SARS-CoV-2 Outbreak Dynamics in an Isolated US Military Recruit Training Center With Rigorous Prevention Measures.

BACKGROUND: Marine recruits training at Parris Island experienced an unexpectedly high rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, despite preventive measures including a supervised, 2-week, pre-entry quarantine. We characterize SARS-CoV-2 transmission in this cohort. METHODS: Between May and November 2020, we monitored 2,469 unvaccinated, mostly male, Marine recruits prospectively during basic training. If participants tested negative for SARS-CoV-2 by quantitative polymerase chain reaction (qPCR) at the end of quarantine, they were transferred to the training site in segregated companies and underwent biweekly testing for 6 weeks. We assessed the effects of coronavirus disease 2019 (COVID-19) prevention measures on other respiratory infections with passive surveillance data, performed phylogenetic analysis, and modeled transmission dynamics and testing regimens. RESULTS: Preventive measures were associated with drastically lower rates of other respiratory illnesses. However, among the trainees, 1,107 (44.8%) tested SARS-CoV-2-positive, with either mild or no symptoms. Phylogenetic analysis of viral genomes from 580 participants revealed that all cases but one were linked to five independent introductions, each characterized by accumulation of mutations across and within companies, and similar viral isolates in individuals from the same company. Variation in company transmission rates (mean reproduction number R 0 ; 5.5 [95% confidence interval [CI], 5.0, 6.1]) could be accounted for by multiple initial cases within a company and superspreader events. Simulations indicate that frequent rapid-report testing with case isolation may minimize outbreaks. CONCLUSIONS: Transmission of wild-type SARS-CoV-2 among Marine recruits was approximately twice that seen in the community. Insights from SARS-CoV-2 outbreak dynamics and mutations spread in a remote, congregate setting may inform effective mitigation strategies.

Phage Therapy for Limb-threatening Prosthetic Knee Klebsiella pneumoniae Infection: Case Report and In Vitro Characterization of Anti-biofilm Activity.

BACKGROUND: Prosthetic joint infection (PJI) is a potentially limb-threatening complication of total knee arthroplasty. Phage therapy is a promising strategy to manage such infections including those involving antibiotic-resistant microbes, and to target microbial biofilms. Experience with phage therapy for infections associated with retained hardware is limited. A 62-year-old diabetic man with a history of right total knee arthroplasty 11 years prior who had suffered multiple episodes of prosthetic knee infection despite numerous surgeries and prolonged courses of antibiotics, with progressive clinical worsening and development of severe allergies to antibiotics, had been offered limb amputation for persistent right prosthetic knee infection due to Klebsiella pneumoniae complex. Intravenous phage therapy was initiated as a limb-salvaging intervention. METHODS: The patient received 40 intravenous doses of a single phage (KpJH46Φ2) targeting his bacterial isolate, alongside continued minocycline (which he had been receiving when he developed increasing pain, swelling, and erythema prior to initiation of phage therapy). Serial cytokine and biomarker measurements were performed before, during, and after treatment. The in vitro anti-biofilm activity of KpJH46Φ2, minocycline and the combination thereof was evaluated against a preformed biofilm of the patient's isolate and determined by safranin staining. RESULTS: Phage therapy resulted in resolution of local symptoms and signs of infection and recovery of function. The patient did not experience treatment-related adverse effects and remained asymptomatic 34 weeks after completing treatment while still receiving minocycline. A trend in biofilm biomass reduction was noted 22 hours after exposure to KpJH46Φ2 (P = .063). The addition of phage was associated with a satisfactory outcome in this case of intractable biofilm-associated prosthetic knee infection. Pending further studies to assess its efficacy and safety, phage therapy holds promise for treatment of device-associated infections.

Successful Treatment of Antibiotic-resistant, Poly-microbial Bone Infection With Bacteriophages and Antibiotics Combination.

A patient with a trauma-related left tibial infection associated with extensively drug-resistant Acinetobacter baumannii and multidrug-resistant Klebsiella pneumoniae was treated with bacteriophages and antibiotics. There was rapid tissue healing and positive culture eradication. As a result, the patient's leg did not have to be amputated and he is undergoing rehabilitation.

