B029 (D021)

# Relevance of quantitative determination of IGG antibodies against VIsE as an activity marker in the monitoring of treated Lyme borreliosis: a retrospective study

Holger Blenk¹, Marion Straubinger¹, Diana Schuster¹, Müzeyyen Ünsal-Kirici¹, Katja Steinhagen², Stefanie Schütt² and Lars Komorowski²

1. Laboratory and Centre for Infectiology, EuromedClinic*, Fuerth, Germany

2. EUROIMMUN AG, Luebeck, Germany

**Introduction:** The first step in the serological diagnosis of Lyme borreliosis comprises a sensitive screening test containing a selected spectrum of native and/or recombinant antigens, including the highly specific recombinant VIsE. From the literature, it is well known that high levels of anti-VIsE antibodies in particular can be detected in the acute phase of a Lyme infection. The goal of our retrospective study was to systematically and quantitatively monitor anti-VIsE IgG antibodies before and after treatment using a specially adapted test system.

**Methods:** We used primarily a classic indirect ELISA from EUROIMMUN containing recombinant VIsE antigen from *Borrelia burgdorferi* sensu stricto for the quantitative *in vitro* determination of human IgG antibodies against the VIsE antigen. A special version of the test which is optimized for titer monitoring was also used. The titer level is measured in relative units (RU/ml). The detection limit of the test is 1 RU/ml and the cut-off is 16 RU/ml. Archived (–80°C) sera from patients with confirmed or suspected Lyme disease from our Lyme outpatient clinic were used as sample material.

**Results:** A significant drop in the titer of anti-VIsE antibodies could be detected as early as 6–8 weeks after successful treatment in all active chronic Lyme infections, including Lyme arthritis, acrodermatitis chronica atrophicans, acute neuroborreliosis, and other active chronic stages of Lyme infection. The decrease in anti-VIsE correlated with a reduction in clinical symptoms. The absence of an anti-VIsE titer virtually excludes a florid chronic Lyme infection (control panel of healthy individuals, *n* = 105). Strongly positive anti-VIsE values (>1000 RU/ml) in untreated patients are to 99% an indication of an active chronic Lyme infection. Some patients with erythema migrans (EM), in contrast, showed only low anti-VIsE values, which increased slowly and/or became negative again a short while after treatment.

**Conclusion:** Quantitative determination of anti-VIsE IgG is suitable – always under consideration of clinical symptoms – for confirmation of diagnosis and as an activity marker for monitoring patients with active chronic Lyme borreliosis before and after treatment.
B030 (D023)

CDC Lyme disease serum repository; collection, analysis, and availability

Martin E. Schriefer, Christopher Sexton and Claudia R. Molins

Division of Vector-Borne Disease, Centers for Disease Control and Prevention, Fort Collins, CO, USA

In the United States, nationally recommended laboratory confirmation for Lyme borreliosis is based largely on
assessment of antibody responses to infection with *Borrelia burgdorferi* sensu stricto. Development and evaluation of
diagnostic tests for Lyme disease is dependent upon analysis of samples from well-documented patients representing
a full spectrum of disease presentations and negative controls composed of likely-to-been countered healthy subjects
and patients with alternate disease etiologies. Comparative test performance also depends on analysis of these types of
samples and can be greatly strengthened by parallel sample set testing. In practice, however, access to such samples is
often restricted to unique clinical settings, where these types of patients and controls are seen; well-documented and
widely available patient sample sets are generally not available. In 2008, NIH and CDC committed to establishing
such an archive for Lyme disease and between 2009 and 2013, a serum repository, collected through funded contracts,
was built. Samples from over 400 US Lyme disease patients, healthy endemic and non-endemic subjects, and persons
with other diseases that may overlap with those of Lyme disease in clinical presentation or antibody reactivity were
collected. Non-Lyme disease patients included those with multiples sclerosis, fibromyalgia, infectious mononucleosis,
severe periodontitis, syphilis, and rheumatoid arthritis. Patient documentation and inclusion targeted nationally
standardized case definitions and laboratory support that avoided serological test outcome for Lyme disease wherever
possible. Alternate proofs for Lyme disease included culture, PCR, clinical history, and epidemiological risk. Upon
receipt by CDC, all samples were evaluated by a variety of Lyme disease serological tests including standardized
two-tier testing. Performance in these tests is presented and highlights the strengths and weaknesses of currently
available tests and test strategies as well as the utility and limitations of the described serum archive. Sera from
well-documented Lyme disease patients and controls along with test results and clinical documentation are now
available. Samples are provided in limited number and volume sets, coded or un-coded, for the purpose of Lyme
disease serological test validation and research toward development of improved Lyme disease diagnostics and FDA
clearance.
B031 (D024)

High incidence of atypical erythema migrans in PCR proven Lyme disease

Steven E. Schutzer¹, Bernard W. Berger², James G. Krueger³, George P. Dempsey⁴,
John N. Aucott⁵, Christopher D. Crowder⁶ and Mark W. Eshoo⁶

1. UMDNJ-New Jersey Medical School, Newark, NJ, USA

2. Department of Dermatology, Southampton, NY, USA

3. Laboratory for Investigative Dermatology, Rockefeller University, New York, NY, USA

4. Urgent Care, East Hampton, NY, USA

5. Johns Hopkins University, Baltimore, MD, USA

6. Ibis Biosciences, Carlsbad, CA, USA

The ability to unequivocally diagnosis Lyme disease early is advantageous to the patient and the research field as it
can provide a validated group for basic and clinical studies. The best diagnostic sign in early Lyme disease when it
occurs, is a skin lesion, erythema migrans (EM). However, it may not occur or be recognized in 30% of cases. Among
those that do occur, it has been reported that approximately 20-30% of EM rash may not display a classic bull's-eye,
ring-within-a ring, appearance, fact that may be underappreciated. Atypical variants of EM have included a blue-red
appearance and occasionally a vesicular central region 4.5. Other cases with annular lesions may have a differential of
fixed drug eruptions, granuloma annulare, cellulitis, dermatophytosis, or systemic lupus erythematosus. We began to
assess the incidence of atypical EM in cases in whom we could provide definitive microbiological proof of a *Borrelia
burgdorferi* infection. We used an enhanced multilocus PCR assay for blood. In a set of 21 patients, we identified 10
PCR-positive cases with non-classic EM lesions and 4 PCR-positive subjects had classic ring-within-a ring target
lesions. Thus among PCR+ cases (also met CDC criteria), 10/14 (71%) had a non-classic EM. All lesions were
>5 cm and were categorized by an experienced panel including dermatologists who published in the field. Atypical
presentations ranged from those close to classic EM to those resembling lesions more common in other conditions
such as insect or spider bites and consequently prone to misdiagnosis. Separately in another 12 subjects from a
different endemic area, we analyzed tick, blood, and skin for genotypes of *B. burgdorferi* that have been reported
to disseminate and cause systemic disease. In no case, where blood was PCR, did we found a disseminating-OspC
genotype. In summary, the occurrence of atypical EM is high enough that clinicians should consider Lyme disease in
patients with endemic area exposure and a rash that is not the classic EM. (For presentation, we will show Photos and
Figures.)
B032 (D029)

# Development of an algorithm for rational *Borrelia* serology in a high endemic European area

D. Nyman, S.-A. Carlsson, M. Nordberg and C. Jansson

The Åland Borreliosis Research Group, Bimelix Laboratory, Åland, Finland

In an endemic high-risk area as the Åland Islands, with annual incidence of Lyme borreliosis of >1000/10⁵, a rational use of diagnostic serology is most important. In this setting, with a high background seropositivity rate of 20–60%, serology is applied to rule out the presence of disseminated Lyme borreliosis, rather than for ruling-in the disease. High sensitivity assays are therefore necessary without reduction of specificity. A two-step serology is most important for preserving the specificity, but also costly. We have previously demonstrated that a combination of two ELISA assays may be useful for reduction of immunoblotting. We have studied 15,490 blood samples from a clinical material using C6 Lyme ELISA Kit (Immunetics, USA) and recomWell IgG (Mikrogen, Germany). Of these, we found positive recomWell in 54.3%, equivocal in 4.1% and negative in 41.6% while C6 antibodies was positive in 40.5%, equivocal in 2.9%, and negative in 57.8%. The difference in the positive fractions was 15% (95% CI 13.5–16.5) and highly significant *p* < 0.0001. In an aliquot of 3380 samples, we confirmed the ELISA results by IgG immunoblot, recomLine (Mikrogen, Germany). The confirmation of positive results in the C6-antibody test by standard immunoblotting against a panel of recombinant *Borrelia* antigens is not recommendable. The ELISA tests used did not perform identically in detecting *Borrelia*-specific antibodies and we find definite cases of disseminated LB with either test negative in a clinical material. An algorithm for rational, cost-saving serology for Lyme borreliosis will be presented. Evaluation of this algorithm during 1 year has shown a reduction of ELISA tests by 27% and immunoblots by 20%, which transforms into 26.8% cost reduction.
B033 (D030)

Evaluation of Mikrogen recombead *Borrelia*-specific IGG and IGM antibodies

Ram B. Dessau, J. K. Møller and B. Kolmos

Department of Clinical Microbiology, Slagelse Hospital, Region Sjælland and Vejle Hospital,
Region Syddanmark, Denmark

Introduction: This new technology where many biomarkers may be assessed simultaneously is promising compared to traditional immunoblots. The laboratory procedures are easier, more or less like an ELISA. Reading of results is quantitative and not based on manual and visual interpretation of bands of varying intensity on a narrow strip. This study presents: (1) result of a new quantitative multiplex assay (2) discussion of methods for scoring multivariate diagnostic data.

Materials: Serum samples from 49 patients with neuroborreliosis and 217 blood donors. The patients with neuroborreliosis were laboratory defined by pleocytosis and a positive antibody index.

Methods: Mikrogen recomBead *Borrelia* IgG and IgM on the Luminex platform, a technology that combines flow cytometry, microspheres, and laser technology. The same 13 different recombinant antigens were included in both the IgG and IgM assays.

Results: Qualitative results using the evaluation software supplied by the manufacturer 24 of 49 patients with Neuroborreliosis were found positive or borderline (Table 1) in IgM or IgG and thus the sensitivity was 48% (36–63). The sensitivity of IgG or IgM alone was 33 and 31%, respectively. The specificity measured in 217 blood donors was 97% (94–99) for IgG and 99.5% (97.4–100.0) for IgM. Quantitative analysis by creating a logistic regression model sensitivity of the assay was improved to 91% when choosing the specificity at 98%. Only IgG VIsE and IgM OspC had significant diagnostic importance. Most of the other included antigens had little if any reactivity in this group of patients with neuroborreliosis were slightly more reactive in the blood donor control group and were subtracted instead of added in the regression model. Technical performance was fairly easily implemented in the laboratory without any failed runs.

Discussion: The excellent results of the regression modeling needs to be confirmed in patients with other disease manifestations of Lyme borreliosis, especially patients with late disease like acrodermatitis or arthritis. Patients with late disease should be expected to have antibody reactivity to many of the antigens, which were “negative” in this group representative of patients with early disseminated Lyme disease. Also, geographical variations in patterns of seroreactivity to different antigens could be important for interpretative scoring of results. The results should be confirmed in an independent group of samples.

Conclusion: The scoring system provided by the manufacturer gave an inadequate sensitivity of 48%. However, combining the quantitative results in a logistic regression model and only the IgG VIsE and the IgM OspC antigens had important discriminatory power. The optimized sensitivity was 91% at a specificity of 98%.
B034 (D031)

# Determination of the CSF/serum antibody index for the diagnosis of Lyme neuroborreliosis: methodological considerations

R. B. Dessau

Department of Clinical Microbiology, Slagelse Hospital, Region Sjaelland, Denmark

In the European case definitions for Lyme borreliosis (Stanek et al., 2011), it is stated that the analysis of paired serum and cerebrospinal fluid (CSF) samples obtained simultaneously is key to determining the specific CSF/serum antibody index (AI). A positive AI together with typical signs of inflammation in the CSF confirms a clinical diagnosis of Lyme neuroborreliosis. The purpose of the study was to assess the analytical performance and the strategy for choice of cutoff.

**Methods:** Results of 1510 index determinations, consecutive routine, from October 2001 to September 2010 were extracted from the laboratory information system. All determinations were performed by the Oxoid IDEIA Neuroborreliosis kit based on native flagella antigen. The main tool for assessing performance and choice of cutoff was the XY plot of the paired OD readings.

**Results:** About 1388 were negative and 122 (8.1%) were determined as index positive in IgG or IgM. The use of paired serum and spinal fluid samples has two advantages. (1) Increase of sensitivity, as the analytical cutoff may be lowered. In this assay, an OD of 0.150 seems well chosen to exclude the negative majority population. (2) Increase of specificity. Due to the blood-brain barrier, only patients with intrathecal antibody production may have a relatively increased reactivity compared to serum (the index principle). In this case, the calibrated index value of 0.3 also seems to be well chosen. These properties are shown in the XY plots together with the chosen cutoffs. Both a lower cutoff and an index cutoff are needed. For IgM, a cutoff for highly positive samples is also specified.

**Discussion and Conclusion:** This is an analytical exercise without clinical data, but nevertheless an important step in test evaluation before performing any clinical evaluation. The results show the practical and theoretical advantages of index determination. When the relative reactivity in serum and spinal fluid is considered for each patient, the variation between patients is filtered out. Thus, the serum for each patient is used as his own control. This allows for a low analytical cutoff to increase sensitivity and at the same time maintain a high specificity as patients with serum antibodies only are not reported positive. These results confirm the importance of using simultaneous determination of serum and spinal fluid when diagnosing Lyme neuroborreliosis.
B035 (D032)

# Development of an IgG Western blot avidity assay to identify antigenic markers for the different stages of *Borrelia burgdorferi* infection in Scottish patients

Sally Mavin<fnref n="1" />,<fnref n="2" />, Roger Evans<fnref n="1" />,<fnref n="2" /> and Alan S. Bowman<fnref n="1" />,<fnref n="2" />

Immunoblot assays have been used to measure IgG antibody avidity to aid the timing of infection for a range of organisms, but not *Borrelia burgdorferi*. Serology is used to support a clinical diagnosis of Lyme borreliosis, but it is unnecessary in the use of antibiotics and may delay diagnosis of the actual cause of a patient's symptoms. The aim of this project was to develop an IgG Western blot avidity assay to identify potential markers for the different stages of Lyme borreliosis. An in-house IgG Western blot assay utilizing *B. burgdorferi* sensu stricto and *Borrelia afzelii* lysates was adapted by substituting the first wash step with 8 M urea/PBST washes. Densitometry was used to measure differences in band intensity between Western blot and avidity Western blot strips for each sample to assess antibody avidity to individual antigens. A decrease in band intensity of <75% (avidity index <25%) in the avidity strip was considered as a high-avidity antibody response. The optimized avidity assay was tested against 50 sera, including patients with confirmed early (n = 15) and late (n = 15) disease. Analysis of results identified potential markers of early (43 kDa antigen) and late Lyme borreliosis (46, 39, 32, 30, and 22 kDa antigens). These antigens were later characterized by 2D electrophoresis and mass spectrometry. Criteria to interpret avidity results based on the number of antigens (including the proposed early and late markers) detected with high avidity were devised to differentiate early, late, and past infection with *B. burgdorferi*. When prospective sera from 55 patients with clinically suspected Lyme borreliosis were tested with the Western blot avidity assay, the avidity results improved the overall result interpretation. The avidity interpretation in 28 patients was consistent with the clinical information provided and the Western blot results. A more definitive serological diagnosis of past infection was indicated in five patients. Twenty patients had insufficient clinical information to determine the stage of infection but the avidity assay indicated 8 early and 12 late Lyme borreliosis. The avidity results may have altered the management of five of the seven patients as they did not agree with the original result interpretation. The Western blot avidity assay has the potential to differentiate early, late, and past Lyme borreliosis, improving the management of Scottish patients. Detailed clinical follow-up and the investigation of the avidity time course of marker antigens will help confirm these initial findings.

