Emerging Tick-borne Infections in the Upper Midwest and Northeast United States Among Patients With Suspected Anaplasmosis

Abstract Background Emerging tick-transmitted illnesses are increasingly recognized in the United States (US). To identify multiple potential tick-borne pathogens in patients from the Upper Midwest and Northeast US with suspected anaplasmosis, we used state-of-the-art methods (polymerase chain reaction [PCR] and paired serology) to test samples from patients in whom anaplasmosis had been excluded. Methods Five hundred sixty-eight patients without anaplasmosis had optimal samples available for confirmation of alternative tick-borne pathogens, including PCR and/or paired serology (acute-convalescent interval ≤42 days). Results Among 266 paired serology evaluations, for which the median acute-convalescent sampling interval was 28 (interquartile range, 21–33) days, we identified 35 acute/recent infections (24 [9%] Borrelia burgdorferi; 6 [2%] Ehrlichia chaffeensis/Ehrlichia muris subsp eauclairensis [EC/EME]; 3 [1%] spotted fever group rickettsioses [SFGR], and 2 [<1%] Babesia microti) in 33 (12%) patients. Two had concurrent or closely sequential infections (1 B burgdorferi and EC/EME, and 1 B burgdorferi and SFGR). Using multiplex PCR and reverse-transcription PCR, we identified 7 acute infections (5/334 [1%] Borrelia miyamotoi and 2/334 [1%] B microti) in 5 (1%) patients, including 2 with B microti–B miyamotoi coinfection, but no Borrelia mayonii, SFGR, Candidatus Anaplasma capra, Heartland virus, or Powassan virus infections. Thus, among 568 patients with ruled-out anaplasmosis, 38 (6.7%) had ≥1 agent of tick-borne illness identified, with 33 patients (35 infections) diagnosed by paired serology and 5 additional patients (7 infections) by PCR. Conclusions By identifying other tick-borne agents in patients in whom anaplasmosis had been excluded, we demonstrate that emerging tick-borne infections will be identified if specifically sought.

First identified in 1990, Anaplasma phagocytophilum causes human granulocytic anaplasmosis (HGA or anaplasmosis), presents as a nonspecific acute febrile illness, and is transmitted by the deer or black-legged tick Ixodes scapularis [1].Ixodes scapularis, the most common tick vector in the United States (US), causes tick-borne diseases in the Upper Midwest (UM) and Northeast (NE) US.However, novel agents, changing epidemiologic patterns, and additional vectors have been discovered when explicitly sought.We report a range of tickborne illnesses that mimic anaplasmosis when suspected.

Patients and Samples
We studied archived specimens from 568 patients residing in the Upper Midwest (UM) and Northeast (NE) US who presented May 1991-July 2009 with suspected anaplasmosis.Although additional clinical and laboratory information was provided for a subset of patients (85 from the UM), etiologic diagnoses were not.Specimens included 266 paired sera for antibody testing and 343 whole blood specimens for polymerase chain reaction (PCR) analysis.We excluded those with A phagocytophilum infection confirmed by ≥1 Centers for Disease Control and Prevention (CDC) criteria: 4-fold immunoglobulin G (IgG) titer rise by indirect fluorescent antibody (IFA) between acute and convalescent (paired) sera, PCR positive, isolation from blood, and acute-phase blood smear positive [2].We used available sera and DNA from blood (archived at −20°C) and/ or ethylenediaminetetraacetic acid (EDTA)-anticoagulated Tick-borne Febrile Illness in the US • OFID • 1 Open Forum Infectious Diseases M A J O R A R T I C L E blood (preserved at −80°C) to diagnose acute tick-borne infections by paired serology and PCR (multiplex DNA PCR [mPCR] or reverse-transcription RNA PCR [RT-PCR]).Our primary analysis included those with optimal (13-42 days) convalescent sampling; others were included in a secondary analysis.We tested for Babesia microti, Borrelia burgdorferi, Borrelia mayonii, Borrelia miyamotoi, Candidatus Anaplasma capra, Ehrlichia chaffeensis (EC)/Ehrlichia muris subsp eauclairensis (EME), Heartland virus, Powassan virus, and spotted fever group rickettsioses (SFGR).Testing positive control sample aliquots stored at −20°C for similar durations regularly confirmed continued seroreactivity.

