Borrelia miyamotoi Infections in Humans and Ticks, Northeastern China.

We conducted an investigation of Borrelia miyamotoi infections in humans and ticks in northeastern China. Of 984 patients reporting recent tick bites, 14 (1.4%) were found to be infected with B. miyamotoi by PCR and genomic sequencing. The 14 patients had nonspecific febrile manifestations, including fever, headache, anorexia, asthenia, and arthralgia. Rash, eschar, and regional lymphadenopathy were each observed in 1 patient. Four (28.6%) patients were hospitalized because of severe disease. B. miyamotoi was detected in 3.0% (19/627) of Ixodes persulcatus, 1 (2.8%) of 36 Haemaphysalis concinna, and none of 29 Dermacentor silvarum ticks. Phylogenetic analyses based on sequences of a nearly entire 16s rRNA gene, a partial flagellin gene, and the glycerophosphodiester phosphodiesterase gene revealed that B. miyamotoi identified in patients and ticks were clustered in the group of the Siberian type. These findings indicate that B. miyamotoi is endemic in northeastern China and its public health significance deserves further investigation.

In Vitro Antimicrobial Susceptibility of Clinical Isolates of Borrelia miyamotoi.

Borrelia miyamotoi is an emerging relapsing fever (RF) Borrelia species that is reported to cause human disease in regions in which Lyme borreliosis is endemic. We recently showed that B. miyamotoi tick isolates are resistant to amoxicillin in vitro; however, clinical isolates have not been studied. Therefore, our aim was to show the antimicrobial susceptibility of recently obtained clinical isolates of B. miyamotoi A dilution series of various antibiotics was made in modified Kelly-Pettenkofer medium with 10% fetal calf serum. The susceptibilities of different B. miyamotoi clinical, B. miyamotoi tick, RF Borrelia, and Borrelia burgdorferisensu lato isolates were tested by measuring MICs through colorimetric changes and by counting motile spirochetes by dark-field microscopy after 72 h of incubation. The ceftriaxone and azithromycin MIC ranges of the six B. miyamotoi clinical isolates tested were 0.03 to 0.06 mg/liter and 0.0016 to 0.0032 mg/liter, respectively. These values are similar to MICs for RF Borrelia strains and B. miyamotoi tick isolates. All tested RF Borrelia strains were susceptible to doxycycline (microscopic MIC range, 0.0625 to 0.25 mg/liter). In contrast to the MICs of the tested B. burgdorferi sensu lato strains and in line with our previous findings, the amoxicillin MICs (range, 8 to 32 mg/liter) of all RF Borrelia strains, including B. miyamotoi clinical isolates, were above the clinical breakpoint for resistance (≤4 mg/liter). Clinical isolates of B. miyamotoi are highly susceptible to doxycycline, azithromycin, and ceftriaxone in vitro Interestingly, as described previously for tick isolates, amoxicillin shows poor in vitro activity against B. miyamotoi clinical isolates.

Case control study: Serological evidence that Borrelia miyamotoi disease occurs nationwide in Japan.

Since 2011, Borrelia miyamotoi disease (BMD) has been reported in five countries in the northern hemisphere. The causative agent of BMD is transmitted by Ixodes ticks, which are also vectors of Lyme disease borreliae. In this study, we examined 459 cases of clinically suspected Lyme disease (LD group), and found twelve cases that were seropositive for the glycerophosphodiester phosphodiesterase (GlpQ) antigen derived from B. miyamotoi. The retrospective surveillance revealed that the seroprevalence of anti-GlpQ in the LD group was significantly higher than in a healthy cohort. Seropositive cases were observed from spring through autumn when ticks are active, and the cases were geographically widespread, being found in Hokkaido-Tohoku, Kanto, Chubu, Kinki, and Kyushu-Okinawa regions. Seropositive cases for GlpQ were most frequent in the Chubu region (6.3%) where B. miyamotoi has been found in Ixodes ticks. Out of the twelve cases that were found in the LD group, three cases exhibited concomitant seropositivity to Lyme disease borreliae by western blot assay. This is the first report of serological surveillance for BMD in Japan, and we conclude that BMD occurs nationwide.

In Vitro Susceptibility of the Relapsing-Fever Spirochete Borrelia miyamotoi to Antimicrobial Agents.

