Mycology: Dermatophytes, Molds, Pneumocystis, and Molecular Detection

Submit tissue for both microbiology and histopathology when invasive mold disease is suspected. Place the microbiology portion in a sterile container without formalin and the pathology portion in formalin. Direct potassium hydroxide (KOH)-calcofluor examination can show fungal elements within minutes. Hematoxylin and eosin (H&E) staining demonstrates tissue injury and host response. Gomori methenamine silver (GMS) and periodic acid-Schiff (PAS) stains improve fungal contrast. A negative direct examination or culture does not exclude infection, particularly after antifungal exposure or when little specimen was collected.

Handle every filamentous or fuzzy colony in a certified Class II biological safety cabinet, including molds first noticed on bacteriology media. Keep plates closed and sealed until work begins in the cabinet. Never smell a culture. Do not prepare open-bench tape mounts, slide cultures, or other procedures that can aerosolize conidia. If an unidentified mold may be a thermally dimorphic pathogen or another high-risk organism, stop routine manipulation and follow your laboratory's containment and referral procedure.^{22 Miller JM, et al. Guidelines for safe work practices in human and animal medical… 2012.}

Dermatophytes infect keratinized skin, hair, and nails. The traditional clinical genera Trichophyton, Microsporum, and Epidermophyton remain useful, but molecular taxonomy also recognizes Nannizzia, Lophophyton, Paraphyton, and Arthroderma. Some organisms formerly reported as Microsporum, including M. gypseum, now belong to Nannizzia. Report the name supported by your laboratory's validated database and include a familiar former name when it prevents confusion.^{3,43 Tille PM. Bailey & Scott's Diagnostic Microbiology. 2021.4 Moskaluk AE, et al. Current topics in dermatophyte classification and clinical… 2022.}

Tinea names describe the infected site and do not indicate the causative genus: capitis involves the scalp, barbae the beard area, corporis glabrous skin, cruris the groin, pedis the foot, manuum the hand, and unguium the nail. Trichophyton species may infect all three keratinized sites. Microsporum species primarily infect hair and skin, and Epidermophyton floccosum infects skin and nails but not hair.

Collect skin scale from the active border, affected hair stubs with scalp scale, and nail plate with subungual debris from the advancing diseased area. KOH with or without a fluorescent brightener demonstrates hyaline septate hyphae and arthroconidia but does not identify a species. In hair, arthroconidia within the shaft indicate endothrix invasion. A sheath around the shaft indicates ectothrix invasion. Some ectothrix infections caused by M. canis, M. audouinii, or M. ferrugineum fluoresce blue-green to yellow-green under a Wood lamp. Fluorescence is inconsistent, and a negative examination does not exclude tinea capitis. T. tonsurans produces endothrix infection and does not fluoresce.

Inoculate a nonselective fungal medium and, when appropriate, a medium containing antibacterial agents and cycloheximide. Cycloheximide suppresses many environmental molds but can also inhibit clinically important nondermatophyte fungi, so never use it as the only medium. Dermatophyte test medium color change is presumptive because nondermatophyte molds can also alkalinize the medium. Direct microscopy and culture are complementary. Either can be negative after treatment or with a poor specimen.

Dematiaceous fungi contain melanin in their cell walls and usually form brown to black colonies. Pigment in tissue may be obvious or subtle. Fontana-Masson staining can support the presence of melanin. Species identification requires culture or sequencing.

Mucormycosis most often occurs with uncontrolled diabetes, especially ketoacidosis, corticosteroid exposure, neutropenia, hematopoietic or solid-organ transplantation, severe trauma or burns, prematurity, or deferoxamine therapy. Clinical forms include rhino-orbital-cerebral, pulmonary, cutaneous, gastrointestinal, and disseminated disease. The major human pathogens belong to Rhizopus, Mucor, Lichtheimia, Rhizomucor, Cunninghamella, Apophysomyces, and Saksenaea.

In tissue, Mucorales usually appear as broad, ribbon-like, irregularly branching hyphae with few septa (pauciseptate). Width and branch angle vary, and folds or fragmentation during tissue processing can create false septa or misleading angles. Report "broad, irregular, pauciseptate hyphae consistent with Mucorales" when that pattern is present. Do not identify Rhizopus, Mucor, or another genus from tissue geometry alone. H&E shows necrosis and angioinvasion. GMS and PAS improve detection of fungal walls.^{77 Cornely OA, et al. Global guideline for the diagnosis and management of… 2019.}

Divide tissue before processing. Mince or slice the culture portion with minimal crushing. Do not grind it. Place separate pieces directly onto media without cycloheximide. Culture may remain negative despite visible tissue invasion, so a negative culture does not exclude the infection seen on histopathology. Molecular testing of fresh or formalin-fixed tissue can support identification when culture fails, but performance depends on the assay, fungal burden, fixation, and contamination control.

These structures support genus-level identification only when the culture sporulates typically. ITS or D1/D2 sequencing with a curated database provides stronger identification. Galactomannan and serum (1→3)-β-D-glucan are usually negative in isolated mucormycosis. A negative result does not exclude disease. A positive result does not exclude mucormycosis, because mixed infection and assay cross-reactivity occur.