The yeast 5'-3' exonuclease Rat1p functions during transcription elongation by RNA polymerase II.

Termination of RNA polymerase II (RNAPII) transcription of protein-coding genes occurs downstream of cleavage/polyadenylation sites. According to the "torpedo" model, the 5'-3' exonuclease Rat1p/Xrn2p attacks the newly formed 5' end of the cleaved pre-mRNA, causing the still transcribing RNAPII to terminate. Here we demonstrate a similar role of S. cerevisiae Rat1p within the gene body. We find that the transcription processivity defect imposed on RNAPII by the rpb1-N488D mutation is corrected upon Rat1p inactivation. Importantly, Rat1p-dependent transcription termination occurs upstream the polyadenylation site. Genetic and biochemical evidence demonstrate that mRNA capping is defective in rpb1-N488D cells, which leads to increased levels of Rat1p all along the gene locus. Consistently, Rat1p-dependent RNAPII termination is also observed in the capping-deficient ceg1-63 strain. Our data suggest that Rat1p serves to terminate RNAPII molecules engaged in the production of uncapped RNA, regardless of their position on the gene locus.

Benefits of Emergency Departments' Contribution to Stroke Prophylaxis in Atrial Fibrillation: The EMERG-AF Study (Emergency Department Stroke Prophylaxis and Guidelines Implementation in Atrial Fibrillation).

BACKGROUND AND PURPOSE: Long-term benefits of initiating stroke prophylaxis in the emergency department (ED) are unknown. We analyzed the long-term safety and benefits of ED prescription of anticoagulation in atrial fibrillation patients. METHODS: Prospective, multicenter, observational cohort of consecutive atrial fibrillation patients was performed in 62 Spanish EDs. Clinical variables and thromboprophylaxis prescribed at discharge were collected at inclusion. Follow-up at 1 year post-discharge included data about thromboprophylaxis and its complications, major bleeding, and death; risk was assessed with univariate and bivariate logistic regression models. RESULTS: We enrolled 1162 patients, 1024 (88.1%) at high risk according to CHA2DS2-VASc score. At ED discharge, 935 patients (80.5%) were receiving anticoagulant therapy, de novo in 237 patients (55.2% of 429 not previously treated). At 1 year, 48 (4.1%) patients presented major bleeding events, and 151 (12.9%) had died. Anticoagulation first prescribed in the ED was not related to major bleeding (hazard ratio, 0.976; 95% confidence interval, 0.294-3.236) and was associated with a decrease in mortality (hazard ratio, 0.398; 95% confidence interval, 0.231-0.686). Adjusting by the main clinical and sociodemographic characteristics, concomitant antiplatelet treatment, or destination (discharge or admission) did not affect the results. CONCLUSIONS: Prescription of anticoagulation in the ED does not increase bleeding risk in atrial fibrillation patients at high risk of stroke and contributes to decreased mortality.

Rat1p maintains RNA polymerase II CTD phosphorylation balance.

In S. cerevisiae, the 5'-3' exonuclease Rat1p partakes in transcription termination. Although Rat1p-mediated RNA degradation has been suggested to play a role for this activity, the exact mechanisms by which Rat1p helps release RNA polymerase II (RNAPII) from the DNA template are poorly understood. Here we describe a function of Rat1p in regulating phosphorylation levels of the C-terminal domain (CTD) of the largest RNAPII subunit, Rpb1p, during transcription elongation. The rat1-1 mutant exhibits highly elevated levels of CTD phosphorylation as well as RNAPII distribution and transcription termination defects. These phenotypes are all rescued by overexpression of the CTD phosphatase Fcp1p, suggesting a functional relationship between the absence of Rat1p activity, elevated CTD phosphorylation, and transcription defects. We also demonstrate that rat1-1 cells display increased RNAPII transcription kinetics, a feature that may contribute to the cellular phenotypes of the mutant. Consistently, the rat1-1 allele is synthetic lethal with the rpb1-E1103G mutation, causing increased RNAPII speed, and is suppressed by the rpb2-10 mutation, causing slowed transcription. Thus, Rat1p plays more complex roles in controlling transcription than previously thought.

