Mycology Laboratory Methods, Yeasts, and Dimorphic Pathogens

Fungi are eukaryotes that reproduce sexually and/or asexually. Molecular sequencing separates morphologically indistinguishable cryptic species, so modern identification combines morphology, physiology, and molecular or proteomic data.

Yeasts are unicellular and reproduce asexually by budding. Their colonies resemble bacterial colonies and, because yeasts are catalase-positive, a yeast colony can be mistaken for a bacterial colony unless a Gram stain or wet preparation is performed. Molds are multicellular and filamentous. Dimorphic pathogens switch between a mold form in the environment at 25-30 °C and a yeast or yeastlike tissue form at 37 °C: Blastomyces, Histoplasma, and Sporothrix form yeasts, whereas Coccidioides forms spherules.

A hypha is the filamentous mold unit; a mass of hyphae is a mycelium. Hyphae with cross-wall pores are septate; those without are aseptate. Mucormycetes have broad, ribbon-like, haphazardly branching hyphae; hyaline molds like Aspergillus have narrow hyphae that branch at acute (dichotomous) angles. Genus-level identification from hyphal morphology alone in a smear or tissue section is only a probability statement. Report the morphology descriptively rather than naming a genus. When daughter yeast cells fail to fully separate, a pseudohypha results, distinguished from a true hypha by constriction at each cell junction. Melanin-containing fungi are dematiaceous; their colonies appear dark on both obverse and reverse. Hyaline molds may have an obverse colored by pigmented spores, but the colony reverse remains pale.

Asexual sporangiospores form by cleavage within a sporangium (mucormycetes). Conidia form by differentiation of a fertile hypha. Thallic conidiogenesis walls off the whole parent cell as an arthroconidium; dermatophytes and Coccidioides use this pathway. Alternating barrel-shaped arthroconidia separated by empty disjunctor cells fragment easily and are a major biosafety concern for Coccidioides. Blastic conidiogenesis buds out the conidium, as in Aspergillus’s phialide-borne chains.

Specimen collection and direct examination

Swabs are inferior to scrapings, curettings, aspirates, and biopsies for essentially every site, and swab fibers can be mistaken for hyphae. Laboratories should discourage swab submission for fungal culture. Hair infected with a dermatophyte (for example Microsporum canis) fluoresces under a Wood’s lamp. Scrape dermatophytosis lesions from the active advancing edge rather than the healing center.

Ten percent potassium hydroxide dissolves keratinized debris so fungal elements stand out; gentle heating speeds clearing. Most yeasts stain partially-to-fully gram-positive and are distinguished from bacteria by size and budding. Giemsa/Wright demonstrates intracellular Histoplasma yeasts within macrophages. India ink negative-stains the Cryptococcus polysaccharide capsule; sensitivity is under 50% because some strains are poorly encapsulated, and it has largely been replaced by antigen testing. PAS shows internal detail; GMS gives the best contrast for sparse organisms; H&E is best for judging host response and whether a fungus is hyaline or dematiaceous. Calcofluor white binds fungal-wall chitin and fluoresces under UV.

Culture and identification

General-purpose media are Sabouraud dextrose agar (pH 5.5-5.6) or Emmons’ modified Sabouraud (pH 6.8-7.0, now more widely used). Nonsterile-site specimens should be plated in parallel on a nonselective medium and a selective medium (cycloheximide 400-500 mg/L plus chloramphenicol or gentamicin). Tissue specimens where dimorphic fungi are possible warrant brain-heart infusion agar plus 5-10% sheep blood. CHROMagar Candida differentiates common Candida species by colony color (C. albicans green, C. krusei pink, C. tropicalis metallic blue) and helps flag mixed cultures.

Incubate at 25-30 °C; parallel 37 °C incubation is not routinely needed except when a dimorphic pathogen is specifically suspected. Typical windows are 7 days for yeast in mouth/throat/vaginal specimens and 21 days for tissue/sterile-fluid fungal pathogens (longer for slow-growing dimorphic and dematiaceous fungi). Blood cultures use a biphasic broth-agar bottle or lysis-centrifugation (Isolator); lysis-centrifugation is more sensitive for fungemia but more prone to contamination.

