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A Surviving Case of Acanthamoeba Granulomatous Amebic Encephalitis in a Hematopoietic Stem Cell Transplant Recipient

Patient: Male, 32-year-old Final Diagnosis: Granulomatous amebic encephalitis Symptoms: — Medication:— Clinical Procedure: — Specialty: — Objective: Challenging differential diagnosis Background: Acanthamoeba are free-living amoebae with potential to infect immunocompromised hosts. The mortality rate of granulomatous amebic encephalitis (GAE) due to Acanthamoeba exceeds 90% and there are currently no reports of survival of this infection in recipients of hematopoietic stem cell transplant. Case Report: We report herein the case of a 32-year-old man presenting to our service with abrupt neurological deterioration and seizures 5 months after allogeneic stem cell transplantation for Hodgkin lymphoma. Clinical and imaging findings were non-specific at presentation. Multiple circumscribed, heterogenous, mass-like lesions were identified on MRI. Brain biopsy was performed and revealed multiple cysts and trophozoites suggesting a diagnosis of granulomatous amebic encephalitis. PCR testing confirmed Acanthamoeba. Treatment with miltefosine, metronidazole, azithromycin, fluconazole, pentamidine isethionate, and co-trimoxazole was instituted and the patient survived and shows continued improvement with intensive rehabilitation. Conclusions: We report the first successful outcome in this setting. The diagnosis would have been missed on cerebrospinal fluid analysis alone, but was rapidly made by histological analysis of brain biopsy. This diagnostically challenging infection is likely under-recognized. Early brain biopsy and commencement of a prolonged miltefosine-containing anti-ameba regimen can be curative.


Background
Granulomatous amebic encephalitis is a rare central nervous system infection that is usually rapidly fatal [1,2]. Symptoms are myriad, and reflect the area of brain infected. Typically, the onset of symptoms is insidious until overwhelming infection results in rapid severe neurological decline, including seizures, altered levels of consciousness, coma, and death. Diagnosis is challenging due to the non-specific clinical presentation and radiological features. Brain biopsy is generally required for diagnosis, with cerebrospinal fluid analysis and culture yielding false-negative results in the majority of cases [2]. The optimal treatment for this infection has not been described. Herein, we report a case in which diagnosis was promptly made by early brain biopsy and a prolonged combination anti-amebic treatment approach was instituted with successful outcome.

