Detection of IFN-γ Secretion in Blood Samples Collected Before and After Treatment of Varying Stages of Lyme Disease.

BACKGROUND: QuantiFERON enzyme-linked immunosorbent assay (ELISA; Qiagen) with Borrelia burgdorferi peptide antigens was previously shown to reliably detect interferon-γ (IFN-γ) in blood samples from adult patients with early Lyme disease and the response disappeared rapidly after treatment. We evaluated the response before and after appropriate antibiotic therapy in adolescent and adult subjects with more diverse stages of the illness. METHODS: Blood was obtained from patients with clinician-identified Lyme disease with constitutional complaints, erythema migrans, nerve palsy, cardiac abnormality, or arthritis before (n = 68) and 6 weeks (n = 46) and 6 months (n = 45) after therapy. The sera were tested for Lyme disease by standard 2-tiered testing (STTT) and anti-C6 antibodies by ELISA and the levels of IFN-γ in the blood samples were detected by QuantiFERON ELISA. RESULTS: A positive STTT result supported the clinical diagnosis of 37 (54%) subjects and anti-C6 antibodies were detected in 45 (66%) subjects, including 36 (97%) STTT-positive subjects, and the responses often persisted or expanded after antibiotic therapy. IFN-γ was detected in 49 (72%) subjects prior to treatment and the response most often significantly decreased 6 weeks (P = .007) or 6 months (P = .001) after treatment. CONCLUSIONS: The QuantiFERON ELISA reliably detected IFN-γ in blood samples from adult and adolescent patients with varying stages of Lyme disease and the response disappeared rapidly after treatment. Additional studies to more critically evaluate clinical utility as a laboratory test for diagnosis and confirmation of effective therapy are warranted.

Detection of antibodies to decorin-binding protein A (DbpA) and DbpB after infection of dogs with Borrelia burgdorferi by tick challenge.

We characterized the antibody response to decorin-binding protein A (DbpA) or DbpB from immune serum samples collected from 27 dogs infected with Borrelia burgdorferi by Ixodes scapularis ticks. Immunoglobulin M (IgM) antibodies to DbpA or DbpB were rarely detected, but high levels of IgG antibodies to DbpA were detected in 16 of 27 of the immune sera collected 1 mo after infection, 20 of 25 of the sera collected after 2 mo, and each of the 23, 17, or 11 serum samples evaluated after 3, 4, or 5 mo, respectively. In addition, IgG antibodies to DbpB were detected in 22 of 27 (p = 0.005) tested dogs after 1 mo, and the frequency of detecting the antibodies thereafter closely mimicked the antibody responses to DbpA. Moreover, antibodies to DbpA or DbpB were not produced by dogs vaccinated with a whole-cell B. burgdorferi bacterin; removing the antibodies to DbpA by adsorption to recombinant DbpA (rDbpA) did not affect the reactivity detected by a rDbpB ELISA. Therefore, the findings from our preliminary study showed that antigenically distinct antibodies to DbpA or DbpB are produced reliably during canine infection with B. burgdorferi, and the response is not confounded by vaccination with a Lyme disease bacterin. Larger studies are warranted to more critically evaluate whether detecting the antibody responses can improve serodiagnostic confirmation of canine Lyme disease.

Evaluation of a Novel Multiplex High-Definition PCR Assay for Detection of Tick-Borne Pathogens in Whole-Blood Specimens.

The prevalence of tick-borne infections has been steadily increasing in both number and geographic distribution in the United States and abroad. This increase, in conjunction with the continued recognition of novel pathogens transmitted by ticks, has made accurate diagnosis of these infections challenging. Mainstay serologic tests are insensitive during the acute phase of infection and are often cross-reactive with similar pathogenic and nonpathogenic organisms. Further, they are unable to reliably differentiate active versus past infection which can lead to misdiagnosis and incorrect understanding of the epidemiology and incidence of specific tick-borne pathogens. We evaluated a novel multiplexed high-definition PCR (HDPCR) Tickborne Panel (TBP) assay (ChromaCode, Carlsbad, CA) for the detection of nine tick-borne pathogens or groups associated with human illness. The HDPCR technology enables multiplex identification of multiple targets in a single fluorometric channel based on fluorescent signal modulation using a limiting probe design. A collection of 530 whole-blood specimens collected from patients being evaluated for tick-borne infections, in addition to a panel of 93 simulated specimens, were used to challenge the HDPCR TBP. The results were compared to a clinically validated traditional multiplexed PCR test with additional sequence analysis and clinical history collected to aid in resolving discrepancies. Among clinical specimens the TBP demonstrated 100% sensitivity for the identification of Anaplasma phagocytophilum, Borrelia miyamotoi, Borrelia mayonii, and Rickettsia rickettsii The sensitivity for identification of B. burgdorferi was 44.4% compared to a composite gold standard. Among simulated specimens containing single or multiple targets present at 103 to 105 copies/PCR, the sensitivity of TBP was 100% for all targets, with a combined specificity of 99.5%. Of note, an increased rate of false-positive results was observed among simulated specimens that contained multiple targets. Based on these data, we find the HDPCR TBP to be a useful adjunct for the diagnosis of tick-borne infections in patients with suspected tick-borne illness.