Hard-tick-borne relapsing fever (HTBRF) is an emerging infectious disease throughout the temperate zone caused by the relapsing-fever spirochete Borrelia miyamotoi Antibiotic treatment of HTBRF is empirically based on the treatment of Lyme borreliosis; however, the antibiotic susceptibility of B. miyamotoi has not been studied to date. Thus, we set out to determine the in vitro antimicrobial susceptibility of B.miyamotoi A microdilution method with 96-well microtiter plates was used to determine the antibiotic susceptibilities of two B.miyamotoi strains isolated on two different continents (Asia and North America), two Borrelia burgdorferisensu lato strains, and one Borrelia hermsii isolate for purposes of comparison. The MIC and minimal bactericidal concentration (MBC) were determined by both microscopy and colorimetric assays. We were able to show that relative to the B. burgdorferi sensu lato isolates, both B.miyamotoi strains and B. hermsii demonstrated greater susceptibility to doxycycline and azithromycin, equal susceptibility to ceftriaxone, and resistance to amoxicillin in vitro The MIC and MBC of amoxicillin for B. miyamotoi evaluated by microscopy were 16 to 32 mg/liter and 32 to 128 mg/liter, respectively. Since B. miyamotoi is susceptible to doxycycline, azithromycin, and ceftriaxone in vitro, our data suggest that these antibiotics can be used for the treatment of HTBRF. Oral amoxicillin is currently used as an alternative for the treatment of HTBRF; however, since we found that the B. miyamotoi strains tested were resistant to amoxicillin in vitro, this issue warrants further study.

Statewide surveillance of Ixodes scapularis (Acari: Ixodidae) for the presence of the human pathogen Borrelia miyamotoi (Spirochaetales: Spirochaetaceae), a relapsing fever spirochete in Pennsylvania, USA, 2019-2020.

Borrelia miyamotoi disease is an emerging tick-borne human illness in the United States caused by Borrelia miyamotoi (Spirochaetales: Spirochaetaceae) bacterium. With Pennsylvania reporting thousands of tick-borne disease cases annually, determining the minimum infection rate (MIR) of B. miyamotoi in Ixodes scapularis (Say, Acari: Ixodidae) adults within Pennsylvania is of utmost importance. Active surveillance was performed from October 2019 to April 2020 to collect a minimum of 50 I. scapularis ticks from every county within Pennsylvania and then screened for B. miyamotoi via qPCR. Ticks were collected from all 67 counties with the majority of those being adult I. scapularis. Additional ticks collected were Dermacentor albipictus (Packard, Acari: Ixodidae), Haemaphysalis longicornis (Neumann, Acari: Ixodidae), and immature I. scapularis. Adult I. scapularis were pooled and tested for B. miyamotoi. MIR for positive B. miyamotoi pools and density of infected adult I. scapularis varied by county, with positive pools from 38 Pennsylvania counties. This is the first statewide evaluation of B. miyamotoi in Pennsylvania in questing adult I. scapularis. These prevalence and distribution data will aid health care practitioners within the state of Pennsylvania and the northeast United States to understand potential risk and bring awareness to the lesser known human Borrelia illness, Borrelia miyamotoi disease.

Spatiotemporal distribution of Borrelia miyamotoi (Spirochaetales: Spirochaetaceae) and coinfection with other tick-borne pathogens in host-seeking Ixodes scapularis (Acari: Ixodidae) from New York State, USA.

Blacklegged ticks (Ixodes scapularis Say, Acari: Ixodidae) were collected from 432 locations across New York State (NYS) during the summer and autumn of 2015-2020 to determine the prevalence and geographic distribution of Borrelia miyamotoi (Spirochaetales: Spirochaetaceae) and coinfections with other tick-borne pathogens. A total of 48,386 I. scapularis were individually analyzed using a multiplex real-time polymerase chain reaction assay to simultaneously detect the presence of Bo. miyamotoi, Borrelia burgdorferi (Spirochaetales: Spirochaetaceae), Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), and Babesia microti (Piroplasmida: Babesiidae). Overall prevalence of Bo. miyamotoi in host-seeking nymphs and adults varied geographically and temporally at the regional level. The rate of polymicrobial infection in Bo. miyamotoi-infected ticks varied by developmental stage, with certain co-infections occurring more frequently than expected by chance. Entomological risk of exposure to Bo. miyamotoi-infected nymphal and adult ticks (entomological risk index [ERI]) across NYS regions in relation to human cases of Bo. miyamotoi disease identified during the study period demonstrated spatial and temporal variation. The relationship between select environmental factors and Bo. miyamotoi ERI was explored using generalized linear mixed effects models, resulting in different factors significantly impacting ERI for nymphs and adult ticks. These results can inform estimates of Bo. miyamotoi disease risk and further our understanding of Bo. miyamotoi ecological dynamics in regions where this pathogen is known to occur.

