We retrospectively reviewed results of 18 months (May 2013–November 2014) of testing for human ehrlichiosis by real-time PCR. All samples submitted to Associated Regional and University Pathologists Laboratories (Salt Lake City, UT, USA) for Ehrlichia and Anaplasma species by real-time PCR were included in the analysis. PCR-positivity rates for Ehrlichia spp. were calculated on the basis of results for individual patients.

We used a real-time PCR for Ehrlichia and Anaplasma species that detects E. chaffeensis, E. muris–like pathogen, E. ewingii, and E. canis (without differentiating E. ewingii and E. canis) and A. phagocytophilum. In brief, nucleic acids extracted from whole blood specimens by using the Chemagic MSM I Automated Extraction Platform and the Chemagen Blood Extraction Kit (Perkin Elmer, Waltham, MA, USA) were amplified by using primers specific for 16S rRNA gene and Ehrlichia and Anaplasma species–specific probes for identification (Table 1). The reaction was prepared by using a 5× custom real-time Master Mix and 4 mmol/L MgCl₂ (Promega, Madison, WI, USA) with the following amplification parameters: 50.0°C for 2 min; denaturation at 95.0°C for 2 min; and 50 cycles at 95.0°C for 5 s, 56.0°C for 20 s, and 76.0°C for 20 s on the Rotor-Gene Q apparatus (QIAGEN, Hilden Germany). Melting curve analysis was performed at 95.0°C for 15 s and then from 45°C through 75°C at 1.0°C/step at 5 s/step with continuous fluorescence acquisition.

We retained samples positive for E. ewingii/E. canis from the initial tests for further analysis for this study. Nucleic acids were extracted from these residual blood specimens, and another real-time PCR specific for a region within the 16S rRNA gene was performed to differentiate E. ewingii from E. canis. We contacted healthcare providers for E. ewingii–positive patients to collect case histories. The study was approved by the University of Utah Institutional Review Board (no. 76713).

Of 4,177 patients from 41 states who had samples submitted to Associated Regional and University Pathologists Laboratories for detection of Ehrlichia and Anaplasma species by real-time PCR during an 18-month study period, 99 (2.4%) were positive for E. chaffeensis, 10 (0.2%) for E. ewingii/E. canis, and 0 for E. muris-like pathogen. A total of 179 (4.3%) patients were positive for A. phagocytophilium. Positivity rates were calculated by state (Table 2).

All 10 E. ewingii/E. canis–positive cases were subsequently identified as E. ewingii, accounting for 10 (9.2%) of 109 ehrlichiosis cases during the study period. 
E. ewingii–positive samples were from 9 men and 1 woman (median age 58 years, range 24–74 years). The samples were from Missouri (n = 4), Indiana (n = 3), Arkansas (n = 1), New Jersey (n = 1), and New York (n = 1) and were collected in June (n = 1), July (n = 3), August (n = 4), and September (n = 2). Case histories and laboratory results were obtained for 5 patients (Table 3).

Case-patient 1 was 24-year-old man (landscaper) from New Jersey who had nausea, vomiting, and fevers in August 2014. The patient had no known tick bites and no major underlying concurrent illnesses. At the time of presentation, his temperature was 102°F; results of a physical examination were otherwise unremarkable. Laboratory testing showed thrombocytopenia, and blood cultures obtained at presentation were negative for bacterial growth. Serologic testing for Lyme disease by ELISA showed antibodies against Borrelia burgdorferi. Western blot showed a positive result for IgM and a negative result for IgG. Serologic testing for Ehrlichia spp. was not performed. The patient received a 2-week course of doxycycline, and his symptoms resolved.

Case-patient 2 was a 38-year-old man from Missouri who had a history of headache and fever for 10 days in August 2014. The patient had no known tick exposure, and his medical history was unremarkable. At the time of presentation, his temperature was 102°F. He had an eczematous rash on his left upper leg and increased levels of liver enzymes. Results of serologic testing for E. chaffeensis were negative. The patient received a 1-week course of doxycycline, and his symptoms resolved.

Case-patient 3 was a 73-year-old man from Indiana who had subjective fevers, mild left lower abdominal pain, myalgia, and malaise. The patient reported a tick bite on his abdomen 2–3 weeks before presentation. His medical history included type 1 diabetes, hypertension, and stroke. Results of a physical examination were unremarkable. He was afebrile at presentation but had leukopenia and thrombocytopenia. Results of serologic testing for Lyme disease (total antibodies against B. burgdorferi by ELISA) were negative. Serologic testing for Ehrlichia spp. was not performed. The patient received a 10-day course of doxycycline, and his symptoms resolved.

Case-patient 4 was a 74-year-old man from Arkansas who had malaise, arthralgia, fever, and nonbloody diarrhea in for 2 weeks in July 2014. The patient reported 2 tick bites 2 weeks before presentation. His medical history included diffuse large B-cell lymphoma (treated with gemcitabine), lung cancer (treated with a right lobectomy), chronic obstructive pulmonary disease that required supplemental oxygen, coronary artery disease, and hypothyroidism. At presentation, he had hypotension (79/46 mm Hg), tachycardia (142 beats/min), and a fever (temperature 100.1°F). Laboratory testing showed leukopenia, thrombocytopenia, and morulae in neutrophils on a peripheral blood smear. Results of serologic testing for E. chaffeensis were negative. The patient received a 3-week course of doxycycline and showed clinical improvement; however, he died 1 month later of unrelated bacterial sepsis.

Case-patient 5 was a 73-year-old man from Missouri who had headache, fever, and nausea in August 2014. The patient reported a tick bite ≈2 months before presentation. His medical history included bladder cancer (treated with immunotherapy), a melanoma on his right arm (treated with surgical excision), type 1 diabetes, and hypothyroidism. He had leukopenia, thrombocytopenia, and increased levels of liver enzymes. Blood cultures at presentation and serologic results for E. chaffeensis were negative. The patient received a 10-day course of doxycycline, and his symptoms resolved.

In our study, E. ewingii accounted for 10 (9.2%) of 109 cases of Ehrlichia spp. infections, compared with only 31 (2.0%) of 1,518 reported human ehrlichiosis cases in the United States (4). Underreporting might be caused by successful empirical treatment without etiologic diagnosis; missed E. ewingii infections by serologic tests for E. chaffeensis in the acute phase of illness (case-patients 2, 4, and 5); and limited availability of molecular tests. Even with molecular testing, cases might be missed because of non-optimal timing of specimen collection and limited sensitivity of the assay, such as with testing after resolution of bacteremia.

Human infections with E. ewingii naturally acquired have been reported in Arkansas, Missouri, Oklahoma, and Tennessee (9,10,12). We now describe cases of infection in Indiana and New Jersey. The location of these cases is consistent with the known range of the vector (A. americanum ticks). E. ewingii has previously been reported in A. americanum ticks collected in New Jersey (14).

The 5 case-patients reported in this study had symptoms of classical ehrlichiosis, including fever, myalgia, malaise, and headache. Thrombocytopenia and leukopenia were the most consistent associated laboratory findings. All patients improved after treatment with doxycycline treatment, despite in some instances, major underlying disease. E. ewingii should be considered as an etiologic agent of tickborne febrile illness in the central and eastern United States that may be missed by serologic testing.