A Cost-Effective Approach for Detection of Toxigenic Clostridium difficile: Toxigenic Culture Using ChromID Clostridium difficile Agar

Abstract

We evaluated the performance and the cost of toxigenic culture using a commercial chromogenic medium (CDIF) for 538 stool specimens. Compared with real-time PCR, this method was found to detect an additional 9% of positive specimens and result in 61% reduction in material costs, with a trade-off increase in turnaround time of 1 day.A ccurate and reliable laboratory diagnosis of Clostridium difficile infection (CDI) remains a challenge to microbiologists 35 years after its discovery (1). While toxin enzyme immunoassays (EIA) have unacceptably low sensitivities (2), the cytotoxin neutralization assay (CTN) is too time-consuming and labor-intensive to perform. The performance of more recently developed PCR tests for toxin gene detection is promising, with mean sensitivity ranging from 90% to 100% and specificity ranging from 96% to 99% (3, 4). The hand-on time of some commercial assays can be minimal, since extraction and PCR are all carried out in a self-contained cartridge (4). However, PCR has not been widely utilized in clinical laboratories, presumably due to budgetary issues (5). Alternatively, C. difficile isolates can be recovered on selective media. When combined with a sensitive and specific toxin detection method, such as CTN or PCR, toxigenic culture is regarded as one of the gold standards of CDI diagnosis (6). The prototype selective culture medium cycloserine-cefoxitin-fructose-egg yolk agar (CCFA) requires 48 h of incubation and alcohol treatment (7). As a result, toxigenic culture is advocated as part of a diagnostic algorithm to increase the yield, after initial screening by glutamate dehydrogenase (GDH) EIA (8-11). With the availability of a commercial chromogenic selective medium, ChromID C. difficile agar (CDIF) (bioMérieux, France), that allows direct recovery of C. difficile within 24 h of incubation (12, 13), coupled with rapid identification by matrix-assisted laser desorption ionization time-of-fight (MALDI-TOF) mass spectrometry and inference of toxigenicity by PCR for the toxin gene (9), toxigenic culture as a routine diagnostic test is no longer impractical. The aim of the present study was to compare the performance and the cost of toxigenic culture against those of real-time PCR performed on stool specimens.Briefly, 538 soft or liquid stool samples were plated directly onto CDIF medium, which was then incubated in an anaerobic chamber for 48 h according to standard laboratory methods. Suspected flat and irregular colonies were confirmed to be C. difficile by MALDI-TOF mass spectrometry (Biotyper 3.0; Bruker Dal-

Clinical Significance of Toxigenic Clostridioides difficile Growth in Stool Cultures during the Era of Nonculture Methods for the Diagnosis of C. difficile Infection

“…In another study, toxigenic stool cultures recovered 24.4% of stool samples harboring toxigenic C. difficile isolates that were missed by a fecal cytotoxin EIA ( 17 ). Compared with real-time PCR, the culture-based method using a commercial chromogenic medium could detect an additional 9% of positive specimens ( 30 ). Although real-time PCR has been suggested as the confirmatory tool when the results of GDH tests and toxin EIAs are inconsistent, according to the IDSA/SHEA guidelines, the higher cost of commercial real-time PCR tests means that the tests are not often available in resource-limited settings.…”

High Prevalence and Frequent Acquisition of Clostridium difficile Ribotype 002 Among Nursing Home Residents in Hong Kong

“…Suspected flat and irregular colonies were confirmed to be C. difficile using mass-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (Biotyper 3.0, Bruker Daltonik, Germany). All C. difficile isolates were tested for the presence of toxin gene using real-time polymerase chain reaction (PCR, LightMix C. difficile kit; TIBMOLBIOL GmbH, Germany) 10 . The PCR ribotyping and slpA typing were performed for all first isolated C. difficile strains at the baseline sampling or sampling upon nursing home residence, as described previously 11 …”

“…All C. difficile isolates were tested for the presence of toxin gene using real-time polymerase chain reaction (PCR, LightMix C. difficile kit; TIBMOLBIOL GmbH, Germany). 10 The PCR ribotyping and slpA typing were performed for all first isolated C. difficile strains at the baseline sampling or sampling upon nursing home residence, as described previously. 11,12 Definitions A resident who had no history of hospitalization within the past 90 days was defined as having newly acquired C. difficile in the nursing home if a negative baseline stool culture was converted to positive on subsequent sampling.…”

Clostridioides difficile Infection: Diagnosis and Treatment Challenges

“…Culturing viable organisms from stool followed by the confirmation of toxin production is considered the “gold standard” for diagnosing CDI [ 25 ]. Cycloserine–cefoxitin–fructose–egg yolk agar (CCFA), or a modified version, is the standard media used for the isolation of C. difficile [ 9 , 25 , 26 ].…”

“…Culturing viable organisms from stool followed by the confirmation of toxin production is considered the “gold standard” for diagnosing CDI [ 25 ]. Cycloserine–cefoxitin–fructose–egg yolk agar (CCFA), or a modified version, is the standard media used for the isolation of C. difficile [ 9 , 25 , 26 ]. Fresh stool samples should be treated with alcohol or heat shock to facilitate the conversion of spores to their vegetative forms prior to inoculation on CCFA or a similar selective media [ 9 , 26 ].…”