Philip J. Molloy

ASSOCIATION OF ANTIBIOTIC TREATMENT-RESISTANT LYME ARTHRITIS WITH T CELL RESPONSES TO DOMINANT EPITOPES OF OUTER SURFACE PROTEIN A OF BORRELIA BURGDORFERI

OBJECTIVE To explore further the association of antibiotic treatment-resistant Lyme arthritis and T cell reactivity with outer surface protein A (OspA) of Borrelia burgdorferi, including the identification of T cell epitopes associated with this treatment-resistant course. METHODS The responses of peripheral blood and, if available, synovial fluid lymphocytes to B burgdorferi proteins, fragments, and synthetic peptides, as determined by proliferation assay and interferon-gamma production, were compared in 16 patients with treatment-responsive and 16 with treatment-resistant Lyme arthritis. RESULTS The maximum severity of joint swelling correlated directly with the response to OspA. Moreover, the only significant difference between patients with treatment-resistant and treatment-responsive arthritis was in reactivity with N-terminal and C-terminal fragments of OspA, OspA1 (amino acids [aa] 16-106), and OspA3 (aa 168-273). Epitope mapping showed that 14 of the 16 patients with treatment-resistant arthritis had responses to OspA peptides (usually 4 or 5 epitopes), whereas only 5 of the 16 patients with treatment-responsive arthritis had reactivity with these peptides (usually 1 or 2 epitopes) (P = 0.003). Patients with HLA-DRB1 alleles associated with treatment-resistant arthritis were more likely to react with peptide 15 (aa 154-173) and, to a lesser degree, with peptide 21 (aa 214-233) than patients with other alleles, whereas the responses to other epitopes were similar in both groups. CONCLUSION The maximum severity of joint swelling and the duration of Lyme arthritis after antibiotic treatment are associated with T cell responses to specific epitopes of OspA.