Aspergillus causes allergic disease, airway colonization, noninvasive fungus balls, chronic pulmonary disease, tracheobronchitis, and invasive disease. A respiratory isolate therefore has different significance in a patient with an aspergilloma, a lung transplant recipient, a neutropenic patient with nodules, and a patient without compatible disease. Repeated recovery, direct hyphae, positive BAL galactomannan, compatible imaging, and host risk increase the likelihood of infection. Tissue invasion establishes invasive mold disease. Hyaline septate hyphae with acute-angle branching indicate only an Aspergillus-like pattern, because Fusarium, Scedosporium, and other hyaline molds can look the same in tissue.^{11 Patterson TF, et al. Practice guidelines for the diagnosis and management of… 2016.}

An Aspergillus conidial head consists of a conidiophore ending in a vesicle, with phialides directly on the vesicle or on metulae. Colony color, conidial-head arrangement, surface ornamentation, and growth temperature support a provisional section or species complex.

Morphology alone does not reliably separate cryptic species. ITS commonly identifies the section or complex. Beta-tubulin or calmodulin sequencing may be needed for species-level resolution. MALDI-TOF can identify an isolate when the extraction method and reference library cover that organism. Report the complex when the validated method cannot support a species.

Hyalohyphomycosis describes tissue infection by hyaline septate molds other than the agents assigned a more specific disease name. Histology establishes hyaline mold invasion but rarely distinguishes among these organisms. Culture and a validated identification method are needed.^{66 Hoenigl M, et al. Global guideline for the diagnosis and management of rare mould… 2021.}

Biomarkers provide mycologic evidence but do not demonstrate an organism in tissue. Their value depends on the population, specimen, assay threshold, prior antifungal exposure, and pretest probability. Correlate an isolated positive result, and often repeat it or confirm it with an orthogonal test.

Microsporidia are obligate intracellular, spore-forming eukaryotes closely related to fungi. Their formal taxonomic placement has changed over time. Enterocytozoon bieneusi and Encephalitozoon intestinalis are major causes of chronic diarrhea in people with advanced immunosuppression. Other species can involve the eye, respiratory tract, urinary tract, muscle, or disseminated sites.

Medically important spores usually measure 1 to 4 µm. E. bieneusi spores are about 0.8 to 1.4 µm. Encephalitozoon and several other genera are commonly 1.5 to 4 µm. Modified trichrome with Chromotrope 2R or a quick-hot Gram-chromotrope method stains spores pink-red or violet and may show an equatorial belt. Calcofluor or another fluorescent brightener is a sensitive screen but can also stain yeast and debris. Routine light microscopy does not identify the species. Species-specific polymerase chain reaction (PCR) or broad-range PCR with sequencing is preferred when species identification affects interpretation. Concentrate stool or other fluids according to the validated procedure, use a positive control, and examine enough fields because organism burden may be low.^{99 Centers for Disease Control and Prevention. DPDx: microsporidiosis. 2019.}

Process specimens that may contain microsporidia under biosafety level 2 conditions in a biological safety cabinet, with eye protection. Spores can infect through splash exposure, and laboratory-acquired ocular infection has been reported.^{99 Centers for Disease Control and Prevention. DPDx: microsporidiosis. 2019.}

Pneumocystis jirovecii infects humans. P. carinii refers to a rat-associated species. Routine clinical culture is not available. Lower respiratory material is required because oral and upper-airway specimens have lower and less consistent diagnostic yield. BAL generally yields more organisms than induced sputum. Organism burden may be lower in patients without HIV than in those with HIV.

GMS stains the walls of the 4 to 8 µm asci, historically called cysts, but does not reliably stain trophic forms. Giemsa, Wright-Giemsa, Diff-Quik, and related stains show nuclei and trophic forms with less specific morphology. Direct or indirect immunofluorescence with monoclonal antibodies is more sensitive than conventional colorimetric stains. A negative stain does not exclude disease when specimen quality or organism burden is low.

Prototheca species are achlorophyllous green algae that produce yeast-like colonies, so the mycology laboratory often recovers them. P. wickerhamii causes most reported human disease, including cutaneous infection, olecranon bursitis, and disseminated infection. Other species occur less often, and older reports that use P. zopfii may not correspond to current molecular species.

Direct preparations and tissue sections show round to oval, nonbudding cells with internal cleavage into autospores. Mature sporangia have a morula-like or cartwheel appearance and stain with H&E, PAS, GMS, and calcofluor. These structures distinguish Prototheca from most yeasts.^{33 Tille PM. Bailey & Scott's Diagnostic Microbiology. 2021.} Morphology and commercial yeast assimilation systems may misidentify the species. MALDI-TOF can identify isolates only if the validated library contains current Prototheca entries. Cytochrome b sequencing provides the strongest species resolution. D1/D2 sequencing is a useful alternative. Interpret growth from a nonsterile site cautiously because environmental contamination or colonization is possible.^{1111 Jagielski T, et al. The genus Prototheca (Trebouxiophyceae, Chlorophyta) revisited. 2019.}

Choose the method that fits the question. Direct molecular detection shows whether fungal DNA is present in a specimen. Sequencing or MALDI-TOF of a culture identifies the recovered isolate. Neither result alone proves tissue invasion.