Borrelia spp and Babesia microti Enzyme-Linked Immunosorbent Assay
We used enzyme-linked immunosorbent assay (ELISA) to test for Borrelia spp (C6 peptide ELISA) and B microti (Immunetics, Boston, Massachusetts).Based on consideration of a 4-fold antibody rise as "significant" and that optical density (OD) can estimate antibody quantity, we applied a statistical approach to define a significant rise in OD by ELISA [3].Those specimens above a plate-to-plate normalized OD cutoff were considered positive (Lyme index value [C6IV] >0.9 or B microti signal-to-noise cutoff ratio [SCR] ≥1.6 [4]), as per the manufacturer.Paired sera in which the convalescent serum was positive and the convalescent minus acute-phase OD was greater than mean +3 standard deviations of the OD change in paired-negative samples were considered to have a significant C6IV or SCR increase akin to a 4-fold rise in titer [3].

B burgdorferi Western Blot
We used a 2-tier test to confirm acute/recent B burgdorferi infection.Since C6 peptide ELISAs cannot distinguish B burgdorferi from B miyamotoi or B mayonii [5,6], we tested available convalescent-phase sera by B burgdorferi IgG/immunoglobulin M (IgM) Western blot (WB) (MarDx/Trinity Biotech, Wicklow, Ireland) if either serum was C6 peptide ELISA positive.

Indirect Fluorescent Antibody for B mayonii
Sera with significant C6IV antibody rises were examined for acute/recent B mayonii infection if a B burgdorferi WB was negative or not done.Borrelia mayonii was cultivated in BSK-II medium to supply antigen for IgG-IFA.Positive controls included immune mouse serum and 2 human anti-B.mayonii immune sera (courtesy Martin Schriefer and Jeannine Petersen, CDC, Fort Collins, Colorado).Borrelia burgdorferi cross-reactivity was assessed using sera from patients with culture-and serologically confirmed Lyme disease.Specificity tests used sera from uninfected mice; B miyamotoi, SFGR, EC, and A phagocytophilum infection; and healthy subjects.Sera were tested by B mayonii IFA at a 1:80 dilution; those with acute-phase titers ≤80 and convalescent-phase titers ≥80 were considered seroconversions.

IFA for Ehrlichia spp
We tested for EC and EME by IgG IFA [7] (cultivating EC and EME in DH82 and RF/6A cells, respectively) [8].Sera reactive at the 1:80 dilution were titrated to ≥2560.Those with 4-fold IgG antibody rises to ≥160 for EC and 10 EC IgG-negative controls were tested for EME.A ≥2-fold higher convalescent titer distinguished acute EME from EC.

IFA for SFGR
Paired sera were screened at 1:80 by IgG IFA using acetonefixed Rickettsia parkeri (Portsmouth strain)-infected human brain microvascular endothelial cells on multiwell glass slides.Reactive samples were titrated to 2560.

