I Clin Pathol 1996;49:95-99 95 Leaders J Clin Pathol: first published as 10.1136/jcp.49.2.95 on 1 February 1996. Downloaded from Mycological techniques K G Davey, C K Campbell, D W Wamock Introduction possible from the free edge of the nail and Laboratory tests are essential to establish the should include its full thickness. Specimens diagnosis of a fungal infection. The methods should be taken from several nails if more than used are based on three broad approaches: (1) one is affected. Debris beneath infected nails the detection ofthe pathogen in tissue on direct often contains the fungus. microscopic examination; (2) its isolation and It is sometimes helpful to use a Wood's lamp identification in culture; and (3) the detection to select infected scalp hairs for investigation. of an immunological response to the pathogen. Ifnone ofthe hairs gives the green fluorescence This Broadsheet describes the collection of which is characteristic of some microsporum clinical specimens from patients with suspected infections, a search should be made for lustre- fungal infection and outlines appropriate test less hairs or stumps and for hairs broken off procedures for the detection, isolation and at follicle mouths. Hairs should be extracted identification of fungal pathogens. It does not from the scalp using flat-ended forceps. Cut cover the serological diagnosis of fungal in- hairs without roots are unsuitable for my- fection. cological examination. Skin scrapings should also be collected from sites where fungal in- fection of hairs is suspected. Collection of specimens Another method which is useful for the col- It is important to provide the laboratory with lection of adequate material from patients with adequate specimens for investigation together suspected scalp infection not detectable under with sufficient clinical information about un- a Wood's lamp is to brush the scalp with a derlying illness, recent travel or previous res- plastic massage pad (available from most chem- idence abroad, animal contacts, and the ists), which is then pressed into the surface of patient's occupation if considered relevant. an agar plate. The pad should be sterilised in http://jcp.bmj.com/ With the exception of skin, nails and hair, all 1 % chlorhexidine for one hour, rinsed in sterile samples should be collected in sterile con- water and dried before being reused. tainers. MUCOUS MEMBRANES SKIN, NAILS AND HAIR Swabs moistened in sterile water or saline nails and hair should be Skin, collected into should be used to collect exudate or discharge. on September 25, 2021 by guest. Protected copyright. folded squares of stiff black paper (about The swabs should be sent to the laboratory in 10 x 10 cm). These can now be purchased from a standard transport medium. a number of commercial suppliers (Dermaco, PO Box 470, Toddington, Bedfordshire LU5 6BK; MycoTrans, PO Box 1172, Biggar, Lan- EAR arkshire ML12 6NN). It is essential to clean Scrapings of material from the ear canal are to superficial lesions with 70% alcohol prior to be preferred, although swabs can also be used. This Broadsheet has been sampling if ointments, creams or powders have prepared by the authors at the been applied. Ifpityriasis versicolor is a possible invitation of the Association of EYE Clinical Pathologists who diagnosis, the affected sites should be examined Material from a corneal ulcer with a suspected reserve the copyright. Further under ultraviolet light (Wood's lamp). The copies of this Broadsheet may fungal cause should be scraped with a sterile be obtained from the Publishing characteristic golden yellow fluorescence often platinum spatula, sampling both the base of Manager, 3rournal of Clinical associated with this infection can help in the Pathology, BMA House, the ulcer and the edges. This procedure should Tavistock Square, selection of skin scrapings. London WClH 9JR. be performed by an eye specialist. Because the Material should be collected from cutaneous amount of material that can be obtained will PHLS Mycology lesions by scraping outwards from the margin be small, it is best transferred to a glass slide Reference Laboratory, of the lesion with a blunt scalpel. If there is for microscopic examination and to an agar Public Health minimal clear tape as Laboratory, scaling, adhesive such plate for culture at the bedside. The culture Kingsdown, sellotape can be used to remove material for plate should be marked to indicate the point Bristol BS2 8EL examination. The sellotape strip is pressed of inoculation. K G Davey against the lesion, peeled off and placed, ad- Swabs are not suitable for sam- C K Campbell pling comeal lesions. D W Wamock hesive side down, on a clean glass microscope slide for transportation to the laboratory. Correspondence to: Dr D W Wamock. Nail scrapings or clippings should be taken BLOOD Accepted for publication from discoloured or dystrophic parts of the Blood culture is an important part of the in- 26 September 1995 nail. Specimens should be cut as far back as vestigation of suspected deep fungal infection. 