Serial MRI and CSF Analysis in a Dog Treated with Intrathecal Amphotericin B for Protothecosis

Introduction

Protothecosis is a rare to uncommon disease of dogs, cats, cattle, and people.^1,2 Since its discovery in 1964, there have been 31 cases reported in the dog and 107 in humans.³ Prototheca spp. are unicellular achlorophylic aerobic algae found worldwide. In the United States they are most commonly found in the southeastern United States.¹ There are three recognized species of Prototheca. P. zopfii and P. wickerhami are most commonly identified in dogs with disseminated disease.⁴ Prototheca spp. are often a transient contaminant found in both raw and treated sewage, slime-flux of trees, and human and animal waste.^1,2 Infection in humans and cats often results in cutaneous lesions only, which are treatable.^1,2,4–7 In contrast, dogs develop a fatal form of infection that starts with gastrointestinal signs (typically hemorrhagic diarrhea) that disseminates to multiple organ systems, including the central nervous system, heart, eyes, and kidneys.^1,7 Diagnosis is made based on isolation of the organism from tissues or body fluids. Species differentiation can be made by either sugar and alcohol assimilation tests or fluorescent antibody testing.¹ Disseminated protothecosis treatments in veterinary medicine have focused on the use of antifungal agents used either alone or in combinations. Most commonly these agents are amphotericin B (AMB) and itraconazole. Protothecosis is difficult to treat, and a cure has not been reported.^1,8,10,13 Infection with P. wickerhami in species other than the dog seems to be more responsive to treatment. The combination of surgery, antifungals, and antibiotics have proven succesful.^1,14 Fluconazole has increased central nervous system penetration compared with AMB and itraconazole; however, it has not been used effectively against P. zopfii.¹⁵ The use of intrathecal AMB has been mentioned in the treatment of fungal disease in both human and veterinary literature.^1,14–16 Specifically, protothecal meningitis has been successfully treated in a human patient with intrathecal AMB.¹⁴ The purpose of this case report is to report the MRI and CSF findings as well as the response to intrathecal AMB in a dog with protothecosis.

Case History

A 3 yr old spayed female English setter mixed-breed dog presented for evaluation of a right head tilt, ataxia, lethargy, inappetence, loss of muscle mass, weight loss, and chronic diarrhea/mixed bowel diarrhea. The patient lived in Virginia and was known to have ingested stagnant water prior to the development of clinical signs. The patient's vaccines for rabies and distemper were current. Six weeks prior to presentation the dog was treated by the primary veterinarian with metronidazole (23.4 mg/kg per os [PO] q 12 hr) and an unknown brand of probiotic. No response was noted over the ensuing 2 wk. Two weeks prior to referral, the primary veterinarian prescribed meclizine (1.5 mg/kg PO q 6 hr) and enrofloxacin (8.5 mg/kg PO q 24 hr) for ataxia and a right head tilt. Ten days prior to referral the meclizine and enrofloxacin were discontinued and doxycycline (9.3 mg/kg PO q 24 hr) and IV dexamethasone (dose unknown) were initiated. A transient improvement in the degree of the head tilt, anorexia, and activity level were noted. The doxycycline and probiotics were continued until the time of referral.

