Cell Culture and Serum Testing

We inoculated recipient urine specimens onto monkey kidney cells (Vero E6) and human embryonic lung fibroblasts by using established methods (5). Cultures were treated with 2% sodium dodecyl sulfate and passaged to eliminate cytomegalovirus. For IFA, Encephalitozoon cuniculi reference strain cultures were suspended in phosphate-buffered saline (PBS) at a concentration of 10⁸ spores/mL. We added 10 μL of this suspension to each well (10⁶ spores/well), allowed the slides to air dry, and stored them at −80°C before use.

Serum samples were diluted 1:2 in 25 μL PBS along with previously established positive (titer 1:4,096) and negative (titer 1:32) control serum obtained from the CDC free-living amebic infections laboratory. The serum samples were then diluted to 1:4,096 by 2-fold dilution. We added 10 μL from each dilution to the wells of the previously made E. cuniculi IFA slides, which were then incubated at 37°C for 30 min. The slides were washed 3 times in PBS before the addition of a 1:100 dilution of fluorescein isothiocyanate–conjugated goat anti-human IgG (Cappel Laboratories, Cochranville, PA, USA) in PBS with 3 μL/mL Evans Blue (Fisher Scientific, Pittsburgh, PA, USA) counterstain. After another 30-min incubation at 37°C, the slides were again washed 3 times in PBS, dried, and mounted with glycerol mounting media. Serum titers were determined by observing the slides under a fluorescence microscope. Antibody titers were considered positive at cutoffs of >1:16 for immunocompromised and >1:64 for immunocompetent persons (6).

Histopathology, Immunohistochemistry, and Transmission Electron Microscopy

We processed formalin-fixed paraffin-embedded tissues by using standard histologic methods. IHC was performed by using a polymer-based indirect immunoalkaline phosphatase detection system with a fast red chromogen kit (Biocare Medical, Concord, CA, USA). The microsporidia IHC assay used rabbit anti–E. cuniculi serum at a 1:1,000 dilution as previously described (7). In addition, IHC assays for lymphocytic choriomeningitis virus, measles virus, and Trypanosoma cruzi were performed as previously described (8–10).

For transmission electron microscopic examination, we evaluated a paraffin-embedded section by on-slide embedding as previously described (11). The urine cell pellet and microsporidia culture isolates were transferred to buffered 2.5% glutaraldehyde and 1% osmium tetroxide, embedded in a mixture of Epon substitute and Araldite (Ted Pella, Inc., Redding, CA, USA), sectioned, and stained with uranyl acetate and lead citrate.

Molecular Techniques

DNA was extracted from unpreserved clinical samples by using a DNeasy Blood & Tissue DNA extraction kit (QIAGEN, Valencia, CA, USA). DNA was extracted from the formalin-fixed paraffin-embedded tissues by using a QIAamp DNA Mini Kit (QIAGEN) as previously described (7,12). PCR primers (concentration 15 μmol/L each) specific for E. cuniculi small subunit ribosomal RNA gene were used (7,12). We performed PCR by using AmpliTaq Gold PCR Master Mix (ThermoFisher Scientific, Grand Island, NY, USA) and 5 μmol of each primer at an annealing temperature of 65°C. All DNA extracts were subjected to PCR. Positive and negative controls were included in every PCR run. Any specimen that resulted in a 551-bp fragment was considered positive for the presence of E. cuniculi DNA.

Right Kidney Recipient

The right kidney recipient was a man with history of end-stage renal disease resulting from type 2 diabetes mellitus. He received basiliximab to induce immunosuppression, and at discharge he received tacrolimus, mycophenolate mofetil, and prednisone to maintain immunosuppression. His immediate postoperative course was complicated by delayed graft function and anemia. Approximately 10 weeks after transplantation, generalized weakness and confusion developed, and he was admitted to the hospital for evaluation. Initially his clinical condition was thought to result from noninfectious causes, such as medication side effects. However, during his hospitalization, fever, pancytopenia, and acute renal failure developed, and his mental status worsened. Lumbar puncture was performed, and cerebrospinal fluid analysis revealed 10 leukocytes/μL, which led to concern for viral encephalitis. An extensive evaluation for CNS infection was initiated, and test results were negative for bacterial, fungal, viral, and parasitic causes (Table 1). Despite empirically prescribed broad-spectrum antimicrobial drugs, the recipient’s illness progressed, his obtundation worsened, and hypotension required vasopressors. He died ≈15 weeks after transplantation. An autopsy was performed at the local hospital, and tissues were submitted to CDC for examination. Four days after the recipient’s death, examination at CDC of the deceased recipient’s renal allograft demonstrated intracellular organisms consistent with microsporidia. Clinicians caring for the other recipients were immediately notified.

Figure 2

Figure 2. Transmission electron microscopy of microsporidia identified in allograft samples from right kidney recipient. The organism shows cross-sections through the polar tube with up to 6 coils and a unikaryotic nucleus, which...

Subsequent PCR of DNA extracts from the right renal allograft revealed the species to be E. cuniculi, and electron microscopy showed a polar tubule arrangement characteristic for E. cuniculi (Figure 2). The recipient’s CNS tissue also was positive for E. cuniculi by histopathology and PCR, which showed microsporidia associated with glial nodules and the leptomeninges, the latter with perivascular inflammation. No arteritis or aneurysmal change in the vessels of the CNS were observed. Immunohistochemistry of CNS tissue also provided positive results (Table 2). IHC results were negative for lymphocytic choriomeningitis virus, measles virus, and T. cruzi.