mPCR to Detect Tick-borne Pathogens
We adapted our mPCR assay including SFGR (sca0) and EC [9] to include B microti (18S rRNA gene) [10], B miyamotoi, B mayonii, and Candidatus A capra (gltA).The EC trp32 (vlpt) PCR also detected EME; thus, results were categorized as EC/ EME.We used glpQ PCR for B miyamotoi with primers and probes common among B miyamotoi strains, but not other relapsing fever Borrelia, and adapted oppA PCR for B mayonii [11] by comparing oppA sequences for B burgdorferi sensu stricto and sensu lato strains.Candidatus A capra gltA PCR was adapted [12] to mPCR.AlleleID, IDT PrimerQuest, or NCBI Primer-BLAST were used to design assays that uniquely identified B microti, B miyamotoi, B mayonii, and Candidatus A capra.For RNA viruses, we targeted the RNA-dependent RNA polymerase gene of Powassan/deer tick virus (POWV; isolate DTV-MN-2008; HM991145.1)and the small segment nonstructural protein gene of the Bandavirus Heartland virus (HRTV).All primer/probe combinations were optimized for default parameters so that multiplexing had minimal impact on analytical sensitivity and retained high analytical specificity (Supplementary Table 1).
The QIAsymphony Midi DNA blood kit was used to extract DNA from 1 mL of EDTA-anticoagulated blood (final volume 200 µL).RNA was extracted from 200 µL archived acute-phase serum using the ZR-96 Viral RNA kit (Zymo Research).
We used a Bio-Rad CFX 384 Multicolor Real Time PCR instrument (default parameters, 40 cycles) for mPCR with duplicate or triplicate reactions.Single-step reverse-transcription mPCR (RT-PCR) was performed in quadruplicate; 2 wells received reverse transcriptase and 2 did not.All reactions included known positive, negative, and no-template controls and cloned amplicon quantitation standards to assure analytical sensitivity (≤10 copies/µL), linearity (R 2 > 0.95), and efficiency (between 0.70 and 1.20).We used human ACTB as an amplification control in mPCR and RT-PCR assays; ACTB-negatives were repeated once.Positives were required to be in duplicate.
No reproducible false positives were demonstrated among specificity controls (Supplementary Table 1).

Reference Case Definitions
Acute/recent infections were confirmed by paired serology and/or PCR.Acute/recent B burgdorferi infection required (1) a positive convalescent C6IV result with a significant C6IV antibody rise or a high stable C6IV (both acute and convalescent C6IV in the top 50th percentile) and ( 2) a positive convalescent-phase B burgdorferi IgG and/or IgM WB.Acute/recent B mayonii infection required a significant C6IV antibody rise, a negative convalescent B burgdorferi IgG/IgM WB, and B mayonii IgG IFA seroconversion.We defined other acute/recent tick-borne infections as a significant SCR antibody increase (B microti) [13]; 4-fold IgG IFA rise (EC/EME or SFGR); a positive B miyamotoi, EC/EME, SFGR, Candidatus A capra, or B microti mPCR; or a positive POWV or HRTV RT-PCR.Seropositives without acute/recent infection were classified as past tick-borne infections.For Borrelia, those unconfirmed by WB were classified as possible acute/recent or past Borrelia based on the presence or absence, respectively, of a significant C6IV antibody rise.

Summary of Acute Tick-borne Infections Identified
In total, 38 of 568 (7%) patients with suspected anaplasmosis in whom that etiology was ruled out had ≥1 agent of acute tick-borne illness identified, including 33 patients (35 infections) diagnosed by paired serology and 5 patients (7 infections) by PCR.These 42 acute/active infections included 24 B burgdorferi, 4 B microti, 5 B miyamotoi, 6 EC/EME, and 3 SFGR (Table 2).Four (10%) had paired serologic or molecular evidence of concurrent or closely sequential infections with distinct tick-borne pathogens.

Correlation of Paired Serology and mPCR
Of 41 patients tested by both paired serology and mPCR, 4 (10%) had evidence of acute/recent infection; none were confirmed by both serology and PCR.Acute/recent infection was confirmed by paired serology in 3 patients (1 B burgdorferi, 1 EC/EME, and 1 SFGR) and by mPCR in 1 (B miyamotoi and B microti).

