Equine Cryptococcal Meningitis: A Case Report and Literature Review

Allen Morris, DVM; Kyung-Il Kang, DVM; Bruce E. LeRoy, DVM, PhD, Diplomate ACVP; Frederic S. Almy, DVM, MS, Diplomate ACVP

Class of 2006 (Morris) and Department of Pathology (Kang, LeRoy, Almy) College of Veterinary Medicine, University of Georgia, Athens, GA 30602-7388

Signalment - Equine, Tennessee Walking Horse, 4-year-old Gelding

Presenting Problem - Acute blindness

History - The patient became acutely blind 5 days prior to presentation to the Veterinary Medical Teaching Hospital (VMTH) at the College of Veterinary Medicine at the University of Georgia. A period of fever and depression with possible mild ataxia was noted approximately two weeks prior to presentation at the VMTH. The fever and ataxia were treated by the referring veterinarian with flunixin meglumine for approximately one week. The fever and ataxia resolved during this time period. However, upon the development of blindness, the patient was immediately referred to the VMTH without additional therapeutic intervention.

Physical Exam - The patient was mildly febrile (37.5° C). Pulse and respiratory rates were within reference intervals. The pupillary light reflex was inconsistent in the right eye; menace responses were absent bilaterally. Both pupils were moderately dilated. These findings, together with the patient’s inability to avoid fixed objects, supported a diagnosis of acute blindness. Intraocular examination was unremarkable. The patient showed intermittent mild (I/IV) ataxia in the hindlimbs. However, a complete neurologic assessment was not undertaken due to the blindness. All other physical examination findings were unremarkable. An electroretinogram performed at the conclusion of the physical exam indicated normal retinal function in both eyes.

Additional diagnostics included a complete blood count, chemistry profile, CSF analysis and thoracic radiographs.

Diagnostics -

Laboratory Data:

Cerebrospinal Fluid (Lumbar)

CSF Cytologic Examination: A 200-cell differential was performed: 92% of the nucleated cells were neutrophils, 7% were small lymphocytes and 1% were large mononuclear cells. A few of the latter cells included leukophagocytic macrophages. Rare mast cells were present along with occasional erythrocytes. Scattered variably-sized yeasts with a thin to occasionally thick, clear capsule and narrow-based budding were observed in moderate numbers. No bacteria were seen. Based upon the morphologic characteristics of the yeasts, a poorly encapsulated variant of Cryptococcus neoformans was suspected. (See Figure 1.)

Figure 1. Cerebrospinal fluid from a horse. Note several yeasts (arrows) with clear capsule surrounded by numerous nondegenerate neutrophils. Wright stain, 1000X.

Radiographic Findings: Thoracic radiographs revealed a mild interstitial pattern with several focal soft tissue opacities in the hilar region. The opacities were presumed to be vessels, but fungal granulomas could not be excluded. Based upon both the thoracic radiographs and the cytologic features of the CSF, radiographs of the frontal sinus were performed but were unremarkable.

Transtracheal Wash: A transtracheal wash followed by upper airway endoscopy were performed due to the suspicious opacities noted on radiography and the presence of yeast in the cerebrospinal fluid. Several highly cellular direct smears were examined from the transtracheal wash fluid. The smears contained many poorly preserved respiratory epithelial cells, foamy macrophages, nondegenerate to mildly degenerate neutrophils and occasional lymphocytes. Neutrophils and macrophages contained phagocytosed bacteria. Extracellular bacteria including diploid cocci and bacilli were also observed. There were a few septate fungal hyphae. Overall findings were consistent with purulent inflammation with mixed bacterial sepsis and fungal hyphae. Fungal yeasts consistent with Cryptococcus spp. were not observed. Bacterial and fungal cultures were recommended to further characterize the etiologic agents.

Fungal Culture: No growth after 5 days.

Bacterial Culture: Not performed.

Cryptococcal antigen latex agglutination test (serum and CSF): Positive

Upper airway endoscopy: Within normal limits

Master Problem List -

1. Acute bilateral blindness

2. History of fever

3. Normocytic, normochromic anemia

4. Cryptococcal meningitis

5. Septic purulent bronchial exudate

Diagnosis

• Cryptococcal meningitis with acute bilateral blindness
• Septic purulent bronchial exudate

Discussion

The anemia was normocytic, normochromic and very mild (decreased hematocrit and red blood cell count). Anemia of established inflammation was considered most likely and was attributed to infection and inflammation of both the CNS and respiratory tract. The remainder of the CBC and biochemical profile were unremarkable; however, this is not an unusual finding with cryptococcal infections of the CNS.¹ The marked neutrophilic pleocytosis and very high protein concentration in the cerebrospinal fluid were consistent with an acute, active infection and inflammation. The presence of round yeast with a clear capsule and narrow-based budding in the CSF was consistent with cryptococcal meningitis. Radiographs indicated nodules in the lungs suggestive of fungal granulomas or prominent blood vessels. Cytologic examination and fungal culture of the transtracheal wash fluid failed to demonstrate cryptococcal yeast. However, these results do not exclude a concurrent respiratory infection since Cryptococcus often causes granuloma formulation in which the organisms are firmly adherent and sequestered in the tissue. Thus, organisms may be present but may not exfoliate during a transtracheal wash or bronchoalveolar lavage. Based upon the lack of fungal growth in the culture from the transtracheal wash, the fungal hyphae observed during cytologic examination were considered to be environmental contaminants. The significance of the bronchial bacterial sepsis is unclear since a bacterial culture was not performed and there were no clinical signs attributable to primary respiratory tract disease. Immunocompromise or opportunistic infection secondary to the cryptococcal meningitis were diagnostic considerations.

