Successful Resolution of Cryptococcal Meningitis and Optic Neuritis in an Adult Horse with Oral Fluconazole

A 4-year-old Tennessee Walking Horse gelding presented to the University of Georgia's Veterinary Teaching Hospital for evaluation of episodic fever and acute bilateral blindness of approximately 7 days' duration. Historically, the fever resolved with administration of flunixin meglumine (1.1 mg/kg PO q24h for 3 days) but the visual deficit persisted. Attitude and appetite were reported to be normal. Annual vaccinations against eastern equine encephalitis and western equine encephalitis viruses, rabies virus, equine herpesvirus-1, equine influenza virus, and tetanus were performed 6 months before presentation. Routine deworming consisted of monthly administration of ivermectin. The horse was kept on grass pasture with 3 other horses and supplemented with grass hay and a commercial pelleted feed. The horse had not traveled from the owner's farm since birth, and no previous medical problems were reported. There were no recent diet or routine changes, and no other horses on the farm were ill. At presentation, the horse was quiet, alert, and responsive. General body condition was excellent, with a body condition score of 5/9 and a weight of 500 kg. Rectal temperature was 99.5°F, heart rate was 44 beats/min, and respiratory rate was 16 breaths per minute. General physical examination was within normal limits, with the exception of the eyes. Abnormalities on initial ophthalmic examination included absent menace responses with intact palpebral reflexes OU. Bilateral mydriasis with mild anisocoria (OD > OS) was present in room light. Direct and consensual pupillary light reflexes (PLRs) were present but sluggish OS and absent OD. Dazzle reflexes were inconsistent OS and absent OD. No other abnormalities were identified on complete ophthalmic and fundoscopic examination OU. Initial neurologic examination was consistent with bilateral blindness as above. Occasional hesitation in the stride at a walk was observed, but was most consistent with uncertainty because of acute blindness rather than a clinically relevant gait or proprioceptive deficit. The remainder of the neurologic examination was within normal limits. Bilateral blindness accompanied by mydriasis with no evidence of ocular abnormalities on slit lamp and fundoscopic examination indicated an afferent lesion in the retinas, optic nerves, optic chiasm, optic tracts, optic radiations, or visual cortices. The presence of PLR deficits excluded an isolated cortical lesion. Aniscocoria (OD > OS) indicated an asymmetric lesion that spared the function of some ganglion cell axons until the pretectal nucleus on the left side. The results of an electroretinogram were within normal limits OU, further localizing the lesion to the retrobulbar afferent pathway (ie, optic nerves, optic chiasm, or optic tracts). However, concurrent involvement of cortical visual centers could not be fully excluded. Additional diagnostic tests were performed to investigate the potential etiologies of acute blindness and fever. Rebreathing examination and rectal examination were within normal limits. Initial CBC and serum biochemistry results were within reference intervals. Cerebrospinal fluid (CSF) was collected by lumbosacral puncture on day 2 of hospitalization. Grossly, the CSF was cloudy and xanthochromic, and a markedly increased protein concentration and severe neutrophilic pleocytosis were present (Table 1). Cytologic evaluation of the CSF identified many fungal organisms resembling poorly encapsulated Cryptococcus neoformans (Fig 1). Serum latex agglutination titer for C. neoformans capsular antigen was negative, but CSF titers were strongly positive (Table 2). Bacterial and fungal culture of the CSF yielded no growth. These findings supported a diagnosis of cryptococcal meningitis and optic neuritis. Cerebrospinal fluid containing a cluster of poorly encapsulated C. neoformans yeasts (arrow) engulfed by large mononuclear cells. There is also a marked neutrophilic pleocytosis present. Inset illustrates a large mononuclear cell containing a phagocytized yeast. Modified Wright's stain, 1000x magnification. In this case, exposure to C. neoformans was presumed to be environmental, because contact with a variety of wild water fowl near a pond in the horse's pasture was reported. The respiratory tract was further evaluated to determine whether it was the primary source of disseminated fungal infection, and immunologic testing was conducted to evaluate for a primary immunodeficiency predisposing the horse to opportunistic fungal infection. No clinically relevant abnormalities were found on thoracic radiography, transthoracic ultrasonography, sinus radiography, upper airway endoscopic examination, and