Mycology: Dermatophytes, Molds, Pneumocystis, and Molecular Detection

Dermatophytes are keratinophilic molds that infect skin, hair, and nails. Three genera cause the overwhelming majority of human disease.²

Clinical nomenclature follows the Latin tinea plus body site: capitis (scalp), barbae (beard), corporis (trunk/limbs), pedis (foot), cruris (groin), unguium (nail). Zoophilic species point to an animal-contact history (for example T. verrucosum facial infection in dairy farmers). Diagnosis starts with KOH demonstration of hyphae in scrapings, but culture confirms that the organism is truly a dermatophyte, identifies the likely source, and flags an increased likelihood of chronic or relapsing disease, particularly with T. rubrum. Sabouraud dextrose agar, with or without cycloheximide, is the standard isolation medium. ITS-region PCR sequencing has become the preferred confirmatory method in many laboratories.

Dematiaceous fungi

Melanin-walled soil and plant saprobes produce three histologically distinct syndromes.

Eumycotic mycetoma is a chronic, painless, draining subcutaneous mass with granule formation that extends to bone, usually in the hand or foot (Madura foot) after traumatic inoculation. Madurella mycetomatis is the most common cause worldwide; the Scedosporium apiospermum complex is the most common cause in the United States. Bacterial (actinomycotic) mycetoma is a separate, nonfungal entity. A fungus ball in a paranasal sinus is a related but granule-free presentation more properly classified as fungal rhinosinusitis.

Chromoblastomycosis is slowly progressive tropical subcutaneous disease with a painless verrucous plaque at the inoculation site, defined histologically by large, muriform, thick-walled sclerotic bodies (“copper pennies”).

Phaeohyphomycosis is a catch-all histopathologic category for infections showing dark-walled dematiaceous septate hyphal elements in tissue. It spans allergic disease (Alternaria/Cladosporium sinusitis; Bipolaris/Curvularia bronchopulmonary disease), superficial infection, deep local infection (Exophiala jeanselmei, E. dermatitidis, Bipolaris/Curvularia), and disseminated disease. Lomentospora prolificans causes an aggressive, largely pan-antifungal-resistant disseminated form. Cladophialophora bantiana is neurotropic (able to grow at 40 °C) and a recognized cause of cerebral phaeohyphomycosis even in immunocompetent hosts. A 2012 multistate outbreak traced to contaminated compounded methylprednisolone showed that organisms usually regarded as culture contaminants can be pathogenic when introduced by direct inoculation.

Diagnosis requires characteristic tissue histopathology (Masson-Fontana melanin stain highlights hyphal elements even when they appear unpigmented) plus culture. Because many causative species are common environmental contaminants, culture growth must be correlated with histopathology before assigning etiologic significance.

Mucormycetes

The disease-causing fungi in the order Mucorales are called mucormycetes. Rhizopus spp. (especially R. arrhizus, R. microsporus) cause the majority of identified cases. Risk factors involve impaired leukocyte function or iron/glucose dysregulation: uncontrolled diabetes, especially ketoacidosis, corticosteroid use, neutropenia, transplantation, prematurity, and deferoxamine therapy. The six recognized clinical syndromes are rhino-orbital-cerebral, pulmonary, cutaneous, gastrointestinal, disseminated, and uncommon presentations such as endocarditis. Rhino-orbital-cerebral and pulmonary disease predominate.

The tissue hallmark is broad (typically 5-15 μm, reported up to 20 μm), sparsely septate hyphae with wide-angle, haphazard branching. These angioinvasive hyphae cause thrombosis and coagulative necrosis and stain better with H&E than with GMS, the opposite pattern from most other pathogenic molds. Culture can fail even with clear tissue evidence because homogenization damages the fragile hyphae. Mince or tease tissue rather than homogenizing it, and avoid cycloheximide-containing selective media. Rhizopus produces unbranched sporangiophores with rootlike rhizoids arising directly opposite them. Lichtheimia has rudimentary rhizoids not directly opposite the sporangiophore. Mucor produces no rhizoids and often grows poorly at 37 °C. Rhizomucor is intermediate, with rudimentary rhizoids and branched sporangiophores, and is thermotolerant (up to about 54 °C). Species-level identification increasingly relies on ITS/28S sequencing.

Hyaline septate molds

Aspergillus spp. cause the most common life-threatening opportunistic mold infection in immunosuppressed hosts, spanning noninvasive (allergic, fungus ball), locally invasive, and disseminated disease. A. fumigatus complex predominates overall; A. flavus complex predominates in some fungal-sinusitis/keratitis series; A. terreus complex is notably amphotericin-B-refractory. Neutropenia, graft-versus-host disease, and transplantation are the dominant risk factors; nosocomial clusters have been linked to hospital construction dust. A. fumigatus produces a fluffy blue-green colony and a short (300-500 μm), smooth conidiophore with a flask-shaped vesicle and a single row of phialides. A. flavus has a velvety yellow-green colony and a longer (400-700 μm) rough conidiophore. A. terreus has a cinnamon-brown colony and a short (<250 μm) conidiophore with biseriate phialides. Diagnosis combines galactomannan and (1→3)-β-D-glucan serology, molecular targets (β-tubulin and calmodulin), and the definitive standard of positive sterile-tissue culture with histologic evidence of invasion.