<footnote n="1">1. *National Lyme Borreliosis Testing Laboratory, Raigmore Hospital, Inverness, UK*</footnote>

<footnote n="2">2. *University of Aberdeen, Aberdeen, UK*</footnote>
B036 (D033)

Evaluation of SpiroFind, a novel cellular-based assay for the diagnosis of active
Lyme disease

M. Oosting, H. Ter Hofstede, B. Kullberg, M. G. Netea and L. A. B. Joosten

Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands

Objective: The presence of IgG/IgM antibodies directed against *Borrelia* antigens is used as standard diagnosis for Lyme disease, but diagnosis based solely on these serological assays is often difficult. In the present study, the potential value of a cellular-based assay (SpiroFind) for the diagnosis of active Lyme disease was evaluated. The SpiroFind test is based on the capacity of peripheral blood mononuclear cells (PBMCs) from patients to build up a cellular-based immunological memory and produce enhanced cytokine levels upon re-stimulation with intact *Borrelia* species.

Methods: Patients suspected of Lyme disease were seen at the outpatient clinic for Lyme disease from the Department of Medicine, Radboud University Nijmegen Medical Centre. The diagnosis of Lyme disease was evaluated using standard *Borrelia* serology (Enzyme-linked immunosorbent assay or Western blot) or SpiroFind assay.

Results: The overall prevalence of *Borrelia* exposure in the tested individuals was similar in SpiroFind compared to both serological tests. The specificity of the SpiroFind was found to be comparable with the current serological tests. The similarity between serology and SpiroFind was 69%. However, the percentages of false-positive or false-negative outcomes using the SpiroFind were markedly lower as compared to serology.

Conclusion: SpiroFind revealed similar specificity for diagnosis of Lyme disease when compared to serology. Compared to serological tests, false-positive or false-negative results were found less often when using SpiroFind. Therefore, SpiroFind can be considered as alternative test next to the standard *Borrelia* IgG and IgM serology assays.
B037 (D035)

# Development of a novel nucleic acid-based diagnostic assay for Lyme disease

Kamfai Chan, Salvatore A. E. Marras, Steven Schutzer and Nikhat Parveen

NJ Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ, USA

The infection with *Borrelia burgdorferi* can result in acute to chronic Lyme disease. The serological tests currently employed for its diagnosis are not very sensitive and are often difficult to interpret in a uniform manner by different laboratories. In addition, antibodies to *B. burgdorferi* persist long after the spirochete clearance, making it difficult to ascertain the cure of the disease after completion of the treatment regime. In addition, variation in the adaptive immunological response to different strains may affect the sensitivity. Finally, the test cannot be used for early disease before the adaptive immune response is induced. Therefore, there is a desperate need for a technically simple, rapid, and accurate assay to unequivocally diagnose active Lyme disease. Since nucleic acids of the pathogens usually do not persist after cure of the disease, DNA-based diagnostic tests are becoming highly useful for various infectious diseases. However, currently available PCR assays for Lyme disease are not very sensitive. Previously, we developed a quantitative real-time polymerase chain reaction (rt-PCR) technique using state of the art molecular beacon probes for detection of Lyme spirochetes presence in the infected mice tissues. Recently, we were able to successfully expand this strategy to detect the presence of *B. burgdorferi* even when they were present in very low numbers in the human DNA. Interestingly, this rt-PCR technique is also able to differentiate the simultaneous presence of all three major Lyme spirochete species, *B. burgdorferi*, *Borrelia afzelii*, and *Borrelia garinii* when present together by utilizing a post-PCR denaturation profile analysis, during which one molecular beacon probe distinguishes all the three species. This could be very useful for diagnosis and discrimination of various Lyme spirochetes in the European countries where all three species are prevalent. This novel diagnostic method will be able to use a variety of patient samples including blood, biopsy samples, cerebrospinal fluid, and synovial fluid for diagnosis of Lyme disease. We anticipate that pre-enrichment of the spirochetes present in the blood by culture before conducting the rt-PCR will further improve the sensitivity and specificity of the assay such that it can be used as a diagnosis of early to chronic stages of infection by Lyme spirochetes in the patients.
B038 (D038)

Performance of United States' serologic assays in the diagnosis of Lyme borreliosis acquired in Europe

John A. Branda¹,², Franc Strle¹,², Klemen Strle¹,², Nikhil Sikand¹,², Mary Jane Ferraro¹,²
and Allen C. Steere¹,²

1. Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA

2. University Medical Centre Ljubljana, Ljubljana, Slovenia

Background: American physicians sometimes need to evaluate a patient for suspected Lyme borreliosis (LB) who may have acquired the infection in Europe. Using serum samples from European LB patients, we compared the performance of European and U.S. serodiagnostic tests, including newer-generation assays containing VlsE or its C6 peptide.

Methods: The sensitivity of each assay was determined using 64 serum samples from LB patients with early or late disease manifestations who acquired the infection in Europe. Specificity was measured using 100 sera from healthy subjects from a non-endemic area.

Results: For the detection of European-acquired infection, conventional two-tiered testing (ELISA followed by immunoblotting) using U.S. assays had an overall sensitivity and specificity of 52 and 100% compared with 81% (*P* = 0.0007) and 99% (*P* = 1.0) using analogous European tests. The sensitivity of a U.S. C6 ELISA used as a stand-alone test (88% overall) was statistically comparable to that of conventional two-tiered testing using European tests (*P* = 0.47) and was 100% specific. Similarly, an alternative two-tiered algorithm using a standard U.S. ELISA followed by a C6 ELISA was comparably sensitive (84% overall) compared with conventional two-tiered testing using European assays (*P* = 0.82), and specificity remained 100%.

Conclusion: European assays outperformed analogous U.S. assays in a conventional two-tiered testing algorithm. However, a C6 ELISA used as a stand-alone test or in the second-tier of a two-tiered algorithm performed comparably to conventional two-tiered testing using European assays, and can be used for evaluation of any patient, regardless of travel history.
B039 (D042)

# Analytical sensitivity of different PCR amplification protocols for detection of Borrelia burgdorferi sensu lato

J. Brenauer, M. Teusser, C. Hizo-Teufel, A. Sing and V. Fingerle

German National Reference Centre for Borrelia, Bavarian Health and Food Safety Authority, Munich, Germany

**Background:** Molecular detection and characterization of *Borrelia burgdorferi s.l.* (Bbsl) is important and widely used for research and diagnostics purposes. These methods enabled major progresses in virtually all fields regarding Lyme borreliosis, like diagnostic, pathogenesis, epidemiology, or prevention. However, one should be aware that standardization of the whole procedure and evaluation of the numerous methods are poor so far, which is especially crucial for diagnostic purposes. This pilot study aimed at testing the sensitivity of different Bbsl-DNA amplification protocols using identical DNA-material of a broad variety of Bbsl strains.

**Materials and Methods:** Sixteen different Bbsl strains – including all known human pathogenic species – *Treponema phagedenis*, *Borrelia hermsii*, *Leptospira biflexa* – Aliquots (n = 30 a 50 ml/each dilution step) of serial diluted DNA extractions [10,000-0.1 genome equivalents/PCR reaction (5 ml)] were frozen at 80°C – five different PCR protocols targeting OspA (n = 2), p41 (n = 2), or Hbb were included.

**Results and Conclusion:** Detection limit for a single PCR may vary from 1 to >10,000 genome equivalents/PCR reaction when testing different strains – detection limit for a single strain may vary from 1 to >10,000 genome equivalents/PCR reaction when using different amplification protocols – none of the protocols showed optimal amplification of all strains tested; therefore, none is suited as stand-alone test for diagnostic purposes There is urgent need for improvement and standardization of Bbsl PCR protocols, e.g., studies that identify well-suited PCR protocols and studies to further improve such protocols.
B040 (D043)

Xenodiagnosis using *Ixodes scapularis* larva in humans

Adriana Marques¹, Sam Telford², Siu-Ping Turk¹, Erin Chung³, Carla Williams⁴, Kenneth Dardick⁵, Peter Krause⁶, Christina Brandeburg³, Chris Crowder⁷, Mark Eshoo⁷ and Linden Hu³

1. LCID/NIAID/NIH, Bethesda, MD, USA

2. Cummings School of Veterinary Medicine, Tufts University, Medford, MA, USA

3. Department of Medicine, Tufts Medical Center, Boston, MA, USA

4. SAIC-Frederick Inc., Frederick, MD, USA

5. Mansfield Family Practice, Mansfield, OH, USA

6. Yale School of Medicine, New Haven, CT, USA

7. Ibis Biosciences, Carlsbad, CA, USA

Background: *Borrelia burgdorferi*, the causative agent of Lyme disease, has been shown to persist in infected animals after antibiotic therapy. Whether *B. burgdorferi* persists in humans with Lyme disease after antibiotic therapy is unknown. One method for successfully detecting *Borrelia* in animals after antibiotic therapy is xenodiagnosis, which involves feeding uninfected ticks on animals in order to draw bacteria into the tick. Because this is part of the natural lifecycle, *B. burgdorferi* may be particularly adapted to recognizing the presence of a feeding tick and re-entering that tick. Xenodiagnosis using *Ixodes scapularis* ticks has not been studied in humans.

Methods: We examined the safety of xenodiagnosis in humans. Laboratory-reared *I. scapularis* larval ticks were placed on 33 subjects and allowed to feed to repletion. Ticks and skin biopsies were tested for the presence of *B. burgdorferi* by PCR, culture, fluorescent microscopy, and mass spectroscopy. Xenodiagnosis was repeated in seven individuals.

Results: Xenodiagnosis was well tolerated with no severe adverse events. The most common adverse event was mild itching at the site. Data from the testing of xenodiagnostic ticks are currently being analyzed, and we anticipate presenting these results at the meeting.

Conclusion: Xenodiagnosis is a safe method for recovering *B. burgdorferi* from subjects after treatment for Lyme disease. Further studies will be needed to determine the sensitivity of xenodiagnosis and whether the presence of organisms after antibiotic therapy is associated with symptoms in patients after treatment for Lyme disease.
B041 (D044)

Development of loop-mediated isothermal amplification (lamp) for relapsing fever

Matilda Ornstein Mecklenburg¹,², Alan G. Barbour¹,² and Sven Bergström¹,²

1. Department of Medicine and Microbiology and Molecular Genetics, University of California Irvine, Irvine, CA, USA

2. Department of Molecular Biology, Umeå University Sweden, Umeå, Sweden

Patients with relapsing fever (RF) are often misdiagnosed with malaria in areas where the diseases coexist. In an effort to address this issue, we have developed a novel tool for detection of RF using loop-mediated isothermal amplification (LAMP) for diagnosis in a basic healthcare setting. In LAMP, amplification and detection of a target gene can be completed under isothermal conditions in a single step. Two assays were designed, one for the North American pathogen *Borrelia hermsii* and the other for the African pathogens *Borrelia duttonii*, *Borrelia recurrentis*, and *Borrelia crocidurae*. Primers targeted the glycerophosphodiester phosphodiesterase (glpQ) gene. Primer specificity was evaluated in three ways: (i) restriction enzyme digestion of LAMP product, (ii) PCR with LAMP primers, and (iii) by running the assays with other *Borrelia* species. Based on these results, the assays were determined to be specific. The sensitivity of the assays was evaluated using qPCR. The African RF assay was highly sensitive, having a detection limit of six gene copies. The *Borrelia hermsii* assay, the North American pathogen, had a detection limit of 400 gene copies. We used a crude DNA extraction method to extract *Borrelia* DNA from whole blood. The LAMP assay was not inhibited by blood products in the crude DNA extract. During method development, issues with amplification products contamination arose. Further development to reduce the risk of amplification products contamination is necessary. Clinical sensitivity and specificity are under investigation using blood samples from patients in Rwanda. LAMP is a simple and reliable method for detecting RF and has the potential to improve diagnosis of RF in basic healthcare settings.
B042 (D046)

Autoantibodies to human endothelial cell growth factor in American and European patients with various manifestations of Lyme disease

Allen C. Steere¹, Klemen Strle¹, Gail McHugh¹, Catherine E. Costello², Dasa Stupica³,
Katarina Ogrinc³, Franc Strle³ and Elise E. Drouin¹

1. *Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA*

2. *Boston University School of Medicine, Boston, MA, USA*

3. *University Medical Center, Ljubljana, Slovenia*

**Background:** In a search for autoantigens in antibiotic-refractory Lyme arthritis, we recently identified endothelial cell growth factor (ECGF) as a target of T and B cell responses in patients with erythema migrans (EM) or Lyme arthritis. Moreover, ECGF, an IFN-inducible protein, was expressed at significantly higher levels in synovial fluid of antibiotic-refractory versus antibiotic-responsive patients, and is also upregulated in neuronal cells under inflammatory conditions. Here, we tested for anti-ECGF autoantibodies in serum samples from American and European (Slovenian) patients with a range of Lyme disease manifestations.

**Methods:** Anti-ECGF antibodies were sought in serum samples from 344 American and 58 Slovenian patients with EM, Lyme neuroborreliosis (LNB), acrodermatitis (ACA), or responsive or refractory arthritis. Additionally, sera were tested from 27 non-antibiotic-treated American patients seen in the 1970s, who were followed longitudinally from early through late infection. A positive response was defined as >3 SD above the mean value in 74 healthy control subjects.

**Results:** Among antibiotic-treated American patients, 15% with EM, 22% with LNB, 8% with responsive arthritis, and 17% with refractory arthritis had positive ECGF antibody responses. Among patients with EM, symptoms resolved more slowly after treatment in those with ECGF reactivity (7 versus 3 days, *P* = 0.01), and ECGF responses were more common in patients with refractory versus responsive arthritis (*P* = 0.1). Of the 27 non-antibiotic-treated American patients followed from EM through the course of arthritis, 7 (26%) had ECGF antibody responses, which often appeared early in the illness. Moreover, during early disseminated infection, 4 of the 7 ECGF-positive patients (57%) developed meningoencephalitis compared with 2 of the 20 anti-ECGF antibody-negative patients (10%) (*P* = 0.02). During the period of persistent infection, the duration of active arthritis was significantly longer in those with ECGF reactivity (67 versus 17 weeks, *P* = 0.004). In contrast, a smaller percentage of Slovenian patients had ECGF antibody responses (7-9% with EM, LNB, or arthritis), except for those with ACA in whom 38% had ECGF reactivity.

