Question

1. Describe the differences between hyaline and dematiaceous fungi.

2.Candida albicans is the most commonly recovered yeast, describe the simple test used to rapidly identify this yeast.

3. Cryptococcus neoformans is another yeast seen generally in immunocompromised hosts. How can this yeast be rapidly identified in the lab microscopically?

Answer 1

1.

Hyaline Hyphomycetes include those conidial fungi which are not darkly pigmented, colonies may be colorless or brightly colored. These include the agents of hyalohyphomycosis, Aspergillosis, dermatophytosis and the dimorphic pathogens, like Histoplasma capsulatum.

The term "dematiaceous" refers to the characteristic dark appearance of this group of fungi as it grows on agar. Colonies are dark gray, brown, or black and, importantly, have a black reverse when the bottom of the agar plate is examined. This distinguishes the dematiaceous fungi from fungi with black conidia but an otherwise pale mycelium, such as A. niger.

Dematiaceous Fungi: Direct Examination

• Hyphae are usually regular, 3-6 μ, and have parallel walls, irregular branching, and septae
• Hyphae are dark yellow or brown when unstained or poorly stained

Dematiaceous fungi can be conveniently divided into three groups based on morphology in slide culture:

1. macroconidia produced and are divided by longitudinal and transverse septae (these are referred to as muriform macroconidia
2. macroconidia produced and are divided transverse septae only
3. microconidia (one celled conidia) produced

2.

We developed the simple, rapid (1 h), and accurate PNA FISH method for the identification of Candida albicans. The method exploits unique in solution in situ hybridization conditions under which the cells are simultaneously fixed and hybridized. This method facilitates the accurate identification of clinical yeast isolates using two scoring techniques: flow cytometry and fluorescence microscopy.

Candida albicans is a common cause of severe health care-associated bloodstream infections. In the routine diagnostic microbiology laboratory, C. albicans can be identified presumptively with simple, rapid, and inexpensive methods such as germ tube or colorimetric tests, as well as the use of selective chromogenic agar media. A germ tube test is often used to exclude C. albicans before applying other yeast species level identification schemes. However, up to 5% of the C. albicans isolates have been reported as germ tube negative , and non-C. albicans isolates can be misinterpreted as germ tube positive

We developed the simple, rapid (1 h), and accurate PNA FISH method for the definitive identification ofC. albicans isolated from solid or liquid media (excluding blood cultures). This method facilitates the use of two scoring techniques: flow cytometry (FC) and fluorescence microscopy (FM). The main goal of this preclinical study was to determine the feasibility, specificity, and sensitivity of the C. albicans PNA FISH method using appropriate laboratory strains and clinical isolates. The peptide nucleic acid (PNA) probe used in the present study was previously rigorously tested by using glass side-based fluorescence in situ hybridization (FISH) assays , which are now approved by the U.S. Food and Drug Administration for in vitro diagnostic use. Clinical application of this test led to substantial cost savings for hospitals

FC Scoring

Guava EasyCyte (Guava Technologies, Hayward, CA) equipped with 488-nm laser line, forward-scatter (FSC) and side-scatter (SSC) detectors and a fluorescence (Fl-1) detector with 525/30 filter was used for FC scoring. Microbial cells were first visualized by using FSC and SSC profiles, and the visualized cell population was gated. The gated population was analyzed further in FSC/FL-1 dot blots. Border lines of 1,000 (FSC axis) and 100 (FL-1 axis) were established so that the population of C. albicans cells (positive control) was above 100 and the population of C. tropicalis (negative control) was below 100 on the FL-1 scale. Positive results were reported for samples which had ≥60% of events in upper left quadrant; negative results were reported for samples which had ≥60% events in the lower left quadrant. Cell populations with <60% of the cells in culture identifying quadrants were reported as inconclusive.

3.

Cryptococcosis is a chronic, subacute to acute pulmonary, systemic or meningitic disease, initiated by the inhalation of basidiospores and/or desiccated yeast cells of Cryptococcus neoformans. Primary pulmonary infections have no diagnostic symptoms and are usually subclinical. On dissemination, the fungus usually shows a predilection for the central nervous system, however skin, bones and other visceral organs may also become involved. Although C. neoformans is regarded as the principle pathogenic species, C. albidus and C. laurentii have on occasion also been implicated in human infection.

Laboratory diagnosis:

1. Clinical material: Cerebrospinal fluid (CSF), biopsy tissue, sputum, bronchial washings, pus, blood and urine.

2. Direct Microscopy: (a) For exudates and body fluids make a thin wet film under a coverslip using India ink to demonstrate encapsulated yeast cells. Sputum and pus may need to be digested with 10% KOH prior to India ink staining. (b) For tissue sections use PAS digest, GMS and H&E, mucicarmine stain is also useful to demonstrate the polysaccharide capsule. Examine for globose to ovoid, budding yeast cells surrounded by wide gelatinous capsules. Note, non-encapsulated variants, although rare, may also occur.

Interpretation: The demonstration of encapsulated yeast cells in CSF, biopsy tissue, blood or urine should be considered significant, even in the absence of clinical symptoms. Positive sputum specimens should be considered potentially significant, even though Cryptococcus may also occur in respiratory secretions as a saprophyte. Basically, all patients with a positive microscopy for cryptococci, from any site should be investigated for disseminated disease, especially by culture and antigen detection.

3. Culture: Inoculate specimens onto primary isolation media, like Sabouraud's dextrose agar. Look for translucent, smooth gelatinous colonies, later becoming very mucoid and cream in color.

Interpretation: The isolation of C. neoformans or C. gattii from any site should be considered significant and patients without clinical symptoms should be thoroughly investigated for disseminated disease. Positive culture of CSF is definitive. However, positive culture of respiratory secretions, especially in patients without clinical symptoms, needs to be interpreted with some caution, until additional supporting evidence is available. Isolation of Cryptococcus albidus orC. laurentii, should also be interpreted with caution as these species are infrequent pathogens and once again, additional supporting clinical and microscopic evidence is necessary.

4. Serology: It should be noted that the detection of cryptococcal capsular polysaccharide antigen in spinal fluid is now the method of choice for diagnosing patients with cryptococcal meningitis. In AIDS patients, cryptococcal antigen can be detected in the serum in nearly 100% of cases. However, in non-AIDS patients antigen detection in serum is less sensitive with only about 60% of patients with cryptococcosis reported as being positive. Note, serum specimens should be pretreated with pronase to enhance detection of antigen and avoid false negative results.

5. Identification: The genus Cryptococcus is characterized by globose to elongate yeast-like cells or blastoconidia that reproduce by multilateral budding. Pseudohyphae are absent or rudimentary. On solid media the cultures are generally mucoid or slimy in appearance. Red, orange or yellow carotenoid pigments may be produced, but young colonies of most species are usually non-pigmented, and are cream in color. Most strains have encapsulated cells with the extent of capsule formation depending on the medium. Under certain conditions of growth the capsule may contain starch-like compounds which are released into the medium by many strains. Within the genus Cryptococcus, fermentation of sugars is negative, assimilation of nitrate is variable and assimilation of inositol is positive. The genus Cryptococcus is similar to the genus Rhodotorula. The distinctive difference between the two is the assimilation of inositol, which is positive in Cryptococcus.