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Background: The novel respiratory virus SARS-CoV-2, responsible for over 380,000 COVID-19 related deaths, has caused significant strain on healthcare infrastructure and clinical laboratories globally. The pandemic's initial challenges include broad diagnostic testing, consistent reagent supply lines, and access to laboratory instruments and equipment. In early 2020, primer/probe sets distributed by the CDC utilized the same fluorophore for molecular detection - requiring multiple assays to be run in parallel - consuming valuable and limited resources., Methods: Nasopharyngeal swabs submitted to UW Virology for SARS-CoV-2 clinical testing were extracted, amplified by our laboratory developed test (LDT) - a CDC-based quantitative reverse transcriptase PCR reaction - and analyzed for agreement between the multiplexed assay. Laboratory- confirmed respiratory infection samples were included to evaluate assay cross-reaction specificity., Results: Triplexing correctly identified SARS-CoV-2 in 98.4% of confirmed positive or inconclusive patient samples by single-plex LDT (n = 183/186). All 170 SARS-CoV-2 negative samples tested by single-plex LDT were negative by triplexing. Other laboratory-confirmed respiratory infections did not amplify for SARS-CoV-2 in the triplex reaction., Conclusions: Multiplexing two virus-specific gene targets and an extraction control was found to be comparable to running parallel assays independently, while significantly improving assay throughput., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Importance: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused epidemic spread of coronavirus disease 2019 (COVID-19) in the Seattle, Washington, metropolitan area, with morbidity and mortality concentrated among residents of skilled nursing facilities. The prevalence of COVID-19 among older adults in independent/assisted living is not understood., Objectives: To conduct surveillance for SARS-CoV-2 and describe symptoms of COVID-19 among residents and staff of an independent/assisted living community., Design, Setting, and Participants: In March 2020, public health surveillance of staff and residents was conducted on site at an assisted and independent living residence for older adults in Seattle, Washington, after exposure to 2 residents who were hospitalized with COVID-19., Exposures: Surveillance for SARS-CoV-2 infection in a congregate setting implementing social isolation and infection prevention protocols., Main Outcomes and Measures: SARS-CoV-2 real-time polymerase chain reaction was performed on nasopharyngeal swabs from residents and staff; a symptom questionnaire was completed assessing fever, cough, and other symptoms for the preceding 14 days. Residents were retested for SARS-CoV-2 7 days after initial screening., Results: Testing was performed on 80 residents; 62 were women (77%), with mean age of 86 (range, 69-102) years. SARS-CoV-2 was detected in 3 of 80 residents (3.8%); none felt ill, 1 male resident reported resolved cough and 1 loose stool during the preceding 14 days. Virus was also detected in 2 of 62 staff (3.2%); both were symptomatic. One week later, resident SARS-CoV-2 testing was repeated and 1 new infection detected (asymptomatic). All residents remained in isolation and were clinically stable 14 days after the second test., Conclusions and Relevance: Detection of SARS-CoV-2 in asymptomatic residents highlights challenges in protecting older adults living in congregate settings. In this study, symptom screening failed to identify residents with infections and all 4 residents with SARS-CoV-2 remained asymptomatic after 14 days. Although 1 asymptomatic infection was found on retesting, a widespread facility outbreak was avoided. Compared with skilled nursing settings, in assisted/independent living communities, early surveillance to identify asymptomatic persons among residents and staff, in combination with adherence to recommended preventive strategies, may reduce viral spread.

We evaluated the performance of the BioFire® Respiratory Panel 2.1 (RP2.1) in the detection of SARS CoV-2 in comparison against three other SARS CoV-2 EUA assays. In these studies, the RP2.1 panel had 98 % positive percent agreement (48/49) and 100 % negative percent agreement (49/49). Since 30 % of nasopharyngeal swab specimens have a SARS CoV-2 Ct >30 and thus detection of virus in low titers is clinically relevant, a sample with a high titer was diluted and each 10 fold dilution was tested in triplicate and compared against 6 other EUA approved SARS CoV-2 assays. These data suggested that the BioFire® RP2.1 panel, along with four other SARS CoV-2 assays (Roche cobas, Cepheid Xpert Xpress, BioFire® Defense COVID19, and NECoV19), consistently detected viral RNA at the 10-7 dilution. Overall, these studies suggest that the BioFire® RP2.1 assay can be used to detect acute cases of SARS CoV2 in addition to patients with low viral titer later in disease presentation., Competing Interests: Declaration of Competing Interest The Corresponding author has acted as a consultant and received grant funding from BioFire Diagnostics., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally, with >365,000 cases in California as of 17 July 2020. We investigated the genomic epidemiology of SARS-CoV-2 in Northern California from late January to mid-March 2020, using samples from 36 patients spanning nine counties and the Grand Princess cruise ship. Phylogenetic analyses revealed the cryptic introduction of at least seven different SARS-CoV-2 lineages into California, including epidemic WA1 strains associated with Washington state, with lack of a predominant lineage and limited transmission among communities. Lineages associated with outbreak clusters in two counties were defined by a single base substitution in the viral genome. These findings support contact tracing, social distancing, and travel restrictions to contain the spread of SARS-CoV-2 in California and other states., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Multiple laboratory-developed tests (LDTs) and commercially available assays have emerged to meet diagnostic needs related to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. To date, there is limited comparison data for these different testing platforms. We compared the analytical performance of a LDT developed in our clinical laboratory based on CDC primer sets and four commercially available, FDA emergency use authorized assays for SARS-CoV-2 (Cepheid, DiaSorin, Hologic Panther, and Roche Cobas) on a total of 169 nasopharyngeal swabs. The LDT and Cepheid Xpert Xpress SARS-CoV-2 assays were the most sensitive assays for SARS-CoV-2 with 100% agreement across specimens. The Hologic Panther Fusion, DiaSorin Simplexa, and Roche Cobas 6800 failed to detect positive specimens only near the limit of detection of our CDC-based LDT assay. All assays were 100% specific, using our CDC-based LDT as the gold standard. Our results provide initial test performance characteristics for SARS-CoV-2 reverse transcription-PCR (RT-PCR) and highlight the importance of having multiple viral detection testing platforms available in a public health emergency., (Copyright © 2020 Lieberman et al.)

