAL REGISTRATION IDENTIFIER: NCT04141930, Date of registration 28/10/2019.

CASCADIA: a prospective community-based study protocol for assessing SARS-CoV-2 vaccine effectiveness in children and adults using a remote nasal swab collection and web-based survey design.

INTRODUCTION: Although SARS-CoV-2 vaccines were first approved under Emergency Use Authorization by the Food and Drug Administration in late 2020 for adults, authorisation for young children 6 months to <5 years of age did not occur until 2022. These authorisations were based on clinical trials, understanding real-world vaccine effectiveness (VE) in the setting of emerging variants is critical. The primary goal of this study is to evaluate SARS-CoV-2 VE against infection among children aged >6 months and adults aged <50 years. METHODS: CASCADIA is a 4-year community-based prospective study of SARS-CoV-2 VE among 3500 adults and paediatric populations aged 6 months to 49 years in Oregon and Washington, USA. At enrolment and regular intervals, participants complete a sociodemographic questionnaire. Individuals provide a blood sample at enrolment and annually thereafter, with optional blood draws every 6 months and after infection and vaccination. Participants complete weekly self-collection of anterior nasal swabs and symptom questionnaires. Swabs are tested for SARS-CoV-2 and other respiratory pathogens by reverse transcription-PCR, with results of selected pathogens returned to participants; nasal swabs with SARS-CoV-2 detected will undergo whole genome sequencing. Participants who test positive for SARS-CoV-2 undergo serial swab collection every 3 days for 21 days. Serum samples are tested for SARS-CoV-2 antibody by binding and neutralisation assays. ANALYSIS: The primary outcome is SARS-CoV-2 infection. Cox regression models will be used to estimate the incidence rate ratio associated with SARS-CoV-2 vaccination among the paediatric and adult population, controlling for demographic factors and other potential confounders. ETHICS AND DISSEMINATION: All study materials including the protocol, consent forms, data collection instruments, participant communication and recruitment materials, were approved by the Kaiser Permanente Interregional Institutional Review Board, the IRB of record for the study. Results will be disseminated through peer-reviewed publications, presentations, participant newsletters and appropriate general news media.

Results from a test-and-treat study for influenza among residents of homeless shelters in King County, WA: A stepped-wedge cluster-randomized trial.

BACKGROUND: Persons experiencing homelessness face increased risk of influenza as overcrowding in congregate shelters can facilitate influenza virus spread. Data regarding on-site influenza testing and antiviral treatment within homeless shelters remain limited. METHODS: We conducted a cluster-randomized stepped-wedge trial of point-of-care molecular influenza testing coupled with antiviral treatment with baloxavir or oseltamivir in residents of 14 homeless shelters in Seattle, WA, USA. Residents ≥3 months with cough or ≥2 acute respiratory illness (ARI) symptoms and onset <7 days were eligible. In control periods, mid-nasal swabs were tested for influenza by reverse transcription polymerase chain reaction (RT-PCR). The intervention period included on-site rapid molecular influenza testing and antiviral treatment for influenza-positives if symptom onset was <48 h. The primary endpoint was monthly influenza virus infections in the control versus intervention periods. Influenza whole genome sequencing was performed to assess transmission and antiviral resistance. RESULTS: During 11/15/2019-4/30/2020 and 11/2/2020-4/30/2021, 1283 ARI encounters from 668 participants were observed. Influenza virus was detected in 51 (4%) specimens using RT-PCR (A = 14; B = 37); 21 influenza virus infections were detected from 269 (8%) intervention-eligible encounters by rapid molecular testing and received antiviral treatment. Thirty-seven percent of ARI-participant encounters reported symptom onset < 48 h. The intervention had no effect on influenza virus transmission (adjusted relative risk 1.73, 95% confidence interval [CI] 0.50-6.00). Of 23 influenza genomes, 86% of A(H1N1)pdm09 and 81% of B/Victoria sequences were closely related. CONCLUSION: Our findings suggest feasibility of influenza test-and-treat strategies in shelters. Additional studies would help discern an intervention effect during periods of increased influenza activity.

