Cryptococcosis is caused by a Gram-positive yeast that has worldwide distribution with an increased incidence in temperate regions such as southern California and Australia. It affects numerous mammalian species, including dogs, cats, ferrets and humans. Cryptococcus neoformans is the species that primarily causes this disease in domestic pets. C. neoformans is a saprophytic, round to oval, yeast-like fungus measuring 3.5-7.0 microns in diameter. It also has a characteristic microscopic "soap-bubble" appearance in lesions due to the large heteropolysaccharide capsule that protects the organism from desiccation when invading tissues. Two variants, Cryptococcus neoformans var. neoformans and Cryptococcus neoformans var. gattii, and five serotypes (A, B, C, D, AD) have been identified.
Several sources of C. neoformans exist, including bird (especially pigeon) excreta which contain high amounts of nitrogen-containing compounds such as creatinine, soil and fruits. Cryptococcus sp. can survive in faeces for up to two years, unless viability is reduced by dry conditions or UV light. It does not spread via direct contact but instead is transmitted by inhalation of the aerosolised organism from the soil or feces. Cryptococcus neoformans var. gattii is primarily found in tropic and subtropic areas due to its very specific habitat of Eucalyptus trees.
Cryptococcal reproduction occurs by the formation of blastoconidia, which are buds connected by a narrow isthmus to the parent cell. Cryptococcus neoformans differs from other dimorphic fungi in that it is always found in the yeast phase in laboratory cultures and infected tissues. Inhalation of the aerosolised organism is the most probable source of infection; however, the exact mode of transmission is unknown. The organism can deposit in the upper or lower respiratory tract. However, the organism primarily remains in the upper airways since the encapsulated organisms (5-20 micrometers in diameter) are larger than the diameter of the terminal airways. This leads to a predominance of upper respiratory infections rather than pulmonary disease. Once established in the airways, the infection can spread hematogenously or by tissue invasion through the cribriform plate to the brain. Mammals with normal immune systems usually clear the cryptococcal infection prior to its spread. The majority of resistance is provided by cell-mediated immunity. Infections with Cryptococcus neoformans may indicate immunosuppression in cats with FIV and FeLV.
Cryptococcosis is an important disease of man and animals and the most common systemic mycosis of cats. The infection is thought to be acquired from the environment, with no reported cases of disease transmission from one affected animal to another. Thus, cryptococcosis is not a contagious or anthropozoonotic disease.
Cryptococcosis is most commonly caused by two species of the genus Cryptococcus; C. neoformans and C. bacillisporus. The ability of C. neoformans and C. bacillisporus to grow at 37oC may in part explain their pathogenicity, because other members of the genus grow poorly at this temperature. Elaboration of a polysaccharide capsule and the enzymes laccase and phospholipase by C. neoformans and C. bacillisporus are additional virulence factors that contribute to pathogenicity. C. neoformans and C. bacillisporus are dimorphic, basidiomycetous fungi. They exist in animal tissues as the yeast form (Cryptococcus spp), but are capable of transforming under special laboratory conditions into a filamentous form (Filobasidiella spp). Thus far, the filamentous phase has been demonstrated only under strictly controlled laboratory conditions, but this perfect state is likely to exist in certain natural environments. The importance of the perfect state is that spores resulting from sexual or asexual filamentous reproduction likely represent the infectious propagules that give rise to mammalian disease.
In animal tissues, Cryptococcus neoformans and C. bacillisporus exists as a round, yeast-like organism, with a variably-sized polysaccharide capsule as its distinguishing feature. The capsule provides protection from environmental insults (e.g., desiccation) and the phagocytic response of the host. In tissues, Cryptococcus reproduces by forming one or two daughter cells (buds) that are connected to the parent cell by a narrow isthmus. Buds may break off when small and thus the cell population varies in size.