Coronavirus disease 2019 (COVID-19), the novel respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is associated with severe morbidity and mortality. The rollout of diagnostic testing in the United States was slow, leading to numerous cases that were not tested for SARS-CoV-2 in February and March 2020 and necessitating the use of serological testing to determine past infections. Here, we evaluated the Abbott SARS-CoV-2 IgG test for detection of anti-SARS-CoV-2 IgG antibodies by testing 3 distinct patient populations. We tested 1,020 serum specimens collected prior to SARS-CoV-2 circulation in the United States and found one false positive, indicating a specificity of 99.90%. We tested 125 patients who tested reverse transcription-PCR (RT-PCR) positive for SARS-CoV-2 for whom 689 excess serum specimens were available and found that sensitivity reached 100% at day 17 after symptom onset and day 13 after PCR positivity. Alternative index value thresholds for positivity resulted in 100% sensitivity and 100% specificity in this cohort. We tested specimens from 4,856 individuals from Boise, ID, collected over 1 week in April 2020 as part of the Crush the Curve initiative and detected 87 positives for a positivity rate of 1.79%. These data demonstrate excellent analytical performance of the Abbott SARS-CoV-2 IgG test as well as the limited circulation of the virus in the western United States. We expect that the availability of high-quality serological testing will be a key tool in the fight against SARS-CoV-2., (Copyright © 2020 Bryan et al.)

In early March 2020, the University of Washington Medical Center clinical virology laboratory became one of the first clinical laboratories to offer testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). When we first began test development in mid-January, neither of us believed there would be more than 2 million confirmed SARS-CoV-2 infections nationwide or that we would have performed more than 150,000 real-time PCR (RT-PCR) tests, with many more to come. This article will be a chronological summary of how we rapidly validated tests for SARS-CoV-2, increased our testing capacity, and addressed the many problems that came up along the way., (Copyright © 2020 American Society for Microbiology.)

Background: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused considerable disruption across the world, resulting in more than 235,000 deaths since December 2019. SARS-CoV-2 has a wide tropism and detection of the virus has been described in multiple specimen types, including various respiratory secretions, cerebrospinal fluid, and stool., Objective: To evaluate the accuracy and sensitivity of a laboratory modified CDCbased SARS-CoV-2 N1 and N2 assay across a range of sample types. Study Design We compared the matrix effect on the analytical sensitivity of SARS-CoV-2 detection by qRT-PCR in nasal swabs collected in viral transport medium (VTM), bronchoalveolar lavage (BAL), sputum, plasma, cerebral spinal fluid (CSF), stool, VTM, phosphate buffered saline (PBS), and Hanks' Balanced Salt Solution (HBSS). Initial limits of detection (LoD) were subsequently narrowed to confirm an LoD for each specimen type and target gene., Results: LoDs were established using a modified CDC-based laboratory developed test and ranged from a mean CT cut-off of 33.8-35.7 (10-20 copies/reaction) for the N1 gene target, and 34.0-36.2 (1-10 copies/reaction) for N2. Alternatives to VTM such as PBS and HBSS had comparable LoDs. The N2 gene target was found to be most sensitive in CSF., Conclusion: A modified CDC-based laboratory developed test is able to detect SARSCoV- 2 accurately with similar sensitivity across all sample types tested., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: More than 2 months separated the initial description of SARS-CoV-2 and discovery of its widespread dissemination in the United States. Despite this lengthy interval, implementation of specific quantitative reverse transcription (qRT)-PCR-based SARS-CoV-2 tests in the US has been slow, and testing is still not widely available. Metagenomic sequencing offers the promise of unbiased detection of emerging pathogens, without requiring prior knowledge of the identity of the responsible agent or its genomic sequence., Methods: To evaluate metagenomic approaches in the context of the current SARS-CoV-2 epidemic, laboratory-confirmed positive and negative samples from Seattle, WA were evaluated by metagenomic sequencing, with comparison to a 2019 reference genomic database created before the emergence of SARS-CoV-2., Results: Within 36 h our results showed clear identification of a novel human Betacoronavirus, closely related to known Betacoronaviruses of bats, in laboratory-proven cases of SARS-CoV-2. A subset of samples also showed superinfection or colonization with human parainfluenza virus 3 or Moraxella species, highlighting the need to test directly for SARS-CoV-2 as opposed to ruling out an infection using a viral respiratory panel. Samples negative for SARS-CoV-2 by RT-PCR were also negative by metagenomic analysis, and positive for Rhinovirus A and C. Unlike targeted SARS-CoV-2 qRT-PCR testing, metagenomic analysis of these SARS-CoV-2 negative samples identified candidate etiological agents for the patients' respiratory symptoms., Conclusion: Taken together, these results demonstrate the value of metagenomic analysis in the monitoring and response to this and future viral pandemics., (© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The novel coronavirus SARS-CoV-2 was first detected in the Pacific Northwest region of the United States in January 2020, with subsequent COVID-19 outbreaks detected in all 50 states by early March. To uncover the sources of SARS-CoV-2 introductions and patterns of spread within the United States, we sequenced nine viral genomes from early reported COVID-19 patients in Connecticut. Our phylogenetic analysis places the majority of these genomes with viruses sequenced from Washington state. By coupling our genomic data with domestic and international travel patterns, we show that early SARS-CoV-2 transmission in Connecticut was likely driven by domestic introductions. Moreover, the risk of domestic importation to Connecticut exceeded that of international importation by mid-March regardless of our estimated effects of federal travel restrictions. This study provides evidence of widespread sustained transmission of SARS-CoV-2 within the United States and highlights the critical need for local surveillance., Competing Interests: Declaration of Interests A.L.W. is the principal investigator on a research grant from Pfizer to Yale University and has received consulting fees for participation in advisory boards for Pfizer., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (Copyright © 2020 Nalla et al.)

