Correlation of Disease Severity with Fecal Toxin Levels in Patients with Clostridium difficile -Associated Diarrhea and Distribution of PCR Ribotypes and Toxin Yields In Vitro of Corresponding Isolates

Abstract

We investigated in vivo and in vitro yields of toxins A and B from and PCR ribotypes of Clostridium difficile isolates from 164 patients with differing severities of C. difficile-associated diarrhea (CDAD) (patients were grouped as follows: <3 loose stools per day, n ‫؍‬ 45; 3 to 10 per day, n ‫؍‬ 97; >10 per day, n ‫؍‬ 22). The median fecal toxin levels in each group were 0.5, 6.8, and 149 U/g feces (P < 0.001), respectively. Patients with severe diarrhea also had more-frequent occurrence of blood in stool and vomiting, but there was no association with fecal toxin levels per se. There was no correlation between fecal toxin level and toxin yield in vitro for the corresponding C. difficile isolate or between its PCR ribotype and disease severity. A broad range of toxin yields among isolates belonging to major PCR ribotypes indicated a presence of many subtypes. We hypothesize that bacterial and host factors that affect C. difficile toxin levels in feces are important determinants of symptoms in CDAD patients. An inverse correlation between toxin yield and spore count (r ‫؍‬ 0.66) in stationary-phase cultures supported the notion that toxin production and sporulation represent opposite alternative survival strategies for C. difficile cells facing nutrient shortage.Most strains of Clostridium difficile produce two toxins, A and B, that cause C. difficile-associated diarrhea (CDAD) with symptoms ranging from mild diarrhea to pseudomembranous colitis. CDAD is associated mainly with the use of antibiotics that reduce the protective microflora, which allows for overgrowth and toxin production by C. difficile (21), and chemostat and animal studies have verified that certain antibiotics induce C. difficile growth, toxin production, and toxin release (11,29,31). Furthermore, the age of the patient, underlying diseases, and levels of toxin-neutralizing antibodies are factors that affect attack rate, severity of disease, and the risk of relapse of CDAD (17,18,21,38). In addition, factors that differ between toxin-producing C. difficile strains, e.g., the amount of toxin produced, the serogroup, and the surface layer protein composition, may affect symptoms (12,14,20,25,32,33). For example, strains of C. difficile belonging to specific serogroups are highly virulent in animal models (3,8), but whether C. difficile strain types differ in terms of virulence in humans is less clear.The epidemiology and disease pattern of C. difficile strains have been studied by several methods, e.g., serotyping, toxinotyping, and PCR ribotyping (4). A cohort study showed no difference in the distributions of C. difficile immunoblot types between asymptomatic carriers and CDAD patients, suggesting that patient factors or bacterial numbers contribute to disease more than the properties of specific C. difficile strain types (24). In addition, for a total of 62 C. difficile strains isolated from 17 patients with CDAD and 11 carriers and typed by PCR ribotyping and by use of randomly amplified polymorphic DNA, no significant correlation between ge...

Performance of Clostridium difficile Toxin Enzyme Immunoassay and Nucleic Acid Amplification Tests Stratified by Patient Disease Severity

“…As toxin expression is thought to be one factor related to CDI severity (10), we sought to determine if false-negative toxin EIA results occurred in all patients with CDI or only in those patients with mild disease and presumably a low fecal concentration of C. difficile toxins (11). In contrast to this supposition, our data demonstrate that toxin EIA was negative in 42% of patients with severe CDI.…”

Cefoperazone-treated mice as an experimental platform to assess differential virulence ofClostridium difficilestrains

“…Our results are also in accord with clinical observations that the severity of CDI is proportional to the in vivo production of these C. difficile toxins. 36 The most severe disease encountered in cefoperazone treated mice was seen in animals that were infected with VPI 10463 and BI1. Much greater cytotoxic activity was found in the tissues of animals infected with these strains compared with animals infected with strain 630 during early infection.…”

Clostridium difficile Infection: Pathogenesis, Diagnosis and Treatment

“…Akerlund and colleagues observed an inverse relationship between toxin production and spore counts, which suggests that premature sporulation in the stationary phase shortens the time required for toxin production [18]. Merrigan et al observed that some hypervirulent strains underwent early sporulation and produced large amounts of toxin, with greater efficiency than other strains.…”