Genomic surveillance of SARS-CoV-2 Omicron variants on a university campus.

Novel variants continue to emerge in the SARS-CoV-2 pandemic. University testing programs may provide timely epidemiologic and genomic surveillance data to inform public health responses. We conducted testing from September 2021 to February 2022 in a university population under vaccination and indoor mask mandates. A total of 3,048 of 24,393 individuals tested positive for SARS-CoV-2 by RT-PCR; whole genome sequencing identified 209 Delta and 1,730 Omicron genomes of the 1,939 total sequenced. Compared to Delta, Omicron had a shorter median serial interval between genetically identical, symptomatic infections within households (2 versus 6 days, P = 0.021). Omicron also demonstrated a greater peak reproductive number (2.4 versus 1.8), and a 1.07 (95% confidence interval: 0.58, 1.57; P < 0.0001) higher mean cycle threshold value. Despite near universal vaccination and stringent mitigation measures, Omicron rapidly displaced the Delta variant to become the predominant viral strain and led to a surge in cases in a university population.

Human Parainfluenza Virus in Homeless Shelters before and during the COVID-19 Pandemic, Washington, USA.

To determine the epidemiology of human parainfluenza virus in homeless shelters during the COVID-19 pandemic, we analyzed data and sequences from respiratory specimens collected in 23 shelters in Washington, USA, during 2019-2021. Two clusters in children were genetically similar by shelter of origin. Shelter-specific interventions are needed to reduce these infections.

The clinical and genomic epidemiology of seasonal human coronaviruses in congregate homeless shelter settings: A repeated cross-sectional study.

Background: The circulation of respiratory viruses poses a significant health risk among those residing in congregate settings. Data are limited on seasonal human coronavirus (HCoV) infections in homeless shelter settings. Methods: We analysed data from a clinical trial and SARS-CoV-2 surveillance study at 23 homeless shelter sites in King County, Washington between October 2019-May 2021. Eligible participants were shelter residents aged ≥3 months with acute respiratory illness. We collected enrolment data and nasal samples for respiratory virus testing using multiplex RT-PCR platform including HCoV. Beginning April 1, 2020, eligibility expanded to shelter residents and staff regardless of symptoms. HCoV species was determined by RT-PCR with species-specific primers, OpenArray assay or genomic sequencing for samples with an OpenArray relative cycle threshold <22. Findings: Of the 14,464 samples from 3281 participants between October 2019-May 2021, 107 were positive for HCoV from 90 participants (median age 40 years, range: 0·9-81 years, 38% female). HCoV-HKU1 was the most common species identified before and after community-wide mitigation. No HCoV-positive samples were identified between May 2020-December 2020. Adults aged ≥50 years had the highest detection of HCoV (11%) among virus-positive samples among all age-groups. Species and sequence data showed diversity between and within HCoV species over the study period. Interpretation: HCoV infections occurred in all congregate homeless shelter site age-groups with the greatest proportion among those aged ≥50 years. Species and sequencing data highlight the complexity of HCoV epidemiology within and between shelters sites. Funding: Gates Ventures, Centers for Disease Control and Prevention, National Institute of Health.

The Clinical and Genomic Epidemiology of Rhinovirus in Homeless Shelters-King County, Washington.

