Borrelia miyamotoi, discovered in Japan in 1995, belongs to the relapsing fever group of Borrelia ( 1 ). Relapsing fever borreliae infections are characterized by influenza-like illness and >1 relapse episode of bacteremia and fever. B. miyamotoi is more distantly related to B. burgdorferi, a group of spirochetes that includes B. burgdorferi s.l. strains (B. afzelii; B. garinii; and B. burgdorferi s.s., the causative agent of Lyme disease) ( 2 , 3 ). In Eurasia and North America, B. miyamotoi is found in a small percentage of all species of ixodid tick vectors of B. burgdorferi, including Ixodes persulcatus ( 1 , 3 , 4 ), I. ricinus ( 5 – 7 ), I. scapularis ( 2 , 3 , 8 , 9 ), and I. pacificus ( 10 ). It is transmitted transovarially and transstadially by ticks and coexists with B. burgdorferi ( 2 , 3 ). Recently, we discovered B. miyamotoi in I. persulcatus and I. ricinus ticks in the European and Asian regions of Russia. In these areas, human ixodid tick-borne infections, including those caused by B. afzelii, B. garinii, and viral tick-borne encephalitis virus (TBEV; genus Flavivirus) are endemic and transmitted by the same tick species. Despite the presence of B. miyamotoi in vector ticks, to our knowledge, human disease caused by this spirochete has not been definitively established. We previously noted presumptive B. miyamotoi infection in residents of central Russia with influenza-like illness but were uncertain whether their clinical manifestations were caused by co-infecting B. burgdorferi s.l. species ( 11 – 13 ). To confirm those findings and develop initial estimates of the prevalence and severity of B. miyamotoi infection, we conducted a comparative cohort study. We used improved antibody assays and PCRs to compare the relative frequency and clinical manifestations of B. miyamotoi infection with those of B. garinii infection in Russia and B. burgdorferi infection in the United States. Methods Study Design We enrolled patients admitted to Municipal Clinical Hospital No. 33 in Yekaterinburg City, Russia, from May 19 through August 25, 2009, for suspected tick-borne infection. Yekaterinburg is in the Asian part of Russia, ≈1,200 miles east of Moscow. Viral tick-borne encephalitis and acute borreliosis are highly endemic to this region. Patients with moderate or severe disease are usually hospitalized. We compared the clinical characteristics of patients experiencing laboratory-confirmed B. miyamotoi infection with those of patients experiencing B. garinii infections from the same area and with those of patients who experienced B. burgdorferi infection in the northeastern United States. The US data came from a tick-borne diseases study conducted during 1991–2008 ( 14 , 15 ). For each patient at all study sites, we recorded the presence or absence of a standard set of 11 clinical manifestations. All patients signed an informed consent form in accordance with the institutional review boards of the Municipal Clinical Hospital in Yekaterinburg City or the University of Connecticut School of Medicine. We also determined the frequency of B. garinii, B. afzelii, B. burgdorferi, and B. miyamotoi in I. persulcatus and I. ricinus ticks in Yekaterinburg and several additional regions of Russia (Figure 1). Ticks were collected by drag cloth, visually identified to species level, and analyzed by PCR to identify specific Borrelia species. Figure 1 Percentage of Ixodes persulcatus (I. p.) and I. ricinus (I. r.) ticks infected with Borrelia miyamotoi in Russia. The number of ticks that were tested is given in parenthesis. Star indicates study location of human B. miyamotoi infection. Case Definitions Diagnosis of B. miyamotoi infection required the report of a tick bite, presence of clinical manifestations consistent with borreliosis, and laboratory evidence of B. miyamotoi infection. Clinical manifestations included fever, headache, chills, fatigue, vomiting, and myalgia. Confirmation of active infection consisted of amplification of B. miyamotoi DNA/RNA in blood by species-specific PCR and detection of anti-borreliae immunoglobulin (Ig) M in acute- and/or convalescent-phase serum samples. In Russia, diagnosis of B. garinii infection required report of a tick bite, physician diagnosis of erythema migrans (EM; an expanding, ring-like erythematous rash >5 cm in diameter), or an influenza-like illness. Confirmation of infection consisted of amplification of B. garinii DNA/RNA in blood by specific PCR, followed by direct sequencing of 5S-23S ribosomal RNA (rRNA) intergenic spacers, and detection of anti-borreliae IgM in acute- and/or convalescent-phase serum samples. In the United States, diagnosis of B. burgdorferi infection required a physician’s diagnosis of EM or an influenza-like illness. For all cases, confirmation of infection consisted of a >4-fold increase in anti–B. burgdorferi antibody in acute- and convalescent-phase serum samples. The diagnosis of TBEV infection was