Diagnostic Mycology: Specimens, Microscopy, Histopathology and Culture
Conventional mycology is still the basis for diagnosis, despite the rise of antigen and molecular tests. It identifies the organism, allows susceptibility testing, and recovers an isolate for epidemiology. Results depend on the quality of the specimen, on timely transport, on the choice of stains and media, on the experience of the reader, and on clear communication between the clinician and the laboratory about the suspected pathogen. Many fungi are hazardous to laboratory staff, so the clinician states the suspicion on the request form.
| Specimen | Collection and handling |
|---|---|
| Tissue and biopsy | The best specimen; a sterile container with a few drops of sterile saline (not formalin, which kills the organism, and not a swab); a separate portion for histology in formalin; Mucorales specimens are minced and not ground, and not refrigerated; sent within 2 hours |
| Blood | Blood culture bottles (aerobic) with automated detection for yeasts; for dimorphic fungi and Histoplasma, lysis centrifugation tubes (Isolator) or fungal bottles improve yield; 10 to 20 mL per set in adults; moulds are rarely recovered from blood (except Fusarium, Scedosporium, and Talaromyces) |
| Cerebrospinal fluid | At least 1 to 5 mL (more for culture of dimorphic fungi and Cryptococcus); centrifuged for culture and India ink; antigen testing on supernatant |
| Respiratory specimens | Sputum (early morning, deep cough, rinsed mouth), bronchoalveolar lavage (at least 20 mL returned), bronchial washings, and transbronchial or open lung biopsy; the specimen is processed within 2 hours or refrigerated |
| Skin, hair, and nail | Skin: scrape the active edge of a cleaned lesion with a blade; hair: pluck infected hairs with their roots, and use a toothbrush or cotton swab for scalp scale; nails: clip and scrape debris from beneath the nail (the nail plate is cleaned with alcohol, and the distal part, the sample of the proximal nail edge in superficial white onychomycosis, is collected) |
| Sterile body fluids | Pleural, peritoneal, synovial, and pericardial fluid in sterile containers, with culture in blood culture bottles for yeasts |
| Urine | Midstream or catheter specimen for yeasts; two or more positive specimens suggest disease only with symptoms |
| Pus and aspirates | Aspirate from deep abscesses, with sulphur granules and grains collected for microscopy |
| Corneal scrapings | Scraping from the ulcer base and margin with a sterile blade onto slides and onto media directly (Sabouraud agar, blood agar, and chocolate agar) |
| Vitreous and aqueous humour | A small volume, inoculated directly to media |
| Stool and swabs | Limited value; swabs for Candida auris screening (axilla and groin) |
| Environmental samples | Air, water, and surface sampling in outbreaks |
General rules: label the specimen with the site and the suspected pathogen, record the antifungal drugs the patient has received, send repeated specimens in suspected disease (a single negative culture does not exclude infection), and transport to the laboratory without delay. Specimens for dermatophytes, Mucorales, and Coccidioides, cerebrospinal fluid, and blood are kept at room temperature, and other specimens are refrigerated when a delay is expected.
| Hazard | Organisms and precautions |
|---|---|
| Biosafety level 2 | Yeasts (Candida, Cryptococcus), dermatophytes, and most moulds |
| Biosafety level 3 | Culture of the mould phase of Coccidioides, Histoplasma, Blastomyces, Paracoccidioides, Talaromyces marneffei, and Emergomyces, with a class II biological safety cabinet |
| Practices | Plates sealed with tape, no sniffing of cultures, specimens opened inside a cabinet, and the laboratory told in advance of suspected endemic mycoses |
| Staff risk | Laboratory acquired coccidioidomycosis and histoplasmosis are well documented |
| Method | Use |
|---|---|
| Potassium hydroxide (10 to 20 percent) | Dissolves keratin and tissue debris to reveal fungal elements in skin, hair, nail, and sputum; dimethyl sulfoxide or glycerol is added to speed clearing of nail; a 5 to 10 minute wait |
| Calcofluor white (with potassium hydroxide) | A fluorescent stain that binds chitin and cellulose in the fungal wall, and is read under a fluorescence microscope; the most sensitive rapid method for yeasts, moulds, and Pneumocystis cysts |
| India ink (or nigrosin) | A negative stain that shows the polysaccharide capsule of Cryptococcus as a clear halo in cerebrospinal fluid; sensitivity about 50 to 80 percent, lower than the antigen test |
| Gram stain | Yeasts are Gram positive; large budding cells and pseudohyphae in sterile fluids, and Candida in blood cultures |
| Lactophenol cotton blue | A mounting medium for moulds in culture (to show conidia and structures) |
| Giemsa and Wright stains | Intracellular yeasts of Histoplasma and Talaromyces in blood, marrow, and smears; Pneumocystis trophic forms |
| Papanicolaou and haematoxylin and eosin | Spherules of Coccidioides, yeasts, and hyphae in cytology |
| Methenamine silver, periodic acid Schiff | Fungal wall stains for smears and tissue |
| Immunofluorescence | Monoclonal antibody staining of Pneumocystis |
| Fontana Masson | Melanin in Cryptococcus (with a capsule deficient strain) and dematiaceous fungi |
| Interpretation | Report the morphology (yeast, pseudohyphae, septate or aseptate hyphae, branching, size) and the presence of tissue invasion; a positive direct examination from a sterile site is significant |
Histology proves invasion, shows the host response, and gives an early morphological diagnosis. The stains are haematoxylin and eosin, Grocott methenamine silver (the most sensitive for fungi, which stain black), periodic acid Schiff (pink), mucicarmine (capsule of Cryptococcus, stains red), Fontana Masson (melanin), and Alcian blue. Immunohistochemistry and in situ hybridisation are used where available.