Case Report
A 32-year-old male engineer presented 5 months after allogeneic hematopoietic stem cell transplant for chemotherapyrefractory Hodgkin lymphoma with a 4-day history of headache, difficulty finding words, and unable to read back cell phone texts which he had just completed. Collateral history confirmed he had driven through a red light he did not notice, and noted episodes of staring with speech arrest. Neurological examination confirmed anomic aphasia with compensatory circumlocution, and alexia without agraphia (unable to read despite retaining the ability to write). Visual field testing revealed right homonymous superior quadrantanopia.
He was diagnosed 2 years prior to this presentation with stage IVB classical Hodgkin lymphoma and international prognostic score (IPS) of 5. Interim positron emission tomography (PET) following 2 cycles of Adriamycin, Bleomycin, Vinblastine, and Dacarbazine (ABVD) chemotherapy was Deauville score 4, and treatment was intensified to the dose-escalated Bleomycin Etoposide Adriamycin Cyclophosphamide Oncovin Procarbazine Prednisolone (BEACOPP) regimen for 2 cycles, resulting in a persistent Deauville 4 score. The patient received salvage therapy with Brentuximab vedotin and Ifosfamide Carboplatin Etoposide (ICE) for 3 cycles, achieving a complete remission (Deauville score 1) which was consolidated with a matched unrelated allograft using Carmustine/BCNU Etoposide Cytarabine Melphalan (BEAM) and Alemtuzumab conditioning. He engrafted on day 12 and his post-transplant course was complicated by steroid-responsive grade 3 skin graft versus host disease. The patient was PET-negative and a 100% total and CD3 donor chimera on day 100.
At the time of this acute neurological presentation on day 160, he remained profoundly immunosuppressed. His medication consisted of valaciclovir 500 mg once daily, atovaquone 750 mg twice daily, phenoxymethylpenicillin 333 mg twice daily, prednisolone 10 mg once daily (for skin graft versus host disease), and ciclosporin 50mg twice daily. He reported adherence to all medications. His absolute lymphocyte count was 0.1×10 9 /L with absolute CD4+ T cell count 51×10 6 /L. MRI brain revealed multiple circumscribed heterogenous masslike lesions in the left occipital and right frontal lobes, with further areas of edema in the splenium of the corpus callosum and left temporal lobe, which correlated with his alexia without agraphia and right superior quadrantanopia, respectively ( Figure 1A). Cerebrospinal fluid (CSF) analysis revealed elevated white cell count of 42/mm 3 (reference 0-5 WBC/mm 3 ) and elevated protein 1.49 mg/dL (reference 0.15-0.45g/L) ( Table 1). CSF microscopy was negative. The patient was rapidly deteriorating neurologically, and electroencephalogram was not possible, but an urgent brain biopsy was performed. The histological features were consistent with GAE, with Acanthamoeba spp. favored as the most likely causative organism based on morphological grounds (Figure 2), in the context of immunosuppression and due to the presence of intra-axial mass lesions. CSF PCR was negative for Acanthamoeba, Naegleria, and Balamuthia, but PCR of brain tissue was positive for Acanthamoeba species.
The Acanthamoeba trophozoites and cysts noted on brain biopsy led to a change in treatment from initial empiric antimicrobial cover to fluconazole 12 mg/kg once daily po, pentamidine isethionate 4 mg/kg 4 times daily iv, metronidazole 500 mg 3 times daily iv, azithromycin 250 mg once daily po, co-trimoxazole 5 mg/kg twice daily IV, and miltefosine 50 mg 3 times daily po.
The patient deteriorated abruptly on day 9 of admission (day 5 of anti-amoebic therapy). Episodes of speech arrest became more frequent and he became disoriented to place and time. On day 8 of admission, serum sodium had been 139 mEq/L (139 mmol/L) (reference range 136-145 mEq/L, 136-145 mmol/L). On the evening of day 9, this fell to 129 mEq/L (129 mmol/L) in tandem with worsening symptomatology. Eslicarbazepine 400 mg qd and clobazam 5 mg bid anticonvulsant therapy was added, but on day 10, the patient had a generalized tonic-clonic seizure associated with a precipitous fall in serum sodium to 110 mmol/L. He was treated with hypertonic (3%) saline with temporary omission of other infusions, including pentamidine isethionate, given its potential to aggravate hyponatremia. Repeat MRI brain revealed significantly worsened vasogenic edema with resultant mass effect ( Figure 1B). Risk of transforaminal herniation was deemed to be high based on the imaging, and dexamethasone 8 mg tid IV was instituted to ameliorate edema. Clinical and laboratory parameters were consistent with Syndrome of Inappropriate Anti-Diuretic Hormone secretion (SIADH) as a cause of this abrupt severe hyponatremia -the onset was 9 days after brain biopsy; serum osmolarity was 280 mmol/kg and urinary osmolarity and sodium were both inappropriately high at 556 mmol/kg and 178 mmol/L, respectively. The patient responded to hypertonic saline, with normalization of serum sodium and correction of both urinary osmolarity and sodium over the course of 11 days.
In total, our patient was hospitalized for 53 days. Increasing nausea limited tolerance of the anti-amebic regimen and in total he completed 21 days of pentamidine isethionate, 28 days of co-trimoxazole and azithromycin, 60 days of metronidazole, 120 days of fluconazole, and 150 days of miltefosine. Maintenance therapy of miltefosine 50 mg 3 times weekly was planned but  Figure 1C).
Persistent language deficits, including anomic aphasia and alexia, on discharge from hospital reflected left temporoparietal and callosal injuries. He underwent an outpatient neuro-rehabilitation program, with significant improvement in attention and recall. At 12 months after presentation, he is performing well in language and reading, with minor residual difficulty in naming low-frequency items, but improved fluency and fewer word-finding pauses. He displays excellent procedural memory for complex skills learned previously including engine repair, which he has resumed as a hobby. Our patient shows ongoing improvement and remains in complete remission 1 year after transplant for Hodgkin lymphoma.