Transient reversal of RNA polymerase II active site closing controls fidelity of transcription elongation.

To study fidelity of RNA polymerase II (Pol II), we analyzed properties of the 6-azauracil-sensitive and TFIIS-dependent E1103G mutant of rbp1 (rpo21), the gene encoding the catalytic subunit of Pol II in Saccharomyces cerevisiae. Using an in vivo retrotransposition-based transcription fidelity assay, we observed that rpb1-E1103G causes a 3-fold increase in transcription errors. This mutant showed a 10-fold decrease in fidelity of transcription elongation in vitro. The mutation does not appear to significantly affect translocation state equilibrium of Pol II in a stalled elongation complex. Primarily, it promotes NTP sequestration in the polymerase active center. Furthermore, pre-steady-state analyses revealed that the E1103G mutation shifted the equilibrium between the closed and the open active center conformations toward the closed form. Thus, open conformation of the active center emerges as an intermediate essential for preincorporation fidelity control. Similar mechanisms may control fidelity of DNA-dependent DNA polymerases and RNA-dependent RNA polymerases.

RNase III is required for localization to the nucleoid of the 5' pre-rRNA leader and for optimal induction of rRNA synthesis in E. coli.

It has recently been demonstrated that ribosomes are preferentially localized outside the nucleoid in Escherichia coli, but little is known about the spatial regulation of pre-rRNA processing. In this work, I investigate the cellular distribution of leader pre-rRNAs using RNA-FISH. In contrast to mature rRNA, the 5' proximal leader region associates with the nucleoid, and this association occurs in an RNase III-dependent manner. Moreover, RNase III plays a role in the rapid induction of ribosomal operons during outgrowth and is essential in the absence of the transcriptional regulator Fis, suggesting a linkage of transcription and RNA processing for ribosomal operons in E. coli.

Mutational analysis of the yeast RNA helicase Sub2p reveals conserved domains required for growth, mRNA export, and genomic stability.

Sub2p/UAP56 is a highly conserved DEAD-box RNA helicase involved in the packaging and nuclear export of mRNA/protein particles (mRNPs). In Saccharomyces cerevisiae, Sub2p is recruited to active chromatin by the pentameric THO complex and incorporated into the larger transcription-export (TREX) complex. Sub2p also plays a role in the maintenance of genome integrity as its inactivation causes severe transcription-dependent recombination of DNA. Despite the central role of Sub2p in early mRNP biology, little is known about its function. Here, we report the presence of an N-terminal motif (NTM) conserved specifically in the Sub2p branch of RNA helicases. Mutation of the NTM causes nuclear accumulation of poly(A)(+) RNA and impaired growth without affecting core helicase functions. Thus, the NTM functions as an autonomous unit. Moreover, two sub2 mutants, that are deficient in ATP binding, act in a trans-dominant negative fashion for growth and induce high recombination rates in vivo. Although wild-type Sub2p is prevented access to transcribed loci in such a background, this does not mechanistically explain the phenotype.

Stroke prophylaxis in atrial fibrillation: searching for management improvement opportunities in the emergency department: the HERMES-AF study.

