Peromyscus leucopus , Mus musculus , and humans have distinct transcriptomic responses to larval Ixodes scapularis bites.

Ixodes scapularis ticks are an important vector for at least six tick-borne human pathogens, including the predominant North American Lyme disease spirochete Borrelia burgdorferi . The ability for these ticks to survive in nature is credited, in part, to their ability to feed on a variety of hosts without excessive activation of the proinflammatory branch of the vertebrate immune system. While the ability for nymphal ticks to feed on a variety of hosts has been well-documented, the host-parasite interactions between larval I. scapularis and different vertebrate hosts is relatively unexplored. Here we report on the changes in the vertebrate transcriptome present at the larval tick bite site using the natural I. scapularis host Peromyscus leucopus deermouse, a non-natural rodent host Mus musculus (BALB/c), and humans. We note substantially less evidence of activation of canonical proinflammatory pathways in P. leucopus compared to BALB/c mice and pronounced evidence of inflammation in humans. Pathway enrichment analyses revealed a particularly strong signature of interferon gamma, tumor necrosis factor, and interleukin 1 signaling at the BALB/c and human tick bite site. We also note that bite sites on BALB/c mice and humans, but not deermice, show activation of wound-healing pathways. These data provide molecular evidence of the coevolution between larval I. scapularis and P. leucopus as well as expand our overall understanding of I. scapularis feeding. Significance: Ixodes scapularis tick bites expose humans to numerous diseases in North America. While larval tick feeding enables pathogens to enter the tick population and eventually spread to humans, how larval ticks interact with mammals has been understudied compared to other tick stages. Here we examined the transcriptomic response of a natural I. scapularis rodent host ( Peromyscus leucopus ), a non-native I. scapularis rodent host ( Mus musculus ), and an incidental host (humans). We find that there are differences in how all three species respond to larval I. scapularis , with the natural host producing the smallest transcriptomic signature of a canonical proinflammatory immune response and the incidental human host producing the most robust signature of inflammation in response to the larval tick. These data expand our understanding of the pressures on ticks in the wild and inform our ability to model these interactions in laboratory settings.

A Longitudinal Study of COVID-19 Sequelae and Immunity: Baseline Findings.

BACKGROUND: A substantial proportion of persons who develop COVID-19 report persistent symptoms after acute illness. Various pathophysiologic mechanisms have been implicated in the pathogenesis of postacute sequelae of SARS-CoV-2 infection (PASC). OBJECTIVE: To characterize medical sequelae and persistent symptoms after recovery from COVID-19 in a cohort of disease survivors and controls. DESIGN: Cohort study. (ClinicalTrials.gov: NCT04411147). SETTING: National Institutes of Health Clinical Center, Bethesda, Maryland. PARTICIPANTS: Self-referred adults with laboratory-documented SARS-CoV-2 infection who were at least 6 weeks from symptom onset were enrolled regardless of presence of PASC. A control group comprised persons with no history of COVID-19 or serologic evidence of SARS-CoV-2 infection, recruited regardless of their current health status. Both groups were enrolled over the same period and from the same geographic area. MEASUREMENTS: All participants had the same evaluations regardless of presence of symptoms, including physical examination, laboratory tests and questionnaires, cognitive function testing, and cardiopulmonary evaluation. A subset also underwent exploratory immunologic and virologic evaluations. RESULTS: 189 persons with laboratory-documented COVID-19 (12% of whom were hospitalized during acute illness) and 120 antibody-negative control participants were enrolled. At enrollment, symptoms consistent with PASC were reported by 55% of the COVID-19 cohort and 13% of control participants. Increased risk for PASC was noted in women and those with a history of anxiety disorder. Participants with findings meeting the definition of PASC reported lower quality of life on standardized testing. Abnormal findings on physical examination and diagnostic testing were uncommon. Neutralizing antibody levels to spike protein were negative in 27% of the unvaccinated COVID-19 cohort and none of the vaccinated COVID-19 cohort. Exploratory studies found no evidence of persistent viral infection, autoimmunity, or abnormal immune activation in participants with PASC. LIMITATIONS: Most participants with COVID-19 had mild to moderate acute illness that did not require hospitalization. The prevalence of reported PASC was likely overestimated in this cohort because persons with PASC may have been more motivated to enroll. The study did not capture PASC that resolved before enrollment. CONCLUSION: A high burden of persistent symptoms was observed in persons after COVID-19. Extensive diagnostic evaluation revealed no specific cause of reported symptoms in most cases. Antibody levels were highly variable after COVID-19. PRIMARY FUNDING SOURCE: Division of Intramural Research, National Institute of Allergy and Infectious Diseases.

Antiphospholipid autoantibodies in Lyme disease arise after scavenging of host phospholipids by Borrelia burgdorferi.

