The molecular determinants of classical pathway complement inhibition by OspEF-related proteins of Borrelia burgdorferi.

The complement system serves as the first line of defense against invading pathogens by promoting opsonophagocytosis and bacteriolysis. Antibody-dependent activation of complement occurs through the classical pathway and relies on the activity of initiating complement proteases of the C1 complex, C1r and C1s. The causative agent of Lyme disease, Borrelia burgdorferi, expresses two paralogous outer surface lipoproteins of the OspEF-related protein family, ElpB and ElpQ, that act as specific inhibitors of classical pathway activation. We have previously shown that ElpB and ElpQ bind directly to C1r and C1s with high affinity and specifically inhibit C2 and C4 cleavage by C1s. To further understand how these novel protease inhibitors function, we carried out a series of hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments using ElpQ and full-length activated C1s as a model of Elp-protease interaction. Comparison of HDX-MS profiles between unbound ElpQ and the ElpQ/C1s complex revealed a putative C1s-binding site on ElpQ. HDX-MS-guided, site-directed ElpQ mutants were generated and tested for direct binding to C1r and C1s using surface plasmon resonance. Several residues within the C-terminal region of ElpQ were identified as important for protease binding, including a single conserved tyrosine residue that was required for ElpQ- and ElpB-mediated complement inhibition. Collectively, our study identifies key molecular determinants for classical pathway protease recognition by Elp proteins. This investigation improves our understanding of the unique complement inhibitory mechanism employed by Elp proteins which serve as part of a sophisticated complement evasion system present in Lyme disease spirochetes.

Analysis of bb0556 Expression and Its Role During Borrelia burgdorferi Mammalian Infection.

In Borrelia burgdorferi, BB0556 was annotated as a conserved hypothetical protein. We herein investigated gene expression and the importance of this protein during infection. Our data support that bb0556 forms an operon with five other genes. A transcriptional start site and the associated σ70-type promoter were identified in the sequences upstream of bb0554, and luciferase reporter assays indicated that this promoter is functional in B. burgdorferi. Furthermore, the sequences upstream of bb0556 contain an internal promoter to drive gene expression. bb0556 expression was affected by various environmental factors such as changes in temperature, pH, and cell density when B. burgdorferi was grown in vitro. Surprisingly, significant differences were observed for bb0556 expression between B. burgdorferi strains B31-A3 and CE162, likely due to the different cis- and trans-acting factors in these strains. Moreover, bb0556 was found to be highly expressed by B. burgdorferi in infected mice tissues, suggesting that this gene plays an important role during animal infection. To test this hypothesis, we generated a bb0556 deletion mutant in a virulent bioluminescent B. burgdorferi strain. The mutant grew normally in the medium and displayed no defect in the resistance to environmental stresses such as reactive oxygen species, reactive nitrogen species, and osmotic stress. However, when the infectivity was compared between the mutant and its parental strain using in vivo bioluminescence imaging as well as analyses of spirochete recovery and bacterial burdens in animal tissues, our data showed that, contrary to the parental strain, the mutant was unable to infect mice. Complementation of bb0556 in cis fully restored the infectious phenotype to wild-type levels. Taken together, our study demonstrates that the hypothetical protein BB0556 is a novel virulence factor essential for B. burgdorferi mammalian infection.

Lipoproteome screening of the Lyme disease agent identifies inhibitors of antibody-mediated complement killing.

Spirochetal pathogens, such as the causative agent of Lyme disease, Borrelia burgdorferi sensu lato, encode an abundance of lipoproteins; however, due in part to their evolutionary distance from more well-studied bacteria, such as Proteobacteria and Firmicutes, few spirochetal lipoproteins have assigned functions. Indeed, B. burgdorferi devotes almost 8% of its genome to lipoprotein genes and interacts with its environment primarily through the production of at least 80 surface-exposed lipoproteins throughout its tick vector­vertebrate host lifecycle. Several B. burgdorferi lipoproteins have been shown to serve roles in cellular adherence or immune evasion, but the functions for most B. burgdorferi surface lipoproteins remain unknown. In this study, we developed a B. burgdorferi lipoproteome screening platform utilizing intact spirochetes that enables the identification of previously unrecognized host interactions. As spirochetal survival in the bloodstream is essential for dissemination, we targeted our screen to C1, the first component of the classical (antibody-initiated) complement pathway. We identified two high-affinity C1 interactions by the paralogous lipoproteins, ElpB and ElpQ (also termed ErpB and ErpQ, respectively). Using biochemical, microbiological, and biophysical approaches, we demonstrate that ElpB and ElpQ bind the activated forms of the C1 proteases, C1r and C1s, and represent a distinct mechanistic class of C1 inhibitors that protect the spirochete from antibody-mediated complement killing. In addition to identifying a mode of complement inhibition, our study establishes a lipoproteome screening methodology as a discovery platform for identifying direct host­pathogen interactions that are central to the pathogenesis of spirochetes, such as the Lyme disease agent.