Cryptococcal meniningitis is a rare condition in horses.² It occurs more frequently in small domestic animals, especially the cat. Although uncommon, cryptococcal infections in the horse typically involve the respiratory tract alone, specifically the nasal sinuses.³ Rare involvement of other organ systems in the horse has been reported. In general, fungal infection of nervous tissue in all species is relatively uncommon except for Cryptococcus, which has a predilection for the CNS.⁴ While it is unknown how the yeasts enter the central nervous system, it is thought they pass from the nasal sinuses via the cribiform plate into the CNS and then circulate in the cerebrospinal fluid.^3,5,6 Another proposed but apparently less likely route is hematogenous spread via the lungs.^5,7

Cryptococcus neoformans is the only known pathogenic strain of Cryptococcus and is normally found worldwide in the feces of birds, in the soil, and on certain plant species.⁶ In humans, Cryptococcus neoformans is usually acquired by inhalation. Infected individuals are often immunosuppressed. However, infections have also been reported in otherwise healthy individuals.⁶ Experimental attempts in animals to introduce the infection by other routes such as cutaneous inoculation have been unsuccessful. Therefore, the respiratory tract is still considered the primary route of infection.⁵

Based upon the different capsular polysaccharides, there are four types of Cryptococcus neoformans: Types A, B, C, and D.⁸ The mucopolysaccharide capsule is unique to C. neoformans, bestowing upon it the properties that allow it to not only be pathogenic but also the ability to evade the host immune system.^3,8,9 One of the features or ‘virulence’ factors of Cryptococcus that improves its survival is the production of sialic acids in the cell wall. The sialic acids alter complement binding and prevent effective opsonization. The capsule also stimulates suppressor T- lymphocytes, which further reduces the host immune response.⁸ Additionally, Cryptococcus neoformans produces melanin and mannitol. These products function as free radical scavengers and effectively decrease the toxicity of the beneficial free radicals generated in the phagolysosomes of the host’s immune system.⁸

Another unique component of the Cryptococcus capsule is phospholipase. Phospholipase not only allows the organism to survive within macrophages, but it may also be a facilitator in the yeast’s migration from the respiratory tract into the CNS. However, this mechanism is not well established.⁸ The yeast may also localize in the CNS due to lower complement concentration and due to the increased CSF concentration of catechols. Catechols function as a substrate for phenol oxidase, an enzyme necessary for production of the previously mentioned free radical scavengers, melanin and mannitol.⁸ As further evidence for the predilection of Cryptococcus for the CNS, experiments in cats indicate that within days of parenteral administration of the organism, antigen specific to C. neoformans can be consistently detected in the CSF.⁹ In reports of veterinary patients with any type of Cryptococcus infection, 60% had Cryptococcus organisms detected by cytologic examination of their CSF.¹⁰

The clinical signs generally associated with cryptococcal meningitis include ataxia, convulsions, depression, proprioceptive deficits, and visual deficits. The blindness associated with the cryptococcus infection affecting this patient, although rare in horses, is a common sequelae in other species.^1,8-13

It has been proposed that the proximity of the cribiform plate to the optic nerves readily allows for infection of the optic tract in addition to the CNS.¹⁰ In cats and dogs, the ocular abnormalities include dilated unresponsive pupils and blindness due to exudative retinal detachment, granulomatous chorioretinitis, panophthalmitis, and optic neuritis. In contrast to cats, dogs tend to have severe disseminated disease, and most have CNS or ocular involvement. Clinical signs are usually related to meningoencephalitis, optic neuritis, and granulomatous chorioretinitis. Unlike horses, only a few dogs have been reported with primary lesions in the nasal cavity. Approximately 50% of dogs have lesions in the respiratory tract, usually the lungs, and most have granulomas throughout the body.⁹ Direct retinal infection by the organism in dogs and cats can cause retinitis and/or retinal detachment as discussed above, but neither condition was apparent in the horse of this report. Another suggested mechanism for blindness in horses is extraorbital compression of the optic nerve due to periorbital granuloma formation.⁹ The specific cause of the blindness in the horse of this report was not determined, but an extra-ocular cause such as optic neuritis was considered most likely.

