Fatal pseudomembranous colitis associated with a variant clostridium difficile strain not detected by toxin A immunoassay.

BACKGROUND: Many clinical laboratories use toxin A immunoassays to test for Clostridium difficile. OBJECTIVE: To describe the clinical course of a patient infected with a toxin variant strain of C. difficile that was not detected by toxin A immunoassay; to genetically characterize this strain; and to estimate the number of laboratories that use only toxin A immunoassays. DESIGN: Case report, molecular investigation, and laboratory survey. SETTING: Tertiary care hospital in Chicago, Illinois. PATIENT: An 86-year-old man. MEASUREMENTS: Restriction endonuclease analysis, polymerase chain reaction, and survey of regional clinical laboratories. RESULTS: An elderly hospitalized man died of advanced pseudomembranous colitis. Four stool specimens submitted over a 2-month period had tested negative on toxin A immunoassay, but a strain of C. difficile with a 1.8-kb deletion of the toxin A gene was recovered from each specimen. This strain, identified as restriction endonuclease analysis type CF4, is closely related to a widely disseminated variant, toxinotype VIII. Toxin A immunoassay was the only test being performed for detection of C. difficile at 31 of 67 (46%) regional clinical laboratories. CONCLUSIONS: Toxin A variant strains of C. difficile cause serious disease and are undetectable in clinical laboratories that use only toxin A immunoassays for C. difficile testing.

Infection of hamsters with epidemiologically important strains of Clostridium difficile.

Five different toxigenic strains of Clostridium difficile of known human epidemiologic importance were tested for virulence in hamsters. Three strains-types B1, J9, and K14-have caused hospital outbreaks. Type Y2 is associated with a high rate of asymptomatic colonization in patients. The fifth strain, type CF2, is a toxin A-negative, toxin B-positive strain implicated in multiple human cases of C. difficile-associated diarrhea. Groups of 10 hamsters per strain were given 1 dose of clindamycin, followed 5 days later with gastric inoculation of 100 cfu of C. difficile. Hamsters given types B1, J9, K14, or Y2 showed 90%-100% colonization (albeit at a slower rate with type Y2) and 100% mortality of colonized animals. Hamsters challenged with type CF2 showed 60% (P= .01) colonization and 30% mortality (P= .0003). The hamster model demonstrated pathogenicity differences between a toxin variant strain and standard toxigenic strains but no significant differences among the standard strains.

Toxin gene analysis of a variant strain of Clostridium difficile that causes human clinical disease.

A toxin variant strain of Clostridium difficile was isolated from two patients with C. difficile-associated disease (CDAD), one of whom died from extensive pseudomembranous colitis. This strain, identified by restriction endonuclease analysis (REA) as type CF2, was not detected by an immunoassay for C. difficile toxin A. Culture supernatants of CF2 failed to elicit significant enterotoxic activity in the rabbit ileal loop assay but did produce atypical cytopathic effects in cell culture assay. Southern hybridization, PCR amplification, and DNA sequence analyses were performed on the toxin A (tcdA) and toxin B (tcdB) genes of type CF2 isolate 5340. Type CF2 5340 tcdA exhibited a 1,821-bp truncation, due to three deletions in the 3' end of the gene, and a point mutation in the 5' end of the gene, resulting in a premature stop codon at tcdA position 139. Type CF2 5340 tcdB exhibited multiple nucleotide base substitutions in the 5' end of the gene compared to tcdB of the standard toxigenic strain VPI 10463. Type CF2 5340 toxin gene nucleotide sequences and deduced amino acid sequences showed a strong resemblance to those of the previously described variant C. difficile strain 1470, a strain reported to have reduced pathogenicity and no association with clinical illness in humans. REA of strain 1470 identified this strain as a distinct type (CF1) within the same REA group as the closely related type CF2. A review of our clinical-isolate collection identified five additional patients infected with type CF2, three of whom had documented CDAD. PCR amplification of the 3' end of tcdA demonstrated identical 1. 8-kb deletions in all seven type CF2 isolates. REA type CF2 is a toxin variant strain of C. difficile that retains the ability to cause disease in humans but is not detected in clinical immunoassays for toxin A.

The effect of cold stress on the modulation of vascular adrenergic transmission by acetylcholine.

Acetylcholine acts via presynaptic receptors to inhibit adrenergic neurotransmission in vascular tissue. To test the possibility that this modulation might be altered by hyperactivity of the sympathetic nervous system, rats were exposed to cold stress for 5 days. Rat caudal (tail) arteries were excised, cannulated and perfused at constant flow. Responses to transmural nerve stimulation and/or acetylcholine were measured. In arteries from nonstressed rats, acetylcholine produced a dose-dependent inhibition of responses to nerve stimulation of 10 Hz. Likewise, acetylcholine (32 ng/ml) produced a frequency-dependent inhibition to nerve stimulation. Cold stress elevated sympathetic nerve activity in the tail artery as indicated by increased tyrosine hydroxylase activity. Responses to exogenous norepinephrine alone were not different between arteries from cold-stressed and nonstressed rats. In arteries from cold-stressed rats, acetylcholine inhibited the response to nerve stimulation in a dose-dependent manner and at each dose of acetylcholine the inhibition was greater than in arteries from nonstressed rats. Likewise, the inhibition of the frequency-dependent responses to nerve stimulation by acetylcholine was greater in arteries from cold-stressed than from nonstressed rats. These data show that after chronic elevation of vasomotor tone, acetylcholine is a more effective modulator of neurogenic tone which indicates the development of the functional equivalent of supersensitivity of presynaptic receptor-mediated events.

Reducing elevator energy use: A comparison of posted feedback and reduced elevator convenience.

The effects of two different procedures for reducing elevator energy use were assessed using a multiple-baseline design. In the first procedure, feedback about the amount of energy consumed by the elevators each week was posted on each elevator door. Later, signs advocating the use of stairs to save energy and improve health were posted next to the feedback signs. In the second procedure, the time required to travel between floors was increased by adding a delay to the elevator door closing mechanisms. Results indicated that neither feedback alone nor feedback plus educational signs reduced the amount of energy consumed by the elevators. However, use of the door delay reduced consumption by one-third in all elevators.A second experiment replicated the effect of the door delay on energy consumption and, in addition, demonstrated that the door delay also produced a reduction in the number of persons using the elevator. The second experiment also showed that, following an initial period during which a full delay was in effect, a gradual reduction of the delay interval resulted in continued energy conservation. Reduced convenience as a general strategy for energy conservation is discussed.