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

Context The first known cases of Borrelia miyamotoi infection in North America were reported in 2013. The incidence, prevalence, and clinical spectrum of this infection are currently undefined. Contribution A review of the clinical records of 51 of 97 patients with acute B. miyamotoi infection diagnosed by polymerase chain reaction testing at a reference laboratory provides preliminary data on the clinical presentation, course, and response to antibiotics of this infection. Caution The study was not prospective or population-based. Implication Borrelia miyamotoi disease may be an emerging tickborne infection of clinical significance in the northeastern United States. The public health burden of infectious agents transmitted by the deer tick (Ixodes dammini, also known as the blacklegged tick or I. scapularis), comprising Borrelia burgdorferi, Babesia microti, Anaplasma phagocytophilum, and deer tick virus (Powassan virus lineage II), seems to be increasing in the northeastern United States (14). The first human cases of infection with Borrelia miyamotoi, previously considered nonpathogenic, were identified in Russia and described in 2011 (5, 6). In 2013, the American index case of B. miyamotoi disease (BMD) was described (7), and 2 other case patients (8) were reported with a clinical illness that was initially diagnosed as human anaplasmosis. Cases have subsequently been identified in New England (9), the Netherlands (10), and Japan (11). The prevalence of B. miyamotoi in host-seeking deer ticks (about 1% to 5% [1214]) is such that human exposure is likely; indeed, a serosurvey suggests that 10% of tick-exposed New England residents may have been exposed to B. miyamotoi (15). It is possible that zoonotic transmission has been common but not discriminated from other deer ticktransmitted zoonoses because the acute clinical presentation of BMD cases reported to date is nonspecific. To better define the clinical spectrum of this newly recognized zoonosis, we identified definitive cases of acute BMD by polymerase chain reaction (PCR) analysis of patient blood samples sent to a reference laboratory by clinical practices. We summarized the clinical features of BMD from 51 case patients out of 97 whom we identified as having active B. miyamotoi infection. In addition, we describe relevant associated laboratory findings and compare the observed frequency of BMD with that of human anaplasmosis and babesiosis, thereby providing a preliminary assessment of its relative public health burden. Methods Blood Samples Emergency departments, urgent care clinics, and primary care offices in Massachusetts, Rhode Island, New Jersey, and New York sent whole blood samples (EDTA-anticoagulated) to IMUGEN (Norwood, Massachusetts) as part of the clinical management of patients who were acutely symptomatic with features or laboratory findings suggestive of possible tickborne infection (typically fever, myalgia, flu-like illness, headache, or rash). An active detection protocol was adopted to identify samples that contained evidence of B. miyamotoi DNA during the 2013 and 2014 transmission seasons (1 April to 30 November). A positive B. miyamotoi finding on PCR prompted a request for chart review, and we focused on patients under our care with a goal of obtaining detailed clinical histories on at least 50 patients. The study was approved by the New England Institutional Review Board, and written, informed consent was obtained for clinical and laboratory follow-up of patients with positive PCR findings for Borrelia species. Molecular Detection of Infection Specimen receipt and handling, DNA extraction, and PCR setup were performed with enhanced contamination control practices and precautions at IMUGEN, which operates under good laboratory practice standards, Clinical Laboratory Improvement Amendments and College of American Pathologists certification, and New York State approval (Clinical Laboratory Evaluation Program). The laboratory has environmentally separate specimen processing, archive, and molecular extraction and setup areas. Extracted DNA from whole blood was tested in triplicate for the presence of Borrelia species by real-time PCR (Appendix) (16). Real-time PCR targeting the msp2 gene of A. phagocytophilum and the 18S ribosomal RNA gene of B. microti was performed from whole blood DNA extractions as previously described (1720). Representative samples were independently analyzed by PCR sequencing at the Telford laboratory of Tufts University (Appendix). B. burgdorferi Serology Sera were tested at IMUGEN by antibody capture enzyme immunoassay (EIA) for IgM, IgA, and IgG isotypes to B. burgdorferi sensu stricto strain 49736 and by immunoblot for IgM and IgG isotypes to B. burgdorferi sensu stricto strain G39/40 (2123). Immunoblot findings were interpreted according to the 2-tiered protocol (24). Recombinant B. miyamotoi GlpQ Enzyme-Linked Immunosorbent Assay The glycerophosphodiester phosphodiesterase (GlpQ) antigen has been found to be useful in the serodiagnosis of relapsing fever (25, 26). Given the genetic relatedness of B. miyamotoi to relapsing fever spirochetes, we analyzed the utility of GlpQ for confirmation of a BMD diagnosis. The GlpQ gene sequence from B. miyamotoi (GenBank accession number AY368276) was used as the basis for cloning and expression as a 38-kDa recombinant protein (rGlpQ), which was used in an indirect EIA for detection of antibody to B. miyamotoi (Appendix). Case Definitions A positive blood sample on 2 independent PCR assays (genus- and species-specific) was considered definitive evidence of active B. burgdorferi or B. miyamotoi infection. A blood sample that was repeatedly positive (reextracted and retested from the original sample) for B. microti or A. phagocytophilum on PCR was considered definitive evidence of active infection. Role of the Funding Source Internal funding from IMUGEN enabled the development and validation of test methods, the study design and execution, the clinical analysis, and the laboratory studies. The decision to prepare a manuscript for publication was not predicated on the availability of funding or the funding source. Results Confirmation of B. miyamotoi as the Infecting Agent We attempted to sequence portions of the GlpQ and flagellin genes, as previously described (7), from the 23 BMD cases from 2013 for which we had adequate remaining blood samples for DNA extraction. The organism was confirmed to be B. miyamotoi (GenBank accession numbers KP754938 to KP754960) in 17 samples (GlpQ, fla, or both sequences were obtained from the sample). The other 6 samples contained few spirochetes; those with high cycle threshold values in the real-time GlpQ PCR did not provide sequence (Spearman rank correlation coefficient, 0.67 [P< 0.001]). Frequency of BMD Between 1 April 2013 and 31 October 2014, we engaged in active case detection using specimens submitted to IMUGEN and identified 97 patients whose samples contained B. miyamotoi DNA. Of these, we obtained clinical information on 51 who had not been previously reported (7, 8) from selected practices in Massachusetts, Rhode Island, New Jersey, and New York. The months of onset or diagnosis of these BMD cases (Figure 1) overlapped with those for the other deer ticktransmitted infections, although most cases had onset in July and August. Figure 1. Seasonal distribution of acute Borrelia miyamotoi incident infections. The months of acute blood collection are presented for 97 samples submitted in 20132014 that were determined by active case detection or retrospectively to contain B. miyamotoi DNA. To provide a crude estimate of the incidence of BMD, we compared the frequency of PCR-confirmed findings of B. burgdorferi, B. miyamotoi, B. microti, and A. phagocytophilum in the patient samples submitted during the peak transmission months (May through October) in 2013 and 2014. Of 11515 unique samples that were submitted and tested for all 4 agents, 3.1% (exact binomial 95% CI, 2.7% to 3.4%) contained B. microti DNA, 1.4% (CI, 1.2% to 1.6%) contained A. phagocytophilum DNA, and 2.5% (CI, 2.2% to 2.8%) contained Borrelia species DNA. Borrelia miyamotoi DNA was detected in 0.8% (CI, 0.6% to 1.0%) of all samples; hence, 1.7% (CI, 1.5% to 1.9%) of all samples contained B. burgdorferi or cognate sequences that were not assigned to species. Clinical Spectrum of BMD Basic clinical data were available for 51 case patients with definitive BMD selected out of 97 total. The clinical spectrum of disease was variable, but presenting symptoms were often suggestive of an undifferentiated flu-like illness. Patients presented with acute headache, fever, and chills and were often found to have leukopenia, thrombocytopenia, and elevated aminotransferase levels, mimicking human anaplasmosis infection (Tables 1 and 2). Patients were commonly described as appearing toxic; more than 50% were suspected of having sepsis, and 24% required hospitalization. The headaches were most commonly described as severe, resulting in head computed tomography scans and spinal taps in 5 patients. Two patients presented with recurrent fever, and 1 of them, who was not treated initially, yielded blood samples drawn a month apart that were positive on PCR. Table 1. Clinical Features of the 51 Case Patients With BMD Table 2. Laboratory Findings for the 51 Case Patients With BMD Treatment regimens were typical for suspected tickborne disease in our referral area practices. Of the 51 case patients, 40 received a 2- to 4-week course of oral doxycycline. Seven of 51 received oral amoxicillin, and 3 of these had received 1 or 2 doses of ceftriaxone beforehand. One of 51 received levofloxacin for 10 days. One case patient first received atovaquone and azithromycin for a laboratory-confirmed diagnosis of babesiosis and later received ceftriaxone after the results of laboratory assays were reported. Of the 42 patients for whom information on outcome was available, 40 had prompt resolution of