| Morphology in tissue | Organisms |
|---|---|
| Hyaline, septate hyphae of 3 to 6 micrometres, acute angle (45 degrees) dichotomous branching | Aspergillus; look alikes are Fusarium, Scedosporium, Lomentosporium, and other hyaline moulds (cannot be separated by morphology) |
| Broad (6 to 25 micrometres), ribbon like, aseptate or pauciseptate hyphae with right angle branching | Mucorales |
| Pigmented (brown) hyphae, yeast forms, or muriform (sclerotic) bodies | Dematiaceous fungi (Fonsecaea, Cladophialophora, Exophiala) |
| Round yeasts of 4 to 10 micrometres with single narrow based buds and a mucicarmine positive capsule | Cryptococcus |
| Narrow based budding yeasts of 2 to 4 micrometres inside macrophages | Histoplasma, Emergomyces |
| Large (8 to 15 micrometres) thick walled yeasts with a single broad based bud | Blastomyces, Histoplasma var. duboisii (smaller buds) |
| Multiple budding yeasts ("ship's wheel") of 5 to 60 micrometres | Paracoccidioides |
| Spherules of 20 to 80 micrometres with endospores | Coccidioides |
| Oval yeasts with a central septum (fission) | Talaromyces marneffei |
| Yeasts with pseudohyphae and budding | Candida |
| Cigar shaped yeasts, or small yeasts with an apical bud | Sporothrix |
| Round yeast with a collarette | Malassezia |
| Cysts of 5 to 8 micrometres that appear as crushed ping pong balls on silver stain, with foamy intra-alveolar exudate | Pneumocystis |
| Thick walled spherules of 200 to 400 micrometres with endospores | Rhinosporidium |
| Grains in abscesses (black, white, or red) | Eumycetoma |
| Adiaspores (thick walled, large and non budding, up to 400 micrometres) | Emmonsia crescens and Emmonsia parva (adiaspiromycosis) |
Inflammatory patterns include suppuration, necrosis, and angioinvasion (moulds), granulomas (endemic mycoses and Cryptococcus), pyogranulomas (Blastomyces), and minimal inflammation (cryptococcosis in the immunocompromised, and Histoplasma in HIV).
Media and conditions
| Medium | Use |
|---|---|
| Sabouraud dextrose agar (pH 5.6) | The standard primary medium for most fungi, with chloramphenicol or gentamicin to inhibit bacteria |
| Sabouraud dextrose agar with cycloheximide (and chloramphenicol) | Selection of dermatophytes, Candida, and dimorphic fungi from contaminated specimens (inhibits Mucorales, Aspergillus, Cryptococcus, Scedosporium prolificans, and some Candida); not used for specimens in which saprophytic moulds are sought |
| Potato dextrose agar and potato flake agar | Sporulation and pigment of moulds and dermatophytes |
| Brain heart infusion agar (with blood, with or without antibacterials) | Enriched medium for dimorphic fungi (yeast conversion at 37 degrees Celsius) and Histoplasma |
| Dermatophyte test medium | Colour change from yellow to red with dermatophytes |
| Cornmeal agar with Tween 80 | Morphology of Candida (chlamydospores of Candida albicans, pseudohyphae) |
| Chromogenic Candida agar | Colour identifies Candida albicans (green), Candida tropicalis (blue), Candida krusei (pink), and others; shows mixed cultures |
| Birdseed (Niger seed) agar and caffeic acid medium | Brown colonies of Cryptococcus neoformans (phenol oxidase) |
| Canavanine glycine bromothymol blue agar | Separates Cryptococcus gattii (blue) from Cryptococcus neoformans |
| Modified Dixon agar (with lipids) | Growth of Malassezia |
| Czapek Dox agar and malt extract agar | Identification of Aspergillus and Penicillium |
| SceSel plus agar (with benomyl) | Selection of Scedosporium from cystic fibrosis sputum |
| Blood culture bottles and lysis centrifugation | Yeasts in blood; fungaemia |
Incubation
| Aspect | Details |
|---|---|
| Temperature | Two sets: 25 to 30 degrees Celsius (moulds and dimorphic fungi in mould phase) and 35 to 37 degrees Celsius (yeasts, thermotolerant moulds, and conversion of dimorphic fungi) |
| Duration | Yeasts: 2 to 5 days; moulds and dermatophytes: 2 to 4 weeks (dermatophytes up to 4 weeks, and nails 4 to 6 weeks); dimorphic fungi: up to 4 to 8 weeks; blood cultures for yeasts: 5 to 7 days and fungal cultures: 14 to 28 days |