Background: Community transmission of coronavirus 2019 (Covid-19) was detected in the state of Washington in February 2020., Methods: We identified patients from nine Seattle-area hospitals who were admitted to the intensive care unit (ICU) with confirmed infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Clinical data were obtained through review of medical records. The data reported here are those available through March 23, 2020. Each patient had at least 14 days of follow-up., Results: We identified 24 patients with confirmed Covid-19. The mean (±SD) age of the patients was 64±18 years, 63% were men, and symptoms began 7±4 days before admission. The most common symptoms were cough and shortness of breath; 50% of patients had fever on admission, and 58% had diabetes mellitus. All the patients were admitted for hypoxemic respiratory failure; 75% (18 patients) needed mechanical ventilation. Most of the patients (17) also had hypotension and needed vasopressors. No patient tested positive for influenza A, influenza B, or other respiratory viruses. Half the patients (12) died between ICU day 1 and day 18, including 4 patients who had a do-not-resuscitate order on admission. Of the 12 surviving patients, 5 were discharged home, 4 were discharged from the ICU but remained in the hospital, and 3 continued to receive mechanical ventilation in the ICU., Conclusions: During the first 3 weeks of the Covid-19 outbreak in the Seattle area, the most common reasons for admission to the ICU were hypoxemic respiratory failure leading to mechanical ventilation, hypotension requiring vasopressor treatment, or both. Mortality among these critically ill patients was high. (Funded by the National Institutes of Health.)., (Copyright © 2020 Massachusetts Medical Society.)

Human herpesvirus 6 (HHV-6) is an important cause of meningitis and meningoencephalitis. As testing for HHV-6 in cerebrospinal fluid (CSF) is more readily available using the FilmArray Meningitis/Encephalitis panel (FA-ME; BioFire Diagnostics, Salt Lake City, UT), we aimed to determine the clinical significance of detecting HHV-6 in order to identify true infections and to ensure appropriate antiviral initiation. Chart review on 25 patients positive for HHV-6 by FA-ME was performed to determine clinical presentation, comorbidity, treatment, and outcome. The presence of chromosomally integrated HHV-6 (ciHHV-6) DNA was also investigated. Of 1,005 children tested by FA-ME, HHV-6 was detected in 25 (2.5%). Five patients were diagnosed with either HHV-6 meningitis or meningoencephalitis based on HHV-6 detection in CSF, clinical presentation, and radiographic findings. Detection of HHV-6 by FA-ME led to discontinuation of acyclovir within 12.0 h in all 12 patients empirically treated with acyclovir. Six of the 12 patients were started on ganciclovir therapy within 6.8 h; 4 of these were treated specifically for HHV-6 infection, whereas therapy was discontinued in the remaining 2 patients. CSF parameters were not generally predictive of HHV-6 positivity. The presence of ciHHV-6 was confirmed in 3 of 18 patients who could be tested. Five of the 25 patients included in the study were diagnosed with HHV-6 meningitis/meningoencephalitis. FA-ME results led to discontinuation of empirical antiviral treatment in 12 patients and appropriate initiation of ganciclovir in 4 patients. In our institution, detection of HHV-6 using FA-ME led to faster establishment of disease etiology and optimization of antimicrobial therapy., (Copyright © 2020 American Society for Microbiology.)

In the Seattle, Washington metropolitan area, where the first case of novel coronavirus 2019 disease (COVID-19) in the United States was reported (1), a community-level outbreak is ongoing with evidence of rapid spread and high morbidity and mortality among older adults in long-term care skilled nursing facilities (SNFs) (2,3). However, COVID-19 morbidity among residents of senior independent and assisted living communities, in which residents do not live as closely together as do residents in SNFs and do not require skilled nursing services, has not been described. During March 5-9, 2020, two residents of a senior independent and assisted living community in Seattle (facility 1) were hospitalized with confirmed COVID-19 infection; on March 6, social distancing and other preventive measures were implemented in the community. UW Medicine (the health system linked to the University of Washington), Public Health - Seattle & King County, and CDC conducted an investigation at the facility. On March 10, all residents and staff members at facility 1 were tested for SARS-CoV-2, the virus that causes COVID-19, and asked to complete a questionnaire about their symptoms; all residents were tested again 7 days later. Among 142 residents and staff members tested during the initial phase, three of 80 residents (3.8%) and two of 62 staff members (3.2%) had positive test results. The three residents had no symptoms at the time of testing, although one reported an earlier cough that had resolved. A fourth resident, who had negative test results in the initial phase, had positive test results 7 days later. This resident was asymptomatic on both days. Possible explanations for so few cases of COVID-19 in this residential community compared with those in several Seattle SNFs with high morbidity and mortality include more social distancing among residents and less contact with health care providers. In addition, early implementation of stringent isolation and protective measures after identification of two COVID-19 cases might have been effective in minimizing spread of the virus in this type of setting. When investigating a potential outbreak of COVID-19 in senior independent and assisted living communities, symptom screening is unlikely to be sufficient to identify all persons infected with SARS-CoV-2. Adherence to CDC guidance to prevent COVID-19 transmission in senior independent and assisted living communities (4) could be instrumental in preventing a facility outbreak., Competing Interests: All authors have completed and submitted the International Committee of Medical Journal Editors form for disclosure of potential conflicts of interest. Alexander L. Greninger reports personal fees from Abbott Molecular outside the submitted work. No other potential conflicts of interest were disclosed.

