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.

Evaluation of Modified 2-Tiered Serodiagnostic Testing Algorithms for Early Lyme Disease.

Background: The conventional 2-tiered serologic testing protocol for Lyme disease (LD), an enzyme immunoassay (EIA) followed by immunoglobulin M and immunoglobulin G Western blots, performs well in late-stage LD but is insensitive in patients with erythema migrans (EM), the most common manifestation of the illness. Western blots are also complex, difficult to interpret, and relatively expensive. In an effort to improve test performance and simplify testing in early LD, we evaluated several modified 2-tiered testing (MTTT) protocols, which use 2 assays designed as first-tier tests sequentially, without the need of Western blots. Methods: The MTTT protocols included (1) a whole-cell sonicate (WCS) EIA followed by a C6 EIA; (2) a WCS EIA followed by a VlsE chemiluminescence immunoassay (CLIA); and (3) a variable major protein-like sequence, expressed (VlsE) CLIA followed by a C6 EIA. Sensitivity was determined using serum from 55 patients with erythema migrans; specificity was determined using serum from 50 patients with other illnesses and 1227 healthy subjects. Results: Sensitivity of the various MTTT protocols in patients with acute erythema migrans ranged from 36% (95% confidence interval [CI], 25%-50%) to 54% (95% CI, 42%-67%), compared with 25% (95% CI, 16%-38%) using the conventional protocol (P = .003-0.3). Among control subjects, the 3 MTTT protocols were similarly specific (99.3%-99.5%) compared with conventional 2-tiered testing (99.5% specificity; P = .6-1.0). Conclusions: Although there were minor differences in sensitivity and specificity among MTTT protocols, each provides comparable or greater sensitivity in acute EM, and similar specificity compared with conventional 2-tiered testing, obviating the need for Western blots.

Babesia microti from humans and ticks hold a genomic signature of strong population structure in the United States.

BACKGROUND: Babesia microti is an emerging tick-borne apicomplexan parasite with increasing geographic range and incidence in the United States. The rapid expansion of B. microti into its current distribution in the northeastern USA has been due to the range expansion of the tick vector, Ixodes scapularis, upon which the causative agent is dependent for transmission to humans. RESULTS: To reconstruct the history of B. microti in the continental USA and clarify the evolutionary origin of human strains, we used multiplexed hybrid capture of 25 B. microti isolates obtained from I. scapularis and human blood. Despite low genomic variation compared with other Apicomplexa, B. microti was strongly structured into three highly differentiated genetic clusters in the northeastern USA. Bayesian analyses of the apicoplast genomes suggest that the origin of the current diversity of B. microti in northeastern USA dates back 46 thousand years with a signature of recent population expansion in the last 1000 years. Human-derived samples belonged to two rarely intermixing clusters, raising the possibility of highly divergent infectious phenotypes in humans. CONCLUSIONS: Our results validate the multiplexed hybrid capture strategy for characterizing genome-wide diversity and relatedness of B. microti from ticks and humans. We find strong population structure in B. microti samples from the Northeast indicating potential barriers to gene flow.

Borrelia miyamotoi Disease in the Northeastern United States: A Case Series.

