Cladophialophora encephalitis in an alpaca

Abstract

A 4-year-old Huacaya hembra was evaluated for acute neurologic signs including recumbency and a left head tilt. Cranial nerve examination revealed a left ear droop, muzzle deviation to the right, mydriasis of the left eye, an absent menace response, bilateral absent pupillary light reflex when light was directed into the left eye, and bilateral horizontal nystagmus with fast phase to the right. Multifocal intracranial lesions were suspected. Computed tomography revealed an intracranial mass. Postmortem examination, histopathology, and sequencing of a polymerase chain reaction product confirmed a diagnosis of phaeohyphomycotic meningoencephalitis caused by Cladophialophora bantiana.

Key clinical message:

Advanced diagnostic imaging (computed tomography) was useful in achieving a diagnosis of an intracranial mass in an alpaca with acute neurological signs, later confirmed to be central nervous system (CNS) phaeohyphomycosis. Although uncommon, intracranial fungal infection should be considered as a differential diagnosis in camelid patients exhibiting CNS signs, particularly if they do not respond to initial antimicrobial and anthelmintic therapy.

Phaeohyphomycosis is a serious and life-threatening infection that has been reported in several species. This report describes the clinical and diagnostic assessment, and advanced imaging findings from an alpaca with an unusual cause of neurological disease. The alpaca did not respond to treatment for common camelid neurological disorders. Ultimately, postmortem histopathological and fungal polymerase chain reaction (PCR) provided a definitive diagnosis of central nervous system phaeohyphomycosis.

Case description

A 4-year-old white Huacaya hembra (80 kg) was referred to the University of Georgia’s Veterinary Teaching Hospital Large Animal Emergency Service for evaluation of acute onset neurologic signs. No abnormalities were noted the evening before presentation when the herd was observed. Approximately 4 h before presentation, the alpaca was reported to be recumbent with a left head tilt, altered mentation, anorexia, and possible blindness. The owners administered flunixin meglumine (88 mg, IM), fenbendazole (50 mg, PO) and brought the alpaca to the University of Georgia Veterinary Teaching Hospital for further evaluation. The alpaca was presumed to be in month 9 of gestation. She resided in a fenced, treeless pasture with a herd of 26 other alpacas, none of which exhibited similar clinical signs. She was born in Georgia and had no travel history outside the state. She ate primarily orchard grass hay and was regularly dewormed and had been vaccinated for viral encephalitidies including West Nile virus and Eastern equine encephalitis. She was not vaccinated for rabies virus.

Physical examination at admission revealed a rectal temperature of 37.8°C [reference range (RR): 37.5°C to 38.9°C], a heart rate of 56 beats/min (RR: 60 to 90 beats/min), and a respiratory rate of 24 breaths/min (RR: 10 to 30 breaths/min). The alpaca had moist, pink mucous membranes with a capillary refill time of < 2 s. Cardiothoracic auscultation did not reveal any cardiac murmurs, crackles, or wheezes. Gastrointestinal borborygmi were present but reduced, and fecal passage was not noted.

Neurologic examination revealed a recumbent, but alert and responsive patient with a left head tilt. She had a left ear droop and muzzle deviation to the right. Cranial nerve examination revealed mydriasis of the left eye, an absent menace response on the left, an absent direct and consensual pupillary light reflex (PLR) when light was directed into the left eye, and bilateral horizontal nystagmus with fast phase to the right. Direct and consensual PLRs were normal when light was directed in the right eye. Palpebral reflexes were normal bilaterally.