Interference with LTßR signaling by tick saliva facilitates transmission of Lyme disease spirochetes.

Lyme spirochetes have coevolved with ticks to optimize transmission to hosts using tick salivary molecules (TSMs) to counteract host defenses. TSMs modulate various molecular events at the tick-host interface. Lymphotoxin-beta receptor (LTßR) is a vital immune receptor and plays protective roles in host immunity against microbial infections. We found that Ltbr knockout mice were more susceptible to Lyme disease spirochetes, suggesting the involvement of LTßR signaling in tick-borne Borrelia infection. Further investigation showed that a 15-kDa TSM protein from Ixodes persulcatus (I. persulcatus salivary protein; IpSAP) functioned as an immunosuppressant to facilitate the transmission and infection of Lyme disease spirochetes. IpSAP directly interacts with LTßR to block its activation, thus inhibiting the downstream signaling and consequently suppressing immunity. IpSAP immunization provided mice with significant protection against I. persulcatus-mediated Borrelia garinii infection. Notably, the immunization showed considerable cross-protection against other Borrelia infections mediated by other ixodid ticks. One of the IpSAP homologs from other ixodid ticks showed similar effects on Lyme spirochete transmission. Together, our findings suggest that LTßR signaling plays an important role in blocking the transmission and pathogenesis of tick-borne Lyme disease spirochetes, and that IpSAP and its homologs are promising candidates for broad-spectrum vaccine development.

Comparative Evaluation of Commercial Test Kits Cleared for Use in Modified Two-Tiered Testing Algorithms for Serodiagnosis of Lyme Disease.

BACKGROUND: Modified 2-tiered testing (MTTT) for Lyme disease utilizes automatable, high throughput immunoassays (AHTIs) in both tiers without involving western immunoblots, offering performance and practical advantages over standard 2-tiered testing (STTT; first-tier AHTI followed by immunoglobulin M (IgM) and immunoglobulin G (IgG) western immunoblots). For MTTT, Centers for Disease Control and Prevention recommends using AHTI test kits that have been cleared by Food and Drug Administration (FDA) specifically for this intended use. We evaluated performance of FDA-cleared MTTT commercial test kits from 3 manufacturers by comparing with STTT results. METHODS: We performed MTTT (total antibody AHTI with reflex to separate IgM and IgG AHTIs) using test kits from Diasorin, Gold Standard Diagnostics (GSD), and Zeus Scientific on 382 excess serum samples submitted to the clinical laboratory for routine Lyme disease serologic testing in July 2018, measuring agreement between MTTT and STTT using the κ statistic. RESULTS: Overall agreement with STTT was 0.87 (95% confidence interval [CI], .77-.97) using Diasorin assays (almost perfect agreement), 0.80 (95% CI, .68-.93) using GSD assays (substantial agreement) and 0.79 (95% CI, .68-.90) using Zeus assays (substantial agreement). For detection of IgM reactivity, agreement between MTTT and STTT was 0.70 (.51-.90; substantial), 0.63 (95% CI, .44-.82; substantial) and 0.56 (95% CI, .38-.73; moderate), respectively. For detection of IgG reactivity, MTTT/STTT agreement was 0.73 (95% CI,.58-.88), 0.78 (95% CI, .62-.94), and 0.75 (95% CI, .60-.90), respectively (substantial agreement in all cases). CONCLUSIONS: MTTT results obtained using commercial test kits from 3 different manufacturers had substantial to almost perfect agreement with STTT results overall and moderate to substantial agreement for IgM and IgG detection independently. Commercial MTTT tests can be used broadly for the diagnosis of Lyme disease.

Genome-wide DNA Methylation Profiling in Lyme Neuroborreliosis Reveals Altered Methylation Patterns of HLA Genes.