Background: The influence of humoral immunity on the prevention of primary cytomegalovirus (CMV) infection after hematopoietic cell transplantation (HCT) is poorly understood., Methods: To determine whether neutralizing antibodies (nAbs) against CMV pentameric complex (PC)-mediated epithelial cell entry decrease CMV infection after HCT, samples were analyzed from a randomized controlled trial of CMV intravenous immunoglobulin (IVIG) prophylaxis. Weekly serum from 61 CMV donor-positive/recipient-negative (D+/R-) HCT patients (33 control, 28 CMV IVIG) was tested using a PC-entry nAb assay and quantitative CMV polymerase chain reaction (PCR)., Results: There was a trend toward higher weekly PC-entry nAb titers (P = .07) and decreased CMV infection by PCR at viral load cutoffs of ≥1000 and ≥10 000 IU/mL in the CMV IVIG arm. High nAb titers were not significantly protective against CMV infection later after HCT in both study arms. Among CMV-infected patients, each log2 increase in nAb titer was associated with an average 0.2 log10 decrease in concurrent CMV viral load after infection (P = .001; adjusted for study arm)., Conclusions: This study provides initial support that CMV IVIG prophylaxis moderately enhances PC-entry nAB activity in D+/R- HCT recipients., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: The ubiquitous human pathogens, herpes simplex virus (HSV)-1 and HSV-2, are distinct viral species that diverged approximately 6 million years ago. At least 4 small, ancient HSV-1 × HSV-2 interspecies recombination events have affected the HSV-2 genome, with recombinants and nonrecombinants at each locus circulating today. However, it is unknown whether interspecies recombination can affect other loci and whether new recombinants continue to be generated., Methods: Using 255 newly sequenced and 230 existing HSV genome sequences, we comprehensively assessed interspecies recombination in HSV., Results: Our findings show that the sizes and locations of interspecies recombination events in HSV-2 are significantly more variable than previously appreciated and that they can impact species-specific T-cell recognition of HSV., Conclusions: We describe 2 large (>5 kb) recombination events, one of which arose in its current host, demonstrating that interspecies recombination continues to occur today. These results raise concerns about the use of live-attenuated HSV-2 vaccines in high HSV-1 prevalence areas., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

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 3 rd 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.

BK polyomavirus (BKPyV) has been associated with hemorrhagic cystitis (HC) after allogeneic hematopoietic cell transplantation (HCT), but the natural history of HC and factors associated with the clinical course are incompletely understood. We retrospectively analyzed allogeneic HCT patients transplanted from 2007-2017 who presented after platelet engraftment or after day 28 post-HCT with BKPyV-associated HC (BKPyV-HC), which was defined as a positive urine BKPyV PCR, ≥1 plasma BKPyV viral load result, and macroscopic hematuria (Bedi grade ≥2). Factors associated with resolution of macroscopic hematuria and resolution of all cystitis symptoms within 90 days after HC diagnosis were investigated in multivariable models. In 128 patients with BKPyV-HC, the median times from diagnosis to resolution of all symptoms, macroscopic hematuria, and urinary clots (present in 55% [71/128]) were 24 days (15-44), 17 days (10-30), and 14 days (5-26), respectively. Ninety percent of patients had BKPyV viremia at the onset of HC with a median viral load of 1850 copies/mL (interquartile range, 240-8550). In multivariable models, high plasma viral load (≥10 000 copies/mL) and cytopenias at the beginning of BKPyV-HC were significantly associated with longer macroscopic hematuria and cystitis symptoms. Use of cidofovir was not associated with shorter duration of illness. In conclusion, BKPyV-HC after allogeneic HCT is characterized by prolonged and severe symptoms and requires improved management strategies. High-grade viremia and cytopenias were associated with a longer duration of BKPyV-associated HC. Accurate descriptions of disease and factors associated with prolonged recovery will inform end points of future clinical trials., (© 2020 by The American Society of Hematology.)

Acute graft-versus-host-disease (aGVHD) is a major complication following hematopoietic cell transplantations (HCTs). We have shown that HCT recipients in whom either the donor or patient had inherited chromosomally integrated human herpesvirus 6 (iciHHV-6) have a higher incidence of developing more severe aGVHD. Previous studies established that increased proinflammatory cytokines are associated with increased risk for aGVHD and nonrelapse mortality post-HCT. We hypothesized that HCT recipients with donor or recipient iciHHV-6 (iciHHV-6 pos HCT cases) will have higher cytokine levels compared with HCT recipients without iciHHV-6 (iciHHV-6 neg HCT controls). We identified 64 iciHHV-6 pos HCT cases with plasma from days 7, 14, and/or 21 post-HCT and before aGVHD onset in patients who developed aGVHD. We identified 64 iciHHV-6 neg HCT controls matched for aGVHD risk factors. We also identified 28 donors with iciHHV-6 and 56 matched donors without iciHHV-6. We measured plasma cytokine concentrations for IL-6, suppression of tumorigenicity 2, T cell immunoglobulin and mucin-domain containing 3, TNFα, soluble TNF receptor 1 (TNFRp55), and C-reactive protein (CRP). We used Mann-Whitney tests and repeated-measures models to compare cytokine levels. iciHHV-6 pos HCT cases had higher CRP levels on day 7 and day 21 and higher TNFRp55 levels on day 14 and day 21 compared with iciHHV-6 neg HCT controls. These findings were recapitulated in a repeated-measures model. The differences were most evident among patients who subsequently developed aGVHD grades 2 to 4. Additionally, iciHHV-6 pos HCT cases had earlier-onset aGVHD (median, 20 versus 27 days post-HCT; P = .02). There were no differences in cytokine levels among healthy donors with or without iciHHV-6. This study demonstrates that HCT recipients with iciHHV-6 have higher proinflammatory cytokines that may be associated with increased risk for aGVHD., (Copyright © 2019 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Objective: This study aimed to assess quantitative human papillomavirus (HPV) type 16 and HPV18 detection in oral rinses obtained in dental offices in Seattle, Washington., Methods: We evaluated oral rinses collected during dental visits from 2016 to 2018. Multiplex TaqMan quantitative polymerase chain reaction was used to determine HPV16 and HPV18 viral load (VL)., Results: Of 15,313 persons, 152 (1%) had detectable oral HPV16/18. Men were at higher risk of oral HPV16/18 infection than women (1.6% vs. 0.6%; odds ratio, 3.2; 95% confidence interval, 2.1-4.4). Compared with women, men with HPV16 were older (median, 55 vs. 48 years; P < 0.001) and had higher VL (39.7 vs. 1.1 copies/mL, P < 0.001). Of 39 with HPV16 at baseline and a second oral rinse, 13 remained positive at subsequent rinse; of 8 with HPV18 at baseline, 2 remained positive at subsequent rinse. Persons with consecutive positive test results were all men and had higher baseline VL compared with those with first positive and second negative samples., Conclusion: Oral rinse is an acceptable method of HPV testing, and persons are interested in testing. Overall HPV16/18 prevalence was low, and detection was more frequent among men than women, especially at higher copy numbers. HPV16 persistence was more common in men with high VL at baseline test. Future studies are needed to evaluate the feasibility of an effective secondary prevention strategy for oropharyngeal cancer using quantitative oral HPV detection.

