A Guide to the Histological Identification of Fungi in Tissues

Home , Coccidioides immitis, Histoplasma duboisii, Phialophora verrucosa, Phycomycosis, Rhinosporidium seeberi

J. clin. Path., 1973, 26, 828-831

A guide to the histological identification of fungi in tissues

P. P. ANTHONY From the Bland-Sutton Institute ofPathology, The Middlesex Hospital, London

SYNOPSIS Infections with fungi and fungus-like organisms have increased in recent years. The presence of a fungus is often unsuspected clinically and it may only come to light in the course of microscopic examination oftissues removed by biopsy or at necropsy. Subsequent culture is desirable but not always possible. A simple scheme for identifying fungi and fungus-like organisms is presented based on general morphology, staining, and other special characteristics with notes on types of tissue reactions and common pitfalls.

Diseases caused by fungi may be divided into or as both. Terminology is cumbersome and the superficial mycoses and deep mycoses. Superficial exact taxonomic position of some is in doubt. mycoses are confined to the epidermis, hairs, and Actinomycetes, for example, resemble filamentous nails and can readily be diagnosed by clinical ap- fungi morphologically and mycobacteria bio- pearance and culture. Deep mycoses are usually chemically. Pneumocystis carinii is thought by some cutaneous, pulmonary, or disseminated. They are to be a protozoon rather than a fungus. These most commonly seen in Britain in (1) patients academic considerations apart, the problem that whose immune defence mechanisms have been presents itself to the clinical histopathologist is how impaired either by disease or treatment; (2) immi- to identify the organism from its shape, size, grants or travellers; and (3) drug addicts who inject staining reactions, or other characteristics and the themselves. The existence of a deep fungal infection type of tissue reaction associated with it. A scheme is often unsuspected and it may only come to light for this process is given below. This necessarily is in the course of microscopic examination of a an oversimplification of a very complex subject biopsy or of tissues removed at necropsy. Culture but it should help to reduce a particular problem is desirable for a precise diagnosis but it may not to practicable limits. The appearances described be possible to obtain further material for this. The are those usually seen in the tissues. histopathologist must be prepared, therefore, to establish the presence of fungi in the tissues, to What Is It Like? attempt identification of the species, and to exclude laboratory contamination and artefacts. ROUND BODIES (YEASTS) Diagnosis is made easier and the choice of Cryptococcus neoformans; Histoplasma capsulatum; alternatives narrowed by reasonable clinical inform- Histoplasma duboisfi; 'Chromomycosis' species: ation, the presence of any predisposing factors, Phialophora verrucosa and pedrosoi, Cladosporium and a working knowledge of the world-wide dis- carrionii; Blastomyces dermatitidis; Paracoccidioides tribution of the various fungi. Descriptions of brasiliensis; Rhinosporidium seeberi; Coccidioides mycotic diseases are to be found in most standard immitis; Sporothrix schenckii; Pneumocystis carinii. textbooks of pathology. A bibliography is given at the end of this text. FILAMENTS (TRUE HYPHAE AND Fungi of medical importance belong to the PSEUDOHYPHAE) fungi imperfecti, that is, they have no discernible Actinomyces israelii; Nocardia asteroides; Asper- phase of sexual reproduction but produce asexual gillus species: fumigatus and niger. spores of various kinds. They grow either as budding yeasts or as filaments (hyphae or pseudohyphae) Phycomycetes Received for publication 21 August 1973. Rhizopus, Mucor, and Absidia species (deep and 828 A guide to the histological identification offungi in tissues '829 systemic phycomycosis), Basidiobolus meristosporus REGULAR, SEPTATE HYPHAE WITH (subcutaneous phycomycosis), Entomophthora DICHOTOMOUS BRANCHING coronata (submucous phycomycosis). Aspergillus fumigatus and niger: these may form large masses ('aspergillomas') in tuberculous or 'Mycetoma' species bronchiectatic cavities in the lung. True fungi ('maduromycetoma'): Madurella Candida species also form regular, septate hyphae. mycetomi, Allescheria boydii, Leptosphaeria senega- These are often club shaped, branch little, and yeast lensis. Actinomycetes ('Actinomycetoma') Strepto- forms are always present. myces and Nocardia species. IRREGULAR, NON-SEPTATE HYPHAE WITH ROUND BODIES AND FILAMENTS HAPHAZARD BRANCHING Candida albicans Phycomycetes: Rhizopus, Mucor, and Absidia species responsible for deep and systemic phycomy- What Size? (Round Bodies) cosis. The hyphae of Basidiobolus meristosporus in subcutaneous and those of Entomophthora coro- SMALL (1-5 MICRONS) nata in submucous phycomycosis are clear and Histoplasma capsulatum, Pneumocystis carinii, difficult to see. They are, however, outlined by a Sporothrix schenckii eosinophilic precipitate.