DISCUSSION
Tick-transmitted infections in the US are increasingly recognized [14,15].Molecular tools have revolutionized taxonomy, identified new pathogens, and improved epidemiologic understanding of pathogens.Investigation of atypical presentations of SFGR or typical presentations in new regions led to the identification of E chaffeensis in 1986, A phagocytophilum in 1990, Ehrlichia ewingii in 1999, EME in 2009, and Candidatus A capra in China in 2015 [7,12,16].Lyme disease, caused by B burgdorferi sensu stricto, is the most frequently reported tick-borne disease in the US; 2 other Borrelia species, B miyamotoi and B mayonii, are now reported as causes of relapsing fever-or Lyme disease-like illnesses in the US, respectively [5,17].Babesia microti cases increased 4-to 20-fold in the last decade [14].Tick-borne viruses, including Powassan and deer tick viruses, can cause fever and severe neurologic illness in the NE and UM [18].In 2012, a new tick-borne virus, "Heartland" Bandavirus, was identified in >60 patients in the US [19]; the tick vector and disease distribution in the US remains understudied [20].Recent identification of new Rickettsia spp, with known and unknown pathogenicity, complicates testing, reporting, and epidemiologic study [21,22].
We used state-of-the-art methods (paired serology and PCR) to test patients living in tick-borne disease-endemic areas with suspected anaplasmosis for other tick-borne agents after excluding anaplasmosis.Importantly, this approach can (1) identify previously undetected pathogens (eg, Candidatus A capra), (2) better define the date of pathogen introduction in an area (eg, B miyamotoi), and/or (3) determine the relative frequency of tick-borne infections in a specific population.
An alternative tick-borne infection (B burgdorferi, EC/EME, B microti, B miyamotoi, and SFGR) was identified in 38 of 568 patients on primary analysis, and 51 on secondary analysis (Supplementary Tables 2 and 3).This is an important proof-of-concept that if extensive testing is performed in patients at high risk for tick-borne infection with a compatible clinical syndrome, a definitive diagnosis can be achieved in a small but nontrivial percentage.Furthermore, it provides additional data that between 1991 and 2009, tick-borne diseases were underdiagnosed in a high-prevalence setting.The substantial number of patients with Borrelia C6 peptide antibodies in patients for whom WB was not positive could represent incomplete evolution of B burgdorferi antibody response, B miyamotoi or B mayonii infections, unknown infections, or, less likely, false positives [5,17].We omitted B burgdorferi PCR, since it is generally insensitive due to transient low-level bacteremia [23].In contrast, we applied B mayonii PCR since that pathogen often achieves higher-level bacteremia; however, we did not detect any confirmed infections by PCR or serology [5].
We identified evidence of acute/recent, simultaneous or closely sequential infections in 4 (10%) patients; 2 were PCR positive for both B microti and B miyamotoi and 2 had paired serology consistent with rapid succession or simultaneous B burgdorferi and EC/EME (1) or SFGR (1) infection.Coinfections with Ixodes tick-transmitted agents (eg, B burgdorferi, B miyamotoi, B mayonii, B microti, and EME) are biologically plausible and documented [24], but presumed to be infrequent [25].A Borrelia C6 peptide-positive result in a B miyamotoi PCR-positive patient could reflect B miyamotoi alone, since Borrelia spp, like SFGR, stimulate cross-reactive antibodies [17].The coinfection prevalence of 10% (95% confidence interval, 4%-22%) is consistent with other studies [25,26].Since patients with anaplasmosis were excluded from the study, we likely underestimate the number with coinfection.
To decrease misclassification of sequential infections as coinfections and past infections as acute, we used reference standard definitions, including a 4-fold rise in IgG antibody titer by IFA and/or positive mPCR, to confirm acute SFGR and Ehrlichia infections, and our primary analysis included those with optimal (≤42 days) convalescent sampling.The problem of resolving coinfections versus rapidly sequential infections is illustrated by "coinfections" of B burgdorferi, transmitted by I scapularis, with EC and SFGR that are transmitted largely by Amblyomma americanum and Dermacentor variabilis, respectively, with overlapping distributions [27].Although historically rare in both regions, SFGR are plausible as single infections, since the most common vector, D variabilis, is well-documented in both regions [28].Although also plausible, SFGR are not known to transmitted by I scapularis tick bites.As precedent, transmission of Rickettsia australis from Ixodes holocyclus is accepted but not proven [29], and human SFGR infection from other Ixodes spp, such as Rickettsia monacensis and Rickettsia helvetica from Ixodes ricinus, is controversial [30,31].Moreover, since PCR for SFGR is poorly sensitive [9], cases here were identified only by serology.The acute SFGR infections here occurred during a 2-decade national trend of increased reporting [32], which may reflect infections with less pathogenic species such as R parkeri, subclinical infections, immune stimulation triggered by nonpathogenic spotted fever group tick endosymbionts [22], or immune stimulation rather than increased incidence or reporting of Rickettsia rickettsii infection (Rocky Mountain spotted fever) in much of the US [33].Since most reported SFGR cases are based on testing single sera [32] and many healthy individuals have detectable antibody [34], we rigorously tested paired sera to confirm acute SFGR infections.
