Transmission from vaccinated individuals in a large SARS-CoV-2 Delta variant outbreak.

An outbreak of over 1,000 COVID-19 cases in Provincetown, Massachusetts (MA), in July 2021-the first large outbreak mostly in vaccinated individuals in the US-prompted a comprehensive public health response, motivating changes to national masking recommendations and raising questions about infection and transmission among vaccinated individuals. To address these questions, we combined viral genomic and epidemiological data from 467 individuals, including 40% of outbreak-associated cases. The Delta variant accounted for 99% of cases in this dataset; it was introduced from at least 40 sources, but 83% of cases derived from a single source, likely through transmission across multiple settings over a short time rather than a single event. Genomic and epidemiological data supported multiple transmissions of Delta from and between fully vaccinated individuals. However, despite its magnitude, the outbreak had limited onward impact in MA and the US overall, likely due to high vaccination rates and a robust public health response.

Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant.

The SARS-CoV-2 spike (S) protein variant D614G supplanted the ancestral virus worldwide, reaching near fixation in a matter of months. Here we show that D614G was more infectious than the ancestral form on human lung cells, colon cells, and on cells rendered permissive by ectopic expression of human ACE2 or of ACE2 orthologs from various mammals, including Chinese rufous horseshoe bat and Malayan pangolin. D614G did not alter S protein synthesis, processing, or incorporation into SARS-CoV-2 particles, but D614G affinity for ACE2 was reduced due to a faster dissociation rate. Assessment of the S protein trimer by cryo-electron microscopy showed that D614G disrupts an interprotomer contact and that the conformation is shifted toward an ACE2 binding-competent state, which is modeled to be on pathway for virion membrane fusion with target cells. Consistent with this more open conformation, neutralization potency of antibodies targeting the S protein receptor-binding domain was not attenuated.

Whole genome sequencing of human Borrelia burgdorferi isolates reveals linked blocks of accessory genome elements located on plasmids and associated with human dissemination.

Lyme disease is the most common vector-borne disease in North America and Europe. The clinical manifestations of Lyme disease vary based on the genospecies of the infecting Borrelia burgdorferi spirochete, but the microbial genetic elements underlying these associations are not known. Here, we report the whole genome sequence (WGS) and analysis of 299 B. burgdorferi (Bb) isolates derived from patients in the Eastern and Midwestern US and Central Europe. We develop a WGS-based classification of Bb isolates, confirm and extend the findings of previous single- and multi-locus typing systems, define the plasmid profiles of human-infectious Bb isolates, annotate the core and strain-variable surface lipoproteome, and identify loci associated with disseminated infection. A core genome consisting of ~900 open reading frames and a core set of plasmids consisting of lp17, lp25, lp36, lp28-3, lp28-4, lp54, and cp26 are found in nearly all isolates. Strain-variable (accessory) plasmids and genes correlate strongly with phylogeny. Using genetic association study methods, we identify an accessory genome signature associated with dissemination in humans and define the individual plasmids and genes that make up this signature. Strains within the RST1/WGS A subgroup, particularly a subset marked by the OspC type A genotype, have increased rates of dissemination in humans. OspC type A strains possess a unique set of strongly linked genetic elements including the presence of lp56 and lp28-1 plasmids and a cluster of genes that may contribute to their enhanced virulence compared to other genotypes. These features of OspC type A strains reflect a broader paradigm across Bb isolates, in which near-clonal genotypes are defined by strain-specific clusters of linked genetic elements, particularly those encoding surface-exposed lipoproteins. These clusters of genes are maintained by strain-specific patterns of plasmid occupancy and are associated with the probability of invasive infection.

Inferring selection effects in SARS-CoV-2 with Bayesian Viral Allele Selection.