Unlike other dimorphic fungi, the yeast phase of Cryptococcus is found under routine laboratory conditions and in infected tissues. Cryptococcus has a worldwide distribution and, in addition to people, infects a variety of domestic and native mammals. In contrast to the other systemic mycoses, the prevalence of cryptococcosis in cats exceeds that in dogs by an order of magnitude. Historically, five serotypes (A, B, C, D, AD) have been identified on the basis of antigenic differences in capsular polysaccharide. Recent advances in the taxonomy of the genus Cryptococcus have led to a new nomenclature that was proposed at the 5th International Conference on Cryptococcus and cryptococcosis. C. neoformans and C. bacillisporus differ biochemically, genetically, ecologically and epidemiologically. C. neoformans has a world wide distribution, while C bacillisporus is largely restricted to tropical and subtropical climates. C. neoformans can be divided into two varieties based on serotyping, C. n var grubii and C. n var neoformans. Both varieties are strongly associated with disease in immunocompromised human patients, although the same may not be true for companion animals. C n var grubii is by far the most common isolate from cryptococcosis in people and animals worldwide, although C. bacillisporus is important in certain geographical regions such as Australia, Papua New Guinea, South East Asia and Central Africa. There is strong evidence that several of Australian eucalyptus trees provide a natural environmental niche for C. bacillisporus. Interestingly, koalas seem capable of amplifying the number of cryptococci in certain environments. The definitive environmental niche for C. neoformans has not been determined, although there is a strong 'historic' association with weathered bird (especially pigeon) guano and more recent evidence for growth in decaying plant matter in hollows of certain trees. The organism passes through the gut of pigeons, but systemic infection of pigeons is extremely rare. Perhaps the pigeon's high body temperature protects it from infection. Pigeon guano provide an alkaline, hyperosmolar environment that is rich in many nitrogen-containing compounds including creatinine that favour cryptococcal growth. Cryptococci may remain viable for at least two years in accumulations of pigeon guano protected from drying or sunlight; pigeon lofts provide such an environment.
Most basidiomycetes reproduce sexually in their natural environment, and the teleomorphs of C. neoformans (Filobasidiella neoformans) and C. bacillisporus (F. bacillisporus) can be induced to undergo sexual reproduction in the laboratory and produce dikaryotic hyphae, blastoconidia, basidia and basidiospores. The recent documentation of both α and a-mating types of C. bacillisporus in Eucalyptus trees suggests that this may occur in nature. However, recent work has suggested that C. neoformans may be evolving into an asexual fungus and that basidiospores may result from haploid (monokaryotic) fruiting as well as by sexual recombination. In either case, the notion that the basidiospore is the infectious propagule for Cryptococcus is attractive, as this stage is suited to dispersal by air currents and has physical properties that favour penetration into the respiratory system, thereby facilitating primary infection of mammalian hosts.
The exact mode of infection is unproven, but the most likely route is via inhalation of air-borne organisms. These may be basidiospores or yeast cells desiccated by environmental exposure. Shrunken, poorly capsulated cryptococci that are small enough for alveolar deposition have been isolated from pigeon guano and soil. Although human patients with cryptococcosis typically present with neurological signs referable to meningoencephalitis, there is strong circumstantial evidence that the infection starts in the lungs and subsequently spreads to the nervous system hematogenously via macrophages. Respiratory involvement usually does not result in clinical signs, although lesions can be detected in thoracic radiographs, thoracic computed tomography scans or at necropsy. The small particle size of infectious propagules is said to be the reason the lung is primary site of infection, as only very small particles are capable of penetrating deep into the lower respiratory tract.
In cats, dogs, koalas and psittacine birds, the nasal cavity is usually the primary site of infection. The reason(s) for this difference (compared to humans) are a matter of conjecture, but possibly the increased development of the nasal passages in animals, with more efficient filtering of small particles, may provide part of the explanation. It is our belief that most cases of feline cryptococcosis begin as mycotic rhinitis following colonization of the nasal cavity without causing clinical signs. C. n var grubii has been shown to be a transient coloniser of the nasal mucus of cats, dogs and koalas in Australia, while C. bacillisporus can be isolated in sufficient numbers and with sufficient frequency to be actually considered part of sinonasal normal flora of koalas in certain environments. Studies in koalas have shown that self-limiting, subclinical infection is common, with limited invasion, granuloma formation and successful eradication or containment of organisms.
