Clostridium difficile

Abstract

Clostridium difficile has re-emerged as a major hospital-acquired infection since 2001. Despite development of polymerase chain reaction-based testing, no single clinical diagnostic test has emerged with sufficient sensitivity, specificity, and turnaround time to be entirely reliable for disease diagnosis. The importance of C difficile acquired outside the hospital environment remains an unknown factor and awaits further epidemiologic investigation. This article discusses the changing epidemiology, clinical presentation, and pathogenesis of C difficile infection and highlights the ongoing challenges of laboratory diagnosis, treatment, and disease relapse.

Fig. 1

(A) Gram stain of C. difficile from 24-hour growth on trypticase soy agar with 5% sheep blood. The vegetative cell bodies are often gram negative during early growth; note the abundant subterminal endospores that do not swell the parent cell. ( B ) Malachite green stain of 48-hour growth of C. difficile. The s afranin counterstain renders vegetative cells pink, whereas the endospores stain green, revealing their ovoid shape. (C) A 48-hour growth of C. difficile on typical selective agar medium, C. difficile basal agar with moxalactam and norfloxacin, CDMN, that uses norfloxacin and moxalactam as selective antibiotics to allow primary isolation from stool specimens. Other media use combinations of cycloserine and cefoxitin as selective antibiotics (cycloserine cefoxitin fructose agar). Neutral red is turned yellow by C. difficile growth. (D) Typical appearance of C. difficile colonies on trypticase soy agar with 5% sheep blood at 48 hours. Colonies are nonhemolytic. (E) A selective broth medium for isolation of C. difficile, cycloserine cefoxitin mannitol broth with taurocholate and lysozyme. The tube at right shows turbidity and growth of C. difficile at 24 hours, with obvious alkalinization of the neutral red indicator. The tube at left is a negative control.

Fig. 2

(A) Negative cell culture cytotoxicity assay. Human fibroblasts remain spindle-shaped and in contact with each other. (B) Positive cell culture cytotoxicity assay revealing cytopathic effects of C. difficile toxin B causing cell rounding and separation. (Courtesy of Ray Hariri, PhD.)