Differences in outcome according to Clostridium difficile testing method: a prospective multicentre diagnostic validation study of C difficile infection.

BACKGROUND: Diagnosis of Clostridium difficile infection is controversial because of many laboratory methods, compounded by two reference methods. Cytotoxigenic culture detects toxigenic C difficile and gives a positive result more frequently (eg, because of colonisation, which means that individuals can have the bacterium but no free toxin) than does the cytotoxin assay, which detects preformed toxin in faeces. We aimed to validate the reference methods according to clinical outcomes and to derive an optimum laboratory diagnostic algorithm for C difficile infection. METHODS: In this prospective, multicentre study, we did cytotoxigenic culture and cytotoxin assays on 12,420 faecal samples in four UK laboratories. We also performed tests that represent the three main targets for C difficile detection: bacterium (glutamate dehydrogenase), toxins, or toxin genes. We used routine blood test results, length of hospital stay, and 30-day mortality to clinically validate the reference methods. Data were categorised by reference method result: group 1, cytotoxin assay positive; group 2, cytotoxigenic culture positive and cytotoxin assay negative; and group 3, both reference methods negative. FINDINGS: Clinical and reference assay data were available for 6522 inpatient episodes. On univariate analysis, mortality was significantly higher in group 1 than in group 2 (72/435 [16·6%] vs 20/207 [9·7%], p=0·044) and in group 3 (503/5880 [8·6%], p<0·001), but not in group 2 compared with group 3 (p=0·4). A multivariate analysis accounting for potential confounders confirmed the mortality differences between groups 1 and 3 (OR 1·61, 95% CI 1·12-2·31). Multistage algorithms performed better than did standalone assays. INTERPRETATION: We noted no increase in mortality when toxigenic C difficile alone was present. Toxin (cytotoxin assay) positivity correlated with clinical outcome, and so this reference method best defines true cases of C difficile infection. A new diagnostic category of potential C difficile excretor (cytotoxigenic culture positive but cytotoxin assay negative) could be used to characterise patients with diarrhoea that is probably not due to C difficile infection, but who can cause cross-infection.

Selective testing of ß-galactosidase activity in the laboratory identification of Salmonella and Shigella species.

BACKGROUND: Salmonella and Shigella species are pathogens of great medical and public health importance. However, laboratory identification of these organisms is time consuming. Using current standard laboratory algorithms, the vast majority of organisms submitted for serological typing with Salmonella-specific and Shigella-specific antisera are not clinically significant. AIMS: To assess the addition of the O-nitrophenyl-ß-d-galactopyranoside (ONPG) test to the standard screening protocol for identification of Salmonella and Shigella species, and to describe a revised algorithm for the identification of these pathogens. METHODS: 71 non-lactose-fermenting isolates that were urease negative, indole negative, and produced acid and gas in triple sugar iron agar, with no H(2)S production in the agar, were tested for ß-galactosidase activity using the ONPG test. The test results were read at half-hourly intervals over a 4 h incubation period in O(2) at 37°C. RESULTS: 42 isolates (59.2%) were found to be Hafnia alvei, of which 36 strains (85.7%) were ONPG positive. All 18 of the Enterobacter species (25.4%) were ONPG positive. Overall, about 79% of the ONPG-positive isolates were positive at the end of the first half-hour of incubation. The two pathogenic isolates obtained during this study were both identified as Salmonella enterica serovar Paratyphi A, and they were ONPG negative. CONCLUSIONS: Incorporation of a 30 min ONPG test for non-lactose-fermenting organisms that are indole negative, urease negative, producers of acid and gas from glucose, oxidase negative and non-H(2)S gas producers eliminates the need for further serological testing of 79% of isolates, substantially improving the efficiency of the identification protocol.