A close association with its vertebrate and tick hosts allows Borrelia burgdorferi, the bacterium responsible for Lyme disease, to eliminate many metabolic pathways and instead scavenge key nutrients from the host. A lipid-defined culture medium was developed to demonstrate that exogenous lipids are an essential nutrient of B. burgdorferi, which can accumulate intact phospholipids from its environment to support growth. Antibody responses to host phospholipids were studied in mice and humans using an antiphospholipid ELISA. Several of these environmentally acquired phospholipids including phosphatidylserine and phosphatidic acid, as well as borrelial phosphatidylcholine, are the targets of antibodies that arose early in infection in the mouse model. Patients with acute infections demonstrated antibody responses to the same lipids. The elevation of antiphospholipid antibodies predicted early infection with better sensitivity than did the standardized 2-tier tests currently used in diagnosis. Sera obtained from patients with Lyme disease before and after antibiotic therapy showed declining antiphospholipid titers after treatment. Further study will be required to determine whether these antibodies have utility in early diagnosis of Lyme disease, tracking of the response to therapy, and diagnosis of reinfection, areas in which current standardized tests are inadequate.

Comparison of Lyme Disease in the United States and Europe.

Lyme disease, or Lyme borreliosis, is the most common tickborne disease in the United States and Europe. In both locations, Ixodes species ticks transmit the Borrelia burgdorferi sensu lato bacteria species responsible for causing the infection. The diversity of Borrelia species that cause human infection is greater in Europe; the 2 B. burgdorferi s.l. species collectively responsible for most infections in Europe, B. afzelii and B. garinii, are not found in the United States, where most infections are caused by B. burgdorferi sensu stricto. Strain differences seem to explain some of the variation in the clinical manifestations of Lyme disease, which are both minor and substantive, between the United States and Europe. Future studies should attempt to delineate the specific virulence factors of the different species of B. burgdorferi s.l. responsible for these variations in clinical features.

Characterization of a Monanema nematode in Ixodes scapularis.

BACKGROUND: Metagenomic studies have revealed the presence of a filarial nematode in Ixodes scapularis. The phylogeny of this agent, and its potential for human infection, are unknown. METHODS: We used existing metagenomic data from I. scapularis to determine the phylogeny of this tick-associated nematode and employed quantitative PCR to determine if the presence of this agent had an effect on the burden of Borrelia burgdorferi. We also developed a Luciferase Immunoprecipitation System assay using the Av33 antigen as a target to investigate the presence of antibodies against this nematode in 128 serum specimens from patients with Lyme disease and babesiosis. To demonstrate assay utility, we used 15 sera from patients with onchocerciasis as controls. RESULTS: We show that this agent is a new species in the genus Monanema and its presence in vector ticks does not impact the burden of B. burgdorferi. We did not detect IgG antibodies to this agent in 127 of 128 sera from patients with Lyme disease or babesiosis. One sample had reactivity above the threshold, but at the low-level equivalent to the least reactive onchocerciasis sera. This low positive signal could be a result of cross-reacting antibodies, antibodies from a previous infection with a filarial nematode, or, less likely, a exposure to the Ixodes scapularis-associated nematode. CONCLUSIONS: We found no evidence that this nematode contributes to the spectrum of human tick-borne infections.

Protective Immunity and New Vaccines for Lyme Disease.

Lyme disease, caused by some Borrelia burgdorferi sensu lato, is the most common tick-borne illness in the Northern Hemisphere and the number of cases, and geographic spread, continue to grow. Previously identified B. burgdorferi proteins, lipid immunogens, and live mutants lead the design of canonical vaccines aimed at disrupting infection in the host. Discovery of the mechanism of action of the first vaccine catalyzed the development of new strategies to control Lyme disease that bypassed direct vaccination of the human host. Thus, novel prevention concepts center on proteins produced by B. burgdorferi during tick transit and on tick proteins that mediate feeding and pathogen transmission. A burgeoning area of research is tick immunity as it can unlock mechanistic pathways that could be targeted for disruption. Studies that shed light on the mammalian immune pathways engaged during tick-transmitted B. burgdorferi infection would further development of vaccination strategies against Lyme disease.

A Multiplexed Serologic Test for Diagnosis of Lyme Disease for Point-of-Care Use.