96 Davey, Campbell, Warnock However, microbiologists should not expect Characteristics of some common dermatophytes invading blood cultures taken for bacterial isolation to hair detect fungal pathogens other than Candida Arthrospore size J Clin Pathol: first published as 10.1136/jcp.49.2.95 on 1 February 1996. Downloaded from spp., Cryptococcus neoformans, or Thichosporon Fungus (oIm) Arrangement spp. Isolation of fungi from blood depends on Microsporunm audouinii Small (2-5) Ectothrix a number of factors including the amount of Microsporum canis Small (2-5) Ectothrix Trichophyton mentagrophytes Small (3-5) Ectothrix blood sampled and the method of processing. Trichophyton tonsurans Large (4-8) Endothrix Commercial biphasic and Bactec blood culture Tichophyton violaceuni Large (4-8) Endothrix systems will often permit the detection offungal Tnchophyton verrucosum Large (5-10) Ectothrix infection. SPUTUM AND BRONCHOALVEOLAR LAVAGE Fresh early morning samples of sputum are men consists ofmore than one piece ofmaterial, ideal. These should be collected in sterile con- it is advisable to use some of each for mi- tainers and processed within two hours of col- croscopic examination and culture. Hairs lection. If delay in processing is unavoidable, should first be examined under a Wood's lamp specimens should be stored at 4°C. More in- and fluorescent parts, if present, used for pro- vasive sampling methods are often required to cessing. Hairs should be cut about 5 mm above obtain specimens from neutropenic patients. the root and the upper portion discarded. Se- Broncheoalveolar lavage (BAL) provides useful lected fragments or roots are placed in a large samples for direct microscopic examination and drop of 20% potassium hydroxide (KOH) on culture. a microscope slide and a coverslip placed on top. The slide should be set to one side to soften and clear the material. Skin material will CEREBROSPINAL FLUID, URINE AND OTHER clear in 15 to 20 minutes, as will thin nail FLUIDS parings and sub-ungual debris, but thicker nail Cerebrospinal fluid samples of 3-5 ml are ideal, fragments can take 30 to 60 minutes to soften. but are usually smaller than this. Samples can Gentle warming ofpreparations will reduce the be centrifuged and the supematant fluid used time required for clearing, but care must be for serological tests. The sediment can be cul- taken to avoid boiling. tured, but is also useful for direct microscopic Once skin or nail material has digested, the examination. In non-catheterised patients, coverslip should be pressed down to squash fresh mid-stream specimens of urine are ad- out the fragments and render them transparent, equate for mycological investigation, provided blotting off the excess KOH. Hair specimens care is taken to ensure that vaginal or perineal should not be heated or squashed as infected infection does not lead to contamination. In hairs will disintegrate and the diagnostic ar- http://jcp.bmj.com/ infants, suprapubic aspiration is the best rangement of the arthrospores will be lost. method of urine collection. Other fluids, Low power magnification ( x 10 objective) is whether aspirated or drained, should be col- adequate for the detection of fungal hyphae in lected into sterile containers which include a KOH preparations, but a higher power ( x 40 small amount of sterile heparin diluted 1 in objective) is often required to confirm their 1000 to prevent clotting. These specimens presence. Septate hyphae which may consist of should be centrifuged and the sediment cul- chains of rectangular spores (termed ar- on September 25, 2021 by guest. Protected copyright. tured. Drain fluid from patients receiving con- throspores) are typical of dermatophyte in- tinuous peritoneal dialysis should be collected fection. In cases of pityriasis versicolor, the into sterile containers without heparin. fungus (Malassezia furfur) appears as clusters of round cells together with short, unbranched hyphae. If a hair specimen is found to be PUS, BONE MARROW AND TISSUE infected, the size of the arthrospores and their If possible, swabs should not be used to collect arrangement should be noted (table). material from draining abscesses or ulcers. If a Care must be taken to distinguish fungal swab must be used, then material should be hyphae from the artefact known as "mosaic". taken from as deep as possible within the lesion. This is thought to be caused by deposition of Pus from undrained subcutaneous abscesses or cholesterol and, unlike true fungus, it tends to sinus tracts should be collected with a sterile follow the outlines of the epidermal cell walls. needle and syringe. If grains are visible in the Methods for selective staining of fungal hyphae pus (as in mycetoma), these must be collected. are available, but are seldom required. Bone marrow specimens should be collected The recognition of fungal hyphae and/or ar- into sterile containers which include a small throspores by direct microscopic examination amount of sterile heparin diluted 1 in 1000.