COVID-19-neutralizing antibodies predict disease severity and survival.

Coronavirus disease 2019 (COVID-19) exhibits variable symptom severity ranging from asymptomatic to life-threatening, yet the relationship between severity and the humoral immune response is poorly understood. We examined antibody responses in 113 COVID-19 patients and found that severe cases resulting in intubation or death exhibited increased inflammatory markers, lymphopenia, pro-inflammatory cytokines, and high anti-receptor binding domain (RBD) antibody levels. Although anti-RBD immunoglobulin G (IgG) levels generally correlated with neutralization titer, quantitation of neutralization potency revealed that high potency was a predictor of survival. In addition to neutralization of wild-type SARS-CoV-2, patient sera were also able to neutralize the recently emerged SARS-CoV-2 mutant D614G, suggesting cross-protection from reinfection by either strain. However, SARS-CoV-2 sera generally lacked cross-neutralization to a highly homologous pre-emergent bat coronavirus, WIV1-CoV, which has not yet crossed the species barrier. These results highlight the importance of neutralizing humoral immunity on disease progression and the need to develop broadly protective interventions to prevent future coronavirus pandemics.

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.

Lyme neuroborreliosis: known knowns, known unknowns.

Lyme borreliosis affects the nervous system in three principal ways-mononuclear cell meningitis, cranial neuropathies and radiculoneuropathies-the last a broad term encompassing painful radiculopathy, unifocal and multifocal peripheral nerve involvement. Diagnostic tools have been significantly refined-including improved peripheral blood and CSF serodiagnostics-and much has been learned about the interactions between the causative pathogen and the nervous system. Despite these advances in our understanding of this disease, a broad range of other disorders continue to be misattributed to nervous system Lyme borreliosis, supported by, at best, limited evidence. These misattributions often reflect limited understanding not only of Lyme neuroborreliosis but also of what constitutes nervous system disease generally. Fortunately, a large body of evidence now exists to clarify many of these issues, establishing a clear basis for diagnosing nervous system involvement in this infection and, based on well performed studies, clarifying which clinical disorders are associated with Lyme neuroborreliosis, which with non-neurologic Lyme borreliosis, and which with neither.

Vasculopathy and Increased Vascular Congestion in Fatal COVID-19 and Acute Respiratory Distress Syndrome.

Rationale: The leading cause of death in coronavirus disease 2019 (COVID-19) is severe pneumonia, with many patients developing acute respiratory distress syndrome (ARDS) and diffuse alveolar damage (DAD). Whether DAD in fatal COVID-19 is distinct from other causes of DAD remains unknown. Objective: To compare lung parenchymal and vascular alterations between patients with fatal COVID-19 pneumonia and other DAD-causing etiologies using a multidimensional approach. Methods: This autopsy cohort consisted of consecutive patients with COVID-19 pneumonia (n = 20) and with respiratory failure and histologic DAD (n = 21; non-COVID-19 viral and nonviral etiologies). Premortem chest computed tomography (CT) scans were evaluated for vascular changes. Postmortem lung tissues were compared using histopathological and computational analyses. Machine-learning-derived morphometric analysis of the microvasculature was performed, with a random forest classifier quantifying vascular congestion (CVasc) in different microscopic compartments. Respiratory mechanics and gas-exchange parameters were evaluated longitudinally in patients with ARDS. Measurements and Main Results: In premortem CT, patients with COVID-19 showed more dilated vasculature when all lung segments were evaluated (P = 0.001) compared with controls with DAD. Histopathology revealed vasculopathic changes, including hemangiomatosis-like changes (P = 0.043), thromboemboli (P = 0.0038), pulmonary infarcts (P = 0.047), and perivascular inflammation (P < 0.001). Generalized estimating equations revealed significant regional differences in the lung microarchitecture among all DAD-causing entities. COVID-19 showed a larger overall CVasc range (P = 0.002). Alveolar-septal congestion was associated with a significantly shorter time to death from symptom onset (P = 0.03), length of hospital stay (P = 0.02), and increased ventilatory ratio [an estimate for pulmonary dead space fraction (Vd); p = 0.043] in all cases of ARDS. Conclusions: Severe COVID-19 pneumonia is characterized by significant vasculopathy and aberrant alveolar-septal congestion. Our findings also highlight the role that vascular alterations may play in Vd and clinical outcomes in ARDS in general.