Several hours after presentation, the alpaca was able to ambulate but circled to the left. Mild ataxia was noted in all 4 limbs but upon handling, the patient’s neurologic signs severely worsened and she became recumbent, precluding a more detailed gait analysis. Based on neurological evaluation, multifocal localization of lesions was suspected. Deficits suggested lesions involving the left retina or left optic nerve and less likely right rostral optic tract (based on mydriasis, absent menace, and absent PLR in the left eye); left cranial nerve VII (based on left ear droop and muzzle deviation to the right); and the left vestibular system (based on left head tilt, circling to the left, and horizontal nystagmus with fast phase to the right). Further localization to the central vestibular system was suspected because multifocal localization typically results from a central rather than peripheral localization. However, confirmation of peripheral versus central vestibular system localization based on examination was not possible. Although the owner described an abnormal mentation, which would suggest a central localization, the alpaca’s mentation was thought to be normal in the hospital. Additionally, gait and postural reactions could not be assessed. Although the presence of a vestibular ataxia would not differentiate a peripheral versus central localization, a concurrent proprioceptive ataxia would have been suggestive of a central lesion affecting the brainstem.

Differential diagnoses included infection of the meninges or brain (bacterial, viral, or parasitic), neoplasia, trauma, otitis interna/media, and thiamine deficiency. Of infectious diseases, the most common causes of neurological abnormalities in alpacas include the meningeal worm Parelaphostrongylus tenuis, meningoencephalitis caused by the bacterium Listeria monocytogenes, viral infections such as West Nile virus and equine herpesvirus 1, or bacterial otitis media. Thiamine deficiency was considered unlikely as a primary cause because of the asymmetry of clinical signs and a responsive mentation.

On presentation, complete blood (cell) count (CBC), serum biochemical analysis, packed cell volume, and total solids were determined. Abnormalities included an elevated lactate (6.3 mmol/L, RR: 0 to 1.9 mmol/L), an increased glucose (12.66 mmol/L, RR: 4.11 to 8.55 mmol/L), and an increased creatinine (336 μmol/L, RR: 123.8 to 282.9 μmol/L). Results were consistent with a stress response in a struggling, recumbent alpaca (increased lactate and glucose), and prerenal or renal azotemia (increased creatinine).

Transabdominal ultrasound was performed to assess the pregnancy but a fetus could not be visualized; loss of the pregnancy was later confirmed by the theriogenology service. Both eyes were stained with fluorescein and a small area of focal uptake was visualized in the left eye, consistent with a superficial corneal ulcer likely from presumptive self-trauma. Tear production appeared adequate but was not objectively measured; therefore, the presence of neurogenic keratoconjunctivitis sicca was not ruled out.

Medical treatment was initiated to target the top differential diagnoses of listeriosis, Parelaphostrongylus tenuis, and otitis media. Treatment with antimicrobials Penicillin G potassium (Sandoz, Princeton, New Jersey, USA), 22 000 IU/kg body weight (BW), IV, q6h, florfenicol (Nuflor; Merck Animal Health, Madison, New Jersey, USA), 20 mg/kg BW, IM, q48h, and the anthelmintic fenbendazole (Panacur; Merck Animal Health, Summit, New Jersey, USA), 50 mg/kg BW, PO, q24h, was instituted. For the treatment of Listeria monocytogenes, monotherapy with penicillin is considered efficacious and combination therapy with an amphenicol and a beta-lactam antibiotic is generally avoided because of antagonistic effects. However, broad-spectrum activity was desired for treatment of possible otitis and penicillin did not provide adequate Gram-negative coverage. Gentamicin was not used as it is contraindicated in an animal with increased creatinine. Enrofloxacin was not used because of potential adverse effects on a fetus. A recent study in horses suggests that adverse musculoskeletal effects do not occur in foals from mares treated with enrofloxacin (1), but to the authors’ knowledge no data are available in camelid species. Thiamine (Henry Schein, Dublin, Ohio, USA), 10 mg/kg BW, IV, q12h, was administered for the prevention of polioencephalomalacia, as the alpaca’s appetite was reduced. Additionally, a gastroprotectant was administered (pantoprazole sodium, Protonix injection; Pfizer, Philadelphia, Pennsylvania, USA), 1.1 mg/kg BW, IV, q24h, and transfaunate from the hospital’s ruminant donor was administered via a nasogastric tube once daily. An ophthalmic ointment containing neomycin, polymyxin, and bacitracin was instilled in the left eye every 6 h. An intravenous jugular catheter was placed and the alpaca was maintained on balanced isotonic crystalloid fluids (30 mL/kg BW per day).