**Conclusion:** American and European patients with various manifestations of Lyme disease had ECGF antibody responses, but their frequencies tended to be greater in Americans. We postulate that patients who experience more severe and prolonged inflammation (Americans with meningoencephalitis or refractory arthritis or Europeans with ACA) have higher IFN levels and greater ECGF expression. This may lead to enhanced presentation of ECGF self-epitopes and autoantibody production. In individuals susceptible to immune dysregulation, these auto-antibodies may contribute to persistent inflammation and more severe and prolonged disease.
B043 (D047)

*Ixodes tick-borne borreliosis with antibody response to glpQ antigen of
Borrelia miyamotoi*

Liudmila S. Karan¹, Mikhail L. Markelov², Denis S. Sarksyan³, Tatiana A. Chekanova² and
German A. Shipulin¹

1. Central Research Institute of Epidemiology, Moscow, Russia

2. Research Institute for Occupational Medicine, Moscow, Russia

3. State Medical Academy, Izhevsk, Russia

Human cases of *Ixodes* tick-borne borrelioses (ITBB) in Russia may be caused either by *Borrelia burgdorferi* s.l. or by *Borrelia miyamotoi*. These infections may be detected by amplification of *Borrelia* DNA in blood using specific PCR. The primary acute clinical manifestation of *B. burgdorferi* infection is the erythema migrans (EM) rash, whereas *B. miyamotoi* infection usually presents as a flu-like disease with high fever but without EM. Serological methods can support and supplement PCR-based diagnostics. Unfortunately, commercial ELISA and Western blot assays using *B. burgdorferi* antigens cross-react with some antigens of *B. miyamotoi*, so they are not useful in discriminating between these two diseases. *B. miyamotoi* is part of the relapsing fever group of *Borrelia* species that produce glpQ protein, which is absent in *B. burgdorferi* s.l. species. We have developed an immunofluorescence microarray that includes eight recombinant antigens (p100, VlsE, p58, p41, p39, BBK32, OspC, and p17) of *Borrelia afzelii* and *Borrelia garinii* from Russian tick isolates, and a *B. miyamotoi* glpQ recombinant antigen from blood of patient in the Ural Region of Russia. We tested this array on serum samples collected from ITBB patients living in Udmurtia in 2012. The first group included nine patients without EM who had fever, headache, and fatigue. *B. miyamotoi* DNA was amplified from admission blood samples using *B. miyamotoi*-specific PCR. Anti-glpQ IgM antibody was found in six of the nine serum samples collected 2 days after disease onset, all the nine serum samples collected at 12 days after disease onset, and all the six serum samples collected 35 days after a disease onset. Anti-glpQ IgG antibody was found in one of the nine samples, three of the nine samples, and five of the six samples, respectively. A second group of study subjects consisted of 12 patients with EM. *B. miyamotoi* DNA was not detected in their blood. The level of anti-glpQ IgM and IgG antibodies was below threshold in all serum samples taken from this group of patients at days 6 and 15 after disease onset, with the exception of one IgM positive sample and another IgG positive sample. The pattern of response to eight non-glpQ antigens was more diverse. The patients from both groups produced IgM antibody to at least two of the four antigens (VlsE, OspC, p41, and p17) and IgG antibody to at least two of the eight non-glpQ antigens. Patients with PCR evidence of *B. miyamotoi* infection have detectable anti-glpQ IgM and IgG antibodies in their acute and convalescent sera while patients with *B. burgdorferi* s.l. do not.
B044 (D048)

Unusual clinical presentations of *acrodermatitis chronica atrophicans*

Nora Wutte, Surtov-Pudar Milana, Bokanovic Danijela and Aberer Elisabeth

Department of Dermatology and Venereology, Medical University of Graz, Graz, Austria

**Background:** Acrodermatitis chronica atrophicans (ACA) is a late cutaneous manifestation of Lyme disease defined as erythematous to livid discoloration and skin atrophy, mostly at the extensor surfaces of the legs and rarely in the face, initially with oedematous swelling, occurring months to years after infection with *Borrelia burgdorferi*. High IgG antibody titres are typically found. We report the cases of two ACA patients with unusual clinical and laboratory presentations. Patient 1: an 80-year-old patient showed diffuse large erythema with teleangicatasias on the trunk extending on the back, the legs showed no such lesions. The patient recalled a tick bite 5 years earlier, but had no history of erythema migrans. Laboratory results revealed highly positive *Borrelia*-specific antibody titres for both IgM and IgG. Histology showed lymphoplasmocytic infiltrates without fibrotic changes. PCR from histology was positive for *B. burgdorferi*-DNA. The patient was treated with doxycycline 200 mg for 30 days, and the lesions resolved almost completely within 6 months. Patient 2: a 76-year-old woman presented to her dermatologist with a livid plaque on the right ankle, 7 cm in size. The patient did not recall a tick bite or an erythema migrans. Histology was compatible with ACA; however, serology showed positive IgM antibodies by ELISA, but was negative for IgG antibodies. Immunoblot diagnosis was negative. PCR from histology was carried out and revealed *B. burgdorferi*-DNA. Treatment with doxycycline for 30 days leads to complete resolution of the plaque. At the end of the treatment, IgG antibodies became positive by ELISA; however, confirmatory immunoblot remained negative.

**Conclusion:** ACA typically occurs at the extremities, while morphea is also found on the trunk. However, ACA may also occur in regions other than the distal extremities, although only a few cases have been reported in the literature so far. Seronegative ACAs are a very rare finding and might be due to variety of borrelial strains or might be associated with HLA-specificity.
B045 (D049)

Development of a new multiplex early diagnostic assay for Lyme borreliosis
based on antigens expressed in the skin of the mammalian host

Alexia A. Belperron¹, Oriana Perez², Elizabeth Peterson-Roth², Sahar Usmani-Brown², Radha Iyer³, Susan J. Wong⁴, Peter J. Krause¹,⁵, Ira Schwartz³, Linda K. Bockenstedt¹ and Michel Ledizet²

1. Yale University School of Medicine, New Haven, CT, USA

2. L2 Diagnostics, LLC, New Haven, CT, USA

3. New York Medical College, Valhalla, NY, USA

4. Wadsworth Center, New York State Department of Health, Albany, NY, USA

5. Yale University School of Public Health, New Haven, CT, USA

Current immunological assays used to diagnose exposure to *Borrelia burgdorferi* suffer from low sensitivity during the early stages of infection. Our hypothesis is that we could improve early detection of Lyme borreliosis by detecting antibodies recognizing antigens expressed by spirochetes as they infect and begin to disseminate within the mammalian host. Toward this end, we used whole *B. burgdorferi* genome microarrays to identify genes expressed by *B. burgdorferi* isolated from the skin of MyD88⁻ mice infected with *B. burgdorferi* strain 297. These immunodeficient mice mount an antibody response against the bacterium that appears identical on immunoblot to wild-type mice, but the pathogen can be found in higher numbers in the skin and other tissues after infection. We selected 11 proteins for initial study based on a variety of criteria: (1) conservation among strains of *Borrelia*; (2) lack of homology to proteins from unrelated organisms; (3) presence of a lipobox; and (4) higher level of expression in the skin compared to liquid culture in BSK-II medium. These proteins were expressed in recombinant form in *Escherichia coli* and coupled to Luminex beads, along with recombinant OspC from *B. burgdorferi* strains 297 and B31. The prototype Luminex assay containing 12 different antigens detected IgG and IgM antibodies in a cohort of 200 human sera that had previously tested positive using a C6 ELISA + immunoblot assay, proving that the selected proteins were expressed and immunogenic in infected human patients. Antibodies against individual antigens were detected in 25–89% of samples. More than 85% of patient serum samples were positive for at least three antigens while serum samples from patients with no history of Lyme borreliosis were positive for 0–2 antigens. We also analyzed serum samples from 15 rheumatoid arthritis patients with no history of Lyme disease. With one exception, all patients tested negative. We next analyzed 134 serum samples collected during a serosurvey in Block Island, RI, in 2012. Thirty-three of 134 samples were IgG-positive in our prototype multiplex assay, and 17 of those were confirmed positive by immunoblotting. Results of C6 ELISA (Immunetics) and immunoblot analysis (Viralab) on the same specimens are pending. Over the next several months, we will be conducting a prospective study of sera from patients presenting with erythema migrans to compare the sensitivity and specificity of our assay with that of existing commercial assays for the detection of early Lyme disease.
B046 (D050)

Diagnosis of Lyme neuroborreliosis by combination of antigen-specific antibody index with CXCL13 cytokine determination

Thorsten Zacher¹, Gereon Göttner¹, Bernd Krämer¹, Dag Nyman², Harald Hlobil³,
Oliver Böcher¹ and Erwin Soutschek¹

1. Mikrogen GmbH, Neuried, Germany

2. Bimelix Biomedical Laboratory, Mariehamn, Åland, Finland

3. Laborärzte Sindelfingen, Sindelfingen, Germany

Lyme neuroborreliosis (LNB) is a nervous system infection caused by *Borrelia burgdorferi sensu lato* (Bb).
The diagnosis of defined LNB is supported by: (i) neurological symptoms, (ii) a lymphocytic pleocytosis in the
cerebrospinal fluid (CSF) and (iii) intrathecally produced *Borrelia burgdorferi* (Bb)-specific antibodies. Bb-specific
antibodies in serum and CSF can be detected with IgG- and IgM-differentiating assays but antibody determination has
to deal with low antibody titers in the very early phase of the disease and delayed antibody titer decrease following
therapy. Recent studies have suggested that the B-cell attracting cytokine CXCL13 is reliably increased in the CSF of
patients with well-defined early LNB, thus determination of CXCL13 might be helpful in seronegative patients during
early disease and for control of therapy. We present a rapid and convenient particle immunoassay (PIA) combining the
calculation of an elevated antibody index (AI) with the quantitative detection of CXCL13. On the basis of the xMAP-
Technology, a serological test-system for diagnosis of Lyme borreliosis including CSF/serum antibody index (AI)
calculation was developed. In this assay, 13 recombinant, immunodominant antigens (p100, VlsE, p58, p39, OspA,
OspC, p18) of the five pathogenic genospecies: *B. burgdorferi sensu strictu*, *Borrelia garinii*, *Borrelia afzelii*, *Borrelia
spielmanii* and *Borrelia bavariensis*, were immobilized to a single color coded population of polystyrene beads
each. In addition, antibodies against the cytokine CXCL13 were immobilized to a 14th bead population. CSF/serum
antibody index (AI) and CXCL13 concentration were determined in three different patient panels: 30 samples with
normal AI, 148 routine samples with suspected neuroborreliosis and 38 samples from clinically well-defined early
neuroborreliosis. Each single antibody/antigen reaction could be measured quantitatively with high reproducibility.
The linear range covers three log levels. The lower detection limit of CXCL13 was 50 pg/ml and the system showed
a linear range up to 10,000 pg/ml. We compared the CXCL13 concentrations in all the three groups for significant
correlation. The evaluation of the selected patient panels show an increasing number of CXCL13 positives depending
on the stage of infection. The combination of antigen-specific antibody index calculation with CXCL13 cytokine
determination is a promising tool for the diagnosis of Lyme neuroborreliosis.
B047 (D053)

PCR-based diagnostics of Lyme disease using capillary blood obtained from
erythema migrans lesion

Denis S. Sarksyan¹,², Ludmila S. Karan¹,², Alexander E. Platonov¹,², Oleg V. Malinin¹,²
and Victor V. Maleev¹,²

1. Central Research Institute of Epidemiology, Moscow, Russia

2. State Medical Academy, Izhevsk, Russia

Introduction: Routine laboratory diagnosis of Lyme disease (LD) is based on the detection of specific *Borrelia burgdorferi* antibodies by ELISA, Western blot, or microarrays. Antibody testing is of limited value because anti-borrelial IgM (and particularly IgG) usually only appears after several weeks after the onset of infection or may not be produced at all. PCR-based assays are an option for early detection of *B. burgdorferi* infection; however, in the absence of systemic infection, *B. burgdorferi* DNA is usually not found in venous blood. In our laboratory, only 15-30% of blood samples taken on admission from patients with confirmed LD are PCR positive. The proportion of positive PCR results may increase to 40-80% if skin biopsy specimens are used. The skin biopsy procedure may be traumatic and is not recommended for LD diagnosis in Russia. We have therefore developed and tested a novel approach using capillary blood sample (CBS) that are obtained from erythema migrans (EM) lesions.

Methods: Twenty patients with LD living in Izhevsk City, Russia, in 2012 were enrolled in the study. First sampling was done at admission, from 2 to 20 days after the appearance of a typical EM (³5 cm in diameter) and before the start of antibiotic treatment. Venous blood was taken for standard ELISA and PCR testing. Simultaneously, 0.1 ml of CBS was taken from the EM lesion, supplemented by EDTA, and subjected to DNA extraction and PCR. Commercial PCR test-system “AmpliSens® TBEV, *B. burgdorferi* s.l., Anaplasma phagocytophilum, Ehrlichia chaffeensis/Ehrlichia muris-FL” was used according to manufacturer’s instructions (www.interlabservise.ru).

Results: Of 20 patients with LD, *B. burgdorferi* DNA was amplified from the venous blood of four patients using specific real-time PCR, whereas three of these patients tested positive using the CBS assay and one tested negative. Six additional PCR-positive results were obtained using the CBS assay. Seroconversion for specific IgM and/or IgG was noted in 14 of the 20 serum samples taken about 10 days after the first sampling, including 8 of 10 samples from PCR-positive patients. The proportion of PCR-positive results in our CBS assay (9 of 20 samples) was similar to that when we tested 2 mm skin biopsy specimens in 2010-2011 (9 of 17 patients) using PCR.

