Performance of a Modified Two-Tiered Testing Enzyme Immunoassay Algorithm for Serologic Diagnosis of Lyme Disease in Nova Scotia

ABSTRACT Compared to the standard two-tiered testing (STTT) algorithm for Lyme disease serology using an enzyme immunoassay (EIA) followed by Western blotting, data from the United States suggest that a modified two-tiered testing (MTTT) algorithm employing two EIAs has improved sensitivity to detect early localized Borrelia burgdorferi infections without compromising specificity. From 2011 to 2014, in the Canadian province of Nova Scotia, where Lyme disease is hyperendemic, sera submitted for Lyme disease testing were subjected to a whole-cell EIA, followed by C6 EIA and subsequently IgM and/or IgG immunoblots on sera with EIA-positive or equivocal results. Here, we evaluate the effectiveness of the MTTT algorithm compared to the STTT approach in a Nova Scotian population. Retrospective chart reviews were performed on patients testing positive with the whole-cell and C6 EIAs (i.e., the MTTT algorithm). Patients were classified as having Lyme disease if they had a positive STTT result, a negative STTT result but symptoms consistent with Lyme disease, or evidence of seroconversion on paired specimens. Of the 10,253 specimens tested for Lyme disease serology, 9,806 (95.6%) were negative. Of 447 patients who tested positive, 271 charts were available for review, and 227 were classified as patients with Lyme disease. The MTTT algorithm detected 25% more early infections with a specificity of 99.56% (99.41 to 99.68%) compared to the STTT. These are the first Canadian data to show that serology using a whole-cell sonicate EIA followed by a C6 EIA (MTTT) had improved sensitivity for detecting early B. burgdorferi infection with specificity similar to that of two-tiered testing using Western blots.

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Evaluation of bioMérieux's Dissociated Vidas Lyme IgM II and IgG II as a First-Tier Diagnostic Assay for Lyme Disease

Journal of Clinical Microbiology ◽

10.1128/jcm.02407-16 ◽

2017 ◽

Vol 55 (6) ◽

pp. 1698-1706 ◽

Cited By ~ 15

Author(s):

Claudia R. Molins ◽

Mark J. Delorey ◽

Adam Replogle ◽

Christopher Sexton ◽

Martin E. Schriefer

Keyword(s):

United States ◽

Lyme Disease ◽

Enzyme Immunoassay ◽

The United States ◽

Statistical Analyses ◽

Diagnostic Approach ◽

Diagnostic Assay ◽

Western Immunoblotting ◽

Testing Strategies ◽

Second Tier

ABSTRACTThe recommended laboratory diagnostic approach for Lyme disease is a standard two-tiered testing (STTT) algorithm where the first tier is typically an enzyme immunoassay (EIA) that if positive or equivocal is reflexed to Western immunoblotting as the second tier. bioMérieux manufactures one of the most commonly used first-tier EIAs in the United States, the combined IgM/IgG Vidas test (LYT). Recently, bioMérieux launched its dissociated first-tier tests, the Vidas Lyme IgM II (LYM) and IgG II (LYG) EIAs, which use purified recombinant test antigens and a different algorithm than STTT. The dissociated LYM/LYG EIAs were evaluated against the combined LYT EIA using samples from 471 well-characterized Lyme patients and controls. Statistical analyses were conducted to assess the performance of these EIAs as first-tier tests and when used in two-tiered algorithms, including a modified two-tiered testing (MTTT) approach where the second-tier test was a C6 EIA. Similar sensitivities and specificities were obtained for the two testing strategies (LYT versus LYM/LYG) when used as first-tier tests (sensitivity, 83 to 85%; specificity, 85 to 88%) with an observed agreement of 80%. Sensitivities of 68 to 69% and 76 to 77% and specificities of 97% and 98 to 99% resulted when the two EIA strategies were followed by Western immunoblotting and when used in an MTTT, respectively. The MTTT approach resulted in significantly higher sensitivities than did STTT. Overall, the LYM/LYG EIAs performed equivalently to the LYT EIA in test-to-test comparisons or as first-tier assays in STTT or MTTT with few exceptions.