Lyme neuroborreliosis (LNB) is a complex neuroinflammatory disorder caused by Borrelia burgdorferi, which is transmitted through tick bites. Epigenetic alterations, specifically DNA methylation (DNAm), could play a role in the host immune response during infection. In this study, we present the first genome-wide analysis of DNAm in peripheral blood mononuclear cells from patients with LNB and those without LNB. Using a network-based approach, we highlighted HLA genes at the core of these DNAm changes, which were found to be enriched in immune-related pathways. These findings shed light on the role of epigenetic modifications in the LNB pathogenesis that should be confirmed and further expanded upon in future studies.

Lyme Arthritis: A 50-Year Journey.

Lyme arthritis (LA) was recognized as a separate entity in 1975 because of geographic clustering of children often diagnosed with juvenile rheumatoid arthritis in Lyme, Connecticut. After identification of erythema migrans as a common early feature of the illness, a prospective study of such patients implicated Ixodes scapularis ticks in disease transmission. In 1982, the causative agent, now called Borrelia burgdorferi, was cultured from these ticks and from Lyme disease patients. Subsequently, it was shown that LA could usually be treated successfully with oral antibiotics but sometimes required intravenous antibiotics. Yet, a small percentage of patients developed a dysregulated, proinflammatory immune response leading to persistent postinfectious synovitis with vascular damage, cytotoxic and autoimmune responses, and fibroblast proliferation, a lesion similar to that of rheumatoid arthritis. The message from postinfectious LA for other autoimmune arthritides is that a complex immune response with autoimmune features can begin with a microbial infection.

Lyme Disease Surveillance and Epidemiology in the United States: A Historical Perspective.

In the 40 years since Steere and colleagues first described Lyme disease, the illness has increased in incidence and distribution to become the most common vector-borne disease in the United States. Public health officials have developed, implemented, and revised surveillance systems to describe and monitor the condition. Much has been learned about the epidemiology of the illness, despite practical and logistical constraints that have encumbered the collection and interpretation of surveillance data. Future development of automated data collection from electronic health records as a source of surveillance and clinical information will address practical challenges and help answer ongoing questions about complications and persistent symptoms. Robust surveillance will be essential to monitor the effectiveness and safety of future vaccines and other preventive measures.

BadR directly represses the expression of the glycerol utilization operon in the Lyme disease pathogen.

Glycerol utilization as a carbohydrate source by Borreliella burgdorferi, the Lyme disease spirochete, is critical for its successful colonization and persistence in the tick vector. The expression of the glpFKD (glp) operon, which encodes proteins for glycerol uptake/utilization, must be tightly regulated during the enzootic cycle of B. burgdorferi. Previous studies have established that the second messenger cyclic di-GMP (c-di-GMP) is required for the activation of glp expression, while an alternative sigma factor RpoS acts as a negative regulator for glp expression. In the present study, we report identification of a cis element within the 5´ untranslated region of glp that exerts negative regulation of glp expression. Further genetic screen of known and predicted DNA-binding proteins encoded in the genome of B. burgdorferi uncovered that overexpressing Borrelia host adaptation regulator (BadR), a known global regulator, dramatically reduced glp expression. Similarly, the badR mutant significantly increased glp expression. Subsequent electrophoretic mobility shift assay analyses demonstrated that BadR directly binds to this cis element, thereby repressing glp independent of RpoS-mediated repression. The efficiency of BadR binding was further assessed in the presence of c-di-GMP and various carbohydrates. This finding highlights multi-layered positive and negative regulatory mechanisms employed by B. burgdorferi to synchronize glp expression throughout its enzootic cycle.IMPORTANCEBorreliella burgdorferi, the Lyme disease pathogen, must modulate its gene expression differentially to adapt successfully to its two disparate hosts. Previous studies have demonstrated that the glycerol uptake and utilization operon, glpFKD, plays a crucial role in spirochetal survival within ticks. However, the glpFKD expression must be repressed when B. burgdorferi transitions to the mammalian host. In this study, we identified a specific cis element responsible for the repression of glpFKD. We further pinpointed Borrelia host adaptation regulator as the direct binding protein to this cis element, thereby repressing glpFKD expression. This discovery paves the way for a deeper exploration of how zoonotic pathogens sense distinct hosts and switch their carbon source utilization during transmission.

Hitchhiker's Guide to Borrelia burgdorferi.

Don't Panic. In the nearly 50 years since the discovery of Lyme disease, Borrelia burgdorferi has emerged as an unlikely workhorse of microbiology. Interest in studying host-pathogen interactions fueled significant progress in making the fastidious microbe approachable in laboratory settings, including the development of culture methods, animal models, and genetic tools. By developing these systems, insight has been gained into how the microbe is able to survive its enzootic cycle and cause human disease. Here, we discuss the discovery of B. burgdorferi and its development as a model organism before diving into the critical lessons we have learned about B. burgdorferi biology at pivotal stages of its lifecycle: gene expression changes during the tick blood meal, colonization of a new vertebrate host, and developing a long-lasting infection in that vertebrate until a new tick feeds. Our goal is to highlight the advancements that have facilitated B. burgdorferi research and identify gaps in our current understanding of the microbe.