BACKGROUND: Rhinovirus (RV) is a common cause of respiratory illness in all people, including those experiencing homelessness. RV epidemiology in homeless shelters is unknown. METHODS: We analyzed data from a cross-sectional homeless shelter study in King County, Washington, October 2019-May 2021. Shelter residents or guardians aged ≥3 months reporting acute respiratory illness completed questionnaires and submitted nasal swabs. After 1 April 2020, enrollment expanded to residents and staff regardless of symptoms. Samples were tested by multiplex RT-PCR for respiratory viruses. A subset of RV-positive samples was sequenced. RESULTS: There were 1066 RV-positive samples with RV present every month of the study period. RV was the most common virus before and during the coronavirus disease 2019 (COVID-19) pandemic (43% and 77% of virus-positive samples, respectively). Participants from family shelters had the highest prevalence of RV. Among 131 sequenced samples, 33 RV serotypes were identified with each serotype detected for ≤4 months. CONCLUSIONS: RV infections persisted through community mitigation measures and were most prevalent in shelters housing families. Sequencing showed a diversity of circulating RV serotypes, each detected over short periods of time. Community-based surveillance in congregate settings is important to characterize respiratory viral infections during and after the COVID-19 pandemic. CLINICAL TRIALS REGISTRATION: NCT04141917.

Host-pathogen dynamics in longitudinal clinical specimens from patients with COVID-19.

Rapid dissemination of SARS-CoV-2 sequencing data to public repositories has enabled widespread study of viral genomes, but studies of longitudinal specimens from infected persons are relatively limited. Analysis of longitudinal specimens enables understanding of how host immune pressures drive viral evolution in vivo. Here we performed sequencing of 49 longitudinal SARS-CoV-2-positive samples from 20 patients in Washington State collected between March and September of 2020. Viral loads declined over time with an average increase in RT-QPCR cycle threshold of 0.87 per day. We found that there was negligible change in SARS-CoV-2 consensus sequences over time, but identified a number of nonsynonymous variants at low frequencies across the genome. We observed enrichment for a relatively small number of these variants, all of which are now seen in consensus genomes across the globe at low prevalence. In one patient, we saw rapid emergence of various low-level deletion variants at the N-terminal domain of the spike glycoprotein, some of which have previously been shown to be associated with reduced neutralization potency from sera. In a subset of samples that were sequenced using metagenomic methods, differential gene expression analysis showed a downregulation of cytoskeletal genes that was consistent with a loss of ciliated epithelium during infection and recovery. We also identified co-occurrence of bacterial species in samples from multiple hospitalized individuals. These results demonstrate that the intrahost genetic composition of SARS-CoV-2 is dynamic during the course of COVID-19, and highlight the need for continued surveillance and deep sequencing of minor variants.

Phylogenomics of Severe Acute Respiratory Syndrome Coronavirus 2 in Emergency Shelters for People Experiencing Homelessness.

BACKGROUND: Residents and staff of emergency shelters for people experiencing homelessness (PEH) are at high risk of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The importance of shelter-related transmission of SARS-CoV-2 in this population remains unclear. It is also unknown whether there is significant spread of shelter-related viruses into surrounding communities. METHODS: We analyzed genome sequence data for 28 SARS-CoV-2-positive specimens collected from 8 shelters in King County, Washington between March and October, 2020. RESULTS: We identified at least 12 separate SARS-CoV-2 introduction events into these 8 shelters and estimated that 57% (16 of 28) of the examined cases of SARS-CoV-2 infection were the result of intrashelter transmission. However, we identified just a few SARS-CoV-2 specimens from Washington that were possible descendants of shelter viruses. CONCLUSIONS: Our data suggest that SARS-CoV-2 spread in shelters is common, but we did not observe evidence of widespread transmission of shelter-related viruses into the general population.

Diagnosis of infectious diseases in immunocompromised hosts using metagenomic next generation sequencing-based diagnostics.

The diagnosis of infectious diseases in immunocompromised hosts presents unique challenges for the clinician. Metagenomic next generation sequencing (mNGS) based diagnostics that identify microbial nucleic acids in clinical samples (mNGS for pathogen identification or mNGSpi) may be a useful tool in addressing some of these challenges. Studies of mNGSpi in immunocompromised hosts have demonstrated that these diagnostics are capable of identifying causative organisms in a subset of patients for whom conventional testing has been negative. While these studies provide proof of concept for mNGSpi utility, they have a number of limitations, which make it difficult to confidently assess test performance and clinical impact based on current data. Future studies will likely feature larger cohort sizes and controlled interventional study designs that assess the impact of mNGSpi on clinical endpoints. They will also likely include assessments of the clinical value of data generated by mNGS beyond pathogen identification.

