A total of 2,150 DNA samples were obtained from blood of patients evaluated during 2014–2015 for illness and tested for either A. phagocytophilum or B. microti because of clinical complaints or abnormalities suggestive of tickborne illness. Because our current laboratory procedures for confirming either infection require extraction of DNA from a blood sample before PCR testing, we also evaluated the extracted DNA for B. miyamotoi. Ethics approval for detecting and sequencing unique bacterial DNA from routine clinical samples and linkage to patient data without individual informed consent was obtained from the Gundersen Institutional Review Board with the stipulation that patient identifiers be appropriately redacted and information be used only as surveillance data for public health purposes.

We initially screened the DNA samples with a modification of a previously described B. miyamatoi screening PCR (13), which amplified a 70-bp DNA fragment of the 16S ribosomal RNA gene specific for B. miyamatoi. In brief, we combined 5 μL of extracted DNA with 20 μL of a master mix that contained 12.5 μL of buffer (AmpliTaq Gold DNA Polymerase with GeneAmp 10X PCR Gold Buffer; Life Technologies, Austin, TX, USA); 2.5 mmol/L magnesium chloride, deoxynucleoside triphosphates; 7 μL of a primer/probe mix comprising forward primer 5′-GCTGTAAACGATGCACACTTGGT-3′, reverse primer 5′-GGCGGCACACTTAACACGTTAG-3′, and probe 5′-HEX-CGGTACTAACCTTTCGATTA-3′; and 0.5 μL (1.5 U) AmpliTaq Gold DNA Polymerase under the following conditions: 1 cycle at 95°C for 10 min, 45 cycles at 95°C for 15 s, 63°C for 1 min, and a final cycle at 25°C for 5 s. For a positive control, we used strain HS1 (ATCC 35209) of Borrelia hermsii because B. miyamotoi DNA or viable spirochetes are not easily obtained.

We amplified B. miyamotoi DNA from 7 patients (1 male, 6 female) whose previous anaplasmosis and babesiosis test results were negative. Mean patient age was 51 years (range 3–70 years), and 3 patients confirmed that they had been bitten by a tick suspected to be I. scapularis within the 10 days before becoming ill. The patients also resided in or near La Crosse; although none reported recent travel outside the region, we cannot rule out the possibility that the infections were acquired elsewhere. Further supporting local acquisition, however, we also recently detected B. miyamotoi DNA by the PCR used in this study in a small number (≈2%) of I. scapularis ticks collected locally (data not shown).

The clinical signs and symptoms strongly supported PCR positivity for B. miyamotoi (5–8). The most common clinical sign was fever; 1 patient reported additional episodes of fever during the 2 months before seeking treatment (Table). In addition, skin lesions were not detected, although 1 patient may have been co-infected with B. burgdorferi; an IgM Western blot test performed in the clinical laboratory also yielded reactivity sufficient to provide serodiagnostic confirmation of early Lyme disease (14). Moreover, elevated levels of serum alanine aminotransferase (n = 2) or aspartate aminotransferase (n = 1) were detected in the only 2 patients for whom B. miyamotoi DNA was identified and who were tested for these liver enzyme abnormalities. In addition, the infected patients received doxycycline therapy for at least 7 days (mean 10 days; range 7–14 days), which uniformly resulted in complete resolution of clinical signs and symptoms.

For final confirmation of infection with B. miyamotoi, we then reamplified a 142-bp fragment (bp 636–777) of the glycerophosphodiester phosphodiesterase (glpQ) gene from the remaining DNA sample from each patient positive for B. miyamotoi by PCR. The glpQ gene was targeted because the GlpQ protein is absent in the Borrelia species that cause Lyme disease (15), which are detected relatively frequently in patients from the region (1). In brief, 5 μL of DNA was combined with 20 μL of a master mix that contained 12.5 μL of buffer, 0.5 μmol/L of forward (5′-GATAATATTCCTGTTATAATGC-3′) and reverse (5′-CACTGAGATTTAGTGATTTAAGTTC-3′) primers, and 0.5 μL (1.5 U) of AmpliTaq Gold DNA polymerase. The DNA was then amplified under the following conditions: 1 cycle at 95°C for 3 min followed by 50 cycles at 95°C for 45 s, 56°C for 30 s, and 72°C for 60 s, followed by a final extension at 72°C for 7 min. Amplified material was purified (QIAquick PCR Purification Kit; QIAGEN, Hilden, Germany) and forwarded to Laragen Inc. (Culver City, CA, USA) for sequencing. In each instance, the sequence of the amplified fragment was 100% homologous with that of B. miyamotoi LB-2001 (GenBank accession no. CP006647.2).

Researchers have documented human illness caused by B. miyamotoi transmitted from I. scapularis ticks (5,7–9,11), but infections in patients from the United States have to date been described only for residents of the northeastern part of the country (10). In this study, however, we confirmed characteristic illness caused by infection with B. miyamotoi in 7 patients who resided in the I. scapularis tick–endemic focus surrounding La Crosse, Wisconsin. Given these findings, clinicians in the upper Midwest must now also consider the possibility of B. miyamotoi infection in patients with suspected tickborne illness, especially because even more cases will probably be detected as appropriate methods, such B. miyamotoi-specific anti-GlpQ antibody testing (8,15) or B. miyamotoi DNA detection, become more widely available. Studies to provide additional insight into human infection with B. miyamotoi remain necessary because the prevalence of the illness will probably increase even more as I. scapularis ticks continue to disperse.