Conclusion: CBS is an effective non-traumatic method for early detection of the *B. burgdorferi* pathogen. It may be
particularly useful if the EM is <5 cm in diameter or has an ambiguous appearance.
B048 (D055)

Direct molecular detection of *Borrelia burgdorferi* from whole blood and CSF
of patients with acute and chronic Lyme disease (revised)

Mark W. Eshoo<sup>1,2,3</sup>, Christopher C. Crowder<sup>1,2,3</sup>, Heather E. Matthews<sup>1,2,3</sup>, Joachim Blocher<sup>1,2,3</sup>, Holger Schmidt<sup>1,2,3</sup>, Steven E. Schutzer<sup>1,2,3</sup> and John N. Aucott<sup>1,2,3</sup>

1. Johns Hopkins School of Medicine, Ibis Biosciences Inc., Carlsbad, CA, USA

2. University of Medicine and Dentistry of New Jersey, Newark, NJ, USA

3. University Medical Center Gottingen, Gottingen, Germany

The early detection and treatment of Lyme disease is crucial to prevent late sequelae and to improve long-term
prognosis. However, infection is often difficult to diagnose because of the variability of clinical manifestations and
the biologically delayed antibody production upon which current serologic tests are based. Furthermore, serological
tests cannot be used to show response to treatment as a patient can remain sero-positive for years following exposure.
Direct molecular tests have the ability to detect *Borrelia* both before and after patients seroconvert, thus aiding in the
earlier diagnosis and subsequent assessment of the response to treatment. However, one of the challenges of direct
molecular tests is that even during acute infection, there are relatively few *Borrelia* present in the blood of the patient.
To address this challenge, we have developed an “ultra-sensitive” molecular assay to detect *B. burgdorferi* directly
from whole blood, CSF, or other specimens collected as early as the initial patient visit. This molecular assay can
detect less than a single genome of *Borrelia* DNA in a specimen. Furthermore, this approach also has the potential
to detect *Borrelia* in patients who have already been treated for Lyme disease. To achieve increased sensitivity,
we employed a larger than typical sample volume (1.25 vs. 0.2 mL). Secondly, we performed an isothermal pre-
amplification of the *Borrelia* DNA (akin to a “molecular culture”) to increase the copies of *Borrelia* DNA in the
sample above the limits of detection of PCR. Thirdly, we detected the enriched *Borrelia* DNA with a broad-range
PCR and electro-spray ionization mass spectrometry assay that targets eight *Borrelia* loci each of which is diagnostic
for *Borrelia*. This assay not only identifies the presence of *Borrelia* DNA but can also indentify the infecting *Borrelia*
species and frequently its genotype. To test this technology, we evaluated the assay on blood that had been collected
at the initial presentation of 21 endemic-area patients who had both physician-diagnosed erythema migrans (EM)
and positive two-tiered serology either at the initial visit or at a follow-up visit after 3 weeks of antibiotic therapy.
The results of this study demonstrated the ability to detect *B. burgdorferi* in 13 of the 21 patients (62%). In 5 of the
21 cases, we demonstrated the ability to detect *B. burgdorferi* prior to seroconversion. Results will also be presented
on the analysis of a large set (>200) of CSF specimens collected in Germany from patients with acute and chronic
neuroborreosis.
B049 (D056)

The C6 assay – a Scottish perspective

Sally Mavin, Roger Evans and Emma Watson

National Lyme Borreliosis Testing Laboratory, Raigmore Hospital, Inverness, UK

The C6 Lyme ELISA has generated a great deal of interest throughout Europe and the USA, where it has been proposed as a sensitive screening assay, an alternative to two-tier testing, and a marker of treatment response or resolving infection. The aim of this pilot study was to assess these roles of the C6 assay in a routine diagnostic laboratory. Sera (n = 252) from patients with clinically suspected Lyme borreliosis were tested by both the C6 Lyme ELISA (Immunetics) and our current screening test, the Enzygnost Lyme link VlsE/IgG ELISA (Siemens), as per the manufacturer's instructions. Reactive samples were confirmed by commercial EU Lyme IgG Western blot (Trinity biotech) and/or in-house IgG Western blot. Overall, the Enzygnost assay was more sensitive than the C6 ELISA (182/252 vs. 168/252 reactive sera, respectively). Since the C6 assay is less sensitive and more expensive, it would not replace the current screening test. Detection of positive sera with single-tiered testing with both EIAs was equivalent to two-tiered testing with in-house IgG Western blot confirmation of equivocal/positive screening sera but was more sensitive than two-tiered testing with Trinity IgG Western blot confirmation. The decrease in sensitivity with the two-tiered testing utilizing the Trinity Western blot (85/152 vs. 55/152 and 79/152 vs. 62/152 positive results with the C6 and Enzygnost assays, respectively) may be due in part to the sensitivity of this particular assay, or a reflection of superior specificity. Analysis of the clinical information from these patients will determine the performance of the single- and two-tiered testing protocols. Paired sera from 18 patients were tested in parallel with the C6 and Enzygnost assays and either the Trinity or in-house IgG Western blots to monitor treatment response or indicate resolving infection. There was no difference in paired sera results for 11 patients. In five patients, there were quantitative differences in the both the C6 and Enzygnost assays and Western blot results. One patient had a quantitative decrease with the Enzygnost ELISA and Western blot result between subsequent samples but no difference with the C6 ELISA. A further patient had a quantitative increase with both the C6 and Enzygnost assays in subsequent samples but no difference in Western blot pattern. Although the numbers of samples in this study is small, the early indication suggests that there is no additional advantage to utilizing the C6 assay as a tool for monitoring treatment response or resolving infection.
B051 (P003)

Antigenic analysis of North American *Borrelia burgdorferi sensu lato* isolates

Brian Leydet and Fang-Ting Liang

Louisiana State University, Baton Rouge, LA, USA

A large number of spirochete species belonging to *Borrelia burgdorferi sensu lato* (s.l.) complex have been identified worldwide. At least eight different species have been isolated in the United States and to date only *B. burgdorferi sensu stricto* (s.s.) is believed to cause human Lyme borreliosis. In this study, antigenic analysis was conducted for six of the US *B. burgdorferi* s.l. species. Mice were inoculated with *Borrelia americana*, *Borrelia andersonii*, *Borrelia bissettii*, *Borrelia carolinensis*, *Borrelia kurtenbachii* and *B. burgdorferi* s.s. Immunoblots of *Borrelia* exposed mouse sera against each of the *Borrelia* whole cell lysates revealed multiple common immunogenic proteins, yet also revealed immunogenic proteins specific to *Borrelia* exposure history. Western blots to specific proteins will allow us to determine the specific response to the more common immunogenic proteins, such as VlsE, OspA, OspC, Flagellin, DpbA, DpbB, and BBK32. *B. bissettii* also stimulated an antibody response to the diagnostic C6 peptide as strong as *B. burgdorferi* s.s. These preliminary results indicate that *B. bissettii* expresses VlsE variable antigen and that the C6 peptide ELISA would remain to be an effective serological tool if 1 day *B. bissettii* is confirmed to cause human disease. And because most of these *Borrelia* have yet to be associated with human disease, characterization of the immune response to these spirochetes may help identify *Borrelia*-specific immunogenic proteins.
B052

Enzyme-linked immunosorbent assay to detect anti-Lyme IgM and IgG antibodies
in sera of patients resident in Kuwait

Mustafa Abu Salim

Department of Microbiology, Faculty of Medicine, Kuwait University, Safat, Kuwait

Lyme disease caused by *Borrelia burgdorferi* is uncommon in Kuwait, but people visiting endemic countries may get infected and develop symptoms when they come back to Kuwait. The aim of this study was to determine the serological evidence of infection in people who returned back from endemic countries and were clinically suspected of Lyme disease. A total of 80 sera, obtained from patients attending five major hospitals in Kuwait, during 2009–2013, were tested for anti-Lyme IgM and IgG antibodies using enzyme-linked immunosorbent assay (ELISA). The samples were tested using a commercial assay (Serion ELISA Classic, Germany). The test employs a positive control, a negative control, and a cut off serum. To achieve a high level of sensitivity, a combination of a European strain of the genus *Borrelia garini* along with *Borrelia afzelli* PKo was used. The antigen cocktail had the antigen OspC and VisE, which are particularly important for the diagnosis of early stage *Borrelia* disease, in addition to the late stage critical protein P100. In order to further increase the sensitivity, a recombinant VisE was added to the coating antigen complex. Specificity was optimized by the addition of a lysate of *Treponema phagedenis* to the dilution buffer which selectively absorbs any cross reacting *Treponema* antibodies, which may be present. The results showed that 32/80 (40%) sera were antibody positive. Out of these, 20 were positive for IgM only, 6 for IgG only and 6 for both IgM and IgG. The results suggest that ELISA could be useful for serological evidence of infection with *B. burgdorferi* due to its immunoglobulin specific analysis of the immune response. The study was supported by Kuwait University Research Sector grant GM01/01.
B053

From prevalence studies to the development of novel diagnostic tests
for Lyme disease

A. L. Reye, J. Kirpach, J. M. Hübschen and C. P. Muller

National Public Health Laboratory, CRP Santé, Institute of Immunology, Luxembourg, Luxembourg

Worldwide, tick-borne diseases seem to become increasingly important for public health, of which Lyme borreliosis is of main importance for Europe. Strategies to assess the risk for contracting tick-borne diseases are, e.g., pathogen surveillance in ticks and determination of seroprevalence in risk groups. Here, we analyzed 5638 ticks that were collected in Luxembourg between 2007 and 2009 and screened blood samples from more than 250 forestry workers collected in the years 2010, 2012, and 2013. Pathogen-specific detection PCRs revealed that 16.3% of questing ticks were infected with *Borrelia burgdorferi* sensu lato, the causative agent of Lyme borreliosis. Other tick-borne pathogens were less prevalent, e.g., Spotted fever group *Rickettsiae* (6.7%), *Babesia* species (1.8%), *Anaplasma phagocytophilum* (0.9%), and *Bartonella* species (0.1%). Tick-borne encephalitis virus *Coxiella burnetii* and *Francisella tularensis* were not found in ticks from Luxembourg. Seroprevalence rates of *B. burgdorferi* s.l. in forestry workers were on average 31% for IgG and 13% for IgM antibodies. Only a proportion of the seropositive forestry workers reported to have been previously diagnosed for Lyme borreliosis, which is in line with reports of several infections with borrelial spirochetes remaining asymptomatic. Within the period from 2010 to 2012, seroconversions were observed for about 1.7% for IgG and 8.7% for IgM. None of the IgM positive candidates analyzed in 2010 (n = 14) converted back to negative within the 2 years period of screening. In contrast, 8.1% of the IgG initially positive individuals were negative in 2012. In an aged and sex matched control group from 2011, 4% displayed IgG and 3% IgM antibodies against *B. burgdorferi* s.l.. These data suggest that Lyme borreliosis is an important health concern in Luxembourg, especially among professional high risk groups. As the diagnosis of Lyme borreliosis is complicated by the often unspecific symptoms and the absence of a reliable diagnostic test of the active disease, our institute currently investigates strategies to shed light on the complex immune response elicited by the pathogen, thus contributing to the development of novel diagnostic tests. So far, the response of the adaptive immune system to *Borrelia* infection is not completely understood. Therefore, antigen-specific B-cells will be isolated and compared on a single cell level between acute, chronic, and resolved patients. With this approach, we expect to identify *Borrelia* specific B-cell clones, which may be valuable tools for epidemiology, symptomatology, and perhaps even diagnosis.
B054 (Ep005)

Emergence of babesiosis in Maine: entomological correlates of human seroprevalence and disease

R. P. Smith Jr., R. Kessler, B. Missaghi, T. Borelli, M. Coulter, S. P. Elias, C. Lubelczyk, E. H. Lacombe and P. W. Rand

Vector-Borne Disease Laboratory, Maine Medical Center Research Institute, South Portland, ME, USA

*Babesia microti* was first documented in *Ixodes scapularis* ticks in Maine in 1996, and the first human case was reported in 2001. Despite presence of vector ticks in all Maine counties, and reports of 900–1000 cases of Lyme disease per year, human babesiosis emerged relatively recently with more geographic focality. We sought to determine whether human cases correlated with *I. scapularis* abundance and tick infection prevalence of *Babesia microti* and *Borrelia burgdorferi*.

**Methods:** We reviewed cases of human babesiosis reported to the Maine CDC from 2001 to present and complete dentomologic surveys at several established field sites from 1995 to 2012 to determine abundance of *I. scapularis* ticks and infection prevalence by *B. burgdorferi* and/or *B. microti*. In addition, we completed a serosurvey for antibody positivity to *Babesia* in healthy blood donors from an area highly endemic for *B. burgdorferi*-infected ticks. Infection by *B. burgdorferi* was detected by direct fluorescent antibody and/or PCR directed against *ospC*. *Babesia* was detected by PCR and sequenced to distinguish *B. microti* from *Babesia odocoilei*. Antibody (FITC fluorescent antibabesial specific IgG) was detected in serum samples from human serosurvey participants by IFA examination at a dilution of 1:64.

**Results:** Reported human cases increased from 0–5 cases/years in 2000–2004 to 5–11 cases/years in 2005–2011. Cases were marginally correlated with *I. scapularis* tick abundance (Spearman correlation coefficient = 0.61, *P* = 0.11), and *B. microti* infection percent (0.66, *P* = 0.07), and correlated with *B. burgdorferi* infection percent (0.91, *P* = 0.007). Also, *B. microti* infection percent was correlated with abundance of *I. scapularis* ticks (0.73, *P* = 0.04). In one study site, we documented a shift in presence of *Babesia* spp. from *B. odocoilei* to *B. microti* between 1996 and 2011. Seroprevalence of anti-*Babesia* antibodies was 3.2% in healthy blood donors (n = 311), but no active infections were identified by PCR.

**Conclusion:** Human babesiosis was recently recognized in Maine 5 years after first detection in vector ticks, and 15 years after recognition of Lyme disease in the state. Prevalence of *B. microti* infection in ticks is correlated with abundance of *I. scapularis* and with prevalence of tick infections by *B. burgdorferi*. Our data are consistent with the hypothesis that human babesiosis may occur after attainment of higher tick densities than are associated with emergence of Lyme disease, possibly due to differences in mechanisms of pathogen dispersal and in enzootic transmission dynamics.
B055 (Ep007)

# Self-reported Lyme disease diagnosis, treatment, and recovery: results from 2009, 2011, and 2012 HealthStyles nationwide surveys

Sarah Hook, Christina Nelson and Paul Mead

Bacterial Diseases Branch, Division of Vector-Borne Diseases, CDC, Atlanta, GA, USA

**Background:** Lyme disease (LD) is the most common vector-borne disease in the U.S. and the sixth most common nationally notifiable disease. The Centers for Disease Control and Prevention (CDC) has a number of projects underway to evaluate key measures of LD burden in the U.S. As part of this overall effort, we utilized several nationwide HealthStyles surveys to investigate patterns of LD diagnosis, treatment, and recovery.

**Methods:** HealthStyles surveys are annual, cross-sectional surveys containing questions on knowledge, attitudes, and behaviors for health-related topics. These surveys, conducted by mail or online, are designed to be demographically representative of the U.S. population, and samples are weighted based on census regions and several demographic variables. We included a total of seven questions related to self-reported LD diagnosis, treatment received, recovery time, and persistence of symptoms in the 2009, 2011, and 2012 HealthStyles surveys. Analysis of responses was primarily descriptive.

**Results:** Response rates were 73% (4728/6504) in 2009, 69% (4050/5868) in 2011, and 80% (3503/4371) in 2012. Overall, 51% of respondents were female, 71% were white, 65% had an annual household income >$40,000, and 64% reported some college education. Median respondent age was 51 years. Fifty-nine (1.3%) respondents in 2009 and 32 (0.9%) in 2012 reported having ever been diagnosed with LD; 10 (0.3%) respondents in 2012 were diagnosed in the last year. Among 26 respondents in 2012 with a past diagnosis of LD (defined as diagnosis >6 months earlier), 15 (58%) reported recovering fully in <6 months, 8 (31%) recovered in 7 months to 2 years, and 3 (12%) remained ill for >3 years. The most common persistent symptoms experienced by 2012 respondents who had not recovered fully were fatigue, muscle aches, joint swelling, and numbness or tingling; none reported receiving intravenous antibiotics or other alternative treatments. In 2009, 23 (39%) of 56 respondents receiving antibiotics were treated <4 weeks, while 21 (36%) were treated >8 weeks. Overall, 426 (10.5%) respondents in 2011 reported personally knowing someone described as having “chronic LD.”