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Thermal history and subsidence of rifted continental margins—evidence from wells on the Nova Scotian and Labrador Shelves

Canadian Journal of Earth Sciences ◽

10.1139/e79-046 ◽

1979 ◽

Vol 16 (3) ◽

pp. 505-522 ◽

Cited By ~ 65

Author(s):

C. E. Keen

Keyword(s):

Nova Scotia ◽

Time History ◽

The United States ◽

Tectonic Subsidence ◽

Continental Margins ◽

Thermal Cooling ◽

Nova Scotian ◽

Temperature Time ◽

Good Agreement ◽

Cooling Model

The subsidence histories of the Labrador and Nova Scotian rifted continental margins have been determined from biostratigraphic data for 11 deep exploratory wells off Nova Scotia, for five wells off Labrador, for three wells northeast of Newfoundland, and for one well off the northeast coast of the United States of America. The components of subsidence, due to sediment loading, and when possible due to loading by changes in eustatic sea level, were removed, leaving that part of the subsidence, the tectonic subsidence, caused by cooling of the lithosphere or by other deep seated processes. The thermal cooling model theoretically predicts a linear relationship between tectonic subsidence and t½, where t is the time since subsidence began. This relationship should be obeyed during the first tens of Ma of subsidence. The slope of this curve depends upon the temperature to which the crust and upper mantle were heated during the initial rifting stage and can be used to derive the temperature–time history within the sediments, the present temperature distribution, and geothermal gradient. The data show that the observed subsidence curves behave in accordance with the thermal cooling model, at least during the first 80 Ma after subsidence began and obey the equation y = 300(± 80)t1/2 m, where y is the tectonic subsidence. The slopes of the subsidence curves are similar for the Labrador Shelf, the Nova Scotian Shelf, and the shelf off the northeastern U.S.A. More rapid and variable subsidence occurs northeast of Newfoundland and this may be associated, in a way yet to be established, with the anomalous foundered continental crust near the Orphan Knoll and Flemish Cap micro-continents which lie close to this area. After about 80 Ma, the subsidence appears to depart from the linear t1/2 law in a manner similar to the subsidence curves for oceanic crust, but this is not well established by the data. The present temperatures and temperature gradients computed using the slope of the subsidence curves show good agreement with measured values; geothermal gradients of 17.5 °C km−1 and 26 °C km−1 are calculated off Nova Scotia and Labrador respectively, and mean values of about 23 °C km−1 are observed. The computed temperature–time history within the sediments was used to estimate values of vitrinite reflectance, an indicator of the degree of organic metamorphism. These values show reasonable agreement with the measured values and suggest that only the Upper Jurassic and Lower Cretaceous sediments off Nova Scotia and the Paleocene sediments off Labrador are sufficiently mature to be good sources of petroleum. The linear t1/2 behaviour of the subsidence, and the good agreement between predicted and observed temperatures support the contention that cooling is largely responsible for the observed tectonic subsidence. The similarity of results from different areas suggests that the usefulness of the method is not restricted to a particular geographical area and may be applied to other rifted continental margins. Comparisons between the subsidence rates, thermal histories, and crustal structure at rifted margins on a worldwide scale may provide insights concerning the processes controlling their development. The temperature–time histories of the sediments estimated from the subsidence may be useful in establishing the potential of a rifted margin area for petroleum generation when little other information is available.

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Lyme Disease Testing by Large Commercial Laboratories in the United States

Clinical Infectious Diseases ◽

10.1093/cid/ciu397 ◽

2014 ◽

Vol 59 (5) ◽

pp. 676-681 ◽

Cited By ~ 238

Author(s):

Alison F. Hinckley ◽

Neeta P. Connally ◽

James I. Meek ◽

Barbara J. Johnson ◽

Melissa M. Kemperman ◽

...

Keyword(s):

United States ◽

Lyme Disease ◽

The United States ◽

Disease Testing

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Antibodies to Borrelia burgdorferi OspA, OspC, OspF, and C6 Antigens as Markers for Early and Late Infection in Dogs

Clinical and Vaccine Immunology ◽

10.1128/cvi.05653-11 ◽

2012 ◽

Vol 19 (4) ◽

pp. 527-535 ◽

Cited By ~ 32

Author(s):

Bettina Wagner ◽

Heather Freer ◽

Alicia Rollins ◽

David Garcia-Tapia ◽

Hollis N. Erb ◽

...