Borrelia miyamotoi: A Comprehensive Review.

Borrelia miyamotoi is an emerging tick-borne pathogen in the Northern Hemisphere and is the causative agent of Borrelia miyamotoi disease (BMD). Borrelia miyamotoi is vectored by the same hard-bodied ticks as Lyme disease Borrelia, yet phylogenetically groups with relapsing fever Borrelia, and thus, has been uniquely labeled a hard tick-borne relapsing fever Borrelia. Burgeoning research has uncovered new aspects of B. miyamotoi in human patients, nature, and the lab. Of particular interest are novel findings on disease pathology, prevalence, diagnostic methods, ecological maintenance, transmission, and genetic characteristics. Herein, we review recent literature on B. miyamotoi, discuss how findings adapt to current Borrelia doctrines, and briefly consider what remains unknown about B. miyamotoi.

Development and Validation of a Protein Array for Detection of Antibodies against the Tick-Borne Pathogen Borrelia miyamotoi.

Current serological tests for the emerging tick-borne pathogen Borrelia miyamotoi lack diagnostic accuracy. To improve serodiagnosis, we investigated a protein array simultaneously screening for IgM and IgG reactivity against multiple recombinant B. miyamotoi antigens. The array included six B. miyamotoi antigens: glycerophosphodiester phosphodiesterase (GlpQ), multiple variable major proteins (Vmps), and flagellin. Sera included samples from cases of PCR-proven Borrelia miyamotoi disease (BMD), multiple potentially cross-reactive control groups (including patients with culture-proven Lyme borreliosis, confirmed Epstein-Barr virus, cytomegalovirus, or other spirochetal infections), and several healthy control groups from regions where Ixodes is endemic and regions where it is nonendemic. Based on receiver operating characteristic (ROC) analyses, the cutoff for reactivity per antigen was set at 5 µg/mL for IgM and IgG. The individual antigens demonstrated high sensitivity but relatively low specificity for both IgM and IgG. The best-performing single antigen (GlpQ) showed a sensitivity of 88.0% (95% confidence interval [CI], 78.9 to 93.5) and a specificity of 94.2% (95% CI, 92.7 to 95.6) for IgM/IgG. Applying the previous published diagnostic algorithm-defining seroreactivity as reactivity against GlpQ and any Vmp-revealed a significantly higher specificity of 98.5% (95% CI, 97.6 to 99.2) but a significantly lower sensitivity of 79.5% (95% CI, 69.3 to 87.0) for IgM/IgG compared to GlpQ alone. Therefore, we propose to define seroreactivity as reactivity against GlpQ and any Vmp or flagellin which resulted in a comparable sensitivity of 84.3% (95% CI, 74.7 to 90.8) and a significantly higher specificity of 97.9% (95% CI, 96.9 to 98.7) for IgM/IgG compared to GlpQ alone. In conclusion, we have developed and validated a novel serological tool to diagnose BMD that could be implemented in clinical practice and epidemiological studies. IMPORTANCE This paper describes the protein array as a novel serological test for the diagnosis of Borrelia miyamotoi disease (BMD), by reporting the methodology, the development of a diagnostic algorithm, and its extensive validation. With rising numbers of ticks and tick bites, tick-borne diseases, such as BMD, urgently deserve further societal and medical attention. B. miyamotoi is prevalent in Ixodes ticks across the northern hemisphere. Humans are exposed to, and infected by, B. miyamotoi and develop BMD in Asia, in North America, and to a lesser extent in Europe. However, the burden of infection and disease remains largely unknown, due to the noncharacteristic clinical presentation, together with the lack of awareness and availability of diagnostic tools. With this paper, we offer a novel diagnostic tool which will assist in assessing the burden of disease and could be implemented in clinical care.