Treponema pallidum genome sequencing from six continents reveals variability in vaccine candidate genes and dominance of Nichols clade strains in Madagascar.

In spite of its immutable susceptibility to penicillin, Treponema pallidum (T. pallidum) subsp. pallidum continues to cause millions of cases of syphilis each year worldwide, resulting in significant morbidity and mortality and underscoring the urgency of developing an effective vaccine to curtail the spread of the infection. Several technical challenges, including absence of an in vitro culture system until very recently, have hampered efforts to catalog the diversity of strains collected worldwide. Here, we provide near-complete genomes from 196 T. pallidum strains-including 191 T. pallidum subsp. pallidum-sequenced directly from patient samples collected from 8 countries and 6 continents. Maximum likelihood phylogeny revealed that samples from most sites were predominantly SS14 clade. However, 99% (84/85) of the samples from Madagascar formed two of the five distinct Nichols subclades. Although recombination was uncommon in the evolution of modern circulating strains, we found multiple putative recombination events between T. pallidum subsp. pallidum and subsp. endemicum, shaping the genomes of several subclades. Temporal analysis dated the most recent common ancestor of Nichols and SS14 clades to 1717 (95% HPD: 1543-1869), in agreement with other recent studies. Rates of SNP accumulation varied significantly among subclades, particularly among different Nichols subclades, and was associated in the Nichols A subclade with a C394F substitution in TP0380, a ERCC3-like DNA repair helicase. Our data highlight the role played by variation in genes encoding putative surface-exposed outer membrane proteins in defining separate lineages, and provide a critical resource for the design of broadly protective syphilis vaccines targeting surface antigens.

SARS-CoV-2 Epidemiology on a Public University Campus in Washington State.

BACKGROUND: We aimed to evaluate a testing program to facilitate control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission at a large university and measure spread in the university community using viral genome sequencing. METHODS: Our prospective longitudinal study used remote contactless enrollment, daily mobile symptom and exposure tracking, and self-swab sample collection. Individuals were tested if the participant was exposed to a known SARS-CoV-2-infected person, developed new symptoms, or reported high-risk behavior (such as attending an indoor gathering without masking or social distancing), if a member of a group experiencing an outbreak, or at enrollment. Study participants included students, staff, and faculty at an urban public university during the Autumn quarter of 2020. RESULTS: We enrolled 16 476 individuals, performed 29 783 SARS-CoV-2 tests, and detected 236 infections. Seventy-five percent of positive cases reported at least 1 of the following: symptoms (60.8%), exposure (34.7%), or high-risk behaviors (21.5%). Greek community affiliation was the strongest risk factor for testing positive, and molecular epidemiology results suggest that specific large gatherings were responsible for several outbreaks. CONCLUSIONS: A testing program focused on individuals with symptoms and unvaccinated persons who participate in large campus gatherings may be effective as part of a comprehensive university-wide mitigation strategy to control the spread of SARS-CoV-2.

Genetic variants associated with cytomegalovirus infection after allogeneic hematopoietic cell transplantation.