Keyword(s):

Lyme Disease ◽

Borrelia Burgdorferi ◽

The United States ◽

Sensu Stricto ◽

Antibody Responses ◽

Surface Proteins ◽

Early Infection ◽

Enzyme Linked Immunosorbent Assay ◽

Content Type ◽

Antibody Levels

ABSTRACTLyme disease in the United States is caused byBorrelia burgdorferisensu stricto, which is transmitted to mammals by infected ticks.Borreliaspirochetes differentially express immunogenic outer surface proteins (Osp). Our aim was to evaluate antibody responses to Osp antigens to aid the diagnosis of early infection and the management of Lyme disease. We analyzed antibody responses during the first 3 months after the experimental infection of dogs using a novel multiplex assay. Results were compared to those obtained with two commercial assays detecting C6 antigen. Multiplex analysis identified antibodies to OspC and C6 as early as 3 weeks postinfection (p.i.) and those to OspF by 5 weeks p.i. Antibodies to C6 and OspF increased throughout the study, while antibodies to OspC peaked between 7 and 11 weeks p.i. and declined thereafter. A short-term antibody response to OspA was observed in 3/8 experimentally infected dogs on day 21 p.i. Quant C6 enzyme-linked immunosorbent assay (ELISA) results matched multiplex results during the first 7 weeks p.i.; however, antibody levels subsequently declined by up to 29%. Immune responses then were analyzed in sera from 125 client-owned dogs and revealed high agreement between antibodies to OspF and C6 as robust markers for infection. Results from canine patient sera supported that OspC is an early infection marker and antibodies to OspC decline over time. The onset and decline of antibody responses toB. burgdorferiOsp antigens and C6 reflect their differential expression during infection. They provide valuable tools to determine the stage of infection, treatment outcomes, and vaccination status in dogs.

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Delineating Surface Epitopes of Lyme Disease Pathogen Targeted by Highly Protective Antibodies of New Zealand White Rabbits

Infection and Immunity ◽

10.1128/iai.00246-19 ◽

2019 ◽

Vol 87 (8) ◽

Cited By ~ 2

Author(s):

Artem S. Rogovskyy ◽

Salvador Eugenio C. Caoili ◽

Yurij Ionov ◽

Helen Piontkivska ◽

Pavel Skums ◽

...

Keyword(s):

New Zealand ◽

Lyme Disease ◽

Protective Efficacy ◽

Empirical Studies ◽

Surface Expression ◽

The United States ◽

Mammalian Host ◽

Content Type ◽

Random Peptide ◽

New Zealand White Rabbits

ABSTRACTLyme disease (LD), the most prevalent vector-borne illness in the United States and Europe, is caused byBorreliella burgdorferi. No vaccine is available for humans. Dogmatically,B. burgdorferican establish a persistent infection in the mammalian host (e.g., mice) due to a surface antigen, VlsE. This antigenically variable protein allows the spirochete to continually evade borreliacidal antibodies. However, our recent study has shown that theB. burgdorferispirochete is effectively cleared by anti-B. burgdorferiantibodies of New Zealand White rabbits, despite the surface expression of VlsE. Besides homologous protection, the rabbit antibodies also cross-protect against heterologousB. burgdorferispirochetes and significantly reduce the pathology of LD arthritis in persistently infected mice. Thus, this finding that NZW rabbits develop a unique repertoire of very potent antibodies targeting the protective surface epitopes, despite abundant VlsE, prompted us to identify the specificities of the protective rabbit antibodies and their respective targets. By applying subtractive reverse vaccinology, which involved the use of random peptide phage display libraries coupled with next-generation sequencing and our computational algorithms, repertoires of nonprotective (early) and protective (late) rabbit antibodies were identified and directly compared. Consequently, putative surface epitopes that are unique to the protective rabbit sera were mapped. Importantly, the relevance of newly identified protection-associated epitopes for their surface exposure has been strongly supported by prior empirical studies. This study is significant because it now allows us to systematically test the putative epitopes for their protective efficacy with an ultimate goal of selecting the most efficacious targets for development of a long-awaited LD vaccine.

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Improved Serodiagnostic Performance for Lyme Disease by Use of Two Recombinant Proteins in Enzyme-Linked Immunosorbent Assay Compared to Standardized Two-Tier Testing

Journal of Clinical Microbiology ◽

10.1128/jcm.01004-17 ◽

2017 ◽

Vol 55 (10) ◽

pp. 3046-3056 ◽

Cited By ~ 4

Author(s):

Gary L. Bradshaw ◽

R. Kelley Thueson ◽

Todd J. Uriona

Keyword(s):