The morning after presentation, CBC, serum biochemical analysis, and triglycerides were determined. Results were all within normal reference intervals excluding an increased creatinine (300 μmol/L) and an increased creatine kinase (4368 IU/L, reference range: 14 to 238 IU/L). The patient was continued on the aforementioned medical treatment. Serial creatinine levels were performed daily (274 μmol/L and 212 μmol/L) until they were normal.

Three days after presentation, the alpaca showed no additional improvement despite therapy. A non-contrast and contrast-enhanced computed tomography (CT) study of the head was performed with a 64-slice helical CT scanner (contiguous 2.0 mm-thick slices). Computed tomography revealed an ill-defined mass in the region of the right thalamus that extended caudally, ventral to the falx tentorium, to the level of the cerebellum (3.8 cm L × 2.7 cm H × 2.1 cm W) causing a leftward midline shift (Figure 1A). The mass was heterogeneous and slightly hyperdense (HU 45) compared to surrounding gray and white matter on pre-contrast images. Contrast agent (Iohexol, Omnipaque350, 2.2 mL/kg; GE Healthcare, Oslo, Norway), 350 mg/mL, IV, was administered. Following contrast administration, there was peripheral contrast enhancement (HU 60) and an irregularly marginated central area that was non-enhancing (HU 34) (Figure 1B). The mass caused dorsal and lateral compression of the right lateral ventricle. Additionally, there was an oblique fracture of the right first mandibular incisor at the level of the gingival margin, likely due to trauma the alpaca suffered during hospitalization from ataxia and blindness.

Figure 1.

A — Transverse CT image (soft tissue displace algorithm) shows an ill-defined mass (yellow arrows) in the region of the right thalamus that extended caudally, ventral to the falx tentorium to the level of the cerebellum and caused a leftward midline shift. The mass was heterogenous, slightly hyperdense and caused dorsal and lateral compression of the right lateral ventricle. B — Transverse, C — sagittal, and D — dorsal CT images (soft tissue displace algorithm) showing the ill-defined mass previously noted in A is now contrast enhancing, consistent with an abscess or a granuloma.

Following CT, differential diagnoses were modified to include abscess or granuloma (bacterial, parasitic, fungal) or neoplasia. While under general anesthesia, cerebrospinal fluid was collected from the lumbosacral space. The fluid was visually normal and was within normal reference values excluding a mildly elevated protein content (0.53 g/L; RR: 0.38 to 0.48 g/L) (2). The fluid had 0 WBC/μL and 197 RBC/μL. A cytospin of the fluid had 5 large mononuclear cells, 1 small lymphocyte, and 1 non-degenerate neutrophil in 1 field (1000× magnification). These findings were consistent with blood contamination in the sample and therefore did not assist in achieving a diagnosis.

