A multiplex PCR toxigenic culture approach was designed for simultaneous identification and toxigenic type characterization of Clostridium difficile isolates. Three pairs of primers were designed for the amplification of (i) a species-specific internal fragment of the tpi (triose phosphate isomerase) gene, (ii) an internal fragment of the tcdB (toxin B) gene, and (iii) an internal fragment of the tcdA (toxin A) gene allowing distinction between toxin A-positive, toxin B-positive (A+B+) strains and toxin A-negative, toxin B-positive (A-B+) variant strains. The reliability of the multiplex PCR was established by using a panel of 72 C. difficile strains including A+B+, A-B-, and A-B+ toxigenic types and 11 other Clostridium species type strains. The multiplex PCR assay was then included in a toxigenic culture approach for the detection, identification, and toxigenic type characterization of C. difficile in 1,343 consecutive human and animal stool samples. Overall, 111 (15.4%) of 721 human samples were positive for C. difficile; 67 (60.4%) of these samples contained A+B+ toxigenic isolates, and none of them contained A-B+ variant strains. Fifty (8%) of 622 animal samples contained C. difficile strains, which were toxigenic in 27 (54%) cases, including 1 A-B+ variant isolate. Eighty of the 721 human stool samples (37 positive and 43 negative for C. difficile culture) were comparatively tested by Premier Toxins A&B (Meridian Bioscience) and Triage C. difficile Panel (Biosite) immunoassays, the results of which were found concordant with toxigenic culture for 82.5 and 92.5% of the samples, respectively. The multiplex PCR toxigenic culture scheme described here allows combined diagnosis and toxigenic type characterization for human and animal C. difficile intestinal infections.

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In this study, we have used genome-wide expression profiling to categorise synovial sarcomas, leiomyosarcomas and malignant fibrous histiocytomas (MFHs). Following hierarchical clustering analysis of the expression data, the best match between tumour clusters and conventional diagnosis was observed for synovial sarcomas. Eight of nine synovial sarcomas examined formed a cluster that was characterised by higher expression of a set of 48 genes. In contrast, sarcomas conventionally classified as leiomyosarcomas and MFHs did not match the clusters defined by hierarchical clustering analysis. One major cluster contained a mixture of both leiomyosarcomas and MFHs and was defined by the lower expression of a set of 202 genes. A cluster containing a subgroup of MFHs was also detected. These results may have implications for the classification of soft tissue sarcomas, and are consistent with the view that sarcomas conventionally defined as MFHs do not represent a separate diagnostic category.

Microarray analysis using sets of known human genes provides a powerful platform for identifying candidate oncogenes involved in DNA amplification events but suffers from the disadvantage that information can be gained only on genes that have been preselected for inclusion on the array. To address this issue, we have performed comparative genome hybridization (CGH) and expression analyses on microarrays of clones, randomly selected from a cDNA library, prepared from a cancer containing the DNA amplicon under investigation. Application of this approach to the BT474 breast carcinoma cell line, which contains amplicons at 20q13, 17q11-21, and 17q22-23, identified 50 amplified and expressed genes, including genes from these regions previously proposed as candidate oncogenes. When considered together with data from microarray expression profiles and Northern analyses, we were able to propose five genes as new candidate oncogenes where amplification in breast cancer cell lines was consistently associated with higher levels of RNA expression. These included the HB01 histone acetyl transferase gene at 17q22-23 and the TRAP100 gene, which encodes a thyroid hormone receptor-associated protein coactivator, at 17q11-21. The results demonstrate the utility of this microarray-based CGH approach in hunting for candidate oncogenes within DNA amplicons.