The global effort to sequence millions of SARS-CoV-2 genomes has provided an unprecedented view of viral evolution. Characterizing how selection acts on SARS-CoV-2 is critical to developing effective, long-lasting vaccines and other treatments, but the scale and complexity of genomic surveillance data make rigorous analysis challenging. To meet this challenge, we develop Bayesian Viral Allele Selection (BVAS), a principled and scalable probabilistic method for inferring the genetic determinants of differential viral fitness and the relative growth rates of viral lineages, including newly emergent lineages. After demonstrating the accuracy and efficacy of our method through simulation, we apply BVAS to 6.9 million SARS-CoV-2 genomes. We identify numerous mutations that increase fitness, including previously identified mutations in the SARS-CoV-2 Spike and Nucleocapsid proteins, as well as mutations in non-structural proteins whose contribution to fitness is less well characterized. In addition, we extend our baseline model to identify mutations whose fitness exhibits strong dependence on vaccination status as well as pairwise interaction effects, i.e. epistasis. Strikingly, both these analyses point to the pivotal role played by the N501 residue in the Spike protein. Our method, which couples Bayesian variable selection with a diffusion approximation in allele frequency space, lays a foundation for identifying fitness-associated mutations under the assumption that most alleles are neutral.

Analysis of the Borreliaceae Pangenome Reveals a Distinct Genomic Architecture Conserved Across Phylogenetic Scales.

The family Borreliaceae contains arthropod-borne spirochetes that cause two widespread human diseases, Lyme disease and relapsing fever. Lyme disease is a subacute, progressive illness with variable stage and tissue manifestations. Relapsing fever is an acute febrile illness with prominent bacteremia that may recur and disseminate, particularly to the nervous system. Clinical heterogeneity is a hallmark of both diseases. While human clinical manifestations are influenced by a wide variety of factors, including immune status and host genetic susceptibility, there is evidence that Borreliaceae microbial factors influence the clinical manifestations of human disease caused by this family of spirochetes. Despite these associations, the spirochete genes that influence the severity and manifestations of human disease are, for the most part, unknown. Recent work has identified lineage-specific expansions of lipoproteome-rich accessory genome elements in virulent clones of Borrelia burgdorferi. Using publicly available genome assemblies, it is shown that all Borreliaceae lineages for which sufficient sequence data are available harbor a similar pattern of strongly structured, lineage-specific expansions in their accessory genomes, particularly among lipoproteins, and that this pattern holds across phylogenetic scales including genera, species, and genotypes. The relationships among pangenome elements suggest that infrequent episodes of marked genomic change followed by clonal expansion in geographically and enzootically structured populations may account for the unique lineage structure of Borreliaceae. This analysis informs future genotype-phenotype studies among Borreliaceae and lays a foundation for studies of individual gene function guided by phylogenetic patterns of conservation, diversification, gain, and/or loss.

SARS-CoV-2 Virologic Rebound With Nirmatrelvir-Ritonavir Therapy : An Observational Study.

BACKGROUND: Data are conflicting regarding an association between treatment of acute COVID-19 with nirmatrelvir-ritonavir (N-R) and virologic rebound (VR). OBJECTIVE: To compare the frequency of VR in patients with and without N-R treatment for acute COVID-19. DESIGN: Observational cohort study. SETTING: Multicenter health care system in Boston, Massachusetts. PARTICIPANTS: Ambulatory adults with acute COVID-19 with and without use of N-R. INTERVENTION: Receipt of 5 days of N-R treatment versus no COVID-19 therapy. MEASUREMENTS: The primary outcome was VR, defined as either a positive SARS-CoV-2 viral culture result after a prior negative result or 2 consecutive viral loads above 4.0 log10 copies/mL that were also at least 1.0 log10 copies/mL higher than a prior viral load below 4.0 log10 copies/mL. RESULTS: Compared with untreated persons (n = 55), those taking N-R (n = 72) were older, received more COVID-19 vaccinations, and more commonly had immunosuppression. Fifteen participants (20.8%) taking N-R had VR versus 1 (1.8%) who was untreated (absolute difference, 19.0 percentage points [95% CI, 9.0 to 29.0 percentage points]; P = 0.001). All persons with VR had a positive viral culture result after a prior negative result. In multivariable models, only N-R use was associated with VR (adjusted odds ratio, 10.02 [CI, 1.13 to 88.74]; P = 0.038). Virologic rebound was more common among those who started therapy within 2 days of symptom onset (26.3%) than among those who started 2 or more days after symptom onset (0%) (P = 0.030). Among participants receiving N-R, those who had VR had prolonged shedding of replication-competent virus compared with those who did not have VR (median, 14 vs. 3 days). Eight of 16 participants (50% [CI, 25% to 75%]) with VR also reported symptom rebound; 2 were completely asymptomatic. No post-VR resistance mutations were detected. LIMITATIONS: Observational study design with differences between the treated and untreated groups; positive viral culture result was used as a surrogate marker for risk for ongoing viral transmission. CONCLUSION: Virologic rebound occurred in approximately 1 in 5 people taking N-R, often without symptom rebound, and was associated with shedding of replication-competent virus. PRIMARY FUNDING SOURCE: National Institutes of Health.

