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A routine protocol for diagnosing Clostridium difficile-associated diarrhoea (CDAD) based on both faecal-cytotoxin detection and toxigenic culture was adopted by the microbiology laboratory of the St Luc-UCL University Hospital in Brussels in 1997. A toxigenic culture is a faecal culture followed, in the case of positivity, by a direct immunoassay on colonies to detect toxin A production. The results obtained over the past 7 years in the hospital are reviewed here. A total of 10 552 diarrhoeal stools from 7042 patients were analysed, of which 9494 were negative for all tests. A total of 1058 samples (10 %) from 794 patients were culture-positive, of which 460 (4 . 4 %) were positive for a faecal cytotoxin. The remaining 598 cultures were tested for toxin A on colonies; 355 of them were positive, which is 3 . 4 % of the total, and the remaining 243 (2 . 3 %) were negative. The positivity of the faecalcytotoxin assay was statistically linked to the number of colonies observed on the culture plate. In conclusion, over a 7 year period, toxigenic culture allowed the diagnosis of 355 cases of CDAD that would have been missed by a protocol using a faecal-cytotoxin assay alone. In terms of both patient care, prevention of environmental contamination and prevention of risk of a hospital outbreak, it is proposed that these results justify the recommendation to perform both faecal-toxin assay and culture in routine medical practice.

Susceptibilities to 11 antimicrobial agents were determined by Etest for 93 Nocardia isolates from clinical specimens and 15 type strains belonging to different Nocardia spp. All isolates were susceptible to trimethoprim-sulphamethoxazole, amikacin and linezolid, but susceptibilities of the various Nocardia spp. to beta-lactams, aminoglycosides, ciprofloxacin and clarithromycin varied markedly. Overall, there was a good correlation between the drug resistance patterns and the species identification established by conventional phenotypic tests and 16S rDNA sequencing. Among the different species encountered, Nocardia farcinica and Nocardia brasiliensis displayed the most multiresistant profiles, with resistance to imipenem occurring mainly among isolates of N. brasiliensis and Nocardia abscessus. The species variability in susceptibility profiles and the numerous recent taxonomic changes means that in-vitro susceptibility tests may be a complementary tool for the identification of Nocardia isolates from human clinical specimens. Further studies on a larger number of species from more diverse geographical sources, including species that are found less commonly among clinical isolates, are required to validate and extend the results.

The identification of Nocardia species, usually based on biochemical tests together with phenotypic in vitro susceptibility and resistance patterns, is a difficult and lengthy process owing to the slow growth and limited reactivity of these bacteria. In this study, a panel of 153 clinical and reference strains of Nocardia spp., altogether representing 19 different species, were characterized by matrix-assisted laser desorption ionizationtime of flight mass spectrometry (MALDI-TOF MS). As reference methods for species identification, fulllength 16S rRNA gene sequencing and phenotypical biochemical and enzymatic tests were used. In a first step, a complementary homemade reference database was established by the analysis of 110 Nocardia isolates (pretreated with 30 min of boiling and extraction) in the MALDI BioTyper software according to the manufacturer's recommendations for microflex measurement (Bruker Daltonik GmbH, Leipzig, Germany), generating a dendrogram with species-specific cluster patterns. In a second step, the MALDI BioTyper database and the generated database were challenged with 43 blind-coded clinical isolates of Nocardia spp. Following addition of the homemade database in the BioTyper software, MALDI-TOF MS provided reliable identification to the species level for five species of which more than a single isolate was analyzed. Correct identification was achieved for 38 of the 43 isolates (88%), including 34 strains identified to the species level and 4 strains identified to the genus level according to the manufacturer's log score specifications. These data suggest that MALDI-TOF MS has potential for use as a rapid (<1 h) and reliable method for the identification of Nocardia species without any substantial costs for consumables.

Eighty-six. A gallery of nine conventional and enzymatic tests was developed for the rapid identification of the most common species isolated during this survey. Pyrrolidonyl aminopeptidase, ␥-glutamyl aminopeptidase, ␣-mannosidase, and ␣-glucosidase were found to be highly discriminating and could be used to develop an identification scheme.Nocardia species are isolated with an increased frequency from clinical specimens, especially in specimens from immunocompromised patients (19). The taxonomy of the genus has dramatically been revised during the last decade, and at least 30 valid species have been reported, besides a number of unnamed genomospecies (18). Not all of them have been found in humans, and Nocardia asteroides, previously most frequently isolated from clinical specimens, has proved to be heterogeneous and has been divided into several species (18). More recently, additional species of human origin have been described (6, 8, 9, 11, 12, 17, 28).The routine identification of Nocardia strains at the species level is difficult in the laboratory. This, and the nomenclature changes, may explain that species distribution in clinical isolates has been poorly documented up to now, and even recent surveys still report the "N. asteroides complex" as the most frequent Nocardia species isolated in humans (7,10,16,19). Identification studies have not been systematically carried out since the several recent taxonomic changes.The aim of this study was to assess the species distribution of a large number of Nocardia isolates and to propose simple and rapid identification tests that may be helpful to identify the species most commonly encountered in clinical material. MATERIALS AND METHODSBacterial strains. Eighty-six Nocardia strains isolated from clinical specimens in Belgium were collected for the study. Most strains were isolated during the past decade, but a few were received before 1990. They were isolated by several laboratories in different parts of the country. All the Nocardia isolates were included in the study to avoid any bias in the selection of the strains. Only one strain per patient was considered. The clinical origins of the isolates were as follows: 36 strains were isolated from the respiratory tract, 18 from pyogenic lesions and wounds, 8 from blood, 4 from brain abscesses, and 1 from cerebral fluid. Nineteen were of unknown origin.The type strains of the most relevant species were included for phenotypic comparison as well as reference strains of some less common species. Nocardia species and strains are listed in Table 1.Sequencing of the 16S rRNA gene. The full-length 16S rRNA gene sequences (Ϯ1,400 nucleotides) of all strains were determined as described previously (24), and sequences were compared to those of the type strains deposited in the GenBank database.Cellular fatty acids were analyzed by gas-liquid chromatography as outlined elsewhere (22).Phenotypic characterization. Strains were cultured on tryptic soy blood agar plates at 35°C. They were examined for partial acid-fastness, presenc...