On initial physical examination at the time of referral, the patient had a body condition score of 1/5 (body weight was 15.9 kg), a dull hair coat, and fecal staining of the perineal area. Neurologic examination revealed a dull mentation, mild tetraparesis, side stepping to the right, a right head tilt, and a poor gag response. Together, these findings suggested a predominantly right lateralizing caudal brainstem lesion. No other significant neurologic abnormalities were noted. A multifocal infectious, noninfectious inflammatory, or neoplastic process affecting both the gastrointestinal system and brain were thought to be more likely than two distinct disease processes. A complete blood cell count was normal and serum chemistry showed only a moderate hyperglobulinemia (4.3 g/dL, reference range, 1.6–3.6 g/dL). Three-view thoracic radiographs revealed no obvious abnormalities. MRI of the brain was performed with a 1.5 T high field magnet^a. The dog was induced with IV propofol^b administered to effect before being intubated and maintained on isoflurane^c with oxygen. The following sequences were obtained: T1-weighted pre- and postcontrast after IV administration of gadolinium^d; and T2-weighted gradient recalled echo and fluid-attenuated inversion recovery. The abnormalities observed on the T2-weighted images consisted of a mild to moderate ventriculomegaly (most notable in the fourth ventricle, but also involving the third and lateral ventricles), a mild increase of T2-weighted signal intensity in the left caudate nucleus and caudodorsal medulla, mild protrusion of the cerebellar vermis through the foramen magnum, and syringomyelia of the cranial cervical spinal cord (Figure 1 and Figures 2A, B). The T1 postcontrast images did not show contrast-enhancement of the left caudate nucleus; however, there was abnormal contrast-enhancement of the meninges. These findings were interpreted as being suggestive of an infectious, inflammatory, or infiltrative disease process leading to secondary hydrocephalus.

View Figure

• Download as Powerpoint
• Download Figure

View Figure

• Download as Powerpoint
• Download Figure

Cerebrospinal fluid (CSF) was collected from the cisterna magna immediately following MRI, and analysis revealed an elevated protein of 2,000 mg/dL (reference range, <30 mg/dL) measured using a Multistix 10 SG^k. The sample was submitted to Antech Diagnostic Laboratory for cytology. Pending the results of infectious disease titers and CSF cytology, the following treatment was initiated: prednisone^e (1.25 mg/kg PO q 24 hr), clindamycin^f (9.3 mg/kg PO q 12 hr), and famotidine^g (1.25 mg/kg PO q 12 hr). CSF titers for Toxoplasma IgG and IgM, Neospora IgG, and Cryptococcus antigen were all within the reference laboratory's normal range and considered negative. CSF analysis and cytology revealed a severe mixed pleocytosis with 111 WBCs/cm² (reference range, 0–5 WBCs/cm²) composed of 32.8% neutrophils, 40.6% lymphocytes, 24.4% monocytes, and 2.2% plasma cells. Additionally, many intracellular oval organisms each with a thin halo, granular basophilic staining cytoplasm, and measuring approximately 10–15 μm in length and 5 μm in diameter consistent with Prototheca spp. were also noted (Figure 3). Similar organisms were also noted on fecal and rectal cytology. An aerobic fungal culture of CSF submitted to North Carolina State Univeristy VTH microbiology lab demonstrated growth of P. zopfii, confirming the diagnosis of disseminated protothecosis. At this time the patient was examined by an ophthalmologist and did not have any intraocular lesions consistent with protothecosis. The prednisone dose was reduced to 0.3 mg/kg PO q 12 hr to minimize the side effects of polyuria and polydipsia and itraconazole^h (6.25 mg/kg PO q 12 hr) was initiated to address systemic infection. To increase concentrations in the CSF and to reduce nephrotoxicity, intrathecal lipid complex amphotericin Bⁱ was also initiated. The patient was anesthetized with propofol^b administered IV to effect and maintained on isoflurane^c with oxygen following intubation. The cisterna magna was clipped and prepared, a 1.5 inch 20 gauge needle was placed into the cerebellomedullary cistern, and 2 mL of CSF were removed prior to instilling AMB (0.5 mg in 5 mL 10% dextrose^j). Following injection of the AMB solution, the patient's head was held below the level of the heart and spinal cord for a period of 10 min, which has been suggested as a counter to the predominant rostral to caudal flow of CSF.¹ This process was repeated q 48 hr for a total of five treatments. A total dose of 2.5 mg of AMB was delivered intrathecally. The basis for a 0.5 mg dose/treatment was drawn from the intrathecal treatment of fungal meningitis in veterinary patients.¹⁵ Following this initial course, the owner declined further intrathecal AMB due to a perceived improvement and financial concerns.