Borrelia miyamotoi Infection in Patients from Upper Midwestern United States, 2014-2015.

We confirmed Borrelia miyamotoi infection in 7 patients who had contracted an illness while near La Crosse, Wisconsin, USA, an area where Ixodes scapularis ticks are endemic. B. miyamatoi infection should now be considered among differential diagnoses for patients from the midwestern United States who have signs and symptoms suggestive of tickborne illness.

Detection of IFN-γ Secretion by T Cells Collected Before and After Successful Treatment of Early Lyme Disease.

BACKGROUND: Current serodiagnostics for Lyme disease lack sensitivity during early disease, and cannot determine treatment response. We evaluated an assay based on QuantiFERON technology utilizing peptide antigens derived from Borrelia burgdorferi to stimulate interferon-gamma (IFN-γ) release as an alternative to serodiagnosis for the laboratory detection of Lyme disease. METHODS: Blood was obtained from patients with erythema migrans before (n = 29) and 2 months after (n = 27) antibiotic therapy. IFN-γ release was measured by enzyme-linked immunosorbent assay (ELISA) following overnight stimulation of whole blood with the peptide antigens, and compared to the results of standard serological assays (C6, ELISA, and Western blot). RESULTS: IFN-γ release was observed in pretreatment blood of 20 of 29 (69%) patients with Lyme disease. Following antibiotic treatment, IFN-γ was significantly reduced (P = .0002), and was detectable in only 4 of 20 (20%) initially positive patients. By contrast, anti-C6 antibodies were detected in pretreatment sera from 17 of 29 (59%) subjects, whereas only 5 of 29 (17%) patients had positive Western blot seroreactivity. Antibody responses persisted and expanded following treatment. CONCLUSIONS: Our findings suggest that measurement of IFN-γ after incubating blood with Borrelia antigens could be useful in the laboratory diagnosis of early Lyme disease. Also, after antibiotic treatment, this response appears to be short lived.

The Emergence of Clinically Relevant Babesiosis in Southwestern Wisconsin.

OBJECTIVE: To determine the frequency and characteristics of babesiosis cases, and to assess the impact of the introduction of a tick-borne infection diagnostic panel on babesiosis diagnosis in the region surrounding La Crosse, Wisconsin, where babesiosis in non-travelers was previously rare. METHODS: In the spring of 2013, we conducted a point-in-time survey of Ixodes scopuloris ticks for the presence of Babesia microti. We also conducted a retrospective study of all babesiosis cases diagnosed in our health system between January 1, 2004, and November 1, 2013. Finally, we compared the number of babesiosis cases diagnosed during the study period before and after the June 1, 2012, introduction of a tick-borne infection diagnostic panel in our organization. RESULTS: Babesia microti was present in 5% of ticks surveyed in our region. Twenty-two cases. of babesiosis were diagnosed in our organization during the study period-19 since 2010. The tick-borne infection diagnostic panel was used widely by clinicians, with an attendant increase in babesiosis diagnoses. CONCLUSION: Babesiosis should be considered endemic in southwestern Wisconsin, and testing should be considered for patients with compatible clinical and laboratory features.

Antibody responses to Borrelia burgdorferi outer surface proteins C and F in experimentally infected Beagle dogs.

Antibody levels to outer surface proteins C and F (OspC and OspF, respectively) in sera collected from laboratory Beagle dogs at 1, 2, and 4 months after challenge with infected black-legged ticks (Ixodes scapularis) were determined. Each dog was confirmed by culture to harbor Borrelia burgdorferi in the skin (n = 10) or the skin and joints (n = 14). Significant levels of immunoglobulin M (Ig)M or IgG anti-OspC antibodies were detected in single serum samples from only 3 (13%) dogs. Similarly, IgM anti-OspF antibodies were detected in only 1 (4%) serum sample collected from a dog with B. burgdorferi in the skin and joints. In contrast, 4 (29%) dogs with skin and joint infections produced IgG anti-OspF antibodies after 2 months, and the response expanded to include 2 (20%) dogs with skin infection and 4 additional dogs with skin and joint infections (overall sensitivity = 62%) after 4 months. The findings failed to support the utility of OspC-based antibody tests for diagnosing canine Lyme disease, but demonstrated that dogs with B. burgdorferi colonizing joint tissue most often produced significant levels of IgG anti-OspF antibodies. Therefore, additional studies to more thoroughly evaluate the clinical utility of OspF-based antibody tests are warranted.