Following extubation during recovery from anesthesia, the alpaca developed acute cardiopulmonary arrest. Cardiopulmonary resuscitation was attempted but efforts to revive the alpaca were unsuccessful. A post-mortem examination revealed no external abnormalities. In the lungs, alveolar septa were diffusely congested, consistent with attempted cardiopulmonary resuscitation following arrest. On cross section of the brain, there was a brown to black, necrotic lesion within the right brainstem, extending from the thalamus to the caudal pons/rostral medulla (Figure 2A). Histopathology of the CNS mass confirmed a large central area of necrosis that unilaterally effaced 60% of the gray and white matter and compressed the adjacent neuroparenchyma (Figure 2B). Centrally, admixed with the necrosis and extending out into surrounding parenchyma were numerous brown yeast and hyphae, surrounded by numerous neutrophils, epithelioid macrophages, and rare multinucleated giant cells. The yeasts were ovoid, 7 to 20 μm in diameter, with 2 to 3 μm-thick, dark brown cell walls, clear to pale brown cytoplasm, and a central basophilic nucleus. Hyphae were 5 to 10 μm wide, septate with irregular, dichotomous and nondichotomous, acute angled to right angled branching and thin, pigmented, nonparallel walls. Adjacent to the previously described area, the vessels were surrounded by a moderate number of lymphocytes and plasma cells. The previously described inflammation was also present within the meninges. The inflammation and necrosis extended from the level of the hippocampus to the level of the pons. Fungal PCR and sequencing were performed from the fixed brain mass as previously described (3). Forward and reverse sequences were aligned to the NCBI non-redundant nucleotide database using the online NCBI BLAST, and were aligned to the CBS-KNAW database using its online alignment tool. Results indicated a 100% similarity of the 611-base pair (bp) internal transcribed sequence 1 (ITS1) region sequence and a 99% similarity of the 581 bp large-subunit ribosomal D1/D2 region sequence to Cladophialophora bantiana. Results were consistent with the organisms seen histologically. Postmortem examination, histopathology, and PCR sequencing confirmed a diagnosis of phaeohyphomycotic encephalitis caused by Cladophialophora bantiana.

Figure 2.

A — Prosencephalon at the level of the thalamus and hippocampus. There is a large, brown to black central area of necrosis that unilaterally effaces 60% of the gray and white matter and compresses the adjacent neuroparenchyma. B — Thalamus 1000 × H&E stain; bar = 50 μm. Yeast cells (arrow) are ovoid, 7 to 20 μm in diameter, with dark brown cell walls, clear to pale brown cytoplasm, and a central basophilic nucleus. Hyphae (arrowhead) are 5 to 10 μm wide, septate with irregular, dichotomous and nondichotomous with acute angled to right angled branching, and thin, pigmented, nonparallel walls.

Discussion

This case demonstrates that advanced diagnostic imaging of the brain of camelids is a feasible clinical option, and in this case imaging was useful in identifying a mass within the brainstem. In camelid species, advanced imaging may not be frequently pursued, and financial constraints may be a concern. However, when intracranial disease is suspected, when there is lack of resolution of clinical signs with medical management, or when CSF analysis does not assist in achieving a diagnosis, advanced imaging should be considered. Computed tomography and magnetic resonance imaging are common imaging modalities of the head used in small animal and equid species (4,5) and are less commonly described in clinically affected camelids (6). Computed tomography has been used in these species to successfully diagnose intracranial masses or abscesses, otitis media/intern, head trauma, encephalitis, and hydrocephalus (6).

In this case, imaging with CT and post-mortem evaluation of the alpaca’s brain revealed a right-sided abscess (with corresponding midline shift) extending from the thalamus to the rostral medulla. It is difficult to fully resolve the expected neuroanatomic lesion localization based on the alpaca’s clinical signs with the actual location of the lesion. The lack of menace response on the left and lack of direct or consensual PLR when light was directed into the left eye would typically be the result of a lesion of the left retina or optic nerve, although it is possible the lesion in the right thalamus caused these signs by affecting the right rostral optic tract. Thalamic infarcts in dogs have also been described to cause contralateral vestibular signs (7). However, such a thalamic lesion still cannot explain the left-sided facial paresis seen in this alpaca. Ultimately, the left retina and optic nerve as well as rostral medulla were not evaluated histopathologically so it is possible that the clinical presentation is the result of unidentified multifocal lesions.

The imaging characteristics and location of the lesion provided useful diagnostic information as the lesion was not consistent with viral encephalitides, primary meningitis, trauma, otitis interna/media, or thiamine deficiency and allowed us to rule out these potential causes. Based on previous descriptions in human patients, early diagnosis and treatment of fungal granulomas is key for achieving a better prognosis (8); in this alpaca, the extent of the lesion likely indicated that the infection was advanced, and the prognosis was considered poor. This patient went into cardiopulmonary arrest following anesthesia and treatment unfortunately could not be attempted. If she had not succumbed, CT-guided aspiration, though risky, could have been attempted and may have been useful to definitively identify the etiology of the mass and direct therapy.