Laboratory Diagnosis of Lyme Borreliosis.

Lyme borreliosis is caused by a growing list of related, yet distinct, spirochetes with complex biology and sophisticated immune evasion mechanisms. It may result in a range of clinical manifestations involving different organ systems, and can lead to persistent sequelae in a subset of cases. The pathogenesis of Lyme borreliosis is incompletely understood, and laboratory diagnosis, the focus of this review, requires considerable understanding to interpret the results correctly. Direct detection of the infectious agent is usually not possible or practical, necessitating a continued reliance on serologic testing. Still, some important advances have been made in the area of diagnostics, and there are many promising ideas for future assay development. This review summarizes the state of the art in laboratory diagnostics for Lyme borreliosis, provides guidance in test selection and interpretation, and highlights future directions.

Multicenter Clinical Evaluation of Modified Two-Tiered Testing Algorithms for Lyme Disease Using Zeus Scientific Commercial Assays.

Modified two-tiered testing (MTTT) algorithms for Lyme disease (LD), which involve the sequential use of orthogonal enzyme immunoassays (EIAs) without immunoblotting, are acceptable alternatives to standard two-tiered testing (STTT; EIA followed by immunoblots) provided the EIAs have been FDA-cleared for this intended use. We evaluated four Zeus Scientific LD EIAs used in two distinct MTTT algorithms for FDA review. MTTT 1 used a VlsE1/pepC10 polyvalent EIA followed by a whole-cell sonicate (WCS) polyvalent EIA. MTTT 2 used the same first-tier EIA followed by separate IgM and IgG WCS EIAs. In a retrospective phase, we compared each MTTT algorithm to STTT using archived samples from LD patients or control subjects. In a prospective phase, we used the same algorithms to analyze consecutive excess samples submitted for routine LD serology to three clinical laboratories. For the retrospective phase, MTTTs 1 and 2 were more sensitive (56% and 74%) than STTT (41%; P ≤ 0.03) among 61 patients with acute erythema migrans (EM). In LD patients with neuroborreliosis, carditis, or arthritis (n = 75), sensitivity was comparable between algorithms (96 to 100%; P = 1.0). Among 190 control subjects without past LD, all algorithms were highly and comparably specific (≥99%, P = 0.48). For the prospective phase, (n = 2,932), positive percent-agreement (PPA), negative percent-agreement (NPA), and overall agreement of MTTT 1 with STTT were 93%, 97.7% and 97.4% (kappa 0.80). MTTT 2 yielded higher PPA (98%) but lower NPA (96.1%) and overall agreement (96.2%, kappa 0.74; all P < 0.05). Compared with STTT, both MTTT algorithms provided increased sensitivity in EM patients, comparable sensitivity in later disease and non-inferior specificity.

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.

Clinical Practice Guidelines by the Infectious Diseases Society of America (IDSA): 2020 Guideline on Diagnosis and Management of Babesiosis.

The purpose of this guideline is to provide evidence-based guidance for the most effective strategies for the diagnosis and management of babesiosis. The diagnosis and treatment of co-infection with babesiosis and Lyme disease will be addressed in a separate Infectious Diseases Society of America (IDSA), American Academy of Neurology (AAN), and American College of Rheumatology (ACR) guideline [1]. Recommendations for the diagnosis and treatment of human granulocytic anaplasmosis can be found in the recent rickettsial disease guideline developed by the Centers for Disease Control and Prevention [2]. The target audience for the babesiosis guideline includes primary care physicians and specialists caring for this condition, such as infectious diseases specialists, emergency physicians, intensivists, internists, pediatricians, hematologists, and transfusion medicine specialists.