The germ-tube test (yeast in serum, 35-37 °C up to 3-4 hours) presumptively identifies C. albicans/C. dubliniensis when combined with cornmeal-Tween-80 morphology (pseudohyphae plus chlamydoconidia). Mold morphology is assessed by teased-mount or cellophane-tape preparations stained with lactophenol cotton blue. A slide culture preserves fragile conidial arrangements but must never be used for a suspected dimorphic pathogen. Examine only after formalin fixation or a nail-polish-sealed coverslip prepared inside a certified biosafety cabinet.

Urease positivity differentiates Cryptococcus (positive) from most Candida (negative) and T. mentagrophytes (positive within 3-5 days) from T. rubrum (negative). Rapid trehalose assimilation flags Candida glabrata. Standard biochemical assays and early MALDI-TOF databases misidentify Candida auris, most often as C. haemulonii. Morphologic or molecular confirmation is required when an identification is clinically inconsistent; CDC currently lists MALDI-TOF with a C. auris-containing library, D1-D2 or ITS sequencing, or an appropriate molecular assay as accurate methods.¹

Cryptococcal polysaccharide antigen testing (latex agglutination or lateral flow, CSF/serum) is now standard and far more sensitive than India ink; a 2014 CDC-linked comparison found LFA sensitivity about 99% in CSF and serum. Histoplasma antigen testing (urine, serum, or BAL) is especially useful for disseminated disease (up to 92% sensitive) and acute pulmonary disease (75-80%), and tracks with treatment response. Galactomannan (serum/BAL) is a moderately sensitive but highly specific marker for invasive aspergillosis; false positives have been reported with some piperacillin-tazobactam lots. (1→3)-β-D-glucan is a nonspecific pan-fungal cell-wall marker present in most pathogenic fungi except Cryptococcus and the Mucorales.

Ribosomal DNA targets (18S, 28S, ITS1/ITS2) dominate molecular fungal identification. Probe-based assays confirm cultured Histoplasma, Blastomyces, and Coccidioides faster and more safely than morphologic conversion. Peptide nucleic acid FISH assays identify Candida species directly from positive blood-culture bottles. MALDI-TOF compares a ribosomal-protein mass-spectrum fingerprint against a reference database.

CLSI reference broth microdilution methods exist for yeasts (M27) and molds (M38). Disk diffusion, Etest, Sensititre YeastOne, and VITEK 2 are practical alternatives. Indications include institutional antibiogram surveillance, refractory oropharyngeal candidiasis, and invasive candidiasis where azole efficacy is uncertain.

Mold cultures should always be manipulated in a certified biosafety cabinet. Coccidioides immitis/posadasii and Histoplasma capsulatum mold-phase cultures are risk-group-3 pathogens requiring BSL-3 practices. Use screw-capped vessels rather than plates when either is suspected. The tissue (yeast/spherule) phase is not airborne-infectious, so clinical specimens from patients with histoplasmosis, blastomycosis, or coccidioidomycosis carry little biohazard. A Coccidioides lung cavity communicating with the bronchial tree is a narrow exception because it permits in vivo mold-phase sporulation.

Candida

Candida species are part of the normal GI, mucosal, and skin flora. Disease occurs when broad-spectrum antibiotics suppress competing bacteria or when host defenses fail. Non-albicans species matter because resistance patterns differ: C. glabrata shows emerging azole and echinocandin resistance, C. krusei is intrinsically fluconazole-resistant, and C. lusitaniae may be or become amphotericin B-resistant. C. auris is an emerging multidrug-resistant pathogen.

Clinical syndromes include cutaneous disease, oral thrush (especially in HIV, where C. dubliniensis is overrepresented), esophageal or gastrointestinal disease, vaginal disease, urinary disease (colonization and infection are often hard to distinguish), and invasive or bloodstream disease. Candidemia is defined by isolation from at least one blood culture. Candida endocarditis is uncommon but is the most frequent fungal cause of endocarditis.

In tissue, organisms appear as mats of budding yeast (3-5 μm) mixed with pseudohyphae. C. glabrata produces smaller (3 μm) yeast cells without pseudohyphae, a pattern that must be distinguished from Histoplasma. Germ-tube positivity with confirmatory cornmeal-agar chlamydoconidia presumptively identifies C. albicans/C. dubliniensis; the two species are further separated by growth at 42 °C, extent of chlamydoconidia formation, sugar assimilation, or MALDI-TOF. Absent germ tubes/chlamydoconidia, presumptive genus identification requires pseudohyphae with no arthroconidia.