When infection ensues, clinical signs of rostral nasal cavity disease such as sneezing, epistaxis and nasal discharge are conspicuous, and sometimes granulomatous protuberances can be seen at the nares. In some cases, destruction of adjacent facial bones facilitates spread of infection to contiguous regions, such as the bridge and side of the nose, the planum nasale or hard palate. When facial distortion develops, the clinical presentation is strongly suggestive of either fungal rhinosinusitis or nasal neoplasia. On the other hand, when infection begins in the caudal portion of the nasal cavity, signs of mycotic rhinitis may be subtle or absent, although it is possible to confirm the sinonasal region as the primary site of infection using cytology, culture, endoscopy or cross-sectional imaging. In some cases, infection spreads through the cribriform plate into the olfactory bulbs and olfactory tract, giving rise to meningoencephalitis. In these cases, the anatomical proximity of the optic nerves frequently results in concurrent cryptococcal optic neuritis, and secondary retinitis. Clinically, this is manifest as widely dilated pupils that respond poorly to light, swelling of the optic disc and focal retinal haemorrhage. There may also be anterior uveitis. The invasion of the meninges results in an extensive leptomeningitis throughout the CNS. This is often accompanied by a ventriculitis. Unilateral strabismus or nystagmus occur as the infection extends caudally along the brain stem. In other cases, caudal nasal cavity involvement gives rise to a mass lesion which occludes one or both choanae, resulting in nasopharyngeal signs viz. stertor, snoring, dyspnea or open mouth breathing. In occasional cases the infection spreads to the middle ear via the auditory tube. A focal granuloma may occur in the brain and clinically mimic a neoplasm.
Cutaneous involvement, if multifocal, reflects hematogenous dissemination from the primary site of infection, as do lesions in bone (e.g., digits) or periarticular soft tissues. In ferrets, and the exceptional cat, localized cutaneous cryptococcosis can develop following penetrating injury of the skin. In some cats, infection spreads to the mandibular lymph nodes, presumably via the lymphatics from the nasal cavity. Rarely, mandibular lymphadenomegaly can be massive and require surgical debridement. Occasionally salivary gland infection has been documented, although how organisms reach this site is a mystery.
In eastern Australia, about 20-30% of human cryptococcosis cases are caused by C. bacillisporus, and a similar proportion is observed in cats. There is a tendency for animals in rural environments to be infected with C. bacillisporus, presumably due to increased exposure to eucalyptus material, and all infections recorded in koalas have been attributable to C. bacillisporus. The development of granulomatous intracranial or pulmonary mass lesions (cryptococcomas), are strongly associated with immunocompetence. Thus, in human patients, cryptococcomas are more typical of C. bacillisporus infections, whereas infections in immunodeficient patients typically result in meningitis with little involvement of the brain parenchyma.
In cats, infections with feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) have been thought to predispose to cryptococcosis. In North America, there is evidence that some FeLV-positive cats develop cryptococcosis as a result of immune dysfunction because these cats are slower to respond or fail to respond to treatment, and are much more likely to suffer relapses. In Australia, FeLV-positive cats with cryptococcosis are exceedingly rare, probably because of the very low prevalence of persistent FeLV infection in the cat population. Although there are several reports of one or two cats with cryptococcosis allegedly secondary to feline immunodeficiency virus (FIV) infection, large studies in Australia have failed to produce convincing evidence that cryptococcosis is a feline AIDS-defining infection. Rather, it is considered that co-infection usually reflects the high prevalence of FIV infection in Australia. Leukocyte and lymphocyte subset numbers in FIV-positive and FIV-negative cats with cryptococcosis were not different and a positive FIV status did not impart an unfavourable prognosis. Indeed, many FIV-positive cats with cryptococcosis can be cured and do not relapse despite cessation of therapy. Cryptococcosis has very rarely been reported in cats receiving immunosuppressive therapy or chemotherapy for malignancy. Thus, underlying diseases are typically not detected in cats with cryptococcosis, and factors predisposing to infections remain elusive. The authors have treated two FIV-negative cats with cryptococcosis which subsequently developed malignant lymphoma and although no relationship was established between the two conditions; a similar association has been the subject of a previous report. We have also seen two FIV-positive cats with cryptococcosis who then developed lymphoma and mast cell neoplasia, respectively, subsequent to other opportunistic infections; in these cats, long-standing FIV infection may have predisposed to both cryptococcosis and the terminal malignancies. Genetic factors may be involved in the predisposition towards development of cryptococcosis, as Siamese, Birman and Ragdoll breeds are significantly over represented amongst reported cases.
Cryptococcosis is the most common fungal disease in cats and should be an important differential diagnosis when a cat presents with sneezing and nasal discharge that is unresponsive to antibiotics. The age range of infected cats varies widely (1-13 yrs), with a mean age of 5 years. There is a predilection for infection in male cats, most likely due to the increased roaming of toms. Siamese cats are also overrepresented in most studies. Outdoor cats also have an increased predominance of cryptococcosis as compared with indoor cats. However, keeping cats indoors does not prevent them from contracting the disease. Potting soil in house plants and mouldy, unfinished basements may be sources of infection in indoor cats. The four primary systems affected are the respiratory, central nervous, ocular, and cutaneous systems.
