17.3 Mucorales, Hyaline Molds, Pneumocystis, and Identification Methods

17.3 Mucorales, Hyaline Molds, Pneumocystis, and Identification Methods

Quick Answer: Broad, ribbon-like, sparsely septate hyphae in a diabetic sinus or infarct = Mucorales (Rhizopus if rhizoids sit under the sporangiophore; Mucor if they do not). Narrow septate hyphae at 45° = Aspergillus (read the fruiting head: A. fumigatus vs flavus vs niger) or another hyaline mold (Fusarium, Penicillium, Scedosporium apiospermum complex). Pneumocystis jirovecii is not cultured — use silver stain, DFA, or PCR on BAL. Identify molds by colony rate and temperature, tape or slide culture, antigen/molecular assays, biochemicals, automation, and MALDI-TOF. Dematiaceous species lists and antifungal AST are SM-only.

This section is the rest of M-tested mycology after yeasts and dimorphs: the fast dirty molds, the common hyaline opportunists, the unculturable lung fungus, and the methods that turn a colony into a name. Official outline III.D.2.f (dematiaceous molds) and III.D.2.h (Microsporidium) are SM only, as is III.D.7 antifungal AST. Mention them only so you do not study the wrong list. Do not memorize Alternaria vs Curvularia vs Bipolaris conidia, microsporidial polar tubes, or CLSI mold MIC breakpoints for the M exam.

Mucorales (historically Zygomycetes)

Rhizopus and Mucor cause mucormycosis: rhino-orbito-cerebral disease in diabetic ketoacidosis, pulmonary or disseminated disease in neutropenia, and wound infections after trauma or contaminated dressings. Tissue shows broad (6–25 µm), ribbon-like, thin-walled hyphae that are aseptate or sparsely septate, with irregular 90-degree branching and a tendency to invade vessels (infarct, black eschar). That picture is the opposite of Aspergillus (narrower, regularly septate, 45°).

Colonies are rapid — often filling the plate in 24–48 hours — cottony, and "lid lifters." Microscopically a sporangium stuffed with spores sits on a sporangiophore.

• Rhizopus: rhizoids (root-like holdfasts) arise directly opposite / under the sporangiophore (nodal). Sporangiophores are unbranched; a columella and collapsing sporangial wall are typical. R. arrhizus (R. oryzae) is the usual clinical species.
• Mucor: no rhizoids (or rare, not aligned under the stalk). Sporangiophores may branch.

A tease or tape mount from a young colony is usually enough; these molds grow so fast that a full slide culture is optional. Do not wait a week. Report a sterile-site or sinus isolate immediately as a Mucorales mold; species can follow. β-D-glucan is typically negative because Mucorales cell walls are poor in that glucan — a negative fungal biomarker does not rule out mucormycosis.

Hyaline molds

Hyaline (clear-walled, non-pigmented) molds include Aspergillus, Fusarium, Penicillium, and the Scedosporium apiospermum complex. In tissue they all look like septate hyphae; culture morphology of the fruiting head is what names them. Incubate at 25–30°C (and 37°C if invasive disease is suspected) on media without cycloheximide, because cycloheximide inhibits many of these opportunists. A mycology bench therefore sets a cycloheximide plate (for dermatophytes/dimorphs) and a plain Sabouraud or inhibitory-mold agar plate.

Aspergillus

Septate hyphae, dichotomous branching at approximately 45°. A fruiting head is a vesicle covered with phialides that produce chains of phialoconidia.

A thermotolerant blue-green mold that fruits as a columnar uniseriate head is A. fumigatus until proven otherwise. Galactomannan antigen (serum or BAL) supports invasive aspergillosis but cross-reacts with Fusarium and some other molds and with certain beta-lactam lots — culture still matters.

Fusarium

Banana- or canoe-shaped multicelled macroconidia (and often microconidia) on banana-boat phialides. Colonies are woolly and may become pink, purple, or yellow. Unique among common molds, Fusarium can grow in blood-culture bottles and cause true fungemia in neutropenia, plus keratitis (contact lenses, trauma) and onychomycosis. In tissue it mimics Aspergillus (septate hyphae); only culture or molecular ID separates them. That is why a mold growing from blood is not "just a contaminant" in a neutropenic patient.

Penicillium

The penicillus (paintbrush) of flask-shaped phialides producing chains of round conidia is the ID. Colonies are often green-blue and granular. Most species are contaminants or causes of keratitis/chronic sinus disease; do not spend M time on every species. (The Southeast Asian dimorph historically called P. marneffei is now Talaromyces marneffei and is not the ordinary laboratory Penicillium contaminant.)