Borrelia miyamotoi-An Emerging Human Tick-Borne Pathogen in Europe.

Borrelia miyamotoi is classified as a relapsing fever spirochete. Although B. miyamotoi is genetically and ecologically distinct from Borrelia burgdorferi sensu lato, both microorganisms are transmitted by the same Ixodes tick species. B. miyamotoi was detected in I. persulcatus ticks in 1994 in Japan. A phylogenetic analysis based on selected sequences of B. miyamotoi genome revealed genetic differences between isolates from Asia, North America, and Europe, which are clearly separated into three genotypes. Symptomatic human cases of Borrelia miyamotoi disease (BMD) were first reported in 2011 in Russia and then in North America, Europe, and Asia. The most common clinical manifestation of BMD is fever with flu-like symptoms. Several differences in rare symptoms (thrombocytopenia, monocytosis, cerebrospinal fluid pleocytosis, or symptoms related to the central nervous system) have been noted among cases caused by Asian, European, and American types of B. miyamotoi. BMD should be considered in the diagnosis of patients after tick bites, particularly with meningoencephalitis, without anti-Borrelia antibodies in the cerebrospinal fluid. This review describes the biology, ecology, and potential of B. miyamotoi as a tick-borne pathogen of public health concern, with particular emphasis on Europe.

Borrelia miyamotoi infection leads to cross-reactive antibodies to the C6 peptide in mice and men.

OBJECTIVES: Borrelia miyamotoi is a relapsing fever Borrelia, transmitted by hard (Ixodes) ticks, which are also the main vector for Borrelia burgdorferi. A widely used test for serodiagnosis of Lyme borreliosis is an enzyme immunoassay (EIA) based on the C6 peptide of the B. burgdorferi sl VlsE protein. We set out to study C6 reactivity upon infection with B. miyamotoi in a large well-characterized set of B. miyamotoi disease (BMD) patient sera and in experimental murine infection. METHODS: We performed in silico analyses, comparing the C6-peptide to immunodominant B. miyamotoi variable large proteins (Vlps). Next, we determined C6 reactivity in sera from mice infected with B. miyamotoi and in a unique longitudinal set of 191 sera from 46 BMD patients. RESULTS: In silico analyses revealed similarity of the C6 peptide to domains within B. miyamotoi Vlps. Cross-reactivity against the C6 peptide was confirmed in 21 out of 24 mice experimentally infected with B. miyamotoi. Moreover, 35 out of 46 BMD patients had a C6 EIA Lyme index higher than 1.1 (positive). Interestingly, 27 out of 37 patients with a C6 EIA Lyme index higher than 0.9 (equivocal) were negative when tested for specific B. burgdorferi sl antibodies using a commercially available immunoblot. CONCLUSIONS: We show that infection with B. miyamotoi leads to cross-reactive antibodies to the C6 peptide. Since BMD and Lyme borreliosis are found in the same geographical locations, caution should be used when relying solely on C6 reactivity testing. We propose that a positive C6 EIA with negative immunoblot, especially in patients with fever several weeks after a tick bite, warrants further testing for B. miyamotoi.

Characterization of a Borrelia miyamotoi membrane antigen (BmaA) for serodiagnosis of Borrelia miyamotoi disease.

Borrelia miyamotoi is a tick-borne pathogen that causes Borrelia miyamotoi disease (BMD), an emerging infectious disease of increasing public health significance. B. miyamotoi is transmitted by the same tick vector (Ixodes spp.) as B. burgdorferi sensu lato (s.l.), the causative agent of Lyme disease, therefore laboratory assays to differentiate BMD from Lyme disease are needed to avoid misdiagnoses and for disease confirmation. We previously performed a global immunoproteomic analysis of the murine host antibody response against B. miyamotoi infection to discover antigens that could serologically distinguish the two infections. An initial assessment identified a putative lipoprotein antigen, here termed BmaA, as a promising candidate to augment current research-based serological assays. In this study, we show that BmaA is an outer surface-associated protein by its susceptibility to protease digestion. Synthesis of BmaA in culture was independent of temperature at either 23 °C or 34 °C. The BmaA gene is present in two identical loci harbored on separate plasmids in North American strains LB-2001 and CT13-2396. bmaA-like sequences are present in other B. miyamotoi strains and relapsing fever borrelia as multicopy genes and as paralogous or orthologous gene families. IgM and IgG antibodies in pooled serum from BMD patients reacted with native BmaA fractionated by 2-dimensional gel electrophoresis and identified by mass spectrometry. IgG against recombinant BmaA was detected in 4 of 5 BMD patient serum samples as compared with 1 of 23 serum samples collected from patients with various stages of Lyme disease. Human anti-B. turicatae serum did not seroreact with recombinant BmaA suggesting a role as a species-specific diagnostic antigen. These results demonstrated that BmaA elicits a human host antibody response during B. miyamotoi infection but not in a tested group of B. burgdorferi-infected Lyme disease patients, thereby providing a potentially useful addition for developing BMD serodiagnostic tests.