The clinical signs, therefore, depend on the organ system(s) affected. Respiratory infections (seen in >80% of cases) are characterized by sneezing, nasal discharge (mucopurulent, hemorrhagic, or serous), subcutaneous swelling over the nasal planum and bridge, oral lesions, mandibular lymphadenopathy, and destruction of the nasal turbinates.
Neurological signs vary with the locale of the lesion and can include depression, ataxia and paresis, seizures, and blindness. Ocular abnormalities predominately affect the retina, choroid, and optic nerve. Eye infections can arise hematogenously or via extension from the brain by the optic nerve. Clinical presentation can range from dilated, unresponsive pupils and blindness to chorioretinitis, anterior uveitis, and retinal damage. While the prognosis for survival with the ocular form of Cryptococcus is fair to good using triazole antifungals, the prognosis for return of vision is guarded to poor due to retinal damage. Dermal lesions are seen in approximately 45% of infected animals and often occur with additional organs simultaneously being affected. The skin lesions are more likely caused by previous dissemination rather than infection by direct trauma to the skin. A more rare presentation of cryptococcosis may include anorexia, peripheral lymphadenopathy, bone lysis, chronic cough, and acute renal failure. Dogs are not infected by C. neoformans nearly as often as their feline counterparts. The prevalence in dogs is .00013%, as compared to cats that are 7-10 times more likely to be infected. The average age of infected dogs is 3.5 years and, unlike cats, there is no gender predisposition. The clinical signs are similar to those found in cats except that fever (103-105° F) is seen more often in affected dogs (25% of cases).
Additional clinical findings, other than those observed on physical examination, are rare. Endoscopy (specifically rhinoscopy), cannot provide clear evidence of a fungal infection and is more beneficial for biopsies of the swollen nasal dorsum or granulomas, as opposed to visualization of the infected tissue. The thick, mucinous nasal material can also be obtained using an otoscope speculum. Cryptococcus neoformans also differs from most other mycotic agents in that radiographs rarely aid in diagnosis. Moth-eaten nasal turbinates with an osteolytic appearance are rarely seen because C. neoformans is usually non-destructive. More commonly, a fluid opacity in the nasal passages and frontal sinuses is seen with surrounding soft-tissue swelling. Thoracic radiographs are almost always normal, although occasional small nodular lesions may be seen in addition to a secondary bronchopneumonia. Gross pathologic findings may range from a gelatinous mass (with several organisms and low numbers of inflammatory cells) to granulomas. An influx of macrophages, giant cells, and some plasma cells and lymphocytes often surrounds the encapsulated organisms, and these cells may be seen with cytologic or histopathologic examination of lesions. Complete blood counts on infected animals may show mild anaemia, eosinophilia, and monocytosis. Serum biochemical test results are usually within reference intervals.
The most rapid and practical means of diagnosing Cryptococcus neoformans is by cytological evaluation of nasal exudates, tissue aspirates, urine, or CSF using one of many stains. Wright’s stain can be used; however, the methyl alcohol can dehydrate and distort the characteristic capsule. Additional diagnostic stains include new methylene blue, Gram stain, and India ink. Caution must also be exercised when examining India ink preparations to prevent confusing lymphocytes, fat droplets, or aggregated ink particles with the organisms.
A negative cytological examination does not exclude cryptococcosis. The next logical choice for diagnostic testing is latex agglutination which detects the cryptococcal capsular antigen of all known serotypes found in serum, urine, or CSF. Commercial kits are very reliable and have 90-100% sensitivity and 97-100% specificity. The specificity is highest when the serum is pre-treated with a protease (pronase). The latex agglutination test is useful for diagnostic purposes Italic textand also for monitoring response to treatment. Effective therapies should cause the initial titre to decrease to 10% or less within 2 months of drug administration. Definitive diagnosis can also be determined via examination of tissue biopsies and fungal cultures. Histologically, the organism can be stained with hematoxylin and eosin, periodic acid-Schiff, Gomori's methenamine silver, Masson-Fontana, or Mayer’s mucicarmine techniques; the latter staining technique is considered definitive. C. neoformans has a thick capsule, thin cell wall, budding, and lack of endospores which differentiate it from Blastomycoides spp and Coccidioides spp. Cryptococcus neoformans can be cultured from exudates, cerebrospinal fluid, urine, joint fluid, and tissue samples on Sabouraud’s agar with antibiotics at 25-37°C. MR imaging may reveal thickened meninges or focal granulomas.