The prognosis for horses with cryptococcal infections is not well defined due to their seemingly infrequent occurrence and the low number of reported cases.^5,12,14 In order for treatment to be instituted in a timely fashion, a definitive diagnosis of Cryptococcus infection at an early stage is probably critical to a positive outcome. As with this case, the most definitive diagnostic test for cryptococcal meningitis is via cerebrospinal fluid analysis with visual identification of the organisms. Confirmation of a cryptococcal infection in CSF is most commonly performed with India ink applied to a cytologic preparation. India ink readily demonstrates the thick capsule unique to Cryptococcus. Furthermore, Cryptococcus is the only yeast that will stain positive with India ink.⁷ (See Figure 2.) In human medicine, a positive India ink preparation is considered diagnostic in the majority of cases.⁴ Stains such as Periodic Acid Schiff’s(PAS) and Grocott’s Methanamine Silver(GMS), although more commonly utilized with histologic preparations, may also be performed on air-dried cytologic specimens to further aid in identification of yeast in CSF. Fungal cultures for Cryptococcus and latex agglutinization tests for Cryptococcus antigen are routinely performed to ensure specific identification.^8,11

Figure 2. Cryptococcus neoformans. India ink stain, 400X. http://www.doctorfungus.org/thefungi/ img/cry1_l.jpg

After definitive diagnosis, a treatment regimen of antifungal agents, most commonly involving Amphoteracin B, fluconazole, or itraconazole, is recommended.^5,11 Therapy is generally continued until clinical signs resolve and the antigen is not detectable in the CSF or serum.

Case Outcome/Conclusion

The patient was administered fluconazole (14mg/kg PO loading dose, then 5mg/kg PO q 24 h x 6 weeks) due to its reported efficacy against C. neoformans and because of its relatively lower cost compared to other antifungal agents. Additional supportive treatments included DMSO (1g/kg in 5L Normosol RÒ IV q 24 h), thiamine (10mg/kg IV q 12 h), Vitamin E (10,000 IU PO q 24 h), and flunixin meglumine (1.1mg/kg IV q 12 h). During the first two days of treatment, the horse exhibited mild ataxia in all four limbs. The ataxia was attributed to CNS inflammation secondary to cryptococcal organism death. After a week of treatment, the horse began to regain some visual function along with complete resolution of the ataxia. The patient was discharged from the VMTH with instructions to continue the fluconazole and flunixin meglumine every 12 hours.

The patient returned for a recheck appointment after 14 days. At the recheck appointment, the patient was considered visual and had normal menace responses bilaterally. A repeat CSF analysis was performed at this time. A mild neutrophilic pleocytosis persisted with rare cryptococcal organisms present. The patient was sent home with instructions to continue the fluconazole for an additional four weeks or until the next recheck appointment. At the time of this report, the patient was doing well at home with no clinical signs of cryptococcal meningitis. The six-week recheck appointment was pending.

References

1. Dickson J, Meyer EP. Cryptococcus in horses in Western Australia. Australian Vet J. 1970;46:558.

2. Cho DY, Pace LW, Beadle RW. Cerebral Cryptococcus in a horse. Vet Pathol. 1986;23:207-209.

3. Carter GR, Wise PJ. Essentials of Veterinary Bacteriology and Mycology. 6^th ed. Ames, Iowa: Blackwell Publishing; 2004:258-259.

4. Bailey CL, Vernau W. Cerebrospinal Fluid. In: Kaneko JJ, Harvey JW, Bruss ML, eds. Clinical Biochemistry of Domestic Animals. San Diego, Calif: Academic Press; 1997:785-827.

5. Steckel RR, Adams SB, Long GG, Rebar AH: Antemortem diagnosis and treatment of Cryptococcal meningitis in a horse. J Am Vet Med Assoc. 1982;80:1085-1089.

6. Ming PR, Baron EG. Manual of Clinical Microbiology 8^th ed. Washington, D.C.: ASM Press; 2003.

7. Reed SM, Bayly WM, Sellon DC. Equine Internal Medicine. St.Louis, Missouri: W.B. Saunders Company; 2004:329.

8. Hirsh DC, MacLachlan NJ, Walker RL. Veterinary Microbiology. 2^nd ed. Ames, Iowa: Blackwell Publishing; 2004:265-268.

9. Jacobs GL, Medleau L. Cryptococcosis. In: Greene CE, ed. Infectious Diseases of the Dog and Cat. Philadelhia, PA: W.B. Saunders Company; 1998:383-390.

10. Chretien F, Lortholary O, Kansau I, et al. Pathogenesis of cerebral Crytococcus neoformans infection after fungemia. J Infectious Dis. 2002;186:522-530.

11. Kobluk CN, Ames TR, Goer RJ. The Horse: Disease and Clinical Management, vol. 1. Philadelphia, PA: WB Saunders Company; 1995:421-422.

12. Scott EA, Duncan JR, McCormack JE. Cryptococcus involving the post-orbital area and frontal sinus in a horse. J Am Vet Med Assoc. 1974;165:626-627.

13. Riley CB, Bolton JR, Mills JN. Cryptococcus in 7 Horses. Australian Vet J. 1992;69:135-139.

14. Pearson EG, Watrous BJ, Schmitz JA, Sonn RJ. Cryptococcal pneumonia in a horse. J Am Vet Med Assoc. 1970;183:557-559.

Acknowledgement

The image "Wind Spirits" (c) 2003 by artist Kathleen Marie is from her website Kathleen Marie Studio and is used with permission.

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