Human cytomegalovirus (CMV) reactivation is a frequent complication of allogeneic hematopoietic cell transplantation (HCT). Despite routine screening for CMV reactivation and early antiviral treatment, the rates of CMV-related complications after HCT remain high. Genetic variants in both the donor and recipient have been associated with the risk of CMV reactivation and disease after HCT, but these associations have not been validated, and their clinical importance remains unclear. In this study, we assessed 117 candidate variants previously associated with CMV-related phenotypes for association with CMV reactivation and disease in a cohort of 2169 CMV-seropositive HCT recipients. We also carried out a genome-wide association study (GWAS) for CMV reactivation and disease in the same cohort. Both analyses used a prespecified discovery and replication approach to control the risk of false-positive results. Among the 117 candidate variants, our analysis implicates only the donor ABCB1 rs1045642 genotype as a risk factor for CMV reactivation. This synonymous variant in P-glycoprotein may influence the risk of CMV reactivation by altering the efflux of cyclosporine and tacrolimus from donor lymphocytes. In the GWAS analysis, the donor CDC42EP3 rs11686168 genotype approached the significance threshold for association with CMV reactivation, although we could not identify a mechanism to explain this association. The results of this study suggest that most genomic variants previously associated with CMV phenotypes do not significantly alter the risk for CMV reactivation or disease after HCT.

Evaluating Specimen Quality and Results from a Community-Wide, Home-Based Respiratory Surveillance Study.

While influenza and other respiratory pathogens cause significant morbidity and mortality, the community-based burden of these infections remains incompletely understood. The development of novel methods to detect respiratory infections is essential for mitigating epidemics and developing pandemic-preparedness infrastructure. From October 2019 to March 2020, we conducted a home-based cross-sectional study in the greater Seattle, WA, area, utilizing electronic consent and data collection instruments. Participants received nasal swab collection kits via rapid delivery within 24 hours of self-reporting respiratory symptoms. Samples were returned to the laboratory and were screened for 26 respiratory pathogens and a housekeeping gene. Participant data were recorded via online survey at the time of sample collection and 1 week later. Of the 4,572 consented participants, 4,359 (95.3%) received a home swab kit and 3,648 (83.7%) returned a nasal specimen for respiratory pathogen screening. The 3,638 testable samples had a mean RNase P relative cycle threshold (Crt ) value of 19.0 (SD, 3.4), and 1,232 (33.9%) samples had positive results for one or more pathogens, including 645 (17.7%) influenza-positive specimens. Among the testable samples, the median time between shipment of the home swab kit and completion of laboratory testing was 8.0 days (interquartile range [IQR], 7.0 to 14.0). A single adverse event occurred and did not cause long-term effects or require medical attention. Home-based surveillance using online participant enrollment and specimen self-collection is a safe and feasible method for community-level monitoring of influenza and other respiratory pathogens, which can readily be adapted for use during pandemics.

Dynamics of Neutralizing Antibody Titers in the Months After Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

Most individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop neutralizing antibodies that target the viral spike protein. In this study, we quantified how levels of these antibodies change in the months after SARS-CoV-2 infection by examining longitudinal samples collected approximately 30-152 days after symptom onset from a prospective cohort of 32 recovered individuals with asymptomatic, mild, or moderate-severe disease. Neutralizing antibody titers declined an average of about 4-fold from 1 to 4 months after symptom onset. This decline in neutralizing antibody titers was accompanied by a decline in total antibodies capable of binding the viral spike protein or its receptor-binding domain. Importantly, our data are consistent with the expected early immune response to viral infection, where an initial peak in antibody levels is followed by a decline to a lower plateau. Additional studies of long-lived B cells and antibody titers over longer time frames are necessary to determine the durability of immunity to SARS-CoV-2.

Herpes Simplex Virus Mistyping due to HSV-1 × HSV-2 Interspecies Recombination in Viral Gene Encoding Glycoprotein B.