Left Kidney and Heart Recipient

The recipient of the left kidney and heart was a woman in whom coronary vasculopathy and calcineurin inhibitor–induced nephropathy had developed after a cardiac transplant 21 years earlier. To induce immunosuppression before the upcoming kidney and heart transplant, she received a 5-day course of antithymocyte globulin; to maintain immunosuppression, she received tacrolimus, mycophenolate mofetil, and prednisone. Her immediate posttransplant course was complicated by delayed renal graft function, as well as delirium and tremors thought to be associated with uremia and excessive levels of tacrolimus. The delirium and tremors resolved with improved graft function, and her immunosuppression regimen was changed to include cyclosporine rather than tacrolimus. Two months after transplantation, she experienced low-grade fever, fatigue, and headache. Testing for infectious causes, including herpesviruses, enterovirus, West Nile virus, Borrelia burgdorferi, and Cryptococcus, were negative. Mycophenolate mofetil, valganciclovir, and trimethoprim/sulfamethoxazole prophylaxis were temporarily discontinued because of leukopenia. A week later, she experienced continuing fevers up to 102°F; worsening frontal headache; fatigue; pain in her shins, wrists, and elbows; cognitive slowing; prominent myoclonus; expressive aphasia; and visual hallucinations. She was markedly leukopenic; absolute neutrophil count was 430 cells/μL. Brain magnetic resonance imaging did not reveal mass lesions, infarct, or hydrocephalus. Cerebrospinal fluid testing revealed 17 leukocytes/μL and a protein level of 80.4 mg/dL; however, results of extensive testing for an infectious etiology were negative (Table 1). Routine endomyocardial biopsy samples showed no evidence of rejection; a renal allograft biopsy sample showed evidence of Banff type IIa acute cellular rejection, for which she received 3 doses of methylprednisolone.

After CDC communicated the finding of microsporidiosis in the deceased recipient’s allograft, this recipient empirically received treatment with albendazole at 400 mg twice daily. E. cuniculi was detected by PCR from urine obtained at the time of albendazole initiation. Her neurologic symptoms resolved, and after 4 months of albendazole therapy, PCR of urine for E. cuniculi was negative. Therapy with albendazole was continued for 1 year. As of July 2016, she remained well without any symptoms of microsporidial infection 1 year after stopping albendazole therapy.

Liver Recipient

Figure 3

Figure 3. Microsporidia identified in urine samples from liver recipient. A) Urine trichrome stain. Original magnification ×100. B) Cell culture showing microsporidium (arrow). Original magnification ×200. C) Transmission electron microscopic image of infected...

The liver recipient was a man with hepatitis C–associated cirrhosis and hepatocellular carcinoma. At the time of transplantation, he received intravenous methylprednisolone to induce immunosuppression; at the time of discharge, he received tacrolimus, prednisone, and mycophenolate mofetil to maintain immunosuppression. Thirteen weeks after transplant, the patient visited an outpatient clinic and reported bilateral upper extremity tremor, which was thought to result from elevated tacrolimus levels. A week later, the patient was readmitted to the hospital after an outpatient visit for right lower extremity swelling and shortness of breath; the ultimate diagnosis was deep vein thrombosis and pulmonary embolism. During this hospitalization, the recipient reported lightheadedness, headache, blurry vision, and continued tremor. Because of his neurologic symptoms and identification of microsporidiosis in the deceased right kidney recipient, this patient was also empirically given albendazole. The recipient declined lumbar puncture. Subsequently, microsporidia were identified in a urine specimen by trichrome staining performed at the patient’s local hospital (Figure 3, panel A). PCR of the urine specimen confirmed infection with E. cuniculi. Microsporidia were also isolated from the urine specimen at CDC after 2 months of cell culture (Figure 3, panel B), and identification was confirmed by IHC and transmission electron microscopy (Figure 3, panel C). The patient’s neurologic symptoms resolved, and subsequent urine PCR for E. cuniculi was negative. Because of financial constraints, albendazole was discontinued after 4 months of therapy. Repeated PCRs of urine over the next year remained negative for microsporidia. As of December 2015 (14 months after stopping albendazole), the patient remained well without symptoms of microsporidial infection.

Organ Donor

The donor was a middle-aged woman, originally from Mexico but a resident of the United States for several decades. In December 2013, she experienced recurrent and persistent headache, which she attributed to a known history of migraine. The headaches persisted and progressed over several weeks to include nausea, pulsatile tinnitus, and diplopia. Approximately 4 weeks later, she sought care at an emergency department, where brain computed tomography and magnetic resonance imaging revealed several arteriovenous malformations and a right internal carotid saccular aneurysm. She underwent endovascular repair with stenting and coil embolization. This repair was complicated by several episodes of intracranial hemorrhage requiring craniotomy. Despite surgical intervention, her neurologic function continued to decline, and she was eventually declared brain dead. No other symptoms were reported before organ donation. Review of her medical record revealed no reports of gastrointestinal illness during her hospitalization. Interviews with next of kin revealed no clear risk factors for microsporidiosis (e.g., exposure to potentially contaminated water, travel to areas with potentially contaminated drinking water), no gastrointestinal illness before her death, and no travel outside the United States within the previous 12 months.

Because no autopsy was performed on the donor, the only donor specimen available for retrospective testing was archived serum. This sample was tested by microsporidia IFA; a positive titer of 1:2,048 suggested active infection (13,14).