Background: Hematopoietic cell transplant (HCT) recipients are frequently infected with respiratory viruses (RVs) in the upper respiratory tract (URT), but the concordance between URT and lower respiratory tract (LRT) RV detection is not well characterized., Methods: Hematopoietic cell transplant candidates and recipients with respiratory symptoms and LRT and URT RV testing via multiplex PCR from 2009 to 2016 were included. Logistic regression models were used to analyze risk factors for LRT RV detection., Results: Two-hundred thirty-five HCT candidates or recipients had URT and LRT RV testing within 3 days. Among 115 subjects (49%) positive for a RV, 37% (42 of 115) had discordant sample pairs. Forty percent (17 of 42) of discordant pairs were positive in the LRT but negative in the URT. Discordance was common for adenovirus (100%), metapneumovirus (44%), rhinovirus (34%), and parainfluenza virus type 3 (28%); respiratory syncytial virus was highly concordant (92%). Likelihood of LRT detection was increased with URT detection (oods ratio [OR] = 73.7; 95% confidence interval [CI], 26.7-204) and in cytomegalovirus-positive recipients (OR = 3.70; 95% CI, 1.30-10.0)., Conclusions: High rates of discordance were observed for certain RVs. Bronchoalveolar lavage sampling may provide useful diagnostic information to guide management in symptomatic HCT candidates and recipients., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: The PCR and its variant, quantitative PCR (qPCR), have revolutionized the practice of clinical microbiology. Continued advancements in PCR have led to a new derivative, digital PCR (dPCR), which promises to address certain limitations inherent to qPCR., Content: Here we highlight the important technical differences between qPCR and dPCR, and the potential advantages and disadvantages of each. We then review specific situations in which dPCR has been implemented in clinical microbiology and the results of such applications. Finally, we attempt to place dPCR in the context of other emerging technologies relevant to the clinical laboratory, including next-generation sequencing., Summary: dPCR offers certain clear advantages over traditional qPCR, but these are to some degree offset by limitations of the technology, at least as currently practiced. Laboratories considering implementation of dPCR should carefully weigh the potential advantages and disadvantages of this powerful technique for each specific application planned., (© 2019 American Association for Clinical Chemistry.)

Despite the adaptation of international standards, quantitative viral load testing of transplant-associated viruses continues to be limited by interlaboratory disagreement. Studies have suggested that this disagreement and the poor commutability of standards may, in some cases, be linked to amplicon size and the fragmentation of circulating viral DNA. We evaluated target fragmentation as a cause of noncommutability and pretest fragmentation of quantitative standards as a potential means of increasing commutability and interassay agreement. Forty-two cytomegalovirus (CMV)-positive and 41 Epstein-Barr virus (EBV)-positive plasma samples, together with two different quantitative standards for each virus, were tested as unknowns using 10 different quantitative PCR assays at 5 different laboratories. Standards were tested both intact and after intentional fragmentation by ultrasonication. Quantitative agreement between methods was assessed, together with commutability, using multiple statistical approaches. Most assays yielded results within 0.5 log 10 IU/ml of the mean for CMV, while for EBV a greater variability of up to 1.5 log 10 IU/ml of the mean was shown. Commutability showed marked improvement following fragmentation of both CMV standards but not after fragmentation of the EBV standards. These findings confirm the impact of amplicon size and target fragmentation on commutability for CMV and suggest that for some (but not all) viruses, interlaboratory harmonization can be improved through the use of fragmented quantitative standards., (Copyright © 2019 American Society for Microbiology.)