Assessment of Borrelia miyamotoi in febrile patients and ticks in Alsace, an endemic area for Lyme borreliosis in France.

BACKGROUND: Borrelia miyamotoi is a relapsing fever Borrelia species transmitted by ticks of the Ixodes ricinus complex. Human disease caused by B. miyamotoi was first described in Russia and later in the USA and Japan. Additionally, five cases of meningoencephalitis in immunocompromised patients and one case in an apparently immunocompetent patient were described. METHODS: We investigated the presence of B. miyamotoi in I. ricinus nymphs and in patients suspected of human granulocytic anaplasmosis, in Alsace (France), an endemic area for I. ricinus ticks and Lyme borreliosis, using direct (PCR) and indirect diagnosis (glycerophosphoryldiester-phosphodiesterase (GlpQ) serology). RESULTS: Borrelia miyamotoi was found in 2.2% of 4354 ticks collected between 2013 and 2016. None of the 575 blood samples, collected from the patients suspected of HGA, was found positive for B. miyamotoi by PCR. Acute and late sera from 138 of these 575 patients were available. These paired sera were tested for IgM and IgG antibodies against the B. miyamotoi GlpQ antigen. A total of 14 out of 138 patients had at least one positive parameter (i.e. anti-GlpQ IgG and/or IgM). One patient seroconverted for IgG, and three had isolated IgM in the acute serum. These three patients were treated with doxycycline which could have prevented seroconversion. After reviewing clinical data and other biological tests performed, co-exposure among different microorganisms vectored by ticks or serological cross-reactivity could not be ruled out in these different cases. One patient had persistent IgG, which strongly suggests previous exposure to B. miyamotoi. CONCLUSIONS: Humans can be exposed to B. miyamotoi through tick bites in Alsace. We present serological data for possible B. miyamotoi exposure or infection of patients with fever after tick bite. Future studies should determine the incidence, clinical course and burden of this emerging tick-borne disease in other parts of Western Europe.

Evaluation of Disease Causality of Rare Ixodes ricinus-Borne Infections in Europe.

In Europe, Ixodes ricinus ticks transmit pathogens such as Borrelia burgdorferi sensu lato and tick-borne encephalitis virus (TBEV). In addition, there is evidence for transmission to humans from I. ricinus of Anaplasma phagocytophilum, Babesia divergens, Babesia microti, Babesia venatorum, Borrelia miyamotoi, Neoehrlichia mikurensis, Rickettsia helvetica and Rickettsia monacensis. However, whether infection with these potential tick-borne pathogens results in human disease has not been fully demonstrated for all of these tick-borne microorganisms. To evaluate the available evidence for a causative relation between infection and disease, the current study analyses European case reports published from 2008 to 2018, supplemented with information derived from epidemiological and experimental studies. The evidence for human disease causality in Europe found in this review appeared to be strongest for A. phagocytophilum and B. divergens. Nonetheless, some knowledge gaps still exist. Importantly, comprehensive evidence for pathogenicity is lacking for the remaining tick-borne microorganisms. Such evidence could be gathered best through prospective studies, for example, studies enrolling patients with a fever after a tick bite, the development of specific new serological tools, isolation of these microorganisms from ticks and patients and propagation in vitro, and through experimental studies.

Borrelia miyamotoi Serology in a Clinical Population With Persistent Symptoms and Suspected Tick-Borne Illness.

