Specimen collection

Specimen collection and processing

Amir Seyedmousavi

National Institutes of Health, Bethesda, U.S.A.

Laboratory diagnosis of fungal infections relies on proper collection of appropriate material and quick delivery of the specimens to the clinical laboratory. Selection of specimens for culture and microscopic examination is based on information obtained from clinical examination and radiographic findings and consideration of the most likely fungal pathogen that may cause a specific type of infection. The laboratory depends on clinical information in making decisions as to the best way to process the specimen to ensure recovery of the etiologic agent. Therefore, the clinical history is useful in interpreting the results of culture and other mycology laboratory testing, especially when dealing with specimens from nonsterile sites such as sputum (human), saliva (animal) and skin (human/animal). In addition, clinical information alerts laboratory personnel that they may be dealing with a potentially dangerous pathogen such as primary pathogenic dimorphs Coccidioides or Histoplasma or an epidemiologically significant organism such as Candida auris.

In principle, specimens should be submitted whenever possible in a sterile leak-proof container and be accompanied by a relevant clinical history. Specimens should be collected aseptically or after proper decontamination of sampling site. An adequate amount of clinical material must be submitted promptly for culture and microscopy. Transportation of specimens to the laboratory should follow immediately. If processing is delayed, the specimens for fungal culture may be stored at 4°C for a short time without loss of organism viability. Twenty-four–hour collections of sputum or urine are not appropriate for mycologic examination because they typically become overgrown with both bacterial and fungal contaminants.

There are some specimens that are better than others for the diagnosis of fungal infections. For example, cultures of blood and other normally sterile body fluids should be done if clinical indications suggest a hematogenous process or involvement of a closed space such as the central nervous system. Skin lesions should be biopsied, and collected specimens should be sent for both histopathology examination and culture. Oral and vaginal mucosal infections are generally best diagnosed by clinical presentation and direct microscopic examination of secretions or mucosal scrapings. Cultures often yield growth that represents normal flora or even contaminants on these sites. Similarly, diagnosis of gastrointestinal fungal infections is best made by biopsy, histopathology, and nucleic acid amplification rather than by culture.

Site of infection	Suspected fungus	Specimen of choice	Collection method(s)	Recommended diagnostic procedure
Blood	Candida, Cryptococcus, Histoplasma, Fusarium, Scedosporium, Lomentospora, Aspergillus terreus, Talaromyces marneffei, Trichosporon, Malassezia, Saccharomyces	Whole blood	EDTA blood collection tubes	Culture, lysis-centrifugation, PCR
		Serum	Serum blood collection tube	Antigen detection (Aspergillus, Candida, Cryptococcus, Histoplasma), PCR, β-D-glucan
		Urine	Sterile	Antigen detection (Histoplasma)
Bone marrow	Histoplasma, Talaromyces marneffei	Bone marrow aspirate	Sodium polyanethol sulfonate (SPS) tube	Histopathology, culture, PCR
Central nervous system	Candida, Cryptococcus, Aspergillus, Scedosporium, melanized fungi, Mucorales, Histoplasma, Coccidioides	Cerebrospinal fluid	Sterile	Microscopy (Gram stain, India ink, fluorescent staining) culture, Cryptococcus antigen, PCR
Bone / joint fluid	Candida, Fusarium, Aspergillus, Histoplasma, Coccidioides, Blastomyces, Talaromyces marneffei, Sporothrix	Aspirate	Sterile	Microscopy (Gram stain, fluorescent staining), culture
Bone / joint fluid		Biopsy	Sterile	Microscopy (Gram stain, fluorescent staining), culture (ground and mince tissue if suspected mucormycosis)
Eye	Fusarium, Candida, Cryptococcus, Aspergillus, Mucorales	Cornea	Scraping	Microscopy (Gram stain, fluorescent staining), culture
Eye	Fusarium, Candida, Cryptococcus, Aspergillus, Mucorales	Vitreous fluid	Sterile aspirate	b
Urogenital system	Candida, Cryptococcus, Trichosporon, Rhodotorula, Histoplasma, Blastomyces, Coccidioides, Microsporidia	Urine, secretions / discharge (vaginal, urethral, prostatic)	Sterile saline swab	Microscopy (Gram stain, fluorescent staining), antigen detection (Histoplasma), culture; specific stain, PCR
Respiratory tract	Cryptococcus. Aspergillus, Fusarium, Mucorales, Scedosporium, Lomentospora, melanized fungi, endemic dimorphs, Pneumocystis	Sputum	Induced (preferred)	Microscopy (fluorescent staining), culture, PCR
		Tracheal	Aspirate	Microscopy (fluorescent staining), culture
		Bronchoalveolar lavage	Sterile	Microscopy (fluorescent staining), culture, PCR, Aspergillus galactomannan, β-D glucan
		Lung biopsy	Sterile	Microscopy (Gram stain, fluorescent staining), culture (direct, concentrated pellets), PCR
Skin and mucosal surfaces	Candida, Cryptococcus, Trichosporon, Aspergillus, Mucorales, Fusarium, melanized fungi, endemic dimorphs, Sporothrix	Biopsy / skin scraping	Sterile	Microscopy (fluorescent staining), culture (ground and mince tissue if suspected mucormycosis), PCR
Skin and mucosal surfaces		Mucosal	Sterile swab	b