There is little doubt that both the incidence and severity of CDAD are increasing (9
). The reasons for this are not entirely clear but probably reflect both patient-related issues, such as changing demographics and underlying disease (41
), and pathogen-related factors, such as the spread of the BI/027 strains of C. difficile
). Warny and coworkers reported the in vitro production of TcdA and TcdB to be 16- and 23-fold greater, respectively, than that of reference strains of toxinotype O (55
). Titers of the BI/027 C. difficile
culture supernatants analyzed here (Fig. ) were ~15-fold greater than the average of the other strains examined, which is similar to the values reported by Warny. In addition to the BI/027 strains associated with recent outbreaks, our panel included two strains (8864 and Dipersio) which are TcdA−
and one strain (CD196) that produces high amounts of binary toxin.
Tolevamer neutralized toxins in the titered C. difficile culture supernatants with efficacies similar for all strains, though the absolute amount of tolevamer required appeared to be slightly higher for the BI/027 strains (Fig. ). However, the limit of discrimination in dose-down experiments depends upon the dilution factor—in this case, 2. Therefore, in this study, the absolute values can vary by a factor of 2, suggesting that the amount of tolevamer required for neutralization was functionally equivalent for all the strains examined, including the BI/027 strains.
Assessing the clinical relevance of the observed in vitro increase in toxin production among BI/027 strains is difficult. C. difficile
toxin production in vitro varies widely, depending upon the composition of the medium (13
), the strain, and the presence of some antibiotics with sub-MIC levels (including metronidazole) (12
). Few studies have examined toxin levels in vivo, in part because assessing toxin titers in fecal samples is not trivial; however, some studies have attempted to correlate toxin production in vitro and in vivo with disease severity (1
). While there appears to be a trend toward a correlation between high toxin production in vivo and disease severity, the largest study to date comparing in vitro and in vivo toxin production demonstrated no correlation between the amount of toxin recovered in stool samples and the capacity of the recovered clinical isolates to produce toxin in vitro (1
). This lack of correlation is not limited to clinical observations, since studies with hamster models have also failed to yield a correlation between virulence and the amount of toxin produced in in vitro cultures (6
The reasons for this lack of concordance are not clear. C. difficile
isolates recovered from stool samples by microbial culture may not represent the prevalent toxin-producing phenotype strains in vivo (53
). Alternatively, the lack of concordance could reflect the complexity and variability of the factors affecting bacterial growth and toxin production in the colon (6
, 58, 60). However, until definitive clinical studies are performed comparing disease severity with levels of fecal toxin production by BI/027 strains of C. difficile
, assessing the clinical consequence of altered in vitro TcdA and TcdB production by these strains remains uncertain.
In addition to carrying TcdA and TcdB, the BI/027 strains have also been shown to carry genes for the binary toxins CdtA and CdtB (33
). Together, these introduce an actin-specific ADP-ribosyltransferase (related to the iota toxins produced by C. perfringens
) into the cytosol (7
). Unlike the large clostridial toxins TcdA and TcdB, the role of binary toxins in disease pathology is not clear. In vitro, cytotoxicity due to binary toxins cannot be detected unless supernatants are concentrated ~40-fold by ammonium sulfate precipitation (45
). Perhaps because of this requirement, the great majority of studies identify the presence of the binary toxin genes but do not assay either cytotoxicity or enzymatic activity (19
). The few studies that have done so identified the strain CD196 as a high toxin producer (compared with the production of binary toxins by other strains) (49
). Notably, the C. difficile
culture supernatant cytopathic activity of this strain was neutralized by tolevamer in the present study (Fig. ); however, this was most likely due to TcdA and TcdB rather than binary toxin.
In vivo, purified binary toxin produces vigorous secretion in rabbit ileal loops (18
). However, while TcdA−
Cdt+ C. difficile
strains colonize hamsters following clindamycin treatment, these strains do not cause disease or altered pathology (18
). Some clinical studies have suggested that C. difficile
isolates bearing CdtA and CdtB genes appear to be associated with increased disease severity (3
), but other studies have noted that at least some BI/027 isolates containing these genes were not associated with more severe clinical disease (26
). Taking these results together, it has been hypothesized that while binary toxins may exacerbate disease caused by TcdA and TcdB, it is not clear that binary toxins are capable of inducing disease on their own (26
). Studies examining whether tolevamer neutralizes binary toxins have been initiated.
Two large, controlled international phase 3 clinical trials evaluating the efficacy of tolevamer compared to that of vancomycin and metronidazole in the treatment of CDAD were completed in 2007. The primary efficacy variable was clinical success, defined as the presence of explicit data indicating diarrhea resolution and the absence of severe abdominal discomfort due to CDAD on day 10. To date, the results of the first study have been presented, and tolevamer was shown to be inferior to both vancomycin and metronidazole for the resolution of CDAD, though it was associated with a lower recurrence rate than antibiotic therapy (29
). The reasons for the disconnect between tolevamer's efficacy in both in vitro and in vivo models and the disappointing results seen in the phase 3 clinical program are unknown, but possible explanations include inadequate polymer concentration or toxin-binding affinity at relevant anatomic sites, either through interfering substances in the gut lumen occupying binding sites, impaired access to the mucosal surface, or insufficient clinical dosing. In the phase 2 study (30
), the 6 g/day dose of tolevamer used was calculated (8
) to be sufficient to neutralize the ~1 μg/ml of toxin estimated by McFarland et al. (35
). Due to technical constraints, and the limited correlation between toxin concentration and manifestations of clinical disease, neither toxin nor tolevamer fecal concentrations were measured in the phase 3 studies. Therefore, whether tolevamer was present at a high enough concentration in the phase 3 studies cannot be ascertained.