Eighty-two patients seeking consultation for long-term sequalae after suspected tick-borne illness were consecutively tested for Borrelia miyamotoi antibodies using a recombinant glycerophosphodiester phosphodiesterase (GlpQ) enzyme immunoassay. Twenty-one of the 82 patients (26%) tested positive on the GlpQ IgG ELISA. Nearly all of the patients (98%) had no prior B. miyamotoi testing, indicating that clinicians rarely test for this emerging tick-borne pathogen. Compared to patients who solely tested positive for Lyme disease antibodies, patients with B. miyamotoi antibodies presented with significantly more sleepiness and pain. A prospective study is needed to ascertain the relationship between the presence of B. miyamotoi antibodies and persistent symptoms.

Serodiagnosis of Borrelia miyamotoi disease by measuring antibodies against GlpQ and variable major proteins.

OBJECTIVES: Borrelia miyamotoi disease (BMD) is an emerging tick-borne disease in the Northern hemisphere. Serodiagnosis by measuring antibodies against glycerophosphodiester-phosphodiesterase (GlpQ) has been performed experimentally but has not been extensively clinically validated. Because we had previously shown the differential expression of antigenic variable major proteins (Vmps) in B. miyamotoi, our aim was to study antibody responses against GlpQ and Vmps in PCR-proven BMD patients and controls. METHODS: We assessed seroreactivity against GlpQ and four Vmps in a well-described, longitudinal cohort of sera from BMD patients (n=182), healthy blood donors (n=136) and controls (n=68). All samples were tested by ELISA and positive sera were tested by western blot, and antibody dynamics and diagnostic value were assessed. RESULTS: IgM antibodies against GlpQ and Vmps peaked between 11 and 20 days, and IgG between 21 and 50 days, after disease onset. Various combinations of GlpQ and Vmps increased sensitivity and/or specificity compared to single antigens. Notably, the GlpQ or variable large protein (Vlp)-15/16 combination yielded a sensitivity of 94.7% (95% CI: 75.4-99.7) 11-20 days after disease onset and a specificity of 96.6% (92.7-98.4) for IgM. A specificity of 100% (97.8-100) for IgM, and 98.3% for IgG (95.2-100), was found when positivity was defined as reactivity to GlpQ and any Vmp, with maximum sensitivities of 79% (56.7-91.5) for IgM and 86.7% (62.1-97.6) for IgG. CONCLUSIONS: We clearly demonstrate here the diagnostic potential of these seromarkers. Our findings will facilitate future epidemiological and clinical studies on BMD and lead to the development of a serologic test to be used in clinical practice.

Development and optimization of an in vitro cultivation protocol allows for isolation of Borrelia miyamotoi from patients with hard tick-borne relapsing fever.

OBJECTIVES: Borrelia miyamotoi has been shown to infect humans in Eurasia and North America causing hard tick-borne relapsing fever (HTBRF). In vitro cultivation of B. miyamotoi was described recently; but clinical isolation of relapsing fever Borrelia is cumbersome. Our aim was to develop a straightforward protocol enabling B. miyamotoi isolation directly from the blood of patients. METHODS: Modified Kelly-Pettenkorfer (MKP-F) medium, with or without anticoagulants, or blood from healthy human volunteers, was spiked with B. miyamotoi spirochaetes in vitro. Subsequently, either media or plasma was used for cultivation directly, or after an additional centrifugation step. This isolation protocol was tested in a clinical setting on patients suspected of HTBRF. RESULTS: Dipotassium-EDTA, trisodium citrate and lithium heparin inhibited growth of B. miyamotoi at concentrations ≥250 µg/mL, 2.5 mM and 1 IU/mL, respectively. However, when plasma originating from human blood containing B. miyamotoi spirochaetes was subjected to an additional centrifugation step at 8000 g, suspended and inoculated into fresh MKP-F media, positive cultures were observed within 2 weeks. Of importance, this straightforward protocol allowed for isolation of B. miyamotoi from six out of nine patients with confirmed HTBRF. CONCLUSIONS: Direct culture from K2-EDTA, trisodium citrate and lithium heparin plasma containing B. miyamotoi is hampered due to anticoagulants. Using a simple centrifugation protocol we were able to circumvent this detrimental effect, allowing for the first clinical isolation of B. miyamotoi. This will be of value for future research on the pathogenesis, genetics, diagnosis, therapy and epidemiology of HTBRF and other tick-borne relapsing fevers.