Characterization of Virologic Rebound Following Nirmatrelvir-Ritonavir Treatment for Coronavirus Disease 2019 (COVID-19).

We enrolled 7 individuals with recurrent symptoms or antigen test conversion following nirmatrelvir-ritonavir treatment. High viral loads (median 6.1 log10 copies/mL) were detected after rebound for a median of 17 days after initial diagnosis. Three had culturable virus for up to 16 days after initial diagnosis. No known resistance-associated mutations were identified.

Distinguishing Severe Acute Respiratory Syndrome Coronavirus 2 Persistence and Reinfection: A Retrospective Cohort Study.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfection is poorly understood, partly because few studies have systematically applied genomic analysis to distinguish reinfection from persistent RNA detection related to initial infection. We aimed to evaluate the characteristics of SARS-CoV-2 reinfection and persistent RNA detection using independent genomic, clinical, and laboratory assessments. METHODS: All individuals at a large academic medical center who underwent a SARS-CoV-2 nucleic acid amplification test (NAAT) ≥45 days after an initial positive test, with both tests between 14 March and 30 December 2020, were analyzed for potential reinfection. Inclusion criteria required having ≥2 positive NAATs collected ≥45 days apart with a cycle threshold (Ct) value <35 at repeat testing. For each included subject, likelihood of reinfection was assessed by viral genomic analysis of all available specimens with a Ct value <35, structured Ct trajectory criteria, and case-by-case review by infectious diseases physicians. RESULTS: Among 1569 individuals with repeat SARS-CoV-2 testing ≥45 days after an initial positive NAAT, 65 (4%) met cohort inclusion criteria. Viral genomic analysis characterized mutations present and was successful for 14/65 (22%) subjects. Six subjects had genomically supported reinfection, and 8 subjects had genomically supported persistent RNA detection. Compared to viral genomic analysis, clinical and laboratory assessments correctly distinguished reinfection from persistent RNA detection in 12/14 (86%) subjects but missed 2/6 (33%) genomically supported reinfections. CONCLUSIONS: Despite good overall concordance with viral genomic analysis, clinical and Ct value-based assessments failed to identify 33% of genomically supported reinfections. Scaling-up genomic analysis for clinical use would improve detection of SARS-CoV-2 reinfections.

Determining the Incidence of Asymptomatic SARS-CoV-2 Among Early Recipients of COVID-19 Vaccines (DISCOVER-COVID-19): A Prospective Cohort Study of Healthcare Workers Before, During and After Vaccination.

The impact of coronavirus disease 2019 vaccination on viral characteristics of breakthrough infections is unknown. In this prospective cohort study, incidence of severe acute respiratory syndrome coronavirus 2 infection decreased following vaccination. Although asymptomatic positive tests were observed following vaccination, the higher cycle thresholds, repeat negative tests, and inability to culture virus raise questions about their clinical significance.

Microfluidic Assays for Probing Neutrophil-Borrelia Interactions in Blood During Lyme Disease.

