First Reported Case of Ehrlichia ewingii Involving Human Bone Marrow

Abstract

A 65-year-old female with a history of multiple tick bites presented with fever and pancytopenia. Intracytoplasmic rickettsial morulae were detected on peripheral smear and bone marrow biopsy specimens, and PCR amplified Ehrlichia ewingii DNA from both specimens. To our knowledge, this is the first report of E. ewingii infection of human bone marrow.

CASE REPORT

A 65-year-old female from rural north-central Arkansas presented in July to an emergency department with weakness and fatigue. She had a 5-day history of subjective fever, productive cough, generalized myalgia, and progressive fatigue. She denied nausea, diarrhea, bleeding, and rash but stated that she had had a urinary tract infection (UTI) 1 month prior that had been treated with sulfamethoxazole-trimethoprim without resolution. She reported multiple recent tick bites while working in her yard and was in close contact with several pet dogs.

Past medical history was significant for type 2 diabetes mellitus, hypertension, hypothyroidism, and laryngeal squamous carcinoma that had been in remission since treatment with chemotherapy and radiation 2 years prior to presentation. The physical examination was significant for fever of 101.3°F and bilateral upper extremity petechiae and bruising.

Initial laboratory studies revealed a white blood count (WBC) of 2,000/μl (reference range, 3,000 to 12,000/μl), a hemoglobin level of 8.9 g/dl (reference range, 11.5 to 16 g/dl), and a platelet count of 32,000/μl (reference range, 150,000 to 500,000/μl). Each of these values had been within normal limits during an evaluation for her UTI that had been performed 3 weeks earlier. The total bilirubin level was 1.3 mg/dl (reference range, 0.2 to 1.2 mg/dl), and the lactate dehydrogenase level was 314 IU/liter (reference range, 100 to 248 IU/liter). Iron studies showed a decreased total iron binding capacity of 234 μg/dl (reference range, 250 to 425 μg/dl) and an increased peripheral blood ferritin level of 732 μg/dl (reference range, 11 to 306 μg/dl) with normal iron and folate levels. The antinuclear antibody (ANA) titer was increased at 1:160. Random-inpatient blood glucose levels ranged from 102 to 111 mg/dl, and her diabetes was adequately controlled with metformin and glipizide by her treatment as an outpatient. HIV serology results were negative.

The patient was admitted to the hospital and was started on vancomycin and cefepime because of fever and neutropenia and was started on doxycycline to address the potential for tick-borne illness. Since recurrent malignancy, myelodysplastic syndrome following chemotherapy, and other marrow processes were in the differential diagnosis for her pancytopenia, peripheral smear and bone marrow aspirate and core biopsy procedures were performed. Review of the peripheral blood smear revealed leukopenia with neutrophilic bands containing intracytoplasmic morulae (Fig. 1), pancytopenia with left-shifted granulopoiesis, reactive lymphocytes, a relative monocytosis, thrombocytopenia, and mild erythrocyte anisopoikilocytosis. The marrow aspirate showed occasional intracytoplasmic morulae within cells of the myeloid lineage and plasma cells (Fig. 1 and 2, respectively). Histology revealed hypercellular marrow with mild erythroid dyspoiesis. The core biopsy was performed 2 days after antibiotic treatment was initiated, and a few morulae seen demonstrated morphological features consistent with treatment response (Fig. 2) (1).

FIG 1.

Bone marrow aspirate at a magnification of ×1,000. Wright-Giemsa staining shows a granulocytic band with an intracellular morula (arrow). (Inset) Peripheral blood at a magnification of ×1,000 (CellaVision microscopy, Lund, Sweden). Wright-Giemsa staining shows a band cell in peripheral blood with an intracellular morula (arrow).

FIG 2.

Bone marrow aspirate at a magnification of ×1,000. Wright-Giemsa staining shows a plasma cell with an intracellular morula (arrow). (Inset) Myeloid precursor with an intracellular morula (arrow) demonstrating treatment effect “halo.”

Given the microscopic findings and the extent of tick exposure, peripheral blood specimens were referred to outside laboratories for further characterization of the tick-borne infection using serologic and molecular methods. Results of serologic studies (ARUP Laboratories, Salt Lake City, UT) were incongruous. A positive Anaplasma phagocytophilum IgG titer of 1:640 (reference range, <1:80) suggested recent or past infection. This result was supported by the finding of granulocytotropic morulae on a peripheral smear; however, A. phagocytophilum is not endemic to Arkansas and the IgM titer was negative at <1:16 (reference range, <1:16). “Rickettsia rickettsii (Rocky Mountain spotted fever)” serology was reactive for IgM at a low titer (1:64; reference range, <1:64) but negative for IgG (<1:64; reference range, <1:64), suggesting possible infection with a spotted fever group Rickettsia species. Francisella tularensis serology was negative (IgM = 2 U/ml and IgG = 9 U/ml; reference range, ≤9 U/ml). Ehrlichia serology was not pursued due to an initial negative PCR result for E. chaffeensis (Arkansas Children's Hospital Clinical Laboratory, Little Rock, AR).