Single multiplexed assays could replace the standard 2-tiered (STT) algorithm recommended for the laboratory diagnosis of Lyme disease if they perform with a specificity and a sensitivity superior or equal to those of the STT algorithm. We used human serum rigorously characterized to be sera from patients with acute- and convalescent-phase early Lyme disease, Lyme arthritis, and posttreatment Lyme disease syndrome, as well as the necessary controls (n = 241 samples), to select the best of 12 Borrelia burgdorferi proteins to improve our microfluidic assay (mChip-Ld). We then evaluated its serodiagnostic performance in comparison to that of a first-tier enzyme immunoassay and the STT algorithm. We observed that more antigens became positive as Lyme disease progressed from early to late stages. We selected three antigens (3Ag) to include in the mChip-Ld: VlsE and a proprietary synthetic 33-mer peptide (PepVF) to capture sensitivity in all disease stages and OspC for early Lyme disease. With the specificity set at 95%, the sensitivity of the mChip-Ld with 3Ag ranged from 80% (95% confidence interval [CI], 56% to 94%) and 85% (95% CI, 74% to 96%) for two panels of serum from patients with early Lyme disease and was 100% (95% CI, 83% to 100%) for serum from patients with Lyme arthritis; the STT algorithm detected early Lyme disease in the same two panels of serum from patients with early Lyme disease with a sensitivity of 48.5% and 75% and Lyme arthritis in serum from patients with Lyme arthritis with a sensitivity of 100%, and the specificity was 97.5% to 100%. The mChip-Ld platform outperformed the STT algorithm according to sensitivity. These results open the door for the development of a single, rapid, multiplexed diagnostic test for point-of-care use that can be designed to identify the Lyme disease stage.

Antimicrobial action of calprotectin that does not involve metal withholding.

Calprotectin is a potent antimicrobial that inhibits the growth of pathogens by tightly binding transition metals such as Mn and Zn, thereby preventing their uptake and utilization by invading microbes. At sites of infection, calprotectin is abundantly released from neutrophils, but calprotectin is also present in non-neutrophil cell types that may be relevant to infections. We show here that in patients infected with the Lyme disease pathogen Borreliella (Borrelia) burgdorferi, calprotectin is produced in neutrophil-free regions of the skin, in both epidermal keratinocytes and in immune cells infiltrating the dermis, including CD68 positive macrophages. In culture, B. burgdorferi's growth is inhibited by calprotectin, but surprisingly, the mechanism does not involve the classical withholding of metal nutrients. B. burgdorferi cells exposed to calprotectin cease growth with no reduction in intracellular Mn and no loss in activity of Mn enzymes including the SodA superoxide dismutase. Additionally, there is no obvious loss in intracellular Zn. Rather than metal depletion, we find that calprotectin inhibits B. burgdorferi growth through a mechanism that requires physical association of calprotectin with the bacteria. By comparison, calprotectin inhibited E. coli growth without physically interacting with the microbe, and calprotectin effectively depleted E. coli of intracellular Mn and Zn. Our studies with B. burgdorferi demonstrate that the antimicrobial capacity of calprotectin is complex and extends well beyond simple withholding of metal micronutrients.

Revisiting the Lyme Disease Serodiagnostic Algorithm: the Momentum Gathers.

Lyme disease is a tick-borne illness caused by Borreliella (Borrelia) burgdorferi, and it is the most common vector-borne disease in the United States, with an estimated incidence of 300,000 cases per year. The currently recommended approach for laboratory support of the diagnosis of Lyme disease is a standard two-tiered (STT) algorithm comprised of an enzyme-linked immunoassay (EIA) or immunofluorescence assay (IFA), followed by Western blotting (WB). The STT algorithm has low sensitivity in early infection, and there are drawbacks associated with the WB use in practice. Modified two-tiered (MTT) algorithms have been shown to improve the sensitivity of the testing in early disease while maintaining high specificity. In this issue of the Journal of Clinical Microbiology, A. Pegalajar-Jurado et al. (J Clin Microbiol 56:e01943-17, 2018, https://doi.org/10.1128/JCM.01943-17) report the results of their evaluation of the Liaison VlsE CLIA, the Captia B. burgdorferi IgG/IgM EIA, and the C6 B. burgdorferi (Lyme) EIA as MTT algorithms compared with results with the STT algorithm using the same tests as the first-tier test and the ViraStripe IgM and IgG WBs as the second-tier test. The results showed that all MTT algorithms had higher sensitivities than STT algorithms and were highly specific. These results showed that MTT approaches are a valid alternative to the currently recommended STT algorithm for serodiagnosis of Lyme disease, opening the door for the development of rapid diagnostics and point-of-care testing that can provide diagnostic information during the initial patient visit.

Early Disseminated Lyme Disease Causing False-Positive Serology for Primary Epstein-Barr Virus Infection: Report of 2 Cases.

False-positive serology for Lyme disease was reported in patients with acute infectious mononucleosis. Here we describe 2 patients with early disseminated Lyme disease who were misdiagnosed with infectious mononucleosis based on false-positive tests for primary Epstein-Barr virus infection.

Insights into Borrelia miyamotoi infection from an untreated case demonstrating relapsing fever, monocytosis and a positive C6 Lyme serology.