Likewise, since A americanum, the EC vector, was infrequent in the UM when these samples were obtained, serologic reactivity in samples from the UM could reflect EME infection.Examples include 2 patients from the UM, 1 with an EC 4-fold titer increase from <80 to 320 concurrent with a ≥16-fold EME titer increase from <80 to 1280, and another with at least a 2-fold EME titer increase (1280 to ≥2560) but stable EC titers of 160.The lack of EME serologic responses in the NE is consistent with its apparent restriction to the UM.However, although EC infection is now established in the NE [35], A americanum and EC were rarely identified there during the interval of this study.
Using molecular tools, we sought evidence for 2 new Borrelia spp (B mayonii, only identified in the UM, and B miyamotoi), a new Ehrlichia spp (EME, only found in the UM), a novel Candidatus Anaplasma spp (identified in Asia and recently in Europe [12,36]), and 2 tick-borne viruses emerging in the US [37].We would expect B miyamotoi to cause anaplasmosislike acute febrile illness in the UM and NE [38]; we identified cases in the NE US between 1995 and 1997, more than a decade prior to the first description of B miyamotoi in humans in Russia and subsequently in the US [38][39][40].We found that B miyamotoi comprised 17% of the clearly identified Borrelia infections.Although PCR on blood for POWV and HRTV is rarely done for those without encephalitis, these infections could have antecedent or accompanying viremia.That we did not identify POWV or HRTV in our 1991-2009 cohorts suggest these viruses were not unrecognized causes of illness in our study population.Similarly, Candidatus A capra infections, a novel form of human anaplasmosis not previously identified as a cause of infections in humans in the US, was not found using PCR.Unavailability of control samples precluded determination of clinical sensitivity for new molecular assays; however, analytical sensitivity at ≤10 genomic copies/µL and clinical specificity >90%-95% appeared high.
Limitations for this study include the small number of individual diagnoses for several key infections identified in this 1991-2009 cohort, the lack of consensus for considering some serologic tests positive [41], the inability to validate the statistically plausible ELISA-based estimation of 4-fold antibody rise for the C6 peptide and B microti assays, the unknown duration of illness before acute-phase sampling, the lack of PCR testing for B burgdorferi and insufficient sera to test all by B burgdorferi WB, the lack of specific PCR and serologic assays to differentiate EC from EME, difficulties diagnosing specific SFGRs even with gold-standard paired IFA, and the unavailability of robust B miyamotoi and B mayonii serological tests to discriminate them from B burgdorferi (which might be overcome by using antigens more unique to B miyamotoi).Other infectious agents that emerged since 2009 include Anaplasma bovis in humans, which might have been detected if an Anaplasma genus-level nucleic acid amplification test were used.Similarly, if more recent samples had been available, it is possible that the pathogens sought might have been detected more often.Importantly, this study highlights the imperfect state-of-the-art regarding diagnosis of tick-borne infections: (1) serology is intrinsically nonspecific, since human immune responses vary greatly and cannot differentiate some closely related organisms, and (2) PCR-based tools, though highly sensitive and specific for detecting some pathogens (eg, A phagocytophilum, B microti, and B miyamotoi) early after illness onset, may be less sensitive later in illness.Furthermore, PCR assays for other pathogens, especially for B burgdorferi sensu stricto and SFGR, remain insensitive due to low-level bacteremia.Currently, newer technologies that employ relatively unbiased approaches for identifying unsuspected pathogens, such as metagenomics, need further optimization and validation [42].However, such approaches offer promise for identifying additional novel tick-borne infections that mimic anaplasmosis.
In conclusion, after excluding those with anaplasmosis, we used reference standard testing (paired serology and/or PCR) to identify other tick-borne infections in 38 of 568 patients.Although most diagnoses were accomplished with paired serology, our results suggest that PCR and paired serology are complementary for the detection of acute/recent tick-borne infections.Since the epidemiologic landscape of tick-borne infections in the US has changed considerably recently, these data may not be generalizable to 2023-2024.However, the findings are highly relevant, since the spectrum of tick-borne pathogens has only increased, and limitations in state-of-the-art testing approaches remain major impediments to accurate diagnosis.Thus, epidemiologic and clinical studies using improved and state-of-the-art assays will be required to define the breadth and relative frequency of emerging tick-borne agents in the US as well as the identification and ecology of relevant vectors.Prospective studies to validate and extend these findings should be performed to improve clinical understanding and management of tick-borne infections, discern trends in tick-borne disease epidemiology, and to search for novel agents.support and Gary P. Wormser for supportive and detailed discussion and advice about data interpretation.
Patient consent.The institutional review boards of Johns Hopkins Medicine (Baltimore, Maryland), Duluth Clinic/St Mary's Hospital (Duluth, Minnesota), St Luke's Hospital (Duluth, Minnesota), New York

