The cryptococcal capsule, largely composed of the polysaccharides glucuronoxylomannan and glucuronoxylomannogalactan, is an important virulence factor for infection and can morph in size depending on environmental pressures exerted by the host or artificial growth media. In vivo, the protective capsule allows for immune evasion through prevention of phagocytosis [2, 6] and numerous immunomodulating properties, including downregulation of pro-inflammatory cytokines and complement depletion. Phenotypic switching and differential capsular expression are associated with changes in virulence, with unencapsulated cells being relatively avirulent and rarely associated with clinical isolates [7]. In vitro, decreased expression of capsular phenotypes in the absence of environmental pressure may result in variable culture morphologies depending on growth conditions [3].
India ink is traditionally used to detect cryptococcal organisms in clinical specimens by revealing the polysaccharide capsule which appears as a "halo" around the organism due to the inability of the ink to penetrate the capsule (Fig. 1D). However, poor sensitivity in clinical samples [8] and morphological variability in culture media limits its utility [2, 3]. Use beyond CSF samples is not established. Case reports describe its use in urine samples [9].
The initial isolate from the urine culture for our patient appeared dry and non-mucoid, consistent with a lack of visible capsules on India ink preparation (Fig. 1A and B). Repeat urine culture while on antifungal therapy revealed an isolate that appeared mucoid and was India ink positive. Capsular expression is regulated by complex host environmental cues. The initial morphology may have been due to a reduced capsular expression in vitro versus selective isolation of a phenotypically capsule deficient strain at the time of collection. Antifungal therapy is also associated with modulation of capsule size while specific host immune responses can induce or select for a larger capsule [10, 11]. It is possible that the distinct change in morphology was related to the host response to treatment initiation.
Identification of Cryptococcus in this case was achieved through urine culture plated to solid media followed by MALDI-TOF MS. Additional confirmation was sought using ITS and D1/D2 sequencing. In general, samples plated on 5% Sheep's Blood agar or Sabouraud dextrose agar can readily grow Cryptococcus sp. at 37 °C as early as 1–2 days after culture inoculation, however some isolates may take up to 5 days [12]. Historically, selective and differential media, were used to identify Cryptococcus sp. Compared to Candida sp., which remain white, Cryptococcal colonies appear dark brown on agar containing Niger seed extract (Fig. 2). Further differentiation of C. neoformans and C. gattii can be accomplished using L-Canavanine glycine bromothymol blue containing media, with C. gattii metabolizing the glycine, increasing the pH and turning the colonies blue via the bromothymol blue indicator [13].
Less labor-intensive phenotypic methods have subsequently emerged. Biochemical assays, such as those employed by the API 20C AUX test strip by bioMerieux are an essential part of yeast identification in many microbiology labs. The use of MALDI-TOF MS for rapid proteomic identification of Cryptococcus from cultured isolates has increasingly replaced traditional phenotypic techniques with a high degree of accuracy, especially for the most common species of Cryptococcus (C. neoformans and C. gattii). Further improvements in commercial database curations are still required to discriminate among less common species [4]. Though sequencing remains the gold standard for species identification, costs and turnaround time limit its use in the routine clinical laboratory.
Given the limitations of microscopy, reduced availability and prolonged turnaround time for culture-based methods, clinicians world-wide still rely heavily on the use of rapid, point of care, lateral flow assays (LFA) to quickly and accurately detect Cryptococcus antigen in serum and cerebrospinal (CSF). These dipstick tests use monoclonal antibodies embedded on an immunochromatographic test strip to signal the presence of cryptococcal capsular polysaccharide glucuronoxylomannan antigen. In a meta-analysis of 12 studies (primarily of HIV-positive individuals) Huang et al. reported the pooled sensitivity and specificity of CSF and serum CrAg to be ~ 98–99% respectively for cryptococcosis [14]. Serum cryptococcal antigen may be less sensitive, however, for localized forms of the disease [15] and other specimen sources (such as from urine) have not been well established or FDA approved. Huang et al. reported a sensitivity of 85% from urine CrAg with insufficient information for specificity calculation [14]. The poor performance of urine CrAg precludes its current use in routine laboratories [16].
A negative serum CrAg may be due to localized disease due to reduced sensitivity in this setting. A negative CrAg LFA may also occur in the setting of capsule deficient, or capsule reduced organisms, as this method is specific for the polysaccharide Cryptococcus capsule. Though this is a rare phenomenon it has been described in the literature [17]. False negative antigen testing can occur in two additional scenarios. In cases of overwhelming cryptococcosis and appropriate immune response, high levels of endogenous antibody may sequester free antigen and thus interfere with antigen testing, called the “Prozone Phenomenon”. Alternatively, the “Post-Zone Phenomenon” may also occur where excessive antigen relative to immunoassay antibodies prevent antibody-antigen cross linking required for immunochromatographic detection [18].
Our patient presenting with new AML had over a month of urological symptoms, an abnormal urinalysis, multiple negative routine blood and sputum cultures, negative serum CrAg, and urine cultures growing non-Candida yeast ultimately identified as C. neoformans. Cryptoccocuria is a rare clinical entity that is typically associated with systemic cryptococcal disease and has been described in both immunocompromised and in immunocompetent hosts [19]. In one retrospective analysis of 58 cases of cryptococcuria, 46 (79.3%) had a CrAg positive in CSF and 39 (67.2%) in blood. In 8 cases, cryptococcuria preceded the diagnosis of disseminated cryptococcosis. As such, identification in the urine may be an early indication of disseminated disease [1, 20]. Cryptococcuria has also been described in cases of chronic prostatitis diagnosed by prostatic biopsy and may represent a potential reservoir for infection in males [21, 22]. Cases of pyelonephritis [23] and epididymitis [24] have also been reported.
We demonstrate a rare case of urinary C. neoformans initially missed due to a laboratory protocol that precluded the workup of non-Candida yeasts from urine and lack of clinical suspicion. The microscopically observed variability in colony morphology and India ink staining may be attributed to variable in vitro growth conditions, the presence of multiple strains, or possibly altered capsular expression in the setting addition of antifungal agents and subsequent immune response. India ink is not routinely used in clinical samples other than CSF and urine microscopy is not sensitive for this organism. Urine cryptococcal antigen testing is not recommended and serum antigen testing may be negative in localized disease or rarely in the setting of capsule deficient strains. False negatives may also occur due to both the “Prozone Phenomenon” and “Post-Zone Phenomenon” and clinicians should understand the limitations of antigen testing. Proteomic techniques, which are increasingly being incorporated into clinical laboratories, can reliably confirm the diagnosis from pure culture.
Cryptococcuria is a rare clinical entity and can be associated with systemic disease or a harbinger for systemic involvement as described in the literature. Clinicians should consider Cryptococcus in cases of treatment-refractory urinary tract infections with non-Candida yeast in immunocompromised hosts, even when serum antigen testing and blood cultures are negative. In these cases, clinicians should request further workup of non-Candida yeasts from urine if not routinely performed.