transtracheal aspiration. Serum immunoglobulin concentrations were measured on day 5 by radial immunodiffusion, and normal concentrations of IgM and IgA and an increased IgG concentration were identified (Table 3a). Peripheral blood lymphocyte immunophenotyping was performed with flow cytometric analysis with monoclonal antibodies for equine leukocyte surface markers as described previously,1 and revealed a CD4+ T lymphocytopenia and a decreased CD4+/CD8+ T-lymphocyte ratio (Table 3b). Lymphocyte proliferation assay was performed by 3[H]-thymidine incorporation as described previously,2 and lymphocyte response to phytohemagglutinin, pokeweed mitogen, and concanavalin A was remarkably low in the patient cells in comparison with cells from control healthy horses. Initial treatment included systemic antifungal treatment with fluconazole (14 mg/kg PO once, and then 5 mg/kg PO q24h). Flunixin meglumine (1.1 mg/kg IV q12h) and dimethylsulfoxide (1 g/kg in 5 L Normosol-R IV q12h) were also administered for their anti-inflammatory effects. After 48 hours of treatment, the horse developed symmetrical grade 2/5 ataxia in all 4 limbs. Development of ataxia was attributed to diffuse CNS inflammation caused by the death of cryptococcal organisms with antifungal treatment. Ataxia persisted unchanged during the remainder of hospitalization. Resolution of anisocoria was noted on day 3 of fluconazole treatment. Visual deficits OS resolved by day 7 of treatment. PLRs were present but consistently sluggish OD at this time, but blindness OD persisted throughout hospitalization. Dimethylsulfoxide was discontinued and the flunixin meglumine dose halved on day 7 without complication. Re-evaluation of the CBC on day 3 of hospitalization revealed development of transient hyperfibrinogenemia (500 mg/dL; reference range, 100–400 mg/dL) that resolved by day 9. The horse was discharged on day 11 to continue treatment with fluconazole as above, and a tapering course of flunixin meglumine (0.5 mg/kg PO q12h for 5 days, 0.5 mg/kg PO q24h for 5 days). The horse was re-evaluated on days 25, 53, 90, and 157 of treatment. CBC and serum biochemistry test results remained within reference intervals for the remainder of the treatment. Blindness and PLR deficits resolved OU by day 53. Serial neurologic examinations revealed resolution of thoracic limb deficits by day 53, but inconsistent grade 1/5 pelvic limb ataxia persisted on day 157. CSF fluid pleocytosis and increased protein concentration resolved by day 157 of treatment (Table 1), but rare cryptococcal organisms and capsular fragments were still seen in the CSF at this time. Periodic CSF fungal cultures were consistently negative. Serial evaluation of serum and CSF cryptococcal antigen titers revealed an initial increase, followed by a steady decrease (Table 2). Fluconazole treatment was ultimately discontinued after 197 days in this horse. The appropriate duration of antifungal treatment for cryptococcal meningitis is not well established in any species. In humans and small animals, an 8–10-week course of treatment is often recommended, until cryptococcal capsular latex agglutination titers or CSF fungal cultures are negative.3–5 In immunocompromised patients, subsequent long-term (often life-long) antifungal prophylaxis with lower dose oral fluconazole is often recommended to prevent recurrence of cryptococcosis.4,5 Discontinuation of antifungal treatment in this case was based on clinical improvement, stable low CSF cryptococcal antigen titers, serial negative CSF fungal cultures, and eventual financial constraints. At re-evaluation on day 279 (82 days after discontinuation of fluconazole), the horse was clinically normal. CSF fluid leukocyte count and protein concentration remained within reference intervals, but rare, acapsular fungal yeast cells were still seen cytologically (Table 1). Serum and CSF cryptococcal antigen titers were negative (Table 2). However, the CD4+ T lymphocytopenia and decreased CD4+/CD8+ ratio persisted. C. neoformans is a basidiomycetous fungus with a worldwide distribution, commonly found in soil contaminated with avian feces.4 Two pathogenic variants of C. neoformans have been identified: C. neoformans var. neoformans and C. neoformans var. gattii. These variants are now recognized as separate species by DNA sequence analysis, but are not distinguishable by the routinely performed and rapidly available C. neoformans capsular antigen latex agglutination titer.4 Typically, C. neoformans var. gattii infection has been reported in a more narrow environmental niche (primarily in tropical and subtropical regions) than C. neoformans var. neoformans.4 Recently, however, reports of clinical cryptococcosis caused by C. neoformans var. gattii infection in both humans and animals are