Clinical Practice Guidelines by the Infectious Diseases Society of America (IDSA): 2020 Guideline on Diagnosis and Management of Babesiosis.

The purpose of this guideline is to provide evidence-based guidance for the most effective strategies for the diagnosis and management of babesiosis. The diagnosis and treatment of co-infection with babesiosis and Lyme disease will be addressed in a separate Infectious Diseases Society of America (IDSA), American Academy of Neurology (AAN), and American College of Rheumatology (ACR) guideline [1]. Recommendations for the diagnosis and treatment of human granulocytic anaplasmosis can be found in the recent rickettsial disease guideline developed by the Centers for Disease Control and Prevention [2]. The target audience for the babesiosis guideline includes primary care physicians and specialists caring for this condition, such as infectious diseases specialists, emergency physicians, intensivists, internists, pediatricians, hematologists, and transfusion medicine specialists.

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.

Clinical Practice Guidelines by the Infectious Diseases Society of America (IDSA), American Academy of Neurology (AAN), and American College of Rheumatology (ACR): 2020 Guidelines for the Prevention, Diagnosis and Treatment of Lyme Disease.

This evidence-based clinical practice guideline for the prevention, diagnosis, and treatment of Lyme disease was developed by a multidisciplinary panel representing the Infectious Diseases Society of America (IDSA), the American Academy of Neurology (AAN), and the American College of Rheumatology (ACR). The scope of this guideline includes prevention of Lyme disease, and the diagnosis and treatment of Lyme disease presenting as erythema migrans, Lyme disease complicated by neurologic, cardiac, and rheumatologic manifestations, Eurasian manifestations of Lyme disease, and Lyme disease complicated by coinfection with other tick-borne pathogens. This guideline does not include comprehensive recommendations for babesiosis and tick-borne rickettsial infections, which are published in separate guidelines. The target audience for this guideline includes primary care physicians and specialists caring for this condition such as infectious diseases specialists, emergency physicians, internists, pediatricians, family physicians, neurologists, rheumatologists, cardiologists and dermatologists in North America.

Clinical Practice Guidelines by the Infectious Diseases Society of America (IDSA), American Academy of Neurology (AAN), and American College of Rheumatology (ACR): 2020 Guidelines for the Prevention, Diagnosis and Treatment of Lyme Disease.

This evidence-based clinical practice guideline for the prevention, diagnosis, and treatment of Lyme disease was developed by a multidisciplinary panel representing the Infectious Diseases Society of America (IDSA), the American Academy of Neurology (AAN), and the American College of Rheumatology (ACR). The scope of this guideline includes prevention of Lyme disease, and the diagnosis and treatment of Lyme disease presenting as erythema migrans, Lyme disease complicated by neurologic, cardiac, and rheumatologic manifestations, Eurasian manifestations of Lyme disease, and Lyme disease complicated by coinfection with other tick-borne pathogens. This guideline does not include comprehensive recommendations for babesiosis and tick-borne rickettsial infections, which are published in separate guidelines. The target audience for this guideline includes primary care physicians and specialists caring for this condition such as infectious diseases specialists, emergency physicians, internists, pediatricians, family physicians, neurologists, rheumatologists, cardiologists and dermatologists in North America.

Clinical sensitivity and interpretation of PCR and serological COVID-19 diagnostics for patients presenting to the hospital.