based on a viral-like illness, including headache (with or without meningitis or encephalitis), amplification of TBEV RNA in blood by species-specific PCR, and/or detection of anti-TBEV IgM in an acute-phase serum sample. Laboratory Assays PCR The PCR we used enabled detection of DNA and RNA sequences. DNA/RNA was extracted from 2 mL of whole venous blood with EDTA or from tick suspensions by using an AmpliSens Riboprep Kit (Central Institute of Epidemiology, Moscow, Russia) according to the manufacturer’s instructions. Of the blood samples used for PCR, 81% were obtained at the time of hospital admission and 96% within 2 days of admission. To assay the inhibitory effect of blood and tick extracts on the PCR, all samples were spiked with a universal RNA recombinant control having a known number of RNA copies per milliliter. Reverse transcription of RNA to cDNA was performed by using an Amplisens Reverta-L Kit (Central Institute of Epidemiology). The cDNA samples were assayed for B. miyamotoi and other tick-borne pathogens by using real-time quantitative PCR (qPCR) assays in a Rotor Gene 6000 cycler (Corbett Life Science, Concorde, New South Wales, Australia). The cDNA samples were divided into 2 aliquots, and different types of real-time qPCR were performed on each. The first used in-house primers and a probe that targeted the 16S rRNA gene of B. miyamotoi. The inclusion of the reverse transcription procedure improved the detection sensitivity because the 16S rRNA that also became detectable is present in higher copy numbers than the 16S rRNA gene. The detection limit of at least 5 × 103 copies/mL was determined by using positive recombinant DNA of the B. miyamotoi 16S rRNA gene fragment with a known number of copies. The B. miyamotoi–specific forward and reverse primers at 360 nmol/L were, respectively, Brm1 5′-CGCTGTAAACGATGCACACTTGGTGTTAATC-3′ and Brm2 5′-CGGCAGTCTCGTCTGAGTCCCCATCT-3′. The corresponding dye-labeled probe (final concentration 100 nmol/L) was R6G-CCTGGGGAGTATGTTCGCAAGAATGAAACTC-BQH1. The PCR conditions were 95°C for 15 min; followed by 10 cycles at 95°C for 20 s, 67°C for 50 s, and 72°C for 20 s; then by 40 cycles at 95°C for 20 s, 60°C for 50 s, and 72°C for 20 s. The fluorescence signal was recorded at the 60°C step for the last 40 cycles. Each run included negative controls and positive recombinant control DNA of the B. miyamotoi 16S rRNA gene fragment as a standard. PCR-based detection of B. burgdorferi s.l., Anaplasma phagocytophilum, Ehrlichia chaffeensis, Ehrlichia muris, and TBEV was performed on the second cDNA aliquot by using a commercial multiplex PCR (AmpliSens TBEV, B. burgdorferi s.l., A. phagocytophillum, E. chaffeensis/E. muris-FL; Central Institute of Epidemiology) ( 16 ), according to the manufacturer’s instructions. This assay was designed to detect, but not discriminate between, B. afzelii, B. burgdorferi s.s., and B. garinii. The same assays were used to detect specific DNA/RNA in ticks and humans. The specificity of B. miyamotoi and B. burgdorferi s.l. assays was confirmed by direct sequencing of flagellin gene fragments and/or 16S rRNA gene fragments and/or 5S-23S rRNA intergenic spacer amplified from blood samples of the same patients or from the same ticks (GenBank accession nos. GU797331–GU797350, JF951378–JF951392). Of the 97 borreliae sequenced, results of DNA amplification using species-specific PCR were entirely consistent with the sequencing results. Absence of false-positive PCR results means that our PCRs were highly specific. Amplification and further direct sequencing of the B. miyamotoi flagellin gene were performed by using degenerate primers FLA120F 5′-AGAATTAATMGHGCWTCTGATGATG-3′ and FLA920R 5′-TGCYACAAYHTCATCTGTCATT-3′ ( 2 , 5 ). The 16S rRNA gene fragment was amplified and sequenced by using 2 primers pairs: first Bf1 5′-GCTGGCAGTGCGTCTTAAGC-3′ and Brsp2 5′-CCTTACACCAGGAATTCTAACTTCCYCTAT-3′, second Brsp1 5′-GGGGTAAGAGCCTACCAAGGCTATGATAA-3′ and Br1 5′-GCTTCGGGTACTCTCAACTC-3′ ( 5 ). Borrelial 5S-23S rRNA intergenic spacer was amplified and sequenced by using nested PCR with outer primers pairs IGSa 5′-CGACCTTCTTCGCCTTAAAGC-3′ and IGSb 5′-AGCTCTTATTCGCTGATGGTA-3′ and inner primers pair IGSe 5′-CCTTAAAGCTCCTAGGCATTCACCA-3′ and IGSd 5′-CGCGGGAGAGTARGTTATTGCGA-3′ ( 17 ). Nucleotide sequences were aligned, compared, and analyzed by using MEGA4.1 (www.megasoftware.net), ClustalW (www.clustal.org), and BLAST (www.ncbi.nlm.nih.gov/blast/Blast.cgi). ELISA Serum samples collected at the time of admission and 1–2 weeks later were tested for anti-borrelial IgM and IgG. Serologic evidence of exposure to borreliae was detected by ELISA EUROIMMUN EI 2132–9601 M and EI 2132–9601–2 G (EUROIMMUN AG, Lübeck, Germany). The ELISA consisted of a mixture of whole antigens from B. afzelii, B. burgdorferi, and B. garinii and thus could detect but not discriminate specific antibody against any of these species. Seroconversion in patients