Human herpesviruses 6A and 6B (HHV-6A and HHV-6B) are human viruses capable of chromosomal integration. Approximately 1% of the human population carries one copy of HHV-6A/B integrated into every cell in their body, referred to as inherited chromosomally integrated human herpesvirus 6A/B (iciHHV-6A/B). Whether iciHHV-6A/B is transcriptionally active in vivo and how it shapes the immunological response are still unclear. In this study, we screened DNA sequencing (DNA-seq) and transcriptome sequencing (RNA-seq) data for 650 individuals available through the Genotype-Tissue Expression (GTEx) project and identified 2 iciHHV-6A- and 4 iciHHV-6B-positive candidates. When corresponding tissue-specific gene expression signatures were analyzed, low levels HHV-6A/B gene expression was found across multiple tissues, with the highest levels of gene expression in the brain (specifically for HHV-6A), testis, esophagus, and adrenal gland. U90 and U100 were the most highly expressed HHV-6 genes in both iciHHV-6A- and iciHHV-6B-positive individuals. To assess whether tissue-specific gene expression from iciHHV-6A/B influences the immune response, a cohort of 15,498 subjects was screened and 85 iciHHV-6A/B + subjects were identified. Plasma samples from iciHHV-6A/B + and age- and sex-matched controls were analyzed for antibodies to control antigens (cytomegalovirus [CMV], Epstein-Barr virus [EBV], and influenza virus [FLU]) or HHV-6A/B antigens. Our results indicate that iciHHV-6A/B + subjects have significantly more antibodies against the U90 gene product (IE1) than do non-iciHHV-6-positive individuals. Antibody responses against EBV and FLU antigens or HHV-6A/B gene products either not expressed or expressed at low levels, such as U47, U57, and U72, were identical between controls and iciHHV-6A/B + subjects. CMV-seropositive individuals with iciHHV-6A/B + have more antibodies against CMV pp150 than do CMV-seropositive controls. These results argue that spontaneous gene expression from integrated HHV-6A/B leads to an increase in antigenic burden that translates into a more robust HHV-6A/B-specific antibody response. IMPORTANCE HHV-6A and -6B are human herpesviruses that have the unique property of being able to integrate into the telomeric regions of human chromosomes. Approximately 1% of the world's population carries integrated HHV-6A/B genome in every cell of their body. Whether viral genes are transcriptionally active in these individuals is unclear. By taking advantage of a unique tissue-specific gene expression data set, we showed that the majority of tissues from iciHHV-6 individuals do not show HHV-6 gene expression. Brain and testes showed the highest tissue-specific expression of HHV-6 genes in two separate data sets. Two HHV-6 genes, U90 (immediate early 1 protein) and U100 (glycoproteins Q1 and Q2), were found to be selectively and consistently expressed across several human tissues. Expression of U90 translates into an increase in antigen-specific antibody response in iciHHV-6A/B + subjects relative to controls. Future studies will be needed to determine the mechanism of gene expression, the effects of these genes on human gene transcription networks, and the pathophysiological impact of having increased viral protein expression in tissue in conjunction with increased antigen-specific antibody production., (Copyright © 2019 Peddu et al.)

Problem: Rhinoviruses (RVs), the most common causes of acute respiratory infections in young children and infants, are highly diverse genetically., Objective: To characterize the RV types detected with respiratory illness episodes in infants in Nepal., Study Methods: Infants born to women enrolled in a randomized trial of maternal influenza immunization in rural, southern Nepal were followed with household-based weekly surveillance until 180 days of age. Infants with respiratory symptoms had nasal swabs tested for twelve respiratory viruses. A subset with RV alone was selected for sequencing of the VP4/2 gene to identify RV types., Results: Among 547 RV-only positive illnesses detected from December 2012 to April 2014, 285 samples (52%) were sequenced. RV-A, B, and C species were detected in 193 (68%), 18 (6%), and 74 (26%) specimens, respectively. A total of 94 unique types were identified from the sequenced samples, including 52 RV-A, 11 RV-B, and 31 RV-C. Multiple species and types circulated simultaneously throughout the study period. No seasonality was observed. The median ages at illness onset were 88, 104, and 88 days for RV-A, B, and C, respectively. The median polymerase chain reaction cycle threshold values did not differ between RV species. No differences between RV species were observed for reported respiratory symptoms, including pneumonia, or for medical care-seeking., Conclusions: Among very young, symptomatic infants in rural Nepal, all three species and many types of RV were identified; RV-A was detected most frequently. There was no association between RV species and disease severity., (© 2019 Wiley Periodicals, Inc.)

Adenoviruses (AdV) have been associated with a variety of human diseases and are recognized as causing significant morbidity and mortality in immunocompromised or transplant patients. Quantification of AdV DNA in plasma is notoriously difficult due to the genetic diversity of the 71 different serotypes identified to date. There is no World Health Organization standard available to harmonize quantitative data, so results between labs vary widely. In this study, we compared a laboratory-developed multiplex PCR assay with primers and probes specific for each group (A to G) and subgroup E4 (Octaplex) to one with a single primer and probe set (modified from N. Jothikumar et al., Appl Environ Microbiol 71:3131-3136, 2005) and one utilizing bisulfite pretreatment of DNA to reduce variation prior to amplification (Genetic Signatures). Our Octaplex assay detected all low-copy-number clinical samples, while the other two assays had subsets of samples that did not amplify. The modified Jothikumar assay failed to efficiently amplify three of the high-copy-number cultured strains, while the Genetic Signatures 3base assay had a positive bias, resulting in higher copies/ml (>0.5 log 10 ) for all culture fluids tested. All three assays resulted in endpoint detection of the available 51 AdV types. Using two different materials to generate a standard curve revealed that the Octaplex TaqMan assay and the modified Jothikumar assay both consistently gave adenovirus levels lower than the commercial platform for AdV culture fluids but not patient samples. This study highlights the differences in detection of AdV between laboratories that can be attributed to both the PCR method, as well as the reference material used for quantitation., (Copyright © 2019 American Society for Microbiology.)

Purpose: Human herpesvirus 6B (HHV-6B) DNA is frequently detected in bronchoalveolar lavage fluid (BALF) from immunocompromised subjects with lower respiratory tract disease (LRTD). Whether HHV-6B is a pulmonary pathogen is unclear., Methods: We tested BALF for HHV-6B DNA using polymerase chain reaction in allogeneic hematopoietic cell transplantation (HCT) recipients who underwent a BAL for evaluation of LRTD from 1992 to 2015. We used multivariable proportional hazards models to evaluate the association of HHV-6B + BALF with overall mortality, death from respiratory failure, and the effect of anti-HHV-6B antivirals on these outcomes. We used branched-chain RNA in situ hybridization to detect HHV-6 messenger RNA ( U41 and U57 transcripts) in lung tissue., Results: We detected HHV-6B + BALF from 147 of 553 (27%) individuals. Subjects with HHV-6B + BALF, with or without copathogens, had significantly increased risk of overall mortality (adjusted hazard ratio [aHR], 2.18; 95% CI, 1.41-3.39) and death from respiratory failure (aHR, 2.50; 95% CI, 1.56-4.01) compared with subjects with HHV-6B - BALF. Subjects with HHV-6B + BALF who received antivirals within 3 days pre-BAL had an approximately 1 log 10 lower median HHV-6B BALF viral load, as well as a lower risk of overall mortality (aHR, 0.42; 95% CI, 0.16-1.10), compared with subjects with HHV-6B + BALF not receiving antivirals. We detected intraparenchymal HHV-6 gene expression by RNA in situ hybridization in lung tissue in all three tested subjects with HHV-6B + BALF and sufficient tissue RNA preservation., Conclusion: These data provide evidence that HHV-6B detection in BALF is associated with higher mortality in allogeneic hematopoietic cell transplantation recipients with LRTD. Definitive evidence of causation will require a randomized prevention or treatment trial.

