SN7 SHORT NOTE 5M

Describe the collection of specimens and the laboratory methods used in mycology.

Microbiology · 2nd Professional — Para-Clinical

Specimen Collection

Site	Specimen and technique
Skin	Scrapings with a blunt scalpel from the active advancing edge, not the cleared centre; clean the site with 70% alcohol first to remove bacteria and ointment; collect onto dark paper, which also allows dry storage for weeks
Hair	Plucked with forceps to include the root and follicle — cut hairs are useless, since the fungus is in the base of the shaft. Use a Wood lamp to select fluorescent hairs where available
Nail	Clippings taken as proximally as possible together with subungual debris, where the viable fungus lies; the distal free edge contains mostly dead material
Respiratory	Early-morning sputum, bronchoalveolar lavage, or a lung biopsy; a 24-hour collection is not used, as it overgrows with bacteria
Cerebrospinal fluid	Large volume, since organisms are scanty; process promptly and do not refrigerate
Blood	Standard bottles detect most yeasts; lysis-centrifugation improves recovery of dimorphic fungi and moulds; overlay with oil if Malassezia is suspected
Tissue	Send fresh and sterile, and not all of it in formalin; for mucormycosis the tissue must be minced rather than ground, since grinding destroys the aseptate hyphae and the culture is then falsely negative
Cornea	Scrapings from the base and edge of the ulcer, inoculated directly onto media at the slit lamp

CLINICAL PEARL

Grinding the tissue is why mucormycosis cultures come back negative. The hyphae are broad and aseptate — a single continuous cytoplasm with no cross-walls — so any disruption kills the whole filament. Routine homogenisation, which is correct for most specimens, destroys exactly this organism. The instruction to MINCE rather than grind is small, easily overlooked, and decides whether the diagnosis is made.

Direct Microscopy

Method	Use
KOH mount (10–20%; 40% for nails)	Digests keratin, cells and debris, leaving the fungal wall intact and visible. Cheap, takes minutes, and is the single most valuable test in mycology
Calcofluor white with KOH	Binds chitin and fluoresces bright blue-white under ultraviolet light; far more sensitive than KOH alone
INDIA ink / nigrosin	Negative staining for the capsule of Cryptococcus in cerebrospinal fluid
Gram stain	Yeasts stain gram-positive; useful for Candida
Lactophenol cotton blue	For examining a culture, not a clinical specimen — lactic acid preserves, phenol kills, and cotton blue stains the chitin, allowing the conidia and hyphae to be identified
Giemsa	Intracellular Histoplasma and Talaromyces in marrow or blood films
Histopathology — PAS and methenamine silver	Demonstrate fungi in tissue and, crucially, whether there is invasion

Culture and Identification

Sabouraud dextrose agar is the standard medium — a high sugar concentration at acid pH 5.6, which suits fungi and suppresses bacteria
Additives — chloramphenicol to suppress bacteria, and cyclohexide to suppress saprophytic moulds. But cycloheximide also inhibits Cryptococcus, Aspergillus, the mucorales and Candida krusei, so a plate without it must always be inoculated in parallel
Incubate at both 25°C and 37°C to demonstrate dimorphism, for up to 4 weeks; discarding at 48 hours is a common cause of a falsely negative result
Identification — colony rate of growth, texture, surface and reverse pigment; microscopic morphology of conidia in a lactophenol cotton blue mount; the germ tube test and chlamydospore formation for Candida; urease and CHROMagar; and MALDI-TOF or DNA sequencing for definitive identification

Non-culture Methods and Applied Aspects

Antigen detection — cryptococcal antigen (CrAg), highly sensitive and specific, available as a lateral flow test usable at the bedside; galactomannan for Aspergillus; Histoplasma antigen in urine and serum
Beta-D-glucan — a broad "pan-fungal" marker, positive in Candida, Aspergillus and Pneumocystis, but negative in mucormycosis and cryptococcosis, which is a useful discriminator; false positives occur with haemodialysis, some antibiotics, albumin and surgical gauze
Serology — immunodiffusion and complement fixation for the dimorphic fungi and ABPA; but it is unreliable in the immunosuppressed, who may not produce antibody
Molecular methods — panfungal and species-specific PCR, and sequencing for identification and resistance
Antifungal susceptibility testing by broth microdilution or gradient strip, which is increasingly necessary as resistance rises but is available in few Indian laboratories
Tell the laboratory what is suspected; the medium, the additives, the incubation temperature and the duration all differ between fungi, and a request marked only "fungal culture" will not detect a dimorphic fungus or Malassezia
Do not put the whole biopsy in formalin — send part fresh for microscopy and culture, or the organism and its susceptibilities are lost
A positive KOH mount with a negative culture still means fungal infection; culture is insensitive, and treatment should follow the microscopy
Distinguish colonisation from infection — fungi grown from sputum, wound swabs or catheter urine are frequently passengers, and only isolation from a sterile site or demonstration of tissue invasion proves disease
Diagnostic mycology is underdeveloped in much of India, and the commonest reason a fungal infection is missed is that nobody looked for it

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