**Conclusion:** Our results are subject to several limitations, including self-reported diagnoses and wide confidence intervals associated with the outcomes of interest. Nevertheless, applied to a population of >300 million, our findings from these nationally representative surveys suggest that a very large number of individuals in the U.S. have been diagnosed with LD. Although most patients recovered in <6 months, a significant proportion received antibiotics for >8 weeks.
B056 (Ep008)

Investigation of tick-borne encephalitis virus in ticks detached from humans with
follow-up of clinical symptoms and serological response

Pontus Lindblom¹, Peter Wilhelmsson¹, Linda Fryland², Johanna Sjöwall³, Mats Haglund⁴, The TBD STING-Study Group, Andreas Matussek⁵, Jan Ernerudh², Sirkka Vene⁶, Åshild Andreassen⁵, Pia Forsberg³ and Per-Eric Lindgren¹

1. Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Linköping University,
Linköping, Sweden

2. Division of Clinical Immunology, Department of Clinical and Experimental Medicine, Linköping University, Sweden

3. Division of Infectious Diseases, Department of Clinical and Experimental Medicine, Linköping University, Sweden

4. Clinic of Infectious Diseases, County Hospital, Kalmar, Sweden

5. NIPH, Oslo, Norway

6. SMI, Stockholm, Sweden

Tick-borne encephalitis virus (TBEV) is a serious cause of acute central nervous system disease in large parts of Europe and Asia. In Sweden at present, 200–300 cases of tick-borne encephalitis (TBE) are registered each year, showing an increasing trend and a spread to new areas. In order to investigate the risk of acquiring TBE after a single tick-bite, the tick-borne diseases (TBD) STING study was set up to collect ticks, blood samples, and questionnaires from newly tick-bitten humans at participating primary health care centers in Sweden and at the Åland Islands, Finland. We received a total of 2167 ticks from 1886 newly tick-bitten individuals during 2008–2009. Using quantitative real-time PCR, we found five ticks infected with TBEV, with highly varying virus copy numbers. Three individuals from the Åland Islands bitten by TBEV-infected ticks were vaccinated. One person bitten by a tick carrying a high viral load was not vaccinated, and experienced a short episode of fever consistent with the viremic phase of European subtype TBEV infection. The other non-vaccinated person was bitten by a tick carrying few TBEV copies, but did neither develop any symptoms nor anti-TBEV antibodies. Two other persons developed new anti-TBEV antibodies during the 3 month study period, but TBEV could not be detected in the ticks from these two persons. One of these two persons reported headache, fatigue, neck pain, stiffness, dizziness, and concentration difficulties during the study time. In conclusion, very few ticks were infected with TBEV in the investigated regions in the TBD STING study, and the risk of acquiring TBE after a single tick-bite seems to be low.
B058 (Ep010)

# The epidemiology of Lyme disease in Ontario, 2007–2011

Curtis Russell, Karen Johnson, Dawn Williams, Badal Dhar, Erik Kristjanson and Samir N. Patel

Public Health Ontario, Toronto, ON, Canada

**Background:** Lyme disease is reportable in Ontario and provincial notification of human cases occurs through the integrated Public Health Information System (iPHIS). Complementary passive tick surveillance, whereby the public submits ticks to public health for identification and testing, and active tick surveillance, whereby public health authorities actively look for and test ticks, are also conducted in Ontario. This poster will describe the epidemiology of Lyme disease in both the human and blacklegged tick populations in Ontario.

**Methods:** Human Lyme disease cases were obtained from iPHIS and tick data were obtained from the tick submission database for the years 2007–2011. Using SPSS 18, changes in incidence over time were evaluated using Poisson regression and differences in proportions were tested using the chi-square statistic, with both assessed at the 95% level of significance.

**Results:** In Ontario, 515 human cases of Lyme disease were reported from 2007 to 2011 for an average of 103 cases per year. The annual incidence rate of Lyme disease increased steadily over the period from 0.60 cases per 100,000 population in 2007 to 1.01 cases per 100,000 population in 2011. Cases ranged in age from 2 to 84 years, with a mean age of 43.1 years; over half (56%) of the cases were female. The majority of cases reported an exposure, including 173 travel-related and 277 locally acquired cases. Of the locally acquired cases, 157 (57%) were associated with exposures in non-endemic areas in Ontario, 98 (35%) with exposures in established endemic areas, and 22 (8%) with unspecified exposures within Ontario. Blacklegged tick submissions to public health also increased from 1,171 submissions in 2007 to 2,286 in 2011.

**Discussion:** Since 2007, Ontario has experienced an increase in the incidence of human Lyme disease cases, as well as an increase in the number and geographic range of the blacklegged tick vector. This trend may be explained by greater public awareness that has been resulted in increased tick submissions. A simultaneous increase in the demand for Lyme disease testing also occurred over this period as evidenced by the 50% increase in the number of human specimens submitted to public health labs for diagnosis. In Ontario, the geographic distribution of human cases and blacklegged ticks show strong geographic correlation, indicating increased risk of human cases.
B059 (Ep011)

Low risk of seroconversion and clinical disease after bite by an *Anaplasma phagocytophilum*-infected tick

Paula Gyllemark<sup>1,2,3,4,5,6</sup>, Linda Fryland<sup>1,2,3,4,5,6</sup>, Peter Wilhelmsson<sup>1,2,3,4,5,6</sup>, Monika Kozak<sup>1,2,3,4,5,6</sup>, Andreas Matussek<sup>1,2,3,4,5,6</sup>, Dag Nyman<sup>1,2,3,4,5,6</sup>, Per-Eric Lindgren<sup>1,2,3,4,5,6</sup>, Pia Forsberg<sup>1,2,3,4,5,6</sup>, Anna J. Henningsson<sup>1,2,3,4,5,6</sup> and The Tick-Borne Diseases Sting Study Group

1. Department of Infectious Diseases, County Hospital Ryhov, Jönköping, Sweden

2. Department of Clinical Immunology, Linköping University, Linköping, Sweden

3. Department of Medical Microbiology, Linköping University, Linköping, Sweden

4. Department of Clinical Microbiology, County Hospital Ryhov, Jönköping, Sweden

5. Åland Group for Borrelia Research, Åland, Finland

6. Department of Infectious Diseases, Linköping University, Linköping, Sweden

Background: The tick-transmitted bacteria *Anaplasma phagocytophilum* is known to occasionally cause human granulocytic anaplasmosis (HGA). HGA is typically a febrile illness with headache, myalgia, and malaise. The symptoms are generally mild, and subclinical infections are common. Since *A. phagocytophilum* share vectors with *Borrelia burgdorferi* sensu lato (s.l.), co-infections occur. Data from animal studies suggest that such co-infections are more severe, possibly due to immunosuppression induced by *A. phagocytophilum*. However, the importance of *A. phagocytophilum* as a human pathogen is still uncertain, and little is known about co-infections with *B. burgdorferi* s.l., as well as the risk of contracting HGA after a tick-bite. The aim of this study was to evaluate the risk for humans of developing HGA or subclinical seroconversion after being bitten by an *A. phagocytophilum*-infected tick.

Methods: This study was part of the on-going Sting project, in which tick-bitten individuals are asked to donate the tick along with a blood sample. Three months later, the participants are asked to give a second blood sample. Participants are also asked to fill in questionnaires concerning symptoms, etc. 2154 ticks collected in 2008–2009 from Sweden and on the Åland Islands, Finland, were analyzed for *A. phagocytophilum* and *B. burgdorferi* s.l. DNA using real time PCR. Feeding time was estimated using the coxal and scutal indices. Paired serum samples from the tick-bitten individuals were analyzed for IgG-antibodies against *A. phagocytophilum* using indirect immunofluorescence.

Preliminary Results: Thirty (1.4%) ticks were positive for *A. phagocytophilum*, seven of these were co-infected with *B. burgdorferi* s.l., 556 (26%) ticks were positive for *B. burgdorferi* s.l. Anaplasma serology was performed on serum pairs from the 30 individuals bitten by *A. phagocytophilum*-infected ticks (group A), 30 individuals bitten by *B. burgdorferi* s.l.-infected ticks (group B), and 30 individuals bitten by ticks negative for both pathogens (group C). The overall seroprevalence in groups A–C was 18%, with no significant difference between groups. Seven individuals in group A reported symptoms (headache, neck pain, fatigue, vertigo), but none displayed significant increase in Anaplasma antibody titer or seroconversion. The estimated feeding time of the ticks in group A was 15–69 h (mean 33 h).

Conclusions: The risk of contracting HGA after a tick-bite is low, even if the tick is infected with *A. phagocytophilum* and has been feeding >24 h. The reported symptoms in group A is probably not related to HGA. The seroprevalence of 18% is in accordance with previous studies from Sweden.
B060 (Ep012)

# Clustering of human Lyme disease by residential location in an endemic county, 2001–2011

Kiersten J. Kugeler¹,², Jennifer L. Peel¹,², Katherine A. Feldman¹,² and Paul S. Mead¹,²

1. Division of Vector Borne Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA

2. Maryland Department of Health and Mental Hygiene, Colorado State University, Fort Collins, CO, USA

**Background:** Lyme disease (LD) incidence differs in space and time even within highly endemic states. Spatial dependence and clustering of human cases have not been quantified on a sub-county scale. To describe the clustering of LD cases and evaluate environmental and demographic risk factors for LD, we conducted a detailed analysis in a single, highly endemic county in Maryland.

**Methods:** We used human LD surveillance information from Howard County, Maryland, for 2001–2011. Residential address for all county households was available from the county Geographic Information Systems Division. Land use/land cover and census block group datasets were obtained from United States Geological Survey GAP Analysis Program, the State of Maryland, and ESRI Inc. Residence was assumed to be the exposure location. Spatial clusters were identified using SaTScan software, which takes into account the background population and calculates a relative risk (RR) based on observed versus expected cases. Spatial data were extracted and analyzed using ArcGIS and SAS. Initially, maximum cluster size was the software default of 50% of the population; subsequent analysis used a 2 km maximum cluster radius.

**Results:** Geocoded residence was available for 1684 (87%) of 1934 LD cases reported during 2001–2011. Using liberal cluster size criteria, elevated risk was evident for the entire western and central part of the county, an area with substantial agriculture and low density development (RR: 2.0, *p* < 0.0001). The eastern part of the county, with higher density development, displayed lower than expected risk (RR: 0.41, *p* < 0.0001). To identify more informative fine-scale risk factors, we restricted analyses to the western and central portion of the county. Six smaller areas of elevated risk (radius: 0.25–2 km) were identified (median RR: 3.17). Preliminary analyses indicate that when compared to other areas in this part of the county, these higher risk clusters had significantly more red and white-oak forest (median: 38 vs. 8%), regardless of amount of total forest. There were no differences in density of development or in census demographic factors. Soil type and forest fragmentation will also be examined.

**Conclusions:** We quantified the degree of spatial clustering of human LD risk within a single highly endemic county. High risk clusters were very similar to immediately surrounding areas except they contained a higher proportion of a single forest type. A better understanding of the factors driving the spatial distribution of human disease is needed to guide the development and implementation of effective prevention measures.
B061 (Ep013)

Lyme disease at a tertiary Lyme referral center in the Netherlands

E. A. Herkes<sup>1,2,3,4,5</sup>, J. Coumou<sup>1,2,3,4,5</sup>, M. W. Tanck<sup>1,2,3,4,5</sup>, M. C. Brouwer<sup>1,2,3,4,5</sup>,
D. van de Beek<sup>1,2,3,4,5</sup>, S. W. Tas<sup>1,2,3,4,5</sup>, M. van Vugt<sup>1,2,3,4,5</sup>, L. Spanjaard<sup>1,2,3,4,5</sup>
and J. W. Hovius<sup>1,2,3,4,5</sup>

1. Center for Experimental and Molecular Medicine, Amsterdam, Netherlands

2. Clinical Epidemiology Biostatistics Bioinformation, Amsterdam, Netherlands

3. Department of Neurology, Academic Medical Center; University of Amsterdam, Amsterdam, Netherlands

4. Department of Internal Medicine, Academic Medical Center; University of Amsterdam, Amsterdam, Netherlands

5. Department of Medical Microbiology, Academic Medical Center; University of Amsterdam, Amsterdam, Netherlands

Introduction: Among patients and physicians, anxiety about Lyme disease (LD) leads to unnecessary diagnostic testing, antibiotic treatment, and misdiagnosis. In an attempt to provide better care to LD patients in the Netherlands, the Amsterdam Multidisciplinary Lyme Center (AMLC) was instituted consisting of various medical specialists (infectious diseases, neurology, dermatology, rheumatology, psychiatry and microbiology).

Methods: We retrospectively reviewed the first 100 adults referred to the AMLC. LD definitions were based on Stanek et al. (2011) and Wormser et al. (2006).

Results: Pretreatment characteristics were gender (56% female) and age (45.1 ± 13.9). The most frequently reported symptoms were fatigue (71%), arthralgia (47%), and tingling sensations (29%). Furthermore, 59% had symptoms lasting more than a year and 55% recollected tick bites. In 81% of patients *Borrelia* serology was performed prior to referral and 83 courses of antibiotics were prescribed (to 50% of the patients). At the AMLC, testing for *Borrelia* antibodies with C6 EIAs and/or immunoblots demonstrated a significantly higher C6-index in LD patients compared to non-LD patients. Of all patients, 49% were diagnosed with current LD – 6% EM, 4% early disseminated LD (two neuroborreliosis and two multiple EM), 11% late disseminated LD (two ACA, three Lyme arthritis, two neuroborreliosis, and four non-specified cases), and 7% putative persisting *B. burgdorferi* infection – or preceding LD [21% post-treatment LD syndrome (PTLDS)]. Using logistical regression analysis, OR's predictive for current LD were calculated for arthralgia [3.768 (1.046–13.566)], tingling sensations [0.133 (0.023–0.785)], fatigue [0.169 (0.45–0.632)], female gender [0.235 (0.067–0.825)], duration of symptoms in weeks [0.994 (0.990–0.999)], and recollection of tick bites [8.922 (2.047–38.887)]. Of the 51% without LD, 23% was explained by another diagnosis, mostly osteoarthritis or allergy/eczema, but incidentally by multiple sclerosis, paraneoplastic syndrome, polymyalgia rheumatica, or delusional parasitosis. We treated 25% of patients with recommended antibiotic therapy. In 65% of the patients, we were informed about the clinical course and subjective improvement was reported by all local and early LD and PTLDS patients. However, non-, late and putative persisting LD patients improved in, respectively, 53, 57 and 67%.

Discussion: In agreement with others, we confirm that the majority of referred patients had no current LD and patients with local and early LD respond well to antibiotic treatment. PTLDS patients reported improvement over time without additional antibiotic retreatment. Interestingly, 50% of PTLDS patients received additional antibiotic retreatment before referral. We will continue evaluating patients referred to the AMLC to determine the burden of LD.
B062 (Ep014)

Human *Borrelia miyamotoi* infection in the United States

P. J. Krause¹,², S. Narasimhan¹,², G. Wormser¹,², L. Rollend¹,², E. Fikrig¹,², T. Steeves¹,²,

J. Brancato¹,², T. Lepore¹,², K. Dardick¹,², A. Barbour¹,² and D. Fish¹,²

1. Yale School of Public Health, New York Medical College, Mansfield Family Practice, Nantucket Cottage Hospital, University of California Irvine, Irvine, CA, USA

2. Yale School of Medicine, New York Medical College, Mansfield Family Practice, Nantucket Cottage Hospital, University of California Irvine, Irvine, CA, USA

Introduction: *Borrelia miyamotoi*, a spirochete that is genetically related to the species of *Borrelia* that cause relapsing fever, has been detected in all tick species that are vectors of Lyme disease. It was detected in *Ixodes scapularis* ticks from Connecticut in 2001 and subsequently has been found in all areas of the United States where Lyme disease is endemic, including 15 states in the northeast and northern Midwest and in California. The first human cases of *B. miyamotoi* infection were reported in central Russia in 2011. We now provide evidence of human *B. miyamotoi* infection and prevalence of this infection among people in the United States.