View Figure

• Download as Powerpoint
• Download Figure

During the first 2 wk of treatment the owner reported the patient was gaining weight and muscle mass, becoming more playful, and going for longer walks. However, she continued to have intermittent episodes of bloody diarrhea, episodes of ataxia, and a subtle head tilt. Repeat neurologic examination revealed the patient had an intermittent, spontaneous, and inducible horizontal and vertical nystagmus. The owners at this time declined further intrathecal treatments with AMB due to cost. They were also concerned about the polyuria and polydipsia associated with the prednisone. The prednisone was therefore reduced to 0.3 mg/kg PO q 24 hr 10 days after treatment had been initiated. Daily IV AMB (2 mg/kg diluted in 15 mL of 5% dextrose) given in a 250 mL fluid bolus was administered until the patient reached a cumulative total dose of 30 mg/kg. During and after AMB treatment the patient was monitored by a physical examination, urinalysis, and serum biochemistry. She did not show signs of either renal or hepatic disease.

During the fourth week of treatment the owner perceived the patient to have regressed because she was anorexic, obtunded, disengaged, persistently tetraparetic, side stepping to the right, and had a more pronounced head tilt. Due to the progression of clinical signs, a second MRI study of the patient was performed using the same sequences as previously described. CSF analysis was also repeated. The CSF analysis revealed an elevated protein (1,360 mg/dL), an elevated cell count (111 WBCs/cm²), and similar numbers of Prototheca organisms on cytology; however, the cell population was predominantly lymphocytic (97% lymphocytes and 3% monocytes). MRI showed further dilation of the fourth ventricle, progressive compression of the cerebellum and brainstem, and an increased T2 signal within the left caudate nucleus and (now) within the cerebellum. There was T1 postcontrast-enhancement of the meninges and the left caudate nucleus. The left caudate nucleus was also creating a mass effect, causing deformation of left lateral ventricle (Figures 4, 5A, B). At this time the owners were given the option of reinitiating the serial intrathecal AMB treatment at a higher dose for a longer time course. Due to the recrudescence of clinical signs, progression on MRI, persistence of the organism in the CSF, and quality of life concerns, the owners elected humane euthanasia. The brain was submitted to the Department of Pathology, University of Pennsylvania, Philadelphia, PA for histopathology, which showed a moderate to severe multifocal granulomatous lymphoplasmacytic meningoencephalitis with intralesional algae consistent with infection with Prototheca (Figures 6, 7).

View Figure

• Download as Powerpoint
• Download Figure

View Figure

• Download as Powerpoint
• Download Figure

View Figure

• Download as Powerpoint
• Download Figure

View Figure

• Download as Powerpoint
• Download Figure

Discussion

Prototheca infections, although rare to uncommon in both the human and the veterinary literature, should be considered in dogs with clinical signs of persistent unresponsive diarrhea and brainstem disease, particularly in endemic areas. MRI and CSF analysis are commonly used to better characterize brainstem disease. In this case, MRI showed contrast-enhancement within the brain, dilation of the fourth ventricle, cerebellar herniation, and syringomyelia. The hydrocephalus was consistent with either an obstruction of outflow or decreased absorption of CSF, which would be expected with infection and inflammation within the meninges and ventricular system. With the clinical history and MRI findings, CSF analysis and rectal mucosal scraping and cytology are indicated. In the case of systemic Prototheca infections, these tests will likely aid in the diagnosis. Prototheca infection has also been diagnosed in the vitreous fluid and fine-needle aspiration of organs or granulomas. In this case metronidazole toxicity was considered based on the history; however, the progression of clinical signs was not consistent with toxicity, and the MRI and CSF did not support this as a possible cause. Risks associated with CSF tap may be increased in the face of cerebellar herniation or increased intracranial pressure; however, in this case the rapid progression and severe nature of the neurologic dysfunction warranted a more specific characterization of the disease process. The procedure was therefore performed with caution.