Discussion
Granulomatous amoebic encephalitis (GAE) is a rare central nervous system infection caused by free-living amoebae and is associated with a high mortality rate. Three genera of pathogenic free-living amoebae are known to infect human hosts: Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri [1,2]. Only the former 2 genera cause GAE and have 2 distinct life-cycle stages -the infectious trophozoite and dormant cyst stages [1,2]. Although Acanthamoeba are found ubiquitously in the environment, there are fewer than 50 reported cases of acanthamebic GAE in the literature, although it is likely that the true incidence is underestimated due to a failure to recognize and diagnose the infection. At the time of writing, we are aware of only 10 reported cases of acanthamebic GAE in patients after hematopoietic stem cell transplant [3][4][5][6][7][8][9][10][11]. In each of these cases, the infection proved fatal, and in 8 of the 10 cases the diagnosis was established only following histologic examination of autopsy specimens [3][4][5][6][7][8][9].
The source of the Acanthamoeba infection was not obvious in the case reported herein. Our patient previously used disposal contact lenses for correction of myopia but ceased using them prior to undergoing hematopoietic stem cell transplantation and had no symptoms or signs of keratitis. Acanthamoeba spp. are thought to enter the host via the lower respiratory tract or olfactory epithelium and spread hematogenously to the central nervous system [2] and this is the most likely mode of infection in our patient. The amoebae induce an inflammatory response and adhere to endothelial cells in the microvasculature via mannose binding protein to disrupt tight junctions and permeate the blood brain barrier [12,13]. Acanthamoeba spp. also produce proteases and toxins that damage tight junctions, connective tissue, and neurons [1,14]. The onset of clinical symptoms is insidious. Typical features include headache, nausea, irritability, and low-grade fever. Other clinical features depend on the area of brain affected, with a literature review indicating that the frontal and temporal lobes are the most commonly affected areas in Acanthamoeba GAE [15]. GAE is progressive, resulting in seizures, raised intracranial pressure, altered mental status, coma, and death. Diagnosis is challenging, with culture or isolation of Acanthamoeba from CSF of affected individuals proving difficult [2] and, in this case, had biopsy not been performed, the diagnosis would have been missed based on CSF analysis alone.
The optimal approach to the management of Acanthamoeba GAE is unknown. Drugs with in vitro activity that have resulted in successful outcomes for a small number of patients include rifampicin, azole anti-fungal drugs, pentamidine isethionate, sulfadiazine, flucytosine, azithromycin, caspofungin, and miltefosine. The Center for Disease Control and Prevention recommend a combination approach with miltefosine, pentamidine isethionate, and fluconazole. Miltefosine is an alkylphosphocholine drug that was initially developed as an anticancer drug but found a niche in the treatment of parasitic infections, in particular leishmaniasis [16]. Later, in vitro activity against Acanthamoeba was observed [17]. The mechanism of activity is thought to be the disruption of lipid-dependent cell signalling pathways in the parasite cell membrane.
The introduction of miltefosine-containing combinations for the treatment of acanthamebic GAE is thought to have improved survival, at least in immunocompetent patients [18,19] and a case of successful treatment of a heart transplant recipient is reported [20]. In general, miltefosine is well tolerated, with manageable gastrointestinal symptoms constituting the chief adverse effect reported. As yet, there has been no clear guidance regarding the optimal duration of therapy, but continued treatment until radiological evidence of the absence of active disease and a normal CSF examination is advisable. It is likely that, as in our patient's case, several months of treatment will be required to secure a successful outcome.
Our patient is the first reported case of survival following acanthamebic GAE in the setting of profound immunosuppression after hematopoietic stem cell transplantation. Prompt biopsy and institution of an appropriate miltefosine-containing regimen led to a successful outcome following a prolonged treatment course and intensive rehabilitation.

Conclusions
This case illustrates the importance of early brain biopsy in a profoundly immunosuppressed patient with unexplained neurological deterioration, as the diagnosis would not have been made if relying on CSF analysis alone. Granulomatous amebic encephalitis is a rapidly fatal infection that is likely under-recognized. Early recognition and prompt institution of appropriate miltefosine-containing anti-amebic regimen can secure a successful outcome, even in profoundly immunosuppressed patients.

Conflicts of interest
None.