STUDY OBJECTIVE: We determine the prevalence of stroke prophylaxis prescription in emergency department (ED) patients with atrial fibrillation and the factors associated with a lack of prescription of anticoagulation in high-risk patients without contraindications. METHODS: This was a multicenter, observational, cross-sectional study with prospective standardized data collection carried out in 124 Spanish EDs. Clinical variables, risk factors for stroke, type of prophylaxis prescribed, and reasons for not prescribing anticoagulation in high-risk patients (congestive heart failure/left ventricular dysfunction, hypertension, age >75 years, diabetes and previous stroke/transient ischemic attack/systemic embolism [CHADS2] score ≥2 and the congestive heart failure/left ventricular dysfunction, hypertension, age >75 years, diabetes, previous stroke/transient ischemic attack/systemic embolism, vascular disease age 65 to 74 years and sex category [CHA2DS2-VASc] score ≥2) without contraindications were collected. RESULTS: Of 3,276 patients enrolled, 71.5% were at high risk according to CHADS2; 89.7% according to CHA2DS2-VASc. At discharge from the ED, 2,255 patients (68.8%) were receiving anticoagulants, 1,691 of whom (75%) were high-risk patients. Of the 1,931 patients discharged home, anticoagulation was prescribed for 384 patients (19.9%) de novo and for 932 patients (48.3%) previously receiving anticoagulation. The main reasons for not prescribing anticoagulation to eligible patients were considering antiplatelet therapy as adequate prophylaxis (33.1%), advanced age (15%), and considering stroke risk as low (8.3%). Advanced age (odds ratio 0.46; 95% confidence interval 0.30 to 0.69) and female sex (odds ratio 0.50; 95% confidence interval 0.36 to 0.71) were significantly associated with the lack of prescription of anticoagulation to eligible patients. CONCLUSION: In Spain, most patients with atrial fibrillation treated in EDs who do not receive anticoagulation are at high risk of stroke, with relevant differences with regard to the risk stratification scheme used. Anticoagulation is underused, mainly because the risk of stroke is underestimated by the treating physicians and the benefits of antiplatelets are overrated, principally in female patients and the elderly. Efforts to increase the prescription of anticoagulation in these patients appear warranted.

The fidelity of transcription: RPB1 (RPO21) mutations that increase transcriptional slippage in S. cerevisiae.

The fidelity of RNA synthesis depends on both accurate template-mediated nucleotide selection and proper maintenance of register between template and RNA. Loss of register, or transcriptional slippage, is particularly likely on homopolymeric runs in the template. Transcriptional slippage can alter the coding capacity of mRNAs and is used as a regulatory mechanism. Here we describe mutations in the largest subunit of Saccharomyces cerevisiae RNA polymerase II that substantially increase the level of transcriptional slippage. Alleles of RPB1 (RPO21) with elevated slippage rates were identified among 6-azauracil-sensitive mutants and were also isolated using a slippage-dependent reporter gene. Biochemical characterization of polymerase II isolated from these mutants confirms elevated levels of transcriptional slippage.

Immune and behavioral correlates of protection against symptomatic post-vaccination SARS-CoV-2 infection.

Introduction: We sought to determine pre-infection correlates of protection against SARS-CoV-2 post-vaccine inzfections (PVI) acquired during the first Omicron wave in the United States. Methods: Serum and saliva samples from 176 vaccinated adults were collected from October to December of 2021, immediately before the Omicron wave, and assessed for SARS-CoV-2 Spike-specific IgG and IgA binding antibodies (bAb). Sera were also assessed for bAb using commercial assays, and for neutralization activity against several SARS-CoV-2 variants. PVI duration and severity, as well as risk and precautionary behaviors, were assessed by questionnaires. Results: Serum anti-Spike IgG levels assessed by research assay, neutralization titers against Omicron subvariants, and low home risk scores correlated with protection against PVIs after multivariable regression analysis. Commercial assays did not perform as well as research assay, likely due to their lower dynamic range. Discussion: In the 32 participants that developed PVI, anti-Spike IgG bAbs correlated with lower disease severity and shorter duration of illness.

A Recombinant Chimeric Cedar Virus-Based Surrogate Neutralization Assay Platform for Pathogenic Henipaviruses.

The henipaviruses, Nipah virus (NiV), and Hendra virus (HeV) can cause fatal diseases in humans and animals, whereas Cedar virus is a nonpathogenic henipavirus. Here, using a recombinant Cedar virus (rCedV) reverse genetics platform, the fusion (F) and attachment (G) glycoprotein genes of rCedV were replaced with those of NiV-Bangladesh (NiV-B) or HeV, generating replication-competent chimeric viruses (rCedV-NiV-B and rCedV-HeV), both with and without green fluorescent protein (GFP) or luciferase protein genes. The rCedV chimeras induced a Type I interferon response and utilized only ephrin-B2 and ephrin-B3 as entry receptors compared to rCedV. The neutralizing potencies of well-characterized cross-reactive NiV/HeV F and G specific monoclonal antibodies against rCedV-NiV-B-GFP and rCedV-HeV-GFP highly correlated with measurements obtained using authentic NiV-B and HeV when tested in parallel by plaque reduction neutralization tests (PRNT). A rapid, high-throughput, and quantitative fluorescence reduction neutralization test (FRNT) using the GFP-encoding chimeras was established, and monoclonal antibody neutralization data derived by FRNT highly correlated with data derived by PRNT. The FRNT assay could also measure serum neutralization titers from henipavirus G glycoprotein immunized animals. These rCedV chimeras are an authentic henipavirus-based surrogate neutralization assay that is rapid, cost-effective, and can be utilized outside high containment.