Table 2 . Results of Testing 568 Patients by Paired Serology (Convalescent Sample ≤42 Days; n = 266 Paired Sera) and by Multiplex Polymerase Chain Reaction (PCR) (n = 343 Acute-Phase Blood DNA) and Reverse-Transcription PCR (n = 233 Acute-Phase Serum RNA for Viral Reverse-Transcription PCR) for Tick-borne Infections That Mimic Anaplasmosis
Includes C6 positive tests in either acute or convalescent serum sample.cIncludes only paired sera with a significant increase in C6IV from acute to convalescent sample.Includes 7 patients with positive acute and convalescent C6 peptide enzyme-linked immunosorbent assay (ELISA) results consistent with stable high titer that were confirmed by Borrelia burgdorferi Western blot; n = 230 because Western blot could not be performed in 7 C6 peptide ELISA-positives with significant increase in C6IV and 29 without significant increase in C6IV antibodies.
b d

Table 3 . Agents of Tick-borne Disease Identified by Paired Serology and/or Polymerase Chain Reaction From Patients With Suspected Anaplasmosis
Tick-borne Febrile Illness in the US • OFID• 7Medical College (Valhalla, New York), and New York State Department of Health (Albany, New York) reviewed and either approved the research or declared the research exempt.Disclaimer.The opinions expressed herein are those of the author(s) and are not necessarily representative of those of the Uniformed Services University of the Health Sciences; the Department of Defense; or the US Army, Navy, or Air Force.The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.Financial support.This work and testing were supported by a Fisher Center Discovery Program (https://hopkinsinfectiousdiseases.jhmi.edu/research/research-areas/environmental-id/fisher-center-discovery-programgrants/)award to M. E. R. Additional laboratory support was provided through the National Institute of Allergy and Infectious Diseases (grant numbers R01 AI044102 and R21 AI09606) to J. S. D.Potential conflicts of interest.All authors: No reported conflicts.