This is the second reported case of CNS Cladophialophora bantiana in an alpaca, and the first report to describe clinical signs associated with the infection. There is 1 other case report in a large animal (9,10). In Frank et al (9), the alpaca was found dead in a pasture and post-mortem testing identified a CNS abscess caused by C. bantiana. The C. bantiana infection reported in the horse was an infection in uterine fluid which resulted in fungal endometritis that was unsuccessfully treated (10).

Dematiaceous infections are caused by darkly pigmented fungi with melanin-containing cell walls. Infections caused by this group of fungi, although uncommon and opportunistic, can cause multiple syndromes and a variety of clinical signs. Three distinct syndromes of dematiaceous fungal infections, with specific gross and histological characteristics in the host tissue, have been described. These syndromes include chromoblastomycosis (pigmented fungi but no hyphae), dematiaceous eumycotic mycetoma (pigmented hyphae in aggregates inside discrete granulomas), and phaeohyphomycosis (pigmented hyphae that are widely separated and often in macrophages) (9,11–13). Case reports describe various clinical presentations in small animal domesticated species and humans including cutaneous or subcutaneous granulomas to disseminated infections and brain abscesses (11–15).

The route of infection is still poorly understood, but inhalation of spores and exposure from ocular or cutaneous infections leading to eventual hematogenous spread has been suggested (14,16). Central nervous system phaeohyphomycosis has been attributed to at least 24 fungal organisms, the most common of which are Cladophialophora bantiana, Exophilia dermatitidis, Ramichloridium mackenziei, and Ochroconis gallopavum (14). The most recognized CNS phaeohyphomycosis in dogs, cats, and humans is caused by C. bantiana (14). Cladophialophora bantiana is considered primarily neurotropic, and infection with this fungus accounts for up to 48% of human cases and most in dogs and cats (14). Unlike other fungal infections, patients that suffer from these CNS fungal infections are often immunocompetent and have no known underlying disease. This is especially true in cases of brain abscesses, which are typically fatal (12,14).

Treatment of CNS phaehyphomycosis in any large animal species has not been reported. In other species, described treatments of intracranial lesions are consistent regardless of the type of inflammation associated with the lesion. In small animal species and humans, CNS phaeohyphomycosis is often fatal, but successful surgical debulkment combined with antifungal therapy has been reported (12,15,17,18). In 1 study, surgical debulkment of cerebral phaeohyphomycosis combined with antifungal treatment (fluconazole for 4 mo followed by voriconazole for an additional 10 mo) was successful in treatment of a dog (17). In a case of eumycetoma caused by C. bantiana, surgical debulkment with oral itraconazole for 6 wk, followed by oral flucytosine and itraconazole for 4 wk subsequently showed no evidence of recurrence at the 10-month follow-up (15). Itraconazole is the most effective antifungal agent for treatment in chromoblastomycosis and subcutaneous phaeohyphomycosis; voriconazole, or posaconazole showed the best ability to treat CNS fungal abscesses (18,19). In vitro antifungal susceptibility of 13 isolates showed good susceptibility to voriconazole, posaconazole, and itraconazole in 1 study (20). Outcome data in large numbers of clinical patients treated with these agents are lacking. When treating infections, it is recommended that a sample be obtained to perform in vitro susceptibility tests, but antifungal susceptibility tests may not correspond well with clinical efficacy. Additionally, obtaining samples from patients with intracranial lesions can pose significant risk.

The case described herein provides clinical and diagnostic assessment, advanced imaging of the lesion, and postmortem histopathological and fungal PCR results. Notable features of this case include lack of response to empirical treatment directed at commonly diagnosed neurological diseases of alpacas. This case serves as a reminder to consider phaeohyphomycosis as a differential diagnosis in alpacas with central neurological disease, especially in cases refractory to treatment for other more common causes. Advanced diagnostic imaging should be considered in cases in which there is concern for intracranial disease, as imaging can provide additional valuable diagnostic information.