Genomic Surveillance of Rabies Virus in Georgian Canines.

Rabies is a fatal zoonosis that is considered a re-emerging infectious disease. Although rabies remains endemic in canines throughout much of the world, vaccination programs have essentially eliminated dog rabies in the Americas and much of Europe. However, despite the goal of eliminating dog rabies in the European Union by 2020, sporadic cases of dog rabies still occur in Eastern Europe, including Georgia. To assess the genetic diversity of the strains recently circulating in Georgia, we sequenced seventy-eight RABV-positive samples from the brain tissues of rabid dogs and jackals using Illumina short-read sequencing of total RNA shotgun libraries. Seventy-seven RABV genomes were successfully assembled and annotated, with seventy-four of them reaching the coding-complete status. Phylogenetic analyses of the nucleoprotein (N) and attachment glycoprotein (G) genes placed all the assembled genomes into the Cosmopolitan clade, consistent with the Georgian origin of the samples. An amino acid alignment of the G glycoprotein ectodomain identified twelve different sequences for this domain among the samples. Only one of the ectodomain groups contained a residue change in an antigenic site, an R264H change in the G5 antigenic site. Three isolates were cultured, and these were found to be efficiently neutralized by the human monoclonal antibody A6. Overall, our data show that recently circulating RABV isolates from Georgian canines are predominantly closely related phylogroup I viruses of the Cosmopolitan clade. Current human rabies vaccines should offer protection against infection by Georgian canine RABVs. The genomes have been deposited in GenBank (accessions: OQ603609-OQ603685).

Establishment of SARS-CoV-2 genomic surveillance within the Military Health System during 1 March-31 December 2020.

This report describes SARS-CoV-2 genomic surveillance conducted by the Department of Defense (DoD) Global Emerging Infections Surveillance Branch and the Next-Generation Sequencing and Bioinformatics Consortium (NGSBC) in response to the COVID-19 pandemic. Samples and sequence data were from SARS-CoV-2 infections occurring among Military Health System (MHS) beneficiaries from 1 March to 31 December 2020. There were 1,366 MHS samples sequenced from 10 countries, 36 U.S states or territories, and 5 Geographic Combatant Commands, representing approximately 2% of DoD cases in 2020. Genomes from these samples were compared with other public sequences; observed trends were similar to those of Centers for Disease Control and Prevention national surveillance in the U.S. with B.1, B.1.2, and other sub-lineages comprising the dominant variants of SARS-CoV-2. Sequence data were used to monitor transmission dynamics on U.S. Navy ships and at military training centers and installations. As new variants emerge, DoD medical and public health practitioners should maximize the use of genomic surveillance resources within DoD to inform force health protection measures.

Genomic and virologic characterization of samples from a shipboard outbreak of COVID-19 reveals distinct variants within limited temporospatial parameters.

