PCR Assays

We tested DNA isolated from the plasma or buffy coat fractions of whole blood by using real-time reverse transcription PCR for the detection of Borrelia burgdorferi (ospA and 16S rRNA genes), Borrelia miyamotoi (16S rRNA gene), Anaplasma phagocytophilum (groEL gene), Rickettsia spp. (gltA gene), and N. mikurensis (groEL gene, named CNM-II in the reference article) at the Department of Medical Microbiology at Sørlandet Hospital (Kristiansand, Norway) using primers and probes as described previously (8). We performed Babesia subspecies DNA PCR at Statens Serum Institut (Copenhagen, Denmark). We established a diagnosis of hemolysis on the basis of reduced hemoglobin, reduced plasma haptoglobin, increased plasma lactate dehydrogenase, and increased blood reticulocytes.

Patient 1

A 61-year-old woman sought care for rapid-onset dyspnea, fatigue, palpitations, dizziness, and a 6-month history of infrequent febrile episodes. Her medical history included rheumatoid arthritis for 17 years, breast cancer treated 16 years previously, and episodic severe depression for 1.5 years. Her arthritis initially was treated with oral methotrexate, salazopyrine, and hydroxychloroquine. Methotrexate temporarily was withdrawn because of cytopenias after a year and permanently withdrawn when she had breast cancer diagnosed. Her breast cancer was treated with surgery after 6 courses of neoadjuvant chemotherapy with fluorouracil, epirubicin hydrochloride, and cyclophosphamide, followed by postoperative radiotherapy. After completion of breast cancer treatment, treatment for rheumatoid arthritis commenced with rituximab infusions (1,000 mg 2×/y). She also was treated with venlafaxine for depression and anxiety.

At admission, we diagnosed severe transfusion-dependent direct antiglobulin test (DAT)–negative hemolytic anemia in the patient (Table). We suspected drug-induced hemolytic anemia and discontinued venlafaxine and rituximab, which later were reinstated because of lack of improvement. Two months after initially seeking care, the patient was admitted to a hospital with acute appendicitis and worsened hemolysis. After appendectomy, we initiated prednisolone (1 mg/kg/d), which resulted in partial reduction of hemolysis. We introduced the interleukin 1 receptor inhibitor anakinra 7 months later as a steroid-sparing agent after relapses of fever and worsened hemolysis during the tapering of prednisolone.

Figure

Figure. Hemoglobin levels (blue line) for patient 1 in a case series of 3 patients with neoehrlichiosis and hemolytic anemia at admission, during disease course, and after treatment with antibiotics, Norway,...

Diagnostic tests of blood and bone marrow revealed no evidence of congenital or acquired erythrocyte membrane defects, including paroxysmal nocturnal hemoglobinuria (PNH), lymphoproliferative disorders, or other hematologic neoplasia. Results of a 184-gene panel of hereditary causes of anemia and bone-marrow failure (e.g., Fanconi anemia, Diamond-Blackfan anemia, and hereditary stomatocytosis) were negative. No findings were suggestive of Wilson disease (i.e., normal ceruloplasmin and copper serum and urine levels) or of zinc- or arsenic-related poisoning. Computed tomography (CT) scans of the neck to pelvis were unremarkable, whereas a fluorodeoxyglucose (FDG) positron emission tomography (PET)–CT scan showed a slight uptake in the spleen initially deemed to be reactive. When a repeat FDG PET–CT scan 3 months later showed a maintained slight uptake in the spleen, the patient underwent diagnostic splenectomy because of a continued suspicion of underlying lymphoproliferative disorder. Before splenectomy, the patient had thrombophlebitis in the right cubital fossa and right ankle diagnosed. After splenectomy, hemoglobin levels improved for a period, but evidence of hemolysis remained. Histopathologic examination of the spleen showed depletion of B cells in the white pulp, secondary to rituximab treatment, and expanded red pulp with extramedullary hematopoiesis but no evidence of lymphoma. Within 2 months, the patient’s hemolytic anemia relapsed. Because of a history of tick exposure and erythema migrans a few years prior, we sent EDTA blood samples for PCR testing for tickborne microbes, including Rickettsia spp., B. miyamotoi, B. burgdorferi, A. phagocytophilum, N. mikurensis (8), and Babesia subsp. The patient tested positive for N. mikurensis, upon which we initiated oral doxycycline (100 mg 2×/d for 21 d). Her symptoms, most prominently fever, night sweats and fatigue, as well as all signs of hemolysis, rapidly improved and cleared completely (Table; Figure). She tested negative for N. mikurensis DNA by PCR after the course of antibiotics. We also detected N. mikurensis DNA in historical serum samples drawn 22, 11, and 6 months before the time of diagnosis. At a 12-month follow-up, no evidence of hemolytic anemia remained.

