Fungal Stains and Culture

Fungal stains and culture detect yeasts, moulds and dimorphic fungi from skin, hair, nails, mucosa, respiratory samples, blood, CSF, sterile fluids and tissue.

Direct microscopy is rapid, with potassium hydroxide (KOH) 10 to 20% clearing keratin and tissue, calcofluor white or Blankophor fluorescence (the most sensitive), lactophenol cotton blue, Gram stain for yeasts, Grocott-Gomori methenamine silver, periodic acid-Schiff, India ink for Cryptococcus and Giemsa for Histoplasma and Talaromyces.

Culture is on Sabouraud dextrose agar (with chloramphenicol and with or without cycloheximide), brain heart infusion agar, chromogenic Candida agar, and special media (dermatophyte test medium, birdseed agar), incubated at 25 to 30 °C and 35 to 37 °C for 2 to 4 weeks (up to 6 weeks for dimorphic fungi).

Blood culture systems detect Candida, but are poor for moulds and Histoplasma (lysis centrifugation is better). Identification uses morphology, MALDI-TOF mass spectrometry and DNA sequencing, followed by antifungal susceptibility testing.

Fungal infections affect over a billion people, with superficial infections (tinea, candidiasis) the commonest, and invasive infections (candidaemia, invasive aspergillosis, cryptococcosis, histoplasmosis, mucormycosis, Pneumocystis pneumonia, talaromycosis) causing about 1.5 million deaths each year.

Invasive disease concentrates in immunocompromised patients (haematological malignancy, transplantation, prolonged neutropenia, corticosteroids, HIV, intensive care, diabetic ketoacidosis, broad spectrum antibiotics, central venous catheters and total parenteral nutrition).

Indications. Suspected dermatophyte infection of skin, hair or nails (before oral therapy, since treatment is prolonged and toxic). Oral and oesophageal candidiasis not responding to treatment. Fever in a neutropenic patient unresponsive to antibiotics. Pulmonary nodules, cavities or infiltrates in an immunocompromised patient.

Sinusitis with black necrotic tissue or palate lesions (mucormycosis). Fungal keratitis, endophthalmitis, sporotrichosis, mycetoma and chromoblastomycosis. Chronic meningitis, osteomyelitis or lymphadenitis. Endemic mycoses after exposure (histoplasmosis, coccidioidomycosis, blastomycosis, talaromycosis).

Specimen. Skin: scrape scales from the active edge of the lesion after cleaning with 70% alcohol. Hair: plucked hairs with roots. Nails: clippings and subungual debris from the deep proximal portion. Mucosal swabs.

Sputum, bronchoalveolar lavage, tissue biopsy (the best specimen for invasive disease, sent in a sterile container with saline and not in formalin for culture), aspirates, CSF, blood (at least two sets, with an adequate volume), and urine. Transport promptly. Never refrigerate specimens for dimorphic fungi or Mucorales, and do not grind tissue, which damages the hyphae of Mucorales.

Reference values. Direct microscopy: no fungal elements. Culture: no fungal growth, or only commensal yeasts (Candida in the mouth or vagina). No growth of moulds from sterile sites. Moulds isolated from non-sterile samples are often contaminants (Penicillium, Cladosporium, Alternaria) unless seen repeatedly or in direct microscopy.

Interpretation.