LARGE (20-200 MICRONS) Any Special Features? Rhinosporidium seeberi, Coccidioides immitis STAINING MEDIUM (5-20 MICRONS) The use of control material is essential with special The rest. stains. Histoplasma capsulatum has to be distinguished Haematoxylin-eosin cannot be relied upon to show from the protozoa leishmania and toxoplasma (see all fungi. Nocardia species, Phycomycetes, and under staining below). The yeast forms of candida Pneumocystis carinii may remain invisible whilst species are egg shaped, 3-5 microns in size, bud many yeasts stain poorly. The asteroid body of frequently and also form hyphae. The large sporangia Sporothrix schenckii is virtually never seen in tissue of Rhinosporidium seeberi and Coccidioides immitis sections. It is wise to do periodic-acid-Schiff and are quite characteristic. The differential diagnosis methenamine silver stains on all suspect sections. of medium-sized yeasts rests on staining and other Periodic-acid-Schiff stain, or one of its modifi- properties and may be very difficult indeed. cations, suchas Gridley'smethod, isuniversally useful. It stains fungi a purple colour of varying intensity, What Shape? (Filaments) sometimes rather faint in postmortem tissues. It also stains many tissuecomponents andtheresults are Slender filaments or pseudohyphae, generally less not always clear cut. than 2 microns in diameter, are formed by organisms Grocott's modification of Gomori's methenamine that are not true fungi but bacteria (Actinomyces, (hexamine) silver stain shows all fungi black and Streptomyces, Nocardia species). True filamentous is superior to methods employed for the demon- fungi consist of broad filaments or hyphae, usually stration of reticulin. The results are bright and crisp, more than 2 microns in diameter. These may be an ideal method for demonstration and micro- septate or non-septate and branching is a feature. photography. It also differentiates Histoplasma Various types ofspores are formed, mostly in culture capsulatum (positive) from leishmania and toxo- and rarely in the tissues. plasma (both negative). Gram's stain has a limited usefulness for staining FELT-LIKE MASSES (GRAINS) the grains of Actinomyces and Nocardia species Actinomyces israelii (usually), Nocardia asteroides and Candida albicans. (rarely), various 'mycetoma' species. Southgate's mucicarmine stains Cryptococcus neo- The grains of actinomycosis, nocardiosis, and formans intensely brilliant red. This is practically 'actinomycetoma' are made up of slender pseudo- specific for this fungus. hyphae. Grains made up of broad hyphae are formed Giemsa's or Wright's stains show Histoplasma by true fungi responsible for 'maduromycetoma'. capsulatum in better detail. They also stain leish- The distinction is a clinically important one in that mania and toxoplasma. the former may respond to chemotherapy but the Ziehl-Neelsen stain may show species of Nocardia latter have to be treated surgically. to be weakly acid-fast. '830 P. P. Anthony