Cryptococcus and Malassezia

The C. neoformans/C. gattii species complex causes systemic infection in both immunocompetent and immunocompromised hosts. C. neoformans is globally distributed, with pigeon guano as its main environmental reservoir; C. gattii is associated with eucalyptus. Primary infection is pulmonary and may disseminate, most importantly to the CNS. Cryptococcal meningitis is often insidious, with headache, personality or mental-status change, low-grade or absent fever, and often no classic meningismus.

Poorly encapsulated strains provoke a granulomatous response. Heavily encapsulated organisms elicit little inflammation and are separated from host tissue by a clear halo. Mucicarmine and GMS highlight the organism; Fontana-Masson demonstrates melanin. Laboratory clues include growth on blood agar at 35-37 °C, mucoid colonies, round yeast cells without pseudohyphae, no growth on cycloheximide-containing media, positive urease, and a positive India ink or polysaccharide antigen test. Most clinical laboratories report “Cryptococcus neoformans/C. gattii species complex.”²

The Malassezia genus contains one non-lipid-dependent species (M. pachydermatis) and 13 lipid-dependent species. Cutaneous disease occurs in normal hosts as commensal overgrowth. Systemic infection is essentially confined to neonates receiving intravenous lipid infusions. Pityriasis (tinea) versicolor shows hyper- or hypopigmented trunk/upper-arm macules; KOH prep shows the classic spaghetti-and-meatballs pattern of short hyphae plus yeast forms. Culture of lipid-dependent species requires an oil overlay. Cells are 3-7 μm with enteroblastic broad-based budding and a visible collarette.

Trichosporon spp., Saprochaete capitata, and other emerging non-Candida yeasts cause invasive disease disproportionately in severely immunosuppressed hosts. Establishing pathogenic significance requires histopathologic tissue demonstration with culture confirmation, or repeated isolation from sterile-site specimens.

Dimorphic systemic pathogens

Thermally dimorphic fungi can cause disease in healthy hosts, occupy specific ecologic niches, and are geographically restricted except for Sporothrix.

Histoplasma capsulatum grows in guano-enriched soil without infecting the bird or bat itself. The clinical spectrum includes asymptomatic seroconversion, acute flu-like pulmonary infection, granulomatous or fibrosing mediastinitis, chronic pulmonary disease associated with COPD, and disseminated disease at the extremes of age or with immunosuppression, including HIV and TNF-α blocker therapy. An oropharyngeal ulcer is a classic finding. Serology is limited by a 2-6-week seroconversion delay, cross-reaction with Blastomyces, and antibody persistence for years after cure.

Blastomyces dermatitidis disease begins in the lung and disseminates most often to skin or bone. Tissue response is a mixed acute-microabscess/granulomatous pattern.

Coccidioides arthroconidia are easily aerosolized from disturbed soil. Most infections are asymptomatic; symptomatic disease mimics bacterial pneumonia. Erythema nodosum or multiforme accompanying primary infection are favorable prognostic markers. Disseminated disease favors skin, skeleton, and meninges and is more frequent among Black and Filipino patients and among immunosuppressed hosts. Unlike in histoplasmosis and blastomycosis, complement-fixation serology is useful for gauging disease extent; titer height and trend track dissemination risk.

Sporothrix infection typically follows traumatic inoculation rather than inhalation (thorn, splinter, sphagnum moss, or a cat scratch). The dominant pattern is lymphocutaneous spread: an ulcerating papule at the inoculation site followed by a chain of new lesions ascending the limb’s regional lymphatics. Disseminated disease is rare and concentrated in patients with alcohol use disorder or immunosuppression.

Paracoccidioidomycosis ranges from acute lymphadenopathic disease to chronic pulmonary/mucocutaneous/lymph-node/adrenal disease that can mimic tuberculosis. Talaromyces marneffei is an important AIDS-associated pathogen in Southeast Asia; its tissue form divides by transverse fission, which distinguishes it from budding yeast.