An AP-3-dependent pathway directs phagosome fusion with Rab8 and Rab11 vesicles involved in TLR2 signaling.

Intracellular compartmentalization of ligands, receptors and signaling molecules has been recognized as an important regulator of inflammation. The toll-like receptor (TLR) 2 pathway utilizes the trafficking molecule adaptor protein 3 (AP-3) to activate interleukin (IL)-6 signaling from within phagosomal compartments. To better understand the vesicular pathways that may contribute to intracellular signaling and cooperate with AP-3, we performed a vesicular siRNA screen. We identified Rab8 and Rab11 GTPases as important in IL-6 induction upon stimulation with the TLR2 ligand Pam3 CSK4 or the pathogen, Borrelia burgdorferi (Bb), the causative agent of Lyme disease. These Rabs were recruited to late and lysosomal stage phagosomes and co-transported with TLR2 signaling adaptors and effectors, such as MyD88, TRAM and TAK1, in an AP-3-dependent manner. Our data support a model where AP-3 mediates the recruitment of recycling and secretory vesicles and the assembly of signaling complexes at the phagosome.

Single-domain antibodies reveal unique borrelicidal epitopes on the Lyme disease vaccine antigen, outer surface protein A (OspA).

Camelid-derived, single-domain antibodies (VHHs) have proven to be extremely powerful tools in defining the antigenic landscape of immunologically heterogeneous surface proteins. In this report, we generated a phage-displayed VHH library directed against the candidate Lyme disease vaccine antigen, outer surface protein A (OspA). Two alpacas were immunized with recombinant OspA serotype 1 from Borrelia burgdorferi sensu stricto strain B31, in combination with the canine vaccine RECOMBITEK Lyme containing lipidated OspA. The phage library was subjected to two rounds of affinity enrichment ("panning") against recombinant OspA, yielding 21 unique VHHs within two epitope bins, as determined through competition enzyme linked immunosorbent assays (ELISAs) with a panel of OspA-specific human monoclonal antibodies. Epitope refinement was conducted by hydrogen exchange-mass spectrometry. Six of the monovalent VHHs were expressed as human IgG1-Fc fusion proteins and shown to have functional properties associated with protective human monoclonal antibodies, including B. burgdorferi agglutination, outer membrane damage, and complement-dependent borreliacidal activity. The VHHs displayed unique reactivity profiles with the seven OspA serotypes associated with B. burgdorferi genospecies in the United States and Europe consistent with there being unique epitopes across OspA serotypes that should be considered when designing and evaluating multivalent Lyme disease vaccines.

Assessment of the hypothetical protein BB0616 in the murine infection of Borrelia burgdorferi.

bb0616 of Borrelia burgdorferi, the Lyme disease pathogen, encodes a hypothetical protein of unknown function. In this study, we showed that BB0616 was not surface-exposed or associated with the membrane through localization analyses using proteinase K digestion and cell partitioning assays. The expression of bb0616 was influenced by a reduced pH but not by growth phases, elevated temperatures, or carbon sources during in vitro cultivation. A transcriptional start site for bb0616 was identified by using 5' rapid amplification of cDNA ends, which led to the identification of a functional promoter in the 5' regulatory region upstream of bb0616. By analyzing a bb0616-deficient mutant and its isogenic complemented counterparts, we found that the infectivity potential of the mutant was significantly attenuated. The inactivation of bb0616 displayed no effect on borrelial growth in the medium or resistance to oxidative stress, but the mutant was significantly more susceptible to osmotic stress. In addition, the production of global virulence regulators such as BosR and RpoS as well as virulence-associated outer surface lipoproteins OspC and DbpA was reduced in the mutant. These phenotypes were fully restored when gene mutation was complemented with a wild-type copy of bb0616. Based on these findings, we concluded that the hypothetical protein BB0616 is required for the optimal infectivity of B. burgdorferi, potentially by impacting B. burgdorferi virulence gene expression as well as survival of the spirochete under stressful conditions.

The plasmid-encoded members of paralogous gene family 52 are dispensable to the enzootic cycle of Borrelia burgdorferi.