increasing in more diverse locations worldwide (eg, Vancouver Island, Canada).4,5 In the few cases of equine cryptococcosis described in the literature, the specific cryptococcal species involved has not been reported. Cryptococcal infection most often occurs after inhalation of small yeasts or basidiospores released from soil organisms, but infection via ingestion of fungal organisms can also occur.4,7,8 The most common clinical manifestation of cryptococcosis in animals is pneumonia or upper respiratory tract granulomas, but multisystemic disease caused by a hematogenous spread of the organisms from the respiratory tract to extrapulmonary sites such as the CNS, eye, and skin is not infrequent.8–10 Disease is also described in these systems in the absence of pulmonary lesions.3 CNS cryptococcosis may also occur because of direct cryptococcal invasion of the CNS via the cribiform plate.8 Cryptococcal organisms induce a granulomatous inflammatory reaction, often resulting in the formation of discrete granulomas (cryptococcomas).4,7,8 Adaptive immunity orchestrated by CD4+ and CD8+ T lymphocytes producing a Th-1 response is the primary mechanism of host defense against cryptococcal infection.4 Serologic surveys in humans suggest that many people are exposed to cryptotoccal antigens and do not develop clinical disease.4 In human medicine, C. neoformans is most important as an opportunistic pathogen in immunocompromised hosts (eg, patients with human immunodeficiency virus infection or on long-term immunosuppressive therapy), although disease is also reported in immunocompetent individuals.4,5 In addition, recent evidence in both humans and small animals suggests that infection with C. neoformans var. gattii is more common in immunocompetent hosts than infection with other variants, suggesting that the organism may be more virulent in some settings.5,11,12 Concurrent or underlying immunodeficiency is not typically documented in veterinary patients with cryptococcosis, but immunologic testing is not routinely performed in affected animals. Cryptococcal yeasts typically express a thick polysaccharide capsule that is important for virulence (Fig 2).4,7 Because of the capsule's role in inhibiting the host immune response, acapsular variants of C. neoformans typically are considered avirulent.7 Capsular morphology, however, can vary with the environmental conditions,13 and clinical disease caused by poorly encapsulated or nonencapsulated forms of C. neoformans is occasionally reported, even in immunocompetent patients.14 In this horse, cytologic evidence of infection with a poorly encapsulated form of C. neoformans in conjunction with a positive C. neoformans capsular antigen latex agglutination titer in the CSF suggested that the infecting organism most likely was exhibiting variable capsule expression, as is described for C. neoformans both in vitro and in clinical cases.13,14 Equine airway washing from a different horse containing a cluster of typical, heavily encapsulated C. neoformans yeasts with a stippled proteinaceous background and scattered degenerate neutrophils. The non-staining areas (arrows) depict the limits of the polysaccharide capsules of individual yeasts. Wright's stain, 1000 × magnification. In animals, cryptococcosis is uncommon in most geographic regions, especially in North America.15 In small animals, nasal cavity or CNS infection is described most often, but ocular, pulmonary, dermal, and disseminated disease are also reported in both dogs and cats.9,10,15,16 Cryptococcosis is rare in horses, and is usually confined to the upper or the lower respiratory tract.8,17 Six cases of equine cryptococcal meningitis are described in the literature,18–23 but an antemortem diagnosis was made in only 1 case.21 Although resolution of respiratory cryptococcosis after local or systemic antifungal treatment (amphotericin B) is described in horses,17 successful treatment of equine cryptococcal meningitis is not reported.21 In humans and small animals, curative treatment of cryptococcal meningitis with a variety of systemic antifungal agents has been described.3,16 In humans, current treatment recommendations include a short course of fungicidal treatment with amphotericin B with or without flucytosine, followed by long-term fluconazole treatment.4,5 However, successful management of cryptococcal meningitis with single-agent therapy with azole antifungals (eg, itraconazole, ketoconazole, fluconazole) is reported in small animals,3,16,24 suggesting that these agents alone can be effective in clearing cryptococcal infection, despite their predominantly fungistatic effects. Although many different established and emerging azole antifungals are effective treatments for cryptococcosis, fluconazole is considered an optimal agent for