A systematic evaluation of methods to optimize culture-based recovery of Clostridium difficile from stool specimens

With the increasing prevalence of Clostridium difficile infection among hospitalized patients, a clear understanding of C. difficile epidemiology is needed to evaluate current prevention policies, and to create new and effective policies. To determine the epidemiology of C. difficile, the most sensitive methods for detection of C. difficile are required. The purpose of this study was to systematically assess multiple methods to determine the most sensitive method to recover C. difficile from stool and rectal swabs. Stool samples from healthy asymptomatic individuals that were collected and confirmed to be culture negative for C. difficile were spiked with known concentrations of C. difficile ranging from 104 CFU of C. difficile per ml of stool to 10 CFU per ml. Two sets of experiments (A and B) were performed and each involved multiple combinations of untreated and treated stool/rectal swab specimens and selective and non-selective broth and agar. Overall, recovery of C. difficile was increased with the use of an initial broth enrichment followed by plating to solid medium. The most sensitive method of C. difficile detection for both stools and swab specimens was heat shock prior to inoculation of cycloserine-cefoxitin mannitol broth with taurocholate lysozyme cysteine (CCMB, Anaerobe Systems, Morgan Hill, CA) followed by isolation from pre-reduced TSA II with 5% sheep blood (BAP, BBL BD and Co., Sparks, MD). Identifying the most sensitive method of recovery will allow for further study of asymptomatic C. difficile carriers and their role in the epidemiology of C. difficile.

► A lack of consensus exists on the most sensitive method for Clostridium difficile culture.
► We systematically evaluated numerous culture conditions.
► C. difficile recovery is enhanced with broth enrichment.
► Heat shock prior to broth enrichment is a cost effective method to optimize recovery.