infected with the relapsing fever borrelia B. persica also has been detected by EUROIMMUN assay ( 18 ). Serum from most B. miyamotoi–positive patients reacted to the antigen(s) in this assay. Anti-TBE IgM was detected by the semiquantitative EUROIMMUN ELISA EI 2661–9601 M. Statistical Analyses Comparisons were performed by using the Mann-Whitney U test (independent numeric interval variables), χ2 test (categorical variables), and corresponding exact tests, if necessary; p 0.99 Multiple EM 0 14 7 0.03 0.18 0.36 Fever† 98 67 32 0.001 0.99 0.99 >0.99 >0.99 Neck stiffness 2 0 38 >0.99 37.2°C for patients in Russia and maximum oral temperature >37.7°C for patients in the United States. Although mean peripheral leukocyte and platelet counts were lower for patients with B. miyamotoi than B. garinii infection, they were within the reference range. Proteinuria and transient elevation of serum alanine aminotransferase and aspartate aminotransferase concentrations were found for 3× more B. miyamotoi patients than B. garinii patients (51% and 68% vs. 15% and 20%, respectively, p 1,000 B. miyamotoi cases might occur in Russia each year. More studies are necessary to determine if this projection is accurate. Acute B. miyamotoi infection was more severe than early stage B. burgdorferi infection. The time from symptom onset to hospital admission was shorter, and the number of clinical manifestations was greater for patients with B. miyamotoi infection than with B. garinii infection. Relapsing febrile episodes were only reported for B. miyamotoi patients. Such multiple disease episodes not only have an adverse effect on a patient’s health but also may result in costly medical bills, many days or weeks of lost wages, and medical misdiagnosis ( 19 – 22 ). Co-infection of B. miyamotoi with other ixodid tick–transmitted agents may increase disease severity ( 15 , 23 ). Additional problems that might occur with B. miyamotoi infection are ocular, neurologic, respiratory, cardiac, and pregnancy complications associated with relapsing fever ( 19 – 22 ). Our study had several limitations. Attempts to detect B. miyamotoi on blood smear or in culture were not successful, although we confirmed B. miyamotoi infection with a combination of qPCR, genetic sequencing, clinical, and seroconversion evidence. The comparison of clinical manifestations of Borrelia spp. infection of patients from Russia and the United States was complicated by enrollment at different times and from different locations, although we assessed the same 11 clinical manifestations at each location. The possibility that the clinical description of our B. miyamotoi cases was compromised by unrecognized co-infection with B. burgdorferi s.l. is unlikely. The expected number of cases of co-infection depends on the prevalence of the pathogens in ticks in the region ( 3 , 11 , 24 ), and this number is even fewer than the 4 B. miyamotoi patients with EM we found. Inclusion or exclusion of these 4 cases had no effect on our comparative analysis with patients who did not have B. miyamotoi infection. We limited our description of B. garinii cases to those that were confirmed by detection of amplifiable B. garinii DNA/RNA, although such cases may be more severe than those in which such DNA/RNA cannot be detected ( 25 , 26 ). Patients with B. burgdorferi s.l. PCR–negative results experienced fewer symptoms and milder fever than did patients with B. burgdorferi s.l. PCR–positive results. Our analysis of patients with B. miyamotoi and B. garinii infection was limited to those who were hospitalized, although hospital admission policy in these regions of Russia is liberal because of concern about TBE and problems associated with B. burgdorferi infection. The geographic dispersion and extent of B. miyamotoi disease in humans are unclear, but the infection probably occurs outside of Russia, given the comparative infection rates of vector ticks in Russia and at several locations in Europe and the United States ( 2 – 8 ). In the northeastern United States, ≈15% of all spirochetes carried by I. scapularis ticks are B. miyamotoi ( 2 ). Cases may remain undiagnosed because of the nonspecific nature of the illness, which might be confused with viral infections or such tick-borne infections as Lyme disease, babesiosis, anaplasmosis, or ehrlichiosis, and because of the lack of laboratory tests for confirmatory diagnosis ( 19 – 22 ). B. miyamotoi infection may have negative health consequences, including relapsing disease that may last for months and may not respond to inappropriate antimicrobial drug therapy. The discovery of a Borrelia sp. that is pathogenic in humans and transmitted by an array of ixodid ticks greatly expands the potential geographic distribution of this disease ( 1 – 11 ). Further investigation of possible B. miyamotoi infection in humans is warranted wherever I. pacificus, I. persulcatus, I. ricinus, and I. scapularis ticks are found.