The diagnosis of COVID-19 requires integration of clinical and laboratory data. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostic assays play a central role in diagnosis and have fixed technical performance metrics. Interpretation becomes challenging because the clinical sensitivity changes as the virus clears and the immune response emerges. Our goal was to examine the clinical sensitivity of two most common SARS-CoV-2 diagnostic test modalities, polymerase chain reaction (PCR) and serology, over the disease course to provide insight into their clinical interpretation in patients presenting to the hospital. We conducted a single-center, retrospective study. To derive clinical sensitivity of PCR, we identified 209 PCR-positive SARS-CoV-2 patients with multiple PCR test results (624 total PCR tests) and calculated daily sensitivity from date of symptom onset or first positive test. Clinical sensitivity of PCR decreased with days post symptom onset with >90% clinical sensitivity during the first 5 days after symptom onset, 70%-71% from Days 9 to 11, and 30% at Day 21. To calculate daily clinical sensitivity by serology, we utilized 157 PCR-positive patients with a total of 197 specimens tested by enzyme-linked immunosorbent assay for IgM, IgG, and IgA anti-SARS-CoV-2 antibodies. In contrast to PCR, serological sensitivity increased with days post symptom onset with >50% of patients seropositive by at least one antibody isotype after Day 7, >80% after Day 12, and 100% by Day 21. Taken together, PCR and serology are complimentary modalities that require time-dependent interpretation. Superimposition of sensitivities over time indicate that serology can function as a reliable diagnostic aid indicating recent or prior infection.

Use of Routine Complete Blood Count Results to Rule Out Anaplasmosis Without the Need for Specific Diagnostic Testing.

BACKGROUND: Anaplasmosis presents with fever, headache, and laboratory abnormalities including leukopenia and thrombocytopenia. Polymerase chain reaction (PCR) is the preferred diagnostic but is overutilized. We determined if routine laboratory tests could exclude anaplasmosis, improving PCR utilization. METHODS: Anaplasma PCR results from a 3-year period, with associated complete blood count (CBC) and liver function test results, were retrospectively reviewed. PCR rejection criteria, based on white blood cell (WBC) and platelet (PLT) counts, were developed and prospectively applied in a mock stewardship program. If rejection criteria were met, a committee mock-refused PCR unless the patient was clinically unstable or immunocompromised. RESULTS: WBC and PLT counts were the most actionable routine tests for excluding anaplasmosis. Retrospective review demonstrated that rejection criteria of WBC ≥11 000 cells/µL or PLT ≥300 000 cells/µL would have led to PCR refusal in 428 of 1685 true-negative cases (25%) and 3 of 66 true-positive cases (5%) involving clinically unstable or immunocompromised patients. In the prospective phase, 155 of 663 PCR requests (23%) met rejection criteria and were reviewed by committee, which endorsed refusal in 110 of 155 cases (71%) and approval in 45 (29%), based on clinical criteria. PCR was negative in all 45 committee-approved cases. Only 1 of 110 mock-refused requests yielded a positive PCR result; this patient was already receiving doxycycline at the time of testing. CONCLUSIONS: A CBC-based stewardship algorithm would reduce unnecessary Anaplasma PCR testing, without missing active cases. Although the prospectively evaluated screening approach involved medical record review, this was unnecessary to prevent errors and could be replaced by a rejection comment specifying clinical situations that might warrant overriding the algorithm.

Pediatric Lyme Disease Biobank, United States, 2015-2020.

In 2015, we founded Pedi Lyme Net, a pediatric Lyme disease research network comprising 8 emergency departments in the United States. Of 2,497 children evaluated at 1 of these sites for Lyme disease, 515 (20.6%) were infected. This network is a unique resource for evaluating new approaches for diagnosing Lyme disease in children.

Evaluation of Three Commercial SARS-CoV-2 Serologic Assays and Their Performance in Two-Test Algorithms.

Sensitive and specific severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serologic assays are needed to inform diagnostic, therapeutic, and public health decision-making. We evaluated three commercial serologic assays as stand-alone tests and as components of two-test algorithms. Two nucleocapsid antibody tests (Abbott IgG and Roche total antibody) and one spike protein antibody test (DiaSorin IgG) were included. We assessed sensitivity using 128 serum samples from symptomatic PCR-confirmed coronavirus disease 2019 (COVID-19)-infected patients and specificity using 1,204 samples submitted for routine serology prior to COVID-19's emergence, plus 64 pandemic-era samples from SARS-CoV-2 PCR-negative patients with respiratory symptoms. Assays were evaluated as stand-alone tests and as components of a two-test algorithm in which positive results obtained using one assay were verified using a second assay. The two nucleocapsid antibody tests were more sensitive than the spike protein antibody test overall (70% and 70% versus 57%; P ≤ 0.003), with pronounced differences observed using samples collected 7 to 14 days after symptom onset. All three assays were comparably sensitive (≥89%; P ≥ 0.13) using samples collected >14 days after symptom onset. Specificity was higher using the nucleocapsid antibody tests (99.3% and 99.7%) than using the spike protein antibody test (97.8%; P ≤ 0.002). When any two assays were paired in a two-test algorithm, the specificity was 99.9% (P < 0.0001 to 0.25 compared with the individual assays), and the positive predictive value (PPV) improved substantially, with a minimal effect on the negative predictive value (NPV). In conclusion, two nucleocapsid antibody tests outperformed a spike protein antibody test. Pairing two different serologic tests in a two-test algorithm improves the PPV, compared with the individual assays alone, while maintaining the NPV.