Vaccination with the ospA- and ospB-Negative Borrelia burgdorferi Strain 50772 Provides Significant Protection against Canine Lyme Disease.

Beagles received placebo or ospA- and ospB-negative Borrelia burgdorferi before a tick challenge. A total of 28 (41%) ticks and skin biopsy specimens from each control dog (n = 10) contained B. burgdorferi. In contrast, 12 (19%) ticks recovered from the vaccine recipients (n = 10) were infected (P = 0.0077), and 5 dogs yielded spirochetes from the skin biopsy specimens (P = 0.0325). In addition, 9 (90%) placebo recipients and 4 (40%) vaccine recipients developed joint abnormalities (P = 0.0573). Therefore, vaccination with the ospA- and ospB-negative spirochete provided significant protection against Lyme disease.

ECOG phase II trial of graded-dose peginterferon α-2b in patients with metastatic melanoma overexpressing basic fibroblast growth factor (E2602).

PURPOSE: We investigated the use of graded-dose peginterferon α-2b (Peg-IFN) in patients with stage IV melanoma overexpressing basic fibroblast growth factor (FGF-2). The primary objective was suppression of plasma FGF-2 to within reference range (≤ 7.5 pg/mL). EXPERIMENTAL DESIGN: Plasma FGF-2 was measured at baseline (step 1), and patients with concentrations of 15 pg/mL or more were eligible for study treatment (step 2). Peg-IFN was given weekly at a starting dose of 0.5 µg/kg/wk with increment every 3 weeks based on serial FGF-2 concentrations. RESULTS: Two hundred seven patients entered step 1; 45 (22%) overexpressed FGF-2 (median = 22 pg/dL). Twenty-nine eligible patients entered step 2 and received treatment. Patients' median age was 64 years (range, 29-84 years). Most had more than two prior therapies. FGF-2 decreased in 28 (97%) patients, with suppression to reference range in 10 (35%). Median time to FGF-2 suppression was 30 days. The best clinical responses were partial response (7%) and stable disease (17%). Median progression-free survival (PFS) and overall survival (OS) were 2.0 and 9.7 months, respectively. Patients who achieved FGF-2 suppression were more likely than those who did not to have a response or stable disease (P = 0.03). VEGF concentrations decreased in 27 patients (93%) during treatment and paralleled those of FGF-2 over time. We found no compensatory increase in VEGF among those with FGF-2 suppression. CONCLUSIONS: Graded-dose Peg-IFN suppresses FGF-2 in patients with metastatic melanoma who overexpress FGF-2. Over one third of patients had complete suppression of plasma FGF-2, which correlated with clinical response to this therapy.

Expansion of the Midwestern focus for human granulocytic anaplasmosis into the region surrounding La Crosse, Wisconsin.

Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis (HGA), shares the same enzootic life cycle as Borrelia burgdorferi, the causative agent of Lyme disease. Although La Crosse, WI, is a well-recognized Lyme disease focus with an abundance of Ixodes scapularis vector ticks and the first documentation of HGA occurred in patients from northwestern Wisconsin, local transmission of A. phagocytophilum has not to date been documented. In this study, we evaluated DNA extracted from 201 ticks captured locally by a real-time PCR that targeted a unique region within msp2, and 24 samples (12%) yielded positive results. The PCR also detected A. phagocytophilum DNA in blood samples obtained from 53 patients with clinical abnormalities consistent with HGA, and sequencing confirmed that the DNA was recovered from the Ap-ha variant of A. phagocytophilum, associated exclusively with human infection. The findings therefore confirmed that the upper Midwestern focus for HGA endemicity now includes the regions immediately surrounding La Crosse, WI. The results also validated the utility of the real-time msp2 PCR test for confirming acute HGA in the clinical setting.

Rapid decline of OspC borreliacidal antibodies following treatment of patients with early Lyme disease.