Lyme disease, the leading vector-borne disease in the United States and Europe, develops after infection with Borrelia burgdorferi sensu lato bacteria. Transmission of the spirochete from the tick vector to a vertebrate host requires global changes in gene expression that are controlled, in part, by the Rrp2/RpoN/RpoS alternative sigma factor cascade. Transcriptional studies defining the B. burgdorferi RpoS regulon have suggested that RpoS activates the transcription of paralogous family 52 (PFam52) genes. In strain B31, PFam52 genes (bbi42, bbk53, and bbq03) encode a set of conserved hypothetical proteins with >89% amino acid identity that are predicted to be surface-localized. Extensive homology among members of paralogous families complicates studies of protein contributions to pathogenicity as the potential for functional redundancy will obfuscate findings. Using a sequential mutagenesis approach, we generated clones expressing a single PFam52 paralog, as well as a strain deficient in all three. The single paralog expressing strains were used to confirm BBI42, BBK53, and BBQ03 surface localization and RpoS regulation. Surprisingly, the PFam52-deficient strain was able to infect mice and complete the enzootic cycle similar to the wild-type parental strain. Indeed, the presence of numerous pseudogenes that contain frameshifts or internal stop codons among the PFam52 genes suggests that they may be subjected to gene loss in B. burgdorferi's reduced genome. Alternatively, the lack of phenotype might reflect the limitations of the experimental mouse infection model.

Suppression of host humoral immunity by Borrelia burgdorferi varies over the course of infection.

Borrelia burgdorferi, the spirochetal agent of Lyme disease, utilizes a variety of strategies to evade and suppress the host immune response, which enables it to chronically persist in the host. The resulting immune response is characterized by unusually strong IgM production and a lack of long-term protective immunity. Previous studies in mice have shown that infection with B. burgdorferi also broadly suppresses host antibody responses against unrelated antigens. Here, we show that mice infected with B. burgdorferi and concomitantly immunized with recombinant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein had an abrogated antibody response to the immunization. To further define how long this humoral immune suppression lasts, mice were immunized at 2, 4, and 6 weeks post-infection. Suppression of host antibody production against the SARS-CoV-2 spike protein peaked at 2 weeks post-infection but continued for all timepoints measured. Antibody responses against the SARS-CoV-2 spike protein were also assessed following antibiotic treatment to determine whether this immune suppression persists or resolves following clearance of B. burgdorferi. Host antibody production against the SARS-CoV-2 spike protein returned to baseline following antibiotic treatment; however, anti-SARS-CoV-2 IgM remained high, comparable to levels found in B. burgdorferi-infected but untreated mice. Thus, our data demonstrate restored IgG responses following antibiotic treatment but persistently elevated IgM levels, indicating lingering effects of B. burgdorferi infection on the immune system following treatment.

Electronic Health Record Data for Lyme Disease Surveillance, Massachusetts, USA, 2017-2018.

Lyme disease surveillance based on provider and laboratory reports underestimates incidence. We developed an algorithm for automating surveillance using electronic health record data. We identified potential Lyme disease markers in electronic health record data (laboratory tests, diagnosis codes, prescriptions) from January 2017-December 2018 in 2 large practice groups in Massachusetts, USA. We calculated their sensitivities and positive predictive values (PPV), alone and in combination, relative to medical record review. Sensitivities ranged from 57% (95% CI 47%-69%) for immunoassays to 87% (95% CI 70%-100%) for diagnosis codes. PPVs ranged from 53% (95% CI 43%-61%) for diagnosis codes to 58% (95% CI 50%-66%) for immunoassays. The combination of a diagnosis code and antibiotics within 14 days or a positive Western blot had a sensitivity of 100% (95% CI 86%-100%) and PPV of 82% (95% CI 75%-89%). This algorithm could make Lyme disease surveillance more efficient and consistent.

Age- and Sex-Specific Differences in Lyme Disease Health-Related Behaviors, Ontario, Canada, 2015-2022.

We investigated differences in risk factors and preventive behaviors by age and sex among persons with reported Lyme disease in Ontario, Canada, during 2015-2022. Incidence rates peaked among children 5-9 and adults 50-79 years of age. Median age was higher for female than male case-patients (54 vs. 51 years). Male case-patients reported more activity in wooded and tall grass areas than did female case-patients; fewer male case-patients reported sharing living space with outdoor-exposed companion animals. As age increased, more case-patients reported activity in blacklegged tick habitats, exposure to ticks, and wearing adequate clothing, but fewer reported sharing living space with outdoor-exposed companion animals. Adoption of preventive behaviors was relatively low and did not differ by sex. Male case-patients, children 5-9 years of age and their parents or caregivers, and adults >59 years of age represent populations that would benefit from tailored public health messaging on Lyme disease prevention.