• KOH or calcofluor positive for hyphae in skin, hair or nail scrapings: dermatophyte infection (tinea, onychomycosis), confirmed by culture (Trichophyton, Microsporum, Epidermophyton), which takes up to 4 weeks.
• Yeasts and pseudohyphae in a sterile site, or any Candida in blood: invasive candidiasis (candidaemia is never a contaminant). Species identification and susceptibility testing are required, since C. glabrata and C. krusei are fluconazole resistant, and C. auris is multidrug resistant.
• Septate hyphae branching at 45 degrees in tissue or respiratory samples: Aspergillus. Broad, ribbon like, pauciseptate hyphae with branching at right angles: Mucorales, a surgical and medical emergency.
• Galactomannan, beta-D-glucan and Aspergillus PCR support diagnosis and complement culture. Culture growth of Aspergillus from a respiratory sample in a high risk patient has a high predictive value.
• Small intracellular yeasts in macrophages (Histoplasma capsulatum), broad based budding yeasts (Blastomyces), spherules (Coccidioides), and intracellular yeasts with a central septum (Talaromyces marneffei): endemic mycoses, with laboratory biosafety warnings.
• Cryptococcus: encapsulated yeast on India ink, with positive antigen.
• Pneumocystis jirovecii is detected by microscopy (Grocott or immunofluorescence) or PCR and does not grow in culture.
• Black grains (eumycetoma) and white or yellow grains (actinomycetoma, Nocardia) in a draining swelling: mycetoma, with species identification driving treatment.
• Yeast in urine from a catheter is mostly colonisation, and treatment is for symptoms or high risk patients.

Critical values and findings needing immediate action.

• Candida in a blood culture: start echinocandin, remove the central venous catheter, repeat cultures daily until negative, perform an ophthalmology review and echocardiography.
• Broad non-septate hyphae in tissue or necrotic sinus or palate lesions: invasive mucormycosis, immediate liposomal amphotericin B, urgent surgical debridement, control of diabetes and reversal of immunosuppression.
• Aspergillus in a respiratory sample from a neutropenic or transplant patient: voriconazole or isavuconazole, CT chest, galactomannan.
• Histoplasma, Talaromyces, Coccidioides or Cryptococcus in a sample: notify the laboratory staff (biosafety level 3 for cultures) and start antifungal therapy.
• Candida auris: strict isolation, contact precautions and environmental decontamination.

Interferences and pitfalls.

• Low sensitivity of culture for invasive disease: blood cultures are positive in only 50% of candidaemia, and under 10% for Aspergillus, so biomarkers and imaging are used together.
• Prior antifungal treatment (topical or oral) lowers yield, and dermatophyte specimens are best taken 2 weeks after stopping topical agents and 4 weeks after oral agents.
• Contamination by airborne moulds in cultures, with growth on only one plate or one specimen and no matching microscopy, is usually insignificant.
• Cycloheximide inhibits some pathogenic fungi (Aspergillus, Cryptococcus, Candida species other than albicans), so plates with and without it are used.
• Slow growth means early reports are preliminary, and cultures are held for 4 to 6 weeks (dimorphic fungi).
• Laboratory acquired infection with dimorphic fungi is a recognised hazard, and cultures are handled in a biological safety cabinet.
• Grocott silver stain may fail to show Mucorales clearly, and histology with periodic acid-Schiff is complementary.
• Susceptibility testing for yeasts and moulds uses broth microdilution, and breakpoints exist only for some species and drugs.

Special groups.

• Neonates and premature infants: candidaemia, with meningitis and kidney involvement.
• Children: tinea capitis (Trichophyton, Microsporum), commonly in African and Caribbean children, needs oral treatment.
• Patients with HIV: oral and oesophageal candidiasis, cryptococcosis, histoplasmosis, talaromycosis and Pneumocystis pneumonia.
• Diabetics and patients on corticosteroids: mucormycosis and invasive aspergillosis.
• Regions: histoplasmosis in the Americas, Africa and Asia, coccidioidomycosis in the south western Americas, talaromycosis in Southeast Asia, paracoccidioidomycosis in Latin America, mycetoma in the "mycetoma belt" (Sudan, India, Mexico), and chromoblastomycosis and sporotrichosis in tropical regions.

Follow up tests. Fungal culture with identification and antifungal susceptibility testing, serum galactomannan and beta-D-glucan, cryptococcal antigen, Histoplasma and Coccidioides antigen or serology, Pneumocystis PCR, fungal PCR and sequencing on tissue, histopathology (Grocott and periodic acid-Schiff), CT chest and sinuses, MRI for CNS disease, ophthalmology review,

echocardiography in candidaemia, azole drug level monitoring, and tests of liver function and renal function during treatment.