Investigational screening for Babesia microti in a large repository of blood donor samples from nonendemic and endemic areas of the United States.

Babesia microti, a transfusion‐transmissible intraerythrocytic parasite, is increasing in frequency in the United States with no available FDA‐licensed donor screening assay. We utilized investigational arrayed fluorescence immunoassay (AFIA) and polymerase chain reaction (PCR) to detect B. microti antibodies and DNA in blood donors.

Borrelia miyamotoi Disease: Neither Lyme Disease Nor Relapsing Fever.

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

Detection of Multiple Reactive Protein Species by Immunoblotting after Recombinant Outer Surface Protein A Lyme Disease Vaccination

Laboratory confirmation of the diagnosis of Lyme disease is based on the detection of an immune response to Borrelia burgdorferi. The serodiagnosis of B. burgdorferi infection is complex and may be further confounded by the immune response to the recombinant outer surface protein A (OspA) Lyme disease vaccine. To describe how the serological response to the recombinant OspA Lyme disease vaccine affects testing for antibody to B. burgdorferi, 240 specimens from 80 study subjects were obtained at defined intervals after recombinant OspA Lyme disease vaccination. Samples were tested by indirect enzyme-linked immunosorbent assay (ELISA), antibody capture enzyme immunoassay (EIA), and Western blotting (WB). After recombinant OspA Lyme disease vaccination, ELISA for 98% of the study subjects revealed reactivity. WB with use of OspA-containing B. burgdorferi strains as sources of antigens demonstrated multiple bands. Results of testing with a US Food and Drug Administration-approved WB kit showed homogeneous reactivity in the molecular weight region >30 kDa. Testing with OspA-free strains completely eliminated all vaccine-associated reactivity by both antibody capture EIA and WB.

Zoonotic Babesia microti in the northeastern U.S.: Evidence for the expansion of a specific parasite lineage

The recent range expansion of human babesiosis in the northeastern United States, once found only in restricted coastal sites, is not well understood. This study sought to utilize a large number of samples to examine the population structure of the parasites on a fine scale to provide insights into the mode of emergence across the region. 228 B. microti samples collected in endemic northeastern U.S. sites were genotyped using published Variable number tandem repeat (VNTR) markers. The genetic diversity and population structure were analysed on a geographic scale using Phyloviz and TESS, programs that utilize two different methods to identify population membership without predefined population data. Three distinct populations were detected in northeastern US, each dominated by a single ancestral type. In contrast to the limited range of the Nantucket and Cape Cod populations, the mainland population dominated from New Jersey eastward to Boston. Ancestral populations of B. microti were sufficiently isolated to differentiate into distinct populations. Despite this, a single population was detected across a large geographic area of the northeast that historically had at least 3 distinct foci of transmission, central New Jersey, Long Island and southeastern Connecticut. We conclude that a single B. microti genotype has expanded across the northeastern U.S. The biological attributes associated with this parasite genotype that have contributed to such a selective sweep remain to be identified.