Human herpes simplex viruses (HSV) 1 and 2 are extremely common human pathogens with overlapping disease spectra. Infections due to HSV-1 and HSV-2 are distinguished in clinical settings using sequence-based "typing" assays. Here we describe a case of HSV mistyping caused by a previously undescribed HSV-1 × HSV-2 recombination event in UL27, the HSV gene that encodes glycoprotein B. This is the first documented case of HSV mistyping caused by an HSV-1 × HSV-2 recombination event and the first description of an HSV interspecies recombination event in UL27, which is frequently used as a target for diagnostics and experimental therapeutics. We also review the primer and probe target sequences for a commonly used HSV typing assay from nearly 700 HSV-1 and HSV-2 samples and find that about 4% of HSV-1 samples have a single nucleotide change in at least one of these loci, which could impact assay performance. Our findings illustrate how knowledge of naturally occurring genomic variation in HSV-1 and HSV-2 is essential for the design and interpretation of molecular diagnostics for these viruses.

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. In the course of optimizing SARS-CoV-2 testing performed by the University of Washington Clinical Virology Lab (UW Virology Lab), we tested assays using seven different primer/probe sets and one assay kit. We found that the most sensitive assays were those the used the E-gene primer/probe set described by Corman et al. (Eurosurveillance 25(3), 2020, https://doi.org/10.2807/1560-7917.ES.2020.25.3.2000045) and the N2 set described by the CDC (Division of Viral Diseases, Centers for Disease Control and Prevention, 2020, https://www.cdc.gov/coronavirus/2019-ncov/downloads/rt-pcr-panel-primer-probes.pdf). All assays tested were found to be highly specific for SARS-CoV-2, with no cross-reactivity with other respiratory viruses observed in our analyses regardless of the primer/probe set or kit used. These results will provide invaluable information to other clinical laboratories who are actively developing SARS-CoV-2 testing protocols at a time when increased testing capacity is urgently needed worldwide.

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. In the course of optimizing SARS-CoV-2 testing performed by the University of Washington Clinical Virology Lab (UW Virology Lab), we evaluated assays using seven different primer-probe sets and one assay kit. We found that the most sensitive assays were those that used the E-gene primer-probe set described by Corman et al. (V. M. Corman, O. Landt, M. Kaiser, R. Molenkamp, et al., Euro Surveill 25:2000045, 2020, https://doi.org/10.2807/1560-7917.ES.2020.25.3.2000045) and the N2 set developed by the CDC (Division of Viral Diseases, Centers for Disease Control and Prevention, 2020, https://www.cdc.gov/coronavirus/2019-ncov/downloads/rt-pcr-panel-primer-probes.pdf). All assays tested were found to be highly specific for SARS-CoV-2, with no cross-reactivity with other respiratory viruses observed in our analyses regardless of the primer-probe set or kit used. These results will provide valuable information to other clinical laboratories who are actively developing SARS-CoV-2 testing protocols at a time when increased testing capacity is urgently needed worldwide.

High-resolution profiling of human cytomegalovirus cell-free DNA in human plasma highlights its exceptionally fragmented nature.

Human cytomegalovirus (CMV) infections comprise a leading cause of newborn impairments worldwide and are pervasive concerns among the immunocompromised. Quantification of CMV viral loads is increasingly used to guide definitions of CMV disease but standardization of CMV quantitation remains problematic, mostly due to differences in qPCR amplicon sizes between clinical laboratories. Here, we used plasma cfDNA sequencing data from 2,208 samples sent for non-invasive prenatal aneuploidy screening to detect CMV and precisely measure the length of CMV fragments in human plasma. CMV reads were identified in 120 (5.4%) samples. Median cfDNA fragment size derived from CMV was significantly shorter than cfDNA derived from human chromosomes (103 vs 172 bp, p < 0.0001), corresponding to the 3rd percentile of human cfDNA. Sequencing of cfDNA from seven plasma samples from transplant patients positive for CMV confirmed the extraordinarily short nature of CMV cfDNA fragment size with a median length of 149 bp. We further show that these high-resolution measurements of CMV DNA fragment size accurately predict measured discrepancies in serum viral load measurements by different qPCR assays. These results highlight the exceptionally fragmented nature of CMV cfDNA and illustrate the promise of plasma cfDNA sequencing for quantitating viral loads through detection of fragments that would be unrecoverable by qPCR.