Lyme Disease ◽

Recombinant Proteins ◽

Immunosorbent Assay ◽

Negative Control ◽

Full Length ◽

Test Method ◽

Stage I ◽

Enzyme Linked Immunosorbent Assay ◽

Western Blots ◽

Content Type

ABSTRACTThe most reliable test method for the serological confirmation of Lyme disease (LD) is a 2-tier method recommended by the CDC in 1995. The first-tier test is a low-specificity enzyme-linked immunosorbent assay (ELISA), and the second-tier tests are higher-specificity IgG and IgM Western blots. This study describes the selection of twoBorrelia burgdorferirecombinant proteins and evaluation of their performance in a simple 1-tier test for the serological confirmation of LD. These two proteins were generated from (i) the full-lengthdbpAgene combined with the invariable region 6 of thevlsEgene (DbpA/C6) and (b) the full-lengthospCgene (OspC). The expressed DbpA/C6 and OspC proteins were useful in detecting anti-BorreliaIgG and IgM antibodies, respectively. A blind study was conducted on a well-characterized panel of 279 human sera from the CDC, comparing ELISAs using these two recombinant antigens with the 2-tier test method. The two methods (DbpA/C6-OspC versus 2-tier test) were equivalent in identifying sera from negative-control subjects (99% and 100% specificity, respectively) and in detecting stage II and III LD patient sera (100% and 100% sensitivity). However, the DbpA/C6-OspC ELISA was markedly better (80% versus 63%) than the 2-tier test method in detecting anti-Borreliaantibodies in stage I LD patients. The findings suggest that these antigens could be used in a simple 1-tier ELISA that is faster to perform, easier to interpret, and less expensive than the 2-tier test method and which is better at detectingBorrelia-specific antibodies in sera from patients with stage I LD.

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Variation in the Microbiota of Ixodes Ticks with Regard to Geography, Species, and Sex

Applied and Environmental Microbiology ◽

10.1128/aem.01562-15 ◽

2015 ◽

Vol 81 (18) ◽

pp. 6200-6209 ◽

Cited By ~ 76

Author(s):

Will Van Treuren ◽

Loganathan Ponnusamy ◽

R. Jory Brinkerhoff ◽

Antonio Gonzalez ◽

Christian M. Parobek ◽

...

Keyword(s):

New York ◽

North Carolina ◽

South Carolina ◽

Lyme Disease ◽

Ixodes Scapularis ◽

Illumina Miseq ◽

The United States ◽

Upper Midwest ◽

Content Type ◽

Ixodes Ticks

ABSTRACTIxodes scapularisis the principal vector of Lyme disease on the East Coast and in the upper Midwest regions of the United States, yet the tick is also present in the Southeast, where Lyme disease is absent or rare. A closely related species,I. affinis, also carries the pathogen in the South but does not seem to transmit it to humans. In order to better understand the geographic diversity of the tick, we analyzed the microbiota of 104 adultI. scapularisand 13 adultI. affinisticks captured in 19 locations in South Carolina, North Carolina, Virginia, Connecticut, and New York. Initially, ticks from 4 sites were analyzed by 454 pyrosequencing. Subsequently, ticks from these sites plus 15 others were analyzed by sequencing with an Illumina MiSeq machine. By both analyses, the microbiomes of female ticks were significantly less diverse than those of male ticks. The dissimilarity between tick microbiomes increased with distance between sites, and the state in which a tick was collected could be inferred from its microbiota. The genusRickettsiawas prominent in all locations.Borreliawas also present in most locations and was present at especially high levels in one site in western Virginia. In contrast, members of the familyEnterobacteriaceaewere very common in North CarolinaI. scapularisticks but uncommon inI. scapularisticks from other sites and in North CarolinaI. affinisticks. These data suggest substantial variations in theIxodesmicrobiota in association with geography, species, and sex.

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The Past, Present, and (Possible) Future of Serologic Testing for Lyme Disease

Journal of Clinical Microbiology ◽

10.1128/jcm.03394-15 ◽

2016 ◽

Vol 54 (5) ◽

pp. 1191-1196 ◽

Cited By ~ 31

Author(s):

Elitza S. Theel

Keyword(s):

United States ◽

Lyme Disease ◽

Borrelia Burgdorferi ◽

Metabolic Profiling ◽

Historical Perspective ◽

The United States ◽

Performance Characteristics ◽

Content Type ◽

The Past ◽

Testing Algorithm

Lyme disease prevails as the most commonly transmitted tick-borne infection in the United States, and serologic evaluation for antibodies toBorrelia burgdorferiremains the recommended modality for diagnosis. This review presents a brief historical perspective on the evolution of serologic assays for Lyme disease and provides a summary of the performance characteristics for the currently recommended two-tiered testing algorithm (TTTA). Additionally, a recently proposed alternative to the traditional TTTA is discussed, and novel methodologies, including immuno-PCR and metabolic profiling for Lyme disease, are outlined.