Patient 2

A 62-year-old man was referred to our outpatient clinic with a 4-month history of gradually progressive symptomatic anemia, prominent fatigue, and night sweats. He did not have fever, weight loss, nor any current or previous vascular events. His medical history included myasthenia gravis for 14 years that had been treated with rituximab infusions (1,000 mg 2×/y) for the previous 5 years. One year earlier, he had a prolonged SARS-COV-2 infection, evaluated with a chest CT, with an incidental finding of a slightly enlarged spleen. Laboratory findings at referral showed mild DAT-negative hemolytic anemia (Table). A peripheral blood smear showed no evidence of cellular dysplasia, blasts, or schistocytes. A test result for PNH was negative. We performed coagulation tests because of easy bruising, but we detected no coagulation defect. The patient had had no recall of tick bites. We performed no testing for SARS-COV-2. Because of several similarities to the first patient case (e.g., a probable nonimmunologic hemolytic anemia, treatment with rituximab, and slight splenomegaly), we performed PCR testing for tickborne microbes, including Rickettsia spp., B. miyamotoi, B. burgdorferi, A. phagocytophilum, N. mikurensis, and Babesia subsp. The patient was positive by PCR for N. mikurensis, and we initiated oral doxycycline (100 mg 2×/d for 21 d). A month later, his anemia had improved, although haptoglobin still was suppressed slightly, indicating low-grade hemolysis. Two months after the patient finished doxycycline treatment, hemoglobin and haptoglobin levels were normal (Table). At 12-month follow-up, no evidence of hemolytic anemia remained.

Patient 3

A 48-year-old man was referred our care with splenomegaly and profuse night sweats that had started 1 year earlier. His medical history included relapsing or remitting multiple sclerosis 5 years prior, which had been treated with rituximab infusions (1,000 mg 1×/y) for the previous 2 years. CT imaging of the thorax, abdomen, and pelvis done a few months before referral revealed no pathologic lymphadenopathy but substantial splenomegaly (largest diameter 17.7 cm). One month before his first episode of night sweats, he had traveled to the southern United States, Caribbean, and South America, none of which warranted malarial chemoprophylaxis. He had not contracted fever, insect bites, or rashes during his travels. He had a history of tick bites after hiking trips in southeastern Norway but no erythema migrans. Blood chemistry taken a week before the appointment at our facility showed mild and fully compensated DAT-negative hemolytic anemia (Table). The levels of haptoglobin, reticulocytes, and lactate dehydrogenase normalized spontaneously 10 days later.

An ultrasound at the time of the first consultation showed a reduction of spleen size to 15.1 cm (largest diameter). PNH test result was negative, and peripheral blood smears results were normal. Bone marrow trephine biopsy showed a very slight increase of intrasinusoidal T-lymphocytes of uncertain importance. Repeated bone marrow trephine biopsies and bone marrow aspirate analyses by flow cytometry yielded similar results. A FDG PET or CT scan showed no pathologic FDG uptake in the liver, spleen, or bone marrow and a maximal spleen diameter of 15.8 cm. We noted only a slight clonal T-cell receptor rearrangement in the bone marrow and normal results in peripheral blood, consistent with a reactive cause with no conclusive evidence of lymphoproliferative disorder. A repeat ultrasound of the spleen a few months later showed a spleen size of 14.3 cm. Six months after the initial diagnostic work-up and 2 months after receiving his yearly rituximab infusion, the patient experienced a relapse of symptoms with increasing night sweats, increase in spleen size to 17.4 cm on ultrasound, and hemolysis with a substantial reduction in hemoglobin levels and signs of intravascular hemolysis (Table).

We performed testing for tickborne microbes, including Rickettsia spp., B. miyamotoi, B. burgdorferi, A. phagocytophilum and N. mikurensis, and the patient was positive by PCR for N. mikurensis. We did not test for Babesia subsp. We initiated treatment of the patient with oral doxycycline (100 mg 2×/d for 21 d). A repeat ultrasound of the spleen performed 6 weeks later showed a reduction in size to 14 cm. His symptoms improved and resolved within a week of antibiotic treatment. A PCR test for N. mikurensis was negative after treatment. We also detected N. mikurensis DNA in stored serum samples drawn 12 and 8 months before the time of diagnosis. At 12-month follow-up, no evidence of hemolytic anemia remained.