treatment of cryptococcal meningitis both in humans and in small animals because of the high concentrations achieved in the CNS3,4 and the sensitivity of most North American cryptococcal isolates to the drug.25 Recent work suggests that fluconazole has excellent oral bioavailability in horses and reaches therapeutic concentrations in the CSF with a once-daily dosing regimen, with no clinically relevant adverse effects reported.26 In addition, recent availability of generic fluconazole has made this drug substantially more affordable than other azole antifungals for long-term use in horses. Thus, PO administered fluconazole may provide an effective, safe, and economically viable alternative to other systemic antifungals for use in horses. In this horse, the clinical relevance of persistent rare, acapsular fungal organisms on CSF analysis during and after treatment is unclear in light of the horse's clinical improvement. Given the horse's excellent clinical status, ongoing immune clearance of dead or avirulent fungal organisms from the CNS in resolving disease was presumed, although recurrence of cryptococcal meningitis caused by reinfection or incomplete resolution of cryptococcal meningitis or CNS cryptococcoma could not be ruled out. Treatment with a predominantly fungistatic rather than a fungicidal agent may also have contributed to incomplete clearance of fungal elements from this horse's CSF. However, consistently negative CSF fungal cultures in this horse suggest that clinically relevant numbers of live, virulent cryptococcal organisms were not present during or after treatment. Also, in 1 horse successfully treated for cryptococcal pneumonia, a strongly positive serum cryptococcal antigen latex agglutination titer persisted for at least 2.5 years after cessation of treatment despite complete resolution of clinical signs and the horse's return to normal use.17 Because this test detects the presence of cryptococcal antigen, it is possible that low numbers of cryptococcal organisms may persist in some horses even after a clinical cure is obtained with systemic antifungal treatment. The immunologic findings were suggestive of compromised cell-mediated immunity, which could have been primary or secondary to the cryptococcal infection. Specific T-lymphocyte deficiencies are rarely described in horses, and their incidence and importance in equine medicine are not fully understood.27,28 Absolute lymphopenia was not present at any point during the clinical course of disease in this horse, but lymphocyte numbers were not assessed in the earlier stages of disease before the development of clinical signs. A component of the CD4+ T lymphocytopenia and poor lymphoproliferative response to ex vivo mitogens in this horse could have been associated with transient T-lymphocyte downregulation secondary to cryptococcal infection, because this organism is known to induce regulatory T cells.7,29,30 Failure to eliminate organisms from the CSF after 197 days of antifungal treatment provides the strongest evidence for impaired immune function in this horse. Further investigation into the potential role of immunodeficiency in veterinary patients with systemic mycotic infections is warranted in the future. Continued evaluation of this horse's T-lymphocyte counts and lymphoproliferative responses may have provided clarification of the role of primary immunodeficiency versus fungal-induced immunosuppression in this case. Periodic immunologic testing, repeat CSF analysis, and CNS imaging to evaluate for persistence of a CNS cryptococcoma were offered in this case but were declined because of resolution of clinical disease. The horse was reported to be clinically normal and in work 16 months after discontinuation of antifungal treatment, suggesting that an underlying immunodeficiency or persistent cryptococcal meningitis were unlikely. To the authors' knowledge, this is the first reported case of equine cryptococcal meningitis to show successful long-term clinical resolution with systemic antifungal treatment. Thus, although the prognosis for systemic mycoses in general and cryptococcal meningitis in particular remains guarded in horses, orally administered fluconazole may represent an economically effective treatment. Because of the failure to fully eliminate cryptococcal organisms from the CSF in this horse, inclusion of a short course of fungicidal treatment (eg, amphotericin B) in addition to fluconazole treatment, as is recommended in humans,4,5 may also warrant further consideration in future cases of equine cryptococcal meningitis. Furthermore, based on the evidence supporting immunosuppression in this case, evaluation of the patient's immune function should be performed in similar cases.

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