Early in the pandemic, in March of 2020, an outbreak of COVID-19 occurred aboard the aircraft carrier USS Theodore Roosevelt (CVN-71), during deployment in the Western Pacific. Out of the crew of 4,779 personnel, 1,331 service members were suspected or confirmed to be infected with SARS-CoV-2. The demographic, epidemiologic, and laboratory findings of service members from subsequent investigations have characterized the outbreak as widespread transmission of virus with relatively mild symptoms and asymptomatic infection among mostly young healthy adults. At the time, there was no available vaccination against COVID-19 and there was very limited knowledge regarding SARS-CoV-2 mutation, dispersal, and transmission patterns among service members in a shipboard environment. Since that time, other shipboard outbreaks from which data can be extracted have occurred, but these later shipboard outbreaks have occurred largely in settings where the majority of the crew were vaccinated, thereby limiting spread of the virus, shortening duration of the outbreaks, and minimizing evolution of the virus within those close quarters settings. On the other hand, since the outbreak on the CVN-71 occurred prior to widespread vaccination, it continued over the course of roughly two months, infecting more than 25% of the crew. In order to better understand genetic variability and potential transmission dynamics of COVID-19 in a shipboard environment of immunologically naïve, healthy individuals, we performed whole-genome sequencing and virus culture from eighteen COVID-19-positive swabs collected over the course of one week. Using the unique variants identified in those genomes, we detected seven discrete groups of individuals within the population aboard CVN-71 infected with viruses of distinct genomic signature. This is in stark contrast to a recent outbreak aboard another U.S. Navy ship with >98% vaccinated crew after a port visit in Reykjavik, Iceland, where the outbreak lasted only approximately 2 weeks and the virus was clonal. Taken together, these results demonstrate the utility of sequencing from complex clinical samples for molecular epidemiology and they also suggest that a high rate of vaccination among a population in close communities may greatly reduce spread, thereby restricting evolution of the virus.

SARS-CoV-2 Infections in Vaccinated and Unvaccinated Populations in Camp Lemonnier, Djibouti, from April 2020 to January 2022.

The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the disparity between developed and developing countries for infectious disease surveillance and the sequencing of pathogen genomes. The majority of SARS-CoV-2 sequences published are from Europe, North America, and Asia. Between April 2020 and January 2022, 795 SARS-CoV-2-positive nares swabs from individuals in the U.S. Navy installation Camp Lemonnier, Djibouti, were collected, sequenced, and analyzed. In this study, we described the results of genomic sequencing and analysis for 589 samples, the first published viral sequences for Djibouti, including 196 cases of vaccine breakthrough infections. This study contributes to the knowledge base of circulating SARS-CoV-2 lineages in the under-sampled country of Djibouti, where only 716 total genome sequences are available at time of publication. Our analysis resulted in the detection of circulating variants of concern, mutations of interest in lineages in which those mutations are not common, and emerging spike mutations.

Prospective Assessment of Symptoms to Evaluate Asymptomatic SARS-CoV-2 Infections in a Cohort of Health Care Workers.

BACKGROUND: The frequency of asymptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is unclear and may be influenced by how symptoms are evaluated. In this study, we sought to determine the frequency of asymptomatic SARS-CoV-2 infections in a prospective cohort of health care workers (HCWs). METHODS: A prospective cohort of HCWs, confirmed negative for SARS-CoV-2 exposure upon enrollment, were evaluated for SARS-CoV-2 infection by monthly analysis of SARS-CoV-2 antibodies as well as referral for polymerase chain reaction testing whenever they exhibited symptoms of coronavirus disease 2019 (COVID-19). Participants completed the standardized and validated FLU-PRO Plus symptom questionnaire scoring viral respiratory disease symptom intensity and frequency at least twice monthly during baseline periods of health and each day they had any symptoms that were different from their baseline. RESULTS: Two hundred sixty-three participants were enrolled between August 25 and December 31, 2020. Through February 28, 2021, 12 participants were diagnosed with SARS-CoV-2 infection. Symptom analysis demonstrated that all 12 had at least mild symptoms of COVID-19, compared with baseline health, near or at time of infection. CONCLUSIONS: These results suggest that asymptomatic SARS-CoV-2 infection in unvaccinated, immunocompetent adults is less common than previously reported. While infectious inoculum doses and patient factors may have played a role in the clinical manifestations of SARS-CoV-2 infections in this cohort, we suspect that the high rate of symptomatic disease was due primarily to participant attentiveness to symptoms and collection of symptoms in a standardized, prospective fashion. These results have implications for studies that estimate SARS-CoV-2 infection prevalence and for public health measures to control the spread of this virus.