RESUMO
AIM: To examine the occurrence of Candida spp. in refractory periapical granulomas. METHODOLOGY: One hundred and three surgically removed periapical granulomas were subjected to molecular analysis for the occurrence of Candida albicans. DNA was extracted from the samples using a modified phenol/chloroform/isoamyl alcohol method and was subjected to polymerase chain reaction (PCR) with OPA-03 and repetitive sequence (GACA)4 primers. The PCR products were separated in agarose gel electrophoresis, stained with ethidium bromide, visualized using UV light and the sequences were analysed. Samples indicating possible occurrence of Candida were further investigated by histological and immunohistological methods. Periodic acid-Shiff staining (PAS) was used to detect yeast cells and hyphae, and specific monoclonal antibodies to recognize high molecular mass mannoproteins present in the C. albicans cell wall. DNA extraction was controlled by running PCR using beta-actin primers (a housekeeping gene). C. albicans CCUG19915, C. tropicalis ATCC750, C. krusei ATCC6258, C. guilliermondii ATCC6260 and C. glabrata CCUG32725 served as positive controls in PCR. A tissue preparation of chronic atrophic candidosis in oral buccal mucosa served as a positive control for histological and immunohistological examinations. RESULTS: Polymerase chain reaction with beta-actin primers indicated successful DNA extraction in 68 out of 103 samples. The majority of the samples (50) were negative whereas 18 of the samples showed PCR products indicating possible occurrence of Candida spp. PAS-staining and immunohistological examination of these samples were, however, negative. Further analysis of the PCR products revealed sequences not typical for Candida spp. CONCLUSIONS: Candida spp. do not seem to occur in periapical granuloma.