Fusarium spp. cause keratomycosis, burn-wound infection, and invasive disease in immunosuppressed hosts, the second most common mold infection after aspergillosis. Histopathology mirrors invasive aspergillosis, but positive fungemia is a distinguishing clinical clue: Fusarium sporulates in tissue and readily seeds the bloodstream; Aspergillus blood cultures are rare even in disseminated disease. Characteristic canoe-shaped, often septate macroconidia are diagnostic.

Scedosporium/Lomentospora spp. are soil saprobes. S. apiospermum complex is the most common cause of invasive disease in immunocompromised hosts and of eumycotic mycetoma. Lomentospora prolificans causes rarer but often pan-antifungal-resistant disseminated disease. Colonies mature to a mousy brownish-gray; the asexual phase bears light-brown, oval annelloconidia. Other septate hyaline saprobes (Acremonium, Paecilomyces, Purpureocillium) are typically low-virulence environmental molds; establishing true pathogenicity requires demonstrating hyphae directly in tissue, not culture growth alone.

Microsporidia, Pneumocystis, and Prototheca

Microsporidia are fungi. Enterocytozoon bieneusi and Encephalitozoon intestinalis cause most human disease. Transmission is via contaminated food or water, or occasionally sexual or parenteral exposure. Disease occurs mainly with immunocompromise and causes chronic diarrhea and wasting in AIDS, similar to cryptosporidiosis. Spores are small (E. bieneusi/Encephalitozoon, 0.8-1.5 × ~1 μm), stain pink on H&E, are positive with GMS and Ziehl-Neelsen, and have a characteristic polar dot-like PAS pattern that helps separate them from similarly sized small yeasts. The main clinical test is a modified trichrome stain with a high chromotrope 2R content and extended staining time, performed on centrifuged stool or body fluid.

Pneumocystis jirovecii is the human-specific species. In vitro culture is unavailable, so definitive diagnosis requires direct cyst or trophozoite demonstration in respiratory specimens (BAL, induced sputum, or tracheal aspirate) stained with modified Papanicolaou, Gram-Weigert, Wright-Giemsa, or the diagnostic-standard GMS, which colors the 4-6 μm oval, cup, crescent, or helmet-shaped cysts brown to black. Immunofluorescent monoclonal antibody staining is also a recognized standard. Classic histology shows alveolar septal widening with a foamy honeycombed intra-alveolar exudate. PCR is highly sensitive, but a positive result cannot reliably distinguish infection from asymptomatic colonization.¹

The achlorophyllous alga Prototheca wickerhamii (rarely P. zopfii) causes protothecosis and is worked up in the mycology laboratory because it mimics fungal infection. Infection is acquired through superficial wounds exposed to fresh or marine water. Large, nonbudding, spheroid-to-ovoid cells with a thick double-layered wall and a morula-like internal structure packed with endospores are diagnostic on PAS/GMS. The organism grows readily on standard fungal media (white-to-tan colonies within 48 hours) and can be speciated with commercial yeast identification systems.

Molecular diagnostics for fungi and parasites

Smear and culture remain the primary fungal detection methods despite limitations in sensitivity, organism viability, and growth rate. For the dimorphic pathogens and Cryptococcus neoformans, gene-probe confirmation of a culture isolate is faster and safer than morphologic conversion because it avoids extended handling of a biohazardous mold-phase culture. Broad-range PCR extends molecular testing to direct clinical specimens. Primers targeting sequences conserved across Candida, Aspergillus, the Mucorales, and the dimorphic fungi generate products resolved to genus or species by probe hybridization or sequencing. Molds are typically typed by ITS-region or 28S rRNA sequencing from 24-48-hour cultures. MALDI-TOF is used less often for fungi than for bacteria because thick fungal cell walls require chemical or mechanical disruption; reliable spectra for Candida and Cryptococcus yeasts are now well established.

Morphology-based parasite diagnosis remains the default but is prone to false negatives at low organism density and requires trained microscopists. Technical obstacles include oocyst and microsporidial spore walls that resist lysis, stool matrices that inhibit PCR, and the unusually low GC content of Plasmodium DNA. Laboratory-developed real-time PCR assays target Babesia in blood and ticks, Trichomonas in vaginal specimens, and Encephalitozoon microsporidia. Multiplex real-time PCR panels detect Entamoeba histolytica, Giardia duodenalis, and Cryptosporidium spp. simultaneously in stool. A shared internal control detects PCR inhibition for every target in the panel, which is important with stool specimens.