We determined whether the levels of OspC borreliacidal antibodies declined following treatment of early Lyme disease and whether the OspC7 peptide enzyme-linked immunosorbent assay (ELISA) could be used as an alternative test for detecting the response. Serum samples were collected from 37 subjects at the onset of illness and 2 and 6 months after treatment with doxycycline. The ELISA detected IgM and IgG OspC7 antibodies within 2 months in 18 (49%) and 5 (14%) sera, respectively. Moreover, the sera from 12 subjects who tested positive by the ELISA also showed borreliacidal activity which was completely abrogated when the antibodies to OspC7 were removed. The borreliacidal activity decreased greater than 4-fold in each seropositive patient within 6 months after treatment, and the findings were accurately predicted by the IgM ELISA. The results confirmed that the ELISA was an effective alternative for detection of OspC borreliacidal antibodies produced during early Lyme disease in humans and also provided strong evidence that a significant decline in the response coincides with successful treatment of the illness.

One-year duration of immunity induced by vaccination with a canine Lyme disease bacterin.

Laboratory-reared beagles were vaccinated with a placebo or a bacterin comprised of Borrelia burgdorferi S-1-10 and ospA-negative/ospB-negative B. burgdorferi 50772 and challenged after 1 year with B. burgdorferi-infected Ixodes scapularis ticks. For the placebo recipients, spirochetes were recovered from 9 (60%) skin biopsy specimens collected after 1 month, and the organisms persisted in the skin thereafter. Ten (67%) dogs also developed joint infection (3 dogs), lameness or synovitis (7 dogs), or B. burgdorferi-specific antibodies (8 dogs). For the vaccine recipients, spirochetes were recovered from 6 (40%) skin biopsy specimens collected after 1 month. However, subsequent biopsy specimens were negative, and the dogs failed to develop joint infection (P = 0.224), lameness/synovitis (P = 0.006), or Lyme disease-specific antibody responses (P = 0.002). The bacterin provided a high level of protection for 1 year after immunization, and the addition of the OspC-producing B. burgdorferi 50772 provided enhanced protection.

Bacterin that induces anti-OspA and anti-OspC borreliacidal antibodies provides a high level of protection against canine Lyme disease.

Groups of 15 laboratory-bred beagles were vaccinated and boosted with either a placebo or adjuvanted bivalent bacterin comprised of a traditional Borrelia burgdorferi strain and a unique ospA- and ospB-negative B. burgdorferi strain that expressed high levels of OspC and then challenged with B. burgdorferi-infected Ixodes scapularis ticks. The vaccinated dogs produced high titers of anti-OspA and anti-OspC borreliacidal antibodies, including borreliacidal antibodies specific for an epitope within the last seven amino acids at the OspC carboxy terminus (termed OspC7) that was conserved among pathogenic Borrelia genospecies. In addition, spirochetes were eliminated from the infected ticks that fed on the bacterin recipients, B. burgdorferi was not isolated from the skin or joints, and antibody responses associated specifically with canine infection with B. burgdorferi were not produced. In contrast, B. burgdorferi was recovered from engorged ticks that fed on 13 (87%) placebo-vaccinated dogs (P<0.0001), skin biopsy specimens from 14 (93%) dogs (P<0.0001), and joint tissue specimens from 8 (53%) dogs (P=0.0022). In addition, 14 (93%) dogs developed specific antibody responses against B. burgdorferi proteins, including 11 (73%) with C6 peptide antibodies (P<0.0001). Moreover, 10 (67%) dogs developed Lyme disease-associated joint abnormalities (P<0.0001), including 4 (27%) dogs that developed joint stiffness or lameness and 6 (40%) that developed chronic joint inflammation (synovitis). The results therefore confirmed that the bacterin provided a high level of protection against Lyme disease shortly after immunization.

Significantly improved accuracy of diagnosis of early Lyme disease by peptide enzyme-linked immunosorbent assay based on the borreliacidal antibody epitope of Borrelia burgdorferi OspC.

Highly specific borreliacidal antibodies are induced by infection with Borrelia burgdorferi, and the immunodominant response during early Lyme disease is specific for an epitope within the 7 amino acids nearest the C terminus of OspC. We evaluated the ability of an enzyme-linked immunosorbent assay (ELISA) based on a synthetic peptide (OspC7) that matched the region to detect the response and compared the sensitivity during early Lyme disease to that for an FDA-approved Western blot. When the optical density value was adjusted to 98% specificity based on the results from testing normal or uncharacterized sera (n = 236) or sera from patients with blood factors or illnesses that commonly produce antibodies that cross-react with B. burgdorferi antigens (n = 77), 115 (73%) of 157 sera from patients likely to have early Lyme disease were positive for immunoglobulin M (IgM) antibodies and 17 (11%) also had IgG antibodies. In addition, the IgM ELISA reactivities and the titers of antibodies detected by a flow cytometric borreliacidal antibody test correlated closely (r = 0.646). Moreover, the IgM ELISA was significantly more sensitive (P < 0.001) than the Western blot procedure. The findings therefore confirmed that the peptide IgM ELISA detected OspC borreliacidal antibodies and provided strong evidence that the test can eliminate the necessity for confirming early Lyme disease by a supplementary test such as Western blotting.

