Rev. Inst. Med. Trop. Sao Paulo 52(3):145-149, May-June, 2010 doi: 10.1590/S0036-46652010000300006 (1) Pathologic Anatomy Unit, Medical School, Rovira i Virgili University, Reus, Spain. (2) Mycology Laboratory, Santa Casa-Complexo Hospitalar, Porto Alegre, RS, Brazil. (3) Department of Pathology, Santa Casa-Complexo Hospitalar, Porto Alegre, RS, Brazil. (4) Joan XXIII University Hospital, Rovira i Virgili University, Tarragona, Spain. (5) Microbiology Unit, Medical School, Rovira i Virgili University, Reus, Spain. (6) Researcher 1B from CNPq, Brazil (7) Department of Internal Medicine, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil. Correspondence to: Prof. Dr. Luiz Carlos Severo. Laboratório de Micologia/Hospital Santa Rita, Santa Casa Complexo Hospitalar. Rua Prof° Annes Dias 285, 90020-090 Porto Alegre, RS, Brasil. Phone: +55.51.32148410. E-mail: severo@santacasa.tche.br; severo@pesquisador.cnpq.br UNUSUAL MORPHOLOGIES OF Cryptococcus spp. IN TISSUE SPECIMENS: REPORT OF 10 CASES Alexandra Flávia GAZZONI(1), Flávio de Mattos OLIVEIRA(2), Emily Ferreira SALLES(3), Emilio MAYAYO(1,4), Josep GUARRO(5), Javier CAPILLA(5) & Luiz Carlos SEVERO(2,6,7) SUMMARY Ten cases of cryptococcosis due to unusual microscopic forms of Cryptococcus sp. observed over a twenty-eight year period (1981-2009) are presented. The most important clinicopathological and laboratory data are tabulated. The uncommon forms of cryptococcal cells given are: structures resembling germ tube (one case), chains of budding yeasts (one case), pseudohyphae (two cases) and nonencapsulated yeast-like organisms (eight cases). The diagnosis was based on the histopathological findings. The causative organism was isolated and identified in seven cases; five were due to C. neoformans, and two to C. gattii. In addition, the importance of using staining histochemical techniques - Grocott’s silver stain (GMS), Mayer’s mucicarmine stain (MM) and Fontana-Masson stain (FM) - in the diagnosis of cryptococcosis is argued. KEYWORDS: Cryptococcosis; Pseudohyphae; Chains of budding yeasts; Germ tube-like structures; Nonencapsulated yeast-like organisms. INTRODUCTION In clinical specimens, Cryptococcus species are usually identified as spherical-to-oval yeast cells, range from 4-20 μm in diameter, and are surrounded by a mucopolysaccharide capsule, which is a major virulence factor 5,14 . Single or multiple budding cells with a narrow base are usually observed 14 . In addition to these classical aspects, Cryptococcus may also be present in unusual forms, which include pseudohyphae 1,7 , chains of budding yeasts 7,27 , structures resembling germ tubes 7 and poorly encapsulated cells 8 . Histopathological identification of the cryptococcosis is based on the micromorphological and staining features of the cryptococcal cells, and include histochemical techniques of hematoxylin and eosin (HE), and Grocott’s silver stain (GMS), as well as special histochemical techniques such as Mayer’s mucicarmine method (MM), which stains the capsule magenta, and Fontana-Masson procedure (FM), which stains fungal melanin reddish-brown 7,8 . In this study, we highlight the unusual micromorphological forms of the Cryptococcus species in tissue specimens. It also emphasizes the use of histochemical techniques in the diagnosis of cryptococcosis. MATERIAL AND METHODS Through database analysis, we retrospectively reviewed all cases of cryptococcal infections diagnosed between January 1981 and May 2009 at the Mycology Laboratory of Santa Casa Complexo Hospitalar (Porto Alegre, RS), in Southern Brazil. Clinical-epidemiological and laboratory records of the cases diagnosed by histopathological examination were reviewed, and we were primarily interested in collecting data such as: sex, age, underlying diseases, titers of cryptococcal antigens (CrAg) in sera, urine and cerebrospinal fluid (CSF) and species of Cryptococcus recovered in cultures. Cultures: The identification was confirmed by: (a) colony morphology - by isolation of yeast colonies with white mucoid aspect (depending on the capsule thickness) after cultivation on fungal media (within 48-72 h), namely Sabouraud’s (SAB) at 25 °C, and brain-heart infusion (BHI) agar at 35 °C; (b) microscopy morphology - by demonstration of spherical-to- oval encapsulated yeast cells and budding on a narrow base. After identity of an isolate had been established, such as Cryptococcus, we proceeded to determine its species status. Canavanine-glycine-broothymol blue (CGB) agar was successfully used for this purpose. In one to five days, isolates of C. gattii cause the CGB medium to turn blue, whereas those of C. neoformans do not. CrAg detection: For serological diagnosis, latex agglutination for cryptococcal polysaccharide antigens was perfomed a specific and sensitive alternative for rapid diagnosis. In this study, the commercial kit IMMY test was used, which has a vital component in Detacher Enzyme (DE), Pronase®. DE eliminates the rheumatoid factor, which can produce false positives.