BACKGROUND: The first recognized cases of Borrelia miyamotoi disease (BMD) in North America were reported in the northeastern United States in 2013. OBJECTIVE: To further describe the clinical spectrum and laboratory findings for BMD. DESIGN: Case series. SETTING: Patients presenting to primary care offices, emergency departments, or urgent care clinics in 2013 and 2014. PARTICIPANTS: Acutely febrile patients from the northeastern United States in whom the treating health care providers suspected and ordered testing for tick-transmitted infections. MEASUREMENTS: Whole-blood polymerase chain reaction (PCR) testing was performed for the presence of specific DNA sequences of common tickborne infections (including BMD). Serologic testing for B. miyamotoi was performed using a recombinant glycerophosphodiester phosphodiesterase (rGlpQ) protein. Clinical records were analyzed to identify the major features of acute disease. RESULTS: Among 11,515 patients tested, 97 BMD cases were identified by PCR. Most of the 51 case patients on whom clinical histories were reviewed presented with high fever, chills, marked headache, and myalgia or arthralgia. Twenty-four percent were hospitalized. Elevated liver enzyme levels, neutropenia, and thrombocytopenia were common. At presentation, 16% of patients with BMD were seropositive for IgG and/or IgM antibody to B. miyamotoi rGlpQ. Most (78%) had seropositive convalescent specimens. Symptoms resolved after treatment with doxycycline, and no chronic sequelae or symptoms were observed. LIMITATION: Findings were based on specimens submitted for testing to a reference laboratory, and medical records of only 51 of the 97 case patients with BMD were reviewed. CONCLUSION: Patients with BMD presented with nonspecific symptoms, including fever, headache, chills, myalgia, and arthralgia. Laboratory confirmation of BMD was possible by PCR on blood from acutely symptomatic patients who were seronegative at presentation. Borrelia miyamotoi disease may be an emerging tickborne infection in the northeastern United States. PRIMARY FUNDING SOURCE: IMUGEN.

Development of a real-time polymerase chain reaction assay for sensitive detection and quantitation of Babesia microti infection.

BACKGROUND: Babesia microti, the most frequently implicated pathogen in transfusion-transmitted babesiosis, is widely endemic in the Northeast and upper Midwestern United States. High seroprevalence in endemic areas limits antibody-based donor screening. A high-performance molecular test is needed to identify donors in the preseroconversion window phase as well as to discriminate past serologic exposure with parasite clearance from continued parasitemia. STUDY DESIGN AND METHODS: Frozen Babesia-spiked whole blood was microcentrifuged, and the supernatant transferred and microcentrifuged again to concentrate the parasite. The DNA was extracted and amplified using real-time polymerase chain reaction (PCR) using Babesia-specific primers. The assay was employed in three series of experiments: 1) a validation and optimization spiking experiment, 2) a blinded serial dilution probit analysis to determine the limit of detection, and 3) evaluation of two blinded panels of clinical samples from possible babesiosis cases. RESULTS: At a decreasing inoculum of 445, 44.5, and 4.45 copies/mL, the assay had positive rates of 100, 97.5, and 81%, respectively. The blinded probit analysis demonstrated a detection rate of 95 and 50% at 12.92 and 1.52 parasites/2 mL of whole blood, respectively. Evaluation of clinical samples showed 13 of 21 samples to be positive, with a range of 85 to 4.8 million parasites/mL. There were no positives detected among 48 healthy donors CONCLUSION: We have developed a highly sensitive and specific, quantitative real-time PCR-based assay for detection of B. microti that could have a useful role in blood screening. It can also be employed broadly to understand Babesia epidemiology, disease pathogenesis, and host immunology.

Semicentennial of Human Babesiosis, Nantucket Island.

Fifty years ago, the index case of human babesiosis due to Babesia microti was diagnosed in a summer resident of Nantucket Island. Human babesiosis, once called "Nantucket fever" due to its seeming restriction to Nantucket and the terminal moraine islands of southern New England, has emerged across the northeastern United States to commonly infect people wherever Lyme disease is endemic. We review the history of babesiosis on Nantucket, analyze its epidemiology and ecology there, provide summaries of the first case histories, and comment on its future public health burden.

Antigen Discovery, Bioinformatics and Biological Characterization of Novel Immunodominant Babesia microti Antigens.

