DIAGNOSIS AND PATHOLOGY

Diagnosis of cryptococcosis involves a combination of serology, culture and direct observation, sometimes complemented by diagnostic imaging (conventional radi­ography, computed tomography, magnetic resonance imaging) (Hemsley et al.

Low-level positive titres (1:2-1:8) are consistent with subclinical infections or early clinical disease, although typically they are associated with subclinical disease that is generally self-limiting. Reciprocal titres of 1:16-1:32 may be subclinical or clinical, whereas titres >1:64 are strongly suggestive of clinical cryptococcosis and should be evaluated further using endoscopy, imaging or response to therapy.

A positive culture from a normally sterile site (lung, bronchoalveolar lavage specimen, lymph node, bone, pleural fluid, CSF) is diagnostic for cryptococcosis. A positive culture from nasal mucosa, mucus or intact skin should be viewed critically, because of the high preva­lence of colonisation by the C. gattii species complex of koalas in many environments, especially in managed care (Krockenberger et al. 2002b). Culture can be done on standard fungal culture media such as Sabouraud’s dex­trose agar, with or without antibiotics, or on selective and differential media such as birdseed agar, caffeic acid agar and other similar media. On these selective media, the C. neoformans and C gattii species complexes develop a brown colour effect of their yeast colonies (Staib 1962b). Rarely, other Cryptococcus spp. colonies may develop a slight brown colour effect or a green colour effect (Gol­ubev and Staib 2000).

Gross lesions are usually mass lesions but can be ulcerative if mucosal surfaces are involved. The gross appearance can vary substantially, from the classically described gelatinous multinodular lesions of cryptococ- cal pneumonia with large numbers of heavily encapsu­lated fungal cells present and minimal host response to solid granulomatous lesions with a strong host response dominated by macrophages and relatively few fungal cells present. Rarely, early in the pathogenesis of cryptococco­sis a suppurative host response may be noted grossly as pus or even solid organ abscessation. This can persist with chronic abscessation or pyogranulomatous inflam­mation. More frequently, chronic lesions are either gran­ulomatous or comprised predominantly of cryptococci and associated capsule with minimal host response. The most common organs affected are the respiratory tract and the CNS, although the skin is not infrequently affected and sporadic disease affecting other organs occurs although often reflects widespread dissemination (see Fig.

Direct detection of the organism using microscopy of fine needle aspirate cytology or smears made from nasal exudate, nasal washing or bronchoalveolar lavage is rela­tively straightforward, even for inexperienced cyto- pathologists. Rapid Romanowsky-type stains such as Quik-Dip (Muraban Laboratories, Mt Kuringai, NSW) are entirely satisfactory, as is Gram-stain. The organism is present in diseased tissues as a large (typically 10-30 μm diameter), round to ovoid, generally extracellular but sometimes intracellular gram-positive yeast, with a wide poorly staining extracellular capsule (Plate 25.1). Intra­cellular detail is often difficult to ascertain and varies in colour from grey to purple with rapid Romanowsky stains. When budding is evident, it is narrow-necked. The size of the capsule is variable, but as Australian mammals tend to be infected by the C. gattii species complex, a prominent capsule is usually present.

In some cases, where cryptococcosis is not initially sus­pected as the cause of a mass lesion, it may be diagnosed by histopathological examination of tissue sections. Histo­logical diagnosis is relatively simple in Australia, where there are few diagnostic alternatives for large encapsulated budding yeasts. A mucicarmine special stain may be used to highlight the capsule, although the morphological fea­tures of the organism in tissue section are insufficient for speciation. Immunohistochemistry may be used to help in the more definitive identification of the organism (Krock- enberger et al. 2001), although differentiation between C. gattii VGII and the C. neoformans species complex may be problematic using this method (Krockenberger et al. 2010). Molecular methods (panfungal PCR and sequence analy­sis) can be attempted, but have low yields from formalin- fixed paraffin embedded tissues when contact with formalin exceeds 24 hr. A better yield of PCR amplicons occurs using material scraped from glass slides fixed with methanol and stained by a rapid Romanowsky method (e.g. Quik-Dip™) (Reppas et al. 2014).

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