BIREFRINGENCE with Phycomycetes, occasionally Candida albicans. This helps to detect Histoplasma capsulatum and It is well to remember that thrombotic angiitis is duboisii but is not specific: Cryptococcus neoformans also seen in Pseudomonas aeruginosa pneumonia. and Blastomyces dermatitidis may also be bi- refringent. NON-SPECIFIC CHRONIC INFLAMMATION Rhinosporidium seeberi (nose), Cryptococcus neo- PIGMENT formans (lung), Pneumocystis carinfi ('plasma The yeast-like bodies of 'chromomycosis' species cell pneumonia'). are golden-brown in sections stained with haematoxylin-eosin; this colour and an occasional septate SUPPURATION form are quite characteristic. The grains of 'my- Actinomyces, Nocardia, 'Mycetoma' species cetoma' species are coloured whitish-yellow to black, particularly, but many others occasionally. The occasionally even red, when looked at in the block. focus of suppuration may be surrounded by a This helps in species identification: 'actinomycetoma' histiocytic granulomatous border in infections species (yellow-white grains) Nocardia asteroides, with Sporothrix schenckii, Blastomyces dermatitidis, Nocardia brasiliensis, Streptomyces madurae, Strep- and Coccidioides immitis. Cat-scratch fever and tomyces somaliensis (red grains), Streptomyces lymphogranuloma venereum may have to be con- pelletieri; 'maduromycetoma' species (yellow-white sidered in the differential diagnosis. grains) Allescheria boydii (brown-black grains), Madurella mycetomi, Leptosphaeria senegalensis. GIANT CELLED TUBERCULOID GRANULO- The pigment may also be seen in the regional MATOUS REACTION lymph nodes: this is not evidence of spread. Histoplasma duboisii, 'Chromomycosis' species, Cryptococcus neoformans, Blastomyces dermatitidis, CAPSULE and Coccidioides immitis. Caseation may occur, Only Cryptococcus neoformans is truly encapsulated sometimes massive, with Histoplasma capsulatum, which is shown well by staining with mucicarmine or Cryptococcus neoformans, and Coccidioides immitis. in India ink preparations. Histoplasma capsulatum, in spite of its name, has no capsule and the halo-like EOSINOPHILIC PROTEINACEOUS PRECIPITATE appearance is an artefact. OUTLINING FUNGAL BODY Subcutaneous phycomycosis (Basidiobolus meri- BUDDING stosporus), submucousphycomycosis(Entomophthora The following yeasts may be seen budding in the coronata), Sporothrix schenckii ('asteroid boyd'). tissues: Candida albicans (filaments also present); Blastomyces dermatitidis (solitary and broadly PSEUDOEPITHELIOMATOUS HYPERPLASIA OF based); Paracoccidioides brasiliensis (multiple and THE SKIN peripheral, 'marine pilot's wheel' appearance); 'Chromomycosis' species, Blastomyces dermatitidis, Cryptococcus neoformans (rarely); Histoplasma Coccidioides immitis, Sporothrix schenckii, severe capsulatum and duboisii (rarely). 'granulomatous' Candida albicans infection. Any Help from the Type of Tissue Reaction? Pitfalls Reactions to fungi vary widely not only between Many artefacts and other structures may be confused one species and another but in different cases of with fungi. The most important are the common the same disease or even at different stages in the haematoxyphil spherules in the central nervous course of the same infection. The following is a system (brain sand'), Russell bodies of plasma guide to the patterns most commonly seen: cells, conchoid and asteroid bodies of sarcoid granulomas, vegetable fibres and suture material, LITTLE OR NO REACTION particularly when cross cut, partly laked red Cryptococcus neoformans (particularly in the brain), cells, nuclear debris, and the like. Tissues may be Candida albicans (on mucous surfaces), Aspergillus contaminated with starch powder from surgical umigatus (in the lung and brain). gloves used by the pathologist on the cutting-up bench. These starch granules are PAS positive and PREDILECTION FOR BLOOD VESSELS WITH birefrigent. Finally, the possibility of contamination THROMBOSIS AND INFARCTION OF TISSUES of paraffin wax, mounting media, and slides with Aspergillusfumigatus (in the lung), systemic infection real fungi should always be borne in mind. A guide to the histological identification offungi in tissiwes 831

Bibliography PULMONARY INFECTIONS Heard, B. E. (1966). Pulmonary infections compl.cating certain diseases GENERAL ACCOUNTS and treatments ('opportunistic infections'). In Recent Advances Baker, R. D. et al, Eds. (1971). The Pathologic Anatomy of Mycoses: in Pathology, 8th ed., edited by C. V. Harrison, pp. 369-385. Human Infection with Fungi, Actinomycetes and Algae. Churchill, London. (Handbuch der spezielien pathologischen Anatomie und Histo- logie, Bd. 3, Th. 5). Springer, New York, Heidelberg, Berlin. TISSUE REACTIONS TO FUNGI (ILLUSTRATEC) Emmons, C. W., Binford, C. H., and Utz, J. P. (1970). Medical Symmers, W. St. C. (1960). Histological examination in the diagnosis Mycology. Lea and Febiger, Philadelphia. Kimpton, London. of deep-seated fungal infections. In Recent Advances in Clinical Wolstenholme, G. E. W. and Porter, R., Eds. (1968). Systemic Pathology, series III, edited by S. C. Dyke, pp. 304-333. Mycoses: A Ciba Foundation Symposium. Churchill, London. Churchill, London.

FUNGAL INFECTIONS IN THE TROPICS TECHNICAL METHODS Davey, W. W. (1968). Companion to Surgery in Africa, pp. 55-73. Coombs, D. M. (1967). A comparison of histological staining tech- Livingstone, Edinburgh. niques for fungi. In Progress in Medical Laboratory Technique, Edington, G. M., and Gilles, H. M. (1968). Pathology in the Tropics, Vol. 4, edited by F. J. Baker, pp. 170-181. Butterworths, pp. 225-255. Arnold, London. London. Hutt, M. S. R., and Bezjak, V. F. (1972). Deep and systemic fungal Drury, R. A. B., and Wallington, E. A., Eds. (1967). Carleton's infections. In Medicine in a Tropical Environment, edited by Histological Technique, 4th ed., pp. 339-343. Oxford University A. G. Shaper, J. W. Kibukamusoke and M. S. R. Hutt, pp. Press, London, New York, Toronto. 88-103. British Medical Association, London. Rolph, L., and Austwick, P. K. C. (1973). Differential stainingoffungi in tissues. J. Sci. Technol., 17, 22-26.