Background: Early and accurate detection of respiratory viruses (RV) is important for patient management. We have previously shown that self-collected nasal swabs (NS) are feasible and as sensitive as clinician-collected nasal washes for detection of RV, but the additive benefit of self-collected throat swabs is unknown., Objectives: To evaluate the added yield of self-collected nasal to throat swabs for detection of RV by PCR in patients with upper respiratory tract infection (URTI) symptoms., Study Design: Patients with URTI symptoms self-collected paired polyurethane foam NS and nylon flocked throat swabs and completed a symptom survey. Swabs were tested for 12 RV by real-time reverse transcription (RT)-PCR. Descriptive, McNemar's, and Wilcoxon signed rank statistical tests were used., Results: 115 paired nasal and throat swabs were collected from 63 individuals, with 71/115 (62%) positive for a RV by at least one specimen, including 51 positive by both, 17 positive by NS only, and 3 positive by throat swab only. The sensitivity of NS was 96% (95%CI: 88-99) versus 76% (95% CI: 65-85) in throat swabs, p<0.001. The median PCR cycle threshold (Ct) in 51 concordant samples was lower (indicating higher viral concentration) in NS (25.1) versus throat swabs (32.0). The three samples positive only by throat swab had high Ct values (33.8, 36.2, and 38.8, all rhinovirus)., Conclusion: Self-collection of NS was significantly more sensitive than self collection of throat swabs for detection of RV by RT-PCR. The addition of throat sampling does not appear to increase the diagnostic load in the self-testing setting., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Human rhinovirus lower respiratory tract infection (LRTI) is associated with mortality after hematopoietic cell transplantation (HCT); however, risk factors for LRTI are not well characterized. We sought to develop a risk score for progression to LRTI from upper respiratory tract infection (URTI) in HCT recipients. Risk factors for LRTI within 90 days were analyzed using Cox regression among HCT recipients with rhinovirus URTI between January 2009 and March 2016. The final multivariable model included factors with a meaningful effect on the bootstrapped optimism corrected concordance statistic. Weighted score contributions based on hazard ratios were determined. Cumulative incidence curves estimated the probability of LRTI at various score cut-offs. Of 588 rhinovirus URTI events, 100 (17%) progressed to LRTI. In a final multivariable model allogeneic grafts, prior rhinovirus URTI, low lymphocyte count, low albumin, positive cytomegalovirus serostatus, recipient statin use, and steroid use ≥2 mg/kg/day were associated with progression to LRTI. A weighted risk score cut-off with the highest sensitivity and specificity was determined. Risk scores above this cut-off were associated with progression to LRTI (cumulative incidence 28% versus 11% below cut-off; P < .001). The weighted risk score for progression to rhinovirus LRTI can help identify and stratify patients for clinical management and for future clinical trials of therapeutics in HCT recipients., (Copyright © 2018 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

A proposed strategy to cure HIV uses latency-reversing agents (LRAs) to reactivate latent proviruses for purging HIV reservoirs. A variety of LRAs have been identified, but none has yet proven effective in reducing the reservoir size in vivo. Nanocarriers could address some major challenges by improving drug solubility and safety, providing sustained drug release, and simultaneously delivering multiple drugs to target tissues and cells. Here, we formulated hybrid nanocarriers that incorporate physicochemically diverse LRAs and target lymphatic CD4 + T cells. We identified one LRA combination that displayed synergistic latency reversal and low cytotoxicity in a cell model of HIV and in CD4 + T cells from virologically suppressed patients. Furthermore, our targeted nanocarriers selectively activated CD4 + T cells in nonhuman primate peripheral blood mononuclear cells as well as in murine lymph nodes, and substantially reduced local toxicity. This nanocarrier platform may enable new solutions for delivering anti-HIV agents for an HIV cure.