Scedosporium apiospermum complex

(Sexual state historically Pseudallescheria boydii.) Annelloconidia (flask or club-shaped conidia on annellides) and a gray-white colony that later darkens. Causes mycetoma, pneumonia, and CNS infection after near-drowning in polluted water. In tissue it is another septate hyaline mold that can be mistaken for Aspergillus — another reason fruiting-head culture or molecular ID is required before the chart says "aspergillosis."

Pneumocystis jirovecii

A fungus by rRNA phylogeny that cannot be cultured in the clinical laboratory. It causes pneumonia in HIV with low CD4 counts and in other immunocompromised hosts (steroids, transplant, hematologic malignancy). Organisms sit in alveolar spaces. Collect induced sputum or BAL — expectorated sputum is insensitive. Detect:

• GMS (Gomori methenamine silver) or toluidine blue: cysts 5–8 µm, round to cup- or crescent-shaped, often in clusters.
• Giemsa or Wright: trophozoites; cysts show a dot-like nucleus but unstained walls.
• DFA with monoclonal antibody.
• PCR on BAL or sputum, now common and more sensitive than stain.

There is no colony, no germ tube, no MALDI from growth. A negative silver stain does not exclude disease if PCR or DFA is positive. Do not order "fungal culture for Pneumocystis." β-D-glucan in serum can be elevated and is supportive but not specific.

Colony morphology and growth

Read rate, temperature, and incubation length before you tease the colony.

• Rate: Mucorales 1–2 days; Aspergillus/Fusarium/Penicillium 2–4 days; Scedosporium several days; dermatophytes 7–14 days; dimorphs often 1–4 weeks. Hold mold cultures up to 4 weeks when a dimorph is in the differential; do not discard a bone-marrow fungus plate at 72 hours.
• Temperature: 25–30°C for morphology; 37°C to ask "does it convert?" and "does it grow at body temperature?" (invasive opportunists usually can). A. fumigatus growing at 45°C is a useful screen.
• Texture and color: cottony, granular, powdery, glabrous, yeast-like; obverse vs reverse pigment. Record them — they are part of the ID, not decoration.
• Media: Sabouraud dextrose, potato dextrose (spores better), inhibitory mold agar, BHI with blood for conversion, dermatophyte media with cycloheximide. Never a single plate for every question.

Microscopic identification

A transparent tape prep (clear Scotch tape on the colony, stain with lactophenol cotton blue) is the first look at fruiting structures. A slide culture (Riddell: agar block on a slide, coverslip, moist chamber) preserves intact fruiting heads when tape crushes them. Use slide cultures for Aspergillus heads, Penicillium brushes, and dermatophyte macroconidia. Do not slide-culture a suspected Coccidioides or other highly infectious mature mold. Tease mounts distort Mucorales ribbons; tape is gentler. Always correlate with the tissue Gram/GMS: aseptate ribbons vs 45° septate vs yeast vs spherules vs Pneumocystis cysts.

Direct antigen and molecular methods

• Cryptococcal antigen (CrAg) on serum/CSF — yeasts section, still an ID method here.
• Aspergillus galactomannan — invasive aspergillosis support.
• (1→3)-β-D-glucan — positive in many Candida, Aspergillus, Pneumocystis, and Fusarium infections; typically negative in Mucorales and Cryptococcus (poor glucan walls / capsule). A pan-fungal biomarker, not a species name.
• PNA-FISH on blood-culture yeast.
• PCR and multiplex panels on BAL, CSF, and blood; ITS or D2 sequencing when MALDI fails.
• Direct wet prep, KOH, and calcofluor-white remain the fastest "is fungus present?" tests on hair, skin, nails, and tissue.

Biochemical, automated, and MALDI-TOF methods

Yeasts still use urease, germ tube, cornmeal Tween 80, CHROMagar, API 20C AUX, and automated cards (Vitek yeast). Molds are identified morphologically first; MALDI-TOF after a formic-acid or tube-extraction protocol names many yeasts superbly and an expanding list of molds if the library contains the species. Update the library for C. auris. When MALDI says "no identification" on a healthcare yeast, do not call it C. haemulonii from an old biochemical overlay — escalate. Automated systems do not replace fruiting-head morphology for Aspergillus vs Fusarium vs Mucorales.

Out of scope as tested on M: do not drill dematiaceous (phaeoid) mold species, Microsporidium, or antifungal susceptibility methods and MIC interpretation. You may know that C. krusei is intrinsically fluconazole-resistant as an identification fact; you will not be asked to set a mold microbroth MIC panel on this exam.