Babesia microti is an intraerythrocytic parasite and the primary causative agent of human babesiosis. It is transmitted by Ixodes ticks, transfusion of blood and blood products, organ donation, and perinatally. Despite its global public health impact, limited progress has been made to identify and characterize immunodominant B. microti antigens for diagnostic and vaccine use. Using genome-wide immunoscreening, we identified 56 B. microti antigens, including some previously uncharacterized antigens. Thirty of the most immunodominant B. microti antigens were expressed as recombinant proteins in E. coli. Among these, the combined use of two novel antigens and one previously described antigen provided 96% sensitivity and 100% specificity in identifying B. microti antibody containing sera in an ELISA. Using extensive computational sequence and bioinformatics analyses and cellular localization studies, we have clarified the domain architectures, potential biological functions, and evolutionary relationships of the most immunodominant B. microti antigens. Notably, we found that the BMN-family antigens are not monophyletic as currently annotated, but rather can be categorized into two evolutionary unrelated groups of BMN proteins respectively defined by two structurally distinct classes of extracellular domains. Our studies have enhanced the repertoire of immunodominant B. microti antigens, and assigned potential biological function to these antigens, which can be evaluated to develop novel assays and candidate vaccines.

Early evening questing and oviposition activity by the Culex (Diptera: Culicidae) vectors of West Nile virus in northeastern North America.

To determine whether the Culex (Diptera: Culicidae) mosquitoes that transmit West Nile virus (family Flaviviridae, genus Flavivirus, WNV) in the northeastern United States seek hosts and oviposit contemporaneously, we recorded when these mosquitoes attacked caged birds and when they deposited eggs. They traversed oviposition sites most frequently approximately 2 h after astronomical sunset, and eggs generally were deposited at that time. Although they most frequently approached avian hosts approximately 2 h after sunset during midsummer, they are more opportunistic during mid- to late fall. Because the Culex mosquitoes that serve as the main vectors of West Nile virus in the northeastern United States quest for hosts and seek to oviposit well after sunset, insecticidal aerosols would be most effective when applied at that time.

Efficacy of resmethrin aerosols applied from the road for suppressing Culex vectors of West Nile virus.

We determined whether aerosol applications of resmethrin, delivered from the road, suppress the reproductive activity of Culex pipiens pipiens and Cx. restuans mosquitoes in suburban sites located near Boston. Oviposition implies a prior blood-feeding event and hence a potential West Nile virus (WNV) transmission-related event. Droplet size, rate of delivery and meteorological conditions were monitored. The target populations proved to be fully susceptible to the insecticide that was used. The roads in the test sites generally gave adequate opportunity for insecticidal coverage. We found that the aerosol plume may have failed to contact the target mosquitoes and conclude that such insecticidal aerosols, delivered from the road, may not effectively reduce the force of transmission of WNV in our test sites.

Borrelia miyamotoi Disease: Neither Lyme Disease Nor Relapsing Fever.

Borrelia miyamotoi disease (BMD) is a newly recognized borreliosis globally transmitted by ticks of the Ixodes persulcatus species complex. Once considered to be a tick symbiont with no public health implications, B miyamotoi is increasingly recognized as the agent of a nonspecific febrile illness often misdiagnosed as acute Lyme disease without rash, or as ehrlichiosis. The frequency of its diagnosis in the northeastern United States is similar to that of human granulocytic ehrlichiosis. A diagnosis of BMD is confirmed by polymerase chain reaction analysis of acute blood samples, or by seroconversion using a recombinant glycerophosphodiester phosphodiesterase enzyme immunoassay. BMD is successfully treated with oral doxycycline or amoxicillin.

A readily constructed lard-can trap for sampling host-seeking mosquitoes.

Although lard-can traps have been used for sampling host-seeking mosquitoes for at least a half-century, the materials from which they originally were constructed no longer are available. We therefore devised a method for constructing such devices from parts available in the ventilation industry. These traps, baited with birds and mounted near the tops of trees, were employed to monitor the host-seeking activity of Culex spp. mosquitoes. Lard-can traps, constructed in this manner, are economical and sturdy and effectively sample the Culex mosquitoes that appear to perpetuate West Nile virus in North America.