Background: Orolabial herpes simplex virus type 1 (HSV-1) infection has a wide spectrum of severity in immunocompetent persons. To study the role of viral genotype and host immunity, we characterized oral HSV-1 shedding rates and host cellular response, and genotyped viral strains, in monozygotic (MZ) and dizygotic (DZ) twins., Methods: A total of 29 MZ and 22 DZ HSV-1-seropositive twin pairs were evaluated for oral HSV-1 shedding for 60 days. HSV-1 strains from twins were genotyped as identical or different. CD4+ T-cell responses to HSV-1 proteins were studied., Results: The median per person oral HSV shedding rate was 9% of days that a swab was obtained (mean, 10.2% of days). A positive correlation between shedding rates was observed within all twin pairs, and in the MZ and DZ twins. In twin subsets with sufficient HSV-1 DNA to genotype, 15 had the same strain and 14 had different strains. Viral shedding rates were correlated for those with the same but not different strains. The median number of HSV-1 open reading frames recognized per person was 16. The agreement in the CD4+ T-cell response to specific HSV-1 open reading frames was greater between MZ twins than between unrelated persons (P = .002)., Conclusion: Viral strain characteristics likely contribute to oral HSV-1 shedding rates., (© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Human herpesvirus 6B (HHV-6B) DNA is frequently detected in human samples. Diagnostic assays distinguishing HHV-6B reactivation from latency are limited. This has impaired strategies to diagnose and treat HHV-6B-associated diseases. We used RNA sequencing to characterize and compare the HHV-6B transcriptome in multiple sample types, including (i) whole blood from hematopoietic cell transplant (HCT) recipients with and without HHV-6B plasma viremia, (ii) tumor tissue samples from subjects with large B cell lymphoma infected with HHV-6B, (iii) lymphoblastoid cell lines (LCLs) from subjects with inherited chromosomally integrated HHV-6B or latent infection with HHV-6B, and (iv) HHV-6B Z29 infected SupT1 CD4 + T cells. We demonstrated substantial overlap in the HHV-6B transcriptome observed in in vivo and in vitro samples, although there was variability in the breadth and quantity of gene expression across samples. The HHV-6B viral polymerase gene U38 was the only HHV-6B transcript detected in all next-generation RNA sequencing (RNA-seq) data sets and was one of the most highly expressed genes. We developed a novel reverse transcription-PCR assay targeting HHV-6B U38, which identified U38 mRNA in all tested whole-blood samples from patients with concurrent HHV-6B viremia. No HHV-6B U38 transcripts were detected by RNA-seq or reverse transcription-real-time quantitative PCR (RT-qPCR) in whole-blood samples from subjects without HHV-6B plasma detection or from latently infected LCLs. A RT-qPCR assay for HHV-6B U38 may be useful to identify lytic HHV-6B infection in nonplasma samples and samples from individuals with inherited chromosomally integrated HHV-6B. This study also demonstrates the feasibility of transcriptomic analyses for HCT recipients. IMPORTANCE Human herpesvirus 6B (HHV-6B) is a DNA virus that infects most children within the first few years of life. After primary infection, HHV-6B persists as a chronic, latent infection in many cell types. Additionally, HHV-6B can integrate into germ line chromosomes, resulting in individuals with viral DNA in every nucleated cell. Given that PCR to detect viral DNA is the mainstay for diagnosing HHV-6B infection, the characteristics of HHV-6B infection complicate efforts to distinguish between latent and active viral infection, particularly in immunocompromised patients who have frequent HHV-6B reactivation. In this study, we used RNA sequencing to characterize the HHV-6B gene expression profile in multiple sample types, and our findings identified evidence-based targets for diagnostic tests that distinguish between latent and active viral infection., (Copyright © 2019 American Society for Microbiology.)

Parainfluenza virus (PIV) infection can progress from upper respiratory tract infection (URTI) to lower respiratory tract disease (LRTD) in immunocompromised hosts. Risk factors for progression to LRTD and presentation with LRTD without prior URTI are poorly defined. Hematopoietic cell transplant (HCT) recipients with PIV infection were retrospectively analyzed using standardized definitions of LRTD. PIV was detected in 540 HCT recipients; 343 had URTI alone and 197 (36%) had LRTD (possible, 76; probable, 19; proven, 102). Among 476 patients with positive nasopharyngeal samples, the cumulative incidence of progression to probable/proven LRTD by day 40 was 12%, with a median time to progression of 7 days (range, 2 to 40). In multivariable analysis monocytopenia (hazard ratio, 2.22; P = .011), steroid use ≥1mg/kg prior to diagnosis (hazard ratio, 1.89; P = .018), co-pathogen detection in blood (hazard ratio, 3.21; P = .027), and PIV type 3 (hazard ratio, 3.57; P = .032) were associated with increased progression risk. In the absence of all 4 risk factors no patients progressed to LRTD, whereas progression risk increased to >30% if 3 or more risk factors were present. Viral load or ribavirin use appeared to have no effect on progression. Among 121 patients with probable/proven LRTD, 64 (53%) presented LRTD without prior URTI, and decreased lung function before infection and lower respiratory co-pathogens were risk factors for this presentation. Mortality was unaffected by the absence of prior URTI. We conclude that the risk of progression to probable/proven LRTD exceeded 30% with ≥3 risk factors. To detect all cases of LRTD, virologic testing of lower respiratory samples is required regardless of URTI symptoms., (Copyright © 2018. Published by Elsevier Inc.)

HIV drug resistance genotyping is a critical tool in the clinical management of HIV infections. Although resistance genotyping has traditionally been conducted using Sanger sequencing, next-generation sequencing (NGS) is emerging as a powerful tool due to its ability to detect low-frequency alleles. However, the clinical value added from NGS approaches to antiviral resistance testing remains to be demonstrated. We compared the variant detection capacity of NGS versus Sanger sequencing methods for resistance genotyping in 144 drug resistance tests (105 protease-reverse transcriptase tests and 39 integrase tests) submitted to our clinical virology laboratory over a four-month period in 2016 for Sanger-based HIV drug resistance testing. NGS detected all true high-frequency drug resistance mutations (>20% frequency) found by Sanger sequencing, with greater accuracy in one instance of a Sanger-detected false positive. Freely available online NGS variant callers HyDRA and PASeq were superior to Sanger methods for interpretations of allele linkage and automated variant calling. NGS additionally detected low-frequency mutations (1 to 20% frequency) associated with higher levels of drug resistance in 30/105 (29%) protease-reverse transcriptase tests and 4/39 (10%) integrase tests. In clinical follow-up of 69 individuals for a median of 674 days, we did not find a difference in rates of virological failure between individuals with and without low-frequency mutations, although rates of virological failure were higher for individuals with drug-relevant low-frequency mutations. However, all 27 individuals who experienced virological failure reported poor adherence to their drug regimen during the preceding follow-up time, and all 19 who subsequently improved their adherence achieved viral suppression at later time points, consistent with a lack of clinical resistance. In conclusion, in a population with low antiviral resistance emergence, NGS methods detected numerous instances of minor alleles that did not result in subsequent bona fide virological failure due to antiviral resistance., (Copyright © 2018 Dalmat et al.)