Human neutrophils are highly sensitive to the presence of Borrelia burgdorferi (Bb), the agent of Lyme disease (LD), in tissues. Although Bb is also found in the blood of LD patients, far less is known about how neutrophils respond to Bb in the presence of blood. In this study, we employed microfluidic tools to probe the interaction between human neutrophils and Bb and measured the activation of human neutrophils in blood samples from patients. We found that neutrophils migrate vigorously toward Bb in the presence of serum, and this process was complement-dependent. Preventing complement factor 5 cleavage or blocking complement receptors decreased neutrophil's ability to interact with Bb. We also found that spiking Bb directly into the blood from healthy donors induced spontaneous neutrophil motility. This response to Bb was also complement-dependent. Preventing complement factor 5 cleavage decreased spontaneous neutrophil motility in Bb-spiked blood. Moreover, we found that neutrophils in blood samples from acute LD patients displayed spontaneous motility patterns similar to those observed in Bb-spiked samples. Neutrophil motility was more robust in blood samples from LD patients than that measured in healthy and ill controls, validating the utility of the microfluidic assay for the study of neutrophil-Bb interactions in the presence of blood.

Detection of Borrelia burgdorferi Cell-free DNA in Human Plasma Samples for Improved Diagnosis of Early Lyme Borreliosis.

BACKGROUND: Laboratory confirmation of early Lyme borreliosis (LB) is challenging. Serology is insensitive during the first days to weeks of infection, and blood polymerase chain reaction (PCR) offers similarly poor performance. Here, we demonstrate that detection of Borrelia burgdorferi (B.b.) cell-free DNA (cfDNA) in plasma can improve diagnosis of early LB. METHODS: B.b. detection in plasma samples using unbiased metagenomic cfDNA sequencing performed by a commercial laboratory (Karius Inc) was compared with serology and blood PCR in 40 patients with physician-diagnosed erythema migrans (EM), 28 of whom were confirmed to have LB by skin biopsy culture (n = 18), seroconversion (n = 2), or both (n = 8). B.b. sequence analysis was performed using investigational detection thresholds, different from Karius' clinical test. RESULTS: B.b. cfDNA was detected in 18 of 28 patients (64%) with laboratory-confirmed EM. In comparison, sensitivity of acute-phase serology using modified 2-tiered testing (MTTT) was 50% (P = .45); sensitivity of blood PCR was 7% (P = .0002). Combining B.b. cfDNA detection and MTTT increased diagnostic sensitivity to 86%, significantly higher than either approach alone (P ≤ .04). B.b. cfDNA sequences matched precisely with strain-specific sequence generated from the same individual's cultured B.b. isolate. B.b. cfDNA was not observed at any level in plasma from 684 asymptomatic ambulatory individuals. Among 3000 hospitalized patients tested as part of clinical care, B.b. cfDNA was detected in only 2 individuals, both of whom had clinical presentations consistent with LB. CONCLUSIONS: This is the first report of B.b. cfDNA detection in early LB and a demonstration of potential diagnostic utility. The combination of B.b. cfDNA detection and acute-phase MTTT improves clinical sensitivity for diagnosis of early LB.

Coronavirus Disease 2019 (COVID-19) Diagnostic Clinical Decision Support: A Pre-Post Implementation Study of CORAL (COvid Risk cALculator).