A cure for protothecosis of the gastrointestinal system and central nervous system in dogs has not been reported. There are reports of management of the disease with the combined use of AMB and itraconazole.^1,8–10 In people the disease is more commonly associated with skin infection from P. wickerhami, and successful treatment has been achieved with AMB, itraconazole, fluconazole, amikacin, and tetracyclines. Amphotericin B has been the most effective drug for control in dogs with gastrointestinal protothecosis; however, canine patients that have responded to this treatment have relapsed following discontinuation of treatment.^1,10 One case report of gastrointestinal protothecosis was treated with success while treatment was maintained; however, the disease relapsed with the discontinuation of treatment. The authors of that case report speculated that direct contact of AMB to the protothecal agent may improve the medical management of this disease.¹³ Fluconazole has increased central nervous system penetration compared with AMB and itraconazole, but fluconazole has not been reported as a successful treatment in the management of either P. zopfii or with disseminated infection.

Intrathecal AMB should provide direct exposure of the infectious agent to AMB and higher concentrations of the drug in the CNS with less risk of nephrotoxicity. The use of intrathecal AMB has been reported for the successful treatment of fungal and algal disease in both humans and animals.^13,15,16 One human patient in Japan with confirmed P. wickerhami was treated initially for intra-abdominal protothecosis with AMB, but the disease then spread to the central nervous system causing protothecal meningitis. The patient was subsequently treated with intrathecal AMB for 16 wk. The clinical signs resolved, and the patient recovered. The total dose of AMB was 9.25 mg in that patient.¹⁴ In a report of 11 human patients with coccidioidal meningitis, intrathecal AMB was also used and provided enhanced survival rates when administered at a dose of 12 mg of AMB/mo for 2 mo.¹⁵

The patient described in this report initially improved after several medications, including intrathecal AMB, were administered. The intrathecal AMB was discontinued due to cost and was instead continued IV at a higher dose. The prednisone was reduced to minimize its side effects, most notably polyuria and polydipsia. The cessation of intrathecal AMB and the reduction of prednisone correlated with the relapse of clinical signs. The transient improvement noted in this case may have been due to either the steroid therapy or from the intrathecal AMB. Prednisone and dexamethasone can reduce CSF production and diminish the inflammatory response to infectious disease; however, the CSF concentration of AMB was likely higher with intrathecal delivery, which may have reduced the number of Prototheca organisms present and been responsible for the initial improvement.¹⁷ The change in route of AMB administration would undoubtedly have reduced the CSF concentration of AMB and may have been the cause of treatment failure. A future study to evaluate intrathecal AMB CSF concentrations and its effect on Prototheca spp. may provide further insight.

MRI and CSF analysis were repeated when the patient relapsed. Persistence of the organism was noted in the CSF, and the MRI revealed progressive disease that was most notable along the ventricular system and the meninges. The MRI was important to evaluate the response to therapy and to show an apparent predilection for the ventricular system and meninges. This same predilection has been noted with fungal infections, and further studies are indicated to show if this is a repeatable MRI pattern that is consistent with central nervous system Prototheca infections. The changes in the cell lineage noted in the second CSF analysis most likely represents maturation of the immune response to include more lymphocytes than neutrophils. Serial MRI and CSF analysis has been used in human medicine to follow both novel medical treatment and disease course.^11,12 MRI allows a noninvasive and more specific evaluation of intracranial disease. To the authors’ knowledge, this is the first report describing these MRI changes and the use of intrathecal AMB in central nervous system protothecosis. Although cost may be prohibitive, serial evaluation with MRI in veterinary medicine to track the progress and response of rare and uncommon diseases may provide better insight into treatments and the disease process.

Conclusion

Infection with Prototheca should be considered whenever a patient presents for intractable diarrhea and brainstem signs. The diagnosis can be made via visualization of the organism on either rectal or CSF cytology or culture. An inflammatory disease can be suspected based on MRI findings of an obstructive ventriculomegaly, syringomyelia, and intraparenchymal contrast-enhancing lesions. Serial MRI and CSF analysis are useful for evaluating response to therapy and disease progression. Intrathecal lipid complex AMB appeared to be well tolerated in this case; however, further evaluation is warranted. Higher doses, sustained intrathecal delivery, or alternative drug therapies will likely be needed to control central nervous system protothecosis in dogs.