Methods: Enzyme-linked immunosorbent assays and confirmatory Western blot assays of archived serum samples obtained from three groups of patients, who were living in areas where Lyme disease was endemic between 1990 and 2010, were used to detect antibody against *B. miyamotoi* GlpQ protein (an antigen that is nonreactive to *Borrelia burgdorferi* antibody). Group 1 consisted of 584 healthy subjects from southern New England, who participated in serologic surveys for tick-borne infections. Group 2 included 277 patients from southern New England, who were evaluated for suspected Lyme disease. Group 3 consisted of 14 patients from southern New York, who were evaluated at a Lyme disease clinic with a viral-like illness in the late spring or summer.

Results: The *B. miyamotoi* seroprevalence among healthy subjects (group 1) was 1.0%. Three patients who experienced a viral-like illness had a *B. miyamotoi* GlpQ seroconversion where the antibody titer was at least four times as high in the convalescent serum samples as in the acute serum samples and confirmed by Western blot assay. Two of these patients were co-infected, one with Lyme disease and one with babesiosis. One patient, who had no erythema migrans skin lesion and no laboratory evidence of human granulocytic anaplasmosis co-infection, experienced a fever of 39.4°C, chills, sweats, a headache, neck stiffness, fatigue, myalgias, arthralgias, abdominal pain, cough, sore throat, and right inguinal lymphadenopathy. The other two patients also presented with a viral-like syndrome. All patients became asymptomatic after treatment with doxycycline.

Conclusion: These data suggest that *B. miyamotoi* infection may be prevalent in areas where Lyme disease is endemic in the United States and can be associated with viral-like symptoms. Additional research is needed to determine the frequency of infection, spectrum of clinical symptoms, and most appropriate diagnostic testing and treatment for this newly appreciated infection.
B063 (Ep015)

# Immobilization, agglutination, and lysis of *Borrelia burgdorferi* spirochetes in horse serum: implications for diagnosis of equine borreliosis

Mark D. Massaro and R. Jory Brinkerhoff

*University of Richmond, Richmond, VA, USA*

Equine borreliosis is poorly understood and substantial controversy surrounds its existence. A number of vertebrate species are innately immune to the Lyme disease (LD) agent, *Borrelia burgdorferi* and inclusion of horses in this group might reduce the number of false positive equine LD diagnoses. To determine if horses display innate immunity to *B. burgdorferi* and if the same immunological mechanisms that exist in other species for this pathogen are likely to be important in horses, we conducted *in vitro* assays of live *B. burgdorferi* cultures incubated with and without horse serum and analyzed rates of spirochete immobilization, lysis, and agglutination. To identify potential mechanisms of borreliacidal activity, we used equine sera with functional complement as well as those in which complement pathways had been heat-inactivated. We quantified changes in spirochete viability in both heat-treated and untreated sera by linear regression. Spirochetes incubated in horse serum suffered significant mortality over time (>80% within 4 h) when compared to control samples incubated in the absence of horse sera. We detected no difference in borreliacidal activity between heat-treated and untreated sera suggesting that the complement system is not responsible for agglutination and lysis of *B. burgdorferi* spirochetes. As a point of reference, we conducted identical assays with white-tailed deer (*Odocoileus virginianus*) serum; heat-treated deer sera failed to kill substantial numbers of spirochetes but untreated serum was hostile to *B. burgdorferi*, a finding which is consistent with xenodiagnostic analysis showing white-tailed deer to be non-competent hosts for this pathogen. Our finding that horse serum is apparently hostile to *B. burgdorferi* spirochetes is consistent with low numbers of confirmed clinical diagnoses of equine borreliosis and suggests that horses might have a relatively high level of innate immunity to this pathogen.
B064 (Ep016)

Risk of Lyme borreliosis and human exposure to ticks of chosen
Landscape Park in South Poland

Lukasz Pindel and Marek Asman

Department of Parasitology, Medical University of Silesia in Katowice, Katowice, Poland

Lyme borreliosis (LB) is the most common vector-borne disease in Europe. LB is caused by spirochete *Borrelia burgdorferi* sensu lato (BBsl), which is transmitted to humans by ticks, in Europe and Poland mainly the species *Ixodes ricinus* – commonly called the sheep tick or castor bean tick. The objective of this study was determination of the risk of human exposure to vector ticks and LB during high tourist season in recreational area (Zywiec Landscape Park) in the populous Zabnica region of South Silesia. Four different places were sampled (picnic areas, hunting tree stands areas, hiking trails, forest workplaces) by dragging a woolen flag over lower vegetation in June and September 2012. Human-biting ticks were enumerated (n = 198) along all trails: *Ixodes ricinus* (100%), including females (14.14% of total), males (16.16% of total), nymphs (68.18% of total), and larvae (1.52% of total). Sixty ticks were randomly selected with equal to number of each tick stages, from the above-mentioned study places. All *I. ricinus* ticks were investigated individually. Bacterial DNA was isolated by ammonium hydroxide method. The isolates obtained from *I. ricinus* ticks were examined for the presence of BBsl by PCR using primers FLA1 and FLA2 specific for fragment of the *fla* gene sequence. Only 1/60 (1.66%) of examined ticks pathogen was detected. The study showed high risk of human exposure to ticks and presumably there is no risk of LB in forest workplaces where BBsl (1.66%) was detected. Future studies should be continued by taking into account the spring season, the other regional parks in South Poland and incorporate tests for multiple pathogens.
B065 (Ep019)

Study on Lyme neuroborreliosis in Transylvania, Romania

Violeta T. Briciu¹,², Mirela Flonta¹,², Violeta Nastase¹,², Daniel Leucuta¹,², Mihaela Lupse¹,², Cristian Jianu¹,², Dumitru Carstina¹,² and Doina F. Tatulescu¹,²

1. University of Medicine and Pharmacy "Iuliu Hatieganu," Cluj Napoca, Romania

2. Clinic of Infectious Diseases, Cluj Napoca, Romania

Introduction: Lyme neuroborreliosis (LNB) may mimic almost any neurological disease and patients suffering from other neurological disorders may be misdiagnosed with Lyme borreliosis (LB).

Aims: Although in the Clinic of Infectious Diseases, Cluj-Napoca, Romania, patients are treated for LNB, no study has been performed so far following the European diagnostic criteria that recommend CSF analysis. We have addressed the question if the apparent poor response to antibiotic treatment in our patients is due to an incorrect diagnosis.

Patients and Method: A prospective study included hospitalized patients with the suspicion of LNB between March 2011 and October 2012. Inclusion criteria were central nervous system manifestations, positive serology for B. burgdorferi, lumbar puncture, and no previous parenteral antibiotic treatment. A questionnaire was completed for each patient by a medical investigator regarding previous or present co-existing diseases, tick bites history or erythema migrans, neurological, musculoskeletal, cutaneous, cardiac and ocular signs, and symptoms at admission, at the end of treatment and 3 month later. Two-tiered testing was used for screening Genzyme Virotech ELISA kit in 2011 and Euroimmun ELISA kit in 2012, followed by EUROLINE-RN-AT Western blot (Euroimmun). The intrathecal antibody index (IAI) was calculated, using a Genzyme Virotech GmbH, Germany kit in 2011 and an Euroimmun AG, Germany kit in 2012. Patients were treated as LNB with parenteral therapy (ceftriaxone 2 g/day or cefotaxime 6 g/day in case of cholelithiasis) for 21 days, indifferent of the CSF analysis results. Clinical and serological evaluation (ELISA) was repeated 3 month post-treatment. Statistical analysis used Fisher exact test and Poisson mixed regression models.

Results: Forty-two patients were included and epidemiological, clinical, and serological data of the patients will be presented. According to European guidelines no case fulfilled the criteria for definite LNB, 7 cases were possible LNB and in 33 cases LNB was infirmed. Two cases could not be classified due to insufficient amount of CSF. Adverse reactions to medication were present in 13 patients, including one *Clostridium difficile* associated disease. The evolution of signs and symptoms in possible/infirmed LNB subgroups will be presented.

Conclusion: (1) This study brings evidence that LNB is over diagnosed in our patients; (2) It is the first proof to stop the antibiotics overuse in not confirmed LNB patients in our region.
B066 (Ep022)

Geographic and genospecies distribution of *Borrelia burgdorferi sensu lato*
strains detected in humans in the United States

Kerry L. Clark

*University of North Florida, Jacksonville, FL, USA*

The present study sought to determine the cause of Lyme-like illness inhuman patients primarily in the southern U.S. with suspected Lyme disease based upon EM-like skin lesions and/or symptoms consistent with early or late stage Lyme borreliosis; the study also included patients from other states throughout the USA. Using several polymerase chain reaction assays (PCR) specific for either *Borrelia* spp. or only for Lyme group *Borrelia* spp., followed by DNA sequencing for confirmation, we identified *Borrelia burgdorferi* sensu lato DNA in blood and skin biopsy samples from patients residing in southern states and elsewhere in the U.S., but no evidence of *Borrelia lonestari* infection. PCR and DNA sequence data showed that *B. burgdorferi* sensu stricto, *Borrelia. americana*, and *Borrelia. andersonii* appear to be associated with Lyme disease-like signs and symptoms in patients from southern states, as well as some states in other regions of the country. Based on these findings, we suggest that human cases of Lyme-like illness often referred to as STARI in the southern U.S. or given other diagnoses may be attributable to previously undetected *B. burgdorferi sensu lato* infection.
B067 (Ep023)

Lyme borreliosis in human patients in Florida and Georgia, USA

Kerry L. Clark¹, Brian Leydet² and Shirley Hartman³

1. University of North Florida, Jacksonville, FL, USA

2. Louisiana State University, Baton Rouge, LA, USA

3. Mandarin Wellness Center Jacksonville, FL, USA

The present study sought to determine the cause of Lyme-like illness in several human patients residing in Florida and Georgia, USA, with suspected Lyme disease based upon EM-like skin lesions and/or symptoms consistent with early or persistent Lyme borreliosis. Using polymerase chain reaction assays (PCR) for either *Borrelia* spp. or specifically for Lyme group *Borrelia* spp., followed by DNA sequencing for confirmation, we identified *Borrelia burgdorferi* sensu lato DNA in blood, skin biopsy samples, or attached lone star ticks (*Amblyomma americanum*) from several patients residing, or with tick exposure history, in Florida or Georgia. This report presents and discusses the first specific molecular evidence of *Lyme Borrelia* spp. infection in human patients in the southern U.S., including several following lone star tick bites. Furthermore, the evidence shows that several *B. burgdorferi* sensu lato species may be associated with Lyme disease-like signs and symptoms in patients from southern states. Based on the findings of this study, we suggest that human *Lyme borreliosis* occurs in Florida and Georgia, and that cases of Lyme-like illness often referred to as STARI in the southern U.S. may be attributable to previously undetected *B. burgdorferi* sensu lato infections.
B068 (Ep024)

# Risk factors associated with seropositivity against Lyme borreliosis: results from a representative serosurvey of adults in Germany

Hendrik Wilking<sup>1,2,3</sup>, Volker Fingerle<sup>1,2,3</sup>, Christiane Klier<sup>1,2,3</sup>, Michael Thamm<sup>1,2,3</sup> and Klaus Stark<sup>1,2,3</sup>

1. *Gastrointestinal Infections, Zoonoses and Tropical Infections*, Robert Koch-Institute, Berlin, Germany

2. *Central Laboratory, Institute for Laboratory Medicine, Northwest Medical Centre, Academic Teaching Hospital, Medical Faculty, Johann Wolfgang Goethe-University, Steinbacher Hohl 2-26, 60488 Frankfurt am Main, Germany*

3. *German National Reference Centre for Borrelia, Bavarian Health and Food Safety Authority, Erlangen, Bavaria, Germany*

**Background:** Lyme borreliosis (LB), caused by *Borrelia burgdorferi* (Bb) sensu lato is the most common tick-borne disease of the northern hemisphere. Major clinical manifestations include Erythema migrans, Neuroborreliosis and Lyme arthritis. Central Europe including Germany is regarded a highly endemic area; however, data on the extent of endemicity and on risk factors are limited.

**Objectives:** Our objectives were to conduct a representative seropidemiological survey among adults in Germany to assess the seroprevalence of LB indifferent geographical regions and to identify potential risk factors for seropositivity.

**Methods:** Sera from a nationwide representative survey for adults (DEGS) (2008–2011) were analyzed by ELISA to assess the prevalence of anti-Bb IgG-antibodies in adults (18–79 years). An immunoblot was carried out as a confirmatory test. Data collected by interview were used to evaluate possible associated factors. Univariable and multivariable statistical analyses used sampling weights and accounted for the cluster structure of the survey design.

**Results:** Out of a total of 6,965 individuals 741 were seropositive. Seroprevalence in men [13.0%; 95% confidence interval (95%-CI: 11.4–14.8%)] was twice as high as in women (5.8%; 95% CI: 4.9–6.7%). Seropositivity increased from 6.0% in the 18–29-year-age-group to 20.0% in the 70–79-year-age-group without effect modification by sex. The largest increase was observed in the elderly (60–79 years). Residence in rural areas [Odds ratio (OR) = 2.50; 95% CI: 1.85–3.30] and living in Southern Germany (OR = 1.43; 95% CI: 1.08–1.88) were independent risk factors for seropositivity.