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Fluorescent Proteins, Promoters, and Selectable Markers for Applications in the Lyme Disease SpirocheteBorrelia burgdorferi

Applied and Environmental Microbiology ◽

10.1128/aem.01824-18 ◽

2018 ◽

Vol 84 (24) ◽

Cited By ~ 3

Author(s):

Constantin N. Takacs ◽

Zachary A. Kloos ◽

Molly Scott ◽

Patricia A. Rosa ◽

Christine Jacobs-Wagner

Keyword(s):

Lyme Disease ◽

Fluorescent Proteins ◽

The United States ◽

Fine Tuning ◽

Human Pathogen ◽

Molecular Tools ◽

Antibiotic Selection ◽

Content Type ◽

Biological Studies ◽

Selection Markers

ABSTRACTLyme disease is the most widely reported vector-borne disease in the United States. Its incidence is rapidly increasing, and disease symptoms can be debilitating. The need to understand the biology of the disease agent, the spirocheteBorrelia burgdorferi, is thus evermore pressing. Despite important advances inB. burgdorferigenetics, the array of molecular tools available for use in this organism remains limited, especially for cell biological studies. Here, we adapt a palette of bright and mostly monomeric fluorescent proteins for versatile use and multicolor imaging inB. burgdorferi. We also characterize two novel antibiotic selection markers and establish the feasibility of their use in conjunction with extant markers. Last, we describe a set of promoters of low and intermediate strengths that allow fine-tuning of gene expression levels. These molecular tools complement and expand current experimental capabilities inB. burgdorferi, which will facilitate future investigation of this important human pathogen. To showcase the usefulness of these reagents, we used them to investigate the subcellular localization of BB0323, aB. burgdorferilipoprotein essential for survival in the host and vector environments. We show that BB0323 accumulates at the cell poles and future division sites ofB. burgdorfericells, highlighting the complex subcellular organization of this spirochete.IMPORTANCEGenetic manipulation of the Lyme disease spirocheteB. burgdorferiremains cumbersome, despite significant progress in the field. The scarcity of molecular reagents available for use in this pathogen has slowed research efforts to study its unusual biology. Of interest,B. burgdorferidisplays complex cellular organization features that have yet to be understood. These include an unusual morphology and a highly fragmented genome, both of which are likely to play important roles in the bacterium’s transmission, infectivity, and persistence. Here, we complement and expand the array of molecular tools available for use inB. burgdorferiby generating and characterizing multiple fluorescent proteins, antibiotic selection markers, and promoters of varied strengths. These tools will facilitate investigations in this important human pathogen, as exemplified by the polar and midcell localization of the cell envelope regulator BB0323, which we uncovered using these reagents.

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Evolutionary Aspects of Emerging Lyme Disease in Canada

Applied and Environmental Microbiology ◽

10.1128/aem.01671-15 ◽

2015 ◽

Vol 81 (21) ◽

pp. 7350-7359 ◽

Cited By ~ 15

Author(s):

N. H. Ogden ◽

E. J. Feil ◽

P. A. Leighton ◽

L. R. Lindsay ◽

G. Margos ◽

...

Keyword(s):

Public Health ◽

United States ◽

At Risk ◽

North America ◽

Lyme Disease ◽

Severe Disease ◽

The United States ◽

Sensu Stricto ◽

Content Type ◽

Evolutionary Aspects

ABSTRACTIn North America, Lyme disease (LD) is a tick-borne zoonosis caused by the spirochete bacteriumBorrelia burgdorferisensu stricto, which is maintained by wildlife. Tick vectors and bacteria are currently spreading into Canada and causing increasing numbers of cases of LD in humans and raising a pressing need for public health responses. There is no vaccine, and LD prevention depends on knowing who is at risk and informing them how to protect themselves from infection. Recently, it was found in the United States that some strains ofB. burgdorferisensu strictocause severe disease, whereas others cause mild, self-limiting disease. While many strains occurring in the United States also occur in Canada, strains in some parts of Canada are different from those in the United States. We therefore recognize a need to identify which strains specific to Canada can cause severe disease and to characterize their geographic distribution to determine which Canadians are particularly at risk. In this review, we summarize the history of emergence of LD in North America, our current knowledge ofB. burgdorferisensu strictodiversity, its intriguing origins in the ecology and evolution of the bacterium, and its importance for the epidemiology and clinical and laboratory diagnosis of LD. We propose methods for investigating associations betweenB. burgdorferisensu strictodiversity, ecology, and pathogenicity and for developing predictive tools to guide public health interventions. We also highlight the emergence ofB. burgdorferisensu strictoin Canada as a unique opportunity for exploring the evolutionary aspects of tick-borne pathogen emergence.

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