The current drugs used to treat cryptococcosis are the polyenes, azoles, terbinafine and flucytosine.
- Amphotericin B (AMB), alone or in combination with other antifungal drugs, has been previously used to treat cryptococcosis. However, this drug is usually reserved for life-threatening and previously unresponsive cases due to its toxicity. Several disadvantages of AMB make it less desirable than other drugs. Intravenous treatment is difficult and causes many adverse reactions, AMB has side-effects including nephrotoxicity and severe azotemia, it has poor efficacy in dogs, and is ineffective with cryptococcal meningitis because AMB does not cross the blood-brain barrier (BBB). Malik et al. have devised a new subcutaneous infusion method of administering AMB which is less toxic and less expensive. New attempts at decreasing toxicity have also involved changing the drug into lipid and lipidsomal-encapsulated formulations. However, these lipid drugs are extremely expensive. The drug flucytosine can also be added to AMB to increase the success of therapy.
- Ketoconazole (KTZ) (Nizoral®) is another treatment option for cryptococcosis but causes large numbers of side-effects including anorexia, vomiting, diarrhoea, weight loss and icnreased level of liver enzymes. Like AMB, KTZ does not cross the BBB and is not as effective in dogs. It should be noted that this decreased efficacy in dogs could be due to the higher prevalence of disseminated and neurologic disease.
- Itraconazole (ITZ) is a triazole antifungal drug similar to KTZ but with fewer adverse side-effects. If gastrointestinal or liver disease is seen, ITZ can be discontinued for 2 weeks and then re-administered at half the original dose. ITZ also does not penetrate the BBB well.
- Fluconazole (FCZ) is another triazole agent that does have the ability to cross the BBB and has fewer side effects than the aforementioned antifungal compounds. FCZ has been reported to have the highest success rates in cats, including those with advanced, longstanding, or disseminated disease. Liver and kidney function should be checked routinely. It should be noted that FCZ is only FDA approved for use in humans.
- Terbinafine - recommended dose of 25mg/kg orally once daily for 4 - 6 weeks
Fluconazole and itraconazole are the current drugs of choice for this disease, used separately or in combination with amphotericin B.
- Flucytosine - recommended dose is 150mg daily for 4 - 6 weeks
|Amphotericin B||0.25 mg/kg IV three times a week or 0.5 - 0.8 mg/kg SQ twice weekly|
|Fluconazole||50 mg orally every 12 -24 hours|
|Ketoconazole||10 mg/kg orally once daily|
|Itraconazole||5 mg/kg orally twice daily for 4 days, then 5 mg/kg once daily for 4 weeks after cessation of clinical signs|
Outcomes of treatment of cryptococcosis are quite varied. Drug therapy is long-term (average of 8.5 months) and relapses occur frequently. Patients with the CNS form of cryptococcosis will require lifelong treatment maintenance. It is recommended that treatment continue for one month after resolution of clinical signs in combination with decrease in antigen titre by at least two orders of magnitude or until serum cryptococcal antigen is undetectable. The prognosis is much worse if the patient has the neurologic form of disease or is immunocompromised by FeLV or FIV infections. Since Cryptococcus neoformans is ubiquitous, the best means of prevention is to decrease contact with areas containing a high concentration of organisms (pigeon droppings, damp buildings or basements).
- Wilson, DE, Bennett, JE & Bailey, JW (1968) Serologic grouping of Cryptococcus neoformans Proc Soc Exp Biol Med 127:820-823
- Ellis, DH & Pfeiffer, TJ (1990) J Clin Micro 28(7):1642-1644
- Malik, R (2003) Feline cryptococcosis. 28th meeting, World Small Animal Veterinary Association, Bangkok
- Gionfriddo, JR (2000) Feline systemic fungal infections. ...Vet Clin North Am Small Anim Pract 30:1029
- Davies, C & Troy, GC (1996) Deep mycotic infections in cats. J Am Anim Hosp Assoc 32:380
- Medleau, L et al (1990) Clinical evaluation of a cryptococcal antigen latex agglutination test for diagnosis of cryptococcus in cats. J Am Vet Med Assoc 196:1470
- Laurenson, IF, Ross, JD & Milne, LJ (1998) Microscopy and latex antigen negative cryptococcal meningitis. J Infect 36:329
- O'Brien CR et al (2006) Long-term outcome of therapy for 59 cats and 11 dogs with cryptococcosis. Aust Vet J 84(11):384-392