We describe a patient from the United States with PCR- and serology-confirmed Borrelia miyamotoi infection who recovered without antibiotics. Our findings suggest that B. miyamotoi infection may cause relapsing fever, blood monocytosis and antibody reactivity to the C6 peptide. Further studies are required to better define the spectrum of clinical and laboratory findings for this emerging tick-transmitted infection.

Epitope-Specific Evolution of Human B Cell Responses to Borrelia burgdorferi VlsE Protein from Early to Late Stages of Lyme Disease.

Most immunogenic proteins of Borrelia burgdorferi, the causative agent of Lyme disease, are known or expected to contain multiple B cell epitopes. However, the kinetics of the development of human B cell responses toward the various epitopes of individual proteins during the course of Lyme disease has not been examined. Using the highly immunogenic VlsE as a model Ag, we investigated the evolution of humoral immune responses toward its immunodominant sequences in 90 patients with a range of early to late manifestations of Lyme disease. The results demonstrate the existence of asynchronous, independently developing, Ab responses against the two major immunogenic regions of the VlsE molecule in the human host. Despite their strong immunogenicity, the target epitopes were inaccessible to Abs on intact spirochetes, suggesting a lack of direct immunoprotective effect. These observations document the association of immune reactivity toward specific VlsE sequences with different phases of Lyme disease, demonstrating the potential use of detailed epitope mapping of Ags for staging of the infection, and offer insights regarding the pathogen's possible immune evasion mechanisms.

Lyme Neuroborreliosis.

PURPOSE OF REVIEW: Lyme disease, caused by the spirochete Borrelia burgdorferi, is the most common tick-borne illness in the United States and Europe. Lyme disease usually begins with the characteristic skin lesion, erythema migrans, at the site of the tick bite. Following hematogenous dissemination, neurologic, cardiac, and/or rheumatologic involvement may occur. Neurologic involvement occurs in up to 15% of untreated B. burgdorferi infection and neurologists should be familiar with its diagnosis and management. RECENT FINDINGS: The most common early neurologic manifestations of Lyme disease are cranial neuropathy (particularly facial palsy), lymphocytic meningitis, and radiculoneuritis, which often occur in combination. Late neuroborreliosis occurs much less frequently than early disease. A combination of clinical and laboratory findings is recommended for the diagnosis of Lyme neuroborreliosis. Treatment with recommended antibiotic regimens is effective in Lyme neuroborreliosis, and patients with early disease usually have excellent outcomes. Recovery is slower and may be incomplete in patients with late disease. SUMMARY: Nervous system involvement occurs in up to 15% of patients with untreated B. burgdorferi infection. This article reviews clinical aspects of the diagnosis and treatment of Lyme neuroborreliosis, with focus on the United States.

DNA Barcoding Identifies Illegal Parrot Trade.

Illegal trade threatens the survival of many wild species, and molecular forensics can shed light on various questions raised during the investigation of cases of illegal trade. Among these questions is the identity of the species involved. Here we report a case of a man who was caught in a Brazilian airport trying to travel with 58 avian eggs. He claimed they were quail eggs, but authorities suspected they were from parrots. The embryos never hatched and it was not possible to identify them based on morphology. As 29% of parrot species are endangered, the identity of the species involved was important to establish a stronger criminal case. Thus, we identified the embryos' species based on the analyses of mitochondrial DNA sequences (cytochrome c oxidase subunit I gene [COI] and 16S ribosomal DNA). Embryonic COI sequences were compared with those deposited in BOLD (The Barcode of Life Data System) while their 16S sequences were compared with GenBank sequences. Clustering analysis based on neighbor-joining was also performed using parrot COI and 16S sequences deposited in BOLD and GenBank. The results, based on both genes, indicated that 57 embryos were parrots (Alipiopsitta xanthops, Ara ararauna, and the [Amazona aestiva/A. ochrocephala] complex), and 1 was an owl. This kind of data can help criminal investigations and to design species-specific anti-poaching strategies, and demonstrate how DNA sequence analysis in the identification of bird species is a powerful conservation tool.

Association of immune response to endothelial cell growth factor with early disseminated and late manifestations of Lyme disease but not posttreatment Lyme disease syndrome.

Endothelial cell growth factor has been recently proposed as a potential autoantigen in manifestations of Lyme disease that are thought to involve immune-mediated mechanisms. Our findings indicate that a humoral immune response to this protein is not associated with posttreatment Lyme disease syndrome.

Laboratory diagnosis of Lyme disease: advances and challenges.

The majority of laboratory tests performed for the diagnosis of Lyme disease are based on detection of the antibody responses against B burgdorferi in serum. The sensitivity of antibody-based tests increases with the duration of the infection. Patients early in their illness are more likely to have a negative result. There is a need to simplify the testing algorithm for Lyme disease, improving sensitivity in early disease while still maintaining high specificity and providing information about the stage of infection. The development of a point of care assay and biomarkers for active infection would be major advances for the field.