RAPID DETECTION OF CANDIDA-ALBICANS IN CLINICAL-SAMPLES BY DNA AMPLIFICATION OF COMMON REGIONS FROM C-ALBICANS-SECRETED ASPARTIC PROTEINASEGENES

Laboratory diagnosis based on genomic amplification methods such as PC R may provide an alternative and more sensitive method than convention al culture for the early detection of deep-seated candidiasis, an incr easing cause of morbidity and mortality among immunocompromised patien ts. A novel method of DNA extraction from clinical samples based on tr eatment,vith proteinase K and isolation of DNA on a silica membrane wa s developed. The targets used for DNA amplification were the Candida a lbicans-secreted aspartic proteinase (SAP) genes, a multiple-gene fami ly of at least seven members in C. albicans. A single pair of primers was designed in order to detect six of these SAP genes and, subsequent ly, to increase the sensitivity of the test, Detection of the PCR prod uct by enzyme-linked immunosorbent assay was found to be as sensitive as Southern blotting with an SAP-labeled probe. The sensitivity of the assay was 1 cell/ml from serially diluted Candida cultures and 1 to 4 cells/ml from seeded blood specimens. The sensitivity and specificity of the present assay were tested in a retrospective study performed b lindly with 156 clinical samples and were 100 and 98%, respectively, c ompared with the results of culture. For the subset of blood culture s amples (n = 124), the sensitivity and the specificity were 100%. The t wo false-positive PCR samples came from patients treated with azole an tifungal agents, indicating that PCR was probably able to detect damag ed organisms that could not be recovered by culture.