**Conclusions:** LB is endemic in all over Germany. The seroprevalence seen in the different age-groups reflects the cumulative incidence proportion. A previous study recognized children and adolescents (1–17 years) as a risk-group, this study identified elderly (60–79 years) as a second risk-group. This is compatible with recreational behavior. Alternatively, this might be explained by a birth cohort effect of a previous time frame of lower risk of seroconversion in Germany resulting in less increase in 18–59 year-old. Seropositivity is not equivalent with clinical manifestation. Thus, differences between groups may not necessarily be reflected by the real disease burden. Identified risk factors are useful for targeted campaigns. Furthermore, the knowledge on seroprevalence in the population is a basic requirement to adjust pre-test probabilities in context of clinical diagnoses.
B069 (Ep025)

Environmental proxies for tick density and human behavioral risk factors for Lyme disease infection on block Island, Rhode Island

M. A. Diuk-Wasser, C. Finch, T. Steeves, P. Shea and P. Krause

Yale School of Public Health, New Haven, CT, USA

Lyme disease, caused by the spirochete *Borrelia burgdorferi*, is the most commonly reported vector-borne disease in the US, with >20,000 cases reported annually. The black-legged tick, *Ixodes scapularis*, serves as the principal vector in maintaining the spirochete's zoonotic transmission, as well as transmission to humans. Human risk of infection with tick-borne pathogens is determined primarily by the interaction between environmental or "acarological" risk (density of infected *I. scapularis* nymphs that are actively host-seeking) and human behaviors increasing exposure risk to infected ticks. The goal of this study was to assess the relative significance of acarological risk and protective behaviors in reducing Lyme infection risk. To assess behavioral modifiers of risk, we examined data from a longitudinal cohort study of 1132 records of Block Island residents who participated in biannual serosurveys from 2005 to 2011. We identified remotely sensed landscape characteristics of people's yards, which were associated with higher density of *I. scapularis* nymphs and used these metrics as proxies for infection exposure risk. Using a negative binomial regression, we found statistically significant positive unadjusted associations between positive Lyme serology and a number of landscape metrics, hours spent outdoors, and owning a cat that spends time indoors and outdoors; using protective clothing was found to be protective. Based on these findings, we constructed a multivariate logistic regression model including wearing protective clothing (protective) and occupational exposure, hours spent in tick habitat, and the total property edge consisting of shrubs (increasing risk). Results from this study suggest that both environmental risk exposure to ticks and human behaviors should be examined in concert in risk assessment studies and to design optimal interventions.
B070 (Ep028)

Risk of Lyme borreliosis and human exposure to ticks of chosen Landscape Park
in South Poland

L. Pindel and M. Asman

*Department of Parasitology, Mediacal University of Silesia in Katowice, Katowice, Poland*

Lyme borreliosis (LB) is the most common tick-borne disease in Europe. LB is caused by spirochete *Borelia burgdorferi* sensu lato (Bbsl), which is transmitted in Europe and Poland mainly by the castorbean tick *Ixodes ricinus* (Ir). The objective of the study was to determine the risk of human exposure to ticks and Bbsl during a high tourist season on recreational areas of the Zydwiec Landscape Park, South Poland. A total of 198 (n = 198) Ir ticks were collected from picnic areas (n = 52, 26.26% ticks), hunting blind places (n = 44, 22.22%), hiking trail (n = 34, 17.17%), and forest workplace (n = 68, 34.34%) by flagging lower vegetation in June and September 2012, including females (14.14% of total), males (16.16%), nymphs (68.18%) and larvae (1.52%). Next, 60 ticks were randomly selected with equal to number of each tick stage, from the above-mentioned study places. All ticks were investigated individually. Bacterial DNA was isolated by ammonium hydroxide method. The isolates obtained from these ticks were examined for the presence of Bbsl by PCR using primers FLA1 and FLA2 specific for the fragment of fla gene sequence. Among 60 examined ticks, only one female found in "forest work place" was positive for Bbsl (1.66%). The study showed high risk of human exposure to ticks in all studied areas. Possibility of the Bbsl infection at the forest work place, where positive Bbsl was found, is higher than on other studied places, where tested ticks were negative for Bbsl. Simultaneously, we cannot ascertain that risk of Bbsl infection at the Zydwiecki Landscape Park is possibly the same at different areas not included into this study. Wherefore, these surveys should be continued taking into account other seasons of the year and other regional parks of South Poland.
B071 (Ep029)

Ecological and climatic conditions matched with human cases of *Borrelia miyamotoi* infection in Russia

Alexander E. Platonov¹,²,³,⁴,⁵, Sergei A. Bartalev¹,²,³,⁴,⁵, Anton V. Titkov¹,²,³,⁴,⁵, Denis S. Sarksyan¹,²,³,⁴,⁵, Natalya A. Makhneva¹,²,³,⁴,⁵, Olga V. Platonova¹,²,³,⁴,⁵, Nadezhda M. Kolyasnikova¹,²,³,⁴,⁵, Ludmila S. Karan¹,²,³,⁴,⁵, Durland Fish¹,²,³,⁴,⁵ and Evgeny A. Loupian¹,²,³,⁴,⁵

1. Central Research Institute of Epidemiology, Moscow, Russia

2. Space Research Institute, Moscow, Russia

3. State Medical Academy, Izhevsk, Russia

4. Municipal Clinical Hospital No. 33, Yekaterinburg, Russia

5. Yale School of Public Health, New Haven, CT, USA

The main clinical characteristics of a “new” borreliosis caused by *Borrelia miyamotoi* in Russia previously have been reported (Platonov et al., 2011). The objective of this presentation is to describe ecological conditions favoring this infection.

**Methods:** Acute *Borrelia miyamotoi* cases were identified as previously described (Platonov et al., 2011). Patients self-reported the date and location of tick bite within about 1–3 km. The ecological and climatic conditions in *B. miyamotoi* infection locations (BMIL) were estimated from satellite remote sensing and meteorological observations using the VEGA web-based monitoring system developed by the Space Research Institute.

**Results:** We analyzed 77 human cases of *B. miyamotoi* infection diagnosed in the Republic of Udmurtia in 2010–2012. All but three BMIL were between 56°N, 58°N, 52°E, and 54°E. Most BMIL were inside the area of 56°30¢ N, 57°15¢ N, 52°45¢ E, and 53°30¢ E, with the center in Izhevsk City. Although there are numerous farms and meadows in Udmurtia, this area is covered mainly by forests. BMIL were surrounded by deciduous forest (36%), coniferous forest (11%), or mixed forest (51%). Sixteen BMIL in Izhevsk were in city parks with mixed forest. Patients were bitten by *Ixodes persulcatus* ticks between May 1 and July 30 (from 18th to 27th weeks of an year) with a peak in 22nd week (18 cases). In this period, the mean daily temperature (MDT) and the Normalized Difference Vegetation Index (NDVI) varied from 5 to 25°C (median 16°) and 0.57 to 0.90 (median 0.82), respectively. Sixty seven (87%) of 77 *B. miyamotoi* cases occurred when MDT was higher than 10°C and the NDVI was more than 0.68. Only 10 of 77 days of infection occurred when precipitation was more than 0.6 g/cm² with a relative humidity >80%. We also analyzed 39 human cases of *B. miyamotoi* infection diagnosed in Sverdlovsk Region in 2009. All but two BMIL were inside the area 56°20¢N, 57°20¢N, 60°00¢E, and 61°30¢E, with the center in Yekaterinburg City. BMIL were surrounded by deciduous, mixed, or coniferous forest (7, 24, and 67%, respectively). Twelve BMIL in industrial Yekaterinburg were in city parks/forests. *B. miyamotoi* infections occurred between 19th and 28th weeks of an year with a peak in 22nd week (seven cases). The MDT varied from 3 to 26°C (median 16°C) and the NDVI from 0.55 to 0.87 (median 0.75).

**Conclusion:** Similar ecological conditions exist where *B. miyamotoi* is endemic.
B072 (Ep030)

Accuracy of reporting of Lyme disease in Connecticut

M. Vázquez¹,², M. L. Cartter¹,² and E. D. Shapiro¹,²

1. Yale School of Medicine, New Haven, CT, USA

2. Connecticut Department of Public Health, Hartford, CT, USA

Background: Connecticut has a consistently high incidence of Lyme disease. The Connecticut Department of Public Health implemented a system of active surveillance for Lyme disease in selected medical practices, but the accuracy of reporting is unknown.

Objective: To assess the accuracy of diagnosis of Lyme disease among cases reported through the active surveillance case reporting system in Connecticut.

Methods: Study patients were persons who were reported to the Connecticut Department of Public Health as having Lyme disease through an active surveillance case reporting system between January 2000 and June 2003. Information in the active surveillance case report forms was reviewed. Medical records of all subjects were also reviewed. All reported cases were classified into the following categories: definite Lyme disease, cases that met the national surveillance case definition for Lyme disease; possible Lyme disease, cases that met most criteria but were missing some information (e.g., size of the erythema migrans rash); unlikely to have Lyme disease, all others.

Results: Of the first 680 cases that were reviewed, based on the active surveillance case reports 666 (98%) had definite Lyme disease, 4 (<1%) had possible Lyme disease, and 10 (2%) were unlikely to have Lyme disease; however, based on reviews of the medical records, only 444 (65%) had definite Lyme disease, 16% had possible Lyme disease, and 19% were unlikely to have Lyme disease (*P* < 0.001).

Conclusion: We conclude that even with an active surveillance system for Lyme disease in a highly endemic area, misdiagnosis of the disease in reports to a state Department of Public Health is common.
B073 (Ep032)

The emergence of Lyme disease in China

Qiong Wu, Zhijie Liu, Jianxun Luo and Hong Yin

Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China

More than 20 years since its description, the occurrence of Lyme disease has been confirmed in 30 provinces of China. The etiologic agent has been identified and grown, molecular diagnostic methods have been developed, and the epidemiology and ecology were understood sufficiently to establish avoidance and prevention strategies. In this review, we integrate the information about recent diagnosis methods, epidemiological investigation, and disease distribution in China. It will provide the measures for the control and prevention of Lyme disease in China and to make advantageous wizard for neighboring countries and the world.
B074 (D006)

Laboratory testing for tick-borne diseases in the United States:
results of a TickNET survey

Neeta Connally¹, Alison Hinckley², James Meek J³, Katherine Feldman⁴, Melissa Kemperman⁵,
Siok-Bi Wee⁴, Jennifer White⁶ and Paul Mead²

1. Western Connecticut State University, Danbury, CT, USA

2. DVBD, CDC, Fort Collins, CO, USA

3. Connecticut Emerging Infections Program, New Haven, CT, USA

4. Maryland Department of Health and Mental Hygiene, Baltimore, MD, USA

5. Minnesota Department of Health, St. Paul, MN

6. New York State Department of Health, New York, NY, USA

Background: Little is known about current laboratory practices and testing volume for Lyme disease and other tick-borne diseases. A CDC TickNET assessment of major clinical laboratories in the United States (U.S.) was conducted to better understand laboratory practices and testing burden for eight tick-borne diseases in order to better inform public health surveillance and practice.

Methods: Paper-based surveys were sent to the 10 major commercial clinical laboratories most frequently identified in tick-borne disease surveillance records. Each laboratory was asked to report 2008 national laboratory testing volume data for eight tick-borne infections in the U.S.: Lyme disease, rocky mountain spotted fever, babesiosis, anaplasmosis, ehrlichiosis, tick-borne relapsing fever, Colorado tick fever, and Powassan virus.

Results: Seven (70%) large commercial laboratories in the U.S. reported testing volume for tick-borne diseases. All seven responding laboratories reported testing specimens for Lyme disease and babesiosis, six (86%) laboratories reported testing for anaplasmosis, ehrlichiosis, and rocky mountain spotted fever. Two (29%) laboratories reported testing for tick-borne relapsing fever, and one (14%) tested for Colorado tick fever. No commercial laboratories reported testing for Powassan virus. Responding laboratories tested 2,927,881 specimens for tick-borne infections. Tick-borne disease diagnostic tests were conducted most commonly for Lyme disease (83%), followed by ehrlichiosis (6%), rocky mountain spotted fever (5%), babesiosis (3%), anaplasmosis (2%), tick-borne relapsing fever (<1%), and Colorado tick fever (<1%).

Discussion: As Lyme and other tick-borne diseases are likely underreported by physicians in endemic states, laboratory testing data may more accurately estimate the incidence of laboratory confirmed tick-borne disease in the U.S. In addition, laboratory surveillance may present a more sustainable methodology for tracking U.S. tick-borne diseases. However, since positive tests may only indicate seropositivity, it is yet unclear how many confirmed and probable incident tick-borne disease cases could truly be ascertained through laboratory surveillance alone. Nevertheless, these results provide baseline test utilization data for continued evaluation of tick-borne disease testing trends in the United States.
B075 (D013)

Serological diagnosis of Lyme borreliosis in suspected patients in Korea, 2008–2012

Ji-Hye Eom, Seon-Do Hwang, Su-Yeon Kim, Sang-Hee Park, Kyu-Jam Hwang, MI-Yeoun Park, Yoon-Tae Noh and Young-Ran Ju

Division of Zoonoses, Center for Immunology and Pathology, Korea Centers for Disease Control and Prevention, National Institute of Health, Chungcheongbuk-do, Republic of Korea

Lyme borreliosis (LB) is a tick-borne disease caused by *Borrelia* spp. Since 2010, LB has been designated as a legal infectious disease in Korea. In this study, we collected 3,296 samples (sera and CSF) from LB suspected patients to investigate LB infection and incidence during 2008–2012 in Korea. We performed indirect immunofluorescence assay (IFA) as a screening test and Western blot as a confirmatory test. Total 1,299 (39.41%) cases were seropositive by IFA, and 32 (0.97%) cases were positive by Western blot. Western blot assay can detect IgG and IgM antibodies directed against *B. burgdorferi* using recombinant antigens of *B. burgdorferi*, *B. afzelii*, and *B. garinii* like p100, p41, p39, p18, OspC, OspA, and VlsE. The main symptoms were showed neuropathy, fever, atrophoderma, and erythema migrans. Among the positive results by Western blots, 13 cases were infected abroad and the remaining 19 cases suspected to have been infected in Korea. The isolation of *Borrelia* spp. from human has not yet been reported in Korea. However, based on cases of LB diagnosed serological diagnosis, broad and active surveillance program is necessary to prevent continuous occurrence of LB in Korea.
B076 (D018)

# Lyme neuroborreliosis is highly prevalent in tertiary care hospitals of Central-southeast Mexico

Gordillo-Perez Guadalupe, Cervantes-Castillo Almudena, Solorzano-Santos Fortino, G. Sánchez-Vaca, A. García, I. García and Torres Javier

Unidad de Investigacion Medica de Enfermedades Infecciosas, Hospital de Pediatria Centro Médico Nacional SXXI, IMSS, Mexico

Lyme disease is considered the number one disease transmitted by tick bite worldwide, and is a multisystemic disease where neurologic manifestations may cause severe disabling episodes. In 2011, the CDC reported more than 24,000 confirmed cases with neurological manifestation 14%. In Mexico, the presence of Lyme disease has been documented since 1999, infected vector and studies conducted in patients with neurologic manifestations of Guillain-Barre syndrome and facial palsy found an infection rate of 14 and 12.5% respectively. Currently, there are no reports on the frequency and diversity of neurologic cases attributed to Lyme disease in Mexico.

**Objective:** To determine the prevalence of Lyme Neuroborreliosis in tertiary care hospitals of Instituto Mexicano del Seguro Social (IMSS) attending patients from central and southeast Mexico.

**Materials and Methods:** Cross-sectional study made in the period from 1999 to 2011, and approved by the ethics committee of IMSS. Blood and cerebral spinal fluid (CSF) samples were taken from patients with neurological symptoms suggestive of Lyme disease according to the CDC criteria (ELISA and Western blot IgG+ vs. *Borrelia burgdorferi* in serum and/or positive in CSF). Results were analyzed with chi square, Fisher exact test, U Mann-Whitney, and student T test.

**Results:** We studied 650 patients and confirmed Lyme Neuroborreliosis in 132 (20.3%). The acute disseminated stage was documented in 100 (75.7%) with facial palsy (38), lymphocytic meningitis (35), poliradiculopathy 27 cases, and the chronic stage in 32 (24.3%) of the cases with encephalomyelitis. Were 91 adults (67%) aged 42 ± 14.7 years and 44 children (33%) aged 9.6 ± 5 years, and a sex ratio male: female 1:1.3. In 28/132 cases (20.7%) presented tick bite and four of the negatives OR 5.2 (95% CI: 3.2–10.6 *p* < 0.001). Among the neurological signs, predominant facial palsy unilateral, paresthesias (*p* < 0.05; OR 3.6, 95% CI: 1.39–9.49), distal weakness (*p* < 0.001; OR 14.7, 95% CI: 7–32), and pleocytosis in CSF (*p* < 0.05) were more frequent in cases.