| Atmosphere | Aerobic; humid; tubes with a loose cap or tape (not tightly sealed) to allow growth |
| Reading | The plates are examined at 2 to 3 days and then weekly, with subcultures of suspicious colonies |
Identification
| Approach | Details |
|---|---|
| Yeast phenotype | Colony colour on chromogenic agar; germ tube test (Candida albicans and Candida dubliniensis form germ tubes within 2 to 3 hours in serum at 37 degrees Celsius); chlamydospores on cornmeal agar; urease (positive in Cryptococcus, Trichosporon, Rhodotorula); nitrate; carbohydrate assimilation (API 20C and Vitek); Candida auris is misidentified by some biochemical systems (as Candida haemulonii, Rhodotorula, or Saccharomyces) |
| Mould phenotype | Colony growth rate, colour, reverse pigment, texture, and microscopy with structures (conidiophores, phialides, vesicles, macroconidia, sporangia); lactophenol cotton blue mounts and slide cultures to show arrangement |
| Mass spectrometry | Matrix assisted laser desorption ionisation time of flight mass spectrometry gives species identification of yeasts and many moulds in minutes from a colony, with an extended fungal database; extraction is needed for moulds |
| DNA sequencing | The internal transcribed spacer region (universal fungal barcode) for most fungi, the D1/D2 large subunit ribosomal DNA region for yeasts, and secondary markers (beta tubulin, calmodulin for Aspergillus; translation elongation factor for Fusarium) to separate cryptic species in complexes |
| Other methods | Exoantigen and DNA probes for dimorphic fungi; thermal conversion; polymerase chain reaction based identification; whole genome sequencing for outbreaks |
Characteristic colony and microscopic features of common moulds:
| Organism | Colony | Microscopy |
|---|---|---|
| Aspergillus fumigatus | Blue green to grey green, powdery | Uniseriate; columnar conidial heads with phialides on the upper two thirds of the vesicle |
| Aspergillus flavus | Yellow green | Uniseriate and biseriate; rough walled conidiophores |
| Aspergillus niger | Black, with a white underside | Biseriate; dark globose conidial heads |
| Aspergillus terreus | Cinnamon brown | Biseriate; compact columnar heads; accessory conidia on hyphae |
| Mucorales | Fast, cottony, grey, filling the plate | Broad aseptate hyphae; sporangiophores; rhizoids (Rhizopus) |
| Fusarium | Woolly, white to pink or violet | Sickle shaped macroconidia |
| Scedosporium apiospermum | Grey brown, woolly | Single celled conidia on annellides |
| Trichophyton rubrum | White, downy, with a red reverse | Teardrop microconidia along hyphae |
| Microsporum canis | Yellow, with a golden reverse | Spindle shaped, thick walled macroconidia with terminal knob |
| Dematiaceous fungi | Olive to black | Pigmented hyphae and conidia (Exophiala, Cladophialophora, Curvularia) |
Interpretation
| Issue | Guidance |
|---|---|
| Contaminants | Common environmental moulds (Penicillium, Cladosporium, Alternaria, and Aspergillus species) from non sterile sites may be contaminants; repeated isolation, a high burden, and a compatible clinical setting favour disease |
| Colonisation of the airways | Candida in sputum is nearly always colonisation, while a positive respiratory culture for Aspergillus in a high risk host has a high positive predictive value |
| Sterile sites | Any yeast or mould from blood, cerebrospinal fluid, or deep tissue is considered significant (except Penicillium and some skin contaminants) |
| Candidaemia | Always significant; never ignored as a contaminant |
| Negative culture | Does not exclude disease: sensitivity of blood cultures for invasive candidiasis is about 50 percent, and for invasive aspergillosis it is below 10 percent |
| Susceptibility testing | Done for Candida from sterile sites and invasive disease, and for moulds in refractory disease or with expected resistance |