BACKGROUND: Isolation of hospitalized persons under investigation (PUIs) for coronavirus disease 2019 (COVID-19) reduces nosocomial transmission risk. Efficient evaluation of PUIs is needed to preserve scarce healthcare resources. We describe the development, implementation, and outcomes of an inpatient diagnostic algorithm and clinical decision support system (CDSS) to evaluate PUIs. METHODS: We conducted a pre-post study of CORAL (COvid Risk cALculator), a CDSS that guides frontline clinicians through a risk-stratified COVID-19 diagnostic workup, removes transmission-based precautions when workup is complete and negative, and triages complex cases to infectious diseases (ID) physician review. Before CORAL, ID physicians reviewed all PUI records to guide workup and precautions. After CORAL, frontline clinicians evaluated PUIs directly using CORAL. We compared pre- and post-CORAL frequency of repeated severe acute respiratory syndrome coronavirus 2 nucleic acid amplification tests (NAATs), time from NAAT result to PUI status discontinuation, total duration of PUI status, and ID physician work hours, using linear and logistic regression, adjusted for COVID-19 incidence. RESULTS: Fewer PUIs underwent repeated testing after an initial negative NAAT after CORAL than before CORAL (54% vs 67%, respectively; adjusted odd ratio, 0.53 [95% confidence interval, .44-.63]; P < .01). CORAL significantly reduced average time to PUI status discontinuation (adjusted difference [standard error], -7.4 [0.8] hours per patient), total duration of PUI status (-19.5 [1.9] hours per patient), and average ID physician work-hours (-57.4 [2.0] hours per day) (all P < .01). No patients had a positive NAAT result within 7 days after discontinuation of precautions via CORAL. CONCLUSIONS: CORAL is an efficient and effective CDSS to guide frontline clinicians through the diagnostic evaluation of PUIs and safe discontinuation of precautions.

Lyme Disease in Humans.

Lyme disease (Lyme borreliosis) is a tick-borne, zoonosis of adults and children caused by genospecies of the Borrelia burgdorferi sensu lato complex. The ailment, widespread throughout the Northern Hemisphere, continues to increase globally due to multiple environmental factors, coupled with increased incursion of humans into habitats that harbor the spirochete. B. burgdorferi sensu lato is transmitted by ticks from the Ixodes ricinus complex. In North America, B. burgdorferi causes nearly all infections; in Europe, B. afzelii and B. garinii are most associated with human disease. The spirochete's unusual fragmented genome encodes a plethora of differentially expressed outer surface lipoproteins that play a seminal role in the bacterium's ability to sustain itself within its enzootic cycle and cause disease when transmitted to its incidental human host. Tissue damage and symptomatology (i.e., clinical manifestations) result from the inflammatory response elicited by the bacterium and its constituents. The deposition of spirochetes into human dermal tissue generates a local inflammatory response that manifests as erythema migrans (EM), the hallmark skin lesion. If treated appropriately and early, the prognosis is excellent. However, in untreated patients, the disease may present with a wide range of clinical manifestations, most commonly involving the central nervous system, joints, or heart. A small percentage (~10%) of patients may go on to develop a poorly defined fibromyalgia-like illness, post-treatment Lyme disease (PTLD) unresponsive to prolonged antimicrobial therapy. Below we integrate current knowledge regarding the ecologic, epidemiologic, microbiologic, and immunologic facets of Lyme disease into a conceptual framework that sheds light on the disorder that healthcare providers encounter.

GATA2 haploinsufficiency caused by mutations in a conserved intronic element leads to MonoMAC syndrome.

Previous reports of GATA2 mutations have focused on the coding region of the gene or full gene deletions. We recently identified 2 patients with novel insertion/deletion mutations predicted to result in mRNA nonsense-mediated decay, suggesting haploinsufficiency as the mechanism of GATA2 deficient disease. We therefore screened patients without identified exonic lesions for mutations within conserved noncoding and intronic regions. We discovered 1 patient with an intronic deletion mutation, 4 patients with point mutations within a conserved intronic element, and 3 patients with reduced or absent transcription from 1 allele. All mutations affected GATA2 transcription. Full-length cDNA analysis provided evidence for decreased expression of the mutant alleles. The intronic deletion and point mutations considerably reduced the enhancer activity of the intron 5 enhancer. Analysis of 512 immune system genes revealed similar expression profiles in all clinically affected patients and reduced GATA2 transcript levels. These mutations strongly support the haploinsufficient nature of GATA2 deficiency and identify transcriptional mechanisms and targets that lead to MonoMAC syndrome.