Identification of Clostridium difficile in stool specimens by culture-enhanced gas-liquid chromatography

Abstract

We have developed a sensitive and specific method for the identification of Clostridium difficile in stool specimens based on the detection of metabolic breakdown products of the organism by gas-liquid chromatography after incubation of stool samples in a selective broth medium containing cefoxitin. Use of this approach to test samples from two different populations of patients at separate medical centers showed this method to be superior to plate cultures or cytotoxin testing alone for both populations. The combined results from the two patient populations showed that 225 of 226 confirmed isolates were identified correctly, resulting in a sensitivity of 99.6% and a specificity of 99.0%. This method eliminates the delay caused by subculturing for tests requiring a pure isolate. The culture phase amplifies even low numbers of C. difficile in fecal samples (due to low in vivo concentrations or delayed transport) and thus increases sensitivity. Other advantages include the ability to detect C. difficile in the mixed flora of the stool and the ability of most clinical laboratories to use this procedure. Given the complexities of the detection of C. difficile toxins and the increasing importance of this organism as a nosocomical agent, culture-based methods remain the preferred approach to screening and routine workup for cases of diarrhea.

Arbitrarily primed-polymerase chain reaction (AP-PCR) typing of Clostridium difficile isolated from different sources of Imam Reza hospital, Tabriz, Iran

“…Gas-liquid chromatography is a more rapid and simpler technique and widely is used in bacteriology laboratories to identify anaerobes and to detect their metabolic products directly in clinical specimens. Detection of C. difficile by GLC has been reported by microbiology laboratories at different times (Johnson et al, 1989;Pepersack et al, 1983). A high sensitivity (99.6%) and specificity (99%) of GLC in detecting isocaproic and isovaleric acids produced by C. difficile in a mixed culture medium has been reported (Johnson et al, 1989) which had a high consistency with the results obtained for identifying of C. difficile in our study.…”

“…Detection of isocaproic and isovaleric acids has been suggested as a screening test for C. difficile in stools (Johnson et al, 1989;Pepersack et al, 1983). Thus, gas chromatographic results for 84 C. difficile isolates, already identified by conventional standard tests, were evaluated with respect to isocaproic and isovaleric acids production.…”

“…Gas chromatography as complementary test was also used to further identification of C. difficile by determination of the volatile short-chain fatty acids (isocaproic acid and isovaleric acid) produced by the growth of C. difficile (Johnson et al, 1989;Pepersack et al, 1983). A 48 h growth in thioglycolate broth medium were used to extract volatile fatty acids by adding 0.2 ml of H 2 S0 4 (50%) and 1 ml of ethyl ether to 1 ml of the well-mixed culture broth medium.…”

“…Helium was used as the carrier gas and column linear velocity was set at 20.0 cm/s and the oven temperature at 150°C. The oven temperature program was isothermal for 20 min (Johnson et al, 1989;Pepersack et al, 1983) retention times were identified by injecting known standards (Sigma chemicals).…”

Comparison of a dot immunobinding assay, latex agglutination, and cytotoxin assay for laboratory diagnosis of Clostridium difficile-associated diarrhea

“…Since many clinical microbiology laboratories do not perform tissue cell culture, alternative diagnostic tests have been evaluated. Counterimmunoelectrophoresis, enzyme-linked immunosorbent assay, gas-liquid chromatography, and latex agglutination have been used in a research setting (1,5,6,8,9,12). In 1986, Marion Laboratories, Inc. (Kansas City, Mo.)…”