Peripheral blood sent for molecular testing by PCR (Mayo Medical Laboratories, Rochester, MN) was positive for E. ewingii using real-time multiplex PCR (2) and negative for A. phagocytophilum, E. chaffeensis, and the recently described E. muris-like organism. Given the E. ewingii-positive PCR result on peripheral blood, PCR was also performed on the decalcified, paraffin-embedded bone marrow core block shavings (Mayo Medical Laboratories) using the same Ehrlichia/Anaplasma PCR. E. ewingii was successfully amplified, and morulae within myeloid precursor cells were stained by an immunohistochemical method for Ehrlichia spp. using an immunoalkaline phosphatase technique and dog hyperimmune anti-Ehrlichia canis antiserum at the Centers for Disease Control and Prevention, Atlanta, GA (3, 4) (Fig. 3).

FIG 3.

Bone marrow core biopsy specimen at a magnification of ×1,000. An Ehrlichia morula (pink) within a myeloid precursor was identified using an immunohistochemical stain.

Vancomycin and cefepime were discontinued, and a 10-day course of doxycycline was completed. At discharge, the patient was afebrile and her pancytopenia had resolved.

Ehrlichiosis is a tick-borne rickettsial illness seen during the summer months most commonly in the southeastern and central United States. Species of Ehrlichia associated with human illnesses in the United States include E. chaffeensis (5), E. ewingii (6, 7), and an E. muris-like organism (8). Patients with ehrlichiosis may present with fever, headache, myalgia, rash, nausea, vomiting, and laboratory findings of thrombocytopenia, leukopenia, anemia, and elevated liver enzyme numbers (4, 6, 9, 10). In peripheral blood, E. chaffeensis generally has tropism for monocytes and E. ewingii tropism for granulocytes (1, 4, 6). Anaplasmosis has similar symptoms and was formerly referred to as human granulocytic ehrlichiosis (11, 12). Although such an interpretation is implied by this old nomenclature, leukocyte tropism (i.e., monocytic versus granulocytic) is not specific, as A. phagocytophilum has demonstrated in vitro growth in both monocytes and granulocytes (13). Additionally, E. chaffeensis, commonly considered specific for monocytes, can be identified in vivo within monocytes, granulocytes, lymphocytes (14), and histiocytes (15). On peripheral smears made using a Wright-Giemsa stain, E. chaffeensis, E. ewingii (Fig. 1), and A. phagocytophilum appear as basophilic clusters of intacytoplasmic bacteria referred to as morulae (1, 11, 15). E. muris-like bacteria have yet to be morphologically described in vivo but infect granulocytic and monocytic cell lineages in cell culture (8).

E. chaffeensis invasion of myeloid cells in human bone marrow and other organs has been previously described (3, 4, 15), and A. phagocytophilum in association with bone marrow infection has also been previously described (16). Prior to this case report, E. ewingii infection of human marrow cells had yet to be documented. However, we observed E. ewingii within myeloid precursors and plasma cells in the bone marrow aspirate (Fig. 2) and in myeloid precursors of the immunohistochemically stained bone marrow core biopsy specimen (Fig. 3). In animal studies, A. phagocytophilum and E. muris have been linked to cytopenias and dyshematopoiesis (17, 18). We postulate that a similar bone marrow process in humans may have been responsible for the pancytopenia observed in this case involving E. ewingii.

As demonstrated in the description of this case, identification of the specific organism causing an ehrlichiosis-like illness can pose a challenge for the clinician. A history of fever and tick bite during summer months in areas of endemicity is commonly diagnosed as “tick fever,” empirical treatment with doxycycline is administered, the patient generally recovers, and no further testing is performed. Thus, these tick-borne diseases go under- or misreported to public health departments. Obstacles to identification to the species level include the turnaround time for PCR and serology and the fact that morulae are seen on peripheral blood smears in only 22% to 38% of ehrlichiosis cases (1, 19). Additionally, serologic studies can be misleading, as cross-reactivity is seen among Ehrlichia spp., A. phagocytophilum, and Rickettsia spp. (6, 9, 20). Further, serologic tests for E. ewingii and the E. muris-like agent are presently not available and these species may be reported as E. chaffeensis due to cross-reactivity (6, 7, 21). Specific to E. ewingii reporting, this cross-reactivity may partially explain statistics from the Centers for Disease Control and Prevention where only 28 cases of E. ewingii infection were reported between 2008 and 2010 compared to 2,645 cases of E. chaffeensis infection (22). Unfortunately, identification of E. ewingii is readily available only to clinicians using PCR. Finally, there is accumulating evidence of an association between the severity of ehrlichiosis disease and treatment with sulfa-containing antimicrobials (23–25). It is noteworthy that the patient reported here was treated prior to hospital admission with sulfamethoxazole-trimethoprim for UTI.

In summary, we report the first recognized case of E. ewingii infection in human bone marrow and demonstrate some diagnostic challenges that may arise when identifying human-infecting Rickettsiales isolates to the species level. As other methodologies may yield confounding results, PCR is currently the only modality available for definitive identification of E. ewingii.