Rapid Increase in Seroprevalence of Borrelia burgdorferi Antibodies among Dogs, Northwestern North Carolina, USA, 2017-20211.

We evaluated spatial-temporal risk for Lyme disease in northwestern North Carolina, USA, by using individual-level canine Borrelia burgdorferi seroprevalence data collected during 2017-2021 at routine veterinary screenings for tickborne diseases. Seroprevalence in dogs increased from 2.2% (47/2,130) in 2017 to 11.2% (339/3,033) in 2021. The percentage of incident seropositivity increased from 2.1% (45/2,130) in 2017 to 7.6% (231/3,033) in 2021. Exploratory geographic analyses found canine seroprevalence shifted from clustered (2017, Moran's I = 0.30) to dispersed (2021, Moran's I = -0.20). Elevation, slope, aspect, and forest land cover density were associated with canine seroprevalence within various household buffer regions in 2017. Slope was associated with seroprevalence at the household level in 2021. Results support the use of individual-level canine seroprevalence data for monitoring human risk for Lyme disease. Establishing sentinel veterinary clinics within Lyme disease-emergent communities might promote prevention and control efforts and provide opportunities for educational and behavioral interventions.

c-di-GMP regulates activity of the PlzA RNA chaperone from the Lyme disease spirochete.

PlzA is a c-di-GMP-binding protein crucial for adaptation of the Lyme disease spirochete Borrelia (Borreliella) burgdorferi during its enzootic life cycle. Unliganded apo-PlzA is important for vertebrate infection, while liganded holo-PlzA is important for survival in the tick; however, the biological function of PlzA has remained enigmatic. Here, we report that PlzA has RNA chaperone activity that is inhibited by c-di-GMP binding. Holo- and apo-PlzA bind RNA and accelerate RNA annealing, while only apo-PlzA can strand displace and unwind double-stranded RNA. Guided by the crystal structure of PlzA, we identified several key aromatic amino acids protruding from the N- and C-terminal domains that are required for RNA-binding and unwinding activity. Our findings illuminate c-di-GMP as a switch controlling the RNA chaperone activity of PlzA, and we propose that complex RNA-mediated modulatory mechanisms allow PlzA to regulate gene expression during both the vector and host phases of the B. burgdorferi life cycle.

Performance of two modified two-tier algorithms for the serologic diagnosis of Lyme disease.

We compared the performance of a new modified two-tier testing (MTTT) platform, the Diasorin Liaison chemiluminescent immunoassay (CLIA), to the Zeus enzyme-linked immunoassay (ELISA) MTTT and to Zeus ELISA/Viramed immunoblot standard two-tier testing (STTT) algorithm. Of 537 samples included in this study, 91 (16.9%) were positive or equivocal by one or more screening tests. Among these 91 samples, only 57 samples were concordant positive by first-tier screening tests, and only 19 of 57 were concordant by the three second-tier methods. For IgM results, positive percent agreement (PPA) was 68.1% for Diasorin versus 89.4% for Zeus compared to immunoblot. By contrast, the PPA for IgG for both Diasorin and Zeus was 100%. Using a 2-out-of-3 consensus reference standard, the PPAs for IgM were 75.6%, 97.8%, and 95.6% for Diasorin, Zeus, and immunoblot, respectively. The difference between Zeus MTTT and Diasorin MTTT for IgM detection was significant (P = 0.0094). PPA for both Diasorin and Zeus MTTT IgG assays was 100% but only 65.9% for immunoblot STTT (P = 0.0005). In total, second-tier positive IgM and/or IgG results were reported for 57 samples by Diasorin MTTT, 63 by Zeus MTTT, and 54 by Viramed STTT. While Diasorin CLIA MTTT had a much more rapid, automated, and efficient workflow, Diasorin MTTT was less sensitive for the detection of IgM than Zeus MTTT and STTT including in 5 early Lyme cases that were IgM negative but IgG positive. IMPORTANCE: The laboratory diagnosis of Lyme disease relies upon the detection of antibodies to Borrelia species. Standard two tier testing (STTT) methods rely upon immunoblots which have clinical and technical limitations. Modified two-tier testing (MTTT) methods have recently become available and are being widely adopted. There are limited independent data available assessing the performance of MTTT and STTT methods.