The specimen for the laboratory diagnosis of fungal diseases depends on the site of infection. Hair, nail clippings, skin scrapings, blood, CSF, and sputum are the most common clinical specimens for the diagnosis of fungal infections.
Selection and collection of appropriate clinical specimens and their timely transport to the laboratory for processing is a prerequisite to ensure the recovery/identification of fungal pathogens.
As much cerebrospinal fluid (CSF) as possible should be used for the culture of fungi. If processing is to be delayed, samples should be left at room temperature or placed in a 30°C incubator because CSF is an adequate fluid culture medium in which fungal elements can survive until subcultured in this environmental condition. Generally, CSF is filtered through 0.45-μm filter paper and filter paper is placed onto the surface of the appropriate culture medium with the inoculum side down. Culture media should contain no antibacterial or antifungal agents.
Respiratory Tract Secretions
Sputum, induced sputum, bronchial washings, bronchoalveolar lavage (BAL), and tracheal aspirations are commonly sent respiratory tract specimens for the diagnosis of fungal infections.
Sputum should be collected early-morning after rinsing but before breakfast.
Instruct patients to rinse their mouths vigorously with water immediately before coughing ½ to 1 oz of sputum into a sterile, screw-capped container. If adequate sputum is not obtained, induced sputum with a heated aerosol saline suspension can be used. Bronchial washings, biopsy, or bronchoalveolar lavage fluid should be transported promptly to the laboratory in sterile sealed containers.
Fungal infections of the respiratory tract clinically resemble mycobacterial infections, and as the same specimen can be used for both fungal and mycobacterial infection diagnosis, Middlebrook 7H11 broth can be used as a transport medium.
Choice of culture media: Culture media should contain antibacterial agent, to prevent overgrowth of contaminating bacteria and antifungal agent such as cycloheximide, to prevent overgrowth by rapidly growing molds.
Blood is the preferred sample to diagnose disseminated fungal infections. Biphasic agar-broth bottles designed specifically for fungal cultures are superior to routine bottles used for the recovery of bacterial pathogens.
Clinical microbiology laboratories mostly rely on automated blood culture systems such as BACTEC (Becton Dickinson), Bact/ALERT (BioMerieux) for the recovery of yeasts. Lysis-centrifugation systems, such as the Isolator (Wampole Laboratories) are also highly recommended, particularly for the recovery of dimorphic fungi especially Histoplasma capsulatum.
The first early-morning urine sample is preferred; random samples are acceptable. Specimens should be collected aseptically in sterile, screw-cap containers and sent immediately for processing. If a delay in processing beyond 2 hours is anticipated, the urine sample should be refrigerated at 4°C to inhibit over-growth by rapidly growing bacteria. Urine samples are centrifuged and sediment is inoculated onto the culture media.
Choice of media: Media containing antibacterial agents (to suppress the growth of gram-negative contaminants) are used to ensure optimal recovery of fungal pathogens.
Twenty-four-hour urine samples are unacceptable for fungal culture.
Some deep-seated mycoses, notably blastomycosis and, less commonly, histoplasmosis or coccidioidomycosis, may be diagnosed by collecting prostatic secretions. The bladder is first emptied, followed by prostatic massage. Secretions should be inoculated directly into appropriate fungal culture media; also, 5-10mL of urine should be collected in a separate container.
The skin over pustular lesions should be disinfected and exudates aspirated using a sterile needle and syringe. The syringe may also serve as transport container if the needle is capped. Biopsy of the lesion may be necessary if the aspirate fails to yield fungi.
Skin, nails, hair
Skin scrapings or biopsies, hair and nail clippings are usually submitted in the suspected cases of dermatophyte infections. These specimens should be placed in a sterile container and should never be refrigerated.
- Skin: First, swab the area of skin to be sampled with 70% alcohol to remove surface bacterial contaminants. Sample the peripheral, erythematous, growing margin of typical “ringworm” lesions, scraping with the side of a glass microscope slide or the edge of a scalpel blade.
- Nail: Infected nails should be sampled from beneath the nail plate to obtain softened material from the nail bed. If this is not possible, scrape away the surface of the nail before collecting shavings from the deeper portions.
- Hairs: Hairs should be collected from areas of scaling or alopecia, or those that fluoresce when viewed under a Wood’s (long-wave-length) ultraviolet lamp. Infected hairs are removed by plucking them with forceps.
Culture media: Mycosel agar (Sabouraud’s dextrose agar with cycloheximide and chloramphenicol) and dermatophyte test medium (DTM) can be used for the isolation of dermatophytes. To ensure the growth of slow-growing fungal pathogens, these media should be cultured for a minimum of 21 days before reporting as negative.
Tissue biopsies of suspected sites of infection should be transported in a sterile gauze moistened with physiologic, nonbacteriostatic, sterile saline solution in a screw-cap container. All tissue biopsies are processed by mincing or grinding before culturing. The specimen should be cultured as soon as possible, not be frozen or allowed to dehydrate prior to culture.
Culture media: At least 1 mL of a processed sample should be spread onto the surface of appropriate culture media and incubated for 30°C for 21 days.
References and Further Readings
- Procop, G. W., & Koneman, E. W. (2016). Koneman’s Color Atlas and Textbook of Diagnostic Microbiology (Seventh, International edition). Lippincott Williams and Wilkins.
- Tille, P. (2017). Bailey & Scott’s Diagnostic Microbiology (14 edition). Mosby.