Circulating endothelial cells in patients with chronic lymphocytic leukemia.

Angiogenesis is increased in B-cell chronic lymphocytic leukemia (B-CLL). We wanted to quantify and characterize the circulating endothelial cells (CECs) in patients with B-CLL and correlate with plasma angiogenesis-related factors. Using a four-color flow cytometry, we prospectively analyzed the CEC in the whole blood of 20 healthy controls and 20 patients with B-CLL. We quantified (CD45-/CD31+/CD146+) and characterized the CECs according to whether they were apoptotic (annexin stain) or activated (CD106+). We also measured plasma levels of vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), and thrombospondin-1 (TSP-1). Most patients (90%) had Rai stages 0-2 at the time of diagnosis. As a group, B-CLL patients had higher number of CECs (median of 26.5 cells/ml) compared (P = 0.04) to healthy controls (18.5 cells/ml). However, only four (20%) patients had elevated CEC counts, defined as >/=2 SD of the control mean (>/=53 cells/ml). The proportions of apoptotic (P = 0.83) and activated (P = 0.12) CECs were similar in both groups. B-CLL patients had higher FGF-2 (P < 0.001), lower TSP-1 (P = 0.004), and similar VEGF (P = 0.27) plasma levels. The number of CECs was not associated with Rai stage, absolute lymphocyte count, or levels of angiogenesis-related factors. CECs are increased in only a small fraction of B-CLL patients in our cohort with low rates of apoptosis and activation. While no correlation was found between CECs and clinical features, more studies in a larger patient sample size and advanced disease are necessary.

Borreliacidal OspC antibody response of canines with Lyme disease differs significantly from that of humans with Lyme disease.

Humans reliably produce high concentrations of borreliacidal OspC antibodies specific for the seven C-terminal amino acids shortly after infection with Borrelia burgdorferi. We show that dogs also produce OspC borreliacidal antibodies but that their frequencies, intensities, and antigenicities differ significantly. The findings therefore confirm a major difference between the borreliacidal antibody responses of humans and canines with Lyme disease.

Borreliacidal OspC antibodies specific for a highly conserved epitope are immunodominant in human lyme disease and do not occur in mice or hamsters.

Humans produce highly specific borreliacidal antibodies against outer surface protein C (OspC) shortly after infection with Borrelia burgdorferi sensu stricto. We previously demonstrated the epitope recognized by immunoglobulin M (IgM) and IgG OspC borreliacidal antibodies was located within the 50 amino acids nearest the carboxy (C) terminus. In this study, we show the immunodominant epitope is located in the highly conserved region within the seven C-terminal amino acids. Six early Lyme disease sera that contained borreliacidal activity and IgM and/or IgG OspC antibodies were chosen randomly and adsorbed with truncated OspC containing the 16 or 7 amino acids nearest the C terminus. Adsorptions with each truncated protein abrogated the borreliacidal activity completely. In addition, only small concentrations of OspC antibodies remained detectable by enzyme-linked immunosorbent assay and Western blotting. Moreover, borreliacidal OspC antibodies were not induced in laboratory mice or hamsters despite heavy infections with B. burgdorferi spirochetes. These findings confirm that borreliacidal antibodies comprise the majority of the IgM and IgG OspC antibody response in human Lyme disease and that the epitope is located in the highly conserved C terminus. In addition, rodent animal models appear to be inappropriate subjects for assessing the effectiveness of the epitope for serodiagnosis or as a human Lyme disease vaccine.

Detection of borreliacidal antibodies by flow cytometry.

Lyme disease is a multisystem disorder that usually begins with a skin lesion called erythema migrans and with constitutional symptoms. If the disease is left untreated or treated inappropriately, dissemination of the organism can lead to more severe sequelae, including nervous system disorders or arthritis. Vaccinations with B. burgdorferi or several individual B. burgdorferi proteins induce borreliacidal antibodies that provide protection against infection by inducing a complement cascade that kills the spirochetes without the necessity of scavenging by phagocytic cells. Detection of borreliacidal antibodies is therefore useful for serodiagnosing Lyme disease and monitoring immune status after vaccination. This unit provides a technique for detecting anti-B. burgdorferi antibodies, as well as for preparing and determining the quality of Barbour-Stoenner-Kelly (BSK medium) and complement. In addition, methods are provided for preparation of a B. burgdorferi stock and Mueller-Hinton agar containing Bacillus subtilis spores.