Direct Diagnostic Tests for Lyme Disease.

Borrelia burgdorferi was discovered to be the cause of Lyme disease in 1983, leading to seroassays. The 1994 serodiagnostic testing guidelines predated a full understanding of key B. burgdorferi antigens and have a number of shortcomings. These serologic tests cannot distinguish active infection, past infection, or reinfection. Reliable direct-detection methods for active B. burgdorferi infection have been lacking in the past but are needed and appear achievable. New approaches have effectively been applied to other emerging infections and show promise in direct detection of B. burgdorferi infections.

Evaluation of the Modified Two-Tiered Testing Method for Diagnosis of Lyme Disease in Children.

Conventional two-tiered testing (CTTT) for Lyme disease includes a first-tier enzyme immunoassay (EIA) followed by a supplemental immunoblot, and modified two-tiered testing (MTTT) relies on two different sequential EIAs without the inclusion of an immunoblot. MTTT has shown promising results as an alternative strategy for the diagnosis of Lyme disease in adults but has not yet been evaluated in children. We performed a cross-sectional study of children and adolescents ≤21 years of age undergoing clinical investigation for suspected Lyme disease. Serum specimens were analyzed with both a whole-cell sonicate (WCS) and a C6 EIA, with a supplemental immunoblot if either EIA was positive or equivocal. We compared CTTT (WCS EIA followed by supplemental immunoblot) to MTTT (WCS EIA followed by C6 EIA) using McNemar's test to evaluate for agreement beyond chance alone. We then used a kappa statistic to measure level of the agreement between testing strategies. We included 1,066 serum specimens, of which 156 (14.6%) had a positive CTTT and 165 (15.5%) had a positive MTTT. There were no significant differences between MTTT and CTTT (P = 0.16). Although the overall agreement between MTTT and CTTT was high (kappa, 0.88; 95% confidence interval, 0.84 to 0.92), 33 children had discordant test results. In a cohort of children and adolescents undergoing investigation for suspected Lyme disease, CTTT and MTTT results agreed in most cases. Since immunoblots are time-consuming, laborious, and challenging to interpret, MTTT provides a promising alternate Lyme disease testing strategy for children.

Advances in Serodiagnostic Testing for Lyme Disease Are at Hand.

The cause of Lyme disease, Borrelia burgdorferi, was discovered in 1983. A 2-tiered testing protocol was established for serodiagnosis in 1994, involving an enzyme immunoassay (EIA) or indirect fluorescence antibody, followed (if reactive) by immunoglobulin M and immunoglobulin G Western immunoblots. These assays were prepared from whole-cell cultured B. burgdorferi, lacking key in vivo expressed antigens and expressing antigens that can bind non-Borrelia antibodies. Additional drawbacks, particular to the Western immunoblot component, include low sensitivity in early infection, technical complexity, and subjective interpretation when scored by visual examination. Nevertheless, 2-tiered testing with immunoblotting remains the benchmark for evaluation of new methods or approaches. Next-generation serologic assays, prepared with recombinant proteins or synthetic peptides, and alternative testing protocols, can now overcome or circumvent many of these past drawbacks. This article describes next-generation serodiagnostic testing for Lyme disease, focusing on methods that are currently available or near-at-hand.