**Conclusions:** This is the first report of Lyme neuroborreliosis in central of Latin-American, and presents as disseminated and a chronic stage. The scientist and the physician must consider that LD is distributed more extensive in America. Only the obligatory report by the Public Health ministry in Latin-America will define the real impact.
B077 (D025)

# Prevalence of tick-borne pathogens field collected ticks from the United States and Europe

Chris D. Crowder¹, Heather E. Carolan¹, Megan A. Rounds¹, Steven E. Schutzer² and Mark W. Eshoo¹

1. *Ibis Biosciences Inc.*, an Abbott Company, Carlsbad, CA, USA

2. *New Jersey Medical School, UMDNJ, Newark, NJ, USA*

**Background:** Tick-borne diseases, many of which have similar symptoms, are caused by a wide range of bacteria, protozoa, and viruses, and can be transmitted by the same vector. Active surveillance of ticks can provide health agencies with data for population risk assessments and inform physicians as to the possible pathogens patients may encounter. Performing risk-assessment and determining the prevalence of the pathogens previously required a specific test for each distinct pathogen resulting in costly and time-consuming processes, which could not always detect novel species or strains. In this ongoing long term study, we demonstrate the use of multi-locus broad-range PCR and electrospray ionization mass spectrometry assays (PCR/ESI-MS) to detect numerous pathogens as well as various strains of *Borrelia burgdorferi* from ticks across the United States and Europe.

**Methods:** We used a multi-locus broad-range PCR/ESI-MS assay designed to detect a wide range of vector-borne bacterial, viral, and protozoal pathogens. Additionally, we utilized a more specific assay for genotyping *Borrelia*. Total nucleic acids were extracted from ticks collected at various sites across North America and Europe and screened on both the assays. Electrospray ionization mass spectrometry of the PCR amplicons was used to determine their base composition. These base composition signatures were subsequently used to identify the organisms found in the samples.

**Results:** We examined field collected ticks from the United States and Europe for pathogens and potential pathogens. We found numerous pathogens present both as single infections and co-infections in these ticks including bacterial, viral, and protozoal co-infections. In instances where *Borrelia* was detected, we often got a genotype signature and in many instances could determine that multiple strains of *Borrelia* were present as a co-infection in the tick. We will show data from hundreds of ticks from various species of *Ixodes*, *Dermacentor*, and *Amblyomma* ticks.

**Conclusion:** We have demonstrated here broad range detection of tick-borne pathogens and potential pathogens PCR/ESI-MS. This has use in public health surveillance for risk assessment of potential vector-borne disease as well as potential applications for animal health and clinical diagnostics.
B078 (D040)

Investigation of Lyme disease in China during 2010–2012 diagnosis and distribution

Qiong Wu, Zhijie Liu, Jifei Yang, Youquan Li, Ze Chen, Guiquan Guan, Jianxun Luo and Hong Yin

Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Science, Beijing, China

Lyme borreliosis, caused by spirochetes belonging to the *Borrelia burgdorferi* sensu lato group, and transmitted through *Ixodes* ticks. Because there is currently no vaccine available to prevent Lyme borreliosis in humans, early diagnosis and prevention is particularly important, one should be aware of atypical presentations and be able to formulate an efficient and effective diagnosis plan. In this update, we will discuss the diagnosis of *Borrelia burgdorferi*, the epidemiology of its vectors and hosts, the transmission of Lyme disease, and the distribution of *Borrelia burgdorferi* in China during 2010–2012.
B079 (M001)

Genespecies of *Borrelia burgdorferi* in ticks of the Northeast of México

Guadalupe Gordillo-Perez¹, Margarita Vargas-Sandoval², Alexandra Brown³, Abha Grover³,
Javier Torres¹ and Maria D. Esteve-Gassent³

1. Unidad de Investigacion Medica de Enfermedades Infecciosas, Centro Medico Nacional SXXI, IMSS, Mexico

2. Facultad de Agrobiología, Universidad Michoacana, Uruapan, Mexico

3. Pathobiology Laboratory, Veterinary Faculty Texas AM, College Station, TX, USA

Lyme disease represents the most frequent tick-borne infectious disease in the United States and Europe (1). The incidence of human Lyme disease correlates with the prevalence and abundance of *Ixodes* ticks infected with *Borrelia burgdorferi* sensu lato in the region (2). *Ixodes scapularis* and *I. pacificus* are the competent vectors of *B. burgdorferi* in the US but, Recently other species of infected ticks are reported in Texas and the Northeast of Mexico. Since 1999, we reported a seroprevalence of 1.1% to *B. burgdorferi* in general population of Mexico with prevalence of 6.4% in the states of the Northeast border with Texas, where we also reported the presence of *Ixodes* ticks infected with *B. burgdorferi* sensu stricto. Clinical cases with cutaneous and neurological manifestation reported exclusively in Europe have been reported in Mexico, but unknown if the species of *B. burgdorferi* is more diverse that reported in North America.

Objective: In this study we aimed to search the genospecies of *B. burgdorferi* that infected ticks of the Northeast of Mexico.

Materials and Methods: We selected *Ixodes* ticks preserved in ethanol of a collection of Emerging Infectious Disease laboratory, UIMEIP, Centro Medico Nacional SXXI, IMSS. The ticks were collected on mammals and plants from 18 forest and rural localities of Tamaulipas and Nuevo Leon, border states with Texas during the period 1990–2007. Infections were determined in extracted DNA by PCR amplification with the Fla B, OspA and intergenic 23S genes for *B. burgdorferi*. The positives ticks were sequencing to genotyping.

Results: Were positive for *B. burgdorferi* 36 (10.2%) ticks of eleven localities near of three big cities. All bacteria were identified how *B. b.* sensu stricto with homology of 96–100%. The infected ticks were identified as *I. scapularis* 4 (1.1%), *Dermacentor andersoni* 4 (1.1%), *Amblyomma americanum* 2 (0.6%) *Rhipicephalus sanguineus* 14 (4%), *Amblyomma cajennense* (11 3.1%), and *Boophylus microplus* (1.1%).

Conclusions: *B. burgdorferi* sensu stricto was prevalent in ticks of the Northeast of Mexico. *R. sanguineus* and *A. cajennense* were the ticks frequently infected, although its role as a competent vector is unknown. Is possible that potential vectors are more diverse in Mexico and Texas. The physician must consider Lyme disease in the differential diagnosis from these endemic areas.
B080 (M011)

Investigation of the *Borrelia* content in ticks that bites humans

Peter Wilhelmsson¹, Pontus Lindblom¹, Linda Fryland², The TBD STING-Study Group, Christina Ekerfelt², Pia Forsberg³,⁴ and Per-Eric Lindgren¹

1. Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden

2. Division of Clinical Immunology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden

3. Division of Infectious Diseases, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden

4. Clinic of Infectious Diseases, University Hospital, Linköping, Sweden

Lyme borreliosis (LB) risk assessments and epidemiology has previously been studied by investigating *Borrelia* prevalence in field-collected ticks. However, more accurate risk assessments and epidemiology would be achieved if ticks that actually bite humans were investigated. The risk of acquiring a LB infection increases with time of tick feeding. However, the knowledge about the impact of spirochetal load in a feeding tick is limited. Therefore the present tick-borne diseases (TBD) Sting study aimed to explore the prevalence and phylogeography of *Borrelia* spirochetes in *Ixodes ricinus* collected from newly tick-bitten humans from highly endemic areas for LB and from areas with unknown LB status in Sweden and at the Åland Islands, Finland. Ticks were identified morphologically to determine species, life stage, sex, and estimated feeding-time by using aUSB-microscope. The ticks were homogenized and total nucleic acids were extracted. cDNA was analyzed with a LUX real-time PCR assay to detect and quantify the *Borrelia* 16S rRNA gene. *Borrelia* species in positive samples were determined by a nested, conventional PCR assay using primers targeting the non-coding intergenic spacer regions (IGS) 5S–23S or the 16S–23S IGS rRNA genes, respectively. A total of 2,154 ticks, including 15 adult males, 496 adult females, 1,510 nymphs, 87 larvae were collected. All ticks were determined as *Ixodes ricinus*, except for 46 undetermined ticks. Approximately one-fourth of the collected ticks harbored *Borrelia* spirochetes. Adult ticks had a higher *Borrelia* prevalence (36%) compared to nymphs (25%, *p* < 0.001). No *Borrelia* was detected in any of the larvae. PCR sequencing identified seven different *Borrelia* species with *Borrelia afzelii* as the predominant species in the collected ticks followed by *Borrelia garinii*, *Borrelia valaisiana*, *Borrelia burgdorferi* sensu stricto (s.s.), *Borrelia miyamotoi*, *Borrelia spielmanii*, and *Borrelia lusitaniae*. The median number of spirochetes per tick ranged was 1.6 ´ 103. Ticks that were infected with *B. miyamotoi* harbored more spirochetes (median of 2.1 ´ 105) than ticks infected with *B. afzelii*, *B. garinii*, *B. valaisiana*, and *B. burgdorferi* s.s., among ticks that had fed for ~ 24 h. In conclusion, one-fourth of the ticks that had bitten humans in Sweden and at Åland Islands were infected with *Borrelia*. The *Borrelia* species *B. spielmanii*, *B. lusitaniae* and *B. miyamotoi* were, to our knowledge, detected for the first time in ticks detached from humans, in Sweden and at the Åland Islands.
B081 (M017)

Molecular detection and identification of *Borrelia burgdorferi* sensu lato
in rodents in Slovenia

T. Cerar, U. Glinšek Biškup, M. Kodre, N. Knap, M. Korva, T. Avšič-Županc, E. Ružić-Sabljić

Faculty of Medicine, Institute of Microbiology and Immunology, University of Ljubljana, Ljubljana, Slovenia

Objective: *Borrelia burgdorferi* sensu lato, the agent of Lyme borreliosis, is mainly maintained in natural foci through transmission cycles of competent tick vectors and vertebrate reservoir. Specific rodents were identified as the principal reservoir of *Borrelia burgdorferi* sensu lato in Europe. In the present study, molecular epidemiological survey was performed to determine the presence of *Borrelia burgdorferi* sensu lato in rodents present in seven different geographic area of Slovenia.

Materials and Methods: A total of 386 rodents were live-trapped. After identification of species, organs were removed and stored at -80°C until tested. DNA from lung was isolated and the presence of *B. burgdorferi* sensu lato DNA was determined using two different PCRs, “in house” nested and commercial real-time PCR (LightMix® Kit for detection of *Borrelia* spp., TIB MOLBIOLGMBh, Germany) both targeting ospA gene. Identification of *Borrelia* species was done by melting temperature (Tm) analysis included in LightMix® protocol and by sequencing of PCR product of “in house” PCR.

Results: Borrelial DNA was detected in 41/386 (10.6%) and 13/386 (3.4%) lungs with LightMix® and "in house" PCR, respectively. Concordance of both tests was 93%. Prevalence of *Borrelia burgdorferi* sensu lato infected rodents differed regarding to particular geographic region and ranged from 0 to 30% according to LightMix® and 0-15% according to "in house" PCR. Identification according to Tm using LightMix® revealed the presence of *Borrelia afzelii* or *Borrelia valaisiana*, while analysis of Tm does not distinguish between these two species. Sequencing of "in house" PCR products revealed the presence of *B. afzelii*, only.

Conclusion: Prevalence of with *Borrelia burgdorferi* sensu lato infected rodents depends on geographic region and used molecular test; it ranges from 0 to 30%. LightMix® was found to be more sensitive in comparison to “in house” PCR. Although LightMix® does not allow distinction of *B. afzelii* and *B. valaisiana*, sequencing of “in house” PCR products of ospA gene reveals presence of *B. afzelii* only, which is in concordance with the results from the literature.
B082 (M018)

Molecular typing of flab gene of a *Borrelia garinii* 935T isolated from *Ixodes persulcatus* in Korea

Yoon-Tae Noh, Su-Yeon Kim, Ji-Hye Eom, Young-Ran Ju and Kyu-Jam Hwang

Division of Zoonoses, Center for Immunology and Pathology, Korea Centers for Disease Control and Prevention, National Institute of Health, Chungcheongbuk-do, Republic of Korea

Lyme borreliosis (LB) is a tick-borne zoonotic disease caused by genus *Borrelia*. *Borrelia burgdorferi* sensu lato complex, the main pathogen group of the disease, is composed of 11 species including *B. burgdorferi* sensu stricto, *Borrelia afzelii*, *Borrelia garinii*, *Borrelia andersonii*, *Borrelia bissenttii*, *Borrelia joponica*, *Borrelia valaisiana*, *Borrelia lusitaniae*, *Borrelia tanukii*, *Borrelia turdi*, and *Borrelia sinica*. Genome of *B. burgdorferi* sensu lato has one linear chromosome and 17–21 plasmids. Differences in genetic features 16S rRNA, ospA, flaB, noncoding genome rrfA-rrlB, and rrs-rrlA for intergenic regions have been used for distinct identification of *Borrelia* species. flaB encoding flagellin is a major antigen inducing strong IgG and IgM immune responses in early infection. Because flaB has a genetic variation, it has been considered as a target gene for identification of *Borrelia* species by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using HpyF31 and Ecl36II restriction enzymes. In Korea, *B. garinii* 935T strain was isolated from *Ixodes* persulcatus in 1993. The protein profiles and reactivities to monoclonal antibodies for FlaB, OspA and OspB were analyzed in this strain. In this study, flaB of the strain was amplified by PCR with 220f/823r primers as previously described and then the amplified DNA was sequenced. The result of PCR-RFLP of flaB clustered V pattern by Beata Wodecka (2011). We will further investigate the characterization of other virulence factors, which may lead to a better understanding 935T strain.
B083 (M025)

Borrelia burgdorferi isolated from *Peromyscus maniculatus* and *Myodes gapperi*
in Eastern North Dakota

Brandee L. Stone, Angela M. Floden and Catherine A. Brissette

Department of Microbiology and Immunology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND, USA

Eastern North Dakota is traditionally viewed as a non-endemic area for Lyme disease, though the emergence of confirmed Lyme disease cases among residents suggests eastern North Dakota is minimally a transition zone for *Borrelia burgdorferi* and *Ixodes scapularis*. Indeed, based upon the isolation of infected nymphal *Ixodes scapularis* ticks, Diuk-Wasser et al. (*Am J Trop Med Hyg* 2012 86:320-7), described eastern North Dakota as a transitional zone. In our study, five spirochetes, based on cell morphology, were isolated from the hearts of *Peromyscus maniculatus* and *Myodesgapperi* around the Turtle River and Forest River areas of Grand Forks, ND. The spirochetes underwent not more than six passages *in vitro* before genetic analyses were performed. Sequence analysis of 16S, flaB, ospA, ospC, p66, and the 16S-23S intergenic spacer region (IGS) indicates the spirochetes are *B. burgdorferi*. 16S sequences (ca. 136 nt) aligned to multiple *Borrelia* spp. using the Ribosomal Database Project website. BLAST results and alignments of ca. 40 residues of flaB showed 100% identity to multiple *B. burgdorferi* strains. p66 showed 100% sequence identity across 239 residues to *B. burgdorferi* B31. Two of the five spirochetes were negative for ospA despite multiplex PCR results showing all possessed the plasmid carrying ospA. Multiplex PCR for 13 linear and 9 circular plasmids revealed a unique complement of linear and circular plasmids among the isolates, suggesting either the arrival of multiple strains of *B. burgdorferi* or rapid divergence of a single strain. All five isolates are missing linear plasmid 25, suggesting they have a low-infectivity phenotype. Future work includes alignment of our eastern North Dakota *B. burgdorferi* sequences with *B. burgdorferi* from western Minnesota to determine directionality and evolutionary history of *B. burgdorferi* in eastern North Dakota; collection of *Ixodes* ticks and comparisons of *B. burgdorferi* to those found in local small mammals; and infection studies to determine the infectivity phenotype of the local isolates. In total, our results confirm that *B. burgdorferi* is present in the small mammal population of eastern North Dakota, further corroborating the eastern progression of *B. burgdorferi*.
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